Cyclin-dependent kinase 12 is a drug target for visceral leishmaniasis.

Visceral leishmaniasis causes considerable mortality and morbidity in many parts of the world. There is an urgent need for the development of new, effective treatments for this disease. Here we describe the development of an anti-leishmanial drug-like chemical series based on a pyrazolopyrimidine scaffold. The leading compound from this series (7, DDD853651/GSK3186899) is efficacious in a mouse model of visceral leishmaniasis, has suitable physicochemical, pharmacokinetic and toxicological properties for further development, and has been declared a preclinical candidate. Detailed mode-of-action studies indicate that compounds from this series act principally by inhibiting the parasite cdc-2-related kinase 12 (CRK12), thus defining a druggable target for visceral leishmaniasis.

Simulation-based mentalizing generates a "proxy" self-reference effect in memory.

The self-reference effect (SRE) in memory is thought to depend on specialized mechanisms that enhance memory for self-relevant information. We investigated whether these mechanisms can be engaged "by proxy" when we simulate other people, by asking participants to interact with two virtual partners: one similar and one dissimilar to self. Participants viewed pairs of objects and picked one for themselves, for their similar partner, or their dissimilar partner. A surprise memory test followed that required participants to identify which object of each pair was chosen, and for whom. Finally, participants were shown both partners' object pairs again, and asked to indicate their personal preference. Four key findings were observed. Overlap between participants' own choice and those made for their partner was significantly higher for the similar than the dissimilar partner, revealing participants' use of their own preferences to simulate the similar partner. Recollection of chosen objects was significantly higher for self than for both partners and, critically, was significantly higher for similar than dissimilar partners. Source confusion between self and the similar partner was also higher. These findings suggest that self-reference by proxy enhances memory for non-self-relevant material, and we consider the theoretical implications for functional interpretation of the SRE.

Pharmacokinetics of oral sitamaquine taken with or without food and safety and efficacy for treatment of visceral leishmaniais: a randomized study in Bihar, India.

This randomized, open-label study of patients in India with visceral leishmaniasis (VL) investigated the effect of food on sitamaquine and desethyl-sitamaquine pharmacokinetics. Patients were randomized to receive oral sitamaquine, 2 mg/kg/day, once a day for 21 days across four cohorts (n = 41) (fasted/fed, fed/fasted, fed/fed, and fasted/fasted) over two periods (days 1-10 and 11-21), or intravenous amphotericin B (AmB), 1 mg/kg every other day for 30 days (n = 20). Mean day 21 pharmacokinetics across the four cohorts were sitamaquine, area under curve (AUC)((0-τ)) = 6,627-8,903 ng.hr/mL, AUC((0-16)) = 4,859-6,633 ng.hr/mL, maximum plasma concentration (C(max)) = 401-570 ng/mL, apparent terminal half-life (t(1/2)) = 18.3-22.8 hr, time to reach C(max) (t(max)) = 3.5-6 hr; and desethyl-sitamaquine, AUC((0-τ)) = 2,307-3,163 ng.hr/mL, C(max) = 109-154 ng/mL, t(1/2) = 23.0-27.9 hr, t(max) = 2-10 hr, with no significant food effect. On-therapy adverse events were observed for sitamaquine in 4 (10%) of 41 patients and for AmB in 17 (85%) of 20 patients. The final clinical cure (day 180) was 85% (95% confidence interval = 70.8-94.4%) for sitamaquine and 95% (95% confidence interval = 75.1-99.9) for AmB. Sitamaquine can be taken regardless of food intake, was generally well tolerated, and showed potential efficacy in patients with visceral leishmaniasis.

Extended treatment with lamivudine and adefovir dipivoxil in chronic hepatitis B patients with lamivudine resistance.

PURPOSE: We and others have reported that adding adefovir dipivoxil (adefovir) to lamivudine results in virological and biochemical improvement in cases of lamivudine resistance. The current study assessed the efficacy and safety of combined therapy after 104 weeks of combined treatment and analyzed the frequency of persistent lamivudine resistant HBV. METHODS: A total of 78 patients with compensated CHB (Group A) were maintained on either adefovir 10 mg daily (n = 38) or placebo (n = 40) while continuing lamivudine. An additional 38 patients with decompensated cirrhosis or post liver transplantation (Group B) received lamivudine plus adefovir. The primary endpoint was HBV DNA response at year 2. RESULTS: At week 104 of therapy, a significantly greater proportion of patients in Group A on combination therapy (76%) had a decline in serum HBV DNA to ≤10(5) copies or >2 log(10) reduction from baseline compared to those receiving lamivudine alone (13%; p < 0.001). Fifty-two percent of Group A patients on combination treatment continued to have the M204V/I HBV mutation compared to 92% receiving lamivudine alone (p = 0.0013). Virologic response occurred less frequently in patients expressing persistent lamivudine resistant HBV. In Group B, 87% of patients had HBV DNA response at week 104 (median change from baseline of -5.84 log(10) copies/mL). CONCLUSIONS: The combination of lamivudine and adefovir for 2 years generally proved effective in lamivudine-resistant cases, but there was a persistently high rate of detection of lamivudine resistant mutants and impaired virologic response in compensated patients.

Oleandrin-mediated inhibition of human tumor cell proliferation: importance of Na,K-ATPase alpha subunits as drug targets.

Cardiac glycosides such as oleandrin are known to inhibit the Na,K-ATPase pump, resulting in a consequent increase in calcium influx in heart muscle. Here, we investigated the effect of oleandrin on the growth of human and mouse cancer cells in relation to Na,K-ATPase subunits. Oleandrin treatment resulted in selective inhibition of human cancer cell growth but not rodent cell proliferation, which corresponded to the relative level of Na,K-ATPase alpha3 subunit protein expression. Human pancreatic cancer cell lines were found to differentially express varying levels of alpha3 protein, but rodent cancer cells lacked discernable expression of this Na,K-ATPase isoform. A correlation was observed between the ratio of alpha3 to alpha1 isoforms and the level of oleandrin uptake during inhibition of cell growth and initiation of cell death; the higher the alpha3 expression relative to alpha1 expression, the more sensitive the cell was to treatment with oleandrin. Inhibition of proliferation of Panc-1 cells by oleandrin was significantly reduced when the relative expression of alpha3 was decreased by knocking down the expression of alpha3 isoform with alpha3 siRNA or increasing expression of the alpha1 isoform through transient transfection of alpha1 cDNA to the cells. Our data suggest that the relative lack of alpha3 (relative to alpha1) in rodent and some human tumor cells may explain their unresponsiveness to cardiac glycosides. In conclusion, the relatively higher expression of alpha3 with the limited expression of alpha1 may help predict which human tumors are likely to be responsive to treatment with potent lipid-soluble cardiac glycosides such as oleandrin.

Autophagic cell death of human pancreatic tumor cells mediated by oleandrin, a lipid-soluble cardiac glycoside.

Lipid-soluble cardiac glycosides such as bufalin, oleandrin, and digitoxin have been suggested as potent agents that might be useful as anticancer agents. Past research with oleandrin, a principle cardiac glycoside in Nerium oleander L. (Apocynaceae), has been shown to induce cell death through induction of apoptosis. In PANC-1 cells, a human pancreatic cancer cell line, cell death occurs not through apoptosis but rather through autophagy. Oleandrin at low nanomolar concentrations potently inhibited cell proliferation associated with induction of a profound G(2)/M cell cycle arrest. Inhibition of cell cycle was not accompanied by any significant sub G1 accumulation of cells, suggesting a nonapoptotic mechanism. Oleandrin-treated cells exhibited time- and concentration-dependent staining with acridine orange, a lysosomal stain. Subcellular changes within PANC-1 cells included mitochondrial condensation and translocation to a perinuclear position accompanied by vacuoles. Use of a fluorescent oleandrin analog (BODIPY-oleandrin) revealed co-localization of the drug within cell mitochondria. Damaged mitochondria were found within autophagosome structures. Formation of autophagosomes was confirmed through electron microscopy and detection of green fluorescent protein-labeled light chain 3 association with autophagosome membranes. Also observed was a drug-mediated inhibition of pAkt formation and up-regulation of pERK. Transfection of Akt into PANC-1 cells or inhibition of pERK activation by MAPK inhibitor abrogated oleandrin-mediated inhibition of cell growth, suggesting that the reduction of pAkt and increased pERK are important to oleandrin's ability to inhibit tumor cell proliferation. The data provide insight into the mechanisms and role of a potent, lipid-soluble cardiac glycoside (oleandrin) in control of human pancreatic cancer proliferation.

Oleandrin-mediated oxidative stress in human melanoma cells.

While certain cardiac glycoside compounds such as oleandrin, bufalin and digitoxin are known to be associated with potent cytotoxicity to human tumor cells, the mechanisms by which this effect is produced are not clear. We now demonstrate that incubation of human malignant melanoma BRO cells with oleandrin results in a time-dependent formation of reactive oxygen species (ROS). Use of Mito-SOX and dihydroethidine dyes revealed the presence of oleandrin-mediated superoxide anions. Formation of superoxide anions correlated with a loss in cellular viability, proliferation and cellular defense mechanisms such as GSH content. Oleandrin also resulted in an unusual time-dependent mitochondrial condensation in BRO cells that could be blocked with use of N-acetyl cysteine (NAC). NAC was also shown to block ROS formation and partially prevent oleandrin-mediated loss of cellular GSH. Taken as a whole, the data suggest that exposure of human tumor cells such as BRO to oleandrin results in the formation of superoxide anion radicals that mediate mitochondrial injury and loss of cellular GSH pools. These mechanisms play a role in cardiac glycoside mediated tumor cell injury. Conversely, incubation of NAC, a precursor to GSH, largely prevents oleandrin-mediated inhibition of proliferation and mitochondria structural changes.

Risk factors for an intensive care unit admission in children with asthma.

An admission to an intensive care unit (ICU) with asthma is a marker of asthma severity and may be a precursor of asthma death. The aim of this study was to investigate risk factors for acute severe asthma needing an ICU admission. We hypothesized that children admitted to the ICU represent a severe phenotype with identifiable premorbid clinical features. The study was case-control in design. One hundred and forty-one children were studied. Seventy children admitted to the ICU and 71 children admitted to the general medical ward served as cases and controls, respectively. Children were aged between 1-16 years. They underwent skin prick allergy testing, and had a nasopharyngeal aspirate and serology performed to screen for respiratory pathogens. Their parents completed an asthma and allergy symptom questionnaire and the Newcastle Asthma Knowledge Questionnaire (NAKQ). On univariate analysis, an admission to the ICU was more likely in children with 1) "frequent episodic" or "persistent" background asthma; 2) three or more previous admissions for asthma; 3) one or more asthma admissions in the previous 12 months; 4) three or more presentations to the Emergency Department (ED) in the preceding 12 months; 5) three or more positive responses on skin prick allergy testing; 6) an elevated IgE level; 7) oxygen saturation on presentation < or =91%; 8) longer duration of asthma; 9) lower level of maternal education; 10) an admission during autumn; 11) three or more siblings; and 12) being prescribed antibiotics. Risk factors that remained significant on multivariate analysis were three or more presentations to the ED in the preceding 12 months (P=0.003), an elevated IgE level (P=0.01), oxygen saturation on presentation < or =91% (P=0.003), and longer asthma duration (P=0.02). ICU patients took longer to see a doctor and to commence oral steroids. No differences were found between cases and controls in the proportion taking preventer therapy (58% vs. 52%), provided with a written asthma action plan (32% vs. 25%), or in whom spirometry or peak flow was measured (28% vs. 42%). However, rates were low in both groups. Parental asthma knowledge was generally poor. This study identified risk factors for an ICU admission in children with asthma. A potentially preventable risk factor is a history of multiple ED presentations in the past year. Specialist referral of children with multiple ED presentations may improve asthma control and reduce the risk of an ICU admission. Background asthma management remains suboptimal in children needing hospitalization.

Free flap complications: when is enough, enough?

The reconstruction of head and neck defects, be they traumatic, oncologic, or iatrogenic, has been revolutionized in the last two decades by the development and refinement of microvascular soft tissue transfer. What was once an onerous and often unpredictable procedure has become standardized and safe. The result is that free tissue transfer is becoming the standard of care for more and more patients, to the point that the profession must now readdress the role of more traditional techniques of reconstruction. This article reviews the recent literature on free flap-related complications to assess the appropriateness of widespread free flap reconstruction and discusses the changing indications and benefits of free tissue transfer and nonmicrovascular reconstruction.