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Dive into the research topics where Michael Naso is active.

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Featured researches published by Michael Naso.


Journal of Immunology | 2011

Distinct Roles of IL-23 and IL-17 in the Development of Psoriasis-Like Lesions in a Mouse Model

Kimiko Nakajima; Takashi Kanda; Mikiro Takaishi; Takeo Shiga; Ken Miyoshi; Hideki Nakajima; Reiko Kamijima; Masahito Tarutani; Jacqueline Benson; M. Merle Elloso; Lester L. Gutshall; Michael Naso; Yoichiro Iwakura; John DiGiovanni; Shigetoshi Sano

Psoriasis is an inflammatory disease with dynamic interactions between the immune system and the skin. The IL-23/Th17 axis plays an important role in the pathogenesis of psoriasis, although the exact contributions of IL-23 and IL-17 in vivo remain unclear. K5.Stat3C transgenic mice constitutively express activated Stat3 within keratinocytes, and these animals develop skin lesions with histological and cytokine profiles similar to those of human plaque psoriasis. In this study, we characterized the effects of anti-mouse IL-17A, anti-mouse IL-12/23p40, and anti-mouse IL-23p19 Abs on the development of psoriasis-like lesions in K5.Stat3C transgenic mice. Treatment with anti–IL-12/23p40 or anti–IL-23p19 Abs greatly inhibited 12-O-tetradecanoylphorbol-13-acetate–induced epidermal hyperplasia in the ears of K5.Stat3C mice, whereas the inhibitory effect of an anti–IL-17A Ab was relatively less prominent. Treatment with anti–IL-12/23p40 or anti–IL-23p19 Abs markedly lowered transcript levels of Th17 cytokines (e.g., IL-17 and IL-22), β-defensins, and S100A family members in skin lesions. However, anti–IL-17A Ab treatment did not affect mRNA levels of Th17 cytokines. Crossing IL-17A–deficient mice with K5.Stat3C mice resulted in partial attenuation of 12-O-tetradecanoylphorbol-13-acetate–induced lesions, which were further attenuated by anti–IL-12/23p40 Ab treatment. FACS analysis of skin-draining lymph node cells from mice that were intradermally injected with IL-23 revealed an increase in both IL-22–producing T cells and NK-22 cells. Taken together, this system provides a useful mouse model for psoriasis and demonstrates distinct roles for IL-23 and IL-17.


mAbs | 2010

Engineering host cell lines to reduce terminal sialylation of secreted antibodies

Michael Naso; Susan H. Tam; Bernard Scallon; T. Shantha Raju

Covalently-linked glycans on proteins have many functional roles, some of which are still not completely understood. Antibodies have a very specific glycan modification in the Fc region that is required for mediating immune effector functions. These Fc glycans are typically highly heterogeneous in structure, and this heterogeneity is influenced by many factors, such as type of cellular host and rate of Ab secretion. Glycan heterogeneity can affect the Fc-dependent activities of antibodies. It has been shown recently that increased Fc sialylation can result in decreased binding to immobilized antigens and some Fcγ receptors, as well as decreased antibody-dependent cell-mediated cytotoxicity (ADCC) activity. In contrast, increased Fc sialylation enhances the anti-inflammatory activity of antibodies. To produce antibodies with increased effector functions, we developed host cell lines that would limit the degree of sialylation of recombinantly-expressed antibodies. Towards this end, the catalytic domain of the Arthrobacter ureafaciens sialidase (sialidase A) was engineered for secreted expression in mammalian cell lines. Expression of this sialidase A gene in mammalian cells resulted in secreted expression of soluble enzyme that was capable of removing sialic acid from antibodies secreted into the medium. Purified antibodies secreted from these cells were found to possess very low levels of sialylation compared with the same antibodies purified from unmodified host cells. The low sialylated antibodies exhibited similar binding affinity to soluble antigens, improved ADCC activity, and they possessed pharmacokinetic properties comparable to their more sialylated counterparts. Further, it was observed that the amount of sialidase A expressed was sufficient to thoroughly remove sialic acid from Abs made in high-producing cell lines. Thus, engineering host cells to express sialidase A enzyme can be used to produce recombinant antibodies with very low levels of sialylation.


BioDrugs | 2017

Adeno-Associated Virus (AAV) as a Vector for Gene Therapy

Michael Naso; Brian Tomkowicz; William L. Perry; William R. Strohl

There has been a resurgence in gene therapy efforts that is partly fueled by the identification and understanding of new gene delivery vectors. Adeno-associated virus (AAV) is a non-enveloped virus that can be engineered to deliver DNA to target cells, and has attracted a significant amount of attention in the field, especially in clinical-stage experimental therapeutic strategies. The ability to generate recombinant AAV particles lacking any viral genes and containing DNA sequences of interest for various therapeutic applications has thus far proven to be one of the safest strategies for gene therapies. This review will provide an overview of some important factors to consider in the use of AAV as a vector for gene therapy.


PLOS ONE | 2015

TIM-3 Suppresses Anti-CD3/CD28-Induced TCR Activation and IL-2 Expression through the NFAT Signaling Pathway.

Brian Tomkowicz; Eileen Walsh; Adam Cotty; Raluca Verona; Nina Chi Sabins; Fred Kaplan; Sandy Santulli-Marotto; Chen-Ni Chin; Jill Mooney; Russell B. Lingham; Michael Naso; Timothy McCabe

TIM-3 (T cell immunoglobulin and mucin-domain containing protein 3) is a member of the TIM family of proteins that is preferentially expressed on Th1 polarized CD4+ and CD8+ T cells. Recent studies indicate that TIM-3 serves as a negative regulator of T cell function (i.e. T cell dependent immune responses, proliferation, tolerance, and exhaustion). Despite having no recognizable inhibitory signaling motifs, the intracellular tail of TIM-3 is apparently indispensable for function. Specifically, the conserved residues Y265/Y272 and surrounding amino acids appear to be critical for function. Mechanistically, several studies suggest that TIM-3 can associate with interleukin inducible T cell kinase (ITK), the Src kinases Fyn and Lck, and the p85 phosphatidylinositol 3-kinase (PI3K) adaptor protein to positively or negatively regulate IL-2 production via NF-κB/NFAT signaling pathways. To begin to address this discrepancy, we examined the effect of TIM-3 in two model systems. First, we generated several Jurkat T cell lines stably expressing human TIM-3 or murine CD28-ECD/human TIM-3 intracellular tail chimeras and examined the effects that TIM-3 exerts on T cell Receptor (TCR)-mediated activation, cytokine secretion, promoter activity, and protein kinase association. In this model, our results demonstrate that TIM-3 inhibits several TCR-mediated phenotypes: i) NF-kB/NFAT activation, ii) CD69 expression, and iii) suppression of IL-2 secretion. To confirm our Jurkat cell observations we developed a primary human CD8+ cell system that expresses endogenous levels of TIM-3. Upon TCR ligation, we observed the loss of NFAT reporter activity and IL-2 secretion, and identified the association of Src kinase Lck, and PLC-γ with TIM-3. Taken together, our results support the conclusion that TIM-3 is a negative regulator of TCR-function by attenuating activation signals mediated by CD3/CD28 co-stimulation.


Molecular Immunology | 2014

IgG variable region and VH CDR3 diversity in unimmunized mice analyzed by massively parallel sequencing.

Jin Lu; Tadas Panavas; Kim Thys; J Aerssens; Michael Naso; Jamie Fisher; Michael Rycyzyn; Raymond Sweet

Most antigen-specific mouse antibodies have been derived by hybridoma technology, predominantly through use of the Balb/c strain. Much of the Balb/c germline repertoire of variable genes (V regions) is known. However, there is little information about the background expressed repertoire of IgG antibodies in mice, which reflects the baseline against which antigen-specific antibodies are generated through immunization. To assess this baseline repertoire, RNA was isolated from splenic B-cells enriched for expression of IgG from three mice. The RNA was individually amplified with three distinct PCR primer sets for comprehensive recovery of the heavy and light chain variable regions. Each PCR product was independently subjected to deep sequencing using 454 pyro-sequencing technology and analysed for redundancy, open reading frame, germline representation, and CDR3 sequence of the heavy chain variable region (VH CDR3) within and across the primer sets and mice. A highly skewed abundance of heavy and light chain variable gene usage was observed for all three primers in all three mice. While showing considerable overlap, there were differences among these profiles indicative of primer bias and animal-to-animal variation. VH CDR3 sequences were likewise highly skewed indicating that the heavy chain genes profiles substantially reflected individual antibodies. This observation was confirmed through analysis of randomly selected complete heavy chain variable sequences. However, there was very little redundancy in VH CDR3 sequences across the different mice. We conclude that the background IgG repertoire in young, unimmunized mice is highly skewed within individual mice and is diverse among them, a pattern similar to that observed in highly immunized mice.


Cytokine | 2011

Expression, refolding and purification of a human interleukin-17A variant.

Bingyuan Wu; Jennifer F. Nemeth; Dariusz J. Janecki; Brian M. Jones; Galina Obmolova; Thomas J. Malia; Audrey Baker; Deidra Bethea; M. Merle Elloso; Michael Naso; Susann Taudte

A human interleukin-17A (IL-17A) variant was overexpressed in Escherichia coli BL21 (DE3) under the control of a T(7) promoter. The resulting insoluble inclusion bodies were isolated and solubilized by homogenization with 6 M guanidine HCl. The denatured recombinant human IL-17A variant was refolded in 20 mM Tris-HCl, pH 9.0, 500 mM arginine, 500 mM guanidine HCl, 15% glycerol, 1 mM cystamine, and 5 mM cysteine at 2-8°C for 40 h. The refolded IL-17A variant was subsequently purified using a combination of cation-exchange, reversed-phase and fluoroapatite chromatography. The final purified product was a monodisperse and crystallizable homodimer with a molecular weight of 30,348.3 Da. The protein was active in both receptor binding competition assay and IL-17A-dependent biological activity assay using human dermal fibroblasts.


BMC Biotechnology | 2015

Optimizing production of Fc-amidated peptides by Chinese hamster ovary cells

Kristina R. Carlson; Steven C. Pomerantz; Omid Vafa; Michael Naso; William R. Strohl; Richard E. Mains; Betty A. Eipper

BackgroundAmidation of the carboxyl terminal of many peptides is essential for full biological potency, often increasing receptor binding and stability. The single enzyme responsible for this reaction is peptidylglycine α-amidating monooxygenase (PAM: EC 1.14.17.3), a copper- and ascorbate-dependent Type I membrane protein.MethodsTo make large amounts of high molecular weight amidated product, Chinese hamster ovary (CHO) cells were engineered to express exogenous PAM. To vary access of the enzyme to its substrate, exogenous PAM was targeted to the endoplasmic reticulum, trans-Golgi network, endosomes and lysosomes or to the lumen of the secretory pathway.ResultsPAM was equally active when targeted to each intracellular location and assayed in homogenates. Immunocytochemical analyses of CHO cells and a pituitary cell line demonstrated that targeting of exogenous PAM was partially successful. PAM substrates generated by expressing peptidylglycine substrates (glucagon-like peptide 1-Gly, peptide YY-Gly and neuromedin U-Gly) fused to the C-terminus of immunoglobulin Fc in CHO cell lines producing targeted PAM. The extent of amidation of the Fc-peptides was determined by mass spectrometry and amidation-specific enzyme immunoassays. Amidation was inhibited by copper chelation, but was not enhanced by the addition of additional copper or ascorbate.ConclusionsPeptide amidation was increased over endogenous levels by exogenous PAM, and targeting PAM to the endoplasmic reticulum or trans-Golgi network increased peptide amidation compared to endogenous CHO PAM.


Cytokine | 2014

The role of interchain disulfide bond in a recombinant human interleukin-17A variant

Bingyuan Wu; Salman Muzammil; Brian Jones; Jennifer F. Nemeth; Dariusz J. Janecki; Audrey Baker; M. Merle Elloso; Michael Naso; Jill Carton; Susann Taudte

Interleukin-17A (IL-17A) is the prototype of IL-17 family and has been implicated in the pathogenesis of a variety of autoimmune diseases. Therefore its structural and functional properties are of great medical interest. During our research on a recombinant human IL-17A (rhIL-17A) variant, four isoforms were obtained when it was refolded. While isoforms 1 and 2 represented non-covalent dimers, isoforms 3 and 4 were determined to be covalent dimers. All four isoforms were structurally similar by Circular Dichroism and fluorescence spectroscopy studies, but differential scanning calorimetry demonstrated thermal stability in the order of isoform 1=isoform 2<isoform 4<isoform 3. In addition, compared to covalent dimers (isoform 3 and 4), the non-covalent dimers (isoforms 1 and 2) are slightly less active in a receptor-binding assay but at least 5-fold less active in a cell-based assay.


Molecular Cancer Therapeutics | 2013

Abstract A190: Colon tumor cells expressing CD24 have oncogenic properties and are inhibited by monoclonal antibody immunotherapy.

Luciana F. Macedo; Elizabeth Kaiser; Haiyan Jiang; Hillary Millar; Diana Wiley; Adam Cotty; Fred Kaplan; Barry Morse; Jill Carton; Michael Naso; Randall J. Brezski; Allison Oberholtze; E. Christine Pietsch; Li Yingzhe; Debbie Marshall; Linda A. Snyder

CD24 is a heavily glycosylated glycosylphosphatidylinositol- anchored protein that is overexpressed in many different tumor types, including colon cancer (>80 %), and has been shown to correlate with shortened patient survival. CD24 plays a role in regulating cancer cell proliferation and tumor microenvironment interactions. The mechanism by which CD24 regulates cell survival and proliferation is not very well understood. What is better known is that CD24 modulates cancer cell adhesion to the vasculature wall and cancer cell-platelet thrombi formation by its binding to P-selectin expressed on activated platelets and endothelial cells. CD24 was also reported to be a functional marker for liver, colon and pancreatic cancer stem cells. All of these functions may contribute to tumor growth and metastasis. The aim of this study was to investigate the function of CD24 as a target for colon cancer therapy. In order to confirm that CD24 expressing cells acquire oncogenic properties, we transfected the CD24 gene into the CD24− colon cancer line SW480. We confirmed that CD24 overexpression induces SW480 tumor growth in vivo. In order to understand the mechanism by which CD24 promotes tumorigenesis, we found that CD24 overexpression activates several oncogenic pathways. Previous publications have shown that CD24 increases expression of p-Raf, p-ERK, and p-JNK in SW480CD24+ cells. Here, using a reporter assay, we show that CD24 expression activates not only ERK and JNK but also the Wnt pathway. It was also shown in the past that treatment of HT-29 colon cancer cells with the antiproliferative anti-CD24 mAb SWA11 caused a decrease in hypoxia and VEGF pathways. We show here that CD24 induced VEGF as well as FGF-2, IL-10, and MMP2 expression and activation of the hypoxia pathway. Finally, we have tested the tumor inhibitory effect of two different anti-CD24 antibodies, SWA11 (mouse IgG2a) and ALB9 (mouse IgG1). Results suggest that both antibodies bind specifically to the CD24 protein core as shown by competition assays with CD24 Fc and binding assays to the deglycosylated CD24. We show that SWA11 significantly inhibited both HT-29 and SW480CD24+ tumor growth in vivo while ALB9 inhibited SW480CD24+ tumor growth only. SWA11 didn9t show any neutralizing activity in vitro in inhibiting cell proliferation or CD24 expression but showed effector function activity that may account for its mechanism of action in vivo. ALB9 mechanism of action was not explored in this study, but as it is a mouse IgG1 antibody, ADCC activity is not likely to be part of its mechanism of action. Together, these results provide support for the hypothesis that CD24 has oncogenic properties and that CD24-expressing tumor can be inhibited with antibody immunotherapy. Citation Information: Mol Cancer Ther 2013;12(11 Suppl):A190. Citation Format: Luciana F. Macedo, Elizabeth Kaiser, Haiyan Jiang, Hillary Millar, Diana Wiley, Adam Cotty, Fred Kaplan, Barry Morse, Jill M. Carton, Michael F. Naso, Randall Brezski, Allison Oberholtze, E. Christine Pietsch, Li Yingzhe, Debbie Marshall, Linda A. Snyder. Colon tumor cells expressing CD24 have oncogenic properties and are inhibited by monoclonal antibody immunotherapy. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2013 Oct 19-23; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2013;12(11 Suppl):Abstract nr A190.


Protein Expression and Purification | 2008

Expression and characterization of a human BMP-7 variant with improved biochemical properties.

Bethany Swencki-Underwood; Juliane Mills; Joe Vennarini; Ken Boakye; Jinquan Luo; Steve Pomerantz; Mark R. Cunningham; Francis X. Farrell; Michael Naso; Bernard Amegadzie

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Bernard Scallon

Dresden University of Technology

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M. Merle Elloso

University of Pennsylvania

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Mark Rutz

Janssen Pharmaceutica

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