Distinct sources and targets of IL-10 during dendritic cell-driven Th1 and Th2 responses in vivo

Dendritic cells (DC) can both initiate an immune response and dictate its character. Cytokines are critically involved in this process and, although interleukin (IL)-10 is known as a potent immunosuppressant, the impact of its release from DC remains unclear. Here, we transfer pathogen-conditioned murine DC in vivo and show that, while DC-derived IL-10 can act to limit Th1 development, it is not required for Th2 induction. In both Th2 and Th1 settings, however, IL-10 from cells other than the initiating DC dominates the regulation of the emerging effector cell populations. Surprisingly, the critical source of IL-10 in this process is neither T nor B cells. These data illustrate the distinct actions of IL-10 during differently polarised, pathogen-focussed, DC-driven immune responses in vivo.     

Genetic association between the phospholipase A2 gene and unipolar affective disorder: a multicentre case-control study

The co-segregation in one pedigree of bipolar affective disorder with Darier's disease whose gene is on chromosome 12q23-q24.1, and findings from linkage and association studies with the neighbouring gene of phospholipase A2 (PLA2) indicate that PLA2 may be considered as a candidate gene for affective disorders. All relevant genetic association studies, however, were conducted on bipolar patients. In the present study, the possible association between the PLA2 gene and unipolar affective disorder was examined on 321 unipolar patients and 604 controls (all personally interviewed), recruited from six countries (Belgium, Bulgaria, Croatia, Germany, Greece, and Italy) participating in the European Collaborative Project on Affective Disorders. After controlling for population group and gender, one of the eight alleles of the investigated marker (allele 7) was found to be more frequent among unipolar patients with more than three major depressive episodes than among controls (P<0.01); genotypic association was also observed, under the dominant model of genetic transmission (P<0.02). In addition, presence of allele 7 was correlated with a higher frequency of depressive episodes (P<0.02). These findings suggest that structural variations at the PLA2 gene or the chromosomal region around it may confer susceptibility for unipolar affective disorder.     

Expression of nuclear factor-kappaB in human astrocytomas: relation to pI kappa Ba, vascular endothelial growth factor, Cox-2, microvascular characteristics, and survival

Although NF-kappaB has been reported to be constitutively activated in various neoplasms, little information is available about its clinical significance in astrocytomas. In this study, we investigated the association of NF kappa B1/p50 and pI kappa Ba immunohistochemical expression with clinicopathologic features, vascular endothelial growth factor, Cox-2, and microvascular parameters in paraffin-embedded tissue from 82 patients with astrocytomas. pI kappa Ba expression was positively correlated with nuclear (P = .0010) and negatively with cytoplasmic (P = .0008) NF kappa B1/p50 expression. Nuclear NF kappa B1/p50 and pI kappa Ba expression increased with tumor grade (P = .0001 and P < .0001). Nuclear NF kappa B1/p50 was associated with vascular endothelial growth factor (P = .0079), Cox-2 (P = .0500), and total vascular surface area (P = .0430), although the latter was significant only in grades II and III. pI kappa Ba was also positively correlated with microvessel caliber (pI kappa Ba/area; P = .0087). Multivariate analysis selected NF kappa B/p50 expression as an independent prognosticator not only for the entire cohort (P = .0220), but also for grades II and III (P = .0029) and grade IV cases (P = .0310). Our results suggest that nuclear NF kappa B1/p50 expression is dictated by its interaction with I kappa Ba in astrocytomas and is associated with tumor grade and angiogenic factors, denoting the importance of nuclear NF kappa B/p50 expression in patients' prognosis.     

Modified CLSI Extended-Spectrum β-Lactamase (ESBL) Confirmatory Test for Phenotypic Detection of ESBLs among Enterobacteriaceae Producing Various β-Lactamases

The worldwide dissemination of Enterobacteriaceae producing AmpC β-lactamases and carbapenemases makes difficult the phenotypic detection of extended-spectrum β-lactamases (ESBLs), as they may be masked by these additional enzymes. A modification of the CLSI ESBL confirmatory test was developed and evaluated in a comparative study for its ability to successfully detect ESBLs among Enterobacteriaceae producing various carbapenemases (Klebsiella pneumoniae carbapenemase [KPC], VIM, NDM, and OXA-48) and plasmidic or derepressed AmpCs. The modified CLSI ESBL confirmatory test was performed with cefotaxime and ceftazidime disks with and without clavulanate, on which both boronic acid (BA) and EDTA were dispensed. A total of 162 genotypically confirmed ESBL-positive Enterobacteriaceae isolates (83 carbapenemase/ESBL producers, 25 AmpC/ESBL producers, and 54 ESBL-only producers) were examined. For comparison, 139 genotypically confirmed ESBL-negative Enterobacteriaceae isolates (94 of them possessed carbapenemases and 20 possessed AmpCs) were also tested. The standard CLSI ESBL confirmatory test was positive for 106 of the 162 ESBL producers (sensitivity, 65.4%) and showed false-positive results for 4 of the 139 non-ESBL producers (specificity, 97.1%). The modified CLSI ESBL confirmatory test detected 158 of 162 ESBL producers (sensitivity, 97.5%) and showed no false-positive results for non-ESBL producers (specificity, 100%). The findings of the study demonstrate that the modified CLSI ESBL confirmatory test using antibiotic disks containing both BA and EDTA accurately detects ESBLs in Enterobacteriaceae regardless of the coexistence of additional β-lactam resistance mechanisms.     

Cross-reactive monoclonal antibodies to multiple HIV-1 subtype and SIVcpz envelope glycoproteins

The extraordinarily high level of genetic variation of HIV-1 env genes poses a challenge to obtain antibodies that cross-react with multiple subtype Env glycoproteins. To determine if cross-reactive monoclonal antibodies (mAbs) to highly conserved epitopes in HIV-1 envelope glycoproteins can be induced, we immunized mice with wild-type or consensus HIV-1 Env proteins and characterized a panel of ten mAbs that reacted with varying breadth to subtypes A, B, C, D, F, G, CRF01_AE, and a highly divergent SIVcpzUS Env proteins by ELISA and Western blot analysis. Two mAbs (3B3 and 16H3) cross-reacted with all tested Env proteins, including SIVcpzUS Env. Surface plasmon resonance analyses showed both 3B3 and 16H3 bound Env proteins with high affinity. However, neither neutralized primary HIV-1 pseudoviruses. These data indicate that broadly reactive non-neutralizing monoclonal antibodies can be elicited, but that the conserved epitopes that they recognize are not present on functional virion trimers. Nonetheless, such mAbs represent valuable reagents to study the biochemistry and structural biology of Env protein oligomers.     

Identification and functional characterization of imatinib‐sensitive DTD1‐PDGFRB and CCDC88C‐PDGFRB fusion genes in eosinophilia‐associated myeloid/lymphoid neoplasms

Eosinophilia‐associated myeloid neoplasms with rearrangement of chromosome bands 5q31‐33 are frequently associated with PDGFRB fusion genes, which are exquisitely sensitive to treatment with imatinib. In search for novel fusion partners of PDGFRB, we analyzed three cases with translocation t(5;20)(q33;p11), t(5;14)(q33;q32), and t(5;17;14)(q33;q11;q32) by 5′‐rapid amplification of cDNA ends polymerase chain reaction (5′‐RACE‐PCR) and DNA‐based long‐distance inverse PCR (LDI‐PCR) with primers derived from PDGFRB. LDI‐PCR revealed a fusion between CCDC88C exon 25 and PDGFRB exon 11 in the case with t(5;17;14)(q33;q11;q32) while 5′‐RACE‐PCR identified fusions between CCDC88C exon 10 and PDGFRB exon 12 and between DTD1 exon 4 and PDGFRB exon 12 in the cases with t(5;14)(q33;q32) and t(5;20)(q33;p11), respectively. The PDGFRB tyrosine‐kinase domain is predicted to be retained in all three fusion proteins. The partner proteins contained coiled‐coil domains or other domains, which putatively lead to constitutive activation of the PDGFRB fusion protein. In vitro functional analyses confirmed transforming activity and imatinib‐sensitivity of the fusion proteins. All three patients achieved rapid and durable complete hematologic remissions on imatinib. © 2014 Wiley Periodicals, Inc.     

Accurate molecular diagnosis of phenylketonuria and tetrahydrobiopterin-deficient hyperphenylalaninemias using high-throughput targeted sequencing

Genetic diagnostics of phenylketonuria (PKU) and tetrahydrobiopterin (BH4) deficient hyperphenylalaninemia (BH4DH) rely on methods that scan for known mutations or on laborious molecular tools that use Sanger sequencing. We have implemented a novel and much more efficient strategy based on high-throughput multiplex-targeted resequencing of four genes (PAH, GCH1, PTS, and QDPR) that, when affected by loss-of-function mutations, cause PKU and BH4DH. We have validated this approach in a cohort of 95 samples with the previously known PAH, GCH1, PTS, and QDPR mutations and one control sample. Pooled barcoded DNA libraries were enriched using a custom NimbleGen SeqCap EZ Choice array and sequenced using a HiSeq2000 sequencer. The combination of several robust bioinformatics tools allowed us to detect all known pathogenic mutations (point mutations, short insertions/deletions, and large genomic rearrangements) in the 95 samples, without detecting spurious calls in these genes in the control sample. We then used the same capture assay in a discovery cohort of 11 uncharacterized HPA patients using a MiSeq sequencer. In addition, we report the precise characterization of the breakpoints of four genomic rearrangements in PAH, including a novel deletion of 899 bp in intron 3. Our study is a proof-of-principle that high-throughput-targeted resequencing is ready to substitute classical molecular methods to perform differential genetic diagnosis of hyperphenylalaninemias, allowing the establishment of specifically tailored treatments a few days after birth.     

Evaluation of PCR-based preimplantation genetic diagnosis applied to monogenic diseases: a collaborative ESHRE PGD consortium study

Preimplantation genetic diagnosis (PGD) for monogenic disorders currently involves polymerase chain reaction (PCR)-based methods, which must be robust, sensitive and highly accurate, precluding misdiagnosis. Twelve adverse misdiagnoses reported to the ESHRE PGD-Consortium are likely an underestimate. This retrospective study, involving six PGD centres, assessed the validity of PCR-based PGD through reanalysis of untransferred embryos from monogenic-PGD cycles. Data were collected on the genotype concordance at PGD and follow-up from 940 untransferred embryos, including details on the parameters of PGD cycles: category of monogenic disease, embryo morphology, embryo biopsy and genotype assay strategy. To determine the validity of PCR-based PGD, the sensitivity (Se), specificity (Sp) and diagnostic accuracy were calculated. Stratified analyses were also conducted to assess the influence of the parameters above on the validity of PCR-based PGD. The analysis of overall data showed that 93.7% of embryos had been correctly classified at the time of PGD, with Se of 99.2% and Sp of 80.9%. The stratified analyses found that diagnostic accuracy is statistically significantly higher when PGD is performed on two cells versus one cell (P=0.001). Se was significantly higher when multiplex protocols versus singleplex protocols were applied (P=0.005), as well as for PGD applied on cells from good compared with poor morphology embryos (P=0.032). Morphology, however, did not affect diagnostic accuracy. Multiplex PCR-based methods on one cell, are as robust as those on two cells regarding false negative rate, which is the most important criteria for clinical PGD applications. Overall, this study demonstrates the validity, robustness and high diagnostic value of PCR-based PGD.     

A Delphi study on research priorities in radiation therapy: The Australian perspective

Radiation therapists (RTs) need to engage more in research to establish an evidence base for their daily practice. However, RTs world-wide conduct little research themselves, although positive moves have been made in some countries.This project is the second stage of a Delphi process aimed at prioritising RT areas of research interest. A questionnaire was constructed using responses to a previous questionnaire which identified the research interests of Australian RTs. Fifty-three Research Areas were identified from these responses and grouped into 12 categories such as “imaging in radiation therapy”, “symptom management”, “accuracy of patient positioning” and “techniques/equipment”. The survey was sent to all Australian departments of radiation oncology, and RTs were asked to form interest groups to discuss and prioritise the Research Areas.There was a 50% response rate (18 of 36 departments surveyed). The highest ranked research Category was “imaging in radiation therapy”. Six of the top 10 ranked Research Areas were within Central RT practice (“imaging in radiation therapy”; “symptom management”; “accuracy of patient positioning” and “techniques/equipment”) and the other four were within broader RT practice (“diversification, recognition and other professional issues”; and “management and staff issues”). Patient Care was also considered to be an area requiring more research.This prioritization of Research Areas and categories provides a useful list of future research for RTs, which will enable them to decide whether their research ideas are a high priority, and spend less time deciding on a relevant research topic that needs investigation in their own workplaces.