Safety assessment of a proprietary fermented soybean solution, Symbiota®, as an ingredient for use in foods and dietary supplements: Non-clinical studies and a randomized trial.

A safety evaluation was performed of Symbiota®, which is made by a proprietary anaerobic fermentation process of soybean with multistrains of probiotics and a yeast. The battery of genotoxicity studies showed that Symbiota® has no genotoxic effects. Safety and tolerability were further assessed by acute or repeated dose 28- and 90-day rodent studies, and no alterations in clinical observations, ophthalmological examination, blood chemistry, urinalysis, or hematology were observed between the control group and the different dosing groups (1.5, 5, and 15 mL/kg/day). There were no adverse effects on specific tissues or organs in terms of weight and histopathology. Importantly, the Symbiota® treatment did not perturb hormones and other endocrine-related endpoints. Of note, the No-Observed-Adverse-Effect-Level was determined to be 15 mL/kg/day in rats. Moreover, a randomized, double-blind, placebo-controlled clinical trial was recently conducted with healthy volunteers who consumed 8 mL/day of placebo or Symbiota® for 8 weeks. Only mild adverse events were reported in both groups, and the blood chemistry and blood cell profiles were also similar between the two groups. In summary, this study concluded that the oral consumption of Symbiota® at 8 mL/day by the general population does not pose any human health concerns.

New Horizons of Macrophage Immunomodulation in the Healing of Diabetic Foot Ulcers.

Diabetic foot ulcers (DFUs) are one of the most costly and troublesome complications of diabetes mellitus. The wound chronicity of DFUs remains the main challenge in the current and future treatment of this condition. Persistent inflammation results in chronic wounds characterized by dysregulation of immune cells, such as M1 macrophages, and impairs the polarization of M2 macrophages and the subsequent healing process of DFUs. The interactive regulation of M1 and M2 macrophages during DFU healing is critical and seems manageable. This review details how cytokines and signalling pathways are co-ordinately regulated to control the functions of M1 and M2 macrophages in normal wound repair. DFUs are defective in the M1-to-M2 transition, which halts the whole wound-healing machinery. Many pre-clinical and clinical innovative approaches, including the application of topical insulin, CCL chemokines, micro RNAs, stem cells, stem-cell-derived exosomes, skin substitutes, antioxidants, and the most recent Phase III-approved ON101 topical cream, have been shown to modulate the activity of M1 and M2 macrophages in DFUs. ON101, the newest clinically approved product in this setting, is designed specifically to down-regulate M1 macrophages and further modulate the wound microenvironment to favour M2 emergence and expansion. Finally, the recent evolution of macrophage modulation therapies and techniques will improve the effectiveness of the treatment of diverse DFUs.

Restoring Prohealing/Remodeling-Associated M2a/c Macrophages Using ON101 Accelerates Diabetic Wound Healing.

Diabetic wounds exhibit chronic inflammation and delayed tissue proliferation or remodeling, mainly owing to prolonged proinflammatory (M1) macrophage activity and defects in transition to prohealing/proremodeling (M2a/M2c; CD206+ and/or CD163+) macrophages. We found that topical treatment with ON101, a plant-based potential therapeutic for diabetic foot ulcers, increased M2c-like (CD163+ and CD206+) cells and suppressed M1-like cells, altering the inflammatory gene profile in a diabetic mouse model compared with that in the controls. An in vitro macrophage-polarizing model revealed that ON101 directly suppressed CD80+ and CD86+ M1-macrophage polarization and M1-associated proinflammatory cytokines at both protein and transcriptional levels. Notably, conditioned medium collected from ON101-treated M1 macrophages reversed the M1-conditioned medium‒mediated suppression of CD206+ macrophages. Furthermore, conditioned medium from ON101-treated adipocyte progenitor cells significantly promoted CD206+ and CD163+ macrophages but strongly inhibited M1-like cells. ON101 treatment also stimulated the expression of GCSF and CXCL3 genes in human adipocyte progenitor cells. Interestingly, treatment with recombinant GCSF protein enhanced both CD206+ and CD163+ M2 markers, whereas CXCL3 treatment only stimulated CD163+ M2 macrophages. Depletion of cutaneous M2 macrophages inhibited ON101-induced diabetic wound healing. Thus, ON101 directly suppressed M1 macrophages and facilitated the GCSF- and CXCL3-mediated transition from M1 to M2 macrophages, lowering inflammation and leading to faster diabetic wound healing.

CYR61 regulates BMP-2-dependent osteoblast differentiation through the {alpha}v{beta}3 integrin/integrin-linked kinase/ERK pathway.

Osteoporosis is one of the most common bone pathologies. A number of novel molecules have been reported to increase bone formation including cysteine-rich protein 61 (CYR61), a ligand of integrin receptor, but mechanisms remain unclear. It is known that bone morphogenetic proteins (BMPs), especially BMP-2, are crucial regulators of osteogenesis. However, the interaction between CYR61 and BMP-2 is unclear. We found that CYR61 significantly increases proliferation and osteoblastic differentiation in MC3T3-E1 osteoblasts and primary cultured osteoblasts. CYR61 enhances mRNA and protein expression of BMP-2 in a time- and dose-dependent manner. Moreover, CYR61-mediated proliferation and osteoblastic differentiation are significantly decreased by knockdown of BMP-2 expression or inhibition of BMP-2 activity. In this study we found integrin α(v)ß(3) is critical for CYR61-mediated BMP-2 expression and osteoblastic differentiation. We also found that integrin-linked kinase, which is downstream of the α(v)ß(3) receptor, is involved in CYR61-induced BMP-2 expression and subsequent osteoblastic differentiation through an ERK-dependent pathway. Taken together, our results show that CYR61 up-regulates BMP-2 mRNA and protein expression, resulting in enhanced cell proliferation and osteoblastic differentiation through activation of the α(v)ß(3) integrin/integrin-linked kinase/ERK signaling pathway.

Apoptotic insults to human chondrocytes induced by sodium nitroprusside are involved in sequential events, including cytoskeletal remodeling, phosphorylation of mitogen-activated protein kinase kinase kinase-1/c-Jun N-terminal kinase, and Bax-mitochondria-mediated caspase activation.

Nitric oxide (NO) can regulate chondrocyte activities. This study was aimed to evaluate the molecular mechanisms of NO donor sodium nitroprusside (SNP)-induced insults to human chondrocytes. Exposure of human chondrocytes to SNP increased cellular NO levels but decreased cell viability in concentration- and time-dependent manners. SNP time dependently induced DNA fragmentation and cell apoptosis. Treatment with 2-phenyl-4,4,5,5-tetramethyl-imidazoline-1-oxyl 3-oxide, an NO scavenger, significantly lowered SNP-induced cell injuries. Administration of SNP interrupted F-actin and microtubule cytoskeletons and stimulated phosphorylation of mitogen-activated protein kinase kinase kinase-1 (MEKK1) and c-Jun N-terminal kinase (JNK). Similar to SNP, cytochalasin D, an inhibitor of F-actin formation, disturbed F-actin polymerization and increased MEKK1 and JNK activations. Overexpression of a dominant negative mutant of MEKK1 (dnMEK1) in human chondrocytes significantly ameliorated SNP-induced cell apoptosis. Exposure to SNP promoted Bax translocation from the cytoplasm to mitochondria, but application of dnMEKK1 lowered the translocation. SNP time dependently decreased the mitochondrial membrane potential, complex I NADH dehydrogenase activity, and cellular ATP levels, but increased the release of cytochrome c from mitochondria to the cytoplasm. Activities of caspase-9, -3, and -6 were sequentially increased by SNP administration. This study shows that SNP can induce apoptosis of human chondrocytes through sequential events, including cytoskeletal remodeling, activation of MEKK1/JNK, Bax translocation, mitochondrial dysfunction, cytochrome c release, caspase activation, and DNA fragmentation.

Glutamine-supplemented total parenteral nutrition attenuates plasma interleukin-6 in surgical patients with lower disease severity.

AIM: Previous reports have shown that decrease in plasma glutamine (Gln) level following major surgery may contribute to the state of immunosuppression. Gln supplementation improves the depletion of body Gln pool, and may have indirect effect on reducing proinflammatory mediator release. This study evaluated whether the effect of Gln dipeptide-enriched total parenteral nutrition (TPN) on postoperative cytokine alteration depended on the disease severity of surgical patients. METHODS: Forty-eight patients with major abdominal surgery were allocated to two groups to receive isonitrogenous (0.228 g nitrogen/kg per d) and isocaloric (30 kcal/kg per d) TPN for 6 d. Control group (Conv) using conventional TPN solution received 1.5 g amino acids/kg per day, whereas the test group received 0.972 g amino acids/kg per day and 0.417 g L-alanyl-L-glutamine (Ala-Gln)/kg per day. Blood samples were collected on d 1 and d 6 postoperatively for plasma interleukin (IL)-2, IL-6, IL-8, and interferon (IFN)-gamma analysis. RESULTS: Plasma IL-2 and IFN-gamma were not detectable. IL-6 concentrations were significantly lower on the 6(th) postoperative day in the Ala-Gln group than those in the Conv group in patients with APACHE II <=6, whereas no difference was noted in patients with APACHE II >6. There was no difference in IL-8 levels between the two groups. No difference in cumulative nitrogen balance was observed on d 2-5 after the operation between the two groups (Ala-Gln -3.2+/-1.6 g vs Conv -6.5+/-2.7 g). A significant inverse correlation was noted between plasma IL-6 levels and cumulative nitrogen balance postoperatively in the Ala-Gln group, whereas no such correlation was observed in the Conv group. CONCLUSION: TPN supplemented with Gln dipeptide had no effect on plasma IL-8 levels after surgery. However, Gln supplementation had a beneficial effect on decreasing systemic IL-6 production after surgery in patients with low admission illness severity, and lower plasma IL-6 may improve nitrogen balance in patients with abdominal surgery when Gln was administered.

Activation of phosphoinositide 3-kinase in response to high glucose leads to regulation of reactive oxygen species-related nuclear factor-kappaB activation and cyclooxygenase-2 expression in mesangial cells.

Hyperglycemia causes glomerular mesangial cell proliferation and increases matrix synthesis, contributing to early diabetic glomerulopathy. Immunohistochemical and functional correlations of renal cyclooxygenase-2 in experimental diabetes have been identified. However, the role of cyclooxygenase-2 in early diabetes-induced mesangial cell proliferation remains unknown. The authors tested the hypothesis that hyperglycemia modulates an intrarenal cyclooxygenase-2 expression, which might mediate the mesangial cell proliferation via a possible phosphoinositide 3-kinase/Akt pathway. Expression of cyclooxygenase-2, but not cyclooxygenase-1, could be induced in mesangial cells cultured under high glucose. Antioxidants (pyrrolidine dithiocarbamate and N-acetyl-l-cysteine) and phosphoinositide 3-kinase inhibitors [2-(4-morpholinyl)-8-phenyl-1(4H)-benzopyran-4-one hydrochloride (LY294002) and wortmannin] effectively inhibited this high glucose-induced response. Moreover, high glucose markedly triggered the activation of phosphoinositide 3-kinase and Akt in mesangial cells, suggesting that a phosphoinositide 3-kinase/Akt pathway is involved in the high glucose-induced responses. Phosphoinositide 3-kinase inhibitors could also effectively attenuate the high glucose-triggered intracellular reactive oxygen species generation and nuclear factor-kappaB activation. Likewise, blocking the phosphoinositide 3-kinase or Akt activity with the dominant-negative vectors DN-p85 or DN-Akt, respectively, also greatly diminished the high glucose-triggered reactive oxygen species generation and nuclear factor-kappaB activation. Treatment of mesangial cells with LY294002 and cyclooxygenase-2 inhibitors [N-[2-(cyclohexyloxyl)-4-nitrophenyl]-methane sulfonamide (NS398) and aspirin] effectively inhibited the high glucose-induced mesangial cell proliferation. These results suggest that high glucose may trigger the reactive oxygen species-regulated nuclear factor-kappaB activation and cyclooxygenase-2 expression and cell proliferation in mesangial cells through a phosphoinositide 3-kinase-dependent pathway.

Ajoene-induced cell death in human promyeloleukemic cells does not require JNK but is amplified by the inhibition of ERK.

Treatment of human promyeloleukemic HL-60 cells with the experimental antileukemic drug ajoene induces the activation of the mitogen-activated protein kinases (MAPKs) c-Jun NH(2)-terminal kinase (JNK), p38 and extracellular signal-regulated kinases (ERK) 1/2 as well as the survival kinase Akt. JNK activation occurred in HL-60/neo, HL-60/bcl-x(L), and in HL-60 cells pretreated with the pan-caspase inhibitor zVAD-fmk, indicating that JNK activation is not dependent on ajoene-induced mitochondria perturbation and subsequent caspase activation. Cells overexpressing a dominant-negative JNK showed no altered sensitivity towards ajoene suggesting that the activation of JNK is not necessary for ajoene-induced cell death. Inhibition of p38 MAPK by SB 203580 had no influence on ajoene-mediated apoptosis. In contrast, inhibition of ERK1/2 vastly enhanced ajoene-induced cell death. The survival kinase Akt, in contrast, did not participate in ajoene-induced death signaling as shown by the use of the phosphatidylinositol-3-kinase inhibitor wortmannin. Thus in contrast to the previous findings regarding stress-induced cell death, ajoene-mediated activation of JNK and p38 has no impact on ajoene-induced apoptosis in HL-60 cells. Blockade of ERK1/2 but not Akt pathways leads to sensitization of cells against ajoene-mediated apoptosis supporting the view that inhibition of ERK1/2 is a valuable strategy to increase the sensitivity of promyeloleukemic cells towards ajoene.