A Novel Immunological Assay for Hepcidin Quantification in Human Serum

Background

Hepcidin is a 25-aminoacid cysteine-rich iron regulating peptide. Increased hepcidin concentrations lead to iron sequestration in macrophages, contributing to the pathogenesis of anaemia of chronic disease whereas decreased hepcidin is observed in iron deficiency and primary iron overload diseases such as hereditary hemochromatosis. Hepcidin quantification in human blood or urine may provide further insights for the pathogenesis of disorders of iron homeostasis and might prove a valuable tool for clinicians for the differential diagnosis of anaemia. This study describes a specific and non-operator demanding immunoassay for hepcidin quantification in human sera.

Methods and Findings

An ELISA assay was developed for measuring hepcidin serum concentration using a recombinant hepcidin25-His peptide and a polyclonal antibody against this peptide, which was able to identify native hepcidin. The ELISA assay had a detection range of 10–1500 µg/L and a detection limit of 5.4 µg/L. The intra- and interassay coefficients of variance ranged from 8–15% and 5–16%, respectively. Mean linearity and recovery were 101% and 107%, respectively. Mean hepcidin levels were significantly lower in 7 patients with juvenile hemochromatosis (12.8 µg/L) and 10 patients with iron deficiency anemia (15.7 µg/L) and higher in 7 patients with Hodgkin lymphoma (116.7 µg/L) compared to 32 age-matched healthy controls (42.7 µg/L).

Conclusions

We describe a new simple ELISA assay for measuring hepcidin in human serum with sufficient accuracy and reproducibility.     

Crystallographic studies on N-azidoacetyl-beta-D-glucopyranosylamine, an inhibitor of glycogen phosphorylase: comparison with N-acetyl-beta-D-glucopyranosylamine

N-acetyl-beta-D-glucopyranosylamine (NAG) is a potent inhibitor (Ki=32 microM) of glycogen phosphorylase b (GPb), and has been employed as a lead compound for the structure-based design of new analogues, in an effort to utilize its potential as a hypoglycaemic agent. Replacement of the acetamido group by azidoacetamido group resulted in an inhibitor, N-azidoacetyl-beta-D-glucopyranosylamine (azido-NAG), with a Ki value of 48.7 microM, in the direction of glycogen synthesis. In order to elucidate the mechanism of inhibition, we determined the ligand structure in complex with GPb at 2.03 A resolution, and the structure of the fully acetylated derivative in the free form. The molecular packing of the latter is stabilized by a number of bifurcated hydrogen bonds of which the one involving a bifurcated C-H...N...H-C type hydrogen bonding is rather unique in organic azides. Azido-NAG can be accommodated in the catalytic site of T-state GPb at approximately the same position as that of NAG and stabilizes the T-state conformation of the 280 s loop by making several favourable contacts to residues of this loop. The difference observed in the Ki values of the two analogues can be interpreted in terms of desolvation effects, subtle structural changes of protein residues and changes in water structure.     

Foods of hunter-killed Black Francolins <Emphasis Type="Italic">(Francolinus francolinus)</Emphasis> in Cyprus

Using stomach content analysis, we studied the diet of the Black Francolin (Francolinus francolinus) on the island of Cyprus during November and December of 2004 and 2005. The purpose of this study was to estimate the most important sources of food on its diet. Stomachs of Black Francolins were obtained from two study areas on Cyprus. We sampled 53 freshly hunter-killed specimens for dietary analysis. Our results showed that dietary intake reflected a generalist and omnivorous diet with seeds of cultivated crops and insects of the Coleoptera and Hymenoptera orders being the most commonly identified. Differences in food composition between the two study areas reflected differences in land use, suggesting that human management of Black Francolin habitat is critical in understanding and managing this important game species.     

A segmental radiological study of the spine and rib – cage in children with progressive Infantile Idiopathic Scoliosis

Background

The role of rib cage in the development of progressive infantile idiopathic scoliosis (IIS) has not been studied previously. No report was found for rib growth in children with IIS. These findings caused us to undertake a segmental radiological study of the spine and rib-cage in children with progressive IIS. The aim of the present study is to present a new method for assessing the thoracic shape in scoliotics and in control subjects and to compare the findings between the two groups.

Materials and methods

In the posteroanterior (PA) spinal radiographs of 24 patients with progressive IIS, with a mean age of 4.1 years old, the Thoracic Ratios (TRs) (segmental convex and concave TRs), the Cobb angle, the segmental vertebral rotation and vertebral tilt were measured. In 233 subjects, with a mean age of 5.1 years old, who were used as a control group, the segmental left and right TRs and the total width of the chest (left plus right TRs) were measured in PA chest radiographs. Statistical analysis included Mann-Whitney, Spearman correlation coefficient, multiple linear regression analysis and ANOVA.

Results

The comparison shows that the scoliotic thorax is significantly narrower than that of the controls at all spinal levels. The upper chest in IIS is funnel-shaped and the vertebral rotation at T4 early in management correlates significantly with the apical vertebral rotation at follow up.

Conclusion

The IIS thorax is narrower than that of the controls, the upper chest is funnel-shaped and there is a predictive value of vertebral rotation at the upper limit of the thoracic curve of IIS, which reflects, impaired rib control of spinal rotation possibly due to neuromuscular factors, which contribute also to the funnel-shaped chest.     

Indications for conservative management of scoliosis (guidelines)

We report a 15-year-old girl who presented with spinal malsegmentation, associated with other skeletal anomalies. The spinal malsegmentation was subsequently discovered to be part of the spondylocarpotarsal synostosis syndrome. In addition, a distinctive craniocervical malformation was identified, which included atlanto-axial rotatory fixation. The clinical and the radiographic findings are described, and we emphasise the importance of computerised tomography to characterize the craniocervical malformation complex. To the best of our knowledge, this is the first clinical report of a child with spondylocarpotarsal synostosis associated with atlanto-axial rotatory fixation.     

Association between adolescent idiopathic scoliosis prevalence and age at menarche in different geographic latitudes

Background

Age at menarche is considered a reliable prognostic factor for idiopathic scoliosis and varies in different geographic latitudes. Adolescent idiopathic scoliosis prevalence has also been reported to be different in various latitudes and demonstrates higher values in northern countries. A study on epidemiological reports from the literature was conducted to investigate a possible association between prevalence of adolescent idiopathic scoliosis and age at menarche among normal girls in various geographic latitudes. An attempt is also made to implicate a possible role of melatonin in the above association.

Material-methods

20 peer-reviewed published papers reporting adolescent idiopathic scoliosis prevalence and 33 peer-reviewed papers reporting age at menarche in normal girls from most geographic areas of the northern hemisphere were retrieved from the literature. The geographic latitude of each centre where a particular study was originated was documented. The statistical analysis included regression of the adolescent idiopathic scoliosis prevalence and age at menarche by latitude.

Results

The regression of prevalence of adolescent idiopathic scoliosis and age at menarche by latitude is statistically significant (p < 0.001) and are following a parallel declining course of their regression curves, especially in latitudes northern than 25 degrees.

Conclusion

Late age at menarche is parallel with higher prevalence of adolescent idiopathic scoliosis. Pubarche appears later in girls that live in northern latitudes and thus prolongs the period of spine vulnerability while other pre-existing or aetiological factors are contributing to the development of adolescent idiopathic scoliosis. A possible role of geography in the pathogenesis of idiopathic scoliosis is discussed, as it appears that latitude which differentiates the sunlight influences melatonin secretion and modifies age at menarche, which is associated to the prevalence of idiopathic scoliosis.     

15-MC-5 manganese metallacrowns hosting herbicide complexes. Structure and bioactivity

Interaction of manganese with salicylhydroxamic ligands leads to the formation of a series of 15-membered metallacrown Mn(II)(L)(2)[15-MC(Mn(III)N(shi))-5](py)(6) (L=alkanoato ligand). The crystal structure contains a neutral 15-membered metallacrown ring of the type [15-MC(Mn(III)N(shi))-5]. The metallacrown core consists of five Mn(III) and five shi(-3) ligands. The 15-membered metallacrown ring is formed by the succession of five structural moieties of the type [Mn(III)-N-O]. The diversity in the configuration (planar or propeller) for the ring Mn(III) ions gives to the metallacrown core flexibility and simultaneously allows the encapsulation of the sixth Mn(II). The encapsulated Mn(II) is seven-coordinate and is bound to the five hydroximate oxygen donors provided by the metallacrown core, and two oxygen atoms from the carboxylate herbicide ligands. Antibacterial screening data showed that among all the compounds tested, manganese metallacrowns are more active than the simple manganese herbicide or carboxylate complexes while an increase in the efficiency of [15-MC(Mn(III)N(shi))-5] towards the analogous [12-MC(Mn(III)N(shi))-4] can be observed.     

Small and large unilamellar vesicle membranes as model system for bile acid diffusion in hepatocytes.

Uptake of bile acids into the liver cell occurs via active transport or passive diffusion. In a model system, passive diffusion was studied in liposomes using pyranine fluorescence. Rate constants for the diffusion of diverse more polar or more apolar bile acids were examined. Hydrophobic lithocholic acid (LCA) revealed a maximal rate constant of 0.057 s(-1); with the polar ursodeoxycholic acid (UDCA), the value was 0.019 s(-1). UDCA (3 mol%) effectively decreased the rate constant of 0.1 mM chenodeoxycholic acid (CDCA), whereas cholesterol reached a similar decrease only between 5 and 10 mol%. At higher concentrations of CDCA (above 1 mM) or LCA (0.3-0.4 mM), breaking up of liposomal structure was confirmed by light-scattering decrease and increase of carboxyfluorescein fluorescence. Changes in lipid composition of phosphatidylcholine (PC)- small unilamellar vesicles (SUVs) or large unilamellar vesicles (LUVs) also caused decreasing rate constants. For a cardiolipin (CL):PC ratio of 1:20 the CDCA (0.1 mM) rate constant was 71% lower (0.015 s(-1)) and for a sphingomyelin (SM):PC ratio of 2:1 the rate constant was 50% lower (0.026 s(-1)). Changes in membrane fluidity were detected using membrane anisotropy measurements with the 1,6-diphenyl-1,3, 5-hexatriene (DPH) method. Membrane fluidity was reduced with cholesterol- but not with CL- or SM-containing SUVs (ratio: cholesterol, CL, SM:PC of 1:5). This model system is currently used for the analysis of more complex lipid vesicles resembling the plasma/hepatocyte membrane, which is either stabilized or destabilized by appropriate conditions. The results should become clinically relevant.     

ApoA-I mimetics reduce systemic and gut inflammation in chronic treated HIV

Novel therapeutic strategies are needed to attenuate increased systemic and gut inflammation that contribute to morbidity and mortality in chronic HIV infection despite potent antiretroviral therapy (ART). The goal of this study is to use preclinical models of chronic treated HIV to determine whether the antioxidant and anti-inflammatory apoA-I mimetic peptides 6F and 4F attenuate systemic and gut inflammation in chronic HIV. We used two humanized murine models of HIV infection and gut explants from 10 uninfected and 10 HIV infected persons on potent ART, to determine the in vivo and ex vivo impact of apoA-I mimetics on systemic and intestinal inflammation in HIV. When compared to HIV infected humanized mice treated with ART alone, mice on oral apoA-I mimetic peptide 6F with ART had consistently reduced plasma and gut tissue cytokines (TNF-α, IL-6) and chemokines (CX3CL1) that are products of ADAM17 sheddase activity. Oral 6F attenuated gut protein levels of ADAM17 that were increased in HIV-1 infected mice on potent ART compared to uninfected mice. Adding oxidized lipoproteins and endotoxin (LPS) ex vivo to gut explants from HIV infected persons increased levels of ADAM17 in myeloid and intestinal cells, which increased TNF-α and CX3CL1. Both 4F and 6F attenuated these changes. Our preclinical data suggest that apoA-I mimetic peptides provide a novel therapeutic strategy that can target increased protein levels of ADAM17 and its sheddase activity that contribute to intestinal and systemic inflammation in treated HIV. The large repertoire of inflammatory mediators involved in ADAM17 sheddase activity places it as a pivotal orchestrator of several inflammatory pathways associated with morbidity in chronic treated HIV that make it an attractive therapeutic target.     

Hydroxylated N-alkyl-4-piperidinyl-2,3-diarylpyrrole derivatives as potent broad-spectrum anticoccidial agents

Diaryl-(4-piperidinyl)-pyrrole derivatives bearing hydroxylated N-alkyl substituents have been synthesized and evaluated as anticoccidial agents. High potency in Et-PKG inhibition and broad-spectrum anticoccidial activities have been observed on compounds, such as 4b and 5h, which are fully efficacious in vivo at 50 ppm in feed.