No abstract

MiRNAs (microRNAs) are a group of endogenous, small noncoding RNA with the length of 18-25 nucleotides, which have recently been demonstrated to play important roles in a wide range of biological processes. In this work, we developed a simple, sensitive, specific, and inexpensive assay through the combination of enzymatic probe ligation and real-time PCR amplification for the measurement of mature miRNAs. A couple of novel DNA probes with a stem-loop structure were implemented to reduce nonspecific ligation by at least 100-fold. The assay has several remarkable features including wide dynamic range, low total RNA input (0.02-0.2 ng), distinct anti-interference from precursor miRNAs (signal-to-noise ratio > 500), and single-base mismatch discrimination among miRNA sequences. In addition, a one-tube assay could be accomplished by designing a couple of universal probes, which makes it feasible to examine the expression of a whole family of miRNA (such as let-7) at one time. Finally, we validated the method for quantifying the expression of four mature miRNAs including miR-122, miR-1, miR-34a, and let-7a across 10 mouse tissues, where U6 snRNA could be simultaneously examined as an endogenous control. Thus, this method revealed a great potential for miRNA quantitation in ordinary laboratory studies and clinical diagnoses.

GB virus type C (GBV-C) is an apparently nonpathogenic virus that replicates in T and B lymphocytes and is

To investigate the influence of antibody structure and specificity on antibody efficacy against Streptococcus pneumoniae, human monospecific antibodies (MAbs) to serotype 3 pneumococcal capsular polysaccharide (PPS-3) were generated from transgenic mice reconstituted with human immunoglobulin loci (XenoMouse mice) vaccinated with a PPS-3-tetanus toxoid conjugate and their molecular genetic structures, epitope specificities, and protective efficacies in normal and complement-deficient mice were determined. Nucleic acid sequence analysis of three MAbs (A7, 1A2, and 7C5) revealed that they use two different V H 3 genes (A7 and 1A2 both use V3-15) and three different V gene segments. The MAbs were found to have similar affinities for PPS-3 but different epitope specificities and CDR3 regions. Both A7 and 7C5 had a lysine at the V H -D junction, whereas 1A2 had a threonine. Challenge experiments with serotype 3 S. pneumoniae in BALB/c mice revealed that both 10-and 1-g doses of A7 and 7C5 were protective, while only a 10-g dose of 1A2 was protective. Both A7 and 7C5 were also protective in mice lacking either an intact alternative (FB ؊/؊ ) or classical (C4 ؊/؊ ) complement pathway, but 1A2 was not protective in either strain. Our data suggest that PPS-3 consists of epitopes that can elicit both highly protective and less protective antibodies and that the superior efficacies of certain antibodies may be a function of their structures and/or specificities. Further investigation of relationships between structure, specificity, and efficacy for defined MAbs to PPS may identify antibody features that might be useful surrogates for antibody (and vaccine) efficacy.

Severe acute respiratory syndrome (SARS) is a novel infectious disease with disastrous clinical consequences, in which the lungs are the major target organs. Previous studies have described the general pathology in the lungs of SARS patients and have identified some of the cell types infected by SARS coronavirus (SARS-CoV). However, at the time of this writing, there were no comprehensive reports of the cellular distribution of the virus in lung tissue. In this study, we have performed double labeling combining in situ hybridization with immunohistochemistry and alternating each of these techniques separately in consecutive sections to evaluate the viral distribution on various cell types in the lungs of seven patients affected with SARS. We found that SARS-CoV was present in bronchial epithelium, type I and II pneumocytes, T lymphocytes, and macrophages/monocytes. For pneumocytes, T lymphocytes, and macrophages, the infection rates were calculated. In addition, our present study is the first to demonstrate infection of endothelial cells and fibroblasts in SARS. (Am J Pathol

GB virus type C (GBV-C

Viral infections alter host cell homeostasis and this may lead to immune evasion and/or interfere with the replication of other microbes in coinfected hosts. Two flaviviruses are associated with a reduction in HIV replication or improved survival in HIV-infected people (dengue virus (DV) and GB virus type C (GBV-C)). GBV-C infection and expression of the GBV-C nonstructural protein 5A (NS5A) and the DV NS5 protein in CD4+ T cells inhibit HIV replication in vitro. To determine whether the inhibitory effect on HIV replication is conserved among other flaviviruses and to characterize mechanism(s) of HIV inhibition, the NS5 proteins of GBV-C, DV, hepatitis C virus, West Nile virus, and yellow fever virus (YFV; vaccine strain 17D) were expressed in CD4+ T cells. All NS5 proteins inhibited HIV replication. This correlated with decreased steady-state CD4 mRNA levels and reduced cell surface CD4 protein expression. Infection of CD4+ T cells and macrophages with YFV (17D vaccine strain) also inhibited HIV replication and decreased CD4 gene expression. In contrast, mumps virus was not inhibited by the expression of flavivirus NS5 protein or by YFV infection, and mumps infection did not alter CD4 mRNA or protein levels. In summary, CD4 gene expression is decreased by all human flavivirus NS5 proteins studied. CD4 regulation by flaviviruses may interfere with innate and adaptive immunity and contribute to in vitro HIV replication inhibition. Characterization of the mechanisms by which flaviviruses regulate CD4 expression may lead to novel therapeutic strategies for HIV and immunological diseases.

The features of protective murine antibodies to the Cryptococcus neoformans capsular polysaccharide glucuronoxylomannan (GXM) have been rigorously investigated; however, the characteristics of protective human antibodies to GXM have not been defined. We produced monoclonal antibodies (MAbs) from XenoMouse mice (transgenic mice that express human immunoglobulin M [IgM], IgG2, and ) which were immunized with a C. neoformans serotype D strain 24067 GXM-diphtheria toxoid conjugate. This study reports the specificity and efficacy of three human IgM MAbs, G14, G15, and G19, generated from these mice. Each MAb was specific for GXM, but G14 and G19 had different specificity based on their binding to serotype A strain H99 and SB4 GXMs, to which G15 did not bind. Nucleic acid sequence analysis revealed that G15 uses V H 3-64 in the germ line configuration. G14 and G19 use V H 6-1, which has somatic mutations. All of the MAbs use V DPK22/A27. Studies of MAb efficacy in BALB/c mice showed that administration of 0.1 mg, but not 1 or 0.01 mg, of G15 prolonged survival against lethal C. neoformans strain 24067 challenge, whereas G14 and G19 were not protective at any dose. This panel of MAbs illustrates that serotype D GXM has epitopes that elicit human antibodies that can be either protective or nonprotective. Our findings suggest that V H gene use may influence GXM specificity and efficacy, and they provide insights into the possible contribution that V H gene use may have in resistance and susceptibility to cryptococcosis.