Killer-cell immunoglobulin-like receptor (KIR) proteins are expressed on natural killer (NK) cells and appear important in innate and adaptive immunity. There are about 14 KIR genes on chromosome 19q13.4, composed of those that inhibit and those that activate NK cell killing. Haplotypes have different combinations of these genes meaning that not all genes are present in a subject. There are two main classes of cognate human leukocyte antigen (HLA) ligands (HLA-Bw4 and HLA-C1/C2) that bind to the inhibitory/activating receptors. As a general rule, the inhibitory state is maintained except when virally infected or tumor cells are encountered; however, both increased activation and inhibition states have been associated with susceptibility and protection against numerous disease states including cancer, arthritis, and psoriasis.
Utilizing DNA from 158 Caucasian subjects with autism and 176 KIR control subjects we show for the first time a highly significant increase in four activating KIR genes (2DS5, 3DS1, 2DS1 and 2DS4) as measured by chi square values and odds ratios. In addition, our data suggests a highly significant increase in the activating KIR gene 2DS1 and its cognate HLA-C2 ligand (2DS1+C2; p=0.00003 [Odds Ratio=2.87]). This information ties together two major immune gene complexes, the Human Leukocyte Complex and the Leukocyte Receptor Complex, and may partially explain immune abnormalities observed in many subjects with autism.
killer-cell immunoglobulin-like receptor; KIR genes; KIR haplotypes; human leukocyte antigen; HLA ligands; leukocyte receptor complex; autism; immune dysfunction; natural killer cells
Hypoxia inducible factor-1 (HIF-1) is a transcription factor that is a major regulator of energy homeostasis and cellular adaptation to low oxygen stress. HIF-1 is also activated in response to bacterial pathogens and supports the innate immune response of both phagocytes and keratinocytes. In this work, we show that a new pharmacological compound AKB-4924 (Akebia Therapeutics) increases HIF-1α levels and enhances the antibacterial activity of phagocytes and keratinocytes against both methicillin-sensitive and -resistant strains of Staphylococcus aureus in vitro. AKB-4924 is also effective in stimulating the killing capacity of keratinocytes against the important opportunistic skin pathogens Pseudomonas aeruginosa and Acinitobacter baumanii. The effect of AKB-4924 is mediated through the activity of host cells, as the compound exerts no direct antimicrobial activity. Administered locally as a single agent, AKB-4924 limits S. aureus proliferation and lesion formation in a mouse skin abscess model. This approach to pharmacologically boost the innate immune response via HIF-1 stabilization may serve as a useful adjunctive treatment for antibiotic-resistant bacterial infections.
Hypoxia inducible factor-1 (HIF-1); innate immunity; Staphylococcus aureus; bacterial infection; antibiotic-resistant bacteria
The overall CD8 T cell response to human/simian immunodeficiency virus (HIV/SIV) targets a collection of discrete epitope specificities. Some of these epitope-specific CD8 T cells emerge in the weeks and months following infection and rapidly select for sequence variants, whereas other CD8 T cell responses develop during the chronic infection phase and rarely select for sequence variants. In this study, we tested the hypothesis that acute-phase CD8 T cell responses that do not rapidly select for escape variants are unable to control viral replication in vivo as well as those that do rapidly select for escape variants. We created a derivative of live attenuated SIV (SIVmac239Δnef) in which we ablated five epitopes that elicit early CD8 T cell responses and rapidly accumulate sequence variants in SIVmac239-infected Mauritian cynomolgus macaques (MCMs) that are homozygous for the M3 major histocompatibility complex (MHC) haplotype. This live attenuated SIV variant was called m3KOΔnef. Viremia was significantly higher in M3 homozygous MCMs infected with m3KOΔnef than in either MHC-mismatched MCMs infected with m3KOΔnef or MCMs infected with SIVmac239Δnef. Three CD8 T cell responses, including two that do not rapidly select for escape variants, predominated during early m3KOΔnef infection in the M3 homozygous MCMs, but these animals were unable to control viral replication. These results provide evidence that acute-phase CD8 T cell responses that have the potential to rapidly select for escape variants in the early phase of infection are needed to establish viral control in vivo.
The human APOBEC3 family of cytidine deaminases provides intrinsic immunity to retroviral infection. A naturally occurring 29.5-kb deletion removes the entire APOBEC3B gene. We examined the impact of the APOBEC3B gene deletion in >4000 individuals from five HIV-1 natural history cohorts. The hemizygous genotype had no effect on either infection or progression. However, the homozygous deletion was significantly associated with unfavorable outcomes for HIV-1 acquisition (OR=7.37, P=0.024), progression to AIDS (RH = 4.01, P=0.03), and viral set-point (P=0.04). These findings suggest that the loss of APOBEC3B may increase host susceptibility to HIV-1/AIDS and warrant further study.
Continuous monitoring and download of endotracheal tube cuff pressure for a 12-hour period were required to collect data for an ongoing program of research related to airway management of the critically ill patient. On the basis of reports from the anesthesia literature, continuous monitoring of cuff pressure via a traditional pressure transducer and monitor was identified as the best method to collect data. Although continuous pressure monitoring of many physiologic variables is routine in critical care settings, application of the technology to measurement of endotracheal tube cuff pressure has not been reported outside the operating room. The research team conducted bench testing and pilot testing in human subjects to establish feasibility, accuracy, and safety of continuous cuff pressure monitoring. Monitoring was feasible with stringent procedures applied to ensure safety. A bias of 0.5 cm H2O between continuous and intermittent measures was obtained in both in vitro and in vivo testing.
endotracheal tube; monitoring; nursing; pressure; research
Hypoxia inducible factors (HIFs) are broadly expressed in human cancers, and HIF1α and HIF2α were previously suspected of promoting tumor progression through largely overlapping functions. However, this relatively simple model has now been challenged in light of recent data from genome-wide analyses of human tumors, genetically engineered mouse models of cancer, and systems biology approaches that reveal unique and sometimes opposing HIFa activities in both normal physiology and disease. These effects are mediated in part through regulation of unique target genes, as well as direct and indirect interactions with important oncoproteins and tumor suppressors, including MYC and p53. As HIF inhibitors are currently under clinical evaluation as cancer therapeutics, a more thorough understanding of unique roles performed by HIF1α and HIF2α in human neoplasia is warranted. This Review summarizes our rapidly changing understanding of shared and independent HIF1α and HIF2α activities in tumor growth and progression, and the implications for using selective HIF inhibitors as cancer therapeutics.
Background. Host genetic variation influences human immunodeficiency virus (HIV) infection and progression to AIDS. Here we used clinically well-characterized subjects from 5 pretreatment HIV/AIDS cohorts for a genome-wide association study to identify gene associations with rate of AIDS progression.
Methods. European American HIV seroconverters (n = 755) were interrogated for single-nucleotide polymorphisms (SNPs) (n = 700,022) associated with progression to AIDS 1987 (Cox proportional hazards regression analysis, co-dominant model).
Results. Association with slower progression was observed for SNPs in the gene PARD3B. One of these, rs11884476, reached genome-wide significance (relative hazard = 0.3; P =3. 370 × 10−9) after statistical correction for 700,022 SNPs and contributes 4.52% of the overall variance in AIDS progression in this study. Nine of the top-ranked SNPs define a PARD3B haplotype that also displays significant association with progression to AIDS (hazard ratio, 0.3; P = 3.220 × 10−8). One of these SNPs, rs10185378, is a predicted exonic splicing enhancer; significant alteration in the expression profile of PARD3B splicing transcripts was observed in B cell lines with alternate rs10185378 genotypes. This SNP was typed in European cohorts of rapid progressors and was found to be protective for AIDS 1993 definition (odds ratio, 0.43, P = .025).
Conclusions. These observations suggest a potential unsuspected pathway of host genetic influence on the dynamics of AIDS progression.
The von Hippel–Lindau tumor suppressor and the hypoxia-inducible factor-αs are essential for the transition from embryonic hyaloidal vascular system to adult retinal vasculature in the mouse eye.
Successful transition from embryonic to adult circulation is critical for survival of mammalian organisms. This shift occurs in the central cardiovascular circulation and in the eye as oxygen tension increases. However, its regulation is not well understood. We have used combinatorial gene deletion and overexpression assays to assess the effect of astrocyte-targeted deletion of von Hippel–Lindau tumor suppressor (Vhl), hypoxia-inducible factor-αs (Hif-αs), and Vegf on the normal regression of the hyaloidal vessels, the fetal ocular circulation system. Astrocytic Vhl deletion induced accelerated hyaloidal regression and subsequent massive secondary outgrowth. Combinatorial gene deletion involving Vhl, Hif-αs, and Vegf genes revealed that HIF-2α/vascular endothelial growth factor signaling induces secondary outgrowth in Vhl mutants. Conversely, HIF-1α regulated macrophage migration inhibitory factor and promoted macrophage infiltration that accelerates hyaloidal vessel regression. The phenotype observed in Vhl mutants strongly resembles human persistent hyperplastic primary vitreous cases and may provide insights into vascular remodeling mechanisms in other systems.
The hypoxia inducible transcription factors (HIFs) control many mediators of vascular response, including both angiogenic factors and small molecules such as nitric oxide (NO). In studying how endothelial HIF response itself affects metastasis, we found that loss of HIF-1α in endothelial cells reduces NO synthesis, retards tumor cell migration through endothelial layers, and restricts tumor cell metastasis, and that loss of HIF-2α has in each case the opposite effect. This results from differential regulation of NO homeostasis that in turn regulates vascular endothelial growth factor expression in an NO-dependent feedback loop. These opposing roles for the two HIF factors indicate that both they and endothelial cells regulate metastasis as malignancy progresses.
► Endothelial cell hypoxic response is a critical determinant of metastatic success ► HIF isoforms differentially regulate NO homeostasis in endothelium ► iNOS is essential for hypoxic NO production in endothelial cells ► HIF isoforms in endothelium differentially promote and hinder metastasis
Caenorhabditis elegans ftn-1 and ftn-2, which encode the iron-storage protein ferritin, are transcriptionally inhibited during iron deficiency in intestine. Intestinal specific transcription is dependent on binding of ELT-2 to GATA binding sites in an iron-dependent enhancer (IDE) located in ftn-1 and ftn-2 promoters, but the mechanism for iron regulation is unknown. Here, we identify HIF-1 (hypoxia-inducible factor -1) as a negative regulator of ferritin transcription. HIF-1 binds to hypoxia-response elements (HREs) in the IDE in vitro and in vivo. Depletion of hif-1 by RNA interference blocks transcriptional inhibition of ftn-1 and ftn-2 reporters, and ftn-1 and ftn-2 mRNAs are not regulated in a hif-1 null strain during iron deficiency. An IDE is also present in smf-3 encoding a protein homologous to mammalian divalent metal transporter-1. Unlike the ftn-1 IDE, the smf-3 IDE is required for HIF-1–dependent transcriptional activation of smf-3 during iron deficiency. We show that hif-1 null worms grown under iron limiting conditions are developmentally delayed and that depletion of FTN-1 and FTN-2 rescues this phenotype. These data show that HIF-1 regulates intestinal iron homeostasis during iron deficiency by activating and inhibiting genes involved in iron uptake and storage.
Due to its presence in proteins involved in hemoglobin synthesis, DNA synthesis, and mitochondrial respiration, eukaryotic cells require iron for survival. Excess iron can lead to oxidative damage, while iron deficiency reduces cell growth and causes cell death. Dysregulation of iron homeostasis in humans caused by iron deficiency or excess leads to anemia, diabetes, and neurodegenerative disorders. All organisms have thus developed mechanisms to sense, acquire, and store iron. We use Caenorhabditis elegans as a model organism to study mechanisms of iron regulation. Our previous studies show that the iron-storage protein ferritin (FTN-1, FTN-2) is transcriptionally inhibited in intestine during iron deficiency, but the mechanisms regulating iron regulation are not known. Here, we find that hypoxia-inducible factor 1 (HIF-1) transcriptionally inhibits ftn-1 and ftn-2 during iron deficiency. We also show that HIF-1 activates the iron uptake gene smf-3. Transcriptional activation and inhibition by HIF-1 is dependent on an iron enhancer in the promoters of these genes. HIF-1 is a known transcriptional activator, but its role in transcriptional inhibition is not well understood. Our data show that HIF-1 regulates iron homeostasis by activating and inhibiting iron uptake and storage genes, and they provide insight into HIF-1 transcriptional inhibition.
In the early neonatal stage, the fetal circulatory system undergoes dramatic transition to the adult circulatory system. Normally, embryonic connecting vessels such as ductus arteriosus and the foramen ovale close and regress. In the neonatal retina, hyaloid vessels maintaining blood flow in the embryonic retina regress, and retinal vessels take over to form adult-type circulatory system. This process is regulated by the programmed cell death switch mediated by macrophages via Wnt and Angiopoietin-2 pathways. In this study, we seek for the other mechanisms that regulate this process, and focus on the dramatic change in oxygen environment at the point of birth. The von Hippel–Lindau tumor suppressor protein (pVHL) is a substrate recognition component of an E3-ubiquitin ligase that rapidly destabilizes hypoxia-inducible factor-αs (HIF-αs) under normoxic conditions, but not hypoxic conditions. To examine the role of oxygen-sensing mechanisms in the retinal circulatory system transition, we generated retina-specific conditional knockout mice for VHL (VHLα-Cre KO mice). These mice exhibit arrested transition from fetal to adult circulatory system: persistence of hyaloid vessels and poorly formed retinal vessels. These defects are suppressed by intraocular injection of Flt1-Fc protein (vascular endothelial growth factor (VEGF) Receptor-1 (Flt1)/Fc chimeric protein that can bind VEGF and inhibit its activity), or by inactivating the HIF-1 α gene. Our results suggest that not only macrophages but also tissue oxygen-sensing mechanisms regulate the transition from fetal to adult circulatory system in retina.
Angiogenesis; Circulatory system; Hypoxia-inducible factor 1
T cells can inhibit tumor growth, but their function in the tumor microenvironment is often suppressed. Many solid tumors exhibit abundant macrophage infiltration and low oxygen tension, yet how hypoxic conditions may affect innate immune cells and their impact on tumor progression is poorly understood. Targeted deletion of the hypoxia responsive transcription factor HIF-1α in macrophages in a progressive murine model of breast cancer resulted in reduced tumor growth, although VEGF-A and vascularization was unchanged. Tumor associated macrophages can suppress tumor infiltrating T cells by several mechanisms, and we found that hypoxia powerfully augmented macrophage-mediated T cell suppression in vitro in a manner dependent on macrophage expression of HIF-1α. Our findings link the innate immune hypoxic response to tumor progression through induction of T cell suppression in the tumor microenvironment.
Vascular/parenchymal crosstalk is increasingly recognized as important in the development and maintenance of healthy vascularized tissues. The retina is an excellent model in which to study the role of cell type-specific contributions to the process of blood vessel and neuronal growth. During retinal vascular development, glial cells such as astrocytes provide the template over which endothelial cells migrate to form the retinal vascular network, and hypoxia-regulated vascular endothelial growth factor (VEGF) has been demonstrated to play a critical role in this process as well as pathological neovascularization. To investigate the nature of cell-specific contributions to this process, we deleted VEGF and its upstream regulators, the hypoxia-inducible transcription factors HIF-1α and HIF-2α, and the negative regulator of HIFα, von Hippel-Lindau protein (VHL), in astrocytes. We found that loss of hypoxic response and VEGF production in astrocytes does not impair normal development of retinal vasculature, indicating that astrocyte-derived VEGF is not essential for this process. In contrast, using a model of oxygen-induced ischemic retinopathy, we show that astrocyte-derived VEGF is essential for hypoxia-induced neovascularization. Thus, we demonstrate that astrocytes in the retina have highly divergent roles during developmental, physiological angiogenesis and ischemia-driven, pathological neovascularization.
Stem cells reside in specialized microenvironments or “niches” which regulate their function. In vitro studies employing hypoxic culture conditions (≤ 5% O2) have revealed strong regulatory links between O2 availability and stem/precursor cell functions1–6. Therefore, while some stem cells are perivascular, others may occupy hypoxic niches and be regulated by O2 gradients. However, the underlying mechanisms remain unclear. Here, we show that Hypoxia Inducible Factor-1α (HIF-1α), a principal mediator of hypoxic adaptations, modulates Wnt/β-catenin signalling in hypoxic embryonic stem (ES) cells by enhancing β-catenin activation and expression of downstream effectors LEF-1 and TCF-1. This regulation extends to primary cells, including isolated neural stem cells (NSCs), and is not observed in differentiated cells. In vivo, Wnt/β-catenin activity is closely associated with low O2 regions in the subgranular zone (SGZ) of the hippocampus, a key NSC niche7. Hif-1α deletion impairs hippocampal Wnt-dependent processes, including NSC proliferation, differentiation and neuronal maturation. This decline correlates with reduced Wnt/β-catenin signalling in the SGZ. Therefore, O2 availability may have a direct role in stem cell regulation via HIF-1α modulation of Wnt/β-catenin signalling.
Embryonic stem cells; hypoxic niche; HIF-1α/ARNT; Wnt/β-catenin signalling; neurogenesis
Single nucleotide polymorphisms (SNPs) in MYH9 and APOL1 on chromosome 22 (c22) are powerfully associated with non-diabetic end-stage renal disease (ESRD) in African Americans (AAs). Many AAs diagnosed with type 2 diabetic nephropathy (T2DN) have non-diabetic kidney disease, potentially masking detection of DN genes. Therefore, genome-wide association analyses were performed using the Affymetrix SNP Array 6.0 in 966 AA with T2DN and 1,032 non-diabetic, non-nephropathy (NDNN) controls, with and without adjustment for c22 nephropathy risk variants. No associations were seen between FRMD3 SNPs and T2DN before adjusting for c22 variants. However, logistic regression analysis revealed seven FRMD3 SNPs significantly interacting with MYH9—a finding replicated in 640 additional AA T2DN cases and 683 NDNN controls. Contrasting all 1,592 T2DN cases with all 1,671 NDNN controls, FRMD3 SNPs appeared to interact with the MYH9 E1 haplotype (e.g., rs942280 interaction p-value = 9.3E−7 additive; odds ratio [OR] 0.67). FRMD3 alleles were associated with increased risk of T2DN only in subjects lacking two MYH9 E1 risk haplotypes (rs942280 OR = 1.28), not in MYH9 E1 risk allele homozygotes (rs942280 OR = 0.80; homogeneity p-value = 4.3E−4). Effects were weaker stratifying on APOL1. FRMD3 SNPS were associated with T2DN, not type 2 diabetes per se, comparing AAs with T2DN to those with diabetes lacking nephropathy. T2DN-associated FRMD3 SNPs were detectable in AAs only after accounting for MYH9, with differential effects for APOL1. These analyses reveal a role for FRMD3 in AA T2DN susceptibility and accounting for c22 nephropathy risk variants can assist in detecting DN susceptibility genes.
African Americans have high rates of kidney disease attributed to type 2 diabetes mellitus. However, approximately 25% of patients are misclassified and have non-diabetic kidney disease on renal biopsy. The APOL1-MYH9 gene region on chromosome 22 is powerfully associated with non-diabetic kidney diseases in African Americans. Therefore, we tested for interactions between single nucleotide polymorphisms across the genome with APOL1 and MYH9 non-diabetic nephropathy risk variants in African Americans with presumed diabetic nephropathy. Markers in FRMD3, a gene associated with type 1 diabetic nephropathy in Caucasians, appeared to interact with MYH9; however, increased nephropathy risk was seen in diabetic cases lacking two MYH9 risk haplotypes, and protective effects were seen in those with two MYH9 risk haplotypes. Stratified analyses based on the chromosome 22 nephropathy risk haplotypes demonstrated that FRMD3 variants were associated with diabetic nephropathy risk in cases without two MYH9 (or APOL1) risk haplotypes. It appears that African Americans with diabetes and kidney disease who are not chromosome 22 nephropathy risk variant homozygotes are enriched for the presence of diabetic nephropathy and FRMD3 risk alleles. This genetic dissection ultimately allowed for detection of the FRMD3 diabetic nephropathy gene association in a subset of cases enriched for this disorder.
Angiogenesis and the development of a vascular network is required for tumor progression, and involves release of angiogenic factors, including vascular endothelial growth factor (VEGF), from both malignant and stromal cell types1. Infiltration by cells of the myeloid lineage is a hallmark of many tumors, and in many cases the macrophages in these infiltrates express VEGF2. Here we show that deletion of inflammatory cell-derived VEGF attenuates the formation of a typical high-density vessel network, thus blocking the angiogenic switch in solid tumors. Vasculature in tumors lacking myeloid cell-derived VEGF was less tortuous, with increased pericyte coverage and decreased vessel length, indicating vascular normalization. In addition, loss of myeloid-derived VEGF decreases VEGFR2 phosphorylation in tumors, even though overall VEGF levels in the tumors are unaffected. However, myeloid deletion of VEGF resulted in an accelerated tumor progression in multiple subcutaneous isograft models and an autochthonous transgenic model of mammary tumorigenesis, with less overall tumor cell death and decreased tumor hypoxia. Furthermore, loss of myeloid cell VEGF increased tumor susceptibility to chemotherapeutic cytotoxicity. This demonstrates that myeloid-derived VEGF is essential for tumorigenic alteration of vasculature and signaling to VEGFR2, and that these changes act to retard, not promote, tumor progression.
Factor Inhibiting HIF-1α (FIH) is an asparaginal hydroxylase. Hydroxylation of HIF-α proteins by FIH blocks association of HIFs with the transcriptional co-activators CBP/p300, thus inhibiting transcriptional activation. We have created mice with a null mutation in the FIH gene, and found that it has little or no discernable role in mice in altering classical aspects of HIF function, e.g., angiogenesis, erythropoiesis, or development. Rather, it is an essential regulator of metabolism: mice lacking FIH exhibit reduced body weight, elevated metabolic rate, hyperventilation, improved glucose and lipid homeostasis, and are resistant to high fat diet-induced weight gain and hepatic steatosis. Neuron-specific loss of FIH phenocopied some of the major metabolic phenotypes of the global null animals: those mice have reduced body weight, increased metabolic rate, enhanced insulin sensitivity, and are also protected against high fat diet-induced weight gain. These results demonstrate that FIH acts to a significant degree through the nervous system to regulate metabolism.
Angiogenesis and osteogenesis are tightly coupled during bone development and regeneration. Mesenchymal cells in the developing stroma elicit angiogenic signals to recruit new blood vessels into bone. Reciprocal signals, likely emanating from the incoming vascular endothelium, stimulate mesenchymal cell specification through additional interactions with cells within the vascular stem cell niche. The hypoxia-inducible factor-1 alpha (HIF-1) pathway has been identified as a key component in this process. We demonstrated that overexpression of HIF-1 in mature osteoblasts through disruption of the von Hippel-Lindau protein profoundly increases angiogenesis and osteogenesis; these processes appear to be coupled by cell nonautonomous mechanisms involving the action of vascular endothelial growth factor (VEGF) on the endothelial cells. The same occurred in the model of injury-mediated bone regeneration (distraction osteogenesis). Surprisingly, manipulation of HIF-1 does not influence angiogenesis of the skull bones, where earlier activation of HIF-1 in the condensing mesenchyme upregulates osterix during cranial bone formation.
knockout mice; osteoblasts; hypoxia-inducible factor; angiogenesis
The regulation of neutrophil lifespan by induction of apoptosis is critical for maintaining an effective host response and preventing excessive inflammation. The hypoxia-inducible factor (HIF) oxygen-sensing pathway has a major effect on the susceptibility of neutrophils to apoptosis, with a marked delay in cell death observed under hypoxic conditions. HIF expression and transcriptional activity are regulated by the oxygen-sensitive prolyl hydroxylases (PHD1–3), but the role of PHDs in neutrophil survival is unclear. We examined PHD expression in human neutrophils and found that PHD3 was strongly induced in response to hypoxia and inflammatory stimuli in vitro and in vivo. Using neutrophils from mice deficient in Phd3, we demonstrated a unique role for Phd3 in prolonging neutrophil survival during hypoxia, distinct from other hypoxia-associated changes in neutrophil function and metabolic activity. Moreover, this selective defect in neutrophil survival occurred in the presence of preserved HIF transcriptional activity but was associated with upregulation of the proapoptotic mediator Siva1 and loss of its binding target Bcl-xL. In vivo, using an acute lung injury model, we observed increased levels of neutrophil apoptosis and clearance in Phd3-deficient mice compared with WT controls. We also observed reduced neutrophilic inflammation in an acute mouse model of colitis. These data support what we believe to be a novel function for PHD3 in regulating neutrophil survival in hypoxia and may enable the development of new therapeutics for inflammatory disease.
As we enter an era when testing millions of SNPs in a single gene association study will become the standard, consideration of multiple comparisons is an essential part of determining statistical significance. Bonferroni adjustments can be made but are conservative due to the preponderance of linkage disequilibrium (LD) between genetic markers, and permutation testing is not always a viable option. Three major classes of corrections have been proposed to correct the dependent nature of genetic data in Bonferroni adjustments: permutation testing and related alternatives, principal components analysis (PCA), and analysis of blocks of LD across the genome. We consider seven implementations of these commonly used methods using data from 1514 European American participants genotyped for 700,078 SNPs in a GWAS for AIDS.
A Bonferroni correction using the number of LD blocks found by the three algorithms implemented by Haploview resulted in an insufficiently conservative threshold, corresponding to a genome-wide significance level of α = 0.15 - 0.20. We observed a moderate increase in power when using PRESTO, SLIDE, and simpleℳ when compared with traditional Bonferroni methods for population data genotyped on the Affymetrix 6.0 platform in European Americans (α = 0.05 thresholds between 1 × 10-7 and 7 × 10-8).
Correcting for the number of LD blocks resulted in an anti-conservative Bonferroni adjustment. SLIDE and simpleℳ are particularly useful when using a statistical test not handled in optimized permutation testing packages, and genome-wide corrected p-values using SLIDE, are much easier to interpret for consumers of GWAS studies.
The importance of a broad CD8-T lymphocyte (CD8-TL) immune response to HIV is unknown. Ex vivo measurements of immunological activity directed at a limited number of defined epitopes provide an incomplete portrait of the actual immune response. Here we examined viral loads in SIV-infected MHC homozygous and heterozygous Mauritian cynomolgus macaques (MCM). Chronic viremia in MHC homozygous macaques was 80-fold greater than in MHC heterozygous macaques. Virus from MHC homozygous macaques accumulated 11 to 14 variants consistent with escape from CD8-TL responses after one year of SIV infection. The pattern of mutations detected in MHC heterozygous macaques suggests that their epitope-specific CD8-TL responses are a composite of those present in their MHC homozygous counterparts. These results provide the clearest example of MHC heterozygote advantage among individuals infected with the same immunodeficiency virus strain, suggesting that broad recognition of multiple CD8-TL epitopes should be a key feature of HIV vaccines.
MHC; heterozygosity; CD8; SIV
Vascular endothelial growth factor (VEGF) plays a critical role in normal development as well as retinal vasculature disease. During retinal vascularization, VEGF is most strongly expressed by not yet vascularized retinal astrocytes, but also by retinal astrocytes within the developing vascular plexus, suggesting a role for retinal astrocyte-derived VEGF in angiogenesis and vessel network maturation. To test the role of astrocyte-derived VEGF, we used Cre-lox technology in mice to delete VEGF in retinal astrocytes during development. Surprisingly, this only had a minor impact on retinal vasculature development, with only small decreases in plexus spreading, endothelial cell proliferation and survival observed. In contrast, astrocyte VEGF deletion had more pronounced effects on hyperoxia-induced vaso-obliteration and led to the regression of smooth muscle cell-coated radial arteries and veins, which are usually resistant to the vessel-collapsing effects of hyperoxia. These results suggest that VEGF production from retinal astrocytes is relatively dispensable during development, but performs vessel stabilizing functions in the retinal vasculature and might be relevant for retinopathy of prematurity in humans.
Feline immunodeficiency virus (FIV) causes AIDS in the domestic cat (Felis catus) but has not been explicitly associated with AIDS pathology in any of the eight free-ranging species of Felidae that are endemic with circulating FIV strains. African lion (Panthera leo) populations are infected with lion-specific FIV strains (FIVple), yet there remains uncertainty about the degree to which FIV infection impacts their health. Reported CD4+ T-lymphocyte depletion in FIVple infected lions and anecdotal reports of lion morbidity associated with FIV sero-prevalence emphasize the concern as to whether FIVple is innocuous or pathogenic. Here we monitored clinical, biochemical, histological and serological parameters among FIVple-positive (N=47) as compared to FIVple negative (N=17) lions anesthetized and sampled on multiple occasions between 1999 and 2006 in Botswana. Relative to uninfected lions, FIVple infected lions displayed a significant elevation in the prevalence of AIDS defining conditions: lymphandenopathy, gingivitis, tongue papillomas, dehydration, and poor coat condition, as well as displaying abnormal red blood cell parameters and elevated liver enzymes and serum proteins. Spleen and lymph node laparoscopic biopsies from free-ranging FIVple infected lions (N=8) revealed evidence of lymphoid depletion, the hallmark pathology documented in immunodefieciency virus infections of humans (HIV-1), macaques, and domestic cats. We conclude that over time FIVple infections in free-ranging lions can lead to adverse clinical, immunological, and pathological outcomes in some individuals that parallel sequelae caused by lentivirus infection in humans (HIV), Asian macaques (SIV) and domestic cats (FIVfca).
Ischemia exists in many diseased tissues including arthritic joints, atherosclerotic plaques and malignant tumors. Macrophages accumulate in these sites and upregulate hypoxia-inducible transcription factors (HIFs) 1 and 2 in response to the hypoxia present. Here we show that the gene expression profile in primary human and murine macrophages changes markedly when they are exposed to hypoxia for 18h. For example, they were seen to upregulate the cell surface receptors, CXCR4 and GLUT1, and the potent, tumor-promoting cytokines, VEGFA, interleukins 1β and 8, adrenomedullin, CXCR4 and angiopoietin-2. Hypoxia also stimulated their expression and/or phosphorylation of various proteins in the NF-κB signalling pathway. We then used both genetic and pharmacological methods to manipulate the levels of HIFs 1α and 2α or NF-κB in primary macrophages in order to elucidate their role in the hypoxic induction of many of these key genes. These studies showed that both HIFs 1 and 2, but not NF-κB, are important transcriptional effectors regulating the responses of macrophages to such a period of hypoxia. Further studies using experimental mouse models are now warranted to investigate the role of such macrophage responses in the progression of various diseased tissues like malignant tumors.
macrophage; hypoxia; HIF; NF-κB
Skin plays an essential role in adaptation to environmental stimuli that is mediated in part by its remarkable vascular plasticity. Certain vertebrates, such as amphibians, respond to hypoxia in part through the skin; but it is unknown whether this tissue can influence mammalian systemic adaptation to low oxygen levels. We have found that epidermal deletion of the hypoxia responsive transcription factor HIF-1α inhibits renal erythropoietin (EPO) synthesis in response to hypoxia. Conversely, mice with an epidermal deletion of the von Hippel Lindau (VHL) factor, a negative regulator of HIF, have increased EPO synthesis and polycythemia. We show that nitric oxide release induced by the HIF pathway acts on cutaneous vascular flow to increase systemic erythropoietin expression. These results demonstrate that in mice the skin is a critical mediator of systemic responses to environmental oxygen.