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Castiello L.,U.S. National Institutes of Health | Sabatino M.,U.S. National Institutes of Health | Zhao Y.,U.S. National Cancer Institute | Tumaini B.,U.S. National Institutes of Health | And 7 more authors.
Molecular Therapy | Year: 2013

Cell-based immunotherapies are among the most promising approaches for developing effective and targeted immune response. However, their clinical usefulness and the evaluation of their efficacy rely heavily on complex quality control assessment. Therefore, rapid systematic methods are urgently needed for the in-depth characterization of relevant factors affecting newly developed cell product consistency and the identification of reliable markers for quality control. Using dendritic cells (DCs) as a model, we present a strategy to comprehensively characterize manufactured cellular products in order to define factors affecting their variability, quality and function. After generating clinical grade human monocyte-derived mature DCs (mDCs), we tested by gene expression profiling the degrees of product consistency related to the manufacturing process and variability due to intra-and interdonor factors, and how each factor affects single gene variation. Then, by calculating for each gene an index of variation we selected candidate markers for identity testing, and defined a set of genes that may be useful comparability and potency markers. Subsequently, we confirmed the observed gene index of variation in a larger clinical data set. In conclusion, using high-throughput technology we developed a method for the characterization of cellular therapies and the discovery of novel candidate quality assurance markers. © The American Society of Gene &Cell Therapy.


News Article | November 7, 2016
Site: www.eurekalert.org

Researchers at Vanderbilt University Medical Center and Washington University School of Medicine in St. Louis, Missouri, have isolated a human monoclonal antibody that in a mouse model "markedly reduced" infection by the Zika virus. The antibody, called ZIKV-117, also protected the fetus in pregnant mice infected with the virus, the researchers reported today in the journal Nature. Zika is believed to cause microcephaly, unusually small heads, and other congenital malformations in children born to infected women. Similar protection studies in primates are warranted, and if the findings hold up, ZIKV-177 could be developed as a protective antibody treatment for pregnant women at risk of Zika infection, the researchers concluded. The findings may also aid efforts to develop an effective anti-Zika vaccine, said James Crowe Jr., M.D., director of the Vanderbilt Vaccine Center and co-corresponding author of the paper with Michael S. Diamond, M.D., Ph.D., at Washington University. "These naturally occurring human antibodies isolated from humans represent the first medical intervention that prevents Zika infection and damage to fetuses," said Crowe, who also is Ann Scott Carell Professor in the Departments of Pediatrics and Pathology, Microbiology & Immunology in the Vanderbilt University School of Medicine. "We're excited because the data suggests we may have antibody treatments in hand that could be developed for use in pregnant women," he said. "The remarkable potency and breadth of inhibition by ZIKV-117 has great promise," Diamond said, "as it was able to inhibit infection by strains from both Africa and America in cell culture and in animals, including during pregnancy." Diamond is associate director of The Andrew M. and Jane M. Bursky Center for Human Immunology & Immunotherapy Programs at Washington University. Zika is a mosquito-borne virus that has emerged as a global public health threat. In addition to its association with congenital birth defects, Zika has been linked to Guillain-Barre syndrome, a neurological disorder that can lead to paralysis and death. Since a major outbreak was reported in Brazil last year, Zika infections transmitted by mosquitoes have been reported throughout Africa, Asia, the Pacific, and the Americas, including Miami-Dade County, Florida. During the past 15 years, Crowe and his colleagues have developed a high-efficiency method for isolating human monoclonal antibodies that has enabled to them identify neutralizing antibodies against a wide range of viral infections, from Ebola to HIV. The Crowe and Diamond laboratories have collaborated recently on several projects including the generation of protective human monoclonal antibodies against Dengue, West Nile, Chikungunya and now Zika viruses. Monoclonal antibodies are made from a single clone of B cells, a type of white blood cell, that have been fused to myeloma (cancer) cells to form fast-growing "hybridomas." This allows researchers to quickly generate large quantities of antibodies against specific viral targets. In the current study, the researchers isolated antibodies from the blood of people who'd been previously infected with the Zika virus in different parts of the world. The antibodies reacted to the envelope or "E" protein on the surface of the virus. The researchers then generated a variety of monoclonal antibodies. In cell culture studies, they identified one, ZIKV-117, which broadly neutralized several different strains of the virus. In mice infected by the Zika virus, injection of the antibody markedly reduced disease and mortality, and reduced transmission from mother to fetus. The paper's first authors were Gopal Sapparapu, Ph.D., research assistant professor of Pediatrics in the Crowe lab, and by Estefania Fernandez, a graduate student in Diamond's lab. Nurgun Kose, senior research specialist in Crowe's lab, made the antibodies. The research was supported in part by National Institutes of Health grants AI073755 and AI104972.


News Article | November 7, 2016
Site: www.sciencedaily.com

Researchers at Vanderbilt University Medical Center and Washington University School of Medicine in St. Louis, Missouri, have isolated a human monoclonal antibody that in a mouse model "markedly reduced" infection by the Zika virus. The antibody, called ZIKV-117, also protected the fetus in pregnant mice infected with the virus, the researchers reported in the journal Nature. Zika is believed to cause microcephaly, unusually small heads, and other congenital malformations in children born to infected women. Similar protection studies in primates are warranted, and if the findings hold up, ZIKV-177 could be developed as a protective antibody treatment for pregnant women at risk of Zika infection, the researchers concluded. The findings may also aid efforts to develop an effective anti-Zika vaccine, said James Crowe Jr., M.D., director of the Vanderbilt Vaccine Center and co-corresponding author of the paper with Michael S. Diamond, M.D., Ph.D., at Washington University. "These naturally occurring human antibodies isolated from humans represent the first medical intervention that prevents Zika infection and damage to fetuses," said Crowe, who also is Ann Scott Carell Professor in the Departments of Pediatrics and Pathology, Microbiology & Immunology in the Vanderbilt University School of Medicine. "We're excited because the data suggests we may have antibody treatments in hand that could be developed for use in pregnant women," he said. "The remarkable potency and breadth of inhibition by ZIKV-117 has great promise," Diamond said, "as it was able to inhibit infection by strains from both Africa and America in cell culture and in animals, including during pregnancy." Diamond is associate director of The Andrew M. and Jane M. Bursky Center for Human Immunology & Immunotherapy Programs at Washington University. Zika is a mosquito-borne virus that has emerged as a global public health threat. In addition to its association with congenital birth defects, Zika has been linked to Guillain-Barre syndrome, a neurological disorder that can lead to paralysis and death. Since a major outbreak was reported in Brazil last year, Zika infections transmitted by mosquitoes have been reported throughout Africa, Asia, the Pacific, and the Americas, including Miami-Dade County, Florida. During the past 15 years, Crowe and his colleagues have developed a high-efficiency method for isolating human monoclonal antibodies that has enabled to them identify neutralizing antibodies against a wide range of viral infections, from Ebola to HIV. The Crowe and Diamond laboratories have collaborated recently on several projects including the generation of protective human monoclonal antibodies against Dengue, West Nile, Chikungunya and now Zika viruses. Monoclonal antibodies are made from a single clone of B cells, a type of white blood cell, that have been fused to myeloma (cancer) cells to form fast-growing "hybridomas." This allows researchers to quickly generate large quantities of antibodies against specific viral targets. In the current study, the researchers isolated antibodies from the blood of people who'd been previously infected with the Zika virus in different parts of the world. The antibodies reacted to the envelope or "E" protein on the surface of the virus. The researchers then generated a variety of monoclonal antibodies. In cell culture studies, they identified one, ZIKV-117, which broadly neutralized several different strains of the virus. In mice infected by the Zika virus, injection of the antibody markedly reduced disease and mortality, and reduced transmission from mother to fetus. The paper's first authors were Gopal Sapparapu, Ph.D., research assistant professor of Pediatrics in the Crowe lab, and by Estefania Fernandez, a graduate student in Diamond's lab. Nurgun Kose, senior research specialist in Crowe's lab, made the antibodies.


News Article | November 7, 2016
Site: www.chromatographytechniques.com

Researchers at Vanderbilt University Medical Center and Washington University School of Medicine in St. Louis, Missouri, have isolated a human monoclonal antibody that in a mouse model "markedly reduced" infection by the Zika virus. The antibody, called ZIKV-117, also protected the fetus in pregnant mice infected with the virus, the researchers reported today in the journal Nature. Zika is believed to cause microcephaly, unusually small heads, and other congenital malformations in children born to infected women. Similar protection studies in primates are warranted, and if the findings hold up, ZIKV-177 could be developed as a protective antibody treatment for pregnant women at risk of Zika infection, the researchers concluded. The findings may also aid efforts to develop an effective anti-Zika vaccine, said James Crowe Jr., M.D., director of the Vanderbilt Vaccine Center and co-corresponding author of the paper with Michael S. Diamond, M.D., Ph.D., at Washington University. "These naturally occurring human antibodies isolated from humans represent the first medical intervention that prevents Zika infection and damage to fetuses," said Crowe, who also is Ann Scott Carell Professor in the Departments of Pediatrics and Pathology, Microbiology & Immunology in the Vanderbilt University School of Medicine. "We're excited because the data suggests we may have antibody treatments in hand that could be developed for use in pregnant women," he said. "The remarkable potency and breadth of inhibition by ZIKV-117 has great promise," Diamond said, "as it was able to inhibit infection by strains from both Africa and America in cell culture and in animals, including during pregnancy." Diamond is associate director of The Andrew M. and Jane M. Bursky Center for Human Immunology & Immunotherapy Programs at Washington University. Zika is a mosquito-borne virus that has emerged as a global public health threat. In addition to its association with congenital birth defects, Zika has been linked to Guillain-Barre syndrome, a neurological disorder that can lead to paralysis and death. Since a major outbreak was reported in Brazil last year, Zika infections transmitted by mosquitoes have been reported throughout Africa, Asia, the Pacific, and the Americas, including Miami-Dade County, Florida. During the past 15 years, Crowe and his colleagues have developed a high-efficiency method for isolating human monoclonal antibodies that has enabled to them identify neutralizing antibodies against a wide range of viral infections, from Ebola to HIV. The Crowe and Diamond laboratories have collaborated recently on several projects including the generation of protective human monoclonal antibodies against Dengue, West Nile, Chikungunya and now Zika viruses. Monoclonal antibodies are made from a single clone of B cells, a type of white blood cell, that have been fused to myeloma (cancer) cells to form fast-growing "hybridomas." This allows researchers to quickly generate large quantities of antibodies against specific viral targets. In the current study, the researchers isolated antibodies from the blood of people who'd been previously infected with the Zika virus in different parts of the world. The antibodies reacted to the envelope or "E" protein on the surface of the virus. The researchers then generated a variety of monoclonal antibodies. In cell culture studies, they identified one, ZIKV-117, which broadly neutralized several different strains of the virus. In mice infected by the Zika virus, injection of the antibody markedly reduced disease and mortality, and reduced transmission from mother to fetus.


Ruan X.,Center for Human Immunology | Loyola D.E.,University of Santiago de Chile | Marolda C.L.,Center for Human Immunology | Perez-Donoso J.M.,University of Santiago de Chile | And 2 more authors.
Glycobiology | Year: 2012

WaaL is a membrane enzyme that catalyzes a key step in lipopolysaccharide (LPS) synthesis: the glycosidic bonding of a sugar at the proximal end of the undecaprenyl-diphosphate (Und-PP) O-antigen with a terminal sugar of the lipid A-core oligosaccharide (OS). Utilizing an in vitro assay, we demonstrate here that ligation with purified Escherichia coli WaaL occurs without adenosine-5′-triphosphate (ATP) and magnesium ions. Furthermore, E. coli and Pseudomonas aeruginosa WaaL proteins cannot catalyze ATP hydrolysis in vitro. We also show that a lysine substitution of the arginine (Arg)-215 residue renders an active protein, whereas WaaL mutants with alanine replacements in the periplasmic-exposed residues Arg-215, Arg-288 and histidine (His)-338 and also the membrane-embedded aspartic acid-389 are nonfunctional. An in silico approach, combining predicted topological information with the analysis of sequence conservation, confirms the importance of a positive charge at the small periplasmic loop of WaaL, since an Arg corresponding to Arg-215 was found at a similar position in all the WaaL homologs. Also, a universally conserved H[NSQ]X 9GXX[GTY] motif spanning the C-terminal end of the predicted large periplasmic loop and the membrane boundary of the transmembrane helix was identified. The His residue in this motif corresponds to His-338. A survey of LPS structures in which the linkage between O-antigen and lipid A-core OS was elucidated reveals that it is always in the β-configuration, whereas the sugars bound to Und-PP are in the-configuration. Together, our biochemical and in silico data argue that WaaL proteins use a common reaction mechanism and share features of metal ion-independent inverting glycosyltransferases. © 2011 The Author.


Patel K.B.,Center for Human Immunology | Ciepichal E.,Polish Academy of Sciences | Swiezewska E.,Polish Academy of Sciences | Valvano M.A.,Center for Human Immunology | Valvano M.A.,University of Western Ontario
Glycobiology | Year: 2012

Two families of membrane enzymes catalyze the initiation of the synthesis of O-antigen lipopolysaccharide. The Salmonella enterica Typhimurium WbaP is a prototypic member of one of these families. We report here the purification and biochemical characterization of the WbaP C-terminal (WbaPCT) domain harboring one putative transmembrane helix and a large cytoplasmic tail. An N-terminal thioredoxin fusion greatly improved solubility and stability of WbaPCT allowing us to obtain highly purified protein. We demonstrate that WbaPCT is sufficient to catalyze the in vitro transfer of galactose (Gal)-1-phosphate from uridine monophosphate (UDP)-Gal to the lipid carrier undecaprenyl monophosphate (Und-P). We optimized the in vitro assay to determine steady-state kinetic parameters with the substrates UDP-Gal and Und-P. Using various purified polyisoprenyl phosphates of increasing length and variable saturation of the isoprene units, we also demonstrate that the purified enzyme functions highly efficiently with Und-P, suggesting that the WbaPCT domain contains all the essential motifs to catalyze the synthesis of the Und-P-P-Gal molecule that primes the biosynthesis of bacterial surface glycans. © 2011 The Author.


Nikoopour E.,Center for Human Immunology | Krougly O.,Center for Human Immunology | Lee-Chan E.,Center for Human Immunology | Mansour Haeryfar S.M.,Center for Human Immunology | And 2 more authors.
Clinical and Experimental Immunology | Year: 2016

Chromogranin A (ChgA) is an antigenic target of pathogenic CD4+ T cells in a non-obese diabetic (NOD) mouse model of type 1 diabetes (T1D). Vasostatin-1 is a naturally processed fragment of ChgA. We have now identified a novel H2-Kd-restricted epitope of vasostatin-1, ChgA 36-44, which elicits CD8+ T cell responses in NOD mice. By using ChgA 36-44/Kd tetramers we have determined the frequency of vasostatin-1-specific CD8+ T cells in pancreatic islets and draining lymph nodes of NOD mice. We also demonstrate that vasostatin-1-specific CD4+ and CD8+ T cells constitute a significant fraction of islet-infiltrating T cells in diabetic NOD mice. Adoptive transfer of T cells from ChgA 36-44 peptide-primed NOD mice into NOD/severe combined immunodeficiency (SCID) mice led to T1D development. These findings indicate that vasostatin-1-specific CD8+ T cells contribute to the pathogenesis of type 1 diabetes in NOD mice. © 2016 British Society for Immunology


PubMed | University of Western Ontario and Center for Human Immunology
Type: Journal Article | Journal: Journal of leukocyte biology | Year: 2014

The G-CSF is best known for its activity in the generation and activation of neutrophils. In addition, studies on G-CSF(-/-) or G-CSFR(-/-) mice and BMC cultures suggested a role of G-CSF in macrophage generation. However, our understanding on the role of G-CSF in macrophage development is limited. Here, using in vitro BMC models, we demonstrated that G-CSF promoted the generation of Gr-1(high)/F4/80(+) macrophage-like cells in M-BMCs, likely through suppressing cell death and enhancing generation of Gr-1(high)/F4/80(+) macrophage-like cells. These Gr-1(high) macrophage-like cells produced M2-like cytokines and surface markers in response to LPS and IL-4/IL-13, respectively. Adoptive transfer of EGFP-expressing (EGFP(+)) M-BMCs showed a dominant, gut-homing phenotype. The small intestinal lamina propria of G-CSFR(-/-) mice also harbored significantly reduced numbers of Gr-1(high)/F4/80(+) macrophages compared with those of WT mice, but levels of Gr-1(+)/F4/80(-) neutrophil-like cells were similar between these mice. Collectively, these results suggest a novel function of G-CSF in the generation of gut-homing, M2-like macrophages.


PubMed | Copenhagen University, NCI Inc, National Human Genome Research Institute, Center for Human Immunology and 3 more.
Type: Clinical Trial, Phase II | Journal: Clinical cancer research : an official journal of the American Association for Cancer Research | Year: 2016

Chronic lymphocytic leukemia (CLL) cells depend on microenvironmental interactions for proliferation and survival that are at least partially mediated through B-cell receptor (BCR) signaling. Ibrutinib, a Bruton tyrosine kinase inhibitor, disrupts BCR signaling and leads to the egress of tumor cells from the microenvironment. Although the on-target effects on CLL cells are well defined, the impact on the microenvironment is less well studied. We therefore sought to characterize the in vivo effects of ibrutinib on the tumor microenvironment.Patients received single-agent ibrutinib on an investigator-initiated phase II trial. Serial blood and tissue samples were collected pretreatment and during treatment. Changes in cytokine levels, cellular subsets, and microenvironmental interactions were assessed.Serum levels of key chemokines and inflammatory cytokines decreased significantly in patients on ibrutinib. Furthermore, ibrutinib treatment decreased circulating tumor cells and overall T-cell numbers. Most notably, a reduced frequency of the Th17 subset of CD4(+)T cells was observed concurrent with reduced expression of activation markers and PD-1 on T cells. Consistent with direct inhibition of T cells, ibrutinib inhibited Th17 differentiation of murine CD4(+)T cells in vitro Finally, in the bone marrow microenvironment, we found that ibrutinib disaggregated the interactions of macrophages and CLL cells, inhibited secretion of CXCL13, and decreased the chemoattraction of CLL cells.In conjunction with inhibition of BCR signaling, these changes in the tumor microenvironment likely contribute to the antitumor activity of ibrutinib and may impact the efficacy of immunotherapeutic strategies in patients with CLL. See related commentary by Bachireddy and Wu, p. 1547.

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