Impact of cyanogen iodide in killing of Escherichia coli by the lactoperoxidase-hydrogen peroxide-(pseudo)halide system.

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-H2O2-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-H2O2-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-H2O2-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.

Resistance to oxidative damage but not immunosuppression by organic tin compounds in natural populations of Daubenton's bats (Myotis daubentonii).

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.

The real-time-based assessment of the microbial killing by the antimicrobial compounds of neutrophils.

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 longitudinal study of serum 25-hydroxyvitamin D and intact parathyroid hormone levels indicate the importance of vitamin D and calcium homeostasis regulation in multiple sclerosis.

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.

High sensitivity measurement of CRP and disease progression in multiple sclerosis.

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).

Ambient air quality and occurrence of multiple sclerosis relapse.

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.

Development of anti-bovine TNF-alpha mAb and ELISA for quantitating TNF-alpha in milk after intramammary injection of endotoxin.

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.

Modulation of IgG and complement receptor expression of phagocytes in kidney cancer patients during treatment with interferon-alpha.

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.

The role of colostral antibodies in prevention of microbial infections.

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.

Production of reactive oxygen species in neutrophils after repeated bouts of exercise in standardbred trotters.

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.

Colostral proteins from cows immunised with Streptococcus mutans/S. sobrinus support the phagocytosis and killing of mutans streptococci by human leucocytes.

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.

Milk hypersensitivity in young adults.

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.

Mode of delivery directs the phagocyte functions of infants for the first 6 months of life.

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.

Effect of florfenicol on the immune response of rainbow trout (Oncorhynchus mykiss).

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%.

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 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.

Determination of complement-mediated killing of bacteria by viability staining and bioluminescence.

Complement-mediated killing of bacteria was monitored by flow cytometric, luminometric, and conventional plate counting methods. A flow cytometric determination of bacterial viability was carried out by using dual staining with a LIVE/DEAD BacLight bacterial viability kit. In addition to the viable cell population, several other populations emerged in the fluorescence histogram, and there was a dramatic decrease in the total cell count in the light-scattering histogram in the course of the complement reaction. To permit luminometric measurements, Bacillus subtilis and Escherichia coli were made bioluminescent by expressing an insect luciferase gene. Addition of substrate after the complement reaction resulted in bioluminescence, the level of which was a measure of the viable cell population. All three methods gave essentially the same killing rate, suggesting that the bacteriolytic activity of serum complement can be measured rapidly and conveniently by using viability stains or bioluminescence. In principle, any bacterial strain can be used for viability staining and flow cytometric analysis. For the bioluminescence measurements genetically engineered bacteria are needed, but the advantage is that it is possible to screen automatically a large number of samples.

Probiotic bacteria down-regulate the milk-induced inflammatory response in milk-hypersensitive subjects but have an immunostimulatory effect in healthy subjects.

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.

Purification and characterization of bovine complement component C3 and its cleavage products.

OBJECTIVE: To purify complement component C3 from bovine serum, characterize and analyze NH2-terminal amino acid sequences from its various cleavage products, and do cross-species homology comparisons. ANIMALS: 2 healthy lactating Holstein cows, and 2 healthy adult female New Zealand White rabbits. PROCEDURE: Bovine C3 was isolated from serum, and was cleaved to C3b. The resulting protein was analyzed to determine apparent molecular mass of resulting protein segments. Bands were electroblotted onto a membrane and excised, then NH2-terminal amino acid sequences were determined. RESULTS: The C3 preparation consisted of 6 segments, with molecular mass of 30, 40 (2 bands, a and b), 70, 75, and 115 kd. Via sequence comparisons, the 115-kd band was identified as the alpha chain; the 75-kd segment was determined to be the NH2-terminal portion of alpha chain; the 70-kd piece was identified as the intact beta chain; and the two 40-kd bands are believed to be located at the C-terminal portion of the alpha chain, at the cleavage site that yields C3f. The 30-kd band is the NH2-terminal portion of the alpha chain (minus the C3a segment). Sequence analysis of each band revealed a high degree of homology with human, rat, mouse, and horse C3. Polyclonal antibodies raised in rabbits yielded sera that reacted to the purified sample in manner similar to that of commercially available antibodies. CONCLUSIONS: The purified preparation contained intact C3, C3b, and the degradation products iC3b and C3c, which had high sequence homology with those of other species. The C3a and C3d, and C3g segments of protein were not detected and may have been lost during purification, lyophilization, or transfer steps. Structure and cleavage characteristics of bovine C3 can be used to better understand immune responses to bacterial pathogens in the mammary gland.

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.

Kinetic measurement of the membranolytic activity of serum complement using bioluminescent bacteria.

A method for studying the kinetics of serum complement activity is presented. The assay utilises Escherichia coli and Bacillus subtilis cells which have been made bioluminescent by expressing an insect luciferase gene from Pyrophorus plagiophthalamus. The diffusion of the luciferase enzyme substrate through the cell membranes is very slow, and therefore a change in membrane permeability is seen as a change of the in vivo luminescence of the cells. Treatment of the bacteria with human serum resulted in a bell-shaped curve of in vivo luminescence since this procedure facilitates passage of the substrate to the cytoplasm. The time point of maximum light emission was dependent on serum dilution. In vivo luminescence also proved to be a good indicator of the viability of bacterial cells. Using C1q deficient serum, or following treatment of normal serum with EGTA and Mg2+ it was possible to separate the membranolytic activities of alternative and classical pathways.