Jari Nuutila

Flow cytometric quantitative determination of ingestion by phagocytes needs the distinguishing of overlapping populations of binding and ingesting cells

The use of flow cytometry with fluorescently labeled particles provides the means to examine quantitatively the phagocytotic capacity of an individual phagocyte. This report describes an improved flow cytometric method of analysis for kinetic measurement of phagocytosis of fluorescein isothiocyanate (FITC)–labeled zymosan particles by human leukocytes.

Green fluorescent protein-propidium iodide (GFP-PI) based assay for flow cytometric measurement of bacterial viability.

Several staining protocols have been developed for flow cytometric analysis of bacterial viability. One promising method is dual staining with the LIVE/DEAD BacLight bacterial viability kit. In this procedure, cells are treated with two different DNA‐binding dyes (SYTO9 and PI), and viability is estimated according to the proportion of bound stain. SYTO9 diffuses through the intact cell membrane and binds cellular DNA, while PI binds DNA of damaged cells only. This dual‐staining method allows effective separation between viable and dead cells, which is far more difficult to achieve with single staining. Although SYTO9‐PI dual staining is practical for various bacterial viability analyses, the method has a number of disadvantages. Specifically, the passage of SYTO9 through the cell membrane is a slow process, which is significantly accelerated when the integrity of the cell membrane is disrupted. As a result, SYTO9 binding to DNA is considerably enhanced. PI competes for binding sites with SYTO9 and may displace the bound dye. These properties diminish the reliability of the LIVE/DEAD viability kit. In this study, we investigate an alternative method for measuring bacterial viability using a combination of green fluorescent protein (GFP) and PI, with a view to improving data reliability.

Milk hypersensitivity – key to poorly defined gastrointestinal symptoms in adults

Lactose intolerance is a common adverse reaction to milk in adults, while milk hypersensitivity is a disorder of infancy. We hypothesized that milk hypersensitivity may cause many unspecific gastrointestinal disorders in adults. Twenty adults were subjected to double‐blind, placebo‐controlled milk challenge. Phagocyte activity, and Fcγ and complement receptor expression of phagocytes were assayed, and serum total IgE. milk‐specific IgE, and serum reactivity to milk protein were determined. The challenge increased phagocyte activity and complement receptor expression of phagocytes in subjects designated miUc‐hypersensitive, who had gastrointestinal symptoms from milk ingestion but normal lactose tolerance. The increase was not detected in lactose‐intolerant or control subjects. The milk‐hypersensitive group was also distinguished from the lactose‐intolerant group by enhanced serum reactivity to milk protein. Only two out of nine milk‐hypersensitive subjects had detectable milk‐specific serum IgE. It is concluded that milk hypersensitivity in adults, occurring as gastrointestinal reactions, may be more common than previously thought.

Simultaneous quantitative analysis of FcγRI (CD64) and CR1 (CD35) on neutrophils in distinguishing between bacterial infections, viral infections, and inflammatory diseases

A flow cytometric quantitative analysis of receptors on neutrophils can be exploited in distinguishing between inflammatory and infectious diseases. In this prospective comparative study, simultaneous quantitative analysis of CD64 and CD35 on peripheral blood neutrophils was performed in febrile patients in order to differentiate between bacterial infections (n=89), viral infections (n=46), and inflammatory diseases (n=21). The patient data was compared to 60 healthy controls. We could divide patients into three groups depending on how they express CD35 and CD64 on neutrophils: (1) patients with a high probability of viral infection (low CD35/low CD64 and low CD35/high CD64), (2) patients with a high probability of inflammatory disease (high CD35/low CD64), and (3) patients with a high probability of bacterial infection (high CD35/high CD64). In summary, simultaneous quantitative analysis of CD64 and CD35 on neutrophils could potentially assist physicians to distinguish between inflammatory and infectious diseases.

Distinction between bacterial and viral infections.

Purpose of review To commence proper treatment as rapidly as possible and to reduce unnecessary antibiotic treatments, timely knowledge of whether the infection is bacterial or viral in origin would be beneficial for the clinician. As a reliable prediction of the causative agent of bacterial infection is not possible based on clinical features, there is an ongoing need for sensitive and specific markers of bacterial infection. Recent findings The most common differential diagnosis methods are reviewed here. It is also demonstrated that the measurement of the expression of complement receptors, particularly CR1 (CD35), on neutrophils can be a useful preliminary test to differentiate between bacterial and viral infections. In addition, a novel marker of local and systemic bacterial infections designated ‘clinical infection score (CIS) point’, which incorporates quantitative analysis of complement receptors on neutrophils and standard clinical laboratory data and displays 98% sensitivity and 97% specificity in distinguishing between bacterial and viral infections, is presented. Summary We conclude that the diagnostic yield of measured individual variables in distinguishing between bacterial and viral infections increases upon combination.

The novel applications of the quantitative analysis of neutrophil cell surface FcγRI (CD64) to the diagnosis of infectious and inflammatory diseases.

Purpose of review Several studies show that the number of FcγRI (CD64) on the surface of neutrophils increases in infections. However, in spite of increased research interest in recent years, there is no clear general view on the usability of neutrophil FcγRI in clinical infection diagnostics. This review tries to bring the clarity to this matter. Recent findings It is shown here that although the high number of FcγRI on neutrophils is a sensitive marker of bacterial infection, it is highly expressed also in DNA virus infections. As a consequence, neutrophil FcγRI cannot be used in distinguishing between bacterial and viral infections. It is also clear that FcγRI on neutrophils cannot be used in distinguishing between Gram-positive and Gram-negative bacterial infections or between microbiologically confirmed and clinically diagnosed bacterial infections. In addition, neutrophil FcγRI cannot be used to reliably detect RNA virus infections, inflammatory diseases, or cancer. Summary The best clinical benefit from the quantitative analysis of FcγRI on neutrophils will be obtained when it is used simultaneously with a reliable bacterial infection marker. DNA virus score point is an efficient novel method in differentiating between DNA and RNA virus infections.

Susceptibility of Fusobacterium nucleatum to killing by peroxidase–iodide–hydrogen peroxide combination in buffer solution and in human whole saliva

Some Gram-negative anaerobic bacteria have been associated with the infection of tooth supporting tissues, i.e. periodontitis. Of these bacteria, Fusobacterium nucleatum is sensitive to lactoperoxidase/myeloperoxidase-iodide-hydrogen peroxide system in vitro, but salivary concentrations of thiocyanate abolishes the bactericidality. These bacteria are located in periodontal pockets, on oral mucosa and in saliva. Although F. nucleatum most probably does not belong to the group of main periodontal pathogens, it sustains its proportion in the periodontal flora when gingivitis progresses to periodontitis. In this study, the sensitivity of F. nucleatum to different horseradish peroxidase-iodide-hydrogen peroxide combinations was tested both in buffer and in sterilized human whole saliva. Horseradish peroxidase was chosen because it does not bind thiocyanate at pH > or = 6. After 1h incubation at 37 degrees C, the cell viability was estimated by plate count and with flow cytometer using LIVE/DEAD BacLight kit (Molecular Probes, USA). In saliva, the horseradish peroxidase (50 microg/mL)-iodide (2.5 mM)-hydrogen peroxide (2.5 mM) combination decreased the amount of viable bacteria to 37% compared to 85% in the control without any of the components when measured with flow cytometer. Replacement of buffer by saliva decreased the bactericidality of the peroxidase system. However, in buffer less iodide and hydrogen peroxide was needed to produce significant decrease in the number of viable bacteria when measured by plate count than with flow cytometer. Our study shows that horseradish peroxidase-iodide-hydrogen peroxide combination is able to kill F. nucleatum cells in saliva. Horseradish peroxidase-iodide-hydrogen peroxide combination may be useful to diminish the degree of re-colonization of periodontitis-associated bacteria after periodontal therapy and to inhibit the transmission of these bacteria via saliva.

Altered expression of IgG and complement receptors indicates a significant role of phagocytes in atopic dermatitis

BACKGROUND Strict dietary precautions against allergic sensitization may benefit a group of predisposed children. OBJECTIVE To develop new strategies for identifying these children, a better understanding of the processes that initiate sensitization and regulate and perpetuate the inflammatory response is needed. METHODS We measured the expression of the receptors for the constant (Fc) region of IgG (Fc gamma RI, Fc gamma RII, and Fc gamma RIII) and that for the complement fragments C3b and C3bi (CR1 and CR3) in neutrophils and monocytes from 39 children with atopic dermatitis, 17 disease control patients with acute infections, and 17 healthy control subjects. The capacity of phagocytes to produce reactive oxygen species was also determined. To find the best way of discriminating the patients with atopic dermatitis from control subjects, a stepwise logistic binary regression model was made. RESULTS The stepwise logistic regression analysis was based on differences in individual receptor expression between the study groups. Because acute infections strongly affected receptor expression in both neutrophils and monocytes, to avoid diagnostic bias, children with acute infections were excluded from the analysis. The combination of the receptors CR1 in neutrophils and Fc gamma RI and Fc gamma RII in monocytes was the best indicator of atopic dermatitis. A significant correlation between the expression of CR1 in neutrophils and in monocytes, as well as reactive oxygen species production of phagocytes, and the severity of the eczema was detected. CONCLUSIONS These results suggest that a distinct receptor profile of phagocytic cells can be characterized in patients with atopic dermatitis, providing a new direction to the search for early identification of children predisposed to allergic sensitization.

Measurement of complement receptor 1 on neutrophils in bacterial and viral pneumonia.

BackgroundA reliable prediction of the causative agent of community-acquired pneumonia (CAP) is not possible based on clinical features. Our aim was to test, whether the measurement of the expression of complement receptors or Fcγ receptors on neutrophils and monocytes would be a useful preliminary test to differentiate between bacterial and viral pneumonia.MethodsSixty-eight patients with CAP were studied prospectively. Thirteen patients had pneumococcal pneumonia; 13 patients, influenza A pneumonia; 5 patients, atypical pneumonia, and 37 patients, aetiologically undefined pneumonia. Leukocyte receptor expression was measured within 2 days of hospital admission.ResultsThe mean expression of complement receptor 1 (CR1) on neutrophils was significantly higher in the patients with pneumococcal pneumonia than in those with influenza A pneumonia. The mean expression of CR1 was also significantly higher in aetiologically undefined pneumonia than in influenza A pneumonia, but there was no difference between pneumococcal and undefined pneumonia.ConclusionOur results suggest that the expression of CR1 is higher in classical bacterial pneumonia than in viral pneumonia. Determination of the expression of CR1 may be of value as an additional rapid tool in the aetiological diagnosis, bacterial or viral infection, of CAP. These results are preliminary and more research is needed to assess the utility of this new method in the diagnostics of pneumonia.

Identification of a novel bacterial outer membrane interleukin-1Β-binding protein from Aggregatibacter actinomycetemcomitans.

Aggregatibacter actinomycetemcomitans is a gram-negative opportunistic oral pathogen. It is frequently associated with subgingival biofilms of both chronic and aggressive periodontitis, and the diseased sites of the periodontium exhibit increased levels of the proinflammatory mediator interleukin (IL)-1β. Some bacterial species can alter their physiological properties as a result of sensing IL-1β. We have recently shown that this cytokine localizes to the cytoplasm of A. actinomycetemcomitans in co-cultures with organotypic gingival mucosa. However, current knowledge about the mechanism underlying bacterial IL-1β sensing is still limited. In this study, we characterized the interaction of A. actinomycetemcomitans total membrane protein with IL-1β through electrophoretic mobility shift assays. The interacting protein, which we have designated bacterial interleukin receptor I (BilRI), was identified through mass spectrometry and was found to be Pasteurellaceae specific. Based on the results obtained using protein function prediction tools, this protein localizes to the outer membrane and contains a typical lipoprotein signal sequence. All six tested biofilm cultures of clinical A. actinomycetemcomitans strains expressed the protein according to phage display-derived antibody detection. Moreover, proteinase K treatment of whole A. actinomycetemcomitans cells eliminated BilRI forms that were outer membrane specific, as determined through immunoblotting. The protein was overexpressed in Escherichia coli in both the outer membrane-associated form and a soluble cytoplasmic form. When assessed using flow cytometry, the BilRI-overexpressing E. coli cells were observed to bind 2.5 times more biotinylated-IL-1β than the control cells, as detected with avidin-FITC. Overexpression of BilRI did not cause binding of a biotinylated negative control protein. In a microplate assay, soluble BilRI bound to IL-1β, but this binding was not specific, as a control protein for IL-1β also interacted with BilRI. Our findings suggest that A. actinomycetemcomitans expresses an IL-1β-binding surface-exposed lipoprotein that may be part of the bacterial IL-1β-sensing system.