Tenascin-C in chronic canine hepatitis: immunohistochemical localization and correlation with necro-inflammatory activity, fibrotic stage, and expression of alpha-smooth muscle actin, cytokeratin 7, and CD3+ cells.

During fibrosis, the extracellular matrix (ECM) is continuously remodeled and increases in volume due to the production of various proteins. We studied the distribution of tenascin-C (TN-C) and the correlation of TN-C with the necro-inflammatory activity and expression of alpha-smooth muscle actin (alpha-SMA), cytokeratin 7 (CK7), and CD3+ T-lymphocytes in canine chronic hepatitis. This was analyzed using immunohistochemistry and semiquantitative scoring. We used 3 groups (n = 19) of dogs: group 1 (n = 5) with neonatal hepatitis/lobular dissecting hepatitis (NH/LDH), group 2 (n = 8) with chronic hepatitis/cirrhosis (CH/CIRR), and group 3 (n = 6) consisting of healthy animals. In normal livers, TN-C was localized in Disse's space and around bile ducts and blood vessels. In CH/CIRR livers, TN-C was localized at the periphery of the regenerating nodules and was conspicuous in the bridging fibrous bands. In NH/LDH, TN-C was diffusely distributed along the reticular fibers that dissected between single cells or groups of hepatocytes. alpha-SMA in the normal hepatic parenchyma showed an irregular distribution along the perisinusoidal linings. In other groups, alpha-SMA was increased in fibrotic septa and perisinusoidal linings. In normal livers, CK7 was positive in bile ducts. In other groups, CK7-expressing cells were conspicuous in the portal-parenchymal interface, the periphery of the regenerative nodules, and the degenerated parenchyma. The pattern of CD3+ lymphocytes was inversely proportional to that of TN-C. These results also showed that TN-C is strongly correlated with increased fibrotic stage, inflammatory activity, and expression of CK7 and alpha-SMA. TN-C, CK7, and CD3 expression did not differ between diagnostic groups.

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.

Expression of versican in relation to chondrogenesis-related extracellular matrix components in canine mammary tumors.

Versican plays a role in tumor cell proliferation and adhesion and may also regulate cell phenotype. Furthermore, it is one of the pivotal proteoglycans in mesenchymal condensation during prechondrogenesis. We have previously demonstrated accumulation of versican protein in myoepithelial-like spindle cell proliferations and myxoid tissues of complex and mixed mammary tumors of dogs. The objective of this study was to investigate whether the high expression of versican relates to prechondrogenesis in these tissues. Therefore, we aimed to identify cartilage markers, such as collagen type II and aggrecan both at mRNA and protein level in relation to versican. The neopitope of chondoitin-6-sulphate (3B3) known to be generated in developing cartilage has been investigated by immunohistochemisty and a panel of antibodies were used to characterize the phenotype of cells that are involved in cartilage formation. In addition, co-localization of versican with hyaluronan and link protein was studied. RT-PCR revealed upregulation of genes of versican, collagen type II and aggrecan in neoplastic tissues, especially in complex and mixed tumors. Immunohistochemistry showed the expression of cartilage biomarkers not only in the cartilagenous tissues of mixed tumors, but also in myoepitheliomas and in the myoepithelial-like cell proliferations and myxoid areas of complex and mixed tumors. The results show the cartilagenous differentiation of complex tumors and myoepitheliomas and indicate that the myxoid tissues and myoepithelial-like cell proliferations are the precursor tissues of the ectopic cartilage in mixed tumors. Furthermore, we suggest that cartilage formation in canine mammary tumors is a result of (myo)epithelial to mesenchymal transition.

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.

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.

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.

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.

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.

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.

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.

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.

Factors involved in the early pathogenesis of bovine Staphylococcus aureus mastitis with emphasis on bacterial adhesion and invasion. A review.

Staphylococcus aureus is the most important and prevalent contagious mammary pathogen; it causes clinical and subclinical intramammary infection with serious economic loss and herd management problems in dairy cows. In vitro studies have shown that Staphylococcus aureus adheres to mammary epithelial cells and extracellular matrix components and invades into mammary epithelial as well as other mammary cells. Staphylococcus aureus strains from intramammary infection produce several cell surface-associated and extracellular secretory products. The exact pathogenic roles of most of the products and their effects on adhesion and invasion are not well evaluated. It is also known that mammary epithelial cell-associated molecules and extracellular matrix components interact with S. aureus during the pathogenesis of mastitis, but their roles on adhesion and invasion have not been characterized. The adhesion of S. aureus to epithelial cells may involve non-specific physicochemical interactions and/or specific interactions between bacterial cell-associated ligands and host cell surface receptors. In vitro adhesion depends on the S. aureus strain, the growth phase of the bacteria, the growth medium and the origin of the epithelial cells. Adhesion is hypothesized to be a prerequisite and crucial early step for mammary gland infection. Staphylococcus aureus invades mammary epithelial cells. It also invades other cells such as endothelial cells and fibroblasts. Bacteria are found enclosed in membrane bound vacuoles in the cytoplasm of mammary epithelial cells. Recent observations indicate that S. aureus escapes from the phagosome into the cytoplasm and induces apoptosis. The invasion into mammary epithelial cells may occur through an endocytic process that requires involvement of elements of the cytoskeleton or by direct binding of bacteria to epithelial cells through a process mediated by specific receptors that needs de novo protein synthesis by both cells. Thus, the recurrent subclinical infection may result from this intracellular existence of bacteria that are protected from host defenses and effects of antibiotics. This review emphasizes on recent findings on S. aureus adhesion to mammary epithelial cells and extracellular matrix components and invasion into mammary epithelial cells.

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.

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.

The biomedical disciplines and the structure of biomedical and clinical knowledge.

The relation between biomedical knowledge and clinical knowledge is discussed by comparing their respective structures. The knowledge of a disease as a biological phenomenon is constructed by the interaction of facts and theories from the main biomedical disciplines: epidemiology, diagnostics, clinical trial, therapy development and pathogenesis. Although these facts and theories are based on probabilities and extrapolations, the interaction provides a reliable and coherent structure, comparable to a Kuhnian paradigma. In the structure of clinical knowledge, i.e. knowledge of the patient with the disease, not only biomedical knowledge contributes to the structure but also economic and social relations, ethics and personal experience. However, the interaction between each of the participating "knowledges" in clinical knowledge is not based on mutual dependency and accumulation of different arguments from each, as in biomedical knowledge, but on competition and partial exclusion. Therefore, the structure of biomedical knowledge is different from that of clinical knowledge. This difference is used as the basis for a discussion in which the place of technology, evidence-based medicine and the gap between scientific and clinical knowledge are evaluated.

Stromal accumulation of chondroitin sulphate in mammary tumours of dogs.

To contribute to the investigation of the composition of the extracellular matrix in epithelial tumours, mammary gland tissues of dogs (including tumours, hyperplasias and normal tissue as well as metastatic lesions in lymph nodes and lung) were studied histochemically and immunohistochemically for distribution of sulphated glycosaminoglycans (s-GAGs). The formaline-fixed tissue was stained by alcian blue at pH 5.8, using the 'critical electrolyte concentration' to study the degree of sulphation of s-GAGs. s-GAGs were characterized by degradation with enzymes and nitrous acid and by immunohistochemistry with two anti-chondroitin sulphate monoclonal antibodies. The light microscopic investigation of s-GAG deposits revealed a limited number of patterns of their distribution. The main s-GAGs found in the mammary gland tumours of dogs and in metastatic lesions were chondroitin sulphate (CS) and heparin/heparan sulphate (HEP/HS). CS accumulated in diffuse structures between epithelial cells as well as around clusters of tumour cells. The latter pattern, possibly representing a mesenchymal reaction to the tumour, was present in 74% of the tumours, and in 67% of these, highly sulphated CS was present. A diffuse accumulation of CS was present almost exclusively in complex and mixed tumours; because of the expression of the 3B3 epitope for CS in immature cartilage the spindle cells of complex tumours are argued to be the precursors of the cartilage in mixed tumours. HEP/HS was stored mainly in mast cells that were found in increased numbers in hyperplasias and tumours. By pretreatment of microscopic slides with chondroitinase AC or ABC immunostaining of fibronectin could be made possible in areas in which CS was abundantly present, suggesting that CS may mask fibronectin epitopes. It is concluded that CS with different degrees of sulphation is the most important s-GAG in the extracellular matrix of mammary tumours of dogs. CS and other s-GAGs accumulate at different sites and may have a different pathogenetic significance.

Expression of growth hormone in canine mammary tissue and mammary tumors. Evidence for a potential autocrine/paracrine stimulatory loop.

The role of progestins in the pathogenesis of breast cancer in women remains controversial. To advance this discussion, we report the demonstration and localization of progestin-induced biosynthesis of growth hormone (GH) in canine mammary gland tissue. Nontumorous mammary tissues and tumors, both benign and malignant, were obtained from private household dogs. Immunoreactive GH was localized in mammary epithelial cells and correlated with the presence of GH mRNA. Local synthesis of GH was also proven immunoelectron microscopically by demonstrating GH-containing secretory granules. Cellular GH production in nontumorous tissues was more extensive during the progesterone-dominated luteal phase of the ovarian cycle or during exposure to synthetic progestins than during anestrus. GH was also associated with areas of hyperplastic mammary epithelium, which may indicate that locally produced GH enhances proliferation, acting in an autocrine and/or paracrine manner. In 41 of 44 tumors, GH was present. Of 3 GH-negative tumor samples, 2 were from progestin-depleted, castrated bitches. In nonmalignant mammary tissues, GH production is stimulated by progesterone and synthetic progestins interacting with progesterone receptors. In some progesterone-receptor-negative malignant tumors, GH expression was found, indicating loss of this control. Progestin-induced GH probably participates in the cyclic development of the mammary gland but may promote mammary tumorigenesis by stimulating proliferation of susceptible, and sometimes transformed, mammary epithelial cells.

Hierarchical organization of biological systems and the structure of adaptation in evolution and tumorigenesis.

Biological systems are structurally organized according to patterns repeated at each hierarchical level. Complex units are composed of so-called interactors, systems that by cooperative interaction maintain the structure of the complex unit. Interactors are composed of large numbers of assemblies of complex units of a limited number of types of a lower hierarchical level. Thus, macromolecules, cells, organisms and ecological communities should be defined as complex units, and cellular organelles, organs and oligospecies populations as the interactors between those units. The similarity of organization at each level should make it possible to describe patterns of structure at one level and apply it to organization at another level. This was tested for the structural aspects of adaptation as viewed in a pathobiological context. Adaptation is then viewed as the result of stress seen at the level of the interactors or at the level of the lower complex units related to the type of stress. Subsequently, this structure of adaptation was applied to adaptation in biological evolution and tumorigenesis, which has led to the conclusion that stress is a driving force for both and that an increase in number of organisms or cells may precede heritable changes or mutations, respectively.