Vol. 42, No. 1, 1994

Mini-Review

Discovery, Mechanism and Expected Clinical Significance of Selective Cytotoxicity of 2-Chloro-2′-Deoxyadenosine (2-CdA)

PAWEL GRIEB

Abstract: 2-Chloro-2′-deoxyadenosine (2-CdA) is a new promising antileukemic and immusuppressive agent. Discovery of 2-CdA and its unique properties, methods of synthesis, data explaining mechanism of its selective cytotoxicity, and current clinical status of the drug are briefly reviewed.

Keywords: 2-chloro-2′-deoxyadenosine; antileukemic drugs; clinical immusuppression.

2-Chloro-2′-Deoxyadenosine – Clinical, Biochemical and Pharmacokinetic Considerations

JAN LILIEMARK and GUNNAR JULIUSSON

Abstract: 2-Chloro-2′-deoxyadenosine (CdA) is a new antimetabolite with very promising effect in the treat- ment of chronic lymphoproliferative diseases. It has also been shown to have good activity in acute myeloge- nous leukemia in children. CdA induces DNA single strand breaks and poly(ADP)-ribosylation, apoptosis and is cytotoxic to non-proliferating as well as proliferating cells. Earlier, CdA has only been given as continuous infusion. Recently, however, pharmacokinetic studies have shown that the bioavailability of oral CdA is 50% and of s.c. injection 100%. Furthermore, studies of the intracellular pharmacokinetics shows that there is a prolonged retention of its nucleotide metabolites supporting intermittent dosing.

Keywords: 2-chloro-2′-deoxyadenosine; antileukemic treatment; pharmacokinetics; mechanism of biological effect

Cladribine (2-Chloro-2′-Deoxyadenosine) : New Perspectives in Clinical Immunosuppression

ANDRZEJ GÓRSKI and PAWEŁ GRIEB

Abstract: Cladribine (2-chloro-2′-deoxyadenosine) has been recently introduced in the treatment of hemato- logic malignancies. We have shown that CdA has potent inhibitory activity in vitro and in vivo in concent- rations readily achievable in the sera of treated patients. The immunosuppressive efficacy of CdA was at least equal to that of cyclosporine. Furthermore, CdA is particularly efficacious inhibitor of B cell activation.

Keywords: 2-chloro-2′-deoxyadenosine; immunosuppressive effect

Stability of 2-Chloro-2′-Deoxyadenosine at Various pH and Temperature

ANDRZEJ TARASIUK ,TANUSZ SKIERSKI and ZYGMUNT KAZIMIERCZUK

Abstract. In this paper data concerning the stability of 2-chloro-2′-deoxyadenosine (2-CdA) solutions at various pH and temperature conditions are presented. Decomposition of 2-CdA was measured as content of its hydrolysis product – 2-chloroadenine, using the HPLC method. We found that 2-CdA is stable at basic and neutral pH and temperatures between 37 and 80″ C. At acidic pH, decomposition markedly increased with time, even in physiological temperatures. At pH 2 and 37″, after 6 h, only 13% of 2-CdA remained in solution. At pH 1 – after 2 h of heating to 37o 2-CdA content was as low as 2%. Calculated half time T1/2 was equal to 1.6 h (pH 2, temperature = 37o) and 0.37 h (pH l, temperature = 37″).

Keywords: 2-chloro-2′-deoxyadenosine; various pH and temperature stability.

Apoptotic Death of Lymphocytes upon Treatment with 2-Chloro-2′-Deoxyadenosine (2-CdA)

PIOTR LASSOTA, ZYGMUNT KAZIMIERCZUK and ZBIGNIEW DARZYNKIEWICZ

Abstract. In this work we addressed the question if the difference in the mechanism by which 2-CdA kills resting and proliferating cells could be responsible for the therapeutic window of the drug. We show that 2-CdA triggers programmed cell death in proliferating human promyelocytic cell line, HL-60, human lym- phocytic cell line, MOLT-4, and human peripheral blood lymphocytes stimulated to proliferation by PHA. Under our experimental conditions 2-CdA failed to induce apoptosis in the resting human peripheral blood lymphocytes despite induction of massive apoptosis in the same lymphocytes stimulated to proliferation by PHA. We also show that 2-CdA-induced apoptosis in HL-60 and MOLT-4 cells can not be prevented by addition of nicotinamide or inhibiting poly(ADP-ribose) synthetase by 3-aminobenzamide. In the case of HL-60 cells apoptosis is specific to the S phase of the cell cycle. Taking together these data suggest that selective induction of apoptosis in proliferating cells may be responsible for the therapeutic value of 2-CdA.

Keywords: 2-chloro-2′-deoxyadenosine; therapeutic in vitro effect; apoptosis.

Treatment of Patients with Hairy Cell Leukemia with 2-Chloro-2′-Deoxyadenosine (2-CdA)

TADEUSZ ROBAK, EUZEBIUSZ KRYKOWSKI, MARIA BLASIIVSKA-MORAWIEC and HALINA URBATISKA-RYS

Abstract. Six patients with hairy cell leukemia have been treated with 2-chloro-2′-deoxyadenosine in a dose 0.05 mg/kg/daily in 2 h intravenous infusions during 7 consecutive days. Two patients underwent splenectomy with no clinical or hematologic improvement. Complete remission has been achieved in 4 patients and partial remission in one. 2-CdA was not effective in a patient with HCL-variant. The longest unmaintained CR lasted 1 I months. The drug was well tolerated. Only slight side effects were observed. The drug exerted, however, myelosuppressive action resulting in cytopenias in the peripheral blood. Clinical consequences of this activity require further investigation. 2-CdA seems to be a drug of choice in the management of NCL.

Keywords: 2-chloro-2′-deoxyadenosine; hairy cell leukemia; clinical evaluation

Treatment of Hairy Cell Leukemia Patients with 2-Chloro-2′-Deoxyadenosine (2-CdA)

MIECZYSLAW KOMARNICKI, MACIEJ KAZMIERCZAK and JANUSZ HANSZ

Abstract. A preliminary study of six hairy cell leukemia patients treated with one course of 2-chloro-2′-deoxya- denosine (2-CdA) is presented. 2-CdA was administered 0.1 mg/kg/daily by intravenous infusion over 7 days. Two patients achieved CR and four PR.

Keywords: hairy cell leukemia; 2-chloro-2′-deoxyadenosine.

Systemic Administration of 2-Chloro-2′-Deoxyadenosine (2-CdA) in Patients with Systemic Scleroderma

SLAWOMIR MAJEWSKI, MAGDALENA SKOPINSKA, MARIA BLASZCZYK, MIROSLAW RYBA, PAWEL GRIEB, TADEUSZ CHORZELSKI and STEFANIA JABLOINSKA

Abstract. Three patients with diffuse variety of systemic scleroderma were treated with 2-chloro-2′-deoxyade- nosine. In two of them a moderate improvement of skin involvement was observed. No significant effects of the drug on the immunological parameters have been found.

Keywords: 2-chloro-2′-deoxyadenosine; scleroderma

2-Chloro-2-Deoxyadenosine (2-CdA) Treatment in a Patient with Waldenström’s Macroglobulinaemia Complicated by Severe Neutrocytopenia

J. MACIEJ ZAUCHA, DOMINIK CIESIELSKI, PAWEL GRIEB and ANDRZEJ HELLMANN

Abstract. A 68-year-old male patient with newly diagnosed Waldenström’s macroglobulinaemia complicated by severe neutrocytopenia at presentation of the disease was treated with 2-CdA. Partial remission with an increase in neutrocyte count was achieved.

Keywords: 2-chloro-2′-deoxyadenosine; Waldenström’s macroglobulinaemia; neutrocytopenia

Influence of 2-Chloro-2′-Deoxyadenosine Alone and in Combination with Cyclophosphamide or Methotrexate on Murine Leukemia L1210

JOANNA GÓRA-TYBOR, TADEUSZ ROBAK, KRZYSZTOF WARZOCHA and PAWEL GRIEB

Abstract. The influence of 2-CdA administrated alone and in combination with CY or MTX on the survival time of mice bearing L1210 leukemia was investigated. 2-CdA treated mice lived longer than control animals. Survival times of mice receiving combined therapy of 2-CdA and CY were significantly prolonged as com- pared with mice treated with these agents separately. Survival times of mice treated with 2-CdA and MTX were not significantly prolonged, compared with animals receiving 2-CdA alone.

Keywords: 2-chloro-2′-deoxyadenosine; mouse L1210 leukemia; combination treatment

2-Chloro-2′-Deoxyadenosine (2-CdA) Combined with Cyclosporine A Successfully Prevents Rejection of Fetal Brain Stem Allograft in Rabbits

PAWEL GRIEB, MIROSLAW RYBA, WIKTOR JANCZEWSKI, JAN SAWICKI, JANUSZ JAGIELSK and JAROSLAW ANDRYCHOWSKI

Abstract. Allografts of brain stem from 20-day-old fetuses to nucleus caudatus of adult rabbits were performed. To prevent graft rejection immunosuppression with 2-CdA and cyclosporine A was transiently induced. Graft survival were assessed by histological and electrophysiological techniques. Both morphological (synaptogenesis, myelinization) and functional (generation of rhythmic neuronal activity) signs of graft maturation were found after nine weeks. The data suggest that transient immunosuppression used is sufficient to induce tolerance to neural graft, and no interference with maturation of implanted fetal tissue occurs.

Keywords: 2-chloro-2′-deoxyadenosine; cyclosporine A; immunosuppression; rabbits; fetal brain allografts; brain tissues maturation.

Modulation of Lymphocyte-Fibroblast Interactions by 2-Chloro-2′-Deoxyadenosine (2-CdA) in Subarachnoid Hemorrhage

MIROSLAW RYBA, PAWEL GRIEB, SLAWOMIR MAJEWSKI, BARBARA MAKIELA, MAREK PASTUSLKO, JAROSLAW ANDRYCHOWSKI and PIOTR BOJARSKI

Abstract. The adherence to monolayers of human fibroblasts of chromium-51 labelled peripheral blood mononuclear cells (PBMC) from normal subjects, and from patients after single or multiple subarachnoid hemorrhage (SAH), and the effect of 2-CdA on cell adhesion have been quantitated. The fraction of cells adhering to fibroblasts were the lowest for multiple SAH patients and the highest for normal subjects, which can be explained by depletion of activated lymphocytes from peripheral blood of SAH patients. 2-CdA decreased the fraction of adhering cells isolated from normal subjects, did not change the adherence of cells from single SAH patients and increased the fraction of adhering cells isolated from multiple SAH patients. We conclude that the influence of 2-CdA on the adherence of PBMC to fibroblasts is inversly related to the degree of immune system activation.

Keywords: 2-chloro-2′-deoxyadenosine; subarachnoid hemorrhage; lymphocyte adhesion

Various

Comparative Evaluation of Toxicity and Antitumor Activity of Original cline Drugs and Reproduced Anthracy

HALINA KUSNIERCZYK, CZESLAW RADZIKOWSKI, PIOTR GÓRECKI and KATARZYNA CICHOCKA

Abstract. Two anthracycline drugs – doxorubicin and 4′-epidoxorubicin – synthesized at the Institute of Biotechnology and Antibiotics were tested in vivo for their biological activity in comparison with original Farmitalia drugs. In parallel experiments similar properties of both drugs and referentials were displayed: comparable subacute and delayed toxicity from i.p. and i.v. routes of administration as well as antitumor effectiveness in two mouse transplantable tumor models – P388 leukemia and B16 melanoma. No statistically significant differences between reproduced and original drugs were found.

Keywords: doxorubicin; epidoxorubicin; reproduced drugs; toxicity; antitumor activity

Immunosuppressive and Cytogenetic Effects of Pelvic Irradiation on the Peripheral Lymphocytes of Patients with Cervical Cancer. One Year Follow-Up

LJILJANA VUCKOVIC-DEKIC, BILJANA SPREMO, NEVENKA STANOJEVIC-BAKIC, BRANIMIR GARZICIC and MIRJANA DURBABA

Abstract. The circulating lymphocytes of patients treated for cervical cancer were examined by four independent manners: by evaluation of T-cell proportion in peripheral blood, proliferative response upon PHA stimulation, PHA-induced leukocyte migration inhibition, and by concomitant chromosome aberration frequency. The immediate and longer-term effects of pelvic irradiation on T lymphocytes were investigated in 19 patients prior to, during, and immediately after radiotherapy, and then at subsequent intervals of two, three and five months. Radiotherapy caused profound depression of already diminished T-cell number and their proliferative response; both parameters gradually recovered during post-treatment period, and achieved their pretreatment values at the end of follow-up. The leukocyte migration inhibition was much less affected; it slightly deteriorated in the middle of post-treatment period, but reached the pretreatment level at the end of monitoring. The chromo- some aberration frequency increased during irradiation in dose-dependent manner; it decreased gradually thereafter, but remained high during follow-up. Their elimination rate correlated with the recovery of T-cell number and proliferative response. However, at the end of monitoring, when all immunological parameters were completely recovered from harmful effect of irradiation, the percentage of chromosome aberrations remained high (12.5%), although significantly lower than the post-treatment one.

Keywords: cervical cancer; pelvic irradiation; peripheral lymphocytes

Standardization of HeLa Cells Preparations as the Source of snRNA’s for Preliminary Study on the Effectors of pre-mRNA Splicing

BOGUSLAW HŁADON, JACEK KRAJEWSKI, ANNA SLODEKBACH and PRZEMYSLAW RADOSH

Abstract. Physical, chemical and biological parameters of HeLa cell cultures, which ensure obtaining standard nuclear material to be used as a source of snRNP and snRNA, have been determined. The secondary structure of U1 snRNA as the result of the conformational studies performed using S1 nuclease, A and T1 ribonuclease and Pb(II) ions, and reproducibility of the results obtained after 6-48 h exposure of the cells to actinomycin D at high concentration permit to accept the proposed method of standardization as satisfactory for investigation into snRNP and snRNA.

Keywords: HeLa cells standardization; source of snRN

Heparin Enhances Generation of Natural Killer Activity in Vitro

MARIA WASIK and ANDRZEJ GÓRSKI

Abstract. The effect of heparin on generation and activity of NK cells in vitro were studied. Heparin decreased NK cytotoxicity when present in the assay. In contrast, the agent increased generation of NK cytotoxicity and showed synergistic action with recombinant IL2.

Keywords: heparin; NK cytotoxicity; interleukin 2

In Vitro Primary Antitumor Screen of Imidazole Platinum Complexes

JANINA KUDUK-JAWORSKA

Abstract. The cytostatic activity in vitro of six cis-platinum analogs with imidazole ligands was examined. The tests were performed on 60 lines of human neoplastic cells in the NCI Preclinical Antitumor Drug Screening Program. The results obtained from 10 the most sensitive lines were analyzed and the structure-activity relationship was deduced. The cytotoxic activity of the active complexes seems to correlate with the hydro- phobic properties of neutral imidazole ligands and with kinetic properties of anionic ligands.

Keywords: imidazole platinum complexes; in vitro cytotoxity

Vol. 42, No. 2, 1994

Mini-Review

Molecular Mimicry Between Fc Receptors and Viral Antigens
EMILIA L. OLESZAK

Abstract. Molecular mimicry has been characterized as the presence of common epitopes, either linear or conformational, shared by host and microbial determinants. Such cross-reactivity may lead to an autoimmune disease. On the other hand molecular mimicry between certain viral proteins and host determinant may protect invadin virus to be eliminated by immune system and may promote persistence. In this mini-review p discuss the molecular mimicry of S peplomer protein of mouse hepatitis virus, strain JHM (MHV-JHM to the host Fc gamma receptor (FcyR). MHV-JHM induces in rodents acute encephalomyelitis and surviving animals develop demyelinating disease with concomitant persistent infection. We have demonstrated that rabbit IgG, but not is F(ab’)fragments, monoclonal rat and mouse IgG and the rat 2.4G2 anti-FcyR mab immunoprecipitated natural and recombinant S peplomer protein of several strains of MHV. Furthermore, MHV-JHM infected cells formed rosettes with anti-sheep red blood cell SRBC – antibody coated SRBC. The 2.4G2 anti-FcyR mab are able to neutralize several strains of MHV, presumably by binding to S pep- lomer protein. Therefore, the Fc binding site of S is present on the surface of MHV-infected cells. This molecular mimicry between S peplomer protein of MHV-JHM and FcyR has been extended to other members of Coronaviridae, namely bovine coronavirus and transmissible gastroenteritis virus but not to infectious bronchitis virus. The molecular mimicry of viral antigens to Fc receptors has been described also for members of Herpesviridae, namely Herpes simplex , cytomegalovirus and Varicella zoster. A complex of glycoproteins gE and 1 of Herpes simplex virus mimics host Fc receptors and binds human and rabbit but not mouse IgG. It has been suggested that the presence of an FcR on HSV-infected cells results in protection of the virus (or virus infected cell) through antibody bipolar bridging. Thus, viruses which belong to two unrelated families (Corona- viridae and Herpesvirida ) but which easily establish persistent infections in the central nervous system show molecular mimicry of their viral proteins to FcR.This morecular mimicry may play a role in the pathogenesis of the disease induced by MHV and HSV and maintenance of persistent infection.

Keywords: molecular mimicry; Fc receptors; mouse hepatitis virus; monoclonal antibodies.

Tubulovesicular Structures (TVS): Virus-Like Particles Specific for All Subacute Spongiform Virus Encephalopathies- What Are They Really?
PAWEL P. LIBERSKI

Abstract. Tubulovesicular structures (TVS) are virus-like particles specific for all the subacute spongiform virus encephalopathies (SSVE). I report here the presence of TVS in the highest range of naturally occurring and experimentally induced SSVE studied so far: natural and experimental Creutzfeld-Jakob disease (CJD), Gerst- mann-Sträussler-Scheinker syndrome, natural bovine spongiform encephalopathy (BSE) and BSE transmitted to pigs and four models of experimental scrapie in hamsters. TVS are spherical particles, approximately 30 nm in diameter. They are observed at both sides of synaptic cleft (i.e. in dendrites and axonal preterminals and terminals). By the ultrastructural criteria, TVS are easily differentiated from other spherical or tubular ele- ments of the nervous system: microtubules, synaptic vesicles, multivesicular bodies and glycogen granules. The number of affected processes roughly correlates with infectivity titre. Their significance is unknown!

Keywords: subacute spongiform virus encephalopathies; tubulovesicular structures; electron microscopy.

Cellular Immunology

An Improved Experimental Model for the Study of in Vitro Release of Nitric Oxide by Murine Peritoneal Macrophages
JANUSZ MARCINKIEWICZ, MARZENA PATER and AGNIESZKA GRABOWSKA

Abstract. In the immune system macrophages are the cells responsible for nitric oxide (NO) production. The synthesis of NO by activated macrophages correlates with their cytotoxic effect on neoplastic cells as well as kiling of intracellular parasites. In the present paper we test several parameters that may influence (in vitro) NO production by murine peritoneal macrophages previously stimulated in vivo by intraperitoneal injection of thioglycollate. In our system the maximum NO/NO-2 release was obtained in the culture containing 10″ M0/m1 after 24 h incubation. For macrophage activation we used lipopolysaccharide (LPS) and several recombinant cytokines (IFN-y, TNF-alfa, IL-2, IL-3, IL-6). We also tested the influence of latex phagocytosis on NO production by simultaneously activated macrophages.

Keywords: nitric oxide; peritoneal macrophages; lipopolysaccharide; cytokines.

The Reduced Expression of HLA-Class II Antigens and Adhesion Molecules on Monocyte Surface after Phagocytosis of Bacteria
JAROSLAW BARAN and JULIUSZ PRYJMA

Abstract. The reduced expression of HLA-class II antigens (DQ and DP) and some intercellular adhesion molecules (CDllb, CDS4 and CDSB) was found on monocytes after phagocytosis of bacteria (S. aureus, E. coli, P. aeruginosa, S. enteritidis) but not of latex particles. In contrast, the expression of HLA-DR, CDlla and CDlB was not changed in the course of phagocytosis. The observed changes were related to the amount of phagocytosed bacteria but not to their viability or phagocytosis induced physical changes expressed as the reduction of FSC signal during flow cytometry analysis.

Keywords: phagocytosis; bacterial infections; HLA-class II antigens; adhesion molecules.

Kinetics of Distribution of Recirculating Lymphocytes during Whole Body Hyperthermia
ERIC P. AMANING and WALDEMAR L. OLSZEWSKI

Abstract. Whole body hyperthermia (WBH) not only enhances lymphocyte migration to the bone marrow and skin, but also prolongs their subsequent transit through these organs, while inhibiting homing to the lymphoid organs. The effect of WBH is transitory, it subsides within 16 h after a 8 h WBH period. The accumulation of circulating lymphocytes in lymph nodes, Payer’s patches and spleen dropped significantly between 4 h and 8 h in WBH, but rose during subsequent 16 h normothermia. In the absence of the adrenal glands, the enhanced bone marrow localization observed in non-adrenalectomised rats during WBH did not occur. Skin localization increased while splenic localization fell in adrenalectomised as compared to non-adrenalectomised rats. These results strongly support the hypothesis that WBH-induced enhancement of lymphocyte migration to the bone marrow is adrenal-hormone dependent. The above mentioned changes can not be attributed to changes in blood flow to the tissues during WBH. Blood flow to the bone marrow was not significantly different from flow to this organ in normothermic rats. Flow to the skin fell significantly and yet localization in this tissue during WBH was higher than during normothermia.

Keywords: lymphocytes; hyperthermia; recalculation.

Presentation of Antigen by B Cells Subsets. I. Lyb-5+ and Lyb-5- B Cells Differ in Ability to Stimulate Antigen Specific T Cells
MICHAL ZIMECKI, PHYLLIS J WHITELEY, CARL W. PIERCE and IUDITH A. KAPP

Abstract. We have examined the antigen presenting cell (APC) function of different B cells. Resident, peritoneal B cells from normal mice were more effcient than splenic B cells in presenting antigen to CD4+ T cell lines. Peritoneal B cells from X-linked immunodeficient (Xid) mice, by contrast, stimulated no detectable responses. Xid splenic B cells were much less efficient APC than normal splenic B cells. B cells from neonatal mice also were very poor APC until the mice were 3 to 4 weeks old. Xid B cells presented antigen to T cell hybridomas as well as normal B cells showing that they process antigen normally. Thus, the defect is most likely in providing secondary signals. The ability of B cells to present antigen efficiently correlates with the percentage of B cells reported to express the Lyb-5 antigen. Anti-Lyb-5 serum and complement abrogated the APC activity of B cells suggesting that Lyb-5+, but not Lyb-5- cells are efficient APC. We also found that activated and resting normal splenic B cells, separated by buoyant density, presented antigen equally. Both populations also contained Lyb-5+ B cells although they were a larger fraction of the activated cells. Lyb-5 is now thought to be an activation antigen rather than a differentiation antigen. If this idea is correct, then our data indicate that anti-Lyb-5 more cleanly separates activated and resting B cells than buoyant density techniques.

Keywords: Lyb-5-; Lyb-5+ B cells; Xid mice; neonatal mice antigen presentation.

Immunochemistry

Study on the Role of Urine Gamma-Glutamyl Transpeptidase Activity during Investigation of Nephrotoxicity
SÜKRAN KOCAOGLU, TAYFUN BERKAN, AYSEN KARAN and GÜLCIN BASDEMIR

Abstract. In this study, gamma-glutamyl transpeptidase activities of the 24 h urine samples taken at the end of the fourth day from the rats to which 160 mg/kg/day gentamicin was applied i.p. for 4 days, were measured. Glucose determination in urine by the use of the glucose hexokinase method was also applied in order to control the reabsorption potentials of the tubules. The formation of necrosis in the kidneys was investigated by histological examinations of the damage occurred by the nephrotoxic effect. All the results were compared with the values obtained from the control group. The average gamma-glutamyl transpeptidase activity for the control group (n = 22) was determined as 5.68±0.26 IU/24 h whereas this level was detected as 15.6± 1.0 IU/24 h in the drug applied group (n = 15). The measurement of gamma-glutamyl transpeptidase activity in urine can be applied as a useful parameter on determination of nephrotoxicity, especially for indicating the dimensions of this toxic effect.

Keywords: nephrotoxicity; gamma-glutamyl transpeptidase; gentamicin

Chemiluminescence Immunosorbent Assay (CLISA) and a Possibility of the Specific Detection of Soluble Antigens of Clostridium botulinum Type A
JERZY LIGIĘZA, JULIUSZ REISS and MARIUSZ MICHALIK

Abstract. A double antibody version of CLISA was demonstrated to be a rapid method (1 h) for detection and quantitative determination of Clostridium botulinum toxin antigens in biological samples. The sensitivity of this assay is about ten-fold higher than both ELISA and passive hemagglutination test. Thus, the double antibody version of CLISA appeared to be useful for the control of food products contaminated with Cl. botulinum type A bacteria.

Keywords: chemiluminescence immunosorbent assay; Clostridium botulinum toxin type A.

Various

Studies on Transplantation Immunity of the Yellow-Bellied Toad Bombina variegata
ELŻBIETA GÓRKALIK, DOMINIKA ŁABUZ, ALIJA JÓZKOWICZ and BARBARA PŁYTYCZ

Abstract. Adults and tadpoles of the yellow-bellied toad Bomhina variegata reacted in a typically chronic manner to skin allografts and to xenografts from closely related fire-bellied toads B. bombina but they rejected quickly skin xenografts from evolutionary distant anuran species (Bufo and Rana). Adult individuals reacted to allografts slowly not only in the laboratory where their mating was ceased and the weight of lymphoid organs significantly diminished but also in the outdoor enclosure where they bred successfully. Breeding activity in captivity can be induced at any season by Biogonadyl injections. However, any hormonal manipulation (gonadectomy or Biogonadyl treatment) performed during winter/spring on animals housed in the laboratory for several months did not influence their transplantation immunity and the weights of thymuses and spleens. These results lead to conclusion that chronic allograft rejection was not a laboratory artifact caused by a hormonal imbalance but rather reflected a weak donor-host genetic disparity connected with the low MHC polymorphism of Bomhina species.

Keywords: evolutionary immunology; immunoendocrinology; allograft; xenograft; amphibia.

Immunotropic Activity of Lupin Seeds Extracts and Fractions from Lupinus angustifolius and Lupinus albus
BARBARA BŁASZCZYK, MACIEJ STOBIECKI, STEFANIA H. KOWALCZYK-BRONISZ, KRZYSZTOF GULEWICZ and STANISLAW SZYMANIEC

Abstract. The results demonstrated immunotropic activity of seeds extracts and fractions from Lupinus angus- tifolius and Lupinus albus. Plaque forming cells (PFC) number to SRBC (sheep red blood cells) were elevated by an extract from Lupinus angustifolius and lowered by extracts from Lupinus albus. All preparations obtained from Lupinus angustifolius reduced the number of rosette forming cells (E-RFC). These preparations suppres- sed also the intensity of graft versus host reaction (GVHR) in case when the donors were treated. Lupinus albus extract suppressed GVH reaction when recipients were treated. Lupin extracts stimulated draining popliteal lymph nodes in PLN assay.

Keywords: lupin; immunotropic activity; plaque forming cells; rosette forming cells; delayed type hy- per-sensitivity; graft versus host reaction; popliteal lymph nodes assay.

Mechanism of Antigenic Variation in Shigella flexneri. II. Sensitivity to Complement as a Selection Factor for Antigenic Mutant 3b in lb Stereotype
WŁODZIMIERZ DOROSZKIEWICZ, RENATA POKŁADEK and TADEUSZ M. LACHOWICZ

Abstract. In the first report we stated that the antigenic mutant of Shigella flexneri 6713 3b serovar with antigenic formula III: 3, 4, 6 was less sensitive to bactericidal action of normal calf serum in comparison to its parent strain S. flexneri 6713 lb with antigenic formula III: 3, 4, 6. In this paper we show that the phenomenon is rather a general one; the difference in sensitivity was observed in three other strains S. ,flexneri lb of independent clinical origin and its antigenic mutants 3b respectively. As the result of serial treatment of these strain with the serum among the survivors the antigenic mutants have been found. In the artificial mixtures of the mutants and isogenic original strains treated with the serum the later show clear cut higher survival.

Keywords: S. flexneri; antigenic mutants; complement.

Vol. 42, No. 3, 1994

Cellular Immunology

Effect of Outer Membrane Proteins (OMP) of Shigella on Interleukin 2 (IL-2) Production by Spleen Cells of Mice
ANNA CZARNY, DANUTA WITKOWSKA and MARIAN MULCZYK

Abstract. The kinetics of interleukin (IL-2) release from mouse spleen cells incubated with different doses of outer membrane proteins (OMP) from Shigella was investigated. OMP induced very low activity of IL-2 after 2 and 4 h, and only a slightly higher level of the cytokine was detected after 6 h. However, IL-2 activity increased markedly after 20 and 24 h of incubation, and doses of 5 and 10 µg of OMP were found to be the most effective. Spleen cells cultured with OMP for 48 h contained reduced concentration of IL-2.

Keywords: Shigella outer membrane protein; interleukin 2.
Thymocyte-Macrophage Interactions. Interleukin 6 Produced by Activated Thymocytes Induces Interleukin 1 Production by Macrophages
MICHAŁ ZIMECKI, ZBIGNIEW WIECZOREK and JUDITH A. KAPP

Abstract. Coculture of paraformaldehyde-fixed macrophages with syngeneic thymocytes resulted in synthesis of a lymphokine which triggered IL-1 production in untreated, peritoneal macrophages. The release of that lymphokine was MHC restricted, however, the stimulation of IL-1 production was not MHC-controlled event. Preliminary studies showed that the lymphokine had molecular weight (MW) of around 10-30 kDa, was heat stable (56′ C), resistant to reduction to DTT and exhibited a weak pyrogenic activity. Fusion of activated thymocytes with the thymoma BW5147 resulted in obtaining several hybridoma clones producing the lym- phokine. Preparation of culture supernatants by means of Amicon membranes and Sephedex G-100 filtration allowed to determine the MW of the lymphokine as 25 kDa. Lastly, the lymphokine was identified as IL-6 by means of anti-IL-6 antibodies which blocked its function and by the use of IL-6-sensitive cell line 7TDl.

Keywords: thymocytes; macrophages; cell interaction; interleukin 1; interleukin 6.
Lactoferrin Inhibits the Effector Phase of the Delayed Type Hypersensitivity to Sheep Erythrocytes and Inflammatory Reactions to M. bovis (BCG)
MICHAŁ ZIMECKI and MICHAŁ MACHNICKI

Abstract. Bovine lactoferrin (BLF) given into mice, sensitized to SRBC, together with the eliciting dose of antigen, inhibits very strongly the DTH reaction measured after 24 h by foot pad swelling. Administration of BLF at 48 or 24 h before eliciting the DTH reaction was not effective, however, BLF suppressed the reaction when given at the peak of the inflammatory process. The effects of BLF were strongest when the protein was injected intravenously. Intraperitoneal or intramuscular administrations of BLF were less inhibitory. In ad- dition, BLF diminishes, although to a much lesser degree, the inflammatory reactions induced by BCG. The inhibitory action of BLF does not involve liver since treatment of mice with galactosamine does not reverse the inhibition. Studies on cytokine production revealed that peritoneal macrophages, derived from mice pretreated with LF, have an increased ability to produce in in vitro IL-6 after induction with LPS. In addition, we demonstrated that inhibition of macrophage migration, mediated by migration inhibition factor, is abolished by BLF. Lastly, the inhibitory effect of BLF could not be transferred with serum from donors treated with BLF. In summary, the data reveal the inhibitory properties of LF, administered systematically, in relation to locally induced inflammation.

Keywords: lactoferrin; delayed hypersensitivity reaction; sheep red blood cells; M. bovis; interleukin 6.
Interleukin 3-Dependent, Asialo-GMl-Positive Cell Line with Natural Cytotoxic but without Natural Killer Cytotoxic Activity
LEON STRZĄDAŁA, EWA ZIOŁO and JANUSZ MATUSZYK

Abstract. IL-3-dependent cell line (designated as A-3) from adherent spleen cells was developed. The surface phenotype of A-3 cells was analysed by flow cytometry and determined as ASGM 1 +, FcR+, Ia+. There was no expression of T cell markers (Thy 1 – CD3 – CD4 – CD8 – ), B cell marker (surface Ig) as well as macrophage marker Mac-1 antigen. Although A-3 cells expressed ASGM1, they are lacking NK cytotoxic activity in 4 h Cr release assay with YAC-1 targets. In contrast, A-3 cells are endowed with NC cytotoxic activity as shown in 18 h Cr release assay with WEHI-164 target cells.

Keywords: interleukin 3-dependent; asialo-GM 1; natural killer antitumor; natural cytotoxic.
Generation of Anti-Hapten T Cell Cytotoxicity in vivo. Relationship to Contact Sensitivity and the Role of Contrasuppression
WŁODZIMIERZ PTAK, ALAN M. FRIEDMAN and PATRICK M. FLOOD

Abstract. Immunization procedures that induce contact sensitivity to the trinitrophenyl (TNP) hapten in vivo were investigated for their ability to induce TNP-specific cytotoxic T lymphocytes in vivo. Spleen cells from C3H/HeN mice primed for CS responses either by the topical application of picryl chloride or by the adoptive transfer of PCL immune cells show little or no cytolytic activity in vivo against TNP-coupled target cells. Intravenous immunization with TNP-substituted syngeneic spleen cells, a procedure known to make animals unresponsive to agents normally inducing CS, also failed to induce cytolytic activity in spleen cells. However, both PCL sensitization and adoptive transfer, when combined with the injection of TNP-substituted syngeneic spleen cells, induce significant cytolytic activity against TNP-haptenated BW5147 target cells in vivo. Further- more, i.v. injection of TNP-spleen cells with surface-bound immune complexes of the IgM or IgGl isotypes, or with a monoclonal TNP-specific contrasuppressor T cell factor also induces strong antigen-specific cytolytic activity against TNP modified targets. TcsF bears serological determinants of T cell receptor a and ß chains and adheres to specific antigen columns. All these immunization regimens were shown to induce CS to TNP as well as the generation of contrasuppressor T cells. The CTL generated in the spleens of immunized mice are Thyl + CD8+ T cells and are antigen-specific and genetically restricted. The implications of these results with respect to the mechanisms by which cytolytic responses are controlled in vivo is discussed.

Keywords: cytotoxic T lymphocytes; contrasuppression; contact sensitivity; antigen-antibody complexes.

Immunochemistry

Guinea Pig Antibody to C3 Component of the Sheep Complement Produced by Immunization with a Heat- -Inactivated Sheep Serum – Alcian Blue Complex
WARREN JONAS and MIROSLAW STANKIEWICZ

Abstract. Normal sheep serum or serum heated at 56′ C for 30 min were added to alcian blue solutions. The optical density of alcian blue-heated sheep serum complexes formed were 2-4 times greater than those obtained with unheated serum. Most guinea pigs immunized with alcian blue-heated sheep serum complexes produced antibodies that precipitated the third component (C3) of sheep complement (C’) from electro- phoresed sheep serum. These antisera also hemagglutinated sheep erythrocyte – rabbit antibody complexes treated with sheep serum (C’). Guinea pigs immunized with alcian blue-normal sheep serum complexes did not react with electrophoresed sheep serum nor with sheep C’ in the hemagglutination test.

Keywords: sheep complement C3; anti-C3; glycosaminoglycans; alcian blue.
Inhibitory Activity of Soluble Material from Sheep Eosinophils for Sheep Complement
WARREN JONAS and MIROSŁAW STANKIEWICZ

Abstract. Soluble material from frozen and thawed sheep eosinophils and sonicated extracts of eosinophils or their granules inhibited the alternative activation pathway dependent action of sheep complement for human erythrocytes sensitized by sheep antibody. Sephadex G-50 fractions of the sonicated eosinophil and granule extracts showed that inhibitory activity was associated with the void volume fraction, the fraction possessing the eosinophil peroxidase activity.

Keywords: sheep complement; eosinophils; anti-complementary activity.
Immunosuppressive Activity of C-Terminal Fragments of Tachykinins
IGNACY Z. SIEMION , ALEKSANDRA KUBIK , MICHAŁ ZIMECKI and ZBIGNIEW WIECZOREK

Abstract. The immunosuppressive activity of three pentapeptides related to the C-terminal fragments of tachykinins was investigated using the PFC (plaque forming cell) test. The peptides were of the general structure Phe-X-Gly-Leu-Met-NH2, with Tyr, Val and Ile in X position, respectively. The results were com- pared with those obtained previously for the substance P (SP) C-terminal pentapeptide. It was found that peptides containing aromatic residues in X position were more potent immunosuppressors than peptides containing aliphatic residues in the same position. The results also suggest that in mammalian organisms substance SP may be involved in immunomodulation which is rather not the case with other mammalian tachykinins (neurokinin A and neurokinin B).

Keywords: peptide immunosuppressors; fragments of tachykinins.
Immunosuppressive Activity of Analogs of Tripeptide Lys-Arg-Pro with D-Amino Acid Residues
IGNACY Z. SIEMION , ALICJA KLUCZYK , JACEK J. SŁOTŃ, MICHAŁ ZIMECKI and ZBIGNIEW WIECZOREK

Abstract. Synthesis of four analogs, containing D-amino acid residues, of the tripeptide immunosuppressor Lys-Arg-Pro is described. In addition, acetyl and amide derivatives of this peptide were synthesized. Biological effects of all peptides were studied using a delayed type hypersensitivity (DTH) test. It was found that incorporation of D-amino acid residues in definite positions of the peptide chain gives the substances with immunosuppressive activity. A strong immunosuppressive activity was observed also for all-D-analogs (D-Lys-D-Arg-D-Pro).

Keywords: tripeptides; immunopeptides; immunosuppressors; immune response
Structural and Immunochemical Studies on O-Specific Polysaccharide of Proteus penneri Strain 14
ZYGMUNT SIDORCZYK, ANNA ŚWIERZKO, EVGENY V. VINOGRADOV, YURIY A. KNIREL and ALEXANDER S. SHASHKOV

Abstract. The complete structure of the O-antigen of Proteus penneri strain 14, containing D-alanine and _L-alanine was established using methylation, solvolysis with anhydrous hydrogen fluoride, partial acid hydro- lysis, ’ H- and ’ 3C-NMR spectroscopy. The role of partial structures of the pentasaccharide repeating unit in manifesting serological specificity and cross-reactivity of this strain with some other bacteria is discussed.

Keywords: Proteus penneri; O-polysaccharide; lipopolysaccharide.

Transplantation

Influence of Different Temperature of Cryopreservation on the Proliferative Potential of Human Bone Marrow Progenitor Cells. Transplantological Implications
MARIUSZ Z. RATAJCZAK, WOJCIECH I. KUCZYŃSKI and JANINA RATAJCZAK

Abstract. The influence of two different temperatures of cryopreservation of human bone marrow on the proliferative potential of granulocyto-monocytic (GM-CFU), erythroid (BFU-E) and megakaryocytic (CFU-Meg) progenitors was investigated. For this purpose, the human bone marrow cells were cryopreserved in the standard freezing medium supplemented with 10% DMSO and stored at – 80oC in a freezer or at -196oC in a liquid nitrogen tank. Subsequently, cells were thawed, plated and stimulated in vitro to grow myeloid colonies. Unexpectedly, bone marrow cells stored at -80oC formed about 60%, and those stored at -196oC formed barely 8% of CFU-GM and BFU-E colonies respectively, in comparison to control non-frozen cells. CFU-Meg progenitors appeared to be the most sensitive to cryopreservation among all clonogeneic cells tested. The number of CFU-Meg derived colonies decreased to 20% in marrow cryopreser- ved at -80oC and to about 2% in cells stored in liquid nitrogen at -196oC. These data demonstrate the advantages of bone marrow storage at -80oC over freezing in liquid nitrogen at -196oC. Moreover, the increased sensitivity of CFU-Meg to cryopreservation could explain, at least partially, the clinical phenome- non of protracted thrombocytopenia observed in patients transplanted with the cryopreserved bone marrow cells.

Keywords: bone marrow transplantation; cryopreservation; hemopoietic progenitors.
Absorption with K562 Erythroblastoma Cells as a Means for Discrimination between Auto- and Alloantibodies in Sera of Hypersensitized Renal Patients
KATARZYNA BALDY-CHUDZIK, BEATA NOWAKOWSKA, PIOTR KUŚNIERCZYK and HENRYK MATEJ

Abstract. We applied K562 erythroblastoma cell line as a substitute for autologous lymphocytes in the absorption of autoantibodies from sera of 26 hypersensitized renal patients exhibiting high degree of cytotoxic reactivity with a panel of lymphocytes from unrelated individuals (panel reactivity, PRA). All of analysed sera contained allo-reactivity before absorption. After removal of autoantibodies, 85% of the sera still contained alloantibodies whereas in 15% of the sera no alloreactivity was detectable. Absorption of high PRA sera with K562 cells and with autologous lymphoblastoid cell lines derived by transformation with Epstein-Barr virus (EBV-LCL) gave substantially the same results. As autoantibodies have reportedly no effect on kidney graft survival, the autoabsorption of high PRA sera may increase the chance of patients in whom only autoan- tibodies were detected to receive the transplant. Application of K562 cells, instead of autologous lymphocy- tes/lymphoblasts, is recommended whenever high numbers of patient’s cells are not available. It may be particularly suitable for routine clinical laboratories, not equipped and prepared for derivation and propaga- tion of EBV-LCL.

Keywords: hypersensitized renal patients; panel reactivity antibodies; autoantibodies; alloantibodies.
Determination of Immunoglobulin Class of Alloantibodies in Sera from Hypersensitized Renal Patients
KATARZYNA BALDY-CHUDZIK, BEATA NOWAKOWSKA, HENRYK MATEJ and PIOTR KUŚNIERCZYK

Abstract. 26 sera from hypersensitized renal patients were analysed for immunoglobulin class of alloantibo- dies. To assess the participation of IgM and IgG in alloreactivity, sera were treated with dithiothreitol (DTT) or Staphylococcus aureus protein A (SpA), respectively. Treatment with both DTT and SpA was also perfor- med to test the possible contribution of other immunoglobulin classes. Immunological complexes which may mimic alloreactivity were excluded by polyethylene glycol (PEG) treatment. Immunochemically modified sera were tested against a panel of allogeneic B and T lymphocytes. IgG class alloantibodies reactive with B lym- phocytes were found two times more frequently than those reactive with T lymphocytes. In 22% of sera only IgM alloantibodies were detected. No contribution of other immunoglobulin classes or immune complexes was seen. Our results show that DTT and SpA treatment are technically simple and reproducible methods of discriminating between IgG and IgM alloantibodies and can be recommended for routine use in clinical analysis of sera from hypersensitized renal patients awaiting kidney graft.

Keywords: hypersensitized renal patient; alloantibodies; immunoglobulin class.

Cytokines

Interferon Production by Peripheral Blood Cells of Patients with Alcoholic Liver Disease (ALD)
JADWIGA DANILUK and MARTYNA KANDEFER-SZERSZEŃ

Abstract. Blood samples from 29 healthy individuals (control), 16 chronic alcohol abusers without clinical symptoms of liver disease, 22 patients with alcoholic steatosis and 31 patients with alcoholic cirrhosis were obtained. The antiviral activity, only partially neutralized by polyclonal and monoclonal anti-HuIFN-a and monoclonal anti-HuIFN-y antibodies, was detected in sera of 38% healthy controls and of 64-77% alcoholic abusers without or with ALD. To measure the activity of the IFN system, leukocyte cultures of chronic alcohol abusers and control individuals were set up using a whole blood technique. Common IFN inducers: NDV, PHA and ConA were used. Blood leukocytes of patients with alcoholic steatosis and cirrhosis respond- ed to NDV, PHA and ConA with significantly lower IFN levels than controls, whereas IFN response of alcoholics without liver diseases was lower only after stimulation with ConA. Regarding the lymphoprolifera- tive test performed with PHA, a remarkably diminished lymphocyte response of patients with cirrhosis was observed as compared to control. Our data indicate that long term ethanol intake impairs the ability of leukocytes and their subpopulation to produce IFNs. The correlation between ALD progression and the defect in IFN production suggests that suppressive action of ethanol, liver injury and immune reactions involved in liver damage may be responsible for impairment of the IFN system in heavy drinkers.

Keywords: alcoholic liver disease; human interferon production; interferon inducers; heavy drinkers.
Influence of Acute and Chronic Ethanol Treatment on Interferon Production in Mice
JADWIGA DANILUK and MARTYNA KANDEFER-SZERSZEŃ

Abstract. The effect of acute (single dose) and long-term ethanol treatment of Swiss Albino and C3H/He mice on IFN production stimulated in vitro by NDV, PHA and ConA was examined. Liver organ cultures and spleen cells of mice which received intraperitoneally a single dose (2 g/kg of body weight) of ethanol (II group) or were drinking ethanol solutions instead of water for 4 weeks (III group) or for 8 weeks (IV group), and cells of control mice not treated with ethanol (I group) were cultivated in vitro. Some of the cultures were treated with 0.4% (w/v) ethanol or several IFN inducers NDV-R, PHA and ConA or incubated without any stimula- tors. The titers of IFN in supernatant fluids were measured. The results showed that cells from mice, which received a single ethanol injection, produced IFN spontaneously and this production was enhanced in the presence of 0.4% ethanol in culture medium. When spleen cell cultures of acute treated mice were induced with PHA and ConA, IFN titers lower than in cells of the control mice were produced. In contrast to these results, liver and spleen cells of mice after long term ethanol consumption produced significantly lower IFN titers than controls after NDV-R induction. Also spleen cells responded poorly both to PHA and ConA induction. These results demonstrate that long-term ethanol treatment can destroy IFN response to viral infection and can impair mitogen-induced IFN production. This suppression of IFN production by long term ethanol was similar in both strains of the mice examined.

Keywords: acute and long-term ethanol treatment; mouse IFN production.

Vol. 42, No. 4, 1994

Mini-Review

Involvement of Heat Shock Proteins and Cellular Membranes in the Development of Thermotolerance
ZOFIA JÓZWIAK

Abstract. A variety of cells subjected to hyperthermia acquire a transient increase in resistance (thermotoleran- ce) to a subsequent heat treatment. The development of thermotolerance is nearly always accompanied by the presence of heat shock proteins (HSPs). However, other states of thermotolerance, some of which are independent of HSPs, have also been postulated. Recently, several studies have shown that cellular membranes, among other cellular structures, may be involved in the response of cells to heat and may constitute a target for the acquisition of thermal resistance.

Keywords: heat; hyperthermia; heat shock proteins; membranes; thermotolerance.

Allergy

Specific Pollen Allergen Activates Eosinophils of the Patient with Chronic Allergic Contact Urticaria
BERNARD PANASZEK, JÓZEF MAŁOLEPSZY, JAN KURYSZKO Z and MARTA LITWA

Abstract. The aim of the study was to evaluate the activation of eosinophils in an unique case of a young man with atopy manifested as chronic pollen contact urticaria. In order to reveal the role of eosinophils in that case, the study was performed by means of monoclonal antibodies EGz and chemiluminescence. In addition, comparative electron microscopic study of peripheral blood and skin infiltrating eosinophils were performed for which the name ultrastructural morphometric analysis of intracytoplasmic eosinophil granules has been proposed. The results indicated, that 40% of peripheral blood eosinophils were activated spontaneously and they were more active than those in skin infltrates. Specific pollen allergen caused activation of 100% of peripheral blood eosinophils. The study suggests presence of a systemic pattern of eosinophil activation in atopy.

Keywords: chronic allergic contact urticaria; pollen allergen; activation of eosinophils; morphometric analysis of intracytoplasmic granules.
High Serum Levels of Allergen Specific IgG-4 (asIgG-4) for Common Food Allergens in Healthy Blood Donors
JERZY KRUSZEWSKI, ALICJA RĄCZKA, MIROSLAW KŁOS and WIESŁAW WIKTOR-JĘDRZEJCZAK

Abstract. High serum levels of asIgG-4 against common food allergens are found in many patients with symptoms suggesting food allergy. The same patients are frequently negative for allergen specific IgE (asIgE) against the same allergens. These data were frequently interpreted as suggestive of a role of asIgG-4 in food allergy. In order to evaluate this hypothesis we tested serum levels of asIgG-4 against food allergens in young blood donors without any signs or history of food allergy. Fifty young healthy male donors were evaluated. The serum levels of IgE, and asIgE and IgG-4 against 14 common food allergens were determined. The studies were carried out using commercially available 3M Diagnostics Systems kits. AsIgG-4 against food allergens were found in sera of 92% blood donors, and in 62% of these healthy persons the levels of asIgG-4 were higher than 10.0µg/ml. In a small proportion of patients, high serum levels of IgE and asIgE against the same food and/or inhalant allergens were found. Common occurrence of asIgG-4 against food allergens in healthy persons (without any symptoms which could suggest allergy or food intolerance) argues against the possible participation of these antibodies in the pathogenesis of food allergy. It is possible that their occurence is the result of immunization against food antigens (allergens). It remains to be resolved whether the presence of these antibodies represents an epiphenomenon or may have some other biological role.

Keywords: food allergy; IgG subclasses.

Cytostatics

Immunological Status of Patients with Denture Stomatitis and Yeast Infection after Treatment of Maxillofacial Tumors
EUGENIUSZ SPIECHOWICZ, KRYSTYNA RUSINIAK-KUBIK, EWA SKOPIŃSKA-RÓŻEWSKA, IRENA SOKOLNICKA,KRYSTYNA ZABUSKA-JABŁOŃSKA, WANDA BRAJCZEWSKA-FISCHER, DARIUSZ ROLSKI, BARBARA CIECHOWICZ, MACIEJ GIL and ROBERT P. RENNER

Abstract. Therapeutic modalities employed in the treatment of maxillofacial malignancy, tumor resection, radiation and chemotherapy, can result in local and systemic changes in healthy oral tissues many years after anticancer therapy has been stopped. The immunologic status of a select group of nine individuals who underwent surgery of the maxillofacial region and after head and neck irradiation, with severe and persistent denture stomatitis was studied. A significant impairment of granulocyte and lymphocyte functions and increased concentrations of serum IgG, as compared to healthy controls, were found in the patients of the study group. It is concluded, that this group of patients show immunological abnormalities many years post therapy, which must be recognised by the clinician in order to effect a cure.

Keywords: immunological status; denture stomatitis; anticancer therapy patients.
Synergistic Antiproliferative Factor Alpha (TNF-a) and Activity of Tumor Necrosis Lovastatin
MAGDALENA K. SORA, ADAM A. KRUSZEWSKI, TOMASZ STOKŁOSA, JAN CZYŻYK, WITOLD LASEK, JACEK MALEJCZYK and MAREK JAKÓBISIAK

Abstract. We assessed the antiproliferative effect of tumor necrosis factor alpha (TNF-a) and lovastatin, an inhibitor of 3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) reductase, alone and in combination, on two murine tumor cell lines. Recombinant TNF-a inhibited proliferation of murine MmB 16 melanoma cells in a concentration-dependent fashion but stimulated growth of murine L1210 leukemia cells at 0.1 ng/ml concentration. Lovastatin inhibited proliferation both of murine MmB 16 melanoma cells and of murine L 1210 leukemia cells in a concentration-dependent fashion. In combination with tumor necrosis factor alpha lovastatin inhibited synergistically growth of both cell lines as assessed by isobologram analysis. Our data show that lovastatin, a cholesterol synthesis inhibitor, introduced to the clinic to treat hypercholesterolemia, used either as a single agent or in combination with TNF-a inhibits growth of MmBl6 melanoma and L1210 leukemia cells.

Keywords: tumor necrosis factor alpha; antiproliferative activity; lovastatin.
Effect of Cyclosporine A (CsA), Cyclophosphamide (Cy) and Azathioprine (Aza) on the Survival of Listeria innocua in Mice
MARIA WALENCKA, MONIKA BLOCH and TERESA GOŚCICKA

Abstract. Immunosuppressors reducing the defensive mechanisms of the organism make favourable conditions for the development of different infections, particularly for intracellular facultative bacteria such as Listeria. In this paper, the effects of different doses of cyclosporine A (CsA), cyclophospamide (Cy) and azathioprine (Aza) on the survival of Listeria innocua, a nonpathogeneic strain, in the peritoneal cavity and spleen of BALB/c mice, were investigated. It was found that CsA and Aza, applied on total doses: 50-250 µg/g or 20-100µg/g respectively, before or on the day of bacteria injection, prolong their longer survival as a result of reduced elimination. In CsA treated animals the number of survived Listeria is higher in the spleen while in Aza treated animals – in the peritoneal cavity. The Cy effect appeared to be the weakest because only the immunosuppres- sive (300 µg/g) dose was able to significantly enhance the survival of L. innocua both in the peritoneal cavity and the spleen. The results are a warning for clinicists applying the immunosuppressor treatment.

Keywords: immunosuppressors; Listeria innocua; bacterial survival.
Effect of H202 on Adriamycin-Induced Cytotoxicity in Vitro
ELŻBIETA L. ANUSZEWSKA

Abstract. Normal and malignant cell lines, sensitive and resistant to adriamycin, have been treated with H202 to look for the possibility of protection from the cytotoxic action of adriamycin on the cells. Pre-treatment of adriamycin-resistant cells with H202 partially protects them against the cytotoxicity of adriamycin.

Keywords: adriamycin; hydrogen peroxide; sensitive and resistant cell lines; cytotoxicity.

Cellular Immunology and Immunopharmacology

Lymphocyte Phenotype Studies of Rheumatoid Arthritis Patients Treated with Methotrexate
JAN K. ŁĄCKI, STEFAN H. MACKIEWICZ and KRZYSZTOF E. WIKTOROWICZ

Abstract. Seventy nine patients with rheumatoid arthritis (RA) were monitored for 12 months. Forty eight individuals were treated weekly with a low dose of methotrexate (MTX), and 31 received nonsteroid antiinflammatory drugs (NSAID) only. The mean total dose of MTX was 413 mg with a range from 250 to 735 mg. The phenotype of lymphocytes was analyzed using double immunofluorescence and a panel of monoclonal antibodies. The clinical status of 33 MTX-treated patients improved, in 9 neither clinical nor laboratory improvement was noticed. Six patients were withdrawn from the study because of adverse reactions. We observed no changes in the percentage of CD3, CD4, CD8 and CD2S positive lymphocytes in both MTX and NSAID-treated groups. However, the percentage of CDl9 positive cells significantly decreased during 12 month observation (15.1 ±6.5% v.s. 10.2±5.0, p < 0.01) in MTX-treated subjects. Moreover, this percentage increased (9.4±6.7% v.s. 17.1 ± I.1 %, p < 0.01) in NSAID-treated patients.

Keywords: rheumatoid arthritis; methotrexate; lymphocyte phenotype.
Contrasting Effect of Oral and Intravenous Cyclophosphamide Treatment on Phenotypes of Human Peripheral Blood Lymphocytes
JAN K. ŁĄCKl, STFFAN H. MACKIEWICZ and KRZYSZTOF E. WIKTOROWICZ

Abstract. Twenty three rheumatoid arthritis (RA) patients treated with cyclophosphamide (CTX) were observed for 6 months. Eight patients received CTX in a single intravenous dose (group I) and 15 orally in a single daily dose (group II). The surface antigens of lymphocytes, isolated from peripheral blood, were determined using immunofluorescence method. The clinical improvement was observed both in group I and in group l I of patients. However, in patients receiving CTX intravenously amelioration of the disease appeared quicker. The percentage of CD3+ and CD8+ T cells remained unchanged. In group I we observed decrease in the number of CD4+ T cells (60.1 ± 11.5% and 43.8± 12.5%, before and after treatment respectively, p < 0.01), in group II this level remained unchanged. In both groups the percentage of CDl9+ B cells decreased (14.7 ± 9.2 % and 8.0 ± 6.1 %, before and after treatment respectively in group I, p < 0.01; 17.4 ± 12.3 % and 11.0 ± 7.1 %, before and after treatment respectively in group II, p < 0.01). Moreover, the percentage of activated T cells (CD2S+ cells and HLA-DR+ cells) was reduced in both groups.

Keywords: rheumatoid arthritis; cyclophosphamide; lymphocyte phenotype.
Influence of Various Forms of Dialyzable Leukocyte Extracts on Rat Adjuvant Arthritis
MÁRIA STANĆIKOVÁ , JOZEF ROVENSKÝ, JÁN PEKÁREK , EDUARD ORVISKÝ, STANISLAVA BLAZÍCKOVÁ and KAREL CECH

Abstract. Adjuvant-induced arthritis in rats is a chronic inflammatory disease, widely used as an animal model for rheumatoid arthritis. In our study the effect of various fractions of dialyzable leukocyte extract (DLE): DLE I-molecular weight below 10 kDa (commercial preparation), DLE II-molecular weight below 5 kDa (suppressor fraction), DLE III-molecular weight 5 -10 kDa on rat adjuvant-induced arthritis was studied. The adjuvant arthritic (AA) rats were treated with DLE fractions i.p. in solutions containing an active substance isolated from 12.5 x 106 and 6.25 x 106 leukocytes from day 1 (adjuvant injected) through day 18, every second day (total 9 times). Various markers of inflammation, immune function and joint destruction were evaluated: hindpaw volume, serum hyaluronic acid, serum albumin and biopterin in urine. All these markers showed a significant improvement after using fraction DLE II in comparison with AA controls. Fractions DLE I and DLE III influenced only some markers of inflammation and immune function. Our results demonstrated a therapeutical effect of fraction DLE II on rat adjuvant-induced arthritis.

Keywords: dialyzable leukocyte extracts; transfer factor; adjuvant arthritis
Changes in the Ultrastructure and Functions of Immunocompetent Cells under the Influence of External Heating, Interleukin 1 ß and Leukocytic Pyrogen
PETR P. MURZENOK and NINA I. NETUKOVA

Abstract. The structural and functional organization of the rabbit immunocompetent cells after injection of human recombinant interleukin 1 ß (IL-I ß), partially purified leukocytic pyrogen (LP), or after external heating (36 C, 1.5 h) was compared. A rectal temperature increase being the same, there were similar and transient changes in epithelial and endothelial cells and macrophages, a rise in the proliferative activity of thymocytes and synthesis of IL-1-like activity, and a decrease in the interleukin 2 (IL-2) production by cultured splenic cells.

Keywords: ultrastructure and proliferation of immunocompetent cells; hyperthermia; interleukin 1 ß.
Influence of the Perinatal Period on Bovine Interferon Production
MARTYNA KANDEFER-SZERSZEŃ, MARIAN KONDRACKI, AGNIESZKA SZUSTER-CIESIELSKA, WOJCIECH RZESKI, BARBARA ZDZISIŃSKA and STANISLAW CĄKAŁA

Abstract. Interferon (IFN) production in cultures of leukocytes of calves immediately after birth, 2-3-day-old calves, dams and nonpregnant lactating cows was compared. For IFN induction Radom strain of Newcastle disease virus (NDV-R), phytohemagglutinin (PHA) and concanavalin A (ConA) were used. Cultures of newborn-calf peripheral blood mononuclear cells (PBMC) separated from blood by Ficoll-Hypaque density centrifugation or leukocytes cultivated by whole blood technique responded to all inducers used with a significantly lower IFN level than those of 2 – 3-day-old calves, dams and lactating cows. Although the mechanism of this phenomenon is not clear, our data connot rule out a defect of IFN synthesis in blood leukocytes of newborn calves. Spontaneous antiviral activity was detected in fetal membranes and in colostral leukocytes. Colostral leukocytes were effcient producers of IFN after induction with NDV-R, but less efficient than blood leukocytes after induction with PHA and ConA. Low response to PHA and ConA correlated with low number of lymphocytes in colostrum.

Keywords: newborn calves; interferon production.
Effect of Macrophage-Modulating Agents on in vivo Growth of Transplantable Lewis Lung Cancer in Mice
ANDRZEJ NOWICKI, GRAŻYNA OSTROWSKA, SHARON L. AUKERMAN and WIESLAW WIKTOR-JĘDRZEJCZAK

Abstract. C57B1/6 mice, bearing transplantable Lewis lung cancer (non-metastatic subline) implanted either subcutaneously or intraperitoneally were treated with macrophage colony stimulating factor (M-CSF, 106 units per mouse, per day for 19 days), Escherichia coli lipopolysaccharide or both. Lipopolysaccharide (5 µ/g per mouse) administered daily once a day for up to 30 days impaired both subcutaneous and intraperitoneal tumor growth and prolonged survival of tumor bearing mice. Macrophage colony stimulating factor, administered daily, inhibited only subcutaneous tumor growth, both when administered alone and in combination with lipopolysaccharide, and had no effect on intraperitoneal tumor. Moreover, it did not prolong survival of tumor bearing mice, when administered alone, and nullified the effects of lipopolysaccharide when administered concomitantly. These data suggest that macrophage colony stimulating factor, at least in this tumor model and in this dose schedule, offers little benefit. In contrast, the present data confirm earlier suggestions on therapeutic usefulness of bacterial lipopolysaccharide in neoplastic disease, which makes this compound an interesting candidate for future clinical trials.

Keywords: macrophages, lipopolysaccharide; colony stimulating factor; lung cancer.
Cytotoxic T Lymphocyte Response to Herpes Simplex Virus Type 1 Is Composed of Both CD8 + and CD4 + T Cell Phenotypes in Acute and Memory States
MAKEK G. NIEMIALTOWSKI and BARRY T. ROUSE

Abstract. Mice were infected via the cornea with HSV-1. Next, draining lymph nodes (DI.N) and spleen cells were analyzed at various times post infection for the presence of cytotoxic T lymphocyte precursors (CTL-p) of both the CD8+ and CD4+ phenotypes. Responses were greatest in the DLN, but memory CTL persisted in the spleen and were undetectable in DLN by 60 days. On all occasions, the frequency of CD8+ CTL outnumbered CD4+ CTL. The murine CTL responses to HSV-1 differ from those in man where CD4+ MHC class II restricted CTL appear to dominate the response at least in the memory phase.

Keywords: herpes simplex virus type 1; cytotoxic T lymphocytes; acute and memory response.
Effect of Carbon Tetrachloride Intoxication and Ethanol Ingestion on Interferon Production in Mice
JADWIGA DANILUK, DANIEL CHIBOWSKI, AGNIESZKA SZUSTER-CIESIELSKA and MARTYNA KANDEFER-SZERSZEŃ

Abstract. Sixty C3H/He male mice were divided into 6 groups (10 mice per group). Control mice (group I) received three injections of PBS and drinking water. Mice of group II were injected with PBS but drinking water was substituted by ethanol solutions with increasing concentration of ethanol (w/v): 6% during the first week of experiment, IO% during the second and 20% during the third week. Group III received three intraperitoneal injections of CCl4 (1 ml of CCl4 per 1 kg of body weight) and water for drinking. Group IV was treated with CCl4 as group III, but drinking water was substituted by ethanol solutions with increasing concentrations as in the group II. Samples of blood, liver and spleen were taken 24 h after the third acute CCl4 intoxication. Group IIIa and IVa were treated as group III and IV, respectively, but samples of blood and organs were taken a week after the last CCl4 injection. A typical increase in serum ALT and necrosis of hepatocytes as confirmed by the histological examination, was observed 24 h after CCl4 injections (group III and IV). A week later (group IIIa and IVa) regenerative changes in the liver and a significant decrease in ALT serum activity was observed. Acute CCl4 intoxication (group III) significantly decreased IFN production in liver and spllen cells isolated 24 h after the last CCl4 injection. Combined CCl4 and ethanol administration affected very strongly IFN production (group IV). Despite the fact that ethanol itself did not cause any histological changes in mouse liver and did not influence ALT serum activity, treatment of mice only with ethanol for three weeks decreased IFN production by liver and spleen cells after induction in vitro either with the virus or PHA and ConA (group II). When liver and spleen cells were examined seven days later after CCl4 and ethanol treatment (group IIIa and IVa) the IFN titers were sligthly higher than in group III and IV. It appears that both acute liver injury with CCl4 and a long-term ethanol consumption may act synergistically in depressing IFN production in mice.

Keywords: carbon tetrachloride; ethanol; interferon production; liver necrosis.
N-Acetyl-ß-D-Glucosaminidase Enzymuria as an Indicator in Monitoring the Therapy of Some Rheumatic Diseases with Potentially Nephrotoxic D rugs
PIOTR WILAND and JACEK SZECHIŃSKI

Abstract. The measurement of NAG enzymuria by the spectrofluorimetric assay according to Merle et al. enables an early diagnosis of tubular dysfunction. Urinary NAG activity was determined in 13 patients with various rheumatic diseases during administration of potentially nephrotoxic drugs like nonsteroidal anti-inflammatory drugs (NSAID), aminoglycosides, gold salts, cyclosporin and steroids. The significant decrease of urinary NAG activity was common in patients with recently diagnosed rheumatic diseases, who received steroids. The decrease of NAG enzymuria was correlated with biochemical indices of inflammation like ESR or hemoglobin. The use of two types of potentially nephrotoxic drugs, like NSAID and gentamicin or NSAID with cyclosporin induced significant augmentation of NAG excretion. This occurrence may precede azotemia. The recognition of high NAG enzymuria permits to reduce dosage or discontinue treatment with potentially nephrotoxic drug prior to irreversible renal insufficiency as shown in the case of a patient with psoriatic arthritis treated with simultaneously administered cyclosporin and diclofenac.

Keywords: N-acetyl-ß-D-glucosaminidase in urine; nephrotoxic drugs; nonsteroidal anti-inflammatory drugs; rheumatic diseases; tubular dysfunction.
Opsonic Activity of Sera in Children with Hematological Malignancies Treated with Sandoglobulin
MARIA MELEŻYŃSKA-MATEJ, GRAŻYNA WRÓBEL, JANINA BOGUSLAWSKA-JAWORSKA and KRYSPINA GRZYBEK-HRYNCEWICZ

Abstract. Twenty seven children with hematological malignancies were treated with Sandoglobulin for life threatening infections due to severe granulocytopenia. We have studied the opsonic activity of sera in patients before and 7,14 and 21 days after the infusion of Sandoglobulin. Before the therapy a decrease of serum opsonic activity at the stage of ingestion and intracellular killing of bacteria has been shown. It was due to a deficiency of opsonizing factors. After treatment with Sandoglobulin the significant improvement of the opsonic activity of tested sera was found, but only at the stage of the ingestion of bacteria. The optimal interrelationship between opsonizing capacity of sera at the ingestion and intracellular killing phase was observed in the group of children treated with the relatively low Sandoglobulin dose (0.3-0.6 g/kg). In patients with the longest infection duration, who received the high Sandoglobulin doses ( > 0.6 g/kg), the largest percentage of sera containing immune complexes was detected. These data demonstrate that high doses of globulins should be administered with certain care.

Keywords: opsonic activity of sera; hematological malignancies; intravenous immunoglobulin; infections.

Vol. 42, No. 5-6, 1994

Mini-Review

Is Sex Worth Searching for?
MICHAL WITT

Abstract. Several methods of molecular sexing (i.e. detection of Y-chromosomal sequences) have been briefly presented. In addition to other applications they can be used for monitoring of patients after sex-mismatched bone marrow transplantation for mixed chimerism and for detection of fetal cells in maternal circulation. Clinical significance of mixed chimerism and possibilities of non-invasive prenatal diagnosis with the use of fetal cells isolated from peripheral blood of pregnant women has been described.

Keywords: molecular sexing; mixed chimerism after bone marrow transplantation; fetal cells in maternal circulation.

Cellular Immunology

Presentation of Antigen by B Cell Subsets. II. The Role of CDS 5 Cells in the Presentation of Antigen to Antigen-Specific T Cells
MICHAŁ ZIMECKI and JUDITH A. KAPP

Abstract. We demonstrate that peritoneal B cells have a much higher ability to present antigen to antigen- -specific T cell lines than splenic B cells. Presentation of antigen by B cells is abrogated or drastically reduced after removal of Lyb-5+ cells from the population of splenic or peritoneal B cells. Peritoneal B cells, precul- tured for 7 days prior to the antigen presentation assay, retain their antigen presenting cell (APC) function. Enrichment for CDS+ cells in the peritoneal B cell population results in a more effective antigen presentation. Lastly, stimulation of B cells via CDS antigen, by treatment of cells with anti-CD5 antibodies or cross-linking of CDS receptors, enhances APC function of these cells. The results indicate, both indirectly and directly, that CDS+ B cells play a predominant role in the presentation of conventional antigens to antigen-specific T cells.

Keywords: Lyb-5 B cells; antigen presentation.
Presentation of Antigen by B Cell Subsets. III. Effects of Interleukins on Antigen Presenting Function and Phenotype of Immature B Cells
MICHAŁ ZIMECKI and JUDITH A. KAPP

Abstract. In this report we demonstrate that B cells from CBA/N or (CBA/N x BALB/c)Ft male mice with x-linked immunodeficiency, that have very limited ability to present antigen to antigen-specific T cells, acquire this function following preincubation with IL-l, IL-4 and to a lesser degree with IL-6 and IL-5. Preincubation of normal B cells with these B-tropic interleukins does not lead to enhancement of their APC function. Incubation of B cells from the peritoneal cavity and spleen of xid mice with B cell tropic interleukins (IL-1, 4, 5 and 6), but not with IL-2 or IL-3, induces appearance of Lyb-5 antigen on these cells. The study demonst- rates that the property of inducing APC activity in immature B cells is correlated with the acquirement of Lyb-5 antigen.

Keywords: antigen presentation; xid mice; immature B cells; interleukins; Lyb-5 antigen.
Presentation of Antigen by B Cell Subsets IV. Defective T-B Cell Signalling Causes Inability to Present Antigen by B Cells from Immunodeficient Mice
MICHAŁ ZIMECKI and JUDITH A. KAPP

Abstract. The purpose of this study was to investigate differences in T-B cell signalling between B cells from normal and immunodeficient mice. B cell blasts from normal and immunodeficient mice expressed comparable levels of membrane-associated IL-l. B cells from normal, but not immunodeficient mice, prefixed with glutar- aldehyde and cultured with thymocytes or a T cell line BK33, induce in T cells production of a factor which causes release of IL-1 by macrophages. This factor, preincubated with B cells from immunodeficient mice significantly enhances their APC function. Furthermore, this cytokine induces expression of Lyb-5 alloantigen on B cells from immunodeficient mice. This effect could be blocked by neutralizing antibodies to I L-6 but not to IL-2, IL-4 or GM-CSF. We conclude that immature B cells from immunodeficient (CBA/N x BALB/c)F, mice are unable to stimulate interacting T cells to produce IL-6 and therefore are inefficient antigen presenting cells.

Keywords: antigen presentation; xid mice; immature B cells; surface interleukin 1; interleukin 6.
Human Immunoglobulin Production in Scid Mice Following Primary Sensitisation of Human Lymphocytes in Situ
MACIEJ KURPISZ, WILLIAM WALKER, MAX FIELD and GRANT GALLAGHER

Abstract. We have grafted human peripheral blood mononuclear cells to the peritoneal cavity of SCID mice, exposed them to soluble antigens in the absence of adjuvant and examined their ability to make specific antibody. The effect of in vitro pre-priming of donor cells on antibody production was determined. Human IgM and IgG antibodies were found in the SCID serum following in situ antigenic challenge and there was good evidence of antigen specificity. The total Ig levels and the relative antigen binding ability were diminished by in vitro pre-priming. The results demonstrate that primary human antibodies against antigens to which the donor is probably naive can be generated by in situ sensitisation in SCID mice, but that optimisation for each antigen may be required.

Keywords: vaccination; human monoclonal antibodies; in vitro sensitization; severe combined immunodeficiency mice.

Clinical Immunology

Development of IgG Subclasses in Healthy Polish Children
HANNA GREGOREK, DOROTA IMIELSKA, JAROSLAW GORNICKI, JOLANTA MIKOLAJEWICZ, BARBARA PRZERADZKA and KAZIMIERZ MADALŃSKI

Abstract. To determine the ontogeny of IgG subclasses in healthy Polish children IgGl, IgG2, IgG3, IgG4 concentrations were measured by enzyme-linked immunosorbent assay in 30 cord blood sera and sera from 152 healthy children aged 4 months to 16 years. The geometric mean of concentrations all of IgG subclasses in age-related groups of children were calculated. In cord sera, four IgG subclasses were found with the IgG 1 level reaching about 90%, IgG2 – 52%, IgG3 – 80% and IgG4 – 33% of the mean adult values. In the group of youngest children (4-6 months of age) the concentration of all IgG subclasses were significantly decreased in comparison to initial values. Then, IgGl and IgG3 levels increased rapidly reaching ca. 50% of the mean adult values by 1 year of age. We also demonstrated much slower maturation of IgG2 and IgG4 which lagged behind IgGl and IgG3 development, especially in younger children.

Keywords: ELISA; IgG subclasses; normal values in children.
Effect of Hydrocortisone Treatment on Oxygen Metabolism and Phagocytosis of Peripheral Blood Neutrophils in Asthmatic Patients, Sensitive and Resistant to Glucocorticosteroid Therapy
RENATA JANKOWSKA, TERESA ŻAK-NEJMARK, JÓZEF MAŁOLEPSZY, GRAŻYNA NADOBNA, MARIA OSOS and WOJCIECH MĘDRALA

Abstract. The inflammatory activity of peripheral blood neutrophils of patients suffering from atopic asthma and treated with glucocorticosteroids was investigated. Two tests were performed: l. chemiluminescence test of neutrophils, and 2. phagocytosis test of latex and bacteria (Staphylococcus aureus 209 P) by neutrophils before and after incubation of the cells with hydrocortisone. The tests were carried out in 3 selected groups of (1) asthmatic steroid sensitive patients, (2) asthmatic steroid resistant patients, and (3) healthy individuals. The neutrophil chemiluminescence stimulation index was found to be significantly lower in the asthmatic steroid sensitive group as compared to the other groups. The index increased in this group after neutrophil incubation with hydrocortisone. The results of phagocytosis of latex by neutrophils before and after their incubation with hydrocortisone were similar in all the 3 groups. The results of phagocytosis of bacteria by neutrophils in the asthmatic steroid sensitive group were significantly lower after incubation of neutrophils with hydrocortisone only.

Keywords: asthma; glucocorticosteroid treatment; chemiluminescence; phagocytosis; neutrophils.
Essential Edema-Proteinuria-Hypertesion (EPH) Gestosis and Gestosis Superimposing Pre-Existing Renal Disease: Comparison of Cellular Immunity Parameters
ANDRZEJ MALINOWSKI, MARIAN SZPAKOWSKI, HENRYK TCHÓRZEWSKI, KRZYSZTOF ZEMAN, PRZEMYSLAW OSZUKOWSKI and LECH PODCIECHOWSKI

Abstract. The paper is aimed at evaluation of certain indices of cellular immunity in pregnant women with gestosis superimposing pre-existing renal diseases and comparing them with the immunological condition of women with essential gestosis. T lymphocyte subpopulations and lymphocyte proliferative responses to mito- gens (phytohemagglutinin – PHA, concanavalin A – Con A and pokeweed mitogen – PWM) in the fetal calf serum and autological sera were studied. The groups of examined pregnant women in the 3rd trimester did not vary with regard to gestation age, calendar age and to the severity of edema (E), proteinuria (P) and hypertension (H) gestosis symptoms. It has been found that equal changes occur in both groups of women, regarding the examined indices of cellular immunity, when referred to normal pregnant women. In the gestotic women a decreased absolute and percentage content of CD8+ T cells and increased percentage of CD3+ and CD4+ lymphocytes were found in comparison with the normal pregnant women, which led to an almost 2.5-fold increase of CD4+/CD8+ ratio. The sera of gestotic women, in comparison with the healthy pregnant group, strongly increased the proliferation of Con A and PWM-induced lymphocytes. It may then be assumed that the observed immunological changes do not coincide with the primary causes of gestosis. We suggest that immunological factors largely contribute to the development of essential gestosis and gestosis superimposing pre-existing renal disease.

Keywords: essential gestosis; superimposed gestosis; immunity
Polymorphonuclear Leukocytes (PMN) Activity in Peripheral Blood in Patients with Esophageal Carcinoma
EWA JABŁOŃSKA, ZOFIA PIETRUSKA, MARIAN FURMAN and MIROSŁAW KOZŁOWSKI

Abstract. The aim of this study was to evaluate some of the PMN functions connected with phagocytosis in 34 patients with esophageal carcinoma, confronted with a clinical progression of the neoplastic disease, according to TNM classification. The level of the neoplastic markers – SCC and CEA antigens were determined also. In the examined group of patients significant decrease in the mean values of FcR and CR receptors expression, migration, chemotaxis and phagocytosis of PMN were observed. No correlation was found between the percentage cells with FcR and CR and phagocytosis and clinical progression of neoplasma. Significant dif- ferences were in mean values of chemotaxis and migration in group with IV stage TNM. There was no found correlation between granulocytic tests and levels of SCC and CEA.

Keywords: esophageal carcinoma; polymorphonuclear leukocytes; receptor for Fc IgG; receptor for C3 component of complement; chemotaxis; migration; phagocytosis; carcinoembryonic antigen; squamous cell carcinoma antigen.

Cytostatics

Effect of Blood Transfusion and Cyclophosphamide on Cardiac Allograft Survival in Sensitized Mice
WITOLD LASEK, MAVDALENA SORA and MAREK JAKÓBISIAK

Abstract. In the H-2-incompatible donor-recipient model in mice (BALB/c -> CBA/H), combination of do- nor-specific blood transfusion (DST) on the day -9 before transplantation with both pre- and posttransplant immunosuppression with cyclophosphamide (CY) interacted beneficially to produce significant donor-specific prolongation of cardiac graft survival. However, in recipients presensitized with donor-specific blood on the day -21, combination of DST with pre- and posttransplant CY immunosuppression did not act synergistical- ly and graft survival in this group did not differ from that in presensitized mice treated with 2 doses of CY alone.

Keywords: blood transfusion; cyclophosphamide; cardiac transplantation; sensitized mice.
Pharmacological Properties of Racemic Chlorobromofosfamide
MARIA JUSZKIEWICZ, ZDZISLAW KLEINROK and ZYGMUNT SAWINIEC

Abstract. Racemic chlorobromofosfamide (CBM-4A) is a bromine substituted analog of ifosfamide. The stu- dies reported here were aimed at establishing the general pharmacological properties and toxicologic profile of this compound. Acute and cumulative toxicity, behavioral screening tests and effects of subchronic and chronic treatment (influence on body weight, mortality, blood morphology, proteins level, urea and creatinine concentration, enzymes activity) were taken into account. Summarized data revealed that CBM-4A possessed similar properties as other oxazaphosphorine cytostatics.

Keywords: oxazaphosphorine cytostatics; chlorobromofosfamide
Experimental Antitumor Activity and Toxicity of the Selected Triazolo- and Imidazoacridinones
HALINA KUŚNIERCZYK, WIESŁAW M. CHOŁODY, JOLANTA PARADZIEJ-ŁUKOWICZ, CZESŁAW RADZIKOWSKI and JERZY KONOPA

Abstract. Toxicity and antitumor effects of four compounds from the groups of triazoloacridinones and imidazoacridinones were evaluated in transplantable tumor systems in mice, including P388 leukemia, B 16 melanoma and 2 colon adenocarcinomas C26 and C38. Tested compounds had moderate antileukemic activity but were active against B16 melanoma and 3 of them were very efficacious against colon tumors, providing high percentages of „cures”. Toxicity for healthy mice, as well as antitumor activity, were found to depend on a treatment protocol. The compounds were better tolerated and gave higher antitumor effects when given as fractionated treatment. They displayed also sex-dependent toxicity and activity.

Keywords: triazoloacridinones; imidazoacridinones; antitumor activity; transplantable mouse tumors; toxicity; sex-dependent effects.

Cytokines

Seasonal Modulation of Interferon Response in Spotted Sousliks (Spermophilus suslicus)
MARTYNA KANDEFER-SZERSZEŃ, STEFAN MĘCZYŃSKI, LILIANA BOROWSKA’ AGNIESZKA SZUSTER-CIESIELSKA and WOJCIECH RZESKI

Abstract. Interferon production in spotted sousliks in different physiological states: in summer activity, summer torpor, winter hibernation and awaked from winter hibernation was studied. Three different interferon inducers: poly rI:rC complexed with DEAE-dextran, lipopolysaccharide from E. coli (LPS) and tilorone hydrochloride were used. In comparison with sousliks active in summer, the interferon levels in serum and different organs of sousliks in winter hibernation after induction with tilorone hydrochloride, LPS and poly rI : rC were significantly lower. Decreased interferon response was also observed in sousliks in summer torpor and awaked from winter hibernation. In contrast to hibernation, artificial acidosis induced by placing sousliks in the atmosphere with a high concentration of COz enhanced interferon production.

Keywords: hibernation; torpor; interferon production
Treatment of Chemotherapy-Induced or Idiopathic Bone Marrow Aplasia with Granulocyte Colony-Stimulating Factor (G-CSF)
TADEUSZ ROBAK, EUZEBIUSZ KRYKOWSKI and KRZYSZTOF WARZOCHA

Abstract. We report on our experience with the application of recombinant human granulocyte colo- ny-stimulating factor (G-CSF, Neupogen, Hoffmann La Roche) in patients with various types of hemato- logical malignancies, who had chemotherapy-induced myelosuppression, and in patients with idiopathic aplastic anemia. The administration of G-CSF was associated with marked increase in white blood cells counts (WBC) in twelve out of 14 treated patients. In one patient with aplastic anemia the WBC decreased rapidly to the initial value after the cessation of cytokine therapy. Significant increase of platelet number was observed in 6 patients. No toxicity was encountered with the hematopoietic growth factor therapy. Our study points to the fact that G-CSF have a stimulating effect on the regeneration of hematopoiesis, particularly within the granulopoietic compartment. The effect can be obtained both in a case of idiopathic- and cytostatic-dependent marrow aplasia.

Keywords: bone marrow aplasia; granulocyte colony-stimulating factor
Interferon and Tumor Necrosis Factor Responses of HIV + Patients as Markers for Monitoring of the AIDS Progression
EGBERT PIASECKI, TOMASZ K. LEDWOŃ, ANNA D. INGLOT, BRYGIDA KNYSZ, KRZYSZTOF SIMON, MAŁGORZATA INGLOT and ANDRZEJ GŁADYSZ

Abstract. The blood samples taken from 31 HIV+ and AIDS patients were used to study interferon (IFN) and tumor necrosis factor (TNF) responses. The IFN and TNF levels in plasma were determined. In the whole blood assay (whole blood diluted 1:10 with culture medium) Newcastle disease virus (NDV) and phytohemagglutinin (PHA) were used as cytokine inducers. Blood leukocytes of HIV+ patients produced significantly less IFN-a after NDV stimulation than the cells of healthy (HIV-) individuals. On the other hand, the production of IFN-y in response to PHA was impaired only in AIDS patients with stage CDC IV and CD4+ cell number < 200/µl. These patients had also increased IFN levels in plasma. Particularly, the high level of IFN in plasma was frequently detected in patients with progressing AIDS with CD4+ cell number < 50/µl. This type of IFN was identified as a mixture of acid-labile and acid-stable IFN-a. The IFN responses of HIV+ patients may be considered as markers for monitoring progression of AIDS and therapy.

Keywords: interferon; tumor necrosis factor; cytokines; human leukocytes; AIDS; HIV; HIV-positive patients.
Attempts to Isolation and Characterization of Autolymphocytotoxins from Syphilitic Sera
JADWIGA PODWIŃSKA, MARIA CHOMIK and ANDRZEJ GAMIAN

Abstract. To solve problem if autolymphocytotoxins (AL) present in syphilitic sera are not circulating immune complexes (CIC), sera containing strong AL activity and weak CIC were precipitated with polyethylene glycol (PEG). Received PEG-precipitates were filtrated on Bio-Gel A-1.5 column to separate CIC from AL. The A1 were found in the first three after void volume fractions of the column. The fractions were examined on content of protein, sugar and sialic acid. The only correlation seen refers to the sugar and protein. The fraction which display the AL activity have the ratios of sugar to protein higher then non active fractions. The data indicated besides that AL is not CIC because it is not immunoglobulin nor Treponema pallidum antigen. For further biochemical characterization of these factors more material is needed.

Keywords: autolymphocytotoxins; circularing immune complexes

Various

Search of Antibodies for Immunoenzymatic Assay of Human Lutropin
APOLINARY SZEWCZUK , MARIANNA KUROPATWA , IWONA KOCHANOWSKA , ALICJA MAJEWSKA, ZBIGNIEW MACKIEWICZ, GOTFRYD KUPRYSZEWSKI and ANDRZEJ RAPAK

Abstract. Five peptides corresponding to human lutropin (hLH) subunit fragments were synthesized by a solid phase method and their physicochemical characteristics are presented. Antibodies induced in rabbits with 3 peptides of ßhLH fragments coupled to thyrotropin did not bind hLH and human chorionic gonadotropin (hCG). Also 2 synthetic peptides of ahLH fragments did not react with rabbit anti-hLH, anti-hCG and anti-ahCG antibodies. Using 2 monoclonal anti-ahCG and anti-ßhLH antibodies a sensitive sandwich ELISA technique was elaborated. Using this technique stability of hLH was investigated. The ELISA was also applied for assays of urine hLH in normal and ovulation days.

Keywords: lutropin; human luteinizing hormone (hLH); ELISA of hLH; stabilization of hLH; assays of hLH in urine
Synthesis and Biological Studies on Analogs of Cyclolinopeptide A with a Shortened Peptide Chain
IGNACY Z. SIEMION , ARTUR PĘDYCZAK , IWONA STRUG and ZBIGNIEW WIECZOREK

Abstract. Continuing our work on the immunosuppressive activity of cyclolinopeptide A (CLA), a cyclic nonapeptide of the sequence: c-(-Leu-Ile-Ile-Leu-Val-Pro-Pro-Phe-Phe), we synthesized a series of 6 linear analogs of CLA, successively shortened at the N-terminus of the peptide chain. Immunosuppressive activity of acetates as well as trifluoroacetates of these peptides was investigated using PFC (humoral immunity) and DTH (cellular immunity) tests. It was found that the immunosupressive potency of the peptides examined diminishes with shortening of the peptide chain. Octapeptide I with the sequence Ile-Ile-Leu-Val-Pro- -Pro-Phe-Phe was found to be the most active of the whole series. The immunosuppressive activity increased again for a tripeptide fragment of CLA of the sequence Pro-Phe-Phe. The immunosuppressive activity of octapeptide probably depends on the supression of IL-1, IL-6 and TNF production by the cells involved in immune and inflammatory response.

Keywords: cyclolinopeptide A; peptide immunosuppressors; shortened cyclolinopeptide A analogs.

Vol. 42, No. 1, 1994

CONTENTS

Mini-Review

Discovery, Mechanism and Expected Clinical Significance of Selective Cytotoxicity of 2-Chloro-2′-Deoxyadenosine (2-CdA)

2-Chloro-2′-Deoxyadenosine – Clinical, Biochemical and Pharmacokinetic Considerations

Cladribine (2-Chloro-2′-Deoxyadenosine) : New Perspectives in Clinical Immunosuppression

Treatment of Hairy Cell Leukemia Patients with 2-Chloro-2′-Deoxyadenosine (2-CdA)

Systemic Administration of 2-Chloro-2′-Deoxyadenosine (2-CdA) in Patients with Systemic Scleroderma

2-Chloro-2-Deoxyadenosine (2-CdA) Treatment in a Patient with Waldenström’s Macroglobulinaemia Complicated by Severe Neutrocytopenia

Influence of 2-Chloro-2′-Deoxyadenosine Alone and in Combination with Cyclophosphamide or Methotrexate on Murine Leukemia L1210

2-Chloro-2′-Deoxyadenosine (2-CdA) Combined with Cyclosporine A Successfully Prevents Rejection of Fetal Brain Stem Allograft in Rabbits

Modulation of Lymphocyte-Fibroblast Interactions by 2-Chloro-2′-Deoxyadenosine (2-CdA) in Subarachnoid Hemorrhage

Various

Comparative Evaluation of Toxicity and Antitumor Activity of Original cline Drugs and Reproduced Anthracy

Immunosuppressive and Cytogenetic Effects of Pelvic Irradiation on the Peripheral Lymphocytes of Patients with Cervical Cancer. One Year Follow-Up

Standardization of HeLa Cells Preparations as the Source of snRNA’s for Preliminary Study on the Effectors of pre-mRNA Splicing

Heparin Enhances Generation of Natural Killer Activity in Vitro

In Vitro Primary Antitumor Screen of Imidazole Platinum Complexes

Vol. 42, No. 2, 1994

CONTENTS

Mini-Review

Cellular Immunology

Immunochemistry

Various

Vol. 42, No. 3, 1994

CONTENTS

Cellular Immunology

Immunochemistry

Transplantation

Cytokines

Vol. 42, No. 4, 1994

CONTENTS

Mini-Review

Allergy

Cytostatics

Cellular Immunology and Immunopharmacology

Vol. 42, No. 5-6, 1994

CONTENTS

Mini-Review

Cellular Immunology

Clinical Immunology

Cytostatics

Cytokines

Various