Integrative and comparative genomic analyses of mammalian macrophage responses to intracellular mycobacterial pathogens.

Mycobacterium tuberculosis, the causative agent of human tuberculosis (hTB), is a close evolutionary relative of Mycobacterium bovis, which causes bovine tuberculosis (bTB), one of the most damaging infectious diseases to livestock agriculture. Previous studies have shown that the pathogenesis of bTB disease is comparable to hTB disease, and that the bovine and human alveolar macrophage (bAM and hAM, respectively) transcriptomes are extensively reprogrammed in response to infection with these intracellular mycobacterial pathogens. In this study, a multi-omics integrative approach was applied with functional genomics and GWAS data sets across the two primary hosts (Bos taurus and Homo sapiens) and both pathogens (M. bovis and M. tuberculosis). Four different experimental infection groups were used: 1) bAM infected with M. bovis, 2) bAM infected with M. tuberculosis, 3) hAM infected with M. tuberculosis, and 4) human monocyte-derived macrophages (hMDM) infected with M. tuberculosis. RNA-seq data from these experiments 24 h post-infection (24 hpi) was analysed using three computational pipelines: 1) differentially expressed genes, 2) differential gene expression interaction networks, and 3) combined pathway analysis. The results were integrated with high-resolution bovine and human GWAS data sets to detect novel quantitative trait loci (QTLs) for resistance to mycobacterial infection and resilience to disease. This revealed common and unique response macrophage pathways for both pathogens and identified 32 genes (12 bovine and 20 human) significantly enriched for SNPs associated with disease resistance, the majority of which encode key components of the NF-κB signalling pathway and that also drive formation of the granuloma.

Integrative genomics of the mammalian alveolar macrophage response to intracellular mycobacteria.

BACKGROUND: Bovine TB (bTB), caused by infection with Mycobacterium bovis, is a major endemic disease affecting global cattle production. The key innate immune cell that first encounters the pathogen is the alveolar macrophage, previously shown to be substantially reprogrammed during intracellular infection by the pathogen. Here we use differential expression, and correlation- and interaction-based network approaches to analyse the host response to infection with M. bovis at the transcriptome level to identify core infection response pathways and gene modules. These outputs were then integrated with genome-wide association study (GWAS) data sets to enhance detection of genomic variants for susceptibility/resistance to M. bovis infection. RESULTS: The host gene expression data consisted of RNA-seq data from bovine alveolar macrophages (bAM) infected with M. bovis at 24 and 48 h post-infection (hpi) compared to non-infected control bAM. These RNA-seq data were analysed using three distinct computational pipelines to produce six separate gene sets: 1) DE genes filtered using stringent fold-change and P-value thresholds (DEG-24: 378 genes, DEG-48: 390 genes); 2) genes obtained from expression correlation networks (CON-24: 460 genes, CON-48: 416 genes); and 3) genes obtained from differential expression networks (DEN-24: 339 genes, DEN-48: 495 genes). These six gene sets were integrated with three bTB breed GWAS data sets by employing a new genomics data integration tool-gwinteR. Using GWAS summary statistics, this methodology enabled detection of 36, 102 and 921 prioritised SNPs for Charolais, Limousin and Holstein-Friesian, respectively. CONCLUSIONS: The results from the three parallel analyses showed that the three computational approaches could identify genes significantly enriched for SNPs associated with susceptibility/resistance to M. bovis infection. Results indicate distinct and significant overlap in SNP discovery, demonstrating that network-based integration of biologically relevant transcriptomics data can leverage substantial additional information from GWAS data sets. These analyses also demonstrated significant differences among breeds, with the Holstein-Friesian breed GWAS proving most useful for prioritising SNPS through data integration. Because the functional genomics data were generated using bAM from this population, this suggests that the genomic architecture of bTB resilience traits may be more breed-specific than previously assumed.

Clinical Outcomes and Immunologic Characteristics of Coronavirus Disease 2019 in People With Human Immunodeficiency Virus.

We performed a retrospective study of coronavirus disease 2019 (COVID-19) in people with human immunodeficiency virus (PWH). PWH with COVID-19 demonstrated severe lymphopenia and decreased CD4+ T cell counts. Levels of inflammatory markers, including C-reactive protein, fibrinogen, D-dimer, interleukin 6, interleukin 8, and tumor necrosis factor α were commonly elevated. In all, 19 of 72 hospitalized individuals (26.4%) died and 53 (73.6%) recovered. PWH who died had higher levels of inflammatory markers and more severe lymphopenia than those who recovered. These findings suggest that PWH remain at risk for severe manifestations of COVID-19 despite antiretroviral therapy and that those with increased markers of inflammation and immune dysregulation are at risk for worse outcomes.

A Population Genomics Analysis of the Native Irish Galway Sheep Breed.

The Galway sheep population is the only native Irish sheep breed and this livestock genetic resource is currently categorised as 'at-risk'. In the present study, comparative population genomics analyses of Galway sheep and other sheep populations of European origin were used to investigate the microevolution and recent genetic history of the breed. These analyses support the hypothesis that British Leicester sheep were used in the formation of the Galway. When compared to conventional and endangered breeds, the Galway breed was intermediate in effective population size, genomic inbreeding and runs of homozygosity. This indicates that, although the Galway breed is declining, it is still relatively genetically diverse and that conservation and management plans informed by genomic information may aid its recovery. The Galway breed also exhibited distinct genomic signatures of artificial or natural selection when compared to other breeds, which highlighted candidate genes that may be involved in production and health traits.

Alveolar Macrophage Chromatin Is Modified to Orchestrate Host Response to Mycobacterium bovis Infection.

Bovine tuberculosis is caused by infection with Mycobacterium bovis, which can also cause disease in a range of other mammals, including humans. Alveolar macrophages are the key immune effector cells that first encounter M. bovis and how the macrophage epigenome responds to mycobacterial pathogens is currently not well understood. Here, we have used chromatin immunoprecipitation sequencing (ChIP-seq), RNA-seq and miRNA-seq to examine the effect of M. bovis infection on the bovine alveolar macrophage (bAM) epigenome. We show that H3K4me3 is more prevalent, at a genome-wide level, in chromatin from M. bovis-infected bAM compared to control non-infected bAM; this was particularly evident at the transcriptional start sites of genes that determine programmed macrophage responses to mycobacterial infection (e.g. M1/M2 macrophage polarisation). This pattern was also supported by the distribution of RNA Polymerase II (Pol II) ChIP-seq results, which highlighted significantly increased transcriptional activity at genes demarcated by permissive chromatin. Identification of these genes enabled integration of high-density genome-wide association study (GWAS) data, which revealed genomic regions associated with resilience to infection with M. bovis in cattle. Through integration of these data, we show that bAM transcriptional reprogramming occurs through differential distribution of H3K4me3 and Pol II at key immune genes. Furthermore, this subset of genes can be used to prioritise genomic variants from a relevant GWAS data set.

Interleukin 8 haplotypes drive divergent responses in uterine endometrial cells and are associated with somatic cell score in Holstein-Friesian cattle.

Interleukin 8 is a proinflammatory chemokine involved in neutrophil recruitment and activation in response to infection and also in the resolution of inflammation. Our previous studies identified a number of genetic polymorphisms in the bovine IL8 promoter region which segregate into two haplotypes, with balanced frequencies in the Holstein-Friesian (HF). We subsequently showed that these haplotypes confer divergent IL8 activity both in vitro in mammary epithelial cells and in vivo in response to LPS. In this study, we hypothesised that the balanced frequency of IL8 haplotype in HF could be explained by divergent selection pressures acting on this locus. To address this hypothesis, an association study was carried out aiming to identify a putative link between the IL8 haplotype and somatic cell score (SCS) in 5746 Holstein-Friesian dairy cows. In addition, the basal and inducible promoter activity of the two IL8 haplotypes was characterised in bovine endometrial epithelial (BEND) cells and in monocyte-derived macrophages. Results showed a significant association between IL8 haplotype 2 (IL8-h2) with increased SCS (P<0.05). Functional analysis showed that the same haplotype was a more potent inducer of IL8 expression in BEND cells in response to LPS and TNFα stimulation. In contrast, co-transfection of the BEND cells with a DNA construct encoding a bovine herpesvirus 4 antigen, induced significantly higher IL8 expression from IL8-h1. The present study sheds light on the molecular mechanisms underlying selection for SCS and provides evidence that the balanced frequencies of the two IL8 haplotypes in HF cattle may occur as a result of opposing directional selection pressures of both bacterial and viral infection.

Prothymosin α variants isolated from CD8+ T cells and cervicovaginal fluid suppress HIV-1 replication through type I interferon induction.

Soluble factors from CD8(+) T cells and cervicovaginal mucosa of women are recognized as important in controlling human immunodeficiency virus type 1 (HIV-1) infection and transmission. Previously, we have shown the strong anti-HIV-1 activity of prothymosin α (ProTα) derived from CD8(+) T cells. ProTα is a small acidic protein with wide cell distribution, to which several functions have been ascribed, depending on its intracellular or extracellular localization. To date, activities of ProTα have been attributed to a single protein known as isoform 2. Here we report the isolation and identification of 2 new ProTα variants from CD8(+) T cells and cervicovaginal lavage with potent anti-HIV-1 activity. The first is a splice variant of the ProTα gene, known as isoform CRA_b, and the second is the product of a ProTα gene, thus far classified as a pseudogene 7. Native or recombinant ProTα variants potently restrict HIV-1 replication in macrophages through the induction of type I interferon. The baseline expression of interferon-responsive genes in primary human cervical tissues positively correlate with high levels of intracellular ProTα, and the knockdown of ProTα variants by small interfering RNA leads to downregulation of interferon target genes. Overall, these findings suggest that ProTα variants are innate immune mediators involved in immune surveillance.

Predicting response to vascular endothelial growth factor inhibitor and chemotherapy in metastatic colorectal cancer.

BACKGROUND: Bevacizumab improves progression free survival (PFS) and overall survival (OS) in metastatic colorectal cancer patients however currently there are no biomarkers that predict response to this treatment. The aim of this study was to assess if differential protein expression can differentiate patients who respond to chemotherapy and bevacizumab, and to assess if select proteins correlate with patient survival. METHODS: Pre-treatment serum from patients with metastatic colorectal cancer (mCRC) treated with chemotherapy and bevacizumab were divided into responders and nonresponders based on their progression free survival (PFS). Serum samples underwent immunoaffinity depletion and protein expression was analysed using two-dimensional difference gel electrophoresis (2D-DIGE), followed by LC-MS/MS for protein identification. Validation on selected proteins was performed on serum and tissue samples from a larger cohort of patients using ELISA and immunohistochemistry, respectively (n = 68 and n = 95, respectively). RESULTS: 68 proteins were identified following LC-MS/MS analysis to be differentially expressed between the groups. Three proteins (apolipoprotein E (APOE), angiotensinogen (AGT) and vitamin D binding protein (DBP)) were selected for validation studies. Increasing APOE expression in the stroma was associated with shorter progression free survival (PFS) (p = 0.0001) and overall survival (OS) (p = 0.01), DBP expression (stroma) was associated with shorter OS (p = 0.037). Increasing APOE expression in the epithelium was associated with a longer PFS and OS, and AGT epithelial expression was associated with a longer PFS (all p < .05). Increasing serum AGT concentration was associated with shorter OS (p = 0.009). CONCLUSIONS: APOE, DBP and AGT identified were associated with survival outcomes in mCRC patients treated with chemotherapy and bevacizumab.

Direct evidence on the contribution of a missense mutation in GDF9 to variation in ovulation rate of Finnsheep.

The Finnish Landrace (Finnsheep) is a well known high-prolificacy sheep breed and has been used in many countries as a source of genetic material to increase fecundity of local breeds. Analyses to date have indicated that mutations with a large effect on ovulation rate are not responsible for the exceptional prolificacy of Finnsheep. The objectives of this study were to ascertain if: 1) any of 12 known mutations with large effects on ovulation rate in sheep, or 2) any other DNA sequence variants within the candidate genes GDF9 and BMP15 are implicated in the high prolificacy of the Finnish Landrace breed; using material from lines developed by divergent selection on ovulation rate. Genotyping results showed that none of 12 known mutations (FecBB, FecXB, FecXG, FecXGR, FecXH, FecXI, FecXL, FecXO, FecXR, FecGE, FecGH, or FecGT) were present in a sample of 108 Finnsheep and, thus, do not contribute to the exceptional prolificacy of the breed. However, DNA sequence analysis of GDF9 identified a previously known mutation, V371M, whose frequency differed significantly (P<0.001) between High and Low ovulation rate lines. While analysis of ovulation rate data for Finnsheep failed to establish a significant association between this trait and V371M, analysis of data on Belclare sheep revealed a significant association between V371M and ovulation rate (P<0.01). Ewes that were heterozygous for V371M exhibited increased ovulation rate (+0.17, s.e. 0.080; P<0.05) compared to wild type and the effect was non-additive (ovulation rate of heterozygotes was significantly lower (P<0.01) than the mean of the homozygotes). This finding brings to 13 the number of mutations that have large effects on ovulation rate in sheep and to 5, including FecBB, FecGE, FecXO and FecXGR, the number of mutations within the TGFß superfamily with a positive effect on prolificacy in the homozygous state.

Global gene expression in endometrium of high and low fertility heifers during the mid-luteal phase of the estrous cycle.

BACKGROUND: In both beef and dairy cattle, the majority of early embryo loss occurs within the first 14 days following insemination. During this time-period, embryos are completely dependent on their maternal uterine environment for development, growth and ultimately survival, therefore an optimum uterine environment is critical to their survival. The objective of this study was to investigate whether differences in endometrial gene expression during the mid-luteal phase of the estrous cycle exist between crossbred beef heifers ranked as either high (HF) or low fertility (LF) (following four rounds of artificial insemination (AI)) using the Affymetrix® 23 K Bovine Gene Chip. RESULTS: Conception rates for each of the four rounds of AI were within a normal range: 70-73.3%. Microarray analysis of endometrial tissue collected on day 7 of the estrous cycle detected 419 differentially expressed genes (DEG) between HF (n = 6) and LF (n = 6) animals. The main gene pathways affected were, cellular growth and proliferation, angiogenesis, lipid metabolism, cellular and tissue morphology and development, inflammation and metabolic exchange. DEG included, FST, SLC45A2, MMP19, FADS1 and GALNT6. CONCLUSIONS: This study highlights, some of the molecular mechanisms potentially controlling uterine endometrial function during the mid-luteal phase of the estrous cycle, which may contribute to uterine endometrial mediated impaired fertility in cattle. Differentially expressed genes are potential candidate genes for the identification of genetic variation influencing cow fertility, which may be incorporated into future breeding programmes.

Effects of systemic progesterone during the early luteal phase on the availabilities of amino acids and glucose in the bovine uterine lumen.

The uterine histotroph provides essential nutrition to the developing conceptus during the preimplantation period of pregnancy. The objective of the present study was to examine the effects of cycle stage and progesterone (P4) concentrations in the blood on the recoverable quantities of amino acids and glucose in the histotroph during the preimplantaion period of conceptus development. Following oestrus, dairy heifers were assigned to low, control or high P4 groups (n=6 heifers per treatment and time point). The uterine horn ipsilateral to the corpus luteum was flushed on either Day 7 or Day 13. The present study quantified 24 amino acids and glucose in the uterine flushings using HPLC and fluorometry, respectively. Heifers in the low P4 group had lower plasma concentrations of P4 throughout the cycle, whereas heifers in the high group had higher plasma concentrations of P4 between Days 3 and 7 compared with the control group (P<0.05). Total recoverable neutral (Ser, Gln, Gly, Thr, Cit, ß-Ala, Tau, Ala, Tyr, Trp, Met, Val, Phe, Ile, Leu, Pro and Cys), acidic (Glu) and basic (His, Arg, Orn and Lys) amino acids were greater (P<0.05) on Day 13 than on Day 7. There was no significant difference in the amount of Asp or Asn between Day 7 and Day 13. The amount of amino acids recovered on Day 7 was similar across treatment groups. On Day 13, the amount of Asn, His and Thr was lower (P<0.05) in the low P4 heifers compared with the controls and/or high P4 heifers. Quantities of glucose were not altered by cycle stage or P4 treatment. In conclusion, the stage of oestrous cycle and P4 play important roles in modulating amino acids in the histotroph, a potentially critical factor for early embryonic and/or conceptus survival.

Telaprevir in the treatment of acute hepatitis C virus infection in HIV-infected men.

BACKGROUND: There is an international epidemic of hepatitis C virus (HCV) infection among human immunodeficiency virus (HIV)-infected men who have sex with men. Sustained virologic response (SVR) rates with pegylated interferon and ribavirin treatment are higher in these men during acute HCV than during chronic HCV, but treatment is still lengthy and SVR rates are suboptimal. METHODS: We performed a pilot study of combination therapy with telaprevir, pegylated interferon, and ribavirin in acute genotype 1 HCV infection in HIV-infected men. Men who were treated prior to the availability of, or ineligible for, telaprevir were the comparator group. The primary endpoint was SVR12, defined as an HCV viral load <5 IU/mL at least 12 weeks after completing treatment. RESULTS: In the telaprevir group, 84% (16/19) of men achieved SVR12 vs 63% (30/48) in the comparator group. Among men with SVR, median time to undetectable viral load was week 2 in the telaprevir group vs week 4 in the comparator group, and 94% vs 53% had undetectable viral loads at week 4. Most patients (81%) who achieved SVR in the telaprevir group received ≤12 weeks of treatment and there were no relapses after treatment. The overall safety profile was similar to that known for telaprevir-based regimens. CONCLUSIONS: Incorporating telaprevir into treatment of acute genotype 1 HCV in HIV-infected men halved the treatment duration and increased the SVR rate. Larger studies should be done to confirm these findings. Clinicians should be alert to detect acute HCV infection of HIV-infected men to take advantage of this effective therapy and decrease further transmission in this epidemic.

A feedback regulatory pathway between LDL and alpha-1 proteinase inhibitor in chronic inflammation and infection.

Dietary lipids are transported via lymph to the liver and transformed to lipoproteins which bind to members of the low density lipoprotein receptor family (LDL-RFMs). Certain LDL-RFMs, e.g., very low density lipoprotein receptor (VLDLR), are also bound by inactivated proteinase inhibitors, the most abundant being α1proteinase inhibitor (α1PI, α1antitrypsin). Inflammation/infection, including HIV-1 infection, is accompanied by low levels of CD4+ T cells and active α1PI and high levels of inactivated α1PI. By inducing LDL-RFMs-mediated cellular locomotion, active α1PI regulates the number of CD4+ T cells. We sought to investigate whether CD4+ T cells and α1PI directly impact lipoprotein levels. At the cellular level, we show that active α1PI is required for VLDLR-mediated uptake of receptor-associated cargo, specifically CD4-bound HIV-1. We show that active α1PI levels linearly correlate with LDL levels in HIV-1 infected individuals (P<0.001) and that therapeutic, weekly infusions of active α1PI elevate the number of CD4+ T cells and HDL levels while lowering LDL levels in patients on antiretroviral therapy with controlled HIV-1. Based on the unusual combination of lipodystrophy and low levels of α1PI and CD4+ T cells in HIV-1 disease, we reveal that LDL and α1PI participate in a feedback regulatory pathway. We demonstrate integral roles for sequentially acting active and inactive α1PI in the uptake and recycling of receptors and cargo aggregated with VLDLR including CD4 and chemokine receptors. Evidence supports a role for α1PI as a primary sentinel to deploy the immune system as a consequence of its role in lipoprotein transport.

Investigation of prolific sheep from UK and Ireland for evidence on origin of the mutations in BMP15 (FecX(G), FecX(B)) and GDF9 (FecG(H)) in Belclare and Cambridge sheep.

This paper concerns the likely origin of three mutations with large effects on ovulation rate identified in the Belclare and Cambridge sheep breeds; two in the BMP15 gene (FecX(G) and FecX(B)) and the third (FecG(H)) in GDF9. All three mutations segregate in Belclare sheep while one, FecX(B), has not been found in the Cambridge. Both Belclare and Cambridge breeds are relatively recently developed composites that have common ancestry through the use of genetic material from the Finnish Landrace and Lleyn breeds. The development of both composites also involved major contributions from exceptionally prolific ewes screened from flocks in Ireland (Belclare) and Britain (Cambridge) during the 1960s. The objective of the current study was to establish the likely origin of the mutations (FecX(G), FecX(B) and FecG(H)) through analysis of DNA from Finnish Landrace and Lleyn sheep, and Galway and Texel breeds which contributed to the development of the Belclare breed. Ewes with exceptionally high prolificacy (hyper-prolific ewes) in current flocks on Irish farms were identified to simulate the screening of ewes from Irish flocks in the 1960s. DNA was obtained from: prolific ewes in extant flocks of Lleyn sheep (n = 44) on the Lleyn peninsula in Wales; hyper-prolific ewes (n = 41); prolific Galway (n = 41) ewes; Finnish Landrace (n = 124) and Texel (n = 19) ewes. The FecX(G) mutation was identified in Lleyn but not in Finnish Landrace, Galway or Texel sheep; FecX(B) was only found among the hyper-prolific ewes. The FecG(H) mutation was identified in the sample of Lleyn sheep. It was concluded from these findings that the Lleyn breed was the most likely source of the FecX(G) and FecG(H) mutations in Belclare and Cambridge sheep and that the FecX(B) mutation came from the High Fertility line that was developed using prolific ewes selected from commercial flocks in Ireland in the 1960's and subsequently used in the genesis of the Belclare.

Alterations in systemic concentrations of progesterone during the early luteal phase affect RBP4 expression in the bovine uterus.

Systemic progesterone affects the timing and duration of uterine endometrial gene and protein expression and has significant effects on conceptus development. The objective of the present study was to examine how changes in progesterone concentrations during the early luteal phase affect retinol-binding protein (RBP4) mRNA and protein concentrations in the uterus. Endometrial tissue and uterine flushings were recovered on Days 7 and 13 of the oestrous cycle in heifers with high, normal and low progesterone concentrations. RBP4 mRNA and protein concentrations were higher (P<0.05) on Day 13 compared with Day 7 in heifers with high and control progesterone concentrations. However, there was no difference in RBP4 protein concentrations between Days 7 and 13 in heifers with low progesterone (P>0.05). On Day 7, although heifers with low progesterone had lower RBP4 mRNA expression compared with controls (P<0.05) there was no difference in protein concentrations between treatment groups. On Day 13, RBP4 mRNA was 2-fold higher (P<0.001) in heifers with high and control progesterone compared with their low-progesterone counterparts and RBP4 protein concentrations were over 2-fold higher (P<0.001) in heifers with high compared to low progesterone. In conclusion, progesterone modulates uterine RBP4 mRNA and protein abundance in a time- and concentration-dependent manner.

A comparison of the bovine uterine and plasma proteome using iTRAQ proteomics.

Early embryo loss is a key factor affecting fertility in dairy and beef herds. Prior to implantation, the bovine embryo spends around 16 days free-floating in the uterine environment and is dependent on the composition of uterine fluid for normal growth and development. However, there is a lack of information regarding the protein composition of the bovine uterus and how it relates to plasma. In this study, uterine flushings (UF) (n = 6) and blood plasma (n = 4) were collected from beef heifers on day 7 of the oestrous cycle, albumin depleted and compared using iTRAQ proteomics. A total of 35 proteins were higher and 18 were lower in UF including metabolic enzymes, proteins with anti-oxidant activity and those involved in modulation of the immune response. This study confirms the dynamic nature of the bovine uterine proteome and that it differs from plasma. Factors affecting the uterine proteome and how it impacts on embryo survival warrant further study.

Proteomic characterization of histotroph during the preimplantation phase of the estrous cycle in cattle.

Uterine secretions, or histotroph, are a critical component for early embryo survival, functioning as the sole supply of vitamins, minerals, enzymes, and other myriad of nutrients required by the developing conceptus before implantation. Histotroph is therefore a promising source for biomarkers of uterine function and for enhancing our understanding of the environment supporting early embryo development and survival. Utilizing label-free liquid chromatography-tandem mass spectrometry (LC-MS/MS) shotgun proteomics, we characterized the uterine proteome at two key preimplantation stages of the estrous cycle in high fertility cattle. We identified 300 proteins on Day 7 and 510 proteins on Day 13 including 281 proteins shared between days. Five proteins were more abundant (P < 0.05) on Day 7 compared with Day 13 and included novel histotroph proteins cytokeratin 10 and stathmin. Twenty-nine proteins were more abundant (P < 0.05) including 13 unique on Day 13 compared with Day 7 and included previously identified legumain, metalloprotease inhibitor-2, and novel histotroph proteins chromogranin A and pyridoxal kinase. Functional analysis of the 34 differentially expressed proteins (including 14 novel to histotroph) revealed distinct biological roles putatively involved in early pregnancy, including remodelling of the uterine environment in preparation for implantation; nutrient metabolism; embryo growth, development and protection; maintenance of uterine health; and maternal immune modulation. This study is the first reported LC-MS/MS based global proteomic characterization of the uterine environment in any domesticated species before implantation and provides novel information on the temporal alterations in histotroph composition during critical stages for early embryo development and uterine function during the early establishment of pregnancy.

α1Proteinase inhibitor regulates CD4+ lymphocyte levels and is rate limiting in HIV-1 disease.

BACKGROUND: The regulation of adult stem cell migration through human hematopoietic tissue involves the chemokine CXCL12 (SDF-1) and its receptor CXCR4 (CD184). In addition, human leukocyte elastase (HLE) plays a key role. When HLE is located on the cell surface (HLE(CS)), it acts not as a proteinase, but as a receptor for α(1)proteinase inhibitor (α(1)PI, α(1)antitrypsin, SerpinA1). Binding of α(1)PI to HLE(CS) forms a motogenic complex. We previously demonstrated that α(1)PI deficiency attends HIV-1 disease and that α(1)PI augmentation produces increased numbers of immunocompetent circulating CD4(+) lymphocytes. Herein we investigated the mechanism underlying the α(1)PI deficiency that attends HIV-1 infection. METHODS AND FINDINGS: Active α(1)PI in HIV-1 subjects (median 17 µM, n = 35) was significantly below normal (median 36 µM, p<0.001, n = 30). In HIV-1 uninfected subjects, CD4(+) lymphocytes were correlated with the combined factors α(1)PI, HLE(CS) (+) lymphocytes, and CXCR4(+) lymphocytes (r(2) = 0.91, p<0.001, n = 30), but not CXCL12. In contrast, in HIV-1 subjects with >220 CD4 cells/µl, CD4(+) lymphocytes were correlated solely with active α(1)PI (r(2) = 0.93, p<0.0001, n = 26). The monoclonal anti-HIV-1 gp120 antibody 3F5 present in HIV-1 patient blood is shown to bind and inactivate human α(1)PI. Chimpanzee α(1)PI differs from human α(1)PI by a single amino acid within the 3F5-binding epitope. Unlike human α(1)PI, chimpanzee α(1)PI did not bind 3F5 or become depleted following HIV-1 challenge, consistent with the normal CD4(+) lymphocyte levels and benign syndrome of HIV-1 infected chimpanzees. The presence of IgG-α(1)PI immune complexes correlated with decreased CD4(+) lymphocytes in HIV-1 subjects. CONCLUSIONS: This report identifies an autoimmune component of HIV-1 disease that can be overcome therapeutically. Importantly, results identify an achievable vaccine modification with the novel objective to protect against AIDS as opposed to the current objective to protect against HIV-1 infection.

Polymorphism discovery and allele frequency estimation using high-throughput DNA sequencing of target-enriched pooled DNA samples.

BACKGROUND: The central role of the somatotrophic axis in animal post-natal growth, development and fertility is well established. Therefore, the identification of genetic variants affecting quantitative traits within this axis is an attractive goal. However, large sample numbers are a pre-requisite for the identification of genetic variants underlying complex traits and although technologies are improving rapidly, high-throughput sequencing of large numbers of complete individual genomes remains prohibitively expensive. Therefore using a pooled DNA approach coupled with target enrichment and high-throughput sequencing, the aim of this study was to identify polymorphisms and estimate allele frequency differences across 83 candidate genes of the somatotrophic axis, in 150 Holstein-Friesian dairy bulls divided into two groups divergent for genetic merit for fertility. RESULTS: In total, 4,135 SNPs and 893 indels were identified during the resequencing of the 83 candidate genes. Nineteen percent (n = 952) of variants were located within 5' and 3' UTRs. Seventy-two percent (n = 3,612) were intronic and 9% (n = 464) were exonic, including 65 indels and 236 SNPs resulting in non-synonymous substitutions (NSS). Significant (P < 0.01) mean allele frequency differentials between the low and high fertility groups were observed for 720 SNPs (58 NSS). Allele frequencies for 43 of the SNPs were also determined by genotyping the 150 individual animals (Sequenom® MassARRAY). No significant differences (P > 0.1) were observed between the two methods for any of the 43 SNPs across both pools (i.e., 86 tests in total). CONCLUSIONS: The results of the current study support previous findings of the use of DNA sample pooling and high-throughput sequencing as a viable strategy for polymorphism discovery and allele frequency estimation. Using this approach we have characterised the genetic variation within genes of the somatotrophic axis and related pathways, central to mammalian post-natal growth and development and subsequent lactogenesis and fertility. We have identified a large number of variants segregating at significantly different frequencies between cattle groups divergent for calving interval plausibly harbouring causative variants contributing to heritable variation. To our knowledge, this is the first report describing sequencing of targeted genomic regions in any livestock species using groups with divergent phenotypes for an economically important trait.