Extracellular matrix metabolism by chondrocytes. 5. The proteoglycans and glycosaminoglycans synthesized by chondrocytes in high density cultures.

Proteoglycans were extracted from the extracellular matrix of cultures of embryonic chick chondrocytes grown at high density and were purified by CsC1 density gradient centrifugation. The chemical, physical and hyaluronate binding properties of the proteoglycans were similar to those observed in proteoglycans from other hyaline cartilages. Proteoglycans in the media were also purified and on analysis showed three populations of proteoglycans to be present. One population had the physical characteristics of a typical proteoglycan subunit and bound hyaluronate, the other two populations were unable to complex with hyaluronate but one had the physical characteristics of the proteoglycan subunit and the other was of smaller molecular weight. The small molecular weight appears to be a product of the enzymatic degradation of the larger molecular weight species.

Inhibition of proteoglycan biosynthesis by hyaluronic acid in chondrocytes in cell culture.

The depression of proteoglycan synthesis in ten-day-old high density chondrocyte cultures was shown to be dependent on both the concentration and time of exposure of the cells to hyaluronic acid. Hyaluronic acid had no effect on the overall protein synthesis by the cultured cells. Using benzyl-beta-D-xyloside an exogenous acceptor, it was shown that glycosaminoglycan biosynthesis by the chondrocytes was not affected by hyaluronic acid. It was concluded that hyaluronic acid was effecting glycosaminoglycan chain initiation, hence proteoglycan biosynthesis, either by specifically depressing the synthesis of the core protein or by repressing the activity of the xylosyltransferase.

Extracellular matrix metabolism by chondrocytes. III. Modulation of proteoglycan synthesis by extracellular levels of proteoglycan in cartilage cells in culture.

Proteoglycan biosynthesis by cultured chondrocytes was shown to be depressed by extracellular concentrations of proteoglycan and partially degraded proteoglycan. This reduction in proteoglycan synthesis was reversible on removal of the added proteoglycan. Benzyl-beta-D-xyloside, an exogenous acceptor of glycosaminoglycan synthesis, was used and it was shown that proteoglycan was inhibiting glycosaminoglycan synthesis. Proteoglycan had no effect on the overall protein synthesis by the cultured cells. It was concluded that the exogenous proteoglycan was inhibiting proteoglycan synthesis at the level of initiation or elongation of the glycosaminoglycan chains.

Effect of dibutyryl cyclic AMP on the sulphation of proteoglycans by chondrocytes.

The addition of a 1 mM N6, O2'-dibutyryl cyclic AMP to incubations of foetal calf articular cartilage for a 4 h period resulted in a 13 and a 15% decrease, respectively, in the rate of incorporation of [3H]glucosamine and [14C]glucose into glycosaminoglycans. Under the same conditions, a 41% increase in the rate of incorporation of [35S]sulphate into glycosaminoglycans was observed. Dibutyryl cyclic AMP had no effect on protein synthesis over a 4 h period or on the hydrodynamic size of the proteoglycan subunits or glycosaminoglycans synthesized by the cartilage. When the glycosaminoglycans were digested with chondroitin ABC lyase and the resulting disaccharides analysed on Dowex 1 (formate form), it was observed that dibutyryl cyclic AMP increased the degree of sulphation of the disaccharides. Furthermore, an over-sulphated disaccharide corresponding to chondroitin 4,6-disulphate was identified. It was concluded that dibutyryl cyclic AMP was stimulating the process of sulphation of glycosaminoglycans by cartilage.

Extracellular matrix metabolism by chondrocytes. 4. Role of glutamine in glycosaminoglycan synthesis in vitro by chondrocytes.

The rate of synthesis of glycosaminoglycans by cartilage was shown to be dependent on an exogenous source of L-glutamine. In the absence of L-glutamine the tissue and cellular levels of this amino acid were rapidly depleted. The levels of nucleotide sugars and their precursors were measured after separation on Dowex 1 (formate form) in cartilage incubated with and without L-glutamine. It was found that the levels of N-acetylhexoamine 6-phosphate and UDP-N-acetylhexosamine were decreased by 27 and 40% respectively. This demonstrates that L-glutamine is required as the amido group donor in the synthesis of glucosamine 6-phosphate and that the decrease in glycosaminoglycan synthesis is due to the limitation in synthesis of UDP-N-acetylhexoamine.

Molecular size distribution of proteoglycan subunits and glycosaminoglycans synthesized by chondrocytes under conditions of reduced proteoglycan synthesis.

The rate of proteoglycan synthesis by chondrocytes in vitro was depressed by either omitting L-glutamine from the incubation medium or by addition of proteoglycan subunit to the medium. The molecular size distribution on Sepharose 2B of the proteoglycan subunits synthesized by the chondrocytes under these conditions of reduced proteoglycan synthesis was found to be the same as those synthesized by the control cells. Likewise, the molecular size distribution on Sepharose 6B CL of the glycosaminoglycan chains synthesized by the depressed cells was found to be similar to that observed in untreated chondrocytes. This work demonstrates that, under conditions of reduced proteoglycan synthesis, fewer proteoglycan subunits are synthesized by chondrocytes and that the molecular size distribution of these macromolecules is similar to those synthesized by untreated cells.

Extracellular matrix metabolism by chondrocytes. VI. Concomitant depression by exogenous levels of proteoglycan of collagen and proteoglycan synthesis by chondrocytes.

The synthesis of collagen and proteoglycans by cultured chondrocytes, as measured by the incorporation of L-[3H]proline into hydroxyproline and [3H]acetate into glycosaminoglycans, was shown to be depressed by 58% and 39%, respectively, by the addition of exogenous proteoglycan at a concentration of 10 mg/ml growth media. The incorporation of L-[3H]proline into acid-insoluble protein remained unaltered in the presence of the proteoglycan. It was concluded that the effect was depressing the activity on the enzymatic steps, associated with the endoplasmic reticulum, which are responsible for the post-translational modification of collagen and proteoglycan.

Extracellular matrix metabolism by chondrocytes. 7. Evidence that L-glutamine is an essential amino acid for chondrocytes and other connective tissue cells.

The incorporation of [3H]glycine into acid-insoluble protein and of [3H]acetate into glycosaminoglycans by cultured chick chondrocytes was stimulated by the addition of L-glutamine to the incubation medium. The effect of exogenous L-glutamine on protein synthesis was studied further by examining changes in the sedimentation patterns on sucrose gradients of ribosomes isolated from chondrocytes incubated in presence and absence of L-glutamine. It was found that the absence of L-glutamine caused a disaggregation of polyribosomes that was revered by the addition of this amino acid to the culture medium. No detectable glutamine synthetase activity could be measured in avian articular cartilage. These results indicate that L-glutamine is an essential amino acid for cartilage in that an extracellular supply of this amino acid is required for the maintenance of protein and glycosaminoglycan synthesis. A dependence of L-glutamine was also demonstrated for other avain connective tissues.

Inhibition of proteoglycan synthesis by hydrogen peroxide in cultured bovine articular cartilage.

Oxygen-derived reactive species, generated enzymatically by the action of xanthine oxidase upon hypoxanthine, significantly inhibit proteoglycan synthesis by cultured bovine articular cartilage (Bates, E.J., Lowther, D.A. and Handley, C.J. (1984) Ann. Rheum. Dis. 43, 462-469). Here we extend these investigations and show, through the use of catalase and the specific iron chelator diethylenetriaminepentaacetic acid, that the active species involved is H2O2 and not the hydroxyl radical. Incubations of cartilage with H2O2 at concentrations of 1 X 10(-4) M and above are also inhibitory to proteoglycan synthesis. Subsequent recovery of the tissue is dependent upon the initial dose of xanthine oxidase or H2O2. Xanthine oxidase at 84 mU per incubation results in a prolonged inhibition of proteoglycan synthesis which is still apparent after 14 days in culture. Lower concentrations of xanthine oxidase (21-66 mU) are inhibitory to proteoglycan synthesis, but the tissue is able to synthesise proteoglycans at near normal rates after 3 days in culture. The inhibition of proteoglycan synthesis by 1 X 10(-4) M H2O2 is completely reversed after 5 days in culture, whereas 1 X 10(-3) M H2O2 results in a more prolonged inhibition. The synthesis of the proteoglycan core protein is inhibited, but the ability of the newly formed proteoglycans to aggregate with hyaluronic acid is unimpaired.

Characterization of the collagen synthesized by cultured cartilage cells.

Cartilage cells from embryonic chick cartilage were grown in primary cultures. The cell layer was sequentially extracted with neutral saline, mercaptoethylamine and pepsin which revealed that these cells produced salt-soluble and salt-insoluble collagen. The alpha1- to alpha2-chain ratio was determined for the collagen extracted from the cultured cells and was found to be 13 to 1. Further analysis of the molecule was carried out by CNBr cleavage of the salt-extracted collagen and separation of resulting peptides by ion-exchange chromatography. It was shown that the cultured cartilage cells synthesize collagen of the type (alpha1[II])3.

Degradation of proteoglycan in articular cartilage.

Adult rabbit articular cartilage was labelled in vivo over 48 h with [35S]sulphate and was then incubated in organ culture at pH 7.2. Approx. 65% of the tissue content of [35S]proteoglycan was released into the culture medium during the first 48 h of incubation. The average molecular size of the released proteoglycans, as assessed by fractionation on Sepharose 2B/CL and 4B/Cl, was only slightly smaller than that of the proteoglycans extracted from non-cultured cartilage with 4 M guanidine HCl. The percentage of released proteoglycans and extracted proteoglycans which formed aggregates with hyaluronic acid was approx. 25% and 75%, respectively. The results indicate that proteoglycan degradation in adult articular cartilage is initiated by a limited proteolysis of subunit core protein, with the production of non-aggregating species which diffuse readily from the tissue.

The oxidant hypochlorite (OCl-), a product of the myeloperoxidase system, degrades articular cartilage proteoglycan aggregate.

The myeloperoxidase-derived oxidant, hypochlorite (OCl-) was shown to be able to degrade proteoglycan aggregate prepared from bovine articular cartilage. Exposure of proteoglycan aggregate to OCl- concentrations less than 10(-4) M resulted in a decrease in the size of the constituent proteoglycan monomers, which were unable to reaggregate with hyaluronate due to the loss of the hyaluronic acid binding region as indicated by immunoblotting using the monoclonal 1-C-6 antibody. Analysis of the [35S]-labeled core proteins by SDS/polyacrylamide electrophoresis and fluorography indicated a decrease in the size of the core protein. These data suggest that concentrations of OCl- below 10(-3) M results in the cleavage of the proteoglycan core protein in or near the hyaluronic acid binding region. The physiological consequences of these data are discussed. Exposure to higher concentrations (greater than 10(-3)) of OCl- caused more extensive degradation of the core protein; however, there was no evidence to suggest that OCl- cleaves glycosaminoglycan (GAG) chains.

Clinically evident fat necrosis in women treated with high-dose-rate brachytherapy alone for early-stage breast cancer.

PURPOSE: To investigate the incidence of and variables associated with clinically evident fat necrosis in women treated on a protocol of high-dose-rate (HDR) brachytherapy alone without external-beam whole-breast irradiation for early-stage breast carcinoma. METHODS AND MATERIALS: From 6/1997 until 8/1999, 30 women diagnosed with Stage I or II breast carcinoma underwent surgical excision and postoperative irradiation via HDR brachytherapy implant as part of a multi-institutional clinical Phase I/II protocol. Patients eligible included those with T1, T2, N0, N1 (< or = 3 nodes positive), M0 tumors of nonlobular histology with negative surgical margins, no extracapsular lymph-node extension, and a negative postexcision mammogram. Brachytherapy catheters were placed at the initial excision, re-excision, or at the time of axillary sampling. Direct visualization, surgical clips, ultrasound, or CT scans assisted in delineating the target volume defined as the excision cavity plus 2-cm margin. High activity (192)Ir (3-10 Ci) was used to deliver 340 cGy per fraction, 2 fractions per day, for 5 consecutive days to a total dose of 34 Gy to the target volume. Source position and dwell times were calculated using standard volume optimization techniques. Dosimetric analyses were performed with three-dimensional postimplant dose and volume reconstructions. The median follow-up of all patients was 24 months (range, 12-36 months). RESULTS: Eight patients (crude incidence of 27%) developed clinically evident fat necrosis postimplant in the treated breast. Fat necrosis was determined by clinical presentation including pain and swelling in the treated volume, computed tomography, and/or biopsy. All symptomatic patients (7 of 8 cases) were successfully treated with 3 to 12 months of conservative management. Continuous variables that were found to be associated significantly with fat necrosis included the number of source dwell positions (p = 0.04), and the volume of tissue which received fractional doses of 340 cGy, 510 cGy, and 680 cGy (p = 0.03, p = 0.01, and p = 0.01, respectively). Other continuous variables including patient age, total excised tissue volume, tumor size, number of catheters, number of days the catheters were in place, planar separation, dose homogeneity index (DHI), and uniformity index (UI) were not significant. Discrete variables including the presence/absence of DCIS, sentinel versus full axillary nodal assessment, receptor status, presence/absence of diabetes, and the use of chemotherapy or hormone therapy were not found to have a significant association with the risk of fat necrosis. CONCLUSIONS: In this study of HDR brachytherapy of the breast tumor excision cavity plus margin, treatment was planned and delivered in accordance with the dosimetric parameters of the protocol resulting in a high degree of target volume dose homogeneity. Nonetheless, at a median follow-up of 24 months, a high rate of clinically definable fat necrosis occurred. The overall implant volume as reflected in the number of source dwell positions and the volume of breast tissue receiving fractional doses of 340, 510, and 680 cGy were significantly associated with fat necrosis. Future dosimetric optimization algorithms for HDR breast brachytherapy will need to include these factors to minimize the risk of fat necrosis.

Evaluation of ultrasound-based prostate localization for image-guided radiotherapy.

To evaluate the use of the ultrasound-based BAT system for daily prostate alignment. Prostate alignments using the BAT system were compared with alignments using radiographic images of implanted radiopaque markers. The latter alignments were used as a reference. The difference between the BAT and marker alignments represents the displacements that would remain if the alignments were done using ultrasonography. The inter-user variability of the contour alignment process was assessed. On the basis of the marker alignments, the initial displacement of the prostate in the AP, superoinferior, and lateral direction was -0.9 +/- 3.9, 0.1 +/- 3.9, and 0.2 +/- 3.4 mm respectively. The directed differences between the BAT and marker alignments in the respective directions were 0.2 +/- 3.7, 2.7 +/- 3.9, and 1.6 +/- 3.1 mm. The occurrence of displacements >/=5 mm was reduced by a factor of two in the AP direction after the BAT system was used. Among eight users, the average range of couch shifts due to contour alignment variability was 7, 7, and 5 mm in the antero-posterior (AP), superoinferior, and lateral direction, respectively. In our study, the BAT alignments were systematically different from the marker alignments in the superoinferior, and lateral directions. The remaining random variability of the prostate position after the ultrasound-based alignment was similar to the initial variability. However, the occurrence of displacements >/=5 mm was reduced in the AP direction. The inter-user variation of the contour alignment process was significant.

Carrageenin-induced arthritis: V. A morphologic study of the development of inflammation in acute arthritis.

Following a single injection of the polysaccharide carrageenin into the rabbit knee joint, a rapid inflammatory process occurs in the joint space and synovial membrane, followed by changes in the articular cartilage. Initially there is an influx of cells, mainly PMNs, into the synovial fluid, accompanied by proliferation of the synovial lining cells and infiltration of the synovial membrane. The numbers of synovial fluid cells decline gradually after 24 hr. The reaction in the synovial membrane is greatest at day 7, and inflammation is still evident at day 21. Initially, the infiltrate consists mainly of PMNs, but by day 7 it is predominantly mononuclear, with small clusters of lymphocytes. The articular cartilage shows loss of metachromasia with toluidine blue at 3-14 days after injection, but stains normally after day 21. Electron microscopy shows damage to the chondrocytes at day 1 and 7, with complete destruction of cells in the surface layer. At day 7 cells in the deeper layers have lost the apparatus required for proteoglycan synthesis, but at day 21 the cells appear virtually normal. There was no evidence for a direct inhibitory effect of carrageenin on proteoglycan biosynthesis. Most labeled carrageenin was rapidly cleared from the joint space, but about 10% was retained in the synovial membrane and 0.6% in articular cartilage at 48 hr after injection. Since the increase and decline in PMN numbers respectively precede the cartilage damage and recovery, it is suggested that there may be a correlation between the clinical activity of arthritis and the number of PMNs in the synovial fluid.

Fertility issues and their management in men with testis cancer.

Although a curable malignancy, testis cancer and its treatment have unique associated morbidities that largely affect reproductive dysfunction. In this focused review, the factors that contribute to infertility in men with testis cancer are outlined. The treatment-specific risks to fertility that accompany cancer management are also discussed. Contemporary methods of overcoming infertility in testis cancer patients are addressed, and several exciting and promising experimental approaches to the preservation or restoration of fertility for men with testis cancer are presented.

Biochemical studies on pulmonary response to inhalation of methylene chloride.

Biochemical response to the toxic lung damage induced by inhalation of methylene chloride was studied. Significant increases in protein, hexose, sialic acid, lactate dehydrogenase, acid and alkaline phosphatase content were observed in the cell-free lavage effluents from lungs of exposed rats compared to the control animals. This was interpreted as increased cell damage accompanied by enhanced pulmonary secretions, perhaps of glycoproteins and mucins, as a result of inhalation toxicity.

Biochemical response of rat lung to inhaled n-hexane.

Biochemical response of the rat lung to inhaled n-hexane was investigated. Dose-dependent increase in protein, lipid, sialic acid, lactate dehydrogenase, angiotensin-converting enzyme, acid and alkaline phosphatase was observed in the cell-free lavage effluent of the lungs of exposed rats compared to the control animals. This was interpreted as enhanced pulmonary secretions accompanied by increased cell damage.

An eye irritation test protocol and an evaluation and classification system.

An in vivo test protocol and an evaluation and classification system for the determination of eye irritation potential of chemicals and mixtures (substances) is proposed. The protocol uses two or three rabbits and reduces distress in test animals. The test substances are classified as non-irritant, irritant or severe irritant to meet regulatory needs. They may be classified on the basis of past experience with similar compounds or mixtures. Screens such as structure-activity relationships, pH extremes, validated and accepted in vitro tests, severe dermal irritation (primary dermal irritation index > or = 5) or severe dermal toxicity (lethality at < 200 mg/kg body weight) should be used to classify irritant or severe irritant materials when one or more of the screens can provide convincing evidence. For suspected severe irritant materials, the proposed in vivo test permits the use of one rabbit and instillation of 0.01 ml (0.01 g) of the test material into the cornea. Materials that are not classified irritant or severe irritant by screens or severe irritant by one rabbit test are tested in two or three rabbits; 0.1 ml (0.1 g) is instilled into the conjunctival sac. The responses (corneal opacity, iritis and conjunctival redness) are scored according to the modified Draize scoring system at 24, 48 and 72 hr and 7 days post-instillation. A rabbit is considered positive when corneal opacity of 1 or above, iritis of 1 or above or conjunctival redness of 2 or above is present at 24, 48 or 72 hr post-instillation. The material is classified as a severe irritant when the rabbit in the one-animal test or two or more rabbits in the standard test have responses of corneal opacity of 3 or above and iritis of 2 at 24, 48 or 72 hr, or positive responses on day 7 after instillation. The material is classified as an eye irritant when two or more rabbits are positive but the responses are not severe and they clear 7 days after instillation. The material is classified as a non-irritant when no more than one rabbit is positive. The opinions expressed in this article are those of the authors and do not necessarily reflect the views of US Federal agencies.

Criteria for in vitro alternatives for the eye irritation test.

A proposal encompassing considerations and criteria for the development of in vitro alternatives to the eye irritation test has been developed and is presented here. Two factors need to be considered initially in developing an alternative test. The first is to determine whether the alternative assay is to be used as a screen or as a replacement for the eye irritation test. Less stringent acceptance criteria are required for an assay used as a screen than for that used as a replacement test. A screen is a preliminary test for the assessment of eye irritation. It is used for making preliminary decisions or establishing the direction for further testing. Screens answer fewer and less complex questions than a replacement test would, since the results from screens are usually confirmed by more definitive testing. A replacement test, however, must provide the same answers as in vivo methods for the assessment of eye irritation and must provide data for making a definitive toxicological assessment of eye irritation. The second factor to be considered is knowledge of the in vivo assay intended to be replaced. This knowledge should include the procedural aspects of the test and the regulatory information it provides. The following may be considered as criteria for in vitro tests used as screens or as replacements for the eye irritation test in rabbits: rationale (there should be a clear statement regarding the rationale for the use of a particular test in relation to the availability of other tests); relevance (the in vitro endpoint should have biological or physiological relevance to the effect to be detected in vivo); and validational (intralaboratory as well as interlaboratory validation must be conducted).