Identification ofSchistosoma haematobium soluble egg antigens that elicit human granuloma formation in vitro

Schistosoma haematobium soluble egg antigens (SH SEAs) induce intense granulomas in human hosts that often culminate in severe disease. In an attempt to identify the SH SEA fractions that are responsible for pathology, we combined T-cell Western blotting and an in vitro model of granuloma formation. Whole SH SEAs were dotted onto nitrocellulose pieces or were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and electrotransferred onto nitrocellulose paper. Horizontal strips bearing the separated antigens were solubilized in dimethylsulfoxide and precipitated in carbonate/bicarbonate buffer. Antigen-free and antigen-bearing particles were used to stimulate peripheral blood mononuclear cells (PBMCs) obtained fromS. haematobium-infected patients and sex- and agematched healthy controls to form granulomas in vitro. Whole SH SEA-bearing nitrocellulose particles elicited in vitro formation of granulomas by PBMCs from infected donors. The response was similar in sensitivity, specificity, and reproducibility to that evoked by SH SEA-bound polyacrylamide beads. The results obtained in samples from 30 patients and 10 controls tested with SH SEA-separated fractions revealed that SEA bands of 84 000, 63 000, 57 000, 55 000, 40 000, 30 000, and 28 000 Da elicited in vitro granuloma reactions by PBMCs of almost all infected patients. Conversely, separated soluble adult-worm antigens failed to stimulate PBMCs of infected patients to form granulomas. This study is the first to identify the SH SEA fractions that evok in vitro granuloma formation and represents an initial step toward the development of an anti-urinary schistosomiasis pathology vaccine.     

Dysferlin mutations in LGMD2B, Miyoshi myopathy, and atypical dysferlinopathies

DYSF encoding dysferlin is mutated in Miyoshi myopathy and Limb-Girdle Muscular Dystrophy type 2B, the two main phenotypes recognized in dysferlinopathies. Dysferlin deficiency in muscle is the most relevant feature for the diagnosis of dysferlinopathy and prompts the search for mutations in DYSF. DYSF, located on chromosome 2p13, contains 55 coding exons and spans 150 kb of genomic DNA. We performed a genomic analysis of the DYSF coding sequence in 34 unrelated patients from various ethnic origins. All patients showed an absence or drastic decrease of dysferlin expression in muscle. A primary screening of DYSF using SSCP or dHPLC of PCR products of each of 55 exons of the gene was followed by sequencing whenever a sequence variation was detected. All together, 54 sequence variations were identified in DYSF, 50 of which predicting either a truncated protein or one amino-acid substitution and most of them (34 out of 54) being novel. In 23 patients, we identified two pathogenic mutations, while only one was identified in 11 patients. These mutations were widely spread in the coding sequence of the gene without any mutational "hotspot."     

A randomized trial of ceftriaxone versus trimethoprim-sulfamethoxazole to prevent ventriculoperitoneal shunt infection.

BACKGROUND AND PURPOSE: Shunt infection represents a particularly morbid condition, which can also result in mortality. In order to decrease the high morbidity and mortality rates, prevention is an essential step. The purpose of this study was to compare the prophylactic use of ceftriaxone and trimethoprim-sulfamethoxazole (SXT) for the prevention of ventriculoperitoneal (VP) shunt infection. METHODS: In this prospective, single-institution, randomized clinical trial, 107 children with hydrocephalus and an indication for shunting were randomly assigned to prophylaxis with ceftriaxone (n = 50) or SXT (55), each administered as a single dose during anesthesia and two divided doses postoperatively. Patients were followed up for at least one year. RESULTS: The mean age of patients was 15 months, and 85% were aged 6 months or younger. During the first postoperative year, meningitis occurred in 13.5% of patients receiving ceftriaxone and 14.5% of the SXT group, with no statistically significant difference between the groups. Younger age, presence of cerebrospinal fluid leakage and aqueductal stenosis as a cause of hydrocephalus showed significant correlation with meningitis occurrence on univariate analysis. However, only the latter 2 factors were associated with meningitis on multivariate analysis. The risk of shunt infection did not correlate with the gender of the patient, time of VP shunt surgery, or duration of hospitalization for shunting. CONCLUSION: Ceftriaxone and SXT showed similar efficacy in preventing shunt infection. Cerebrospinal fluid leakage before or after VP shunt placement and aqueductal stenosis were independent risk factors for meningitis after VP shunt.     

Homocysteine,lipoprotein (a): risk factors for coronary heart disease

Our data suggest that the hyperhomocysteinemia and/or increased plasma level of lipoprotein Lp(a) are risk factors for coronary heart disease. We investigated 178 patients who underwent complete cardiac examination comprising coronary angiography and biological analysis (CT, HDL-c, LDL-c, TG, and apoAI, apoB, homocysteine and Lp(a)). Patients presenting a significant stenosis of the coronary artery ( 50% of the vascular lumen) were considered as cases (113 patients). Those without stenosis or with non-significant stenosis (< 50% of the vascular lumen) were used as controls (65 subjects). Homocysteinemia was significantly higher in cases than in control subjects (8.26 mol/L (2.34 versus 17.85 (2.34, p < 0.001). A strong association between coronary heart disease and homocystein has been found (Eta(2) = 0.76). The OR were 0.16 when homocystein level was lower than 15 mol/L, and 27.78 when homocysteine level was upper than or equal to 15 mol/L. The RR was 5.16 (95% IC = 3.66-6.66, p < 0.001). Even though there was a significant correlation between tabagic impregnation and homocysteinemia (Spermann's rho = 0.37, p < 0.05), there was no interactive effect between these two factors and coronary disease (Wald khi2 = 0.086, p > 0.05). Therefore, no association was found between homocyteinemia and other coronary heart disease risk factors. The Lp(a) levels were significantly higher in cases than in controls subjects (188 (84 mg/L in control subjects versus 590 (199 in cases, p < 0.001). A stronger relationship was noted between coronary heart disease and Lp(a) (Eta (2) = 0.66). The OR were 0.09 when lipoprotein (a) levels were lower than 350 mg/L, and 5,88 when Lp(a) levels were higher than or equal to 350 mg/L. The estimate RR was 6.47 (95% IC = 4.39-8.55, p < 0.001). The level of Lp(a) was positively correlated with the severity of coronary heart disease (Spermann's rho = 0.95, p < 0.001). A weak correlation between Lp(a) and LDL-c was observed (Spermann's rho = 0.12, p = 0.048). But the multivariate analysis didn't show interactive effect between these two factors and coronary disease (khi2 de Wald = 0.264, p > 0.05). No association was noted between Lp(a) and the others risk factors. Moreover, a positive correlation between the levels of homocysteine and those of Lp(a) was found (Spermann's rho = 0.54, p < 0.001). In contrast their effect on coronary heart disease seems to be independant (Wald khi2 = 2.957, p > 0.05). Thus, these two parameters appear as independant risk factors for coronary heart disease.     

Functional consequences of ROMK mutants linked to antenatal Bartter's syndrome and implications for treatment

The antenatal variant of Bartter's syndrome is an autosomal recessive kidney disease characterized by polyhydramnios, premature delivery, hypokalemic alkalosis and hypercalciuria. It is genetically heterogeneous, having been linked recently to mutations in an ATP- sensitive, renal outer medullary K+channel, ROMK, and earlier to mutations in the Na-K-2Cl co-transporter, NKCC2. We characterized four of the mutations reported in three heterozygous ROMK variants of antenatal Bartter's and found that each expressed a distinct phenotype in Sf9 cells. One mutation expressed normal function and appears to be an allelic polymorphism. The other three mutations produced channels with significantly reduced K+fluxes. However, the mechanisms in each case were different and reflected abnormalities in phosphorylation, proteolytic processing or protein trafficking. The different mechanisms may be important in the design of appropriate therapy for patients with this disease.      

Uterine myomata and outcome of assisted reproduction

The aim of this work was to study the effect of uterine myomata on the implantation rate and outcome in in-vitro fertilization (IVF) and intracytoplasmic sperm injection (ICSI). Among 406 patients, 51 (12.6%) were found to have uterine corporeal myomata. Twelve patients were excluded from the study as they had large myomata, submucous myomata or intramural myomata encroaching on the cavity. These patients were advised to have myomectomy before being enrolled in the IVF/ICSI programme. The remaining patients (n = 39) were sorted according to the number, site and size of the myomata as assessed by transvaginal sonography. Three patients had more than one myoma. Most of the myomata were subserous (72.7%) and the mean diameter of the myomata was 3.5 +/- 0.9 cm. A control group (n = 367) was chosen with normal uteri and no history of uterine reconstruction surgery. The mean age of myoma patients was 34.7 +/- 3.6 years as compared to 34.0 +/- 4.4 years in the control group. The age, period of infertility, body mass index, duration and number of human menopausal gonadotrophin ampoules needed for stimulation, oestradiol levels, number of oocytes retrieved and the fertilization rate were not significantly different in the myoma patients compared to the control group. Fifteen myoma patients (38.5%) subsequently showed one or more pregnancy sacs on ultrasonography of which three (20%) spontaneously aborted during the first trimester and two (13.3%) had preterm labour, as compared to 123 (33.5%), 19 (15.5%) and nine (7.3%) respectively, among the control group (P = 0.27, 0.33 and 0.21). In conclusion, uterine corporeal myomata, not encroaching on the cavity and <7 cm in mean diameter, do not affect the implantation or miscarriage rates in IVF or ICSI.      

Germline mutations of the CDKN2 gene in UK melanoma families

Germline mutations in CDKN2 on chromosome 9p21, which codes for the cyclin D kinase inhibitor p16, and more rarely, mutations in the gene coding for CDK4, the protein to which p16 binds, underlie susceptibility in some melanoma families. We have sequenced all exons of CDKN2 and analysed the CDK4 gene for mutations in 27 UK families showing evidence of predisposition to melanoma. Five different germline mutations in CDKN2 were found in six families. Three of the mutations (Met53Ile, Arg24Pro and 23ins24) have been reported previously. We have identified two novel CDKN2 mutations (88delG and Ala118Thr) which are likely to be associated with the development of melanoma, because of their co-segregation with the disease and their likely functional effect on the CDKN2 protein. In binding assays the protein expressed from the previously described mutation, Met53Ile, did not bind to CDK4/CDK6, confirming its role as a causal mutation in the development of melanoma. Ala118Thr appeared to be functional in this assay. Arg24Pro appeared to bind to CDK6, but not to CDK4. No mutations were detected in exon 2 of CDK4, suggesting that causal mutations in this gene are uncommon. The penetrance of these mutant CDKN2 genes is not yet established, nor is the risk of non-melanoma cancer to gene carriers.      

Determination of the parent of origin in nine cases of prenatally detected chromosome aberrations found after intracytoplasmic sperm injection

Prenatal cytogenetic analysis of 71 fetuses conceived by intracytoplasmic sperm injection (ICSI) resulted in the detection of nine (12.7%) chromosome aberrations including two cases of 47,XXY, four cases involving a 45,X cell line and three autosomal trisomies. Molecular analysis of the parental origin of the deleted or supernumerary chromosome was performed by using polymorphic microsatellite markers. Six cases involving a sex chromosome abnormality were found to be of paternal origin while the two trisomic cases that could be analysed were of maternal origin. Two cases involved the same infertile couple who had two consecutive ICSI pregnancies terminated because of a chromosome abnormality. The replaced embryos in both cases originated from a single batch of ICSI fertilized oocytes of which part was used to initiate the first pregnancy and part was cryopreserved and used to initiate the second pregnancy.