Vol. 46, 1998

Vol. 46, No. 1, 1998

CONTENTS

Mini-Review

  • Mechanism of Joint Destruction in Rheumatoid Arthritis
    GAYE CUNNANE, KLAUS M. HUMMEL, ULF MULLER-LADNER, RENATE E. GAY and STEFFFN GAY

    Abstract. The synovium in rheumatoid arthritis (RA) is characterized by an increase in lining layer thickness and infiltration of inflammatory cells into the sublining area. Fibroblasts in the lining layer develop the appearance of “transformed cells”, under the influence of proto-oncogenes involved in the regulation of the cell cycle. Fibroblast and macrophage-derived cytokines such as IL-1 and TNF-a are present abundantly in the rheumatoid synovium and stimulate these cells to produce destructive enzymes. Other cytokines such as IL-4 and IL-10 represent a physiological attempt to reverse the inflammatory process. Adhesion molecules facilitate both the migration of cells to the joint as well as the attachment of synovium to bone and cartilage. Joint destruction is mediated by enzymes such as serine proteases, matrix metalloproteinases (MMPs) and the cathepsins. Treatments directed against various components of the inflammatory cascade have shown promise. Inhibition of MMPs or adhesion molecules, blockade of IL-1 or TNF-a and the use of anti-Fas antibodies to induce apoptosis offer new possibilities for the treatment of RA. More recently, the employment of genes with antlarthritic properties has shown therapeutic potential.

    Keywords: rheumatoid arthritis; synovium; oncogenes; cytokines; adhesion molecules.

Cellular Immunology

  • Surface Phenotypes of Human T Cell Lines Generated after WGA- and PHA-Stimulation
    JAN KONRAD SIWICKI, HENRYK SKURZAK and JAN A. STEFFEN

    Abstract. We have reported earlier that human T lymphoeytes from blood or spleens of normal donors can be propagated in long-term culture in the presence of IL-2, after stimulation either with wheat germ agglutinin (WGA) or phytohemagglutinin (PHA) and with the use of repeated restimulation with supernatants collected after 24 h of such cultures. In the present study we demonstrate that lymphoblast lines generated with this method contain predominantly the cells expressing CD3 molecule and a b heterodimer of T cell receptor (TcRa b ). In PHA-initiated lymphoblast lines CD4– CD8+ cells prevailed, whereas in lymphoblast lines generated after WGA-stimulation various proportion of CD4– CD8+, CD+ CD8– and CD4– CD8– cells were found. In some cell lines a gradual selective outgrowth of TcRa b CD4+ CD8– or CD4– CD8– cells was observed. These data indicate that the long-term growth potential of human normal T lymphoblasts is not restricted to a single subset and that the surface phenotypes acquired during differentiation of the cells can be retained even after more than 100 population doublings in culture.

    Keywords: T cell; long-term growth; phenotype.

  • Effect of Heparin on the Course of Sulphur Dioxide Induced Bronchitis in Rats
    MARYLA KRASNOWSKA, ARTUR KWAŚNIEWSKI, JERZY RABCZYŃSKI, ANDRZEJ FAL and JAN KURYSZKO

    Abstract. The aim of this study was to investigate the effect of heparin on the rat bronchial mucosa changes induced by sulphur dioxide (S02) inhalation. Sixty five rats were used in this experiment. Five of them constituted a control group, while 60 were exposed to SO2. Forty of the latter subgroup were additionally treated with low molecular weight heparin (LMWH), either during or after terminating exposure to SO2. In all animals exposed to S02 inflammatory cells were found in broncho-alveolar lavage fluid (BALf) in numbers significantly higher from those observed in healthy controls. The rats exposed to S02 and treated with LMWH showed intermediate cell pattern in the bronchi between healthy and S02– exposed animals. When comparing histological picture of the bronchi, we noted extensive changes in irritated rats. These changes were either less expressed or totally absent in animals treated with heparin. The activity of enzymes: acid phosphatase (ACP), alkaline posphatase (AP) and lactate dehydrogenase (LDH) rose in BALF, although the rise was not parallel and did not correlate with the magnitude of cellular influx or histological changes. Heparin did not influence this changes.

    Keywords: heparin; sulphur dioxide; bronchitis in rats; bronchoalveolar lavage fluid enzymes.nzymes.

Clinical Immunology

  • Changes in Soluble IL-6 Receptor and IL-6 Production by Polymorphonuclear Cells and Whole Blood Cells of Breast Cancer Patients
    EWA JABŁOŃSKA and ZOFIA PIETRUSKA

    Abstract. The release of soluble interleukin 6 receptor (IL-6sR) and interleukin 6 (IL-6) was investigated in polymorphonuelear and whole blood cell cultures from untreated patients with breast cancer. IL-6sR and IL-6 were determined in the culture supernatants by a sensitive enzymoimmunological assay. We have shown a decreased ability of unstimulated and stimulated polymorphonuclear cells (PMNs) and whole blood cells (WBC) of cancer patients to release IL-6sR in comparison to a control group. Simultaneously, we have also found an increased capacity of unstimulated cells from the patients to produce of IL-6. The used stimulators zymosan and lipopolysaccharide (LPS) resulted in increased IL-6sR secretion by PMNs and WBC of patients but did not influence IL-6 production by the same cells. We have also determined the levels of IL-6sR and IL-6 in the sera of cancer patients. The mean concentrations of IL-6sR and IL-6 in the sera of patients were higher than those in the control group. We have not noticed any correlations in the culture supernatant and serum levels of IL-6sR or IL-6 between control and cancer patient groups.

    Keywords: breast cancer; polymorphonuclear cells; whole blood cells; interleukin 6 (IL-6); soluble IL-6 receptor.IL-6 receptor.

  • Association between Longevity and Allelic Forms of Human Leukocyte Antigens (HLA): Population Study of Aged Italian Human Subjects
    GIORGIO RICCI, CLARA COLOMBO, BARBARA GHIAZZA and MARIA TERESA ILLENI

    Abstract. Several arguments support the idea of a link between longevity and heredity, both in humans and in experimental animals. We have therefore investigated the possibility of an association between the human leukocyte antigens (HLA) and longevity in two groups of Italian subjects: 108 healthy subjects over 85 years old, and 749 healthy blood donors (controls). Only four antigens showed a higher frequency in the elder group: HLA-A31(19), B7, Cw7 and DQ1 These findings suggest an involvement of HLA antigens in human longevity, but the real biological meaning of these results is still unclear.

    Keywords: longevity; human leucocyte antigens (HLA). (HLA).

  • Lymphocyte Interactions with Extracellular Matrix Proteins and Endothelium in Renal Allograft Recipients
    MARIA NOWACZYK, ANDRZEI GÓRSKI, GRAŻYNA KORCZAK-KOWALSKA, PIOTR WIERZBICKI, JANUSZ WYZGAL, MAGDALENA DURLIK and ANNA ORŁOWSKA

    Abstract. Recent data indicate that extracellular matrix (ECM) proteins can provide costimulatory signals during the process of T cell activation. Those proteins accumulate in situ during allograft rejection; therefore, it may be expected that local ECM: T cell interactions may be relevant in the immunopathology of rejection. T cell adhesion from allograft of recipients with stable renal function (RAR-S) and patients with biopsy-proven chronic rejection (RAR-CH) to ECM proteins (collagen type IV, fibronectin, elastin) was measured. Furthermore, T cell: endothelial interactions in vitro were studied. Adhesion of PHA-activated T cells from both groups of allograft recipients to fibronectin, collagen type IV and elastin was significantly lower than in healthy blood donors. Moreover, similar pattern of activity was observed when T cell attachment to resting and activated endothelium was studied. There were no significant differences in the number of circulating CD45RO and CD4 positive T cells. We observed a higher (although not significantly) adhesion of the T cells to resting human dermal microvascular endothelial cells (HMEC) in the chronic stages of rejection, which can suggest that the immunosuppressive protocol used in the treatment of chronic rejection is insufficient to control immunopathologic phenomena occurring in that process. Therefore, it may be argued that too low immunosuppression can be one of the factors responsible for the development of this complication.

    Keywords: interactions; lymphocyte; extracellular matrix; endothellum; allograft.um; allograft.

  • Application of Microsatellite Markers for Monitoring of Reconstitution Pattern in Patients after Bone Marrow Transplantation/Peripheral Blood Progenitor Cell Transplantation
    MALGORZATA JUNGERMAN, MARIOLA SĘDZIMIRSKA, ANDRZEI LANGE, and MICHAŁ WITT’

    Abstract. We present data on application of microsatellite markers for detection of complete vs. mixed chimerism in individuals after bone marrow transplantation (BMT) and peripheral blood progenitor cell transplantation (PBPCT). Genotypes of the peripheral blood or bone marrow cells of three donor/recipient pairs were studied at several microsatellite loci. In two cases we identified complete chimerism in recipients’ peripheral blood and bone marrow after transplantation. These two patients were alive with no symptoms of disease relapse and/or transplant rejection long time after BMT or PBPCT. In the third patient we observed mixed chimerism with the prevalence of donor’s genotype while she was alive and well and the prevalence of recipient’s genotype when she relapsed and required interferonotherapy. Results of our molecular studies correlated with the patients’ clinical outcome. We show that molecular detection of mixed chimerism may be used for prediction of relapse in patients after BMT or PBPCT.

    Keywords: bone marrow transplantation; mixed chimerism; microsatellite markers.llite markers.

Various

  • Reappraisal of Amiloride Action on Transepithelial Electrical Potential Difference of Isolated Tracheal Wall
    TOMASZ TYRAKOWSKI, BOLESŁAW BANACH, ARTUR MO?CIBRODA, MIROSŁAWA BARTŁOMOWICZ and MAŁGORZATA WOJCIECHOWSKA

    Abstract. The action of amiloride on two different components of airway electrical potential difference, one stable (PD) and the other transiently hyperpolarizable after gentle mechanical stimulation (dPD), was studied by means of isolated tracheal wall mounted in a modified Ussing apparatus. The immediate effect of amiloride, when added to the bathing and stimulating medium, was a partial depolarization, and a diminution or elimination of dPD. After at least 60 min incubation of the tracheal wall with amiloride in the presence of the drug in the bathing and stimulating fluid, both the PD, and dPD were no different from the control. This difference between the immediate and the sustained action of amiloride on airway PD suggests that there is an efficient regulatory system in the airway walls which stabilizes the transepithelial PD. The usefulness of amiloride as a drug for cystic fibrosis patients is discussed in the light of these findings.

    Keywords: airway; amiloride; cystic fibrosis; isolated trachea; mechanically sensitive receptor; transepithelial potential difference.al difference.

  • Culture of Human Nasal Epithelial Cells from Nasal Polyps on Collagen Matrix
    MAREK L. KOWALSKI, RAFAŁ PAWLICZAK, JANINA WOŹNIAK, KATARZYNA SIUDA, JANINA GRZEGORCZYK, MARTA PIETRZAK, ZBIGNIEW KOZŁOWSKI and KRYSTYNA MAREK

    Abstract. Epithelial cell cultures became an experimental model employed for both animal and human studies. We established a modified method of culture of human nasal epithelial cells from nasal polyps using serum-free, hormonally supplemented Ham’s F-12 medium and Vitrogen 100 collagen matrix. Cells reached confluent monolayer structures in 5 to 7 days (mean time 6 + 0.89 days) of culture. The confluent cultures consisted of pure epithelial cells, which was confirmed by light and electron microscopy, and immunohistochemical staining with anticytokeratin antibody. The success ratio of cultures was 61.5%. The culture system described is efficient enough to provide pure epithelial cells for further functional studies.

    Keywords: nasal polyps; human nasal epithelial cells; culture, in vitro; collagen matrix.llagen matrix.

  • Average Quantitative Concentration of Cotinine within the System Pregnant Woman-Baby
    DOROTA DOBEK, ANDRZEJ KARMOWSKI, KRZYSZTOF A. SOBIECH, ŁUKASZ TERPIŁOWSKI and MAGDALENA MI?-MICHAŁEK

Abstract. The findings presented by the authors of this report contribute to the diagnosis of pathology of pregnancy i.e. they assess the influence of nicotine on the organisms of a mother and a new-born child, estimated by concentration of cotinine, the most important metabolise of nicotine. The mean proportional share of citinine in the fluids and organs in the pregnant women smoking actively was as follows: urine 72.1 %, amniotic fluids 14.3%, colostrum 8.9% and placenta 4.7%. The authors pay a particular attention to the “colostrum-milk way” in the mother-child relationship what is emphasized in this report.

Keywords: cotinine; pregnancy; biological fluids.ogical fluids.

Vol. 46, No. 2, 1998

CONTENTS

Mini-Review

  • Trials of Protecting Poland from Acquiring Bovine Spongiform Encephalopathy
    JERZY KITA and HENRYK MACIOŁEK

    Abstract. The article contains a short review on transmissible spongiform encephalopathies, Creutzfeld-Jakob disease (CJD) and bovine spongiform encephalopathy (BSE), technology of meat and bone meal production, regulations and monitoring. Poland has imposed a ban on the importation of live cattle, sheep, and food products of ruminant origin from countries where BSE has been identified. Imported meat should be documented with a valid certificate stating that is does not originate from cattle whose parents were imported from Holland, United Kingdom, Ireland and Switzerland. Currently no licences are being issued for importation of live cattle, bovine semen or bovine embryos. The Polish Ministry of Agriculture has appeal to born meal producers to observe the Polish Norm PN-92R-64809.

    Keywords: bovine spongiform encephalopathy; Creutzfeld-Jakob disease (CJD); new variant CJD; meat and bone meal technology and regulations in Poland.Poland.

Immunochemistry

  • Purification of Human Anti-TF (Thomsen-Friedenreich) and Anti-Tn Antibodies by Affinity Chromatography on Glycophorin A Derivatives and Characterization of the Antibodies by Microtiter Plate ELISA
    MARIA DUK and ELWIRA LISOWSKA

    Abstract. The TF and Tn antigens were obtained from glycophorin A (GPA) by desialylation under mild acidic conditions and by desialylation followed by Smith degradation, respectively. A method of purification of anti-TF and anti-Tn antibodies from human sera by affinity chromatography on the immobilized asialoGPA (TF atigen) and on asialo-agalactoGPA (Tn antigen), respectively, is described. Purity of the antibodies was demonstrated by SDS-polyacrylamide gel electrophoresis and their specific reactivity with TF or Tn antigens was shown using hemagglutination and the microtiter plate ELISA. A high unspecific binding of human immunoglobulins to the ELISA plates was encountered, therefore optimal conditions for the most specific binding of the antibodies to the target antigens were selected. Problems of the unspecific binding of immunoglobulins were more difficult to overcome when the antibodies were determined in whole sera by their binding to.antigen-coated ELISA plates.

    Keywords: glycophorin; Thomsen-Friedendreich (TF) antigen; Tn antigen; anti-TF antibodies; anti-Tn antibodies, affinity chromatography; ELISA. ELISA.

  • Serum IgG Antibodies in Children and Adults Reacting with Helicobacter pylori Lipopolysaccharides
    MACIEJ CEDZYŃSKI, KEN-ICHI AMANO, MAGDALENA CHMIELA, MAGDALENA ŁAWNIK, ELŻBIETA CZKWIANIANC, IZABELA PŁANETA-MAŁECKA, WIESŁAWA RUDNICKA and WIESŁAW KACA

    Abstract. The presence of IgG antibodies reacting with Helicobacter pylori lipopolysaccharides (LPSS) in sera from children and adults diagnosed as H. pylori-infected, as well as healthy persons, was tested. There was no correlation between the production of antibodies reacting with H. pylori surface proteins and LPSS. Also no correlation between reactivity of tested sera with H. pylori antigens and deep rough mutant (Re types) enterobacterial LPSs was revealed. The prevalence of anti-LPS IgG in randomly selected children was relatively high.

    Keywords: Helicobacter pylori: lipopolysaccharide; IgG.ccharide; IgG.

  • Influence of Some Factors on Immunoassays of Human Myoglobin
    IWONA E. KOCHANOWSKA, MARIANNA KUROPATWA and APOLINARY SZEWCZUK

    Abstract. Six ELISA variants exploiting two monoclonal antibodies, one rabbit antibody and their peroxidase conjugates were applied in assays of purified human myoglobin, apomyoglobin and the protein in human muscle extracts. The myoglobin was accurately determined with monoclonal antibody no. 82 used for coating of ELISA plates while assays performed with monoclonal antibody no. 49 or rabbit antibody used for coating were weak or none. Determinations of human apomyoglobin with ELISA variants were somewhat more sensitive than those of myoglobin. Obtained in this work results were compared with those done usiner commercial Seratec kit for immunoassav of human myoglobin. Addition to the muscle extracts not only7 concentrated salts but also acetone, ethanol, sodium dodecyl sulfate or some other denaturing agents markedly increased assays of myoglobin by ELISA with monoclonal antibody no. 49 and antibody no. 82 conjugated with peroxidase. Removal of acetone or ammonium sulfate from extracts resulted in dramatic decrease of the estimated myoglobin. Filtration of the extract through Bio-Gel A5m column did not affect low assays of myoglobin in fractions without pretreatment with acetone. Myoglobin was isolated from human heart extract by immunoaffinity chromatography on Sepharose-antibody no. 82 column and the isolated protein was identified by gel electrophoresis and Western blot.

    Keywords: human myoglobin; immunoassay of myoglobin; ELISA; muscle extracts; complexes of myoglobin.globin.

Cytokines

  • Serum Levels of Tumor Necrosis Factor a and Production of this Cytokine by Polymorphonuelear Cells in Breast Cancer Patients
    EWA JABŁOŃSKA

    Abstract. Unstimulated as well as zymosan and LPS-stimulated production of tumor necrosis factor Y. (TNF-oc) by polymorphonuclear cells (PMNS) was investigated in breast cancer patients. Serum levels of TNF-ot were also determined. Spontaneous TNF-cz production was lower, comparable with that found in healthy persons. Zymosan and LPS-stimulated PMNs of cancer patients were found to produce significantly lower amounts of TNF-cz, as compared with PMNs of healthy control. There was no significant differences iii the serum levels of TNF-oc between breast cancer patients and healthy persons. Because the peripheral blood cells contain more than 50% PMNS, the altered TNF-ot production by PMNs may have a series of relevant implications during malignant process and seems to be one of the factors helpful in determining the cellular immunology potency of breast cancer patients.

    Keywords: polymorphonuclear cells; tumor necrosis factor a .

  • Lactoferrin Lowers Serum Interieukin 6 and Tumor Necrosis Factor a Levels in Mice Subjected to Surgery
    MICHAł ZIMECKI, ANDRZEJ WŁASZCZYK, TADEUSZ ZAGULSKI, and ANDRZEJ KÜBLER

    Abstract. Mice subjected to thymectomy or splenectomy in general anesthesia release interleukin 6 (IL-6) and tumor necrosis factor a . (TNF-a ) into circulation reaching high concentrations after 4 h following operation. In the case of thymectomy IL-6 can be detected only on the day of operation and TNF-a attains a maximal value on day 3 postoperation. Splenectomy, which is a more extensive surgical operation, results in a higher, and more prolonged existence of IL-6 in circulation accompanied by higher levels of TNF-a . Bovine lactoferrin (BLF; 10 mg/mouse), given intravenously (i.v.) 24 h before thymectomy, reduced, on avarage, the level of serum IL-6 by 70% as measured 4 h after operation. The inhibiting effect of BLF on TNF-a production was smaller with a mean 30% reduction. The effects of BLF (i.v.) administration on the cytokine levels following splenectomy were less inhibitory. BLF caused an approximate 35% fall in IL-6 levels and even weaker effects (20% inhibition) on TNF-a release. Application of much lower (1 -0.2 mg) per os doses of BLF was even more effective in lowering IL-6 levels after thymectomy (up to 90%) after 5 BLF doses, and by 55% of TNF-a . The data suggest that lactoferrin may find therapeutical application for diminishing manifestations of shock caused by clinical insults.

    Keywords: interleukin 6; tumor necrosis factor a ; thymectomy; splenectomy; bovine lactoferrin.

  • Interferon and Tumor Necrosis Factor Production during Endotoxemia in Sheep
    KAZIMIERZ BIENIEK, AGNIESZKA SZUSTER-CIESIELSKA, TERESA KAMIŃSKA, MARIAN KONDRACKI, IRENA ŁOKAJ and MARTYNA KANDEFER-SZERSZEŃ

    Abstract. In humans endotoxemia has often been associated with the development of adult respiratory distress syndrome (ARDS). Sheep have an abundant population of pulmonary intravascular macrophages, therefore they are a popular animal model for ARDS. In this study we characterized the temporal sequence and duration of the release of two cytokines: tumor necrosis factor (TNF) and interferon (IFN) and evaluated the effect of lipopolysaccharide (LPS) dose. Rectal temperature and white blood cell (WBC) count were also measured. Twenty four adult sheep were given E. coli endotoxin at a dose of 0 (saline solution) 0.05, 0.1 and 1.0 m g/kg of body weight by intravenous (i.v.) bolus. In all groups, TNF-a was produced earlier (3 – 4.5 h) after injection than IFN (4 – 5 h). No correlation between increased rectal temperature, the magnitude of leukopenia and time course of both cytokines production was observed. No straight relationship between LPS dose and the titer of cytokines was seen, but lower doses of LPS-induced delayed cytokine response in comparison to the dose 1 m g/kg of LPS. As 1FN, present in the circulation of sheep, was mainly a /b type, the role of this class of IFN in endotoxemia is discussed.

    Keywords: sheep tumor necrosis factor; interferon; endotoxemia.

Immunopharmacology

  • Interactions of 2′,2′-Difluorodeoxycytidine (Gemcitabine) with Tumr Necrosis Factor a or Its Mutein VI in Murine Leukemias L1210 and P388
    EWA MARAŃDA and TADEUSZ ROBAK

    Abstract. The interactions of 2′,2′-difluorodeoxycytidine (gemcitabine) with tumor necrosis factor a (TNF-a ) or its mutein VI on the survival time of mice bearing L1210 and P388 leukemia was investigated. Four hundred eighty male CD2F1 mice were used in the experiments. They were given gemcitabine (20 mg/kg) on days 1, 4, 7 and 10 after i.p. inoculation with leukemic cells (day 0). Cytokines were administered i.p. at a dose of 250 m g/kg as daily injections for 8 days or at a dose of 400 m g/kg given 2, 4, 6 and 8 days after day 0. Drugs were administered alone and in combination. Our study indicates that TNF-a and its mutein VI decrease the antileukemic effect of gemcitabine on murine leukemias L1210 and P388.

    Keywords: gemcitabine; tumor necrosis factor a ; mutein VI; leukemia L1210; leukemia P388; interactions.

  • Effect of Serine Proteinase Inhibitors on Intracellular Free Calcium Rise in Human Polymorphonuclear Leukocytes
    DARIUSZ NOWAK, MACIEJ KRÓL and PIOTR BIAŁASIEWICZ

Abstract. The respiratory burst of polymorphonuclear leukocytes depends on activation of many enzymes and generation of variety of second messengers. One of important links leading to polymorphonuclear leukoeytes (PMNL) activation is a transient rise in intracellular free calcium concentration ([Ca2+]i). Serine proteinase inhibitors such as a -1-proteinase inhibitor (a 1PI), phenylmethylsulphonylfiuoride (PMSF) and soybean trypsin inhibitor (SBTI) were reported to inhibit human PMNL respiratory burst. In this study we tested the hypothesis whether these inhibitors can inhibit the rise in [Ca2+]i after stimulation of human PMNL with 10-7 M n-formyl-methionyl-leucyl-phenylalanine (FMLP), leukotriene B4 (LTB4) or platelet activating factor (PAF). [Ca2+]i was measured with use of a fluorescent probe Fura-2AM under conditions of 100 nM and 1 mM extracellular Ca2+ concentration. Preincubation of PML with 600 mg/dl of a lPI or SBTI for 30 min at 37o C had no influence on Ca2+ response to all agonists in comparison with cells treated with the same concentration of human serum albumin. However, 0.5 mM PMSF enhanced 1.7-fold (p < 0.002) [Ca2+]i rise after challenge with FMLP while did not affect significantly Ca2+ response to PAF and LTB4. The stimulatory effect of PMSF after addition of FMLP was dependent on increased Ca2+ influx from extracellular space. Our results suggest that suppression of PMNL respiratory burst by serine proteinase inhibitors is not mediated via Ca2+ pathway and that some proteases take part in Ca2+ response to FMLP.

Keywords: intracellular calcium; polymorphonuclear leukocytes; a -1-proteinase inhibitor; phenylmethylsul-phonylfluoride; n-formyl-methionyl-leucyl-phenylalanine; leukotriene B4.

Vol. 46, No. 3, 1998

CONTENTS

Mini-Review

  • Carcinoembryonic Antigen as an Adhesion Molecule
    ANNA KROP-WĄTOREK and ELWIRA LISOWSKA

    Abstract. Carcinoembryonic antigen (CEA), one of the most widely studied tumor markers, shows homotypic (CEA-CEA) intercellular adhesion and heterotypic adhesion to other members of CEA family, receptors on Kupffer cells, macrophages, microorganisms, and lectins. This review is focused on those adhesive properties of CEA which may pertain to the role of CEA in tumor progression.

    Keywords: carcinoembryonic antigen; cell adhesion; tumor progression; immunoglobulin superfamily.family.

  • MHC Molecules and Lymphocytes: Evolutionary Perspective
    BARBARA PŁYTYCZ and ROLF SELJELID

    Abstract. The classical polymorphic MHC molecules of class I or class II bind peptides derived from the processed cytosolic or endosomal antigens and export them to the cell surface for presentation to the T cell receptors (TcR) of CD8 or CD4 T lymphoeytes, respectively. The classical MHC molecules are unstable when peptides are not bound. The MHC-peptide-TcR interactions constitute a molecular basis of thymic selection of the major streams of a b and gd T lymphocytes. The monomorphic MHC class I-like molecules (class Ib) bind peculiar peptides or nonpeptide antigens or can keep proper conformation even without antigenic peptides. They are recognized by the specialized subsets of nonconventional lymphocytes, mainly extrathymic g d T or natural killer (NK) T lymphocytes. The most unorthodox T lymphoeytes can see antigens directly without the participation of MHC or MHC-like molecules or can see MHC-like molecules not loaded with peptides. The conventional B2 lymphocytes are indirectly dependent on MHC-peptide-TcR interactions as they can bind the epitopes of native antigens via Ig surface receptors, to be activated they must present the processed antigens via the MHC class II molecules to the Th2 lymphocytes. In contrast, the B1 lymphocytes can be activated directly without cooperation with T cells via MHC molecules. It seems that both MHC molecules and lymphocyte antigen receptors arose by the expansion of Ig-superfamily genes at the early steps of vertebrate phylogeny. The nonconventional T lymphocytes (gd T cells and NK T lymphoeytes) and Bl cells which support innate immunity at the body surfaces or cavities as well as the MHC-like molecules might appear earlier, creating a proper microenvironment for development of the conventional T and B2 subsets of lymphocytes.

    Keywords: MHC molecules; MHC-like molecules; B2 lymphocytes; B1 lymphocytes; a b T cells; g d T cells; NK T lymphocytes; phylogeny of immunity.

  • Cytokines in Inflammatory Bowel Disease
    ZBIGNIEW KMIEĆ

    Abstract. Inflammatory bowel disease (IBD) denotes chronic inflammatory disorders of gastrointestinal tract of unknown etiology that comprises 2 major groups: ulcerative colitis (UC) and Crohn’s disease (CD). Disregulation of the intestinal immune system both at humoral and cellular level constitutes an important element in the multifactorial pathogenesis of IBD. The expression of pro-inflammatory cytokines, most notably IL-1, IL-6, TNF-( and chemokines (IL-8, ENA-78, MCP-1, RANTES) in intestinal mucosa from IBD patients is markedly enhanced, however, it is not always accompanied by increases in cytokines’ serum levels. In IBD also significant changes occur in the tissue expression of immunoregulatory cytokines: increased levels of IL-2 mRNA and IFN-( mRNA, and decreased expression of IL-4 were found in affected intestinal mucosa. Chronic intestinal lesions of patients with Crohn’s disease are associated with a Th1 type cytokine profile. The clinical effectiveness of anti-TNF-( antibodies and of IL-10 has been demonstrated in steroid-refractory Crohn’s disease patients. The data demonstrating the role of cytokines in the pathogenesis of IBD should be carefully analyzed because of limitations imposed by the patient- and sample-related parameters. Further investigations will clarify the significance of the impairments in cytokine network for the initiation and progression of the IBD.

    Keywords: inflammatory bowel disease; cytokines; interleukins; anti-TNF-( therapy).NF-( therapy).

  • Immunoregulatory Role of Soluble HLA Molecules: a New Skin for an Old Subject?
    FRANCESCO PUPPO, MASSIMO GHIO, PAOLA CONTINI, SABRINA BRENCI, DONATELLA BIGNARDI, GILBERTO FILACI, MARCO SCUDELETTI and FRANCESCO INDIVERI

    Abstract. The available evidence suggests that measurement of the level of total sHLA-1 antigens and of donor-derived and recipient-derived allospecificities as well as the characterization of their variants in recipient’s serum may provide useful information to differentiate graft rejections from infections in allograft recipients. Moreover, a significant progress has been made in our understanding of the functional properties of sHLA-I antigens in serum and of their potential role in the modulation of immune responses. If these preliminary results will be confirmed, then sHLA-I antigens are likely to become important reagents to monitor and treat graft recipients.

    Keywords: HLA soluble molecules; graft recipients; graft rejections.ctions.

Clinical Immunology

  • Systemic Humoral Response to Helicobacter pylori in Children and Adults
    MAGDALENA CHMIELA, MAGDALENA ŁAWNIK, EL(BIETA CZKWIANIANC, TOMASZ RECHCIŃSKI, IZABELA PŁANETA-MAŁECKA and WIESŁAWA RUDNICKA

    Abstract. In this study we compared the development of anti-H. pylori humoral response in adults and children. Two antigens: H. pylori acid glycine extract (GE) and recombinant caga were used for ELISA and Western blot. Anti-GE IgG were detected in all and anti-caga IgG in about 50% of H. pylori infected adults and children. The prevalence of anti-GE and anti-caga IgG in the sera from H.pylori-uninfected children with gastritis/gastroduodenitis was lower than in the sera from healthy adult blood donors. Serum IgA were demonstrated for 71% of H. pylori-infected adults and for a smaller proportion (about 30%) of uninfected adult patients or normal subjects. Such antibodies were detected neither for infected nor for uninfected children. There was an evident difference between the proteins of H. pylori glycine extract recognized by antibodies present in the sera from H. pylori-infected children and adults. The antigen of molecular weight over 107 kDa was recognized exclusively by the sera from 30% of H. pylori-infected adults. The 80- 107 kDa bands were recognized more frequently by the sera from adults than from children. In contrast, sera from infected children more frequently than sera from infected adults reacted with the bands of 14 kDa, 19 kDa and 26 kDa. The H. pylori antigens recognized by IgG, produced by infected children and adult patients, should be taken into consideration in the developing of tests for serodiagnosis of H. pylori infection.

    Keywords: Helicobacter pylori; patient’s age; ELISA; Western blot. Western blot.

  • Lactoferrin Effects on the in Vitro Immune Response in Critically Ill Patients
    BARBARA ADAMIK, MICHAŁ ZIMECKI, ANDRZEJ WŁASZCZYK, PAWEŁ BEREZOWICZ and ANDRZEJ KÜBLER

    Abstract. The aim of the study was to investigate whether lactoferrin can improve the immune competence of cells from patients with systemic inflammatory response syndrome (SIRS). We studied the effect of lactoferrin (LF) on the proliferative response of peripheral blood mononuclear cells (PBMC) to lipopolisaccharide (LPS) in vitro and its influence on production of 2 proinflammatory cytokines: interleukin – 6 (IL-6) and tumor necrosis factor a . (TNF-a ). Three groups of patients: septic survivors, septic nonsurvivors and multiple trauma patients, were investigated. Blood samples were taken upon admission to intensive care unit and after 2, 3 and 5 days. The proliferative response of PBMC in vitro was tested using 3-day culture with LPS. Cell proliferation/death was measured using MTT calorimetric method. The spontaneous and LPS-induced activity of TNF-a and IL-6 were measured with bioassays using indicator cell lines WEHI-164.13 and 7TD1, respectively. We demonstrated that LF inhibited the proliferative response, both spontaneous and LPS-induced, in all groups of patients. Lactoferrin alone was a good inducer of IL-6 and TNF-a production by monoclear cells in vitro. Addition of LF to the cultures of LPS-activated mononuclear cells stimulated IL-6 production, most markedly in the group of septic survivor patients (mean 1479, 1452, 1728, 1980 pg/ml on day 1, 2, 3 and 6 respectively). Lactoferrin also unregulated TNF-a production. That effect was very significant in the septic survivor patients (mean 7407, 6739, 7498 and 8509 pg/ml on day 1, 2, 3 and 5 respectively) and less pronounced in the group of trauma patients. We conclude that lactoferrin exhibited regulatory actions on the altered reactivity of PBMC from patients with sepsis and multiple injury. Lactoferrin is a good inducer of IL-6 and TNF-a production. However, in most cases of septic nonsurvivors LF could not abolish low reactivity of cells with regard to cytokine production.

    Keywords: lactoferrin; sepsis; multiple trauma; interleukin 6; tumor necrosis factor a .

  • Apolipoprotein E Genotypes in Sporadic Early and Late-Onset Alzheimer’s Disease
    ANNA KOWALSKA, JOLANTA FLORCZAK, DANUTA PRUCHNIK-WOLIŃSKA, ADAM KRASZEWSKI and MIECZYSŁAW WENDER

    Abstract. Recent data have demonstrated that presence of apolipoprotein E (APOE) e *4 allele is a major risk factor of Alzheimer’s disease (AD). We determined the APOE genotypes in 64 patients with sporadic probable AD and 43 non-demented aged controls selected from Poznań region and the western part of Poland using the polymerase chain reaction (PCR) followed by the restriction fragment length polymorphism (RFLP) analysis. We confirmed a strong correlation of the APOE e *4 allele with sporadic late-onset AD. In contrast to manv previous reports we did not found an association between the APOE e *4 allele and sporadic early-onset AD.

    Keywords: Alzheimer’s disease; apolipoprotein E genotypes; association analysis.

  • Effect of General and Regional Anesthesia on Some Neutrophil Functions
    JOANNA CIEPICHAŁ and ANDRZEJ KÜBLER

    Abstract. Transient immunosuppression in patients during anesthesia and surgery may cause postoperative infections. In the immunity against pathogens the processes of non-specific defence are of special importance and engage neutrophils as the major cell type. The aim of this study was to monitor activity of neutrophils during and after surgery, applying different methods of anesthesia. The status of neutrophils was determined by evaluation of chemotactic, phagocytic and chemiluminescence (bactericidal) activities. The study included 78 patients, who underwent surgery due to pathological injury-effected changes or due to varicose veins of extremities. Blood samples for determination of the neutrophil activity were taken just before anesthesia, when the symptoms of anesthesia occurred, 15 min after starting of surgery, 24 h and 5-7 day after surgery. In majority of patients we observed neutrophilic leukoeytosis. There was a statistically significant correlation between leukoeytosis and percentage of neutrophils in the subsequent days after surgery, depending on a kind and extension of surgical intervention. In addition, we have found a decrease in chemotactic, phagocytic and bactericidal activity after all kinds of anesthesia. The decrease in the phagoeytic activity lasted longer, usually at least 24 h after surgery, whereas chemotaxis and chemiluminescence returned quickly to control levels. In the spinal anesthesia the chemiluminescence was not altered; this phenomenon could result from blockade of the sympathetic nerve system and activation of neutrophils metabolism. The brachial plexus blockade had the statistically smallest effect on the function of neutrophils. In the long-term anesthesia (longer than 2 h), particularly using halothane, we have observed depressed values of chemotaxis, phagocytosis and chemilumineseence which lasted longer than in other studied group of patients. We conclude that monitoring of the neutrophil functions is a useful form of studying immune reactions of patients to anesthesia and surgery.

    Keywords: general and regional anesthesia; surgery; neutrophils; chemotaxis; phagocytosis; chemiluminescence.iluminescence.

Various

  • Effect of Colostrinin, an Immunomodulatory Proline-Rich Polypeptide from Ovine Colostrum, on Sialidase and b -Galactosidase Activities in Murine Thymocytes
    IZABELA SOKAL, MARIA JANUSZ, HANNA MIECZNIKOWSKA, GOTFRYD KUPRYSZEWSKI and JÓZEF LISOWSKI

Abstract. Colostrinin: a proline-rich polypeptide (PRP) from ovine colostrum and its nonapeptide active fragment (NP) induce maturation and differentiation of murine thymocytes, formation of helper cells from PNAhigh thymocytes and cytotoxic T cells from PNAlow thymocytes. These processes are accompanied by changes in expression of receptors for peanut agglutinin (PNA), PNAhigh thymocytes were transformed into PNAlow cells, and vice versa. It was shown, in various laboratories, that sialyltransferases are involved in the transformation of PNAhigh thymocytes into PNAlow cells. To find out whether the expression of receptors for PNA on murine thymocytes might also be influenced by other enzymes, we decided to study the effect of PRP and NP on sialidase and b -galactosidase activities in these cells. The results obtained showed that the most of sialidase. activity of murine thymocytes is present in the plasma membrane compartments. Both thymocyte subpopulations PNAhigh and PNAlow, showed similar sialidase activity, which was not affected either by PRP or NP. In contrast to sialidases, most of b -galactosidase activity was present in the cytosol. PNAhigh thymocytes showed a higher b -galactosidase activity than PNA lowcells. Incubation of immature, PNAhigh thymocytes with PRP or NP enhanced the b -galactosidase activity in these cells. The presented results suggest that sialidases seem not to be involved in modulation of surface sialic acid content during murine thymocyte maturation. On the other hand, stimulation of activity of b -galactosidase in PNAhigh immature thymocytes by PRP and NP suggests that b -galactosidase in murine thymocytes might be involved in transformation of PNAhigh into PNAlow cells.

Keywords: proline-rich polypeptide; murine thymocytes; peanut agglutinin; sialidase; b -galactosidase.

Vol. 46, No. 4, 1998

CONTENTS

Review

  • Human Natural Killer Cells
    ISABEL BARĂO and JOĂO L. ASCENSĂO

    Abstract. Human natural killer (NK) cells comprise 10 to 15% of peripheral blood lymphocytes, characterized by their morphologic appearance of large granular lymphocytes (LGLs) and phenotype CD3– CD56+ CD16+ or CD16–. Functionally, these cells are defined by their ability to lyse target cells without prior sensitization and without restriction by major histocompatibility (MHC) antigens. These cells play an important role in immune defenses, especially after hematopoietic transplantation. They contribute to the defenses against virus-infected cells, graft rejection, and neoplasias; they also participate in the regulation of hematopoiesis through cytokine production and cell to cell interaction. In this mini-review we attempt to summarize the most relevant findings about these cells in terms of their origin and differentiation, their cell surface characteristics including activation and their cytolytic pathway. We have also provided a brief approach of their potential clinical use. Increased knowledge of NK cell differentiation, ontogeny and regulatory mechanisms may be of use for the planning of immunotherapeutic strategies.

    Keywords: ontogeny; NK cells; cytokines; transplantation; cytotoxicity. cytotoxicity.

Immunopharmacology

  • Immunoregulatory Effects of a Nutritional Preparation Containing Bovine Lactoferrin Taken Orally by Healthy Individual
    MICHAL ZIMECKI, ANDRZEJ WŁASZCZYK, PASCAlE CHENEAU, ANNE-SYLVIE BRUNEL, JÖEL MAZURIER, GENEVIEVE SPIK and ANDRZEJ KÜBLER

    Abstract. . The aim of this study was to monitor several immune parameters in 17 healthy volunteers taking orally commercially available capsules containing bovine lactoferrin (BLFT) for 10 days (40 mg of BLFT daily). We determined leukocyte number and content of main blood cell types, spontaneous and phytohemagglutinin A (PHA)-Induced proliferation of lymphocytes, plasma levels of interleukin 6 (IL-6) and tumor necrosis factor ( (TNF-() as well as spontaneous and lipopolysaccharide (LPS)-Induced production of these cytokines in peripheral blood cell cultures. All measurements were performed before, one day and 14 days following cessation of BLFT treatment. We established some, transient drop in the percentage of neutrophils accompanied by an opposite phenomenon with regard to lymphoeyte levels. More profound changes were registered in the percentage of other cell types, for example a 100% increase in the level of immature cell forms (bands) was noted. At the same time the percentages of eosinophils and monocytes declined significantly. All these changes were, however, more individual and regulatory, the direction of these changes depended on initial picture of blood cells. Although the proliferative response of lymphoeytes showed, on avarage, a transient decrease, differentiated effects of BLFT treatment were observed depending on initial ability of lymphoeytes to proliferate. TNF-( serum levels showed a tendency to decrease during the monitoring time, the changes of IL-6 levels were, however, not significant. As in the case of the proliferative response, the treatment with BLFT was regulatory with respect to serum TNF-( levels. When we analyzed spontaneous and LPS-induced cytokine production in cell cultures we found that mainly the mean spontaneous response was affected (Inhibition). We also observed a typical, regulatory action of BLFT on the level of spontaneously produced IL-6. This kind of regulatory action of BLFT was also found in the case of spontaneously produced TNF-(. In cell cultures. The influence of other ingredients such as selenium or vitamins, contained in the capsules, cannot be excluded, although our latest data showed that orally taken bovine lactoferrin alone can induce identical changes as BLFT. Taken together, we revealed regulatory effects of oral treatment with commercially available capsules, containing BLFT. The results indicate that oral administration on BLFT-coiitaining capsules mav regulate some immune parameters in healthy individuals. In addition, the data suggest that bovine lactoferrin may be applied in clinic to improve the immune status of patients.

    Keywords: : bovine lactoferrin; immunoregulation; blood cells; proliferation; interleukin 6; tumor necrosis factor .rosis factor .

  • Does Moxonidine Increase Cytotoxic Activity of Ethanol in Rats? Preliminary Report on the Base of the Ultrastructural Study
    KRZYSZTOF MARCZEWSKI, JANINA HALLIOP, PIOTR DZIEMIDOK, ELŻBIETA KORCZAK, DANIELA OBUCHOWSKA-PRZEBIROWSKA, ALINA TOCHMAN and AGNIESZKA SIEMIONOW

    Abstract. Combined effects of moxonidine and ethanol on ultrastructure of selected Wistar-Kyoto rat organs (kidney, liver, heart) were investigated. In cells of the animals, which received moxonidine alone, no morphological changes were found. In the group of rats which received alcohol, several typical submicroscopical changes were found, especially related to the mitochondria. After the combined administration of ethanol and moxonidine the pathological changes were bigger than those found in animals which received alcohol alone. Those changes were particularly significant in kidney and liver. The results might indicate that moxonidine increased cytotoxic activity of ethanol on several organs in rats.

    Keywords: kidney; liver; heart; electron microscopy; moxonidine; ethanol; Wistar rats.r rats.

Cellular Immunology

  • Epithelial-Mesenchymal Cell Interactions and Participation of the Neuroendocrine Markers in Tumor Development. Immunohistochemical Study
    ALBINA ŻÓŁTOWSKA, JAN STĘPIŃSKI, JARZY KUCZKOWSKI, MACIEJ KAMIŃSKI, BOGUSŁAWA CYNOWSKA and WIESŁAW KRUSZEWSKI

    Abstract. Existence of the organ stem cell population seems to decide on ability of the tissue to regenerate and, likewise, on carcinogenesis. The source of the organ specific stem cells may be perivascular mesenchyma of thin-walled vascular channels. Our previous study on the breast cancer indicates that the perivascular mesenchyma of thin-walled vessels appears to be source of myoid cells (myofibroblasts) from which cancer cells arise. Similar results have been observed in the cancers of lung, salivary gland and colon, investigated in the current study. The perivascular cells of thin-walled channels are the source of myoid cells with expression of synaptophysin (Syn) and/or chromogranin A (Chg A), and from these cells neoplastic cells could originate. Syn and/or Chg A positive neoplastic cells were particularly well visible in connection with the vascular channels on the peripheries of tumors while other parts of tumors were only weak positive or negative for those neuroendocrine markers. Similarly as in breast cancers, the S100-protein positive dendritic cells with various of distribution were seen, expressing intimate connection with neoplastic cells. The epithelial pearls especially abundant in non-small cell lung carcinomas demonstrated immunohistochemical analogy to Hassall’s bodies: they had monocytogenic cell inside and they displayed thymosin alpha 1 (TA1), as well as mucin secretion and minute calcification. Some epithelial cells expressed desmin and Syn. All types of investigated cancers demonstrated TA1. Results of our present study suggest that the perivascular cells have a differentiation defect. Such defect may initiate abnormal stromal environment, commonly observed in neogenesis, however, the presence of thymic growth factors may favor tumor growth.

    Keywords: : stromal environment; synaptophysin; chromogranin A; S100-protein; epithelial pearls.helial pearls.

  • Myoid Cells and Neuroendocrine Markers in Myasthenic Thymuses
    ALBINA ŻÓŁTOWSKA, TADEUSZ PAWEŁCZYK, MACIEJ STOPA, JAN SKOKOWSKI, JAN STĘPIŃSKI, ANDRZEJ ROSZKIEWICZ and WALENTY NYKA

    Abstract. We have studied myoid cells in normal and myasthenic thymuses as well as in thymomas. For the presence of neuroendocrine maikers-producing cells and identification of synaptophysin (Syn) the immunohistochemical method and immunoblot analysis were used. Myoid cells can be demonstrated in the thymus of myasthenic patients in high number. These cells occur in the vicinity of Hassall’s bodies but also within them. Some regenerated Hassall’s bodies displayed majority of myold cells with their concentric arrangment around the centrally situated lacunar-like cell with nuclei of monocytogenic origin. Such phenomenon may suggest cooperation of myoid cells and their epithelial transitional forms with monocytogenic cells in various thymic hormone production. It is likely that myoid cells are the source of some thymic epithelial cells. According to some authors, thymomatous epithelial cells and skeletal muscle share a common epitope defined by a monoclonal antibody (mAb), whereas thymic epithelial cells possess acetylocholine receptor (AChR) on their surface. The epithelial cells of some thymomas express also desmin. In normal thymuses of children, Syn and chromogranin A (Chg A) were demonstrated in some cells of Hassall’s bodies by immunohistochemical method. In addition, antibodies to Syn stained nerve structures surrounding the thymic blood vessels. In myasthenic thymuses, Syn expression was in cortical and medullary epithelial cells, in myoid cells and only scanty and focal in keratinized epithelial cells of Hassall’s bodies. The epithelial cells of some thymomas also express Syn. In some thymuses of all groups investigated in this study Chg A was seen in single cells of Hassall’s bodies and focally in cortical epithelial cells. Our results show that in normal thymuses of cardiac surgery patients and in the adult myasthenic thymuses antibody raised against Syn recognized protein with molecular weight of 48 000 but not normal (38 000) Syn. It remains to be elucidated if the overexpression of synaptophysin-like protein in myasthenic thymuses is a compensatory phenomenon to the defect in normal synaptic function.

    Keywords: myasthenia gravis; thymus; myoid cells; synaptophysin; immunohistochemical and immunoblot analysis.blot analysis.

Experimental Oncology

  • Insertion of the IL-2 Gene Decreases Tumorigenicity of Murine Fibrosarcoma
    HALINA KU?NIERCZYK, PIOTR ZAŁĘCKI, ELŻBIETA PAJTASZ-PIASECKA and CZESŁAW RADZIKOWSKI

Abstract. Murine interleukin 2 (mIL-2) cDNA was introduced through lipofection into cells of murine F-69-3 fibrosarcoma line established in vitro from tumors induced chemically in athymic mice. Using a modified MTT bloassay in CTLL-2 indicator line the F-69-3/IL-2 transfectants were found to secrete between 650 – 1750 laboratory units (LU) of IL-2/5 x 105 cells/48 h in restricted culture conditions. When inoculated subcutaneously to immunocompetent BALB/c or CD2F1 mice, the transfected cells showed reduced tumorigenic potential as compared with parental F-69-3/wt or control F-69-3/neo cells. The rejection of F-69-3/IL-2 tumors required an intact immune system as they grew progressively in athymic mice. The majority of immunocompetent mice that rejected IL-2 secretors were found to be protected against subsequent challenge with parental cells. These preliminary results suggest that IL-2-transfected murine fibrosarcoma could be used as a model for studying mechanisms underlying the antitumor immune response.

Keywords: F-69-3 fibrosarcoma; parental tumor cells; interleukin 2-secreting tumor cells; rejection.ls; rejection.

Vol. 46, No. 5, 1998

CONTENTS

Mini-Review

  • T Cell Immunobiology of Alloimmune Thrombocytopenias
    JACK GORSKI, MARYAM YASSAI, SHENHONG WU and KRYSTYNA MAŚLANKA

    Abstract. Allorecognition and generation of alloantisera depend on T cell help. Here we summarize our recent work on identyfying the T cells that recognize a specific platelet alloantigen generated by a Pro to Leu polymorphism in b -chain of the platelet integrin a Iibb 3. The ability to generate alloantibodies is restricted by HLA-DRB3*0101. By measuring peptide binding to HLA-DRB3*0101, we have shown that the polymorphism controls the generation of the T cell epitope. This is not a result of the polymorphism changing a T cell contact residue, but rather by its generating a peptide anchor. The result is a directional antibody response in which only the b -chain that is antigenic is the one that produces a peptide that binds to the HLA-DR. This mechanism of generating alloantibodies may be a paradigm for a whole class of responses.

    Keywords: MHC restriction; peptide binding; alloantibodies.

  • Onset and Persistence of the Allergic Inflammation: a Molecular Explanation ?
    FABRIZIO SPINOZZI

    Abstract. Allergic respiratory inflammation in target organs does not occur in any atopic (getically susceptible) subject, since other not fully chracterized factors can influence the subsequent development of overt clinical disease. Here are presented some recent developments in experimental animal and human research that can offer a new “non-classical” interpretation about the way by which allergens are recognized and allergic inflammation persists. These aspects of the immunopathogenesisof allergic diseases can now be viewed as organ-specific pathways, acting independently from other peropheral lymphoid organs. This is a consequence of new knowledge about the function of, and molecular interactions by, intraepithelial gd T cells and CD1+ dendritic cells. The allergic subject, unlike the normal one, is equipped at the mucosal surface by particular T cell and antigen-presenting cell (APC) subsets that enable them to recognize undernatured proteic and non-proteic (glycolipidic) external structures of aerodispersed particles, presented in the context ofCD1 molecules. Once initiated, the mucosal allergic reaction cannot be turned off in atopic individuals because CD4+ allergen-specific T cells lack surface Fas receptor. This defect, yhat impairs the socalled activation-induced programmed cell death (detrmined by Fas/FasL inetraction), is caused by the local Th2-type cytokine milieu.

    Keywords: T cells; allergic inflammation; programmed cell death.

  • MHC Class II and Negative Regulators of Hematopoiesis
    H. JOACHIM DEEG and VLADIMIR LESNIKOV

    Abstract. HLA-DR antigens a side from their antigen-presenting function have recently been shown to allow for transmembrane signaling. Findings in several model suggest that such a signaling function may occur in hematpoietic tissues. Signals generated via HLA-DR result in upregulation of tumor necrosis factor a (TNF-a ) and nitric oxide as well as an enhanced expression of Fas and its natural ligand (Fas-L), all molecules associated with programmed cell death (apoptosis). While a role of HLA-DR in hematopietic failure remains hypothetical, the data presented into a model of hematopoietic failure initiated by HLA-DR mediated signals.

    Keywords: HLA-DR; hematopoiesis; tumor necrosis factor a ; Fas; nitric oxide.

Experimental Oncology

  • Therapeutic potential of Biological Response Modifiers Against Transplantable Mouse Tumors of Spontaneous Origin. II. Local Interleukin 2 Treatment of Tumors of Different Immunogenic Strength
    ŁUCJA FISZER-MALISZEWSKA, WILLEM DEN OTTER, JANUSZ A. MADEJ and MARIAN MORDARSKI

    Abstract. This report describes tumor susceptibility to inetrleukin 2 (IL-2) therapy in ralation to tumor immunogenicity. The following lines recently established from spontaneous tumors were used: X5,X6, and X9 mammary tumors of DBA/2, BALB/c and CBA origin, respectively, X7 cacinoma of BALB/c and X18 papilloma of CBA mice. Two spontaneous tumors of long transplantation history, SL2 lymphoma (SL2) of DBA/2 and M109 Madison lung carcinoma (M109) of BALB/c origin, were used as control system. Mice were transplanted with different inocula of tumor cells at day O; IL-2 treatment was initiated on day 1 – 3 or 10 and consisted of daily injections of 5000 or 20 000 IU/mouse given 5 times a week for 3 weeks. SL2 (i.p. – 2 ´ 104 cells) treatment cosisted of i.p. injestions of 5000 or 20 000 IU IL-2 given on days 10 – 14. IL-2 therapy of SL2 bearing DBA/2JIco mice resulted in the significant proportion of cures; however, no response to IL-2 treatment was achieved in SL2 bearing DBA/2Crliw mice. BALB/c mice with the i.p. transplant of M109 responded to IL-2 treatment with 40% inrease in lifespan.. The low-dose IL-2 therapy of the 5 recently established tumors resulted, in general, in transient growth inhibition of the i.m. transplants of line X5, X6, and X7 provided IL-2 was administerde locally. The therapeutic effect depended on the numberof transplanted tumor cells, and the best results begin achieved at cell numbers close to the dose inducing tumor growth in 50% of animals (TD50). We found that the tumors responding to IL-2 treatment were all slowly growing and immunogenic (X6 and X7) or might have viral association (X5) and as such might express foregin antigens.

    Keywords: biological response modifiers; interleukin 2; spontaneous mouse tumors; tumor immunogenecity.

  • 5-Aminolevulinic Acid Photodynamic Therapy of Transplantable Colon Adenocarcinoma in BALB/c Mice
    PIOTR ZIÓŁKOWSKI, KRZYSZTOF SYMONOWICZ, BEATA J. OSIECKA and JERZY RABCZYŃSKI

    Abstract. We present results of preliminary studies on 5-aminolevulinic acid (5-ALA)-photodynamic therapy (PDT). In order to assess the effectiveness of 5-ALA-PDT we have used BALB/c mice transplnted subcutaneously with mouse colon adenocarcinoma C51. 5-ALA in the dose of 50 mg/kg was given intraperitoneally and 5 h later tumors were exposed to light at total doses from 50 to 75 J/cm2 , wavelength 630 ± 20 nm and light intensity 200 mW/cm2. Several time points following 5-ALA injections have been used to measure protoporphyrin IX (PpIX) concentration in different tissues from unirradiated mice. PDT effects were evaluated with regard to survival time, tumor response and necrosis depth. The main finding in our tumor model was that the optimum tumor to skin ratio of PpIX occurs within 5 h after sensitizer injection. 5-ALA-PDT resulted in prolongation of survival time of treated mice as compared to control animals and, in some cases, in complete response of tumors. PDT also caused increase in tumor necrosis, while no skin photodamage was observed.

    Keywords: photodynamic therapy; 5-aminolevulinic acid; colon adenocarcinoma; protoporphyrin IX concentration.

Clinical Immunology

  • Interleukin 6 and Its Soluble Receptor in Periodontitis
    MAŁGORZATA PIETRUSKA, JAN K. PIETRUSKI, WANDA STOKOWSKA and EWA JABŁOŃSKA

    Abstract. The paper presents the evaluation of interleukin 6 (IL-6) and interleukin 6 soluble receptor (sIL-6R) values in the blood serum of patients with various forms of periodontitis (P), as confronted with local state. The study involved a group of 15 patients with P, including 4 patients with rapidly progressive periodontitis (RPP), 10 with adult periodontitis (AP) and 1 with juvenile periodontitis (JP). The concentration of IL-6 and its soluble receptor in blood serum were assessed using the immunoenzymatic ELISA method. The clinical state was estimated with the following periodontal indices: plaque index (PI), oral hygiene index (OHI), sulcus bleeding index (SBI), probing pocket depth and probing attachment loss. The P group showed an insignificant decrease in the IL-6 level in serum and a significant increase in sIL-6R concentration, compared with controls. A correlation was found between the level of IL-6 and PI, OHI and SBI.

    Keywords: periodontitis; interleukin 6; soluble interleukin 6 receptor.in 6 receptor.

  • Concentrations of Leptin and Neuropeptide Y in Maternal Plasma, Umbilical Cord Blood and in Amniotic Fluid in Pregnant Women with EPH-Gestosis
    FRANCISZEK KOKOT, IZABELA ULMAN, ANDRZEJ WIĘCEK, TOMASZ IRZYNIEC and JÓZEF ULMAN

    Abstract. Leptin is presumed to be related to body mass index (BMI) and body fat stores and is involved together with neuropeptid Y (NPY) in the regulation of appetite. As pregnancy is accompanied both by changes of BMI and appetite, performance of studies presented in this paper were fully justified. In 43 healthy pregnant women and in 18 pregnant women with mild or moderate EPH-gestosis, concentrations of leptin and NPY were estimated in maternal venous blood, umbilical cord blood and in amniotic fluid. The control group consisted of 26 healthy nonpregnant women. Healthy nonpregnant women showed a BMI of 23.08 + 0.65 kg/m2 which was significantly lower than in healthy pregnant women (26.9 + 0.4 kg/m2, p < 0.001) and in women with EPH-gestosis (29.7+0.9, p < 0.0001). Also in healthy pregnant women the BMI was significantly lower than in EPH-gestosis subjects (p < 0.001). In healthy nonpregnant women plasma leptin levels were significantly lower than in healthy pregnant and EPH-gestosis women (10.9 + 1.68 vs 14.99 + 1.28 vs 21.89 + 2.58 ng/ml, respectively). In umbilical cord blood plasma leptin levels were significantly lower than in maternal blood only in healthy pregnant women (7.37 + 0.69 vs 14.99 + 1.28 ng/ml) but not in EPH-gestosis subjects (18.06 + 3.38 vs 21.89 + 2.58 ng/rnl). Leptin levels in amniotic fluid were significantly lower than in umbilical cord blood both in healthy pregnant women (2.25 + 0.20 vs 7.37 + 0.69 ng/ml) and EPH-gestosis women (6.58 + 2.62 vs 18.06 + 3.38 ng/ml). In EPH-gestosis women leptin levels were significantly higher than in healthy pregnant women in maternal blood (21.89 + 2.58 vs 14.99 + 1.28 ng/ml), umbilical cord blood (18.06 + 3.38 vs 7.37 + 0.69 ng/ml) and amniotic fluid (6.58 + 2.62 vs 2.25 + 0.2 ng/ml). In both examined pregnant groups plasma NPY levels were nonsignificantly lower in healthy pregnant and EPH-gestosis women (42.28 + 4.09 and 43.68 + 8.45 pg/rnl, respectively) than in nonpregnant women (50.65 + 6.13 pg/ml). In nortnal pregnancy a significantly higher NPY level was found in umbilical cord blood as compared with respective values in EPH-gestosis women (116.28 + 17.0 vs 49.65 + 7.01 pg/ml). Finally in both examined groups of pregnant women the amniotic fluid NPY level was of similar magnitude (13.85 + 1.52 and 13.89 + 2.46 pg/ml in healthy pregnant and EPH-gestosis women respectively). No significant correlation was found between fetal birth weight and cord-serum leptin and NPY levels respectively.

    Keywords: : leptin; neuropeptide Y; normal pregnancy; EPH-gestosis; umbilical cord blood; amniotic fluid.mniotic fluid.

  • CD3+ T Lymphoeytes and Interieukin 2 in Myelomatous Blood Hyperviscosity Syndrome Treated with Plasmapheresis
    LIDIA USNARSKA-ZUBKIEWICZ

Abstract. The study was aimed at testing changes of absolute CD3+ T lymphocyte counts and serum interleukin 2 (IL-2) concentrations in 20 patients suffering from myelomatous and macroglobulinemic hyperviscosity syndrome. Mean values have been calculated from assays made before treatment, immediately after plasmapheresis (PP) and after 1 month of subsequent chemotherapy. Patients showing hyperviscosity displayed initial IL-2 values ranging from 529 to 2722 pg/ml. These levels were significantly (p = 0.001) higher than in healthy subjects, in whom IL-2 was undetectable. Absolute CD3+ counts in patients with hyperviscosity ranged from 600 to 1290 G/1 and were significantly (p = 0.05) lower than in healthy controls with a range of 1110 – 1520 G/1. In 6 patients in coma or precoma serum IL-2 concentrations were insignificantly and absolute CD3+ counts significantly (p = 0.02) lower than in hyperviscosity patients with less severe symptoms. After PP the previously elevated serum IL-2 levels diminished by 20%, but absolute CD3+ counts remained unchanged. After 1 month of chemotherapy IL-2 levels increased again in 15 multiple myeloma (MM) patients, but in all patients with Waldenström’s macroglobulinemia (WM) a further decrease of serum IL-2 level was observed. By that time CD3+ counts increased significantly (p = 0.01) being higher than initial values. Effects of combined myeloma treatment and prognostic value of serum IL-2 level and absolute number of CD3+ etermination are discussed.

Keywords: : CD3+ lymphocytes; interleukin 2; hyperviscosity syndrome; plasmapheresis; multiple myeloma; Waldenström’s macroglobulinemia.inemia.

Vol. 46, No. 6, 1998

CONTENTS

Review

  • Minor Transplantation Antigens: Mouse Models for Human Host-Versus-Graft, Graft-Versus-Host and Graft-Versus- Leukemia Reactions
    ELIZABETH SIMPSON

    Abstract. Minor transplantation or histocompatibility (H) antigens are barriers to the transplntation of organs and tissues between genetically non-identical individuals who are matched for transplntation antigens encoded by the major histocompatibility complex (MHC), H2 in mice and HLA in humans. Recognition of minor H antigens by T cells can lead to host-versus-graft (HvG) rejection of organs and, in bone marrow transplantation, to life theatening graft-versus-host (GvH) reactions, although if these can be controlled, clinically useful graft-versus-leukemia (GvL) reactions can be obtained. This review describes the early in vivo studies that laid the foundations of our understanding that minor H antigens are the product of genetic polymorphisms, the subsequent in vitro work which has identified these at the molecular level as peptides presented to T cells by self MHC molecules, and the most recent developments, which have seen the use of minor H specific T cells clones and Sophisticated biochemical and molecular biological approaches to identify genes encoding them.

    Keywords: transplantation; minor histocompatibility antigens; HY; mitochondrial antigens; T cells; expression cloning; graft-versus-host; host-versus-graft; graft-versus-leukemia; peptide epitopes; major histocompatibility complex.omplex.

  • Novel Protein Toxin-Based Strategy for Development of Cytotoxic T Lymphocyte Vaccines
    ANTONI WIĘDŁOCHA

    Abstract. Specific activation of CD8+ cytotoxic T lymphocytes (CTL) iscrucial to elicit immunity against intracellular pathogens including viruses, bacteria and protozoa. CTLs recognize mainly intracellularly processed peptides of pathogen origin associated with histocompatibility complex class I molecules (MHC class I) at the cell surface infected cells. It implicates the way how to induce specific CTL response and develop an efficient vaccine against intracellular pathogens.Therefore, the general strategy is to mimic the infection by introducing thetarget antigen into the cytosol of host cells in vivo. Several approaches have been proposed so far, including self replicating vectors, adjuvants and liposomes. However, none of them are ready for practical use. Recently, it has been shown that a number of protein toxins can be used to carry passenger proteins across cellular membranes into the cytosol. This suggested new possibilities for how to deliver a protein antigen into the cytosol for intracellular processing and presentation by MHC class I and to develop CTL vaccines. Here the use of protein toxins as translocation vehicles for delivery of antigen peptides into the cytosol is discussed. Experimental data already obtained demonstrating in vivo elicited immunity by the toxin systems are reviewed and considered.

    Keywords: CD8+ T cells; protein toxins; MHC class I; antigen; vaccine.igen; vaccine.

  • Interferon a in the Treatment of Chronic Myelogenous Leukemia
    TADEUSZ ROBAK

    Abstract. Fifteen years ago Talpaz and colleagues were the first to determine that natural interferon a (INF-a ) induces hematologic remission in chronic phase patients with chronic myeloid leukemia (CML). Further research revealed that this agent, contrarily to conventional chemotherapy with busulfan or hydroxyurea, eliminates leukemic hematopoietic cells having Philadelphia chromosome (Ph1-positive) in about 20% of patients, leading to the phase of cytogenetic remission. Comparison of the efficiency of IFN-a with conventional chemotherapy was carried out in several randomized clinical trials. It was found, that IFN-a delays the occurrence of blastic phase of CML and prolongs patients life span. It does not create, however, a likely chanse of full recovery. The results of the randomized trails, carried out in France, showed that the combination of IFN-a and cytarabine as compared with INF-a alone, increases the rate of major cytogenetic response and prolongs survival in the chronic phase of CML has not been proven so far, although this drug may be of certain value in combination with hydroxyuera or other cytostatic agents. At present, it is more often considered that IFN-a should be a first line therapy in newly diagnosed CML in it’s chronic phase, if due to absence of appropriate donors or advanced age, allogenic bone marrow transplantation cannot be performed.

    Keywords: chronic myelogenous leukemia; interferon a treatment; mechanism of action; randomized trials.

  • Alterations in Signal Transducing Molecule CD3x in Patients with Neoplastic Diseases
    IRENA FRYDECKA, PAWEŁ KACZMAREK, DOROTA BOĆKO, AGATA KOSMACZEWSKA and LIDIA CISZAK

    Abstract. The impairmentof immune cell functions in cancer patients may result impairment of signal transduction. Recent studies have demonstrated altered expression and function of signal transduction molecule CD3x in T and natural killer cells in patients with neoplastic diseases. Tumor infiltrating lymphocytes are more affected than peripheral blood lymphocytes. CD3x expression is more prominent in advanced stage of the disease and correlates with reduced proliferative response, IFN-g , IL-2, TNF-a production.

    Keywords: CD3x ; neoplastic diseases.

Clinical Immunology

  • Oral Treatment of Rats with Bovine Lactoferrin Inhibits Carrageenan-Induced inflammation; Correlation with Decreased Cytokine Production
    MICHAŁ ZIMECKI, RYSZARD MIĘDZYBRODZKI and STANISŁAW SZYMANIEC

    Abstract. The aim of this study was to investigate an effect of oral treatment of rats with bovine lactoferrin (BLF) on carrageenan-induced inflammation. Rats were given 5 oral does of BLF (10 mg each) on alternate days and 24 h after the last dose a carrageenan inflammation was induced in the hind foot. Control rats were given 0.9% natrium chloride (NaCl) or bovine serum albumin (BSA). The magnitude of the reaction was measured after 2 h (optimal response) and expressed as an increase of the foot pad thickess in milimeters. The evaluation of BLF effects on carrageenan reaction was sopplemented by determination of the ability of spleen cell cultures to produce interleukin 6 (IL-6) and tumor necrosis factor a (TNF-a ) upon lipopolysaccharide (LPS) induction using bioassays. The results revealed an inhibition of the carrageenan-induced inflammation in BLF-treated rats by 50 and 40 % as compared to NaCl and BSA control groups, respectively. The inhibition was also decreased, although to a lessre degree (48 and 35%, respectively). The decreased ability of spleen cells to produce inflammatory cytokines in BLF-treated rats indicates that hyporeactivity of the immune system cells may be the basis for the inhibition of carrageenan-induced inflammation.

    Keywords: bovine lactoferrin; carrageenan inflammation; rats; interleukin 6; tumor necrosis factor a .

  • Exogenous Interleukin 2 Regulates Interleukin 6 and Nitric Oxide but Not Interferon g and Tumor Necrosis Factor a Production in Bronchoalveolar Leukocytes from Patients with Small Cell Lung Cancer
    MONIKA CENBRZYŃSKA-NOWAK, MAŁGORZATA BIEŃKOWSKA and EDWARD SZKLARZ

    Abstract. The relationship and in situ interactions between interleukin 2 (IL-2)-regulated mediators remain unclear, particularly in lung cancer model. The purpose of the present study was to determine in vitro effect of IL-2 on the secretory activity of bronchoalveolar leukocytes from 11 patients with previously unterated small cell lung cancer (SCLC) and 9 patients wuth non-small cell lung cancer (NSCLC). Control group (n = 6) comprised patients who underwent diagnostic investigations and were free of any clinical or radiographic evidence of lung diseases. IL-2 induced secretion of mediators was compared with that following stimulation with lipopolisaccharide (LPS; 5 m g/ml) or Newcastle disease virus (NDV; 640 HU/ml). Obtained from bronchoalveolar lavages (BAL) cells were cultured for 24 – 48 h in the presence or absence of inducers. The levels of cytokines were determined in BAL cell supernatants by bioassays. Nitric oxide (NO) was estimated by colorimetric method in Griess reaction. Compared with normal controls, the spontaneous secretion of the above mediators excluding IFN-g in BAL cultures from NSCLC group was elevated by up to 20 -30-fold and further increase was observed after stimulation with LPS. However, very low secretion of cytokines and NO was found in BAL leukocyte cultures activated by IL-2. In contrast, the cells obtained from SCLC group produced little detectable levels of TNF-a (median 12.0, range 3 – 45 U/ml), IFN-g (median 3, range 3 – 12 U/ml) and IL-6 (median 15, range 6 – 45 U/ml) in response to LPS and interferons, mainly IFN-a ; (median 3, range 3 – 12 U/ml) in response to NDV. Although, upon IL-2-stimulation was observed only noteworthy production of IL-6 (median 405, range 45 – 1215 U/ml). IL-2-induced secretion of IL=6 was accompanied by up to 5-fold augmented secretion of NO in comparison with NSCLC group and healthy controls. These observations suggest that BAL cells from patients with lung cancers express a selective secretory activity and that IL-2 is an important regulatory factor of secondary production of IL-6 and NO. Utilization of IL-2 in therapeutic strategy in SCLC can lead to alterations in synthesis/release of biologically active IL-6 and NO that may contribute to the clinical settings.

    Keywords: interleukin 2; interleukin 6; interferon; tumor necrosis factor; nitric oxide; bronchoalveolar leukocytes; small cell lung cancer; non-small cell lung cancer.

  • Association of Interferon g, Tumor Necrosis Factor a and Interleukin 6 Serum Levels wih Systemic Lupus Erythematosus Activity
    EWA ROBAK, ANNA SYSA-JĘDRZEJEWSKA, BOŻENA DZIANKOWSKA, DOROTA TORZECKA, KRZYSZTOF CHOJNOWSKI and TADEUSZ ROBAK

    Abstract. We investigated serum level of interferon g (IFN-g ), tumor necrosis factor a (TNF-a ) and interleukin 6 (IL=6) using an enzyme-linked immunosorbent assay (ELISA) in 59 patient with systemic lupus erythematosus (SLE) and 16 healthy controls. We examined a possible association between serum levels of these cytokines and SLE activity, as well as correlation between IFN-g concetration and the level of TNF-a and IL-6 and also IL-6 and TNF-a . TNF-a and IL-6 were detectable in all 59 patients and normal individuals and their level was significantly higher in SLE patients than in the control group (p < 0.001 and p < 0.02, respectively). In contrast IFN-g was detectable in 23 (39%) patients and in only 3 (20%) healthy individuals. We found positive correlation between serum concentration of TNF-a and IL-6 with SLE activity and no such correlation with IFN-g . We also observed positive correlation betweenserum levels of IFN-g and TNF-a , IFN-g and IL-6 as well as TNF-a and IL-6. In conclusion, an increase in the serum levels of TNF-a and IL-6 may be useful markers for SLE activity.

    Keywords: interferon g ; tumor necrosis factor a ; interleukin 6; systemic lupus erythematosus; disease activity.

  • TNF-a , IL-6 and IFN-g Secreted by Bronchoalveolar Leukocytes Isolated from Patients with Bronchial Asthma, Complicated by Fungal Airways Infections
    MONIKA CEMBRZYŃSKA-NOWAK, JERZY LIEBHART, BERNARD BANASZEK, RAFAŁ DOBEK, MAŁGORZATA BIEŃKOWSKA and EDWARD SZKLARZ

Abstract. It is widely known that fungal airways infections may deteriorate the course of bronchial asthma. The mechanism of the phenomenom it still unclar. The aim of our studt was to assess the effect of fungal infections on the secretion of selected cytokines by bronchoalveolar leukocytes. Five patients (group FA) with bronchial asthma and Candida albicans or Aspergillus fumigatus airways infections (confirmed by bronchoscopy and culture) were included in the study. All of them were on the chronic treatment with corticosteroids (10 – 20 mg of prednison per day) and underwent everal courses of therapy with antibiotics. The control groups comprised 5 previusly untreated asthmatics without bronchial colonization with fungi (group A) as well as 5 healthy volunteers (group H). Leukocytes were isolated from bronchoalveolar lavage fluid (BALF) and cultured in the presence or absence of cytokine inducers such as phytohemagglutinin L (PHA), lipopolysaccharide (LPS) from E. coli. The activity of TNF-a , IL-6 and IFN-g were measured in the BAL cell culture suprenatans by using specific bioassays. In comparison with healthy controls the spontaneous or induced secretion of cytokines were significantly augmented in patients from group A. In contrast, the asthmatics who represented group FA demonstrated normal levels of spontaneous cytokine secretion. However, the tendency to increase of LPS and PHA-induced production was observed in BAL leukocytes from the patients. The above results support the view that beneficial effect of corticosteroid treatment in bronchial asthma may act, at least in part, by inhibition of the high spontaneous secretion of proinflammatory cytokines. Nevertheless, fungal airwaysinfections may lead to increase of LPS- or PHA-induced production of TNF-a , IL-6 or IFN-g (despite f prednison therapy) by prestimulation of the BAL cells with fungi.

Keywords: bronchial asthma; bronchoalveolar leukocytes; fungal airway infections; IFN-g ; TNF-a; IL-6; prednison.