Publications by authors named "Sheetal Trivedi"

16 Publications

  • Page 1 of 1

A Methyltransferase-Defective Vesicular Stomatitis Virus-Based SARS-CoV-2 Vaccine Candidate Provides Complete Protection against SARS-CoV-2 Infection in Hamsters.

J Virol 2021 09 11;95(20):e0059221. Epub 2021 Aug 11.

Department of Veterinary Biosciences, The Ohio State Universitygrid.261331.4, Columbus, Ohio, USA.

The current pandemic of coronavirus disease 2019 (COVID-19) caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has led to dramatic economic and health burdens. Although the worldwide SARS-CoV-2 vaccination campaign has begun, exploration of other vaccine candidates is needed due to uncertainties with the current approved vaccines, such as durability of protection, cross-protection against variant strains, and costs of long-term production and storage. In this study, we developed a methyltransferase-defective recombinant vesicular stomatitis virus (mtdVSV)-based SARS-CoV-2 vaccine candidate. We generated mtdVSVs expressing SARS-CoV-2 full-length spike (S) protein, S1, or its receptor-binding domain (RBD). All of these recombinant viruses grew to high titers in mammalian cells despite high attenuation in cell culture. The SARS-CoV-2 S protein and its truncations were highly expressed by the mtdVSV vector. These mtdVSV-based vaccine candidates were completely attenuated in both immunocompetent and immunocompromised mice. Among these constructs, mtdVSV-S induced high levels of SARS-CoV-2-specific neutralizing antibodies (NAbs) and Th1-biased T-cell immune responses in mice. In Syrian golden hamsters, the serum levels of SARS-CoV-2-specific NAbs triggered by mtdVSV-S were higher than the levels of NAbs in convalescent plasma from recovered COVID-19 patients. In addition, hamsters immunized with mtdVSV-S were completely protected against SARS-CoV-2 replication in lung and nasal turbinate tissues, cytokine storm, and lung pathology. Collectively, our data demonstrate that mtdVSV expressing SARS-CoV-2 S protein is a safe and highly efficacious vaccine candidate against SARS-CoV-2 infection. Viral mRNA cap methyltransferase (MTase) is essential for mRNA stability, protein translation, and innate immune evasion. Thus, viral mRNA cap MTase activity is an excellent target for development of live attenuated or live vectored vaccine candidates. Here, we developed a panel of MTase-defective recombinant vesicular stomatitis virus (mtdVSV)-based SARS-CoV-2 vaccine candidates expressing full-length S, S1, or several versions of the RBD. These mtdVSV-based vaccine candidates grew to high titers in cell culture and were completely attenuated in both immunocompetent and immunocompromised mice. Among these vaccine candidates, mtdVSV-S induces high levels of SARS-CoV-2-specific neutralizing antibodies (Nabs) and Th1-biased immune responses in mice. Syrian golden hamsters immunized with mtdVSV-S triggered SARS-CoV-2-specific NAbs at higher levels than those in convalescent plasma from recovered COVID-19 patients. Furthermore, hamsters immunized with mtdVSV-S were completely protected against SARS-CoV-2 challenge. Thus, mtdVSV is a safe and highly effective vector to deliver SARS-CoV-2 vaccine.
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http://dx.doi.org/10.1128/JVI.00592-21DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8475528PMC
September 2021

Pathogenesis, MicroRNA-122 Gene-Regulation, and Protective Immune Responses After Acute Equine Hepacivirus Infection.

Hepatology 2021 09 11;74(3):1148-1163. Epub 2021 Jun 11.

Copenhagen Hepatitis C Program (CO-HEP), Department of Infectious Diseases, Hvidovre Hospital and Department of Immunology and Microbiology, University of Copenhagen, Copenhagen, Denmark.

Background And Aims: Equine hepacivirus (EqHV) is phylogenetically the closest relative of HCV and shares genome organization, hepatotropism, transient or persistent infection outcome, and the ability to cause hepatitis. Thus, EqHV studies are important to understand equine liver disease and further as an outbred surrogate animal model for HCV pathogenesis and protective immune responses. Here, we aimed to characterize the course of EqHV infection and associated protective immune responses.

Approach And Results: Seven horses were experimentally inoculated with EqHV, monitored for 6 months, and rechallenged with the same and, subsequently, a heterologous EqHV. Clearance was the primary outcome (6 of 7) and was associated with subclinical hepatitis characterized by lymphocytic infiltrate and individual hepatocyte necrosis. Seroconversion was delayed and antibody titers waned slowly. Clearance of primary infection conferred nonsterilizing immunity, resulting in shortened duration of viremia after rechallenge. Peripheral blood mononuclear cell responses in horses were minimal, although EqHV-specific T cells were identified. Additionally, an interferon-stimulated gene signature was detected in the liver during EqHV infection, similar to acute HCV in humans. EqHV, as HCV, is stimulated by direct binding of the liver-specific microRNA (miR), miR-122. Interestingly, we found that EqHV infection sequesters enough miR-122 to functionally affect gene regulation in the liver. This RNA-based mechanism thus could have consequences for pathology.

Conclusions: EqHV infection in horses typically has an acute resolving course, and the protective immune response lasts for at least a year and broadly attenuates subsequent infections. This could have important implications to achieve the primary goal of an HCV vaccine; to prevent chronicity while accepting acute resolving infection after virus exposure.
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http://dx.doi.org/10.1002/hep.31802DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8435542PMC
September 2021

A safe and highly efficacious measles virus-based vaccine expressing SARS-CoV-2 stabilized prefusion spike.

Proc Natl Acad Sci U S A 2021 03;118(12)

Department of Veterinary Biosciences, The Ohio State University, Columbus, OH 43210;

The current pandemic of COVID-19 caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) highlights an urgent need to develop a safe, efficacious, and durable vaccine. Using a measles virus (rMeV) vaccine strain as the backbone, we developed a series of recombinant attenuated vaccine candidates expressing various forms of the SARS-CoV-2 spike (S) protein and its receptor binding domain (RBD) and evaluated their efficacy in cotton rat, IFNARmice, IFNAR-hCD46 mice, and golden Syrian hamsters. We found that rMeV expressing stabilized prefusion S protein (rMeV-preS) was more potent in inducing SARS-CoV-2-specific neutralizing antibodies than rMeV expressing full-length S protein (rMeV-S), while the rMeVs expressing different lengths of RBD (rMeV-RBD) were the least potent. Animals immunized with rMeV-preS produced higher levels of neutralizing antibody than found in convalescent sera from COVID-19 patients and a strong Th1-biased T cell response. The rMeV-preS also provided complete protection of hamsters from challenge with SARS-CoV-2, preventing replication in lungs and nasal turbinates, body weight loss, cytokine storm, and lung pathology. These data demonstrate that rMeV-preS is a safe and highly efficacious vaccine candidate, supporting its further development as a SARS-CoV-2 vaccine.
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http://dx.doi.org/10.1073/pnas.2026153118DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8000430PMC
March 2021

Vaccination to prevent T cell subversion can protect against persistent hepacivirus infection.

Nat Commun 2019 03 7;10(1):1113. Epub 2019 Mar 7.

Center for Vaccines and Immunity, The Research Institute at Nationwide Children's Hospital, Columbus, OH, 43205, USA.

Efforts to develop an effective vaccine against the hepatitis C virus (HCV; human hepacivirus) have been stymied by a lack of small animal models. Here, we describe an experimental rat model of chronic HCV-related hepacivirus infection and its response to T cell immunization. Immune-competent rats challenged with a rodent hepacivirus (RHV) develop chronic viremia characterized by expansion of non-functional CD8 T cells. Single-dose vaccination with a recombinant adenovirus vector expressing hepacivirus non-structural proteins induces effective immunity in majority of rats. Resolution of infection coincides with a vigorous recall of intrahepatic cellular responses. Host selection of viral CD8 escape variants can subvert vaccine-conferred immunity. Transient depletion of CD8 cells from vaccinated rats prolongs infection, while CD4 cell depletion results in chronic viremia. These results provide direct evidence that co-operation between CD4 and CD8 T cells is important for hepacivirus immunity, and that subversion of responses can be prevented by prophylactic vaccination.
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http://dx.doi.org/10.1038/s41467-019-09105-0DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6405742PMC
March 2019

Viral testing of 18 consecutive cases of equine serum hepatitis: A prospective study (2014-2018).

J Vet Intern Med 2019 Jan 5;33(1):251-257. Epub 2018 Dec 5.

Department of Clinical Sciences, College of Veterinary Medicine, Cornell University, Ithaca, New York.

Background: Three flaviviruses (equine pegivirus [EPgV]; Theiler's disease-associated virus [TDAV]; non-primate hepacivirus [NPHV]) and equine parvovirus (EqPV-H) are present in equine blood products; the TDAV, NPHV, and EqPV-H have been suggested as potential causes of serum hepatitis.

Objective: To determine the prevalence of these viruses in horses with equine serum hepatitis.

Animals: Eighteen horses diagnosed with serum hepatitis, enrolled from US referral hospitals.

Methods: In the prospective case study, liver, serum, or both samples were tested for EPgV, TDAV, NPHV, and EqPV-H by PCR.

Results: Both liver tissue and serum were tested for 6 cases, serum only for 8 cases, and liver only for 4 cases. Twelve horses received tetanus antitoxin (TAT) 4-12.7 weeks (median = 8 weeks), 3 horses received commercial equine plasma 6-8.6 weeks, and 3 horses received allogenic stem cells 6.4-7.6 weeks before the onset of hepatic failure. All samples were TDAV negative. Two of 14 serum samples were NPHV-positive. Six of 14 serum samples were EPgV-positive. All liver samples were NPHV-negative and EPgV-negative. EqPV-H was detected in the serum (N = 8), liver (N = 4), or both samples (N = 6) of all 18 cases. The TAT of the same lot number was available for virologic testing in 10 of 12 TAT-associated cases, and all 10 samples were EqPV-H positive.

Conclusions And Clinical Importance: We demonstrated EqPV-H in 18 consecutive cases of serum hepatitis. EPgV, TDAV, and NPHV were not consistently present. This information should encourage blood product manufacturers to test for EqPV-H and eliminate EqPV-H-infected horses from their donor herds.
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http://dx.doi.org/10.1111/jvim.15368DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6335536PMC
January 2019

New Parvovirus Associated with Serum Hepatitis in Horses after Inoculation of Common Biological Product.

Emerg Infect Dis 2018 02;24(2):303-310

Equine serum hepatitis (i.e., Theiler's disease) is a serious and often life-threatening disease of unknown etiology that affects horses. A horse in Nebraska, USA, with serum hepatitis died 65 days after treatment with equine-origin tetanus antitoxin. We identified an unknown parvovirus in serum and liver of the dead horse and in the administered antitoxin. The equine parvovirus-hepatitis (EqPV-H) shares <50% protein identity with its phylogenetic relatives of the genus Copiparvovirus. Next, we experimentally infected 2 horses using a tetanus antitoxin contaminated with EqPV-H. Viremia developed, the horses seroconverted, and acute hepatitis developed that was confirmed by clinical, biochemical, and histopathologic testing. We also determined that EqPV-H is an endemic infection because, in a cohort of 100 clinically normal adult horses, 13 were viremic and 15 were seropositive. We identified a new virus associated with equine serum hepatitis and confirmed its pathogenicity and transmissibility through contaminated biological products.
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http://dx.doi.org/10.3201/eid2402.171031DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5782890PMC
February 2018

Viral persistence, liver disease, and host response in a hepatitis C-like virus rat model.

Hepatology 2018 08 21;68(2):435-448. Epub 2018 May 21.

Center for Vaccines and Immunity, The Research Institute at Nationwide Children's Hospital, Columbus, OH.

The lack of a relevant, tractable, and immunocompetent animal model for hepatitis C virus (HCV) has severely impeded investigations of viral persistence, immunity, and pathogenesis. In the absence of immunocompetent models with robust HCV infection, homolog hepaciviruses in their natural host could potentially provide useful surrogate models. We isolated a rodent hepacivirus from wild rats (Rattus norvegicus), RHV-rn1; acquired the complete viral genome sequence; and developed an infectious reverse genetics system. RHV-rn1 resembles HCV in genomic features including the pattern of polyprotein cleavage sites and secondary structures in the viral 5' and 3' untranslated regions. We used site-directed and random mutagenesis to determine that only the first of the two microRNA-122 seed sites in the viral 5' untranslated region is required for viral replication and persistence in rats. Next, we used the clone-derived virus progeny to infect several inbred and outbred rat strains. Our results determined that RHV-rn1 possesses several HCV-defining hallmarks: hepatotropism, propensity to persist, and the ability to induce gradual liver damage. Histological examination of liver samples revealed the presence of lymphoid aggregates, parenchymal inflammation, and macrovesicular and microvesicular steatosis in chronically infected rats. Gene expression analysis demonstrated that the intrahepatic response during RHV-rn1 infection in rats mirrors that of HCV infection, including persistent activation of interferon signaling pathways. Finally, we determined that the backbone drug of HCV direct-acting antiviral therapy, sofosbuvir, effectively suppresses chronic RHV-rn1 infection in rats.

Conclusion: We developed RHV-rn1-infected rats as a fully immunocompetent and informative surrogate model to delineate the mechanisms of HCV-related viral persistence, immunity, and pathogenesis. (Hepatology 2018).
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http://dx.doi.org/10.1002/hep.29494DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5832584PMC
August 2018

The Surface Protein SdrF Mediates Staphylococcus epidermidis Adherence to Keratin.

J Infect Dis 2017 06;215(12):1846-1854

1 Division of Infectious Diseases.

Background: Staphylococcus epidermidis, a major component of skin flora, is an opportunist, often causing prosthetic device infections. A family of structurally related proteins mediates staphylococcal attachment to host tissues, contributing to the success of S. epidermidis as a pathogen. We examined the ability of the surface protein SdrF to adhere to keratin, a major molecule expressed on the skin surface.

Methods: A heterologous Lactococcus lactis expression system was used to express SdrF and its ligand-binding domains. Adherence to keratin types 1 and 10, human foreskin keratinocytes, and nasal epithelial cells was examined.

Results: SdrF bound human keratins 1 and 10 and adhered to keratinocytes and epithelial cells. Binding involved both the A and B domains. Anti-SdrF antibodies reduced adherence of S. epidermidis to keratin and keratinocytes. RNA interference reduced keratin synthesis in keratinocytes and, as a result, SdrF adherence. Direct force measurements using atomic force microscopy showed that SdrF mediates bacterial adhesion to keratin 10 through strong and weak bonds involving the A and B regions; strong adhesion was primarily mediated by the A region.

Conclusions: These studies demonstrate that SdrF mediates adherence to human keratin and suggest that SdrF may facilitate S. epidermidis colonization of the skin.
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http://dx.doi.org/10.1093/infdis/jix213DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5853823PMC
June 2017

In vitro effect of temperature on the conformational structure and collagen binding of SdrF, a Staphylococcus epidermidis adhesin.

Appl Microbiol Biotechnol 2015 Jul 17;99(13):5593-603. Epub 2015 Feb 17.

Department of Molecular Medicine, Center for Tissue Engineering (CIT), INSTM UdR of Pavia, University of Pavia, Viale Taramelli 3/b, 27100, Pavia, Italy.

Staphylococcus epidermidis is the leading etiologic agent of device-related infections. S. epidermidis is able to bind, by means of the adhesins of its cell wall, the host matrix proteins filming the artificial surfaces. Thence, bacteria cling to biomaterials and infection develops. The effect of temperature on integrity, structure, and biological activity of the collagen-binding adhesin (SdrF) of S. epidermidis has been here investigated. By cloning in E. coli XL1-Blue, a recombinant of the SdrF binding domain B (rSdrFB), carrying an N-terminal polyhistidine, was obtained. Purification was by HiTrap(TM) Chelating HP columns. Assessment of purity, molecular weight, and integrity was by SDS-PAGE. The rSdrFB-collagen binding was investigated by ELISA. A full three-dimensional reconstruction of rSdrFB was achieved by small-angle X-ray scattering (SAXS). At 25 °C, rSdrFB bound to type I collagen in a dose-dependent, saturable manner, with a Kd of 2.48 × 10(-7) M. When temperature increased from 25 to 37 °C, a strong conformational change occurred, together with the abolition of the rSdrFB-collagen binding. The rSdrFB integrity was not affected by temperature variation. SdrFB-collagen binding is switched on/off depending on the temperature. Implications with the infection pathogenesis are enlightened.
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http://dx.doi.org/10.1007/s00253-015-6456-xDOI Listing
July 2015

207-nm UV light - a promising tool for safe low-cost reduction of surgical site infections. I: in vitro studies.

PLoS One 2013 16;8(10):e76968. Epub 2013 Oct 16.

Center for Radiological Research, Columbia University Medical Center, New York, New York, United States of America.

Background: 0.5% to 10% of clean surgeries result in surgical-site infections, and attempts to reduce this rate have had limited success. Germicidal UV lamps, with a broad wavelength spectrum from 200 to 400 nm are an effective bactericidal option against drug-resistant and drug-sensitive bacteria, but represent a health hazard to patient and staff. By contrast, because of its limited penetration, ~200 nm far-UVC light is predicted to be effective in killing bacteria, but without the human health hazards to skin and eyes associated with conventional germicidal UV exposure.

Aims: The aim of this work was to test the biophysically-based hypothesis that ~200 nm UV light is significantly cytotoxic to bacteria, but minimally cytotoxic or mutagenic to human cells either isolated or within tissues.

Methods: A Kr-Br excimer lamp was used, which produces 207-nm UV light, with a filter to remove higher-wavelength components. Comparisons were made with results from a conventional broad spectrum 254-nm UV germicidal lamp. First, cell inactivation vs. UV fluence data were generated for methicillin-resistant S. aureus (MRSA) bacteria and also for normal human fibroblasts. Second, yields of the main UV-associated pre-mutagenic DNA lesions (cyclobutane pyrimidine dimers and 6-4 photoproducts) were measured, for both UV radiations incident on 3-D human skin tissue.

Results: We found that 207-nm UV light kills MRSA efficiently but, unlike conventional germicidal UV lamps, produces little cell killing in human cells. In a 3-D human skin model, 207-nm UV light produced almost no pre-mutagenic UV-associated DNA lesions, in contrast to significant yields induced by a conventional germicidal UV lamp.

Conclusions: As predicted based on biophysical considerations, 207-nm light kills bacteria efficiently but does not appear to be significantly cytotoxic or mutagenic to human cells. Used appropriately, 207-nm light may have the potential for safely and inexpensively reducing surgical-site infection rates, including those of drug-resistant origin.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0076968PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3797730PMC
August 2014

The role of ionic interactions in the adherence of the Staphylococcus epidermidis adhesin SdrF to prosthetic material.

FEMS Microbiol Lett 2013 Jan 2;338(1):24-30. Epub 2012 Nov 2.

Division of Infectious Diseases, Department of Medicine, College of Physicians and Surgeons, Columbia University, New York, NY, USA.

Staphylococcus epidermidis infections are common complications of prosthetic device implantation. SdrF, a surface protein, appears to play a critical role in the initial colonization step by adhering to type I collagen and Dacron™. The role of ionic interactions in S. epidermidis adherence to prosthetic material was examined. SdrF was cloned and expressed in Lactococcus lactis. The effect of pH, cation concentration, and detergents on adherence to different types of plastic surfaces was assessed by crystal violet staining and bacterial cell counting. SdrF, in contrast with controls and other S. epidermidis surface proteins, bound to hydrophobic materials such as polystyrene. Binding was an ionic interaction and was affected by surface charge of the plastic, pH, and cation concentration. Adherence of the SdrF construct was increased to positively charged plastics and was reduced by increasing concentrations of Ca(2+) and Na(+). Binding was optimal at pH 7.4. Kinetic studies demonstrated that the SdrF B domain as well as one of the B subdomains was sufficient to mediate binding. The SdrF construct also bound more avidly to Goretex™ than the lacotococcal control. SdrF is a multifunctional protein that contributes to prosthetic devices infections by ionic, as well as specific receptor-ligand interactions.
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http://dx.doi.org/10.1111/1574-6968.12018DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3521083PMC
January 2013

Identification of a highly transmissible animal-independent Staphylococcus aureus ST398 clone with distinct genomic and cell adhesion properties.

mBio 2012 28;3(2). Epub 2012 Feb 28.

Department of Medicine, Division of Infectious Diseases, College of Physicians and Surgeons, Columbia University, New York, New York, USA.

Unlabelled: A methicillin-resistant Staphylococcus aureus (MRSA) clone known as ST398 has emerged as a major cause of acute infections in individuals who have close contact with livestock. More recently, the emergence of an animal-independent ST398 methicillin-sensitive S. aureus (MSSA) clone has been documented in several countries. However, the limited surveillance of MSSA has precluded an accurate assessment of the global spread of ST398 and its clinical relevance. Here we provide evidence that ST398 is a frequent source of MSSA infections in northern Manhattan and is readily transmitted between individuals in households. This contrasts with the limited transmissibility of livestock-associated ST398 (LA-ST398) MRSA strains between humans. Our whole-genome sequence analysis revealed that the chromosome of the human-associated ST398 MSSA clone is smaller than that of the LA-ST398 MRSA reference strain S0385, due mainly to fewer mobile genetic elements (MGEs). In contrast, human ST398 MSSA isolates harbored the prophage ϕ3 and the human-specific immune evasion cluster (IEC) genes chp and scn. While most of the core genome was conserved between the human ST398 MSSA clone and S0385, these strains differed substantially in their repertoire and composition of intact adhesion genes. These genetic changes were associated with significantly enhanced adhesion of human ST398 MSSA isolates to human skin keratinocytes and keratin. We propose that the human ST398 MSSA clone can spread independent of animal contact using an optimized repertoire of MGEs and adhesion molecules adapted to transmission among humans.

Importance: Staphylococcus aureus strains have generally been considered to be species specific. However, cross-species transfers of S. aureus clones, such as ST398 methicillin-resistant S. aureus (MRSA), from swine to humans have been reported. Recently, we observed the emergence of ST398 methicillin-susceptible S. aureus (MSSA) as a colonizing strain of humans in northern Manhattan. Here we report that ST398 is a frequent cause of MSSA infections in this urban setting. The ST398 MSSA clone was readily transmitted within households, independent of animal contact. We discovered that human ST398 MSSA genomes were smaller than that of the LA-ST398 strain S0385 due to fewer mobile genetic elements. Human and LA-ST398 strains also differed in their composition of adhesion genes and their ability to bind to human skin keratinocytes, providing a potential mechanism of S. aureus host adaptation. Our findings illustrate the importance of implementing molecular surveillance of MSSA given the evidence for the rapid and clinically undetected spread of ST398 MSSA.
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http://dx.doi.org/10.1128/mBio.00027-12DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3302565PMC
June 2012

Similar effects of selective laser trabeculoplasty and prostaglandin analogs on the permeability of cultured Schlemm canal cells.

Am J Ophthalmol 2010 Aug 8;150(2):254-64. Epub 2010 Jun 8.

Beckman Vision Center, Department of Ophthalmology, University of California-San Francisco, 10 Koret Way, San Francisco, CA 94143-0730, USA.

Purpose: To evaluate whether selective laser trabeculoplasty and prostaglandin analogs regulate the permeability of cultured human Schlemm canal cells by inducing intercellular junction disassembly.

Design: Laboratory investigation.

Methods: Intercellular junctions were made visible in living cells by making them fluoresce after transfection with a plasmid expressing the zonula occludens 1 protein tagged with green fluorescent protein. Schlemm canal cells were treated by direct laser irradiation; by exposure to media conditioned by either lasered Schlemm canal cells or trabecular meshwork cells; by exposure to the prostaglandin analogs latanoprost, bimatoprost, and travoprost; or by the addition of the nonprostaglandin agents brimonidine, timolol, and dorzolamide. Junction disassembly was monitored using fluorescence microscopy, and permeability alterations were measured as changes in conductivity using flow meters.

Results: The direct laser irradiation of Schlemm canal cells caused a 3-fold increase in conductivity. Exposure of the cells to media conditioned by lasered Schlemm canal cells or trabecular meshwork cells induced junction disassembly and a 2- to 4-fold increase in conductivity. Exposure to prostaglandin analogs also induced junction disassembly and a 4- to 16-fold increase in conductivity, whereas the 3 nonprostaglandin agents tested were ineffective in both regards.

Conclusions: Exposure to factors secreted by lasered Schlemm canal cells and lasered trabecular meshwork cells and the application of prostaglandin analogs induced junction disassembly while increasing the permeability of Schlemm canal cells. These findings support our hypothesis that selective laser trabeculoplasty and prostaglandin analogs share a common mechanism that likely mediates their pressure-lowering effects.
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http://dx.doi.org/10.1016/j.ajo.2010.03.012DOI Listing
August 2010

Monocyte modulation of aqueous outflow and recruitment to the trabecular meshwork following selective laser trabeculoplasty.

Arch Ophthalmol 2010 Jun;128(6):731-7

Department of Ophthalmology, University of California, San Francisco, CA 94143-0730, USA.

Objectives: To determine whether selective laser trabeculoplasty (SLT) induces monocyte recruitment to the trabecular meshwork (TM) in human and monkey eyes and whether monocytes increase both aqueous outflow in vivo and the conductivity of human Schlemm canal endothelial cells (SCEs) in vitro.

Methods: Monocyte recruitment was examined morphometrically in control human and monkey eyes and compared with that following SLT applied 1 to 3 days earlier. Outflow facility was measured for up to 4 days after the intracameral infusion of autologous macrophages in rabbits. Schlemm canal endothelial cell conductivity was measured using flow meters after exposing cultured SCEs to monocytes and monocyte-secreted factors for 24 hours.

Results: Our estimates show that the TM in the human eye normally had an average of 15 003 monocytes, while in the monkey eye there were 3181 monocytes, and this number increased 4- to 5-fold following SLT. The intracameral infusion of autologous macrophages in rabbits increased outflow facility 2-fold in a rapid and sustained manner. Human monocytes and monocyte-secreted factors increased SCE conductivity 2-fold in vitro.

Conclusions: The number of monocytes/macrophages in the TM increases substantially after SLT and monocytes augment both outflow facility and SCE conductivity. Clinical Relevance These findings indicate that the innate immune system in general and monocytes in particular play an important role in aqueous outflow homeostasis. The recruitment of monocytes in increased numbers after SLT likely plays a role in lowering the intraocular pressure after this procedure. The intracameral introduction of autologous monocytes harvested from a vein could have therapeutic potential as a cell-based individualized treatment of glaucoma.
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http://dx.doi.org/10.1001/archophthalmol.2010.85DOI Listing
June 2010

Constitutive secretion of chemokines by cultured human trabecular meshwork cells.

Exp Eye Res 2010 Jul 18;91(1):42-7. Epub 2010 Apr 18.

Department of Ophthalmology, University of California, San Francisco, 10 Koret Way, San Francisco, CA 94143-0730, USA.

Trabecular meshwork endothelial (TME) cells secrete a number of factors, such as enzymes and cytokines, which modulate the functions of the cells and the extracellular matrix of the conventional aqueous outflow pathway. TME cells usually secrete these factors in response to stimuli such as mechanical stretching, laser irradiation and pro-inflammatory cytokines. Here, we report that cultured human TME cells isolated from two non-glaucomatous individuals secrete significant quantities of the chemotactic cytokines IL8, CXCL6 and MCP1 in the absence of any stimulation. The secretion of these chemokines was augmented by treatment with the pro-inflammatory cytokines TNFalpha and IL1beta. By way of comparison, there was little or very low production of the three chemokines by human non-pigmented ciliary epithelial cells in the absence of stimulation. Our findings provide support to our recent observations that monocytes, presumably under the influence of chemotactic signals, circulate through the trabecular meshwork in the normal state and also that cytokines regulate the permeability of Schlemm's canal endothelial cells. In addition, the fact that normal TME cells constitutively secrete chemotactic cytokines strengthens the notion that cytokines play a key role in the homeostasis of the outflow of the aqueous humor and, possibly, in the pathogenesis of glaucoma.
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http://dx.doi.org/10.1016/j.exer.2010.04.001DOI Listing
July 2010

Early effects of iodine on DNA synthesis in sulfur mustard-induced skin lesions.

Arch Toxicol 2006 Apr;80(4):212-6

Department of Pharmacology, School of Pharmacy, Institute of Life Sciences, The Hebrew University of Jerusalem, 91904 Givat Ram, Jerusalem, Israel.

Sulfur mustard (SM) is powerful alkylator and highly cytotoxic blisterogen in both humans and animals. This study in male guinea pigs shows that, at an early stage (5 h) after SM exposure, a marked increase occurred in epithelial nuclear vacuolation, epidermal thickening, and dermal acute inflammation. Topical iodine treatment reduced the severity of these parameters. The rate of DNA synthesis expressed by incorporation of bromodeoxyuridine was reduced upon topical treatment with iodine only or SM only by 46 and 72%, respectively. Iodine treatment following SM exposure exerted an effect similar to that of SM only, indicating that DNA synthesis is not directly involved in the mechanism of action of iodine-induced protection.
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http://dx.doi.org/10.1007/s00204-005-0032-6DOI Listing
April 2006
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