pH inactivation of SARS-CoV-2 and SARS-CoV in virus spiked protein A eluates from a mAb purification process.

Biopharmaceutical manufacturing processes may include a low pH treatment step as a means of inactivating enveloped viruses. Small scale virus clearance studies are routinely performed using model enveloped viruses such as murine leukemia virus to assess inactivation at the pH range used in the downstream manufacturing process. Further, as a means of bioburden reduction, chromatography resins may be cleaned and stored using sodium hydroxide and this can also inactivate viruses. The susceptibility of SARS-CoV-2 and SARS-CoV to low pH conditions using protein A eluate derived material from a monoclonal antibody production process as well as high pH cleaning conditions was addressed. SARS-CoV-2 was effectively inactivated at pH 3.0, moderately inactivated at pH 3.4, but not inactivated at pH 3.8. Low pH was less effective at inactivating SARS-CoV. Both viruses were inactivated at a high pH of ca.13.4. These studies provide important information regarding the effectiveness of viral clearance and inactivation steps of novel coronaviruses when compared to other enveloped viruses.

Immunogenicity and efficacy of the COVID-19 candidate vector vaccine MVA-SARS-2-S in preclinical vaccination.

Severe acute respiratory syndrome (SARS) coronavirus 2 (SARS-CoV-2) has emerged as the infectious agent causing the pandemic coronavirus disease 2019 (COVID-19) with dramatic consequences for global human health and economics. Previously, we reached clinical evaluation with our vector vaccine based on modified vaccinia virus Ankara (MVA) against the Middle East respiratory syndrome coronavirus (MERS-CoV), which causes an infection in humans similar to SARS and COVID-19. Here, we describe the construction and preclinical characterization of a recombinant MVA expressing full-length SARS-CoV-2 spike (S) protein (MVA-SARS-2-S). Genetic stability and growth characteristics of MVA-SARS-2-S, plus its robust expression of S protein as antigen, make it a suitable candidate vaccine for industrial-scale production. Vaccinated mice produced S-specific CD8+ T cells and serum antibodies binding to S protein that neutralized SARS-CoV-2. Prime-boost vaccination with MVA-SARS-2-S protected mice sensitized with a human ACE2-expressing adenovirus from SARS-CoV-2 infection. MVA-SARS-2-S is currently being investigated in a phase I clinical trial as aspirant for developing a safe and efficacious vaccine against COVID-19.

Structural Analysis of Plasmodium falciparum Hexokinase Provides Novel Information about Catalysis Due to a Plasmodium-Specific Insertion.

The protozoan parasite Plasmodium falciparum is the causative pathogen of the most severe form of malaria, for which novel strategies for treatment are urgently required. The primary energy supply for intraerythrocytic stages of Plasmodium is the production of ATP via glycolysis. Due to the parasite's strong dependence on this pathway and the significant structural differences of its glycolytic enzymes compared to its human counterpart, glycolysis is considered a potential drug target. In this study, we provide the first three-dimensional protein structure of P. falciparum hexokinase (PfHK) containing novel information about the mechanisms of PfHK. We identified for the first time a Plasmodium-specific insertion that lines the active site. Moreover, we propose that this insertion plays a role in ATP binding. Residues of the insertion further seem to affect the tetrameric interface and therefore suggest a special way of communication among the different monomers. In addition, we confirmed that PfHK is targeted and affected by oxidative posttranslational modifications (oxPTMs). Both S-glutathionylation and S-nitrosation revealed an inhibitory effect on the enzymatic activity of PfHK.

Induced arginine transport via cationic amino acid transporter-1 is necessary for human T-cell proliferation.

Availability of the semiessential amino acid arginine is fundamental for the efficient function of human T lymphocytes. Tumor-associated arginine deprivation, mainly induced by myeloid-derived suppressor cells, is a central mechanism of tumor immune escape from T-cell-mediated antitumor immune responses. We thus assumed that transmembranous transport of arginine must be crucial for T-cell function and studied which transporters are responsible for arginine influx into primary human T lymphocytes. Here, we show that activation via CD3 and CD28 induces arginine transport into primary human T cells. Both naïve and memory CD4(+) T cells as well as CD8(+) T cells specifically upregulated the human cationic amino acid transporter-1 (hCAT-1), with an enhanced and persistent expression under arginine starvation. When hCAT-1 induction was suppressed via siRNA transfection, arginine uptake, and cellular proliferation were impaired. In summary, our results demonstrate that hCAT-1 is a key component of efficient T-cell activation and a novel potential target structure to modulate adaptive immune responses in tumor immunity or inflammation.

Novel proteolytic activation of Ebolavirus glycoprotein GP by TMPRSS2 and cathepsin L at an uncharted position can compensate for furin cleavage.

A multistep priming process involving furin and endosomal cathepsin B and L (CatB/L) has been described for the Orthoebolavirus zairense (EBOV) glycoprotein GP. Inhibition or knockdown of either furin or endosomal cathepsins, however, did not prevent virus multiplication in cell cultures. Moreover, an EBOV mutant lacking the furin cleavage motif (RRTRR→AGTAA) was able to replicate and cause fatal disease in nonhuman primates, indicating that furin cleavage may be dispensable for virus infectivity. Here, by using protease inhibitors and EBOV GP-carrying recombinant vesicular stomatitis virus (VSV) and transcription and replication-competent virus-like particles (trVLPs) we found that processing of EBOV GP is mediated by different proteases in different cell lines depending on the protease repertoire available. Endosomal cathepsins were essential for EBOV GP entry in Huh-7 but not in Vero cells, in which trypsin-like proteases and stably expressed trypsin-like transmembrane serine protease 2 (TMPRSS2) supported wild-type EBOV GP and EBOV GP_AGTAA mutant entry. Furthermore, we show that the EBOV GP_AGTAA mutant is cleaved into fusion-competent GP2 by TMPRSS2 and by CatL at a so far unknown site. Fluorescence microscopy co-localization studies indicate that EBOV GP cleavage by TMPRSS2 may occur in the TGN prior to virus release or in the late endosome at the stage of virus entry into a new cell. Our data show that EBOV GP must be proteolytically activated to support virus entry but has even greater flexibility in terms of proteases and the precise cleavage site than previously assumed.

Warthin-Like Papillary Thyroid Microcarcinoma With Coincidental Ipsilateral Warthin Tumor of the Parotid Gland Detected on 131 I-SPECT/US and 18 F-PET/US Fusion Imaging.

ABSTRACT: A 66-year-old woman was referred with an incidental finding of a bilateral papillary thyroid microcarcinoma after thyroidectomy. On the right side, a Warthin-like variant was observed. After radioiodine therapy, whole-body scan revealed an unclear iodine uptake on the right-sided neck. For further clarification, 131 I-SPECT/US and 18 F-PET/US fusion imaging were performed, unambiguously revealing iodine and glucose uptake within a hypoechoic lesion located in the parenchyma of the right parotid gland. Surgical excision confirmed a Warthin tumor ipsilateral to the Warthin-like variant of the papillary thyroid microcarcinoma. Because the extensive imaging, targeted minimal-invasive surgery was possible.

Liver PET Reloaded: Automated Synthesis of [68Ga]Ga-BP-IDA for Positron Imaging of the Hepatobiliary Function and First Clinical Experience.

Hepatobiliary scintigraphy is a well-established nuclear imaging method for evaluating liver function and displaying the biliary system, but the spatial and temporal resolution is limited, and, there is still no established PET equivalent. Adapted from the work of Schuhmacher et al. in 1983, the production of a 68Gallium-labeled substitute, tetrabromophthalein ([68Ga]Ga-BP-IDA), was undertaken according to current Good Manufacturing Practice (GMP) standards and proved feasible and reproducible. PET/CT with the radiotracer was performed in two complex patients with hepatocellular carcinoma in preparation for transarterial radioembolization. Due to its high spatial and temporal resolution, localization of areas with impaired liver function and visualization of the biliary system were possible. We could demonstrate that this 68Gallium-labeled, IDA-based PET tracer is feasible and could advance hepatic and biliary function PET imaging.

Image-Guided Fine-Needle Aspiration Cytology for BRCA Mutation Assessment of PSMA-Positive Lymph Node Metastases in a Patient With Metastatic Castration-Resistant Prostate Cancer.

ABSTRACT: A 64-year-old man with metastatic castration-resistant prostate cancer presented for prostate-specific membrane antigen (PSMA) PET/CT in preparation for 177 Lu-PSMA radioligand therapy. For precedent BRCA mutation assessment, fine-needle aspiration cytology of 2 PSMA-positive lymph node metastases was conducted. The acquired material was suitable for next-generation sequencing-based gene panel diagnostics and did not show a BRCA1 / 2 mutation, thus PSMA radioligand therapy was initiated. Fine-needle aspiration cytology of lymph node metastases may be a viable option in evaluating further therapeutic alternatives.

The C-terminus of Sudan ebolavirus VP40 contains a functionally important CXnC motif, a target for redox modifications.

The Ebola virus matrix protein VP40 mediates viral budding and negatively regulates viral RNA synthesis. The mechanisms by which these two functions are exerted and regulated are unknown. Using a high-resolution crystal structure of Sudan ebolavirus (SUDV) VP40, we show here that two cysteines in the flexible C-terminal arm of VP40 form a stabilizing disulfide bridge. Notably, the two cysteines are targets of posttranslational redox modifications and interact directly with the host`s thioredoxin system. Mutation of the cysteines impaired the budding function of VP40 and relaxed its inhibitory role for viral RNA synthesis. In line with these results, the growth of recombinant Ebola viruses carrying cysteine mutations was impaired and the released viral particles were elongated. Our results revealed the exact positions of the cysteines in the C-terminal arm of SUDV VP40. The cysteines and/or their redox status are critically involved in the differential regulation of viral budding and viral RNA synthesis.

The anti-inflammatory fungal compound (S)-curvularin reduces proinflammatory gene expression in an in vivo model of rheumatoid arthritis.

In previous studies, we identified the fungal macrocyclic lactone (S)-curvularin (SC) as an anti-inflammatory agent using a screening system detecting inhibitors of the Janus kinase/signal transducer and activator of transcription pathway. The objective of the present study was to investigate whether SC is able to decrease proinflammatory gene expression in an in vivo model of a chronic inflammatory disease. Therefore, the effects of SC and dexamethasone were compared in the model of collagen-induced arthritis (CIA) in mice. Total genomic microarray analyses were performed to identify SC target genes. In addition, in human C28/I2 chondrocytes and MonoMac6 monocytes, the effect of SC on proinflammatory gene expression was tested at the mRNA and protein level. In the CIA model, SC markedly reduced the expression of a number of proinflammatory cytokines and chemokines involved in the pathogenesis of CIA as well as human rheumatoid arthritis (RA). In almost all cases, the effects of SC were comparable with those of dexamethasone. In microarray analyses, we identified additional new therapeutic targets of SC. Some of them, such as S100A8, myeloperoxidase, or cathelicidin, an antimicrobial peptide, are known to be implicated in pathophysiological processes in RA. Similar anti-inflammatory effects of SC were also observed in human C28/I2 chondrocyte cells, which are resistant to glucocorticoid treatment. These data indicate that SC and glucocorticoid effects are mediated via independent signal transduction pathways. In summary, we demonstrate that SC is a new effective anti-inflammatory compound that may serve as a lead compound for the development of new drugs for the therapy of chronic inflammatory diseases.

High-Resolution Splenic Imaging: [68Ga]Ga-Oxine Red Blood Cell PET/CT for Differentiation of Splenosis Mimicking Malignant Lymphoma.

The differentiation of splenic tissue from malignant lesions via imaging may be challenging, particularly considering aberrant or accessory lesions and diseases that are rarely encountered. Functioning splenic tissue can be identified using technetium-99m red blood cell (99mTc-RBC) scintigraphy, but its sensitivity is limited and may not be available. We present the case of a patient in whom disseminated abdomino-pelvic splenosis was diagnosed using PET/CT with gallium-68-oxine-labeled RBCs. The method represents a feasible and probably superior alternative to splenic scintigraphy.

Metabolic regulation of immune responses to cancer.

The tumor microenvironment is an ecosystem composed of multiple types of cells, such as tumor cells, immune cells, and cancer-associated fibroblasts. Cancer cells grow faster than non-cancerous cells and consume larger amounts of nutrients. The rapid growth characteristic of cancer cells fundamentally alters nutrient availability in the tumor microenvironment and results in reprogramming of immune cell metabolic pathways. Accumulating evidence suggests that cellular metabolism of nutrients, such as lipids and amino acids, beyond being essential to meet the bioenergetic and biosynthetic demands of immune cells, also regulates a broad spectrum of cellular signal transduction, and influences immune cell survival, differentiation, and anti-tumor effector function. The cancer immunometabolism research field is rapidly evolving, and exciting new discoveries are reported in high-profile journals nearly weekly. Therefore, all new findings in this field cannot be summarized within this short review. Instead, this review is intended to provide a brief introduction to this rapidly developing research field, with a focus on the metabolism of two classes of important nutrients-lipids and amino acids-in immune cells. We highlight recent research on the roles of lipids and amino acids in regulating the metabolic fitness and immunological functions of T cells, macrophages, and natural killer cells in the tumor microenvironment. Furthermore, we discuss the possibility of "editing" metabolic pathways in immune cells to act synergistically with currently available immunotherapies in enhancing anti-tumor immune responses.

l-Citrulline ameliorates pathophysiology in a rat model of superimposed preeclampsia.

BACKGROUND AND PURPOSE: Preeclampsia, characterized by hypertension, proteinuria and restriction of fetal growth, is one of the leading causes of maternal and perinatal mortality. So far, there is no effective pharmacological therapy for preeclampsia. The present study was conducted to investigate the effects of supplementation with l-citrulline in Dahl salt-sensitive rats, a model of superimposed preeclampsia. EXPERIMENTAL APPROACH: Parental Dahl salt-sensitive rats were treated with l-citrulline (2.5 g·L-1 in drinking water) from the day of mating to the end of lactation period. Blood pressure was monitored throughout pregnancy and markers of preeclampsia were assessed. Endothelial function of the pregnant Dahl salt-sensitive rats was assessed by wire myograph. KEY RESULTS: In Dahl salt-sensitive rats, l-citrulline supplementation significantly reduced maternal blood pressure, proteinuria and levels of circulating soluble fms-like tyrosine kinase 1. l-Citrulline improved maternal endothelial function by augmenting the production of nitric oxide in the aorta and improving endothelium-derived hyperpolarizing factor-mediated vasorelaxation in resistance arteries. l-Citrulline supplementation improved placental insufficiency and fetal growth, which were associated with an enhancement of angiogenesis and reduction of fibrosis and senescence in the placentas. In addition, l-citrulline down-regulated genes involved in the TLR4 and NF-κB signalling pathways. CONCLUSION AND IMPLICATIONS: This study shows that l-citrulline supplementation reduced gestational hypertension and improved placentation and fetal growth in a rat model of superimposed preeclampsia. l-Citrulline supplementation may provide an effective and safe therapeutic strategy for preeclampsia that benefits both the mother and the fetus.

Intranasal Administration of a Monoclonal Neutralizing Antibody Protects Mice against SARS-CoV-2 Infection.

Despite the recent availability of vaccines against severe acute respiratory syndrome coronavirus type 2 (SARS-CoV-2), there is an urgent need for specific anti-SARS-CoV-2 drugs. Monoclonal neutralizing antibodies are an important drug class in the global fight against the SARS-CoV-2 pandemic due to their ability to convey immediate protection and their potential to be used as both prophylactic and therapeutic drugs. Clinically used neutralizing antibodies against respiratory viruses are currently injected intravenously, which can lead to suboptimal pulmonary bioavailability and thus to a lower effectiveness. Here we describe DZIF-10c, a fully human monoclonal neutralizing antibody that binds the receptor-binding domain of the SARS-CoV-2 spike protein. DZIF-10c displays an exceptionally high neutralizing potency against SARS-CoV-2, retains full activity against the variant of concern (VOC) B.1.1.7 and still neutralizes the VOC B.1.351, although with reduced potency. Importantly, not only systemic but also intranasal application of DZIF-10c abolished the presence of infectious particles in the lungs of SARS-CoV-2 infected mice and mitigated lung pathology when administered prophylactically. Along with a favorable pharmacokinetic profile, these results highlight DZIF-10c as a novel human SARS-CoV-2 neutralizing antibody with high in vitro and in vivo antiviral potency. The successful intranasal application of DZIF-10c paves the way for clinical trials investigating topical delivery of anti-SARS-CoV-2 antibodies.

Inhibition of Arginase 1 Liberates Potent T Cell Immunostimulatory Activity of Human Neutrophil Granulocytes.

Myeloid cell arginase-mediated arginine depletion with consecutive inhibition of T cell functions is a key component of tumor immune escape. Both, granulocytic myeloid-derived suppressor cells (G-MDSC) and conventional mature human polymorphonuclear neutrophil granulocytes (PMN) express high levels of arginase 1 and can act as suppressor cells of adaptive anti-cancer immunity. Here we demonstrate that pharmacological inhibition of PMN-derived arginase 1 not only prevents the suppression of T cell functions but rather leads to a strong hyperactivation of T cells. Human PMN were incubated in cell culture medium in the absence or presence of an arginase inhibitor. T cells from healthy donors were then activated either polyclonally or in an antigen-specific manner in the supernatants of the PMN cultures at different PMN-T cell ratios. T cell proliferation was completely suppressed in these supernatants in the absence of an arginase inhibitor. Arginase inhibition led to a strong hyperinduction of T cell proliferation, which exceeded control activation conditions up to 25-fold. The hyperinduction was correlated with higher PMN-T cell ratios and was only apparent when PMN arginase activity was blocked sufficiently. The T cell stimulatory factor was liberated very early by PMN and was present in the < 3 kDa fraction of the PMN supernatants. Increased T cell production of specific proinflammatory cytokines by PMN supernatant in the presence of arginase inhibitor was apparent. Upon arginase inhibition, downregulation of important T cell membrane activation and costimulation proteins was completely prevented or de novo induction accelerated. Antigen-specific T cell cytotoxicity against tumor cells was enhanced by PMN supernatant itself and could be further increased by PMN arginase blockade. Finally, we analyzed anergic T cells from multiple myeloma patients and noticed a complete reversal of anergy and the induction of strong proliferation upon T cell activation in PMN supernatants by arginase inhibition. In summary, we discovered a potent PMN-mediated hyperactivation of human T cells, which is apparent only when PMN arginase-mediated arginine depletion is concurrently inhibited. Our findings are clearly relevant for the analysis and prevention of human tumor immune escape in conjunction with the application of arginase inhibitors already being developed clinically.

Effects of inorganic nitrate and vitamin C co-supplementation on blood pressure and vascular function in younger and older healthy adults: A randomised double-blind crossover trial.

BACKGROUND: Vitamin C and inorganic nitrate have been linked to enhanced nitric oxide (NO) production and reduced oxidative stress. Vitamin C may also enhance the conversion of nitrite into NO. AIMS: We investigated the potential acute effects of vitamin C and inorganic nitrate co-supplementation on blood pressure (BP) and peripheral vascular function. The secondary aim was to investigate whether age modified the effects of vitamin C and inorganic nitrate on these vascular outcomes. METHODS: Ten younger (age 18-40 y) and ten older (age 55-70 y) healthy participants were enrolled in a randomised double-blind crossover clinical trial. Participants ingested a solution of potassium nitrate (7 mg/kg body weight) and/or vitamin C (20 mg/kg body weight) or their placebos. Acute changes in resting BP and vascular function (post-occlusion reactive hyperemia [PORH], peripheral pulse wave velocity [PWV]) were monitored over a 3-h period. RESULTS: Vitamin C supplementation reduced PWV significantly (vitamin C: -0.70 ± 0.31 m/s; vitamin C placebo: +0.43 ± 0.30 m/s; P = 0.007). There were significant interactions between age and vitamin C for systolic, diastolic, and mean arterial BP (P = 0.02, P = 0.03, P = 0.02, respectively), with systolic, diastolic and mean BP decreasing in older participants and diastolic BP increasing in younger participants following vitamin C administration. Nitrate supplementation did not influence BP (systolic: P = 0.81; diastolic: P = 0.24; mean BP: P = 0.87) or vascular function (PORH: P = 0.05; PWV: P = 0.44) significantly in both younger and older participants. However, combined supplementation with nitrate and vitamin C reduced mean arterial BP (-2.6 mmHg, P = 0.03) and decreased PWV in older participants (PWV: -2.0 m/s, P = 0.02). CONCLUSIONS: The co-administration of a single dose of inorganic nitrate and vitamin C lowered diastolic BP and improved PVW in older participants. Vitamin C supplementation improved PWV in both age groups but decreased systolic and mean BP in older participants only. CLINICAL TRIAL REGISTRATION: Current Controlled Trials (ISRCTN98942199).

Determination of effective half-life of 131I in patients with differentiated thyroid carcinoma: comparison of cystatin C and creatinine-based estimation of renal function.

PURPOSE: Renal function and effective half-life (t1/2,eff) of I-131 have not been fully elucidated in patients undergoing radioiodine therapy (RAIT) for differentiated thyroid cancer (DTC). Aim of the present analysis was to evaluate the potential of cystatin C-based estimated glomerular filtration rate (eGFRCysC) in comparison to conventional creatinine (eGFRCrea) and to verify which methods to determine t1/2,eff are most accurate to predict t1/2,eff. METHODS: Forty-eight patients receiving whole-body I-131-scintigraphy were included. eGFRCysC was compared to eGFRCrea with regard to accuracy of t1/2,eff prediction. Three different methods (i.e. blood-based, gamma camera-based and probe-based) and two protocols with either three (short period,SP; up to 42 h) or four (long period,LP; up to 114 h) time points were compared using the Akaike's information criterion. RESULTS: The eGFRCysC measurement is more likely than eGFRCrea in predicting the t1/2,eff. High correlation coefficients were found between t1/2,eff assessed by gamma camera and probe measurements and blood-based determination revealed lower values. Patients with normal eGFR showed higher values of t1/2,eff of LP compared to SP. CONCLUSIONS: eGFRCysC should be included in further study protocols. As camera and probe measurements lead to almost superimposable results, one of the methods is expendable. Blood-based results of t1/2,eff were lower, presumably due to unspecific iodine retention, whereas the lower correlation with renal function may be caused by individual differences in intestinal iodine resorption. SP-protocols up to 42 h after I-131 administration are sufficient to determine t1/2,eff. Further studies are necessary for specific recommendations regarding I-131 activity reduction during RAIT in patients with DTC and renal insufficiency.

Cationic Amino Acid Transporter-1-Mediated Arginine Uptake Is Essential for Chronic Lymphocytic Leukemia Cell Proliferation and Viability.

Interfering with tumor metabolism by specifically restricting the availability of extracellular nutrients is a rapidly emerging field of cancer research. A variety of tumor entities depend on the uptake of the amino acid arginine since they have lost the ability to synthesize it endogenously, that is they do not express the rate limiting enzyme for arginine synthesis, argininosuccinate synthase (ASS). Arginine transport through the plasma membrane of mammalian cells is mediated by eight different transporters that belong to two solute carrier (SLC) families. In the present study we found that the proliferation of primary as well as immortalized chronic lymphocytic leukemia (CLL) cells depends on the availability of extracellular arginine and that primary CLL cells do not express ASS and are therefore arginine-auxotrophic. The cationic amino acid transporter-1 (CAT-1) was the only arginine importer expressed in CLL cells. Lentiviral-mediated downregulation of the CAT-1 transporter in HG3 CLL cells significantly reduced arginine uptake, abolished cell proliferation and impaired cell viability. In a murine CLL xenograft model, tumor growth was significantly suppressed upon induced downregulation of CAT-1 in the CLL cells. Our results suggest that inhibition of CAT-1 is a promising new therapeutic approach for CLL.

Isolation and cultivation of canine corneal cells for in vitro studies on the anti-inflammatory effects of dexamethasone.

OBJECTIVE: This study was conducted to establish a protocol for the isolation and culture of canine corneal cells (i.e. endothelium, keratocytes, and epithelium) to be used for in vitro studies on the effects of dexamethasone in corneal inflammation. ANIMAL MATERIAL: Corneal endothelial cells, epithelial cells and keratocytes from enucleated eyes of dogs (euthanized not related to this study) were isolated and cultured. PROCEDURE: Canine corneal cells were isolated using a combined enzymatic and mechanical technique and separately taken into culture. The three different cell types were verified by phase contrast microscopy, immunofluorescence, and Western blot using antivimentin and anticytokeratin antibodies. The mRNA for the glucocorticoid receptor (GR) was detected using reverse transcriptase polymerase chain reaction (RT-PCR). To study dexamethasone effects, primary cells were stimulated with lipopolysaccharides (LPS) to induce production of inflammatory mediators, particularly prostaglandin E(2) (PGE(2)). The concentration of PGE(2) in cell culture supernatant was determined utilizing an ELISA assay. Results were compared between control, stimulated as well as stimulated and dexamethasone treated cells. RESULTS: A protocol for the isolation and culture of canine corneal endothelium, keratocytes, and epithelium was successfully established. Using morphological criteria as well as immunocytochemistry and Western blotting the identity of the cells could be verified. RT-PCR of the primary cells showed mRNA for the GR in all three cell types of the canine cornea. Furthermore, stimulation with LPS led to an increased PGE(2)-production in epithelial cells and fibroblasts, which was significant for epithelial cells. The PGE(2)-concentration was decreased in a dose dependent manner by the addition of dexamethasone. CONCLUSION: The three major cell types of the canine cornea (i.e. endothelium, keratocytes, and epithelium) can be isolated and cultured in vitro. The mRNA for the GR is shown in all three cell types, its functionality is demonstrated by the dose dependent reduction of PGE(2)-production following dexamethasone treatment.