Differential effects of histamine on Leydig cell and testicular macrophage activities in wall lizards: precise role of H1/H2 receptor subtypes

The present study in the wall lizard, Hemidactylus flaviviridis, was aimed to understand the role of histamine (HA) in the regulation of Leydig cell and testicular macrophage activities, for the first time, in ectothermic vertebrates. Although HA did not affect the testosterone production from unstimulated Leydig cells, it had dual concentration-related effects, stimulatory at a low concentration of 10(-10) M while inhibitory at a high concentration of 10(-5) M, on FSH-induced testosterone production. This suggests that HA did not influence the basal Leydig cell steroidogenesis, but modulated the FSH-stimulated testosterone production in a biphasic manner depending upon its concentration. However, HA failed to affect the FSH-stimulated Leydig cell proliferation, indicating that HA modulated the testosterone production from Leydig cells without influencing their proliferation in wall lizards. HA, apart from Leydig cells, differentially regulated the testicular macrophage immune responses. It inhibited phagocytosis and superoxide production at high concentration (10(-5) M), while stimulated superoxide production and could not affect phagocytosis at low concentration (10(-10) M). Using selective H1 and H2 antagonists, pyrilamine and famotidine respectively, H1 receptor subtype was seen responsible for mediating the inhibitory effect of HA on Leydig cell steroidogenesis and testicular macrophage immune responses at high concentration, while H2 receptors were involved for the stimulation at low concentration.     

Multiplexed assay panel of cytotoxicity in HK-2 cells for detection of renal proximal tubule injury potential of compounds

Proximal tubules of the kidneys are one of the most common targets of nephrotoxic drugs and chemicals. Screens to predict nephrotoxic potential of compounds with insights to mechanisms of toxicity facilitate lead optimization, guide structure-activity relationships, minimize risks of clinical nephrotoxicity and therefore are valuable in the process of drug discovery. We developed and characterized an in vitro assay multiplexed to measure several endpoints of cytotoxicity using HK-2 cells. Assays for lactate dehydrogenase, cellular caspase 3/7 activation, resazurin dye reduction and Hoechst 33342 DNA staining were multiplexed to maximize the ability to detect cell injury. Assays were performed after 5- or 24-h incubations to further enhance the sensitivity of detection of toxicity. Individual assays were optimized for cell density, assay linearity and assay performance under multiplexed conditions. Inducers of apoptosis (staurosporine) and necrosis (perhexiline) were used to validate the mechanistic aspects of cell death. Nephrotoxic compounds (5-fluorouracil, gentamicin, cisplatin, acetaminophen, para-aminophenol, potassium dichromate, ibuprofen, doxorubicin, cyclosporine, citrinin, puromycin) were used to determine the potential of this method to detect proximal tubule toxicity of compounds. Overall, this cost-effective multiplexed platform is more sensitive than a single endpoint assay, provides mechanistic cues of toxicity and is amenable for higher throughput screening.     

On designing non-saccharide, allosteric activators of antithrombin

Antithrombin, a plasma glycoprotein serpin, requires conformational activation by heparin to induce an anticoagulant effect, which is mediated through accelerated factor Xa inhibition. Heparin, a highly charged polymer and an allosteric activator of the serpin, is associated with major adverse effects. To design better, but radically different activators of antithrombin from heparin, we utilized a pharmacophore-based approach. A tetrahydroisoquinoline-based scaffold was designed to mimic four critical anionic groups of the key trisaccharide DEF constituting the sequence-specific pentasaccharide DEFGH in heparin. Activator IAS(5) containing 5,6-disulfated tetrahydroisoquinoline and 3,4,5-trisulfated phenyl rings was found to bind antithrombin at pH 7.4 with an affinity comparable to the reference trisaccharide DEF. IAS(5) activated the inhibitor nearly 30-fold, nearly 2- to 3-fold higher than our first generation flavanoid-based designs. This work advances the concept of antithrombin activation through non-saccharide, organic molecules and pinpoints a direction for the design of more potent molecules.     

Broadening the age restriction for initiating rotavirus vaccination in regions with high rotavirus mortality: Benefits of mortality reduction versus risk of fatal intussusception

Introduction

Recently developed rotavirus vaccines have the potential to reduce diarrhea mortality in children in developing countries. Available data to date do not indicate risk of intussusception with these new vaccines. To avoid a potential unanticipated risk post-licensure, it is recommended that rotavirus immunization be initiated before 12 weeks of age when background intussusception rates are low. This policy could exclude a substantial number of children from vaccination, especially in developing countries where delays in vaccination are common.

Methods

We conducted a scenario analysis to assess the potential benefits of mortality reduction from rotavirus versus the risk of fatal intussusception when the first dose of the vaccine is strictly administered by 12 weeks of age compared with a free strategy with vaccine administered before 1 year of age using data on rotavirus disease, vaccine safety and efficacy, and current diphtheria–tetanus–pertussis vaccination rates, and by incorporating hypothetical risks of intussusception.

Results

In developing countries, assuming vaccine efficacy of 50% and 75% for doses 1 and 2, respectively, and a hypothetical sixfold and threefold increased relative risk of intussusception within 7 days of doses 1 and 2, respectively, initiating rotavirus immunization before 12 weeks of age would prevent 194,564 of the 517,959 annual rotavirus-associated deaths among children <5 years, while potentially resulting in 1106 fatal intussusception events. Administration of the first dose to infants up to 1 year of age would prevent an additional 54,087 rotavirus-associated deaths (total = 248,651) while potentially resulting in an additional 1226 intussusception deaths (total = 2332).

Conclusion

In developing countries, the additional lives saved by broadening the age restrictions for initiation of rotavirus vaccination would far outnumber the hypothetical excess intussusception deaths that would accompany such an approach.     

Development and implementation of a pediatric cardiac anesthesia/intensive care database

Systematic collection and electronic storage of data can assist in improving quality and efficiency of patient care and can provide a data set to interrogate for subsequent performance improvement and clinical research purposes. In this article, an electronic perioperative pediatric cardiac surgery database to be used by a multidisciplinary care team was designed, developed, and implemented. Technical goals for the design included low cost, rapid development and implementation, adequate security, and potential for internal and external distribution. Implementation of the described database has proved to be invaluable for quality assurance and statistical analysis of data relevant to patient care. From the overall positive experience, it was concluded that the electronic data management does not always need major cost investment.