Biomedical subjects

A J da Silva

Publications and source records attributed to A J da Silva.

At least 19 recordsLinked to original sources

PubMed2005

Cellular and complement-dependent cytotoxicity of Ep-CAM-specific monoclonal antibody MT201 against breast cancer cell lines.

MT201 is a fully human monoclonal IgG1 antibody with moderate affinity for epithelial cell adhesion molecule (Ep-CAM) being clinically developed for the treatment of carcinomas. Like many other clinically validated IgG1 monoclonal antibodies, MT201 primarily acts by antibody-dependent cellular cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC). Here, we analysed ADCC and CDC induced by MT201 and, as reference, trastuzumab against a panel of nine human breast cancer cell lines expressing distinct surface levels of Ep-CAM and human epithelial growth factor receptor type 2 antigen. Maximal cell lysis by ADCC by MT201 and trastuzumab in the presence of peripheral mononuclear cells did not significantly differ when averaged over the nine cell lines, but showed marked differences with respect to individual cell lines. The extent of cell lysis at intermediate surface target density was highly variable, suggesting a dominant influence of other susceptibility factors. Only one breast cancer cell line was eliminated via CDC, but only by MT201. Resistance to CDC appeared to correlate with high expression levels of complement resistance factors. Our present data as well as recent data on the prevalence and prognostic relevance of Ep-CAM expression in metastatic breast cancer suggest that Ep-CAM-specific monoclonal IgG1 antibodies may have a significant therapeutic potential in the treatment of breast cancer.

Animals↗

PubMed2001

How do gold nanowires break?

Suspended gold nanowires have recently been made in an ultrahigh vacuum and were imaged by electron microscopy. Using realistic molecular dynamics simulation, we study the mechanisms of formation, evolution, and breaking of these atomically thin Au nanowires under stress. We show how defects induce the formation of constrictions that eventually will form the one-atom chains. We find that these chains, before breaking, are five atoms long, which is in excellent agreement with experimental results. After the nanowire's rupture, we analyze the structure of the Au tip, which we believe will be universally present due to its highly symmetric nature.

Journal Article↗

PubMed2001

O2 diffusion in SiO2: triplet versus singlet.

We address the diffusion of the oxygen molecule in SiO2, using first-principles spin-polarized total-energy calculations. We find that the potential energy surfaces for the singlet and triplet states are very different in certain regions, and that the O2 molecule preserves its spin-triplet ground state not only at its most stable interstitial position inside the solid but also throughout its diffusion pathway. Therefore, the singlet state is not a good approximation to describe the behavior of O2 inside SiO2, and spin-polarization effects are fundamental to understand the properties of this system.

Journal Article↗

PubMed2001

Two-atom structures of Ge on Si(100): dimers versus adatom pairs.

We present an ab initio study of the properties of structures composed of two and four Ge atoms adsorbed on the troughs of the Si(100) surface, and we conclude that these structures are all composed of dimers, with a chemical bonding between the adatoms. We compare our calculated local density of states with scanning tunneling microscope (STM) images, and we show that these Ge dimers adsorbed on the troughs between the substrate dimer rows can be identified with the adatom pairs observed experimentally. We also show that the local buckling of the substrate dimers can give rise to similar structures with very different STM images.

Journal Article↗

PubMed2001

Outbreak of cyclosporiasis associated with basil in Missouri in 1999.

During the summer of 1999, an outbreak of cyclosporiasis occurred among attendees of 2 events held on 24 July in different counties in Missouri. We conducted retrospective cohort studies of the 2 clusters of cases, which comprised 62 case patients. The chicken pasta salad served at one event (relative risk [RR], 4.25; 95% confidence interval [CI], 1.80-10.01) and the tomato basil salad served at the other event (RR, 2.95; 95% CI, 1.72-5.07) were most strongly associated with illness. The most likely vehicle of infection was fresh basil, which was included in both salads and could have been grown either in Mexico or the United States. Leftover chicken pasta salad was found to be positive for Cyclospora DNA by means of polymerase chain reaction analysis, and 1 sporulated Cyclospora oocyst was found by use of microscopy. This is the second documented outbreak of cyclosporiasis in the United States linked to fresh basil and the first US outbreak for which Cyclospora has been detected in an epidemiologically implicated food item.

Adolescent↗

PubMed2000

Chemical constituents of two Mollinedia species.

A butanolide, marliolide, was isolated from the hexane extract of leaves of Mollinedia marliae (Monimiacae) along with the long chain fatty alcohol hexacosanol. Phytol and a mixture of sitosterol and stigmasterol were also isolated from leaves of both M. marliae and M. gilgiana. trans-N-Feruloyltyramine, a ferulic acid derivative, is the major constituent of the stems of M. gilgiana.

Magnetic Resonance Spectroscopy↗

PubMed2000

Morphologic, host specificity, and molecular characterization of a Hungarian Cryptosporidium meleagridis isolate.

This study was undertaken in order to characterize Cryptosporidium meleagridis isolated from a turkey in Hungary and to compare the morphologies, host specificities, organ locations, and small-subunit RNA (SSU rRNA) gene sequences of this organism and other Cryptosporidium species. The phenotypic differences between C. meleagridis and Cryptosporidium parvum Hungarian calf isolate (zoonotic genotype) oocysts were small, although they were statistically significant. Oocysts of C. meleagridis were successfully passaged in turkeys and were transmitted from turkeys to immunosuppressed mice and from mice to chickens. The location of C. meleagridis was the small intestine, like the location of C. parvum. A comparison of sequence data for the variable region of the SSU rRNA gene of C. meleagridis isolated from turkeys with other Cryptosporidium sequence data in the GenBank database revealed that the Hungarian C. meleagridis sequence is identical to a C. meleagridis sequence recently described for a North Carolina isolate. Thus, C. meleagridis is a distinct species that occurs worldwide and has a broad host range, like the C. parvum zoonotic strain (also called the calf or bovine strain) and Cryptosporidium felis. Because birds are susceptible to C. meleagridis and to some zoonotic strains of C. parvum, these animals may play an active role in contamination of surface waters not only with Cryptosporidium baileyi but also with C. parvum-like parasites.

Animals↗

PubMed1999

Novel isoform of lymphoid adaptor FYN-T-binding protein (FYB-130) interacts with SLP-76 and up-regulates interleukin 2 production.

T-cell activation involves the participation of protein-tyrosine kinases p56(lck) and ZAP-70/SYK as well as lymphoid proteins such as SLP-76 and FYB/SLAP. FYB/SLAP has the hallmarks of an adaptor protein that binds to the SH2 domains of the Src kinase FYN-T and SLP-76. Whereas two forms of FYB at 120 and 130 kDa have been identified biochemically, a cDNA encoding only the lower molecular weight isoform has been cloned (termed FYB-120 or SLAP-130). In this study, we report the isolation of an alternative isoform of FYB with a molecular mass of 130 kDa (FYB-130) that has the same structure as FYB-120 except for an insertion of 46 amino acids toward the carboxyl-terminal region of the protein. FYB-120 and FYB-130 share an ability to bind to the SH2 domains of FYN-T and SLP-76, to act as substrates for p59(FYN-T), and to be expressed in the cytoplasm and nucleus of T-cells. Differences were noted between the isoforms in the efficiency of binding to SLP-76 and in the preferential expression of FYB-130 in mature T-cells. When co-expressed together with FYN-T and SLP-76, FYB-130 caused a significant increase in anti-CD3-driven NF-AT transcription. Finally, fluorescence in situ hybridization analysis localized the FYB gene to human chromosome 5 at position p13.1. FYB-130 therefore represents a novel variant of FYB protein that can up-regulate T-cell receptor-driven interleukin 2 production in mature T-cells.

Adaptor Proteins, Signal Transducing↗

PubMed1999

Immunosuppressive and anti-inflammatory effects of methanolic extract and the polyacetylene isolated from Bidens pilosa L.

The immunomodulatory effect of the methanolic extract obtained from dried leaves of Bidens pilosa L. (Asteraceae) and the polyacetylene 2-O-beta-D-glucosyltrideca-11 E-en-3,5,7,9-tetrayn-1,2-diol (PA-1) isolated from it was investigated. The extract inhibited the proliferative response in two in vitro models: human lymphocytes stimulated by 5 microg ml(-1) phytohemagglutinin (PHA) or to 100 nM 12-O-tetradecanoyl phorbol-13-acetate (TPA) plus 0.15 microM ionomycin and murine lymphocytes stimulated by 5 microg ml(-1) concanavalin A (Con A) or in the mixed leukocyte reaction (IC50 = 12.5 to 25 microg ml(-1)). PA-1 was 10-told more potent than the original extract in blocking both human and murine lymphocyte proliferation (IC50 = 1.25 to 2.5 microg ml(-1)). In mice, the intraperitoneal (i.p.) administration of methanolic extract of B. pilosa significantly reduced the size of the popliteal lymph node (PLN) after the inflammation induced by zymosan. One week after the injection of zymosan (150 microg) in the foot pad, PLN weighed 4.6 +/- 0.6 mg in comparison with 0.5 +/- 0.07 mg of the contralateral non-inflamed foot pad. The i.p. treatment with 10 mg extract from day 2 to day 6 after zymosan injection reduced the PLN weight to 1.8 +/- 0.3 mg. The data suggest an immunosuppressive activity of components of B. pilosa that may explain its popularly perceived anti-inflammatory effect.

Acetylene↗

PubMed1999

Fast and reliable extraction of protozoan parasite DNA from fecal specimens.

BACKGROUND: Polymerase chain reaction (PCR) detection of intestinal protozoa in fecal specimens is hampered by poor recovery of DNA and by the presence of PCR inhibitors. In this study we describe a novel method for DNA extraction from such specimens containing spores and oocysts of Enterocytozoon bieneusi and Cryptosporidium parvum, respectively. METHODS AND RESULTS: Extraction was done using commercial kits modified to maximize the recovery and purity of extracted DNA. In comparison with a procedure we previously reported, we estimate that this method may increase the sensitivity of parasite DNA detection in fecal specimens up to tenfold. An additional advantage of this method is that up to 12 samples may be processed simultaneously within 2 hours. CONCLUSIONS: By using this method, we were able to increase reproducibility of PCR amplification on fecal specimens and significantly reduce the hands-on time required to process the samples.

Animals↗

PubMed1999

Identification of Cryptosporidium felis in a cow by morphologic and molecular methods.

Apicomplexan Cryptosporidium parasites infect a wide range of vertebrate hosts. While some species are limited to a single host group, such as Cryptosporidium baileyi, which infects chickens, other species of this genus, such as C. parvum, infect a wide range of mammalian species from mice to humans. During an investigation of Cryptosporidium infection in cattle on a farm in northern Poland, we identified an infection caused by C. felis, in addition to known infections with C. muris and C. parvum. This new infection was identified based on the size of the oocysts (mean size, 4.3 +/- 0.4 micrometer; range, 3.5 to 5.0 micrometer), as well as by analysis of the molecular sequence of the variable region of the small-subunit rRNA. This finding demonstrates the complex host specificity and circulation in the environment of Cryptosporidium species.

Animals↗

PubMed1999

Cyclospora cayetanensis infections in Haiti: a common occurrence in the absence of watery diarrhea.

Stool samples from a population-based cohort of mothers and children living in Leogane, Haiti were tested for Cyclospora cayetanensis from January 1997 through January 1998. Data on gastrointestinal symptoms were also collected. During the winter months of January to March, the infection was detected in 15-20% of the persons sampled. Most infections did not appear to be causing diarrhea and most infected persons had few oocysts detectable in concentrates of stool. The infection appears to have marked seasonality, with highest rates during the driest and coolest time of the year. It may be that in this tropical setting, high summer temperature is the critical environmental factor that influences the seasonality of infection. This study demonstrates that Cyclospora infections in Haiti are common in the general population.

Adolescent↗

PubMed1998

FYB (FYN binding protein) serves as a binding partner for lymphoid protein and FYN kinase substrate SKAP55 and a SKAP55-related protein in T cells.

TcRzeta/CD3 ligation initiates a signaling cascade involving CD4/CD8-p56(lck), p59(fyn), and ZAP-70, as well as lymphoid downstream proteins VAV, SLP-76, and FYB/SLAP. A current question concerns the nature of the downstream binding partner(s) of FYB in T cells. In this study, using a two-hybrid screen with FYB as bait, we have identified eight clones, four of which correspond to the recently published lymphoid protein SKAP55, and two which correspond to a related protein with some 44% homology to SKAP55 (termed SKAP55-related protein, SKAP55R). The SKAP55 clones showed only minor differences (two substitutions and one residue deletion) from SKAP55. SKAP55R has the same overall structure as SKAP55 except for the presence of a unique N terminus with a well-defined coiled-coil domain. Both SKAP55 and SKAP55R were found to bind FYB through their SH3 domains and to act as substrates for the FYN kinase in T cells. Furthermore, immunofluorescence confocal microscopy showed that FYB and SKAP55 colocalize in the perinuclear region of cells. SKAP55 also colocalizes with another FYB binding protein, SLP-76. Taken together, these observations demonstrate that FYB is part of an interactive matrix with SKAP55 and a SKAP55-related protein.

Adaptor Proteins, Signal Transducing↗

PubMed1998

Detection of Enterocytozoon bieneusi in two human immunodeficiency virus-negative patients with chronic diarrhea by polymerase chain reaction in duodenal biopsy specimens and review.

Intestinal microsporidiosis has been associated traditionally with severely immunocompromised patients with AIDS. We describe two new cases of intestinal microsporidiosis due to Enterocytozoon bieneusi in human immunodeficiency virus-negative adults. Both patients presented with chronic nonbloody diarrhea, and one had intestinal lymphangiectasia as well. Intestinal microsporidiosis was diagnosed by evaluation of stool samples, and the specific species was determined by use of polymerase chain reaction (PCR) in duodenal biopsy specimens. To our knowledge, this is the first report of confirmation of E. bieneusi in the intestinal epithelium of HIV-negative individuals by use of PCR in duodenal biopsy specimens. Cases of intestinal microsporidiosis in HIV-negative individuals reported in the English-language literature are reviewed. These two new cases along with those described previously corroborate the need to evaluate for microsporidia in HIV-negative individuals with unexplained diarrhea.

Adult↗

PubMed1998

Immunologic, microscopic, and molecular evidence of Encephalitozoon intestinalis (Septata intestinalis) infection in mammals other than humans.

Encephalitozoon intestinalis (Septata intestinalis) is the second most prevalent microsporidian species infecting humans, but it has not been described in other animal species. This investigation examined 10 domestic animal stool samples (8 mammalian, 2 avian) containing spores detected by anti-Encephalitozoon monoclonal antibody immunofluorescence (FA). The presence of E. intestinalis but not Encephalitozoon hellem or Encephalitozoon cuniculi was confirmed in 6 of 8 mammalian stool samples by species-specific FA and polymerase chain reaction. Clusters of spores inside epithelial cells were observed in feces of five mammals (donkey, dog, pig, cow, and goat) using "quick-hot" Gram-chromotrope stain. None of the 10 samples reacted with anti-E. hellem or anti-E. cuniculi sera, nor were they amplified with species-specific primers for E. hellem and E. cuniculi. To our knowledge, this is the first identification of E. intestinalis in animals other than humans. The data shown herein suggest the possibility that E. intestinalis infection may be zoonotic in origin.

Animals↗

PubMed1998

Characterization of natural occurring Pneumocystis carinii pneumonia in pigs by histopathology, electron microscopy, in situ hybridization and PCR amplification.

Macroscopic, histologic, ultrastructural, microbiologic, in situ hybridization (ISH) and PCR detection results in three 8-week-old pigs naturally infected with Pneumocystis carinii (PC) are described. All animals had a nonsuppurative interstitial pneumonia and intra-alveolar Pneumocystis organisms with foamy eosinophilic and PAS positive appearance. Ultrastructurally, PC trophozoites and cysts were observed in pigs No. 2 and No. 3, with the former being much more numerous. PC organisms were located on the alveolar surface or within the alveolar septa. Trophozoites had numerous filopodia and were thick-walled. Cysts had no or few filopodia, were thick-walled and contained intracystic bodies. Using non-isotopic ISH on formalin-fixed, paraffin-embedded lung tissue sections, PC DNA from pigs No. 2 and No. 3 hybridized with a probe specific for PC ribosomal RNA (rRNA). Using primers specific for mitochondrial rRNA gene (pAZ102-E/pAZ102-H), and for the internal transcriber spacers of ribosomal gene of PC, PCR methods amplified a product in the lung of pigs No. 2 and No. 3 using either frozen or formalin-fixed and paraffin-embedded lung tissue. DNA from Pig No. 1 samples did not amplify with any primer. This is the first time that molecular biology techniques (in situ hybridization and PCR) have been applied to the study of porcine pneumocystosis.

Animals↗

PubMed1997

Cloning of a novel T-cell protein FYB that binds FYN and SH2-domain-containing leukocyte protein 76 and modulates interleukin 2 production.

T cell receptor zeta (TcRzeta)/CD3 ligation initiates a signaling cascade that involves src kinases p56(lck) and zeta-associated protein 70, leading to the phosphorylation of substrates such as TcRzeta, Vav, SH2-domain-containing leukocyte protein 76 (SLP-76), cbl, and p120/130. FYN binding protein (FYB or p120/130) associates with p59(fyn), the TcRzeta/CD3 complex, and becomes tyrosine-phosphorylated in response to receptor ligation. In this study, we report the cDNA cloning of human and murine FYB and show that it is restricted in expression to T cells and myeloid cells and possesses an overall unique hydrophilic sequence with several tyrosine-based motifs, proline-based type I and type II SH3 domain binding motifs, several putative lysine/glutamic acid-rich nuclear localization motifs, and a SH3-like domain. In addition to binding the src kinase p59(fyn), FYB binds specifically to the hematopoietic signaling protein SLP-76, an interaction mediated by the SLP-76 SH2 domain. In keeping with this, expression of FYB augmented interleukin 2 secretion from a T cell hybridoma, DC27.10, in response to TcRzeta/CD3 ligation. FYB is therefore a novel hematopoietic protein that acts as a component of the FYN and SLP-76 signaling cascades in T cells.

Adaptor Proteins, Signal Transducing↗

PubMed1997

Biochemical analysis of p120/130: a protein-tyrosine kinase substrate restricted to T and myeloid cells.

T cell activation is mediated by a cascade of intracellular events involving protein-tyrosine kinases and their substrates. p56(lck) and p59(fyn) are protein-tyrosine kinases that associate with CD4/CD8 and the TCRzeta/CD3 complex, respectively. We previously reported the appearance of a protein doublet at 120 and 130 kDa that preferentially associates with p59(fyn) and undergoes tyrosine phosphorylation upon receptor ligation. In this paper, we demonstrate that p120/130 is a novel protein that is restricted in expression to T cells, thymocytes and myeloid cells. Internal peptide sequencing and immunoblotting using an anti-p120/130 antisera showed that p120/130 is a unique protein that is distinct from p130(cas) and p125(cbl). By contrast, p120 and p130 shared similar peptide patterns and are structurally related. Alkaline phosphatase digestion of precipitates showed that they are not related due to phosphorylation. p120/130 was found to associate constitutively with a 55-kDa protein of unknown identity, but which is distinct from p56(lck) and Shc. p120/130 also undergoes a unique kinetics of phosphorylation and associates with the Ag receptor in response to TCR ligation. In keeping with the association with p59(fyn), T cells from p59(fyn)-negative mice exhibit reduced phosphorylation of the protein. p120/130 therefore represents a novel TCR associated intracellular molecule with potential to play a role in T cell signaling.

Amino Acid Sequence↗