Exploring the longitudinal dynamics of herd BVD antibody test results using model-based clustering.

Determining the Bovine Viral Diarrhoea (BVD) infection status of cattle herds is a challenge for control and eradication schemes. Given the changing dynamics of BVD  virus (BVDV) antibody responses in cattle, classifying herds based on longitudinal changes in the results of BVDV antibody tests could offer a novel, complementary approach to categorising herds that is less likely than the present system to result in a herd's status changing from year to year, as it is more likely to capture the true exposure dynamics of the farms. This paper describes the dynamics of BVDV antibody test values (measured as percentage positivity (PP)) obtained from 15,500 bovines between 2007 and 2010 from thirty nine cattle herds located in Scotland and Northern England. It explores approaches of classifying herds based on trend, magnitude and shape of their antibody PP trajectories and investigates the epidemiological similarities between farms within the same cluster. Gaussian mixture models were used for the magnitude and shape clustering. Epidemiologically meaningful clusters were obtained. Farm cluster membership depends on clustering approach used. Moderate concordance was found between the shape and magnitude clusters. These methods hold potential for application to enhance control efforts for BVD and other infectious livestock diseases.

Quasi-Isotropic and Pseudo-Ductile Highly Aligned Discontinuous Fibre Composites Manufactured with the HiPerDiF (High Performance Discontinuous Fibre) Technology.

Conventional composite materials reinforced with continuous fibres display high specific strength but have a number of drawbacks including: the elastic-brittle behaviour, difficulties in producing defect-free components of complex shape with high-volume automated manufacturing processes, and inherent lack of recyclability. Highly aligned, discontinuous fibre-reinforced composites (ADFRCs) are truly beneficial for mass production applications, with the potential to offer better formability and comparable mechanical properties with continuous fibre-reinforced composites. In previous publications, the High Performance Discontinuous Fibre (HiPerDiF) technology has been shown to offer the possibility to intimately hybridise different types of fibres, to achieve pseudo-ductile tensile behaviour, and remanufacture reclaimed fibres into high-performance recycled composites. However, to date, the work has been conducted with unidirectional (UD) laminates, which is of limited interest in engineering applications with mechanical stresses acting across many directions; this paper reports, for the first time, the mechanical behaviour of quasi-isotropic (QI) ADFRCs. When compared with randomly-oriented discontinuous fibre composites (RODFRCs), QI ADFRCs offer enhanced stiffness (+26%) and strength (+77%) with higher consistency, i.e., a reduction of the coefficient of variance from the 25% of RODFRCs to the 6% of ADFRCs. Furthermore, hybrid QI ADFRCs retain the pseudo-ductility tensile behaviour previously observed in unidirectional (UD) lay-up.

A cross-sectional study of 329 farms in England to identify risk factors for ovine clinical mastitis.

The aims of this study were to estimate the incidence rate of clinical mastitis (IRCM) and identify risk factors for clinical mastitis in suckler ewes to generate hypotheses for future study. A postal questionnaire was sent to 999 randomly selected English sheep farmers in 2010 to gather data on farmer reported IRCM and flock management practices for the calendar year 2009, of which 329 provided usable information. The mean IRCM per flock was 1.2/100 ewes/year (CI:1.10:1.35). The IRCM was 2.0, 0.9 and 1.3/100 ewes/year for flocks that lambed indoors, outdoors and a combination of both, respectively. Farmers ran a variety of managements before, during and after lambing that were not comparable within one model, therefore six mixed effects over-dispersed Poisson regression models were developed. Factors significantly associated with increased IRCM were increasing percentage of the flock with poor udder conformation, increasing mean number of lambs reared/ewe and when some or all ewes lambed in barns compared with outdoors (Model 1). For ewes housed in barns before lambing (Model 2), concrete, earth and other materials were associated with an increase in IRCM compared with hardcore floors (an aggregate of broken bricks and stones). For ewes in barns during lambing (Model 3), an increase in IRCM was associated with concrete compared with hardcore flooring and where bedding was stored covered outdoors or in a building compared with bedding stored outdoors uncovered. For ewes in barns after lambing (Model 4), increased IRCM was associated with earth compared with hardcore floors, and when fresh bedding was added once per week compared with at a frequency of ≤2 days or twice/week. The IRCM was lower for flocks where some or all ewes remained in the same fields before, during and after lambing compared with flocks that did not (Model 5). Where ewes and lambs were turned outdoors after lambing (Model 6), the IRCM increased as the age of the oldest lambs at turnout increased. We conclude that the reported IRCM is low but highly variable and that the complexity of management of sheep around lambing limits the insight into generating hypotheses at flock level for risks for clinical mastitis across the whole industry. Whilst indoor production was generally associated with an increased IRCM, for ewes with large litter size indoor lambing was protective, we hypothesise that this is possibly because of better nutrition or reduced exposure to poor weather and factors associated with hygiene.

Collagen-like peptide sequences inhibit bacterial invasion of root dentine.

AIM: To investigate the effects of peptides derived from the sequence of collagen to inhibit penetration of human or bovine dentine by species of streptococci and enterococci. METHODOLOGY: Blocks of human or bovine root dentine were infected for 14 days with bacterial cultures, in the presence or absence of various collagen-like peptide sequences. Invasion of dentinal tubules was determined from microscopic images of histochemically stained dentine thin sections. Extent of invasion was expressed as tubule invasion index (TI), or tubule invasion factor (TIF) which, in addition to the density of invasion, took into account the depth of invasion. Data were analysed by two-way anova. RESULTS: Streptococcus gordonii, Streptococcus mutans and Enterococcus faecalis were associated with heavy invasion (TI >2.5, TIF >4) of human or bovine root dentinal tubules, with E. faecalis being the most penetrative. Incorporation of peptides Gly-Pro-Ala or Gly-Pro-Hyp into the in vitro model system significantly reduced (P < 0.05) dentine invasion by the three species of highly invasive organisms. Inhibition of bacterial invasion by the peptides was dose dependent, and the peptides did not inhibit bacterial growth in culture. CONCLUSION: Specific collagen-like peptide sequences inhibited the invasion of dentine in vitro by a range of oral bacteria. The peptides likely act as competitive inhibitors blocking bacterial collagen receptors and could potentially allow for target-specific control of dentine infections.

A longitudinal study of risk factors for teat lesions in 67 suckler ewes in a single flock in England.

A longitudinal study of 67 suckler ewes on a commercial sheep farm near Wolverhampton, England was carried out from March to July 2010. Data on ewe teat lesions and udder and teat conformation were collected together with data on lamb health and weight at lambing and at 14-day intervals until lambs were 8-10 weeks old. Each ewe was examined on 4-5 occasions. Teat lesions were categorised as traumatic or non-traumatic based on appearance. Risk factors for the development of each type of teat lesion were investigated using mixed effect binomial regression models. The incidence of traumatic teat lesions was greatest 3-4 weeks after lambing with 27.9% new cases by udder half. There was an increased risk of traumatic teat lesions in 2 year old ewes (OR 3.5, CI 1.2, 10.5) compared with 6 year old ewes and in ewes with abnormal compared with normal teat placement, a wide or narrow udder width compared with a normal udder width, a heavy total litter weight, body condition score (BCS)<2.5, compared with BCS of 2.5 14-days previously, and in ewes with abnormal udder skin (waxy, dry or chapped) compared with normal udder skin. The incidence of non-traumatic teat lesions was greatest 7-10 weeks after lambing with 21.4% new cases. Non-traumatic lesions were more likely to occur in ewes with BCS=3 compared with BCS of 2.5. The risk factors for traumatic and non-traumatic teat lesions differ. Traumatic lesions are associated with teat and udder morphology, ewe age and BCS, and lamb growth rate, whereas non-traumatic lesions are associated with ewe BCS.

A cohort study of the associations between udder conformation, milk somatic cell count, and lamb weight in suckler ewes.

A cohort study of 67 suckler ewes from 1 farm was carried out from January to May 2010 to investigate associations between udder conformation, udder half milk somatic cell count (SCC), and lamb weight. Ewes and lambs were observed at lambing. Ewe health and teat condition and lamb health and weight were recorded on 4 to 5 further occasions at 14-d intervals. At each observation, a milk sample was collected from each udder half for somatic cell counting. Two weeks after lambing, ewe udder conformation and teat placement were scored. Low lamb weight was associated with ewe SCC >400,000 cells/mL (-0.73 kg), a new teat lesion 14 d previously (-0.91 kg), suboptimal teat position (-1.38 kg), rearing in a multiple litter (-1.45 kg), presence of diarrhea at the examination (-1.19 kg), and rearing by a 9-yr-old ewe compared with a 6-yr-old ewe (-2.36 kg). High lamb weight was associated with increasing lamb age (0.21 kg/d), increasing birth weight (1.65 kg/kg at birth), and increasing number of days the ewe was given supplementary feed before lambing (0.06 kg/d). High udder half SCC was associated with pendulous udders (9.6% increase in SCC/cm of drop) and greater total cross-sectional area of the teats (7.2% increase of SCC/cm(2)). Low SCC were associated with a heavier mean litter weight (6.7% decrease in SCC/kg). Linear, quadratic, and cubic terms for days in lactation were also significant. We conclude that poor udder and teat conformation are associated with high levels of intramammary infection, as indicated by increased SCC and that both physical attributes of the udder and SCC are linked to lamb growth, suggesting that selection of suckler ewes with better udder and teat conformation would reduce intramammary infection and increase lamb growth rate.

Short communication: Preliminary investigation into the effect of freezing and a cryopreservant on the recovery of mastitis pathogens from ewe milk.

The objective of this study was to investigate the recovery of bacteria from ewe milk after freezing for 4 or 8 wk with and without the addition of glycerol as a cryopreservant. A total of 50 udder-half milk samples with a known range of bacterial species were selected, stored, and analyzed in 5 treatment groups: time zero; frozen for 4 wk with, and without, glycerol; and frozen for 8 wk with, and without, glycerol. A lower recovery was observed in all bacterial species studied after freezing. Samples containing fewer than 100 cfu/mL came from ewes with a lower somatic cell count and were more likely to be bacteriologically negative after freezing than those above this threshold. The addition of glycerol increased recovery of gram-negative bacteria after freezing, although this requires further study to draw strong conclusions. The effects on gram-positive species were inconsistent. We conclude that although the addition of glycerol had a small beneficial effect on the sensitivity of detection of bacteria from frozen sheep milk, sensitivity was highest in cultures from fresh milk.

Aedes aegypti in Jamaica, West Indies: container productivity profiles to inform control strategies.

OBJECTIVE: To describe the Aedes aegypti container profile in the three parishes of Portland, St. Anns and St. Catherine, Jamaica. METHOD: Traditional stegomyia and pupae per person indices. RESULTS: A total of 8855 containers were inspected. A. aegypti were breeding in 19.2% of the 4728 containers in Portland, in 6.7% of the 2639 containers in St. Ann, and in 27.2% of the 1488 containers in Tryhall Heights, St. Catherine. Container types differed between Portland (P > 0.02) on one hand and St. Ann and Tryhall Heights, St. Catherine on the other hand: there were with no vases or potted plants with water saucers in St. Ann and St. Catherine. A. aegypti were breeding in more containers in St. Catherine (38%) (38% in wet season and 21% in the dry season) than in Portland (19%) or St. Ann (6%), both of which had more containers but A. aegypti breeding in fewer: 17.7% and 11.2% in the wet and 20.4% and 3.5% in the dry seasons respectively. The daily production of adult mosquitoes in the three study sites was 1.51, 1.29 and 0.66 adult female mosquitoes per person in Portland, St. Ann and St. Catherine during the dry season and 1.12, 0.23 and 1.04 female mosquitoes per person in the wet season respectively. CONCLUSION: All three communities are at risk for dengue outbreaks and vector control should concentrate on reducing the mosquito populations from the most productive containers before a new dengue virus serotype is introduced into Jamaica.

Aedes aegypti in Jamaica, West Indies: container productivity profiles to inform control strategies

OBJECTIVE: To describe the Aedes aegypti container profile in the three parishes of Portland, St. Anns and St. Catherine, Jamaica.METHOD: Traditional stegomyia and pupae per person indices.RESULTS: A total of 8855 containers were inspected. A. aegypti were breeding in 19.2% of the 4728 containers in Portland, in 6.7% of the 2639 containers in St. Ann, and in 27.2% of the 1488 containers in Tryhall Heights, St. Catherine. Container types differed between Portland (P > 0.02) on one hand and St. Ann and Tryhall Heights, St. Catherine on the other hand: there were with no vases or potted plants with water saucers in St. Ann and St. Catherine. A. aegypti were breeding in more containers in St. Catherine (38%) (38% in wet season and 21% in the dry season) than in Portland (19%) or St. Ann (6%), both of which had more containers but A. aegypti breeding in fewer: 17.7% and 11.2% in the wet and 20.4% and 3.5% in the dry seasons respectively. The daily production of adult mosquitoes in the three study sites was 1.51, 1.29 and 0.66 adult female mosquitoes per person in Portland, St. Ann and St. Catherine during the dry season and 1.12, 0.23 and 1.04 female mosquitoes per person in the wet season respectively.CONCLUSION: All three communities are at risk for dengue outbreaks and vector control should concentrate on reducing the mosquito populations from the most productive containers before a new dengue virus serotype is introduced into Jamaica.

Survey of the prevalence, diagnosis and treatment of dermatological conditions in small animals in general practice.

A survey was made of the prevalence, diagnosis and treatment of dermatological conditions in small animals in general practice in the UK. Out of 3707 small animal consultations in general practice that were observed and recorded, 795 (21.4 per cent) involved animals that had a dermatological problem. In dogs and exotic species, pruritus was the most common presenting sign, accounting for 30 to 40 per cent of the dermatological consultations. In cats, cutaneous swellings were the most common presentation (36 per cent). A diagnosis or recommendation for treatment was made on the basis of the presenting clinical signs and physical examination alone in 576 (72 per cent) of the cases, and various diagnostic tests were performed in the other cases. In dogs, parasitic infestations, bacterial infections and neoplasia accounted for the majority of the diagnoses. In cats, parasites and bacterial infections were the most common. In exotic species, parasites accounted for over 80 per cent of the dermatological diagnoses. In dogs, the most common final diagnoses were otitis, pyoderma, anal sac impaction, flea infestation and atopic dermatitis. In cats, abscesses, flea infestation, and otitis were the most common diagnoses. In exotic species, the most common diagnosis was an unspecified mite infestation. Systemic antibiotics were prescribed in 196 cases (25 per cent), systemic glucocorticoids were prescribed in 162 cases (20 per cent) and treatment with an ectoparasiticide was prescribed in 167 cases (21 per cent).

Metastatic dissemination of human malignant oral keratinocyte cell lines following orthotopic transplantation reflects response to TGF-beta 1.

This study examined the behaviour of nine human malignant oral keratinocyte cell lines following orthotopic transplantation to the floor of the mouth of athymic mice. Tumourigenesis, local spread, and metastatic dissemination were correlated with known cellular responses to transforming growth factor-beta 1 (TGF-beta 1). Six of nine cell lines were tumourigenic; four of these cell lines showed local spread which was characterized by vascular and bone invasion. Metastatic spread was uncommon, with only 9% of animals with primary tumours developing metastases and these were almost exclusively found in the regional lymph nodes; there was one pulmonary metastasis and no liver deposits. Tumour cell behaviour did not reflect the clinical stage of the original tumours. Cell lines that were resistant to TGF-beta 1-induced growth inhibition were more likely to form primary tumours, exhibit local spread, and metastasize than cells that were growth-inhibited by the ligand. The data demonstrate that tumourigenicity and tumour behaviour in this orthotopic mouse model varied between cell lines and that the pattern of local invasion and metastasis was similar to that seen in human oral cancer. Furthermore, cell lines that were refractory to the growth inhibitory effects of TGF-beta 1 behaved more aggressively than cells that underwent ligand-induced cell-cycle arrest.

Functional significance of MMP-2 and MMP-9 expression by human malignant oral keratinocyte cell lines.

This study examined the expression of MMP-2 and MMP-9 in normal and human malignant oral keratinocytes. The expression of pro-MMP-2 and pro-MMP-9 was heterogeneous in the malignant cell lines. Normal oral keratinocytes expressed less pro-MMP-2 and more pro-MMP-9 than their malignant counterparts. Cells that expressed high levels of both MMP-2 and MMP-9 showed the greatest degree of invasion through Matrigel in vitro compared to cells with either low or variable levels of these enzymes; normal keratinocytes were non-invasive in these conditions. The degree to which the cells invaded through Matrigel was similar to their motility in the absence of Matrigel and was not influenced by the activation of the pro-enzymes or the inhibition of enzyme activity using a chemical inhibitor of gelatinases. Cells were transplanted orthotopically to athymic mice and demonstrated a variable capacity not only to form tumours at the site of inoculation but, also, to metastasise; normal oral keratinocytes were non-tumorigenic. There was no correlation between the expression of either MMP-2 or MMP-9 and the tumorigenic/metastatic phenotype. The results emphasise the limitations of correlating in vitro and in vivo assays of tumour cell behaviour and suggest that invasion/motility in vitro may be a distinct phenotype from tumorigenicity/metastasis in vivo.

Distribution of MUC1 in the normal human oral cavity is localized to the ducts of minor salivary glands.

The change in expression of MUC1 from health to disease forms the basis of its use as a potential disease marker. Previous attempts at isolating MUC1 from normal, healthy human oral mucosa have, however, drawn conflicting conclusions as to its presence. Furthermore, when MUC1 was detected in the oral glycocalyx, it was not clear which cells were synthesising it. We examined human oral glycocalyx using pooled buccal smears from 50 normal individuals. Following isopycnic density centrifugation and membrane extraction with octyl glucoside and saponin, MUC1 was detected with the polyclonal antibody CT1. Immunohistochemistry using antibodies CT1 and BC2 was performed on sections from eight labial, seven palatal, four buccal, three retromolar pad, three dorsum of tongue and two ventral surface of tongue biopsies. In-situ hybridisation using MUC1 and cytoplasmic tail oligoprobes on sections from four palatal, seven labial and two retromolar pad biopsies was also carried out. MUC1 mRNA could only be detected in the minor salivary mucous glands. MUC1 has already been identified in the ducts of normal parotid and submandibular gland, and our findings demonstrate a similar distribution in minor salivary glands. We conclude that when present in the normal oral glycocalyx, the only oral source of MUC1 is from cell membranes of the minor salivary glands.

Decreased expression of TGF-beta cell surface receptors during progression of human oral squamous cell carcinoma.

This study examined the immunocytochemical expression of the transforming growth factor-beta (TGF-beta) isoforms TGF-beta1, TGF-beta2, and TGF-beta3, together with the TGF-beta cell surface receptors TbetaR-I and TbetaR-II, in patient-matched tissue pairs of normal human oral epithelium, primary squamous cell carcinomas, and metastatic lymph node tumour deposits. There were no significant differences in the intensity of TGF-beta isoform specific staining between the normal oral epithelium, the primary tumours, and the lymph node metastases. By contrast, there was significantly less TbetaR-II in the metastases than in the primary tumour and between the primary tumour and the normal oral epithelium. Similar trends were evident with TbetaR-I, but not at a statistically significant level. This study also examined the structure of TbetaR-I and TbetaR-II in normal human oral keratinocytes in vitro and in 14 human oral carcinoma cell lines with known responses to TGF-beta1. No structural abnormalities of TbetaR-II were present in the normal keratinocytes or in 13 of 14 malignant cell lines; in one line, there were both normal and mutant forms of TbetaR-II, the latter being in the form of a frameshift mutation with the insertion of a single adenine base (bases 709-718, codons 125-128), predicting a truncated receptor having no kinase domain. No defects were present in TbetaR-I. The structures of TbetaR-I and TbetaR-II did not correlate with growth inhibition by TGF-beta1. The data suggest that decreased expression of TGF-beta receptors, rather than structural defects of these genes, may be important in oral epithelial tumour progression. In order to examine the functional significance of a specific decrease in TbetaR-II expression, a dominant-negative TbetaR-II construct (dnTbetaR-II) was transfected into a human oral carcinoma cell line with a normal TGF-beta receptor profile and known to be markedly inhibited by TGF-beta1. In those clones that overexpressed the dnTbetaR-II, growth inhibition and Smad binding activity were decreased, whilst the regulation of Fra-1 and collagenase-1 remained unchanged following treatment with TGF-beta1. The results demonstrate that a decrease in TbetaR-II relative to TbetaR-I leads to selective gene regulation with loss of growth inhibition but continued transcription of AP-1-dependent genes that are involved in the regulation of the extracellular matrix.

Overexpression of JunB in undifferentiated malignant rat oral keratinocytes enhances the malignant phenotype in vitro without altering cellular differentiation.

Our study examined the expression of AP-1 family members in keratinocytes derived from the rat-4NQO model of oral carcinogenesis in which extremes of epithelial differentiation and tumour cell aggressiveness are evident. The constitutive expression of JunB was diminished in the undifferentiated, more aggressive tumour phenotype compared with the well-differentiated, less aggressive keratinocytes, whereas the expression of other AP-1 family members (c-jun, junD, c-fos, fra1, fra2 and fosB) was either very weak or variable. After transfection of the undifferentiated keratinocytes with junB cDNA, clonal populations were isolated that expressed similar levels of JunB protein as the well-differentiated cells. Both untransfected and transfected cell lines were keratin negative and vimentin positive. Increased expression of JunB in the transfected cells resulted in up-regulation of c-Jun and Fra1 and an enhanced AP-1 activity as demonstrated by transcriptional activation of the prototypic AP-1 dependent promoter, MMP-1. JunB transfected cells grew more quickly than vector-only controls and were refractory to the growth inhibitory effects of TGF-beta1. Over-expression of JunB resulted in the elevated expression of the AP-1 dependent proteinase, MMP-9, whereas the expression of the AP-1 independent enzyme, MMP-2, was unaffected. JunB transfected keratinocytes were highly invasive in an in vitro assay of tumour cell invasion compared with vector controls. The results indicate that increased expression of JunB above baseline levels in undifferentiated rat keratinocytes does not alter epithelial differentiation but enhances the malignant phenotype in vitro, possibly by altering the dynamics of the AP-1 complex.

Endogenous TGF-beta1 inhibits the growth and metastatic dissemination of rat oral carcinoma cell lines but enhances local bone resorption.

This study examined the effect of stable transfection of latent transforming growth factor-beta1 (TGF-beta1) cDNA into a predominantly polygonal, 4 nitroquinoline N-oxide (4NQO)-induced rat oral keratinocyte cell line. Seven polygonal and five spindle clonal populations were isolated that overexpressed TGF-beta1 protein by approximately two- to four-fold compared to vector-only transfected controls. Neutralisation experiments indicated that the majority of protein was in the latent form. There was no change in the proportion of polygonal and spindle cells in vitro after transfection with TGF-beta1 cDNA. Polygonal and spindle cells that overexpressed TGF-beta1 produced similar amounts of protein and grew more slowly in vitro than controls. The parent cell line and all transfected cells were growth inhibited (60-75%) by exogenous TGF-beta1. Orthotopic transplantation of the parent and the vector-only control cell lines resulted in primary tumours in the floor of the mouth in almost 100% (20/21) of athymic mice, with no evidence of bone resorption at the site of the primary tumour and pulmonary metastatic tumour deposits in some 40% (7/20) of these animals. The polygonal and spindle cells that overexpressed TGF-beta1 behaved similarly following orthotopic transplantation. A 96% (23/24) primary tumour take was evident following transplantation of cells that overexpressed TGF-beta1, with a significantly (P<0.02) higher number of animals showing bone resorption at the site of the primary tumour (35%; 8/23) compared to controls. By contrast, there was a significant (P<0.03) decrease in the number of animals with pulmonary metastases (4%; 1/23) following transplantation of TGF-beta1 overexpressing cells compared to controls. Overexpression of TGF-beta1 did not alter tumour cell differentiation in vivo. The results demonstrate that endogenous TGF-beta1 functions as a tumour suppressor in the rat-4NQO model of oral carcinogenesis without altering tumour cell morphology or differentiation but can also act to promote local bone resorption.

Loss of differentiation of 4NQO-induced rat malignant oral keratinocytes correlates with metastatic dissemination and is associated with a reduced cellular response to TGF-beta1 and an altered receptor profile.

This study examined the metastatic capacity of clonal populations of 4NQO-induced rat malignant oral keratinocytes following orthotopic transplantation to athymic mice. Polygonal and spindle cells formed well-differentiated squamous cell carcinomas (keratin positive and vimentin negative) and undifferentiated spindle cell tumours (keratin negative and vimentin positive), respectively, in almost 100% of animals at the site of inoculation (floor of mouth). Transplantation of 5x 10(6) cells of either cell type at high cell density resulted in approximately 50% of animals forming pulmonary metastases. By contrast, inoculation of 2x 10(6) differentiated polygonal cells resulted in the formation of significantly fewer pulmonary metastases than the undifferentiated spindle cells. A single well-differentiated clone of polygonal cells and 3 of 4 of the undifferentiated spindle cell lines produced comparable levels of TGF-beta1. One undifferentiated spindle cell line expressed significantly more TGF-beta1 and, following transplantation orthotopically, fewer animals formed pulmonary metastases despite the formation of primary tumours in almost all grafted animals, suggesting that TGF-beta1 can act as a tumour suppressor in this cell type. All cell lines produced comparable amounts of TGF-beta2. The clones of polygonal cells were markedly inhibited and the spindle cells were only partially inhibited by exogenous TGF-beta1. Both cell types expressed high-affinity TGF-beta cell surface receptors; the ratio of type I to type II TGF-beta receptors was 1.0:<3.0 in the spindle cells and 1.0:17.9 in the polygonal clone. The results suggest that differentiated rat malignant oral keratinocytes are less aggressive and have a decreased potential to metastasise than their undifferentiated spindle cell counterparts. This may be attributable, in part, to a change in TGF-beta receptor profile leading to the partial loss of response to exogenous TGF-beta1.

Destabilization and denaturation of cellular protein by glutathione depletion.

This investigation tested the hypothesis that depletion of intracellular glutathione, in contrast to its oxidation could lead to non-native oxidation of protein thiols, thereby trapping proteins in an unstable conformation. Chinese hamster cells were exposed to the alpha, beta-unsaturated dicarboxylic acid diethylmaleate in order to produce rapid glutathione (GSH) depletion without oxidation. Measurement of the fluorescence of oxidized 2',7'-dichlorofluorescein diacetate indicated that reactive oxygen species accumulated in GSH depleted cells. Glutathione depletion was found to alter protein thiol/disulfide exchange ratios such that 17 to 23 nmol of protein SH/mg protein underwent oxidation. Differential scanning calorimetry (DSC) of glutathione depleted cells yielded a profile of specific heat capacity versus temperature that was characteristic of cells containing destabilized and denatured protein. In addition, cells depleted of glutathione exhibited a two-fold increase in NP-40 insoluble protein. These results indicate that depletion of intracellular glutathione caused oxidation of protein thiols, protein denaturation and aggregation and provide a mechanism to explain how GSH depletion can initiate stress responses.

Overexpression of autocrine TGF-beta 1 suppresses the growth of spindle epithelial cells in vitro and in vivo in the rat 4NQO model of oral carcinogenesis.

We examined the effect of the stable transfection of latent TGF-beta 1 cDNA, under the control of a cytomegalovirus promoter in the expression vector pcDNA3, into a 4NQO-induced clonal rat oral keratinocyte cell line that formed undifferentiated spindle cell tumours following subcutaneous transplantation to athymic mice. Test cells containing latent TGF-beta 1 cDNA produced a 2.3-fold increase in TGF-beta 1 protein compared to pcDNA3 controls as demonstrated by ELISA. Neutralisation experiments indicated that the majority of the protein was in the latent form. Untransfected and transfected (containing either TGF-beta 1 cDNA or pcDNA3) cell lines were keratin negative and vimentin positive. Cells transfected with TGF-beta 1 were inhibited more than pcDNA3 controls when cultured in an anchorage dependent or independent environment. Subcutaneous transplantation of cells overproducing TGF-beta 1 resulted in tumours of significantly smaller volume than vector-only controls. Further, orthotopic transplantation of cells containing TGF-beta 1 cDNA to the floor of the mouth in athymic mice markedly inhibited the development of pulmonary metastases compared to vector-only controls. Both test and control cell lines in athymic mice formed undifferentiated tumours with a complete absence of keratin elaboration. Subcutaneous xenografts were recultured and cells containing the TGF-beta 1 cDNA produced a similar amount of TGF-beta 1 peptide as the cells containing pcDNA3 only. The production of TGF-beta 1 by both of the xenograft-derived cell lines was significantly less than the parent, pre-transplanted cell lines and the untransfected cell line. All of the cell lines were inhibited by exogenous TGF-beta 1. Our results demonstrate that autocrine TGF-beta 1 functions as a tumour suppressor in vitro and in vivo in 4NQO-induced spindle tumour cells that are growth inhibited by the ligand. Furthermore, tumour formation in athymic mice is associated with selection for a cell phenotype with diminished autocrine TGF-beta 1 production.