Monoclonal antibodies to bovine platelet factor 4: species interactions to platelets and megakaryocytes using indirect immunocytofluorescence.

Murine monoclonal antibodies (mAb) were raised to a purified product of bovine PF-4, a 9,500 dalton protein with heparin neutralization activity comparable to that of human PF-4. Using a non-radioactive slide immunoenzymatic assay, four major classes of mAb could be identified when comparisons were made between purified antigens of PF-4 and beta-TG-like protein from both bovine and human species. Type 1 cross-reacted with all four antigens; type 2 reacted with PF-4s; type 3 reacted with only bovine PF-4 and beta-TG-like protein; and type 4 reacted only with bovine PF-4. Differences in immunoreactivities of types 1, 2 and 3 were retained throughout the growth of succeeding clones and in ascitic fluids. Using a modified factor Xa, S-2222 chromogenic substrate-heparin inhibition assay, no mAb was found to block PF-4's ability to neutralize heparin. mAbs representative of types 1, 2 and 3 were successfully raised in stable cell lines from at least second generation clones. These were purified with protein A agarose and found to be IgG1. By indirect immunocytofluorescence a purified type 2 mAb, 2E7, was found to specifically stain granules of human platelets and megakaryocytes, as well as masses (putative platelets within late stage megakaryocytes) without staining other cellular types in either bone marrow or peripheral blood. Species comparisons displayed positive staining for human, rat, and rabbit platelets and megakaryocytes, and negative staining for mouse, guinea pig and dog platelets and megakaryocytes. It seems likely that mAb, 2E7, is directed against an epitope, common to PF-4 of bovine, human, rabbit and rat.

Surface ultrastructure of human megakaryocytes sorted on the basis of DNA content.

The relationship of polyploidization (DNA content) to differentiation is not well defined. We have developed centrifugal elutriation and Percoll density gradient centrifugation to obtain large numbers of highly-purified megakaryocytes which subsequently were stained for DNA content with Hoechst 33342 and sorted by FACS into 8C, 16C and 32C ploidy classes for correlated analysis of cell surface structures by scanning electron microscopy. Each ploidy class revealed unique surface characteristics that reflect differentiation occurring in megakaryocytes independent of their DNA content.

A study of free lung cells from young rats chronically exposed to carbon monoxide from birth.

Carbon monoxide (CO) is a well know constituent of cigarette smoke and automobile exhaust. The purpose of this study was to determine the effect of CO on the first line of lung defense, the alveolar macrophage (AM). Free lung cells were obtained by bronchoalveolar lavage with Dulbecco's phosphate buffered saline from rats exposed to 500 ppm CO from birth until 33 days of age and from littermate controls. Morphological and functional parameters of the exposed cells showed minor changes. Hemoglobin concentration and hematocrit were significantly elevated in the CO-exposed group. Recovery yield of lavage fluid, wet lung weight, and lung displaced volume were identical to controls. Neither number of AM, viability, maximal diameter, surface area nor acid phosphatase activity of exposed AM appeared to be different from controls. However, in a test with fluorescent latex spheres, the phagocytic ability of AM was found to be enhanced in CO-exposed rats and was correlated with an increased percentage of spread forms of AM adhering to glass coverslips. The surface features of the AM were somewhat modified. Number and percentage of granular leukocytes were statistically increased in lavages following chronic exposure to CO. These alterations contrast with the effects of diesel exhaust and cigarette smoke.

Species comparisons of bronchoalveolar lavages from guinea pigs and rats exposed in vivo to diesel exhaust for one year.

Male Hartly guinea pigs and Fischer rats 344 were exposed to diesel exhaust (DE) concentrations at 0, 250, and 1500 micrograms/m3 in short terms, as well as long term experiments up to one year. The effects of inhaled DE on these rodents were evaluated using bronchoalveolar lavage technique. Both the morphological and functional studies of free lung cells and the biochemical and immunologic studies of the supernatant lavage fluid provided the basis for a quantitative species comparison of the pulmonary responses of exposed guinea pigs and rats versus age matched controls. Following inhalation of 250 micrograms DE/m3, there were little or no significant changes in either species. In contrast, at higher DE concentration, leukocytic infiltration and elevation of specific proteins in lavage fluids were observed in both species. The findings occurred and persisted in both species. Some of the responses were species specific (e.g., the specific type of exudative leukocytes, appearance of reactive monocytes, and different amounts of free DE particles and debris in the lavage fluid). Other responses were similar in both species. Among them, the emergence and increase of lymphocytes was evidence of immunologic responses. Biochemical data from the supernatant fluid correlates with the changes in cellular population in the lavage. The responses appear to be dose and duration dependent. These data indicate that species differences occur. However, it is clear that the alveolar macrophage and granulocytic leukocytes continue to exert effective defense at the DE dose-durations studied. In general, rats appeared more resistant to DE exposure than guinea pigs.

Effects of Diesel engine exhaust on pulmonary alveolar macrophages.

The in vivo effects of inhalation of diesel engine exhaust (DEE) on pulmonary alveolar macrophages (PAM) was studied in 73 guinea pigs and 48 rats. Animals were exposed in individual cages in special chambers to 3 different dose levels of DEE expressed in terms of the concentation of soot or carbon particles (-P); 250, 1500, 6000 micrograms DEE-P/M3. Exposure durations for guinea pigs were 1 and 3 days, 1 and 2 weeks, 2, 4, 8 and 12 months while rats were exposed 1, 2, 4, 8 and 12 months. Age matched controls were similarly exposed concurrently to "clean" air. PAM obtained by bronchopulmonary lavage from exposed animals had viabilities comparable to controls. PAM diameters and relative surface areas increased 2--3 fold over controls and in relation to both the dose of DEE-P given and the exposure duration. Most of the in vivo exposed PAM had phagocytized DEE-P which did not appear to be cytotoxic and remained confined in phagosomes as discrete particles with diameters of 0.014--0.072 micrometer. Ability of PAM to adhere and spread on test surfaces was greater in the DEE-P sets than in controls. DEE-P containing PAM were still able to phagocytize latex particles when fed in vitro. However, such PAM had defective phagocytosis ability, and did not in the same time interval take up as much fluorescent latex as controls when studied by flow system technology. Absolute numbers of PAM in guinea pig lavages from exposures to 250 and 1500 microgram DEE-P/M3 for 2 months were not significantly changed over concurrent controls. Exudative leukocytes (eosinophils in guinea pigs and neutrophils in rats) appeared in the lavage in greater numbers as dose and duration of exposure increased. Another species difference was the appearance in DEE-P exposed guinea pig lavages of "reactive" monocytes.

Two knots in the umbilical cord.

A case of two true knots occurring in an umbilical cord is reported because of its relative rarity. The literature is reviewed. The cause of this potentially dangerous phenomenon is discussed, and a recommendation for further study made.