ALBERT

Description: The majority of sinonasal non-Hodgkin's lymphomas (NHLs) are thought to originate from T-cell lineage. However, they often express natural killer (NK)-cell markers so that their origin still remains obscure. In this study, cell type of sinonasal NHLs were characterized by immunohistochemical and Southern blot analyses. We examined nine patients with sinonasal NHL. Six patients with tonsillar or pharyngeal non-B-cell lymphomas served as a control group. Immunohistochemical study showed that all nine cases of sinonasal NHL were CD56+CD2+, whereas controls were CD56-CD2+. According to the rearrangement of T- cell receptors (TCRs) and expression of CD3 markers, the sinonasal NHL cases were classified into three groups: TCR-CD56(Leu-19)+CD3(Leu4)- NHL (three patients), TCR-CD56+CD3+ NHL (five patients), and TCR+CD56+CD3+ NHL (one patient). In contrast, control patients' NHLs were TCR+CD56-CD3+. These results imply that eight cases of TCR-CD56+ sinonasal NHL are of NK-cell lineage. Among these eight cases, TCR- CD56+CD3+ cases (five of eight patients) were rather similar to the phenotype of fetal NK cells. From these results, the majority of sinonasal NHLs seem to originate from varying maturation stages of NK- cell lineage.

Description: In an attempt to clarify the mechanisms of cytotoxicity mediated by CD4+ cytotoxic T lymphocytes (CTL), the expression of perforin and membrane-bound lymphotoxin (LT) (tumor necrosis factor-beta) in herpes simplex virus (HSV)-specific CD4+ human cytotoxic and noncytotoxic T- cell clones was examined. Three HSV-specific CD4+ human CTL clones that showed HLA-DR-restricted cytotoxicity and proliferative response were established. The cytotoxicity of these clones in 5-hour 51Cr release assays was found to be mediated by the directional target cell lysis and not by the release of cytotoxic soluble factors, ie, “innocent bystander” killing. Northern blot analysis showed that messenger RNAs for perforin and LT, which were both considered to be important mediators for cytotoxicity of CD8+ CTL and natural killer cells, were abundantly expressed in HSV-specific CD4+ CTL clones. Expression of perforin in the cytoplasm of CD4+ CTL clones was also detected by immunohistochemical staining using a monoclonal antibody against perforin. In addition, LT bound to the cell surface of CD4+ CTL clones was detected by flow cytometry. In contrast, little or no expression of perforin and LT was detected in three HSV-specific CD4+ noncytotoxic T- cell clones. Although the cytotoxicity mediated by lymphokine-activated killer cells was partly inhibited by addition of anti-LT antibody, it did not show any effect on the cytotoxicity of HSV-specific CD4+ CTL clones. In addition, it was found that cytotoxicity mediated by these CD4+ CTL clones was Ca2(+)-dependent. These data thus suggest that perforin and membrane-bound LT are both expressed in HSV-specific CD4+ CTL, although perforin might be the more important mediator in short- term culture.

Description: The Ws mutant allele of rats represents a 12-base deletion at the tyrosine kinase domain of the c-kit gene. Although homozygous Ws/Ws rats were deficient in both connective tissue-type mast cells (CTMC) and mucosal-type mast cells (MMC), mast cells did develop when bone marrow cells of Ws/Ws rats were cultured in the presence of concanavalin A-stimulated spleen cell conditioned medium (ConA-SCM). Although the proliferative response of rat cultured mast cells (RCMC) derived from Ws/Ws rats to ConA-SCM was comparable to that of RCMC derived from control normal (+/+) rats, the proliferative response of Ws/Ws RCMC to rat recombinant stem cell factor (rrSCF; a ligand for the c-kit receptor tyrosine kinase) was much lower than that of +/+ RCMC. However, a slight c-kit kinase activity was detectable in Ws/Ws RCMC, and the proliferation of Ws/Ws RCMC was accelerated when rrSCF was added to ConA-SCM. Because CTMC contain rat mast cell protease-I (RMCP- I) and MMC contain RMCP-II, the phenotype of +/+ and Ws/Ws RCMC in various culture conditions was evaluated by immunohistochemistry of RMCPs. Both +/+ and Ws/Ws RCMC showed the MMC-like phenotype (RMCP-I- /II+) when they were cultured with ConA-SCM alone. Most +/+ RCMC and about half of Ws/Ws RCMC acquired a novel protease (RMCP-I+/II+) phenotype when they were cultured with rrSCF alone. However, because the number of Ws/Ws RCMC dropped to one-tenth in the medium containing rrSCF alone, the absolute number of Ws/Ws RCMC with the RMCP-I+/II+ phenotype did not increase significantly. The effect of rrSCF in inducing the novel phenotype was suppressed when ConA-SCM was added to rrSCF. In contrast, +/+ and Ws/Ws RCMC cocultured with +/+ fibroblasts showed the RMCP-I+/II+ phenotype even in the presence of ConA-SCM. Moreover, a fibroblast cell line derived from SI/SI mouse embryos that did not produce SCF did not support the survival of both +/+ and Ws/Ws RCMC but did induce the RMCP-I+/II+ phenotype in about half of +/+ and Ws/Ws RCMC when their survival was supported by the addition of ConA- SCM. The normal signal transduction through the c-kit receptor did not appear to be prerequisite for the acquisition of the RMCP-I+/II+ phenotype.

Description: Analyses of the kininogen (KGN) molecule and KGN gene status in five Japanese families with high-molecular-weight (HMW) KGN deficiency were performed by the immunoblotting method with monoclonal antibodies to HMW-KGN, and by the Southern blotting method with the cDNA for human low-molecular-weight prekininogen. No molecular abnormality of KGN was detected in the DNA from four patients with total KGN deficiency or one patient with isolated HMW-KGN deficiency. In the former, the KGN gene appeared to be grossly normal at the level of the whole genome on Southern blotting. In isolated HMW-KGN deficiency, a partial deletion in intron 7 was found by restriction analyses of EcoRI, BamHI, HindIII, Sca I, and Bgl II fragments. This partial deletion is assumed to be related to an abnormality of the alternative RNA splicing events for HMW-prekininogen mRNA.

Description: Protein C (PC) is considered to be an important regulator of blood coagulation and fibrinolysis. During the production of monoclonal antibodies (MoAbs) against human PC in mouse ascitic fluid, one hybridoma was found to induce heavy thrombus in mice, resulting in severe hemorrhage. Intravenous infusion of the purified MoAb (PC01) from this hybridoma also caused thrombosis in mice. The crossreacting substance was then isolated from mouse plasma with PC01 immunoaffinity column, which was identified as mouse PC by several criteria. Mouse PC prolonged the activated partial thromboplastin time of mouse plasma, and PC01 neutralized this in vitro anti-coagulant activity. Therefore, heavy thrombosis observed in PC01-treated mice is likely to be ascribed to the defect of PC caused by PC01.

Description: Sodium dodecyl sulfate-polyacrylamide gel electrophoresis followed by immunoblot analysis of plasma thrombomodulin concentrate revealed that four degraded forms of thrombomodulin with different molecular weights are present in plasma. Plasma concentrations of thrombomodulin in patients with various diseases were measured by two methods of enzyme- linked immunosorbent assay using monoclonal antibodies. One method measures intact thrombomodulin and degraded forms of thrombomodulin; the other does not detect the two smaller degraded forms of thrombomodulin present in plasma. The results indicated that thrombomodulin was increased in the circulating blood of patients with disseminated intravascular coagulation syndrome, pulmonary thromboembolism, adult respiratory distress syndrome, chronic renal failure, or acute hepatic failure. The different values obtained by the two methods indicate that the increase of plasma thrombomodulin found in these patients was mainly due to an increase of the smaller fragments of degraded forms, suggesting that the release of thrombomodulin from endothelial cells was accelerated in various disease states by proteolytic activity generated on the surface of the endothelium and may be removed from the circulation mostly by the kidneys and liver.

Description: Protein C (PC) is considered to be an important regulator of blood coagulation and fibrinolysis. During the production of monoclonal antibodies (MoAbs) against human PC in mouse ascitic fluid, one hybridoma was found to induce heavy thrombus in mice, resulting in severe hemorrhage. Intravenous infusion of the purified MoAb (PC01) from this hybridoma also caused thrombosis in mice. The crossreacting substance was then isolated from mouse plasma with PC01 immunoaffinity column, which was identified as mouse PC by several criteria. Mouse PC prolonged the activated partial thromboplastin time of mouse plasma, and PC01 neutralized this in vitro anti-coagulant activity. Therefore, heavy thrombosis observed in PC01-treated mice is likely to be ascribed to the defect of PC caused by PC01.

Description: Sodium dodecyl sulfate-polyacrylamide gel electrophoresis followed by immunoblot analysis of plasma thrombomodulin concentrate revealed that four degraded forms of thrombomodulin with different molecular weights are present in plasma. Plasma concentrations of thrombomodulin in patients with various diseases were measured by two methods of enzyme- linked immunosorbent assay using monoclonal antibodies. One method measures intact thrombomodulin and degraded forms of thrombomodulin; the other does not detect the two smaller degraded forms of thrombomodulin present in plasma. The results indicated that thrombomodulin was increased in the circulating blood of patients with disseminated intravascular coagulation syndrome, pulmonary thromboembolism, adult respiratory distress syndrome, chronic renal failure, or acute hepatic failure. The different values obtained by the two methods indicate that the increase of plasma thrombomodulin found in these patients was mainly due to an increase of the smaller fragments of degraded forms, suggesting that the release of thrombomodulin from endothelial cells was accelerated in various disease states by proteolytic activity generated on the surface of the endothelium and may be removed from the circulation mostly by the kidneys and liver.

Description: The expression of the hematopoietic transcription factors GATA-1, GATA- 2, and GATA-3 was studied in eosinophils and basophils. Eosinophils express mRNA for GATA-1, GATA-2, and GATA-3. Basophils express GATA-2 and GATA-3. Treatment of HL-60 eosinophilic sublines with either interleukin-5 or butyric acid increased the expression of GATA-1 mRNA concomitant with the expression of eosinophil-specific genes, whereas levels of GATA-2 mRNA remained relatively constant. The presence of mRNA for these proteins in eosinophils and basophils suggests that gene transcription in these lineages may be regulated by GATA-binding proteins.

Description: We administered recombinant human interleukin-1 beta (IL-1 beta), the common mediator of inflammation process, to C57B1/6 male mice (0.5 microgram, every 12 hours over five times) intraperitoneally and consequently induced a remarkable thrombocytosis. Day 1 was designated as the following day of the last injection in the morning. A significant thrombocytosis was observed on days 1 through 5 with a peak on day 2 (162 +/- 9 x 10(4)/mm3) compared with the control mice injected with heated IL-1 beta (101 +/- 11 x 10(4)/mm3). A striking increase in mean size of marrow megakaryocytes was noted on days 1 and 2. The incorporation of 75Se-selenomethionine into circulating platelets as a measure of platelet production was about 2.3 times higher in IL-1 beta-treated mice than in control mice. To determine which factor(s) is responsible for elicited thrombocytosis, the in vitro studies and bioassays for several hematopoietic factors were performed. IL-1 beta by itself did not stimulate megakaryocytopoiesis in vitro, suggesting that the thrombocytosis is attributed to other factor(s) via IL-1 beta stimulation. Serum colony-stimulating factor (CSF) activity after a single IL-1 beta (0.5 microgram) injection, monitored by colony assay with 10% tested serum, peaked at 3 hours. Formed colonies were mostly granulocyte (G) and granulocyte-macrophage (GM)-types, and studies using rabbit anti-mouse GM-CSF serum or using human marrow as target cells showed that the CSF activity of the tested serum consisted of, at least, GM-CSF and G-CSF. Addition of IL-3 concomitantly with the tested serum gave rise to a greater number of megakaryocytic colonies. Serum IL-3, monitored by IL-3-dependent cell line 32D clone 5, and erythropoietin activities were not detected at serum level in IL-1 beta-treated mice. Serum IL-6 assay by IL-6- dependent mouse hybridoma cell line MH-60.BSF2 showed high levels of the tested serum with a peak at 2.5 hours with no detection at 10 hours after the injection. Heated IL-1 beta caused an increase of neither IL- 6 nor CSF activities. Our data suggest that the thrombocytosis induced by IL-1 beta is mediated by IL-6 or a combination of IL-6 and other cytokine(s), and that IL-6 may play a regulatory role in platelet production in vivo.