Nox2 is a mediator of chronic CsA nephrotoxicity.

We hypothesized that Nox2, the classical phagocytic NADPH oxidase, plays an important role in calcineurin inhibitor (CNI)-induced renal fibrosis. We tested this hypothesis in vitro, in animal and in human studies. Cyclosporine A (CsA) and tacrolimus (TAC) were associated with greater levels of Nox2 mRNA and epithelial to mesenchymal transition (EMT) in NRK52E cells. CsA increased Nox2, α-SMA and phosphorylated-p38MAPK, Smad3 and NFκB proteins. Nox2 upregulation and EMT were inhibited in TGF-ß1 knockout cells suggesting that TGF-ß1 is required for Nox2 activation. Fisher344 rats treated with high dose CsA showed increased Nox2 in the tubulointerstitium and greater Nox2, α-SMA, phosphorylated Smad3 and nitrotyrosine by immunoblot analyses. Inhibition of Nox2 by coadministration of apocynin or diphenyleneiodonium was associated with reduced fibrogenesis. We validated these findings by treating wild type and Nox2 null (B6.129S-Cybb(Tm1Din)/J) mice with high dose CsA. Western blot analyses confirmed the absence of Nox2 and significantly lower levels of α-SMA and 4-hydroxynonenal (HNE) in CsA-treated knockout mice. These findings were clinically relevant since Nox2 and α-SMA were increased in the tubulointerstitium of kidneys from 15 liver transplant recipients with biopsy-confirmed chronic CsA or TAC nephrotoxicity. In conclusion, specific Nox2 inhibition strategies may improve chronic CNI nephrotoxicity in solid organ transplantation.

Neurotrophic factors modulate hair cells and their potassium currents in chick otocyst explants.

Neurotrophins, retinoids and their receptors are present in the sensory epithelia of the inner ear during development. We show that these factors modulate the proliferation of hair cells and their K+-currents when the embryonic day 3 (ED 3) presumptive inner ear (i.e. otocyst) is maintained in organ culture. All trans-retinoic acid (RA) increases hair cell differentiation and enhances the acquisition of outward currents, including a delayed rectifier and a fast activating, transient type, voltage-gated potassium current. In contrast, brain-derived neurotrophic factor (BDNF) and neurotrophin-3 (NT-3) decrease ionic current activity, and the addition of RA with the neurotrophins enhances this inhibitory response in an age-dependent manner. We measured the total number of cells per explant over time to determine precisely when and how these factors inhibit explant growth. We found that high concentrations of BDNF and NT-3 administered together, and low concentrations of both neurotrophins combined and administered with RA suppress otocyst cell numbers after 24 h in vitro. This suppressive response is induced by RA and NT-3, not by RA and BDNF. The suppressive or inhibitory influence of NT-3 and RA is the result of NT-3 binding to the low affinity receptor, p75NTR, not the result of RA increasing mRNA levels for the high affinity receptor, trkC. However, trk may act with p75NTR, as disruption of trk signalling alleviates the inhibitory response induced by NT-3 and RA. Our data suggest that various combinations and/or concentration gradients of these factors can differentially regulate inner ear development and hair cell excitability.

Aminolevulinate synthase: lysine 313 is not essential for binding the pyridoxal phosphate cofactor but is essential for catalysis.

5-Aminolevulinate synthase is the first enzyme of the heme biosynthetic pathway in animals and some bacteria. Lysine-313 of the mouse erythroid aminolevulinate synthase was recently identified to be linked covalently to the pyridoxal 5'-phosphate cofactor (Ferreira GC, Neame PJ, Dailey HA, 1993, Protein Sci 2:1959-1965). Here we report on the effect of replacement of aminolevulinate synthase lysine-313 by alanine, histidine, and glycine, using site-directed mutagenesis. Mutant enzymes were purified to homogeneity, and the purification yields were similar to those of the wild-type enzyme. Although their absorption spectra indicate that the mutant enzymes bind pyridoxal 5'-phosphate, they bind noncovalently. However, addition of glycine to the mutant enzymes led to the formation of external aldimines. The formation of an external aldimine between the pyridoxal 5'-phosphate cofactor and the glycine substrate is the first step in the mechanism of the aminolevulinate synthase-catalyzed reaction. In contrast, lysine-313 is an essential catalytic residue, because the K313-directed mutant enzymes have no measurable activity. In summary, site-directed mutagenesis of the aminolevulinate synthase active-site lysine-313, to alanine (K313A), histidine (K313H), or glycine (K313G) yields enzymes that bind the pyridoxal 5'-phosphate cofactor and the glycine substrate to produce external aldimines, but which are inactive. This suggests that lysine-313 has a functional role in catalysis.

Efficient assessment of filariasis endemicity by screening for filarial antigenaemia in a sentinel population.

We have previously reported that a monoclonal antibody-based antigen detection assay (AD12) is sensitive and specific for Bancroftian filariasis in Egypt. The purpose of the present study was to demonstrate the use of this assay in a sentinel population as a means of efficiently screening for filariasis endemicity. Antigen testing was performed with finger-prick blood collected during the day from 743 schoolchildren (ages 11-16 years). The school draws students from 5 villages in Qalubia Governorate, 35 km north-east of Cairo, Egypt. The prevalence of filarial antigenaemia in the school was 17.2%. Antigenaemia rates in children from the 5 villages were 29, 20, 18, 17, and 10% (non-uniformity significant by chi 2 analysis, P = 0.02). These data agree with Ministry of Health rankings of relative endemicity for these villages based on prior night blood surveys. The village with the highest antigen prevalence in children was surveyed one year before the present study. Prevalence rates of antigenaemia and microfilaraemia at that time for a different sample of children aged 11-16 years were 33% and 22%, respectively. We conclude that antigen detection in schoolchildren of this age group is an efficient means of assessing filariasis endemicity in Egypt.

The resurgence of lymphatic filariasis in the Nile delta.

A study of 325,000 residents of 314 villages in six governorates of the Nile delta area of Egypt revealed that the prevalence of lymphatic filariasis increased from < 1% in 1965 to > 20% in 1991, especially in the governorates of Qalyubiya, Monufiya, Dakhaliya, and Giza. The distribution of the communites with endemic filariasis is focal. Clusters of villages with high prevalences are surrounded by others in which the disease is absent, although their environmental, social, and agricultural features appear similar. The article analyses why the significant decline in filariasis between 1945 and 1965 in Egypt has been followed by a resurgence of the disease.