In Vivo Downregulation of Innate and Adaptive Immune Responses in Corneal Allograft Rejection by HC-HA/PTX3 Complex Purified From Amniotic Membrane

Citation: He H, Tan Y, Duffort S, Perez VL, Tseng SCG. In vivo downregulation of innate and adaptive immune responses in corneal allograft rejection by HC-HA/PTX3 complex purified from amniotic membrane. Invest Ophthalmol Vis Sci. 2014;55:164755: -165655: . DOI:10.1167 PURPOSE. Heavy chain-hyaluronic acid (HC-HA)/PTX3 purified from human amniotic membrane (AM) was previously observed to suppress inflammatory responses in vitro. We now examine whether HC-HA/PTX3 is able to exert a similar effect in vivo, using murine models for keratitis and corneal allograft rejection. METHODS. The in vitro effect of HC-HA/PTX3 was tested using OTII ovalbumin (OVA) transgenic, purified CD4 þ T cells, or IFN-c/lipopolysaccharide (LPS)-stimulated RAW264.7 cells. Cytokine production was measured by ELISA, while cell surface markers and cell proliferation were determined by flow cytometry. In vivo effects of HC-HA/PTX3 were analyzed by quantifying the recruitment of enhanced green fluorescence-labeled macrophages and by measuring the expression of arginase 1 (Arg-1), IL-10, and IL-12 in LPS-induced keratitis in the macrophage Fas-induced apoptosis (Mafia) mouse. The effect of corneal allograft survival in a complete major histocompatibility complex (MHC) mismatched mouse model was assessed by grading corneal opacification. RESULTS. In vitro studies demonstrated that HC-HA/PTX3 significantly enhanced the expansion of FOXP3 T cells and suppressed cell proliferation and protein expression of IFN-c, IL-2, CD25, and CD69 in activated CD4 þ T cells. Furthermore, immobilized HC-HA/PTX3 significantly upregulated IL-10 gene expression but downregulated that of IL-12 and IL-23 in activated RAW264.7 cells. Finally, in vivo subconjunctival injection of HC-HA/PTX3 significantly prolonged corneal allograft survival, suppressed macrophage infiltration, and promoted M2 polarization by upregulating Arg-1 and IL-10 but downregulating IL-12. CONCLUSIONS. HC-HA/PTX3 can suppress inflammatory responses in vivo by modulating both innate and adaptive immunity of macrophages and CD4 þ T cells

Synthesis of a Dual Functional Anti-MDR Tumor Agent PH II-7 with Elucidations of Anti-Tumor Effects and Mechanisms

Multidrug resistance mediated by P-glycoprotein in cancer cells has been a major issue that cripples the efficacy of chemotherapy agents. Aimed for improved efficacy against resistant cancer cells, we designed and synthesized 25 oxindole derivatives based on indirubin by structure-activity relationship analysis. The most potent one was named PH II-7, which was effective against 18 cancer cell lines and 5 resistant cell lines in MTT assay. It also significantly inhibited the resistant xenograft tumor growth in mouse model. In cell cycle assay and apoptosis assay conducted with flow cytometry, PH II-7 induced S phase cell cycle arrest and apoptosis even in resistant cells. Consistently revealed by real-time PCR, it modulates the expression of genes related to the cell cycle and apoptosis in these cells, which may contributes to its efficacy against them. By side-chain modification and FITC-labeling of PH II-7, we were able to show with confocal microscopy that not only it was not pumped by P-glycoprotein, it also attenuated the efflux of Adriamycin by P-glycoprotein in MDR tumor cells. Real-time PCR and western blot analysis showed that PH II-7 down-regulated MDR1 gene via protein kinase C alpha (PKCA) pathway, with c-FOS and c-JUN as possible mediators. Taken together, PH II-7 is a dual-functional compound that features both the cytotoxicity against cancer cells and the inhibitory effect on P-gp mediated drug efflux

Infiltration of Proinflammatory M1 Macrophages into the Outer Retina Precedes Damage in a Mouse Model of Age-Related Macular Degeneration

Age-related macular degeneration (AMD) is a major cause of blindness in the developed world. Oxidative stress and inflammation are implicated in AMD, but precise mechanisms remain poorly defined. Carboxyethylpyrrole (CEP) is an AMD-associated lipid peroxidation product. We previously demonstrated that mice immunized with CEP-modified albumin developed AMD-like degenerative changes in the outer retina. Here, we examined the kinetics of lesion development in immunized mice and the presence of macrophages within the interphotoreceptor matrix (IPM), between the retinal pigment epithelium and photoreceptor outer segments. We observed a significant and time-dependent increase in the number of macrophages in immunized mice relative to young age-matched controls prior to overt pathology. These changes were more pronounced in BALB/c mice than in C57BL/6 mice. Importantly, IPM-infiltrating macrophages were polarized toward the M1 phenotype but only in immunized mice. Moreover, when Ccr2-deficient mice were immunized, macrophages were not present in the IPM and no retinal lesions were observed, suggesting a deleterious role for these cells in our model. This work provides mechanistic evidence linking immune responses against oxidative damage with the presence of proinflammatory macrophages at sites of future AMD and experimentally demonstrates that manipulating immunity may be a target for modulating the development of AMD

Integrated motor drives: state of the art and future trends

With increased need for high power density, high efficiency and high temperature capabilities in Aerospace and Automotive applications, Integrated Motor Drives (IMD) offers a potential solution. However, close physical integration of the converter and the machine may also lead to an increase in components temperature. This requires careful mechanical, structural and thermal analysis; and design of the IMD system. This paper reviews existing IMD technologies and their thermal effects on the IMD system. The effects of the power electronics (PE) position on the IMD system and its respective thermal management concepts are also investigated. The challenges faced in designing and manufacturing of an IMD along with the mechanical and structural impacts of close physical integration is also discussed and potential solutions are provided. Potential converter topologies for an IMD like the Matrix converter, 2-level Bridge, 3-level NPC and Multiphase full bridge converters are also reviewed. Wide band gap devices like SiC and GaN and their packaging in power modules for IMDs are also discussed. Power modules components and packaging technologies are also presented

The immune system and gastrointestinal stromal tumor: a wealth of opportunities

This article reviews the current literature on tumor-infiltrating immune cells in gastrointestinal stromal tumor (GIST), and the current status and prospects of effective immunotherapeutic strategies. Tumor-infiltrating immune cells populate the microenvironment of GISTs; the most numerous are tumor-associated macrophages (TAMs) and CD3 T cells. TAMs have not been shown to have a relationship with the biological behavior of GISTs; however, the number of CD3 T cells correlates with better outcomes. The prognostic significance of tumor-infiltrating neutrophils, natural killer cells, CD4 T cells, CD8 T cells, and Treg cells remains unknown.Imatinib mesylate achieves a clinical response in 80% of patients with GIST. Its antitumor mechanism is partially immune mediated. The combination of imatinib and interferon-α has been shown to be effective against GIST - it eradicates tumor cells including those that are drug resistant. Preclinical trials including cytotoxic T lymphocyte-associated antigen 4 blockade, anti-KIT antibody, and the generation of designer T cells have shown promising therapeutic effect in animal models of GIST. GIST contains many tumor-infiltrating immune cells and should be susceptible to immunotherapy; early clinical and preclinical trials have shown promising results that should lead to new investigations and effective forms of direct and synergistic therapies

In Vivo Downregulation of Innate and Adaptive Immune Responses in Corneal Allograft Rejection by HC-HA/PTX3 Complex Purified From Amniotic Membrane

PURPOSE. Heavy chain–hyaluronic acid (HC-HA)/PTX3 purified from human amniotic membrane (AM) was previously observed to suppress inflammatory responses in vitro. We now examine whether HC-HA/PTX3 is able to exert a similar effect in vivo, using murine models for keratitis and corneal allograft rejection. METHODS. The in vitro effect of HC-HA/PTX3 was tested using OTII ovalbumin (OVA) transgenic, purified CD4(+) T cells, or IFN-γ/lipopolysaccharide (LPS)-stimulated RAW264.7 cells. Cytokine production was measured by ELISA, while cell surface markers and cell proliferation were determined by flow cytometry. In vivo effects of HC-HA/PTX3 were analyzed by quantifying the recruitment of enhanced green fluorescence–labeled macrophages and by measuring the expression of arginase 1 (Arg-1), IL-10, and IL-12 in LPS-induced keratitis in the macrophage Fas-induced apoptosis (Mafia) mouse. The effect of corneal allograft survival in a complete major histocompatibility complex (MHC) mismatched mouse model was assessed by grading corneal opacification. RESULTS. In vitro studies demonstrated that HC-HA/PTX3 significantly enhanced the expansion of FOXP3 T cells and suppressed cell proliferation and protein expression of IFN-γ, IL-2, CD25, and CD69 in activated CD4(+) T cells. Furthermore, immobilized HC-HA/PTX3 significantly upregulated IL-10 gene expression but downregulated that of IL-12 and IL-23 in activated RAW264.7 cells. Finally, in vivo subconjunctival injection of HC-HA/PTX3 significantly prolonged corneal allograft survival, suppressed macrophage infiltration, and promoted M2 polarization by upregulating Arg-1 and IL-10 but downregulating IL-12. CONCLUSIONS. HC-HA/PTX3 can suppress inflammatory responses in vivo by modulating both innate and adaptive immunity of macrophages and CD4(+) T cells

Benzyl and naphthalene methylphosphonic acid inhibitors of autotaxin with anti-invasive and anti-metastatic activity

Autotaxin (ATX, NPP2) is a member of the nucleotide pyrophosphate phosphodiesterase enzyme family. ATX catalyzes the hydrolytic cleavage of lysophosphatidylcholine (LPC) by lysophospholipaseD activity, which leads to generation of the growth-factor-like lipid mediator lysophosphatidic acid (LPA). ATX is highly upregulated in metastatic and chemotherapy-resistant carcinomas and represents a potential target to mediate cancer invasion and metastasis. Herein we report the synthesis and pharmacological characterization of ATX inhibitors based on the 4-tetradecanoylaminobenzylphosphonic acid scaffold, which was previously found to lack sufficient stability in cellular systems. The new 4-substituted benzylphosphonic acid and 6-substituted naphthalen-2-ylmethylphosphonic acid analogues block ATX activity with Ki values in the low micromolar to nanomolar range against FS3, LPC, and nucleotide substrates through a mixed-mode inhibition mechanism. None of the compounds tested inhibit the activity of related enzymes (NPP6 and NPP7). In addition, the compounds were evaluated as agonists or antagonists of seven LPA receptor (LPAR) subtypes. Analogues 22 and 30b, the two most potent ATX inhibitors, inhibit the invasion of MM1 hepatoma cells across murine mesothelial and human vascular endothelial monolayers invitro in a dose-dependent manner. The average terminal half-life for compound 22 is 10±5.4h and it causes a long-lasting decrease in plasma LPA levels. Compounds 22 and 30b significantly decrease lung metastasis of B16-F10 syngeneic mouse melanoma in a post-inoculation treatment paradigm. The 4-substituted benzylphosphonic acids and 6-substituted naphthalen-2-ylmethylphosphonic acids described herein represent new lead compounds that effectively inhibit the ATX-LPA-LPAR axis both invitro and invivo. Inhibiting the ATX-LPA-LPAR axis: New 4-substituted benzylphosphonic acid and 6-substituted naphthalen-2-ylmethylphosphonic acid analogues were synthesized, and the most potent ATX inhibitors, 22 and 30b, show outstanding invivo profiles by diminishing lung metastases of B16-F10 syngeneic mouse melanoma in a post-inoculation treatment model. These two lead compounds effectively inhibit the ATX-LPA-LPAR axis both invitro and invivo. © 2011 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

Recruitment Of T Cells and Macrophages To The Eyes In Recipients Of Allogeneic Hematopoietic Stem Cell Transplants Correlate With Inflammatory Cytokine Presence In Ocular Gvhd

Abstract Allogeneic hematopoietic stem cell transplantation (HSCT) has become the standard of care for the treatment of several life-threatening hematologic malignancies as well as certain immunodeficiency disorders. Unfortunately, as the survival rate of patients with these diseases is improved, the quality of life is negatively impacted by the development of Graft vs. Host Disease (GVHD). GVHD is a complex, multi-organ disorder arising from an immunological attack by donor allo-reactive T cells that results in damage to vital organs including the liver, skin, hematopoietic compartment and the ocular surface of the eye. Ocular GVHD occurs in >60% of these patients and is characterized by dry eye, conjunctiva damage, punctate keratopathy, corneal ulceration and perforation. Despite the high frequency of eye involvement in patients undergoing GVHD, little is known regarding the underlying immune mechanisms responsible for ocular GVHD, limiting the ophthalmic care of these patients to palliative therapies and global anti-inflammatory drugs. In this study, we examined the ocular and immunological changes occurring in recipients of MHC-matched, minor antigen mis-matched donor HSCT. C3H.SW (H-2b, Ly9.1+) mice transplanted with EGFP+ B6 (H-2b, Ly9.1-) T cell depleted bone marrow cells (TCD-BM) supplemented with T cells: a) underwent weight loss and began exhibiting clinical signs of GVHD ∼3wks post-HSCT, b) contained damaged thymuses, c) expressed an inverted CD4/CD8 ratio in the peripheral lymphoid compartments, d) contained activated effector cells and e) low CD19 levels. Importantly, these mice also developed ocular surface disease evidenced by progression of ocular surface damage characterized by increased corneal fluorescein staining and ulceration by week 6 (Figure). Furthermore, histological analyses demonstrated that only mice that developed systemic GVHD exhibited corneal thickening and epithelial irregularity. Ocular pathology was also associated with conjunctiva involvement indicated by significant goblet cell destruction as well as dense inflammatory cell infiltrates identified by intra-vital fluorescent microscopy (Figure). IHC and flow analyses demonstrated donor EGFP+ Ly9.1- CD4+ and CD8+ T cells. Notably, significant levels of Ly9.1- EGFP-CD11b+ macrophages had also infiltrated the ocular surface. In contrast to the systemic CD8>CD4 GVHD associated phenotype, the T cell infiltrate in the ocular compartment was reversed; i.e. CD8<CD4. We detected IFNγ and TNFα mRNA from corneal tissue, which is consistent with Th1 effector allo-reactive cells and M1 inflammatory macrophages involvement in ocular GVHD. In total, the present findings have identified alterations and pathology in the eye and adnexa reflective of ocular GVHD and unequivocally demonstrate the presence of donor T cells in the ocular surface. We hypothesize that T cell–macrophage interactions underlie the pathology detected in this pre-clinical model and studies are underway to develop local therapeutic modalities targeting these infiltrative populations. Disclosures: No relevant conflicts of interest to declare