Refined acquisition of high-resolution chest CTs in macaques by free breathing.

The use of medical imaging as a non-invasive or minimally invasive method to assess disease or treatment response continues to grow. A similar trend is observed in pre-clinical research, in general, and more specifically in macaques, enabling longitudinal assessment of disease in individual animals. Computed tomography (CT) is such an imaging technique used to obtain clinically applicable data. To acquire a chest CT using a cone beam tomography system, some kind of respiration control is needed. A commonly used technique for this is endotracheal intubation and mechanical ventilation. However, although routinely performed this can increase the risk of impact on welfare in comparison with non-invasive imaging. Therefore, we studied the option of retrospectively gated CTs: acquiring high resolution chest CTs in freely breathing macaques. For this, we compared 748 CTs obtained during free breathing with 881 CTs obtained with mechanical ventilation in combination with a breath-hold procedure predominantly on the appearance of misregistration artifacts. The scans were obtained during different stages of multiple experimentally induced respiratory diseases. The comparison shows that although there are still streaking artifacts present in the retrospective gated scans, the amount of shading artifacts is reduced to such a level that it possibly dominates underlying lesions, causing misdiagnosis. Our data reveal that the use of retrospective gating in high resolution CTs for macaques can be successfully applied. With the use of this technique, artifacts due to free breathing are reduced to a diagnostically appropriate level. Most importantly, this technique makes chest CTs with this instrumentation a non-invasive modality.

Myeloablative conditioning for allo-HSCT in pediatric ALL: FTBI or chemotherapy?-A multicenter EBMT-PDWP study.

Although most children with acute lymphoblastic leukemia (ALL) receive fractionated total body irradiation (FTBI) as myeloablative conditioning (MAC) for allogeneic hematopoietic stem cell transplantation (allo-HSCT), it is an important matter of debate if chemotherapy can effectively replace FTBI. To compare outcomes after FTBI versus chemotherapy-based conditioning (CC), we performed a retrospective EBMT registry study. Children aged 2-18 years after MAC for first allo-HSCT of bone marrow (BM) or peripheral blood stem cells (PBSC) from matched-related (MRD) or unrelated donors (UD) in first (CR1) or second remission (CR2) between 2000 and 2012 were included. Propensity score weighting was used to control pretreatment imbalances of the observed variables. 3.054 patients were analyzed. CR1 (1.498): median follow-up (FU) after FTBI (1.285) and CC (213) was 6.8 and 6.1 years. Survivals were not significantly different. CR2 (1.556): median FU after FTBI (1.345) and CC (211) was 6.2 years. Outcomes after FTBI were superior as compared with CC with regard to overall survival (OS), leukemia-free survival (LFS), relapse incidence (RI), and nonrelapse mortality (NRM). However, we must emphasize the preliminary character of the results of this retrospective "real-world-practice" study. These findings will be prospectively assessed in the ALL SCTped 2012 FORUM trial.

Performance Evaluation of a High-Resolution Nonhuman Primate PET/CT System.

The LFER 150 PET/CT device (large-field-of-view extreme-resolution portable research imager) is a system for nonhuman primate (NHP) imaging. The objective of this study was to evaluate the performance of the system using the National Electrical Manufacturers Association NU 4-2008 standard protocol. As a preliminary in vivo evaluation of the system, a PET measurement in an NHP was also performed. Methods: Resolution, sensitivity, image quality, and noise-equivalent count rate (NECR) were measured. NECR measurement was performed with a ratlike phantom and a monkeylike phantom. A Derenzo phantom experiment was performed to test the resolution using 3-dimensional ordered-subset expectation maximization reconstruction. One cynomolgus monkey (4.5 kg, intravenous ketamine/xylazine anesthesia) was examined with the dopamine transporter radioligand 18F-FE-PE2I (94 MBq) to evaluate the in vivo performance of the system. List-mode PET data acquired for 93 min were reconstructed into 38 frames with the Tera-Tomo 3-dimensional engine. Binding potential for caudate nucleus, putamen, and substantia nigra was evaluated using the simplified reference tissue model. Results: Radial full-width half-maximum resolution using Fourier rebinning and a 2-dimensional filtered backprojection algorithm was less than 2.2 mm and less than 3.2 mm in the central 60-mm-diameter and 140-mm-diameter regions, respectively. Maximum sensitivity in the 400- to 600-keV and 250- to 750-keV energy windows was 30.03 cps/kBq (3.3%) and 49.11 cps/kBq (5.4%), respectively. The uniformity in the image-quality phantom was 3.3%, and the spillover ratio for air and water was 0.1. The peak of the NECR curve was 430 kcps (at 115 MBq) with the ratlike phantom and 78 kcps (at 139 MBq) with the monkeylike phantom. Rods of the Derenzo phantom with 1-mm diameter could be distinguished by eye. In the NHP experiment, binding potentials in the caudate, putamen, and substantia nigra (4.9, 4.9, and 1, respectively) were similar to those previously reported using the same radioligand and a high-resolution research tomograph. Conclusion: The results obtained from phantom experiments and 1 representative PET measurement in an NHP confirm that the LFER 150 is a high-resolution PET/CT system with suitable performance for brain imaging in NHPs.

Development of clinical simultaneous SPECT/MRI.

There is increasing clinical use of combined positron emission tomography and MRI, but to date there has been no clinical system developed capable of simultaneous single-photon emission computed tomography (SPECT) and MRI. There has been development of preclinical systems, but there are several challenges faced by researchers who are developing a clinical prototype including the need for the system to be compact and stationary with MRI-compatible components. The limited work in this area is described with specific reference to the Integrated SPECT/MRI for Enhanced stratification in Radio-chemo Therapy (INSERT) project, which is at an advanced stage of developing a clinical prototype. Issues of SPECT/MRI compatibility are outlined and the clinical appeal of such a system is discussed, especially in the management of brain tumour treatment.

Experimental Fusion of Contrast Enhanced High-Field Magnetic Resonance Imaging and High-Resolution Micro-Computed Tomography in Imaging the Mouse Inner Ear.

OBJECTIVE: Imaging cochlear, vestibular, and 8th cranial nerve abnormalities remains a challenge. In this study, the membranous and osseous labyrinths of the wild type mouse inner ear were examined using volumetric data from ultra high-field magnetic resonance imaging (MRI) with gadolinium contrast at 9.4 Tesla and high-resolution micro-computed tomography (µCT) to visualize the scalae and vestibular apparatus, and to establish imaging protocols and parameters for comparative analysis of the normal and mutant mouse inner ear. METHODS: For in vivo MRI acquisition, animals were placed in a Milleped coil situated in the isocenter of a horizontal 9.4 T Varian magnet. For µCT examination, cone beam scans were performed ex vivo following MRI using the µCT component of a nanoScan PET/CT in vivo scanner. RESULTS: The fusion of Gd enhanced high field MRI and high-resolution µCT scans revealed the dynamic membranous labyrinth of the perilymphatic fluid filled scala tympani and scala vestibule of the cochlea, and semicircular canals of the vestibular apparatus, within the µCT visualized contours of the contiguous osseous labyrinth. The ex vivo µCT segmentation revealed the surface contours and structural morphology of each cochlea turn and the semicircular canals in 3 planes. CONCLUSIONS: The fusion of ultra high-field MRI and high-resolution µCT imaging techniques were complementary, and provided high-resolution dynamic and static visualization of the complex morphological features of the normal mouse inner ear structures, which may offer a valuable approach for the investigation of cochlear and vestibular abnormalities that are associated with birth defects related to genetic inner ear disorders in humans.

Performance evaluation of the small-animal nanoScan PET/MRI system.

UNLABELLED: nanoScan is a high-resolution integrated system for consecutive PET and MR imaging of small laboratory animals. We evaluated the performance of the system, using the NEMA NU 4-2008 protocol for the PET component and the NEMA MS 1-2007, MS 2-2008, and MS 3-2007 standards for the MR imaging component. METHODS: The imaging system uses magnetically shielded position-sensitive photomultiplier tubes and a compact 1-T permanent-magnet MR imaging platform. Spatial resolution, sensitivity, counting rate capabilities, and image quality parameters were evaluated in accordance with the aforementioned NEMA standards. Further in vivo evaluation experiments complement the physical validation results. RESULTS: The spatial resolution of the PET system enabled the 0.8-mm rods of a Derenzo phantom to be resolved. With point source and 2-dimensional filtered backprojection reconstruction, the resolution varied from 1.50 to 2.01 mm in full width at half maximum in the radial direction and from 1.32 to 1.65 mm in the tangential direction within the radius of 25 mm. Peak absolute sensitivity was 8.41%. Scatter fraction was 17.3% and 34.0%, and maximum noise-equivalent counting rate was 406 and 119 kcps in the mouselike and ratlike phantom, respectively. The image quality test found a nonuniformity of 3.52% and a spillover ratio of 6.2% and 5.8% in water and air, respectively. In testing of the MR imaging component, artifact-free images with high signal-to-noise ratio were recorded. Geometric distortion was below 5%, and image uniformity was at least 94.5% and 96.6% for the 60- and 35-mm radiofrequency coils, respectively. CONCLUSION: The nanoScan integrated small-animal PET/MR imaging system has excellent spatial resolution and sensitivity. The performance characteristics of the PET and the MR imaging components are not compromised as a result of their integration onto a single platform. Because of its combination of features and performance parameters, the system provides crucial advantages for preclinical imaging studies over existing PET/CT systems, especially in neurologic and oncologic research.

Detection of early precursors of t(12;21) positive pediatric acute lymphoblastic leukemia during follow-up.

DNA-, RNA-, and cell-based methods provide different biologic information for determining the presence of minimal residual disease (MRD). We monitored the responses of patients with pediatric acute lymphoblastic leukemia (pALL) using DNA markers, TEL/AML1 expression, and scanning fluorescence microscopy (SFM). Using SFM, 36% of patients exhibited 1.5-3.1 log and 2.9-4.2 log higher MRD levels compared with those based on DNA and RNA markers, respectively. CD10+ ancestor cells with germline antigen receptors, but silent TEL/AML1 expression, may reside in the lymphoid stem cell compartment of treated t(12;21)-positive patients and might act as a potential source of cells for late relapses.

Genetic and demographic features of X-linked agammaglobulinemia in Eastern and Central Europe: a cohort study.

Primary immunodeficiency disorders are a recognized public health problem worldwide. The prototype of these conditions is X-linked agammaglobulinemia (XLA) or Bruton's disease. XLA is caused by mutations in Bruton's tyrosine kinase gene (BTK), preventing B cell development and resulting in the almost total absence of serum immunoglobulins. The genetic profile and prevalence of XLA have not previously been studied in Eastern and Central European (ECE) countries. We studied the genetic and demographic features of XLA in Belarus, Croatia Hungary, Poland, Republic of Macedonia, Romania, Russia, Serbia, Slovenia, and Ukraine. We collected clinical, immunological, and genetic information for 122 patients from 109 families. The BTK gene was sequenced from the genomic DNA of patients with a high susceptibility to infection, almost no CD19(+) peripheral blood B cells, and low or undetectable levels of serum immunoglobulins M, G, and A, compatible with a clinical and immunological diagnosis of XLA. BTK sequence analysis revealed 98 different mutations, 46 of which are reported for the first time here. The mutations included single nucleotide changes in the coding exons (35 missense and 17 nonsense), 23 splicing defects, 13 small deletions, 7 large deletions, and 3 insertions. The mutations were scattered throughout the BTK gene and most frequently concerned the SH1 domain; no missense mutation was detected in the SH3 domain. The prevalence of XLA in ECE countries (total population 145,530,870) was found to be 1 per 1,399,000 individuals. This report provides the first comprehensive overview of the molecular genetic and demographic features of XLA in Eastern and Central Europe.

Natural course of isolated pulmonary langerhans' cell histiocytosis in a toddler. 3-year follow-up.

Isolated pulmonary Langerhans' cell histiocytosis (LCH) is distinctly rare under the age of 15 years, since the majority of patients are young adult males with heavy smoking habits. Isolated pulmonary involvement suggests that antigens inhaled from cigarette smoke are involved. Here we present a case of LCH restricted to the lungs in a toddler whose parents were heavy smokers. Since LCH was not medically treated for 3 years due to parental refusal, the disease can be regarded as having followed its natural course. During the 3-year follow-up, the disease progressed to severe pulmonary fibrosis resulting in honeycomb lungs. Based on the comparative immunohistochemical analyses of the cells obtained from bronchoalveolar lavages during the disease course, it appears that the evolution of fibrosis is rather a result from the accumulating alveolar macrophages than from the persistence of the Langerhans' cells. Passive cigarette smoking may be considered a significant risk factor in both the pathogenesis and development of pulmonary LCH in a small child.

A randomized, placebo-controlled trial of infliximab plus methotrexate for the treatment of polyarticular-course juvenile rheumatoid arthritis.

OBJECTIVE: To evaluate the safety and efficacy of infliximab in the treatment of juvenile rheumatoid arthritis (JRA). METHODS: This was an international, multicenter, randomized, placebo-controlled, double-blind study. One hundred twenty-two children with persistent polyarticular JRA despite prior methotrexate (MTX) therapy were randomized to receive infliximab or placebo for 14 weeks, after which all children received infliximab through week 44. Patients received MTX plus infliximab 3 mg/kg through week 44, or MTX plus placebo for 14 weeks followed by MTX plus infliximab 6 mg/kg through week 44. RESULTS: Although a higher proportion of patients in the 3 mg/kg infliximab group than in the placebo group had achieved responses according to the American College of Rheumatology (ACR) Pediatric 30 (Pedi 30) criteria for improvement at week 14 (63.8% and 49.2%, respectively), the between-group difference in this primary efficacy end point was not statistically significant (P = 0.12). By week 16, after the crossover from placebo to infliximab 6 mg/kg when all patients were receiving infliximab, an ACR Pedi 30 response was achieved in 73.2% of all patients. By week 52, ACR Pedi 50 and ACR Pedi 70 responses had been reached in 69.6% and 51.8%, respectively, of patients. Infliximab was generally well tolerated, but the safety profile of infliximab 3 mg/kg appeared less favorable than that of infliximab 6 mg/kg, with more frequent occurrences of serious adverse events, infusion reactions, antibodies to infliximab, and newly induced antinuclear antibodies and antibodies to double-stranded DNA observed with the 3 mg/kg dose. CONCLUSION: While infliximab at 3 mg/kg and 6 mg/kg showed durable efficacy at 1 year, achievement of the primary efficacy end point at 3 months did not differ significantly between infliximab-treated and placebo-treated patients. Safety data indicated that the 6-mg/kg dose may provide a more favorable risk/benefit profile. These results warrant further investigation in children with JRA.

Prevention and treatment of hyperuricemia with rasburicase in children with leukemia and non-Hodgkin's lymphoma.

To prevent acute renal failure in children at risk for developing tumor lysis syndrome due to acute lymphoblastic leukemia or non-Hodgkin's lymphoma treated according to international BFM protocols, we investigated recombinant urate oxidase (rasburicase) in the first Central European openlabeled, prospective, multicenter phase IV trial. Rasburicase was administered intravenously, at 0.2 mg/kg for 5 consecutive days to 36 patients. Blood levels of uric acid, creatinine, phosphorus, calcium, lactate dehydrogenase and complete blood count were measured daily during rasburicase treatment and on days 6, 7 and 12. Initial uric acid level decreased significantly by 4 hours (from 343 micromol/L to 58 micromol/L, p<0.001), except for one steroid-resistant patient who required hemodialysis on day 14 after having introduced combined cytostatic treatment. Comparing the data of a subgroup of 12 patients receiving rasburicase with that of a historic cohort of 14 patients treated with allopurinol indicated the superiority of rasburicase over allopurinol in prophylaxis and treatment of hyperuricemia in children with leukemia and lymphoma.

Development of spherical iron(II) sulfate heptahydrate-containing solid particles with sustained drug release.

The aim of this work was to develop a simple, economic procedure for the manufacturing of coated iron(II) sulfate particles by using a crystallization technique for the development of round particles, followed by coating with a lipophilic material. Several batches of iron(II) sulfate heptahydrate were produced by a cooling crystallization, with variation of the crystallization parameters. The spherical crystals were coated with stearin. The products were characterized for particle size, roundness, bulk density and in vitro drug dissolution. Crystallization was performed from deionized water with no addition of seed crystals and by cooling by applying a linear cooling rate. The developed iron(II) sulfate crystals were round with average diameter of 729+/-165 microm. The best form for the sustained release of iron(II) sulfate was the sample HTP-2 which contained 11% of stearin relative to the iron(II) sulfate. The spherical crystallization of iron(II) sulfate is simple and fast, and does not require a dangerous, expensive solvent. The round particles can coat directly with lipophilic material which results in slow release of iron(II) sulfate and protects the iron(II) from oxidation and inhibits the loss of crystal water. The coated crystals can be filled into capsules to yield the final dosage form.

DNA content heterogeneity in neuroblastoma analyzed by means of image cytometry and its potential significance.

The aim of this study was to determine the frequency and significance of the tumor DNA content heterogeneity in 33 previously untreated human neuroblastomas. We used image cytometry to selectively analyze neuroblasts by excluding karyorrhectic or stromal cells from cytometric measurements. DNA content heterogeneity with more than one clonal subpopulation on DNA histogram was found in 8 of 33 cases. Of these 8 cases, 4 showed MYCN amplification. Double labeling fluorescent in situ hybridization with probes for the centromeric region of chromosome 2 and MYCN gene was used to confirm the DNA content heterogeneity. DNA content heterogeneity was associated with poorer prognosis in this study (P<0.05). There was a significant correlation between euploidy (di- and tetraploidy) and worse prognosis, but only when heterogeneous neuroblastomas with euploid cell population were assigned to euploid tumors (P=0.006). Our results may explain the conflicting data in the literature regarding ploidy and suggest that DNA content heterogeneity and the presence of a euploid population may predict worse prognosis in neuroblastoma patients.

[Treatment results with ALL-BFM-95 protocol in children with acute lymphoblastic leukemia in Hungary]. / Az ALL-bFM-95 protokollal szerzett hazai eredmények akut lymphoblastos leukaemiás gyermekek kezelésében.

BACKGROUND: In Hungary children (from 1 to 18 years of age) with de novo acute lymphoblastic leukemia were treated from January 1996 to October 2002, according to protocol ALL-BFM-95. AIM: The aim of this study was to evaluate the experience with this protocol, the treatment results according to the risk groups and to compare the Hungarian data with the international results. METHODS: Patients were stratified into 3 risk groups, based on initial white blood cell count, age, immunology, cytogenetics and response to treatment: standard, medium and high risk group. RESULTS: Three hundred sixty eight children entered the study (male-female ratio was 1.27:1, median age 6 years and 4 months). 110 (29.9%) children were in the standard, 210 (57.1%) in the medium and 48 (13%) in the high risk group. Duration of the chemotherapy was 2 years, except of the boys in the standard risk group, their maintenance therapy was 1 year longer. The overall complete remission rate was 93.2%. 20 (5.4%) children died in induction and 5 (1.4%) were non-responders. The 5-year overall survival for all patients was 78.5%, in the standard risk group 93.2%, in the medium risk group 78.4% and in the high risk group 44.5% with a minimum follow up of 1.19 years and median follow up of 4.85 years. From the 368 patients 272 (73.9%) are still in their first complete clinical remission and other 18 children are alive after relapse. In 14.7% of the patients relapse was diagnosed; the most common site was the bone marrow. In one patient second malignancy occurred. The 5-year event free survival for all patients was 72.6%, in the standard risk group 87.6%, in the medium risk group 72.1% and in the high risk group 39.9%. CONCLUSION: The treatment outcome of children with acute lymphoblastic leukemia improved remarkably over the last decades. 78% of children suffering from acute lymphoblastic leukemia could be cured with the ALL-BFM-95 protocol. The Hungarian results are comparable to those achieved by other leukaemia study groups in the world regarding the ALL-BFM-95 protocol.

Detection of n-myc gene amplification in neuroblastoma by comparative, in situ,and real-time polymerase chain reaction.

We have used semiquantitative comparative and real-time quantitative polymerase chain reactions (PCR) to detect n-myc gene-amplification in 20 frozen neuroblastoma biopsies and IMR 32 cell line to predict biological behavior of the tumors. Two primer pairs were used for the semiquantitative method to co-amplify a 520-bp fragment of the beta-globin gene--used as a single copy reference standard--and a 258-bp fragment of the n-myc gene. After 30 cycles the PCR products were electrophoresed through an agarose gel and were compared to each other with use of a gel-densitometer. Real-time quantitative analyses were performed in a LightCycler instrument. A single primer pair was used to amplify a 120-bp fragment of the n-myc oncogene and a LC640-labeled fluorescent probe pair to detect the product. Calibration curve, set up from a serial dilution including samples with 1, 2, 10, 13, 25-fold n-myc oncogene amplification, was used for quantitative analysis. The semiquantitative method did not show distinct difference between tumor groups with no amplification and less than 10-fold amplification, whereas quantitative LightCycler analysis was able to detect even 2-fold amplification. Differentiated neuroblastomas seldom show n-myc amplification. In spite of this, we have found two partly differentiated tumor samples that contained n-myc amplification. In these cases in situ PCRs were performed to examine the tumor heterogeneity. We used biotinated ATP labeling and the same primer pair as for the LightCycler analysis. In both cases differentiated cells did not show n-myc gene amplification, whereas considerable amplification was detected in the neuroblasts.

Studies on the mechanisms of allergen-induced activation of the classical and lectin pathways of complement.

Allergen extracts are efficient activators of the complement system trough the classical pathway. Involvement of the lectin pathway was not previously studied. To further examine the mechanism of complement activation by allergens, in vitro experiments, which covered early steps both of classical and lectin pathways, were performed. Two types of allergens used in these studies: parietaria (PA) and house dust (HD) mite extracts. These allergen extracts bound to the globular head of C1q and interacted with purified mannan-binding lectin (MBL) as measured by solid-phase ELISA. None of the allergen extracts was able to activate human C1 in vitro, as measured by the determination of the split products of C1s in a reconstituted precursor C1 preparation. Neither the HD nor the PA extracts induced C4d generation above background in the serum of three subjects with hypogammaglobulinaemia but normal complement haemolytic activity. After reconstitution to normal level with purified human IgG, allergen extracts induced C4d formation above control at a level comparable to that measured in normal serum incubated with the same amounts of the extracts. HD-induced C4d generation was about the same comparable in MBL-depleted serum and in normal sera. In contrast PA induced no C4d formation in the MBL-depleted serum, whereas reconstitution with purified MBL restored C4d generation. These in vitro findings indicate that although the allergen extracts can bind purified C1q and MBL, they require IgG for efficient complement activation. Depending on the allergens, this activation may be initiated through C1, MBL, or both.

[Early recognition and successful treatment of an infant with severe combined immune deficiency]. / Súlyos kombinált immundeficiencia korai felismerése és eredményes kezelése.

A male infant of three month presented with recurrent mucosal and fungal infections, diarrhoea and failure to thrive from the age of three weeks. Laboratory test revealed T-B-NK + severe combined immunodeficiency (SCID). Family history and immunolaboratory findings suggested autosomal recessive form of the disease. Haploidentical maternal bone marrow transplantation (BMT) was carried out at five and half months of age. Over the two years after BMT, the patient's somatomotoric and mental development is normal. Cellular immune responses and the substantial immunoglobulin production suggest immunoreconstruction in the child born with complete lack of adaptive immunity. According to the author's knowledge, this is the first T-B-NK + patient in Hungary, whose disease was diagnosed and adequately treated in infancy.