Metello Iacobini

Clinical, genetic, and cellular analysis of 49 osteopetrotic patients: implications for diagnosis and treatment

Background: Osteopetrosis, a genetic disease characterised by osteoclast failure, is classified into three forms: infantile malignant autosomal recessive osteopetrosis (ARO), intermediate autosomal recessive osteopetrosis (IRO), and autosomal dominant osteopetrosis (ADO). Methods: We studied 49 patients, 21 with ARO, one with IRO, and 27 with type II ADO (ADO II). Results: Most ARO patients bore known or novel (one case) ATP6i (TCIRG1) gene mutations. Six ADO II patients had no mutations in ClCN7, the only so far recognised gene implicated, suggesting involvement of yet unknown genes. Identical ClCN7 mutations produced differing phenotypes with variable degrees of severity. In ADO II, serum tartrate resistant acid phosphatase was always elevated. Bone alkaline phosphatase (BALP) was generally low, but osteocalcin was high, suggesting perturbed osteoblast differentiation or function. In contrast, BALP was high in ARO patients. Elevated osteoclast surface/bone surface was noted in biopsies from most ARO patients. Cases with high osteoclasts also showed increased osteoblast surface/bone surface. ARO osteoclasts were morphologically normal, with unaltered formation rates, intracellular pH handling, and response to acidification. Their resorption activity was greatly reduced, but not abolished. In control osteoclasts, all resorption activity was abolished by combined inhibition of proton pumping and sodium/proton antiport. Conclusions: These findings provide a rationale for novel therapies targeting pH handling mechanisms in osteoclasts and their microenvironment.

Mechanisms of Osteoclast Dysfunction in Human Osteopetrosis: Abnormal Osteoclastogenesis and Lack of Osteoclast-Specific Adhesion Structures

Osteoclasts from a patient affected by osteopetrosis were examined in vivo and in vitro. Iliac crest biopsy revealed an osteosclerotic pattern, with prominent numbers of osteoclasts noted for hypernuclearity and incomplete adherence to the bone surface. A population comprising tartrate‐resistant acid phosphatase (TRAP)‐positive, multinucleated and mononuclear cells, and alkaline phosphatase‐positive stromal fibroblasts was obtained in vitro from bone marrow. Mononuclear TRAP‐positive precursors spontaneously fused in culture to form giant osteoclast‐like cells. These cells expressed the osteoclast marker MMP‐9 and calcitonin receptor, and lacked the macrophage marker, Fc receptor. Expression and distribution of c‐src, c‐fms, and CD68, and response to steroid hormones relevant to osteoclast differentiation and function were apparently normal, whereas cell retraction in response to calcitonin was impaired. TRAP‐positive multinucleated cells did not form osteoclast‐specific adhesion structures (clear zone, podosomes, or actin rings). Bone resorption rate was severely reduced in vitro. Focal adhesions and stress fibers were observed en lieu of podosomes and actin rings. Adhesion structures contained low levels of immunoreactive vitronectin receptor, most of this integrin being retained in cytoplasmic vesicles. These data provide the first characterization of abnormal differentiation and function of human osteopetrotic osteoclast‐like cells.

Type II benign osteopetrosis (Albers-Schönberg disease) caused by a novel mutation in CLCN7 presenting with unusual clinical manifestations.

AbstractA 16-year-old male patient with type II autosomal dominant benign osteopetrosis (ADO) was genotyped and found to harbor a novel mutation in exon 25 of the gene encoding for the osteoclast-specific chloride channel, CLCN7, inherited from the father, who was asymptomatic. The patient had normal biochemical findings and acid-base balance, except for increased serum levels of creatine kinase, lactic dehydrogenase, and the bone formation markers bone alkaline phosphatase isoenzyme, osteocalcin and N-terminal type I collagen telopeptide/creatinine ratio. Unusual generalized osteosclerosis was observed together with a canonical increase in vertebral and pelvis bone mass. An affected first grade cousin presented with normal biochemical findings and a milder osteosclerotic pattern of the pelvis. At the cellular level, cultured osteoclasts from the patient showed increased motility, with lamellipodia, membrane ruffling and motile pattern of podosome distribution, all of which could have contributed to functional impairment of bone resorption. The present report documents a novel mutation of the CLCN7 gene causing osteopetrosis in a radiologically uncertain form of the diseases, with apparent incomplete penetrance.

Involvement of oxygen radicals in cytarabine-induced apoptosis in human polymorphonuclear cells.

We investigated apoptosis in polymorphonuclear neutrophils (PMNs) induced by cytarabine (Ara-C). This drug increased apoptosis by 100% with respect to the controls after 3 hr of incubation. This increase was inhibited by N-acetyl-L-cysteine (NAC) or diphenyleneiodonium chloride (DPI). Ara-C alone caused an early increase (after a 30-min incubation) in intracellular oxidant generation (inhibitable by rotenone, fumonisin b1, and DPI) and in protein tyrosine phosphorylations (inhibitable by NAC). The drug also affected the observed reduction of dimethylthiazol diphenyltetrazolium bromide (MTT). No extracellular release of reactive oxygen species (ROS) was elicited by the addition of Ara-C, while the drug increased the release of ROS by N-formyl-leucyl-phenylalanine-(f-MLP) but not phorbol 12-myristate 13-acetate-stimulated PMNs. This phenomenon was abolished by the addition of genistein, whereas such an effect was not observed following the addition of 1-(5-isoquinolynilsulfonyl)-2-methylpiperazine (H7). Ara-C induced ROS release from PMNs in the presence of subthreshold concentrations of f-MLP (priming effect). These results indicate that intracellular ROS production from mitochondria promotes Ara-C-induced apoptosis. Ara-C primes plasma membranes by a mechanism involving protein tyrosine phosphorylations and may also contribute to ROS generation from the granules.

Apparent Cure of a Newborn with Malignant Osteopetrosis Using Prednisone Therapy

A newborn girl with hemorrhagic purpura, suspected neonatal sepsis, and pale and dry skin was lethargic with remarkable hepatosplenomegaly, convergent strabismus, severe anemia, and elevated alkaline phosphatase activity. Radiographs showed a generalized increase in bone density, small medullary cavities, sclerosis of the skull and vertebrae, transverse wavy stripes of sclerotic bone in the metaphyses, and bone‐in‐bone appearance in phalanges of hands and feet. On this basis, she was diagnosed with malignant infantile osteopetrosis. On the first day of life, the infant was given a blood transfusion and vitamin K (1 mg intravenously [iv]). Corticosteroid therapy was started with prednisone (2 mg/kg per day). She showed marked improvement of symptoms. On the 26th day and 42nd day of life, she received additional blood transfusions. On the 49th day, the patient was discharged and corticosteroid therapy was continued at a regimen of 5 mg/day. Subsequent blood sample analyses revealed normal values for age. At 1 year of life, a bone marrow sample showed normal white and red cell lineages. X‐ray confirmed attenuation of the bone sclerosis; therefore, bone marrow transplantation (BMT) was not implemented. At the age of 1.5 years, prednisone therapy was discontinued gradually and withdrawn before the age of 2 years. Subsequent follow‐up showed normalization of all radiological and hematologic parameters. At present, the patient is 3 years old and appears healthy with apparently complete regression of the disease.

Chemokine concentrations in nasal washings of infants with rhinovirus illnesses.

We determined RANTES (regulated on activation, normal T cell expressed and secreted) and interleukin-8 (IL-8) concentrations, and total white blood cell (WBC) and differential counts in nasal wash samples from rhinovirus-infected infants presenting with wheezing or acute upper respiratory illness alone and compared them with those from healthy infants. RANTES concentrations were significantly greater in acute samples from wheezy patients than in those from patients with acute upper respiratory illness only, or in control samples. IL-8 concentrations and WBC and neutrophil counts were significantly greater in acute samples from wheezy infants and patients with upper respiratory illness alone than in control samples, but they did not differ significantly between the 2 patient groups.

Transient Hypogammaglobulinemia of Infancy: Intravenous Immunoglobulin as First Line Therapy

IVIG (Intravenous immunoglobulin) have significantly improved the prognosis and the quality of life of immunodeficient patients and are routinely used as substitutive therapy. Transient hypogammaglobulinemia of infancy (THI) is a primary humoral immunodeficiency characterized by a transient IgG defect, but is not considered as a disease that justifies substitutive treatment and thus the use of IVIG as an alternative to antibiotic prophylaxis remains controversial also in symptomatic children. We treated 13 THI children severely symptomatic with IVIG (400mg/kg/every 3 weeks) for a limited period (2 or 3 months) and followed them for 1 to 3 years. During the follow-up, the frequency of overall infections decreased approximately tenfold (from 0.39 to 0.047 infection/month per child) and no severe infections were reported. Although this study lacks untreated controls, the results suggest that the observed clinical improvement is correlated to IVIG therapy. Furthermore, our study suggests that the infused IVIG have no long-term effect on endogenous IgG production and do not lengthen the immunodeficiency condition since all children produced a normal amount of specific IgG in response to vaccination carried out 5 months after the end of infusions. In conclusion, our results suggest that IVIG may stop the vicious circle of infection-immunodeficiency and should be considered as a first line therapy in highly symptomatic THI children.

Chronic granulomatous disease mimicking early-onset Crohn’s disease with cutaneous manifestations

BackgroundChronic granulomatous disease is a rare inherited disorder of the innate immune system. In patients with a clinical history of recurrent or persistent infections, especially infections caused by uncommon species, chronic granulomatous disease should be considered.Case presentationWe report the case of a 5-year-old boy with a presumptive diagnosis of Crohn’s disease with extraintestinal manifestations. Chronic granulomatous disease was suspected in this case after Serratia marcescens was isolated from a skin ulcer culture. Granulomas were confirmed on histology and chronic granulomatous disease was diagnosed.ConclusionThis case emphasizes the importance of high clinical suspicion of an alternative diagnosis of immune deficiency in patients with presumed inflammatory bowel disease and opportunistic infections, especially when disease occurs in early life.

Taurine deficiency in thalassemia major-induced osteoporosis treated with neridronate

The aetiology of thalassemia major-induced osteoporosis is multifactorial. Up to now, bisphosphonates seem to be a promising therapy. Taurine is found in a high concentration in bone cells enhancing bone tissue formation and inhibiting bone loss. Recently we found a decrease taurine plasma level in children affected by osteogenesis imperfecta during neridronate (amino-bisphosphonate) therapy suggesting a possible interaction between pharmacological effect of this drug and taurine availability. On the basis of these results, we performed plasma and urine amino acid (AA) analysis in thalassemia major-induced osteoporosis before and after 12 months of neridronate treatment. Twelve patients, five males and seven females, aged from 20 to 29 years following a hypertransfusion treatment protocol were enrolled in the study. Patients were treated with neridronate infusion every one month (30 mg in 100ml of saline). Plasma and urine specimens for AA analysis, bone mineral density, bone mineral content and vertebral project area were examined at baseline (T0) and after 12 months of treatment (T12). A significant decrease was observed for plasma level and urinary excretion of taurine (T0 vs. T12=p<0.01) whereas bone mineral content and vertebral projection area showed a statistical significant increase (T0 vs. T12=p<0.05). These results and other experimental researches warrant further studies examining the long-term effect of taurine supplementation in association with neridronate treatment.

Severe warm autoimmune haemolytic anaemia due to anti-Jk(a) autoantibody associated with Parvovirus B19 infection in a child.

Dear Sir, Autoimmune haemolytic anaemia (AIHA) is an uncommon clinical condition in which endogenous antibodies are directed against the patient’s own red blood cells coated by immunoglobulin and/or complement. Anaemia appears when the destruction of red blood cells overwhelms the bone marrow’s capacity to produce new cells. People of all ages can be affected, although some reports suggest that, in childhood, secondary cases are more common than idiopathic forms, and viral or bacterial agents are frequently the only recognisable stimuli. Indeed, AIHA follows viral infection or vaccination much more often in children than in adults. However, in the majority of cases of AIHA the pathogen remains undefined. Parvovirus B19 infections have frequently been implicated as a trigger of several forms of autoimmune diseases both in children and adults. Here we report the case of a 5-year old girl who was referred to our hospital because of anaemia and mild jaundice. The patient was previously healthy until 15 days prior to admission, when she developed a fever, weakness, lack of appetite, diarrhoea and vomiting for about 4 days. On admission, the main clinical signs and symptoms noted during a general physical examination were pallor, jaundice and tachycardia (heart rate: 150 bpm). Haematological tests showed a haemoglobin (Hb) level of 4.1 g/dL, mean corpuscular volume 83 fL, reticulocyte count 147×109/L and normal leucocyte and platelet counts. Marked polychromasia with spherocytosis and nucleated red blood cells were noted on the peripheral blood smear, without atypical cells. The serum lactate dehydrogenase (LDH) was raised at 1,047 IU/L, total bilirubin was 2.61 mg/dL, direct bilirubin 0.61 mg/dL, haptoglobin 10 mg/dL, C-reactive protein 10.8 mg/L, aspartate amino transferase 68 IU/L, alanine amino transferase 24 IU/L and ferritin level 354 ng/mL. Tests for anti-nuclear, anti-double-stranded DNA, and anti-smooth muscle antibodies and anti-phospholipids were negative. Abdominal ultrasonography revealed hepatosplenomegaly. An immunohaematological study was performed. A direct antiglobulin test (DAT) was performed with a broad-spectrum antiserum and with monospecific anti-IgG, -IgA, -IgM, -C3d and -C3b antisera, in liquid phase and by column agglutination (reagents from Ortho Clinical Diagnostics, Raritan, New Jersey, USA and Diamed, Cressier sur Morat, Switzerland). Eluate testing was performed by Rubin’s method and with low pH glycine buffer using a commercial kit (ELU-KIT™ II, Immucor, Norcross, Georgia, USA). An indirect antiglobulin test (IAT) with untreated and treated (ficin/papain) homologous red blood cells (Resolve C - Ortho Clinical Diagnostics and ID-Diamed Panel- DiaMed) was also performed. On admission, the DAT was strongly positive for an IgG autoantibody which was also present in the patient’s serum. Both the eluate and the serum, investigated using a broad panel of reagent red blood cells, showed an anti-Jka antibody. Kidd typing of the erythrocytes, performed using a monoclonal IgM reagent (Ortho Clinical Diagnostics), showed a Jk(a) positive, Jk(b) negative phenotype so the anti-Jka antibodies found in the blood of the patient were presumed to be autoantibodies. AIHA was diagnosed and therapy was started with intravenous methylprednisolone (20 mg/kg/die) and folic acid (20 mg/die). From the fifth day the steroid treatment was continued in the form of oral prednisone (2 mg/kg/die). Due to severe, symptomatic anaemia the child was transfused with a compatible unit (150 mL) of Jk(a) negative, Jk(b) positive red blood cells. Bacterial culture of stools for Campylobacter, Rotavirus, Yersinia, and Escherichia coli were negative, as was the search for lactate-positive, coagulase-negative Enterobacteria, Staphylococcus and Enterococci spp. Other causative agents were investigated: cytomegalovirus, Epstein-Barr virus, hepatitis B and C viruses, human immunodeficiency viruses 1/2, herpes simplex virus 1–2 were negative, while Parvovirus B19 IgM and IgG were found. The patient’s clinical conditions gradually improved and she was discharged from hospital. About 2 weeks after admission the immunohaematological tests became negative and the patient had a Hb level of 11.5 g/dL; LDH levels returned to the normal range. One month after the onset, the child developed a transient thrombocytopenia (platelet count of 96×109/L and 106×109/L on two different occasions). The prednisone treatment was maintained at a dose of 2 mg/kg/die for 15 days, and then gradually reduced to 2.5 mg/die and discontinued 40 days after the initial presentation because the patient had normal haematological findings. She remains healthy at follow-up (6 months). In paediatric patients AIHA is often associated with infections or organ-specific autoimmune diseases1–4. Human Parvovirus B19 may cause a widespread benign and self-limiting disease in children and adults, but this viral infection has also been associated with the production of autoantibodies against many autoantigens (nuclear antigens, neutrophil cytoplasmic antigens, phospholipids) and with rheumatoid factor.2. It has previously been shown that chronic B19 infection can induce anti-viral antibodies with autoantigen-binding properties. The autoimmune sequelae have a multifactorial and complex origin: the involvement of molecular mimicry between self-antigens and viral proteins, the induction of enhanced cytokine production via the viral transactivator protein NS1 and the phospholipase A2-like activity of the capsid protein VP1 seem to contribute to the induction of autoimmune diseases3. Parvovirus B19 interacts extensively with human red blood cells; in vitro studies have shown changes in capsid conformation following B19 binding to red blood cells, leading to exposure of a region (VP1 “unique region”) that seems to play a central role in the induction of autoimmune processes. Antibodies derived from the exposed VP1 “unique region” would not neutralise free infectious particles in the blood, but would instead target receptor-attached virus4. An interesting finding in our case was the rarely occurring specific complement-binding warm auto-antibodies against the Jk(a) antigen. Generally autoantibodies with single specificity are produced against Rh system antigens. Warm anti-Jka autoantibodies have been rarely described, in association or not with haemolysis; most of the cases reported in the literature were in patients with autoimmune disorders, such as ulcerative colitis or systemic lupus erythematosus. In our patient the simultaneous disappearance of the anti-Jka autoantibodies and the haemolysis strongly suggests that the anti-Jka was responsible for the haemolysis. It is noteworthy that the first manifestations of infection in our patient were in the gastrointestinal system and no infectious agent was identified other than the B19 virus. The gastrointestinal symptoms were followed 2 weeks later by acute haemolysis. Antigen sharing between the gastrointestinal tract and red blood cells has been described by Hinoue et al.5, who identified and characterised a Kidd antigen/UT-B urea transporter expressed in the human colon, encoded by the Slc14A1 gene, with a sequence identical to that reported for the Kidd/UT-B present on the red blood cells. In the light of these data, we can hypothesise a cross-reactivity of autoantibodies between autoantigens of the colon and the red blood cells. The severe, transient haemolysis observed in our patient occurred after the acute phase of infection. Our patient also showed a transient mild thrombocytopenia, while her leucocyte count remained normal. A meta-analysis that included 516 cases of childhood autoimmune thrombocytopenia and 246 healthy controls showed that human B19 infection is closely associated with childhood autoimmune thrombocytopenia. Parvovirus B19, which can be a primary cause of reticulocytopenic post-infection anaemia in childhood, is strongly suspected to have been responsible for the haemolytic anaemia in our patient. This case is unusual and interesting because of the association of B19 infection with warm AIHA and because of the rare specificity of the autoantibodies. Besides its scientific interest, the authors recommend identification of antibody specificities in AIHA since this will play an important role in the appropriate transfusion therapy if the severity of the clinical course is such as to impose this therapeutic strategy.