The fine needle aspiration of translocation sarcomas.

INTRODUCTION: Soft tissue sarcomas comprise a heterogeneous group of clinically aggressive cancers that are often hard to classify on limited cytological samples. "Translocation sarcomas" (TS) are a diverse subset of such cancers, different from pleomorphic sarcomas, and characterised by unique single chromosomal translocations in each sarcoma subtype. Interestingly, despite their high-grade biological behaviour, TS have deceptively monotonous and bland cytomorphology, therefore creating diagnostic issues on limited samples. MATERIALS AND METHODS: A retrospective search was conducted of the cytopathology archives of The Johns Hopkins Hospital revealing 147 translocation sarcoma cases over a 25-year period. RESULTS: The common morphological denominators for most translocation sarcomas were: hypercellularity, cellular monotony, mostly discohesive and single cells, round-to-oval or short spindled cells and a lack of necrosis. The exceptions were an inflammatory myofibroblastic tumour, in which cellular monotony was not present owing to the prominence of lymphocytes and plasma cells, and low-grade fibromyxoid sarcoma, in which the specimens were generally hypocellular. Ancillary testing, especially immunoperoxidase staining, was often required for primary lesions. CONCLUSION: Distinct morphological clues and subsequent ancillary testing (particularly immunoperoxidase staining) provide an accurate diagnosis on cytological interpretation of both, primary and recurrent/metastatic lesions.

Cellular prion protein (PrPC) in the development of Merlin-deficient tumours.

Loss of function mutations in the neurofibromatosis Type 2 (NF2) gene, coding for a tumour suppressor, Merlin, cause multiple tumours of the nervous system such as schwannomas, meningiomas and ependymomas. These tumours may occur sporadically or as part of the hereditary condition neurofibromatosis Type 2 (NF2). Current treatment is confined to (radio) surgery and no targeted drug therapies exist. NF2 mutations and/or Merlin inactivation are also seen in other cancers including some mesothelioma, breast cancer, colorectal carcinoma, melanoma and glioblastoma. To study the relationship between Merlin deficiency and tumourigenesis, we have developed an in vitro model comprising human primary schwannoma cells, the most common Merlin-deficient tumour and the hallmark for NF2. Using this model, we show increased expression of cellular prion protein (PrPC) in schwannoma cells and tissues. In addition, a strong overexpression of PrPC is observed in human Merlin-deficient mesothelioma cell line TRA and in human Merlin-deficient meningiomas. PrPC contributes to increased proliferation, cell-matrix adhesion and survival in schwannoma cells acting via 37/67 kDa non-integrin laminin receptor (LR/37/67 kDa) and downstream ERK1/2, PI3K/AKT and FAK signalling pathways. PrPC protein is also strongly released from schwannoma cells via exosomes and as a free peptide suggesting that it may act in an autocrine and/or paracrine manner. We suggest that PrPC and its interactor, LR/37/67 kDa, could be potential therapeutic targets for schwannomas and other Merlin-deficient tumours.

Cue-conditioned alcohol seeking in rats following abstinence: involvement of metabotropic glutamate 5 receptors.

BACKGROUND AND PURPOSE: The current study was designed to: (i) examine whether functional interactions occur between receptors known to regulate alcohol self-administration; and (ii) characterize relapse to alcohol seeking following abstinence. EXPERIMENTAL APPROACH: The selective cannabinoid CB(1) receptor antagonist SR141716A (0.03-1.0 mg.kg(-1) i.p.) resulted in a dose-dependent reduction in ethanol self-administration in ethanol-preferring Indiana-preferring rats. SR141716A was then co-administered with either the selective glutamate metabotropic glutamate 5 (mGlu(5)) receptor antagonist 3-[(2-methyl-1,3-thiazol-4-yl)ethynyl]pyridine (MTEP) or the selective adenosine A(2A) receptor antagonist SCH58261. KEY RESULTS: When administered at individually sub-threshold doses, a combination of SR141716A (0.1 mg.kg(-1)) and SCH58261 (0.5 mg.kg(-1) i.p.) produced a reduction (28%) in ethanol self-administration. Combinations of threshold doses of SR141716A (0.3 mg.kg(-1)) and SCH58261 (2.0 mg.kg(-1), i.p.) caused an essentially additive reduction (68%) in alcohol self-administration. A combination of individually sub-threshold doses of CB(1) and mGlu(5) receptor antagonists did not affect alcohol self-administration; however, combined threshold doses of SR141716A (0.3 mg.kg(-1)) and MTEP (1.0 mg.kg(-1) i.p.) did reduce ethanol self-administration markedly (80%). Cue-conditioned alcohol seeking was attenuated by pretreatment with MTEP (1.0 mg.kg(-1)) co-administered with SR141716A (0.3 mg.kg(-1) i.p.). In contrast, SCH58261 (2.0 mg.kg(-1)) co-administered with SR141716A (0.3 mg.kg(-1) i.p.) did not reduce cue-conditioned alcohol seeking. CONCLUSIONS AND IMPLICATIONS: Adenosine A(2A) and cannabinoid CB(1) receptors regulated alcohol self-administration additively, but combined low-dose antagonism of these receptors did not prevent cue-conditioned alcohol seeking after abstinence. In contrast, combined low-dose antagonism of mGlu(5) and CB(1) receptors did prevent relapse-like alcohol seeking after abstinence, suggesting a prominent role for mGlu(5) receptors in this paradigm.

Survey of veterinary technical and professional skills in students and recent graduates of a veterinary college.

OBJECTIVES: To determine perceptions of veterinary technical and professional skills among veterinary students and recent graduates. DESIGN: Cross-sectional study. SAMPLE POPULATION: 281 students and 142 recent graduates from the Ontario Veterinary College. PROCEDURE: A survey was designed and administered to first- through fourth-year students and veterinarians who had graduated either 1 or 6 years before survey administration. RESULTS: Overall response rate was 70%. Learning about technical and professional skills was highly valued. Most participants felt they had not received instruction about professional skills, but those who had felt more competent about them. Perceptions of competence increased slightly with increased comfort discussing emotional veterinary issues with instructors. Neither gender nor increased age was related to increased feelings of competence. Almost all fourth-year students felt competent and comfortable about examining an animal with the client present, assessing suffering, diagnosing parvovirus infection, performing surgery, and working as group members. However, many did not feel competent or comfortable about delivering bad news, setting time limits yet providing quality service, helping clients with limited funds make treatment decisions, dealing with demanding people, and euthanasia. Feelings of competence and comfort were closely related but were not identical. CONCLUSIONS AND CLINICAL RELEVANCE: In the interests of best preparing entry-level veterinarians, technical and professional skills need to be emphasized in a learning environment where students feel comfortable discussing emotional veterinary issues. A professional skills curriculum addressing underlying self-awareness, communication, and interpersonal issues, as well as procedural matters, would likely increase the proportion of fourth-year students who feel competent and comfortable about professional skills by the end of their undergraduate training.

Cytomechanics of cadherin-mediated cell-cell adhesion.

Cadherin-mediated adhesion regulates transitions from initial cell-cell recognition to loosely adherent cell clusters and ultimately, to strongly compacted groups of cells in colonies. Recent studies have described distinct roles for intermolecular clustering of cadherins as well as interactions of cadherin with the actin cytoskeleton in establishing cell-cell adhesion. Integrating cytomechanical roles of cadherin-mediated adhesion will lead to a greater understanding of how cadherins regulate tissue morphogenesis.

Quantitative analysis of cadherin-catenin-actin reorganization during development of cell-cell adhesion.

Epithelial cell-cell adhesion requires interactions between opposing extracellular domains of E-cadherin, and among the cytoplasmic domain of E-cadherin, catenins, and actin cytoskeleton. Little is known about how the cadherin-catenin-actin complex is assembled upon cell-cell contact, or how these complexes initiate and strengthen adhesion. We have used time-lapse differential interference contrast (DIC) imaging to observe the development of cell-cell contacts, and quantitative retrospective immunocytochemistry to measure recruitment of proteins to those contacts. We show that E-cadherin, alpha-catenin, and beta-catenin, but not plakoglobin, coassemble into Triton X-100 insoluble (TX-insoluble) structures at cell-cell contacts with kinetics similar to those for strengthening of E-cadherin-mediated cell adhesion (Angres, B., A. Barth, and W.J. Nelson. 1996. J. Cell Biol. 134:549-557). TX-insoluble E-cadherin, alpha-catenin, and beta-catenin colocalize along cell-cell contacts in spatially discrete micro-domains which we designate "puncta," and the relative amounts of each protein in each punctum increase proportionally. As the length of the contact increases, the number of puncta increases proportionally along the contact and each punctum is associated with a bundle of actin filaments. These results indicate that localized clustering of E-cadherin/catenin complexes into puncta and their association with actin is involved in initiating cell contacts. Subsequently, the spatial ordering of additional puncta along the contact may be involved in zippering membranes together, resulting in rapid strengthening of adhesion.

The adenomatous polyposis coli tumor suppressor protein localizes to plasma membrane sites involved in active cell migration.

Mutations in the adenomatous polyposis coli (APC) gene are linked to polyp formation in familial and sporadic colon cancer, but the functions of the protein are not known. We show that APC protein localizes mainly to clusters of puncta near the ends of microtubules that extend into actively migrating regions of epithelial cell membranes. This subcellular distribution of APC protein requires microtubules, but not actin filaments. APC protein-containing membranes are actively involved in cell migration in response to wounding epithelial monolayers, addition of the motorgen hepatocyte growth factor, and during the formation of cell-cell contacts. In the intestine, APC protein levels increase at the crypt/villus boundary, where cell migration is crucial for enterocyte exit from the crypt and where cells accumulate during polyp formation that is linked to mutations in the microtubule-binding domain of APC protein. Together, these data indicate that APC protein has a role in directed cell migration.

The use of light-directed combinatorial peptide synthesis in epitope mapping.

The application of light-directed combinatorial peptide synthesis to epitope mapping is described. Photolithography and solid phase peptide synthesis were combined in an automated fashion to assemble arrays containing 1024 peptide sequences on a glass support in ten steps with the precise location of each peptide known. The simultaneous synthesis of two slides containing three arrays of peptides each allowed for the independent screening of both a monoclonal antibody (mAb) and its Fab fragment at two different concentrations. A binary synthesis strategy was used to assemble the arrays, resulting in all deletions and truncations possible within the FLRRQFKVVT sequence being present and available for screening. The relative binding interactions of each peptide was determined by incubating the arrays with either mAb D32.39 and goat antimouse immunoglobulin G-FITC or mAb D32.39 Fab-FITC conjugate, followed by scanning the surface for fluorescence with an epifluorescence microscope. The fragment RQFKVVT was found to bind tightly to both the mAb and Fab fragment while tethered to the surface, and was measured to have 0.49 nM affinity in solution. The frame-shifted RRQFKVV sequence was found to have lower affinity both in solution (1.3 mM) and on the surface. The fragment RQFKVV was determined to be responsible for antibody recognition and was found to bind tightly when tethered to the surface, yet exhibited no binding in solution as the free acid, suggesting the requirement of an amidated C-terminus or an additional flanking residue. A deletion analysis revealed that the novel RQFKVT sequence exhibited higher affinity than the RQFKVV sequence while tethered to the surface.

Evaluation of swinepox virus as a vaccine vector in pigs using an Aujeszky's disease (pseudorabies) virus gene insert coding for glycoproteins gp50 and gp63.

Pigs were vaccinated by scarification or intramuscular injection with a swinepox virus-Aujeszky's disease (pseudorabies) recombinant (rSPV-AD) constructed by inserting the linked Aujeszky's disease virus genes coding for glycoproteins gp50 and gp63, attached to a vaccinia virus p7.5 promoter, into the thymidine kinase gene of swinepox virus. By 21 days after vaccination, 90 and 100 per cent of the animals vaccinated by scarification or intramuscular injection, respectively, had developed serum neutralising antibodies to Aujeszky's disease virus. Upon challenge with virulent virus, significantly fewer vaccinated pigs developed clinical Aujeszky's disease, nasal shedding of challenge virus was markedly reduced, and the vaccinated groups of pigs maintained or gained weight during the week after challenge whereas the unvaccinated control group lost weight. No transmission of rSPV-AD to in-contact controls was detected during the three weeks before challenge. In a second experiment, serum neutralising antibodies to Aujeszky's disease virus persisted for 150 days after the pigs were vaccinated with rSPV-AD by scarification or intramuscular injection and all the pigs showed an anamnestic response when they were revaccinated.

Multiplexed biochemical assays with biological chips.

High density peptide and oligonucleotide chips are fabricated using semiconductor-based technologies. These chips have a variety of biological applications.