We have shown previously that administration of endotoxin induces a smaller decrease of body temperature in spontaneously hypertensive rats (SHR) than in normotensive Brown Norway (BN) rats. Several studies have suggested that tumor necrosis factor đ (TNFđ) is one of the mediators of the body-temperature response to endotoxin. To test whether the TNFđ gene could be involved in determination of the observed difference in the body-temperature response to endotoxin, we studied SHR (n = 6) and a congenic strain, SHR.1N (n = 5), which differs from SHR by a segment of chromosome 20 originating from BN and containing the TNFđ gene. Body temperature was recorded continuously by means of radiotelemetry. We showed that, in both strains, an intraperitoneal injection of endotoxin (500 Ţg/kg of body weight) induces a rapid hyperthermic phase (20--40 minutes post-injection), which is followed, first, by a hypothermic phase (100--120 minutes post-injection) and, then, by a late hyperthermic phase (seven hours). Although both strains demonstrated a similar trend in the response, a significant difference was observed between the two response curves (P = 0.0001). Further analysis at each time point revealed that the two strains differed significantly at a peak of the hypothermic phase (P = 0.035) and the late hyperthermic phase (P = 0.035). In conclusion, these data indicate that the differential chromosomal segment of SHR.1N contains a gene(s) causally related to the body-temperature response to endotoxin. In the light of previously published data, the TNFđ gene appears to be the most likely candidate gene within the segment.
IL-2 kinetics was assessed in mice vaccinated with irradiated syngeneic tumour vaccines carrying an inserted IL-2 gene and producing constitutively IL-2. For comparison, the kinetics of i.v. administered recombinant IL-2 was also examined. During regular time intervals after the vaccination or administration of recombinant IL-2, samples of serum and peritoneal fluid were collected and examined, using CTLL bioassay or its MTT modification. After i.p. administration of irradiated IL-2-producing plasmacytoma (X63-m-IL-2) vaccine, the levels of IL-2 were substantially higher in the peritoneal fluid than in the serum. Both in the peritoneal fluid and in the serum, the IL-2 level was increasing up to 60 min after administration and then it gradually decreased. The last time point when IL-2 was still detectable both in the peritoneal fluid and in the serum was 30 hrs. Almost identical results were obtained when the IL-2 levels were detected by the conventional CTLL assay, in which DNA synthesis was monitored by 3H-thymidine labeling, and by the isotope-free MTT modification of the CTLL assay, in which the DNA synthesis was monitored by staining. The MTT modification has the advantage of an isotope-free method. Comparison of two different IL-2-producing vaccines, a murine plasmacytoma X63-m-IL-2, with high IL-2 production, and murine sarcoma MC12-IL-2, with low IL-2 production, revealed that whereas after i.p. administration of the high producers, the peak of IL-2 was reached both in the peritoneal fluid and in the serum after 1 h, the administration of low producers gave the peak level of IL-2 later, 5 h after i.p. administration.
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Changes in Immunochemical Localization of Cytoskeletal Proteins in Human and Boar Spermatozoa before and after Acrosome Reaction
J. PALEČEK, J. PĚKNICOVÁ, M. VÍTŮ
Several cytoskeletal proteins (đ-tubulin, Đ-tubulin, actin, spectrin, tropomyosin, vimentin and cytokeratin) were studied in human and boar spermatozoa. Their localization was observed by means of specific antibodies using indirect immunofluorescence technique. Immunocytochemical results were confirmed by the Western blot technique. Cytoskeletal proteins were examined in ejaculated spermatozoa before and after acrosome reaction induced by the ionophore A23187. The immunofluorescence assay revealed that localization of the studied cytoskeletal proteins in human and boar spermatozoa differ remarkably. In human spermatozoa, the localization of actin and spectrin changed after acrosome reaction; on the other hand, in boar spermatozoa, the changes of localization concerned đ-tubulin, Đ-tubulin, actin and spectrin. The type of changes differs in the studied species and follows probably the course of acrosome reaction. The results suggest that cytoskeleton participates in the process of acrosome reaction of mammalian spermatozoa.
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Corneas from mice incompatible at both major histocompatibility complex (MHC) and non-MHC antigens were grafted orthotopically to unmodified and high-risk recipients. Production of interleukin-2 (IL-2) and IL-10 by cells from spleens and draining lymph nodes from corneal graft recipients was determined in vitro. Over 90% of corneal allografts suffered alloantigen-induced inflammatory reaction within the second or third week after surgery. However, only 56% of grafts in unmodified and 75% of grafts in high-risk recipients were irreversibly rejected. Cells obtained from draining lymph nodes from the vicinity of the eye of the corneal graft recipients produced significantly elevated amounts of IL-10 and decreased amounts of IL-2. This shift to the Th2 type cytokine response was observed after stimulation of the cells with graft donor MHC antigens, but not after stimulation with donor non-MHC or third-party alloantigens. No changes in cytokine production were detected in spleen. The enhancement of IL-10 production in the vicinity of the eye was a consequence of corneal grafting and did not correlate with the fate of the graft. The results thus show that orthotopic corneal transplantation induces a local shift to the Th2 type cytokine response, which might be considered another factor that contributes to the unique characteristics of the immunity in the eye and to the tolerance of an unusually high percentage of corneal allografts.
Enhanced IL-10 and Decreased IL-2 Production after Orthotopic Corneal Transplantation in Mice
Z. HAŠKOVÁ, M. FILIPEC, V. HOLÁŇ
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Frequencies of HLA-DRB1, -DQB1 and -DPB1 Alleles in Czech PopulationThe frequencies of phenotypes, alleles and allelic subtypes of DRB1 and DQB1 HLA loci in 420 unrelated individuals from the Czech population were determined. The frequencies of DPB1 alleles of the HLA locus were determined in 92 individuals. The assays were performed using the polymerase chain reaction (PCR) method or the restriction fragment length polymorphism (RFLP) analysis. The most frequent DRB1 allele was *07, the most frequent DQB1 allele was *03 and the most frequent DPB1 allele detected was *04. These assays define the extent of polymorphism of the HLA system and are useful for determining the selection strategy of HLA-identical donor-recipient pair suitable for bone marrow transplantation.
M. LOUDOVÁ, I. ŠRÁMKOVÁ, V. CUKROVÁ, Z. SIEGLOVÁ, R. BRDIČKA
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Short CommunicationThe distribution of HLA class II DRB1 and DQB1 alleles in cervical carcinoma patients was compared with the frequency of these alleles found in healthy population living in the Czech Republic. The RFLP analysis and PCR-SSP were used for DNA typing. Although the differences in the frequency of DRB1*03, DQB1*02 and DQB1*0303 alleles between the cases and the controls were rather large, corrected P values did not reach significance.
The Frequency of HLA-DRB1 and -DQB1 Alleles in Cervival Cancer Cases in the Czech Republic
E. HAMŠÍKOVÁ, Y. ŠRÁMKOVÁ, M. LOUDOVÁ, V. LUDVÍKOVÁ, L. ROB, V. VONKA
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Cytoskeleton of mammalian cells is a complex filamentous network of microtubules, microfilaments and intermediate filaments. Intermediate filament proteins can be divided into six types according to their sequence homologies, immunological cross-reactivities, compatibilities of self-assembly, and with respect to their occurrence in cell types (Franke, 1993). Vimentin, desmin, glial fibrillary acidic protein and peripherin (intermediate filament proteins of type III) are the most similar in terms of primary structure, and the genes encoding them have the same basic intron/exon structure (Klymkowsky, 1995). Vimentin appears mainly in mesenchymal cells (Franke, 1993). Vimentin filaments form, in cultured cell lines, extended networks that are partially coaligned with microtubules. Disruption and redistribution of microtubules induced by microtubule-acting drugs result in coiling of vimentin filaments (Traub, 1985). Monoclonal antibodies against vimentin are useful markers of cells of mesenchymal origin and can be used to study the interaction of vimentin filaments with other cytoskeletal structures (Dráberová and Dráber, 1993).
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