Purpose Radiation-induced lymphopenia (RIL) is associated with inferior survival in patients with glioblastoma, lung cancer, and pancreatic cancer. We asked whether stereotactic body radiation therapy (SBRT) decreases severity of RIL compared to conventional chemoradiation therapy (CRT) in locally advanced pancreatic cancer (LAPC). Methods and Materials Serial total lymphocyte counts (TLCs) from patients enrolled in a prospective trial of SBRT for LAPC were compared to TLCs from an existing database of LAPC patients undergoing definitive CRT. SBRT patients received 33 Gy (6.6 × Gy 5 fractions). CRT patients received a median dose of 50.4 Gy (1.8 Gy × 28 fractions) with concurrent 5-fluorouracil (77%) or gemcitabine (23%) therapy. Univariate and multivariate analyses (MVA) were used to identify associations between clinical factors and post-treatment TLC and between TLC and survival. Results Thirty-two patients received SBRT and 101 received CRT. Median planning target volume (PTV) was smaller in SBRT (88.7 cm3) than in CRT (344.6 cm3; P<.001); median tumor diameter was larger for SBRT (4.6 cm) than for CRT (3.6 cm; P=.01). SBRT and CRT groups had similar median baseline TLCs. One month after starting radiation, 71.7% of CRT patients had severe lymphopenia (ie, TLC <500 cells/mm3 vs 13.8% of SBRT patients; P<.001). At 2 months, 46.0% of CRT patients remained severely lymphopenic compared with 13.6% of SBRT patients (P=.007). MVA demonstrated that treatment technique and baseline TLCs were significantly associated with post-treatment TLC at 1 but not 2 months after treatment. Higher post-treatment TLC was associated with improved survival regardless of treatment technique (hazard ratio [HR] for death: 2.059; 95% confidence interval: 1.310–3.237; P=.002). Conclusions SBRT is associated with significantly less severe RIL than CRT at 1 month in LAPC, suggesting that radiation technique affects RIL and supporting previous modeling studies. Given the association of severe RIL with survival in LAPC, further study of the effect of radiation technique on immune status is warranted.

HL. Characterization of large deletions in the F8 gene using multiple competitive amplification and the genome walking technique. J Thromb Haemost 2013; 11: 1103-10.Summary. Background: Large deletions in the F8 gene are responsible for approximately 3% of severe hemophilia A (HA) cases. However, only a few breakpoints in large deletions have been characterized. Objectives: To identify large deletions in the F8 gene and to characterize the molecular mechanisms leading to these deletions. Patients and methods: We used AccuCopy technology, a copy number variation (CNV) genotyping method based on multiplex competitive amplification, to confirm deletions in index patients and to screen potential female carriers in 10 HA families. Also, breakpoints of these large deletions were characterized by a primer walking strategy and genome walking technique. Results: Ten large deletions and four female carriers were identified by AccuCopy. The extents of deleted regions ranged from 1.3 to 68.5 kb. Exact breakpoints of these deletions were successfully characterized. Eight of them presented microhomologies at breakpoint junctions and several recombination-associated elements (repetitive elements, non-B conformation forming motifs and sequence motifs) were also observed in close proximity to the junctions. Conclusions: AccuCopy technology is a reliable and efficient tool for detecting large deletions in the F8 gene and identifying HA female carriers. The genome walking technique is a highly specific, efficient and versatile method for characterizing the deletion breakpoints. Molecular characterization of deletion breakpoints revealed that non-homologous end joining and microhomology-mediated replicationdependent recombination were the major causative mechanisms of the 10 large deletions in the F8 gene.

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Mutations of vacuolar protein sorting-associated protein 33b (VPS33B) cause arthrogryposis, renal dysfunction, and cholestasis syndrome, and a lack of platelet α-granules in the affected patients. Conditional Vps33b knockout mice were developed to investigate the function(s) of Vps33b in platelet α-granule formation. We found that early embryonic deletion of Vps33b was lethal. PF4-Cre-driven megakaryocyte-targeted Vps33b gene deletion greatly diminished Vps33b expression in platelets, but had no effect on platelet α-granule formation and protein content. Tamoxifen-induced, haematopoietic stem cell (HSC)-specific Vps33b deletion completely depleted Vps33b in platelets, caused the absence of α-granules, and increased the number of vacuoles in platelets and megakaryocytes. VPS33B association with VIPAS39, α-tubulin, and SEC22B was identified by co-immunoprecipitation, mass spectra, and immunoblotting in human embryonic kidney 293T (HEK293T) cells. Also, pull-down experiments revealed that VIPAS39 bound to intact VPS33B; in contrast, α-tubulin and SEC22B separately interacted with the sec1-like domains of VPS33B. Vps33b deficiency in megakaryocytes disturbs the redistribution of Vipas39 and Sec22b to proplatelets, and interrupted the co-localization of Sec22b with Vwf-positive vesicles. The data presented in this study suggest that Vps33b is involved in α-granule formation possibly by facilitating the Vwf-positive vesicular trafficking to α-granule-related vacuoles in megakaryocytes. Copyright © 2016 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

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Key Points• Paired immunoglobulin-like receptor B negatively regulates platelet activation.Murine paired immunoglobulin-like receptors B (PIRB), as the ortholog of human leukocyte immunoglobulin-like receptor B2 (LILRB2), is involved in a variety of biological functions. Here, we found that PIRB and LILRB2 were expressed in mouse and human platelets, respectively. PIRB intracellular domain deletion (PIRB-TM) mice had thrombocythemia and significantly higher proportions of megakaryocytes in bone marrow. Agonist-induced aggregation and spreading on immobilized fibrinogen were facilitated in PIRB-TM platelets. The rate of clot retraction in platelet-rich plasma containing PIRB-TM platelets was also increased. Characterization of signaling confirmed that PIRB associated with phosphatases Shp1/2 in platelets. The phosphorylation of Shp1/2 was significantly downregulated in PIRB-TM platelets stimulated with collagen-related peptide (CRP) or on spreading. The results further revealed that the phosphorylation levels of the linker for activation of T cells, SH2 domain-containing leukocyte protein of 76kDa, and phospholipase C were enhanced in PIRB-TM platelets stimulated with CRP. The phosphorylation levels of FAK Y397 and integrin b3 Y759 were also enhanced in PIRB-TM platelet spread on fibrinogen. The PIRB/LILRB2 ligand angiopoietin-like-protein 2 (ANGPTL2) was expressed and stored in platelet a-granules. ANGPTL2 inhibited agonist-induced platelet aggregation and spreading on fibrinogen. The data presented here reveal that PIRB and its ligand ANGPTL2 possess an antithrombotic function by suppressing collagen receptor glycoprotein VI and integrin aIIbb3-mediated signaling. (Blood. 2014;124(15):2421-2430 Introduction Platelets, which are derived from megakaryocytes, circulate in mammalian blood and play essential roles in hemostasis, angiogenesis, inflammation, and metastasis, 1-3 contain a variety of receptors on their surface. The immunoglobulin superfamily (IgSF) is a large group of cell surface proteins that are involved in the adhesion, binding, or recognition of cells. 4 Several IgSF members expressed on the platelet surface regulate platelet adhesion, activation, and aggregation. Among those receptors, platelet collagen receptor glycoprotein VI (GPVI) has short cytoplasmic domains lacking signaling motifs, but it transmits activating signals by linking to immunoreceptor tyrosine-based activation motif (ITAM) of the Fc receptor g chain (FcRg chain). 5 The GPVI/FcRg chain complex can propagate potent signaling causing aIIbb3 activation and platelet aggregation and thereby play an important role in hemostasis and thrombosis formation. 6 In contrast to GPVI, platelet endothelial cell adhesion molecule-1, a platelet surface IgSF member with 6 extracellular Ig domains and a cytoplasmic immunoreceptor tyrosinebased inhibitory motif (ITIM), mildly inhibits human or mouse platelet activation by collagen, adenosine 59-diphosphate (ADP), or thrombin. 7 Similarly, antibody-mediated cross-linking of G6B, a platelet surface IgS...

Severe hypocalcemia (SH) is a common and serious complication in dialysis patients with secondary hyperparathyroidism (SHPT) after parathyroidectomy (PTX). The aim is to explore the risk predictors of SH in post-PTX dialysis ESRD patients with SHPT. 129 consecutive dialysis patients with SHPT underwent PTX were retrospectively reviewed. A total of 22 clinical parameters were included in the study. SH was defined as the minimum values of serum calcium lower than 1.875 mmol/L (7.5 mg/dL) after surgery. Univariate analysis showed that pruritus, lumbar X-ray changes of renal osteodystrophy, pre- and post-operative intact parathyroid hormone (iPTH), Calcium, alkaline phosphatase, and gland mass were significantly different between SH and non-SH groups. In the multivariate logistic regression model, the pre-operative serum iPTH, calcium, and pruritus were independent risk predictors of SH. AUCs for pre-operative serum iPTH, calcium and pruritus were 0.810, 0.714 and 0.591, respectively. Patients with higher level of pre-operative serum iPTH, lower level of serum calcium and with no/mild symptoms of pruritus are at greater risk of developing SH after PTX.

Background: Severe hypocalcemia is the most dangerous complication occurring after total parathyroidectomy without autotransplantation (TPTX) for secondary hyperparathyroidism (SHPT). We aim to identify the prevalence and potential risk factors of very severe hypocalcemia in patients with SHPT undergoing TPTX. Methods: From April 2012 to August 2015, 157 patients with SHPT undergoing TPTX were reviewed. The critical value of hypocalcemia (CVH) was postoperative serum Ca 2þ levels of 1.5 mmol/L. Results: Univariate analysis showed that patients in the CVH group were significantly younger than those in the non-CVH group. Sex ratio was significantly different between the two groups. The CVH group had significantly higher levels of preoperative PTH and ALP. Male sex and preoperative levels of PTH and ALP were significant independent risk factors by logistic regression analysis. Conclusions: Male sex, preoperative PTH and ALP were significantly associated with CVH in patients with SHPT undergoing TPTX.