Molecular sensitization patterns to cat and dog allergens in Lithuanian children population.

Background: Over the last few decades, there was observed an increase of asthma and allergic rhinitis cases caused by allergy to pets. Objective: This study aimed to analyze molecular sensitization patterns to dog and cat allergens in Lithuanian children who were experiencing allergy-like symptoms. Materials and methods: A total of 574 children (0-18 years) were tested for allergen-specific immunoglobulin E (sIgE) levels with ALEX2 (ALEX2®, Allergy Explorer Test System). Positive sera were further analyzed for sensitization to cat (Fel d 1, Fel d 2, Fel d 4, and Fel d 7) and dog (Can f 1, Can f 2, Can f 3, Can f 4, Can f 5, and Can f 6) allergen components. Results: Two hundred forty-seven children tested positive (sIgE ≥0.3 kUA/L) to at least 1 dog or cat allergen component. There were 61.1% children sensitized to components from both sources, 29.2% - exclusively to cat, and 9.7% - to dog components. The major sensitizers were Fel d 1 (84.8%) and Can f 1 (59.4%). There were 42.9% patients sensitized to 3 or more different mammalian protein families and 40.4% - to 3 or more lipocalins. There were 5.7% of children sensitized both to Fel d 1 + Fel d 4 and Can f 1/2 + Can f 5, indicating the high risk of severe asthma. Monosensitization to Fel d 1 was the dominant pattern among Lithuanian children (26.3%). Conclusion: The majority of children were cat/dog-polysensitized, although sensitization only to cat allergens was most observed. Extensive molecular profiling can be an useful tool for accurate true sensitization diagnosis and prognosis of disease severity.

Lessons from Animal Models in Sjögren's Syndrome.

Primary Sjögren's syndrome (pSS) is a connective tissue disease characterized by a wide spectrum of clinical features, extending from a benign glandular disease to an aggressive systemic disorder and/or lymphoma. The pathogenesis of Sjögren's syndrome (SS) is not completely understood, but it is assumed that pathogenesis of SS is multifactorial. The studies based on the animal models of SS provided significant insight in SS disease pathogenesis and management. The aim of this review is to summarize current studies on animal models with primary SS-like symptoms and discuss the impact of these studies on better understanding pathogenesis and management of Sjögren's syndrome. Databases PubMed, Web of Science, Scopus and Cochrane library were searched for summarizing studies on animal models in SS. Available data demonstrate that animal models are highly important for our understanding of SS disease.

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 ß-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.

Native matrix-based human lung alveolar tissue model in vitro: studies of the reparatory actions of mesenchymal stem cells.

Studies of lung diseases in vitro often rely on flat, plastic-based monocultures, due to short lifespan of primary cells, complicated anatomy, lack of explants, etc. We hereby present a native 3D model with cues for repopulating epithelial cells. Abilities of mesenchymal stem cells (MSC) to modulate bacterial lipopolysaccharide (LPS) and cigarette smoke-induced injury to pulmonary epithelium were tested in our model. Post-mortem human lung tissue was sliced, cut and decellularized. Resulting matrix pads were reseeded with pulmonary epithelium (A549 line). Markers of the layer integrity and certain secreted proteins in the presence of cigarette smoke extract (CSE) and LPS were assessed via Western blot, ELISA and RT-PCR assays. In parallel, the effects of MSC paracrine factors on exposed epithelial cells were also investigated at gene and protein levels. When cultured on native 3D matrix, A549 cells obtain dual, type I- and II-like morphology. Exposure to CSE and LPS leads to downregulation of several epithelial proteins and suppressed proliferation rate. MSC medium added to the model restores proliferation rate and some of the epithelial proteins, i.e. e-cadherin and beta-catenin. CSE also increases secretion of pro-inflammatory cytokines by epithelial cells and upregulates transcription factor NFκB. Some of these effects might be counteracted by MSC in our model. We introduce repopulated decellularized lung matrix that highly resembles in vivo situation and is convenient for studies of disease pathogenesis, cytotoxicology and for exploring therapeutic strategies in the human lung context in vitro. MSC paracrine products have produced protecting effects in our model.

Bioluminescence Imaging of Adoptively Transferred Lymphocytes During Allogeneic Tumor Rejection.

AIM: The aim of the present study was to analyze the survival, spatial distribution and proliferation of adoptively transferred lymphocytes in allogeneic tumor rejection. MATERIALS AND METHODS: Transgenic ß-actin-luc mice that express luciferase were sensitized against SL2 tumors and were used as lymphocyte donors to study the anti-tumor effect in SL2 tumor-bearing lymphocyte-deficient RAG(-/-) mice. Whole-body bioluminescence images of recipient mice were obtained to track the adoptively transferred lymphocytes. Proliferation of lymphocytes was estimated by quantification of photon emission. RESULTS: T lymphocytes sensitized against allogeneic SL2 tumors cured the majority of SL2 tumor-bearing RAG(-/-) mice. Bioluminescence imaging showed that transferred T lymphocytes survived in the spleen and lymph nodes. Tumor rejection was associated with lymphocyte proliferation and migration to the tumor site. CONCLUSION: Sensitized T lymphocytes from transgenic ß-actin-luc mice reject allogeneic SL2 tumors in RAG(-/-) mice and can be tracked in vivo using bioluminescence imaging.

Distribution of Peripheral Lymphocyte Populations in Primary Sjögren's Syndrome Patients.

Purpose of this study was to evaluate the lymphocyte populations' distribution changes in peripheral blood of patients with primary Sjögren's syndrome (pSS). Lymphocyte populations' distribution changes in peripheral blood of pSS patients were investigated in 52 patients with pSS and in 28 healthy controls by flow cytometry. We found decreased absolute count of CD3(+) T cell population in pSS patients. Analysis of CD4(+) T cell population showed significant proportion and absolute count differences in pSS patient's blood with SSA/SSB antibodies (Abs) in comparison to controls. No significant differences were observed analyzing CD4(+) and CD8(+) Treg subpopulation. Proportion and absolute counts of Th17 cells were significantly lower in pSS patient's blood. Absolute counts of CD8(+) T cells were significantly lower in pSS patients in comparison to controls and also impaired proportion and absolute counts of CD8(+) subpopulations according to CD27(+) and CD57(+) were observed. Absolute counts of NKT and NK cells were decreased in pSS with Abs. B cells proportion was increased only in blood of pSS with Abs. Lymphocyte distribution impairment can be due to genetically determined lymphopenia or lymphocyte migration from periphery to inflammatory sites or/and increased susceptibility to apoptosis.

Could the complement component C4 or its fragment C4d be a marker of the more severe conditions in patients with primary Sjögren's syndrome?

Our aim is to evaluate the complement component C4 (C4) and its fragment C4d (C4d) levels, focusing on their associations with other markers of B cells' activity in patients with primary Sjögren's syndrome (pSS). Humoral factors C4, C4d, B cell-activating factor (BAFF), κ and λ free light chains (FLCs) and IgG (by immunoassay) were investigated in 58 patients with pSS and in 28 healthy controls. We observed significantly higher levels of BAFF, κ and λ FLC and IgG, and significantly lower level of C4 in pSS patients, while the level of C4d was similar in the both groups. Significantly higher levels of BAFF, κ and λ FLCs, IgG, and significantly lower C4 level were found in anti-SSA/SSB antibodies (Abs) seropositive pSS patients' group comparing with healthy controls. Level of C4d was significantly lower in anti-SSA/SSB Abs seropositive pSS patients comparing with seronegative pSS patients and healthy controls. C4d correlated with C4, anti-SSB Abs level and κ/λ ratio. Significantly higher κ FLC and IgG levels were found in anti-SSA/SSB Abs seronegative pSS patients comparing with healthy controls. Anti-SSA/SSB seropositivity in pSS patients is associated with the decreased level of C4d. These results show that C4d can be an appropriate marker of antibody response and complement activation in pSS patients with Abs, and possibly may show the more severe condition-exhaustion of C4. Further studies are required to determine whether C4d assessment could be a relevant biomarker for the more severe condition and the worse prognosis of pSS.

Activity of T-helper cells in patients with primary Sjogren's syndrome.

AIM: To investigate the T-helper (Th1, Th2 and Th17) cell activity in the peripheral blood of patients with primary Sjögren's syndrome (pSS), non-Sjögren's sicca syndrome (nSS) and healthy controls. PATIENTS AND METHODS: Peripheral blood mononuclear cells from 34 pSS, 13 nSS patients and 13 healthy controls were stimulated, labeled for cluster of differentiation-4 (CD4), interferon-γ (IFN-γ), interleukin-4 (IL-4) and IL-17A and analyzed by flow cytometry. RESULTS: The activities of Th1 and Th17 cells in patients with pSS were similar to those of the control group. The percentage of both IFN-γ- and IL-17-producing Th17/Th1-like cells was significantly higher in the pSS, as compared to the control group, whereas that of Th2 cells was lower. A significant correlation was found between all Th-subset activities in the control group. However, in the pSS group, a correlation was found only between Th1 with Th2 and Th17 and Th17 with Th17/Th1-like. CONCLUSION: The imbalance in Th-subset activities in peripheral blood may play a role in the pathogenesis of pSS.

CD4+CD25(high) Treg cells in peripheral blood during remission and exacerbation of allergic asthma in children.

AIM: To determine the percentage of CD4+CD25(high) Treg cells in peripheral bloodCD4+ T cells of allergic asthmatic children during disease remission and exacerbation. METHODS: Peripheral blood mononuclear cells (PBMC) and serum samples were collected from 6- to 11-year-old children with mild-to-moderate allergic asthma (n = 34)and from healthy controls (n = 15). CD4+CD25(high) T cells in PBMC were detected by flow cytometry. Total and specific IgE in serum were analysed by enzyme-amplified chemiluminescence, and IL-2 was measured by ELISA. RESULTS: There was no significant difference in CD4+CD25(high) T-cell proportions between asthmatic children in exacerbation and remission as compared with controls.CD4+CD25(high) T-cell percentages were not correlated with total and specific IgE. IL-2 was elevated in both disease remission and exacerbation but did not correlate significantly with CD4+CD25(high) T-cell percentages. CONCLUSION: CD4+CD25(high) T-cell proportion in the peripheral blood of total CD4+T cells is not reduced in children with allergic IgE-mediated asthma and does not differ between disease remission and exacerbation.

An anti-inflammatory effect of murine fetal liver cells in BALB/c mouse contact hypersensitivity model.

Anti-inflammatory effects of murine fetal liver (FL) cells were studied using BALB/c mouse contact hypersensitivity (paw edema) model. Paw weight differences, lymphatic organ weights, hematological and histological indices as well as proinflammatory (TNF-alpha) and anti-inflammatory (IL-10) cytokine levels in sera were evaluated. Immunophenotyping revealed that both murine FL homogenate cells (HC) and FL hematopoietic stem cells (HSC) express CD117 and CD38 surface markers. Single doses of 1x10(6) cells/mouse and 2x10(6) cells/mouse of FL HC as well as of FL HSC, when used separately, all statistically significantly (p<0.05) inhibited paw edema, but the lower dose was more effective and giving results similar to that of prednisolone. Either dose of FL HC or FL HSC studied had no significant influence on lymphatic organ weights; no significant changes were also observed in blood indices. The data of cytokine studies showed that TNF-alpha concentration in sera of mice treated with either FL HC or FL HSC at a dose of 1x10(6) cells/mouse was statistically significantly (p<0.001) lower than that of the control mice. A concentration of IL-10 was statistically significantly higher (p<0.01) in mice treated with a dose of 1x10(6) cells/mouse of FL HC but not with the same dose of FL HSC as compared to the control group. Histological examination revealed better effects of a dose of 1x10(6) cells/mouse of FL HC when compared with the same dose of FL HSC as in regard to reduction of edema thickness and cell infiltration.

Aberrant expression of CD95 and CD69 molecules among CD56 cells in women with endometriosis.

PROBLEM: Activated lymphocytes can be eliminated by Fas/Fas ligand (FasL)-induced cell death. Endometrial cells express FasL. The aim of our study was to determine the expression of CD56+ cells (natural killer and natural killer T cells) Fas antigen CD95 and the early activation molecule CD69 in the peritoneal fluid of women with endometriosis. METHOD: Two-colour flow cytometry was used. RESULTS: In the early stages of endometriosis, more CD56+ cells expressed CD69 and CD95 molecules when compared with the control group. However, in case of severe endometriosis the percentage of CD95+CD56+ cells in peritoneal fluid was similar to that of the control group, but the expression of CD69 molecules remained high. CONCLUSION: Because of Fas/FasL mechanisms, in the initial stages of endometriosis the activated peritoneal fluid CD56+ cells can be intensively eliminated, thus providing conditions for the survival of ectopic endometrial cells and the development of the disease.

Oral tryptic casein hydrolysate enhances phagocytosis by mouse peritoneal and blood phagocytic cells but fails to prevent induced inflammation.

Mouse experiments were conducted in order to find whether oral application of tryptic casein hydrolysate (TCH) results in enhancement of phagocytosing capacity of murine phagocytic cells as well as whether such application might be of use for prevention of inflammatory processes. Phagocytosing capacity of phagocytic cells of mice that received oral TCH once daily in a dose of 1.0 mg/g body weight dissolved in 0.5 ml of distilled water for five successive days was significantly higher (p < 0.05) than that of mice given equivalent volumes of distilled water, with a phagocytosing capacity enhancement index being 1.39 and 1.34 regarding peritoneal macrophages and blood phagocytic cells, respectively. Taken on the other hand, the immunostimulatory effects of oral TCH were found to be not enough to prevent mice from inflammation that was induced experimentally using acute (paw edema) and contact hypersensitivity models. A possibility for development of food protein enzymatic hydrolysates as antimicrobial immunostimulants acting through improvement of phagocytic cell functioning is discussed.

Differential expression of KIR/NKAT2 and CD94 molecules on decidual and peripheral blood CD56bright and CD56dim natural killer cell subsets.

OBJECTIVE: To investigate killer inhibitory receptor (KIR) expression by natural killer (NK) cells in early pregnancy. DESIGN: Case-control study of immunologic markers. SETTING: University hospital. PATIENT(S): Thirty pregnant women and 22 nonpregnant women. INTERVENTION(S): Peripheral venous blood sampling and decidual tissue collection after elective abortion. MAIN OUTCOME MEASURE(S): Flow cytometry was used to assess expression of KIR by NK cells in the cell samples. RESULT(S): In contrast to CD56(bright) peripheral blood NK cells, CD56(dim) cells express killer cell Ig-like receptor KIR/NKAT2. However, KIR/NKAT2 and lectin-like CD94 are present on both subsets of decidual NK cells. We found no differences between peripheral blood NK cell subsets from pregnant and nonpregnant women. CONCLUSION(S): Our findings demonstrate that NK cell subsets, distributed in accordance with CD56 molecule density on cell surface, express killer inhibitory receptors CD94 and KIR/NKAT2 in a different way. Our data support the view that CD56(bright)KIR/NKAT2+CD94+ decidual NK cells are specialized NK cells that have an important role to play in early pregnancy.