Efficacy and Safety of a Recombinant Plant-Based Adjuvanted Covid-19 Vaccine.

BACKGROUND: Coronavirus-like particles (CoVLP) that are produced in plants and display the prefusion spike glycoprotein of the original strain of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) are combined with an adjuvant (Adjuvant System 03 [AS03]) to form the candidate vaccine. METHODS: In this phase 3, multinational, randomized, placebo-controlled trial conducted at 85 centers, we assigned adults (≥18 years of age) in a 1:1 ratio to receive two intramuscular injections of the CoVLP+AS03 vaccine or placebo 21 days apart. The primary objective of the trial was to determine the efficacy of the CoVLP+AS03 vaccine in preventing symptomatic coronavirus disease 2019 (Covid-19) beginning at least 7 days after the second injection, with the analysis performed after the detection of at least 160 cases. RESULTS: A total of 24,141 volunteers participated in the trial; the median age of the participants was 29 years. Covid-19 was confirmed by polymerase-chain-reaction assay in 165 participants in the intention-to-treat population; all viral samples that could be sequenced contained variants of the original strain. Vaccine efficacy was 69.5% (95% confidence interval [CI], 56.7 to 78.8) against any symptomatic Covid-19 caused by five variants that were identified by sequencing. In a post hoc analysis, vaccine efficacy was 78.8% (95% CI, 55.8 to 90.8) against moderate-to-severe disease and 74.0% (95% CI, 62.1 to 82.5) among the participants who were seronegative at baseline. No severe cases of Covid-19 occurred in the vaccine group, in which the median viral load for breakthrough cases was lower than that in the placebo group by a factor of more than 100. Solicited adverse events were mostly mild or moderate and transient and were more frequent in the vaccine group than in the placebo group; local adverse events occurred in 92.3% and 45.5% of participants, respectively, and systemic adverse events in 87.3% and 65.0%. The incidence of unsolicited adverse events was similar in the two groups up to 21 days after each dose (22.7% and 20.4%) and from day 43 through day 201 (4.2% and 4.0%). CONCLUSIONS: The CoVLP+AS03 vaccine was effective in preventing Covid-19 caused by a spectrum of variants, with efficacy ranging from 69.5% against symptomatic infection to 78.8% against moderate-to-severe disease. (Funded by Medicago; ClinicalTrials.gov number, NCT04636697.).

ART Adherence Among Malawian Youth Enrolled in Teen Clubs: A Retrospective Chart Review.

To improve outcomes among HIV-positive adolescents, the Malawi Ministry of Health is supporting scale-up of "Teen Clubs," a facility-based antiretroviral treatment (ART) delivery model. Teen Clubs are monthly ART clinics for adolescents (10-19 years old) that provide clinical services and peer psychosocial support. This paper assesses ART adherence among Teen Club attendees in Malawi. We performed a retrospective analysis of medical records and Teen Club attendance data on 589 HIV-positive adolescents at 16 Partners in Hope (PIH)-Extending Quality Improvement for HIV/AIDS in Malawi (EQUIP) supported facilities across Malawi, from January to June of 2017, who attended at least two Teen Club sessions. Multi-level logistic regression models were used to examine the role of gender and age on optimal ART adherence (≥ 95% based on pill count) among HIV-positive adolescents enrolled in Teen Clubs. The median age of adolescents in this sample was 14 years, and 47% were male. Older adolescent males (15-19 years) were 64% more likely to achieve ≥ 95% ART adherence (aOR 1.64, 95% CI 1.16-2.31, p < 0.01) compared to younger (10-14 years) males. The effect of age on adherence was smaller and not significant among females (aOR 1.36, 95% CI 0.96-1.94, p = 0.08). In the full model including males and females, older adolescence was associated with higher odds of optimal adherence (aOR 1.48, 95% CI 1.16-1.90, p < 0.01). These results reinforce the need for age-specialized programming for adolescents, and future research should evaluate this in achieving optimal ART adherence.

Efficacy of intravaginal dehydroepiandrosterone (DHEA) on moderate to severe dyspareunia and vaginal dryness, symptoms of vulvovaginal atrophy, and of the genitourinary syndrome of menopause.

OBJECTIVE: The aim of this study is to confirm the local beneficial effects of intravaginal dehydroepiandrosterone (DHEA, Prasterone) on moderate to severe dyspareunia or pain at sexual activity, the most frequent symptom of vulvovaginal atrophy due to menopause or genitourinary syndrome of menopause (GSM). METHODS: In a prospective, randomized, double-blind, and placebo-controlled phase III clinical trial, the effect of daily intravaginal 0.50% DHEA (6.5 mg) (Prasterone, EndoCeutics) was examined on four coprimary objectives, namely percentage of parabasal cells, percentage or superficial cells, vaginal pH, and moderate to severe pain at sexual activity (dyspareunia) identified by the women as their most bothersome vulvovaginal atrophy symptom. The intent-to-treat population included 157 and 325 women in the placebo and DHEA-treated groups, respectively. RESULTS: After daily intravaginal administration of 0.50% DHEA for 12 weeks, when compared to baseline by the analysis of covariance test, the percentage of parabasal cells decreased by 27.7% over placebo (P < 0.0001), whereas the percentage of superficial cells increased by 8.44% over placebo (P < 0.0001), vaginal pH decreased by 0.66 pH unit over placebo (P < 0.0001), and pain at sexual activity decreased by 1.42 severity score unit from baseline or 0.36 unit over placebo (P = 0.0002). On the other hand, moderate to severe vaginal dryness present in 84.0% of women improved at 12 weeks by 1.44 severity score unit compared to baseline, or 0.27 unit over placebo (P = 0.004). At gynecological evaluation, vaginal secretions, epithelial integrity, epithelial surface thickness, and color all improved by 86% to 121% over the placebo effect (P < 0.0001 for all comparisons with placebo). Serum steroid levels remained well within the normal postmenopausal values according to the involved mechanisms of intracrinology. The only side effect reasonably related to treatment is vaginal discharge due to melting of the vehicle at body temperature and this was reported in about 6% of the participants. CONCLUSIONS: The daily intravaginal administration of 0.50% (6.5 mg) DHEA (Prasterone) has shown clinically and highly statistically significant effects on the four coprimary parameters suggested by the US Food and Drug Administration. The strictly local action of Prasterone is in line with the absence of significant drug-related adverse events, thus showing the high benefit-to-risk ratio of this treatment based upon the novel understanding of the physiology of sex steroids in women.

M#1045: Training Course in Focused Assessment with Sonography for HIV/TB in HIV Prevalent Medical Centers in Malawi.

PURPOSE: Training medical providers of different backgrounds the "focused assessment with sonography for HIV-associated TB" (FASH) exam to expand the availability of ultrasound for TB diagnosis in resource poor settings in the central region of Malawi. METHODS AND MATERIALS: A survey was completed by the 19 eligible participants before and after a 4-day training course regarding the utility of the FASH exam. A six-question quiz was used to assess knowledge of the use of ultrasound in the FASH exam before and after the course. RESULTS: Participants' knowledge of the FASH technique significantly improved after the four-day course with a 32% increase in total quiz questions answered correctly (p<0.001).Ninety-five percent (n= 18) of participants answered that they would "likely" incorporate FASH in their clinical practice. Furthermore, 100% (n=19) of participants agreed that the FASH exam would improve their ability to diagnose TB and 95% (n=18) agreed that FASH would improve patient care in their clinic. CONCLUSIONS: After completing a 4-day training course, medical providers were more knowledgeable about the FASH exam and its findings, and felt more comfortable using ultrasound for the diagnosis of TB. Participants were also unanimous in opinion that the FASH ultrasound exam would improve their ability to diagnose TB.

Provider perspectives on barriers to reproductive health services for HIV-infected clients in Central Malawi.

Despite widespread availability of Depo-Provera in HIV clinics in Malawi, coverage of family planning (FP) remains low. We sought to understand provider perspectives about the challenges of providing reproductive health services to HIV-infected clients in antiretroviral therapy (ART) clinics in Central Malawi by conducting surveys and semi structured in-depth interviews with 31 ART providers across 16 clinical sites. Additionally, site surveys were performed to assess contraceptive resources. Major barriers to the provision of FP in ART clinics were inadequate staff in the facility, shortage of trained providers, limited time to counsel on FP, and lack of private space for the provision of FP services. These barriers limit the direct delivery of FP in ART clinics. Strategies to integrate FP with HIV/ART services and task shifting FP service provision to non-ART providers should be explored in Malawi as a means to improve coverage of services to HIV-infected clients.

Factors associated with retention in Option B+ in Malawi: a case control study.

INTRODUCTION: There are limited data on factors associated with retention in Option B+. We sought to explore the characteristics of women retained in Option B+ in Malawi, with a focus on the role of HIV disclosure, awareness of partner HIV status, and knowledge around the importance of Option B+ for maternal-child health. Methods We performed a case-control study of HIV-infected women in Malawi initiated on antiretroviral therapy (ART) under Option B+. Cases were enrolled if they met criteria for default from Option B+ (out of ART for >60 days), and controls were enrolled in approximately 3:1 ratio if they were retained in care for at least 12 months. We surveyed socio-demographic characteristics, HIV disclosure and awareness of partner HIV status, self-report about receiving pre-ART education, and knowledge of Option B+. Univariate logistic regression was performed to determine factors associated with retention. Multivariate logistic regression model was used to evaluate the relationship between HIV disclosure, Option B+ knowledge, and retention after adjusting for age, schooling, and travel time to clinic. RESULTS: We enrolled 50 cases and 153 controls. Median age was 30 years (interquartile range (IQR) 25-34), and the majority (82%) initiated ART during pregnancy at a median gestational age of 24 weeks (IQR 16-28). Ninety-one per cent of the cases (39/43) who started ART during pregnancy defaulted by three months postpartum. HIV disclosure to the primary sex partner was more common among women retained in care (100% versus 78%, p < 0.001). Odds of retention were significantly higher among women with: age >25 years (odds ratio (OR) 2.44), completion of primary school (OR 3.06), awareness of partner HIV status (OR 5.20), pre-ART education (OR 6.17), higher number of correct answers to Option B+ knowledge questions (OR 1.82), and support while taking ART (OR 3.65). Pre-ART education and knowledge were significantly correlated (r = 0.43, p < 0.001). In multivariate analysis, awareness of partner HIV status (OR 4.07, 95% confidence interval (CI) 1.51-10.94, p = 0.02) and Option B+ knowledge (OR 1.60, 95% CI 1.15-2.23, p = 0.004) remained associated with retention. CONCLUSIONS: Interventions that address partner disclosure and strengthen pre-ART education around the benefits of ART for maternal and child health should be evaluated to improve retention in Malawi's Option B+ programme.

CD4 Variability in Malawi: Implications for Use of a CD4 Threshold of 500 Cells/mm3 Versus Universal Eligibility for Antiretroviral Therapy.

Background. Given the uncertainty about the ability of a single CD4 count to accurately classify a patient as antiretroviral therapy (ART) eligible, we sought to understand the extent to which CD4 variability results in misclassification at a CD4 threshold of 500 cells/mm3. Methods. We performed a prospective study of CD4 variability in Malawian human immunodeficiency virus-infected, ART-naive, World Health Organization (WHO) stage 1 or 2, nonpregnant adults. CD4 counts were performed daily for 8 days. We fit a Bayesian linear mixed-effects model of log-transformed CD4 cell counts to the data. We used Monte Carlo approximations to estimate misclassification rates for different observed values of CD4. The misclassification rate was calculated based on the conditional probability of true CD4 given the geometric mean of observed CD4 measurements. Results. Fifty patients were enrolled from 2 sites. The median age was 33.5 years (interquartile range, 27.5-40.0) and 34 (68%) were female. Misclassification rates were <1% when the observed CD4 counts were ≤250 or ≥750 cells/mm3. Rates of misclassification were high at observed CD4 counts between 350 and 650 cells/mm3, particularly when a single measurement was used (up to 46.7%). Conclusions. Our data show that ART eligibility based on a single CD4 count results in highest risk of misclassification when observed CD4 counts are in the range of 350-650 cells/mm3. Given the benefits of early ART, countries should weigh the costs and complexity of CD4 testing using a 500 cell/mm3 threshold against the cost savings and public health benefits of universal eligibility.

Efficacy of intravaginal dehydroepiandrosterone (DHEA) on moderate to severe dyspareunia and vaginal dryness, symptoms of vulvovaginal atrophy, and of the genitourinary syndrome of menopause.

OBJECTIVE: The aim of this study is to confirm the local beneficial effects of intravaginal dehydroepiandrosterone (DHEA, Prasterone) on moderate to severe dyspareunia or pain at sexual activity, the most frequent symptom of vulvovaginal atrophy due to menopause or genitourinary syndrome of menopause (GSM). METHODS: In a prospective, randomized, double-blind, and placebo-controlled phase III clinical trial, the effect of daily intravaginal 0.50% DHEA (6.5 mg) (Prasterone, EndoCeutics) was examined on four coprimary objectives, namely percentage of parabasal cells, percentage or superficial cells, vaginal pH, and moderate to severe pain at sexual activity (dyspareunia) identified by the women as their most bothersome vulvovaginal atrophy symptom. The intent-to-treat population included 157 and 325 women in the placebo and DHEA-treated groups, respectively. RESULTS: After daily intravaginal administration of 0.50% DHEA for 12 weeks, when compared to baseline by the analysis of covariance test, the percentage of parabasal cells decreased by 27.7% over placebo (P < 0.0001), whereas the percentage of superficial cells increased by 8.44% over placebo (P < 0.0001), vaginal pH decreased by 0.66 pH unit over placebo (P < 0.0001), and pain at sexual activity decreased by 1.42 severity score unit from baseline or 0.36 unit over placebo (P = 0.0002). On the other hand, moderate to severe vaginal dryness present in 84.0% of women improved at 12 weeks by 1.44 severity score unit compared to baseline, or 0.27 unit over placebo (P = 0.004). At gynecological evaluation, vaginal secretions, epithelial integrity, epithelial surface thickness, and color all improved by 86% to 121% over the placebo effect (P < 0.0001 for all comparisons with placebo). Serum steroid levels remained well within the normal postmenopausal values according to the involved mechanisms of intracrinology. The only side effect reasonably related to treatment is vaginal discharge due to melting of the vehicle at body temperature and this was reported in about 6% of the participants. CONCLUSIONS: The daily intravaginal administration of 0.50% (6.5 mg) DHEA (Prasterone) has shown clinically and highly statistically significant effects on the four coprimary parameters suggested by the US Food and Drug Administration. The strictly local action of Prasterone is in line with the absence of significant drug-related adverse events, thus showing the high benefit-to-risk ratio of this treatment based upon the novel understanding of the physiology of sex steroids in women.

Effect of Intravaginal Prasterone on Sexual Dysfunction in Postmenopausal Women with Vulvovaginal Atrophy.

INTRODUCTION: Previous data have shown that intravaginal dehydroepiandrosterone (DHEA, prasterone) improved all the domains of sexual function, an effect most likely related to the local formation of androgens from DHEA. AIMS: To confirm in a placebo-controlled, prospective, double-blind and randomized study the benefits of daily intravaginal DHEA for 12 weeks on sexual function using the Female Sexual Function Index (FSFI) questionnaire. METHODS: Placebo was administered daily to 157 women while 325 women received 0.50% (6.5 mg) DHEA daily for 12 weeks. All women were postmenopausal meeting the criteria of vulvovaginal atrophy (VVA), namely moderate to severe dyspareunia as their most bothersome symptom of VVA in addition to having ≤5% of vaginal superficial cells and vaginal pH > 5.0. The FSFI questionnaire was filled at baseline (screening and day 1), 6 weeks and 12 weeks. Comparison between DHEA and placebo of the changes from baseline to 12 weeks was made using the analysis of covariance test, with treatment group as the main factor and baseline value as the covariate. MAIN OUTCOME MEASURES: The six domains and total score of the FSFI questionnaire were evaluated. RESULTS: The FSFI domain desire increased over placebo by 0.24 unit (+49.0%, P = 0.0105), arousal by 0.42 unit (+56.8%, P = 0.0022), lubrication by 0.57 unit (+36.1%, P = 0.0005), orgasm by 0.32 unit (+33.0%, P = 0.047), satisfaction by 0.44 unit (+48.3%, P = 0.0012), and pain at sexual activity by 0.62 unit (+39.2%, P = 0.001). The total FSFI score, on the other hand, has shown a superiority of 2.59 units in the DHEA group over placebo or a 41.3% greater change than placebo (P = 0.0006 over placebo). CONCLUSION: The present data show that all the six domains of the FSFI are improved over placebo (from P = 0.047 to 0.0005), thus confirming the previously observed benefits of intravaginal DHEA on female sexual dysfunction by an action exerted exclusively at the level of the vagina, in the absence of biologically significant changes of serum steroids levels.

Pregnancy desire and dual method contraceptive use among people living with HIV attending clinical care in Kenya, Namibia and Tanzania.

AIM: To describe factors associated with pregnancy desire and dual method use among people living with HIV in clinical care in sub-Saharan Africa. DESIGN: Sexually active HIV-positive adults were enrolled in 18 HIV clinics in Kenya, Namibia and Tanzania. Demographic, clinical and reproductive health data were captured by interview and medical record abstraction. Correlates of desiring a pregnancy within the next 6 months, and dual method use [defined as consistent condom use together with a highly effective method of contraception (hormonal, intrauterine device (IUD), permanent)], among those not desiring pregnancy, were identified using logistic regression. RESULTS: Among 3375 participants (median age 37 years, 42% male, 64% on antiretroviral treatment), 565 (17%) desired a pregnancy within the next 6 months. Of those with no short-term fertility desire (n=2542), 686 (27%) reported dual method use, 250 (10%) highly effective contraceptive use only, 1332 (52%) condom use only, and 274 (11%) no protection. Respondents were more likely to desire a pregnancy if they were from Namibia and Tanzania, male, had a primary education, were married/cohabitating, and had fewer children. Factors associated with increased likelihood of dual method use included being female, being comfortable asking a partner to use a condom, and communication with a health care provider about family planning. Participants who perceived that their partner wanted a pregnancy were less likely to report dual method use. CONCLUSIONS: There was low dual method use and low use of highly effective contraception. Contraceptive protection was predominantly through condom-only use. These findings demonstrate the importance of integrating reproductive health services into routine HIV care.

Psychosocial functioning and depressive symptoms among HIV-positive persons receiving care and treatment in Kenya, Namibia, and Tanzania.

In sub-Saharan Africa, the prevalence of depressive symptoms among people living with HIV (PLHIV) is considerably greater than that among members of the general population. It is particularly important to treat depressive symptoms among PLHIV because they have been associated with poorer HIV care-related outcomes. This study describes overall psychosocial functioning and factors associated with depressive symptoms among PLHIV attending HIV care and treatment clinics in Kenya, Namibia, and Tanzania. Eighteen HIV care and treatment clinics (six per country) enrolled approximately 200 HIV-positive patients (for a total of 3,538 participants) and collected data on patients' physical and mental well-being, medical/health status, and psychosocial functioning. Although the majority of participants did not report clinically significant depressive symptoms (72 %), 28 % reported mild to severe depressive symptoms, with 12 % reporting severe depressive symptoms. Regression models indicated that greater levels of depressive symptoms were associated with: (1) being female, (2) younger age, (3) not being completely adherent to HIV medications, (4) likely dependence on alcohol, (5) disclosure to three or more people (versus one person), (6) experiences of recent violence, (7) less social support, and (8) poorer physical functioning. Participants from Kenya and Namibia reported greater depressive symptoms than those from Tanzania. Approximately 28 % of PLHIV reported clinically significant depressive symptoms. The scale-up of care and treatment services in sub-Saharan Africa provides an opportunity to address psychosocial and mental health needs for PLHIV as part of comprehensive care.

The emphysematous lung is abnormally sensitive to TRAIL-mediated apoptosis.

BACKGROUND: Alveolar apoptosis is increased in the emphysematous lung. However, mechanisms involved are not fully understood. Recently, we demonstrated that levels of TRAIL receptor 1 and 2, levels of p53, and Bax/Bcl-xL ratio were elevated in the lung of subjects with emphysema, despite smoking cessation. Thus, we postulate that due to chronic pulmonary oxidative stress, the emphysematous lung would be abnormally sensitive to TRAIL-mediated apoptosis. METHODOLOGY: A549 cells were exposed to rTRAIL, cigarette smoke extract, and/or H2O2 prior to caspase-3 activity measurement and annexin V staining assessment. In addition, freshly resected lung samples were obtained from non-emphysematous and emphysematous subjects and exposed ex vivo to rTRAIL for up to 18 hours. Lung samples were harvested and levels of active caspase-3 and caspase-8 were measured from tissue lysates. RESULTS: Both cigarette smoke extract and H2O2 were able to sensitize A549 cells to TRAIL-mediated apoptosis. Moreover, following exposure to rTRAIL, caspase-3 and -8 were activated in lung explants from emphysematous subjects while being decreased in lung explants from non-emphysematous subjects. SIGNIFICANCE OF THE STUDY: Alveolar sensitivity to TRAIL-mediated apoptosis is strongly increased in the emphysematous lung due to the presence of oxidative stress. This might be a new mechanism leading to increased alveolar apoptosis and persistent alveolar destruction following smoking cessation.

Alveolar epithelial and endothelial cell apoptosis in emphysema: what we know and what we need to know.

Emphysema is mainly caused by cigarette smoking and is characterized by the loss of alveolar integrity and an enlargement of the alveolar space. However, mechanisms involved in its development are not fully understood. Alveolar cell apoptosis has been previously investigated in the lung of emphysematous subjects as a potential contributor to the loss of alveolar cell and has been found abnormally elevated. Though, mechanisms involved in the increased alveolar apoptosis that occurs in emphysema have now become a prolific field of research. Those mechanisms are reviewed here with special focus on how they affect cell viability and how they may be implicated in emphysema. Moreover, we suggest a model that integrates all those mechanisms to explain the increased alveolar apoptosis observed in emphysema. This review also includes some reflections and suggestions on the research to come.

Increased p53 level, Bax/Bcl-x(L) ratio, and TRAIL receptor expression in human emphysema.

RATIONALE: Emphysema is mainly known for the complex inflammatory processes associated with its development. In addition to lung inflammation, it is now accepted that increased alveolar cell apoptosis is also part of emphysema pathophysiology. However, little is known about the mechanisms involved in alveolar apoptosis. We postulate that oxidative stress and proinflammatory cytokines could lead to p53 accumulation, Bax/Bcl-x(L) ratio elevation, and higher tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL) receptor levels in the emphysematous lung. OBJECTIVES: To evaluate the expression of p53, Bax, Bcl-x(L), TRAIL, and TRAIL receptors in lung parenchyma from nonemphysematous nonsmokers and smokers and emphysematous smokers and ex-smokers and to determine whether H2O2 and/or TNF can modulate the expression of these apoptotic proteins. METHODS: p53, Bax, Bcl-x(L), and TRAIL receptor protein levels in lung parenchyma were measured by Western blot, and TRAIL mRNA levels were measured by real-time polymerase chain reaction. Changes in TRAIL receptor, Bax, Bcl-x(L), and p53 protein levels after in vitro H2O2 and/or TNF stimulation of A549 cells were also assessed by Western blot. MEASUREMENTS AND MAIN RESULTS: The p53 protein levels, the Bax/Bcl-x(L) ratio, and TRAIL receptors 1, 2, and 3 protein levels were significantly higher in subjects with emphysema. Moreover, they were also increased after H2O2 and TNF treatments of A549 cells. CONCLUSIONS: These findings suggest that oxidative stress and proinflammatory cytokines may be involved in the elevation of p53 levels, the Bax/Bcl-x(L) ratio, and TRAIL receptor levels, new mechanisms that may be implicated in the increased alveolar cell apoptosis that occurs in emphysema.

Perforin, granzyme B, and FasL expression by peripheral blood T lymphocytes in emphysema.

BACKGROUND: It is generally accepted that emphysematous lungs are characterized by an increase in the numbers of neutrophils, macrophages, and CD8+ T lymphocytes, the lasts having increased cytotoxic activity. Because systemic inflammation is also a component of emphysema, we hypothesize that peripheral CD8+ T lymphocytes of emphysematous smokers who show evidence of systemic inflammation will have higher expression of cytotoxic molecules. METHODS: We assessed parameters of systemic inflammation in normal individuals (smokers or non-smokers) and in emphysematous subjects with an active smoking history by measuring serum interleukine-6, C-reactive protein, and tumor necrosis factor. Expression of perforin, granzyme B, and FasL protein by CD8+ T lymphocytes, CD4+ T lymphocytes, and natural killer cells were assessed by flow cytometry while perforin, granzyme B, and FasL mRNA expression were measured on purified systemic CD8+ T lymphocytes by real-time PCR. RESULTS: Emphysematous smokers had higher levels of serum interleukine-6 than normal subjects. Even with the presence of systemic inflammation in emphysematous smokers, the percentage of peripheral CD8+ T lymphocytes, CD4+ T lymphocytes, and NK cells expressing perforin and granzyme B protein was not different between the three groups. CONCLUSION: Despite evidence of systemic inflammation, peripheral T lymphocytes of emphysematous smokers did not show higher levels of cytotoxic markers, suggesting that increase of activated T lymphocytes in the emphysematous lung may be due to either activation in the lung or specific peripheral recruitment.

Molecular cloning and tissue distribution of microsomal-1 and cytosolic prostaglandin E synthases in macaque.

Prostaglandins derived from arachidonic acid are involved in a wide variety of physiological and pathological processes. The primary enzymes involved in the production of PGE2 from arachidonic acid are cyclooxygenases and prostaglandin E synthases. These enzymes have been identified in human, but only partially in the monkey where microsomal PGES-1 and cytosolic PGES have not been characterized. The present study was undertaken to clone these enzymes and to study their tissue distribution, along with mPGES-2. The coding sequence of Macaque mPGES-1 is 98% homologous to human mPGES-1 at the nucleic acid level and the deduced amino acid sequence has 98% homology with the human protein. The Macaque cPGES cDNA is more than 99% homologous to the human and the deduced amino acids sequence is identical to that of the human cPGES. By Northern blot analysis, we found that mPGES-2 and cPGES mRNA were expressed in the endometrium, myometrium, ovary and oviduct, albeit at different levels, while mPGES-1 mRNA was detected at a weak level, mainly in the oviduct. Western Blot analysis revealed that mPGES-2, mPGES-1 and cPGES proteins were present in all tissues tested. These results suggest that production of PGE2 in Macaque may involve more than one PGES and that further studies will be needed to fully understand the conditions under which each PGES contributes to PGE2 production.

Expression and contribution of three different isoforms of prostaglandin E synthase in the bovine endometrium.

Prostaglandins are involved in the regulation of several reproductive processes such as ovulation, luteolysis, and establishment of pregnancy. Prostaglandin E(2) (PGE(2)) appears to favor establishment of pregnancy in most mammals studied so far. The primary enzymes involved in the production of PGE(2) from arachidonic acid are cyclooxygenases and prostaglandin E synthases (PGES). Three PGES have been identified in humans, but in the bovine, microsomal PGES2 and cytosolic PGES genes have neither been cloned nor associated to any physiological processes. The present study was undertaken to clone bovine MPGES2 and CPGES and to report on their regulation in the endometrium during the estrous cycle. CPGES mRNA expression declines progressively during the cycle; its protein is not modulated according to a precise pattern. MPGES2 mRNA and protein expression decrease from the beginning of the cycle until Days 13-15 and then increase until ovulation. Immunohistochemical analysis reveals that both enzymes are located in luminal epithelial and glandular epithelial cells and at a lower level in stromal cells. In addition, using the bovine endometrial cell line BEND, where higher accumulation of PGE(2) is observed following treatment with phorbol 12-myristate 13-actetate (PMA) and tumor necrosis factor-alpha (TNF-alpha), we have found an associated increase of MPGES1 and COX2 but not CPGES or MPGES2 protein expression. Together, our results suggest that MPGES1 is not the only PGES present in the bovine endometrium but is the main enzyme associated with increased PGE(2) production in vitro.

Expression of human prostaglandin transporter in the human endometrium across the menstrual cycle.

Prostaglandins (PGs) are important regulators of reproductive function. The mechanism by which PGs are transported across the biological membrane is a new emerging field of investigation. Prostaglandin transporter (PGT) has been identified as a functional PG carrier. The aim of our study was to outline the expression of PGT in the human endometrium across the menstrual cycle. Quantitative RT-PCR showed human PGT (hPGT) expression to be strong in the proliferative and early secretory phases and low in the middle to late secretory phase. Northern blot analysis revealed hPGT mRNA transcript of 4 kb in the human endometrium. A peptide-directed polyclonal antibody was generated in rabbits against the 22 amino acids forming the C terminus of hPGT. Antibody specificity was demonstrated by Western blot. Immunoblots of endogenous hPGT in the human endometrium revealed a 70-kDa protein in endometrial cells. Endometrial biopsies collected across the menstrual cycle were used to assess hPGT protein expression by immunohistochemistry. hPGT was immunolocalized to luminal, glandular epithelial, and stromal cells. Because it was observed at the mRNA level, semiquantitative analysis showed a higher protein expression in proliferative and early secretory phases than in the mid-late secretory phase. In conclusion, our study revealed that hPGT expression is modulated in epithelial and stromal cells of the human endometrium at both mRNA and protein levels during the menstrual cycle. These findings support a role for hPGT as an important new player in the regulation of PG action in the human endometrium.

The effects of estrogen and progesterone on prostaglandins and integrin beta 3 (beta3) subunit expression in primary cultures of bovine endometrial cells.

In cattle, endometrial expression of integrin alphavbeta3 is reduced on day 16 of the estrous cycle, coinciding with the critical period during which the decision is made to initiate luteolysis or continue with pregnancy. The objective of these experiments was to examine the relationship between estrogen and progesterone treatments, endometrial integrin alphavbeta3 expression, and prostaglandin F2alpha (PGF2alpha) and E2 (PGE2) production. Epithelial and stromal cells from intercaruncular (ICAR) and caruncular (CAR) bovine endometrium were treated with 17beta-estradiol (0.1 and 1.0 nM) and/or progesterone (1.0 and 10 nM) in a manner designed to mimic the steroid fluctuations of the estrous cycle. All cell types expressed estrogen receptor and progesterone receptor mRNA and protein. Intercaruncular stromal cells were the most responsive to steroidal regulation. Estrogen suppressed expression of integrin subunit beta3 mRNA in ICAR stromal cells (P< or =0.05). Progesterone and estrogen + progesterone treated cells did not differ in beta3 expression from controls (P> or =0.05). Steroid treatment did not affect PGF2alpha production in any cell type (P> or =0.05), however, estrogen decreased PGE2 production in all cells except CAR stroma (P< or =0.05). The results indicate that in bovine endometrium expression of integrin alphavbeta3 and production of PGE2 is influenced by estrogen.

Influence of different isoforms of recombinant trophoblastic interferons on prostaglandin production in cultured bovine endometrial cells.

In ruminants, interferon produced by the trophectoderm (IFN-tau) is recognized as the embryonic signal responsible for maternal recognition of pregnancy. IFN-tau is believed to act by down-regulating estrogen receptors, thus preventing appearance of oxytocin receptors responsible for the release of prostaglandin F(2alpha) (PGF(2alpha)) by the endometrium. The present study was undertaken to determine in vitro the biological activities of different IFN-tau isoforms and document putative alternate luteotrophic mechanisms. Endometrial cells in primary cultures were treated with five different rIFN-tau isoforms: two ovine isoforms (ro-4 and ro-11) and three bovine isoforms (rb-1a, rb-2b and rb-3b). Their effect was quantified by measurement of PGE(2) and PGF(2alpha) production by ELISA and induction of cyclooxygenase (COX-2) by Western and Northern analysis and correlated with antiviral activity previously reported. The overall pattern of response to the IFNs tested suggests that low concentrations (<1 microg/ml) reduced the production of both PGs and higher concentrations (>1 microg/ml) stimulated preferentially PGE(2); however, exceptions were noted. Isoform rb-2b with high antiviral activity inhibited PG production in both cell types at all concentrations tested. IFNs rb-1a and ro-11 had similar antiviral activities, inhibiting PG at low concentrations and stimulating them at high concentrations. Isoform rb-3b stands out relative to the other IFNs tested because it induced a variable non-dose-dependent effect on PG production and low antiviral activity. An increase in COX-2 protein expression and messenger was correlated with increased PG production. The results showing two distinct responses to IFN-tau depending on its concentration and/or isoform and the absence of correlation with antiviral activity suggest that complex transduction mechanisms are involved.