Lamellar particles(lamellae) were prepared by non-solvent precipitation from crystalic poly-L-lactic acid (PLLA). The PLLA lamellae exhibite a diamond or stepped irregular shape with a size range between 3-5 microns. Prepared without any surfactants and dispersing agents, the lamellar particles have clean surface, which is advantageous for the adsorption of proteins. The PLLA lamellar particles adsorbed protein cytokine interferon-alpha (IFN-alpha) with an adsorption efficiency of > or = 95%. The release of loaded IFN could continue for more than 10 days. The cell incubation experiments showed that the PLLA lamellar particles were easy to be phagocytosed by macrophages. The immunological experiments showed that the biological activity of IFN-alpha loaded on the PLLA lamellar particle was effectively retained.
Adsorption ; Chemistry, Pharmaceutical ; Drug Carriers ; Interferon-alpha ; chemistry ; pharmacology ; Lactic Acid ; chemistry ; Particle Size ; Polyesters ; Polymers ; chemistry

This study was aimed to examine the expression of apoptosis-associated gene Fas in HeLa cell, explore the effects of the co-immobilized cytokines (tumor necrosis factor-alpha and interferon-gamma), and probe the potential mechanism of action. The preparation and application of the research couple IFN-gamma and TNF-alpha to the polystyrene cell culture plate were performed using the Photo-immobilization method, with different doses (20 ng/well and 200 ng/well) and synthesized optical active material. HeLa cells were treated with cytokines for two dose and 1, 3, 6 days. The result showed that the free cytokines induced HeLa apoptosis quickly, yet the HeLa apoptosis induced by co-immobilized cytokines had longer effect.
Apoptosis ; drug effects ; genetics ; Drug Synergism ; HeLa Cells ; Humans ; Immobilized Proteins ; chemistry ; pharmacology ; Interferon-gamma ; chemistry ; pharmacology ; Tumor Necrosis Factor-alpha ; chemistry ; pharmacology ; Up-Regulation ; fas Receptor ; metabolism

BACKGROUNDHaptoglobin (Hp) is one of the acute-phase proteins. Recent studies have demonstrated that Hp exerts immunoregulatory and anti-inflammatory actions and may be one of the inhibitory factors of immune reactions in the skin. In this study we investigated the regulation of Hp expression in a human keratinocyte cell line HaCaT by various cytokines and glucocorticoid.

METHODSHaCaT cells were cultured with IL-6 (50 ng/ml), TNF-alpha (20 ng/ml), IFN-gamma (20 ng/ml) or IL-4 (20 ng/ml) with or without 1 micromol/L dexamethasone in 6-well plates for 12, 24 and 48 hours. Both the cells and the supernatants were collected to detect the changes of Hp expression by reverse-transcription PCR, ELISA and immunohistochemistry.

RESULTSThe results showed that Hp expression were elevated at both the mRNA and protein level by the combination of IL-6, TNF-alpha or IL-4 with dexamethasone, whereas the three cytokines alone did not upregulate the Hp expression. IFN-gamma showed no effect on the Hp expression in HaCaT cells.

CONCLUSIONSThese findings suggest that different inflammatory cytokines as well as glucocorticoid may be involved in the regulation of Hp expression in keratinocytes, and this may be one of the negative feedback mechanisms in inflammatory skin diseases.

Cell Line ; Dexamethasone ; pharmacology ; Glucocorticoids ; pharmacology ; Haptoglobins ; analysis ; Humans ; Interferon-gamma ; pharmacology ; Interleukin-4 ; pharmacology ; Interleukin-6 ; pharmacology ; Keratinocytes ; chemistry ; drug effects ; Tumor Necrosis Factor-alpha ; pharmacology

In order to create a novel recombinant human interferon alpha2b (rh-IFN alpha2b) with higher biological activity, we subjected the rational designed sequence of rh-IFN alpha2b to direct evolution by strategy of the combinatorial mutagenesis. The amino acid residues at multiple sites of 52-53-55, 103-107, and 121-125 were simultaneously mutated. The resulted gene of the mutated rh-IFN a2b was cloned into the pET28a and expressed in E. coli BL21 Condon plus (RIL). The anti-virus activity of the novel interferon alpha2b was 9.3 x 10(7) IU/mg, 93 times higher than the wild type (1 x 10(6) IU/mg). The results showed that the multiple point mutation used in this study could effectively combine the site effects of rh-IFN alpha2b and increase its biological activity.
Antiviral Agents ; pharmacology ; Base Sequence ; Combinatorial Chemistry Techniques ; Humans ; Interferon-alpha ; genetics ; pharmacology ; Molecular Sequence Data ; Mutagenesis, Site-Directed ; methods ; Recombinant Proteins ; genetics ; pharmacology

Interferon (IFN) has therapeutic potential for a wide range of infectious and proliferative disorders. However, the half-life of IFN is too short to have a stable therapeutic effect. To overcome this problem, serum immunoglobulin has been fused to IFN. In this study, the efficacy of serum immunoglobulin fused INFs (si-IFN1 and si-IFN2) was evaluated on athymic mice bearing colon 26 adenocarcinoma cells. Seven days after the implantation of tumor cells, each group of mice was injected once a week with si-IFN1 and si-IFN2 at two different concentrations (10 x : 30 microgram/kg and 50 x : 150 microgram/kg). A slight anti-tumoral effect was observed in all 10 x groups compared to the control. In the 50 x groups, however, si-IFN1 and si-IFN2 showed significant anti- tumoral effects compared to the control. To gain more information on the mechanisms associated with the decrease of tumor size, a Western blot assay of apoptosis-related molecules was performed. The protein expression of cytochrome c, caspase 9, 6, and 3 were increased by si-IFN1 and si-IFN2. These 2 IFNs also increased the expressions of p53, p21, Bax and Bad. Interestingly, si-IFN1 and si-IFN2 decreased the expression of VEGF-beta. Taken together, serum immunoglobulin fused IFNs increased therapeutic efficacy under current experimental condition.
Adenocarcinoma/*drug therapy ; Alanine Transaminase/blood ; Animals ; Antineoplastic Agents/chemistry/pharmacology ; Blood Urea Nitrogen ; Dose-Response Relationship, Drug ; Immunoglobulins/*chemistry/*pharmacology ; Interferon Alfa-2a/chemistry/pharmacology ; Interferon-alpha/*chemistry/*pharmacology ; Mice ; Mice, Nude ; Neoplasms, Experimental/*drug therapy ; Polyethylene Glycols/chemistry/pharmacology ; Recombinant Fusion Proteins/chemistry/pharmacology

To explore the effect of the co-photo-immobilization of Recombinant Human Interferon-r (IFN-gamma) and Recombinant Tumor Necrosis Factor (TNF-alpha) on the Hela cell cultivated in vitro. Co-immobilize the photoactive IFN-gamma and TNF-alpha on the polystyrene membrane; Cultivate the Hela cell on the cell culture polystyrene plate surface and set up the cultivation of the photo-immobilization IFN-gamma+TNF-alpha, Photo-immobilization IFN-gamma, nature (IFN-gamma, TNF-alpha, IFN-gamma+TNF-alpha). After 48 hours, the inhibition effect against Hela cells was studied in vitro by SEM and flow cytometry. It showed that the co-immobilization IFN-gamma+TNF-alpha has a more obvious inhibition effect on the Hela cell. And IFN-gamma can cooperate with TNF-alpha obviously.
Antineoplastic Agents ; chemistry ; immunology ; pharmacology ; Apoptosis ; drug effects ; Cell Proliferation ; drug effects ; Drug Synergism ; HeLa Cells ; Humans ; Interferon-gamma ; chemistry ; pharmacology ; Photochemistry ; Recombinant Proteins ; Tumor Necrosis Factor-alpha ; chemistry ; pharmacology

OBJECTIVETo compare several schemes of inducing and expanding the antibody-mediated high efficiency CIK (AMHE-CIK) in vitro, so as to find out a method that can acquire a large number of cells capable to kill the tumor cells in a short time.

METHODSPeripheral blood mononuclear cells (PBMNC) from healthy volunteers was isolated and activated with CD3 antibody, then were cultured with the addition of different cytokines (IL-2, IL-4, G-CSF, GM-CSF, IFN-γ, TNF-α) for 14 days in vitro. The morphological changes of cells were observed by light microscopy. Based on the immunophenotypes of cells in each groups analyzed by flow cytometry, the cytokines capable to induce the dendritic cells and killer cells were screened out, respectively. According to different combination of cytokines, the cells were divided 4 groups: control, IL-2, group 1 (componant A included IL-2, IL-4, and GM-CSF. Componant B included IL-2, G-CSF, IFN-γ, and TNF-α), and group 2 (componant A included IL-2, IL-4, and GM-CSF. Componant B included IL-2, IL-4, G-CSF, IFN-γ, and TNF-α). The proliferation and differentiation of CD3(+) CD8(+) and CD3(+) CD56(+) cells were measured by flow cytometry after culture in vitro for 7 days.

RESULTSAfter inducing and expanding in vitro for 7 days, the cell proliferation rate of control group, IL-2 group, group 1 and group 2 were 1.57 ± 0.01, 4.17 ± 0.16, 5 ± 0.47, 7.17 ± 0.24-folds, respectively. The differences between IL-2 group, group 1, group 2 and control group were statistically significant (P < 0.05). The immunophenotype analysis showed that the proportion of CD3(+) CD8(+) induced by each protocol was 13.96 ± 0.23%, 26.33 ± 0.55%, 36.83 ± 0.34% and 35.88 ± 0.16%, respectively. The proportion of CD3(+) CD8(+) in group 1 and 2 was higher than that in IL-2 group (P < 0.05), but the difference between them was not significant (P < 0.05). The proportions of CD3(+) CD56(+) induced by each protocol were 11.03 ± 0.28%, 29.31 ± 0.60%, 39.96 ± 0.38% and 29.33 ± 0.54%, respectively, the proportion of group 1 was higher than that of IL-2 group and group 2 (P < 0.05), but the difference between IL-2 group and group 2 was not significant (P < 0.05).

CONCLUSIONThe group 1 protocol obtained from this study can promote the proliferation of DC-CIK and also increase the proportion of the tumor killing cells (CD3(+) CD8(+) and CD3(+) CD56(+)).

Cell Culture Techniques ; Cells, Cultured ; Culture Media ; chemistry ; Cytokine-Induced Killer Cells ; cytology ; Granulocyte Colony-Stimulating Factor ; pharmacology ; Granulocyte-Macrophage Colony-Stimulating Factor ; pharmacology ; Humans ; Immunophenotyping ; Interferon-gamma ; pharmacology ; Interleukin-2 ; pharmacology ; Interleukin-4 ; pharmacology ; Tumor Necrosis Factor-alpha ; pharmacology

OBJECTIVETo determine whether advanced glycosylation end products modified bovine serum albumin (AGEs-BSA) affects endothelial cell lateral junction protein, platelet-endothelial cell adhesion molecule-1 (PECAM-1) in the presence or absence of inflammatory mediators.

METHODSCultured human umbilical vein endothelial cells (HUVECs) were exposed to AGEs-BSA for 6, 12, 24, and 36 hours, and exposed to AGEs-BSA glycosylated with different concentrations of glucose, tumor necrosis factor-alpha (TNF-alpha), interferon (IFN-gamma), TNF-alpha + IFN-gamma and AGEs-BSA + TNF-alpha for 24 hours, respectively. Expression of PECAM-1 mRNA was measured by semi-quantitative reverse transcription-polymerase chain reaction (RT-PCR) with beta-actin as an internal standard, and sequencing of RT-PCR products was performed to confirm the specificity of amplification for PECAM-1 gene. The endothelial cell surface expression of PECAM-1 was determined by flow cytometry (FCM).

RESULTSThere were no significant changes in the expression of PECAM-1 mRNA and protein when the cells were exposed to AGEs-BSA with different concentrations or periods (P > 0.05). However, PECAM-1 expression was reduced in the cells treated with TNF-alpha, IFN-gamma, TNF-alpha + IFN-gamma and AGEs-BSA + TNF-alpha. The level of PECAM-1 treated with AGEs-BSA + TNF-alpha was lower than that of TNF-alpha treated alone (P < 0.01).

CONCLUSIONSAGEs-BSA had no effect on the expression of PECAM-1 mRNA and protein in cultured HUVEC. With the presence of inflammatory mediator TNF-alpha, AGEs-BSA decreased the level of PECAM-1, which might reduce the adhesion interaction between adjacent endothelial cells, enhance the permeability of endothelial cells, and might be implicated in the endothelial dysfunction and pathogenesis of atherosclerosis in patients with diabetes mellitus. The significance of this phenomenon in intracellular signal transduction remains to be determined.

Cells, Cultured ; Endothelial Cells ; chemistry ; drug effects ; Glycation End Products, Advanced ; pharmacology ; Humans ; Interferon-gamma ; pharmacology ; Platelet Endothelial Cell Adhesion Molecule-1 ; analysis ; Serum Albumin, Bovine ; pharmacology ; Tumor Necrosis Factor-alpha ; pharmacology ; Umbilical Veins

OBJECTIVETo investigate the effect of huanglian jiedu tang on Alzheimer' s disease, and its influence on cytokines.

METHODAD rats models were made by A beta injection, and then water-maze tests were made to investigate the study and memory ability. TNF-alpha, INF-gamma and IL-2 were examined by ELISA. After treatment with huanglian jiedu tang, the rats were evaluated again.

RESULTAfter A beta injection, the study and memory ability of the rats was decreased. By the treatment with huanglian jiedu tang, it was improved (P < 0.01); TNF-alpha, INF-gamma and IL-2 levels of AD rats were higher than those in control group (P < 0.01); after treatment the levels came down (P < 0.01).

CONCLUSIONHuanglian jiedu tang can improve the study and memory ability of AD rats by changing their immune state.

Alzheimer Disease ; blood ; psychology ; Animals ; Coptis ; chemistry ; Cytokines ; blood ; Drug Combinations ; Drugs, Chinese Herbal ; isolation & purification ; pharmacology ; Gardenia ; chemistry ; Interferon-gamma ; blood ; Interleukin-2 ; blood ; Male ; Memory ; drug effects ; Phellodendron ; chemistry ; Plants, Medicinal ; chemistry ; Rats ; Rats, Sprague-Dawley ; Scutellaria ; chemistry ; Tumor Necrosis Factor-alpha ; metabolism

OBJECTIVETo observe the effect of volatile oil of Schizonepetae Herba (VOSH), and its essential components-menthone and pulegone against anti-influenza virus A/PR/8/34 (H1N1) in vivo and in vitro, as well as the signaling mechanism of its toll-like receptor/interferon (TLR/IFN).

METHODThe lung-adapted PR-8 virus model was prepared in mice. They were administered with preventive and therapeutic drugs, and the hemagglutination titer of model animals was determined to evaluate in vivo effect against H1N1. ELISA test was conducted to observe the effect on IFN-alpha, IFN-beta, IL-2, IL-6 and TNF-alpha in serum, as well as IFN-beta secretion in H1N1 infected MDCK supernatant. Real-time RT-PCR was employed to observe the expression levels of IRAK4 and TLR3 mRNA.

RESULTThe in vivo experiment shows that the hemagglutination titer was significantly decreased when the mice were treated with VOSH (0.266 mg x kg(-1)), menthone(0.5 mg x kg(-1)) and pulegone (0.19 mg x kg(-1)) in therapeutic way; VOSH (0.226 mg x kg(-1)) had a significant effect on increasing serum levels of IFN-alpha, IL-2; Methone (0.5 mg x kg(-1)) had a significant effect on increasing serum levels of IFN-beta; Methone (0.5 mg x kg(-1)) and pulegone (0.19 mg x kg(-1)) had a significant effect on decreasing serum levels of IL-6; VOSH (0.452, 0.226 mg x kg(-1)) and pulegone (0.19 mg x kg(-1)) had a significant effect on decreasing serum levels TNF-alpha. The in vitro experiment showed that the expression levels of IRAK4 mRNA and IFN-beta were significantly increased in VOHS (0.1 g x L(-1)) and pulegone (0.1 g x L(-1)) groups; and the menthone (0.25 g x L(-1)) group showed a significant rise in the expression levels of IRAK4 mRNA, but a notable decline in TLR3 mRNA.

CONCLUSIONThe administration with VOSH, methone and pulegone in therapeutic way can significantly decrease the hemagglutination titer, which demonstrates the anti-virus effect of the administration in therapeutic way, but no notable efficacy of the administration in preventive way. The in vivo anti-virus mechanism is related to regulation of IFN-alpha, IFN-beta and IL-2.

Animals ; Drugs, Chinese Herbal ; pharmacology ; Female ; Humans ; Influenza A Virus, H1N1 Subtype ; drug effects ; physiology ; Influenza, Human ; drug therapy ; genetics ; immunology ; virology ; Interferon-alpha ; genetics ; immunology ; Interleukin-1 Receptor-Associated Kinases ; Interleukin-2 ; genetics ; immunology ; Interleukin-6 ; genetics ; immunology ; Lamiaceae ; chemistry ; Male ; Mice ; Oils, Volatile ; pharmacology ; Tumor Necrosis Factor-alpha ; genetics ; immunology