Ludwik Hirszfeld Institute of Immunology and Experimental Therapy
Polish Academy of Sciences
Rudolfa Weigla 12, 53-114 Wrocław

Research Report – 2001

DEPARTMENT OF IMMUNOCHEMISTRY
Head: Professor Czesław Ługowski, Ph.D.

The aim of our studies in the year 2001 was the immunochemical characterization of endotoxins - lipopolysaccharides, major surface antigens and important virulence factors of Gram-negative bacteria. Endotoxins were isolated from opportunistic pathogens such as Hafnia alvei, and Citrobacter gillenii.

By utilizing two-dimensional NMR spectroscopy in conjunction with chemical and immunological methods, we have established the structures of H. alvei O-specific polysaccharides present in the strains PCM 1196 and PCM 1223. An acidic O-specific polysaccharide isolated from strain 1196 is consists of pentasaccharide repeating units containing hexoses, N-acetylhexosamines and C-4 substituted galacturonic acid. O-antigen of H. alvei strain 1223 is a neutral a -mannan composed of pentasaccharide repeating units. Immunoblotting showed a strong cross-reactivity between the investigated LPS and the LPS of E. coli O9 and K. pneumoniae O3. The serological relationship of the LPSs of these bacteria is due to the structural identity of their O-specific polysaccharides, though the LPSs differ in their core regions. A neutral O-specific polysaccharide isolated from the LPS of C. gillenii strain 1544 was studied by specific degradations and chemical and instrumental analyses. It is composed of neutral, tetrasaccharide repeating units substituted with O-acetyl groups on C-2 of b-L-rhamnose residues. (In collaboration with the N.D. Zielinsky Institute of Organic Chemistry, Russian Academy of Sciences, Moscow, the Russian Federation).

During the last years we have investigated the presence of the blood-group ABH antigens in human glycophorin A (in collaboration with prof. Bo Nilsson, Umea, Sweden); this year we analyzed N-glycans of GPA, originating from blood group O erythrocytes. Using ES mass spectrometry of permethylated oligosaccharide alditols, native and desialylated, we confirmed the presence of blood group H antigen in these N-glycans: it resides on one branch of the N-glycan, while the other is sialylated. Studies like these are important in view of the emerging knowledge on the role of GPA as a marker of glycosylation changes under pathological conditions.

This year we participated in the 4^th Int. Workshop on Monoclonal Antibodies Against Human Red Blood Cells and Related Antigens. The aim of our studies was to investigate the specificity of the monoclonal antibodies against human glycophorin A (GPA) and C (GPC), against variant glycophorin Mi^a, and against Duffy antigen. The general method we used was Pepscan-analysis, which is based on the chemical synthesis of defined short peptides on plastic pins and determining the reactivity of the antibodies using the ELISA method. We identified 10 peptidic epitopes of a defined sequence.

We also investigated the polyagglutination of the NOR erythrocytes by antibodies, commonly present in human sera. We found that a neutral glycosphingolipid fraction from NOR erythrocytes inhibited this polyagglutination. Using ES mass spectrometry (in collaboration with Dr. V. Reinhold, New Hampshire, USA), we isolated and analyzed the structure of a neutral glycosphingolipid from NOR erythrocytes, which was shown to be a1-4 galactosylated globoside (Gala 1-4GalNAcb1-3Gala 1-4Galb1-4Glc-Cer). This is a new carbohydrate structure, never before described. We also found extended neutral glycosphingolipid in NOR erythrocytes (Gala 1-4GalNAcb1-3Gala 1-4GalNAcb 1-3Gala1-4Galb1-4Glc-Cer).

A proline-rich polypeptide (PRP) complex was isolated in our Laboratory by Janusz et al.(1974) from ovine colostrum. The polypeptide complex shows immunoregulatory and procognitive activities. PRP, in the form of orally administered tablets called Colostrinin ^Ò (containing 100 mg PRP), improves the outcome of Alzheimer’s disease (AD) patients and prevents further deterioration of their health status.

The mechanism of action of Colostrinin^Ò in AD patients is not yet fully clarified. Recently, the involvement of the immune system in the pathogenesis of AD and the role of cytokines in the regulation of neurodegenerative processes have been recognized. Other factors which can affect the development of AD are oxidative stress and an overproduction of nitric oxide (NO).

We have found that Colostrinin^Ò may regulate the secretion of an array of cytokines in cultures of human blood cells and that it inhibits overproduction of NO and superoxide anion (O₂^-) in murine macrophages. The results obtained suggest that the beneficial effects of Colostrinin ^Ò in AD patients might be caused, among others, by its regulatory effects on cytokine secretion and its inhibition of an overproduction of reactive oxygen radicals and NO.

In our studies on new carriers for gene transfer, the effect of surface charge on the association of liposomes with human colon CX-1 cancer cells was analyzed. When phospatidylserine was incorporated into a lipid bilayer, the amount of liposomes associated with cells tended to increase along with the amount of negatively charged lipid present in the liposomal lipid bilayer. When the cationic lipid dioleyol-1,2-diacyl-3-trimethylammonium-propane (DOTAP) was included in the liposome formula, uptake by the cells was also increased. Maximum binding occurred when the amount of positively charged lipids in liposomes was about 10 mol% of lipids.

The condensation of chemically well-defined DNA plasmids was studied by the use of fluorescence correlation spectroscopy (FCS). It became clear that the DNA molecule exhibits discrete conformational change between the coil and globule states with the addition of a small amount (the order of magnitude being 10^-5 M) of cationic surfactants, such as spermine and hexadecyltrimethyl ammonium bromide (HTAB).

DEPARTMENT OF EXPERIMENTAL THERAPY
Head: Professor Michał Zimecki, Ph.D.

Studies on the role of lactoferrin (LF) in immune response were continued. We demonstrated that a receptor, exhibiting affinity to mannose, plays a critical role in the mediation of the regulatory activity of LF in cellular immune response in mice. Other studies in vitro revealed that LF regulated the proliferative response of mononuclear blood cells to phytohemagglutinin (PHA) and mixed lymphocyte reaction. The first clinical trial on patients, subjected to minor surgery, showed that LF given per os before operation enhanced the response of mononuclear blood cells to PHA and lipopolysaccharide.

Other studies demonstrated immunosuppressory activities of Vratizolin®, a registered antiviral drug, in several models in vivo and in vitro, in both mice and humans. Interesting, was the differential action of cyclosporine A on the generation of humoral and cellular immune responses in mice, which were also described.

Other investigations involved the immunotropic properties of 10 derivatives of 5-amino-3-methylizoxazolo-4 carboxyl acid hydrazide in models of humoral and cellular immune response in mice.

Studies were also conducted on the mechanism of bone formation by injection of Hela cells into the thigh muscles of mice. We proved that the induction of bone formation was conditioned by the secretion of bone morphogenic proteins by the grafted cells.

The biological activity of lipopolysaccharide (LPS) containing sphingosine was investigated in the rat model of experimental induced uveitis (EIU). A glucose-nonfermenting Gram-negative strain was isolated from a routinely disinfected bronchofiberoscope. LPS isolated from the bacterial mass contained sphingosine, a rare component in bacteria compared with those typically occuring in LPS (Department of Immunology of Infectious Disease, Institute of Immunology and Experimental Therapy).

The LPS isolated from the clinical strain was twice as active in EIU induction as that from Shigella sonnei Phase I and Escherichia coli K12 strains. Polymyxin B (PmB) did not inhibit the EIU induced by this LPS, in contrast to the complete inhibition of Eschericha coli K12 lipopolysaccharide activity by PmB. The results indicate that the LPS from the investigated clinical isolate has potent endotoxic activity. The structural difference of lipid A in the LPS containing sphingosine may be responsible for the lack of inhibitory effect of Polymyxin B.

A series of new nociceptin analogues containing cysteine (synthesized by Dr H. Bartosz-Bechowski, Wroclaw University) was tested for their nociceptive effects in tail-flick tests on 129/Iiw strain female mice after icv (intracerebroventricular) injection. The cysteines were introduced in order to get irreversibly binding analogues, based on the assumption that the cysteines in the ligand can interact with those from the receptor to form S-S bridges. In vivo tests revealed that Cys¹-nociceptin (1-13)-NH₂ (Cys1-NC) is an antagonist, whereas Cys7-NC is an agonist. Gly¹,[Phe(p-NO₂)]⁴-NC was less active, indicating that the antagonist properties of Cys1-NC are associated with the presence of the sulfhydryl group of cysteine. The analogues D-Cys2 and Cys3 were also almost inactive.

Several mechanisms have been suggested to account for the poor immune response of T lymphocytes in B chronic lymphocytic leukemia (B-CLL). Recently, there has been increasing interest in costimulatory and inhibitory regulators of immune activation, in particular in the CD28 homologue CTLA-4 (CD152) molecule, which is transiently expressed on T lymphocytes after activation.

We evaluated the kinetics and levels of expression of down-regulatory CTLA-4 and costimulatory CD28 molecule on peripheral blood T lymphocytes stimulated with anti-CD3+rIL-2. There were different kinetics and levels of T-cell CTLA-4 expression in healthy controls and B-CLL patients. The percentage of CD3+/CTLA-4+ cells among the unstimulated cells of all the persons studied was negligible. The highest percentages of CD3+/CTLA-4+ cells were obtained in normal subjects after 72 h and in B-CLL patients after 24 h of stimulation. The mean percentage of CD3+/CTLA-4+ cells returned to undetectable levels after 72 h of stimulation in the control group and after 120 h of stimulation in B-CLL patients. There was a statistically higher percentage of CD3+/CTLA-4+cells in B-CLL patients at each time point tested compared with the control group. There was also a statistically lower percentage of CD3+/CD28+ cells in B-CLL patients compared with the controls.

Our study provided evidence of different kinetics, increased CTLA-4 and decreased CD28 expression on peripheral blood T lymphocytes in B-CLL, which, in consequence, may result in a stronger, earlier and longer-lasting down-regulation of T-cell activation and may be one of the mechanisms of immunological impairment in this disease.

We also studied the expression of the universal cdk inhibitor p27^KIP1 in B-CLL. This protein was present in nearly 100% of lymphocytes in all B-CLL populations tested. Its cellular content, estimated semi-quantitatively by specific mean fluorescence intensity (MIF) and the percentage of p27+/CD19+/CD5+ cells, was significantly higher in B-CLL patients than in the control group. There was no correlation between the level of p27 expression and clinical stage of the disease. Apoptosis of neoplastic cells, estimated by the TUNNEL method, did not correlate with p27 expression.

DEPARTMENT OF MICROBIOLOGY
Head: Professor Jolanta Zakrzewska-Czerwińska, Ph.D.

Initiation of Helicobacter pylori chromosome replication. The sequence analysis of the H. pylori genome revealed a few surprising features: (i) the typical eubacterial block of replication genes, dnaA-dnaN-recF-gyrB, does not exist; (ii) an origin of DNA replication is not evident from the genome sequence; (iii) the dnaC gene encoding DnaC protein that delivers the DnaB helicase to the prepriming complex is absent. We charecterized the key elements of the initiation of H. pylori chromosome replication: DnaA protein and a putative oriC region. The H. pylori oriC region containing five DnaA boxes was cloned as an autonomously replicating minichromosome.

The interactions between DnaA protein and the oriC region were analyzed by competition gel retardation assay. The H. pylori DnaA protein shows an interesting DNA binding feature: being a protein of rather low sequence specificity it forms nucleoprotein complexes preferentially with the H. pylori oriC region.

Type II thioesterase from Streptomyces coelicolor A3(2). Many type I polyketide synthases (PKS) clusters contain additional TE genes located adjacent to PKS genes. These are discrete proteins called type II thioesterases (TE II) to distinguish them from the chain-terminating thioesterase domains, (TE I), which are usually fused to the terminal PKS module. A gene of a new, type II thioesterase, scoT, associated with the cluster of putative type I polyketide synthase genes from Streptomyces coelicolor A3(2) was found. When expressed in a heterologous host, Streptomyces fradiae, scoT successfully complemented the resident TE II gene (tylO) and proved to be catalytically equivalent to the TylO protein.

The intracellular level of cyclic GMP (cGMP) depends on the activity of opposite enzymes: guanylyl cyclases (GC), which synthesize the nucleotide from GTP, and phosphodiesterases (PDE), which hydrolyze it to GMP.

Two distinct forms of guanylyl cyclases (GC), particulate (pGC) and soluble (sGC), have been recognized in vertebrates. They differ in structure, subcellular localization, and mechanism of activation. The aim of our studies was to determine which form of GC is responsible for cGMP synthesis in cells of mouse lymphatic organs. Using known activators of pGCs (ANP, BNP, STa) and sGC (SNP), we found that although both forms of GCs contributed to cGMP synthesis in the thymus, lymph nodes, and spleen, the activity of soluble GC was markedly higher.

Continued studies on the mechanisms that regulate the metabolism of cGMP in rat macrophages showed that the activity of pGC in the cells depends on environmental conditions and can be modulated by other intracellular signaling pathways. Since phosphodiesterases (PDEs) belonging to seven different families are able to hydrolyze cGMP, we asked which of them might be responsible for the nucleotide degradation in macrophages. We found that in rat macrophages at least three isoforms of PDEs are possibly involved in cGMP hydrolysis. Detailed studies are in progress to determine which of these are in fact expressed in the cells.

DEPARTMENT OF CLINICAL IMMUNOLOGY
Head: Professor Andrzej Lange, M.D.

Allogeneic stem cell transplantation is performed across the barrier of histocompatibility antigens. In family HLA matched transplantation, the difference between the recipient and donor depends rather on minor than on major HLA specificities. In alternative donor transplantation, genetic difference is apparent in one haplotype in family setting and in both haplotypes in unrelated donors transplantation. Therefore, in a group of transplanted patients there are a variety of individuals with different degrees of incompatibility in histocompatibility specificities. This was investigated with the use of DNA techniques which describe the specificities at a high resolution. A limit of permissive incompatibility was assesed by following the fates of patients transplanted with the use of manipulated (elutriation for T cell depletion) and unmanipulated grafting material. For immunological outcome, probably the most important cytokine is IFN-gamma. Cytokine genes are polymorphic, and we documented an association between the risk of aGvHD and IFN-gamma gene polyumorphism.

The outcome of transplantation with a given extent of HLA compatibility largely depends on the number of CD34+ cells transplanted. It was shown that the contribution of CD34+CXCR4- cells to the PBPC was positive correlated with the yield of hematopoietic progenitors mobilization to the blood. The architecture of the marrow was investigated in some detail by immunostaining trephine biopsy specimens taken from cadaveric organ donors. It was found that the reactive nodules in the marrow were organized according to the interaction of CXCR3 and CXCR4 with their respective ligands.

This work was done in collaboration with the Clinic of Dermatology and Venerology, Medical University of Wrocław (Prof. F. Wąsik, Prof. E. Baran, Prof. M. Cisło, and Dr. P. Nockowski).

Psoriasis vulgaris is the only human disease associated with HLA-C. The association of the HLA-Cw*0602 allele with this condition was described for many populations, but to varying degree, from strong to almost negligible. In addition, it is still not known whether it is the HLA-C gene itself or rather a closely linked gene that is causatively linked with psoriasis. Several genes from this region were examined in addition to HLA-C and -B. However, HLA-linked complement component polymorphic genes were not studied extensively. Therefore, we typed 67 psoriatic patients for alleles of the HLA-linked complement components BF, C4A and C4B. Alleles of C3, encoded on another chromosome, were established in parallel as a negative control. The frequencies in patients were compared with those in unrelated healthy controls: 100 individuals for C4A and C4B, 890 for BF, and 4719 for C3. We found no association of BF alleles with disease, nor of C3. Among the C4 alleles, C4B*3 was present in 13.4% of patients as compared with 1% of controls (OR, 15.36; 95% CI, 1.897 to 124.42; p=0.0009), and C4A*6 was present in 19.4% of patients versus 7% of controls (OR, 3.20; 95% CI, 1.202 to 8.508; p=0.0155). The high frequency of C4B*3 in psoriatics has not been described so far. These results suggest a contribution of C4 genes themselves or a closely linked gene to the susceptibility to psoriasis (Cisło et al., Immunol. Letters, 2001, 80,
139-143).

In last years we have described a new B lymphoblastoid cell line (B-LCL), HAJ, that did not express HLA class II antigens HLA-DR, -DQ and –DP, although the genes for these molecules were present (Mańczak et al., Arch. Immunol. Ther. Exp., 1996, 44, 171-178; Kość et al., Centr. Europ. J. Immunol., 1998, 23, 26-29). Here, we examined the effect of interferong, a potent stimulator of HLA gene expression, on HLA class I and class II expression on HAJ cells. We found that culture of HAJ cells in the presence of interferon g upregulated HLA class I expression that was otherwise much lower than on B-LCLs without the HLA defect. However, interferon g did not restore HLA class II expresssion on HAJ cells, similar to its lack of effect on bare lymphocyte syndrome-derived B-LCLs and bare lymphocyte syndrome-like B-LCL mutants (Nowak et al., Arch. Immunol. Ther. Exp., 2001, 49, 453-460).

DEPARTMENT OF MEDICAL IMMUNOLOGY
Head: Professor Andrzej Górski, M.D.

Extensive studies on phage therapy were continued in 2001. Specific phages were applied in 341 patients with different suppurative infections caused by multi-drug-resistant bacteria. The majority of infections were long persisting ones and antibiotic therapy had failed. Phage therapy was found to be highly effective in 81% of cases.

Bacteriophage treatment was also performed in 20 cancer patients with solid tumor and hematological malignances. Recovery from infections was achieved in all cases.

In addition, the bacterial flora of the posterior laryngeal wall, larynx and surgical wounds of the patients treated surgically for carcinoma of the larynx was investigated. A similarity of some bacterial strains isolated from various habitats was demonstrated by comparing the biochemical characteristics, drug resistance patterns, and susceptibility to bacteriocins and bacteriophages. Bacteriophagotyping confirmed the similarity of rods belonging to the genera Enterobacter and Pseudomonas.

Apoptosis has been proposed as a mechanism for maintaining immune privilege. Expression of Fas ligand (FasL) by the human trophoblast has been recently accepted as a mechanism providing protection against the lytic action of activated decidual immune cells. Transient expression of extracellular matrix (ECM) proteins reflects trophoblast differentiation. Therefore, the purpose of the study was to determine the role in pregnancy maintenance of T-cell apoptosis in an ECM environment in women with a history of recurrent spontaneous abortion (RSA). We studied the apoptosis of peripheral blood T cells after culture with mAb OKT-3 alone and with mAb OKT-3 and the following ECM: fibronectin and collagen IV in 10 women with a history of RSA, examined before and during pregnancy, and in 10 normal, healthy women with previous successful pregnancy outcome. We used Cell Death Detection ELISA (Boehringer Mannheim), a photometric enzyme immunoassay for the quantitative in vitro determination of cytoplasmic histone-associated DNA fragments after induced cell death. In addition, the apoptosis of T cells isolated from the decidua of women with subsequent pregnancy failure was studied. Cells undergoing DNA fragmentation were identified by DNA analysis using flow cytometry. This method was based on the accumulation of ethanol-fixed apoptotic cells in the sub-G0/G1 peak of the DNA content as a result of the loss of DNA fragments from the cells and because of the reduced ability of DAN to be stained by propidium iodide. The highest values of enrichment factor, i.e. mU of the sample (dying/dead cells) per mU of the corresponding control (viable cells), were observed after peripheral T-cell culture with fibronectin in samples of women with subsequent pregnancy success. The apoptotic index (the percentage of positive cells per total number of cells) of decidual T cells was lowest in women with a history of recurrent spontaneous abortion compared with women after sporadic or elective abortion. Apoptosis of activated maternal immune cells in the human decidua may be a defense mechanism against rejection of the fetal allograft by the maternal immune system. Preliminary data suggest that disturbances in the programmed cell death of activated T cells in the human decidua can be responsible for recurrent pregnancy loss. Therefore, the apoptosis of activated cytotoxic T cells might be an interesting possible explanation of successful pregnancy outcome.

1,25-Dihydroxyvitamin D₃ induces differentiation and inhibits proliferation of human promyelocytic leukemia cells. PD 98059, the specific inhibitor of MEK1 and MEK2, was found to inhibit D₃-induced monocytic differentiation of HL-60 cells. This finding proves that activation of the raf/MEK1,2/erk1,2 signal transduction pathway is essential for monocytic differentiation of human leukemia cells. On the other hand, inhibition of cytosolic phospholipase A₂ accelerated differentiation of HL-60 cells induced by D₃, suggesting the possible existence of a feedback loop between extracellular-signal regulated kinases and phospholipase A₂. PRI-1906, an analog of vitamin D₂, and PRI-2191, an analog of vitamin D₃, bind the nuclear vitamin D receptor (nVDR) with substantially lower affinity than D₃, but retain a higher differentiation-inducing activity, as estimated in a HL-60 leukemia-cell model. To examine how their increased differentiation-inducing activity is regulated, we tested the hypothesis that membrane-mediated events, unrelated to nVDR, are necessary for differentiation in response to PRI-1906 and PRI-2191. Induction of leukemia-cell differentiation in response to analogs of vitamin D was inhibited by LY294002 (PI 3-K inhibitor), PD98059 and rapamycin (p70^S6K inhibitor), indicating that activation of signal transduction pathways unrelated to nVDR is necessary for differentiation.

Among the five non-sex-linked minor histocompatibility antigens, a recipient's disparity for HA-1 is associated with the occurrence of grades II-IV acute GvHD. HA-1 antigen is a nonapeptide encoded by a gene designated as KIAA0223 located on chromosome 19. HLA-A*0201 molecules have high affinity for the HA-1^H peptide, and this complex is recognized by HA-1-specific cytotoxic T cells. The HLA-A*0201/HA-1^R complex does not generate a detectable immune response.

In our study we determined the allele frequency of HA-1 in 58 unrelated HLA-A*0201 individuals from the south-western area of Poland. The HA-1^H allele frequency was 0.405 and that of HA-1^R 0.595. The genotype distribution was: homozygotes H/H 14% and R/R 33%, heterozygote H/R 53%. The distribution of HA-1 alleles and genotypes fits well with the distribution expected under Hardy-Weinberg equilibrium (c ² =0.668).

Genetic factors associated with increased risk for cancer: the p53 Arg/Pro polymor-phism at codon 72

The p53 Arg/Pro polymorphism at codon 72 (CGC vs. CCC) is in a proline-rich functional domain (aa 64-92) necessary for efficient growth suppression. It has been suggested to be involved in susceptibility to various types of cancers. To examine whether it could represent a risk factor in the Polish population, we analyzed exon 4 codon 72 in constitutional DNA.

The frequency of p53 Pro allele was 0.276, compared with 0.29 to 0.35 reported for Caucasians in general. The genotype distribution was: Pro – 0.054, Arg – 0.503 and Pro/Arg – 0.443. A similar distribution was observed in cancer patients with familial cancer history. In patients with multiple primary neoplasms, no Pro genotype was found, and the frequencies of Arg and Pro/Arg were 0.482 and 0.519, respectively. In pediatric neoplastic and non-neoplastic patients, the respective frequencies of Pro allele were 0.285 and 0.352. In both these groups, the respective genotype patterns were as follows: Pro: 0.1 and 0.11, Arg: 0.53 and 0.47, Pro/Arg: 0.37 and 0.482.

We found that murine resident peritoneal cells exhibit innate antiviral immunity. During in vitro culture of the cells for several days, this type of immunity was gradually reduced. As the viruses VSV, EMCV, HSV-1 belong to different taxonomic groups, the immunity expressed by the leukocytes appears to be non-specific. Cyclosporine A, given to BALB/c mice, reduced the innate antiviral immunity in the mice.

Viral and bacterial infections are among the risk factors for the vertical transmission of HIV. Therefore, the effect of experimental double infection with unrelated viruses (VSV and EMCV) and the effect of VSV and sonicated treponema pallidum or bacterial LPS on innate antiviral immunity was studied. A reduction of the innate immunity of placenta and amniotic membrane by the agents was observed, which may contribute to an increased vertical transmission of viruses.

The effect of paramyxovirus (NDV) on the nitric oxide (NO) production by pulmonary leukocytes was studied on the bronchial asthma model. Paramyxovirus enhanced NO production in pulmonary leukocytes, mainly in neutrophils, in the cases of overlapping, mixed airway infections coexisting with bronchial asthma.

Acid-labile IFN levels in sera from HIV-infected patients were measured. The correlations with clinical status and standard diagnostic parameters were analyzed. In the case of patients on HAART therapy, acid-labile IFN was usually absent. Therefore, a high level of IFN suggests unsuccessful therapy.

The antiviral properties of organoselenium compounds, analogs of ebselen, were determined using EMCV, VSV and HSV-1 viruses. Virucidal activity against EMCV and HSV-1 was shown for ebselen and several of its derivatives. Some of them were able to reduce VSV replication in human cord blood leukocytes.

Antiviral activity of free and immobilized photosensitizers was studied in culture medium and in the presece of red blood cell concentrate. The enveloped viruses (HSV-1 and VSV) were photoinactivated by some of the compounds. However, there was no activity against EMCV, a non-enveloped virus.

The organ specific phenotype of endothelial cells influences cell trafficking and extravasation. A necessary step of the extravasation process are the adhesive interactions of endothelial cell adhesion molecules with their ligands, presented by a partner cell surface molecules. In our previous study we demonstrated that the acquisiton of tissue specific high metastatic phenotype by several selected variants LS-180 human colon carcinoma cells was accompanied by an increased expression of glycosylated tumor antigens, which correlated with their differential adhesion pattern to endothelial cells.

The studies in 2001 concerned endothelial cells phenotypic characteristics which determine the organ specificity of metastatic secondary growth. The subject of the study was a panel of eight human endothelial cell lines from distinct tissue origin that we immortalized and characterized (Patent No. 99 16169, 21/12/99, CNRS, France). Their organ specificity was confirmed by the differential display of endogenous lectins and cytokine receptors, as well as tissue specific addressins and other adhesion molecules expression (Kieda et al., Endothelium, in press). In collaboration with the group of Prof. S. Szala from the Institute of Oncology in Gliwice, two of the endothelial cell lines were examined for the presence of KDR/flk-1 gene promoter, specific for endothelial cells. It was revealed that the promoter was present in some tumors, i.e. human ovary carcinoma and in mouse sarcoma, which makes it a candidate for use in antitumor gene therapy (Szary J. et al., Anticancer Res.).

DEPARTMENT OF INFECTIOUS DISEASE MICROBIOLOGY
Head: Associate Professor Andrzej Gamian, Ph.D.

The current activity of this laboratory is focused on studies of the mechanisms of pathogenicity in autoimmune diseases with bacterial etiology, the role of molecular mimicry, bacterial proteins in pathogenicity, and the structures and functions of bacterial capsular antigens and endotoxins (Post. Hig. Med. Dośw., 2001, 55, 211-232).

The general strategy for the elaboration of protective tools against invading bacteria involves determining the structures of the molecules involved in infection and immune processes and understanding their biological activities. Thus, the structures of several such antigens have been established (Carbohydr. Res., 2001, 330, 523-528 and 331, 331-336, FEMS Immunol. Med. Microbiol., 2001, 30, 223-227).Sialic acid is one of the key molecules on the surface of tissue cells participating in immunological functions. In some bacteria, sialic acid may be a constituent of its surface antigens, and the occurrence of sialic acid is associated with an increased pathogenicity of the bacteria, particularly as it is involved in autoimmune processes. Sialic acid is structurally similar to 3-deoxy-octulosonic acid, an inherent component of endotoxin. Thus, the interference of these bacterial components with the functions of tissue sialic acid may contribute to the mechanisms of pathogenicity (FEMS Immunol. Med. Microbiol., 2001, 31, 169-173). Due to the structural mimicry of sialic acid-containing structures, care should be taken when antibacterial vaccines are constructed to avoid the induction of autoantibodies.

DEPARTMENT OF CANCER IMMUNOLOGY
Acting head: Associate Professor Leon Strządała, Ph.D.

Studies on the effects of experimental antitumor immunogenotherapy have been performed. After multiple peritumoral injections of IL-2-producing plasmocytoma cells into C38 colon cancer-bearing mice, an increase of NO production in tumor tissue, correlating with significant therapeutic effect was observed.

In the search for models allowing study of the pathogenesis of metastasis, tumor angiogenesis, and the effectiveness of antitumor and antiangiogenic (genistein) treatment we have been shown that: 1) The distribution of experimental tumor metastases depends on the route of tumor cells inoculation. 2) Genistein inhibits the proliferation in vitro of all mouse tumor cell lines applied, but its activity in vivo is differential and depends on the tumor type and on the route of tumor cells transplantation. 3) The antimetastatic effect of genistein in mice which have undergone surgical removal of a primary tumor depends on the histological type of cancer. 4) The influence of genistein on the antitumor effect of cyclophosphamide depends on the route of tumor cells transplantation and on the histological type of cancer.

An original model to determine the amount of blood in the tumor tissue, corresponding to the degree of its vascularization was established. The results obtained in this model (I¹²⁵-labeled mouse albumin) did not differ from those obtained with the use of the referential technique applying FITC-dextran.

A new original procedure for the conjugation of antitumor drugs with macromolecular carriers was elaborated. The procedure was tested for synthesis of the following conjugates: BSA-tomudex, BSA-methotrexate, immunoglobulins-antitumor drugs, fibrinogen-fluorescein-antitumor drugs, and lysozyme-haptens.

We have been searching for novel thymus-specific genes and proteins that change their expression during the intrathymic development of T lymphocytes. The identification, characterization and functional analysis of such genes and proteins may contribute to a better understanding of the processes that are central to the function of the immune system, such as the generation and selection of the self MHC-restricted and self tolerant repertoire of T-cell receptor specificities. Using such techniques as cDNA-RDA (Representational Difference Analysis) and ‘cDNA microarrays’, we have identified a number of new thymocyte-specific sequences that are currently under investigation.

Cell lines derived from fetal mouse brain were established in our laboratory. The cells were characterized with the use of antibodies against beta-tubulin III (neuronal marker) and GFAP (marker of astroglial cells) and visualized with the use of immunofluorescent confocal microscopy. Preliminary experiments using dual-labeled cells showed that beta-tubulin III and GFAP were expressed in the same cells. A panel of neuronal and glia markers (nestin, MAP2, neurofilaments, beta-tubulin III, Gap43, p75NTR, and GFAP) are currently under investigation using the RT-PCR technique.