Seropositivity for and Intestinal Colonization with Entamoeba histolytica and Entamoeba dispar in Individuals in Northeastern Brazil

In a slum community in northeastern Brazil 20% of a sample population was colonized with Entamoeba histolytica or Entamoeba dispar and 10.6% was colonized with E. histolytica alone. No correlation between seropositivity for anti-GalNAc lectin antibody and colonization was found. These results suggest that colonization does not necessarily produce immunity to reinfection.     

Immunoglobulins and other serological parameters in Chagas'' disease: evidence for increased IgA levels in the chronic digestive form.

Immunoglobulin levels were measured in serum samples from 36 patients with different clinical forms of chronic Chagas' disease. Increased IgA levels were observed in 50% of the patients in the chronic digestive group and there was a significant correlation with the severity of the disease. IgG and IgM levels were within the normal range. Anti-ssDNA antibodies and EVI (endothelium, vessels and interstitium) antibodies were found in some patients with different clinical forms of the disease.     

Kinetics of estimated human muscle capillary blood flow during recovery from exercise

The kinetic characteristics of muscle capillary blood flow (Qcap) during recovery from exercise are controversial (e.g. one versus two phases). Furthermore, it is not clear how the overall Qcap kinetics are temporally associated with muscle oxygen uptake (VO2m) kinetics. To address these issues, we examined the kinetics of Qcap estimated from the rearrangement of the Fick equation (Qcap=VO2m/C(a-v)O2) using the kinetics of pulmonary VO2 (VO2p, primary component) and deoxy-haemoglobin concentration ([HHb]) as indices of VO2m and C(a - v)O2 (arterio-venous oxygen difference) kinetics, respectively. VO2p (l min-1) was measured breath by breath and [HHb] (microm) was measured by near infrared spectroscopy during moderate (M; below lactate threshold, LT) and heavy exercise (H, above LT) in nine subjects. The kinetics of Qcap were biphasic, with an initial fast phase (tauI; M=9.3+/-4.9 s and H=6.0+/-3.8 s) followed by a slower phase 2 (tauP; M=29.9+/-8.6 s and H=47.7+/-26.0 s). For moderate exercise, the overall kinetics of Qcap (mean response time [MRT], 36.1+/-8.6 s) were significantly slower than the kinetics of VO2p (tauP; 27.8+/-5.3 s) and [HHb] (MRT for [HHb]; 16.2+/-6.3 s). However, for heavy exercise, there was no significant difference between MRT-[HHb] (34.7+/-10.4 s) and tauP for VO2p (32.3+/-6.7 s), while MRT for Qcap (48.7+/-21.8 s) was significantly slower than MRT for [HHb] and tauP for VO2p. In conclusion, during recovery from exercise the estimated Qcap kinetics were biphasic, showing an early rapid decrease in blood flow. In addition, the overall kinetics of Qcap were slower than the estimated VO2m kinetics.     

Determination of Blomia tropicalis-specific IgE and IgG subclasses in atopic dermatitis patients

BACKGROUND: To verify the importance of Blomia tropicalis in atopic dermatitis (AD), we determined the cutaneous reactivity and the serum level of B. tropicalis-specific IgE and IgG subclasses in AD patients. METHODS: B. tropicalis-specific IgE and IgG subclasses were determined in AD patients and compared with bronchial asthma (BA) patients and a control group (CG) of nonatopic subjects. Specific IgE was obtained by skin prick test and RAST. B. tropicalis-specific IgG subclasses were determined by ELISA. The data were statistically analyzed by chi-square test (Mantel-Haenszel) and odds ratio (OR). RESULTS: We detected positive skin prick tests in 61.76% of AD and 83.33% of BA patients, and in 12.5% of the CG. RAST was positive in 44.12% of AD and in 61.90% of BA patients, but not in the CG. B. tropicalis-specific IgG1 and IgG2 subclasses showed no significant differences between the three groups. IgG3 subclass positivity was statistically significant in AD patients (41.17%) when compared to BA patients (14.29%) and the CG (16.67%). The determination of B. tropicalis-specific IgG4 was positive in 32.35% of AD patients, 21.43% of BA patients, and 8.33% of the CG. CONCLUSIONS: These results confirm that the storage mite B. tropicalis is an important allergen in AD. It is possible that IgG3 activates the complement in AD patients, releasing vasoactive amines that further amplify the allergic reaction. The positive results of the B. tropicalis-specific IgG4 found in AD and BA were probably due to chronic exposure to this storage mite in the home environment.     

Immunomodulation Induced by Ascaris suum Extract in Mice: Effect of Anti-Interleukin-4 and Anti-Interleukin-10 Antibodies

Simultaneous immunization of mice with an Ascaris suum extract (Asc) and ovalbumin (OA) markedly affects the immune response to OA. The role of interleukin (IL)-4 and IL-10 induced by Asc immunization on the modulation of antigen-specific and mitogen-induced responses was investigated following single or combined cytokine-specific monoclonal antibody (MoAb) treatment of mice before immunization with OA + Asc. Immediate hypersensitivity reactions to aggregated OA and OA-specific immunoglobulin (Ig)G2a antibody production were completely restored only when both IL-4 and IL-10 were neutralized. These findings were associated with enhanced interferon (IFN)-γ secretion by OA-stimulated lymph node (LN) cells. In addition, the Asc-specific cytokine response in anti-IL-4 plus anti-IL-10 MoAb treated mice was shifted towards a Th1 phenotype, with an increase in IFN-γ and IL-2 levels and a decrease in IL-4, but not in IL-10, levels. Consequently, Asc-specific IgG2a antibody production increased, whereas IgE titres diminished in these animals. These results indicate that IL-4 and IL-10 act together in the Asc-induced mechanism of antigen-specific pansuppression. In contrast, modulation of Concanavalin A (Con A)-induced cytokine responses in Asc-immunized mice appears to be essentially mediated by an IL-4-dependent mechanism, since the neutralization of just IL-4 (and not of IL-10), either in vivo or in vitro , changed the cytokine profile from a Th2 towards a Th1 type. However, OA and Asc-specific cell responses were not modified by either anti-IL-4 or by anti-IL-4 + anti-IL-10 MoAbs in vitro treatments, suggesting that the induction of a Th2 response to Asc components concomitant to OA immunization has a strong suppressive effect on the priming stage of OA-specific Th1 type response.     

Molecular cloning and characterization of actin genes from Echinococcus granulosus

An Echinococcus granulosus genomic library has been screened with a mouse β-actin cDNA probe. Two clones carrying DNA fragments of about 15 kb, possibly derived from the same genome region, have been isolated. This 15-kb genomic region includes 2 actin-related sequences (EgactI and EgactII) separated by about 4 kb. The nucleotide sequences of both genes were determined. The EgactI sequence presents no introns, but an intron of 591 bp was observed in the EgactII sequence. The genes potentially encode 375 and 376 amino-acid-long actins, respectively, with a homology of 85.3%. The deduced amino acid sequences from both genes were compared to the actin sequences from other organisms, showing similarities ranging from 63.5% to 90.6%. The nucleotide sequence of a partial actin cDNA clone has been determined. The deduced amino acids sequence showed a homology of 90.3% and 88.0% in relation to the EgactI and EgactII sequences respectively, suggesting the existence of at least one more actin gene in E. granulosus. This hypothesis is reinforced by the number of bands detected in the Southern blot analysis. Experiments based on the amplification of DNA segments using 3′-specific actin primers indicate that the EgactI gene is transcribed in protoscoleces.     

Presence of the cfxA gene in Bacteroides distasonis

In this study we investigated the presence of the cfxA gene (encoding a class A beta-lactamase) in 73 strains of the Bacteroides fragilis group belonging to the species B. distasonis (34), B. vulgatus (14), B. thetaiotaomicron (8), B. merdae (6), B. caccae (9) and B. ovatus (2) isolated from human intestinal microflora of healthy children and adults. Employing specific primers to the cfxA gene, a 312-bp amplified fragment was obtained in 2 strains of B. vulgatus and 9 strains, the majority from children, of B. distasonis. The expression of this enzyme was analysed by determining the MICs to cefoxitin and cefotaxime and values varied from 2 to >256 microg/ml of both cefoxitin and cefotaxime. Sequence analysis of the amplicons corresponding to the cfxA gene from B. distasonis and B. vulgatus revealed identical sequences between these isolates and high similarity with other beta-lactamase genes of anaerobes such as cfxA of B. vulgatus (99%) and cfxA2 of Prevotella intermedia (99%), both sequences of which deposited in Genbank under accession numbers U38243 and AF118110, respectively. However, a fragment obtained from a B. distasonis strain (EC17-4) showed a unique RFLP profile and 87% nucleotide similarity with cfxA and cfxA2 genes. These results seem to suggest a dissemination of these resistance determinants among Bacteroides species.     

Molecular analysis of Bruton’s tyrosine kinase gene in Spain

Mutations in Bruton’s tyrosine kinase (BTK) gene result in X linked agammaglobulinemia (XLA). Using Single Strand Conformation Polymorphism (SSCP) followed by direct sequencing 21 mutations were found in 27 patients with an XLA phenotype from 21 unrelated families. We identified 13 novel and 8 known mutations: seven missense (R288W, R544G, P566S, K430E; K374N, L512P, R544S), 5 nonsense (Q196X, Y361X, L249X, Q612X, Q466X), 2 deletions of one nucleotide (A207fsX216, Q612fsX648), 2 deletion‐insertions (V219fsX227, K218fsX228), one insertion of two nucleotides (S572fsX587) and 4 point mutations in donor/acceptor splice sites (g.IVS1+1G>C, g.IVS6+5G>A, g.IVS10+1G>T, g.IVS13‐1GG>CT). Carrier detection was performed in 18 mothers. Only in one case the mutation was found to be de novo. Additionally, BTK mutations were not found in four patients without family history, but with XLA‐compatible phenotype. Hum Mutat 18:84, 2001. © 2001 Wiley‐Liss, Inc.     

E-cadherin germline missense mutations and cell phenotype: evidence for the independence of cell invasion on the motile capabilities of the cells

In Hereditary Diffuse Gastric Cancer syndrome, E-cadherin germline mutations of the missense type harbour significant functional consequences. In this study, we have characterised the effect of T340A, A617T, A634V and V832M E-cadherin germline missense mutations on cell morphology, motility and proliferation. Wild-type E-cadherin and A617T expressing cells have an epithelial-like morphology, with polarised cells migrating unidirectionally. T340A and A634V expressing cells, fibroblast-like, have a high motile phenotype. We show that this phenotype is dependent on an increased level of active RhoA. V832M expressing cells grow in piled-up structure of round cells, as an effect of the disturbance of the binding between alpha-catenin and beta-catenin. The destabilisation of the adhesion complex is shown to hamper the motile capabilities of these cells. We did not observe any effect of the E-cadherin mutations on cell proliferation. We show the existence of a genotype-phenotype correlation between different E-cadherin mutations and cell behaviour. However, we demonstrate that the ability of cells expressing the different E-cadherin mutations to invade is independent on their motile capabilities, providing evidence that motility is neither necessary nor sufficient for cells to invade. Our data give new insights into the understanding of the mechanisms linking invasion and E-cadherin mutations in diffuse gastric cancer.     

Sequential Flow Cytometry and Fluorescence In Situ Hybridization for the Study of Formalin-Fixed, Paraffin-Embedded Breast Cancer Cells

Both flow cytometry and fluorescence in situ hybridization (FISH) are useful techniques in the analysis of cancer tissues. When the two are used in the study of the same specimens, they are usually performed in parallel, separately. This is problematic where there is a scarcity of material, making completion of both studies impossible. Fluorescence in situ hybridization procedures that will utilize excess material discarded from flow cytometry would be advantageous. The present report describes an optimized protocol for performing sequential flow cytometry and FISH using formalin-fixed paraffin-embedded archival material. Although breast cancer tissues were used in this initial study, the protocol is applicable to other cancer tissues as well.