T. V. Darnule

Solid-phase radioimmunoassay for estimation of elastin peptides in human sera

Abstract A sensitive, reproducible, and rapid radioimmunoassay was developed to determine clastin peptide content of human serum in nanogram amounts. Significant differences have been determined between normal volunteers, both smokers and nonsmokers, and patients wth chronic obstructive pulmonary disease. The average concentration of clastin peptides in 9 normal nonsmokers was 19.88 ± 10.46 μg/ml serum, indistinguishable from that in 7 normal smokers which was 17.64 ± 8.54 μg/ml. Average concentration of clastin peptides in 17 patients with lung disease was 53.38 ± 22.46 μg/ml, significantly different (P

Synthesis of crosslinked elastin by an endothelial cell culture

Summary Synthesis of crosslinked elastin by a major lung cell has not previously been reported. Elastin production by an established clone of rat lung endothelial cells was detected by two separate, highly sensitive methods. The first procedure involved isolation and identification of the labelled, elastin-specific crosslinking amino acids desmosine and isodesmosine by thin layer electrophoresis and radioautography. The second procedure involved detection of elastin by immunofluorescence, using anti-rat lung elastin peptide serum.

Immune response to peptides produced by enzymatic digestion of microfibrils and elastin of human lung parenchyma.

Antibodies were raised in rabbits against human lung elastic fiber and peptides derived from human lung elastin and microfibrils. Elastic fiber produced antibodies with very low liter. Elastin peptides had low immunogenicity and multiple injections of at least 100 mg per rabbit per injection were required to produce antibodies. Microfibril peptides had a much higher immunogenicity and produced antibodies with multiple injections of only 10 mg per rabbit per injection.Anti-elastic fiber serum did not react with peptides derived from microfibrils or elastin at concentrations of 5 mg or 10 mg per ml solution. At 25 mg/ml solution microfibril peptides gave a faint precipitin line in Ouchterlony double diffusion tests, but elastin peptides did not react. There was no cross reactivity between microfibril peptides and anti-elastin serum or elastin peptides and anti-microfibril serum at low concentration. At higher concentration, i.e. 25 mg/ml, some cross reactivity was observed with both antigens and both antise...

Enhancement of humoral immune response against human lung elastin peptides.

When guinea pigs instead of rabbits were used as the host animals, 8–16 times higher antibody titers against human lung elastin peptides were produced with only 1/20 the amount of antigen per unit body weight. This corresponds to a 200-fold enhancement of the immune response.

Detection of factor VIII related antigens in long term cultures of rat endothelial cells

Rat endothelial cells in culture can be distinguished from fibroblasts and epithelial cells by their reaction with antisera against human factor VIII (AHF) associated proteins.

Antiserum to surface antigens as a marker for cultured rat lung endothelial cells (1).

Antibodies were developed in rabbits against an established line of endothelial cells from normal adult rat lung. Pre- and postimmunization sera were tested for antibody activity by the indirect immunofluorescence technique. Preimmunization serum failed to react with the endothelial cells, whereas the antibody titer of postimmunization serum from two rabbits was 1:512. Organ specificity and species specificity were assessed by absorbing the serum with packed dissociated cells from different organs of the rat and lung cells of other species. Only cells obtained from rat lung absorbed the antibodies completely. The antiserum showed some crossreactivity with the other cultured cells but the pattern of fluorescence was different. In the presence of complement the antiserum was found to be cytotoxic to cultured rat lung endothelial cells but not to the other crossreacting cells.

Proteolytic Mechanisms and Pulmonary Emphysema

Pulmonary emphysema causes between 40.000 and 45.000 deaths per year in the United States. However. most people with this chronic illness. whose natural history extends over many years. are physically disabled by severe breathlessness for much of their late adult life. It is estimated that in the United States. several million people are disabled from some form of airway obstructive disease — including pulmonary emphysema (Task Force Report 1980).

Induction of Cell-Mediated Immune Response to Peptides Produced by Enzymatic Digestion of Elastin from Human Lung Parenchyma

Abstract Guinea pigs were sensitized with elastin peptides dissolved in complete Freunds adjuvant, and with complete Freunds adjuvant alone. Four weeks after sensitization, the guinea pigs were challenged intradermally with elastin peptides. Only guinea pigs sensitized and challenged with elastin peptides produced erythema and induration. Histological studies of the indurated skin showed heavy infiltration of mononuclear cells. Migration of peritoneal exudate cells of those guinea pigs was strongly inhibited in the presence of elastin peptides, whereas other proteins such as serum albumin did not inhibit the migration. These observations suggest that the reaction is specific for elastin peptides, although a slight but significant effect was shown by peptides derived from other connective tissue proteins of human lung, i.e., microfibrils and collagen. Cell-mediated immunity could also be induced in normal guinea pigs by the transfer of spleen cells obtained from guinea pigs sensitized and challenged with elastin peptides.

Identification of surface antigens of endothelial cells

A monolayer of a clone of endothelial cells derived from rat lung cells (RLE) was overlaid with 1 M urea to extract the surface proteins. Hydrolysis and SDS-gel electrophoresis of the urea extracted cell surface proteins (UCSP) yielded four peptides of 350,000, 84,000, 66,000 and 18,500 molecular weight. Of these only the 66,000 and 18,500 molecular weight peptides reacted with antibodies raised in rabbit against rat lung endothelial cells (RLE). The 18,500 mol. wt. antigenic peptide was a serum protein associated with the cell surface, whereas the 66,000 mol. wt. peptide was the surface antigen synthesized and released into the medium by the rat lung endothelial cells. On rocket immunoelectrophoresis, the 66,000 mol. wt. rat kidney fibroblast surface peptide produced only a single rocket whereas peptides of RLE produced two rockets, suggesting the presence of an additional antigenic peptide which could serve as a marker for endothelial cells.