Immunohistochemical expression of tenascin in melanocytic tumours of dogs.

The aim of this study was to investigate tenascin-C (TN) immunolabelling and labelling for endothelium by von Willebrand Factor (vWF) in melanocytic tumours of dogs as compared with normal tissues, to evaluate the TN distribution in these types of tumours and to investigate whether a relation could be established between TN and angiogenesis in different types of tumour. Samples of normal dog skin (n=8), benign skin melanocytomas (n=10), malignant oral melanomas (n=9) and malignant toe melanomas (n=5) were studied. The percentages of TN and vWF immunolabelling per total microscopical area were analysed by morphometric methods. In normal skin, TN was found at dermo-epidermal junctions, around hair follicles, in the smooth muscles of hair follicles, and in the walls of blood vessels. TN immunolabelling (distribution and intensity) in melanocytomas was comparable with that found in normal skin. In melanomas, TN expression was considerably increased, its intensity in toe melanomas being twice that observed in oral melanomas. The degree of TN immunolabelling was not related to the histological malignancy of the melanomas. In melanomas, TN was found in the connective tissue surrounding the tumour cell nests and in narrow stromal strands inside the tumour. Regions infiltrated with lymphocytes were devoid of TN. The presence of TN around capillaries in melanocytomas and melanomas was investigated by double-immunolabelling (for TN and vWF). The intensity of vWF and TN immunolabelling was higher in melanomas than in melanocytomas, and higher in toe melanomas than in oral melanomas; however, no clear relation between TN expression and immunolabelling for vWF was found.

Reversal of methotrexate inhibition of colony growth of L1210 leukemia cells in semisolid medium.

L1210 mouse leukemia cells were grown in a methylcellulose-based medium, and the inhibitory effect of methotrexate (MTX) on colony formation and its reversal were examined. The effect on colony formation was studied in order to compare the results with those obtained with normal mouse bone marrow cells grown in a similar manner in previous studies and in additional experiments presented. Light microscopy could not be used for colony counting of L1210 cells because MTX did not inhibit colony formation and only affected further colony growth. Therefore, it was necessary to evaluate the toxic effect of MTX by analysis of colony size distribution using an electronic image analyzer. Results show that the reversal of MTX toxicity to L1210 cells with leucovorin is competitive and is similar to that with normal mouse bone marrow cells. Thymidine in combination with a purine prevents MTX toxicity as well. The optimal concentration of thymidine is 10(-5) M, whereas at least 10(-4) M purine is required. Reversal of MTX toxicity by thymidine and purines is independent of the MTX concentration and is possible at drug concentrations as high as 10(-4) M. Compared to mouse bone marrow cells, L1210 cells appear to require more purines to prevent the toxic effects of MTX. MTX toxicity towards bone marrow myeloid precursor cells can be reversed by 10(-4) M inosine alone. These cells are better protected against MTX toxicity when 10(-6) to 10(-5) M thymidine is added. The results suggest that the use of a high-purine-low-thymidine combination has advantages over the use of leucovorin in controlling toxicity over a wide range of MTX concentrations and in providing some degree of selective protection to normal proliferating cells.

E-cadherin expression and in vitro invasion of canine mammary tumor cells.

E-cadherin is considered to be an invasion suppressor molecule. We have studied the expression and function of E-cadherin in three cell lines derived from a dog mammary tumor, namely SH15, SH24, and SH27. In monolayer culture the cell lines can be distinguished by their morphotype: epithelioid (SH15), fibroblast-like (SH24) and intermediate type (SH27). SH27 was unable to form colonies in collagen gel in contrast to SH15 and SH24. All three cell lines expressed the E-cadherin antigen, as evident from immunocytochemistry, and alpha-, beta-, and gamma-catenins as evident from immunoprecipitation with E-cadherin antibody. Only SH27 showed E-cadherin-dependent aggregation, and little invasion into collagen type 1 gels, in contrast to SH15 and SH24 cells. However, in the precultured embryonic chick heart assay all three cell lines were invasive, demonstrating that invasion depends upon the microenvironment. We assume that in the embryonic chick heart, factors were present or were induced by the SH27 cells, interfering with the function of E-cadherin.

Effect of the glutathione-depleting agents diethylmaleate, phorone and buthionine sulfoximine on biliary copper excretion in rats.

The involvement of glutathione (GSH) in the biliary excretion of Cu was investigated in bile-cannulated inbred WAG/Rij and BN rats, pretreated with diethylmaleate (DEM), phorone or buthionine sulfoximine (BSO) and injected with Cu doses of 10 or 30 micrograms/100 g body wt. DEM reduced liver GSH to 27-56% and biliary GSH excretion to 18-38%; phorone reduced GSH in the liver to 55% and increased it in the bile (113%) followed by a slight decrease (79%); BSO reduced liver GSH to 50% and bile GSH to 20%. After injection of Cu to control rats a profile of biliary Cu excretion was found, composed of a slowly (SCuE) and a rapidly (RCuE) disappearing component, the latter only present after the dose of 30 micrograms Cu. DEM had no effect on SCuE after a 10 micrograms dose and a temporary effect on SCuE after a 30 micrograms dose in both WAG/Rij and BN rats. Phorone reduced SCuE after both Cu doses to 50%. Both agents abolished RCuE and reduced endogenous biliary Cu excretion to less than 50%. Release of injected Cu from plasma and uptake by the liver was inhibited by DEM and phorone in both rat strains; in BN rats basal plasma Cu level of DEM-treated rats was increased as well. BSO reduced SCuE after both Cu doses but had no influence on RCuE. Endogenous Cu excretion was reduced by BSO in BN rats but not in WAG/Rij rats. The results show that biliary Cu excretion proceeds by a pattern, the components of which can be affected differently by the various drugs. They also indicate that GSH is not directly involved in biliary Cu excretion but suggest that it may play a role in the metabolism of Cu in the liver.

Validation of the use of proliferation markers in canine neoplastic and non-neoplastic tissues: comparison of KI-67 and proliferating cell nuclear antigen (PCNA) expression versus in vivo bromodeoxyuridine labelling by immunohistochemistry.

In order to evaluate the suitability of Ki-67 and proliferating cell nuclear antigen (PCNA) for determination of proliferative activity, the immunohistochemically determined nuclear expression of these antigens in canine non-neoplastic and neoplastic tissues was compared with the results of in vivo bromodeoxyuridine (BrdU) labelling, which - by measurement of the fraction of S-phase cells - is considered as the standard in the analysis of proliferative activity. The samples investigated consisted of non-neoplastic mammary and lymphoid tissues, and of benign and malignant (primary/metastatic) mammary tumours, and malignant lymphomas. Great regional heterogeneity prevented determination of an overall labelling index (LI) in normal lymphoid tissues. In the remaining combined group of samples, LI values were significantly ranked in the order PCNA>Ki-67>BrdU. However, the correlation of Ki-67 or PCNA as compared to BrdU LI values was only moderate in the combined group [approximately 0.5, Spearman rank test] as well as in most subgroups, whilst it was very poor in the group of primary mammary cancers. These observations indicate that Ki-67 or PCNA LIs as markers of proliferation do not evenly match in vivo BrdU labelling.

Stromal cells and extracellular matrix components in spontaneous canine transmissible venereal tumour at different stages of growth.

Stromal cells and extracellular matrix (ECM) components are important for tumour cell behaviour. Little is known about the role of stromal cells and ECM components in the progression and regression of spontaneous canine transmissible venereal tumour (CTVT). In this study, the stromal cell type was determined by immunohistochemical labelling with antibodies to desmin, vimentin and alpha-smooth muscle actin (alpha-SMA) during the progressive and regressive stages of spontaneous CTVT. The distribution of ECM components tenascin-C, chondroitin sulphate and versican were determined immunohistochemically, and hyaluronan distribution was determined using a biotinylated protein complex with specific affinity for hyaluronan. Stromal cells of tumours in both the progressive and regressive stage were positive for vimentin and negative for desmin. The number of stromal cells expressing alpha-SMA was significantly higher (P=0.001) in regressing tumours, than progressing tumours. These results suggest that the modulation of stromal cells that occurs during the regression of CTVT is similar to that occurring during wound healing. Tenascin-C was weakly expressed in the stroma of tumours in the progressive stage and in regions of the regressing tumours with tumour infiltrating lymphocytes (TILs), but intensely expressed in the stroma of tumours in late regressive stage. In addition, tenascin-C was also expressed in the cytoplasm of some tumour cells in the late regressive stage. A strong stromal tenascin-C intensity was significantly associated with regressing tumours (P=0.001). Strong stromal hyaluronan intensity and a high proportion of hyaluronan-positive tumour cells were significantly associated with progressing tumours (P=0.001). This suggests that hyaluronan is involved in the growth of the tumour. There was no significant difference in the expression of chondroitin sulphate and versican in progressing and regressing tumours.

Immunohistochemical evaluation of versican, in relation to chondroitin sulphate, in canine mammary tumours.

The expression of increased amounts of versican, a chondroitin sulphate proteoglycan, in neoplastic tissues may play a role in promoting tumour cell proliferation and migration. This study investigated the immunolocalization of versican in normal and neoplastic canine mammary tissues, using antibodies 12C5 and 2B1, against different epitopes of the protein core of versican. Antibody CS56, recognising chondroitin sulphate (CS), was used to investigate the relation between versican and CS, which accumulates in canine mammary tumours. We found enhanced versican expression in both benign and malignant tumours, appearing in three main patterns: in periductal tissues, probably in association with basement membranes of ducts; in peripheral invasive areas of malignant tumours; and in spindle cell proliferations and myxoid areas of complex and mixed tumours. The 12C5 and 2B1 immunoreactivities co-localised in all types of tumours, and could be improved by chondroitinase digestion. The only exception was the abundant extracellular matrix (ECM) of spindle cell proliferations, particularly in myxoid areas of complex and mixed tumours, which displayed intense and diffuse 12C5 immunoreactivity and patchy or absent 2B1 and CS56 immunoreactivities; versican immunoreactivity could not be enhanced by chondroitinase digestion. The results indicate that versican is one of the extracellular matrix components characteristic of canine mammary tumours. It appears likely that in complex and mixed tumours versican exists in at least two forms, one of them lacking the CS attachment domain and the 2B1 epitope. Furthermore, the enhanced versican expression in the invasive areas of malignant tumours indicates the involvement of this proteoglycan in tumour cell invasion.

Influence of dietary molybdenum on the metabolism of intravenously injected radioactive copper in the rat.

Control and molybdenum-supplemented rats (Mo rats) were injected i.v. with 64Cu or 67Cu. The distribution of radioactivity over plasma, liver, and kidney, as well as the intracellular distribution in these organs, was studied as a function of time. Compared with control rats, the level of radioactive Cu in the plasma of Mo rats was increased. The results for liver and kidney of Mo rats had to be corrected for a decrease in specific activity of 64Cu; they suggested that the 64Cu release from the liver and the 64Cu uptake in the kidney of Mo rats ran parallel to that in control rats for up to 8 hr; after which, an increase in both organs of the Mo-rats followed. 64Cu in the subcellular particles and in a high molecular weight (MW) protein of the cytoplasma of the organs of Mo rats was increased compared with control rats. In control rats the transport of 64Cu from a 10,000 MW protein to a 30,000 MW protein of the cytoplasma of the kidney seemed to be much slower compared with that of the liver, but Mo had no influence on this process in either liver or kidney.

Changes in the binding of copper in the plasma of molybdenum supplemented rats.

After incubating plasma of Mo-supplemented rats (Mo-plasma) with 64Cu only part of it could be removed by dialysis against EDTA or histidine or by treatment with dithiocarbamate; this nondialyzable Cu was shown to be bound to albumin. The maximal amount of 64Cu bound this way equaled the Mo-induced increase in total plasma Cu. After addition of stable Cu, dialysis of Mo-plasma against a histidine solution showed that no extra Cu became tightly bound, suggesting that the 64Cu binding was due to an exchange between added 64Cu and stable Cu already present. Incubating Mo-plasma with Hg compounds prevented 64Cu binding and released stable Cu, indicating that Cu in Mo-plasma was sulfhydryl bound. Part of the Mo in Mo-plasma was freely dialyzable. The remaining part was shown to be SH bound as well. The estimated atomic ratio of SH-bound Cu and Mo was unity. Molybdenum increased the number of SH groups in plasma, and for each Cu atom at least one SH group was calculated to be present.

Studies about the mechanism of internalization by mammary epithelial cells of Escherichia coli isolated from persistent bovine mastitis.

The purpose of this study was to investigate the interaction between Escherichia coli and primary mammary epithelial cell cultures derived from cows with persistent intramammary infection (IMI). Two strains of E. coli, isolated from the milk of two different cows suffering from persistent E. coli IMI were tested for adhesion to and invasion of three primary mammary epithelial cell cultures derived from mammary biopsies of the two infected cows. Intracellular E. coli were detected during five days post infection in vitro. Both strains of E. coli adhered to and invaded monolayers of all three primary mammary epithelial cell cultures. One strain adhered less but invaded more than the other. Comparison with other mammary pathogens indicated that E. coli invaded the cells less efficiently than Staphylococcus aureus, about as efficiently as Streptococcus dysgalactiae and more efficiently than Streptococcus uberis. The mechanism of E. coli invasion was studied using the cytoskeleton disrupting agents colchicine and cytochalasin D. These compounds inhibited the invasion of E. coli. Invasion of E. coli could also be inhibited by the phosphokinase inhibitors genistein and staurosporin in a dose-dependent fashion. Phorbol-myristyl-acetate (PMA) had no effect on the invasion of E. coli. Histology of mammary tissue revealed chronic inflammatory changes in quarters that were persistently infected by E. coli. Intracellular bacteria were not detected in mammary tissue sections. Polymerase chain reaction (PCR) analysis suggested that the two strains of E. coli lacked genes encoding for bundle-forming pili (bfpA), intimin (eae) and translocated intimin receptor (tir), which are characteristic for enteropathogenic E. coli (EPEC).

Adhesion and invasion of Escherichia coli from single and recurrent clinical cases of bovine mastitis in vitro.

Seven strains of Escherichia coli, originating from clinical cases of bovine mastitis, and one Salmonella typhimurium control strain were tested for their ability to adhere to, and invade, bovine mammary epithelial cells (MAC-T cells) in vitro. Four of the seven strains were isolated from cows with chronic intramammary infections with recurrent episodes of clinical mastitis and three strains were isolated from single cases of clinical mastitis. Both adhesion and invasion of all strains were dose and time dependent. The four E. coli strains isolated from recurrent cases of clinical mastitis invaded twice as frequently as and three times faster than the strains isolated from single cases of clinical mastitis. By contrast, there was no difference in the amount or speed of adhesion between the two types of strains of E. coli. Adhesion and invasion curves of E. coli resembled a two-step chain reaction, where invasion was the rate-limiting step. Although adhesion and invasion of E. coli has not been demonstrated in vivo yet, the results of the present study may contribute to an understanding of the pathogenesis of chronic intramammary infections caused by E. coli.

Lack of effect of extracellular matrix or 3T3 feeder layer on the maintenance of differentiation or survival time of cultured rat hepatocytes.

Cultured rat hepatocytes are often used in in vitro studies. In this culture system the lack of the normal environment to which the cells would be exposed in vivo contributes to a change in differentiation. To study this differentiation problem we compared the effects of culturing primary hepatocytes on an extracellular matrix (ECM) isolated from normal rat livers and on a 3T3 (mouse fibroblast) feeder layer. The use of the two coatings did not result in changes in survival time, as measured by leakage of lactate dehydrogenase, differentiation (activities of pyruvate kinase and gamma-glutamyltransferase) or specific functions (albumin production and concentration of cytochrome P-450) in comparison with cells cultured on plastic. Since no positive effects of the coatings were found it is proposed that more attention should be paid to methods of isolating and culturing hepatocytes, rather than to obtaining conditions for the hepatocytes that are completely comparable with the in vivo situation.

Phenobarbital pretreatment in vivo and in vitro and the effect of hepatotoxicity of d-galactosamine in rat hepatocytes in culture.

Galactosamine (GalN) is a known hepatotoxic compound, acting by depletion of uracil nucleotides. The relation between an active cytochrome P-450 system (CYP) and the hepatotoxicity of GalN was studied in rat hepatocytes that were pretreated with phenobarbital (PB) in vivo or in vitro. A 24-hr in vitro pretreatment of cultured hepatocytes with PB resulted in a significant decrease in GalN toxicity as measured by lactate dehydrogenase (LDH) leakage. Furthermore, GalN treatment resulted in an increase in the activity of the PB-induced forms of CYP (namely CYP 2B1/2) as measured by 7-pentoxyresorufin O-depentylase (PROD) activity. This increase was not found after GalN treatment of microsomes. GalN had no effect on the concentration of the apoenzymes. GalN administration to hepatocytes of in vivo PB-pretreated rats resulted in a similar effect of GalN on the activity of the CYP enzymes but PB in vivo had no effect on GalN toxicity. These results suggest that GalN treatment may result in a significant increase in the specific activity of CYP 2B1/2 enzymes (PROD), without an obvious increase in the amount of PB-induced apoenzymes. This phenomenon was measurable only in intact cells. No direct relation is assumed between the activity of the CYP apoenzymes and the decrease in GalN toxicity after PB treatment. The toxicity of Galn was inhibited by PB treatment in vitro.

Versican and hyaluronan expression in canine colonic adenomas and carcinomas: relation to malignancy and depth of tumour invasion.

Changes in the production and structure of glycosaminoglycans and proteoglycans have been reported in many neoplastic tissues, and versican and hyaluronan (extracellular matrix components) are frequently increased in tumours and promote tumour progression. The distribution of chondroitin sulphate, versican and hyaluronan in normal canine colonic wall (n=10), and normal colonic lymph nodes (n=10), colonic adenomas (n=22), colonic adenocarcinomas (n=28), colonic undifferentiated carcinomas (n=7), and colonic lymph node metastases (n=8), was examined, with antibodies against chondroitin sulphate and versican, and a specific biotinylated probe for hyaluronan. The epithelial cells of the normal colonic mucosa were negative for all three substances, whereas the stromal tissue and lamina propria were moderately positive for chondroitin sulphate and hyaluronan, and weakly positive for versican. Chondroitin sulphate expression was increased in adenomas and carcinomas. However, there was no significant correlation between grade of tumour and degree of chondroitin sulphate expression. Versican expression was increased in the peritumoral stroma of adenocarcinomas and reduced in adenomas. A significant correlation was observed between grade of tumour and degree of versican expression. In 13 adenocarcinomas and undifferentiated carcinomas with invasion into all layers of the colorectum, the intensity of stromal versican expression was significantly related to the depth of invasion; the intensity was increased in the stroma of tumour islands in deep layers of the colonic wall. Unlike versican expression, hyaluronan expression was increased in the stromal tissue of both adenomas and carcinomas. However, the degree of stromal hyaluronan expression was unrelated to tumour grade and depth of tumour invasion. Hyaluronan was also expressed in the membrane and in the cytoplasm of tumour cells in 3/22 (14%) adenomas, 18/28 (64%) adenocarcinomas and 2/7 (29%) undifferentiated carcinomas. These results suggest that altered levels of both versican and hyaluronan in canine colonic tumours affect tumour progression.

Tenascin expression in relation to stromal tumour cells in canine gastrointestinal epithelial tumours.

The expression of tenascin, alpha-smooth muscle actin (alpha-SMA), desmin and vimentin was investigated immunohistochemically in the stroma of normal canine stomach, small intestine and colon, and in 30 epithelial tumours of the canine stomach, small intestine or colon. In addition, "co-localization" of tenascin and alpha-SMA was investigated by double immunohistochemistry. Tenascin was absent in the normal gastric mucosa but present in the normal intestine, with a gradual increase in immunolabelling intensity from the cryptal glands to the surface epithelium. Tenascin expression was greater in all adenomas and carcinomas than in normal tissues. Two different patterns of tenascin expression were observed in all carcinomas, irrespective of their site. In well-differentiated tumour regions of both gastric and intestinal tumours, a fibrillary sub-glandular expression was observed; in poorly differentiated tumour regions, however, the expression pattern was diffuse. Incomplete invasion of the muscularis mucosae was accompanied by thickening and increased tenascin expression. In normal stomach and intestines, alpha-SMA and desmin were demonstrated in pericryptal myofibroblasts and smooth muscle cells of the muscle layers. In colonic adenomas and gastric and intestinal carcinomas, alpha-SMA was demonstrated in all stromal cells surrounding tumour cells. In contrast to alpha-SMA labelling, desmin labelling was negative in tumour stromal cells (in both gastric and intestinal tumours), except in tumour regions close to the muscularis mucosae. This suggested that myofibroblasts in gastric and intestinal tumours originated from pre-existing fibroblasts, except in tumour regions close to the muscularis mucosae, where the myofibroblasts seemed to originate from smooth muscle cells of the muscularis mucosae. There was a strong co-localization of tenascin and alpha-SMA-expressing myofibroblasts, suggesting that myofibroblasts are responsible for tenascin secretion.

Tenascin expression in normal, hyperplastic, dysplastic and neoplastic canine mammary tissues.

Mammary tumours are the most common neoplasias of female dogs and may have a complex histological pattern with both epithelial and spindle cells participating in the transformation process. A frequent feature of these tumours is chondroid or bone metaplasia of the extracellular matrix, which mainly occurs in areas of proliferated spindle-shaped cells, probably of myoepithelial origin. The present study evaluates immunohistochemically the expression of tenascin in 186 surgical samples of canine mammary tissues, ranging from normality to neoplasia. Tenascin was present in all mammary tissues studied, with an increased expression in remodelling situations and in neoplastic lesions. Basement membrane was the most frequently labelled structure, but stromal tissue was more often and widely labelled in neoplastic lesions. The extracellular matrix was positive in solid and anaplastic carcinomas as well as in spindle cell proliferation areas. Tenascin expression in extracellular matrix was also abundant in areas of initial chondroid metaplasia and, with variable extension, in almost all cartilage islands of mixed tumours. In well differentiated secretory areas only apical granules of luminal cells were positive, suggesting a different pattern of tenascin expression during secretory differentiation. The digestion of chondroitin sulphate significantly improved the labelling for tenascin when a co-expression of these two molecules was present. Although our results suggest that tenascin cannot be used as a marker of transformation or of malignancy in canine mammary oncology, it is clear that this molecule plays an important role in proliferation and differentiation processes in the canine mammary gland.

Tenascin and proteoglycans: the role of tenascin and proteoglycans in canine tumours.

Tenascin is a high molecular weight, extracellular matrix glycoprotein, subject to complex spatial and temporal patterns of expression during embryogenesis, wound healing and neoplastic processes. Proteoglycans are complex macromolecules, containing one or more glycosaminoglycans attached to a core protein, which are involved in cell-cell and cell-matrix interaction. Altered expression of both tenascin and proteoglycans has been found in tumours and expression of these two extracellular matrix proteins seems to be modulated in the same way in human and canine tumours. The quantitative and qualitative changes in tenascin and proteoglycan composition may significantly affect behaviour of tumour cells. While tenascin and proteoglycans have many biological functions likely to influence tumour development and progression, their exact role in regulation of tumour cell-cell interaction, proliferation, invasion and metastasis remains to be established. This review focuses on the role of tenascin and proteoglycans in neoplasia and recent developments in canine tumours.

Relationship between cell proliferation and tenascin-C expression in canine gastrointestinal tumours and normal mucosa.

Tenascin-C is an extracellular matrix glycoprotein that has been implicated in cell proliferation and adhesion by in vitro experiments. Its expression is known to be increased in canine and human gastrointestinal tumours. The aim of this study was to investigate the possible relationship between cell proliferation and tenascin expression in these tumours. In tissue sections of normal stomach, small intestine and colon, and gastrointestinal epithelial tumours, the monoclonal antibody Ki-67, which is directed against a proliferation-associated nuclear antigen, was used to identify proliferating cells. Serial sections were also stained for tenascin. Serial sections stained for tenascin and Ki-67 were compared to determine whether there is a correlation between tenascin expression and tumour cell proliferation. In the normal gastric mucosa, Ki-67 positive cells were confined to the neck region and in the normal small intestinal mucosa positive cells were confined to the lower parts of the crypts. In adenomas and carcinomas, the frequency of positive cells was increased at the edges of adenomas and invasive tumour margins of carcinomas and there was inter- and intra-tumoural heterogeneity. Carcinomas with lymphatic invasion showed a high Ki-67-index. There was no relation between cell proliferation and tenascin expression in both normal tissues and tumours studied. The absence of a correlation between tenascin and Ki-67 expression suggests that the main function of tenascin in both normal tissues and tumours of the canine gastrointestinal tract is antiadhesion rather than proliferation.

Differences in wound contraction between horses and ponies: the in vitro contraction capacity of fibroblasts.

The contribution of wound contraction to wound closure determines the speed of second intention wound healing and it has been shown that significant differences exist with regard to both contraction and inflammatory response between horses and ponies and between various areas of the body. In this study, the contraction capacity of fibroblasts from limbs and buttocks of 4 Dutch Warmblood horses and 4 Shetland ponies was studied in vitro, in order to determine whether differences in wound contraction are due to differences in the inherent contraction capacity of the fibroblasts or to differences in tissue environmental factors, such as the inflammatory response. Fibroblasts were harvested from subcutaneous tissue, cultured and then suspended in both floating and anchored collagen gels. Contraction capacity was assessed by measuring the decrease in area of the floating gels and by measuring the microforces generated in the anchored gels using a custom-built measuring device. In the floating gels, no difference existed in the contraction capacity of fibroblasts from horses and ponies, or from limbs and buttocks. In the anchored gels, no differences existed between horse and pony fibroblasts, but the fibroblasts from the limbs started to contract significantly sooner and produced significantly higher forces than those from the buttocks. It is concluded that the in vivo differences in wound contraction between horses and ponies and between different sites of the body are not caused by differences in the inherent contraction capacity of fibroblasts. The in vitro differences between fibroblasts from limbs and buttocks are thought to be due to the lower proliferation rate and the longer culture time of the fibroblasts originating from the limbs, because mature fibroblasts can develop higher contraction forces than immature fibroblasts. This means that tissue environmental factors, such as cytokine profiles during the inflammatory response, determine the extent of contraction during wound healing. Further research should be directed towards the role of the inflammatory response in wound healing.

Strain differences in kidney copper concentrations of male rats.

The copper concentrations of the kidneys of male rats of six inbred (BN, F344, LEW, SHR, WAG/Cpb, and WAG/Rij) and one random-bred Wistar strain were determined.In inbred rats the mean concentration varied between strains and ranged from 7.10 µg/g for F 344 to 23.48 µg/g for WAG/Cpb. The calculated coefficient of genetic determination (g(2)) was 0.88. A remarkable discrepancy was found between the two WAG inbred strains; the WAG/Cpb had a 2.5 times higher kidney Cu concentration than the WAG/Rij.Kidney Cu concentrations of random-bred rats varied considerably; the coefficient of variation of the means was 28 and 34% in two samples taken with a 1-yr interval, respectively, indicating inhomogeneity within the population.The results indicate that the individual differences in kidney Cu concentration have a genetic basis.