Blastocystis in Côte d'Ivoire: molecular identification and epidemiological data.

Blastocystis is an enteric protozoan infecting humans and animals in both developed and developing countries at all latitudes. Despite this, data on Blastocystis infection are not available for several geographical areas, including many African countries. In this study, a survey was conducted on Blastocystis among humans and domestic animals in rural and urban localities in Côte d'Ivoire, in order to investigate the prevalence, the subtype distribution, and the zoonotic potential in association with sociodemographic factors, seasonality, symptoms, and co-infections. A total of 110 fecal samples were collected from patients living in four localities. Molecular and phylogenetic analyses were conducted for Blastocystis detection and subtyping. Positive samples from symptomatic patients were tested by Luminex xTAG® Gastrointestinal Pathogen Panel (GPP) to evidence the presence of other common intestinal pathogens. Overall, a prevalence of 58.2% was observed in humans and subtypes ST1(50.0%), ST2 (22.0%) and ST3 (28.1%) were identified. The prevalence values varied significantly among the sites but not in relation to the subtype. The seasonal rains significantly increase the infection rate in all localities. No significant differences in the ST distribution between asymptomatic and symptomatic subjects were observed. As regard the zoonotic transmission, an additional sampling was conducted in another village where fecal samples were simultaneously collected from humans and animals. Blastocystis STs 1-3 and ST7 were identified in eight humans and four chickens, respectively. This study provides the first exhaustive data on the prevalence and molecular epidemiology of Blastocystis in Côte d'Ivoire.

Social determinants associated with Giardia duodenalis infection in southern Côte d'Ivoire.

The purpose of this investigation was to analyze the association between different social determinants of health at the local scale and Giardia duodenalis infection in diverse settlements in southern Côte d'Ivoire. Stool samples from 306 individuals aged 1-16 years were collected from six rural villages and a small town. Five variables were categorized to classify the increasing risk levels of acquiring intestinal parasites. Giardia prevalences (%) and odds ratios (ORs) were evaluated. Correlation and regression analyses were conducted to determine the correlation coefficients and to propose predictive models based on social determinants to forecast the risk of giardiasis. The overall observed prevalence of Giardia was 21.6 %. When the analysis was conducted at the local level, the percentage of infected people varied from a minimum of 12.7 up to 36.4 %. A significant association (p < 0.001) was found between the selected social determinants and G. duodenalis prevalence in the different localities. Correlation and regression analyses allowed us to describe two predictive models to estimate the OR of Giardia transmission. This study helps to clarify the possible impact of different social determinants of health on the risk of giardiasis at the local scale. Both predictive models could be suitable in order to assess the likelihood of the transmission of intestinal parasitic infection in developing countries.

Variant sequences of insulin receptor substrate-1 in patients with noninsulin-dependent diabetes mellitus.

The pathophysiology of noninsulin-dependent diabetes mellitus (NIDDM) is characterized by insulin resistance and insulin deficiency. To search for genetic defects causing NIDDM, we have screened for mutations in the gene encoding insulin receptor substrate-1 (IRS-1), an intracellular protein that is phosphorylated by the insulin receptor and is thought to play an important role in mediating insulin action. The coding sequence of the IRS-1 gene (divided into 12 overlapping fragments) was amplified by polymerase chain reaction and screened for the presence of single stranded conformational polymorphisms. This led to the identification of 6 variants in the nucleotide sequence. There were 3 nonconservative amino acids substitutions: Gly819-->Arg, Gly972-->Arg, and Arg1221-->Cys. In addition, there were three silent polymorphisms: GAC vs. GAT encoding Asp90, GGG vs. GGA encoding Gly235, and GCA vs. GCG encoding Ala805. The previously reported Arg972 substitution was identified in 7 of 31 patients with NIDDM, 4 of 32 normal subjects, and 4 of 16 nondiabetic obese individuals. The 2 novel amino acid substitutions (Arg819 and Cys1221) were both detected in 1 patient with NIDDM, but not in either of the other 2 groups of nondiabetic individuals. All 3 amino acid residues are identically conserved in the amino acid sequences of human, mouse, and rat IRS-1, suggesting that Gly819, Gly972, and Arg1221 are important for the normal function of IRS-1. Furthermore, the prevalence of amino acid substitutions in IRS-1 is increased in patients with NIDDM. These observations suggest that mutations in the IRS-1 gene may play a causal role in the pathogenesis of NIDDM.

Tissue-specific expression of two alternatively spliced isoforms of the human insulin receptor protein.

Two insulin receptor mRNA species are expressed in human tissues as a result of alternative splicing of exon 11. This event is regulated in a tissue-specific manner. To date, there is little information about the relative abundance of the two receptor protein isoforms on the cell surface. The aim of the present investigation was to assess whether the tissue-specific expression of the two insulin receptor mRNA species is paralleled by a similar pattern of expression of the two receptor protein isoforms. To this end, we assessed the relative distribution of the two receptor variants in various human tissues at the mRNA and protein levels. A PCR-based technique was used to measure the relative abundance of the two mRNA species, and two immunological assays were used to measure the relative steady-state expression of the two receptor protein isoforms. The expression of the two insulin receptor protein isoforms followed the tissue-specific pattern of expression of the two mRNA species.

Molecular analysis based on mtLSU-rRNA and DHPS sequences of Pneumocystis jirovecii from immunocompromised and immunocompetent patients in Italy.

Pneumocystis jirovecii is an opportunistic fungus predominantly reported in immunocompromised individuals, who develop severe interstitial pneumonia (PcP). However, it is known that asymptomatic or mild pulmonary infections, defined as colonization, are widely observed in the general adult population. So far, genetic and epidemiological data of P. jirovecii infections in Italy are rather scarce and limited to defined geographical regions, mainly regarding isolates from HIV-infected patients. The aim of this study was to evaluate the polymorphisms at the mtLSU-rRNA and the DHPS loci by the screening and genotyping of a cohort of patients from two major hospitals in Rome (Italy). The study included 263 patients divided into two groups, all enrolled consecutively from January 2006 to December 2010: (i) 38 immunocompromised subjects including 25 HIV-infected; (ii) 225 immunocompetent patients. Sixty-seven out of 263 patients (25.5%) were found positive after PCR amplification of the mtLSU-rRNA gene. Overall, genotyping at mtLSU-rRNA locus revealed that the genotype 2 was the most frequent. Sequences of the DHPS gene were obtained from 21 patients, 9 from immunocompromised patients (6 from HIV infected individuals), 12 from immunocompetent ones. Considering the most common DHPS mutations usually detected at amino acid positions 55 and 57 and potentially related to drug resistance, all samples analyzed showed the wild-type signatures. These are the first data in Italy on prevalence and genotypes of P. jirovecii regarding colonized immunocompetent adults. Further multicenter analyses on P. jirovecii infection will be necessary to better define the specific epidemiology of the disease in the Italian populations.

The sulfonylurea glimepiride regulates intracellular routing of the insulin-receptor complexes through their interaction with specific protein kinase C isoforms.

Sulfonylureas may stimulate glucose metabolism by protein kinase C (PKC) activation. Because interaction of insulin receptors with PKC plays an important role in controlling the intracellular sorting of the insulin-receptor complex, we investigated the possibility that the sulfonylurea glimepiride may influence intracellular routing of insulin and its receptor through a mechanism involving PKC, and that changes in these processes may be associated with improved insulin action. Using human hepatoma Hep-G2 cells, we found that glimepiride did not affect insulin binding, insulin receptor isoform expression, and insulin-induced receptor internalization. By contrast, glimepiride significantly increased intracellular dissociation of the insulin-receptor complex, degradation of insulin, recycling of internalized insulin receptors, release of internalized radioactivity, and prevented insulin-induced receptor down-regulation. Association of PKC-betaII and -epsilon with insulin receptors was increased in glimepiride-treated cells. Selective depletion of cellular PKC-betaII and -epsilon by exposure to 12-O-tetradecanoylphorbol-13-acetate (TPA) or treatment of cells with PKC-betaII inhibitor G06976 reversed the effect of glimepiride on intracellular insulin-receptor processing. Glimepiride increased the effects of insulin on glucose incorporation into glycogen by enhancing both sensitivity and maximal efficacy of insulin. Exposing cells to TPA or G06976 inhibitor reversed these effects. Results indicate that glimepiride increases intracellular sorting of the insulin-receptor complex toward the degradative route, which is associated with both an increased association of the insulin receptor with PKCs and improved insulin action. These data suggest a novel mechanism of action of sulfonylurea, which may have a therapeutic impact on the treatment of type 2 diabetes.

Peptide-based radioimmunoassay for the two isoforms of the human insulin receptor.

The insulin receptor exists in two isoforms differing by the absence (HIR-A) or presence (HIR-B) of 12 amino acids in the COOH-terminus of the alpha-subunit as a consequence of alternative splicing of exon 11. In this study, we developed a radioimmunoassay for the two isoforms employing antibodies raised against two peptides, one (Pep-12) corresponding to residues encoded by exon 11, and the other (Pep-13) corresponding to a COOH-terminal domain of the alpha-subunit which is common to both HIR-A and HIR-B isoforms. These peptides were iodinated and used as both ligands and standards. The assay is specific, highly reproducible, and sensitive with a detection limit of 10 fmol of receptor. One mole of purified insulin receptor, measured by Scatchard analysis, is read as one mole of receptor in the radioimmunoassay with either Pep-12 or Pep-13 as standards. The radioimmunoassay is applicable to the measurement of total content and relative abundance of the two isoforms in extracts from various tissues. We applied the radioimmunoassay to measure the relative abundance of the two isoforms in fat and muscle from normal, obese non-diabetic and non-insulin-dependent diabetic (NIDDM) subjects. Results demonstrate that expression of the low-affinity HIR-B form is significantly increased in obese and NIDDM subjects compared with control subjects. In addition, the increased expression of the HIR-B isoform was significantly correlated with both body mass index (r = 0.52; p = 0.006) and fasting glucose levels (r = 0.59; p = 0.001).

Delayed intracellular dissociation of the insulin-receptor complex impairs receptor recycling and insulin processing in cultured Epstein-Barr virus-transformed lymphocytes from insulin-resistant subjects.

Insulin-receptor internalization and processing are defective in insulin-resistant subjects. To assess the reversibility of these defects, we cultured Epstein-Barr virus-transformed-lymphoblasts from six normal, six obese, and six non-insulin-dependent diabetic (NIDDM) subjects in media containing low (5 mmol/l) or high (25 mmol/l) glucose concentrations, and studied the insulin-receptor internalization and processing in vitro. In cells from normal, obese, and NIDDM subjects cultured in low glucose concentrations, exposure to 100 nmol/l insulin for 30 min at 37 degrees C reduced cell-surface 125I-insulin binding to a similar extent (82 +/- 2, 77 +/- 5, and 82 +/- 5% of initial values, respectively). The same results were obtained with cells cultured in high glucose concentrations. In cells cultured under both glucose conditions, and exposed to 100 nmol/l insulin for 30 min at 37 degrees C, a complete recovery of the initial 125I-insulin binding was observed in normal but not in obese and NIDDM subjects. Release of intracellular insulin and its degradation in vitro was determined by incubating cells with 600 pmol/l of 125I-insulin for 60 min at 37 degrees C, acid washing cells, and re-incubating in insulin-free buffer at 37 degrees C. The radioactivity released by cells was characterized by trichloroacetic acid precipitability, Sephadex G-50 column chromatography, and re-binding to fresh cells. Rates of release of internalized radioactivity were reduced in obese and NIDDM subjects (t1/2 = 61 +/- 9 min, p < 0.02; 58 +/- 10 min, p < 0.05; and 38 +/- 4 min in obese, NIDDM, and normal subjects, respectively). The percentage of intact insulin released from cells was significantly higher in obese and NIDDM subjects than in the normal subjects. The t1/2 of intracellular dissociation of insulin-receptor complexes measured by a polyethylene glycol assay was lower in normal (6 +/- 1 min) than in obese (12 +/- 2 min, p < 0.03) and NIDDM subjects (14 +/- 3 min, p < 0.02). The results suggest that in insulin-resistant subjects a primary defect in intracellular dissociation of insulin is responsible for alterations of receptor recycling and insulin processing.