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Biomedical subjects

A Fasano

Publications and source records attributed to A Fasano.

At least 55 records · Page 3Linked to original sources
PubMed2000

All that scallops is not celiac disease.

BACKGROUND: Scalloping of duodenal folds as well as a mosaic mucosal pattern, decreased folds, and increased vascularity are markers of duodenal mucosal injury, the most common cause being celiac disease. We have recognized scalloping in patients with a variety of conditions other than celiac disease. METHODS: Clinical, endoscopic and histologic data were reviewed from selected patients with endoscopically visualized scalloped folds along with testing for endomysial antibodies. Biopsy specimens were examined histologically for villous:crypt ratio, intraepithelial lymphocytes, and inflammation. RESULTS: Thirteen patients with scalloped folds underwent endoscopy for the following reasons: family history of celiac disease and osteoporosis, gastrointestinal bleeding, dyspepsia (2), B(12)/ folate deficiency (4), and diarrhea (8). Histologic examination was abnormal in all but 1 patient. Villous atrophy or flattening as evidenced by reduced villous:crypt ratio was seen in 11 of 13 patients. Other abnormalities were edematous or broadened villi (10), intraepithelial lymphocytosis (7), and infiltration of lamina propria (6). An infectious organism was identified in 6 patients (46%). Celiac disease was excluded by the lack of specific biopsy findings combined with endomysial antibody testing. Final diagnoses were normal (1), eosinophilic enteritis (1), giardiasis (1), tropical sprue (4), human immunodeficiency virus-related diseases (6) including human immunodeficiency virus enteropathy (1). CONCLUSION: We conclude that scalloping is not specific for celiac disease but rather a predictor of mucosal disease as evidenced by villous atrophy, widening, and edema.

Biopsy↗
PubMed2000

Human recombinant tissue transglutaminase ELISA: an innovative diagnostic assay for celiac disease.

OBJECTIVE: Tissue transglutaminase is the autoantigen recognized by the sera of celiac patients. An enzyme-linked immunosorbent assay (ELISA) based on guinea-pig tissue transglutaminase was recently used to measure serum tissue transglutaminase antibodies for the diagnosis of celiac disease. We determine the sensitivity and specificity of an ELISA test based on the use of human recombinant transglutaminase, compared with the guinea pig transglutaminase ELISA and IgA antiendomysium antibodies. METHODS: Serum samples were tested from 65 patients with intestinal biopsy proven celiac disease, from 10 patients with Crohn's disease, and from 150 healthy blood donors. RESULTS: Human transglutaminase ELISA identified 64 of 65 celiac patients, whereas the guinea pig transglutaminase ELISA and IgA antiendomysium antibodies identified 58 of 65 and 60 of 65 subjects, respectively. The three tests showed comparable specificity. CONCLUSIONS: These results proved that the human tissue transglutaminase-based ELISA represents a cost-effective strategy for identifying both symptomatic and atypical forms of celiac disease and could mean that intestinal biopsy need no longer be the gold standard for diagnosing this clinical condition. Furthermore, early identification and treatment of patients with celiac disease in an outpatient setting could have significant implications for reducing long-term morbidity and can produce major savings in future health care costs.

Adolescent↗
PubMed2000

Regulation of intercellular tight junctions by zonula occludens toxin and its eukaryotic analogue zonulin.

The intestinal epithelium represents the largest interface between the external environment and the internal host milieu and constitutes the major barrier through which molecules can either be absorbed or secreted. There is now substantial evidence that tight junctions (tj) play a major role in regulating epithelial permeability by influencing paracellular flow of fluid and solutes. Tj are one of the hallmarks of absorptive and secretory epithelia. Evidence now exists that tj are dynamic rather than static structures and readily adapt to a variety of developmental, physiological, and pathological circumstances. These adaptive mechanisms are still incompletely understood. Activation of PKC either by Zonula occludens toxin (Zot) or by phorbol esters increases paracellular permeability. Alteration of epithelial tj is a recently described property for infectious agents. Clostridium difficile toxin A and B and influenza and vesicular stomatitis viruses have been shown to loosen tj in tissue culture monolayers. Unlike what occurs after the Zot stimulus, these changes appear to be irreversible and are associated with destruction of the tj complex. On the basis of this observation, we postulated that Zot may mimic the effect of a functionally and immunologically related endogenous modulator of epithelial tj. We were able to identify an intestinal Zot analogue, which we named zonulin. It is conceivable that the zonulins participate in the physiological regulation of intercellular tj not only in the small intestine, but also throughout a wide range of extraintestinal epithelia as well as the ubiquitous vascular endothelium, including the blood-brain barrier. Disregulation of this hypothetical zonulin model may contribute to disease states that involve disordered intercellular communication, including developmental and intestinal disorders, tissue inflammation, malignant transformation, and metastasis.

Cholera Toxin↗
PubMed2000

IS1414, an Escherichia coli insertion sequence with a heat-stable enterotoxin gene embedded in a transposase-like gene.

Enteroaggregative Escherichia coli (EAEC) heat-stable enterotoxin 1 (EAST1) was originally discovered in EAEC but has also been associated with enterotoxigenic E. coli (ETEC). Multiple genomic restriction fragments from each of three ETEC strains of human origin showed homology with an EAST1 gene probe. A single hybridizing fragment was detected on the plasmid of ETEC strain 27D that also encodes heat-stable enterotoxin Ib and colonization factor antigen I. We isolated and characterized this fragment, showing that it (i) carries an allele of astA nearly identical to that originally reported from EAEC 17-2 and (ii) expressed enterotoxic activity. Sequence analysis of the toxin coding region revealed that astA is completely embedded within a 1,209-bp open reading frame (ORF1), whose coding sequence is on the same strand but in the -1 reading frame in reference to the toxin gene. In vitro expression of the predicted M(r)- approximately 46,000 protein product of ORF1 was demonstrated. ORF1 is highly similar to transposase genes of IS285 from Yersinia pestis, IS1356 from Burkholderia cepacia, and ISRm3 from Rhizobium meliloti. It is bounded by 30-bp imperfect inverted repeat sequences and flanked by 8-bp direct repeats. Based on these structural features, pathognomonic of a regular insertion sequence, this element was designated IS1414. Preliminary experiments to show IS1414 translocation were unsuccessful. Overlapping genes of the type suggested by the IS1414 core region have heretofore not been described in bacteria. It seems to offer a most efficient mechanism for intragenomic and horizontal dissemination of EAST1.

Alleles↗
PubMed2000

Enterotoxicity and cytotoxicity of Vibrio parahaemolyticus thermostable direct hemolysin in in vitro systems.

Vibrio parahaemolyticus is a marine bacterium known to be a common cause of seafood gastroenteritis worldwide. The thermostable direct hemolysin (TDH) has been proposed to be a major virulence factor of V. parahaemolyticus. TDH causes intestinal fluid secretion as well as cytotoxicity in a variety of cell types. In this study, we investigated the interplay between the hemolysin's enterotoxic and cytotoxic effects by using both human and rat cell monolayers. As revealed by microspectrofluorimetry, the toxin causes a dose-dependent increase in intracellular free calcium in both Caco-2 and IEC-6 cells. This effect was reversible only when low toxin concentrations were tested. The TDH-activated ion influx pathway is not selective for calcium but admits ions such sodium and manganese as well. Furthermore, in the same range of concentration, the hemolysin triggers a calcium-dependent chloride secretion. At high concentrations, TDH induces a dose-dependent but calcium-independent cell death as assessed by functional, biochemical, and morphological assays.

Animals↗
PubMed2000

Development of a novel rapid non-invasive screening test for coeliac disease.

BACKGROUND: Coeliac disease is one of the commonest underdiagnosed diseases in general practice. The autoantigen recognised by the sera of patients with coeliac disease has recently been identified as tissue transglutaminase. AIMS: We evaluated a simple non-invasive immunological dot blot assay for coeliac disease, suitable for use by the general physician in the ambulatory setting. The sensitivity and specificity of this dot blot assay based on recognition of recombinant human transglutaminase were compared with those of antiendomysial antibodies and an enzyme linked immunosorbent assay. METHODS: Serum samples were analysed from 64 healthy controls, 58 first degree relatives of coeliacs, 74 diseased controls, and 70 biopsy confirmed untreated patients with coeliac disease. Dot blot assay and enzyme linked immunosorbent assay were performed using recombinant human transglutaminase as antigen. RESULTS: The dot blot assay, which can be performed in 20 minutes, was positive in all 70 untreated coeliacs (sensitivity 100%). Among the three control groups, there were three false positive tests by dot blot (specificity 98%), all belonging to the group of healthy subjects. The antiendomysial antibodies test missed five untreated coeliac patients (sensitivity 93%) and was negative in all three control groups (specificity 100%). The specificity of the immunosorbent assay was 99% for IgA and 98% for IgG, while sensitivity was 93% for IgA, 47% for IgG, and 100% for IgA and IgG combined. CONCLUSIONS: The dot blot assay is highly accurate in detecting untreated subjects with coeliac disease and can be performed in the general physician's medical office during the course of a routine examination. This innovative test is a practical, reliable alternative to both the immunofluorescent based antiendomysial test and immunosorbent assay for detection of transglutaminase antibodies for the diagnosis of coeliac disease.

Adolescent↗
PubMed2000

Human zonulin, a potential modulator of intestinal tight junctions.

Intercellular tight junctions are dynamic structures involved in vectorial transport of water and electrolytes across the intestinal epithelium. Zonula occludens toxin derived from Vibrio cholerae interacts with a specific intestinal epithelial surface receptor, with subsequent activation of a complex intracellular cascade of events that regulate tight junction permeability. We postulated that this toxin may mimic the effect of a functionally and immunologically related endogenous modulator of intestinal tight junctions. Affinity-purified anti-zonula occludens toxin antibodies and the Ussing chamber assay were used to screen for one or more mammalian zonula occludens toxin analogues in both fetal and adult human intestine. A novel protein, zonulin, was identified that induces tight junction disassembly in non-human primate intestinal epithelia mounted in Ussing chambers. Comparison of amino acids in the active zonula occludens toxin fragment and zonulin permitted the identification of the putative receptor binding domain within the N-terminal region of the two proteins. Zonulin likely plays a pivotal role in tight junction regulation during developmental, physiological, and pathological processes, including tissue morphogenesis, movement of fluid, macromolecules and leukocytes between the intestinal lumen and the interstitium, and inflammatory/autoimmune disorders.

Animals↗
PubMed1999

Expression of Vibrio cholerae zonula occludens toxin and analysis of its subcellular localization.

Vibrio cholerae elaborates zonula occludens toxin (Zot), a protein that increases the permeability of small intestinal mucosa by opening intercellular tight junctions. The zot gene is located, together with the genes encoding CT and Ace enterotoxins, within the genome of V. cholerae filamentous phage CTXsmall ef, Cyrillic. Interestingly, Zot appears to be structurally and functionally related to the gene I product of other filamentous phages and it has been shown to be required for CTXsmall ef, Cyrillic morphogenesis. In this study we described the cloning of zot in several expression plasmid systems and we examined the subcellular localization of Zot by using affinity purified anti-Zot antibodies. We found that Zot localizes in the V. cholerae cell envelope with M(r);45 kDa which is consistent with the predicted primary translation product from the first methionine of zot (44.8 kDa). A second molecule, corresponding to the 33 kDa N-terminal region of Zot, was also detected. Both molecules are exposed at the bacterial cell surface. The production of the 33 kDa Zot, that might represent a processing product, was abolished in mutant ZotG59. N-terminal tagged 6xHis-Zot fusion protein retained the capability to reach the outer membrane and the 6xHis tag was not cleaved off during the translocation to the periplasm, whereas the presence of the tag partially blocked the formation of the 33 kDa molecule. Zot secretion and anchorage to the bacterial outer membrane was also observed in E. coli strains expressing Zot, suggesting that the toxin may be directed to the outer membrane via the same pathway in E. coli and V. cholerae. Zot cleavage might be due to a V. cholerae specific protease activity, since the 33 kDa protein was not efficiently produced in E. coli. On the basis of these data and Zot amino acid sequence analysis, we suggest that while the N-terminal part of the molecule is involved in the morphogenesis of CTXsmall ef, Cyrillic, the C-terminal region might carry the domain(s) responsible for Zot enterotoxic activity.

Animals↗
PubMed1999

Conformation of bovine myelin basic protein purified with bound lipids.

The basic protein of myelin (called MBP) is an extrinsic protein of the myelin membrane. Its structure and function are still unknown. MBP has been extensively studied in its water-soluble form, but it is also known in a detergent-soluble form, which is purified with endogenous myelin lipids and should correspond to the native form of the protein in the membrane. In order to acquire insight into the structure of MBP, we have carried out circular dichroism (CD) experiments on the protein both in the lipid-free and in the lipid-bound form. Our data clearly show that lipid-free MBP is mainly disordered with only a small amount having alpha-helix and beta-sheet motifs. On the other hand, the lipid-bound form of MBP appears to have a consistent amount of ordered secondary structure. Theoretical predictions, made using different computational methods, substantially confirm the tendency of the protein to assume an ordered secondary structure in accordance with our CD results.

Amino Acid Sequence↗
PubMed1999

Effect of small bowel bacterial overgrowth on the immunogenicity of single-dose live oral cholera vaccine CVD 103-HgR.

Several live oral vaccines (polio, bovine rotavirus, CVD 103-HgR cholera) are less immunogenic in developing than in industrialized countries. It was hypothesized that proximal small bowel bacterial overgrowth (common in children in less developed countries but rare in industrialized settings) diminishes the vibriocidal antibody response to CVD 103-HgR. In total, 202 fasting Santiago schoolchildren aged 5-9 years had lactulose breath H2 tests to detect proximal small bowel bacteria 1 day before ingesting CVD 103-HgR. Florid small bowel overgrowth was observed in 10 (5.6%) of 178 analyzable children. In children with florid overgrowth, vibriocidal seroconversion differed little from other children (60% vs. 67%), but the geometric mean titer was lower (160 vs. 368; P=.25). By logistic regression, increased peak breath H2 at small bowel time points was associated with diminished seroconversion (P=.04), as was the interaction of H2 value and weight (children >25 kg had lower seroconversion rates among subjects with heaviest overgrowth).

Administration, Oral↗
PubMed1999

Intestinal toxins.

The application of molecular techniques to the study of bacterial pathogenesis has made possible discoveries that are changing the way scientists view the bacterium-host interaction. Today, research on the molecular basis of the pathogenesis of infective diarrheal diseases of necessity transcends established boundaries between cell biology, bacteriology, intestinal pathophysiology, and immunology. A comprehensive approach has been taken here to outline the most recent findings on the interaction between enteric pathogens and their target eukaryotic cells through the elaboration of toxins.

Journal Article↗
PubMed1999

Production of enterotoxin by Yersinia bercovieri, a recently identified Yersinia enterocolitica-like species.

Yersinia bercovieri, a recently identified Y. enterocolitica-like species, produces a heat-stable enterotoxin (designated YbST) which has biologic activity in infant mice and increases short circuit current in Ussing chambers. Although YbST has some properties in common with the heat-stable enterotoxins of Y. enterocolitica (YST I and YST II), it appears to be a novel toxin because (i) it was not neutralized by anti-YST I antiserum, (ii) YbST-neutralizing antiserum did not neutralize YST I, and (iii) Y. bercovieri strains did not hybridize with genetic probes for yst I, yst II, and other known enterotoxins.

Animals↗
PubMed1999

Vibrio parahaemolyticus thermostable direct hemolysin modulates cytoskeletal organization and calcium homeostasis in intestinal cultured cells.

Vibrio parahaemolyticus is a marine bacterium known to be the leading cause of seafood gastroenteritis worldwide. A 46-kDa homodimer protein secreted by this microorganism, the thermostable direct hemolysin (TDH), is considered a major virulence factor involved in bacterial pathogenesis since a high percentage of strains of clinical origin are positive for TDH production. TDH is a pore-forming toxin, and its most extensively studied effect is the ability to cause hemolysis of erythrocytes from different mammalian species. Moreover, TDH induces in a variety of cells cytotoxic effects consisting mainly of cell degeneration which often leads to loss of viability. In this work, we examined the cellular changes induced by TDH in monolayers of IEC-6 cells (derived from the rat crypt small intestine), which represent a useful cell model for studying toxins from enteric bacteria. In experimental conditions allowing cell survival, TDH induces a rapid transient increase in intracellular calcium as well as a significant though reversible decreased rate of progression through the cell cycle. The morphological changes seem to be dependent on the organization of the microtubular network, which appears to be the preferential cytoskeletal element involved in the cellular response to the toxin.

Animals↗
PubMed1999

Zonula occludens toxin is a powerful mucosal adjuvant for intranasally delivered antigens.

Zonula occludens toxin (Zot) is produced by toxigenic strains of Vibrio cholerae and has the ability to reversibly alter intestinal epithelial tight junctions, allowing the passage of macromolecules through the mucosal barrier. In the present study, we investigated whether Zot could be exploited to deliver soluble antigens through the nasal mucosa for the induction of antigen-specific systemic and mucosal immune responses. Intranasal immunization of mice with ovalbumin (Ova) and recombinant Zot, either fused to the maltose-binding protein (MBP-Zot) or with a hexahistidine tag (His-Zot), induced anti-Ova serum immunoglobulin G (IgG) titers that were approximately 40-fold higher than those induced by immunization with antigen alone. Interestingly, Zot also stimulated high anti-Ova IgA titers in serum, as well as in vaginal and intestinal secretions. A comparison with Escherichia coli heat-labile enterotoxin (LT) revealed that the adjuvant activity of Zot was only sevenfold lower than that of LT. Moreover, Zot and LT induced similar patterns of Ova-specific IgG subclasses. The subtypes IgG1, IgG2a, and IgG2b were all stimulated, with a predominance of IgG1 and IgG2b. In conclusion, our results highlight Zot as a novel potent mucosal adjuvant of microbial origin.

Adjuvants, Immunologic↗
PubMed1999

Cellular microbiology: can we learn cell physiology from microorganisms?

Cellular microbiology is a new discipline that is emerging at the interface between cell biology and microbiology. The application of molecular techniques to the study of bacterial pathogenesis has made possible discoveries that are changing the way scientists view the bacterium-host interaction. Today, research on the molecular basis of the pathogenesis of infective diarrheal diseases of necessity transcends established boundaries between cell biology, bacteriology, intestinal pathophysiology, and immunology. The use of microbial pathogens to address questions in cell physiology is just now yielding promising applications and striking results.

Amino Acid Sequence↗
PubMed1998

Purification of bovine P2 myelin protein with bound lipids.

The P2 protein is a neuritogenic, small basic protein present in PNS myelin. It belongs to the family of the cytoplasmic lipid-binding proteins and can be incorporated in lipidic bilayers. P2 has been purified and crystallized only in the lipid-free form. Here we show that the P2 protein can be purified with bound lipids by applying to PNS myelin the same procedure that as used to purify lipid-bound myelin basic protein from CNS myelin. SDS-PAGE showed a single band of 16.5 kDa, and TLC showed the presence of most of the myelin lipids associated with the protein. Lipid-bound P2 revealed different circular dichroism spectra from the corresponding lipid-free form, indicating that lipids influence P2 conformation.

Animals↗