Different patterns of 11q allelic losses in digestive endocrine tumors

Most foregut digestive endocrine neoplasms may be associated with the multiple endocrine type 1 (MEN-1) syndrome. In contrast, midgut/hindgut carcinoids never show such association. To investigate the pathogenetic involvement of the MEN-1 gene and of putative additional oncosuppressor gene(s) distal to it, a comparative analysis of loss of heterozygosity (LOH) at chromosome 11q13 to 11qter was performed in 27 foregut (pancreatic endocrine tumors [PETs]), 23 midgut (ileal and appendiceal), and 3 hindgut (rectal) endocrine tumors. LOH at the MEN-1 gene locus at 11q13 was observed in 52% of the 23 sporadic and in all 4 MEN-1-associated PETs and was found to consistently and continuously span to the most distal marker investigated at 11qter. In contrast, only occasional, discontinuous, and mostly interstitial LOH for 11q markers was observed in ileal (midgut) carcinoids, whereas no LOH was found in all appendiceal (midgut) and rectal (hindgut) carcinoids. The consistent extension of LOH from the MEN-1 region to 11qter in sporadic PETs suggests a mechanism of gene inactivation via chromosomal breakage and complete loss of chromosome 11q; furthermore, these results expand beyond the 11q13 region the search for additional oncosuppressor gene(s) potentially involved in the genesis of these neoplasms. The low frequency, limited extension, and discontinuous distribution of 11q deletions in midgut/hindgut carcinoids suggest that MEN-1 gene is not involved in the pathogenesis of these tumors.     

Basic fibroblast growth factor: effects on matrix remodeling, receptor expression, and transduction pathway in human periosteal fibroblasts with FGFR2 gene mutation.

The Crouzon syndrome, which is associated with fibroblast growth factor receptor (FGFR2) mutations, is characterized by premature fusion of cranial sutures. We used an in vitro model of cultured periosteal fibroblasts from normal subjects and from Crouzon patients with FGFR2 mutation. We analyzed the matrix turnover rate and the effects of adding FGF2 by evaluating fibronectin synthesis and the activity of some proteolytic enzymes. To assess the role of some FGF signaling molecules involved in FGFR2 regulation, we studied Grb2 tyrosine phosphorylation and the phosphotyrosine proteins associated with Grb2. The iodinate FGF binding assay was performed to quantify FGFR expression. Compared with normal fibroblasts, fibronectin synthesis was decreased in Crouzon fibroblasts, and protease activities in cells and medium were enhanced, suggesting that excess fibronectin catabolism is present. Differences were more marked when FGF2 was added. Very few phosphoproteins were visible in anti-Grb2 immunoprecipitations from Crouzon fibroblasts, which showed a significant increase in the number of high-affinity and low-affinity FGF2 receptors. These results suggest that the abnormal genotype and the Crouzon cellular phenotype are related. To compensate the low levels of tyrosine phosphorylation, Crouzon cells might increase the numbers of FGFR2, thus increasing the cell surface binding sites for FGF2.     

Identification of the basic subunit of Ara h 3 as the major allergen in a group of children allergic to peanuts.

BACKGROUND: Several proteins have been identified as peanut allergens; among them, Ara h 1 (7S globulin) and Ara h 2 (2S globulin) are usually considered the major allergens. OBJECTIVE: To identify the major allergens in a group of children selected for their specific pattern of immunoreactivity. METHODS: We identified the dominant allergen by using (1) amino acid sequencing of the bands that show the strongest IgE immunoreactivity in 1-dimensional electrophoresis and immunoblotting and (2) specific animal IgGs raised against the dominant immunoreactive band to pinpoint the allergen(s) in peanut proteins separated by 2-dimensional electrophoresis and immunoblotting. To confirm these data, we further examined the peanut proteome using serum samples from the children with the unusual immunoreactivity. RESULTS: We found a group of children with marked peanut allergy who are specifically sensitized to the basic subunit of Ara h 3 (11S globulin family). CONCLUSION: That the dominant immunoreactivity in these patients is in a basic subunit of Ara h 3 was unexpected, because previous studies had indicated that Ara h 3 was only a minor peanut allergen and that the identified allergenic epitopes occurred mainly in the acidic Ara h 3 subunit.     

Mild mental retardation in a child with a de novo interstitial deletion of 15q21.2q22.1: a comparison with previously described cases

We report on a child with mild mental retardation, hypotelorism, blepharophimosis, face slight asymmetry and partial hypoplasia of corpus callosum, with an interstitial deletion of a chromosome 15. The deletion was molecularly characterized by array-CGH and FISH techniques. This rearrangement has a 7.18 Mb extension and maps to 15q21.2q22.1. To date, there have been only six individuals reported with a deletion of 15q21; in three cases, the rearrangement was characterized by molecular cytogenetic techniques. After a comparison with these three cases, it appeared that the deletion we found is one of the smallest and it overlaps the distal portion of the ones taken into account. Finally, we tried to delineate the genotype–phenotype correlation in patients with a deletion of 15q21.     

Vaccine vectors derived from a large collection of simian adenoviruses induce potent cellular immunity across multiple species

Replication-defective adenovirus vectors based on human serotype 5 (Ad5) induce protective immune responses against diverse pathogens and cancer in animal models, as well as elicit robust and sustained cellular immunity in humans. However, most humans have neutralizing antibodies to Ad5, which can impair the immunological potency of such vaccines. Here, we show that rare serotypes of human adenoviruses, which should not be neutralized in most humans, are far less potent as vaccine vectors than Ad5 in mice and nonhuman primates, casting doubt on their potential efficacy in humans. To identify novel vaccine carriers suitable for vaccine delivery in humans, we isolated and sequenced more than 1000 adenovirus strains from chimpanzees (ChAd). Replication-defective vectors were generated from a subset of these ChAd serotypes and screened to determine whether they were neutralized by human sera and able to grow in human cell lines. We then ranked these ChAd vectors by immunological potency and found up to a thousandfold variation in potency for CD8+ T cell induction in mice. These ChAd vectors were safe and immunologically potent in phase 1 clinical trials, thereby validating our screening approach. These data suggest that the ChAd vectors developed here represent a large collection of non-cross-reactive, potent vectors that may be exploited for the development of new vaccines.     

Toll-like receptors 3 and 4 are expressed by human bone marrow-derived mesenchymal stem cells and can inhibit their T-cell modulatory activity by impairing Notch signaling

Bone marrow (BM)-derived mesenchymal stem cells (MSCs) are multipotent, nonhemopoietic progenitors that also possess regulatory activity on immune effector cells through different mechanisms. We demonstrate that human BM-derived MSCs expressed high levels of Toll-like receptors (TLRs) 3 and 4, which are both functional, as shown by the ability of their ligands to induce nuclear factor kappaB (NF-kappaB) activity, as well as the production of interleukin (IL)-6, IL-8, and CXCL10. Of note, ligation of TLR3 and TLR4 on MSCs also inhibited the ability of these cells to suppress the proliferation of T cells, without influencing their immunophenotype or differentiation potential. The TLR triggering effects appeared to be related to the impairment of MSC signaling to Notch receptors in T cells. Indeed, MSCs expressed the Notch ligand Jagged-1, and TLR3 or TLR4 ligation resulted in its strong downregulation. Moreover, anti-Jagged-1 neutralizing antibody and N[N-(3,5-difluorophenacetyl-l-alanyl)]-S-phenylglycine t-butyl ester (DAPT), an inhibitor of Notch signaling, hampered the suppressive activity of MSCs on T-cell proliferation. These data suggest that TLR3 and TLR4 expression on MSCs may provide an effective mechanism to block the immunosuppressive activity of MSCs and therefore to restore an efficient T-cell response in the course of dangerous infections, such as those sustained by double-stranded RNA viruses or Gram-negative bacteria, respectively.     

Oral sensitization with shrimp tropomyosin induces in mice allergen-specific IgE, T cell response and systemic anaphylactic reactions

Appropriate murine models of shrimp tropomyosin (ST) allergy would be useful in investigating the mechanisms underlying food allergy in human subjects, as well as for the pre-clinical evaluation of efficacy and safety of novel therapeutic approaches. These models should mimic immune and clinical features of human disease, including anaphylactic response. We sensitized C3H/HeJ mice by the oral route with purified ST using cholera toxin (CT) as adjuvant. ST-specific IgE, IgG1, IgG2a and IgA responses were evaluated by ELISA. Spleen cell proliferation and cytokine production by allergen-specific activation were assessed. Jejunum and colon fragments were collected to evaluate the local expression of cytokine genes by PCR. Local and systemic anaphylactic reactions induced by oral ST challenge were scored according to symptoms observed. Faecal samples were collected to assess local IgA production and histamine levels. Oral sensitization with ST plus CT induced in mice significant levels of serum IgE and IgG1 and faecal IgA. ST-specific cell proliferation and IL-4, IL-13 and IFN-gamma cytokine production were induced in the spleen. After oral challenge, 100% of mice had anaphylactic symptoms while no symptoms were observed in challenged naive mice. Faecal histamine content after ST challenge appeared significantly increased in sensitized mice when compared with that observed in pre-immune mice. Jejunum mRNA expression of T(h)2 cytokines was up-regulated by ST sensitization. These results support the importance of the oral way of sensitization and of the in-depth characterization of the anaphylactic response for the development of a suitable in vivo model of food allergy.     

Vascular endothelial growth factor stimulates endothelial colony forming cells proliferation and tubulogenesis by inducing oscillations in intracellular Ca2+ concentration

Endothelial progenitor cells (EPCs) home from the bone marrow to the site of tissue regeneration and sustain neovascularization after acute vascular injury and upon the angiogenic switch in solid tumors. Therefore, they represent a suitable tool for cell-based therapy (CBT) in regenerative medicine and provide a novel promising target in the fight against cancer. Intracellular Ca(2+) signals regulate numerous endothelial functions, such as proliferation and tubulogenesis. The growth of endothelial colony forming cells (ECFCs), which are EPCs capable of acquiring a mature endothelial phenotype, is governed by store-dependent Ca(2+) entry (SOCE). This study aimed at investigating the nature and the role of VEGF-elicited Ca(2+) signals in ECFCs. VEGF induced asynchronous Ca(2+) oscillations, whose latency, amplitude, and frequency were correlated to the growth factor dose. Removal of external Ca(2+) (0Ca(2+)) and SOCE inhibition with N-(4-[3,5-bis(trifluoromethyl)-1H-pyrazol-1-yl]phenyl)-4-methyl-1,2,3-thiadiazole-5-carboxamide (BTP-2) reduced the duration of the oscillatory signal. Blockade of phospholipase C-γ with U73122, emptying the inositol-1,4,5-trisphosphate (InsP(3))-sensitive Ca(2+) pools with cyclopiazonic acid (CPA), and inhibition of InsP(3) receptors with 2-APB prevented the Ca(2+) response to VEGF. VEGF-induced ECFC proliferation and tubulogenesis were inhibited by the Ca(2+)-chelant, BAPTA, and BTP-2. NF-κB activation by VEGF was impaired by BAPTA, BTP-2, and its selective blocker, thymoquinone. Thymoquinone, in turn, suppressed VEGF-dependent ECFC proliferation and tubulogenesis. These data indicate that VEGF-induced Ca(2+) oscillations require the interplay between InsP(3)-dependent Ca(2+) release and SOCE, and promote ECFC growth and tubulogenesis by engaging NF-κB. This novel signaling pathway might be exploited to enhance the outcome of CBT and chemotherapy.