NHLBI Workshop summary. Current concepts in idiopathic pulmonary fibrosis: a road map for the future.
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Biomedical subjects
Publications and source records attributed to A R Kalica.
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The application of the method of bronchoalveolar lavage to an increasing array of pulmonary diseases was evident, and the use of sophisticated technology to study cells and measure minute amounts of protein and other components in bronchoalveolar lavage fluid indicated that more meaningful information may be gained about diseased airways and alveolar spaces than suspected. That new techniques are being developed to make assays more sensitive and specific was evident. The popularity of this research approach was underscored by the interest and participation of colleagues in the United States and especially in Europe (notably France, England, Italy, and West Germany) and in Japan. This meeting was an opportune time to reflect on what bronchoalveolar lavage analysis has contributed to date and to focus attention on new applications that will be forthcoming and will improve our understanding of immunopathogenic mechanisms in an expanding number of pulmonary diseases.
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The possibility of immunizing human infants against rotaviruses, which cause severe dehydrating diarrheal disease, may depend on the use of a related rotavirus, derived from another animal species, as a source of antigen. To test the feasibility of this approach, calves were infected in utero with a bovine rotavirus and challenged with bovine or human type 2 rotavirus shortly after birth. Infection in utero with bovine rotavirus induced resistance to diarrheal disease caused by the human virus as well as the homologous bovine virus. These data suggest that the bovine virus is sufficiently related antigenically to the human type 2 virus to warrant further evaluation of the former as a source of vaccine.
Simian rotavirus (SA-11) isolated from infected African green monkey kidney cells was separated into two virus fractions in a CsCl density gradient and their proteins analysed on a continuous phosphte buffered polyacrylamide gel electrophoresis system. One peak (buoyant density 1.37 g/ml) contained double capsid virus particles which were radioimmunoassay (RIA)- and haemagglutinin (HA)- positive and yielded eight polypeptides whose mol. wt. ranged from 48,000 to 128,000. The second peak (buoyant density 1.39 g/ml) which contained 70% single capsid particles and was RIA-positive but HA-negative, yielded only five polypeptides. The three polypeptides missing in the second peak are associated presumably with the outer capsid of SA-11 virus particles and one or more of these is assumed to be the HA of SA-11 rotavirus.
Infection of cell cultures with human rotavirus preparations was attempted and the effects of trypsin and low-speed centrifugation on antigen incorporation, as demonstrated by immunofluorescence and radioimmunoassay, were determined. In addition, the effect of viral aggregation on antigen incorporation was investigated by filtering viral preparations. Four strains of human rotavirus were employed, and the results were compared to those obtained with two tissue culture-adapted animal rotaviruses. Centrifugation and trypsin appeared to have little or no effect on infectivity of the tissue culture-adapted (simian rotavirus) or -adaptable (Nebraska calf diarrhea virus) strains, whereas centrifugation and viral aggregation appeared to be essential for the human viruses. In addition, trypsin enhanced antigen incorporation of the human strains to some extent. Infectivity for cell cultures and in vitro human rotavirus protein formation was demonstrated by [35S]methionine incorporation, and the specificity of this human viral protein was established by radio-immunoprecipitation.
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Rotaviruses cause gastroenteritis in man and a wide variety of animal species. They cross-react in many immunologic tests and have a similar appearance by electron microscopy, making differentiation among them difficult. Rotaviruses derived from different host species were distinguished by postinfection serum blocking virus activity in an enzyme-linked immunosorbent assay (ELISA). Thirty-three rotavirus isolates from children living in three different parts of the world could not be differentiated by this technique, but they were distinct from four strains recovered from calves, and a series of strains isolated from piglets, foals, monkeys, and infant mice. The four bovine strains were similar, but they could be differentiated from the other animal strains, each of which exhibited a distinct pattern when tested by the ELISA blocking technique.
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The development of microtiter solid-phase radioimmunoassays for the detection of Norwalk antigen and its antibody is described. The tests are simple to perform and are sensitive and specific. The test for antigen can be used on crude stool filtrates and suspensions. Both tests are at least as sensitive as immune electron microscopy and more sensitive than immune adherence assay.
An immune adherence hemagglutination assay (IAHA) for the detection of antibody to the Norwalk agent of acute epidemic nonbacterial gastroenteritis was developed using as antigen virus purified from stool from an experimentally infected volunteer. The assay was sensitive and specific and was efficient for detecting Norwalk antibody seroresponses. The prevalence of Norwalk antibody in various groups in the United States was studied. Antibody to the Norwalk agent was acquired gradually, beginning slowly in childhood and accelerating in the adult period so that by the fifth decade 50% possessed antibody. This pattern of antibody acquisition contrasted sharply with that for the human rotavirus of infantile gastroenteritis. Rotavirus antibody was acquired during early childhood by almost all individuals in the pediatric groups studied. Antibody to the Norwalk agent was also found in rural Bangladesh; in a small prevalence survey of 39 children and adults 21% possessed Norwalk IAHA antibody, whereas 95% possessed antibody to the human rotavirus.
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A simian rotavirus (SA-11) was shown to hemagglutinate human group "O" and guinea pig erythrocytes. The hemagglutinin appeared to be associated with the outer capsid of the SA-11 virus and was inhibited by specific hyperimmune anti-SA-11 guinea pig serum.
An enzyme-linked immunosorbent assay (ELISA) was developed for the detection of the human reovirus-like agent of infantile gastroenteritis in human stools. The results of the assay can be read either with a simple colorimeter or the naked eye. Investigations with 143 samples from children with gastroenteritis and 75 samples from children with other illnesses showed that the ELISA was as sensitive as electron microscopy or radioimmunoassay for detection of this agent. In addition, the ELISA was simple to perform and, when read visually, did not require sophisticated technical equipment. These advantages make it suitable for field work.
Viruslike particles 26-27 nm in size were detected by immune electron microscopy in stools of volunteers who were ill after administration of bacteria-free fecal filtrates derived from two separate family outbreaks of acute epidemic nonbacterial gastroenteritis. Fluorocarbon treatment and concentration of the filtrates were necessary to provide enough antigen to test sera by immune electron microscopy. Serum antibody responses were detected in both naturally occurring and experimentally induced cases of illness. The Montgomery County viruslike particle appeared to be related to the previously described Norwalk particle, whereas the Hawaii particle appeared to be unrelated to the Norwalk particle.
Human reovirus-like agent (HRVLA) is a major cause of gastroenteritis in infants and young children in many parts of the world. Detection of HRVLA in stools is impractical with the techniques currently available. We describe a rapid immunofluorescence assay for detection of HRVLA in stools. Results with this assay agreed well with results obtained from examination of stool specimens by electron microscopy.