Bruno Nobili

Anti-CD20 monoclonal antibody (Rituximab) for life-threatening autoimmune haemolytic anaemia in a patient with systemic lupus erythematosus.

Summary. Innovative approaches are needed for patients with systemic lupus erythematosus (SLE) who develop autoimmune haemolytic anaemia (AIHA) that does not respond to conventional treatment. Rituximab, a chimaeric anti‐CD20 monoclonal antibody, has been demonstrated to be highly effective for in vivo B‐cell depletion. We report an 18‐year‐old‐girl with SLE and life‐threatening AIHA that did not respond to steroids, intravenous immunoglobulin and cyclosporin A. Rituximab was given weekly at 375 mg/m2 for two doses. The drug was well tolerated and the patient had no adverse effects. Her haemolytic disorder markedly ameliorated, with a progressive increase of haemoglobin levels, starting a few days after therapy. The patient remains disease‐free 7 months later.

The endovanilloid/endocannabinoid system in human osteoclasts: Possible involvement in bone formation and resorption

Recent studies suggest a role for the endocannabinoid/endovanilloid anandamide in the regulation of bone resorption/formation balance in mice. Here, we examined the co-expression of the transient receptor potential vanilloid type 1 (TRPV1) and the cannabinoid CB1/CB2 receptors together with N-acylphosphatidylethanolamine-hydrolizing phospholipase D (NAPE-PLD) and fatty acid amide hydrolase (FAAH), the two enzymes responsible of the synthesis and catabolism of anandamide respectively, in human osteoclasts. Co-expression of TRPV1, CB1/CB2, NAPE-PLD and FAAH was found in both human osteoclast cultures and in native osteoclasts from human bone biopsies. Moreover, agonist-evoked calcium entry indicated that the TRPV1 receptor is functionally active in vitro. Consistently, biomolecular and functional experiments showed that resiniferatoxin (RTX), a selective TRPV1 receptor agonist, increased the expression and the activity of TRAP and cathepsin K, two specific osteoclast biomarkers. The evidence that cannabinoid and vanilloid receptors are co-expressed in human osteoclasts suggests that they might cross-talk to modulate the intrinsic balance of bone mineralization and resorption by different actions of anandamide through TRPV1 and cannabinoid receptors. The presence of the endocannabinoid/endovanilloid proteins in human osteoclasts will likely have implications for the management of bone demineralization associated syndrome (i. e. osteoporosis).

ROBO2 Gene Variants Are Associated with Familial Vesicoureteral Reflux

The SLIT2 receptor ROBO2 plays a key role in the formation of the ureteric bud, and its inactivation in mice leads to supernumerary ureteric bud development, lack of ureter remodeling, and improper insertion of the ureters into the bladder. Recently, two heterozygous ROBO2 missense mutations were identified in two families with primary vesicoureteral reflux occurring in combination with congenital anomalies of the kidney and urinary tract (VUR/CAKUT). This study investigated a possible causal role of ROBO2 gene variants in 95 unrelated patients with primary VUR (n = 78) or VUR/CAKUT. Eighty-two percent of all patients had a family history of genitourinary anomalies. Twenty-four ROBO2 gene variants were identified by direct sequencing of all 26 exons and the exon-intron boundaries. Of these, four led to amino acid substitutions: Gly328Ser, Asn515Ile, Asp766Gly, and Arg797Gln. When the families were examined, the missense variants co-segregated with VUR (three families) or VUR/CAKUT (one family). These variants were not found in 190 control subjects, and the affected amino acids have been conserved through evolution. In conclusion, a relatively high frequency of ROBO2 variants (5.1%) was found in familial cases; however, functional studies and validation in other cohorts are warranted.

Oxidative stress as a multiple effector in Fanconi anaemia clinical phenotype

Abstract:  Fanconi anaemia (FA) is a genetic disease characterised by bone marrow failure with excess risk of myelogenous leukaemia and solid tumours. A widely accepted notion in FA research invokes a deficiency of response to DNA damage as the fundamental basis of the ‘crosslinker sensitivity’ observed in this disorder. However, such an isolated defect cannot readily account for the full cellular and clinical phenotype, which includes a number of other abnormalities, such as malformations, endocrinopathies, and typical skin spots. An extensive body of evidence pointing toward an involvement of oxidative stress in the FA phenotype includes the following: (i) In vitro and ex vivo abnormalities in a number of redox status endpoints; (ii) the functions of several FA proteins in protecting cells from oxidative stress; (iii) redox‐related toxicity mechanisms of the xenobiotics evoking excess toxicity in FA cells. The clinical features in FA and the in vivo abnormalities of redox parameters are here reconsidered in view of the pleiotropic clinical phenotype and known biochemical and molecular links to an in vivo prooxidant state, which causes oxidative damage to biomolecules, resulting in an excessive number of acquired abnormalities that may overwhelm the cellular repair capacity rather than a primary deficiency in DNA repair. FA may thus represent a unique model disease in testing the integration between the acquisition of macromolecular damage as a result of oxidative stress and the ability of the mammalian cell to respond effectively to such damage.

The outcome of children with Fanconi anemia given hematopoietic stem cell transplantation and the influence of fludarabine in the conditioning regimen: a report from the Italian pediatric group

Background and Objectives Hematopoietic stem cell transplantation (HSCT) still represents the only treatment potentially able to prevent/rescue the development of marrow failure and myeloid malignancies in patients with Fanconi anemia (FA). While in the past HSCT from an HLA-identical sibling was proven to cure many patients, a higher incidence of treatment failure has been reported in recipients of an unrelated donor (UD) or HLA-partially matched related allograft. Design and Methods We analyzed the outcome of 64 FA patients (age range, 2–20 years) who underwent HSCT between January 1989 and December 2005. Patients were transplanted from either an HLA-identical sibling (n=31), an UD (n=26), or an HLA-partially matched relative (n=7). T-cell depletion of the graft was performed in patients transplanted from an HLA-disparate relative. Results The 8-year estimate of overall survival (OS) for the whole cohort was 67%; it was 87%, 40% and 69% when the donor was an HLA-identical sibling, an UD and a mismatched relative, respectively (p<0.01). The outcome of recipients of grafts from an UD improved over time, the probability of survival being 10% and 72% for patients transplanted before and after 1998, respectively (p<0.05). The OS probability of children who did or did not receive fludarabine in preparation for the allograft was 86% and 59%, respectively (p<0.05). Interpretation and Conclusions These data, useful for counselling, provide support to the concept that a relevant proportion of FA patients undergoing HSCT can now be successfully cured, even in the absence of an HLA-identical sibling, especially if the conditioning regimen includes fludarabine.

Erythrocyte membrane protein alterations underlying clinical heterogeneity in hereditary spherocytosis

Summary. Hereditary spherocytosis (HS) is a very heterogenous condition both at clinical and biochemical level. To establish the relationship between these aspects we performed a clinical and biochemical study in 87 Italian HS subjcts. Patients were divided into three groups based on clinical severity (mild, typical and severe) and into five subgroups based on specific membrane abnormalities identified by polyacrylamide gel electrophoresis (isolated spectrin deficiency, spectrin deficiency combined with mild ankyrin reduction, spectin deficiency combined with severe ankyrin reduction, band 3 reduction and isolated protein 4.2 reduction). We were not able to assess any alteration in six HS patients. A good correlation between clinical HS forms and memberane protein defects is shown. We conclude that erythrocyte memberane analysis should be carried out after diagnosis of HS in order to predict the clinical course of the disease.

Effect of eradication of Helicobacter pylori in children with chronic immune thrombocytopenia: A prospective, controlled, multicenter study†

The eradication of Helicobacter pylori has been associated with remission of immune thrombocytopenia (ITP) in approximately half of eradicated patients. Data on children are limited to small case series.

Long‐term follow‐up analysis after rituximab therapy in children with refractory symptomatic ITP: identification of factors predictive of a sustained response

We report the long‐term follow‐up (median 39·5 months) of 49 paediatric patients (33 females and 16 males) with refractory symptomatic immune thrombocytopenic purpura (ITP) treated with rituximab. The overall response rate was 69% (34/49 patients). Twenty‐one responders had a platelet count >50 × 109/l at a median 20·2 months from treatment. Kaplan–Meier analysis showed a probability of relapse‐free survival (RFS) of 60% at 36 months from the first rituximab infusion. The number of infusions and a previous splenectomy did not influence overall response rate. Patients who achieved complete response were significantly older at diagnosis and first rituximab infusion than partial responders (P = 0·027). Older children displayed a significantly greater probability of sustained response (RFS) at 36 months than younger children (88·9% vs. 56·7%, P = 0·037). Earlier responses (within 20 d from treatment) were significantly associated with both complete (P = 0·004) and sustained response (P = 0·002). Only mild and transient side‐effects were observed in 9/49 children; no major infections nor delayed toxicities were recorded during the follow‐up.

Clinical and molecular evaluation of non‐dominant hereditary spherocytosis

About 75% of hereditary spherocytosis (HS) patients have the autosomal dominant form of the disease, whereas both parents of the remaining HS patients are clinically and haematologically normal. These patients could have either the autosomal recessive form of the disease or a de novo mutation. We studied 80 randomly chosen, Italian HS children with normal parents. They had different clinical phenotypes (16 mild, 40 moderate, 16 moderately severe and eight severe). These patients were screened for the occurrence of ankyrin or β‐spectrin de novo mutations. To search for ankyrin de novo mutations affecting mRNA accumulation, we studied a (AC)n microsatellite located in the non‐coding sequence of the last exon of the ankyrin gene, and four different exonic polymorphisms in the β‐spectrin gene were utilized for the detection of de novo mutations influencing β‐spectrin mRNA stability. They were also screened for the presence of α‐spectrinLEPRA as well as for the mutation −108T→C in the ankyrin promoter, two variants previously found in some cases of genuinely recessive HS. Twenty‐five patients showed ankyrin de novo mutations and 10 HS subjects had β‐spectrin de novo mutations. Furthermore, we found five patients to be heterozygous for α‐spectrinLEPRA and one heterozygous for the mutation in the ankyrin promoter. Therefore, a molecular diagnosis was achieved in about 50% of the cases. Our data demonstrate that, among HS patients with normal parents, de novo dominant mutants are six times more common than recessive mutations. These results should be considered in view of the genetic counselling of a normal couple with a HS child.

Association Between a Polymorphism in Cannabinoid Receptor 2 and Severe Necroinflammation in Patients With Chronic Hepatitis C

BACKGROUND & AIMS The cannabinoid receptor 2 (CB2) has been implicated in liver disease. The single-nucleotide polymorphism rs35761398 in cannabinoid receptor 2 gene (CNR2), which encodes the CB2, substitutes glutamine (Q) 63 with arginine (R), and reduces the function of the gene product. We investigated the effects of CNR2 rs35761398 in patients with hepatitis C virus (HCV) infection. METHODS We studied 169 consecutive patients with asymptomatic chronic hepatitis (tested positive for anti-HCV and HCV RNA) at 2 liver units in southern Italy. First, liver biopsy samples were collected from July 2009 through December 2011. All patients were naive to antiviral therapy; CNR2 genotype was determined by polymerase chain reaction analysis. RESULTS Patients with the CB2-63 QQ variant had higher serum levels of aminotransferase than those with the CB2-63 QR or RR variants; they also had higher histologic activity index (HAI) scores (8.6 ± 3.8) than patients without the CB2-63 RR variant (5.3 ± 3.6; P < .005) or those with the CB2-63 QR variant (5.8 ± 3.3; P < .001). Patients with the different variants of CNR2 did not differ in fibrosis stage or steatosis score. Moderate or severe chronic hepatitis (HAI score, >8) was identified more frequently (55.5%) in patients with the CB2-63 QQ variant than in those with the 63 QR (20%; P < .005) or RR variants (17.4%; P < .005). In logistic regression analysis, the CB2-63 QQ variant and fibrosis score were independent predictors of moderate or severe chronic hepatitis (HAI score, >8; P < .0001). CONCLUSIONS The CB2-63 QQ variant of CNR2 is associated with more severe inflammation and hepatocellular necrosis in patients with HCV infection.