The Excitatory Action of Acetylcholine on Intradental Sensory Units

In order to test the hypothesis that ACh mediates the transmission of pain stimuli from dentin to sensory intradental nerve endings the following experiments were performed. Intradental nerve impulses were recorded by means of low impedance electrodes inserted in dentinal cavities in the tooth of the cat. An air blast proved to be an efficient physical stimulus to excite the intradental nerves. Local application of acetylcholine caused a similar response. This response to acetylcholine was followed by a transient blockage to repeated application. The response to acetylcholine could be blocked by d-tubocurarine, atropine, succinylcholine and hexamethonium administered locally. In contrast, the response to physical stimuli (air blasts) could not be blocked by these drugs. Moreover, during the period of depression following acetylcholine the preparation responded to physical stimuli. These findings suggest that acetylcholine is not a mediator in the intradental pain transmission provoked by physical stimuli.     

Performance characteristics of HIV-1 culture and HIV-1 DNA and RNA amplification assays for early diagnosis of perinatal HIV-1 infection

A plasma HIV-1 RNA amplification assay (RNA assay), a quantitative peripheral blood mononuclear cell (PBMC) microculture (culture), and a PBMC HIV-1 DNA amplification assay (DNA assay) were compared for diagnosis of HIV-1 infection in infants receiving zidovudine in Pediatric AIDS Clinical Trials Group protocol 185; assays were performed for all 24 infected and 100 uninfected infants. HIV-1 infection was defined as >or=2 positive cultures or positive antibody to HIV-1 at >or=18 months. Cultures were performed at birth and 6 and 24 weeks of age; DNA and RNA assays were performed on cryopreserved specimens. The sensitivity of culture and DNA and RNA assays at birth was 20.8%, 10.5%, and 26.7%, respectively. At older ages, sensitivity typically exceeded 80%, remaining highest for the RNA assay (>85%). Assay specificity was >99%. Positive predictive values exceeded 93% for each assay at each age; negative predictive values were highest (>90%) for the RNA assay. At birth (P < 0.005) and age 6 weeks (P < 0.001), a significantly larger proportion of infected infants were identified by means of the RNA assay than by the other assays. The diagnostic performance of the RNA assay matched or exceeded that of culture and the DNA assay. Given that RNA assays require less blood volume and yield rapid results, our study adds to existing data suggesting that RNA assays may be used for early diagnosis of HIV-1 infection in infants.     

Inflammatory cytokine response to Vibrio vulnificus elicited by peripheral blood mononuclear cells from chronic alcohol users is associated with biomarkers of cellular oxidative stress

Vibrio vulnificus is the leading cause of death in the United States associated with the consumption of raw seafood, particularly oysters. In epidemiological studies, primary septicemia and inflammation-mediated septic shock caused by V. vulnificus is strongly associated with liver disease, often in the context of chronic alcohol abuse. The present study was undertaken to determine whether clinical biomarkers of liver function or cellular oxidative stress are associated with peripheral blood mononuclear cell inflammatory cytokine responses to V. vulnificus. Levels of interleukin-1 beta (IL-1 beta), IL-6, IL-8, and tumor necrosis factor alpha elicited in response to V. vulnificus and measured in cell supernatants were not associated with the liver biomarkers aspartate aminotransferase (AST) or alanine aminotransferase (ALT) or the AST/ALT ratio. In contrast, reduced glutathione (GSH) levels were associated with the release of all four cytokines (IL-1 beta [R(2) = 0.382; P = 0.006], IL-6 [R(2) = 0.393; P = 0.005], IL-8 [R(2) = 0.487; P = 0.001], and TNF-alpha [R(2) = 0.292; P = 0.021]). Those individuals with below-normal GSH levels produced significantly less proinflammatory cytokines in response to V. vulnificus. We hypothesize that persons with markers for cellular oxidative stress have increased susceptibility to V. vulnificus septicemia.     

Multilocus sequence typing for characterization of clinical and environmental salmonella strains

Multilocus sequence typing (MLST) based on the 16S RNA, pduF, glnA, and manB genes was developed for Salmonella, and its discriminatory ability was compared to those of pulsed-field gel electrophoresis (PFGE) and serotyping. PFGE differentiated several strains undifferentiable by serotyping, and 78 distinct PFGE types were identified among 231 Salmonella isolates grouped into 22 serotypes and 12 strains of undetermined serotype. The strains of several PFGE types were further differentiated by MLST, which suggests that the discriminatory ability of MLST for the typing of Salmonella is better than that of serotyping and/or PFGE typing. manB-based sequence typing identified two distinct genetic clusters containing 32 of 54 (59%) clinical isolates whose manB gene sequences were analyzed. The G+C contents and Splitstree analysis of the manB, glnA, and pduF genes of Salmonella indicated that the genes differ in their evolutionary origins and that recombination played a significant role in their evolution.     

Alterations in Lung Macrophage Antimicrobial Activity Associated with Viral Pneumonia

Secondary bacterial infections are a common sequelae of viral pneumonias. To study 2 functions of the phagocytic defenses of the lung, macrophages were obtained by lung lavage from parainfluenza 1 virus-infected and noninfected mice. The phagocytic capacities (binding, ingestion, and killing) of these cells were assessed in vitro against viable Candida krusei. Viral pneumonia resulted in a progressive suppression (through day 7 of the infection) of the ability of macrophages to bind candida to their surfaces by nonimmunological or complement receptors; ingestion and intracellular killing of candida were also decreased. After day 7, all these functions returned and, in fact, cells with enhanced activities were present on day 17. After introduction of virus into the lungs, the lung macrophage population increased significantly between days 3 and 7 of infection. This resulted in an increase in the phagocytic potential of the lung, despite the virus-associated suppression of the phagocytic activity in a portion of the macrophages. However, the ability of the macrophages to kill ingested microorganisms was also reduced, resulting in an overall deficiency in the lung macrophage defenses. It was concluded that viral pneumonia was associated with at least two suppressive effects on the lung macrophage-decreased receptor activity and microbicidal activity-resulting in a deficiency in the lung phagocytic defenses represented by these cells. These effects were maximal 1 week after infection and could account for the increased susceptibility of these lungs to secondary bacterial pneumonias. In contrast, during the period of convalescence, the lung macrophage antimicrobial activities were increased and reflected in enhanced resistance of the lungs to infections.     

Safety and immunogenicity of a prototype enterotoxigenic Escherichia coli vaccine administered transcutaneously

Transcutaneous immunization (TCI) is a new method for vaccine delivery that has been shown to induce immunity relevant to enteric disease vaccines. We evaluated the clinical safety and immunogenicity of a recombinant subunit vaccine against enterotoxigenic Escherichia coli (ETEC) delivered by TCI. Adult volunteers received patches containing the recombinant ETEC colonization factor CS6, either with heat-labile enterotoxin (LT) or patches containing CS6 alone. The vaccine was administered at 0, 1, and 3 months, and serum antibodies and antibody-secreting cells (ASCs) were assessed. Among the 26 volunteers that completed the trial, there were no responses to CS6 in the absence of LT. In the groups receiving both CS6 and LT, 68 and 53% were found to have serum anti-CS6 immunoglobulin G (IgG) and IgA, respectively; 37 and 42% had IgG and IgA anti-CS6 ASCs. All of the volunteers receiving LT had anti-LT IgG, and 90% had serum anti-LT IgA; 79 and 37% had anti-LT IgG and IgA ASCs. Delayed-type hypersensitivity (DTH), suggesting T-cell responses, was seen in 14 of 19 volunteers receiving LT and CS6; no DTH was seen in subjects receiving CS6 alone. This study demonstrated that protein antigens delivered by a simple patch could induce significant systemic immune responses but only in the presence of an adjuvant such as LT. The data suggest that an ETEC vaccine for travelers delivered by a patch may be a viable approach worthy of further evaluation.     

Genetic mediation of infanticide and parental behavior in male and female domestic and wild stock house mice

Infanticide is a reproductive strategy found in many mammals, especially rodents. The proportion of male and female house mice (Mus domesticus) that are either infanticidal or noninfanticidal is strain specific and varies widely from stock to stock. Male house mice also show strain-specific variation in the behavioral mechanisms that inhibit infanticidal individuals from killing their own offspring. The adult offspring generated from reciprocally crossed CF-1 and Wild stock house mice were tested for their behavior toward newborn pups. In male CF-1xWild hybrids, the proportion of infanticidal and noninfanticidal males matched with their maternal phenotype, whereas female CF-1xWild hybrids exhibited a proportion of behaviors typical of the CF-1 phenotype, regardless of their mother's genotype. Our results suggest three conclusions: first, that infanticide is a highly labile and heritable behavior in both sexes; second, that there is a sex difference in the genetic substrate that regulates the inheritance of infanticidal behavior; and third, that selection pressures in male mice may operate independently on the mechanisms that promote spontaneous infanticidal behavior versus the mechanisms that inhibit infanticide.     

Temperature-mediated heteroduplex analysis for detection of pncA mutations associated with pyrazinamide resistance and differentiation between Mycobacterium tuberculosis and Mycobacterium bovis by denaturing high- performance liquid chromatography

The goal of this study was to apply temperature-mediated heteroduplex analysis using denaturing high-performance liquid chromatography to identify pyrazinamide (PZA) resistance in Mycobacterium tuberculosis isolates and simultaneously differentiate between M. tuberculosis and Mycobacterium bovis. Features that contributed to an optimal assay included the use of two different reference probes for the pncA gene targets from wild-type M. tuberculosis and wild-type M. bovis, optimization of the column temperature, increasing the starting concentration of the elution buffer, and reducing the rate of elution buffer increase (slope). A total of 69 strains were studied, including 48 wild-type M. tuberculosis strains (13 were PZA-resistant strains) and 21 M. bovis strains (8 were BCG strains). In all isolates tested, wild-type M. tuberculosis generated a single-peak pattern when mixed with the M. tuberculosis probe and a double-peak pattern with the M. bovis probe. In contrast, all M. bovis isolates generated a double-peak pattern when mixed with the M. tuberculosis probe and a single-peak pattern with the M. bovis probe. PZA-resistant mutant M. tuberculosis isolates generated characteristic patterns that were easily distinguishable from both wild-type M. tuberculosis and M. bovis isolates. Chromatographic patterns generated by the two reference probes allowed the rapid detection of PZA resistance with the simultaneous ability to distinguish between M. tuberculosis and M. bovis. This approach may allow the detection of drug resistance-associated mutations, with potential application to clinical and epidemiological aspects of tuberculosis control.     

Essential role for cyclic AMP and its receptor protein in Yersinia enterocolitica virulence

Insertion mutations were isolated in cya and crp of Yersinia enterocolitica, which encode adenylate cyclase and the cyclic AMP (cAMP) receptor protein (CRP). The cya and crp mutants were affected for the production of proteins exported by the Ysc, Ysa, and flagellar type III secretion systems (TTSS). Protein production by each TTSS was restored when the respective mutation was complemented by a plasmid-encoded copy of the wild-type gene. Both cya and crp mutants exhibited reduced virulence for orally infected BALB/c mice in a 50% lethal dose analysis. Examination of bacterial survival in host tissues showed that cya and crp mutants colonized Peyer's patches and, to a lesser extent, mesenteric lymph nodes. However, the mutants did not appear to disseminate to the liver and spleen of infected mice. An initial examination of the effectiveness of Y. enterocolitica cya and crp mutants to stimulate protective immunity against subsequent challenge with virulent bacteria in mice was promising. The results indicate that the cAMP-CRP regulatory system is required for Y. enterocolitica virulence.