Calcium exchange and ionized cytoplasmic calcium in resting and activated human monocytes.

We have performed a comprehensive study of calcium tracer flux, distribution, content, and ionized cytoplasmic calcium during monocyte activation. A model of monocyte calcium was developed from 45Ca uptake and exodus curves which indicated that cell calcium was partitioned between three compartments. The magnitude of the time constants for each pool lead us to propose cellular locations for these three compartments: a surface plasma membrane pool, a cytoplasmic pool, and an organelle pool. 45Ca uptake and exodus experiments were analyzed using a nonlinear least squares fit of compartmental exchange rates and sizes. The production of superoxide was used as a reflection of the state of activation of the monocytes treated with Concanavalin A (Con A). We found that Con A-treated monocytes have an increase in the calcium exchange rate with the cytoplasmic pool from 0.04 to 0.07/min (P less than 0.05), and an increase in the size of the cytoplasmic pool from 0.08 to 0.13 pmol/cell (P less than 0.05). There were no significant changes in the exchange rates or sizes associated with either of the other two compartments. The cytoplasmic ionized calcium was measured with the fluorescent probe, Quin 2, which indicated a resting level of 83 nM free calcium in unadhered monocytes. Con A stimulation caused a doubling of the cytoplasmic free calcium to 163 nM within 45 s. This increment in cytoplasmic free calcium preceded the onset of superoxide following Con A treatment. These studies indicate that Con A binding to the plasma membrane increases the monocyte plasma membrane permeability to calcium. External calcium enters the cell at an increased rate and contributes to both internally bound and free calcium. The magnitude of the increase in free calcium is proportional to the concentration of Con A and stimulates calcium extrusion via the calcium transport ATPase. Moreover, there is an increased concentration of ionized cytoplasmic calcium which has the potential to interact with other cellular regulators that modulate cell activation and superoxide production.

Relationship of superoxide production to cytoplasmic free calcium in human monocytes.

Calcium has been proposed as an intracellular second messenger for activation of secretion, phagocytosis, and the oxidative burst of neutrophils. We have examined the role of calcium in human monocyte activation. Concanavalin A (Con A)-stimulated monocytes displayed an increment in cytoplasmic ionized calcium at 31 +/- 6 s and the onset of superoxide production at 61 +/- 9 s. The increase in cytoplasmic calcium invariably preceded the onset of superoxide production. If the external calcium concentration was reduced to less than 28 nM by the addition of 10 mM EGTA, superoxide production was not diminished at 5 min; however, superoxide production decreased thereafter. The Con A-evoked increment in cytoplasmic ionized calcium was blunted upon the addition of EGTA and decreased further with time. Both the production of superoxide and the Con A-evoked increment in cytoplasmic ionized calcium displayed a 50% inhibition after 15 min of calcium depletion and were completely inhibited after 60 min. Total cell calcium fell from 0.7 to 0.5 fmol/cell, and the basal level of ionized calcium fell from 83 to 30 nM after 60 min. Histidine, a strong chelator of divalent cations other than calcium and magnesium, had no effect on monocyte superoxide production or on ionized calcium concentrations, indicating that EGTA inhibition was due to cell calcium depletion. In calcium-depleted cells, Con A did not evoke superoxide production until calcium was restored to the incubation medium. The restoration of calcium to Con A-treated, calcium-depleted monocytes permitted a rapid rise in the cytoplasmic ionized calcium, and the production of superoxide within 9 s. These data suggest that an increase in ionized cytoplasmic calcium is necessary for the activation of monocyte superoxide production by Con A. The rise in ionized calcium in response to Con A results, in part, from an internal redistribution of calcium, which is sufficient to permit superoxide generation.

Radiation therapy and hyperthermia improve the oxygenation of human soft tissue sarcomas.

The adverse prognostic impact of tumor hypoxia has been demonstrated in human malignancy. We report the effects of radiotherapy and hyperthermia (HT) on soft tissue sarcoma oxygenation and the relationship between treatment-induced changes in oxygenation and clinical treatment outcome. Patients receiving preoperative radiotherapy and HT underwent tumor oxygenation measurement pretreatment after the start of radiation/pre-HT and one day after the first HT treatment. The magnitude of improvement in tumor oxygenation after the first HT fraction relative to pretreatment baseline was positively correlated with the amount of necrosis seen in the resection specimen. Patients with <90% resection specimen necrosis experienced longer disease-free survival than those with > or = 90% necrosis. Increasing levels of tumor hypoxia were also correlated with diminished metabolic status as measured by P-31 magnetic resonance spectroscopy.

Tumor oxygenation predicts for the likelihood of distant metastases in human soft tissue sarcoma.

This study was performed to explore the relationship between tumor oxygenation and treatment outcome in human soft tissue sarcoma. Twenty-two patients with nonmestastatic, high-grade, soft tissue sarcomas underwent preoperative irradiation and hyperthermia and pretreatment measurement of tumor oxygenation. The 18-month actuarial disease-free survival was 70% for patients with tumor median oxygen pressure (pO2) values of >10 mm Hg but only 35% for those with median pO2 values of <10 mm Hg (P=0.01). There were eight treatment failures; the first site of recurrence was lung in all patients. Median pO2 was 7.5 mm Hg for metastasizing tumors versus 20 mm Hg for nonmetastasizing tumors (P=0.03). Potential mechanisms and implications for clinical trial design are discussed.

Role of surgical resection in pelvic Ewing's sarcoma.

PURPOSE: The improved survival in patients with Ewing's sarcoma over the past two decades has placed increased importance on achievement of local disease control. Ewing's sarcoma that arises in the pelvis has been recognized to have a worse prognosis than that in the appendicular skeleton, and the role of surgical resection in these cases remains controversial. The current study attempts to identify a benefit to surgical resection in these patients. METHODS: We retrospectively examined 39 patients who presented with Ewing's sarcoma in a pelvic location, all of whom were treated systemically with chemotherapy. Twenty patients received radiation only as a means of local control, and 19 underwent resection with or without radiation therapy. The patients were evaluated with end points of disease-free survival and overall survival for a minimum of 24 months and a mean of 58 months. RESULTS: There was an even distribution among patients who underwent surgical resection for local control as compared with those who received only radiation therapy with respect to age, site, date of treatment, and stage of disease. Despite uncontrolled biases including tumor size and response to chemotherapy that would be expected to favor patients who undergo resection, surgery in addition to or in substitution for radiation therapy did not result in a statistically significant increase in disease-free survival or overall survival. Local disease control was comparable between those who underwent resection and those who did not: three patients in each group developed a local recurrence. CONCLUSION: Currently, morbidity of surgical resection should be weighed against the efficacy and secondary complications of radiation therapy in the decision-making process for local disease control. The issue of whether overall survival and local disease control is improved in patients who undergo surgical resection remains controversial and may require a prospective randomized trial to be answered definitively.

Transforming growth factor beta 1 stimulates type II collagen expression in cultured periosteum-derived cells.

Chondrogenesis can occur during a bone repair process, which is related to several growth factors. Transforming growth factor beta 1 (TGF-beta 1) downregulates the expression of type II collagen by chondrocytes in vitro, but injection of TGF-beta 1 into the periosteum in vivo increases type II collagen mRNA levels and initiates chondrogenesis. We examined the effect of TGF-beta 1 on collagen gene expression in a bovine periosteum-derived cell culture system to evaluate its direct effect on the periosteum. Cultured cells expressed alkaline phosphatase and collagen pro alpha 1(I) and pro alpha 1(II) mRNAs. A low level of type II collagen synthesis was demonstrated by immunoprecipitation. TGF-beta 1 had no effect on periosteal cell proliferation. Expression of collagen pro alpha 1(I) mRNA did not change with TGF-beta 1 treatment, but alkaline phosphatase mRNA showed a dose-dependent decrease. Expression of collagen pro alpha 1(II) mRNA was stimulated 2.7-fold by TGF-beta 1. TGF-beta 1 also caused a 2.6-fold increase in type II collagen synthesis by immunoprecipitation. These findings indicate that TGF-beta 1 is an enhancer of the expression of the chondrocyte phenotype of the periosteal cells and suggest that TGF-beta 1 is important in initiating and promoting cartilage formation in vivo.

Plasma membrane vesicles prepared from unadhered monocytes: characterization of calcium transport and the calcium ATPase.

We have purified unadhered human monocytes in sufficient quantities to prepare monocyte plasma membrane vesicles and study vesicular calcium transport. Monocytes were isolated from plateletpheresis residues by counterflow centrifugal elutriation. By combining this source and procedure, 7 x 10(8) monocytes of over 90% purity were obtained. The membranes, isolated on a sucrose step gradient, had an 18-fold enrichment in Na,K-ATPase, a 29-fold diminution of succinate dehydrogenase activity and were vesicular on transmission electron micrographs. The membrane vesicles loaded with oxalate accumulated calcium only in the presence of Mg and ATP. Calcium uptake did not occur if ATP was replaced by any of five nucleotide phosphates or if Mg was omitted. Calcium transport had a maximal velocity of 4 pmoles calcium/micrograms vesicle protein/min and a Km for calcium of 0.53 microM. The ionophore A23187 completely inhibited calcium accumulation while 5 mM sodium cyanide and 10 microM ouabain had no effect. A calcium-activated ATPase was present in the same plasma membrane vesicles. The calcium ATPase had a maximal velocity of 18.0 pmoles calcium/micrograms vesicle protein/min and a Km for calcium of 0.60 microM. Calcium-activated ATPase activity was absent if Mg was omitted or if (gamma - 32P) GTP replaced (gamma - 32P) ATP. Monocyte plasma membranes that were stripped of endogenous calmodulin by EGTA treatment showed a reduced level of calcium uptake and calcium ATPase activity. The addition of exogenous calmodulin restored the transport activity to that of unstripped monocyte plasma membranes. Thus, monocyte plasma membrane vesicles contain a highly specific, ATP-dependent calcium transport system and a calcium-ATPase with similar high calcium affinities.

The treatment of high-grade soft tissue sarcomas with preoperative thermoradiotherapy.

PURPOSE: To explore the use of a novel program of preoperative radiation and hyperthermia in the management of high-grade soft tissue sarcomas (STS). METHODS AND MATERIALS: Eligible patients were adults over 18 with Grade 2 or 3 STS, surgically resectable without a local excision prior to referral to Duke University Medical Center and without distant metastases. Patients were staged generally with CT and/or MR imaging. The diagnosis was established with fine needle aspiration or incisional biopsy. Patients were then treated with 5000 to 5040 cGy, 180-200 cGy per fraction. Chemotherapy was usually not employed. Generally two hyperthermia treatments per week were given with a planned thermal dose of 10-100 CEM 43 degrees T90. Invasive thermometry and thermal mapping were done in all patients. Surgical resection was planned 4-6 weeks after the completion of radiation and hyperthermia. RESULTS: Ninety-seven patients were treated on study between 1984 and 1996. Follow-up ranged from 12 to 155 months (median 32). All tumors were high-grade in nature, 44 greater than 10 cm in size (maximum tumor diameter), 43 5-10 cm in size, 10 less than 5 cm. Seventy-eight of the 97 tumors were located in an extremity. Of the 97 patients, 48 remain alive and continually free of disease following initial therapy. Of the remaining 49 patients, 44 have relapsed (34 dead, 10 living with disease), 3 have died secondary to complications of therapy, and 2 have died of unrelated causes. Ten-year actuarial overall survival, cause-specific survival, and relapse-free survival are 50, 47, and 47% respectively. The predominant pattern of failure has been distant metastases with only 2 patients developing local failure alone. Ten-year actuarial local control for extremity tumors is 94%, 63% for the 19 patients with tumors at sites other than the extremity. Of the 78 patients with extremity lesions, 63 have had limb preservation and remain locally controlled. Overall 38 patients experienced 57 major complications. There were 3 deaths, one due to adriamycin cardiomyopathy and two secondary to wound infections. Four patients required amputation secondary to postoperative wound healing problems. Complications directly attributable to hyperthermia occurred in 15 patients with 11 instances of second- or third-degree burns and two instances of subcutaneous fat necrosis. The hyperthermia complications were generally not severe and either healed readily or were excised at the time of surgical resection of the primary tumor. CONCLUSIONS: For these aggressive high-grade soft tissue sarcomas, this treatment program of preoperative thermoradiotherapy provided excellent local regional control for extremity lesions (95%) and satisfactory local regional control (63%) of nonextremity sarcomas, but did not appear to influence the rate of distant metastases or survival. Complications were frequent but apart from the direct thermal burns, not too different from those reported for preoperative radiotherapy alone. More effective adjuvant systemic therapy is necessary to impact favorably on survival.

Monitoring of neoadjuvant therapy response of soft-tissue and musculoskeletal sarcoma using fluorine-18-FDG PET.

UNLABELLED: The purpose of this study was to investigate the potential role of FDG-PET in the monitoring of neoadjuvant therapy of soft-tissue and musculoskeletal sarcomas. METHODS: Nine patients were studied. Neoadjuvant therapy consisted of either chemotherapy or combined radiotherapy and hyperthermia. The FDG-PET studies were obtained, when possible, prior to therapy, 1-3 wk after commencement of therapy, and prior to surgery after completion of neoadjuvant therapy. In two patients, all three studies were completed. The remainder of patients underwent one or two studies at varying timepoints. RESULTS: In tumors treated with combined radiotherapy and hyperthermia, well-defined regions of absent uptake developed within responsive tumors, correlating pathologically with necrosis. Following treatment, a peripheral rim of FDG accumulation was found to correlate pathologically with the formation of a fibrous pseudocapsule. In tumors treated with chemotherapy, FDG accumulation decreased more homogeneously throughout the tumor, in responsive cases. Despite 100% tumor cell kill in some patients, persistent tumor FDG uptake was observed which correlated pathologically with uptake within benign therapy-related fibrous tissue. Significant FDG accumulation was also observed at the site of an uncontaminated incisional biopsy. CONCLUSION: These initial results demonstrate changes in tumor accumulation of FDG during and after neoadjuvant therapy; these changes are dependent on the type of neoadjuvant therapy administered. Prominent FDG accumulation was observed in benign tissues both within and adjacent to the treated tumor.

Decrease in particle-induced osteolysis in obese (ob/ob) mice.

There may be variability in the susceptibility of different individuals to osteolysis from wear debris, and it is not clear whether some individuals may have a genetic predisposition for a more marked osteolytic response. The purpose of this study in mice was to determine whether genetically determined obesity can alter the response to particulate debris. Polyethylene particles were implanted onto the calvaria of seven wild-type mice and seven obese mice (ob/ob). Calvaria from unimplanted wild-type and obese mice served as controls. Calvaria were harvested after 7 days, stained with toluidine blue and for tartrate-specific alkaline phosphatase, and analyzed by histomorphometry. The osteoclast number per mm total bone perimeter was 8.000+/-3.464 in wild-type animals with particles and 2.857+/-1.676 in ob/ob animals with particles (p=0.002; Fisher's PLSD). Bone resorption was 1.895+/-0.713 mm/mm(2) in wild-type animals with particles and 1.265+/-0.494 mm/mm(2) in ob/ob animals with particles (p=0.0438; Fisher's PLSD). Particles induced a diminished osteolytic response in genetically determined obese mice, suggesting that obesity may have a protective role against particle-induced bone resorption-similar to obesity and osteoporosis. These important new findings may help to stimulate clinical studies which may define criteria to better identify patients at risk to develop particle-induced osteolysis.

Extracellular collagen regulates expression of transforming growth factor-beta1 gene.

A complex interrelationship exists between the extracellular matrix and cytokine signaling in articular chondrocytes. We sought to determine whether the extracellular matrix serves as a regulatory component of transforming growth factor-beta1 expression. Bovine articular chondrocytes were isolated and resuspended in alginate, yielding final extracellular protein concentrations of 0 to 1.5% (wt/vol) for type-II or type-I collagen. Cultures were maintained for 7 days in the presence or absence of transforming growth factor-beta1-supplemented medium (10 ng/ml). The amount of transforming growth factor-beta1 mRNA was examined with quantitative competitive reverse transcription-polymerase chain reaction analysis. The results indicate that exogenous transforming growth factor-beta1 stimulates endogenous transforming growth factor-beta1 mRNA expression approximately 8-fold. This effect depended on the concentration of extracellular type-II collagen. As the concentration of extracellular type-II collagen is increased, the expression of transforming growth factor-beta1 mRNA decreases in both basal and transforming growth factor-beta1-stimulated cultures. Exogenous extracellular type-I collagen also served to negatively modulate transforming growth factor-beta1 gene expression but with a different concentration profile. The results demonstrate that transforming growth factor-beta1 mRNA expression was upregulated by exogenous transforming growth factor-beta1 and was downregulated by extracellular type-I and type-II collagens. The profoundly different effects on transforming growth factor-beta1 expression by the two collagens are consistent with those reported for mammary epithelial cells and likely serve as a negative feedback mechanism to preserve tissue homeostasis.

Extracellular collagen modulates the regulation of chondrocytes by transforming growth factor-beta 1.

This article describes the modulation, by extracellular collagen, of DNA and proteoglycan synthesis in articular chondrocytes stimulated with transforming growth factor-beta 1. Type-I and type-II collagen, heat-denatured type-II collagen, and bovine serum albumin were each incorporated into alginate in increasing concentrations. Bovine articular chondrocytes were isolated and were resuspended in the alginate, yielding alginate beads with final extracellular protein concentrations of 0-1.5% (wt/vol) for the collagens and 0-2.5% (wt/vol) for bovine serum albumin. Cultures of beads were maintained for 7 days in basal Dulbecco's modified Eagle medium or in medium supplemented with 10 ng/ml transforming growth factor-beta 1. Subsequently, the synthesis of DNA and proteoglycan was measured by radiolabel-incorporation methods with [35S]sulfate and [3H]thymidine, and the values were normalized to the DNA content. Transforming growth factor-beta 1 stimulated the synthesis of both DNA and proteoglycan in a bimodal fashion. The presence of extracellular type-II collagen increased the rate of DNA and proteoglycan synthesis in a dose-dependent fashion in cultures stimulated by transforming growth factor-beta 1, whereas heat-inactivated type-II collagen abrogated the effects observed with type-II collagen for synthesis of both DNA and proteoglycan. In contrast, the presence of extracellular type-I collagen caused a dose-dependent inhibition of synthesis of both DNA and proteoglycan in cultures stimulated with transforming growth factor-beta 1. Extracellular bovine serum albumin brought about a limited increase in synthesis rates, presumably by blocking nonspecific cytokine binding. These results suggest that type-II collagen has a specific role in chondrocyte regulation and serves to mediate the response of chondrocytes to transforming growth factor-beta 1.

Articular chondrocyte tenascin-C production and assembly into de novo extracellular matrix.

Tenascin-C is an oligomeric glycoprotein of the extracellular matrix that is expressed in a variety of processes including development, tissue remodeling, wound healing, cell adhesion/antiadhesion, and cell/matrix interactions. Tenascin has recently been acknowledged as a component of the extracellular matrix of articular cartilage, but its function remains unclear. In this study, bovine articular chondrocytes were grown in alginate beads for 35 days to examine the kinetics of tenascin synthesis and incorporation into de novo extracellular matrix. During the culture period, 6 harvest days were established in which culture medium was recovered, alginate beads were dissociated with an EDTA solution, and chondrocytes were collected and lysed by sonication. Total DNA determination performed on the cell lysates demonstrated chondrocyte survival and proliferation. Western blotting performed on the medium, EDTA/alginate, and lysate samples demonstrated the production of both the 220 and 320 kDa tenascin size variants and their differential compartmentalization within the culture system. Tenascin was incorporated into the alginate bead matrix at a constant rate of 3.8 micrograms/day. The 320 kDa variant was produced in higher quantity, but the 220 kDa fragment was twice as likely to be incorporated into the de novo matrix. Methylene blue/acid fuchsin staining and tenascin immunohistochemistry demonstrated the incorporation of tenascin into a progressively expanding matrix surrounding the chondrocytes. The results suggest a role for tenascin in the assembly of the chondrocyte matrix and as a soluble mediator of chondrocytes with possible diverse functions for the tenascin size variants.

Transforming growth factor-beta 1 and fibroblast growth factors in rat growth plate.

Chondrocytes in the growth plate progress in an orderly fashion from resting through proliferating to hypertrophic cells. In the region of hypertrophic chondrocytes, the cartilage is invaded by capillary loops and endochondral ossification is initiated. It is currently believed that growth factors may regulate the proliferation and maturation of chondrocytes and the synthesis of extracellular matrix in the growth plate. The ordered sequence of proliferation and differentiation observed in the growth plate provides a unique opportunity to study the role of acidic fibroblast growth factor, basic fibroblast growth factor, and transforming growth factor-beta 1 in the regulation of these processes. In this study, expression of the mRNA of these growth factors was examined using total RNA extracted from the physis and epiphysis of rat tibias. Transforming growth factor-beta 1 mRNA was detected by Northern hybridization. Expression of the genes encoding acidic and basic fibroblast growth factors was demonstrated by polymerase chain reaction amplification. In addition, using polyclonal antibodies against these growth factors, we localized them by immunohistochemical analysis. Strong intracellular staining with a predominantly nuclear pattern was observed in chondrocytes from the proliferating and upper hypertrophic zones. In contrast, chondrocytes in the resting zone stained only faintly for the presence of these growth factors. Some chondrocytes in the resting zone adjacent to the proliferating zone stained with these antibodies, and the antibodies also stained cells in the zone of Ranvier, which regulates latitudinal bone growth. Lastly, the location of transforming growth factor-beta 1 was examined further with use of a polyclonal antipeptide antibody specific for its extracellular epitope.(ABSTRACT TRUNCATED AT 250 WORDS)

Survival analysis of hips treated with core decompression or vascularized fibular grafting because of avascular necrosis.

Avascular necrosis of the femoral head is a multifaceted process that leads to articular incongruity and subsequent osteoarthrosis of the joint. Clinicians concur that primary treatment should focus on preservation of the natural surface of the joint; however, there has not been a consensus on how best to accomplish this. While a number of therapeutic interventions have been reported, the efficacy has varied markedly and there have been few statistical comparisons. The purpose of the current study was to use statistical analysis to compare the results of two widely used procedures, vascularized fibular grafting (614 hips; 480 patients) and core decompression (ninety-eight hips; seventy-two patients), for the treatment of avascular necrosis. The patients were stratified according to age and the stage of disease, and a survival analysis was performed with total hip arthroplasty as the end point for failure. None of the eleven hips that had Ficat stage-I disease needed a total joint replacement after being treated with either regimen. Analysis of the hips that had stage-II disease revealed rates of survival, at fifty months, of 65 per cent (twenty-eight of forty-three hips) after core decompression and 89 per cent (ninety-nine of 111 hips) after vascularized fibular grafting. For the hips that had Ficat stage-III disease, the rates of survival at fifty months were 21 per cent (ten of forty-seven hips) after core decompression and 81 per cent (405 of 500 hips) after vascularized fibular grafting. Among the hips that had Ficat stage-II or III disease, the rate of eventual total joint arthroplasty after vascularized fibular grafting was significantly lower than that after core decompression (p < 0.0001). The results indicate that the increased morbidity associated with vascularized fibular grafting is justified by the associated delay in or prevention of articular collapse in hips that have stage-II or III disease.

Association between ratio of matrix metalloproteinase-1 to tissue inhibitor of metalloproteinase-1 and local recurrence, metastasis, and survival in human chondrosarcoma.

Chondrosarcoma, a malignant cartilage-forming mesenchymal tumor, displays a wide range of clinical behavior that can be difficult to predict with histological analysis. Matrix metalloproteinases contribute to the processes of local invasion and metastasis by controlling the ability of a tumor to transverse tissue boundaries. The specificity of matrix metalloproteinase-1 (interstitial collagenase) for fibrillar collagen may be central to those processes. Matrix metalloproteinase-2 facilitates invasion by degradation of such basement-membrane structures as type-IV collagen. The balance between the activity of tissue inhibitors of metalloproteinase and the activity of matrix metalloproteinase determines the proteolytic activity and may, in part, determine the overall invasiveness and potential for metastasis. The measurement of the ratio of matrix metalloproteinase to tissue inhibitor of metalloproteinase may have prognostic value for determining whether individual chondrosarcomas are locally invasive or will metastasize. Furthermore, there may be a specific pattern of expression of matrix metalloproteinase and tissue inhibitor of metalloproteinase in chondrosarcomas that is related to local invasion and probability of metastasis. Sixteen paraffin-embedded archival specimens of tumors were examined. Six twenty-micrometer-thick sections were cut from each tumor, and the amounts of cDNA formed from the mRNA were determined with reverse transcription-polymerase chain reaction with use of novel primers for matrix metalloproteinase-1, matrix metalloproteinase-2, tissue inhibitor of metalloproteinase-1, and tissue inhibitor of metalloproteinase-2. The amounts of cDNA for the matrix metalloproteinases and their inhibitors were determined by chemiluminescence and band densitometry. The ratio of the amount of cDNA for matrix metalloproteinase-1 to that for its tissue inhibitor and the ratio of the amount of cDNA for matrix metalloproteinase-2 to that for its tissue inhibitor were calculated, and the results were compared with use of the Student t test, enabling log-rank analysis of Kaplan-Meier survival curves. These ratios as well as the age and gender of the patient; the grade, size, and location of the tumor; the type of adjuvant therapy; and the operative margins were examined for significance with use of stepwise logistic-regression analysis. The patients who had recurrent disease had a significantly higher (p < 0.003) ratio of matrix metalloproteinase-1 to tissue inhibitor of metalloproteinase-1 (mean, 0.939; range, 0.647 to 1.101) than the patients who were free of disease (mean, 0.703; range, 0.629 to 0.772). Moreover, there was a striking difference between the Kaplan-Meier survival curve associated with a high ratio (more than 0.8) and that associated with a low ratio (p = 0.0015). The mean ratio of matrix metalloproteinase-2 to tissue inhibitor of metalloproteinase-2 was 1.814 (range, 1.206 to 3.77) in the patients who had recurrent disease compared with 1.473 (range, 1.073 to 2.390) in those who were free of disease; this difference was not found to be significant, with the numbers available. Analysis of the survival curves indicated that a worse prognosis was associated with a high ratio, but again this relationship was not found to be significant. Regression analysis revealed that a high ratio of matrix metalloproteinase-1 to its tissue inhibitor was a moderately significant independent predictor of a poor outcome (alpha = 0.07).

Collagenase specificity in chondrosarcoma metastasis.

The treatment of some mesenchymal malignancies has made significant gains over the past few decades with the development of effective systemic therapies. In contrast, the treatment of chondrosarcoma has been limited to surgical resection, with the most significant prognostic indicators being surgical margins and histologic grade. We have reported that MMP-1/TIMP-1 gene expression serves to prognosticate for tumor recurrence in this group of patients. This led to the hypothesis that collagenase activity facilitates cell egression from the cartilaginous matrix. In the current study we examine the specificity of collagenase gene expression in archival human chondrosarcoma samples using semi-quantitative PCR. Messenger RNA was affinity extracted and subject to reverse transcription. The subsequent cDNA was amplified using novel primers and quantitated by densitometry. Ratios of gene expression were constructed and compared to disease-free survival. The data demonstrate that the significance of the MMP-1/TIMP-1 ratio as a predictor of recurrence is confirmed with a larger number of patients. Neutrophil collagenase or MMP-8 was observed in only 5 of 29 samples. Collagenase-3 or MMP-13 was observed in all samples but the level did not correlate with disease-free survival. Since the collagenases have similar activity for fibrillar collagens and cleave the peptide in the same location, post-transcriptional regulatory mechanisms may account for the observed specificity. The determination of the MMP-1/TIMP-1 gene expression ratio not only serves to identify those patients at risk for recurrence but may also serve as a novel therapeutic avenue as an adjunct to surgical resection.

Cytologic findings in tenosynovial giant cell tumors investigated by fine-needle aspiration cytology.

Tenosynovial giant cell tumor is a relatively common benign proliferation affecting the articular and periarticular soft tissues. Cytologic findings on smears obtained by fine-needle aspiration are rather characteristic and include a mixture of oval or polygonal mononuclear cells showing vacuolation and/or pigment deposition along with a population of multinucleated giant cells. Separation from other giant cell lesions including true giant cell tumor of bone, chondroblastoma, aneurysmal bone cyst, and granulomatous inflammation must be made. Careful attention to the cytologic findings and correlation with clinical and radiographic data should result in the appropriate diagnosis of tenosynovial giant cell tumor in most cases.

Pigmented villonodular synovitis of the foot and ankle: a review of six cases.

Pigmented villonodular synovitis is a rare but well recognized proliferative lesion of synovial tissue. It has been most commonly described in the knee and hip, with most series reporting <5% occurrence in the foot and ankle. Six patients with pigmented villonodular synovitis of the foot and ankle that was treated between 1978 and 1997 were reviewed. Four of the patients had not been previously diagnosed, and two patients presented with recurrent disease. All six were women. Two patients' disease presented isolated to the ankle joint. The other four involved more than one joint: subtalar joint and midfoot in two, and the metatarsal region in two. The histology of the primary and recurrent lesions did not differ. The recurrent lesions were more diffuse and locally destructive. Five were found on magnetic resonance imaging to be a low-to-medium signal intensity mass on T1- and T2-weighted images. Surgical management ranged from simple excision to synovectomy to Lisfranc amputation. Average follow-up was 13 months (range, 3 weeks to 51 months). One of the lesions recurred 4 years later.

Giant cell tumor of bone in the foot and ankle.

Giant cell tumor of bone has been shown to behave more aggressively when located in the wrist and hand. Although nearly 4% of giant cell tumors arise in the foot and ankle, biological features specific to this location have not been identified. In our experience with more than 300 cases of giant cell tumor, 12 arose in the foot and ankle and were followed for more than 2 years. These included nine females and three males ranging in age from 15 to 52 years (mean age, 29.5 years). All patients presented with pain of 5.0 months' mean duration and 9 of 12 tumors demonstrated aggressive radiographic features, including bone erosion and destruction; five had either invasion of a joint or a soft tissue mass present. Unlike the hand, where metacarpal and phalangeal lesions are common, no tumors arose in the forefoot and nine of the tumors were present in the ankle region. Four patients were treated with resection (no recurrence), two with curettage and cement packing (one recurrence), and six with curettage and autologous bone graft (two recurrences), which resulted in an overall recurrence rate of 25%. None of the recurrent tumors have returned after additional treatment, which consisted of curettage and cement packing in two cases and resection in one case. Five tumors (four primary, one recurrent) were treated with local resection and reconstruction with no major complications and with no amputations performed. Thus, giant cell tumors of the foot and ankle can be treated with local procedures, which result in recurrence rates similar to those found in more common locations.