Pectin-coated titanium implants are well-tolerated in vivo.

Multiform coated titanium implants are widely used in orthopedic and dental surgery. In this study, we have investigated the reactivity of pectin-coated titanium samples implanted under the latissimus dorsi-muscle fascia of rats. Samples were coated with two enzyme treated apple pectins; modified hairy regions (MHR-A and MHR-B) that differed in chemical structure. Aminated (AMI) and uncoated titanium (Ti) served as controls. The thicknesses of the peri-implant fibrous tissue capsules formed 1 or 3 weeks after implantation were measured as indicative of possible inflammatory reactions toward the biomaterials. After 1 week, the MHR-B implant was surrounded by a thicker fibrous capsule (42.9 microm) than any of the other sample types: MHR-A (33.2 microm), AMI (32.5 microm), and Ti (32.3 microm), the last one being the only statistically significant difference. After 3 weeks, however, this difference disappeared; the capsule thicknesses around MHR-B and Ti implants had decreased to the values found for AMI and MHR-A. Additionally, the capsule formation represents merely a stromal rather than an inflammatory reaction, as indicated by the absence of activated macrophages or foreign body giant cells in the capsules. These results indicate for the first time the in vivo tolerability of covalently linked pectins, and suggest the feasibility of pectin-coated bone and dental implants for clinical use.

The terminal prostaglandin synthases mPGES-1, mPGES-2, and cPGES are all overexpressed in human gliomas.

Prostaglandin E2 has been connected to processes promoting tumor growth in several human malignancies including gliomas. The terminal prostaglandin synthases mPGES-1, mPGES-2, and cPGES convert PGH2 into prostaglandin E2. The inhibition of their function could significantly reduce PGE2 levels in tumors while avoiding some side effects related to the inhibition of the upstream enzymes COX-1 and COX-2. In this study, the immunohistochemical staining of mPGES-1 and, for the first time, the staining of mPGES-2 and cPGES are characterized and compared with COX-1 and COX-2 staining in the same tumor samples of 94 human gliomas. The main results demonstrate over-expression of all three proteins, including cPGES and mPGES-2 that are commonly considered noninducible, in both low- and high-grade tumors. For all three proteins, average expression in tumor cells was higher in grade III tumors than grade II tumors. The analysis showed no correlation between tumor grade and staining of tumor cells or vascular endothelium with any of the antibodies except in oligodendrogliomas where moderate correlation (linear correlation coefficient 0.6; P < 0.01) could be found between tumor grade and tumor cell staining with mPGES-1 and cPGES. In grade II tumors which recurred and were reoperated upon during the data gathering period, average expression of COX-2, mPGES-1, and cPGES was higher than in tumors that were operated on only once. Our results demonstrate the significance of all three terminal prostaglandin synthases, mPGES-1, mPGES-2, and cPGES, as a possible future target of inhibition in glioma therapy.

Angiogenesis and expression of angiogenic agents in uterine and ovarian carcinosarcomas.

Carcinosarcomas of the female genital tract are a heterogeneous group of aggressive malignant neoplasms characterized by poor prognosis that contain elements expressing both carcinomatous and sarcomatous characteristics. In this study specimens from 25 patients were treated with labeled antibodies to vascular endothelium (FVIII), and to vascular endothelial growth factor (VEGF) for analysis of angiogenesis, and to vascular endothelial growth factor receptor 3 (VEGFR-3) for analysis of lymphangiogenesis, in 11,099 vessels. Automated quantitative image analysis was used and the results were compared with clinical data. Microvessel density increased from a median value of 18.32 vessels/mm(2) in non-neoplastic stroma to 131.25 vessels/mm(2) in neoplasms. In areas around tumor islets expressing predominantly epithelial carcinomatous characteristics, microvessel density was increased three-fold compared with the islets themselves. Vessels were arranged in a garland-type pattern, or in bursts, and they exhibited directional angiogenesis. Clinical indicators of poor survival were high tumor stage (p=0.002) and age above 65 (p=0.0769). A high number of small vessels (16-300 mum(2) in cross-sectional area) predicted poor survival (p=0.0149), and more so in tumors exhibiting predominantly sarcomatous characteristics (p=0.0087). Tumor tissue area above the median exhibiting VEGF expression was also a sign of poor survival (p=0.0267), as was an area of positive staining for VEGFR-3 exceeding the median (p=0.00487). In this study, active angiogenesis (increased number of vessels, variable in shape and exhibiting decreased antibody staining intensity) was a distinct feature of carcinosarcomas, its extent and distribution depending upon neoplasm morphology. Increased vessel numbers and increased VEGF and VEGFR-3 expression indicated poor survival.

Increased angiogenesis is associated with poor prognosis of squamous cell carcinoma of the vulva.

BACKGROUND: Increased angiogenesis is associated with an increased risk of malignant transformation in many organs. The occurrence, characteristics and significance of angiogenesis in vulvar squamous cell carcinoma are less well known. METHODS: In this study, vessel number as well as vessel size, vessel shape and vessel antibody staining intensity were determined in 23,091 vessels in CD34-stained histological specimens of 42 patients by computer-assisted quantitative image analysis methods and findings were related to tumor morphology, clinical characteristics and patient survival. RESULTS: The 2- and 5-year survival rates were 81.1 and 64.1%, respectively. High disease stage and high histological grade indicated poor prognosis. In an age-adjusted multivariate analysis of stage, grade and extent of invasion, these variables compiled a statistically significant prognostic factor (p=0.001). Tumor histopathological type was a prognostic factor in our study and keratinising tumors did significantly better. Large tumor size, but not depth of invasion, and spray type growth pattern also indicated poor survival. In an age-adjusted multivariate analysis of stage, grade and extent of invasion, these variables compiled a statistically significant prognostic factor (p=0.001), but none of these variables alone proved to be an independent prognostic factor. High vessel number and increased vessel size were also significant indicators of poor survival, as were vessel characteristics: vessel number, vessel size, vessel shape and staining intensity together. CONCLUSION: Increased angiogenesis and altered vessel characteristics were prognostic factors in determining patient survival in this study. Analysis of angiogenesis provided new information on tumor behaviour and provided markers for survival analysis in neoplasms classified by tumor type and growth pattern.

Altered extent, pattern and characteristics of microvascular density are indicators of neoplastic progression in the endometrium.

The occurrence, structure and extent of microvascular density were examined in normal endometria, hyperplasia of different types and adenocarcinomas of different degrees of differentiation to determine their biologic and clinical significance in tumor development and progression. Computer-assisted quantitative image analysis was carried out on 12,500 vessels in regard to vessel number, vessel volume, size, shape and extent of vessel antibody staining, with sensitivity and reproducibility exceeding 99%. The results showed the extent, pattern and characteristics of microvascular density to be intimately associated with extent of tumor development and degree of differentiation of the tumor. Vessel number increased with superficial location in normal endometrium, with increased degree of hyperplasia and atypia and with increased degree of dedifferentiation of adenocarcinoma. Increased vessel shape alterations were characteristic of atypical complex hyperplasia when compared to other types of hyperplasia. Vessel number, size and shape were similar in proliferative endometrium and simplex type hyperplasia, and microvascular density in atypical complex hyperplasia was similar to that in well-differentiated adenocarcinoma. The results indicated that vessel shape alterations occur during progression of hyperplasia and vessel size increase occurs in complex-type hyperplasia and in moderately differentiated adenocarcinomas. We conclude that microvascular density is associated with endometrial location and with specific patterns of alteration in different stages of endometrial disease. The results suggest potential clinical applications of vessel analysis for determination of clinical behavior of endometrial preneoplastic and neoplastic alterations.

Synthesis and maturation of type I and type III collagens in endometrial adenocarcinoma.

OBJECTIVE: The structure and distribution of type I and type III collagens in the extracellular matrix of malignant endometrium was evaluated for their roles in the development and progression of this neoplasm. STUDY DESIGN: Collagen synthesis and deposition in endometrial adenocarcinomas was determined by immunohistochemical analysis of type I and type III procollagen and verified by computer-assisted morphometry and in situ hybridization. RESULTS: In the stroma of well-differentiated adenocarcinomas increased intracellular collagen synthesis was observed in fibroblastic cells as well as increased extracellular formation of newly synthesized type I and type III procollagen. Collagen maturation was also rapid. In moderately differentiated tumors, destruction and dissolution occurred around invading islets, concomitantly with decreased deposits of both collagens, despite increases in corresponding mRNAs. In poorly differentiated neoplasms, solid epithelial islets coexisted with sparse and distinctly collagen-positive stroma. Poorly differentiated neoplasms also contained tumor cells exhibiting intracellular collagen staining as well as in situ hybridization signals. In highly malignant papillary adenocarcinomas, the tumor cells induced distinctly increased collagen synthesis and deposition of newly synthesized collagen but not the mature cross-linked protein. CONCLUSIONS: In malignancy, compression of surrounding stroma and a fibroproliferative response with increased collagen synthesis and deposition may prevent tumor growth. In more advanced lesions, stromal dissolution may permit tumor spread and in highly malignant lesions an abnormal stroma may promote neoplasm progression.

Stage-dependent expression of an angiogenic agent and vascular organization in experimental skin tumor development.

Increased angiogenesis and expression of antibodies to vascular endothelial growth factor (VEGF), an angiogenic agent, have been shown in the tumor development of many tissues. Areas of skin expressing VEGF and total volume of vessels expressing laminin in the wall were measured in chemical carcinogen-exposed mice using CAS-200 morphometry apparatus having a sensitivity exceeding 99% and reproducibility exceeding 99%. The area of VEGF expression was increased in carcinogen-exposed skin, dysplasia and in well-differentiated squamous cell carcinomas, but decreased in squamous cell carcinomas with decreased degree of differentiation. The vessel volume increased prior to the formation of tumors in carcinogen-exposed skin as well as in highly malignant neoplasms. In well-differentiated squamous cell carcinomas with an expansive growth pattern, the vessels were parallel to the basal membrane, in moderately differentiated tumors the vessels were in the direction of tumor invasion, and in poorly differentiated tumors, active angiogenesis consisted of numerous, enlarged vessels within the tumor. This study showed increased VEGF expression and number of vessels occurring in early stages of skin tumor development, pointing to a role of angiogenesis in chemical risk assessment and in cancer prevention. Altered vessel structure and vessel arrangement were distinct in later stages of tumor growth and in malignant neoplasms, pointing to the utility of detailed vessel analysis in neoplasm characterization.

Automated image analysis of proliferating cells in carcinoma of the larynx.

OBJECTIVE: To determine the role of cell proliferation neoplastic progression in the larynx and possibly derive criteria of clinical significance using automated quantitative image analysis. MATERIAL AND METHODS: In a retrospective study involving archival material, the occurrence and location, size, shape and staining intensity of proliferating cell nuclear antigen (PCNA)-positive cells (12,538 cells in total) were analyzed in squamous cell carcinoma (SCC), as well as in pre- and non-neoplastic conditions, using computer-assisted morphometry with reproducibility and sensitivity exceeding 99%. RESULTS: Immunohistochemically detectable PCNA-positive cells were located in the basal layer in non-neoplastic states, in well-differentiated SCCs in layers adjacent to the basal membrane and in poorly differentiated neoplasms in the neoplastic epithelial islets. An increased degree of dysplasia was associated with an increased number of PCNA-immunoreactive cells of increased nuclear size and staining intensity. There was a significant difference between carcinomas and dysplasia in terms of altered nuclear shape. With increasing malignancy of SCCs, nuclear shape alterations and PCNA staining intensity increased, whilst nuclear size decreased. CONCLUSIONS: Automated image analysis of cell populations allowed the identification of populations of malignant cells and provided information on the severity of preneoplastic and neoplastic conditions of use in studies of tumor behavior and with potential clinical application.

Apoptosis and apoptosis-related factors Bcl-2, Bax, tumor necrosis factor-alpha, and NF-kappaB in human endometrial hyperplasia and carcinoma.

BACKGROUND: Apoptosis controls cell homeostasis in the endometrium during normal menstrual cycles, and morphologic studies have suggested its association with the development of endometrial carcinoma. Apoptosis is regulated by several genes, especially those of the Bcl-2 gene family, but their significance in endometrial pathologies is not well understood. METHODS: To study the role and regulation of apoptosis in endometrial hyperplasia and carcinoma, human endometrial specimens were analyzed using in situ 3'-end labeling of apoptotic cells and in situ hybridization and immunohistochemistry of apoptosis-related factors. RESULTS: Apoptosis was scarce in normal proliferating endometrium as well as in simplex, complex, and atypical hyperplasia and was low in Grade I adenocarcinoma. In Grade II adenocarcinoma a significant increase in the rate of apoptosis was observed. Apoptosis decreased in Grade III adenocarcinoma, but it was still higher than in normal or hyperplastic endometrium. Bcl-2 and Bax were expressed in normal and hyperplastic endometrium, and the Bcl-2/Bax ratio was lower in endometrial carcinoma. Tumor necrosis factor-alpha was expressed in normal endometrium and simplex and complex hyperplasia, but it was down-regulated in atypical hyperplasia and endometrial carcinoma. The transcription factor NF-kappaB was present in proliferating endometrium and in endometrial hyperplasia, but its expression was lower in carcinoma. CONCLUSIONS: In endometrial proliferation and hyperplasia a low rate of apoptosis is present. In Grade I carcinoma the rate of apoptosis is decreased, but the rate is subsequently increased in advanced carcinoma. The decrease in the rate of apoptosis in Grade III adenocarcinoma may reflect loss of control of cell homeostasis, decreased differentiation, and increased malignancy.

Collagen formation in extracellular matrix of transplants of human transformed keratinocyte cell lines.

BACKGROUND, MATERIALS AND METHODS: The role of epithelial cell growth and neoplastic transformation on collagen formation and deposition in the extracellular matrix (ECM) was analyzed by culturing immortalized human epidermal cell lines and Ras-transformed benign and malignant clones on collagen gels as transplants. The lesions were analyzed for extent of growth and morphology of epithelial and mesenchymal components as well as synthesis and deposition of different collagens. RESULTS: Immortalized cell lines required up to 5 weeks of growth for a well-organized mesenchyme to develop; transplants of Ras-transformed benign clones needed 3 weeks and transplants of highly malignant clones only 2 weeks to form an organized stroma. In transplants of immortalized cells after 2 weeks of growth newly-synthesized collagen type I and type III were deposited in the mesenchyme adjacent to the muscle, forming a mature ECM, while ECM was absent adjacent to growing, differentiated, immortalized cells. In transplants of Ras-transformed benign clones the subepithelial ECM was immature at day 14, but it was forming fibers at the same time in transplants of malignant clones. These were seen as thin irregular fibers in immunohistochemistry, ultimately organized into fibrillar structures in similar locations to active synthesis detected by in situ hybridization. Depositions of crosslinked mature type I collagen occurred later in similar locations. Type III collagen synthesis and deposition was most prominent in transplants of malignant cell clones, with degradation and destruction of the extracellular matrix around invading islets of malignant cells. CONCLUSION: The development of mesenchyme was directly related to duration of growth of transplants and degree of malignancy; mesenchyme organization was inversely related to differentiation of the epithelial cells. The results showed the usefulness of the transplant model in studies on cell and tissue growth and organization.

Downregulation of estrogen-metabolizing 17 beta-hydroxysteroid dehydrogenase type 2 expression correlates inversely with Ki67 proliferation marker in colon-cancer development.

The 17HSDs are a group of isozymes that catalyze the interconversion between high-activity 17 beta-hydroxysteroids and low-activity 17-ketosteroids. In the present study, we characterized the expression of 17HSD types 1 and 2 in normal and malignant gastrointestinal tissues and cells. Using the colon as a model for cancer of the gastrointestinal tract, expression of the 17HSD enzymes in cancer development was studied and correlated with proliferation and differentiation markers as assessed by Ki67 and mucin staining, respectively. In normal colon and small intestine, 17HSD type 2 mRNA was expressed in the surface epithelial cells and, to a lesser extent, in the cryptal epithelial cells. In colon-cancer specimens, 17HSD type 2 expression was downregulated both in the tissues and in the cell lines and correlated inversely with the proliferation marker. No expression for the 17HSD type 1 enzyme was observed in normal or cancerous gastrointestinal tract tissues. In line with the expression studies, 17HSD activity measurements with colon cells showed that only the oxidative conversion of E2 to E1 was present, and Northern blot analysis showed the signal only for 17HSD type 2. Localization of the ERs alpha and beta, assessed by immunohistochemistry and in situ hybridization, showed the presence of ER beta in the lamina propria of the colon. Our study shows that 17HSD type 2 expression is associated with the functional integrity of the gastrointestinal tract. The decrease in expression of the type 2 enzyme may increase estrogen influence in colon cancer.