Mary Ann Accavitti

CD28 costimulation can promote T cell survival by enhancing the expression of Bcl-xL

T cell activation through the TCR can result in either cell proliferation or cell death. The role of costimulatory receptors in regulating T cell survival has not been defined. Here, we present data demonstrating that CD28 costimulation enhances the in vitro survival of activated T cells. One mechanism for this enhancement is the ability of CD28 costimulation to augment the production of IL-2, which acts as an extrinsic survival factor for T cells. In addition, CD28 costimulation augments the intrinsic ability of T cells to resist apoptosis. Although CD28 signal transduction had no effect on Bcl-2 expression, CD28 costimulation was found to augment the expression of Bcl-XL substantially. Transfection experiments demonstrated that this level of Bcl-XL could prevent T cell death in response to TCR cross-linking, Fas cross-linking, or IL-2 withdrawal. These data suggest that an important role of CD28 costimulation is to augment T cell survival during antigen activation.

Immunization with undenatured bovine zonular fibrils results in monoclonal antibodies to fibrillin

Microfibrils were dissected from the zonular apparatus of the bovine eye, homogenized and used as an immunogen to prepare monoclonal antibodies. Initial screening of hybridomas was performed by immunoblotting to a sonicate of zonular fibrils and by immunolocalization to frozen sections of the zonular apparatus. Subsequently, monoclonal antibodies with strong immunoreactivity to zonular fibrils were shown to recognize microfibrils in a wide range of connective tissues both by immunofluorescent staining and by electron microscopic immunolocalization. All antibodies were found to recognize a single protein of 350 kDa on Western blotting of the proteins secreted by bovine aortic smooth muscle cells. A protein of the same molecular weight and properties was recognized by an antibody previously prepared by another group against fibrillin. A member of the fibrillin family therefore represents the major immunogen of intact zonular fibrils, and the results support previous evidence for a close relationship between zonular fibrils and other connective tissue microfibrils. The zonular apparatus is a suitable system to obtain purified preparations of microfibrils in order to investigate their composition and structural organization.

Monoclonal Antibodies that Distinguish Avian Type I and Type III Collagens: Isolation, Characterization and Immunolocalization in Various Tissues

Monoclonal antibodies were prepared that were specific for chicken type I and type III collagens. The specificity of these antibodies was determined by ELISA, inhibition ELISA, and immunoblot assays. The results showed that the monoclonal antibodies were specific for their respective antigens without significant cross reactivity to other types of collagen. An analysis of the location of the epitopes by rotary shadowing that a monoclonal antibody for type I collagen (called DD4) recognized type I procollagen close to the large globular domain at the carboxyl terminus of the molecule. A monoclonal antibody for type III collagen (called 3B2) recognized both the intact type III molecule and also the TCA fragment of type III collagen after mammalian collagenase digestion. The epitope was located approximately one-fifth of the distance from the amino-terminus of the intact molecule. The monoclonal antibodies were used for immunolocalization of type I and type III collagens in cryosections of heart, aorta, kidney, liver, thymus, skin, gizzard and myotendinous junction. In heart, aorta, kidney, liver, thymus and skin, type I and III collagens were colocalized in the connective tissue of each organ. In contrast, gizzard and myotendinous junction showed distinctly different staining patterns for the distribution of type I and type III collagen. The two monoclonal antibodies reported here are potentially useful reagents to study fibril formation involving type I and type III collagens.

Monoclonal antibody to the aminotelopeptide of type II collagen: loss of the epitope after stromelysin digestion.

A monoclonal antibody was prepared to the aminotelopeptide of type II collagen after immunization of DBA/1 mice with lathyritic type II collagen and subsequent screening for antibodies that recognize lathyritic but not pepsin-digested type II collagen. One antibody (called 5B2) was identified that recognized a short peptide sequence in the aminotelopeptide of chicken type II collagen but did not recognize other collagen types. Further characterization of the epitope was achieved using a Multipin system and the epitope was localized to a short linear sequence of six amino acids. The antibody recognized type II collagen from a variety of species including man and mouse. The epitope for 5B2 was found to be susceptible to cleavage with recombinant stromelysin without cleavage of the major collagen triple helix. Comparison was made between MAb 5B2 and two other antibodies (called MAb 2B1 and MAb 6B3) that recognize separate epitopes located along the triple helix of the type II collagen molecule.