Acute release of cytokines is proportional to tissue injury induced by surgical trauma and shock in rats

Cytokines are important mediators of the inflammatory reaction and microvascular injury after trauma and tissue ischemia. The plasma activity of a cytokine reflects the net effect of positive and negative signals. We examined the sequential serum activity of IL-1, IL-2, IL-6, and TNF in a severe model of splanchnic artery occlusion (SAO) shock induced in rats by total occlusion of the superior mesenteric and the celiac arteries for 40 min. A control group with negligible surgical intervention and two sham-shock groups, one with minor operation and another with major surgery employed in SAO rats, both without vascular occlusion, were also studied. No IL-1 activity was detected throughout the 190-min experimental protocol in any of the groups. Low activity of IL-2 was measured only in SAO rats (1 U/ml at the peak). We found graded increases in serum TNF and IL-6 activities which were proportional to the surgical trauma and were highest in SAO rats (IL-6 up to 30 U/ml,P<0.01 from both sham groups; TNF, 2500 pg/ml 30 min after reperfusion,P<0.01 from both sham groups). These data further support the role played by cytokines in the early mediation of surgical trauma and shock.This study was carried out in adherence with the NIH guidelines for the use of experimental animals.     

CD40/CD154 system and pro-inflammatory cytokines in young healthy male smokers without additional risk factors for atherosclerosis

Objective and design:  Atherosclerosis, as an inflammatory disease, is characterized by pathologically altered levels of cytokines. We investigated whether smoking affects the CD40/CD154 system and pro-inflammatory cytokines in young males without other risk factors for atherosclerosis. Subjects:  Young male smokers (n=13) and 14 non-smoking controls were investigated. Methods:  The differences in CD40/CD154 system and serum cytokines between the groups were measured using flow cytometry and ELISA. Results:  In smokers, there was a strong trend (P<0.06) for increased CD40 expression on platelets as compared with non-smokers. However, there were no significant differences in CD40 expression on monocytes or in CD154 expression on platelets and T-cells between smokers and non-smokers. There was a strong trend for increased platelet-monocyte aggregates in smokers (P<0.06). Also, smokers had slightly but not significantly elevated hsCRP and IL-6 levels, and slightly decreased TNF-α and MCP-1. Interestingly, IL-18, a cytokine which has the ability to promote both Th1 and Th2 responses, was significantly decreased in smokers group (P=0.03 vs controls). Conclusions:  In young healthy males, smoking is not associated with dramatic changes in CD40/CD154 system. However, cigarette smoke alters the secreted cytokine profile, leading to significant decrease in systemic IL-18 levels. Received 1 October 2007; returned for revision 5 November 2007; received from final revision 28 April 2008; accepted by G. Wallace 9 June 2008 C. D. Garlichs, I. Cicha: These authors contributed equally to this work.     

In vivo effects of cytokines on psoriatic skin grafted on nude mice: involvement of the tumour necrosis factor (TNF) receptor

Following engraftment of human involved psoriatic skin to nude mice there is a partial normalization of pathology associated with a loss of inflammatory leucocytes. However, the epidermis remains hyperproliferative, which may reflect a primary defect. The roles of TNF-α, IL-1 and IL-6 in epidermal hyperproliferation of grafted psoriatic lesions were investigated. Before and after treatment, grafts were analysed to determine epidermal thickness and labelling index (LI). HLA-DR, intercellular adhesion molecule-1 (ICAM-1), and TNF receptor (TNF-R; p75 and p55) expression were determined by immunoperoxidase staining. Psoriatic epidermis was found consistently to be negative for p55 TNF-R and p75 TNF-R before grafting. Following engraftment, TNF-R-positive cells (i.e. p55 by keratinocytes; p75 by epidermal dendritic cells) were identified throughout the epidermis. Higher numbers of p75 TNF-R epidermal dendritic cells were found in grafts following a course of TNF-α, IL-6 or IL-1 treatment. The p55 form of the TNF-R expressed by keratinocytes was significantly elevated after treatment with TNF-α or IL-6. HLA-DR and ICAM-1 were also expressed in these grafts. TNF-α, anti-IL-1, and anti-IL-6 treatment induced a marked decrease in the epidermal thickness and LI of psoriatic graft tissue, correcting the hyperproliferation associated with psoriatic epidermis. Supraphysiological levels of TNF-α may saturate and consequently down-regulate their own receptors, leading to a paradoxical inhibitory effect.     

Ivermectin inhibits LPS-induced production of inflammatory cytokines and improves LPS-induced survival in mice

Objective and Design:  To investigate whether ivermectin, a semi-synthetic derivative of a family of macrocyclic lactones could inhibit lipopolysaccharide (LPS)-induced inflammation in vivo and in vitro. Materials and Methods:  C57BL/6 mice were administered ivermectin (or saline) orally and challenged intraperitoneally with LPS at a lethal dose of 32 mg/kg. RAW 264.7 murine macrophages were stimulated with LPS at 1 μg/ml, with or without ivermectin for 6, 12 and 24 h. The production of tumor necrosis factor-α (TNF-α), interleukin-1? (IL-1?) and interleukin-6 (IL-6) in serum from mice and supernatants from cells were measured by ELISA. Nuclear factor-kB (NF-kB) translocation with subunit p65 was evaluated by immunocytochemical analysis. Results:  Ivermectin improved mouse survival rate induced by a lethal dose of LPS. In addition, ivermectin significantly decreased the production of TNF-α, IL-1? and IL-6 in vivo and in vitro. Furthermore, ivermectin suppressed NF-kB translocation induced by LPS. Conclusions:  The results indicate that ivermectin may inhibit LPS-induced production of inflammatory cytokines by blocking NF-kB pathway and improve LPS-induced survival in mice. This finding might provide a new strategy for the treatment of endotoxemia and associated inflammation. Xuemei Zhang and Yu Song contributed equally to this work Received 13 January 2008; returned for revision 7 February 2008; received from final revision 3 April 2008; accepted by M. Parnham 4 April 2008 Xuemei Zhang and Yu Song contributed equally to this work     

Role of lymphocytes in the course of murine zymosan-induced peritonitis

OBJECTIVE AND DESIGN: To investigate a putative role of lymphocytes in a murine model of zymosan peritonitis. MATERIAL OR SUBJECTS: Rag-deficient mice (KO) and their counterparts (WT) (13 animals in each group). TREATMENT: Mice were injected i. p. with zymosan (2 mg/ml, 0.5 ml/mouse) and sacrificed either 30 min or 6 h post-treatment. METHODS: At 30 min of inflammation vascular permeability was assessed by peritoneal leakage of i. v. injected Evans blue. At 6 h of peritonitis leukocyte numbers were estimated (Turk's staining), and MMP-2 and -9 presence (zymography). Levels of inflammatory mediators were evaluated by either ELISA (PGE(2), KC) or Cytometric Bead Array (IL-6, IL-10, MCP-1, IFN-gamma, TNF-alpha, and IL-12p70). The Amount of nitric oxide (NO) was measured by the Greiss reaction. Differences between WT and KO mice were analyzed by Student's t-test (p 相似文献   

Neutrophil elastase inhibitor (sivelestat) reduces the Levels of inflammatory mediators by inhibiting NF-kB

Objective:  Sivelestat sodium hydrate (sivelestat) is a specific synthetic inhibitor of neutrophil elastase (NE). Various studies suggest that sivelestat treatment reduces inflammation. In this study, we tested the hypothesis that sivelestat acts as an inhibitor of inflammatory mediators and prevents nuclear factor-kB (NF-kB) activation. Methods:  In the presence and absence of sivelestat, the mouse macrophage cell line RAW 264.7 was stimulated with lipopolysaccharide (LPS) and the levels of inflammatory mediators (TNF-α, IL-6 and high mobility group box 1 (HMGB1)) and nitrite in the cell supernatant were measured, along with inducible nitric oxide synthase (iNOS) expression. Results:  While LPS administration increased the secretion of inflammatory mediators and nitric oxide (NO), sivelestat decreased the secretion of these mediators. Cell signaling studies demonstrated that sivelestat decreased NF-kB activation by inhibiting IkB phosphorylation. Conclusion:  Sivelestat may inhibit the various inflammatory mediators through NF-kB inhibition. Received 14 June 2008; returned for revision 11 July 2008; received from final revision 4 September 2008; accepted by H. Katori 9 September 2008     

Regulation of innate and adaptive immune responses by the related cytokines IL-12, IL-23, and IL-27

The functional characterization and subsequent purification of T cell growth factor/interleukin (IL)-2 in the early 1980s established this secreted protein as a key mediator of immune cell activation and provided the prototype that enabled the discovery of numerous cytokines over the ensuing two decades. While soluble immunoregulatory factors were initially identified functionally as biological activities present in the culture supernatants of activated lymphocytes/monocytes, this methodology shifted radically following the completion of the human genome sequence. Computer-generated structural modeling algorithms have replaced functional assays and biochemical purification as the initial means of discovering new cytokines. To date, a total of 31 interleukins, as well as over a dozen other related hematopoietic factors, have been identified. These cytokines and their receptors may be grouped on the basis of structural homologies as well as by shared ligand and receptor subunits. The challenge now at hand is to define the biological functions of the newly identified cytokines and to elucidate the common and divergent roles of related family members. This point is well illustrated by the IL-12/IL-23/IL-27 family, whose members share ligand and receptor subunits and play somewhat overlapping roles in innate and adaptive immune responses. These three cytokines are not entirely redundant, as they may preferentially activate na?ve or memory T cells, induce discrete T cell cytokine profiles, contribute to distinct stages of host immune responses to infectious agents, and differentially promote autoimmunity. Further elucidation of the unique functions of the IL-12 family members may lead to improved immunodiagnostics and therapies.     

Age-related changes in glutamate release in the CA3 and dentate gyrus of the rat hippocampus

The present studies employed a novel microelectrode array recording technology to study glutamate release and uptake in the dentate gyrus, CA3 and CA1 hippocampal subregions in anesthetized young, late-middle aged and aged male Fischer 344 rats. The mossy fiber terminals in CA3 showed a significantly decreased amount of KCl-evoked glutamate release in aged rats compared to both young and late-middle-aged rats. Significantly more KCl-evoked glutamate release was seen from perforant path terminals in the DG of late-middle-aged rats compared young and aged rats. The DG of aged rats developed an increased glutamate uptake rate compared to the DG of young animals, indicating a possible age-related change in glutamate regulation to deal with increased glutamate release that occurred in late-middle age. No age-related changes in resting levels of glutamate were observed in the DG, CA3 and CA1. Taken together, these data support dynamic changes to glutamate regulation during aging in subregions of the mammalian hippocampus that are critical for learning and memory.     

Altered expression of pro-inflammatory and developmental genes in the fetal brain in a mouse model of maternal infection

Human studies of unexplained cerebral palsy (CP) suggest an association with maternal infection. We used an established model of maternal infection, lipopolysaccharide (LPS) administration, to investigate the molecular changes in the fetal brain that may link maternal infection and CP. We compared gene expression in brains from mouse pups exposed to LPS in utero to those from saline-treated controls. Dams were injected with 50 microg LPS or saline on E18 with surgical delivery from 0.5 to 6h later. Differential gene expression was analyzed in the whole mouse brain using RT-PCR. When compared to control mice, pups exposed to LPS showed increased expression of pro-inflammatory genes monocyte chemoattractant protein-1 (MCP-1), interleukin-6 (IL-6), and interleukin-1beta (IL-1beta), as well as VEGF, a regulator of vascular development and permeability, the anti-apoptotic protein Y-box-binding protein-1 (YB-1), and the neuronal differentiation factor necdin. LPS-exposed mice also showed downregulation of semaphorin 5b and groucho, involved in axon guidance and neurogenesis, respectively, providing evidence that LPS may disrupt normal developmental pathways. These data suggest possible mechanisms for adverse neurological outcomes following maternal infection involving elevated cytokine levels and altered expression of developmental genes in the fetal brain.     

Presence and distribution of biogenic amines (histamine, serotonin and epinephrine) in immunophenotyped human immune cells

Objective:  In animal experiments many hormones were demonstrated in immune cells. However, very few data are at our disposal in the case of human immune cells. In an earlier experiment, ACTH, endorphin and T₃ were studied and found in different subsets of human immune cells. Here, three biogenic amines (histamine, serotonin and epinephrine) were studied. Methods:  Biogenic amine content of immunophenotyped human lymphocytes from 15 blood donors were investigated by multicolor flow cytometry using anti-biogenic amine antibodies. Monocytes and granulocytes separated by size and granularity were also studied. Results:  Each biogenic amine could be detected in each subset of leukocytes, except epinephrine and serotonin in granulocytes. Activated T cells contained a higher amount of the amines, and CD19+B cells a higher amount of histamine, related to the whole lymphocyte population and to other subsets. Monocytes contained more histamine and epinephrine than lymphocytes and granulocytes contained twice as much histamine as monocytes and three times as much as lymphocytes. Conclusion:  Human lymphocytes contain the three biogenic amine, similar to rat. However, while each amine was present in monocytes, in granulocytes serotonin and epinephrine were not demonstrated. The results call attention to the possible extrapolation of animal data to human lymphocytes and monocytes, but in the case of granulocytes, caution is needed. Taking into consideration earlier results, activated T cells appear to have an important role in the loss or production of hormones inside the immune system. Received 14 January 2008; returned for revision 11 February 2008; received from final revision 20 February 2008; accepted by I. Ahnfelt-R?nne 18 March 2008     

Antigen-nonspecific activation of CD8+ T lymphocytes by cytokines: relevance to immunity, autoimmunity, and cancer

Development of T lymphocytes and their survival in the periphery are dependent on signals emanating from cytokine receptors as well as the T cell antigen receptor (TCR). These two signaling pathways play distinct and complementary roles at various stages of T cell development, maturation, survival, activation and differentiation. During immune response to foreign antigens initiated by TCR signaling, cytokines play a key role in the expansion of activated T cells. Even though the initial activation of T cells occurs via the TCR, this requirement can be overcome under certain circumstances. During lymphopenia, cytokines trigger memory CD8⁺ T cells to undergo antigen non-specific homeostatic expansion, whereas naïve CD8⁺ T cells require both cytokines and TCR signaling. Recent reports show certain combinations of cytokines can induce proliferation and effector functions of naïve CD8⁺ T cells without concomitant stimulation via the TCR. While such antigen non-specific stimulation of naïve T cells might significantly boost the adaptive immune response, it could also have an undesirable effect of triggering potentially autoreactive cells. Understanding the mechanisms and the regulation of cytokine-driven stimulation of naïve CD8⁺ T cells may lead to novel strategies of intervention for autoimmune diseases. On the other hand, in vitro expansion of naïve CD8⁺ T cells by certain combinations of cytokines could be used to generate tumor-specific cells with ideal properties for cellular immunotherapy of cancer.     

Reduced production of histamine-induced suppressor factor (HSF) by atopic mononuclear cells and decreased prostaglandin E2 output by HSF-stimulated atopic monocytes

To characterize the defect responsible for abnormal histamine-induced suppressor cell function observed in atopic subjects, we studied histamine-induced suppressor factor (HSF) production and augmented prostaglandin E2 production. In addition, we exogenously provided interleukin 1 to determine whether abnormal histamine-induced suppressor cell function could be corrected by this monokine. Mononuclear cells from 16 asymptomatic atopic subjects generated significantly less (p less than 0.01) histamine-induced suppressor activity than cells from 10 nonatopic controls, and mean suppressor-factor production was also significantly reduced (p less than 0.01). The latter two responses were not corrected by interleukin 1. Atopic monocytes produced significantly less (p less than 0.01) prostaglandin E2 in response to suppressor factor than did control monocytes. The atopic group had phenotypically normal numbers of T-helper cells, T-suppressor cells, and ratios of these cells. These data indicate that despite having phenotypically adequate numbers, T-suppressor cells from atopics produce fewer suppressor signals than cells from nonatopics and their monocytes produce less prostaglandin E2 even if the suppressor signals are provided.     

Interactions between the serotonergic and histaminergic systems in the central cardiovascular regulation in haemorrhage-shocked rats: involvement of 5-HT1A receptors

Aim and objective:  The aim of the work was to characterise the nAChRs on human PBMC. Method:  PBMC were isolated from human blood buffy coats provided by the blood transfusion service and were used for radioligand binding studies with [³H]-nicotine. RT-PCR experiments were used to determine nAChR subunit expression while immunoblotting experiments were used to confirm that nAChR subunits identified by RT-PCR were translated into protein. Results:  Binding studies suggested the presence of one binding site for (-)- nicotine on human peripheral blood lymphocytes. Competition studies showed that only (-)- nicotine, epibatidine and α-bungarotoxin, displaced radiolabelled nicotine from cells. RT-PCR studies demonstrated mRNA for α4, α5, α7, β1 and β2 nAChRs subunits in PBMC. Expression of mRNA for the a5 subunit of nAChR was observed in all lymphocyte samples tested. In contrast, the expression pattern of mRNAs for α4, α7, β1, and β2 mRNAs subunits of nAChRs, varied between samples. Western blot analysis showed that protein for α4, α5, and α7 and β2 nAChR subunits was expressed in most, but not all of the PBMC samples tested but some of the bands obtained were faint. Conclusion:  The results obtained suggest that human PBMC contain nAChRs containing α4β2, α4β2α5, and/or α7 subunits. Received 6 August 2008; returned for revision 3 September 2008; received from final revision 3 October 2008; accepted by M. Parnham 6 October 2008     

Differential distribution of fibroblast growth factor (FGF)-7 and FGF-10 in L-arginine-induced acute pancreatitis

The regenerative process of the pancreas after acute pancreatitis (AP) is characterized by acinar and ductal cell proliferation with synthesis and transient deposition of extracellular matrices. Various growth factors were reported to be highly expressed in AP, but their regulation has not yet been clarified. Fibroblast growth factor (FGF)-7, also known as keratinocyte growth factor (KGF), and FGF-10 are members of the FGF family and show high structural homology and similar biological characteristics. Both are mainly synthesized by mesenchymal cells and stimulate epithelial cells via KGF receptor (KGFR) which is a splice variant of FGFR-2. In the present study, we attempted to immunohistochemically determine the localization of FGF-7 and FGF-10 in pancreatic tissues of an L-arginine-induced rat pancreatitis model. Furthermore, highly specific KGFR antibodies were prepared and used for Western blot analysis and immunohistochemistry. In the normal pancreas, FGF-7 was localized in alpha cells of islets, but FGF-10 was not detected. KGFR was also localized in islet cells, ductal cells, and centroacinar cells in the normal pancreas. In the pancreatic tissues of rats with L-arginine-induced pancreatitis, FGF-7 was localized in alpha cells, whereas FGF-10 was expressed in vascular smooth muscle cells (VSMCs). KGFR was not expressed in centroacinar cells and its level decreased after L-arginine treatment. However, KGFR was detected instead in some acinar cells and VSMCs in addition to islet cells. These findings suggest that FGF-7 and FGF-10 contribute to the regeneration and differentiation of acinar cells and angiogenesis in AP through KGFR.     

Inhibitory effect of ZPDC glycoprotein on the expression of inflammation-related cytokines through p38 MAP kinase and JNK in lipopolysaccharide-stimulated RAW 264.7 cells

Objective:  This study was carried out to investigate the anti-inflammatory potentials of 24 kDa glycoprotein isolated from Zanthoxylum piperitum DC fruit (ZPDC glycoprotein) in lipopolysaccharide (LPS)-stimulated murine macrophage cell line (RAW 264.7 cells). Material and Methods:  RAW 264.7 cells were treated with ZPDC glycoprotein (50–200 μg/ml) in presence of LPS (2 μg/ml). The changes of the levels of inflammation-related factors were determined by using Western blot, EMSA, and RT-PCR. Results:  ZPDC glycoprotein has inhibitory effects on the phosphorylation of p38 mitogen-activated protein (MAP) kinase and c-Jun N-terminal kinase (JNK), on the DNA binding activity of activator protein-1 (AP-1), and on the expression of c-Jun and c-Fos in LPS-stimulated RAW 264.7 cells. Interestingly, the DNA binding activity of AP-1 was attenuated by treatment with inhibitors of p38 MAP kinase and JNK. In addition, ZPDC glycoprotein (200 μg/ml) not only diminished the production of superoxide anion, hydrogen peroxide, and nitric oxide, but also suppressed the expression of pro-inflammatory cytokines (IL-1β, IL-6, and TNF-α) and proteins (iNOS, COX-2, and MMP-9) in LPS- stimulated RAW 264.7 cells. Conclusions:  The present study demonstrates that ZPDC glycoprotein is a natural anti-oxidant and one of the modulators of pro-inflammatory signal transduction pathways in RAW 264.7 cells. Received 2 June 2008; returned for revision 12 August 2008; received from final revision 15 August 2008; accepted by G. Wallace 18 September 2008     

Involvement of proinflammatory factors, apoptosis, caspase-3 activation and Ca2+ disturbance in microglia activation-mediated dopaminergic cell degeneration

Increasing evidences suggest that activated microglia may contribute to neurodegeneration in Parkinson's disease (PD). In the present study, primary ventral mesencephalic (VM) cultures from E14 rats and PC12 cells were utilized as in vitro models to examine the mechanism underlying microglia activation mediated dopaminergic neurodegeneration. Using lipopolysaccharide (LPS) (1-100 ng/ml) as a tool, we observed that microglia activation-mediated a selective dopaminergic neurodegeneration in VM neuron-glia cultures, which was supported by the further study showing that conditioned medium (CM) from microglia-enriched cultures treated with LPS (10-100 ng/ml) decreased PC12 cell viability. The results from antibody neutralization, NO inhibition and superoxide neutralization suggested that the dopaminergic cell death was due to the production of microglia-derived proinflammatory factors (TNF-alpha, NO and superoxide), among which reactive oxygen species (ROS) might outweigh proinflammatory cytokines. Apoptosis assay on PC12 cells and primary dopaminergic neurons showed that apoptosis was a mechanism for both microglia activation-mediated dopaminergic cell death. Through Western blot and immunocytochemistry, we found that caspase-3 activation was involved in both dopaminergic cell injuries. Finally, the results from laser scanning confocal microscope demonstrated that PC12 cell intracellular free Ca(2+) ([Ca(2+)](i)) increased early after CM treatment. [Ca(2+)](i) increase involved influx of calcium from the extracellular milieu and release from intracellular stores and participated in the CM-induced PC12 cell apoptosis. Further investigations indicated that TNF-alpha, IL-1beta, NO and superoxide contributed at different degrees to CM-induced [Ca(2+)](i) increase and apoptosis in PC12 cells. Using primary VM cultures and PC12 cells, our study shows the roles of proinflammatory factors, apoptosis, caspase-3 activation and Ca(2+) disturbance in microglia activation-mediated dopaminergic cell degeneration. Understanding the mechanism for microglia activation-mediated dopaminergic neurodegeneration may contribute to the development of new neuroprotective strategies against PD.     

Tumor-associated lymphocytes (TAL) are competent to produce higher levels of cytokines in neoplastic pleural and peritoneal effusions than those found in sera and are able to release into culture higher levels of IL-2 and IL-6 than those released by PBMC

This work was designed to study the proliferative response of tumor-associated lymphocytes (TAL) from neoplastic effusions against autologous tumor cells and the immunophenotype pattern of TAL from neoplastic effusions and that of PBMC of the same patients. We also compared the serum levels of the cytokines interleukin (IL) 1, 2 and 6, tumor necrosis factor- (TNF) and soluble IL-2 receptor (sIL-2R) with those present in neoplastic effusions of the same patients. Moreover, we examined the ability of TAL and peripheral blood mononuclear cells (PBMC) to produce and release the cytokines and sIL-2R and to express membrane CD25 following their stimulation with phytohemagglutinin (PHA) in vitro. Finally, we compared the cytokines/sIL-2R production and membrane CD25 expression by PHA-stimulated PBMC of the patients with neoplastic effusions with a series of 90 cancer patients without neoplastic effusions and 20 normal healthy subjects. Thirteen neoplastic pleural and eight peritoneal effusions were collected from 11 patients with primary lung cancer, 7 with primary epithelial ovarian cancer, 1 with breast cancer, 1 with pleural mesothelioma, and 1 with pancreatic cancer. The proliferative response of TAL from neoplastic effusions against autologous tumor cells was lower than the response to PHA, IL-2, and anti-CD3, but significant. The percentage distribution of CD3⁺ and CD8⁺ lymphocyte subpopulations was higher in peritoneal than in pleural effusions, while the CD16⁺ subset was higher in pleural than in peritoneal effusions. The percentage distribution of CD16⁺ was significantly lower in pleural effusions than in PBMC of patients with pleural effusions. The CD39 antigen was higher on TAL from peritoneal effusions than on PBMC of the same patients. The levels of IL-1 and sIL-2R in peritoneal effusions did not differ from those measured in the sera of the same patients, while the levels of IL-2, IL-6, and TNF were higher in the peritoneal effusions. The levels of IL-2, IL-6, TNF, and sIL-2R, but not IL-1, in pleural effusions were significantly higher than those found in the sera of the same patients. The amounts of IL-2 and IL-6 produced by TAL were generally higher than those released by PBMC. The secretion of cytokines IL-1, IL-2, and sIL2R by PHA-stimulated PBMC was lower, but IL-1 and IL-6 secretion was higher in cancer patients with neoplastic effusions than in either cancer patients without neoplastic effusions or normal subjects. The CD25 expression on PHA-stimulated PBMC derived from cancer patients with neoplastic effusions was in the same range as that of cancer patients without neoplastic effusions and normal subjects. These findings suggest that TAL may be able to produce cytokines and may be amenable to immune manipulation.Abbreviations FITC Fluorescein-isothiocyanate - IL Interleukin - mAb Monoclonal antibody - MHC Major histocompatibility complex - NK Natural killer - PBMC Peripheral blood mononuclear cells - PHA Phytohemagglutinin - TAL Tumor-associated lymphocytes - TIL Tumor-infiltrating lymphocytes - TNF Tumor necrosis factor- - sIL-2R Soluble interleukin-2 receptor     

Serological and structural characterization of the O-antigens of the unclassified <Emphasis Type="Italic">Proteus mirabilis</Emphasis> strains TG 83, TG 319, and CCUG 10700 (OA)

INTRODUCTION: Lipopolysaccharide (endotoxin, LPS) is an important potential virulence factor of Proteus rods. The serological specificity of the bacteria is defined by the structure of the O-polysaccharide chain (O-antigen) of the LPS. Until now, 76 O-serogroups have been differentiated among Proteus strains. MATERIALS AND METHODS: LPSs were isolated from Proteus mirabilis TG 83, TG 319, and CCUG 10700 (OA) strains by phenol/water extraction. Antisera were raised by immunization of rabbits with heat-killed bacteria. Serological investigations were performed using enzyme immunosorbent assay, passive immunohemolysis, inhibition of both assays, absorption of antisera, and Western blot. RESULTS: The cross-reactive epitope shared by these strains and P. penner O72a,O72b is located on the O-polysaccharide and is most likely associated with an alpha-D-Glcp-(1-->6)-beta-D-GalpNAc disaccharide fragment. The serological data indicated the occurrence of two core types in the LPSs studied, one characteristic for P. mirabilis TG 319 and CCUG 10700 (OA) and the other for P. mirabilis TG 83 and O57. CONCLUSIONS: The serological and structural data showed that P. mirabilis TG 83, TG 319, CCUG 10700 (OA), and O57 have the same O-antigen structure and could be qualified to the Proteus O57 serogroup.     

Synthesis,characterization, and in vitro evaluation of targeted gold nanoshelled poly(d,l-lactide-co-glycolide) nanoparticles carrying anti p53 antibody as a theranostic agent for ultrasound contrast imaging and photothermal therapy

Breast cancer is the leading cause of cancer-related deaths in women and earlier detection can substantially reduce deaths from breast cancer. Polymers with targeted ligands are widely used in the field of molecular ultrasound imaging and targeted tumor therapy. In our study, the nanotheranostic agent was fabricated through filling perfluoropropane (C₃F₈) into poly(d,l-lactic-co-glycolic acid) nanoparticles (PLGA NPs), followed by the formation of gold nanoshell on the surface, then conjugated with anti p53 antibody which has high specificity with the p53 protein overexpressing in breast cancer. The average diameter of the gold nanoshelled PLGA NPs carrying anti p53 antibody (p53-PLGA@Au NPs) was 247 ± 108.2 nm. The p53-PLGA@Au NPs had well-defined spherical morphology and hollow interiors observed by electron microscope, and had a good photothermal effect under the irradiation of an 808 nm laser. The results of laser scanning confocal microscope (LSCM) and flow cytometer (FCM) indicated the specific targeting of p53-PLGA@Au NPs conjugating with breast cancer MCF-7 cells overexpressing p53 protein in vitro. Also the ultrasound imaging experiments in vitro showed that p53-PLGA@Au NPs were suitable for ultrasound contrast imaging. In conclusion, the p53-PLGA@Au NPs are demonstrated to be novel targeted UCAs and may have potential applications in the early diagnosis and targeted near-infrared (NIR) photothermal therapy of breast cancer in the future.     

Fabrication and characterization of poly (ethylenimine) modified poly (l-lactic acid) nanofibrous scaffolds

Abstract

Bone tissue engineering aims to construct biological substitutes for repairing bone defects. Nanofibrous (NF) scaffolds are commonly utilized to mimic the extracellular matrix (ECM) environment and promote tissue regeneration in tissue engineering process. Poly (lactic acid) (PLA) has attracted much attention in the field of tissue engineering because of its biocompatibility, biodegradability and so on. However, the intrinsic hydrophobicity and the lacking of active functional groups limit its practical application to some extent. In this study, poly(ethylenimine) (PEI) modified PLLA nanofibrous scaffolds were fabricated in a one step process by aminolysis combined with thermally induced phase separation technique for introducing more functional groups, PEI acting as the modifier. The morphology of PEI-modified PLLA scaffolds prepared under different experimental conditions was analyzed by scanning electron microscope (SEM). The suitable conditions to fabricate scaffolds with a homogeneous nanofibrous structure, good hydrophilicity and excellent mechanical properties were determined according to the results of SEM, water contact angle (WCA) and mechanical properties testing. Besides, Fourier transform infrared spectroscopy (FTIR), proton nuclear magnetic resonance spectroscopy (¹H NMR), X-ray Photoelectron Spectroscopy (XPS) and gel permeation chromatography (GPC) were used to confirm the occurrence of the ammonolysis reaction between PLLA and PEI. The in vitro biomineralization study showed that the PEI-modified PLLA scaffolds had a greater ability to induce the formation of apatite in 1.5SBF than PLLA scaffolds, indicating that the bone-bioactivity of PLLA scaffolds was significantly improved after modification with PEI. Furthermore, cell culture assay revealed that MC3T3-E1 osteoblasts exhibited better proliferation performance on the PEI-modified PLLA scaffolds. All the results implied that the synthesized modified PLLA nanofibrous scaffolds may provide promising applications in bone tissue engineering.