Determinants of Survival of HIV Patients Receiving Dolutegravir: A Prospective Cohort Study in Conflict-Affected Bunia, Democratic Republic of Congo.

This study aims to determine the factors influencing HIV-related mortality in settings experiencing continuous armed conflict atrocities. In such settings, people living with HIV (PLHIV), and the partners of those affected may encounter specific difficulties regarding adherence to antiretroviral therapy (ART), and retention in HIV prevention, treatment, and care programs. Between July 2019 and July 2021, we conducted an observational prospective cohort study of 468 PLHIV patients treated with Dolutegravir at all the ART facilities in Bunia. The probability of death being the primary outcome, as a function of time of inclusion in the cohort, was determined using Kaplan-Meier plots. We used the log-rank test to compare survival curves and Cox proportional hazard modeling to determine mortality predictors from the baseline to 31 July 2021 (endpoint). The total number of person-months (p-m) was 3435, with a death rate of 6.70 per 1000 p-m. Compared with the 35-year-old reference group, older patients had a higher mortality risk. ART-naïve participants at the time of enrollment had a higher mortality risk than those already using ART. Patients with a high baseline viral load (≥1000 copies/mL) had a higher mortality risk compared with the reference group (adjusted hazard ratio = 6.04; 95% CI: 1.78-20.43). One-fourth of deaths in the cohort were direct victims of armed conflict, with an estimated excess death of 35.6%. Improving baseline viral load monitoring, starting ART early in individuals with high baseline viral loads, the proper tailoring of ART regimens and optimizing long-term ART, and care to manage non-AIDS-related chronic complications are recommended actions to reduce mortality. Not least, fostering women's inclusion, justice, peace, and security in conflict zones is critical in preventing premature deaths in the general population as well as among PLHIV.

Determinants of Patients' Adherence to Malaria Treatment in the Democratic Republic of the Congo.

(1) Background: Malaria heavily affects the Democratic Republic of the Congo (DRC) despite the use of effective drugs. Poor adherence to malaria treatment may contribute to this problem. (2) Methods: In one rural and one urban health area in each of the 11 former provinces of the DRC, all households with a case of malaria in the 15 days preceding the survey were selected and the patients or caregivers were interviewed. Adherence to malaria treatment was assessed by self-declaration about its completion. Logistic regression was used to assess predictors. (3) Results: 1732 households participated. Quinine was the most used drug; adherence to artesunate-amodiaquine was the lowest and the main reason for treatment discontinuation was adverse reactions. Predictors of adherence were residence in an urban area, university education, catholic religion, and adoption of recommended behaviour towards a malaria case. Adherence was significantly lower for responders who obtained information on antimalarials from Community Health Workers (CHW). (4) Conclusions: Usage of recommended drugs and adherence to malaria treatment need to be promoted, especially in rural areas, and CHW involvement needs to be improved. Awareness messages need to be made accessible and comprehensible to poorly educated populations and churches need to be involved.

Incidence and Predictors of Loss to Follow Up among Patients Living with HIV under Dolutegravir in Bunia, Democratic Republic of Congo: A Prospective Cohort Study.

This study aimed to examine the incidence and predictors of loss to follow up (LTFU) in the context of ongoing atrocities caused by armed conflict, where HIV treatment programs and HIV-infected patients may face unique challenges in terms of ART adherence and retention in care. We conducted an observational prospective cohort study of 468 patients living with HIV (PLWHIV) under dolutegravir (DTG) in all health facilities in Bunia between July 2019 and July 2021. Kaplan-Meier plots were used to determine the probability of LTFU as a function of time as inclusive of the cohort. The main outcome variable was LTFU, defined as not taking an ART refill for a period of 3 months or longer from the last attendance for refill, and not yet classified as 'dead' or 'transferred-out.' The log-rank test was used to compare survival curves based on predictors. Cox proportional hazard modeling was used to measure predictors of LTFU from the baseline until 31 July 2021 (the endpoint). A total of 3435.22 person-months (p-m) were involved in follow up, with an overall incidence rate of 33.48 LTFU per 1000 p-m. Patients who had less experience with ART at enrolment and the ethnically Sudanese, had a higher hazard of being LTFU compared to their reference groups. This study reports a high LTFU rate in this conflict setting. An ART program in such a setting should pay more attention to naive patients and other particularly vulnerable patients such as Sudanese during the pre-ART phase. The study implies the implementation of innovative strategies to address this high risk of being LTFU, reducing either the cost or the distance to the health facility.

Predictors of Viral Non-Suppression among Patients Living with HIV under Dolutegravir in Bunia, Democratic Republic of Congo: A Prospective Cohort Study.

The Democratic Republic of the Congo adopted the integrase inhibitor dolutegravir (DTG) as part of its preferred first-line HIV treatment regimen in 2019. This study aimed to identify predictors of viral non-suppression among HIV-infected patients under a DTG-based regimen in the context of ongoing armed conflict since 2017 in the city of Bunia in the DRC. We conducted a cohort study of 468 patients living with HIV under DTG in all health facilities in Bunia. We calculated the proportion of participants with an HIV RNA of below 50 copies per milliliter. About three in four patients (72.8%) in this cohort had a viral load (VL) of <50 copies/mL after 6-12 months. After controlling for the effect of other covariates, the likelihood of having non-suppression remained significantly lower among the 25-34 age group and self-reported naïve patients with a baseline VL of ≥50 copies/mL. The likelihood of having non-suppression remained significantly higher among those who were at advanced stages of the disease, those with abnormal serum creatinine, those with high baseline HIV viremia over 1000 copies/mL, and the Sudanese ethnic group compared to the reference groups. This study suggests that we should better evaluate adherence, especially among adolescents and economically vulnerable populations, such as the Sudanese ethnic group in the city of Bunia. This suggests that an awareness of the potential effects of DTG and tenofovir is important for providers who take care of HIV-positive patients using antiretroviral therapy (ART), especially those with abnormal serum creatinine levels before starting treatment.

Knowledge of Antimalarials and Health Seeking Behaviour of Households in Case of Suspected Malaria in Democratic Republic of the Congo.

(1) Background: The Democratic Republic of the Congo (DRC) is heavily affected by malaria despite availability of effective treatments. Ignorance and unrecommended behaviour toward a suspected malaria case in households may contribute to this problem. (2) Method: In communities of one rural and one urban Health Centres in each of the 11 previous provinces of DRC, all households with a case of malaria in the 15 days prior to the survey were selected. The patient or caregiver (responder) were interviewed. Logistic regression was used to assess predictors of knowledge of recommended antimalarials and adequate behaviour in case of suspected malaria. (3) Results: 1732 households participated; about 62% (1060/1721) of the responders were informed about antimalarials, 70.1% (742/1059) knew the recommended antimalarials and 58.6% (995/1699) resorted to self-medication. Predictors of knowledge of antimalarials were education to secondary school or university, information from media and smaller households. Predictors of good behaviour were Catholic religion and smaller households. Receiving information from Community Health Workers (CHWs) failed to be determinants of knowledge or adequate behaviour. (4) Conclusion: malaria control in DRC is hampered by ignorance and non-adherence to national recommendations. These aspects are influenced by unsuccessful communication, size of households and level of education.

Drug use in the management of uncomplicated malaria in public health facilities in the Democratic Republic of the Congo.

BACKGROUND: Malaria the first causes of death from parasitic infection worldwide. Interventions to reduce the burden of malaria have produced a tremendous drop in malaria morbidity and mortality. However, progress is slower in DRC, which shares with Nigeria 39% of deaths related to malaria globally. Inappropriate use of drugs may be one of the factors of this below-average performance. The aim of this study was to describe the use of drugs in the management of uncomplicated malaria in public health facilities in DRC. METHODS: A drug use study was carried out in DRC from January to March 2014. In each of the former 11 provinces of DRC, one Rural Health Centre, one Urban Health Centre and one General Hospital were selected. In each of them, 100 patient's files containing prescription of anti-malarials from January to December 2013 were randomly selected. Among them, all of the files with diagnosis of uncomplicated malaria were included in this study. Prescribed anti-malarials, co-prescribed drugs and their indications were collected. Descriptive analyses were performed. RESULTS: A total of 2300 files out of 3300 (69.7%) concerned uncomplicated malaria and were included in analysis. Malaria treatment was initiated after a positive RDT or microscopy in 51.5% of cases, upon suspicion without requesting biological confirmation in 37% and despite negative results in 11%. Twenty-nine (29) different treatment regimens were used. The drugs recommended by the National Malaria Control Programme were used in 54.3% of cases (artesunate-amodiaquine 37.4% or artemether-lumefantrine 16.9%). The second most used anti-malarial was quinine (32.4%). Apart from anti-malarials, an average of 3.1 drugs per patient were prescribed, among which antibiotics (67.9%), analgesics and non-steroidal anti-inflammatory (NSAIDs) (all abbreviations to be explicated on first use) (70.6%), vitamins (29.1%), anaemia drugs, including blood transfusion (9.1%) and corticosteroids (5.7%), In 51.4% of cases there was no indication for the concomitant medication. CONCLUSION: Management of uncomplicated malaria in DRC is characterized by a low adherence to treatment policy, numerous treatment regimens, and abundant concomitant medication potentially harmful to the patient. This may contribute to the low performance of DRC in malaria control. Determinant of this irrational use of drugs need to be assessed in order to formulate and implement efficient corrective measures.

Thyroid hormone in the regulation of hepatocellular carcinoma and its microenvironment.

Hepatocellular carcinoma (HCC) commonly arises from a liver damaged by extensive inflammation and fibrosis. Various factors including cytokines, morphogens, and growth factors are involved in the crosstalk between HCC cells and the stromal microenvironment. Increasing our understanding of how stromal components interact with HCC and the signaling pathways involved could help identify new therapeutic and/or chemopreventive targets. It has become increasingly clear that the cross-talk between tumor cells and host stroma plays a key role in modulating tumor growth. Emerging reports suggest a relationship between HCC and thyroid hormone signaling (dysfunction), raising the possibility that perturbed thyroid hormone (TH) regulation influences the cancer microenvironment and cancer phenotype. This review provides an overview of the role of thyroid hormone and its related pathways in HCC and, specifically, its role in regulating the tumor microenvironment.

Tendances de la tuberculose pulmonaire bactériologiquement confirmée et issues thérapeutiques en République Démocratique du Congo : 2007-2017

Contexte et objectifs. La République Démocratique du Congo compte parmi les pays à lourd fardeau pour la tuberculose (TB), l'incidence réelle de la maladie n'est pas formellement connue. La présente étude vise à décrire les tendances de l'incidence notifiée des patients atteints de tuberculose pulmonaire bactériologiquement confirmée (TP+) et leurs issues thérapeutiques. Méthodes. Cette étude documentaire, analyse les données des patients diagnostiqués et traités pour tuberculose de 2007 à 2017 en RDC. L'incidence notifiée des patients TP+, le taux d'accroissement annuel, les issues thérapeutiques ont été recherchés. Les variations du nombre de patients sont exprimées par les proportions. Les tendances sont présentées à travers les courbes de régression linéaire. Les issues thérapeutiques sont comparées à l'aide du z-score avec un seuil significatif de p˂ 0,05. Résultats. Au total 884 458 patients TP+ ont été rapportés, dont 820 858 nouveaux patients (NP TP+) et 63 600 déjà traités. Le taux d'accroissement au cours de cette décade était de 28,95%, soit de 66099 en 2007 à 93767 en 2017 pour les NP TP+. L'augmentation annuelle moyenne était de 2,41% +/- 3,28 pour les NP TP+ et de 5,7% +/- 0,26 par an pour les rechutes. La notification des échecs de traitement initial et repris après abandon de traitement ont une tendance à la baisse. L'évaluation thérapeutique de tous les cas cumulés a concerné 848 163 patients dont 789 716 NP TP+ et 58447 en retraitement. Le succès thérapeutique était de 88,0 % pour les NP TP+ et 70,0 % pour les rechutes, de 64,3 % pour les échecs et de 67,8% pour les repris en traitement après abandon. En somme 70 515 (8,3%) patients ont gardé des expectorations positives. Conclusion. Cette étude montre une tendance à la hausse de notification des cas incidents dont l'issue de traitement répond aux standards de l'OMS. En outre, un nombre des personnes demeurent porteurs de germes persistants précurseurs d'une TB pharmacorésistante acquise

Tendances de la tuberculose pulmonaire bactériologiquement confirmée et issues thérapeutiques en République Démocratique du Congo : 2007-2017

Contexte et objectifs. La République Démocratique du Congo compte parmi les pays à lourd fardeau pour la tuberculose (TB), l'incidence réelle de la maladie n'est pas formellement connue. La présente étude vise à décrire les tendances de l'incidence notifiée des patients atteints de tuberculose pulmonaire bactériologiquement confirmée (TP+) et leurs issues thérapeutiques. Méthodes. Cette étude documentaire, analyse les données des patients diagnostiqués et traités pour tuberculose de 2007 à 2017 en RDC. L'incidence notifiée des patients TP+, le taux d'accroissement annuel, les issues thérapeutiques ont été recherchés. Les variations du nombre de patients sont exprimées par les proportions. Les tendances sont présentées à travers les courbes de régression linéaire. Les issues thérapeutiques sont comparées à l'aide du z-score avec un seuil significatif de p˂ 0,05. Résultats. Au total 884 458 patients TP+ ont été rapportés, dont 820 858 nouveaux patients (NP TP+) et 63 600 déjà traités. Le taux d'accroissement au cours de cette décade était de 28,95%, soit de 66099 en 2007 à 93767 en 2017 pour les NP TP+. L'augmentation annuelle moyenne était de 2,41% +/- 3,28 pour les NP TP+ et de 5,7% +/- 0,26 par an pour les rechutes. La notification des échecs de traitement initial et repris après abandon de traitement ont une tendance à la baisse. L'évaluation thérapeutique de tous les cas cumulés a concerné 848 163 patients dont 789 716 NP TP+ et 58447 en retraitement. Le succès thérapeutique était de 88,0 % pour les NP TP+ et 70,0 % pour les rechutes, de 64,3 % pour les échecs et de 67,8% pour les repris en traitement après abandon. En somme 70 515 (8,3%) patients ont gardé des expectorations positives. Conclusion. Cette étude montre une tendance à la hausse de notification des cas incidents dont l'issue de traitement répond aux standards de l'OMS. En outre, un nombre des personnes demeurent porteurs de germes persistants précurseurs d'une TB pharmacorésistante acquise

Vascular hepoxilin and trioxilins mediate vasorelaxation through TP receptor inhibition in mouse arteries.

AIM: 12/15-lipoxygenase (12/15-LO) metabolizes arachidonic acid (AA) into several vasoactive eicosanoids. In mouse arteries, we previously characterized the enzyme's 15-LO metabolites 12(S)-hydroxyeicosatetraenoic acid (HETE), 15-HETE, hydroxyepoxyeicosatrienoic acids (HEETAs) and 11,12,15-trihydroxyeicosatrienoic acids (11,12,15-THETAs) as endothelium-derived relaxing factors. However, the observed 12-LO metabolites remained uncharacterized. The purpose of this study was to determine the structure and biological functions of eicosanoids generated by the enzyme's 12-LO activity. METHODS: Metabolites extracted from aortas of C57BL/6 male mice were separated using a series of reverse and normal phase chromatographic steps and identified as hepoxilin A3 , trioxilin A3 and trioxilin C3 by mass spectrometry. Activities of these natural compounds were tested on isometric tension and intracellular calcium release. The role of thromboxane (TP) receptor was determined in HEK293 cells overexpressing TPα receptor (TPα -HEK). RESULTS: All identified vascular 12-LO metabolites were biologically active. In mouse mesenteric arteries, trioxilin A3 , C3 and hepoxilin A3 (3 µm) relaxed arteries constricted with the thromboxane mimetic, U46619-constricted arteries (maximum relaxations of 78.9 ± 3.2, 29.7 ± 4.6, 82.2 ± 5.0 and 88.0 ± 2.4% respectively), but not phenylephrine-constricted arteries. In TPα-HEK cells, trioxilin A3 , C3 and hepoxilin A3 (10 µm) inhibited U46619 (10 nM)-induced increases in intracellular calcium by 53.0 ± 7.2%, 32.8 ± 5.0% and 37.9 ± 13.5% respectively. In contrast, trioxilin B3 and hepoxilin B3 were not synthesized in arteries and exhibited little biological activity. CONCLUSION: Trioxilin A3 and C3 and hepoxilin A3 are endogenous vascular relaxing factors. They are not endothelium-derived hyperpolarizing factors but mediate vascular relaxation by inhibiting TP agonist-induced increases in intracellular calcium. Thus, they regulate vascular homeostasis by acting as endogenous TP antagonists.

Antimalarial activity of medicinal plants from the Democratic Republic of Congo: A review.

ETHNOPHARMACOLOGICAL RELEVANCE: Malaria is the most prevalent parasitic disease and the foremost cause of morbidity and mortality in the Democratic Republic of Congo. For the management of this disease, a large Congolese population recourses to traditional medicinal plants. To date the efficacy and safety of many of these plants have been validated scientifically in rodent malaria models. In order to generate scientific evidence of traditional remedies used in the Democratic Republic of Congo for the management of malaria, and show the potential of Congolese plants as a major source of antimalarial drugs, this review highlights the antiplasmodial and toxicological properties of the Congolese antimalarial plants investigated during the period of 1999-2014. In doing so, a useful resource for further complementary investigations is presented. Furthermore, this review may pave the way for the research and development of several available and affordable antimalarial phytomedicines. MATERIALS AND METHODS: In order to get information on the different studies, a Google Scholar and PubMed literature search was performed using keywords (malaria, Congolese, medicinal plants, antiplasmodial/antimalarial activity, and toxicity). Data from non-indexed journals, Master and Doctoral dissertations were also collected. RESULTS: Approximately 120 extracts and fractions obtained from Congolese medicinal plants showed pronounced or good antiplasmodial activity. A number of compounds with interesting antiplasmodial properties were also isolated and identified. Some of these compounds constituted new scaffolds for the synthesis of promising antimalarial drugs. Interestingly, most of these extracts and compounds possessed high selective activity against Plasmodium parasites compared to mammalian cells. The efficacy and safety of several plant-derived products was confirmed in mice, and a good correlation was observed between in vitro and in vivo antimalarial activity. The formulation of several plant-derived products also led to some clinical trials and license of three plant-derived drugs (Manalaria(®), Nsansiphos(®), and Quinine Pharmakina(®)). CONCLUSION: The obtained results partly justify and support the use of various medicinal plants to treat malaria in folk medicine in the Democratic Republic of Congo. Antimalarial plants used in Congolese traditional medicine represent an important source for the discovery and development of new antimalarial agents. However, in order to ensure the integration of a larger number of plant-derived products in the Congolese healthcare system, some parameters and trends should be considered in further researches, in agreement with the objectives of the "Traditional Medicine Strategy" proposed by the World Health Organization in 2013. These include evaluation of geographical and seasonal variation, investigation of reproductive biology, assessment of prophylactic antimalarial activity, evaluation of natural products as adjuvant antioxidant therapy for malaria, development of plant-based combination therapies and monitoring of herbal medicines in pharmacovigilance systems.

Malaria epidemic outbreaks in the Democratic Republic of Congo, part I: cross-sectional survey in Mweka District.

Background: A series of outbreaks of fever has previously been reported in the DR Congo. The occurrence of similar outbreaks in Mweka district presented the opportunity to investigate these occurrences. Materials and Methods: Health facilities and communities were visited. Permission was obtained to access to health records and a questionnaire was competed in the community. Blood samples for malaria, salmonellosis, Chikungunya, dengue and filovirus testing were obtained both in health facilities and the communities. Capture of mosquitoes and larvae in breeding sites was done and used bednets were collected. Excel, SPSS and Stats Direct were used for analyses of epidemiological data and malaria case management, with the Chi-square test and Fisher's Exact test used for assessing relationships resulting from contingency table analyses. Results: An increase in the number of malaria cases beyond the expected number for the study period was observed in the two health districts located in the savannah zone (p<0.05) and in one health centre among sixteen located in the forest zone (p<0.05). In the health facilities and households visited (653 people), 141 persons had fever of which 82.2% was attributed to Plasmodium falciparum malaria. An incidence of 5.87% was recorded in the first half of 2013. Hundred and sixty patients (6.9%) died among 2,304 admitted for severe malaria in the three referral hospitals, 118 of them were children of under five years old. PCR testing of the blood samples obtained during home visits revealed malaria parasites in 63 (73.3%) of the 86 analysed samples. The test was negative for other parasites and bacteria and one dengue virus case was detected. Anopheles gambiae from Mweka were found to be resistant to permethrin using the WHO susceptibility test, with a knock down rate of ≤ 50% and mortality of ≤ 30%. Conclusion: These investigations confirmed epidemic outbreaks in Mweka District caused by malaria with a high mortality rate in children below five years of age.

Inhibition of 2-arachidonoylglycerol catabolism modulates vasoconstriction of rat middle cerebral artery by the thromboxane mimetic, U-46619.

BACKGROUND AND PURPOSE: Cerebrovascular smooth muscle cells express the CB1 cannabinoid receptor and CB1 agonists produce vasodilatation of the middle cerebral artery (MCA). The thromboxane A2 mimetic, U-46619, increased the content of the endocannabinoid, 2-arachidonoylglycerol (2-AG) in the MCA and 2-AG moderated the vasoconstriction produced by U46619 in this tissue. The purposes of this study were to examine the extent to which 2-AG is catabolized by cerebral arteries and to determine whether blockade of 2-AG inactivation potentiates its feedback inhibition of U-44619-mediated vasoconstriction. EXPERIMENTAL APPROACH: The diameters of isolated, perfused MCA from male rats were measured using videomicroscopy. KEY RESULTS: Exogenous 2-AG produces a CB1 receptor-dependent and concentration-related increase in the diameter of MCA constricted with 5-HT. The E (max) for 2-AG dilation is increased 4-fold in the presence of the metabolic inhibitors 3-(decylthio)-1,1,1-trifluropropan-2-one (DETFP), URB754 and URB597. To examine the role of catabolism in the effects of endogenous 2-AG, vasoconstriction induced by U-46619 was studied. DETFP and URB754, but not the fatty acid amide hydrolase inhibitor, URB597, significantly increased the EC(50) for U-46619. These data support a physiological role for endocannabinoid feedback inhibition in the effects of U-46619 and indicate that endogenously produced 2-AG is also efficiently catabolized within the MCA. CONCLUSIONS AND IMPLICATIONS: MCA express mechanisms for the efficient inactivation of 2-AG, providing further support for an endocannabinoid feedback mechanism that opposes thromboxane-mediated vasoconstriction. These data suggest that potentiation of endogenously produced 2-AG could be a novel therapeutic approach to the treatment of thrombotic stroke.

Regulation of lipoprotein lipase by the oxysterol receptors, LXRalpha and LXRbeta.

Lipoprotein lipase (LPL) is a key enzyme for lipoprotein metabolism and is responsible for hydrolysis of triglycerides in circulating lipoproteins, releasing free fatty acids to peripheral tissues. In liver, LPL is also believed to promote uptake of high density lipoprotein (HDL)-cholesterol and thereby facilitate reverse cholesterol transport. In this study we show that the Lpl gene is a direct target of the oxysterol liver X receptor, LXRalpha. Mice fed diets containing high cholesterol or an LXR-selective agonist exhibited a significant increase in LPL expression in the liver and macrophages, but not in other tissues (e.g. adipose and muscle). Studies in Lxr-deficient mice confirmed that this response was dependent more on the presence of LXRalpha than LXRbeta. Analysis of the Lpl gene revealed the presence of a functional DR4 LXR response element in the intronic region between exons 1 and 2. This response element directly binds rexinoid receptor (RXR)/LXR heterodimers and is sufficient for rexinoid- and LXR agonist-induced transcription of the Lpl gene. Together, these studies further distinguish the roles of LXRalpha and beta and support a growing body of evidence that LXRs function as key regulators of lipid metabolism and are anti-atherogenic.

Role of epoxyeicosatrienoic acids as endothelium-derived hyperpolarizing factor in bovine coronary arteries.

BACKGROUND: Acetylcholine and bradykinin cause endothelium-dependent hyperpolarization and relaxation of vascular smooth muscle. These responses are mediated in part by an endothelium-derived hyperpolarizing factor (EDHF). Epoxyeicosatrienoic acids (EETs) are cytochrome P450 metabolites of arachidonic acid and appear to function as EDHFs in the coronary artery. This conclusion is based on the findings that EETs are synthesized by the vascular endothelium, open calcium-activated potassium (KCa) channels, hyperpolarize and relax vascular smooth muscle. METHODS: Bovine coronary arteries were precontracted with U46619 and isometric tension was measured to agonists. Whole cell K currents were measured in coronary smooth muscle cells by the patch clamp method. RESULTS: Bradykinin caused a concentration-related relaxation (ED50=10(-10) M) which was not affected by inhibitors of cytochrome P450. Inhibition of NO synthase and cyclooxygenase reduced the response to bradykinin (ED(50)=2 x 10(-9) M) and these responses were then completely blocked by cytochrome P450 inhibitors. 11, 12-EET relaxed the precontracted bovine coronary artery with an ED50 of 3 x 10(-8) M in the presence and absence of the endothelium. 14, 15-EET caused an increase in outward potassium current that was blocked by the KCa channel inhibitor iberiotoxin. A series of 14, 15-EET analogs were tested for agonist activity. CONCLUSIONS: These data indicate that cytochrome P450 metabolites contribute to bradykinin-induced, endothelium-dependent relaxations but only following inhibition of NO synthase and cyclooxygenase. EETs relax coronary arteries in an endothelium-independent manner. Carbon-1 carboxyl, 8, 9 and 11, 12-double bonds and 14, 15-epoxy groups are required for full agonist activity. These studies provide further support for the hypothesis that EETs represent EDHFs in the coronary artery and mediate the relaxation responses to bradykinin.

Genetic analysis reveals different functions for the products of the thyroid hormone receptor alpha locus.

Thyroid hormone receptors are encoded by the TRalpha (NR1A1) and TRbeta (NR1A2) loci. These genes are transcribed into multiple variants whose functions are unclear. Analysis by gene inactivation in mice has provided new insights into the functional complexity of these products. Different strategies designed to modify the TRalpha locus have led to strikingly different phenotypes. In order to analyze the molecular basis for these alterations, we generated mice devoid of all known isoforms produced from the TRalpha locus (TRalpha(0/0)). These mice are viable and exhibit reduced linear growth, bone maturation delay, moderate hypothermia, and reduced thickness of the intestinal mucosa. Compounding TRalpha(0) and TRbeta(-) mutations produces viable TRalpha(0/0)beta(-/-) mice, which display a more severe linear growth reduction and a more profound hypothermia as well as impaired hearing. A striking phenotypic difference is observed between TRalpha(0/0) and the previously described TRalpha(-/-) mice, which retain truncated TRDeltaalpha isoforms arising from a newly described promoter in intron 7. The lethality and severe impairment of the intestinal maturation in TRalpha(-/-) mice are rescued in TRalpha(0/0) animals. We demonstrate that the TRDeltaalpha protein isoforms, which are natural products of the TRalpha locus, are the key determinants of these phenotypical differences. These data reveal the functional importance of the non-T3-binding variants encoded by the TRalpha locus in vertebrate postnatal development and homeostasis.

Functional interference between thyroid hormone receptor alpha (TRalpha) and natural truncated TRDeltaalpha isoforms in the control of intestine development.

Thyroid hormone is known to participate in the control of intestine maturation at weaning. Its action is mediated by the thyroid hormone nuclear receptors, encoded by the TRalpha and TRbeta genes. Since previous studies have shown that TRbeta plays a minor role in the gut, we focused here our analysis on the TRalpha gene. The TRalpha locus generates the TRalpha1 receptor together with the splicing variant TRalpha2 and the truncated products TRDeltaalpha1 and TRDeltaalpha2, which all lack an intact ligand binding domain. The TRDeltaalpha isoforms are transcribed from an internal promoter located in intron 7, and their distribution is restricted to a few tissues including those of the intestine. In order to define the functions of the different isoforms encoded by the TRalpha locus in the intestinal mucosa, we produced mice either lacking all known TRalpha products or harboring a mutation which inactivates the intronic promoter. We performed a detailed analysis of the intestinal phenotypes in these mice and compared it to that of the previously described TRalpha(-/-) mice, in which TRalpha isoforms are abolished but the TRDeltaalpha isoforms remain. This comparative analysis leads us to the following conclusions: (i) the TRalpha1 receptor mediates the T3-dependent functions in the intestine at weaning time and (ii) the TRDeltaalpha products negatively control the responsiveness of the epithelial cells to T3. Moreover, we show that TRDeltaalpha proteins can interfere with the transcription of the intestine-specific homeobox genes cdx1 and cdx2 and that their activity is regulated by TRalpha1. Altogether these data demonstrate that cooperation of TRalpha and TRDeltaalpha products is essential to ensure the normal postnatal development of the intestine and that mutations in the TRalpha locus can generate different phenotypes caused by the disruption of the equilibrium between these products.

Increased sensitivity to thyroid hormone in mice with complete deficiency of thyroid hormone receptor alpha.

Only three of the four thyroid hormone receptor (TR) isoforms, alpha1, beta1, and beta2, bind thyroid hormone (TH) and are considered to be true TRs. TRalpha2 binds to TH response elements on DNA, but its role in vivo is still unknown. We produced mice completely deficient in TRalpha (TRalpha(o/o)) that maintain normal serum thyroid-stimulating hormone (TSH) concentration despite low serum thyroxine (T(4)), suggesting increased sensitivity to TH. We therefore examined the effects of TH (L-3,3',5-triiodothyronine, L-T3) given to TH-deprived and to intact TRalpha(o/o) mice. Controls were wild-type (WT) mice of the same strain and mice resistant to TH due to deficiency in TRbeta (TRbeta(-/-)). In liver, T3 produced significantly greater responses in TRalpha(o/o) and smaller responses in TRbeta(-/-) as compared with WT mice. In contrast, cardiac responses to L-T3 were absent or reduced in TRalpha(o/o), whereas they were similar in WT and TRbeta(-/-) mice, supporting the notion that TRalpha1 is the dominant TH-dependent TR isoform in heart. 5-Triiodothyronine (L-T3) given to intact mice produced a greater suppression of serum T(4) in TRalpha(o/o) than it did in WT mice and reduced by a greater amount the TSH response to TSH-releasing hormone. This is an in vivo demonstration that a TR deficiency can enhance sensitivity to TH. This effect is likely due to the abrogation of the constitutive "silencing" effect of TRalpha2 in tissues expressing the TRbeta isoforms.