Persistent productive HIV infection in EBV-transformed B lymphocytes

The susceptibility to HIV infection of 14 B-cell lines established from five healthy HIV seronegative and from six HIV seropositive subjects by lymphocyte transformation with EBV and/or by lymphocyte cultivation with cyclosporin A was studied. Although the cell lines contained different proportions of CD4-positive cells, as shown by flow cytometry, all of them could be infected with the SF-2 strain of HIV. Infection was blocked by a monoclonal antibody directed against the viral attachment site of the CD4 molecule, even in a line that lacked demonstrable CD4 receptors. B-cell lines with high proportions of CD4-expressing cells produced HIV p24 antigen more rapidly and at higher concentrations than cell lines with low CD4 expression. Although HIV infection resulted in some cytopathic effects, it was possible to cultivate the infected cells for more than 8 months without refeeding the cultures with uninfected cells. Even in long-term cultures, there was a continuous production of infectious HIV, as detected by transfer of culture supernatants to other susceptible cell lines. The production of viral antigens was consistently more pronounced in the B-cell line with the highest CD4 positivity than it was in a permissive T-cell line (HUT-78) infected in the same manner. These results indicate that HIV can chronically and productively infect transformed B cells via interaction with CD4 molecules. Thus it is possible that B cells may constitute a source of infectious virus in HIV-infected EBV-positive individuals.     

Intimal changes associated with arterial spasm induced by periarterial application of calcium chloride

Arterial spasm was induced by application of calcium chloride to the adventitial surface of the rabbit common carotid artery in vivo. Sodium chloride (NaCl) was applied to the contralateral vessel as control. Vessels were fixed in situ by intravascular perfusion after 15 min, 1 hr, or 24 hr and prepared for light and scanning electron microscopy (SEM). With SEM, the luminal surface at the site of calcium application showed severe longitudinal folding accompanied by endothelial desquamation with extensive platelet deposition on exposed subendothelium. The luminal cross-sectional area was reduced by 53 +/- 19.5% after 15 min and by 44 +/- 12% after 1 hr as compared with the contralateral control. Furthermore, the luminal area at the site of calcium application was found to be reduced by 42 +/- 8% after 1 hr when compared with segments of the same vessel distal to the site of calcium application. Blood flow rate, as measured by electromagnetic flow probe, was not reduced. Vessels examined after 24 hr showed a significant increase in luminal cross-sectional area as compared with contralateral control vessels (136 +/- 70%). Control vessels (NaCl) showed no significant change in luminal cross-sectional area and no endothelial desquamation or platelet deposition after 15 min, 1 hr, or 24 hr. Examination of histologic sections showed calcium precipitation within the attached thrombus after 15 min with calcium deposits also adherent to the adjacent luminal aspect of the internal elastic lamina (IEL). By 24 hr, this precipitation extended throughout the media. Marked deposition of leukocytes was seen after 24 hr which showed a preferential attachment for areas of endothelial damage and discontinuity of IEL.     

Parameters of cellular immunity in perimenopausal patients with glandular and adenomatous endometrial hyperplasia

No appreciable disorders of cellular immunity were detected in patients with glandular cystic endometrial hyperplasia. Atypical endometrial hyperplasia was associated with quantitative changes in T lymphocytes and their subpopulations, decreased level of lymphocytes carrying activation antigens, and increased count of natural killers. These changes can be characterized as immunocompensation.     

Nucleotide mutations associated with hepatitis B e antigen negativity

One hundred and forty four patients with chronic hepatitis B were tested to identify new mutations associated with hepatitis B e antigen (HBeAg) negativity, using a full genome sequence analysis. All the patients were Chinese and had hepatitis B virus infection of genotype C. Patients with none of the pre-core or core promoter mutations were significantly (P < 0.001) less common in the group with anti-HBe (13%) than in the group with HBeAg (56%). The complete nucleotide sequence was determined in four anti-HBe-positive patients who had neither pre-core nor core promoter mutations and in five HBeAg-positive patients who also had neither of these mutations (the groups were matched for age and sex). Six mutations were found to be significantly more common in the former group than in the latter: G529A (3/4 vs. 0/5), C934A (4/4 vs. 1/5), A1053G (4/4 vs. 1/5), G1915T/A (4/4 vs. 0/5), T2005C/A (4/4 vs. 0/5), and C3026T (3/4 vs. 0/5). Three of the six mutations were significantly more common in the four anti-HBe-positive patients who had neither pre-core nor core promoter mutations, compared to 11 HBeAg-positive patients who had pre-core and core promoter mutations, and also compared to 15 anti-HBe-positive patients who had pre-core and core promoter mutations, suggesting further the specificity of these mutations. Of the six mutations, two resulted in amino acid substitution in the polymerase protein, and one is located near the enhancer I region. The results suggest that the six newly discovered mutations are associated with HBeAg negativity.     

C and V proteins of Sendai virus target signaling pathways leading to IRF-3 activation for the negative regulation of interferon-beta production

We here report a molecular basis for downregulation of interferon (IFN)-beta production by V and C proteins of Sendai virus (SeV). The infection of HeLa cells with SeV poorly induced IFN-beta even if the expression of C/C' was disrupted. In contrast, when the expression of C/C'/Y1/Y2 or V/W was disrupted, SeV infection strongly induced IFN-beta production and significantly activated the interferon regulatory factor (IRF)-3 pathway. The independent expression of C or V inhibited the double-stranded (ds) RNA- or Newcastle disease virus (NDV)-induced activation of IRF-3 and NF-kappa B, as well as the IFN-beta promoter. This inhibitory effect was also observed when Y1, Y2, or a C-terminal half fragment (aa 85-204) of C was independently expressed. Phosphorylation and homodimer formation of IRF-3 were suppressed not only in cells infected with SeV capable of expressing both C/C'/Y1/Y2 (or Y1/Y2) and V/W, but also in HeLa cells constitutively expressing Y1. These results suggest that C, Y1, Y2, and V block signaling pathways leading to IRF-3 activation to downregulate IFN-beta production.     

A Novel Lymphocyte Signaling Defect: trk A Mutation in the Syndrome of Congenital Insensitivity to Pain and Anhidrosis (CIPA)

Congenital insensitivity to pain with anhidrosis is a syndrome characterized by loss of pain and sensation. The condition frequently evolves into deep wounds and prolonged healing times. Anhidrosis is another prominent component of the disorder. Often associated with recurrent episodes of unexplained fever, it can result in patient mortality. Recent investigations point to Trk A, the high affinity receptor for nerve growth factor (NGF), as a candidate for the site of the mutation that causes the disorder. Functional NGF receptors, such as Trk A and the Trk family of tyrosine kinases, are essential for NGF signaling of human lymphocytes. In this study, we demonstrated that the presence of a trk A mutation in patient B cells results in a novel lymphocyte signaling defect. In these B cells, NGF failed to induce Trk A phosphorylation, cytoskeleton assembly, or MAP kinase activation. These abnormalities may explain some of the clinical features of the disease.     

Antibodies against the extracellular enveloped virus B5R protein are mainly responsible for the EEV neutralizing capacity of vaccinia immune globulin

In the event of smallpox bioterrorism, widespread vaccination may be required. Vaccinia immune globulin (VIG) has been used to treat complications from the smallpox vaccine. While the potency of VIG was defined by its ability to neutralize intracellular mature virus, a second form of vaccinia called the extracellular enveloped virus (EEV) is critical for virus spread in the host. The B5R-protein is one of many EEV-specific proteins. Immunoprecipitation and ELISA revealed that VIG recognizes the B5R-protein. An EEV plaque-reduction assay using a recombinant vaccinia that lacks the majority of the extracellular domain of B5R showed that the ability of VIG to neutralize EEV is principally directed at B5R. In addition, absorbing out the anti-B5R antibody present in VIG through the addition of recombinant B5R protein abrogated VIG's ability to significantly neutralize wild-type EEV. This work demonstrates the prominent role of B5R as a target of EEV-neutralizing activity of human antibodies.     

Molecular analysis of streptogramin resistance in enterococci

The new semi-synthetic streptogramin antibiotic combination quinupristin/dalfopristin (Synercid) is a promising alternative for a treatment of infections with multiple resistant gram-positive pathogens, e.g. glycopeptide- and multi-resistant Enterococcus faecium. Streptogramins consist of two unrelated compounds, a streptogramin A and B, which act synergistically when given in combination. Mechanisms conferring resistance against both components are essential for resistance against the combination in E. faecium. In this species resistance to streptogramin A compounds is mediated via related acetyltransferases VatD and VatE. Resistance against streptogramins B is either encoded by the widespread ermB gene cluster conferring resistance to macrolide-lincosamide-streptogramin B antibiotics or via expression of the vgbA gene, which encodes a staphylococcal-type lactonase. E. faecalis is intrinsically resistant to streptogramins. Due to a wide use of streptogramins (virginiamycins S/M) in commercial animal farming a reservoir of streptogramin-resistant E. faecium isolates had already been selected. Determinants for streptogramin resistance are localized on plasmids that can be transferred into an E. faecium recipient both in vitro in filter-matings and in vivo in the digestive tracts of rats. Hybridization and sequencing experiments revealed a linkage of resistance determinants for streptogramins A and B on definite plasmid fragments.     

Interaction of B7RP-1 with ICOS Negatively Regulates Antigen Presentation by B Cells

Stimulation of T cells through the T cell receptor is insufficient for optimal T cell activation. A second activation signal is necessary, being usually provided by the costimulatory molecule CD28. Recently, additional costimulatory pathways have been identified, including inducible costimulator (ICOS) and its ligand B7RP-1. We have examined the role of the B7RP-1/ICOS costimulatory pathway on antigen presentation by B cells, using the I-A^k and I-E^k-positive CH27 B cell line and several different T cell lines. We found that CH27 expressed B7RP-1 and PD-L1 whereas the T cell lines expressed ICOS and PD-1. In the presence of HEL, the T cell hybridomas C10 and 3A9 released IL-2, which is indicative of antigen-specific T cell activation by the CH27 cells. Unexpectedly, blocking antibodies for B7RP-1 and ICOS enhanced the IL-2 response in both T cells. As expected, an increase in the production of IL-2 was seen when blocking antibodies for PD-1 were used. Blocking with antibodies for I-A^k, CD28, B7.1, and B7.2 lead to a decrease in IL-2 production. Additionally we tested a Th1 and a Th2 T cell clone. Blockade of B7RP-1/ICOS lead to an increased IFN- response in Th1 cells (A.E7) and an increased IL-4 response in Th2 cells (D10.G4.1). Intracellular staining also showed an increase in cytokine production when the B7RP-1/ICOS pathway was blocked. In conclusion, the B7RP-1/ICOS pathway is negatively regulating T cell activation by B cells and may play a role similar to that of the PD-L1/PD-1 pathway.     

Stress-induced impairment of inhibitory avoidance learning in female neuromedin B receptor-deficient mice

Neuromedin B (NMB) is a mammalian bombesin (BN)-like peptide that exerts its function via the neuromedin B receptor (NMB-R). The NMB/NMB-R system is involved in stress response, and therefore we examined behavioral properties in female mice lacking NMB-R using a restraint-induced stress paradigm. Thirty minutes of restraint in a wire mesh cage constituted a sufficient stress stimulus for mice as evidenced by elevated blood glucose concentrations in stressed wild-type and NMB-R-deficient mice. Using a one-trial passive avoidance test, stressed NMB-R-deficient mice exhibited a marked reduction in memory performance. NMB-R-deficient mice exhibited elevated spontaneous activity in a novel environment compared to non-stressed mutant mice after 30-min stress, and a similar difference was also observed between stressed/non-stressed wild-type mice. An elevated plus maze test showed that the stress stimulus had no effect on anxiety in either wild-type or NMB-R-deficient mice. Furthermore, pain response of wild-type and NMB-R-deficient mice induced by electric foot shock was not affected under either stressed or non-stressed conditions. These results indicate that impaired memory performance in stressed NMB-R-deficient mice is not a consequence of changes in spontaneous activity, anxiety, or pain response, and suggest that the NMB/NMB-R pathway may play a role in regulating the stress response via the neural system that controls learning and memory.