Optimizing viable leukocyte sampling from the female genital tract for clinical trials: an international multi-site study

BACKGROUND: Functional analysis of mononuclear leukocytes in the female genital mucosa is essential for understanding the immunologic effects of HIV vaccines and microbicides at the site of HIV exposure. However, the best female genital tract sampling technique is unclear. Methods and FINDINGS: We enrolled women from four sites in Africa and the US to compare three genital leukocyte sampling methods: cervicovaginal lavages (CVL), endocervical cytobrushes, and ectocervical biopsies. Absolute yields of mononuclear leukocyte subpopulations were determined by flow cytometric bead-based cell counting. Of the non-invasive sampling types, two combined sequential cytobrushes yielded significantly more viable mononuclear leukocytes than a CVL (p<0.0001). In a subsequent comparison, two cytobrushes yielded as many leukocytes (∼10,000) as one biopsy, with macrophages/monocytes being more prominent in cytobrushes and T lymphocytes in biopsies. Sample yields were consistent between sites. In a subgroup analysis, we observed significant reproducibility between replicate same-day biopsies (r = 0.89, p = 0.0123). Visible red blood cells in cytobrushes increased leukocyte yields more than three-fold (p = 0.0078), but did not change their subpopulation profile, indicating that these leukocytes were still largely derived from the mucosa and not peripheral blood. We also confirmed that many CD4 + T cells in the female genital tract express the α4β7 integrin, an HIV envelope-binding mucosal homing receptor. CONCLUSIONS: CVL sampling recovered the lowest number of viable mononuclear leukocytes. Two cervical cytobrushes yielded comparable total numbers of viable leukocytes to one biopsy, but cytobrushes and biopsies were biased toward macrophages and T lymphocytes, respectively. Our study also established the feasibility of obtaining consistent flow cytometric analyses of isolated genital cells from four study sites in the US and Africa. These data represent an important step towards implementing mucosal cell sampling in international clinical trials of HIV prevention

Dietary assessment in food environment research: A systematic review

The existing evidence on food environments and diet is inconsistent, potentially due in part to heterogeneity in measures used to assess diet. The objective of this review, conducted in 2012–2013, was to examine measures of dietary intake utilized in food environment research

Thorough in silico and in vitro cDNA analysis of 21 putative BRCA1 and BRCA2 splice variants and a complex tandem duplication in BRCA2 allowing the identification of activated cryptic splice donor sites in BRCA2 exon 11

For 21 putative BRCA1 and BRCA2 splice site variants, the concordance between mRNA analysis and predictions by in silico programs was evaluated. Aberrant splicing was confirmed for 12 alterations. In silico prediction tools were helpful to determine for which variants cDNA analysis is warranted, however, predictions for variants in the Cartegni consensus region but outside the canonical sites, were less reliable. Learning algorithms like Adaboost and Random Forest outperformed the classical tools. Further validations are warranted prior to implementation of these novel tools in clinical settings. Additionally, we report here for the first time activated cryptic donor sites in the large exon 11 of BRCA2 by evaluating the effect at the cDNA level of a novel tandem duplication (5 breakpoint in intron 4; 3 breakpoint in exon 11) and of a variant disrupting the splice donor site of exon 11 (c.6841+1G>C). Additional sites were predicted, but not activated. These sites warrant further research to increase our knowledge on cis and trans acting factors involved in the conservation of correct transcription of this large exon. This may contribute to adequate design of ASOs (antisense oligonucleotides), an emerging therapy to render cancer cells sensitive to PARP inhibitor and platinum therapies

Trauma memory characteristics and neurocognitive performance in youth exposed to single-event trauma

Cognitive models of posttraumatic stress disorder (PTSD) highlight characteristics of trauma memories, such as disorganisation, as key mechanisms in the aetiology of the disorder. However, studies investigating trauma memory in youth have provided inconsistent findings. Research has highlighted that PTSD in youth may be accompanied by difficulties in neurocognitive functioning, potentially impacting ability to recall the trauma memory. The present study sought to investigate both trauma memory characteristics and neurocognitive functioning in youth aged 8–17 years. Youths exposed to single-event trauma, with (N = 29, Mage = 13.6, 21 female) and without (N = 40, Mage = 13.3, 21 female) a diagnosis of PTSD, completed self-report measures of trauma memory, a narrative memory task and a set of neurocognitive tests two to six months post-trauma. A group of non trauma-exposed youths (N = 36, Mage = 13.9, 27 female) were compared on narrative and neurocognitive tasks. Results indicated that trauma memories in youth with, versus without, PTSD were more sensory-laden, temporally disrupted, difficult to verbally access, and formed a more ‘central’ part of their identity. Greater differences were observed for self-reported memory characteristics compared to narrative characteristics. No between group differences in neurocognitive function were observed. Self-reported trauma memory characteristics highlight an important factor in the aetiology of PTSD. The observed lack of significant differences in neurocognitive ability potentially suggests that cognitive factors represent a more relevant treatment target than neurocognitive factors in single-event PTSD. Further research to understand the cognitive factors represented by self-reported trauma memory characteristics is recommended

Mammalian Neurogenesis Requires Treacle-Plk1 for Precise Control of Spindle Orientation, Mitotic Progression, and Maintenance of Neural Progenitor Cells

The cerebral cortex is a specialized region of the brain that processes cognitive, motor, somatosensory, auditory, and visual functions. Its characteristic architecture and size is dependent upon the number of neurons generated during embryogenesis and has been postulated to be governed by symmetric versus asymmetric cell divisions, which mediate the balance between progenitor cell maintenance and neuron differentiation, respectively. The mechanistic importance of spindle orientation remains controversial, hence there is considerable interest in understanding how neural progenitor cell mitosis is controlled during neurogenesis. We discovered that Treacle, which is encoded by the Tcof1 gene, is a novel centrosome- and kinetochore-associated protein that is critical for spindle fidelity and mitotic progression. Tcof1/Treacle loss-of-function disrupts spindle orientation and cell cycle progression, which perturbs the maintenance, proliferation, and localization of neural progenitors during cortical neurogenesis. Consistent with this, Tcof1+/− mice exhibit reduced brain size as a consequence of defects in neural progenitor maintenance. We determined that Treacle elicits its effect via a direct interaction with Polo-like kinase1 (Plk1), and furthermore we discovered novel in vivo roles for Plk1 in governing mitotic progression and spindle orientation in the developing mammalian cortex. Increased asymmetric cell division, however, did not promote increased neuronal differentiation. Collectively our research has therefore identified Treacle and Plk1 as novel in vivo regulators of spindle fidelity, mitotic progression, and proliferation in the maintenance and localization of neural progenitor cells. Together, Treacle and Plk1 are critically required for proper cortical neurogenesis, which has important implications in the regulation of mammalian brain size and the pathogenesis of congenital neurodevelopmental disorders such as microcephaly

Intra- and Inter-clade Cross-reactivity by HIV-1 Gag Specific T-Cells Reveals Exclusive and Commonly Targeted Regions: Implications for Current Vaccine Trials

The genetic diversity of HIV-1 across the globe is a major challenge for developing an HIV vaccine. To facilitate immunogen design, it is important to characterize clusters of commonly targeted T-cell epitopes across different HIV clades. To address this, we examined 39 HIV-1 clade C infected individuals for IFN-γ Gag-specific T-cell responses using five sets of overlapping peptides, two sets matching clade C vaccine candidates derived from strains from South Africa and China, and three peptide sets corresponding to consensus clades A, B, and D sequences. The magnitude and breadth of T-cell responses against the two clade C peptide sets did not differ, however clade C peptides were preferentially recognized compared to the other peptide sets. A total of 84 peptides were recognized, of which 19 were exclusively from clade C, 8 exclusively from clade B, one peptide each from A and D and 17 were commonly recognized by clade A, B, C and D. The entropy of the exclusively recognized peptides was significantly higher than that of commonly recognized peptides (p = 0.0128) and the median peptide processing scores were significantly higher for the peptide variants recognized versus those not recognized (p = 0.0001). Consistent with these results, the predicted Major Histocompatibility Complex Class I IC50 values were significantly lower for the recognized peptide variants compared to those not recognized in the ELISPOT assay (p<0.0001), suggesting that peptide variation between clades, resulting in lack of cross-clade recognition, has been shaped by host immune selection pressure. Overall, our study shows that clade C infected individuals recognize clade C peptides with greater frequency and higher magnitude than other clades, and that a selection of highly conserved epitope regions within Gag are commonly recognized and give rise to cross-clade reactivities

Human germline heterozygous gain-of-function STAT6 variants cause severe allergic disease

STAT6 (signal transducer and activator of transcription 6) is a transcription factor that plays a central role in the pathophysiology of allergic inflammation. We have identified 16 patients from 10 families spanning three continents with a profound phenotype of early-life onset allergic immune dysregulation, widespread treatment-resistant atopic dermatitis, hypereosinophilia with esosinophilic gastrointestinal disease, asthma, elevated serum IgE, IgE-mediated food allergies, and anaphylaxis. The cases were either sporadic (seven kindreds) or followed an autosomal dominant inheritance pattern (three kindreds). All patients carried monoallelic rare variants in STAT6 and functional studies established their gain-of-function (GOF) phenotype with sustained STAT6 phosphorylation, increased STAT6 target gene expression, and TH2 skewing. Precision treatment with the anti-IL-4Rα antibody, dupilumab, was highly effective improving both clinical manifestations and immunological biomarkers. This study identifies heterozygous GOF variants in STAT6 as a novel autosomal dominant allergic disorder. We anticipate that our discovery of multiple kindreds with germline STAT6 GOF variants will facilitate the recognition of more affected individuals and the full definition of this new primary atopic disorder

Pathogenetics of alveolar capillary dysplasia with misalignment of pulmonary veins.

Alveolar capillary dysplasia with misalignment of pulmonary veins (ACDMPV) is a lethal lung developmental disorder caused by heterozygous point mutations or genomic deletion copy-number variants (CNVs) of FOXF1 or its upstream enhancer involving fetal lung-expressed long noncoding RNA genes LINC01081 and LINC01082. Using custom-designed array comparative genomic hybridization, Sanger sequencing, whole exome sequencing (WES), and bioinformatic analyses, we studied 22 new unrelated families (20 postnatal and two prenatal) with clinically diagnosed ACDMPV. We describe novel deletion CNVs at the FOXF1 locus in 13 unrelated ACDMPV patients. Together with the previously reported cases, all 31 genomic deletions in 16q24.1, pathogenic for ACDMPV, for which parental origin was determined, arose de novo with 30 of them occurring on the maternally inherited chromosome 16, strongly implicating genomic imprinting of the FOXF1 locus in human lungs. Surprisingly, we have also identified four ACDMPV families with the pathogenic variants in the FOXF1 locus that arose on paternal chromosome 16. Interestingly, a combination of the severe cardiac defects, including hypoplastic left heart, and single umbilical artery were observed only in children with deletion CNVs involving FOXF1 and its upstream enhancer. Our data demonstrate that genomic imprinting at 16q24.1 plays an important role in variable ACDMPV manifestation likely through long-range regulation of FOXF1 expression, and may be also responsible for key phenotypic features of maternal uniparental disomy 16. Moreover, in one family, WES revealed a de novo missense variant in ESRP1, potentially implicating FGF signaling in the etiology of ACDMPV