RESEARCH REPORT 1996-1997

Recently we investigated the immunomodulatory activity of the peptides related to the two exposed loops of the molecules of proteins belonging to the transforming growth factor b (TGFb) family. We found that these proteins may differ in their immunomodulatory activities; whereas TGFb2 seems to be the strongest immunosuppressor of the whole family, TGFb3 may produce immunostimulative effects rather, and TGFb1 possesses both immunostimulative and immunosuppressive potency, which reside in two different loops of the protein molecule. The immunosuppressive activity of TGFb2 is connected mainly to its Lys-Thr-Pro-Lys-Ile (94-98) fragment. Continuing our research on the immunomodulatory activity of the peptides related to the proteins of TGFb family, we have synthesized and investigated by Jerne’s plaque forming cell (PFC) test and delayed type hypersensitivity (DTH) test the following linear peptides and their cyclic (disulphide bridged) analogs:

KTPKI (Ia) and CKTPKIC (Ib)

YIGKTP (IIIa) and CYIGKTPC (IIIb)

YIGKTPKI (IVa) and CYIGKTPKIC (IVb)

YYIGKTPKIE (Va) and CYYIGKTPKIEC (Vb)

It has been found that the insertion of the sequence of Ia into the disulphide bridged Ib leads to the product with enhanced immunosuppressive activity. The similar procedure applied to IIIa, IVa, and Va produces the cyclic peptides devoided of activity or of diminished activity in DTH test. The exchange of L-Pro residue of Ia for D-Pro resulted in the analog IIa with preserved immunosuppressive activity. The disulphide bridged peptide IIb, a cyclic analog of IIa, demonstrated the same activity as IIa in both PFC and DTH tests.

*Department of Chemistry, University of Wrocław, Wrocław, Poland

Publication no. 3431

In the past few years we started a search program for new peptide immunomdulators, looking for thymopentin-like (TP5-like) structures within the sequences of important immunoregulatory and defense proteins. Thymopentin is a pentapeptide with the sequence: Arg-Lys-Asp-Val-Tyr. It is an active fragment of a thymus polypeptide - thymopoietin II, a very well known immune system stimulant. Taking into account the sequence homology existing between thymopoietin II and the DNA-binding domain of p53 protein a series of octapeptides related to the wild p53 type protein, as well as to its mutated forms, appearing in some human tumors were synthesized. The wild-type octapeptide has immunostimulative activity with regard to the humoral immune response, but is inactive in the cellular immune response. The mutated peptides of p53 differ in their immunomodulatory activity from the wild-type octapeptide. The Ser5 analog of the wild-type peptide is a strong stimulant of the humoral immune response and enhances TNF-a production while and at the same time suppressing the cellular immune response. The data suggest that the mutations of p53, which favour the tumor development and growth, may also change the immune activity of respective p53 fragments.

*Department of Chemistry, University of Wrocław, Wrocław, Poland

Publication no. 3352

Thymopentin (TP5), a pentapeptide fragment of thymopoietin II with a sequence: Arg-Lys-Asp-Val-Tyr, is used in the clinics for the infection prophylaxis in cancer patients. Therefore, a search for the active analogs of TP5 is an important goal of the peptide chemistry.

The immunomodulatory activity of the disulphide bridged heptapeptide Mpa-Arg-Lys-Asp-Val-Tyr-Cys-NH₂ (CTP-1), containing the thymopentin (TP5) sequence (Arg-Lys-Asp-Val-Tyr), was investigated by different immunological tests. The activity of CTP-1 was compared with the activities of two other disulphide bridged TP5 analogs: Mpa-Arg-Lys-D-Pro-Val-Tyr-Cys-NH2 (CTP-2) and Mpa-Arg-Lys-Asp-Val-Pro-Cys-NH2 (CTP-3). The results obtained indicate that from the whole series of TP5 analogs CTP-1 may be of interest from the point of view of its prospective medical use. The results of autologous rosette forming cells (ARFC-test), as well as the direct examination of the influence of CTP-1 on the interleukin-1 (IL-1) production by P-388 D1 cell line evidence that the mechanism of CTP-1 action consists in the stimulation of IL-1 production. The results of E-rosette assay, ARFC test, and imuran test show that the activity of CTP-1 is even stronger than that of TP5.

*Ferring AB, Malmö, Sweden;

#Department of Chemistry, University of Wrocław, Wrocław, Poland

Publication no. 3432

A few years ago we have found that cyclolinopeptide A (CLA) possesses strong immunosuppressive activity. Following this finding we synthesized and examined for immunosuppressive activity a substantial number of CLA analogs containing, e.g., the threonine, glycine, and alanine residues substituted in definite positions of the peptide chain. The linear analogs resulting from the splitting of the CLA ring in nine successive amide bond positions, as well as the analogs in which the CLA sequence was installed into the heterodetic ring by disulphide bridge formation possesses also immunosuppressive activity.

A series of nine linear and nine cyclic analogs of CLA, in which the Phe residues were substituted by the aromatic D-residues (D-Phe, D-Tyr, D-Trp) or by the L-Trp residue, was synthesized by solid phase peptide synthesis (SPPS) method. The peptides were tested for their influence on humoral and cellular immune response in mice. Most of the analogs show some immunosuppressive activity similar to that of CLA. High biological activity was observed for the cyclic: peptides modified in position 9, especially [D-Phe9]CLA. This corresponds well with the peptide circular dichroism (CD) spectrum, which shows that backbone conformation of this analog resembles most the conformation of cyclolinopeptide A.

*Department of Chemistry, University of Wrocław, Wrocław, Poland

Publication no. 3442

Cyclolinopeptide A (CLA) is a highly hydrophobic cyclic nonapeptide of the sequence c-(Leu1-Ile2-Ile3-Leu4-Val5-Pro6-Pro7-Phe8-Phe9), isolated from linseed oil. We found that the cyclolinopeptide possesses a strong immunosuppressive activity. The use of this peptide in biological experiments or as a possible therapeutic agent is limited owing to its low solubility in water solutions. In our search for better soluble CLA analogs we have synthesized a series of six linear and cyclic nonapeptides in which one or both phenylalanine residues were substituted by their sulfonated analogs.

Linear and cyclic analogs of CLA in which one or both phenylalanine residues in fragment Pro6-Pro7-Phe8-Phe9 were substituted by their sulfonated derivatives have been synthesized by SPPS method and cyclization with the BOP reagent. The peptides were examined for their immunosuppressive activity in the humoral and cellular immune response by PFC and DTH tests. All of the analogs retain some immunosuppressive activity of native CLA. Their CD spectra confirm that the optical activity of aromatic residues in CLA depends on their position in the peptide chain. Only the residue in position 8 seems to be optically active. CD spectrum of the cyclic analog modified in position 9 is very similar to that of native CLA which correlates with its high biological activity. The chiroptical properties of the p-sulfonated Phe-residue are established.

*Department of Chemistry, University of Wrocław, Wrocław, Poland

Publication no. 3443

Thymopoietins I and II are two closely related 49-amino acid peptides, which were isolated from bovine thymus. They influence differentiation of thymocytes, which leads to the emergence of different subclasses of T cells after contact with antigen. Thymopoietins affect also the neuromuscular transmission, evoking the abnormalities similar to those observed in the human disease, myasthenia gravis.

During our search for thymopentin-like sequences present in the molecules of some regulatory and defence proteins we found in the molecule of the muscle contractile protein, G-actin, a discontinuous thymopentin-like motif. A discontinuous thymopoietin-like motif, composed of the fragments 97-111 and 277-307 of the molecule, as well as of residues Arg178 and Asn163 of G-actin. It was established that G-actin has an immunosuppressive activity regarding the humoral immune response. This activity is probably connected to the thymopentin-like sequence RKDLY, which is present in the 277-307 fragment of G-actin. The immunomodulatory activity of a series of peptide-partial sequences of G-actin was tested using plaque forming cell (PFC) and delayed type hypersensitivity (DTH) test. The investigated series consisted of five peptides: RKDLY (I), RKDLYANT (II), DVDIRKDLY (III), DVDIR (NO2)KDLY (IV), DVDIRKDLYANT (V). The peptides have the immunosuppressive activity with regard to the humoral and cellular immune response.

* Department of Chemistry, University of Wrocław, Wrocław, Poland

# Institute of Experimental Biology, Polish Academy of Sciences, Warszawa, Poland

Publication no. 3501

Recently, we initiated a search for new oligopeptide immunomodulators by looking for thymopentin-like fragments of the molecules of important immunoregulatory proteins. From such fragments, these exposed on the surfaces of the protein molecules, which may create the interaction sites with the cellular receptors, were selected by us in lactoferrin (LF) and in the proteins of the transforming growth factor b (TGFb) family. It has been found that some of the fragments possess immunosuppressive activity. Similar studies of class II human leukocyte antigens (HLA-II) were undertaken. Class II human leukocyte antigens (HLA-II) are cell surface ab heterodimers (Mr » 60 000) that play a pivotal role in the immune response by presenting peptides derived from environmental antigens to the T-cell receptor. A 167-171 fragment of the b2-chain of the HLA-DQ molecule consists of the sequence RGDVY, which is very similar to thymopentin pentapeptide RKDVY (an active fragment (32-36) of thymopentin, an immune system activator produced in thymi), and at the same time contains the RGD sequence, known as an inhibitor of adhesion processess. We synthesized and investigated the immunomodulatory activity of series of peptide fragments of HLA-DQ containing thymopentin-like sequences. All synthesized peptides suppress the cellular immune response. However, RGDV, RGDVY, and QRGDVY show very weak stimulatory activity in the humoral immunological response tests. In contrast to the shorter peptides, the nonapeptide fragment of HLA-DQ, TPQRGDVYT, shows significant immunosuppressive activity in all tests. The smallest size fragment of HLA-DQ, having both cellular and humoral immunosuppressive activity, is the hexapeptide: RGDVYT. We also found that linear and cyclic fragments of HLA-DQ do not affect cell line production of various cytokines, which suggests that the mechanism of interactions of these peptides with the immunological system is different as compared with most other known immunosuppressors. We tested an influence of the nonapeptide and its shorter fragments on binding of activated platelets and K562 cells to fibrinogen and fibronectin, respectively. We also designed and synthesized a cyclic thymopentin-like peptide. C*RGDVYC* (where C* indicates Cys participating in disulphide bridge) to restrict its conformation. The cyclization product strongly suppresses the humoral and cellular immune response and selectively inhibits the adhesion of K562 cells to fibronectin. The results are discussed in the light of CD conformational studies. A possible role of the fragments of the polypeptide chain of HLA-DQ in the regulation of HLA functions is suggested.

*Department of Chemistry, University of Wrocław, Wrocław, Poland

#Institute of Physiology and Biochemistry, Medical Academy, Łódź, Poland

Publications no. 3422, 3423, 3506

The interleukin-1 family consists of three different but structurally related proteins, interacting with the same cell surface receptors. Two of them, interleukin-1a (IL-1a) and b (IL-1b), elicit biological response, whereas the third protein, interleukin-1 receptor antagonist (IL-1ra), acts as a competitive inhibitor of IL-1 action.

IL-1 plays a key role in many immune and inflammatory processes. The inflammatory effects of IL-1 consist mainly in the induction of IL-2 production by the target cells; IL-2 together with tumor necrosis factor a (TNF-a) plays the main role in inflammation. Therefore, the down-regulation of IL-1 production in living organisms or the inhibition of IL-1 cellular receptors by specific inhibitors, like IL-1ra, may be used in anti-inflammatory therapy.

In order to find the low-molecular-weight interleukin 1 (IL-1) inhibitors we synthesized a series of peptides, derived from three regions of interleukin-1 receptor antagonist (IL-1ra); N-terminal (residues 5-9), central (90-98) and C-terminal (143-148). The decision was based on the thorough analysis of structural and functional properties of IL-1 proteins and the resemblance of some fragments of IL-1ra to well-known immunomodulators, like thymopentin and tuftsin. The competition between our peptides and IL-1 was measured as the inhibition of IL-1-induced IL-2 production in LBRM/CTLL cell line system. The activity of tuftsin (TKPR), a peptide immunomodulator derived from IgG molecule, was also examined. All peptides presented some activity, although the most interesting results (when the range of activity and dose-dependence were taken into account) were obtained for tuftsin and peptide VTKFYF from the C-terminal part of IL-1ra, which is in agreement with the latest reports on the structure of IL-1ra - receptor complex.

We also examined the immunomodulatory properties of the peptides from interleukin 1 receptor antagonist (IL-1ra) with regard to the humoral (plaque forming cells - PFC) and cellular (delayed type hypersensitivity - DTH) immune response and GvH reaction. It was found that peptide RKSSK (II) from the N-terminal part of IL-1ra, although inactive with regard to the inhibition of IL-1-IL-1 receptor interaction, reduces immune response in a manner similar to cyclosporin A (DTH, PFC in vivo). Peptide GRKSSK (III) was even more potent, whereas peptides from respective fragment of mouse IL-1ra were weaker immunosuppressant than II. Peptide VTKFYF (VII) from the C-terminal part of IL-1ra, very active as IL-1 inhibitor, and its analog VIII with Asp residue, characteristic for IL-1, instead of Lys from IL-1ra, showed only limited activity despite the previously observed competition with IL-1 for the cellular receptor. Thus, no correlation between the inhibitory and immunomodulatory properties of peptides derived from IL-1ra was observed.

*Department of Chemistry, University of Wrocław, Wrocław, Poland

Publications no. 3458, 3513

Demand for therapeutic agents, able to restore a normal immune response in immunocompromised patients (primary and acquired immunodeficiency), has led to the discovery of a number of substances, collectively defined as immunomodulators.

Search for new bioactive compounds of heterocyclic, isoxazole structure has accelerated in the recent years owing to introduction of new drugs from this group and advanced clinical studies. Our investigations led us to synthesis and demonstration of biological activity among isoxazole derivatives.

A series of 5-aminomethinimino-3-methyl-4-isoxazolecarboxylic acid phenylamides has been prepared by condensation of 5-amino-3-methyl-4-isoxazolecarboxylic acid phenylamides with trichloroacetic aldehyde, alcoholysis of obtained trichloro derivatives and reaction of the products with an appropriate amine. The compounds obtained were evaluated for their immunological activity. The properties of three compounds inhibited the immune response in all possible ways diminishing humoral immune response, cellular immune response or both types of immune response. The last compound is comparable in its effectiveness to cyclosporine A (CsA), so it may be potentially used as an agent for prolongation of the function of transplanted organs. Two other compounds may potentially be used in cases where only one type the immune response is required for combating pathogen invasion.

Five new 5-amino-3-methyl-4-isoxazolecarboxylic acid amides were also obtained. All new structures were equipped with markedly different groups of electron acceptor character, different special structure and nitrogen heteroatom enabling formation of salts and, and the same time, a higher biological accessibility. They were examined for immunomodulating activity in comparison with CsA. We investigated effects of the compounds on the lipopolysaccharide (LPS)-induced production of TNF-a and IL-6 by human peripheral blood cells. A higher, than CsA, suppressory action was found which correlated with stronger electronoacceptor nature of amide substituent. Quite different properties were exhibited by two compounds, characterized by flat aromatic rings. Much higher activity was demonstrated by compounds containing -NH- group, which conditioned immunostimulatory activity in other compounds.

*Department of Organic Chemistry, Medical Academy, Wrocław, Poland

#Department of Chemistry, University of Wrocław, Wrocław, Poland

Publication no. 3498

Quinazolinone-4 derivatives have been found to be biologically versatile compounds, having antimalarial, hypnotic, anticonvulsant, antipyretic, analgesic, antiinflammatory, diuretic, antihypersensive, antitubercular (febrifugine), bronchodilator and other diverse activities. Over 30 drugs, derivatives of quinazolinone-4, have been registered all over the world so far, most of them are diuretics and drugs of/for the Central Nervous System.

The aim of this study was to examine a possibility of the synthesis of novel derivatives of 6-oxo-1,4,5-thiadiazin[2,3-b]quinazoline and the products of the reaction of 3-amino-2(1H)-thioxo-4(3H)-quinazolinone (I) with selected a,b unsaturated carbonyl compounds.

Another goal of this study was to investigate effects of the synthesized compounds on the development of humoral and cellular immune response.

The synthesis of two series of derivatives containing the quinazilinone-4 moiety was undertaken. 3-Amino-2(1H)-thioxo-4(3H)-quinazoline was subjected to reactions with halogenoketones and halogenoaldehydes under different experimental conditions and the following products were obtained: ketones aldehydes, Schiff bases and 6-oxo-1,4,5-thiadiazin[2,3-b]quinazoline derivatives. I was condensed with selected a,b-unsaturated carbonyl compounds, aldehydes, ketones, acid chlorides, esters to yield different final products depending on the substituent at the double bond, carbonylic groups and experimental conditions. The compounds were also tested for their potential activity in the model of humoral and cellular immune response. Immunological tests showed that the compounds exhibited differential immunotropic activities. Of a particular interest was one compound, exhibiting a strong stimulatory activity with regard to cellular immune response, and another one exerting a strong inhibitory action in both types of the immune responses. These drugs may find a potential application in therapy.

*Department of Technology of Drugs, Medical Academy, Wrocław, Poland

Publication no. 3485

Our studies on the seleno-organic compounds were focused at their activities as modest cytokine inducers in human peripheral blood leukocyte culture. Our bioassays used in the screening methods were based on the quantitative determinations of mainly two types of cytokines: interferons (IFNs) and tumor necrosis factors (TNFs). More recently we have found that several of the compounds have direct immunotropic actions in vitro and in vivo, in mice and in chickens. The data related to the cytokine-inducing activity of 65 seleno--organic compounds divided into 4 groups according to their chemical structures. The reference compound was ebselen, the well known experimental drug with various biological activities. Approximately 50% of the compounds were found to be active in our bioassays. The selected compounds induced also IL-6 and GM-CSF. Their activities were clearly correlated with defined chemical structures as well as with the presence of selenium. We suggest that some of the selected by us compounds, other than ebselen, are interesting as immunostimulants and potential antiviral agents and cytokine inducers active in humans.

* Institute of Organic Chemistry, Biochemistry and Biotechnology, Technical University of Wrocław, Poland

Publication no. 3369

We have found that many synthetic seleno-organic compounds including ebselen have immunotropic activity. These studies were designed to assess the effect of the analog of ebselen bis[2-pyridyl (2-carbamoyl) phenyl]diselenide (AE-22) on human leukocytes that may express various activation state. The cells were obtained from bronchoalveolar lavages (BAL cells) of patients with different inflammatory lung diseases. The AE-22-treated BAL cells from patients with bronchial asthma (n=6) and with small cell lung cancer (SCLC) (n=6) were compared with these in the peripheral blood leukocytes (PBL) from the same donors. Control group comprised five patients who underwent diagnostic examination and were free of any cancer or concomitant diseases. The secretion of TNF-a, IL-6 and IFN-g was considered as markers of BAL or PBL cells activation. Different response of the cells and various effects of AE-22 were observed in relation to the origin and functional state of leukocytes. It was established that AE-22 can induce TNF-a, IL-6 and IFN-g in the dose dependent manner in BAL cells and PBL isolated from healthy individuals. However, BAL cells were found to be less reactive as the cytokine producers than PBL. In contrast, AE-22 had no effect on the BAL cells obtained from patients with lung cancer that were found to be hyporeactive to phytohemagglutinin and bacterial lipopolysaccharide also and did not produce TNF-a, IL-6 or IFNs, spontaneously. On the other hand, the spontaneous release of cytokines by BAL cells from bronchial asthma patients, but not by PBL from the same individuals, was significantly (p<0.01) higher than that from the control cultures of healthy subjects. The high secretion of cytokines by the locally activated BAL cells was significantly (p<0.01) reduced after the administration of AE-22. The results suggest that AE-22 has the immunomodulatory activity. AE-22 can down-regulate the hyporeactive BAL cells from asthmatics but it appears to be inactive in the BAL cell of cancer patients that are tolerant to the cytokine inducers.

*Department of Lung Diseases, Military District Clinical Hospital, Wrocław, Poland

Publication no. 3444

A convenient synthesis of the 2-carboxyalkyl-1,2-benzisoselenazol-3(2H)--ones and their esters from 2-(chloroseleno)benzoyl chloride and amino acids or their carboxy esters is reported. In similar way other 2-substituted 1,2-benzisoselenazol-3(2H)-ones were synthetized. The related bis[2-(carbaomyl) phenyl] diselenides were obtained by reductive conversion of 1,2-benzisoselenazol--3(2H)-ones or directly by the reaction of bis[2-(chlorocarbonyl) phenyl] diselenide with compounds having a primary amino group. It was found that some of compounds and are modest cytokine (TNF, IFN) inducers in human peripheral blood leukocyte cultures and block the constitutive endothelial nitric oxide synthase (ce NOS).

*Institute of Organic Chemistry, Biochemistry and Biotechnology, Technical University of Wrocław, Poland

#Collegium Medicum Jagiellonian University, Department of Pharmacology, Kraków, Poland

Publication no. 3400

A proline-rich polypeptide (PRP), now named colostrinine, molecular weight 18 000, was isolated from ovine colostrum and characterized by Janusz, Lisowski et al. The nonapeptide (NP) which is an active fragment of PRP was obtained by chemical synthesis. In mice, PRP has many regulatory effects on the humoral and cellular immune response. The present work describes PRP as a cytokine inducer. PRP at concentration of 1-100 mg/ml was found to induce production of interferon (IFN) and tumor necrosis factor (TNF) in human peripheral blood leukocytes and in whole blood cultures. The effects were dose related. The identified till now cytokines induced by PRP were IFN-g and TNF-a but many other cytokines may be also stimulated. NP was considerably less active as the cytokine inducer than the natural PRP. Two volunteers given orally once daily for two to three weeks 100 or 200 mg PRP in tablets were found to develop a tolerance to IFN induction and had a modified TNF response. Taken together, our observations suggest that ovine PRP is active in humans and may have therapeutic value as an immunostimulant.

Publication no. 3368

We studied the effects of ovine colostrinine (proline-rich polypeptide, PRP) on interferon (IFN) and tumor necrosis factor (TNF) production by murine resident peritoneal cells (RPC). The cells from several mouse strains have been found to produce small amounts of IFN-b and TNF-a, constitutively. The colostrinine at concentration of 1-100 mg/ml of cell suspension containing 1x106 RPC isolated from BALB/c mice, enhanced the IFN and TNF production by 3-30 folds. Upregulation of TNF and IFN production has been observed in the RPC cultures that produced spontaneously less than 16 units of the cytokines only. Synthetic nonapeptide fragment of the colostrinine (Val-Glu-Ser--Tyr-Val-Pro-Leu-Phe-Pro) at concentration of 1-100 mg/ml stimulated TNF synthesis but not IFN production.

Publication no. 3438

It was previously shown that resident peritoneal cells (RPC) from BALB/c female mice express a constitutive, non-specific antiviral immunity. The immunity is reduced progressively during several days of cell culture in vitro. Now we studied the effect of a proline rich polypeptide (PRP), isolated from ovine colostrum, on the kinetics of VSV replication in freshly isolated and one day cultured RPC. The polypeptide was added to cell culture either immediately after virus adsorption, one day before or after viral infection. Independently on time of PRP addition, the effect of inhibition (up to 4 log) of VSV replication was observed. Occasionally, however, a weak effect of stimulation (1-2 log) of VSV replication by PRP was noticed in cells constitutively resistant to the infection.

Publication no. 3545

Immunoregulatory proline-rich polypeptide(s) - PRP isolated from ovine colostrum and its active nonapeptide (NP) fragment induce in murine thymocytes changes in expression of surface glycoconjugates (e.g. transformation of PNAhigh into PNAlow thymocytes, and vice versa; changes in autologous rosette formation). This may affect intercellular interactions and adhesive properties of thymocytes. The aim of our work was to study effects of PRP and NP on adhesive properties of murine thymocytes. We have studied adhesion of the cells to plastic plates, interthymocyte interaction, and interaction of thymocytes with the endothelial cells. The experiments were performed both on PNAhigh and PNAlow thymocytes. The results obtained showed no significant effect of the peptides on intercellular interactions and on the effect on adhesive properties to plastic plates. The peptides increased the number of PNAlow cells interacting with fibronectin whereas they lowered the number of PNAhigh thymocytes interacting with fibronectin. However, we assume that adhesive properties of only a small subpopulation of thymocytes was affected by PRP and NP.

A proline-rich polypeptide(s) - PRP isolated from ovine colostrum shows immunoregulatory activity. PRP induces secretion of TNF and IFN-g. It was of interest to investigate whether there are in human colostrum factors resembling PRP from the ovine colostrum. It was found that human colostral IgG are accompanied by polypeptides with Mr of 6-12 KDa. The concentration of the polypeptides is the highest on a third day after the delivery. Determination of the amino acid compositions showed that the human peptides also contain high proportion of proline residues, similarly as in the case of ovine PRP. The human peptides also have the ability to induce secretion of IFN-g and TNF. The human peptides, similarly as in the case of ovine PRP, might be important for the regulation of the maturation, differentiation lymphocytes, and the development of the immune system. The results obtained suggest that colostra of various species may contain peptides important for the development of their immune systems.

We have tentatively identified colostrinines as novel cytokines produced by the mammary gland after delivery and detectable in colostrum. The primary colostrinine, the proline-rich polypeptide, was isolated from ovine colostrum in 1974. It is generally understood that various factors, present in colostrum, play a pivotal role in transmitting passive or active immunity from mother to child. We have previously found that both ovine and human colostrinines are inducers of interferon (IFN) gamma and other cytokines. Now we found that the leukocytes isolated from human colostrum donated by healthy mothers at 1-9 days after delivery, produced interferons (IFNs) and tumor necrosis factors (TNFs), spontaneously. The release of IFNs and TNFs coincided with production of a colostrinine that has been isolated from the human colostrum samples and partially characterized. Our results suggest that the maximal production of colostrinine occurs 3 days after delivery. The tolerance/hyporeactivity of the colostral leukocytes to IFN inducers and the modulation of the TNF response may be the late effects of the colostrinine release.

*Department of Obstetrics and Gynecology, Regional Specialistic Hospital, Wrocław, Poland

Publication no. 3492

Lactoferrin (LF) is an iron-binding protein with a broad spectrum of biological activities. LF present in secretory fluids of mammals and secondary granules of neutrophils plays an important role in host defence against microorganisms and bacterial products and in the regulation of the immune response. LF has been shown to promote maturation and functional development of T and B cells. The protein modulates also activities of several cytokines such as IL-1, TNF-a, GM-CSF and IL-6 and activity of natural killer cells.

The aim of this study was to find out whether LF will inhibit effector functions and phenotype of antigen specific TH1 and TH2 cells. We found that bovine (BLF) and human (HLF) lactoferrin inhibit proliferation and cytokine production by an antigen specific TH1 cell line stimulated with antigen presenting cells (APC) and antigen. Beside the inhibitory effects on TH1 cells we observed a decrease of interleukin 2 receptor (IL-2R) expression on these cells contacted with LF. Lactoferrin did not inhibit proliferation of indicator HT-2 cells to IL-2 suggesting lack of interference with IL-2/IL-2R interaction by LF. No inhibitory effect was observed on proliferation and cytokine production by TH2 cell lines and no changes in interleukin 4 receptor (IL-4R) levels were found with regard to TH2 cell line. We conclude that LF has differential activity with regard to effector functions of antigen-specific T cells by inhibiting TH1 but not TH2-mediated immune responses.

*Laboratory of Biological Chemistry, University of Sciences and Technology, Lille, Villeneuve d’Asq., France

#Winship Cancer Center, Emory University, School of Medicine, Atlanta, GA, USA

Publication no. 3434

The factors affecting proliferation of lymphoid progenitor cells are important in normal cell differentiation as well as in developing hematological malignancies. The lactoferrin (LF) binds to human activated lymphocytes, the cells of macrophage/monocyte lineage and to other cell types. However, the biological response due to LF binding to cell receptors has not been definitely established yet.

The effect of different forms of human LF on proliferative response and differentiation of Jurkat human lymphoblastic T cell line was studied. Lactoferrin enhanced cell proliferation in serum-reduced growth medium, but not in the presence of 10% fetal calf serum. The stimulatory effect depended slightly on the degree of iron saturation of LF. These results suggest that LF can substitute for Fe-transferrin in a low serum environment. Presence of iron saturated LF resulted in the appearance of CD4 and downregulation of CD71 antigens on the cell surface. It may be concluded that, in the presence of LF, entry of a quiescence state of previously proliferating cells may be accompanied by cell differentiation. The mechanism of induction of T cell differentiation by LF still remains to be elucidated.

*Laboratory of Biological Chemistry and UMR no. 111 CNRS, University of Sciences and Technology, Lille, Villeneuve d’Asq., France

Publication no. 3437

Interaction of ligands with receptors induces, in many systems, activation of kinases and phosphoprotein phosphatases. Similar effects are also observed in the case of Fc receptors.

The aim of the presented studies was investigation of a role of kinases and phosphoprotein phosphatases in the process of insolubilization of the guinea pig peritoneal macrophage Fcg receptors, in induction of the signal transmission mediators, and in the effector activity of macrophages. The effect of cross-linking of the Fcg receptors on phosphorylation and dephosphorylation of macrophage proteins was also studied.

Cross-linking of cell receptors induces in many ligand-receptor systems an association of the receptors with the membrane skeleton and/or cytoskeleton. This association is reflected in a decrease of solubility of the receptors in nonionic detergents. This effect was used as an indication of an interaction of membrane proteins with the skeleton.

Studies on insolubilization of the macrophage Fcg receptors carried out on cells preincubated with various metabolic inhibitors: okadaic acid (2 mM) and genistein (100 mM) showed that insolubilization of complexes of guinea pig IgG1 and of rabbit IgG is inhibited by genistein, an inhibitor of tyrosine kinases. The results showed an important role of phosphorylation in association of the receptors with the cytoskeleton. The presence of genistein enhanced the association of the receptors with the membrane skeleton and lowered the interaction with the cytoskeleton.

Cross-linking of the guinea pig peritoneal macrophage Fcg receptors induced a release of free Ca2+ ions in macrophages. The release, in the case of guinea pig IgG2 and rabbit IgG, was inhibited by genistein. In the case of guinea pig IgG1, the release of Ca2+ ions was inhibited by okadaic acid, an inhibitor of phosphoprotein phosphatases.

Phosphorylation of macrophage proteins was dependent on the degree of cross-linking of a receptor binding guinea pig IgG2 or rabbit IgG. However, the cross-linking of the FcgR by ligands containing guinea pig IgG1 did not affect the phosphorylation of the macrophage proteins.

All immune complexes were endocytosed by macrophages in a similar degree. Genistein inhibited endocytosis of complexes containing guinea pig IgG1 or rabbit IgG, only.

The results obtained showed differential effect of kinases and phosphoprotein phsphatases in signal transmission and effector function of the guinea pig peritoneal macrophage Fcg receptors. Moreover, our results indicated that, depending on a subclass and origin of immunoglobulins and on the type of an Fcg receptor on macrophages, phosphorylation and dephosphorylation processes of cell proteins play more or less important role in signal transmission and in effector function of macrophages.

Sialylated Le strucures present on the surface of tumor cells are carried by the carbohydrate chains of glycoproteins and glycolipids. In contrast to leukocytes, no specific counter-receptors were found so far, which mediate the attachment of cancer cells to E- and P-selectins. The effective binding seems to be dependent on the total number of sialylated Lewis oligosaccharides available on the cell surface. To evaluate which cellular glycoconjugates carrying sialosyl Lea antigen play a role in adhesion of colon cancer cells, glycoproteins as well as glycolipids of human bladder cancer and colon carinoma cells were studied. It was found that human urothelial cell line Hu 1703He, which specifically binds to E-selectin-expressing CHO cells, is characterized by the presence of several sialosyl Lea-carrying glycoproteins with apparent molecular masses from 100-250 kDa (it was found previously that this cell line contains large amount of sialosyl Lea-ganglioside). Using O-sialoglycoprotease from Pasteurella haemolytica it was shown that protein-linked sialosyl Lea structures are carried mostly by mucin-type glycoproteins. However, treatment of Hu 1703He cells did not decrease either their binding to E-selectin-expressing CHO cells, or binding of anti-sialosyl Lea antibodies to the cell surface, suggesting that cleveage of sialomucins uncovered cryptic sialosyl Lea-ganglioside, which were unaccessible for the antibody and E-selectin in untreated cells. Essentially the same results were obtained with CX-1.1 colon carcinoma cells with high content of mucins-type glycoproteins and ganglioside carrying sialosyl Lea. In addition, this suggestion was confirmed by higher accessibility of gangliosides to galactose oxidase on the surface of O-sialoglycoprotease--treated CX-1.1 cells, comparing to untreated cells. We propose that glycoproteins as well as gangliosides carrying sialosyl Lea structures, when properly exposed and present in high density on surface of cancer cells can effectively support the adhesion of cancer cells to E-selectin.

Publications no. 3376, 3534

Sialosyl Lea antigen is well known tumor-associated antigen used in diagnosis and evaluation of prognosis in several types of human cancers. It has been found recently that this oligosaccharide is specifically recognized by the members of selectin family, and is involved in adhesion of cancer cells to endothelium. To study whether the adhesion of colon cancer cells to E-selectin can be directly affected by changes in the expression level of sialosyl Lea antigen we created a specific “loss of function” phenotype. Stable subclone (CX-1.1) with high expression of sialosyl Lea structure, obtained from heterogenous population of colon carcinoma CX-1 cells, was transfected with expression vector containing fragment of cDNA for a1,3/4-fucosyltransferase (FT III) in antisense orientation. Among obtained cell colonies resistant to G418, two were characterized by the lack of expression of sialosyl Lea structure, which suggested that these cell clones did not express FT III enzymatic activity. The specificity of enzyme inhibition was analysed on the level of (i) oligosaccharide (product) formation, (ii) enzymatic activity (translation), and (iii) mRNA expression. It was found that the specific lack of expression of sialosyl Lea structure on the surface of colon cancer cells completely abolishes their adhesion to E-selectin. Our data confirmed the thesis that sialosyl Lea oligosaccharides are directly involved in adhesion of colon cancer cells, and point to the a1,3/4--fucosyltransferase as a key enzyme responsible for binding of these cells to E-selectin.

Several lines of accumulating evidence indicate that sialosyl Lea is involved in formation of metastases. To study the role of this carbohydrate structure in development of metastases, we have used the clone (CX-1.1AS5) of human sialosyl Lea-negative colon carcinoma cells transfected with antisense expression vector. The formation of liver metastases by CX-1.1AS5 cells was analyzed after their orthotopic or intraspenic implantation in athymic nu/nu mice. After orthotopic implantation of sialosyl Lea-negative cells, the number of mice with liver metastases was markedly lower (21% of mice) in comparison with their number after implantation of the parental CX-1.1 cells (86% of mice). However, no differences in ability to form colonies in liver were observed between parental CX-1.1 cells and antisense-transfected CX-1.1AS5 cells after intrasplenic injection. The liver metastases were formed in 89% and 84% of mice, respectively. Our data support the thesis on the importance of sialosyl Lea antigen expression in development of liver metastases by colon cancer cells, and indicate the role of transplantation route and primary tumor localization in formation of metastases.

Publication no. 3534

Aberrant glycosylation is a common phenomenon observed in cancer progression. As a consequence, the increased expression of Lewis blood group family antigens, particularly Lex, sialyl Lex, and sialyl Lea has been reported. The aim of the following work was to study the relationship between the Lewis antigens expression, tumorigenicity and metastatic potential of colon carcinoma cells.

The cells of the subline, HT-29EB3, selected in vitro on the basis of their affinity toward human endothelial cells showed (relatively high) expression of Lex, sialyl Lex and Ley antigens. HT-29EB3 cells were successively passaged in nu/nu mice. Transplanted cells and tissue fragments were derived either from liver or lymph nodes metastases. This approach resulted in selection of highly metastatic variants of human colon cancer cells. After intravenous (i.v.) passaging, a LNL2N* variant was obtained which formed tumors selectively in lymph nodes. Another variant, LN3L, obtained after 5 successive passages of cells from liver metastases was growing selectively in liver after i.s. inoculation. The third, LN2L metastatic variant was transplanted by ortothopic route, giving metastatic spread into different organs (liver, lymph nodes, peritoneal cavity).

Our results, together with those described in Report No. 24 imply that the route of cancer cells inoculation in nude mice can influence both local tumor growth and metastases distribution.The orthotopic transplantation of human cancer cells to nude mice offers the experimental conditions more close to clinical situation and proves its usefulness for studies on the pathogenesis of metastasis formation.

*Laboratory of Glycobiology, Center of Molecular Biophysics, CNRS
and University of Orléans, Orléans, France

#Bartholin Institute, Copenhagen, Denmark

Publication no. 3544

Adhesive interactions between cancer cells and microvasculature endothelium are a crucial step preceeding cancer cell extravasation and metastatic growth. The aim of the work was study on target organ specificity of metastasis. Adhesive interactions of cancer cells with endothelial cells of different tissue origin were measured by flow cytometry according to a quantitative method developed by us.

As a model of tumor cells, HT-29 human colon adenocarcinoma cells were used. The first, HT-29B3 variant cells were selected in vitro on the basis of their affinity to HPLNEC.B3 endothelial cells, presented increased expression of Lex, sialyl Lex and Ley antigens. The in vivo selection, by serial xenotransplantation of HT-29B3 cells in athymic nu/nu NCR mice supplied further highly metastatic variants: LN3L, LNL2N and LN2L (described in the Report No. 25). During in vivo passaging the cell variants acquired increased metastatic ability and differential metastatic spread pattern, preserving their relatively high expression of Lex, sialyl Lex and Ley antigens, which arose after the first in vitro selection. It has been shown recently, that these antigenic epitopes may play an important role in cell endogenous lectin-specific carbohydrate ligand adhesive interactions.

As partner cells, human endothelial cells from surgical biopsies of metastatic lymph nodes or other tissues were isolated and established as in vitro cell lines. In culture, these cells retained their morphological as well as phenotypic features of endothelial cells, such as the expression of von Willebrand’s factor, angiotensin converting enzyme (ACE), E selectin as well as their endogenous cell surface lectins. These cell lines constitute an useful tool to study of carbohydrate-dependent organ specific cancer cell-endothelial cell interactions.

Flow cytometry study of adhesion between these metastatic variants and endothelial cell lines cells, originated from lymph nodes or other (lung, ovary) tissue, revealed differential endothelial-cancer cells adhesive interactions. The adhesion pattern correlated with the origin of endothelial cells, with a way of metastatic spread and with the level of expression of cell surface carbohydrate antigens: sialyl Lex, Ley, Tn and sialyl Tn, of cancer cells. These findings show that tissue-specific cell-cell interactions as well as a level of expression of carbohydrate antigens may play an important role in localization of metastasis.

*Laboratory of Glycobiology, Center of Molecular Biophysics, CNRS
and University of Orléans, Orléans, France

#Lower Silesia Oncological Centre, Wrocław, Poland

Preclinical trials indicated that cytokine gene transfected cells appeared to be an usefull tool for anticancer therapy. In our study we have used IL-2 gene transfected fibrosarcoma cells to test this cytokine as a modulator of host’s antitumor response. The IL-2 gene transfectants synthesize mIL-2 mRNA and secrete this cytokine. Transfection did not influence FGFR1mRNA expression and synthesis of basic FGF known angiogenic and tumor promoting factor. Release of IL-2 by IL- 2 gene transfected fibrosarcoma F 69-3 cells resulted in the decrease of their tumorigenicity in euthymic, syngeneic recipients and in delay of growth in athymic mice.

FGF-1 stimulated DNA synthesis and induced expression of IL-2 receptors in the murine fibrosarcoma cell line, F69-3. Concomitant treatment with IL-2 abolished the stimulation of DNA synthesis, but not binding of FGF-1 to the FGF-receptors or subsequent endocytosis of the bound growth factor. Also, it did not inhibit activation of the FGF-receptor tyrosine kinase or stimulation of the downstream effector, MAP kinase. Treatment with IL-2 prevented transport of FGF-1 to the nuclear fraction in a time- and dose-dependent manner that paralleled the inhibition of FGF-1 stimulated DNA synthesis. The data support our earlier finding that transport of FGF-1 to the nucleus is required for stimulation of DNA synthesis, and they demonstrate that treatment with a cytokine can modulate the cellular response to a growth factor.

*Department of Biochemistry, Institute for Cancer Research,
The Norwegian Radium Hospital, Oslo, Norway

Tumorigenesis is a multistep process gradually freeing cells from social constraints by accumulating mutations, that interfere with normal cellular responses to the regulatory signals of the microenvironment. This view implies that cells undergoing oncogenic transformation are initially susceptibile to most physiological signals regulating growth and differentiation of their normal, nontransformed counterparts. In the thymus, survival and differentiation of immature thymocytes is regulated by positive and negative selection processes involving interaction of the ab T cell receptor with major histocompatibility complex (MHC) molecules. The influence of ab TCR-mediated selection on the development of spontaneous thymic lymphoma which appear in mice expressing a transgenic TCR specific for male antigen (HY) in the context of H-2Db molecules was analyzed. The molecular mechanism underlying the observed oncogenicity of TCR transgens is not yet known. Phenotypic and functional analysis suggested, that lymphomas originated from the stage of pre-TCR-dependent transition of immature CD4-CD8- to CD4+CD8+ thymocytes, which is accompanied by vigorous proliferation of cells expressing functional TCR b chain and beginning to rearange the endogenous TCR a locus. Transforming events initiated by TCR transgens do not seem to “fix” the affected cell at its developmental stage, as evidenced by the ability of some CD4-8- and CD4-CD8+ lymphoma cells to change their phenotype according to the differentiation program of T cell lineage. Lymphoma cells in TCR anty--HY/Db transgenic H-2b females (positive selection) and males (negative selection) developed into tumors under different environmental pressures. The results obtained in the TCR transgenic mouse model suggest that self antigen--induced negative selection, playing an important role during normal development as a central mechanism establishing self tolerance, can also act as a tumor surveillance mechanism by eliminating or suppressing growth of thymocytes undergoing oncogenic transformation.

*Basel Institute for Immunology, Basel, Switzerland

Publications no. 3396, 3482, 3503, 3504

Mice with transgenic T cell receptor (TCR), recognizing H-Y male antigen, developed spontaneous lymphomas originated from immature thymocytes, with the surface expression of transgenic TCR and CD4/CD8 co-receptors. The role of the antigen receptor-mediated signals in disclosing or suppressing the oncogenic potential of genetically altered (i.e. preleukemic) lymphocytes is poorly understood. It has been suggested that antigen receptor-driven expansion and selection may play a role in the pathogenesis of certain B and T cell lymphomas. Therefore, we were attracted by the observation that some lymphoma cell lines lost the surface expression of TCR/CD3 complex and CD4/CD8 co-receptors during long term culture in vitro. It indicates that surface expression of transgenic TCR is not necessary for the in vitro proliferation. Interestingly, the proteins of transgenic receptor were expressed intracellularly but TCR was not detectable on the surface of the in vitro selected subline in contrast to TCR-positive parental cell maintained in vivo. TCR-negative subline has been found to be slowly growing in vivo and less tumorigenic than parental TCR-positive lymphoma. It seems that the in vivo interactions of lymphoma cells with microenvironment preserve their TCR expression and endow with growth advantage, while the selected in vitro TCR-negative cells lose the tumorigenic potential.

In conclusion, TCR-negative lymphoma cells can be indicative of a background oncogenic potential of TCR transgen. On the other hand, TCR expression in the membrane of genetically altered thymocytes could be important in further steps of tumor progression, playing a role in disclosing in vivo of a full expression of malignant phenotype of transformed cells.

Publications no. 3377, 3459

Plasmocytomas can be induced in susceptible strains of BALB/c mice by the intraperitoneal injection of the chemically defined oil pristane (2.6.10.14--tetramethylpentadecane). This hydrocarbon induces chronic inflammation followed by development of plasmocytoma.

Attempts to study the direct biological influence of pristane on cells in vitro fail because it is completely insoluble in aqueous cell culture media. Therefore, in current experiments the complex of b-cyclodextrin (b-CyD) with pristane (P) for delivery of this hydrocarbon to cells was used.

It was found, that after solubilization of pristane, b-CyD-P complex used in sub-toxic concentration had several effects on the studied cells. It promoted proliferation and differentiation of human promyelocytic HL-60 leukemia cells to acquire a phagocytic activity of macrophages. Secondly, it activated cells of murine B lymphocyte line (P-388D1) to produce high amounts of IL-6 into culture medium. Thirdly, it stimulated in vitro proliferation of spleen cells from pristane-primed BALB/c mice in short-term culture, whereas in the same concentrations it was toxic for spleen cells from intact mice. The spleen cells from pristane primed mice acquired attributes of tumor cells when treated in vitro with b-CyD-P complex in long term cultures. Namely, they have become immortal and grew as the tumor when transplanted intraperitoneally in syngeneic mice.

Two lines of tumor cells (SN-1 and SN-2) were obtained by this procedure. They behave differently: the cells of SN-1 line grow as solid tumor after intraperitoneal injection to mice while cells of SN-2 line grow as ascitic tumor. The cells of SN-2 line produced constitutively IL-6.

Inherited defects in tumor suppressor genes predispose to cancer. Carriers of defective alleles can be identified by molecular techniques, and that facilitates detection of neoplastic changes in target tissues. p53 germline mutations are associated with a very complex cancer phenotype, difficult to be earlier diagnosed in routine clinical examination, and molecular techniques may be particularily useful for identification of individuals carrying mutated allele. Our studies included patients, both of high risk groups: 1. patients from families with increased risk for cancers typical for Li-Fraumeni syndrome, 2. patients with multiple tumors and pediatric patients with randomly arising tumors. Up to now we screened 100 tumor specimens and around 250 blood samples. In the studied tumor samples, we found somatic mutations in exons 5-9, covering all five hot spot regions of p53 gene. Mutations were found e.g. in hepatoblastoma, fibrohistiocytoma mal., lymphoma mal. Mutations found by us correspond well with the spectrum of p53 mutations and tumor types noted in p53 data base, which at present contained 7000 mutations. It has been reported that in pediatric sarcomas the frequency of p53 gene mutations is high in rhabdomyosarcomas and osteosarcomas. In our material in these tumors we did not found mutations. To find out whether in some of the studied tumors p53 function was changed due to other mechanisms, we are now examining the expression of p53.

Studies performed in cultures of normal human fibroblasts, AF1, that carry constitutive (germline) mutation of p53, showed that the presence of single mutated p53 allele is not sufficient to cause malignant transformation of cells or immortal phenotype. However, introduction of mutated p53 to immortal fibroblasts KMST-6 induced their malignant transformation. As most germline p53 mutations seemed to be the effect of 5-methylcytosine deamination, all disturbances in DNA methylation might affect the frequency of gene mutations and cell transformation in general. To study the effect of DNA methylation on transformation, we transfected KMST-6 cells with a plasmid containing a fragment of DNA: methylase in antisense orientation. After transplantation into nude mice transfected cells appeared to be tumorigenic. Changes in the genome caused by transfected sequence of DNA: methylase are now studied.

*Department and Clinic of Pediatric Surgery, Medical Academy, Wrocław, Poland

#Department of Oncology and Clinic of Oncological Gynecology,
Medical Academy, Wrocław, Poland

‡Clinic of Pediatric Oncology, Institute of Mother and Child, Warszawa,
Poland

The prognostic factors predicting the outcome of patients with urinary bladder tumors are still not satisfactory. There are a few reports concerning the correlation between DNA ploidy and the biologic behavior of bladder tumors.

The aim of the study was to look for a relationship between DNA ploidy and the established prognostic factors such as histologic grade, tumor stage (T) and survival. The nuclear DNA content of bladder tumor biopsies and cells from bladder irrigation was evaluated by flow cytometry.

In prospective studies, of 105 patients with bladder carcinoma, 53 tumor tissue specimens were classified as aneuploid, with one or more aneuploid cell populations. A significant correlation was found between T category and DNA ploidy as well as between DNA ploidy and clinical course of the disease: patients with diploid tumors had low recurrence and progression rate during 22 month follow-up.

In retrospective analysis DNA ploidy was checked on formalin-fixed/paraffin-embedded tissue specimens obtained after transurethral tumor resection from 53 patients. Thirty-three patients were found to possess aneuploid tumor cells and 65% of them suffered progression, metastatic disease and died, whereas patients with diploid tumors developed almost no local tumor progression during up to 5 years of follow-up.

The results obtained show that DNA ploidy status could be an useful additional prognostic marker in urinary bladder tumors. DNA ploidy evaluation is more useful in prognosis of survival than histological grading, comparable only with clinical classification of disease.

*Clinic of Urology, Medical Academy, Wrocław, Poland

Publication no. 3380, 3461

The cytotoxic activity in vitro of new agents, both of natural origin and synthetic compounds was tested against the following human tumor cell lines:

Antiproliferative assay was done by SRB and MIT techniques as described by Skehan et al. (J. Natl. Cancer Inst., 82:1107-1112, 1990) and by Mossman (J. Immunol. Meth., 65:55, 1983) in 72-96 hour cultures in vitro.

The following agents and chemicals were tested:

1) Derivatives of indolo[2,3-b]quinolines representing the products of biotransformation by Cunninghamella elegans. All of them appeared to be cytotoxic in vitro against tumor lines used (a-c, e-h) with ED50 varying from 0.04 to 5 mg/ml.

2) Analogs of chimaphilline extracted from traditional medical plant Haerba Chimaphilae used in ethnopharmacology of Vilnius region in Lithuania. All drugs have shown antiproliferative activity in vitro against tumor lines used (a-d) with ED50 varying from 3 to 10 mg/ml.

3) New daunomycine derivatives with expected lower cardiotoxicity and higher antitumor activity synthesized by group of Dr Borowicz. All of them revealed cytotoxic activity in vitro against tumor cell lines used (a-c) similar to those of daunomycine (ED50 varying from 0.12 to 0.36 mg/ml). One of them was selected for further studies in vivo using P388 mice leukemia screening model. These studies are actually in course.

4) New rhodamine derivatives synthetised by group of Dr Pruchnik. Some of them revealed cytotoxic activity in vitro against tumor cell line used (i).

5) New derivatives of pyrymido[4,5-b]quinolines. Some of them have shown cytotoxic activity against tumor cell lines used (e, j-l).

6) New derivatives of dacarbazine. Some of them have shown cytotoxic activity against tumor cell lines used (e, j-l).

*Institute of Organic Chemistry, Biochemistry and Biotechnology,
Technical University, Wrocław, Poland

#Polish University of Vilnius, Vilnius, Lithuania

‡Institute of Biotechnology and Antibiotics, Warszawa, Poland

§Department of Chemistry, University of Wrocław, Poland

$Department of Organic Chemistry, Medical Academy, Wrocław, Poland

Publications no. 3359, 3416, 3499, 3529

1,25-Dihydroxyvitamin D3 in addition to calcium mobilizing activity in vivo, is able to induce differentiation of human promyelocytic leukemia cells in vitro. The cell differentiating activity of four new analogs of 1,25-dihydroxyvitamin D3 has been studied, using HL60 cell line as a model. We have also analyzed the influence of these compounds on the proliferation of HL60 cells, normal human keratinocytes, normal fibroblasts from human skin and human keratinocytes transfected with human papilloma virus type 16. Both side--chain extended analogs studied appeared to be similar to 1,25-dihydroxyvitamin D3 in cell differentiating and anti-proliferative effects. Analogs with shortened side-chain and with additional hydroxyl in the side chain revealed substantially lower activity than 1,25-dihydroxyvitamin D3. Remarkable differences in sensitivity of cells of different origin to anti-proliferative effect of 1,25-dihydroxyvitamin D3 and its analogs were observed.

*Pharmaceutical Institute, Warszawa, Poland

1,25-Dihydroxyvitamin D3 (1,25(OH)2D3), in addition to its classical role in calcium homeostasis, regulates cell differentiation. The mechanisms involved in mediating numerous functions of 1,25(OH)2D3 are not clearly understood. Besides genomic actions involving nuclear vitamin D receptor (VDR) some rapid nongenomic responses have been observed, but the full signalling pathway activated by 1,25(OH)2D3 has still not been described. Our recent data allow for better understanding of nongenomic effects evoked by 1,25(OH)2D3. In this work we show that mitogen activated protein kinase (MAPK) is activated in HL-60 promyelocytic leukemia cells and in normal human keratinocytes under exposure to differentiation inducing concentrations of 1,25(OH)2D3. The MAPK is then transported to the cell nucleus in active form, which is different from the activation evoked by fetal calf serum (FCS). Experiments utilising tyrosine kinase inhibitor suggested that the postulated putative membrane vitamin D receptor (mVDR), if exists, does not have tyrosine kinase activity. Usage of protein kinase C (PKC) inhibitor allowed to state that PKC is an upstream element in MAPK signalling pathway.

*Department of Biochemistry, Institute for Cancer Research,
The Norwegian Radium Hospital, Oslo, Norway

Publication no. 3480

We have previously found that by means of two peritumoral injections of murine plasmocytoma cells, engineered to secrete mIL-2 (subline X63-mIL-2, non-tumorigenic in BALB/c mice), a significant antitumor response could have been obtained in genetically compatible mice (CD2F1 hybrids) bearing autologous tumors (wild type plasmocytoma, X63/0). When transfected cells were administered to mice with nonrelated tumor (BFS1 fibrosarcoma) a weak antitumor effect was observed which, however, could have been augmented if mice were pretreated with single dose of a cytostatic agent and then with multiple (4-6) injections of transfected cells.

A similar approach was used in our current experiments, conducted in B6D2F1 mice, with subcutaneously growing murine transplantable colon adenocarcinoma 38 (MC38), chemically induced in C57Bl/6 mice. Plasmocytoma cells, as genetically incompatible for these mice, can be regarded as an allogeneic vaccine, or a cellular adjuvant. IL-2 producing cells were administered alone or in combination with chemotherapy which consisted of one or two doses of 200 mg/kg of cyclophosphamide i.p., given 3 days before peritumoral immunotherapy. Usually a series of 5 to 7 p.t. injections (107 cells/mouse in the first injection and 5-7x106 cells in subsequent) were given once per week. The treatment with X63-mIL-2 cells was continued for 4-6 weeks. The therapeutic effects were evaluated using tumor regrowth delay assay (TGD) and life-span prolongation (ILS) parameters, in relation to either untreated mice, or injected with cell vehicle, PBS(-), or with the wild type (non-transfected) plasmocytoma cells. These effects depended on the stage of tumor progression at the moment of treatment beginning. The majority of mice treated with transfected cells on day 3rd after C38 inoculation, that is before formation of visible tumor nodules, remained tumor-free for more than three months (long-term survivors, LTS). When the treatment was delayed to the day 9 or 11 (the volume of palpable tumors was 40-60 mm3) or started later, on the day 16, tumor growth rate was delayed in a fraction of mice (44-80% in different experiments) and complete regressions were observed in 10-22% of mice. These mice remained LTS for more than 6 months. The combination therapy, i.e. CY plus X63-mIL-2 cells, applied in mice bearing tumors of 60-200 mm3 was more effective than the application of transfected plasmocytoma cells or chemotherapy alone and resulted in 10-37% of LTS and significant, up to >170%, life-span prolongation over control. These mice appeared also to be resistant to a second challenge with MC38 tumor. This observation indicates that systemic antitumor immunity had developed.

The cells of non-tumorigenic X63-Ag8.653 mouse plasmocytoma line transfected with murine interleukin-2 cDNA (X63-mIL-2) were used as the slow-release system of IL-2 for immunotherapy and chemoimmunotherapy in mice challenged with subcutaneous injections of different non-related tumors (MCA induced fibrosarcomas BFS1 and F-69-3, colon adenocarcinoma C38) The combination of one dose of the cytostatic agent (bromoanalog of ifosfamide) administration with subsequent peritumoral injections of the cytokine--producing cells was observed to be more efficient in tumor growth inhibition as compared with the cytostatic alone.

Publication no. 3367

Bromofosfamides, the group of novel compounds closely related to ifosfamide, are currently in the stage of advanced preclinical evaluation. Ifosfamide, although itself the effective antineoplastic drug, useful in situations which have proved refractory to cyclophosphamide therapy, has the side-effect toxicities caused by its metabolites that pose clinically a very real problem. One of their manifestations is the the severe urinary tract toxicity which now could be adequately managed by conjuctive administration of mesna (sodium 2-mercaptoethan sulphonate). In this study we have compared the magnitude of urotoxic effects elicited by ifosfamide and two bromofosfamide compounds, racemate and S(-) isomer of chlorobromofosfamide (ClBrs), selected previously on the base of their superior antitumor activity in advanced animal tumor models. The urotoxic effects, expressed by the increase of urinary bladder weight and histopathologically defined organ wall edema, were estimated in healthy mice 24 h following single intraperitoneal or oral administration of tested compounds, applied in amounts equal to curative, sublethal or lethal doses. It was found that the expression of toxic effects revealed by both ClBrs was statistically significantly lower as compared to ifosfamide. Mesna administration prevented urotoxic effects almost completely in mice treated with ifosfamide or racemic ClBr. Somewhat lower efficacy of uroprotection was observed in the case of S(-) isomer of ClBr.

*Department of Pathological Anatomy, Medical Academy, Wrocław, Poland

Publication no. 3468

Targeting of cytostatics to tumor cells with the help of tumor-specific monoclonal antibodies is one of the ways to fulfil the idea of “magic bullet”, gave by Paul Erlich. The aim of the study was to obtain selectively cytotoxic immunoconjugate for experimental tumor therapy. Monoclonal antibody 17-1A, directed towards colon and breast adenocarcinoma cells, was coupled directly or indirectly, using poly-L-lysine as an intermediate, with cytotoxic nicroacridine compound C921, synthesized by the group of Professor J. Konopa from Technical University in Gdańsk.

Directly coupled conjugates retained antibody specificity towards several human adenocarcinoma cell lines but were not cytotoxic in in vitro assay. On the other hand, conjugates obtained with the use of poly-L-lysine as an intermediate, demonstrated only low, nonspecific cytotoxicity, proportional to the poly-L-lysine content. The work may help to solve the problem of construction of specific antitumor immunoconjugates.

Publication no. 3486

Our involvement in HLA typing is bidirectional (i) to make an optimal matching for blood/marrow transplantation in family and unrelated transplantation settings and (ii) to study HLA associated features as factors with some diagnostic and predictive significand for the course of the disease. In both these situations the accuracy and a high resolution of typing is required. Therefore, a substantial time was spent for developing new techniques and quality control studies. Advantages and drawbacks of DNA typing of HLA class II antigens were described including our original application of the ARMS technique for DRB3 specificity typing in sarcoidosis. This activity was also seen in organization of conferences with worthmentioning workshops covering PCR-SSO, PCR-SSP, reverse PCR-SSO techniques and sequence--based typing (SBT). The latter workshop was run with the use of two systems (Perkin Elmer ABI-PRISM 377 and Pharmacia A.L.F. Express) and was organized as a part of the 3rd Central European Transplant Conference which was held in our Institute in December 1997.

Activity in the field of the quality control workshops is associated with our involvement in the National Polish Marrow Donor Registry which is active in our Unit. Confirmatory typing performed in 73 potential marrow recipients and members of their families revealed the 34% of class II typings were erroneous. The most frequent errors were associated with the typing of DRw52 group of antigens and DR13 appeared as especially difficult antigen.

TNF locus is situated in a close vicinity of class I MHC. The recognized associations between the class I and class II MHC specificities can be in some diseases entities indirect via association between TNFa and b encoding genes and class I or class II antigens. It has been described that PCR-RFLP technique can help in discrimination of individuals respectively to the sensitivity of amplified TNF locus genes to NcoI restriction enzyme. Accordingly to the pattern of digestion two alleles are described 1 and 2 of both TNFa and TNFb genes. The PCR-RFLP technique was succesfully introduced and the pattern of a link between TNFa and TNFb NcoI allels was assessed. In addition, an analysis was performed to validate the associations between DRB1 and TNFa and b allelic specificities. Interestingly, the opposite pattern of association between some DR antigens (DR3, DR6 and DR7 vs DR1 and DR4) and TNFa and TNFb specificities was found. Therefore, TNF alleles cluster with DR specificities in a characteristic manner what may have biological significance.

*BMT Unit, K. Dłuski Hospital, Wrocław, Poland

Publications no.: 3439, 3440

We typed 41 individuals for HLA-C using Dynal “low resolution” PCR--SSP system. Additionally, 25 of them were typed for HLA in complement--dependent microcytotoxicity test and results of both typings were compared. We found discordance between serology and PCR in 18 out of 25 individuals (72%). This result was not surprising, since anti-Cw6, -Cw7 and -Cw8 sera were not included in most serological tests. Discrepancies consisted of incorrectly assigned serological specificities, serological antigen misses, and in one case a serological heterozygote turned out to be homozygote at the molecular level. Although 19 out of 41 individuals in our sample were selected for positive reactions with anti-Cw3 sera, there was a striking similarity in frequency of HLA-C phenotypes determined in PCR by us in our small sample and those reported for other European Caucasian populations represented by large groups of about 600 individuals each. Major deviations in our group were in Cw*05 (undetected), in Cw*06 and Cw*08, which were underrepresented, and in Cw*17 which was overrepresented. These results should be confirmed on random and much larger group of individuals. Nevertheless, they already show that “low resolution” PCR-SSP system is more reliable for HLA-C typing than serological test because many HLA-C alleles are undetectable by serology (“blank”), and antisera to some serologically detectable alleles are hardly commercially available now. To our knowledge, this is the first report on HLA-C typing using Dynal PCR-SSP system.

Publication no.: 3540

B-lymphoblastoid cell line HAJ was derived from peripheral blood lymphocytes of an HLA-DR-positive donor. Nevertheless, in flow cytofluorimetry, neither cell surface nor intracellular HLA-DR molecules were detectable, whereas these molecules were abundant on the surface of positive control PAJ and Daudi cells. This phenotype resembled that of a panel of in vivo and in vitro transcription factor mutants (bare lymphocyte syndrome-derived cell lines BLS-1, BLS-2, RJ2.2.5, 6.1.6, and SJO; in vitro mutants: 721.174 and T2) analyzed in parallel by flow cytofluorimetry. Although HLA-DRB1 gene was detected in HAJ cell genomic DNA, no transcript was revealed by RT-PCR using two different primer pairs. In contrast, PAJ cells were clearly positive. We have described earlier similar findings for DQA1 and DPA1 genes [Mańczak et al., 1996]. Thus, HAJ cells seem to have a defect of a transcription factor, similar to defects in bare lymphocyte syndrome, a severe combined immunodeficiency disease characterized by the presence of HLA class II genes but lack of their transcription. Fusion of HAJ cells with bare lymphocyte syndrome-derived cell lines and in vitro mutants should provide information whether the defect in HAJ cells happened in a gene already described or in so far unknown gene.

Publications no. 3394, 3536, 3541

The frequencies of HLA class II alleles differ between populations. The information about their distribution is important for improving the accuracy of HLA typing and donor-recipient matching in transplantation, for the studies of HLA antigens associations with some diseases, as well as for ethnic studies. We selected a random sample of 100 Polish, healthy individuals, to analyze the SSP polymorphism of DRB1 locus. We observed that DR2 (encoded by DRB1* 15 or *16), DR5 (DRB1*11 or *12) and DR6 (DRB1*13 or *14) are the most frequent alleles in population - 44%, 28%, 26%, respectively. After high--resolution subtyping alleles DRB1*15 and *16, we found four, among ten known, subtypes: DRB1*1501, *1502, *1601 and *1605. The highest frequency showed DRB1* 1501 (63,3%) and *1601 (26,7%). Allelic comparison between the Polish and other Europeans for DRB1 polymorphism showed that some alleles like DRB1*03, *11, *12, *09 and *10 have the distribution analogy to those in other West-Europeans and such like DRB1* 01, *04, *07 to South populations. The frequency of DRB1*08 is quite different from other ethnic European groups.

Successful bone marrow transplantation is largely dependent upon the degree of HLA matching between donor and recipient. HLA -A, -B, -C and -DR, -DQ serology or DNA typing is used to select donors, with mixed lymphocyte culture (MLC) as the functional test of compatibility. Recently, the utility of MLC test has come under review because more precise methods on the DNA level have been introduced. In our study we investigated whether results, matched or not matched, from PCR fingerprinting of HLA -DRB, -DQA, -DPB test agree with the degree of stimulation in MLC. The comparison of results for 13 related and 15 unrelated donor-recipient pairs, showed 4 various situations. Two of them were expected: (i) matched pairs with negative MLC (mainly with related donors), (ii) mismatched pairs with positive MLC (the degree of incompatibilities was reflected in relative response index). But we could show other results: (i) matched pairs with positive MLC (mainly unrelated donors), (ii) mismatched pairs with negative MLC (certain incompatibilities cannot lead to a positive MLC reaction). We conclude that MLC test still has a significant role as biological test in the selection of donors, but must be associated with other tests, on DNA level, for better matching BMT donor--recipient.

A beneficial effect of HLA matching on cadaver organ transplant outcome has been demonstrated in many multicenter studies. The subject of this study were 151 cadaver kidney recipients, transplanted between 1986-1995. We estimated the impact of matching on one and two year graft survival and on number of acute graft rejection. We had insufficient number of well matched grafts, the average number of mismatches in HLA-A,B,DR was 3 (32%) or 4 (30.4%). Our analysis showed that HLA-A,B,DR matching had no impact on 1 and 2 year graft survival. When 0-2 HLA-B, DR mismatches were compared with 3-4 mismatches, we showed improved success rates, with better matches 10% higher at 1 year and 15% at two years. We found a correlation between DR mismatching and number of acute rejections. Dialysed patients waiting, especially for the second or third renal transplantation became allosensitized. Eighty percent of our potential recipients of second graft are highly sensitized. Therefore, now we are studying the character of these antibodies, which are present in the sera of patients. Our efforts are directed to identification of the protective (like IgA or anti-idiotypic) or destructive antibodies and to the evaluation and relevance of autolymphocytotoxic antibodies.

Publication no. 3354

Allogeneic bone marrow transplantation (BMT) in hematological malignancies depends largely on alloreactivity. Therefore, studies on immunological phenomena operating after transplantation help not only to understand development of a tolerance but also the mechanisms responsible for graft-versus--host (GvH) reaction. The latter is very close to graft-versus-leukemia effect. Long-term follow-up of immunological reconstitution after transplantation has been conducted which provided a solid piece of evidence that CD57+ cells, if present in a substantial proportion after transplantation (>20%), are associated with a better prognosis and survival. Cytokine work in patients having and lacking an acute graft-versus-host disease (aGvHD) revealed that both serum IFNg levels and the number of CD4+ cells were lower in patients with severe aGvHD than in those without or with mild manifestation of aGvHD prior to the overt clinical manifestation of this complication. This suggests that the immune system depression may favour aGvHD, likely due to the reactivation of viral infections. In later stages after transplantation, elevation of CRP is predictive of gut manifestation of aGvHD. This in turn shows that microbial complications after BMT may promote gut involvement. Cytokines are generated at the site of aGvHD as shown by the presence of cytokine mRNA. Generated IFNg exertes its biological activity that was documented by the identification of IP-10 mRNA. The TUNNEL technique was succesfully introduced, documenting apoptosis of epithelial cell at the site of aGvHD lesions.

Clinical involvement in hematopoietic cell transplantation prompted us to develop techniques relevant for treatment of transplanted patients. Cell separation in in vivo setting offers purification of platelets when collected from healthy donors. Similar technique has been extensively used for separation of hematopoietic cells from oncological patients to use them at later stages of the treatment to support hematopoietic reconstitution after myeloablative therapy. This ability was helpful in our work on the use of blood progenitor cells as a transplant material in allogeneic setting. We were one of the first European teams to introduce this technique in the clinic.

Last year we celebrated 10th year anniversary of our BMT Unit activity. Our Unit is proud of its longest activity in bone marrow transplantation in Central-East Europe. Altogether, 91 allogeneic transplantations were performed. This group of patients included 43 children and 48 adults. In most cases they were matched sibling transplantations, but also 3 transplantations from unrelated donors, 4 from family alternative donors and one cord blood transplantation were performed. Long-term survival differed accordingly to the disease entity and stage of the disease being 100% in aplastic anemia cases in children, 64% in early stage leukemias and 21% in advanced stage cases. Incidence and fatality of aGvHD was higher among cases with advanced disease and in older patients and was the main cause of mortality after BMT. This prompted us to continue our work on immunological predictors of aGvHD. The factors of a predictive significance were found among HLA antigens and TNF gene allelic specificities. To understand the mechanism favouring the survival, a follow-up study was conducted.

*BMT Unit, K. Dłuski Hospital, Wrocław, Poland

#J. Korczak Childrens Hospital, Wrocław, Poland

‡Department of Pediatrics, Hematology and Oncology, Medical Academy,
Warszawa, Poland

Cooperating EBMT centres:

1Department of Internal Medicine II, University of Kiel, Kiel, Germany

2Department of Hematology, Hopital Saint Antoine, Paris, France

3Department of Hematology, Ospedale San Martino, Genova, Italy

4Department of Hematology, Ospedale V. Cervello, Palermo, Italy

5Rikshospitalet, Oslo, Norway

6Department of Hematology, Glasgow Royal Infirmary, Glasgow, UK

7Istituto Scienze Mediche, Univeristy of Milano, Milano, Italy

8Postgraduate School for Hematology, Hospital Clinic, Barcelona, Spain

9Servicio de Hematologia, Valencia, Spain

10Department of Medicine-Hematology/Oncology, University of Freiburg, Freiburg, Germany

11Department of Hematology, Hospital Santa Creu i Sant Pau, Barcelona, Spain

12Kantonsspital Basel, Basel, Switzerland

Publicatons no: 3358, 3385, 3387, 3426

Our present ongoing study deals with genetical predisposition to the alloreactivity exemplified by the sensitivity to the GvHD. It has been shown that DR6 and DR5 are associated with the severity of GvHD with a positive association with respect to DR6 and rather protective role of DR5-associated features. The latter associations were validated with the use of the discriminant analysis which showed that these DRB1 specificities contribute to the risk of GvHD in addition to the known factors, namely age of the recipient and the donor, blood group compatibility, donor-recipient gender relationship and stage of the disease. GvHD apparently is a consequence of donor-recipient matching and the environmental factors which include the advert effect of the conditioning regimen toxicity. The outcome of the advert effect of conditioning regimen may depend on the inflammatory cytokine generation potential of an individual. Therefore, it was of interest to study the possible association between TNFa and b allelic specificities and the outcome of transplantation. It was found that some TNFb alleles constitute a risk factor of the incidence of aGvHD but only in patients receiving more toxic conditioning regimen due to the more advance stage of the disease. Sarcoidosis is a chronic granulomatous disease characterized by a strong association with some HLA specificities. In addition, some cytokines including TNFa and b are involved in a granuloma formation. Therefore, it was of interest to study whether of allelic specificities TNF genes constitute risk factors in the course of sarcoidosis. Our study conducted with Forschungszentrum Borstel documented such an association and showed a complex influence of MHC associated features on the course of the disease. Rheumatoid arthritis was also studied together with a group from the Institute of Rheumatology in Warszawa. DR1 was identified as a risk factor of the progression of undifferentiated chronic arthritis to rheumatoid arthritis.

All these studies are at present in progress.

*BMT Unit, K. Dłuski Hospital, Wrocław, Poland

#Forschungszentrum für Experimentelle Biologie und Medizin,
Borstel, Germany

‡Institute of Rheumatology, Warszawa, Poland

Publications no.: 3500, 3546

High-dose chemotherapy is still an investigated part of medical oncology. All efforts are made to validate this approach and to establish optimal indications for this treatment. Our work on high dose chemotherapy in solid tumors began as early as in 1987 and since that time we constantly contribute to the program of the EBMT Solid Tumours Working Party. Together with a number of European centers a study was conducted on sequential high-dose chemotherapy in small cell lung carcinoma. This work documented feasibility of this approach and as a consequence a phase II study has been already launched. Independently on this multicentre study our work, on high-dose chemotherapy in SCLC has been continued since 1987. This study showed that sequential high-dose chemotherapy offers long-term survival of a proportion of cases and the success rate is higher in patients having tumors with a high fraction of Ki67+ cells and showing at later date after this therapy a higher proportion of CD57+ cells.

In an effort to standardize indications and protocols of high-dose chemotherapy several papers as chapters or separate articles were published. Therapy of lung cancer, ovarian carcionoma, lymphomas and other hematological malignancies was covered.

*BMT Unit, K. Dłuski Hospital, Wrocław, Poland

Cooperating EBMT centres:

1C.H.R. Citadelle, Liege, Belgium

2Ospedale Civile, Maggiore, Verona, Italy

3Centre de Sante, Chimay, Belgium

4H.U. St. Luc, Bruxelles, Belgium

5Centre Pluridisciplinaire d’Oncologie, Lausanne, Switzerland

6Ospedale Civile, Ravenna, Italy

Publications no.: 3409, 3426, 3474, 3475, 3510

Our activity included two Registries:

1. Polish National Marrow Donor Registry

2. Polish National Transplant Registry.

This work helped to analyze critically our abilities and activity in the field of marrow/blood transplantation in Poland. Altogether, 352 allogeneic and 515 autologous transplantations were performed in Poland between 1984-1997.

In our unrelated donor transplantation out-patient clinic 281 patients were seen and 576 patients and family members were typed. It enabled us to analyze critically typing ability in Poland.

Analysis of indications and results were reported at meetings and symposia.

*Polish Ministry of Health and Welfare, Warszawa, Poland

#BMT Unit, K. Dłuski Hospital, Wrocław, Poland

Most patients with multiple myeloma (MM) develop severe immune deficiency. Patients with MM are highly susceptible to infections with encapsulated gram positive microorganisms.

A major contributor to the increased risk of infections is the acquired inability to mount an effective antibody response to invading pathogens. Patients with MM usually develop “effective hypogammaglobulinemia” of normal immunoglobulins, manifesting a poor effective antibody response to invading pathogens. In addition, patients with MM often are noted to have impaired cellular immune defects. Special interest has been recently paid to the development of methods for the detection in serum, urine and other biological fluids of soluble extracellular fragments of cell differentiation/activation antigens which may serve as natural blockers of their respective ligants. Their increased levels in body fluids have been implicated as one of the possible causes of depressed cellular immunity associated with neoplastic diseases.

The aim of our study was to evaluate serum level of soluble interleukin-2 receptor alfa (sIL-2Ra-p55) and soluble CD8 (sCD8) in 35 patients with MM in correlation with the stage of the diseases and renal function tests. The mean absolute numbers of CD3+ and CD4+ lymphocytes as well as CD4/CD8 ratio in patients in stage II and III as well as substage A and B were significantly lower compared to control group. The serum level of sIL-2Ra significantly correlated with the stage and substage of the disease.

The highest levels were found in patients with renal failure probably as a result of impaired renal excretion of serum sIL-2Ra. The results of our study suggest that all measures which may improve renal function may improve immune status of these patients. The number of CD8+ lymphocytes and serum sCD8 levels did not differ from normal values in all groups of MM patients. The results of our study have shown that sCD8 serum level in MM patients is not an indicator of suppressor cell activation in this disease.

*Department of Hematology, Medical Academy, Wrocław, Poland

Publication no. 3449

Impairment of interferon (IFN) system in human immunodeficiency virus (HIV) infection and acquired immunodeficiency syndrome (AIDS) became a basis for searching IFN responses to monitor the disease progression. For detailed investigations 16 HIV+/AIDS patients with at least four successively taken blood samples available for IFN determinations were selected. IFN responses were tested in two ways. Firstly, IFN level in plasma was measured. Secondly, capacity of IFN production by leukocytes was evaluated. The latter was determined in the whole blood assay, in which Newcastle disease virus (NDV) and phytohemagglutinin (PHA) were used as IFN-a and IFN-g inducers, respectively. The levels of IFN induced in whole blood leukocytes varied considerably in all individuals that had been tested. Nevertheless, two patterns of IFN responses were observed. In pattern I, patients had low levels of IFN in plasma and high levels of induced IFN-a and IFN-g. It was characteristic for eight patients in good clinical condition. On the contrary, severe disease found in two patients was correlated with high levels of IFN in plasma and low levels of induced IFNs (pattern II). In six patients IFN responses were classified as intermediate pattern I/II suggesting transition from pattern I to pattern II. A variation of pattern I was found in the case of a patient defined as long-term survivor having relatively low levels of all IFN tested. The results suggested that interferon measurements reflected clinical condition of HIV+ patients showing not only past but also current immune changes.

*Clinic of Infectious Diseases, Medical Academy, Wrocław, Poland

Publication no. 3410

The aim of this study was to determine immunological status of septic survivor, nonsurvivor and trauma patients and the in vitro effects of bovine lactoferrin (BLF). Plasma samples were taken upon admission, then on days 1, 2 and 5 following admission. The results showed that the kinetics of the cytokine release in septic patients differed significantly between survivors and nonsurvivors. In survivors interleukin 6 (IL-6) concentration were initially high, fell down rapidly on day 1 after admission, and persisted very low throughout the monitoring time. In contrast, relatively low IL-6 levels in nonsurvivors, registered upon admission, rose significantly with peak values on day 3 of observation, declining thereafter. Tumor necrosis factor alpha (TNF-a) levels were similar in both groups, with a tendency to increase in the survivor group. IL-6 levels in the trauma patients were very low which contrasted with progressively increasing concentration of TNF-a. In this category of patients over 90% of patients survived. From this part of the study we concluded that high IL-6 serum levels are associated with high mortality. We have also studied proliferative response to phytohemagglutinin (PHA) and bacterial lipopolysaccharide (LPS) of peripheral blood mononuclear cells (PBMC) and their ability to produce IL-6 and TNF-a in vitro.

The proliferative response of lymphocytes, both spontaneous as well as PHA- and LPS-induced, was generally higher in septic nonsurvivor patients compared to other groups. Markedly elevated LPS-induced proliferation (particularly on day 3) was a bad prognostic sign for survival. The ability of PBMC from the patients to produce IL-6 in culture was depressed in nonsurvivor septic patients and elevated in trauma patients, compared with control donors. More profound differences were found with respect to TNF-a production which was deeply depressed (both spontaneous and induced) in septic nonsurvivors. In contrast, TNF-a production in septic survivors was significantly higher with a peak response on day 3. Trauma patients, on the other hand, had significantly increased ability to produce TNF-a on day 1 and 2, declining there after. Addition of BLF to the PBMC cultures significantly increased the depressed ability of cells from septic patients to produce cytokines. A better enhancing effect of BLF was seen in the group of surviving patients. The data presented in these reports revealed that low production (or even anergy) with respect to synthesis of proinflammatory cytokines in vitro, as well as increased spontaneous proliferation of PBMC and proliferation in response to LPS as well as increased serum IL-6 levels, are significantly correlated with the fatal outcome of the septic shock. Bovine lactoferrin was found to increase the depressed reactivity of PBMC from septic patients in vitro.

*Department of Anesthesiology and Intensive Therapy, Medical Academy,
Wrocław, Poland

Publications no. 3435, 3436, 3521

The immune response of patients to general anesthesia and surgery is beneficial and necessary for generation of local defence processes and wound healing. An optimal postoperative immune response is desirable but lack of the response or an excessive one may harm the patient. The aim our studies was to monitor plasma interleukin 6 and tumor necrosis factor alpha levels in patients subjected to cardiac surgery. In addition, the reactivity of peripheral mononuclear blood cells as assessed by proliferation to phytohemagglutinin and lipopolysaccharide-induced IL-6 and TNF-a production, was measured. Most significant phenomenon was increase of IL-6 level on 1 day after operation, associated with an inhibition of TNF-a concentration. Control, healthy donors did not exhibit measurable levels of these cytokines. Both the proliferative response of PBMC, as well as the ability of these cells to produce cytokines in vitro, depended strongly on the initial reactivity of cells, tested before operation. Low response of cells was usually progressively elevated and vice versa. We also investigated an effect of bovine lactoferrin (BLF) on the studied immune parameters in vitro. In patients, the proliferation of PBMC and the ability to produce IL-6 and TNF-a by these cells underwent characteristic changes depending on preoperative immune reactivity. In general, low, preoperative reactivity of PBMC showed a tendency to increase within the monitoring period whereas moderate/high responsiveness was diminishing. BLF had, in majority of cases, a down-regulatory effect on the proliferative response, best pronounced in patients of high/moderate preoperative response. Similarly, BLF exhibited in general, an inhibitory effect on LPS-induced IL-6 production. In terms of TNF-a production, a considerable up-regulatory effect of BLF, particularly in low responding patients was of a special interest. In summary, we suggest that lactoferrin may play a role in lowering the immune response of patients to surgery and promoting tissue regeneration.

Our studies were also extended to an animal model where we decided to evaluate effects of BLF administration on surgery-elicited shock. We found that BLF given intravenously or per os to mice before thymectomy or splenectomy reduced release of IL-6 and TNF-a into circulation. That effect was better pronounced in case of thymectomy and IL-6 (inhibition up to 90%). TNF-a production was affected to a lesser degree. Taken together, we anticipate that lactoferrin may be applied in clinic to reduce consequences of surgery-induced shock and in optimalization of immunological status of patients prior to operation.

*Department of Anesthesiology and Intensive Therapy, Medical Academy, Wrocław, Poland

Publications no. 3433, 3516, 3553

Monoclonal antibodies (MAbs) against glycophorin A (GPA) and glycophorin C (GPC) were characterized within the Third Workshop on Monoclonal Antibodies against Red Blood Cell and Related Antigens (Nantes, 1996).

Among anti-GPA antibodies, 10 and 8 MAbs showed anti-M and anti-N specificity, respectively. To characterize the epitopes, these antibodies were tested by the microtiter plate ELISA and immunoblotting with untreated and chemically modified GPA-M and GPA-N (differing in amino acid residues 1 and 5). The results obtained confirmed and extended our earlier observations on diversified subspecificities of blood group MN-related antibodies. The anti-M MAbs reacted with epitopes dependent on Ser1 or Gly5 residues of GPA-M, while all anti-N recognized epitopes dependent on Leu1 residue of GPA-N. Moreover, the epitopes differed in their dependence on sialic acid residues and amino group of NH2-terminal amino acid residue. As found previously, the blood group M and N epitopes dependent on the first amino acid residue of GPA were inactivated by periodate oxidation, while the blood group M epitopes dependent on Gly5 were resistant to this treatment. Other anti-GPA MAbs were specific for internal, mostly peptidic, epitopes of GPA. The peptidic epitopes of 12 MAbs were identified by means of peptides synthesized on multiple pins. The antipeptidic MAbs could be divided into 4 groups, each reactive with similar regions of GPA polypeptide chain: a.a. residues 38-43/44 (4 MAbs), 49-56 (2 MAbs), 52/53-56/58 (4 MAbs) and 119-124 (cytoplasmic portion, 2 MAbs).

Several anti-GPC MAbs recognized amino-terminal glycopeptidic epitopes of GPC, dependent on Met1 residue and showing different degrees of dependence on sialic acid residues. Two other MAbs were specific for epitopes located within a.a. residues 16-23 of the GPC polypeptide chain, one MAb was specific for the residues 36-39, and one recognized the residues 110-115 in the cytoplasmic tail of GPC.

Some anti-GPA and anti-GPC MAbs recognizing internal epitopes did not react with synthetic peptides, most likely due to dependence of epitopes in native glycoproteins on adjacent glycosylation. Identification of epitopes recognized by multiple antibodies allowed characterization of most immunogenic regions of GPA and GPC molecules. Moreover, application of antibodies with precisely defined specificities for studies on normal and variant glycophorins allows to draw more precise conclusions.

Publications no. 3446, 3495, 3496, 3511

Our earlier studies with the use of Molluccella laevis anti-Tn lectin (MLL) showed that glycophorin A (GPA) contains a small number of GalNAc residues not substituted with galactose (Tn and sialyl-Tn) and that a stronger reaction of MLL (and other anti-Tn lectins) with GPA-N than with GPA-M results from the presence in GPA-N of a higher number of these truncated oligosaccharide chains. These results suggested that efficiency of in vivo galactosylation of GalNAc residues in GPA is modulated by amino acid residues at positions 1 or/and 5 which are different in GPA-M and GPA-N. To examine whether this difference is restricted to a defined domain of GPA, the amino-terminal tryptic glycopeptides of GPA-M and GPA-N (a.a. residues 1-39) and their fragments obtained by degradation with CNBr (a.a. residues 1-8 and 9-39) were obtained (* denotes glycosylated residues):

The untreated and desialylated glycopeptides were tested as inhibitors of MLL (microtiter plate ELISA), and the content of free GalNAc-ol was determined by gas-liquid chromatography/mass spectrometry in the products of
b-elimination of the asialoglycopeptides. The glycopeptides 1-39 and 1-8 derived from GPA-N showed 2-4-times higher content of non-galactosylated GalNAc residues and higher reactivity with MLL than their counterparts derived from GPA-M, while the glycopeptides 9-39 did not show such differences. These results demonstrated that higher expression of non-galactosylated GalNAc in GPA-N than in GPA-M is confined to GalNAc residues located in the amino-terminal portion of GPA polypeptide chain, between the blood group M- and N-specific amino acid residues 1 and 5. Such localization of this subtle but persistent difference in galactosylation between GPA-M and GPA-N shows that amino acid residues can modulate biosynthesis of proximal oligosaccharide chains.

*The Weizmann Institute of Science, Rehovot, Israel

Publication no. 3464

We have previously reported that glycophorin A (GPA) of human erythrocytes is preferentially digested by incubation of erythrocytes with human neutrophil elastase (HNE) or cathepsin G (CathG). The GPA fragments which remained bound to the membranes in digested erythrocytes were detected after SDS-PAGE and blotting with the monoclonal antibody PEP80 directed against the epitope in the cytoplasmic tail of GPA. The release of neutrophil proteinases under in vivo conditions has been detected in patients with various hematological proliferative disorders, but it is not known whether proteinase--mediated damage of erythrocyte components occurs under these conditions. To approach this problem, erythrocytes were incubated with HNE and CathG at low enzyme concentrations, similar to those found in vivo, or with granulocytes in the presence of Ca2+ and calcium ionophore. Characteristic electrophoretic pattern of bands derived from a partial GPA digestion was observed for each enzyme, and granulocytes produced a pattern similar to that obtained with low doses of HNE. No GPA digestion was observed after treatment of erythrocytes with plasmin or kallikrein. Untreated erythrocytes of 21 patients with various myelo- or lymphoproliferative disorders were tested and a partial degradation of GPA (resembling that given by low doses of CathG) was detected in 9 patients. No distinct relation was found between occurrence of GPA degradation and increases in plasma levels of HNE-a1-proteinase inhibitor complex. However, the results suggested that a partial GPA degradation may occur under pathological conditions due to limited proteolysis by neutrophil proteinases.

*Institute of Hematology and Blood Transfusion, Warszawa, Poland

Publication no. 3441

Blood-group ABO(H) antigens are nonreducing fragments of oligosaccharides, present both in glycolipids and glycoproteins. The ABO(H) blood--group determinants are present in glycoproteins and glycolipids of erythrocyte membranes, they are also present in other tissues and are found in the secreted glycoproteins and oligosaccharides. Glycophorin A (GPA) is the major sialoglycoprotein of erythrocyte membranes, it contains one N-linked and about 15 O-linked oligosaccharides per molecule. There were few reports in the literature indicating the presence of H structures in oligosaccharides of glycophorin originating from O erythrocytes. Therefore, we decided to check if accordant blood-group antigens are present in glycophorins originating from A (GPA-A) and B (GPA-B) erythrocytes. Respective glycophorins were isolated from the membranes of A and B erythrocytes by a phenol-water extraction, and purified by gel filtration in the presence of SDS. Preliminary experiments with immunostaining of glycophorin samples, separated in SDS-PAGE and transferred onto nitrocellulose confirmed the presence of respective blood--group antigens. The two glycophorins were chemically degraded (b-elimination) and two fractions, containing O-linked and N-linked chains, were isolated by gel filtration from both of them. These fractions exhibited inhibitory activity in agglutination test with appropriate monoclonal antibodies, indicating presence of blood-group antigens. Better inhibitors were fractions containing N-linked chains. In collaboration with Dr Bo Nilsson (National Defence Research Establishment, Umea, Sweden) an analysis of the fractions containing O-linked chains, using ES mass spectrometry in a negative mode, was performed. In the fraction of O-linked chains, originating from GPA-A, an ion of m/z 1389 was obtained, which corresponds to a reduced heptasaccharide, containing blood-group A determinant. Investigations will be continued in order to confirm the presence of blood-group determinants in the N-linked chains and to check the presence of B-determinants in the O-linked chains from GPA-B.

Carbohydrate-deficient glycoprotein (CDG) syndrome is an inherited, multisystemic disorder with severe nervous system involvement, CDGS type I is the most frequent form of this illness. Many serum glycoproteins were shown to have a lower carbohydrate content in CDGS, among them transferrin was the most investigated. The aim of this study was to check if the membrane glycoproteins are also affected in CDGS; human glycophorin A (GPA), a major sialoglycoprotein of erythrocyte membranes, was chosen as an example. Several GPA preparations were isolated from healthy individuals of blood group A, B and O; one sample, designated GPA-CDGS, was isolated from O erythrocytes of a patient with CDG syndrome. When run in SDS-PAGE, the GPA-CDGS showed slightly higher mobility in comparison with glycophorin from normal erythrocytes and almost the same mobility as the normal GPA sample, digested with a mixture of enzymes: endo F/glycopeptidase F. These data, taken together, suggested that GPA-CDG has a slightly lower molecular mass which could indicate a lower carbohydrate content. The glycophorin samples were analyzed, therefore, for the sugar content using GLC-MS method after hydrolysis and derivatization of monosugars into peracetylated alditols. The results showed that GPA-CDG had lower content of all sugars, present in glycophorin molecule, which could indicate partial deficiency in both N-linked and O-linked chains. Preliminary experiments with binding the lectins by glycophorins immobilized on nitrocellulose showed that in GPA-CDGS there are no desialylated O-linked chains (no reaction with peanut agglutinin) and the N-linked chain is still present. The experiments will be continued with more GPA--CDGS samples.

*Department of Biochemistry, Medical Academy, Wrocław, Poland

#Department of Clinical Chemistry, University Hospital, Linköping, Sweden

Anti-TF (directed against Galb1-3GalNAca-Ser/Thr) and anti-Tn (directed against GalNAca-Ser/Thr) antibodies, present in most human sera and showing decreased levels in cancer patients, are heterogeneous with respect to structure and fine specificity. Anti-TF and anti-Tn antibodies agglutinate erythrocytes containing asialoglycophorins (TF antigen) or asialo-agalactoglycophorins (Tn antigen), respectively. The anti-TF antibodies were purified by absorption onto and elution from asialoerythrocytes, or by affinity chromatography on the acid-treated agarose or on the immobilized glycophorin isolated from asialoerythrocytes. However, no convenient method of purification of anti-Tn antibodies has been described so far. Tn erythrocytes, which could be used for absorption, are extremely rare, and transformation of relatively easily available asialoglycophorins into Tn glycophorins by enzymatic release of galactose residues is not an efficient process. In our previous studies on anti-TF and anti-Tn monoclonal antibodies and lectins we applied the TF and Tn antigens obtained by efficient and less expensive chemical modifications of glycophorin A (GPA). GPA is desialylated by mild acid hydrolysis to give GPA-TF which in turn is degalactosylated by periodate oxidation followed by mild acid hydrolysis (Smith degradation) that gives GPA-Tn. Now we applied affinity chromatography on the Sepharose 4B-linked GPA-TF and GPA-Tn for purification of human anti-TF and anti-Tn antibodies, respectively. Human immunoglobulins (precipitated from pooled sera with ammonium sulfate) were consecutively passed through GPA-TF and GPA-Tn columns, and absorbed antibodies were eluted with KCNS. Purity of the antibodies (mixture of IgG and IgM) was demonstrated by SDS-PAGE and their specific reactivity with various TF or Tn antigens was shown by hemagglutination and the microtiter plate ELISA. A high, unspecific binding of human immunoglobulins to ELISA plates was found. For testing the purified antibodies it was possible to find the conditions (coating the plates in deionized water) which allowed to minimize the unspecific binding. However, this problem is more difficult to overcome when the antibodies are determined in whole sera. Similar binding of immunoglobulins was found when diluted human sera were incubated in GPA-, GPA-TF-. GPA-Tn-coated or deionized water-treated wells of ELISA plates.

Publication no. 3524

The Duffy antigen of human erythrocyte membranes is an interesting protein, since it is involved in the invasion of human erythrocytes by some malaria parasites and binds CC and CXC chemokines. The Duffy polypeptide chain is composed of 338 a.a. residues, contains 7 membrane-spanning segments (similarly as chemokine receptors) and an N-terminal extracellular domain of over 60 a.a. residues containing three potential N-glycosylation sites. Only a few anti-Duffy monoclonal antibodies (MAbs) have been reported. Some ot them are directed against a trypsin-resistant and chymotrypsin-sensitive epitope (called Fy6) located in the N-terminal extracellular domain. This epitope is interesting, since anti-Fy6 MAbs block both, the invasion of red blood cells by malaria parasites and binding of chemokines to erythrocytes. The epitope Fy6 recognized by two MAbs, i3A and BG6, was characterized by means of peptides synthesized on plastic pins and deletion mutagenesis. Both MAbs showed very similar specificity. They recognized a linear epitope, the essential portion of which was the heptapeptide QLDFEDV comprising a.a. residues 21-27, located between two glycosylation sites (Asn20 and Asn30) of the Duffy protein. All the amino acid residues of the epitope, except Glu, were essential for the antibody binding, since they could not be replaced by any or most other amino acid residues. The Glu residue could be replaced by most other amino acid residues, and its replacement by 10 amino acid residues gave a distinct increase in the antibody binding. The results were in full agreement with the finding that the mutant of the Duffy antigen, lacking a.a. residues 23-25 (DFE) stably expressed in K562, cells did not bind the anti-Fy6 MAb (i3A), but bound the unrelated anti-Fy3 Mab, similarly to the wild-type of the recombinant Duffy antigen. The apparent affinity constants of the MAbs i3A and BG6, determined by surface plasmon resonance, using Duffy protein immunopurified from erythrocytes as a ligand, were 1.1x109 M-1 and 1.4x108 M-1,
respectively. Identification of the Fy6 epitope may be helpful in identification ot the chemokine-binding site of the Duffy antigen.

*Etablissement de Transfusion Sanguine de Loire Atlantique/Vendee, Nantes, France

#Banque du Sang, Paris, France

‡University of Louisville, Louisville, KY, USA

Publication no. 3429

This work is a part of our systematic studies of enterobacterial lipopolysaccharides. Structures of over fifteen H. alvei O-specific polysaccharides, as well as the common hexasaccharide core, have been elucidated.

The O-specific polysaccharide of the lipopolysaccharide produced by Hafnia alvei strain 1220 is composed of D-glucose, D-galactose, N-acetyl-D-glucosamine, N-acetyl-L-fucosamine, glycerol, and phosphate. It was proven by sugar and methylation analyses, Smith degradation, and one- and two-dimensional 1H NMR spectroscopy to be a teichoic acid-like polymer with a branched repeating unit of the following structure:

a-D-Glcp-(1®6)-a-D-Galp a-D-Glcp
1 1
Ż Ż
3 6
®6)-b-D-Glcp-(1®4)-a-L-FucpNAc-(1®3)-b-D-GlcpNAc-(1®1)-Gro-(3-PO-4®

*Max-Planck-Institut für Medizinische Forschung, Heidelberg, Germany

Publication no. 3357

The O-specific polysaccharide of H. alvei PCM 1185 contains D-glucose, D-glucuronic acid, N-acetyl-D-glucosamine, and 3,6-dideoxy-3-[(R)-3-hydroxybutyramido]-D-glucose (Qui3NAcyl) in the ratios 2:1:1:1, as well as O-acetyl groups. On the basis of sugar and methylation analyses of the polysaccharide before and after chemical modyfications, and 1H and 13C NMR spectroscopy, it was found that the polysaccharide has a pentasaccharide repeating unit with the following structure:

a-D-Glcp
1
Ż
4
®4)-b-D-GlcpA-(1®3)-a-D-GlcpNAc-(1®2)-b-D-Quip3NAcyl-(1®6)-a-D-Glcp-(1®

with O-acetyl groups present in nonstoichiometric amounts, mainly in position 2 of GlcA and position 6 of GlcNAc or lateral Glc. Serological study (double immunodiffusion, quantitative microprecipitation and immunoblotting) showed that H. alvei strain PCM 1185 can be placed in a new serotype D and that O-acetyl group is a part of its epitope.

*N. D. Zelinsky Institute of Organic Chemistry, Russian Academy of Sciences,
Moscow, Russia

Publication no. 3375

The O-specific polysaccharide of Hafnia alvei PCM 1222 has a branched hexasaccharide repeating unit containing D-galactose, L-rhamnose, D-ribose, D-galacturonic acid and N-acetyl-D-glucosamine in the ratios 1:2:1:1:1, as well as 2-aminoethyl phosphate (EtNP) and O-acetyl groups in nonstoichiometric amounts. The polysaccharide was modified by carboxyl reduction, O-deacetylation, and dephosphorylation with 48% HF. The last reaction was accompanied by removal of the terminal residue of b-D-glalactofuranose. The modified polysaccharides were studied by methylation analysis and 1H and 13C NMR spectroscopy. The following structure of the O-deacetylated polysaccharide was established:

b-D-Galf
1 EtNP
Ż ˝
3 3
®2)-a-L-Rhap-(1®2)-a-L-Rhap-(1®2)-b-D-Ribf-(1®4)-a-D-GalpA-(1®3)-a-D-GlcpNAc-(1®

In different batches of the polysaccharide, the content of EtNP varied from 0.35 to 0.55 and that of O-acetyl groups from 0.05 to 0.4 per repeating unit. It was tentatively suggested that the O-acetyl group is located at position 4 of a rhamnosyl residue.

*N. D. Zelinsky Institute of Organic Chemistry, Russian Academy of Sciences,
Moscow, Russia

Publication no. 3427

The O-specific polysaccharide of Hafnia alvei PCM 1199 is composed of pentasaccharide repeating units containing O-galactose, 4-acetamido-4,6--dideoxy-D-glucose (DQui4NAc), two N-acetylglucosamines, glycerol phosphate and O-acetyl groups.

On the basis of chemical analyses and NMR spectroscopic studies it was found that the repeating unit of the O-specific polysaccharide has the following structure:

®3)-b-D-Quip4NAc-(1®1)-Gro-3P-(O®3)-b-D-Galp-(1®3)-a-D-GlcpNAc(1®
2 | 6
˝ 1 OAc
b-D-GlcpNAc
| 6
OAc

It was established that the biological repeating unit has Qui4NAc at its nonreducing end.

Comparative analysis revealed that the structure of H. alvei PCM 1199
O-specific polysaccharide is similar to that of H. alvei PCM 1205 (Carbohydr. Res. 231, 187-195, 1992), which differs in the presence of an additional terminal a-D-Glcp residue and the position of the O-acetyl groups only.

The results of serological tests confirmed a high degree of structural similarity between O-specific polysaccharides isolated from H. alvei 1199 and 1205 lipopolysaccharides.

*N. D. Zelinsky Institute of Organic Chemistry, Russian Academy of Sciences,
Moscow, Russia

Publication no. 3533

The structure of the O-specific polysaccharides of the lipopolysaccharides produced by Hafnia alvei strains ATCC 13337 and 1187 was reinvestigated. The position of phosphate group in the repeating units of the polysaccharides was established with the aid of 1H detected, 31P edited NMR spectra. According to the results obtained, the polysaccharides are teichoic acid-like polymers with the repeating units of the following structure:

OAc a-D-Glcp
Ż 3 Ż 6
®2)-a-D-Glcp-(1®PO4®6)-a-D-GlcNAcylp-(1®4)-a-D-GalNAcp-(1®3)-b-D-GalNAcp-(1®

®2)-a-D-Glcp-(1®PO4®6)-a-D-GlcNAcylp-(1®4)-a-D-GalNAcp-(1®3)-b-D-GalNAcp-(1®

where Acyl=D-3-hydroxylbutyryl, and 3-O-acetylation was approximately 30%.

*Max-Planck-Institut für Medizinische Forschung, Heidelberg, Germany

Publication no. 3525

The structures of the O-specific side-chain of the Hafnia alvei strain 32, 744, PCM 1190, PCM 1194, PCM 1206 and PCM 1210 lipopolysaccharides have been investigated. Methylation analysis, partial acidic hydrolysis, dephosphorylation, Smith degradation, 1H-NMR and 13C-NMR spectroscopy, MALDI-TOF and FAB mass spectrometry in combination with collision induced decomposition MS/MS were the principal methods used. It is concluded that the investigated polysaccharides are composed of heptasaccharide (PCM 1190) and pentasaccharide repeating-units (32, 744, PCM 1194, PCM 1206, PCM 1210). O-specific polysaccharide of strain 32 is partially O-acetylated in the 2- (20%) and 3-position (50%) of the ®4)-a-D-GalpA-(1® residue. D-allo-threonine, amide-linked to the D-galacturonic acid, was identified as a constituent in the polysaccharide of strain PCM 1206.

Analysis of the chemical composition of the O-specific polysaccharides of H. alvei strain 744 and PCM strains 1194 and 1210, showed the presence of phosphate in addition to sugar components. This type of teichoic acid like structures has been found in some capsular polysaccharides, it has not been found among the O-specific polysaccharides. The repeating units of H. alvei strains 32, 744, PCM 1190, PCM 1194, PCM 1206 and PCM 1210 have the following structures:

Hafnia alvei 32

®4)-a-D-GalpA-(1®2)-a-L-Rhap-(1®4)-b-D-Galp-(1®3)-b-D-GalpNAc-(1®4)-a-D-GlcpNAc-(1®
2,3
:
OAc

Hafnia alvei 744 and PCM 1194

®6)-a-D-GlcpNAcyl-(1®4)-a-D-GalpNAc-(1®3)-b-D-GalpNAc-(1®2)-a-D-Glcp-(1-P®
6
­
1
a-D-Glcp

Acyl=[(R)-3-hydroxybutyryl

Hafnia alvei PCM 1190

a-D-Glcp
1
Ż
5
®4)-a-D-GalpA-(1®3)-b-D-GlcpNAc-(1®3)-a-L-Rhap-(1®2)-b-D-Ribf-(1®
2
­
1
a-D-Galf-(1®2)-a-L-Rhap

Hafnia alvei 1206

D-allo-Thr
Ż
6
®4)-a-D-GalpA-(1®2)-a-L-Rhap-(1®2)-b-D-Ribf-(1®4)-b-D-Galp-(1®3)-b-D-GalpNAc-(1®

Hafnia alvei PCM 1210

®6)-a-D-Galp-(1®4)-b-D-Galp-(1®3)-b-D-GlcpNAc-(1®3)-a-D-GlcpNAc-(1-P®
4
­
1
b-L-Rhap

*Swedish University of Agricultural Sciences, Uppsala, Sweden

Publications no. 3370, 3488, 3489, 3490

Structural analysis using 13C NMR spectroscopy and methylation showed that lipopolysaccharides (LPSs) of Citrobacter freundii O35 and Salmonella arizonae O59 have identical O-specific polysaccharide chains, and those of Citrobacter freundii O38 and Salmonella kentucky differ only in the presence of O-acetyl groups in the former. Serological relationships between the structurally similar LPSs were demonstrated using inhibition of ELISA, rocket immunoelectrophoresis, double gel diffusion, and immunoblotting. The O-acetyl groups present in C. freundii O38 LPS are of little importance for its serological specificity. A cross-reaction was observed in immunoblotting between O-antisera to C. freundii O35 and S. arizonae O59 and a structurally related LPS of Pseudomonas aeruginosa O11a, 11b.

*N. D. Zelinsky Institute of Organic Chemistry, Russian Academy of Sciences,
Moscow, Russia

#I. I. Mechnikov Institute of Vaccines and Sera, Moscow, Russia

Publication no. 3379

A glucose-nonfermenting Gram-negative bacterial strain isolated from bronchofiberoscope used for examination of the patients suffering from pulmonary diseases was subjected to phenol-water extraction. Lipopolysaccharides (LPS) isolated from the water and the phenol phase differed in fatty acid composition. Both contained xylose, glucose, glucosamine and components typical for LPS, namely heptose, 3-deoxyoctulosonic acid (Kdo) and 3-hydroxymyristic acid. The presence of sphingosine in all LPS preparations classified the strain to the genus Sphingomonas.

Publication no. 3484

Lipopolysaccharides are known to be responsible for the initiation of endotoxic shock, therefore they can be targets for new preventive and therapeutic strategies. The traditional antibiotic therapy in endotoxemia is not efficient because these substances are not able to decrease the amount of lipopolysaccharide in the bloodstream. These limitations have led to investigation of protective antibodies as candidates for therapeutic interventions. It was found that immunization with endotoxin core structures protected animals against bacterial infections and endotoxic shock. These antisera are of great interest because they are directed against conserved parts of LPS and therefore could have cross-reactive and cross-protective properties with respect to many Gram--negative rods. We described earlier the possibility of obtaining a good non-toxic immunogen which did not contain lipid A, by conjugation of lipopolysaccharide core with tetanus toxoid.

The aim of this study was to prepare immunogenic conjugates of core oligosaccharides E. coli type R1, R2, R3, J5 and Salmonella Ra with tetanus toxoid and to use them for production of monospecific sera. The neoglycoconjugates were good immunogens in rabbits yielding a high level of antilipopolysaccharide antibodies of IgG class. The antibodies were used to examine the possibility of their reactions with smooth lipopolysaccharides. We have found that all antisera were able to react with the lipopolysaccharide molecules of identical or related core type, possessing core oligosaccharides substituted with O-specific chains. These reactions were shown in both ELISA assay and immunoblotting test.

Escherichia coli strains are the most frequent clinical isolates in episodes of sepsis. Among them 60-80 % belong to R1 lipopolysaccharide core type. The reactivity of anti-OS R1-tetanus toxoid serum with intact, live smooth bacterial cells have been determined in flow cytometry. It has been shown in flow cytometry that anti-core antibodies labelled more than 95% of bacterial cells with high intensity of fluorescence. The anticonjugate serum was able to react with free form of smooth lipopolysaccharides and to inhibit their TNF stimulation activity in in vitro and in vivo models.

Publications no. 3390, 3391, 3502

Most Hafnia alvei strains are sensitive to the bactericidal action of normal bovine serum (NBS) as well as to a serum in which the alternative pathway of complement activation has been thermally blocked. Introduction of polysaccharides (LPS) to NBS lowers the bactericidal effect. In a serum in which the alternative pathway of complement activation is blocked, LPS completely cancels the bactericidal effect.

*Department of Microbiology, Medical Academy, Wrocław, Poland

Publication no. 3371

An opportunistic actinomycete was isolated as the only etiological agent of a severe, suppurative pulmonary infection. The strain was rapidly recognized as Nocardiopsis by the taxonomically important and immunologically active glycolipid markers (G1 and G2). Identification of the clinical isolate, from a group of actinomycetes mainly known as soil habitants, was definitely proved by chemotaxonomic studies (cell wall/sugar, phospholipid and fatty acid types) as well as by genomic data (GC content, DNA-DNA reassociation). The level of DNA-DNA homology of the clinical actinomycete, in comparison with other reference members of this genus, revealed the highest (88%) relationship to Nocardiopsis dassonvillei. The results seem to confirm the value and generic specificity of glycolipid markers from Nocardiopsis, the first time used for rapid recognition of a clinical strain causing a nocardiosis like disease.

Publication no. 3543

Permanently growing number of new cases of syphilis and number of patients difficult for curing caused that syphilis is again very important medical problem. Expansion of this disease is promoted by frequent use of different drugs, particularly cocaine, which increases sexual activity and risk of the disease. The central nervous system is often involved in early syphilis what previously was very rare. The reason for development of this stage of syphilis may be an inadequate treatment as well as a weakening of the immunological responses. It is known from our previous studies that cell-mediated immune response of importance in protection against T. pallidum infection is distinctly suppressed in some stages of syphilis. This was found as a diminished ability of lymphocytes to produce MIF, antitreponemal lymphotoxin (ATL) and IL-2. Taking into account that IL-2 induces production of TNF, which may take part in cell-mediated immune response and resistance as well as in dysfunction of organs and high mortality, we examined if lymphocytes of syphilitic patients are able to produce this cytokine. In addition, the ability of cells to IL-6 production, which may inhibit TNF secretion, was determined. The studies were carried out on the blood samples taken from 31 patients with different stages of syphilis and from 6 healthy persons used as a control. The level of TNF and IL-6 was examined in culture supernates of peripheral blood mononuclear cells stimulated with T. pallidum antigen and ConA, as well as in sera of the same people. It was found that T. pallidum antigen was better stimulator of TNF than ConA. The highest level of TNF was found in stages with clear clinical symptoms of disease. There was some relationship between high level of IL-6 and low level of TNF. The data suggest that TNF takes part in development of syphilitic lesions, whereas high level of IL-6 may inhibit TNF production.

*Department of Dermatology, Medical Academy, Poznań, Poland

Publication no. 3493

Acne is a disease which occurs in 70% of teenagers. It is connected with excessive functional activity of sebaceous glands. In etiopathogenesis of this disease the role of genetic factors, hormonal, bacterial and environmental was examined. Not very much is known about immunological response, especially about circulating immune complexes (CIC) formation, in this disease. Besides, the data are controversial. CIC were found only in patients with acne fulminans, conglobata and hidradenitis suppurativa, coexisting with arthritis. No CIC were found in acne vulgaris. The aim of this work was to elucidate these contradictory results on material with different forms of acne treated in our Department. CIC were examined in sera of 40 patients hospitalized in years 1986-1994. The sera were obtained from 8 patients with acne inversa, 2 with acne fulminans, 19 with acne conglobata and 11 with acne papulo-pustulosa. As a control 25 healthy people sera were used. CIC were detected by two methods: a) precipitation of sera with 3.5% polyethylene-glycol; b) immunoelectrophoresis. CIC were found in 6 from 8 patients with acne inversa, in both patients with acne fulminans and in 18 from 19 patients with acne conglobata. In sera of patients with acne papulo-pustulosa (11 persons) CIC were found only in one patient’s serum and only in minimal amount. The data suggest that CIC are involved in pathogenesis of severe forms of acne.

*Department of Dermatology, Medical Academy, Wrocław, Poland

Publication no. 3415

The subject of this report is the case of purulent meningitis in a newborn caused by Klebsiella pneumoniae. As the intensive antibiotic therapy turned out to be ineffective, phage therapy was applied. Oral administration of specific phage preparate for the period of 5 weeks resulted in complete sterilization of cerebrospinal fluid and unquestionable improvement of child’s health.

*Children Hospital, Sosnowiec, Poland

Publication no. 3550

During last years data have been accumulated showing that specific bacteriophages are highly effective in the treatment of bacterial infections. We now report on phage therapy which was applied in 22 patients with chronic purulent otitis media caused by Staphylococcus aureus, Staphylococcus epidermidis, Staphylococcus saprophyticus, Pseudomonas, Proteus and Klebsiella. All bacterial strains isolated from patients were resistant to available antibiotics. In all cases the antibiotic therapy was ineffective. Application of specific bacteriophages, locally and orally, eliminated completely the infection in 19 casees. In the remaining 3 cases marked improvement was noted. The results obtained have indicated that specific bacteriophages deserve a special attention as a very important and effective preparation in the treatment of chronic purulent otitis media.

*Clinic of Laryngology, Medical Academy, Wrocław, Poland

Publication no. 3512

The antiviral immunity of human placenta and amniotic membrane in an organ culture (OC) system was studied. Freshly isolated explants of most of the placentas at term and the amniotic membranes were found to be relatively resistant to herpes simplex virus type 1 (HSV-1), encephalomyocarditis virus (EMCV), and vesicular stomatitis virus (VSV) infections. After in vitro culture, however, the OC acquired a sensitivity to the viruses. In about 66%-90% of placentas, resistance of freshly isolated explants to the infection was observed. This indicates that the placentas displayed a constitutive immunity against the viruses. To study the role of endogenous cytokines in antiviral immunity, we added specific antibodies neutralizing IFN and TNF activities to VSV--infected OC and checked their influence on viral replication. Increases of 10-fold to 100-fold of VSV replication in the OC-treated with anti-TNF-a, anti- -IFN-a, anti-IFN-g or anti-IFN-b sera were observed. The results indicate the importance of the endogenous cytokines in placental and amniotic membrane immunity. However, we did not observe a simple correlation between the spontaneous IFN and TNF production and the level of resistance against viruses. In view of the results, the participation of TNF and IFN in the constitutively expressed immunity of human placenta is of a more complex nature.

*Department of Obstetrics, Regional Specialistic Hospital, Wrocław, Poland

Publication no. 3407

We have studied the effect of culturing in vitro of BALB/c mouse resident peritoneal cells (RPC) on their ability to multiply vesicular stomatitis virus (VSV). Results of 36 experiments performed on RPC, freshly isolated from individual female mice, showed that over 70% of them expressed constitutive antiviral immunity against VSV infection. The virus multiplied to low titers (<103 TCID50/106 cells) only in 10 out of 36 cases. After several days in culture, the RPC were found to lose the resistance and VSV multiplied in the cells to high titers (up to 107 TCID50/106 cells). The time period, required for the cultures to reach a full ability to replicate the virus, was individually differentiated and ranged from 24 h to over 96 h.

Publication no. 3404

We have reported that cultured human umbilical cord vein endothelial cells (HUVEC) differ from endothelium present on vein surface of organ culture (OC) in production of cytokines and susceptibility to viral infections. We present the effect of viral infections on interferon (IFN), tumor necrosis factor (TNF) and interleukin 6 (IL-6) production in two culture systems: HUVEC and OC. Infection of 24-48 h HUVEC cultures with herpes simplex type 1 (HSV-1) and vesicular stomatitis virus (VSV) reduced the amounts of IL-6 and TNF in comparison to those released spontaneously by uninfected cells. No IFN was detected in media from infected and uninfected HUVEC. Limited viral infections of 3h HUVEC and OC usually diminished their efficiency of IL-6 and TNF production. In the case of IL-6 synthesis by OC, effect of viral infection depended, however, on the constitutive synthesis of the cytokine. When spontaneous production was high (>800 U/ml), VSV and HSV-1 infection reduced IL-6 level by 2-50 times; in the case of low production (<150 U/ml) the stimulation effect (2-4 fold) was observed. OC released spontaneously some IFN activity (2-32 U/ml). HSV-1 infection of OC reduced IFN level, while VSV in single cases slightly upregulated IFN synthesis.

*Department of Obstetrics, Regional Specialistic Hospital, Wrocław, Poland

Publication no. 3517

Results of our previous study on the immunity of human placenta and amniotic membranes revealed that in majority of cases these organs present constitutive non-specific antiviral immunity in the organ culture (OC) system. It is possible that interferons (IFNs), tumour necrosis factors (TNFs) and interleukin 6 (IL-6) may be responsible for the antiviral effect. Here, the constitutive and lipopolysaccharide (LPS)-induced production of these cytokines and additionally, interleukin 10 (IL-10) were determined in OC of chorionic villi, decidua and amniotic membranes. Significant amounts of constitutive TNF-a (2-64 U/ml), IL-6 (200-12000 U/ml) and IL-10 (1-70 ng/ml) were detected in the maternal decidua and chorionic villi of placenta. Amniotic membranes produced lower concentrations of the cytokines. LPS increased the production of cytokines from 2-8 times. In contrast, activity of IFN released spontaneously was found only in 4 out of 50 placenta and amniotic membranes. LPS and Newcastle disease virus (NDV) induced IFN production in the OC system. However, the increase of IFN after induction was also very small (up to 32 U/ml). Individual differentiation in the cytokine production was observed among placentas and amniotic membranes. TNF was identified as type a with addition of TNF-b, IFN as type a, b and g.

*Department of Obstetrics, Military District Clinical Hospital, Wrocław,
Poland

Publication no. 3487

Single-stranded DNA (ssDNA) is a preferred template for dideoxy DNA sequencing. It is most commonly obtained by cloning of the studied sequences into an M13 vector; however, large inserts tend to be unstable. Plasmid-phage chimeric vectors (phagemids) offer an alternative. They replicate as normal plasmids in E. coli, but upon infection with a helper phage produce phagemid ssDNA and package it into phage-like particles. Single-stranded DNA can be easily recovered from the culture supernatant by a standard procedure. Some helper phage constructs like M13K07 and its derivatives such as VCSM13 were designed to ensure that phagemid ssDNA is packaged and exported preferentially over phage ssDNA. However, helper phage ssDNA can contaminate the phagemid ssDNA to various extent. This causes ambiguities in sequencing reactions. In our work additional problems were encountered in sequencing Streptomyces spp. DNA (G+C content of 70%-74%). Double-strand sequencing of Streptomyces coelicolor A3(2) DNA gave poor results. Sequencing of a single--stranded template obtained after VCSM13 helper phage infection was more successful, but the use of recommended methods of phagemid ssDNA production resulted in a variable amount of DNA rescue, often with an abundance of a helper phage ssDNA.

We examined the following factors influencing phagemid ssDNA production: initial density of cell cultures, multiplicity of infection (m.o.i.) and insert length of a recombinant. The quantity of ssDNA was measured by densitometry.

Examining conditions for single-stranded DNA production from phagemid recombinants with inserts of high G+C content yielded DNA templates of reasonable quality for dideoxy DNA sequencing. Only cultures in exponential growth should be used to inoculate cultures infected with helper phage. Initial cell density is the most important factor influencing phagemid ssDNA production and should be relatively low (105-106 cells/ml). This differs from protocols in procedures previously recommended. Multiplicities of phage infection can vary without strongly influencing phagemid ssDNA production.

Publication no. 3538

Streptomycete are known as producers of a variety of secondary metabolites. Among these, biologically active compounds of a polyketide structure, most of which are antibiotics, are of a great concern.

Streptomyces coelicolor A3(2), genetically the most characterized member of the genus, produces two aromatic polyketides: antibiotic actinorhodin and a spore pigment. The physical structure, organization and functions of the respective genes for these so called type II polyketides are known.

In our study we searched for a DNA sequence thought to be a part of a larger fragment comprising a new ketosynthase-homologous sequence from Streptomyces coelicolor A3(2) chromosome (Kuczek et al., 1994, FEMS Microbiol. Lett. 118, 317-326). Based on hybridization of the S. coelicolor A3(2) genomic DNA library with the ketosynthase probe, four cosmid clones, located in the AseI B fragment of the chromosome, were selected. The cosmid clones were screened further for the presence of putative polyketide synthase genes. Primers for PCR reaction were designed by comparison of sequences of several known polyketide synthase gene clusters, based on the conservative sequences of beta-ketoacyl synthase and acyltransferase active sites. The PCR product was amplified from all four cosmid clones and was sequenced. The putative amino acid sequence of the fragment resembles acyl-CoA:ACP acyltransferase domains specific for malonyl:CoA from several bacterial enzymatic complexes of polyketide synthase. There is a high similarity with acyltarnsferase domains from type I polyketide synthase complexes. Hybridization of the cosmids and chromosomal DNA with the probe confirms the sequencing data. The amplified fragment as well as at least two distinct ketosynthase-homologous sequences are considered to be a part of a larger, type I polyketide synthase gene complex.

Publicaton no. 3466

Staphylococcus saprophyticus is one of the most frequently encountered clinically significant members of the coagulase-negative staphylococci. A set of species-specific PCR primers was defined for the detection of Staphylococcus saprophyticus. These primers target variable regions (V3 and V6) of the 16S rRNA gene. Primer-specific PCR has potential applications in epidemiological studies and diagnosis of Staphylococcus saprophyticus.

Publication no. 3450

Forty six CNS isolates from neonates with nosocomial bacteremia were studied. The results of the S. epidermidis-specific PCR were compared with those obtained using ribotyping and API ID32 STAPH system. Excellent congruence was found between primer-specific PCR and ribotyping. Primer--specific PCR, turned out to be fast and reliable method for the identification of S. epidermidis strains. According to the primer-specific PCR and ribotyping analysis, few CNS isolates were found to be incorrectly identified by API ID32 STAPH system. Primer-specific PCR is fast and reliable method for the identification of S. epidermidis. Primer-specific PCR in combination with the ribotyping is a promising approach for studying the epidemiology of S. epidermidis and other CNS species in hospital.

*Department of Microbiology, Medical Academy, Wrocław, Poland

Publication no. 3527

Streptomyces differ from other prokaryotic organisms in their mycelial life cycle and in possessing a large, linear, GC-rich chromosome. In order to deduce structural features of the Streptomyces origin of chromosomal replication, the oriC sequences of three Streptomyces species (S. antibioticus, S. chrysomallus and S. lividans) have been compared. In the Streptomyces, the oriC region contains 19 DnaA boxes whose location, orientation and spacing are conserved. The preferred sequence of DnaA box indentified within the Streptomyces oriC is (T/C)(T/C(G/A/C)TCCACA. The interactions of the DnaA protein with DNA fragments containing single, two or three DnaA boxes were studied using surface plasmon resonance. The dissociation constant (KD) for specific binding of individual DnaA boxes varied between 12 and 78 nM. The Streptomyces oriC does not contain the three AT-rich 13-mer direct repeats present in the left part of the E. coli oriC region. However, short AT-rich sequences are distributed among the DnaA boxes of Streptomyces oriC. Repeated attemps to unwind the Streptomyces oriC have been unsuccessful. It remains to be elucidated whether the DnaA protein interacts with putative accessory proteins which help unwinding of the Streptomyces oriC.

*Max-Planck Institute of Molecular Genetics, Berlin, Germany

#Departamento de Biologia Funcional e Instituto Universitario
de Biotecnologia de Asturias, Universidad de Oviedo, Spain

‡Max-Volmer-Institut für Biophysikalische Chemie und Biochemie,
Fachgebiet Biochemie und Molekulare Biologie, Technische Universität
Berlin, Germany

§Universität Osnabrück, FB Biologie/Chemie, Osnabrück, Germany

Publication no. 3530

The Streptomyces lividans DnaA protein (73 kDa) consists, like the Escherichia coli DnaA protein (52 kDa), of four domains. The larger size of the S. lividans protein is due to an additional stretch of 120 predominantly acidic amino acids within domain II. The S. lividans protein was overproduced as His-tagged fusion protein. The purified protein (isoelectric point 5.7) has a weak ATPase activity. Using DNaseI footprinting studies, each of the 17 DnaA boxes (consensus sequence TTGTCCACA) in the S. lividans oriC region was found to be protected by the DnaA fusion protein. Purified mutant proteins carrying a deletion of the C-terminally located helix-loop-helix (HLH) motif, or amino acid substitutions in helix A (L577G) or helix B (R595A) no longer interact with DnaA boxes. A substitution of basic amino acids in the loop of the HLH motif (R587A or R589A) entailed the formation of S. lividans mutant DnaA proteins with little or no capacity of binding to DnaA boxes. Thus, like in E. coli, the C-terminally located domain IV is absolutely necessary for the specific binding of DnaA. A mutant protein lacking a stretch of acid amino acids, corresponding to domain II, is not affected in its DNA binding capacity. Whether the acid domain II interacts with accessory proteins remains to be elucidated.

*Max-Planck Institute of Molecular Genetics, Berlin, Germany

#Universität Osnabrück, FB Biologie/Chemie, Osnabrück, Germany

Publication no. 3479

We described a DNA binding assay for isolation of specific sequence(s) recognized by protein of interest directly from genomic or cosmid DNA. In our assay, the protein is fused to the glutathione-S-transferase and bound to glutathione-Sepharose beads. Then the immobilized fusion protein can be used to search for DNA fragment(s) that interact specifically with the protein of interest. As an example of such an approach, we identified and cloned a few prokaryotic oriC regions using the initiator DnaA protein fused to the glutathione-S-transferase.

Publication no. 3478

Streptomyces lividans contains six ribisomal RNA (rRNA) gene sets, rrnA-F. We have cloned the rrnB gene cluster. Physical maping revealed that rrnB gene set is located on a 290 kb AseI fragment in the 11 to 12 o’clock region of the S. coelicolor A3(2) chromosome. The complete nucleotide (nt) sequence of S. lividans 23S rRNA has been determined. The structural gene of the 23S rRNA codes for the 3108 nt RNA chain. The G+C content of the 23S rRNA is 57.3 mol%. The length of the spacer region between the 23S and 5S genes is 99 bp. Analysis of the sequence between the 16S and 23S genes and downstream of the 5S rRNA gene failed to identify any tRNA-like sequences. A secondary structure model of S. lividans 23S rRNA is proposed.

Publication no. 3519

Reexamination of the morphological cultural, and physiological characteristics of Streptomyces spitsbergensis Wieczorek et al. 1993 revealed that this organism belongs to the whorl-forming streptomycetes, and DNA-DNA hybridization data confirmed that S. spitsbergensis is a later subjective synonym of Streptomyces baldacci (Farina and Locci 1996) Witt and Stackebrandt 1991. Phenotypic characteristics of the new isolate, in comparison with whorl--forming streptomycetes, having red aerial mycelia, its typical recti-flexibilis spore chains and the levels of DNA relatedness between the compared strains were presented.

*Institute for Fermentation, Osaka, Japan

Publication no. 3452

Microbial transformation of cytotoxic 5,11-dimethyl-5H-indolo[2,3-b] quinoline (a compound displaying antitumor activity and affecting the activity of calf thymus DNA topoisomerase II) was performed by the Rhizopus arrhizus strain and yielded a 9-hydroxy derivative. The metabolite obtained displayed a stronger cytotoxity against KB cells than the parent compound (ID50 = 0.001 mmol/mL), and stimulated also the formation of calf thymus topoisomerase II mediated pSP65 DNA cleavage in vitro at the concentration of 3 mM. Being analogous to 9-hydroxyellipticine (which is an antitumor alkaloid), this novel indolo[2,3-b]quinoline derivative can be regarded as a novel potential antitumor agent.

*Institute of Organic Chemistry, Biochemistry, and Biotechnology, Technical University of Wrocław, Wrocław, Poland

#Pharmaceutical Institute, Warszawa, Poland

Publication no. 3408

Juvenile hormone (JH) is one of the major regulators of growth, development and reproduction of insects. JH is produced in Corpora allata and transferred to target tissues by JH-binding proteins (JHBP). Among several types of JHBPs, a special attention has been payed to a group of low relative molecular mass (~30 kDa) single-chain proteins with high specificity and affinity for JH, which, so far, were not considered to be glycosylated. Glycosylation of this type of JHBP from Galleria mellonella hemolymph has been shown by several lines of evidence. Carbohydrate gas-liquid chromatography analysis of the purified JHBP preparations showed the presence of a low amount of sugars (Man and GlcNAc were the major components). The JHBP electrophoretic band blotted onto nitrocellulose was stained with GlycoTrack (a reagent kit used the detection of protein glycosylation) and showed a strong binding of Concanavalin A (ConA). JHBP was fractionated on a ConA-Sepharose 4B column into ConA--bound (strongly stained with ConA) and ConA-unbound (hardly stained with ConA) portions. Both fractions showed JH-binding activity and were glycosylated, as revealed by staining both of them with GlycoTrack. Electrospray--ionization mass spectropmetry of FHBP suggested the presence of a small amount of of presumably nonglycosylated protein (24988 Da) and five glycoforms, two of which (containing Man2GlcNAc2 or Man2Fuc1GlcNAc2 chain) were not bound or weakly bound to ConA, and three (with Man3GlcNAc2, Man3Fuc1GlcNAc2, or Man5GlcNAc2 chain) were present in the fraction strongly bound to ConA. In conclusion, the monosugar composition, GlycoTrack staining, ConA-binding properties and molecular mass analyses of JHBP supplied convincing evidence for its glycosylation and gave some information on the character of the oligosaccharide chains.

*Institut de Biologie Structurale, CEA/CNRS, Grenoble, France

#Institute of Organic Chemistry, Biochemistry and Biotechnology,
Technical University of Wrocław, Wrocław, Poland

Publication no. 3360

Human lutropin (human luteinizing hormone, hLH) is a heterodimer glycoprotein composed of a and b subunits. Demonstration of hLH peak in woman urine is useful for ovulation prediction. Therefore, assay of hLH is a valuable diagnostic tool. The aim of this work was to elaborate effective immunoassay of hLH and to compare pituitary hLH with urine hormone. Two newly obtained monoclonal anti-bhLH antibodies were applied for immunoassays of hLH, however, sensitivities of these assays were much lower than those with two other monoclonal antibodies obtained earlier in our laboratory. No significant differences between assays of pituitary hLH and urinary hormone with 6 various pairs of monoclonal antibodies (4 anti-b and 1 anti-a subunits) were noted. The highest hLH concentration in urine portions collected during 53 menstrual cycles of 6 women was demonstrated in various time of the day. In 14 from other 52 preovulatory urine portions, preserved for several weeks, over 80% increases of assayed hLH were shown. The highest assay values of pituitary and urinary hLH were noted at pH 7.5-9.0. Using affinity chromatography the whole pituitary hLH was bound to ConA-Sepharose column and eluted with a-methylmannoside (aMM) as a single peak. However, a small part of urinary hLH was not retarded on the lectin column, and the hormone bound to the column was eluted with aMM as two peaks. When HPLC with DEAE column was applied, pituitary hLH was separated in two fractions while urinary lutropin was eluted as a single peak.

Publication no. 3467

Myoglobin is a 17.3 kDa protein occurring mainly in heart and skeletal muscles. Its assay in human and horse serum is useful for medical and veterinary diagnosis. To elaborate immunoassays of myoglobins, rabbit and two monoclonal antibodies were obtained. These three antibodies react with human myoglobin but rabbit and monoclonal No. 49 antibody recognized also horse myoglobin. The last two antibodies reacted also with apo-, FITC- and treated with hydrochloric acid or TPCK-trypsin horse myoglobin. Their binding to horse myoglobin pretreated with NaOH was reduced. Thirteen overlapping peptides covering the sequence of human myoglobin were synthesized on polyethylene pins. Rabbit and mouse polyclonal antibodies reacted with some of these peptides but no reaction was noted with purified mouse monoclonal antibodies. Two monoclonal antibodies were applied for specific immunoassay of human myoglobin.

Publication no. 3378

It was noted that human and horse sera as well as human heart and skeletal muscle homogenates or extracts distinctly decrease immunoassays of purified human and horse myoglobins. The sensitivity of assays of the myoglobins in homogenate and extract could be many times increased by precipitation certain proteins with concentrated ammonium sulfate or sodium chloride. Also in homogenates and extracts incubated for several days increased assay values of myoglobins were noted. The obtained results suggest that both myoglobins occur in complex with other tissue component(s).

Publication no. 3460

Six ELISA variants with the use two monoclonal and one rabbit antibodies were applied for determination of purified commercial human myoglobin and myoglobin in human muscle extracts. Monoclonal antibody No. 82, used for coating of ELISA plates, gave high assay values of the analyzed samples of muscle extracts while low assay values were acquired with monoclonal No. 49 or rabbit antibodies. Determinations of apomyoglobin with ELISA variants were somewhat more sensitive than that of commercial myoglobin. The obtained results were compared with those by Seratec kit for immunoassay of human myoglobin. Addition to the extracts not only concentrated salts but also acetone, ethanol, some other reagents or sodium dodecyl sulfate markedly increased assay sensitivity of myoglobin by ELISA with monoclonal antibody No. 49 and antibody No. 82 conjugated with peroxidase. Removal of acetone or ammonium sulfate from extracts caused dramatic decrease of estimated myoglobin. Filtration of the extract through Bio-Gel A5m or Sephadex G-100 columns did not affect low sensitivity of myoglobin determination in fractions without pretreatment with acetone. Purified myoglobin was isolated from human heart extract by immunoaffinity chromatography on the Sepharose-antibody No. 82 column. The isolated myoglobin and muscle extracts were investigated by gel electrophoresis and Western blot.

Publication no. 3535

Two different modifications of CTLL techniques for IL-2 detection, the routine 3H-thymidine incorporation assay (described by Gillis et al., 1978) and the colorimetric MTT method (a modification of Mosmann’s methods - Berg et al., 1990), were compared. In colorimertic assay, one laboratory unit (LU;
1.0 LU = 1.21 International unit) of IL-2 activity was defined as the concentration of supernatant that induced 50% of maximal cell growth.

The results of the measurements of the supernatants from in vitro propagated transfected cells were reproducible. The colorimetric methods used by other authors were found to be more sensitive and useful for detection of low levels of cytokines than the isotopic test. We have observed similar activities of supernatants in both assays. Thus, the employed methods appeared to be comparable and are used simultaneously in our co-operating laboratories for estimation of the levels of IL-2 secreted by the transfectants that are applied as the source of the cytokine in therapeutic experiments.

*Institute of Molecular Genetics, Academy of Sciences of the Czech Republic, Prague, Czech Republic

Publication no. 3453

Characterization of oligosaccharide structures present on electrophoretically fractionated proteins of cell extracts or other biological materials is frequently done on blots with the use of lectins or anti-carbohydrate antibodies. Some oligosaccharide structures can by carried by N-linked or O-linked glycans and information on the type of oligosaccharide chain can be obtained by selective removal of one type of glycan. While there are several endoglycosidases or asparagine amidases which remove N-glycans, the choice of such enzymes specific for O-glycans is limited. Moreover, an enzymatic release of oligosaccharide chains is frequently not complete and all such enzymes are expensive. An alternative method widely used for glycoprotens in solution is a chemical release of O-glycans by b-elimination which is carried out under mild alkaline conditions in the presence of an excess of NaBH4. This method has been adapted to glycoproteins on blots. The experiments were performed with erthrocyte membrane proteins in which glycophorins are the major poly-O--glycosylated components, and with lysates of colon cancer cells CX-1.1. Lectins and monoclonal antibodies against peptidic, glycopeptidic and carbohydrate epitopes were used to examine the effect of degradation. The proteins should be transferred after electrophoresis to Immobilon P membranes and not to nitrocellulose which is mechanically unstable under alkaline conditions. Moreover, NaBH4 (which is used for protection of released O-glycans by their reduction and is not necessary for experiments on blots) has to be omitted, since it strongly decreased (for unknown reasons) the binding of anti-peptidic antibodies to de-O-glycosylated proteins. The optimal results were obtained by heating the blots in 0.055 M NaOH for 16 h at 40°C. Under these conditions the O-glycans of glycophorins were undetectable, while N-glycans and peptidic epitopes of glycophorins and other membrane proteins were detected with unchanged or even increased intensity, comparing to untreated blots. Anti-M and anti-N antibodies, which recognize O-glycosylation-dependent epitopes of glycophorin A (GPA) did not detect GPA on NaOH-treated blots. Some less frequent anti-M antibodies cross-reacting with the rare GPA variant of Mg type are specific for a peptidic epitope which is unrelated to the MN blood group--specific amino acid sequence in unglycosylated peptides, but is recognized in GPA-M only in the glycosylated antigen. These antibodies showed specificity for GPA-M on untreated blots, but detected GPA-M, GPA-N and glycophorin B bands on NaOH-treated blots, which is the easiest way for identification of the anti-M/Mg specificity. The method was also used to show that most protein--linked sialyl-Lea epitopes present on CX-1.1 cancer cells are located on O-glycans. Summing up, b-elimination on blots can be used for identification of the type of glycan carrying a defined oligosaccharide structure and for investigation of the role of O-glycosylation in interaction of glycoproteins with antibodies and other ligands.

Publication no. 3447

The carbohydrate moiety of glycoproteins consists of sugar chains of different length and structure. The N-linked oligosaccharides of glycoproteins may consist of as many as about 70 monosugar residues, whereas O-linked chains, which in general are much shorter, may consist even of a single monosugar bound to a peptide. A method, which enables to determine such single monosugars bound to a peptide, was investigated and simplified. The procedure is as follows: a glycoprotein or a glycopeptide (50-400 mg) is chemically degraded under modified conditions of Carlson degradation (b-elimination performed in 0.1 M NaOH/1 M sodium borohydride in the presence of Cd2+ ions). The reaction mixture is neutralized and an aliquot, supplemented with a known amount of a standard monosugar alditol, is peracetylated, extracted with chloroform and directly analyzed by GLC-MS. All the O-linked oligosaccharides are split off from the peptide and are derivatized under described conditions, but under applied parameters of gas-liquid chromatography only monosugar peracetylated alditols are determined. By comparing the retention times of appropriate peaks with standards and by checking their mass spectra the monosugar alditols are unequivocally identified. The detectable amount of a reduced monosugar in the analyzed sample is about 0.3 mg.

Several glycoproteins were analyzed using this method, but only in two cases degradation products contained reduced monosugars: GalNAc-ol and Gal-ol was obtained after degradation of human glycophorin A (GPA) and GalNAc-ol was obtained after degradation of ovine submaxillary mucin (OSM). The method is versatile and may be used for any type of glycoprotein.

*Department of Biochemistry, Medical Academy, Wrocław, Poland

Publication no. 3465

A new procedure was used to conjugate lactose, dextran and bacterial hexasacharide with proteins without using coupling or activating reagents. The method is simple, rapid and cheap. Reducing sugars covalently bind to proteins when lyophilized together and briefly heated to a high temperature.

Summing up, the existence of a stabile protein-saccharide bond has been confirmed by the following findings:

*Department of Chemistry, University of Ottawa, Canada

Publication no. 3522

Aqueous mixtures of glucose and fructose produce red solutions when treated with 2% (w/v) phenol in 5% (v/v) aqueous acetone in the presence of concentrated sulfuric acid. The color is stable for days, and the red chromophore has an absorbance maximum at 568 nm. When the concentration of phenol is raised to 25%, fructose, but not glucose, produces red solutions, allowing for the selective detection of ketoses. Two complementary methods have been developed to remove the interference of ketoses in solutions containing glucose. The first one relies on the selective reduction of ketoses with sodium borohydride in the presence of cerium (III) chloride prior to the addition of the phenol-acetone reagents. The second method is based on the differential specific determination of glucose using 2% versus 25% levels of phenol. The relative sensitivities of different sugars are also presented as well as the applicability of the methods using bacterial polysaccharides for immunochemical analyses. The quantitative determination of glucose or ketoses in the polysaccharides does not require hydrolysis prior to the estimation.

*Department of Chemistry, University of Ottawa, Canada

Publication no. 3364