DNA-based selection and screening of peptide ligands.

Phage display selection strategies rely on the physical link between the displayed heterologous protein ligand and the DNA encoding it. Thus, genes expressing a ligand with a specific binding affinity can be selected rapidly. To improve the specificity and sensitivity of this technology for potential use in identifying ligands to a specific antibody present in a complex mixture, we incorporated a DNA selection step along with the phage display technology. Ligands for hepatitis C virus (HCV) antibodies present in serum were identified by panning a phage-displayed random peptide library against pools of serum HCV antibodies. An additional DNA hybridization screening step using single-stranded DNA isolated from one of the pools increased the specificity and sensitivity, resulting in the selection of an HCV antibody ligand with diagnostic potential.

Double intestinal duplication and incidental neuroendocrine tumor of appendix, a rare case of acute abdomen.

INTRODUCTION: Intestinal duplication is rarely reported in adulthood and often remains undiagnosed until onset of complications. We describe the case of a 39 year old woman who came to our observation for acute abdomen due to a combination of double intestinal duplication (colon and ileum) and an incidental neuroendocrine tumor of the appendix. MATERIALS AND METHODS: A 39 year old woman who was admitted at with upper abdominal pain. Multisliced spiral CT scan showed a cystic lesion suggestive of an inflammed Meckel's diverticulum.The patient was underwent an urgent explorative laparoscopy. The intraoperative findings revealed a cystic lesion of the anti-mesenteric side of transverse colon, apparently dissectable from the bowel and a second lesion with a strongly adherent and unresectable from the anti-mesenteric aspect of the small bowel. A combined appendectomy was also performed. The histological diagnosis was consistent with a typical intestinal duplication for both intestinal lesionsand an incidental 2mm carcinoid tumor was also found in the appendix. The postoperative course was uneventful and the patient was discharged on p.o. day 5. At the presenttime she is well and following a regular oncologic follow-up. DISCUSSION: The rarity of this case is due to the concomitant presence of an incidental, sincronous, appendiceal NET. The elective treatment is surgical resection. CONCLUSION: Intestinal duplication in the adulthood is extremely rare and may either have an acute presentation as acute abdomen or represents an incidental finding of mass. We suggest that, once the diagnosis is suspected patient must undergo surgery.

Human CD4+ T lymphocytes with remarkable regulatory functions on dendritic cells and nickel-specific Th1 immune responses.

The contribution of T helper (Th) and T cytotoxic (Tc) type 1 lymphocytes in the expression of allergic contact dermatitis to haptens has been amply documented. Conversely, the existence of T cell-based regulatory mechanisms has been poorly investigated. Here, we examined the properties of a subset of nickel-specific CD4+ T cells displaying the cytokine profile (IL-10 , IL-5 , IFN-gamma+/-, IL-4+/-) of T regulatory cells 1 (Tr1) and with the potential to down-modulate immune responses to nickel. Tr1 clones were isolated from skin challenged with NiSO4 and peripheral blood of nickel-allergic patients, and from the blood of healthy individuals. Tr1 clones expressed CD25, CD28, CD30, CD26, and the IL-12 receptor beta2 chain upon activation, whereas the lymphocyte activation antigen-3 was present on 50% of the clones. Monocytes precultured with Tr1 cells in the presence of nickel, or treated with Tr1-derived supernatant, exhibited a markedly diminished capacity to stimulate nickel-specific Th1 responses. Tr1 supernatants also blocked the differentiation of dendritic cells (DC) from monocytes, as well as DC maturation and IL-12 production induced by lipopolysaccharide. As a consequence, the ability of DC to stimulate nickel-specific Th1 and Tc1 responses was greatly impaired. These inhibitory effects were completely prevented by IL-10, but not IL-5, neutralization. In aggregate, the results indicate that Tr1 cells can potently regulate the expression of Th1-mediated allergic diseases via release of IL-10.

[Evaluation of the tolerance of a lipid emulsion administrated during total parenteral nutrition in surgical patients]. / Valutazione della tollerabilità di una emulsione lipidica somministrata in corso di nutrizione parenterale totale in pazienti chirurgici.

Tolerance's evaluation of a lipid emulsion given in course of parenteral total nutrition in surgical patient's. The first fat emulsions for intravenous application were thrown on the market in the 1920's years. Authors make a study on a limited sample of surgical patients about type and incidence of both immediate and late adverse reactions versus intravenous administration of Lipofundin S. They also suggest, on the same time, a protocol for the survey of these reactions. The results suggest a good tolerance to Lipofundin S intravenous administration and no influence on haematic biochemical parameters.

[Retroperitoneal liposarcoma]. / I liposarcomi retroperitoneali.

Liposarcoma is the most frequent histotype of the rare and malignant retroperitoneal tumours. This neoplasm has a remarkable tendency to recurrence after surgical excision, rarely to metastasize. Recurrence usually shows a more aggressive behaviour than primitive disease with a higher tendency to penetrate into adjacent organs. The symptomatology often appears late and the first sign is frequently a palpable abdominal mass. Preoperative study involves using CT and MRI. The surgical resection is the only tool able to modify natural history with regard to survival and local recurrence. Prognosis is severe, with a survival of 5 years, variable from 12 to 50% in the different series. A very important prognostic factor is the degree of tumour and radical surgical treatment. From 1990 to 2001, 32 operations for malignant retroperitoneal tumors were performed by our surgical unit in 19 patients; in 7 of them the tumor was a liposarcoma (4 male and 3 female). Total operations for retroperitoneal liposarcoma were 15, in 4 patients a second operation was performed for recurrence, in 3 a third operation and in 1 a fourth. Eleven cases out of 15 have undergone exeresis of sarcoma. Average survival consisted in 4 years and 2 months. Intraoperative radiotherapy, performed in 3 patients, perhaps is able to offer some advantages in relation to local control of the disease and of the survival. A possible resection of eventual recurrence justifies a early follow up with CT and MRI.

[Gastrointestinal stromal tumors]. / I tumori stromali gastrointestinali.

Gastrointestinal stromal tumors (GIST) are generally found in the stomach or small intestine and less commonly in the colon or rectum. Complete surgical removal remains the best current therapy for GISTs. The treatment of advanced GIST patients is with imatinib, a selective tyrosine kinase inhibitor. In our series, 23 patients observed between 1994 and 2004 and affected by GIST were treated with complete negative margin resections (three cases by laparoscopy).

Human dendritic cells are superior to B cells at presenting a major histocompatibility complex class II-restricted heterologous antigen expressed on recombinant Streptococcus gordonii.

Bacteria are being actively investigated as vaccine carriers for inducing or boosting protective immune responses. In this study, human monocyte-derived dendritic cells (DCs) and normal B cells were compared for their capacity to present the C fragment of tetanus toxin (TTFC), expressed on the surface of recombinant Streptococcus gordonii, to specific CD4(+) T lymphocytes. DCs were more efficient than B cells at presenting soluble TTFC and remarkably more capable of presenting bacterium-associated TTFC both in terms of the amount of antigen required to obtain a given T-cell response and on a per-cell basis. This difference was associated with a much lower capacity of B cells to endocytose soluble TTFC and phagocytose recombinant S. gordonii. In addition, S. gordonii induced the phenotypic maturation of DCs but not of B cells. The results thus indicate that DCs but not B cells play a crucial role in the amplification of class II-restricted immune responses induced by immunization with recombinant gram-positive bacteria.

Identification of HCV core mimotopes: improved methods for the selection and use of disease-related phage-displayed peptides.

Disease-specific epitope discovery from random peptide libraries displayed on phage using sera from patients involves a number of screening steps with many immune and non-immune sera. To rapidly identify mimotopes of the human hepatitis C virus (HCV) core protein, we have used an anti-core human monoclonal antibody (mAb; B12.F8) as a probe in screening phage that were affinity-selected using a serum from an HCV infected patient. Three different positive phage were isolated displaying low or no homology with the natural antigen, but which still efficiently bound to the antigen binding site of the B12.F8 antibody. Testing the reactivity of these phage with forty-five sera from HCV infected patients showed that antibodies recognizing them are present in more than 80% of this population. These antibodies showed distinct fine specificity, as they bound the selected phage in a mutually exclusive fashion. Co-expression of two mimotopes in the same cells led to chimeric particles which were recognized by antibodies of different specificity. These data provide novel information on the potential use of the phage display technology for the characterization of antibody specificity as well as disease diagnosis and prevention.

Selection of antigenic and immunogenic mimics of hepatitis C virus using sera from patients.

Using sera from hepatitis C virus (HCV)-infected patients and noninfected subjects to screen random peptide libraries displayed on phage, we selected peptides specifically reacting with sera from infected patients. These phage- borne peptides were shown to mimic distinct HCV determinants. They detected in all cases the presence of anti-HCV Abs in a large panel of patients' sera, thus demonstrating the high sensitivity of the selected peptides as diagnostic markers. In addition, this diagnostic approach allowed a detailed characterization of the individual humoral response to viral infection. Phage-displayed HCV mimics were substitutes for the authentic HCV epitopes in inducing a strong specific response against HCV when used as immunogens in mice. These results support the search for HCV mimics with the potential to elicit a protective immune response as leads for the development of a mimotope-based vaccine against viral infection.

Virus-specific CD8(+) T cells with type 1 or type 2 cytokine profile are related to different disease activity in chronic hepatitis C virus infection.

The present study demonstrates that the quality of the virus-specific CD8(+) T cell responses, as detected by both enzyme-linked immunospot assay and specific MHC-peptide tetramers, changed in relation to the different disease activity in chronically hepatitis C virus-infected patients. Indeed, both the serum alanine transaminase and the hepatic flogosis levels were related directly to the frequencies of peripheral memory effector CD8(+) T cells producing IFN-gamma (Tc1), but inversely to the frequencies of those producing both IL-4 and IL-10 (Tc2). Longitudinal studies highlighted that Tc1 or Tc2 responses fluctuate in relation to the different phases of the disease in the same individual. Furthermore, the Tc1 or Tc2 phenotype correlates with tetramer-positive cells expressing either CXCR3 or CCR3, promoting differential tissue localization of these cells and the maintenance of T cell homeostasis. Finally, studies at the level of liver-infiltrating lymphocytes indicated that they produced both IFN-gamma and IL-4 with an evident bias towards the Tc1-like phenotype. Our studies suggest that the progressive fluctuation of Tc1 and Tc2 responses may play a fundamental role in maintaining a long-lasting low-level liver inflammation, and may constitute the basis for new therapeutic strategies of immune regulation.