Evaluation of the beta-Neocept test for early pregnancy.

We tested the validity of the beta-Neocept test for early pregnancy against that of the plasma human chorionic gonadotropin beta-subunit radioimmunoassay (beta-HCG RIA). The beta-Neocept test had a sensitivity of 88%, a specificity of 93%, a positive predictive value of 95%, a negative predictive value of 84% and an accuracy of 90%. In view of these performance characteristics, its low cost and its ease of use, the beta-Neocept test could be used as the initial pregnancy test when there is a high probability of pregnancy, as there was in this study population, which consisted of 111 women attending endocrine infertility clinics. The more expensive beta-HCG RIA could be reserved for special indications and for patients in whom the results of the urinary hemagglutination inhibition tests are inconsistent with the clinical signs and symptoms.     

Characterization of the Vibrio cholerae VolA Surface-Exposed Lipoprotein Lysophospholipase

Bacterial lipases play important roles in bacterial metabolism and environmental response. Our laboratory recently discovered that a novel lipoprotein lysophospholipase, VolA, localizes on the surface of the Gram-negative aquatic pathogen Vibrio cholerae. VolA functions to cleave exogenous lysophosphatidylcholine, freeing the fatty acid moiety for use by V. cholerae. This fatty acid is transported into the cell and can be used as a nutrient and, more importantly, as a way to alter the membrane architecture via incorporation into the phospholipid biosynthesis pathway. There are few examples of Gram-negative, surface-exposed lipoproteins, and VolA is unique, as it has a previously undercharacterized function in V. cholerae membrane remodeling. Herein, we report the biochemical characterization of VolA. We show that VolA is a canonical lipoprotein via mass spectrometry analysis and demonstrate the in vitro activity of VolA under a variety of conditions. Additionally, we show that VolA contains a conserved Gly-Xaa-Ser-Xaa-Gly motif typical of lipases. Interestingly, we report the observation of VolA homologs in other aquatic pathogens. An Aeromonas hydrophila VolA homolog complements a V. cholerae VolA mutant in growth on lysophosphatidylcholine as the sole carbon source and in enzymatic assays. These results support the idea that the lipase activity of surface-exposed VolA likely contributes to the success of V. cholerae, improving the overall adaptation and survival of the organism in different environments.     

Harmonization guidelines for HLA-peptide multimer assays derived from results of a large scale international proficiency panel of the Cancer Vaccine Consortium

Purpose  The Cancer Vaccine Consortium of the Cancer Research Institute (CVC-CRI) conducted a multicenter HLA-peptide multimer proficiency panel (MPP) with a group of 27 laboratories to assess the performance of the assay. Experimental design  Participants used commercially available HLA-peptide multimers and a well characterized common source of peripheral blood mononuclear cells (PBMC). The frequency of CD8+ T cells specific for two HLA-A2-restricted model antigens was measured by flow cytometry. The panel design allowed for participants to use their preferred staining reagents and locally established protocols for both cell labeling, data acquisition and analysis. Results  We observed significant differences in both the performance characteristics of the assay and the reported frequencies of specific T cells across laboratories. These results emphasize the need to identify the critical variables important for the observed variability to allow for harmonization of the technique across institutions. Conclusions  Three key recommendations emerged that would likely reduce assay variability and thus move toward harmonizing of this assay. (1) Use of more than two colors for the staining (2) collect at least 100,000 CD8 T cells, and (3) use of a background control sample to appropriately set the analytical gates. We also provide more insight into the limitations of the assay and identified additional protocol steps that potentially impact the quality of data generated and therefore should serve as primary targets for systematic analysis in future panels. Finally, we propose initial guidelines for harmonizing assay performance which include the introduction of standard operating protocols to allow for adequate training of technical staff and auditing of test analysis procedures. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Epitope mapping of full-length glycoprotein D from HSV-2 reveals a novel CD4+ CTL epitope located at the transmembrane-cytoplasmic junction

The glycoprotein D of HSV-2 (gD2) is currently a leading candidate vaccine target for genital herpes vaccines as both cellular and humoral responses can be generated against it. However, little is known about how vaccine composition will affect T cell epitope selection. A panel of 15-mer peptides (with 11 amino acid overlap) spanning full-length gD2 was used to investigate the fine specificity of T cell responses to gD2 as well as the role of vaccine composition on epitope selection. Spleen cells from BALB/c mice (H-2(d)) immunized with gD2, formulated with or without AlPO(4) and/or IL-12, were stimulated in vitro with overlapping gD2 peptides. Cellular responses (lymphoproliferation and IFN-gamma expression) were mapped to four epitopes within the gD2 molecule: gD2(49-63), gD2(105-119), gD2(245-259), and gD2(333-347). CTL analysis of these four epitopes indicated that not all of them could serve as a CTL epitope. Mice immunized with gD2 expressed from a viral vector mounted CTL responses primarily to one epitope located in the extracellular domain of gD2 (gD2(245-259)). More importantly, mice immunized with gD2 co-administered with IL-12 mounted CTL responses to an additional epitope located at the transmembrane-cytoplasmic junction of gD2 (gD2(333-347)). The location of this novel epitope emphasizes the benefit of using full-length versions of glycoproteins when designing vaccine components.     

Comparisons of clustered regularly interspaced short palindromic repeats and viromes in human saliva reveal bacterial adaptations to salivary viruses

Explorations of human microbiota have provided substantial insight into microbial community composition; however, little is known about interactions between various microbial components in human ecosystems. In response to the powerful impact of viral predation, bacteria have acquired potent defences, including an adaptive immune response based on the clustered regularly interspaced short palindromic repeats (CRISPRs)/Cas system. To improve our understanding of the interactions between bacteria and their viruses in humans, we analysed 13?977 streptococcal CRISPR sequences and compared them with 2?588?172 virome reads in the saliva of four human subjects over 17 months. We found a diverse array of viruses and CRISPR spacers, many of which were specific to each subject and time point. There were numerous viral sequences matching CRISPR spacers; these matches were highly specific for salivary viruses. We determined that spacers and viruses coexist at the same time, which suggests that streptococcal CRISPR/Cas systems are under constant pressure from salivary viruses. CRISPRs in some subjects were just as likely to match viral sequences from other subjects as they were to match viruses from the same subject. Because interactions between bacteria and viruses help to determine the structure of bacterial communities, CRISPR-virus analyses are likely to provide insight into the forces shaping the human microbiome.     

Relation between trunk fat volume and reduction of total lung capacity in obese men

Reduction in total lung capacity (TLC) in obese men is associated with restricted expansion of the thoracic cavity at full inflation. We hypothesized that thoracic expansion was reduced by the load imposed by increased total trunk fat volume or its distribution. Using MRI, we measured internal and subcutaneous trunk fat and total abdominal and thoracic volumes at full inflation in 14 obese men [mean age: 52.4 yr, body mass index (BMI): 38.8 (range: 36-44) kg/m(2)] and 7 control men [mean age: 50.1 yr, BMI: 25.0 (range: 22-27.5) kg/m(2)]. TLC was measured by multibreath helium dilution and was restricted (<80% of the predicted value) in six obese men (the OR subgroup). All measurements were made with subjects in the supine position. Mean total trunk fat volume was 16.65 (range: 12.6-21.8) liters in obese men and 6.98 (range: 3.0-10.8) liters in control men. Anthropometry and mean total trunk fat volumes were similar in OR men and obese men without restriction (the ON subgroup). Mean total intraabdominal volume was 9.41 liters in OR men and 11.15 liters in ON men. In obese men, reduced thoracic expansion at full inflation and restriction of TLC were not inversely related to a large volume of 1) intra-abdominal or total abdominal fat, 2) subcutaneous fat volume around the thorax, or 3) total trunk fat volume. In addition, trunk fat volumes in obese men were not inversely related to gas volume or estimated intrathoracic volume at supine functional residual capacity. In conclusion, this study failed to support the hypotheses that restriction of TLC or impaired expansion of the thorax at full inflation in middle-aged obese men was simply a consequence of a large abdominal volume or total trunk fat volume or its distribution.     

Relief of asthma in two patients receiving danazol for endometriosis.

This paper describes two patients with endometriosis in whom treatment with danazol resulted in a concomitant remission of asthma. Neither patient reported an association of clinical symptoms with phases of their menstrual cycle. Whether the remission was fortuitous or related to suppression of the activity of the endometriotic tissue or to the ability of danazol to increase the serum levels of two protease inhibitors that are deficient in patients with asthma remains to be determined.