Prognostic value of O-6-methylguanine-DNA methyltransferase (MGMT) protein expression in glioblastoma excluding nontumour cells from the analysis.

AIMS: It is important to predict response to treatment with temozolomide (TMZ) in glioblastoma (GBM) patients. Both MGMT protein expression and MGMT promoter methylation status have been reported to predict the response to TMZ. We investigated the prognostic value of quantified MGMT protein levels in tumour cells and the prognostic importance of combining information of MGMT protein level and MGMT promoter methylation status. METHODS: MGMT protein expression was quantified in tumour cells in 171 GBMs from the population-based Region of Southern Denmark (RSD)-cohort using a double immunofluorescence approach. Pyrosequencing was performed in 157 patients. For validation we used GBM-patients from a Nordic Study (NS) investigating the effect of radiotherapy and different TMZ schedules. RESULTS: When divided at the median, patients with low expression of MGMT protein (AF-low) had the best prognosis (HR = 1.5, P = 0.01). Similar results were observed in the subgroup of patients receiving the Stupp regimen (HR = 2.0, P = 0.001). In the NS-cohort a trend towards superior survival (HR = 1.6, P = 0.08) was seen in patients with AF-low. Including MGMT promoter methylation status, we found for both cohorts that patients with methylated MGMT promoter and AF-low had the best outcome; median OS 23.1 and 20.0 months, respectively. CONCLUSION: Our data indicate that MGMT protein expression in tumour cells has an independent prognostic significance. Exclusion of nontumour cells contributed to a more exact analysis of tumour-specific MGMT protein expression. This should be incorporated in future studies evaluating MGMT status before potential integration into clinical practice.

Chorioamniontis in preterm delivery is associated with degradation of decorin and biglycan and depletion of hyaluronan in fetal membranes.

OBJECTIVE: The proteoglycan decorin stabilizes collagen whereas biglycan and hyaluronan disrupt well-organized collagen. The aim was to determine the concentrations of these constituents in fetal membranes in relation to gestational age, preterm labour, PPROM and chorioamnionitis. STUDY DESIGN: Preterm fetal membranes (24-34 weeks gestation) were obtained from elective caesarean deliveries (N = 4), from PPROM (N = 14), and from preterm labour (N = 14). Term fetal membranes from elective caesarean deliveries (N = 9) and spontaneous vaginal deliveries (N = 11) were used for comparison. Chorioamnionitis was assessed histologically. The proteoglycans were analysed using alcian blue precipitation, SDS-PAGE and immunostaining. Hyaluronan was estimated by a radioimmunoassay. RESULTS: Preterm amniotic membranes with chorioamnionitis displayed a 8-fold decrease in hyaluronan concentration as well as a pronounced (88%) degradation of decorin and biglycan (p < 0.05). The amnion from preterm elective caesarean sections had higher decorin (3.2 vs. 1.7 µg/mg, p < 0.05) and lower biglycan (0.4 vs. 1.0 µg/mg, p < 0.05) concentrations as compared to similar term amnion (p < 0.05), whereas the hyaluronan concentrations were not associated with gestational age. Also the chorio-decidua from preterm caesarean sections had higher decorin concentrations (1.8 vs. 1.0 µg/mg, p < 0.05) whereas the biglycan concentration was unchanged. Labour (term as well as preterm) was characterized by increased hyaluronan and biglycan concentrations in the amnion (not statistically significant). CONCLUSION: The biglycan/decorin balance increases during third trimester of pregnancy and during active labour. This relation might contribute to mechanical weakening of the membranes. Chorioamnionitis induces dramatic degradation of both proteoglycans and hyaluronan, which can explain the decreased biomechanical strength.

Pegylated liposomal doxorubicin as first-line monotherapy in elderly women with locally advanced or metastatic breast cancer: novel treatment predictive factors identified.

We investigated the efficacy and safety of single-agent pegylated liposomal doxorubicin (PLD) as first-line treatment for elderly women with advanced breast cancer and evaluated predictive markers for response and toxicity. Twenty-five women ≥ 65 years received 40 mg/m(2) PLD every 28 days. Time to treatment failure (TTF), response rate, time to progression (TTP) and overall survival (OS) was calculated. The ABCB1 single nucleotide polymorphisms (SNP), tumor MRN complex, and TOPOIIα were analyzed. A mean of 7.4 cycles PLD were administered and TTF was 5.5 months and OS 20.6 months. ABCB1 SNPs were found to correlate to both efficacy and toxicity, while tumor expression of the MRN complex and TOPOIIα correlated to TTP. PLD is a safe and effective treatment for elderly breast cancer patients. Also potential predictive markers were identified.

Different secretion patterns of matrix metalloproteinases and IL-8 and effect of corticotropin-releasing hormone in preterm and term cervical fibroblasts.

The aims of the present study were to compare the levels of mRNA and protein expression of matrix metalloproteinase (MMP)-1, -3, -8 and -9 in human cervical tissue in preterm and term labor as well as not in labor and to determine if corticotropin-releasing hormone (CRH) has an effect on MMP-1, -3 and interleukin (IL)-8 secretion in both preterm and term cervical fibroblasts. Cervical biopsies were taken from 60 women: 18 at preterm labor, 7 at preterm not in labor, 18 at term labor and 17 at term not in labor. ELISA and Immulite were used for protein and real-time RT-PCR for mRNA analysis. Cervical fibroblast cultures were incubated for 18 h with different CRH concentrations (10(-13)-10(-6) M). The mRNA expression of MMP-1, -3 and -9 was higher in laboring groups compared with term not in labor. Protein levels of MMP-8 and -9 were higher in term in labor group compared with non-laboring groups. There were no significant differences in mRNA and protein expression between the preterm and respective term control groups. CRH significantly increased secretion of IL-8 in preterm and term cervical fibroblasts compared with controls. The secretion of IL-8 and MMP-1 was significantly higher and MMP-3 secretion lower in preterm cervical fibroblasts. In conclusion, cervical ripening at preterm seems to be a similar inflammatory process as at term with CRH involved. However, preterm and term cervical fibroblasts might have different phenotypes based on different secretion patterns of IL-8, MMP-1 and MMP-3.

Long-term follow-up of acupuncture and hormone therapy on hot flushes in women with breast cancer: a prospective, randomized, controlled multicenter trial.

OBJECTIVE: To evaluate the effects of electro-acupuncture (EA) and hormone therapy (HT) on vasomotor symptoms in women with a history of breast cancer. METHODS: Forty-five women were randomized to EA (n = 27) for 12 weeks or HT (n = 18) for 24 months. The number of and distress caused by hot flushes were registered daily before, during and up to 24 months after start of treatment. RESULTS: In 19 women who completed 12 weeks of EA, the median number of hot flushes/24 h decreased from 9.6 (interquartile range (IQR) 6.6-9.9) at baseline to 4.3 (IQR 1.0-7.1) at 12 weeks of treatment (p < 0.001). At 12 months after start of treatment, 14 women with only the initial 12 weeks of EA had a median number of flushes/24 h of 4.9 (IQR 1.8-7.3), and at 24 months seven women with no other treatment than EA had 2.1 (IQR 1.6-2.8) flushes/24 h. Another five women had a decreased number of flushes after having additional EA. The 18 women with HT had a baseline median number of flushes/24 h of 6.6 (IQR 4.0-8.9), and 0.0 (IQR 0.0-1.6; p = 0.001) at 12 weeks. CONCLUSION: Electro-acupuncture is a possible treatment of vasomotor symptoms for women with breast cancer and should be further studied for this group of women.

Labour induces increased concentrations of biglycan and hyaluronan in human fetal membranes.

OBJECTIVE: The proteoglycan decorin stabilizes collagen whereas biglycan and hyaluronan disrupt well-organized collagen. The aim was to compare hyaluronan and proteoglycans in human fetal membranes obtained before and after spontaneous labour at term. STUDY DESIGN: Prelabour samples of fetal membranes (N=9) were obtained from elective caesarean sections and regionally sampled from over the cervix (cervical membranes) and mid-zone samples between this area and the placental edge. Postlabour samples (N=11) were obtained from spontaneous vaginal delivery and also regionally sampled. Amnion and chorio-decidua were analysed separately. The proteoglycans decorin and biglycan were analysed using alcian blue precipitation, SDS polyacrylamide gel electrophoresis and immunostaining. Hyaluronan was analysed using a radioimmunoassay and by histochemistry. Collagen was measured by estimating hydroxyproline content. RESULTS: In prelabour membranes the biglycan concentration (microg/mg wtw) in the cervical amnion was 40% lower than in the mid-zone amnion (P<0.05). After delivery the cervical amnion showed a twofold increase in biglycan (P<0.05), a 30% decrease in collagen (P<0.05), and a 50% decrease in decorin concentration (P<0.05). In mid-zone samples after delivery the concentrations of hyaluronan showed an increase form 1.0 to 4.9 microg/mg wtw (P<0.05). Histology demonstrated a gelatinous substance, which separated amnion and chorio-decidua, in particular at the cervical site. This gelatinous substance contained hyaluronan at a concentration of 3.0 microg/mg wtw. CONCLUSION: It is well established that prelabour fetal membranes are considerably stronger than postlabour fetal membranes. Two features may explain this; a weakening of the amnion combined with a separation of amnion and chorio-decidua. The biomechanical changes are consistent with the decrease in collagen and decorin, and the increase in hyaluronan and biglycan demonstrated in this study. The separation of the membranes is caused by the formation of a gelatinous substance, rich in hyaluronan. The results indicate that the biomechanical changes are not merely secondary to the stress of labour but that an active maturation process is involved.

The glucuronyl C5-epimerase activity is the limiting factor in the dermatan sulfate biosynthesis.

An early step in the biosynthesis of dermatan sulfate is polymerization to chondroitin, which then is modified by the D-glucuronyl C5-epimerase and mainly 4-O-sulfotransferase. The final structure of the dermatan sulfate side chains varies and our aim was to identify, which of the two enzymes that are crucial to generate dermatan sulfate copolymeric structures in tissues. Dermatan sulfate side chains of biglycan and decorin were prepared from fibroblasts and nasal and articular chondrocytes and characterized regarding detailed structure. Microsomes were prepared from these cells and the activities of D-glucuronyl C5-epimerase and 4-O-sulfotransferase were determined. Chondrocytes from nasal cartilage synthesized biglycan and decorin containing 10%, articular chondrocytes 20--30%, and fibroblast 80% of the uronosyl residues in the l-iduronyl configuration. All three tissues contained high amount of 4-O-sulfotransferase activity. The activity of d-glucuronyl C5-epimerase showed different relationships. Fibroblasts contained a high level of the epimerase activity, articular chondrocytes intermediary activity, and in nasal cartilage it was barely detectable. The data indicate that the activity of the d-glucuronyl C5-epimerase is the main factor for formation of dermatan sulfate in tissues.

Proteoglycans and hyaluronan in human fetal membranes.

OBJECTIVE: The aim of this study was to describe the distributions of major extracellular matrix components, such as proteoglycans, collagen and hyaluronan, in the fetal membranes at term. STUDY DESIGN: Fetal membranes were obtained from elective cesarean deliveries at term. Guanidinium extracts were analyzed for proteoglycans with alcian blue precipitation, sodium dodecyl sulfate- polyacrylamide gel electrophoresis, and Western blotting and for hyaluronan with a radioimmunoassay. Collagen was measured by estimating hydroxyproline content. Tissue sections were immunostained for decorin and biglycan and stained for hyaluronan with a biotin-labeled hyaluronan-binding protein. RESULTS: The fetal membranes contained predominantly smaller proteoglycans, such as biglycan and decorin. The amnion consisted of typical fibrous connective tissue with a high concentration of collagen. The amnion was dominated by decorin located in close connection with the collagen fibrils. The chorion was composed of a fibroblastic part containing collagen and decorin and a trophoblastic part mainly containing biglycan. In addition, large amounts of hyaluronan were found, especially in the amnion and in the decidual cell layers. CONCLUSION: The distributions of proteoglycans, collagen, and hyaluronan in human fetal membranes may explain the biomechanical properties of this tissue. We suggest that changes in the relative proportions of these extracellular molecules are crucial for the proposed maturation process in the fetal membranes during the last weeks of pregnancy.

Cd40 expression in uterine tissues: a key regulator of cytokine expression by fibroblasts.

CD40 is a cell surface receptor initially discovered on cells of the hemopoietic lineage. Its primary role on immune cells is to enhance their activation and hence their production of cytokines and immunomodulatory molecules. Recently, CD40 has also been detected on human fibroblasts. An emerging view of the fibroblast is that it is far more than a structural cell, being capable of intimate interaction with cells of the immune system. In fibroblasts from several tissues, the engagement of CD40 with its ligand (CD40L) resulted in the secretion of proinflammatory molecules such as interleukin-6 (IL-6) and IL-8. Currently, there are few data about the presence of the CD40-CD40L system in female reproductive tissues. This study investigates the expression of CD40 by human endometrium, myometrium, and cervix both in situ and in tissue explant-derived fibroblasts. CD40 was detected mainly in the perivascular region of endometrium, myometrium, and cervix. Light staining for CD40 was observed in stromal elements. Additionally, the basal epithelium of cervix expressed CD40. Fibroblastic cells derived from all three sources express low levels of CD40, and this is up-regulated with interferon-gamma treatment (500 U/mL; 72 h). When activated with interferon-gamma and CD40L, the fibroblasts secreted increased amounts of IL-6, IL-8, and MCP-1. These data suggest that the CD40-CD40L system may provide a link between the resident structural cells of these reproductive tissues and the infiltrating immune cells or activated platelets that may express CD40L. The possible interaction of CD40 with CD40L may be particularly important during events such as menstruation and cervical ripening, where up-regulation of the proinflammatory molecules IL-6 and IL-8 is viewed as critical for these processes. In addition, dysregulation of this system may be a contributory factor to problems such as menstrual dysfunction and preterm labor.

Identification of the major proteoglycans from human myometrium.

BACKGROUND: During pregnancy and parturition a remodeling within the extracellular matrix of the cervix and the corpus uteri occurs, which is of fundamental importance to a normal labor. The aim of this study is to identify the major proteoglycans in corpus uteri of non-pregnant subjects. METHODS: From human uterine tissue proteoglycans were extracted and purified using CsCl-density gradient centrifugation, gel and ion-exchange chromatography. The proteoglycans were quantified and identified by Alcian Blue before and after ABC-digestion and by Western blotting. RESULTS: The results showed that the corpus uteri contains a substantial amount of proteoglycans, 1.825 microg/mg wet weight. Decorin is dominating, constituting 63% of the total amount of proteoglycans. Heparan sulphate proteoglycans accounted for 20% and biglycan for 16%. Less than 1% consisted of the large proteoglycan versican. CONCLUSIONS: Further investigations must be performed to provide more information of the biological role of the proteoglycans in the uterus, especially during labor, by the presence of heparan sulphate proteoglycans and the minute presence of versican which indicate that the proteoglycan composition and organization is different to that of the cervix.

Human cervical ripening, an inflammatory process mediated by cytokines.

An extensive remodelling process, referred to as cervical ripening, takes place in the cervical tissue during pregnancy and labour. It is recognized as softening and dilation of the cervical canal, and starts as a slow process during pregnancy, becoming rapid close to partum. In this study we focus on cytokines as possible mediators of this final remodelling. mRNA levels for interleukin (IL)-8, IL-6 and granulocyte colony-stimulating factor (G-CSF) were upregulated in the ripe postpartum cervical tissue (n = 8) compared to the unripe state (n = 9). Likewise, released cytokine concentrations increased from non-pregnant (n = 11) to the term-pregnant group (n = 13) with a further increase at partum (n = 16). IL-8 concentrations increased 4-fold from non-pregnant to term-pregnant (P<0.01), and a further 10-fold to postpartum state (P<0.0001). Concentrations of IL-6 and G-CSF were similarly increased. Specific IL-8 immunostaining was identified in the epithelia of pregnant cervical tissue (n = 7) and was most pronounced in the epithelia and stroma of postpartum tissue (n = 4). In conclusion, IL-8, IL-6 and G-CSF increase in the human cervix during the ripening process, indicating their important role in the cervical remodelling. These data demonstrate that cervical ripening is similar to an inflammatory process.

Dermatan is a better substrate for 4-O-sulfation than chondroitin: implications in the generation of 4-O-sulfated, L-iduronate-rich galactosaminoglycans.

The biosynthesis of dermatan sulfate is a complex process that involves, inter alia, formation of L-iduronic acid residues by C5-epimerization of D-glucuronic acid residues already incorporated into the growing polymer. It has been shown previously that this reaction is promoted by the presence of the sulfate donor 3'-phosphoadenosine-5'-phosphosulfate. In the present investigation, the role of sulfation in the biosynthesis of L-iduronic acid-rich galactosaminoglycans was examined more closely by a study of the substrate specificities and kinetic properties of the sulfotransferases involved in dermatan sulfate biosynthesis. Comparison of the acceptor reactivities of oligosaccharides from chondroitin and dermatan, in an in vitro system containing microsomes from cultured human skin fibroblasts and 3'-phosphoadenosine-5'-phosphosulfate, showed that Km values for the dermatan fragments were substantially lower than those for their chondroitin counterparts. Calculation of Vmax values likewise showed that dermatan was the better substrate. Whereas dermatan incorporated [35S]sulfate exclusively at the C4 position of N-acetylgalactosamine residues, approximately equal amounts of radioactivity were found at the C4 and C6 positions in the labelled chondroitin. Under standard assay conditions, the 4-O-sulfation of dermatan proceeded about six times faster than the 4-O-sulfation of chondroitin. On the basis of these results, we propose that L-iduronic acids, formed in the course of the biosynthesis of dermatan sulfates, enhance sulfation of their adjacent N-acetylgalactosamine residues, and will thereby be locked in the L-ido configuration.

Isolation and characterization of proteoglycans from human follicular fluid.

Two proteoglycans differing in size and composition were isolated from human follicular fluid. The larger one of high density had a molecular mass of 3.0x10(6) Da, as determined by laser light-scattering, and was substituted with 15-20 chondroitin sulphate (CS) chains (Mr 60000-65000). Half of the CS disaccharides were 6-sulphated, whereas the remaining ones were non-sulphated. Digestion of the CS proteoglycan with chondroitinase ABC lyase, followed by SDS/PAGE, yielded a protein core of 600 to 700 kDa including substituted oligosaccharides, and a band of 70 kDa that was identified as the heavy-chain component of the inter-alpha-trypsin inhibitor (ITI). Western blotting of the CS proteoglycan showed that this had reactivity with antibodies raised against human versican. Electron microscopy (EM) of the CS proteoglycan also revealed a versican-like structure, with one globular domain at each end of a long extended segment substituted with CS side chains, as well as a structure interpreted as being the heavy chain of ITI attached to CS chains. Laser light-scattering revealed that the smaller proteoglycan had a molecular mass of 1. 1x10(6) Da, and EM demonstrated that it had a globular-protein core structure. The core protein, which showed immunological reactivity with perlecan antibodies, was substituted with approximately seven heparan sulphate (HS) and CS chains of similar size (50-55 kDa), the CS disaccharides being mainly 6-sulphated (68%), with a small proportion being 4-sulphated. The protein core was shown to be heterogeneous, with bands occurring at 215, 330 and 400 kDa after enzymic degradation of the glycosaminoglycan chains followed by SDS/PAGE analysis. The demonstration of intact molecules and fragments obtained after stepwise degradations, as shown by gel chromatography, supported a 'composite' structure of this proteoglycan.

High-dose chemotherapy with autologous stem cell support in patients with responding stage IV breast cancer.

Ninety-four patients underwent high-dose chemotherapy with stem cell support for stage IV breast cancer. The high-dose chemotherapy consisted of the Stamp V regimen in all patients comprising cyclophosphamide, thiotepa and carboplatin (CTCb). Twenty-three patients received sequential high-dose therapies with the first consisting of high-dose melphalan and the second of Stamp V. Two patients died from chemotherapy-related complications resulting in a transplant-related mortality at 100 days of 2.2%. The progression-free survival at 3 years was 36% in patients with no evidence of disease at the first course of high-dose therapy compared with 17% in patients with remaining disease at time of the high-dose therapy (P = 0.03). There was no difference in overall survival between patients with no evidence of disease and other patients. The source of stem cells, single or double courses of high-dose therapy, positive selection of CD34+ cells, or number of involved sites had no influence on either progression-free survival or overall survival. Further studies of more intensive induction chemotherapy followed by high-dose therapy with stem cell support are indicated.

Differential expressions of mRNA for proteoglycans, collagens and transforming growth factor-beta in the human cervix during pregnancy and involution.

During pregnancy and involution, an extensive remodelling of the human cervical connective tissue occurs. This cervical ripening is one of the most pronounced physiological remodelling processes known in human connective tissue. To investigate how the remodelling is accomplished, the levels of mRNA for collagen I and III, versican and three small proteoglycans, biglycan, decorin and fibromodulin, were evaluated using Northern blots at different stages of cervical ripening. In the corresponding biopsies the concentration of collagen and of small and large proteoglycans were determined. The role of transforming growth factor-beta (TGF-beta) as a mediator of the remodelling process was also investigated. The concentration of collagen decreased and 1 week before partus, 50% of the nonpregnant level was attained. No further decrease was noted after partus. The mRNA for collagen I and III did, however, not decrease in the term pregnant cervix 1 week before partus. Only 20-30% decrease during the final ripening just before partus was recorded. Neither did the mRNA levels of the small proteoglycans change significantly during the ripening, despite an almost 50% decrease in the concentration of the small proteoglycans. The message for versican was, however, 5-fold increased at partus and then gradually returned to nonpregnant levels within 4 days after delivery. These changes corresponded to similar changes in the concentration of the large proteoglycan. Thus, the remodelling of the cervical connective tissue is achieved by two different mechanisms, on one hand an increased turnover of collagen and the small proteoglycans, on the other a changed transcription followed by an increased production of versican. During the involution 2- to 3-fold increases in the messages for collagen I and III, and the small proteoglycans, biglycan and decorin, corresponded to increases in the concentration of the small proteoglycans and non-extractable collagen. The message for TGF-beta was increased 2-fold immediately after delivery compared with the term pregnant state. Thus, TGF-beta may be of importance for the reconstruction of the cervix, which starts immediately after partus.

Cervical fetal fibronectin correlates to prostaglandin E2-induced cervical ripening and can be identified in cervical tissue.

OBJECTIVE: Our purpose was to investigate whether prostaglandin E2-induced cervical ripening can be related to changes in fetal fibronectin levels and whether fetal fibronectin can be detected by immunohistochemistry in amniotic and cervical tissue. STUDY DESIGN: Fetal fibronectin levels in cervical mucus were quantitated in 28 nulliparous term pregnant women with unfavorable cervical states before and after intracervical application of prostaglandin E2 gel. The concentration of fetal fibronectin was determined with use of an enzyme immunoassay. Cervical biopsy specimens and amniotic tissue for immunohistochemical analysis were obtained from three term pregnant women and after parturition in three women. Cervical biopsy specimens from two nonpregnant women served as controls. Immunohistochemical analysis was performed with antibodies directed toward fetal fibronectin. RESULTS: The fetal fibronectin level in cervical mucus was low in all women before prostaglandin E2 application. In women with a successful prostaglandin E2-induced ripening (i.e., an increase of cervical score with > or =3 points), a tenfold increase in the fetal fibronectin level was registered. In women with an insufficient cervical ripening after prostaglandin E2 treatment no significant increase in the fetal fibronectin level was registered. The immunohistochemical analyses have identified fetal fibronectin in the epithelial cells of the cervix uteri. CONCLUSION: Successful prostaglandin E2-induced cervical ripening seems to be related to a significant increase in cervical fetal fibronectin levels. Fetal fibronectin can be detected immunohistochemically in the pregnant human cervix.

Heparan/chondroitin/dermatan sulfate primer 2-(6-hydroxynaphthyl)-O-beta-D-xylopyranoside preferentially inhibits growth of transformed cells.

Xylose forms the direct carbohydrate-protein link in extra- or pericellular proteoglycans (PGs) that are substituted with either chondroitin sulfate (CS)/dermatan sulfate (DS) and/or heparan sulfate (HS). Cell surface PGs carrying HS are important regulators of cell growth. Xylose coupled to an aromatic compound can enter cells and initiate either CS/DS synthesis or both HS and CS/DS synthesis, depending on the nature of the aromatic adduct. Here, we show that 2-(6-hydroxynaphthyl)-O-beta-D-xylopyranoside, which can prime both types of glycan chains, inhibits growth of a set of normal and transformed cells. Transformed cells are preferentially inhibited, and at a concentration of 0.15-0.20 mM xyloside, transformed cells are totally growth arrested, whereas normal cells are only < or = 50% inhibited. No inhibition of growth is observed with the stereoisomeric 2-(6-hydroxynaphthyl)-O-beta-L-xylopyranoside, which does not prime glycosaminoglycan synthesis at all; with the nonhydroxylated 2-naphthyl-O-beta-D-xylopyranoside, which only primes CS/DS synthesis under these conditions; or with p-nitrophenyl-O-beta-D-xylopyranoside, which is known to prime only CS/DS synthesis. We conclude that growth inhibition is due to priming of HS and/or CS/DS synthesis, which may either lead to the formation of specific antiproliferative glycans or glycan fragments or to interference with endogenous PG synthesis and turnover.

Different organization of collagen fibrils in stress-incontinent women of fertile age.

OBJECTIVE: The objective was to test the hypothesis that stress urinary incontinence in women is correlated to changes in the paraurethral connective tissue ultrastructure and metabolism. METHODS: Transvaginal biopsies were obtained from the paraurethral connective tissue in women of fertile age with stress urinary incontinence and in matched continent controls. All the stress-incontinent women were characterized with urodynamic investigation. In the biopsies, collagen concentration, measured as hydroxyproline, and the degree of extraction by pepsin digestion were quantified. Proteoglycan composition and concentration were analyzed using Alcian blue precipitation, followed by electrophoretic separation and quantification. Using Northern blots mRNA levels for the collagens I and III, the small proteoglycans decorin and biglycan, and the large proteoglycan versican, were quantified. Collagen organization was examined with transmission electron microscopy and the diameters of collagen fibrils were analyzed with an interactive image analysis system (IBAS, Zeiss/Kontron). RESULTS: The biochemical and morphological analyses exposed a significant difference in the paraurethral connective tissue between stress urinary incontinent women before menopause and comparable controls. The collagen concentration was almost 30% higher and the diameters of the collagen fibrils were 30% larger in the incontinent group of women. Also the organization of the collagen fibrils differed, with considerably higher cross-linking. A higher level of mRNA for collagen I and III in the incontinent group indicates that the differences can be related to an altered collagen metabolism. No change of proteoglycan amount or composition was observed, resulting in a significantly lower proteoglycan/collagen ratio in the incontinent group of women. CONCLUSION: Stress urinary incontinence in fertile women is associated with a change in collagen metabolism resulting in an increased concentration of collagen and larger collagen fibrils. These alterations should result in a more rigid form of extracellular matrix, suggesting a connective tissue with impaired mechanical function.

Paraurethral connective tissue in stress-incontinent women after menopause.

OBJECTIVE: To study whether stress urinary incontinence after menopause is correlated to changes in the paraurethral connective tissue ultrastructure and metabolism. METHODS: Transvaginal biopsies were obtained from the paraurethral connective tissue in stress urinary incontinent women after menopause with and without estrogen replacement therapy, and from comparable controls. All the stress-incontinent women underwent urodynamic investigation. In the specimens, collagen concentration, measured as hydroxyproline, and the degree of extractability by pepsin digestion, were quantified. Proteoglycan composition and concentration were analyzed using Alcian Blue precipitation, followed by electro-phoretic separation and quantification. Using Northern blots, mRNA levels for the collagens I and III, the small proteoglycans decorin and biglycan, and the large proteoglycan versican, were quantified. Collagen structure was examined with transmission electron microscopy, and the diameters of collagen fibrils were analyzed with an interactive image analysis system (IBAS, Zeiss/Kontron). RESULTS: No significant difference in paraurethral connective tissue biochemistry or ultrastructure was registered between women with stress incontinence and controls. Estrogen replacement therapy resulted in a lower collagen concentration both between the controls (p = 0.02) and between the incontinent women (0.02). In the women with stress incontinence also the extractability by pepsin digestion was higher in the group with estrogen treatment (p = 0.004), indicating a decrease in cross-linking. The proteoglycan/collagen ratio was higher in the control group with estrogen treatment compared to untreated (p = 0.02), but no difference was found between estrogen treated and untreated incontinent women. The median collagen fibril diameter was 15% larger in the incontinent group of women without estrogen therapy compared to the control group and 5% larger when comparing the incontinent group on estrogen replacement therapy to the corresponding control group. CONCLUSION: The extracellular matrix of paraurethral connective tissue in stress urinary incontinent women after menopause reacted differently to estrogen replacement therapy compared to continent controls. In contrast to incontinent women of fertile age no major changes in collagen metabolism were found in stress urinary incontinent women after menopause.