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Biomedical subjects

F Castellino

Publications and source records attributed to F Castellino.

At least 19 recordsLinked to original sources
PubMed2005

Therapeutic ERCP in paediatric patients.

BACKGROUND: Major limitations of endoscopic retrograde cholangiopancreatography in paediatric populations are a low incidence of biliopancreatic disease among children, the equipment dimensions (size of endoscopes and devices) and the increasing role of MR-cholangiopancreatography in the field of diagnostic indications. Aim of this study was to evaluate the diagnostic and therapeutic yields of endoscopic retrograde cholangiopancreatography for biliopancreatic diseases in a paediatric population. METHODS: Between 1996 and 2002, 48 endoscopic retrograde cholangiopancreatographies were performed in 38 children aged 4 weeks to 17 years as part of the diagnostic evaluation for suspected pancreatic or biliary tract disease. Endoscopic retrograde cholangiopancreatography was carried out under general anaesthesia, using prototype paediatric duodenoscopes or standard duodenoscopes in children younger or older than 18 months, respectively. RESULTS: The indications to perform endoscopic retrograde cholangiopancreatography were common bile duct stones (14 children), biliopancreatic abnormalities (8), primary sclerosing cholangitis (2), Wirsung disruption (1), biliary leakage (1), cholestasis (4) and pancreatitis (8). Cannulation was successful in all patients but one. Sphincterotomy together with stone extraction or stent insertion was performed in 30/38 patients. Immediate complications were mild and treated conservatively. CONCLUSIONS: Diagnostic and therapeutic endoscopic retrograde cholangiopancreatography can be used safely and effectively in the management of biliopancreatic diseases in childhood as well. Indications, endoscopic techniques and complications are similar to those reported for adult patients.

Acute Disease↗
PubMed2002

Tissue factor deficiency causes cardiac fibrosis and left ventricular dysfunction.

Exposure of blood to tissue factor (TF) activates the extrinsic (TF:FVIIa) and intrinsic (FVIIIa:FIXa) pathways of coagulation. In this study, we found that mice expressing low levels of human TF ( approximately 1% of wild-type levels) in an mTF(-/-) background had significantly shorter lifespans than wild-type mice, in part, because of spontaneous fatal hemorrhages. All low-TF mice exhibited a selective heart defect that consisted of hemosiderin deposition and fibrosis. Direct intracardiac measurement demonstrated a 30% reduction (P < 0.001) in left ventricular function in 8-month-old low-TF mice compared with age-matched wild-type mice. Mice expressing low levels of murine FVII ( approximately 1% of wild-type levels) exhibited a similar pattern of hemosiderin deposition and fibrosis in their hearts. In contrast, FIX(-/-) mice, a model of hemophilia B, had normal hearts. Cardiac fibrosis in low-TF and low-FVII mice appears to be caused by hemorrhage from cardiac vessels due to impaired hemostasis. We propose that TF expression by cardiac myocytes provides a secondary hemostatic barrier to protect the heart from hemorrhage.

Animals↗
PubMed2002

Review article: medical treatment of severe ulcerative colitis.

Approximately 15% of patients with ulcerative colitis have a severe attack requiring hospitalization at some time during their illness. This treatment leads to a remission in 60-80% of patients and non-responders may require a total colectomy. Mortality in severe episodes of ulcerative colitis decreased from 31-61% in the 1950s to 5-9% in the 1960s thanks to the introduction of steroids and to a policy of early colectomy. Recently, some new drugs have been shown to be effective in the treatment of severe steroid-refractory ulcerative colitis. This review concentrates on the clinical evaluation, prognostic factors and new developments in medical therapy in severe ulcerative colitis. A retrospective evaluation of a consecutive series of patients with severe ulcerative colitis admitted to a Gastroenterology Department in Torino, Italy, is also reported.

Anti-Bacterial Agents↗
PubMed2001

The transmembrane segment of invariant chain mediates binding to MHC class II molecules in a CLIP-independent manner.

Invariant chain (Ii) association with MHC class II molecules is strongly dependent upon interaction of CLIP (Ii exon 3, residues 81 - 104) with the peptide binding groove of the class II dimer. This dominant interaction does not adequately explain, however, the efficient association of Ii with class II molecules of diverse allelic and isotypic origin, which have markedly different affinities for synthetic peptides corresponding to CLIP. In agreement with other recent observations, we demonstrate here that class II molecules with occupied binding sites unable to engage CLIP maintain association with Ii in mild detergent. The association is direct and not mediated through unoccupied class II chains bound to properly assembled and loaded class II dimers, nor is it mediated through chaperones. The site of this CLIP-independent binding has been mapped using truncation mutants and an Ii-human transferrin receptor chimeric protein to the transmembrane segment of Ii. The existence of multiple low-affinity sites of interaction between MHC class II and Ii helps explain how effective occupancy of all newly synthesized class II molecules can occur despite substantial variations in the strength of CLIP-dependent association that arise from class II binding domain polymorphism. These data establishing a site of Ii-MHC class II association N-terminal to CLIP also provide new insight into the possible functional relationship between the sequential endocytic proteolysis of Ii from its C terminus and a series of contact sites with MHC class II molecules spread from the transmembrane region through to the tip of the lumenal segment of Ii.

Animals↗
PubMed2001

Detection of anti-Saccharomyces cerevisiae antibodies in Crohn's disease: is it a reliable diagnostic and prognostic marker?

BACKGROUND: In the past few years, serologic markers have been proposed in inflammatory bowel disease. Anti-Saccharomyces cerevisiae antibodies showed high specificity for Crohn's disease. A prognostic role for serology has also been hypothesised. AIMS: To evaluate anti-Saccharomyces cerevisiae antibody distribution in an unselected Italian inflammatory bowel disease population. To analyse whether anti-Saccharomyces cerevisiae antibody status (positive/negative) and/or anti-Saccharomyces cerevisiae antibody titres are associated with clinical variables and outcome measures in Crohn's disease patients. PATIENTS AND METHODS: A series of 299 inflammatory bowel disease patients were evaluated; serum samples were taken and a short clinical history was recorded. anti-Saccharomyces cerevisiae antibodies IgG enzyme-linked immunosorbent assay Medilab (Milan, Italy) kit was used in order to determine anti-Saccharomyces cerevisiae antibody status. RESULTS: Sensitivity, specificity and likelihood ratio for positive test in the differential diagnosis of inflammatory bowel disease was 59%, 89%, 8.1, respectively. Clinical variables significantly associated with anti-Saccharomyces cerevisiae antibody status in logistic regression were found to be ileal location (p=0.01) and earlier age at diagnosis (p<0.01). Among ileal Crohn's disease patients, there was a trend in concordance between anti-Saccharomyces cerevisiae antibody titres and higher number of surgical procedures which was not statistically significant applying more complex statistics. CONCLUSIONS: In an Italian inflammatory bowel disease population, anti-Saccharomyces cerevisiae antibodies status showed characteristics similar to those previously reported. Anti-Saccharomyces cerevisiae antibody positivity is associated with ileal involvement and with earlier onset of Crohn's disease.

Adult↗
PubMed2000

Receptor-mediated uptake of antigen/heat shock protein complexes results in major histocompatibility complex class I antigen presentation via two distinct processing pathways.

Heat shock proteins (HSPs) derived from tumors or virally infected cells can stimulate antigen-specific CD8(+) T cell responses in vitro and in vivo. Although this antigenicity is known to arise from HSP-associated peptides presented to the immune system by major histocompatibility complex (MHC) class I molecules, the cell biology underlying this presentation process remains poorly understood. Here we show that HSP 70 binds to the surface of antigen presenting cells by a mechanism with the characteristics of a saturable receptor system. After this membrane interaction, processing and MHC class I presentation of the HSP-associated antigen can occur via either a cytosolic (transporter associated with antigen processing [TAP] and proteasome-dependent) or an endosomal (TAP and proteasome-independent) route, with the preferred pathway determined by the sequence context of the optimal antigenic peptide within the HSP-associated material. These findings not only characterize two highly efficient, specific pathways leading to the conversion of HSP-associated antigens into ligands for CD8(+) T cells, they also imply the existence of a mechanism for receptor-facilitated transmembrane transport of HSP or HSP-associated ligands from the plasma membrane or lumen of endosomes into the cytosol.

Amino Acid Sequence↗
PubMed2000

A novel method for detecting IgA endomysial antibodies by using human umbilical vein endothelial cells.

BACKGROUND: Although tissue transglutaminase was recently identified as the main autoantigen recognized by endomysial antibodies in coeliac patients, anti-endomysium antibody detection still persists as the gold standard for coeliac disease screening and diagnosis. OBJECTIVES: (1) To evaluate human umbilical vein cells (HUVEC) as an alternative source of endomysial antigen and to assess their suitability in the diagnosis of coeliac disease. (2) To verify whether tissue transglutaminase is one target antigen eliciting the endomysial antibody fraction of coeliac serum IgA. SETTING: University teaching hospital. PATIENTS AND METHODS: Sera from 123 untreated adults with biopsy-proven coeliac disease and 84 controls (40 healthy and 44 diseased) were assessed by indirect immunofluorescence, using HUVEC on glass slides prepared by cytocentrifugation and permeabilized by using Triton X (0.5%). Indirect immunofluorescence was performed: (1) using coeliac disease serum samples on HUVEC with or without prior incubation with tissue transglutaminase; and (2) incubating both HUVEC and monkey oesophagus with goat anti-guinea pig tissue transglutaminase antibody. RESULTS: All the coeliac patients, who were also positive on monkey oesophagus, showed the typical fluorescent homogeneous cytoplasmic stain on HUVEC. All control sera were negative both on HUVEC and on monkey oesophagus. IgA antibodies did not react with non-permeabilized cells, with intact membrane. Preincubation of coeliac sera with tissue transglutaminase abolished the typical fluorescent pattern. The incubation of anti-tissue transglutaminase antibody with monkey oesophagus and HUVEC resulted in an immunofluorescence staining pattern identical to that obtained with positive coeliac sera. CONCLUSIONS: (1) As a substrate for anti-endomysial antibody, HUVEC may provide the same diagnostic accuracy as monkey oesophagus, thus bypassing economical and ethical problems. The HUVEC antigen reacting with IgA from coeliac disease sera is an intracellular rather than a cell-surface antigen, as IgA antibodies reacted only with permeabilized cells. (2) Pretreatment of untreated coeliac sera with tissue transglutaminase abolished almost completely the specific staining; incubation with anti-tissue transglutaminase antibody elicited the characteristic fluorescent pattern, thus confirming that tissue transglutaminase represents the prominent autoantigen in coeliac disease.

Adult↗
PubMed1999

Cellular distribution of a mixed MHC class II heterodimer between DRalpha and a chimeric DObeta chain.

Human MHC class II antigens include HLA-DR, -DQ, and -DP molecules that present antigens to CD4+ T cells, as well as the non-classical molecules HLA-DM and -DO. HLA-DM promotes peptide binding to class II molecules in endocytic compartments and HLA-DO, which is physically associated with HLA-DM in B lymphocytes, regulates HLA-DM function. Antibodies specific for the DObeta chain were obtained by immunization of mice with a heterodimer consisting of a chimeric DObeta chain (DR/DObeta), containing 18 N-terminal residues of DRbeta, paired with the DRalpha chain and isolated from transfected murine fibroblasts. The specificity of this serum for the DObeta chain and the lysosomal expression of the HLA-DO protein was confirmed using mutant human B cell lines lacking DR or DO molecules. The lysosomal localization of HLA-DO in human B cells contrasts with the cell surface expression of the mixed pair in transfected murine fibroblasts and raises questions concerning the role of the putative targeting motifs in HLA-DO. Transfection of the chimeric DR/DObeta chain along with DRalpha into human epithelial HeLa cells resulted in high levels of expression of the mixed isotypic pair at the surface of transfectants as well as in lysosomes. The same pattern was observed in HeLa cells transfected with the DObeta chimera and a DRa chain lacking the cytoplasmic tail. Taken together, these results suggest that functional sorting motifs exist in the DObeta chain but that the tight compartmentalization of HLA-DO observed inside B lymphocytes is controlled by the HLA-DOalpha chain and HLA-DM.

Animals↗
PubMed1998

Large protein fragments as substrates for endocytic antigen capture by MHC class II molecules.

Although the binding sites of MHC class II molecules can accommodate longer ligands, peptides of 15 to 20 residues are the primary form of processed Ag recovered from class II dimers isolated from living cells. These peptides are derived from intact Ags by proteolysis in endocytic organelles, where binding to class II dimers also occurs. Whether generation of these short peptides typically precedes association with class II molecules, or whether class II molecules initially bind to unfolded proteins or large protein fragments, followed by degradation of the unprotected regions, remains unknown. Here we report the identification of an SDS-stable, long-lived, 120-kDa complex composed of two class II dimers bound to a common large Ag fragment. This complex is produced within the endocytic pathway from newly synthesized MHC class II molecules following exposure of the cells to exogenous hen egg lysozyme. These data suggest that a major pathway of Ag processing involves the initial binding of class II heterodimers to large protein substrates upon exposure of regions with suitable motifs, followed by cleavage and/or trimming of the exposed protein around this bound region. This sequence of events during Ag processing may provide a partial molecular explanation for the immunodominance of certain determinants in protein Ags.

Animals↗
PubMed1997

Antigen presentation by MHC class II molecules: invariant chain function, protein trafficking, and the molecular basis of diverse determinant capture.

Major histocompatibility complex class II molecules are heterodimeric integral membrane proteins whose primary function is the presentation of antigenic peptides derived from proteins entering the endocytic pathway to CD4+ T lymphocytes. To accomplish this physiologic function, class II molecules must assemble in the secretory pathway without undergoing irreversible ligand association at that site, traffic efficiently to the endocytic pathway, and productively interact with protein ligands in these organelles before their ultimate expression on the plasma membrane. Here we review our work describing how invariant chain promoters the assembly and transport process, the complex itinerary of class II-invariant chain complexes through the endocytic pathway, the role of large protein fragments as substrates for class II binding, and the existence of a second pathway for antigen capture by mature class II molecules that complements that involving newly synthesized dimers. We integrate these observations into a coherent model for the operation of a class II-dependent antigen processing and presentation system able to capture diverse antigenic determinants present in proteins of varying structure.

Antigen Presentation↗
PubMed1997

IgM antibody against measles virus in patients with inflammatory bowel disease: a marker of virus-related disease?

OBJECTIVE: Viral infections of the mesenteric microvascular endothelium have been hypothesized as pathogenetic factors in inflammatory bowel diseases. The aim of this study was to determine whether immunoglobulin M (IgM) antibody against measles virus is associated with disease. PATIENTS AND METHODS: The IgM antibody was detected by indirect antibody test in 36 patients with evidence of Crohn's disease (23 males and 13 females, median age 40 years, range 20-66), 22 patients with ulcerative colitis (14 males and 8 females, median age 42 years; range 19-65), 59 patients with a chronic active hepatitis (35 males and 24 females, median age 56 years, range 38-77) and 30 blood donors (20 males and 10 females, median age 45 years, range 29-62). RESULTS: Twenty-eight of 36 patients (78%) with Crohn's disease and 13 of 22 patients (59%) with ulcerative colitis tested positive as compared to only 3 of 89 (3.3%) controls (P < or = 0.001). CONCLUSION: The detection of IgM anti-measles virus in the majority of patients with Crohn's disease and in about half of ulcerative colitis patients as compared to a very low prevalence in patients with other chronic inflammatory disease is consistent with the hypothesis that the measles virus has pathogenetic implications in inflammatory bowel diseases.

Adult↗
PubMed1996

Evidence that binding site occupancy is necessary and sufficient for effective major histocompatibility complex (MHC) class II transport through the secretory pathway redefines the primary function of class II-associated invariant chain peptides (CLIP).

Invariant chain (Ii) associates with newly synthesized class II molecules in the endoplasmic reticulum (ER), an interaction that has been shown to interfere with peptide binding to class II molecules. The class II-associated invariant chain peptide (CLIP) region (residues 81-104) of Ii is believed to mediate this inhibition by engaging the binding domain of class II like an antigenic peptide. Together, these findings have given rise to a model in which CLIP association with the class II groove acts to prevent inappropriate presentation of peptides imported into the ER for association with major histocompatibility complex class I molecules. However, the properties of class II molecules synthesized by cells lacking coexpressed Ii are at least superficially inconsistent with this paradigm in that they do not show clear evidence of peptide acquisition. At the same time, we have previously shown the shortest form of Ii still containing CLIP to play an essential role in regulation of early class II molecule assembly and transport in the secretory pathway. Using covalent peptide technology, we now show that occupancy of the class II binding site in the ER regulates class II trafficking to the Golgi complex, an event that is the locus of the major defect in cells of Ii-deficient mice. These data argue that CLIP occupies the class II binding site, not to prevent interaction with short peptides meant for class I, but rather to maintain the structural integrity of class II molecules that are labile without engaged binding regions, and that would also associate with intact proteins in the ER if left unoccupied. By these means, CLIP occupancy of the class II binding site promotes effective export of useful class II molecules for endocytic peptide acquisition.

Amino Acid Sequence↗
PubMed1995

Extensive trafficking of MHC class II-invariant chain complexes in the endocytic pathway and appearance of peptide-loaded class II in multiple compartments.

Major histocompatibility complex class II molecules bind and present to T cells fragments of protein antigens entering the endocytic pathway. Using normal B lymphoblasts, we have combined metabolic pulse-chase labelling, high resolution organelle fractionation, and immunoprecipitation to examine class II trafficking and antigen loading in a physiological model system. Most newly synthesized class II-invariant chain complexes first entered early endosomes, then accessed multiple discrete endocytic subcompartments cofractionating with late endosomes and immature lysosomes. Invariant chain was removed and peptide-loaded class II molecules appeared in each of these latter distinct organelles. These findings suggest that class II molecules traffic through much of the endocytic pathway, permitting capture of distinct determinants made available under differing conditions of pH and proteolytic activity.

Animals↗
PubMed1995

Essential role of caldesmon in the actin filament reorganization induced by glucocorticoids.

Glucocorticoids induce the remodeling of the actin cytoskeleton and the formation of numerous stress fibers in a protein synthesis-dependent fashion in a variety of cell types (Castellino, F., J. Heuser, S. Marchetti, B. Bruno, and A. Luini. 1992. Proc. Natl. Acad. Sci. USA. 89:3775-3779). These cells can thus be used as models to investigate the mechanisms controlling the organization of actin filaments. Caldesmon is an almost ubiquitous actin- and calmodulin-binding protein that synergizes with tropomyosin to stabilize microfilaments in vitro (Matsumura, F., and Yamashiro, S. 1993. Current Opin. Cell Biol. 5:70-76). We now report that glucocorticoids (but not other steroids) enhanced the levels of caldesmon (both protein and mRNA) and induced the reorganization of microfilaments with similar time courses and potencies in A549 cells. A caldesmon antisense oligodeoxynucleotide targeted to the most abundant caldesmon isoform in A549 cells dramatically inhibited glucocorticoid-induced caldesmon synthesis and actin reorganization with similar potencies. Several control oligonucleotides were inactive. These results demonstrate that caldesmon has a crucial role in vivo in the organization of the actin cytoskeleton and suggest that hormone-induced changes in caldesmon levels mediate microfilament remodeling.

Actins↗
PubMed1995

Anti-neutrophil cytoplasmic antibodies in inflammatory bowel disease: do they recognize different subsets of a heterogeneous disease?

OBJECTIVE: To evaluate the prevalence of anti-neutrophil cytoplasmic antibodies in a series of patients with inflammatory bowel disease, the discriminatory value of these antibodies in differentiating between ulcerative colitis and Crohn's disease, their antigen specificity and their correlation with epidemiological and clinical variables. METHODS: Serum anti-neutrophil cytoplasmic antibodies were evaluated by indirect immunofluorescence and immunoblotting using neutrophils isolated from peripheral blood and by enzyme-linked immunosorbent assays (ELISAs) using proteinase 3 and myeloperoxidase as antigens. RESULTS: Anti-neutrophil cytoplasmic antibodies were detected by immunofluorescence in 43 (39.8%) of 108 patients with ulcerative colitis, in 11 (11.9%) of 92 patients with Crohn's disease (P < 0.001) and 5 (6.8%) of 73 control patients. The predominant pattern was perinuclear staining around neutrophil nuclei (44 of 59, 75%); a homogeneous cytoplasmic staining was present in 15 (25%) of 59 sera, mainly among Crohn's disease and control patients. The ELISAs gave no positive results. Recognition of proteins of relative molecular masses 27,000 and 49,000 at immunoblotting was common to ulcerative colitis, Crohn's disease and control sera. The proteins of relative molecular masses 32,000 and 106,000 were recognized exclusively by 11% of anti-neutrophil-positive ulcerative colitis sera. No significant correlation was found between the presence of anti-neutrophil cytoplasmic antibodies and the demographic and clinical characteristics of the patients. CONCLUSION: Anti-neutrophil cytoplasmic antibodies are detectable in a large proportion of patients with ulcerative colitis, but their prevalence in a limited proportion of patients with Crohn's disease reduces their discriminatory capability. The persistence of anti-neutrophil cytoplasmic antibodies after total colectomy and the absence of a correlation between the activity of the disease and the presence or titre of these antibodies support the hypothesis that anti-neutrophil cytoplasmic antibodies are not simply an epiphenomenon of colonic inflammation.

Adult↗
PubMed1992

Glucocorticoid stabilization of actin filaments: a possible mechanism for inhibition of corticotropin release.

The mechanism by which glucocorticoids induce various cellular responses in different tissues is only partially understood. Here we demonstrate that glucocorticoids stabilize the actin cytoskeleton of several cell types, as revealed by increased resistance of actin filaments to the disrupting effect of cytochalasin and by visible thickening of actin filament bundles. These effects require several hours to develop, require protein synthesis, and are accompanied by increased expression of the actin-binding protein caldesmon. These data may help to explain why glucocorticoids inhibit corticotropin release from pituitary cells, if interpreted in terms of the current idea that an actin filament "barrier" modulates exocytotic secretion in various cell types. In support of this idea, we find that in "model" corticotrophs (AtT-20 cells), glucocorticoids stabilize actin filaments and inhibit corticotropin release with similar potencies. Furthermore, we show here that glucocorticoid inhibition is overcome by exposing AtT-20 cells to concentrations of cytochalasin B or D that disrupt their stabilized actin filaments. On the other hand, our freeze-etch electron microscopy of AtT-20 cells has shown that actin filaments do not, in fact, create a dense submembranous barrier that might prevent corticotropin secretory droplets from discharging; instead, they form open networks near the membrane that appear to hold secretory droplets in their interstices. We propose that the delicate physical crosslinks maintaining this actin-mediated membrane "docking" of secretory droplets may need to disconnect in order to permit corticotropin discharge and that these crosslinks may be stabilized along with the actin filaments in dexamethasone-treated cells.

Actin Cytoskeleton↗
PubMed1990

Intrathymic deficient expansion of T cell precursors in Down syndrome.

To correlate the intrinsic cellular immunodeficiency, which is a major cause of increased susceptibility to polytopic infections in Down syndrome (DS) patients, with the histologic abnormalities observed in the thymus of these patients, we have studied thymus fragments and thymocyte cell suspensions from 15 non-institutionalized DS subjects. Comparing to the control age-matched samples, a reduced thymic cortex and a distinct depletion of CD1-positive (+) cells was observed by immuno-histologic examination. The phenotypic analysis of unselected thymocytes showed a significant reduction of CD3+, CD1+, CD4+, and CD8+ cells. When the total thymocyte population was separated into 10 fractions, using a continuous Percoll density gradient, a difference in cell distribution was observed. DS thymuses are almost devoid of high-density thymocytes (fractions 6-9) while more than 75% of the cells were recovered in the lightest 3 fractions (Frs). In addition, these thymuses were characterized by a marked depletion of CD1+ cells and by a conspicuous reduction of CD3+ cells normally present in the high-density Frs. On the other hand, the lightest 3 Frs of DS subjects were enriched in low-density CD1+ cells. Although enriched in these cells, normally characterized by a high mitotic activity, Fr1 DS thymocytes showed a reduced spontaneous proliferative capacity. When the expression of T cell receptor alpha- and beta-subunits was studied, the percentages of cells stained with anti-alpha and anti-beta antisera were found to be reduced in DS unfractionated thymocytes. The reduced number of high-density CD1+ thymocytes associated with a reduced spontaneous proliferative activity of low-density CD1+ thymocytes suggests that in DS thymuses there is a deficient expansion of immature T cells, resulting in a reduction of the various thymocyte subpopulations, including the thymocyte pool which differentiates into functionally mature T cells expressing the alpha-beta T cell receptor.

Child, Preschool↗