Publications

BACKGROUND

Non-dependent alcohol and substance use patterns are prevalent among men who have sex with men (MSM), yet few effective interventions to reduce their substance use are available for these men. We evaluated whether an adapted brief counseling intervention aimed at reducing HIV risk behavior was associated with secondary benefits of reducing substance use among episodic substance-using MSM (SUMSM).

METHODS

326 episodic SUMSM were randomized to brief Personalized Cognitive Counseling (PCC) intervention with rapid HIV testing or to rapid HIV testing only control. Both arms followed over 6 months. Trends in substance use were examined using GEE Poisson models with robust standard errors by arm. Reductions in frequency of use were examined using ordered logistic regression.

RESULTS

In intent-to-treat analyses, compared to men who received rapid HIV testing only, we found men randomized to PCC with rapid HIV testing were more likely to report abstaining from alcohol consumption (RR=0.93; 95% CI=0.89-0.97), marijuana use (RR=0.84; 95% CI=0.73-0.98), and erectile dysfunction drug use (EDD; RR=0.51; 95% CI=0.33-0.79) over the 6-month follow-up. PCC was also significantly associated with reductions in frequency of alcohol intoxication (OR=0.58; 95% CI=0.36-0.90) over follow-up. Furthermore, we found PCC was associated with significant reductions in number of unprotected anal intercourse events while under the influence of methamphetamine (RR=0.26; 95% CI=0.08-0.84).

CONCLUSION

The addition of adapted PCC to rapid HIV testing may have benefits in increasing abstinence from certain classes of substances previously associated with HIV risk, including alcohol and EDD; and reducing alcohol intoxication frequency and high-risk sexual behaviors concurrent with methamphetamine use.

Human induced pluripotent stem cells (iPSCs) have the capability of revolutionizing research and therapy of liver diseases by providing a source of hepatocytes for autologous cell therapy and disease modelling. However, despite progress in advancing the differentiation of iPSCs into hepatocytes (iPSC-Heps) in vitro, cells that replicate the ability of human primary adult hepatocytes (aHeps) to proliferate extensively in vivo have not been reported. This deficiency has hampered efforts to recreate human liver diseases in mice, and has cast doubt on the potential of iPSC-Heps for liver cell therapy. The reason is that extensive post-transplant expansion is needed to establish and sustain a therapeutically effective liver cell mass in patients, a lesson learned from clinical trials of aHep transplantation. Here, as a solution to this problem, we report the generation of human fibroblast-derived hepatocytes that can repopulate mouse livers. Unlike current protocols for deriving hepatocytes from human fibroblasts, ours did not generate iPSCs but cut short reprogramming to pluripotency to generate an induced multipotent progenitor cell (iMPC) state from which endoderm progenitor cells and subsequently hepatocytes (iMPC-Heps) could be efficiently differentiated. For this purpose we identified small molecules that aided endoderm and hepatocyte differentiation without compromising proliferation. After transplantation into an immune-deficient mouse model of human liver failure, iMPC-Heps proliferated extensively and acquired levels of hepatocyte function similar to those of aHeps. Unfractionated iMPC-Heps did not form tumours, most probably because they never entered a pluripotent state. Our results establish the feasibility of significant liver repopulation of mice with human hepatocytes generated in vitro, which removes a long-standing roadblock on the path to autologous liver cell therapy.

RATIONALE

Calmodulin (CaM) mutations are associated with an autosomal dominant syndrome of ventricular arrhythmia and sudden death that can present with divergent clinical features of catecholaminergic polymorphic ventricular tachycardia (CPVT) or long QT syndrome (LQTS). CaM binds to and inhibits ryanodine receptor (RyR2) Ca release channels in the heart, but whether arrhythmogenic CaM mutants alter RyR2 function is not known.

OBJECTIVE

To gain mechanistic insight into how human CaM mutations affect RyR2 Ca channels.

METHODS AND RESULTS

We studied recombinant CaM mutants associated with CPVT (N54I and N98S) or LQTS (D96V, D130G, and F142L). As a group, all LQTS-associated CaM mutants (LQTS-CaMs) exhibited reduced Ca affinity, whereas CPVT-associated CaM mutants (CPVT-CaMs) had either normal or modestly lower Ca affinity. In permeabilized ventricular myocytes, CPVT-CaMs at a physiological intracellular concentration (100 nmol/L) promoted significantly higher spontaneous Ca wave and spark activity, a typical cellular phenotype of CPVT. Compared with wild-type CaM, CPVT-CaMs caused greater RyR2 single-channel open probability and showed enhanced binding affinity to RyR2. Even a 1:8 mixture of CPVT-CaM:wild-type-CaM activated Ca waves, demonstrating functional dominance. In contrast, LQTS-CaMs did not promote Ca waves and exhibited either normal regulation of RyR2 single channels (D96V) or lower RyR2-binding affinity (D130G and F142L). None of the CaM mutants altered Ca/CaM binding to CaM-kinase II.

CONCLUSIONS

A small proportion of CPVT-CaM is sufficient to evoke arrhythmogenic Ca disturbances, whereas LQTS-CaMs do not. Our findings explain the clinical presentation and autosomal dominant inheritance of CPVT-CaM mutations and suggest that RyR2 interactions are unlikely to explain arrhythmogenicity of LQTS-CaM mutations.