Sulfonylurea-responsive neonatal diabetes mellitus diagnosed through molecular genetics in two children and in one adult after a long period of insulin treatment.

A permanent neonatal diabetes mellitus has finally been diagnosed through molecular genetics in two children and one adult after 9 to 35 years of uninterrupted insulin treatment. These patients developed diabetes before 6 months of age and were autoantibody negative. In one boy, a mutation in the KCNJ11 gene was identified at 9 years of age. In the other two patients (daughter and father, 12.6 and 25 years old respectively) the new gene variant (ABCC8/L213P) was found. Switching from insulin to sulfonylurea treatment leads to the definitive discontinuance of insulin therapy, improving metabolic control as well as the amelioration of the associated neurodevelopmental disabilities in the young girl in which an intermediate Development Delay, Epilepsy, Neonatal Diabetes syndrome was diagnosed.

Severe combined immunodeficiency (SCID): from molecular basis to clinical management.

Primary immune deficiency diseases (PID) comprise a genetically heterogeneous group of disorders that affect distinct components of the innate and adaptive immune system, such as neutrophils, macrophages, dendritic cells, complement proteins, natural killer cells, as well as T and B lymphocytes. Severe combined immunodeficiency (SCID) is a group of disorders characterized by increased susceptibility to severe infections and early death. The diagnosis of SCID is supported by the demonstration of low absolute lymphocyte count and T cell lymphopenia (variably associated with numerical defects of B and NK cells). In the last two decades, advances in the characterization of the molecular pathophysiology of SCID, have permitted the development of novel diagnostic assays based on analysis of the expression of the disease-associated proteins and mutation analysis. More recently, pilot newborn screening programs for the identification of infants with SCID have been initiated in the United States. Prompt and aggressive treatment of infections, antimicrobial prophylaxis (in particular against Pneumocystis jiroveci) and regular administration of immunoglobulins are essential to reduce the risk of early death. However, survival ultimately depends on reconstitution of immune function, that is usually achieved by means of hematopoietic cell transplantation (HCT). Gene therapy and enzyme replacement therapy have also been used successfully is selected forms of SCID. Here we review the molecular and cellular pathophysiology and the mainstay of treatment of SCID.

Celiac disease in children with type 1 diabetes: impact of gluten free diet on diabetes management.

UNLABELLED: Background and aim of the work the coexistence of Type 1 Diabetes (T1D) and celiac disease (CD) has been long established. METHODS: Between January 2000 and December 2009, biopsy-proven CD was diagnosed in 12 children with T1D, giving a prevalence of 4.8 % in our out-patient clinic population. For each patient with coexisting T1D and CD, two control subjects with T1D and without CD who matched for age, sex and duration of diabetes were chosen. Prospective study follow up lasted 24 months. At the enrolment time, and at 2-month intervals, time from diagnosis of T1D to diagnosis of CD, presence of gastrointestinal symptoms, HbA1c value, body mass index (BMI), Height and Weight SDS were collected by a single observer. Daily insulin requirements were also retained. RESULTS: In 3 children, CD predated the onset of T1D and these children were excluded from the analysis. The 9 children who subsequently developed CD became earlier diabetic than control group (p=0.002). Eight of these children had CD diagnosis within 1 year after T1D onset. Seven out of 9 children were positive for TTG antibodies and all were positive for EMA. A significant increase in insulin requirement was found in CD children after 1 year of GFD (p= 0.02). The mean HbAlc value in CD children was higher than in the control subjects (p<0.01).A significant increase in the insulin requirement after 1 year in the GFD compliant children was found. There was a significant improvement in height-SDS after institution of GFD in the GFD-compliant children. Families of children with both T1D and CD reported higher burden than those affected by T1D only (p=0.001). The health care providers perceived family burden to increase with CD appearance (p<0.05). CONCLUSION: Our study supports the importance of screening for CD in children with T1D 1. The early treatment with GFD of biopsy-confirmed CD children promotes a significant catch-up growth and prevents a growth failure during the follow-up.

Sedentary lifestyle in active children admitted to a summer sport school.

Aim of this study was to investigate the sedentary patterns of school-aged active children admitted to a summer sport school. One hundred-twelve children aged 9-11 years were interviewed through a questionnaire about sedentary behaviours and nutrition habits. Seventy-one per cent of children reported they watch TV seven days a week, girls less than boys (84 +/- 45 minutes vs. 110 +/- 75 minutes) (t = 2.056; p = 0.042). The habit of TV viewing during meals was widespread (38% breakfast, 31% lunch, 62% dinner, 18% every meal). The prevalence of overweight or obesity (58.5%) was significantly higher among boys watching TV at dinner compared to the boys viewing TV only in the afternoon (35%) (chi2 = 4.976; p = 0.026). Fifty-seven per cent of children (65% boys) were accustomed to nibble snacks during TV viewing, and this habit was widespread in overweight or obese boys (chi2 = 4.546; p = 0.033). The dietary patterns of children watching TV include more snack foods and fewer fruits than the dietary patterns of the same children exercising (chi2 = 4.199 p = 0.040). Also in active children the habit to watch television is widespread and, in spite of the tendency to physical activity, 46% of them were overweight or obese; in fact the time spent looking at a TV may be associated to overweight/obesity and this relationship could be explained by the amount of high-density foods consumption during inactivity. Playing video games, read a book and listening to music are sedentary lifestyle patterns but these seem not to represent a risk factor for an increased BMI.

TRAIL-induced apoptosis of FHIT-negative lung cancer cells is inhibited by FHIT re-expression.

Fragile histidine triad (FHIT) is a tumor suppressor gene whose allelic loss is associated to a number of human cancers. FHIT protein acts as a diadenosine oligophosphate hydrolase, but its tumor suppressive activity appears as independent from its enzymatic activity. Tumor necrosis factor (TNF)-related apoptosis inducing ligand (TRAIL) can induce apoptosis in the FHIT-negative non-small lung cancer cell line Calu-1. We generated four FHIT-inducible Calu-1 cell clones and demonstrated that FHIT expression was able to protect cells from TRAIL-induced apoptosis, without affecting TRAIL-receptors surface expression. FHIT-specific small interference RNA transfection of SV40-immortalized normal bronchial BEAS cells that show levels of FHIT protein comparable to those of normal bronchial cells, resulted in a significant increase of TRAIL-induced apoptosis. Of note, suramin-mediated inhibition of FHIT enzymatic activity also enhanced TRAIL-induced apoptosis. We conclude that FHIT expression in lung cancer cells is protective from TRAIL-induced apoptosis. Our data suggest that FHIT exerts this protective effect downstream TRAIL-receptors and likely requires its dinucleoside-triphosphate hydrolase activity. As TRAIL represents in the near future a good candidate for death ligands-based anticancer therapy, its potential therapeutic use should be envisaged as preliminary to molecular genetics interventions or drug-induced epigenetic modulations aimed to restoring FHIT gene expression levels in non-small cells lung tumors.

Aberrant expression of B203.13 antigen in acute lymphoid leukemia of B-cell origin.

The B203.13 monoclonal antibody was developed by immunizing mice with the B/monocyte biphenotypic cell line B1b. During normal hematopoiesis B203.13 is expressed on a fraction of CD34+ cells, while on mature cells it is only present on B-lymphocytes. We tested this antibody as a marker of childhood B-acute lymphoblastic leukemia (B-ALL). Bone marrow aspirates from 139 cases of early B-ALL and 25 controls were studied. About 40% of the B-ALL patients expressed B203.13. In these patients, B203.13 was constantly co-expressed with CD10, but never co-expressed with CD20, contrary to the controls. The CD10(+)/B203.13(+) phenotype was specific to B-ALL, since CD10(+)/CD20(+) cells from common acute lymphoblastic leukemia (c-ALL) did not express B203.13. We concluded that the use of B203.13 in association with CD10 and CD20 provides meaningful information for distinguishing normal residual B-cells from leukemic B-lymphoblasts and that recurrence of a CD10(+)/B203.13(+) phenotype after transplantation may be a very early relapse indicator of early B-acute lymphoblastic leukemia.

Timing and expression level of protein kinase C epsilon regulate the megakaryocytic differentiation of human CD34 cells.

Protein kinase C (PKC)-mediated intracellular signaling participates in several key steps of hematopoietic cell differentiation. The epsilon isoform of PKC has been associated with erythroid differentiation as well as with the early phases of megakaryocytic (MK) lineage commitment. Here, we worked on the hypothesis that PKCepsilon expression levels might be modulated during MK differentiation, with a specific role in the early as well as in the late phases of thrombopoiesis. We demonstrate that--at variance with the erythroid lineage development--PKCepsilon is completely downmodulated in TPO-induced CD34 cells from day 6 onward. The forced expression of PKCepsilon in the late phases of MK differentiation delays the phenotypic differentiation of progenitors likely via Bcl-xL upregulation. Moreover, tumor necrosis factor-related apoptosis-inducing ligand (TRAIL), known as a negative regulator of early erythroid expansion, is not apoptogenic for thrombopoietin-induced CD34 cells, but rather accelerates their maturation. However, PKCepsilon levels negatively interfere also with the effects of TRAIL in MK differentiation. PKCepsilon can therefore be considered a signaling intermediate whose expression levels are finely tuned, with a virtually opposite kinetic, in erythroid versus megakaryocytic lineages, to adequately respond to the signaling requirements of the specific hematopoietic lineage.

Exogenous hydrogen sulfide induces functional inhibition and cell death of cytotoxic lymphocytes subsets.

The toxic effects of exogenous hydrogen sulfide on peripheral blood lymphocytes have been investigated in detail. Hydrogen sulfide is now considered as a gasotransmitter with specific functional roles in different cell types, like neurons and vascular smooth muscle. Here we show that exogenous hydrogen sulfide induces a caspase-independent cell death of peripheral blood lymphocytes that depends on their intracellular glutathione levels, with a physiologically relevant subset specificity for CD8+ T cells and NK cells. Although lymphocyte activation does not modify their sensitivity to HS-, after 24 h exposure to hydrogen sulfide surviving lymphocyte subsets show a dramatically decreased proliferation in response to mitogens and a reduced IL-2 production. Overall, our data demonstrate that HS- reduces the cellular cytotoxic response of peripheral blood lymphocytes as well as their production of IL-2, therefore de-activating the major players of local inflammatory responses, adding new basic knowledge to the clinically well known anti-inflammatory effects of sulfur compounds.

Efficient platelet delta-granule release induced by [Ca2+]i elevation is modulated by GPIIbIIIa.

Intracellular Ca2+ elevation generates a cascade of events that leads to platelet activation and degranulation. The GPIIbIIIa-ligand molecular complex plays a central role in several aspects of platelet activation. Taking advantage of the flow cytometric simultaneous analysis of surface GPIIbIIIa expression and intracellular serotonin content, we demonstrate here that the functional inhibition of GPIIbIIIa generates an impairment of delta-granule release even upon maximal intracellular Ca2+ elevation. In healthy subjects, the GPIIbIIIa inhibitor tirofiban impairs platelet delta-granule release. Analogously, Glanzmann thrombasthenia patients show an impairment of delta-granule release that is proportional to their residual expression of platelet GPIIbIIIa. These data show that platelet surface expression of functional GPIIbIIIa is required for a fully efficient secretion of delta-granules and serotonin release. The implications of our findings are discussed in the light of the complex interplay between vesicle release and ligand-receptor triggering during platelet activation.

Down-regulation of human leukocyte antigen class I and II and beta 2-microglobulin expression in human herpesvirus-7-infected cells.

Human herpesvirus (HHV)-7 is a ubiquitous virus that infects >90% of adults. We show that HHV-7 impairs beta2-microglobulin and human leukocyte antigen (HLA) class I and II expression in lymphoid cells in vitro. Accordingly, infected cells were more sensitive to peripheral blood natural cytotoxic activity than were uninfected cells. Analysis of beta2-microglobulin and HLA expression in biopsy specimens from human submandibular glands confirmed the ability of HHV-7 to modulate the expression of these antigens in vivo. We demonstrate that the down-modulation of HLA by HHV-7 is linked to viral replication and is not merely a consequence of the interaction of virions with the cell surface. Infected cells can therefore efficiently escape host immune pressure, which might explain the persistence of HHV-7-positive cells in several kinds of tumors and chronic infectious diseases.

PKCepsilon controls protection against TRAIL in erythroid progenitors.

Apoptosis plays a central role in the regulation of the size of the hematopoietic stem cell pool as well as in the processes of cell differentiation along the various hematopoietic lineages. TRAIL is a member of the TNF family of cytokines with a known apoptogenic role against a variety of malignant cells and an emerging role in the modulation of normal hematopoiesis. Here we worked on the hypothesis that PKCepsilon could act as a switch of the cellular response to TRAIL during erythropoiesis. We demonstrate that EPO-induced erythroid CD34 cells are insensitive to the apoptogenic effect of TRAIL at day 0 due to the lack of specific receptor expression. From day 3 onward, erythroid cells express surface death receptors and become sensitive to TRAIL up to day 7/8 when, notwithstanding death-receptor expression, the EPO-driven up-regulation of PKCepsilon intracellular levels renders differentiating erythroid cells resistant to TRAIL likely via Bcl-2 up-regulation. Our conclusion is that in human CD34 cells, EPO promotes a series of events that, being finely regulated in their kinetics, restricts the sensitivity of these cells to TRAIL to a specific period of time, which therefore represents the "TRAIL window" for the negative regulation of erythroid-cell numbers.

Anticancer agents sensitize osteosarcoma cells to TNF-related apoptosis-inducing ligand downmodulating IAP family proteins.

Although TNF-related apoptosis-inducing ligand (TRAIL) usually induces cell death in tumor cells, there are some tumor cell types that are resistant to its apoptogenic effects. Some chemotherapeutic drugs, however, can sensitize resistant cancer cells to TRAIL by either upregulating surface TRAIL death receptor expression or by modulating intracellular signalling pathways emanating from TRAIL receptors. U2OS human osteosarcoma cells express TRAIL-R2 but are resistant to TRAIL-induced apoptosis. however, the genotoxic drugs, Doxorubicin and Cisplatin, are able to sensitize U2OS cells to TRAIL, without affecting their surface expression of either death or decoy TRAIL receptors. We demonstrate that Doxorubicin and Cisplatin downmodulate X-IAP, while not affecting FLIP levels in U2OS cells. Selective downmodulation of X-IAP protein synthesis by specific small interference RNA transfection induced a sensitization of U2OS cells to TRAIL comparable to that induced by pharmacological treatment with genotoxic drugs. TRAIL-R2 downmodulation by siRNAs completely abolished the TRAIL-induced apoptosis of genotoxin-treated U2OS cells. Our findings demonstrate that Doxorubicin and Cisplatin do not sensitize U2OS osteosarcoma cells to TRAIL by surface receptor modulation but rather by the removal of the intracellular signalling inhibition generated by X-IAP, suggesting a foreseeable relevant advantage to the therapy of these tumors by the combined regimen of genotoxin-based chemotherapy and TRAIL.

Expression of HLA class I antigen and proteasome subunits LMP-2 and LMP-10 in primary vs. metastatic breast carcinoma lesions.

Malignant transformation of breast epithelia is frequently associated with an altered expression of MHC products and of antigen processing molecular machinery. The consequent impairment of tumor immune recognition is thought to confer to tumor cells a selective advantage with respect to survival and metastatization. In order to understand if metastatic breast cancer lesions might be associated with a defective proteasome subunit expression that, in turn, might limit the peptide availability and prevent stable cell surface HLA class I-tumor antigen expression, we studied by immunostaining the expression of beta2-microglobulin, HLA class I antigens and proteasome subunits LMP-2 and LMP-10 in 35 matched primary and metastatic human breast carcinoma lesions. Overall, we found a downregulation of LMP-2 in 51.4% of the lesions, of LPM-10 in 45.7% of the lesions, of HLA class I heavy chain in 40.0% of the lesions, while beta2-microglobulin was downregulated in 25.7% of the lesions studied. In most primary and metastatic lesions the downmodulation of each antigen examined was coordinated. In the cases where a selective downmodulation of antigens was observed in the primary or in the metastatic lesion (with the exception of beta2-microglobulin), it was rather observed in the primary lesions. However, LMP-10 showed a significant selective downmodulation in the metastases as well. Antigen downmodulation does not appear therefore to represent a strategy for the primary tumor to metastasize successfully.

Activated human NK and CD8+ T cells express both TNF-related apoptosis-inducing ligand (TRAIL) and TRAIL receptors but are resistant to TRAIL-mediated cytotoxicity.

The expression of tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL) and TRAIL receptors was investigated in resting and cytokine-activated purified primary human natural killer (NK) and CD8(+) T cells. Resting NK and CD8(+) T cells expressed the mRNA for all TRAIL receptors, but TRAIL-R4 was the only receptor clearly detectable on the surface of both cell types. NK cells were activated by interleukin 2 (IL-2) or IL-15, whereas CD8(+) T cells were activated by phytohemagglutinin (PHA) + IL-2 followed by IL-2 alone for up to 10 days. On activation, both cell types rapidly expressed TRAIL-R2 and TRAIL-R3, whose expression peaked at day 10 of culture. TRAIL-R1, however, was never expressed at any time point examined, whereas the expression of TRAIL-R4, which showed a progressive increase in CD8(+) T cells, remained constant in NK cells. Notwithstanding the expression of TRAIL-R2, recombinant TRAIL did not show any cytotoxic activity on either NK or CD8(+) T cells. Both resting and activated NK and CD8(+) T cells were found to express high levels of the 2 isoforms of c-FLIP (cellular Fas-associated death domain protein [FADD]-like IL-1-converting enzyme [FLICE]-inhibitory protein). Small interference RNA-mediated inhibition of c-FLIP expression in NK cells abrogated their resistance to the apoptotic effect of soluble TRAIL. Thus, once activated the major cytotoxic effector cells are potentially sensitive to TRAIL but are physiologically protected from its apoptotic action by intracellular level of c-FLIP.