Association between cannabinoid receptor type 2 Q63R variant and oligo/polyarticular juvenile idiopathic arthritis.

OBJECTIVES: To investigate whether the functional variant Q63R of the cannabinoid 2 (CB2) receptor is associated with susceptibility to oligo/poly-articular juvenile idiopathic arthritis (JIA) and with its clinical features. METHOD: A total of 171 Italian children with oligoarticular/rheumatoid factor negative poly-articular JIA and 600 healthy controls were enrolled in the study and genotyped. RESULTS: A significant difference in genotype distribution of the CB2 Q63R variant (CNR2 rs35761398) between oligo/poly-articular JIA patients and controls was found (p = 0.001). The R63 variant was associated with increased rates of relapse (p = 0.0001). CONCLUSIONS: This study indicates that the CB2 receptor contributes to susceptibility to oligo/polyarticular JIA and to the severity of its clinical course.

The genetic ablation or pharmacological inhibition of TRPV1 signalling is beneficial for the restoration of quiescent osteoclast activity in ovariectomized mice.

BACKGROUND AND PURPOSE: Osteoporosis is a condition characterized by a decrease in bone density, which decreases its strength and results in fragile bones. The endocannabinoid/endovanilloid system has been shown to be involved in the regulation of skeletal remodelling. The aim of this study was to investigate the possible modulation of bone mass mediated by the transient receptor potential vanilloid type 1 channel (TRPV1) in vivo and in vitro. EXPERIMENTAL APPROACH: A multidisciplinary approach, including biomolecular, biochemical and morphological analysis, was used to investigate the involvement of TRPV1 in changes in bone density in vivo and osteoclast activity in vitro, in wild-type and Trpv1(-/-) mice, that had undergone ovariectomy or had a sham operation. KEY RESULTS: Genetic deletion of Trpv1 as well as pharmacological inhibition/desensitization of TRPV1 signalling dramatically reduced the osteoclast activity in vitro and prevented the ovariectomy-induced bone loss in vivo, whereas the expression of cannabinoid type 2 (CB2 ) receptors was increased. CONCLUSIONS AND IMPLICATIONS: These findings highlight the pivotal role TRPV1 channels play in bone resorption and suggest a possible cross-talk between TRPV1 and CB2 receptors. Based on these results, hybrid compounds acting on both TRPV1 and CB2 receptors in an opposite manner could provide a future pharmacological tool for the treatment of diseases associated with disturbances in the bone remodelling process.

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).

Successful T-cell-depleted, related haploidentical peripheral blood stem cell transplantation in a patient with Fanconi anaemia using a fludarabine-based preparative regimen without radiation.

Haematopoietic stem cell transplantation (HSCT) represents the treatment of choice for severe bone marrow failure in patients with Fanconi anaemia (FA). When the donor is a compatible relative, the chance of being cured with an allograft is in the order of 70%. However, for FA children lacking an HLA-identical sibling, the results of HSCT from an alternative donor are less satisfactory because of a higher risk of graft rejection, graft-versus-host-disease (GVHD) and regimen-related toxicity. We report on a 12-year-old girl with FA, who was treated by T-cell-depleted (TCD) peripheral blood HSCT from her haploidentical uncle, using a novel fludarabine-based preparative regimen without radiation. She had rapid engraftment with no toxicity and no GVHD. Progressive recovery of both numbers of lymphocyte and of proliferative response to polyclonal activators occurred over time. At 18 months after transplantation, she is well with 100% donor chimerism and has recovered normal immune function.

Inadequate leptin level negatively affects body fat loss during a weight reduction programme for childhood obesity.

UNLABELLED: Obesity is a typical example of a complex multifactorial disease arising from behavioural, environmental and genetic factors that may affect individual responses to dietary intake and physical activity. Observational, longitudinal dietary interventional studies in obese patients present contrasting reports on the predictive value of baseline leptin levels. We report on the effect of a weight reduction programme in three different groups of obese children (82 patients in all) assembled on the basis of their baseline leptin levels adjusted for body mass index (BMI), gender and pubertal development. The effectiveness of this programme was decreased in patients with relative hyperleptinaemia or hypoleptinaemia compared to children with baseline leptin levels appropriate to BMI gender and pubertal development. CONCLUSION: Information gained from leptin assays could provide predictive insight into an individual's ability to lose body fat and may therefore have important implications for our approach to the treatment and prevention of childhood obesity.

Factors influencing post-transfusional platelet increment in pediatric patients given hematopoietic stem cell transplantation.

Patients undergoing allogeneic hematopoietic stem cell transplantation (HSCT) always require platelet transfusions, but the increase in platelet count is often less than expected. Since factors responsible for poor response to platelet transfusions in this clinical setting are largely unknown, we performed a prospective study in 87 consecutive children transplanted in a single institution. The mean 16-h corrected count increment (CCI) of 598 platelet transfusions was 5.76 +/- 8.32 x 10(9)/l. Both before and after HSCT, 13.8% of patients had antibodies against HLA and/or platelet-specific antigens. Univariate analysis identified 12 factors significantly associated with a lower post-transfusion CCI, but only four reached statistical significance in the multivariate analysis. These four factors were concomitant therapy with vancomycin, alloimmunization, use of an Autopheresis cell separator for preparation of platelet concentrates and cytomegalovirus infection. We, therefore, suggest that a better response to platelet transfusions could be obtained by choosing a suitable cell separator, by avoiding the use of vancomycin and by adopting measures that reduce alloimmunization and CMV infection. Moreover, screening patients for HLA and platelet-specific antibodies before HSCT would identify the majority of subjects who will develop alloimmune refractoriness after transplantation and would allow the search for a compatible donor in advance.

Effects of nebivolol on human platelet aggregation.

It has been documented that beta-adrenergic antagonists can influence platelet aggregation by a mechanism independent of their ability to antagonize beta-adrenoceptors. Nebivolol, a selective beta1-adrenergic receptor antagonist with additional hemodynamic effects, is able to vasodilate human forearm vasculature by acting on the L-arginine/nitric oxide pathway. Constitutive nitric oxide synthase is present also in human platelets, resulting in the formation of nitric oxide, an endogenous inhibitor of platelet aggregation. The aim of this study was to investigate the effects of nebivolol on platelet aggregation and in particular to determine the involvement of the platelet L-arginine/nitric oxide pathway. Propranolol, a nonselective beta-adrenergic antagonist, and carvedilol, a beta-blocker with vasodilating properties, were compared with nebivolol on platelet activity. Plasma from healthy male subjects was used. Platelet aggregation was achieved with adenosine diphosphate (ADP) (3 microM) and collagen (1 microg/ml), using the Born turbidimetric method to measure platelet aggregation. Our results showed that nebivolol, propranolol, and carvedilol all had an inhibitory effect on both ADP- and collagen-induced platelet aggregation. Nebivolol exhibited the greatest inhibition effect on platelet aggregation. The mechanism responsible for the inhibitory effect of nebivolol appeared to involve a nitric oxide-dependent pathway. Indeed, L-arginine augmented the inhibitory effects of nebivolol on platelet aggregation induced by collagen and ADP. Furthermore, the inhibitory effect of nebivolol on platelet aggregation was reduced in the presence of the nitric oxide synthase inhibitor N(G)-monomethyl-L-arginine (L-NMMA). In conclusion, we have demonstrated in this study that nebivolol's mechanism of platelet aggregation inhibition differs from that of other beta-adrenergic antagonists by being partially dependent on nitric oxide production.

Interaction between metabotropic and NMDA glutamate receptors in the periaqueductal grey pain modulatory system.

The formalin test was used to investigate the interactive role of periaqueductal grey (PAG) N-methyl-D-aspartate (NMDA) and metabotropic glutamate (mGlu) receptors in the modulation of persistent noxious stimulation in mice. Intra-PAG microinjections of 1 or 3 nmol NMDA, a selective agonist at NMDA-subtype receptors, decreased the nociceptive response (-94+/-5% with 3 nmol) during the latter phase of the test. This effect was antagonized by MK-801, a selective antagonist at NMDA receptors. No change in the early nociceptive phase was observed after NMDA injection. Pretreatment either with 2-methyl-6-phenylethynylpyridine (MPEP, 25 nmol/mouse), a selective antagonist at mGlu5 receptors, or with (2S)-alpha-ethylglutamic acid [(2S)-alpha-EGlu, 30 nmol/mouse], a selective antagonist at group-II mGluRs, prevented the NMDA-induced antinociceptive effect during the late hyperalgesic phase. Pretreatment with (R,S)-alpha-methylserine-O-phosphate [(R,S)-alpha-MSOP, 70 nmol/mouse], a selective antagonist at group-III mGlu receptors, had no effect on the NMDA-induced antinociception. None of the antagonists changed the formalin-induced nociceptive behaviour per se with the dosages used in combination with NMDA. MPEP at 50 nmol/mouse, however, potentiated the early nociceptive phase whilst 100 nmol/mouse attenuated the late phase. Similarly, at the higher dose of 140 nmol/mouse, (R,S)-alpha-MSOP decreased the late hyperalgesic phase. These results provide additional evidence that NMDA and mGlu receptors participate in modulating the hyperalgesia induced by peripheral noxious stimulation. In particular, mGlu receptors may modulate the NMDA receptors in the PAG since their physiological stimulation seems to be required for the NMDA-induced effect. This suggests that, together with ionotropic glutamate receptors, mGlu receptors also play a role in modulating a type of spinal cord neuroplasticity (i.e. wind-up) that has been proposed to mediate hyperalgesia.

Anti-CD20 monoclonal antibody for the treatment of severe, immune-mediated, pure red cell aplasia and hemolytic anemia.

Immune-mediated, acquired pure red cell aplasia (PRCA) is a rare disorder frequently associated with other autoimmune phenomena. Conventional immunosuppressive treatment is often unsatisfactory. Rituximab is a monoclonal antibody against the CD20 antigen, highly effective for in vivo B-cell depletion. An 18-month-old girl with both severe PRCA and autoimmune hemolytic anemia, refractory to immunosuppressive treatment, received 2 doses of rituximab, 375 mg/m(2) per week. The drug was well tolerated. After anti-CD20 therapy, substitutive treatment with intravenous immunoglobulin was started. The treatment resulted in marked depletion of B cells; a striking rise in reticulocyte count ensued, with increasing hemoglobin levels, finally leading to transfusion independence. The child is now 5 months off-therapy, with normal hemoglobin and reticulocyte levels. This case suggests a role of anti-CD20 monoclonal antibody for treatment of patients with antibody-mediated hematologic disorders. (Blood. 2001;97:3995-3997)

Flow-cytometric analysis of erythrocytes and reticulocytes in congenital dyserythropoietic anaemia type II (CDA II): value in differential diagnosis with hereditary spherocytosis.

Congenital dyserythropoietic anaemia type II (CDA II) is the most common congenital dyserythropoietic anaemia. CDA II is frequently misdiagnosed as Hereditary Spherocytosis (HS) due to the presence of mild chronic haemolytic anaemia with splenomegaly, increased osmotic fragility, and presence of microspherocytes. Accurate diagnosis of CDA II is important to prevent severe iron overload. Erythrocyte and reticulocyte indices were assessed in 10 patients from six families with CDA II, 18 patients from eight families with HS, and 50 normal controls. Characteristic increases in distribution width were present in CDA II for cell volume (RDW, anisocytosis) and in HS for cell haemoglobin concentration (HDW, anisochromia), resulting in an RDW/HDW ratio which was significantly greater in CDA than HS (P < 0.0002). A cut-off value for RDW/HDW of 5.34 resulted in 89% sensitivity and 70% specificity in distinguishing CDA II from HS. Distribution width for cell haemoglobin content of reticulocytes (CHDWr) was characteristically increased in CDA II, resulting in a CHDW/CHDWr ratio significantly lower in CDA II than HS (P < 0.0002). A cut-off value of 0.98 provided 89% sensitivity and 80% specificity in distinguishing CDA II from HS. These differences in distribution widths of flow-cytometric parameters of reticulocytes and mature erythrocytes reflect the different pathogeneses of the two diseases and are helpful for the differential diagnosis of these two conditions.

Reversible erythrocyte skeleton destabilization is modulated by beta-spectrin phosphorylation in childhood leukemia.

The erythrocyte skeleton plays an essential role in determining the shape and deformability of the red cell. Disruption of the interaction between components of the red cell membrane skeleton may cause loss of structural and functional integrity of the membrane. Several observations based on studies in vitro strongly suggest that phosphorylation may modify interactions between proteins, leading to a reduced affinity. In particular, increased phosphorylation of beta-spectrin decreases membrane mechanical stability. In order to investigate the presence of membrane protein defects we investigated the erythrocyte membrane protein composition and phosphorylation in 22 children with leukemia at diagnosis and during the remission phase. Sixteen children had acute lymphoblastic leukemia (ALL), three had chronic myeloid leukemia (CML) and three had acute myeloid leukemia (AML). Ten patients (eight ALL and two CML) displayed elliptocytosis and poikilocytosis, an increase of spectrin dimers (41.8 +/- 15.6) and an enhanced phosphorylation of beta-spectrin (108 +/- 15%) at diagnosis. These alterations disappeared during the remission phase. This is the first demonstration of a reversible erythrocyte membrane alteration in leukemia. Since the beta-spectrin phosphate sites are located near the C-terminal region and close to the head of the beta-chain that is involved in dimer-dimer interaction, we supposed that the beta-chain phosphorylation has an effect upon the interactions between spectrin dimers, ie the tetramerization process. The weakening of this process should be responsible for the presence of elliptocytes and poikilocytes as reported in hereditary elliptocytosis and pyropoikilocytosis.

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 beta-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 beta-spectrin gene were utilized for the detection of de novo mutations influencing beta-spectrin mRNA stability. They were also screened for the presence of alpha-spectrin(LEPRA) 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 beta-spectrin de novo mutations. Furthermore, we found five patients to be heterozygous for alpha-spectrin(LEPRA) 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.

Gilbert's syndrome accounts for the phenotypic variability of congenital dyserythropoietic anemia type II (CDA-II).

The molecular basis for the considerable variation of serum bilirubin levels and the incidence of gallstone formation in patients with congenital dyserythropoietic anemia (CDA) type II are unknown. We show that the combined effect of an increased bilirubin load caused by dyserythropoiesis in CDA II and decreased bilirubin conjugation caused by reduced expression of uridine diphosphate glucuronosyl transferase (UGT1A) would increase the risk of hyperbilirubinemia (P <.005) and gallstone formation (chi(2): P <. 001). The rate of gallstone formation in patients with CDA II is 4. 75-fold the rate of patients without Gilbert's syndrome, and gallstone diagnosis occurs at a younger age (P < 0.01). These findings should be considered during the follow-up of patients with CDA II.

Recombinant erythropoietin therapy as an alternative to blood transfusions in infants with hereditary spherocytosis.

INTRODUCTION: In hereditary spherocytosis, erythropoiesis has been described as 'sluggish' during the first months of life. The lack of appropriate erythropoietic response to compensate for increased red cell destruction necessitates blood transfusions in 70-80% of hereditary spherocytosis-affected infants during their first year of life. After this period, less than 30% require regular transfusion support. This transient requirement for transfusion led us to wonder whether anemic hereditary spherocytosis infants, like anemic premature infants, could benefit from recombinant erythropoietin therapy (rHu-Epo). MATERIAL AND METHODS: In 16 hereditary spherocytosis infants (age range 16-119 days) with severe anemia, a compassionate open preliminary study was performed. rHu-Epo treatment (1000 IU/kg/week) was instituted together with iron supplementation. Hemoglobin values and reticulocyte counts were repeatedly assessed. RESULTS: In 13 out of 16 infants, prompt increases in reticulocyte counts were noted after the first week of treatment with 1000 IU/kg/week of rHu-Epo. During treatment with Epo these infants maintained clinically acceptable levels of hemoglobin and did not require blood transfusions. As the infants grew and began to mount an adequate erythropoietic response, the rHu-Epo dose could be tapered and the treatment could be discontinued before the age of nine months. CONCLUSION: Epo treatment in most hereditary spherocytosis infants appears to be effective in the management of anemia and could serve as a valuable alternative to packed RBC transfusions.

Coinheritance of Gilbert syndrome increases the risk for developing gallstones in patients with hereditary spherocytosis.

The precocious formation of bilirubinate gallstones is the most common complication of hereditary spherocytosis (HS), and the prevention of this problem represents a major impetus for splenectomy in many patients with compensated hemolysis. Because Gilbert syndrome has been considered a risk factor for gallstone formation, there are reasons for postulating that the association of this common inherited disorder of hepatic bilirubin metabolism with HS could increase cholelithiasis. To test this hypothesis, 103 children with mild to moderate HS who, from age 1, have undergone a liver and biliary tree ultrasonography every year, were retrospectively examined. The 2-bp (TA) insertion within the promoter of the uridine diphosphate-glucuronosyltransferase gene (UGT1A1), associated with Gilbert syndrome, was screened. The risk of developing gallstones was statistically different among the 3 groups of patients: homozygotes for the normal UGT1A1 allele, heterozygotes, and homozygotes for the allele with the TA insertion. Fitting a Cox regression model, in fact, a statistically significant hazard ratio of 2.19 (95% confidence interval: 1.31 to 3.66) was estimated from one to the next of these genetic classes. The individual proneness to form gallstones from TA insertion in the TATA-box of the UGT1A1 promoter should be considered during the follow-up of patients with HS. Although patients with HS were the only ones studied, extrapolating these data to patients who have different forms of inherited (eg, thalassemia, intraerythrocytic enzymatic deficiency) or acquired (eg, autoimmune hemolytic anemia, hemolysis from mechanical heart valve replacement) chronic hemolysis can be warranted.

Diamond-Blackfan anaemia in the Italian population.

Diamond-Blackfan anaemia (DBA) is a congenital disease characterized by defective erythroid progenitor maturation: 30% of patients have congenital malformations. The link between these malformations and defective erythropoiesis is unclear: a defect in a molecule acting both on embryo development and haemopoiesis has been proposed. Inheritance is autosomal dominant in most familial cases, but recessive families have also been reported. Many cases are sporadic. A DBA locus has been mapped on chromosome 19q13.2 (Gustavsson et al, 1997), but several families unlinked to this locus have also been reported (Gustavsson et al, 1998). This paper presents clinical, epidemiological and molecular data for DBA in the Italian population. Segregation analysis of 19q markers in patients with DBA showed exclusion of this locus in 5/12 families with inherited DBA. There was evidently locus heterogeneity for DBA in this population. A new microdeletion was identified in one patient. Other families, in which DBA segregates concordantly with the 19q critical region, suggest incomplete penetrance and expressivity of the DBA gene.

Frequent de novo monoallelic expression of beta-spectrin gene (SPTB) in children with hereditary spherocytosis and isolated spectrin deficiency.

This report represents an attempt to define the rate of beta-spectrin de novo mutations affecting mRNA accumulation in patients with hereditary spherocytosis (HS). 19 HS children with haematologically normal parents and varying degrees of spectrin deficiency were studied. 13 of the 19 cases who were heterozygous at the genomic level for polymorphisms in the beta-spectrin coding region were further studied. However, in an analysis of reverse-transcripted amplified cDNA from the regions of the polymorphisms, seven patients appeared to be homozygous, suggesting the occurrence of de novo mutational events affecting expression of one beta-spectrin allele. We conclude that in HS patients with isolated spectrin reduction and normal parents the apparently recessive pattern of inheritance may frequently be associated with de novo monoallelic expression of beta-spectrin.

High frequency of de novo mutations in ankyrin gene (ANK1) in children with hereditary spherocytosis.

OBJECTIVE: To evaluate the frequency of de novo monoallelic expression of the ANK1 gene in hereditary spherocytosis individuals appearing as recessive. STUDY DESIGN: We studied 40 unrelated children with spherocytosis and their normal parents. The genomic distribution of the ankyrin (AC)n dinucleotide repeats was evaluated in the patients showing combined ankyrin and spectrin deficiency. To search for the absence of mRNA derived from one of the two ANK1 genes, cDNA from the heterozygous patients was amplified using polymerase chain reaction. This was analyzed for the (AC)n dinucleotide repeats. RESULTS: Thirty-three hereditary spherocytosis subjects had variable degrees of combined ankyrin and spectrin reduction; 19 were found to be heterozygous for the AC repeat lengths and were further studied. In 12, we found a cDNA polymerase chain reaction product from one ankyrin gene alone. These findings strongly suggested the nonexpression of one of the two ANK1 genes because of the de novo mutational events. CONCLUSION: The de novo loss of an ankyrin allele expression is a frequent cause of hereditary spherocytosis in children with normal parents. Therefore the category of genuinely recessive hereditary spherocytosis cases is further reduced compared with spherocytosis cases because of de novo mutations. The determination of the (AC)n microsatellite polymorphisms appears as a helpful and reliable tool for the discrimination between these two categories.