Publications by authors named "Heuy-Ching Wang"

35 Publications

Neurotrophic Factors Secreted by Induced Pluripotent Stem Cell-Derived Retinal Progenitors Promote Retinal Survival and Preservation in an Adult Porcine Neuroretina Model.

J Ocul Pharmacol Ther 2021 Mar 3. Epub 2021 Mar 3.

Ocular and Sensory Trauma Task Area, United States Army Institute of Surgical Research, Fort Sam Houston, Texas, USA.

Paracrine factors released by pluripotent stem cells have shown great potential as therapeutic agents in regenerative medicine. The purpose of this study was to characterize trophic factor secretion of retinal progenitor cells (RPCs) derived from human induced pluripotent stem cells (iPSCs) and to assess its impact on retinal survival . RPCs were generated from human 3D1 iPSCs following previously established protocols with modifications. Conditioned medium (CM) was harvested from iPSC-derived retinal progenitors and analyzed for trophic factor composition through multiplex enzyme-linked immunosorbent assay. Retina-preserving capability of the collected CM was examined using a degenerative porcine neuroretina model. Viability of the CM-treated retina explants was evaluated using the resazurin-based PrestoBlue reagent, whereas the lactate dehydrogenase (LDH) assay was used to assess retinal cytotoxicity. Retina explants were also analyzed morphologically through immunohistochemistry for glial cell activation and apoptosis. We have successfully generated and characterized iPSC-derived RPCs that secreted an array of neuroprotective factors, including osteopontin, hepatocyte growth factor, stromal cell-derived factor 1, and insulin-like growth factor-1. Retina explants cultured in CM derived from iPSC-RPCs (iPSC-RPC-CM) showed better preservation of the retinal microarchitecture and fewer terminal deoxynucleotidyl transferase dUTP nick-end labeling (TUNEL) nuclei, and reduced reactive gliosis. Furthermore, we saw a reduction in extracellular LDH levels in CM-treated retina explants, which also exhibited higher metabolic activity than the untreated controls. iPSC-derived RPCs secrete many trophic factors that have been shown to promote neuroprotection, tissue repair, and regeneration in the retina. Overall, we have demonstrated the neuroprotective effects of iPSC-RPC-CM through a degenerative neuroretina model .
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http://dx.doi.org/10.1089/jop.2020.0088DOI Listing
March 2021

Topical delivery of a small molecule RUNX1 transcription factor inhibitor for the treatment of proliferative vitreoretinopathy.

Sci Rep 2020 11 30;10(1):20554. Epub 2020 Nov 30.

Schepens Eye Research Institute of Massachusetts Eye and Ear and the Department of Ophthalmology at Harvard Medical School, Boston, USA.

Proliferative vitreoretinopathy (PVR) is the leading cause of retinal detachment surgery failure. Despite significant advances in vitreoretinal surgery, it still remains without an effective prophylactic or therapeutic medical treatment. After ocular injury or retinal detachment, misplaced retinal cells undergo epithelial to mesenchymal transition (EMT) to form contractile membranes within the eye. We identified Runt-related transcription factor 1 (RUNX1) as a gene highly expressed in surgically-removed human PVR specimens. RUNX1 upregulation was a hallmark of EMT in primary cultures derived from human PVR membranes (C-PVR). The inhibition of RUNX1 reduced proliferation of human C-PVR cells in vitro, and curbed growth of freshly isolated human PVR membranes in an explant assay. We formulated Ro5-3335, a lipophilic small molecule RUNX1 inhibitor, into a nanoemulsion that when administered topically curbed the progression of disease in a novel rabbit model of mild PVR developed using C-PVR cells. Mass spectrometry analysis detected 2.67 ng/mL of Ro5-3335 within the vitreous cavity after treatment. This work shows a critical role for RUNX1 in PVR and supports the feasibility of targeting RUNX1 within the eye for the treatment of an EMT-mediated condition using a topical ophthalmic agent.
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http://dx.doi.org/10.1038/s41598-020-77254-0DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7705016PMC
November 2020

Proliferative Vitreoretinopathy After Combat Ocular Trauma in Operation Iraqi Freedom and Operation Enduring Freedom: 2001-2011.

Ophthalmic Surg Lasers Imaging Retina 2020 10;51(10):556-563

Background And Objective: To determine the risk factors associated with developing proliferative vitreoretinopathy (PVR) from combat ocular injuries in U.S. service members.

Patients And Methods: Retrospective review of associated risk factors and outcomes of PVR within the Walter Reed Ocular Trauma Database. Ocular injuries in U.S. service members wounded during Operation Enduring Freedom and Operation Iraqi Freedom from 2001 to 2011 were assessed, and of these all cases of PVR were studied. Principal outcome measures were the risk factors associated with PVR development and rate of final visual acuity (VA) less than 20/200.

Results: Eight hundred ninety eyes of 651 U.S. service members were evaluated. A total of 76 eyes (8.5%) of 66 patients developed PVR. Five patients had bilateral PVR. Nineteen patients had bilateral eye injuries. Sixty-one eyes (80.2%) had a final VA less than 20/200. PVR was found to be a significant risk factor for a poor final VA (P < .001). Retinal detachment (RD) was found in 52 eyes (68.4%) of patients. In patients with a RD, intraocular foreign bodies (IOFBs) (P < .001), unsuccessful repair (P = .002), and macular hemorrhage (P = .04) were significant risk factors for the development of PVR. Time to initial retina surgery was not found to be a risk factor for PVR development (P = .5). Time to initial retina surgery was available in 41 patients and the time to surgery on average was 22.56 days (range: 3 to 87 days).

Conclusions: PVR occurs frequently in combat trauma and is a significant cause of poor final VA. In patients with PVR and RD, injuries caused by an IOFB, macular hemorrhage, or unsuccessful repair were significant risk factors for the development of PVR. [Ophthalmic Surg Lasers Imaging Retina. 2020;51:556-563.].
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http://dx.doi.org/10.3928/23258160-20201005-03DOI Listing
October 2020

Multidrug-Resistant Organisms from Ophthalmic Cultures: Antibiotic Resistance and Visual Acuity.

Mil Med 2020 08;185(7-8):e1002-e1007

Department of Ophthalmology, Brooke Army Medical Center, 3551 Roger Brooke Drive, San Antonio, TX 78234.

Introduction: There is a growing trend of multidrug-resistant organisms (MDRO). The goal of this study was to characterize MDRO at a single center from ophthalmic cultures to better understand how treatments were tailored and to assess effect on visual acuity.

Materials And Methods: The MDRO data were collected by the Multidrug-Resistant Organism Repository and Surveillance Network from the Brooke Army Medical Center clinical laboratory. Both patient- and isolate-specific data were collected and qualitatively analyzed. Primary outcome measures were organism and type of resistance, anatomic location of isolate, initial and final antibiotic choice, and visual acuity.

Results: Thirty-one bacterial culture samples were analyzed from 29 patients. Twenty-two (72%) were Gram-positive and all were methicillin-resistant Staphylococcus aureus (MRSA). Nine (29%) were Gram-negative and of these five were Pseudomonas spp. Fourteen (45%) isolates were cultured from the cornea, nine (29%) from the lid, four (13%) from the conjunctiva, and four (13%) from other locations. The majority (66.6%) required adjustment of initial antibiotics following ocular culture results. Sixteen adult patients had recorded initial and final visual acuities. Fifteen of those 16 patients had stable or improved visual acuities following treatment of the infection, but five patients had a final visual acuity less than 20/200.

Conclusion: This study demonstrated a high frequency of corneal MDRO infections and specifically MRSA and Pseudomonas spp. isolates. Antibiotic treatments frequently required adjustment. Further prospective study of visual outcomes from ophthalmic MDRO cultures is needed.
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http://dx.doi.org/10.1093/milmed/usaa111DOI Listing
August 2020

Detection of Retinal Fibrosis in a Rabbit Model of Penetrating Eye Injury.

Mil Med 2020 01;185(Suppl 1):443-447

Sensory Trauma, United States Army Institute of Surgical Research, 3698 Chambers Pass, Fort Sam Houston, TX 78234.

Introduction: To establish a rabbit model of posterior penetrating eye injury as a platform to test potential therapeutics.

Materials And Methods: Anesthetized rabbits received posterior penetrating eye injury in one eye, whereas contralateral eyes were maintained as uninjured controls. Rabbits were randomized into two experimental groups. Group A was euthanized on Day 14 postinjury to determine retinal fibrosis at an early phase of disease progression. Group B was euthanized on Day 28 postinjury to examine retinal fibrosis at a late phase of disease progression. We examined animals on postinjury Days 7, 14, 21, and 28 with indirect ophthalmoscope and fundus photography. After euthanasia, eyes were processed for histology and immunofluorescence labeling of fibrotic proteins α-smooth muscle actin and collagen I.

Results: Early fibrosis was detected by Day 14, as indicated by indirect ophthalmoscopy and fundus imaging. Fibrotic membranes were visible at sites of injury. Immunofluorescence analysis detected α-smooth muscle actin and collagen I within the fibrotic membranes.

Conclusions: These data show that ocular fibrosis can be detected within 14 days after initial injury, with more severe fibrosis detected at 28 days postinjury. These results will be used to determine the optimal time points for later studies designed to test treatment strategies.
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http://dx.doi.org/10.1093/milmed/usz221DOI Listing
January 2020

Utility of Induced Pluripotent Stem Cell-Derived Retinal Pigment Epithelium for an In Vitro Model of Proliferative Vitreoretinopathy.

Adv Exp Med Biol 2019 ;1186:33-53

Ocular Trauma Task Area, US Army Institute of Surgical Research, JBSA Fort Sam Houston, Houston, TX, USA.

The advent of stem cell technology, including the technology to induce pluripotency in somatic cells, and direct differentiation of stem cells into specific somatic cell types, has created an exciting new field of scientific research. Much of the work with pluripotent stem (PS) cells has been focused on the exploration and exploitation of their potential as cells/tissue replacement therapies for personalized medicine. However, PS and stem cell-derived somatic cells are also proving to be valuable tools to study disease pathology and tissue-specific responses to injury. High-throughput drug screening assays using tissue-specific injury models have the potential to identify specific and effective treatments that will promote wound healing. Retinal pigment epithelium (RPE) derived from induced pluripotent stem cells (iPS-RPE) are well characterized cells that exhibit the phenotype and functions of in vivo RPE. In addition to their role as a source of cells to replace damaged or diseased RPE, iPS-RPE provide a robust platform for in vitro drug screening to identify novel therapeutics to promote healing and repair of ocular tissues after injury. Proliferative vitreoretinopathy (PVR) is an abnormal wound healing process that occurs after retinal tears or detachments. In this chapter, the role of iPS-RPE in the development of an in vitro model of PVR is described. Comprehensive analyses of the iPS-RPE response to injury suggests that these cells provide a physiologically relevant tool to investigate the cellular mechanisms of the three phases of PVR pathology: migration, proliferation, and contraction. This in vitro model will provide valuable information regarding cellular wound healing responses specific to RPE and enable the identification of effective therapeutics.
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http://dx.doi.org/10.1007/978-3-030-28471-8_2DOI Listing
October 2019

CLIC4 regulates late endosomal trafficking and matrix degradation activity of MMP14 at focal adhesions in RPE cells.

Sci Rep 2019 08 22;9(1):12247. Epub 2019 Aug 22.

Department of Ophthalmology, Weill Medical College of Cornell University, New York, NY, USA.

Dysregulation in the extracellular matrix (ECM) microenvironment surrounding the retinal pigment epithelium (RPE) has been implicated in the etiology of proliferative vitreoretinopathy and age-related macular degeneration. The regulation of ECM remodeling by RPE cells is not well understood. We show that membrane-type matrix metalloproteinase 14 (MMP14) is central to ECM degradation at the focal adhesions in human ARPE19 cells. The matrix degradative activity, but not the assembly, of the focal adhesion is regulated by chloride intracellular channel 4 (CLIC4). CLIC4 is co-localized with MMP14 in the late endosome. CLIC4 regulates the proper sorting of MMP14 into the lumen of the late endosome and its proteolytic activation in lipid rafts. CLIC4 has the newly-identified "late domain" motif that binds to MMP14 and to Tsg101, a component of the endosomal sorting complex required for transport (ESCRT) complex. Unlike the late domain mutant CLIC4, wild-type CLIC4 can rescue the late endosomal sorting defect of MMP14. Finally, CLIC4 knockdown inhibits the apical secretion of MMP2 in polarized human RPE monolayers. These results, taken together, demonstrate that CLIC4 is a novel matrix microenvironment modulator and a novel regulator for late endosomal cargo sorting. Moreover, the late endosomal sorting of MMP14 actively regulates its surface activation in RPE cells.
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http://dx.doi.org/10.1038/s41598-019-48438-0DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6706427PMC
August 2019

Current Advancements in the Development and Characterization of Full-Thickness Adult Neuroretina Organotypic Culture Systems.

Cells Tissues Organs 2018 15;206(3):119-132. Epub 2019 Mar 15.

Ocular and Sensory Trauma Task Area, U.S. Army Institute of Surgical Research, Fort Sam Houston, Texas, USA.

Retinal degenerative diseases such as macular degeneration, glaucoma, and diabetic retinopathy constitute the leading cause of blindness in the industrialized world. There is a continuous demand in investigative ophthalmic research for the development of new treatment modalities for retinal therapy. Unfortunately, efforts to identify novel neuroprotective and neuroregenerative agents have often been hindered by an experimental model gap that exists between high-throughput methods via dissociated cells and preclinical animal models. Even though dissociated cell culture is rapid and high-throughput, it is limited in its ability to reproduce the in vivo conditions. In contrast, preclinical animal models may offer greater fidelity, albeit they lack efficiency and experimental control. Retina explant cultures provide an ideal bridge to close this gap and have been used to study an array of biological processes such as retinal development and neurodegeneration. However, it is often difficult to interpret findings from these studies due to the wide variety of experimental species and culture methods used. This review provides a comprehensive overview of current ex vivo neuroretina culture methods and assessments, with a focus on their suitability, advantages, and disadvantages, along with novel insights and perspectives on the organotypic culture model as a high-throughput platform for screening promising molecules for retinal regeneration.
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http://dx.doi.org/10.1159/000497296DOI Listing
September 2019

Quantitative Assessment of Retina Explant Viability in a Porcine Ex Vivo Neuroretina Model.

J Ocul Pharmacol Ther 2018 09 20;34(7):521-530. Epub 2018 Jun 20.

Ocular and Sensory Trauma Task Area, United States Army Institute of Surgical Research , Fort Sam Houston, Texas.

Purpose: Given that porcine and human retinas have similar structures and characteristics, ex vivo culture of porcine neuroretina provides an attractive model for studying mechanisms of human retinal injury and degenerative disease. Here, we describe the method that was used to establish and characterize an adult porcine retina culture system as a rapid screening tool for retinal survival in real time.

Methods: Neuroretina explants 8 mm in diameter were harvested from adult swine and cultured on porous cell culture inserts with adjustable heights. Retina explant viability was evaluated at 1, 4, 7, 11, and 14 days of culture using a resazurin-based metabolic assay. The explants were analyzed morphologically through immunohistochemistry for glial activation and apoptosis. Morphometric analysis was also performed on hematoxylin and eosin-stained retina sections from each time point.

Results: The viability of retina explants gradually decreased over time in culture. The laminar structure of the neuroretina was well preserved during the first 7 days. However, by day 14, most explants showed significant loss of cells in each laminar layer and obvious thinning. Overall, the progressive loss of retinal lamination and thickness, and increase in apoptotic nuclei with activated hypertrophic Müller cells were well correlated with the metabolic activity of the ex vivo neuroretina explants.

Conclusions: This study was the first report to describe the use of a high-throughput and quantitative method for monitoring retina explant viability in real time. Ex vivo neuroretina cultures closely mimic the functional dynamics of the organ, and can be used efficiently to screen novel therapeutics for retinal neurodegenerative disease.
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http://dx.doi.org/10.1089/jop.2018.0021DOI Listing
September 2018

Sealing of Corneal Lacerations Using Photoactivated Rose Bengal Dye and Amniotic Membrane.

Cornea 2018 Feb;37(2):211-217

San Antonio Uniformed Services Health Education Consortium, San Antonio, TX.

Purpose: Watertight closure of perforating corneoscleral lacerations is necessary to prevent epithelial ingrowth, infection, and potential loss of the eye. Complex lacerations can be difficult to treat, and repair with sutures alone is often inadequate. In this study, we evaluated a potentially sutureless technology for sealing complex corneal and scleral lacerations that bonds the amniotic membrane (AM) to the wound using only green light and rose bengal dye.

Methods: The AM was impregnated with rose bengal and then sealed over lacerations using green light to bond the AM to the deepithelialized corneal surface. This process was compared with suture repair of 3 laceration configurations in New Zealand White rabbits in 3 arms of the study. A fourth study arm assessed the side effect profile including viability of cells in the iris, damage to the blood-retinal barrier, retinal photoreceptors, retinal pigment epithelium, and choriocapillaris in Dutch Belted rabbits.

Results: Analyses of the first 3 arms revealed a clinically insignificant increase in polymorphonuclear inflammation. In the fourth arm, iris cells appeared unaffected and no evidence of breakdown of the blood-retinal barrier was detected. The retina from green light laser-treated eyes showed normal retinal pigment epithelium, intact outer segments, and normal outer nuclear layer thickness.

Conclusions: The results of these studies established that a light-activated method to cross-link AM to the cornea can be used for sealing complex penetrating wounds in the cornea and sclera with minimal inflammation or secondary effects.
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http://dx.doi.org/10.1097/ICO.0000000000001389DOI Listing
February 2018

Polarized Secretion of Matrix Metalloproteinases and Their Inhibitors by Retinal Pigment Epithelium Derived from Induced Pluripotent Stem Cells During Wound Healing.

J Ocul Pharmacol Ther 2017 04 22;33(3):132-140. Epub 2017 Feb 22.

Ocular Trauma Task Area, U.S. Army Institute of Surgical Research , JBSA Fort Sam Houston, San Antonio, Texas.

Purpose: To characterize the secretion of matrix metalloproteinases (MMPs) and tissue inhibitors of matrix metalloproteinases (TIMPs) by induced pluripotent stem cell-derived retinal pigment epithelium (iPS-RPE) during wound healing. We hypothesize that iPS-RPE secretes mediators of tissue remodeling such as MMPs and TIMPs to promote migration and proliferation of cells during wound healing.

Methods: iPS-RPE was grown on transwells until fully confluent and pigmented. The monolayers were scratched to induce a wound. Conditioned media were collected from the apical and basolateral sides of the transwells every 72 h for 12 days. The media were analyzed by multiplex ELISA assays to detect secreted MMPs and TIMPs. Activity assays were performed to detect the active form of MMP-2 in conditioned media.

Results: MMP-2 and TIMP-1, -2, -3, and -4 were detected in conditioned media from iPS-RPE. The proteins were found to be secreted in a polarized manner. The apical secretion and activation of MMP-2 was elevated from days 3 to 12 after wounding. TIMP-1, -2, -3, and -4 were detected in conditioned media from both the apical and basolateral sides of wounded cells. Apical secretion of all 4 TIMPs increased within 3 days after wounding.

Conclusions: These results indicate that iPS-RPE secretes MMP-2 and all 4 TIMPs in a polarized manner. After wounding, apical secretion of MMP-2 was higher compared to control. Apical secretion of all 4 TIMPs increased compared to control, while only TIMP-1 showed increased basolateral secretion compared to control.
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http://dx.doi.org/10.1089/jop.2016.0070DOI Listing
April 2017

Secretion Profile of Induced Pluripotent Stem Cell-Derived Retinal Pigment Epithelium During Wound Healing.

Invest Ophthalmol Vis Sci 2016 Aug;57(10):4428-4441

Ocular Trauma Task Area US Army Institute of Surgical Research, Joint Base San Antonio-Fort Sam Houston, Texas, United States.

Purpose: The purpose of this study was to characterize the secretion profile of induced pluripotent stem cell-derived retinal pigment epithelium (iPS-RPE) during wound healing. iPS-RPE was used to develop an in vitro wound healing model. We hypothesized that iPS-RPE secretes cytokines and growth factors which act in an autocrine manner to promote migration and proliferation of cells during wound healing.

Methods: iPS-RPE was grown in transwells until fully confluent and pigmented. The monolayers were scratched to induce a wound. Levels of Ki-67, β-catenin, e-cadherin, n-cadherin, and S100A4 expression were analyzed by immunofluorescent labeling. Cell culture medium samples were collected from both the apical and basolateral sides of the transwells every 72 hours for 21 days. The medium samples were analyzed using multiplex ELISA to detect secreted growth factors and cytokines. The effects of conditioned medium on collagen gel contraction, cell proliferation, and migration were measured.

Results: iPS-RPE underwent epithelial-mesenchymal transition (EMT) during wound healing as indicated by the translocation of β-catenin to the nucleus, cadherin switch, and expression of S100A4. GRO, GM-CSF, MCP-1, IL-6, and IL-8 were secreted by both the control and the wounded cell cultures. VEGF, FGF-2, and TGFβ expression were detected at higher levels after wounding than those in control. The proteins were found to be secreted in a polarized manner. The conditioned medium from wounded monolayers promoted collagen gel contraction, as well as proliferation and migration of ARPE 19 cells.

Conclusions: These results indicate that after the monolayer is wounded, iPS-RPE secretes proteins into the culture medium that promote increased proliferation, contraction, and migration.
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http://dx.doi.org/10.1167/iovs.16-19192DOI Listing
August 2016

Trichostatin A Inhibits Retinal Pigmented Epithelium Activation in an In Vitro Model of Proliferative Vitreoretinopathy.

J Ocul Pharmacol Ther 2016 09 5;32(7):415-24. Epub 2016 Aug 5.

Ocular Trauma, U.S. Army Institute of Surgical Research , JBSA-Fort Sam Houston, Texas.

Purpose: Proliferative vitreoretinopathy (PVR) is a blinding disorder that develops after a retinal tear or detachment. Activation of the retinal pigmented epithelium (RPE) is implicated in PVR; however, the mechanisms leading to enhanced RPE proliferation, migration, and contraction remain largely unknown. This study utilized an in vitro model of PVR to investigate the role of acetylation in RPE activation and its contribution to the progression of this disease.

Methods: ARPE-19 cells, primary cultures of porcine RPE, and induced pluripotent stem cell-derived RPE (iPS-RPE) were utilized for cellular and molecular analyses. Cells treated with transforming growth factor beta 2 (TGFβ2; 10 ng/mL) alone or in the presence of the broad-spectrum histone deacetylase (HDAC) inhibitor, trichostatin A (TSA; 0.1 μM), were assessed for contraction and migration through collagen contraction and scratch assays, respectively. Western blotting and immunofluorescence analysis were performed to assess α-smooth muscle actin (α-SMA) and β-catenin expression after TGFβ2 treatment alone or in combination with TSA.

Results: TGFβ2 significantly increased RPE cell contraction in collagen matrix and this effect was inhibited in the presence of TSA (0.1 μM). In agreement with these data, immunofluorescence analysis of TSA-treated iPS-RPE wounded monolayers revealed decreased α-SMA as compared with control. Scratch assays to assess wound healing revealed TSA inhibited TGFβ2-mediated iPS-RPE cell migration.

Conclusions: Our findings indicate a role of acetylation in RPE activation. Specifically, the HDAC inhibitor TSA decreased RPE cell proliferation and TGFβ2-mediated cell contraction and migration. Further investigation of pharmacological compounds that modulate acetylation may hold promise as therapeutic agents for PVR.
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http://dx.doi.org/10.1089/jop.2016.0038DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5011631PMC
September 2016

Recombinant Xeno-Free Vitronectin Supports Self-Renewal and Pluripotency in Protein-Induced Pluripotent Stem Cells.

Tissue Eng Part C Methods 2016 02 15;22(2):85-90. Epub 2016 Jan 15.

1 Ocular Trauma Task Area, U.S. Army Institute of Surgical Research, JBSA Fort Sam Houston, Texas.

Patient safety is a major concern in the application of induced pluripotent stem cells (iPSCs) in cell-based therapy. Efforts are being made to reprogram, maintain, and differentiate iPSCs in defined conditions to provide a safe source of stem cells for regenerative medicine. Recently, human fibroblasts were successfully reprogrammed into pluripotent stem cells using four recombinant proteins (OCT4, c-Myc, KLF4, and SOX2) fused with a cell-penetrating peptide (9R). These protein-induced pluripotent stem cells (piPSCs) are maintained and propagated on a feeder layer of mouse embryonic fibroblasts. Use of animal-derived products in maintenance and differentiation of iPSCs poses risks of zoonotic disease transmission and immune rejection when transplanted into humans. To avoid potential incorporation of xenogenic products, we cultured piPSCs on recombinant human matrix proteins. We then tested whether recombinant human matrix proteins can support self-renewal and pluripotency of piPSCs. After long-term culture on recombinant human vitronectin in xeno-free conditions, piPSCs retained the expression of pluripotent markers. The pluripotency of these cells was further evaluated by differentiating toward ectoderm, mesoderm, and endoderm lineages in vitro. In conclusion, recombinant human vitronectin can support the long-term culture and maintain the stemness of piPSCs in defined nonxenogenic conditions.
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http://dx.doi.org/10.1089/ten.TEC.2015.0180DOI Listing
February 2016

Deriving retinal pigment epithelium (RPE) from induced pluripotent stem (iPS) cells by different sizes of embryoid bodies.

J Vis Exp 2015 Feb 4(96). Epub 2015 Feb 4.

Ocular Trauma, U.S. Army Institute of Surgical Research;

Pluripotent stem cells possess the ability to proliferate indefinitely and to differentiate into almost any cell type. Additionally, the development of techniques to reprogram somatic cells into induced pluripotent stem (iPS) cells has generated interest and excitement towards the possibility of customized personal regenerative medicine. However, the efficiency of stem cell differentiation towards a desired lineage remains low. The purpose of this study is to describe a protocol to derive retinal pigment epithelium (RPE) from iPS cells (iPS-RPE) by applying a tissue engineering approach to generate homogenous populations of embryoid bodies (EBs), a common intermediate during in vitro differentiation. The protocol applies the formation of specific size of EBs using microwell plate technology. The methods for identifying protein and gene markers of RPE by immunocytochemistry and reverse-transcription polymerase chain reaction (RT-PCR) are also explained. Finally, the efficiency of differentiation in different sizes of EBs monitored by fluorescence-activated cell sorting (FACS) analysis of RPE markers is described. These techniques will facilitate the differentiation of iPS cells into RPE for future applications.
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http://dx.doi.org/10.3791/52262DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4354611PMC
February 2015

Profiling the microRNA Expression in Human iPS and iPS-derived Retinal Pigment Epithelium.

Cancer Inform 2014 15;13(Suppl 5):25-35. Epub 2014 Oct 15.

Department of Biology, University of Texas at San Antonio, San Antonio, TX, USA. ; South Texas Center for Emerging Infectious Diseases, University of Texas at San Antonio, San Antonio, TX, USA.

The purpose of this study is to characterize the microRNA (miRNA) expression profiles of induced pluripotent stem (iPS) cells and retinal pigment epithelium (RPE) derived from induced pluripotent stem cells (iPS-RPE). MiRNAs have been demonstrated to play critical roles in both maintaining pluripotency and facilitating differentiation. Gene expression networks accountable for maintenance and induction of pluripotency are linked and share components with those networks implicated in oncogenesis. Therefore, we hypothesize that miRNA expression profiling will distinguish iPS cells from their iPS-RPE progeny. To identify and analyze differentially expressed miRNAs, RPE was derived from iPS using a spontaneous differentiation method. MiRNA microarray analysis identified 155 probes that were statistically differentially expressed between iPS and iPS-RPE cells. Up-regulated miRNAs including miR-181c and miR-129-5p may play a role in promoting differentiation, while down-regulated miRNAs such as miR-367, miR-18b, and miR-20b are implicated in cell proliferation. Subsequent miRNA-target and network analysis revealed that these miRNAs are involved in cellular development, cell cycle progression, cell death, and survival. A systematic interrogation of temporal and spatial expression of iPS-RPE miRNAs and their associated target mRNAs will provide new insights into the molecular mechanisms of carcinogenesis, eye differentiation and development.
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http://dx.doi.org/10.4137/CIN.S14074DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4218680PMC
November 2014

Pathophysiology of blast-induced ocular trauma in rats after repeated exposure to low-level blast overpressure.

Clin Exp Ophthalmol 2015 Apr 13;43(3):239-46. Epub 2014 Sep 13.

Ocular Trauma Task Area, U.S. Army Institute of Surgical Research, JBSA Fort Sam Houston, Texas, USA.

Background: The incidence of blast-induced ocular injury has dramatically increased due to advances in weaponry and military tactics. A single exposure to blast overpressure (BOP) has been shown to cause damage to the eye in animal models; however, on the battlefield, military personnel are exposed to BOP multiple times. The effects of repeated exposures to BOP on ocular tissues have not been investigated. The purpose of this study is to characterize the effects of single or repeated exposure on ocular tissues.

Methods: A compressed air shock tube was used to deliver 70 ± 7 KPa BOP to rats, once (single blast overpressure [SBOP]) or once daily for 5 days (repeated blast overpressure [RBOP]). Immunohistochemistry was performed to characterize the pathophysiology of ocular injuries induced by SBOP and RBOP. Apoptosis was determined by quantification activated caspase 3. Gliosis was examined by detection of glial fibrillary acidic protein (GFAP). Inflammation was examined by detection of CD68.

Results: Activated caspase 3 was detected in ocular tissues from all animals subjected to BOP, while those exposed to RBOP had more activated caspase 3 in the optic nerve than those exposed to SBOP. GFAP was detected in the retinas from all animals subjected to BOP. CD68 was detected in optic nerves from all animals exposed to BOP.

Conclusion: SBOP and RBOP induced retinal damage. RBOP caused more apoptosis in the optic nerve than SBOP, suggesting that RBOP causes more severe optic neuropathy than SBOP. SBOP and RBOP caused gliosis in the retina and increased inflammation in the optic nerve.
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http://dx.doi.org/10.1111/ceo.12407DOI Listing
April 2015

Pathophysiology of blast-induced ocular trauma with apoptosis in the retina and optic nerve.

Mil Med 2014 Aug;179(8 Suppl):34-40

Ocular Trauma Task Area, U.S. Army Institute of Surgical Research, 3698 Chambers Pass, Building 3611, JBSA Fort Sam Houston, TX 78234.

Background: Blast-induced ocular trauma is a frequent cause of morbidity for survivors of improvised explosive devices. Blast overpressure (BOP) of 120 ± 7 KPa has been shown to cause damage to lungs, brain, and gut in a rat model; however, the effects of BOP on ocular tissues have not been characterized. To elucidate the pathophysiology of blast-induced ocular trauma, ocular tissues from rats subjected to blast were examined for evidence of apoptosis by the detection of activated caspase 3 and TUNEL assay in their ocular tissues.

Methods: A compressed air shock tube was used to deliver 120 ± 7 KPa of BOP for duration of 2 msec to the right side of the rats. Rats were then euthanized at specific time points after blast exposure (3 hours, 24 hours, 48 hours). Ocular tissues were processed for immunohistochemistry to detect activated caspase 3 and TUNEL assay. Tissues were evaluated for relative levels of positive signal as compared to nonblast exposed controls.

Results: Activated caspase 3 was detected in the optic nerve, ganglion layer, and inner nuclear layer post blast exposure. At 24 and 48 hours, the inner nuclear layer from the right side had more cells with activated caspase 3. In the optic nerve, the highest levels of activated caspase 3 were detected on the right side at 24 hours post blast.

Conclusion: BOP of 120 ± 7 KPa induces optic neuropathy and retinal damage. In both the optic nerve and retina, caspase 3 was activated in the right and left sides following blast exposure. The results of this study reveal that blast exposure induces apoptosis in both the optic nerve and retinal tissues.
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http://dx.doi.org/10.7205/MILMED-D-13-00504DOI Listing
August 2014

MicroRNA expression profiles of human iPS cells, retinal pigment epithelium derived from iPS, and fetal retinal pigment epithelium.

J Vis Exp 2014 Jun 24(88):e51589. Epub 2014 Jun 24.

Ocular Trauma, U.S. Army Institute of Surgical Research, JBSA Fort Sam Houston;

The objective of this report is to describe the protocols for comparing the microRNA (miRNA) profiles of human induced-pluripotent stem (iPS) cells, retinal pigment epithelium (RPE) derived from human iPS cells (iPS-RPE), and fetal RPE. The protocols include collection of RNA for analysis by microarray, and the analysis of microarray data to identify miRNAs that are differentially expressed among three cell types. The methods for culture of iPS cells and fetal RPE are explained. The protocol used for differentiation of RPE from human iPS is also described. The RNA extraction technique we describe was selected to allow maximal recovery of very small RNA for use in a miRNA microarray. Finally, cellular pathway and network analysis of microarray data is explained. These techniques will facilitate the comparison of the miRNA profiles of three different cell types.
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http://dx.doi.org/10.3791/51589DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4204998PMC
June 2014

Retinoid uptake, processing, and secretion in human iPS-RPE support the visual cycle.

Invest Ophthalmol Vis Sci 2014 Jan 9;55(1):198-209. Epub 2014 Jan 9.

Ocular Trauma, U.S. Army Institute of Surgical Research, JBSA Fort Sam Houston, Houston, Texas.

Purpose: Retinal pigmented epithelium derived from human induced pluripotent stem (iPS) cells (iPS-RPE) may be a source of cells for transplantation. For this reason, it is essential to determine the functional competence of iPS-RPE. One key role of the RPE is uptake and processing of retinoids via the visual cycle. The purpose of this study is to investigate the expression of visual cycle proteins and the functional ability of the visual cycle in iPS-RPE.

Methods: iPS-RPE was derived from human iPS cells. Immunocytochemistry, RT-PCR, and Western blot analysis were used to detect expression of RPE genes lecithin-retinol acyl transferase (LRAT), RPE65, cellular retinaldehyde-binding protein (CRALBP), and pigment epithelium-derived factor (PEDF). All-trans retinol was delivered to cultured cells or whole cell homogenate to assess the ability of the iPS-RPE to process retinoids.

Results: Cultured iPS-RPE expresses visual cycle genes LRAT, CRALBP, and RPE65. After incubation with all-trans retinol, iPS-RPE synthesized up to 2942 ± 551 pmol/mg protein all-trans retinyl esters. Inhibition of LRAT with N-ethylmaleimide (NEM) prevented retinyl ester synthesis. Significantly, after incubation with all-trans retinol, iPS-RPE released 188 ± 88 pmol/mg protein 11-cis retinaldehyde into the culture media.

Conclusions: iPS-RPE develops classic RPE characteristics and maintains expression of visual cycle proteins. The results of this study confirm that iPS-RPE possesses the machinery to process retinoids for support of visual pigment regeneration. Inhibition of all-trans retinyl ester accumulation by NEM confirms LRAT is active in iPS-RPE. Finally, the detection of 11-cis retinaldehyde in the culture medium demonstrates the cells' ability to process retinoids through the visual cycle. This study demonstrates expression of key visual cycle machinery and complete visual cycle activity in iPS-RPE.
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http://dx.doi.org/10.1167/iovs.13-11740DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4588713PMC
January 2014

Stem cells in large animal models of retinal and neurological disease.

Stem Cells Int 2012 10;2012:460504. Epub 2012 May 10.

Gavin Herbert Eye Institute and Stem Cell Research Center, University of California-Irvine, Irvine, CA 92697, USA.

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http://dx.doi.org/10.1155/2012/460504DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3359744PMC
August 2012

Transplantation of quantum dot-labelled bone marrow-derived stem cells into the vitreous of mice with laser-induced retinal injury: survival, integration and differentiation.

Vision Res 2010 Mar 25;50(7):665-73. Epub 2009 Sep 25.

US Army Medical Research Detachment, Walter Reed Army Institute of Research, 7965 Dave Erwin Drive, Brooks City-Base, TX 78235, USA.

Accidental laser exposure to the eyes may result in serious visual impairment due to retina degeneration. Currently limited treatment is available for laser eye injury. In the current study, we investigated the therapeutic potential of bone marrow-derived stem cells (BMSCs) for laser-induced retinal trauma. Lineage negative bone marrow cells (Lin(-) BMCs) were labelled with quantum dots (Qdots) to track the cells in vivo. Lin(-) BMCs survived well after intravitreal injection. In vivo bromodeoxyuridine (BrdU) labelling showed these cells continued to proliferate and integrate into injured retinas. Furthermore, they expressed markers that distinguished retinal pigment epithelium (RPE), endothelium, pericytes and photoreceptors. Our results suggest that BMSCs participate in the repair of retinal lesions by differentiating into retinal cells. Intravitreal transplantation of BMSCs is a potential treatment for laser-induced retinal trauma.
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http://dx.doi.org/10.1016/j.visres.2009.09.003DOI Listing
March 2010

Cryptosporidium infection of human intestinal epithelial cells increases expression of osteoprotegerin: a novel mechanism for evasion of host defenses.

J Infect Dis 2008 Mar;197(6):916-23

Infectious Disease Section, Department of Medicine, Baylor College of Medicine, Houston, TX, USA.

Cryptosporidium parasites are pathogens of human intestinal epithelial cells. To determine which genes are regulated during early infection, human ileal mucosa cultured as explants was infected with C. parvum or C. hominis, and gene expression was analyzed by microarray. The gene for osteoprotegerin (OPG) was up-regulated by both parasites. OPG mRNA was also significantly increased in biopsy specimens obtained from a volunteer experimentally infected with C. meleagridis, compared with levels in a prechallenge biopsy specimen. After in vitro infection of HCT-8 cells, there was an early peak in production of OPG mRNA protein. Treatment of infected cells with the OPG ligand tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) induced epithelial cell apoptosis and reduced parasite numbers, and recombinant OPG blocked these effects. These results suggest a novel TRAIL-mediated pathway for elimination of Cryptosporidium infection and a role for OPG in modulating this host response.
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http://dx.doi.org/10.1086/528374DOI Listing
March 2008

Intestinal immune response to human Cryptosporidium sp. infection.

Infect Immun 2008 Jan 29;76(1):23-9. Epub 2007 Oct 29.

Infectious Diseases Division, Department of Internal Medicine, University of Texas Medical Branch, 301 University Boulevard, Rt. 0435, Galveston, TX 77555-0435, USA.

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http://dx.doi.org/10.1128/IAI.00960-07DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2223661PMC
January 2008

Seropositive human subjects produce interferon gamma after stimulation with recombinant Cryptosporidium hominis gp15.

Am J Trop Med Hyg 2007 Sep;77(3):583-5

Translational Biology and Molecular Medicine Program, Infectious Disease Section Department of Medicine, Department of Immunology, and Department of Pathology, Baylor College of Medicine, Houston, Texas, USA.

Cryptosporidiosis is an important cause of diarrhea worldwide. In normal hosts, infection is self-limited and associated with seroconversion and partial immunity to reinfection. Immunity is associated with interferon gamma (IFNgamma) production. Cryptosporidium surface proteins gp15 and gp40 are among the immunodominant proteins in terms of antibody responses. We asked the question of whether these antigens also stimulate production of IFNgamma in patients who have serologic evidence of prior infection. Whole blood from seropositive donors was stimulated with recombinant gp15 and gp 40 from Cryptosporidium hominis and Cryptosporidium parvum or His-tag controls. C. hominis gp15 stimulated increased production of IFNgamma. By contrast, there was no significant increase after stimulation with C. parvum gp15 or either gp40 preparation. IFNgamma production in response to C. hominis gp15 was noted in both CD4(+) and CD8(+) cells. This highlights the potential for C. hominis gp15 as a vaccine candidate for human cryptosporidiosis.
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September 2007

Stimulatory and costimulatory effects of IL-18 directed to different small intestinal CD43 T cell subsets.

J Leukoc Biol 2007 Nov 16;82(5):1166-73. Epub 2007 Aug 16.

Department of Diagnostic Sciences, University of Texas Health Science Center at Houston, Dental Branch, Houston, TX 77030, USA.

This study has examined the stimulatory and costimulatory effects of IL-18 on two subsets of murine small intestinal intraepithelial lymphocytes (IELs) defined by the expression of the CD43 S7 glycoform. Data from gene array studies and real-time PCR indicated that S7(+) IELs had significantly higher levels of gene expression for the IL-18 receptor and the IL-18R accessory protein than S7(-) IELs. IL-18 costimulation of IELs in conjunction with CD3-induced activation resulted in significantly greater proliferation than CD3 stimulation alone. In CFSE dilution experiments, IL-18 costimulation favored the S7(+) IEL population. IL-18 costimulation did not affect apoptosis of either S7(-) or S7(+) IELs compared with CD3 stimulation alone. Although IL-18 costimulation did not alter the total number of IFN-gamma-producing cells relative to CD3 stimulation alone, twice as many S7(+) IELs were IFN-gamma -secreting cells than S7(-) IELs in both CD3-stimulated and IL-18-costimulated cultures. Notably, direct IL-18 stimulation in the absence of CD3 activation induced an IFN-gamma response that was predominantly directed to the S7(+) population, indicating that IL-18 is itself an IFN-gamma activational signal for intestinal T cells. In contrast, direct IL-18 stimulation of IELs did not generate TNF-alpha-producing cells, indicating a differential response in the activation of proinflammatory cytokines following IL-18 exposure. These findings point to distinctly different activational effects of IL-18 on IELs, both with regard to the type of functional responses elicited and with respect to the IEL subsets affected.
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http://dx.doi.org/10.1189/jlb.0207108DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2582815PMC
November 2007

High levels of CXCL10 are produced by intestinal epithelial cells in AIDS patients with active cryptosporidiosis but not after reconstitution of immunity.

Infect Immun 2007 Jan 16;75(1):481-7. Epub 2006 Oct 16.

Infectious Disease Section, Department of Medicine, Baylor College of Medicine, One Baylor Plaza, 525D, Houston, TX 77030, USA.

Chemokines play key roles in attracting immune cells to sites of infections. However, few data on chemokine expression in the gut during human infections are available. We examined expression of chemokines in intestinal tissues of AIDS patients during active Cryptosporidium infection and during resolution of such an infection. The chemokines and cytokines in cell lysates from jejunal biopsy tissues were assayed by a 22-multiplex bead immunoassay. CXCL10 (IP-10) and its receptor, CXCR3, in sections were studied by immunohistochemistry. In biopsies from AIDS patients with active cryptosporidiosis, four chemokines (CXCL10, CCL11 [eotaxin], CCL5 [RANTES], and CCL2 [monocyte chemoattractant protein 1]) and three cytokines (interleukin-1alpha [IL-1alpha], IL-10, and granulocyte colony-stimulating factor) were detected. The level of CXCL10 was significantly increased in AIDS patients with cryptosporidiosis compared to the level in AIDS patients without cryptosporidiosis or in normal volunteers (median in AIDS patients with cryptosporidiosis, 508 pg/mg protein, compared to 111 pg/mg and 72 pg/mg protein in AIDS patients without cryptosporidiosis and in normal volunteers, respectively [P < 0.05 and P < 0.005, respectively, as determined by a Mann-Whitney test]). The level of CXCL10 correlated with the parasite burden (as measured by the number of Cryptosporidium oocysts in the stools) and also with the IL-1alpha concentration (Pearson correlation values, 0.961 [P < 0.01] and 0.737 [P < 0.05]). As determined by immunohistochemistry, CXCL10 localized to epithelial cells at the site of infection. Following effective antiparasite and antiretroviral therapy, Cryptosporidium infections resolved, and the levels of CXCL10 decreased to normal levels. We hypothesized that CXCL10 plays an important role in the resolution of cryptosporidiosis by attracting immune effector cells to the site of infection. By contrast, in AIDS patients lacking effector cells, CXCL10 may contribute to the immunopathogenesis by recruiting inflammatory cells.
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http://dx.doi.org/10.1128/IAI.01237-06DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1828373PMC
January 2007

Interleukin-15 activates human natural killer cells to clear the intestinal protozoan cryptosporidium.

J Infect Dis 2005 Oct 23;192(7):1294-302. Epub 2005 Aug 23.

Department of Medicine, Baylor College of Medicine, Houston, TX 77030, USA.

Intracellular protozoans of the genus Cryptosporidium are a major cause of diarrheal illness worldwide, but little is known about the mechanisms that control intestinal infection. We have previously demonstrated interleukin (IL)-15 expression in the intestinal mucosa of seronegative symptomatic volunteers after oral challenge with C. parvum, which suggests a role for IL-15 in the control of acute infection. We hypothesize that IL-15 activates an innate cytolytic cell response that contributes to the clearance of initial C. parvum infection. We report here that IL-15 activates peripheral blood mononuclear cells to lyse Cryptosporidium-infected epithelial cells in a dose-dependent manner. Lysis was due to CD3(-)CD16(+)CD56+ cells (i.e., natural killer [NK] cells). Furthermore, flow cytometry revealed that IL-15 increased expression of the activation receptor NKG2D on NK cells, particularly among the CD16Hi cytolytically active cells. Major histocompatibility complex class I-related molecules A and B (MICA and MICB), ligands for NKG2D, were increased after infection of epithelial cell lines and human ileal tissue. These data suggest that IL-15 has an important role in activating an NK cell-mediated pathway that leads to the elimination of intracellular protozoans from the intestines, which is a previously unrecognized feature of NK cell function.
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http://dx.doi.org/10.1086/444393DOI Listing
October 2005

Maximum immunobioactivity of murine small intestinal intraepithelial lymphocytes resides in a subpopulation of CD43+ T cells.

J Immunol 2004 Nov;173(10):6294-302

Department of Diagnostic Sciences, Dental Branch, University of Texas Health Science Center at Houston, 6516 M.D. Anderson Boulevard, Houston, TX 77030, USA.

CD43 has been linked to many function-associated T cell activities. Using mAbs that recognize two different CD43 determinants, we show that, although mouse small intestinal intraepithelial lymphocytes (IELs) expressed the CD43 core molecule reactive with mAb R2/60, only about one-half of the total IELs-including some but not all of the TCRalphabeta and TCRgammadelta cells-expressed the CD43 S7(-) reactive determinant. CD43 S7(+) IELs secreted more IL-2, IL-4, IL-10, IL-17, and IFN-gamma following anti-CD3 stimulation, and were >4-fold more cytotoxic in fresh isolates and >16-fold more cytotoxic after anti-CD3 stimulation, than S7(-) IELs. S7(+) but not S7(-) IELs from the ileum of IL-10(-/-) mice spontaneously produced IFN-gamma. In vivo BrdU uptake by IELs in non-Ag-primed mice was greatest in the S7(+) population, indicating that significantly more S7(+) IELs than S7(-) IELs undergo cell expansion under normal homeostatic conditions. DNA microarray analyses showed that S7(+) IELs expressed higher levels of genes associated with activated T cells, whereas S7(-) IELs expressed genes used in the regulation of NK cells. These findings define two functionally distinct populations of IELs based on CD43 expression independent of TCR class, and they identify a subset of IELs that may serve as a target to better control intestinal inflammation.
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http://dx.doi.org/10.4049/jimmunol.173.10.6294DOI Listing
November 2004

Characterization of a novel set of resident intrathyroidal bone marrow-derived hematopoietic cells: potential for immune-endocrine interactions in thyroid homeostasis.

J Exp Biol 2004 Jan;207(Pt 1):55-65

Department of Basic Sciences, Dental Branch, University of Texas Health Science Center at Houston, Houston, TX 77030, USA.

Immunofluorescent staining of thyroid tissues was done using monoclonal antibodies to dendritic cell (DC), lymphocyte, macrophage and granulocyte markers. Despite the presence of occasional CD11c+ cells, CD11b+ cells, morphologically characteristic of DCs, were abundant in thyroid of normal mice, at a density of approximately 2.0 cells per thyroid follicle, and were >tenfold more frequent than CD11c+ cells. Thyroid tissues were non-reactive with antibodies to F4/80, CD8alpha, CD40, CD80, Gr-1, CD3, or CD19, indicating that the CD11b+ cells were not macrophages, activated DCs, granulocytes, plasmacytoid DCs, T cells or B cells. Following systemic immune activation, DCs in secondary lymphoid tissues but not in the thyroid, upregulated CD80 expression. Using radiation chimeras made from bone marrow from enhanced green fluorescent protein (EGFP) transgenic mice, EGFP+ DC-like cells were present in the thyroid from 1-20 weeks after bone marrow transfer, but were rare in the kidney and liver, although EGFP+ cells were present in secondary lymphoid tissues. Additionally, DCs generated from EGFP+ bone marrow cells localized in the thyroid of EGFP- mice following adoptive transfer. Double staining of thyroid tissue sections with antibodies to the thyroid stimulating hormone (TSH)-beta molecule and to CD11b revealed co-expression of TSHbeta and CD11b among intrathyroidal DCs. Moreover, RT-PCR analyses indicated expression of the TSHbeta gene in thyroid tissues. These findings define a novel bone marrow-derived hematopoietic cell population that resides in the thyroid of normal mice, which may have a unique role in the microregulation of thyroid physiology and homeostasis.
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http://dx.doi.org/10.1242/jeb.00710DOI Listing
January 2004