CONTENTS
Cellular Immunology
- Effect of Interferon a on HLA-DR Expression by Human Buccal Epithelial Cells
J. KELLY SMITH, DAVID S. CHI, GUHA KRISHNASWAMY, SUJATA SRIKANTH, SCOTT REYNOLDS and STEVEN L. BERKAbstract. We have studied the effect of interferon a (IFN-a) on MHC class II expression by human buccal epithelial cells (BEC), and mRNA expression by BEC and mucosal-associated mononuclear cells (MAMC). In 6 experiments, freshly collected BEC were suspended at a concentration of 1.0 x 105/ml in RPMI 1640 and incubated in the presence of 0-10000 IU/ml of human lymphoblastoid IFN-a (HuIFN-a ). Zero and six hour samples were analyzed by single color flow cytometry using FITC-labeled murine IgG1 monoclonal antibody to HLA-DR. Preparations were also analyzed for expression of cytokine transcripts (IL-2, IL-4, IL-5, IL-6, IL-8, IFN-g , GM-CSF) by reverse transcriptase polymerase chain reaction (RT-PCR). Increasing concen- trations of IFN-a resulted in proportionate increases in the percentage of HLA-DR+BEC (r = 0.7897, p = 0.0627) and in the percentage of HLA-DR+staining at higher intensities (101 to 10z log fluorescence intensity) (LFI) (r = 0.4010, p = 0.0424). The percentage of HLA-DR + BEC rose from a mean of 1.5 % with no IFN-a to 7 % with 10 000 IU/ml IFN-a (p < 0.05). The percentage of HLA-DR + BEC staining at 101 to 10z LFI rose from a mean of 8.3o/o with no added IFN-a to 19.2% with 10000 IU/ml IFN-a (p < 0.05). Unstimulated BEC constitutively expressed IL-8 and GM-CSF. IFN-a stimulated preparations also expressed IFN-g , possibly due to the presence of MAMC, which comprised 2-9% of the total cell population. These data indicated that HuIFN-a upregulates MHC class II expression by human BEC, possibly by enhancing IFN-g production by MAMC present in the culture preparations.
Keywords: human lymphoblastoid interferon; human buccal epithelial cells; MHC class II antigens; cytokine mRNA transcripts; mucosal-associated mononuclear cells.
- Analysis of Tolerance Inducing Mechanism after Application of Oncogen-Transformed Macrophages
ANA MARIA WAAGA, JAN-MICHAEL OTTE, MACIEJ KRZYMAŃSKI, VOLKER ECKSTEIN and WOLFGANG MÜLLER-RUCHHOLTZAbstract. The purpose of this study was to examine the expression of T cell receptors (TCR) and their Vß subclasses under the influence of the parental cell line P388D1 and its clones mos2 and mos3, using a mouse model. It was shown, that v-mos oncogene-transformed cells of this line (mos2) induced selective immunological unresponsiveness in vitro. Because the induction of tolerance is of a central importance for the organ transplantation, this phenomenon, found in vitro, was also studied in vivo. We found that the in vivo injection of mos2 cells into mice induced a state of selective noncreactivity. To further analyse these effects, we studied whether specific tolerance is the consequence of a decreased number of essential receptors or receptor families. For this purpose C57BL/6 mice were immunized with cells of the parental line P388D1 or mos2 and mos3 clones. Their spleen and thymus cells were examined phenotypically. The most impressive result of this study was a clearly changed amount of T cells receptors in mos2 immunized mice, in which a state of tolerance was induced. In these mice only the expression of CD3 T receptors as well as that of the Vß11 chains was reduced. In spleen of these mice the CD3 expression was decreased, compared to D1 or nonimmunized control animals by 54-58% and compared to mos3 mice by 38-40%. Even though the differences in the thymus were not very prononced, we still saw a decrease in CD3 stained cells selective in mos2 immunized C57B1/6. The expression of Vß11 chains on the surface of spleen cells of mos2 animals was reduced by 33.3%, on the thymocytes even by 50% comparying to that in nonimmunized mice. Whether the reduced expression of T receptor Vß families is due to changes in the genetic material (cDNA), has to be studied.
Keywords: tolerance induction; oncogene-transformed macrophages; polymerase chain reaction; T cell receptor; P388D1 (D1) transformed with v-mos oncogene; clones mos2 and mos3.mos2 and mos3.
- Tumor Necrosis Factor and Interferon Production by Peripheral Blood Leukocytes of Patients with Alcoholic Cirrhosis
JADWIGA DANILUK, MARTYNA KANDEFER-SZERSZEŃ and LILIANA BOROWSKAAbstract. Sixteen male and female patients with alcoholic cirrhosis and 13 healthy controls were included in the study. The level of interferon (IFN) and tumor necrosis factor (TNF) activity was examined in the sera and also in cultures in peripheral blood leukocytes (PBL) after in vitro stimulation with the cytokine inducers. In sera of patients with alcoholic cirrhosis higher IFN and TNF levels were detected than in controls. However, after induction with Newcastle disease virus (NDV), phytohemagglutinin M (PHA) and lipopolysaccharide from E. coli (LPS), PBL from cirrhotic patients produced lower IFN levels in comparison to controls. In contrast to depressed ability to produce IFNs, TNF production was higher in PBL of cirrhotic patients induced by PHA and a low dose of LPS (1 m g/ml). NDV induced comparable levels of TNF in both groups. It appears likely that cells of cirrhotic patients were suppressed by an unknown factor or were hyporeactive for IFN production, but synthesis and release of TNF was enhanced, suggesting that cells producing TNF were preactivated in vivo. Mechanism of such preactivation is discussed.
Keywords: alcoholic cirrhosis; interferon; tumor necrosis factor.
- Unexpected Reaction of Anti-H-2d Serum with Thymic Lymphoma Cells Derived from Transgenic B6-H-2b TCR anti-HY/Db Mice
JANUSZ MATUSZYK, EWA ZIOŁO, MARCIN KOBZDEJ and LEON STRZĄDAŁAAbstract. Transgenic mice with a ß TCR specific for male antigen (HY) in the context of H-2Db MHC class I were prepared by introduction of transgens into (CS7BL/6J x DBA/2J)F1 hybrid mice (H-2b/d), where only H-2b is selective for maturating thymocytes. Founder transgenic mouse was backcrossed to H-2b MHC background. Serological typing of H-2 antigens revealed that transgenic mice do not express H-2d antigens. Unexpectedly, cells of thymic lymphomas, spontaneously developed in about 45% of old B6 (H-2b) transgenic mice and peripheral blood lymphocytes of about 40% of transgenic mice, reacted with anti-H-2d serum (strain 129 mice (H-2b) immunized with thymocytes and splenocytes of BALB/c mice) but not with monoclonal antibodies against H-2d MHC class I antigens. Anti-H-2d serum has been shown to react with thymocytes but not peripheral blood lymphocytes from non-transgenic H-2b mice. The lymphocytes of transgenic mice reacting with anti-H-2d serum could represent disseminating preneoplastic or neoplastic cells expressing antigen encoded outside MHC region and present on the cell surface of immature thymocytes but not lymphocytes in healthy mice.
Keywords: TCR anti-HY/Db transgenic mice; thymocytes; thymic lymphomas.
- Early Supernatants of Mitogen-Stimulated Cells Promote Long-Term IL-2-Dependent of Human T Lymphocytes
JAN KONRAD SIWICKI and JAN A. STEFFENAbstract. Human wheat germ agglutinin (WGA)- or PHA-activated T lymphocytes from either peripheral blood or normal spleens can be propagated in vitro for several months in the presence of IL-2 but not IL-4 when periodically restimulated with supernatants collected from 24 h cultures of mitogen-stimulated human blood or spleen mononuclear cells. Following several rounds of stimulation with these supernatans some of the spleen-derived T lymphoblast lines acquire the ability of continuous growth (over 100 population doublings) in the presence of IL-2 alone. We postulate that the ability of 24 h supernatants from mitogen stimulated mononuclear cells to maintain T lymphoblast IL-2-dependent growth depends upon yet undefined cytokine(s) released early after activation.
Keywords: wheat germ agglutinin; T cell; long-term growth.growth.
Experimental Oncology
- Interferon Production by Peripheral Blood Leukocytes of Patients with Acute Myeloid Leukemia
MARTYNA KANDEFER-SZERSZEŃ, ANNA DMOSZYŃSKA, LILIANA BOROWSKA, MARIA ADAMCZYK-CIOCH, MAREK HUS, IWONA HUS and JACEK ROLIŃSKIAbstract. The ability of peripheral blood leukocytes (PBL) of 13 selected patients with acute myeloid leukemia (AML), and of 10 healthy subjects to produce interferon (IFN) spontaneously and after in vitro induction was tested. Spontaneous IFN production was detected in supernatants of PBL cultures of healthy subjects but was not present in cultures of leukemic cells (leukocytes density 1 x 106 ml). After induction with Newcastle disease virus (NDV) PBL cultures from 5 patients with AML exhibited a low IFN response, while in others IFN titers were similar to controls. The IFN titers induced with lipopolysaccharide (LPS) were lower in leukemic cells than those detected in control cells. The absence or low IFN levels after induction with phytohemagglutinin (PHA) correlated with very small numbers of normal lymphocytes in blood of leukemic patients.
Keywords: acute myeloid leukemia; interferon production.on production.
- Serum Soluble Interleukin 2 Receptor a and Soluble CD8 Levels in Patients with Gynecological Undergoing Radiotherapy
IRENA FRYDECKA, MAŁGORZATA RUSIECKA, KAZIMIERZ KULICZKOWSKI and JAN KORNAFELAbstract. The phenotype peripheral blood lymphocytes, soluble interleukin 2 receptor a (sIL-2Ra) and soluble CD8 (sCD8) were studied in 30 patients with gynecological malignancies before and after ratiotherapy. The absolute count of CD3+, CD4+, CD8+, CDl9+ and CD2S+ lymphocytes before treatment was within normal range. The levels of serum sIL-2Ra and sCD8 were significantly increased before treatment compared to normal controls. After treatment the absolute number of CD3+, CD4+ and CDl9+ lymphocyte subsets significantly decreased. The levels of sIL-2a and sCD8 corrected according to absolute lymphocyte count reached normal range. The decrease of tumor mass correlated with the serum levels of sIL-2Ra and sCD8. Further studies are needed to evaluate the usefulness of these tests in the prognosis and treatment of these diseases. Our study suggests that sIL-2Ra and sCD8 in our patients probably has not been originating from the proteolytic cleavage of membrane bound receptors from peripheral blood mononuclear cells (PBMC). It can be specutaled that sIL-2Ra and sCD8 originated in these patients from activated tumor-infiltrating lymphocytes or may arise from separate alternatively spliced mRNAs coding for soluble vs. membrane forms.
Keywords: soluble interleukin 2 receptor a; soluble CD8; gynecological malignancies.
- Impaired CD2S Expression and Soluble CD2S-IL-2Ra Receptor Release by Anti-CD3 Stimulated Peripheral Blood Mononuclear Cells in Hodgkin’s Disease Patients
IRENA FRYDECKA and GRZEGORZ MAZURAbstract. We examined 22 patients in active phase of Hodgkin’s disease (HD),12 patients in complete clinical remission and 16 healthy subjects for their responsiveness to anti-CD3 stimulation as measured by CD2S antigen expression on peripheral blood mononuclear cells (PBMC) and production of CD2S-IL-2Ra . Simultaneously, the serum levels of soluble interleukin 2 receptor a (sIL-2Ra ) were estimated.0ur studies indicate that PBMC in patients in active phase and in clinical remission have impaired ability to express CD2S antigen after stimulation with anti-CD3 monoclonal antibody. Similarly, the levels of sIL-2Ra in culture supernatants were significantly lower in both groups of patients: in active phase and clinical remission compared with the controls. In contrast, the mean serum level of sIL-2Ra was significantly higher in active phase of the disease compared to that found in patients in clinical remission and controls.0ur studies suggest that sIL-2Ra , derived from PBMC, is not the source of increased levels of sIL-2Ra found in the sera of patients in active phase of HD. Their synthesis and secretion/shedding most probably takes place in the involved tissues.
Keywords: soluble interleukin 2 receptor a ; anti-CD3; Hodgkin’s disease.
- Mouse Multivalent IgG(2a, b) Antibody – Ricin A-Chain Immunotoxins Combined with Homologous or Heterologous Interleukin 2 in the Treatment of a Murine Malignant Lymphoproliferation (EL4)
GABRIELA MOTA, RADU MARCHES, VALENTIN CIALACU, VIVIANA ROMAN, ECATERINA KOZMA, MARINELA MĂRGINEANU, SILVIA DIMA and ION I. MORARUAbstract. Two immunotoxins containing ricin A-chain, staphylococcal protein A and mouse anti-Thy 1.2 antibodies of IgG(2a,b) subclass, were prepared. The two multivalent immunotoxins of 750 and 370 kDa and molar ratio A-chain: IgG of 1:2, were used for the treatment of mice bearing ascitic EL4 lymphoma. The immunotoxin-treatment, performed intraperitoneally, was combined or not with homologous or heterologous interleukin 2. The antitumor effects expressed by increase of mice survival time (p < 0.001 as compared with nontreated animals) corresponded to a proportion of 88-90% lymphoma cell-kill by immunotoxin and 93-95a/o by combination treatment (immunotoxin+interleukin 2), as calculated from the relationship between the survival time of nontreated mice and the number of tumor cells inoculated.
Keywords: immunotoxins; ricin A-chain; interleukin 2; experimental tumors.mental tumors.
Immunopharmacology
- Antiviral Activity of 2-Chloro-2′ -Deoxyadenosine (2-CdA)
RENATA ZAHORSKA, NATALIA MAZUR, JANUSZ S. SKIERSKI and PAWEŁ GRIEBAbstract. The effect of 2-chloro-2′-deoxyadenosine (cladribine,2-CdA) on replication of herpes simplex type 1(HSV) virus in Vero cells (green african monkey kidney line) was evaluated and compared with that of acyclovir.2-CdA up to 600m g/ml was not cytotoxic. ED50 for 2-CdA was approximately 5 m g/ml and ED 1 oo was approximately 70 tlg/ml, compared with 0.16m g/ml and 1.6 gg/ml for acyclovir, respectively. DNA cell cytometry suggested the progressive S-phase block at concentrations of 2-CdA 0.14m g/ml and higher. The results suggest that 2-CdA is not activated by HSV-encoded kinase, and a weak antiviral effect of the drug results from its interference with DNA replication following activation by kinases of host cells.
Keywords: 2-chloro-2′-deoxyadenosine; cladribine; herpes simplex virus; cell cycle; DNA content; Vero cells.
- Induction of Specific Tolerance by 15-Deoxyspergualin (DOS) Treatment after Rat Skin and Kidney Transplantation in Rats. Analysis of Effectivity of Various Protocols of DOS Application
ANA MARIA WAAGA, MACIEJ KRZYMAŃSKI, KARIN ULRICHS, ANDRZEJ OKO and WOLFGANG MÜLLER-RUCHHOLTZAbstract. The purpose of this study was to analyse the effect of various protocols of 15-deoxyspergualin (DOS) application on skin or kidney graft survival. Following rat strain combinations were used: AS ® LEW (MHC identical/non-MHC-different) and DA ® LEW (MHC-different/non-MHC-different). Reference DOS dose was 2.5 mg/kg, i.p. It was shown that the effect of DOS depended on multiple factors, such as: type of tissue or organ, onset of treatment, drug dose and length of drug application. In skin transplantation graft survival was 32-34 days in AS ® LEW and 24-26 days in DA ® LEW. Kidney graft survived more than 150 days. DOS prolonged skin survival when the application was started earlier than day 8, whereas kidney graft survived only when DOS treatment was started not later than 3-4 days after transplantation. In skin transplantation a dose of 0.3 mg/kg had a small effect – prolongation graft survival up to 4 days. Higher doses induced longer graft survival, however, maximal survival of allogeneic skin was 22 days. In kidney transplantation a dose of 0.3 mg/kg led to prolonged graft survival – up to 150 days. Doses of 2.0-2.5 mg/kg were able to induce specific tolerance. The optimal skin or kidney graft survival was obtained when DOS was applied for 14 days. Shorter than 12-day treatment with DOS led to a shorter graft survival. When donor was pretreated with DOS prolongation of non-allogeneic graft survival was observed. Our results showed that short-term application of DOS is safe and effective. To obtain optimal DOS effect the drug application must be started directly after transplantation.
Keywords: 15-deoxyspergualin; experimental transplantation; rat kidney transplantation; skin transplantation.
- In vitro Analysis of the Mode of Action of the Immunosuppressive Drug 15-Deoxyspergualin
ANA MARIA WAAGA, MACIEJ KRZYMAŃSKI, KARIN ULRICHS, JÖRGEN TREUMER, ANDRZEJ OKO and WOLFGANG MÜLLER-RUCHHOLTZAbstract. The purpose of the study was to analyse the action of the immunosuppressive drug I 5-deoxysper- gualin (DOS) in vitro. We studied: a) the influence of DOS alone and DOS in combination with various monoclonal antibodies on alloantigen stimulation in the mixed lymphocyte culture (MLC), b) the influence of DOS treatment on the MHC class I and II expression of splenocytes, lymph node cells and peritoneal macrophages, c) the influence of DOS treatment on a suppressor cell population. Our study showed that: a) DOS inhibits interleukin 1 (IL-1) secretion by macrophages, leading to reduction of immune response to alloantigens. This effect was neutralized by addition of IL-l; b) DOS treatment has no influence on MHC class II antigen expression, but induces changes of MHC class I expression. After DOS application in a population of spleen macrophages a subpopulation of cells with reduced MHC class I antigen expression appeared. Down-regulation of these molecules was also observed in immunomorphological studies of kidney graft sections of rats treated with DOS after transplantation; c) after DOS treatment suppressor cells were detected in „suppressor” MLC,16-33 days after kidney transplantation. Their activity was confirmed 137 days after treatment with DOS, but were inactive in the case of third party cells. These results suggest that DOS action is based on a blockade of antigen presentation by reducing IL-1 production, down-regulation of MHC class I antigen and by inducing suppressor cell population.
Keywords: 15-deoxyspergualin; interleukin l; MHC antigens; in vitro studies; mixed lymphocyte culture; monoclonal antibodies; suppressor cells. cells.
- Monoclonal Antibody Analysis of Glomerular and Renal Interstitial Immune Cells in Steroid-Resistant and Steroid-Responsive Minimal Change Disease in Children
MAŁGORZATA WĄGROWSKA-DANILEWICZ and MARIAN DANILEWICZAbstract. Glomerular and interstitial leukocyte infiltrates were studied by indirect immunoperoxidaze technique using monoclonal antibodies against all leukocytes, pan-T lymphocytes, helper/inducer CD4 cells, suppressor/cytotoxic CD8 cells, pan-B lymphocytes, monocytes/macrophages and polymorphs in 23 renal biopsy samples obtained from children with minimal change disease (MCD). The cases were classified on the basis of light, electron microscopy and immunofluorescence. Nephrotic syndrome was steroid-sensitive in 15 patients (group I) and steroid-resistant in 8 subjects (group II). Both groups did not differ in the type or number of glomerular immune cells, quantified as immunopositive cells per glomerular cross section. Intraglomerular cellular infiltrates were mainly constituted of monocytes/macrophages; among T lymphocytes T helper/inducer cells predominated over T cytotoxic/suppressor cells. Enumeration of interstitial immune cells showed significantly higher number of all leukocytes and T lymphocytes, quantified as immunopositive cells/mm2, in steroid-resistant group. T helper/inducer cells predominated over T cytotoxic/suppressor cells in the renal interstitium in steroid-responsive and in steroid-resistant MCD. The leukocyte infiltrates did not correlate with a degree of proteinuria at the tine of biopsy and with the renal cortical interstitial volume in both studied groups. Our data suggest, that steroid resistance in minimal change nephropathy in children seems to be related to interstitial T lymphocytes.
Keywords: minimal change disease; monoclonal antibodies; immunohistochemistry; leukocyte subsets.ocyte subsets.
Various
- A New Lymphoblastoid Cell Line Defective in Class II HLA Expression
MARIA MAŃCZAK, JOANNA DUBIS, ANITA KOŚĆ, KATARZYNA BALDY-CHUDZIK, MICHAŁ MACHNICKI, RAFAŁ PACHOLCZYK, MAGDALENA PRUSSAK, BEATA NOWAKOWSKA and PIOTR KUŚNIERCZYKAbstract. A lymphoblastoid cell line, HAJ, was derived by in vitro transformation with Epstein-Barr virus of peripheral blood lymphocytes (PBL) from a patient with renal insufficiency awaiting kidney graft. Cell surface expression of class I and class II HLA molecules was determined by flow cytofluorimetry using monoclonal antibodies and compared with that of cell line PAJ similarly derived from a healthy donor. HAJ cells expressed class I antigens at levels comparable with PAJ cells. In contrast, class II antigens were absent from the cell surface of HAJ cells while they were abundant on PAJ cells. Permeabilization and fixation of cells with acetone/formaldehyde solution revealed intracellular Ki-67 antigen but not class II HLA molecules. The genes for HLA-DRß, DQa and DPa were present in the HAJ genome as detected with polymerase chain reaction (PCR) using locus-specific primers amplifying a second exon. In RT (reverse transcriptase) – PCR, transcripts of DQA1 and DPA1 genes were easily detectable in PAJ (positive control) but not in HAJ cells. These results suggest a defect in HAJ cells of transcription of genes for all class II antigens. The cell line HAJ may prove to be an interesting model for in vitro studies of molecular mechanisms of the regulation of class II expression.
Keywords: HLA class II genes/antigens; gene expression; lymphoblastoid cell line; polymerase chain reaction.
- Structure of the O-Specific Polysaccharide Chain and Serological Characterization of the Proteus penneri 62 Lipopolysaccharide Compared with the Lipopolysaccharides of the P. penneri Strains
ZYGMUNT SIDORCZYK, ANNA ŚWIERZKO, KRYSTYNA ZYCH NIKOLAY A. PARAMONOV, EUGENY V. VINOGRADOV, ALEKSANDER S. SHASHKOV and YURIY A. KNIRELAbstract. The chemical structure of the O-specific polysaccharide chain of Proteus penneri 62 lipopolysaccharide (LPS) containing N-acetylisomuramic acid was established using acid hydrolysis, solvolysis with anhydrous hydrogen fluoride and 1H and 13C NMR spectroscopy. Cross reactivity of the anti-O-serum P. penneri 62 with a number of other strains of the same species isolated in the USA, Canada, Germany and Poland is discussed.
Keywords: Proteus penneri; O-polysaccharide; lipopolysaccharide.haride.
- Natural Human Interferon a Given Orally Has Different Effects on Patients with Distinct Forms of Chronic Viral Hepatitis B
JERZY A. GEORGIADES, JERZY CABAN, TEOFILA ŻYRKOWSKA-BIEDA, MALGORZATA ZEJC, URSZULA JONAS-SKULINA and ANDRZEJ CIEŚLAAbstract. During therapy of chronic viral hepatitis B (CVHB), some patients treated with natural human interferon a (nHuIFN-a ) lozenges failed to respond. These observations triggered studies aimed to determine whether there are markers predicting patients’ response to therapy with nHuIFN-a lozenges. In these studies, 32 patients with CVHB were involved: 20 males and 12 females, 16-61 years of age with proven persistent hepatitis B viremia (HBV). Patients were evaluated for clinical, biochemical liver function, and virological markers of disease. During 300 days of treatment the patients received 75 -150 IU nHuIFN-a daily in form of lozenges. The responders to oral interferon therapy were those who had initially alanine amino transferase (ALAT) level higher than 100 IU (85.7% cure rate) and weak responses were observed among patients who had an initial ALAT level below 100 IU (9.0% response rate). Therefore, ALAT test in patients with CVHB may serve as a predicting indicator of the outcome of IFN lozenges therapy.
Keywords: natural interferon a ; oral administration; chronic viral hepatitis B; therapeutical efficacy.
- Influence of Uro-Vaxom on sIgA Level in Urine in Children with Recurrent Urinary Tract Infections
MIECZYSŁAWA CZERWIONKA-SZAFLARSKA and MAŁGORZATA PAWŁOWSKA
Abstract. We have examined the influence of Uro-Vaxom on secretory IgA (sIgA) level in urine of children with recurrent urinary tract infections. In the group of children treated with antibiotics and Uro-Vaxom, a significant increase of sIgA in the urine was found which persisted for at least 3 months. During this period no infection appeared in 92% examined children.
Keywords: Uro-Vaxom; recurrent urinary tract infection.ection.