Combined Effect of HPV and Several Gene SNPs in Laryngeal Cancer.

Background and objectives: Laryngeal squamous cell carcinoma (LSCC) is one of the most common head and neck tumors. The molecular mechanism of LSCC remains unclear. The aim of this study was to evaluate the prevalence of Human papillomavirus (HPV) and single nucleotide polymorphisms (SNPs) of TP53, MDM2, MDM4, MTHFR, CASP8, and CCR5 genes in LSCC, and to assess their correlations with patient survival. Materials and Methods: 49 LSCC patients were enrolled in this study. PCR and qRT-PCR were used to detect, identify, and quantify HPV. SNPs were genotyped using PCR and PCR-RFLP. Results: By analyzing the interactions of the SNPs of the genes with clinical parameters, the majority of patients with lymph node status (N1,2) were identified as carriers of MDM2 T/G, CASP8 ins/del, CCR5 wt/wt SNP. Cluster analysis showed that patients with MDM2 T/T SNP survive longer than patients identified as CASP8 ins/ins, MTHFR C/C, and MDM4 A/A variant carriers; meanwhile, LSCC patients with MDM2 T/T polymorphic variant had the best survival. Multivariate analysis showed that HPV-positive patients without metastasis in regional lymph nodes (N0) and harboring CASP8 ins/del variant had the best survival. Meanwhile, HPV-negative patients with identified metastasis in lymph nodes (N1 and N2) and CASP8 ins/del variant had poor survival. Conclusions: This finding suggests patients survival prognosis and tumor behavior are different according HPV status, SNP variants, and clinical characteristics of the LSCC.

Single-Cell Tracking Reveals a Role for Pre-Existing CCR5+ Memory Th1 Cells in the Control of Rhinovirus-A39 After Experimental Challenge in Humans.

Background: Little is known about T cells that respond to human rhinovirus in vivo, due to timing of infection, viral diversity, and complex T-cell specificities. We tracked circulating CD4+ T cells with identical epitope specificities that responded to intranasal challenge with rhinovirus (RV)-A39, and we assessed T-cell signatures in the nose. Methods: Cells were monitored using a mixture of 2 capsid-specific major histocompatibility complex II tetramers over a 7-week period, before and after RV-A39 challenge, in 16 human leukocyte antigen-DR4+ subjects who participated in a trial of Bifidobacterium lactis (Bl-04) supplementation. Results: Pre-existing tetramer+ T cells were linked to delayed viral shedding, enriched for activated CCR5+ Th1 effectors, and included a minor interleukin-21+ T follicular helper cell subset. After RV challenge, expansion and activation of virus-specific CCR5+ Th1 effectors was restricted to subjects who had a rise in neutralizing antibodies, and tetramer-negative CCR5+ effector memory types were comodulated. In the nose, CXCR3-CCR5+ T cells present during acute infection were activated effector memory type, whereas CXCR3+ cells were central memory type, and cognate chemokine ligands were elevated over baseline. Probiotic had no T-cell effects. Conclusions: We conclude that virus-specific CCR5+ effector memory CD4+ T cells primed by previous exposure to related viruses contribute to the control of rhinovirus.

Alterations of CCR5 and CCR7 expression on donor peripheral blood T cell subsets after mobilization with rhG-CSF correlate with acute graft-versus-host disease.

To investigate the effects of recombinant human granulocyte colony-stimulating factor (rhG-CSF) on chemokine receptors and explore the potential mechanism of rhG-CSF inducing immune tolerance, ninety-seven donor and recipient pairs undergoing family-donor allogeneic hematopoietic stem cell transplantation were studied. The results indicated that different donors showed great disparities in expression changes after mobilization. Multivariate analysis revealed that both HLA mismatching and CCR7 downregulation on donors' CD4+ T cells after mobilization were independent risk factors for acute graft-versus-host disease (GVHD). In contrast, CCR5 downregulation on CD4+ T cells was associated with reduced incidence of acute GVHD. In conclusion, rhG-CSF mobilization could lead to differential regulation of chemokine receptors expression on T cell subsets in different donors. Downregulation of CCR5 and upregulation of CCR7 expression on donor CD4+ T cells might protect recipients from acute GVHD. This finding may provide a promising new strategy for the prevention and treatment of acute GVHD.

Surveying GPCR solubilisation conditions using surface plasmon resonance.

Biophysical screening techniques, such as surface plasmon resonance, enable detailed kinetic analysis of ligands binding to solubilised G-protein coupled receptors. The activity of a receptor solubilised out of the membrane is crucially dependent on the environment in which it is suspended. Finding the right conditions is challenging due to the number of variables to investigate in order to determine the optimum solubilisation buffer for any given receptor. In this study we used surface plasmon resonance technology to screen a variety of solubilisation conditions including buffers and detergents for two model receptors: CXCR4 and CCR5. We tested 950 different combinations of solubilisation conditions for both receptors. The activity of both receptors was monitored by using conformation dependent monoclonal antibodies and the binding of small molecule ligands. Despite both receptors belonging to the chemokine receptor family they show some differences in their preference for solubilisation conditions that provide the highest level of binding for both the conformation dependent antibodies and small molecules. The study described here is focused not only on finding the best solubilisation conditions for each receptor, but also on factors that determine the sensitivity of the assay for each receptor. We also suggest how these data about different buffers and detergents can be used as a guide for selecting solubilisation conditions for other membrane proteins.

T cells are involved in the induction of macrophage phenotypes in oral leukoplakia and squamous cell carcinoma-a preliminary report.

BACKGROUND: The prognosis of human malignancies has been shown to depend on immunological parameters, such as macrophage polarization (M1 and M2). In this study, we identify the phenotype of macrophages, and investigate an involvement of infiltrated T cells that participate in the polarization of macrophages, in oral leukoplakia (OLK), and oral squamous cell carcinoma (OSCC). METHODS: Immunohistochemical method was used to examine the number of CD68+ , CD163+ (M2), iNOS+ (M1) macrophages, and CD4+ , CD8+ , CCR4+ (Th2), CCR5+ (Th1) cells in 102 cases of OSCC: without metastases-OSCC M(-) (n = 54), and with metastases-OSCC M(+) (n = 48), 23 cases of OLK, and 18 control cases. RESULTS: The mean number of CD68+ , CD163+ , iNOS+ , CD4+ , CCR4+ , CCR5+ cells was significantly increased in OSCC M(+) group compared with OLK, OSCC M(-) and control group. We found positive correlations between the number of CD4+ T cells and CD163+ and iNOS+ macrophages as well as CCR4+ and CCR5+ cells in both OSCC groups. The mean number of CD8+ cells was significantly increased in OSCC M(-) and OLK compared with OSCC M(+) and control group. In OSCC M(+) and OSCC M(-) groups, a negative correlation between the number of CD8+ cells and CD163+ and iNOS+ macrophages was found. CONCLUSIONS: The number and co-localization of lymphocytes and macrophages in OLK and OSCC may indicate that infiltrating cells influence the early and subsequent stage of oral carcinogenesis.

Microparticles derived from obese adipose tissue elicit a pro-inflammatory phenotype of CD16+, CCR5+ and TLR8+ monocytes.

Macrophage infiltration into adipose tissue (AT) is a hallmark of the chronic inflammatory response in obesity and is supported by an intense monocyte migration towards AT. Although it has been detected an increased proportion of circulating CD16+ monocyte subsets in obese subjects, the mechanisms underlying this effect and the contribution of these cells to the inflamed profile of obese AT are still poorly understood. We investigated whether factors secreted by human obese omental AT could polarize monocytes to CD16+ enriched phenotype, and how these changes could modify their migratory capacity towards adipose tissue itself. We show that explants of human obese omental AT, obtained during bariatric surgery, released higher levels of MIP1-α, TNFα, leptin and also VEGF, together with increasing amounts of microparticles (MP), when compared to explants of lean subcutaneous AT. A higher content of circulating MP derived from preadipocytes and leukocytes was also detected in plasma of obese subjects. Conditioned media or MP released from obese omental AT increased CD16 and CCR5 expression on CD14+CD16- monocytes and augmented their migratory capacity towards the conditioned media from obese omental AT, itself. This effect was inhibited when MIP1-α was neutralized. Additionally, we demonstrate that MP derived from obese omental AT carry and transfer TLR8 to monocytes, thus triggering an increase in CD16 expression in those cells. Our data shows a positive feedback loop between blood monocytes and obese omental AT, which releases chemotactic mediators and TLR8-enriched MP, thus inducing an up-regulation of CD16+ monocytes, favoring leukocyte infiltration in the obese omental AT.

CD25+ FoxP3+ Memory CD4 T Cells Are Frequent Targets of HIV Infection In Vivo.

UNLABELLED: Interleukin 2 (IL-2) signaling through the IL-2 receptor alpha chain (CD25) facilitates HIV replication in vitro and facilitates homeostatic proliferation of CD25(+) FoxP3(+) CD4(+) T cells. CD25(+) FoxP3(+) CD4(+) T cells may therefore constitute a suitable subset for HIV infection and plasma virion production. CD25(+) FoxP3(+) CD4(+) T cell frequencies, absolute numbers, and the expression of CCR5 and cell cycle marker Ki67 were studied in peripheral blood from HIV(+) and HIV(-) study volunteers. Different memory CD4(+) T cell subsets were then sorted for quantification of cell-associated HIV DNA and phylogenetic analyses of the highly variable EnvV1V3 region in comparison to plasma-derived virus sequences. In HIV(+) subjects, 51% (median) of CD25(+) FoxP3(+) CD4(+) T cells expressed the HIV coreceptor CCR5. Very high frequencies of Ki67(+) cells were detected in CD25(+) FoxP3(+) memory CD4(+) T cells (median, 27.6%) in comparison to CD25(-) FoxP3(-) memory CD4(+) T cells (median, 4.1%; P < 0.0001). HIV DNA content was 15-fold higher in CD25(+) FoxP3(+) memory CD4(+) T cells than in CD25(-) FoxP3(-) T cells (P = 0.003). EnvV1V3 sequences derived from CD25(+) FoxP3(+) memory CD4(+) T cells did not preferentially cluster with plasma-derived sequences. Quasi-identical cell-plasma sequence pairs were rare, and their proportion decreased with the estimated HIV infection duration. These data suggest that specific cellular characteristics of CD25(+) FoxP3(+) memory CD4(+) T cells might facilitate efficient HIV infection in vivo and passage of HIV DNA to cell progeny in the absence of active viral replication. The contribution of this cell population to plasma virion production remains unclear. IMPORTANCE: Despite recent advances in the understanding of AIDS virus pathogenesis, which cell subsets support HIV infection and replication in vivo is incompletely understood. In vitro, the IL-2 signaling pathway and IL-2-dependent cell cycle induction are essential for HIV infection of stimulated T cells. CD25(+) FoxP3(+) memory CD4 T cells, often referred to as regulatory CD4 T cells, depend on IL-2 signaling for homeostatic proliferation in vivo Our results show that CD25(+) FoxP3(+) memory CD4(+) T cells often express the HIV coreceptor CCR5, are significantly more proliferative, and contain more HIV DNA than CD25(-) FoxP3(-) memory CD4 T cell subsets. The specific cellular characteristics of CD25(+) FoxP3(+) memory CD4(+) T cells probably facilitate efficient HIV infection in vivo and passage of HIV DNA to cell progeny in the absence of active viral replication. However, the contribution of this cell subset to plasma viremia remains unclear.

Effects of conventional CPB and mini-CPB on neutrophils CD162, CD166 and CD195 expression.

OBJECTIVE: Cardiac surgery is known to trigger a systemic inflammatory response. While the use of conventional cardiopulmonary bypass (CPB) results in profound inflammation, modified mini-CPB is considered less harmful. We evaluated the impact of cardiac surgery on the expression of CD162, CD166, CD195 molecules and their association with the type of CPB used. METHODS AND RESULTS: Twenty-four patients were enrolled in our study. Twelve of them were operated using conventional CPB while the other twelve patients underwent surgery with mini-CPB. Blood samples were analysed by flow cytometry. We observed a significant increase in median fluorescence intensity of CD162 and CD195 that peaked instantly after surgery and normalized to the baseline value on the 1st day post surgery, whereas CD166 was initially down-regulated and its median fluorescence intensity (MFI) value increased to the baseline in the next few days. CONCLUSION: We observed immediate changes in the expression of CD162, CD166, and CD195 molecules on the neutrophils after surgery in both study groups of patients. The intensity of the observed changes was significantly greater in the group of patients who underwent conventional CPB compared to patients who underwent mini-CPB cardiac surgery.

HIV Disrupts Human T Cells That Target Mycobacterial Glycolipids.

Single-cell analysis captures the heterogeneity of T-cell populations that target defined antigens. Human immunodeficiency virus (HIV) infection results in defects of antimycobacterial immunity, which remain poorly defined. We therefore recruited a small number of subjects, including those with latent and active M. tuberculosis infection, with or without concomitant HIV infection, and tracked the mycobacterial glycolipid-reactive T-cell repertoire by using CD1b tetramers. Glycolipid-reactive T cells expressed memory markers and the HIV coreceptors CD4 and CCR5; they were not detected in subjects with HIV-associated active M. tuberculosis infection. HIV infection may affect T cells that recognize mycobacterial glycolipids and influence immunity.

Plasmacytoid Dendritic Cell Infection and Sensing Capacity during Pathogenic and Nonpathogenic Simian Immunodeficiency Virus Infection.

UNLABELLED: Human immunodeficiency virus (HIV) in humans and simian immunodeficiency virus (SIV) in macaques (MAC) lead to chronic inflammation and AIDS. Natural hosts, such as African green monkeys (AGM) and sooty mangabeys (SM), are protected against SIV-induced chronic inflammation and AIDS. Here, we report that AGM plasmacytoid dendritic cells (pDC) express extremely low levels of CD4, unlike MAC and human pDC. Despite this, AGM pDC efficiently sensed SIVagm, but not heterologous HIV/SIV isolates, indicating a virus-host adaptation. Moreover, both AGM and SM pDC were found to be, in contrast to MAC pDC, predominantly negative for CCR5. Despite such limited CD4 and CCR5 expression, lymphoid tissue pDC were infected to a degree similar to that seen with CD4(+) T cells in both MAC and AGM. Altogether, our finding of efficient pDC infection by SIV in vivo identifies pDC as a potential viral reservoir in lymphoid tissues. We discovered low expression of CD4 on AGM pDC, which did not preclude efficient sensing of host-adapted viruses. Therefore, pDC infection and efficient sensing are not prerequisites for chronic inflammation. The high level of pDC infection by SIVagm suggests that if CCR5 paucity on immune cells is important for nonpathogenesis of natural hosts, it is possibly not due to its role as a coreceptor. IMPORTANCE: The ability of certain key immune cell subsets to resist infection might contribute to the asymptomatic nature of simian immunodeficiency virus (SIV) infection in its natural hosts, such as African green monkeys (AGM) and sooty mangabeys (SM). This relative resistance to infection has been correlated with reduced expression of CD4 and/or CCR5. We show that plasmacytoid dendritic cells (pDC) of natural hosts display reduced CD4 and/or CCR5 expression, unlike macaque pDC. Surprisingly, this did not protect AGM pDC, as infection levels were similar to those found in MAC pDC. Furthermore, we show that AGM pDC did not consistently produce type I interferon (IFN-I) upon heterologous SIVmac/HIV type 1 (HIV-1) encounter, while they sensed autologous SIVagm isolates. Pseudotyping SIVmac/HIV-1 overcame this deficiency, suggesting that reduced uptake of heterologous viral strains underlays this lack of sensing. The distinct IFN-I responses depending on host species and HIV/SIV isolates reveal the host/virus species specificity of pDC sensing.

Differences in T cell distribution and CCR5 expression in HIV-positive and HIV-exposed seronegative persons who inject drugs.

Some individuals remain uninfected despite repeated exposure to HIV. This protection against HIV has been partly associated with altered T cell subset distributions and CCR5 expression levels. However, the majority of studies have been conducted in sexually exposed subjects. We aimed to assess whether HIV infection and intravenous drug use were associated with differences in CCR5 expression, immune activation on the CD4+ and CD8+ T cells and T cell distribution among Caucasian persons who inject drugs (PWIDs). Analyses of the data from 41 HIV-positive PWIDs, 47 HIV-exposed seronegative PWIDs (ESNs) and 47 age- and gender-matched HIV-negative non-drug users are presented. Of all of the study subjects, 111 (82 %) were male, and the median age was 29 years. T cell phenotyping was performed in peripheral blood mononuclear cells with multicolour flow cytometry using anti-CD3, CD4, CD8, CD45RA, CD45RO, HLA-DR and CCR5 antibodies. The ESNs exhibited greater levels of immune activation and higher percentages of CD4+ CD45RA+RO+ and CD8+ CD45RA+RO+ cells compared to the controls but not the HIV-positive people. The CCR5 expression on the CD4+ T cell subsets in the ESNs was lower than that in the controls but similar to that the HIV positives. The percentages of CCR5+ T cells were similar in all study groups and in most of the studied cell populations. Intravenous drug use was similarly associated with differences in T cell subset distributions and CCR5 expression among both the HIV-positive and HIV-negative PWIDs compared with the controls.

Cytomegalovirus upregulates expression of CCR5 in central memory cord blood mononuclear cells, which may facilitate in utero HIV type 1 transmission.

Administration of combination antiretroviral therapy to human immunodeficiency virus type 1 (HIV-1)-infected pregnant women significantly reduces vertical transmission. In contrast, maternal co-opportunistic infection with primary or reactivated cytomegalovirus (CMV) or other pathogens may facilitate in utero transmission of HIV-1 by activation of cord blood mononuclear cells (CBMCs). Here we examine the targets and mechanisms that affect fetal susceptibility to HIV-1 in utero. Using flow cytometry, we demonstrate that the fraction of CD4(+)CD45RO(+) and CD4(+)CCR5(+) CBMCs is minimal, which may account for the low level of in utero HIV-1 transmission. Unstimulated CD4(+) CBMCs that lack CCR5/CD45RO showed reduced levels of HIV-1 infection. However, upon in vitro stimulation with CMV, CBMCs undergo increased proliferation to upregulate the fraction of T central memory cells and expression of CCR5, which enhances susceptibility to HIV-1 infection in vitro. These data suggest that activation induced by CMV in vivo may alter CCR5 expression in CD4(+) T central memory cells to promote in utero transmission of HIV-1.

HIV-1 replicates in human osteoclasts and enhances their differentiation in vitro.

BACKGROUND: HIV-1 infected patients frequently have osteolytic bone disease, which is caused by the dysregulation of the bone remodeling system that involves the interaction between osteoblasts and osteoclasts, but the relationship between osteolytic disease and HIV-1 infection remains unclear. In this study we tested whether HIV-1 infection of osteoclasts affects their differentiation. RESULTS: We prepared human osteoclasts from CD14+ monocytes and examined them for their susceptibility to HIV-1. Furthermore, we investigated the effect of HIV-1 infection on osteoclast differentiation. CD14-derived osteoclasts were shown to express CD4, CCR5, and CXCR4 each at the similar level to that shown with macrophages. R5-tropic HIV-1 and X4-tropic HIV-1 were found to infect CD14-derived osteoclasts and replicate in them. Furthermore, HIV-1 infection induced formation of larger osteoclastst, enhanced the expression of mRNAs for three osteoclast specific marker molecules (tartrate-resistant acid phosphatase, cathepsin K, and the calcitonin receptor), and up-regulated osteoclast bone resorption activity. CONCLUSIONS: Our results suggest that osteoclasts serve as a novel target for HIV-1 infection, which may enhance the osteoclast differentiation contributing to the development of osteolytic disease in HIV-1-infected patients.

Innate immune interleukin-1 receptor-associated kinase 4 exacerbates viral myocarditis by reducing CCR5(+) CD11b(+) monocyte migration and impairing interferon production.

BACKGROUND: Viral myocarditis follows a fatal course in ≈30% of patients. Interleukin-1 receptor-associated kinase 4 (IRAK4), a major nodal signal transducer in innate immunity, can play a pivotal role in host inflammatory response. We sought to determine how IRAK4 modulates inflammation and outcome in a mouse model of viral myocarditis. METHODS AND RESULTS: Myocarditis was induced after intraperitoneal inoculation of coxsackievirus B3 into C57Bl/6 IRAK4-deficient mice and their littermate controls. Mortality and viral proliferation were markedly reduced in IRAK4(-/-) mice compared with their IRAK4(+/+) littermates. Disease resistance of IRAK4(-/-) mice paralleled increased amounts of protective heart-infiltrating CCR5(+) monocytes/macrophages and enhanced interferon-α and interferon-γ production 2 days after infection. Competitive bone marrow chimera demonstrated that intact IRAK4 function inhibited heart-specific migration of bone marrow-derived CCR5(+) cells. Mechanistically, lack of IRAK4 resulted in interferon regulatory factor 5 homodimerization via reduced melanoma differentiation-associated protein 5 degradation and enhanced Stat1 and Stat5 phosphorylation. Consequently, antiviral interferon-α and interferon-γ production, as well as CCR5(+) cell recruitment, increased, whereas the overall proinflammatory response was drastically reduced in the absence of IRAK4. CONCLUSIONS: Innate immunity signal transducer IRAK4 exacerbates viral myocarditis through inhibition of interferon production and reduced mobilization of protective CCR5(+) monocytes/macrophages to the heart. The combination of IRAK4 inhibitors and antiviral adjuvants may become an attractive therapeutic approach against viral myocarditis in the future.

In vitro effects of the CCR5 inhibitor maraviroc on human T cell function.

BACKGROUND: Several potential immunological benefits have been observed during treatment with the CC chemokine receptor 5 (CCR5) antagonist maraviroc, in addition to its antiviral effect. Our objective was to analyse the in vitro effects of CCR5 blockade on T lymphocyte function and homeostasis. METHODS: Peripheral blood mononuclear cells (PBMCs) from both HIV-negative (n=28) and treated HIV-positive (n=27) individuals were exposed in vitro to different concentrations of maraviroc (0.1-100 µM). Effects on T cell activation were analysed by measuring the expression of the CD69, CD38, HLA-DR and CD25 receptors as well as CCR5 density using flow cytometry. Spontaneous and chemokine-induced chemotaxis were measured by transwell migration assays, and polyclonal-induced proliferation was assessed by a lymphoproliferation assay and carboxyfluorescein succinimidyl ester staining. RESULTS: Maraviroc increases CCR5 surface expression on activated T cells, even at low doses (0.1 µM). Slight differences were detected in the frequency and mean fluorescence intensity of activation markers at high concentrations of maraviroc. Expression of CD25, CD38 and HLA-DR tended to decrease in both CD4+ and CD8+ T lymphocytes, whereas expression of CD69 tended to increase. Maraviroc clearly inhibits T cell migration induced by chemokines in a dose-dependent manner. Moreover, at 100 µM, maraviroc tends to inhibit T cell proliferation. CONCLUSIONS: These data showed that in vitro exposure to maraviroc decreases some activation expression markers on T lymphocytes and also migration towards chemoattractants. These results support the additional immunological effects of CCR5 blockade and suggest that maraviroc might have potential capacity to inhibit HIV-associated chronic inflammation and activation, both by directly affecting T cell activation and by reducing entrapment of lymphocytes in lymph nodes.

Sequential staining improves detection of CCR2 and CX3CR1 on monocytes when simultaneously evaluating CCR5 by multicolor flow cytometry.

Chemokines and their receptors play an essential role within the immune system by dictating cellular migration. In vivo, receptor-ligand interactions rarely occur in isolation as cellular recruitment and migration are complex and highly coordinated processes often involving networks of multiple chemokines and multiple receptors. Simultaneous detection of multiple chemokine receptors on the single cell level is necessary to allow immunophenotyping studies that will help understand the intricacies of these networks. Chemokine receptors undergo a basal level of ongoing internalization, intracellular trafficking, and recycling back to the cell surface, even in the absence of the ligand. In the presence of ligand, receptor-ligand interactions enhance receptor internalization, reducing the cell surface receptor concentration, making precise determination of intrinsic levels challenging. Using multicolor flow cytometry, we sought to evaluate and optimize the simultaneous detection of cell surface expression levels of CCR2, CX3CR1, and CCR5 in primary human monocytes using a single antibody panel. We observed that staining for CCR2 alone or for CX3CR1 alone showed greater expression levels than when the cells were stained with the full panel of antibodies. Fluorescent-minus-one (FMO) controls revealed that ligation of the CCR5 monoclonal antibody to the receptor interfered with detection of CX3CR1 and CCR2. Sequential addition of antibodies during the staining procedure was sufficient to restore the detection levels, suggesting close proximity and possible functional interactions between CCR2/CCR5 and CX3CR1/CCR5 in monocytes. This study highlights the importance of optimizing staining procedures and using proper controls when simultaneously evaluating expression levels of multiple chemokine receptors by flow cytometry. Concurrent assessment of multiple receptors will provide insight and greater understanding of the complex interactions involved in cellular migration.

Circulating human CD4 and CD8 T cells do not have large intracellular pools of CCR5.

CC Chemokine Receptor 5 (CCR5) is an important mediator of chemotaxis and the primary coreceptor for HIV-1. A recent report by other researchers suggested that primary T cells harbor pools of intracellular CCR5. With the use of a series of complementary techniques to measure CCR5 expression (antibody labeling, Western blot, quantitative reverse transcription polymerase chain reaction), we established that intracellular pools of CCR5 do not exist and that the results obtained by the other researchers were false-positives that arose because of the generation of irrelevant binding sites for anti-CCR5 antibodies during fixation and permeabilization of cells.

The CD14(bright) CD16+ monocyte subset is expanded in rheumatoid arthritis and promotes expansion of the Th17 cell population.

OBJECTIVE: Circulating monocytes contain a subpopulation of CD14+CD16+ cells; this subpopulation of cells has been described to be proinflammatory and to have an increased frequency in rheumatoid arthritis (RA). New evidence suggests that this subpopulation can be further subdivided into CD14(dim) CD16+ and CD14(bright) CD16+ cells. The aim of this study was to determine which of the two CD16+ monocyte subpopulations is expanded in patients with RA and to investigate their possible role in disease pathogenesis. METHODS: The frequencies of monocyte subpopulations in the peripheral blood of healthy donors and patients with RA were determined by flow cytometry. Monocyte subpopulations were sorted and cocultured with CD4+ T cells. Cytokines were determined in the supernatant, and Th17 cell frequencies were measured by flow cytometry. RESULTS: In comparison with the other monocyte subpopulations, CD14(bright) CD16+ cells showed higher HLA-DR and CCR5 expression and responded with higher tumor necrosis factor production to direct cell contact with preactivated T cells. They were observed at increased frequencies in the peripheral blood of patients with RA, while CD14(dim) CD16+ monocyte frequencies were not increased. CD14(bright) CD16+ cells were extremely potent inducers of Th17 cell expansion in vitro. Their frequency in the peripheral blood of patients with RA correlated closely with Th17 cell frequencies determined ex vivo. CONCLUSION: This study is the first to provide a link between the increased frequency of the CD14(bright) CD16+ monocyte subpopulation in RA and the expansion of Th17 cells, which are likely to have a role in the pathogenesis of autoimmunity.

Characterization of a human cervical CD4+ T cell subset coexpressing multiple markers of HIV susceptibility.

The HIV pandemic disproportionately affects women, with most infections acquired through receptive vaginal sex. Although the target cells by which HIV establishes infection in the female genital tract remain poorly defined, it is known that immune activation results in CD4(+) T cells with enhanced susceptibility, as does expression of the mucosal integrin α4ß7 and the HIV coreceptor CCR5. Blood and cervical cytobrush specimens were collected from female sex workers (FSWs) in Nairobi, Kenya. Genital infection diagnostics were performed, T cell populations were defined by multiparameter flow cytometry based on their expression of surface receptors relevant to mucosal homing and/or HIV acquisition, and cytokine production was assayed by intracellular cytokine staining. The integrin α4ß7 was expressed on 26.0% of cervical CD4(+) T cells, and these cells were more likely to express both the HIV coreceptor CCR5 (p < 0.0001) and the early activation marker CD69 (p < 0.0001) but not CXCR4 (p = 0.34). Cervical Th17 frequencies were enhanced compared with blood (7.02 versus 1.24%; p < 0.0001), and cervical IL-17A(+) CD4(+) T cells preferentially coexpressed α4ß7 and CCR5. Expression of IFN-γ and IL-22 was greater in cervical Th17 cells than in blood Th17 cells. In keeping with the hypothesis that these cells are preferential HIV targets, gp120 preferentially bound CCR5(+) cervical T cells, and cervical Th17 cells were almost completely depleted in HIV(+) FSWs compared with HIV(-) FSWs. In summary, a subset of Th17 CD4(+) T cells in the cervical mucosa coexpresses multiple HIV susceptibility markers; their dramatic depletion after HIV infection suggests that these may serve as key target cells during HIV transmission.

Bone marrow-derived monocyte lineage cells recruited by MIP-1ß promote physiological revascularization in mouse model of oxygen-induced retinopathy.

Recent clinical observations have indicated that vascular endothelial growth factor (VEGF) is a key factor that stimulates the development of preretinal pathological neovascularization (NV). However, it has not been established how intraretinal physiological revascularization of hypoxic avascular areas is regulated. Our earlier study on the gene expression profile of hypoxic retinas in a mouse model of oxygen-induced retinopathy (OIR) showed that macrophage inflammatory protein-1ß (MIP-1ß) was the most upregulated protein. The purpose of this study was to investigate the role played by MIP-1ß in recruiting bone marrow-derived monocyte lineage cells (BM-MLCs) in a mouse model of OIR. Our results showed that MIP-1ß was upregulated, and its receptor, CCR5, was expressed in BM-MLCs in the hypoxic inner retina. Neutralizing Ab against MIP-1ß reduced the infiltration of BM-MLCs into the OIR retinas and increased the avascular area and preretinal neovascular tufts. A very strong significant correlation was found between the area of the preretinal neovascular tufts and the avascular area, regardless of the extent of BM-MLC infiltration into the OIR retinas. Additional treatment with VEGF-A-neutralizing Ab showed that the MIP-1ß-regulated pathological NV strongly depended on VEGF-A, which was probably secreted by the hypoxic avascular retinas. These results indicate that MIP-1ß is involved in the recruitment of BM-MLCs, which have a significant role in the physiological revascularization of hypoxic avascular retinas. Overall, these findings indicate that the MIP-1ß induction of BM-MLCs might possibly be used to promote intraretinal revascularization and thus prevent the abnormal NV in ischemic vision-threatening retinal diseases.