Intensified Tuberculosis Case Finding among Malnourished Children in Nutritional Rehabilitation Centres of Karnataka,India: Missed Opportunities

Background

Severe acute malnutrition (SAM) is the most serious form of malnutrition affecting children under-five and is associated with many infectious diseases including Tuberculosis (TB). In India, nutritional rehabilitation centres (NRCs) have been recently established for the management of SAM including TB. The National TB Programme (NTP) in India has introduced a revised algorithm for diagnosing paediatric TB. We aimed to examine whether NRCs adhered to these guidelines in diagnosing TB among SAM children.

Methods

A cross-sectional study involving review of records of all SAM children identified by health workers during 2012 in six tehsils (sub-districts) with NRCs (population: 1.8 million) of Karnataka, India.

Results

Of 1927 identified SAM children, 1632 (85%) reached NRCs. Of them, 1173 (72%) were evaluated for TB and 19(2%) were diagnosed as TB. Of 1173, diagnostic algorithm was followed in 460 (37%). Among remaining 763 not evaluated as per algorithm, tuberculin skin test alone was conducted in 307 (41%), chest radiography alone in 99 (13%) and no investigations in 337 (45%). The yield of TB was higher among children evaluated as per algorithm (4%) as compared to those who were not (0.3%) (OR: 15.3 [95%CI: 3.5-66.3]). Several operational challenges including non-availability of a full-time paediatrician, non-functioning X-ray machine due to frequent power cuts, use of tuberculin with suboptimal strength and difficulties in adhering to a complex diagnostic algorithm were observed.

Conclusion

This study showed that TB screening in NRCs was sub-optimal in Karnataka. Some children did not reach the NRC, while many of those who did were either not or sub-optimally evaluated for TB. This study pointed to a number of operational issues that need to be addressed if this collaborative strategy is to identify more TB cases amongst malnourished children in India.     

Effect of postnatal development on calcium currents and slow charge movement in mammalian skeletal muscle

Single- (whole-cell patch) and two-electrode voltage-clamp techniques were used to measure transient (Ifast) and sustained (Islow) calcium currents, linear capacitance, and slow, voltage-dependent charge movements in freshly dissociated fibers of the flexor digitorum brevis (FDB) muscle of rats of various postnatal ages. Peak Ifast was largest in FDB fibers of neonatal (1-5 d) rats, having a magnitude in 10 mM external Ca of 1.4 +/- 0.9 pA/pF (mean +/- SD; current normalized by linear fiber capacitance). Peak Ifast was smaller in FDB fibers of older animals, and by approximately 3 wk postnatal, it was so small as to be unmeasurable. By contrast, the magnitudes of Islow and charge movement increased substantially during postnatal development. Peak Islow was 3.6 +/- 2.5 pA/pF in FDB fibers of 1-5-d rats and increased to 16.4 +/- 6.5 pA/pF in 45-50-d-old rats; for these same two age groups, Qmax, the total mobile charge measurable as charge movement, was 6.0 +/- 1.7 and 23.8 +/- 4.0 nC/microF, respectively. As both Islow and charge movement are thought to arise in the transverse-tubular system, linear capacitance normalized by the area of fiber surface was determined as an indirect measure of the membrane area of the t-system relative to that of the fiber surface. This parameter increased from 1.5 +/- 0.2 microF/cm2 in 2-d fibers to 2.9 +/- 0.4 microF/cm2 in 44-d fibers. The increases in peak Islow, Qmax, and normalized linear capacitance all had similar time courses. Although the function of Islow is unknown, the substantial postnatal increase in its magnitude suggests that it plays an important role in the physiology of skeletal muscle.     

Molecular evolution of voltage-sensitive ion channel genes: on the origins of electrical excitability

We have analyzed nucleic acid and amino acid sequence alignments of a variety of voltage-sensitive ion channels, using several methods for phylogenetic tree reconstruction. Ancient duplications within this family gave rise to three distantly related groups, one consisting of the Na+ and Ca++ channels, another the K+ channels, and a third including the cyclic nucleotide-binding channels. A series of gene duplications produced at least seven mammalian homologues of the Drosophila Shaker K+ channel; clones of only three of these genes are available from all three mammalian species examined (mouse, rat, and human), pointing to specific genes that have yet to be recovered in one or another of these species. The Shaw-related K+ channels and the Na+ channel family have also undergone considerable expansion in mammals, relative to flies. These expansions presumably reflect the needs of the high degree of physiological and neuronal complexity of mammals. Analysis of the separate domains of the four-domain channels (Ca++ and Na+) supports their having evolved by two sequential gene duplications and implies the historical existence of a functional two-domain channel.      

GNA11 Q209L Mouse Model Reveals RasGRP3 as an Essential Signaling Node in Uveal Melanoma

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Immune Modulating Effects of NKT Cells in a Physiologically Low Dose Leishmania major Infection Model after αGalCer Analog PBS57 Stimulation

Leishmaniasis is a parasitic infection affecting ∼12 million people worldwide, mostly in developing countries. Treatment options are limited and no effective vaccines exist to date. Natural Killer T (NKT) cells are a conserved innate-like lymphocyte population with immunomodulating effects in various settings. A number of reports state a role of NKT cells in different models of Leishmania infection. Here, we investigated the effect of NKT cells in a physiologically relevant, intradermal low dose infection model. After inoculation of 10³ infectious-stage L. major, comparable numbers of skin-immigrating NKT cells in both susceptible BALB/c mice and resistant C57BL/6 mice were noted. Compared to their wild type counterparts, NKT cell-deficient mice on a C57BL/6 background were better able to contain infection with L. major and showed decreased IL-4 production in cytokine analysis performed 5 and 8 weeks after infection. Low doses of the NKT cell stimulating αGalCer analog PBS57 applied at the time of infection led to disease exacerbation in C57BL/6 wild-type, but not NKT-deficient mice. The effect was dependent both on the timing and amount of PBS57 administered. The effect of NKT cell stimulation by PBS57 proved to be IL-4 dependent, as it was neutralized in IL-4-deficient C57BL/6 or anti-IL-4 antibody-treated wild-type mice. In contrast to C57BL/6 mice, administration of PBS57 in susceptible BALB/c mice resulted in an improved course of disease. Our results reveal a strain- and cytokine-dependent regulatory role of NKT cells in the development of immunity to low dose L. major infections. These effects, probably masked in previous studies using higher parasite inocula, should be considered in future therapy and immunization approaches.     

Phase II DeCOG-Study of Ipilimumab in Pretreated and Treatment-Na?ve Patients with Metastatic Uveal Melanoma

Purpose

Up to 50% of patients with uveal melanoma (UM) develop metastatic disease with limited treatment options. The immunomodulating agent ipilimumab has shown an overall survival (OS) benefit in patients with cutaneous metastatic melanoma in two phase III trials. As patients with UM were excluded in these studies, the Dermatologic Cooperative Oncology Group (DeCOG) conducted a phase II to assess the efficacy and safety of ipilimumab in patients with metastatic UM.

Patients and Methods

We undertook a multicenter phase II study in patients with different subtypes of metastatic melanoma. Here we present data on patients with metastatic UM (pretreated and treatment-naïve) who received up to four cycles of ipilimumab administered at a dose of 3 mg/kg in 3 week intervals. Tumor assessments were conducted at baseline, weeks 12, 24, 36 and 48 according to RECIST 1.1 criteria. Adverse events (AEs), including immune-related AEs were graded according to National Cancer Institute Common Toxicity Criteria (CTC) v.4.0. Primary endpoint was the OS rate at 12 months.

Results

Forty five pretreated (85%) and eight treatment-naïve (15%) patients received at least one dose of ipilimumab. 1-year and 2-year OS rates were 22% and 7%, respectively. Median OS was 6.8 months (95% CI 3.7–8.1), median progression-free survival 2.8 months (95% CI 2.5–2.9). The disease control rate at weeks 12 and 24 was 47% and 21%, respectively. Sixteen patients had stable disease (47%), none experienced partial or complete response. Treatment-related AEs were observed in 35 patients (66%), including 19 grade 3–4 events (36%). One drug-related death due to pancytopenia was observed.

Conclusions

Ipilimumab has very limited clinical activity in patients with metastatic UM. Toxicity was manageable when treated as per protocol-specific guidelines.

Trial Registration

ClinicalTrials.gov NCT01355120     

Single-nucleotide polymorphism,linkage disequilibrium and geographic structure in the malaria parasite <Emphasis Type="Italic">Plasmodium vivax</Emphasis>: prospects for genome-wide association studies

Background

The ideal malaria parasite populations for initial mapping of genomic regions contributing to phenotypes such as drug resistance and virulence, through genome-wide association studies, are those with high genetic diversity, allowing for numerous informative markers, and rare meiotic recombination, allowing for strong linkage disequilibrium (LD) between markers and phenotype-determining loci. However, levels of genetic diversity and LD in field populations of the major human malaria parasite P. vivax remain little characterized.

Results

We examined single-nucleotide polymorphisms (SNPs) and LD patterns across a 100-kb chromosome segment of P. vivax in 238 field isolates from areas of low to moderate malaria endemicity in South America and Asia, where LD tends to be more extensive than in holoendemic populations, and in two monkey-adapted strains (Salvador-I, from El Salvador, and Belem, from Brazil). We found varying levels of SNP diversity and LD across populations, with the highest diversity and strongest LD in the area of lowest malaria transmission. We found several clusters of contiguous markers with rare meiotic recombination and characterized a relatively conserved haplotype structure among populations, suggesting the existence of recombination hotspots in the genome region analyzed. Both silent and nonsynonymous SNPs revealed substantial between-population differentiation, which accounted for ~40% of the overall genetic diversity observed. Although parasites clustered according to their continental origin, we found evidence for substructure within the Brazilian population of P. vivax. We also explored between-population differentiation patterns revealed by loci putatively affected by natural selection and found marked geographic variation in frequencies of nucleotide substitutions at the pvmdr-1 locus, putatively associated with drug resistance.

Conclusion

These findings support the feasibility of genome-wide association studies in carefully selected populations of P. vivax, using relatively low densities of markers, but underscore the risk of false positives caused by population structure at both local and regional levels.See commentary: http://www.biomedcentral.com/1741-7007/8/90

     

Calcium currents in embryonic and neonatal mammalian skeletal muscle

The whole-cell patch-clamp technique was used to study the properties of inward ionic currents found in primary cultures of rat and mouse skeletal myotubes and in freshly dissociated fibers of the flexor digitorum brevis muscle of rats. In each of these cell types, test depolarizations from the holding potential (-80 or -90 mV) elicited three distinct inward currents: a sodium current (INa) and two calcium currents. INa was the dominant inward current: under physiological conditions, the maximum inward INa was estimated to be at least 30-fold larger than either of the calcium currents. The two calcium currents have been termed Ifast and Islow, corresponding to their relative rates of activation. Ifast was activated by test depolarizations to around -40 mV and above, peaked in 10-20 ms, and decayed to baseline in 50-100 ms. Islow was activated by depolarizations to approximately 0 mV and above, peaked in 50-150 ms, and decayed little during a 200-ms test pulse. Ifast was inactivated by brief, moderate depolarizations; for a 1-s change in holding potential, half-inactivation occurred at -55 to -45 mV and complete inactivation occurred at -40 to -30 mV. Similar changes in holding potential had no effect on Islow. Islow was, however, inactivated by brief, strong depolarizations (e.g., 0 mV for 2 s) or maintained, moderate depolarizations (e.g., -40 mV for 60 s). Substitution of barium for calcium had little effect on the magnitude or time course of either Ifast or Islow. The same substitution shifted the activation curve for Islow approximately 10 mV in the hyperpolarizing direction without affecting the activation of Ifast. At low concentrations (50 microM), cadmium preferentially blocked Islow compared with Ifast, while at high concentrations (1 mM), it blocked both Ifast and Islow completely. The dihydropyridine calcium channel antagonist (+)-PN 200-110 (1 microM) caused a nearly complete block of Islow without affecting Ifast. At a holding potential of -80 mV, the half-maximal blocking concentration (K0.5) for the block of Islow by (+)-PN 200-110 was 182 nM. At depolarized holding potentials that inactivated Islow by 35-65%, K0.5 decreased to 5.5 nM.     

Genetic and molecular characterization of uveal melanoma cell lines

The recent identification of frequent activating mutations in GNAQ or GNA11 in uveal melanoma provides an opportunity to better understand the pathogenesis of this melanoma subtype and to develop rational therapeutics to target the cellular effects mediated by these mutations. Cell lines from uveal melanoma tumors are an essential tool for these types of analyses. We report the mutation status of relevant melanoma genes, expression levels of proteins of interest, and DNA fingerprinting of a panel of uveal melanoma cell lines used in the research community.