Mykolas Mauricas

Regulation of T-cell-independent and T-cell-dependent antibody production by circadian rhythm and melatonin

Melatonin is a hormone that has immunomodulatory activity and is believed to influence the production of antibodies in mammals. The aim of the present study was to investigate the effect of suppressed melatonin synthesis on the antibody production. BALB/c mice were immunized with T-cell-dependent (TD) and T-cell-independent (TI) antigens and kept under (i) normal lighting, (ii) constant exposure to light, (iii) exposed to light and treated daily with melatonin. It was revealed that melatonin modulated TD and TI antibody production. Suppressed melatonin synthesis increased the amount of IgM, IgG1, IgG2b and IgG3 antibodies after immunization with TI antigen. The level of TD antibodies IgM, IgG2a, IgG2b and IgG3 also increased, however, the antigen-specific antibodies of IgG1 isotype significantly decreased in mice exposed to light. Daily melatonin treatment brought the antibody level back to normal. The antibody concentration in the sera of mice kept at normal lighting was significantly higher when the immunizations were performed in the evening. The action of melatonin on B cells via MT2 receptor was shown in vitro and in vivo.

A method to estimate cell cycle time and growth fraction using bromodeoxyuridine-flow cytometry data from a single sample

BackgroundPresently available flow cytometric methods of bromodeoxyuridine (BrdUrd) labelling do not provide information on the cell cycle time (TC) and the growth fraction (GF). In this paper, we describe a novel and simple method to estimate TC and GF from flow cytometric analysis of a single tumour sample after BrdUrd labelling.MethodsThe proposed method is based on two assumptions: (1) the number of labelled cells traversing the cell cycle per unit time is constant and (2) the total number of labelled cells is constant throughout the cycle, provided that cells produced after division are excluded. The total numbers of labelled divided G1 cells, labelled divided S cells, labelled undivided S cells, and labelled undivided G2 cells were obtained for DNA histograms of BrdUrd-positive cells in a collected sample. These cell numbers were used to write equations to determine the durations of cell cycle phases, TC and GF. To illustrate the application of the proposed formulae, cell cycle kinetic parameters were analysed in solid SL2 tumours growing in DBA/2 mice and in human T-leukaemia Jurkat cells in culture.ResultsThe suitability of the proposed method for estimating durations of the cell cycle phases, TC and GF was demonstrated. TC in SL2 tumours was found to be relatively constant at 4 and 10 days after tumour implantation (20.3 ± 1.1 h and 21.6 ± 0.9 h, respectively). GF in tumours at day 10 was lower than GF at day 4 (54.2 ± 7.7% vs. 79.2 ± 5.9%, p = 0.0003). Approximate values of TC and GF of cultured Jurkat cells were 23.9 h and 79.3%, respectively.ConclusionThe proposed method is relatively simple and permits estimation of the cell cycle parameters, including TC and GF, from a single tumour sample after labelling with BrdUrd. We have shown that this method may be useful in preclinical studies, allowing estimation of changes in GF during growth of murine tumours. Experiments with human Jurkat cells suggest that the proposed method might also prove suitable for measurement of cell kinetics in human tumours. Development of suitable software enabling more objective interpretation of the DNA profile in this method would be desirable.

Melatonin inhibits granulocyte adhesion to ICAM via MT3/QR2 and MT2 receptors

Neutrophils are cells of the innate immune system that first respond and arrive to the site of infection. Melatonin modulates acute inflammatory responses by interfering with leukocyte recruitment. It is known that melatonin modulates granulocyte migration though the endothelial layer thereby acting on the endothelial cell. Here we investigated whether melatonin could modulate granulocyte infiltration by acting directly on granulocytes. Granulocyte infiltration into the peritoneal cavity was investigated in mice kept at normal light/dark conditions and mice kept under constant lighting. To induce migration of neutrophils from the blood into the injury site via the endothelial layer, a bacterial product N-formyl-l-methionyl- l-leucyl- l-phenylalanine (fMLP) was injected into the peritoneal cavity. We found that the number of infiltrated granulocytes during the dark time was lower than that during the light time. It did not depend on circadian time. Moreover, the expression of an adhesion molecule, CD18, on granulocytes, was also lower during the dark time as compared with the light time. We have found that melatonin inhibited fMLP-induced CD18 up-regulation. Importantly, melatonin also inhibited the integrin-mediated granulocyte adhesion to intercellular adhesion molecule-coated plates. This study additionally showed that melatonin receptors MT2 and MT3/quinone reductase 2 (QR2) are expressed on granulocytes. Interestingly, melatonin increases the expression of its MT3/QR2 receptor. The fMLP-mediated CD18 up-regulation was inhibited by melatonin via MT2 receptor and the integrin-mediated granulocyte adhesion was inhibited by melatonin via MT3/QR2 and MT2 receptors. In conclusion, we show that melatonin suppresses granulocyte migration via endothelium by acting directly on granulocytes.

Frequency and significance of parvovirus B19 infection in patients with rheumatoid arthritis

The present study aims to clarify the possible involvement of parvovirus B19 (B19V) infection in rheumatoid arthritis (RA) pathogenesis by investigating the presence of B19V infection markers (genomic sequences and virus-specific antibodies) in association with the level of cytokines and RA clinical activity and aggressiveness. A total of 118 RA patients and 49 age- and sex-matched healthy volunteers were enrolled in the study. Nested PCR was used to detect B19V sequences in whole blood and cell-free plasma DNA, ELISA to detect virus-specific antibodies and cytokine levels in plasma and recomLine dot blot assay for antibodies to separate B19V antigens. The detection frequency of B19V DNA was higher in patients with RA (25.4 %) in comparison with healthy persons (18.4 %). B19V DNA in cell-free plasma (B19+p) was detected significantly often in RA patients in comparison with healthy controls (13.6 vs 2 %; P=0.0002). RA B19+p patients had higher disease activity and aggressiveness, decreased haemoglobin and increased erythrocyte sedimentation rates. IL-6 plasma levels were significantly higher in RA patients than in controls. Within the RA patients’ group the IL-6 level was significantly increased in B19+p patients with disease activity scores of DAS28>5.2, high C-reactive protein and low haemoglobin. Contrary to the healthy controls, the majority of RA B19+p patients did not have antibodies to VP-1S (VP1u) and VP-N (N-terminal half of structural proteins VP1 and VP2), which correspond to the epitopes of neutralizing antibodies. These results indicate that B19V infection at least in some patients is involved in RA pathogenesis.

The use of high-resolution melting analysis for Salmonella spp. CRISPR sequence genotyping@@@Aukštos rezoliucijos lydymosi temperatūrą nustatančio metodo pritaikymas Salmonella spp. CRISPR sekų genotipavimui

Correspondence to: Maksim Bratcikov, Institute of Immunology, Vilnius University, Molėtų pl. 29, LT-08409, Vilnius, Lithuania. E-mail: bratchikov@gmail.com Background. Clustered Regularly Interspaced Short Palindromic Repeats (CRISPRs) of Salmonella spp. and of other bacteria continuously evolve due to the rearrangements of fragments. Th is phenomenon forms a high degree of strain polymorphism. Real-time PCR and high-resolution DNA melting (HRM) analysis of CRISPR regions provide a possibility for a rapid evaluation of strain polymorphism. Our intent was to apply the HRM technique for Salmonella spp. genotyping. Materials and methods. Salmonella spp. strains DNA were amplifi ed and analyzed with HRM using Rotor-Gene 65H0-100. Results. Forty-nine Salmonella spp. strains were analyzed, and 23 genotypes were identifi ed for each CRISPR (CRISPR1 and CRISPR2). We have found the genotypes of both CRISPRs to be related to the same Salmonella spp. serotypes. Conclusions. Th e CRISPRs HRM method is a valuable tool for Salmonella spp. genotyping.

Intratumoral Accumulation and Clonal Expansion May Not Be Decisive for Rejection of Allogeneic Tumors by CD8+T-Lymphocytes

Background: The aim of this study was to analyze the spatial distribution and proliferation of adoptively transferred CD8+ T-lymphocytes sensitized against allogeneic tumors. Materials and Methods: Transgenic β-actin-luc mice that express luciferase were sensitized against allogeneic SL2 lymphoma. CD8+ T-lymphocytes from these mice were transferred to lymphocyte-deficient, recombination activating gene-deficient (Rag−/−) mice bearing SL2 tumors and were tracked using bioluminescence imaging. Results: Two out of six Rag−/− mice rejected their tumors. There were no apparent differences in spatial distribution and proliferative intensity of adoptively-transferred CD8+ T-lymphocytes between the two Rag−/− mice that rejected allogeneic SL2 tumors and the four Rag−/− mice that did not. Conclusion: The pattern of distribution in the mouse body and proliferative intensity of CD8+ T-lymphocytes do not seem to be decisive factors influencing allogeneic tumor rejection.

The effect of Saccharomyces cerevisiae β-glucan on proliferation, phagocytosis and cytokine production of murine macrophages and dendritic cells

Abstract β-Glucan is a natural carbohydrate, which is produced by the variety of different organisms – bacteria, fungi, plants, etc. β-Glucan from different sources has been recognized as an active material, which is an immune stimulator for plants, invertebrates and mammals. Saccharomyces cerevisiae, the baker’s yeast, is one of the commonly used sources of β-1,3-glucan. The aim of the present work was to investigate how the different S. cerevisiaeβ-glucan preparations affect proliferation, phagocytosis and cytokine production of murine macrophages and dendritic cells. In our experiments, BALB/c macrophages and dendritic cells were treated with different β-glucan preparations in vitro. Then cell proliferation (AlamarBlue reagent), ability to phagocytose zymosan particles and production of IL-12 and IFNγ (Western blot) were investigated. Our results showed that β-glucan preparations stimulated proliferation of BALB/c macrophages and dendritic cells in vitro, but acted on phagocytosis and cytokine synthesis in different ways. This study demonstrated that S. cerevisiae β-glucan preparations propelled proliferation of the murine macrophages and dendritic cells and influenced phagocytosis and cytokine production of these cells. These effects can depend on the size of β-glucan molecules.

Different saccharomices cerevisiae β-glucans preparation's effect on murine dendritic cells

Nowadays non-cellulosic, β-glucans from the different source are intensively investigated due to the fact that these biological polymers are recognized as potent immunological stimulators for the mammals immune system. Dendritic cells are the most important antigen presenting cells for activating naive T cells. The research goals of the recent investigation were to determine how the different molecular weight Saccharomyces cerevisiae β- glucan preparations affect such properties of the murine DC as phagocytosis, proliferation and cytokine synthesis in vitro.