Establishment of IL-5-dependent early B cell lines by long-term bone marrow cultures

We established two different IL-5-dependent Ly1+ early B cell lines in long-term bone marrow culture system. One of them (J-87) is stromal cell (ST2) dependent and the other (T-88) is ST2 independent. Both J-87 and T-88 are B220+, Ly1+, sIgM-, Ia-, Thy1-, and IL-2R+, and respond to IL-3 and IL-5 in the presence of ST2. The T-88 can proliferate only in response to IL-5 in the absence of ST2. Southern blot analysis using JH probe revealed that configuration of IgH gene of both cell lines shows rearranged pattern. Binding assay for radiolabeled IL-5 to T-88 demonstrated that T-88 has two classes of IL-5 binding sites (low and high affinity) on the membrane. These data strongly suggest that there are IL-5-sensitive stages at both stromal cell-dependent and stromal cell-independent phases in early B cell development.     

Transforming growth factor beta is protective in host resistance against Listeria monocytogenes infection in mice.

The role of transforming growth factor beta (TGF-beta) in host resistance against Listeria monocytogenes infection was studied with mice. The constitutive expression of TGF-beta 1 mRNA was observed in the spleens and livers of mice before and after infection. Injecting the mice with anti-TGF-beta 1 peptide serum resulted in diminished antilisterial resistance, whereas the administration of human platelet-derived TGF-beta 1 enhanced the resistance. Moreover, mice were protected against lethal infection when treated with TGF-beta 1. These results suggest the TGF-beta 1 might be involved in antilisterial resistance. On the other hand, injecting the mice with TGF-beta 1 resulted in a decrease in the titers of endogenous gamma interferon, tumor necrosis factor alpha, and interleukin-6, which are crucial in antilisterial resistance, in sera and in extracts of spleen and liver. Thus, a complicated mechanism might be involved in the role of TGF-beta 1 in host resistance against L. monocytogenes infection.     

Invasion of human respiratory epithelial cells by Bordetella pertussis: possible role for a filamentous hemagglutinin Arg-Gly-Asp sequence and alpha5beta1 integrin

Bordetella pertussis, the agent of whooping cough, is capable of invading human respiratory epithelial cells. In this study, we investigated the mechanisms by which B. pertussis invades the human lung epithelial cell line A549 and normal human bronchial epithelial (NHBE) cells. In vitro adhesion and invasion assays using both cell types with a virulent B. pertussis strain and its isogenic mutants revealed profound defects in a mutant deficient in filamentous hemagglutinin (FHA) expression. In addition, a mutant in which an FHA Arg-Gly-Asp (RGD) site had been changed to Arg-Ala-Asp had significantly diminished invasiveness, although its adhesiveness was comparable to that of the parental strain. Furthermore, a synthetic RGD-containing hexapeptide inhibited invasion of both cell types by the virulent strain. These results demonstrate that an RGD sequence of FHA is involved in B. pertussis invasion of epithelial cells in vitro. Monoclonal antibodies directed against human alpha5beta1 integrin, but not other integrins, blocked invasion, indicating that this integrin is involved in B. pertussis invasion. Taken together, these findings suggest that B. pertussis FHA may promote invasion of human respiratory epithelial cells through the interaction of its RGD sequence with host cell alpha5beta1 integrin.     

Intracellular pH measurement in frog muscle by means of 31P-nuclear magnetic resonance.

The 31P-NMR technique was used for the monitoring of intracellular pH and studying its heterogeneity in the femoral biceps muscle of Rana catesbiana under anaerobic conditions. The value of intracellular pH of fresh muscle calculated from the chemical shift of intracellular inorganic phosphate (P1) was 7.3 on average and the line width of P1 was about 0.2 ppm. As the line width determined by the relaxation mechanism was 0.099 ppm, the P1 signal in fresh muscle was concluded to consist of overlapped narrow components, which indicated the heterogeneity of muscular pH (about 0.2 pH unit). Living muscle showed gradual acidification due to glycolysis and the decrease in heterogeneity. When glycolysis was suppressed by iodoacetic acid, slight alkalization due to the breakdown of creatine phosphate was observed. When the Lohmann reaction was suppressed by 2, 4-dinitro-1-fluorobenzene, rapid acidification accompanied by the appearance of a new acidic component was observed with the onset of ATP decrease. This new component was not detected in the muscle pretreated with glycerol to disrupt the transverse tubules. Therefore, it is likely that this new acidic component originates in the intracellular compartment, and not in the cellular difference.     

Rapid Detection of Species of the Opportunistic Yeast Trichosporon by PCR

Trichosporon species are opportunistic pathogens, associated with a high mortality rate in immunocompromised patients. Oligonucleotide primers were used to amplify a 170-bp fragment of small-subunit ribosomal DNA of all species in the genus Trichosporon by PCR. The primers amplify DNAs of all species in the genus Trichosporon, including six causative agents of trichosporonosis. DNAs of other medically important yeasts, such as Candida albicans and Cryptococcus neoformans, are not amplified by this detection system.     

Bruceine B, A Potent Inhibitor of Leukocyte-Endothelial Cell Adhesion

Leukocyte adhesion to vascular endothelial cells is an essential step in the development of inflammatory diseases. We have searched for inhibitors of leukocyte-endothelial cell adhesion that could be used as anti-inflammatory drugs and found that bruceine B (0.2 g/ml; 0.44 M) inhibited human neutrophil or T cell adhesion to tumor necrosis factor- (TNF) stimulated human umbilical vein endothelial cells (HUVEC). The inhibition of neutrophil adhesion to TNF-stimulated HUVEC by bruceine B was not derived from cytotoxic effects, as determined by measurement of the level of lactate dehydrogenase (LDH) activity in conditioned medium. The effect of bruceine B on neutrophil adhesion to HUVEC was not seen when the neutrophils were preincubated with bruceine B. However, inhibitory effects were evident when the HUVEC were preincubated with bruceine B. Bruceine B also inhibited neutrophil adhesion to lipopolysaccharide-stimulated HUVEC and T cell adhesion to TNF-stimulated HUVEC. These findings suggest that bruceine B may have anti-inflammatory activity.     

Twenty-six-Week carcinogenicity study of chloroform in CB6F1 rasH2-transgenic mice

The carcinogenic potential of chloroform was evaluated in a short-term carcinogenicity testing system using CB6F1 rasH2-Tg (rasH2-Tg) mice. Chloroform was administered to rasH2-Tg males at doses of 28, 90, or 140 mg/kg and rasH2-Tg females at 24, 90, or 240 mg/kg by oral gavage for 26 weeks. Wild-type (non-Tg) male and female mice received doses of 140 mg/kg and 240 mg/kg, respectively. N-methyl-N-nitrosourea was administered to rasH2-Tg mice by single intraperitoneal injection (75 mg/kg) as a positive control. In both the rasH2-Tg and non-Tg mice, there was no significant increase in the incidence of neoplastic lesions by chloroform treatment. The incidence of hepatocellular foci in the rasH2- and non-Tg females receiving 240 mg/kg was increased. Forestomach tumors and malignant tumors occurred in most of the rasH2-mice in the positive control group. Swelling or vacuolation of hepatocytes, a toxic change induced by chloroform, occurred in both the rasH2-Tg and non-Tg mice. It is concluded that chloroform, a putative human noncarcinogen, did not show evidence of carcinogenic potential in the present study using rasH2-Tg mice. This study suggests that the rasH2-Tg mouse model may not be appropriate for detecting nongenotoxic carcinogens. However, the sensitivity of rasH2-Tg mice to nongenotoxic carcinogens should be assessed with consideration of the results from the other ILSI-HESI project studies.