Screening for Fabry Disease in Left Ventricular Hypertrophy: Documentation of a Novel Mutation

AbstractBackground:Fabry disease is a lysosomal storage disease caused by enzyme α-galactosidase A deficiency as a result of mutations in the GLA gene. Cardiac involvement is characterized by progressive left ventricular hypertrophy.Objective:To estimate the prevalence of Fabry disease in a population with left ventricular hypertrophy.Methods:The patients were assessed for the presence of left ventricular hypertrophy defined as a left ventricular mass index ≥ 96 g/m2 for women or ≥ 116 g/m2 for men. Severe aortic stenosis and arterial hypertension with mild left ventricular hypertrophy were exclusion criteria. All patients included were assessed for enzyme α-galactosidase A activity using dry spot testing. Genetic study was performed whenever the enzyme activity was decreased.Results:A total of 47 patients with a mean left ventricular mass index of 141.1 g/m2 (± 28.5; 99.2 to 228.5 g/m2] were included. Most of the patients were females (51.1%). Nine (19.1%) showed decreased α-galactosidase A activity, but only one positive genetic test − [GLA] c.785G>T; p.W262L (exon 5), a mutation not previously described in the literature. This clinical investigation was able to establish the association between the mutation and the clinical presentation.Conclusion:In a population of patients with left ventricular hypertrophy, we documented a Fabry disease prevalence of 2.1%. This novel case was defined in the sequence of a mutation of unknown meaning in the GLA gene with further pathogenicity study. Thus, this study permitted the definition of a novel causal mutation for Fabry disease - [GLA] c.785G>T; p.W262L (exon 5).

Morphological aspects of cell reabsorption in laying queens and workers of Apis mellifera (Hymenoptera, Apidae)

The occurrence of cell reabsorption in the ovaries of queens in several rates of laying eggs, artificially impeded of laying, and in nurse workers, of Apis mellifera (Linnaeus, 1758), was studied with light (LM) and transmission electron microscopy (TEM). Two types of structures were described and named by analogy with vertebrates ovarian structures, as corpus luteus, when resulting from the reabsorption of the follicular cells after ovulation, and corpus atresicus when resulting from total follicular reabsorption at any oocyte developmental stage. These structures have the same morphological characteristics and physiological signification in both castes. The corpus luteus occurrence indicates ovulation and its number is correspondent to the queen's rates of oviposition. The presence of this structure in nurse workers ovarioles shows that this caste may lay eggs. The incidence of corpus atresicus in queens decay with the increasing of the oviposition indicating that the inhibition of the normal sequence of oocyte maturation in the ovaries is deleterious. Both, corpus luteus and corpus atresicus incidence may be influenced by environmental factors.

Studies on Trypanosoma rangeli Tejera, 1920: V - Developmental pattern in the alimentary canal of Rhodnius prolixus

The morphological sequence of Trypanosoma rangeli development in the alimentary canal of Rhodnius prolixus, is described, with observation made in dissected guts from 6 hours to 45 days post-infection. No metacyclic-forms are produced in the digestive tract at any time, and transmission by the contaminative route must be considered atypical. Amastigotes appear to be an essential stage in the development of T. rangeli in the gut of R. prolixus. The epidemiological importance of the developmental pattern of T. rangeli in the vector´s gut is discussed, and its usefulness for aging infection is considered.

Studies on Trypanosoma rangeli Tejera 1920: VI. Developmental pattern in the haemolymph of Rhodnius prolixus

The morphological sequence of Trypanosoma rangeli development in the body cavity of Rhodnius prolixus is described. The metacyclic trypanosome is the product of successive division and transformation during the intra and extracellular development in the haemocoele. The significance of the early invasion of T. rangeli into the haemolymph is discussed. The epidemiological importance of the developmental pattern of T. rangeli in the vectors haemolymph and the host-response to the parasite are considered.

Behavior of triatomines (Hemiptera: Reduviidae) vectors of Chagas' disease: I. Courtship and copulation of Panstrongylus megistus (Burm-1835) in the laboratory

A study of the courship and copulation behaviour of Panstrongylus megistus was carried out in the laboratory. fifty-five newly-fed virgin couples were used. Experiments were performed during the day (9:00 to 12:00 a.m.) and at night (7:00 to 10:00 p.m). Behaviour was recorded by direct observation and was found to consist of the following sequence of behavioral patterns: the male approached the female and jumped on her or mounted her; he took on a dorsolateral position and immobilized the female dorsally and ventrally with his three pairs of legs; the male genital was placed below those of the female; the paramers of the male immobilized the female's genitals; copulation started. The couple joined by the iniciative of the male. The female could be receptive and accept copulation, or nonreceptive and reject the male. Copulation occurred more often on the occasion of the first attempt by the male. Duration of copulation was X = 29.3 ± 9.3 min (CV = 83%). No behavioral differences were observed couples tested during the day or at night.

A new repetitive DNA sequence from Trypanosoma cruzi

Tandemly repeated DNA sequences are found in the genome of higher eukaryotes, and have also been demonstrated in Trypanosoma cruzi. Repeated DNA sequences are potentially useful for the diagnostic detection of T. cruzi (A. Gonzales et al., 1984, Proc. Natl. Acad. Sci. USA, 81: 3356-3360). We have isoleted two clones from a genomic library of T. cruzi (Y strain) that contain, in one clone a family of at least seven copies of a repetitive sequence of approximately 600 base pairs, and in the other an independent copy of the same sequence. One copy of the repetition (HSP) and the independent clone (HCR) were sequenced by the Sanger procedure (Fig.). This sequence hybridized to four strains of T. cruzi tested and did not hybridize to eleven species of trypanosotids from five different Genera, being a good candidate for diagnostic assays. GenBank accession numbers: HSP#m31919, HCR#31920.

New methods for the diagnosis of Babesia bigemina infection

Accurate diagnosis of Babesia bigemina infection, an economically important tick-transmitted protozoan parasite of cattle, is essential in the management of disease control and in epidemiological studies. The currentlyused methods of diagnosis are blood smear examination and serological tests which include agglutination and immunofluorescence tests. These testes have been used the fild but because they lack sensitivity and specificity, never and improved methods of diagnosis are being developed. The quantitative buffy coat (OBC) method, using microhaematocrit tubes and acridine orange staining allows rapid and quicker diagnosis of B. bigemina and other blood parasites compared to light microscopic examination of stained smears. Parasite specific monoclonal antibodies have been used in antigen/antibody capture enzymelinked immunosorbent assays with grater sensitivity and specificity than previously described serological tests. Similary, DNA probes, derived from a repetitive sequence of the B. bigemina genome, offer a method of detecting very small numbers of parasites which are undetectable by conventional microscopy. An extrachromosomal DNA element, present in all the tick-borne protozoan parasites so far tested, provides an accurate means of diferentiating mixed parasite populations in infected animals. These improved methods will greatly facilitate epidemiological studies.

Protection of aouts monkeys after immunization with recombinant antigens of Plasmodium falciparum

The genus Aotus spp. (owl monkey) is one of the WHO recommended experimental models for Plasmodium falciparum blood stage infection, especially relevant for vaccination studies with asexual blood stage antigens of this parasite. For several immunization trials with purified recombinant merozoite/schizont antigens, the susceptible Aouts kenotypes II, III, IV and VI were immunized with Escherichia coli derived fusion proteins containg partial sequences of the proteins MSAI (merozoite surface antigen I), SERP (serine-strech protein) and HRPII (histidine alanine rich protein II) as well as with a group of recombinant antigens obtained by an antiserum raised against a protective 41 kD protein band. The subcutaneous application (3x) of the antigen preparations was carried out in intact animals followed by splenectomy prior to challange, in order to increase the susceptibility of the experimental hosts to the parasite. A partial sequence of HRPII, the combination of three different fusion proteins of the 41 kD group and mixture of two sequences of SERP in the presence of the modified Al(OH)3 adjuvant conferred significant protection against a challange infection with P. falciparum blood stages (2-5 x 10 (elevado a sexta potência) i. RBC). Monkey immunized with the MS2-fusion protein carrying the N-terminal part of the 195 kD precursor of the major merozoite surface antigens induced only marginal protection showing some correlation between antibody titer and degree of parasitaemia. Based on the protective capacity of these recombinant antigens we have expressed two hybrid proteins (MS2/SERP/HRPII and SERP/MSAI/HRPII) in E. coli containing selected partial sequences of SERP, HRPII and MSAI. Antibodies raised against both hybrid proteins in rabbits and Aotus monkeys recognize the corresponding schizont polypeptides. In two independent immunization trials using 13 animals (age 7 months to 3 years) we could show that immunization of Aotus monkeys with either of the two hybrid proteins administered in an oil-based well tolerated formulation protected the animals frm a severe experimental P. falciparum (strain Palo Alto) infection.

Cytological and isoenzyme analysis of the Bucay and Quevedo cytotypes of the onchocerciasis vector Simulium exiguum (Diptera: Simuliidae) in Ecuador

Four cytotypes of Simulium exiguum occur in Ecuador, where this morphospecies is the primary vector of onchocerciasis. In this paper, we give the first full description of the banding pattern of the larval polytene chromosomes of the Quevedo cytotypes differ from the chromosomal standard sequence (of the Cayapa cytotype) by the fixed inversions IIL-5 and IIL-6. The Quevedo cytotype additionally differs from the standard and Bucay cytotypes by processing a differentiated X chromosome, wich is indicated by the inversion IIS-A. As the degree of reproductive isolation between the Bucay and Quevedo cytotypes has not yet been estabilished, they must be regarded as intraspecific variants of the same species. In fact, isoenzyme characterizations showed that the Bucay and Quevedo cytotypes are differentiated only to the extent expected of incipient species or geographical populations. Moreover, the sibiling species status previously given to the Bucay cytotype needs be reassessed, there being inadequate analysis from areas in Ecuador where Bucay occurs in sympatry with the standard Cayapa cytotype. No isoenzyme electromorphs were discovered that identified all or mostadult females of any one (cytotype-pure) collection.

A novel merozoite surface antigen of Plasmodium falciparum (MSP-3) identified by cellular-antibody cooperative mechanism antigenicity and biological activity of antibodies

We report the identification of a 48kDa antigen targeted by antibodies which inhibit Plasmodium falciparum in vitro growth by cooperation with blood monocytes in an ADCI assay correlated to the naturally acquired protection. This protein is located on the surface of the merozoite stage of P. falciparum, and is detectable in all isolates tested. Epidemiological studies demonstrated that peptides derived from the amino acid sequence of MSP-3 contain potent B and T-cell epitopes recognized by a majority of individuals living in endemic areas. Moreover human antibodies either purified on the recombinant protein, or on the synthetic peptide MSP-3b, as well as antibodies raised in mice, were all found to promote parasite killing mediated by monocytes.

Comparison of purified 12 kDa and recombinant 15 kDa Fasciola hepatica antigens related to a Schistosoma mansoni fatty acid binding protein

Vaccines in schistosomiasis using homologous antigens have been studied extensively in experimentally infected mammalian hosts. Vaccines using heterologous antigens have received comparatively less attention. This review summarizes recent work on a heterologous 12 kDa Fasciola hepatica antigenic polypeptide which cross reacts with Schistosoma mansoni. A cDNA has been cloned and sequenced, and the predicted amino acid sequence of the recombinant protein has been shown to have significant (44) identity with a 14 kDa S. mansoni fatty acid binding protein. Thus in the parasitic trematodes fatty acid binding proteins may be potential vaccine candidates. The F. hepatica recombinant protein has been overexpressed and purified and denoted rFh15. Preliminary rFh15 migrates more slowly (i.e. may be slightly larger) than nFh12 on SDS-PAGE and has a predicted pI of 6.01 vs. observed pI of 5.45. Mice infected with F. hepatica develop antibodies to nFh12 by 2 weeks of infection vs. 6 weeks of infection to rFh15; on the other hand, mice with schistosomiasis mansoni develop antibodies to both nFh12 and rFh15 by 6 weeks of infection. Both the F. hepatica and S. mansoni cross-reactive antigens may be cross-protective antigens with the protection inducing capability against both species.

An Experimental Approach to the Pathogenesis of "Pipestem" Fibrosis (Symmers' Fibrosis of the Liver)

Pathogenesis of schistosomal hepatic fibrosis ("pipestem" fibrosis of the liver) was investigated by means of the murine model. Although worm load appears as the main pathogenetic factor, alone it is not sufficient to produce that characteristic lesion. By comparing the findings in animals with heavy and prolonged Schistosoma mansoni infection, which developed or not" pipestem" fibrosis, it was observed that the lesion was more frequent in intact animals than in the splenectomized one. However, the size of the spleen, the number of recovered worms, the number of eggs per gram of liver tissue, the level of serum idiotype and anti-idiotype antibodies, the size and volume of periovular granulomas formed in the liver, all that failed to show statistically significant differences between the two groups. After analysing all these data, other factors, that apparently have been hitherto negleted, rested to explain the findings. Among them, the timing and sequence of the egg-induced intrahepatic vascular changes seemed crucial. The sequential development of intrahepatic portal vein obstruction, followed by the opening of periportal collateral veins and the continous arrival of schistosome eggs going to be lodged into the latter, appeared as essential steps in the pathogenesis of "pipestem" fibrosis

The Amazonia Variant of Vibrio cholerae: Molecular Identification and Study of Virulence Genes

The pathogenic O1 Amazonia variant of Vibrio cholerae has been shown previously to have a cytotoxin acting on cultured Vero and Y-1 cells, and to lack important virulence factors such as the cholera toxin (Coelho et al. 1995a). This study extends the molecular analysis of the Amazonia strains, detecting the presence of the toxR gene, with a very similar sequence to that of the El Tor and classical biotypes. The outer membrane proteins are analyzed, detecting a variation among the group of Amazonia strains, with three different patterns found. As a by-product of this work a polymerase chain reaction fragment was sequenced, reading part of the sequence of the Lon protease of the Amazonia strains. This gene was not previously described in V. cholerae, but its sequence is present in the TIGR database specific for this species.

Restriction fragment length polymorphism of 195 bp repeated satellite dna of Trypanosoma cruzi supports the existence of two phylogenetic groups

The restriction fragment length polymorphism of the 195 bp repeated DNA sequence of Trypanosoma cruzi was analyzed among 23 T. cruzi stocks giving a reliable picture of the whole phylogenetic variability of the species. The profiles observed with the enzymes Hinf I and Hae III were linked together and supported the existence of two groups. Group 1 shows a 195 bp repeated unit (Hinf I) and high molecular weight DNA (Hae III), while group 2 presents a ladder profile for each enzyme, which is a characteristic of tandemly repeated DNA. The two groups, respectively, clustered stocks pertaining to the two principal lineages evidenced by isoenzyme and RAPD markers. The congruence among these three independent genomic markers corroborates the existence of two real phylogenetic lineages in T. cruzi. The specific monomorphic profiles for each major phylogenetic lineage suggest the existence of ancient sexuality and cryptic biological speciation.

Plasmodium yoelii: identification of a gene encoding a putative ADP-ribosylation factor-1 GTPase-activating Protein, PyAG1

The PyAG1 gene, identified by the screening of a Plasmodium yoelii genomic DNA library with a rhoptry-specific Mab, encodes a protein with a zinc finger structure immediately followed by the consensus sequence of the Arf GAP catalytic site. The serum of mice immunized with the recombinant protein recognized specifically the rhoptries of the late infected erythrocytic stages. Blast analysis using the Genbank database gave the highest scores with four proteins presenting an Arf1 GAP activity. If presenting also this activity, the PyAG1 protein could be involved in the regulation of the secreted protein vesicular transport and, consequently, in the rhoptry biogenesis.