The college viewpoint.

Our changing health care environment presents new challenges to the surgical profession. This article examines those concerns, including strains on surgeons' professional values, financial pressures resulting from federal laws and regulations, the expansion of managed care, and demands for improved continuing education opportunities. This article illustrates how the American College of Surgeons has been helping surgeons cope with the shifts in balance taking place around them. It further highlights how the American College of Surgeons is moving forward in all areas of concern to surgeons by promoting standards for surgical care, educating third-party payors about the effects of financial constraints, expanding its educational programs, and seeking innovative methods to fulfill its mission.

Endodontic pathogens stimulate monocyte chemoattractant protein-1 and interleukin-8 in mononuclear cells.

Microbial infection of the dental pulp leads to the recruitment of leukocytes and the formation of lesions of endodontic origin. The chemokines interleukin-8 (IL-8) and monocyte chemoattractant protein-1 (MCP-1) are relatively specific chemoattractants for neutrophils and monocytes, respectively. In the present studies, peripheral blood mononuclear cells were stimulated by Streptococcus mutants, Porphyromonas endodontalis, and Peptostreptococcus anaerobius, which are associated with lesions of endodontic origin. Each of these bacteria induced a dose-dependent increase in IL-8 and MCP-1, determined by ELISA. The levels induced are physiologically relevant. However, low doses of P. endodontalis were less effective in inducing IL-8 or MCP-1 expression, compared with S. mutants or P. anaerobius. Thus, these bacteria can induce significant levels of the chemokines IL-8 and MCP-1, which could contribute to the recruitment of neutrophils or monocytes in vivo. The expression of these mediators may contribute to the development of endodontic infections, particularly with regard to inflammatory leukocyte recruitment.

A whole blood alternative to traditional methods for CD4+ T lymphocyte determination.

Flow cytometry, the method of choice for determining subset cell populations, requires sophisticated instrumentation and highly trained operators. An alternative method for subpopulation determinations is presented that is fast, simple, and readily interpreted for absolute subset counts. This method uses antibody labeled microspheres added to whole blood and after lysing, read on a hemocytometer using an ordinary light microscope. The total time of testing is less than 10 min with results comparable to that obtained using a flow cytometer and a hematology analyzer to calculate absolute subpopulation values. This method is demonstrated for CD4+ lymphocytes which have considerable value in following HIV-infected individuals.

A simple and rapid method for removal of specific cell populations from whole blood.

A method is presented in which specific cells can be rapidly separated from whole blood by the use of antibody labelled dense particles utilizing gravity as a means of separation. This method specifically removes the targeted cells with minimal non-specific cell loss. The method is simple to perform and rapid, requiring less than 10 min for cell/particle binding and separation. The method is robust so that exact ratios of particles to cells are not needed. This method could be used in any separation scheme to remove specific cell populations without significant modification.

Monocyte chemoattractant protein 1 and interleukin-8 production in mononuclear cells stimulated by oral microorganisms.

Chemokines are a family of low-molecular-weight proinflammatory cytokines that stimulate recruitment of leukocytes. The chemokines interleukin-8 (IL-8) and monocyte chemoattractant protein 1 (MCP-1) are relatively specific chemoattractants for neutrophils and monocytes, respectively. Chemokine expression contributes to the presence of different leukocyte populations observed in normal and pathologic states. In the present studies, peripheral blood mononuclear cells (PBMC) were stimulated by microbes (Candida albicans, Streptococcus mutans, Porphyromonas gingivalis, and Actinobacillus actinomycetemcomitans) selected based upon their importance as oral pathogens. IL-8 and MCP-1 gene expression and protein release were determined by Northern blot (RNA blot) analysis and enzyme-linked immunosorbent assay. C. albicans, P. gingivalis, and A. actinomycetemcomitans induced high levels of production of both MCP-1 and IL-8. S. mutans was a strong inducer of MCP-1, but it did not stimulate significant production of IL-8. C. albicans, S. mutans, and A. actinomycetemcomitans were 500 to 5,000 times more potent than P. gingivalis in terms of MCP-1 production. In general, the microbe-to-PBMC ratios required for maximum gene expression of MCP-1 were lower than those for IL-8. However, for actual protein release of MCP-1 versus IL-8, differences in the effects of various microbe concentrations were observed only for A. actinomycetemcomitans. These results demonstrate that different oral pathogens induce specific dose-dependent patterns of chemokine gene expression and release. Such patterns may help explain the immunopathology of oral infections, particularly with regard to inflammatory leukocyte recruitment.

Flow cytometric immunofluorescence and DNA analysis: using a 1.5 mW helium-neon laser (544 nm).

We evaluate a 1.5 mW HeNe laser (544 nm) for use on an EPICS Elite with a 76 microns Sortsense flow cell. The two applications chosen were immunofluorescence and DNA analysis. We measured the fluorescence threshhold of phycoerytherin calibration beads to be approximately 336 MESF. Cell analysis with a HeNe laser and Argon laser correlated well for the CD4PE, CD56PE, CD19PE conjugates, with correlation coefficients of 0.98, 0.99, 0.94, respectively. The % positive and mean channel fluorescence were comparable to the results obtained with a 15 mW Argon laser. In addition, a three-color configuration yielded excellent results. Cell analysis of CD4PE, CD3ECD and CD19Cy-Chrome with the HeNe laser and Argon laser correlated well with correlation coefficients of 0.96, 0.95, and 0.92, respectively. The histograms showed good separation between the negative cells, the dimly staining cells and the brightly staining cells. Propidium Iodide was chosen for DNA analysis. Full CV values for whole blood DNA fluorescence using the green laser were good at 2.6%. These data indicate the low power 544 nm laser is sufficient to do immunophenotyping and DNA analysis. Results may be explained by higher quantum efficiency and lower background fluorescence. The wavelength of the 544 nm laser is much closer to the excitation peaks of PI, PE, and the tandem dyes ECD and Cy-Chrome. Also, the Raman scattering of water for the 544 nm laser has a longer wavelength maximum than the emission peaks of PI, PE, and ECD. The major advantages of this laser for the research laboratory are small size, no cooling fan, low power requirements and low cost.

Lymphocyte subset determination using a hematology analyzer.

Anti-CD4 antibody (T4)-coated microspheres were used to label CD4 cells in whole blood. The mixture was lysed and analyzed by a modified Coulter VCS hematology analyzer, which differentiated microsphere-labeled cells by a change in Coulter volume, conductance, and light scatter. %CD3+/CD4+ fluorescent values from a profile were compared to %CD4 values using the VCS-microsphere method. CD3 gating was used to exclude CD4+ monocytes from the 90LS-FALS lymphocyte gate. The results correlated well (R = 0.996). The percentage of CD4+ lymphocytes from profile scatterplots and VCS scatterplots showed a line of regression close to the equivalence line (n = 76, slope = 0.96) when CD3 gating was used for the profile. These results suggest that CD3 gating, though necessary for 90LS-FALS scatterplots, may not be necessary for volume-conductance-light scatterplots.