Home About us Contact
|
Home About us Contact | ||
|
|
||
Type IV Collagen (type + iv_collagen)
Selected AbstractsSynthesis and degradation of type IV collagen in rat skeletal muscle during immobilization in shortened and lengthened positionsACTA PHYSIOLOGICA, Issue 4 2003A. M. Ahtikoski Abstract Aim:, Type IV collagen is a major protein in basement membranes surrounding and supporting skeletal muscle cells. In the present study, we tested the hypotheses that immobilization down-regulates synthesis and up-regulates degradation of type IV collagen in skeletal muscle. Methods:, mRNA level and concentration of type IV collagen as well as mRNA levels and activities of proteins involved in its degradation were analysed from soleus (SOL), gastrocnemius (GAS) and extensor digitorum longus muscles after immobilization in shortened and lengthened positions for 1, 3 and 7 days. Results:, Following immobilization, type IV collagen mRNA level was decreased in SOL and GAS suggesting down-regulated synthesis of this protein. The mRNA level and activity of matrix metalloproteinase-2 (proMMP-2) were increased in all muscles, while the activity of tissue inhibitor of metalloproteinase-2 was decreased in SOL and GAS. These findings reflect an increased capacity for degradation of type IV collagen. Conclusions: As a consequence of decreased synthesis/degradation ratio immobilization reduced the concentration of type IV collagen in all muscles. The regulation of type IV collagen through synthesis and/or degradation seems, however, to be muscle specific. Immobilization in lengthened position seems to delay and partly decrease the net degradation of type IV collagen. [source] In vitro reconstructed mucosa-integrating Langerhans' cellsEXPERIMENTAL DERMATOLOGY, Issue 4 2003P. Sivard Abstract:, All three-dimensional in vitro mucosal models constructed, thus far, have only been reconstituted by epithelial cells. We have developed a reconstructed oral and vaginal epithelium that integrates Langerhans' cells (LC), the dendritic cells (DC) of malpighian epithelia. The epithelium was composed of gingival or vaginal keratinocytes seeded on a de-epidermized dermis (DED) and grown in submerged culture for 2 weeks. LC precursors, obtained after differentiation of cord blood-derived CD34+ hematopoietic progenitor cells (CD34+HPC) by granulocyte macrophage-colony stimulating factor (GM-CSF), tumor necrosis factor-, (TNF-,), transforming growth factor-, (TGF-,) and Flt3-ligand (Flt3-L), were introduced after 6,8 days of culture into the reconstituted epithelium. The in vitro reconstituted mucosal epithelium formed a multilayered, well-differentiated epithelial structure, confirmed by the immunohistochemical expression of cytokeratins 4, 6, 10, 13, 14, 16 and involucrin. LC were identified in the basal and suprabasal epithelial layers by CD1a antigen, S100 protein and Langerin/CD207 expression, and by transmission electron microscopy. Type IV collagen was expressed at the chorio,epithelial junction, and most ultrastructural features of this junction were visualized by electron microscopy. This in vitro reconstructed gingiva or vagina integrating LC represents interesting models very similar to native tissues. Because LC play an important role in the mucosal immune system, our models could be useful for conducting studies on interactions with pathogenic agents (viruses, bacteria etc.), as well as in pharmacological, toxicological and clinical research. [source] ,6 integrin subunit mediates laminin enhancement of cisplatin-induced apoptosis in testicular tumor germ cellsINTERNATIONAL JOURNAL OF CANCER, Issue 1 2005Maoulana Andjilani Abstract Our study demonstrates that laminin potentiates cisplatin-induced apoptosis in NCCIT, a testicular tumor germ cell line. When cultured on laminin, NCCIT cells displayed a significantly higher susceptibility to cisplatin-induced apoptosis than on plastic or on other ECM components including fibronectin, Type IV collagen and vitronectin. This high cisplatin sensitivity observed on NCCIT cell cultured on laminin was mediated by the ,6-integrin signaling. The knockdown of the ,6-integrin subunit by small interfering RNAs suppressed the higher cisplatin-sensitivity supporting the existence of a crosstalk between laminin-,6-integrin signaling and cisplatin-induced apoptosis. Our findings indicate that in cisplatin-treated NCCIT cells, the laminin-,6-integrin signaling induces the activation of executioner procaspase-3 and -6 as well as apoptosis-inducing factor (AIF) transcription and expression. The ability of integrin-mediated specific stroma,tumor cell interactions to modulate the chemosensitive phenotype of a tumor cell might provide new insights to overcome cisplatin resistance of tumor cells. © 2005 Wiley-Liss, Inc. [source] Cutaneous sclerosing perineurioma of the digitINTERNATIONAL JOURNAL OF DERMATOLOGY, Issue 9 2006Toshitsugu Nakamura MD An 11-year-old Japanese girl noticed a small nodule, with mild tenderness, on the right index finger 5 years before visiting our outpatient clinic. She had no familial history of neurofibromatosis or past history of traumatic injury at the site of the tumor. Physical examination revealed a slightly elevated, subcutaneous, nodular tumor in the volar aspect between the proximal and distal interphalangeal joints of the digit (Fig. 1A). By magnetic resonance imaging examination, the tumor showed low density on both T1- and T2-weighted images, and was located just adjacent to the tendon with no invasive signs. The tumor was extirpated; at operation, it was well circumscribed and mobile without adhesion to adjacent tendon or nerve, and was easily removed. Figure 1. (a) Slightly elevated subcutaneous tumor (arrow) on the volar aspect of the right index finger. (b) gross appearance of the extirpated tumor, showing a well-circumscribed, whitish solid nodule Grossly, the tumor was a well-circumscribed, firm nodule (10 mm × 8 mm × 5 mm in size) (Fig. 1B). The cut surface was whitish, homogeneous, and solid without cystic lesions. Histologically, it was an unencapsulated, paucicellular dense, fibrous nodule with a concentric circular arrangement of collagen bundles (Fig. 2A). Amongst the fibrous bundles, a small number of ovoid/epithelioid or plump spindle cells were arranged in a corded, trabecular, or whorled (onion bulb-like) pattern (Fig. 2B); a storiform pattern was not noted. These cells were relatively uniform and had a somewhat elongated, slightly hyperchromatic nucleus with fine granular chromatin. Neither nuclear pleomorphism nor multinucleated cells were evident, and necrosis and mitotic figures were not observed. Periodic acid,Schiff (PAS) stain after diastase digestion highlighted the corded or whorled pattern of the tumor cells by encasing them. For immunohistochemical examination, formalin-fixed, paraffin-embedded serial tissue sections were stained by a labeled streptavidin,biotin method. The tumor cells were positive for vimentin and epithelial membrane antigen (EMA) (Fig. 3A), and negative for pan-cytokeratin, carcinoembryonic antigen (CEA), CD34, ,-smooth muscle actin, desmin, and CD68. Type IV collagen and laminin (Fig. 3B) were detected along the cords or whorls of the tumor cells, similar to the staining pattern of the diastase-PAS reaction. Schwann cells and axonal components, immunoreactive for S100 protein and neurofilament, respectively, were focally detected just adjacent to the cords or whorls, although the tumor cells per se did not express these proteins. Consequently, the tumor was found to be perineurial in origin and was diagnosed as cutaneous sclerosing perineurioma. Figure 2. (a) Low-power view of the tumor, showing an unencapsulated, paucicellular, dense, fibrous nodule with a concentric circular arrangement of collagen bundles (hematoxylin and eosin stain: original magnification, ×15). (b) Higher magnification of the tumor, showing ovoid or epithelioid cells arranged in cords or whorls in the abundant collagen bundles (hematoxylin and eosin stain: original magnification, ×150) Figure 3. Immunohistochemical profiles of the tumor. The tumor cells are positive for epithelial membrane antigen (a) and are surrounded by laminin (b) (original magnification, ×150) [source] Abnormal basement membrane type IV collagen ,-chain composition in labial salivary glands in Sjögren's syndromeARTHRITIS & RHEUMATISM, Issue 4 2009P. Poduval Objective Sjögren's syndrome (SS) is characterized by atrophy and malfunction of the acinar cells. The aim of this study was to investigate whether type IV collagen ,-chain composition of acinar cell compartments could be abnormal in diseased glands. Methods Messenger RNA (mRNA) from human submandibular gland (HSG) cells, cultured with or without growth factor,depleted Matrigel, was analyzed using quantitative reverse transcription,polymerase chain reaction (RT-PCR). Labial salivary glands were analyzed using quantitative RT-PCR and immunohistochemistry. Results HSG cells of both the ductal and acinar phenotypes synthesized all ,-chain mRNA, in particular those of the ,1 and ,2 chains. Labial salivary glands (LSGs) contained ,1/2 chains but also contained mRNA of all the other ,-chains, although the mRNA copy numbers for the ,3 and ,4 chains were low, and the corresponding proteins were absent. Type IV collagen ,1/2-chains were observed in all tubuloalveolar basement membranes. In healthy glands, ,5 and ,6 chains were continuous around ducts but discontinuous around acini. In SS glands, these chains were absent or patchy around the ducts and absent around the acini. Conclusion Ductal and acinar epithelial cells are able to locally produce mRNA for all 6 different ,-chains. Type IV collagen ,1/2-chains seem to form the backbone in the tubuloalveolar basement membrane in salivary glands. Type IV collagen ,3 and ,4 chain mRNA were found in cultured salivary epithelial cells and LSG explants but were not translated to the corresponding ,-chains in LSGs. Both ,5 and ,6 mRNA were observed in salivary epithelial cells and glands. In healthy glands, immunolabeling always disclosed corresponding ,-chains around ducts, but their synthesis and/or degradation seemed to be locally regulated around acinar cells. [source] Synthesis and degradation of type IV collagen in rat skeletal muscle during immobilization in shortened and lengthened positionsACTA PHYSIOLOGICA, Issue 4 2003A. M. Ahtikoski Abstract Aim:, Type IV collagen is a major protein in basement membranes surrounding and supporting skeletal muscle cells. In the present study, we tested the hypotheses that immobilization down-regulates synthesis and up-regulates degradation of type IV collagen in skeletal muscle. Methods:, mRNA level and concentration of type IV collagen as well as mRNA levels and activities of proteins involved in its degradation were analysed from soleus (SOL), gastrocnemius (GAS) and extensor digitorum longus muscles after immobilization in shortened and lengthened positions for 1, 3 and 7 days. Results:, Following immobilization, type IV collagen mRNA level was decreased in SOL and GAS suggesting down-regulated synthesis of this protein. The mRNA level and activity of matrix metalloproteinase-2 (proMMP-2) were increased in all muscles, while the activity of tissue inhibitor of metalloproteinase-2 was decreased in SOL and GAS. These findings reflect an increased capacity for degradation of type IV collagen. Conclusions: As a consequence of decreased synthesis/degradation ratio immobilization reduced the concentration of type IV collagen in all muscles. The regulation of type IV collagen through synthesis and/or degradation seems, however, to be muscle specific. Immobilization in lengthened position seems to delay and partly decrease the net degradation of type IV collagen. [source] Serum markers of lamellar basement membrane degradation and lamellar histopathological changes in horses affected with laminitisEQUINE VETERINARY JOURNAL, Issue 6 2000P. J. JOHNSON Summary In order better to evaluate the extent to which degradation of the lamellar basement membrane (LBM) by matrix metalloproteinases (MMP) occurs in equine laminitis, we determined the concentration of type IV collagen and laminin in normal and laminitic horses, using specific immunoassays. Blood samples were obtained from both the jugular and the cephalic veins of horses (n = 10) before and after the induction of acute alimentary laminitis by carbohydrate overload. Jugular and cephalic venous blood samples were also obtained from horses affected with naturally occurring laminitis (n = 16) and nonlaminitic controls (n = 8). The serum collagen IV concentration was not changed following the induction of laminitis in the experimental group. Serum collagen IV concentration was increased in jugular venous blood obtained from cases of naturally occurring laminitis (mean ± s.e. 218.04 ± 18.59 ng/ml) compared with nonlaminitic controls (157.50 ± 10.93 ng/ml) (P<0.05). Serum collagen IV concentration was also increased in jugular venous blood obtained from severely laminitic horses (219.50 ± 18.18 ng/ml) compared with nonlaminitic controls (157.50 ± 10.93 ng/ml) (P<0.05). A difference in serum concentration of collagen IV was not identified based on chronicity of naturally occurring laminitis. Serum laminin concentration did not differ between laminitic and nonlaminitic horses. Differences in serum laminin concentration were not identified based on sampling location (jugular orcephalic vein), severity of laminitic pain, or chronicity of spontaneous laminitis. In conclusion, the circulating concentration of collagen IV was increased in horses affected with naturally occurring laminitis. The potential role for serum collagen IV assay for characterisation of equine laminitis warrants further investigation. [source] Efficacy of ezetimibe for the treatment of non-alcoholic steatohepatitis: An open-label, pilot studyHEPATOLOGY RESEARCH, Issue 6 2010Masato Yoneda Aim:, Non-alcoholic steatohepatitis (NASH) is considered a hepatic manifestation of metabolic syndrome. However, effective drug therapy for NASH has not been established yet. In the present study, we evaluated the efficacy of 6 months of ezetimibe treatment for NASH patients with dyslipidemia for the comparison of improvement of the clinical parameters and histological alterations. Methods:, We prospectively evaluated 10 consecutive NASH patients with dyslipidemia who agreed to participate in this study. The patients were given ezetimibe (10 mg/day) for 6 months, and clinical parameters and histological alterations were comparatively evaluated before and after treatment. All the patients were given standard calorie diet (30 kcal/kg per day, carbohydrate 50,60%, fat 20,30%, protein 15,20%) and exercise counseling from 3 months before the ezetimibe treatment. Results:, The serum aspartate aminotransferase, alanine aminotransferase, ,-glutamyl transpeptidase, low-density lipoprotein cholesterol, high-sensitivity C-reactive protein and type IV collagen 7 s levels were significantly improved by the treatment with ezetimibe for 6 months. In histological observations, follow-up liver biopsies revealed that the NAS score and steatosis grade were also significantly improved. The fibrosis stage did not change significantly, but six of the 10 patients exhibited an improvement in their fibrosis stage. Conclusion:, Major clinical parameters and histological observations were significantly improved by the treatment with ezetimibe. Our pilot study demonstrated the efficacy of ezetimibe for drug therapy of NASH and may lead to a large-scale clinical trial in the future. [source] Hepatic differentiation of human bone marrow-derived UE7T-13 cells: Effects of cytokines and CCN family gene expressionHEPATOLOGY RESEARCH, Issue 12 2007Takashi Shimomura Aim:, Bone marrow-derived mesenchymal stem cells (MSC) are expected to be an excellent source of cells for transplantation. We aimed to study the culture conditions and involved genes to differentiate MSC into hepatocytes. Methods:, The culture conditions to induce the efficient differentiation of human bone marrow-derived UE7T-13 cells were examined using cytokines, hormones, 5-azacytidine and type IV collagen. Results:, We found that combination of acidic fibroblast growth factor (aFGF), basic fibroblast growth factor (bFGF) and hepatocyte growth factor (HGF) with type IV collagen coating induced hepatic differentiation of UE7T-13 cells at over 30% frequency, where expression of albumin mRNA was increased over 20-fold. The differentiated cells had functions of albumin production, glycogen synthesis and urea secretion as well as expressing hepatocyte-specific genes. In addition, these cellshave binuclear and cuboidal morphology, which is a characteristic feature of hepatocytes. During hepatic differentiation, UE7T-13 cells showed depressed expression of WISP1 and WISP2 genes, members of the CCN family. Conversely, knockdown of WISP1 or WISP2 gene by siRNA stimulated hepatic differentiation. The effect of aFGF/bFGF/HGF/type IV collagen coating and WISP1-siRNA on hepatic differentiation was additive. Conclusion:, The present study suggests that aFGF/bFGF/HGF/type IV collagen coating is the efficient condition for hepatic differentiation of UE7T-13 cells, and that WISP1 and WISP2 play an important role in hepatic transdifferentiation of these cells. [source] Mesenchymal stem cell interaction with a non-woven hyaluronan-based scaffold suitable for tissue repairJOURNAL OF ANATOMY, Issue 5 2008G. Pasquinelli Summary The fabrication of biodegradable 3-D scaffolds enriched with multipotent stem cells seems to be a promising strategy for the repair of irreversibly injured tissues. The fine mechanisms of the interaction of rat mesenchymal stem cells (rMSCs) with a hyaluronan-based scaffold, i.e. HYAFF®11, were investigated to evaluate the potential clinical application of this kind of engineered construct. rMSCs were seeded (2 × 106 cells cm,2) on the scaffold, cultured up to 21 days and analysed using appropriate techniques. Light (LM), scanning (SEM) and transmission (TEM) electron microscopy of untreated scaffold samples showed that scaffolds have a highly porous structure and are composed of 15-µm-thick microfibres having a rough surface. As detected by trypan blue stain, cell adhesion was high at day 1. rMSCs were viable up to 14 days as shown by CFDA assay and proliferated steadily on the scaffold as revealed by MTT assay. LM showed rMSCs in the innermost portions of the scaffold at day 3. SEM revealed a subconfluent cell monolayer covering 40 ± 10% of the scaffold surface at day 21. TEM of early culture showed rMSCs wrapping individual fibres with regularly spaced focal contacts, whereas confocal microscopy showed polarized expression of CD44 hyaluronan receptor; TEM of 14-day cultures evidenced fibronexus formation. Immunohistochemistry of 21-day cultures showed that fibronectin was the main matrix protein secreted in the extracellular space; decorin and versican were seen in the cell cytoplasm only and type IV collagen was minimally expressed. The expression of CD90, a marker of mesenchymal stemness, was found unaffected at the end of cell culture. Our results show that HYAFF®11 scaffolds support the adhesion, migration and proliferation of rMSCs, as well as the synthesis and delivery of extracellular matrix components under static culture conditions without any chemical induction. The high retention rate and viability of the seeded cells as well as their fine modality of interaction with the substrate suggest that such scaffolds could be potentially useful when wide tissue defects are to be repaired as in the case of cartilage repair, wound healing and large vessel replacement. [source] Changes in concentrations of type IV collagen and tissue inhibitor of metalloproteinase-1 in patients with paraquat poisoningJOURNAL OF APPLIED TOXICOLOGY, Issue 6 2001Tsukasa Nakamura Abstract Respiratory failure is one of the major causes of death in patients with paraquat poisoning. In paraquat-poisoned lungs, abnormal extracellular matrix regulation occurs. The aim of the present study is to determine whether serum concentrations of type IV collagen and tissue inhibitor of metalloproteinase-1 (TIMP-1) are altered during the course of paraquat poisoning and whether haemoperfusion therapy affects these concentrations. Twenty-one patients were admitted within 3 h after ingestion of paraquat and all patients received direct haemoperfusion therapy. Five out of 21 patients survived and 16 patients died within 28 days. Plasma paraquat concentrations in non-survivors (5740 ± 380 µg l,1) were not significantly different from those in survivors ( 5920 ± 280 µg l,1) before treatment. Haemoperfusion reduced these concentrations in both non-survivors (120 ± 7 µg l,1) as well as survivors (136 ± 9 µg l,1) on day 5. Serum concentrations of type IV collagen and TIMP-1 in survivors showed little change between day 1 (type IV collagen, 90.4 ± 3.6 ng ml,1; TIMP-1, 172.2 ± 7.0 ng ml,1) and day 5 (type IV collagen, 92.6 ± 4.2 ng ml,1; TIMP-1, 174.2 ± 7.2 ng ml,1). In contrast, these concentrations in non-survivors on day 5 (type IV collagen, 143.6 ± 7.8 mg ml,1; TIMP-1, 246.8 ± 13.6 ng ml,1) were significantly higher than those on day 1 (type IV collagen, 88.4 ± 4.2 ng ml,1, P < 0.01; TIMP-1, 170.6 ± 9.2 ng ml,1, P < 0.05). These data suggest that serum concentrations of type IV collagen and TIMP-1 may be useful indicators for the development of respiratory failure in patients with paraquat poisoning. Copyright © 2001 John Wiley & Sons, Ltd. [source] Relationship between levels of urinary type IV collagen and renal injuries in patients with IgA nephropathyJOURNAL OF CLINICAL LABORATORY ANALYSIS, Issue 1 2004Hiroaki Io Abstract Because type IV collagen is synthesized by podocytes and mesangial cells, we investigated the relationship between levels of urinary type IV collagen (uIV) and renal injuries in patients with IgA nephropathy. uIV was measured by a highly sensitive one-step sandwich enzyme immunoassay prior to renal biopsy. Patients with IgA nephropathy were classified into four grades (grade 1 = good prognosis, grade 2 = relatively good prognosis, grade 3 = relatively poor prognosis, and grade 4 = poor prognosis) by the prognostic criteria of the Ministry of Health, Labor, and Welfare of Japan. Levels of uIV in grade 4 were significantly higher than those in grades 1,3. These levels tended to increase gradually due to progression of renal injuries. The grades were further divided into two groups: group I (good or relatively good prognoses) and group II (relatively poor or poor prognoses). Patients with proteinuria of <1.0 g/day were defined as groups Ip and IIp. The levels of uIV in group II were significantly higher than those in group I, and those in group IIp were significantly higher than those in group Ip. It appears that the level of uIV can be a useful marker for detection of renal injuries in IgA nephropathy. J. Clin. Lab. Anal. 18:14,18, 2004. © 2004 Wiley-Liss, Inc. [source] Validity of FibroScan values for predicting hepatic fibrosis stage in patients with chronic HCV infectionJOURNAL OF DIGESTIVE DISEASES, Issue 2 2009Ryosuke TAKEMOTO OBJECTIVE: The aim of this study was to validate the FibroScan system compared with liver histology and serum markers for the diagnosis of hepatic fibrosis. We also tried to determine the cut-off levels and assess the feasibility of using FibroScan values to predict the fibrosis stage. METHODS: In 44 patients with HCV infection, liver stiffness was evaluated by FibroScan, serum fibrosis markers and a liver biopsy. Associations between these indices were also analyzed. RESULTS: FibroScan values showed a good correlation with serum levels of type IV collagen, hyaluronic acid and procollagen-III-peptide, and with the platelet count. Compared with liver histology, the FibroScan values increased proportionally with the progression of the histological fibrosis stage. Advanced fibrosis (F3 or F4) could be efficiently predicted by a FibroScan cut-off value of 15 kPa. The FibroScan sensitivity, specificity, positive predictive value, negative predictive value and accuracy were 100%, 73.9%, 77.8%, 100%, and 86.4%, respectively. CONCLUSION: FibroScan values gave a good correlation with various markers of fibrosis and increased proportionally with the progression of the hepatic fibrosis stage. A FibroScan value of 15 kPa was found to be a significant separation limit for differentiating advanced fibrosis stages (F3 and F4) from the milder stages (F0,F2). FibroScan values are clinically useful for predicting the fibrosis stages and helpful in managing interferon therapy in patients with chronic hepatitis C. [source] Serum amino-terminal propeptide of type III procollagen and 7S domain of type IV collagen correlate with hepatic iron concentration in patients with chronic hepatitis C following ,-interferon therapyJOURNAL OF GASTROENTEROLOGY AND HEPATOLOGY, Issue 2 2001Ichiro Shimizu Abstract Background: It has been reported that chronic infection with hepatitis C virus is associated with excess iron deposits in the liver of subjects who are neither alcoholics nor recipients of blood transfusions. However, little is known about the relationship between hepatic iron concentration (HIC) and the serum levels of hepatic fibrogenesis markers, which were caused by interferon therapy for chronic hepatitis C. Therefore, changes in the serum amino-terminal propeptide of type III procollagen (P-III-P) and the 7S domain of type IV collagen (7S-IV) in 16 patients treated with ,-interferon (IFN-,) were studied, and their HIC and histological assessment evaluated. Hepatic iron concentrations were measured by using liver biopsy specimens obtained before and 6 months after the cessation of treatment. Methods and Results: Eight subjects (50%) who had normal alanine transaminase levels at 6 months after therapy showed significantly lowered HIC, and attenuated hepatic iron staining with decreased serum levels of P-III-P and 7S-IV compared to the remaining subjects. The HIC was significantly correlated with the serum levels of P-III-P and 7S-IV in all subjects. Conclusions: These findings suggest that IFN-, treatment may decrease stimuli for fibrogenesis, at least in part, by reducing the hepatic iron deposition in patients with chronic hepatitis C. [source] Induction of MMP-2 at the interface between epithelial cells and fibroblasts from human periodontal ligamentJOURNAL OF PERIODONTAL RESEARCH, Issue 3 2010M. Shimonishi Shimonishi M, Takahashi I, Terao F, Komatsu M, Kikuchi M. Induction of MMP-2 at the interface between epithelial cells and fibroblasts from human periodontal ligament. J Periodont Res 2010; 45: 309,316. © 2009 John Wiley & Sons A/S Background and Objective:, MMP-2 can degrade type IV collagen and MMP-14 can activate pro MMP-2. The present study was undertaken to examine the expression of MMP-2 and MMP-14 with respect to interaction between the cells of the epithelial rests of Malassez and fibroblasts from human periodontal ligament. Material and Methods:, Explants of human periodontal ligament tissues produced outgrowths containing both putative epithelial rests of Malassez cells and human periodontal ligament fibroblasts after incubation in a modified serum-free medium. The distribution and expression of MMP-2 and MMP-14 were analysed using immunohistochemistry, in situ hybridization and RT-PCR analysis. The conditioned media and cell extracts were collected for western blot analysis for MMP-2. Results:, Putative epithelial rests of Malassez cells at the interface between the cells of the epithelial rests of Malassez and fibroblasts expressed MMP-2 and MMP-14 strongly. However, in situ hybridization analysis revealed that human periodontal ligament fibroblasts expressed MMP-2 mRNA while putative epithelial rests of Malassez cells expressed MMP-14 mRNA at the interface. The RT-PCR analysis showed that the expression of MMP-2 mRNA was significantly higher when putative epithelial rests of Malassez cells and human periodontal ligament fibroblasts were cultured together than when cultured alone. Western blot analysis showed that the active form of MMP-2 was detected at higher levels in the conditioned medium of the co-cultured cells. Conclusion:, These findings indicate that putative epithelial rests of Malassez cells stimulate the production of MMP-2 in human periodontal ligament fibroblasts. Up-regulated proMMP-2 bound by MMP-14 expressed in epithelial rests of Malassez cells can degrade matrix molecules, such as type IV collagen, in the basal membrane between putative epithelial rests of Malassez cells and human periodontal ligament fibroblasts. [source] Basement membrane changes in lichen planopilarisJOURNAL OF THE EUROPEAN ACADEMY OF DERMATOLOGY & VENEREOLOGY, Issue 11 2009K Al-Refu Abstract Background, Lichen planopilaris (LPP) is an inflammatory disease that affects the scalp and tends to produce cicatricial alopecia. The inflammatory process frequently results in the disruption of the basal cell of the external root sheath of the hair follicle. Objectives, To investigate the alterations in the basement membrane zone (BMZ) in LPP by immunohistochemistry. Methods, Skin biopsies from six patients with LPP plus six normal controls were studied by immunohistochemistry with antibodies to the following BMZ components: cytokeratin 5, cytokeratin 14, BP230 (bullous pemphigoid), BP180, plectin, laminin 5, collagen IV and collagen VII. Results, The localization and staining of the hemidesmosome, laminin and collagen components were strikingly different in the inflamed follicular epithelium when compared to the uninvolved follicles or interfollicular epithelium in active LPP lesions. The hemidesmosome-associated complexes were weakly expressed and discontinuous in involved hair follicles. The expression of laminin-5, type IV collagen and type VII collagen was disrupted and not linear along the BMZ with finger-like projections of the staining protruding into the dermis. The expression of the intermediate filaments was normal. Conclusion, These alterations in the BMZ in LPP may explain the abnormal healing at follicular level which leads to irreversible hair loss and scarring in this condition. [source] Investigating the importance of flow when utilizing hyaluronan scaffolds for tissue engineeringJOURNAL OF TISSUE ENGINEERING AND REGENERATIVE MEDICINE, Issue 2 2010Gail C. Donegan Abstract Esterified hyaluronan scaffolds offer significant advantages for tissue engineering. They are recognized by cellular receptors, interact with many other extracellular matrix proteins and their metabolism is mediated by intrinsic cellular pathways. In this study differences in the viability and structural integrity of vascular tissue models cultured on hyaluronan scaffolds under laminar flow conditions highlighted potential differences in the biodegradation kinetics, processes and end-products, depending on the culture environment. Critical factors are likely to include seeding densities and the duration and magnitude of applied biomechanical stress. Proteomic evaluation of the timing and amount of remodelling protein expression, the resulting biomechanical changes arising from this response and metabolic cell viability assay, together with examination of tissue morphology, were conducted in vascular tissue models cultured on esterified hyaluronan felt and PTFE mesh scaffolds. The vascular tissue models were derived using complete cell sheets derived from harvested and expanded umbilical cord vein cells. This seeding method utilizes high-density cell populations from the outset, while the cells are already supported by their own abundant extracellular matrix. Type I and type IV collagen expression in parallel with MMP-1 and MMP-2 expression were monitored in the tissue models over a 10 day culture period under laminar flow regimes using protein immobilization technologies. Uniaxial tensile testing and scanning electron microscopy were used to compare the resulting effects of hydrodynamic stimulation upon structural integrity, while viability assays were conducted to evaluate the effects of shear on metabolic function. The proteomic results showed that the hyaluronan felt-supported tissues expressed higher levels of all remodelling proteins than those cultured on PTFE mesh. Overall, a 21% greater expression of type I collagen, 24% higher levels of type IV collagen, 24% higher levels of MMP-1 and 34% more MMP-2 were observed during hydrodynamic stress. This was coupled with a loss of structural integrity in these models after the introduction of laminar flow, as compared to the increases in all mechanical properties observed in the PTFE mesh-supported tissues. However, under flow conditions, the hyaluronan-supported tissues showed some recovery of the viability originally lost during static culture conditions, in contrast to PTFE mesh-based models, where initial gains were followed by a decline in metabolic viability after applied shear stress. Proteomic, cell viability and mechanical testing data emphasized the need for extended in vitro evaluations to enable better understanding of multi-stage remodelling and reparative processes in tissues cultured on biodegradable scaffolds. This study also highlighted the possibility that in high-density tissue culture with a biodegradable component, dynamic conditions may be more conducive to optimal tissue development than the static environment because they facilitate the efficient removal of high concentrations of degradation end-products accumulating in the pericellular space. Copyright © 2009 John Wiley & Sons, Ltd. [source] Roxithromycin inhibits transforming growth factor-, production by cultured human mesangial cellsNEPHROLOGY, Issue 6 2006HIDEAKI YAMABE SUMMARY: Background: Transforming growth factor-, (TGF-,) plays an important role in progression of renal injury. However, few materials which inhibit TGF-, have been known. Roxithromycin (ROX), macrolide antibiotics, is known to have anti-inflammatory, immunomodulatory and tissue reparative effects besides its bacteriostatic activity, although the exact mechanism of its anti-inflammatory and immunomodulatory effects was not defined. We examined the effect of ROX on production of TGF-, and type IV collagen by cultured human mesangial cells (HMC). Methods: Human mesangial cells were incubated with several concentrations of ROX and TGF-, and type IV collagen levels in the culture supernatants were measured by enzyme-linked immunoassay. Amount of TGF-, mRNA was also quantified by using a colourimetric mRNA quantification kit and semiquantitative reverse transcriptase polymerase chain reaction. We also examined the effect of ROX on tyrosine kinase, MAP kinase and NF-,B stimulated by thrombin. Results: Roxithromycin (0.1,10.0 µg/mL) inhibited TGF-, production by HMC in a dose- and time-dependent manner without inducing cell injury. ROX (10.0 µg/mL) also inhibited mRNA expression of TGF-, in HMC. Thrombin (5 U/mL) stimulated TGF-, production by HMC and ROX significantly inhibited the stimulating effect of thrombin on TGF-, production. ROX also inhibited the increment of type IV collagen production stimulated by thrombin. ROX (10.0 µg/mL) suppressed the thrombin-induced NF-,B activation, although ROX did not inhibit the activation of tyrosine kinase and MAP kinase by thrombin. Conclusion: Roxithromycin has an inhibitory effect on TGF-, production by HMC possibly via inhibition of NF-,B. ROX may be a potential agent for the treatment of glomerulosclerosis. [source] Familial fibronectin glomerulopathy: analysis of chromosome 1q32 and uteroglobin gene loci in a large New Zealand familyNEPHROLOGY, Issue 5 2001Robert Walker SUMMARY: Recently, a newly recognized familial glomerulopathy with predominant fibronectin deposits has been reported. This is the first report of a family with this condition in Australasia and spans two generations over a 30-year period, with the histologically confirmed glomerulopathy present in the father and five out of eight siblings. The clinical presentations have ranged from asymptomatic proteinuria, pregnancy-associated proteinuria and the nephrotic syndrome to hypertension and proteinuria with progressive renal failure. The time-course from presentation to renal failure was over a 20 years. Histology demonstrated global and diffuse thickening of capillary loops, but no cellular proliferation. Immunofluorescence demonstrated granular positivity for IgM in the capillary loops only. Electron microscopy demonstrated massive electron-dense subendothelial granular deposits with occasional small fibrils and unremarkable epithelial cell foot processes. Immunohistochemical staining was strongly positive for fibronectin and negative for type I or type IV collagen and transforming growth factor , in all biopsies. Genetic studies of familial fibronectin glomerulopathy have recently highlighted two genetic loci. Firstly, a large five-generation pedigree has been described with linkage of fibronectin glomerulopathy to chromosome 1q32. Secondly, fibronectin glomerulopathy has been reported in uteroglobin gene knockout mice. In our studies, DNA sequence analysis of the uteroglobin gene showed that it was normal in all family members, and a DNA polymorphism in the uteroglobin gene did not co-segregate with the disease. In addition, DNA microsatellite markers at the 1q32 locus did not co-segregate with the disease in our family. We presume that the underlying abnormality involves as yet undefined glomerular extracellular matrix regulation and is inherited as an autosomal dominant condition. These data favour genetic heterogeneity for the aetiology of fibronectin glomerulopathy. [source] Large cell calcifying Sertoli cell tumor of the testis: Comparative immunohistochemical study with Leydig cell tumorPATHOLOGY INTERNATIONAL, Issue 6 2005Katsuaki Sato Large cell calcifying Sertoli cell tumor is a rare type of testicular, tumor., Reported, herein, is, a, Japanese, patient with this tumor not associated with Carney's complex. An 11-year-old boy was admitted to hospital because of left testicular enlargement, and radical orchiectomy was performed. Macroscopically, the tumor was well circumscribed and had a maximum diameter of approximately 2 cm. The cut surface showed a yellow-white solid mass. Histologically, the tumor was composed of large neoplastic cells with abundant eosinophilic cytoplasm with a tubular, trabecular, and solid arrangement and loose myxoid stroma with irregularly shaped calcification. Immunohistochemically, the tumor cells were positive for vimentin, S-100 protein, calretinin, inhibin-,, melan-A, and CD10, and type IV collagen and laminin were observed in the extracellular matrix around the tumor cells. The distributions of melan-A, CD10, and mitochondria were characteristically patchy; in contrast, they were diffusely distributed in the cytoplasm in a control case of Leydig cell tumor. The differences in immunostaining patterns for melan-A, CD10, and mitochondria as well as positivity for S-100 protein-, might be useful diagnostic hallmarks of large cell calcifying Sertoli cell tumor for discrimination from Leydig cell tumor. [source] Abnormal basement membrane type IV collagen ,-chain composition in labial salivary glands in Sjögren's syndromeARTHRITIS & RHEUMATISM, Issue 4 2009P. Poduval Objective Sjögren's syndrome (SS) is characterized by atrophy and malfunction of the acinar cells. The aim of this study was to investigate whether type IV collagen ,-chain composition of acinar cell compartments could be abnormal in diseased glands. Methods Messenger RNA (mRNA) from human submandibular gland (HSG) cells, cultured with or without growth factor,depleted Matrigel, was analyzed using quantitative reverse transcription,polymerase chain reaction (RT-PCR). Labial salivary glands were analyzed using quantitative RT-PCR and immunohistochemistry. Results HSG cells of both the ductal and acinar phenotypes synthesized all ,-chain mRNA, in particular those of the ,1 and ,2 chains. Labial salivary glands (LSGs) contained ,1/2 chains but also contained mRNA of all the other ,-chains, although the mRNA copy numbers for the ,3 and ,4 chains were low, and the corresponding proteins were absent. Type IV collagen ,1/2-chains were observed in all tubuloalveolar basement membranes. In healthy glands, ,5 and ,6 chains were continuous around ducts but discontinuous around acini. In SS glands, these chains were absent or patchy around the ducts and absent around the acini. Conclusion Ductal and acinar epithelial cells are able to locally produce mRNA for all 6 different ,-chains. Type IV collagen ,1/2-chains seem to form the backbone in the tubuloalveolar basement membrane in salivary glands. Type IV collagen ,3 and ,4 chain mRNA were found in cultured salivary epithelial cells and LSG explants but were not translated to the corresponding ,-chains in LSGs. Both ,5 and ,6 mRNA were observed in salivary epithelial cells and glands. In healthy glands, immunolabeling always disclosed corresponding ,-chains around ducts, but their synthesis and/or degradation seemed to be locally regulated around acinar cells. [source] A structural study of the interaction between the Dr haemagglutinin DraE and derivatives of chloramphenicolACTA CRYSTALLOGRAPHICA SECTION D, Issue 6 2009David M. Pettigrew Dr adhesins are expressed on the surface of uropathogenic and diffusely adherent strains of Escherichia coli. The major adhesin subunit (DraE/AfaE) of these organelles mediates attachment of the bacterium to the surface of the host cell and possibly intracellular invasion through its recognition of the complement regulator decay-accelerating factor (DAF) and/or members of the carcinoembryonic antigen (CEA) family. The adhesin subunit of the Dr haemagglutinin, a Dr-family member, additionally binds type IV collagen and is inhibited in all its receptor interactions by the antibiotic chloramphenicol (CLM). In this study, previous structural work is built upon by reporting the X-ray structures of DraE bound to two chloramphenicol derivatives: chloramphenicol succinate (CLS) and bromamphenicol (BRM). The CLS structure demonstrates that acylation of the 3-hydroxyl group of CLM with succinyl does not significantly perturb the mode of binding, while the BRM structure implies that the binding pocket is able to accommodate bulkier substituents on the N -acyl group. It is concluded that modifications of the 3-hydroxyl group would generate a potent Dr haemagglutinin inhibitor that would not cause the toxic side effects that are associated with the normal bacteriostatic activity of CLM. [source] Use of Green Fluorescent Protein-Conjugated ,-Actin as a Novel Molecular Marker for in Vitro Tumor Cell Chemotaxis AssayBIOTECHNOLOGY PROGRESS, Issue 6 2000Louis Hodgson To study the dynamics of actin cytoskeleton rearrangement in living cells, an eukaryotic expression vector expressing a ,-actin-GFP fusion protein was generated. The expression construct when transfected into NIH3T3 fibroblast, A2058 human melanoma and 293T human embryonic kidney carcinoma cell lines expressed ,-actin-GFP fusion protein, which colocalized with endogenous cellular actin as determined by histoimmunofluorescence staining. The ,-actin-GFP was also observed to be reorganized in response to treatments with the chemoattractant type IV collagen. Cells extended pseudopodial protrusions and altered the morphology of their cortical structure in response to type IV collagen stimulation. More importantly, ,-actin-GFP accumulated in areas undergoing these dynamic cytoskeleton changes, indicating that ,-actin-GFP could participate in actin polymerization. Although ectopic expression of ,-actin-GFP lead to minor side effects on cell proliferation, these studies suggest that this strategy provides an alternative to the invasive techniques currently used to study actin dynamics and permits real-time visualization of actin rearrangements in response to environmental cues. [source] Low-energy helium,neon laser induces melanocyte proliferation via interaction with type IV collagen: visible light as a therapeutic option for vitiligoBRITISH JOURNAL OF DERMATOLOGY, Issue 2 2009C-C.E. Lan Summary Background, The treatment of vitiligo remains a challenge for clinical dermatologists. We have previously shown that the helium,neon laser (He,Ne laser, 632·8 nm) is a therapeutic option for treatment of this depigmentary disorder. Objectives, Addressing the intricate interactions between melanocytes, the most important cellular component in the repigmentation scheme of vitiligo, and their innate extracellular matrix collagen type IV, the current study aimed to elucidate the effects of the He,Ne laser on melanocytes. Methods, Cultured melanocytes were irradiated with the He,Ne laser. Relevant biological parameters including cell attachment, locomotion and growth were evaluated. In addition, the potentially involved molecular pathways were also determined. Results, Our results show that in addition to suppressing mobility but increasing attachment to type IV collagen, the He,Ne laser stimulates melanocyte proliferation through enhanced ,2,1 integrin expression. The expression of phosphorylated cyclic-AMP response element binding protein (CREB), an important regulator of melanocyte growth, was also upregulated by He,Ne laser treatment. Using a specific mitochondrial uncoupling agent [carbonyl cyanide m-chlorophenyl-hydrazone (CCCP)], the proliferative effect of the He,Ne laser on melanocytes was abolished and suppression of melanocyte growth was noted. Conclusions, In summary, we have demonstrated that the He,Ne laser imparts a growth stimulatory effect on functional melanocytes via mitochondria-related pathways and proposed that other minor pathways including DNA damage may also be inflicted by laser treatment on irradiated cells. More importantly, we have completed the repigmentation scheme of vitiligo brought about by He,Ne laser light in vitro and provided a solid theoretical basis regarding how the He,Ne laser induces recovery of vitiligo in vivo. [source] The effect of mechanical strain on protease production by keratinocytesBRITISH JOURNAL OF DERMATOLOGY, Issue 2 2008N. Bhadal Summary Background, Intact skin is under constant tension, transmitted from the underlying dermis, but when tension is lost (i.e. upon wounding) protease activity is upregulated. Objectives, To investigate the effect of mechanical strain on protease production by both normal and transformed keratinocytes in vitro. Methods, Keratinocytes were seeded on to membranes precoated with either type I or type IV collagen. After 48 h medium was replaced with serum-free medium and mechanical strain was applied. Results, Mechanical strain resulted in decreased urokinase-type plasminogen activator (uPA) production by normal human keratinocytes (P < 0·05) but increased production by transformed keratinocytes (P < 0·05) cultured on type I and type IV collagen. Conclusions, Differential production of uPA by normal and transformed keratinocytes is relevant in the context of normal function, wound healing and tumorigenesis. [source] | ||||||||||||||||