Evaluation of a Quantitative Real-Time PCR Assay to Measure HIV-Specific Mucosal CD8+ T Cell Responses in the Cervix

Chege, Duncan; Chai, Yijie; Huibner, Sanja; McKinnon, Lyle R.; Wachihi, Charles; Kimani, Murungaru; Jaoko, Walter; Kimani, Joshua; Ball, T. Blake; Plummer, Francis A.; Kaul, Rupert; Rebbapragada, Anu

doi:10.1371/journal.pone.0013077

Cited by 17 publications

(21 citation statements)

References 41 publications

(67 reference statements)

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“…PCR fragments were cloned into pGEM-T vector (Promega, Madison, WI) and used to generate standard curves for corresponding genes. The expression of glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was used for normalization of gene expression (58). All amplification reactions were performed in duplicate and an average mRNA quantity is expressed as copy number per 1,000 copies of GAPDH.…”

Section: Methodsmentioning

confidence: 99%

The Selective Serotonin Reuptake Inhibitor Citalopram Decreases Human Immunodeficiency Virus Receptor and Coreceptor Expression in Immune Cells

Greeson

Gettes

Spitsin

et al. 2016

Biological Psychiatry

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Background This study investigated whether a selective serotonin reuptake inhibitor (SSRI), citalopram, downregulates the expression of HIV receptor (CD4) and coreceptors (CCR5 and CXCR4) on peripheral blood mononuclear cells (PBMC) and macrophages ex vivo as a potential mechanism of reducing susceptibility to HIV infection. Methods The sample included 150 participants ages 18–58 (59% women, 65% African American, 61% depressed). Monocyte depleted PBMC were treated with phytohemagglutinin (PHA) for 72-hours and then cultured in the presence of IL-2 with vehicle control or SSRI (10−6 M) for 2-hours. To generate monocyte derived macrophages (MDM), monocytes were cultured for 7-days, after which either vehicle control or SSRI (10−6 M) was added for 2-hours. RNA was then collected from both cell types, and CD4, CCR5 and CXCR4 mRNA expression was measured by real-time PCR. Results In PBMC, SSRI treatment decreased expression of CD4 (p=.009), CCR5 (p=.008), and CXCR4 (p<.0001). In MDM, SSRI decreased expression of CD4 (p<.0001) and CXCR4 (p=.0003), but not CCR5 (p=.71). The suppressive effects of SSRI on receptor expression did not differ as a function of depression diagnosis or depressive symptom severity. Conclusions SSRI treatment at a physiologic dose decreased CD4, CCR5 and CXCR4 expression on PBMC and macrophages ex vivo. These findings suggest that SSRI treatment, independent of depression status, downregulates HIV receptor and coreceptor expression and thus may reduce susceptibility of immune cells to HIV infection and decrease inflammation. If clinical trials confirm the present findings, ultimately there may be a role for using SSRI treatment adjunctively in HIV/AIDS.

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Section: Methodsmentioning

confidence: 99%

The Selective Serotonin Reuptake Inhibitor Citalopram Decreases Human Immunodeficiency Virus Receptor and Coreceptor Expression in Immune Cells

Greeson

Gettes

Spitsin

et al. 2016

Biological Psychiatry

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“…Samples were processed and assayed via quantitative real-time PCR (qPCR) as previously described (24). RNA was extracted from cell pellets, and genomic DNA was eliminated using the Qiagen RNeasy Plus Kit (Qiagen).…”

Section: Intracellular Cytokine Stainingmentioning

confidence: 99%

“…Specific primers were designed to target IL-17A (forward, 59-CATGAACTCTGTCCCCATCC-39; reverse, 59-CCCACGGACACCAGTATCTT-39) and IL-22 (forward, 59-TGCATTT-GACCAGAGCAAAG-39; reverse, 59-AGTTTGGCTTCCCATCTTCC-39) mRNA induction. SYBR green fluorescent dye was used to detect amplification under previously published amplification conditions (24). All reactions were run in triplicate.…”

Section: Intracellular Cytokine Stainingmentioning

confidence: 99%

“…Quantitative PCR values crossing threshold were obtained during the exponential amplification phase using SDS 2.3 Software (Applied Biosystems). The housekeeping genes GAPDH and b-actin were used to assess target gene expression in the cervix and blood, respectively (24). Gene quantities were calculated from standard curves in arbitrary units and normalized by dividing the amplified gene target by the housekeeping gene for each sample and reported as the normalized gene induction ratio.…”

Section: Intracellular Cytokine Stainingmentioning

confidence: 99%

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Characterization of a Human Cervical CD4+ T Cell Subset Coexpressing Multiple Markers of HIV Susceptibility

McKinnon

Nyanga

Chege

et al. 2011

The Journal of Immunology

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161

158

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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 α4β7 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 α4β7 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 α4β7 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.

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“…The GAPDH gene was chosen for its constitutive expression pattern because it is stably expressed in PBMCs according to Chege et al [33]. GAPDH was found to be stably expressed under our laboratory conditions in PBMC cells.…”

Section: Methodsmentioning

confidence: 99%

Changes in Gene Expression in PBMCs Profiles of PPARα Target Genes in Obese and Non-Obese Individuals During Fasting

Felicidade¹,

Marcarini

Carreira³

et al. 2014

Ann Nutr Metab

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Background: The prevalence of obesity has risen dramatically and the World Health Organization estimates that 700 million people will be obese worldwide by 2015. Approximately, 50% of the Brazilian population above 20 years of age is overweight, and 16% is obese. Aim: This study aimed to evaluate the differences in the expression of PPARα target genes in human peripheral blood mononuclear cells (PBMCs) and free fatty acids (FFA) in obese and non-obese individuals after 24 h of fasting. We first presented evidence that Brazilian people exhibit expression changes in PPARα target genes in PBMCs under fasting conditions. Methods: Q-PCR was utilized to assess the mRNA expression levels of target genes. Results: In both groups, the FFA concentrations increased significantly after 24 h of fasting. The basal FFA mean concentration was two-fold higher in the obese group compared with the non-obese group. After fasting, all genes evaluated in this study showed increased expression levels compared with basal expression in both groups. Conclusion: However, our results reveal no differences in gene expression between the obese and non-obese, more studies are necessary to precisely delineate the associated mechanisms, particularly those that include groups with different degrees of obesity and patients with diabetes mellitus type 2 because the expression of the main genes that are involved in β-oxidation and glucose level maintenance are affected by these factors. © 2014 S. Karger AG, Basel

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Evaluation of a Quantitative Real-Time PCR Assay to Measure HIV-Specific Mucosal CD8+ T Cell Responses in the Cervix

Cited by 17 publications

References 41 publications

The Selective Serotonin Reuptake Inhibitor Citalopram Decreases Human Immunodeficiency Virus Receptor and Coreceptor Expression in Immune Cells

The Selective Serotonin Reuptake Inhibitor Citalopram Decreases Human Immunodeficiency Virus Receptor and Coreceptor Expression in Immune Cells

Characterization of a Human Cervical CD4+ T Cell Subset Coexpressing Multiple Markers of HIV Susceptibility

Changes in Gene Expression in PBMCs Profiles of PPARα Target Genes in Obese and Non-Obese Individuals During Fasting

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