MAB1430 Sigma-AldrichAnti-Collagen Type IV Antibody, clone 24.12.8 (PHM-12)

Cutaneous squamous cell carcinoma (SCC) is the second most common type of skin cancer in the Caucasian population worldwide, having a propensity for invasion, local recurrence and metastasis. Stromal cancer-associated fibroblasts (CAFs) are suspected to play an important role in SCC carcinogenesis. In this study, we characterized CAFs isolated from primary cutaneous SCCs and compared them to normal fibroblasts (NFs) isolated from healthy dermis. Human skin CAFs in monolayers displayed different morphology, increased proliferation and migration compared to NFs. CAFs caused strong contraction of collagen matrices in which they were seeded and released high levels of the extracellular matrix component pro-collagen I. CAFs decreased proliferation and differentiation in the epidermis of human skin equivalents (HSEs) seeded with SCC cell lines, without affecting basement membrane composition. Finally, CAFs significantly increased invasion and dermal-epidermal detachment of SCC cell lines SCC-12B2 and SCC-13, respectively, when cultured in HSEs. These distinct features of CAFs point out a specific role in cutaneous SCC development.

Epithelial-mesenchymal interactions play an important role in controlling epidermal morphogenesis and homeostasis but little is known about the mechanisms of these interactions. To examine whether diffusible factors produced by fibroblasts and/or keratinocytes support epidermal morphogenesis and basement membrane formation, organotypic keratinocyte monocultures were established in media collected either from organotypic fibroblast or keratinocyte-monocultures or from keratinocyte-fibroblast cocultures, and the expression of keratin 10, 16, and 17 and basement membrane components (types IV and VII collagen, laminin 5, nidogen, BP 180, LAD-1) were examined. We found that diffusible factors released by keratinocytes were not sufficient to support the establishment of normalized epidermal phenotype and deposition of basement membrane components in contrast to fibroblast- or keratinocyte/fibroblast-derived factors. Keratinocytes appear to affect the spectrum of secreted soluble factors, as keratinocyte/fibroblast-derived factors were more effective to accomplish continuous linear deposition of laminin 5 and of nidogen. The finding that released amounts of keratinocyte growth factor and granulocyte macrophage colony stimulating factor were not sufficient to fully support epidermal morphogenesis and deposition of basement membrane components is suggestive for the involvement of other released diffusible factors. Generation of organotypic keratinocyte monocultures in the presence of fibroblast- or keratinocyte/fibroblast-derived soluble factors resulted in enhanced expression of keratins K16 and K17 and the absence of type IV collagen. This observation indicates that next to paracrine acting factors, epidermal homeostasis is controlled by mutual keratinocyte-fibroblast interaction.

The re-epithelialization of the wound involves the migration of keratinocytes from the edges of the wound. During this process, keratinocyte migration and proliferation will depend on the interaction of keratinocytes with dermal fibroblasts and the extracellular matrix. The present study aimed to investigate (1) the role of fibroblasts in the re-epithelialization process and on the reconstitution of the dermal-epidermal junction (DEJ) and (2) differential protein expression during re-epithelialization. For both purposes, three-dimensional human skin equivalents (HSE) were used. A full-thickness wound in HSE was introduced by freezing with liquid nitrogen and a superficial wound by linear incision with a scalpel. The closure of the wound in the absence or presence of exogenous growth factors was followed by monitoring the rate of re-epithelialization and regeneration of the DEJ. The results obtained in this study demonstrate that fibroblasts facilitate wound closure, but they differentially affected the deposition of various basement membrane components. The deposition of laminin 5 at the DEJ was delayed in superficial wounds as compared to the full-thickness wounds. During freeze injury, some basement membrane (BM) components remain associated with the dermal compartment and probably facilitate the BM reconstitution. The re-epithelialization process in full-thickness but not in superficial wounds was accelerated by the presence of keratinocyte growth factor and especially by epidermal growth factor. In addition, we have examined the deposition of various basement membrane components and the differences in protein expression in a laterally expanding epidermis in uninjured HSE. Laminin 5, type IV and VII collagen deposition was decreased in the laterally expanding epidermis, indicating that the presence of these proteins is not required for keratinocyte migration to occur in vitro. Using two-dimensional polyacrylamide gel electrophoresis, we have identified DJ-1, a protein not earlier reported to be differently expressed during the epithelialization process of the skin.

The architecture of the basement membranes is essential for proper function. This architecture is based on interactions among its components, which assemble in a complex network. Entactin-1 appears to be the mastermind of this assembling. In entactin-1-null transgenic mice, immunocytochemistry established the absence of entactin-1 in the glomerular basement membrane, and morphological thickening of this membrane was demonstrated. This prompted us to investigate the organization of other components of the glomerular basement membrane in the transgenic animals. The distribution of type IV collagen and laminin remained unchanged, whereas that of anionic charges was significantly altered. We also evaluated the impact of the absence of entactin-1 on cell relays by studying the alpha(3)- and the alpha(v)-integrins along the endothelial and epithelial glomerular cell plasma membranes. Only the density of alpha(v) was found to be increased. Finally, the filtration properties of the glomerular wall were evaluated by revealing endogenous albumin distribution across the basement membrane. This was altered in transgenic animals, suggesting changes in permselectivity properties. Entactin-1 appears to be an essential component in basement membranes because its absence appears to modify the molecular organization leading to alterations in functional properties.

Severe combined immunodeficiency (SCID) mice were engrafted with rheumatoid arthritis (RA) synovium and evaluated to determine whether RA synovial morphology and function were maintained in the RA-SCID grafts. The four major components of RA synovitis, inflammation, immune reactivity, angiogenesis, and synovial hyperplasia persisted in RA-SCID grafts for 12 weeks. Retention of chronic inflammatory infiltrates was demonstrated by histological evaluation and by immunohistology for CD3, CD20, and CD68. Staining for CD68 also revealed that the grafts had undergone reorganization of the tissue, possibly as a result of fibroblast hyperplasia. Immune and inflammatory components were confirmed by the detection of human immunoglobulins and human interleukin-6 in serum samples obtained from grafted animals. Human blood vessels were detected by dense expression of CD31. Small vessels persistently expressed the vitronectin receptor, alpha v beta 3, a marker of angiogenesis. All vessels expressed VAP-1, a marker of activated endothelial cells. Finally, the grafts retained the ability to support immigration by human leukocytes, as demonstrated by the functional capacity to recruit adoptively transferred 5- (and -6)-carboxyfluorescein diacetate succinimidyl ester-labeled T cells. T cells entering the RA-SCID grafts became activated and produced interferon-gamma, as detected by reverse transcriptase-polymerase chain reaction analysis. These studies demonstrate that the RA-SCID model maintains many of the phenotypic and functional features of the inflamed RA synovium.

Records of 1,530 Japanese quail were used to estimate heritabilities and genetic correlations based on a derivative-free restricted maximum likelihood (REML) method with an animal model and ANOVA. The animal model included fixed effects of hatch and sex, random effects of additive genetic value of the bird, and common environmental effect of the dam. Heritabilities estimated from REML for body weights at hatch, 7, 14, 21, and 28 d of age were .38, .12, .31, .12, and .44, respectively. Heritabilities estimates from the sire component of variance for the same traits were .57, .08, .28, .15, and .47. These values indicate that genetic progress can be made by selecting for either 14-d or 28-d body weight. Genetic correlation (REML) of .76 between body weights at 14 and 28 d of age indicates the possibility of improving body weight at 28 d of age by selecting for body weight at 14 d of age.

A series of monoclonal antibodies to human glomerular antigens was prepared by immunisation of a mouse with isolated whole glomeruli, followed by boosting with particulate glomerular basement membrane and fusion of murine spleen cells with the NSI-myeloma line. Hybridoma supernatants were screened jointly by a radioimmunoassay involving binding to isolated glomeruli, and by a 4-layer immunoperoxidase technique applied to polyester wax-embedded sections. Seven monoclonal antibodies with different specificities (PHM7-PHM13) were established and repeatedly cloned. Each antibody displayed a distinctive distribution within the glomerulus, including different patterns of staining of mesangial cells, mesangial matrix and glomerular basement membrane, in addition to extra-glomerular basement membranes and extracellular matrix. All antibodies also stained cellular outgrowths of isolated glomeruli cultured in vitro, and showed additive binding to cultured cells by radioimmunoassay. Physical characterization using absorptions with purified substrates, plus specific chemical and enzymatic digestions, indicated that PHM12 is directed against type IV collagen. PHM13 is directed against fibronectin as shown by absorption with purified fibronectin and immunoprecipitation of a 220 000 MW glycoprotein. The remaining 5 monoclonal antibodies, which react with carbohydrate (PHM7) or protein (PHM8-PHM11) determinants, were shown to be nonreactive with type IV collagen, fibronectin or other known glomerular components including sialic acid, laminin, amyloid P-component or various glycosaminoglycans. These monoclonal antibodies therefore appear to define a new series of human glomerular antigens, or possibly closely related antigenic determinants, which are synthesized by glomerular cells and incorporated into the mesangium and glomerular basement membrane. These antibodies, by providing markers for at least 2 antigens known to be important in glomerular cell-matrix interactions, should prove useful in research into the mechanisms involved in renal pathology.

A monoclonal antibody to human glomerular type IV collagen has been characterized and used in an immunohistochemical study of the distribution of this basement membrane protein in dysplasias, intraepithelial carcinomas, and infiltrating squamous cell carcinomas. Four squamous carcinoma cell lines established as xenografts in nude mice were also examined. In normal epidermis and mucosae, the basement membrane was clearly defined and intact. In areas of intraepidermal carcinoma, the basal lamina as defined by the antibody was usually continuous, and defects were only present in areas associated with an inflammatory infiltrate. Invasive squamous cell carcinomas, regardless of the degree of differentiation had a clearly delineated basement membrane at the epithelial stromal interphase. In areas of invasion, far removed from the in situ component, there were protrusions of tumor cells through the basement membrane into the stroma. Other abnormalities of basement membrane production such as aggregation of basement membrane and reduplication of the basal lamina were also associated with the carcinomas. There was a similar distribution of basal lamina material in involved lymph nodes and in squamous cell carcinomas that were growing as xenografts in nude mice. Our studies suggest that the loss of basement membrane type IV collagen is not generally associated with invasive squamous cell carcinomas and is not likely to be useful in the assessment of early invasion in this tumor. The similar distribution of type IV collagen in the xenografts and in the infiltrating tumors suggests that this system, in conjunction with the use of the same cell lines in vitro, will provide a model with which to study the control of deposition of type IV collagen.