Dapsone-induced methemoglobinemia in renal transplant recipients: more prevalent than previously thought.

BACKGROUND: After an outbreak of Pneumocystis pneumonia (PCP) in our nephrology unit, dapsone was used as the second-line chemoprophylactic agent. Dapsone is the most common cause of drug-induced methemoglobinemia (MHb). Its prevalence is poorly described in the renal transplant population. Because dapsone is excreted by the kidneys, we hypothesized that the rate of MHb in these patients would be higher than previously reported. We aimed to describe the demographics, risk factors, and presenting features of MHb in these renal transplant patients. METHODS: Twenty-six transplant recipients commenced on dapsone for chemoprophylaxis against PCP from February to September 2011. All patients had normal glucose-6-phosphate dehydrogenase levels before treatment. Characteristics of patients with MHb were compared with those of the rest of the cohort to determine potential risk factors. RESULTS: Twelve (46%) patients developed MHb (levels 6.4 ± 4.1%). Six (50%) of the patients with MHb were asymptomatic on presentation. Cases had a mean drop in hemoglobin of 19 ± 7%. MHb led to five admissions (median length of stay 5 days, range 1-10 days). MHb level showed a strong correlation with the length of stay (correlation coefficient 0.762, P = 0.002). CONCLUSION: This is the highest reported prevalence of MHb, to our knowledge, in patients receiving dapsone, and its use led to significant hospitalization in this population. This study raises concerns about the use of dapsone as chemoprophylaxis in renal transplant recipients.

Clinical and laboratory features of invasive community-onset methicillin-resistant Staphylococcus aureus infection: a prospective case-control study.

Differences between the features of invasive community-onset methicillin-resistant Staphylococcus aureus (cMRSA) and methicillin-susceptible S. aureus (cMSSA) infections are incompletely understood. Fifty-seven patients with invasive cMRSA infection were prospectively identified at two teaching hospitals; for each cMRSA case, two cases of invasive cMSSA infection acted as controls. The primary outcome was 30-day all-cause mortality. Patients with invasive cMRSA infection were more likely to be Aboriginal (25% vs. 14%, age-adjusted odds ratio [OR] 2.5, p = 0.037), reside in a long-term care facility and/or have been hospitalised in the previous year (51% vs. 34%, p = 0.04) and less likely to have endocarditis (2% vs. 12%, p = 0.02) or require admission to an intensive care unit or high-dependency area (7% vs. 21%, p = 0.02). All-cause mortality at 30 days was similar in the cMRSA and cMSSA groups (9% vs. 7%, p = 0.68). Panton-Valentine leukocidin (PVL) genes were detected in a similar proportion of cMRSA and cMSSA isolates (32% vs. 27%, p = 0.49) and the presence of PVL genes was associated with younger age (35 years vs. 55 years, p < 0.001), Aboriginal ethnicity (38% vs. 10%, p < 0.001), skin and soft-tissue infection (54% vs. 19%, p < 0.001), lower illness severity at presentation (SAPS II score 9 vs. 21, p = 0.001) and shorter hospitalisation (9 days vs. 24 days, p < 0.001). Patients with "PVL-positive" and "PVL-negative" S. aureus infection had similar 30-day all-cause mortality (4% vs. 9%, p = 0.28). Few clinical features differentiated patients with invasive cMRSA infection from those with infection caused by cMSSA. Invasive "PVL-positive" S. aureus infection was associated with less morbidity but similar mortality to "PVL-negative" infection.

Opossum fetuses grown in culture.

Opossum fetuses explanted at limb bud stages have been successfully grown in culture for periods up to 20 hours. Blood circulation was maintained, and organogenesis continued at about the same rate as in vivo.

Tumor necrosis factor alpha-induced apoptosis in human neuronal cells: protection by the antioxidant N-acetylcysteine and the genes bcl-2 and crmA.

Tumor necrosis factor alpha (TNF-alpha) is a candidate human immunodeficiency virus type 1-induced neurotoxin that contributes to the pathogenesis of AIDS dementia complex. We report here on the effects of exogenous TNF-alpha on SK-N-MC human neuroblastoma cells differentiated to a neuronal phenotype with retinoic acid, TNF-alpha caused a dose-dependent loss of viability and a corresponding increase in apoptosis in differentiated SK-N-MC cells but not in undifferentiated cultures. Importantly, intracellular signalling via TNF receptors, as measured by activation of the transcription factor NF-kappa B, was unaltered by retinoic acid treatment. Finally, overexpression of bcl-2 or crmA conferred resistance to apoptosis mediated by TNF-alpha, as did the addition of the antioxidant N-acetylcysteine. These results suggest that TNF-alpha induces apoptosis in neuronal cells by a pathway that involves formation of reactive oxygen intermediates and which can be blocked by specific genetic interventions.

GABAB heterodimeric receptors promote Ca2+ influx via store-operated channels in rat cortical neurons and transfected Chinese hamster ovary cells.

The GABAB receptors are generally considered to be classical Gi-coupled receptors that lack the ability to mobilize intracellular Ca2+ without the aid of promiscuous G proteins. Here, we report the ability of GABAB receptors to promote calcium influx into primary cultures of rat cortical neurons and transfected Chinese hamster ovary cells. Chinese hamster ovary cells were transfected with GABAB1(a) or GABAB1(b) subunits along with GABAB2 subunits. In experiments using the fluorometric imaging plate reader platform, GABA and selective agonists promoted increases in intracellular Ca2+ levels in transfected Chinese hamster ovary cells and cortical neurons with the expected order of potency. These effects were fully antagonized by selective GABAB receptor antagonists. To investigate the intracellular pathways responsible for mediating these effects we employed several pharmacological inhibitors. Pertussis toxin abolished GABAB mediated Ca2+ increases, as did the phospholipase Cbeta inhibitor U73122. Inhibitor 2-aminethoxydiphenyl borane acts as an antagonist at inositol 1,4,5-trisphosphate receptors and at store-operated channels. In all cell types, 2-aminethoxydiphenyl borane prevented Ca2+ mobilization. The selective store-operated channel inhibitor 1-[2-(4-methoxyphenyl)-2-[3-(4-methoxyphenyl)propoxy]ethyl-1H-imidazole hydrochloride prevented increases in intracellular Ca2+ levels as did performing the assays in Ca2+ free buffers. In conclusion, GABAB receptors expressed in Chinese hamster ovary cells and endogenously expressed in rat cortical neurons promote Ca2+ entry into the cell via the activation of store-operated channels, using a mechanism that is dependent on Gi/o heterotrimeric proteins and phospholipase Cbeta. These findings suggest that the neuronal effects mediated by GABAB receptors may, in part, rely on the receptor's ability to promote Ca2+ influx.

Prolonged development of normal and parthenogenetic postimplantation mouse embryos in vitro.

Parthenogenetic mammalian embryos show reduced placental development and do not develop beyond the 25-somite stage. But non-parthenogenetic embryos in culture, without a functional placenta, can develop to 40 somites or more. We have therefore examined the possibility that parthenogenetic embryos might also show prolonged development in culture. After parthenogenetic activation and diploidization, 23% of CBA and 56-58% of hybrid (CBAxC57BL/6) F1 mouse eggs developed in culture to blastocysts. When transferred to pseudopregnant recipients: 60% of the CBA blastocysts implanted and 26% of these developed to somite stage embryos; 71-72% of the hybrid blastocysts implanted and 11-17% of these developed to somite stage embryos. Improved development of postimplantation embryos explanted into culture at about the 15-20 somite stage was obtained by opening the visceral yolk sac (without exteriorizing the embryo). All the normal (non-parthenogenetic) embryos cultured in this way developed to more than 35 somites and many reached 45-55 somites. Under the same conditions, 11/17 diploid parthenogenetic CBA embryos developed in culture to more than 35 somites and 5 of these reached 45 somites; and 9/28 diploid parthenogenetic (CBAxC57BL/6) F1 embryos developed to 35 somites or more and 5 of these reached 45 somites. The size and protein content of the parthenogenetic embryos after culture was less than that of the normal embryos of equivalent stages. These results raise new possibilities for the analysis of parthenogenesis and genomic imprinting, including studies of the effects of adding to the culture medium specific growth factors and demethylating agents.

Design and synthesis of heterofunctional V1a-selective vasopressin receptor ligands with lysine at position 9.

A peptide analogue of [8-arginine]vasopressin (AVP) with Lys substituted for Gly at position 9 ([d(CH2)5Tyr(Me)2LysNH2(9)]AVP; ALVP) has been synthesized as a precursor for the production of heterofunctional vasopressin receptor ligands. Three heterofunctional ligands have been prepared by attaching biotin and a photoreactive cross-linker capable of iodination, either alone or in combination, to the epsilon-amino group of Lys at position 9 in ALVP. The binding characteristics of these novel ligands have been determined at the V1a and V2 vasopressin receptors by employing membrane preparations of rat liver and kidney respectively. All of the analogues synthesized during the course of this study bound selectively, and with high affinity, to the V1a vasopressin receptor subtype. Our results demonstrate that the strategies described in this paper provide a convenient means of synthesizing heterofunctional vasopressin receptor ligands with preservation of subtype-specific, high affinity binding characteristics. These parameters establish the potential value of the analogues as probes for investigating V1a receptor structure and function.

Techniques for assessment of teratologic effects: embryo culture.

A simple method is described for growing rat embryos in vitro for 48 hr from head-fold to early limb-bud stages at rates of development and protein synthesis indistinguishable from those in vivo. Culture of the embryos can be continued for longer periods but at a reduced growth rate. Preheating the culture serum to 56 degrees C for 30 min improves embryonic development, but raising the culture temperature 2-3 degrees C or exposing the presomite embryos to 20% O2 (160 mm Hg) causes malformations, particularly of the brain and spinal cord. The value of such culture methods for teratology is briefly discussed.

Effects of fibroblast growth factor 2 and insulin-like growth factor II on the development of parthenogenetic mouse embryos in vitro.

Most parthenogenetic embryos (PEs) in mammals die shortly after implantation, and this failure to develop is associated with genomic imprinting. We have examined the influence of human recombinant basic fibroblast growth factor 2 (FGF-2) and human recombinant insulin-like growth factor II (ICF-II) on the development of (CBA x C57BL/6)F1 parthenogenetic mouse embryos. Embryos were treated in vitro at the morula stage with different doses of FGF-2 and, after their development to blastocysts, transferred to pseudopregnant recipients. The optimal doses of FGF-2 did not affect the number of forming and implanting blastocysts, but increased, from 20 to 42%, the number of embryos developing to somite stages. PEs (18-21 somites) treated with an optimal dose of FGF-2 were explanted for further development in culture by treatment with the second growth factor, IGF-II. Eighty-three percent of those embryos cultured with IGF-II (2.5 microg/ml) developed to 35 or more somites, as compared with 36% of embryos cultured without any growth factors (P < 0.01). Also, a significantly higher proportion of PEs developed to 40-50 somites in this case. These results show that the in vitro treatment of PEs with FGF-2 at the morula stage increases the number of somite embryos, and the second treatment of somite PEs with IGF-II in culture medium prolongs their development significantly.

Effects of the anti-progestin RU 38486 on rat embryos growing in culture.

RU 38486 is a steroid currently manufactured as an abortifacient. In view of the lack of conclusive information about teratogenic risk, it is current practice for most human embryos surviving RU 38486 to be surgically aborted. In this study the effects were examined of different concentrations of RU 38486 on postimplantation rat embryos in culture. No statistically significant effects were observed until the doses reached x 7 the 'standard' dose (x 1). The experiments provide no evidence of a teratogenic effect of RU 38486 at the levels currently used in clinical practice.

[Effects of growth factors FGF2 and IGF2 on the development of parthenogenetic mouse embryos in utero and in vitro]. / Deistvie rostovykh factorov FGF2 i IGF2 na razvitie partenogeneticheskikh c'mbrionov myshei in utero i in vitro.

We studied the effects of fibroblast growth factor 2 (FGF2) and insulin-like growth factor 2 (IGF2) on the development of parthenogenetic mouse embryos (CBA x C57BK/6)F1. The parthenogenetic embryos were treated in vitro during the preimplantation period and, at the blastocyst stage, transplanted into the uterus of pseudopregnant females. The addition of FGF2 at an optimal dose (2.5 ng/ml) to the culture medium increased twofold the number of embryos developed in utero to the somite stages as compared to the control: 18 and 43%, respectively. The parthenogenetic embryos (18-21 somites), treated and nontreated with FGF2 during the preimplantation period, were explanted for further development in vitro and treated with IGF2 at 2.5 micrograms/ml. As a result, many more parthenogenetic embryos (> 87%) of both groups developed in vitro to the stage of 30 or more somites as compared to the control (59%). The treatment of the parthenogenetic embryos with FGF2 alone at the preimplantation stages did not improve their development in vitro at the postimplantation stages. The results we obtained suggest that the treatment of parthenogenetic embryos in vitro with FGF2 during the preimplantation period increased twofold the number of somite embryos in utero, while their subsequent treatment in vitro with IGF2 leads to a significant prolongation of their development, as compared to the control.

Stabilization of HIF-2α through redox regulation of mTORC2 activation and initiation of mRNA translation.

Hypoxia inducible factor-2α (HIF-2α) has a critical role in renal tumorigenesis. HIF-2α is stabilized in von Hippel-Lindau (VHL)-deficient renal cell carcinoma through mechanisms that require ongoing mRNA translation. Mammalian target of rapamycin (mTOR) functions in two distinct complexes: Raptor-associated mTORC1 and Rictor-associated mTORC2. Rictor-associated mTORC2 complex has been linked to maintaining HIF-2α protein in the absence of VHL; however, the mechanisms remain to be elucidated. Although Raptor-associated mTORC1 is a known key upstream regulator of mRNA translation, initiation and elongation, the role of mTORC2 in regulating mRNA translation is not clear. Complex assembly of the mRNA cap protein, eukaryotic translation initiation factor 4 (eIF4)E, with activators (eIF4 gamma (eIF4G)) and inhibitors (eIF4E-binding protein 1 (4E-BP1)) are rate-limiting determinants of mRNA translation. Our laboratory has previously demonstrated that reactive oxygen species, mediated by p22(phox)-based Nox oxidases, are enhanced in VHL-deficient cells and have a role in the activation of Akt on S473, a site phosphorylated by the mTORC2 complex. In this study, we examined the role of Rictor-dependent regulation of HIF-2α through eIF4E-dependent mRNA translation and examined the effects of p22(phox)-based Nox oxidases on TORC2 regulation. We demonstrate for the first time that mTORC2 complex stability and activation is redox sensitive, and further defined a novel role for p22(phox)-based Nox oxidases in eIF4E-dependent mRNA translation through mTORC2. Furthermore, we provide the first evidence that silencing of p22(phox) reduces HIF-2α-dependent gene targeting in vitro and tumor formation in vivo. The clinical relevance of these studies is demonstrated.

The culture of postimplantation embryos.

Culture methods for postimplantation embryos are now widely used in studies of embryo physiology, growth and development. Available methods support growth and development of rat and mouse embryos at all stages of organogenesis. The best results are obtained from embryos between head fold and early limb bud stages; overall growth and differentiation of these embryos in vitro is almost identical to that in vivo. Some examples of the application of postimplantation embryo culture to specific lines of study are given and some possible future developments of the technique are discussed.

Whole embryo culture, teratogenesis, and the estimation of teratologic risk.

Mammalian whole-embryo culture systems are now widely used and have proved useful in many studies of normal and abnormal development. The main advantages of these systems are that they allow precise control of experimental conditions and can often provide information unobtainable from in vivo studies; the main disadvantages are the rather short period of embryonic development that can be supported in culture and the present restriction of the techniques to very few species. The possibility of using whole-embryo culture systems for screening for new teratogenic agents remains controversial, but there are indications that the systems may have more potential in this area than has sometimes been supposed.

Effects of temporary cooling, and of different explantation and storage conditions, on the subsequent development of post-implantation rat embryos in vitro.

Rat embryos explanted at head fold stage were stored under various levels of hypothermia prior to culture. The storage media were Hanks' Balanced Salt Solution (BSS), 50% rat serum with 50% Dulbecco's Modification of Eagle's Medium (standard medium), or 100% rat serum. The media were gassed with 5% O2/5% CO2/90% N2 or 20% O2/5% CO2/75% N2. Subsequent development of embryos after storage at temperatures between 10 degrees C and 30 degrees C for 5 hr in Hanks' BSS, or for 5-10 hr in standard medium or serum, was similar to that of controls. Some embryos developed well even after storage for 48 hr in standard medium. Development was poorer after storage at 0 degrees C or 5 degrees C, and after storage at all temperatures in ungassed Hanks' or standard medium (pH greater than 8.0). Differences in oxygen level had little effect. For routine explantation at room temperature in (ungassed) phosphate-buffered saline solutions such as Hanks', it is recommended that the delay before transferring the embryos to the culture incubator not exceed 2-3 hr.

A rotating bottle culture method with continuous replacement of the gas phase.

A 'rotator' culture method is described which provides a continuous flow of oxygenating gas to cultures in rotating bottles. The system maintains constant O2 and CO2 levels in the culture medium throughout the incubation period. It also provides a more stable pH than systems with sealed culture bottles.

Abnormalities induced in cultured rat embryos by hyperthermia.

Rat embryos were explanted at nine and one-half days of gestation and cultured for 48 hours in rotating bottles containing rat serum and a gas phase, at temperatures of 38, 40, 40.5 and 41 degrees C. The embryo cultured at 40.5 degrees C were retarded and many of them were abnormal, and at 41 degrees C, all the embryos were malformed and retarded. The most frequent abnormalities occurring at both these temperatures were microcephaly and oedema of the pericardium. Development of the embryos cultured at 40 degrees C was similar to that of the controls at 38 degrees C, and superficially they appeared to be normal. However, measurement of the head dimensions, and separate determinations of head and body protein contents showed that the 40 degrees C embryos were microcephalic.

Effects of ultrafiltration on solute clearances in hollow fiber artificial kidneys.

Although solute clearances in artificial kidney coils increase with ultrafiltration (UF), we have previously shown that increases are usually less than UF rate (most likely because of decreases in diffusive transport with UF coils and, for larger solutes, molecular sieving). The present studies demonstrate the effects of UF on clearances of Na and bromsulphalein (BSP) (mol. wt. 838) in hollow fiber dialyzers. Clearances were measured at increasing transmembrane hydrostatic pressures at perfusion rates of 200 and 500 ml. per minute. Fractions of total clearance attributable to diffusion as compared to solvent drag forces were calculated. Sieving coefficients were determined in studies where diffusion was minimized and clearance was primarily by solvent drag. Clearance increases were less than UF rate only for BSP; molecular sieving most likely accounts for the difference at high perfusion rates. Only at 200 ml. per minute was slight decrease of diffusion with UF suggested. Thus, in contrast to coils, there is minimal or no decrease in diffusion with UF in hollow fiber dialyzers.