Endotoxin Neutralization as a Biomonitor for Inflammatory Bowel Disease

Gram-negative bacterial endotoxin is a potent immunostimulant implicated in the development and/or progression of a variety of diseases. The mammalian immune system has both innate and adaptive immune responses to neutralize endotoxin. In this study, a system was developed to monitor bacterial exposure by measuring the extent and nature of endotoxin neutralization in plasma. In control patients, females had higher levels of endotoxin neutralization than males, mirroring clinical outcomes from bacterial infection and sepsis. In addition to the total amount of neutralization, we used inactivation techniques to elucidate the nature of this activity and develop a system to compare early and late immune responses. Using this method to monitor patients with inflammatory bowel disease, we found a more robust total response that relies more on long-term, adaptive components of the immune system and less on early, innate components. Our results indicate that endotoxin neutralization is a valuable method to discern inflammatory bowel disease patients from a control population. Additionally, the nature of neutralization may be valuable in monitoring disease severity and/or the role of medication.     

Consumption of Dental Treatment in Patients with Inflammatory Bowel Disease,a Register Study

Objective

The aim of this study was to compare the consumption of dental treatment among patients with Crohn´s disease (CD) or ulcerative colitis (UC) compared to age and gender matched control groups.

Design

The study group comprised 2085 patients with CD and 3161 with UC from the Uppsala-Örebro region and from the Stockholm region. The patients in the cohort were diagnosed between 1960 and 1989. Patients up to 70 years of age were included in the study. The two patients groups were compared to age- and gender-matched, randomly selected control groups from the same geographic area comprising a corresponding number of participants.

Results

CD patients had significantly higher total number of procedures registered (p < 0.000). The difference was most pronounced for removable dentures (+65%), fillings in front teeth (+52%) and endodontic treatment (+46%) when Crohn’s patients were compared to controls (p<0.001). The corresponding figures for UC patients were also a significantly higher total number of procedures (p < 0.005), more clinical examinations (p<0.000), fillings in canines and incisors (p < 0.001) and fillings in bicuspids and molars (p < 0.000).

Conclusion

This study demonstrate that CD and UC individuals use more dental treatment compared to an age-gender matched control group, and more caries-related treatments. The difference was most pronounced for restorative treatment in patients with Crohn’s.     

Identification of gp96 as a Novel Target for Treatment of Autoimmune Disease in Mice

Heat shock proteins have been implicated as endogenous activators for dendritic cells (DCs). Chronic expression of heat shock protein gp96 on cell surfaces induces significant DC activations and systemic lupus erythematosus (SLE)-like phenotypes in mice. However, its potential as a therapeutic target against SLE remains to be evaluated. In this work, we conducted chemical approach to determine whether SLE-like phenotypes can be compromised by controlling surface translocation of gp96. From screening of chemical library, we identified a compound that binds and suppresses surface presentation of gp96 by facilitating its oligomerization and retrograde transport to endoplasmic reticulum. In vivo administration of this compound reduced maturation of DCs, populations of antigen presenting cells, and activated B and T cells. The chemical treatment also alleviated the SLE-associated symptoms such as glomerulonephritis, proteinuria, and accumulation of anti-nuclear and –DNA antibodies in the SLE model mice resulting from chronic surface exposure of gp96. These results suggest that surface translocation of gp96 can be chemically controlled and gp96 as a potential therapeutic target to treat autoimmune disease like SLE.     

Serum CD121a (Interleukin 1 Receptor,Type I): A Potential Novel Inflammatory Marker for Coronary Heart Disease

Inflammation is now believed to be responsible for coronary heart disease (CHD). This belief has stimulated the evaluation of various inflammatory markers for predicting CHD. This study was designed to investigate the association between four inflammatory cytokines (CD121a, interleukin [IL]-1β, IL-8, and IL-11) and CHD. Here, we evaluated 443 patients with CHD and 160 CHD-free controls who underwent coronary angiography. Cytokines were evaluated using flow cytometry, and statistical analyses were performed to investigate the association between cytokine levels and the risk of CHD. Patients with CHD had significantly higher levels of CD121a. The odds ratios for CHD according to increasing CD121a quartiles were 1.00, 1.47 [95% confidence interval (CI): 0.79–2.72], 2.67 (95% CI: 1.47–4.84), and 4.71 (95% CI: 2.65–8.37) in an age- and sex-adjusted model, compared to 1.00, 1.48 (95% CI: 0.70–3.14), 2.25 (95% CI: 1.10–4.62), and 4.39 (95% CI: 2.19–8.79) in a model that was adjusted for multiple covariates. A comparison of the stable angina, unstable angina, and acute myocardial infarction (AMI) subgroups revealed that patients with AMI had the highest CD121a levels, although IL-1β levels were similar across all groups. IL-8 levels were also increased in AMI patients, and IL-11 levels were higher in CHD patients than in non-CHD patients. Correlation analysis revealed a positive association between CD121a, IL-8, and the Gensini score. Together, the significant increase in CD121a levels among CHD patients suggests that it may be a novel inflammatory marker for predicting CHD.     

Hydrogen Sulfide: A Novel Gaseous Molecule for Plant Adaptation to Stress

Hydrogen sulfide (H₂S) has emerged as a novel gaseous signal molecule with multifarious effects on seed germination, plant growth, development, and physiological processes. Due to its dominant role in plant stress tolerance and cross-adaptation, it is getting more attention nowadays, although it has been largely referred as toxic and environmental hazardous gas. In this review work, we are highlighting the importance of H₂S as an essential gaseous molecule to help in signaling, metabolism, and stress tolerance in plants. Firstly, production of H₂S from different natural and artificial sources were discussed with its transformation from sulfur (S) to sulfate (SO₄²⁻) and then to sulfite (SO₃²⁻). The importance of different kinds of transporters that helps to take SO₄²⁻ from the soil solution was presented. Mainly, these transporters are SULTRs (H⁺/SO₄²⁻ cotransporters) and multigene family encodes them. Furthermore, these SULTRs have LAST (Low affinity transport proteins), HAST (High affinity transport proteins), vacuole transporters, and plastid transporters. Since it is well known that there is strong relationship between SO₄²⁻ and synthesis of hydrogen sulfide or dihydrogen sulfide or sulfane in plant cells. Thus, cysteine (Cys) metabolism through which H₂S could be generated in plant cell with the role of different enzymes has been presented. Furthermore, H₂S in interaction with other molecules could help to mitigate biotic and abiotic stress. Based on this review work, it can be concluded that H₂S has potential to induce cross-adaptation to biotic and abiotic stress; thus, it is recommended that it should be considered in future studies to answer the questions like what are the receptors of H₂S in plant cell, where in plants the physiological concentration of H₂S is high in response to multiple stress and how it induces cross-adaptation by interaction with other signal molecules.

     

No Induction of Thiopurine Methyltransferase During Thiopurine Treatment in Inflammatory Bowel Disease

The aim of this study was to follow, during standardized initiation of thiopurine treatment, thiopurine methyltransferase (TPMT) gene expression and enzyme activity and thiopurine metabolite concentrations, and to study the role of TPMT and ITPA 94C > A polymorphisms for the development of adverse drug reactions. Sixty patients with ulcerative colitis or Crohn's disease were included in this open and prospective multi-center study. Thiopurine naïve patients were prescribed azathioprine (AZA), patients previously intolerant to AZA received 6-mercaptopurine (6-MP). The patients followed a predetermined dose escalation schedule, reaching target dose at Week 3; 2.5 and 1.25 mg/kg body weight for AZA and 6-MP, respectively. The patients were followed every week during Weeks 1–8 from baseline and then every 4 weeks until 20 weeks. TPMT activity and thiopurine metabolites were determined in erythrocytes, TPMT and ITPA genotypes, and TPMT gene expression were determined in whole blood. One homozygous TPMT-deficient patient was excluded. Five non compliant patients were withdrawn during the first weeks. Twenty-seven patients completed the study per protocol; 27 patients were withdrawn because of adverse events. Sixty-seven percent of the withdrawn patients tolerated thiopurines at a lower dose at Week 20. There was no difference in baseline TPMT enzyme activity between individuals completing the study and those withdrawn for adverse events (p = 0.45). A significant decrease in TPMT gene expression (TPMT/huCYC ratio, p = 0.02) was found, however TPMT enzyme activity did not change. TPMT heterozygous individuals had a lower probability of remaining in the study on the predetermined dose (p = 0.039). The ITPA 94C > A polymorphism was not predictive of adverse events (p = 0.35).     

微波联合抗生素治疗盆腔炎疗效观察

目的观察微波联合抗生素治疗盆腔炎的临床疗效。方法采用抗生素加微波治疗,对照组采用抗生素进行治疗,比较两组临床疗效。结果观察组30例治愈27例,好转2例。治愈率90．0％．总有效率96．67％。对照组30患者治愈24例,好转3倒,治愈率80．0％。总有效率90．0％。两组差异有统计学意义。结论微波联合抗生素治疗盆腔炎性的治疗效果肯定,值得基层医疗机构推广应用。     

Genomic Characterization of Japanese Macaque Rhadinovirus,a Novel Herpesvirus Isolated from a Nonhuman Primate with a Spontaneous Inflammatory Demyelinating Disease

Japanese macaque rhadinovirus (JMRV) is a novel gamma-2 herpesvirus that was isolated from a Japanese macaque (JM) with an inflammatory demyelinating encephalomyelitis referred to as Japanese macaque encephalomyelitis, a disease that possesses clinical and histopathological features resembling multiple sclerosis in humans. Genomic DNA sequence analysis reveals that JMRV is a gammaherpesvirus closely related to rhesus macaque rhadinovirus (RRV) and human herpesvirus 8. We describe here the complete nucleotide sequence and structure of the JMRV genome, as well as the sequence of two plaque isolates of this virus. Analysis of the JMRV genome not only demonstrates that this virus shares a number of genes with RRV that may be involved in pathogenesis but also indicates the presence of unique JMRV genes that could potentially contribute to disease development. The knowledge of the genomic sequence of JMRV, and the ability to easily propagate the virus in vitro, make JMRV infection of JM an attractive model for examining the potential role of an infectious viral agent in the development of demyelinating encephalomyelitis disease in vivo.     

Chronic Inflammatory Disease and Osteopathy: A Systematic Review

Background

Chronic inflammatory diseases (CID) are globally highly prevalent and characterized by severe pathological medical conditions. Several trials were conducted aiming at measuring the effects of manipulative therapies on patients affected by CID. The purpose of this review was to explore the extent to which osteopathic manipulative treatment (OMT) can be benefi-cial in medical conditions also classified as CID.

Methods

This review included any type of experimental study which enrolled sub-jects with CID comparing OMT with any type of control procedure. The search was conducted on eight databases in January 2014 using a pragmatic literature search approach. Two independent re-viewers conducted study selection and data extraction for each study. The risk of bias was evaluated according to the Cochrane methods. Heterogeneity was assessed and meta-analysis performed where possible.

Results

10 studies met the inclusion criteria for this review enrolling 386 subjects. The search identified six RCTs, one laboratory study, one cross-over pilot studies, one observation-al study and one case control pilot study. Results suggest a potential effect of osteopathic medicine on patients with medical pathologies associated with CID (in particular Chronic Obstructive Pul-monary Disease (COPD), Irritable Bowel Syndrome, Asthma and Peripheral Arterial Disease) com-pared to no treatment or sham therapy although data remain elusive. Moreover one study showed possible effects on arthritis rat model. Meta-analysis was performed for COPD studies only show-ing no effect of any type of OMT applied versus control. No major side effects were reported by those receiving OMT.

Conclusion

The present systematic review showed inconsistent data on the effect of OMT in the treatment of medical conditions potentially associated with CID, however the OMT appears to be a safe approach. Further more robust trials are needed to determine the direction and magnitude of the effect of OMT and to generalize favorable results.     

Methotrexate Treatment Causes Early Onset of Disease in a Mouse Model of Ross River Virus-Induced Inflammatory Disease through Increased Monocyte Production

Part of the Togaviridae family, alphaviruses, including chikungunya virus (CHIKV), Sindbis virus (SINV) and Ross River virus (RRV), are able to cause significant inflammatory pathologies ranging from arthritis to encephalitis. Following symptomatic infection with arthritis-associated alphaviruses, patients often experience severe joint pain, affecting distal and small joints, which can last six months or longer. Recently, methotrexate (MTX), a disease modifying anti-rheumatic drug (DMARD), was used to treat patients experiencing chronic rheumatic symptoms following infection with CHIKV. Here, the effect of MTX on Ross River virus disease (RRVD) in mice was examined to better understand its therapeutic potential for alphaviral-induced musculoskeletal disease and to further our knowledge of the development of alphaviral pathologies. Using a mouse model, we analyzed the effect of MTX on RRVD. RRV disease pathogenesis in response to MTX treatment was determined by measuring levels of proinflammatory factors, cellular infiltrates, viral titer and histological analysis of infected tissues. RRV-infected mice receiving MTX treatment rapidly developed musculoskeletal disease, which correlated with a significant influx of inflammatory cell infiltrates into the skeletal muscle tissue. Although no difference was observed in the level of proinflammatory cytokines and chemokines, the viral load increased at early time points post infection in the serum and quadriceps of MTX treated mice, possibly contributing to disease pathogenesis. Results suggest that MTX treatment of acute RRVD in mice provides no therapeutic benefit and underline the importance of inflammatory monocytes in alphaviral induced arthritides.     

Small Molecule Inhibitors of Phospholipase C from a Novel High-throughput Screen

Phospholipase C (PLC) isozymes are important signaling molecules, but few small molecule modulators are available to pharmacologically regulate their function. With the goal of developing a general approach for identification of novel PLC inhibitors, we developed a high-throughput assay based on the fluorogenic substrate reporter WH-15. The assay is highly sensitive and reproducible: screening a chemical library of 6280 compounds identified three novel PLC inhibitors that exhibited potent activities in two separate assay formats with purified PLC isozymes in vitro. Two of the three inhibitors also inhibited G protein-coupled receptor-stimulated PLC activity in intact cell systems. These results demonstrate the power of the high-throughput assay for screening large collections of small molecules to identify novel PLC modulators. Potent and selective modulators of PLCs will ultimately be useful for dissecting the roles of PLCs in cellular processes, as well as provide lead compounds for the development of drugs to treat diseases arising from aberrant phospholipase activity.     

Discovery and Preclinical Characterization of Novel Small Molecule TRK and ROS1 Tyrosine Kinase Inhibitors for the Treatment of Cancer and Inflammation

Receptor tyrosine kinases (RTKs), in response to their growth factor ligands, phosphorylate and activate downstream signals important for physiological development and pathological transformation. Increased expression, activating mutations and rearrangement fusions of RTKs lead to cancer, inflammation, pain, neurodegenerative diseases, and other disorders. Activation or over-expression of ALK, ROS1, TRK (A, B, and C), and RET are associated with oncogenic phenotypes of their respective tissues, making them attractive therapeutic targets. Cancer cDNA array studies demonstrated over-expression of TRK-A and ROS1 in a variety of cancers, compared to their respective normal tissue controls. We synthesized a library of small molecules that inhibit the above indicated RTKs with picomolar to nanomolar potency. The lead molecule GTx-186 inhibited RTK-dependent cancer cell and tumor growth. In vitro and in vivo growth of TRK-A-dependent IMR-32 neuroblastoma cells and ROS1-overexpressing NIH3T3 cells were inhibited by GTx-186. GTx-186 also inhibited inflammatory signals mediated by NFκB, AP-1, and TRK-A and potently reduced atopic dermatitis and air-pouch inflammation in mice and rats. Moreover, GTx-186 effectively inhibited ALK phosphorylation and ALK-dependent cancer cell growth. Collectively, the RTK inhibitor GTx-186 has a unique kinase profile with potential to treat cancer, inflammation, and neuropathic pain.     

Anti-Tumor Activity of a Novel HS-Mimetic-Vascular Endothelial Growth Factor Binding Small Molecule

The angiogenic process is controlled by variety of factors of which the vascular endothelial growth factor (VEGF) pathway plays a major role. A series of heparan sulfate mimetic small molecules targeting VEGF/VEGFR pathway has been synthesized. Among them, compound 8 (2-butyl-5-chloro-3-(4-nitro-benzyl)-3H-imidazole-4-carbaldehyde) was identified as a significant binding molecule for the heparin-binding domain of VEGF, determined by high-throughput-surface plasmon resonance assay. The data predicted strong binding of compound 8 with VEGF which may prevent the binding of VEGF to its receptor. We compared the structure of compound 8 with heparan sulfate (HS), which have in common the functional ionic groups such as sulfate, nitro and carbaldehyde that can be located in similar positions of the disaccharide structure of HS. Molecular docking studies predicted that compound 8 binds at the heparin binding domain of VEGF through strong hydrogen bonding with Lys-30 and Gln-20 amino acid residues, and consistent with the prediction, compound 8 inhibited binding of VEGF to immobilized heparin. In vitro studies showed that compound 8 inhibits the VEGF-induced proliferation migration and tube formation of mouse vascular endothelial cells, and finally the invasion of a murine osteosarcoma cell line (LM8G7) which secrets high levels of VEGF. In vivo, these effects produce significant decrease of tumor burden in an experimental model of liver metastasis. Collectively, these data indicate that compound 8 may prevent tumor growth through a direct effect on tumor cell proliferation and by inhibition of endothelial cell migration and angiogenesis mediated by VEGF. In conclusion, compound 8 may normalize the tumor vasculature and microenvironment in tumors probably by inhibiting the binding of VEGF to its receptor.     

Rescuing Trafficking Mutants of the ATP-binding Cassette Protein,ABCA4, with Small Molecule Correctors as a Treatment for Stargardt Eye Disease

Stargardt disease is the most common form of early onset macular degeneration. Mutations in ABCA4, a member of the ATP-binding cassette (ABC) family, are associated with Stargardt disease. Here, we have examined two disease-causing mutations in the NBD1 region of ABCA4, R1108C, and R1129C, which occur within regions of high similarity with CFTR, another ABC transporter gene, which is associated with cystic fibrosis. We show that R1108C and R1129C are both temperature-sensitive processing mutants that engage the cellular quality control mechanism and show a strong interaction with the chaperone Hsp 27. Both mutant proteins also interact with HDCAC6 and are degraded in the aggresome. We also demonstrate that novel corrector compounds that are being tested as treatment for cystic fibrosis, such as VX-809, can rescue the processing of the ABCA4 mutants, particularly their expression at the cell surface, and can reduce their binding to HDAC6. Thus, our data suggest that VX-809 can potentially be developed as a new therapy for Stargardt disease, for which there is currently no treatment.     

In Vivo Evaluation of a Novel Oriented Scaffold-BMSC Construct for Enhancing Full-Thickness Articular Cartilage Repair in a Rabbit Model

Tissue engineering (TE) has been proven usefulness in cartilage defect repair. For effective cartilage repair, the structural orientation of the cartilage scaffold should mimic that of native articular cartilage, as this orientation is closely linked to cartilage mechanical functions. Using thermal-induced phase separation (TIPS) technology, we have fabricated an oriented cartilage extracellular matrix (ECM)-derived scaffold with a Young''s modulus value 3 times higher than that of a random scaffold. In this study, we test the effectiveness of bone mesenchymal stem cell (BMSC)-scaffold constructs (cell-oriented and random) in repairing full-thickness articular cartilage defects in rabbits. While histological and immunohistochemical analyses revealed efficient cartilage regeneration and cartilaginous matrix secretion at 6 and 12 weeks after transplantation in both groups, the biochemical properties (levels of DNA, GAG, and collagen) and biomechanical values in the oriented scaffold group were higher than that in random group at early time points after implantation. While these differences were not evident at 24 weeks, the biochemical and biomechanical properties of the regenerated cartilage in the oriented scaffold-BMSC construct group were similar to that of native cartilage. These results demonstrate that an oriented scaffold, in combination with differentiated BMSCs can successfully repair full-thickness articular cartilage defects in rabbits, and produce cartilage enhanced biomechanical properties.     

Fenretinide: A Novel Treatment for Endometrial Cancer

Resistance to progestin treatment is a major hurdle in the treatment of advanced and reoccurring endometrial cancer. Fenretinide is a synthetic retinoid that has been evaluated in clinical trials as a cancer therapeutic and chemo-preventive agent. Fenretinide has been established to be cytotoxic to many kinds of cancer cells. In the present study, we demonstrate that fenretinide decreased cell viability and induced apoptosis in Ishikawa cells, which are an endometrial cancer cell line, in dose dependent manner in-vitro. This effect was found to be independent of retinoic acid nuclear receptor signaling pathway. Further, we have shown that this induction of apoptosis by fenretinide may be caused by increased retinol uptake via STRA6. Silencing of STRA6 was shown to decrease apoptosis which was inhibited by knockdown of STRA6 expression in Ishikawa cells. Results of an in-vivo study demonstrated that intraperitoneal injections of fenretinide in endometrial cancer tumors (created using Ishikawa cells) in mice inhibited tumor growth effectively. Immunohistochemistry of mice tumors showed a decrease in Ki67 expression and an increase in cleaved caspase-3 staining after fenretinide treatment when compared to vehicle treated mice. Collectively, our results are the first to establish the efficacy of fenretinide as an antitumor agent for endometrial cancer both in-vitro and in-vivo, providing a valuable rationale for initiating more preclinical studies and clinical trials using fenretinide for the treatment of endometrial cancer.     

In-Silico Analysis of Inflammatory Bowel Disease (IBD) GWAS Loci to Novel Connections

Genome-wide association studies (GWASs) for many complex diseases, including inflammatory bowel disease (IBD), produced hundreds of disease-associated loci—the majority of which are noncoding. The number of GWAS loci is increasing very rapidly, but the process of translating single nucleotide polymorphisms (SNPs) from these loci to genomic medicine is lagging. In this study, we investigated 4,734 variants from 152 IBD associated GWAS loci (IBD associated 152 lead noncoding SNPs identified from pooled GWAS results + 4,582 variants in strong linkage-disequilibrium (LD) (r² ≥0.8) for EUR population of 1K Genomes Project) using four publicly available bioinformatics tools, e.g. dbPSHP, CADD, GWAVA, and RegulomeDB, to annotate and prioritize putative regulatory variants. Of the 152 lead noncoding SNPs, around 11% are under strong negative selection (GERP++ RS ≥2); and ~30% are under balancing selection (Tajima’s D score >2) in CEU population (1K Genomes Project)—though these regions are positively selected (GERP++ RS <0) in mammalian evolution. The analysis of 4,734 variants using three integrative annotation tools produced 929 putative functional SNPs, of which 18 SNPs (from 15 GWAS loci) are in concordance with all three classifiers. These prioritized noncoding SNPs may contribute to IBD pathogenesis by dysregulating the expression of nearby genes. This study showed the usefulness of integrative annotation for prioritizing fewer functional variants from a large number of GWAS markers.