Vol. 43, 1995

Vol. 43, No. 1, 1995

CONTENTS

Review

  • HIV-1 Prevention: Interdisciplinary Studies on the Efficacy of Bleach and Development of Prevention Protocols
    CLYDE B. MCCOY, PAUL SHAPSHAK, LISA R. METSCH, JAMES E. RIVERS, H. V. MCCOY, NORMAN L. WEATHERBY, SFED M. SHAH and DALE D. CHITWOOD

    Abstract. In the United States, a major federally-funded approach to HIV-1 prevention for injecting drug users (IDUs) includes teaching them to always rinse their needles/syringes with household bleach and water before use. This report describes interdisciplinary studies of the extent to which HIV-1 can be found in injection equipment and the efficacy of bleach as a disinfectant, under simulated field conditions. Bloody needle/syringe units collected from Miami, Florida, shooting galleries or from community outreach prevention participants were selected for these studies. Groups of needle/syringe units were cleansed with bleach using a standard technique taught to IDUs in community outreach programs. Cleansed and uncleansed groups of need- les/syringe units were then tested for the presence of HIV-1. The data demonstrate the efficacy of bleach rinses in reducing the risks of HIV-1 infection from needle/syringe units and indicate that the teaching of a bleach cleansing method to IDUs should be part of a total AIDS prevention protocol.

    Keywords: HIV-1; prevention; injecting drug user (IDU); bleach; needles; syringes.ringes.

Clinical Immunology

  • Interleukin 10 Inhibits Interleukin 6 Production and Acute Phase Response in Rheumatoid Arthritis
    JAN K. ŁĄCKI, KAMILLA KLAMA, WIESŁAWA PORAWSKA, STEFAN H. MACKIEWICZ, WOLFGANG MÜLLER and KRZYSZTOF WIKTOROWICZ

    Abstract. To evaluate the effect of interleukin 10 (IL-10) on acute phase response, we determined serum levels of IL-10, interleukin 6 (IL-6), C-reactive protein (CRP), alpha-1-acid glycoprotein (AGP), alpha-1-anti- chymotrypsin (ACT) in 34 rheumatoid arthritis (RA) patients. IL-10 and IL-6 levels were evaluated using an enzyme-linked immunoassay (ELISA). CRP, AGP and ACT levels were measured using rocket immunoelect- rophoresis. The results showed that IL-10 serum level was increased in RA patients as compared to controls (60.0 ± I 7.5 pg/ml vs. 7.2 ± 1.9 pg/ml). IL-6 level was significantly elevated (87.3 ± 32.7 pg/ml vs. 45 ± 19 pg/ml, p < 0.05). CRP was significantly increased as compared to healthy controls (34± 19 mg/1 vs. 3±2 mg/l, p < 0.05). AGP and ACT serum levels were increased in RA patients, but we did not find these changes to be statistically significant. A good negative correlation between IL-10 and IL-6 serum level was found (r = -0.73, p < 0.05). A positive significant correlation between IL-6 serum level and CRP (r = 0.62, p < 0.05), AGP (r = 0.74, p < 0.05) and ACT (r = 0.45, p < 0.05) was established. Moreover, a negative correlation between IL-10 and serum level of CRP (r = -0.76, p < 0.05), AGP (r = -0.60, p < 0.05) and ACT (r = -0.37, p < 0.05) was also shown. According to the data thus far obtained it seems that IL-10 decreases IL-6 produc- tion, and by that indirectly affects acute phase response, decreasing CRP, AGP and ACT synthesis.

    Keywords: rheumatoid arthritis; interleukin 10; interleukin 6; acute phase proteins.oteins.

  • Immunohistochemical Comparative Investigations of Lymphatic Tissue in Reactive Processes, Myasthenic Thymuses and Hodgkin’s Disease
    ALBINA ZÓŁTOWSKA

    Abstract. To evaluate the effect of interleukin 10 (IL-10) on acute phase response, we determined serum levels of IL-10, interleukin 6 (IL-6), C-reactive protein (CRP), alpha-1-acid glycoprotein (AGP), alpha-1-anti- chymotrypsin (ACT) in 34 rheumatoid arthritis (RA) patients. IL-10 and IL-6 levels were evaluated using an enzyme-linked immunoassay (ELISA). CRP, AGP and ACT levels were measured using rocket immunoelect- rophoresis. The results showed that IL-10 serum level was increased in RA patients as compared to controls (60.0 ± I 7.5 pg/ml vs. 7.2 ± 1.9 pg/ml). IL-6 level was significantly elevated (87.3 ± 32.7 pg/ml vs. 45 ± 19 pg/ml, p < 0.05). CRP was significantly increased as compared to healthy controls (34± 19 mg/1 vs. 3±2 mg/l, p < 0.05). AGP and ACT serum levels were increased in RA patients, but we did not find these changes to be statistically significant. A good negative correlation between IL-10 and IL-6 serum level was found (r = -0.73, p < 0.05). A positive significant correlation between IL-6 serum level and CRP (r = 0.62, p < 0.05), AGP (r = 0.74, p < 0.05) and ACT (r = 0.45, p < 0.05) was established. Moreover, a negative correlation between IL-10 and serum level of CRP (r = -0.76, p < 0.05), AGP (r = -0.60, p < 0.05) and ACT (r = -0.37, p < 0.05) was also shown. According to the data thus far obtained it seems that IL-10 decreases IL-6 produc- tion, and by that indirectly affects acute phase response, decreasing CRP, AGP and ACT synthesis.

    Keywords: rheumatoid arthritis; interleukin 10; interleukin 6; acute phase proteins.oteins.

  • Reactivity of Monoclonal Antibodies OK-CLL (Anti-CD5) with Peripheral Blood Cells of Patients with B Cell Lymphoproliferative Disorders
    DUŚANKA MILOŚEVIĆ, MILICA MARINKOVIĆ, SVETISLAV JELIĆ, MILICA ĆOLOVIĆ and IVAN SPUŻIĆ

    Abstract. Reactivity of OK-CLL monoclonal antibodies that can identify CD5 antigen on peripheral blood mononuclear cells, was investigated in 172 patients with B cell chronic lymphocytic leukemia (B-CLL) in clinical stages RAI O – I and RAI II – I V and in patients with non-Hodgkin’s lymphomas, classified according to the Working formulation into high and low grade histologic type. The OK-CLL reactivity with B-CLLs in the initial (RAI O – I), as well as in advanced stages of disease (RAI II – IV) was significantly higher than in controls. Peripheral blood cells of lymphoma patients, regardless of histological type, showed a much lower values of the CD5 positive population than chronic lymphocytic leukemia (CLL) patients, and a non sig- nificant discrepancy between the number of cells stained with anti-CD5 and those stained with other T cell markers. In spite of the showed considerable decrease of CD5 positive cells in CLL patients during therapy, elevated number of this population compared to normal individuals, after chemotherapy, was found. However, in lymphoma patients of both types of malignancies, CD5 positive population increased concomitantly with therapy. These results may suggest that analysis of CD5 antigen expression on peripheral blood cells of patients with B cell lymphoproliferative malignancies may have diagnostic, or, in correlation with some relevant clinical parameters, a potential predictive value in the treatment of those patients.

    Keywords: CD5 expression; non-Hodgkin’s lymphomas; chemotherapy; B cell chronic lymphocytic leukemia.ukemia.

Cellular Immunology

  • Preactivation and Phenotype of Monocytes Have No Influence on Their Elimination from Culture by Activated T Lymphocytes
    JULIUSZ PRYJMA, MAREK ZEMBALA, MARTIN ERNST and HANS-DIETER FLAD

    Abstract. T lymphocytes can kill antigen-presenting cells (APC) in the presence of antigen or lectin. The subject of this study was to investigate whether the state of activation or phenotype of monocytes, influence their susceptibility to killing by T cells activated with pokeweed mitogen (PWM) or anti-CD3 monoclonal antibody. The data are presented which show that monocytes activation with cytokines (IFN-y, IL-4, or IL-2), PPD, phorbol ester or phagocytic stimulus, have no influence on monocyte susceptibility to killing by T lym- phocytes. Furthermore, flow cytometry data suggest that monocytes eliminated from culture have no charac- teristic phenotype. In conclusion, our data indicate that elimination of monocytes by activated T lymphocytes does not depend on the state of activation of monocytes.

    Keywords: monocytes; cytotoxic T cells; cytokines.okines.

  • Effect of Peritoneal Cells on Tumor Cells Growth in vitro
    IAN SALWA

    Abstract. The cytotoxic and cytostatic activity of PMA-treated macrophages, obtained from pristane-primed BALB/c mice, was analyzed in vitro. The activated macrophages were cytotoxic and cytostatic for YAC-1 lymphoma, P-388 leukemia and P-815 mastocytoma target cells. However, the RPC-5 plasmacytoma target cells appeared to be resistant to their cytotoxicity. The observed cytotoxic or cytostatic effects of macrophages in vitro were not correlated with their ability to produce the superoxide ion. Cytotoxic activity of NK cells, obtained from pristane-primed mice, was also studied. No differences in cytotoxicity of NK cells obtained from pristane-treated and untreated donors, were found. However, only the effector cells from untreated mice were able to respond to stimulatory effect of polyinosinic acid-polycytidylic acid-poly-L-lysine (poly ICLC).

    Keywords: macrophages; plasmacytoma; cytotoxic and cytostatic activity.tivity.

  • Influence of Theophylline on Interferon Production and cAMP Level in Lpa Cells
    RENATA ZAHORSKA, MICHAŁ KORBECKI and JAN BARCISZEWSKI

    Abstract. In the presence and absence of methionine initiator tRNA (tRNMe Met/i) theophylline applied for I h starting 6 h after induction increased significantly (2-fold) interferon (IFN) yield. Theophylline did not change the kinetics of IFN production in the presence of tRNA Met/i. The shut-off stage was present and the production was prolonged to 24 h after IFN induction. In comparison with the values obtained in untreated cells, poly I:C plus DEAE dextran (inducing complex) caused an about 2-2.5 fold increase of cyclic adenosine 3’5′-monophosphate (cAMP) level. The similar results were observed in the cells treated with inducing comp- lex plus tRNA Met/i. About 2-fold higher cAMP level occurred in the cells under the influence of the inducing complex plus theophylline in comparison to the level obtained without theophylline. The addition of tRNA Met/i did not influence. the above presented data.

    Keywords: theophylline; methionine initiator tRNA; cyclic adenosine 3’5′-monophosphate.sphate.

  • Effect of Captopril on the Proliferation Cell Lines and Their Influence on Cytostatic Activity of Interferon ( or Cytotoxic Activity of Doxorubicin
    JAN KOWALSKI, DARIUSZ BELOWSKI, JACEK WIELGUS, BOŻENA GABRYEL, MARIUSZ KLIN and ZBIGNIEW S. HERMAN

    Abstract. We have assessed the effect of thiorphan or captopril on proliferation of two human tumor cell lines, A549 and HL60 including their influence on the cytostatic activity of interferon a or doxorubicin. The results showed that captopril inhibits the proliferation of both A549 and HL6o cell lines but thiorphan has anti- proliferative effect only on A549 cells in a dose-dependent manner. However, neither captopril nor thiorphan administered in combination with interferon a or doxorubicin enhanced cytotoxic potential of doxorubicin and cytostatic activity of interferon a.

    Keywords: thiorphan; captopril; tumor cell proliferation.ration.

  • Accumulation of Inositol l, 4, 5-Triphosphate after Cholinergic Stimulation of Human Lymphocytes
    URSZULA BANY, HANNA LASKOWSKA-BOŻEK and JAN RYŻEWSKI

    Abstract. The interrelationships between the immune and neurohormonal system are the subject of this work. The formation of inositol 1,4,5-triphosphate (IP3) in response to cholinergic stimulation was studied in the human peripheral blood mononuclear cells (PBMC) and lymphocytes from the lymphoblastoid cell lines Jurkat and Raji. The cells were stimulated with mitogen and then treated with cholinergic agonist-carbachol or nicotine. The radioreceptor assay was used to determine the amount of IP3 in all studied lymphocytes. The results, combined with the presence of muscarinic and nicotinic receptors on the surface of these lymphocytes, suggest that the parasympathetic nervous system is involved in the immune-neurohormonal crosstalk.

    Keywords: inositol 1,4,5-triphosphate; cholinergic receptors; human lymphocytes.ocytes.

  • Influence of Cyclosporine A on Thyroid Function in the Experimental Autoimmune Thyroiditis (EAT) in Mice
    JAN KAZIMIERZ CZYŻYK and MAREK JAKÓBISIAK

    Abstract. We have examined the influence of cyclosporine A (CsA) on thyroid gland function during develop- ment of experimental autoimmune thyroiditis (EAT) in CBA (H-2″) mice, delivering the agent in different regimens and doses, either as commercial intravenous preparation or encapsulated into liposomes. The evalu- ation of autoimmunity was done by the assessment of serum concentration of thyroxine (T4). We showed that a high dose CsA (360 mg/kg) prevented the development of thyroid deficiency whereas a small dose (25 – 60 mg/kg) potentiated the decrease of T4 serum level. Moreover, we found that the same dose of CsA enclosed in liposomes and of its commercial preparation can have different effect on T4 level in mice with EAT.

    Keywords: thyroiditis; cyclosporine.porine.

Various

  • Depression of Interferon Production in Leukocytes of Cows with Fat Mobilization Syndrome
    AGNIESZKA SZUSTER-CIESIELSKA, JÓZEF FILAR and MARTYNA KANDEFER-SZERSZEŃ

    Abstract. White and Black dairy cows were classified on the basis of clinical symptoms, hematological data, biochemical tests of blood and histological examination into two groups: I – with fat mobilization syndrome (n =1 I), II – healthy cows (control) (n = 6). Interferon (IFN) induction in leukocytes of cows with fatty liver, elevated ketone bodies, free fatty acids, bilirubin concentration and high aspartate aminotransferase (AST) activity, revealed impaired IFN production which was not improved by using known immunomodulators such as isoprinosine, levamisole, TFX and lactoferrin. The defciency of IFN production observed in cows with fat mobilization syndrome may result in increased susceptibility of cows to different infections.

    Keywords: fat mobilization syndrome; interferons.ferons.

  • Mechanism of Antigenic Variation in Shigella flexneri Bacilli. III Complement Sensitivity of S. flexneri 1b Strains and Their 3b Variants Selected Either as Lac+ Recombinants or Phage F2 Resistant Mutants
    WLODZIMIERZ DOROSZKIEWICZ, BOLESLAW KOPOCIŃSKI and TADEUSZ M. LACHOWICZ

Abstract. Several S. flexneri lb serotype strains of clinical isolation were compared with their 3b serotype variants isolated either as Lac+ recombinants from crosses of S. flexneri x E. coli Hfr C, or F2-resistant mutants. The studied variants 3b were deprived of type I antigen but acquired type III antigen. They appeared to be less sensitive to killing action of complement present in normal bovine serum although kinetics of killing and its effectiveness differed between the strains.

Keywords: killing action of complement; S. flexneri lb; antigenic variation.iation.

Vol. 43, No. 2, 1995

CONTENTS

Transplantation

  • Incidence of Bone Marrow Transplantation in Europe. Report from the European Group for Blood and Marrow Transplantation
    ALOIS GRATWOHL

    Abstract. The European Group of Blood and Marrow Transplantation (EBMT) is collecting information on incidence of bone marrow transplantation in Europe. 203 teams in 26 European countries performed in 1992 a total of 6065 bone marrow transplants (BMT). There were 2666 (44%) allogeneic transplants from 2171 HLA identical sibling donors, 170 non-identical family donors, 29 twin donors and 296 unrelated volunteer donors. There were 3399 autologous transplants (56%): 2494 autologous BMT, 644 autologous peripheral blood stem cell transplants and 261 combined autologous bone marrow peripheral blood stem cell trans- plants. Transplant indications were leukemias in 2963 patients (49%) (1987 allogeneic, 976 autologous), lymphoproliferative disorders in 1890 patients (31 %) (201 allogeneic, 1689 autologous), solid tumors in 739 patients (12%) (10 allogeneic, 739 autologous), aplastic anemia in 194 patients (3%) (193 allogeneic, 1 auto- logous), thalassemia in 128 patients (2%) (all allogeneic), inborn errors in 115 patients (2%) (all allogeneic) and miscellaneous disorders in 36 patients (32 allogeneic, 4 autologous). Main differences were observed in the number of transplanting teams, and in the number of allogeneic and autologous transplants performed in the different European countries compared to the number of inhabitants.

    Keywords: bone marrow transplantation; allogeneic; autologous.logous.

  • HLA and Bone Marrow Transplantation
    THOMAS H. EIERMANN

    Abstract. Bone marrow transplantation between HLA-identical siblings is an established therapeutic strategy in leukemia and a variety of lympho-hematopoetic disorders. In recent years unrelated donors were increasin- gly used for patients lacking a family donor. Here we report on current progress in HLA class II typing by molecular methods which have influenced donor search and improved matching.

    Keywords: bone marrow transplantation; mixed lymphocyte culture; polymerase chain reaction sequence-specific oligonucleotide.eotide.

  • DRB Polymerase Chain Reaction Fingerprinting, DQA Oligotyping and Mixed Lymphocyte Culture Results In related Bone Marrow Donors and Recipients
    EWA BROJER, MONIKA SANKOWSKA, JANUSZ J. STAŃCZAK, ANNA GRONKOWSKA, BARBARA STĘPIEŃ-SOPNIEWSKA, ANDRZEJ GÓRSKI and BARBARA ŻUPAŃSKA

    Abstract. Donor and recipient are usually regarded as well matched for bone marrow transplantation when they are compatible in HLA class I and II antigens and in mixed lymphocyte culture (MLC). However, results of serological typing of class II antigens may be unreliable. Hence, polymerase chain reaction fingerprinting of HLA DRB (PCR FP) was introduced for the screening of related donors for 29 patients awaiting bone marrow transplantation. In addition, the sequence-specific oligonucleotide (SSO) typing of DQA alleles was performed. In 18 pairs the results of DNA analysis methods were compared with the results of MLC. 72% of pairs were HLA DQA compatible and 59% showed compatibility in PCR FP. MLC compatibility was found in 61%. A high correlation of PCR FP and MLC results was observed.

    Keywords: bone marrow transplantation; HLA; polymerase chain reaction; fingerprinting.inting.

  • Analysis of Reconstituting T Cell Receptor Repertoires in Bone Marrow Transplant Recipients
    JACK GORSKI, MARYAM YASSAI, CAROLYN KEEVER and NEAL FLOMENBERG

    Abstract. Allogeneic bone marrow transplantation (BMT) has become the treatment of choice for a variety of hematopoetic disorders. An important factor limiting the use of allogeneic BMT is delayed restoration of immune function. A quantitative understanding of immune reconstitution of the T cell compartment based on an efficient method of analysis would be of benefit. Distinct lineages of T cells which have resulted from previous and ongoing clonal expansion can be identified by their unique T cell receptors (TCR). Thus, TCR complexity can be used as a measure of repertoire complexity. Here we use a polymerase chain reaction-based approach which visualizes the size heterogeneity of the third complementarity determining region (CDR3) to study T cell reconstitution in adult bone marrow transplant recipients. We find that repertoire complexity, as determined by the number of bands of different length for each V family, reflects the general immune status of individuals tested. Contractions and gaps in repertoires are revealed in individuals suffering from recurrent infections associated with T cell impairment. This approach provides a new tool in the analysis of recons- titution of a/ß T cell repertoires and it can also be applied to B cells and y/â T cells.

    Keywords: T cell receptor; polymerase chain reaction; bone marrow transplantation.tation.

  • Cytokines, Adhesion Molecules (E-Selectin and VCAM-1) and Graft-Versus-Host Disease
    ANDRZEJ LANGE, ALEKSANDRA KLIMCZAK, LIDIA KARABON and KRZYSZTOF SUCHNICKI

    Abstract. Analysis of skin biopsy specimens for the presence of adhesion molecules, composition of cellular rofiltrates, Ki-67 antigen expression, and examination of serum for interleukin 6 (IL-6) and tumor necrosis factor a (TNF-a) levels, was performed in patients after allogeneic bone marrow transplantation (alloBMT), to study the pathomechanism of acute graft-versus-host disease (aGvHD). It was found that: 1) early hemato- bgical recovery constitutes a risk factor for grade IV GvHD, 2) vascular cell adhesion molecule-1 (VCAM-1) is present in the matrix organizing the cells in the bone marrow and in aGvHD infiltrates, 3) HLA DR antigens aberrant expression in epithelial cells, as well as 4) strong expression of Ki-67 is seen in early stages of aGvHD. These immunopathomorfological lesions are cytokine-dependent. High levels of IL-6 and TNF-a were found in sera of patients affected with the aGvHD process and infectious complications. An increase of IL-6 in the course of aGvHD is a sign of poor prognosis. These data support the notion that cytokines facilitate the cell accumulation at the site of aGvHD at the beginning of this process and again, at the final stage of the disease, cytokines high levels are associated with the organ damage.

    Keywords: bone marrow transplantation; cytokines; graft-versus-host disease; immunopathomorphology.hology.

  • Statistical Evaluation of Bone Marrow Transplantation Data
    RYSZARD M. SZYDŁO

    Abstract. The analysis of data generated from a transplant programme is fundamental to assessing both the etfectiveness of different treatments, and the prognostic role of disease and patient variables. Therefore, the use of life-table analyses, methods of comparing survival curves, and a brief discussion of multivariate methods will be presented.

    Keywords: bone marrow transplantation; survival analysis; life-table; logrank; proportional hazards regres- sion analysis.alysis.

Molecular Immunology

  • Use of Spontaneously Mutated Human DNA as Competitive Internal Standard for Nucleic Acid Quantification by Reverse Transcription-Polymerase Chain Reaction (RT-PCR)
    LIDIA RUDNICKA, ARTURO DIAZ, JOHN VARGA, ANGELO CHRISTIANO, JOUNI UITTO and SERGIO A. JIMENEZ

    Abstract. Quantification of gene expression is of increasing interest in many medical sciences. Methods based on reverse transcription-polymerase chain reactions (RT-PCRs) are timesaving and require only very small amounts of RNA. A limiting factor, however, is the significant fluctuation in the efficacy of reverse transcrip- tion as well as in the polymerase chain reactions. Various external and internal standards have been suggested for correcting these fluctuations. We describe á novel way of creating an internal standard for assessing the expression of type VII collagen in human cells. The total RNA of a patient with hereditary epidermolysis bullosa dystrophica associated with a homozygous T to A point mutation in type VII collagen gene was reverse transcribed and a 382bp fragment of type VII collagen cDNA containing the mutation was amplified. The mutated cDNA, unlike normal type VII collagen cDNA could be cleaved by Ear1 endonuclease into 244bp and 138bp fragments. Semiquantitative PCR was performed with the mutated cDNA as internal standard and the studied cDNA sample in the same tube in the presence of a3zP-labeled dCTP. The reaction was followed by Ear1 digestion, electrophoresis on a polyacrylamide gel and exposure to a X-ray film. In conclusion, we describe a timesaving method for creating internal standards for semiquantitative RT-PCR.

    Keywords: polymerase chain reaction; internal standard; transforming growth factor ß.ctor ß.

  • Cell Proliferation-Associated Nuclear Antigen Defined by Antibody Ki-67: a New Kind of Cell Cycle-Maintaining Proteins
    MICHAEL DUCHROW, CARSTEN SCHLÜTER, GORAN KEY, MICHAEL H. G. KUBBUTAT, CLAUDIA WOHLENBERG, HANS-DIETER FLAD and JOHANNES GERDES

    Abstract. A decade of studies on the human nuclear antigen defined by monoclonal antibody Ki-67 (the „Ki-67 protein”) has made it abundantly clear that this structure is strictly associated with human cell proliferation and that the expression of this protein can be used to assess the growth fraction of a given cell population. Until recently the Ki-67 protein was described as a nonhistone protein that is highly susceptible to protease treatment. We have isolated and sequenced cDNAs encoding for this antigen and found two isoforms of the full length cDNA of 11.5 and 12.5 kb, respectively, sequence and structure of which are thus far unique. The gene encoding the Ki-67 protein is organized in 15 exons and is localized on chromosome 10. The center of this gene is formed by an extraordinary 6845 bp exon containing 16 successively repeated homologous segments of 366 bp („Ki-67 repeats”), each containing a highly conserved new motif of 66 bp („Ki-67 motif’). The deduced peptide sequence of this central exon possesses 10 ProGluSerThr (PEST) motifs which are associated with high turnover proteins such as other cell cycle-related proteins, oncogenes and transcription factors, etc. Like the latter proteins the Ki-67 antigen plays a pivotal role in maintaining cell proliferation because Ki-67 protein antisense oligonucleotides significantly inhibit 3H-thymidine incorportation in per- manent human tumor cell lines in a dose-dependent manner.

    Keywords: Ki-67; Mib-1; monoclonal antibody; cell proliferation; nuclear antigen; cDNA.; cDNA.

  • Analysis of T Cell Receptor aß Variability in Tumor-Infiltrating Lymphocytes in Primary and Metastatic Melanoma
    JESPER ZEUTHEN, ANETTE BIRCK and PER THOR STRATEN

    Abstract. The T cell receptor (TCR) aß variable (V) gene family usage of tumor-infiltrating lymphocytes (TIL) in different primary human malignant melanomas and corresponding metastatic lesions were characterized using a recently developed method using the reverse transcription coupled polymerase chain reaction (RT-PCR). This semiquantitative RT-PCR method could be adapted to analysis of formalin-fixed, paraffin-embedded histopathological samples of primary tumor material and demonstrated to be reproducible and to be useful for the assessment of V( and Vß-gene family usage in tumor samples. The TIL in primary tumors were observed to preferentially express certain TCR V( and Vß-gene families: V(4, V(22 and Vß8 were highly expressed in several of the primary tumors analyzed using this method. With respect to V(22 and VßB, the preferential expression of these V-gene families was demonstrated to be due in situ clonal expansion of T cells by means of cloning and sequencing. of the CDR3 regions (V-J or V-D-J, respectively) corresponding to the RT-PCR products from one of the primary tumors. The observed preferential usage of certain TCR V( and Vß-genes strongly suggest the in situ clonal expansion of specific populations of T cells in accordance with recent results from others. These clonal T cell populations probably react with certain melanoma-associated peptides presen- ted by specific HLA molecules. The preferential usage of certain V(- and Vß-gene families observed in several tumors further supports the involvement of a limited number of shared melanocyte or melanoma-associated peptides. Since the HLA status of the patients is obviously important to interpret these results, some of the patients were typed for HLA-A1 and -A2, the two most well-characterized restriction elements for melano- ma-associated antigens, either serologically or by a newly developed RT-PCR method which similarly could be applied directly to the tumor material. In TIL in primary melanomas, a possible correlation was suggested between HLA-A2 and the preferential usage of the TCR V-gene families V(4, V(5, V(22 and VßB, whereas the Vß3-gene family appeared to be expressed together with HLA-A1. The V-gene families which were highly expressed in the primary tumors were generally not, or only very weakly, expressed in the corresponding metastases and vice versa, possibly reflecting a substantial change in the phenotype of the metastatic melano- ma target cells. Continued studies of larger patient materials will be necessary to extend and validate these conclusions and of obvious interest for the further analysis of the T cell response in melanoma.

    Keywords: T cell receptor; tumor-infiltrating lymphocytes; melanoma.lanoma.

  • HLA-B27 Subtypes Among the Chukotka Native Groups
    MIKHAIL Y. KRYLOV, .IOHN D. REVEILLE, LUDMILA I. ALEXEEVA, SHANDOR ERDESZ, FRANK C. ARNETT and LIDIYA I. BENEVOLENSKAYA

    Abstract. The purpose of this study was to assess the relative frequency of the known HLA=B27 subtypes in HLA-B27 positive Chukotka natives, which have higher frequencies of HLA-B27 (to 40%) and spondyloarth- ropathies (to 2%) than the Russian Caucasian population. Using oligotyping of the polymerase-chain reaction amplified second and third exons of the HLA-B27 gene in 86 DNA samples from HLA-B27 positive in- dividuals were successfully typed. All had HLA-B*2705, including 4 patients with Reiter’s syndrome and 5 with ankylosing spondyloarthritis, except one Eskimo who had HLA-B*2702. None had HLA-B*2704 a frequent subtype in Orientals. With respect to HLA-B27 subtypes the indigenous populations from the eastern part of the Chukotka Peninsula are genetically more closely related to Caucasians than to Orientalis.

    Keywords: HLA-B27 subtypes; Chukotka natives; Eskimos.skimos.

  • Role of Hydrophobic Amino Acids at Position 74 o DRB1 Chain in Reumatoid Arthritis
    MARIE ĆERNA, STANISLAV HAVELKA and EVA IVAŚKOVA

    Abstract. We have analyzed HLA class II alleles in a group of 153 Czech children with rheumatoid arthritis by PCR and hybridization with oligonucleotide probes. When we try to find a common sequence for all DRB1 alleles involved in juvenile and adult arthritis, we can notice hydrophobic amino acid at position 74, which is present in all these alleles, but not in nonsusceptible alleles, where is the hydrophilic amino acid at position 74. In our model, we speculate that the hydrophilic amino acid at position 74 creates a such kind of epitope which is not suitable for rheumatoid-associated peptides or T cells, and only hydrophobic amino acid can permit binding of these peptides or recognition by certain T cells. Analyses of the DPBI sequences have shown that alleles which have a negatively charged amino acid at position 69, are more frequent in pauciarticular patients while those with a positively charged amino acid are more frequent in polyarticular patients. A positively charged amino acid at position 69 might present the same rheumatoid associated peptide as susceptible DRB1 alleles. The presence of more rheumatoid-associated peptide on the cell surface may cause conversion to more severe polyarticular forms. A negatively charged amino acid at position 69 could not present this peptide and a low concentration of the peptide on the cell surface presented just by DRB1 molecules keeps disease in a relatively benign condition of pauciarticular forms.

    Keywords: HLA; DRB1; amino acid sequences; peptide presentation.tation.

  • Use of Specific Anti-HLA Antibodies in Clinical Nephrology
    SOPHYA E. BUGLOVA, VALERY I. LEVIN, VALERY S. PILOTOVICH, GENNADY T. KOZLOV and LUDMILA N. DRAPEZO

    Abstract. In order to optimize the regimen of pathogenetic therapy of glomerulonephritis with nephrotic syndrome we used transfusions of plasma containing specific anti-HLA antibodies against the patient’s pheno- type as a part of the treatment in the acute stages of disease. The method was theoretically based on the conception of autoimmune genesis of glomerular lesion as well as on the assumption that the highest expres- sion of H LA-antigens are on the surface of activated immunocompetent cells. In previous experiments in vitro we demonstrated immunosuppressive activity of cytotoxic serum on lymphocytes in the absence of comp- lement. Stable remission that was associated with the recovery of serum IgG/IgM balance and normalization of cellular immunity indices was achieved in 72% of 50 treated patients after 2-4 transfusions. Immuno- therapeutic properties of anti-HLA antibodies were also confirmed with preliminary results of plasma tran- sfusions in 5 kidney graft recipients. Posttransfusion reactions of a non-haemolytic type were slight and transient. The data presented allow us to conclude that our method may be useful in clinical cases associated with undesirable immune activation induced by both auto- and alloantigens.

    Keywords: anti-HLA antibodies; immunotherapy; glomerulonephritis; kidney transplantation.tation.

Cytokines

  • Cytokine Regulation of Monocyte Recruitment
    ALBERTO MANTOVANI, BARBARA BOTTAZZI, SILVANO SOZZANI, GIUSEPPE PERI, PAOLA ALLAVENA, QUIANG(GANG DONG, ANNUNCIATA VECCHI and FRANCESCO COLOTTA

    Abstract. Phagocytes infiltrating neoplastic tissues have peculiar membrane phenotype and functional proper- ties. Tumor-associated macrophages (TAM) play a complex, ambiguous role in the regulation of primary tumor grwoth and metastasis (a `macrophage balance”). Yet these cells are strategically located at the very interface between tumor and host and represent a potential target for immonomodulation. A better undrestan- ding of the regulation and function of TAM may provide a less empirical basis of or rational design of therapeutic approaches, as vividly illustrated by the antitumor activity of i.p. in IFN ovarian cancer patients with minimal residual disease resistant to chemotherapy.

    Keywords: tumor-associated macrophages; cytokine; ovarian cancer.cancer.

  • Cytokines in Mycobacterial Infections in vitro and ex vivo Studies
    HANS-DIETER FLAD 1, JENS GERCKEN 1, LORENZ HUBNER t, CARSTEN SCHLUTER t JULIUSZ PRYJMA 2 and MARTIN ERNST 1,

Abstract. Different species of mycobacteria differ in their capacity to induce the production of tumor necrosis factor-a (TNF-alpha) by human monocytes in vitro. Whereas M. tuberculosis is a potent inducer of TNF-a, M. leprae is much less potent. TNF-a production is found to be associated with the availability of H2O2 genera- ted by activated monocytes, as superoxide enhancing H202 concentration increases and catalase degrading H2O2 decreases TNF( production. Furthermore, M. kansasii with high intrinsic catalase induce less TNF-a than mycobacteria with low intrinsic catalase. In vitro infection of monocytes with M. tuberculosis leads to an impairment of the antigen-presenting capacity, as determined by a reduction of antigen-induced T cell prolife- ration and interferon y (IFN-y) production. Of crucial importance in this impairment is the M. tuberculo- sis-induced down-modulation of MHC class II antigens. The role of TNF(alpha in vivo is reflected in patients with various forms of leprosy. In skin lesions of lepromatous leprosy patients TNF(alpha, interleukin lß (IL-lß), and IFN-(y production are found to be rare, whereas these cytokines are well expressed in skin lesions of patients with tuberculoid leprosy. After multidrug chemotherapy an increase of local cytokine production is found. Taken together, these findings suggest that components of mycobacteria may interfere with local cell-mediated immune reactions in vivo. The molecular mechanisms involved in these local responses need to be defined.

Keywords: tumor necrosis factor; cytokines; mycobacterial infections.ctions.

 

Vol. 43, No. 3-4, 1995

CONTENTS

Mini-Review

  • Alterations in Glycoconjugates Related to the Transformation Grade of Human Uroepithelial Cell Lines
    MACIEJ UGORSKI and DANUTA DUŚ

    Abstract. To study the changes in carbohydrate expression associated with the progression of malignant phenotype, we have used the series of human urinary bladder epithelial cell lines. Non-tumorigenic and non-invasive cell lines, with infinite life span, were defined as transformation grade II (TGr II): Cell lines producing tumors in nude mice and invading fragments of embryonic chick hearts in vitro were defined as TGr III. In the present review the results obtained from our immunochemical, biochemical and structural studies on glycosphingolipids and glycoproteins of 10 human urothelial cell lines representing both grades of transfor- mation are summarized and discussed in relation to the literature data. TGr III cell lines are characterized by decreased level of the GMZ ganglioside, presence of sialyl-Lea ganglioside, increased amount of more highly branched, tri- and tetraantennary N-acetyllactosamine-type glycans and higher number of binding sites for peanut lectin and wheat germ agglutinin lectin, as compared to non-tumorigenic and non-invasive TGr II cell lines.

    Keywords: human uroepithelial cell lines; transformation grade; glycoconjugates.ugates.

  • Mouse Monoclonal IgA Antibodies Lack Interchain Disulfide Bonds
    HUBERT KROTKIEWSKI, ANNA LASKOWSKA and BOŻENA KROTKIEWSKA

    Abstract. A mouse monoclonal IgA antibody (A008), anti-blood group A antigen, was shown to dissociate into heavy (H) and light (L) polypeptide chains under non-reducing conditions. The dissociation occurred in the presence of 02% sodium dodecyl sulphate (2 h at room temperature) or at elevated temperature (1 h at 70C). The free H and L polypeptide chains could be separated in SDS-polyacrylamide gel electrophoresis or by gel filtration in the presence of SDS. The dissociation of IgA (A008) antibody into heavy and light polypeptide chains was reversible, since the fractions containing gel filtration-isolated chains, after removing the detergent, pooling together and incubation at 4oC created again the immunoglobulin molecules with activity close to the native value. Similarly, the same IgA antibody heated at 70oC for 1 h recovered its antibody activity after keeping at 4″C. Three other mouse monoclonal IgA antibodies showed the same ability to dissociate into heavy and light polypeptide chains after exposure to SDS or elevated temperature, which suggests that this outstanding property is a chracteristic feature of the mouse monoclonal IgA antibodies and it comes out from the lack of H-H, L-L and H-L interchain disulfide bonds in these molecules.

    Keywords: immunoglobulin polypeptide chains; non-reducing conditions; H-H dimers; disulfide bonds; re-association.iation.

  • Human Serum Immunoglobulins Binding to Synthetic Fragments of Human Chorionic Gonadotropin
    IWONA E. KOCHANOWSKA and APOLINARY SZEWCZUK

    Abstract. Human sera obtained from healthy individuals, patients with cancer diseases, infertile and pregnant women were investigated for the presence of immunoglobulins (Ig) binding to synthetic fragments correspon- ding to sequence of human chorionic gonadotropin (hCG). Four synthetic peptides corresponding to some regions of (hCG and ßhCG subunits, coupled to polystyrene beads and 16 peptides overlapping sequences of (- and ßhCG subunits, synthesized on polyethylene pins, were used. IgG and IgM bound to hCG-synthetic peptides were assayed using goat or mouse monoclonal antibodies anti-human IgG labelled with horseradish peroxidase. Most of the investigated sera contained IgG which bound to examined synthetic peptides, but obtained results were dependent on serum donors. Some relationships between results obtained with peptides synthesized on modified pins according to Geysen et al. method and corresponding peptides synthesized by Merrifield method and coupled to beads were noted.

    Keywords: ELISA; human chorionic gonadotropin (hCG); IgG and IgM binding to hCG fragments; synthetic peptides of hCG sequence.quence.

  • Stability of Human Lutropin Studied with Immunoenzymatic Technique
    EWA KUROWSKA, IWONA E. KOCHANOWSKA, MARIANNA KUROPATWA, .IANUSZ BORATYŃSKI and APOLINARY SZEWCZUK

    Abstract. Characteristics of two monoclonal antibodies against human lutropin and their use in enzyme immunoassays of the hormone are presented. Diluted solution of pituitary lutropin was unstable in buffered saline and could be stabilized with some proteins. Concentration of the lutropin in woman urine, collected during „lutropin-peak”, was markedly increased during long time storage at 5` C or at room temperature. Using HPLC it was demonstrated that pituitary lutropin, normal urine lutropin and „increased” urinary lutropin were eluted from ion exchange column almost with the same retention time.

    Keywords: human lutropin; human luteinizing hormone (hLH); pituitary hLH, urine hLH, stability of hLH.of hLH.

Clinical Immunology

  • Comparison of the Influence of Stress Exercise Test and Transesophageal Cardiac Pacing on Polymorphonuclear Neutrophils Functions
    HENRYK WYSOCKI, ANDRZEJ MINCZYKOWSKI, ANDRZEJ WYKRETOWICZ, GRZEGORZ JUNICZAK and JULIUSZ SMIELECKI

    Abstract. Peripheral polymorphonuclear neutrophils (PMN) number, percent of PMN bearing IgG Fc recep- tors as well as PMN adherence were evaluated in 29 patients submitted to an exercise test. The peripheral PMN count significantly increased at the maximal work load. The increase in number of neutrophils bearing IgG Fc receptors was also noticed at that point, while PMN adherence to nylon wool columns did not change significantly. Nine subjects were additionally submitted to diagnostic transesophageal atrial pacing with a rate similar to maximal heart rate observed during the exercise test. It was revealed that atrial pacing in these patients had no influence on the peripheral PMN count, PMN adherence as well as number of neutrophils bearing IgG Fc receptors. We conclude, that granulocytosis observed during exercise can not be sollely attributed to the increased heart rate and increased cardiac output but other mechanisms like muscle work play a significant role in this process.

    Keywords: stress; neutrophils; exercise test; cardiac pacing.pacing.

  • Autologous Peripheral Blood Stem and Progenitor Cells Transplantation as a Valid Treatment Approach in Recurrent, Refractory Lymphoma
    JERZY HOŁOWIECKI, JERZY WOJNAR, MIROSŁAW MARKIEWICZ, ANNA WACŁAWIK, KRYSTYNA JAGODA, HANNA KAMINSKA, ŁUCJA KACHEL, MARIA WOJCIECHOWSKA and DARIUSZ KATA

    Abstract. Autologous peripheral blood stem cell transplantation (APBSCT) is used similarly to autologous bone marrow transplantation (ABMT) to reconstitute bone marrow following myeloablative therapy in pa- tients with proliferative diseases of the blood. Eight patients with recurrent and refractory lymphoma (3 HD, 4 NH L) and multiple myeloma aged 17 – 55 were included into the study. Peripheral blood stem cells following their prior mobilisation with cyclophosphamide 4-7 g/m2 and/or G-CFS or Dexa- -BEAM + G-CSF were collected by subsequent leukaphereses on Fenwal CS3000. Nucleated cells were sepa- rated by sedimentation, cryopreserved in a programmed freezer and then stored at -196oC. Bone marrow has been additionally collected in one patient. Conditioning treatment prior to transplantation consisted of BCNU, etoposide and cyclophosphamide (CBV) in lymphomas and melphalan in multiple myeloma. Collec- ted material with mean cellularity 5.52 x l Os/kg and mean CD34+ contents 627 x 10(6/kg was reinfused by central line. G-CSF was given in 5 patients to hasten the bone marrow recovery. All patients fully recovered and left hospital on average 35.5 days following transplantation. No signs of relapse were seen throughout the observation period (mean 349.5 days). Neutrophils > 0.5 G/I were obtained on day +20, > 1.0 G/1 on day 30, platelets > 50 G/1 on day 29, > 100 G/1 on day 53, reticulocytes ) 0.015 on day 30, erythrocytes transfusions were needed up to day 39. Presented outcomes together with other reports indicate, that APBSCT is a highly efficient way to rescue repeatedly relapsing patients with proliferative diseases of the lymphatic systems, even those presenting with changes in the bone marrow (neoplasmatic infiltrate, hypoplasia or fibrosis).

    Keywords: autologous peripheral blood stem cell transplantation; cytokines; malignant lymphomasmphomas

  • Influence of Gold Salts Treatment on the Serum Concentration of IL-1 and IL-2 in Patients with Rheumatoid Arthritis
    JOLANTA LEWANDOWICZ, BOŻENA DĄBKOWSKA and DARIUSZ NOWAK

    Abstract. Eighteen subjects (2 males, 16 females) with rheumatoid arthritis were treated with gold salts for 6 months. The statistically significant decrease of IL-1 and IL-2 levels during the treatment was observed. The mean serum concentrations of IL-1 and IL-2 decreased from 41.0± 11.5 to 28.6±7.3 (p < 0.001) and 98.3 ± 57.6 to 29.3 ± 20.5 fmol/ml (p < 0.001 ), respectively. This decrease was accompanied by a decline of serum concentration of acute phase proteins and immunoglobulins. These results can explain a strong an- ti-inflammatory action of gold salts in patients with rheumatoid arthritis.

    Keywords: interleukins; gold salts; rheumatoid arthritis; non-steroid anti-inflammatory drugs. drugs.

Immunotherapy

  • Synthesis and Immunological Activity of Mono- and Disubstituted Derivatives of 2,5-Dimercapto-1,3,4-Thiodiazoles
    STEFANIA H. KOWALCZYK-BRONISZ, JULIAN POMORSKI, BARBARA BUBAK, MACIEJ PREGIEL and BARBARA BŁASZCZYK

    Abstract. It is presented that the newly synthesized mono- and disubstituted 2,5-dimercapto-1,3,4-thiodiazole derivatives exert immunotropic activity. This effect was evaluated by measuring plaque forming cell number, circulating immunoglobulins level, autologous rosette number, mitogenic activity and by performing popliteal lymph node assay. It is shown that intensity of immunotropic activity depends on chemical structure: mono- substituted derivatives had a strong suppressive effect, disubstituted compounds showed the variable activity. One compound of this group exerted a stimulatory effect. The tested compounds were active in popliteal lymph node assay and had no mitogenic activity.

    Keywords: plaque forming cells; immunoglobulins; autologous rosettes; mitogenic activity; popliteal lymph node assay. assay.

  • Lactoferrin Lowers the Incidence of Positive Coombs’ Test in New Zealand Black Mice
    MICHAŁ ZIMECKI, ZBIGNIEW WIECZOEK, JOEL MAZURIER and GENËVIEVE SPIK

    Abstract. The results presented in this communication show that New Zealand Black (NZB) mice treated for a prolonged period with bovine lactoferrin (BLF) exhibit a decreased frequency of positive Coombs’ reaction. This effect was dose dependent and best pronounced at a dose of 50 µg/dose. We found, in addition, that incubation of peritoneal cells with BLF resulted in a decreased number of cells recognising Hb antigen on autologous erythrocytes. The data indicated that lactoferrin may be of therapeutical value in treatment of autoimmune disorders.

    Keywords: lactoferrin; New Zealand Black mice; autoimmune hemolytic anemia.anemia.

  • Influence of Lys-Residue Deletion on the Immunomodulatory Activity of Peptides Related to Immunosuppressive Region of Lactoferrin
    JACEK I. SŁOŃ, IGNACY Z. SIEMION, MICHAŁ ZIMECKI and ZBIGNIEW WIECZOREK

    Abstract. The effects of Lys residue deletion from peptides, related to immunosuppressive regions of lactoferrin (LF), were studied in the humoral and cellular immune responses to sheep erythrocytes (SRBC) in mice. We found that the deletion of Lys residue, vicinal to Pro, influences mostly the immunomodulatory activity of short fragments (tetra-, and pentapeptides) of immunosuppressive 231-245 mini-domain of LF. The activity of longer peptides, related to this mini-domain, is influenced to much lower extent by Lys removal. In particular, the tetrapeptide derived from Arg-Lys-Pro-Val-Asp sequence by deletion of Lys, exhibits strongly reduced immunosuppressive activity. On the contrary, the similar change within Arg-Lys-Pro-Val-Thr sequen- ce, derived of 575-589 loop of LF, produces a tetrapeptide Arg-Pro-Val-Thr, that is active as immunosupp- ressor both in humoral and cellular immune response. A quite strong immunosuppressive activity of tripeptide Lys-Arg-Pro is also worth consideration, especially when compared with the loss of activity observed for Thr-Arg-Pro tripeptide. The data presented herein indicate that exclusion of Lys residue from the LF-derived immunomodulatory peptides differentially alters their effects on the immune response to SRBC in mice.

    Keywords: lactoferrin; immunoregulatory fragments of; lysine.lysine.

Various

  • Lymphocyte Mitogen-Induced Proliferation in Patients with Allergic Rhinitis
    JOANNA CHOROSTOWSKA-WYNIMKO, DANUTA KLENIEWSKA, IRENA SOKOLNICKA, EWA ROGALA and EWA SKOPIŃSKA-RÓŻEWSKA

    Abstract. Lymphocytes play a central regulatory role in mechanisms contributing to impairied function of immune system in atopy. The aim of our study was evaluate the mitogen-induced proliferation of lymphocytes in a group of asymptomatic, seasonal allergic rhinitis patients. A highly significant lower mitogen-induced proliferation and, in contrast to other studies, significantly lower background proliferative activity of lym- phocytes were found in the atopic persons, comparing to the controls. We concluded that the decreased mitogen-induced proliferation of lymphocytes observed in allergic patients reflects abnormal T cell function, which is due to the atopic status, and not only as it was believed to the antigen-induced lymphocyte activation.

    Keywords: lymphocyte mitogen-induced proliferation; allergic rhinitis.initis.

  • Effect of Domperidone-Induced Hyperprolactinemia on Selected Immune Parameters in Healthy Women
    JOZEF ROVENSKÝ, MILAN BUC, ZDENEK LOJDA, MARIE RUZICKOVÁ, STANISLAVA BLAZÍCKOVA, LŮBICA RAUOVÁ, TIMOTEJ MIŚTINA, MILAN VIGAŚ and VLADIMÍR LAĆKOVIĆ

    Abstract. Domperidone, anti-emetic drug, given to healthy female volunteers, induced an elevation of plasma prolactin (PRL) concentration with the peak in 1-4 h. The release of prolactin had a transient stimulating effect on theophylline sensitive T lymphocytes and on concanavalin A induced mitogenic activity, suggesting an enhanced activity of T suppressor lymphocytes. The relative number of CD4+ lymphocytes decreased markedly one hour after domperidone administration and returned to normal values within 2 h (that means 3 h after taking the drug). The number of lymphocytes positive for dipeptidyl peptidase IV exhibited similar transient increase and normalization of activity. No change was observed in the number of CD8+ lym- phocytes. The production of interferon by leukocytes treated with Newcastle disease virus was found to be significantly increased 2 h after domperidone administration. The results suggest that prolactin can selectively stimulate some functions of cellular immunity as well as the release of cytokines (IFN). The present study may contribute to the understanding of the role of the immune system in endogenous hyperprolactinemia.

    Keywords: domperidone; hyperprolactinemia; cellular immunity.munity.

  • Cell Migration between Graft and Host – an Analysis with Monoclonal Antibodies after Allogeneic Rat Kidney Transplantation
    MACIEJ KRZYMAŃSKI, ANDRZEJ OKO, ANA MARIA WAAGA,JAN ŻEROMSKI, KARIN ULRICHS and WOLFGANG MÜLLER-RUCHHOLTZ

Abstract. In order to analyse migration patterns of donor MHC class II cells out of transplanted kidney and accumulation of host cells within the graft, immunomorphological studies were performed using monoclonal antibodies in rat allogeneic kidney transplantation model. To answer the question of how many donor cells migrate out of the renal cortex MRC 0 x 3 monoclonal antibody (MoAb) against LEW MHC class II antigens was used. In the grafts explanted after 4, 24 48 and 73 h, a slow reduction of donor class II cells was observed and some areas in cortex showed only very few, if any, donor cells. At the same time, starting from day 2 after transplantation accumulation of donor cells was found in perivascular spaces. Spleen sections stained at 24, 48, 72 and 96 h after transplantation revealed donor cells present in recipient’s spleen. They were detected up to day 3 after surgery. Their numbers, however, decreased after day 2. After 2 and 3 days, accumulations of recipient’s cells between tubules were detected. It was found that many cells in infiltrations were stained with anti-T lymphocyte MoAb. Expression of class II antigen on rat kidney cells increases significantly from the day 4 after transplantation.

Keywords: kidney transplantation; immunomorphology; monoclonal antibodies; cell migration; experimental model. model.

Vol. 43, No. 5-6, 1995

CONTENTS

Cellular Immunology

  • Participation of IL-2 and Direct Cell Cytotoxicity in the Modulation of GvH Reaction in Mice Infected with Listeria innocua
    TERESA GOŚCICKA, MARIA WALENCKA, JACEK SZELIGA, MONIKA BLOCH and BOŻENA DZIADEK

    Abstract. Listeria innocua can intensify the development of local GvH reaction in a semiallogeneic system in mice. This phenomenon is observed in (BALB/c x AKR)F1 mice intraperitoneally injected with the live bac- teria on the 7th day before the transfer of BALB/c spleen cells. The local GvH reaction develops as strongly as in normal hybrid recipients given the parental cell graft in the presence of exogenous interleukin 2 (IL-2). The development of the reaction is associated with an intensive increase of the direct cytotoxicity of lymph node cells which is also markedly enhanced after the bacteria injection itself IL-2 seems to play a relevant role in the development of local GvH reaction in L. innocua-injected mice.

    Keywords: GvH reaction; bacterial immunomodulation; Listeria.steria.

  • Characterization of Cellular Components in the Peritoneal Fluid and in the Endometrial Tissue of Women with Endometriosis
    KRZYSZTOF KAMIŃSKI, JAN KOTARSKI and MAREK GOGACZ

    Abstract. The presence of leukocyte subpopulations was demonstrated in peritoneal fluid and in endometrial tissue of women with (n =16) and without (n =20) endometriosis. Peritoneal fluid samples were also assayed for effects on lymphocyte proliferalion in vitro. Peritoneal fluid leukocyte profiles were observed to be different between these groups. The most significant elevations in total number of leukocytes, macrophages and lym- phocytes and natural killer (NK) cells were observed in women with endometriosis. In normal eutopic en- dometrium T lymphocytes (CD3 ), macrophages (Ki-M 1 ) and NK cells (CD 16- CD56+) were present. In contrast aggregates of NK cells (CDl6+ CD56+) were only identified in ectopic endometrial tissue. Mito- gen-induced lymphocyte proliferation was significantly higher in the presence of peritoneal fluid from patients with severe stage of endometriosis as compared with other samples.0ur data indicate that disturbances of the cellular immune system may lead to progression of endometriosis in female peritoneal cavity.

    Keywords: endometriosis; leukocyte subpopulations; peritoneal fluid; endometrial tissue.tissue.

  • Presentation of Antigen by B Cell Subsets. V. Effect of Interleukin 7 (IL-7) and Interleukin 10 (IL-10) on Phenotype and Antigen Presenting Function of Immature B Cells
    MICHAŁ ZIMECKI and JUDITH A. KAPP

    Abstract. In this communication we demonstrate that B cells from CBA/N mice with x chromosome-linked immunodeficiency and B cells from newborn mice, that have very limited ability to present antigen to an- tigen-specific T cell lines, acquire this function following preincubation with IL-7 or IL-10. These interleukins do not affect the antigen presenting function of B cells from normal, adult CBA mice. Incubation of B cells from xid and newborn mice with IL-7 or IL-10 but not with IL-2 induces expression of Lyb-5 marker on these cells. The study shows that the property of IL-7 and IL-10 to induce antigen presenting cell (APC) activity in immature B cells is associated with appearance of Lyb-5 antigen on these cells.

    Keywords: antigen presentation; B cells; newborn mice; xid mice; interleukin 7; interleukin 10; Lyb-5 antigen.ntigen.

  • CD4 + T Cells from Mice Primed for Humoral Immunity to Sheep Erythrocytes Specifically Inhibit Cell-Mediated Immunity and Vice versa
    MICHAŁ ZIMECKI, JUDITH A. KAPP and ZBIGNIEW WIECZOREK

    Abstract. The aim of this study was to demonstrate whether development of the humoral or cellular immunity to sheep erythrocytes (SRBC) can be inhibited by a transfer of antigen-specific CD4+ T cells isolated from mice exhibiting cellular or humoral response. For generation of the humoral immune response and suppressor T cells for the cellular immunity, mice were immunized with a high dose of SRBC intraperitoneally (i.p.). On the other hand, for development of the cellular response and generation of T cells with inhibitory activity in relation to the humoral response, mice were immunized subcutaneously (s.c.) with SRBC emulsified with complete Freund’s adjuvant or intravenously (i.v.) with low dose of SRBC (105). We showed that depletion of CD4+ but not CD8+ T cells in the populations of transferred T cells abolished their inhibitory activity. The phenomenon of this suppression was dependent on a dose of T cells transferred and was antigen-specific, since T cells specific for chicken erythrocytes did not inhibit anti SRBC-response. The data indicate the importance of antigen specific CD4+ T cells in the mutual regulation of the two major type of the immune response.

    Keywords: humoral response; cellular response; sheep red blood cells; CD4+; CD8+; T lymphocytes; sup- pression.ession.

Experimental Oncology

  • Antitumor Activity of Bestatin and Thiorphan in Mice
    JAN KOWALSKI, DARIUSZ BELOWSKI, ANDRZEJ MADEJ and ZBIGNIEW S. HERMAN

    Abstract. We have investigated the effect of bestatin and thiorphan on growth of murine transplantable tumors, survival time, activity of natural killer (NK) cells and macrophages in mice. The injections of thior- phan at the doses of 0.5 or 5 µg per mouse retarded tumor growth and prolonged survival period in B16 melanoma bearing animals. Pretreatment with naloxone (an unspecific antagonist of opioid receptors) blocked the tumor growth inhibition induced by the treatment with bestatin and thiorphan what could suggest a contribution of endogenous enkephalin in this antitumor effect. The. percentage of mice bearing B 16 melano- ma tumor in the group treated with thiorphan at doses of 0.5, 5 or 50 µg per mouse was lower in comparison with control animals in a dose-dependent manner. The treatment with thiorphan at concentrations of 0.4-1.6 mg/ml inhibited growth of cultured in vitro B16 melanoma cells in comparison with control culture. Thiorphan added to the medium at concentration of 0.5 µg/ml or administered 4 times at the dose of 0.5 µg/mouse augmented NK lymphocyte activity.

    Keywords: thiorphan; bestatin; tumor; natural killer cell activity; macrophages.phages.

  • Peanut Agglutinin (PNA) Binding Glycoproteins on Human Urothelial Cell Lines of Different Grades of Transformation
    DANUTA DUŚ, MACIEJ UGORSKI, WOJCIECH GORCZYCA and CZESŁAW RADZIKOWSKI

    Abstract. PNA-reactive sites, representing mainly unmasked Thomsen-Friedenreich (TF) antigen, are predic- tors of invasive transitional cell carcinoma. The present studies were undertaken in order: 1) to quantify the expression of PNA-reactive sites on well-characterized human urinary bladder cell lines belonging to two different transformation grades (TGr II and TGr III); 2) to identify PNA-binding glycoproteins that are restricted in their expression to tumorigenic and invasive urothelial cell lines. Flow cytomery studies revealed significant differences between TGr II and TGr III cell lines. The mean fluorescence intensities of TGr III, tumorigenic and invasive cells, were in the range of 28.4 to 57.3 arbitrary units. The TGr II cells exhibited several fold lower fluorescence intensity: 9.8 arbitrary units for HCV 29 cell line and 13.1 arbitrary units for Hu 609 cells. Neuraminidase treatment, increasing PNA-binding in TGr II as well as in TGr III cell lines, revealed the presence of cryptic PNA-binding sites. The number of cryptic PNA-binding sites seemed to be similar in TGr II and in TGr III cells and, therefore, only add to the total number of PNA-binding sites on native, untreated cells. Binding of 125 I-PNA to all cellular proteins resolved by SDS-PAGE and transferred to nitrocellulose showed multiple bands. The TGr III cell lines, after desialylation, were characterized by the presence of two major PNA-binding components represented by diffuse bands with apparent molecular mass about 68 – 79 k Da and 116 -156 k Da, respectively, and two weakly stained bands with apparent molecular mass of 51 kDa and 60 kDa. Both cell lines representing TGr II expressed high molecular mass PNA-binding component of 207 kDa. They were further characterized by the weaker staining intensity of 116-156 kDa glycoproteins as compared to TGr III cell lines.

    Keywords: transitional cell carcinoma; transformation grade; Thomsen-Friedenreich antigen; peanut ag- glutinin binding; membrane glycoproteins.oteins.

  • Growth Inhibition of Transplantable Tumors in Mice by mIL-2-Secreting Murine Plasmocytoma Cells Used Alone or in Combination with a Cytostatic Agent
    ELŻBIETA PAJTASZ-PIASECKA, HALINA KUŚNIERCZYK, JAN SALWA, LESZEK KONARSKI and CZESŁAW RADZIKOWSKI

    Abstract. The non-tumorigenic cells of X63-Ag8.653 mouse plasmocytoma line tfansfected with murine inter- leukin 2 cDNA (X63-mIL-2) served us as the source of the cytokine to induce or to augment antitumor response in syngeneic BALB/c or semisyngeneic (CD2F1) mice challenged subcutaneously with either „wild” line tumor cells (X63/0) or with non-related methylcholantrene induced BFS1 fibrosarcoma of BALB/c mice. When applied peritumorally in several injections (2 to 6) to mice with non-advanced stages of the tumors, IL-2-secreting cells were able to cause tumor growth retardation in most of the treated mice and to induce tumor rejection in some of them. The combination chemoimmunotherapy was attempted in mice with advan- ced BFS1 fibrosarcoma using compound CBM-4A (the bromoanalog of ifosfamide) administered at various time (4 h or 3, 5 or 7 days) before the first of two local injections of transfected cells. The strategy proved to be more effcient in the tumor growth inhibition as compared with the cytostatic alone.

    Keywords: murine tumors; murine interleukin 2 secreting cells; tumor vaccine; antitumor response; chemoim- munotherapy.herapy.

Immunopharmacology

  • Influence of a Rat Kidney Perfusion Using the Anti-MHC Class II Monoclonal Antibody and Reduced Dose of 15-Deoxyspergualine on the Allogeneic Graft Survival
    ANDRZEJ OKO, MACIEJ KRZYMAIŃSKI, ADAM DEJA, KARIN ULRICHS, ANA MARIA WAAGA and WOLFGANG MULLER-RUCHHOLTZ

    Abstract. One of the main factors inducing rejection of the allogenic graft are the donor MHC-class II antigens. In this study, the allogenic rat renal graft survival after the blockade of MHC-class II positive cells was analyzed and compared with the effectiveness of the recipient treatment with 15-deoxyspergualine (IS-DOS). It was found that the DA (RT1 a) rat kidney perfusion with the anti-MHC class II monoclonal antibody (MoAb 29A1 – Kiel) allowed to prolong survival of the graft in the LEW (RT1 1) recipient (9.6±0.8 vs. 7.7±0.5 days). Our another study demonstrated that the 14 day treatment of the LEW recipient with 15-DOS at the dose of 0.5 mg/kg body weight can induce tolerance to the grafted kidney from the DA strain. The dose of 02 mg/kg body weight of 15-DOS prolonged the graft survival only to a small extend (16.5±0.5 days). In contrast, the combination of the graft pretreatment with MoAb 29A1 with the application of the reduced dose of 15-DOS to the LEW recipient allowed to further prolong the graft surwival (97.4±59.0 days, n = 5). In 3 cases, the long-time (close to 150 days) graft survival was obtained. The above presented results suggest that the blockade of the MHC-class II antigens can reduce the immunogenicity of the graft. Although this procedure was not sufficient to induce tolerance, it allowed to minimize the immunosuppressive treatment.

    Keywords: experimental transplantation; organ perfusion; immunosuppressive treatment; monoclonal an- tibody; graft survival.rvival.

  • Effect of Benzisoselenazolones and Organic Diselenides on Graft Versus Host Reaction and Immunoglobulins Levels in Chickens
    BARBARA BLASZCZYK, ANNA D. INGNOT, PAAVO TOIVANEN, JACEK MŁOCHOWSKI and STANISŁAW SZYMANIEC

    Abstract. Two week old chickens were treated once daily for 5 days with AE8 – I-pyridyl-1,2-benzisoselena- zol-3(2H)-one, AE22 – bis-2-(N-phenyle-carboxamido) 1 pyridyl diselenide and AE31 – bis(phenylo-diseleni- de with R3 = CONHC18H37). Their whole blood alone or blood mixed with thymus cells were used to generate graft versus host (GvH) reaction in 15 day old chicken embryos. The treatment of the chickens with the compounds stimulated the GvH reaction modifying activity of the donor cells as measured by increase of the spleen weight of the recipient chicken embryos. On the other hand, treatment with these compounds inhibited the IgG or IgM production in chickens immunized with human albumin.

    Keywords: seleno-organic compounds; graft versus host reactions in chickens; immunoglobulins; enzy- me-linked-immunosorbent-assay.-assay.

  • Immunotropic Activities of Benzisoselenazolones and Organic Diselenides in Mice
    BARBARA BŁASZCZYK, ANNA D. INGLOT, STEFANIA H. KOWALCZYK-BRONISZ, STANISLAW SZYMANIEC and JACEK MŁOCHOWSKI

    Abstract. We have investigated the immunotropic effects of 23 seleno-organic compounds (8 benzisoselenazo- lones, 3 benzisoselenazolone oxides and 12 organic diselenides). All of the compounds increased the rosette formation of sheep red blood cells (SRBC) with spleen cells obtained from thymectomized CS3BL/6 mice and incubated in vitro in the presence of imuran. Furthermore,16 of the compounds were also assayed in vitro in the hydrocortisone test performed with CS7BL/6 mouse thymocytes. It was found that all of them significantly protected the cells against hydrocortisone induced cytotoxicity. Also in the Jerne’s assay, performed in 129Ao/ Boy mice pretreated in vivo with 3 selected compounds 5 days before immunization with SRBC, the stimula- tion of plaque forming cells (PFC) was observed. Only one compound (AE22, an analog of piroxicam) was found to be inhibitory in this assay. In contrast, in the graft versus host (GvH) assay performed in hybrid mice the donor lymphoid cells obtained from CS7BL/6 mice pretreated with 9 selected seleno-organic compounds, suppressed the GvH reaction in the recipient hybrid mice. Thus, in all of the immunotropic assays except the GvH reaction in adult mice, the seleno-organic compounds were found to have immunostimulating activities.

    Keywords: seleno-organic compounds; hydrocortisone toxicity for thymocytes; rosette forming cells; plaque forming cells; graft versus host reaction in mice; immunostimulants.ulants.

  • Oral Heparin in the Treatment of Rheumatoid Arthritis
    JACEK IMIELA, JERZY NOSARZEWSKI and ANDRZEJ GÓRSKI

    Abstract. Heparin was administered orally in 10 patients with rheumatoid arthritis. The treatment resulted in clinical improvement in all cases (significant reduction in the number of tender and swollen joints and morning stiffness). This was associated with a decrease in erythrocyte sedimentation rate (8/10 patients) and cholesterol in all patients, although the differences were not significant. Recent data indicate that heparin can bind cytokines with potent immune modulatory action (e.g. TNF-a, interferon y etc.). Thus, neutralization of their action on target cells could be at partially responsible for the beneficial effects reported. Moreover, heparin inhibits T cell traffic to a site of antigen by blocking enzymes digesting the extracellular matrix and interfering with selection activity. The results of our preliminary study suggest that heparin, given per os, may have immunomodulatory properties with potential application in human disease.

    Keywords: heparin; rheumatoid arthritis.hritis.

  • 2-Chlorodeoxyadenosine (2-CdA) in the Treatment of Patients with Relapsed Chronic Lymphocytic Leukemia
    EUZEBIUSZ KRYKOWSKI, KRZYSZTOF WARZOCHA and TADEUSZ ROBAK

    Abstract. This study attempts to characterize the response of patients with chronic lymphocytic leukemia (CLL) to the purine analog 2-chlorodeoxyadenosine (2-CdA). We have treated 10 patients with 2-CdA, at a dose 0.05 – 0.1 mg/kg/daily, for 7 days as a 2-hour infusion. Mean age was 54.6 years (range, 34 – 68 years). Mean time from diagnosis to treatment with 2-CdA was 37.0 months (range, 8 – 84 months). All the studied patients had received preliminary therapy consisting of other than 2-CdA chemotherapeutic regimens. Eight out of 10 patients had Rai stage III – IV disease. Four patients had Coombs positive hemolytic anemia before 2-CdA treatment. Seven patients responded to 2-CdA. Two complete remission (CR) and 5 partial remission (PR) were achieved. All patients but one with Coombs positive autoimmune positive hemolytic anemia achieved complete resolution of hemolysis. Severe neutropenia was frequent, and serious infections were noted in 20%, 43% and 50% of cases during the first, second and third course of 2-CdA, respectively. We conclude that 2-CdA is an effective agent in relapsed CLL patients, particularly in cases complicated by autoimmune hemolytic anemia.

    Keywords: 2-chlorodeoxyadenosine; chronic lymphocytic leukemia; treatment.atment.

  • Effect of Repeated Treatments with Cladribine (2-Chlorodeoxyadenosine) on Blood Counts in Multiple Sclerosis Patients
    PAWEŁ GRIEB, ZBIGNIEW STELMASIAK, JANUSZ SOLSKI, JACEK NOWICKI, BEATA JAKUBOWSKA and MIROSŁAW RYBA

    Abstract. We report the results of blood morphology monitoring of 11 remitting-relapsing multiple sclerosis patients who received repeated treatments with cladribine (2-chlorodeoxyadenosine). The drug was given once, daily, subcutaneously (5 mg) or orally (10 mg) for 5 consecutive days, as 6 monthly courses followed by one or two additional courses at 3 or 6 month intervals. The treatments were well tolerated, although many patients suffered from incidental upper respiratory tract infections, most of which occured during the last 6 months of the observation period. One patient had recurrent infections, including an episode of urosepsis. All infections responded to standard therapy with antibiotics. Progressive lymphocyte reduction to 1000/µl on average, and clear but clinically insignificant drop in thrombocytes, was observed. Granulocyte counts were sometimes markedly elevated. A few patients developed macrocytosis, but none required transfusion. With our dosing and schedule, cladribine seems relatively safe in multiple sclerosis patients.

    Keywords: 2-chlorodeoxyadenosine; blood counts; toxicity.xicity.

  • 2-Chlorodeoxyadenosine: Lack of Synergism with Cyclosporine A and Tacrolimus (FK 506)
    MARIA NOWACZYK, BARBARA STĘPIEŃ-SOPNIEWSKA, GRAŻYNA KORCZAK-KOWALSKA, TOMASZ MROWIEC and ANDRZEJ GÓRSKI

    Abstract. New clinically useful drug, 2-chlorodeoxyadenosine (2-CdA), was found to be a potent immunosupp- ressant both in vitro and in vivo. The present study examines the in vitro interactions of classical immunosupp- ressive agents – FK 506 and Cyclosporine A (CsA) with 2-CdA at the level of T and B cells proliferation, expression receptor for interleukin 2 (R-IL-2) and Ig synthesis. No synergism between 2-CdA and either FK 506 or CsA was demonstrable.

    Keywords: 2-chlorodeoxyadenosine; cyclosporine A; tacrolimus; proliferation; receptor for IL-2; Ig synthesis.thesis.

  • Continuous Versus Split-Course Irradiation for Lung Cancer. Immunological Implications
    LJILJANA VUĆKOVIĆ-DEKIĆ, NEVENKA STANOJEVIĆ-BAKIĆ, MIODRAG DEKIĆ and OLIVERA FRIM

Abstract. The therapeutical irradiation for lung cancer causes profound disturbances of host’s general im- munocompetence, the cellular immunodepression being the dominant finding. It is thought that split-course technique holds certain advantage over the continuous irradiation, since the former includes an interruption of 4 week duration, thus allowing the lymphopoietic system to recover to a certain degree. In this report, we compared the radiotherapy-due alterations of several parameters of cellular immunity (the number and func- tion of total T cells, active T cells and the cells of monocyte/macrophage lineage), immediately after the completion of therapy in either continuously (n =13) or split-course-irradiated (n =12) lung cancer patients. All patients had received the total dose of 60 Gy. Both therapeutical techniques caused alterations of the parameters tested: the significant decrease of the total and active T cells and their proliferative responses, while the phagocytic activity and the number of mononuclear phagocytes were increased, the latter being affected to a lesser extent in split-course-treated patients. Our results suggest that both techniques have similar im- munodepressant effect on the cellular immunity of lung cancer patients.

Keywords: cellular immunity; iatrogenic immunosuppression; radiotherapy; lung cancer.cancer.