Generation and functional characterisation of dendritic cells from patients with pancreatic carcinoma with special regard to clinical applicability.

Background: We studied dendritic cell (DC) function in patients affected by pancreatic carcinoma, and the possibility of obtaining DC adequate for immunological treatment modalities.

Methods: Leucocytes were isolated from buffy coats obtained by autotransfusion of six patients undergoing pancreatico-duodenectomy. The leucocytes were cryopreserved and, after thawing, were purified by density gradient and/or plastic adhesion.

p95-APP1 links membrane transport to Rac-mediated reorganization of actin.

Motility requires protrusive activity at the cellular edge, where Rho family members regulate actin dynamics. Here we show that p95-APP1 (ArfGAP-putative, Pix-interacting, paxillin-interacting protein 1), a member of the GIT1/PKL family, is part of a complex that interacts with Rac. Wild-type and truncated p95-APP1 induce actin-rich protrusions mediated by Rac and ADP-ribosylation factor 6 (Arf6).

Acquisition of intact allogeneic human leukocyte antigen molecules by human dendritic cells.

In an attempt to transduce monocyte-derived dendritic cells (DCs) by a retroviral vector coding for a cell surface marker, we were confronted by the observation of high transfer of the surface molecule in the absence of vector proviral DNA in the treated cells. Indeed, DCs acquired the surface marker by a mechanism independent of the vector machinery, requiring cell-to-cell contact and involving transfer of lipids and a variety of intact membrane proteins. Most important, this property of DCs also includes acquisition of foreign human leukocyte antigen (HLA) molecules.

Rac GTPases localize at sites of actin reorganization during dynamic remodeling of the cytoskeleton of normal embryonic fibroblasts.

Rac GTP-binding proteins are implicated in the dynamic organization of the actin cytoskeleton, and the mechanisms utilized for this purpose are not understood yet. In this paper we have analysed the effects of the expression of Rac proteins on the organization of the cytoskeleton, and their subcellular distribution in chicken embryo fibroblasts. In these cells, overexpression of wild-type Rac GTPases induces disassembly of stress fibers, and production of long, highly branched actin-rich protrusions, with consequent dramatic changes in cell morphology.

Mechanisms of coordination of Ca2+ signals in pancreatic islet cells.

Within pancreatic islet cells, rhythmic changes in the cytosolic Ca2+ concentration have been reported to occur in response to stimulatory glucose concentrations and to be synchronous with pulsatile release of insulin. We explored the possible mechanisms responsible for Ca2+ signal propagation within islet cells, with particular regard to gap junction communication, the pathway widely credited with being responsible for coordination of the secretory activity. Using fura-2 imaging, we found that multiple mechanisms control Ca2+ signaling in pancreatic islet cells.

Restoration of bacterial killing activity of human respiratory cystic fibrosis cells through cationic vector-mediated cystic fibrosis transmembrane conductance regulator gene transfer.

In vitro and in vivo studies have demonstrated that gene transfer of the CFTR (cystic fibrosis transmembrane conductance regulator) cDNA into human respiratory cells through nonviral vectors can occur safely and can be done repeatedly. Although functional evaluation of CFTR in cystic fibrosis (CF) patients enrolled in phase I clinical trials using cationic liposomes has shown a partial correction of nasal potential difference, a biological assay indicating a therapeutic relevance of CFTR gene transfer is still missing. Our aims were to study the induction of killing activity toward Pseudomonas aeruginosa (PA) in CF cells by cationic vector-mediated CFTR gene transfer and to use this assay as a therapeutic end point.

Glucocorticoids increase the endocytic activity of human dendritic cells.

We have investigated the effect of glucocorticoids (GC) on antigen uptake molecule expression and on endocytic activity of human dendritic cells (DC). Human monocyte-derived DC were differentiated in vitro for 7 days with granulocyte macrophage colony stimulating factor and IL-4 in the presence or absence of dexamethasone 10(-8) M (Dex). Dex-treated DC showed an enhancement of mannose receptor (MR)-mediated endocytosis (measured as uptake of FITC-dextran) and of fluid-phase endocytosis [measured as uptake of Lucifer yellow (LY)] The effect was dose dependent and correlated with the length of exposure to Dex.

Ruptured hepatic adenoma in liver adenomatosis: a case report of emergency surgical management.

In hepatic adenomatosis, multiple liver cell adenomas (usually > or = 10) generally affects patients with no prior history of oral contraceptive use, androgenic steroid use, or glycogen storage disease. We report a rare case of a 44 year-old female who underwent emergency surgery for hemoperitoneum due to spontaneous rupture of a liver cell adenoma in hepatic adenomatosis, after prolonged use of two different contraceptives (Gestodene and Ethinylestradiol).

Survival after repeat hepatic resection for recurrent colorectal metastases.

Background/aims: This is a retrospective study examining survival of patients undergoing repeat hepatic resection for recurrent colorectal metastases.

Methodology: The records of 41 patients undergoing hepatic resection for metastatic colorectal cancer were reviewed. Curative resections (negative resection margin and no extrahepatic disease) were attempted in all patients.

The use of Matrigel at low concentration enhances in vitro blastocyst formation and hatching in a mouse embryo model.

Objective: To determine the effect of Matrigel at a low concentration on the growth of mouse embryos in culture.

Design: Randomized case-control study of mouse embryos.

Setting: An academic research environment.

Glucocorticoids affect human dendritic cell differentiation and maturation.

Because dendritic cells (DC) play a major role in the initiation of T cell-mediated immunity, we studied the effects of glucocorticoids, well-known inhibitors of the immune and inflammatory response, on the differentiation and maturation of human DC. DC were differentiated from human monocytes by culture with GM-CSF and IL-4 for 7 days with and without dexamethasone (Dex). Cells treated with Dex (10-8 M) (Dex-DC) developed a characteristic dendritic morphology; however, membrane phenotype analysis demonstrated that they were not fully differentiated.

The frog neuromuscular junction revisited after quick-freezing-freeze-drying: ultrastructure, immunogold labelling and high resolution calcium mapping.

Until now, most ultrastructural studies on the neuromuscular junction have been carried out on samples first exposed to chemical treatments--with fixatives and/or dehydration agents--that are known to induce, or to be inadequate to prevent, artefactual changes of the native state. We report here on the potential of a physical approach to the preparation of samples that combines quick-freezing and freeze-drying (with or without exposure to OsO4 vapours) followed by direct embedding of the samples in various resins. Thin sections from physically processed frog neuromuscular junctions, when compared to their chemically fixed counterparts, exhibit an overall excellent preservation, with the organelles retaining their native density and shape.

Atrial fibrillation in elite athletes.

Introduction: Atrial fibrillation (AF) is a rare event in people younger than 25 years of age, but is probably more frequent in competitive athletes. We analyzed the presence of AF, paroxysmal or chronic, in a population of young elite athletes, including previous Olympic and World champions, who were studied for arrhythmias that endangered their athletic careers.

Methods And Results: From 1974 to June 1977, 1,772 athletes identified with arrhythmias (1,464 males and 308 females; mean age 21 years) underwent individualized work-ups.

Cardiac arrest and sudden death in competitive athletes with arrhythmogenic right ventricular dysplasia.

Arrhythmogenic right ventricular dysplasia (ARVD) is a predisposing factor for sport-related cardiac arrest (CA), sudden cardiac death (SD), and life-threatening ventricular tachyarrhythmias (VT). The aim of this study was the assessment of athletes with ARVD, particularly the CA survivors. From 1974 to January 1996, 1642 competitive athletes (aver.

Inactivation of the zebrafish homologue of Chx10 by antisense oligonucleotides causes eye malformations similar to the ocular retardation phenotype.

We report the cloning of a zebrafish paired-type homeobox gene, Alx, closely related to the murine Chx10 and the gold fish Vsx-I homeodomain proteins. Alx is first expressed at about 12 h post-fertilization (hpf) when optic vesicles appear. Its expression is restricted to the early retinal neuroepithelium, whereas no signal can be detected in the optic placode.

Preferential localization of tyrosine-phosphorylated paxillin in focal adhesions.

Focal adhesions are sites for integrin-mediated attachment of cultured cells to the extracellular matrix. Localization studies have shown that focal adhesions can be stained by antiphosphotyrosine antibodies, but the role of tyrosine-phosphorylated proteins in focal adhesions is not known. By using ventral plasma membranes prepared from chicken embryo fibroblasts spread on the substrate, we present evidence for the preferential localization of a minor pool of tyrosine-phosphorylated paxillin in focal adhesions.

Integrin-mediated tyrosine phosphorylation and redistribution of paxillin during neuronal adhesion.

Integrins are important receptors for neuronal adhesion to laminin, which is one of the best promoters of neurite outgrowth. The present study was carried out to understand some of the intracellular mechanisms which allow integrin-mediated neurite extension on laminin. In chicken retinal neurons, integrin-mediated adhesion to laminin and antibody-induced integrin clustering caused an increase in tyrosine phosphorylation of paxillin and focal adhesion kinase.

Restricted TCR repertoire and long-term persistence of donor-derived antigen-experienced CD4+ T cells in allogeneic bone marrow transplantation recipients.

We investigated the contribution of transfer of Ag-experienced donor T cells to the immune reconstitution of allogeneic bone marrow transplantation (BMT) recipients. To this purpose, we used a combination of cell culture methods to isolate tetanus toxoid (TT)-specific T cell clones, and a sensitive and specific heteroduplex analysis to monitor the presence of a particular clonotype using TCR N region sequences. We document that patients after BMT display a small response to TT, entirely accounted for by few donor-derived clones.

Role of HLA class I in HIV type 1-induced syncytium formation.

Neutralization of HIV-1 in vitro by anti-HLA class I antibodies suggests that class I molecules are involved in HIV-1 infection. HIV-infected cells can fuse with uninfected cells in a process that leads to the formation of multinucleated syncytia, involving an interaction between host and viral antigens expressed at the cell surfaces. We used a syncytium assay between the 8E5 cell line chronically infected with a pol-defective variant of LAV IIIb, and the CD4-positive cell line MOLT3, to study the role of HLA class I in HIV-1-induced cell fusion.

Multiple HLA-A alleles can present an immunodominant peptide of the human melanoma antigen Melan-A/MART-1 to a peptide-specific HLA-A*0201+ cytotoxic T cell line.

The majority of HLA-A*0201-restricted tumor-infiltrating lymphocytes from melanoma patients recognize a peptide, MT(27-35), derived from the Melan-A/MART-1 Ag. This study reports that six variants of HLA-A2 and the HLA-A28 subtype A*6901 can present peptide MT(27-35). A CTL line specific for peptide MT(27-35) was generated by in vitro stimulation of PBL of an HLA-A*0201+, healthy donor with peptide-pulsed, activated autologous B lymphoblasts.

Tyrosine phosphorylation induced by integrin-mediated adhesion of retinal neurons to laminin.

Integrin alpha 6 beta 1 is a laminin receptor involved in adhesion and neurite extension of retinal neurons on laminin. The present study was carried out to understand some of the intracellular mechanisms which allow integrin-mediated neurite extension on laminin in primary neuronal cultures. Both integrin-mediated adhesion to laminin and antibody-induced integrin clustering resulted in the increased tyrosine phosphorylation of a 120 kDa polypeptide which was identified as the focal adhesion kinase.

HIV-1-specific immunity in persistently seronegative individuals at high risk for HIV infection.

A growing number of reports indicates that certain groups of individuals who almost certainly have been exposed to human immunodeficiency virus (HIV), yet continue to exhibit no signs or symptoms of infection, often have subtle evidence of specific immunity. We studied such a high-risk (HR) cohort of persistently seronegative individuals with histories of long-term sexual exposure to an HIV-infected partner to look for evidence of both humoral and cellular immunity that might have been induced by exposure to the virus. Twenty-three heterosexual and four homosexual monogamous couples with discordant HIV status were included in the study.

Human immunodeficiency virus type 1 (HIV-1)-seronegative injection drug users at risk for HIV exposure have antibodies to HLA class I antigens and T cells specific for HIV envelope.

The question of whether persistently seronegative persons at high risk for human immunodeficiency virus type 1 (HIV-1) infection exhibit HIV-1-specific T cell responses and antibodies to HIV-1 envelope epitopes shared with selected HLAs was assessed. These antibodies are not detectable by conventional serologic methods. Envelope-specific helper T (Env-Th) cell responses and antibodies specific for the HIV/HLA epitopes were studied in 21 HIV-1-negative injection drug users (IDUs).

(Ca+Mg)ATPase and calcium influx in erythrocytes of patients with idiopathic hypercalciuria.

Increased erythrocyte (Ca+Mg)ATPase activity was previously observed in idiopathic hypercalciuria. In order to verify if this alteration is a primary or a secondary event, we studied Sr influx in erythrocytes from subjects with idiopathic hypercalciuria. (Ca+Mg)ATPase activity was significantly higher in hypercalciuric than in hypercalciuric than in normocalciuric subjects whereas no difference in Sr influx was found between the two groups.