Robert Burrell

Purification of haptoglobin and its effects on lymphocyte and alveolar macrophage responses.

Clinical observations made on a patient with acute hypersensitivity pneumonitis revealed that the patients serum contained a mitogenic inhibitor and an extremely high haptoglobin (Hp) level; this led to an investigation of the role of Hp in lymphocyte function. Hp was isolated and purified from acute phase rabbit serum by a new method using DE-52 anion exchange chromatography and preparative isoelectric focusing in a range of pH 3.0–5.0. This technique produced a homogenous, biologically active product in fewer steps and in higher yields than existing techniques. Purified rabbit Hp significantly inhibited polyclonal lymphocyte mitogenic responses to PHA or Con A in a dose-response fashion. Hp also significantly inhibited B-cell mitogenesis at a high concentration (200 mg/100 ml) in response to LPS while it enhanced B-cell mitogenesis at lower concentrations (50 and 100 mg/100 ml). Purified Hp had no effect on monoclonal, antigen-specific (BSA) mitogenesis. Rabbit alveolar macrophages produced significant amounts of prostaglandin E in vitro in response to LPS but Hp had no effect on this production. However, Hp alone caused approximately the same amount of stimulation of PGE production by alveolar macrophages as did LPS alone. The ability of Hp to modulate lymphocyte as well as macrophage function seems to indicate that Hp plays a role in the moderation of inflammation. This ability to moderate inflammation, especially in the lungs, may play an important role in regulating tissue damage and disease following inhalation of inflammatory aerosols.

The induction of fibrogenesis by silica-treated alveolar macrophages.

Abstract An in vitro model for the study of the pathogensis of silicosis is described. In it, filtered supernates from alveolar macrophage cultures incubated in contact with silica particles are added to target cultures of lung fibroblasts. The effects of this system are easily studied by observation for cytotoxicity and hydroxyproline production. These results confirm and extend the observations of Heppleston and Styles and point to the use of such a system for the in vitro study of the effects of silica.

The role of precipitins and complement activation in the etiology of allergic lung disease

An experimental model of allergic lung disease has been described that is monitored by analysis of arterial oxygen tension following aerosol challenge with antigen. Rabbits immunized to a classical soluble antigen, human serum albumin (HSA), to the point where severe Arthus skin reactivity was demonstrable, were aerosol-challenged with antigen. Arterial oxygen tension measurements made on pre- and post-challenge samples yielded early, late, and continuous response patterns, reminiscent of those obtained in humans following provocation testing. Aerosol challenge of unimmunized animals with HSA resulted in no change from baseline conditions. Unimmunized rabbits exposed to small and massive (10X) aerosols of Aspergillus spores also demonstrated various postchallenge depressions in arterial oxygen tension as well as decreased levels in hemolytic complement activity, depending on the species of fungus and dose of spores used. Unimmunized animals pretreated with cobra venom factor in a manner known to achieve complement depletion failed to respond with altered arterial oxygen tensions following similar aerosol challenge. It is postulated that although precipitins may play a role in artificial disease initiated by soluble antigens, nonspecific complement activation may be more important in understanding the etiology of spontaneous disease in humans brought about by inhalation of moldy particulate matter.

Morphometric changes of the lung induced by inhaled bacterial endotoxin.

Due to the ubiquitous nature of airborne endotoxin, an understanding of pulmonary alterations which follow inhalation of environmentally realistic concentrations of purified bacteria derived lipopolysaccharide (LPS) is important. Using LPS derived from Enterobacter agglomerans, a bacterium found in cotton and cotton mill dust, aqueous aerosols (effective LPS concentration 4 micrograms/m3) were generated and used to expose either normal hamsters (N = 6) or those rendered endotoxin tolerant by pre-ip injection of 0.1 LD50 LPS. Control groups (normal--N = 6; tolerant--N = 6) received saline aerosol only. At 6 hr after 5-hr aerosol exposure, lungs of all animals were fixed, processed for light and transmission electron microscopy, and subject to qualitative and to multitiered morphometric analysis using standard point counting techniques. Qualitative evaluation of TEM micrographs from LPS aerosolized-nontolerant hamsters showed endothelial alteration (focal disruption, subendothelial space formation, and cytoplasmic blebbing) but volume and number of endothelial cells were not changed indicating only slight, focal endothelial damage. Quantitatively, septal capillary blood space in nontolerant, LPS aerosolized hamsters showed increased Vv of PMNs and platelets. These changes were not seen in tolerant induced-LPS aerosolized hamsters. Independent of tolerization treatment, LPS inhalation led to a decrease in fixed lung volume and an increase in numerical density of endothelial pinocytotic vesicles. It is concluded that the inhalation of realistic, environmental levels of bacterial endotoxin may induce significant changes in distal lung and may be important in the pathogenesis of byssinosis and adult respiratory distress syndrome.

Ultrastructure of the Endotoxin Produced by Gram-negative Bacteria Associated with Organic Dusts

Summary The isolates of two out of four examined bacterial species associated with organic dusts (Erwinia herbicola and Enterobacter sp.) showed an ability to shed the membrane vesicles while growing on a solid medium. The formation of such vesicles was not observed in strains of other two species (Acinetobacter calcoaceticus and Alcaligenes faecalis) that revealed a lower degree of the outer membrane development. The preparations of crude lipopolysaccharide isolated with two methods from strains of all four species were found to be composed of vesicular particles resembling those shed from the outer membrane of E. herbicola and Enterobacter sp. The similar structures were found in natural environment, using wood as a model substrate. It has been shown by electron microscopy and specific immunolabelling that wood dust from American basswood contained numerous gram-negative bacteria of the species Erwinia herbicola that were shedding large amounts of the endotoxin-containing membrane vesicles, measuring about 30 nm. Bacteria were not found in the samples of wood dust from other kinds of timber. These results were in full accord with those received by the examination of the wood dust samples by culture and by Limulus test for the presence of endotoxin. The presented results suggest that the significant portion of an environmental “dust-borne” endotoxin occurs in the form of microvesicles, what may respresent an increased risk for the people exposed to various organic dusts.

Effects of inhaled endotoxin-containing bacteria

Abstract Results of a study of pulmonary response-producing mechanisms resulting from the inhalation of endotoxin are reported. A number of experimental variations in challenging unimmunized rabbits with Escherichia coli without envelope antigens was attempted. Changes in arterial blood gases, leukocytes, platelets, and peripheral complement were monitored as indices of inhalation response. When either purified endotoxin or heat-treated E. coli cells were used for a single challenge, consistent elevations in P aO 2 with concurrent depressions in P aCO 2 were noted 4 hr after challenge. Experimental animals also exhibited a leukocytosis 10 min post challenge, which persisted up to 4 hr, but no evidence of fever could be documented. Since occupational exposure to endotoxin-contaminated dusts would involve exposures of varying concentration and duration, a comparison was made between single and dual challenges with the same dose given 24 hr apart, in an effort to see if something analogous to a pulmonary Shwartzman reaction might take place in unimmunized animals. When an aerosol sensitizing dose of E. coli was followed 24 hr later by an intravenous provoking dose of the same cell suspension, marked decreases in P aO 2 occurred within several hours after challenge and continued for up to 8 hr. Moreover, a decline in the number of circulating platelets coincided with the observed depressions of P aO 2 . A portion of the animals died with hemorrhagic lung injury, but in those surviving, P aO 2 values returned to normal within 24 hr. Indomethacin was shown to be effective in inhibiting these changes observed following dual challenge, thus pointing to the role of prostaglandins in the response. These experiments demonstrate that common endotoxin-containing microorganisms may cause pulmonary reactions in at least two ways. The ubiquity of gram-negative organisms allows for possible pulmonary injury in individuals associated with a wide variety of occupational exposures.

Keyhole limpet hemocyanin immunotherapy of murine bladder cancer

SummaryThe current treatment of choice for superficial bladder cancer, bacillus Calmette-Guérin, has significant adverse side effects. We have compared two alternative immunotherapies—crude keyhole limpet hemocyanin (KLH) and Immucothel, a KLH modified for clinical use (Biosyn)-in an intralesional mouse model of bladder cancer (MBT2). Crude KLH required either immunization before tumor transplant or frequent intralesional therapy after transplantation to be effective. In addition, Immucothel required pre-immunization to be effective, and increasing the frequency and dosage of post-transplant immunization was not effective without pre-immunization. Preliminary investigations into the KLH-induced anti-tumor mechanism(s) suggest that natural killer cell activity may be involved. Both crude KLH and Immucothel appear to be effective immunotherapies of use in the treatment of transitional cell carcinoma.

IMMUNOLOGICAL ASPECTS OF COAL WORKERS' PNEUMOCONIOSIS*

Several factors indicate that coal workers pneumoconiosis (CWP) , silicosis, and other chronic pulmonary diseases involve immunological components. In these diseases, the immunological system participates in several different ways during the interaction of host and offending agent. Our interest in immunological aspects of these diseases stems from studies 1 e 2 on experimental tuberculosis, wherein antibodies reactive with host lung tissue components were produced. That these antibodies participate in the pathogenesis of the disease was evidenced by the demonstration of the altered course of the infection in tuberculous animals passively administered with antilung serum. Such animals tended to show less defense against the tubercle bacillus and developed a more miliary form of the disease. These observations were later extended to other chronic pulmonary diseases, including experimental silicosis. It is the purpose of this paper to present various lines of evidence that indicate immunological involvement in the pneumoconioses.

Further studies on inhaled endotoxin-containing bacteria

Abstract A highly sensitive, discriminatory, and reproducible assay for changes produced by inhalation of gram-negative, endotoxin-containing bacteria was developed. The assay measures the production of lactic dehydrogenase (LDH) of mice in pulmonary lavages at various time intervals after standardized aerosol exposure. Aerosols of four such bacteria, Enterobacter cloacae, Escherichia coli, Citrobacter freundii , and a heavily mucoid species of Flavobacterium , chosen because of their occurrence in aerosols in agricultural work environments, produce distinctly different patterns of LDH evaluation above those of control mice exposed to saline aerosols. Comparative results obtained from endotoxin-responsive and C3H HeJ endotoxin-resistant mice indicated that endotoxins differed in their ability to induce the LDH release. In the case of the response to Flavobacterium , the enzyme release was due more to a synergistic effect between the glycoprotein slime layer and the endotoxin. More detailed studies with the most toxic strain studied, C. freudii , indicated the LDH response was in part due to a bound endotoxin recognition trigger, partly due to complement activation, and most likely independent of antibodies.

Detection of antibodies to sinus tissue in chronic bronchial asthma

Abstract A comparative study of the incidence and specificities of sinus reactive antibodies in humans is presented. The sera from twenty-four patients, most of them suffering from chronic asthma, have been studied using the antiglobulin consumption test. Sixty-two per cent of the sera showed the presence of antibodies to lung and forty-six per cent reacted to sinus tissue antigens. Adsorption studies and correlations with clinical findings are presented. Possible interpretations of these findings are discussed in relation to the etiology of intrinsic asthma.