How evolution of the nailing system improves results and reduces orthopedic complications: more than 2000 cases of trochanteric fractures treated with the Gamma Nail System.

PURPOSE: The incidence of trochanteric fractures has increased significantly during the last few decades, especially in elderly patients with osteoporosis. The dynamic/sliding hip screw and the cephalomedullary nail are the most commonly used fixation methods to treat trochanteric fractures. The improvements in the Gamma Nail System (GNS) associated with a correct surgical technique reduced the postoperative orthopedic complications. The purpose of this study was to compare the results of the different Gamma Nails. METHODS: The present study is a retrospective analysis of 2144 patients treated with GNS between January 1997 and December 2011 for trochanteric fractures, classified according to AO classification method. The patients were divided into three groups according to the nailing system: 525 were treated with Standard Gamma Nail (SGN), 422 with Trochanteric Gamma Nail (TGN) and 1197 with Gamma3 Nail. RESULTS: The overall incidence of intra-operative complications was 1.21 %; the incidence of intra-operative complications for each group was 1.71 % for SGN group, 0.47 % for TGN group and 1.25 % for Gamma3 Nail group. The overall incidence of postoperative complications was 5.48 %, and the incidence for each group was 10.73 % for SGN group, 9.92 % for TGN group and 2.92 % for Gamma3 Nail group. CONCLUSION: The GNS is a safe device with a low rate of intra-operative complications. The evolution of this nail system reduces postoperative complications, thus improving the results at follow-up and confirming that the Gamma3 Nail is a safe and predictable device to fix trochanteric fracture.

Induction of a multifactorial resistance phenotype by high paclitaxel selective pressure in a human ovarian carcinoma cell line.

Paclitaxel (PTX) is a potent anti-neoplastic agent that is highly effective in treating ovarian cancer. Nevertheless, the emergence of PTX resistance has limited the control of this disease. To gain insight into the molecular alterations accompanying drug resistance in ovarian cancer, we generated a new stable PTX-resistant ovarian carcinoma cell line. CABA I cells, which display an intrinsic PTX resistance (IC50 = 800 ng/ml), were subjected to continuous exposure to PTX. From the residual surviving cells, the highly PTX-resistant line CABA-PTX (IC50 = 256000 ng/ml) was generated and stably maintained in vitro. Analysis of beta-tubulin expression indicated that only the HM40 and Hbeta9 isotypes were expressed in both parental and resistant cells. No specific point mutations in the HM40 were detected in either cell line, but expression levels of this isotype were significantly reduced (40%) in CABA-PTX cells. Hbeta9 levels were unchanged. In those cells, PTX resistance was associated with cross-resistance to vinblastine but not to methotrexate or 5-fluorouracil. Verapamil treatment did not reverse the intrinsic drug resistance of parental cells, but partially modulated the sensitivity of CABA-PTX cells to PTX and induced total sensitivity to vinblastine. No changes in the cell surface expression of the drug efflux pumps MRP1, MRP2 and P-glycoprotein were observed. PTX influx, monitored using a fluorescent drug derivative, was significantly reduced and delayed in CABA-PTX cells as compared to the parental cells. Together, these findings suggest that more than one mechanism is involved in PTX resistance, making CABA-PTX cell line a potentially valuable in vitro tool to study multifactorial acquired drug resistance in ovarian cancer.

Matrix-degrading proteinases are shed in membrane vesicles by ovarian cancer cells in vivo and in vitro.

The in vitro release of matrix-degrading proteinases from breast cancer cells is associated in part with shed membrane vesicles. To determine whether shed vesicles might play a similar role in ovarian cancer cells, we analyzed the shedding phenomenon in vivo and in vitro as well as the enzymatic content of their vesicles. This is the first time that an immunoelectron microscopical analysis revealed membrane vesicles carrying tumor-associated antigen alpha-Folate Receptor (alpha-FR), circulating in biological fluids (ascites and serum) of an ovarian carcinoma patient. These vesicles were trapped in a fiber network with characteristic fibrin periodicity. An ovarian cancer cell line (CABA I) established from ascitic fluid cells of this patient, grew in Matrigel and formed tubular structures suggesting invasive capability. Immunofluorescence analysis demonstrated strong cytoplasmic staining of CABA I cells with anti-matrix metalloproteinase-9 (MMP-9) and anti-urokinase-type plasminogen activator (uPA) antibodies. CABA I cells shed membrane vesicles, which were morphologically similar to those identified in vivo, as determined by electron microscopy. Gelatin zymography of vesicles isolated both in vivo and in vitro revealed major gelatinolytic bands of the MMP family, identified as the zymogen and active forms of gelatinase B (MMP-9) and gelatinase A (MMP-2). By casein-plasminogen zymography we observed high-molecular weight (HMW)-uPA and plasmin bands. Incubation of purified vesicles from CABA I cells with Matrigel led to cleavage of Matrigel components. Taken together, our results point to a possible role of shed vesicles, both in vivo and in vitro, in proteolysis that mediates invasion and spread of ovarian epithelial carcinoma cells.

Selective localization of matrix metalloproteinase 9, beta1 integrins, and human lymphocyte antigen class I molecules on membrane vesicles shed by 8701-BC breast carcinoma cells.

The shedding of membrane vesicles from the cell surface is a vital process considered to be involved in cell-cell and cell-matrix interactions and in tumor progression. By immunoelectron microscopic analysis of surface replicas of 8701-BC human breast carcinoma cells, we observed that membrane vesicles shed from plasma membranes contained densely clustered gelatinase B [matrix metalloproteinase 9 (MMP-9)], beta1 integrins, and human lymphocyte antigen class I molecules. By contrast, alpha-folate receptor was uniformly distributed on the smooth cell membrane and shedding areas. Both cell surface clustering of selected molecules and membrane vesicle release were evident only when cells were cultured in the presence of serum. Vesicle shedding occurred preferentially at the edge or along narrow protrusions of the cell. Specific accumulation of proMMP-9 and active forms of MMP-9 in shed vesicles was also demonstrated by gelatin zymography. In addition, Western blotting analysis showed the presence of a large amount of proMMP-9/tissue inhibitor of metalloproteinase 1 complex. The release of selected areas of plasma membranes enriched with MMP-9 and beta1 integrins indicates that membrane vesicle shedding from tumor cells plays an important role in the directional proteolysis of the extracellular matrix during cellular migration. The presence of human lymphocyte antigen class I antigens suggests a mechanism for tumor cells to escape from immune surveillance.

Plasminogen activator system modulates invasive capacity and proliferation in prostatic tumor cells.

The malignant phenotype of prostatic tumor cells correlates with the expression of both uPA and its cell-membrane receptor (uPAR); however, there is little information concerning the role of cell-bound uPA in matrix degradation and invasion. Our results suggest that cell-associated uPA plays a key role in regulating the amount of plasmin present at the surface of prostatic carcinoma (PRCA) cells and show that differential production of uPA corresponds with the capacity to bind and activate plasminogen. In addition, we provide direct evidence that both uPA secretion and the presence of uPA-uPAR complexes characterize the invasive phenotype of PRCA cells and suggest the existence of several pathways by which tumor cells acquire plasmin activity. LNCaP cells (which do not produce uPA but express uPAR) may activate plasmin through exogenous uPA. In vivo, the source of uPA may be infiltrating macrophages and/or fibroblasts as observed in several other systems. PAI-1 accumulation in the conditioned medium (CM) limits plasmin action to the pericellular microenvironment. Our results indicate that MMP-9 and MMP-2 are also activated by plasmin generated by cell-bound but not by soluble, extracellular uPA. Plasmin activation and triggering of the proteolytic cascade involved in Matrigel invasion is blocked by antibodies against uPA (especially by anti- A-chain of uPA which interacts with uPAR) and by PA inhibitors such as p-aminobenzamidine which may regulate levels of cell-bound uPA. uPA may also regulate growth in PRCA cells. Indeed, antibodies against uPA A-chain (and also p-aminobenzamidine treatment) interfere with the ATF domain and inhibit cell growth in uPA-producing PC3 and DU145 prostate cancer cell lines, whereas exogenous uPA (HMW-uPA with ATF) induces growth of LNCaP prostate tumor cell line. These data support the hypothesis that in prostatic cancer patients at risk of progression, uPA/plasmin blockade may be of therapeutic value by blocking both growth of the primary tumor and dissemination of metastatic cells.

Ultrastructural and phenotypic characterization of CABA I, a new human ovarian cancer cell line.

We have established an ovarian cancer cell line (CABA I) from ascitic fluid obtained from a patient with papillary adenocarcinoma of the ovary prior to drug treatment. The epithelial origin of the cell line was confirmed by morphology and by immunofluorescence analysis using anticytokeratin antibodies. Ultrastructural analysis revealed a very irregular membrane surface and a clear cytoplasm rich in electron-lucent vesicles. CABA I cells grow rapidly in culture (doubling time 18 h) in an anchorage-independent manner. Exogenously added beta-estradiol and epidermal growth factor (EGF) treatments did not influence cell growth rate. FACS analysis to determine the phenotypic profile of tumor-associated antigen, membrane receptor, and adhesion molecule expression indicated that the cell line was positive for different members of the c-erbB family, for alpha 6 and beta 1 integrin receptors, and intensively positive for HLA class I antigens and the folate receptor. Molecular characterization revealed no mutations for c-myc and c-k-ras genes, but did detect an exon 5 mutation in the p53 gene. CABA I cells grew poorly as heterotransplants in nude mice, and tumors showed long latency periods. Because early (15-20) and late (55-60) passage cells maintain the same growth and phenotypic characteristics, the CABA I cell line might provide a good in vitro model system to investigate the cellular and molecular events involved in ovarian carcinogenesis.

Increased matrix metalloproteinase-9 secretion in short-term tissue cultures of prostatic tumor cells.

We have examined the expression of 2 tumor-associated metalloproteinases, MMP-2 and MMP-9, in 48 primary cultures of prostatic carcinoma (PRCA) and 33 cultures of benign prostatic hyperplasia (BPH). PRCA cultures secrete significantly more MMP-9 than their benign counterparts. Secreted MMP-2 did not differ significantly in cultures but was lower in PRCA cultures. Two cultures of benign origin exhibited high MMP-9 secretion and growth patterns consistent with a malignancy. Both cases were followed and successively re-evaluated histologically and rediagnosed as organ-confined PRCA. MMP expression in culture may be of predictive value in the identification of incidental PRCA. MMP-9 secretion and its ratio with MMP-2 were highest in epithelial cultures from invasive, metastatic tumors when compared both to disease confined to prostate gland and to locally extensive disease. MMP-9 secretion was greatest also in cultures derived from tissues of high Gleason histological grade. Active MMP-9 species were detected in 15 cultures (31%) of PRCA. Active MMP-2 species were observed in cultures of both BPH and PRCA origin in almost the same amounts. Although average levels were not significantly different, as a ratio to proform species, a significant elevation was observed in cultures of PRCA origin. We propose, therefore, that an elevated expression of MMP-9 and a high ratio of MMP-9 to MMP-2 in short-term prostate epithelial cultures is of potential diagnostic and prognostic significance.