Variability in distribution and use of tuberculosis diagnostic tests in Kenya: a cross-sectional survey.

BACKGROUND: Globally, 40% of all tuberculosis (TB) cases, 65% paediatric cases and 75% multi-drug resistant TB (MDR-TB) cases are missed due to underreporting and/or under diagnosis. A recent Kenyan TB prevalence survey found that a significant number of TB cases are being missed here. Understanding spatial distribution and patterns of use of TB diagnostic tests as per the guidelines could potentially help improve TB case detection by identifying diagnostic gaps. METHODS: We used 2015 Kenya National TB programme data to map TB case notification rates (CNR) in different counties, linked with their capacity to perform diagnostic tests (chest x-rays, smear microscopy, Xpert MTB/RIF®, culture and line probe assay). We then ran hierarchical regression models for adults and children to specifically establish determinants of use of Xpert® (as per Kenyan guidelines) with county and facility as random effects. RESULTS: In 2015, 82,313 TB cases were notified and 7.8% were children. The median CNR/100,000 amongst 0-14yr olds was 37.2 (IQR 20.6, 41.0) and 267.4 (IQR 202.6, 338.1) for ≥15yr olds respectively. 4.8% of child TB cases and 12.2% of adult TB cases had an Xpert® test done, with gaps in guideline adherence. There were 2,072 microscopy sites (mean microscopy density 4.46/100,000); 129 Xpert® sites (mean 0.31/100,000); two TB culture laboratories and 304 chest X-ray facilities (mean 0.74/100,000) with variability in spatial distribution across the 47 counties. Retreatment cases (i.e. failures, relapses/recurrences, defaulters) had the highest odds of getting an Xpert® test compared to new/transfer-in patients (AOR 7.81, 95% CI 7.33-8.33). Children had reduced odds of getting an Xpert® (AOR 0.41, CI 0.36-0.47). HIV-positive individuals had nearly twice the odds of getting an Xpert® test (AOR 1.82, CI 1.73-1.92). Private sector and higher-level hospitals had a tendency towards lower odds of use of Xpert®. CONCLUSIONS: We noted under-use and gaps in guideline adherence for Xpert® especially in children. The under-use despite considerable investment undermines cost-effectiveness of Xpert®. Further research is needed to develop strategies enhancing use of diagnostics, including innovations to improve access (e.g. specimen referral) and overcoming local barriers to adoption of guidelines and technologies.

Finding the missing children for TB care and prevention in Kenya.

SETTING: One hundred high TB burden facilities in nine counties in Kenya.OBJECTIVES: 1) To increase uptake of TB preventive therapy (TPT) among child contacts aged <5 years, and 2) to increase TB diagnosis in children aged <15 years presenting to health facilities for routine care.DESIGN: For objective 1, a clinic-based child contact management strategy incorporating transport/healthcare cost reimbursement, monitoring and evaluation tools, and healthcare worker education was utilized. For objective 2, community health screeners were established in pediatric outpatient departments to perform verbal screening, flagging symptomatic children for further evaluation.RESULTS: Over 15 months, identification of 8,060 individuals diagnosed with bacteriologically confirmed TB led to 2,022 child contacts. Of these, 1,848 (91%) were evaluated; 149 (8%) were diagnosed with TB disease, leaving 1,699 (92%) eligible for TPT; 1,613 (95%) initiated TPT and 1,335 (83%) completed TPT. In outpatient settings, 140,444 children were screened; 54,236 (39%) had at least two TB symptoms; 2,395 (4%) were diagnosed with TB diseaseCONCLUSION: Health system strengthening supporting a clinic-based child contact management program increased the number of children initiating TPT. Systematic screening in outpatient clinics can lead to increased TB case notifications; however, optimal screening tools and clearer diagnostic pathways for the evaluation of these children are needed.

TB training in Kenya: building capacity for care and prevention.

BACKGROUND: Devolution of healthcare services in Kenya resulted in a large number of newly recruited tuberculosis (TB) coordinators. We describe a unique collaboration between a national tuberculosis program (NTP), a local, and an international non-governmental organization to build human resource capacity in TB care and prevention. METHODS: From 2016 to 2021, the Kenya Division of National Tuberculosis, Leprosy and Lung Disease Program, Centre for Health Solutions-Kenya, and the International Union Against Tuberculosis and Lung Disease developed and conducted a series of 7-day training courses. A key focus of training was the introduction of TBData4Action, an approach involving the local use of routinely available data to strengthen decision-making and support supervision. RESULTS: Implementation outcomes included training 331 (96%) coordinators out of 344, representing all 47 counties, 37 national officers and 21 other stakeholders using the country-tailored curriculum, including hands-on group work by county teams and field practicals. Thirty-five national facilitators were identified and mentored as local faculty. Training costs were reduced by 75% compared with international alternatives. CONCLUSION: The collaboration resulted in the training of the majority of the coordinators in a standardized approach to TB care. A sustainable approach to capacity building in local data use was found feasible; the model could be adapted by other NTPs.

CONTEXTE: La décentralisation des services de santé au Kenya a conduit au recrutement d'un grand nombre de nouveaux coordinateurs TB. Nous décrivons une collaboration unique entre un programme national de lutte contre la TB (NTP), une organisation non gouvernementale locale et une organisation non gouvernementale internationale visant à renforcer les capacités humaines en matière de prévention et de soins de la TB. MÉTHODES: De 2016 à 2021, la division kényane du programme national de lutte contre la tuberculose, la lèpre et les maladies respiratoires, le Centre for Health Solutions-Kenya et l'Union internationale contre la tuberculose et les maladies respiratoires ont développé et dispensé une série de formations en 7 jours. La formation mettait l'accent sur l'introduction de l'approche TBData4Action, qui promeut une utilisation locale des données disponibles en routine afin de renforcer la prise de décision et d'épauler les activités de supervision. RÉSULTATS: Les résultats de la mise en place de cette formation comprenaient la formation de 331 (96%) coordinateurs sur 344, représentant l'ensemble des 47 pays, 37 administrateurs nationaux et 21 autres acteurs formés à l'aide du programme adapté aux besoins du pays concerné (dont travail de groupe pratique par les équipes nationales et travaux pratiques sur le terrain). Trente-cinq facilitateurs nationaux ont été identifiés et formés comme enseignants locaux. Les coûts de la formation ont été réduits de 75% par rapport aux alternatives internationales. CONCLUSION: La collaboration a permis de former la majorité des coordinateurs à l'aide d'une approche standardisée de soins de la TB. Une approche durable de renforcement des capacités en matière d'utilisation des données locales s'est avérée réalisable. Ce modèle peut être adapté à d'autres NTP.

Improving TB case notification and treatment coverage through data use.

BACKGROUND: This was a study on national TB data. OBJECTIVE: To assess improvement in TB case notification and treatment coverage through improved data use for action in Nigeria. DESIGN: We analysed pre- and post-intervention secondary TB programme data comprising data on increased supervisory visits, incentives for health workers, DOTS expansion, outreaches and geo-code monitoring. Trend analysis was performed using Cochran-Armitage χ2 test for linear trends. RESULTS: Case-finding increased from 104,904 cases in 2017 to 138,591 in 2020. There was an increase of 2.0% from 2017 to 2018, 13.0% in 2018 to 2019 and 15.0% in 2019 to 2020 (P < 0.001). Facility DOTS coverage increased from 7,389 facilities in 2017 to 17,699 in 2020. There was an increase of 30.0% in 2018, 31.0% in 2019 and 40.0% in 2020 (P < 0.001). The number of reporting facilities increased from 5,854 in 2017 to 12,775 in 2020. Compared with 2017, there were an increase of 20.0% in 2018, 38.0% in 2019 and 32.0% in 2020 (P < 0.001). Treatment coverage rate increased from 24% in 2018 to 27% in 2019 and 30% in 2020. CONCLUSION: TB service expansion, improved monitoring and the use of data for decision making are key in increasing TB treatment coverage.

CONTEXTE: Il s'agit d'une étude sur les données nationales relatives à la TB. OBJECTIF: Évaluer l'amélioration de la notification des cas de TB et de la couverture du traitement grâce à une meilleure utilisation des données pour l'action au Nigéria. MÉTHODE: Nous avons analysé les données du programme secondaire de lutte contre la TB avant et après l'intervention, y compris les données sur l'augmentation des visites de supervision, les mesures incitatives pour les travailleurs de la santé, l'expansion du système DOTS, les activités de proximité et la surveillance des codes géographiques. L'analyse des tendances a été réalisée à l'aide du test du χ2 de Cochran-Armitage pour les tendances linéaires. RÉSULTATS: La recherche de cas est passée de 104 904 cas en 2017 à 138 591 en 2020. On observe une augmentation de 2,0% de 2017 à 2018, de 13,0% de 2018 à 2019 et de 15,0% de 2019 à 2020 (P < 0,001). La couverture DOTS des établissements est passée de 7 389 établissements en 2017 à 17 699 en 2020. On observe une augmentation de 30,0% en 2018, 31,0% en 2019 et 40,0% en 2020 (P < 0,001). Le nombre d'installations déclarantes est passé de 5 854 en 2017 à 12 775 en 2020. Par rapport à 2017, il y a eu une augmentation de 20,0% en 2018, 38,0% en 2019 et 32,0% en 2020 (P < 0,001). Le taux de couverture du traitement est passé de 24% en 2018 à 27% en 2019 et 30% en 2020. CONCLUSION: L'expansion des services de lutte contre la TB, l'amélioration de la surveillance et l'utilisation des données pour la prise de décision sont essentielles pour augmenter la couverture du traitement de la TB.

Can isoniazid preventive therapy be scaled up rapidly? Lessons learned in Kenya, 2014-2018.

BACKGROUND: TB is the leading cause of mortality among people living with HIV (PLHIV), for whom isoniazid preventive therapy (IPT) has a proven mortality benefit. Despite WHO recommendations, countries have been slow in scaling up IPT. This study describes processes, challenges, solutions, outcomes and lessons learned during IPT scale-up in Kenya.METHODS: We conducted a desk review and analyzed aggregated Ministry of Health (MOH) IPT enrollment data from 2014 to 2018 to determine trends and impact of program activities. We further analyzed IPT completion reports for patients initiated from 2015 to 2017 in 745 MOH sites in Nairobi, Central, Eastern and Western Kenya.RESULTS: IPT was scaled up 75-fold from 2014 to 2018: the number of PLHIV covered increased from 9,981 to 749,890. The highest percentage increases in the cumulative number of PLHIV on IPT were seen in the quarters following IPT pilot projects in 2014 (49%), national launch in 2015 (54%), and HIV treatment acceleration in 2016 (158%). Among 250,069 patients initiating IPT from 2015 to 2017, 97.5% completed treatment, 0.2% died, 0.8% were lost to follow-up, 1.0% were not evaluated, and 0.6% discontinued treatment.CONCLUSIONS: IPT can be scaled up rapidly and effectively among PLHIV. Deliberate MOH efforts, strong leadership, service delivery integration, continuous mentorship, stakeholder involvement, and accountability are critical to program success.

Impact of a national nutritional support programme on loss to follow-up after tuberculosis diagnosis in Kenya.

SETTING: Undernourishment is prevalent among tuberculosis (TB) patients. Nutritional support is given to TB patients to prevent and treat undernourishment; it is also used to improve treatment outcomes and as an incentive to keep patients on treatment. OBJECTIVE: To determine whether nutritional support is associated with a reduction in the risk of loss to follow-up (LTFU) among TB patients in Kenya. DESIGN: This was a retrospective cohort study using national programmatic data. Records of 362 685 drug-susceptible TB patients from 2012 to 2015 were obtained from Treatment Information from Basic Unit (TIBU), a national case-based electronic data recording system. Patients who were LTFU were compared with those who completed treatment. RESULTS: Nutrition counselling was associated with an 8% reduction in the risk of LTFU (RR 0.92, 95%CI 0.89-0.95), vitamins were associated with a 7% reduction (adjusted RR [aRR] 0.93, 95%CI 0.90-0.96) and food support was associated with a 10% reduction (aRR 0.90, 95%CI 0.87-0.94). Among patients who received food support, the addition of nutrition counselling was associated with a 23% reduction in the risk of LTFU (aRR 0.77, 95%CI 0.67-0.88). CONCLUSION: Nutritional support was associated with a reduction in the risk of LTFU. Providing nutrition counselling is important for patients receiving food support.

Effects of relaxin on mast cells. In vitro and in vivo studies in rats and guinea pigs.

The results of the current study demonstrate that relaxin inhibits histamine release by mast cells. This effect is related to the peptide concentrations, and could be observed in both isolated rat serosal mast cells stimulated with compound 48/80 or calcium ionophore A 23187, and in serosal mast cells isolated from sensitized guinea pigs and challenged with the antigen. The morphological findings agree with the functional data, revealing that relaxin attenuates calcium ionophore-induced granule exocytosis by isolated rat serosal mast cells. Similar effects of relaxin have also been recognized in vivo by light microscopic and densitometric analysis of the mesenteric mast cells of rats which received the hormone intraperitoneally 20 min before local treatment of the mesentery with calcium ionophore. Moreover, evidence is provided that relaxin stimulates endogenous production of nitric oxide and attenuates the rise of intracellular Ca2+ concentration induced by calcium ionophore. The experiments with drugs capable of influencing nitric oxide production also provide indirect evidence that the inhibiting effect of relaxin on mast cell histamine release is related to an increased generation of nitric oxide. It is suggested that relaxin may have a physiological role in modulating mast cell function through the L-arginine-nitric oxide pathway.

Nitric oxide mediates angiogenesis in vivo and endothelial cell growth and migration in vitro promoted by substance P.

We evaluated the effects of nitric oxide (NO) generators and endogenous production of NO elicited by substance P (SP) in the angiogenesis process. Angiogenesis was monitored in the rabbit cornea in vivo and in vitro by measuring the growth and migration of endothelial cells isolated from coronary postcapillary venules. The angiogenesis promoted in the rabbit cornea by [Sar9]-SP-sulfone, a stable and selective agonist for the tachykinin NK1 receptor, and by prostaglandin E1 (PGE1), was potentiated by sodium nitroprusside (SNP). Conversely, the NO synthase inhibitor N omega-nitro-L-arginine methyl ester (L-NAME), given systemically, inhibited angiogenesis elicited by [Sar9]-SP-sulfone and by PGE1. Endothelial cells exposed to SNP exhibited an increase in thymidine incorporation and in total cell number. Exposure of the cells to NO generating drugs, such as SNP, isosorbide dinitrate, and glyceryl trinitrate, produced a dose-dependent increase in endothelial cell migration. Capillary endothelial cell proliferation and migration produced by SP were abolished by pretreatment with the NO synthase inhibitors N omega-mono-methyl-L-arginine (L-NMMA), N omega-nitro-L-arginine (L-NNA), and L-NAME. Exposure of the cells to SP activated the calcium-dependent NO synthase. Angiogenesis and endothelial cell growth and migration induced by basic fibroblast growth factor were not affected by NO synthase inhibitors. These data indicate that NO production induced by vasoactive agents, such as SP, functions as an autocrine regulator of the microvascular events necessary for neovascularization and mediates angiogenesis.

Diabetes and pre-diabetes in tuberculosis patients in western Kenya using point-of-care glycated haemoglobin.

Setting: The tuberculosis (TB) clinics of five health facilities in western Kenya. Objective: To assess the prevalence and associated determinants of diabetes mellitus (DM) and pre-diabetes hyperglycaemia among adult TB patients using point-of-care DCA Vantage glycated haemoglobin (HbA1c) devices. Design: This was a cross-sectional study. Results: Of 454 patients, 272 (60%) were males, the median age was 34 years, 175 (39%) were co-infected with the human immunodeficiency virus (HIV), and the median duration of anti-tuberculosis treatment was 8 weeks; 180 (40%) patients reported at least one classical symptom suggestive of DM. The prevalence of DM (HbA1c ⩾6.5%) was 5.1% (95%CI 3.2-7.5), while that of pre-diabetes (HbA1c 5.7-6.4%) was 37.5% (95%CI 33.1-42.2). The number needed to screen (NNS) was 19.6 for DM and 2.7 for pre-diabetes. Combined, 42.6% (95%CI 38.0-47.3) of the patients had either pre-diabetes or DM (NNS 2.3). Seven of the 23 patients with DM knew their prior DM status. Higher rates of DM were associated with age ⩾40 years and a family history of DM, but not obesity, type of TB, HIV status or suggestive symptoms. Conclusions: High rates of pre-diabetes and DM were found in adult TB patients. This study supports the need for routine screening of all patients with TB for DM in Kenya.

Contexte: Centres tuberculose (TB) de cinq structures de santé dans l'Ouest du Kenya.Objectif: Evaluer la prévalence et les déterminants associés du diabète (DM) et de l'hyperglycémie pré-diabète chez des patients adultes atteints de TB grâce aux appareils DCA Vantage de dosage d'hémoglobine glyquée (HbA1c) utilisés sur place.Schéma: Une étude transversale.Résultats: Sur 454 patients, 272 (60%) ont été des hommes, leur âge médian a été de 34 ans et 175 (39%) ont été co-infectés par le virus de l'immunodéficience humaine (VIH) ; la durée médiane du traitement de la TB a été de 8 semaines et 180 (40%) patients ont fait état d'au moins un symptôme classique suggérant un DM. La prévalence du DM (HbA1c ⩾ 6,5%) a été de 5,1% (IC95% 3,2­7,5) tandis que celle du pré-diabète (HbA1c 5,7­6,4%) a été de 37,5% (IC95% 33,1­422). Le nombre de dépistages requis (NNS) a été de 19,6 pour diagnostiquer un DM et de 2,7 pour un pré-diabète. En combinant les deux, 42,6% (IC95% 38,0­47,3) des patients avaient soit un pré-diabète soit un DM (NNS 2,3). Sept des 23 patients atteints de DM étaient au courant de leur statut. Des taux de DM plus élevés ont été associés avec un âge ⩾ 40 ans et des antécédents de DM dans la famille, mais pas avec l'obésité, le type de TB, le statut du VIH ou des symptômes suggestifs du VIH.Conclusions: Des taux élevés de pré-diabète et de DM ont été découverts chez des patients TB adultes. Cette étude est en faveur du dépistage de routine du DM chez tous les patients atteints de TB au Kenya.

Marco de referencia: Los consultorios de atención de la tuberculosis (TB) en cinco establecimientos de salud en el occidente de Kenya.Objetivo: Evaluar la prevalencia de diabetes (DM) e hiperglucemia prediabética y los factores determinantes asociados en los pacientes adultos con diagnóstico de TB, mediante la utilización de dispositivos de diagnóstico (DCA Vantage) que determinan la glucohemoglobina (HbA1c) en el lugar de atención.Método: Un estudio transversal.Resultados: De los 454 pacientes, 272 fueron de sexo masculino (60%), la mediana de la edad fue 34 años, 175 (39%) sufrían coinfección por el virus de la inmunodeficiencia humana (VIH), la mediana de la duración del tratamiento antituberculoso fue 8 semanas y 180 pacientes notificaron por lo menos un síntoma patognomónico de DM (40%). La prevalencia de DM (HbA1c ⩾ 6,5%) fue 5,1% (IC 95% 3,2­7,5) y la prevalencia de pre-diabetes (HbA1c 5,7­6,4%) fue 37,5% (IC95% 33,1­42,2). El número de pacientes que es necesario cribar (NNC) para detectar un caso de DM fue 19,6 y 2,7 para un caso de prediabetes. Combinados, el 42,6% de los pacientes presentaba ya sea prediabetes o DM (NNC 2,3; IC95% 38,0­47,3). Siete de los 23 pacientes con DM conocían ya su diagnóstico. Las tasas más altas de DM se asociaron con una edad de 40 años o más y el antecedente familiar de DM, pero no con la obesidad, el tipo de TB, la situación frente al VIH ni la presencia de síntomas indicativos.Conclusión: Se encontraron altas tasas de prediabetes y DM en los pacientes adultos con diagnóstico de TB. El presente estudio respalda la práctica de la detección sistemática de la DM en los pacientes con TB en Kenya.

A topical nitric oxide-releasing dexamethasone derivative: effects on intraocular pressure and ocular haemodynamics in a rabbit glaucoma model.

BACKGROUND: Topical nitric oxide-releasing dexamethasone (NCX1021) may avoid the negative effects of dexamethasone phosphate. AIMS: To obtain more information on the role of nitric oxide in glaucoma and to compare a nitric oxide-releasing dexamethasone with dexamethasone phosphate with regard to intraocular pressure (IOP) and ocular haemodynamics in an experimental rabbit model. METHODS: Six rabbits were treated with dexamethasone phosphate 0.1% in the right eye and with NCX1021 in the left eye for 5 weeks. The parameters considered were IOP, nitric oxide marker levels in aqueous humour, ocular haemodynamics of ophthalmic artery (by means of colour Doppler imaging), expression of endothelial nitric oxide synthase (eNOS)in ciliary processes and histology of ciliary bodies. RESULTS: Dexamethasone increased IOP levels, NCX1021 did not. Nitrite and cyclic guanosine monophosphate levels in aqueous humour were lowered by dexamethasone and increased by NCX1021. Resistivity index of the ophthalmic artery was increased, eNOS expression was reduced and ciliary bodies showed histological lesions in dexamethasone-treated eyes, not in NCX1021-treated ones. CONCLUSIONS: NCX1021 may avoid the IOP increase, impairment of ocular blood flow and the morphological changes in the ciliary bodies possibly induced by corticosteroid treatment.

Role of nitric oxide in angiogenesis and tumor progression in head and neck cancer.

BACKGROUND: Angiogenesis (formation of new blood vessels) is associated with tumor growth and metastasis in patients with solid tumors, including those of the head and neck. Nitric oxide (NO) production may contribute to these processes. We assessed the role of the NO pathway in angiogenesis and tumor progression in patients with head and neck cancer. METHODS: Biochemical assays were used to measure NO synthase (NOS) activity and cyclic guanosine monophosphate (cGMP) levels in specimens of tumor and normal mucosa obtained from 27 patients. Microvessels in tumor specimens were identified by CD-31-specific immunohistochemical staining. Associations between microvessel densities, levels of NOS, and cGMP were examined by use of two-sided statistical tests. Tumor specimens and human squamous carcinoma A-431 cells were grown as explants on the corneas of rabbits, and the effect of the NOS inhibitor N(omega)-nitro-L-arginine-methyl ester (L-NAME) was tested. RESULTS: Levels of total NOS, inducible NOS, and cGMP were higher in tumor specimens than in specimens of normal mucosa (all P<.0001). Tumor specimens from patients with lymph node metastases presented a higher total NOS activity (P = .005) and were markedly more vascularized than tumor specimens from patients with no lymph node involvement (P = .0002). Microvessel density at the tumor edge was an independent predictor of metastasis for this series of patients (odds ratio = 1.19; 95% confidence interval = 1.07-2.89; P = .04). A-431 cells and tumor specimens exhibiting high levels of NOS activity induced angiogenesis in the rabbit cornea assay; when NO production was blocked, tumor angiogenesis and growth were repressed. CONCLUSIONS: The NO pathway appears to play a key role in tumor angiogenesis and spread in patients with head and neck cancer.

Relaxin activates the L-arginine-nitric oxide pathway in human breast cancer cells.

Recently, we demonstrated that relaxin (RLX), a peptide hormone of ovarian origin, inhibits growth and promotes differentiation of MCF-7 breast adenocarcinoma cells. We also showed that RLX stimulates the production of nitric oxide (NO) in several cell types. NO has been reported to have antitumor activity by inhibiting proliferation, promoting differentiation, and reducing the metastatic spread of some tumor cell types. In this study, we aimed at evaluating whether RLX influences the L-arginine-NO pathway in MCF-7 cells. The cells were grown in the absence or presence of RLX at different concentrations, and cell proliferation, constitutive and inducible NO synthase activities, nitrite production, and intracellular levels of cyclic GMP were investigated. The results obtained indicate that RLX increases inducible NO synthase activity and potentiates NO production. This was accompanied by an elevation of intracellular cyclic GMP, which is known to mediate the cell response to NO. The RLX-induced activation of the L-arginine-NO pathway in the MCF-7 cells was inversely related to the rate of cell proliferation. These results suggest that the cytostatic effect of RLX on MCF-7 breast cancer cells may rely on its ability to stimulate endogenous production of NO.

Protection from cardiac injury by induction of heme oxygenase-1 and nitric oxide synthase in a focal ischaemia-reperfusion model.

The enzymes heme oxygenase (HO) generate carbon monoxide (CO) in living organisms during heme degradation. Carbon monoxide has recently been shown to dilate blood vessels and to possess anti-inflammatory properties. It is also known that nitric oxide (NO) donors ameliorate cardiac ischaemia-reperfusion injury in experimental models of global or focal ischaemia-reperfusion (FIR). The two gaseous mediators share the same mechanism of action via the stimulation of soluble guanylyl cyclase and the increase in cellular levels of cyclic GMP. We studied the effects of manipulating the HO system and the possible interaction between CO and NO in an experimental in vivo model of FIR in the rat heart. FIR-subjected rats had necrotic area in the left ventricle, ventricular arrhythmias and a shortening of survival time in comparison to sham-operated animals. Resident mast cells underwent a heavy degranulation, malonyldialdehyde was produced by myocardial cell membranes, and tissue calcium levels were increased. High levels of myeloperoxidase were also detected, suggesting a FIR-related inflammatory process. In animals pre-treated with the HO-1 inducer, hemin, all the biochemical and morphometric markers of FIR were minimized or fully abated. Consistently, the biochemical and morphometric markers of FIR were reversed in rats treated with the HO-1 blocker, ZnPP-IX, prior to hemin administration. Pre-treatment with hemin significantly increases the expression and activity of both cardiac HO-1 and iNOS, suggesting that CO and NO cooperate in the cardioprotective effect against FIR-induced damage, and that there is a therapeutic synergism between NO-donors and CO-releasing molecules, via the common stimulation of increase in cGMP levels and decrease in calcium overload.

Virus-induced gene silencing in Catharanthus roseus by biolistic inoculation of tobacco rattle virus vectors.

Catharanthus roseus constitutes the unique source of several valuable monoterpenoid indole alkaloids, including the antineoplastics vinblastine and vincristine. These alkaloids result from a complex biosynthetic pathway encompassing between 30 and 50 enzymatic steps whose characterisation is still underway. The most recent identifications of genes from this pathway relied on a tobacco rattle virus-based virus-induced gene silencing (VIGS) approach, involving an Agrobacterium-mediated inoculation of plasmids encoding the two genomic components of the virus. As an alternative, we developed a biolistic-mediated approach of inoculation of virus-encoding plasmids that can be easily performed by a simple bombardment of young C. roseus plants. After optimisation of the transformation conditions, we showed that this approach efficiently silenced the phytoene desaturase gene, leading to strong and reproducible photobleaching of leaves. This biolistic transformation was also used to silence a previously characterised gene from the alkaloid biosynthetic pathway, encoding iridoid oxidase. Plant bombardment caused down-regulation of the targeted gene (70%), accompanied by a correlated decreased in MIA biosynthesis (45-90%), similar to results obtained via agro-transformation. Thus, the biolistic-based VIGS approach developed for C. roseus appears suitable for gene function elucidation and can readily be used instead of the Agrobacterium-based approach, e.g. when difficulties arise with agro-inoculations or when Agrobacterium-free procedures are required to avoid plant defence responses.

Cyclooxygenase-2 pathway correlates with VEGF expression in head and neck cancer. Implications for tumor angiogenesis and metastasis.

We evaluated the role of COX-2 pathway in 35 head and neck cancers (HNCs) by analyzing COX-2 expression and prostaglandin E2 (PGE2) production in relation to tumor angiogenesis and lymph node metastasis. COX-2 activity was also correlated to vascular endothelial growth factor (VEGF) mRNA and protein expression. COX-2 mRNA and protein expression was higher in tumor samples than in normal mucosa. PGE2 levels were higher in the tumor front zone in comparison with tumor core and normal mucosa (P<.0001). Specimens from patients with lymph node metastasis exhibited higher COX-2 protein expression (P=.0074), PGE2 levels (P=.0011) and microvessel density (P<.0001) than specimens from patients without metastasis. A significant correlation between COX-2 and tumor vascularization (r(s)=0.450, P=.007) as well as between COX-2 and microvessel density with VEGF expression in tumor tissues was found (r(s)=0.450, P=.007; r(s)=0.620, P=.0001, respectively). The induction of COX-2 mRNA and PGE2 synthesis by EGF and Escherichia coli lipopolysaccharide (LPS) in A-431 and SCC-9 cell lines, resulted in an increase in VEGF mRNA and protein production. Indomethacin and celecoxib reversed the EGF- and LPS-dependent COX-2, VEGF, and PGE2 increases. This study suggests a central role of COX-2 pathway in HNC angiogenesis by modulating VEGF production and indicates that COX-2 inhibitors may be useful in HNC treatment.

Relaxin counteracts asthma-like reaction induced by inhaled antigen in sensitized guinea pigs.

In previous studies, the peptide hormone relaxin (RLX) was found to inhibit mast cell secretion and platelet activation. It has been established that the release of mediators from these cells plays a central pathogenic role in allergic asthma. This prompted us to ascertain whether RLX may counteract the respiratory and histopathological abnormalities of the asthma-like reaction to inhaled antigen in sensitized guinea pigs. Guinea pigs were sensitized with ovalbumin and challenged with the same antigen given by aerosol. Some animals received RLX (30 microg/kg BW, twice daily for 4 days) before antigen challenge. Other animals received inactivated RLX in place of authentic RLX. Respiratory abnormalities, such as cough and dyspnea, were analyzed as were light and electron microscopic features of lung specimens. RLX was shown to reduce the severity of respiratory abnormalities, as well as histological alterations, mast cell degranulation, and leukocyte infiltration in sensitized guinea pigs exposed to ovalbumin aerosol. RLX was also found to promote dilation of alveolar blood capillaries and to reduce the thickness of the air-blood barrier. This study provides evidence for an antiasthmatic property of RLX and raises the possibility of new therapeutic strategies for allergic asthma in humans.

Relaxin counteracts myocardial damage induced by ischemia-reperfusion in isolated guinea pig hearts: evidence for an involvement of nitric oxide.

Relaxin was previously shown to cause coronary vasodilation and to inhibit mast cell activation through a stimulation of endogenous nitric oxide production. This suggests that relaxin may have beneficial effects on ischemia-reperfusion-induced myocardial injury, which is triggered by endothelial damage and impaired nitric oxide generation. In this study, we tested the effect of relaxin on isolated and perfused guinea pig hearts subjected to ischemia and reperfusion. Ischemia was induced by ligature of the left anterior descending coronary artery; removal of the ligature induced reperfusion. Relaxin, at the concentration of 30 ng/ml of perfusion fluid, causes: a significant increase in coronary flow and in nitric oxide generation; a significant decrease in malonyldialdehyde production and in calcium overload, both markers of myocardial injury; an inhibition of mast cell granule exocytosis and histamine release, which are known to contribute to myocardial damage; a reduction of ultrastructural abnormalities of myocardial cells; an improvement of heart contractility. The beneficial effects of relaxin were blunted by the NO synthase inhibitor L-NMMA. The current study provides first experimental evidence that relaxin has a powerful protective effect on the heart undergoing ischemia and reperfusion acting through a nitric oxide-driven mechanism.

Relaxin activates the L-arginine-nitric oxide pathway in vascular smooth muscle cells in culture.

The peptide hormone relaxin (RLX) has been shown to elicit a powerful vasodilatory response in several target organs. This response is mediated by the stimulation of intrinsic nitric oxide (NO) generation. The present study was designed to clarify whether RLX directly promotes the relaxation of vascular smooth muscle cells through stimulation of NO generation. Vascular smooth muscle cells from bovine aortas were incubated with RLX at concentrations ranging from 1 nmol/L to 1 micromol/L. The expression and activity of NO synthase, production of NO, and the intracellular levels of cGMP and Ca2+ were determined. The cell morphology and signal transduction mechanisms of these bovine aortic smooth muscle cells in response to RLX were also studied. RLX stimulated the expression of immunoreactive inducible NO synthase and increased significantly and in a concentration-related fashion inducible NO synthase activity, NO generation, and intracellular cGMP levels. Concurrently, RLX significantly decreased cytosolic Ca2+ concentrations and caused changes in cell shape and the actin cytoskeleton that were consistent with cell relaxation. The signal transduction mechanisms leading to the enhanced expression of inducible NO synthase protein and activity caused by RLX involve the activation of tyrosine kinase, phosphatidylcholine-phospholipase C, and the transcription factor nuclear factor-kappaB, similar to bacterial endotoxins and proinflammatory cytokines. This study suggests that RLX is an endogenous agent capable of regulating vascular tone by activation of the L-arginine-NO pathway in vascular smooth muscle cells.

The release of histamine by free radicals.

An extensive discussion of the available evidence about the release of histamine by free radicals is presented. In addition, the involvement of the free-radical driven release of histamine in human pathophysiology is debated.