Brazilian red propolis exerts a cytotoxic action against prostate cancer cells and upregulates human monocyte functions.

Different propolis samples can be obtained in Brazil, such as green, brown and red. Studies related to Brazilian red propolis (BRP) have increased in the last few years, so the aim of this study was to investigate its effects on the prostate cell lines LNCaP and PC-3 and on human monocytes. BRP chemical composition was analyzed by HPLC-DAD, the viability of monocyte and cancer cell by MTT assay. Cytokine production (TNF-α, IL-1ß, IL-6, IL-10) by monocytes was quantitated by ELISA, the expression of cell markers (TLR-2, TLR-4, HLA-DR, CD80) and reactive oxygen species by flow cytometry. The candidacidal activity and the effects of supernatant of treated monocytes on tumor cells were assessed. BRP affected LNCaP viability after 48 and 72 h, while PC-3 cells were more resistant over time. BRP upregulated CD80 and HLA-DR expression, and stimulated TNF-α, IL-6 and IL-10 production. BRP enhanced the fungicidal activity of monocytes, displayed an antioxidant action and the supernatant of BRP-treated monocytes diminished LNCaP viability. In the search for new immunomodulatory and antitumoral agents, BRP exerted a selective cytotoxic activity on prostate cancer cells and an immunomodulatory action, suggesting its potential for clinical trials with oncological patients and for the discovery of new immunomodulatory and antitumor drugs.

Effect of Omega-3 on HLA-DR Expression by Conjunctival Cells and Tear Cytokine Concentrations in the Dry Eye Assessment and Management Study.

OBJECTIVES: To determine effect of omega-3 supplementation on conjunctival cell HLA-DR expression and tear concentrations of interleukin (IL)-1ß, IL-6, IL-8, IL-10, IL-17A, interferon-γ, and tumor necrosis factor-α in dry eye disease patients in the Dry Eye Assessment and Management study. METHODS: Patients were randomized to receive a daily dose of eicosapentaenoic and docosahexaenoic acids (ω3) or refined olive oil (placebo) for 12 months. At baseline, 6 and 12 months, HLA-DR expression in conjunctival total, epithelial, and white blood cells and cytokine concentration in tears were determined. Differences in change from baseline between treatment groups were assessed using generalized estimating equations (HLA-DR) or Wilcoxon rank-sum test (cytokines). RESULTS: No differences were observed in HLA-DR expression in total, epithelial, or white blood cells between ω3 and placebo groups at 6 months (n=435) or 12 months (n=436). The median concentration percent change differed between ω3 and placebo groups at 6 months for IL-6 (-36.6 vs. 24.5%, P =0.02, n=75) and for IL-8 (3.7% vs. 72.6%, P =0.02, n=68); at 12 months, they did not differ ( P ≥0.18). No other differences between the treatment groups were detected. CONCLUSIONS: ω3 supplementation did not consistently affect ocular inflammatory status as measured by the frequency of HLA-DR expressing conjunctival cells or tear cytokines.

Fatty acids secreted from head and neck cancer induce M2-like Macrophages.

Tumor-infiltrating monocytes can mature into Macrophages that support tumor survival or that display antitumor properties. To explore mechanisms steering Macrophage maturation, we assessed the effects of supernatants from squamous cell carcinoma cell lines (FaDu and SCC) on monocyte-derived Macrophage maturation. Purified monocytes were incubated in medium or medium supplemented with supernatants from FaDu and SCC9 or the leukemia monocytic cell line, THP-1. Macrophages were examined for markers of maturation (CD14, CD68), activation (HLA-DR, CD86, IL15R), scavenger receptor (CD36), toll-like receptor (TLR4), M2 marker (CD206), immune checkpoint (PD-L1), and intracellular chemokine expression (IP-10). Compared to other conditions, cells incubated with FaDu or SCC9 supernatants displayed enhanced survival, down-regulation of cell surface HLA-DR, CD86, IL-15R, CD36, and intracellular IP-10 expression, and increased cell surface PD-L1, CD14, and CD206 expression. Despite expressing TLR4 and CD14, Macrophages matured in tumor supernatants failed to respond to stimulation with the canonical TLR4 agonist, LPS. These changes were accompanied by a decrease in intracellular phospho-p38 expression in tumor supernatant conditioned Macrophages. Depletion of fatty acids from tumor supernatants or treatment of cell cultures with an inhibitor of fatty acid oxidation, Etomoxir, reversed a number of these phenotypic changes induced by tumor supernatants. Additionally, Macrophages incubated with either palmitic acid or oleic acid developed similar phenotypes as cells incubated in tumor supernatants. Together, these data suggest that fatty acids derived from tumor cells can mediate the maturation of Macrophages into a cell type with limited pro-inflammatory characteristics.

Accelerating Lead Identification by High Throughput Virtual Screening: Prospective Case Studies from the Pharmaceutical Industry.

At the onset of a drug discovery program, the goal is to identify novel compounds with appropriate chemical features that can be taken forward as lead series. Here, we describe three prospective case studies, Bruton Tyrosine Kinase (BTK), RAR-Related Orphan Receptor γ t (RORγt), and Human Leukocyte Antigen DR isotype (HLA-DR) to illustrate the positive impact of high throughput virtual screening (HTVS) on the successful identification of novel chemical series. Each case represents a project with a varying degree of difficulty due to the amount of structural and ligand information available internally or in the public domain to utilize in the virtual screens. We show that HTVS can be effectively employed to identify a diverse set of potent hits for each protein system even when the gold standard, high resolution structural data or ligand binding data for benchmarking, is not available.

Effects of perioperative transcutaneous electrical acupoint stimulation on monocytic HLA-DR expression in patients undergoing coronary artery bypass grafting with cardiopulmonary bypass: study protocol for a double-blind randomized controlled trial.

BACKGROUND: Cardiac surgery involving cardiopulmonary bypass (CPB) is known to be associated with a transient postoperative immunosuppression. When severe and persistent, this immune dysfunction predisposes patients to infectious complications, which contributes to a prolonged stay in the intensive care unit (ICU), and even mortality. Effective prevention and treatment methods are still lacking. Recent studies revealed that acupuncture-related techniques, such as electroacupuncture and transcutaneous electrical acupoint stimulation (TEAS), are able to produce effective cardioprotection and immunomodulation in adult and pediatric patients undergoing cardiac surgery with CPB, which leads to enhanced recovery. However, whether perioperative application of TEAS, a non-invasive technique, is able to improve immunosuppression of the patients with post-cardiosurgical conditions is unknown. Thus, as a preliminary study, the main objective is to evaluate the effects of TEAS on the postoperative expression of monocytic human leukocyte antigen (-D related) (mHLA-DR), a standardized "global" biomarker of injury or sepsis-associated immunosuppression, in patients receiving on-pump coronary artery bypass grafting (CABG). METHODS: This study is a single-center clinical trial. The 88 patients scheduled to receive CABG under CPB will be randomized into two groups: the group receiving TEAS, and the group receiving transcutaneous acupoint pseudo-electric stimulation (Sham TEAS). Expression of mHLA-DR serves as a primary endpoint, and other laboratory parameters (e.g., interleukin [IL]-6, IL-10) and clinical outcomes (e.g., postoperative infectious complications, ICU stay time, and mortality) as the secondary endpoints. In addition, immune indicators, such as high mobility group box 1 protein and regulatory T cells will also be measured. DISCUSSION: The current study is a preliminary monocentric clinical trial with a non-clinical primary endpoint, expression of mHLA-DR, aiming at determining whether perioperative application of TEAS has a potential to reverse CABG-associated immunosuppression. Although the immediate clinical impact of this study is limited, its results would inform further large-sample clinical trials using relevant patient-centered clinical outcomes as primary endpoints. TRIAL REGISTRATION: ClinicalTrials.gov, NCT02933996. Registered on 13 October 2016.

Frequency of Circulating CD4+Ki67+HLA-DR- T Regulatory Cells Prior to Treatment for Multidrug Resistant Tuberculosis Can Differentiate the Severity of Disease and Predict Time to Culture Conversion.

Identifying a blood circulating cellular biomarker that can be used to assess severity of disease and predict the time to culture conversion (TCC) in patients with multidrug resistant tuberculosis (MDR-TB) would facilitate monitoring response to treatment and may be of value in the design of future drug trials. We report on the frequency of blood Ki67+HLA-DR- CD4+ T regulatory (Treg) cells in predicting microbiological outcome before initiating second-line treatment for MDR-TB. Fifty-one patients with MDR-TB were enrolled and followed over 18 months; a subset of patients was sputum culture (SC) negative at baseline (n = 9). SC positive patients were divided into two groups, based on median TCC: rapid responders (≤71 days TCC; n = 21) and slow responders (>71 days TCC; n = 21). Whole blood at baseline, months 2 and 6 was stimulated with M tuberculosis (Mtb) antigens and Treg cells were then identified as CD3+CD4+CD25hiFoxP3+CD127-CD69- and further delineated as Ki67+HLA-DR- Treg. The frequency of these cells was significantly enlarged at baseline in SC positive relative to SC negative and smear positive relative to smear negative patients and in those with lung cavitation. This difference was further supported by unsupervised hierarchical clustering showing a significant grouping at baseline of total and early differentiated memory Treg cells in slow responders. Conversely, there was a clustering of a lower proportion of Treg cells and activated IFNγ-expressing T cells at baseline in the rapid responders. Examining changes over time revealed a more gradual reduction of Treg cells in slow responders relative to rapid responders to treatment. Receiver operating curve analysis showed that baseline Mtb-stimulated Ki67+HLA-DR- Treg cells could predict the TCC of MDR-TB treatment response with 81.2% sensitivity and 85% specificity (AUC of 0.87, p < 0.0001), but this was not the case after 2 months of treatment. In conclusion, our data show that the frequency of a highly defined Mtb-stimulated blood Treg cell population at baseline can discriminate MDR-TB disease severity and predict time to culture clearance.

Marina crystal minerals (MCM) activate human dendritic cells to induce CD4+ and CD8+ T cell responses in vitro.

Marina crystal minerals (MCM) are a mixture that contains crystallized minerals along with trace elements extracted from seawater. It is a nutritional supplement that is capable of enhancing natural killer (NK) cell activity and increasing T and B cell proliferation in humans post ingestion. However, its effect on dendritic cells (DCs), the cells that bridge innate and adaptive immunity, is not yet known. In this study, we examine the stimulatory effects of MCM on DCs' maturation and function in vitro. Human monocyte-derived DCs were treated with MCM at two different concentrations (10 and 20 µg/mL) for 24 h. Results showed that MCM treatment activated DCs in a dose-dependent fashion. It caused the upregulation of costimulatory molecules CD80, CD86, and HLA-DR, and prompted the production of DC cytokines, including interleukin (IL)-6, IL-10, tumor necrosis factor (TNF)-α, and IL-1ß, and chemokines (monocyte chemotactic protein-1 (MCP-1)) and interferon-gamma-inducible protein-10 (IP-10). In addition, activated DCs primed CD4+ T cells to secrete significant amounts of interferon gamma (IFN-γ), and they also stimulated CD8+ T cells to express higher amounts of CD107a. These results indicate that MCM is a potentially powerful adjuvant, from natural materials, that activates human DCs in vitro and therefore may suggest its possible use in immune-based therapies against cancer and viral infections.

Effect of phenolic extracts from different extra-virgin olive oil varieties on osteoblast-like cells.

The reported incidence of osteoporosis is lower in countries in which the Mediterranean diet predominates, and this apparent relationship may be mediated by the phenolic compounds present in olive oil. The objective of this study was to determine the effect of phenolic extracts from different varieties of extra-virgin olive oil (Picual, Arbequina, Picudo, and Hojiblanca) on the differentiation, antigenic expression, and phagocytic capacity of osteoblast-like MG-63 cells. At 24 h of treatment a significant increase in phosphatase alkaline activity and significant reductions in CD54, CD80, and HLA-DR expression and in phagocytic activity were observed in comparison to untreated controls. The in vitro study performed has demonstrated that phenolic compounds from different extra virgin olive oil varieties can modulate different parameters related to osteoblast differentiation and function.

Effect of Eucommia ulmoides Oliv., Gynostemma pentaphyllum (Thunb.) Makino, and Curcuma longa L. on Th1- and Th2-cytokine responses and human leukocyte antigen-DR expression in peripheral blood mononuclear cells of septic patients.

ETHNOPHARMACOLOGICAL RELEVANCE: Many traditional Chinese medicines (TCM), such as Eucommia ulmoides Oliv., Gynostemma pentaphyllum (Thunb.) Makino, and Curcuma longa L., have been reported to have various immune-modulatory effects. AIM OF THE STUDY: To determine the effects of extracts from these three TCM on type 1 T help (Th1)- and Th2-cytokine responses and human leukocyte antigen (HLA)-DR expression in peripheral blood mononuclear cells (PBMCs) obtained from septic patients. MATERIALS AND METHODS: Lipopolysaccharide (LPS)-stimulated PBMCs of healthy controls and septic patients were cultured for 48 hs with or without 0.05/0.1 mg/ml of TCM extract. HLA-DR expression in monocytes was detected using flow cytofluorimetry. The interferon [IFN]-γ, tumor necrosis factor [TNF]-α, interleukin (IL)- 2, IL-5, IL-10, and IL-13 levels in supernatants were measured with a human enzyme-linked immunosorbent assay. RESULTS: Treatment with either 0.05 or 0.1 mg/ml of C. longa L. extract significantly restored the percentage of HLA-DR-positive monocytes, which was decreased by LPS in control and patient groups. Treatment with 0.05 or 0.1 mg/ml E. ulmoides Oliv. and C.longa L. extract decreased IL-10 production from LPS-stimulated PBMCs of controls and patients. In patients with sepsis, C. longa L. extract decreased IL-10 production to a greater degree than did E. ulmoides Oliv extract. Although IFN-γ, TNF-α, or IL-13 productions from LPS-stimulated PBMCs were influenced by E. ulmoides Oliv., G. pentaphyllum (Thunb.) Makino, or C. longa L. in control or sepsis groups in this study, only the influence of IL-10 was consistent in both control and sepsis groups. CONCLUSIONS: By enhancing monocyte HLA-DR expression and decreasing IL-10 production, C. longa L. might help restore inflammatory responses in septic patients to eradicate pathogens.

Production of IL-17 by MAIT Cells Is Increased in Multiple Sclerosis and Is Associated with IL-7 Receptor Expression.

Multiple sclerosis (MS) is a T cell-driven inflammatory disease of the CNS. Research on T cell subsets involved in MS pathogenesis has mainly focused on classical CD4+ T cells, especially Th17 cells, as they produce the proinflammatory, MS-associated cytokine IL-17. However, the abundant unconventional mucosal-associated invariant T (MAIT) cells are also able to produce IL-17. MAIT cells are characterized by high CD161 expression and a semi-invariant Vα7.2 TCR, with which they recognize bacterial and yeast Ags derived from the riboflavin (vitamin B2) metabolism. In this study, we characterized MAIT cells from the peripheral blood of MS patients in comparison with healthy individuals with respect to their type-17 differentiation. We found a specific increase of IL-17+ MAIT cells as well as an increased expression of retinoic acid-related orphan receptor (ROR)γt and CCR6 in MAIT cells from MS patients, whereas the expression of T cell activation markers HLA-DR and CD38 was not different. IL-17 production by MAIT cells furthermore correlated with the surface expression level of the IL-7 receptor α-chain (CD127), which was significantly increased on MAIT cells from MS patients in comparison with healthy individuals. In summary, our findings indicate an augmented type-17 differentiation of MAIT cells in MS patients associated with their IL-7 receptor surface expression, implicating a proinflammatory role of these unconventional T cells in MS immunopathology.

Anti-angiogenic effect of hexahydrocurcumin in rat corneal neovascularization.

AIM: This study was to investigate the anti-angiogenic effect of hexahydrocurcumin (HHC) to evaluate gene (p-basic fibroblast growth factor (bFGF)-SAINT-18 & p-vascular endothelial growth factor (VEGF)-SAINT-18 complex)-induced corneal neovascularization (CorNV) in rats. METHODS: CorNV was induced in 24 eyes of 24 rats. Four groups (Group A: 0 µg, B: 0.01 µg, C: 0.1 µg, and D: 1 µg) of HHC were prepared and implanted into the rat subconjunctival substantia propria 1.5 mm from the limbus at temporal side. The 1 µg of p-bFGF-SAINT-18 & p-VEGF-SAINT-18 complex were prepared and implanted into the rat corneal stroma 1.5 mm from the limbus at the same side. Inhibition of CorNV was observed and quantified from day 1 to day 60. bFGF and VEGF protein expression were analyzed by biomicroscopic examination, western blot analysis, and immunohistochemistry. RESULTS: Subconjunctival injection by 1 µg HHC successfully inhibited gene-induced CorNV in rats. bFGF and VEGF protein expression were reduced after 6 days. Meanwhile, the reduction of HLA-DR expression was detected. CONCLUSIONS: Our study showed that the HHC might provide an important anti-angiogenesis factor to inhibit CorNV development at the corneal experimental angiogenesis model.

Xenotransplantation of interferon-gamma-pretreated clumps of a human mesenchymal stem cell/extracellular matrix complex induces mouse calvarial bone regeneration.

BACKGROUND: Three-dimensional cultured clumps of a mesenchymal stem cell (MSC)/extracellular matrix (ECM) complex (C-MSC) consists of cells and self-produced ECM. C-MSC can regulate the cellular function in vitro and induce successful bone regeneration using ECM as a cell scaffold. Potentiating the immunomodulatory capacity of C-MSCs, which can ameliorate the allo-specific immune response, may be helpful in developing beneficial "off-the-shelf" cell therapy for tissue regeneration. It is well reported that interferon (IFN)-γ stimulates the immunosuppressive properties of MSC via upregulation of the immunomodulatory enzyme IDO. Therefore, the aim of this study was to investigate the effect of IFN-γ on the immunomodulatory capacity of C-MSC in vitro and to test the bone regenerative activity of C-MSC or IFN-γ-pretreated C-MSC (C-MSCγ) xenografts in a mice calvarial defect model. METHODS: Human bone marrow-derived MSCs were seeded at a density of 2.0 × 105 cells/well into 24-well plates and cultured with growth medium supplemented with 50 µg/mL L-ascorbic acid for 4 days. To obtain C-MSC, confluent cells that had formed on the cellular sheet were scratched using a micropipette tip and were then torn off. The cellular sheet was rolled to make a round clump of cells. C-MSC was stimulated with IFN-γ and IDO expression, immunosuppressive capacity, and immunophenotype were evaluated in vitro. Moreover, C-MSC or C-MSCγ was xenotransplanted into immunocompetent or immunodeficient mice calvarial defect models without artificial scaffold, respectively. RESULTS: IFN-γ stimulated IDO expression in C-MSC. C-MSCγ, but not C-MSC, attenuated CD3/CD28-induced T cell proliferation and its suppressive effect was reversed by an IDO inhibitor. C-MSCγ showed upregulation of HLA-DR expression, but its co-stimulatory molecule, CD86, was not detected. Xenotransplantation of C-MSCγ into immunocompetent mice calvarial defect induced bone regeneration, whereas C-MSC xenograft failed and induced T cell infiltration in the grafted area. On the other hand, both C-MSC and C-MSCγ xenotransplantation into immunodeficient mice caused bone regeneration. CONCLUSIONS: Xenotransplantation of C-MSCγ, which exerts immunomodulatory properties via the upregulation of IDO activity in vitro, may attenuate xenoreactive host immune response, and thereby induce bone regeneration in mice. Accordingly, C-MSCγ may constitute a promising novel allograft cell therapy for bone regeneration.

Long-term Supplementation With n-6 and n-3 PUFAs Improves Moderate-to-Severe Keratoconjunctivitis Sicca: A Randomized Double-Blind Clinical Trial.

PURPOSE: Supplementation with gamma-linolenic acid (GLA) and omega-3 (n-3) polyunsaturated fatty acids (PUFAs) has been found to decrease the production of disease-relevant inflammatory mediators that are implicated in the pathogenesis of chronic dry eye. This study evaluated the effect of a supplement containing both GLA and n-3 PUFAs on signs and symptoms of moderate-to-severe keratoconjunctivitis sicca in postmenopausal patients. METHODS: This multicenter, double-masked placebo-controlled clinical trial enrolled 38 patients (both eyes) with tear dysfunction who were randomized to supplemental GLA + n-3 PUFAs or placebo for 6 months. Disease parameters, including Ocular Surface Disease Index, Schirmer test, tear breakup time, conjunctival fluorescein and lissamine green staining, and topographic corneal smoothness indexes (surface asymmetry index and surface regularity index), were assessed at baseline and at 4, 12, and 24 weeks. The intensity of dendritic cell CD11c integrin and HLA-DR expression was measured in conjunctival impression cytologies. RESULTS: The Ocular Surface Disease Index score improved with supplementation and was significantly lower than placebo (21 ± 4 vs. 34 ± 5) after 24 weeks (P = 0.05, n = 19 per group). The surface asymmetry index was significantly lower in supplement-treated subjects (0.37 ± 0.03, n = 15) than placebo (0.51 ± 0.03, n = 16) at 24 weeks (P = 0.005). Placebo treatment also significantly increased HLA-DR intensity by 36% ± 9% and CD11c by 34% ± 7% when compared with supplement treatment (n = 19 per group, P = 0.001, 24 weeks). Neither treatment had any effect on tear production, tear breakup time, or corneal or conjunctival staining. CONCLUSIONS: Supplemental GLA and n-3 PUFAs for 6 months improved ocular irritation symptoms, maintained corneal surface smoothness, and inhibited conjunctival dendritic cell maturation in patients with postmenopausal keratoconjunctivitis sicca.Clinical Trial Registration-URL: http://www.clinicaltrials.gov. Unique identifier: NCT00883649.

[Effects of Bushen Jiedu Recipe and Jianpi Jiedu Recipe containing plasma on dendritic cells of chronic hepatitis B virus infection patients under different immune states].

OBJECTIVE: To compare the effects of Bushen Jiedu Recipe (BJR) and Jianpi Jiedu Recipe (JJR) containing plasma on dendritic cells (DCs) of chronic hepatitis B virus (HBV) infection patients under different immune states. METHODS: Recruited were 36 chronic HBV infection outpatients from First Affiliated Hospital of Hunan University of Traditional Chinese Medicine from April 2010 to January 2011. They were assigned to the immune tolerance group (18 cases) and the immune clearance group (18 cases).Another 10 healthy subjects were recruited as the healthy control group. Their anticoagulated peripheral venous blood was respectively collected. The peripheral blood mononuclear cells (PBMCs) were isolated and further extracted for incubating DCs. The DCs were intervened by BJR and JJR containing plasma. The morphology of DCs was identified. The expressions of CD1alpha, CD80, CD86, and HLA-DR were detected. The level of interferon-alpha (IFN-alpha) in the supernatant was observed by ELISA. RESULTS: The CD80 expression level was lower in the immune clear group than in the healthy control group before intervention (P < 0.05). The expression levels of CD80, CD86, and HLA-DR were lower in the immune tolerance group than in the healthy control group before intervention (P < 0.05).The IFN-alpha expression level was lower in the immune tolerance group and the immune clearance group than in the healthy control group before intervention (P < 0.05). The expression levels of CD80, HLA-DR, and IFN-alpha were lower in the immune tolerance group than in the immune clearance group before intervention (P < 0.05). Compared with the same group before intervention, the CD80 expression significantly increased in each treatment group (P < 0.05). After intervention the expression levels of CD80 and HLA-DR were higher in the immune tolerance group than in the immune clearance group in the same time phase, and the CD86 expression level was higher in the BJR group than in the immune clearance group in the same time phase, showing statistical difference (P < 0.05). CONCLUSIONS: The middle dose BJR and the small dose JJR both could promote the recovery of DCs in chronic HBV infection patients. Besides, BJR showed more prominent effects on the function of DCs in chronic HBV infection patients in the immune tolerance stage.

RETRACTED: Increased neuroinflammatory and arachidonic acid cascade markers, and reduced synaptic proteins, in the postmortem frontal cortex from schizophrenia patients.

This article has been retracted: please see Elsevier Policy on Article Withdrawal (https://www.elsevier.com/about/our-business/policies/article-withdrawal). This article has been retracted at the request of the Editors. The National Institutes of Health has found that Dr. Jagadeesh S. Rao engaged in research misconduct by falsifying data. Data in Figures 1A, 1E, 3E and 3F were falsified. Dr. Rao was solely responsible for the falsification. None of the other authors are implicated in any way.

Effect of bFGF on HLA-DR expression of human bone marrow-derived mesenchymal stem cells.

There has been a steady rise in the therapeutic applications of bone marrow mesenchymal stem cells (BM-MSCs) because of their unique properties of multilineage differentiation and immune modulation as well as the ease in isolation. However, up-regulation of surface HLA-DR levels when maintaining MSCs in culture under the influence of mitotic factors such as Basic fibroblast growth factor (bFGF) is an area of concern when considering them for the purpose of clinical applications. Thus, we investigated the association of bFGF supplemented to the culture media and the surface expression levels of HLA-DR in BM-MSCs in order to optimize the yield, while keeping HLA-DR levels under permissible levels. Human BM-MSCs were culture expanded in the absence of bFGF and in the presence of 1 ng/ml or 2 ng/ml bFGF. The HLA-DR profile of the cultures was analyzed at the end of each passage. On comparing the percent HLA-DR+ cell population at different concentrations as well as absence of bFGF, significant differences were not observed in the HLA-DR expression levels of the MSC cultures which had reached complete confluence. However, variations in HLA-DR expressions levels were seen which could be traced to the age of cells in culture with values drastically reduced to below 4% on maintaining MSCs typically two to three days beyond achieving full confluence. On the basis of the findings from this study, no significant correlation could be established on the effect of bFGF in modulating HLA-DR surface expression of BM-MSCs. Instead, the data are suggestive of the reasoning that the duration for which BM-MSCs are maintained in culture directly influences their phenotypic characteristics in terms of HLA-DR expression levels, with lowest levels achieved on their prolonged maintenance in culture.

Endotoxin- and ATP-neutralizing activity of alkaline phosphatase as a strategy to limit neuroinflammation.

BACKGROUND: Alkaline phosphatase (AP) is a ubiquitously expressed enzyme which can neutralize endotoxin as well as adenosine triphosphate (ATP), an endogenous danger signal released during brain injury. In this study we assessed a potential therapeutic role for AP in inhibiting neuroinflammation using three complementary approaches. METHODS: Mice were immunized to induce experimental autoimmune encephalomyelitis (EAE) and treated with AP for seven days during different phases of disease. In addition, serological assays to determine AP activity, endotoxin levels and endotoxin-reactive antibodies were performed in a cohort of multiple sclerosis (MS) patients and controls. Finally, the expression of AP and related enzymes CD39 and CD73 was investigated in brain tissue from MS patients and control subjects. RESULTS: AP administration during the priming phase, but not during later stages, of EAE significantly reduced neurological signs. This was accompanied by reduced proliferation of splenocytes to the immunogen, myelin oligodendrocyte glycoprotein peptide. In MS patients, AP activity and isoenzyme distribution were similar to controls. Although endotoxin-reactive IgM was reduced in primary-progressive MS patients, plasma endotoxin levels were not different between groups. Finally, unlike AP and CD73, CD39 was highly upregulated on microglia in white matter lesions of patients with MS. CONCLUSIONS: Our findings demonstrate that: 1) pre-symptomatic AP treatment reduces neurological signs of EAE; 2) MS patients do not have altered circulating levels of AP or endotoxin; and 3) the expression of the AP-like enzyme CD39 is increased on microglia in white matter lesions of MS patients.

[Clinical trial of treating stress-induced hyperglycemia patients with sepsis by supplementing QI, nourishing yin, and promoting blood flow].

OBJECTIVE: To investigate the effects of the treatment of supplementing qi, nourishing yin, and promoting blood flow (SQNYPBF) on the serum levels of CRP, TNF-alpha, IL-1 and IL-6, as well as the expression of HLA-DR in the peripheral monocytes in septic patients suffering from stress-induced hyperglycemia. METHODS: In the prospective randomized controlled study, eighty-five stress-induced hyperglycemia patients with sepsis were randomly assigned to the experimental group (45 cases) and the control group (40 cases). On the basis of routine therapies, including anti-infection, nutrition support, and the glucose control with insulin pump, patients in the experimental group additionally received the treatment of SQNYPBF (They were intravenously dripped with Shenmai Injection and Sulfotanshinone Sodium Injection, once daily, for 7 successive days). The serum levels of CRP, TNF-alpha, IL-1, and IL-6 and the HLA-DR expression of the peripheral monocytes were detected using ELISA before treatment and on the 8th day of the treatment. The total dose and the duration of insulin used, the morbidity of hypoglycemia, the APACHE II scores, and the mortality within 28-day hospitalization were compared between the two groups. RESULTS: The total dose of insulin used, the duration of insulin used, the morbidity of hypoglycemia, the APACHE II score on the 8th day of treatment, and the mortality within 28-day hospitalization significantly decreased in the experimental group, when compared with the control group (P < 0.05, P < 0.01). There was no difference in the expression of HLA-DR, the serum levels of CRP, TNF-alpha, IL-1, or IL-6 before treatment between the two groups (P > 0.05). After treatment the serum levels of CRP, TNF-alpha, IL-1, and IL-6 significantly decreased (P < 0.05) and the expression of HLA-DR significantly increased in the two groups (P < 0.05). Better effects were shown in the experimental group (P < 0.05, P < 0.01). CONCLUSION: SQNYPBF combined intensive insulin therapy could better improve the sepsis patients' immunity, decrease the plasma glucose level and duration, increase their survival rate, and improve their prognosis.

Randomized pilot trial of a synbiotic dietary supplement in chronic HIV-1 infection.

BACKGROUND: Infection with HIV-1 results in marked immunologic insults and structural damage to the intestinal mucosa, including compromised barrier function. While the development of highly active antiretroviral therapy (HAART) has been a major advancement in the treatment of HIV-1 infection, the need for novel complementary interventions to help restore intestinal structural and functional integrity remains unmet. Known properties of pre-, pro-, and synbiotics suggest that they may be useful tools in achieving this goal. METHODS: This was a 4-week parallel, placebo-controlled, randomized pilot trial in HIV-infected women on antiretroviral therapy. A synbiotic formulation (Synbiotic 2000®) containing 4 strains of probiotic bacteria (10(10) each) plus 4 nondigestible, fermentable dietary fibers (2.5 g each) was provided each day, versus a fiber-only placebo formulation. The primary outcome was bacterial translocation. Secondary outcomes included the levels of supplemented bacteria in stool, the activation phenotype of peripheral T-cells and monocytes, and plasma levels of C-reactive protein and soluble CD14. RESULTS: Microbial translocation, as measured by plasma bacterial 16S ribosomal DNA concentration, was not altered by synbiotic treatment. In contrast, the synbiotic formulation resulted in significantly elevated levels of supplemented probiotic bacterial strains in stool, including L. plantarum and P. pentosaceus, with the colonization of these two species being positively correlated with each other. T-cell activation phenotype of peripheral blood lymphocytes showed modest changes in response to synbiotic exposure, with HLA-DR expression slightly elevated on a minor population of CD4+ T-cells which lack expression of HLA-DR or PD-1. In addition, CD38 expression on CD8+ T-cells was slightly lower in the fiber-only group. Plasma levels of soluble CD14 and C-reactive protein were unaffected by synbiotic treatment in this study. CONCLUSIONS: Synbiotic treatment for 4 weeks can successfully augment the levels of probiotic species in the gut during chronic HIV-1 infection. Associated changes in microbial translocation appear to be absent, and markers of systemic immune activation appear largely unchanged. These findings may help inform future studies aimed at testing pre- and probiotic approaches to improve gut function and mucosal immunity in chronic HIV-1 infection. TRIAL REGISTRATION: Clinical Trials.gov: NCT00688311.

Randomized clinical trial of omega-3 fatty acid-supplemented enteral nutrition versus standard enteral nutrition in patients undergoing oesophagogastric cancer surgery.

BACKGROUND: Oesophagogastric cancer surgery is immunosuppressive. This may be modulated by omega-3 fatty acids (O-3FAs). The aim of this study was to assess the effect of perioperative O-3FAs on clinical outcome and immune function after oesophagogastric cancer surgery. METHODS: Patients undergoing subtotal oesophagectomy and total gastrectomy were recruited and allocated randomly to an O-3FA enteral immunoenhancing diet (IED) or standard enteral nutrition (SEN) for 7 days before and after surgery, or to postoperative supplementation alone (control group). Clinical outcome, fatty acid concentrations, and HLA-DR expression on monocytes and activated T lymphocytes were determined before and after operation. RESULTS: Of 221 patients recruited, 26 were excluded. Groups (IED, 66; SEN, 63; control, 66) were matched for age, malnutrition and co-morbidity. There were no differences in morbidity (P = 0·646), mortality (P = 1·000) or hospital stay (P = 0·701) between the groups. O-3FA concentrations were higher in the IED group after supplementation (P < 0·001). The ratio of omega-6 fatty acid to O-3FA was 1·9:1, 4·1:1 and 4·8:1 on the day before surgery in the IED, SEN and control groups (P < 0·001). There were no differences between the groups in HLA-DR expression in either monocytes (P = 0·538) or activated T lymphocytes (P = 0·204). CONCLUSION: Despite a significant increase in plasma concentrations of O-3FA, immunonutrition with O-3FA did not affect overall HLA-DR expression on leucocytes or clinical outcome following oesophagogastric cancer surgery. REGISTRATION NUMBER: ISRCTN43730758 (http://www.controlled-trials.com).