Initial salivary pellicle formation on solid substrates studied by AFM.

Organic layers of salivary biopolymers adsorbed on soft and hard oral tissues, referred to also as salivary pellicle, play a critical role with respect to all surface phenomena taking place in the oral cavity. The initial stages of pellicle formation are of great interest since they determine the ensuing processes of salivary biopolymer adsorption and subsequent adherence of bacteria. In spite of the important physiological role of the pellicle in protecting the enamel surface against short-term acidic attacks, the composition and ultrastructure of the pellicle layer are not yet understood and resolved in detail. The present study utilized atomic force microscopy (AFM), for the first time, to elucidate the morphogenesis and ultrastructural pattern of initial salivary pellicle formation taking place in situ on solid substrates of mica, silicon wafer and graphite. Using tapping mode AFM, salivary pellicles were found in all intraorally exposed specimens and revealed a globular surface morphology of the adsorbed protein layer. The average diameter and height of the adsorbed salivary proteins were determined to be 15 +/- 3 nm and 2.0 +/- 0.5 nm, respectively. It was also found that the surface energy of the substrates affects the rate of pellicle formation, while the overall size of the adsorbed salivary proteins appears to be identical on all studied substrates.

Cylindrical molecular brushes under poor solvent conditions: microscopic observation and scaling analysis.

Axial contraction of cylindrical molecular brushes of polymethylmethacrylate was observed by static light scattering and scanning force microscopy. Single brush molecules were visualized as worm-like particles whose length was almost three times shorter than the contour length of the backbone. A somewhat larger length was measured by light scattering in a good solvent. A scaling approach has been used to analyze the driving forces for the axial contraction and the conformation of the molecular brushes.

Regulation of alveolar macrophage and type II cell DNA synthesis: effects of ozone inhalation.

A characteristic reaction of the lung to inhaled ozone is an increase in the number of type II epithelial cells and alveolar macrophages (AMs). In the present study, we analyzed mechanisms regulating this response. Acute exposure of rats to ozone (2 parts/million, 3 h) induced expression of proliferating cell nuclear antigen, a marker of cellular proliferation, in both type II cells and AMs. This was maximum 48 h after ozone inhalation. Type II cells and AMs isolated from treated rats at this time also incorporated significantly more [3H]thymidine ([3H]TdR) than cells from control animals. When type II cells and AMs were cocultured, a synergistic increase in [3H]TdR uptake was observed. This appeared to be due to increased DNA synthesis by both cell types. Thus [3H]TdR incorporation by type II cells and AMs cocultured with mitomycin C-treated AMs and type II cells, respectively, was elevated compared with cells cultured alone. Type II cells and AMs plated onto tissue culture inserts, as well as culture supernatants from these cells, were found to stimulate DNA synthesis in AMs and type II cells, respectively. In addition, crude membrane preparations from these cells exhibited growth-promoting activity. Thus the mitogenic effects of both cell types appeared to be mediated by soluble factors and membrane-associated molecules. Ozone inhalation resulted in an increase in the mitogenic activity of AMs treated with mitomycin C and plated on tissue culture inserts toward type II cells and of type II cell culture supernatants toward AMs. These data suggest that type II cell and AM proliferation contributes to the regulation of the number of cells in the lung under normal homeostatic conditions and after ozone-induced injury. Moreover, type II cells and AMs produce paracrine mediators that contribute to cellular proliferative responses.

Distinct inflammatory responses of adherent vascular lung neutrophils to pulmonary irritants.

The nature and the extent of the damage that occurs in the lung following exposure to pulmonary irritants vary with the pathogenic agent. In the present studies we determined if this was due to unique functional responses of adherent vascular neutrophils to different irritants. Because of their location within the lung, these cells may be more relevant than circulating neutrophils to the pathophysiology of irritant-induced lung injury. For our studies we used two model irritants, ozone and endotoxin, which cause distinct pathologic effects in the lung. Treatment of rats with ozone resulted in a transient increase (2-fold) in the number of adherent vascular neutrophils in the lung which was maximum 2 hr after exposure and returned to control levels by 12 hr. In contrast, following endotoxin administration, 10-fold greater numbers of adherent neutrophils were recovered from the lung. Moreover, cell number remained elevated 3-fold for up to 48 hr. Unstimulated neutrophils isolated 2-12 hr after endotoxin treatment of rats produced 3 times more superoxide anion than cells from ozone-treated rats. Cells isolated 12-48 hr after endotoxin administration were also sensitized to produce more nitric oxide than cells from ozone-treated rats and to express inducible nitric oxide synthase protein. These data demonstrate that endotoxin and ozone induce distinct patterns of accumulation and functional changes in adherent vascular neutrophils in the lung which may contribute to different pathological processes observed following exposure to these pulmonary irritants.

Macrophage and interleukin-1 induced nitric oxide production and cytostasis in hamster tumor cells varying in malignant potential.

Nitric oxide has been shown to contribute to cytotoxicity in mouse and rat tumor cells. In these studies we examined the role of nitric oxide in cytostasis in hamster tumor cells varying in their malignant potential. Spontaneously transformed hamster embryonic fibroblasts (STHE cells) with low metastatic activity produced significantly greater amounts of nitric oxide in response to interleukin-1 (IL-1) or lipopolysaccharide (LPS)-activated hamster alveolar macrophages (HAM) than did tumor cell lines with high experimental metastatic activity (HET-SR, HET-SR1, STHE-83/20 cells). HET-SR cells, which exhibit low spontaneous metastastic activity, also produced relatively high levels of nitric oxide in response to IL-1, whereas the response of the spontaneously metastatic lines, HET-SR1 and STHE-83/20 cells, was low. IL-1 and HAM also induced cytostasis in nitric oxide-producing STHE and HET-SR cells. However, the nitric oxide synthase inhibitor, N(G)-monomethyl-L-arginine (L-NMMA), had no effect on this activity. These findings, together with the observation that anti-tumor necrosis factor alpha antibody prevented HAM-mediated cytostasis in all of the tumor cell lines demonstrate that nitric oxide is not involved in hamster macrophage-induced tumor cell growth suppression. In contrast to HAM, rat alveolar macrophages, which produced nitric oxide in response to LPS, exerted similar levels of cytostasis toward all of the hamster tumor cell variants, an action that was blocked by L-NMMA in HET-SR, HET-SR1, and STHE-83/20 cells. Thus production of nitric oxide by hamster tumor cells is inversely correlated with their malignant potential. However, nitric oxide does not appear to be involved in IL-1- or HAM-mediated cytostasis toward hamster tumor cells.

Cytostasis is required for IL-1 induced nitric oxide production in transformed hamster fibroblasts.

Interleukin-1 (IL-1) is known to inhibit proliferation in some tumor cells. This proinflammatory cytokine also induces nitric oxide production in a variety of cell types. In the present studies we determined if nitric oxide is involved in IL-1 induced growth inhibition in spontaneously transformed hamster embryonic fibroblasts (STHE cells). Both IL-1 alpha and IL-1 beta were found to stimulate nitric oxide production and to reduce 3H-thymidine (TdR) incorporation in high density cultures of STHE cells. However, maximal cytostasis was observed at least 24 h before significant amounts of nitric oxide accumulated in the cultures. In addition, doses of IL-1 which were too low to stimulate nitric oxide synthesis were effective in inducing cytostasis. Furthermore, in low density cultures of STHE cells, IL-1 inhibited DNA synthesis without inducing nitric oxide production. The nitric oxide synthase inhibitor NG-monomethyl-1-arginine (L-NMMA) had no effect on proliferation of cells plated at low density. In contrast, L-NMMA treatment resulted in a 40-60% reduction in IL-1 induced cytostasis in high density cultures. Neutralizing antibodies to IL-1 were found to completely block IL-1 induced cytostasis and nitric oxide production in cells plated at both densities. Although anti-IL-1 alpha and anti-IL-1 beta antibodies were highly specific and did not cross react, anti-tumor necrosis factor-alpha (TNF-alpha) antibody was able to partially suppress activation of STHE cells by both IL-1 alpha and IL-1 beta. These data suggest a potential involvement of endogenous TNF-alpha in IL-1 induced cytostasis and nitric oxide production. Exponentially growing STHE cells produced six-times less nitric oxide than non-proliferating cells. A ten-fold excess of 1-arginine was found to stimulate nitric oxide synthesis, an action that was independent of the rate of cellular proliferation. Taken together these data suggest that nitric oxide is not a major mediator of IL-1 induced cytostasis in STHE cells. Moreover, cytostasis appears to be required for nitric oxide synthesis in these cells.

Mechanisms regulating macrophage-induced nitric oxide production by spontaneously transformed hamster fibroblasts.

Nitric oxide has been implicated as an important effector molecule involved in tumor cell growth and cytotoxicity. In these studies we examined mechanisms regulating nitric oxide production by hamster tumor cells. Cocultures of hamster alveolar macrophages (HAM) and spontaneously transformed hamster embryonic fibroblasts (STHE cells) produced significant quantities of nitric oxide in response to lipopolysaccharide (LPS). Culture supernatants from HAM treated with LPS also stimulated nitric oxide production by STHE cells, whereas tumor cell culture supernatants had no effect on HAM. These data, together with the findings that paraformaldehyde treatment of STHE cells, but not macrophages, completely abrogated nitric oxide production in the cocultures demonstrate that the tumor cells were the source of this mediator. In contrast to STHE cells, STHE-83/20 cells, a highly malignant variant, did not produce nitric oxide in response to HAM or HAM culture supernatants even in the presence of LPS. Both anti-tumor necrosis factor-alpha (TNF-alpha) and anti-interleukin-1alpha (IL-1alpha) antibodies inhibited HAM-induced nitric oxide production by STHE cells. However, the kinetics of their effects were different. Moreover, although the nitric oxide stimulating activity in HAM culture supernatants was abrogated by anti-TNF-alpha antibody, it was only minimally reduced by anti-IL-1alpha antibody. These data demonstrate that TNF-alpha and IL-1alpha play distinct roles in induction of nitric oxide synthesis in STHE cells. HAM were also found to suppress proliferation of STHE cells, an effect that was inhibited by anti-TNF-alpha antibody, but not NG-monomethyl-L-arginine, which blocks nitric oxide synthase. Abrogation of macrophage-induced cytostasis in STHE cells by anti-TNF-alpha antibody was associated with decreased nitric oxide production. Thus TNF-alpha released by macrophages may indirectly activate STHE cells for nitric oxide synthesis by suppressing tumor cell proliferation.

[Hygienic evaluation of combined biotechnology for treating sewage from artificial accumulations]. / Gigienicheskaia otsenka kombinirovannoi biotekhnologii ochistki stochnykh vod iskusstvennykh nakopitelei.

Combined biotechnology for treating sewage and industrial wastes with Pseudomonas fluorescens bacteria and Scirpus lacustris L. reed mass was developed. The treated waters can be discharged into the surface reservoirs.

Functional characterization of interstitial macrophages and subpopulations of alveolar macrophages from rat lung.

The specific function of interstitial macrophages (IM) in the lung is poorly understood because of difficulties in isolating these cells in high purity or large number. In the present studies, a pure population of enzymatically isolated IM and lung macrophages obtained mechanically from the lung were compared functionally with alveolar macrophages recovered by lavage (AM). Macrophages isolated mechanically from the tissue and AM displayed similarly high levels of Fc-receptor mediated phagocytosis. In contrast, IM phagocytized significantly fewer opsonized sheep red blood cells per macrophage than AM. In addition, although some variations in the amounts of nitric oxide and superoxide anion produced by AM and macrophages obtained by mechanical tissue disruption were observed, these subpopulations released significantly more of these mediators than IM. These data support the concept that macrophages isolated by mechanical disruption of the tissue represent a subpopulation of AM. We also found that, in contrast to AM, IM did not respond synergistically to combinations of IFN-gamma and lipopolysaccharide (LPS) or tumor necrosis factor alpha in terms of nitric oxide production. Furthermore, regulation of superoxide anion release in AM and IM by LPS and/or IFN-gamma was distinct. Taken together, these studies demonstrate that IM are functionally different from other macrophage subpopulations which might reflect their unique location within the lung.

Isolation and partial characterization of subpopulations of alveolar macrophages, granulocytes, and highly enriched interstitial macrophages from rat lung.

The contribution of interstitial macrophages (IM) to lung defense, homeostasis, and pathophysiology is for the most part unknown. Studies on this cell type are difficult because they are not readily accessible in large numbers or in high purity. In the present work, various nonenzymatic and enzymatic methods were compared with the aim of isolating and characterizing pure populations of lung IM. The results of our studies demonstrate that most procedures currently used to isolate IM yield subpopulations of alveolar macrophages (AM) or IM highly contaminated by AM and granulocytes. We found that lavage of the lung yielded only one half of the total AM present in the tissue. The remainder of the AM could only be obtained by extensive washing of cut and disaggregated lung tissue, which is considered by some investigators to be an effective procedure for IM isolation. According to our results, cells recovered by lavage and washing of cut and disaggregated lung tissue were morphologically and histochemically identical, were strongly positive for nonspecific esterase, highly phagocytic, and appeared to represent subpopulations of AM with apparently varying degrees of adherence to the alveolar walls. We also found that IM could be obtained in high purity by sequential digestion of the remaining lung tissue with 60 and 175 IU/ml of collagenase followed by selective adherence. Digestion of the tissue with 60 IU/ml of collagenase resulted in a highly enriched population of granulocytes and also reduced their contamination in the IM population. The resulting IM were distinct from AM by morphology and histochemistry. Like AM, these cells displayed Fc receptor-mediated phagocytosis.(ABSTRACT TRUNCATED AT 250 WORDS)

[Mechanism of the erythrocyte de-agglutination reaction]. / Nekotorye voprosy mekhanizma reaktsii raskleivaniia éritrotsitov.

The effect on the indication sensitivity of the level of antigenic sensitization of erythrocytes, molecular parameters of sensitin and of the antigen determined was revealed on various models of agglutinates and antigens determined with the aid of erythrocyte deaggregation reaction (EDR). By its dependence on the EDR sensitivity from the hemosensitization level this reaction was analogous to the passive hemagglutination inhibition test described earlier. Specific deaggregation activity of the antigens depended on their molecular parameters in the same way as their specific agglutinating activity in respect to the erythrocytic antibody diagnostic agents--both of these properties were more intensive in larger antigenic molecules.