CD8+ T cells in the central nervous system of mice with herpes simplex infection are highly activated and express high levels of CCR5 and CXCR3.

Herpes simplex virus type 2 (HSV-2) is a neurotropic virus that can cause meningitis, an inflammation of the meninges in the central nervous system. T cells are key players in viral clearance, and these cells migrate from peripheral blood into the central nervous system upon infection. Several factors contribute to T cell migration, including the expression of chemokines in the inflamed tissue that attract T cells through their expression of chemokine receptors. Here we investigated CD8+ T cell profile in the spinal cord in a mouse model of herpes simplex virus type 2 neuroinflammation. Mice were infected with HSV-2 and sacrificed when showing signs of neuroinflammation. Cells and/or tissue from spinal cord, spleen, and blood were analyzed for expression of activation markers, chemokine receptors, and chemokines. High numbers of CD8+ T cells were present in the spinal cord following genital HSV-2-infection. CD8+ T cells were highly activated and HSV-2 glycoprotein B -specific effector cells, some of which showed signs of recent degranulation. They also expressed high levels of many chemokine receptors, in particular CCR2, CCR4, CCR5, and CXCR3. Investigating corresponding receptor ligands in spinal cord tissue revealed markedly increased expression of the cognate ligands CCL2, CCL5, CCL8, CCL12, and CXCL10. This study shows that during herpesvirus neuroinflammation anti-viral CD8+ T cells accumulate in the CNS. CD8+ T cells in the CNS also express chemotactic receptors cognate to the chemotactic gradients in the spinal cord. This indicates that anti-viral CD8+ T cells may migrate to infected areas in the spinal cord during herpesvirus neuroinflammation in response to chemotactic gradients.

Reduced CCR5 Expression and Immune Quiescence in Black South African HIV-1 Controllers.

Unique Individuals who exhibit either suppressive HIV-1 control, or the ability to maintain low viral load set-points and preserve their CD4+ T cell counts for extended time periods in the absence of antiretroviral therapy, are broadly termed HIV-1 controllers. We assessed the extent to which black South African controllers (n=9), differ from uninfected healthy controls (HCs, n=22) in terms of lymphocyte and monocyte CCR5 expression (density and frequency of CCR5-expressing cells), immune activation as well as peripheral blood mononuclear cell (PBMC) mitogen-induced chemokine/cytokine production. In addition, relative CD4+ T cell CCR5 mRNA expression was assessed in a larger group of controllers (n=20) compared to HCs (n=10) and HIV-1 progressors (n=12). Despite controllers having significantly higher frequencies of activated CD4+ and CD8+ T cells (HLA-DR+) compared to HCs, CCR5 density was significantly lower in these T cell populations (P=0.039 and P=0.064, respectively). This lower CCR5 density was largely attributable to controllers with higher VLs (>400 RNA copies/ml). Significantly lower CD4+ T cell CCR5 density in controllers was maintained (P=0.036) when HCs (n=12) and controllers (n=9) were matched for age. CD4+ T cell CCR5 mRNA expression was significantly less in controllers compared to HCs (P=0.007) and progressors (P=0.002), whereas HCs and progressors were similar (P=0.223). The levels of soluble CD14 in plasma did not differ between controllers and HCs, suggesting no demonstrable monocyte activation. While controllers had lower monocyte CCR5 density compared to the HCs (P=0.02), significance was lost when groups were age-matched (P=0.804). However, when groups were matched for both CCR5 promoter haplotype and age (n=6 for both) reduced CCR5 density on monocytes in controllers relative to HCs was highly significant (P=0.009). Phytohemagglutinin-stimulated PBMCs from the controllers produced significantly less CCL3 (P=0.029), CCL4 (P=0.008) and IL-10 (P=0.028) compared to the HCs, which was largely attributable to the controllers with lower VLs (<400 RNA copies/ml). Our findings support a hypothesis of an inherent (genetic) predisposition to lower CCR5 expression in individuals who naturally control HIV-1, as has been suggested for Caucasian controllers, and thus, likely involves a mechanism shared between ethnically divergent population groups.

Alterations in chemokine receptor CCR5 activity influence tumor cell biology in human cholangiocarcinoma cell lines.

INTRODUCTION AND OBJECTIVES: Intrahepatic (I-CCA) and extrahepatic (E-CCA) cholangiocarcinoma (CCA) have different growth patterns and risks for tumor metastasis. Inhibition and/or activation of the chemokine receptor CCR subclasses have been reported to alter tumor cell biology in non-CCA cancers. In this study we documented CCR expression profiles in representative human I-CCA and E-CCA cell lines and the in vitro effects of CCR antagonists and agonists on tumor cell biology. MATERIALS AND METHODS: CCR expression profiles were documented by real-time reverse transcription polymerase chain reaction; cell proliferation by WST-1; spheroid formation by sphere dimensions in anchorage-free medium; cell migration by wound healing and invasion by Transwell invasion chambers. RESULTS: All 10 CCR motifs (CCR1-10) were expressed in the I-CCA, HuCCT1 cell line and six (CCR4, 5, 6, 8, 9 and 10) in the E-CCA, KMBC cell line. In HuCCT1 cells, CCR5 expression was most abundant whereas in KMBC cells, CCR6 followed by CCR5 were most abundant. The CCR5 antagonist Maraviroc significantly inhibited cell proliferation, migration and invasion in HuCCT1 cells, and spheroid formation and invasion in KMBC cells. The CCR5 agonist RANTES had no effect on HuCCT1 cells but increased cell proliferation, migration and invasion of KMBC cells. CONCLUSION: These results suggest that CCR expression profiles differ in I-CCA and E-CCA. They also indicate that CCR5 antagonists and agonists have cell-specific effects but in general, CCR5 inactivation inhibits CCA tumor cell aggressiveness. Additional research is required to determine whether CCR5 inactivation is of value in the treatment of CCA in humans.

Localization-Specific Expression of CCR1 and CCR5 by Mast Cell Progenitors.

Mast cells are powerful immune cells found predominately in barrier tissues. They play an important role in immune surveillance and act as effector cells in allergic reactions. Mast cells develop from mast cell progenitors (MCp), which migrate to the peripheral tissues via the blood circulation. Presumably, the homing of MCp to the peripheral sites and localization is regulated by chemotactic signals. Due to the scarce abundance of these cells, chemotactic receptors have not been previously characterized on primary MCp. Here, mRNA transcripts for CCR1 and CX3CR1 were identified in mouse bone marrow and lung MCp in a gene expression screen of chemotactic receptors. However, surface expression of CCR1 was only found in the bone marrow MCp. Flow cytometry-based screening identified distinct surface expression of CCR5 by mouse peritoneal mast cells and MCp, while surface expression of CXCR2-5, CX3CR1, CCR1-3, CCR6-7, and CCR9 was not detected. Low surface expression of CCR5 was detected in mouse MCp in the bone marrow, spleen, and lung. To translate the findings to human, blood and bone marrow MCp from healthy donors were analyzed for possible CCR1 and CCR5 expression. Human MCp showed distinct surface expression of both CCR1 and CCR5. The expression levels of these chemokine receptors were higher in human bone marrow MCp than in the peripheral blood, suggesting that CCR1 and CCR5 may mediate retention in the bone marrow. In conclusion, mouse and human MCp show differential expression of CCR1 and CCR5 depending on their localization.

Enquiring beneath the surface: can a gene expression assay shed light into the heterogeneity among newborns with neonatal encephalopathy?

BACKGROUND: We aimed to assess whether a gene expression assay provided insights for understanding the heterogeneity among newborns affected by neonatal encephalopathy (NE). METHODS: Analysis by RT-qPCR of the mRNA expression of candidate genes in whole blood from controls (n = 34) and NE (n = 24) patients at <6, 12, 24, 48, 72 and 96 h of life, followed by determination of differences in gene expression between conditions and correlation with clinical variables. RESULTS: During the first 4 days of life, MMP9, PPARG, IL8, HSPA1A and TLR8 were more expressed and CCR5 less expressed in NE patients compared to controls. MMP9 and PPARG increased and CCR5 decreased in moderate/severe NE patients compared to mild. At 6-12 h of life, increased IL8 correlated with severe NE and death, decreased CCR5 correlated with chorioamnionitis and increased HSPA1A correlated with expanded multiorgan dysfunction, severe NE and female sex. CONCLUSIONS: MMP9, PPARG and CCR5 mRNA expression within first days of life correlates with the severity of NE. At 6-12 h, IL8 and HSPA1A are good reporters of clinical variables in NE patients. HSPA1A may have a role in the sexual dimorphism observed in NE. CCR5 is potentially involved in the link between severe NE and chorioamnionitis.

Bidirectional Action of Cenicriviroc, a CCR2/CCR5 Antagonist, Results in Alleviation of Pain-Related Behaviors and Potentiation of Opioid Analgesia in Rats With Peripheral Neuropathy.

Clinical management of neuropathic pain is unsatisfactory, mainly due to its resistance to the effects of available analgesics, including opioids. Converging evidence indicates the functional interactions between chemokine and opioid receptors and their influence on nociceptive processes. Recent studies highlight that the CC chemokine receptors type 2 (CCR2) and 5 (CCR5) seem to be of particular interest. Therefore, in this study, we investigated the effects of the dual CCR2/CCR5 antagonist, cenicriviroc, on pain-related behaviors, neuroimmune processes, and the efficacy of opioids in rats after chronic constriction injury (CCI) of the sciatic nerve. To define the mechanisms of action of cenicriviroc, we studied changes in the activation/influx of glial and immune cells and, simultaneously, the expression level of CCR2, CCR5, and important pronociceptive cytokines in the spinal cord and dorsal root ganglia (DRG). We demonstrated that repeated intrathecal injections of cenicriviroc, in a dose-dependent manner, alleviated hypersensitivity to mechanical and thermal stimuli in rats after sciatic nerve injury, as measured by von Frey and cold plate tests. Behavioral effects were associated with the beneficial impact of cenicriviroc on the activation/influx level of C1q/IBA-1-positive cells in the spinal cord and/or DRG and GFAP-positive cells in DRG. In parallel, administration of cenicriviroc decreased the expression of CCR2 in the spinal cord and CCR5 in DRG. Concomitantly, we observed that the level of important pronociceptive factors (e.g., IL-1beta, IL-6, IL-18, and CCL3) were increased in the lumbar spinal cord and/or DRG 7 days following injury, and cenicriviroc was able to prevent these changes. Additionally, repeated administration of this dual CCR2/CCR5 antagonist enhanced the analgesic effects of morphine and buprenorphine in neuropathic rats, which can be associated with the ability of cenicriviroc to prevent nerve injury-induced downregulation of all opioid receptors at the DRG level. Overall, our results suggest that pharmacological modulation based on the simultaneous blockade of CCR2 and CCR5 may serve as an innovative strategy for the treatment of neuropathic pain, as well as in combination with opioids.

Differential regulatory effects of chemotherapeutic protocol on CCL3_CCL4_CCL5/CCR5 axes in acute myeloid leukemia patients with monocytic lineage.

AIMS: AML (Acute myeloid leukemia) is characterized as a heterogeneous cancer. Chemokines play fundamental roles in the onset, progression cellular, migration, survival and improvement of AML therapy outcomes. The CCR5 receptors together with their ligands have indirect effects on the progression of cancer. In the present study, we have decided to investigate the impact of chemotherapy on the expression of CCR5 and its related ligands (CCL5, CCL4 and CCL3). MAIN METHODS: In this study, peripheral blood and bone marrow specimens were collected prior and post the first stage of (7 + 3) chemotherapy from 25 AML-M4/M5 patients. The expression of CCR by Lymphocytes in peripheral blood was examined by flow cytometry and QRT-PCR. The serum levels of chemokines were measured by ELISA. KEY FINDINGS: There was not observed leukemic blast cells in peripheral blood smear at post first stage of chemotherapy. We found that the expression of CCR5 was attenuated in patients post the first stage of chemotherapy and the healthy control subjects. We have also observed that the serum levels of chemokines were elevated in AML patients prior to chemotherapy. Although in post-chemotherapy stage, only CCL3 was found to reach to the baseline level, CCL5 and CCL4 have not returned to the basal level and were significantly higher than healthy control subjects. SIGNIFICANCE: The current chemotherapy protocol was not able to completely inhibit CCL5 and CCL4. In conclusion, our findings in harmony with previous studies suggest that inhibition of chemokines along with chemotherapy in AML patients may aid therapy.

Transcriptional down-regulation of ccr5 in a subset of HIV+ controllers and their family members.

HIV +Elite and Viremic controllers (EC/VCs) are able to control virus infection, perhaps because of host genetic determinants. We identified 16% (21 of 131) EC/VCs with CD4 +T cells with resistance specific to R5-tropic HIV, reversed after introduction of ccr5. R5 resistance was not observed in macrophages and depended upon the method of T cell activation. CD4 +T cells of these EC/VCs had lower ccr2 and ccr5 RNA levels, reduced CCR2 and CCR5 cell-surface expression, and decreased levels of secreted chemokines. T cells had no changes in chemokine receptor mRNA half-life but instead had lower levels of active transcription of ccr2 and ccr5, despite having more accessible chromatin by ATAC-seq. Other nearby genes were also down-regulated, over a region of ~500 kb on chromosome 3p21. This same R5 resistance phenotype was observed in family members of an index VC, also associated with ccr2/ccr5 down-regulation, suggesting that the phenotype is heritable.

Dopamine and its receptors play a role in the modulation of CCR5 expression in innate immune cells following exposure to Methamphetamine: Implications to HIV infection.

The Human Immunodeficiency Virus (HIV) infects cells in the Central Nervous System (CNS), where the access of antiretrovirals and antibodies that can kill the virus may be challenging. As a result of the early HIV entry in the brain, infected individuals develop inflammation and neurological deficits at various levels, which are aggravated by drugs of abuse. In the non-human primate model of HIV, we have previously shown that drugs of abuse such as Methamphetamine (Meth) increase brain viral load in correlation with a higher number of CCR5-expressing myeloid cells. CCR5 is a chemokine receptor that may be involved in increasing inflammation, but also, it is a co-receptor for viral entry into target cells. CCR5-expressing myeloid cells are the main targets of HIV in the CNS. Thus, the identification of factors and mechanisms that impact the expression of CCR5 in the brain is critical, as changes in CCR5 levels may affect the infection in the brain. Using a well-characterized in vitro system, with the THP1 human macrophage cell line, we have investigated the hypothesis that the expression of CCR5 is acutely affected by Meth, and examined pathways by which this effect could happen. We found that Meth plays a direct role by regulating the abundance and nuclear translocation of transcription factors with binding sites in the CCR5 promoter. However, we found that the main factor that modifies the CCR5 gene promoter at the epigenetic level towards transcription is Dopamine (DA), a neurotransmitter that is produced primarily in brain regions that are rich in dopaminergic neurons. In THP1 cells, the effect of DA on innate immune CCR5 transcription was mediated by DA receptors (DRDs), mainly DRD4. We also identified a role for DRD1 in suppressing CCR5 expression in this myeloid cell system, with potential implications for therapy. The effect of DA on innate immune CCR5 expression was also detectable on the cell surface during acute time-points, using low doses. In addition, HIV Tat acted by enhancing the surface expression of CCR5, in spite of its poor effect on transcription. Overall, our data suggests that the exposure of myeloid cells to Meth in the context of presence of HIV peptides such as Tat, may affect the number of HIV targets by modulating CCR5 expression, through a combination of DA-dependent and-independent mechanisms. Other drugs that increase DA may affect similar mechanisms. The implications of these epigenetic and translational mechanisms in enhancing HIV infection in the brain and elsewhere are demonstrated.

[Protection of Lymphocytes Against HIV using Lentivirus Vector Carrying a Combination of TRIM5α-HRH Genes and microRNA Against CCR5].

Gene therapy is considered a promising approach to treating infections caused by human immunodeficiency virus (HIV). One strategy is to introduce antiviral genes into cells in order to impart resistance to HIV. In this work, the antiviral activity of new anti-HIV lentiviral vector pT has been studied. The vector carries a combination that consists of two identical artificial miRNA mic13lg and the TRIM5α-HRH gene. Two mic13lg microRNAs suppress the expression of the CCR5 gene, which encodes the HIV coreceptor and, thus, prevents the penetration of R5-tropic HIV strains into the cell. It has been shown that pT effectively inhibits the expression of CCR5 in both the HT1080 CCR5-EGFP model cell line and in human primary lymphocytes. The second line of protection against R5- and X4-tropic HIV is provided by the TRIM5α-HRH protein, which binds virus capsids after the virus enters the cell. Indeed, when infecting cells of the SupT1 line, which contains four copies of the vector per cell, with the X-4 tropic HIV, more than 1000-fold suppression of viral replication has been observed. The process of generation of the pT vector and conditions of transduction of CD4^(+) lymphocytes were optimized for testing the antiviral activity of the vector on primary human lymphocytes. As a result, the transduction efficiency for the pT vector was 28%. After infection with the R5-tropic strain of the virus, the survival of cells in the culture of lymphocytes with the vector was significantly higher than in the control. However, the complete suppression of HIV replication was not achieved, presumably due to the inadequate fraction of cells that carry the vector in culture. In the future, it is planned to find the best way to enrich the lymphocyte culture with modified cells to increase resistance to HIV.

Reversing HIV latency via sphingosine-1-phosphate receptor 1 signaling.

OBJECTIVE: In this study, we looked for a new family of latency reversing agents. DESIGN: We searched for G-protein-coupled receptors (GPCR) coexpressed with the C-C chemokine receptor type 5 (CCR5) in primary CD4 T cells that activate infected cells and boost HIV production. METHODS: GPCR coexpression was unveiled by reverse transcriptase-PCR. We used fluorescence resonance energy transfer to analyze the dimerization with CCR5 of the expressed GPCR. Viral entry was measured by flow cytometry, reverse transcription by quantitative PCR, nuclear factor-kappa B translocation by immunofluorescence, long terminal repeat activation using a gene reporter assay and viral production by p24 quantification. RESULTS: Gαi-coupled sphingosine-1-phophate receptor 1 (S1P1) is highly coexpressed with CCR5 on primary CD4 T cells and dimerizes with it. The presence of S1P1 had major effects neither on viral entry nor on reverse transcription. Yet, S1P1 signaling induced NFκB activation, boosting the expression of the HIV LTR. Consequently, in culture medium containing sphingosine-1-phophate, the presence of S1P1 enhanced the replication of a CCR5-, but also of a CXCR4-using HIV-1 strain. The S1P1 ligand FTY720, a drug used in multiple sclerosis treatment, inhibited HIV-1 productive infection of monocyte-derived dendritic cells and of severe combined immunodeficiency mice engrafted with human peripheral blood mononuclear cells. Conversely, S1P1 agonists were able to force latently infected peripheral blood mononuclear cells and lymph node cells to produce virions in vitro. CONCLUSION: Altogether these data indicate that the presence of S1P1 facilitates HIV-1 replicative cycle by boosting viral genome transcription, S1P1 antagonists have anti-HIV effects and S1P1 agonists are HIV latency reversing agents.

CCR5 promoter activity correlates with HIV disease progression by regulating CCR5 cell surface expression and CD4 T cell apoptosis.

CCR5 is the major co-receptor for HIV and polymorphisms in the CCR5 gene as well as promoter region that alter cell surface expression have been associated with disease progression. We determined the relationship between CCR5 promoter polymorphisms and CD4 decline and other immunopathological features like immune activation and CD4+ T cell apoptosis in HIV patients. CCR5 promoter haplotype HHC was significantly associated with higher CD4 counts in patients. The relative promoter activity (RPA) of each haplotype was determined in vitro and combined promoter activity based on both alleles (CRPA) was assigned to each patients. Interestingly, CCR5 CRPA correlated inversely with CD4 counts and CD4:CD8 ratio specifically in viremic patients. In normal individuals, the CRPA correlated with the number of CCR5+ CD4+ T cells in the peripheral blood suggesting an effect on CCR5 expression. In a subset of high viremic patients harboring R5 tropic HIV, there was a strong correlation between CCR5 CRPA and both CD4 counts and CD4 T cell apoptosis. Our study demonstrates that, CCR5 promoter polymorphisms correlate with CD4 T cell loss possibly by regulating CD4 T cell apoptosis in HIV patients. Furthermore, assigning CRPAs to each patient is a new method of translating genotype to phenotype.

Ontogeny of CD4+ T Lymphocytes With Phenotypic Susceptibility to HIV-1 During Exclusive and Nonexclusive Breastfeeding in HIV-1-Exposed Ugandan Infants.

Background: Among infants exposed to human immunodeficiency virus (HIV) type 1, mixed breastfeeding is associated with higher postnatal HIV-1 transmission than exclusive breastfeeding, but the mechanisms of this differential risk are uncertain. Methods: HIV-1-exposed Ugandan infants were prospectively assessed during the first year of life for feeding practices and T-cell maturation, intestinal homing (ß7hi), activation, and HIV-1 coreceptor (CCR5) expression in peripheral blood. Infants receiving only breast milk and those with introduction of other foods before 6 months were categorized as exclusive and nonexclusive, respectively. Results: Among CD4+ and CD8+ T cells, the expression of memory, activation, and CCR5 markers increased rapidly from birth to week 2, peaking at week 6, whereas cells expressing the intestinal homing marker increased steadily in the central memory (CM) and effector memory T cells over 48 weeks. At 24 weeks, when feeding practices had diverged, nonexclusively breastfed infants showed increased frequencies and absolute counts of ß7hi CM CD4+ and CD8+ T cells, including the HIV-1-targeted cells with CD4+ß7hi/CCR5+ coexpression, as well as increased activation. Conclusions: The T-cell phenotype associated with susceptibility to HIV-1 infection (CCR5+, gut-homing, CM CD4+ T cells) was preferentially expressed in nonexclusively breastfed infants, a group of infants at increased risk for HIV-1 acquisition.

Establishment of mouse leukemia cell lines expressing human CD4/CCR5 using lentiviral vectors.

A low-cost rodent model of HIV infection and which presents high application value is an effective tool to investigate HIV infection and pathogenesis. However, development of such a small animal model has been hampered by the unsuitability of rodent cells for HIV-1 replication given that the retrovirus HIV-1 has high selectivity to its host cell. Our study used the mouse leukemia cell lines L615 and L1210 that were induced by murine leukemia virus and transfected with hCD4/CCR5 loaded-lentiviral vector. Lentiviral vectors containing the genes hCD4/CCR5 under the transcriptional control of cytomegalovirus promoter were designed. Transfection efficiencies of human CD4 and CCR5 in L615 and L1210 cells were analyzed by quantitative real-time polymerase chain reaction (RT-PCR) and Western blot assay. Results showed that hCD4 and CCR5 proteins were expressed on the cell surface, demonstrating that the L615 and L1210 cells were humanized and that they possess the characteristics necessary for HIV infection of human host cells. Moreover, the sensitivity of human CD4/CCR5 transgenic mouse cells to HIV infection was confirmed by RT-PCR and ELISA. Mouse leukemia cell lines that could express hCD4 and CCR5 were thus established to facilitate normal entry of HIV-1 so that a human CD4/CCR5 transgenic mice cell model can be used to investigate the transmission and pathogenesis of HIV/AIDS and potential antiviral drugs against this disease.

Cell-Delivered Entry Inhibitors for HIV-1: CCR5 Downregulation and Blocking Virus/Membrane Fusion in Defending the Host Cell Population.

HIV-1 infection requires the presence of the CD4 receptor on the target cell surface and a coreceptor, predominantly CC-chemokine receptor 5 (CCR5). It has been shown that individuals who are homozygous for a defective CCR5 gene are protected from HIV-1 infection. A novel self-inactivating lentiviral vector LVsh5/C46 (Cal-1) has been engineered to block HIV-1 infection with two viral entry inhibitors, conferring resistance to HIV-1 infection from both CCR5 and CXCR4 tropic strains. Cal-1 encodes a short hairpin RNA (sh5) to downregulate CCR5 and C46, an HIV-1 fusion inhibitor. Gene therapy by Cal-1 is aimed at transducing CD4+ T cells and CD34+ hematopoietic stem/progenitor cells in an autologous transplant setting. Pre-clinical safety and efficacy studies in vitro and in vivo (humanized mouse model and nonhuman primates) have shown that Cal-1 is safe with no indication of any toxicity risk and acts to decrease viral load and increase CD4 counts. Two clinical trials are underway using Cal-1: a phase I/II study to assess safety and feasibility in an adult HIV-1-positive population not on antiretroviral therapy (ART); and a second Fred Hutchinson Investigator Initiated phase I study to assess safety and feasibility in adults with HIV-1-associated non-Hodgkin or Hodgkin lymphoma.

Immune response genes receptors expression and polymorphisms in relation to multiple sclerosis susceptibility and response to INF-ß therapy.

Interferon (IFN)-ß is one of the disease modifying drugs used in the treatment of multiple sclerosis. A predictive marker that indicates good or poor response to the treatment is highly desirable. We aimed to investigate the relation between the immune response genes receptors (IFNAR1, IFNAR2, and CCR5) expression and their polymorhic variants and multiple sclerosis (MS) susceptibility as well as the response to IFN-ß therapy. The immune response genes receptors expression and genotyping were analyzed in 80 patients with MS, treated with IFN-ß and in 110 healthy controls. There was a significant decrease of IFNAR1 and IFNAR2 mRNA expression and a significant increase of CCR5 mRNA expression in MS patients compared with the control group. Also, the level of IFNAR1, IFNAR2, and CCR5 mRNA expression was found to be significantly lower in the responders than nonresponders. Carriers of IFNAR1 18417 C/C genotype and C allele had an increased risk of developing MS. There was a significant relation between CCR5 Δ32 allele and IFN-ß treatment response in MS patients. Our results highlighted the significance of IFNAR and CCR5 genes in multiple sclerosis risk and the response to IFN-ß therapy. © 2016 IUBMB Life, 68(9):727-734, 2016.

Phenotypic Coreceptor Tropism in Perinatally HIV-infected Youth Failing Antiretroviral Therapy.

BACKGROUND: Perinatally HIV-infected (PHIV) children and youth are often heavily treatment-experienced, with resultant antiretroviral resistance and limited treatment options. For those with virologic failure (VF), new agents such as CCR5 (R5) antagonists may be useful; however, reports of R5 antagonist susceptibility in children have mostly relied on genotypic testing, which may not accurately reflect the phenotypic tropism of the viral populations. We characterized phenotypic coreceptor usage among PHIV children and youth with VF on antiretroviral treatment to identify predictors of CXCR4 (X4) tropism which preclude R5 antagonist use. METHODS: Plasma samples with >1000 HIV RNA copies/mL were obtained from 73 PHIV antiretroviral treatment-treated children and youth (age 9-21 years) enrolled in the multicenter Pediatric HIV/AIDS Cohort Study. Samples were analyzed using the Trofile phenotypic assay. Multiple logistic regression was performed to identify factors associated with detectable X4 tropism. RESULTS: Tropism results were obtained for 59 (81%) of the 73 children and youth; 32 (54%) had X4-tropism. Persistent viremia (≥80% of HIV RNA measurements >400 copies/mL) was associated with detectable X4 tropism (adjusted odds ratio: 6.6, 95% confidence interval: 1.4, 31.4), while longer cumulative nucleoside reverse transcriptase inhibitor use was associated with lower risk of X4 tropism (adjusted odds ratio: 0.6, 95% confidence interval: 0.5, 0.9). CONCLUSIONS: Using a phenotypic assay, >50% of PHIV children and youth with VF had X4 tropism, similar to that in experienced adults, and higher than the 30% reported for children using genotypic assays. Persistent viremia and shorter nucleoside reverse transcriptase inhibitor exposure are associated with X4-tropism in children and youth and may help target phenotypic testing to those most likely to benefit from R5 antagonist.

In vivo effects of methamphetamine on HIV-1 replication: A population-based study.

BACKGROUND: Although a number of in vitro studies have shown that methamphetamine (METH) can increase HIV-1 replication in human immune cells, a direct link between METH use and HIV-1 pathogenesis remains to be determined among HIV-1 patients. METHODS: According to the status of METH use and HIV-1 infection, we enrolled participants and divided them into four groups: METH+HIV+, METH-HIV+, METH+HIV-, and METH-HIV-. HIV viral loads and HIV-1-related cellular factors were measured and compared among different groups. RESULTS: A total of 60 participants were enrolled into this study, 15 within each group. HIV viral loads in METH+HIV+ group were significantly higher than those in METH-HIV+ group, while CD4+ T cell counts had an inverse trend between the two groups (p<0.05). METH users or HIV-1 infected patients had lower CCR5+, CXCR4+ percentages in CD4+ T cells than METH-HIV- subjects (p<0.01). However, METH use had little effect on CD3 expression in PBMCs and the levels of MIP-1α, MIP-1ß and IL-6 in PBMCs or plasma, which were increased by HIV-1 infection with or without METH. TLR-9 and IFN-α levels in PBMCs of METH users with or without HIV infection were higher than non-METH users (p<0.05). CONCLUSIONS: METH use is associated with higher viral loads and lower CD4+ T cell counts in HIV-infected individuals. This finding may be mediated by activation of innate immunity (TLR-9, IFN-α) by METH use.

Inhibition of HIV Expression and Integration in Macrophages by Methylglyoxal-Bis-Guanylhydrazone.

UNLABELLED: Macrophages are a target for infection with HIV and represent one of the viral reservoirs that are relatively resistant to current antiretroviral drugs. Here we demonstrate that methylglyoxal-bis-guanylhydrazone (MGBG), a polyamine analog and potent S-adenosylmethionine decarboxylase inhibitor, decreases HIV expression in monocytes and macrophages. MGBG is selectively concentrated by these cells through a mechanism consistent with active transport by the polyamine transporter. Using a macrophage-tropic reporter virus tagged with the enhanced green fluorescent protein, we demonstrate that MGBG decreases the frequency of HIV-infected cells. The effect is dose dependent and correlates with the production of HIV p24 in culture supernatants. This anti-HIV effect was further confirmed using three macrophage-tropic primary HIV isolates. Viral life cycle mapping studies show that MGBG inhibits HIV DNA integration into the cellular DNA in both monocytes and macrophages. IMPORTANCE: Our work demonstrates for the first time the selective concentration of MGBG by monocytes/macrophages, leading to the inhibition of HIV-1 expression and a reduction in proviral load within macrophage cultures. These results suggest that MGBG may be useful in adjunctive macrophage-targeted therapy for HIV infection.

Codelivery of Envelope Protein in Alum with MVA Vaccine Induces CXCR3-Biased CXCR5+ and CXCR5- CD4 T Cell Responses in Rhesus Macaques.

The goal of an HIV vaccine is to generate robust and durable protective Ab. Vital to this goal is the induction of CD4(+) T follicular helper (TFH) cells. However, very little is known about the TFH response to HIV vaccination and its relative contribution to magnitude and quality of vaccine-elicited Ab titers. In this study, we investigated these questions in the context of a DNA/modified vaccinia virus Ankara SIV vaccine with and without gp140 boost in aluminum hydroxide in rhesus macaques. In addition, we determined the frequency of vaccine-induced CD4(+) T cells coexpressing chemokine receptor, CXCR5 (facilitates migration to B cell follicles) in blood and whether these responses were representative of lymph node TFH responses. We show that booster modified vaccinia virus Ankara immunization induced a distinct and transient accumulation of proliferating CXCR5(+) and CXCR5(-) CD4 T cells in blood at day 7 postimmunization, and the frequency of the former but not the latter correlated with TFH and B cell responses in germinal centers of the lymph node. Interestingly, gp140 boost induced a skewing toward CXCR3 expression on germinal center TFH cells, which was strongly associated with longevity, avidity, and neutralization potential of vaccine-elicited Ab response. However, CXCR3(+) cells preferentially expressed the HIV coreceptor CCR5, and vaccine-induced CXCR3(+)CXCR5(+) cells showed a moderate positive association with peak viremia following SIV251 infection. Taken together, our findings demonstrate that vaccine regimens that elicit CXCR3-biased TFH cell responses favor Ab persistence and avidity but may predispose to higher acute viremia in the event of breakthrough infections.