Ariel Koren

Red blood cells, platelets and polymorphonuclear neutrophils of patients with sickle cell disease exhibit oxidative stress that can be ameliorated by antioxidants

Sickle cell disease (SCD) is basically a red blood cell (RBC) disorder characterised by sickling and haemolysis, but platelets and polymorphonuclear neutrophils (PMN) are also involved. Oxidative damage may play a role in the pathogenesis of SCD. Using flow cytometry, we measured oxidative‐state markers simultaneously in RBC, platelets and PMN obtained from 25 normal donors, nine homozygous (SS) patients and six SS/beta‐thalassaemia patients. Reactive oxygen species (ROS) and reduced glutathione (GSH) were measured following staining of blood samples with fluorescence probes and gating on specific subpopulations based on size and granularity. Ten‐ to 30‐fold higher ROS production and 20–50% lower GSH content were found in RBC, platelets and PMN from SCD patients versus those of their normal counterparts. This could in part account for the clinical manifestations, such as haemolysis, a hypercoagulable state, recurrent bacterial infections and vaso‐occlusive incidences, in SCD. We further showed that exposure of SCD samples to antioxidants, such as N‐acetyl‐cysteine, vitamin C and vitamin E, decreased their oxidative stress. These results suggest that antioxidant treatment of patients with SCD could reduce oxidative damage to RBC, PMN and platelets, thereby alleviating symptoms associated with their pathology. The flow cytometry techniques presented herein could assist in monitoring the efficacy of such treatment.

Congenital dyserythropoietic anemia type I is caused by mutations in codanin-1.

Congenital dyserythropoietic anemias (CDAs) constitute a rare group of inherited red-blood-cell disorders associated with dysplastic changes in late erythroid precursors. CDA type I (CDAI [MIM 224120], gene symbol CDAN1) is characterized by erythroid pathological features such as internuclear chromatin bridges, spongy heterochromatin, and invagination of the nuclear membrane, carrying cytoplasmic organelles into the nucleus. A cluster of 45 highly inbred Israeli Bedouin with CDAI enabled the mapping of the CDAN1 disease gene to a 2-Mb interval, now refined to 1.2 Mb, containing 15 candidate genes on human chromosome 15q15 (Tamary et al. 1998). After the characterization and exclusion of 13 of these genes, we identified the CDAN1 gene through 12 different mutations in 9 families with CDAI. This 28-exon gene, which is transcribed ubiquitously into 4738 nt mRNA, was reconstructed on the basis of gene prediction and homology searches. It encodes codanin-1, a putative o-glycosylated protein of 1,226 amino acids, with no obvious transmembrane domains. Codanin-1 has a 150-residue amino-terminal domain with sequence similarity to collagens and two shorter segments that show weak similarities to the microtubule-associated proteins, MAP1B (neuraxin) and synapsin. These findings, and the cellular phenotype, suggest that codanin-1 may be involved in nuclear envelope integrity, conceivably related to microtubule attachments. The specific mechanisms by which codanin-1 underlies normal erythropoiesis remain to be elucidated.

Urinary tract infection: is there a need for routine renal ultrasonography?

Aims: To assess the yield of routine renal ultrasound (RUS) in the management of young children hospitalised with first uncomplicated febrile urinary tract infection (UTI). Methods: All children aged 0–5 years who had been hospitalised over a two year period with first uncomplicated febrile UTI in a medium size institutional regional medical centre were included. Children with known urinary abnormalities and/or who had been treated with antibacterial agents within seven days before admission were excluded. All included children underwent renal ultrasonography during hospitalisation and voiding cystouretrography (VCUG) within 2–6 months. The yield of RUS was measured by its ability to detect renal abnormalities, its sensitivity, specificity, and positive and negative predictive values for detecting vesicoureteral reflux (VUR), and by its impact on UTI management. Results: Of 255 children that were included in the study, 33 children had mild to moderate renal pelvis dilatation on RUS suggesting VUR, of whom only nine had VUR on VCUG. On the other hand, in 36 children with VUR on VCUG the RUS was normal. The sensitivity, specificity, positive predictive value, and negative predictive value of abnormal RUS for detecting VUR were 17.7%, 87.6%, 23.5%, and 83.2% respectively. In none of the patients with abnormal RUS was a change in the management at or following hospitalisation needed. Conclusion: Results show that the yield of RUS to the management of children with first uncomplicated UTI is questionable.

Procalcitonin as a marker of acute pyelonephritis in infants and children

Abstract In the absence of specific symptomatology in children, the early diagnosis of acute pyelonephritis is a challenge, particularly during infancy. In an attempt to differentiate acute pyelonephritis from lower urinary tract infection (UTI), we measured serum procalcitonin (PCT) levels and compared these with other commonly used inflammatory markers. We evaluated the ability of serum PCT levels to predict renal involvement, as assessed by dimercaptosuccinic acid (DMSA) scintigraphy. Serum C-reactive protein (CRP), leukocyte counts, and PCT levels were measured in 64 children admitted for suspected UTI. Renal parenchymal involvement was assessed by 99mTc-DMSA scintigraphy in the first 7 days after admission. In acute pyelonephritis, the median PCT level was significantly higher than in the lower UTI group (3.41, range 0.36–12.4 μg/l vs. 0.13, range 0.02–2.15 μg/l, P<0.0001). In these two groups, respectively, median CRP levels were 120 (range 62–249 mg/l) and 74.5 (range 14.5–235 mg/l, P=0.012) and leukocyte counts were 15,910/mm3 (range 10,200–26,900) and 14,600/mm3 (range 8,190–26,470, P=0.34). For the prediction of acute pyelonephritis, the sensitivity and specificity of PCT were 94.1% and 89.7%, respectively; CRP had a sensitivity of 100%, but a specificity of 18.5%. We conclude that serum PCT may be an accurate marker for early diagnosis of acute pyelonephritis.

Procalcitonin as a diagnostic aid in osteomyelitis and septic arthritis

Objectives: Plasma procalcitonin (PCT) increases rapidly during bacterial infections but remains low in viral infections and other inflammatory processes. High plasma PCT typically occurs in children with bacterial meningitis, severe bacterial infections, particularly in cases of septic shock or bacteremia, and in renal parenchymal damage. The aim of this study was to test the usefulness of plasma PCT analysis in the diagnosis of osteomyelitis, septic arthritis, and other skeletal inflammatory diseases in pediatric patients admitted because of fever and limping. Methods: White blood cell count, erythrocyte sedimentation rate, C-reactive protein, and PCT levels were measured in children admitted to the pediatric department with fever, limping, and suspected osteomyelitis or septic arthritis. PCT levels were measured by an immunochromatography assay, based on monoclonal and polyclonal antibodies against katacalcin. Results: Forty-four children were evaluated: 12 (27.3%) were diagnosed with osteomyelitis, 11 (25%) had septic arthritis, 5 children (11.4%) were diagnosed as a soft tissue infection, and transient synovitis or reactive arthritis was diagnosed in another 6 children (13.6%). Four children (9.1%) were diagnosed as having juvenile rheumatoid arthritis, and 6 (13.6%) with different diseases. PCT value was elevated in 7 patients (58.3%) with osteomyelitis, and only 3 children (27.2%) with the diagnosis of septic arthritis had a mildly elevated value. Among the children with other diagnosis, there were no positive PCT values (P < 0.001 between skeletal infection and all other diagnosis). Conclusions: In this study, PCT was found to be a useful marker in the diagnosis of osteomyelitis and not in septic arthritis. A larger group of patients needed to be studied to confirm our findings.

Downregulation of hepcidin and haemojuvelin expression in the hepatocyte cell-line HepG2 induced by thalassaemic sera

β‐Thalassaemia represents a group of diseases, in which ineffective erythropoiesis is accompanied by iron overload. In a mouse model of β‐thalassaemia, we observed that the liver expressed relatively low levels of hepcidin, which is a key factor in the regulation of iron absorption by the gut and of iron recycling by the reticuloendothelial system. It was hypothesised that, despite the overt iron overload, a putative plasma factor found in β‐thalassaemia might suppress liver hepcidin expression. Sera from β‐thalassaemia and haemochromatosis (C282Y mutation) patients were compared with those of healthy individuals regarding their capacity to induce changes the expression of key genes of iron metabolism in human HepG2 hepatoma cells. Sera from β‐thalassaemia major patients induced a major decrease in hepcidin (HAMP) and lipocalin2 (oncogene 24p3) (LCN2) expression, as well as a moderate decrease in haemojuvelin (HFE2) expression, compared with sera from healthy individuals. A significant correlation was found between the degree of downregulation of HAMP and HFE2 induced by β‐thalassaemia major sera (r = 0·852, P < 0·0009). Decreased HAMP expression was also found in HepG2 cells treated with sera from β‐thalassaemia intermedia patients. In contrast, the majority of sera from hereditary haemochromatosis patients induced an increase in HAMP expression, which correlated with transferrin (Tf) saturation (r = 0·765, P < 0·0099). Our results suggest that, in β‐thalassaemia, serum factors might override the potential effect of iron overload on HAMP expression, thereby providing an explanation for the failure to arrest excessive intestinal iron absorption in these patients.

Response to hydroxyurea therapy in β-thalassemia

Although a relatively small number of previous studies suggest a modest response to hydroxyurea (HU) therapy in β‐thalassemia, more recent investigations have revealed that some transfusion‐dependent patients can become transfusion‐independent following HU therapy. Patients with Gγ XmnI polymorphism, several β‐globin mutations, and α‐thalassemia deletions were inconsistently reported to have significant responses to HU therapy. To better predict who may respond, we retrospectively evaluated the clinical response and the molecular background of 18 β‐thalassemia patients treated with HU for a mean of 46 months. The majority of transfusion‐dependent patients responded to HU therapy with 9 out of 11 (82%) becoming transfusion‐independent. Five thalassemia intermedia (TI) patients receiving occasional blood transfusion did not require any additional transfusions following therapy and two TI patients who had never received transfusions had a 2 g/dl increase in their hemoglobin level. The majority of β‐thalassemia major patients who became transfusion‐independent (7/9) were either homozygous (5) or heterozygous (2) for the XmnI polymorphism. No correlation was identified between response to therapy and the presence of specific β‐thalassemia mutations or α‐globin deletions. We conclude that further analysis of the degree of response of transfusion‐dependent β‐thalassemia patients to HU therapy, as well as, the impact of their genetic background on this response is required to identify patients likely to have significant response. Am. J. Hematol., 2008.

Effect of hydroxyurea in sickle cell anemia: a clinical trial in children and teenagers with severe sickle cell anemia and sickle cell beta-thalassemia.

This study evaluated the efficacy of hydroxyurea treatment in the prevention of vaso-occlusive crises among children and teenagers with severe sickle cell anemia and sickle cell beta-thalassemia. Nineteen children and young adults with severe sickle cell disease were enrolled to the hydroxyurea treatment trial. The incidence of vaso-occlusive crises, acute chest syndrome, hemolytic crises, splenic sequestration episodes, blood transfusions, and hospital days in the 2 years before hydroxyurea (HU) treatment were compared with the same parameters in the first 2 years of treatment. The patients received a mean dose of 21.3 mg/kg/day daily and were treated during a mean period of 40.3 +/- 14 months (range 20 to 68 months). Significant increases were observed after 1 month in the Hgb, MCV, MCH, and MCHC levels and were more notable after 3 months. The increase in the Hgb F level became important after 3 months of HU therapy and was highly significant (p < .001) beyond 6 months. No differences were observed in the RDW, reticulocyte count, Hgb S, and Hgb A2. Severe neutropenia was observed in one case. A decrease in the frequency of vaso-occlusive crises, acute chest syndrome, hemolytic crises, blood transfusions, and days spent in the hospital was demonstrated during the HU treatment period compared to the same period before. The clinical and laboratory response to HU was dramatic in severely affected sickle cell anemia (SCA) patients. The response to HU in children and teenagers with severe sickle cell anemia is similar to the response in adults, and no severe adverse effects were observed.

Renal function in children with β-thalassemia major and thalassemia intermedia

In β-thalassemia, profound anemia and severe hemosiderosis cause functional and physiological abnormalities in various organ systems. In recent years, there have been few published studies demonstrating proteinuria, aminoaciduria, low urine osmolality, and excess secretion of the tubular damage markers, such as urinary N-acetyl-D-glucosaminidase (UNAG) and β2 microglobulin, in patients with thalassemia. The object of this study was to analyze renal tubular and glomerular function in pediatric patients with β-thalassemia and to correlate the renal findings to iron overload. Thirty-seven patients with β-thalassemia major and 11 with thalassemia intermedia were studied. Twelve children without iron metabolism disorders or renal diseases served as a control group. No difference in blood urea nitrogen (BUN), serum creatinine, creatinine clearance, electrolytes, fractional excretion of sodium and potassium, and tubular phosphorus reabsorption was found. Serum uric acid was equal in the two groups, but its urine excretion was significantly higher in the thalassemic group. UNAG and UNAG to creatinine ratio (UNAG/CR) were elevated in all patients with thalassemia compared with the control group (p < 0.001) and were directly correlated to the amount of transfused iron but not to actual ferritin level. We found that renal tubular function is impaired in children with β- thalassemia major and intermedia. It is not known whether these functional abnormalities would have any long-term effects on the patients. Further studies are needed, and means of preventing these disturbances should be sought.

Frequency and natural history of inherited bone marrow failure syndromes: the Israeli Inherited Bone Marrow Failure Registry

Background Inherited bone marrow failure syndromes are rare genetic disorders characterized by bone marrow failure, congenital anomalies, and cancer predisposition. Available single disease registries provide reliable information regarding natural history, efficacy and side effects of treatments, and contribute to the discovery of the causative genes. However, these registries could not shed light on the true incidence of the various syndromes. We, therefore, established an Israeli national registry in order to investigate the relative frequency of each of these syndromes and their complications. Design and Methods Patients were registered by their hematologists in all 16 medical centers in Israel. We included patients with Fanconi anemia, severe congenital neutropenia, Diamond-Blackfan anemia, congenital amegakaryocytic thrombocytopenia, dyskeratosis congenita, Shwachman-Diamond syndrome, and thrombocytopenia with absent radii. Results One hundred and twenty-seven patients diagnosed between 1966 and 2007 were registered. Fifty-two percent were found to have Fanconi anemia, 17% severe congenital neutropenia, 14% Diamond-Blackfan anemia, 6% congenital amegakaryocytic thrombocytopenia, 5% dyskeratosis congenita, 2% Shwachman-Diamond syndrome, and 2% thrombocytopenia with absent radii. No specific diagnosis was made in only 2 patients. Of the thirty patients (24%) developing severe bone marrow failure, 80% had Fanconi anemia. Seven of 9 patients with leukemia had Fanconi anemia, as did all 6 with solid tumors. Thirty-four patients died from their disease; 25 (74%) had Fanconi anemia and 6 (17%) had severe congenital neutropenia. Conclusions This is the first comprehensive population-based study evaluating the incidence and complications of the different inherited bone marrow failure syndromes. By far the most common disease was Fanconi anemia, followed by severe congenital neutropenia and Diamond-Blackfan anemia. Fanconi anemia carried the worst prognosis, with severe bone marrow failure and cancer susceptibility. Diamond-Blackfan anemia had the best prognosis. The data presented provide a rational basis for prevention programs and longitudinal surveillance of the complications of inherited bone marrow failure syndromes.