ABSTRACT
This study aims to develop a method to detect bovine multi-cytokines based on flow cytometry. Previously we have prepared and screened monoclonal antibodies against bovine cytokines IFN-γ, IL-2, TNF-α, IP-10 and MCP-1. These bovine cytokine monoclonal antibodies were fluorescently labeled, and the combination of antibody and cell surface molecules were used to develop the method for detecting bovine multi-cytokines. Subsequently, the developed method was used to determine the cytokine expression profile of Mycobacterium bovis BCG infected bovine peripheral blood mononuclear cells in vitro, and evaluate the cytokine expression level of peripheral blood CD4+ T cells of tuberculosis-positive cattle. The bovine multi-cytokine flow cytometry detection method can effectively determine the cytokine expression of BCG-infected bovine peripheral blood T lymphocytes. Among them, the expression levels of IFN-γ, IL-2, and TNF-α continue to increase after 40 hours of infection, while the expression levels of IP-10 and MCP-1 decreased. The combined detection of IFN-γ, IL-2, and TNF-α on CD4+ T lymphocytes in peripheral blood of cattle can effectively distinguish tuberculosis-positive and tuberculosis-negative samples. This method may facilitate evaluating the level of cellular immune response after bovine pathogen infection and vaccine injection.
Subject(s)
Cattle , Animals , Cytokines , BCG Vaccine/metabolism , Tumor Necrosis Factor-alpha/metabolism , Interleukin-2 , Flow Cytometry/methods , Chemokine CXCL10/metabolism , Leukocytes, Mononuclear , CD4-Positive T-Lymphocytes/metabolism , Tuberculosis , Antibodies, Monoclonal/metabolismABSTRACT
@#AIM: To investigate the morphology and electrophysiological properties of neurons in the inner nuclear layer(INL)of the adult rat retina slices. The retinal slices were prepared by low-melt agarose embedding and then cutted by vibratome. <p>METHODS: Whole cell patch clamp and intracellular staining with Lucifer Yellow were used in this study to study the morphology and electrophysiological properties of the INL neurons in retinal slices.<p>RESULTS: Retinal slices prepared in this method possessed a very smooth surface. The cells on the retinal slices maitained very good vitality, and some of the cells even retained their dendritic connections with other cells on the slice. According to the size and location of the cell bodies, neurons in the INL were easy to differentiate. Luciifer Yellow contained in the intracellular solution revealed the morphology of the recorded cell very well. Bipolar cells possessed elongated cell bodies and their processes mainly extended along the vertical direction. Horizontal cells and amacrine cells possessed much bigger and round cell bodies, resided in the outermost and inner most of the INL, respectively. The rest membrane potential and membrane capacitance of horizontal cell and amacrine cell were much higher than that of bipolar cells. Under a voltage step from -60mV to +40mV, 10mV per step, 41.7% of the cone bipolar cells and 64.7% of the amacrine cells exhibited inward sodium current and outward potassium current. Other cells only possessed outward potassium current. <p>CONCLUSION: The method of preparing retinal slices was very simple, and the viability of the slices were stable. These facilitated the patch-clamp recording of all the neurons in the INL including horizontal cells. Further investigation of the electrophysiological properties of the neurons in the INL was essential in revealing the mechanism of vision.
ABSTRACT
INTRODUCTION: This study evaluated the intracellular profile of interleukin-2 (IL-2), interleukin-4 (IL-4), interleukin-10 (IL-10) and interferon-γ (IFN-γ) in peripheral blood mononuclear cells (PBMCs) from leprosy patients based on oral infections presence to determine whether these coinfections could be associated with pro-inflammatory activity in leprosy. METHODS:Leprosy patients regardless of clinical form and specific leprosy treatment (n=38) were divided into two groups: Group I - leprosy patients with oral infections (n=19), and Group II - leprosy patients without oral infections (n=19). Non-leprosy patients presenting oral infections were assigned to the control Group (n=10). Intracellular IL-2, IL-4, IL-10 and IFN-γ production was evaluated by flow cytometry (FACS) before and 7 days after controlling the oral infection in the Group I, before and 7 days after dental prophylaxis in the Group II, and during oral infection process in control Group. RESULTS: Low percentages of CD3+ lymphocytes bearing IL-2, IL-10 and IFN-γ were observed in the Group I and Group II at baseline and 7 days after therapy or prophylaxis compared to controls. Group I showed reduced percentages of IL-4 at baseline and 7 days after therapy compared to controls, or at baseline of Group II, and the Group II showed reduced percentages of CD3+ cells bearing IL-4 compared to control. An increase of the percentages of CD3+cells bearing IL-4 was observed in the Group I after the oral infections treatment. CONCLUSIONS: The occurrence of oral infections favors the intracellular cytokines expression and, probably, the inflammatory reaction operating as a stimulatory signal triggering the leprosy reactions.
Subject(s)
Adolescent , Adult , Aged , Aged, 80 and over , Female , Humans , Male , Middle Aged , Young Adult , Coinfection/immunology , Cytokines/immunology , Leprosy/immunology , Lymphocytes/immunology , Periodontal Diseases/immunology , Case-Control Studies , Cytokines/blood , Interferon-gamma/blood , Interferon-gamma/immunology , /blood , /immunology , /blood , /immunology , /blood , /immunology , Leprosy/complications , Periodontal Diseases/complicationsABSTRACT
Objective To prepare recombinant adenovirus pAd-gal-9 containing murine galectin-9 and explore galectin-9's pro-apoptotic effect on T lymphocytes. Methods The recombinant adenovirus plas-mid pAd/CMV/V5-DEST-gal-9 was prepared by conventional molecular cloning and LR reaction. The pAd/ CMV/V5-DEST-gal-9 linearlized by Pac I was transfected into 293A cells with Lipofectin 2000. Eight days after transfection, the 293A cells were subjected to freeze/thraw circle for three times and the supernatant was collected after centrifugation. Higer titer pAd-gal-9 was produced by large-scale infection of 293A cells with the supernatant containing pAd-gal-9. The supernatant was condensed to get purified pAd-gal-9 by CsCl density gradient centrifugation. After titer determination with gradient dilution of harvested pAd-gal-9 infec-tion in 293A-seeded 96-wells, pAd-gal-9 was used to infect the CHO cell line. Immunohistological assay, Western blot and flow cytometry were employed to ascertain the subcellular location expression of galectin-9. We added solid-phase transgenic CHO cells or freshly-cultured supernatant to medium containing activated T cells to detect the pro-apoptotic effect of galectin-9. Results The pAd-gal-9 was prepared successful. Im-munohistochemical staining of CHO infected with pAd-gal-9 confirmed that galectin-9 was expressed in the cytosol. Intercellular staining indicated that mean fluorescence intensity of galectin-9 was significantly higher in pAd-gal-9-infected CHO group than control group. Supernatant from pAd-gal-9-infected CHO promoted the apoptosis of T cells. The percent of apoptotic T cells was higher than the Tim-3 positive T cells. Conclu-sion CHO infected with pAd-gal-9 can secret galectin-9 to promote the apoptosis of activated T cells via Tim-3-independent mechanisms.
ABSTRACT
Objective To study the expression and significance of Th17 cells from peripheral blood of patients with rheumatoid arthritis (RA). Methods Intracelluar flow cytomete detection of IL-17/IFN-γ and IL-17/IL-6 was established using anti-CD3/Anti-CD28/IL-23 as stimulators after isolation of untouched human CD4~+T cells from PBMC. There were three groups in the present study: ①healthy controls group; ② RA stable group; ③RA active group. Results The isolation of untouched human CD4~+T cells from PBMC was effective and its purity was over 90%. The percentage of intracelluar IL-17 in CD4~+ T cells from RA patients was increased significantly. Such percentage in active group (1.54±0.41) was higher than that of stable group (0.70±0.21, P<0.01) and both of them were higher than those of healthy controls (0.42±0.12, P<0.01). Under anti-CD3/Anti-CD28/IL-23 stimulation, the percentage of intracelluar IL-17 was also increased significantly(P<0.01). The porcentage of intracellular IFN-γ was similar to that of IL-17, while that of IL-6 was not significantly different. There is an correlation between IL-17 and IFN-γ or IL-6. Conclusion There is an abnormal expression of IL-17 and IFN-γ in human CD4~+T cells in RA patients, which is related to disease activity . Th17 cells may be used as a new marker for the assessment of RA activity.