Protective Effects of Bromelain against Cadmium-Induced Pulmonary Intoxication in Rats: A Histopathologic and Cytologic Study.

Bromelain is the active substance of pineapple with a variety of therapeutic properties. In this study, the possible protective effects of bromelain were assessed against cadmium acute intratracheal exposure and its bronchopulmonary cytologic and histopathologic consequences. For this purpose, the following treatments were performed on 11 groups of Wistar rats: group 1 was negative control; groups2 and 3 received Cadmium Chloride (CdCl2) 400 µg/rat intratracheally and sampled after 5 and 10 days, respectively; groups4 and 5received bromelain 20 mg/kg orally (PO) from 14 days before until 5 and 10 days after CdCl2 instillation, respectively; groups6 and 7received bromelain 40 mg/kg from 14 days before until 5 and 10 days after CdCl2 instillation, respectively; group 8received bromelain 40 mg/kg for 24 days; groups9 and 10: celecoxib 25 mg/kg PO from 1day before until 5 and 10 days after CdCl2 instillation, respectively; group 11 received celecoxib for 11 days. Cytologic evaluation of bronchoalveolar lavage fluid revealed that intratracheal cadmium administration resulted in a significant rise in total cell count, epithelial cells, neutrophils, and eosinophils, 5- and 10-days post-exposure. Treatment with bromelain either in low or high doses in cadmium-exposed rats resulted in a significant reduction of neutrophil count. Bromelain treatment could not completely prevent or recover interstitial pneumonia and fibrinous bronchopneumonia in cadmium exposed rats. However, administration of low doses resulted in a significant decrease of semi quantitative histopathologic scores, including pneumonia and cellular infiltration indices. In conclusion, bromelain may help to improve the cytological and histopathological complications following cadmium intoxication in the lungs.

Association between Autism Spectrum Disorder and Caregiver-Reported Dental Caries in Children.

INTRODUCTION: There is evidence that dental caries is both increased and decreased in children with autism spectrum disorder (ASD). OBJECTIVES: This study examined the association between ASD and the probability of a child having caregiver-reported dental caries based on a nationally representative sample. We hypothesized that when compared with children without ASD, children with ASD would have greater odds of dental caries. METHODS: We performed a cross-sectional analysis of the 2016 National Survey of Children's Health. Caregivers reported whether a health provider informed them that their children had ASD and "decayed teeth or cavities" during the past 12 mo. We used logistic regression controlling for child characteristics (age, sex, race/ethnicity, insurance, preventive dental use) and family characteristics (education and federal poverty level). RESULTS: Among the 45,155 children in our sample, 1,228 (2.5%) had ASD. The prevalence of caregiver-reported dental caries was 14.7% in children with ASD and 9.5% in children without ASD. The odds of having caregiver-reported child dental caries (adjusted odds ratio = 1.4, 95% CI = 1.2 to 1.7) was greater among children with ASD than children without ASD when controlling for the aforementioned covariates. CONCLUSION: Using a nationally representative sample, we found that children with ASD had significantly greater odds of having caregiver-reported dental caries as compared with children without ASD. Families can be educated on the increased odds of having dental caries in children with ASD. Moreover, this finding highlights a need for oral health services and policies to prevent and treat dental caries, which are tailored to the increasing number of American children with ASD. KNOWLEDGE TRANSFER STATEMENT: The results of this study support the need for policy makers, clinicians, and families to improve oral health services that prevent and treat dental caries in the increasing number of American children with autism spectrum disorder.

Experimental Infection of Turkeys with A Virulent Newcastle Disease Virus Isolated from Broiler Chickens.

Newcastle disease (ND) is a highly contagious infection of many avian species, mainly chickens and turkeys, with a devastating impact on worldwide poultry production. The ND accounts for heavy losses in Iranian poultry flocks. There are some reports regarding the epidemiology of this infection in Iran. This study was performed to investigate the infection of turkeys with a Newcastle disease virus (NDV) isolated from a broiler chicken flock in southwestern Iran during 2013. For the purpose of the study, 70 day-old Wishard bronze poults were allocated into two groups of control (n=25) and infected (n=45). At 32 days of age, each bird in the infected group was inoculated with 0.1 mL (50 μL per eye) of NDV-infected allantoic fluid through an ocular route and received 105 EID50 of viral inoculum. On the other hand, the birds in the control group were inoculated with phosphate buffered saline by the same route. Swab samples were taken from both groups at different time points, namely from 1 to 21 days postinoculation, and verified for NDV infection by using reverse transcription-polymerase chain reaction (RT-PCR). Both groups were also examined serologically by haemagglutination inhibition test. Clinically, the infected turkeys exhibited anorexia, severe depression, sitting on the hock joint, white to greenish (sometimes bloody) diarrhea, neurological disorders, and mild respiratory problems. Out of 45 inoculated birds, 9 (20%) cases died. Based on RT-PCR, virus shedding was observed in the challenged birds 3-8 days postinoculation. The NDV was detected more in tracheal swabs (50%) than in cloacal swabs (12.5%). The infected birds showed a high seroconversion. Therefore, the NDV circulating in Iranian chicken flocks has the potential to cause a serious illness in commercial turkeys. The vaccination of turkeys, as well as biosecurity, should be considered carefully to prevent the ND outbreaks in the future.

Expression and functional characterization of two natural heparin-binding site variants of antithrombin.

Essentials Heparin-binding site (HBS) variants of antithrombin (AT) are associated with thrombosis risk. HSB variants have, in general, normal progressive inhibitory activity but reduced heparin affinity. Thrombosis in HSB carriers has been primarily attributed to the loss of heparin cofactor activity. Results here demonstrate that HSB variants of AT also lack anti-inflammatory signaling functions. SUMMARY: Background Several heparin-binding site (HBS) variants of antithrombin (AT) have been identified that predispose carriers to a higher incidence of thrombosis. Thrombosis in carriers of HBS variants has been primarily attributed to a loss in their heparin-dependent anticoagulant function. Objective The objective of this study was to determine whether HSB mutations affect the anti-inflammatory functions of variants. Methods Two HBS variants of AT (AT-I7N and AT-L99F), which are known to be associated with a higher incidence of thrombosis, were expressed in mammalian cells and purified to homogeneity. These variants were characterized by kinetic assays followed by analysis of their activities in established cellular and/or in vivo inflammatory models. The possible effects of mutations on AT structure were also evaluated by molecular modeling. Results The results indicated that, whereas progressive inhibitory activities of variants were minimally affected, their heparin affinity and inhibitory activity in the presence of heparin were markedly decreased. Unlike wild-type AT, neither AT variant was capable of inhibiting activation of nuclear factor-κB or downregulation of expression of cell adhesion molecules in response to lipopolysaccharide (LPS). Similarly, neither variant elicited barrier protective activity in response to LPS. Structural analysis suggested that the L99F substitution locally destabilizes AT structure. Conclusions It is concluded that the L99F mutation of AT is associated with destabilization of the serpin structure, and that the loss of anti-inflammatory signaling function of the HBS variants may also contribute to enhanced thrombosis in carriers of HBS mutations.

Platform for combined analysis of functional and biomolecular phenotypes of the same cell.

Functional and molecular cell-to-cell variability is pivotal at the cellular, tissue and whole-organism levels. Yet, the ultimate goal of directly correlating the function of the individual cell with its biomolecular profile remains elusive. We present a platform for integrated analysis of functional and transcriptional phenotypes in the same single cells. We investigated changes in the cellular respiration and gene expression diversity resulting from adaptation to repeated episodes of acute hypoxia in a premalignant progression model. We find differential, progression stage-specific alterations in phenotypic heterogeneity and identify cells with aberrant phenotypes. To our knowledge, this study is the first demonstration of an integrated approach to elucidate how heterogeneity at the transcriptional level manifests in the physiologic profile of individual cells in the context of disease progression.

Can three-dimensional high-resolution anorectal manometry detect anal sphincter defects in patients with faecal incontinence?

AIM: Endoanal ultrasound (EAUS) is the gold standard for detecting anal sphincter defects in patients with faecal incontinence (FI), while anorectal manometry evaluates sphincter function. Three-dimensional high-resolution anorectal manometry (3D HRAM) is a newer modality with the potential to assess both sphincter function and anatomy. The purpose of the present study was to compare 3D HRAM with 3D EAUS for the detection of anal sphincter defects in patients with FI. METHOD: A linkage analysis was performed between the 3D HRAM and 3D EAUS databases of a tertiary referral centre to identify patients with FI who underwent both 3D EAUS and 3D HRAM. With 3D HRAM, a defect was defined as any pressure measurement below 25 mmHg at rest with at least 18° of continuous expansion. The 3D HRAM findings were compared with those of 3D EAUS. RESULTS: The study cohort included 39 patients with a mean age of 64.7 ± 15.2 years (SD); and 31 (79%) were female. Eight (21%) patients had an anal sphincter defect on EAUS with a median size of 93° (range 40°-136°). Fourteen (36%) had a defect shown by 3D HRAM with a median size of 144° (36°-180°). The sensitivity, specificity and positive and negative predictive values of 3D HRAM in detecting a sphincter defect were 75%, 74%, 43% and 92%, respectively. CONCLUSION: With a negative predictive value of 92%, 3D HRAM may be a useful screening method for ruling out a sphincter defect in patients with FI, thereby avoiding both EAUS and manometry in selected patients.

Do Jackhammer contractions lead to achalasia? A longitudinal study.

BACKGROUND: Jackhammer esophagus (JE) is a rare esophageal motility disorder defined in the Chicago Classification of Esophageal Motility by presence of excessively high distal contractile integral (DCI) on high-resolution manometry (HRM), with unknown natural manometric course. We examined the development of achalasia over time in patients with JE. METHODS: Through a retrospective longitudinal design, patients with Jackhammer contractions who had more than one HRM between 2005 and 2015 were identified. Any change in manometric finding was assessed for the presence of achalasia. Demographic and manometric risk factors for this progression were then sought in univariate analysis. KEY RESULTS: Of 3363 HRM studies, 229 subjects had multiple manometries, accounting for 528 studies. Twelve subjects met our inclusion criteria for JE on any of the multiple tests, represented by 27 studies for a total of 347 patient-months of manometric follow-up. Subjects with JE whose manometry included impedance demonstrated consistent esophageal bolus clearance. Of 12 subjects with Jackhammer contractions, three subjects progressed to type III achalasia, over a mean of 24 months (range: 19-31 months). At the time of diagnosis with JE, impaired esophago-gastric junction relaxation was seen in all three subjects and was the only risk factor that could predict progression to achalasia (P<.01). CONCLUSIONS & INFERENCES: In this unique study of the natural course of JE, we have shown that JE can progress to achalasia. Manometric findings at the time of JE diagnosis might predict this progression.

Inorganic polyphosphate promotes cyclin D1 synthesis through activation of mTOR/Wnt/ß-catenin signaling in endothelial cells.

Essentials Polyphosphate (polyP) activates mTOR but its role in Wnt/ß-catenin signaling is not known. PolyP-mediated cyclin D1 expression (ß-catenin target gene) was monitored in endothelial cells. PolyP and boiled platelet-releasates induced the expression of cyclin D1 by similar mechanisms. PolyP establishes crosstalk between mTOR and Wnt/ß-catenin signaling in endothelial cells. SUMMARY: Background Inorganic polyphosphate (polyP) elicits intracellular signaling responses in endothelial cells through activation of mTOR complexes 1 and 2. Glycogen synthase kinase 3 (GSK-3) is known to be a negative regulator of mTOR and Wnt/ß-catenin signaling pathways. Objective The objective of this study was to investigate the effect of polyP on the expression, degradation and subcellular localization of the Wnt/ß-catenin target gene, cyclin D1, in endothelial cells. Methods Regulation of cyclin D1 expression, phosphorylation and subcellular localization by polyP or platelet releasates was monitored in the absence and presence of pharmacological inhibitors and/or siRNA for specific molecules of the upstream mTOR/Wnt/ß-catenin signaling network by established methods. Results Both synthetic polyP and boiled-platelet releasates induced the phosphorylation-dependent inactivation of GSK-3, thereby increasing the expression and nuclear localization, but inhibiting the degradation of cyclin D1. Inhibitors of mTORC1 (PI3K, AKT, PLC, PKC), rapamycin and siRNA for raptor (mTORC1-specific component) and ß-catenin, all inhibited polyP-mediated regulation of cyclin D1 expression, phosphorylation and subcellular localization in endothelial cells. The signaling effect of polyP was effectively inhibited by the recombinant extracellular domain of the receptor for advanced glycation end products (RAGE) and/or by the RAGE siRNA. Specific pharmacological inhibitors and siRNA knockdown of ERK1/2 and NF-κB pathways indicated that polyP-mediated cyclin D1 expression and nuclear localization are IKKɑ and ERK1/2 dependent, whereas its inhibitory effect on phosphorylation-dependent degradation of cyclin D1 is IKKß-dependent. Conclusion We conclude that a RAGE-dependent polyP-mediated crosstalk between mTOR and the GSK-3/Wnt/ß-catenin signaling network can modulate important physiological processes in endothelial cells.

Polymorphism of P53-Ets/AP1 transactivation region of MDM2 oncogene and its immunohistochemical analysis in canine tumours.

Mouse Double Minute-2 (MDM2) is an ubiquitin ligase which is overexpressed or its promoter polymorphism has been reported in different tumours. The objective of this study was to examine the MDM2 protein expression and its promoter polymorphism in some canine tumours. Twenty specimens were collected from 20 dogs with 15 mammary gland carcinomas, 3 lymphomas, 1 transmissible venereal tumour and 1 trichoblastoma. Samples were analysed immunohistochemically using human antibody against MDM2 protein. PCR and DNA sequencing were carried out to identify MDM2 promoter polymorphism. MDM2 gene was expressed in 13 of 20 samples including 11 mammary carcinomas, 1 lymphoma and 1 trichoblastoma. We found 94% homology between canine and human sequences. Four mutations including G169C, A177G, G291T and A177G were identified in different types of breast carcinomas. An extra p53 response element was found in a mixed mammary carcinoma.

Inorganic polyphosphate elicits pro-inflammatory responses through activation of the mammalian target of rapamycin complexes 1 and 2 in vascular endothelial cells.

BACKGROUND: Inorganic polyphosphate (polyP) elicits pro-inflammatory signaling responses in endothelial cells through interaction with two receptors, RAGE and P2Y1 . It is known that polyP activates mTOR signaling in breast cancer cells. OBJECTIVES: The objective of this study is to understand the mechanism of the polyP-mediated signaling pathway in endothelial cells and to determine whether polyP exerts its pro-inflammatory effect through activation of mTOR. METHODS: mTOR activation by polyP or platelet releasates in cellular and animal models was monitored in the absence and presence of pharmacological inhibitors and/or siRNA knockdown of specific signaling molecules. RESULTS: PolyP effectively induced phosphorylation of mTOR complex 1 (mTORC1) substrate, p70S6K, in endothelial cells by an AKT-dependent but ERK-independent mechanism. The siRNA knockdown of both RAGE and P2Y1 or specific inhibitors of the PI3K/PLC/PKC/Ca(2+) signaling axis inhibited polyP-mediated p70S6K phosphorylation. Moreover, either rapamycin or siRNA knockdown of raptor (mTORC1-specific component) abrogated polyP-mediated phosphorylation of p70S6K. By contrast, the siRNA knockdown of rictor (mTOR complex 2-specific component) but not raptor eliminated the barrier-disruptive effect of polyP. Specific NF-κB inhibitors abrogated polyP-mediated phosphorylation of p70S6K and rapamycin suppressed polyP-induced activation of NF-κB. Finally, specific inhibitors of the mTOR signaling network eliminated polyP-mediated vascular leakage and leukocyte recruitment in animal models. CONCLUSIONS: PolyP, through interaction with RAGE and P2Y1 , activates both the mTORC1 and mTORC2 signaling network. Both the pro-inflammatory and mTOR signaling functions of polyP are linked.

The incidence rate of colectomy for medically refractory ulcerative colitis has declined in parallel with increasing anti-TNF use: a time-trend study.

BACKGROUND: Medical therapy is standard treatment for ulcerative colitis with colectomy reserved for medically refractory disease or malignancy. The introductions of ciclosporin in 1994 and anti-TNF therapy in 2005 have extended medical management options. AIM: To determine whether the colectomy incidence rate for medically refractory ulcerative colitis has changed since the introduction of anti-TNF therapy. METHODS: Adult patients with a diagnosis of ulcerative colitis and who subsequently underwent an urgent or elective colectomy for medically refractory disease in Edmonton, Canada between 1 January 1998 and 31 December 2011 were identified. Log-linear regression was used to estimate the annual percent change in the total colectomy incidence rate (urgent and elective combined) and the urgent and elective incidence rates individually, before and after 2005, the year infliximab was approved for use in ulcerative colitis. Temporal trends of drug utilisation in this study population were also described. RESULTS: During 1998-2011, 481 patients with ulcerative colitis underwent a colectomy for medically refractory disease. There was negligible change in the total colectomy incidence rate from 1998 to 2005, with an annual percent change of 4.4% (95% confidence interval (CI): -1.12% to 10.16%). From 2005-2011, following the approval and increasing use of anti-TNF therapy, the total colectomy incidence rate decreased by 16.1% (95% CI: -21.32% to -10.54%) every year to 0.9 per 100 ulcerative colitis patients in 2011. CONCLUSION: The total incidence rate of colectomy for medically refractory ulcerative colitis has declined substantially since 2005, paralleling the increased use of anti-TNF therapy in this patient population.

Engineering D-helix of antithrombin in alpha-1-proteinase inhibitor confers antiinflammatory properties on the chimeric serpin.

Antithrombin (AT) is a heparin-binding serpin in plasma which regulates the proteolytic activity of procoagulant proteases of the clotting cascade. In addition to being an anticoagulant, AT also exhibits antiinflammatory activities when it binds to cell surface heparan sulfate proteoglycans (HSPGs) on the endothelium via its basic residues of D-helix to elicit intracellular signalling responses. By contrast to AT, α1-proteinase inhibitor (α1-PI) is a non-heparin-binding serpin that exhibits very slow reactivity with coagulation proteases and possesses no HSPG-dependent antiinflammatory properties. To determine whether the antiinflammatory signaling specificity of AT can be transferred to α1-PI, we replaced the D-helix of human α1-PI with the corresponding sequence of human AT and expressed the chimeric serpin α1-PI/D-helix) in a bacterial expression system. High molecular weight heparin bound to α1-PI/D-helix and accelerated the inhibition of thrombin by the serpin mutant by a template mechanism reminiscent of the cofactor effect of heparin on inhibition of thrombin by AT. Like AT, α1-PI/D-helix exhibited antiinflammatory properties in both cellular and animal models. Thus, α1-PI/D-helix inhibited the barrier-disruptive effect of proinflammatory cytokines and inhibited the activation of nuclear factor-κB transcription factor in lipopolysaccharide-stimulated endothelial cells by a concentration-dependent manner. Furthermore, the chimeric serpin reduced lipopolysaccharide-mediated lethality, elicited a vascular protective effect and inhibited infiltration of activated leukocytes to the peritoneal cavity of mice in an HMGB1-mediated inflammatory model. These results suggest that grafting the D-helix of AT to α1-PI confers antiinflammatory properties on the serpin and that the chimeric serpin may have therapeutic utility for treating inflammatory disorders.

Evaluation of immunopathologic effects of aqueous extract of Echinacea purpurea in mice after experimental challenge with Pasteurella multocida serotype A.

In order to assess the immunopathological effects of aqueous Echinacea purpurea extract (EPE) on mice experimentally challenged with Pasteurella multocida serotype A, forty female BALB/c mice were randomly divided into four groups. The groups included a control group (received sterile distilled water 2 times/week for 2 weeks, intraperitoneally and then 100 µl sterile saline intranasally), a PMA group (received sterile distilled water as the control group and after 2 weeks, 5.6 × 10(3) CFU/ml of P. multocida serotype A, intranasally), an EPE+PMA group (received E. purpurea extract intraperitoneally 2 times/week for 2 weeks and then challenged as the PMA group) and an EPE group (received E. purpurea extract as EPE+PMA group and then 100 µl sterile saline intranasally). After 24 and 48 h post challenge, half of the animals in each group were sacrificed and analyzed for bacterial counts in their lungs and livers, TNFα serum levels and histapathological changes. The results showed significant differences in lung bacterial counts between PMA and EPE+PMA groups. TNFα serum level was significantly higher in the PMA group. Histopathological examination revealed infiltration of neutrophils in alveolar septa and hyperemia in the PMA group. In addition, the criteria of bronchopneumonia were partially recovered in the EPE+PMA compared to the PMA group. According to the results, it seems that E. purpurea extract has an immunomodulatory effect and can be used to prevent or control of pneumonia caused by Pasteurella.

Klossiella equi in a donkey--a first case report from Iran.

Klossiella equi is the only known and rarely reported coccidian parasite of the renal paranchyma of equids. An aged male donkey (Equus asinus asinus) was submitted to necropsy department of veterinary hospital. In histopathological study of renal sections different developmental stages of parasite were observed. These stages were as follow: Trophozoites, microgametes, macrogametes, sporont, budding sporont, sporoblasts, free sporoblasts, mature sporoblast and sporocyst. Parasitic infection with K. equi was encountered in the donkey. According to literature review this is the first report of donkey klossiellosis in Iran.

Designing a minimum data set for breast cancer: a starting point for breast cancer registration in iran.

BACKGROUND: Due to the high incidence of deaths from breast cancer, high cost of treatment and limited resources, the need to formulate and implement effective programs in reducing the burden of disease is obvious. Care, control and creation of cancer information system having an infrastructure from collection of minimum data sets (MDS) are the top priorities of research in Iran's Ministry of Health. METHODS: This is an applied descriptive research with comparative approach implemented in 2010. MDS for breast cancer on selected countries were searched and reviewed and proposed model based on the country's need was designed. Research data were implemented in 2 stages; assessment of MDS on selected countries and the validation of the proposed model through several meetings that has been carried out by the Undersecretary for Research and Technology and several oncologists and pathologists. RESULTS: The MDS is composed of 11 parameters in the form of fields in closed structured arrangements with consideration to coding responses. These parameters include: hospital data, demography, referral, physical examination and investigation, diagnostic information, pathology, treatment, palliative care, completion of primary treatment, clinical trials and follow-up. This form is available for use in the cancer registry database. CONCLUSION: MDS provides an opportunity to strengthen communication between performed researches and research results for the improvement of programs, policies and strategies and provides positive effect on equality in the health system. Although the stages of creating the MDS for breast cancer has been successful, but many challenges has been met until its completion.

Antithrombin is protective against myocardial ischemia and reperfusion injury.

BACKGROUND: Antithrombin (AT) is a plasma serpin inhibitor that regulates the proteolytic activity of procoagulant proteases of the clotting cascade. In addition to its anticoagulant activity, AT also possesses potent anti-inflammatory properties. OBJECTIVES: The objective of this study was to investigate the anti-inflammatory activity of wild-type AT (AT-WT) and a reactive centre loop mutant of AT (AT-RCL) which is not capable of inhibiting thrombin. METHODS: The cardioprotective activities of AT-WT and AT-RCL were monitored in a mouse model of ischemia/reperfusion (I/R) injury in which the left anterior descending coronary artery was occluded and then released. RESULTS: We demonstrate that AT markedly reduces myocardial infarct size by a mechanism that is independent of its anticoagulant activity. Thus, AT-RCL attenuated myocardial infarct size to the same extent as AT-WT in this acute injury model. Further studies revealed that AT binds to vascular heparan sulfate proteoglycans via its heparin-binding domain to exert its protective activity as evidenced by the therapeutic AT-binding pentasaccharide (fondaparinux) abrogating the cardioprotective activity of AT and a heparin-site mutant of AT exhibiting no cardioprotective property. We further demonstrate that AT up-regulates the production of prostacyclin in myocardial tissues and inhibits expression of pro-inflammatory cytokines tumor necrosis factor (TNF)-α and interleukin (IL)-6 in vivo by attenuating ischemia/reperfusion-induced JNK and NF-κB signaling pathways. CONCLUSIONS: The present results suggest that both AT and the non-anticoagulant AT-RCL, through their anti-inflammatory signaling effects, elicit potent cardioprotective responses. Thus, AT may have therapeutic potential for treating cardiac I/R injury.

Expression and functional characterisation of natural R147W and K150del variants of protein C in the Chinese population.

Protein C is a vitamin K-dependent serine protease zymogen in plasma which upon activation to activated protein C (APC) by thrombin down-regulates the clotting cascade by limited proteolysis of the procoagulant cofactors Va and VIIIa. In addition to its anticoagulant activity, APC also exhibits potent cytoprotective and anti-inflammatory activities. While the anticoagulant activity of APC is enhanced by the cofactor function of protein S on membrane phospholipids, the cytoprotective intracellular signalling activity of APC requires complex formation with endothelial protein C receptor (EPCR) expressed on the vascular endothelium. Two natural variants of APC [Arg-147 to Trp substitution (R147W) and Lys-150 deletion (K150del)] have been identified in the Chinese population as hotspot mutants occurring with high frequencies of 27.8% and 13.9%, respectively, among 36 protein C-deficient subjects. The affected individuals exhibit variable thrombotic tendencies. To understand the underlying cause of the thrombotic phenotype in these patients, we expressed these two protein C variants in mammalian cells and characterised their anticoagulant and anti-inflammatory properties using established in vitro and cellular assays. Our results suggest that both R147W and K150del variants have normal amidolytic and proteolytic activities in the absence of cofactors. However, the R147W mutant exhibits ~3 times lower affinity for binding to EPCR and the K150del variant has ~2-3-fold impaired anticoagulant activity in the presence of protein S. These results provide some insight into the possible pathogenic mechanism of protein C deficiency in Chinese patients carrying these mutations.

Activated protein C modulates cardiac metabolism and augments autophagy in the ischemic heart.

BACKGROUND: Modulation of energy substrate metabolism may constitute a novel therapeutic intervention against ischemia/reperfusion (I/R) injury. AMP-activated protein kinase (AMPK) has emerged as a key regulator of favorable metabolic signaling pathways in response to myocardial ischemia. Recently, we demonstrated that activated protein C (APC) is cardioprotective against ischemia/reperfusion (I/R) injury by augmenting AMPK signaling. OBJECTIVES: The objective of this study was to determine whether the APC modulation of substrate metabolism contributes to its cardioprotective effect against I/R injury. METHODS: An ex vivo working mouse heart perfusion system was used to characterize the effect of wild-type APC and its signaling-proficient mutant, APC-2Cys (which has dramatically reduced anticoagulant activity), on glucose transport in the ischemic heart. RESULTS: Both APC and APC-2Cys (0.2 µg g(-1)) augment the ischemic stress-induced translocation of the glucose transporter (GLUT4) to the myocardial cell membrane, leading to increased glucose uptake and glucose oxidation in the ischemic heart (P < 0.05 vs. vehicle). Both APC derivatives increased the autophagic flux in the heart following I/R. The activity of APC-2Cys in modulating these metabolic pathways was significantly higher than APC during I/R (P < 0.05). Intriguingly, APC-2Cys, but not wild-type APC, attenuated the I/R-initiated fatty acid oxidation by 80% (P < 0.01 vs. vehicle). CONCLUSIONS: APC exerts a cardioprotective effect against I/R injury by preferentially enhancing the oxidation of glucose over fatty acids as energy substrates in the ischemic heart. Given its significantly higher beneficial metabolic modulatory effect, APC-2Cys may be developed as a potential therapeutic drug for treating ischemic heart disease without risk of bleeding.