The role of myofibroblasts and interstitial fibrosis in the progression of membranous nephropathy

Renal interstitial fibrosis has been observed in a large number of nephropathies and contributes to the progressive deterioration of renal function. Myofibroblasts have been implicated in the reparative process of tissue injury, including renal scarring secondary to glomerular diseases. We performed a retrospective study on 28 patients with biopsy-proven primary membranous nephropathy, to determine whether interstitial myofibroblasts and tubulointerstitial lesions correlated with renal function at follow-up. Tubulointerstitial pathology was evaluated by morphometric and semiquantitative methods. Interstitial myofibroblasts were counted; 24-hour urinary protein and serum creatinine at the time of diagnosis and at the end of follow-up were available for all the patients. There were 20 males and 8 females, age 2-67 years (mean 42.3±153), most of them with nephrotic syndrome (78.6%). The final renal function had deteriorated in 16 patients (57.1%) and in 5 patients (17.8%) reached end-stage. The renal outcome was correlated with histological changes. We found a positive correlation between the severity of tubulointerstitial damage and the deterioration of the final serum creatinine (r 2=0.185; p=0.016). Myofibroblasts did not predict impaired renal function at the final follow-up. The current data do not support previous suggestions that myofibroblasts are a useful a predictor of end-stage renal disease.

The role of membranous urethral afferent autonomic innervation in the continence mechanism after nerve sparing radical prostatectomy: a clinical and prospective study

Purpose: We evaluated the somatic and autonomic innervation of the pelvic floor and rhabdosphincter before and after nerve sparing radical retropubic prostatectomy using neurophysiological tests and correlated findings with clinical parameters and urinary continence. Materials and Methods: From February 2003 to October 2005, 46 patients with prostate cancer were enrolled in a controlled, prospective study. Patients were evaluated before and 6 months after nerve sparing radical retropubic prostatectomy using the UCLA-PCI urinary function domain and neurophysiological tests, including somatosensory evoked potential, and the pudendo-urethral, pudendo-anal and urethro-anal reflexes. Clinical parameters and urinary continence were correlated with afferent and efferent innervation of the membranous urethra and pelvic floor. We used strict criteria to define urinary continence as complete dryness with no leakage at all, not requiring any pads or diapers and with a UCLA-PCI score of 500. Patients with a sporadic drop of leakage, requiring up to 1 pad daily, were defined as having occasional urinary leakage. Results: Two patients were excluded from study due to urethral stricture postoperatively. We evaluated 44 patients within 6 months after surgery. The pudendo-anal and pudendo-urethral reflexes were unchanged postoperatively (p = 0.93 and 0.09, respectively), demonstrating that afferent and efferent pudendal innervation to this pelvic region was not affected by the surgery. Autonomic afferent denervation of the membranous urethral mucosa was found in 34 patients (77.3%), as demonstrated by a postoperative increase in the urethro-anal reflex sensory threshold and urethro-anal reflex latency (p <0.001 and 0.0007, respectively). Six of the 44 patients used pads. One patient with more severe leakage required 3 pads daily and 23 showed urinary leakage, including 5 who needed 1 pad per day and 18 who did not wear pads. Afferent autonomic denervation at the membranous urethral mucosa was found in 91.7% of patients with urinary leakage. Of 10 patients with preserved urethro-anal reflex latency 80% were continent. Conclusions: Sensory and motor pudendal innervation to this specific pelvic region did not change after nerve sparing radical retropubic prostatectomy. Significant autonomic afferent denervation of the membranous urethral mucosa was present in most patients postoperatively. Impaired membranous urethral sensitivity seemed to be associated with urinary incontinence, particularly in patients with occasional urinary leakage. Damage to the afferent autonomic innervation may have a role in the continence mechanism after nerve sparing radical retropubic prostatectomy.

The Involvement of the Intermediate Chain of Cytoplasmic Dynein in Binding the Motor Complex to Membranous Organelles of Xenopus Oocytes

Cytoplasmic dynein is one of the major motor proteins involved in intracellular transport. It is a protein complex consisting of four subunit classes: heavy chains, intermediate chains (ICs), light intermediate chains, and light chains. In a previous study, we had generated new monoclonal antibodies to the ICs and mapped the ICs to the base of the motor. Because the ICs have been implicated in targeting the motor to cargo, we tested whether these new antibodies to the intermediate chain could block the function of cytoplasmic dynein. When cytoplasmic extracts of Xenopus oocytes were incubated with either one of the monoclonal antibodies (m74–1, m74–2), neither organelle movement nor network formation was observed. Network formation and membrane transport was blocked at an antibody concentration as low as 15 μg/ml. In contrast to these observations, no effect was observed on organelle movement and tubular network formation in the presence of a control antibody at concentrations as high as 0.5 mg/ml. After incubating cytoplasmic extracts or isolated membranes with the monoclonal antibodies m74–1 and m74–2, the dynein IC polypeptide was no longer detectable in the membrane fraction by SDS-PAGE immunoblot, indicating a loss of cytoplasmic dynein from the membrane. We used a panel of dynein IC truncation mutants and mapped the epitopes of both antibodies to the N-terminal coiled-coil domain, in close proximity to the p150Glued binding domain. In an IC affinity column binding assay, both antibodies inhibited the IC–p150Glued interaction. Thus these findings demonstrate that direct IC–p150Glued interaction is required for the proper attachment of cytoplasmic dynein to membranes.

Subcellular colocalization of the cellular and scrapie prion proteins in caveolae-like membranous domains

Results of transgenetic studies argue that the scrapie isoform of the prion protein (PrPSc) interacts with the substrate cellular PrP (PrPC) during conversion into nascent PrPSc. While PrPSc appears to accumulate primarily in lysosomes, caveolae-like domains (CLDs) have been suggested to be the site where PrPC is converted into PrPSc. We report herein that CLDs isolated from scrapie-infected neuroblastoma (ScN2a) cells contain PrPC and PrPSc. After lysis of ScN2a cells in ice-cold Triton X-100, both PrP isoforms and an N-terminally truncated form of PrPC (PrPC-II) were found concentrated in detergent-insoluble complexes resembling CLDs that were isolated by flotation in sucrose gradients. Similar results were obtained when CLDs were purified from plasma membranes by sonication and gradient centrifugation; with this procedure no detergents are used, which minimizes artifacts that might arise from redistribution of proteins among subcellular fractions. The caveolar markers ganglioside GM1 and H-ras were found concentrated in the CLD fractions. When plasma membrane proteins were labeled with the impermeant reagent sulfo-N-hydroxysuccinimide-biotin, both PrPC and PrPSc were found biotinylated in CLD fractions. Similar results on the colocalization of PrPC and PrPSc were obtained when CLDs were isolated from Syrian hamster brains. Our findings demonstrate that both PrPC and PrPSc are present in CLDs and, thus, support the hypothesis that the PrPSc formation occurs within this subcellular compartment.

Membranous catarrh of the intestines (colica mucosa),

Authorized American edition, tr. under the direction of Boardman Reed ...

A predictor of membrane class:discriminating α-helical and β-barrel membrane proteins from non-membranous proteins

Accurate protein structure prediction remains an active objective of research in bioinformatics. Membrane proteins comprise approximately 20% of most genomes. They are, however, poorly tractable targets of experimental structure determination. Their analysis using bioinformatics thus makes an important contribution to their on-going study. Using a method based on Bayesian Networks, which provides a flexible and powerful framework for statistical inference, we have addressed the alignment-free discrimination of membrane from non-membrane proteins. The method successfully identifies prokaryotic and eukaryotic α-helical membrane proteins at 94.4% accuracy, β-barrel proteins at 72.4% accuracy, and distinguishes assorted non-membranous proteins with 85.9% accuracy. The method here is an important potential advance in the computational analysis of membrane protein structure. It represents a useful tool for the characterisation of membrane proteins with a wide variety of potential applications.

Simultaneous Occurrence of Systemic Lupus Erythematosus, Rheumatoid Arthritis, Hashimoto’s Thyroiditis and Membranous Glomerulonephritis

Introduction: Membranous glomerulonephritis is commonly described in systemic lupus erythematosus (SLE) and hypothyroidism. Clinical presentation: We report a case of a 40-year-old woman who presented with a membranous glomerulonephritis associated with SLE, rheumatoid arthritis and hypothyroidism due to Hashimoto’s thyroiditis. Conclusions: The simultaneous occurrence of these three diseases as possible causes of membranous glomerulonephritis is extremely exceptional.

Characterisation of mesenchymal cells from osteophytes in osteoarthritis

Osteophytes form through the process of chondroid metamorphosis of fibrous tissue followed by endochondral ossification. Osteophytes have been found to consist of three different mesenchymal tissue regions including endochondral bone formation within cartilage residues, intra-membranous bone formation within fibrous tissue and bone formation within bone marrow spaces. All these features provide evidence of mesenchymal stem cells (MSC) involvement in osteophyte formation; nevertheless, it remains to be characterised. MSC from numerous mesenchymal tissues have been isolated but bone marrow remains the “ideal” due to the ease of ex vivo expansion and multilineage potential. However, the bone marrow stroma has a relatively low number of MSC, something that necessitates the need for long-term culture and extensive population doublings in order to obtain a sufficient number of cells for therapeutic applications. MSC in vitro have limited proliferative capacity and extensive passaging compromises differentiation potential. To overcome this barrier, tissue derived MSC are of strong interest for extensive study and characterisation, with a focus on their potential application in therapeutic tissue regeneration. To date, no MSC type cell has been isolated from osteophyte tissue, despite this tissue exhibiting all the hallmark features of a regenerative tissue. Therefore, this study aimed to isolate and characterise cells from osteophyte tissues in relation to their phenotype, differentiation potential, immuno-modulatory properties, proliferation, cellular ageing, longevity and chondrogenesis in in vitro defect model in comparison to patient matched bone marrow stromal cells (bMSC). Osteophyte derived cells were isolated from osteophyte tissue samples collected during knee replacement surgery. These cells were characterised by the expression of cell surface antigens, differentiation potential into mesenchymal lineages, growth kinetics and modulation of allo-immune responses. Multipotential stem cells were identified from all osteophyte samples namely osteophyte derived mesenchymal stem cells (oMSC). Extensively expanded cell cultures (passage 4 and 9 respectively) were used to confirm cytogenetic stability and study signs of cellular aging, telomere length and telomerase activity. Cultured cells at passage 4 were used to determine 84 pathway focused stem cell related gene expression profile. Micro mass pellets were cultured in chondrogenic differentiation media for 21 days for phenotypic and chondrogenic related gene expression. Secondly, cell pellets differentiated overnight were placed into articular cartilage defects and cultured for further 21 days in control medium and chondrogenic medium to study chondrogenesis and cell behaviour. The surface antigen expression of oMSC was consistent with that of mesenchymal stem cells, such as lacking the haematopoietic and common leukocyte markers (CD34, CD45) while expressing those related to adhesion (CD29, CD166, CD44) and stem cells (CD90, CD105, CD73). The proliferation capacity of oMSC in culture was superior to that of bMSC, and they readily differentiated into tissues of the mesenchymal lineages. oMSC also demonstrated the ability to suppress allogeneic T-cell proliferation, which was associated with the expression of tryptophan degrading enzyme indoleamine 2,3 dioxygenase (IDO). Cellular aging was more prominent in late passage bMSC than in oMSC. oMSC had longer telomere length in late passages compared with bMSC, although there was no significant difference in telomere lengths in the early passages in either cell type. Telomerase activity was detectable only in early passage oMSC and not in bMSC. In osteophyte tissues telomerase positive cells were found to be located peri vascularly and were Stro-1 positive. Eighty-four pathway-focused genes were investigated and only five genes (APC, CCND2, GJB2, NCAM and BMP2) were differentially expressed between bMSC and oMSC. Chondrogenically induced micro mass pellets of oMSC showed higher staining intensity for proteoglycans, aggrecan and collagen II. Differential expression of chondrogenic related genes showed up regulation of Aggrecan and Sox 9 in oMSC and collagen II in bMSC. The in vitro defect models of oMSC in control medium showed rounded and aggregated cells staining positively for proteoglycan and presence of some extracellular matrix. In contrast, defects with bMSC showed fragmentation and loss of cells, fibroblast-like cell morphology staining positively for proteoglycans. For defects maintained in chondrogenic medium, rounded, aggregated and proteoglycan positive cells were found in both oMSC and bMSC cultures. Extracellular matrix and cellular integration into newly formed matrix was evident only in oMSC defects. For analysis of chondrocyte hypertrophy, strong expression of type X collagen could be noticed in the pellet cultures and transplanted bMSC. In summary, this study demonstrated that osteophyte derived cells had similar properties to mesenchymal stem cells in the expression of antigen phenotype, differential potential and suppression of allo-immune response. Furthermore, when compared to bMSC, oMSC maintained a higher proliferative capacity due to a retained level of telomerase activity in vitro, which may account for the relatively longer telomeres delaying growth arrest by replicative senescence compared with bMSC. oMSC behaviour in defects supported chondrogenesis which implies that cells derived from regenerative tissue can be an alternative source of stem cells and have a potential clinical application for therapeutic stem cell based tissue regeneration.

Conceptualisation of articular cartilage as a giant reverse micelle: A hypothetical mechanism for joint biocushioning and lubrication

Phospholipid (PL) molecules form the main structure of the membrane that prevents the direct contact of opposing articular cartilage layers. In this paper we conceptualise articular cartilage as a giant reverse micelle (GRM) in which the highly hydrated three-dimensional network of phospholipids is electrically charged and able to resist compressive forces during joint movement, and hence loading. Using this hypothetical base, we describe a hydrophilic-hydrophilic (HL-HL) biopair model of joint lubrication by contacting cartilages, whose mechanism is reliant on lamellar cushioning. To demonstrate the viability of our concept, the electrokinetic properties of the membranous layer on the articular surface were determined by measuring via microelectrophoresis, the adsorption of ions H, OH, Na and Cl on phospholipid membrane of liposomes, leading to the calculation of the effective surface charge density. The surface charge density was found to be -0.08 ± 0.002 cm-2 (mean ± S.D.) for phospholipid membranes, in 0.155 M NaCl solution and physiological pH. This value was approximately five times less than that measured in 0.01 M NaCl. The addition of synovial fluid (SF) to the 0.155 M NaCl solution reduced the surface charge density by 30% which was attributed to the binding of synovial fluid macromolecules to the phospholipid membrane. Our experiments show that particles charge and interact strongly with the polar core of RM. We demonstrate that particles can have strong electrostatic interactions when ions and macromolecules are solubilized by reverse micelle (RM). Since ions are solubilized by reverse micelle, the surface entropy influences the change in the charge density of the phospholipid membrane on cartilage surfaces. Reverse micelles stabilize ions maintaining equilibrium, their surface charges contribute to the stability of particles, while providing additional screening for electrostatic processes. © 2008 Elsevier Ireland Ltd. All rights reserved.

An exploratory study of the potential of resurfacing articular cartilage with synthetic phospholipids

This thesis is aimed at further understanding the uppermost lipid-filled membranous layer (i.e. surface amorphous layer (SAL)) of articular cartilage and to develop a scientific framework for re-introducing lipids onto the surface of lipid-depleted articular cartilage (i.e. "resurfacing"). The outcome will potentially contribute to knowledge that will facilitate the repair of the articular surface of cartilage where degradation is limited to the loss of the lipids of the SAL only. The surface amorphous layer is of utmost importance to the effective load-spreading, lubrication, and semipermeability (which controls its fluid management, nutrient transport and waste removal) of articular cartilage in the mammalian joints. However, because this uppermost layer of cartilage is often in contact during physiological function, it is prone to wear and tear, and thus, is the site for damage initiation that can lead to the early stages of joint condition like osteoarthritis, and related conditions that cause pain and discomfort leading to low quality of life in patients. It is therefore imperative to conduct a study which offers insight into remedying this problem. It is hypothesized that restoration (resurfacing) of the surface amorphous layer can be achieved by re-introducing synthetic surface-active phospholipids (SAPL) into the joint space. This hypothesis was tested in this thesis by exposing cartilage samples whose surface lipids had been depleted to individual and mixtures of synthetic saturated and unsaturated phospholipids. The surfaces of normal, delipidized, and relipidized samples of cartilage were characterized for their structural integrity and functionality using atomic force microscope (AFM), confocal microscope (COFM), Raman spectroscopy, magnetic resonance imaging (MRI) with image processing in the MATLAB® environment and mechanical loading experiments. The results from AFM imaging, confocal microscopy, and Raman spectroscopy revealed a successful deposition of new surface layer on delipidized cartilage when incubated in synthetic phospholipids. The relipidization resulted in a significant improvement in the surface nanostructure of the artificially degraded cartilage, with the complete SAPL mixture providing better outcomes in comparison to those created with the single SAPL components (palmitoyl-oleoyl-phosphatidylcholine, POPC and dipalmitoyl-phosphatidylcholine, DPPC). MRI analysis revealed that the surface created with the complete mixture of synthetic lipids was capable of providing semipermeability to the surface layer of the treated cartilage samples relative to the normal intact surface. Furthermore, deformation energy analysis revealed that the treated samples were capable of delivering the elastic properties required for load bearing and recovery of the tissue relative to the normal intact samples, with this capability closer between the normal and the samples incubated in the complete lipid mixture. In conclusion, this thesis has established that it is possible to deposit/create a potentially viable layer on the surface of cartilage following degradation/lipid loss through incubation in synthetic lipid solutions. However, further studies will be required to advance the ideas developed in this thesis, for the development of synthetic lipid-based injections/drugs for treatment of osteoarthritis and other related joint conditions.

MMP-9 and the epidermal growth factor signal pathway in non-small cell lung cancer

Background Matrix metalloproteinase (MMP)-9 is an endopeptidase that digests basement membrane type-IV collagen. Enhanced expression has been related to tumour progression in a number of systems. The control of MMP expression is complex, but recently epidermal growth actor receptor (EGFR) activity has been implicated in up-regulation of MMP-9 in tumour cells in vitro. Aims To evaluate interrelations between MMP-9 and EGFR expression in non-small cell lung cancer (NSCLC) and to assess the impact of expression on survival. Methods This is a retrospective study of 152 patients who underwent resection for stage I-IIIa NSCLC with a post-operative survival >60 days. Minimum follow-up was 2 years. Standard ABC immunohistochemistry was performed on 4μm paraffin-embedded sections from the tumour periphery using monoclonal antibodies to MMP-9 and EGFR. Results: MMP-9 was expressed in the tumour cells of 79/152 (52%) cases. EGFR expression was found in 86/152 (57%) cases [membranous 51/152 (34%), cytoplasmic 35/152 (23%)]. MMP-9 expression was associated with poor outcome (p=0.04). Membranous, cytoplasmic and overall EGFR expression were not associated with outcome (p=0.29, p=0.85 and p=0.41 respectively). There was a strong correlation between MMP-9 expression and EGFR expression (p=0.001) and EGFR membranous expression (p=0.01) but not with cytoplasmic EGFR expression (p=0.28). Co-expression of MMP-9 and EGFR (36%) conferred a worse prognosis (p=0.003). Subset analysis revealed only MMP-9 and membranous EGFR co-expression (22%) was associated with poor outcome (p=0.008). Conclusions Our results show that MMP-9 and EGFR are co-expressed in NSCLC. This finding suggests the EGFR signalling pathway may play an important role in the invasive behaviour of NSCLC via specific upregulation of MMP-9. The co-expression of these markers also confers a poor prognosis.

Herceptest: HER2 expression and gene amplification in non-small cell lung cancer

HER2 is an erbB/HER type I tyrosine kinase receptor that is frequently over-expressed in malignant epithelial tumours. Herceptin, a humanised mouse monoclonal antibody to HER2, is proven therapeutically in the management of metastatic breast cancer, significantly prolonging survival when combined with cytotoxic chemotherapeutic agents. Immunohistochemical studies suggest that non-small-cell lung cancer (NSCLC) tumours may over-express HER2. Our aim was to evaluate HER2 gene amplification and semi-quantitative immuno-expression in NSCLC. A total of 344 NSCLC cases were immunostained for HER2 expression in 2 centres using the HercepTest. Fluorescence in situ hybridisation (FISH) analysis for HER2 gene amplification was performed on most positive cases and a subset of negative cases. Fifteen cases (4.3%) demonstrated 2+ or 3+ membranous HER2 immuno-expression. There was no correlation between immuno-expression and tumour histology or grade. Tumours from higher-stage disease were more often HercepTest-positive (p < 0.001). All 4 HercepTest 3 + cases demonstrated gene amplification. One of the 5 2+ cases tested for gene amplification showed areas of borderline amplification and areas of polyploidy. None of the 19 HercepTest-negative cases demonstrated gene amplification or polyploidy (p < 0.001). Gene amplification was demonstrated in all HercepTest 3+ scoring NSCLC cases. Unlike breast cancer, gene amplification and HER2 protein over-expression assessed by the HercepTest appeared to be uncommon in NSCLC. Herceptin may therefore target only a small proportion of NSCLC tumours and be of limited clinical value in this disease, particularly in the adjuvant setting. © 2001 Wiley-Liss, Inc.

Cyclooxygenase-2 protein levels are independent of epidermal growth factor receptor expression or activation in operable non-small cell lung cancer

Both cyclooxygenase (COX)-2 and epidermal growth factor receptor (EGFR) are thought to play important roles in the pathogenesis of non-small cell lung cancer (NSCLC). A number of in vitro studies have postulated a link between EGFR activation and subsequent COX-2 upregulation. The relationship between these factors has not been established in patients with NSCLC. COX-2 and EGFR expression were studied in 172 NSCLC specimens using standard immunohistochemical techniques. Western blotting was used to determine COX-2 and EGFR levels in five NSCLC cell lines. The effect of treatment with EGF on COX-2 expression in A549 cells was assessed. Results: Both EGFR and COX-2 are overexpressed in NSCLC. The predominant pattern of COX-2 and EGFR staining was cytoplasmic. Membranous EGFR staining was seen in 23.3% of cases. There was no relationship between COX-2 and EGFR expression and survival or any clinicopathological features. No correlation was seen between EGFR expression and COX-2 expression in the immunohistochemical series or in the cell lines. Treatment with EGF did not upregulate COX-2 levels in A549 cells, either in serum free or serum-supplemented conditions. Conclusions: Although COX-2 and EGFR are over-expressed in NSCLC neither was of prognostic significance in this series of cases. There is no correlation between these two factors in either tumour samples or cell lines. Although these factors show no correlation in NSCLC, they remain potential, though independent targets for treatment. © 2004 Elsevier Ireland Ltd. All rights reserved.

Carbonic anhydrase IX expression and outcome after radiotherapy for muscle-invasive bladder cancer

Aims: Carbonic anhydrase IX (CA IX) expression has been described as an endogenous marker of hypoxia in solid neoplasms. Furthermore, CA IX expression has been associated with an aggressive phenotype and resistance to radiotherapy. We assessed the prognostic significance of CA IX expression in patients with muscle-invasive bladder cancer treated with radiotherapy. Materials and methods: A standard immunohistochemistry technique was used to show CA IX expression in 110 muscle-invasive bladder tumours treated with radiotherapy. Clinicopathological data were obtained from medical case notes. Results: CA IX immunostaining was detected in 89 (∼81%) patients. Staining was predominantly membranous, with areas of concurrent cytoplasmic and nuclear staining and was abundant in luminal and perinecrotic areas. No significant correlation was shown between the overall CA IX status and the initial response to radiotherapy, 5-year bladder cancer-specific survival or the time to local recurrence. Conclusions: The distribution of CA IX expression in paraffin-embedded tissue sections seen in this series is consistent with previous studies in bladder cancer, but does not provide significant prognostic information with respect to the response to radiotherapy at 3 months and disease-specific survival after radical radiotherapy. © 2007 The Royal College of Radiologists.