Detection of polyomavirus BK and JC in children with kidney diseases and renal transplant recipients

BACKGROUND: Reactivation of polyomavirus is a known reason for severe renal dysfunction in adult renal transplant recipients. Testing for polyomavirus DNA in plasma has been described as a sensitive and specific method to discover viral nephropathy in adult patients. We were now interested in polyomavirus status in a pediatric patient setting. METHODS: Plasma and urine samples were obtained from 80 children including 38 children after renal transplantation (group 1), 7 children with different kidney diseases receiving immunosuppressive treatment (group 2) and 35 children with different kidney diseases not receiving immunosuppressive treatment (group 3). A nested polymerase chain reaction method was used for amplification of polyomavirus DNA fragments. Differentiation between JC and BK virus was done by digestion with restriction endonucleases. RESULTS: Polyomavirus DNA was detected in the urine sample of 19 of 38 (50%) renal transplant recipients (group 1), of 1 of 7 (14%) patients from group 2 and in none of the 35 patients of group 3. Plasma samples from 3 (8%) of group 1 patients and from 1 child each of group 2 (14%) and group 3 (3%) were tested positive for polyomavirus DNA. CONCLUSION: Urinary polyomavirus excretion seems to be more frequent in pediatric patients with kidney diseases receiving immunosuppressive treatment and after renal transplantation than in children with various kidney diseases without immunosuppressive treatment.     

Validation of the Bech–Rafaelsen Melancholia Scale and the Hamilton Depression Scale in patients with major depression; is the total score a valid measure of illness severity?

OBJECTIVE: Evaluation of antidepressant drug efficacy requires adequate rating scales for measuring the severity of depression. However, to measure the illness severity by such a total score, the scale needs to fulfil criteria of unidimensionality. On this background, we aimed at comparing the unidimensionality of the Bech-Rafaelsen Melancholia Scale (MES) and the 17-item Hamilton Depression Rating Scale (HAM-D(17)). METHOD: A total of 1629 patients aged between 18 and 65 years with a major depressive episode were treated openly with sertraline at a fixed oral dose of 50 mg daily during 4 weeks. The HAM-D(17) and the MES were applied at baseline and at weeks 2 and 4. Unidimensionality was tested with Mokken and Rasch analysis. RESULTS: Unidimensionality of the HAM-D(17) could not be confirmed. However, the 6-item Hamilton Depression Subscale (HAM-D(6)), was accepted by the Rasch analysis both at baseline and after 2 and 4 weeks of therapy. For the MES (as well as for the HAM-D(6)), a Loevinger coefficient of homogeneity above 0.40 (suggesting acceptance) was found at week 4. CONCLUSION: The HAM-D(6) and the MES did fulfil criteria for unidimensionality while the HAM-D(17) did not. Therefore, the extended use of the HAM-D(17) in drug trials may be questioned.     

Endogenous distress in ventilated full-term newborns with acute respiratory failure

OBJECTIVE: The main rationale behind the continuous analgesia/sedation currently practiced in the treatment of neonates with severe respiratory failure in intensive care is an attempt at shielding the sick newborn from exogenous stress and pain caused by diagnostic and therapeutic interventions. Until now, however, the factors which influence endogenous, disease-related distress have been largely ignored. METHOD: We retrospectively studied the daily need for analgesics and sedatives (fentanyl, midazolam, pentobarbital, thiopental) of 40 full-term newborns with severe respiratory failure who had been ventilated for at least 48 h over an observational period of 2-5 days. Dosing of the analgesics and sedatives was based on a neonatal sedation score for ventilated infants. These daily amounts were converted to a normative comparative dose (analgesic/sedative need = ASN) and compared with the oxygenation index (OI) as a measure of the degree of pulmonary insufficiency. RESULTS: Depending on the duration of ventilation, an increasingly close correlation between the ASN and the OI was detected: the index of correlation (r) was detected to be 0.65 on the second day, but increased up to 0.94 after 5 days. The subgroup of patients who had been ventilated for more than 3 days (n = 8) consistently showed a very high correlation, ranging from r = 0.86 to r = 0.94. CONCLUSION: Our results indicate a direct relationship between severity of pulmonary failure (expressed as OI) and degree of disease-related distress (reflected by ASN). This supports the hypothesis that in full-term neonates under mandatory intensive care for severe respiratory failure, endogenous distress caused by the primary disease itself, in addition to exogenous distress caused by therapeutic and diagnostic interventions, is key factor for the determination of the required amount of analgesic and sedative drugs.     

Overexpression of the embryonic-lethal abnormal vision-like protein HuR in ovarian carcinoma is a prognostic factor and is associated with increased cyclooxygenase 2 expression

The human embryonic-lethal abnormal vision-like protein HuR is involved in the regulation of mRNA turnover and serves as a shuttling protein between the nucleus and the cytoplasm that stabilizes mRNAs containing adenine- and uridine-rich elements in their 3' untranslated region. We have shown recently that expression of cyclooxygenase (COX)-2 is related to poor prognosis in ovarian carcinoma. Other studies have shown that the COX-2 mRNA contains an adenine- and uridine-rich element and is stabilized by HuR. In this study, we investigated the expression and cellular distribution of HuR in 83 primary ovarian carcinomas, 16 borderline tumors of the ovary, 3 normal ovaries, and 9 ovarian carcinoma cell lines. Expression of HuR was detected in all cell lines on the mRNA and protein level and showed a predominantly nuclear staining in OVCAR-3 cells by confocal microscopy. In an immunohistochemical evaluation of human ovarian carcinomas, HuR showed a nuclear expression in 81% of tumors. In addition, a cytoplasmic expression of HuR was observed in a subgroup of 45% of ovarian carcinomas. Nuclear as well as cytoplasmic expression of HuR was significantly increased in ovarian carcinomas compared with borderline tumors or normal ovaries. In univariate analysis, a significant association between cytoplasmic HuR expression and increased COX-2 expression (P = 0.025) as well as between histological grade (P = 0.008) and mitotic activity (P = 0.002) was observed, although nuclear expression of HuR was not correlated with COX-2 expression or other clinicopathological parameters. In Kaplan-Meier survival analysis, increased cytoplasmic expression of HuR was a significant prognostic indicator for progression-free survival (P = 0.03) as well as overall survival (P = 0.007). In multivariate analysis using the Cox regression model, cytoplasmic expression of HuR was an independent prognostic parameter for reduced overall survival with a relative risk of 2.62 (95% confidence interval, 1.32-5.19). Our results suggest that there is a dysregulation of cellular distribution of the mRNA stability factor HuR in a subset of invasive ovarian carcinomas. This dysregulation appears to result in an increased expression of COX-2, an increased proliferative rate, and may lead to a reduced survival time. Additional studies are required to analyze the downstream effects of increased cytoplasmic expression of HuR. In addition, it would be interesting to investigate the prognostic role of increased cytoplasmic expression of HuR in prospective studies.     

SPRY2 is an inhibitor of the ras/extracellular signal-regulated kinase pathway in melanocytes and melanoma cells with wild-type BRAF but not with the V599E mutant

BRAF mutations result in constitutively active BRAF kinase activity and increased extracellular signal-regulated kinase (ERK) signaling and cell proliferation. Initial studies have shown that BRAF mutations occur at a high frequency in melanocytic nevi and metastatic lesions, but recent data have revealed much lower incidence of these mutations in early-stage melanoma, implying that other factors may contribute to melanoma pathogenesis in a wild-type (WT) BRAF context. To identify such contributing factors, we used microarray gene expression profiling to screen for differences in gene expression between a panel of melanocytic and melanoma cell lines with WT BRAF and a group of melanoma cell lines with the V599E BRAF mutation. We found that SPRY2, an inhibitor homologous to SPRY4, which was previously shown to suppress Ras/ERK signaling via direct binding to Raf-1, had reduced expression in WT BRAF cells. Using small interfering RNA-mediated SPRY2 knockdown, we showed that SPRY2 acts as an inhibitor of ERK signaling in melanocytes and WT BRAF melanoma cells, but not in cell lines with the V599E mutation. We also show that SPRY2 and SPRY4 directly bind WT BRAF but not the V599E and other exon 15 BRAF mutants. These data suggest that SPRY2, an inhibitor of ERK signaling, may be bypassed in melanoma cells either by down-regulation of its expression in WT BRAF cells, or by the presence of the BRAF mutation.     

Cycle dependency of intrauterine vascular endothelial growth factor levels is correlated with decidualization and corpus luteum function

OBJECTIVE: To determine intrauterine levels of vascular endothelial growth factor (VEGF)-A during the menstrual cycle in the human female and to investigate the impact of decidualization and corpus luteum function. DESIGN: Prospective clinical study. SETTING: Tertiary university center. PATIENT(S): Fifty-four women with infertility problems. INTERVENTION(S): Intrauterine concentrations of VEGF-A were determined at various time points during the secretory phase using a novel intrauterine microdialysis device. Concomitantly, intrauterine insulin-like growth factor binding protein (IGFBP)-1 levels served as a paracrine parameter for decidualization. Serum progesterone (P) and E(2) levels were determined as markers for corpus luteum function.Intrauterine VEGF levels. RESULT(S): The VEGF levels in utero were clearly cycle dependent with increasing levels during the late secretory and premenstrual phases. There was a significant correlation with the decidualization marker IGFBP-1. In contrast, intrauterine VEGF levels showed a significant negative correlation with serum E(2) and P. CONCLUSION(S): Intrauterine VEGF levels are regulated in a cycle-dependent way. Increasing levels in the late secretory phase are clearly correlated with decidualization. In contrast, decreasing serum levels of steroids produced by the regressing corpus luteum are less likely to be responsible for increasing VEGF levels in the premenstrual phase.     

Sensitization of B-cell chronic lymphocytic leukemia cells to recombinant immunotoxin by immunostimulatory phosphorothioate oligodeoxynucleotides

A recombinant anti-CD25 immunotoxin, LMB-2, has shown clinical efficacy in hairy cell leukemia and T-cell neoplasms. Its activity in B-cell chronic lymphocytic leukemia (B-CLL) is inferior but might be improved if B-CLL cells expressed higher numbers of CD25 binding sites. It was recently reported that DSP30, a phosphorothioate CpG-oligodeoxynucleotide (CpG-ODN) induces immunogenicity of B-CLL cells by up-regulation of CD25 and other antigens. The present study investigated the antitumor activity of LMB-2 in the presence of DSP30. To this end, B-CLL cells from peripheral blood of patients were isolated immunomagnetically to more than 98% purity. Incubation with DSP30 for 48 hours augmented CD25 expression in 14 of 15 B-CLL samples, as assessed by flow cytometry. DSP30 increased LMB-2 cytotoxicity dose dependently whereas a control ODN with no CpG motif did not. LMB-2 displayed no antitumor cell activity in the absence of CpG-ODN as determined colorimetrically with an (3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium, inner salt (MTS) assay. In contrast, B-CLL growth was inhibited in 12 of 13 samples with 50% inhibition concentrations (IC(50)) in the range of LMB-2 plasma levels achieved in clinical studies. Two samples were not evaluable because of spontaneous B-CLL cell death in the presence of DSP30. Control experiments with an immunotoxin that does not recognize hematopoietic cells, and an anti-CD22 immunotoxin, confirmed that sensitization to LMB-2 was specifically due to up-regulation of CD25. LMB-2 was much less toxic to normal B and T lymphocytes compared with B-CLL cells. In summary, immunostimulatory CpG-ODNs efficiently sensitize B-CLL cells to a recombinant immunotoxin by modulation of its target. This new treatment strategy deserves further attention.     

The amputee mobility predictor: an instrument to assess determinants of the lower-limb amputee's ability to ambulate

OBJECTIVES: To describe the development of the Amputee Mobility Predictor (AMP) instrument designed to measure ambulatory potential of lower-limb amputees with (AMPPRO) and without (AMPnoPRO) the use of a prosthesis, and to test its reliability and validity. DESIGN: Measurement study using known groups method and concurrence with existing measures. SETTING: Academic medical center. PARTICIPANTS: A convenience sample of 191 lower-limb amputee subjects who had completed prosthetic training, 24 in the reliability study (mean age +/- standard deviation, 68.3+/-17.9y, range, 28-99y) and 167 in the validity study (mean age, 54.8+/-18.6y; range, 18-100y). INTERVENTIONS: Not applicable. MAIN OUTCOME MEASURES: Intra- and interrater reliability; construct validity by known groups method; concurrent validity by comparisons with 6-minute walk test, Comorbidity Index, age, and time since amputation; predictive validity by comparison with 6-minute walk test after controlling for other factors. RESULTS: Interrater reliability was.99 for subjects tested with and without their prosthesis; intrarater reliability was.96 and.97. Both the AMPnoPRO (P<.0001) and the AMPPRO scores (P<.0001) distinguished among the 4 Medicare functional classification levels. The AMP correlated strongly with 6-minute walk scores (AMPnoPRO r=.69, P<.0001; AMPPRO r=.82, P<.0001) and the amputee activity survey (AMPnoPRO r=.67, P<.0001; AMPPRO r=.77, P<.0001), and negatively correlated with age (AMPnoPRO r=-.69, P<.0001; AMPPRO r=.56, P<.0001) and comorbidity (AMPnoPRO r=-.43, P<.0001; AMPPRO r=.38, P<.0001). CONCLUSION: The AMP with and without a prosthesis are reliable and valid measures for the assessment of functional ambulation in lower-limb amputee subjects.