Publications

BACKGROUND

Being a parent, especially a custodial parent, living with HIV was anticipated to increase psychological distress and challenges to self-care.

METHODS

Mental health symptoms, substance use, and health care utilization were assessed among 3818 HIV-infected adults, including custodial parents, noncustodial parents, and nonparents, in 4 AIDS epicenters.

RESULTS

Custodial parents demonstrated significantly poorer medication adherence and attendance at medical appointments but were similar to nonparents and noncustodial parents in mental health symptoms and treatment utilization for mental health and substance use problems. Noncustodial parents demonstrated the highest levels of recent substance use and substance abuse treatment. Other markers of risk, such as African American ethnicity, lack of current employment income, and injection drug use moderated many of the apparent psychosocial disadvantages exhibited by parents.

CONCLUSIONS

Interventions specific to the psychosocial stressors facing families living with HIV are needed.

The nuclear functions of NF-kappaB p50/RelA heterodimers are regulated in part by posttranslational modifications of its RelA subunit, including phosphorylation and acetylation. Acetylation at lysines 218, 221, and 310 differentially regulates RelA's DNA binding activity, assembly with IkappaBalpha, and transcriptional activity. However, it remains unclear whether the acetylation is regulated or simply due to stimulus-coupled nuclear translocation of NF-kappaB. Using anti-acetylated lysine 310 RelA antibodies, we detected p300-mediated acetylation of RelA in vitro and in vivo after stimulation of cells with tumor necrosis factor alpha (TNF-alpha). Coexpression of catalytically inactive mutants of the catalytic subunit of protein kinase A/mitogen- and stress-activated kinase 1 or IKK1/IKK2, which phosphorylate RelA on serine 276 or serine 536, respectively, sharply inhibited RelA acetylation on lysine 310. Furthermore, phosphorylation of RelA on serine 276 or serine 536 increased assembly of phospho-RelA with p300, which enhanced acetylation on lysine 310. Reconstitution of RelA-deficient murine embryonic fibroblasts with RelA S276A or RelA S536A decreased TNF-alpha-induced acetylation of lysine 310 and expression of the endogenous NF-kappaB-responsive E-selectin gene. These findings indicate that the acetylation of RelA at lysine 310 is importantly regulated by prior phosphorylation of serines 276 and 536. Such phosphorylated and acetylated forms of RelA display enhanced transcriptional activity.

We showed recently that short-term increases in stratum corneum (SC) pH are accompanied by minor alterations in permeability barrier homeostasis and SC integrity/cohesion. Since prolonged SC neutralization more closely mirrors clinical situations (i.e., neonatal skin, occupational dermatitis conditions), we assessed here whether sustained elevations of SC pH by long-term application of 1,1,3,3-tetramethylguanidine superbase provoke profound alterations in SC function. Sustained SC neutralization altered not only barrier recovery kinetics but also basal permeability barrier function. These abnormalities were attributable to a decrease in beta-glucocerebrosidase (beta-GlcCer'ase) and acidic sphingomyelinase (aSMase) catalytic activity and enzyme degradation consequent to a pH-induced sustained serine protease (SP) activity. The role of SP in this process was shown by the normalization of enzyme activities/content by co-applied SP inhibitors (SPI). To address whether lipid-processing enzymes are potential substrates for the stratum corneum chymotryptic enzyme (SCCE), protein extracts from human SC were treated for 2 h at 37 degrees C with recombinant active SCCE at pH 7.2. Recombinant SCCE induced a significant decrease in the immunoblotting of both beta-GlcCer'ase or aSMase compared with control experiments performed in the absence of the active SCCE. Finally, with sustained SC neutralization, SC integrity/cohesion deteriorated, attributable to SP-mediated degradation of corneodesmosomes (CD) as well as CD constituent proteins, desmoglein 1. These abnormalities were again reversed by co-applied SPI. In conclusion, prolonged SC neutralization provokes profound abnormalities in SC function, due to pH-induced high SP activity that, in turn, degrades lipid processing enzymes and CD proteins.

OBJECTIVE

Resolution of deep venous thrombosis (DVT) is involved in the pathogenesis of postthrombotic syndrome. Matrix metalloproteinases (MMPs) are a family of proteolytic enzymes that are critical in angiogenesis and tissue remodeling. We hypothesized that MMP-2 and its membrane-bound activator membrane type-1 matrix metalloproteinase (MT1-MMP) expression would be expressed and activated during the resolution of DVT.

METHODS

DVT was generated by caval ligation in wild-type and MMP-2 transgenic reporter mice. Ligated and sham-operated (control) cavae were analyzed for MMP-2 transcription (beta-galactosidase activity in MMP-2 reporter mice) and MT1-MMP mRNA by real-time polymerase chain reaction. MMP-2 activity was determined by zymography, and immunohistochemical staining for beta-galactosidase and MT1-MMP protein was used to localize expression. Human umbilical vascular endothelial cells (HUVEC) were treated with 10 U/mL thrombin and MMP-2 and MT1-MMP mRNA levels and MMP-2 activity was determined.

RESULTS

MMP-2 activity increased 71% (n = 5, P < .05) at day 8 in ligated vs control cavae by zymography. beta-galactosidase activity showed a 1.2-fold (n = 8, P < .05) and 1.7-fold (n = 8, P < .05) induction in MMP-2 transcription at day 3 and day 8, respectively. No significant MT1-MMP gene induction was seen at day 3 in ligated vs control cavae, but MT1-MMP mRNA was upregulated 2.5-fold (n = 8, P < .05) in ligated cavae at day 8. Immunohistochemical staining localized MMP-2 and MT1-MMP expression to the vein wall and cellular infiltrates of the thrombus. Thrombin-treated HUVEC showed differential responses of MMP-2 and MT1-MMP. Zymography of conditioned media and cell lysates illustrated a 220% (152.6 +/- 8.6 vs 69.445 +/- 5.46 pixels/unit area, n = 5, P < .05) and 150% (74.1 +/- 7.3 vs 49.2 +/- 5.7 pixels/unit area, n = 5, P < .05) increases in MMP-2 activity respectively. MMP-2 mRNA levels were downregulated 30% (0.48 +/- 0.023 vs 0.63 +/- 0.035 copies of MMP-2 mRNA/copy GAPDH, n = 5, P < .05), whereas MT1-MMP message was upregulated 250% (0.147 +/- 0.009 vs 0.059 +/- 0.005 copies of MT1-MMP mRNA/copy GAPDH, n = 5, P < .05).

CONCLUSIONS

Resolution of DVT is associated with increased MMP-2 transcription and activity as well as MT1-MMP expression. Thrombin may mediate the increase in MT1-MMP noted in DVT. This is the first article studying MMP-2 and MT1-MMP transcription in DVT. These findings add DVT resolution to the class of inflammatory and fibrotic disorders in which transcriptional activation of the MT1-MMP/MMP-2 genes occurs and identify a potential therapeutic target to modulate this clinically relevant process.

CLINICAL RELEVANCE

Postthrombotic syndrome remains a significant clinical problem after deep venous thrombosis (DVT), but the cellular and molecular mechanisms involved in thrombus resolution and vein wall fibrosis remain undefined. Matrix metalloproteinase (MMP) enzymes are critical to cell migration and matrix breakdown. We identify gene transcription and activity of two MMP isoforms, MMP-2 and MMP-14 (membrane type MMP 1, MT1-MMP) in the resolution phase of experimental DVT and in thrombin-treated endothelial cells. These studies define new proteases potentially important to resolution of DVT and development of postthrombotic syndrome.

Members of the integrin family recognize a variety of spatially-restricted extracellular ligands. Classically, ligation of integrins activates cytoplasmic signals in the integrin-expressing cell and contributes to cell adhesion, migration, proliferation and survival. At least two members of this family, alphavbeta6 and alphavbeta8 perform an additional function, activation of latent complexes of transforming growth factor beta. In effect, this process allows integrins on one cell to activate signals on adjacent (in the case of alphavbeta6) or nearby cells (in the case of alphavbeta8). Integrin-mediated TGFbeta activation has been shown to play important roles in modulating tissue fibrosis, acute lung injury and pulmonary emphysema. Given the important roles that TGFbeta plays in modulating epithelial cell growth, epithelial-to-mesenchymal transformation and tumor invasion and metastasis, integrin-mediated TGFbeta activation is likely to play important roles in tumor growth and metastasis.

BACKGROUND

Preovulatory gonadotropin-releasing hormone and luteinizing hormone (LH) surges depend on activation of estrogen-inducible progesterone receptors (PRs) in the hypothalamus. Although testosterone treatment can suppress LH secretion under some circumstances, how androgens affect the release of preovulatory hormone surges, and the cellular mechanisms by which androgens exert any such effects, remains unknown.

OBJECTIVE

This study examined the hypothesis that testosterone can block the release of estrogen-induced gonadotropin surges via attenuation of estrogen's ability to induce PRs in the preoptic area (POA)-hypothalamus.

METHODS

In experiment 1, proestrus rats were implanted with capsules filled with crystalline testosterone or empty control capsules. Four days later, animals were bled via atrial catheters at 30-minute intervals from noon to 9:00 pm. In experiment 2, proestrus rats received testosterone-filled or empty control capsules, and 3 days later were ovariectomized (OVX) and injected with estradiol benzoate (EB) 30 mug SC or sesame oil vehicle. The next day, blood samples were obtained from the rats. In experiment 3, proestrus rats similarly implanted with testosterone-filled or empty control capsules, OVX, and injected with EB or vehicle were sacrificed, and POA-hypothalamic tissue was collected for quantitative reverse transcription-polymerase chain reaction analysis of PR messenger RNA.

RESULTS

In experiment 1, radioimmunoassay of serum revealed that testosterone completely blocked release of LH surges that were fully evident in the control group. In experiment 2, LH radioimmunoassay revealed that high-physiologic testosterone exposure completely abolished the release of EB-induced LH surges. In experiment 3, although EB treatment was found to induce an increase in PR expression in control animals, no such induction of PR expression was observed in the testosterone-treated rats.

CONCLUSIONS

Our findings are consistent with the hypothesis that hyperandrogenic interference in the release of preovulatory LH surges is mediated by the suppressive effects of androgens on PR expression in POA-hypothalamic tissue. These findings may have important implications in the understanding of reproductive dysregulation in female hyperandrogenic syndromes, including polycystic ovary syndrome.

The role of NK cells following solid organ transplantation remains unclear. We examined NK cells in acute allograft rejection using a high responder model (DA-->Lewis) of rat orthotopic liver transplantation. Recipient-derived NK cells infiltrated liver allografts early after transplantation. Since chemokines are important in the trafficking of cells to areas of inflammation, we determined the intragraft expression of chemokines known to attract NK cells. CCL3 was significantly increased in allografts at 6 h post-transplant as compared to syngeneic grafts whereas CCL2 and CXCL10 were elevated in both syngeneic and allogeneic grafts. CXCL10 and CX3CL1 were significantly upregulated in allografts by day 3 post-transplant as compared to syngeneic grafts suggesting a role for these chemokines in the recruitment of effector cells to allografts. Graft-infiltrating NK cells were shown to be a major source of IFN-gamma, and IFN-gamma levels in the serum were markedly increased, specifically in allograft recipients, by day 3 post-transplant. Accordingly, in the absence of NK cells the levels of IFN-gamma were significantly decreased. Furthermore, graft survival was significantly prolonged. These data suggest that IFN-gamma-producing NK cells are an important link between the innate and adaptive immune responses early after transplantation.

The role of cytotoxic T-lymphocyte (CTL) escape in rapidly progressive infant human immunodeficiency virus type 1 (HIV-1) infection is undefined. The data presented here demonstrate that infant HIV-1-specific CTL can select for viral escape variants very early in life. These variants, furthermore, may be selected specifically in the infant, despite the same CTL specificity being present in the mother. Additionally, pediatric CTL activity may be compromised both by the transmission of maternal escape variants and by mother-to-child transmission of escape variants that originally arose in the father. The unique acquisition of these CTL escape forms may help to explain the severe nature of some pediatric HIV infections.

Formation and transport of glucuronide metabolites were studied in LLC-PK1 cells. Glucuronidation of 17beta-estradiol, 1-naphthol, mycophenolic acid, and 4-methylumbelliferone was examined in microsomes prepared from LLC-PK1 cells, human livers, human kidneys, and human intestines. The rate of glucuronide metabolite formation observed with LLC-PK1 microsomes was comparable to rates observed with various human tissue microsomes. The fate of the glucuronide metabolite formed in the LLC-PK1 cells was studied by examining its extracellular transport using mycophenolic acid as a model substrate. After administration of mycophenolic acid, the amount of the glucuronide metabolite exiting to the extracellular compartments significantly decreased in the presence of MK-571, an inhibitor for the multidrug resistance-associated protein (MRP) transporter. However, the intracellular levels of the glucuronide metabolite did not change, suggesting that MK-571 was probably blocking metabolite efflux. In summary, these results suggest that the glucuronidating enzyme(s) expressed in the LLC-PK1 cells are capable of sufficient glucuronidation activity and that endogenous transporter(s) in LLC-PK1 cells are active and determine the distribution of the formed metabolites. Since these cells have been previously used to study drug transport, they may be a useful tool in future studies to explore the effect of drug transporters on glucuronidation.