Loss of CASK Accelerates Heart Failure Development.

[Figure: see text].

Dried Blood Spots (DBS): A suitable alternative to using whole blood samples for diagnostic testing of visceral leishmaniasis in the post-elimination era.

BACKGROUND: Serum or whole blood collection, processing, transport and storage still present significant challenges in low resource settings where mass surveillance is required to sustain disease elimination. Therefore, in this study, we explored the diagnostic efficacy of dried blood spots (DBS) as a minimally invasive and potentially cost-effective alternative sampling technique to whole blood sampling procedures for subsequent detection of Leishmania donovani antibodies or DNA. METHODOLOGY AND PRINCIPAL FINDINGS: Archived serum, DNA samples from whole blood of visceral leishmaniasis (VL) cases and healthy controls, and DBS from corresponding cases and controls, were used. Both molecular and serological assays were optimized to detect L. donovani antibodies or DNA in DBS elute and results were compared against those obtained with whole blood. Serological assays (both rK28 ELISA and rK39 ELISA) of DBS samples showed sensitivity and specificity of 100% and had excellent agreement with results from whole blood samples (kappa value ranged from 0.98-1). Bland-Altman analysis of OD values from rK28-ELISA with DBS elute and patients' serum showed an excellent agreement (ICC = 0.9) whereas a good agreement (ICC = 0.8) was observed in the case of rK39-ELISA. However, qPCR and RPA of DBS samples had a diminished sensitivity of 76% and 68%, respectively, and poor agreement was observed with the whole blood samples. CONCLUSION: Our results demonstrate that DBS offer excellent diagnostic efficiency for serological assays and represent a viable alternative to whole blood sampling procedures.

Catechol-O-methyltransferase and dopamine receptor D4 gene variants: Possible association with substance abuse in Bangladeshi male.

Genetic risk of substance abuse is encoded mainly by central neurochemical pathways(mostly dopaminergic system) related to reinforcement and reward. In this study a functionalpolymorphism in Catechol-O-methyltransferase (COMT) (Val158Met) and the Dopamine receptor D4 gene (DRD4) (120 bp tandem duplication) has been studied in substance abused subjects. The study was carried out with 183 substance abused subjects and 175 healthy persons with no history of substance abuse. DNA was extracted and polymorphisms were analyzed using allele-specific PCR. The impact of these two polymorphisms was also analyzed on addictive characteristics (age of starting abuse, a pattern of drug habit, and period of addiction). It was found that only the heterozygous variant of COMT polymorphism (Val/Met) (p<0.05, OR = 1.66, 95% CI = 1.044-2.658) and both homozygous (p<0.05, OR = 0.43, 95% CI = 0.193-0.937) and heterozygous (p<0.05, OR = 0.37, 95% CI = 0.172-0.826) derived variants of DRD4 120 bp tandem duplication were significantly associated with risk of substance abuse compared to controls. In case of association of these polymorphisms with an age of onset, no significant difference was found among three different genotypic groups of COMT polymorphism. Whereas, the homozygous derived variant (240 bp/240 bp) of DRD4 gene was found to have a later age of onset (20.5±0.8) for substance abuse compared to heterozygous (120 bp/240 bp) (19.1±0.8) and wild type homozygous variant (120 bp/120 bp) (16.0±0.5), which was statistically significant (p<0.05). Again, in the case of the pattern of drug habit, the frequency of the Val/Val genotype is higher in polysubstance abused (>2 drugs) subjects (p<0.05) compared to the heterozygous Val/Met containing variants. An association of period of addiction was analyzed with an individual type of substance abuse and found that heroin abused subjects have a significantly higher period of addiction (11.6±1.0) compared to other abusers (p<0.01). Further, it was found that Met/Met containing variants of COMT polymorphism has a more extended period of addiction than other genetic variants in heroin abused subjects. These results indicate that genetic variability may influence the susceptibility to the risk of substance abuse and addictive characteristics.

Enhanced Heart Failure in Redox-Dead Cys17Ser PKARIα Knock-In Mice.

Background PKARIα (protein kinase A type I-α regulatory subunit) is redox-active independent of its physiologic agonist cAMP. However, it is unknown whether this alternative mechanism of PKARIα activation may be of relevance to cardiac excitation-contraction coupling. Methods and Results We used a redox-dead transgenic mouse model with homozygous knock-in replacement of redox-sensitive cysteine 17 with serine within the regulatory subunits of PKARIα (KI). Reactive oxygen species were acutely evoked by exposure of isolated cardiac myocytes to AngII (angiotensin II, 1 µmol/L). The long-term relevance of oxidized PKARIα was investigated in KI mice and their wild-type (WT) littermates following transverse aortic constriction (TAC). AngII increased reactive oxygen species in both groups but with RIα dimer formation in WT only. AngII induced translocation of PKARI to the cell membrane and resulted in protein kinase A-dependent stimulation of ICa (L-type Ca current) in WT with no effect in KI myocytes. Consequently, Ca transients were reduced in KI myocytes as compared with WT cells following acute AngII exposure. Transverse aortic constriction-related reactive oxygen species formation resulted in RIα oxidation in WT but not in KI mice. Within 6 weeks after TAC, KI mice showed an enhanced deterioration of contractile function and impaired survival compared with WT. In accordance, compared with WT, ventricular myocytes from failing KI mice displayed significantly reduced Ca transient amplitudes and lack of ICa stimulation. Conversely, direct pharmacological stimulation of ICa using Bay K8644 rescued Ca transients in AngII-treated KI myocytes and contractile function in failing KI mice in vivo. Conclusions Oxidative activation of PKARIα with subsequent stimulation of ICa preserves cardiac function in the setting of acute and chronic oxidative stress.

Th1 and Th17 responses to Helicobacter pylori in Bangladeshi infants, children and adults.

Both Th1 and Th17 cells are important components of the immune response to Helicobacter pylori (Hp) in adults, but less is known about T cell responses to Hp during early childhood, when the infection is often acquired. We investigated Th1 and Th17 type responses to Hp in adults, children and infants in Bangladesh, where Hp is highly endemic. IL-17 and IFN-γ mRNA levels in gastric biopsies from Hp-infected Bangladeshi adults were analyzed and compared to levels in infected and uninfected Swedish controls. Since biopsies could not be collected from infants and children, cytokine responses in Bangladeshi infants (6-12 months), children (3-5 years) and adults (>19 years) were instead compared by stimulating peripheral blood mononuclear cells (PBMCs) with a Hp membrane preparation (MP) and analyzing culture supernatants by ELISA and cytometric bead array. We found significantly higher expression of IL-17 and IFN-γ mRNA in gastric mucosa of Hp-infected Bangladeshi and Swedish adults compared to uninfected Swedish controls. PBMCs from all age groups produced IL-17 and IFN-γ after MP stimulation, but little Th2 cytokines. IL-17 and IFN-γ were primarily produced by CD4+ T cells, since CD4+ T cell depleted PBMCs produced reduced amounts of these cytokines. Infant cells produced significantly more IL-17, but similar levels of IFN-γ, compared to adult cells after MP stimulation. In contrast, polyclonal stimulation induced lower levels IL-17 and IFN-γ in infant compared to adult PBMCs and CD4+ T cells. The strong IL-17 production in infants after MP stimulation was paralleled by significantly higher production of the IL-17 promoting cytokine IL-1ß from infant compared to adult PBMCs and monocytes. In conclusion, these results show that T cells can produce high levels of IL-17 and IFN-γ in response to Hp from an early age and indicate a potential role for IL-1ß in promoting Th17 responses to Hp during infancy.