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Zika virus (ZIKV) is an emerging mosquito-borne flavivirus linked to devastating neurologic diseases. Immune responses to flaviviruses may be pathogenic or protective. Our understanding of human immune responses to ZIKV in vivo remains limited. Therefore, we performed a longitudinal molecular and phenotypic characterization of innate and adaptive immune responses during an acute ZIKV infection. We found that innate immune transcriptional and genomic responses were both cell type- and time-dependent. While interferon stimulated gene induction was common to all innate immune cells, the upregulation of important inflammatory cytokine genes was primarily limited to monocyte subsets. Additionally, genomic analysis revealed substantial chromatin remodeling at sites containing cell-type specific transcription factor binding motifs that may explain the observed changes in gene expression. In this dengue virus-experienced individual, adaptive immune responses were rapidly mobilized with T cell transcriptional activity and ZIKV neutralizing antibody responses peaking 6 days after the onset of symptoms. Collectively this study characterizes the development and resolution of an in vivo human immune response to acute ZIKV infection in an individual with pre-existing flavivirus immunity., Competing Interests: The authors declare that there are no competing interest.

Background: The lack of specific biomarkers makes the diagnosis of hereditary angioedema (HAE) with normal levels of C1-inhibitor (C1INH) protein (HAE-nl-C1INH) and idiopathic non-histaminergic angioedema (INHA) difficult. Confirming or excluding these diagnoses is a significant challenge for clinicians evaluating patients with angioedema., Objective: To develop a reliable biomarker that would aid the diagnosis of HAE-nl-C1INH and INHA., Methods: A total of 154 consecutive patients referred for angioedema at a single centre were enrolled and evaluated. Subjects were clinically phenotyped based on clinical history and response to treatment by clinicians blinded to laboratory assay results. Plasma kallikrein activity was measured by the cleavage of the fluorometric substrate Z-Phe-Arg-AMC-HCL in plasma samples stimulated ex vivo with submaximal doses of dextran sulphate., Results: Stimulated plasma kallikrein activity (mean relative fluorescence units/min ± SD) was significantly increased in both HAE-nl-C1INH (1804 ± 600) and INHA (1579 ± 371) subjects compared to non-swelling controls (171 ± 46) and histaminergic angioedema (133 ± 30) subjects. Using a threshold cut-off based on the normal controls, HAE-nl-C1INH and INHA subjects could be differentiated from histaminergic angioedema subjects with high sensitivity (negative predictive value 86%-89%) and specificity (positive predictive value 80%-100%)., Conclusion and Clinical Relevance: The stimulated kallikrein activity assay allows differentiation of bradykinin- from histamine-mediated angioedema. The assay could feasibly be considered as a potential clinical tool for the diagnosis of bradykinin-mediated angioedema., (© 2018 John Wiley & Sons Ltd.)

We have previously shown that heparin-binding EGF-like growth factor (HB-EGF) protects intestinal epithelial cells (IEC) from necrosis and apoptosis in vitro and from intestinal ischemia/reperfusion injury in vivo; however, the mechanisms of HB-EGF cytoprotection are unclear. Overproduction of iNOS and NO have been implicated in the pathogenesis of several forms of ischemia/reperfusion injury. We therefore studied whether HB-EGF could down-regulate proinflammatory cytokine-induced iNOS and NO production in intestinal epithelial cells in vitro. DLD-1 human intestinal epithelial cells were exposed to the proinflammatory cytokines interleukin-1beta (IL-1beta) (20 ng/ml) and interferon-gamma (IFN-gamma) (10 ng/ml) to stimulate iNOS induction and NO production. Cells were treated with HB-EGF (0-100 ng/ml) either before or with cytokine exposure, and cells and supernatants were harvested 24 and 48 h later. Accumulated NO was measured in supernatants by chemiluminescence. Total RNA was extracted from cell lysates for iNOS mRNA quantification using real-time reverse transcription-polymerase chain reaction (RT-PCR), and total protein was extracted from cell lysates for detection of iNOS protein. HB-EGF significantly decreased cytokine-induced NO production in a dose dependent manner, and NO reduction was associated with iNOS suppression at both the mRNA and protein levels. While cytokine exposure resulted in a significant increase in iNOS mRNA expression in these cells (109 plus minus 9 fold), HB-EGF reduced iNOS expression by 5.7-fold (P < 0.05). These results suggest that HB-EGF may exert its cytoprotective effects, in part, by down-regulating iNOS and NO production, and provides further rationale for additional testing of the effects of HB-EGF in the treatment of intestinal ischemia/reperfusion injury in vivo., ((C)2001 Elsevier Science (USA).)

Background: T cells have been implicated in the pathogenesis of atopic asthma. We have previously shown that memory T helper cells (CD4+CD45RO+) are preferentially activated relative to naïve T helper cells (CD4+CD45RA+) after bronchial allergen challenge. However, specific T helper subpopulations that are activated in atopy and/or asthma remain undefined., Objective: To determine the T helper subpopulations and activation phenotypes relevant to acute and stable asthma that may be common with or distinct from atopy., Methods: Two groups of atopic asthmatics (ten acute and nine stable asthmatics) and two non-asthmatic groups (14 non-asthmatic atopics and eight normal non-atopic controls) were analysed. Ten acute asthmatics were assessed in the emergency room during an acute episode (FEV1 43.6% +/- 18.4). Nine stable asthmatics were assessed during a symptom-free period (FEV1 85% +/- 6). Using multiple colour flow cytometry we analysed T cell subpopulations and the expression of IL-2-receptor (IL-2R) and MHC-class II antigens (MHC II) on naïve and memory T helper cells in the peripheral blood of asthmatic and non-asthmatic groups., Results: Atopic asthmatics (acute and stable) had an increased percentage of memory T helper cells expressing IL-2R compared with normal non-atopics (mean SD 16.1 +/- 6%, 12.4 +/- 2% and 7.7 +/- 1.8%, P < 0.05) but not compared with non-asthmatic atopics (10 +/- 3.5%). Naïve T helper cells had low expression of IL-2R and MHC II in all four groups. MHC II antigen expression was increased in memory T helper cells of asthmatics (acute and stable) compared with normal non-atopics (13.9 +/- 7.5, 10.6 +/- 5 and 4.9 +/- 2.5, P < 0.05) but not compared with non-asthmatic atopics (7.92 4). A novel finding was that IL-2R and the MHC II molecules were mainly expressed in non-overlapping populations and coexpression was found predominantly on memory T helper cells. Asthmatics (acute and stable) had higher proportion of double positive memory T helper cells (IL-2R+MHC II+) compared with both non-asthmatic groups (P < 0.05)., Conclusions: We demonstrate a differential expression of IL-2R+ and MCH II+ on CD45RO+ T helper cells that would suggest that there are three subsets of activated memory T helper cells in asthmatics. Two non-overlapping IL-2R+ or MHC II+ CD45RO+ T helper cells and a third subpopulation of activated cells that coexpress IL-2R and MHC II (double positives). This latter subpopulation is significantly higher in asthmatics (acute or stable) compared with both non-asthmatic groups, suggesting a specific T helper activation phenotype distinct to atopic asthmatics as compared with atopic non-asthmatics.

Vasoactive intestinal peptide (VIP) binding sites have been identified in the human thymus, but the receptor subtype and how these receptors are distributed in the human thymus subsets is unknown. To assess gene expression, distribution, and receptor regulation of the two G-protein-associated VIP receptors, VPAC1 and VPAC2 mRNAs were quantified using a novel fluorometric-based kinetic (real-time) RT-PCR. Bulk and fractionated thymocytes were stimulated via the TCR/CD3 receptor complex and anti-CD28. Our results demonstrate that thymocytes express higher levels of VPAC2 compared to VPAC1 expression in bulk thymocytes, CD4+CD8+ selected double positives (DP), and CD8 depleted thymocytes. Double negative cells express low levels of VPAC2 mRNA. We demonstrate T-cell activation-dependent down-regulation of VPAC1, but not VPAC2, in human thymocytes. This study reports the first direct evidence of a differential distribution and selective regulation of VPAC1 and VPAC2 gene expression in normal human thymocyte subsets.

Background: Genes linked to the major histocompatibility complex (MHC), have been implicated in atopic asthma. Asthma is highly prevalent in the Venezuelan population (estimated at 20%) and genetic markers are needed to identify populations at risk and plan intervention strategies., Objective: To study the influence of the MHC class I and class II genes in the susceptibility to atopic asthma., Methods: MHC-class I HLA-A, -C, -B and MHC-class II HLA-DR, -DQ, -DP gene haplotype frequencies were determined in 135 Venezuelan mestizos, 71 belong to 20 atopic asthmatic families and 64 unrelated controls. The index cases were 20 atopic asthmatics with positive skin-prick tests and specific serum immunoglobulin E (IgE) for Dermatophagoides pteronyssinus (Der p) and Dermatophagoides farinae (Der f). To ascertain the genes associated with susceptibility to atopy and/or asthma, two control groups were studied, 41 non-atopic subjects with skin-prick negative test, and undetectable levels of specific IgE and 23 non-asthmatic atopic subjects with detectable specific IgE to Der p and Der f. A linkage analysis was performed in those families with two or more atopic siblings (with or without asthma)., Results: MHC-class I genes analysis showed that HLA-Cw7 was absent in the asthmatic patients studied, whereas the frequency of this allele was 14.3% in non-atopic controls (P = 0.0 17, PC = 0.19) and 20.8% in the atopic controls (P = 0.0066, PC = 0.07). MHC-class II gene analysis showed a significant increase of the HLA-DRB1*11 in the asthmatic patients compared with non-atopic controls (allele frequencies of 25.6 vs 4.4% P = 0.0017, PC = 0.02). There were no significant differences among asthmatic and atopic controls in the frequency of HLA-DRB1*11 (25.6 vs 17.4%). In contrast, the HLA-DRB1*1101+ haplotypes were significantly higher in asthmatics compared with atopic and non-atopic controls (19.6% vs 2.2% vs 2.3%, PC<0.05). The HLA-DRB1*1101, DQA1*0501, DQB1*0301 haplotype was found significantly increased in the patients vs non-atopic controls (15.4 vs 1.1%, PC< 0.01). The serum levels of specific IgE were detectable in both atopic asthmatics and atopic controls; however, it was higher in atopic asthmatics vs atopic controls Der p (median, 58.7 vs 2.7 kU/L, P<0.001) and Der f (median, 46.9 vs 2.7 kU/L, P<0.001). No linkage between MHC genes and mite-atopy could be documented on informative families with two or more atopic siblings., Conclusions: We have identified an association between the haplotype HLA-DRB1*1101, DQA1*0501, DQB1*0301 and atopic asthma that confers susceptibility to develop mite-sensitive asthma to atopics (relative risk, RR 8.2), and to non-atopic controls (RR = 15.8) that carry this haplotype. Conversely, the allele HLA-Cw7 was absent in the asthmatics studied and had higher frequencies in the atopic (RR = 0.05) and non-atopic (RR = 0.08) controls. Thus, it may have a protective role for developing atopic asthma in the population studied.

Background: T helper cells are a heterogeneous group of cells that have phenotypic and functional differences. Activated T helper cells have been found in peripheral blood after allergen challenge of subjects with atopic asthma, but the phenotypes of specific T helper subpopulation involved remains to be identified., Objective: To characterize the T cell activation markers that may be regulated by allergens, we analyzed peripheral blood lymphocytes obtained before and after allergen challenge from subjects with atopic asthma., Methods: We analyzed the distribution of the cell surface activation markers, interleukin 2 receptor (IL-2R) and major histocompatibility complex class II antigens (MHC II) among T helper subpopulations classified as naive (CD45RA) or memory (CD45RO) phenotypes. Nine adult subjects with atopic asthma underwent bronchoprovacative allergen inhalation and isocapnic cold air hyperventilation (ISH) challenge followed by serial spirometry. Peripheral blood mononuclear cells (PBMC) were isolated at baseline and 2 and 24 hours after challenge. Four-color flow cytometry was used to analyze the expression and distribution in vivo of IL-2R and MHC II activation markers on naive and memory T cell subsets after challenge., Results: At 2 and 24 hours after allergen challenge, there was a significant increase in the CD45RO+IL-2R+ T helper cells compared with baseline (mean +/- SE, baseline, 12.5% +/- 1% versus 2 hours, 18.1% +/- 1% and 24 hours, 17.8% +/- 2%, p < 0.025). MHC II expression was not significantly increased after challenge on naive and memory T helper cells and coexpression of IL-2R and MHC II was only found in a small proportion of CD45RO+ T helper cells (2.7% +/- 1%). No changes of IL-2R or MHC II expression on T helper subsets were observed after ISH challenge in the same patients. We also found that 31% to 46% of T helper cells coexpress CD45RA and CD45RO simultaneously, and upregulation of IL-2-R and MHC II expression occurs only on those T helper cells that express CD45RO., Conclusions: We have found that T helper cells express both CD45RA and CD45RO isoforms, which suggests the existence of a transitional phenotype among naive and memory T helper cells in peripheral blood. In subjects with atopic asthma, our in vivo analysis characterizes two populations of activated memory T helper cells based on the expression of IL-2R or MHC II surface molecules after allergen challenge.

Peripheral blood lymphocytes from nonresponders to hepatitis B vaccine (HBsAg) failed to undergo a proliferative response to recombinant HBsAg in vitro, whereas cells from responders proliferated vigorously. The lack of proliferative response was not due to defective antigen presentation in that MHC-identical responder and nonresponder antigen presenting cells were equally effective in stimulating responder T cells. Nonresponder T cells did not proliferate in response to antigen-pulsed MHC identical responder antigen presenting cells. The present study demonstrated that: 1) there were no detectable (1 in < 20 x 10(4) HBsAg-precursor T cells in any of the nonresponders, while in responders the frequency of HBsAg-precursor T cells ranged from 1 in 3.2 x 10(3) to 1 in 40 x 10(3); 2) nonresponder cell cultures did not secrete IL-2 in response to HBsAg stimulation; 3) exogenous recombinant IL-2 did not restore the proliferative response of the T cells in HBsAg-pulsed cultures of nonresponders. These results suggest that the cellular basis for the lack of response to HBsAg is a defect in HBsAg-specific Th1-like cells; either there is an absence of the Th1 cells or cells with TCR specificity for HBsAg are present but are unresponsive to the HBsAg peptide-MHC complex (i.e., anergy or tolerance).