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Several complement regulatory proteins exist on self-cells to prevent damage by the serum complement system. In the present study, we aimed to perform quantitative analysis of membrane-bound complement regulators, CR1 (CD35), MCP (CD46), DAF (CD55), and MIRL (CD59), on peripheral blood neutrophils, monocytes, and lymphocytes from healthy controls (n=36) and febrile patients diagnosed with either bacterial (n=21) or viral (n=26) infections. Our results show that: (a) increased CD35 and CD55 levels on neutrophils and monocytes present potent markers of bacterial infection, (b) increased expression of CD46 on monocytes is an indicator of viral infection, and (c) increased CD59 expression on neutrophils and monocytes is a general infection marker. Additionally, CD19-positive B-lymphocytes represent practically the only lymphocyte population capable of expressing CD35. We further developed two novel clinical flow cytometric markers (indices), specifically, clinical mononucleosis (CM)-INDEX (incorporating CD35, CD55, and CD59 expression on lymphocytes) and clinical bacterial infection (CBI)-INDEX (incorporating CD35 and CD55 expression on neutrophils and lymphocytes), for the effective detection of viral mononucleosis and bacterial infection, respectively. In summary, bacterial and viral infections induce different expression patterns of membrane-bound complement regulators in human leukocytes, which may be effectively exploited in clinical differential diagnosis., (Copyright © 2013 American Society for Histocompatibility and Immunogenetics. Published by Elsevier Inc. All rights reserved.)

Passive immunisation, based on bovine colostral preparations, is an area of active research. Specific bovine antibodies inhibit the virulence factors of target pathogens but the interactions between whey preparations and human immune defence cells are not well known. Bovine colostrum inhibits the phagocytic activity of bovine leucocytes and this may reflect the biological activity of immunoglobulins in it. Therefore, this study aimed to examine the effects of bovine whey protein preparations from the colostrum of Streptococcus mutans/S. sobrinus-immunised and sham-immunised cows on binding, ingestion and killing of these bacteria by human leucocytes. Binding and ingestion of FITC-labelled bacteria were estimated by flow cytometry and leukocyte activation was measured as chemiluminescence. Killing rate was estimated by plate counting and by measuring bioluminescence from S. mutans- containing the insect luciferase gene. Colostral whey protein preparation from hyperimmunised cows activated human leucocytes by opsonising specific bacteria. Neutrophils, eosinophils and monocytes weakly phagocytosed non-opsonised bacteria and bacteria opsonised with control product. On the contrary, binding and ingestion were efficient in the presence of the preparation from immunised cows. Thus, these results show that bovine colostral whey proteins are able to support the activation of human phagocytes against pathogenic microbes and that this property is related to specific antibodies in whey preparations. These whey proteins may also be clinically useful, especially in preventing the colonisation of newly erupted teeth by mutans streptococci.

A novel quantitative nonlabel immunoassay is described. It is based on the recognition of antigen-antibody complexes by the Fc-receptors of phagocytic leukocytes and the subsequent activation of these cells. Activation which is proportional to the amount of immune complexes present can be detected by measuring the intensity of chemiluminescence emitted by the activated cells. In addition to determinations of an antigen and an antibody, the binding capacity of complement to antigen-antibody complexes can be estimated.

The activity of leukocytes, determined by chemiluminescence (CL) emission, was compared with the somatic cell count (SCC) in 4,883 quarter-milk samples from 132 dairy cows. The presence of bacteria was determined by bacteriologic culture of samples in which SCC and CL were high. Chemiluminescence was measured with an automated illuminometer system at 37 C after separating the leukocytes from milk by allowing them to adhere to cotton-wool swabs. Chemiluminescence emission was induced by opsonized zymosan and enhanced by luminol. After luminol and zymosan were added to the measuring vials containing the swabs, CL emission increased rapidly, reaching its maximum usually at about 15 minutes of reaction time, and decreasing slowly thereafter. In general, good correlation was found between CL and SCC (r = 0.876; P less than or equal to 0.001; n = 4,883). Even milk samples with low SCC gave reliably measurable CL signals. Minor pathogens in the milk caused about a sevenfold increase in both SCC and CL, whereas major pathogens caused 14- and 25-fold increases in SCC and CL, respectively. The diagnostic situation that requires both sensitivity and specificity to be at least 90% was attained only by the CL assay for major pathogens. These results suggest that the measurement of milk leukocyte activity by CL assay applies well to the diagnosis of mastitis, and has the potential to become a large-scale laboratory test, as well as a simple cowside test.

The effect of histamine and histamine antagonists on the respiratory burst activity of leukocytes was studied. The activity was measured as zymosan-induced luminol-dependent chemiluminescence (CL) of isolated leukocytes and 1:2,500 diluted whole blood (WB). Histamine caused a dose-dependent inhibition of CL. For separated leukocytes the ID50 was 8 x 10(-5) M and for WB it was 1.5 x 10(-3) M. Diphenhydramine, an H1-antagonist increased the inhibitory effect of histamine while H2-antagonist cimetidine blocked the inhibition of CL of separated leukocytes. Cimetidine was not capable of reversing the effect of histamine on WB-CL. These data suggest that the histamine receptors on leukocytes are associated with Fc and/or complement receptors and the expression and function of histamine receptors can be studied by measuring the respiratory burst activity. The results also provide evidence of differences in histamine action in vitro and ex vivo conditions in WB.

The luminol-enhanced chemiluminescence (CL) of peripheral blood leukocytes was studied daily in five patients with acute myelogenous leukaemia (AML) in first remission, who were undergoing allogeneic bone marrow transplantation (BMT). The CL was measured after stimulation of leukocytes with opsonized zymosan in highly diluted whole blood. All patients had an undetectable CL level on day +7, post BMT, simultaneously with severe pancytopenia caused by the pre-conditioning for BMT. Subsequently, CL started to rise, reaching the maximum level, twice that of healthy controls, on day +11. This preceded the rise of blood leukocytes above 1.0 x 10(9) l.-1 and that of neutrophils above 0.5 x 10(9) l.-1 by 3-14 days, but coincided with the appearance of large unstained cells (LUC; a parameter given by a Technicon H 6000 blood analyzer). One of the patients later had a transient decline of CL. This preceded the fall in white blood count and platelets by 7 days, suggesting marrow suppression. We conclude that in AML the measurement of leukocyte CL is a more sensitive test for prediction of graft take than the conventional blood counts.