Maria Luz Lara-Marquez

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. Zika virus (ZIKV) is an emerging flaviviral infection that causes significant clinical disease. It is estimated that approximately one half of the world’s population is at risk for ZIKV infection. There are only a limited number of studies describing the human immune response to ZIKV infection. Carlin et al. combined conventional and genomic approaches to longitudinally analyze the innate and adaptive immune responses to acute ZIKV infection and its resolution in a person who was infected while traveling in Venezuela during the 2016 ZIKV epidemic year. Genome-wide sequencing in individual cell types revealed that although many populations respond to interferon stimulation, only specific cell populations within peripheral blood mononuclear cells upregulate important inflammatory cytokine gene expression. Additionally, analysis of open chromatin using ATAC-seq suggests that chromatin remodeling at sites containing cell-type specific transcription factor binding motifs may help us understand changes in gene expression. Consistent with previous reports, this individual with prior exposure to dengue virus (DENV), rapidly developed neutralizing anti-ZIKV responses that were cross-reactive with multiple DENV serotypes. Collectively this study combines traditional and genomic approaches to characterize the cell-type specific development of an in vivo human immune response to acute ZIKV infection.

Summary: 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.

Vasoactive intestinal peptide receptors 1 (VPAC1) and 2 (VPAC2) have been identified in humans. Cell lines expressing only VPAC1 (HT-29) or VPAC2 (Molt-4b) were identified using real-time reverse transcriptase polymerase chain reaction. Vasoactive intestinal peptide (VIP) and related peptides, VIP-6-28, VIP4-28, and VIP10-28, previously isolated from cultures of human leukocytes, were evaluated for their ability to bind to VPAC1 and VPAC2 and to increase the levels of cAMP in HT-29 and Molt-4b cells. VIP bound to membranes of HT-29 colon carcinoma cells and Molt-4b lymphoblasts with high affinity (KD = 1.6 +/- 0.2 and 1.7 +/- 0.9 nM, respectively). VIP4-28 also demonstrated high-affinity binding (KD = 1.7 +/- 0.2 and 1.7 +/- 0.7 nM in HT-29 and Molt-4b, respectively). VIP and VIP4-28 are potent VPAC1 agonists, inducing maximal 200- and 400-fold increases in cAMP, respectively. VIP demonstrated weak VPAC2 agonist activity, inducing a maximal 14-fold increase in cAMP. VIP4-28 had no VPAC2 agonist activity but demonstrated potent VPAC2 antagonist activity. VIP4-28 inhibited VPAC2-mediated increases in cAMP in Molt-4b cells up to 95%, but had no antagonistic effect on VPAC1. Lymphoblasts did not hydrolyze VIP4-28 to a form with VPAC1 antagonist activity. VIP4-28 thus is a lymphocyte-generated VIP fragment with potent agonist activity for VPAC1 and potent antagonist activity for VPAC2.

Heparin-binding epidermal growth factor-like growth factor (HB-EGF) has been shown to protect intestine from ischemia/reperfusion (I/R) injury in vivo and to down-regulate inducible nitric oxide synthase (iNOS) and nitric oxide (NO) production in intestinal epithelial cells in vitro. The present study was undertaken to investigate whether HB-EGF could modulate the iNOS/NO axis after total midgut I/R injury in rats. I/R injury induced a significant increase in iNOS gene expression (quantified by real-time RT-PCR) and protein production (detected by western blots), as well as elevation of serum NO levels (measured by chemiluminescence assay). Nitrotyrosine (NT) and iNOS production colocalized immunohistochemically, with positive staining found mainly in villous and crypt epithelial cells, as well as ganglion cells. Intraluminal administration of HB-EGF 45 min after the start of a 90-min ischemic interval significantly decreased I/R-induced iNOS gene expression and protein production, as well as serum NO levels. Immunohistochemically, HB-EGF administration led to elimination of iNOS and NT staining in crypt epithelial cells and ganglion cells, with only weak staining that remained in villous epithelial cells. Thus, HB-EGF protects the intestine from I/R injury, at least partially, through down-regulation of the iNOS/NO/NT pathway, a mechanism that is central to I/R injury in multiple organ systems.

Vasoactive intestinal peptide (VIP) has potent antiproliferative and anti-inflammatory functions in the immune system. Two structurally distinct G-protein-associated receptors, VIP receptor type 1 (VPAC1) and VIP receptor type 2 (VPAC2), mediate the biological effects of VIP. The regulation of VIP receptor gene expression and the distribution of these receptors in different compartments of the human immune systems are unknown. This study reports, for the first time, a quantitative analysis of VPAC1 and VPAC2 mRNA expression in resting and activated T cells as well as in resting monocytes. Purified human peripheral blood CD4⁺ T cells and CD8⁺ T cells were stimulated via the TCR/CD3 receptor complex. Using the novel fluorometric-based kinetic (real-time) RT-PCR, we determined that VPAC1 is constitutively expressed in resting T cells and monocytes; the levels of expression were significantly higher in monocytes and CD4⁺ T cells than in CD8⁺ T cells. VPAC1 mRNA expression is significantly higher relative to VPAC2 in resting CD4⁺ T cells and CD8⁺ T cells. VPAC2 is expressed at very low levels in resting T cells but is not detectable in resting monocytes. In vitro stimulation of Th cells with soluble anti-CD3 plus PMA induced a T cell activation-dependent down-regulation of VPAC1. VPAC1 is down-regulated under conditions of optimal T cell stimulation. Our results suggest that selective VIP effects on T cell function may be mediated via selective expression of VPAC1 and VPAC2 on T cells and monocytes. Furthermore, down-regulation of VPAC1 in CD4⁺ T cell subpopulations is highly correlated with T cell activation.

Abstract: 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 downregulation 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 = 0.017, 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.