Irute Girkontaite

A rapid ELISA test for detection of human paraproteins

A rapid ELISA test for detection, characterization and quantification of human paraproteins was developed. The proposed method is a sandwich ELISA, where the capture antibody is specific for a given heavy chain (gamma, alpha or mu) and the labelled antibody is specific either for kappa or for lambda light chain. Both standard and patient sera are tested with all six possible antibody combinations. Each paraprotein produces a significant increase in titre (as compared with standard) only when tested with the relevant pair of antibodies. This enables the determination of the isotype and light chain type of the paraprotein and the evaluation of its relative quantity in patient serum. The accuracy of the assay (relative deviation) varies from 0.04 for gamma lambda to 0.19 for alpha kappa. The cut-off values for each type of polyclonal immunoglobulin were determined with 200 healthy donor sera. 103 patient sera were analysed. ELISA data are in good agreement with M-component and other clinical data.

Parvovirus B19 infection modulates the levels of cytokines in the plasma of rheumatoid arthritis patients

Background Parvovirus B19 (B19V) infection is associated with various autoimmune diseases. We investigated the levels of pro‐inflammatory (IFN&ggr;, TNF&agr;, IL‐2, IL‐12) and anti‐inflammatory (IL‐4, IL‐10) cytokines in the plasma of B19V DNA positive (B19+) and negative (B19−) rheumatoid arthritis (RA) patients in comparison with the control group (healthy persons). Methods Blood samples were collected from 118 patients with RA and 49 healthy voluntaries. B19V sequence was determined in whole blood and cell‐free plasma DNA by nested PCR. The levels of cytokines in the plasma and cell culture medium from Concanavalin A (ConA) or B19V VP1 protein stimulated PBMC were determined by ELISA. Results The levels of IL‐4, IL‐10, IL‐12, IL‐2 and TNF&agr; were higher in plasma of RA patients in comparison with control persons. B19+ controls and RA patients had lower levels of IFN&ggr; in comparison with B19− controls and RA patients. Within RA patients the plasma levels of IFN&ggr; were lower in patients with low RA disease activity or remission. Plasma level of IL‐4 was increased and IL‐10 level was decreased in B19+ RA patients in comparison with B19− RA patients and did not differ between B19+ and B19− controls. B19V infection did not affect plasma levels of IL‐12, IL‐2, and TNF&agr;. ConA and B19 VP1 protein stimulated PBMC from RA patients produced less IFN&ggr; than stimulated PBMC from the healthy controls. Conclusions B19V infection could differently modulate the amount of cytokines in the plasma of healthy persons and RA patients. Decreased production of IFN&ggr; and raised level of plasma IL‐4 in RA patients could lower antiviral clearance. HighlightsCytokine levels in parvovirus B19 DNA positive and negative individuals were investigated.Parvovirus B19 reduces INF&ggr; plasma levels in rheumatoid arthritis patients.Parvovirus B19 elevates IL‐4 plasma levels in rheumatoid arthritis patients.Parvovirus B19 reduces IL‐10 plasma levels in rheumatoid arthritis patients.B19V infection did not affect plasma levels of IL‐12, IL‐2, and TNF&agr;.

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.

Reversible Permeabilization of Cancer Cells by High Sub-Microsecond Magnetic Field

Exposure of cells to pulsed electric fields (PEFs) induces a phenomenon known as electroporation, which leads to increase of membrane permeability. Electroporation is applied in biotechnology, food processing, and medicine, including cancer treatment. Recently, a contactless method based on pulsed magnetic fields (PMFs) for the permeabilization of biological cells has been proposed; however, the permeabilization mechanism of the PMF method is still hypothetical. In this paper, we have shown that it is possible to reversibly permeabilize Sp2/0 myeloma cells by sub-microsecond (450 ns) PMF in the range of 0–3.3 T. The PMF methodology was also combined with PEF treatment to evaluate additive effects. The 1.35 kV/cm

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

1 \times 100~\mu \text{s}

Presence of Human Bocavirus 1 in Hospitalised Children with Acute Respiratory Tract Infections in Latvia and Lithuania / Cilvēka Bokavīrusa 1 Klātbūtne Latvijā Un Lietuvā Hospitalizētiem Bērniem Ar Akūtām Elpceīu Slimībām

(PEF) and 3.3 T, 50 pulses, 0.25 Hz (PMF) protocols were applied. The cells were treated in the presence of fluorescence dye YO-PRO-1 and influx into the cells was evaluated by cytometry. Cell viability after the treatment was evaluated by CellEvent Caspase-3/7 assays. A significant (P < 0.05) additive effect of the two pulsed power methodologies was detected, resulting in up to 12% increase of membrane permeabilization. The PMF method is an emerging technique and the results of the study can be used for the development of new effective protocols, while the determined additive effects with PEF are promising in the field of electrochemotherapy.

Immunochemical study of human immunoglobulin G Fc region.

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.

Influence of Circadian Time and Lighting Conditions on Expression of Melatonin Receptors 1 and 2 in Murine Lymphocytes

Abstract Human bocavirus 1 (HBoV1) is a parvovirus recently found to be a possible aetiologic agent of acute respiratory disease in children. We conducted the first clinical and molecular study on this virus in Latvia (LV) and Lithuania (LT). The aim of the study was to determine the occurrence of HBoV1 in respiratory tract samples taken from hospitalised children with acute respiratory tract infections in LV and LT. In total 186 children with age one to 50 months, and who fulfilled criteria of acute respiratory tract infection, including lower respiratory tract infections, with or without fever, were included in this study. A nasopharyngeal aspirate was obtained from each patient on admission. DNA was isolated and polimerase chain reaction (PCR) performed targeting the HBoV1 NS1sequence. HBoV1 positive samples were sequenced and phylogenetic analysis was performed. HBoV1 sequence was detected in 42 (32%) of 130 LV and in 8 (14%) of 56 LT samples. In LV the majority of patients with HBoV1 infection were observed in February while in LT in October. The phylogenetic tree for HBoV1 indicated that isolates of HBoV1 cluster closely and include almost all of the isolates in this study. HBoV1 is common in Latvia and Lithuania and might be a significant pathogen that contributes to acute respiratory tract infections in children.