Ve Controls Research Articles

Identification of Leukocyte Surface P2X7 as a Biomarker Associated with Alzheimer's Disease.

Alzheimer’s disease (AD) has shown altered immune responses in the periphery. We studied P2X7 (a proinflammatory receptor and a scavenger receptor) and two integrins, CD11b and CD11c, on the surface of circulating leukocytes and analysed their associations with Aβ-PET, brain atrophy, neuropsychological assessments, and cerebrospinal fluid (CSF) biomarkers. Total 287 age-matched, sex-balanced participants were recruited in a discovery cohort and two validation cohorts through the AIBL study and studied using tri-colour flow cytometry. Our results demonstrated reduced expressions of P2X7, CD11b, and CD11c on leukocytes, particularly monocytes, in Aβ +ve cases compared with Aβ −ve controls. P2X7 and integrin downregulation was observed at pre-clinical stage of AD and stayed low throughout disease course. We further constructed a polygenic risk score (PRS) model based on 12 P2RX7 risk alleles to assess the genetic impact on P2X7 function in AIBL and ADNI cohorts. No significant association was identified between the P2RX7 gene and AD, indicating that P2X7 downregulation in AD is likely caused by environmental changes rather than genetic factors. In conclusion, the downregulation of P2X7 and integrins at pre-clinical stage of AD indicates altered pro-inflammatory responses, phagocytic functions, and migrating capabilities of circulating monocytes in early AD pathogenesis. Our study not only improves our understanding of peripheral immune involvement in early stage of AD but also provides more insights into novel biomarker development, diagnosis, and prognosis of AD.

The dichotomy of the systemic and local cytokine responses of mucosal leishmaniasis patients in Sudan: A pilot study.

Sudanese mucosal leishmaniasis (ML) is a rare clinical form of leishmaniasis and characterized by persistent ulcer of the oral and/or the nasal mucous membranes caused by Leishmania donovani. No data is available about the systemic and local immune responses in mucosal leishmaniasis. This study aimed to measure the systemic and the local cytokines responses of Sudanese ML patients compared to cured cutaneous leishmaniasis patients (Leishmanin skin test positive, LST+ve) and unexposed healthy controls (Leishmanin skin test negative, LST-ve). Six parasitological confirmed ML patients, 7 LST+ve, and 6 LST-ve were enrolled. Systemic Th-1 (IFN-γ and TNF-α), Th-2 (IL-10 and IL-13), Treg (TGF-β1), and inflammatory cytokines IL-6 and IL-8 concentration were measured in the supernatant of whole blood samples following stimulation with live L. donovani promastigotes using ELISA. Local intralesion IL-10, IFN-γ, and IL-13 expression was measured using Real Time PCR. A significant high concentrations of IFN-γ, TNFα, IL-10, TGFβ, IL-6, and IL-8 were detected in the supernatant of stimulated whole blood samples of ML patients compared with the LST+ve and LST-ve controls. Using Real Time-PCR and primers for various cytokines, a significant high expression of TH2 cytokines IL-10 and IL-13 mRNA was detected in contrast to a low TH1 cytokine IFN-γ mRNA in the mucosal lesion. There is a clear dichotomy in the cytokine response during Mucosal leishmaniasis. A significantly high TH1, inflammatory and Treg cytokines response is produced systemically, in contrast to a significant high TH2 cytokines response in the mucosal lesion.

THE POTENTIAL CONVENTION OF GARLIC AND BLACK SEED DIFFERENT EXTRACTS AS AN EFFECTIVE TREATMENT OF CRYPTOSPORIDIUM SPP. : AN EXPERIMENTAL STUDY

The study investigated the effect of garlic (Allium sativum) and black seed (Nigella sativa) extractsproducts as treatment of Cryptosporidium infection in experimental mice in comparison toNitazoxanide (NTZ). Forty-eight male Swiss Albino mice divided into 8 experimental conditions;G1-8, the first seven groups received 1.5×105 Cryptosporidium spp. oocysts on 1st infectionday. The first group included mice treated with garlic juice; in G2, G4 & G5, mice were treatedwith black seed oil, alcoholic extract of black seed and alcoholic extract of black ariel parts respectively.In G3 mice were treated with garlic & black seed oil mixture, while G6 mice treatedwere with Nitaxanoide. Besides G7 were infected untreated (+ ve control); and G8 were neitherinfectednor treated (– ve control); From 2nd day post-infection, pooled fecal samples of eachgroup were collected daily to assess each extract efficacy as compared with NTZ and both +veand –ve controls by calculating oocyst excretion pattern. The comparison between number ofoocysts shedding rates among different treatment groups showed that the efficacy of NTZ wasthe highest (76.1%) followed by N. sativa oil (75.8%) followed by mixture of garlic and blackseed oil (75.4%), alcoholic black seed extract (75.1%); alcoholic arial extract (74.7%) and thelowest was garlic juice (73.1%). Although both A. sativum and all N. sativa extract productsshowed a great degree of efficacy in reducing Cryptosporidium spp. oocysts excretions in theexperimental animals compared to positive control, but without significance differences betweenthem and preparation of both garlic, black seed and mixture with NTZ.

Latency of tobacco smoking for head and neck cancer among HPV-positive and HPV-negative individuals.

Human papillomavirus (HPV) infection and tobacco smoking are well-known risk factors for head and neck cancers (HNC). Although an effect modification between oral HPV infection and tobacco smoking may exist, evidence is lacking on how they interact temporally. We investigated the latency and life course effects of tobacco smoking on risk of HNC among HPV-positive (HPV+ve ) and negative (HPV-ve ) individuals. We used data from 631 ever-smoker participants of a hospital-based case-control study conducted in four major hospitals in Montréal, Canada. Cases (n = 320), incident, histologically confirmed, primary squamous cell carcinomas, were frequency-matched to controls (n = 311) by age and sex. Sociodemographic and behavioral factors (e.g., tobacco and alcohol use and sexual history) were collected using a structured interview applying a life grid technique. Oral exfoliated cells were used for HPV DNA detection and genotyping. Latency effects were estimated flexibly using a Bayesian relevant exposure model and further extended with a life course approach. Retrospective smoking trajectories for HPV+ve cases and controls had similar shapes. Exposure to tobacco smoking even 40 years before diagnosis was associated with an increased HNC risk among both HPV+ve and HPV-ve participants. The effect of smoking before the start of sexual activity compared to afterwards was higher among HPV+ve individuals. This pattern of association was less profound among HPV-ve participants. Temporal interactions may exists between oral HPV infection and life course smoking trajectories in relation to HNC risk.

Validity of Coagulation Activation Markers in Antiphospholipid Syndrome: A Systematic Review and Meta-analysis with a Short Data Report.

Prothrombin fragment F1 + 2 (F1 + 2) and thrombin-antithrombin (TAT) have been assessed in antiphospholipid syndrome (APS) but without evaluating a direct relationship with antiphospholipid (aPL) antibody titers. This article aims to investigate a direct relationship between aPL and F1 + 2 and perform a systematic review and meta-analysis of F1 + 2 and TAT in APS. Systematic search was performed using EMBASE and PubMed databases from January 1982 to December 2018 and random effects meta-analyses for continuous outcomes. This is a cross-sectional case-control study; immunoglobulin G/immunoglobulin M (IgG/IgM) anticardiolipin (aCL) anti-β2-glycoprotein-I, antiprothrombin (aPT) antibodies, F1 + 2, and lupus anticoagulants (LA) were measured in 25 thrombotic primary APS (PAPS), 9 nonthrombotic carriers of aPL, and 18 controls. The significant effect size (ES) for F1 + 2 between aPL +ve and aPL -ve systemic lupus erythematosus (SLE) and between aPL +ve SLE and control displayed high heterogeneity. The significant ES for F1 + 2 between aPL -ve SLE and controls displayed no heterogeneity. The ES for TAT between aPL +ve and aPL -ve SLE patients and between aPL -ve SLE and controls was low, without heterogeneity. Mean F1 + 2 was greater in PAPS (p < 0.0001), inversely correlated with IgG aCL, IgM aPT, and LA (p = 0.001, 0.03, and 0.01, respectively), though only IgG aCL negatively predicted F1 + 2 (p = 0.01). IgG aCL inversely predicts F1 + 2. IgG aCL positivity introduces heterogeneity in the F1 + 2 ES, whereas the lack of heterogeneity in the ES for TAT may indicate poor TAT formation in aPL +ve group. Thus, F1 + 2 measurements may be unfounded as already demonstrated for TAT in the 1990s.

In Vitro Clot Dissolving Activity of Carbomer Based Gel Containing Ethanolic Extract of Calotropis Gigantea Leaves

Blood clotting is an important and vital process that takes places in humans, animals and birds. At the same time blood clots sometimes considered an issue when developed in the circulatory system due to failure of hemostasis causes vascular blockage and leads to serious consequences in thrombolytic diseases such as acute myocardial or cerebral infarction which may cause death. Certain substances like Alteplase, anistreplase, streptokinase, urokinase, tissue plasminogen activator, Heparin and Aspirin are used as clot dissolving agents. Plants have also been proved to perform such activity. Calotropis gigantea L. is a traditional medicinal plant with several pharmacological properties belongs to the family Apocyanaceae of Asclepiadaceous was investigated for its efficacy for blood clot dissolving activity under in vitro conditions when incorporated into carbomer based gels. The ethanolic extract of the leaves of C. gigantea was prepare by soxhlation which upon analysis reported to contain alkaloids, glycosides, flavonoids, tannins, terpenoids and saponins as usual components as were reported in earlier investigations. There were 3 gels with ethanolic leaf extract C. gigantea with 1 mg, 0.5 mg and 0.25 mg per gram of 1% Carbopol-940 gel containing methyl paraben, glycerol and water. The two gels containing sterile distilled water and 0.1 mg per gram gel streptokinase enzyme served as –ve and positive controls respectively. The in vitro thrombolytic of clot dissolving activity war performed on goat blood collected from slaughter houses. From the results the percentage clot dissolving activity of gels containing 1 mg, 0.5 mg and 0.25 mg C. gigantea extract per gram were reported as 34%, 27% and 14% respectively considered significant when compared to the activity of standard streptokinase in the gel. The activity of these experimental gels describes the possibility of development of new safe, and non reactive topical anti-clot gel products using herbal or plant components like C. gigantea extracts.&#x0D; Keywords: Calotropis gigantea L, Streptokinase, Thrombolytic activity, Carbopol gel, Biopharmaceutical.

Prevalence of early repolarization pattern and its association with sudden cardiac death and arrhythmia over one-year follow-up in an Egyptian cohort.

Background and objectivesEarly repolarization pattern (ERP) is not uncommon electrocardiography (ECG) finding and could be associated with arrhythmia and sudden cardiac death (SCD). We aimed to prospectively determine the prevalence of ERP and its association with arrhythmia and SCD during one-year follow-up in an outpatient Egyptian cohort.MethodsClinical assessment and ECG were performed to 1850 consecutive individuals presented at the outpatient clinic of Suez Canal University Hospital (SCUH). Then, the ERP group and 100 age and gender-matched ERP −ve controls had undergone echocardiography, 24-h Holter ECG and exercise stress ECG.ResultsERP was found in 124 individuals (6.7%); we excluded 24 patients with structural heart disease. ERP group (No. = 100) were relatively young (80% <50 years-old) and showed male preponderance (60%). ERP frequencies were: inferolateral (50%), antero-lateral (38%), inferior (10%), and global (2%). ERP subjects were leaner than controls (BMI was 25.3 vs. 30 kg/m2, P value < 0.001) and achieved more metabolic equivalents (METS) on stress ECG (10.7 vs. 8.5 METS, P value < 0.01). Only 4% in the ERP group had horizontal/descending ST slope, while 8% had ST elevation ≥ 2 ms. No arrhythmia or SCD were reported during 1-year follow-up in both groups. Regression analysis showed that male gender, Sokolow-Lyon criteria and short QTc were significant independent predictors of ERP, P value < 0.05.ConclusionsIn outpatient-based Egyptian cohort, the prevalence of ERP was 6.7%, mostly the inferolateral pattern. Our ERP subjects had low-risk clinical and ECG criteria for malignant ERP. Further epidemiological studies are needed to explore the natural history of ERP.

Immunohistochemical and biochemical alterations following administration of proanthocyanidin extract in rats hepatocellular carcinoma

Grape seed proanthocyanidin extract (GSPE) is known to be effective on broad spectrum of biological pathways in living organisms including oxidative stress. The present study aimed to investigate the effects of proanthocyanidin on preneoplastic lesions and liver cancer induced in rats by Diethylnitrosamine (DEN). 7–8 Week old male Sprague Dawley (S.D.) rats were divided into six groups: The 1st group received no treatment and were −ve controls, the 2nd were treated with a single dose of DEN 200mg/kg intraperitoneally (i.p.) and served as +ve control group. The 3rd and 4th groups were injected with the same dose of DEN as in group 2 and then post treated with 300 or 150mg/kg/b.wt./day GSPE by intrgastroluminal gavage (i.g.) respectively until the end after the 22 weeks. Groups 5 and 6 were treated with the same doses of GSPE as in groups 3 and 4 respectively without DEN administration. The results showed that the immunohistochemical Proliferating Cell Nuclear Antigen (PCNA) labeling indexes (PCNA LI%) were significantly inhibited in liver tissues and tumors by both treatments of GSPE. Furthermore, treatment with GSPE has modified the liver tissue oxidative stress markers levels of SOD, CAT, GSH, GST, GPx, GR and MDA changed by DEN. In conclusion, GSPE has a sufficient therapeutic effect against liver carcinogenesis through their free radical scavenging, inhibition of cellular proliferation.

Variation in Phenolic Profile, β-Carotene and Flavonoid Contents, Biological Activities of Two Tagetes Species from Pakistani Flora.

The objective of present study was to evaluate the variation in phenolic profile, β-carotene, flavonoid contents, antioxidant and antimicrobial properties of Tagetes erecta and Tagetes patula (T. erecta and T. patula) through different in vitro assays. Antioxidant activity was determined through 2,2-diphenyl-1-picrylhydrazyl (DPPH) free radical scavenging and inhibition of linoleic acid peroxidation assays and antibacterial and antifungal activities studied using the disc diffusion and resazurin microtiter-plate assays against bacterial and fungal strains. Moreover, total phenolics (TP), total carotenoids (TC) and total flavonoids (TF) were also determined. Highest (TP 35.8 mg GAE/g) and TF (16.9 mg CE/g) contents were found in MeOH extract of T. patula. T. erecta extract showed higher TC contents (6.45 mg/g) than T. patula extract (6.32 mg/g). T. erecta exhibited the highest DPPH radical-scavenging activity (IC50 ) (5.73 μg/mL) and inhibition of linoleic acid peroxidation (80.1%). RP-HPLC revealed the presence of caffeic acid, sinapic acid and ferulic acid in Tagetes extracts, m-coumaric acid in T. erecta whereas chlorogenic acid in T. patula extract only. Both extracts possessed promising antimicrobial activity compared to the ciprofloxacin and flumequine (+ve controls) against Bacillus subtilis and Alternaria alternate. Both extract were rich source of polyphenols exhibiting excellent biological activities.

Fibrosis Progression and Incidence of Cirrhosis and Hepatic Decompensation in Persons Treated with Paritaprevir/Ritonavir/Ombitasvir/Dasabuvir: Results from ERCHIVES

Background Data are limited regarding the effect of paritaprevir/ritonavir, ombitasvir, dasabuvir regimen (PrOD) upon the rate of liver fibrosis progression and incidence of cirrhosis and hepatic decompensation after treatment for HCV.Methods Within ERCHIVES (Electronically Retrieved Cohort of HCV Infected Veterans), we identified HCV infected persons treated with PrOD and treatment-na•ve controls to determine the effect of PrOD treatment upon subsequent progression of fibrosis and incident cirrhosis and hepatic decompensation. Controls were propensity-score matched based on demographic and clinical characteristics. We excluded those with HIV coinfection, positive HBsAg, hepatocellular carcinoma at baseline and those with missing HCV RNA or FIB-4 scores. Fibrosis progression and liver cirrhosis were assessed using the FIB-4 score.ResultsThe final propensity score matched sample included 1,473 PrOD-treated individuals, and an equal number of matched, untreated controls. PrOD-treated patients had significantly reduced median FIB-4 scores over time, compared with controls (median absolute change in FIB-4 = -0.7 [IQR -1.51, -0.3] vs. +0.06 [IQR -0.38, 0.49]; P < 0.0001). Compared with matched controls, PrOD-treated patients had an 86% relative reduction in the risk of incident cirrhosis over 2,241 patient-years of follow-up (adjusted HR 0.14 [95% CI 0.08-0.23]). Treatment with PrOD was also associated with delayed time to first hepatic decompensation event (P < 0.001). In sensitivity analysis, the exclusion of patients with baseline cirrhosis did not materially alter the estimates of effect.Conclusion Treatment with PrOD is associated with a significant reduction in fibrosis progression, a longer time to the development of cirrhosis, and reduced risk of hepatic decompensation. Our results demonstrate the long-term anti-fibrotic benefits associated with PrOD therapy for chronic HCV.Disclosures J. Kort, Abbvie: Employee and Shareholder, Salary; A. Butt, Merck: Investigator, Grant recipient

Evaluation of Tissue Culture Seedlings for Their Genetic Fidelity and Virus Indexing in Sugarcane

Plant tissue culture technique is a powerful tool for rapid and large scale multiplication of virus free seed material. Clonal fidelity of the in vitro raised plants was carried out using RAPD. A total of fifteen primers were used for detecting polymorphism by RAPD analysis and the results revealed that all the tissue culture plants were grouped into one cluster and indicated that all the tissue culture developed plantlets are true-to-type. There are no somaclonal variations and are genetically identical with the mother plant. Virus indexing through ELISA indicated that the tissue culture developed seedlings does not contain the protein particles of ScMV and are free from the viral disease. The wells coated with leaf samples with suspected ScMV infection (97 A 85, C3) gave visual confirmation by yellow colour change, with OD 405 values of 0.526. As the reading is 2 times more than the negative control (OD 0.215) we can presume mild infection of mosaic in the test sample with lower titre of ScMV. All the ScMV +ve controls showed significant higher OD values of more than 2.515 confirming the earlier observations. Thus, viral detection using DAS-ELISA method can be used for detection and indexation in large scale sugarcane crop especially for in vitro regenerated plants with relatively cheaper cost and faster for supplying virus free seedlings to farming community in a large scale.

Abstract 4688: Overcoming drug resistance and stemness in oncogenic kras driven pancreatic ductal adenocarcinoma through PAK4 inhibition

Abstract Background: In spite of the established role of oncogenic Kras in the development and subsistence of Pancreatic Cancer (PC), no clinically viable agents have been developed to suppress this master regulator. Unlike other druggable proteins, Kras lacks the ideal binding pocket for small molecules. Therefore, there is an unmet need to identify novel druggable sites on Kras or to develop agents that target key effector proteins downstream. The p21-activated kinase 4 (PAK4) is a key effector downstream of Rho family GTPases. PAK4 is over-expressed in most PC cell lines tested but not in normal human pancreatic ductal epithelial cells (HPDE). Gene copy number amplification studies in PC patient cohorts confirmed PAK4 amplification. RNA interference of PAK4 suppresses PC cell proliferation making PAK4 an attractive therapeutic target. In collaboration with Karyopharm Therapeutics, we developed novel PAK4 allosteric modulators (PAMs; KPT-7189, KPT-9274, KPT-9307). Methods: Using multiple molecular biology techniques we tested PAMs activity (in the presence and absence of -ve and +ve controls) on a panel of PC cells lines, PAK4 over-expressing Gemcitabine resistant (GEM-R) PC models and highly resistant flow sorted cancer stem cells (CSC). Pancreatic CSC's are triple positive for CD133+CD44+EpCam+ and undergo epithelial-to-mesenchymal transition (EMT). The toxicity and efficacy of PAMs were evaluated in vitro and in sub-cutaneous mouse models of PC. Results: The novel, orally bioavailable PAMs show anti-proliferative activity in vitro against different PC cell lines (AsPC-1, Colo-357, MiaPaCa-2, L3.6pl and HPAC IC50s &amp;lt;250nM) while sparing HPDE (IC50s 5 fold higher). Cell growth inhibition was concurrent with apoptosis induction and suppression of colony formation in 5 different PC cell lines (not in HPDE). PAMs reduced RNA and PAK4 protein levels along with the inhibition of proliferative and anti-apoptotic signals downstream of PAK4. Co-immunoprecipitation experiments showed disruption of PAK4 complexes (p65, Bcl-2 and vimentin). Confocal, western blot and RT-PCR analysis of PAM-treated CSC spheroids showed reversal of EMT and suppression of stem markers EpCAM, vimentin and snail with re-expression of epithelial phenotype promoter E-cadherin. Additionally PAMs synergize with Gemcitabine and oxaliplatin (CI&amp;lt;1) in vitro. KPT-9274, possesses desirable PK properties and is well tolerated in mice with the absence of any clinical signs of toxicity up to 200 mg/kg oral daily dose. Pre-clinical animal efficacy (as a single agent and in combination with gemcitabine) in a sub-cutaneous, orthotopic (from primary cells) and LSL-K-Ras G12D/+;LSL-Trp53R172H/+;Pdx-1-Cre transgenic mouse models are ongoing. Conclusions: These proof of concept studies demonstrating the anti-proliferative effects of novel PAK4 allosteric modulators in pancreatic cancer warrant further clinical investigations. Citation Format: Asfar S. Azmi, William Senapedis, Erkan Baloglu, Yosef Landesman, Michael Kauffman, Sharon Shacham, Jack Wu, Amro Aboukameel, Irfana Muqbil, Ramzi M. Mohammad. Overcoming drug resistance and stemness in oncogenic kras driven pancreatic ductal adenocarcinoma through PAK4 inhibition. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 4688. doi:10.1158/1538-7445.AM2015-4688

Abstract 1424: F-box protein fbxl5 nuclear retention by specific inhibitors of nuclear export induces snail ubiquitination leading to reversal of EMT

Abstract Background: FBXL5 belonging to SKP1-cullin 1-F-box protein E3 ligase complexes (F-BOX) been shown to promote snail nuclear ubiquitination thereby regulating snail induced epithelial-to-mesenchymal transition (EMT) processes. However, cancer associated enhancement in the nuclear exporter Exportin1 (Xpo1) expression results in nuclear expulsion of FBXL5 causing snail stability and EMT. Here we demonstrate that Xpo1 inhibition by specific inhibitors of nuclear export (SINEs) results in nuclear retention FBXL5, causing nuclear degradation of SNAIL leading to reversal of mesenchymal phenotype to epithelial in HMLE-SNAIL models. Methods: Molecular assays (MTT, Annexin V FITC, Histone DNA ELISA, Spheroid formation, Western Blotting, Confocal microscopy, Co-immunoprecipitation, Xpo1 site directed mutagenesis) and computational techniques (gene expression microarray, pathway analysis) were used. In vivo activity of Selinexor was evaluated in xenografts developed from HMLE-SNAIL cells in ICR-SCID mice. Results: SINEs [Selinexor (KPT-330) and +ve controls KPT-185 and Leptomycin B (LMB)) not KPT-301 (-ve control)] reverse mesenchymal morphology, induce growth inhibition and apoptosis in HMLE-SNAIL and Kras-HMLE-SNAIL cells and prevent spheroid formation (IC50s ∼150 nM). Immunofluorescence analysis demonstrated that SINE treatment resulted in nuclear retention of FBXL5 that was concurrent with nuclear degradation of snail. Co-immunoprecipitation experiments showed nuclear ubiquitination of snail by SINEs. Western blotting analysis verified nuclear enhancement of FBXL5 that was consistent with down-regulation of EMT markers (Vimentin, snail, EpCAM) and enhancement of E-Cadherin. SiRNA against FBXL5 or transfecting cells with cys528 mut-Xpo1 that lacks SINE binding site markedly abrogated SINE activity thereby verifying the Xpo1 and FBXL5 mediated mechanisms of action. Pathways analysis of quadruplet microarray expression arrays from SINE treated HMLE-SNAIL cells demonstrated differential expression of F-Box family proteins [FBXO2, FBXL17, FBXO33, FBXO37, FBXW7 (p&amp;lt;0.001)] and suppression of snail network. Most significantly, oral administration of SINE (Selinexor at 15 mg/kg three times a week for three weeks) resulted in complete cure of HMLE-SNAIL tumors (tumor free at 120 days). SINE exposed animals showed normal spleen size and morphology in comparison to control animals that showed spleen enlargement (p&amp;lt;0.001). Quantitative sandwich ELISA of spleen tissue extracts showed suppression of snail expression in SINE treatment animals. Conclusion: This is the first proof of concept study demonstrating that targeted inhibition of Xpo1 can inhibit EMT through nuclear retention of FBOX protein, particularly FBXL5 and consequent snail ubiquitination and degradation. Our findings open a unique possibility to block EMT at the nuclear pore. Citation Format: Irfana Muqbil, Amro Aboukameel, Yosef Landesman, Michael Kauffman, Sharon Shacham, Ramzi M. Mohammad, Asfar S. Azmi. F-box protein fbxl5 nuclear retention by specific inhibitors of nuclear export induces snail ubiquitination leading to reversal of EMT. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 1424. doi:10.1158/1538-7445.AM2015-1424

Leishmania donovani influenced cytokines and Toll-like receptors expression among Sudanese visceral leishmaniasis patients.

Leishmaniasis remains a serious health problem. The outcome of Leishmania infection depends on the early innate response. In this study, whole blood samples of 40 patients with visceral leishmaniasis (VL), 10 leishmanin skin test-negative (LST-ve) controls and 10 leishmanin skin test-positive (LST+ve) controls were stimulated by live L. donovani promastigotes. Also, THP1 human cell line was infected with L. donovani. The production of interleukin 10 (IL-10), tumour necrosis factor alpha (TNF) and interferon gamma (IFNG) cytokines was measured, and the expression of Toll-like receptors (TLR2, TLR4 and TLR9) was done in the blood samples and also in the THP1 cell line. IL-10 was found to be higher in LST+ve controls compared with VL patients. TNF was moderately produced with no variation between patients, controls and THP1 cells. IFNG was higher in LST+ve controls also in THP1 cells. TLR4 and TLR9 were found to be highly expressed in patients with VL. L. donovani increases the expression of TLR4 and TLR9 in patients with VL and TLR2 in THP1 cells, suggesting a TLRs relation in induction of a mixed cytokine response. TLR9 was markedly recognized by L. donovani DNA.

Cytomegalovirus-associated biliary atresia: An aetiological and prognostic subgroup

Background and aimsPerinatal cytomegalovirus (CMV) infection is a possible cause or trigger of biliary atresia though clinical evidence is scant. We hypothesised that CMV IgM+ve biliary atresia is a separate clinical entity compared to CMV IgM−ve biliary atresia. MethodsProspective single-centre study. 210 infants with histologically confirmed biliary atresia were treated in our institution (Jan. 2004 to Dec. 2011); of these 20 (9.5%) were CMV IgM+ve at presentation. We compared these with 111 infants who were CMV IgM−ve (controls) for clinical features, biochemistry at presentation and outcome following Kasai portoenterostomy (KPE). A blinded comparison of age-matched liver histology was also performed. Data are quoted as median (interquartile range). A P value ≤0.05 was regarded as significant. ResultsInfants with CMV IgM+ve biliary atresia were older at Kasai portoenterostomy (or laparotomy) [70 (60–80) days vs. 56 (44–75)days; P=0.003] and were more jaundiced [175 (147–224) vs. 140 (121–181) μmol/L; P=0.002+ with higher AST*287 (157–403) vs. 180 (133–254) IU/L; P=0.005] and aspartate aminotransferase-to-platelet ratio index [1.1 (0.79–3.0) vs. 0.63 (0.43–0.95)] levels. Liver histology: CMV IgM+ve biliary atresia was characterised by a greater degree of inflammation (P<0.0001) and fibrosis (P=0.02), whereas CMV IgM−ve isolated biliary atresia had a higher degree of lobular cholestasis (P=0.001). This effect was independent of the effects of age at KPE. Outcome: CMV IgM+ve biliary atresia had a poorer outcome with a reduced clearance of jaundice (15% vs. 52.2%; P=0.002), native liver survival (P<0.0001) and increased mortality (P=0.002). ConclusionsCMV IgM+ve biliary atresia is a distinct clinical and pathological entity with a diminished response to Kasai portoenterostomy.

Abstract A24: Novel small molecule PAK4 allosteric modulators show potency against oncogenic K-Ras driven pancreatic cancer

Abstract Introduction: In spite of the well-recognized role of oncogenic K-Ras in the establishment and sustenance of the highly fatal disease, pancreatic cancer (PC), development of clinical agents that can tame this master regulator have been unsuccessful. This is primarily because, unlike other druggable targets, K-Ras lacks an ideal binding pocket that can be used to design small molecule drugs. Therefore, there is an urgent need to identify novel druggable sites in K-Ras or to develop agents that can target key effector proteins downstream of K-Ras signaling. To this end, we are pursuing p21-activated kinase 4 (PAK4) as a novel target for PC. PAK4 acts as a key effector of Rho family GTPases downstream of K-Ras and is found over-expressed in most of the available PC cell lines but not in normal human pancreatic ductal epithelial cells (HPDE). Gene copy number amplification studies in PC patient cohorts has shown amplification of PAK4. Most importantly, RNA interference of PAK4 suppresses PC cell proliferation making PAK4 an attractive therapeutic target within the K-Ras signaling network. Nevertheless, the previously developed PAK4 Type I ATP competitive inhibitor (PF-3758309; tested in non-pancreatic models) was evaluated in a Phase 1 study and showed undesirable pharmacokinetic properties as well as no objective responses and was subsequently discontinued. In order to fill this scientific void, we evaluated a new class of PAK4 allosteric modulators in pancreatic cancer models. Experimental Procedure: Using multiple molecular biology techniques we tested the Pak4 modulators' activities (in the presence and absence of -ve and +ve controls) in a panel of PC cells lines, PAK4 over-expressing Gemcitabine resistant (GEM-R) PC models and highly resistant flow sorted PC stem cells (CSC). CSC's are triple positive for CD133+CD44+EpCam+ and undergo epithelial-to-mesenchymal transition (EMT). The toxicity and efficacy of these PAK4 modulators were evaluated in sub-cutaneous mouse models of PC. Results: The novel, orally available PAK4 allosteric modulators (KPT-7189, KPT-8752) show anti-proliferative activity against different PC cell lines (AsPC-1, Colo-357, MiaPaCa-2, L3.6pl and HPAC IC50s &amp;lt;250nM) sparing HPDE (IC50s 5 fold higher). Cell growth inhibition was concurrent with apoptosis induction and suppression of colony forming abilities in 5 different PC cell lines (not in HPDE). KPT-7189 reduced PAK4 protein levels along with the inhibition of proliferative and anti-apoptotic signals downstream of PAK4. Molecularly, PAK4 RNA interference enhanced KPT-7189 activity and co-immunoprecipitation experiments showed disruption of PAK4 complexes (p65, Bcl-2 and vimentin). KPT-7189 inhibited spheroid forming ability of CSCs and reversed the epithelial-to-mesenchymal (EMT) phenotype. Confocal, western blot and RT-PCR analysis of KPT-7189 treated CD33+CD44+EpCam+ spheroids showed suppression of EMT and CSC markers EpCAM, vimentin and snail with re-expression of epithelial phenotype promoter E-cadherin. A similar compound, KPT-8752, with desirable PK properties was well tolerated in mice with the absence of any clinical signs of toxicity up to 200 mg/kg oral daily dose. Pre-clinical animal efficacy trial in sub-cutaneous, orthotopic (developed from human primary cells) and LSL-K-Ras G12D/+;LSL-Trp53R172H/+;Pdx-1-Cre transgenic mouse models are ongoing. Conclusion: This is the first proof of concept study demonstrating the anti-proliferative effects of novel allosteric modulators of PAK4, a downstream effector of K-Ras, in pancreatic cancer that warrants further clinical investigations. Citation Format: ASFAR S. AZMI, William T. Senapedis, Yosef Landesman, Erkan Baloglu, Bin Bao, Jack Wu, Kalid Ori, Sharon Shacham, Michael Kauffman, Ramzi M. Mohammad. Novel small molecule PAK4 allosteric modulators show potency against oncogenic K-Ras driven pancreatic cancer. [abstract]. In: Proceedings of the AACR Special Conference on RAS Oncogenes: From Biology to Therapy; Feb 24-27, 2014; Lake Buena Vista, FL. Philadelphia (PA): AACR; Mol Cancer Res 2014;12(12 Suppl):Abstract nr A24. doi: 10.1158/1557-3125.RASONC14-A24

Abstract 1771: Novel small molecule pak4 allosteric modulators with activity against pancreatic cancer

Abstract The p21-activated kinase 4 (PAK4) acts as a key effector of Rho family GTPases downstream of K-Ras and is found over-expressed in most of the available pancreatic cancer (PC) cell lines but not in normal human pancreatic ductal epithelial cells (HPDE). Gene copy number amplification studies in PC patient cohorts has shown amplification of PAK4. Most importantly, RNA interference of PAK4 suppresses PC cell proliferation making PAK4 an attractive therapeutic target within the K-Ras signaling network. Nevertheless, the previously developed PAK4 Type I ATP competitive inhibitor (PF-3758309; tested in non-pancreatic models) was evaluated in a Phase 1 study and showed undesirable pharmacokinetic properties as well as no objective responses and was subsequently discontinued. In order to fill this scientific void, we evaluated a new class of PAK4 allosteric modulators in PC. Using multiple molecular biology techniques we tested the Pak4 modulators' activities (in the presence and absence of -ve and +ve controls) in a panel of PC cells lines, PAK4 over-expressing Gemcitabine resistant (GEM-R) PC models and highly resistant flow sorted PC stem cells (CSC). CSC's are triple positive for CD133+CD44+EpCam+ and undergo epithelial-to-mesenchymal transition (EMT). The toxicity and efficacy of these PAK4 modulators were evaluated in sub-cutaneous mouse models of PC. The novel, orally available PAK4 allosteric modulators (KPT-7189, KPT-8752) show anti-proliferative activity against different PC cell lines (AsPC-1, Colo-357, MiaPaCa-2, L3.6pl and HPAC IC50s &amp;lt;250nM) sparing HPDE (IC50s 5 fold higher). Cell growth inhibition was concurrent with apoptosis induction and suppression of colony forming abilities in 5 different PC cell lines (not in HPDE). KPT-7189 reduced PAK4 protein levels along with the inhibition of proliferative and anti-apoptotic signals downstream of PAK4. Molecularly, PAK4 RNA interference enhanced KPT-7189 activity and co-immunoprecipitation experiments showed disruption of PAK4 complexes (p65, Bcl-2 and vimentin). KPT-7189 inhibited spheroid forming ability of CSCs and reversed the epithelial-to-mesenchymal (EMT) phenotype. Confocal, western blot and RT-PCR analysis of KPT-7189 treated CD33+CD44+EpCam+ spheroids showed suppression of EMT and CSC markers EpCAM, vimentin and snail with re-expression of epithelial phenotype promoter E-cadherin. A similar compound, KPT-8752, with desirable PK properties was well tolerated in mice with the absence of any clinical signs of toxicity up to 200 mg/kg oral daily dose. Pre-clinical animal efficacy trial in sub-cutaneous, orthotopic and LSL-K-Ras G12D/+;LSL-Trp53R172H/+;Pdx-1-Cre transgenic mouse models are ongoing. This is the first proof of concept study demonstrating the anti-proliferative effects of novel allosteric modulators of PAK4, a downstream effector of K-Ras, in pancreatic cancer that warrants further clinical investigations. Citation Format: Asfar S. Azmi, William Senapedis, Yosef Landesman, Erkan Baloglu, Ori Kalid, Jack Wu, Bin Bao, Amro Aboukameel, Sharon Shacham, Michael Kauffman, Ramzi M. Mohammad. Novel small molecule pak4 allosteric modulators with activity against pancreatic cancer. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 1771. doi:10.1158/1538-7445.AM2014-1771

Increased Seizure Susceptibility in Mice 30 Days after Fluid Percussion Injury

Traumatic brain injury (TBI) has been reported to increase seizure susceptibility and also contribute to the development of epilepsy. However, the mechanistic basis of the development of increased seizure susceptibility and epilepsy is not clear. Though there is substantial work done using rats, data are lacking regarding the use of mice in the fluid percussion injury (FPI) model. It is unclear if mice, like rats, will experience increased seizure susceptibility following FPI. The availability of a mouse model of increased seizure susceptibility after FPI would provide a basis for the use of genetically modified mice to study mechanism(s) of the development of post-traumatic epilepsy. Therefore, this study was designed to test the hypothesis that, mice subjected to a FPI develop increased seizure susceptibility to a subconvulsive dose of the chemoconvulsant, pentylenetetrazole (PTZ). Three groups of mice were used: FPI, sham, and naïve controls. On day 30 after FPI, mice from the three groups were injected with PTZ. The results showed that FPI mice exhibited an increased severity, frequency, and duration of seizures in response to PTZ injection compared with the sham and naïve control groups. Histopathological assessment was used to characterize the injury at 1, 3, 7, and 30 days after FPI. The results show that mice subjected to the FPI had a pronounced lesion and glial response that was centered at the FPI focus and peaked at 3 days. By 30 days, only minimal evidence of a lesion is observed, although there is evidence of a chronic glial response. These data are the first to demonstrate an early increase in seizure susceptibility following FPI in mice. Therefore, future studies can incorporate transgenic mice into this model to further elucidate mechanisms of TBI-induced increases in seizure susceptibility.

Long-term HIV infection and antiretroviral therapy are associated with bone microstructure alterations in premenopausal women

We evaluated the influence of long-term HIV infection and its treatment on distal tibia and radius microstructure. Premenopausal eumenorrheic HIV-positive women displayed trabecular and cortical microstructure alterations, which could contribute to increased bone fragility in those patients. Bone fragility is an emerging issue in HIV-infected patients. Dual-energy X-ray absorptiometry (DXA) quantified areal bone mineral density (BMD) predicts fracture risk, but a significant proportion of fracture risk results from microstructural alterations. We studied the influence of long-term HIV infection on bone microstructure as evaluated by high-resolution peripheral quantitative computed tomography (HR-pQCT) in 22 HIV-positive (+ve) premenopausal eumenorrheic women and 44 age- and body mass index (BMI)-matched HIV-negative (-ve) controls. All subjects completed questionnaires regarding calcium/protein intakes and physical activity, and underwent DXA and HR-pQCT examinations for BMD and peripheral skeleton microstructure, respectively. A risk factor analysis of tibia trabecular density using linear mixed models was conducted. In HIV+ve women on successful antiretroviral therapy (undetectable HIV-RNA, median CD4 cell count, 626), infection duration was 16.5 ± 3.5 (mean ± SD) years; median BMI was 22 (IQR, 21-26) kg/m². More HIV+ve women were smokers (82 versus 50 %, p = 0.013). Compared to controls, HIV+ve women had lower lumbar spine (spine T-score -0.70 vs -0.03, p = 0.014), but similar proximal femur BMD. At distal tibia, HIV+ve women had a 14.1 % lower trabecular density and a 13.2 % reduction in trabecular number compared to HIV-ve women (p = 0.013 and 0.029, respectively). HR-pQCT differences in distal radius were significant for cortical density (-3.0 %; p = 0.029). Compared with HIV-ve subjects, premenopausal HIV+ve treated women had trabecular and cortical bone alterations. Adjusted analysis revealed that HIV status was the only determinant of between group tibia trabecular density differences. The latter could contribute to increased bone fragility in HIV+ve patients.

Helicobacter pylori Infection and Inflammation: Implications for Pathophysiology of Diabetes Mellitus and Coronary Heart Disease

Asian Indians living in the Indian subcontinent or abroad experience high rate of coronary heart disease (CHD) and type 2 diabetes mellitus (T2DM). Asian Indians are also known to suffer from various infections, particularly during their childhood. One such chronic infection is with Helicobacter pylori (H. pylori). Since H. pylori with its specific virulence factor cytotoxin-associated gene A (cagA) has been suggested to be associated with CHD, a role of this H. pylori infection was investigated in the pathogenesis of CHD in Asian Indians living in Bangladesh. H. pylori (CagA) infected subjects with CHD (HP+ve cases, n=21), and without CHD (HP+ve controls, n=20), and non-infected without CHD (HP-ve normal controls, n=21) were included in this study. Thromboxane (TXB), an index of platelet activation, was found to be significantly higher in the HP+ve cases (p=0.05), but not in the HP+ve controls (p=0.88) when compared with HP-ve controls. Analyses of lipid profiles revealed that while triglycerides, total cholesterol and LDL did not show any significant changes, HDL was significantly lower in both the HP+ve cases (p=0.0003) and controls (p=0.005). The mean fasting glucose level in the HP+ve cases was markedly increased (p>0.0001), while it was intermediate in the HP+ve controls, and lowest in the HP-ve controls. HOMA-IR values, a measure of insulin resistance, did not reflect any substantial differences between the HP+ve and HP-ve controls, but they were highly significantly different between the HP+ve cases and HP-ve controls. HOMA-B, indicating insulin secretory dysfunction (ISD), was significantly higher in both the HP+ve groups when compared with the normal controls. The data indicate that H. pylori infection is associated with impaired insulin secretion, and that a component of insulin resistance that occurs independent of H. pylori can then lead to a worsening of glucose tolerance and the development of CHD. This is the first demonstration to our knowledge that H. pylori (CagA) infection is associated with insulin secretory dysfunction in human subjects. Since many Asian Indians contract various other chronic and acute infections, it is important to investigate the role of H. pylori and other infectious agents in the pathogenesis of T2DM and CHD.