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"Diagnosing, Monitoring and Treating Inflammation" in Patent Application Approval Process

July 28, 2014



By a News Reporter-Staff News Editor at Cancer Gene Therapy Week -- A patent application by the inventor Feinberg, Mark W. (Jamaica Plain, MA), filed on December 17, 2013, was made available online on July 17, 2014, according to news reporting originating from Washington, D.C., by NewsRx correspondents (see also The Brigham and Women's Hospital, Inc.).

This patent application is assigned to The Brigham and Women's Hospital, Inc.

The following quote was obtained by the news editors from the background information supplied by the inventors: "The present invention relates to the field of treating chronic inflammatory diseases.

"T regulatory cells, or T reg cells, are a subclass of T cells, capable of inhibiting Th1- and Th2-driven inflammatory responses that contribute to the development of autoimmune diseases, such as, type I diabetes, multiple sclerosis, inflammatory bowel disease, and atherosclerosis, among others. Of particular interest is the identification of novel molecular mechanisms underlying the development and suppression function of T regs as a means for establishing new strategies for treating autoimmune diseases.

"With respect to atherosclerosis, atherogenesis is the developmental process that results in the formation of atheromatous plaques, which may lead to atherosclerosis. T cell activation plays an important role in atherogenesis (e.g., by macrophage activation/recruitment; SMC proliferation; and collagen formation). For example, it has been shown that injection of Tr1 T regulatory cells--a subset of T regs that have been previously identified, along with natural regulatory CD4+CD25+ cells and Th3 cells--was able to decrease atheroma size in ApoE-/- mice (Mallat Z. et al., 2003). In addition, injection of CD4+CD25+ T regulatory cells was able to reduce lesion size in mouse models of atherosclerosis. The same study also showed that a depletion of T regulatory cells accelerated atherosclerosis in mice (Ait-Oufella et al., 2006). Thus studies have shown an important role for T reg cells in inhibiting the progression of atherosclerosis in mice.

"It has been previously shown that TGF-.beta.1 (transforming growth factor (.beta.1) may play a role in regulating the activity of T reg cells. TGF-.beta.1 is a pleiotropic growth factor important in cell growth, differentiation, and activation in a number of immune and non-immune cell types (Feinberg and Jain, 2005; Li and Flavell, 2008; Shi et al., 1999). TGF-.beta.1 is involved in the maintenance of self-tolerance and homeostasis of several T cell effectors including T regulatory cells (Fantini et al., 2004; Li et al., 2006; Wan and Flavell, 2005). Indeed, disruption of TGF-.beta.1 or its receptors in T cells induces a severe lymphoproliferative response and autoimmunity (Marie et al., 2006; Shull et al., 1992). Thus, tight control of TGF-.beta.1 and its downstream signaling pathways may allow for fine-tuning of the immune response by modulating T regulatory cell development or function. With particular regard to atherosclerosis, the effect of T regulatory cells in limiting atherosclerosis has been shown to be dependent in part on TGF-.beta. signaling. TGF-.beta. has potent immunosuppressive effects on multiple cell types, including effects on T cell activation, SMC proliferation, collagen formation, endothelial proliferation, and macrophage activation.

"Additionally, with regard to CD4+CD25+ T reg cells, TGF-.beta.1 signaling is believed to be required for the differentiation of CD4+CD5- T reg cells to CD4+CD5+ T reg cells. TGF-.beta. is also required for peripheral maintenance of CD4+CD25+ cells and their suppression function, but not for their thymic development (Marie et al., 2005). The suppression function of CD4+CD25+ cells includes inhibition of the inflammation response, e.g., due to the immune response to intracellular pathogens. Because inflammation is also associated with pathologies such as atherosclerosis, type I diabetes, and multiple sclerosis, TGF-.beta.1 signaling may inhibit atherosclerosis by conferring T regulatory function and control of inflammation.

"An additional possible regulator of T reg cells are the Kruppel-like family of proteins, or KLFs, which a family of related zinc-finger transcription factors that have roles in various aspects of cellular growth, development and differentiation, particularly in the hematopoietic system. Several KLFs with these properties have been identified, including KLFs 1-4. KLF1, or EKLF has been shown to be essential for erythropoiesis. KLF2, or LKLF (KLF2) plays a role in T-lymphocyte development. KLF3, or BKLF, has been implicated in the of myeloproliferative disorder. KLF4 or GKLF, is involved in epithelial development, including differentiation of gut, skin, monocyte. Because of the importance of KLFs in different hematopoietic lineages, Kruppel-like zinc-finger protein may regulate the differentiation or function of T reg cells.

"Due to the wide prevalence of autoimmune diseases, including type I diabetes, multiple sclerosis, inflammatory bowel disease, and atherosclerosis, among others, as well as the limited treatment options that are available, there remains a need for new strategies for developing and obtaining useful therapeutics for combating these disorders."

In addition to the background information obtained for this patent application, NewsRx journalists also obtained the inventor's summary information for this patent application: "The purpose and advantages of the present invention will be set forth in and become apparent from the description that follows. Additional advantages of the invention will be realized and attained by the methods and systems particularly pointed out in the written description and claims hereof, as well as from the appended drawings.

"It has been discovered that KLF10 is expressed in T regulatory cells and modulates T regulatory function. Without being bound to a particular theory, KLF10 has at least two activities that modulate T regulatory function, i.e., inducing Foxp3 and negatively regulating NFAT. Applicants' discovery, accordingly, provides for uses of KLF10 as described herein.

"To achieve these and other advantages and in accordance with the purpose of the invention, as embodied herein, the invention includes, among other things, a method of detecting, from a patient's blood sample, expression of KLF10 as a relative marker for inflammatory disease states such as coronary artery disease, among others. Such assays to identify KLF10 may include a chip, plate, liquid, bead, or membrane array and the like. In addition, if desired the KLF10 promoter (5'UTR) may be used as a screen to identify molecules or compounds that may be important for modulating T regulatory cell function or cell number and, as a consequence, the development of inflammatory disease states, autoimmune disease, multiple sclerosis, and cancer, among others. Finally, KLF 10 itself may be used to generate or promote increased number or function of CD4+CD25+ T regulatory cells which may be used in a wide variety of applications in which suppressing inflammation is important for limiting disease progression. Various systems can be used to facilitate localized delivery of compounds to treat inflammation in accordance with the invention.

"In one aspect, the invention provides methods for treating a chronic inflammatory disease in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of a KLF10 polypeptide or fragment thereof, wherein the KLF10 polypeptide or fragment thereof induces a T regulatory phenotype.

"In one embodiment, the KLF10 protein or fragment thereof is systemically administered. In another embodiment, the KLF10 polypeptide or fragment thereof is administered locally at a site of inflammation

"In one embodiment, the KLF10 polypeptide or fragment thereof further comprises an intracellular cargo delivery ligand.

"In one embodiment, the KLF10 polypeptide or fragment thereof is administered by gene therapy. In a further embodiment, administration by gene therapy comprises delivery of an expression vector capable of expressing the KLF10 polypeptide or fragment thereof.

"In one embodiment, the invention provides methods for treating a chronic inflammatory disease in a subject in need thereof, according to one or more of the preceding aspects and embodiments, and further comprising contacting a CD4+/CD25- cell with the KLF10 or fragment thereof, whereby the step of contacting results in a conversion of the CD4+/CD25- cell to a CD4+/CD25+ cell.

"In one embodiment, the inflammatory disease is a coronary artery disease. In another embodiment, the inflammatory disease is atherosclerosis. In another embodiment, the inflammatory disease is type 1 diabetes. In another embodiment, the inflammatory disease is multiple sclerosis.

"In one embodiment, the KLF10 polypeptide or fragment thereof is administered on an implantable stent. In another embodiment, the KLF10 polypeptide or fragment thereof is administered by myocardium injection.

"In a specific embodiment, the KLF10 polypeptide or fragment thereof is administered by gene therapy. The administration by gene therapy comprises delivery of an expression vector capable of expressing the KLF10 polypeptide or fragment thereof and the expression vector is administered by myocardium injection.

"In a specific embodiment, the KLF10 polypeptide or fragment thereof is administered by gene therapy. The administration by gene therapy comprises delivery of an expression vector capable of expressing the KLF10 polypeptide or fragment thereof and the expression vector is administered on an implantable stent.

"In another aspect, the invention provides methods for identifying a candidate compound that may modulate a KLF 10-induced T regulatory phenotype. The methods comprise the steps of contacting a KLF10-sensitive reporter gene with the KLF10 polypeptide both in the presence and the absence of the candidate compound; detecting the expression level of the KLF10-sensitive reporter gene; and comparing the expression level of the KLF10-sensitive reporter gene in the presence and absence of the candidate compound. A modulated level of expression of the KLF10-sensitive reporter gene in the presence of the candidate compound is indicative of a compound that may modulate a KLF 10-induced T regulatory phenotype.

"In one embodiment, the methods further comprise testing the candidate compound in an animal model to determine whether the candidate compound modulates a KLF10-induced T regulatory phenotype.

"In yet another aspect, the invention provides methods for identifying a candidate compound that induces the T regulatory phenotype in CD4+/CD25- cells. The methods comprise the steps of contacting a CD4+/CD25- cell comprising a reporter gene under the control of a KLF10 promoter with a candidate compound; detecting the expression level of the reporter gene, wherein upregulation of the reporter gene is indicative of a compound that induces the T regulatory phenotype.

"In one embodiment, the methods further comprise testing the candidate compound in an animal model to determine whether the candidate compound modulates a KLF10-induced T regulatory phenotype.

"In still another aspect, the invention provides pharmaceutical compositions comprising a therapeutically effective dose of a KLF10 polypeptide or fragment thereof and a pharmaceutically acceptable excipient.

"In one aspect, the invention provides kits comprising a therapeutically effective dose of a KLF10 polypeptide or fragment thereof and instructions for use in treating a chronic inflammatory disease.

"In one embodiment, the chronic inflammatory disease is atherosclerosis, type 1 diabetes, multiple sclerosis, an autoimmune disease, an inflammatory cardiac disease, or cancer.

"In one aspect, the invention provides kits comprising a therapeutically effective dose of an expression vector encoding a KLF10 polypeptide or fragment thereof and instructions for use in treating a chronic inflammatory disease.

"In one embodiment, the chronic inflammatory disease is atherosclerosis, type 1 diabetes, multiple sclerosis, an autoimmune disease, an inflammatory cardiac disease, or cancer.

"In another aspect, the invention provides stents comprising a plurality of interconnected struts, wherein at least one of the struts includes a beneficial agent including a therapeutically effective amount of a KLF10 polypeptide or fragment thereof disposed thereon.

"In one embodiment, the beneficial agent is disposed in a polymeric medium formed on the strut. In another embodiment, the beneficial agent is disposed in a depression formed in the strut. In yet another embodiment, the stent is formed substantially of a polymeric material.

"In yet another aspect, the invention provides a system for treating a patient, comprising a stent delivery catheter; and a stent disposed on the stent delivery catheter. The stent comprises a plurality of interconnected struts, wherein at least one of the struts includes a beneficial agent including a therapeutically effective amount of a KLF10 polypeptide or fragment thereof disposed thereon.

"In one embodiment, the stent delivery catheter is an over the wire catheter. In another embodiment, the stent delivery catheter is a rapid exchange catheter.

"In another embodiment the stent delivery catheter includes a retractable sheath that exposes the stent when the sheath is retracted. In a specific embodiment, the stent is a self-expanding stent.

"It is to be understood that both the foregoing general summary and the following detailed description are exemplary and are intended to provide further explanation of the invention claimed.

BRIEF DESCRIPTION OF THE DRAWINGS

"The following Detailed Description, given by way of example, but not intended to limit the invention to specific embodiments aspects described, may be understood in conjunction with the accompanying drawings, which incorporated herein by reference. Various features and aspects of the present invention will now be described by way of non-limiting examples and with reference to the accompanying drawings, in which:

"FIG. 1 depicts KLF10 expression in CD4.sup.+CD25.sup.+ T regulatory cells. (A) Real-time qPCR analyses of KLF10 mRNA in CD4.sup.+CD25.sup.- and CD4.sup.+CD25.sup.+ T cells isolated from spleens of mice demonstrate that KLF10 (left) is highly expressed in CD4.sup.+CD25.sup.+ T regulatory cells. Expression of the T regulatory marker Foxp3 (right) was used as a positive control. (B) qPCR analyses show that other KLFs (KLF2, KLF4, and KLF5) were either not changed or decreased. (C) CD4.sup.+CD25.sup.- T cells were treated for TGF-.beta.1 at the indicated time points, and total RNA was harvested for qPCR analyses. KLF10 (left) and Foxp3 (right) mRNAs are rapidly induced by TGF-.beta.1 at 1 hr and 6 hrs, followed by reduced expression at 24 hrs. (D and E) TGF-.beta.1 responsiveness of KLF10 was also assessed using the Jurkat T cell line. Total RNA was subjected to Northern analysis (10 .mu.g/lane) after cells were treated with TGF-.beta.1 at the indicated time points (D) or in a dose-dependent manner (E). KLF10 mRNA was induced in a time- and dose-dependent manner in Jurkat cells. (F) Depicts the expression of KLF10 mRNA in response to stimulation by .alpha.-CD3; (G) Depicts the effect of ApoE deficiency on the expression of KLF10 mRNA.

"FIG. 2. Shows the effect of KLF10 overexpression on Foxp3 expression in CD4.sup.+CD25.sup.- T cells. (A) CD4.sup.+CD25.sup.- T cells were isolated as in FIG. 1, transduced with retrovirus GFP-RV-EV (empty-virus, ctrl) or GFP-RV-KLF10 for 72 hrs, and FACS analyses (left) were performed to assess percentage of GFP-positive cells that also expressed intracellular Foxp3. Quantification (right) of Foxp3 intracellular expression by FACS was performed from n=3 separate experiments. (B) Transduced cells from (A) were subjected to qPCR analyses for Foxp3 expression. KLF10 overexpression in CD4.sup.+CD25.sup.- T cells induces Foxp3 mRNA expression. (C) Effects of KLF10-overexpressing CD4.sup.+CD25.sup.- T cells on T-bet (left) and Gata3 (right) mRNA expression by qPCR analysis. (D) The growth rate of EV or KLF10-infected CD4.sup.+CD25.sup.- T cells counted over 5 days. (E) Suppression of co-cultured CD4.sup.+CD25.sup.- T cells by KLF10. EV or KLF10-overexpressing cells were co-cultured with CD4.sup.+CD25.sup.- T cell effectors, .alpha.-CD3 Ab (1 .mu.g/ml), and APCs for 72 hrs and proliferation was assessed by thymidine incorporation during the last 18 hrs. (F) KLF10-overexpressing cells promote contrasting effects on TGF-.beta.1 and IFN-.gamma. expression. KLF10 promotes TGF-.beta.1 mRNA and protein (top) as measured by ELISA from cell culture supernatants, whereas it represses IFN-.gamma. expression (bottom). (G) KLF10 transctivates the Foxp3 promoter and binds to DNA through an evolutionarily conserved KLF site. Transient transfection studies were performed by nucleofection (Amaxa) using CD4.sup.+CD25.sup.- T cells. KLF10 induced the Foxp3 promoter, whereas mutation of the KLF site completely abolished this induction. TGF-.beta.1 induced the Foxp3 promoter; however, mutation of the KLF site prevented the TGF-.beta.1-mediated induction. (H) KLF10 binding to the Foxp3 promoter is dynamically regulated by TGF-.beta.1. CD4.sup.+CD25.sup.- T cells were subjected to chromatin immunoprecipitation (ChIP) assays using antibodies to IgG or KLF10. (I) DNAs isolated from ChIP assays were analyzed in triplicate by quantitative real-time PCR. Values were presented as relative to DNA input. (J) Effect of KLF10 expression on p21 expression and KLF10 modification.

"FIG. 3 shows the effect of KLF10-deficiency in CD4.sup.+CD25.sup.- T cells on Foxp3 expression, T regulatory cell development, and Th1/Th2 differentiation. (A-B) CD4.sup.+CD25.sup.- cells were transfected with siRNA for NS (non-specific scrambled siRNA) or KLF10 (siKLF10) by nucleofection technique (Amaxa) and harvested 48 hrs later for qPCR analysis after 6 hrs of treatment with vehicle Ctrl or TGF-.beta.1 (1 ng/ml). KLF10 'knockdown' (A) reduces Foxp3 mRNA expression in response to TGF-.beta.1 (B). (C) CD4.sup.+CD25.sup.- cells transfected cells (NS or siKLF10) were grown for 72 hrs. KLF10-knockdown promoted cell growth by .about.33% vs. NS Ctrl. (D) In response to TGF-.beta.1, Foxp3 mRNA expression is reduced in KLF10-/- CD4.sup.+CD25.sup.- T cells. WT or KLF10-/- CD4.sup.+CD25.sup.- T cells were treated with anti-CD3 Abs and TGF-.beta.1 for 24 hours followed by qPCR analysis. (E) KLF10-/- mice have markedly reduced peripheral CD4.sup.+CD25.sup.+ and CD4.sup.+CD25.sup.+Foxp3.sup.+ T regulatory cells. Spleen and mesenteric lymph nodes from littermate, day 10 male KLF10-/- and WT mice (n=4/group) were analyzed by FACS for percentage of CD4.sup.+CD25.sup.+ and CD4.sup.+CD25.sup.+Foxp3.sup.+ T regulatory cells. (F) KLF10-/- CD4.sup.+CD25.sup.- T cells differentiate more readily along Th1 and Th2 pathways than WT CD4.sup.+CD25.sup.- T cells. CD4.sup.+CD25.sup.- T cells were treated along Th1- or Th2-skewing conditions for up to 3 days. (Top) The Th1 markers T-Bet and IFN-131 were assessed by qPCR and ELISA, respectively; (bottom) the Th2 markers Gata3 and IL-5 were assessed by qPCR and ELISA, respectively.

"FIG. 4 depicts the effect of KLF10-deficiency on CD4.sup.+CD25.sup.+ T regulatory cell function. (A) KLF10-/- CD4.sup.+CD25.sup.+ T regs have markedly impaired suppression function. Equal numbers of WT or KLF10-/- CD4.sup.+CD25.sup.+ T regs were co-cultured with WT CD4.sup.+CD25.sup.- T cell effectors, .alpha.-CD3 Ab (0.25 .mu.g/ml), and APCs for 72 hrs and proliferation was assessed by thymidine incorporation during the last 18 hrs. (B) Gene-dosage effect of KLF10-deficiency on T reg suppression function. Equal numbers of WT, KLF10+/-, or KLF10-/- CD4.sup.+CD25.sup.+ T regs were subjected to suppression assays as described in (A). (C) Reduced levels of TGF-.beta.1 elaboration in KLF10-/- CD4.sup.+CD25.sup.+ and CD4.sup.+CD25.sup.+Foxp3.sup.+ T regs. FACS-purified T regs were stimulated by .alpha.-CD3 Abs for 24 hrs. After stimulation, culture supernatants were harvested and assessed by SearchLight Proteome Arrays/multiplex sandwich ELISA for TGF-.beta.1. D) Rescue of KLF10-/- T regulatory cell suppression function by exogenous TGF-.beta.1. CD4.sup.+CD25.sup.+ T regs (left) or CD4.sup.+CD25.sup.+Foxp3.sup.+ T regs (right) were isolated from WT or KLF10-/- mice and suppression assays were performed in the presence of .alpha.-CD3 Ab, APCs, and TGF-.beta.1 (1 ng/ml) as described in (A). (E) KLF10-/- T regs have defective TGF-.beta.1 signaling. WT and KLF10-/- CD4.sup.+CD25.sup.+ T regs were stimulated with anti-CD3 Abs and TGF-.beta.1 (1 ng/ml) for 1 hr and Western blot analyses performed for the indicated proteins. KLF10-/- T regs have markedly reduced levels of phosphorylated Smad2. Data are representative of three independent experiments and similar results were obtained as quantitated by densitometry of the bands (right).

"FIG. 5 shows that KLF10-/- CD4.sup.+CD25.sup.- T cells are hyperactivated and promote atherosclerosis. (A) WT or KLF10-/- CD4.sup.+CD25.sup.- T cell effectors were FACS-purified and subjected to suppression assays using WT CD4.sup.+CD25.sup.+ T regs in the presence of anti-CD3 Abs and APCs for 72 hrs and proliferation was assessed by thymidine incorporation during the last 18 hrs. WT T regs suppress KLF10-/- CD4.sup.+CD25.sup.- T cells less effectively than WT CD4.sup.+CD25.sup.- T cell effectors. (B) KLF10-/- CD4.sup.+CD25.sup.- T cells are hyperactivated in response to TCR stimulation. WT or KLF10-/- CD4.sup.+CD25.sup.- T cells were stimulated with anti-CD3 Abs for 24 hrs and subjected to qPCR analyses for the indicated cytokines (C) After stimulation in (B), culture supernatants were harvested and assessed by SearchLight Proteome Arrays/multiplex sandwich ELISA for the indicated cytokines. Each sample was evaluated in triplicate and is representative of two independent experiments. (D) KLF10-/- CD4.sup.+CD25.sup.- T cells promotes atherosclerotic lesion formation in ApoE-/-/scid/scid mice. WT or KLF10-/- CD4.sup.+CD25.sup.- T cells were transferred intravenously into ApoE-/-/scid/scid mice (n=5 recipient mice for WT cells; n=7 recipient mice for KLF10-/- cells), placed on a high fat diet for five weeks, and aortic root lesion size (.mu.m.sup.2) was quantitated morphometrically after Oil Red O staining (left). Quantitation of staining for CD4+ T cells and Mac3+ macrophages (Right). Data were obtained by counting lesions at the aortic root from 5 to 7 mice of each group. (E) Plasma levels of pro-inflammatory mediators increased, whereas TGF-.beta.1 levels decreased in recipient ApoE-/-/scid/scid adoptively transferred with KLF10-/- CD4.sup.+CD25.sup.- T cells. Plasma from ApoE-/-/scid/scid mice receiving WT or KLF10-/- CD4.sup.+CD25.sup.- T cells was collected and assessed by SearchLight Proteome Arrays/multiplex sandwich ELISA as in (B). (F) KLF10-/- CD4.sup.+CD25.sup.- T cells have defective TGF-.beta.1 signaling. WT and KLF10-/- CD4.sup.+CD25.sup.- T cells were stimulated with anti-CD3 Abs and TGF-.beta.1 (1 ng/ml) for 1 hr and Western blot analyses performed for the indicated proteins. KLF10-/- CD4.sup.+CD25.sup.- T cells have markedly reduced levels of phosphorylated Smad2. Data are representative of three independent experiments and similar results were obtained as quantitated by densitometry of the bands (right).

"FIG. 6 depicts the effect of KLF10 overexpressing cells on cytokines/chemokines after stimulation with PMA/ionomycin as measured by ELISA including the cytokines/chemokines. (A) IL-2; (B) IFN-.gamma.; (C) IFN-.alpha.; (D) IL-1.beta.; (E) MIP-1.alpha.; (F) MIP-1.beta.; (G) CD40L; and (H) RANTES.

"FIG. 7 depicts the effect of KLF10 in an NFAT concatamer assay and an IFN.gamma. promoter assay: (A) KLF10 inhibits NFAT concatamer activity in Cos-7 cells; (B) KLF10 inhibits IFN.gamma. promoter activity in Jurkat cells; (C) KLF10 inhibits NFAT concatamer activity in Cos-7 cells; and (D) KLF10 inhibits IFN.gamma. promoter activity in Jurkat cells.

"FIG. 8 depicts KLF10 regulation of NFAT1: (A) KLF10 inhibits NFAT1 expression; and (B) KLF10 inhibits NFAT DNA-protein binding to the IFN.gamma. promoter.

"FIG. 9 depicts that overexpression of KLF10 suppresses cell growth and inhibits pro-inflammatory responses in Jurkat T cells. (A-C) Jurkat cells were retrovirally transduced with GFP-RV-EV (ctrl) or GFP-RV-KLF10 for 48 hrs, FACS-sorted for GFP-positive cells, and in (A), allowed to grow over the indicated time course. KLF10-overexpression repressed cell growth 2-fold up to 5-days in culture and, in (B), was associated with induction of the cyclin-dependent kinase inhibitor, p21.sup.WAF1CIP1. KLF10 (exo) is exogenous KLF10; KLF10 (endo) is endogenous KLF10. EtBr, Ethidium Bromide. (C) KLF10-overexpressing cells suppress PMA (20 ng/ml)/ionomycin (3.5 .mu.g/mL) induction of a variety of cytokines, growth factors, and chemokines by ELISA analysis using Searchlight Multiplex protein arrays.

"FIG. 10 depicts that KLF10-/- CD4.sup.+CD25.sup.- T cells have similar levels of TGF-01 mRNA and secreted protein. WT or KLF10-/- CD4.sup.+CD25.sup.- T cells were stimulated with anti-CD3 Abs for 24 hrs and subjected to qPCR analyses (A) for TGF-.beta.1 mRNA or culture supernatants were harvested and assessed by SearchLight Proteome Arrays/multiplex sandwich ELISA for TGF-.beta.1 protein (B). Each sample was evaluated in triplicate and is representative of two independent experiments."

URL and more information on this patent application, see: Feinberg, Mark W. Diagnosing, Monitoring and Treating Inflammation. Filed December 17, 2013 and posted July 17, 2014. Patent URL: http://appft.uspto.gov/netacgi/nph-Parser?Sect1=PTO2&Sect2=HITOFF&u=%2Fnetahtml%2FPTO%2Fsearch-adv.html&r=1847&p=37&f=G&l=50&d=PG01&S1=20140710.PD.&OS=PD/20140710&RS=PD/20140710

Keywords for this news article include: Angiology, Arterial Occlusive Diseases, Arteriosclerosis, Atherosclerosis, Autoimmune Diseases of the Nervous System, Autoimmune Disorders, Bioengineering, Biotechnology, CNS Demyelinating Autoimmune Diseases, Cancer Gene Therapy, Cardiology, Cardiovascular Diseases, Chemokine Receptors, Chemokines, Chemotactic Factors, Cytokine Receptors, Cytokines, Demyelinating Diseases, G-Protein-Coupled Receptors, Genetics, Hematology, Hematopoietic, Immune System Diseases, Immunology, Inflammation Mediators, Insulin Dependent Diabetes Mellitus, Macrophages, Membrane Proteins, Mononuclear Phagocyte System, Multiple Sclerosis, Neuroimmunology, Neurology, Oncology, Phagocytes, Pharmaceuticals, Small Interference RNAs, The Brigham and Women's Hospital, The Brigham and Women's Hospital Inc., Type 1 Diabetes, Viral DNA, Viral RNA, siRNA.

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