Foetal haemoglobin, erythrocytes containing foetal haemoglobin, and hematological features in congolese patients with sickle cell anaemia.

High HbF levels and F cells are correlated with reduced morbidity and mortality in sickle cell disease (SCD). This paper was designed to determine the HbF and F cells levels in Congolese sickle cell anemia (SCA) patients in order to determine their impact on the expression of SCD. Population and Method. HbF levels were measured in 89 SCA patients (mean age 11.4 yrs) using a standard HPLC method. F cell quantitation was done in a second group of SCA patients (n = 42, mean age 8.9 yrs) and compared with a control group (n = 47, mean age 5 yrs). F cells were quantified by a cytofluorometric system (MoAb-HbF-FITC; cut off at 0.5%). Results. The mean value of HbF was 7.2% ± 5.0 with heterogeneous distribution, most patients (76%) having HbF < 8%. Mean values of F-cells in SCA patients and control group were 5.4% ± 7.6 (median: 2.19%; range 0,0-30,3%) and 0.5% ± 1.6 (median 0.0, range 0-5.18), respectively. SCA patients with F cells >4.5% developed less painful crisis and had higher percentage of reticulocytes. Conclusion. Congolese SCA patients displayed low levels of HbF and F-cells that contribute to the severity of SCD.

BCR/ABL1 fusion transcripts generated from alternative splicing: implications for future targeted therapies in Ph+ leukaemias.

Philadelphia (Ph+) positive leukaemias are an example of haematological malignant diseases where different chromosomal rearrangements involving both BCR and ABL1 genes generate a variety of chimeric proteins (BCR/ABL1 p210, p190 and p230) which are considered pathological "biomarkers". In addition to these three, there is a variety of fusion transcripts whose origin may depend either on diverse genetic rearrangement or on alternative/atypical splicing of the main mRNAs or on the occurrence of single-point mutations. Although the therapy of Ph+ leukaemias based on Imatinib represents a triumph of medicine, not all patients benefit from such drug and may show resistance and intolerance. Furthermore, interruption of Imatinib administration is often followed by clinical relapse, suggesting a failure in the eradication of residual leukaemic stem cells. Therefore, while the targeted therapy is searching for new and implemented pharmacological inhibitors covering all the possible mutations in the kinase domain, there is urge to identify alternative molecular targets to develop other specific and effective therapeutic approaches. In this review we discuss the importance of recent advances based on the discovery of novel BCR/ABL1 variants and their potential role as new targets/biomarkers of Ph+ leukaemias in the light of the current therapeutic trends. The limits of the pharmacological inhibitors used for treating the disease can be overcome by considering other targets than the kinase enzyme. Our evaluations highlight the potential of alternative perspectives in the therapy of Ph+ leukaemias.

X-ray microdiffraction and urine: a new analysis method of crystalluria.

The qualitative and quantitative analyses of crystalluria have clinical significance in the diagnosis and prognosis of urolithiasis. The aim of this paper is to provide a new accurate methodology to get qualitative and quantitative data on urine particulate in patients with renal stone disease.The procedure involves a urine collection, the separation of the solid residual by centrifugation, and its analysis by X-ray diffraction, utilizing a micro-diffractometer in order to analyze very low amounts of residual. The spectrum obtained was converted into 2 θ -I profiles and quantitatively refined by Rietveld method. The proposed methodology has the advantage to accurately quantify all crystalline phases and the amorphous component of the urine; anyway urine samples have to be centrifuged and analysed as soon as possible, because the quantitative results obtained by the X-ray microdiffraction showed that after some days and at room temperature urine increased significantly both amorphous and crystalline phases.

The metabolism and disposition of a potent inhibitor of hepatitis C virus NS3/4A protease.

Compound A ((1aR,5S,8S,10R,22aR)-5-tert-butyl-N-{(1R,2S)-1-[(cyclopropylsulfonyl)carbamoyl]-2-ethenylcyclopropyl}-14-methoxy-3,6-dioxo-1,1a,3,4,5,6,9,10,18,19,20,21,22,22a-tetradecahydro-8H-7,10-methanocyclopropa[18,19][1,10,3,6]dioxadiazacyclononadecino[12,11-b]quinoline-8-carboxamide) is a prototype of a series of subnanomolar inhibitors of genotypes 1, 2, and 3 hepatitis C virus (HCV) NS3/4A proteases. HCV NS3/4A protease inhibitors have demonstrated high antiviral effects in patients with chronic HCV infection and are likely to form a key component of future HCV therapy. Compound A showed excellent liver exposure in rats, which is essential for compounds intended to treat HCV. The compound was mainly eliminated intact in bile and showed greater than dose proportional systemic exposure in rats. Compound A demonstrated time- and temperature-dependent uptake into rat and human hepatocytes and proved to be a substrate for rat hepatic uptake transporter Oatp1b2 and for human hepatic uptake transporters OATP1B1 and OATP1B3. The liver selectivity observed for this compound is likely to be due to transporter-mediated hepatic uptake together with moderate passive permeability. Metabolism was mainly CYP3A-mediated and generated a reactive epoxide on the vinylcyclopropyl sulfonamide moiety that could be quenched by glutathione. Similar metabolic profiles of Compound A were obtained in liver microsomes of rats and humans. The oral bioavailability at 5 mg/kg was low due to extensive hepatic first-pass effect but clearly the intestinal absorption was enough to deliver a high amount of the compound to the liver. The metabolism and disposition properties of Compound A are particularly attractive to support its evaluation as a drug candidate for the treatment of hepatitis C.

[Hemogram findings in Congolese children with sickle cell disease in remission]. / L'hémogramme de l'enfant drépanocytaire congolais au cours des phases stationnaires.

INTRODUCTION: Sickle cell disease is associated with a wide range of clinical and laboratory findings depending on genetic modulators and environmental factors. The most severe forms of sickle cell disease occur in patients with the Bantu haplotype. The purpose of this study was to determine the hematological profile of Congolese patients with homozygous sickle cell disease during periods of remission. PATIENTS AND METHODS: Hemograms were performed in two series of patients with sickle cell disease in remission, i.e., one including 89 patients with a mean age of 8.7 years and the other including 42 patients with a mean age of 8.9 years. Hemograms were performed using an automated counter and reticulocytes were counted manually on peripheral blood smears. Fetal hemoglobin level (HbF) was measured by chromatography (HPLC). The mean values obtained were compared with those obtained in a sickle-cell-disease-free control group. Some parameters were also compared with those obtained in a group of patients exhibiting complications of sickle cell disease. RESULTS: Hemograms in the first series of patients demonstrated the following values: Hb: 7.2 g/dl; Hct 23.1%, red cells: 2.47 tera/L, leukocytes: 14.9 giga/L; VGM: 95.3 fL; CCMH:30.3% L and platelets:345,3 giga/L. Blood count showed 30.4% of polynuclear neutrophils, 33% de lymphocytes, 0.8% of polynuclear basophiles, 14% of monocytes, 7.8% of polynuclear eosinophils and 14% of erythroblasts. Mean HbF level was 7.2% and reticulocytes were at 88%. In the sickle cell disease-free group, the leukocyte rate was almost three fold higher than in the patient group exhibiting sickle cell disease in remission even though rates were higher than during complications. CONCLUSION: Hemogram profiles in Congolese patients with sickle cell disease are similar to those reported in the literature for subjects exhibiting the Bantou haplotype. Leukocytosis was associated with esinophilia and monocytosis suggested a topical state and chronic inflammation.

Studies of metabolism and disposition of potent human immunodeficiency virus (HIV) integrase inhibitors using 19F-NMR spectroscopy.

(19)F-nuclear magnetic resonance (NMR) has been extensively used in a drug-discovery programme to support the selection of candidates for further development. Data on an early lead compound, N-(4-fluorobenzyl)-5-hydroxy-1-methyl-2-(4-methylmorpholin-3-yl)-6-oxo-1,6-dihydropyrimidine-4-carboxamide (compound A (+)), and MK-0518 (N-(4-fluorobenzyl)-5-hydroxy-1-methyl-2-(1-methyl-1-{[(5-methyl-1,3,4-oxadiazol-2-yl)carbonyl]amino}ethyl)-6-oxo-1,6-dihydropyrimidine-4-carboxamide), a potent inhibitor of this series currently in phase III clinical trials, are described. The metabolic fate and excretion balance of compound A (+) and MK-0518 were investigated in rats and dogs following intravenous and oral dosing using a combination of (19)F-NMR-monitored enzyme hydrolysis and solid-phase extraction chromatography and NMR spectroscopy (SPEC-NMR). Dosing with the (3)H-labelled compound A (+) enabled the comparison of standard radiochemical analysis with (19)F-NMR spectroscopy to obtain quantitative metabolism and excretion data. Both compounds were eliminated mainly by metabolism. The major metabolite identified in rat urine and bile and in dog urine was the 5-O-glucuronide.

Short term effect of aldosterone on Na,K-ATPase cell surface expression in kidney collecting duct cells.

Aldosterone controls extracellular volume and blood pressure by regulating Na+ reabsorption, in particular by epithelia of the distal nephron. A main regulatory site of this transcellular transport is the epithelial sodium channel (ENaC) that mediates luminal Na+ influx. The Na,K-ATPase (Na+ pump) that coordinately extrudes Na+ across the basolateral membrane is known to be regulated by short term aldosterone as well. We now show that in the cortical collecting duct (CCD) from adrenalectomized rats, the increase in Na,K-ATPase activity (approximately 3-fold in 3 h), induced by a single aldosterone injection, can be fully accounted by the increase in Na,K-ATPase cell surface expression (+ 497 +/- 35%). The short term aldosterone action was further investigated in cultured mouse collecting duct principal cells mpkCCD(cl4). Within 2 h, maximal Na,K-ATPase function assessed by Na+ pump current (I(p)) measurements and Na,K-ATPase cell surface expression were increased by 20-50%. Aldosterone did not modify the Na+ dependence of the Na+ pumps and induced transcription- and translation-dependent actions on pump surface expression and current independently of ENaC-mediated Na+ influx. In summary, short term aldosterone directly increases the cell surface expression of pre-existing Na+ pumps in kidney CCD target cells. Thus, aldosterone controls Na+ reabsorption in the short term not only by regulating the apical cell surface expression of ENaC (Loffing, J., Zecevic, M., Feraille, E., Kaissling, B., Asher, C., Rossier, B. C., Firestone, G. L., Pearce, D., and Verrey, F. (2001) Am. J. Physiol. 280, F675-F682) but also by coordinately acting on the basolateral cell surface expression of the Na,K-ATPase.

Mediators of aldosterone action in the renal tubule.

The aldosterone-sensitive distal nephron extends from the second part of the distal convoluted tubule to the inner medullary collecting duct. As recently shown, aldosterone increases within two hours the abundance of the alpha-subunit of the epithelial sodium channel along the entire aldosterone-sensitive distal nephron, whereas it induces only in an initial portion of the aldosterone-sensitive distal nephron an apical translocation of all three epithelial sodium channel subunits. This suggests that another factor or factors determines the length of the aldosterone-sensitive distal nephron portion in which aldosterone controls epithelial sodium channel surface expression. Since the glucocorticoid-induced kinase SGK1 was identified as aldosterone-induced protein in 1999, it has been postulated to play a key regulatory role. The in-vivo localization of its induction to segment-specific cells of the aldosterone-sensitive distal nephron, and the in-vitro correlation of the amount of its hyperphosphorylated form with transepithelial sodium transport, support this hypothesis. Other recent studies unravel pathways other than those activated by aldosterone and insulin that impact on SGK1 expression and/or function, and thus shed some light onto the complex network that appears to control sodium transport. In view of the ongoing research, the question of how, and formally also whether, SGK1 acts on the epithelial sodium channel should be resolved in the near future.

Pleiotropic action of aldosterone in epithelia mediated by transcription and post-transcription mechanisms.

The aldosterone-induced increase in sodium reabsorption across tight epithelia can be divided schematically into two functional phases: an early regulatory phase starting after a lag period of 20 to 60 minutes, during which the pre-existing transport machinery is activated, and a late phase (>2.5 h), which can be viewed as an anabolic action leading to a further amplification/differentiation of the Na+ transport machinery. At the transcriptional level, both early and late responses are initiated during the lag period, but the functional impact of newly synthesized regulatory proteins is faster than that of the structural ones. K-Ras2 and SGK were identified as the first early aldosterone-induced regulatory proteins in A6 epithelia. Their mRNAs also were shown to be regulated in vivo by aldosterone, and their expression (constitutively active K-Ras2 and wild-type SGK) was shown to increase the function of ENaC coexpressed in Xenopus oocytes. Recently, aldosterone was also shown to act on transcription factors in A6 epithelia: It down-regulates the mRNAs of the proliferation-promoting c-Myc, c-Jun, and c-Fos by a post-transcriptional mechanism, whereas it up-regulates that of Fra-2 (c-Fos antagonist) at the transcriptional level. Together, these new data illustrate the complexity of the regulatory network controlled by aldosterone and support the view that its early action is mediated by the induction of key regulatory proteins such as K-Ras2 and SGK. These early induced proteins are sites of convergence for different regulatory inputs, and thus, their aldosterone-regulated expression level tunes the impact of other regulatory cascades on sodium transport. This suggests mechanisms for the escape from aldosterone action.

LAT2, a new basolateral 4F2hc/CD98-associated amino acid transporter of kidney and intestine.

Glycoprotein-associated amino acid transporters (gpaAT) are permease-related proteins that require heterodimerization to express their function. So far, four vertebrate gpaATs have been shown to associate with 4F2hc/CD98 for functional expression, whereas one gpaAT specifically associates with rBAT. In this study, we characterized a novel gpaAT, LAT2, for which mouse and human cDNAs were identified by expressed sequence tag data base searches. The encoded ortholog proteins are 531 and 535 amino acids long and 92% identical. They share 52 and 48% residues with the gpaATs LAT1 and y(+)LAT1, respectively. When mouse LAT2 and human 4F2hc cRNAs were co-injected into Xenopus oocytes, disulfide-linked heterodimers were formed, and an L-type amino acid uptake was induced, which differed slightly from that produced by LAT1-4F2hc: the apparent affinity for L-phenylalanine was higher, and L-alanine was transported at physiological concentrations. In the presence of an external amino acid substrate, LAT2-4F2hc also mediated amino acid efflux. LAT2 mRNA is expressed mainly in kidney and intestine, whereas LAT1 mRNA is expressed widely. Immunofluorescence experiments showed colocalization of 4F2hc and LAT2 at the basolateral membrane of kidney proximal tubules and small intestine epithelia. In conclusion, LAT2 forms with LAT1 a subfamily of L-type gpaATs. We propose that LAT1 is involved in cellular amino acid uptake, whereas LAT2 plays a role in epithelial amino acid (re)absorption.