Affordable flow cytometry for enumeration of absolute CD4(+ )T-lymphocytes to identify subtype C HIV-1 infected adults requiring antiretroviral therapy (ART) and monitoring response to ART in a resource-limited setting

BACKGROUND: The World Health Organization (WHO)'s "3 × 5 program" has spurred efforts to place 3 million people on combination antiretroviral therapy (ART) for treatment of AIDS in resource-limited countries. Paradoxically, the cost of CD4(+ )T-lymphocyte count essential for decision-making to commence HIV positive adults on ART as well as for monitoring responses to ART remains unaffordable in most resource-limited countries. Thus, low-cost methods for enumerating CD4(+ )T-lymphocyte are urgently needed. OBJECTIVE: To evaluate Cyflow cytometry (Cyflow SL, Partec, Munster, Germany) for enumeration of absolute CD4(+ )T-lymphocyte in subtype C HIV-1 seropositive subjects using FACSCount (Becton and Dickinson, Immunocytometry Systems, San Jose, CA, USA) as the "predicate method". METHODS: A total of 150 HIV-1 seropositive subjects were included in the evaluation exercise. Fifty-eight specimens were collected from pregnant HIV-1 seropositive women (subtype C drug resistance study). Twenty-seven specimens were collected from women and their spouses with AIDS followed in a Duke ART study to assess the immunologic and virologic responses to generic ART, comprising Stavudine, Lamivudine and Nevirapine (Stalanev, Varichem Labs, Harare, Zimbabwe). Sixty-five specimens were collected from AIDS patients enrolled in an ongoing Kaposi Sarcoma (KS) study to investigate impact of ART on KS progression. Enumeration of CD4(+ )T-lymphocytes using FACSCount is routinely conducted for all the three studies. The Medical Research Council of Zimbabwe and Medicines Control Authority of Zimbabwe approved the studies. Whole blood was collected in EDTA vacutainer tubes and aliquoted into two tubes (200 μL in each). CD4(+ )T-lymphocyte counts were enumerated using a Cyflow counter, in the Department of Immunology and a FACSCount in the Department of Obstetrics and Gynaecology within 6 hours of phlebotomy following manufacturers' instructions. RESULTS: Using linear regression analysis, there was a very strong correlation (R = 0.991) between the overall CD4(+ )T-lymphocyte counts obtained by FACSCount and those obtained by Cyflow. When data analysis was stratified by study groups, there was a strong correlation between the FACSCount and Cyflow CD4(+ )T-lymphocyte counts from subjects in the three independent studies; Subtype C resistance (R(2 )= 0.987), Duke ART (R(2 )= 0.980) and KS (R(2 )= 0.994), Table 1. Using Bland-Altman plots, the overall, absolute CD4(+ )T lymphocytes obtained by the two methods were in excellent agreement (mean difference 1.21, 95% Confidence Interval {CI): -2.1 to 3.3). For the 0–250 CD4(+ )T-lymphocytes range, the CD4 counts obtained using FACSCount were also in good agreement with those obtained using Cyflow counter (mean difference = 2.6 cells/μL, 95% CI: -1.1 to 6.3). Similarly, in the 251–500 (mean difference 1.0, cells/μL, 95% CI: -3.7 to 5.6) and the 501–1200 (mean difference = 0.29 cells/μL, 95% CI: -8.1 to 8.7) CD4 T-lymphocytes range, good agreement was observed. CONCLUSION: The Cyflow counter is as accurate as the FACSCount in enumerating absolute CD4(+ )T-lymphocytes in the range 1–1200 cells/μL. Cyflow cytometry is relatively affordable, easy to use technology that is useful not only in identifying HIV seropositive individuals who require ART but also for monitoring immunologic responses to ART

Differential Effects of Dietary versus Exercise Intervention on Intrahepatic MAIT Cells and Histological Features of NAFLD

Background: Mucosal-associated invariant T (MAIT) cells promote inflammation in obesity and are implicated in the progression of non-alcoholic fatty liver disease (NAFLD). However, as the intrahepatic MAIT cell response to lifestyle intervention in NAFLD has not been investigated, this work aimed to examine circulating and intrahepatic MAIT cell populations in patients with NAFLD, after either 12 weeks of dietary intervention (DI) or aerobic exercise intervention (EI). Methods: Multicolour flow cytometry was used to immunophenotype circulating and intrahepatic MAIT cells and measure MAIT cell expression (median fluorescence intensity, MFI) of the activation marker CD69 and apoptotic marker CD95. Liver histology, clinical parameters, and MAIT cell populations were assessed at baseline (T0) and following completion (T1) of DI or EI. Results: Forty-five patients completed the study. DI participants showed decreased median (interquartile range) expression of the activation marker CD69 on circulating MAIT cells (T0: 104 (134) versus T1 27 (114) MFI; p = 0.0353) and improvements in histological steatosis grade post-intervention. EI participants showed increased expression of the apoptotic marker CD95, both in circulating (T0: 1549 (888) versus T1: 2563 (1371) MFI; p = 0.0043) and intrahepatic MAIT cells (T0: 2724 (862) versus T1: 3117 (1622) MFI; p = 0.0269). Moreover, the percentage of intrahepatic MAIT cells significantly decreased after EI (T0: 11.1 (14.4) versus T1: 5.3 (9.3)%; p = 0.0029), in conjunction with significant improvements in fibrosis stage and hepatocyte ballooning. Conclusions: These data demonstrate independent benefits from dietary and exercise intervention and suggest a role for intrahepatic MAIT cells in the observed histological improvements in NAFLD

Gene processing control loops suggested by sequencing, splicing, and RNA folding

Abstract Background Small RNAs are known to regulate diverse gene expression processes including translation, transcription, and splicing. Among small RNAs, the microRNAs (miRNAs) of 17 to 27 nucleotides (nts) undergo biogeneses including primary transcription, RNA excision and folding, nuclear export, cytoplasmic processing, and then bioactivity as regulatory agents. We propose that analogous hairpins from RNA molecules that function as part of the spliceosome might also be the source of small, regulatory RNAs (somewhat smaller than miRNAs). Results Deep sequencing technology has enabled discovery of a novel 16-nt RNA sequence in total RNA from human brain that we propose is derived from RNU1, an RNA component of spliceosome assembly. Bioinformatic alignments compel inquiring whether the novel 16-nt sequence or its precursor have a regulatory function as well as determining aspects of how processing intersects with the miRNA biogenesis pathway. Specifically, our preliminary in silico investigations reveal the sequence could regulate splicing factor Arg/Ser rich 1 (SFRS1), a gene coding an essential protein component of the spliceosome. All 16-base source sequences in the UCSC Human Genome Browser are within the 14 instances of RNU1 genes listed in wgEncodeGencodeAutoV3. Furthermore, 10 of the 14 instances of the sequence are also within a common 28-nt hairpin-forming subsequence of RNU1. Conclusions An abundant 16-nt RNA sequence is sourced from a spliceosomal RNA, lies in a stem of a predicted RNA hairpin, and includes reverse complements of subsequences of the 3'UTR of a gene coding for a spliceosome protein. Thus RNU1 could function both as a component of spliceosome assembly and as inhibitor of production of the essential, spliceosome protein coded by SFRS1. Beyond this example, a general procedure is needed for systematic discovery of multiple alignments of sequencing, splicing, and RNA folding data

Mechanisms of hypoxic up-regulation of versican gene expression in macrophages

Hypoxia is a hallmark of many pathological tissues. Macrophages accumulate in hypoxic sites and up-regulate a range of hypoxia-inducible genes. The matrix proteoglycan versican has been identified as one such gene, but the mechanisms responsible for hypoxic induction are not fully characterised. Here we investigate the up-regulation of versican by hypoxia in primary human monocyte-derived macrophages (HMDM), and, intriguingly, show that versican mRNA is up-regulated much more highly (>600 fold) by long term hypoxia (5 days) than by 1 day of hypoxia (48 fold). We report that versican mRNA decay rates are not affected by hypoxia, demonstrating that hypoxic induction of versican mRNA is mediated by increased transcription. Deletion analysis of the promoter identified two regions required for high level promoter activity of luciferase reporter constructs in human macrophages. The hypoxia-inducible transcription factor HIF-1 has previously been implicated as a key potential regulator of versican expression in hypoxia, however our data suggest that HIF-1 up-regulation is unlikely to be principally responsible for the high levels of induction observed in HMDM. Treatment of HMDM with two distinct specific inhibitors of Phosphoinositide 3-kinase (PI3K), LY290042 and wortmannin, significantly reduced induction of versican mRNA by hypoxia and provides evidence of a role for PI3K in hypoxic up-regulation of versican expression

Relevance of laboratory testing for the diagnosis of primary immunodeficiencies: a review of case-based examples of selected immunodeficiencies

The field of primary immunodeficiencies (PIDs) is one of several in the area of clinical immunology that has not been static, but rather has shown exponential growth due to enhanced physician, scientist and patient education and awareness, leading to identification of new diseases, new molecular diagnoses of existing clinical phenotypes, broadening of the spectrum of clinical and phenotypic presentations associated with a single or related gene defects, increased bioinformatics resources, and utilization of advanced diagnostic technology and methodology for disease diagnosis and management resulting in improved outcomes and survival. There are currently over 200 PIDs with at least 170 associated genetic defects identified, with several of these being reported in recent years. The enormous clinical and immunological heterogeneity in the PIDs makes diagnosis challenging, but there is no doubt that early and accurate diagnosis facilitates prompt intervention leading to decreased morbidity and mortality. Diagnosis of PIDs often requires correlation of data obtained from clinical and radiological findings with laboratory immunological analyses and genetic testing. The field of laboratory diagnostic immunology is also rapidly burgeoning, both in terms of novel technologies and applications, and knowledge of human immunology. Over the years, the classification of PIDs has been primarily based on the immunological defect(s) ("immunophenotype") with the relatively recent addition of genotype, though there are clinical classifications as well. There can be substantial overlap in terms of the broad immunophenotype and clinical features between PIDs, and therefore, it is relevant to refine, at a cellular and molecular level, unique immunological defects that allow for a specific and accurate diagnosis. The diagnostic testing armamentarium for PID includes flow cytometry - phenotyping and functional, cellular and molecular assays, protein analysis, and mutation identification by gene sequencing. The complexity and diversity of the laboratory diagnosis of PIDs necessitates many of the above-mentioned tests being performed in highly specialized reference laboratories. Despite these restrictions, there remains an urgent need for improved standardization and optimization of phenotypic and functional flow cytometry and protein-specific assays. A key component in the interpretation of immunological assays is the comparison of patient data to that obtained in a statistically-robust manner from age and gender-matched healthy donors. This review highlights a few of the laboratory assays available for the diagnostic work-up of broad categories of PIDs, based on immunophenotyping, followed by examples of disease-specific testing

The Mating Type Locus (MAT) and Sexual Reproduction of Cryptococcus heveanensis: Insights into the Evolution of Sex and Sex-Determining Chromosomal Regions in Fungi

Mating in basidiomycetous fungi is often controlled by two unlinked, multiallelic loci encoding homeodomain transcription factors or pheromones/pheromone receptors. In contrast to this tetrapolar organization, Cryptococcus neoformans/Cryptococcus gattii have a bipolar mating system, and a single biallelic locus governs sexual reproduction. The C. neoformans MAT locus is unusually large (>100 kb), contains >20 genes, and enhances virulence. Previous comparative genomic studies provided insights into how this unusual MAT locus might have evolved involving gene acquisitions into two unlinked loci and fusion into one contiguous locus, converting an ancestral tetrapolar system to a bipolar one. Here we tested this model by studying Cryptococcus heveanensis, a sister species to the pathogenic Cryptococcus species complex. An extant sexual cycle was discovered; co-incubating fertile isolates results in the teleomorph (Kwoniella heveanensis) with dikaryotic hyphae, clamp connections, septate basidia, and basidiospores. To characterize the C. heveanensis MAT locus, a fosmid library was screened with C. neoformans/C. gattii MAT genes. Positive fosmids were sequenced and assembled to generate two large probably unlinked MAT gene clusters: one corresponding to the homeodomain locus and the other to the pheromone/receptor locus. Strikingly, two divergent homeodomain genes (SXI1, SXI2) are present, similar to the bE/bW Ustilago maydis paradigm, suggesting one or the other homeodomain gene was recently lost in C. neoformans/C. gattii. Sequencing MAT genes from other C. heveanensis isolates revealed a multiallelic homeodomain locus and at least a biallelic pheromone/receptor locus, similar to known tetrapolar species. Taken together, these studies reveal an extant C. heveanensis sexual cycle, define the structure of its MAT locus consistent with tetrapolar mating, and support the proposed evolutionary model for the bipolar Cryptococcus MAT locus revealing transitions in sexuality concomitant with emergence of a pathogenic clade. These studies provide insight into convergent processes that independently punctuated evolution of sex-determining loci and sex chromosomes in fungi, plants, and animals

Organogenesis relies on SoxC transcription factors for the survival of neural and mesenchymal progenitors

During organogenesis, neural and mesenchymal progenitor cells give rise to many cell lineages, but their molecular requirements for self-renewal and lineage decisions are incompletely understood. In this study, we show that their survival critically relies on the redundantly acting SoxC transcription factors Sox4, Sox11 and Sox12. The more SoxC alleles that are deleted in mouse embryos, the more severe and widespread organ hypoplasia is. SoxC triple-null embryos die at midgestation unturned and tiny, with normal patterning and lineage specification, but with massively dying neural and mesenchymal progenitor cells. Specific inactivation of SoxC genes in neural and mesenchymal cells leads to selective apoptosis of these cells, suggesting SoxC cell-autonomous roles. Tead2 functionally interacts with SoxC genes in embryonic development, and is a direct target of SoxC proteins. SoxC genes therefore ensure neural and mesenchymal progenitor cell survival, and function in part by activating this transcriptional mediator of the Hippo signalling pathway