Transmission of Streptococcus pneumoniae in adults may occur through saliva.

Of 742 army recruits tested for pneumococcal nasopharyngeal/oropharyngeal carriage, 6·6% were positive. Frequent sharing of a drinking glass/bottle was a common, strong and independent risk factor for pneumococcal carriage. Our findings strongly suggest, for the first time, that in young adults, transmission of pneumococci may occur via saliva and this should be considered when conducting an outbreak investigation and carriage studies.

Serum antibodies to Vibrio vulnificus biotype 3 lipopolysaccharide and susceptibility to disease caused by the homologous V. vulnificus biotype.

In 1996 an outbreak of severe soft tissue infections caused by Vibrio vulnificus unexpectedly erupted in fish consumers in Israel with relatively little morbidity in fish farmers. To test the hypothesis that recurrent exposure of fishermen to the virulent strain may have provided protection against severe or symptomatic disease, we investigated the association between the immune response to V. vulnificus biotype 3 lipopolysaccharide (BT3 LPS) and disease susceptibility in fish farmers and fish consumers. Serum samples were tested for IgA and IgG of anti-BT3 LPS in fishermen and fish consumers who suffered from V. vulnificus BT3 infections and their matched controls. Pre-existing levels of IgG (IgG0) of anti-BT3 LPS were significantly lower in diseased fishermen who developed disease associated with the homologous biotype, compared to controls. In multivariate analysis, levels of IgG0 anti-BT3 LPS remained the only variable significantly associated with disease occurrence in fishermen. Higher levels of pre-existing IgG anti-BT 3 LPS antibodies may be associated with protection against severe or symptomatic disease with the homologous biotype in fishermen but not in subjects from the general public.

Comparative analyses of phenotypic and genotypic methods for detection of enterotoxigenic Escherichia coli toxins and colonization factors.

Enterotoxigenic Escherichia coli (ETEC) is one of the main causes of childhood diarrhea in developing countries and in travelers. However, this pathogen has often not been reported in surveys of diarrheal pathogens, due to lack of simple standardized methods to detect ETEC in many laboratories. ETEC expresses one or both of two different enterotoxin subtypes: heat-stable toxins, a heat-labile toxin (LT), and more than 22 different colonization factors (CFs) that mediate adherence to the intestinal cell wall. Here we compare established phenotypic and genotypic detection methods and newly developed PCR detection methods with respect to sensitivity, specificity, positive predictive value, and ease of performance. The methods include GM1-enzyme-linked immunosorbent assay and dot blot techniques using specific monoclonal antibodies (MAbs) for phenotypic detection of the toxins and CFs, respectively, as well as different PCR and DNA/DNA hybridization techniques, including new PCR assays, for genotypic identification of the toxin and CF genes, respectively. We found very good general agreement in results derived from genotypic and phenotypic methods. In a few strains, LT and CFs were identified genetically but not phenotypically. Based on our analyses, we recommend initial screening for ETEC in clinical samples by multiplex toxin gene PCR. Toxin-positive strains may then be analyzed by dot blot tests for detection of the CFs expressed on the bacterial surface and by PCR for determination of additional CFs for which MAbs are currently lacking as well as for strains that harbor silent CF genes.

Seroepidemiology of Varicella zoster in Israel prior to large-scale use of varicella vaccines.

OBJECTIVES: This large-scale study provides up-to-date estimates of Varicella zoster virus (VZV) age-specific seroprevalence and characteristics of VZV transmission in a representative sample of the Israeli population. METHODS: In 2000-2001, 1,642 sera collected from an agestratified general population sample were tested for VZV antibodies using an indirect IgG ELISA system. RESULTS: The age-weighted VZV overall estimate was 90.2%. Seropositivity increased rapidly with age, from 68.9% at age 4 to 94.4% at age 7 and 96.6% at age 12 years. The highest force of infection was in the 4-5 years age group (0.548 per susceptible year) followed by the 6-9 years age group. Multivariate analysis revealed that VZV seroprevalence estimates were significantly associated with age and place of origin. The highest seroprevalence estimate was found among subjects of Eastern origin. CONCLUSIONS: The seroepidemiology of VZV in Israel shows a pattern corresponding to that described for developed European countries. This study indicates that the highest force of infection is in pre-school children. Knowledge of pre-vaccination seroepidemiology is important to evaluate the effect of vaccination programs on the epidemiology of the disease.

Pronounced acute immunosuppression in vivo mediated by HIV Tat challenge.

HIV infection is accompanied by an early immune dysfunction limiting host control of virus and likely contributing to difficulties in achieving a successful vaccine against HIV. We report here that the HIV Tat protein is strongly immunosuppressive, both immediately after immunization of mice with soluble protein (sTat), and in seroconverting humans, and propose that Tat-induced suppression cripples immune surveillance to HIV infection. We show that macrophages are sensitive to sTat stimulation at concentrations 1,000-fold lower (500 pM) than T cells, and this stimulation is accompanied by the immunosuppressive induction of Fas ligand on the macrophage. T cell proliferative defects induced by sTat in vitro can be completely (at lower concentrations of sTat) or partially (at higher concentrations) reversed by antagonists to Fas/Fas ligand interaction. We further report a method to preserve immunogenicity while inactivating Tat immunosuppression through oxidation, which advances the use of oxidized Tat as a component of an anti-HIV vaccine. These observations define additional methods to study the immunosuppressive functions of sTat that now may be rapidly applied to primary isolates from individuals with differing clinical courses. Our findings have immediate relevance for vaccine development, by describing and supporting a strategy that includes inactivated sTat in a multicomponent, anti-HIV vaccine.

Screening for infection and disease as a tuberculosis control measure among indigents in New York City, 1994-1997.

SETTING: Several social service agencies in New York City, and the Chest Clinic of Bellevue Hospital, a large public hospital. OBJECTIVE: To determine the utility of screening as a preventive and control measure among persons at risk for tuberculosis. DESIGN: Persons seeking social services at several private agencies in New York City were screened, and those with a positive skin test or symptoms suggestive of active tuberculosis were referred to the Chest Clinic for evaluation. RESULTS: Of 3828 persons evaluated, 20 had active tuberculosis, and 33% of the screened cohort were tuberculin skin test positive. Of 466 persons with tuberculosis infection who were evaluated, only 55 persons were given isoniazid (INH), and only 20 completed preventive therapy. Most patients who were not given INH had taken it previously, were older than 35 years, or had continuing alcohol use which made physicians reluctant to prescribe isoniazid. CONCLUSION: Screening for tuberculosis may detect a significant number of cases of active disease when the background prevalence of the disease is very high. However, screening for infection as a means to prevent future cases is unlikely to be effective unless rates of administration and completion of isoniazid preventive therapy are increased.

Highly specific separation of heterogeneous cell populations by lectin-coated beads: application for the isolation of inner medullary collecting duct cells.

Conditions for the highly specific selection of a cell type by the use of lectin-coated magnetic beads are reported for the isolation of inner medullary collecting duct (IMCD) cells from a heterogeneous inner medullary cell suspension, containing both single cells and tubular fragments of variable size. The lectin Dolichos Biflorus Agglutinin (DBA), which binds in rat inner medulla exclusively to IMCD cells, was coupled via the avidin-biotin system to beads. By isolating DBA-bead-IMCD cells in a magnetic field (positive selection) from a suspension containing about 50% IMCD, a fraction of 98 +/- 1% purity was obtained; recovery of cells was up to 90%. Suspensions negative on reverse-transcriptase polymerase chain reaction for vimentin as a marker of contaminating interstitial and vascular cells could be received by repeating this procedure and additional trypsinization. On the other hand, it was possible to reduce the portion of IMCD cells in the suspension by one isolation step to 1.5 +/- 0.9% (negative selection). Performing this step twice resulted in virtually pure suspensions. No significant effects of this isolation technique on cell viability, growth characteristics, and biochemical parameters were observed. Therefore, this method appears to be a powerful tool for the highly specific separation of heterogeneous cell populations.

Alloantigen-stimulated anti-HIV activity.

A number of studies have suggested that an immune response to human leukocyte antigen (HLA) alloantigens may contribute to protection against HIV infection. In the present study, we examined the effect of alloantigen-stimulated cell lines obtained from peripheral blood mononuclear cells (PBMC) of HIV-uninfected (HIV-) individuals and the soluble factors produced by these cell lines on HIV-1 replication. Multiple in vitro restimulation with irradiated allogeneic PBMC from HIV- donors resulted in the expansion of CD8(+) T-cell lines that inhibited HIV-1 replication when cocultured with either autologous or heterologous in vitro-infected phytohemagglutinin (PHA) blasts. Supernatants from the alloantigen-stimulated cell lines also inhibited HIV replication in both PHA blasts and a chronically infected cell line. The alloantigen-stimulated cell lines and the factors they produced inhibited both T-cell-tropic (T) and macrophage-tropic (M) isolates of HIV-1. Blocking experiments using anti-chemokine antibodies suggested that this inhibition of HIV replication was not due to the beta-chemokines present in cocultures of cell lines with HIV-infected blasts. These results indicate that alloantigen-stimulation of PBMC from HIV- individuals activates CD8(+) T cells that produce soluble factor(s) that inhibit HIV replication of a wide spectrum of HIV-1 isolates through a chemokine-independent mechanism. This is a US government work. There are no restrictions on its use.

bca: an activation-related B-cell gene.

We have identified a novel activation related B-cell gene (bca) through differential hybridization screening of a murine B cell cDNA library. The deduced amino acid sequence predicted a protein of 482 amino acids with strong sequence similarity to the SH2 and SH3 domains present within the non-catalytic regions of several protein tyrosine kinases. Northern analysis of RNA from several murine B-cell lines revealed a transcript of 1.8 kb, which was not detected in T-cell and non-lymphoid cell lines. bca was transcribed at low levels in resting spleen cells from a variety of normal mouse strains and was strongly expressed in kidney RNA. bca expression was markedly increased in RNA prepared from mitogen activated B cells, and in freshly isolated spleen and lymph node cells of MRL/lpr and NZB autoimmune strains. The unique sequence of bca, which bears no obvious similarity to any specific class of proteins containing SH2 and SH3 domains, suggests that this gene encodes a novel protein potentially involved in B-cell signal transduction.

Transformation of rat inner medullary fibroblasts to myofibroblasts in vitro.

Renal fibroblasts play a major role in the pathogenesis of renal interstitial fibrosis. This process is associated at least in some forms of interstitial fibrosis with a differentiation of fibroblasts into myofibroblasts, characterized by the de novo expression of alpha-smooth muscle (alpha-sm) actin and/or desmin. Both the mechanisms underlying this differentiation and their effects on cellular function are poorly understood. In vitro studies are difficult since the phenotypes of fibroblasts in culture have as yet not been well defined. We have, therefore, examined the phenotype of inner medullary fibroblasts (IMF) during the transition from in vivo to in vitro in various cell fractions derived from the inner medulla of healthy rats. IMF were positive for the lectin BSL-1 and negative for markers of endothelial cells. IMF first lost their prominent lipid droplets in vitro. Subsequently they developed cytoplasmic processes accompanied by a decrease in their reactivity for the lectin BSL-1 from strong to weak. From day 3 in primary culture, exclusively these weakly positive BSL-1 cells showed a de novo expression of alpha-sm actin (day 4 of primary culture, 75 +/- 4%; day 20, 94 +/- 2%) and desmin (day 4, 43 +/- 8%; day 20, 66 +/- 6%), classifying them as myofibroblasts. This transformation depended on culture conditions. In a mixed coculture with inner medullary collecting duct (IMCD) cells the transformation of IMF was largely absent: a significantly greater number of strong BSL-1 positive cells contained prominent lipid droplets (39 +/- 4 vs. 19 +/- 4%, P < 0.05) on day 4 of primary culture, and the transition of strongly to weakly positive BSL-1 IMF was almost completely blocked. By reducing the seeding density of IMCD cells the effect of this condition on IMF transformation could be largely abolished. This first detailed phenotypic characterization of rat fibroblasts during the transition from in vivo to in vitro demonstrates that these cells-depending on culture conditions-differentiate to myofibroblasts within a few days of primary culture and that subcultured IMF exhibit predominantly this phenotype. The presented model may serve as a useful tool for the in vitro study of myofibroblast formation and the consequences of such a differentiation for the physiological functions of IMF.

A major human immunodeficiency virus type 1-initiated killing pathway distinct from apoptosis.

We have investigated the relative contribution of apoptosis or programmed cell death (PCD) to cell killing during acute infection with T-cell-tropic, cytopathic human immunodeficiency virus type 1 (HIV-1), by employing diverse strategies to inhibit PCD or to detect its common end-stage sequelae. When Bcl-2-transfected cell lines were infected with HIV-1, their viability was only slightly higher than that of control infections. Although the adenovirus E1B 19-kDa protein has been reported to be a stronger competitor of apoptosis than Bcl-2, it did not inhibit HIV-mediated cell death better than Bcl-2 protein. Competition for Fas ligand or inactivation of the Fas pathway secondary to intracellular mutation (MOLT-4 T cells) also had modest effects on overall cell death during acute HIV infection. In contrast to these observations with HIV infection or with HIV envelope-initiated cell death, Tat-expressing cell lines were much more susceptible (200% enhancement) to Fas-induced apoptosis than controls and Bcl-2 overexpression strongly (75%) inhibited this apoptotic T-cell death. PCD associated with FasR ligation resulted in the cleavage of common interleukin-1beta-converting enzyme (ICE)-protease targets, poly(ADP-ribose) polymerase (PARP) and pro-ICE, whereas cleaved products were not readily detected during HIV infection of peripheral blood mononuclear cells or T-cell lines even during periods of extensive cell death. These results indicate that one important form of HIV-mediated cell killing proceeds by a pathway that lacks the characteristics of T-cell apoptosis. Our observations support the conclusion that at least two HIV genes (env and tat) can kill T cells by distinct pathways and that an envelope-initiated process of T-cell death can be discriminated from apoptosis by many of the properties most closely associated with apoptotic cell death.

HIV envelope-directed signaling aberrancies and cell death of CD4+ T cells in the absence of TCR co-stimulation.

HIV-1 infection in CD4(+) T cells initiates a viral cytopathic effect (CPE) that is dependent on the activation of intracellular protein tyrosine kinases (PTK). PTK in T cells are also activated during the course of TCR or CD4 receptor engagement and the manner of receptor engagement may generate signals leading either to cell proliferation, tolerance induction (anergy) or programmed cell death (PCD). We have identified PTK triggered during the interaction of cells stably expressing surface HIV envelope (gp 120/gp41; HIVenv) and CD4(+)T cells, which leads to extensive and rapid individual cell death. We have found that killing is accompanied by tyrosine phosphorylation and activation of the CD4-associated p56(ICK) kinase, and by activation of a second member of the scr family of PTK, p59(fyn) kinase, normally associated with T cell stimulation through the TCR. Interestingly, in contrast with normal T cell signaling, the zeta subunit of the TCR fails to become tyrosine-phosphorylated during signaling accompanying HIV-directed cell killing. Downstream activation of the ZAP-70 PTK also does not occur. Unlike T cell apoptosis triggered by soluble HIVenv glycoproteins, which requires co-stimulation of CD4 and the antigen-specific TCR, T cell killing by membrane-associated HIVenv does not require TCR co-stimulation, because aberrant signaling and cell death are triggered by CD4(+) but TCR- cell lines. These results are the first report where dual activation of the Lck and Fyn PTK does not result in normal downstream signaling through the ZAP PTK, We suggest by analogy to SCID resulting from ZAP-70 mutations, that the dissociation of upstream PTK activation from ZAP-70 signaling contributes to T cell depletion by HIV and to the development of AIDS.

Human immunodeficiency virus 1 envelope-initiated G2-phase programmed cell death.

Despite intensive investigation, no clearly defined mechanism explaining human immunodeficiency virus (HIV)-induced cell killing has emerged. HIV-1 infection is initiated through a high-affinity interaction between the HIV-1 external envelope glycoprotein (gp120) and the CD4 receptor on T cells. Cell killing is a later event intimately linked by in vitro genetic analyses with the fusogenic properties of the HIV envelope glycoprotein gp120 and transmembrane glycoprotein gp41. In this report, we describe aberrancies in cell cycle regulatory proteins initiated by cell-cell contact between T cells expressing HIV-1 envelope glycoproteins and other T cells expressing CD4 receptors. Cells rapidly accumulate cyclin B protein and tyrosine-hyperphosphorylated p34cdc2 (cdk1) kinase, indicative of cell cycle arrest at G2 phase. Moreover, these cells continue to synthesize cyclin B protein, enlarge and display an abnormal ballooned morphology, and disappear from the cultures in a pattern previously described for cytotoxicity induced by DNA synthesis (S phase) inhibitors. Similar changes are observed in peripheral blood mononuclear cells infected in vitro with pathogenic primary isolates of HIV-1.

Intercellular adhesion molecules (ICAM)-1 ICAM-2 and ICAM-3 function as counter-receptors for lymphocyte function-associated molecule 1 in human immunodeficiency virus-mediated syncytia formation.

It has been previously demonstrated that lymphocyte function-associated molecule 1 (LFA-1) plays a major role in human immunodeficiency virus (HIV)-mediated syncytia formation. In the present study we investigated the involvement of intercellular adhesion molecule-1 (ICAM-1), ICAM-2 and ICAM-3 in the process. The ability of monoclonal antibodies (mAb) directed against ICAM-1, ICAM-2 and ICAM-3 to block syncytia was analyzed either in phytohemagglutinin (PHA)-activated lymphocytes infected in vitro with primary or laboratory strains of HIV or by coculturing a T cell line stably expressing HIV envelope with PHA-activated lymphocytes. Complete inhibition of syncytia formation was observed only by the simultaneous addition to the cell cultures of all (i.e. anti-ICAM-1, anti-ICAM-2 and anti-ICAM-3) mAb. These results indicate that the interaction between LFA-1 and ICAM is a critical step in HIV-mediated syncytia formation, and that ICAM-1, ICAM-2 and ICAM-3 are the receptor molecules for the LFA-1-dependent syncytia formation.

Enhanced in vitro human immunodeficiency virus type 1 replication in B cells expressing surface antibody to the TM Env protein.

The human immunodeficiency virus type 1 (HIV-1) external envelope glycoprotein gp120 tightly binds CD4 as its principal cellular receptor, explaining the tropism of HIV-1 for CD4+ cells. Nevertheless, reports documenting HIV infection or HIV binding in cells lacking CD4 surface expression have raised the possibility that cellular receptors in addition to CD4 may interact with HIV envelope. Moreover, the lymphocyte adhesion molecule LFA-1 appears to play an important role in augmenting HIV-1 viral spread and cytopathicity in vitro, although the mechanism of this function is still not completely defined. In the course of characterizing a human anti-HIV gp41 monoclonal antibody, we transfected a CD4-negative, LFA-1-negative B-cell line to express an anti-gp41 immunoglobulin receptor (surface immunoglobulin [sIg]/gp41). Despite acquiring the ability to bind HIV envelope, such transfected B cells could not be infected by HIV-1. These cells were not intrinsically defective for supporting HIV-1 infection, because when directed to produce surface CD4 by using retroviral constructs, they acquired the ability to replicate HIV-1. Interestingly, transfected cells expressing both surface CD4 and sIg/gp41 receptors replicated HIV much better than cells expressing only CD4. The enhancement resided specifically in sIg/gp41, because isotype-specific, anti-IgG1 antibodies directed against sIg/gp41 blocked the enhancement. These data directly establish the ability of a cell surface anti-gp41 receptor to enhance HIV-1 replication.

Normal T cell receptor-mediated signaling in T cell lines stably expressing HIV-1 envelope glycoproteins.

A series of T cell lines transfected to stably express HIV-1 envelope (env) glycoproteins were analyzed for viability and for T cell signaling. One transfectant was distinguished by its stable expression of gp120 and gp41, whereas the remainder of the T cell lines were similar to previously reported env-expressing T cells in synthesizing predominantly unprocessed env glycoprotein gp160. All of the transfectants were additionally constructed to express tat and rev proteins. None of these cell lines displayed growth abnormalities or spontaneous cell fusion, although the cell line synthesizing env gp120/gp41 could be induced to fuse and die when cocultured with a second cell expressing surface CD4. A cell line expressing only gp160 and the transfectant expressing gp160, gp120, and gp41 could be triggered normally via CD3-cross-linking as measured by protein tyrosine phosphorylation and by the induction of the CD69 activation marker. At levels of env protein expression sufficient to mediate syncytium formation and to kill cells, these HIV-1 env transfectants displayed no intrinsic T cell signaling abnormalities, suggesting that mechanisms other than a direct intracellular action of the tat or env proteins may be contributing to the deficit in Ag-specific T cell activation described subsequent to HIV infection in vivo and in vitro.

Participation of tyrosine phosphorylation in the cytopathic effect of human immunodeficiency virus-1.

Protein tyrosine phosphorylation is a common mechanism of signaling in pathways that regulate T cell receptor-mediated cell activation, cell proliferation, and the cell cycle. Because human immunodeficiency virus (HIV) is though to affect normal cell signaling, tyrosine phosphorylation may be associated with HIV cytopathicity. In both HIV-infected cells and transfected cells that stably express HIV envelope glycoproteins undergoing HIVgp41-induced cell fusion, a 30-kilodalton protein was phosphorylated on tyrosine with kinetics similar to those of syncytium formation and cell death. When tyrosine phosphorylation was inhibited by the protein tyrosine kinase inhibitor herbimycin A, envelope-mediated syncytium formation was coordinately reduced. These studies show that specific intracellular signals, which apparently participate in cytopathicity, are generated by HIV and suggest strategies by which the fusion process might be interrupted.

A common V delta 2-D delta 2-D delta 3 T cell receptor gene rearrangement in precursor B acute lymphoblastic leukaemia.

Despite their apparent commitment to the B lymphocytic lineage, human precursor B cell acute lymphoblastic leukaemias (ALL) frequently rearrange their T cell antigen receptor (TCR) alpha, beta and gamma chain genes. Since these three genes are active sites of rearrangement in precursor B cell neoplasms, it seemed that the recently discovered fourth TCR gene, delta, might be similarly rearranged. To investigate this possibility, a series of precursor B cell leukaemias was analysed for rearrangements at the delta chain gene locus, using probes of the variable, joining, and constant regions of the delta chain gene. The majority of precursor B cell ALLs in this series (25/32, 78%) showed rearrangement or deletion of one or more TCR delta genes. This contrasts sharply with a series of 16 mature B cell neoplasms (chronic lymphocytic leukaemia) in which no TCR delta gene rearrangements were detected. An unusual TCR delta rearrangement, rarely observed in normal or neoplastic T cells, was seen in the majority (14/18) of precursor B cell ALLs with TCR delta rearrangements. In contrast to the utilization ov V delta 1 in T cell ALL, detailed restriction mapping of precursor B ALL revealed an incomplete rearrangement without involvement of J delta segments. Direct genomic sequencing was performed on one example and demonstrated a nonproductive V delta 2-D delta 2-D delta 3 recombination in this precursor B ALL. We conclude that the TCR delta chain gene is an active locus in precursor B cell neoplasia, involves an unusual type of rearrangement and provides a clonal tumour marker for diagnosis of precursor B ALL.