Tuberculosis knowledge and awareness in tribal-dominant districts of Jharkhand, India: implications for ACSM.

SETTING: Forty hard-to-reach villages in the East and West Singhbhum Districts of Jharkhand State, India. OBJECTIVES: To document knowledge and awareness of tuberculosis (TB) among the general population, understand gender differences and inform intervention activities for the improvement of TB control programmes in tribal-dominant hard-to-reach areas in India. DESIGN: A cross-sectional community-based survey was carried out among 825 respondents using population proportionate sampling. RESULTS: Most of the respondents were in the 18-35 years age group, tribal and married; 44% were illiterate. The study shows poor knowledge about TB symptoms, causes, modes of transmission and moderate awareness about government TB services. Correct knowledge about the cause of TB was negligible: half of the respondents reported local liquor as the cause, 61% considered TB as transmissible and one third considered sharing of food as the mode of transmission. Awareness about the availability of free treatment services at government health facilities was high, but awareness about DOTS was low. Significant gender differences were observed in knowledge and awareness levels. CONCLUSION: Study findings point to the importance of urgent intensification of culturally congruent and gender-sensitive advocacy, communication and social mobilisation activities.

Contexte : Quarante villages difficiles d'accès de l'Est et de l'Ouest des districts de l'état de Singhbhum, Jharkhand, Inde.Objectifs : Documenter les niveaux de connaissances et de sensibilisation en matière de la tuberculose (TB) parmi les membres de la communauté, de comprendre les différences entre les sexes et de contribuer à l'élaboration d'activités d'intervention afin d'améliorer le programme de lutte contre la TB dans des zones à dominante tribale difficiles d'accès en Inde.Schéma : Une enquête transversale à base communautaire a été réalisée auprès de 825 répondants grâce à une méthode d'échantillonnage proportionnel de la population.Résultats : La majorité des répondants appartenait à la tranche d'âge 18­35 ans, à une minorité ethnique et étaient mariés ; 44% étaient illettrés. L'étude a montré une faible connaissance des symptômes, causes et modes de transmission de la TB et une connaissance modérée des services gouvernementaux de TB. Les connaissances relatives à la cause exacte de la TB était négligeables­la moitié des répondants a attribué la TB à la consommation d'alcool local ; 61% savaient que la TB était transmissible et un tiers affirmait que le partage de nourriture était une voie de transmission. La connaissance de la disponibilité de services gratuits de prise en charge dans des structures de santé étatiques était élevée, mais la stratégie DOTS était très mal connue. Des différences significatives entre les sexes ont été observées en matière de niveau de connaissance et de sensibilisation.Conclusion : Les résultats de l'étude soulignaient l'importance d'une intensification urgente des activités de plaidoyer, de communication et de mobilisation sociale culturellement adaptées et spécifiques au genre.

Marco de referencia: Cuarenta poblaciones de difícil acceso de los distritos de Singhbhum oriental y occidental en el estado de Jharkhand de la India.Objetivos: Documentar los conocimientos y la sensibilización en materia de tuberculosis (TB) de los miembros de la comunidad general, comprender las diferencias asociadas con el sexo y aportar recomendaciones a la formulación de intervenciones destinadas a mejorar el programa contra la TB en las zonas de difícil acceso con predominio de población tribal, en la India.Método: Se llevó a cabo una encuesta transversal comunitaria a 825 personas escogidas mediante un muestreo proporcional a la población.Resultados: La mayoría de las personas que respondieron a la encuesta se encontraba en el grupo etario de 18 años a 35 años, pertenecía a un grupo tribal y su estado civil era casado; el 44% era analfabeto. El estudio puso en evidencia un conocimiento precario de la TB con relación a los síntomas, las causas y los modos de transmisión y una sensibilización moderada a la existencia de servicios gubernamentales de atención de la TB. El grado de conocimientos sobre causa real de la TB era exiguo, pues en la mitad de las respuestas se atribuía la TB al consumo de un licor local. El 61% de quienes respondieron consideraba que la enfermedad era contagiosa y un tercio atribuía el modo transmisión al hecho de compartir los alimentos. Se observó un alto grado de conocimiento de la existencia de servicios de tratamiento sin costo en las instituciones públicas de salud, pero pocos conocían la estrategia DOTS. Se observaron diferencias notables entre los sexos en materia de conocimientos y sensibilización.Conclusión: Los resultados del estudio destacan la urgencia de intensificar las actividades de promoción, comunicación y movilización social que sean culturalmente adaptadas y tomen en consideración las diferencias entre los sexos.

Genes encoding bile acid, phospholipid and anion transporters are expressed in a human fetal cardiomyocyte culture.

OBJECTIVES: To establish a human fetal cardiomyocyte culture and to investigate whether the genes that encode transporters that may influence influx or efflux of bile acids are expressed in human fetal cardiomyocytes. DESIGN: Laboratory study. SETTING: Imperial College London. SAMPLE: Six fetal hearts were obtained at the time of termination of pregnancy at 12-13 weeks of gestation and used to generate primary human cardiomyocyte cultures. METHODS: To confirm the presence of cardiomyocytes, the cells were incubated with monoclonal antibodies to sarcomeric alpha-actinin and anticardiac myosin heavy chain. Real-time reverse transcription polymerase chain reaction was used to establish whether transcripts of genes that may influence bile acid transport are present in the culture (NTCP, BSEP, MDR3, FIC1, MRP2, MRP3, OATP-A, OATP-C, OATP-D, OATP-E) and whether taurocholate administration alters messenger RNA (mRNA) expression. MAIN OUTCOME MEASURES: Relative mRNA expression of genes of interest. RESULTS: Real-time polymerase chain reaction demonstrated the presence of mRNA for BSEP, MDR3, FIC1, OATP-C, OATP-D and OATP-E in fetal heart. Four transcripts remained in the cardiomyocyte culture (BSEP, MDR3, FIC1 and OATP-D), and we demonstrated the influence of taurocholate on gene expression. CONCLUSIONS: We have developed an in vitro model of the fetal heart that may be used for studies of the cardiac effect of endobiotics, e.g. bile acids, or of specific agents that may be used to treat the mother or fetus in pregnancy.

Comparison of the arrhythmogenic effects of tauro- and glycoconjugates of cholic acid in an in vitro study of rat cardiomyocytes.

Obstetric cholestasis is associated with intrauterine death. In obstetric cholestasis, primary bile acids are more commonly conjugated with taurine than glycine, while glycoconjugates predominate in normal pregnancy. Using an in vitro model of rat cardiomyocytes, we compared the effect of tauro- and glycoconjugated cholate on cardiomyocyte rhythm, contraction amplitude and network integrity. We demonstrated that taurocholate had a more marked effect on all of these parameters, and the effects of the glycoconjugates were fully reversible while those of tauroconjugates were not. The increased proportion of tauroconjugated bile acids in obstetric cholestasis may contribute to the aetiology of the intrauterine death associated with the condition.

Rate dependence of mechanically induced electrophysiological changes in right ventricle of anaesthetized lambs during pulmonary artery occlusion.

AIM: Mechanically induced early afterdepolarization (EAD) is morphologically similar but different in the mechanisms with drug-induced EAD, which lead to arrhythmia. Pacing suppresses the drug-induced EAD and arrhythmia, however the effect of pacing on mechanically induced EAD and arrhythmia is not clear. This study addressed this issue in right ventricle (RV) of anaesthetized lambs. METHODS: Six lambs were anaesthetized, and their hearts exposed. Nine monophasic action potential (MAP) electrodes were placed on RV apex, outflow and inflow regions, and recorded before, during, and after a 10 s occlusion of pulmonary artery at a number of pacing rates. RESULTS: Pacing significantly reduced the baseline MAP duration at 90% repolarization (MAPD90), decreased the reduction of MAPD at early repolarization at the peak of occlusion. Nonetheless, the percentage of reduction was not significantly different among them. Pacing was able to reduce the frequencies, size of mechanically induced EADs. MAPD90 at the peak of occlusion was all shortened during pacing rather than some lengthened at intrinsic rate. Therefore, the dispersion of MAPD90 at the peak of occlusion reduced from 86 +/- 6 ms at intrinsic rate to 42 +/- 4 ms at 120 beats min-1, 38 +/- 3 ms at 150 beats min-1 and 26 +/- 3 ms at 170 beats min-1. Ultimately, pacing reduced/suppressed mechanically induced premature ventricular beats. These alterations were inversely related to heart rates. CONCLUSION: Pacing reduces/suppresses both stretch-induced EADs and arrhythmia. These modulations are remarkably similar to those on other EADs by the pacing.

Dexamethasone and ursodeoxycholic acid protect against the arrhythmogenic effect of taurocholate in an in vitro study of rat cardiomyocytes.

OBJECTIVE: To establish whether the therapeutic agents ursodeoxycholic acid and dexamethasone protect cardiomyocytes from taurocholate-induced arrhythmias in an in vitro model. DESIGN: Laboratory study. SETTING: Imperial College London, Hammersmith Campus. SAMPLE: Neonatal rat cardiomyocytes. METHODS: Using scanning ion conductance microscopy, we measured the rate, rhythm, amplitude of contraction and calcium dynamics of ventricular myocytes from one to two day old rats. Cells were pre-incubated for 16 hours in dexamethasone (80 or 800 nM) or 0.1 mM ursodeoxycholic acid before adding taurocholate at different concentrations (0.3-4.5 mM). MAIN OUTCOME MEASURES: Changes in rate and amplitude of contraction, calcium dynamics and rhythm. RESULTS: Taurocholate at concentrations of up to 3 mM induces abnormal changes including reductions in rate, amplitude of contraction, abnormal calcium dynamics and dysrhythmias. Although dexamethasone had no immediate protective effect on these changes, pre-incubation with dexamethasone was protective. Ursodeoxycholic acid pre-incubation was protective at taurocholate concentrations up to 1 mM. CONCLUSION: The therapeutic agents dexamethasone and ursodeoxycholic acid appear protective against the arrhythmogenic effect of taurocholate on cardiomyocytes.

Right ventricular distension alters monophasic action potential duration during pulmonary arterial occlusion in anaesthetised lambs: evidence for arrhythmogenic right ventricular mechanoelectrical feedback.

Abnormal loading and distension of the right ventricle may induce arrhythmia through the process of mechanoelectrical feedback. Nonetheless, the electrophysiological effects of right ventricular distension are ill-defined and the mechanisms which underpin mechanoelectrical feedback in the right ventricle are unknown. We examined the effects of changes in right ventricular load (complete occlusion of both caval veins or the main pulmonary artery) in 14 anaesthetised lambs, instrumented with right ventricular surface electrodes and strain gauges for recording monophasic action potential and segment length, and an integrated conductance and micromanometer-tipped catheter for measurement of right ventricular pressure and volume. Caval occlusion did not alter right ventricular segment length and monophasic action potential duration. By contrast, pulmonary arterial occlusion increased the segment length and decreased the monophasic action potential duration at 25, 50 and 70% repolarisation by 29 +/- 6, 22 +/- 4 and 17 +/- 3 ms, respectively (all P < 0.01). Of the 42 pulmonary arterial occlusions, 38 were associated with early afterdepolarisations (EADs) which increased progressively in magnitude as the occlusion was maintained until, in 32, overt arrhythmia was observed. By contrast, none of the four occlusions in which EADs were not observed resulted in arrhythmia. As a result, the proportion of occlusions which resulted in arrhythmia were greater in those associated with EADs than in those which were not (P = 0.002). Right ventricular distension alters the pattern of repolarisation, precipitates early afterdepolarisations and results in a variety of ventricular arrhythmia, including ventricular tachycardia.

Simultaneous measurement of Ca2+ and cellular dynamics: combined scanning ion conductance and optical microscopy to study contracting cardiac myocytes.

We have developed a distance modulated protocol for scanning ion conductance microscopy to provide a robust and reliable distance control mechanism for imaging contracting cells. The technique can measure rapid changes in cell height from 10 nm to several micrometers, with millisecond time resolution. This has been demonstrated on the extreme case of a contracting cardiac myocyte. By combining this method with laser confocal microscopy, it was possible to simultaneously measure the nanometric motion of the cardiac myocyte, and the local calcium concentration just under the cell membrane. Despite large cellular movement, simultaneous tracking of the changes in cell height and measurement of the intracellular Ca2+ near the cell surface is possible while retaining the cell functionality.

Heterogeneous changes of monophasic action potential induced by sustained stretch in atrium.

INTRODUCTION: Chronic enlargement of atrium is common in atrial fibrillation, and the effects of stretch on atrial action potentials seem inconsistent. As atrial muscle is heterogeneous, we suggest that atrial stretch induces a variable electrophysiologic response and that the effects of stretch are only partially mediated by stretch-activated channels. METHODS AND RESULTS: Sixteen guinea pig hearts were perfused by the Langendorff method using Kreb's solution with and without 80 microM streptomycin, which is a stretch-activated channel blocker. Suction electrodes were used to record monophasic action potentials (MAPs) of the left atrium. Left auricular pressure was monitored by a balloon. We determined the MAP duration at 50% and 90% repolarization (MAPD50 and MAPD90) in basal conditions and after a slow onset but sustained stretch of the atrium in the absence and presence of streptomycin. Stretch induced no overall consistent or significant change in mean MAPD50 and MAPD90. The individual responses were markedly variable. The most frequent response (about 50%) was a decrease in MAPD50 and MAPD90. In 25% of cases, there was no change, and in 25% we observed increases in MAPD50 and MAPD90. Streptomycin did not affect MAPD50 and MAPD90, but it dramatically modified the distribution of MAPD changes induced by stretch. In particular, streptomycin removed stretch-induced shortening of MAPD50 and MAPD90, whereas it did not affect stretch-induced lengthening. CONCLUSION: Sustained stretch of atrium induces variable modifications of MAPD that are only partially inhibited by streptomycin. This suggests the participation of ionic channels other than specific stretch-activated channels in the response of atrial myocardium to sustained stretch.

The bile acid taurocholate impairs rat cardiomyocyte function: a proposed mechanism for intra-uterine fetal death in obstetric cholestasis.

Obstetric cholestasis is a liver disease of pregnancy that can be complicated by sudden, hitherto unexplained, intra-uterine fetal death. Because intra-uterine death occurs suddenly, and because fetal heart rate abnormalities have been reported in obstetric cholestasis, we hypothesized that intra-uterine death is caused by impaired fetal cardiomyocyte function, resulting in fetal cardiac arrest. Obstetric cholestasis is associated with raised levels of maternal and fetal serum bile acids, and we propose that these may alter cardiomyocyte function. It was not possible to investigate the effects of bile acids on the intact human fetal heart at a cellular level. Therefore we used the closest available model of fetal myocardium at term: a primary culture of neonatal rat cardiomyocytes in which cells beat synchronously and develop pacemaker activity. The effect of the primary bile acid taurocholate (0.3 mM and 3 mM) on cultures of single cardiomyocytes, each with its own independent rate of contraction, was a reversible decrease in the rate of contraction and in the proportion of beating cells (P < 0.001). Addition of taurocholate to a network of synchronously beating cells caused a similar decrease in the rate of contraction. Furthermore, the integrity of the network was destroyed, and cells ceased to beat synchronously. Taurocholate also resulted in altered calcium dynamics and loss of synchronous beating. These data suggest that raised levels of the bile acid taurocholate in the fetal serum in obstetric cholestasis may result in the development of a fetal dysrhythmia and in sudden intra-uterine death.

Functional localization of single active ion channels on the surface of a living cell.

The spatial distribution of ion channels in the cell plasma membrane has an important role in governing regional specialization, providing a precise and localized control over cell function. We report here a novel technique based on scanning ion conductance microscopy that allows, for the first time, mapping of single active ion channels in intact cell plasma membranes. We have mapped the distribution of ATP-regulated K+ channels (KATP channels) in cardiac myocytes. The channels are organized in small groups and anchored in the Z-grooves of the sarcolemma. The distinct pattern of distribution of these channels may have important functional implications.

Hybrid scanning ion conductance and scanning near-field optical microscopy for the study of living cells.

We have developed a hybrid scanning ion conductance and scanning near-field optical microscope for the study of living cells. The technique allows quantitative, high-resolution characterization of the cell surface and the simultaneous recording of topographic and optical images. A particular feature of the method is a reliable mechanism to control the distance between the probe and the sample in physiological buffer. We demonstrate this new method by recording near-field images of living cells (cardiac myocytes).

Mechano-electric feedback in right atrium after left ventricular infarction in rats.

Left ventricular myocardial infarction (MI) can lead to alterations in hemodynamic load conditions, thereby inducing right atrial hypertrophy and dilatation associated with phenotypic modulation of cardiomyocytes, electrical abnormalities, rhythm disturbances, and atrial fibrillation. However, there is limited information on the electrophysiological basis for these events. We investigated whether atrial stretch in the setting of chronic MI modulates the electrophysiological properties of cardiomyocytes via "mechano-electric feedback", providing a mechanism for atrial arrhythmia after ventricular infarction. Five weeks after left ventricular MI (n=37), action potentials (AP) were measured in right atrial tissue preparations using a current clamp scheme, and compared to sham-operated rats (SO, n=10). Contractile activity was recorded at a preload of 1 mN, and sustained stretch was applied via a micrometer. In SO, stretch of 1.75 mN shortened repolarization at 50% and prolonged it at 90%. In MI, mechanically-induced electrical alterations were observed at a significantly lower level of stretch than in SO (0.19 mN). Sustained stretch in MI prolonged AP at 90% repolarization giving rise to stretch-activated depolarizations (SAD) near 90% repolarization (SAD90). When reaching threshold for premature APs, electrical phenomena similar to atrial fibrillations were seen in some preparations. Moreover, we observed APs with prolonged duration at 25%, 50%, and 90% repolarization where stretch induced SAD near 50%. Gadolinium used at a concentration to inhibit stretch-activated channels (40microM) suppressed mechanically-induced electrical events. In conclusion, increased susceptibility after MI to mechanical stretch may predispose atrial cardiomyocytes to arrhythmia. These mechano-electrical alterations are sensitive to gadolinium suggesting involvement of stretch-activated ion channels.

Mechanoelectric feedback after left ventricular infarction in rats.

BACKGROUND: Myocardial infarction can lead to electrical abnormalities and rhythm disturbances. However, there is limited data on the electrophysiological basis for these events. Since regional contraction abnormalities feature prominently in infarction, we investigated whether stretch of myocardium from the infarction borderzone can modulate the electrophysiological properties of cardiomyocytes via mechanoelectric feedback providing a mechanism for post-infarction arrhythmia. METHODS: Five weeks after experimental myocardial infarction (MI) in rats due to ligation of the left coronary artery (n = 26) or after sham operation (SO, n = 16), action potentials (AP) were measured in left ventricular preparations from the infarction borderzone. Sustained stretch was applied via a micrometer. RESULTS: Preparations from MI generated spontaneous electrical and contractile activity. Cardiomyocytes from MI had a comparable AP amplitude, a more negative resting membrane potential, and a prolonged AP duration (APD) when compared to SO. In SO, stretch of 150 microns increased the APD90. This was associated with stretch activated depolarizations near APD90 (SAD-90). In MI, significantly lower stretch, of only 20 microns, elicited SAD-90s, or SADs near APD50 (SAD-50). Stretch-induced events were suppressed by gadolinium, at a concentration (40 microM) normally used to inhibit stretch-activated channels. CONCLUSION: After MI, SADs are generated in the infarction borderzone at lower degrees of stretch. Increased sensitivity of the membrane potential of cardiac myocytes to mechanical stimuli may contribute to the high risk of arrhythmia after infarction. These SADs may involve the opening of stretch-activated channels.

Cell volume measurement using scanning ion conductance microscopy.

We report a novel scanning ion conductance microscopy (SICM) technique for assessing the volume of living cells, which allows quantitative, high-resolution characterization of dynamic changes in cell volume while retaining the cell functionality. The technique can measure a wide range of volumes from 10(-19) to 10(-9) liter. The cell volume, as well as the volume of small cellular structures such as lamelopodia, dendrites, processes, or microvilli, can be measured with the 2.5 x 10(-20) liter resolution. The sample does not require any preliminary preparation before cell volume measurement. Both cell volume and surface characteristics can be simultaneously and continuously assessed during relatively long experiments. The SICM method can also be used for rapid estimation of the changes in cell volume. These are important when monitoring the cell responses to different physiological stimuli.

Human papillomavirus detection by non isotopic in situ hybridization, in situ hybridization with signal amplification and in situ polymerase chain reaction.

Classical in situ hybridization (ISH) with biotinylated probes makes it possible to detect and localize human papillomavirus (HPV) nucleic acid sequences in cytological and histological materials. This method is however of limited value in the detection of a few copies of the virus. Moreover the specificity of such a technique is not always convincing when ISH signals are small and/or of low intensity. Recently, much attention has been focused on the utility of the in vitro polymerase chain reaction (PCR) and especially on PCR-single strand conformation polymorphism (SSCP) to amplify small amounts of viral DNA with accurate hybrid specificity. But the latter method requires nucleic acid extraction and tissue destruction. Thus, correlation between the PCR results and histological findings is not possible. Hence, the aim of our current study was to apply to HeLa cells and cervical formalin-fixed and paraffin-embedded biopsies, a novel procedure of ISH signal amplification, the catalyzed signal amplification (CSA). Such a procedure is based on the deposition of streptavidin-horseradish peroxidase catalyzing the deposition of biotinylated tyramide molecules on the location of the probed target. The biotin accumulation is then detected with streptavidin peroxidase and diaminobenzidine. The results were compared with those obtained by direct and indirect in situ PCR. The catalysed signal amplification successfully increased the sensitivity and efficiency of ISH for the detection of rare sequences in HPV infected cells and histological materials. Such a method was found simpler and faster than in situ PCR and tissue morphology was better preserved.

Direct in situ reverse transcriptase-linked polymerase chain reaction with biotinylated primers for the detection of hepatitis C virus RNA in liver biopsies.

To assess the presence and the cellular distribution of hepatitis C virus (HCV) RNA in the liver of 11 patients with confirmed HCV infection, a direct in situ reverse transcriptase-linked polymerase chain reaction (RT-PCR) method was performed on formalin-fixed and paraffin-embedded biopsies. The oligonucleotide primers used were specific to the 5' non coding region. An unlabelled downstream oligonucleotide served as a primer for reverse transcription as well as PCR. The upstream oligonucleotide serving as a primer for PCR was biotinylated, allowing a direct enzymatic detection of PCR products. HCV infected cells revealed cytoplasmic staining mainly concentrated towards the interface of the nucleus and cytoplasm. Most of the stained cells were hepatocytes and sometimes Kupffer cells. The results were compared with those obtained by RT-PCR of RNA extracted from the corresponding tissue block. Extracted HCV RNA could be detected in liver tissues of nine out of 11 (82%) infected patients. The detection rate using in situ RT-PCR was 7/11 (63%). The use of labelled primers improved specificity of direct in situ methods, by preventing non-specific incorporation of labelled dNTPs into fragmented DNA. Further studies are however required in order to increase detection sensitivity of HCV infection by in situ molecular methods.