Challenges in the Application of Glyco-Technology to Hepatitis B Virus Therapy and Diagnosis

Hepatitis B virus (HBV) is a major pathogen that causes acute/chronic hepatitis. Continuous HBV infection can lead to the development of hepatocellular carcinoma (HCC). Although several different anti-HBV treatments are available for chronic hepatitis B patients, discontinuing these medications is difficult. Patients with chronic hepatitis B at high risk for HCC therefore require close observation. However, no suitable biomarkers for detecting high-risk groups for HCC exist, except for serum HBV-DNA, but a number of HCC biomarkers are used clinically, such as alpha-fetoprotein (AFP) and protein induced by vitamin K absence-II (PIVKA-II). Glycosylation is an important post-translational protein modification involved in many human pathologic conditions. HBV surface proteins contain various oligosaccharides, and several reports have described their biological functions. Inhibition of HBV glycosylation represents a potential novel anti-HBV therapy. It is thought that glycosylation of hepatocytes/hepatoma cells is also important for HBV infection, as it prevents HBV from infecting cells other than hepatocytes, even if the cells express the HBV receptor. In this review, we summarize considerable research regarding the relationship between HBV and glycosylation as it relates to the development of novel diagnostic tests and therapies for HBV.     

Perospirone augmentation of paroxetine in treatment of refractory obsessive-compulsive disorder with depression

Obsessive-compulsive disorder (OCD) is often complicated by depression. We report on a patient with treatment-refractory OCD and treatment-refractory major depression who demonstrated a robust response to augmentation of paroxetine with perospirone. Perospirone is a second-generation antipsychotic agent with antagonist effects on both serotonin 5-HT(2A) and dopamine D(2) receptors, as well as a unique agonist effects on serotonin 5-HT(1A) receptors. Future studies would be valuable to elucidate the utility of augmentation therapy of selective serotonin reuptake inhibitors with perospirone in the treatment of refractory OCD with depression.     

Xenografts derived from patients with head and neck cancer recapitulate patient tumour properties

Rodent models mimic the heterogeneity of head and neck cancer (HNC) malignancies and are used to investigate HNC-associated biomarkers and evaluate drug responses. To assess the utility of patient-derived xenografts (PDXs) as an HNC model, 18 tumour samples were obtained from surgical specimens of patients with HNC and implanted into non-obese diabetic severe combined immunodeficient mice. The histological features of PDXs and corresponding patient samples were compared. Furthermore, the present study investigated how PDX responses to anticancer drugs mimic patient clinical responses, as well as the expression of adenosine triphosphate-binding cassette transporters through chemotherapy in an HNC-PDX model. A total of five PDXs from patients with HNC exhibiting high correspondence with histopathological features of the original patient samples were established (establishment rate, 28%). The responses of three PDXs to cisplatin were associated with clinical responses of the patients. ABC transporter expression was augmented in one PDX model after anticancer drug treatment, but not in PBS-treated passaged PDXs. PDX models exhibited similar biological and chemosensitive characteristics to those of the primary tumours. PDXs could be a useful preclinical tool to test novel therapeutic agents and identify novel targets and biomarkers in HNC.     

Roles for hENT1 and dCK in gemcitabine sensitivity and malignancy of meningioma

BackgroundHigh-grade meningiomas are aggressive tumors with high morbidity and mortality rates that frequently recur even after surgery and adjuvant radiotherapy. However, limited information is currently available on the biology of these tumors, and no alternative adjuvant treatment options exist. Although we previously demonstrated that high-grade meningioma cells were highly sensitive to gemcitabine in vitro and in vivo, the underlying molecular mechanisms remain unknown.MethodsWe examined the roles of hENT1 (human equilibrative nucleoside transporter 1) and dCK (deoxycytidine kinase) in the gemcitabine sensitivity and growth of meningioma cells in vitro. Tissue samples from meningiomas (26 WHO grade I and 21 WHO grade II/III meningiomas) were immunohistochemically analyzed for hENT1 and dCK as well as for Ki-67 as a marker of proliferative activity.ResultshENT1 and dCK, which play critical roles in the intracellular transport and activation of gemcitabine, respectively, were responsible for the high gemcitabine sensitivity of high-grade meningioma cells and were strongly expressed in high-grade meningiomas. hENT1 expression was required for the proliferation and survival of high-grade meningioma cells and dCK expression. Furthermore, high hENT1 and dCK expression levels correlated with stronger tumor cell proliferative activity and shorter survival in meningioma patients.ConclusionsThe present results suggest that hENT1 is a key molecular factor influencing the growth capacity and gemcitabine sensitivity of meningioma cells and also that hENT1, together with dCK, may be a viable prognostic marker for meningioma patients as well as a predictive marker of their responses to gemcitabine.     

TMEM16F is required for phosphatidylserine exposure and microparticle release in activated mouse platelets

Phosphatidylserine (PtdSer) exposure on the surface of activated platelets requires the action of a phospholipid scramblase(s), and serves as a scaffold for the assembly of the tenase and prothrombinase complexes involved in blood coagulation. Here, we found that the activation of mouse platelets with thrombin/collagen or Ca²⁺ ionophore at 20 °C induces PtdSer exposure without compromising plasma membrane integrity. Among five transmembrane protein 16 (TMEM16) members that support Ca²⁺-dependent phospholipid scrambling, TMEM16F was the only one that showed high expression in mouse platelets. Platelets from platelet-specific TMEM16F-deficient mice exhibited defects in activation-induced PtdSer exposure and microparticle shedding, although α-granule and dense granule release remained intact. The rate of tissue factor-induced thrombin generation by TMEM16F-deficient platelets was severely reduced, whereas thrombin-induced clot retraction was unaffected. The imaging of laser-induced thrombus formation in whole animals showed that PtdSer exposure on aggregated platelets was TMEM16F-dependent in vivo. The phenotypes of the platelet-specific TMEM16F-null mice resemble those of patients with Scott syndrome, a mild bleeding disorder, indicating that these mice may provide a useful model for human Scott syndrome.Phospholipids are asymmetrically distributed between the inner and outer leaflets of plasma membranes as a result of the activity of flippase(s), which specifically translocates phosphatidylserine (PtdSer) and phosphatidylethanolamine from the outer to the inner leaflet of plasma membranes (1). PtdSer is preferentially exposed on the cell surface during certain physiological processes. During apoptosis, cell-surface PtdSer functions as an “eat me” signal to induce engulfment by phagocytic cells, and, during platelet activation, it serves as a scaffold for the activation of clotting factors. Exposed PtdSer is also implicated in pathological processes and may promote the retention of Ca²⁺ oxalate in kidneys, leading to kidney stone formation (2).PtdSer exposure is accomplished by the inactivation of flippase(s), along with the activation of scramblases (3). We recently identified two protein families (TMEM16 and Xkr) that support phospholipid scrambling (4–6). The TMEM16 family consists of 10 members with 10 transmembrane regions, and TMEM16C, 16D, 16F, 16G, and 16J support the Ca²⁺-dependent scrambling of phospholipids. Scott syndrome is a mild bleeding disorder caused by a defect in platelet procoagulant activity (7, 8). Platelets, red blood cells, and EBV-transformed B cells from patients with Scott syndrome exhibit defective PtdSer exposure following platelet activation or treatment with Ca²⁺ ionophore (9–11). We and others reported that patients with Scott syndrome carry null mutations in the TMEM16F gene (6, 12).The fetal thymocyte cell lines established from TMEM16F^−/− mouse embryos exhibit defective PtdSer exposure upon treatment with Ca²⁺ ionophore (5), reminiscent of the EBV-transformed B-cell lines from patients with Scott syndrome. In contrast, Yang et al. (13) reported that TMEM16F-deficient mouse platelets exhibit only a mild defect in Ca²⁺ionophore–induced PtdSer exposure and tissue factor-induced thrombin generation. Furthermore, in contrast to a human patient with Scott syndrome (14) and dogs with a similar hereditary syndrome (15), neither of which exhibits apparent bleeding-time defects, TMEM16F^−/− mice exhibit a prolonged bleeding time—twice that of WT mice (13).To address the apparent discrepancies between TMEM16F^−/− mouse phenotypes and the clinical presentation of patients with Scott syndrome, and to examine the role of platelet-expressed TMEM16F in blood clotting, we generated a platelet-specific TMEM16F deletion in mice. Our in vitro and in vivo analyses of thrombus formation induced by TMEM16F-null platelets suggested a role for TMEM16F in activation-induced PtdSer exposure, and supported the model in which Ca²⁺-induced PtdSer exposure is involved in the generation of thrombin and fibrin, but not clot retraction (16, 17). Mice with the platelet-specific deletion of TMEM16F exhibited a phenotype similar to that of human patients with Scott syndrome, and may provide a useful model for this human disease.     

Magnetic resonance imaging features of angiomyofibroblastoma-like tumor of the scrotum with pathologic correlates

Various tumors can occur in the scrotum. Among them, angiomyofibroblastoma-like tumors are very rare mesenchymal tumors. We report a case of an angiomyofibroblastoma-like tumor that arose in the right half of the scrotum in a 72-year-old man. It is difficult to separate angiomyofibroblastoma-like tumors from other malignant tumors invading the male genital tract on the basis of clinical characteristics and magnetic resonance imaging findings.     

Combination of thrombin-antithrombin complex,plasminogen activator inhibitor-1, and protein C activity for early identification of severe coagulopathy in initial phase of sepsis: a prospective observational study

Introduction

Current criteria for early diagnosis of coagulopathy in sepsis are limited. We postulated that coagulopathy is already complicated with sepsis in the initial phase, and severe coagulopathy or disseminated intravascular coagulation (DIC) becomes overt after progressive consumption of platelet and coagulation factors. To determine early diagnostic markers for severe coagulopathy, we evaluated plasma biomarkers for association with subsequent development of overt DIC in patients with sepsis.

Methods

A single-center, prospective observational study was conducted in an adult ICU at a university hospital. Plasma samples were obtained from patients with sepsis at ICU admission. Fourteen biomarkers including global markers (platelet count, prothrombin time, activated partial thromboplastin time, fibrinogen and fibrin degradation product (FDP)); markers of thrombin generation (thrombin-antithrombin complex (TAT) and soluble fibrin); markers of anticoagulants (protein C (PC) and antithrombin); markers of fibrinolysis (plasminogen, α₂-plasmin inhibitor (PI), plasmin-α₂-PI complex, and plasminogen activator inhibitor (PAI)-1); and a marker of endothelial activation (soluble E-selectin) were assayed. Patients who had overt DIC at baseline were excluded, and the remaining patients were followed for development of overt DIC in 5 days, and for mortality in 28 days.

Results

A total of 77 patients were enrolled, and 37 developed overt DIC within the following 5 days. Most patients demonstrated hemostatic abnormalities at baseline with 98.7% TAT, 97.4% FDP and 88.3% PC. Most hemostatic biomarkers at baseline were significantly associated with subsequent development of overt DIC. Notably, TAT, PAI-1 and PC discriminated well between patients with and without developing overt DIC (area under the receiver operating characteristic curve (AUROC), 0.77 (95% confidence interval, 0.64 to 0.86); 0.87 (0.78 to 0.92); 0.85 (0.76 to 0.91), respectively), and using the three together, significantly improved the AUROC up to 0.95 (vs. TAT, PAI-1, and PC). Among the significant diagnostic markers for overt DIC, TAT and PAI-1 were also good predictors of 28-day mortality (AUROC, 0.77 and 0.81, respectively).

Conclusions

Severe coagulation and fibrinolytic abnormalities on ICU admission were associated with subsequent development of overt DIC. A single measurement of TAT, PAI-1, and PC activity could identify patients with ongoing severe coagulopathy, early in the course of sepsis.