Improvement of a rapid screening test for chronic granulomatous disease.

Diagnosis of CGD is made by demonstrating absent or markedly reduced oxidase activity in stimulated neutrophils. The screening test proposed is based upon the naked eye evaluation of the reduction of NBT on a solid surface. It seems to be a useful tool for rapid and inexpensive detection of CGD patients, especially for large-scale screening purposes. The test was carried out on forty-five subjects: two males affected by CGD, three female carriers and forty healthy donors. The test confirmed the results obtained with flow cytometric and NBT assays.

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.

Down regulation of CD11b and CD18 expression in children with hypercholesterolemia: a preliminary report.

BACKGROUND AND AIM: Cell adhesion molecules play an important role in the development of atherosclerosis mediating the attachment of monocytes to the endothelium. The aim of our study was to assess the cell surface expression of CD11b/CD18 integrin on the phagocytes of children affected by hypercholesterolemia. METHODS AND RESULTS: Twenty-six children with hypercholesterolemia (15 males, mean age 8.3, range 2-18) with a family history of early cardiovascular disease, as well as 26 children with normocholesterolemia matched for gender and age (15 males, mean age 8.3) were studied. Cell surface expression of CD11b/CD18 on peripheral blood mononuclear cells (PBMC) were analyzed by flow cytometry. The geometric mean percentages of CD11b and CD18 expression were significantly lower in the hypercholesterolemic group [52 (95% confidence intervals, 40-68) and 88 (84-93)] than in the control group [87 (83-91), P<0.0001 and 93 (89-96), P<0.05], respectively. After correction for age, gender, and pubertal status, CD11b cell surface expression on PBMC was inversely and independently correlated with total cholesterol concentrations (r=-0.395; P<0.01) and LDL (r=-0.307; P<0.05), as well as with triglycerides (r=-0.406; P<0.01). CONCLUSIONS: In children with hypercholesterolemia, cell surface expression of CD11b and CD18 on PBMC was significantly decreased. Follow-up studies are necessary to determine the clinical implications of these findings in the context of the natural course and progression of atherosclerosis in high risk children.

[Theoretical considerations and comments on the physiopathology in renovascular hypertension]. / Consideratii teoretice si comentarii privind fiziopatologia hipertensiunii arteriale de origine renovasculara.

The researches performed during the last four decades did not elucidate completely the pathogenic mechanism of the renovascular hypertension. The present knowledge considers that the origins of renovascular hypertension are the imbalance between the renal hypotensive system located in the medullar renal site (antihypertensive and hypotensive substances) and the renal hypertensive system (renin-angiotensin-aldosterone) located cortically. As an additional mechanism in producing hypertension is involved the disorder of hydro electrolytic metabolism, as a result of decreased excretory function, inducing an increase of plasmatic natrium level, of volemia and interstitial liquid.

[Renovascular hypertension: symptoms and signs. Clinical study on 20 patients]. / Hipertensiunea arteriala de origine renala: simptome si manifestari. Studiu clinic pe 20 pacienti.

OBJECTIVES: symptoms in renovascular hypertension can be wrong interpreted, which leads to a late diagnostic, after discover the determination disease. MATERIALS AND METHODS: the study on the renovascular hypertension was made on 20 patients, aged between 2 and 36 years old, diagnosed with reno-ureteral malformations, pyelonephritis, reno-ureteral reflux and renal trauma as a determination disease, leads to manifestation types that guide the diagnostic: neuro-psychiatric signs, weight loss, renal signs and digestive signs. Beginning from these signs the arterial hypertension was diagnosed and the investigations determined the causes. RESULTS: Periodic postoperative evaluation at 3 months, during a period between 4 months and 7 years, individualised 4 evolutional clinical types: AHT with lumbar pain, AHT with no clinical signs, AHT with ophthalmologic signs and AHT with encephalitis like signs. CONCLUSIONS: symptoms in renovascular hypertension don't have pathognomonic clues and the identified signs, one type or all together, enforce the evaluation or even the monitoring of the arterial tension at least 30 days. If the values exceed the normal, complex investigations will be made in order to determine the specific cause of the AHT.

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.

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.

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.

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.

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.

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.

[Disseminated intravascular coagulation in chickenpox. Report of a case in a child]. / CID in corso di varicella. Descrizione di un caso in un bambino.

The case of a healthy and immunocompetent five-year-old boy, who developed a disseminated intravascular coagulation during chickenpox is described. Disseminated intravascular coagulation manifestations were extremely severe and included macroscopic hematuria, necrotic purpura and cerebrovascular thrombosis. The outcome in this patient was a complete recovery. Nevertheless, the possibility of a seriously complicated course of chickenpox even in low-risk children subgroups suggests that the Varicella-Zoster virus infection should not be underestimated. More accurate information about the impact of chickenpox and its complications on the population is needed, in order to provide a contribution for the debate about the costs associated with this disease and the potential benefits of both the early antiviral therapy and the vaccinal prophylaxis.

Superoxide release by human polymorphonuclear leukocytes in the presence of deferoxamine.

BACKGROUND AND OBJECTIVE: Anecdotal reports in patients with acute and chronic iron overload have recently indicated that the efficacy and safety of an alternative chelation program including intravenous and/or continuous delivery of deferoxamine (DFO) may be in contrast with the risk of developing lung injury. Production of oxygen radicals has been postulated to be an important mechanism by which polymorphonuclear leukocytes (PMNs) could cause tissue injury in patients undergoing this alternative treatment method. METHODS: PMNs obtained from healthy donors were incubated at 37 degrees C for 30 min with DFO (across the drug concentration 0.125 to 10 mg/mL). Superoxide (O2) production was measured by superoxide inhibitable cytochrome c reduction as well as by an NBT densitometric kinetic test. In the same run the effect of lipid peroxidation was demonstrated by means of a malonyl-dialdehyde (MDA) assay. RESULTS: Preincubation of PMNs with any study concentration of DFO significantly enhanced O2 release as well as MDA production upon PMA stimulation. Maximal intracellular and extracellular O2-release as well as MDA production occurred at certain drug concentrations. INTERPRETATION AND CONCLUSIONS: Our in vitro findings suggest that O2-release may be an additional detrimental contribution to tissue injury in some patients who develop pulmonary toxic effects while on intravenous and/or continuous DFO administration.

Intestinal obstruction at the onset of acute lymphoblastic leukemia in a child.

Surgical complications need not be fatal in acute leukemia. If these are promptly diagnosed and properly treated, the prognosis will improve. This report deals with a case of acute lymphoblastic leukemia presenting with an acute abdomen following surgery for choledochal cyst. A peripheral blood smear and examination of the bone marrow revealed acute lymphoblastic leukemia. The child received transfusions of blood and platelets. Pretreatment with prednisolone was started as therapy for leukemia, and 2 days later, the patient underwent surgery. Therapy was continued until the general condition allowed a more aggressive form of treatment. Complete remission was achieved, and the patient is still in good health 48 months after diagnosis and 15 months after discontinuation of treatment. The favorable outcome in this child shows that prompt surgery is sometimes an essential step in the treatment of childhood leukemia.

[Purpura fulminans in the newborn. Report of two cases successfully treated with heparin and antithrombin III]. / La purpura fulminans nel neonato. Descrizione di due casi trattati efficacemente con eparina ed antitrombina III.

Purpura fulminans is a rare form of disseminated intravascular coagulation characterized by rapidly progressive purpuric lesions, hypotension and, in some cases, fever. In neonates, purpura fulminans usually develops following deficiency of anticoagulant protein C or S, although acquired forms have been described. The management of disseminated intravascular coagulation is still controversial, with some studies finding a positive effect of anticoagulants and others showing no effect or even a detrimental one. Therefore, at present, management is limited to the treatment of underlying disease and replacement of clotting factors. Personal experience is reported on the efficacy of heparin in combination with antithrombin III in the treatment of purpura fulminans in two preterm neonates who did not have inherited deficiency of protein C or S and developed the disease possibly following prolonged labor (36 hours) in the first case, and maternal neoplasia, in the second. Both neonates presented with widespread cyanotic areas rapidly evolving in purpuric lesions and hemorrhagic bullae. Laboratory tests (prolonged prothrombin and partial thromboplastin time, fibrinogen and antithrombin III concentrations below normal ranges, d-dimer highly positive) were consistent with disseminated intravascular coagulation. In both cases anticoagulant treatment with heparin (50 UI/kg in bolus followed by 15 UI/kg/h) and antithrombin III was associated with resolution of disseminated intravascular coagulation and prompt amelioration of the purpuric lesions, without apparent side effects.

Allogeneic stem cell transplant from HLA-identical sibling for chronic granulomatous disease and review of the literature.

Chronic granulomatous disease (CGD) is a rare primary immunodeficiency caused by an abnormal function of the nicotinamide adenine dinucleotide phosphate (NADPH) oxidase in the phagocytic cells, which results in an increased susceptibility to severe bacterial and fungal infections. We report on a 12-year-old boy with X-linked CGD who was successfully treated with allogeneic bone marrow transplantation from an HLA-identical sibling following a conditioning regimen consisting of busulphan (BU, 16 mg/kg) and cyclophosphamide (CY, 200 mg/kg). At >2 years from transplant, the boy is in excellent clinical and hematological condition with full chimerism. Our patient is the 24th case of CGD transplanted from an HLA-identical sibling. A review of the literature revealed that 20 of 24 CGD patients are alive and disease free 1-7 years after transplant. Most of these patients were conditioned with the BUCY combination, which should be considered the recommended regimen.

Superoxide production by neutrophils in children with malignant tumors treated with recombinant human granulocyte colony-stimulating factor.

BACKGROUND: Human recombinant granulocyte colony-stimulating factor (rhG-CSF), widely used to combat chemotherapy-induced neutropenia, stimulates both in vivo and in vitro intra- and extra-cellular O2- production in human polymorphonuclear cells (PMNs). PATIENTS AND METHODS: Twelve patients with solid tumors or acute lymphoblastic leukemia were treated during induced aplasia with rhG-CSF (5 micrograms/kg/day). Intra- and extracellular O2- production by PMNs isolated from these patients after 5 days of rhG-CSF therapy was assessed following both fMLP and PMA stimulation. RESULTS: All patients showed a rise in PMN count; administration of rhG-CSF enhanced intra- and extracellular O2- release after fMLP but not after PMA stimulation. CONCLUSIONS: rhG-CSF potentiates in vivo O2- production by PMNs stimulated with receptor-mediated agonists via G-protein (e.g. fMLP), but not by those stimulated with agonists that bypass receptors via protein kinase C (e.g. PMA).

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

A 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.