Citations

Abstract Background: Immunoassays specific for 1–84 parathyroid hormone (PTH) reportedly reflect the bioactivity of PTH; however, PTH immunoassays can be susceptible to interference by cross-reacting PTH fragments. In addition, these assays currently lack standardization. A methodology using immunocapture purification with liquid chromatography–tandem mass spectrometry (LC-MS/MS) detection, along with a stable isotope–labeled internal standard, may help address these issues. Methods: We isolated 1–84 PTH from 1 mL serum by immunocapture on a 6.5-mm polystyrene bead. The immobilized PTH was digested in situ and analyzed by LC-MS/MS. For quantification, we used the selected reaction monitoring response from the N-terminal tryptic peptide 1–13 PTH (1SVSEIQLMHNLGK13). Results: The linear range of the assay was 39.1–4560 ng/L, and the limit of detection and limit of quantification were 14.5 ng/L and 39.1 ng/L, respectively. The intraassay CVs ranged from 6% to 11%, and the interassay CVs ranged from 7% to 17%. Interference by PTH fragments 1–44 PTH, 7–84 PTH, 43–68 PTH, 52–84 PTH, 64–84 PTH, and PTH-related protein (PTHrP) was ≤1% to ≤0.001%. Method comparison of LC-MS/MS vs the Roche Cobas® immunoassay yielded Deming fit of LC-MS/MS = 1.01x immunoassay – 13.21. The mean bias by Bland–Altman plot was −9.4%. Conclusions: In patients with hyperparathyroidism, the immunocapture in situ digestion LC-MS/MS method can provide accurate and precise PTH results compared with immunoassay.

Abstract Anti-Smith (Sm) D autoantibodies are specific for systemic lupus erythematosus. In this investigation, the influence of HLA-D genes on immune responses to SmD was investigated. Mice with HLA-DR3, HLA-DR4, HLA-DQ0601, HLA-DQ0604, or HLA-DQ8 transgenes were immunized with recombinant SmD1, and their Ab responses were analyzed. Analysis by ELISA showed that all strains responded well to SmD. However, when synthetic SmD peptides were used as substrate, DR3 mice had the highest Ab response followed by DQ8, DQ0604, DQ0601, and DR4. A similar trend was observed in Western blot analysis using WEHI 7.1 cell lysate as the substrate, with the exception that DR4 mice did not generate detectable amounts of Abs. Only sera from DR3 and DQ0604 mice immunoprecipitated A-ribonucleoprotein (RNP), SmB, and SmD. Intermolecular epitope spreading to A-RNP and SmB was evident in DR3 and DQ0604 mice, as sera depleted of anti-SmD Abs were reactive with these proteins. DR3 mice also generated an immune response to C-RNP. Anti-nuclear Abs were detected in the majority of the DR3 mice, whereas moderate reactivities were seen in DQ0604 and DQ8 mice. Interestingly, only DR3 mice mounted an anti-dsDNA Ab response. Approximately half of the anti-dsDNA Abs were cross-reactive with SmD. Ab responses correlated with the strength of the T cell responses. Thus, HLA-DR3 appears to be the dominant HLA-D gene that determines the magnitude and quality of the anti-SmD immune response. In addition, our findings provide insights into the origin of the anti-dsDNA Abs often detected in patients with systemic lupus erythematosus.

AbstractA paucity of validated kinase targets in human multiple myeloma has delayed clinical deployment of kinase inhibitors in treatment strategies. We therefore conducted a kinome-wide small interfering RNA (siRNA) lethality study in myeloma tumor lines bearing common t(4;14), t(14;16), and t(11;14) translocations to identify critically vulnerable kinases in myeloma tumor cells without regard to preconceived mechanistic notions. Fifteen kinases were repeatedly vulnerable in myeloma cells, including AKT1, AK3L1, AURKA, AURKB, CDC2L1, CDK5R2, FES, FLT4, GAK, GRK6, HK1, PKN1, PLK1, SMG1, and TNK2. Whereas several kinases (PLK1, HK1) were equally vulnerable in epithelial cells, others and particularly G protein–coupled receptor kinase, GRK6, appeared selectively vulnerable in myeloma. GRK6 inhibition was lethal to 6 of 7 myeloma tumor lines but was tolerated in 7 of 7 human cell lines. GRK6 exhibits lymphoid-restricted expression, and from coimmunoprecipitation studies we demonstrate that expression in myeloma cells is regulated via direct association with the heat shock protein 90 (HSP90) chaperone. GRK6 silencing causes suppression of signal transducer and activator of transcription 3 (STAT3) phosphorylation associated with reduction in MCL1 levels and phosphorylation, illustrating a potent mechanism for the cytotoxicity of GRK6 inhibition in multiple myeloma (MM) tumor cells. As mice that lack GRK6 are healthy, inhibition of GRK6 represents a uniquely targeted novel therapeutic strategy in human multiple myeloma.

Purpose No established treatment exists for pancreatic neuroendocrine tumor (NET) progression after failure of chemotherapy. Everolimus (RAD001), an oral inhibitor of mammalian target of rapamycin, in combination with octreotide has demonstrated encouraging antitumor activity in patients with NETs. Patients and Methods This open-label, phase II study assessed the clinical activity of everolimus in patients with metastatic pancreatic NETs who experienced progression on or after chemotherapy. Patients were stratified by prior octreotide therapy (stratum 1: everolimus 10 mg/d, n = 115; stratum 2: everolimus 10 mg/d plus octreotide long-acting release [LAR], n = 45). Tumor assessments (using Response Evaluation Criteria in Solid Tumors) were performed every 3 months. Chromogranin A (CgA) and neuron-specific enolase (NSE) were assessed monthly if elevated at baseline. Trough concentrations of everolimus and octreotide were assessed. Results By central radiology review, in stratum 1, there were 11 partial responses (9.6%), 78 patients (67.8%) with stable disease (SD), and 16 patients (13.9%) with progressive disease; median progression-free survival (PFS) was 9.7 months. In stratum 2, there were two partial responses (4.4%), 36 patients (80%) with SD, and no patients with progressive disease; median PFS was 16.7 months. Patients with an early CgA or NSE response had a longer PFS compared with patients without an early response. Coadministration of octreotide LAR and everolimus did not impact exposure to either drug. Most adverse events were mild to moderate and were consistent with those previously seen with everolimus. Conclusion Daily everolimus, with or without concomitant octreotide LAR, demonstrates antitumor activity as measured by objective response rate and PFS and is well tolerated in patients with advanced pancreatic NETs after failure of prior systemic chemotherapy.

Background and Purpose— We performed a meta-analysis of published studies on the endovascular treatment of very small intracranial aneurysms, including 71 patients treated at our institution. Methods— We conducted a computerized MEDLINE search of the literature for reports on the treatment of intracranial aneurysms with a maximum dimension of ≤3 mm by using the search terms “small,” “tiny,” “intracranial aneurysm,” “endovascular,” and “coil.” A total of 7 studies, including our institution’s consecutive case series of 71 intracranial aneurysms, were included in this study. We extracted information regarding intraoperative complications, procedural mortality and morbidity, immediate- and long-term angiographic outcomes, and retreatment rate. The meta-analysis was performed with the statistical package Comprehensive Meta-Analysis. Results— Approximately 61% of the aneurysms in this meta-analysis presented as ruptured, whereas 39% of the aneurysms were unruptured. Procedural rupture rates for very small aneurysms was 8.3% (95% CI, 6.0% to 11.4%). The mortality rate due to procedural rupture was 2.4% (95% CI, 1.2% to 4.7%). The morbidity rate due to thromboembolic complications was 1.9% (95% CI, 0.9% to 3.9%). Subarachnoid hemorrhage within 1 month of treatment occurred in 1.6% (95% CI, 0.6% to 3.7%) of cases. There was no statistically significant difference between unruptured and ruptured aneurysms for any of these outcomes. Conclusion— Our meta-analysis suggests that treatment of very small aneurysms is feasible and effective in >90% of treated aneurysms. However, the risk of periprocedural rupture is higher than that reported for larger aneurysms. Similarly, the combined rate of periprocedural mortality and morbidity is not negligible (7.3%) and should be considered when considering the best therapeutic option for these aneurysms.

Background— The benefit of primary percutaneous coronary interventions (P-PCI) for patients with ST-elevation myocardial infarction (STEMI) has been well documented. However, controversy still exists as to whether PCI should be expanded to hospitals without coronary artery bypass graft surgery. Methods and Results— Patients who were discharged after PCI for STEMI between January 1, 2003, and December 12, 2006, in P-PCI centers (hospitals with no coronary artery bypass graft surgery, and PCI only for patients with STEMI) were propensity matched with patients in full service centers, and mortality and subsequent revascularization rates were compared. For patients undergoing PCI, there were no differences for in-hospital/30-day mortality (2.3% for P-PCI centers versus 1.9% for full service centers [ P =0.40]), emergency coronary artery bypass graft surgery immediately after PCI (0.06% versus 0.35%, P =0.06), 3-year mortality (7.1% versus 5.9%, P =0.07), or 3-year subsequent revascularization (23.8% versus 21.5%, P =0.52). P-PCI centers had a lower same/next day coronary artery bypass graft rate (0.23% versus 0.69%, P =0.046) and higher repeat target vessel PCI rates (12.1% versus 9.0%, P =0.003). For patients with STEMI who did not undergo PCI, P-PCI centers had higher in-hospital mortality (28.5% versus 22.3%; adjusted odds ratio, 1.38; 95% CI, 1.10 to 1.75). Conclusions— No differences between P-PCI centers and full service centers were found in in-hospital/30-day mortality, the need for emergency surgery, 3-year mortality or subsequent revascularization, but P-PCI centers had higher repeat target vessel PCI rates and higher mortality rates for patients who did not undergo PCI. P-PCI centers should be monitored closely, including the monitoring of patients with STEMI who did not undergo PCI.