Basement Membrane Morphology Research Articles

Baseline reticular basement membrane morphology is related to subsequent spirometry deterioration in pediatric chronic airway inflammation: a follow-up study.

Reticular basement membrane (RBM) thickening may occur in children with allergic bronchial asthma (BA), cystic fibrosis (CF), and primary ciliary dyskinesia (PCD). Its functional consequences remain unknown. We investigated the relationship between baseline RBM thickness and subsequent spirometry. In our cohort follow-up study, patients aged 3-18 yr with BA, CF, and PCD and controls underwent baseline lung clearance index (LCI) measurement, spirometry, and endobronchial biopsy sampling. Total RBM and collagen IV-positive layer thickness were measured. Trends in forced vital capacity (FVC), forced expired volume in 1 s (FEV1), and FEV1/FVC were analyzed during follow-up, and their relationship to baseline characteristics was studied using univariate analysis and multiple regression models. Complete baseline data were available in 19 patients with BA, 30 patients with CF, 25 patients with PCD, and 19 controls. The RBM was thicker in patients with BA (6.33 ± 1.22 μm), CF (5.60 ± 1.39 μm), and PCD (6.50 ± 1.87 μm) than in controls (3.29 ± 0.55 μm) (all P < 0.001). The LCI was higher in patients with CF (15.32 ± 4.58, P < 0.001) and PCD (10.97 ± 2.46, P = 0.002) than in controls (7.44 ± 0.43). The median follow-up times were 3.6, 4.8, 5.7, and 1.9 years in patients with BA, CF, PCD, and controls, respectively. The z-scores of FEV1 and FEV1/FVC deteriorated significantly in all groups except in controls. In patients with CF and PCD, trends in FEV1 z-scores correlated with baseline LCI and RBM; in BA, it correlated with collagen IV. In multiple regression models, RBM morphology and ventilation inhomogeneity could predict up to 84.4% of variability in spirometry trends. In conclusion, baseline LCI value and RBM morphology may predict trends in subsequent spirometry.NEW & NOTEWORTHY This paper deals with the relationship between reticular basement membrane (RBM) morphology at baseline and follow-up spirometry in children with asthma, cystic fibrosis, and primary ciliary dyskinesia. For the first time, to our knowledge, the possibility to predict subsequent lung function development using selected baseline characteristics (reticular basement membrane morphology from endobronchial biopsy and ventilation inhomogeneity from nitrogen multiple breath washout test) is proposed. Corresponding predictive models are presented.

Effects of collagen type ⅩⅦ α1 on epidermal stem cells in aging skin and the microRNA intervention mechanism

Effects of collagen type ⅩⅦ α1 on epidermal stem cells in aging skin and the microRNA intervention mechanism

Collagen type XIX regulates cardiac extracellular matrix structure and ventricular function

The cardiac extracellular matrix plays essential roles in homeostasis and injury responses. Although the role of fibrillar collagens have been thoroughly documented, the functions of non-fibrillar collagen members remain underexplored. These include a distinct group of non-fibrillar collagens, termed, fibril-associated collagens with interrupted triple helices (FACITs). Recent reports of collagen type XIX (encoded by Col19a1) expression in adult heart and evidence of its enhanced expression in cardiac ischemia suggest important functions for this FACIT in cardiac ECM structure and function. Here, we examined the cellular source of collagen XIX in the adult murine heart and evaluated its involvement in ECM structure and ventricular function. Immunodetection of collagen XIX in fractionated cardiovascular cell lineages revealed fibroblasts and smooth muscle cells as the primary sources of collagen XIX in the heart. Based on echocardiographic and histologic analyses, Col19a1 null (Col19a1N/N) mice exhibited reduced systolic function, thinning of left ventricular walls, and increased cardiomyocyte cross-sectional areas—without gross changes in myocardial collagen content or basement membrane morphology. Col19a1N/N cardiac fibroblasts had augmented expression of several enzymes involved in the synthesis and stability of fibrillar collagens, including PLOD1 and LOX. Furthermore, second harmonic generation-imaged ECM derived from Col19a1N/N cardiac fibroblasts, and transmission electron micrographs of decellularized hearts from Col19a1N/N null animals, showed marked reductions in fibrillar collagen structural organization. Col19a1N/N mice also displayed enhanced phosphorylation of focal adhesion kinase (FAK), signifying de-repression of the FAK pathway—a critical mediator of cardiomyocyte hypertrophy. Collectively, we show that collagen XIX, which had a heretofore unknown role in the mammalian heart, participates in the regulation of cardiac structure and function—potentially through modulation of ECM fibrillar collagen structural organization. Further, these data suggest that this FACIT may modify ECM superstructure via acting at the level of the fibroblast to regulate their expression of collagen synthetic and stabilization enzymes.

Abstract P6-06-03: Mechanobiology of cancer cells and tumour microenvironment jointly regulates cancer cell invasion and onset of metastasis

Abstract Mechanical properties of cancer cells and their microenvironment play a critical role in cancer progression and metastatic dissemination. We have established an in vitro assay using native human basement membrane (BM) to study interactions with normal epithelial cells in terms of BM composition, architecture and stiffness. We showed that these are required to act jointly in order to achieve apico-basal polarity, tissue barrier formation and specific stiffness to maintain healthy epithelial tissues. In contrast, in cancer the onset of metastasis occurs when cancer cells invade and breach the BM. For example, it is well known that cancer cells can perforate BM using proteolysis. However, recently we have demonstrated that in presence of carcinoma-associated fibroblasts (CAFs) isolated from cancer patients, moderately invasive cancer cells were able to invade into surrounding stromal matrix in a metalloproteinase-independent manner. In fact cell invasion was associated with decrease in stiffness and correlated to changes in cell and BM morphology. Using live imaging and atomic force microscopy (AFM), we could visualise CAFs actively pulling, stretching and softening the BM, forming gaps through which cancer cells can migrate. By exerting contractile forces, CAFs altered the organisation and physical properties of native BM, making it permissive for cancer cell invasion. Also recently we showed that from the epithelial side, structural centrosome aberrations known to accompany breast cancer progression can create local stiffness heterogeneity that triggers the escape of soft cells undergoing mitosis. In particular, we demonstrated that structural centrosome aberrations can trigger cell dissemination through a non-cell-autonomous mechanism, raising the prospect that centrosome aberrations might facilitate dissemination of soft metastatic cells. In summary, physical interactions between cancer cells and the extracellular matrix (ECM) components contribute to softening of specific cancer cells, physical activation of stromal cells such as CAFs to facilitate cancer invasion and metastases. By extending these findings to a routine clinical setting we measured nanomechanical profiles of biopises from breast cancer patients in a physiological tissue protective solution thus preserving tissue viability and allowing for further pathohistological analysis of the same specimens. Conclusively, we demonstrated how detecting highly deformable cancer cells in patient tissues can serve as a unique marker of cancer aggressiveness thus potentially enabling treatment optimization and development of novel treatment strategies. Citation Format: Marija Plodinec, Philipp Oertle, Alexandre Glentis, Danijela Vignjevic, Olivier Ganier, Eric Nigg, Tobias Appenzeller, Ahmed Jizawi, Flora Scott, Cristian Raez, Marko Loparic, Zlatko Marusic, Ellen C. Obermann, Rosemarie Burian, Roderick Y.H. Lim. Mechanobiology of cancer cells and tumour microenvironment jointly regulates cancer cell invasion and onset of metastasis [abstract]. In: Proceedings of the 2019 San Antonio Breast Cancer Symposium; 2019 Dec 10-14; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2020;80(4 Suppl):Abstract nr P6-06-03.

MON-229 IRX3 and IRX5 Regulate Downstream Targets that Promote Ovarian Follicle Integrity in Mice

Appropriate cell-cell interactions and communication within ovarian follicles are required for their healthy development and maturation and are critical for maintaining female reproductive life quality. Our lab previously showed that two Iroquois transcription factor family genes, Irx3 and Irx5, were important for ovarian follicle integrity. Deletion of Irx3 and Irx5 (Irx3-Irx5EGFP/Irx3-Irx5EGFP, Irx3/5 DKO) resulted in disrupted granulosa cell-oocyte contacts, ectopic gap junction protein expression, and abnormal basement membrane morphology. The mechanisms by which Irx3 and Irx5 promote follicle integrity, however, are unknown. Therefore, we performed RNA-seq experiments on control and Irx3/5 DKO ovarian samples at two critical phases of ovarian development: embryonic day 13.5 when germline nests are established and just after birth when germline nests break down and primordial follicles form. RNA-seq analyses revealed that Inhbb, a TGF-β superfamily gene, was significantly upregulated in Irx3/5 DKO samples at both time points. It was previously reported that Inhbb was also upregulated in Wnt4 and β-catenin mutant ovaries. Notably, similar to the Irx3/5 DKO ovary, cell-cell interaction failure and germ cell death are prominent features of these models and removal of the excessive Inhbb by breeding to Inhbb knockout animals rescued the defects. These data suggest that Inhbb must be blocked to maintain germ cell and follicle survival. Based on the similarities in ovarian phenotype and Inhbb upregulation resulting from both Wnt4/β-catenin and Irx3/5 mutant models, we hypothesized that Irx3 and Irx5 mediate the Wnt4/β-catenin repression of Inhbb while they also regulate the expression of other downstream target genes required for cell contacts and germ cell health within follicles. First, we used gain- and loss-of-function gonad culture paradigms to manipulate β-catenin activity in ovaries and testes, respectively. Results showed that Irx3 and Irx5 expression levels were positively regulated by β-catenin signaling within the canonical Wnt4 pathway. Next, additional analysis of the RNA-seq data using the Database for Annotation, Visualization and Integrated Discovery (DAVID) showed that the differentially expressed genes (DEGs) in Irx3/5 DKO samples included factors critical to extracellular matrix, cell projections, actin-binding and cell adhesion. We are currently validating the DEGs relevant to our Irx3/5 DKO follicle defects and investigating their relationship to Inhbb in ovaries. Results from this study will help us to discover the means by which initial cell contacts are formed between support cells and oocytes in germline nests and new primordial follicles. These interactions provide the foundation for follicle integrity within the ovarian reserve and ultimately ensure that healthy follicles are available for recruitment and maturation.

Integration of Genetic Testing and Pathology for the Diagnosis of Adults with FSGS.

FSGS and nephrotic syndrome studies have shown that single gene causes are more likely to be found in pediatric cases than adults. Consequently, many studies have examined limited gene panels in largely pediatric cohorts. Whole-exome sequencing was performed in adults with FSGS diagnosed between 1976 and 2017 in the Toronto GN Registry. An expanded panel of 109 genes linked to FSGS, glomerular basement membrane abnormalities, as well as causes of pediatric ESKD including congenital abnormalities of the kidney and urinary tract (CAKUT) and nephronophthisis, were examined. The cohort was composed of 193 individuals from 179 families. Nearly half (49%) developed ESKD at a mean age of 47±17 years. The genetic diagnostic rate was 11%. Of definitely pathogenic variants, 55% were in COL4A (A3/A4/A5), 40% were in podocyte genes, and 5% were in CAKUT genes. Many, but not all individuals with COL4A definitely pathogenic variants had some evidence of glomerular basement membrane abnormalities. The estimated mean survival/age of kidney failure for individuals with COL4A definitely pathogenic variants was 58 years (95% confidence interval, 49 to 69), far later than what has been reported in the literature. Likely pathogenic variants were identified in an additional 9% of the cohort, with most in COL4A. Correlation with glomerular basement membrane morphology suggested a causal role for at least some of these likely pathogenic variants. Even with an expanded gene panel, we find that COL4A disorders are the leading monogenic cause in adults diagnosed with FSGS. This article contains a podcast at https://www.asn-online.org/media/podcast/CJASN/2019_01_15_CJASNPodcast_19_02_.mp3.

Abstract 2962: Mechanobiology of epithelia on native basement membrane and relevance for cancer invasion

Abstract The onset of metastasis occurs when cancer cells invade and breach the basement membrane (BM) that provides mechanical support to epithelial tissues. Yet, it remains unclear what triggers cancer cells to breach the BM, and how ‘triggered’ cells in fact breach the BM. We have established an in vitro assay using native BM interface for culturing epithelial cells. Using atomic force microscopy (AFM) with other high-resolution microscopies and TER (trans-epithelial resistance), we have correlated the mechano-cellular attributes of the BM/epithelium interface to its biochemical and structural properties. We demonstrated that the internal limiting membrane (ILM) isolated from human retinas acts as a native substrate for culturing epithelial cells in terms of BM composition, architecture and stiffness. These are required to act jointly in order to achieve apico-basal polarity, tissue barrier formation and stiffness properties of the epithelial layer similarly to secretory epithelia in vivo. The native BM serves several advantageous over reconstituted Matrigel, an ECM extracts that originates from mouse tumor ascites. Besides variations in thickness and biochemical composition, we find that Matrigel is mechanically 100-fold more compliant (i.e., softer) than native BMs. When tumorigenic variants of cells are used, we demonstrated that cancer cell invasion is associated with a decrease in cellular stiffness correlated to changes in cell and BM morphology. In addition, we showed that activation of ß1 integrin by the stiffness and architecture of the native alpha - 5 laminin chain has a key role, not only as previously thought for maintenance of cell polarity but also for the establishment of a physiological mechanophenotype. On the other hand it is well know that during cancer progression in vivo, cancer cells can perforate the BM using proteolysis. Whether stromal cells also play a role and what kind of role in this process is unclear. Therefore, we asked if carcinoma-associated fibroblasts (CAFs) isolated from cancer patients promote cancer cell invasion through a BM. In the presence of CAFs, moderately invasive cancer cells invade in a matrix metalloproteinase-independent manner. Using live imaging and atomic force microscopy, we showed that CAFs actively pull, stretch and soften the BM, forming gaps through which cancer cells can migrate. By exerting contractile forces, CAFs alter the organization and the physical properties of the BM, making it permissive for cancer cell invasion. Finally, we propose that, in addition to proteolysis, mechanical interactions between CAFs and BM represent an alternative mechanism of BM breaching. Given their mechano-biological relevance, native BMs allow us to further understand how mechanosignaling occurs between the epithelia and the surrounding stromal layers at the BM interface during cancer invasion and progression. Note: This abstract was not presented at the meeting. Citation Format: Marija Plodinec, Philipp Oertle, Daphne Assgeirson, Willi Halfter, Serenella Eppenberger Castori, Ellen C. Obermann, Alexandre Glentis, Roderick Y. LIM. Mechanobiology of epithelia on native basement membrane and relevance for cancer invasion [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 2962. doi:10.1158/1538-7445.AM2017-2962

P.1.i.007 - The effect of kainic acid on hippocampal dendritic spine dynamics

Recent work demonstrates that Alport glomerular disease is mediated through a biomechanical strain-sensitive activation of mesangial actin dynamics. This occurs through a Rac1/CDC42 cross-talk mechanism that results in the invasion of the subcapillary spaces by mesangial filopodia. The filopodia deposit mesangial matrix proteins in the glomerular basement membrane, including laminin 211, which activates focal adhesion kinase in podocytes culminating in the up-regulation of proinflammatory cytokines and metalloproteinases. These events drive the progression of glomerulonephritis. Here we test whether endothelial cell-derived endothelin-1 is up-regulated in Alport glomeruli and further elevated by hypertension. Treatment of cultured mesangial cells with endothelin-1 activates the formation of drebrin-positive actin microspikes. These microspikes do not form when cells are treated with the endothelin A receptor antagonist sitaxentan or under conditions of small, interfering RNA knockdown of endothelin A receptor mRNA. Treatment of Alport mice with sitaxentan results in delayed onset of proteinuria, normalized glomerular basement membrane morphology, inhibition of mesangial filopodial invasion of the glomerular capillaries, normalization of glomerular expression of metalloproteinases and proinflammatory cytokines, increased life span, and prevention of glomerulosclerosis and interstitial fibrosis. Thus endothelin A receptor activation on mesangial cells is a key event in initiation of Alport glomerular disease in this model.

Endothelin A receptor activation on mesangial cells initiates Alport glomerular disease

Recent work demonstrates that Alport glomerular disease is mediated through a biomechanical strain-sensitive activation of mesangial actin dynamics. This occurs through a Rac1/CDC42 cross-talk mechanism that results in the invasion of the subcapillary spaces by mesangial filopodia. The filopodia deposit mesangial matrix proteins in the glomerular basement membrane, including laminin 211, which activates focal adhesion kinase in podocytes culminating in the up-regulation of proinflammatory cytokines and metalloproteinases. These events drive the progression of glomerulonephritis. Here we test whether endothelial cell-derived endothelin-1 is up-regulated in Alport glomeruli and further elevated by hypertension. Treatment of cultured mesangial cells with endothelin-1 activates the formation of drebrin-positive actin microspikes. These microspikes do not form when cells are treated with the endothelin A receptor antagonist sitaxentan or under conditions of small, interfering RNA knockdown of endothelin A receptor mRNA. Treatment of Alport mice with sitaxentan results in delayed onset of proteinuria, normalized glomerular basement membrane morphology, inhibition of mesangial filopodial invasion of the glomerular capillaries, normalization of glomerular expression of metalloproteinases and proinflammatory cytokines, increased life span, and prevention of glomerulosclerosis and interstitial fibrosis. Thus endothelin A receptor activation on mesangial cells is a key event in initiation of Alport glomerular disease in this model.

Visualization of basement membranes in normal breast and breast cancer tissues using multiphoton microscopy.

Since basement membranes represent a critical barrier during breast cancer progression, timely imaging of these signposts is essential for early diagnosis of breast cancer. A label-free method using multiphoton microscopy (MPM) based on two-photon excited fluorescence signals and second harmonic generation signals for analyzing the morphology of basement membrane in normal and cancerous breast tissues is likely to enable a better understanding of the pathophysiology of breast cancer and facilitate improved clinical management and treatment of this disease. The aim of this study was to determine whether MPM has the potential for label-free assessment of the morphology of basement membrane in normal and cancerous breast tissues. A total of 60 tissue section samples (comprising 30 fresh breast cancer specimens and 30 normal breast tissues) were first imaged (fresh, unfixed and unstained) with MPM and are then processed for routine hematoxylin and eosin (H&E) histopathology. Comparisons were made between MPM imaging and gold standard sections for each specimen stained with H&E. Simply by visualizing morphological features appearing on multiphoton images, cancerous lesions may be readily identified by the loss of basement membrane and tumor cells characterized by irregular size and shape, enlarged nuclei and increased nuclear-cytoplasmic ratio. These results suggest that MPM has potential as a label-free method of imaging the morphology of basement membranes and cell features to effectively distinguish between normal and cancerous breast tissues.

Identifying three different architectural subtypes of mammary ductal carcinoma in situ using multiphoton microscopy

Ductal carcinoma in situ (DCIS) is often considered as the precursor of invasive breast cancer, and the risk of DCIS progression to IBC has been estimated based on the evaluation of pathological features, among which the architectural subtype is the most common one. In this study, multiphoton microscopy (MPM) is applied to identify three different architectural subtypes of DCIS (solid, cribriform and comedo). It is found that MPM has the capability to visualize the proliferating pattern of tumor cells, the presence of intraluminal necrosis and the morphology of basement membrane, which are all taken into account in subtyping DCIS. In addition, MPM also can be used to quantify the cellular metabolism, for quantitatively identifying tumor staging during tumor progression. This result highlights the potential of MPM as an advanced technique to assess the pathological characters of the breast tumor in real-time and reflect the degree of tumor progression in vivo, by integrating into the intra-fiberoptic ductoscopy or transdermal biopsy needle.

Feasibility and safety of porcine Descemet's membrane as a carrier for generating tissue-engineered corneal endothelium.

The aim of this study was to evaluate the feasibility and safety of porcine Descemet's membrane (DM) as a carrier for the generation of tissue-engineered corneal endothelium by analyzing porcine endogenous retroviruses (PERVs) and the α-gal epitope. The morphology of porcine and human DM was observed by hematoxylin and eosin staining and scanning electron microscopy. Immunohistochemical staining was used to investigate the location of α-gal epitopes on porcine DM used for xenotransplantation. The porcine DM was treated with ethylene glycol diglycidyl ether (EDGE) for 2 weeks, and then the PERV gene sequences in porcine DM and DM-EDGE were detected by polymerase chain reaction (PCR) and real-time PCR, respectively. The porcine DM had tight basement membrane morphology, which was similar to human DM in terms of thickness. No positive immunohistochemical staining of the α-gal epitope was detected in porcine DM. PERV expression of pol, gag, env-A and env-B was noted in porcine DM, but in DM-EDGE it was completely degraded. Based on structural, immunological and etiological studies, porcine DM may be an ideal and viable carrier for the generation of tissue-engineered corneal endothelium.

Absence of the Basement Membrane Component Nidogen 2, but Not of Nidogen 1, Results in Increased Lung Metastasis in Mice

Nidogen 1 and 2 are ubiquitous basement membrane (BM) components. They show a divergent expression pattern in certain adult tissues with a prominent localization of nidogen 2 in blood vessel BMs. Deletion of either nidogen 1 or 2 in mice had no effect on BM formation, suggesting complementary functions. However, studies in these mice revealed isoform-specific functions with nidogen 1-deficient mice showing neurological abnormalities and wound-healing defects not seen in the absence of nidogen 2. To investigate this further nidogen 1- or 2-deficient mice were intravenously injected with B16 murine melanoma cells, and lung metastasis was analyzed. The authors could show that loss of nidogen 2, but not of nidogen 1, significantly promotes lung metastasis of melanoma cells. Histological and ultrastructural analysis of nidogen 1- and 2-deficient lungs did not reveal differences in morphology and ultrastructure of BMs, including vessel BMs. Furthermore, deposition and distribution of the major BM components were indistinguishable between the two mouse strains. Taken together, these results suggest that absence of nidogen 2 might result in subtle changes of endothelial BMs in the lung, which would allow faster passage of tumor cells through these BMs, leading to a higher metastasis rate and more larger tumors.

Relationship among follicular apoptosis, integrin β1 and collagen type IV during early ovarian regression in the teleost Prochilodus argenteus after induced spawning

Early ovarian regression was analyzed in the neotropical freshwater teleost, curimatã-pacu (Prochilodus argenteus), in order to evaluate follicular apoptosis, basement membrane morphology, and integrin beta1 and collagen type IV immunostainning in postovulatory follicles. Mature females were induced to spawn by using carp pituitary extract for study of ovarian regression up to 5 days post-spawning. Morphometric analyses showed that the postovulatory follicle area decreased progressively after spawning and was coupled to the gonadosomatic index (r=0.92). During ovarian regression, follicular cells detached from the neighboring cells and basement membrane and then died by apoptosis. The follicular basement membrane became thicker and diffuse and was breached during regression of the postovulatory follicles. Follicular apoptosis was detected by TUNEL, histology, and electron microscopy. The ladder pattern of apoptotic DNA was revealed by agarose gel electrophoresis. The apoptotic index for the follicular cells increased until 3 days post-spawning and decreased thereafter. Immunohistochemistry reactions detected caspase 3, integrin beta1, and collagen type IV in the follicular layer of the postovulatory follicles. Labeling for integrin beta1 and collagen type IV decreased significantly, whereas a peak in cell death occurred 3 days post-spawning. At 4-5 days post-spawning, the connective theca was more thickened and vascularized. Simultaneously, granulocytes migrated toward the follicular lumen. Thus, follicular apoptosis contributes to early ovarian regression in P. argenteus. Additionally, our findings suggest integrin beta1 and collagen type IV as possible survival factors for follicular cells in teleost ovary.

Integrin α1β1 and Transforming Growth Factor-β1 Play Distinct Roles in Alport Glomerular Pathogenesis and Serve as Dual Targets for Metabolic Therapy

Alport syndrome is a genetic disorder resulting from mutations in type IV collagen genes. The defect results in pathological changes in kidney glomerular and inner-ear basement membranes. In the kidney, progressive glomerulonephritis culminates in tubulointerstitial fibrosis and death. Using gene knockout-mouse models, we demonstrate that two different pathways, one mediated by transforming growth factor (TGF)-beta1 and the other by integrin alpha1beta1, affect Alport glomerular pathogenesis in distinct ways. In Alport mice that are also null for integrin alpha1 expression, expansion of the mesangial matrix and podocyte foot process effacement are attenuated. The novel observation of nonnative laminin isoforms (laminin-2 and/or laminin-4) accumulating in the glomerular basement membrane of Alport mice is markedly reduced in the double knockouts. The second pathway, mediated by TGF-beta1, was blocked using a soluble fusion protein comprising the extracellular domain of the TGF-beta1 type II receptor. This inhibitor prevents focal thickening of the glomerular basement membrane, but does not prevent effacement of the podocyte foot processes. If both integrin alpha1beta1 and TGF-beta1 pathways are functionally inhibited, glomerular foot process and glomerular basement membrane morphology are primarily restored and renal function is markedly improved. These data suggest that integrin alpha1beta1 and TGF-beta1 may provide useful targets for a dual therapy aimed at slowing disease progression in Alport glomerulonephritis.

The prognostic value of basement membrane morphology, tumour histology and morphometry in superficial bladder cancer.

This analysis evaluated the prognostic importance of clinical, histological (WHO grade, papillary status, tumour-infiltrating lymphocytes) and morphometric factors (mitotic index, mean nuclear area, SD of nuclear area) in a cohort of 132 patients with a Ta-T1 transitional cell bladder cancer followed-up for over 8 years. Special attention was given on the state of the subepithelial basement membrane (BM) and subepithelial vascular density as demonstrated by type IV collagen immunostaining. Defects in the BM and dense vascular network were related to nodular growth pattern, high histological grade, rapid cancer cell proliferation and a dense inflammatory cell infiltrate (tumour-infiltrating lymphocytes, TIL) in tumour stroma. The most important predictor of progression was the mitotic index (P < 0.0001) followed by vascular density (P = 0.0054), the state of the BM (P = 0.0056), density of TIL (P = 0.0193), WHO grade (P = 0.0356) and papillary status (P = 0.0448). In univariate survival analysis the mitotic index (P < 0.0001), the state of the BM (P < 0.0001), subepithelial vascular density (P = 0.0013) and WHO grade (P = 0.01) predicted survival. In a multivariate analysis the mitotic index (P < 0.0001) and the state of the BM (P = 0.008) were independent predictors. The results show that the evaluation of the mitotic rate and investigation of the continuity of the BM should be done whenever the prognosis of Ta-T1 tumours of the bladder is assessed.

Histological, microbiological and haematological investigations in denture-induced stomatitis

Biopsy specimens of palatal mucosa from 10 edentulous female subjects exhibiting the signs of chronic atrophic denture-induced stomatitis were fixed and processed to produce sections which were quantitatively analysed by light microscopy. The type and degree of surface keratinization, the epithelial thickness and basement membrane morphology were assessed. Microbiological and haematological evaluations were related to the histological findings. It was found that within this clinical entity of palatal inflammation there was considerable variation in tissue response at a cellular level, and that the numbers of organisms associated with the inflammatory reaction appeared to be fewer in patients with abnormal haematological status.

Rat kidney glomerular basement membrane visualized in situ by embedment‐free sectioning and subsequent platinum‐carbon replication

Following the removal of polyethylene glycol (PEG) from thin sections, and viewing through the endothelial fenestrae and/or the interpedicel spaces, the rat renal glomerular basement membrane in situ was revealed to consist of meshworks and to be electron-transparent when examined at right angles to the plane of the membrane. By subsequent platinum replication of the embedment-free sections, the lamina densa of the basement membrane appeared as a veil composed of rather closely packed particles. The architecture of the split diaphragm and the surface morphology of the endothelial cell membrane were also clearly revealed. The present results indicate that the PEG method, with or without replication, can provide valuable information on basement membrane morphology.

Dermal nerves in monzygotic twins discordant for diabetes: Morphometric study of perineurial cell basement membrane

Nerves in the dermal layer of skin are typically composed of several myelinated and unmyelinated nerve fibers surrounded by a perineurial sheath. Generally, this sheath is composed of one to two perineurial cell layers. The perineurial sheath helps to maintain a suitable environment for proper nerve function (1) and morphological changes in the sheath may cause nerve dysfunction (2). Perineurial cell basement membrane (PCBM) becomes thickened in diabetics and this phenomenon has been viewed as a diagnostic aid in the detection of diabetes (3). Since other factors (e.g. genetics, aging, vascular disease) may play a role in basement membrane morphology, we undertook this study of dermal nerves from identical twins discordant for diabetes to determine the effects of diabetic dysmetabolism on morphological changes in the PCBM. Since monozygotic twins have an identical genetic composition, it is likely that any significant differences in the dermal nerve would be due to factors related to diabetes.

An electron microscope study of kidney basement membrane changes in the mouse by lipoteichoic acid from Streptococcus pyogenes

Mice injected repeatedly, intraperitoneally or intravenously, for approximately 1 month with a total of 1.04 mg lipoteichoic acid from a nephritogenic strain of Streptococcus pyogenes lost weight. Analysis by electron microscopy revealed that they also exhibited extensive kidney changes in basement membrane morphology which resembled, in part, those observed in human poststreptococcal glomerulonephritis. For example, the glomerular basement membrane became electron dense and exhibited at least a twofold increase in width sporadically within the same preparation after exposure to lipoteichoic acid. Also, whereas appreciable loss or reduction in epithelial foot processes as a result of fusion was clearly evident, epithelial slits and slit membranes or diaphragms between normal foot processes were not selectively affected. In addition, another mostly thickened, highly coiled or serpentinelike basement membrane with amorphous nodules appeared in these preparations. This type membrane was not observed surrounding the capillary lumina and was the most pronounced abnormality apparent in almost all preparations from mice exposed to lipoteichoic acid. Likewise, the proximal tubular basement membrane became variable in width and increased in electron density in mice given lipoteichoic acid as compared with controls. In addition, this membrane was often punctuated with various morphological protrusions originating from only its thickened areas and which extended away from, and not into, the capillary space. This change was only associated with the basement membrane of the proximal tubular capillaries. All membrane changes persisted but gradually subsided, with normal kidney membrane morphology reappearing on the 4th day following the last injection of lipoteichoic acid.(ABSTRACT TRUNCATED AT 250 WORDS)