Compositional and Morphological Characterization of 'Sorrento' and 'Chandler' Walnuts.

In Italy, most of the cultivated walnuts belong to the Sorrento ecotype, and they are considered commercially valuable due to their specific organoleptic characteristics. The aim of this study is to evaluate and compare the morphological and compositional characteristics of walnuts sampled from 'Sorrento' trees cultivated in different locations in Campania and trees of both the 'Chandler' and 'Sorrento' varieties derived from the same location. The results demonstrated that 'Sorrento' and 'Chandler' walnuts have different biometric characteristics and a different fat content, with the highest fat content being found in the 'Sorrento' variety. Regarding the fatty acid (FA) composition, the content of monounsaturated and saturated fatty acids (MUFAs and SFAs) was highest in the 'Sorrento' variety (from 13 to 15% for MUFAs and from 11 to 13% for SFAs), while the polyunsaturated fatty acids (PUFAs) content was highest in the 'Chandler' variety (77%). The total phenolics content (TPC) was highest in the 'Sorrento' variety (from 910 to 1230 mg GAE/100 g), while no difference in γ-tocopherol content was found. Furthermore, the influence of walnut area cultivation was shown for fat content, FA composition and TPC. Therefore, both walnut varieties demonstrated good nutritional properties considering the PUFAs and γ-tocopherol content.

Distinct subcellular localization of the neuronal marker HuC/D reveals hypoxia-induced damage in enteric neurons.

BACKGROUND: Correct neuronal identification is essential to study neurons in health and disease. Although commonly used as pan-neuronal marker, HuC/D's expression pattern varies substantially between healthy and (patho)physiological conditions. This heterogenic labeling has received very little attention. We sought to investigate the subcellular HuC/D localization in enteric neurons in different conditions. METHODS: The localization of neuronal RNA-binding proteins HuC/D was investigated by immunohistochemistry in the mouse myenteric plexus using different toxins and caustic agents. Preparations were also stained with Sox10 and glial fibrillary acidic protein (GFAP) antibodies to assess enteric glial cell appearance. KEY RESULTS: Mechanically induced tissue damage, interference with the respiratory chain and oxygen (O2 ) deprivation increased nuclear HuC/D immunoreactivity. This effect was paralleled by a distortion of the GFAP-labeled glial network along with a loss of Sox10 expression and coincided with the activation of a non-apoptotic genetic program. Chemically induced damage and specific noxious stimuli did not induce a change in HuC/D immunoreactivity, supporting the specific nature of the nuclear HuC/D localization. CONCLUSIONS & INFERENCES: HuC/D is not merely a pan-neuronal marker but its subcellular localization also reflects the condition of a neuron at the time of fixation. The functional meaning of this change in HuC/D localization is not entirely clear, but disturbance in O2 supply in combination with the support of enteric glial cells seems to play a crucial role. The molecular consequence of changes in HuC/D expression needs to be further investigated.

Neurotransmitters involved in fast excitatory neurotransmission directly activate enteric glial cells.

BACKGROUND: The intimate association between glial cells and neurons within the enteric nervous system has confounded careful examination of the direct responsiveness of enteric glia to different neuroligands. Therefore, we aimed to investigate whether neurotransmitters known to elicit fast excitatory potentials in enteric nerves also activate enteric glia directly. METHODS: We studied the effect of acetylcholine (ACh), serotonin (5-HT), and adenosine triphosphate (ATP) on intracellular Ca(2+) signaling using aequorin-expressing and Fluo-4 AM-loaded CRL-2690 rat and human enteric glial cell cultures devoid of neurons. The influence of these neurotransmitters on the proliferation of glia was measured and their effect on the expression of c-Fos as well as glial fibrillary acidic protein (GFAP), Sox10, and S100 was examined by immunohistochemistry and quantitative RT-PCR. KEY RESULTS: Apart from ATP, also ACh and 5-HT induced a dose-dependent increase in intracellular Ca(2+) concentration in CRL-2690 cells. Similarly, these neurotransmitters also evoked Ca(2+) transients in human primary enteric glial cells obtained from mucosal biopsies. In contrast with ATP, stimulation with ACh and 5-HT induced early gene expression in CRL-2690 cells. The proliferation of enteric glia and their expression of GFAP, Sox10, and S100 were not affected following stimulation with these neurotransmitters. CONCLUSIONS & INFERENCES: We provide evidence that enteric glial cells respond to fast excitatory neurotransmitters by changes in intracellular Ca(2+). On the basis of our experimental in vitro setting, we show that enteric glia are not only directly responsive to purinergic but also to serotonergic and cholinergic signaling mechanisms.

Proinflammatory stimuli activates human-derived enteroglial cells and induces autocrine nitric oxide production.

BACKGROUND: Enteric glial cells (EGCs) have been recently indicated as key regulators of intestinal inflammation in animals. Whether or not this is true and how these cells participate to inflammatory responses in humans is unknown. METHODS: We isolated primary EGCs from human small bowel and then, we purified and characterized those using specific glial markers, such as S100B and glial fibrillary acidic protein (GFAP). To mimic an inflammatory scenario, we exposed EGCs to exogenous stimuli, such as lipopolysaccharide and interferon-gamma (LPS and IFN-γ), alone or in combination, to evaluate glial activation [measuring GFAP, S100B level together with c-fos, major histocompatibility complex (MHC) class II, inducible nitric oxide (iNOS) proteins expression and nitric oxide (NO) production] and proliferation, respectively. KEY RESULTS: We showed that, when challenged with a combination of LPS and IFN-γ, EGCs are significantly activated, as indicated by their positivity to c-fos and MHC class II. Similarly, pro-inflammatory stimuli significantly increase the cell proliferation rate, the expression of both S100B and GFAP, and the NO production consequent to the induction of EGCs-derived iNOS protein, with the last being dependent on S100B-RAGE (receptor for advanced glycation endproducts) interaction. CONCLUSIONS & INFERENCES: Our data provide the first evidence that human EGCs directly respond to pro-inflammatory stimuli by changing their expression profile and by proliferating. The finding that stimulated EGCs are able to produce NO points to a role of this cell population in the scenario of intestinal inflammation.

Viral-load and B-lymphocyte monitoring of EBV reactivation after allogeneic hemopoietic SCT in children.

EBV-associated post transplant lymphoproliferative disease (EBV-PTLD) is a life-threatening complication that may occur after hemopoietic SCT. We prospectively screened 80 children on a weekly basis using nested quantitative PCR to evaluate EBV genome copies. EBV viral load <1000 copies per 10(5) PBMC was observed in 63% of transplants, whereas it was between 1000 and 9999 copies per 10(5) PBMC in 13%, and between 10 000 and 19 999 in 10%, with no significant increase in percentage of CD20+ lymphocytes. Viral load reached > or = 20 000 copies per 10(5) PBMC in 14% of patients, and rituximab was administered to 75% of them. None of the patients except one developed a lymphoproliferative disease. Our study found that only 13% of unrelated donor HSCT recipients had a very high risk of EBV-PTLD defined as > or = 20 000 geq per 10(5) PBMC associated with an increase in CD20+ lymphocyte. We suggest that rituximab could be administered in the presence of very high levels of EBV-DNA viral load or in the presence of mid levels of EBV-DNA viral load associated with an increase in the percentage of CD20+ lymphocytes. Through this approach, we significantly reduced the number of patients treated with rituximab, and consequently the acute and chronic adverse events related to this treatment.

Increased mucosal nitric oxide production in ulcerative colitis is mediated in part by the enteroglial-derived S100B protein.

In the central nervous system glial-derived S100B protein has been associated with inflammation via nitric oxide (NO) production. As the role of enteroglial cells in inflammatory bowel disease has been poorly investigated in humans, we evaluated the association of S100B and NO production in ulcerative colitis (UC). S100B mRNA and protein expression, inducible NO synthase (iNOS) expression, and NO production were evaluated in rectal biopsies from 30 controls and 35 UC patients. To verify the correlation between S100B and NO production, biopsies were exposed to S100B, in the presence or absence of specific receptor for advanced glycation end-products (RAGE) blocking antibody, to measure iNOS expression and nitrite production. S100B and iNOS expression were evaluated after incubation of biopsies with lipopolysaccharides (LPS) + interferon-gamma (IFN-gamma) in the presence of anti-RAGE or anti-S100B antibodies or budesonide. S100B mRNA and protein expression, iNOS expression and NO production were significantly higher in the rectal mucosa of patients compared to that of controls. Exogenous S100B induced a significant increase in both iNOS expression and NO production in controls and UC patients; this increase was inhibited by specific anti-RAGE blocking antibody. Incubation with LPS + IFN-gamma induced a significant increase in S100B mRNA and protein expression, together with increased iNOS expression and NO production. LPS + IFN-gamma-induced S100B up-regulation was not affected by budesonide, while iNOS expression and NO production were significantly inhibited by both specific anti-RAGE and anti-S100B blocking antibodies. Enteroglial-derived S100B up-regulation in UC participates in NO production, involving RAGE in a steroid insensitive pathway.

Rare viral infections in children receiving hemopoietic stem cell transplant.

Viral infections are a rare complication in autologous hemopoietic stem cell transplant (HSCT) recipients but represent a frequent cause of disease after allogeneic HSCT. In the last years, there has been an increase in the number of viral diseases observed in these patients. This fact may be at least partially due to an improvement in diagnostic facilities, but the increasing number of transplant procedures and the more severe immunosuppression may also have played an important role.

[The Hartmann procedure in colorectal emergencies. Report of 76 cases]. / L'intervento di Hartmann nelle urgenze colo-rettali. Riflessioni su 76 casi.

The Hartmann procedure has, in emergency colo-rectal surgery, many implications. The decision is based on clinical, radiological, instrumental and pathological findings. The authors report the results of 76 Hartmann's procedures performed between 1986 and 1995 and compare their results with those found in the current medical literature on the topic. In particular, they draw attention to an increased use of this procedures in colo-rectal emergencies; the morbility and mortality rates confirm the severity of the clinical cases that can be treated with this operation. To improve results the authors propose a therapeutic plan that uses a score for stratification of the risk (MPI, APA-CHE II, SSI, Hinchey); so the surgeon can choose the best surgical operation. Finally, the authors underline the importance of the principles of oncology surgery in colo-rectal cancer.

Adhesion of human neuroblasts to HIV-1 tat.

Several neuropathologic findings in infants and children with human immunodeficiency virus type-1 (HIV-1) infection are different from those observed in adults, probably related to the fact that the retroviral infection occurs in the setting of neuro-development. This report describes the interaction and biologic activity of tat, the HIV-1 trans-activating protein on human neuroblasts. Two human neuroblastoma cell lines, LAN-5 and GI-CA-N, have been studied for their capability to adhere to tat (full recombinant protein) and to two different peptide residues of it. Both cells adhere to tat and tat46-60 basic domain, although not to tat65-80 residue, which contains the RGD (arginine-glycine-aspartic acid) motif. Adhesion to collagen I was inhibited by preincubating GI-CA-N cells with tat,46-60 although not with tat,65-80 indicating the capability of the basic residue to interfere with collagen I-induced cellular adhesion. The expression of 200-kD neurofilaments induced by collagen I was not induced by tat,46-60 indicating that neural differentiation along the same pathway is not mimicked by this peptide. Neuroblast cell proliferation was not affected by adhesion to tat46-60 nor to tat.65-80 GI-CA-N cells are not permissive to HIV-1 infection. However, proviral DNA was documented in the cell lysate for 14 consecutive in vitro passages, whereas HIV-1 transcription was never detectable. This would exclude the possibility that tat would be transduced by these cells. GI-CA-N stained negative for CD4, although positive for Gal-C, which may explain HIV-1 entry. Results show that immature human neural cells interact with tat protein and/or its basic residue in vitro. A mechanism similar to that herein described would possibly be active in vivo, which may help in clarifying the pathogenic mechanisms of neurologic dysfunction and destruction of the CNS observed in infants infected with HIV-1.

Effect of interferon-alpha therapy in a patient with common variable immunodeficiency and chronic Epstein-Barr virus infection.

We report an 18-year-old boy with common variable immunodeficiency who presented with splenomegaly as well as left axillary and lateral cervical lymphadenopathy. Main laboratory investigations showed severe thrombocytopenia. Epstein-Barr virus (EBV) DNA was detected in the patient's throat-washing specimens and lymph node biopsy. Lymphocytes from the lymph node biopsy were also positive for EBV nuclear antigen. Serology for EBV and cytomegalovirus was negative. A therapeutic attempt with acyclovir did not influence the course of infection. Six months' treatment with human lymphoblastoid interferon-alpha (IFN alfa) brought about the normalization of clinical and hematologic conditions. Detection on throat-washing specimens carried out 1 year after therapy was negative. Our preliminary experience suggests that human lymphoblastoid IFN-alpha is a valid alternative in therapy of immunodeficient EB virus-infected patients.

Dual-centre, double-blind, randomised trial of lymphoblastoid interferon alpha with or without steroid pretreatment in children with chronic hepatitis B.

Thirty-five children with chronic HBV infection, HBV-DNA and eAg serum positivity, and HBcAg in liver tissue were treated with lymphoblastoid human interferon alpha with (16 cases) or without (19 cases) prednisolone pretreatment. The patients were double-blind randomized to receive steroid or placebo for 4 weeks, followed after 2 weeks by 5 or 10 MU/m2 interferon for 12 weeks. The e anti-e seroconversion rate reached 48%, which is much higher than the spontaneous seroconversion rate. The influence of "prednisolone priming" was not statistically significant. HBeAg clearance was similar in both groups (44% after prednisolone/interferon and 53% after interferon alone). The response to either treatment did not correlate with the pretreatment serum transaminase. HBV-DNA or degree of histological activity. Interferon was well tolerated, the side effects being less severe than in adults, and never led to suspension of the treatment.

A human acute leukemia-derived T-cell line produces two inhibitor factors which suppress lymphocyte proliferation: characterization and purification of the molecules.

The crude supernatant of an CD1+/CD8+ T-cell clone (GI-CO-T-9) established from a long standing culture of an acute T-lymphoblastic leukemia (T-ALL) was shown to inhibit the responsiveness of normal PBMC to PHA. This clone does not produce TNF-alpha, TNF-beta, alpha-IFN, tau-IFN, IL-1, IL-2 and has no NK-like activity. The crude supernatant has been subjected to a multi-step chromatographic fractioning. Preparative gel permeation HPLC allowed us to recover two peaks of biologic activity in the range of 100-120 kDa and 75-85 kDa (referred to as "high molecular mass inhibitor factor", HMMIF, and "low molecular mass inhibitor factor", LMMIF, respectively). Both fractions were then subjected to anion exchange HPLC: HMMIF was recovered in fractions eluting at 0.04 M NaCl while LMMIF eluted at higher ionic strength (0.48 M NaCl). The fractions with biologic activity recovered from anion exchange HPLC have been subjected to hydrophobic interaction HPLC (HIC) for final purification. The highly purified material was characterized by polyacrylamide gel electrophoresis with and without sodium dodecyl sulfate (SDS) revealing single bands of 115 kDa and 80 kDa. The isoelectric points (pI), determined by flat-bed isoelectricfocusing, were 7.4 for HMMIF and 3.5-3.6 for LMMIF. Studies on temperature lability indicated that both proteins are stable for 3-4 hours at room temperature (RT), 24-36 hours at +4 degrees C and 7-10 days at -80 degrees C.

Purification to homogeneity and biochemical characterization of two suppressor factors from human malignant T-cells.

A cell clone (GI-CO-T-9) derived from a long term T-cell culture (PF-382), established from a patient affected by acute T-lymphoblastic leukemia (T-ALL), was selected for the presence in the culture medium of factors suppressing T-cell proliferation. The crude supernatant has been subjected to a multi-step chromatographic fractioning, including: preparative gel permeation, anion exchange, and hydrophobic interaction High Performance Liquid Chromatography (HPLC). The highly purified material was characterized by polyacrylamide gel electrophoresis in sodium dodecyl sulfate (SDS-PAGE), revealing single bands of 115 Kd and 80 Kd. The isoelectric points (pI), determined by flat-bed isoelectric-focusing, were 7.4 for High Molecular Weight Suppressor Factor (HMWSF) and 3.5-3.6 for Low Molecular Weight Suppressor Factor (LMWSF).

[Immunosuppressive factors produced by a T cell line derived from acute lymphoblastic leukemia]. / Fattori immunosoppressivi prodotti da una linea cellulare T derivante da una leucemia linfoblastica acuta.

The supernatant of CD8+ cells, isolated from a permanent lymphoblastoid cell clone established from a long term culture of a T cell acute lymphoblastic leukemia, contained two distinct molecules with suppressive activity on PHA1-induced PBMC proliferation. This clone does not produce TNF-alpha, TNF-beta, alpha-IFN, gamma-IFN, IL-1, IL-2 and has not natural killer activity. In the attempt to purify and biochemically characterize the lymphoblastic cell line-derived T-cell SFs, a multi-step chromatographic separation has been used. Two different peaks of biologic activity have been separated by HPLC gel permeation in the range of 100-120 Kd and 75-85 Kd referred to as high molecular weight suppressor factor HMWSF, and low molecular weight suppressor factor LMWSF, respectively. These fractions were then concentrated, dialyzed and further purified by anion exchange HPLC. This chromatographic step allowed us to considerably purify the two SFs. The biologically active fractions derived from the previous chromatographic step were eventually subjected to hydrophobic interaction HPLC (HIC) for final purification. The highly purified material was characterized by polyacrylamide gel electrophoresis in sodium-dodecylsulfate (SDS-PAGE): a single band corresponding to 115 Kd was observed for HMWSF, while LMWSF yielded a single band at 80 Kd. The isoelectric points (pI) of the different SFs was determined by flat-bed isoelectric-focusing: the HMWSF yielded a single band at pI 7.4, while a much lower pI was observed for LMWSF, 3.5-3.6. Studies on temperature lability indicated that both proteins are stable for 3-4 hours at room temperature (RT), 24-36 hours at +4 degrees C and 7-10 days at -80 degrees C.

Effect of cytosine arabinoside on the growth and phenotypic expression of GI-ME-N, a new human neuroblastoma cell line.

Cytosine-arabinoside (ARA-C) effects on a new human neuroblastoma cell line (GI-ME-N), recently established in our laboratory, have been extensively tested. Low doses of ARA-C allowing virtually 100% cell viability induce morphological differentiation and growth inhibition; differentiated cells appear larger and flattened with elongated dendritic processes; such cells appeared within 48 hours after a dose of ARA-C as low as 0.1 microgram/ml. The new morphological aspect reached the maximum expression after 5-6 days of culture being independent of the addition of extra drug to the culture. A decrease in (3H)-thymidine incorporation was also observed within 48 hours being the cell growth completely inhibited at the 6th day. Membrane immunofluorescence with specific monoclonal antibodies showed several dramatic changes in NB-specific antigen expression after 5 days of treatment with ARA-C. These findings suggest that low ARA-C doses promote the differentiation of GI-ME-N neuroblastoma cells resulting in an altered expression of the malignant phenotype.