Advances in urinary proteome analysis and biomarker discovery

Noninvasive diagnosis of kidney diseases and assessment of the prognosis are still challenges in clinical nephrology. Definition of biomarkers on the basis of proteome analysis, especially of the urine, has advanced recently and may provide new tools to solve those challenges. This article highlights the most promising technological approaches toward deciphering the human proteome and applications of the knowledge in clinical nephrology, with emphasis on the urinary proteome. The data in the current literature indicate that although a thorough investigation of the entire urinary proteome is still a distant goal, clinical applications are already available. Progress in the analysis of human proteome in health and disease will depend more on the standardization of data and availability of suitable bioinformatics and software solutions than on new technological advances. It is predicted that proteomics will play an important role in clinical nephrology in the very near future and that this progress will require interactive dialogue and collaboration between clinicians and analytical specialists.     

Immunoglobulin gene analysis reveals 2 distinct cells of origin for EBV-positive and EBV-negative Burkitt lymphomas

The normal counterpart of the neoplastic B cells in Burkitt lymphoma (BL) is still unclear. Based on immunoglobulin gene rearrangement studies, some authors suggest an origin from germinal center cells and others from memory B cells. However, most of these studies rely on cell lines or on a small series of cases. To help clarify the cell of origin of BL, semi-nested polymerase chain reaction (PCR) was performed to amplify the VDJ rearrangements of the immunoglobulin heavy chain (V(H)) genes, and the resultant amplificates were sequenced for comparison with known germline V(H) segments. The results of this approach revealed that all cases (15 endemic BL [eBL], 10 sporadic BL [sBL], and 6 AIDS-related BL) harbor mutated V(H) genes, with different mutation ranges among the 3 types of BL. The eBL and AIDS-related forms showed considerably higher mutation rates than the sBL form (5.1%, 5.4%, and 1.5%, respectively). The mutations in eBL and AIDS-related BL also showed signs of antigen selection, whereas no signs of antigen selection were found in sBL. Finally, after subcloning the amplificates, sequence analysis revealed no signs of ongoing mutations in any of the cases analyzed. Given that one of the main differences between eBL and AIDS-related BL on the one hand and sBL on the other hand is the association with Epstein-Barr virus (EBV), we compared EBV-positive and EBV-negative BLs independently of their geographic origin and HIV status. The differences in the number of somatic mutations and antigen selection were even more evident when this approach was used. According to our molecular results, it appears that EBV-positive and EBV-negative BL may originate from 2 distinct subsets of B cells, pointing to a particular role for the germinal-center reaction in the pathogenesis of these tumors. The different types of C-MYC translocation reported in BL may also be related to the different stages of B-cell maturation.     

Alternating versus concurrent schedules of imatinib and chemotherapy as front-line therapy for Philadelphia-positive acute lymphoblastic leukemia (Ph+ ALL)

The best strategy for incorporating imatinib in front-line treatment of Ph+ acute lymphoblastic leukemia (ALL) has not been established. We enrolled 92 patients with newly diagnosed Ph+ ALL in a prospective, multicenter study to investigate sequentially 2 treatment schedules with imatinib administered concurrent to or alternating with a uniform induction and consolidation regimen. Coadministration of imatinib and induction cycle 2 (INDII) resulted in a complete remission (CR) rate of 95% and polymerase chain reaction (PCR) negativity for BCR-ABL in 52% of patients, compared with 19% in patients in the alternating treatment cohort (P = .01). Remarkably, patients with and without a CR after induction cycle 1 (INDI) had similar hematologic and molecular responses after concurrent imatinib and INDII. In the concurrent cohort, grades III and IV cytopenias and transient hepatotoxicity necessitated interruption of induction in 87% and 53% of patients, respectively; however, duration of induction was not prolonged when compared with patients receiving chemotherapy alone. No imatinib-related severe hematologic or nonhematologic toxicities were noted with the alternating schedule. In each cohort, 77% of patients underwent allogeneic stem cell transplantation (SCT) in first CR (CR1). Both schedules of imatinib have acceptable toxicity and facilitate SCT in CR1 in the majority of patients, but concurrent administration of imatinib and chemotherapy has greater antileukemic efficacy.     

High-Throughput Analysis of Antimalarial Susceptibility Data by the WorldWide Antimalarial Resistance Network (WWARN) In Vitro Analysis and Reporting Tool

Assessment of in vitro susceptibility is a fundamental component of antimalarial surveillance studies, but wide variations in the measurement of parasite growth and the calculation of inhibitory constants make comparisons of data from different laboratories difficult. Here we describe a Web-based, high-throughput in vitro analysis and reporting tool (IVART) generating inhibitory constants for large data sets. Fourteen primary data sets examining laboratory-determined susceptibility to artemisinin derivatives and artemisinin combination therapy partner drugs were collated from 11 laboratories. Drug concentrations associated with half-maximal inhibition of growth (IC₅₀s) were determined by a modified sigmoid E_max model-fitting algorithm, allowing standardized analysis of 7,350 concentration-inhibition assays involving 1,592 isolates. Examination of concentration-inhibition data revealed evidence of apparent paradoxical growth at high concentrations of nonartemisinin drugs, supporting amendment of the method for calculating the maximal drug effect in each assay. Criteria for defining more-reliable IC₅₀s based on estimated confidence intervals and growth ratios improved correlation coefficients for the drug pairs mefloquine-quinine and chloroquine-desethylamodiaquine in 9 of 11 and 8 of 8 data sets, respectively. Further analysis showed that maximal drug inhibition was higher for artemisinins than for other drugs, particularly in ELISA (enzyme-linked immunosorbent assay)-based assays, a finding consistent with the earlier onset of action of these drugs in the parasite life cycle. This is the first high-throughput analytical approach to apply consistent constraints and reliability criteria to large, diverse antimalarial susceptibility data sets. The data also illustrate the distinct biological properties of artemisinins and underline the need to apply more sensitive approaches to assessing in vitro susceptibility to these drugs.     

Joint analysis of individual participants’ data from 17 studies on the association of the IL6 variant -174G>C with circulating glucose levels,interleukin-6 levels,and body mass index

Background. Several studies have investigated associations between the -174G>C single nucleotide polymorphism (rs1800795) of the IL6 gene and phenotypes related to type 2 diabetes mellitus (T2DM) but presented inconsistent results.

Aims. This joint analysis aimed to clarify whether IL6 -174G>C was associated with glucose and circulating interleukin-6 concentrations as well as body mass index (BMI).

Methods. Individual-level data from all studies of the IL6-T2DM consortium on Caucasian subjects with available BMI were collected. As study-specific estimates did not show heterogeneity (P>0.1), they were combined by using the inverse-variance fixed-effect model.

Results. The main analysis included 9440, 7398, 24,117, or 5659 non-diabetic and manifest T2DM subjects for fasting glucose, 2-hour glucose, BMI, or circulating interleukin-6 levels, respectively. IL6 -174 C-allele carriers had significantly lower fasting glucose (?0.091 mmol/L, P=0.014). There was no evidence for association between IL6 -174G>C and BMI or interleukin-6 levels, except in some subgroups.

Conclusions. Our data suggest that C-allele carriers of the IL6 -174G>C polymorphism have lower fasting glucose levels on average, which substantiates previous findings of decreased T2DM risk of these subjects.     

Proteomics applied to the clinical follow-up of patients after allogeneic hematopoietic stem cell transplantation

A phase 1 diagnostic study was performed to evaluate a novel technology for clinical proteomic research based on capillary electrophoresis and mass spectrometry. Urine from 40 patients after hematopoietic stem cell transplantation (HSCT; 35 allogeneic, 5 autologous) and 5 patients with sepsis was collected for a period of 100 days and analyzed. More than 1000 different polypeptides could be detected in individual samples. Polypeptide patterns excreted in the urine of patients were significantly different from those of healthy volunteers. No significant differences were detected comparing different conditioning regimens. The aim of this study was to identify polypeptide patterns functioning as early indicators of graft-versus-host disease (GVHD). Eighteen patients developed GVHD after allogeneic HSCT. Sixteen differentially excreted polypeptides formed a pattern of early GVHD markers, allowing discrimination of GVHD from patients without complications with 82% specificity and 100% sensitivity, cross-validated. Inclusion of 13 sepsis-specific polypeptides allowed us to distinguish sepsis from GVHD with a specificity of 97% and a sensitivity of 100%. Sequencing 2 prominent GVHD-indicative polypeptides led to the identification of a peptide from leukotriene A4 hydrolase and a peptide from serum albumin. The data reveal that capillary electrophoresis and mass spectrometry allow identification of biomarkers for a variety of diseases or related complications.