Blocking the CD40–CD154 interaction is reported to be effective for transplantation management and autoimmune disease models in rodents and nonhuman primates. However, clinical trials with anti-CD154 mAbs were halted because of high incidence of thromboembolic complications. Thus, we generated and characterized a fully human anti-CD40 mAb ASKP1240, as an alternative to anti-CD154 mAb. In vitro ASKP1240 concentration-dependently inhibited human peripheral blood mononuclear cell proliferation induced by soluble CD154. In addition, ASKP1240 did not destabilize platelet thrombi under physiological high shear conditions while mouse anti-human CD154 mAb (mu5C8) did. And ASKP1240 itself did not activate platelet and endothelial cells. In vivo administration of ASKP1240 (1 or 10 mg/kg, intravenously) to cynomolgus monkeys, weekly for 3 weeks, significantly attenuated both delayed-type hypersensitivity and specific antibody formation evoked by tetanus toxoid. The immunosuppressive effect was well correlated with the CD40 receptor saturation. Thus, these results suggest that ASKP1240 is immunosuppressive but not prothromboembolic, and as such appears to be a promising therapeutic candidate for the management of solid organ transplant rejection and autoimmune diseases therapy.
CD40; costimulation; immunosuppressive therapy; mAbs
Ligands of transmembrane receptor tyrosine kinases have important roles in cell proliferation, survival, migration and differentiation in solid tumours. We conducted this study to evaluate the relationship between concentration of serum ligands and prognosis of patients with metastatic colorectal cancer (mCRC) treated with anti-epidermal growth factor receptor (EGFR) antibodies.
Between August 2008 and August 2011, serum samples were obtained from KRAS wild-type patients who met the inclusion criteria and received an anti-EGFR antibody treatment. Serum concentration of ligands was measured by an enzyme-linked immunosorbent assay, and somatic mutations of KRAS, BRAF, PIK3CA and BRAF were analysed by direct sequencing.
A total of 103 patients were enrolled in the present study. At the pretreatment serum levels, patients with high levels of hepatocyte growth factor (HGF) had shorter progression-free survival (PFS) and overall survival (OS) compared with those with low levels of HGF (median PFS: 6.4 months vs 4.4 months; P<0.001, median OS: 15.3 months vs 8.0 months; P<0.001, respectively). Patients with high levels of epiregulin (EREG) also had shorter PFS and OS compared with those with low levels of EREG (median PFS: 6.6 months vs 4.9 months; P=0.016, median OS: 13.8 months vs 7.4 months; P=0.048, respectively). In addition, patients whose serum levels of ligands were elevated at progressive disease had shorter PFS and OS compared with other patients.
Our study indicated that high levels of HGF and EREG were associated with resistance to treatment with anti-EGFR antibodies in KRAS wild-type patients with mCRC. Our findings will contribute to the newly combination therapy on the treatment of anti-EGFR antibodies.
colorectal cancer; HGF; epiregulin; ligands; EGFR
In human cells, the RIPK1–RIPK3–MLKL–PGAM5–Drp1 axis drives tumor necrosis factor (TNF)-induced necroptosis through mitochondrial fission, but whether this pathway is conserved among mammals is not known. To answer this question, we analyzed the presence and functionality of the reported necroptotic axis in mice. As in humans, knockdown of receptor-interacting kinase-3 (RIPK3) or mixed lineage kinase domain like (MLKL) blocks TNF-induced necroptosis in L929 fibrosarcoma cells. However, repression of either of these proteins did not protect the cells from death, but instead induced a switch from TNF-induced necroptosis to receptor-interacting kinase-1 (RIPK1) kinase-dependent apoptosis. In addition, although mitochondrial fission also occurs during TNF-induced necroptosis in L929 cells, we found that knockdown of phosphoglycerate mutase 5 (PGAM5) and dynamin 1 like protein (Drp1) did not markedly protect the cells from TNF-induced necroptosis. Depletion of Pink1, a reported interactor of both PGAM5 and Drp1, did not affect TNF-induced necroptosis. These results indicate that in these murine cells mitochondrial fission and Pink1 dependent processes, including Pink-Parkin dependent mitophagy, apparently do not promote necroptosis. Our data demonstrate that the core components of the necrosome (RIPK1, RIPK3 and MLKL) are crucial to induce TNF-dependent necroptosis both in human and in mouse cells, but the associated mechanisms may differ between the two species or cell types.
RIPK3; MLKL; TNF; apoptosis; necroptosis
Although TRAIL (tumor necrosis factor (TNF)-related apoptosis inducing ligand) is a well-known apoptosis inducer, we have previously demonstrated that acidic extracellular pH (pHe) switches TRAIL-induced apoptosis to regulated necrosis (or necroptosis) in human HT29 colon and HepG2 liver cancer cells. Here, we investigated the role of RIPK1 (receptor interacting protein kinase 1), RIPK3 and PARP-1 (poly (ADP-ribose) polymerase-1) in TRAIL-induced necroptosis in vitro and in concanavalin A (Con A)-induced murine hepatitis. Pretreatment of HT29 or HepG2 with pharmacological inhibitors of RIPK1 or PARP-1 (Nec-1 or PJ-34, respectively), or transient transfection with siRNAs against RIPK1 or RIPK3, inhibited both TRAIL-induced necroptosis and PARP-1-dependent intracellular ATP depletion demonstrating that RIPK1 and RIPK3 were involved upstream of PARP-1 activation and ATP depletion. In the mouse model of Con A-induced hepatitis, where death of mouse hepatocytes is dependent on TRAIL and NKT (Natural Killer T) cells, PARP-1 activity was positively correlated with liver injury and hepatitis was prevented both by Nec-1 or PJ-34. These data provide new insights into TRAIL-induced necroptosis with PARP-1 being active effector downstream of RIPK1/RIPK3 initiators and suggest that pharmacological inhibitors of RIPKs and PARP-1 could be new treatment options for immune-mediated hepatitis.
TRAIL; necroptosis; colon cancer; hepatitis; concanavalin A; RIPK1/RIPK3
Mesothelin is expressed in various types of malignant tumour, and we recently reported that expression of mesothelin was related to an unfavourable patient outcome in pancreatic ductal adenocarcinoma. In this study, we examined the clinicopathological significance of the mesothelin expression in gastric cancer, especially in terms of its association with the staining pattern.
Tissue specimens from 110 gastric cancer patients were immunohistochemically examined. The staining proportion and intensity of mesothelin expression in tumour cells were analysed, and the localisation of mesothelin was classified into luminal membrane and/or cytoplasmic expression.
Mesothelin was positive in 49 cases, and the incidence of mesothelin expression was correlated with lymph-node metastasis. Furthermore, luminal membrane staining of mesothelin was identified in 16 cases, and the incidence of luminal membrane expression was also correlated with pT factor, pStage, lymphatic permeation, blood vessel permeation, recurrence, and poor patient outcome. Multivariate analysis showed that luminal membrane expression of mesothelin was an independent predictor of overall patient survival.
We described that the luminal membrane expression of mesothelin was a reliable prognostic factor in gastric cancer, suggesting the functional significance of membrane-localised mesothelin in the aggressive behaviour of gastric cancer cells.
mesothelin; luminal membrane expression; gastric cancer
Necrostatin-1 (Nec-1) is widely used in disease models to examine the contribution of receptor-interacting protein kinase (RIPK) 1 in cell death and inflammation. We studied three Nec-1 analogs: Nec-1, the active inhibitor of RIPK1, Nec-1 inactive (Nec-1i), its inactive variant, and Nec-1 stable (Nec-1s), its more stable variant. We report that Nec-1 is identical to methyl-thiohydantoin-tryptophan, an inhibitor of the potent immunomodulatory enzyme indoleamine 2,3-dioxygenase (IDO). Both Nec-1 and Nec-1i inhibited human IDO, but Nec-1s did not, as predicted by molecular modeling. Therefore, Nec-1s is a more specific RIPK1 inhibitor lacking the IDO-targeting effect. Next, although Nec-1i was ∼100 × less effective than Nec-1 in inhibiting human RIPK1 kinase activity in vitro, it was only 10 times less potent than Nec-1 and Nec-1s in a mouse necroptosis assay and became even equipotent at high concentrations. Along the same line, in vivo, high doses of Nec-1, Nec-1i and Nec-1s prevented tumor necrosis factor (TNF)-induced mortality equally well, excluding the use of Nec-1i as an inactive control. Paradoxically, low doses of Nec-1 or Nec-1i, but not Nec -1s, even sensitized mice to TNF-induced mortality. Importantly, Nec-1s did not exhibit this low dose toxicity, stressing again the preferred use of Nec-1s in vivo. Our findings have important implications for the interpretation of Nec-1-based data in experimental disease models.
necroptosis; RIPK1; IDO; necrostatin; SIRS; sepsis
IA-2 and IA-2β are dense core vesicle (DCV) transmembrane proteins and major autoantigens in type 1 diabetes. The present experiments were initiated to test the hypothesis that the knockout of these genes impairs the secretion of insulin by reducing the number of DCV.
Insulin secretion, content and DCV number were evaluated in islets from single knockout (IA-2 KO, IA-2β KO) and double knockout (DKO) mice by a variety of techniques including electron and two-photon microscopy, membrane capacitance, Ca2+ currents, DCV half-life, lysosome number and size and autophagy.
Islets from single and DKO mice all showed a significant decrease in insulin content, insulin secretion and the number and half-life of DCV (P < 0.05 to 0.001). Exocytosis as evaluated by two-photon microscopy, membrane capacitance and Ca2+ currents support these findings. Electron microscopy of islets from KO mice revealed a marked increase (P < 0.05 to 0.001) in the number and size of lysosomes and enzymatic studies showed an increase in cathepsin D activity (P < 0.01). LC3 protein, an indicator of autophagy, also was increased in islets of KO as compared to WT mice (P < 0.05 to 0.01) suggesting that autophagy might be involved in the deletion of DCV.
We conclude that the decrease in insulin content and secretion, resulting from the deletion of IA-2 and/or IA-2β, is due to a decrease in the number of DCV.
Protein tyrosine phosphates; Pancreatic islet; Insulin; Dense-core vesicle; Diabetes; Autophagy; Gene knockout; Capacitance; Two-photon microscopy; Electron microscopy
Anthracycline antibiotics are inducers of an immunogenic form of apoptosis that has immunostimulatory properties because of the release of damage-associated molecular patterns. To study the mechanisms used by the innate immune system to sense this immunogenic form of cell death, we established an in vivo model of cell death induced by intraperitoneal injection of doxorubicin, a prototype of anthracyclines. The acute sterile inflammation in this model is characterized by rapid influx of neutrophils and increased levels of IL-6 and monocyte chemotactic protein-1. We demonstrate that acute inflammation induced by doxorubicin is associated with apoptosis of monocytes/macrophages and that it is specific for doxorubicin, an immunogenic chemotherapeutic. Further, the inflammatory response is significantly reduced in mice deficient in myeloid differentiation primary response gene 88 (MyD88), TLR-2 or TLR-9. Importantly, a TLR-9 antagonist reduces the recruitment of neutrophils induced by doxorubicin. By contrast, the acute inflammatory response is not affected in TRIFLps2 mutant mice and in TLR-3, TLR-4 and caspase-1 knockout mice, which shows that the inflammasome does not have a major role in doxorubicin-induced acute inflammation. Our findings provide important new insights into how the innate immune system senses immunogenic apoptotic cells and clearly demonstrate that the TLR-2/TLR-9-MyD88 signaling pathways have a central role in initiating the acute inflammatory response to this immunogenic form of apoptosis.
doxorubicin; TLRs; apoptosis; neutrophils; DAMPs; immunogenic cell death
Physiochemical assessment of the parasite-biomaterial interface is essential in the development of new biomaterials. The purpose of this study was to develop a method to evaluate pH at the bacteria-dental cement interface and to demonstrate physiochemical interaction at the interface. The experimental apparatus with a well (4.0 mm in diameter and 2.0 mm deep) was made of polymethyl methacrylate with dental cement or polymethyl methacrylate (control) at the bottom. Three representative dental cements (glass-ionomer, zinc phosphate, and zinc oxide-eugenol cements) were used. Each specimen was immersed in 2 mM potassium phosphate buffer for 10 min, 24 hrs, 1 wk, or 4 wks. The well was packed with Streptococcus mutans NCTC 10449, and a miniature pH electrode was placed at the interface between bacterial cells and dental cement. The pH was monitored after the addition of 1% glucose, and the fluoride contained in the cells was quantified. Glass-ionomer cement inhibited the bacteria-induced pH fall significantly compared with polymethyl methacrylate (control) at the interface (10 min, 5.16 ± 0.19 vs. 4.50 ± 0.07; 24 hrs, 5.20 ± 0.07 vs. 4.59 ± 0.11; 1 wk, 5.34 ± 0.14 vs. 4.57 ± 0.11; and 4 wks, 4.95 ± 0.27 vs. 4.40 ± 0.14), probably due to the fluoride released from the cement. This method could be useful for the assessment of pH at the parasite-biomaterial interface.
pH; ISFET pH electrode; Streptococcus; glass-ionomer cement; fluoride; interface
Dental caries is initiated by demineralization of the tooth surface through acid production from sugar by plaque biofilm. Fluoride and xylitol have been used worldwide as caries-preventive reagents, based on in vitro-proven inhibitory mechanisms on bacterial acid production. We attempted to confirm the inhibitory mechanisms of fluoride and xylitol in vivo by performing metabolome analysis on the central carbon metabolism in supragingival plaque using the combination of capillary electrophoresis and a time-of-flight mass spectrometer. Fluoride (225 and 900 ppm F−) inhibited lactate production from 10% glucose by 34% and 46%, respectively, along with the increase in 3-phosphoglycerate and the decrease in phosphoenolpyruvate in the EMP pathway in supragingival plaque. These results confirmed that fluoride inhibited bacterial enolase in the EMP pathway and subsequently repressed acid production in vivo. In contrast, 10% xylitol had no effect on acid production and the metabolome profile in supragingival plaque, although xylitol 5-phosphate was produced. These results suggest that xylitol is not an inhibitor of plaque acid production but rather a non-fermentative sugar alcohol. Metabolome analyses of plaque biofilm can be applied for monitoring the efficacy of dietary components and medicines for plaque biofilm, leading to the development of effective plaque control.
metabolome analysis; supragingival plaque; sugar metabolism; fluoride; xylitol; dental caries
Schizophrenia is a serious and chronic mental disorder, in which both genetic and environmental factors have a role in the development of the disease. Neuregulin-1 (NRG1) is one of the most established genetic risk factors for schizophrenia, and disruption of NRG1 signaling has been reported in this disorder. We reported previously that NRG1/ErbB4 signaling is inhibited by receptor phosphotyrosine phosphatase-β/ζ (RPTP β/ζ) and that the gene encoding RPTPβ/ζ (PTPRZ1) is genetically associated with schizophrenia. In this study, we examined the expression of RPTPβ/ζ in the brains of patients with schizophrenia and observed increased expression of this gene. We developed mice overexpressing RPTPβ/ζ (PTPRZ1-transgenic mice), which showed reduced NRG1 signaling, and molecular and cellular changes implicated in the pathogenesis of schizophrenia, including altered glutamatergic, GABAergic and dopaminergic activity, as well as delayed oligodendrocyte development. Behavioral analyses also demonstrated schizophrenia-like changes in the PTPRZ1-transgenic mice, including reduced sensory motor gating, hyperactivity and working memory deficits. Our results indicate that enhanced RPTPβ/ζ signaling can contribute to schizophrenia phenotypes, and support both construct and face validity for PTPRZ1-transgenic mice as a model for multiple schizophrenia phenotypes. Furthermore, our results implicate RPTPβ/ζ as a therapeutic target in schizophrenia.
animal model; dopamine; GABA; glutamate; neuregulin; schizophrenia
Chondrocytes exhibit specific responses to BMPs and TGF-βs. The bioactivity of these growth factors is regulated by numerous mediators. In our previous study, Smad1 was found to interact with the cytoplasmic domain of the hyaluronan receptor CD44. The purpose of this study was to determine the ability of hyaluronan in the pericellular matrix to modulate the chondrocyte responses to BMP-7 or TGF-β1.
Nuclear translocation of Smad1, Smad2 and Smad4 was studied in bovine articular chondrocytes in response to BMP-7 and TGF-β1. The effects of matrix disruption by hyaluronidase treatment and the initiation of matrix repair by the addition of hyaluronan on the nuclear translocation of Smad proteins, Smad1 phosphorylation and luciferase expression by a CD44 reporter construct in response to BMP-7 were also studied.
The disruption of the hyaluronan-dependent pericellular matrix of chondrocytes resulted in diminished nuclear translocation of endogenous Smad1 and Smad4 in response to BMP-7; however, the nuclear translocation of Smad2 and Smad4 in these matrix-depleted chondrocytes in response to TGF-β1 was not diminished. Incubation of the matrix-depleted chondrocytes with exogenous hyaluronan restored Smad1 and Smad4 nuclear translocation and increased pCD44(499)-Luc luciferase expression in response to BMP-7. Both exogenous hyaluronan and matrix re-growth enhanced by HAS2 transfection restored Smad1 phosphorylation.
Disruption of hyaluronan-CD44 interactions has little effect on the TGF-β responses; however, re-establishing CD44-hyaluronan ligation promotes a robust cellular response to BMP-7 by articular chondrocytes. Thus, changes in cell-hyaluronan interactions may serve as a mechanism to modulate cellular responsiveness to BMP-7.
The heart rate (HR) responses after performance of the squatting and standing manoeuvre are thought to be a useful tool to assess autonomic neuropathy in diabetics. Our aim was to develop new simple squatting test indices and to analyse their applicability to the assessment of baroreflex sensitivity (BRS) in patients with diabetes.
Twenty healthy volunteers (mean age 23.2 ± 3.8 years) and 51 patients with diabetes (mean age 55.9 ± 10.6 years) were enrolled in study 1 and study 2, respectively. Each subject stood for 3 min (basal period), then squatted down for 1 min (Sq) and stood up again for 1 min (St). In study 1, the squatting test was performed before and after pharmacological autonomic blockade. In study 2, we measured HR in each period and calculated the difference between basal HR and HRSq (ΔHRSq) and between HRSt and HRSq (ΔHRSt). BRS was also measured using the phenylephrine method in diabetic patients.
In healthy individuals during autonomic blockade, HR changes were mainly controlled by the vagal tone during squatting and by the sympathetic tone during standing. In diabetic patients, ΔHRSq and ΔHRSt positively correlated (r = 0.86, P < 0.0001) and both ΔHRSq and ΔHRSt significantly correlated with BRS (r = 0.66, P < 0.0001 and r = 0.61, P < 0.0001, respectively).
The new squatting test indices provide useful information for assessing autonomic neuropathy and for identifying diabetic patients at high risk of cardiovascular events.
autonomic neuropathy; baroreflex sensitivity; diabetes; squatting test
N-acetylglucosaminyltransferase V (GnT-V) is an enzyme that catalyses β1–6 branching of N-acetylglucosamine on asparagine-linked oligosaccharides of cell proteins. The present study aimed to investigate GnT-V expression and its prognostic significance in endometrial cancer. N-acetylglucosaminyltransferase V expression was studied by immunohistochemistry in 74 surgically resected endometrial cancers, and the staining intensity was evaluated. High GnT-V expression in tumour cells was found in 43 (58.1%) of the 74 cases, and was positively correlated with advanced patient age, histological grade, and lymph vascular space involvement. Patients with high GnT-V expression had significantly impaired overall survival and progression-free survival (PFS) (P=0.0041 and P=0.0023, respectively) compared to patients with low expression of GnT-V. On multivariate analysis, GnT-V expression was an independent prognostic factor for PFS (P=0.0364). β1–6 branching of asparagine-linked oligosaccharides was also detected in GnT-V-positive endometrial cancer cells by leukoagglutinating phytohaemagglutinin (L4-PHA) staining, and the molecular size of the major glycoproteins recognised by L4-PHA was approximately 60–200 kDa by lectin blot analysis. These results suggested that high GnT-V expression was correlated with an unfavourable clinical outcome, and that GnT-V is involved in the malignant potential of endometrial cancer by increasing the synthesis of β1–6 branching of asparagine-linked oligosaccharides.
N-acetylglucosaminyltransferase V; endometrial cancer; prognostic factor; progression-free survival (PFS)
Indoleamine 2,3-dioxygenase (IDO) is a tryptophan-catabolising enzyme inducing immune tolerance. The present study aimed to investigate IDO expression and its prognostic significance in endometrial cancer. Indoleamine 2,3-dioxygenase expression in endometrial cancer tissues (n=80) was immunohistochemically scored as four groups (IDO−, 1+, 2+, and 3+). The high IDO expression (IDO2+ or 3+) in tumour cells was found in 37 (46.3%) of the 80 cases, and was positively correlated with surgical stage, myometrial invasion, lymph-vascular space involvement, and lymph node metastasis, but not with the histological grade. Patients with high IDO expression had significantly impaired overall survival and progression-free survival (PFS) (P=0.002 and P=0.001, respectively) compared to patients with no or weak expression of IDO (IDO− or 1+). The 5-year PFS for IDO−/1+, 2+, and 3+ were 97.7, 72.9, and 36.4%, respectively. Even in patients with early-stage disease (International Federation of Gynecology and Obstetrics I/II, n=64), the PFS for IDO2+/3+ was significantly poor (P=0.001) compared to that for IDO−/1+. On multivariate analysis, IDO expression was an independent prognostic factor for PFS (P=0.020). These results indicated that the high IDO expression was involved in the progression of endometrial cancer and correlated with the impaired clinical outcome, suggesting that IDO is a novel and reliable prognostic indicator for endometrial cancer.
indoleamine 2,3-dioxygenase (IDO); endometrial cancer; prognostic factor; progression-free survival (PFS)
heart rate variability; autonomic nervous system; dysorexia nervosa; anorexia nervosa; bulimia nervosa
A 62-year-old man visited our clinic for dental implantation under intravenous sedation. He demonstrated increased psychomotor activity and incomprehensible verbal contact during intravenous sedation. Although delirium caused by midazolam or propofol in different patients has been reported, the present case represents a delirium that developed from both drugs in the same patient, possibly because of the patient's smaller tolerance to midazolam and propofol.
Delirium; Midazolam; Propofol; Dental treatment
OBJECTIVE—To investigate the clinical value of a new non-invasive method for assessing baroreflex sensitivity using downward tilting.
PATIENTS—34 patients with diabetes mellitus, mean (SD) age, 53.6 (11.8) years.
DESIGN—Arterial blood pressure and ECG were recorded simultaneously while the patients were on a tilt table. After 20 minutes at a 70° upright tilt, the patients were returned to the supine position at a speed of 3.2°/s (downward tilting baroreflex sensitivity test, DT-BRS). A beat to beat systolic blood pressure increase associated with a corresponding lengthening of the RR interval was noted during downward tilting. Baroreflex sensitivity was also assessed using the conventional method of an intravenous injection of phenylephrine (Phe-BRS). Heart rate variability was analysed during rest and tilting.
RESULTS—The slope of the regression line for systolic blood pressure v RR interval during downward tilting was highly correlated with Phe-BRS (r = 0.83, p < 0.0001). Both DT-BRS and Phe-BRS were correlated with the high frequency (HF) component of resting heart rate variability (p < 0.005) and with the ratio of the low frequency to the high frequency component (LF/HF) during upright tilting (p < 0.005). DT-BRS and Phe-BRS were also correlated with the difference between rest and tilting values of HF and LF/HF (p < 0.005).
CONCLUSIONS—DT-BRS provides a physiological, non-invasive method for determining baroreflex sensitivity and may be a useful index of reflex cardiac vagal and sympathetic function in patients with diabetes mellitus.
Keywords: baroreflex sensitivity; downward tilting; heart rate variability; diabetes
The use of caged-Ca2+ compounds to stimulate Ca(2+)-dependent exocytosis has substantially increased our understanding of this complex process. By this approach, the existence of multiple kinetic components of exocytosis has been established. These components may correspond to a series of sequential steps that lead to a single fusion-ready state (sequential mechanism) or, alternatively, to heterogeneity in secretory vesicles or in fusion-ready states (parallel mechanism). It is suggested that both of these mechanisms can underlie exocytosis of a single type of vesicle (mixed sequential-parallel mechanism). Studies with caged-Ca2+ compounds have also indicated that the Ca2+ requirement for exocytosis is substantially greater than that suggested by conventional methodologies. This discrepancy is mainly attributable to the underestimation, by imaging studies with high-affinity Ca2+ indicators (due to dye saturation), of the local increases in cytosolic Ca2+ concentration that trigger the exocytosis of individual vesicles. The effects of local saturation of such indicators are explored by means of a simple theory.