Baldassarre Martire

Hereditary deficiency of gp91(phox) is associated with enhanced arterial dilatation: results of a multicenter study.

Background— NADPH oxidase is believed to modulate arterial tone, but its role in humans is still unclear. The objective of this study was to evaluate whether NADPH oxidase is involved in flow-mediated arterial dilation (FMD). Methods and Results— Twenty-five patients with hereditary deficiency of gp91phox, the catalytic core of NADPH oxidase, (X-CGD), 25 healthy subjects, and 25 obese patients matched for sex and age were recruited. FMD, platelet gp91phox, serum levels of nitrite and nitrate as markers of nitric oxide generation, oxidized low-density lipoprotein, and urinary excretion of isoprostanes as markers of oxidative stress were determined. Platelet gp91phox expression was downregulated in X-CGD patients (1.0±0.8 mean fluorescence; P<0.001) and upregulated in obese patients (4.1±2.2 mean fluorescence; P=0.01) compared with healthy subjects (2.9±1.7 mean fluorescence). Urinary excretion of isoprostanes was reduced in X-CGD patients (41.7±33.3 pg/mg creatinine; P=0.04) and increased in obese patients (154.4±91 pg/mg creatinine; P<0.001) compared with healthy subjects (69.5±52.4 pg/mg creatinine). Obese patients had higher serum oxidized low-density lipoprotein than healthy subjects (35.3±6.7 versus 24.8±9.8 U/L; P<0.001) and X-CGD patients (28.5±7.2 U/L; P<0.001). X-CGD patients had significantly higher FMD (14.7±5.9%) compared with healthy subjects (7.9±2.5%; P<0.001); obese patients had lower FMD (5.3±3.0%; P=0.028) compared with healthy subjects. Serum nitrite and nitrate levels were significantly higher in patients with X-CGD (36.0±10.8 μmol/L; P=0.016) and lower in obese patients (9.3±11.0 μmol/L; P=0.001) compared with healthy subjects (27.1±19.1 μmol/L). Serum nitrite and nitrate levels significantly correlated with FMD (Rs=0.403, P<0.001) and platelet gp91phox (Rs=−0.515, P<0.001). FMD inversely correlated with platelet gp91phox (Rs=−0.502, P<0.001) and isoprostanes (Rs=−0.513, P<0.001). Conclusion— This study provides the first evidence that, in humans, gp91phox is implicated in the modulation of arterial tone.

Clinical Features and Follow-Up in Patients with 22q11.2 Deletion Syndrome.

OBJECTIVE To investigate the clinical manifestations at diagnosis and during follow-up in patients with 22q11.2 deletion syndrome to better define the natural history of the disease. STUDY DESIGN A retrospective and prospective multicenter study was conducted with 228 patients in the context of the Italian Network for Primary Immunodeficiencies. Clinical diagnosis was confirmed by cytogenetic or molecular analysis. RESULTS The cohort consisted of 112 males and 116 females; median age at diagnosis was 4 months (range 0 to 36 years 10 months). The diagnosis was made before 2 years of age in 71% of patients, predominantly related to the presence of heart anomalies and neonatal hypocalcemia. In patients diagnosed after 2 years of age, clinical features such as speech and language impairment, developmental delay, minor cardiac defects, recurrent infections, and facial features were the main elements leading to diagnosis. During follow-up (available for 172 patients), the frequency of autoimmune manifestations (P = .015) and speech disorders (P = .002) increased. After a median follow-up of 43 months, the survival probability was 0.92 at 15 years from diagnosis. CONCLUSIONS Our data show a delay in the diagnosis of 22q11.2 deletion syndrome with noncardiac symptoms. This study provides guidelines for pediatricians and specialists for early identification of cases that can be confirmed by genetic testing, which would permit the provision of appropriate clinical management.

Molecular analysis of the pre-BCR complex in a large cohort of patients affected by autosomal-recessive agammaglobulinemia.

Autosomal-recessive agammaglobulinemia is a rare and heterogeneous disorder, characterized by early-onset infections, profound hypogammaglobulinemia of all immunoglobulin isotypes and absence of circulating B lymphocytes. To investigate the molecular basis of the disease, 23 patients with early-onset disease and no mutations in Bruton tyrosine kinase, the gene responsible for X-linked agammaglobulinemia, were selected and analyzed by direct sequencing of candidate genes. Two novel mutations in the μ heavy chain (μHC) gene (IGHM) were identified in three patients belonging to two unrelated families. A fourth patient carries a previously described G>A nucleotide substitution at the −1 position of an alternative splice site in IGHM; here, we demonstrate that this mutation is indeed responsible for aberrant splicing. Comparison of bone marrow cytofluorimetric profiles in two patients carrying different mutations in the IGHM gene suggests a genotype–phenotype correlation with the stage at which B-cell development is blocked. Several new single nucleotide polymorphisms (SNPs) both in the μHC and in the λ5-like/VpreB-coding genes were identified. Two unrelated patients carry compound heterozygous variations in the VpreB1 gene that may be involved in disease ethiology.

Defective surface expression of attractin on T cells in patients with common variable immunodeficiency (CVID)

The proliferative responses of T lymphocytes of a subset of patients with CVID are abnormally low. This may be due to abnormalities in extracellular interactions or signalling defects downstream from membrane‐associated receptors. Demonstrating that the T cell receptor signalling was normal, we observed no abnormal pattern of activation‐induced tyrosine phosphorylation in cells from CVID patients. Moreover, the addition of exogenous IL‐2 increased the low proliferation to mitogens, thus indicating the integrity of the IL‐2R signalling apparatus. Attractin is a rapidly expressed T cell activation antigen involved in forming an association between T cells and monocytes. Twenty‐four to 48 h after activation by CD3 cross‐linking, attractin expression was not up‐regulated on the cells of CVID patients despite normal up‐regulation of CD25 and CD26. On control cells, however, attractin expression was up‐regulated together with CD25 and CD26. The addition of the purified 175‐kD attractin was capable of restoring the proliferative response of peripheral blood mononuclear cells following CD3 X‐L in the presence of suboptimal concentrations of rIL‐2 (10 and 20 U/ml). The effect was dose‐dependent with the maximal effect at a concentration of 500 ng/ml, and present at a concentration as low as 50 ng/ml. Due to the likely role of attractin in cell guidance and amplification of the immune response, our results indicate that the lack of up‐regulation of the molecule in patients with CVID may in turn affect any further step of productive immune response. Our finding may also imply a potential therapeutic role for this novel molecule.

Does NADPH Oxidase Deficiency Cause Artery Dilatation in Humans

NADPH oxidase is known to modulate the arterial tone, but the role of its specific subunits is still unclear. The objective of this study was to compare the role of p47 and gp91phox (NOX2) on artery dilatation. We conducted a multicenter study enrolling 30 patients with chronic granulomatous disease (CGD) (25 with NOX2 deficiency and 5 with p47(phox) deficiency) and 30 healthy subjects (HS), matched for gender and age, in whom flow-mediated dilation (FMD), serum activity of NOX2 (soluble NOX2-derived peptide [sNOX2-dp]), urinary isoprostanes (8-iso-PGF2α), and platelet production of isoprostanes and NOX2 were determined. Compared to HS, patients with CGD had significantly higher FMD and lower sNOX2-dp and 8-iso-PGF2α levels. Compared to patients with NOX2 deficiency and HS, patients with p47(phox) hereditary deficiency had intermediate FMD and oxidative stress, that is, higher and lower FMD and lower and higher isoprostanes compared to HS and patients with NOX2 deficiency, respectively. In agreement with this finding, an ex vivo study showed higher inhibition of NOX2 activity and lower isoprostane formation in platelets from patients with NOX2 deficiency compared to platelets from ones with p47(phox) deficiency. Our observations lead to the hypothesis that oxidants are implicated in artery vasoconstriction.

Intergenerational and intrafamilial phenotypic variability in 22q11.2 Deletion syndrome subjects

Background22q11.2 deletion syndrome (22q11.2DS) is a common microdeletion syndrome, which occurs in approximately 1:4000 births. Familial autosomal dominant recurrence of the syndrome is detected in about 8-28% of the cases. Aim of this study is to evaluate the intergenerational and intrafamilial phenotypic variability in a cohort of familial cases carrying a 22q11.2 deletion.MethodsThirty-two 22q11.2DS subjects among 26 families were enrolled.ResultsSecond generation subjects showed a significantly higher number of features than their transmitting parents (212 vs 129, P = 0.0015). Congenital heart defect, calcium-phosphorus metabolism abnormalities, developmental and speech delay were more represented in the second generation (P < 0.05). Ocular disorders were more frequent in the parent group. No significant difference was observed for the other clinical variables. Intrafamilial phenotypic heterogeneity was identified in the pedigrees. In 23/32 families, a higher number of features were found in individuals from the second generation and a more severe phenotype was observed in almost all of them, indicating the worsening of the phenotype over generations. Both genetic and epigenetic mechanisms may be involved in the phenotypic variability.ConclusionsSecond generation subjects showed a more complex phenotype in comparison to those from the first generation. Both ascertainment bias related to patient selection or to the low rate of reproductive fitness of adults with a more severe phenotype, and several not well defined molecular mechanism, could explain intergenerational and intrafamilial phenotypic variability in this syndrome.

Congenital and acquired neutropenia consensus guidelines on diagnosis from the Neutropenia Committee of the Marrow Failure Syndrome Group of the AIEOP (Associazione Italiana Emato-Oncologia Pediatrica).

Congenital and acquired neutropenia are rare disorders whose frequency in pediatric age may be underestimated due to remarkable differences in definition or misdiagnosed because of the lack of common practice guidelines. Neutropenia Committee of the Marrow Failure Syndrome Group (MFSG) of the AIEOP (Associazione Italiana Emato‐Oncologia Pediatrica) elaborated this document following design and methodology formerly approved by the AIEOP board. The panel of experts reviewed the literature on the topic and participated in a conference producing a document which includes a classification of neutropenia and a comprehensive guideline on diagnosis of neutropenia. Pediatr Blood Cancer 2011;57:10–17.

Congenital and acquired neutropenias consensus guidelines on therapy and follow‐up in childhood from the Neutropenia Committee of the Marrow Failure Syndrome Group of the AIEOP (Associazione Italiana Emato‐Oncologia Pediatrica)

The management of congenital and acquired neutropenias presents some differences according to the type of the disease. Treatment with recombinant human granulocyte-colony stimulating factor (G-CSF) is not standardized and scanty data are available on the best schedule to apply. The frequency and the type of longitudinal controls in patients affected with neutropenias are not usually discussed in the literature. The Neutropenia Committee of the Marrow Failure Syndrome Group (MFSG) of the Associazione Italiana di Emato-Oncologia Pediatrica (AIEOP) elaborated this document following design and methodology formerly approved by the AIEOP board. The panel of experts reviewed the literature on the topic and participated in a conference producing a document that includes recommendations on neutropenia treatment and timing of follow-up.

Reduced interleukin-5 production by peripheral CD4+ T cells in common variable immunodeficiency patients.

We evaluated the capacity of peripheral CD4+ T helper cells in four Common Variable Immunodeficiency (CVI) patients to secrete interleukin-4 (IL-4) and IL-5. While in control CD4+ T cells, stimulated via CD3 and cultured in presence of IL-2 or IL-15, a 10 fold increased production of IL-5 (146 ± 30; 142 ± 25 pg/ml) was found, a 4 fold increment of this cytokine was, instead, detected in 3 out of 4 CVI patients (34 ± 13; 39 ± 12 pg/ml) (p < 0.05). In conclusion, the reduction of IL-5, involved in the late regulation of B cell differentiation into Ig-secreting plasma cells, may contribute to the defective antibody production in CVI patients.

Paroxysmal nocturnal hemoglobinuria clones in children with acquired aplastic anemia: a multicentre study.

A multicentre study evaluating the presence of glycosil phosphatidyl-inositol (GPI)-negative populations was performed in 85 children with acquired aplastic anemia (AA). A GPI-negative population was observed in 41% of patients at diagnosis, 48% during immune-suppressive therapy (IST), and 45% in patients off-therapy. No association was found between the presence of a GPI-negative population at diagnosis and the response to IST. In addition, the response rate to IST did not differ between the patients who were GPI-positive at diagnosis and later developed GPI-negative populations and the 11 patients who remained GPI-positive. Two patients with a GPI-negative population >10%, and laboratory signs of hemolysis without hemoglobinuria were considered affected by paroxysmal nocturnal hemoglobinuria (PNH) secondary to AA; no thrombotic event was reported. Excluding the 2 patients with a GPI-negative population greater than 10%, we did not observe a significant correlation between LDH levels and GPI-negative population size. In this study monitoring for laboratory signs of hemolysis was sufficient to diagnose PNH in AA patients. The presence of minor GPI-negative populations at diagnosis in our series did not influence the therapeutic response. As occasionally the appearance of a GPI-negative population was observed at cyclosporine (CSA) tapering or AA relapse, a possible role of GPI-negative population monitoring during IST modulation may need further investigation.