Legacy and new halogenated persistent organic pollutants in polar bears from a contamination hotspot in the Arctic, Hudson Bay Canada.

A large and complex suite of 295 legacy and new halogenated persistent organic pollutants (POPs) were investigated in fat or liver tissue samples of polar bears collected in 2013-2014 from Southern (SHB) and Western (WHB) subpopulations of the Canadian Arctic contaminants hotspot of Hudson Bay. A total of 210 POPs were detected and/or quantifiable with some frequency in all fat or liver samples. POP profile and concentration differences were investigated both within (e.g. age and sex) and between the two subpopulations. Two time-point comparisons were made relative to POPs reported for Hudson Bay polar bears harvested in 2007-2008. ΣPolychlorinated biphenyl (PCB) concentrations at both time points were the most concentrated of the POP groups, and were spatially uniform with no detectable influence of sex or age, as were concentrations of the dominant congener CB153. ΣChlordanes (ΣCHLs, 74-79% oxychlordane) and the Σperfluoroalkyl substances (ΣPFASs, ≈60% perfluorooctane sulfonate (PFOS)) had the second greatest POP group concentrations in SHB and WHB respectively, with ΣPFASs and ΣCHLs being significantly influenced by age and/or sex. ΣCHLs were spatially uniform but ΣPFASs were greater in the SHB bears, as were e.g. some flame retardants, due to e.g. local contamination and/or changes in bear behavior and diet. Endosulfans and hexabromocyclododecane were detectable in samples from 2007-2008 but not from 2013-2014, which is consistent with their global POP regulations. ΣPolychlorinated naphthalenes (ΣPCNs) were consistently detected at relatively high concentrations compared to other arctic wildlife, however these concentrations were low relative to legacy POPs. ΣShort-chain chlorinated paraffins (ΣSCCPs) were major contributors to the overall POPs burden with concentrations comparable to other legacy POPs, though there was no significant difference between or within subpopulations for PCNs or SCCPs. Except for octachlorostyrene, POPs concentrations were generally lower in female and male bears from SHB in 2013-2014 relative to 2007-2008, however those of WHB males were greater over the same timeframe for almost all POPs.

MR Imaging Characteristics Associate with Tumor-Associated Macrophages in Glioblastoma and Provide an Improved Signature for Survival Prognostication.

BACKGROUND AND PURPOSE: In glioblastoma, tumor-associated macrophages have tumor-promoting properties. This study determined whether routine MR imaging features could predict molecular subtypes of glioblastoma that differ in the content of tumor-associated macrophages. MATERIALS AND METHODS: Seven internally derived MR imaging features were assessed in 180 patients, and 25 features from the Visually AcceSAble Rembrandt Images feature set were assessed in 164 patients. Glioblastomas were divided into subtypes based on the telomere maintenance mechanism: alternative lengthening of telomeres positive (ALT+) and negative (ALT-) and the content of tumor-associated macrophages (with [M+] or without [M-] a high content of macrophages). The 3 most frequent subtypes (ALT+/M-, ALT-/M+, and ALT-/M-) were correlated with MR imaging features and clinical parameters. The fourth group (ALT+/M+) did not have enough cases for correlation with MR imaging features. RESULTS: Tumors with a regular margin and those lacking a fungating margin, an expansive T1/FLAIR ratio, and reduced ependymal extension were more frequent in the subgroup of ALT+/M- (P < .05). Radiologic necrosis, lack of cystic component (by both criteria), and extensive peritumoral edema were more frequent in ALT-/M+ tumors (P < .05). Multivariate testing with a Cox regression analysis found the cystic imaging feature was additive to tumor subtype, and O6-methylguanine methyltransferase (MGMT) status to predict improved patient survival (P < .05). CONCLUSIONS: Glioblastomas with tumor-associated macrophages are associated with routine MR imaging features consistent with these tumors being more aggressive. Inclusion of cystic change with molecular subtypes and MGMT status provided a better estimate of survival.

CD4 T cells inhibit the CD8 T cell response during low-dose virus infection.

CD4 T cells are not thought to play a significant role in generating an effective primary CD8 T cell response to most viral infections. We have challenged this view by demonstrating that antigen-specific CD4 T cells can indeed suppress the proliferation of antigen-specific naive CD8 T cells in response to low doses of vesicular stomatitis virus. This finding is in contrast to the established observations that at high antigen loads CD4 T cells play little role in generating CD8 T cell responses, and that in non-infectious model systems CD4 T cells actually help the CD8 T cell response. Our results suggest that at low infectious doses, CD4 T cells play a much larger role in controlling infections than previously appreciated.

hRad9 rapidly binds DNA containing double-strand breaks and is required for damage-dependent topoisomerase II beta binding protein 1 focus formation.

Checkpoint proteins protect the genomic integrity of a cell, repeatedly impaired by DNA damage and normal cellular processes, such as replication. Checkpoint proteins hRad9, hRad1, and hHus1 form a heterotrimeric complex that is thought to act as a genomic surveyor of DNA damage. We show here that, when DNA double-strand breaks (DSBs) are specifically generated in a subnuclear area, hRad9 is rapidly retained at the damaged DNA, within 2 min of damage induction. Rapid localization of hRad9 to regions of DNA containing DSBs is most efficient during replication. Furthermore, hRad9 colocalizes with the phosphorylated form of damage-response protein H2AX (gamma H2AX) after DNA damage. This localization is independent of the damage repair kinase ataxia telangiectasia-mutated kinase (ATM), because hRad9/gamma H2AX colocalization still occurs in ATM(-/-) fibroblasts. Secondly, hRad9 interacts with replication and checkpoint protein topoisomerase II beta binding protein 1 (TopBP1) before and after DNA damage, and this interaction is dependent on the COOH-terminal 17 amino acids of hRad9. Overexpression of a COOH-terminally deleted form of hRad9 abolishes the colocalization of TopBP1 to gamma H2AX, ablating TopBP1 but not gamma H2AX foci formation. The loss of TopBP1 containing foci, but not of gamma H2AX containing foci, indicates that hRad9 is required for TopBP1 focus formation after damage, but is not required for gamma H2AX formation at DSBs. These results are consistent with a model in which the hRad9/hHus1/hRad1 complex acts as a checkpoint sensor during S phase by rapidly localizing to sites of DNA damage and transducing checkpoint responses by facilitating proper localization of downstream checkpoint proteins, including TopBP1.

A role for the phosphorylation of hRad9 in checkpoint signaling.

The integrity of the human genome is preserved by signal transduction pathways called checkpoints, which delay progression through the cell cycle when DNA damage is present. Three checkpoint proteins, hRad9, hRad1, and hHus1, form a proliferating cell nuclear antigen-like, heterotrimeric complex that has been proposed to function in the initial detection of DNA structural abnormalities. hRad9 is highly modified by phosphorylation, in a constitutive manner and in response to both DNA damage and cell cycle position. Here we present evidence that Thr292 of hRad9 is subject to Cdc2-dependent phosphorylation in mitosis. Furthermore, our data are also consistent with four other hRad9 phosphorylation sites (Ser277, Ser328, Ser336, and Thr355) being regulated in part by Cdc2. We also identify Ser387 as a novel site of hRad9 constitutive phosphorylation and show that phosphorylation at Ser387 is a prerequisite for one form of DNA damage-induced hyperphosphorylation of hRad9. Characterization of nonphosphorylatable mutants has revealed that hRad9 phosphorylation plays a critical role in checkpoint signaling. Overexpression of these mutants blocks the interaction between hRad9 and the DNA damage-responsive protein TopBP1 and impairs the cellular response to DNA damage during S phase.

The "knowingly and willfully" continuum of the anti-kickback statute's scienter requirement: its origins, complexities, and most recent judicial developments.

Mr. Blair analyzes the evolution of the Anti-Kickback Statute's scienter requirement. The article includes a historical review of the Anti-Kickback Statute and an in-depth discussion of three notable cases in this area, United States v. Greber, Hanlester Network v. Shalala, and United States v. Davis. The author concludes that without further guidance from either Congress or the Department of Health and Human Services, the split among circuits as to the proper definition of the Statute's scienter requirement makes the matter ripe for Supreme Court review.

Human x human hybridomas from patients with myasthenia gravis: possible tools for idiotypic therapy for myasthenia.

Hybridomas secreting monoclonal antibodies directed against the nicotinic acetylcholine receptor have been developed from rats with experimental autoimmune myasthenia gravis and from a patient with myasthenia gravis. Rat monoclonal antibodies were characterized by their ability to bind to electroblotted acetylcholine receptor subunits. Of 34 tested, 22 bound to the alpha subunit. Three bound to other subunits, and the remainder appeared to bind only to the native molecule. The human monoclonal antibodies were analyzed with respect to their binding to membrane-bound and solubilized acetylcholine receptor. Many bound with greater affinity to the membrane-bound form of the antigen. Two rat monoclonal antibodies capable of passively transferring experimental autoimmune myasthenia gravis, and with reactivities to the alpha subunit of the acetylcholine receptor, were employed to produce isogeneic monoclonal antiidiotypic antibodies. When they were injected prior to immunization with acetylcholine receptor, two of the antiidiotypic antibodies directed against framework determinants prevented the development of experimental autoimmune myasthenia gravis. This observation raises the possibility that the human monoclonal antibodies will be useful in the development of idiotypic treatment of the human disease.

Monoclonal antibodies to acetylcholine receptor secreted by human x human hybridomas derived from lymphocytes of a patient with myasthenia gravis.

Peripheral blood lymphocytes of a patient with myasthenia gravis (MG) were fused to the non-secreting human lymphoblastoid line HuNSI to produce human x human hybridomas that secrete monoclonal antibodies (mAb) to acetylcholine receptor (AChR). Screening of hybridomas for antibody production involved an enzyme-linked immunosorbent (ELISA) assay with AChR from Torpedo californica (TAChR). 25 of 302 wells tested (8.3%) were positive for anti-AChR antibody production and have been stable in their secretion of mAb for eleven months. Nine lines have been studied in detail. All produced IgM mAb, and most had greater activity against membrane-bound TAChR, than against solubilized TAChR. For anti-AChR clones, the mAb concentration in culture supernatants ranged from 2 to 33 micrograms/ml. Saturation curves of binding to TAChR performed on 4 lines demonstrated dissociation constants (Kds) estimated to range from 0.1-1.0 nM. The patient whose lymphocytes were used in this study had a serum anti-AChR antibody concentration of 243nM against human AChR and 15nM against AChR from T. californica. The results demonstrate the feasibility of producing stable human x human hybridomas secreting mAb to the autoantigen from the peripheral blood of patients with organ-specific autoimmune diseases. The mAb produced here may prove to be useful in analyzing, and possibly treating, the autoimmune phenomena in MG.