Characterization of soluble dietary fiber from Moringa oleifera seeds and its immunomodulatory effects.

Moringa oleifera (moringa or drumstick) seeds are a potential source of dietary fiber with 6.5% w/w soluble dietary fiber. Biochemical characterization of moringa seed soluble fiber revealed that it is a glycoprotein with 5% neutral sugars. Arabinose and xylose are the major neutral sugars identified by gas liquid chromatography (GLC). Moringa seed soluble fiber was identified as protease resistant-glycoprotein and termed as moringa seed resistant protein (MSRP). MSRP was found to be a homodimer (18kDa) containing two 9kDa monomeric units as revealed by SDS-PAGE analysis with pI 10.8. Immunostimulating activity of MSRP was assessed by murine splenocyte proliferation and production of NO from macrophages. MSRP at low concentration (0.01µg/well) strongly increased proliferation of splenocytes, while MSRP at high concentration weakly responded. MSRP induced 6-fold increase in NO production when compared to the control which indicates the activation of macrophages. MSRP isolated from defatted moringa seed flour is a potent mitogen, enhancing the proliferation of lymphocytes and inducing NO from macrophages. This study concludes that moringa seed is a potential nutritional source to promote the immune system of the host.

Downmodulation of key inflammatory cell markers with a topical Janus kinase 1/2 inhibitor.

BACKGROUND: INCB018424 is a novel, potent Janus kinase (JAK)1/JAK2 inhibitor that blocks signal transduction of multiple proinflammatory cytokines. OBJECTIVES: To evaluate the safety, tolerability, pharmacokinetics, pharmacodynamics and preliminary efficacy of topical INCB018424 phosphate cream in patients with plaque psoriasis. METHODS: Topical INCB018424 phosphate 1·0% or 1·5% cream was applied once daily (QD) or twice daily (BID) for 4 weeks to 2-20% body surface area in five sequential cohorts of five patients aged 18-65 years. Target lesions were scored on a scale of 0-4 for erythema, scaling and thickness. Additionally, the overall disease activity in each patient was measured using Physician's Global Assessment. INCB018424 concentrations were measured in plasma, and cytokine stimulated phosphorylated signal transducer and activator of transcription 3 phosphorylation (pSTAT3) levels in peripheral blood cells were evaluated. Pretreatment and post-treatment skin biopsies were compared with healthy skin, including evaluation of histopathology, immunohistochemistry and mRNA expression. RESULTS: Treatment with INCB018424 phosphate cream either 1·0% QD or 1·5% BID resulted in improvements in lesion scores. No significant inhibition of pSTAT3 in peripheral blood cells was observed following topical application, consistent with the generally low steady-state plasma concentrations of INCB018424 measured. Transcriptional markers of immune cell lineage/activation in lesional skin were reduced by topical INCB018424, with correlations observed between clinical improvement and decreases in markers of T helper 17 lymphocyte activation, dendritic-cell activation and epidermal hyperplasia. INCB018424 treatment reduced epidermal hyperplasia and dermal inflammation in most patient samples, with reductions in CD3, CD11c, Ki67 and keratin 16 observed by immunohistochemical analysis. CONCLUSIONS: Topical INCB018424 dosed for 28 days QD or BID is pharmacologically active in patients with active psoriasis and modulates proinflammatory cytokines in the pathogenesis of psoriatic lesions.

Characterization of onion lectin (Allium cepa agglutinin) as an immunomodulatory protein inducing Th1-type immune response in vitro.

Onion (Allium cepa), a bulb crop of economic importance, is known to have many health benefits. The major objective of the present study is to address the immunomodulatory properties of onion lectin (A. cepa agglutinin; ACA). ACA was purified from onion extract by D-mannose-agarose chromatography (yield: ~1 mg/kg). ACA is non-glycosylated and showed a molecular mass of ~12 kDa under reducing/non-reducing SDS-PAGE; glutaraldehyde cross-linking indicated that ACA is a non-covalent tetramer of ~12 kDa subunits. Its N-terminal sequence (RNVLLNNEGL; UniProt KB Accn. C0HJM8) showed 70-90% homology to mannose-specific Allium agglutinins. ACA showed specific hemagglutination activity of 8200 units/mg and is stable in the pH range 6-10 and up to 45° C. The immunomodulatory activity of ACA was assessed using the macrophage cell line, RAW264.7 and rat peritoneal macrophages; at 0.1 µg/well, it showed a significant increase (6-8-fold vs. control) in the production of nitric oxide at 24h, and significantly stimulated (2-4-fold vs. control) the production of pro-inflammatory cytokines (TNF-α and IL-12) at 24h. ACA (0.1 µg/well) enhanced the proliferation of murine thymocytes by ~4 fold (vs. control) at 24h; however, ACA does not proliferate B cell-enriched rat splenocytes. Further, it significantly elevated the expression levels of cytokines (IFN-γ and IL-2) over the control in murine thymocytes. Taken together, purified ACA induces a Th1-type immune response in vitro. Though present in low amounts, ACA may contribute to the immune-boosting potential of the popular spice onion since considerable amounts are consumed on a daily basis universally.

Biology of TACE inhibition.

Studies conducted over the past decade have demonstrated a central role for tumour necrosis factor alpha (TNFalpha) in inflammatory diseases. As a result of this work, a number of biological agents that neutralise the activity of this cytokine have entered the clinic. The recent clinical data obtained with etanercept and infliximab highlight the relevance of this strategy. TNFalpha converting enzyme (TACE) is the metalloproteinase that processes the 26 kDa membrane bound precursor of TNFalpha (proTNFalpha) to the 17 kDa soluble component. Although a number of proteases have been shown to process proTNFalpha, none do so with the efficiency of TACE. A series of orally bioavailable, selective, and potent TACE inhibitors are currently in clinical development. These inhibitors effectively block TACE mediated processing of proTNFalpha and can reduce TNF production by lipopolysaccharide stimulated whole blood by >95%. Through a series of studies it is shown here that >80% of the unprocessed proTNFalpha is degraded intracellularly. The remainder appears to be transiently expressed on the cell surface. Although, in vitro, TACE inhibition has also been implicated in shedding of p55 and p75 surface TNFalpha receptors, the in vivo data cast doubt on the consequences of this finding. In a mouse model of collagen-induced arthritis, the inhibitors are efficacious both prophylactically and therapeutically. The efficacy seen is equivalent to strategies that neutralise TNFalpha. In many studies greater efficacy is observed with the TACE inhibitors, presumably owing to greater penetration to the site of TNFalpha production.

Increased monocyte MCP-1 production in acute alcoholic hepatitis.

Monocyte chemoattractant protein-1 (MCP-1) is a potent mononuclear cell-specific chemotactic protein. MCP-1 is a candidate chemoattractant for activation and hepatic infiltration of mononuclear cells in alcoholic hepatitis (AH). Blood was collected from 15 patients with AH (mean bilirubin 17.6+/-3.5 mg/dl; normal 0. 2-1.0 mg/dl) on admission and at time points for up to 6 months. Peripheral blood monocytes were isolated and MCP-1 production assessed by measuring MCP-1 concentrations in monocyte culture supernatants after overnight (20 h) incubation. Monocytes from normal subjects did not product detectable MCP-1 unless stimulated with endotoxin (LPS;5 microg/ml). The mean level of constitutive MCP-1 from AH patient monocytes was 4694+/-2432 pg/ml 20 h on admission. The mean MCP-1 level for LPS-treated monocytes was 4903+/-1540 pg/ml 20 h for normal subjects and was significantly elevated in AH patients to 11589+/-3266 pg/ml/20 h. AH patient monocyte MCP-1 production was decreased in vitro when monocytes were treated with N-acetylcysteine (5 mM) and also decreased over the 6-month study as the patients improved clinically. MCP-1 plasma levels were below the detection limits of the assay used in both AH patients and normal subjects. Thus, monocytes from AH patients not only constitutively product MCP-1, but also produce higher levels of MCP-1 with endotoxin stimulation. Further studies are needed to clarify the role of MCP-1 in the activation and hepatic infiltration of mononuclear cells in alcoholic liver disease.

A comparison of the pharmacological properties of carbohydrate remodeled recombinant and placental-derived beta-glucocerebrosidase: implications for clinical efficacy in treatment of Gaucher disease.

The objective of these studies was to characterize the macrophage mannose receptor binding and pharmacological properties of carbohydrate remodeled human placental-derived and recombinant beta-glucocerebrosidase (pGCR and rGCR, respectively). These are similar but not identical molecules that were developed as enzyme replacement therapies for Gaucher disease. Both undergo oligosaccharide remodeling during purification to expose terminal mannose sugar residues. Competitive binding data indicated carbohydrate remodeling improved targeting to mannose receptors over native enzyme by two orders of magnitude. Mannose receptor dissociation constants (Kd) for pGCR and rGCR were each 13 nmol/L. At 37 degrees C, 95% of the total macrophage binding was mannose receptor specific. In vivo, pGCR and rGCR were cleared from circulation by a saturable pathway. The serum half-life (t1/2) was 3 minutes when less than saturable amounts were injected intravenously (IV) into mice. Twenty minutes postdose, beta-glucocerebrosidase activity increased over endogenous levels in all tissues examined. Fifty percent of the injected activity was recovered. Ninety-five percent of recovered activity was in the liver. Parenchymal cells (PC), Kupffer cells (KC), and liver endothelium cells (LEC) were responsible for 75%, 22%, and 3%, respectively, of the hepatocellular uptake of rGCR and for 76%, 11%, and 12%, respectively, of the hepatocellular uptake of pGCR. Both molecules had poor stability in LEC and relatively long terminal half-lives in PC (t1/2 = 2 days) and KC (t1/2 = 3 days).

Modulation of macrophage activation by ammonium metavanadate.

The molecular basis of the immunotoxic effect of ammonium metavanadate on signal transduction involved in macrophage activation was studied in resident peritoneal macrophages (PEM) and a murine macrophage-like cell line, J774. A fourfold elevation in cytosolic free calcium levels was observed within 10 s following lipopolysaccharide (LPS) stimulation of the non-vanadate-exposed controls both in vitro and in vivo; the levels returned to prestimulation values within 70 s. Exposure to phorbol ester (PMA) did not result in any appreciable change in cytosolic free calcium levels. Compared to untreated controls, treatment with vanadate caused a significant elevation in basal cytosolic calcium levels. Such elevation was not enhanced further by LPS. LPS stimulation of macrophages also resulted in a significant elevation of membrane-associated protein kinase C (PKC) activity, which was, however, inhibited in a dose-dependent manner by vanadate in both in vitro and in vivo studies. Exposure to PMA also resulted in a significant elevation of membrane-associated PKC activity; vanadate treatment at lower levels did not cause downregulation, indicating that vanadate at these levels interfered with the receptor-mediated events rather than the enzyme directly. Vanadate at higher exposure levels inhibited the activity even in PMA-stimulated macrophages. No significant difference occurred in cytosolic PKC activities in control macrophages; vanadate treatment at lower levels resulted in a significant elevation of cytosolic PKC activities following stimulation with LPS or PMA, indicating that vanadate might be interfering with the translocation process.

Differential inhibition of IL-1 alpha and TNF-alpha generation by ammonium metavanadate in murine macrophages.

The relationship between immunotoxicity of ammonium metavanadate (NH4VO3) and levels of tumor necrosis factor-alpha (TNF-alpha) and interleukin-1 (IL-1) was studied with a NH4VO3-treated murine macrophage-like cell line, 1774 and resident peritoneal macrophages (PEM) obtained from treated mice. Lipopolysaccharide (LPS)-induced elevation of extracellular TNF-alpha in PEM and 1774 cells was not markedly affected by prior treatment with vanadate. However, PEM from treated mice at 10 mg V/kg (10V) had a significantly lower level of LPS-induced intracellular TNF-alpha. NH4VO3-treated 1774 cells at 3.6 (V1) and 7.2 micrograms V/10(7) cells (V2) had significantly higher levels of intracellular TNF-alpha than the PO4 and V3 (10.8 micrograms V/10(7) cells) groups. Although the four PEM groups showed no marked difference in extracellular IL-1 alpha levels, PEM from treated mice at 2.5V and 10V had significantly lower levels of intracellular IL-1 alpha than those from control groups. 1774 cells from PO4 and NH4Cl groups showed significant increases in intracellular IL-1 alpha following treatment with LPS. However, 1774 cells with prior treatment with vanadate revealed significant reduction in levels of LPS-induced intracellular IL-1 alpha when compared to control groups. Therefore, the previously reported reduced resistance of vanadate-treated mice to Listeria monocytogenes could be attributed to an inhibitory effect on the production of IL-1 alpha in macrophages.

Regulation of monocyte integrin expression by beta-family chemokines.

In the present study we investigated the ability of three monocyte chemokines (MCP-1, MIP-1 alpha, and RANTES) to modulate monocyte adhesion molecules in an attempt to evaluate their potential to induce tissue infiltration of macrophages in vivo. All three chemokines tested induced increased expression of the alpha-chains of two members of beta 2 family of integrins, CD11b and CD11c, and their common beta-chain (CD18). They had no effect on CD11a expression. Enhancement of CD11b and CD11c was dose dependent and followed a distinct time course with peak levels at 4 h. Levels declined to reach basal levels by 24 h. In contrast, IL-1 induced enhancement remained high after 24 h of stimulation. However, the increases caused by chemokines were not mediated by IL-1 as indicated by lack of inhibition by the IL-1R antagonist. Studies on the mechanism of integrin up-regulation showed that mobilization of cytosolic free calcium is an important signaling event in this response and that up-regulation is associated with mobilization from intracellular pools mediated by microtubules. Enhanced CD11b and CD11c expression by chemokines was also found to result in enhancement of monocyte binding to endothelial cells. Further studies indicated that monocyte binding to endothelial cells follows similar dose-response kinetics as the up-regulation of integrins and can be partially blocked by Abs to CD11b and CD11c. These results suggest that modulation of the integrin expression by chemokines may facilitate the tissue trafficking of monocytes during inflammation.

Increased secretion of tumor necrosis factor-alpha and interferon-gamma by mononuclear leukocytes in patients with ischemic heart disease. Relevance in superoxide anion generation.

BACKGROUND: There is growing evidence for a pathogenic role for cytokines in atherogenesis. The presence of certain cytokines has been documented in human atherosclerotic vessels. This study was designed to investigate cytokine production by mononuclear leukocytes from patients with ischemic heart disease. METHODS AND RESULTS: We measured kinetics of secretion of tumor necrosis factor-alpha (TNF-alpha) and interferon-gamma (IFN-gamma) by mononuclear leukocytes from 8 control subjects, 10 patients with stable angina pectoris, and 10 patients with unstable angina pectoris. Mononuclear leukocytes were isolated and incubated with or without the plant lectin mitogen concanavalin A for 48 hours. TNF-alpha and IFN-gamma secretion were measured by ELISA. The effect of TNF-alpha and IFN-gamma on superoxide radical generation by neutrophils was also examined. Secretion of both TNF-alpha and IFN-gamma by mononuclear leukocytes increased progressively over 48 hours, and it was consistently higher (P < .02) in patients compared with control subjects. A similar increase in cytokine secretion was observed in patients with stable or unstable angina pectoris. In addition, there was no relation between the severity of coronary artery disease by angiography and cytokine secretion. Basal neutrophil superoxide radical generation was increased in patients with ischemic heart disease, and incubation with cytokines failed to further stimulate superoxide generation in these patients. CONCLUSIONS: Similar increases in cytokine secretion by mononuclear leukocytes in stable or unstable angina pectoris indicate that the increased cytokine release is not a nonspecific inflammatory response in acute myocardial ischemia. Increased cytokine secretion in ischemic heart disease may play a role in superoxide radical generation, endothelial injury, deposition and activation of cellular elements on the vessel wall, and possibly in the progression of atherosclerosis.

Comparison of biological responses of human monocytes and THP-1 cells to chemokines of the intercrine-beta family.

The biological responses of human monocytes and cells of the monomyelocytic THP-1 cell line to stimulation with members of the beta chemokine family are described in this report. All three chemokines tested, MCP-1, MIP-1 alpha, and RANTES, elicited mobilization of intracellular free calcium in monocytes and THP-1 cells. The magnitude of response was highest with MCP-1 stimulation. MCP-1 desensitized monocyte responses to MIP-1 alpha and RANTES, but no such desensitization was observed in THP-1 cells. MIP-1 alpha or RANTES did not desensitize either monocytes or THP-1 cells to MCP-1 stimulation. All three chemokines elicited a potent chemotactic response in monocytes that was comparable in magnitude to that of f-Met-Leu-Phe. MIP-1 alpha and RANTES required a fivefold higher dose than MCP-1 to elicit a peak response. On the contrary, THP-1 cells showed no significant chemotactic response. Studies of the desensitization of the monocyte chemotactic response indicated that all three chemokines are capable of causing complete homologous desensitization. Heterologous desensitization was observed only when monocytes were treated with MCP-1 followed by MIP-1 alpha or RANTES. Studies of actin polymerization and cell polarization responses of monocytes indicated that these two responses attained peak magnitude after 10 min of stimulation with any of the chemokines. Dose-response kinetics were similar to those of the chemotactic response. THP-1 cells again failed to show either of these two responses. Finally, the activation potential of the chemokines was measured by their ability to induce respiratory burst. A tenfold higher concentration than that causing peak chemotactic response was required to elicit respiratory burst and no heterologous desensitization was noticed. Respiratory burst could be induced in THP-1 cells with a direct protein kinase C activator but not with any of the chemokines. These results indicate that, of the three examples tested, MCP-1 is the most potent member of the beta chemokine family in the biological responses examined. Although a calcium response was elicited in THP-1 cells with chemokines, a lack of subsequent responses indicates some missing links in the downstream signal transduction pathways.

Effect of ammonium metavanadate on the mouse peritoneal macrophage lysosomal enzymes.

Female B6C3F1 mice were injected intraperitoneally with ammonium metavanadate (2.5 or 10 mg V/kg), ammonium chloride, or sodium phosphate buffer (0.1 M, pH 7.2) every 3 d for 6 wk. Resident peritoneal macrophage (PEM) cytolysates were prepared and assayed for intracellular enzyme activities of beta-glucuronidase, N-acetyl-beta-D-glucosaminidase, acid phosphatase, and lysozyme, to investigate possible reasons for the depressive effect of ammonium metavanadate on the intracellular killing of Listeria monocytogenes by murine PEM. Acid phosphatase activity per 10(6) cells for the 2.5 and 10 mg V/kg groups was depressed by 22.8 and 44.7%, respectively, when compared to phosphate buffer controls. No significant effect by vanadium treatment was observed with regard to the other three enzymes. Kinetic studies (in vitro) on the effect of ammonium metavanadate (5, 10, 15, and 20 mM) on the above enzymes showed similar patterns of effect by vanadium. Lineweaver-Burk analysis of acid phosphatase indicated linear noncompetitive inhibition by vanadium with a Kj of 14.8 mM. NH4Cl and 10 mg V/kg treatments also enhanced extracellular secretion of beta-glucuronidase and lysozyme from PEM, which could be attributed to the presence of ammonium ion. The decrease in acid phosphatase activity might contribute, in part through its interference in the phosphorylation/dephosphorylation, to the diminished intracellular killing ability of PEM.

Modulation of Fc tau receptor expression and function in mouse peritoneal macrophages by ammonium metavanadate.

Resident peritoneal macrophages (PEM) harvested from female B6C3F1 mice given an intraperitoneal injection of ammonium metavanadate (2.5 or 10 mg V/kg), an equivalent amount of ammonium in the form of ammonium chloride, or sodium phosphate buffer (0.1 M, pH 7.2) every third day for 6 weeks, were subjected to flow cytometric analysis of Fc tau 2a and Fc tau 2b receptor expression, and photometric microassay to measure receptor mediated binding and phagocytosis of sheep red blood cells (SRBC). The NH4Cl and 10V groups showed 21.7 and 17.2% lower mean fluorescence channel (MFC) values and 7.1 and 5.9% lower values in percentage fluorescence-positive cells than the phosphate buffer control with respect to Fc tau 2a expression. For Fc tau 2b expression, the 10V group showed significantly (P less than 0.05) lower MFC (31.2%) and percentage fluorescence-positive cells (15.7%) than the phosphate buffer control. Though the four groups did not show a significant difference in Fc tau 2a mediated binding and phagocytosis of SRBC, the 10V group showed a significantly lower Fc tau 2b mediated binding and phagocytosis. The results indicate that the reduction in Fc tau 2b expression and function could contribute toward the previously observed depression in phagocytosis, NADPH-oxidase and superoxide generation in peritoneal macrophages obtained from vanadate-treated animals.