Expression of the mdr-1/P-170 gene in patients with acute lymphoblastic leukemia

Increased expression of the multidrug resistance gene (mdr-1/P-170) and the dihydrofolate reductase (DHFR) gene have been implicated in the development of in vitro drug resistance. Overexpression, with or without gene amplification, is seen in the development of drug resistance in culture and it has been postulated that genetic modulation of mdr-1/P-170 and DHFR may also be involved in the development of clinical drug resistance. We screened lymphoblasts from 28 patients with acute lymphoblastic leukemia (ALL) for evidence of overexpression of mdr-1/P-170 using RNAse protection, RNA in situ hybridization and immunohistochemistry. Overexpression of mdr-1/P-170 without gene amplification was detected in samples from four patients (three after multiple relapses, one at presentation). Overexpression of mdr-1/P-170 was heterogeneous within the population of malignant lymphoblasts as demonstrated by RNA in situ hybridization, immunohistochemistry, and drug uptake using daunomycin autofluorescence analysis. There was no evidence of overexpression of DHFR in any of the eight patient samples tested by RNAse protection nor was there any evidence of gene amplification in 11 patient samples on Southern blot analysis. From these observations it appears that overexpression without gene amplification of mdr-1/P-170 may be one mechanism of clinical drug resistance in ALL.     

Expression of the human monocyte membrane antigen gp55 by murine fibroblasts after DNA-mediated gene transfer

Human DNA sequences that contain the gene encoding gp55, a cell surface glycoprotein expressed exclusively on mature human monocytes and monocytic leukemia cells, were isolated in a mouse genetic background. DNA from mature human monocytes was cotransfected with DNA from a molecularly cloned feline sarcoma virus containing the v-fms oncogene into NIH-3T3 cells. Transformed mouse fibroblasts that expressed gp55, based on their reactivity with the MY4, B44.1, or LeuM3 monoclonal antibodies, were selected by fluorescence-activated cell sorting. Regardless of which antibody was used for selection, equivalent binding of all three antibodies was observed for positive transformants. Secondary and tertiary mouse cell transformants were obtained after additional rounds of transfection and cell sorting with the use of DNA from primary and then secondary transformants. Southern blot analysis of the cellular DNA from two independently derived tertiary subclones revealed a limited complement of human sequences, thus indicating that the gene encoding gp55 is included in fewer than 50 kilobases of human DNA. Independently derived tertiary subclones displayed concordant patterns of reactivity with 13 monocyte-specific monoclonal antibodies, thus indicating that each recognized an epitope on the product (gp55) of a single human gene. The 55-kilodalton cell surface polypeptide was specifically immunoprecipitated with a representative monoclonal antibody, 26if, from lysates of enzymatically radioiodinated peripheral blood monocytes and tertiary transformants. We conclude that gp55 is highly immunogenic and that a large number of independently derived monoclonal antibodies specific for human monocytes react with epitopes on this one molecule.     

Detection of CYP1A1 protein in human liver and induction by TCDD in precision-cut liver slices incubated in dynamic organ culture

Cytochrome P4501A1 (CYP1A1) has been implicated in the conversion of numerous polycyclic aromatic hydrocarbons into electrophilic species capable of binding covalently to DNA and has therefore been postulated to be involved in the initiation of carcinogenesis. The expression of CYP1A1 protein appears not to be constitutive, but is readily inducible by aryl hydrocarbon (Ah) receptor ligands in a majority of tissues of experimental animals, especially the liver. To date, there is conflicting evidence for the expression or inducibility of CYP1A1 protein in human liver. In this present study, we report the detection of CYP1A1 in all 20 human liver microsomal samples tested by standard western immunoblotting with chemiluminescent detection using a specific monoclonal antibody (mAb 1-12-3) directed against a marine fish (scup) cytochrome P450E. mAb 1-12-3 has been shown previously to specifically recognize CYP1A1 in mammals. This system consistently demonstrated a detection sensitivity as low as 0.01-0.025 pmol CYP1A1 per lane. In the samples where CYP1A1 protein levels were quantitated, CYP1A1 ranged from approximately 0.4 to 5 pmol CYP1A1/mg microsomal protein. Additionally, the inducibility of CYP1A1 protein was demonstrated by incubating precision-cut human liver slices in dynamic organ culture for up to 96 h in the presence of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). The specificity of mAb 1-12-3 was tested using several purified human and rat cytochrome P450s to ensure that the protein being detected was CYP1A1. mAb 1-12-3 did not cross-react with human CYP1A2 or CYP3A4 or rat CYP1B1, but did strongly recognize CYP1A1. However, there was a very weak cross-reactivity of mAb 1-12-3 with human CYP2E1, approximately 75-fold less compared with CYP1A1. In order to confirm CYP1A1 as the immunoreactive protein detected in human liver, microsomal samples were subjected to two-dimensional electrophoresis involving isoelectric focusing followed by SDS-PAGE and immunoblotting. Utilizing mAb 1-12-3, the human liver microsomal samples displayed an immunoblotting profile matching that obtained from a microsomal preparation from a AHH-1 TK+/- cell line expressing solely human CYP1A1 and differing from the profile obtained using a polyclonal antibody directed against CYP2E1 and cells expressing CYP2E1. Furthermore, mAb 1- 12-3 recognized only one protein of identical mobility on the two- dimensional blots from human liver microsomes and AHH-1 TK+/- cells expressing CYP1A1, while displaying no reaction to cells expressing only CYP2E1. In conclusion, CYP1A1 appears to be expressed in human liver at low levels and is inducible upon exposure to TCDD.      

Acinetobacter junii causes life-threatening sepsis in preterm infants

Acinetobacter junii caused sepsis in six preterm infants in our neonatal unit within 48 h. Each infant with clinical signs of systemic infection and activation of the acute phase response had two positive blood cultures with Acinetobacter junii. The sudden onset, the short duration of the outbreak and the fact that none of the infants were colonized by A. junii suggested a common source of A. junii administered directly into the blood. The only feature shared by all six affected newborns was an intravenous fat emulsion (Intralipid 10%), which was shown to be an excellent growth medium for A. junii. Sepsis did not occur in four infants with 20% fat emulsion or amino acids only. Vaminolact^® did not support growth of the outbreak strain. The immediate source of the outbreak could not be identified: samples of the actual feeds given were not available for investigation, but A. junii was not isolated from parenteral solutions with identical batch numbers used in the septic infants. We conclude that Acinetobacter junii can cause a life-threatening infection in preterm neonates. Contaminated intravenous fat emulsion is implicated as a possible source of the infection. As a part of rigid infection control, intravenous feedings should be prepared under aseptic conditions.     

Fetal cord blood as an alternative source of hematopoietic progenitor cells: immunophenotype, maternal cell contamination, and ex vivo expansion.

The present study was performed to investigate the character of hematopoietic progenitor cells in fetal cord blood (CB). Thirty blood samples from fetuses at a median of 24 weeks of gestation (range 19-29) and 30 neonatal CB samples were analyzed for their immunophenotype by three-color flow cytometry and examined for the presence of female cells by fluorescence in situ hybridization (FISH). We tested the effects of different cytokine combinations (rhIL-1beta, rhIL-3, rhIL-6, rh erythropoietin [rhEPO], rhGM-CSF plus rhSCF, and rhSCF plus rhflt3-ligand) on the differentiation of 100 CD34+-enriched neonatal CB cells for up to 21 days. Ex vivo expansion of 32 unselected fetal blood samples cells was performed in the presence of rhSCF and rhflt3-ligand. The percentage of CD34+ cells in fetal blood was significantly higher compared with neonatal CB (1.24%+/-0.82% versus 0.33%+/-0.18%, p = 0.0001) and inversely correlated with the age of gestation. The contamination of fetal and neonatal CB with maternal cells was low (1.72%+/-0.89%, range 1.0%-4.0%). By using rhflt3-ligand we were able to expand committed progenitor cells while maintaining cells with stem cell function. The use of expanded fetal immature progenitors might have implications for in utero transplantation and autologous gene therapy.     

Obstructed calycocystostomy site in association with a transplanted kidney: percutaneous management

We have used advanced endourologic techniques in the treatment of an obstructed lower-pole calycocystostomy site. The unusual clinical course that led to this problem and the interventional treatment used are discussed.     

Nonpalpable testes in young boys: evaluation with MR imaging

A prospective evaluation of magnetic resonance (MR) imaging for localization of a nonpalpable testis was performed in 24 boys aged 11 months to 6 years. Definitive surgical follow-up was obtained for 15 nonpalpable testes in 14 patients who form the basis of this study. MR imaging correctly indicated the unilateral absence of a testis in six of seven patients prospectively and all seven patients retrospectively. Surgically localized undescended testes were identified with MR imaging in five of eight cases prospectively and seven of eight cases retrospectively. Like scrotal testes, undescended testes were hypointense to fat on sequences with a short repetition time (TR) and echo time (TE) in all cases, and hyperintense or isointense to fat on long TR/TE sequences in all but two cases. Inguinal testes were located along the course of a linear low-signal-intensity structure that extended to the scrotum, which may represent the remnant of the gubernaculum testis. A low-signal-intensity band through the testis, presumably the mediastinum testis, was seen in five of the undescended testes. Although MR imaging can often be used to localize a nonpalpable testis, currently MR is not sensitive enough to allow complete exclusion of the diagnosis of an undescended testis; thus failure to localize a testis with MR imaging should not defer laparoscopy or surgical exploration when indicated.     

Cutaneous reaction to contrast material

Two children are described who developed an apparent cutaneous contact reaction to contrast material in urine. Both children had undergone uneventful voiding cystourethrography with diatrizoate meglumine injection USP 18% followed by intravenous urography with diatrizoate meglumine injection USP 60%. Approximately 1 hour after urography cutaneous bullae and surrounding erythema of the buttocks (one case) or foreskin (one case) were noted. This reaction resembled a superficial chemical burn.     

Is high-resolution ultrasonography suitable for the detection of temporomandibular joint involvement in children with juvenile idiopathic arthritis?

Objectives:

The purpose of this study was to determine the potential of high-resolution ultrasonography for the detection of temporomandibular joint (TMJ) changes in children with juvenile idiopathic arthritis (JIA).

Methods:

We investigated prospectively 20 children (17 female and 3 male; mean age 11.06 years, standard deviation 3.43 years) with TMJ disorders caused by JIA, over a period of 16 months. Using a 12 MHz array transducer, four images in each TMJ (160 images) were acquired. Each image was analysed with regard to five different aspects (condylar erosion, thickness of the condylar disc, synovial thickness, joint effusion and enlargement of the intra-articular space).

Results:

Diagnosis of JIA was ensured for every child and involvement of the TMJ was proven by MRI. Overall 287 changes (35.9%) were detected by using high-resolution ultrasonography. On 124 images (77.5%) condylar erosions were diagnosed; on 55 images (34.4%) synovial thickness was abnormal; on 48 images (30%) we could see higher thickness of the condylar disc; on 40 images (25%) irregularities of the bony surface were detected; and on 20 images (12.5%) we found joint effusion.

Conclusion:

High-resolution ultrasonography could be a sufficient diagnostic method, especially for the detection of condylar involvement in children with JIA, even if not all parts of the TMJ are visible for ultrasonography. High-resolution ultrasonography is a valuable tool in particular situations: (i) when MRI examination is not available; (ii) when children fear MRI examination; (iii) in more advanced stages of JIA; and (iv) for monitoring the progression of TMJ involvement and response of therapy.     

Low skeletal muscle mass is associated with non-alcoholic fatty liver disease in Korean adults：the Fifth Korea National Health and Nutrition Examination Survey

BACKGROUND: Sarcopenia and non-alcoholic fatty liver disease(NAFLD) share similar pathophysiological mechanisms, and the relationship between sarcopenia and NAFLD has been recently investigated. The study investigated whether low skeletal muscle mass is differentially associated with NAFLD by gender in Korean adults.METHODS: We conducted a cross-sectional analysis of the data from the Fifth Korea National Health and Nutrition Examination Survey. The skeletal muscle index(SMI) was obtained by the appendicular skeletal muscle mass divided by the weight. NAFLD was defined as a fatty liver index(FLI) ≥60 in the absence of other chronic liver disease.RESULTS: Among the included subjects, 18.3%(SE: 1.4%) in men and 7.0%(SE: 0.7%) in women were classified as having FLI-defined NAFLD. Most of the risk factors for FLI-defined NAFLD showed a significant negative correlation with the SMI in both genders. Multiple logistic regression analysis showed that low SMI was associated with FLI-defined NAFLD, independent of other metabolic and lifestyle parameters in both genders [males: odds ratio(OR)=1.35; 95% confidence interval(CI): 1.17-1.54; females: OR=1.36; 95% CI: 1.18-1.55]. The magnitude of the association between FLI-defined NAFLD and low SMI was higher in middle aged to elderly males(OR=1.50; 95% CI: 1.22-1.84) than in males less than 45 years of age(OR=1.25; 95% CI: 1.02-1.52) and in premenopausal females(OR=1.50; 95% CI: 1.12-2.03) than in postmenopausal females(OR=1.36; 95% CI: 1.20-1.54).CONCLUSIONS: Low SMI is associated with the risk of FLIdefined NAFLD independent of other well-known metabolic risk factors in both genders. This association may differ according to age group or menopausal status. Further studies are warranted to confirm this relationship.