Which attributes within target product profiles for tuberculosis diagnostics are the most important to focus on?

SETTING: New diagnostic tools for tuberculosis (TB) are urgently needed. To guide investment decisions, sufficient information regarding which test attributes are the most important for users and decision makers interested in introducing TB diagnostics should be made available. OBJECTIVE: To rank test attributes in a target product profile (TPP) in order of importance for a sputum smear replacement test for TB. DESIGN: An online survey was administered among 33 participants representing 14 of the 22 TB high-burden countries. Respondents included laboratory personnel, national TB program managers, donors, technical experts, patients and researchers. Participants were asked to rank 21 TPP test attributes in order of importance using a maximum-differential method that required ranking of relative trade-offs. RESULTS: Sensitivity was ranked as the most important test attribute, followed by maintenance/calibration, reagent kit storage/stability, sample preparation steps and time to results. CONCLUSION: Consulting widely regarding which TPP attributes are valued most by users and decision-makers involved in introducing TB diagnostics can assist TB test developers to prioritize their investments, and guide decision making if trade-offs are necessary.

An Alternative Splice Variant of Zebrafish Cx52.6 is Expressed in Retinal Horizontal Cells.

Retinal horizontal cells (HCs) are inhibitory neurons, which modulate the transmission of light-elicited signals from photoreceptors to bipolar cells in the outer retina. HCs of the same physiological type are extensively coupled via gap junctions. In the zebrafish retina, the population of HCs comprises up to four morphologically distinct subtypes. Four different connexins (Cx52.6, Cx52.7, Cx52.9 and Cx55.5) were detected in these cells with overlapping expression patterns. In this study, we show that Cx52.6 is alternatively spliced in the retina, resulting in an additional isoform, designated as Cx53.4, which differs from the originally described Cx52.6 only by the final C-terminal peptide (12 vs. 4 aa). Further protein sequence alignments revealed that Cx53.4 represents the counterpart of alternatively spliced mouse Cx57 and human Cx62. RT-PCR analyses of mRNA expression in different adult zebrafish tissues showed that Cx53.4 is expressed exclusively in the retina. The localization of Cx53.4 protein within the retina was analyzed using a specific antibody. Immunofluorescence analyses demonstrated that the expression of Cx53.4 is restricted to HCs of all four subtypes. Further, immunoelectron microscopy confirmed the presence of Cx53.4 in gap junctions between HC dendrites and between their axon terminals.

Complexity of gap junctions between horizontal cells of the carp retina.

In the vertebrate retina, horizontal cells (HCs) reveal homologous coupling by gap junctions (gj), which are thought to consist of different connexins (Cx). However, recent studies in mouse, rabbit and zebrafish retina indicate that individual HCs express more than one connexin. To provide further insights into the composition of gj connecting HCs and to determine whether HCs express multiple connexins, we examined the molecular identity and distribution of gj between HCs of the carp retina. We have cloned four carp connexins designated Cx49.5, Cx55.5, Cx52.6 and Cx53.8 with a close relationship to connexins previously reported in HCs of mouse, rabbit and zebrafish, respectively. Using in situ hybridization, Cx49.5 expression was detected in different subpopulations of retinal neurons including HCs, whereas the Cx52.6 transcript was localized exclusively in HCs. Using specific antibodies, Cx55.5 and Cx53.8 were detected on dendrites of all four HC subtypes and axon terminals. Immunoelectron microscopy confirmed the presence of Cx55.5 and Cx53.8 in gap junctions between these processes and Cx55.5 was additionally observed in HC dendrites invaginating cone pedicles, suggesting its participation in the modulation of photoreceptor output in the carp retina. Furthermore, using single-cell RT-PCR, all four connexins were detected in different subtypes of HCs, suggesting overlapping expression patterns. Thus, the composition of gj mediating homologous coupling between subtypes of carp HCs appears to be more complex than expected. Moreover, BLAST searches of the preliminary carp genome, using novel sequences as query, suggest that most of the analyzed connexin genes are duplicated in carp.

Transcriptional repression by the insulator protein CTCF involves histone deacetylases.

The highly conserved zinc-finger protein, CTCF, is a candidate tumor suppressor protein that binds to highly divergent DNA sequences. CTCF has been connected to multiple functions in chromatin organization and gene regulation including chromatin insulator activity and transcriptional enhancement and silencing. Here we show that CTCF harbors several autonomous repression domains. One of these domains, the zinc-finger cluster, silences transcription in all cell types tested and binds directly to the co-repressor SIN3A. Two distinct regions of SIN3A, the PAH3 domain and the extreme C-terminal region, bind independently to this zinc-finger cluster. Analysis of nuclear extract from HeLa cells revealed that CTCF is also capable of retaining functional histone deacetylase activity. Furthermore, the ability of regions of CTCF to retain deacetylase activity correlates with the ability to bind to SIN3A and to repress gene activity. We suggest that CTCF driven repression is mediated in part by the recruitment of histone deacetylase activity by SIN3A.

New screening technologies for type 2 diabetes mellitus appropriate for use in tuberculosis patients.

Type 2 diabetes mellitus (DM), which is epidemic in low- and middle-income countries (LMICs), may threaten gains made in tuberculosis (TB) control, as DM is both a major risk factor for developing active TB and it can lead to adverse TB treatment outcomes. Despite World Health Organization guidance that all TB patients should be screened for DM, most facilities in LMICs that manage TB patients do not currently perform screening for DM, due in part to the cost and complexity involved. DM screening is further complicated by the presentation of transient hyperglycemia in many TB patients, as well as differences in diabetes risk factors (e.g., body mass index) between TB patients and the general public. In this article, we review existing and new technologies for DM screening that may be more suitable for TB patients in LMICs. Such methods should be rapid, they should not require fasting, and they should allow the provider to differentiate between transient and longer-term hyperglycemia, using inexpensive tools that require little training and no specialized infrastructure. Several methods that are currently under development, such as point-of-care glycated hemoglobin and glycated albumin assays, non-invasive advanced glycation end-product readers, and sudomotor function-based screening devices, offer interesting performance characteristics and warrant evaluation in populations with TB.

L'épidémie de diabète sucré (DM) de type 2 dans les pays à revenus faibles et moyens (LMIC) peut constituer une menace pour les progrès de la lutte contre la tuberculose (TB), car le DM est à la fois un facteur majeur de risque et de développement d'une TB active et peut aussi entrainer des résultats défavorables du traitement de la TB. En dépit de la directive de l'Organisation mondiale de la Santé selon laquelle tous les patients TB devraient faire l'objet d'un dépistage pour le DM, la plupart des services des LMIC qui traitent les patients TB ne réalisent pas actuellement le dépistage du DM, en partie en raison du coût et de la complexité qu'il implique. Le dépistage du DM est par ailleurs compliqué par l'existence d'une hyperglycémie transitoire chez beaucoup de patients TB ainsi que par les différences de facteurs de risque de DM (par exemple l'indice de masse corporelle) entre les patients TB et la population générale. Dans cet article, nous révisons les technologies existantes et nouvelles pour le dépistage du DM qui pourraient être les plus applicables aux patients TB dans les LMIC. De telles méthodes devraient être rapides, elles devraient ne pas exiger le jeûne et elles devraient permettre aux pourvoyeurs de soins de distinguer entre des hyperglycémies transitoires et de plus longue durée au moyen d'outils peu coûteux, n'exigeant que peu de formation et aucune infrastructure spécialisée. Différentes méthodes sont actuellement en cours de développement, tels que les tests sur l'hémoglobine glycosylée aux lieux de soins et sur l'albumine glycosylée, les lecteurs des produits finaux d'une glycation avancée non-invasive et les outils de dépistage basés sur la fonction sudomotrice ; elles offrent des caractéristiques intéressantes de performance et méritent une évaluation dans les populations TB.

La diabetes (DM) de tipo 2 presenta características epidémicas en los países con ingresos bajos e intermedios (LMIC) y puede poner en peligro los avances alcanzados en materia de control de la tuberculosis (TB); la DM constituye un factor de riesgo importante de padecer la enfermedad TB activa y también puede tener consecuencias desfavorables en el desenlace del tratamiento antituberculoso. Pese a la recomendación de la Organización Mundial de la Salud de realizar la detección sistemática de la DM en todos los pacientes TB, la mayoría de los establecimientos que atienden a estos pacientes en los LMIC no cumplen con esta práctica, en parte debido a los costos y a la complejidad de la misma. La detección de la DM se complica además por la hiperglucemia transitoria que suele observarse en muchos pacientes TB y por las diferencias en los factores de riesgo de DM, presentes en los pacientes con TB y la población general, por ejemplo el índice de masa corporal. En el presente artículo se analizan las técnicas existentes y los nuevos métodos de detección sistemática de la DM que pueden ser más adaptados a los pacientes con TB de los LMIC. Estos métodos deben ser rápidos, no deben precisar el estado de ayuno y deben permitir al profesional de salud diferenciar entre la hiperglucemia transitoria y la hiperglucemia de largo plazo, mediante la utilización instrumentos de bajo costo, que exijan poco entrenamiento y no necesiten infraestructuras especializadas. En la actualidad, se encuentran en curso de desarrollo varios métodos como las pruebas de hemoglobina glucosilada y albúmina glucosilada realizadas en el punto de atención, los lectores no invasivos de productos finales de la glucosilación avanzada y los dispositivos de detección basados en la función sudomotora; estos métodos ofrecen características de rendimiento interesantes y merecen una evaluación en las poblaciones de pacientes con diagnóstico de TB.

Pharmacokinetics of amlodipine in renal impairment.

Amlodipine was administered as 14 single 5-mg oral daily doses to 27 male subjects with renal function ranging from normal to haemodialysis-dependent. Blood specimens were obtained for measurement of plasma amlodipine concentrations for 24 h following the first dose, for 168 h following the final dose and during daily administration of amlodipine. Amlodipine was well tolerated. Renal impairment had little effect on the pharmacokinetics of amlodipine. The elimination half-life was of the order of 50 h, similar to previously reported values and did not vary with renal function. Steady-state pre-dose concentrations were observed after the ninth dose. Accumulation of amlodipine was not significantly different from that expected on theoretical grounds and did not significantly change with renal function. These results suggest that once daily administration of amlodipine is suitable for all degrees of renal function and that dosage adjustment is not necessary in renal impairment.

Pharmacokinetics of amlodipine in renal impairment.

The pharmacokinetics of amlodipine was studied in 27 subjects with renal function ranging from normal to dialysis-dependent. Amlodipine (as a single 5-mg capsule) was administered once daily for 14 days and its plasma concentrations were measured by gas chromatography during and after treatment. Renal impairment had little or no effect on the pharmacokinetics of amlodipine. The elimination half-life was of the order of 50 h, similar to previously observed values, and did not vary with differences in renal function. Steady-state predose concentrations were observed after the ninth dose. Accumulation of amlodipine to steady-state levels was not significantly different from that expected on theoretical grounds and did not significantly change with renal function. These results suggest that once-daily administration of amlodipine is suitable for all degrees of renal function and that dosage adjustment is not necessary in renal impairment.