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J M Fitch

Publications and source records attributed to J M Fitch.

45 records · Page 3Linked to original sources
PubMed1984

Thermal stability of the helical structure of type IV collagen within basement membranes in situ: determination with a conformation-dependent monoclonal antibody.

To examine the thermal stability of the helical structure of type IV collagen within basement membranes in situ, we have employed indirect immunofluorescence histochemistry performed at progressively higher temperatures using a conformation-dependent antibody, IV-IA8. We previously observed by competition enzyme-linked immunosorbent assay that, in neutral solution, the helical epitope to which this antibody binds undergoes thermal denaturation over the range of 37-40 degrees C. In the present study, we have reacted unfixed cryostat tissue sections with this antibody at successively higher temperatures. We have operationally defined denaturation as the point at which type IV-specific fluorescence is no longer detectable. Under these conditions, the in situ denaturation temperature of this epitope in most basement membranes is 50-55 degrees C. In capillaries and some other small blood vessels the fluorescent signal is still clearly detectable at 60 degrees C, the highest temperature at which we can confidently use this technique. We conclude that the stability of the helical structure of type IV collagen within a basement membrane is considerably greater than it is in solution, and that conformation-dependent monoclonal antibodies can be useful probes for investigations of molecular structure in situ.

Animals↗
PubMed1984

Extracellular matrices in the developing avian eye: type V collagen in corneal and noncorneal tissues.

We have used immunofluorescence histochemistry to examine the temporal and spatial deposition of type V collagen in the extracellular matrices of the developing chick cornea and other selected ocular structures. Tissue sections from animals ranging in age from 4-day-old embryos to 1-day post-hatching were examined by indirect immunofluorescence employing monoclonal antibodies specific for conformational dependent sites in this molecule. In eyes from embryos younger than 6 days of development, no type V staining could be detected. Thus, the epithelially derived primary corneal stroma, which is already well formed at this time, contains little if any of this molecule. Its appearance was concomitant with the physical swelling of the primary stroma and invasion of pericorneal mesenchymal cells. Staining was initially localized in the anterior cornea; subsequently, all corneal matrices showed intense reactions, including the stroma proper and Bowman's and Descemet's membranes. In adjacent noncorneal tissues, the appearance of type V collagen occurred later in development. In some of these, such as in the ciliary body, the pattern of acquisition involved initial deposition at an epithelial-mesenchymal interface with subsequent progression of fibrous strands out into the surrounding mesenchymal tissue. Eventually, all ocular structures with a dense connective tissue component showed staining, but the intensity was appreciably less than that within the cornea. We have previously reported that in all mature tissues except Bowman's membrane, type V collagen is present in an "antigenically masked" form, and that unmasking can be achieved by pretreatment of the tissue sections with dilute acetic acid.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1983

The use of monoclonal antibodies to fragments of chicken type IV collagen in structural and localization studies.

In previous experiments, three pepsin-resistant fragments of type IV collagen were isolated from chicken gizzards and designated 7S, F3, and (F1)2F2 (Mayne, R., and Zettergren, J. G. (1980) Biochemistry 19, 4065-4072). In the present experiments, a series of monoclonal antibodies to type IV collagen were prepared, each one of which recognized an epitope present in only one of the three fragments. A high molecular weight fraction of type IV collagen (designated 7S + arms (215 nm)) was isolated after agarose gel filtration and characterized by electron microscopy after rotary shadowing and by gel electrophoresis. Analysis of 7S + arms (215 nm) by inhibition enzyme-linked immunosorbent assay demonstrated the presence of the epitopes for 7S and F3 but not for (F1)2F2. This result, therefore, provides additional evidence that the order of the pepsin-resistant fragments of chicken type IV collagen is 7S-F3-(F1)2F2.

Animals↗
PubMed1983

Monoclonal antibody analysis of ocular basement membranes during development.

As a first step in a study of the role(s) of basement membranes in ocular morphogenesis, we have produced a variety of monoclonal antibodies against native lens capsule from adult chicks, and have used these reagents to stain histological sections of ocular tissues from 4 1/2- to 18-day-old chicken embryos. Four different patterns of immunofluorescence were observed in sections of corneas of 18-day-old chicken embryos stained with these antibodies. The antibodies in group 1 stained the basement membranes of both the corneal epithelium and the endothelium (as well as Descemet's membrane). Those in groups 2 and 3 stained only the epithelial or endothelial basement membranes, respectively. The group 4 antibody stained the corneal stroma as well as Bowman's membrane and Descemet's membrane. The antibodies in group 1 could be further subdivided into groups 1a and 1b on the basis of temporal differences in the onset of staining in corneas from 4 1/2- to 7-day-old embryos. Thus, this series of monoclonal antibodies appears to recognize at least five different antigenic determinants. When these antibodies were used to stain sections of eyes at different stages of development, we found that the characteristic differential staining of some basement membranes was maintained throughout development, while the staining properties of others changed. This indicates that many of the ocular basement membranes may differ from one another in composition or conformation, and that at least some of them may undergo developmental changes. We also noticed a similarity in the pattern of fluorescence associated with the basement membranes of the limbal blood vessels and the corneal endothelium that is consistent with the hypothesis that the corneal endothelium is derived from the early periocular vascular endothelium. Our observations of developing corneas also revealed that the antigen recognized by the group 4 antibody may be produced by both the corneal epithelium and the stromal fibroblasts. The suitability of monoclonal antibodies for probing basement membrane heterogeneity is discussed.

Age Factors↗
PubMed1983

Monoclonal antibodies against chicken type V collagen: production, specificity, and use for immunocytochemical localization in embryonic cornea and other organs.

Two monoclonal antibodies have been produced against chick type V collagen and shown to be highly specific for separate, conformational dependent determinants within this molecule. When used for immunocytochemical tissue localization, these antibodies show that a major site for the in situ deposition of type V is within the extracellular matrices of many dense connective tissues. In these, however, it is largely in a form unavailable to the antibodies, thus requiring a specific "unmasking" treatment to obtain successful immunocytochemical staining. The specificity of these two IgG antibodies was determined by inhibition ELISA, in which only type V and no other known collagen shows inhibition. In ELISA, mixtures of the two antibodies give an additive binding reaction to the collagen, suggesting that each is against a different antigenic determinant. That both antigenic determinants are conformational dependent, being either in, or closely associated with, the collagen helix is demonstrated by the loss of antibody binding to molecules that have been thermally denatured. The temperature at which this occurs, as assayed by inhibition ELISA, is very similar to that at which the collagen helix melts, as determined by optical rotation. This gives strong additional evidence that the antibodies are directed against the collagen. The antibodies were used for indirect immunofluorescence analyses of cryostat sections of corneas and other organs from 17 to 18-day-old chick embryos. Of all tissues examined only Bowman's membrane gave a strong staining reaction with cryostat sections of unfixed material. Staining in other areas of the cornea and in other tissues was very light or nonexistent. When, however, sections were pretreated with pepsin dissolved in dilute HAc or, surprisingly, with the dilute HAc itself dramatic new staining by the antibodies was observed in most tissues examined. The staining, which was specific for the anti-type V collagen antibodies, was largely confined to extracellular matrices of dense connective tissues. Experiments using protease inhibitors suggested that the "unmasking" did not involve proteolysis. We do not yet know the mechanism of this unmasking; however, one possibility is that the dilute acid causes swelling or conformational changes in a type-V collagen-containing supramolecular structure. Further studies should allow us to determine whether this is the case.

Animals↗
PubMed1983

Developmental acquisition of basement membrane heterogeneity: type IV collagen in the avian lens capsule.

To investigate potential heterogeneity and developmental changes in basement membranes during embryogenesis, we performed immunohistochemical analyses on lens capsules in chicken embryos of different ages using domain-specific monoclonal antibodies against type IV collagen. We found that the capsule of the newly formed lens stained uniformly with antibodies against this component of basement membranes, but with increasing age and differentiation of the lens cells the anterior lens capsule remained brightly fluorescent while staining of the posterior capsule became relatively much less intense. This antero-posterior gradient of anti-type IV collagen antibody reactivity demonstrated that developmentally-regulated changes can occur within a single, continuous basement membrane.

Animals↗
PubMed1982

Domain and basement membrane specificity of a monoclonal antibody against chicken type IV collagen.

A monoclonal antibody, IV-IA8, generated against chicken type IV collagen has been characterized and shown to bind specifically to a conformational-dependent site within a major, triple helical domain of the type IV molecule. Immunohistochemical localization of the antigenic determinant with IV-IA8 revealed that the basement membranes of a variety of chick tissues were stained but that the basement membrane of the corneal epithelium showed little, if any, staining. Thus, basement membranes may differ in their content of type IV collagen, or in the way in which it is assembled. The specificity of the antibody was determined by inhibition ELISA using purified collagen types I-V and three purified molecular domains of chick type IV collagen ([F1]2F2, F3, and 7S) as inhibitors. Only unfractionated type IV collagen and the (F1)2F2 domain bound the antibody. Antibody binding was destroyed by thermal denaturation of the collagen, the loss occurring at a temperature similar to that at which previous optical rotatory dispersion studies had shown melting of the triple helical structure of (F1)2F2. Such domain-specific monoclonal antibodies should prove to be useful probes in studies involving immunological dissection of the type IV collagen molecule, its assembly within basement membranes, and changes in its distribution during normal development and in disease.

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