Jack E. Bodwell’s research while affiliated with Geisel School of Medicine at Dartmouth and other places

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Publications (60)


Figure 1. Evaluation of the protective effect of secretions from endometrial epithelial cells following preincubation with TFV or TAF on HIV infection of blood CD4+ T cells. (a) Dose-dependent increase in intracellular TFV-DP levels following treatment of purified polarized endometrial (EM) epithelial cells with TAF (white bars) or TFV (black bars) for 24 hr. Bars and horizontal lines represent mean and SEM respectively from triplicate cultures of cells from a representative patient. Values are expressed as fmol/million cells. (b,c) Levels of HIV infection (released p24) in CD4+ T cells after incubation with apical and basolateral CM from EM epithelial cells pre-treated with TFV (3277 µM) or TAF (10 µM) as described in Methods. (b) Representative example of secreted p24 levels from a single patient run in quadruplicate. Columns and horizontal lines represent the mean and SEM respectively. (c) Secreted p24 values are normalized to the infection of CD4+ T cells in the absence of CM (media control) which is set to 100%. Each circle represents an individual patient (n = 10) and horizontal lines represent the mean and SEM. Blood CD4+ T cells were isolated from 4 donors. *p < 0.05. **p < 0.01.
Figure 2. Protective anti-HIV effect of basolateral secretions from endometrial (EM) epithelial cells following treatment with TFV or TAF correlates with epithelial levels of intracellular TFV-DP. (a) HIV infection levels in CD4+ T cells after incubation with CM from EM epithelial cells pre-treated with TFV or TAF. EM epithelial cells were pre-treated with either TFV (3277 µM) or TAF (10 µM) for 24 hr, after which ARVs were washed out of cell culture. Cells were then incubated with fresh media that was replaced daily for an initial 24 hr (Day 1), a second 24 hr period (Day 2), and a third 24 hr period (Day 3) after which basolateral conditioned media (CM) was collected. Activated CD4+ T cells were treated for 24 hr with CM recovered at Day 1, 2, and 3. Following washout of CM, CD4+ T cells were infected after which secreted p24 levels measured by p24 ELISA after 5 days as described in Methods. Data are normalized to the infection of CD4+ T cells in the absence of CM (media control) which is set to 100% (dashed line). (n = 4 individual patients). Columns and horizontal lines represent the mean and SEM respectively. *p < 0.05. **p < 0.01. ***p < 0.001. (b and c) Intracellular TFV-DP levels in EM epithelial cells are inversely related to HIV infection of CD4+ T cells treated with EM epithelial CM. Intracellular TFV-DP levels in EM epithelial cells were measured by LC-MS/MS following TFV (b) and TAF (c) treatment (24 hr), followed by incubation for 24 hr intervals with fresh media collection on Day 1, Day 2, and Day 3. CM was collected at each time point along with cell recovery to measure HIV infection as described in Methods. Circles and horizontal lines represent the mean and SEM from triplicates in a single representative patient. (d) Time course of the lack of an effect on transepithelial resistance (TER) of polarized epithelial cells treated with TFV (3277 µM) for 24 hr after which ARV was washed out of cell culture. Cells were then incubated with fresh media that was replaced daily for 3 days. (e) Lack of an effect of TFV and TAF on epithelial cell viability. Polarized EM epithelial cells were apically and basolaterally treated with TFV (3277 µM) or TAF (10 µM) for 24 hr prior to washout and measurement of viability on days 1, 2 and 3. Cell viability was tested with CellTiter 96 AQ ueous One Solution cell proliferation assay. The bars represent the mean and SEM of triplicate cell inserts. (f) Intracellular accumulation of TFV-DP on endometrial epithelial cells following apical or basolateral incubation with TFV or TAF. Intracellular TFV-DP levels were measured by LC-MS/MS in purified polarized endometrial epithelial cells treated with TFV (3277 µM) or TAF (10 µM) apical or basolateral for 24 hr. Values are expressed as fmol/million cells. The bars represent the mean and SEM from 3 patients. **p < 0.01.
Figure 3. Secretions from endocervical and ectocervical epithelial cells treated with TFV or TAF inhibit HIV infection of CD4+ T cells. Apical and basolateral conditioned media (CM) were collected from endocervix (CX) and ectocervix (ECX) epithelial cells pre-treated with TFV (3277 µM) or TAF (10 µM) for 24 hr. CM were collected 24 hr post ARV washout and incubation with fresh media; CM was incubated with activated CD4+ T cells prior to HIV infection. Secreted p24 levels in the culture media after 5 days of infection were measured by p24 ELISA as described in Methods. Data are normalized to the infection of CD4+ T cells in the absence of CM (media control) which is set to 100% using (a) CM from CX epithelial cells from 9 patients, (b) CM from ECX epithelial cells from 4 patients. (c) Time course of HIV protection of CD4+ T cells by basolateral CM from CX (dark circles) and ECX (open circles) epithelial cells pre-treated with either ARVs for 24 hr prior to wash out. Cells were then incubated with fresh media that was replaced each day for 3 days, basolateral CM was collected daily. Activated CD4+ T cells were treated for 24 hr with CM. Following washout of CM, CD4+ T cells were infected after which secreted p24 levels measured by p24 ELISA as described in Methods. Data are normalized to the infection of CD4+ T cells in the absence of CM (media control) which is set to 100% (dashed line). (n = 7 individual patients). Columns and horizontal lines represent the mean and SEM respectively. *p < 0.05. **p < 0.01.
Figure 4. Effect of fibroblasts and endometrial (EM) epithelial cells in decreased HIV infection of CD4+ T cells. Conditioned media (CM) was collected from (a) endometrium (EM), (b) CX/ECX fibroblasts pre-treated with TFV (3277 µM) or TAF (10 µM) for 24 hr. CM were collected 24 hr post ARV washout and incubation with fresh media; CM was incubated with activated CD4+ T cells prior to HIV infection. Secreted p24 levels in the culture media after 5 days of infection were measured by p24 ELISA as described in Methods. CM was collected from EM fibroblasts of 5 patients while CM from CX (dark circle) and ECX (open circle) fibroblasts was from 3 matched patients. Each circle represents an individual patient. Data are normalized in (a,b) to the infection of CD4+ T cells in the absence of CM (media control) and set to 100%. Each circle represents a different patient. Blood CD4+ T cells were isolated from 4 donors. Horizontal lines represent the mean and SEM respectively. *p < 0.05. **p < 0.01. (c) Polarized EM epithelial cells were apically treated with TFV or TAF for 24 hr, after which basolateral CM was immediately collected (ARV-Incubation). Following rinsing, cells were incubated with fresh media (no TFV or TAF) for an additional 24 hr prior to CM collection (Post-Incubation 24 hr). Activated CD4+ T cells from blood were incubated with CM for 24 hr and washed and infected with HIV for 2 hr. Secreted viral p24 levels were measured after 5 days of infection (Methods). Data are normalized to the infection of CD4+ T cells in the absence of CM (media control) which is set to 100%. Bars represent EM tissues from 3 patients. (d) Effect of epithelial cells and fibroblasts on prevention of HIV infection. Polarized EM epithelial cells grown in the upper chamber of cell inserts were treated apically with TFV (3277 µM) or TAF (10 µM) for 24 hr in the presence of confluent fibroblasts (SF) from the same donor grown in the lower chamber (no cell contact). Following incubation, epithelial cells and stromal fibroblasts (EC + SF), basolateral CM was collected for analysis (ARV-Incubation). Following extensive washing to remove extracellular ARVs (PostIncubation), epithelial cells alone (EC) and stromal fibroblasts alone (SF) were incubated in fresh media for 24 hr, after which CM were collected. CD4+ T cell protection against HIV infection for each CM was measured as described in Methods. Data are normalized to the infection of CD4+ T cells in the absence of CM (media control). Bars represent 4 patients. Blood CD4+ T cells were isolated from 2 donors. Column and horizontal lines represents the mean and SEM respectively. *p < 0.05. **p < 0.01.
Figure 5. Blockade of Multidrug resistance-associated proteins (MRP) leads in part to decreased anti-HIV activity in basolateral epithelial CM via increased intracellular accumulation of TFV-DP. (a) MRP (1, 3, 4, 5 and 6) gene expression in epithelial cells from endometrium (EM), endocervix (CX) and ectocervix (ECX). Gene expression was determined by real time RT-PCR and normalized to β-Actin using epithelial cells from 4 patients with matched EM, CX and ECX. Y axis represents gene expression relative to the house keeping gene β-actin. Columns and horizontal lines represent the mean and SEM respectively. *p < 0.05. **p < 0.01. (b) Blockade of MRP transporter function decreases the release of TFV and TAF into the extracellular environment. EM epithelial cells were incubated with TFV (3277 µM) or TAF (10 µM) for 20 hr, followed by the addition of a human MRP-specific inhibitor MK571 (100 µM) to both the apical and basolateral compartments for 4 hr. Following washout, fresh media +/− MK571 (100 uM) was added to both compartments for an additional 24 hr after which the basolateral CM was collected. Activated CD4+ T cells from blood were incubated with basolateral CM for 24 hr prior to HIV infection (Methods). Data were normalized to % of HIV infection of CD4+ T cells infected in the absence of basolateral CM (media control). Each circle represents a different individual patient (n = 6) with dark circles representing basolateral CM collected from cells not treated with MK571, and open circles basolateral CM from cells treated with MK571 Data are normalized to the infection of CD4+ T cells in the absence of CM (media control) and set to 100%. *p < 0.05. (c) Intracellular TFV-DP levels in purified polarized EM epithelial cells increase following incubation with TFV (3277 µM) or TAF (10 µM) for 24 hr in the presence of MK571 (100 µM). Data were normalized to % of intracellular TFV-DP without MK571 in experiments using EM epithelial cells from 4 individual patients. Each circle represents a different patient. Horizontal lines represent the mean and SEM respectively. *p < 0.05.

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Epithelial Cells and Fibroblasts from the Human Female Reproductive Tract Accumulate and Release TFV and TAF to Sustain Inhibition of HIV Infection of CD4+ T cells
  • Article
  • Full-text available

February 2019

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110 Reads

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8 Citations

Zheng Shen

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Marta Rodriguez-Garcia

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Mickey V. Patel

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[...]

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Tenofovir (TFV) treatment of female reproductive tract (FRT) cells results in differential accumulation of intracellular Tenofovir diphosphate (TFV-DP) in different cell types, with greater concentrations in epithelial cells (100-fold) and fibroblasts (10-fold) than in CD4+ T cells. The possibility that TFV-DP accumulation and retention in epithelial cells and fibroblasts may alter TFV availability and protection of CD4+ T cells against HIV infection, prompted us to evaluate TFV and/or Tenofovir alafenamide (TAF) release from FRT cells. Endometrial, endocervical and ectocervical polarized epithelial cells and fibroblasts were pre-loaded with TFV or TAF, and secretions tested for their ability to inhibit HIV infection of activated blood CD4+ T cells. Epithelial cell basolateral secretions (1, 2 and 3 days post-loading), but not apical secretions, suppressed HIV infection of CD4+ T cells, as did secretions from pre-loaded fibroblasts from each site. Intracellular TFV-DP levels in epithelial cells following preloading with TFV or TAF correlated directly with ARV protection of CD4+ T cells from HIV infection. When added apically to epithelial cells, TFV/TAF was released basolaterally, in part through Multidrug Resistant Protein transporters, taken up by fibroblasts and released into secretions to partially protect CD4+ T cells. These findings demonstrate that epithelial cells and fibroblasts release TFV/TAF for use by CD4+ T cells and suggest that the tissue environment plays a major role in the sustained protection against HIV infection.

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Hormonal Contraceptives Differentially Suppress TFV and TAF Inhibition of HIV Infection and TFV-DP in Blood and Genital Tract CD4+ T cells

December 2017

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86 Reads

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10 Citations

HIV prevention research is focused on combining antiretrovirals (ARV) and progestin contraceptives to prevent HIV infection and pregnancy. The possibility that progestins compromise ARV anti-HIV activity prompted us to evaluate the effects of progestins on tenofovir (TFV) and TFV-alafenamide (TAF) on HIV infection and intracellular TFV-diphosphate (TFV-DP) concentrations in blood and genital CD4+ T cells. Following incubation of blood CD4+ T cells with TFV or TAF, Medroxyprogesterone acetate (MPA), but not Levonorgestrel, Norethisterone or progesterone, suppressed the anti-HIV effect of TFV by reducing intracellular TFV-DP, but had no effect on TAF inhibition of infection or TFV-DP. In contrast, with genital CD4+ T cells, MPA suppressed TAF inhibition of HIV infection and lowered of TFV-DP concentrations without affecting TFV protection. These findings demonstrate that MPA selectively compromises TFV and TAF protection in blood and genital CD4+ T cells and suggests that MPA may decrease ARV protection in individuals who use ARV intermittently for prevention.


Tenofovir Inhibits Wound Healing of Epithelial Cells and Fibroblasts from the Upper and Lower Human Female Reproductive Tract

April 2017

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112 Reads

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22 Citations

Disruption of the epithelium in the female reproductive tract (FRT) is hypothesized to increase HIV infection risk by interfering with barrier protection and facilitating HIV-target cell recruitment. Here we determined whether Tenofovir (TFV), used vaginally in HIV prevention trials, and Tenofovir alafenamide (TAF), an improved prodrug of TFV, interfere with wound healing in the human FRT. TFV treatment of primary epithelial cells and fibroblasts from the endometrium (EM), endocervix (CX) and ectocervix (ECX) significantly delayed wound closure. Reestablishment of tight junctions was compromised in EM and CX epithelial cells even after wound closure occurred. In contrast, TAF had no inhibitory effect on wound closure or tight junction formation following injury. TAF accumulated inside genital epithelial cells as TFV-DP, the active drug form. At elevated levels of TAF treatment to match TFV intracellular TFV-DP concentrations, both equally impaired barrier function, while wound closure was more sensitive to TFV. Furthermore, TFV but not TAF increased elafin and MIP3a secretion following injury, molecules known to be chemotactic for HIV-target cells. Our results highlight the need of evaluating antiretroviral effects on genital wound healing in future clinical trials. A possible link between delayed wound healing and increased risk of HIV acquisition deserves further investigation.







Effects of Tenofovir on Cytokines and Nucleotidases in HIV-1 Target Cells and the Mucosal Tissue Environment in the Female Reproductive Tract

October 2014

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51 Reads

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14 Citations

Tenofovir (TFV) is a reverse transcriptase inhibitor used in microbicide Preexposure Prophylaxis trials to prevent HIV infection. Recognizing that changes in cytokine/chemokine secretion and nucleotidase biological activity can influence female reproductive tract (FRT) immune protection against HIV infection, prompted us to test the hypothesis that TFV regulates immune protection in the FRT. Epithelial cells, fibroblasts, CD4(+) T cells and CD14(+) cells were isolated from the Endometrium (Em), Endocervix (Cx) and Ectocervix (Ecx) following hysterectomy. Levels of pro-inflammatory cytokines (MIP3α, IL-8 and TNFα), the expression of specific nucleotidases and nucleotidase biological activity were analyzed in the presence or absence of TFV. TFV influenced mRNA and/or protein cytokines and nucleotidases in a cell- and site-specific manner. TFV significantly enhanced IL-8 and TNFα secretion by epithelial cells from Em and Ecx, but not from Cx. In contrast, in response to TFV, IL-8 secretion significantly decreased in Em and Cx fibroblasts but increased with fibroblasts from the Ecx. When incubated with CD4(+) T cells from the FRT, TFV increased IL-8 (Em and Ecx) and TNFα (Cx and Ecx) secretion. Moreover, when incubated with Em CD14(+) cells, TFV significantly increased MIP3α, IL-8 and TNFα secretion relative to controls. In contrast, nucleotidase biological activity was significantly decreased by TFV in epithelial (Cx) and CD4(+) T cells (Em), but increased in fibroblasts (Em). Our findings indicate that TFV modulates pro-inflammatory cytokines, nucleotidase gene expression and nucleotidase biological activity in epithelial cells, fibroblasts, CD4(+) T and CD14(+) cells at distinct sites within the FRT.


Citations (47)


... Studies with female genital tract (FGT) models have shown that TAF achieves similar protection against HIV infection at concentrations~300-fold lower than TFV [13,14]. However, none has been carried out for insertive sex. ...

Reference:

Pre-Clinical Evaluation of Tenofovir and Tenofovir Alafenamide for HIV-1 Pre-Exposure Prophylaxis in Foreskin Tissue
Epithelial Cells and Fibroblasts from the Human Female Reproductive Tract Accumulate and Release TFV and TAF to Sustain Inhibition of HIV Infection of CD4+ T cells

... ISL-TP competes with the natural nucleotide dATP for incorporation by reverse transcriptase, and some studies suggest that hormonal contraceptives can alter nucleotide metabolism [39][40][41]. To determine whether ENG affected nucleotide metabolism and possibly competition between ISL-TP and dATP, we compared ratios between ISL-TP and dATP among ISL-98 mg-implanted animals before and after ENG-33 mg implantation. ...

Hormonal Contraceptives Differentially Suppress TFV and TAF Inhibition of HIV Infection and TFV-DP in Blood and Genital Tract CD4+ T cells

... The female genital tract (FGT) consists of the uterine cervix, fallopian tubes, vagina and vulva, which perform various functional steps, such as the passage of the spermatozoa during insemination, and the expulsion of the fetus and placenta during delivery, and of the exfoliated endometrium during the menstrual period [1,2]. ...

Tenofovir Inhibits Wound Healing of Epithelial Cells and Fibroblasts from the Upper and Lower Human Female Reproductive Tract

... Different studies have been carried out in different models, such as peripheral blood mononuclear cells (PBMCs) and mouse cells, investigating the inflammatory properties of tenofovir (Fig. 3) [65,[68][69][70][71]. These studies formulated the same conclusion that tenofovir upregulates pro-inflammatory cytokines and downregulates anti-inflammatory cytokines in acute and chronic exposure. ...

Effects of Tenofovir on Cytokines and Nucleotidases in HIV-1 Target Cells and the Mucosal Tissue Environment in the Female Reproductive Tract

... Further, our predicted steady-state plasma concentrations were consistent with literature values, and our predicted time to steady state in colorectal tissue (9 days for TFVdp) was consistent with findings from previous reports [33]. The use of homogenates of tissue biopsy specimens to measure mucosal tissue drug concentrations assumes uniform distribution of drug and endogenous nucleotide concentrations across the biopsy specimen, which is unlikely [42]. However, isolating HIV target cells from vaginal and cervical tissue biopsy specimens has resulted in incomplete PK data sets, owing to small, inconsistent cell yields [39], and previous publications have demonstrated linear relationship between isolated mucosal cells and tissue homogenate concentrations [29]. ...

Female Sex Hormone Regulation of Tenofovir-diphosphate in Human Female Reproductive Tract (FRT) Cells in Culture

AIDS Research and Human Retroviruses

... Moreover, drug distribution in different mucosal cells needs to be considered. In vitro studies using human tissues from the upper and lower FGT suggested that concentrations of active TFV were 100 times higher in epithelial cells than in CD4+ T cells (90), and imaging methods in animal vaginal tissues were used to confirm the preferential TFV concentration in the epithelium (91). Additional studies showed that after first TFV treatment, endometrial, endocervical and ectocervical polarized epithelial cells secreted TFV that partially protected CD4+ T cells in vitro for at least 3 days (92). ...

Sex Hormones Regulate Tenofovir-Diphosphate in Female Reproductive Tract Cells in Culture

... 69 5′,3′-nucleotidase, mitochondrial (NT5M) is distributed in the mitochondrial matrix and is a 5′-nucleotidase, which may regulate immunity. 70,71 In this study, 6-hydroxyrubiadin was applied to CDA and NT5M simultaneously (Table S2). Whether 6-hydroxyrubiadin and munjistin regulated pyrimidine metabolism through UCK2, CDA and NT5E to alleviate immune disorder and deterioration of RA. ...

Effect of Tenofovir on Nucleotidases and Cytokines in HIV-1 Target Cells

... NT5C2 encodes for a 5' nucleotidase that functions in purine metabolism (135), and has a suggested role in type 2 diabetes and hypertension (136). NT5C2 is present on vascular endothelium cells and likely has an inflammatory process relation to CAD (136,137). Knockdown NT5C2 zebrafish had higher blood flow and elevated linear velocity, in addition to increased inflammatory markers such as angiotensin-converting enzyme and C-reactive protein (138). There is some evidence to suggest that NT5C2 may be involved in the estradiol regulation of fibroblasts (137). ...

Estradiol Regulation of Nucleotidases in Female Reproductive Tract Epithelial Cells and Fibroblasts

... Most worthy of mention is that some chemicals do not interact with DBD by themselves but by their metabolites. For example, inorganic As cannot alter GR-GRE interactions directly; however, methylated metabolites can prevent GR-GRE binding in a manner of competing with Zn 2+ for the Cys residues of GR-DBD (Gosse et al., 2014;Zhang et al., 2019). ...

Monomethylated trivalent arsenic species disrupt steroid receptor interactions with their DNA response elements at non-cytotoxic cellular concentrations
  • Citing Article
  • May 2014

Journal of Applied Toxicology

... The two glucocorticoids, dexamethasone and budesonide, used in the current study are both GR-selective and produce synonymous effects on gene expression (Hellal-Levy et al., 1999;Millan et al., 2011;Mostafa et al., 2019). By employing Org34517, a competitive GR antagonist, that unlike RU496 (mifepristone) (Jewell et al., 1995;Chivers et al., 2004;Rider et al., 2018), may not lead to GR translocation (Peeters et al., 2008), and which shows a much-reduced level of partial (Wen et al., 1995;Sadzak et al., 2008), the delay in S727 phosphorylation compared to the rapid increase in Y701 phosphorylation suggests mechanistic differences. It is likely that IFNG This article has not been copyedited and formatted. ...

Immunocytochemical analysis of hormone mediated nuclear translocation of wild type and mutant glucocorticoid receptors
  • Citing Article
  • December 1995

The Journal of Steroid Biochemistry and Molecular Biology