Survival of modern knee tumor megaprostheses: failures, functional results, and a comparative statistical analysis.

BACKGROUND: Modular megaprostheses are now the most common method of reconstruction after segmental resection of the long bones in the lower extremities. Previous studies reported variable outcome and failure rates after knee megaprosthetic reconstructions. QUESTIONS/PURPOSES: The objectives of this study were to analyze the results of a modular tumor prosthesis after resection of bone tumor around the knee with respect to (1) survivorship; (2) failure rate; (3) comparative survivorship against different sites of reconstructions and of primary and revision implants; and (4) functional results on the Musculoskeletal Tumor Society (MSTS) scoring system. METHODS: Between 2003 and 2010, 247 rotating-hinge Global Modular Reconstruction System (GMRS) knee prostheses were implanted in our institute for malignant and aggressive benign tumors. During this time, that group represented 23% of the patients who had oncologic megaprosthesis reconstruction about the knee after resection of primary or metastatic bone tumors (247 of 1086 patients). In the other 77% of cases we used other types of oncologic prostheses. Before 2003 we used the older Howmedica Modular Resection System and Kotz Modular Femur/Tibia Replacement from 2003 we used mostly the GMRS but we continued to use the HMRS in some cases such as patients with poor prognoses, elderly patients, or metastatic patients. Sites included 187 distal femurs and 60 proximal tibias. Causes of megaprosthesis failure were classified according to Henderson et al. in five types: Type 1 (soft tissue failure), Type 2 (aseptic loosening), Type 3 (structural failure), Type 4 (infection), and Type 5 (tumor progression). Followup was at a minimum oncologic followup of 2 years (mean, 4 years; range, 2-8 years). Kaplan-Meier actuarial curves of implant survival to major failures were done. Functional results were analyzed according to the MSTS II system; 223 of the 247 were available for functional scoring (81%). RESULTS: At latest followup, among 175 treated patients for primary reconstruction, 117 are continuously disease-free, 26 have no evidence of disease after treatment of relapse, eight are alive with disease, and 24 died from disease. The overall failure rate of the megaprostheses in our series was 29.1% (72 of 247). Type 1 failure occurred in 8.5% (21 of 247) cases, Type 2 in 5.6% (14 of 247), Type 3 in 0%, Type 4 in 9.3% (23 of 247), and Type 5 in 5.6% (14 of 247). Kaplan-Meier curve showed an overall implant survival rate for all types of failures of 70% at 4 years and 58% at 8 years. Prosthetic survivorship for revisions was 80% at 5 years and for primary reconstructions was 60% at 5 years (p = 0.013). Survivorship to infection was 95% at 5 years for revision patients and 84% at 5 years for primary patients (p = 0.475). The mean MSTS score was 84 (25.2; range, 8-30) with no difference between sites of localization (24.7 in proximal tibia versus 25.4 in distal femur reconstruction; p = 0.306). CONCLUSIONS: Results at a minimum of 2 years with this modular prosthesis are satisfactory in terms of survivorship (both oncologic and reconstructive) and causes and rates of failure. Although these results seem comparable with other like implants, we will continue to follow this cohort, and we believe that comparative trials among the available megaprosthesis designs are called for. LEVEL OF EVIDENCE: Level IV, therapeutic study. See Guidelines for Authors for a complete description of levels of evidence.

Indole-3-carbinol selectively uncouples expression and activity of estrogen receptor subtypes in human breast cancer cells.

Estrogen-responsive breast cancer cells, such as MCF7 and T47D cells, express both estrogen receptor (ER)-alpha (ERalpha) and ERbeta. Indole-3-carbinol (I3C) strongly down-regulated ERalpha protein and transcript levels, without altering the level of ERbeta protein, in both cell lines. In cells transfected with the ERalpha promoter linked to a luciferase gene reporter, I3C ablated ERalpha promoter activity. Propyl pyrazole triol (PPT) is a highly selective ERalpha agonist, whereas, 17beta-estradiol activates both ERalpha and ERbeta. I3C treatment inhibited the PPT- and 17beta-estradiol-induced proliferation of breast cancer cells, disrupted the PPT and 17beta-estradiol stimulation of estrogen response element (ERE)-driven reporter plasmid activity as well as of endogenous progesterone receptor transcripts. Using an in vitro ERE binding assay, I3C was shown to inhibit the level of functional ERalpha and stimulated the level of ERE binding ERbeta even though the protein levels of this receptor remained constant. In ERalpha-/ERbeta+ MDA-MB-231 breast cancer cells, I3C treatment stimulated a 6-fold increase in binding of ERbeta to the ERE. I3C also induced ERE- and activator protein 1-driven reporter plasmid activities in the absence of an ER agonist, suggesting that ERbeta is activated in indole-treated cells. Taken together, our results demonstrate that the expression and function of ERalpha and ERbeta can be uncoupled by I3C with a key cellular consequence being a significantly higher ERbeta:ERalpha ratio that is generally highly associated with antiproliferative status of human breast cancer cells.

Monocyte membrane type 1-matrix metalloproteinase. Prostaglandin-dependent regulation and role in metalloproteinase-2 activation.

Membrane type 1-matrix metalloproteinase (MT1-MMP)-mediated activation of MMP-2 is thought to be important in the proteolysis of extracellular matrix in pathological events in which monocytes/macrophages are found. Here we report on the induction and regulation of human monocyte MT1-MMP and its role in MMP-2 activation. Activation of monocytes by lipopolysaccharide resulted in the induction of MT1-MMP mRNA and protein that was suppressed by inhibitors of prostaglandin synthesis (indomethacin), adenylyl cyclase (SQ 22536), and protein kinase A (Rp-cAMPs). Suppression of MT1-MMP by indomethacin and SQ 22536 was reversed by prostaglandin E(2) and dibutyryl cyclic AMP, respectively, demonstrating that induction of monocyte MT1-MMP is regulated through a prostaglandin-cAMP pathway. Functional analysis revealed that pro-MMP-2 in the supernatants from human bone marrow stromal fibroblasts, normal male-derived fibroblasts and melanoma cells (A2058) was converted to active MMP-2 when cultured with activated but not control monocytes. Antibodies against MT1-MMP blocked the activation of MMP-2. Tissue inhibitor of metalloproteinase-2 regulation of MMP-2 activation was shown through the addition of varying amounts of recombinant tissue inhibitor of metalloproteinase-2 with pro-MMP-2 to MT1-MMP-expressing monocytes. These findings demonstrate that activated monocytes express functionally active MT1-MMP that may play a significant role in the activation of MMP-2 produced by other cells and as such influence developmental and pathological conditions.

Inactivating mutation of the mouse tissue inhibitor of metalloproteinases-2(Timp-2) gene alters proMMP-2 activation.

To understand the biologic function of TIMP-2, a member of the tissue inhibitors of metalloproteinases family, an inactivating mutation was introduced in the mouse Timp-2 gene by homologous recombination. Outbred homozygous mutants developed and procreated indistinguishably from wild type littermates, suggesting that fertility, development, and growth are not critically dependent on TIMP-2. Lack of functional TIMP-2, however, dramatically altered the activation of proMMP-2 both in vivo and in vitro. Fully functional TIMP-2 is essential for efficient activation of proMMP-2 in vivo. No evidence of successful functional compensation was observed. The results illustrate the duality of TIMP-2 function, i.e. at low concentrations, TIMP-2 exerts a "catalytic" or enhancing effect on cell-mediated proMMP-2 activation, whereas at higher concentrations, TIMP-2 inhibits the activation and/or activity of MMP-2.

Glycosylation and NH2-terminal domain mutants of the tissue inhibitor of metalloproteinases-1 (TIMP-1).

Mutants in the tissue inhibitor of metalloproteinases-1 (TIMP-1) protein have been created by site-directed mutagenesis and expressed in HeLa cells, using a recombinant vaccinia virus system. Removal of either or both glycosylation sites yielded proteins which retained wild-type inhibitory activity against both human fibroblast-type collagenase (FIB-CL) and Mr 72000 gelatinase (GL). However, the double glycosylation mutant protein was expressed at a level that was 2-4-fold lower than that of the wild-type or the single site glycosylation mutants. The 'tiny-TIMP' COOH-terminal deletion mutant that lacks the last 57 residues was also inhibitory, but the dose-response curve suggested that the interaction with the Mr 72000 gelatinase had been altered. A number of replacement mutants in the highly conserved NH2-terminal domain, including replacement of P5A and P8A or a double mutation in the VIRAK sequence which is absolutely conserved in all TIMPs in all species (VIRAK to VIAAA), also yielded functional proteins capable of inhibiting FIB-CL and Mr 72000 GL and of forming SDS-resistant complexes with FIB-CL. None of the above manipulations abolished inhibitory function suggesting that binding of the inhibitor by the enzyme involves multiple interactions.

Replacement of conserved cysteines in human tissue inhibitor of metalloproteinases-1.

Tissue inhibitor of metalloproteinases-1 (TIMP-1) is resistant to extremes of temperature and pH. This is thought to be due in part to the presence of six sulfhydryl bridges presumed to maintain the structural integrity of the molecule. As part of a study looking at structure-function relationships, a number of the conserved cysteine residues in TIMP-1 were targeted for replacement with serine. Single and double replacements of these conserved cysteines, as well as replacements around these cysteines, were expressed using a vaccinia virus system and analyzed for functional and structural competence. Analysis by circular dichroism indicated that these mutants maintained secondary structures similar to those of wild-type TIMP-1. Trypsin susceptibility experiments indicated that the tertiary structure of the mutants had not been drastically changed. Analysis of functional competence demonstrated that there were significant changes in some of these mutants. Assays using collagen fibrils or gelatin as substrates indicated that the double mutant C1S/C70S, but not C3S/C99S, had lost inhibitory activity against human fibroblast-type collagenase (FIB-CL) and at high concentrations only had slight activity against Mr 72,000 gelatinase (Mr 72,000 gelatinase). Kinetic analysis of TIMP-1 inhibition of FIB-CL cleavage of a peptide substrate indicated that mutants C1S/C70S, C3S/C99S, and CEEC --> CQQC retained their ability to inhibit FIB-CL in a manner similar to wild-type TIMP-1, while mutants C1S and C70S showed little inhibitory activity. The mutants C99S and C137S could also inhibit FIB-CL cleavage of the peptide substrate. The results indicated that the degree of inhibition by the TIMP-1 mutants varied somewhat depending on the choice of substrates. Interestingly, replacing both cysteines from a disulfide bond in the wild-type molecule resulted in a more competent inhibitor than either of the single site "parent" mutations. Taken together, these experiments indicate that TIMP-1 can be rendered inactive by the loss of a single cysteine.

The mechanism of inhibition of collagenase by TIMP-1.

Tissue inhibitor of metalloproteinase-I (TIMP-1) is a slow, tight-binding inhibitor of fibroblast-type collagenase. Time-course data from inhibition experiments were analyzed by graphic analysis, by nonlinear regression of the analytic integrals of the rate equations and by nonlinear regression with numeric integration of the rate equations. With the same assumptions, approximations and data, all three methods of analysis produced the same model preferences and values for the kinetic parameters. The time-course data for the inhibition of fibroblast-type collagenase by TIMP-1 are best described by the equations for a noncompetitive two-step mechanism, in which an inactive, rapidly formed, reversible complex slowly forms an inactive, tight complex. However, from the analysis of data from experiments at concentrations of TIMP-1 comparable to that of collagenase, it is apparent that free TIMP-1 also functions in the breakdown of the tight complex. The rapidly formed complex has a dissociation constant of 8 nM and reacts to the tight complex with a first-order rate constant of 0.003 s-1. The back reaction of the tight complex to the rapid complex has a second-order rate constant of 5 x 10(4) M-1 s-1. The resulting global dissociation constant of the tight complex is 0.1 nM at 3 nM TIMP-1 and collagenase concentration. Collagenase without the carboxyl-terminal domain (mini-collagenase) is inhibited by TIMP-1 according to a mechanism, in which the rapidly formed complex has such a high dissociation constant (247 nM) that it effectively constitutes a one-step mechanism, in which TIMP-1 binds with an apparent second-order rate constant of 9.6 x 10(4) mol-1 s-1 and the enzyme-TIMP-1 complex dissociates with a first order rate constant of 0.00026 s-1. The apparent global dissociation constant for the tight complex (2.7 nM) is higher than that for the fibroblast-type collagenase. Participation of TIMP-1 in the dissociation is not demonstrable. Therefore, the carboxyl-terminal domain of fibroblast-type collagenase is important for the initial, rapid binding of TIMP-1 and the initial complex contributes to the overall binding.

Functional domains of human TIMP-1 (tissue inhibitor of metalloproteinases).

To define domains of tissue inhibitor of metalloproteinases (TIMP-1) that are important to its ability to inhibit fibroblast-type collagenase (FIB-CL), two different approaches were used: (i) competition with synthetic peptides modeled after the human TIMP-1 sequence and (ii) localization of epitopes of blocking antibodies. TIMP-1 consists of six loops, held in place by six disulfide bonds arranged in three knotlike structures. Several long peptides (n = 20-34), together covering three-fourths of the human TIMP-1 sequence, were able to block inhibition of human FIB-CL by TIMP-1. While most of these peptides were modeled after sequences in the NH2-terminal domain of the molecule (loops 1, 2, and 3), they also included two-thirds of the residues of the COOH-terminal domain including loops 4 and 5 and the COOH-terminal tail but not loop 6. Refinement by competition with shorter peptides (7-10 residues) showed that the region surrounding the second "disulfide knot" (Cys13-Cys124, Cys127-Cys174) plays a major role in the inhibition of FIB-CL. This region consists of two strands, residues 10-25 and 121-129, connected through Cys13-Cys124. Peptides from this region also directly inhibited FIB-CL in the absence of TIMP-1. Additional competing peptides included T2-11 of the NH2-terminal domain and T34-42, a highly conserved region in the middle of loop 1. Among a series of monoclonal and polyclonal antibodies (mAbs and pAbs) to TIMP-1, we identified two, one mAb and one pAb, that neutralized the activity of TIMP-1 against FIB-CL. Both recognized epitopes in loop 3. The epitope for the mAb was located in the sequence that marks the transition between loops 3 and 4, GCEEC127, a region also identified as important by peptide competition experiments. By contrast, the epitope for a nonblocking mAb was located in a short 9-residue segment of loop 4, and a nonblocking pAb recognized epitopes in loop 1, loop 6, and the COOH-terminal tail. Our findings suggest that the FIB-CL-TIMP-1 complex possesses multiple contact sites that involve several different subdomains of the inhibitor.

[Clinico-functional observations on the use of guacetisal in the treatment of chronic obstructive bronchopneumopathy]. / Osservazioni clinico-funzionali sull'impiego del guacetisal nel trattamento delle broncopneumopatie croniche ostruttive.

The results of an approximately 14-day treatment with 500 mg broncaspin capsules in 25 patients with recrudescent chronic bronchitis are presented. There was a marked, constant improvement in the subjective and objective symptomatology, and in respiratory function indices. The expectoration cell parameters displayed changes consonant with the therapeutic effect. Local and general tolerance were good overall.