CONTENTS
Mini-Review
- Trials of Protecting Poland from Acquiring Bovine Spongiform Encephalopathy
JERZY KITA and HENRYK MACIOŁEKAbstract. The article contains a short review on transmissible spongiform encephalopathies, Creutzfeld-Jakob disease (CJD) and bovine spongiform encephalopathy (BSE), technology of meat and bone meal production, regulations and monitoring. Poland has imposed a ban on the importation of live cattle, sheep, and food products of ruminant origin from countries where BSE has been identified. Imported meat should be documented with a valid certificate stating that is does not originate from cattle whose parents were imported from Holland, United Kingdom, Ireland and Switzerland. Currently no licences are being issued for importation of live cattle, bovine semen or bovine embryos. The Polish Ministry of Agriculture has appeal to born meal producers to observe the Polish Norm PN-92R-64809.
Keywords: bovine spongiform encephalopathy; Creutzfeld-Jakob disease (CJD); new variant CJD; meat and bone meal technology and regulations in Poland.Poland.
Immunochemistry
- Purification of Human Anti-TF (Thomsen-Friedenreich) and Anti-Tn Antibodies by Affinity Chromatography on Glycophorin A Derivatives and Characterization of the Antibodies by Microtiter Plate ELISA
MARIA DUK and ELWIRA LISOWSKAAbstract. The TF and Tn antigens were obtained from glycophorin A (GPA) by desialylation under mild acidic conditions and by desialylation followed by Smith degradation, respectively. A method of purification of anti-TF and anti-Tn antibodies from human sera by affinity chromatography on the immobilized asialoGPA (TF atigen) and on asialo-agalactoGPA (Tn antigen), respectively, is described. Purity of the antibodies was demonstrated by SDS-polyacrylamide gel electrophoresis and their specific reactivity with TF or Tn antigens was shown using hemagglutination and the microtiter plate ELISA. A high unspecific binding of human immunoglobulins to the ELISA plates was encountered, therefore optimal conditions for the most specific binding of the antibodies to the target antigens were selected. Problems of the unspecific binding of immunoglobulins were more difficult to overcome when the antibodies were determined in whole sera by their binding to.antigen-coated ELISA plates.
Keywords: glycophorin; Thomsen-Friedendreich (TF) antigen; Tn antigen; anti-TF antibodies; anti-Tn antibodies, affinity chromatography; ELISA. ELISA.
- Serum IgG Antibodies in Children and Adults Reacting with Helicobacter pylori Lipopolysaccharides
MACIEJ CEDZYŃSKI, KEN-ICHI AMANO, MAGDALENA CHMIELA, MAGDALENA ŁAWNIK, ELŻBIETA CZKWIANIANC, IZABELA PŁANETA-MAŁECKA, WIESŁAWA RUDNICKA and WIESŁAW KACAAbstract. The presence of IgG antibodies reacting with Helicobacter pylori lipopolysaccharides (LPSS) in sera from children and adults diagnosed as H. pylori-infected, as well as healthy persons, was tested. There was no correlation between the production of antibodies reacting with H. pylori surface proteins and LPSS. Also no correlation between reactivity of tested sera with H. pylori antigens and deep rough mutant (Re types) enterobacterial LPSs was revealed. The prevalence of anti-LPS IgG in randomly selected children was relatively high.
Keywords: Helicobacter pylori: lipopolysaccharide; IgG.ccharide; IgG.
- Influence of Some Factors on Immunoassays of Human Myoglobin
IWONA E. KOCHANOWSKA, MARIANNA KUROPATWA and APOLINARY SZEWCZUKAbstract. Six ELISA variants exploiting two monoclonal antibodies, one rabbit antibody and their peroxidase conjugates were applied in assays of purified human myoglobin, apomyoglobin and the protein in human muscle extracts. The myoglobin was accurately determined with monoclonal antibody no. 82 used for coating of ELISA plates while assays performed with monoclonal antibody no. 49 or rabbit antibody used for coating were weak or none. Determinations of human apomyoglobin with ELISA variants were somewhat more sensitive than those of myoglobin. Obtained in this work results were compared with those done usiner commercial Seratec kit for immunoassav of human myoglobin. Addition to the muscle extracts not only7 concentrated salts but also acetone, ethanol, sodium dodecyl sulfate or some other denaturing agents markedly increased assays of myoglobin by ELISA with monoclonal antibody no. 49 and antibody no. 82 conjugated with peroxidase. Removal of acetone or ammonium sulfate from extracts resulted in dramatic decrease of the estimated myoglobin. Filtration of the extract through Bio-Gel A5m column did not affect low assays of myoglobin in fractions without pretreatment with acetone. Myoglobin was isolated from human heart extract by immunoaffinity chromatography on Sepharose-antibody no. 82 column and the isolated protein was identified by gel electrophoresis and Western blot.
Keywords: human myoglobin; immunoassay of myoglobin; ELISA; muscle extracts; complexes of myoglobin.globin.
Cytokines
- Serum Levels of Tumor Necrosis Factor a and Production of this Cytokine by Polymorphonuelear Cells in Breast Cancer Patients
EWA JABŁOŃSKAAbstract. Unstimulated as well as zymosan and LPS-stimulated production of tumor necrosis factor Y. (TNF-oc) by polymorphonuclear cells (PMNS) was investigated in breast cancer patients. Serum levels of TNF-ot were also determined. Spontaneous TNF-cz production was lower, comparable with that found in healthy persons. Zymosan and LPS-stimulated PMNs of cancer patients were found to produce significantly lower amounts of TNF-cz, as compared with PMNs of healthy control. There was no significant differences iii the serum levels of TNF-oc between breast cancer patients and healthy persons. Because the peripheral blood cells contain more than 50% PMNS, the altered TNF-ot production by PMNs may have a series of relevant implications during malignant process and seems to be one of the factors helpful in determining the cellular immunology potency of breast cancer patients.
Keywords: polymorphonuclear cells; tumor necrosis factor a .
- Lactoferrin Lowers Serum Interieukin 6 and Tumor Necrosis Factor a Levels in Mice Subjected to Surgery
MICHAł ZIMECKI, ANDRZEJ WŁASZCZYK, TADEUSZ ZAGULSKI, and ANDRZEJ KÜBLERAbstract. Mice subjected to thymectomy or splenectomy in general anesthesia release interleukin 6 (IL-6) and tumor necrosis factor a . (TNF-a ) into circulation reaching high concentrations after 4 h following operation. In the case of thymectomy IL-6 can be detected only on the day of operation and TNF-a attains a maximal value on day 3 postoperation. Splenectomy, which is a more extensive surgical operation, results in a higher, and more prolonged existence of IL-6 in circulation accompanied by higher levels of TNF-a . Bovine lactoferrin (BLF; 10 mg/mouse), given intravenously (i.v.) 24 h before thymectomy, reduced, on avarage, the level of serum IL-6 by 70% as measured 4 h after operation. The inhibiting effect of BLF on TNF-a production was smaller with a mean 30% reduction. The effects of BLF (i.v.) administration on the cytokine levels following splenectomy were less inhibitory. BLF caused an approximate 35% fall in IL-6 levels and even weaker effects (20% inhibition) on TNF-a release. Application of much lower (1 -0.2 mg) per os doses of BLF was even more effective in lowering IL-6 levels after thymectomy (up to 90%) after 5 BLF doses, and by 55% of TNF-a . The data suggest that lactoferrin may find therapeutical application for diminishing manifestations of shock caused by clinical insults.
Keywords: interleukin 6; tumor necrosis factor a ; thymectomy; splenectomy; bovine lactoferrin.
- Interferon and Tumor Necrosis Factor Production during Endotoxemia in Sheep
KAZIMIERZ BIENIEK, AGNIESZKA SZUSTER-CIESIELSKA, TERESA KAMIŃSKA, MARIAN KONDRACKI, IRENA ŁOKAJ and MARTYNA KANDEFER-SZERSZEŃAbstract. In humans endotoxemia has often been associated with the development of adult respiratory distress syndrome (ARDS). Sheep have an abundant population of pulmonary intravascular macrophages, therefore they are a popular animal model for ARDS. In this study we characterized the temporal sequence and duration of the release of two cytokines: tumor necrosis factor (TNF) and interferon (IFN) and evaluated the effect of lipopolysaccharide (LPS) dose. Rectal temperature and white blood cell (WBC) count were also measured. Twenty four adult sheep were given E. coli endotoxin at a dose of 0 (saline solution) 0.05, 0.1 and 1.0 m g/kg of body weight by intravenous (i.v.) bolus. In all groups, TNF-a was produced earlier (3 – 4.5 h) after injection than IFN (4 – 5 h). No correlation between increased rectal temperature, the magnitude of leukopenia and time course of both cytokines production was observed. No straight relationship between LPS dose and the titer of cytokines was seen, but lower doses of LPS-induced delayed cytokine response in comparison to the dose 1 m g/kg of LPS. As 1FN, present in the circulation of sheep, was mainly a /b type, the role of this class of IFN in endotoxemia is discussed.
Keywords: sheep tumor necrosis factor; interferon; endotoxemia.
Immunopharmacology
- Interactions of 2′,2′-Difluorodeoxycytidine (Gemcitabine) with Tumr Necrosis Factor a or Its Mutein VI in Murine Leukemias L1210 and P388
EWA MARAŃDA and TADEUSZ ROBAKAbstract. The interactions of 2′,2′-difluorodeoxycytidine (gemcitabine) with tumor necrosis factor a (TNF-a ) or its mutein VI on the survival time of mice bearing L1210 and P388 leukemia was investigated. Four hundred eighty male CD2F1 mice were used in the experiments. They were given gemcitabine (20 mg/kg) on days 1, 4, 7 and 10 after i.p. inoculation with leukemic cells (day 0). Cytokines were administered i.p. at a dose of 250 m g/kg as daily injections for 8 days or at a dose of 400 m g/kg given 2, 4, 6 and 8 days after day 0. Drugs were administered alone and in combination. Our study indicates that TNF-a and its mutein VI decrease the antileukemic effect of gemcitabine on murine leukemias L1210 and P388.
Keywords: gemcitabine; tumor necrosis factor a ; mutein VI; leukemia L1210; leukemia P388; interactions.
- Effect of Serine Proteinase Inhibitors on Intracellular Free Calcium Rise in Human Polymorphonuclear Leukocytes
DARIUSZ NOWAK, MACIEJ KRÓL and PIOTR BIAŁASIEWICZ
Abstract. The respiratory burst of polymorphonuclear leukocytes depends on activation of many enzymes and generation of variety of second messengers. One of important links leading to polymorphonuclear leukoeytes (PMNL) activation is a transient rise in intracellular free calcium concentration ([Ca2+]i). Serine proteinase inhibitors such as a -1-proteinase inhibitor (a 1PI), phenylmethylsulphonylfiuoride (PMSF) and soybean trypsin inhibitor (SBTI) were reported to inhibit human PMNL respiratory burst. In this study we tested the hypothesis whether these inhibitors can inhibit the rise in [Ca2+]i after stimulation of human PMNL with 10-7 M n-formyl-methionyl-leucyl-phenylalanine (FMLP), leukotriene B4 (LTB4) or platelet activating factor (PAF). [Ca2+]i was measured with use of a fluorescent probe Fura-2AM under conditions of 100 nM and 1 mM extracellular Ca2+ concentration. Preincubation of PML with 600 mg/dl of a lPI or SBTI for 30 min at 37o C had no influence on Ca2+ response to all agonists in comparison with cells treated with the same concentration of human serum albumin. However, 0.5 mM PMSF enhanced 1.7-fold (p < 0.002) [Ca2+]i rise after challenge with FMLP while did not affect significantly Ca2+ response to PAF and LTB4. The stimulatory effect of PMSF after addition of FMLP was dependent on increased Ca2+ influx from extracellular space. Our results suggest that suppression of PMNL respiratory burst by serine proteinase inhibitors is not mediated via Ca2+ pathway and that some proteases take part in Ca2+ response to FMLP.
Keywords: intracellular calcium; polymorphonuclear leukocytes; a -1-proteinase inhibitor; phenylmethylsul-phonylfluoride; n-formyl-methionyl-leucyl-phenylalanine; leukotriene B4.