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This perspective presents a viewpoint on potential methods assessing toxicity of indoor air. Until recently, the major techniques to document moldy environment have been microbial isolation using conventional culture techniques for fungi and bacteria as well as in some instances polymerase chain reaction to detect microbial genetic components. However, it has become increasingly evident that bacterial and fungal toxins, their metabolic products, and volatile organic substances emitted from corrupted constructions are the major health risks. Here, we illustrate how phagocytes, especially neutrophils can be used as a toxicological probe. Neutrophils can be used either in vitro as probe cells, directly exposed to the toxic agent studied, or they can act as in vivo indicators of the whole biological system exposed to the agent. There are two convenient methods assessing the responses, one is to measure chemiluminescence emission from activated phagocytes and the other is to measure quantitatively by flow cytometry the expression of complement and immunoglobulin receptors on the phagocyte surface.

In the presence of hydrogen peroxide, the heme protein lactoperoxidase is able to oxidize thiocyanate and iodide to hypothiocyanite, reactive iodine species, and the inter(pseudo)halogen cyanogen iodide. The killing efficiency of these oxidants and of the lactoperoxidase-H 2 O 2 -SCN - /I - system was investigated on the bioluminescent Escherichia coli K12 strain that allows time-resolved determination of cell viability. Among the tested oxidants, cyanogen iodide was most efficient in killing E. coli, followed by reactive iodine species and hypothiocyanite. Thereby, the killing activity of the LPO-H 2 O 2 -SCN - /I - system was greatly enhanced in comparison to the sole application of iodide when I - was applied in two- to twenty-fold excess over SCN - . Further evidence for the contribution of cyanogen iodide in killing of E. coli was obtained by applying methionine. This amino acid disturbed the killing of E. coli mediated by reactive iodine species (partial inhibition) and cyanogen iodide (total inhibition), but not by hypothiocyanite. Changes in luminescence of E. coli cells correlate with measurements of colony forming units after incubation of cells with the LPO-H 2 O 2 -SCN - /I - system or with cyanogen iodide. Taken together, these results are important for the future optimization of the use of lactoperoxidase in biotechnological applications.

Fish immune systems must be able to cope with pathogens over a wide temperature range. Earlier research suggest that fish are more dependent on innate immune responses based on pattern recognition than acquired functions with specific recognition. If this applies to phagocytes, then opsonins (serum factors that augment phagocytosis e.g. immunoglobulins and complement proteins) attached on zymosan (Z) particles should be recognized better at higher temperatures than Z only. Z is recognized by glucan receptor representing pattern recognition. In this study perch were acclimated to 5 °C or 16 °C for 3-5 weeks. The recognition and activation of respiratory burst reaction of peripheral blood phagocytes was examined at seven different measurement temperatures (5, 10, 16, 20, 24 27, and 30 °C) when the cells were stimulated with Z and serum opsonized zymosan (OZ). Respiratory burst was measured as luminol chemiluminescence (CL) from diluted whole blood. OZ-induced CL per volume of blood was on average approximately 4.6 times higher in 16 °C acclimated fish than 5 °C acclimated perch (P<0.0001). Z-induced CL was approximately 3 times higher at lower temperatures in 16 °C acclimated perch than 5 °C acclimated fish and 6-9 times higher at 27 °C and 30 °C (P<0.001), respectively. CL reaction kinetics were faster in perch acclimated to 5 °C than 16 °C -acclimated fish, especially at low temperatures (P<0.001). Thermal acclimation caused a 3-4 °C shift in temperature response curves of CL towards the acclimation temperature (P<0.0001 and P<0.053 in Z and OZ-induced CL, respectively). Serum opsonins activated perch phagocytes substantially better at higher temperatures in both acclimation groups, which is consistent with an earlier study in rainbow trout (O. mykiss). However, opsonin recognition was significantly better in 16 °C acclimated perch than 5 °C acclimated fish, which was seen as higher CLs for OZ compared to Z, especially at higher temperatures. This is opposite to previously reported results in rainbow trout. Differences between rainbow trout and perch in opsonin recognition by blood phagocytes suggest that the living habits of perch, which prefers approximately a 10 °C higher temperature than rainbow trout, may be reflected in immune cell functions. Results of the present examination suggest that also in fish phagocytes pattern recognition is the prevailing system at low temperatures, and specific recognition is more effective at high temperatures., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

Moniliformin is a Fusarium mycotoxin mainly produced by several species infecting grains in different climatic conditions. According to our previous studies, it is acutely toxic to rats, with an LD50 cut-off value of 25mg/kg b.w. To further assess the possible health risks of low dose exposure to moniliformin, a subacute oral toxicity study was conducted in Sprague-Dawley rats, adapting OECD guideline 407. Five dose groups and two satellite groups, each consisting of five male rats, were daily exposed to moniliformin by gavage. Two rats in the highest dose group, showed decreased activity followed by acute heart failure and death. The rats of the lower doses (<9mg/kg b.w.) showed no signs of toxicity. The daily intake of moniliformin strongly reduced the phagocytic activity of neutrophils in all dose groups. The decrease continued in the satellite group during the follow-up period, indicating a severe impact on the immune system and a LOAEL value of 3mg/kg b.w. for moniliformin. Moniliformin was rapidly excreted into urine, ranging between 20.2 and 31.5% daily and showed no signs of accumulation. The concentration of moniliformin in faeces was less than 2%, which suggests efficient absorption from the gastrointestinal tract., (Copyright © 2014 Elsevier Ireland Ltd. All rights reserved.)

The aim of this study was to develop a rapid and simple flow cytometric bacterial infection marker. In this prospective comparative study, quantitative flow cytometric analysis of CD10, CD35, CD66b, CD282, and MHC Class I molecules on human neutrophils, monocytes, and B-lymphocytes from 141 hospitalized febrile patients with suspected infection and from 50 healthy controls was performed. We developed a flow cytometric marker of local and systemic bacterial infections, designated "bacterial infection (BI)-INDEX", incorporating the quantitative analysis of CD10, CD35, MHCI, CD66b, and CD282 on neutrophils, monocytes, and B-lymphocytes, which displayed 90% sensitivity and 96% specificity in distinguishing between microbiologically confirmed bacterial (n = 31) and viral infections (n = 27) within a 1-h time-frame. We propose that our novel rapid BI-INDEX test will be useful in assisting physicians to ascertain whether antibiotic treatment is required, thus limiting unnecessary antimicrobial usage., (Copyright © 2014 Elsevier Inc. All rights reserved.)

Photorhabdus luminescens luxCDABE genes were integrated into E. coli K-12 using a high copy number plasmid containing modified luxABCDE genes under the control of the powerful Lac promoter. This strain emitted 10 times higher bioluminescence (BL) than P. luminescens. BL production under different growth conditions was studied. In both bacterial strains, the increase in BL signal correlated with the increase in optical density (OD) in a rich growth medium. However, at the logarithmic growth phase, the BL signal was roughly constant. By contrast, in minimal growth media, there was no substantial growth and the BL/cell was approximately five times higher than in the rich medium. The dynamic measurement range of BL was 10(2) -10(7) colony-forming units (CFU) in E. coli and 10(3) -10(7)  CFU in P. luminescens. Because the decrease in the BL signal correlated with the decrease in CFU and OD, i.e. the number of bacterial cells killed, it proved to be very suitable for assessing the antibacterial effects of different antimicrobial agents. Unlike with plate counting, the kinetics of killing can be monitored on a real-time basis using BL measurements. Complement activities in different samples can be estimated using only one serum dilution. The transformed E. coli strain appeared to be superior to P. luminescens in these applications because E. coli was complement sensitive, the detection limit of E. coli was one order lower and the BL-producing system of P. luminescens appeared to be quite unstable., (Copyright © 2012 John Wiley & Sons, Ltd.)

Individually tagged rainbow trout representing 15 full-sibling families were sequentially challenged twice with Aeromonas salmonicida causing furunculosis: first as cohabitation and then as injected intraperitoneally. The bleeding procedure prior to challenges caused the outbreak of cold water disease by Flavobacterium psychrophilum. Before and after the outbreak and challenges, 11 immunological parameters were measured from blood samples. The immunological responses predicted the fate of the fish since nearly all the initial responses were lower in individuals which later died from cold water disease than in survivors. Fish died from furunculosis had impaired respiratory burst (RB) response to A. salmonicida. Fish that had initially the highest responses survived in the outbreak and challenges. The outbreak and challenges resulted in these individuals higher and faster responses compared with initial values. Unlike in mammals, the number of monocytes, but not that of granulocytes, in rainbow trout blood correlated well with the whole blood RB activity. The fish families differed markedly from each other in capacity to resist the induced diseases., (Copyright © 2013 Elsevier B.V. All rights reserved.)

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.)

The acute toxicity of organic tin compounds (OTCs) has been studied in detail. However, due to their complex nature, very little is known about species-specific methods of accumulation and consequences for food-webs. Chironomids, on which e.g. Daubenton's bats feed, may act as vectors for the transport of organic tin compounds from aquatic to terrestrial ecosystems. Bats are prone to environmental toxins because of their longevity and their ecological role as top predators. Organic tin compounds are associated with increased formation of reactive oxygen species and associated oxidative damage as well as suppression of immune function. The present paper investigates whether the OTC, tributyltin (TBT) and its metabolite, dibutyltin (DBT), accumulate in natural populations of Daubenton's bats and whether TBT-associated effects are seen in general body condition, redox balance, redox enzyme activities, associated oxidative damage of red blood cells and complement function. We discovered the concentration of bat fur DBT correlated with local marine sediment TBT concentrations. However, we did not find a correlation between the explanatory factors, bat fur DBT and marine sediment TBT concentrations, and several physiological and physical response variables apart from complement activity. Higher DBT concentrations resulted in weaker complement activity and thus a weaker immune response. Although the observed physiological effects in the present study were not strongly correlated to butyltin concentrations in fur or sediment, the result is unique for natural populations so far and raises interesting questions for future ecotoxicological studies., (Copyright © 2013 Elsevier Inc. All rights reserved.)

Antibiotic resistance due to the inappropriate use of antimicrobials is one of the most critical public health problems worldwide. A major factor underlying the unnecessary use of antibiotics is the lack of rapid and accurate diagnostic tests. Therefore, we aimed to develop a novel rapid flow cytometric method for distinguishing between febrile bacterial and viral infections. In this prospective comparative study, quantitative flow cytometric analysis of FcγRII/CD32, CR1/CD35, MHC Class I receptor (MHCI), and C5aR/CD88 on human phagocytes was performed in 286 hospitalized febrile patients with suspected infection. After using microbiological and serological detection methods, or clinical diagnosis, 205 patients were identified with either bacterial (n=136) or viral (n=69) infection. Receptor data from patients were compared to those of 50 healthy controls. We developed a flow cytometric marker of local and systemic bacterial infections designated "bacterial infection score (BIS)" incorporating the quantitative analysis of FcγRII/CD32, CR1/CD35, C5aR/CD88 and MHCI on neutrophils and/or monocytes, which displays 91% sensitivity and 92% specificity in distinguishing between microbiologically confirmed bacterial (n=77) and serologically confirmed viral infections (n=61) within 1h. The BIS method was effectively applied to distinguish between bacterial and viral (pandemic H1N1 influenza) pneumonia cases with 96% sensitivity and 92% specificity. We propose that the rapid BIS test can assist physicians in deciding whether antibiotic treatment is necessary, thus reducing unnecessary antimicrobial use., (Copyright © 2012 Elsevier B.V. All rights reserved.)

Treating viral illnesses or noninfective causes of inflammation with antibiotics is ineffective and contributes to the development of antibiotic resistance, toxicity, and allergic reactions, leading to increasing medical costs. A major factor behind unnecessary use of antibiotics is, of course, incorrect diagnosis. For this reason, timely and accurate information on whether the infection is bacterial in origin would be highly beneficial. In this paper we will present our recent studies on the expression of opsonin receptors on phagocytes. The analysis of the expression levels of FcγRI, CR1, and CR3, along with CRP and ESR data, provides a novel application to the diagnosis of infectious and inflammatory diseases. The best clinical benefit will be obtained when the individual variables are combined to generate the CIS point method for a bacterial infection marker, DNAVS point for differentiating between DNA and RNA virus infections, and CRP/CD11b ratio for a marker of Gram-positive sepsis.

A recombinant Escherichia coli K-12 strain, transformed with a modified bacterial luciferase gene (luxABCDE) from Photorhabdus luminescens, was constructed in order to monitor the activity of various antimicrobial agents on a real-time basis. This E. coli-lux emitted, without any addition of substrate, constitutive bioluminescence (BL), which correlated to the number of viable bacterial cells. The decrease in BL signal correlated to the number of killed bacterial cells. Antimicrobial activity of hydrogen peroxide (H(2)O(2)) and myeloperoxidase (MPO) was assessed. In high concentrations, H(2)O(2) alone had a bacteriocidic function and MPO enhanced this killing by forming hypochlorous acid (HOCl). Taurine, the known HOCl scavenger, blocked the killing by MPO. When E. coli-lux was incubated with neutrophils, similar killing kinetics was recorded as in H(2)O(2)/MPO experiments. The opsonization of bacteria enhanced the killing, and the maximum rate of the MPO release from lysosomes coincided with the onset of the killing.

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.

The interaction between bacterial cells and activated complement components as a kinetic biological event is described. The bacteriolytic activity of complement in human and fish serum was assayed by measuring the decrease of bioluminescence of Escherichia coli transformed with lux genes. From the kinetic curves, the bacteriolytic CB(50)- and AB(50)-units were derived at any desired time point. It was observed that these values were irregular but decreased as a function of incubation time, and reached equal values during prolonged incubation, suggesting that the difference between the classical and alternative pathway activity is kinetic. From the kinetic curves, entirely new parameters could be derived: rate of the activation phase, rate of killing by the lytic phase and rate of killing by the entire pathway in undiluted serum. The rates of human and fish classical pathway were about five and two times higher than those of the alternative pathway respectively.

The aim of this study was to compare degranulation of easily mobilizable secretory vesicles (SVs) or secretory vesicle-like granules (SVLGs) in neutrophils, monocytes, and eosinophils of healthy controls (n = 60) and febrile patients with microbiologically confirmed or clinically diagnosed bacterial (n = 89) and viral (n = 46) infections. For this purpose, flow cytometric immunophenotyping of isolated phagocytes was performed using monoclonal antibodies against the phagocytosis receptors CR1 (CD35) and CR3 (CD11b) that are predominantly stored in the SVs of resting neutrophils. Similar to neutrophils, monocytes contain easily mobilizable SVLGs that constitute the main intracellular reservoir of CD35 and CD11b. In both neutrophils and monocytes, activation mechanisms leading to degranulation of SV and SVLG appeared dependent on both intra- and extracellular calcium levels. The kinetics of degranulation of SVLGs in control monocytes was significantly faster than that of SVs of control neutrophils. We conclude that phagocytes in patients with bacterial infections can be arranged in order of decreasing magnitude of SV or SVLG degranulation as follows (from left to right): neutrophils > monocytes " eosinophils. However, in viral infections, the corresponding degranulation order is monocytes > neutrophils approximately eosinophils.

To commence proper antibiotic treatment in sepsis, timely knowledge of whether the cause of systemic infection is gram-negative (gram(-)) or gram-positive (gram(+)) bacteria in origin would be beneficial for clinicians. In this clinical prospective study, our objective was to develop a method for distinguishing between gram(+) and gram(-) bacterial infection. In gram(-) bacterial infection (n = 21), the average amount of CD11b on neutrophils was significantly higher than in gram(+) bacterial infection (n = 22). On the contrary, serum C-reactive protein (CRP) level was significantly higher in gram(+) than in gram(-) bacterial infection. By dividing the serum CRP value by the amount of CD11b on neutrophils, we derived a novel marker of gram(+) sepsis, CRP/CD11b ratio, which displayed 76% sensitivity and 80% specificity for the detection of gram(+) sepsis (n = 17) among febrile patients with microbiologically confirmed or clinically diagnosed bacterial infection. The detection of gram(+) sepsis is possible after the combination of neutrophil CD11b data and serum CRP level. In conclusion, our findings indicate that the proposed CRP/CD11b ratio test could potentially assist physicians in determining an appropriate antibiotic treatment in patients with severe bacterial infection.

Background: To commence proper antiviral treatment, timely knowledge of whether the infection is caused by DNA or RNA virus would be beneficial for the clinician., Objectives: Our objective was to develop a method for distinguishing between DNA and RNA virus infections., Study Design: In this prospective study, total and differential count of leukocytes, serum C-reactive protein level, erythrocyte sedimentation rate, and quantitative flow cytometric analysis of FcgammaRI (CD64) on neutrophils and monocytes were obtained from 289 hospitalized febrile patients. After microbiological confirmation, 89 patients (31%) were found to have either bacterial (n=46) or viral (n=43) infection. The patient data was compared to 60 healthy controls., Results: For the first time ever, it was noticed that in dsDNA virus infections (n=21) the average amount of CD64 on neutrophils was over five-fold compared to ssRNA virus infections (n=22)., Conclusions: DNA virus score (DNAVS) point, which incorporates quantitative analysis of CD64 on neutrophils and total and differential count of leukocytes, varied between 0 and 8, and displayed 95% sensitivity and 100% specificity in distinguishing between dsDNA and ssRNA virus infections [average (S.D.); DNAVS points: 5.4 (2.5) vs. 0.3 (0.4); p<0.001].

Background: Past sun exposure and vitamin D3 supplementation have been associated with a reduced risk of multiple sclerosis (MS). There are no previous longitudinal studies of vitamin D in MS., Objectives: To compare regulation of vitamin D and calcium homeostasis between patients with MS and healthy controls. To study the correlation of parameters of vitamin D metabolism with MS activity., Methods: We measured 25-hydroxyvitamin D (25(OH)D), parathyroid hormone (PTH), calcium, phosphate, magnesium, chloride, alkaline phosphatase, albumin and thyroid stimulating hormone in serum every 3 months and at the time of relapse over 1 year in 23 patients with MS and in 23 healthy controls. MRI burden of disease and T2 activity were assessed every 6 months., Results: Vitamin D deficiency (S-25(OH)D < or = 37 nmol/l) was common, affecting half of the patients and controls at some time in the year. Seasonal variation of 25(OH)D was similar in patients and controls, but 25(OH)D serum levels were lower and intact PTH (iPTH) serum levels were higher during MS relapses than in remission. All 21 relapses during the study occurred at serum iPTH levels > 20 ng/l (2.2 pmol/l), whereas 38% of patients in remission had iPTH levels < or = 20 ng/l. Patients with MS had a relative hypocalcaemia and a blunted PTH response in the winter. There was no correlation between serum 25(OH)D and MRI parameters., Conclusions: The endocrine circuitry regulating serum calcium may be altered in MS. There is an inverse relationship between serum vitamin D level and MS clinical activity. The role of vitamin D in MS must be explored further.

We performed simultaneous quantitative flow cytometric analysis of neutrophil and monocyte FcgammaRI (CD64) in 289 hospitalized febrile patients. Microbiological evaluation or clinical diagnosis confirmed bacterial (n=89) or viral (n=46) infection in 135 patients. Patient data were compared with data from 60 healthy controls. The average number of FcgammaRI on the surfaces of both neutrophils and monocytes was significantly increased in patients with febrile viral and bacterial infections, compared to healthy controls. Furthermore, we describe a novel marker of febrile infection, designated 'CD64 score point', which incorporates the quantitative analysis of FcgammaRI expressed on both neutrophils and monocytes, with 94% sensitivity and 98% specificity in distinguishing between febrile infections and healthy controls. By contrast, analysis of FcgammaRI expression on neutrophils and monocytes displayed poor sensitivity (73% and 52%) and specificity (65% and 52%) in distinguishing between bacterial and viral infections, and the levels did not differ significantly between systemic (sepsis), local, and clinically diagnosed bacterial infections. In summary, our results clearly show that the increased number of FcgammaRI on neutrophils and monocytes is a useful marker of febrile infection, but cannot be applied for differential diagnosis between bacterial and viral infections or between systemic and local bacterial infections.

Gram-negative bacteria are resistant to many hydrophobic antibiotics (such as penicillin G) owing to the highly hydrophilic saccharide part of lipopolysaccharide in the cell membrane, whilst most hydrophilic antibiotics (such as ampicillin) are more freely diffused into cells through aqueous porins. In this study, we investigated the possibility of altering the membrane permeability of Escherichia coli with tricyclic cationic compounds, such as the non-antibiotic drug promethazine. We also established the activity of these compounds as modifiers of antibiotic resistance in bacteria by a fluoro-luminometric approach. According to the results, promethazine has no bacteriostatic effect on E. coli at concentrations <64 microg/mL. However, promethazine at these concentrations in combination with penicillin G produced a significant synergistic activity against E. coli. Specifically, a constant promethazine concentration of 32 microg/mL in combination with penicillin G concentrations of 16-128 microg/mL suppressed the growth and viability of bacteria and converted penicillin G-resistant cells to being susceptible to this antibiotic with a minimum inhibitory concentration of 128 microg/mL. In contrast to penicillin G, the efficacy of ampicillin was apparently not increased in the presence of promethazine, suggesting that promethazine directly affects the membrane permeability of bacteria or alternatively inhibits the function of efflux pumps. In conclusion, we conclude that exposing E. coli cells to a given antibiotic in combination with promethazine can increase the susceptibility of bacteria; this effect is reliably assessed on a real-time basis using kinetic fluoro-luminometric measurements.

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 goal of fish vaccination today is to protect fish against multiple bacterial fish pathogens simultaneously using polyvalent vaccines. However, many immunological processes such as antigenic cross-reaction, antigenic competition, affinity maturation and antigen-induced suppression may affect the specificity, avidity and level of antibodies. Consequently, the biological function of antibodies may be markedly different from that predicted by conventional serologic tests. Here, we investigated the effects of vaccination and composition of vaccine on the plasma antibody levels, biological function of antibodies in opsonophagocytosis as well as the effects of vaccination on the blood leucocyte counts. Rainbow trout were vaccinated with saline or with two different polyvalent, mineral oil-adjuvanted vaccines. Vaccine 1 contained Aeromonas salmonicida, Listonella anguillarum and both Th and Fd serotypes of Flavobacterium psychrophilum antigens and vaccine 2 contained A. salmonicida, L. anguillarum and only Fd serotype of Fl. psychrophilum. The antibody-mediated opsonophagocytosis was determined as the respiratory burst (RB) activity of blood monocytes and granulocytes against the tested bacterial antigens. Three weeks after vaccination both vaccine groups and the control group showed increased RB activity against all bacterial strains. However, the increase in RB activities was non-specific and originated from the increased number of circulating granulocytes and monocytes. On the other hand, at 6 weeks post-vaccination both specific antibodies and antibody-dependent opsonophagocytosis appeared in both vaccine groups. However, the composition of the vaccine had a marked effect on the magnitude of specific responses. The Fd+Th vaccine enhanced the target specific opsonophagocytosis, to a lesser extent than the Fd vaccine. Both polyvalent vaccines appeared to mainly affect the numbers of circulating monocytes and our results suggest that the monocytes play a more significant role than the granulocytes in antibody-dependent opsonophagocytosis. Our results also suggest that the presented opsonophagocytic assay is an advantageous method to predict vaccine efficiency and that the number, and properties, of bacterial antigens in polyvalent vaccines should be carefully selected in order to avoid inhibitory effects of antigens on the specific response of fish.

Kinetic measurements of the bacteriostatic, bactericidal and bacteriolytic activities of six model antibiotics (ampicillin, erythromycin, nalidixic acid, polymyxin B, tetracycline, and trimethoprim) against Escherichia coli as target bacteria were performed by bioluminescence, fluorescence, and optical density based real-time assay. Additionally, plate counting was used as a control measurement. The gfp and insect luciferase (lucFF) genes were cloned into cells used for measurements to enable fluoro-luminometric detection. Bacteria were exposed to antibiotics for 10 h, and the effects of antimicrobial agents were established. Inhibitory concentration of 50% (IC(50)), minimum bactericidal concentration (MBC), and bactericidal concentration of 50% (BC(50)) of each antibiotic were calculated from these procedures. Bacteriostatic, bactericidal or bacteriolytic actions of each antibiotic, as well the time interval from exposure to visible effect, were readily observed from kinetic data. No significant differences were observed between data obtained with the different methods employed. Ampicillin and polymyxin B were clearly bacteriolytic, nalidixic acid and tetracycline showed bactericidal effects, and erythromycin and trimethoprim were bacteriostatic drugs. The assay has the advantage of speed and accurately discerns between lytic, cidal and static compounds. Thus, this reliable and fully automated novel kinetic assay with high sample capacity offers new possibilities for real-time detection, making it suitable for diverse high throughput screening (HTS) applications.

There is an ongoing need for sensitive and specific markers of bacterial infection. In this prospective study, standard clinical laboratory data (neutrophil count, serum C reactive protein level, erythrocyte sedimentation rate) and quantitative flow cytometric analysis of neutrophil complement receptors, CR1 and CR3, were obtained from 289 hospitalized febrile patients. After microbiological confirmation or clinical diagnosis, 135 patients were found to have either bacterial (n = 89) or viral (n = 46) infection. The patient data was compared to 60 healthy controls. In bacterial infections, all measured variables were significantly increased, particularly the average amounts of CR1 and CR3 on neutrophils were over three-fold and two-fold higher, respectively, compared to viral infections and controls. We described 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. CIS point varied between 0 and 8, and displayed 98% sensitivity and 97% specificity in distinguishing between bacterial and viral infections [average (S.D.); CIS points: 6.2 (1.7) vs. 0.6 (1.0); p < 0.001]. These findings suggest that the proposed CIS-based diagnostic test could potentially assist physicians in deciding whether antibiotic treatment is necessary.

In the present study we investigated the respiratory burst (RB) activity of Atlantic cod (Gadus morhua L.) blood phagocytes and we evaluated how the RB activity of cod blood cells differ from that of trout. The RB activities were measured directly from highly diluted whole blood as luminol-amplified chemiluminescence (CL) under various conditions. Studies regarding the blood dilutions for cod whole blood chemiluminescence measurements (WBCL) revealed that at a final blood dilution of 1.5 microl ml(-1) or less the CL response was strictly proportional to the number of phagocytes. This range of blood dilution did not markedly differ from that of trout. However, the opsonisation capacity of cod plasma was markedly poorer. The RB activity of phagocytes was most active at 15 degrees C when heterologous cod serum was used as a source of opsonin, whereas at final blood dilution of 8.0 microl ml(-1) (when homologous cod plasma was at a higher concentration) the highest RB activity was observed at 10 degrees C. Aeromonas salmonicida strain MT004 (As MT004) induced higher RB activity than the two known pathogens for cod, atypical A. salmonicida and Vibrio anguillarum. Cod blood phagocytes were more responsive to plastic surfaces and the adhesion response of phagocytes was partly inhibited but did not totally vanish even at a final gelatin concentration of 0.4%. Moreover, cod serum enhanced the adherence of phagocytes and cod blood phagocytes also showed slow spontaneous degranulation. Finally, within the tested anticoagulants (heparin, Na-citrate, EDTA) heparin treated blood phagocytes generated the highest RB activity.

Luminol-amplified chemiluminescence (CL) from phagocytes has previously been shown to be almost completely dependent on the release of myeloperoxidase (MPO) from azurophilic granules. We measured the luminol-amplified chemiluminescence response (WBCL) by using serially diluted whole blood. In these experiments, non-opsonized and serum-opsonized zymosan (NWBCL and OWBCL, respectively) were used concurrently as phagocytosable particles. We found two whole-blood dilution ranges with clinical significance: first, <0.04% of whole blood in the reaction volume, where MPO released by the zymosan-activated leukocyte population came almost totally from neutrophils and the OWBCL response could be exploited as a measure of a neutrophil count in a given blood specimen, despite the pathophysiological state of the host. In contrast, the NWBCL response was two-fold in blood samples from bacterial infection patients compared to those of controls and patients with viral infection, suggesting the use of NWBCL for the differential diagnosis of bacterial infections from viral infections; second, 0.16-1.2% of whole blood in the reaction volume, where the opsonization capacity of plasma (OC(50)) can be determined. We also found that at whole blood content >0.04%, erythrocytes quickly start to absorb chemiluminescence light, and that at whole blood content >1.2%, plasma proteins, most probably albumin and fibrinogen, start to inhibit MPO release., (Copyright (c) 2006 John Wiley & Sons, Ltd.)

Background: A 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 Fcgamma receptors on neutrophils and monocytes would be a useful preliminary test to differentiate between bacterial and viral pneumonia., Methods: Sixty-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., Results: The 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., Conclusion: Our 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.

A rapid and simple method is presented for determining antibody activity following vaccination, directly from diluted fish blood. The proposed method evaluates the effects of specific antibodies on ingestion by blood phagocytes, and may be used for measuring antibody levels following vaccination. The enhancing effect of trout IgM on ingestion was measured by luminol-amplified chemiluminescence (CL) emission of blood phagocytes. Respiratory burst (RB) activity of blood phagocytes was induced with the strain MT004 of bacterial fish pathogen Aeromonas salmonicida. To determine the boosting level of specific IgM on ingestion, various volumes of purified trout IgM containing specific antibodies against A. salmonicida were added to blood samples collected from non-vaccinated fish, and the RB activity of blood phagocytes was measured. The presence of antibodies in plasma of artificially prepared immune blood (AIB) was confirmed using enzyme-linked immunosorbent assay (ELISA). At a final blood dilution of 1:250, the mean RB activity of blood samples boosted with IgM was more than seven times higher, compared to other tested blood dilutions boosted with equal amount of IgM. Accordingly, a dilution of 1:250 was employed in the field study of vaccinated and non-vaccinated fish. The levels of A. salmonicida-specific antibodies in plasma samples of vaccinated and non-vaccinated fish were additionally confirmed with the ELISA assay. Based on these results, it is proposed that the biological activity of elicited antibodies can be assessed directly from diluted fish blood, using homologous blood neutrophils as immune sensors.

The authors measured serum C-reactive protein (CRP) serially in patients with multiple sclerosis (MS) who participated the PRISMS study using a high-sensitivity technique. CRP values were similar in patients with MS and in healthy controls but higher during MS relapses than in remission (p = 0.010). CRP levels were lower during treatment with high-dose interferon beta 1a than placebo (p = 0.035) and higher during first 12 months of study in patients who progressed by year 4 compared with stable patients (p = 0.007).

Background: 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., Methods: FITC-labeled zymosan was incubated with leukocyte suspension, and at selected time intervals fluorescence positive neutrophils were divided by phagocytotic gates into three subpopulations: neutrophils that were neither binding nor ingesting particles, neutrophils that were only binding particles (binding cells), and neutrophils that were binding and ingesting particles (ingesting cells). For the distinction between internalized and surface-bound FITC-labeled zymosan, trypan blue (1.2 mg/ml) was used to quench surface-bound fluorescence., Results: The technical challenges related to settings of phagocytotic gates and derivation of phagocytotic equations were presented. From 28 control samples, numerical values of mean fluorescence intensities and percentages of phagocytotic subpopulations inside phagocytotic gates before and after quenching were inserted into phagocytotic equations and corrected phagocytotic parameters were calculated. Calculated parameters were surprisingly constant across individuals., Conclusions: Essential elements of the present method appeared to be partial quenching of extracellular fluorescence with trypan blue and distinguishing between overlapping populations of binding and ingesting cells. Corrections using derived phagocytotic equations proved necessary for accurate kinetic phagocytotic measurements. Corrections were less necessary when the ingestion process was finished., (Copyright (c) 2005 Wiley-Liss, Inc.)

The phagocytes of fish play an important role in innate host defense against bacterial infection, and participate in various immunoregulatory processes. Here, we investigated the effects of various opsonins in the ingestion and adhesion processes by examining respiratory burst (RB) activity in blood and head kidney (HK) fish phagocytes. RB activity was induced in rainbow trout phagocytes with the bacterium Aeromonas salmonicida (strain MT004) in the presence of various opsonins [purified antibodies (Ab), immune serum (IS), normal serum (NS) and heat-inactivated immune serum (HI-IS)], and measured in terms of luminol-amplified chemiluminescence (CL) emission at 20 degrees C for 210 min. The RB activity of blood phagocytes was measured directly from highly diluted whole blood and compared to that observed in isolated head kidney (HK) phagocytes measured under similar conditions. In addition, the extracellular RB activity of adhesion (extracellular degranulation) and the intracellular RB activity of ingestion were distinguished through their inhibition by gelatin and cytochalasin D. Our results showed that the first CL peak appeared within 50 min, and decreased or vanished when gelatin was added to the reaction or when the active complement was destroyed by heating. The second CL peak appeared after 50 min, depending on the utilized opsonin, and vanished when cytochalasin D was added to the reaction. Our results indicate that adhesion and ingestion compete for consumption of reactive oxygen intermediates. Specific IgM without an active complement was a relatively inefficient opsonin, whereas specific IgM with an active complement increased the magnitude of ingestion-mediated RB activity and accelerated the ingestion of target bacteria. Taken together, these results indicate that adhesion and ingestion responses competed for limited phagocyte resources and that the bacterial uptake by blood phagocytes can be measured directly from highly diluted blood.

Objective: To examine blood neutrophil counts and luminol-enhanced chemiluminescence (CL) responses in dogs undergoing ovariohysterectomy (OH), premedicated with 2 different drugs., Study Design: Randomized clinical study., Animals: Forty-two healthy client-owned bitches., Methods: Dogs had OH under isoflurane anesthesia with either acepromazine or medetomidine, both in combination with butorphanol, administered as preanesthetic medication. Blood samples were collected when the dog was admitted, at the end of surgery, and the next day (approximately 20 hours after surgery). Blood neutrophils were counted automatically, and neutrophil oxidative activity was assessed by measuring blood CL responses (induced by opsonized zymosan and enhanced by luminol) at 37 degrees C for 40 minutes., Results: Number of circulating neutrophils was significantly increased the day after surgery reflected by enhanced blood CL responses. Neutrophil CL, however, was not significantly altered. No significant differences were detected for perioperative Polymorphonuclear neutrophil (PMN) characteristics between the 2 preanesthetic regimens., Conclusions: In conclusion, despite clearly increasing the number of circulating neutrophils, OH did not significantly affect neutrophil respiratory burst, as measured by whole-blood CL responses., Clinical Relevance: Surgical operation of moderate intensity (e.g., OH) did not significantly alter one of the important immune functions, neutrophil oxidative activity. Further studies are warranted to confirm the significance of this finding, and to assess the value of following this variable in different animal patient populations.

Background: 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., Methods: Recombinant Escherichia coli cells with a plasmid containing the gene for jellyfish GFP were stained with PI, and green and red fluorescence were measured by FCM. For comparison, cells containing the plasmid from which gfp was removed were stained with SYTO9 and PI, and analyzed by FCM. Viability was estimated according to the proportion of green and red fluorescence. In addition, bioluminescence and plate counting (other methods to assess viability) were used as reference procedures., Results: SYTO9-PI dual staining of bacterial cells revealed three different cell populations: living, compromised, and dead cells. These cell populations were more distinct when the GFP-PI combination was used instead of dual staining. No differences in sensitivity were observed between the two methods. However, substitution of SYTO9 with GFP accelerated the procedure. Bioluminescence and plate counting results were in agreement with flow cytometric viability data., Conclusions: In bacterial viability analyses, the GFP-PI combination provided better distinction between current viability stages of E. coli cells than SYTO9-PI dual staining. Additionally, the overall procedure was more rapid. No marked differences in sensitivity were observed.

Phagocytosis, complement lytic activity and opsonization capacity of rainbow trout plasma as well as the ability of phagocytes to recognize foreign particles were studied at different temperatures. Respiratory burst (RB) activity and opsonization capacity were assessed as chemiluminescence emission from diluted whole blood of fish which were acclimatized for 57 days at temperatures between 5 and 20 degrees C. RB activity was higher at higher acclimatization and in vitro assay temperatures. The peak time of RB was significantly delayed in fish kept at lower temperatures (5-10 degrees C) as compared to fish kept at 15 or 20 degrees C temperatures. Opsonization capacity of plasma decreased in fish acclimatized at low temperatures and was also affected by in vitro assay temperature. The importance of glucan receptors in RB activity increased in fish kept at higher temperatures and was also affected by the in vitro assay temperature. The higher acclimation temperatures increased the lytic activity of both total and alternative complement pathways., (Copyright 2003 Elsevier Ltd.)

The present study assessed the immune enhancement of fish by a lactic acid bacterium (LAB) Lactobacillus rhamnosus (ATCC 53103). The bacterium was administered orally at five different doses 7.9 x 10(4) (LAB4), 2.1 x 10(6) (LAB6), 2.8 x 10(8) (LAB8), 1.9 x 10(10) (LAB10) and 9.7 x 10(10) (LAB11) CFU/g feed to rainbow trout for two weeks and the feed was changed to un-supplemented diet. From the onset of feeding supplemented diets at 1, 2, 3 and 4 weeks, blood and mucus samples were taken. During the LAB feeding period L. rhamnosus persisted in the fish intestine and in the tank water in high numbers. However, L. rhamnosus disappeared from the intestine, skin mucus and tank water within one week after the change to the non-supplemented feed. In comparison to untreated control fish, respiratory burst activity of blood cells was raised significantly in the LAB4 treated group on week 2. Serum-mediated killing of Escherichia coli was increased significantly in group LAB6 on week 2. Serum immunoglobulin levels were significantly raised only in LAB8 group on week 1 and in LAB4 and LAB8 at the end of the trial. The results show that rainbow trout immune parameters were enhanced by using probiotic bacteria.

The viability and killing of Escherichia coli was measured on a real-time basis using a fluoro-luminometric device, which allows successive measurements of fluorescence and bioluminescence without user intervention. Bacteria were made fluorescent and bioluminescent by expression of gfp and insect luciferase (lucFF) genes. The green fluorescent protein (GFP) is a highly fluorescent, extremely stable protein, which accumulates in cells during growth, and therefore the measured fluorescence signal was proportional to the total number of cells. The luciferase reaction is dependent of ATP produced by living cells, so that the bioluminescence level was a direct measure of the viable cells. In contrast to the bacterial luciferase, the insect luciferase uses a water-soluble and nonvolatile substrate, which makes automated multi-well microplate assay possible. For the validation of the assay, the proportion of living and dead cell populations was experimentally modified by incubating E. coli cells in the presence of various ethanol concentrations. Bacterial viability and killing measured by a fluoro-luminometric assay correlated fairly well with the reference methods: conventional plate counting, optical density measurement and various flow cytometric analyses. The real-time assay described here allows following the changes in bacterial cultures and assessing the bactericidal and other effects of various chemical, immunological and physical agents simultaneously in large numbers of samples.

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.

Infectious viruses and bacteria can trigger multiple sclerosis (MS) exacerbations. Seasonally changing concentrations of ambient air pollutants are known to predispose to transmissible infections, to induce systemic immune responses and to enhance existing peripheral inflammation. Ambient air quality and monthly MS relapse occurrence in south-western Finland were compared by multivariate logistic regression. The odds ratio of the risk of a relapse onset was over fourfold (4.143, p < 0.001) when the concentration of inhalable particulate matter (PM(10)) was at the highest quartile. Inhalable airborne particulate matter concentrations were connected to relapse occurrence. Poor air quality may enhance the seasonal changes in MS relapse occurrence by an increased susceptibility to transmissible infections., (Copyright 2003 S. Karger AG, Basel)

The total bacteriolytic activity comprising of the classical, alternative and possible lectine pathways as well as the bacteriolytic activity of the alternative pathway (AP) of rainbow trout (Oncorhynchus mykiss) complement was assessed in temperatures ranging from 0 to 35 degrees C against a recombinant strain Escherichia coli containing two reporter genes gfp and lucFF. At 35 degrees C there was no difference between the total (TC) activity and the activity of the AP, but at 10 degrees C the TC was notably higher than the AP. Total activity peaked at 30 degrees C and gradually grew smaller towards 0 degrees C. The activity of the AP was similarly temperature-dependent, but CB50 value was found to be beyond measurable range at temperatures below 10 degrees C. When compared to human serum complement, the peak human TC activity at 37 degrees C was four times higher than the TC of rainbow trout at 30 degrees C. Human TC activity was 10.1-fold lower at 25 degrees C when compared to the activity at 37 degrees C. At 37 degrees C the human AP bacteriolytic activity was 4.5-fold less effective than human TC, but at 25 degrees C there was no difference between human TC and AP. In contrast to previous reports where AP activity of fish was assayed as hemolytic activity our study showed that the bacteriolytic activity of AP was lower than that of TC and very low at temperatures below 10 degrees C suggesting that the earlier proposed particular importance of AP in fish should be reconsidered.

Murine mAb reactive with recombinant bovine tumor necrosis factor-alpha (r-boTNF-alpha) were produced. An ELISA using murine mAb and rabbit polyclonal antibodies, each reactive with r-boTNF-alpha to sandwich bovine TNF-alpha was developed. Secretion of TNF-alpha in quarter milk increased 1 h after injection of 0.1 mg (four cows) or 0.5 mg (four cows) Escherichia coli lipopolysaccharide (LPS) into a mammary quarter, peaked 1 to 5 h later, and returned to control levels in 24 h. There were no differences in body temperature, SCC, TNF-alpha, and blood leukocyte responses between 0.1 and 0.5 mg of LPS. To determine effects of repeated injections of LPS into the same udder, a second injection of 0.1 mg of LPS into the same quarter (two cows) 24 h after the first injection produced a strongly attenuated TNF-alpha response. However, a normal TNF-alpha response was observed when LPS was injected into a contralateral quarter (two cows) 24 h after the first LPS injection. Leukocyte counts in blood decreased and body temperature increased substantially after each injection of LPS. Quarter milk SCC increased 200-fold 8 to 12 h after the LPS injections. It would appear that these changes were not regulated by TNF-alpha secretion because the changes were also similar after the second injection of LPS into the same mammary quarter.

Mammalian colostrum offers passive protection to the newborn against a variety of microbial pathogens, in the form of specific immunoglobulin A, G and M antibodies. Sharing maternal immunological memory is in many cases vital for the infant, but may have disastrous consequences, such as involuntary transfer of disease and disturbance of the developing immune system. In most published studies, immune milk preparations are reported to be effective in the prevention of various gastroenteric infections, but not in the treatment of an established infection.

Background: The mode of action of interferon involves both direct cytotoxic and antiproliferative effects on the tumour cell and indirect effects that facilitate immune detection by the host. Among the immunological effects of interferon-alpha is the activation of monocytes. As opsonin receptors are crucial in the function of phagocytes, e.g. monocytes and neutrophils, their modulation by interferon-alpha (INF-alpha) merit to be further clarified. We hypothesised that the role of phagocytes in defence against cancer is reflected in the expression of opsonin receptors for IgG and complement, which further could be modified by INF-alpha., Patients and Methods: The expression of the receptors for IgG and complement was studied in neutrophils and monocytes from blood samples of 18 kidney cancer patients treated with INF-alpha and from 39 healthy individuals. Blood samples were collected prior/to and during treatment with INF-alpha, 4.5 to 13.5 MU t.d.w., subcutaneously. After lysing the red blood cells, the samples were incubated with fluorochrome conjugated monoclonal antibodies specific for IgG (Fc gammaRI, -RII and -RIII) and complement (CR1, CR3) receptors and then analysed in flow cytometry. The results were given as the mean log fluorescence intensity (a measure of receptor number) and as the proportion of receptor-positive cells. In the in vitro experiments, the direct effect of interferon-alpha on the receptors of neutrophils and monocytes was studied., Results: In patients before any treatment, the expression of CR3 and Fc gammaRI receptors in neutrophils and all receptors except Fc gammaRIII in monocytes was significantly raised when compared to the controls. Treatment with INF-alpha, induced statistically significant; transient changes in CR1-receptor expression in neutrophils and Fc gammaRI expression in monocytes. Incubation of blood cells with INF-alpha in vitro confirmed the induction of CR1 receptors in neutrophils., Conclusion: Changes in receptor expression reflect the inflammatory activation of phagocytes in metastatic kidney cancer. The pattern of receptor expression differs from that observed in infectious diseases. Interferon-alpha both in vivo and in vitro modulates the expression of phagocytic receptors.

Six trained Standardbred trotters exercised on a racetrack on 2 days with a 3-day interval. On both exercise days the horses trotted three different exercise bouts with increasing intensity with 60-min intervals. Exercise-induced stress was manifested as leucocytosis, an increase in the neutrophil:lymphocyte (N:L) ratio, and increased capacity to produce reactive oxygen species in the peripheral blood as indicated by an increase in whole blood chemiluminescence. The leucocytosis was mainly due to neutrophilia, which lasted for 6 h. Production of reactive oxygen species per single neutrophil showed no significant change during a day of exercise, but was lower on the second exercise day. The cortisol concentrations and N:L ratio, used as indicators of stress, behaved differently: Cortisol did not change significantly after exercise, whereas the N:L ratio increased. These results suggest that in trained horses, the N:L ratio is a sensitive indicator of stress of short duration, and an attenuated N:L response can be taken as an indicator of adaptation to exercise stress.

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.

Objective: To estimate the prevalence of milk hypersensitivity in Finnish adults., Design: Cross-sectional study., Subjects: Two hundred men and 206 women aged 27 y randomly recruited from the population register in southwestern Finland., Interventions: The subjects were interviewed about their dairy product consumption, abdominal discomfort after dairy product intake and lactose intolerance. From serum samples, serum reactivity to milk protein and milk-specific IgG1, IgG2, IgG3 and IgA were measured., Results: About 20% of the subjects reported abdominal discomfort after dairy product intake, whereas only 6.4% had been diagnosed to have lactose intolerance. The amount of milk consumed correlated well with the serum assay results in subjects reporting abdominal discomfort but not in subjects who were free from these symptoms. Among subjects with no record of dairy product restriction or lactose intolerance, those experiencing abdominal discomfort after dairy product intake had significantly higher serum reactivity to milk protein than those without such discomfort. The concentrations of serum milk-specific antibodies did not differ between these two groups. The prevalence of milk hypersensitivity in this population was estimated to be 3-6%., Conclusions: Milk hypersensitivity may be as common in adults as in infants. The measurement of serum reactivity to milk protein may prove useful in screening milk hypersensitivity in subjects who have not restricted their dairy product consumption.

Factors that direct the immune responsiveness of the newborn beyond the immediate post-natal period are not known. We investigated the influence of mode of delivery and type of feeding on the phagocyte activity during the first 6 months of life. Sixty-four healthy infants (34 delivered vaginally and 30 by elective Caesarean section) were studied at birth and at the ages of 2 and 6 months. Phagocyte functions were studied by measuring the chemiluminescence (CL) activity of whole blood and isolated leucocytes and by investigating the expression of phagocyte receptors (FcgammaRI (CD64), FcgammaRII (CD32), FcgammaRIII (CD16), CR1 (CD35), CR3 (CD11b) and FcalphaR (CD89)) on neutrophils, monocytes and eosinophils by using receptor-specific MoAbs and immunofluorescence flow cytometry. Infants born by elective Caesarean section had significantly higher CL activity than those delivered vaginally during the entire 6-month follow up. In addition, infants who received formula feeds had significantly higher CL activity at 6 months of age and higher expression of FcgammaRI-, Fcalpha- and CR3-receptors on neutrophils than infants exclusively breast-fed. We suggest that stress reaction associated with labour influences the phagocytic activity measured in the cord blood but later during infancy the intraluminal antigens, gut microflora and diet, become important determinants in immune programming of human individuals.

Florfenicol, a drug effective against several bacterial diseases of fish, was tested for possible immunomodulatory effects. The aim of the study was to follow the kinetics of the immune response after vaccination with simultaneous oral antibiotic treatment. The fish were immunised with a commercial oil-based divalent (furunculosis/vibriosis) vaccine and were simultaneously given oral antibiotic treatment. The specific immune response was monitored by analysing the levels of specific antibodies with ELISA. As an indicator of the non-specific immune response the phagocytic activity of circulating leucocytes was measured by a chemiluminescence assay. Total circulating leucocyte counts and differentials were also monitored. The disease resistance was evaluated by challenge tests at the end of the experiment. The results showed that florfenicol did not have any significant effect on antibody production and circulating leucocyte levels but caused a suppression in chemiluminescence response/phagocytic cell 5-6 weeks after vaccination. The survival after challenge was slightly suppressed by the florfenicol treatment. The RPS-value for the vaccinated group was 98% and for the florfenicol-treated group was 88%.

Background: Probiotic bacteria can influence immune responses both specifically by stimulating antibody production and nonspecifically by enhancing phagocytosis of pathogens and modifying cytokine production., Objective: The authors hypothesized that probiotic bacteria can alleviate hypersensitivity by influencing phagocytes. The modulation of phagocytes may be different in healthy subjects compared with hypersensitive subjects., Subjects and Methods: In a double-blind, cross-over study, challenges with milk in milk-hypersensitive and healthy adults with or without an intestinal bacterial strain, Lactobacillus GG (ATCC 53103) were performed. The challenge-induced immunoinflammatory response was recorded by measuring the expression of phagocytosis receptors prior to and after the challenge using flow cytometry., Results: In milk-hypersensitive subjects, milk challenge increased significantly the expression of CR1, FcgammaRI and FcalphaR in neutrophils and CR1, CR3 and FcalphaR in monocytes. Milk with Lactobacillus GG prevented the increase of the receptor expression. In healthy subjects, milk challenge did not influence receptor expression while milk with Lactobacillus GG increased significantly the expression of CR1, CR3, FcgammaRIII and FcalphaR in neutrophils., Conclusion: Probiotic bacteria appear to modulate the nonspecific immune response differently in healthy and hypersensitive subjects. This is seen as an immunostimulatory effect in healthy subjects, and as a down-regulation of immunoinflammatory response in milk-hypersensitive subjects.

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 Fc gamma 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 milk-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.