Human neonates have reduced and delayed CD4+ T-cell immunity to certain pathogens compared to older children and adults, but the mechanisms for these developmental differences in immune function remain poorly understood. We investigated the hypothesis that impaired human neonatal CD4+ T-cell immunity was due to reduced signaling by naive CD4+ T cells following engagement of the αβ-TCR/CD3 complex and CD28. Surprisingly, calcium flux following engagement of CD3 was significantly higher in neonatal naive CD4+ T cells from umbilical cord blood compared to naive CD4+ T cells from adult peripheral blood. Enhanced calcium flux was also observed in adult CD4+ recent thymic emigrants. Neonatal naive CD4+ T cells also had higher activation-induced Erk phosphorylation. The microRNA miR-181a, which enhances activation-induced calcium flux in murine thymocytes, was expressed at significantly higher levels in cord blood naive CD4+ T cells compared to adult cells. Overexpression of miR-181a in adult naive CD4+ T cells increased activation-induced calcium flux, implying that the increased miR-181a levels of cord blood naive CD4+ T cells contributed to their enhanced signaling. In contrast, AP-1-dependent transcription, which is downstream of Erk and required for full T-cell activation, was decreased in cord blood naive CD4+ T cells compared to adult cells. Thus, cord blood naive CD4+ T cells have enhanced activation-dependent calcium flux, indicative of the retention of a thymocyte-like phenotype. Enhanced calcium signaling and Erk phosphorylation are decoupled from downstream AP-1-dependent transcription, which is reduced and likely contributes to limitations of human fetal and neonatal CD4+ T-cell immunity.

Varicella-zoster virus (VZV) causes varicella and establishes latency in sensory nerve ganglia, but the characteristics of VZV latency are not well defined. Immunohistochemical detection of the VZV immediateearly 63 (IE63) protein in ganglion neurons has been described, but there are significant discrepancies in estimates of the frequency of IE63-positive neurons, varying from a rare event to abundant expression. We examined IE63 expression in cadaver ganglia using a high-potency rabbit anti-IE63 antibody and corresponding preimmune serum. Using standard immunohistochemical techniques, we evaluated 10 ganglia that contained VZV DNA from seven individuals. These experiments showed that neuronal pigments were a confounding variable; however, by examining sections coded to prevent investigator bias and applying statistical analysis, we determined that IE63 protein, if present, is in a very small proportion of neurons (<2.8%). To refine estimates of IE63 protein abundance, we modified our protocol by incorporating a biological stain to exclude the pigment signal and evaluated 27 ganglia from 18 individuals. We identified IE63 protein in neurons within only one ganglion, in which VZV glycoprotein E and an immune cell infiltrate were also demonstrated. Antigen preservation was shown by detection of neuronal synaptophysin. These data provide evidence that the expression of IE63 protein, which has been referred to as a latency-associated protein, is rare. Refining estimates of VZV protein expression in neurons is important for developing a hypothesis about the mechanisms by which VZV latency may be maintained.Varicella-zoster virus (VZV), the human alphaherpesvirus that causes varicella during primary infection, establishes a lifelong latency in neurons of the sensory ganglia along the cerebrospinal axis (7). Sensory ganglia are composed of heterogeneous populations of neurons surrounded by satellite cells and other nonneuronal cells; axons extend from these neurons to innervate the skin and mucous membranes (50). Herpes zoster (shingles) results from reactivation of latent virus within ganglion cells and transfer of newly synthesized infectious particles to the skin via axonal transport (7).The number and type of neural cells that harbor latent virus are fundamental to the question of VZV latency. Studies to address this question have used methods to detect viral DNA, RNA, and proteins in cadaver ganglia. Although RNA is especially vulnerable to RNA-degrading enzymes during the postmortem interval between death and fixation of histological specimens at autopsy, DNA and proteins are reasonably stable (16). Nevertheless, reports using either VZV DNA or protein detection to assess the numbers of cells that contain VZV in human cadaver ganglia have yielded estimates that are extremely variable. By in situ hybridization and PCR methods, VZV DNA has been reported in as few as 1.5% of neurons exclusively (none in satellite cells) to as many as 30% of ganglion cells (neurons as well as satellite cells) (24, 26). Most recently,...

Background A first step to combating antimicrobial resistance in enteric pathogens is to establish an objective assessment of antibiotic exposure. Our goal was to develop and evaluate a liquid chromatography–ion trap mass spectrometry (LC/MS) method to determine antibiotic exposure in patients with cholera. Methods A priority list for targeted LC/MS was generated from medication-vendor surveys in Bangladesh. A study of patients with and those without cholera was conducted to collect and analyze paired urine and stool samples. Results Among 845 patients, 11% (90) were Vibrio cholerae positive; among these 90 patients, analysis of stool specimens revealed ≥1 antibiotic in 86% and ≥2 antibiotics in 52%. Among 44 patients with cholera and paired urine and stool specimens, ≥1 antibiotic was detected in 98% and ≥2 antibiotics were detected in 84%, despite 55% self-reporting medication use. Compared with LC/MS, a low-cost antimicrobial detection bioassay lacked a sufficient negative predictive value (10%; 95% confidence interval, 6%–16%). Detection of guideline-recommended antibiotics in stool specimens did (for azithromycin; P = .040) and did not (for ciprofloxacin) correlate with V. cholerae suppression. A nonrecommended antibiotic (metronidazole) was associated with decreases in anaerobes (ie, Prevotella organisms; P < .001). Conclusion These findings suggest that there may be no true negative control group when attempting to account for antibiotic exposure in settings like those in this study.

A fundamental, clinical and scientific concern is how lytic bacteriophage, as well as antibiotics, impact diagnostic positivity. Cholera was chosen as a model disease to investigate this important question because cholera outbreaks enable large enrollment, field methods are well established, and the predatory relationship between lytic bacteriophage and the etiologic agent Vibrio cholerae share commonalities across bacterial taxa. Patients with diarrheal disease were enrolled at two remote hospitals in Bangladesh. Diagnostic performance was assessed as a function of lytic bacteriophage detection and exposure to the first-line antibiotic azithromycin detected in stool samples by mass spectrometry. Among diarrheal samples positive by nanoliter quantitative PCR for V. cholerae (n=78/849), the odds that a rapid diagnostic test (RDT) or qPCR was positive was reduced by 89% (OR 0.108; 95%CI 0.002-0.872) and 87% (OR 0.130; 95%CI 0.022-0.649) when lytic bacteriophage were detected, respectively. The odds that a rapid diagnostic test (RDT) or qPCR was positive was reduced by more than 99% (OR 0.00; 95% CI: 0.00-0.28) and 89% (OR 0.11; 95% CI: 0.03-0.44) when azithromycin was detected, respectively. Analysis of additional samples from South Sudan found similar phage effects on RDTs; antibiotics were not assayed. Cholera burden estimates may improve by accommodating for the negative effects of lytic bacteriophage and antibiotic exposure on diagnostic positivity. One accommodation is using bacteriophage detection as proxy for pathogen detection. These findings have relevance for other diagnostic settings where bacterial pathogens are vulnerable to lytic bacteriophage predation.

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