Publications by authors named "Manoj Tyagi"

34 Publications

Immuno-transcriptomic profiling of extracranial pediatric solid malignancies.

Cell Rep 2021 Nov;37(8):110047

University of Toronto Musculoskeletal Oncology Unit, Sinai Health System; Department of Surgery, University of Toronto, Toronto, ON, Canada.

We perform an immunogenomics analysis utilizing whole-transcriptome sequencing of 657 pediatric extracranial solid cancer samples representing 14 diagnoses, and additionally utilize transcriptomes of 131 pediatric cancer cell lines and 147 normal tissue samples for comparison. We describe patterns of infiltrating immune cells, T cell receptor (TCR) clonal expansion, and translationally relevant immune checkpoints. We find that tumor-infiltrating lymphocytes and TCR counts vary widely across cancer types and within each diagnosis, and notably are significantly predictive of survival in osteosarcoma patients. We identify potential cancer-specific immunotherapeutic targets for adoptive cell therapies including cell-surface proteins, tumor germline antigens, and lineage-specific transcription factors. Using an orthogonal immunopeptidomics approach, we find several potential immunotherapeutic targets in osteosarcoma and Ewing sarcoma and validated PRAME as a bona fide multi-pediatric cancer target. Importantly, this work provides a critical framework for immune targeting of extracranial solid tumors using parallel immuno-transcriptomic and -peptidomic approaches.
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http://dx.doi.org/10.1016/j.celrep.2021.110047DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8642810PMC
November 2021

Impact of the methylation classifier and ancillary methods on CNS tumor diagnostics.

Neuro Oncol 2021 Sep 23. Epub 2021 Sep 23.

Laboratory of Pathology, Center for Cancer Research, National Cancer Institute, National Institutes of Health, Bethesda, Maryland, USA.

Background: Accurate CNS tumor diagnosis can be challenging, and methylation profiling can serve as an adjunct to classify diagnostically difficult cases.

Methods: An integrated diagnostic approach was employed for a consecutive series of 1,258 surgical neuropathology samples obtained primarily in a consultation practice over 2-year period. DNA methylation profiling and classification using the DKFZ/Heidelberg CNS tumor classifier was performed, as well as unsupervised analyses of methylation data. Ancillary testing, where relevant, was performed.

Results: Among the received cases in consultation, a high confidence methylation classifier score (>0.84) was reached in 66.4% of cases. The classifier impacted the diagnosis in 46.5% of these high-confidence classifier score cases, including a substantially new diagnosis in 26.9% cases. Among the 289 cases received with only a descriptive diagnosis, methylation was able to resolve approximately half (144, 49.8%) with high-confidence scores. Additional methods were able to resolve diagnostic uncertainty in 41.6% of the low-score cases. Tumor purity was significantly associated with classifier score (p = 1.15e-11). Deconvolution demonstrated that suspected GBMs matching as control/inflammatory brain tissue could be resolved into GBM methylation profiles, which provided a proof-of-concept approach to resolve tumor classification in the setting of low tumor purity.

Conclusions: This work assesses the impact of a methylation classifier and additional methods in a consultative practice by defining the proportions with concordant vs. change in diagnosis in a set of diagnostically challenging CNS tumors. We address approaches to low-confidence scores and confounding issues of low tumor purity.
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http://dx.doi.org/10.1093/neuonc/noab227DOI Listing
September 2021

Whole-exome sequencing reveals germline-mutated small cell lung cancer subtype with favorable response to DNA repair-targeted therapies.

Sci Transl Med 2021 01;13(578)

Developmental Therapeutics Branch, Center for Cancer Research, NCI, Bethesda, MD 20892, USA.

Because tobacco is a potent carcinogen, secondary causes of lung cancer are often diminished in perceived importance. To assess the extent of inherited susceptibility to small cell lung cancer (SCLC), the most lethal type of lung cancer, we sequenced germline exomes of 87 patients (77 SCLC and 10 extrapulmonary small cell) and considered 607 genes, discovering 42 deleterious variants in 35 cancer-predisposition genes among 43.7% of patients. These findings were validated in an independent cohort of 79 patients with SCLC. Loss of heterozygosity was observed in 3 of 14 (21.4%) tumors. Identification of variants influenced medical management and family member testing in nine (10.3%) patients. Unselected patients with SCLC were more likely to carry germline RAD51 paralog D (), checkpoint kinase 1 (), breast cancer 2 (), and mutY DNA glycosylase () pathogenic variants than healthy controls. Germline genotype was significantly associated with the likelihood of a first-degree relative with cancer or lung cancer (odds ratio: 1.82, = 0.008; and 2.60, = 0.028), and longer recurrence-free survival after platinum-based chemotherapy ( = 0.002), independent of known prognostic factors. Treatment of a patient with relapsed SCLC and germline pathogenic mutation of interacting protein C-terminal helicase 1 (), a homologous recombination-related gene, using agents synthetically lethal with homologous recombination deficiency, resulted in a notable disease response. This work demonstrates that SCLC, currently thought to result almost exclusively from tobacco exposure, may have an inherited predisposition and lays the groundwork for targeted therapies based on the genes involved.
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http://dx.doi.org/10.1126/scitranslmed.abc7488DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8489678PMC
January 2021

Tremelimumab in Combination With Microwave Ablation in Patients With Refractory Biliary Tract Cancer.

Hepatology 2019 05 10;69(5):2048-2060. Epub 2019 Mar 10.

Gastrointestinal Malignancies Section, Center for Cancer Research, National Cancer Institute, National Institutes of Health, Bethesda, MD.

Treatment options for patients with advanced biliary tract cancer are limited. Dysregulation of the immune system plays an important role in the pathogenesis of biliary tract cancer (BTC). This study aimed to investigate whether tremelimumab, an anti-CTLA4 (cytotoxic T-lymphocyte-associated protein 4) inhibitor, could be combined safely with microwave ablation to enhance the effect of anti-CTLA4 treatment in patients with advanced BTC. Patients were enrolled to receive monthly tremelimumab (10 mg/kg, intravenously) for six doses, followed by infusions every 3 months until off-treatment criteria were met. Thirty-six days after the first tremelimumab dose, patients underwent subtotal microwave ablation. Interval imaging studies were performed every 8 weeks. Adverse events (AEs) were noted and managed. Tumor and peripheral blood samples were collected to perform immune monitoring and whole-exome sequencing (WES). Twenty patients with refractory BTC were enrolled (median age, 56.5 years). No dose-limiting toxicities were encountered. The common treatment-related AEs included lymphopenia, diarrhea, and elevated transaminases. Among 16 patients evaluable for efficacy analysis, 2 (12.5%) patients achieved a confirmed partial response (lasting for 8.0 and 18.1 months, respectively) and 5 patients (31.3%) achieved stable disease. Median progression free survival (PFS) and overall survival (OS) were 3.4 months (95% confidence interval [CI], 2.5-5.2) and 6.0 months (95% CI, 3.8-8.8), respectively. Peripheral blood immune cell subset profiling showed increased circulating activated human leukocyte antigen, DR isotype ([HLA-DR] positive) CD8 T cells. T-cell receptor (TCR)β screening showed tremelimumab expanded TCR repertoire, but not reaching statistical significance (P = 0.057). Conclusion: Tremelimumab in combination with tumor ablation is a potential treatment strategy for patients with advanced BTC. Increased circulating activated CD8 T cells and TCR repertoire expansion induced by tremelimumab may contribute to treatment benefit.
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http://dx.doi.org/10.1002/hep.30482DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6461476PMC
May 2019

Keratomycosis due to : A case report from Sub-Himalayan region of Uttarakhand.

Indian J Pathol Microbiol 2018 Oct-Dec;61(4):607-609

Department of Ophthalmology, Bundelkhand Medical College, Sagar, Madhya Pradesh, India.

The fungus Purpureocillium lilacinum previously known as Paceliomyces lilacinus is an emerging pathogen that can cause severe human infections including devastating oculomycosis. Treatment with traditional antifungals often fails, and the organism shows variable susceptibility to novel triazoles. We hereby report a case of keratomycosis caused by Pur. lilacinum in an immunocompetent male patient following trauma. The patient was successfully treated with voriconazole. The drug shows good activity against Pur. lilacinum and could be a promising therapeutic alternative to treat infections caused by this fungus, which generally shows resistance to conventional antifungal agents including novel triazoles.
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http://dx.doi.org/10.4103/IJPM.IJPM_404_17DOI Listing
February 2019

Inhibition of Spontaneous Contractility of Isolated Caprine Ureter by Flupirtine.

Int J Appl Basic Med Res 2018 Apr-Jun;8(2):116-119

Department of Pharmacology and Clinical Pharmacology, Christian Medical College, Vellore, Tamil Nadu, India.

Context: Kv7 potassium channels are expressed in several types of smooth muscles and could mediate physiological responses in the tissues expressed. Flupirtine is an analgesic that acts by opening Kv7 potassium channels. It has been shown to inhibit the contractility of several types of isolated smooth muscle.

Aims: This study investigated the ability of flupirtine to inhibit the spontaneous contractility of isolated distal caprine (goat) ureter.

Settings And Design: Spontaneous contractility of the isolated goat ureter was recorded using a physiograph.

Materials And Methods: The ability of 1, 3, 10, 30, and 90 μM concentrations of flupirtine maleate to inhibit the spontaneous contractility of isolated distal goat ureter was investigated. The ability of the nonspecific potassium channel blocker 4-aminopyridine (4-AP; 1 mM) and the specific Kv7 channel blocker XE-991 (100 μM) to reverse the inhibitory effect of flupirtine on ureteric contractility was also investigated.

Statistical Analysis Used: Both parametric and nonparametric statistical tests were used.

Results: At 10, 30, and 90 μM concentrations, flupirtine significantly inhibited the spontaneous contractility of the isolated goat ureter. The EC of flupirtine for a contact period of 10 min was 17.7 μM. The inhibitory effect of flupirtine on ureteric contractility was significantly reversed by 4-AP and XE-991.

Conclusions: Flupirtine inhibits the spontaneous contractility of the isolated goat ureter by opening Kv7 channels.
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http://dx.doi.org/10.4103/ijabmr.IJABMR_159_17DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5932919PMC
May 2018

An activating mutation of interferon regulatory factor 4 (IRF4) in adult T-cell leukemia.

J Biol Chem 2018 05 14;293(18):6844-6858. Epub 2018 Mar 14.

From the Division of Oncology, Department of Medicine, Washington University School of Medicine, St. Louis, Missouri 63110.

The human T-cell leukemia virus-1 (HTLV-1) oncoprotein Tax drives cell proliferation and resistance to apoptosis early in the pathogenesis of adult T-cell leukemia (ATL). Subsequently, probably as a result of specific immunoediting, Tax expression is down-regulated and functionally replaced by somatic driver mutations of the host genome. Both amplification and point mutations of interferon regulatory factor 4 (IRF4) have been previously detected in ATL., K59R is the most common single-nucleotide variation of IRF4 and is found exclusively in ATL. High-throughput whole-exome sequencing revealed recurrent activating genetic alterations in the T-cell receptor, CD28, and NF-κB pathways. We found that IRF4, which is transcriptionally activated downstream of these pathways, is frequently mutated in ATL. IRF4 RNA, protein, and IRF4 transcriptional targets are uniformly elevated in HTLV-1-transformed cells and ATL cell lines, and IRF4 was bound to genomic regulatory DNA of many of these transcriptional targets in HTLV-1-transformed cell lines. We further noted that the K59R IRF4 mutant is expressed at higher levels in the nucleus than WT IRF4 and is transcriptionally more active. Expression of both WT and the K59R mutant of IRF4 from a constitutive promoter in retrovirally transduced murine bone marrow cells increased the abundance of T lymphocytes but not myeloid cells or B lymphocytes in mice. IRF4 may represent a therapeutic target in ATL because ATL cells select for a mutant of IRF4 with higher nuclear expression and transcriptional activity, and overexpression of IRF4 induces the expansion of T lymphocytes .
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http://dx.doi.org/10.1074/jbc.RA117.000164DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5936815PMC
May 2018

Knowledge-based prediction of protein backbone conformation using a structural alphabet.

PLoS One 2017 21;12(11):e0186215. Epub 2017 Nov 21.

Université de Nantes, Unité Fonctionnalité et Ingénierie des Protéines (UFIP), UMR 6286 CNRS, UFR Sciences et Techniques, 2, chemin de la Houssinière, France.

Libraries of structural prototypes that abstract protein local structures are known as structural alphabets and have proven to be very useful in various aspects of protein structure analyses and predictions. One such library, Protein Blocks, is composed of 16 standard 5-residues long structural prototypes. This form of analyzing proteins involves drafting its structure as a string of Protein Blocks. Predicting the local structure of a protein in terms of protein blocks is the general objective of this work. A new approach, PB-kPRED is proposed towards this aim. It involves (i) organizing the structural knowledge in the form of a database of pentapeptide fragments extracted from all protein structures in the PDB and (ii) applying a knowledge-based algorithm that does not rely on any secondary structure predictions and/or sequence alignment profiles, to scan this database and predict most probable backbone conformations for the protein local structures. Though PB-kPRED uses the structural information from homologues in preference, if available. The predictions were evaluated rigorously on 15,544 query proteins representing a non-redundant subset of the PDB filtered at 30% sequence identity cut-off. We have shown that the kPRED method was able to achieve mean accuracies ranging from 40.8% to 66.3% depending on the availability of homologues. The impact of the different strategies for scanning the database on the prediction was evaluated and is discussed. Our results highlight the usefulness of the method in the context of proteins without any known structural homologues. A scoring function that gives a good estimate of the accuracy of prediction was further developed. This score estimates very well the accuracy of the algorithm (R2 of 0.82). An online version of the tool is provided freely for non-commercial usage at http://www.bo-protscience.fr/kpred/.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0186215PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5697859PMC
December 2017

Somatic Variants in the Human Lens Epithelium: A Preliminary Assessment.

Invest Ophthalmol Vis Sci 2016 08;57(10):4063-75

Department of Ophthalmology and Visual Sciences, Washington University School of Medicine, St. Louis, Missouri, United States.

Purpose: We hypothesize that somatic mutations accumulate in cells of the human lens and may contribute to the development of cortical or posterior sub-capsular cataracts. Here, we used a Next-generation sequencing (NGS) strategy to screen for low-allelic frequency variants in DNA extracted from human lens epithelial samples.

Methods: Next-Generation sequencing of 151 cancer-related genes (WUCaMP2 panel) was performed on DNA extracted from post-mortem or surgical specimens obtained from 24 individuals. Usually, pairwise comparisons were made between two or more ocular samples from the same individual, allowing putative somatic variants detected in lens samples to be differentiated from germline variants.

Results: Use of a targeted hybridization approach enabled high sequence coverage (>1000-fold) of the WUCaMP2 genes. In addition to high-frequency variants (corresponding to homozygous or heterozygous SNPs and Indels), somatic variants with allelic frequencies of 1-4% were detected in the lens epithelial samples. The presence of one such variant, a T > C point substitution at position 32907082 in BRCA2, was verified subsequently using droplet digital PCR.

Conclusions: Low-allelic fraction variants are present in the human lens epithelium, at frequencies consistent with the presence of millimeter-sized clones.
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http://dx.doi.org/10.1167/iovs.16-19726DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4986767PMC
August 2016

Proteinase activated receptor-2-mediated dual oxidase-2 up-regulation is involved in enhanced airway reactivity and inflammation in a mouse model of allergic asthma.

Immunology 2015 Jul;145(3):391-403

Department of Pharmacology & Toxicology, College of Pharmacy, King Saud University, Riyadh, Saudi Arabia.

Airway epithelial cells (AECs) express a variety of receptors, which sense danger signals from various aeroallergens/pathogens being inhaled constantly. Proteinase-activated receptor 2 (PAR-2) is one such receptor and is activated by cockroach allergens, which have intrinsic serine proteinase activity. Recently, dual oxidases (DUOX), especially DUOX-2, have been shown to be involved in airway inflammation in response to Toll-like receptor activation. However, the association between PAR-2 and DUOX-2 has not been explored in airways of allergic mice. Therefore, this study investigated the contribution of DUOX-2/reactive oxygen species (ROS) signalling in airway reactivity and inflammation after PAR-2 activation. Mice were sensitized intraperitoneally with intact cockroach allergen extract (CE) in the presence of aluminium hydroxide followed by intranasal challenge with CE. Mice were then assessed for airway reactivity, inflammation, oxidative stress (DUOX-2, ROS, inducible nitric oxide synthase, nitrite, nitrotyrosine and protein carbonyls) and apoptosis (Bax, Bcl-2, caspase-3). Challenge with CE led to up-regulation of DUOX-2 and ROS in AECs with concomitant increases in airway reactivity/inflammation and parameters of oxidative stress, and apoptosis. All of these changes were significantly inhibited by intranasal administration of ENMD-1068, a small molecule antagonist of PAR-2 in allergic mice. Administration of diphenyliodonium to allergic mice also led to improvement of allergic airway responses via inhibition of the DUOX-2/ROS pathway; however, these effects were less pronounced than PAR-2 antagonism. The current study suggests that PAR-2 activation leads to up-regulation of the DUOX-2/ROS pathway in AECs, which is involved in regulation of airway reactivity and inflammation via oxidative stress and apoptosis.
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http://dx.doi.org/10.1111/imm.12453DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4479538PMC
July 2015

Performance of common analysis methods for detecting low-frequency single nucleotide variants in targeted next-generation sequence data.

J Mol Diagn 2014 Jan 5;16(1):75-88. Epub 2013 Nov 5.

Department of Pathology and Immunology, Washington University, St. Louis, Missouri. Electronic address:

Next-generation sequencing (NGS) is becoming a common approach for clinical testing of oncology specimens for mutations in cancer genes. Unlike inherited variants, cancer mutations may occur at low frequencies because of contamination from normal cells or tumor heterogeneity and can therefore be challenging to detect using common NGS analysis tools, which are often designed for constitutional genomic studies. We generated high-coverage (>1000×) NGS data from synthetic DNA mixtures with variant allele fractions (VAFs) of 25% to 2.5% to assess the performance of four variant callers, SAMtools, Genome Analysis Toolkit, VarScan2, and SPLINTER, in detecting low-frequency variants. SAMtools had the lowest sensitivity and detected only 49% of variants with VAFs of approximately 25%; whereas the Genome Analysis Toolkit, VarScan2, and SPLINTER detected at least 94% of variants with VAFs of approximately 10%. VarScan2 and SPLINTER achieved sensitivities of 97% and 89%, respectively, for variants with observed VAFs of 1% to 8%, with >98% sensitivity and >99% positive predictive value in coding regions. Coverage analysis demonstrated that >500× coverage was required for optimal performance. The specificity of SPLINTER improved with higher coverage, whereas VarScan2 yielded more false positive results at high coverage levels, although this effect was abrogated by removing low-quality reads before variant identification. Finally, we demonstrate the utility of high-sensitivity variant callers with data from 15 clinical lung cancers.
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http://dx.doi.org/10.1016/j.jmoldx.2013.09.003DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3873500PMC
January 2014

Ocular chemical injuries and their management.

Oman J Ophthalmol 2013 May;6(2):83-6

Department of Ophthalmology, V. C. S. G. Government Medical Sciences and Research Institute, Srinagar, Garhwal, Uttarakhand, India.

Chemical burns represent potentially blinding ocular injuries and constitute a true ocular emergency requiring immediate assessment and initiation of treatment. The majority of victims are young and exposure occurs at home, work place and in association with criminal assaults. Alkali injuries occur more frequently than acid injuries. Chemical injuries of the eye produce extensive damage to the ocular surface epithelium, cornea, anterior segment and limbal stem cells resulting in permanent unilateral or bilateral visual impairment. Emergency management if appropriate may be single most important factor in determining visual outcome. This article reviews the emergency management and newer techniques to improve the prognosis of patients with chemical injuries.
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http://dx.doi.org/10.4103/0974-620X.116624DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3779420PMC
May 2013

Cancer missense mutations alter binding properties of proteins and their interaction networks.

PLoS One 2013 14;8(6):e66273. Epub 2013 Jun 14.

National Center for Biotechnology Information, National Library of Medicine, National Institutes of Health, Bethesda, Maryland, United States of America.

Many studies have shown that missense mutations might play an important role in carcinogenesis. However, the extent to which cancer mutations might affect biomolecular interactions remains unclear. Here, we map glioblastoma missense mutations on the human protein interactome, model the structures of affected protein complexes and decipher the effect of mutations on protein-protein, protein-nucleic acid and protein-ion binding interfaces. Although some missense mutations over-stabilize protein complexes, we found that the overall effect of mutations is destabilizing, mostly affecting the electrostatic component of binding energy. We also showed that mutations on interfaces resulted in more drastic changes of amino acid physico-chemical properties than mutations occurring outside the interfaces. Analysis of glioblastoma mutations on interfaces allowed us to stratify cancer-related interactions, identify potential driver genes, and propose two dozen additional cancer biomarkers, including those specific to functions of the nervous system. Such an analysis also offered insight into the molecular mechanism of the phenotypic outcomes of mutations, including effects on complex stability, activity, binding and turnover rate. As a result of mutated protein and gene network analysis, we observed that interactions of proteins with mutations mapped on interfaces had higher bottleneck properties compared to interactions with mutations elsewhere on the protein or unaffected interactions. Such observations suggest that genes with mutations directly affecting protein binding properties are preferably located in central network positions and may influence critical nodes and edges in signal transduction networks.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0066273PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3682950PMC
January 2014

A pharmacological profile of ribavirin and monitoring of its plasma concentration in chronic hepatitis C infection.

J Clin Exp Hepatol 2012 Mar 12;2(1):42-54. Epub 2012 Apr 12.

Department of Pharmacology and Clinical Pharmacology, Christian Medical College, Vellore - 632002, Tamil Nadu, India.

Chronic hepatitis C (CHC) infection, usually an asymptomatic infection, has long-term serious complications such as cirrhosis, hepatocellular carcinoma, and end-stage liver disease requiring liver transplantation (LT). Several novel drugs against hepatitis C which form part of 'specifically targeted antiviral therapy for hepatitis C' (STAT-C) have been developed. These include NS3/4A protease inhibitors telaprevir, boceprevir, and nucleoside/non-nucleoside polymerase inhibitors (NS5A) which hold promise for future therapy. Despite the development of new anti-hepatitis C virus (HCV) drugs, ribavirin (RBV) remains the single most important drug to prevent relapse and is frequently included among newer regimens being developed with novel small molecule anti-HCV drugs. The current approved treatment is a combination therapy of once weekly subcutaneous pegylated-interferon (PEG-IFN)-α plus body-weight-based oral RBV regimen. The most significant dose-dependent side effect of RBV is hemolytic anemia warranting dose reduction or discontinuation in severe cases compromising sustained virological response (SVR). Monitoring RBV plasma concentration has been challenging due to its peculiar pharmacokinetics and has been done to predict both efficacy and toxicity. Herein, we review the pharmacological profile of RBV and the monitoring of its plasma concentration, monitoring in renal impairment, post-LT, and human immunodeficiency virus (HIV)-HCV co-infection in patients being treated with combination therapy of PEG-IFN-α and RBV.
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http://dx.doi.org/10.1016/S0973-6883(12)60090-5DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3940409PMC
March 2012

Homology inference of protein-protein interactions via conserved binding sites.

PLoS One 2012 31;7(1):e28896. Epub 2012 Jan 31.

National Center for Biotechnology Information, National Library of Medicine, National Institutes of Health, Bethesda, Maryland, United States of America.

The coverage and reliability of protein-protein interactions determined by high-throughput experiments still needs to be improved, especially for higher organisms, therefore the question persists, how interactions can be verified and predicted by computational approaches using available data on protein structural complexes. Recently we developed an approach called IBIS (Inferred Biomolecular Interaction Server) to predict and annotate protein-protein binding sites and interaction partners, which is based on the assumption that the structural location and sequence patterns of protein-protein binding sites are conserved between close homologs. In this study first we confirmed high accuracy of our method and found that its accuracy depends critically on the usage of all available data on structures of homologous complexes, compared to the approaches where only a non-redundant set of complexes is employed. Second we showed that there exists a trade-off between specificity and sensitivity if we employ in the prediction only evolutionarily conserved binding site clusters or clusters supported by only one observation (singletons). Finally we addressed the question of identifying the biologically relevant interactions using the homology inference approach and demonstrated that a large majority of crystal packing interactions can be correctly identified and filtered by our algorithm. At the same time, about half of biological interfaces that are not present in the protein crystallographic asymmetric unit can be reconstructed by IBIS from homologous complexes without the prior knowledge of crystal parameters of the query protein.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0028896PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3269416PMC
June 2012

Large-scale mapping of human protein interactome using structural complexes.

EMBO Rep 2012 Mar 1;13(3):266-71. Epub 2012 Mar 1.

National Center for Biotechnology Information, US National Library of Medicine, National Institutes of Health, 8600 Rockville Pike, Bethesda, Maryland 20894, USA.

Although the identification of protein interactions by high-throughput (HTP) methods progresses at a fast pace, 'interactome' data sets still suffer from high rates of false positives and low coverage. To map the human protein interactome, we describe a new framework that uses experimental evidence on structural complexes, the atomic details of binding interfaces and evolutionary conservation. The structurally inferred interaction network is highly modular and more functionally coherent compared with experimental interaction networks derived from multiple literature citations. Moreover, structurally inferred and high-confidence HTP networks complement each other well, allowing us to construct a merged network to generate testable hypotheses and provide valuable experimental leads.
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http://dx.doi.org/10.1038/embor.2011.261DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3296913PMC
March 2012

IBIS (Inferred Biomolecular Interaction Server) reports, predicts and integrates multiple types of conserved interactions for proteins.

Nucleic Acids Res 2012 Jan 18;40(Database issue):D834-40. Epub 2011 Nov 18.

National Center for Biotechnology Information, National Library of Medicine, National Institutes of Health, 8600 Rockville Pike, Building 38A, Bethesda, MD 20894, USA.

We have recently developed the Inferred Biomolecular Interaction Server (IBIS) and database, which reports, predicts and integrates different types of interaction partners and locations of binding sites in proteins based on the analysis of homologous structural complexes. Here, we highlight several new IBIS features and options. The server's webpage is now redesigned to allow users easier access to data for different interaction types. An entry page is added to give a quick summary of available results and to now accept protein sequence accessions. To elucidate the formation of protein complexes, not just binary interactions, IBIS currently presents an expandable interaction network. Previously, IBIS provided annotations for four different types of binding partners: proteins, small molecules, nucleic acids and peptides; in the current version a new protein-ion interaction type has been added. Several options provide easy downloads of IBIS data for all Protein Data Bank (PDB) protein chains and the results for each query. In this study, we show that about one-third of all RefSeq sequences can be annotated with IBIS interaction partners and binding sites. The IBIS server is available at http://www.ncbi.nlm.nih.gov/Structure/ibis/ibis.cgi and updated biweekly.
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http://dx.doi.org/10.1093/nar/gkr997DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3245142PMC
January 2012

A short survey on protein blocks.

Biophys Rev 2010 Aug 5;2(3):137-147. Epub 2010 Aug 5.

DSIMB, Dynamique des Structures et Interactions des Macromolécules Biologiques Université Paris-Diderot - Paris VII INTS INSERM : U665 INTS, 6 rue Alexandre Cabanel, 75739 Paris Cedex 15 FRANCE,FR.

Protein structures are classically described in terms of secondary structures. Even if the regular secondary structures have relevant physical meaning, their recognition from atomic coordinates has some important limitations such as uncertainties in the assignment of boundaries of helical and β-strand regions. Further, on an average about 50% of all residues are assigned to an irregular state, i.e., the coil. Thus different research teams have focused on abstracting conformation of protein backbone in the localized short stretches. Using different geometric measures, local stretches in protein structures are clustered in a chosen number of states. A prototype representative of the local structures in each cluster is generally defined. These libraries of local structures prototypes are named as "structural alphabets". We have developed a structural alphabet, named Protein Blocks, not only to approximate the protein structure, but also to predict them from sequence. Since its development, we and other teams have explored numerous new research fields using this structural alphabet. We review here some of the most interesting applications.
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http://dx.doi.org/10.1007/s12551-010-0036-1DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3124139PMC
August 2010

Knowledge-based annotation of small molecule binding sites in proteins.

BMC Bioinformatics 2010 Jul 1;11:365. Epub 2010 Jul 1.

National Center for Biotechnology Information, 8600 Rockville Pike, Building 38A, Bethesda, MD 20894, USA.

Background: The study of protein-small molecule interactions is vital for understanding protein function and for practical applications in drug discovery. To benefit from the rapidly increasing structural data, it is essential to improve the tools that enable large scale binding site prediction with greater emphasis on their biological validity.

Results: We have developed a new method for the annotation of protein-small molecule binding sites, using inference by homology, which allows us to extend annotation onto protein sequences without experimental data available. To ensure biological relevance of binding sites, our method clusters similar binding sites found in homologous protein structures based on their sequence and structure conservation. Binding sites which appear evolutionarily conserved among non-redundant sets of homologous proteins are given higher priority. After binding sites are clustered, position specific score matrices (PSSMs) are constructed from the corresponding binding site alignments. Together with other measures, the PSSMs are subsequently used to rank binding sites to assess how well they match the query and to better gauge their biological relevance. The method also facilitates a succinct and informative representation of observed and inferred binding sites from homologs with known three-dimensional structures, thereby providing the means to analyze conservation and diversity of binding modes. Furthermore, the chemical properties of small molecules bound to the inferred binding sites can be used as a starting point in small molecule virtual screening. The method was validated by comparison to other binding site prediction methods and to a collection of manually curated binding site annotations. We show that our method achieves a sensitivity of 72% at predicting biologically relevant binding sites and can accurately discriminate those sites that bind biological small molecules from non-biological ones.

Conclusions: A new algorithm has been developed to predict binding sites with high accuracy in terms of their biological validity. It also provides a common platform for function prediction, knowledge-based docking and for small molecule virtual screening. The method can be applied even for a query sequence without structure. The method is available at http://www.ncbi.nlm.nih.gov/Structure/ibis/ibis.cgi.
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http://dx.doi.org/10.1186/1471-2105-11-365DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2909224PMC
July 2010

Inferred Biomolecular Interaction Server--a web server to analyze and predict protein interacting partners and binding sites.

Nucleic Acids Res 2010 Jan 20;38(Database issue):D518-24. Epub 2009 Oct 20.

National Center for Biotechnology Information, National Library of Medicine, National Institutes of Health, Bethesda, MD 20894, USA.

IBIS is the NCBI Inferred Biomolecular Interaction Server. This server organizes, analyzes and predicts interaction partners and locations of binding sites in proteins. IBIS provides annotations for different types of binding partners (protein, chemical, nucleic acid and peptides), and facilitates the mapping of a comprehensive biomolecular interaction network for a given protein query. IBIS reports interactions observed in experimentally determined structural complexes of a given protein, and at the same time IBIS infers binding sites/interacting partners by inspecting protein complexes formed by homologous proteins. Similar binding sites are clustered together based on their sequence and structure conservation. To emphasize biologically relevant binding sites, several algorithms are used for verification in terms of evolutionary conservation, biological importance of binding partners, size and stability of interfaces, as well as evidence from the published literature. IBIS is updated regularly and is freely accessible via http://www.ncbi.nlm.nih.gov/Structure/ibis/ibis.html.
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http://dx.doi.org/10.1093/nar/gkp842DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2808861PMC
January 2010

Protein short loop prediction in terms of a structural alphabet.

Comput Biol Chem 2009 Aug 25;33(4):329-33. Epub 2009 Jun 25.

Laboratoire de Biochimie et Génétique Moléculaire, Université de La Réunion, BP 7151, 15 avenue René Cassin, 97715 Saint Denis Messag Cedex 09, La Réunion, France.

Loops connect regular secondary structures. In many instances, they are known to play crucial biological roles. To bypass the limitation of secondary structure description, we previously defined a structural alphabet composed of 16 structural prototypes, called Protein Blocks (PBs). It leads to an accurate description of every region of 3D protein backbones and has been used in local structure prediction. In the present study, we used our structural alphabet to predict the loops connecting two repetitive structures. Thus, we showed interest to take into account the flanking regions, leading to prediction rate improvement up to 19.8%, but we also underline the sensitivity of such an approach. This research can be used to propose different structures for the loops and to probe and sample their flexibility. It is a useful tool for ab initio loop prediction and leads to insights into flexible docking approach.
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http://dx.doi.org/10.1016/j.compbiolchem.2009.06.002DOI Listing
August 2009

Analysis of loop boundaries using different local structure assignment methods.

Protein Sci 2009 Sep;18(9):1869-81

Laboratoire de Biochimie et Génétique Moléculaire, Université de La Réunion, BP 7151, 15 avenue René Cassin, 97715 Saint Denis Messag Cedex 09, La Réunion, France.

Loops connect regular secondary structures. In many instances, they are known to play important biological roles. Analysis and prediction of loop conformations depend directly on the definition of repetitive structures. Nonetheless, the secondary structure assignment methods (SSAMs) often lead to divergent assignments. In this study, we analyzed, both structure and sequence point of views, how the divergence between different SSAMs affect boundary definitions of loops connecting regular secondary structures. The analysis of SSAMs underlines that no clear consensus between the different SSAMs can be easily found. Because these latter greatly influence the loop boundary definitions, important variations are indeed observed, that is, capping positions are shifted between different SSAMs. On the other hand, our results show that the sequence information in these capping regions are more stable than expected, and, classical and equivalent sequence patterns were found for most of the SSAMs. This is, to our knowledge, the most exhaustive survey in this field as (i) various databank have been used leading to similar results without implication of protein redundancy and (ii) the first time various SSAMs have been used. This work hence gives new insights into the difficult question of assignment of repetitive structures and addresses the issue of loop boundaries definition. Although SSAMs give very different local structure assignments capping sequence patterns remain efficiently stable.
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http://dx.doi.org/10.1002/pro.198DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2777362PMC
September 2009

Exploring functional roles of multibinding protein interfaces.

Protein Sci 2009 Aug;18(8):1674-83

Computational Biology Branch, National Center for Biotechnology Information, National Library of Medicine, National Institutes of Health, Bethesda, Maryland 20894, USA.

Cellular processes are highly interconnected and many proteins are shared in different pathways. Some of these shared proteins or protein families may interact with diverse partners using the same interface regions; such multibinding proteins are the subject of our study. The main goal of our study is to attempt to decipher the mechanisms of specific molecular recognition of multiple diverse partners by promiscuous protein regions. To address this, we attempt to analyze the physicochemical properties of multibinding interfaces and highlight the major mechanisms of functional switches realized through multibinding. We find that only 5% of protein families in the structure database have multibinding interfaces, and multibinding interfaces do not show any higher sequence conservation compared with the background interface sites. We highlight several important functional mechanisms utilized by multibinding families. (a) Overlap between different functional pathways can be prevented by the switches involving nearby residues of the same interfacial region. (b) Interfaces can be reused in pathways where the substrate should be passed from one protein to another sequentially. (c) The same protein family can develop different specificities toward different binding partners reusing the same interface; and finally, (d) inhibitors can attach to substrate binding sites as substrate mimicry and thereby prevent substrate binding.
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http://dx.doi.org/10.1002/pro.181DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2776955PMC
August 2009

HSP20 phosphorylation and airway smooth muscle relaxation.

Cell Health Cytoskelet 2009 Jun;2009(1):27-42

Department of Pharmacology, University of Nevada School of Medicine, Reno, NV, USA.

HSP20 (HSPB6) is a small heat shock protein expressed in smooth muscles that is hypothesized to inhibit contraction when phosphorylated by cAMP-dependent protein kinase. To investigate this hypothesis in airway smooth muscle (ASM) we showed that HSP20 was constitutively expressed as well as being inducible in cultured hASM cells by treatment with 1 μM isoproterenol or 10 μM salmeterol. In contrast, a mixture of proinflammatory mediators (interleukin-1β, tumor necrosis factor α, and interferon γ) inhibited expression of HSP20 by about 50% in 48 hours. To determine whether phosphorylation of HSP20 is sufficient to induce relaxation, canine tracheal smooth muscle was treated with a cell permeant phosphopeptide that mimics the phosphorylation of HSP20. The HSP20 phosphopeptide antagonized carbachol-induced contraction by 60% with no change in myosin light chain phosphorylation. Recombinant full length HSP20 inhibited skeletal actin binding to smooth muscle myosin subfragment 1 (S1), and recombinant cell permeant TAT-HSP20 S16D mutant reduced F-actin filaments in cultured hASM cells. Carbachol stimulation of canine tracheal smooth muscle tissue caused redistribution of HSP20 from large macromolecular complexes (200-500 kDa) to smaller complexes (<60 kDa). The results are consistent with HSP20 expression and macromolecular structure being dynamically regulated in airway smooth muscle. HSP20 is upregulated by beta agonists and downregulated by proinflammatory cytokines. HSP20 is phosphorylated in vivo in a cAMP-dependent manner and the phosphorylated form promotes airway smooth muscle relaxation, possibly through depolymerization of F-actin as well as inhibition of myosin binding to actin.
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http://dx.doi.org/10.2147/chc.s5783DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3113651PMC
June 2009

Small heat shock proteins in smooth muscle.

Pharmacol Ther 2008 Jul 16;119(1):44-54. Epub 2008 May 16.

Department of Neurology, Oregon Health Sciences University, Portland, OR, USA.

The small heat shock proteins (HSPs) HSP20, HSP27 and alphaB-crystallin are chaperone proteins that are abundantly expressed in smooth muscles are important modulators of muscle contraction, cell migration and cell survival. This review focuses on factors regulating expression of small HSPs in smooth muscle, signaling pathways that regulate macromolecular structure and the biochemical and cellular functions of small HSPs. Cellular processes regulated by small HSPs include chaperoning denatured proteins, maintaining cellular redox state and modifying filamentous actin polymerization. These processes influence smooth muscle proliferation, cell migration, cell survival, muscle contraction and synthesis of signaling proteins. Understanding functions of small heat shock proteins is relevant to mechanisms of disease in which dysfunctional smooth muscle causes symptoms, or is a target of drug therapy. One example is that secreted HSP27 may be a useful marker of inflammation during atherogenesis. Another is that phosphorylated HSP20 which relaxes smooth muscle may prove to be highly relevant to treatment of hypertension, vasospasm, asthma, premature labor and overactive bladder. Because small HSPs also modulate smooth muscle proliferation and cell migration they may prove to be targets for developing effective, novel treatments of clinical problems arising from remodeling of smooth muscle in vascular, respiratory and urogenital systems.
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http://dx.doi.org/10.1016/j.pharmthera.2008.04.005DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2581864PMC
July 2008

Newer insights into renal regulation of water homeostasis.

Indian J Exp Biol 2008 Feb;46(2):89-93

Department of Pharmacology, Christian Medical College, Vellore 632002, India.

The regulation of high osmolality is an important driving force for water reabsorption and urinary concentration--the key functions of the kidney for maintaining optimum body fluid volume. New evidence shows that transcription factor tonicity responsive enhancer binding protein (TonEBP) and calcineurin-nuclear factor of activated T cells through cross-talk enhance Aquaporin 2 (AQP2) expression. AQP2 is the predominant vasopressin regulated water channel of the kidney collecting duct and is essential for urinary concentration. The serine/threonine phosphatase calcineurin is an important signaling molecule involved in kidney development and function. One potential target of calcineurin action is the water channel AQP2. The nuclear factor of activated T cells (NFAT) family has recently been expanded by the discovery of a new member, NFAT 5, or Ton EBP. Ton EBP is the only known mammalian transcription factor that regulates gene expression in response to hypertonicity. This review examines the importance of AQP2, calcineurin, NFATc and TonEBP in the renal regulation of water homeostasis.
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February 2008

Acetyl salicylic acid augments functional recovery following sciatic nerve crush in mice.

J Brachial Plex Peripher Nerve Inj 2007 Feb 4;2. Epub 2007 Feb 4.

Department of Pharmacology & Clinical Pharmacology, Christian Medical College, Vellore-632002, India.

Cyclin-dependent kinase 5 (CDK-5) appears to play a significant role in peripheral nerve regeneration as CDK-5 inhibition retards nerve regeneration following nerve crush. Anti-inflammatory drug acetyl salicylic acid elevates CDK-5 and reduces ischemia - reperfusion injury in cultured neurons. In this study we have evaluated the effect of acetyl salicylic acid on functional recovery following sciatic nerve crush in mice. Eighteen Swiss albino mice underwent unilateral sciatic nerve crush. Test animals received acetyl salicylic acid (100 mg/kg/day, n = 6 or 50 mg/kg/day, n = 6) and control animals (n = 6) received normal saline for 14 days following surgery. Functional recovery was assessed with improvement in Sciatic Function Index, nociception and gait. In comparison with normal saline treatment, acetyl salicylic acid (100 mg/kg/day) significantly improved functional recovery following sciatic nerve crush. Anti-inflammatory drug acetyl salicylic acid appears to be a promising agent for treating peripheral nerve injuries and hence elucidation of its neuroprotective pathways is necessary.
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http://dx.doi.org/10.1186/1749-7221-2-3DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1802865PMC
February 2007

PAF antagonism modifies neuroprotective action of histone deacetylase and calcineurin phosphatase inhibitors in mice.

Indian J Exp Biol 2006 Nov;44(11):886-91

Department of Pharmacology and Clinical Pharmacology, Christian Medical College, Vellore 632 002, India.

To evaluate the hypothesis that platelet activating factor (PAF) antagonism may affect the functional recovery following the nerve injuries and also to evaluate the effect of PAF receptor antagonism on the neuroprotective effect of tacrolimus and sodium valproate, effect of PAF receptor antagonist, WEB2086 was evaluated in animal models of sciatic nerve crush and endothelin-1 induced focal cerebral ischemia. WEB2086, per se, while attenuating spontaneous sensory motor recovery after sciatic nerve crush, enhanced functional recovery after focal cerebral ischemia. WEB2086 also attenuated the neuroprotective effect of tacrolimus and sodium valproate subsequent to peripheral nerve injury, while it significantly improved the neuroprotective action of tacrolimus and sodium valproate following cerebral ischemia reperfusion injury. These results suggest that PAF receptor antagonists alone and in combination with tacrolimus/sodium valproate could be used in the treatment of cerebral ischemia reperfusion injuries however, their use following peripheral nerve injuries could be detrimental.
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November 2006

A substitution matrix for structural alphabet based on structural alignment of homologous proteins and its applications.

Proteins 2006 Oct;65(1):32-9

Laboratoire de Biochimie et Génétique Moléculaire, Université de La Réunion, BP 7151, 15 avenue René Cassin, 97715 Saint Denis Messag Cedex 09, La Réunion, France.

Analysis of protein structures based on backbone structural patterns known as structural alphabets have been shown to be very useful. Among them, a set of 16 pentapeptide structural motifs known as protein blocks (PBs) has been identified and upon which backbone model of most protein structures can be built. PBs allows simplification of 3D space onto 1D space in the form of sequence of PBs. Here, for the first time, substitution probabilities of PBs in a large number of aligned homologous protein structures have been studied and are expressed as a simplified 16 x 16 substitution matrix. The matrix was validated by benchmarking how well it can align sequences of PBs rather like amino acid alignment to identify structurally equivalent regions in closely or distantly related proteins using dynamic programming approach. The alignment results obtained are very comparable to well established structure comparison methods like DALI and STAMP. Other interesting applications of the matrix have been investigated. We first show that, in variable regions between two superimposed homologous proteins, one can distinguish between local conformational differences and rigid-body displacement of a conserved motif by comparing the PBs and their substitution scores. Second, we demonstrate, with the example of aspartic proteinases, that PBs can be efficiently used to detect the lobe/domain flexibility in the multidomain proteins. Lastly, using protein kinase as an example, we identify regions of conformational variations and rigid body movements in the enzyme as it is changed to the active state from an inactive state.
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http://dx.doi.org/10.1002/prot.21087DOI Listing
October 2006

Influence of erythrocyte function-enhancing drugs on the bronchoprotective actions of chemokine receptor blockers in mice.

Med Sci Monit 2006 Aug 12;12(8):BR279-82. Epub 2006 Jul 12.

Department of Pharmacology, Christian Medical College, Vellore, India.

Background: CC chemokine receptors are likely to have important roles in the regulation of leukocytes and mast cells. Chemokine receptors are crucial in orchestrating innate and acquired immune responses as well as in allergic inflammation. A disturbance in erythrocyte function can lead to bronchial hyperreactivity and asthma.

Material/methods: This study was conducted on Swiss albino mice weighing between 25 to 35 g. The animals were divided into eight groups (n=8). Groups of mice (n=8) were pretreated with the chemokine receptor blockers A122058 (600 microg/kg i.p.) or cyclophosphamide (20 mg/kg i.p.). Other groups received A122058 or cyclophosphamide in combination with either erythropoietin and quercetin. The effects of these drugs on bronchoconstriction and salivation induced by carbachol were evaluated.

Results: The results of this study suggest that blockade of chemokine receptors significantly potentiated erythropoietin- and quercetin-induced inhibition of carbachol-induced bronchoconstriction (P<0.05) compared with the control group. However, pentoxifylline did not produce significant bronchoprotection against carbachol-induced bronchoconstriction (P>0.05).

Conclusions: The results of this study suggest that blockade of chemokine receptors and enhancement of the erythrocyte function together potentiate the bronchoprotective effects of these drugs. This combination could be a novel strategy in combating bronchial hyper-responsiveness.
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August 2006
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