Esa-Matti Lilius

Immune enhancement in rainbow trout (Oncorhynchus mykiss) by potential probiotic bacteria (Lactobacillus rhamnosus)

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.

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 ⩽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 ⩽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.

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

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

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

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

Febrile infection changes the expression of IgG Fc receptors and complement receptors in human neutrophils in vivo.

We have examined the expression of the IgG Fc receptors (FcRI, FcRII, FcRIII) and complement receptors (CR1, CR3) in neutrophils from 42 patients with febrile bacterial infection, 20 patients with febrile viral infection and 69 non‐febrile healthy individuals. Using receptor‐specific MoAbs and immunofluorescence flow cytometry the relative fluorescence intensity (as a measure of receptor number) and the proportion of receptor‐positive cells were determined in peripheral blood neutrophils exposed to minimal processing, consisting only of erythrolysis. Both the percentage of FcRI‐positive neutrophils and FcRI number per neutrophil were significantly (P<0.001 and P<0.0001) increased in bacterial infected patients compared with controls, whereas in viral infected patients only the FcRI percentage was markedly elevated (P<0.05). In addition, both FcRII and CR1 levels were significantly higher in the bacterial infection group than in the viral infection and control groups (bacterial versus control P<0.001, bacterial versus viral P<0.0001). No changes in expression of FcRIII or CR3 were found in the patient groups. The kinetic analysis of receptor expression in bacterial infection patients revealed a shift in the percentage of FcRI‐bearing neutrophils towards normal values already on day 2 after the first analysis. On the other hand, the levels of FcRI, FcRII and CR1 remained clearly elevated in these patients during 1 weeks follow‐up period. We conclude that febrile infection may cause systemic activation of the entire pool of circulating neutrophils, resulting in alterations in cell surface receptor expression, some of which are characteristic of the nature of the infectious agent.

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 (FcγRI (CD64), FcγRII (CD32), FcγRIII (CD16), CR1 (CD35), CR3 (CD11b) and FcαR (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 FcγRI‐, Fcα‐ 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.

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.

Bacteriolytic activity of rainbow trout (Oncorhynchus mykiss) complement

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.

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 (PM10) 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.

Quantitative extraction and estimation of intracellular nicotinamide nucleotides of Escherichia coli.

Abstract The conditions of extraction of nicotinamide nucleotides from Escherichia coli were systematically investigated. The optimum pH for NAD + and NADP + appeared to be around 1.5 while that for NADH and NADPH was around 12.5. Seven minutes at 60°C was chosen for the extraction of all four nucleotides. Addition of CuCl to the alkaline extract improved considerably the recovery of the reduced forms. Two sensitive (10–100 pmol) assay methods for nicotinamide nucleotides were compared. A spectrophotometric cycling method based on the measurement of reduced thiazolyl blue formation appeared to be more sensitive and more reproducible and gave better recovery than a fluorometric cycling method used earlier in bacterial studies.