Selection of stereotyped VH81X-{micro}H chains via pre-B cell receptor early in ontogeny and their conservation in adults by marginal zone B cells

The pre-B cell receptor (preBCR) plays critical roles in early B cell differentiation. It has been shown that not all muH chains are capable of pairing with surrogate light (SL) chains to form preBCR. Here, we established a novel system to differentially identify two types of early pre-B cell populations in bone marrow and fetal liver of mice, one producing SL-pairing muH chains and the other producing SL-non-pairing muH chains. The former population accounted for 80% of all the early pre-B cells in adult bone marrow, while it accounted for only 20% of those in fetal liver. Comparison of the two types of pre-B cell populations in fetal liver revealed the structural difference between SL-pairing and -non-pairing muH chains encoded by the V(H)81X segment that was most frequently utilized in fetal liver pre-B cells but rarely expressed by B cells generated in adults. PreBCR played an important role in the positive selection of V(H)81X-muH chains carrying the characteristic sequences of the complementarity-determining region 3 with little or no nibbling or N nucleotide addition, leading to their predominance in neonatal splenic B cells. These fetal-type V(H)81X-muH chains were also detected in adult spleen, but almost exclusively in marginal zone (MZ) B cells in contrast to the adult-type V(H)81X-muH chains. This strongly suggests that neonatally generated and selected B cells expressing the stereotyped V(H)81X-muH chains are maintained in the adult MZ and could function as innate-like lymphocytes.     

CD40 ligand-independent B cell activation revealed by CD40 ligand-deficient T cell clones: evidence for distinct activation requirements for antibody formation and B cell proliferation

We report the capacity of CD40 ligand (CD40L)-negative T cell clones to activate human B cells. CD40L-negative T cells induce a level of B cell proliferation 10–20% of that seen with normal T cells. The signal provided by the negative clones is synergistic with that derived from a CD40L transfectant, and restores B cell proliferation to normal levels, showing that CD40L-negative T cell clones are not inherently inhibitory for B cells. Although their capacity to induce proliferation was much reduced, CD40L-negative T cell clones were still strong inducers of B cell differentiation to plasma cells. This differentiation to plasma cells was inhibited by a CD40L transfectant. The data are discussed with regard to the normal in vivo mechanism for maintaining B cell memory and memory antibody responses to T-dependent antigens.     

Non-redundancy of cytidine deaminases in class switch recombination

Class switch recombination (CSR), somatic hypermutation, and gene conversion are immunoglobulin diversification mechanisms that are strictly dependent on the activity of the activation-induced cytidine deaminase (AID). The precise role and substrate(s) of AID in these processes remain to be well defined. The closest homologue of AID is APOBEC-1, a bona fide mRNA-editing enzyme, which shares with AID the ability to deaminate cytidines within single-stranded DNA in vitro and in prokaryotic cells. To determine whether APOBEC-1 can therefore substitute for AID in activated B cells, we expressed human AID, a catalytic mutant thereof, and rat APOBEC-1 in AID-deficient murine B cells. Whereas AID rescued CSR, neither the inactive mutant nor APOBEC-1 could complement AID deficiency. This indicates that cytidine deaminase activity is necessary but not sufficient to initiate CSR, and suggests that AID is specifically targeted to its cognate substrate, the immunoglobulin genes or a distinct mRNA, by an as-yet-unknown mechanism.     

Lysenin: A new tool for investigating membrane lipid organization

Sphingomyelin is a major sphingolipid species in animal cells and is a major lipid constituent of plasma membranes. Recent reports have established important roles for sphingomyelin and its metabolites as second messengers in signal transduction events during development and differentiation. Sphingomyelin is also a major component of sphingolipid, cholesterol-rich plasma membrane microdomains, known as 'lipid rafts'. However, little is known about the organization of sphingomyelin in biological membranes. Lysenin is a recently discovered sphingomyelin-specific toxin. In the present review, we summarize the current characterization of this protein and describe our recent attempt to elucidate the organization of sphingomyelin in cellular membranes using lysenin as a unique tool.     

Mechanism(s) promoting HIV-1 infection of primary unstimulated T lymphocytes in autologous B cell/T cell co-cultures

Resting CD4(+) T cells in the lymphoid tissue (LT) are essential producers of virions at the beginning of HIV infection in vivo. We previously developed a model that allowed in vitro infection of non-prestimulated T lymphocytes in the presence of autologous B lymphocytes and complement. In this study, we try to clarify the mechanism(s) responsible for virus transmission in unstimulated autologous B cell/T cell co-cultures. Ex vivo analyses of patient plasma samples revealed that HIV was opsonized. Flow cytometry showed that opsonized virus preferentially bound to complement receptor (CR)-2 on B lymphocytes in primary B cell/T cell co-cultures. As indicated by cytokine measurements and transwell experiments, soluble factors seemed to play a minor role in enabling infection. Rather, direct interaction between B and T lymphocytes and direct binding of opsonized virus to CR2 on B cells turned out to be essential for productive infection. Antibodies blocking cell-cell adhesion inhibited p24 antigen production. An anti-CR2 antibody blocking C3d-CR2 binding also significantly reduced viral replication. Since the infection of unstimulated T cells by opsonized primary HIV isolates in the presence of B cells was highly efficient independent of the tropism of the virus, this mechanism may be critical in the pathogenesis of HIV.     

Analysis of wild-type and e antigen-defective hepatitis B viruses during the course of a short-term corticosteroid therapy in chronic hepatitis B

Hepatitis B virus (HBV), with a G-to-A point mutation at nucleotide 83 in the precore region (mutant HBV83), accounts for most cases of hepatitis B e antigen (HBeAg)-defective HBV. However, it is still not clear how mutant HBV83 is associated with HBe seroconversion. Twenty-six HBeAgpositive patients with chronic hepatitis B who received oral prednisolone (30 mg/day) for 3 weeks were studied to clarify the prevalence of mutant HBV83 during the treatment using polymerase chain reaction with a restriction fragment length polymorphism assay. Twelve (46%) patients seroconverted to anti-HBe 1 year after treatment, whereas 14 (54%) did not. The proportion of mutant HBV83 to whole HBV remained unchanged in both groups during an acute exacerbation induced by withdrawal of corticosteroids. Among 12 anti-HBe-0seroconverted patients, five (56%) of nine patients with only wild-type HBV at baseline developed detectable levels of mutant HBV83 while all three patients with a mixed viral population of wild-type HBV and mu tant HBV83 at baseline developed a higher pro portion of mutant HBV83 one year after treat ment. In contrast, these changes were observed in only one (14%) of seven who failed to seroconvert. The results indicate that a flare-up of hepa titis precedes emergence or selection of mutant HBV83, followed by HBe seroconversion in patients with chronic hepatitis B. © 1995 WiIey-Liss, Inc.     

Hepatic lysosomal enzymes activity and liver morphology after short-time omeprazole administration

The aim of the study was to establish the influence of short-time omeprazole administration on liver function and morphology. Omeprazole was administered intraperitoneally, twice daily, for 3 days to male Wistar rats in two doses: 0.571 mg/kg and 5.71 mg/kg. Control animals were treated with physiological saline. Half of the animals were sacrificed 12 hours after the last injection. The remaining rats were raised for another 6 weeks, without any xenobiotics, and sacrificed on the 47th day of the experiment. The activity of free and bound fractions of hepatic acid phosphatase, beta-galactosidase, beta-N-acetyl-glucosaminidase, cathepsin B, D and L, lipase, and sulphatase were determined spectrophotometrically in homogenates of the liver. The liver sections were examined by light microscopy with hematoxylin-eosin, azan, and periodic acid-Schiff stains. Marginally significant (p < 0.1) differences in activity of free sulphatase fraction, and free and bound fractions of beta-galactosidase were found in animals exposed to the higher dose of omeprazole and sacrificed 12 hours after the last injection. Enzymatic profiles were normalised during the next 6 weeks. Histological evaluation revealed small degenerative and adaptive changes in all examined groups. It could be concluded that observed differences of hepatic lysosomal enzyme activities were the result of accompanied chemical-induced peritonitis as previously reported, and not a direct drug-toxic effect.     

Double minutes and other chromosomal aberrations in two malignant cell lines of the German cockroach Blattella germanica

Structural anomalies of mitotic chromosomes from two tumorigenic cell lines of the German cockroach (Blattella germanica) is described. Aberrations, such as unpairable marker chromosomes, double minutes, di- and tricentrics, ring chromosomes, tri- and quadriradials, and chromosome and chromatid gaps and breaks, were observed in varying proportions. This study reports that the double minute chromosomes (DMs) are associated with insect tumor cells, similar to the findings in both murine and human tumor cells.     

Virus-inhibiting surgical glove to reduce the risk of infection by enveloped viruses

Needle puncture and other accidents that occur during surgery and other procedures may lead to viral infections of medical personnel, notably by hepatitis C (HCV) and human immunodeficiency virus (HIV), now that hepatitis B can be prevented by vaccination. A new surgical glove called G-VIR, which contains a disinfecting agent for enveloped viruses, has been developed. Herpes simplex type 1 (HSV) was used as a standard enveloped virus in both in vitro and in vivo tests of the virucidal capacity of the glove. Bovine viral diarrhea virus (BVDV) and feline immunodeficiency virus (FIV) were used as models for HCV and HIV, respectively. For in vitro study, a contaminated needle was passed through a glove and residual virus was titrated; for in vivo studies, animals were stuck with a contaminated needle through a glove. Despite variation in virus enumeration inherent in the puncture technique, statistical evaluation showed that infection was reproducibly and substantially reduced by passage through the virucidal layer. For BVDV, the amount of virus passing through the virucidal glove was reduced in 82% of pairwise comparisons with control gloves that lacked the virucidal agent; when plaque counts were adjusted to a common dilution, the median count for the virucidal glove was on the average reduced >10-fold. In experiments in which the proportion of wells infected with FIV was measured, the ratio of TCID(50) values (control glove to G-VIR) was >15, and probably much higher. For HSV, the amount of virus passing through the virucidal glove was reduced in 81% of comparisons with control gloves; the median of adjusted plaque counts was reduced on the average approximately eightfold or ninefold. In vivo tests with FIV and HSV in cats and mice, respectively, found smaller percentage reductions in infection than the in vitro tests but confirmed the virucidal effect of the gloves.