Rice bran hydrolysates induce immunomodulatory effects by suppression of chemotaxis, and modulation of cytokine release and cell-mediated cytotoxicity

Objective: To evaluate the immunomodulatory effects of rice bran hydrolysates on cultured immune cells and their underlying mechanism.Methods: Rice bran hydrolysates were prepared from pigmented rice(Oryza sativa L.) by hydrothermolysis and protease digestion. Rice bran hydrolysates were assayed for phenolic content and antioxidant activity. Cell proliferation of Jurkat, THP-1 and peripheral blood mononuclear cells(PBMC) was evaluated by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay. Chemotaxis was evaluated by transwell chamber methods. Immunoadherence of THP-1 was performed on cultured human umbilical vein endothelial cells(HUVEC). Cytokine released from PBMC was measured by ELISA assay kits. Lymphocyte-mediated cytotoxicity was carried out on KKU-452 cells. Proteins associated with immunomodulation were analyzed by Western immunoblotting assay. Results: Rice bran hydrolysates were rich in phenolic compounds, such as ferulic acid, catechin, quercetin, and quercetin glycosides. Rice bran hydrolysates suppressed phytohemagglutinin(PHA)-stimulated proliferation of PBMC and Jurkat cells, chemotaxis of Jurkat and THP-1 cells, and immunoadherence of THP-1 on HUVEC cultured cells. The cellular mechanism of rice bran hydrolysates involved the activation of AMPK as well as suppression of m TOR, NF-κB and VCAM-1. Rice bran hydrolysates potentiated PBMC on the PHA-stimulated release of IL-2, TNF-α, and IL-4, and enhanced PHA-induced non-MHC-restricted cytotoxicity on KKU-452 cancer cells. Conclusions: The immunomodulatory effect of phytochemicals derived from rice bran hydrolysates suggests its therapeutic potential for further investigation.

Cell lysates and egg white create homeostatic microenvironment for gene expression in cell-free system

Homeostasis widely exists in living systems,and plays essential roles for maintaining normal physiological activities,enabling to preserve their functionalities against variations.Gene expression is a crucial process that allows cells to produce the necessary protein,giving cells the flexibility to adapt to variations.Herein we study homeostasis of gene expression in cell-free system.Heat-inactivated cell lysates and egg white are utilized to create homeostatic microenvironment.Results show that both in cell lysates and egg white,gene expression is maintained at relatively stable levels upon variations including gene amount,magnesium ions and temperature.Our work presents a nascent concept and experimental evidence for the homeostasis in cell-free systems,and provides implication for living systems.

Autologous peripheral blood-derived orthobiologics:Different types and their effectiveness in managing knee osteoarthritis

Knees are the most commonly impacted weight-bearing joints in osteoarthritis(OA),affecting millions of people worldwide.With increasing life spans and obesity rates,the incidence of knee OA will further increase,leading to a significant increase in the economic burden.Conventional treatment modalities utilized to manage knee OA have limitations.Over the last decade,the role of various autologous peripheral blood-derived orthobiologics(APBOs)for the treatment of knee OA has been extensively investigated.This editorial provided an overview and focused on defining and shedding light on the current state of evidence based on the most recent published clinical studies concerning the use of APBO for the management of knee OA.While numerous studies have demonstrated promising results for these preparations,a notable gap exists in the comparative analysis of these diverse formulations.This absence of head-to-head studies poses a considerable challenge for physicians/surgeons in determining the optimal preparation for managing knee OA and achieving sustained longterm results.Thus,more adequately powered,multicenter,prospective,doubleblind,randomized controlled trials with longer follow-ups are needed to establish the long-term efficacy and to aid physicians/surgeons in determining the optimal APBO for the management of knee OA.

Efficacy Evaluation of Bifida Ferment Lysate in Cosmetic

By constructing a cell repair model after photodamage, we evaluated the photoresistance of FermentDFL, a lysate of Bifidus yeast fermentation products, of human immortalized keratinocytes(HaCaT) and human skin fibroblasts(HDF) under UVB and UVA irradiation. The experimental data show that when human cells are damaged by light, Ferment-DFL with a concentration of 5% and 2% can significantly increase the level of cell viability, reduce the content of ROS reactive oxygen species in cells, and promote the secretion of type I collagen. The regeneration experiment evaluates the repair effect of Ferment-DFL, a lysate of two fission yeast fermentation products. At a concentration of 6%, after 48 h, the repairing promotion rate of zebrafish embryo tail fin reaches 15%, which can significantly promote the regeneration of zebrafish embryo tail fin.Promotes repairing effect.

Honokiol-enhanced cytotoxic T lymphocyte activity against cholangiocarcinoma cells mediated by dendritic cells pulsed with damage-associated molecular patterns

BACKGROUND Cholangiocarcinoma or biliary tract cancer has a high mortality rate resulting from late presentation and ineffective treatment strategy. Since immunotherapy by dendritic cells (DC) may be beneficial for cholangiocarcinoma treatment but their efficacy against cholangiocarcinoma was low. We suggest how such antitumor activity can be increased using cell lysates derived from an honokioltreated cholangiocarcinoma cell line (KKU-213L5). AIM To increase antitumour activity of DCs pulsed with cell lysates derived from honokiol-treated cholangiocarcinoma cell line (KKU-213L5). METHODS The effect of honokiol, a phenolic compound isolated from Magnolia officinalis, on choangiocarcinoma cells was investigated in terms of the cytotoxicity and the expression of damage-associated molecular patterns (DAMPs). DCs were loaded with tumour cell lysates derived from honokiol-treated cholangiocarcinoma cells their efficacy including induction of T lymphocyte proliferation, proinflammatory cytokine production and cytotoxicity effect on target cholangiocarcinoma cells were evaluated. RESULTS Honokiol can effectively activate cholangiocarcinoma apoptosis and increase the release of damage-associated molecular patterns. DCs loaded with cell lysates derived from honokiol-treated tumour cells enhanced priming and stimulated T lymphocyte proliferation and type I cytokine production. T lymphocytes stimulated with DCs pulsed with cell lysates of honokiol-treated tumour cells significantly increased specific killing of human cholangiocarcinoma cells compared to those associated with DCs pulsed with cell lysates of untreated cholangiocarcinoma cells. CONCLUSION The present findings suggested that honokiol was able to enhance the immunogenicity of cholangiocarcinoma cells associated with increased effectiveness of DC-based vaccine formulation. Treatment of tumour cells with honokiol offers a promising approach as an ex vivo DC-based anticancer vaccine.

血液内毒素对哺乳仔猪腹泻的影响

前腔静脉采集试验组(腹泻组)与对照组(不腹泻组)母猪和仔猪血液,用LAL试剂(鲎试剂)测定血液内毒素含量,用分光光度法测定D-乳酸浓度和二胺氧化酶活性。结果表明,试验组母猪血液内毒素含量平均为1.978 EU/m L,比对照组1.007 EU/m L高96.43%,说明母猪体内高含量的内毒素是引起新生仔猪腹泻的原因之一。试验组仔猪血液内毒素含量为1.125 EU/m L,比对照组0.276 EU/m L高307.61%,说明仔猪体内较高含量的内毒素是引起仔猪腹泻的直接原因。试验组仔猪血浆D-乳酸含量达12.33μg/m L,比对照组仔猪血浆D-乳酸含量8.17μg/m L高4.16μg/m L。试验组仔猪血浆二胺氧化酶活性9.12 U/L,比对照组仔猪血浆二胺氧化酶活性5.63 U/L高3.49 U/L,说明腹泻仔猪肠黏膜屏障受到一定程度的破坏。

More than a Few LAB Alleviate Common Allergies: Impact of Paraprobiotics in Comparison to Probiotical Live Cells

The evidence in this paper indicates that the alarming increase of common allergies can be reduced by the intake of particular “probiotics” or “paraprobiotics” along with food. This appears to build a consensus in the pharmaceutical and food communities about the role of probiotics in the prevention and treatments of common allergies. Food allergy is one of the common allergies, defined as an adverse health effect arising from a specific immune response that occurs reproducibly on exposure to a given food. Improving the digestion of foods and maintaining a healthy gastrointestinal (GI) tract is certainly critical to controlling food allergens. Therefore, the association between a leaky gut or an impaired intestinal permeability and food-allergenic reactions is explained. Gluten has been found to be somehow a justification for protein allergy, and should, therefore, be avoided by people with celiac disease. In several, in vitro models, surface layer (S- layer) proteins of selective paraprobiotics have shown potential in alleviating food allergies and intestinal disorders. Notably, lactobacilli paraprobiotics have proven to be the immediate immunomodulators against common allergies and other diseases, including viral (flu, hepatitis C), bacterial (bronchitis), asthma, chronic fatigue, fibromyalgia, gastrointestinal distress, and autism disorders in humans.

Antitumor immunity by a dendritic cell vaccine encoding secondary lymphoid chemokine and tumor lysate on murine prostate cancer

Aim： To investigate the antitumor immunity by a dendritic cell （DC） vaccine encoding secondary lymphoid chemokine gene and tumor lysate on murine prostate cancer. Methods： DC from bone marrow of C57BL/6 were transfected with a plasmid vector expressing secondary lymphoid chemokine （SLC） cDNA by Lipofectamine2000 liposome and tumor lysate. Total RNA extracted from SLC＋lysate-DC was used to verify the expression of SLC by reverse transcriptase-polymerase chain reaction （RT-PCR）. The immunotherapeutic effect of DC vaccine on murine prostate cancer was assessed. Results： We found that in the prostate tumor model of C57BL/6 mice, the adminstration of SLC＋lysate-DC inhibited tumor growth most significantly when compared with SLC-DC, lysate-DC, DC or phos- phate buffer solution （PBS） counterparts （P 〈 0.01）. Immunohistochemical fluorescent staining analysis showed the infiltration of more CD4＋, CD8＋ T cell and CD11c＋ DC within established tumor treated by SLC＋lysate-DC vaccine than other DC vaccines （P 〈 0.01）. Conclusion： DC vaccine encoding secondary lymphoid chemokine and tumor lysate can elicit significant antitumor immunity by infiltration of CD4＋, CD8＋ T cell and DC, which might provide a potential immunotherapy method for prostate cancer.

Use of platelet lysate for bone regeneration-are we ready for clinical translation?

Current techniques to improve bone regeneration following trauma or tumour resection involve the use of autograft bone or its substitutes supplemented with osteoinductive growth factors and/or osteogenic cells such as mesenchymal stem cells(MSCs).Although MSCs are most commonly grown in media containing fetal calf serum,human platelet lysate(PL) offers an effective alternative.Bone marrow- derived MSCs grown in PLcontaining media display faster proliferation whilst maintaining good osteogenic differentiation capacity.Limited pre-clinical investigations using PL-expanded MSCs seeded onto osteoconductive scaffolds indicate good potential of such constructs to repair bone in vivo.In an alternative approach,nude PL-coated scaffolds without seeded MSCs have been proposed as novel regenerative medicine devices.Even though methods to coat scaffolds with PL vary,in vitro studies suggest that PL allows for MSC adhesion,migration and differentiation inside these scaffolds.Increased new bone formation and vascularisation in comparison to uncoated scaffolds have also been observed in vivo.This review outlines the state-of-the-art research in the field of PL for ex vivo MSC expansion and in vivo bone regeneration.To minimise inconsistency between the studies,further work is required towards standardisation of PL preparation in terms of the starting material,platelet concentration,leukocyte depletion,and the method of platelet lysis.PL quality control procedures and its "potency" assessment are urgently needed,which could include measurements of key growth and attachment factors important for MSC maintenance and differentiation.Furthermore,different PL formulations could be tailor-made for specific bone repair indications.Such measures would undoubtedly speed up clinical translation of PL-based treatments for bone regeneration.

Experimental Study on Inactivation of Bacterial Endotoxin by Using Dielectric Barrier Discharge

The low-temperature plasma （LTP） generated by dielectric barrier discharge （DBD） was used to sterilize the E.coli endotoxin, which is usually difficult to kill by traditional methods. Three different concentrations of bacterial endotoxin （1 EU/mL, 0.5 EU/mL and 0.25 EU/mL） were treated by LTP for different time （20 s, 40 s and 60 s）. Tachypleus amebocyte lysate （TAL） method was employed to detect the concentration variation of bacterial endotoxin before and af- ter the plasma treatment, and endotoxic shock mice model was used to evaluate the inactivation effects of LTP on endotoxin for further study. Experimental results demonstrated that, DBD plasma can inactivate the bacterial endotoxin quickly and effectively, and when the LTP treatment time was increased, the concentrations of bacterial endotoxin decreased gradually （after 60 s plasma treatment, its inactivation effect was beyond the Chinese pharmacopoeia standard）, and the average survival time of mice gradually extended. The possible inactivation mechanisms are proposed to be related to reactive oxygen species （ROSs）.

<i>Blastomyces dermatitidis</i>Antibody Responses in Serial Serum Specimens from Dogs with Blastomycosis: Comparison of Different Yeast Lysate Antigens

The systemic fungal organism, Blastomyces dermatitidis causes blastomycosis in animals and hu-mans. This study was designed to evaluate antibody detection in 55 serial serum specimens from 9 dogs with blastomycosis using B. dermatitidis yeast lysate antigens produced from two human isolates (B5896;B5931) and two dog isolates (ERC-2;T-58) with the indirect enzyme linked im-munosorbent assay (ELISA;peroxidase system) to determine an optimal lysate antigen(s) for use in the ELISA to detect antibody in the dog serum specimens. The mean absorbance values when the lysate antigens were compared with respect to their ability to detect antibody in the day 0 sera from the 9 dogs were 1.024 (ERC-2), 1.351 (B5896), 1.700 (B5931) and 2.084 (T-58) respectively. All of the reagents exhibited a high level of sensitivity and in all instances the amount of antibody declined as the time interval post-treatment increased, but the T-58 lysate prepared from the dog isolate from Tennessee was the optimal reagent. We continue to evaluate antigens for B. derma-titidis antibody detection in different immunodiagnostic assays.

Sensitivity and Specificity Determinations with Isoelectric Focusing Fractions of <i>Blastomyces dermatitidis</i>for Antibody Detection in Serum Specimens from Infected Dogs

Blastomycosis and histoplasmosis manifest as lung and systemic fungal infections in mammals caused by Histoplasma capsulatum, and Blastomyces dermatitidis. These infections exhibit cross reactivity of antibodies which makes a correct diagnosis potentially elusive. The purpose of this study was to gain an understanding of which isoelectric focusing fractions (RotoforTM) of B. dermatitidis were reactive or cross reactive with serum specimens from dogs infected with B. dermatitidis, H. capsulatum, and Cryptococcus neoformans. Three serum specimens from dogs that were infected with B. dermatitidis, two dogs infected with H. capsulatum, and one dog infected with C. neoformans were assayed against the 20 B. dermatitidis RotoforTM fractions. Reactivity was determined using the indirect enzyme linked immunoassay (ELISA). Reactivity with B. dermatitidis was found predominantly in the protein fractions 1 - 6, and cross reactivity with H. capsulatum, and C. neoformans sera was found within the B. dermatitidis protein fractions 15 - 19.

<i>In vitro</i>analysis of T cell responses induced by breast tumor cell lysate pulsed with autologous dendritic cells

In this In vitro study, T cell responses induced by breast tumor cell lysate pulsed monocyte-derived DCs were analyzed in terms of proliferation, specific cytotoxicity and cytokine-release in order to use in immunotherapeutic settings. Nylon wool enriched T lymphocytes from 5 patients with breast cancer stimulated In vitro with tumor cell lysate pulsed monocyte-derived DCs and their proliferation response were analyzed by [3H] thymidine uptake test. Specific cytotoxic activity of tumor antigen primed T cells after three rounds weekly stimulation was evaluated by flow cytometry, and interferon-γ (IFN-γ) and interleukin-4 (IL-4) cytokines release assay was carried out 24 hours after last stimulation in the supernatant of primed T cells using commercially available ELI-SA kits. T cell proliferation assay revealed that tumor cell lysate pulsed DCs could stimulate autologous T cell proliferation response with stimulation indices 4.9 - 30. T cell mediated cytotoxicity assay demonstrated that tumor antigen primed T cells could significantly kill autologous tumor cells more than normal cells (P γ and IL-4 in response to restimulation by antigen pulsed DCs which were dominated by IFN-γ production in 2 and IL-4 production in 3 out of 5 patients. Our result suggested that breast tumor antigen pulsed DCs could elicit effective specific antitumor T cell responses In vitro, therefore, tumor antigen pulsed DC vaccination may be considered as a novel strategy for immunotherapy of patients with breast cancer refractory to standard modalities.

Validated LC-MS/MS method for simultaneous determination of SIM and its acid form in human plasma and cell lysate: Pharmacokinetic application

Simvastatin （SIM） is a 3-hydroxy-3-methylglutaryl coenzyme A reductase inhibitor widely used in hyperlipidemia therapy. SIM has recently been studied for its anticancer activity at doses higher than those used for the hyperlipidemia therapy. This prompted us to study the pharmacokinetics of high-dose SIM in cancer patients. For this purpose, an LC-MS/MS method was developed to measure SIM and its acid form （SIMA） in plasma and peripheral blood mononuclear cells （PBMCs） obtained from patients. Chromatographic analyte separation was carried out on a reverse-phase column using 75：25 （% v/v） acetonitrile：ammonium acetate （0.1 M, pH 5.0） mobile phase. Detection was performed on a triple quadrupole mass spectrometer, equipped with a turbo ion spray source and operated in positive ionization mode. The assay was linear over a range 2.5-500 ng/mL for SIM and 5-500 ng/mL for SIMA in plasma and 2.5-250 ng/mL for SIM and 5-250 ng/mL for SIMA in cell lysate. Recovery was 〉 58% for SIM and 〉 75% for SIMA in both plasma and cell lysate. SIM and SIMA were stable in plasma, cell lysate and the reconstitution solution. This method was successfully applied for the determination of SIM and SIMA in plasma and PBMCs samples collected in the pharmacokinetic study of high-dose SIM in cancer patients.

LAL test and RPT for endotoxin detection of CPT-11/DSPE-mPEG_(2000) nanoformulation: What if traditional methods are not applicable?

Endotoxin detection is an important step in drug characterization. Herein we found that a chemotherapeutic drug nanoformulation composed of irinotecan hydrochloride(CPT-11) and an amphiphilic molecule DSPE-mPEG_(2000) can interfere with the limulus amebocyte lysate assay(LAL). Furthermore, the rabbit pyrogen test(RPT) results indicated that at a relatively high dosage, the drug irinotecan hydrochloride can induce a hypothermia effect which may render the RPT results ambiguous in determination of the safety of the drug formulation.Our findings demonstrate limitations of endotoxin detection in micellar drugs,and call for the necessity of developing reliable endotoxin detection methods that can overcome the interference of nanomaterials in order to better ensure the drug safety of patients in future pharmaceutical drug development.

Comparison of Antibody Detection with Yeast Lysate Antigens Prepared from Blastomyces dermatitidis Dog Isolates from Wisconsin and Tennessee

Blastomyces dermatitidis, the causative agent of blastomycosis, a potentially lethal dimorphic fungal disease of humans and animals has been difficult to diagnose in the clinical laboratory. We are attempting to develop and improve immunodiagnostic assays by producing novel yeast lysate reagents for the detection of antibodies in blastomycosis. The objective of this study was to use lysate antigens prepared from four B. dermatitidis antigens isolated from dogs infected with blastomycosis from two different endemic areas (Wisconsin and Tennessee) testing for the detection of antibodies in serum specimens from immunized rabbits and infected dogs using the indirect ELISA. In the dog sera, absorbance values ranged from 0.774 to 1.350, while the rabbit sera values ranged from 0.533 to 1.191. Antigen T-58 appeared to lack any geographical specificity in antibody detection, which could prove useful in future immunodiagnostic detection of blastomycosis infections.

Detection of Antibody in Dogs with Blastomycosis Using <i>Blastomyces dermatitidis</i>Yeast Phase Lysate Antigens

The objective of our study was to compare two B. dermatitidis yeast phase lysate antigens [ERC-2, dog Wisconsin;85, soil Georgia, ATCC 56,920] for detecting antibody in 38 serum specimens [pre-treatment, 30-day, and 60-day post treatment] from dogs with diagnosed blastomycosis. The mean absorbance values obtained with the two antigens (N = 38) were ERC-2 = 2.359 and 85 = 2.189. The mean absorbance values when the sera were divided into the three treatment groups were as follows pre-treatment: Isolate ERC-2 had an absorbance value of 2.418;Isolate 85 had an absorbance value of 2.688, 30-day post treatment: ERC-2 had an absorbance value of 2.452;85 had an absorbance value of 2.303 and 60-day post treatment: ERC-2 had an absorbance value of 2.150;85 had an absorbance value of 2.073 with the mean absorbance values of all treatment groups were ERC-2: 2.229 and 85: 2.141. This study indicates the potential for further evaluations of the two lysate antigens with regard to antibody detection in dog sera with the ERC-2 reagent slightly more reactive than the 85 lysate antigen.

Detection of Antibodies in Serum Specimens from Dogs with Blastomycosis with Lysate Antigens Prepared from Four <i>Blastomyces dermatitidis</i>Dog Isolates: Individual Antigens vs Antigen Combinations

Blastomycosis, the systemic fungal infection of humans and animals, has presented a diagnostic challenge to clinicians and laboratory personnel for many years. Our laboratory has been concentrating on attempting to develop antigenic reagents from the yeast phase of various isolates of Blastomyces dermatitidis and to evaluate these lysate antigens with regard to antibody detection in blastomycosis. The aim of this current study was to evaluate yeast phase antigens prepared from four dog isolates of B. dermatitidis and to evaluate their efficacy, when used individually or in combination, for antibody detection in sera from dogs with blastomycosis. Mean absorbance values using the ELISA to assay 24 serum specimens (Trial 1) ranged from 0.588 with an individual lysate antigen to 0.992 when three reagents were combined. Eight of the lysates exhibited mean absorbance values ranging from 0.992 to 0.915 with 7 out of 8 being lysate antigen combinations. Mean absorbance values with the other 6 lysates ranged from 0.899 to 0.588. In Trial 2, the 6 most sensitive reagents from Trial 1 were assayed against 10 highly reactive dog sera. The results of Trial 2 showed that 5 antigen combinations detected antibody to a greater degree than the individual lysate antigen. Combinations of northern and southern antigens were able to detect antibody in serum specimens from either of these geographical regions. Comparative studies are continuing to further evaluate various lysate antigen combinations for antibody detection in blastomycosis.

Antigen Detection in Canine Blastomycosis: Comparison of Different Antibody-Antigen Combinations in Two Competitive ELISAs

This present study was designed to evaluate four different Blastomyces dermatitidis antibody-antigen combinations (B5896 and T-58 antibodies and B5896 and WI-R antigens) for the detection of antigen in 36 urine specimens from dogs with blastomycosis using a standard indirect ELISA (STD) and a biotin-streptavidin ELISA (B-SA). The antigen detection sensitivity values ranged from 81% (B-SA: T-58 Ab + WI-R Ag) to 100% (STD and B-SA: B5896 Ab + WI-R Ag;B5896 Ab + B5896 Ag) with the antibody-antigen combinations in the two assays. Optimal detection was evidenced when the B5896 Ab was allowed to react with the urine specimens for 30 min at 37?C and then placed in the B-SA ELISA plates containing the B5896 Ag. The greatest absorbance value obtained with this antibody-antigen com-bination was 0.903 (range of 0.596 - 0.903) as compared to the control value of 1.246. The difference between the control absorbance and the test absorbance values was 0.343 which was considerably greater than the control-test values with the other combinations. This study thus showed that the results obtained in antigen detection assays are dependent upon the antibody used to react with the urine specimens as well as the antigen used in the enzyme immunoassay.