Publications by authors named "Rauf Bhat"

15 Publications

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Sequence and phylogentic analysis of MERS-CoV in Saudi Arabia, 2012-2019.

Virol J 2021 04 30;18(1):90. Epub 2021 Apr 30.

Department of Botany and Microbiology, College of Science, King Saud University, Riyadh, 11451, Saudi Arabia.

Background: The Middle East Respiratory Syndrome-related Coronavirus (MERS-CoV) continues to exist in the Middle East sporadically. Thorough investigations of the evolution of human coronaviruses (HCoVs) are urgently required. In the current study, we studied amplified fragments of ORF1a/b, Spike (S) gene, ORF3/4a, and ORF4b of four human MERS-CoV strains for tracking the evolution of MERS-CoV over time.

Methods: RNA isolated from nasopharyngeal aspirate, sputum, and tracheal swabs/aspirates from hospitalized patients with suspected MERS-CoV infection were analyzed for amplification of nine variable genomic fragments. Sequence comparisons were done using different bioinformatics tools available.

Results: Several mutations were identified in ORF1a/b, ORF3/4a and ORF4b, with the highest mutation rates in the S gene. Five codons; 4 in ORF1a and 1 in the S gene, were found to be under selective pressure. Characteristic amino acid changes, potentially hosted and year specific were defined across the S protein and in the receptor-binding domain Phylogenetic analysis using S gene sequence revealed clustering of MERS-CoV strains into three main clades, A, B and C with subdivision of with clade B into B1 to B4.

Conclusions: In conclusion, MERS-CoV appears to continuously evolve. It is recommended that the molecular and pathobiological characteristics of future MERS-CoV strains should be analyzed on regular basis to prevent potential future outbreaks at early phases.
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http://dx.doi.org/10.1186/s12985-021-01563-7DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8085657PMC
April 2021

Induction of Immune Responses and Immune Evasion by Human Bocavirus.

Int Arch Allergy Immunol 2021 Apr 19:1-8. Epub 2021 Apr 19.

Virology Research Group, Department of Botany and Microbiology, College of Science, King Saud University, Riyadh, Saudi Arabia.

Respiratory tract infections are the primary cause of morbidity and mortality globally. Human bocavirus 1 (HBoV1), a member of the Parvoviridae family causes a wide spectrum of respiratory diseases in children, and gastroenteritis in adults. The mechanisms of latency, persistence, and reinfection of Bocavirus are poorly understood at present due to the lack of permissive cell lines and efficient animal models. Moreover, the dual infections of HBoV and other respiratory viruses further complicate the study of the pathogenicity of Bocaviruses. The data on immunological consequences of Bocavirus infection are sparse. However, the existing data have highlighted the role of CD4 T cells in Bocavirus infection. High titres of HBoV-specific antibodies have been detected in different populations suggesting its ubiquitous prevalence. Interestingly, the mechanism employed by Bocavirus to evade the immune system mostly targets type I IFN pathways and cause pyroptotic cell death of host cells. This review summarizes the immune responses evoked in response to Bocavirus infection, escape mechanism employed by the virus, and the vaccination strategies, including antisense technology to combat Bocavirus infections.
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http://dx.doi.org/10.1159/000514688DOI Listing
April 2021

Double-edged role of natural killer cells during RSV infection.

Int Rev Immunol 2020 29;39(5):233-244. Epub 2020 May 29.

Virology Research Group, Department of Botany and Microbiology, College of Science, King Saud University, Riyadh, Saudi Arabia.

Natural killer cells play a vital role in the rejection of tumors and pathogen-infected cells. NK cells are indispensable in the early immune response against viral infections by directly targeting infected cells. Furthermore, NK cells influence adaptive immunity by driving virus-specific T-cell responses. Respiratory syncytial virus, a highly contagious virus that causes bronchiolitis, is the main reason for mortality in infants and elderly patients. RSV infection triggers both innate and adaptive immune responses. However, immunity against RSV is ephemeral due to the impaired development of immunological memory. The role of NK cells during RSV infection remains ambiguous. NK cells play a dual role in RSV infection; initially, their role is a protective one as they utilize their intrinsic cytotoxicity, followed by a detrimental one that induces lung injury due to the inhibition of antibody responses and the secretion of pro-inflammatory factors. Noteworthy, IFN-γ released from NK cells play a critical role in promoting a shift to adaptive responses and inhibiting antibody responses in neonates. Indeed, NK cells have a pro-inflammatory and inhibitory role rather than a cytotoxic one that contributes to the severity of the disease. Therapeutic options, including DNA-protein-based vaccines, synthetic peptides, and attenuated strains, are presently under tests. However, there is a need for effective strategies to augment NK cell activity and circumvent the pro-inflammatory activity to benefit the host. In this review, we focused on the role played by NK cells in the immune response and its outcome on the immunopathogenesis of RSV disease.
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http://dx.doi.org/10.1080/08830185.2020.1770748DOI Listing
May 2020

Oncolytic H-1 Parvovirus Shows Safety and Signs of Immunogenic Activity in a First Phase I/IIa Glioblastoma Trial.

Mol Ther 2017 Dec 24;25(12):2620-2634. Epub 2017 Aug 24.

Department of Tumor Virology, German Cancer Research Center (DKFZ), Im Neuenheimer Feld 242, 69120 Heidelberg, Germany. Electronic address:

Oncolytic virotherapy may be a means of improving the dismal prognosis of malignant brain tumors. The rat H-1 parvovirus (H-1PV) suppresses tumors in preclinical glioma models, through both direct oncolysis and stimulation of anticancer immune responses. This was the basis of ParvOryx01, the first phase I/IIa clinical trial of an oncolytic parvovirus in recurrent glioblastoma patients. H-1PV (escalating dose) was administered via intratumoral or intravenous injection. Tumors were resected 9 days after treatment, and virus was re-administered around the resection cavity. Primary endpoints were safety and tolerability, virus distribution, and maximum tolerated dose (MTD). Progression-free and overall survival and levels of viral and immunological markers in the tumor and peripheral blood were also investigated. H-1PV treatment was safe and well tolerated, and no MTD was reached. The virus could cross the blood-brain/tumor barrier and spread widely through the tumor. It showed favorable pharmacokinetics, induced antibody formation in a dose-dependent manner, and triggered specific T cell responses. Markers of virus replication, microglia/macrophage activation, and cytotoxic T cell infiltration were detected in infected tumors, suggesting that H-1PV may trigger an immunogenic stimulus. Median survival was extended in comparison with recent meta-analyses. Altogether, ParvOryx01 results provide an impetus for further H-1PV clinical development.
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http://dx.doi.org/10.1016/j.ymthe.2017.08.016DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5768665PMC
December 2017

Co-Activation of Cultured Human Natural Killer Cells: Enhanced Function and Decreased Inhibition.

J Toxicol Environ Health A 2016 ;79(22-23):1078-1084

a Leibniz Research Center for Working Environment and Human Factors (IfADo), TU Dortmund University , Dortmund , Germany.

Natural killer (NK) cells are important immune effector cells that protect the organism against viral infections and cancer. The cytotoxic activity of NK cells is induced by the engagement of a number of different activating surface receptors and controlled by inhibitory receptors to ensure self-tolerance. Resting NK cells need to be co-activated by involvement of at least two distinct activating receptors in order to induce their functional activity. However, in cultured NK cells, which have been expanded in cytokines such as interleukin (IL)-2, the engagement of a single activating receptor may be sufficient to induce their function. Data demonstrated that also cultured NK cells may be co-activated by involvement of certain combinations of activating receptors. This co-activation results in enhanced activation of Vav-1 and ERK signaling pathways and produces greater degranulation. In addition to enhanced functionality, co-activation makes NK cells more resistant to the effect of inhibitory receptors, thereby inducing more potent and efficient NK cell responses.
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http://dx.doi.org/10.1080/15287394.2016.1219587DOI Listing
May 2017

Antigen-armed antibodies targeting B lymphoma cells effectively activate antigen-specific CD4+ T cells.

Blood 2015 Mar 7;125(10):1601-10. Epub 2015 Jan 7.

German Cancer Research Centre (DKFZ) Unit F100, Heidelberg, Germany; Institut National de la Santé et de la Recherche Médicale Unit U1074, Heidelberg, Germany; German Centre for Infection Research (DZIF), Heidelberg, Germany.

The treatment of non-Hodgkin lymphomas has benefited enormously from the introduction of monoclonal antibody-based therapies. However, the efficacy of these treatments varies with lymphoma subtypes and typically decreases with subsequent relapses. Here, we report on antigen-armed antibodies (AgAbs) as a potential treatment of B-cell lymphoma. AgAbs include antigens from ubiquitous pathogens, such as Epstein-Barr virus (EBV), that persist in their host and elicit strong lifelong T-cell responses. They act as vectors by introducing antigen directly into tumor cells to induce an antigen-specific CD4(+) T-cell response against these cells. We have fused antibodies targeting human B-cell surface receptors (CD19-22) to immunodominant T-cell antigens from EBV proteins, including EBNA1, EBNA3B, and EBNA3C. Exposure of EBV-transformed B cells and of Burkitt lymphoma cells to AgAbs led to antigen presentation, T-cell recognition, and target cell killing. The efficiency of AgAb action paralleled the abundance of the targeted molecules on lymphoma cells as well as their HLA class II expression levels. AgAbs can also induce activation and proliferation of EBV-specific memory CD4(+) T cells ex vivo. These studies show the potential of AgAbs as an effective therapeutic strategy against B-cell lymphomas.
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http://dx.doi.org/10.1182/blood-2014-07-591412DOI Listing
March 2015

Emerging role of Natural killer cells in oncolytic virotherapy.

Immunotargets Ther 2015 31;4:65-77. Epub 2015 Mar 31.

Division of Tumor Virology, German Cancer Research Center, Heidelberg, Germany.

Natural killer (NK) cells constitute a subtype of lymphocytes that initiate innate immune responses against tumors and virus-infected cells. The ability of NK cells to kill target cells or to produce cytokines depends on the balance between signals from activating and inhibitory cell-surface receptors. Therapies with NK cells involve activation of endogenous NK cells and/or exogenous transfer by hematopoietic stem cell transplantation/adoptive cell therapy. To exploit the diverse functional abilities of NK cells for cancer immunotherapy, it is important to understand NK cell biology and the underlying regulatory mechanisms. The state of immune suppression prevalent in malignancies creates the need for innovative therapies. Oncolytic viruses are novel anticancer agents showing selective tropism for tumor cells and lacking pathogenicity in humans, but the use of oncolytic virotherapy (OVT) presents multiple challenges. An increasing body of evidence suggests that the host immune response may critically influence the outcome of OVT. Classically, the immune system is thought to limit the efficacy of therapy through virus clearance mediated by innate immune effectors or through adaptive antiviral immune responses eliminating infected cells. Effective strategies do need to be designed in OVT to circumvent the early antiviral activity of NK cells and to augment late NK-cell-mediated antitumor responses. The intrinsic immunostimulating capacity of oncolytic viruses and the possibility of engineering them to express heterologous immunostimulatory molecules (eg, cytokines) support the use of these agents to enhance antitumor immune responses besides inducing direct oncolytic effects. OVT has indeed shown promising therapeutic outcomes in various clinical trials. Here, we review the biology of NK cells, strategies involving NK cells for achieving cancer therapy, and, more particularly, the emerging role of NK cells in OVT.
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http://dx.doi.org/10.2147/ITT.S55549DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4918259PMC
July 2016

NK-cell-dependent killing of colon carcinoma cells is mediated by natural cytotoxicity receptors (NCRs) and stimulated by parvovirus infection of target cells.

BMC Cancer 2013 Jul 31;13:367. Epub 2013 Jul 31.

German Cancer Research Center (DKFZ), Tumor Virology, F010, Im Neuenheimer Feld 242, Heidelberg D-69120, Germany.

Background: Investigating how the immune system functions during malignancies is crucial to developing novel therapeutic strategies. Natural killer (NK) cells, an important component of the innate immune system, play a vital role in immune defense against tumors and virus-infected cells. The poor survival rate in colon cancer makes it particularly important to develop novel therapeutic strategies. Oncolytic viruses, in addition to lysing tumor cells, may have the potential to augment antitumor immune responses. In the present study, we investigate the role of NK cells and how parvovirus H-1PV can modulate NK-cell mediated immune responses against colon carcinoma.

Methods: Human NK cells were isolated from the blood of healthy donors. The cytotoxicity and antibody-mediated inhibition of NK cells were measured in chromium release assays. Phenotypic assessment of colon cancer and dendritic cells was done by FACS. The statistical significance of the results was calculated with Student's t test (*p <0.05; **, p < 0.01; ***, p < 0.001).

Results: We show that IL-2-activated human NK cells can effectively kill colon carcinoma cells. Killing of colon carcinoma cells by NK cells was further enhanced upon infection of the former cells with parvovirus H-1PV. H-1PV has potent oncolytic activity against various tumors, yet its direct killing effect on colon carcinoma cells is limited. The cytotoxicity of NK cells towards colon carcinoma cells, both mock- and H-1PV-infected, was found to be mostly mediated by a combination of natural cytotoxicity receptors (NCRs), namely NKp30, 44, and 46. Colon carcinoma cells displayed low to moderate expression of NK cell ligands, and this expression was modulated upon H-1PV infection. Lysates of H-1PV-infected colon carcinoma cells were found to increase MHC class II expression on dendritic cells.

Conclusions: Altogether, these data suggest that IL-2-activated NK cells actively kill colon carcinoma cells and that this killing is mediated by several natural cytotoxicity receptors (NCRs) in combination. Additionally, in association with parvovirus H-1PV, IL-2-activated NK cells have the potential to boost immune responses against colon cancer.
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http://dx.doi.org/10.1186/1471-2407-13-367DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3733944PMC
July 2013

Antitumoral activity of parvovirus-mediated IL-2 and MCP-3/CCL7 delivery into human pancreatic cancer: implication of leucocyte recruitment.

Cancer Immunol Immunother 2012 Nov 11;61(11):2113-23. Epub 2012 May 11.

Laboratory of Molecular Immunology, Rega Institute for Medical Research, K.U. Leuven, Leuven, Belgium.

Pancreatic ductal adenocarcinoma (PDAC) represents the fourth leading cause of cancer-related death in western countries. The patients are often diagnosed in advanced metastatic stages, and the prognosis remains extremely poor with an overall 5-year survival rate less than 5 %. Currently, novel therapeutic strategies are being pursued to combat PDAC, including oncolytic viruses, either in their natural forms or armed with immunostimulatory molecules. Natural killer cells are critical players against tumours and infected cells. Recently, we showed that IL-2-activated human NK cells displayed killing activity against PDAC cells, which could further be enhanced through the infection of PDAC cells with the rodent parvovirus H-1PV. In this study, the therapeutic efficacy of parvovirus-mediated delivery of three distinct cyto/chemokines (Il-2, MCP-3/CCL7 and IP-10/CXCL10) was evaluated in xenograft models of human PDAC. We show here that activated NK and monocytic cells were found to be recruited by PDAC tumours upon infection with parvoviruses armed with IL-2 or the chemokine MCP-3/CCL7, resulting in a strong anti-tumour response.
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http://dx.doi.org/10.1007/s00262-012-1279-4DOI Listing
November 2012

Enhancement of NK cell antitumor responses using an oncolytic parvovirus.

Int J Cancer 2011 Feb;128(4):908-19

Division of Tumor Virology, F010, German Cancer Research Center, Heidelberg, Germany.

Natural killer (NK) cells play a vital role in the rejection of tumors. Pancreatic ductal adenocarcinoma (PDAC), however, remains a poor prognosis malignancy, due to its resistance to radio- and chemotherapy, and low immunogenicity. We demonstrate here that IL-2-activated human NK cells are able to kill PDAC cells. Currently, novel strategies are being pursued to combat PDAC. In this regard, oncolytic viruses, in addition to killing tumor cells, may also have the potential to augment antitumor immune responses. We found that, besides having an intrinsic oncolytic activity, parvovirus H-1PV is able to enhance NK cell-mediated killing of PDAC cells. Our results show that H-1PV infection of Panc-1 cells increases NK cell capacity to release IFN-γ, TNF-α and MIP-1α/β. Multiple activating receptors are involved in the NK cell-mediated killing of Panc-1 cells. Indeed, blocking of the natural cytotoxicity receptors-NKp30, 44 and 46 in combination, and NKG2D and DNAM1 alone inhibit the killing of Panc-1 cells. Interestingly, H-1PV infection of Panc-1 cells overcomes the part of inhibitory effects suggesting that parvovirus may induce additional NK cell ligands on Panc-1 cells. The enhanced sensitivity of H-1PV-infected PDAC cells to NK cell-dependent killing could be traced back to the upregulation of the DNAM-1 ligand, CD155 and to the downregulation of MHC class I expression. Our data suggests that NK cells display antitumor potential against PDAC and that H-1PV-based oncolytic immunotherapy could further boost NK cell-mediated immune responses and help to develop a combinatorial therapeutic approach against PDAC.
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http://dx.doi.org/10.1002/ijc.25415DOI Listing
February 2011

Regulation of NK cell activity by 2B4, NTB-A and CRACC.

Front Biosci 2008 Jan 1;13:956-65. Epub 2008 Jan 1.

Institute for Immunology, University Heidelberg, INF 305, 69120 Heidelberg, Germany.

2B4, NTB-A and CRACC are members of the recently defined family of SLAM-related receptors. Here we review the role of these receptors for the regulation of Natural Killer cell function and describe the current knowledge about the signal transduction of these receptors. Finally, we critically analyze some controversial data about the function of 2B4 in mouse and man.
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http://dx.doi.org/10.2741/2735DOI Listing
January 2008

Serial killing of tumor cells by human natural killer cells--enhancement by therapeutic antibodies.

PLoS One 2007 Mar 28;2(3):e326. Epub 2007 Mar 28.

Institute for Immunology, University Heidelberg, Heidelberg, Germany.

Background: Natural killer cells are an important component of the innate immune system. Anti-cancer therapies utilizing monoclonal antibodies also rely on the cytotoxicity of NK cells for their effectiveness. Here, we study the dynamics of NK cell cytotoxicity.

Methodology/principal Findings: We observe that IL-2 activated human NK cells can serially hit multiple targets. Using functional assays, we demonstrate that on an average, a single IL-2 activated NK cell can kill four target cells. Data using live video microscopy suggest that an individual NK cell can make serial contacts with multiple targets and majority of contacts lead to lysis of target cells. Serial killing is associated with a loss of Perforin and Granzyme B content. A large majority of NK cells survive serial killing, and IL-2 can replenish their granular stock and restore the diminished cytotoxicity of 'exhausted' NK cells. IL-2 and IL-15 are equally effective in enhancing the killing frequency of resting NK cells. Significantly, Rituximab, a therapeutic monoclonal antibody increases the killing frequency of both resting and IL-2 activated NK cells.

Conclusion/significance: Our data suggest that NK cell-based therapies for overcoming tumors rely on their serial killing ability. Therefore, strategies augmenting the killing ability of NK cells can boost the immune system and enhance the effectiveness of monoclonal antibody-based therapies.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0000326PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1828617PMC
March 2007

Fine-tuning of immune responses by SLAM-related receptors.

J Leukoc Biol 2006 Mar 19;79(3):417-24. Epub 2005 Dec 19.

Institute for Immunology, University Heidelberg, INF 305, 69120 Heidelberg, Germany.

The modulation of antigen receptor signals is important for a productive immune response. The main function of the recently identified members of the signaling lymphocyte activating molecule (SLAM)-related receptors (SRR) is the fine-tuning of immune cell activation. Disruption of SRR function is the cause for severe immune disorders such as X-linked lymphoproliferative syndrome (XLP), where XLP patients carry a mutation in SLAM-associated protein (SAP) (SH2D1A), an important adaptor molecule for the signal transduction of SRR. Recent data also suggest that SRR may play a role in autoimmune diseases and the function of hematopoietic stem and progenitor cells. Here, we review the current understanding of SRR function in different immune cells.
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http://dx.doi.org/10.1189/jlb.0905537DOI Listing
March 2006

Coordinate expression of CC chemokine ligand 5, granulysin, and perforin in CD8+ T cells provides a host defense mechanism against Mycobacterium tuberculosis.

J Immunol 2005 Dec;175(11):7474-83

Institut für Klinische Mikrobiologie, Immunologie und Hygiene der Friedrich Alexander Universitaet Erlangen-Nuernberg, Erlangen, Germany.

The ability of CD8+ T cells to kill intracellular pathogens depends upon their capacity to attract infected cells as well as their secretion of cytolytic and antimicrobial effector molecules. We examined the Ag-induced expression of three immune effector molecules contained within cytoplasmic granules of human CD8+ T cells: the chemokine CCL5, the cytolytic molecule perforin, and the antimicrobial protein granulysin. Macrophages infected with virulent Mycobacterium tuberculosis triggered the expression of CCL5 in CD8+ T cells only in donors with previous exposure to the tuberculosis bacteria, not in naive donors. Functionally, CCL5 efficiently attracted M. tuberculosis-infected macrophages, but failed to exert direct antibacterial activity. Infected macrophages also triggered the expression of granulysin in CD8+ T cells, and granulysin was found to be highly active against drug-susceptible and drug-resistant M. tuberculosis clinical isolates. The vast majority of CCL5-positive cells coexpressed granulysin and perforin. Taken together, this report provides evidence that a subset of CD8+ T cells coordinately expresses CCL5, perforin and granulysin, thereby providing a host mechanism to attract M. tuberculosis-infected macrophages and kill the intracellular pathogen.
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http://dx.doi.org/10.4049/jimmunol.175.11.7474DOI Listing
December 2005

Inverse correlation of maturity and antibacterial activity in human dendritic cells.

J Immunol 2005 Apr;174(7):4203-9

Institut für Klinische Mikrobiologie, Immunologie und Hygiene, Universität Erlangen-Nürnberg, Erlangen, Germany.

Dendritic cells (DCs) are a key part of host defense against microbial pathogens, being part of the innate immune system, but also instructing the adaptive T cell response. This study was designed to evaluate whether human DCs directly contribute to innate immunity by killing intracellular bacteria, using tuberculosis as a model. DCs were detected in bronchoalveolar lavage samples indicating that DCs are available for immediate interaction with Mycobacterium tuberculosis (M. Tb) after inhalation of the pathogen. The phenotype of DC in bronchoalveolar lavage closely resembles monocyte-derived immature DC (iDC) according to the expression of CD1a, CD83, and CCR7. The antimicrobial activity of iDC against intracellular M. Tb inversely correlated with TNF-alpha-release and was enhanced by treatment with anti-TNF-alpha Abs. Differentiation of iDC into mature DC by addition of TNF-alpha or activation via Toll-like receptors further reduced killing of M. Tb. The antibacterial activity against intracellular M. Tb of all DCs was significantly lower than alveolar macrophages. Therefore, the maintenance of a pool of DCs at the site of disease activity in tuberculosis, and the maturation of these DC by TNF-alpha provides a mechanism by which M. Tb escapes the innate immune system.
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http://dx.doi.org/10.4049/jimmunol.174.7.4203DOI Listing
April 2005