Repeated oral administration of a cathepsin K inhibitor significantly suppresses bone resorption in exercising horses with evidence of increased bone formation and maintained bone turnover.

Our investigations evaluated the effect of VEL-0230, a highly specific irreversible inhibitor of cathepsin K (CatK). The objectives of our study were to determine whether repeated dosing of a CatK inhibitor (CatKI) produced a desired inhibition of the bone resorption biomarker (CTX-1), and document the effect of repeated dosing on bone homeostasis, structure, and dynamics of bone resorption and formation in horses. Twelve young exercising horses were randomized in a prospective, controlled clinical trial and received 4 weekly doses of a CatKI or vehicle. Baseline and poststudy nuclear scintigraphy, blood sampling and analysis of plasma bone biomarkers (CTX-1 and osteocalcin), poststudy bone fluorescent labeling, and bone biopsy were performed. Bone specimens were further processed for microcomputed tomography and bone histomorphometry. Each dose of this CatKI transiently inhibited plasma CTX-1 (reflecting inhibition of bone collagen resorption) and increased bone plasma osteocalcin concentrations, with no detectable adverse effect on normal bone turnover in the face of exercise. Bone morphology, density, and formation rate were not different between control and treated group. Further investigation of CatK inhibition in abnormal bone turnover is required in animals with bone diseases.

Inflammatory effects of autologous, genetically modified autologous, allogeneic, and xenogeneic mesenchymal stem cells after intra-articular injection in horses.

OBJECTIVES: To compare the clinical and inflammatory joint responses to intra-articular injection of bone marrow-derived mesenchymal stem cells (MSC) including autologous, genetically modified autologous, allogeneic, or xenogeneic cells in horses. METHODS: Six five-year-old Thoroughbred mares had one fetlock joint injected with Gey's balanced salt solution as the vehicle control. Each fetlock joint of each horse was subsequently injected with 15 million MSC from the described MSC groups, and were assessed for 28 days for clinical and inflammatory parameters representing synovitis, joint swelling, and pain. RESULTS: There were not any significant differences between autologous and genetically modified autologous MSC for synovial fluid total nucleated cell count, total protein, interleukin (IL)-6, IL-10, fetlock circumference, oedema score, pain-free range-of-motion, and soluble gene products that were detected for at least two days. Allogeneic and xenogeneic MSC produced a greater increase in peak of inflammation at 24 hours than either autologous MSC group. CLINICAL SIGNIFICANCE: Genetically engineered MSC can act as vehicles to deliver gene products to the joint; further investigation into the therapeutic potential of this cell therapy is warranted. Intra-articular MSC injection resulted in a moderate acute inflammatory joint response that was greater for allogeneic and xenogeneic MSC than autologous MSC. Clinical management of this response may minimize this effect.

The value of biomedical research training for veterinary anatomic and clinical pathologists.

Veterinary pathologists traditionally have been actively engaged in research as principal investigators and as collaborators. Pathologists frequently obtain advanced training in research; however, it appears that in the last 10 years there has been a reversal of a previous trend toward increasing numbers of pathologists obtaining PhD degrees. This has arisen despite an established shortage of veterinarians engaged in research. This article evaluates the benefits of research training for individual pathologists, including a wide spectrum of professional opportunities and additional skill development beyond that usually provided by diagnostic pathology training alone. Various training models are discussed, including combined and sequential diagnostic residency and research degree training as well as the nondegree research fellowship programs more commonly pursued in human medicine. Best-practice recommendations for program infrastructure, mentorship, time management, and a team approach to research and research training are advocated to facilitate the development of successful programs and to encourage a continued emphasis on integrated training for pathologists as both clinical diagnosticians and experimentalists. This article is intended to help prospective and active pathology trainees, their mentors, and educational administrators optimize opportunities to ensure the future vitality of veterinary pathologists, and their contributions, in basic and applied research.

Gamma-glutamyl transferase expression in higher-grade astrocytic glioma.

Increased expression of gamma-glutamyltransferase (GGT) has been detected in a range of human malignancies and is thought to be involved in neoplastic proliferation and treatment resistance. Since GGT expression and its role in malignant glioma biology remain largely unknown, we investigated this phenomenon by immunostaining 26 higher-grade human astrocytic gliomas (WHO grades III and IV) with a monoclonal anti-GGT-antibody (138H11). Further, human pancreatic GGT cDNA was used for liposome-mediated transfection of 9L gliosarcoma cells. GGT-expressing and control 9L cells were cultured in media containing different amounts of essential amino acids and/or cytotoxic agents. Cell viability was evaluated by microplate MTT assay. Immunohistochemical staining of tumor specimens demonstrated that GGT expression is a frequent feature of higher-grade human astrocytic gliomas, but not of normal brain tissue. Human tumors were strongly GGT-positive in 6 of 7 cases of grade III astrocytoma, and in 12 of 19 grade IV astrocytoma (glioblastoma multiforme, GBM) cases. In the cell culture model, 9L-GGT cells had a growth advantage over control cells in cysteine-deficient medium. but not in standard or glutamine-free medium. No significant difference in numbers of viable cells of either clone was found in media containing the alkylating drug BCNU (5-200 microg/ml). In conclusion, GGT is expressed in a high percentage of human WHO grade III astrocytomas and GBM, but not in normal brain tissue. This molecule seems to give neoplastic cells a moderate growth advantage under in vivo conditions.

Acivicin induces apoptosis independently of gamma-glutamyltranspeptidase activity.

Inhibition of cellular gamma-glutamyltranspeptidase (GGT) enzyme activity by its specific inhibitor acivicin is frequently used in studies aimed at demonstrating the physiological role of this enzyme. However, because acivicin is a glutamine antagonist, it also inhibits many other glutamine-dependent enzymes involved in purine and pyrimidine biosynthesis. The objective of the present work is to determine whether acivicin exhibits apoptotic properties and the significance of GGT activity level in the response to acivicin treatment. We compared acivicin (0-150 microM) effect on V79 cell lines expressing or not expressing human GGT. Apoptosis was assayed by annexin-V staining, cell cycle analysis, and caspase activation using flow cytometry. We found that acivicin causes a dose- and time-dependent apoptosis in the GGT-negative V79 cell line as well as in its GGT-positive counterpart line. This is the evidence that acivicin induces apoptosis in V79 cell independently of their GGT activity level.

Notification of Papanicolaou smear results: a survey of women's experiences and preferred means of notification.

In a population of Australian women attending a colposcopy clinic, we aimed to establish the method by which they were notified of their Pap smear result, who notified them of their result, and if given the choice, how they would prefer to be notified. Of women attending a colposcopy clinic between October 1998 and June 1999, 59.7% participated in the survey. There was a final sample of 210 women after exclusion of questionnaires with missing data. The doctor most often notified women of their Pap smear result (81.7%). The usual methods of notification were by a follow-up appointment (43.1%), by a telephone call (35.8%) or with a letter (13.8%); 42% of women preferred to be notified with a mailed copy of the report and explanation of follow-up; 29.6% preferred to be telephoned with the result, and 11.3% would prefer a follow-up appointment. Women preferred methods of notification which were initiated by the doctor. Women have varying preferences, which commonly differ from the current services provided by both GPs and colposcopy clinic. To overcome this, women could be asked to nominate their preferred method of notification when completing the cervical smear request form.

Regulation of transcription of the glutathione S-transferase P1 gene by methylation of the minimal promoter in human leukemia cells.

To study the relationship between methylation and the transcriptional activity of the minimal promoter of the glutathione S-transferase GSTP1 gene encoding glutathione S-transferase P1-1, GSTP1 mRNA levels as well as basal promoter activity were compared in human leukemia cell lines. The K562 erythroleukemia cell line presented a strong GSTP1 promoter activity, as measured in transient transfection assays using a luciferase reporter plasmid, and correlated with a high mRNA whereas in Raji cells no mRNA was expressed. In order to establish a relationship between the expression and the methylation status, we used in vitro bisulfite sequencing which indicated that both methylated and unmethylated GSTP1 promoter alleles coexisted in K562 cells, whereas Raji lymphoma cells showed a nearly uniform hypermethylation of the promoter region. To determine the impact of methylation, we used in vitro SssI methylation of the minimal GSTP1 promoter, which led to the silencing of the promoter activity in transient transfection assays in expressing K562 as well as in non-expressing Raji cells. These data are in good agreement with previously obtained results and indicate that methylation of CpG sites of the basal promoter is an essential mechanism in the control of GSTP1 gene expression in human leukemia.

Structure of the 5' sequences of the human gamma-glutamyltransferase gene.

In humans, five distinct mRNAs code for gamma-glutamyltransferase (GGT). Their coding regions are identical and their 5' untranslated regions exhibit both common and type-specific sequences. To elucidate the mecanisms that generate these different mRNAs, we cloned and determined the structure of the 5' region of the human GGT gene. The common regions of the 5' UTR are encoded by five exons, localized within a 2.4-kb region of the genomic DNA. Three of them are separated only by intron-donor or intron-acceptor sites at their boundaries. Alternative splicing of these exons may determine the unique pattern of the different GGT mRNA 5' UTRs in a tissue-specific manner. In addition, we have isolated a genomic fragment containing the most distal 5' sequences of the major GGT mRNA in HepG2 cells. Primer extension analysis revealed one major transcription initiation site while 5' RACE indicated that one more distal initiation site could be present. In the putative promoter sequence neither classical TATA or CAAT boxes were found. However, sites for AP1, AP2, CREB, GRE and SP1 transcription factors were identified. Chimeric plasmids, containing this genomic region fused to the luciferase gene, were transiently expressed in three cell lines of different origin: HeLa cells, ovarian carcinoma A2780 cells and V79 lung fibroblasts. The significant promoter activities obtained indicate a transcription start within this region. However, differences in the level of expression were found between the different cell lines used. These data suggest that the human GGT gene employs regulatory sequences and alternative splicing, and gene expression may therefore be regulated in tissue specific and cell-type-specific manners.

Phorbol ester responsiveness of the glutathione S-transferase P1 gene promoter involves an inducible c-jun binding in human K562 leukemia cells.

Overexpression of the glutathione S-transferase P1 (GSTP1) gene is related to drug resistance in human cancer cells. However, the mechanisms of the transcriptional activation of this gene remain unclear. In this study, we examined the molecular mechanisms underlying phorbol ester mediated gene regulation using human K562 leukemia cells as a model. Promoter deletion analyses revealed that the activator protein-1 (AP-1) transcription factor site was crucial for 12-O-tetradecanoyl phorbol 13-acetate (TPA)-mediated GSTP1 gene transcription. Electrophoretic mobility shift assays and transient transfection analysis demonstrated that both DNA binding and transactivation activities of AP-1 were induced by TPA. By supershift analysis, we identified transcription factors c-jun and fra-1 as well as NF-E2p45 as components of the induced binding complex. These results show for the first time that the phorbol ester TPA is involved in the molecular mechanism(s) mediating the activation of the GSTP1 promoter in a human leukemia model.

Evidence for the pro-oxidant effect of gamma-glutamyltranspeptidase-related enzyme.

It has been previously reported that the metabolism of reduced glutathione (GSH) by gamma-glutamyltranspeptidase (GGT) in the presence of chelated metals leads to free radical generation and lipid peroxidation (LPO). The present study demonstrates for the first time that an established cell line expressing GGT-rel, a human GGT-related enzyme, metabolizes extracellular GSH to cysteinylglycine (CysGly) in a time-dependent manner when cells were incubated in a medium containing 2.5 mM GSH and 25 mM glycylglycine. Supplementation with 150-165 microM Fe(3+)-EDTA resulted in a reactive oxygen species (ROS) generation process. The resulting data showed a significantly higher level (7.6-fold) of ROS production in the GGT-rel positive cells in comparison with the GGT-rel negative control cells. CysGly and Cys, but not GSH, were responsible for the observed ROS production, as we confirmed by measuring the same process in the presence of Fe(3+)-EDTA and different thiols. A higher iron reduction and an increased LPO level determined by malondialdehyde HPLC measurement were also found in GGT-rel-overexpressing cells compared to GGT-rel negative cells. Our data clearly indicate that in the presence of iron, not only GGT, but also GGT-rel has a pro-oxidant function by generation of a reactive metabolite (CysGly) and must be taken into account as a potential physiopathological oxidation system.

Gamma-glutamyltranspeptidase-dependent glutathione catabolism results in activation of NF-kB.

gamma-glutamyltranspeptidase (GGT) is a key enzyme implicated in the homeostasis of intracellular reduced glutathione (GSH) and hence in the regulation of the cellular redox state. Besides, the extracellular cleavage of GSH by GGT leads to reactive oxygen species (ROS) production, depending on the generation and enhanced reactivity of cysteinylglycine (CysGly). Using a model cell line, the V79 GGT, which highly expresses a human GGT transgene, we examined whether the GGT induced oxidant stress could modulate intracellular transcription factors. For the first time, we show that GGT-dependent ROS production induces the NF-kB-binding and transactivation activities. This induction mimicked the one observed by H(2)O(2) and was inhibited by catalase, suggesting the involvement of H(2)O(2) in the NF-kB activation.

Regulation of gamma-glutamyltransferase in cisplatin-resistant and -sensitive colon carcinoma cells after acute cisplatin and oxidative stress exposures.

Glutathione plays an important role in drug resistance of tumor cells and in their ability to resist oxidative stress. Improved salvage of glutathione can be obtained through increased activity of gamma-glutamyltransferase (GGT), which is of importance in the maintenance of cellular glutathione homeostasis. We investigated the regulation of GGT in 2 cisplatin-resistant and 1 cisplatin-sensitive colon carcinoma cell lines. Enzyme activity was induced in all 3 cell lines after acute exposure to cisplatin. The elevation was significantly higher in sensitive cells (3.3-fold) than in resistant (1.6- to 1.7-fold) cells. Exposure of cells to oxidative stress generated by menadione also resulted in enzyme induction but only in cisplatin-sensitive cells. Addition of anti-oxidants had different effects on the 2 inductions: N-acetylcysteine blocked the induction of both cisplatin and menadione, whereas catalase and glutathione-ester blocked only the menadione induction. Glutathione depletion alone was not sufficient to induce GGT in these cells. The data show that GGT is regulated by multiple mechanisms during anti-tumor drug treatment and oxidative stress and that reactive oxygen species were involved in the menadione, but not cisplatin, induction of the enzyme.

Leukoerythroblastosis and normoblastemia in the cat.

Over a six-month period, 6% of 313 cats evaluated hematologically had either leukoerythroblastosis or normoblastemia. Diseases associated with these hematological conditions included haemobartonellosis, hepatic lipidosis, trauma, viral and bacterial infections, myeloproliferative disorders, and hemangiosarcoma. The finding of leukoerythroblastosis or normoblastemia may aid in diagnosing cats presenting with nonspecific signs.

Simultaneous measurement of reactive oxygen species and reduced glutathione using capillary electrophoresis and laser-induced fluorescence detection in cultured cell lines.

A capillary zone electrophoretic (CZE) method coupled with laser-induced fluorescence (LIF) was developed for the simultaneous determination of two important intracellular parameters related to oxidative stress (i.e. reactive oxygen species, ROS, and reduced glutathione, GSH). This rapid and sensitive method was applied to the study of oxidative stress in cultured V79 fibroblast cells. The fluorogenic reagents selected were: (i) dihydrorhodamine-123 (DHR-123) which is converted intracellularly by ROS to the fluorescent rhodamine-123 dye (Rh-123), and (ii) naphthalene-2,3-dicarboxaldehyde (NDA), which reacts quickly with GSH in cell extracts to produce a fluorescent adduct. Separation of Rh-123, GSH-NDA and gamma-glutamylcysteine-NDA adducts was performed using an uncoated fused-silica capillary and a 100 mM borate buffer, pH 9.2, at 20 degrees C and at an applied voltage of 25 kV; LIF detection was operated using an argon laser. The cell line was also tested for its ability to alleviate oxidative stress induced by tert-butylhydroperoxide (t-BuOOH). Exposure to t-BuOOH (up to 3 mm for 2 h) did not affect the intracellular ROS and GSH concentrations. At higher (4-10 mM) t-BuOOH concentrations, an inverse relationship between the concentrations of ROS and GSH was obtained, showing that the present method can readily evaluate the gradual consumption of the primary cellular scavenger of ROS which occurs simultaneously with the increase of oxidative insult.

The role of dehydroepiandrosterone in diabetes mellitus.

Much has been written in the lay literature regarding potential benefits of dehydroepiandrosterone (DHEA). Although it was removed from the over-the-counter market in 1985, the Dietary Supplement Health Education Act of 1994 allowed the drug to be marketed as a food supplement. Because DHEA no longer falls under the scrutiny of the Food and Drug Administration, many unverified claims have been put forth in the press espousing its therapeutic value. This barrage of "infomercials" has left the average American consumer (and health care professional) curious about DHEA and its possible therapeutic utility. One focus of recent research is to define the role of DHEA in diabetes mellitus. Although it has been claimed that decreased levels of endogenous DHEA are associated with diabetes, impaired glucose tolerance, hyperglycemia, and insulin resistance, much of the information is based on cross-sectional studies. Other claims correlate decreased endogenous DHEA levels with adverse cardiovascular effects. Some information is contradictory and indicates high doses of exogenous DHEA may produce adverse cardiovascular effects, an undesirable outcome in patients with diabetes mellitus. At this time, its administration in patients with diabetes is not warranted. Long-term trials evaluating the role of exogenous DHEA and its effect on patients with diabetes should be conducted.

Determination of cellular thiols and glutathione-related enzyme activities: versatility of high-performance liquid chromatography-spectrofluorimetric detection.

A high-performance liquid chromatography (HPLC) method to determine the most important cellular thiols [reduced glutathione (GSH), cysteine, gamma-glutamylcysteine and cysteinylglycine] is described. Separation relies upon isocratic ion-pairing reversed-phase chromatography and detection is operated by spectrofluorimetry coupled with post-column derivatization reactions using either N-(1-pyrenyl)maleimide (NPM) or ortho-phthalaldehyde (OPA). When OPA is used without co-reagent, only GSH and gamma-glutamylcysteine are detected (heterobifunctional reaction). However, either the OPA reaction in the presence of glycine in the mobile phase (thiol-selective reaction) or NPM allows the detection of all the cited thiols. The HPLC system has been validated as concerning linearity, accuracy and precision. The low detection limits reached (in the pmol range for each thiol injected) allow the screening and the quantification of thiols (as NPM derivatives) in V79cl and V79HGGT cells as well as the measurement of two cytosolic enzymes related to the glutathione synthesis, using the heterobifunctional OPA reaction.

5-Azacytidine modulates gamma-glutamyltransferase and glutathione levels in two human prostatic adenocarcinoma cell lines.

Two human prostate adenocarcinoma cell lines, LNCaP and PC-3, were used to study the effect of 5-azacytidine on GGT gene expression, via genomic DNA methylation, and GSH content. When the cells were treated with 5-azaC, the specific GGT activity increased in a dose and time-dependent manner and was accompanied by the elevation of intracellular glutathione content. Southern blot analysis of DNA digested with MspI or HpaII showed negative correlation between the methylation pattern of GGT DNA and GGT activity, either in control or 5-azaC treated cells. Reverse transcription-polymerase chain reaction (RT-PCR) analysis revealed that GGT mRNA types I are expressed and induced in a cell type-specific manner in control and 5-azacC treated cells respectively. Overall, our investigations suggest that DNA methylation and other mechanisms combine to regulate GGT gene expression in studied prostate adenocarcinoma derived cells.