Effects of Benzoxazole Derivative PO- 296 on Dendritic Cell Differentiation and Related Indexes / 中国药房

OBJECTIVE： To investigate the effects of benzoxazole derivative 2-（chlorobenzoxazolyl-2-yl）-4，5，6，7-tetrahydro- dihydro-indazole-3-ol （PO-296） on the differentiation of murine bone marrow-derived dendritic cells（DC） and their related indexes as specific surface molecules and inflammatory cytokines. METHODS： Bone marrow nuclear cells of mice were isolated， and immature DC （imDC） was obtained by recombinant mice granulocyte macrophage colony-stimulating factor and recombinant mice IL-4. After pretreated with low-dose， medium-dose and high-dose （1， 5， 25 μmol/L） of PO-296， DC was obtained by lipopolysaccharide induction. Flow cytometry was used to detect the expression of DC specific surface molecules [i.e. the proportion of class Ⅱ major histocompatibility complex （MHC Ⅱ）， CD80， CD86 and chemokine receptor 7 （CCR7） positive cells]， imDC phagocytosis （i.e. the proportion of dextran positive cells） and DC survival （i.e. the proportion of survival cells）. ELISA method was used to detect the levels of inflammatory cytokines （IL-10， IL-12 and TNF-α） in cell culture medium. RESULTS： Compared with imDC group， the proportion of MHC Ⅱ， CD80 and CD86 positive cells were increased significantly in non-loading group （P＜0.05）. Compared with non-loading group， the levels of IL-10 in cell culture medium were increased significantly in PO-296 groups. The proportions of MHC Ⅱ， CD80 and CD86 positive cells in positive group and PO-296 medium-dose and high-dose groups as well as the levels of IL-12 and TNF-α in cell culture medium in administration groups were decreased significantly （P＜0.05）. There was no statistical significance in the proportion of CCR7 positive cells， dextran positive cells and survival cells in administration groups， compared with non-loading group （P＞0.05）. CONCLUSIONS： PO-296 has no obvious cytotoxicity and does not affect the phagocytic function of imDC. At the same time， the compound can inhibit the expression of DC specific surface molecules and regulate the secretion of inflammatory cytokines.

Transformation of PPS induce M2 subtype to M1 macrophages / 中国免疫学杂志

Objective:To study the positive expression rate of M2 subtype of macrophage cell surface molecules and the inflammatory factors of PPS in IL-4-induced M2 macrophage.Methods:The experiment was divided into 5 groups:blank control group, Model group,PPS groups(50 μg/ml,100 μg/ml and 200 μg/ml).The expression of CD206 and CD23 was used as bio-maker to confirm IL-4 induced macrophages by treating RAW264.7 with 20ng/ml of IL-4.IL-4 induced RAW264.7 cells were treated with PPS of 50μg/ml,100μg/ml and 200μg/ml for 24 h.Then the expression of CD206,CD16/32 and CD40 were analyzed by flow cytometry, and the mRNA expression of IL-1β,TNF-α,IL-10 and iNOS were detect by qRT-PCR.Results: After treated with IL-4,the positive rate of CD206 of RAW264.7 were high.After treated with PPS ,the rate of CD16/32 and CD40 in IL-4 induced RAW264.7 cells were high ,the expression of CD206 decreased,and the mRNA level of IL-1βand TNF-αincreased.Conclusion:RAW264.7 cells can be polarlized to M2 subtype macrophage by using 20 ng/ml IL-4.PPS enhances the mRNA of IL-1β,TNF-αand the expression of CD40, CD16/32 in IL-4-induced RAW264.7 cells .These results indicate that PPS can induce the M2 subtype to become M1 macrophages, can improve immune function of macrophages.

Non-Thermal Atmospheric-Pressure Plasma Possible Application in Wound Healing

Non-thermal atmospheric-pressure plasma, also named cold plasma, is defined as a partly ionized gas. Therefore, it cannot be equated with plasma from blood; it is not biological in nature. Non-thermal atmospheric-pressure plasma is a new innovative approach in medicine not only for the treatment of wounds, but with a wide-range of other applications, as e.g. topical treatment of other skin diseases with microbial involvement or treatment of cancer diseases. This review emphasizes plasma effects on wound healing. Non-thermal atmospheric-pressure plasma can support wound healing by its antiseptic effects, by stimulation of proliferation and migration of wound relating skin cells, by activation or inhibition of integrin receptors on the cell surface or by its pro-angiogenic effect. We summarize the effects of plasma on eukaryotic cells, especially on keratinocytes in terms of viability, proliferation, DNA, adhesion molecules and angiogenesis together with the role of reactive oxygen species and other components of plasma. The outcome of first clinical trials regarding wound healing is pointed out.

CTLA-4-Tg/CD-28-KO Mice Exhibit Reduced T Cell Proliferation in vivo Compared to CD-28-KO Mice in a Graft-versus-host Disease Model

Activated T cells express inhibitory receptors such as CTLA-4 that can downregulate immune responses. Blockade of or genetic deficiency in CTLA-4 can result in autoimmunity. Therefore, strategies to increase the inhibitory function of CTLA-4 may be attractive in settings of undesirable T cell responses such as autoimmunity or transplant rejection. We have tested the hypothesis that transgenic constitutive expression of CTLA-4 can further attenuate immune responses when compared with normal inducible expression. Our results indicate that transgenic expression of CTLA-4 in mouse T cells (CTLA-4-Tg T cells) results in reduced cell cycle progression and increased apoptosis of TCR-stimulated T cells. CTLA-4-Tg T cells display reduced T cell proliferation in an in vivo model of graft versus host disease (GVHD). These results further our understanding of how CTLA-4 can be manipulated to inhibit immune responses and may help development of new therapeutic strategies for clinical settings of autoimmunity and transplantation.

Avaliação da interação in vitro do fungo oportunista, emergente, paecilomyces lilacinus, agente causal da hialohifomicose, com células apresentadoras de antígenos humanas (macrófagos e células dentríticas) / Assessment of in vitro interaction of opportunistic emerging fungus, Paecilomyces lilacinus, causal agent of hyalohyphomycosis with human antigen-presenting cells (macrophages and dendritic cells)

Paecilomyces lilacinus, um dos agentes causais da hialohifomicose, é um fungo filamentoso, assexuado. Está amplamente distribuído pelo mundo e frequentemente é encontrado como contaminante, proveniente do ar, em espécimes clínicos e em soluções consideradas estéreis. Acomete principalmente pacientes comprometidos imunologicamente, porém pacientes imunocompetentes também podem apresentar manifestações clínicas da doença, sendo esse fungo, atualmente, considerado como um importante patógeno oportunista humano. O presente trabalho teve por objetivo a investigação da interação de conídios de P. lilacinus, proveniente de caso clínico de hialohifomicose humana, com células apresentadoras de antígenos (APCs - macrófagos e células dendríticas). Conídios interagiram com macrófagos e células dendritícas de doadores humanos saudáveis, em diferentes concentrações (conídio/APCs) e tempos de incubação para avaliação qualitativa e quantitativa da interação por microscopia óptica e para estudo do percentual de células expressando moléculas de superfície celular que atuam na resposta imune do hospedeiro. Sobrenadantes provenientes da interação das APCs com os conídios foram coletados para o estudo das citocinas IL1-beta e TNF-alfa...

Os resultados demonstraram que o fungo foi capaz de infectar de modo semelhante ambas as APCs e que a concentração de 1:1 (conídio/APCs) foi a melhor proporção entre as células para avaliar o processo de fagocitose e as etapas de desenvolvimento do fungo assim como o tempo de 6 horas de interação foi o melhor tempo para a certificação da internalização dos conídios pelas APCs. Após o processo de internalização dos conídios pelas APCs os mesmos começaram a se dilatar, formar tubos germinativos e hifas septadas que se ramificaram formando um micélio que destruiu as células humanas no período de 24 horas de observação. A avaliação quantitativa da fagocitose dos conídios pelas APCs demonstrou que não houve diferenças significativas entre o percentual fagocítico dos macrófagos e células dendríticas de cada doador como também entre os doadores. A interação do fungo com as APCs na presença de L-Name não modificou o processo de desenvolvimento do conídio no interior das células humanas, quando comparado a interação das células sem L-Name. Em relação aos marcadores de superfície celular a presença dos conídios não aumentou o percentual de macrófagos expressando CD14, porém aumentou as células expressando B7.1 e DC-SIGN. As células dendríticas apresentaram um percentual semelhante de células positivas para B7.1,na ausência e presença do fungo, e um aumento significativo do percentual de células DC-SIGN positivas na presença dos conídios. Os conídios de P. lilacinus foram capazes de estimular a secreção da citocina IL1-beta pelos macrófagos e células dendríticas e é possível que eles tenham inibido a produção de TNF-alfa pelos macrófagos. Então, os dados aqui apresentados demonstram a capacidade invasiva para APCs e de estimulação de moléculas de superfície importantes na resposta imune do hospedeiro por P. lilacinus, até o momento desconhecida...

Method for analysis of surface molecule alteration upon phagocytosis by flow cytometry.

In this study, we introduce an application of flow cytometry for the concurrent detection of phagocytotic cells and surface molecules involved in the phagocytic process. E. coli expressing green fluorescent protein (GFP) were applied as the phagocytosable particles. Blood samples were incubated with E. coli expressing GFP, followed by indirect immunofluorescence using four candidate monoclonal antibodies (mAbs). Granulocytes that had phagocytosed E. coli exhibited high levels of GFP intensity, in contrast to the non-phagocytosed cells. By comparing the level of expression of molecules expressed on phagocytosed granulocytes with that of non-phagocytosed cells by flow cytometry, it enabled the determination of the expression and alteration of the cell surface molecules upon phogocytosis. Of the four mAbs used in this study, upon phagocytosis, molecules recognized by mAbs WK13, COSA5A and COSA33NL were up-regulated. However, CD15 recognized by mAb VIMD5 was down-regulated. The proposed method will benefit the study of phagocytic mechanisms in the future.

Molecular mechanisms of Trypanosoma cruzi infection by oral route

Frequent reports on outbreaks of acute Chagas' disease by ingestion of food contaminated with parasites from triatomine insects illustrate the importance of this mode of transmission. Studies on oral Trypanosoma cruzi infection in mice have indicated that metacyclic trypomastigotes invade the gastric mucosal epithelium. A key molecule in this process is gp82, a stage-specific surface glycoprotein that binds to both gastric mucin and to target epithelial cells. By triggering Ca2+ signalling, gp82 promotes parasite internalisation. Gp82 is relatively resistant to peptic digestion at acidic pH, thus preserving the properties critical for oral infection. The infection process is also influenced by gp90, a metacyclic stage-specific molecule that negatively regulates the invasion process. T. cruzi strains expressing high gp90 levels invade cells poorly in vitro. However, their infectivity by oral route varies considerably due to varying susceptibilities of different gp90 isoforms to peptic digestion. Parasites expressing pepsin-susceptible gp90 become highly invasive against target cells upon contact with gastric juice. Such is the case of a T. cruzi isolate from an acute case of orally acquired Chagas' disease; the gp90 from this strain is extensively degraded upon short period of parasite permanence in the gastric milieu. If such an exacerbation of infectivity occurs in humans, it may be responsible for the severity of Chagas' disease reported in outbreaks of oral infection.

The Role Of Cells, Neurotrophins, Extracellular Matrix And Cell Surface Molecules In Peripheral Nerve Regeneration

Wallerian degeneration is a complicated process whereby axons and myelin sheaths undergo degeneration, and eventually are phagocytosed by macrophages and Schwann cells following nerve damage. Schwann cells proliferate and the endoneural tubes persist. In addition, neurotrophins, neural cell adhesion molecules, cytokines and other soluble factors are upregulated to facilitate regeneration. The important role of cellular components, neurotrophins, and extracellular matrix components, including cell surface molecules involved in this regenerative process, is highlighted and discussed in this review.

Evaluation of ginger (Zingiber officinale Roscoe) bioactive compounds in increasing the ratio of T-cell surface molecules of CD3+CD4+:CD3+CD8+ in-vitro

The potential ability of ginger bioactive compounds in increasing the ratio of T-cell surface molecules of CD3+CD4+:CD3+CD8+ was investigated using dual tagging FITC and PE of monoclonal antibody anti-human with its fluorescence measured by flow cytometer. Oleoresin was extracted using sinkhole distillation technique. Its components namely, gingerol in fraction-1, shogaol in fraction 2 and zingeron in fraction-3 were separated by column vacuum chromatography method. The doses of oleoresin, gingerol, shogaol, and zingeron tested were 50, 100,150, 200, and 250 μg/ml. Lymphocytes (2x106 cell/ml) from human peripheral blood were isolated using ficoll density gradient technique, and cultured in the presence of the compounds in RPMI-1640 medium and phytohemaglutinin (PHA) mitogen for 96 h under normal conditions. Percentages of T-cell surface molecules (CD4+ and CD8+) were determined using dual-tagging FITC and PE fluorescents labeled on monoclonal antibody anti human. The fluorescence-labeled bands on the T-cell surface molecules were counted using flow cytometer. The experiment revealed that oleoresin and its three fractions increased the percentage of CD3+CD4+. The compound in fraction 3 of oleoresin at 200 μg/ml increased by the highest percentage of CD3+CD4+ of 9%, but slightly decreased the percentage of CD3+CD8+. These ginger bioactive compounds increased the ratio of CD3+CD4:CD3+CD8+ T-cells with the highest increment of 30% from effects of 200 μg/ml fraction 3 of oleoresin. This in vitro finding revealed that ginger bioactive compounds potentially increased cellular and humoral immune response. Further clinical studies are needed to confirm the benefits of these ginger bioactive compounds as a potential functional food for testing on HIV infected patients.

Histopaghologic Finding and Modulation of Expression of The Cell Surface Molecules in The Primary Syphilitic Sedions of a Rabbit Infected with Treponema pallidum / 대한피부과학회지

BACKGROUND: When T. pallidum invades through skin and mucous membrane, there may be some changes in the expression of cell surface molecules and cell adhesion molecules on the keratinocytes and vascular endothelial cells. OBJECTIVES: The purpose of this study was to investigate histologic changes and changes in the expression of cell surface molecules and cell adhesion molecules on the keratinocytes and vascular endothelial cells according to the time course of primary syphilic lesion. METHODS: We obtained primary syphilitic lesions by inoculation of T. pallidum into the back skin of the rabbit. Biopsies of the syphilitic lesions were performed according to the stages. and H&E and immunohistochemical stains for cell surface molecules were done. RESULTS: 1. Out of the 39 injected sites(103 T. pallidum were inoculated into the back skin of the rabbit), 24(61.5%) primary syphilitic lesion could be found. The duration for the developement of papules, ulcers, and softenings is an average 15 days, 27 days, and 47 days respectively. 2. H & E findings :Acanthosis, spongiosis, and exocytosis in the epidermis were observed in the papule of the primary syphilitic lesion. Infiltration of inflammatory cells, including many lymphocytes, a few histiocytes and plasma cells was also observed. Some cases showed endothelial cell swelling of vessels. Compared to papules, the number of lymphocytes in the ulcer reduced but the number of histiocytes increased. Softened lesion showed infiltrating cells, consisted of lymphocytes and histiocytes, and fibrosis. 3. Immunohistochemical findings :Keratinocytes of the lower epidermis, upper portion of hair follicles, vessel, and infiltrating inflammatory cells in papules and ulcers showed expression of the MHC class II molecule. Most of the infiltrating cells in all cases of papules showed CD5 expression. Keratinocytes, inflammatory cells, and vascular endothelial cells showed positive reaction to ICAM-1 stain in papules and ulcers. VCAM-1 showed the positive reaction to the vascular endothelial cells in the papules and ulcers. In softened lesions, the intensity of the positive reaction to MHC class II, ICAM-1 and VCAM-1 was weakened. CONCLUSION: The skin of the rabbit which was invaded by T. pallidum increased the expression of the cell surface molecules and cell adhesion molecules of MHC class I, MHC class II, ICAM-1, and VCAM-1. We believe that these expressions of cell surface molecules and cell adhesion molecules by T. pallidum, inflammatory cells, activated keratinocytes, and vascular endothelial cells play important roles in the host defence mechanism and the T. pallidum infection.

Impacts of rhG-CSF on neutrophilic granulocytes and their surface molecules expression / 临床检验杂志

Objective To investigate injecting recombinant human granulocyte-colony stimulating factor(rhG-CSF) on the proportion of peripheral neutrophilic granulocyte and their surface molecules expression in healthy people.Methods The peripheral blood samples were collected from the healthy people who were injected with rhG-CSF.The expressions of CD62L,CD11a,CD44 and CD49d on the surface neutrophilic granulocytes were determined before and after injection with flow cytometery.Results The median percentage of neutrophilic granulocytes in peripheral blood leucocytes after injecting rhG-CSF significantly increased from 60% to 85%(P0.10).Conclusion Injecting rhG-CSF may obviously impact the expression of CD62L and CD49d on neutrophilic granulocytes.