Prevalence, severity and explanatory factors of tooth wear in Arab populations.

OBJECTIVES: The aim of this study was to assess the prevalence and severity of tooth wear and related risk indicators in six Arab countries. METHODS: This multicenter, multinational cross-sectional study was conducted among 2924 participants between the ages of 18-35 years old from six Arab countries. Calibrated dentists assessed tooth wear using the Basic Erosive Wear Examination Index (BEWE). Participants were also asked to complete a questionnaire that assessed their dietary and oral health habits. Each participant was identified by the sextant with the highest BEWE score in the upper and lower jaws. RESULTS: Odds ratios were estimated for correlates of tooth wear anterior and posterior regions. Oman had the highest prevalence of BEWE score 3 (N = 255 (60.2%)). Overall, the highest BEWE score 3 was observed on the incisal edge of the upper anterior teeth (N = 602 (20.6%)) and in the lower left posterior region (sextant 6) on the occlusal surface (N = 466 (15.9%)). Correlates of severe tooth wear in both regions were eating or drinking more than six times per day, age and education. Drinking soft drinks "once a day" was significantly associated with severe tooth wear in the posterior region (OR: 1.3, 95% CI:1.05, 1.6). Brushing teeth more than twice a day was inversely associated with tooth wear in the anterior region (OR:0.75, 95% CI: 0.57,0.97). CONCLUSION: The prevalence of tooth wear in Arab populations is relatively high, specific preventive and therapeutic measures should be developed to target people at higher risk of this condition. CLINICAL SIGNIFICANCE: Tooth wear is now regarded as a significant oral health problems, dentists should advise their patients about dietary and oral health habits that can lead to severe tooth wear.

Dental age estimation of Omani children using Demirjian's method.

Dental age plays a significant role in forensic dentistry, orthodontics and paediatric dentistry, as well as in general diagnosis and treatment planning. Different methods have been developed to determine dental age. One of the most commonly used methods is Demirjian's method, which was developed in 1973 from research on a large number of French-Canadian children. It is based on the degree of tooth mineralisation by examining the radiological appearance of the lower mandibular left quadrant. The purpose of this study was to assess the dental age of Omani children using Demirjian's method and evaluate the applicability of the method in dental age estimation for Omani children. The sample consisted of 485 digital panoramic radiographs of children (264 males, 221 females) aged between 4.6 years and 16.5 years, and obtained from the records of the Military Dental Centre in Oman. The data were analysed using SPSS. Paired t-tests, intraclass correlation coefficients (ICC) and difference-against-mean plots were used to compare the dental age calculated by Demirjian's method with chronological age. A single examiner scored the radiographs, and intra-observer reliability was evaluated using Cronbach's alpha on data from rescoring one out of every 20 radiographs. For boys, the mean difference between chronological age and dental age for all age groups was 0.10 (95% CI -0.03 to 0.24). For girls, the mean difference between chronological age and dental age for all age groups was 0.05 (95% CI -0.11 to 0.22). Difference-against-mean plots showed no evidence of differential bias by age. For boys, the ICC was 0.896 (95% CI 0.869-0.917); for girls, it was 0.886 (95% CI 0.854-0.911). Difference-against-mean plots for boys (Fig. 1) and girls (Fig. 2) showed some evidence of differential bias by age. In conclusion, the extent of the observed differences was sufficient for doubt to be cast upon the utility of Demirjian's method for Oman, particularly when it is considered that the method's most likely application would be in age determination for minors in the workforce.

Testing the Arabic short form versions of the Parental-Caregivers Perceptions Questionnaire and the Family Impact Scale in Oman.

UNLABELLED: Short form versions of the Parental-Caregivers Perception Questionnaire (P-CPQ) and Family Impact Scale (FIS) have been developed for use as measures of oral health-related quality of life in dental research. OBJECTIVES: (1) To translate the original English short form versions of the P-CPQ and FIS and examine their validity, and (2) to describe the impact of early childhood caries on oral health-related quality of life in young Omani children and their families. METHODS: Parents/caregivers of children awaiting treatment for early childhood caries completed the P-CPQ and FIS at the Military Dental Center in Oman. Data were obtained from 191 families (representing a 94.1% participation rate). A global Oral Health Quality of Life (OHRQoL) item was used concurrently to examine the scales' validity. RESULTS: The cross-sectional concurrent validity of the short form version of the P-CPQ was apparent in the significant gradient across the response categories of the global OHRQoL item, but the FIS short form version did not perform as well. CONCLUSION: The P-CPQ appears to be valid, but further investigation of the FIS is required, along with examination of the scales' responsiveness to change.

The impact of periodontitis on oral health-related quality of life: a review of the evidence from observational studies.

Modern population based oral health management requires a complete understanding of the impact of disease in order to provide efficient and effective oral health care and guidance. Periodontitis is an important cause of tooth loss and has been shown to be associated with a number of systemic conditions. The impact of oral conditions and disorders on quality of life has been extensively studied. However, the impact of periodontitis on quality of life has received less attention. This review summarizes the literature on the impact of periodontitis on oral health-related quality of life (OHRQoL). Relevant publications were identified after searching the MEDLINE and EMBASE electronic databases. Screening of titles and abstracts and data extraction was conducted. Only observational studies were included in this review. Most of the reviewed studies reported a negative impact of periodontitis on OHRQoL. However, the reporting standards varied across studies. Moreover, most of the studies were conducted in developed countries.

Evaluation of the impact of highly active antiretroviral therapy on immune recovery in antiretroviral naive patients.

OBJECTIVES: To examine the extent of immune reconstitution in treatment-naive patients with CD4 T-cell counts <500 cells/microL following 48 weeks of highly active antiretroviral therapy (HAART). METHODS: Thirteen antiretroviral naive patients were evaluated longitudinally for 48 weeks on HAART utilizing immune functional and lymphocyte phenotyping assays, including lymphocyte proliferation assay, flow cytometric evaluation of cell surface markers, and delayed type hypersensitivity skin tests. Virologic responses were monitored using commercially available viral load assays and gag/pol mRNA quantification using simultaneous immunophenotyping/UltraSensitive fluorescence in situ hybridization (ViroTect In Cell HIV-1 Detection Kit; Invirion, Frankfort, MI). Thymic function was evaluated for a subset of four patients using real-time polymerase chain reaction (PCR) for T-cell receptor excision circle (TREC) quantification and thymic scans using computerized axial tomography (CT) of the thymus. RESULTS: HAART initiation resulted in a significant decline in plasma viremia and percentage of infected peripheral blood cells, and a rise in CD4 T cells from a baseline median of 207 cells/microL to a week-48 median of 617 cells/microL. The rise was predominately in CD4 memory cells. Naive T cells also increased in number, but at a slower rate. Activated (HLA-DR CD38) CD4 and CD8 T cells were elevated at baseline (24 and 62%, respectively) and declined by week 48 (17 and 36%, respectively) but did not reach normal levels. The number of Fas CD4 T cells increased from a baseline median of 169 to 381 cells/microL at week 48. Both soluble interleukin (IL)-2 and tumour necrosis factor (TNF) II receptors declined by week 48. HIV p24 lymphocyte proliferation assay responses were transiently detected in three patients. TREC values increased from a median 6400 copies/microg at baseline to a week-48 median value of 26 697 copies/microg. CONCLUSION: Immune functional reconstitution was not achieved in these HAART naive patients.

T cell receptor excision circle (TREC) content following maximum HIV suppression is equivalent in HIV-infected and HIV-uninfected individuals.

BACKGROUND: The adult human thymus contributes to de novo T cell synthesis; such synthesis can be assessed by analyzing T cell receptor excision circles (TREC). METHODS: TREC levels were measured in total peripheral blood mononuclear cells (PBMC) and CD4- and CD8-enriched cells of 29 HIV-positive patients with maximal viral suppression. The expression of CD45RA+CD45RO-, CD45RA+CD62L+, CD45RO-CD27+CD95low and HLA-DR+CD38+ was assessed using three-color flow cytometric analysis of whole blood. Thymic index score was based on computed tomographic scans of the thymus. The relationship of TREC with thymic index and the expression of the naive phenotypes was evaluated. RESULTS: TREC expression was not statistically different in these HIV-positive patients from that in age-matched HIV-negative controls. Among HIV-positive patients with CD4 cell count of > 500 x 10(6) cells/l after antiretroviral therapy (n = 15), PBMC TREC levels correlated with the expression of CD45RA+CD45RO- and CD45RA+CD62L+ naive phenotypes, and inversely correlated with the expression of HLA-DR+CD38+. The change between pre- and post-therapy CD4 cell counts for these 15 patients significantly correlated with both thymic index and expression of the CD45RA+CD45RO- phenotype. CONCLUSIONS: The finding that TREC expression was equivalent between HIV-positive patients after therapy and HIV-negative donors suggests that there is no reduction in thymic output among HIV-positive individuals after therapy. Given that TREC is inversely correlated with HLA-DR/CD38 expression, its analysis in studies of thymopoiesis should be evaluated in the context of maximum viral suppression to reduce HIV-mediated immune activation and/or by normalizing for cell turnover.

A menstrual cycle pattern for cytokine levels exists in HIV-positive women: implication for HIV vaginal and plasma shedding.

OBJECTIVES: To evaluate the effect of the menstrual cycle in HIV-positive women on plasma and genital cytokine levels, interrelationships between vaginal and plasma cytokines, CD4 and CD8 T cell fluctuations, and genital and plasma viral loads. METHODS: Plasma and cervicovaginal lavage specimens were collected from 55 HIV-positive women with CD4 cell counts < 350 cells/microl during phases of the menstrual cycle. Samples were assayed for IL-1beta, IL-6, IL-4, IL-8, IL-10, TGFbeta, TNFalpha, INFgamma, MIP1alpha, MIP1beta, RANTES, and TNFR-II using enzyme-linked immunosorbent assays. CD4 and CD8 T cell expression was evaluated by flow cytometry. Repeated measures regression models were used to assess the effect of the menstrual cycle on cytokines and viral load. Multivariate repeated regression models were used to assess the correlation among selected cytokines and between selected cytokines and HIV viral load. RESULTS: Vaginal IL-1beta, IL-4, IL-6, IL-8, IL-10, MIP1beta, RANTES, TGFbeta, and TNFR-II were significantly elevated during menses but were not altered during other phases. Plasma cytokine levels were not altered during the menstrual cycle. A positive Candida culture increased vaginal IL-8 during menses, whereas vaginal discharge was associated with a reduction in vaginal IL-4, IL-10, and RANTES. CD4 and CD8 cell numbers did not vary with the menstrual cycle. Vaginal cytokine levels correlated only with vaginal viral load, in a sampling method-dependent manner. CONCLUSION: We provide evidence of elevated vaginal cytokine levels during menses, which appear to regulate vaginal and not plasma HIV shedding, suggesting that a menstrual cycle pattern exists for cytokine production in HIV-positive women impacting vaginal shedding of HIV.

Upregulation of CD4 on CD8+ T cells: CD4dimCD8bright T cells constitute an activated phenotype of CD8+ T cells.

Aside from an intermediate stage in thymic T-cell development, the expression of CD4 and CD8 is generally thought to be mutually exclusive, associated with helper or cytotoxic T-cell functions, respectively. Stimulation of CD8+ T cells, however, induces the de novo expression of CD4. We demonstrate that while superantigen (staphylococcal enterotoxin B, SEB) and anti-CD3/CD28 costimulation of purified CD8+ T cells induced the expression of CD4 on CD8+ T cells by 30 and 17%, respectively, phytohaemagglutinin (PHA) stimulation did not induce CD4 expression on purified CD8+ T cells but significantly induced the expression of both CD4 on CD8 (CD4dimCD8bright) and CD8 on CD4 (CD4brightCD8dim) T cells in unfractionated peripheral blood mononuclear cells (PBMC). The level of the PHA-mediated induction of CD4dimCD8bright and CD4brightCD8dim was at 27 and 17%, respectively. Depletion of CD4+ T cells from PBMC abrogated this PHA-mediated effect. Autologous CD4+ and CD8+ T-cell co-cultures in the presence of PHA induced this CD4dimCD8bright T-cell expression by 33%, demonstrating a role for CD4 cells in the PHA-mediated induction of the double positive cells. The induction of CD4dimCD8bright was independent of a soluble factor(s). Phenotypic analysis of CD4dimCD8bright T cells indicated significantly higher levels of CD95, CD25, CD38, CD69, CD28, and CD45RO expression than their CD8+CD4- counterparts. CD4dimCD8bright T cells were also negative for CD1a expression and were predominantly T-cell receptor (TCR) alphabeta cells. Our data demonstrate that CD4dimCD8bright T cells are an activated phenotype of CD8+ T cells and suggest that CD4 upregulation on CD8+ T cells may function as an additional marker to identify activated CD8+ T cells.

Evaluation of thymopoiesis using T cell receptor excision circles (TRECs): differential correlation between adult and pediatric TRECs and naïve phenotypes.

To determine whether the thymus is still functional despite age-related involution, we measured a biomarker for thymopoiesis known as the T cell receptor excision circle (TREC) from peripheral blood mononuclear cells (PBMCs) of 148 healthy children and from PBMCs, CD4(+), and CD8(+) cells of 32, 30, and 50 healthy adults, respectively. We demonstrate that during the first 5 years of life, thymic output is decreased (P 0.002) but not dramatically (r = -0. 282). Among adults aged 23-58, thymic output was inversely correlated with age, as measured from PBMCs (r = -0.628, P < 0.0005), CD4(+) (r = -0.530, P 0.003), and CD8(+) fractions (r = -0.385, P 0. 006). A strong correlation existed between pediatric PBMC TRECs and the expression of three naïve phenotypic markers (CD45RA(+)CD45RO(-), CD45RA(+)CD62L(+), and CD45RO(-)CD27(+)CD95(low)). Adult PBMC TRECs correlated only with the expression of CD45RA(+)CD45RO(-) (r = 0.459, P 0.012). Our data suggest that in adults CD45RA(+)CD45RO(-) may be enriched for TRECs and add to a growing body of evidence illustrating intact thymic function in adulthood.

The impact of the ovulatory cycle on cytokine production: evaluation of systemic, cervicovaginal, and salivary compartments.

To understand the impact of the menstrual cycle on immunologic parameters, we measured the level of cytokines and chemokines from plasma, cervicovaginal lavage (CVL), and saliva samples of 6 premenopausal women during the follicular and luteal phases of the ovulatory cycle. We demonstrate that the level of plasma interleukin-8 (IL-8) was 4-fold higher during the follicular phase than the luteal phase (p = 0.004), whereas plasma IL-1beta, IL-4, IL-6, IL-10, interferon-gamma (IFN-gamma), transforming growth factor-beta (TGF-beta), tumor necrosis factor-alpha (TNF-alpha), macrophage inflammatory protein-1alpha (MIP-1alpha), and TNF receptor II (TNFR II) were not altered during the ovulatory cycle. In the vaginal compartment, as measured from CVL samples, the levels of IL-6 and IL-1beta were both 5-fold higher in the follicular than the luteal phase (p = 0.0002 and 0.03, respectively). Salivary cytokine and chemokine samples were similar when measured during the luteal and the follicular phases. Additional analysis of lymphocyte subsets for phenotypic and functional markers indicated that they were not influenced by the ovulatory cycle. Collectively, these data suggest that IL-6, IL-8, and IL-1beta are differentially regulated during the ovulatory cycle.

Maximum suppression of HIV replication leads to the restoration of HIV-specific responses in early HIV disease.

OBJECTIVES: It is predicted that HIV-infected individuals in early HIV disease are the most likely group to achieve immune reconstitution following highly active antiretroviral treatment. We assessed whether suppression of HIV replication in this group would improve immune function. METHODS: Seventeen antiretroviral-naïve patients in early HIV disease were evaluated for immune function and lymphocyte phenotyping using standard immunological assays. RESULTS: Absolute CD4+ T-cell number increased from a median of 550 to 800 x 10(6) cells/l while CD8+ T-cell numbers were reduced. The decrease in CD8+ cells correlated with a decrease in the CD8+ memory phenotype. Kinetics of CD4+ naïve and memory T-cell rise indicated that 80% of the maximum CD4+ naïve increase was achieved within 18 weeks whereas maximum CD4+ memory T-cell rise was achieved within 36 weeks. Activation markers (HLA-DR, CD38) and an apoptosis-related marker (CD95) were reduced on CD4+ and CD8+ T cells. Lymphocyte proliferation responses to tetanus toxoid, alloantigen, and anti-CD3/CD28 were restored in patients that were initially unresponsive. At baseline, 31% of the patients responded to HIV p24, which increased to 69% post-therapy. The inducible RANTES response was normalized following therapy whereas inducible interferon-gamma, interleukin (IL)-12, and MIP1beta were elevated. The depressed inducible IL-10 response, however, was not altered after therapy. CONCLUSIONS: This is one of the first studies to demonstrate the restoration of HIV-1 specific responses in non-acute HIV infection, suggesting early intervention with potent antiretroviral therapy may reverse immune-mediated damage not seen with treated patients who have more advanced disease.

Detection of T cell receptor circles (TRECs) as biomarkers for de novo T cell synthesis using a quantitative polymerase chain reaction-enzyme linked immunosorbent assay (PCR-ELISA).

Currently, phenotypic markers that distinguish between recent thymic emigrants/de novo T cells and the rest of the peripheral T cell pool are lacking. This distinction is critical in studies aimed at evaluating immune reconstitution following intensive chemotherapy, in immunodeficiency-related therapies, or in the elucidation of the kinetics of thymic function. During V(D)J T cell receptor rearrangement, DNA extrachromosomal excision products are generated. These products, known as T cell receptor excision circles (TRECs), are not replicated during mitosis and are thus diluted with each round of cell division. Therefore, TRECs can be used as an indicator of recent thymic emigrants. Thus far, quantitative competitive-polymerase chain reaction (QC-PCR) and real time PCR were used to measure TREC levels. However, QC-PCR relies on radioactivity, is cumbersome when processing many samples at once and the cost of real time PCR does not make it a viable option for many laboratories. We describe here the development of a quantitative PCR-ELISA method for the measurement of coding joint TRECs generated from ValphaJalpha recombination. Our assay is ultra sensitive, relies on biotin labeling rather than radioactivity, is based on a 96-well format making multiple process sampling relatively easy, and is cost effective. Using this PCR-ELISA method, we evaluated thymic output among 22 normal subjects, ranging in age from 22-53 years, and among HIV-infected individuals following highly active antiretroviral therapy (HAART). We demonstrate that an inverse relationship exists between TREC levels and aging in normal individuals and that, among some HIV patients, HAART treatment leads to enhanced thymic output. Our assay has direct relevance in projects examining normal and abnormal thymic function and in immune reconstitution studies.

Bacterial vaginosis-associated microflora isolated from the female genital tract activates HIV-1 expression.

Alteration of cervicovaginal microbial flora can lead to vaginosis, which is associated with an increased risk of HIV-1 transmission. We recently characterized a soluble HIV-inducing factor (HIF) from the cervicovaginal lavage (CVL) samples of women. The goals of this study were to determine the effect of cervicovaginal microflora on HIV-1 expression and to elucidate the relationship between HIF activity and microflora. Physiologically relevant microorganisms, Mycoplasma, diphtheroid-like bacteria, Gardnerella vaginalis, Streptococcus agalactiae, and Streptococcus constellatus, cultured from the CVL of a representative woman with a clinical condition of bacterial vaginosis and possessing HIF activity, induced HIV-1 expression. The magnitude of virus induction varied widely with the greatest stimulation induced by diphtheroid-like bacteria and Mycoplasma. The transcriptional induction by Mycoplasma was mediated by activation of the KB enhancer, an activation mechanism shared with HIF. Also as with HIF, Mycoplasma induced AP-1 dependent transcription. Polymerase chain reaction (PCR)-based speciation showed that the isolate was M. hominis. Our data indicate that bacterial vaginosis-associated microflora can enhance HIV-1 transcription and replication and identify M. hominis as a potential source for HIF activity. The virus-enhancing activities associated with the microflora and HIF may increase genital tract viral load, potentially contributing to HIV transmission.

Human immunodeficiency virus type-1 transcription: role of the 5'-untranslated leader region (review).

Human immunodeficiency virus type-1 (HIV-1) transcription is dependent on the interaction of host-cell transcription factors with cis-regulatory DNA elements within the viral long terminal repeat (LTR). Much attention has focused on the series of sequence elements upstream of the transcriptional initiation site in the U3 region of the LTR including the Sp1 and NF-kappaB binding sites. Recent studies, however, demonstrate that the transcribed 5'-untranslated leader region (5'-UTR) also contains important transcriptional elements. These regulatory elements situated downstream of transcription interact with constitutive and inducible transcription factors, mediate transmission of cellular activation signals, and are important for efficient HIV-1 transcription and replication. The 5'-UTR contains binding sites for the transcription factors AP-1, NF-kappaB, NF-AT, IRF, and Sp1. Mutations in these binding sites can interfere with the viral response to cell activation signals, decrease LTR transcription, and inhibit viral replication. The 5'-UTR also interacts with a specific nucleosome that is rapidly displaced during transcriptional activation of the latent provirus. We propose that the inducible transcription factor binding sites in the 5'-UTR comprise a downstream enhancer domain that can function independent of, or in concert with, the LTR promoter to rapidly increase latent proviral transcription in response to cell activation signals. In this review, we describe the host-cell transcription factors that interact with the 5'-UTR and discuss their role in the transcriptional regulation of HIV-1 gene expression.

Interleukin-10 enhances tumor necrosis factor-alpha activation of HIV-1 transcription in latently infected T cells.

Interleukin-10 (IL-10) is elevated in HIV-1-infected individuals and has been implicated in disease progression. We previously reported that IL-10 cooperates with tumor necrosis factor-alpha (TNF-alpha) to activate HIV-1 expression synergistically in acutely infected monocyte-derived macrophages and the chronically infected U1 promonocytic cell line. To determine whether IL-10 also cooperates with TNF-alpha to activate latent HIV-I expression in lymphocytes, we examined the effects of IL-10 on proviral expression in the chronically infected T-cell line, ACH-2. Although IL-10 inhibited HIV-1 expression acting alone, in combination with suboptimal concentrations of TNF-alpha, IL-10 increased HIV-1 steady-state mRNA expression and p24 core antigen production in ACH-2 cells. Interestingly, IL-10 concentrations that synergistically induced virus also maximally stimulated endogenous TNF-alpha expression, suggesting that cell-derived TNF-alpha may contribute to cytokine synergy. Transfection studies in ACH-2 cells indicated that IL-10 combined with TNF-alpha to activate the HIV-1 long terminal repeat (LTR). IL-10 also cooperated with TNF-alpha to activate HIV-1 LTR in 1G5 cells, a Jurkat T-cell line stably transfected with an LTR-dependent luciferase reporter gene. Pyrrolidine dithiocarbamate, a potent transcriptional inhibitor of the viral LTR, abrogated the cytokine responses in both U1 and ACH-2 cells, suggesting a common TNF-alpha-mediated transcriptional mechanism in these cell types despite their different modes of provirus latency. Taken collectively, these data suggest that IL-10 enhances suboptimal TNF-alpha activation of HIV-1 transcription in chronically infected T-cells at least in part through induction of endogenous TNF-alpha expression.

A human immunodeficiency virus (HIV)-inducing factor from the female genital tract activates HIV-1 gene expression through the kappaB enhancer.

Virus-enhancing factors present in the female genital tract may influence the transmission of human immunodeficiency virus type 1 (HIV-1). Previously, the presence of a heat-stable soluble factor in the cervicovaginal lavage (CVL) fluid of both HIV-infected and -uninfected women that induces HIV-1 expression in T cells and monocytes was reported. Now this CVL factor was shown to increase HIV-1 gene expression through the activation of the kappaB enhancer in the viral long terminal repeat (LTR). DNA binding studies, together with functional studies using mutant LTR reporter constructs, indicate the requirement for an NF-kappaB-dependent pathway in the CVL-mediated activation of HIV-1 expression. CVL samples that activated HIV-1 expression also stimulated AP-1-dependent transcription. These data demonstrate that an HIV-inducing factor, distinct from heat-labile cytokines, present in the female genital mucosa can activate AP-1 and NF-kappaB and increase HIV-1 gene expression through the kappaB enhancer, possibly contributing to HIV-1 transmission.

The cAMP-dependent protein kinase A and protein kinase C-beta pathways synergistically interact to activate HIV-1 transcription in latently infected cells of monocyte/macrophage lineage.

The HIV-1 long terminal repeat (LTR) responds to a variety of cellular signal transduction pathways. We demonstrate that the cAMP-dependent protein kinase A (PKA) and protein kinase C (PKC) signaling pathways synergize to increase HIV-1 LTR-mediated transcription and viral replication in a latently infected promonocytic cell line (U1). The LTR-mediated synergy induced by cholera toxin (Ctx), a potent activator of the cAMP-dependent PKA pathway, and the PKC activator phorbol 12-myristate 13-acetate (PMA) was abrogated by a PKC-beta-specific inhibitor (LY333531). In contrast, the LTR-mediated synergy induced by Ctx and TNF alpha was not affected by LY333531. The synergy induced by Ctx and TNF alpha was also abrogated by mutation of the cAMP-responsive downstream sequence elements (DSE) in the 5' untranslated leader region, whereas the DSE mutations did not affect the synergy induced by Ctx and PMA. These distinctions indicate that Ctx cooperates differently with TNF alpha and PMA to activate the HIV-1 LTR. Ctx and PMA synergistically activated AP-1- and NF-kappa B-dependent transcription, even though no cooperative binding of AP-1 or NF-kappa B was observed in gel shift assays. An extensive mutational analysis of the HIV-1 LTR that included the NF-kappa B and AP-1 binding sites revealed no distinct cis-acting element or region within the HIV-1 LTR that was required for the transcriptional synergy. Ctx and PMA also synergistically interact to activate the HTLV-1 LTR. These results indicate that the transcriptional synergy elicited by Ctx and PMA targets multiple functional elements and promoters, requires a cooperative interaction between the PKA and PKC-beta pathways, and differs mechanistically from the transcriptional synergy induced by Ctx and TNF alpha.

Induction of HIV-1 replication by type 1-like cytokines, interleukin (IL)-12 and IL-15: effect on viral transcriptional activation, cellular proliferation, and endogenous cytokine production.

Cytokine dysregulation is evident in HIV-1 infection and it may play an important role in HIV-1 pathogenesis. Administration of T helper cytokines potentially may restore the functional abnormalities to the HIV-1 immune response. Type 1-like cytokines, IL-2, IL-12, and IL-15, are candidates for immune-based therapy for HIV. Given their potential therapeutic use, we determined the effects of IL-2, IL-12, and IL-15 on HIV-1 replication in both primary blood mononuclear cells (PBMC) and the T-cell line, Kit 225-K6. We demonstrate that both IL-2 and IL-12 induce a similar level of HIV-1 replication (9- and 11-fold, respectively) in mitogen-stimulated PBMC. The effect of IL-2 plateaued by day 6, while that of IL-12 continued to increase HIV-1 expression. IL-15 induced a 2.5-fold increase in HIV-1 expression that remained at the same level through day 6. In Kit 225-K6, an IL-2-dependent T cell line, IL-12 and IL-15 enhanced HIV-1 replication by 5- and 3.5-fold over IL-2-treated cultures, respectively. IL-2-, IL-12-, and IL-15-mediated induction of HIV was independent of direct HIV-1 LTR activation, since none of the cytokines induced LTR activity from transfected reporter gene constructs. The cytokine-mediated induction of HIV-1 expression was also independent of cellular proliferation. In PBMC, the IL-12-mediated effect was partially mediated by endogenous cytokine production of IL-1beta and IL-7, whereas in Kit 225-K6, TNFalpha, INFgamma, IL-1beta, and IL-7 did not contribute significantly to the IL-12-mediated effect. IL-15 effect on HIV-1 in PBMC was independent of endogenous cytokine production. However, in Kit 225-K6, neutralizing antibodies to IL-7 had a significant effect on HIV-1 expression. These data suggest that IL-2, IL-12, and IL-15 increase HIV-1 replication predominantly through a posttranscriptional mechanism that may be enhanced by endogenous cytokine production.