RESEARCH REPORT 1996-1997

Most studies performed at the Institute in the years 1996-1997 concerned the following areas: (i) synthetic and natural immunomodulators, (ii) bacterial antigens and microbial infections, (iii) tumor immunology and experimental therapy and (iv) clinical immunology, with a focus on marrow/blood transplantation. Smaller groups continued the research on HLA antigens, red blood cell antigens, cellular signalling via Fc receptors and molecular biology of microorganisms. The major problems of interest and obtained results are summarized below (numbers in square brackets are the numbers of respective research reports presented on further pages).

A substantial part of the research was continuation of an interesting idea of chemical synthesis and testing an immunomodulatory activity of peptides with sequences corresponding to selected fragments of immunologically active proteins [1-8]. The peptides studied corresponded to fragments of TGFb-family proteins, thymopentin-like sequences of G-actin and HLA-DQ, fragments of IL-1R antagonist. Analogs of these peptides and analogs of cyclinopeptide-A from linseed oil were also synthesized and examined. Most peptides were active in variety of immunologic tests. The accumulating data allow to draw conclusions on the mechanism of action of proteins containing the studied sequences and on the relation of immunomodulatory activity to the peptide structure. The aim of studies is also a selection of peptides with potential therapeutic value.

Several new seleno-organic compounds were synthesized and their cytokine-inducing and other activities were compared with those of ebselen, used as a reference compound [11-13]. Some of these new compounds appeared to be interesting as immunostimulants, potential antiviral agents and cytokine-inducers, active in humans.

Among natural immunoregulatory proteins, the long-term subjects of research at our Institute are lactoferrin (LF), and proline-rich polypeptide (PRP) from ovine colostrum (called also colostrinine), discovered at our Institute over 20 years ago. Investigation of these two proteins was also carried out during last two years.

Studies on lactoferrin were continued with an ultimate goal to apply this immunoregulatory protein in prevention and therapy [20, 21]. LF was found to decrease the postoperative shock in mice as determined by cytokine release into circulation. Furthermore, LF was regulatory with regard to proliferative response of PBMC and their ability to produce interleukin 6 and tumor necrosis factor alpha in several categories of patients such as: septic, trauma and cardiac surgery patients [53, 54].

Continuation of studies on colostrinine [14-19] led to further characterization of its cytokine-inducing properties and to finding an analog in human colostrum. Moreover, a therapeutic form of colostrinine was elaborated and applied successfully in clinical trials as an immunotropic and psychotropic factor.

The results obtained indicated a differential effect of kinases and phosphoprotein phosphatases in transmission of a signal and in effector functions of Fcg receptors on guinea pig peritoneal macrophages. These effects were dependent on a subclass and origin of immunoglobulins bound to the receptors and on a type of receptor [22].

It is well known that cancer cells have profoundly altered glycosylation of surface proteins and some tumor-associated carbohydrate antigens are receptors for human lectins and may play a role in tumor progression and metastases. Examples, which are a subject of research at the Institute, are sialosyl Lewis X (sLe^x) and sialosyl Lewis A (sLea) structures, which are receptors for selectins, and Thomsen-Friedenreich, Tn and related antigens, which are diagnostic and prognostic markers and therapeutic targets.

With the use of antisense RNA approach, sLea-negative subclone was obtained from colon carcinoma CX-1 cells characterized by high expression of sLea antigen. The data confirmed the thesis that sLea antigen is directly involved in adhesion of colon cancer cells, and point to the role of a1,3/4--fucosyltransferase as a key enzyme responsible for binding of these cells to E-selectin. In vivo experiments, using athymic nude mice as the recipients of xenografted human cancer cell lines, revealed that antisense transfected colon carcinoma cells develop liver metastases in markedly lower number of mice after orthotopic implantation, as compared to parental cells [23-24].

In another approach to study the role of intercellular interactions in the mechanism of cancer progression and metastatic spread of cancer cells, HT-29 human colorectal adenocarcinoma cell sublines with increased adhesive and invasive properties were initially selected in vitro and then in vivo in athymic nu/nu mice, inoculated orthotopically, intrasplenically or intravenously [25, 26]. During in vivo passaging the variant cell lines acquired increased metastatic ability and differential metastatic spread pattern, showing the increased expression of Lex, sialyl Lex and Ley antigens. As partner cells, six human endothelial cell lines derived from surgically removed metastatic lymph nodes or other tissue biopsies were established in vitro.

The in vitro selective adhesive interactions of colon carcinoma cells derived from metastatic foci of various localization with endothelial cells of various tissue origin suggest that the endogenous lectins as well as carbohydrate antigens may play a role in homing of cancer cells.

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. The influence of ab TCR-mediated selection on the development of spontaneous thymic lymphoma which appears in mice expressing a transgenic TCR specific for male antigen (HY) in the context of H-2Db molecules was analyzed [29, 30]. The molecular mechanism underlying the observed oncogenictiy of TCR transgens is not yet known. 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. 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.

In search for p53 gene mutations, the screening of blood and tumor samples of patients from families with tumor aggregations characteristic for Li-Fraumeni syndrome, or with multiple or sporadic tumors, was performed [32]. Somatic mutations in tumors were found in exons 5-9, covering hot spot regions of p53 gene. Present data base of p53 mutations has 7000 citations; our mutations are related to spontaneous deamination of 5-methylcytosine, changing it to thymine. Studies done in cultures of AF1 fibroblasts carrying mutated p53 allele showed that p53 germline mutation is not sufficient to cause transformation of the cells. However, transfection of mutated p53 to immortal fibroblasts induced tumorigenic phenotype. As DNA methylation affects frequency of transformation (most germline mutations of p53 gene seem to be the effect of 5-methylcytosine spontaneous deamination), experiments were started on the effect of disturbances in DNA methylation on cell transformation. As expected, cells transfected with a fragment of DNA:methylase in antisense orientation acquired the tumorigenic phenotype.

For screening in vitro of new potential anticancer agents, including both, of plant origin and newly synthesised compounds supplied from collaborative laboratories, the panel of human cancer cell lines representing various organ origin were applied. From among about 150 agents tested, 12 were selected for further studies [34]. The in vivo screening confirmed pharmacological and antitumor acitivity of the selected for phase I clinical studies (-)-(S) bromofosfamide [39]. The antitumor effect of some novel derivatives of triazolo- and imidazoloacridones was demonstrated in mice with transplanted murine colon cancer (MC38, resistant for cytostatics).

The antitumor effects of combined chemoimmunogenotherapy was examined in vivo using, in addition to treatment with cytostatic agent, the plasmocytoma (X63-mIL-2) or fibrosarcoma (F69-3mIL-2) cells transfected with mIL-2 gene [27, 28, 37, 38]. In mice with advanced growth of sc transplanted colon cancer (MC-38), treated with single dose of cytostatic followed by peritumoral injections of IL-2 transfected cells, the retardation of tumor growth and prolongation of survival time was achieved, and in some mice the tumors regressed completely.

The studies on differentiation inducing activity of calcitriol and its new analogs represent a new approach to anticancer therapy. The new analogs studied exerted similar to calcitriol antiproliferative and differentiation--inducing effects on HL60 cells, normal human keratinocytes or human papilloma virus type 16 transfected keratinocytes. In studies on the mechanism of induction of differentiation, the activation of intracellular transduction signals was demonstrated. In particular, the activation and nuclear translocation of mitogen activated protein kinase (MAP) was shown [35, 36].

The immune status of cardiac surgery patients and patients undergoing septic or traumatic shock was investigated [53, 54]. The immune response of operable patients was highly dependent on initial, preoperative reactivity of the immune system in a given patient or category of patients, as determined by proliferative response of peripheral blood mononuclear cells (PBMC) to mitogen and ability of these cells to produce inflammatory cytokines. The reactivity of PBMC in septic patients, particularly in nonsurvivors, was deeply diminished and correlated with a high interleukin 6 serum level.

The main achievements of the Laboratory of Clinical Immunology and Blood/Marrow Transplantation (BMT) Unit can be summarized as follows [41, 47-50]: 1. HLA typing in Poland was ongoing (see also [42, 44-46]); 2. MHC associated features (DR5, DR6 and NcoI TNF allelic specificities) were demonstrated as risk factors of aGvHD; 3. Apoptosis was shown as a characteristic feature of aGvHD, but not toxic lesions; 4. Levels of INFg and CD4+ cells are negatively correlated with a risk of aGvHD; 5. IL-6 and CRP contribute to the pathomechanism of aGvHD; 6. CD57+ cells are associated with a good prognosis after BMT; 7. Clinical application of allogeneic peripheral blood progenitor cells in transplantation was studied; 8. Sequential chemotherapy applied in treatment of small cell lung carcinoma was investigated.

Other clinical studies concerned AIDS. Along with the progression of HIV infection increasing dysfunction of the cytokine system is observed. Interferon (IFN) and tumor necrosis factor (TNF) responses were studied in HIV+ and AIDS patients [52]. It was found that blood leukocytes of HIV+ patients produced in vitro significantly less IFN-a after viral stimulation than HIV- individuals. The production of IFN-g in response to mitogen was impaired only in AIDS patients. These patients had also enhanced IFN-a levels in plasma. Multiple measurements of interferon levels allowed to define two patterns of IFN responses. Pattern I was characteristic for patients in good clinical condition and pattern II correlated with severe disease progression. Since IFN response reflected clinical condition of the HIV+ patients, its determination may be considered as marker for monitoring progression of AIDS and therapy.

Glycophorins, and particularly the major one, glycophorin A (GPA, carrying blood group M and N antigens), were thoroughly studied, also at our Institute. Although the function of GPA is largely unknown, it still serves as a very convenient model for solving various problems.

The epitopes of many new anti-glycophorin monoclonal antibodies were studied within the Third International Workshop on anti-erythrocytic antibodies (Nantes, 1996) [55]. Identification of the glycopeptidic and peptidic epitopes with the use of multi-pin peptide synthesis not only increased the value of the antibodies as research tools, but also supplied information on the effect of glycosylation on peptide antigenic properties.

It was previously shown in our Institute by means of anti-Tn lectins that O-glycans of GPA-N are slightly less galactosylated than those of GPA-M. Both GPAs differ in amino acid residues at positions 1 and 5. Recent studies showed that this difference of galactosylation pertains to 3 O-glycans located between amino acid residues 1 and 5. It is a spectacular example of regulation of O-glycosylation in vivo by the adjacent amino acid residues [56]. These results and new data on A and B blood group determinants in GPA [58] show that although the glycans of GPA were well characterized, not all minor details of glycans structure and its regulation are known. GPA partially deglycosylated by chemical methods supplies ‘multipurpose’ artificial TF and Tn antigens which were used for purification human anti-TF and anti-Tn antibodies [60].

GPA was shown to be a major target on erythrocytes for granulocytic proteases and its partial degradation was observed in red cells of patients with myelo- and lymphoproliferative disorders [57]. Carbohydrate-deficient glycoprotein syndrome (CDGS), a rare but severe inherited disorder, is characterized by decreased glycosylation of plasma proteins. Finding underglycosylated GPA in one CDGS patient [59] suggests that the deffect may concern also cell membrane glycoproteins.

Another interesting red cell membrane component is antigen Duffy which serves as the receptor for chemokines and some malaria parasites. A detailed characterization of peptidic epitope of Duffy antigen recognized by two anti-Fy6 monoclonal antibodies [61] (which inhibit chemokine binding) will serve for characterization of chemokine-binding site of Duffy.

Hafnia alvei, a member of Enterobacteriaceae, is a rare but important opportunistic pathogen which causes typical nosocomial infections. Among these, urinary tract, respiratory tract and wound infections are most common. H. alvei was isolated from some incidences of gastroenteritis, endogenous endophthalmitis and endotoxic shock with disseminated intravascular coagulation in adults with postoperative lung cancer. It has been found that multidrug resistance regulatory chromosomal locus is widespread among enteric bacteria including Escherichia coli and Hafnia alvei. This important finding in connection with data on sharing of virulence associted properties at the phenotypic and genetic levels between enteropathogenic E. coli and H. alvei led to the conclusion that H. alvei should be considered as an important diarrhogenic pathogen.

As the O-specific polysaccharides play an important role in the serology and interaction with the host, the structures of O-specific polysaccharides from 10 new, serologically different H. alvei strains have been elucidated and new constituents, which have not been found in H. alvei O-specific polysaccharides previously, were identified [62-67].

Interesting observation was that one of bacterial lipopolysaccharides contains sphingosine [69]. This LPS was extracted from a glucose-nonfermenting strain isolated from bronchofiberoscope used for examination of the patients suffering from pulmonary diseases. The presence of sphingosine in LPS preparation classifies the strain to the genus Sphingomonas. However, structure of known glycosphingolipid from Sphingomonas differs markedly from that of the clinical isolate.

Endotoxins are responsible for initiation of septic shock, which increases the number of fatalities in Gram-negative bacteremia among hospital patients. The mortality from septic shock is still high despite recent developments in antibiotic therapy, because antibiotics are unable to decrease the level of free lipopolysaccharides in the bloodstream. Moreover, it has been demonstrated that a significant amount of endotoxin can be released from killed bacterial cells following administration of antibiotics. Prevention and treatment of septicemia could involve stimulation of an immune response against lipopolysaccharide (LPS). The antibodies against conserved part of endotoxin core oligosaccharide may be cross-reactive and cross-protective if they can bind to smooth lipopolysaccharides in their natural states, either on bacterial cell surface, or bound to serum components in the circulation.

In 1996-1997 the immunogenic conjugates of core oligosaccharides E. coli and Salmonella with tetanus toxoid were prepared and used for production of monospecific sera. The neoglycoconjugates were good immunogens in rabbits yielding a high level of anti-lipopolysaccharide antibodies of IgG class. All antisera were able to react with the lipopolysaccharide molecules of identical or related core type, possessing core oligosaccharides substituted with O-specific chains [70].

In continuation of studies on mechanism of immunological response in syphilis it was found that PBM cells of syphilitic patients stimulated with T. pallidum antigen are able to produce IL-2, IFN, IL-6 and TNF in primary syphilis [73]. High level of TNF in culture supernates and low in sera in early stages with clear symptoms of disease, suggest that this cytokine is secreted locally and may take part in healing of syphilitic lesions. In late stages of the disease, when disturbance of cytokines secretion exists, TNF may take part in pathology of the disease. Dependence between high level of IL-6 and low level of TNF suggests that IL-6 inhibits secretion of TNF. The presence of circulating immune complexes (CIC) is correlated with decreased ability of cells to produce IL-2, IFN, TNF but not IL-6. The data seem confirm inhibitory effect of CIC on cell-mediated immune response which is very important in protection against T. pallidum infection. CIC were also found in nearly all patients with severe forms of acne [74].

Previous studies showed that specific bacteriophages are highly effective in the treatment of bacterial infections. The results obtained recently revealed that phage therapy may be successfully applied in the treatment of the cases of purulent otitis media and meningitis in which available antibiotics were found to be ineffective [75, 76].

Studies on chemotaxonomic markers from actinomycetes were continued. The most specific markers seemed to be mycolic acids and polar amphipathic glycolipids. Some of them showed to be useful for classification and identification of rare clinical strains isolated form human and animal infections. Rhodococcus equi, a member of the family Nocardiaceae and a common soil actinomycete is known as a cause of periodic epizootia in horse stables as well as an opportunistic etiological agent of severe infections of immunocompromised patients. In the cell wall of the microorganism glucose mycolate was found, with fatty acid chain specific exclusively for genus Rhodococcus (C_36-C42). This amphipathic compound, which appeared to be biologically active, can be useful for recognition of Rhodococcus as well as for rhodococcosis itself. Another opportunistic actinomycete was isolated as the only etiological agent of a severe, suppurative pulmonary nocardiosis-like disease [72]. The isolate was identified as Nocardiopsis by taxonomically important and immunologically active two glycolipid markers: dimannosyldiglyceride and acylated trehalose. Both of them were found useful as chemotaxonomic and immunological markers for recognition of the opportunistic actinomycete as well as for nocardiosis-like infection.

It is currently believed that transplacental fetal infection is important mechanism of vertical transmission of viruses. Human placenta, however, is involved in prevention of viral transmission. It was found that most of human placentas, amniotic membranes and endothelium of umbilical cord vein express constitutive immunity that prevents the organs against viral infection. The immunity has nonspecific character; viruses belonging to different taxonomic groups (EMCV, VSV, HSV-1) could not multiply in freshly isolated explants of the organs. The nonspecific immunity is dependent on endogenous cytokines, that are produced by the organs [77, 79, 80]. Tumor necrosis factor type a (TNF-a), interferons type a, b and g (IFN a, b, g) play most important role in the immunity. Contribution of other mediators, however, could not be excluded. The explants of the organs lost the immunity during their culture one or two days in vitro and laboratory strains of viruses effectively multiplied in them.

The detected nonspecific immunity together with active transport of specific antiviral IgG make the placenta strong barrier protecting human fetus against viral infections.

According to results of the study, 10-30% of human placentas ex vivo have no constitutive immunity and are permissive for experimental infections. The results are in agreement with clinical data concerning vertical transmission of HIV, hepatitis B, C or HCMV, when infections during delivery and breastfeeding are excluded.

Gram-positive soil bacteria of the genus Streptomyces are characterized by their complex life cycle with morphological differentiation. Concomitantly with the development of aerial mycelia, Streptomyces produce a wide variety of secondary metabolites, most of which are bioactive compounds including antibiotics, ionophores, immunomodulators and enzyme inhibitors.

Polyketides are a class of secondary metabolites with a huge range of structures, used as medical and veterinary drugs. All polyketides are synthesized by a mechanism similar to fatty acid biosynthesis. Streptomyces coelicolor A3(2), genetically the most studied member of the genus, synthesizes two aromatic polyketides of the so called type II at different stages of its development cycle. Earlier results obtained at the Institute indicated the presence of a new, type I, polyketide synthase genes in this strain. This has been confirmed by further studies on a putative polyketide synthase gene set. Distinctive sequence homology with the respective domains of other type I polyketide synthases as well as the repetitive manner of the organization of characteristic domains strongly support the idea that a new type I polyketide synthase-encoding gene cluster is present on the chromosome of Streptomyces coelicolor A3(2) [82]. The isolation and characterization of novel polyketide--synthase encoding genes could be useful in creating novel hybrid polyketides through genetic engineering.

Central to the processes regulating prokaryotic DNA replication are the events that occur at the chromosomal replication origin, oriC [85]. The initiator protein DnaA plays a key role in the initiation of DNA replication in bacteria [86]. In E. coli, it interacts with four DnaA boxes of the oriC region and forms the initial replication complex. In the light of the recent discoveries that the Streptomyces oriC region is centrally located in the linear chromosome and shows higher complexity than the oriC regions of unicellular bacteria, it is interesting to gain a better understanding of the initiation of Streptomyces DNA replication. In Streptomyces, the oriC region contains 19 DnaA boxes whose location, orientation and spacing are conserved. The Streptomyces DnaA protein consists, like other bacterial DnaA proteins of four domains; it binds to all 19 DnaA boxes. Contrary to other bacteria, the Streptomyces protein contains an additional stretch of 120 predominantly acidic amino acids within the domain II [86]. Currently, it is examined if the acidic domain II of the Streptomyces DnaA protein interacts with putative accessory basic proteins.

Several projects have been carried out in close cooperation with other Polish and foreign research groups. This highly appreciated cooperation is indicated by the list of coworking institutions (pp. 23-29) and by the addresses of coauthors given under the research reports.