Use of [Cit312,314] filaggrin (306–324) analogue for the diagnosis of rheumatoid arthritis. Conformational study by circular dichroism and fourier transformed infrared spectroscopy

Antifilaggrin autoantibodies (AFA) have described to be the most specific markers of rheumatoid arthritis (RA) and epitopes containing citrulline within the sequence of filaggrin identified as major antigenic sites recognised by AFA. In this paper we confirm that citrulline is an essential constituent of filaggrin related antigenic determinants recognised by RA specific autoantibodies, thus having an important role for the development of filaggrin peptides based serological tests. Moreover, we describe our findings on the comparative conformational analysis of filaggrin (306–324) peptide sequence and an analogue in which two arginines were simultaneously substituted by citrulline carried out by Circular Dichroism (CD) and Fourier-Transform Infrared Spectroscopy (FTIR). Results might contribute to the understanding of the structural features that may be important to explain the enhanced binding characteristics of citrullinated peptides to the autoantibodies in rheumatoid arthritis.     

Optimization of Trichomonas vaginalis Diagnosis during Pregnancy at a University Hospital,Argentina

The aim of this study was to evaluate different methods for Trichomonas vaginalis diagnosis during pregnancy in order to prevent maternal and perinatal complications. A total of 386 vaginal exudates from pregnant women were analyzed. T. vaginalis was investigated by 3 types of microscopic examinations direct wet mount with physiologic saline solution, prolonged May-Grunwald Giemsa (MGG) staining, and wet mount with sodium-acetate-formalin (SAF)/methylene blue method. PCR for 18S rRNA gene as well as culture in liquid medium were performed. The sensitivity and specificity of the microscopic examinations were evaluated considering the culture media positivity or the PCR techniques as gold standard. The frequency of T. vaginalis infection was 6.2% by culture and/or PCR, 5.2% by PCR, 4.7% by culture, 3.1% by SAF/methylene blue method and 2.8% by direct wet smear and prolonged MGG staining. The sensitivities were 83.3%, 75.0%, 50.0%, and 45.8% for PCR, culture, SAF/methylene blue method, and direct wet smear-prolonged MGG staining, respectively. The specificity was 100% for all the assessed methods. Microscopic examinations showed low sensitivity, mainly in asymptomatic pregnant patients. It is necessary to improve the detection of T. vaginalis using combined methods providing higher sensitivity, such as culture and PCR, mainly in asymptomatic pregnant patients, in order to prevent maternal and perinatal complications.     

Population-based carrier screening and prenatal diagnosis of fragile X syndrome in East Asian populations

Identification of carriers of fragile X syndrome (FXS) with the subsequent prenatal diagnosis and knowledge of FXS-associated genetic profiles are essential for intervention in specific populations. We report the results of carrier screening of 39,458 East Asian adult women and prenatal diagnosis from 87 FXS carriers. The prevalence of FXS carriers and full mutation fetuses was estimated to be 1/581 and 1/3124 in East Asian populations, respectively. We confirmed the validity of the current threshold of CGG trinucleotide repeats for FMR1 categorization; the integral risks of full mutation expansion were approximately 6.0%, 43.8%, and 100% for premutation alleles with 55–74, 75–89, and ≥ 90 CGG repeats, respectively. The protective effect of AGG (adenine-guanine-guanine nucleotides) interruption in East Asian populations was validated, which is important in protecting premutation alleles with 75–89 CGG repeats from full mutation expansion. Finally, family history was shown not an effective indicator for FXS carrier screening in East Asian populations, and population-based screening was more cost-effective. This study provides an insight into the largest carrier screening and prenatal diagnosis for FXS in East Asian populations to date. The FXS-associated genetic profiles of East Asian populations are delineated, and population-based carrier screening is shown to be promising for FXS intervention.     

The genetics and expression of an esterase locus inAnopheles gambiae

The main polymorphic system of esterase isoenzymes in adults of the G3 laboratory strain ofAnopheles gambiae consists of two to five major bands of activity per individual. The bands are designated 5S, 5F, 13, 14, and 15. In genetic crosses, the genes which coded for the bands assorted as three codominant alleles, Est A, Est B, and Est C, at a single autosomal locus. Homozygotes for the Est C allele were significantly underrepresented among backcross progeny. The developmental pattern of esterase expression was examined. Esterase gene expression in embryos was first detectable between 2 and 12 hr after oviposition. The initiation or termination of expression of some of the bands corresponded to boundaries between developmental stages. Most of the esterase fractions were not specifically localized within the tissues tested, with the exception of a series of bands which were restricted largely to adult male testes.     

In vitro study of malaria parasite induced disruption of blood-brain barrier

The mechanism of blood-brain barrier breakdown in the complex pathogenesis of cerebral malaria is not well understood. In this study, primary cultures of porcine brain capillary endothelial cells (PBCEC) were used as in vitro model. Membrane-associated malaria antigens obtained from lysed Plasmodium falciparum schizont-infected erythrocytes stimulated human peripheral blood mononuclear cells (PBMC) to secrete tumor necrosis factor alpha. In co-cultivation with the brain endothelial cell model, the malaria-activated PBMC stimulated the expression of E-selectin and ICAM-1 on the PBCEC. Using electric cell-substrate impedance sensing, we detected a significant decrease of endothelial barrier function within 4h of incubation with the malaria-activated PBMC. Correspondingly, immunocytochemical studies showed the disruption of tight junctional complexes. Combination of biochemical and biophysical techniques provides a promising tool to study changes in the blood-brain barrier function associated with cerebral malaria. Moreover, it is shown that the porcine endothelial model is able to respond to human inflammatory cells.     

Seasonal prevalence of mosquitoes collected from light traps in the Republic of Korea, 2006

Adult mosquito surveillance was conducted during 2006 at 22 US Army and Air Force installations and two training sites located in six provinces in the Republic of Korea. Adult mosquitoes were collected in New Jersey light traps from 1 May through 15 October 2006 to determine threshold levels to initiate pesticide applications based on trap indices. In total 16 217 adult mosquitoes [13 612 (83.9%) females and 2605 (16.1%) males] comprising 16 species (including five members of the Anopheles sinensis Group) belonging to eight genera were collected. Females of the most commonly collected species were members of the Anopheles sinensis Wiedemann Group (58.9%), followed by Aedes vexans nipponii (Theobald) (21.3%), Culex tritaeniorhynchus Giles (11.8%) and Culex pipiens Coquillett (5.2%). Trap indices varied widely for species over their range, due in part, to their geographical distribution and degree of association with urban or rural communities.     

Sexing the human fetus and identification of polyploid nuclei by DNA-DNA in situ hybridisation in interphase nuclei

Samples of human adult lymphocytes, fetal lymphocytes, amniotic fluid cells, and chorionic villus cells were sexed independently by cytogenetics and DNA-DNA in situ hybridisation to a tritiated Y probe. For the in situ hybridisation analysis, the presence of Y bodies (hybridisation bodies) in 100 interphase nuclei were scored after autoradiography. In all, 82/83 samples were sexed in this way (one technical failure) and 78/82 were sexed by both in situ hybridisation and cytogenetics. There was complete agreement between the two methods. There was a considerable variation (40-100%) in the percentage of interphase nuclei with a hybridisation body among the male samples, but very few nuclei from female samples showed significant hybridisation. In situ hybridisation could be used to sex the conceptus when males but not females are at risk for various X-linked genetic disorders and may also be useful for detecting 45,X/46,XY mosaicism or polyploid/diploid mosaicism. This would be particularly useful for direct preparations of chorionic villus samples, which often prove difficult to analyse cytogenetically but offer the best means of avoiding maternal contamination. Some interphase nuclei had more than one hybridisation body, and this was most commonly found among amniotic fluid cells. Comparison of sizes of nuclei with one or two hybridisation bodies strongly suggested that most of the amniotic fluid cell nuclei with two hybridisation bodies were tetraploid.     

A high degree of aneuploidy in frozen-thawed human preimplantation embryos

We have studied the chromosomal content in 68 normally fertilised freeze-thawed human embryos of good morphology from 34 patients with an average maternal age of 32,6 years. Forty embryos showed post-thaw cellular division and twenty-eight post-thaw cleavage arrest. After spreading of the embryos on microscope slides, analysis of chromosomes X, Y, 15, 16, 17 and 18 was performed using two rounds of fluorescent in situ hybridisation (FISH). According to the results, the embryos were divided into four groups: (I) normal, all nuclei uniformly diploid, (II) diploid mosaics, normal diploid blastomeres in combination with abnormal blastomeres, (III) abnormal, all nuclei abnormal, (IV) chaotic, the chromosome constitution varies randomly from cell to cell. Approximately 25% of the embryos had normal number of the chromosomes tested, while the majority of the embryos were abnormal. Most of the abnormal embryos were diploid mosaics (57%). This was true for the embryos showing cleavage division as well as the embryos showing cleavage arrest. Our data show a slightly higher incidence of abnormal embryos compared to those obtained with FISH in non-cryopreserved embryos and confirm that the majority of preimplantation embryos fertilised in vitro contain abnormal blastomeres. The results, mechanisms, significance and implications are discussed. Received: 19 November 1998 / Accepted: 4 March 1999