Low castes have poor access to visceral leishmaniasis treatment in Bihar, India.

OBJECTIVES: Bihar, the poorest state in India, concentrates most of the visceral leishmaniasis (VL) cases in the country. A large proportion of the poor rural communities where VL is endemic are marginalized by their socio-economic status, intrinsically related to the caste system. In this study, we evaluated whether people from low socio-economic strata had difficulties accessing VL treatment in Bihar. As a secondary outcome, we evaluated whether people delaying their VL treatment had poorer clinical indicators at admission. METHODS: Data on 2187 patients with VL treated by Médecins Sans Frontières (MSF) in Vaishali district from July 2007 to December 2008 were analysed. Patients who reported having onset of symptoms ≥8 weeks before admission were defined as 'late presenters'. Logistic regression models were used to evaluate whether low castes had higher risk to be 'late presenters' compared to the rest of castes and whether 'late presenters' had poorer indicators at admission (i.e. haemoglobin level, spleen size). RESULTS: After adjusting for age, gender and distance to VL treatment facility, Mushars (the lowest caste in Bihar) had twice the odds to be 'late presenters' compared to the rest of castes (OR 2.05, 95% CI: 1.24-2.38). Subjects that had VL symptoms for ≥8 weeks had a larger spleen and lower haemoglobin level than those that were treated earlier. CONCLUSION: Low castes have poor access to VL treatment in Bihar, and late presenters have poorer clinical indicators at admission. These findings have implications at individual and community levels and should stimulate targeted VL control programmes to ensure that marginalized communities in Bihar are properly treated.

Decreased peripheral health service utilisation during an outbreak of Marburg haemorrhagic fever, Uíge, Angola, 2005.

In 2005, a Marburg haemorrhagic fever (MHF) outbreak occurred in Uíge province, Angola, which had its epicentre in Uíge municipality. Concurrently, a health facility located a considerable distance from the outbreak's epicentre reported a drastic reduction in attendance, possibly due to a remote effect of the ongoing MHF outbreak. Health officials should devise strategies to ensure that communities far from a filovirus haemorrhagic fever epicentre are not adversely affected by interventions at the epicentre and, to the greatest extent possible, ensure that these peripheral communities receive essential medical care during an epidemic.

A new satellite-borne neutral wind instrument for thermospheric diagnostics.

The bulk motion of the neutral gas at altitudes between about 200 and 600 km is an important factor in predicting the onset of plasma instabilities that are known to distort and/or disrupt high frequency radio communications. These neutral winds have historically been quite difficult to measure, especially from a moving spacecraft. A new space science instrument called the ram wind sensor has been developed to measure the component of the neutral gas velocity that lies along the orbit track of a satellite in low Earth orbit. Laboratory tests of an engineering model of the instrument have been carried out using a supersonic neutral argon beam, in order to validate the measurement concept. The results show that the technique is viable for measurements of neutral flow velocities in future satellite missions.

Structural requirements of ceramide and sphingosine based inhibitors of mitochondrial ceramidase.

The effects of structural analogues of ceramide on rat brain mitochondrial ceramidase (mt-CDase) were investigated. Design of target compounds was mainly based on modifications of the key elements in ceramide and sphingosine, including stereochemistry, the primary and secondary hydroxyl groups, the trans double bond in the sphingosine backbone, and the amide bond. Mt-CDase was inhibited by (1) all stereoisomers of D-erythro-ceramide (D-e-Cer) with an IC50 of 0.11, 0.21, and 0.26 mol % for the L-threo, D-threo, and L-erythro isomers, respectively; (2) all stereoisomers of sphingosine with IC50 ranging from 0.04 to 0.14 mol %, N-methyl-D-erythro-sphingosine (N-Me-Sph, IC50 0.13 mol %); and (3) D-erythro-urea-C16-ceramide (C16-urea-Cer IC50 0.33 mol %). The enzyme was not inhibited by N-methyl ceramide (N-Me-C16-Cer), 1-O-methyl ceramide (1-O-Me-C16-Cer), 3-O-methyl ceramide (3-O-Me-C16-Cer), cis-D-erythro ceramide (cis-D-e-C16-Cer) and 3-O-methyl-D-erythro-sphingosine (3-O-Me-Sph). It was less potently inhibited by D-erythro-sphinganine (D-e-dh-Sph, IC50 0.20 mol %), D-erythro-dehydro sphingosine (D-e-deh-Sph, IC50 0.25 mol %), (2S)-3-keto-sphinganine (3-keto-dh-Sph, IC50 0.34 mol %), (2S) 3-keto-ceramide (3-keto-C16-Cer, IC50 0.60 mol %), and ceramine (C18-ceramine, IC50 0.62 mol %), 1-O-methyl-D-erythro-sphingosine (1-O-Me-Sph), cis-D-erythro-sphingosine (cis-D-e-Sph), (2S)-3-ketosphingosine (3-keto-Sph), (2S)-3-keto-dehyrosphingosine (3-keto-deh-Sph), and N,N-dimethyl-D-erythrosphingosine (N,N-diMe-Sph) were weak inhibitors whereas ceramide-1-phosphate (Cer-1-P) and sphingosine-1-phosphate (Sph-1-P) stimulated the enzyme. Thus, for inhibition, the enzyme requires the primary and secondary hydroxyl groups, the C4-C5 double bond, the trans configuration of this double bond, and the NH-protons from either the amide of ceramide or the amine of sphingosine. Therefore, these results provide important information on the requirements for ceramide-enzyme interaction, and they suggest that ligand interaction with the enzyme occurs in a high affinity low specificity manner, in contrast to catalysis which is highly specific for D-erythro-ceramide (D-e-Cer) but occurs with a lower affinity. In addition, this study identifies two competitive inhibitors of mt-CDase; urea-ceramide (C16-urea-Cer) and ceramine (C18-ceramine) that may be further developed and used to understand the mechanism of mt-CDase in vitro and in biologic responses.

Biochemical characterization of the reverse activity of rat brain ceramidase. A CoA-independent and fumonisin B1-insensitive ceramide synthase.

We have previously purified a membrane-bound ceramidase from rat brain and recently cloned the human homologue. We also observed that the same enzyme is able to catalyze the reverse reaction of ceramide synthesis. To obtain insight into the biochemistry of this enzyme, we characterized in this study this reverse activity. Using sphingosine and palmitic acid as substrates, the enzyme exhibited Michaelis-Menten kinetics; however, the enzyme did not utilize palmitoyl-CoA as substrate. Also, the activity was not inhibited in vitro and in cells by fumonisin B1, an inhibitor of the CoA-dependent ceramide synthase. The enzyme showed a narrow pH optimum in the neutral range, and there was very low activity in the alkaline range. Substrate specificity studies were performed, and the enzyme showed the highest activity with d-erythro-sphingosine (Km of 0.16 mol %, and Vmax of 0.3 micromol/min/mg), but d-erythro-dihydrosphingosine and the three unnatural stereoisomers of sphingosine were poor substrates. The specificity for the fatty acid was also studied, and the highest activity was observed for myristic acid with a Km of 1.7 mol % and a Vmax of 0.63 micromol/min/mg. Kinetic studies were performed to investigate the mechanism of the reaction, and Lineweaver-Burk plots indicated a sequential mechanism. Two competitive inhibitors of the two substrates were identified, l-erythro-sphingosine and myristaldehyde, and inhibition studies indicated that the reaction followed a random sequential mechanism. The effect of lipids were also tested. Most of these lipids showed moderate inhibition, whereas the effects of phosphatidic acid and cardiolipin were more potent with total inhibition at around 2.5-5 mol %. Paradoxically, cardiolipin stimulated ceramidase activity. These results define the biochemical characteristics of this reverse activity. The results are discussed in view of a possible regulation of this enzyme by the intracellular pH or by an interaction with cardiolipin and/or phosphatidic acid.

Molecular cloning and characterization of a human mitochondrial ceramidase.

We have recently purified a rat brain membrane-bound nonlysosomal ceramidase (El Bawab, S., Bielawska, A., and Y. A. Hannun (1999) J. Biol. Chem. 274, 27948-27955). Using peptide sequences obtained from the purified rat brain enzyme, we report here the cloning of the human isoform. The deduced amino acid sequence of the protein did not show any similarity with proteins of known function but was homologous to three putative proteins from Arabidospis thaliana, Mycobacterium tuberculosis, and Dictyostelium discoideum. Several blocks of amino acids were highly conserved in all of these proteins. Analysis of the protein sequence revealed the presence at the N terminus of a signal peptide followed by a putative myristoylation site and a putative mitochondrial targeting sequence. The predicted molecular mass was 84 kDa, and the isoelectric point was 6.69, in agreement with rat brain purified enzyme. Northern blot analysis of multiple human tissues showed the presence of a major band corresponding to a size of 3.5 kilobase. Analysis of this major band on the blot indicated that the enzyme is ubiquitously expressed with higher levels in kidney, skeletal muscle, and heart. The enzyme was then overexpressed in HEK 293 and MCF7 cells using the pcDNA3. 1/His-ceramidase construct, and ceramidase activity (at pH 9.5) increased by 50- and 12-fold, respectively. Next, the enzyme was characterized using lysate of overexpressing cells. The results confirmed that the enzyme catalyzes the hydrolysis of ceramide in the neutral alkaline range and is independent of cations. Finally, a green fluorescent protein-ceramidase fusion protein was constructed to investigate the localization of this enzyme. The results showed that the green fluorescent protein-ceramidase fusion protein presented a mitochondrial localization pattern and colocalized with mitochondrial specific probes. These results demonstrate that this novel ceramidase is a mitochondrial enzyme, and they suggest the existence of a topologically restricted pathways of sphingolipid metabolism.

Development of a topically active imiquimod formulation.

The purpose of this work was to develop a topical formulation of imiquimod, a novel immune response modifier, to induce local cytokine production for the treatment of external genital and perianal warts. A pH-solubility profile and titration data were used to calculate a pKa of 7.3, indicative of a weak base. Solubility experiments were conducted to identify a solvent that dissolves imiquimod to achieve a 5% formulation concentration. Studies to select surfactants, preservatives, and viscosity-enhancing excipients to formulate an oil-in-water cream indicated that fatty acids were the preferred solvent for topical imiquimod formulations, and isostearic acid (ISA) was selected. A relationship existed between the fatty acid composition of four commercially available ISA sources and the solubility of imiquimod. A combination of polysorbate 60, sorbitan monostearate, and xanthan gum was used to produce a physically stable cream. The preservative system included parabens and benzyl alcohol to meet the USP criteria for preservative activity. An in vitro method was developed to demonstrate that imiquimod was released from the formulation. Topical application of the formulation induced local cytokine activity in mice.

AIDS orphans in Louisiana in the year 2000: the potential economic impact on the foster care system.

This study estimates the number of AIDS orphans in Louisiana and projects the fiscal demand on the state's foster care system in the year 2000. The Michaels and Levine model is used to estimate the number of orphans, and state foster care data are used to project the placement cost of those AIDS orphans. Between 1992 and the year 2000, the AIDS orphan count in Louisiana is estimated to range between 1,242 and 2,627 infants and children. Black children are estimated to comprise the overwhelming majority of this population. If only 50% of the AIDS orphans this study projects for the year 2000 require foster care services, the state of Louisiana would need additional revenues for that year alone, ranging from $308,112 to $624,546, to serve those new entrants. This study provides a critical starting point for state policy planning. The AIDS orphan population in Louisiana is growing each year and the needs of these infants and children must be anticipated and accommodated.

Two closely linked but separable promoters for human neuronal nitric oxide synthase gene transcription.

In this report we demonstrate that the human cerebellum contains neuronal nitric oxide synthase (nNOS) mRNAs with two distinct 5'-untranslated regions that are encoded through use of closely linked but separate promoters. nNOS cDNA clones were shown to contain different 5' terminal exons spliced to a common exon 2. Genomic cloning and sequence analysis demonstrate that the unique exons are positioned within 300 bp of each other but separated from exon 2 by an intron that is at least 20 kb in length. A CpG island engulfs the downstream 5'-terminal exon. In contrast, most of the upstream exon resides outside of this CpG island. Interestingly, the upstream exon includes a GT dinucleotide repeat. A fusion gene with a 414-bp nNOS genomic fragment that includes a portion of the upstream 5'-terminal exon and its immediate 5'-flanking DNA is expressed in transfected HeLa cells. Also expressed is a fusion gene that contains the luciferase reporter under transcriptional control by a 308-bp genomic fragment that includes the region separating both 5'-terminal exons. These results indicate that expression of these exons is subject to transcriptional control by separate promoters. However, the proximity of these promoters raise the possibility that complex interactions may be involved in regulating nNOS gene expression at these sites.

Kinetics of absorption of a new once-a-day formulation of theophylline in the presence and absence of food.

Two three-way crossover studies were done to characterize the drug release characteristics of Monospan (3M Pharmaceuticals, St. Paul, MN) capsules, a new once-a-day theophylline formulation. In the first study, 22 healthy males received single 450-mg doses of Monospan in the presence and absence of a high-fat breakfast; the same dose of Somophyllin (Fisons, Rochester, NY) immediate-release liquid was given to fasted subjects as a reference. The second study involved 29 healthy males given a single dose of 900 mg of Monospan in the presence and absence of the same high-fat meal; Theo-24 (G. D. Searle and Co., Skokie, IL) capsules were given to fasted subjects as a reference. The results of both studies showed that food did not affect the absorption of theophylline from Monospan; peak concentration, to and area under the serum concentration-time curve were all unchanged. The absorption rates were similar with both strengths and dietary conditions and showed that theophylline was absorbed slowly from Monospan at a constant rate (approximately 3.2%/h) over 24 h. Absorption continued past 24 h, and the extent of absorption from Monospan compared with that from each reference averaged 88% or higher. A good correlation (r > 0.980) was observed for Monospan between the amount absorbed in vivo and the amount released in the in vitro dissolution test, a result that demonstrates the precise rate control of Monospan. We conclude that Monospan is a suitable once-a-day formulation that can be taken without regard to food.

Male-female differences in mental health visits under cost-sharing.

This article, which was prepared as part of a larger study of the impact of the copayment requirement on United Mine Workers of America (UMWA) beneficiaries carried out at the National Center for Health Services Research (NCHSR), compares male to female changes in ambulatory care visits for mental disorders and discusses the implications of these changes for the use of other services and for the quality of care. Figures were derived from aggregate claims data provided by the UMWA for the time periods immediately preceding the introduction of copayment (full coverage for all health care) and the first year following the introduction of copayment. Our findings suggest that, at least as far as visits for mental disorders are concerned, copayment may reduce necessary visits. The men in our population, who sought care for mental disorders more sparingly than women and for more severe complaints, were most affected by copayment.

Supply, need, and distribution of anesthesiologists and nurse anesthetists in the U.S., 1972 and 1980.

This paper examines the relationship between problems in anesthesia care delivery and the availability and utilization of trained anesthesia manpower. The 1972 and 1980 projected supply and mix of anesthesiologists and nurse anesthestists is described and compared to the current and projected need for their services. The need estimates developed for anesthesia manpower are based upon published data on operations, productivity and theoretical team configurations. Utilization data are used since they are quantifiable and the need for anesthesia services is, indeed, supply-induced. Two estimates of need are developed based upon differing degrees to which anesthesia teams can be effectively employed, this being contingent upon the size of the hospital operative workload. While the need estimates developed for anesthesiologists for 1972 and 1980 were fairly close in aggregate number to the actual and projected supply; the need estimates for nurse anesthetists fell far short of their 1972 and 1980 projected supply. The 1972 need estimates are then compared on a statewide basis to the actual supply and mix of anesthesia personnel to reveal a severe maldistribution which is quantified in terms of shortage and execesses of anesthesiologists and nurse anesthetists for each state.