Casein Lactose-Glycation of the Maillard-Type Attenuates the Anti-Inflammatory Potential of Casein Hydrolysate to IEC-6 Cells with Lipopolysaccharide Stimulation

During the thermal processing of dairy products, the Maillard reaction occurs between milk proteins and lactose, resulting in the formation of various products including glycated proteins. In this study, lactose-glycated casein was generated through the Maillard reaction between casein and lactose and then hydrolyzed by a trypsin preparation. The anti-inflammatory effect of the resultant glycated casein hydrolysate (GCH) was investigated using the lipopolysaccharide (LPS)-sitmulated rat intestinal epithelial (IEC-6) cells as a cell model and corresponding casein hydrolysate (CH) as a control. The results indicated that the preformed glycation enabled lactose conjugation to casein, which endowed GCH with a lactose content of 12.61 g/kg protein together with a lower activity than CH to enhance the viability value of the IEC-6 cells. The cells with LPS stimulation showed significant inflammatory responses, while a pre-treatment of the cells with GCH before LPS stimulation consistently led to a decreased secretion of three pro-inflammatory mediators, namely, IL-6, IL-1β and tumor necrosis factor-α (TNF-α) but an increased secretion of two anti-inflammatory mediators, including IL-10 and transforming growth factor-β (TGF-β), demonstrating the anti-inflammatory potential of GCH in LPS-stimulated cells. In addition, GCH up-regulated the expression of TLR4, p-p38, and p-p65 proteins in the stimulated cells, resulting in the suppression of NF-κB and MAPK signaling pathways. Collectively, GCH was mostly less efficient than CH to exert these assessed anti-inflammatory activities in the cells and more importantly, GCH also showed an ability to cause cell inflammation by promoting IL-6 secretion and up-regulating the expression of TLR4 and p-p65. The casein lactose-glycation of the Maillard-type was thereby concluded to attenuate the anti-inflammatory potential of the resultant casein hydrolysate. It is highlighted that the casein lactose-glycation of the Maillard-type might cause a negative impact on the bioactivity of casein in the intestine, because the glycated casein after digestion could release GCH with reduced anti-inflammatory activity.     

Protective Effect of Wheat Peptides against Indomethacin-Induced Oxidative Stress in IEC-6 Cells

Recent studies have demonstrated that wheat peptides protected rats against non-steroidal anti-inflammatory drugs-induced small intestinal epithelial cells damage, but the mechanism of action is unclear. In the present study, an indomethacin-induced oxidative stress model was used to investigate the effect of wheat peptides on the nuclear factor-κB(NF-κB)-inducible nitric oxide synthase-nitric oxide signal pathway in intestinal epithelial cells-6 cells. IEC-6 cells were treated with wheat peptides (0, 125, 500 and 2000 mg/L) for 24 h, followed by 90 mg/L indomethacin for 12 h. Wheat peptides significantly attenuated the indomethacin-induced decrease in superoxide dismutase and glutathione peroxidase activity. Wheat peptides at 2000 mg/L markedly decreased the expression of the NF-κB in response to indomethacin-induced oxidative stress. This study demonstrated that the addition of wheat peptides to a culture medium significantly inhibited the indomethacin-induced release of malondialdehyde and nitrogen monoxide, and increased antioxidant enzyme activity in IEC-6 cells, thereby providing a possible explanation for the protective effect proposed for wheat peptides in the prevention of indomethacin-induced oxidative stress in small intestinal epithelial cells.     

外源核苷酸对IEC-6细胞凋亡的影响

目的探讨外源核苷酸对正常肠上皮细胞系IEC6凋亡的作用。方法体外培养的IEC6细胞经4种单核苷酸及按母乳组成模式配制的核苷酸混合物处理,然后采用流式细胞术、电子显微镜等方法观测细胞凋亡。结果流式细胞检测表明嘌呤核苷酸AMP和GMP均能诱导细胞凋亡,但电子显微镜观察发现仅有AMP诱导典型的细胞凋亡特征。嘧啶核苷酸和核苷酸混合物不诱导细胞凋亡,而且嘧啶核苷酸UMP能解除嘌呤核苷酸诱导细胞凋亡的作用。结论嘌呤核苷酸可诱导肠上皮细胞凋亡,嘧啶核苷酸UMP可解除嘌呤核苷酸的凋亡诱导作用。     

Vit B6对支持细胞乳酸分泌和体外培养睾丸碎块合成睾酮的影响

目的研究Vit B6对原代培养睾丸支持细胞分泌乳酸和体外对睾丸合成睾酮的影响,探讨Vit B6生殖毒性作用机制。方法胰蛋白酶和Ⅳ胶原酶两步分离法分离雄性SD大鼠睾丸支持细胞进行体外培养,分别加入不同浓度的Vit B6(0.1mg/ml,1mg/ml,10mg/ml和20mg/ml),24h后收集培养液测定乳酸含量。利用离体组织培养和放射免疫技术,观察不同浓度的Vit B6是否对大鼠睾丸睾酮的分泌有直接作用。结果与对照组相比,Vit B610mg/ml和20mg/ml剂量组乳酸含量减少。Vit B6各剂量组睾丸培养液中睾酮的含量与对照组相比均没有显著性差异。结论Vit B6可能通过减少了睾丸支持细胞乳酸的分泌而影响生殖细胞的功能。Vit B6对睾丸分泌睾酮没有直接的抑制作用,可能对间质细胞正常功能没有影响。     

HM-Chromanone,a Major Homoisoflavonoid in Portulaca oleracea L., Improves Palmitate-Induced Insulin Resistance by Regulating Phosphorylation of IRS-1 Residues in L6 Skeletal Muscle Cells

This study investigated the effect of (E)-5-hydroxy-7-methoxy-3-(2-hydroxybenzyl)-4-chromanone (HM-chromanone) on palmitate-induced insulin resistance and elucidated the underlying mechanism in L6 skeletal muscle cells. Glucose uptake was markedly decreased due to palmitate-induced insulin resistance in these cells; however, 10, 25, and 50 µM HM-chromanone remarkably improved glucose uptake in a concentration-dependent manner. HM-chromanone treatment downregulated protein tyrosine phosphatase 1B (PTP1B) and phosphorylation of c-Jun N-terminal kinase (JNK) and inhibitor of nuclear factor kappa-B kinase subunit beta (IKKβ), which increased because of palmitate mediating the insulin-resistance status in cells. HM-chromanone promoted insulin receptor substrate-1 (IRS-1) tyrosine phosphorylation and suppressed palmitate-induced phosphorylation of IRS-1 serine. This activated phosphoinositide 3-kinase (PI3K) and stimulated protein kinase B (AKT) phosphorylation. Phosphorylated AKT promoted the translocation of Glucose transporter type 4 to the plasma membrane and significantly enhanced glucose uptake into muscle cells. Additionally, HM-chromanone increased glycogen synthesis through phosphorylating glycogen synthase kinase 3 alpha/beta (GSK3 α/β) via AKT. Consequently, HM-chromanone may improve insulin resistance by downregulating the phosphorylation of IRS-1 serine through inhibition of negative regulators of insulin signaling and inflammation-activated protein kinases in L6 skeletal muscle cells.     

Dietary Flavone Baicalein Combinate with Genipin Attenuates Inflammation Stimulated by Lipopolysaccharide in RAW264.7 Cells or Pseudomonas aeruginosa in Mice via Regulating the Expression and Phosphorylation of AKT

Mounting evidence has shown that single-targeted therapy might be inadequate to achieve satisfactory effects. Thus, drug combinations are gaining attention as they can regulate multiple targets to obtain more beneficial effects. Heat shock protein 90 (HSP90) is a molecular chaperone that assists the protein assembly and folding of client proteins and maintains their stability. Interfering with the interaction between HSP90 and its client proteins by inhibiting the latter’s activity may offer a new approach toward combination therapy. The HSP90 client protein AKT plays an important role in the inflammatory response syndrome caused by infections. In this study, the dietary flavone baicalein was identified as a novel inhibitor of HSP90 that targeted the N-terminal ATP binding pocket of HSP90 and hindered the chaperone cycle, resulting in AKT degradation. Combining baicalein with genipin, which was extracted from Gardenia jasminoides, could inhibit the pleckstrin homology domain of AKT, significantly increasing the anti-inflammatory effects both in vitro and in vivo. This synergistic effect was attributed to the reduction in AKT expression and phosphorylation. Thus, elucidating the mechanism underlying this effect will provide a new avenue for the clinical application and development of synergistic anti-inflammatory drugs.     

Targeting of rotavirus VP6 to DEC-205 induces protection against the infection in mice

Rotavirus (RV) is the primary etiologic agent of severe gastroenteritis in human infants. Although two attenuated RV-based vaccines have been licensed to be applied worldwide, they are not so effective in low-income countries, and the induced protection mechanisms have not been clearly established. Thus, it is important to develop new generation vaccines that induce long lasting heterotypic immunity. VP6 constitutes the middle layer protein of the RV virion. It is the most conserved protein and it is the target of protective T-cells; therefore, it is a potential candidate antigen for a new generation vaccine against the RV infection. We determined whether targeting the DEC-205 present in dendritic cells (DCs) with RV VP6 could induce protection at the intestinal level. VP6 was cross-linked to a monoclonal antibody (mAb) against murine DEC-205 (αDEC-205:VP6), and BALB/c mice were inoculated subcutaneously (s.c.) twice with the conjugated containing 1.5 μg of VP6 in the presence of polyinosinic–polycytidylic acid (Poly I:C) as adjuvant. As controls and following the same protocol, mice were immunized with ovalbumin (OVA) cross-linked to the mAb anti-DEC-205 (αDEC-205:OVA), VP6 cross-linked to a control isotype mAb (Isotype:VP6), 3 μg of VP6 alone, Poly I:C or PBS. Two weeks after the last inoculation, mice were orally challenged with a murine RV. Mice immunized with α-DEC-205:VP6 and VP6 alone presented similar levels of serum Abs to VP6 previous to the virus challenge. However, after the virus challenge, only α-DEC-205:VP6 induced up to a 45% IgA-independent protection. Memory T-helper (Th) cells from the spleen and the mesenteric lymph node (MLN) showed a Th1-type response upon antigen stimulation in vitro. These results show that when VP6 is administered parenterally targeting DEC-205, it can induce protection at the intestinal level at a very low dose, and this protection may be Th1-type cell dependent.     

Investigation of Sensitization Potential of the Soybean Allergen Gly m 4 by Using Caco-2/Immune Cells Co-Culture Model

The soybean allergen Gly m 4 is known to cause severe allergic reactions including anaphylaxis, unlike other Bet v 1 homologues, which induce mainly local allergic reactions. In the present study, we aimed to investigate whether the food Bet v 1 homologue Gly m 4 can be a sensitizer of the immune system. Susceptibility to gastrointestinal digestion was assessed in vitro. Transport through intestinal epithelium was estimated using the Caco-2 monolayer. Cytokine response of different immunocompetent cells was evaluated by using Caco-2/Immune cells co-culture model. Absolute levels of 48 cytokines were measured by multiplex xMAP technology. It was shown that Gly m 4 can cross the epithelial barrier with a moderate rate and then induce production of IL-4 by mature dendritic cells in vitro. Although Gly m 4 was shown to be susceptible to gastrointestinal enzymes, some of its proteolytic fragments can selectively cross the epithelial barrier and induce production of Th2-polarizing IL-5, IL-10, and IL-13, which may point at the presence of the T-cell epitope among the crossed fragments. Our current data indicate that Gly m 4 can potentially be a sensitizer of the immune system, and intercommunication between immunocompetent and epithelial cells may play a key role in the sensitization process.     

Modification of the Interleukin-6 Response to Air Pollution by Interleukin-6 and Fibrinogen Polymorphisms

Background

Evidence suggests that cardiovascular effects of air pollution are mediated by inflammation and that air pollution can induce genetic expression of the interleukin-6 gene (IL6).

Objectives

We investigated whether IL6 and fibrinogen gene variants can affect plasma IL-6 responses to air pollution in patients with cardiovascular disease.

Methods

We repeatedly determined plasma IL-6 in 955 myocardial infarction survivors from six European cities (n = 5,539). We conducted city-specific analyses using additive mixed models adjusting for patient characteristics, time trend, and weather to assess the impact of air pollutants on plasma IL-6. We pooled city-specific estimates using meta-analysis methodology. We selected three IL6 single-nucleotide polymorphisms (SNPs) and one SNP each from the fibrinogen α-chain gene (FGA) and β-chain gene (FGB) for gene–environment analyses.

Results

We found the most consistent modifications for variants in IL6 rs2069832 and FBG rs1800790 after exposure to carbon monoxide (CO; 24-hr average; p-values for interaction, 0.034 and 0.019, respectively). Nitrogen dioxide effects were consistently modified, but p-values for interaction were larger (0.09 and 0.19, respectively). The strongest effects were seen 6–11 hr after exposure, when, for example, the overall effect of a 2.2% increase in IL-6 per 0.64 mg/m³ CO was modified to a 10% (95% confidence interval, 4.6–16%) increase in IL-6 (p-value for interaction = 0.002) for minor homozygotes of FGB rs1800790.

Conclusions

The effect of gaseous traffic-related air pollution on inflammation may be stronger in genetic subpopulations with ischemic heart disease. This information could offer an opportunity to identify postinfarction patients who would benefit more than others from a cleaner environment and antiinflammatory treatment.     

Recruitment of Normal Stem Cells to an Oncogenic Phenotype by Noncontiguous Carcinogen-Transformed Epithelia Depends on the Transforming Carcinogen

Background: Cancer stem cells (CSCs) drive tumor initiation, progression, and metastasis. The microenvironment is critical to the fate of CSCs. We have found that a normal stem cell (NSC) line from human prostate (WPE-stem) is recruited into CSC-like cells by nearby, but noncontiguous, arsenic-transformed isogenic malignant epithelial cells (MECs).Objective: It is unknown whether this recruitment of NSCs into CSCs by noncontact co-culture is specific to arsenic-transformed MECs. Thus, we used co-culture to examine the effects of neighboring noncontiguous cadmium-transformed MECs (Cd-MECs) and N-methyl-N-nitrosourea–transformed MECs (MNU-MECs) on NSCs.Results: After 2 weeks of noncontact Cd-MEC co-culture, NSCs showed elevated metalloproteinase-9 (MMP-9) and MMP-2 secretion, increased invasiveness, increased colony formation, decreased PTEN expression, and formation of aggressive, highly branched duct-like structures from single cells in Matrigel, all characteristics typical of cancer cells. These oncogenic characteristics did not occur in NSCs co-cultured with MNU-MECs. The NSCs co-cultured with Cd-MECs retained self-renewal capacity, as evidenced by multiple passages (> 3) of structures formed in Matrigel. Cd-MEC–co-cultured NSCs also showed molecular (increased VIM, SNAIL1, and TWIST1 expression; decreased E-CAD expression) and morphologic evidence of epithelial-to-mesenchymal transition typical for conversion to CSCs. Dysregulated expression of SC-renewal genes, including ABCG2, OCT-4, and WNT-3, also occurred in NSCs during oncogenic transformation induced by noncontact co-culture with Cd-MECs.Conclusions: These data indicate that Cd-MECs can recruit nearby NSCs into a CSC-like phenotype, but MNU-MECs do not. Thus, the recruitment of NSCs into CSCs by nearby MECs is dependent on the carcinogen originally used to malignantly transform the MECs.     

Improvement of the immunity of pig to Hog cholera vaccine by recombinant plasmid with porcine interleukin-6 gene and CpG motifs

In order to observe the dosage-effect of recombinant pig interleukin-6 gene and CpG motifs on the immune responses of swine to vaccine, a novel recombinant eukaryotic VPIL6C plasmid was packed with chitosan nanoparticles (CNP) prepared by ionic cross linkage, which contains pig interleukin-6 gene and immunostimulatory sequence consisted of 11 CpG motifs. CNP-VRIL6C was then utilized to inoculate 30-day-old piglets intramuscularly at the dosage of 0.5, 1.0 and 1.5 mg/per capita, respectively. Meanwhile, the piglets were injected with attenuated classical Hog cholera vaccine and designated as A1, A2 and A3 group. The blood was weekly collected from the piglets after vaccination to detect the changes of immunoglobulins, specific antibody, interleukins, IFN-γ and immune cells. The results were found that compared to those of the control piglets injected with VR1020-CNP, the content of IgG, IgA and IgM, specific antibodies, IL-2, IL-6 and IFN-γ significantly increased in the sera from the treated three groups from 14 to 70 days after vaccination (P < 0.05); the number of T_H, T_C and CD3⁺ positive T cells raised obviously in the blood of VPIL6C treated piglets (P < 0.05). Also the above immune indexes of A1 group were significantly lower to different extent in comparison with those of A2 and A3 group from 14 to 56 days post inoculation (P > 0.05). Moreover, the lymphocytes also remarkably elevated in the treated groups (P < 0.05). These indicate that VPIL6C entrapped with CNP is a novel effective adjuvant to boost the humoral and cellular immunity of pig to Hog cholera, implying it's potentiality to enhance the resistance of pig against infectious diseases.     

Bifidobacterium animalis subsp. lactis A6 Enhances Fatty Acid β-Oxidation of Adipose Tissue to Ameliorate the Development of Obesity in Mice

Fatty acid β-oxidation (FAO) is confirmed to be impaired in obesity, especially in adipose tissues. We previously proved that Bifidobacterium animalis subsp. lactis A6 (BAA6) had protective effects against diet-induced obesity. However, whether BAA6 enhances FAO to ameliorate the development of obesity has not been explored. After being fed with high-fat diet (HFD) for 9 weeks, male C57BL/6J mice were fed HFD or BAA6 for 8 weeks. In vitro study was carried out using 3T3-L1 adipocytes to determine the effect of BAA6 culture supernatant (BAA6-CM). Here, we showed that administration of BAA6 to mice fed with HFD decreased body weight gain (by 5.03 g) and significantly up-regulated FAO in epididymal adipose tissues. In parallel, FAO in 3T3-L1 cells was increased after BAA6-CM treatment. Acetate was identified as a constituent of BAA6-CM that showed a similar effect to BAA6-CM. Furthermore, acetate treatment activated the GPR43-PPARα signaling, thereby promoting FAO in 3T3-L1 cells. The levels of acetate were also elevated in serum and feces (by 1.92- and 2.27-fold) of HFD-fed mice following BAA6 administration. The expression levels of GPR43 and PPARα were increased by 55.45% and 69.84% after BAA6 supplement in the epididymal fat of mice. Together, these data reveal that BAA6 promotes FAO of adipose tissues through the GPR43-PPARα signaling, mainly by increasing acetate levels, leading to alleviating the development of obesity.     

刺儿菜提取物对诱导人肝癌细胞株的裸鼠移植瘤抑制作用的研究

<正>刺儿菜Cephalanoplos(Bunge)Kitam,是经常采食的一种野生蔬菜。我们的测定结果表明:每百克刺儿菜含Ca378mg,是大白菜的近6倍;含硒高达40.64μg,是一般蔬菜的许多倍;含VE2.34mg,而大白菜仅含0.92mg。含VK1极高,达7.88mg,是菠菜的5倍,西红柿的16.5倍,土豆的82.5倍。美国匹兹堡大学癌症研究所肝癌中心的科学家发现维生素K在组织培养液里,     

Induction of IL-6 and inhibition of IL-8 secretion in the human airway cell line Calu-3 by urban particulate matter collected with a modified method of PM sampling

Exposure to particulate matter (PM) induces inflammatory cytokines. In the present study, we evaluated the secretion of IL-6 and IL-8 by an airway cell line exposed to PM with a mean aerodynamic size equal to or less than 10 or 2.5 μm (PM₁₀ and PM_2.5, respectively) collected in Mexico City, using a modified high-volume sampling method avoiding the use of solvents or introducing membrane components into the samples. PM was collected on cellulose-nitrate (CN) membranes modified for collection on high-volume samplers. Composition of the particles was evaluated by particle-induced X-ray emission (PIXE) and scanning electron microscopy. The particles (10-160 μg/cm²) were tested on Calu-3 cells. Control cultures were exposed to LPS (10 ng/mL to 100 μg/mL) or silica (10-160 μg/cm²). IL-6 and IL-8 secretions were evaluated by ELISA. An average of 10 mg of PM was recovered form each cellulose-nitrate filter. No evidence of contamination from the filter was found. Cells exposed to PM₁₀ presented an increase in the secretion of IL-6 (up to 400%), while IL-8 decreased (from 40% to levels below the detection limit). A similar but weaker effect was observed with PM_2.5. In conclusion, our modified sampling method provides a large amount of urban PM free of membrane contamination. The urban particles induce a decrease in IL-8 secretion that contrasts with the LPS and silica effects. These results suggest that the regulation of IL-8 expression is different for urban particles (complex mixture containing combustion-related particles, soil and biologic components) than for biogenic compounds or pure mineral particles.     

乙醇诱导人脐静脉内皮细胞损伤及ADMA/NOS/NO信号机制

目的探讨乙醇诱导人脐静脉内皮细胞损伤效应及ADMA/NOS/NO信号机制。方法用不同浓度的乙醇与人脐静脉内皮细胞共培养,观察细胞形态和活性,通过检测细胞上清液丙二醛（MDA）、非对称性二甲基精氨酸（AD-MA）、一氧化氮（NO）的含量和一氧化氮合酶（NOS）的活性等指标,观察乙醇对人脐静脉内皮细胞损伤的量-效关系（乙醇处理细胞24 h）和时-效关系（终浓度为100 mmol/L的乙醇分别于处理0、3、6、12、24、48 h后观察检测指标）。实验分为正常对照组（加入与处理组等体积的D-Hank’s液,乙醇浓度为0 mmol/L）、乙醇处理组（终浓度分别为25、50、100、200mmol/L的乙醇）、乙醇＆VitC组（乙醇终浓度为100 mmol/L,VitC终浓度为100 mg/L）和阳性对照组（终浓度为500μmol/L的过氧化氢）。结果50-200 mmol/L乙醇能显著改变细胞形态、降低细胞活性（OD值：0.91±0.10-0.58±0.04,细胞生长抑制率：0.00±0.00-35.57±3.13,P〈0.01）;能显著性诱导内皮细胞MDA升高（258.72±7.36-524.07±8.25,P〈0.01）。50-200 mmol/L的乙醇还能显著性诱导：内皮型一氧化氮合酶（eNOS）活性下降（6.27±0.10-0.47±0.23,P〈0.05）及总NOS活性下降（10.69±0.54-3.77±0.32,P〈0.05）;NO含量降低（191.61±7.96-88.38±5.07,P〈0.05）。而且上述生物学效应均具有显著的时间依赖性,各处理组间两两比较也具有显著性（P〈0.05或P〈0.01）。100 mmol/L乙醇处理时细胞诱导型一氧化氮合酶（iNOS）活性及ADMA含量最高,且分别在处理后24 h到达最高峰。VitC均能扭转上述生物学效应,且具有显著性（P〈0.01）。结论50-200 mmol/L的乙醇能显著诱导人脐静脉内皮细胞损伤;ADMA在乙醇诱导人脐静脉内皮细胞损伤中有着重要作用。     

Response of MUTZ-3 dendritic cells to the different components of the Haemophilus influenzae type B conjugate vaccine: towards an in vitro assay for vaccine immunogenicity

Potency testing is mandatory for vaccine registration and batch release. Due to various limitations to in vivo potency testing, there is need for relevant in vitro alternatives. These alternative tests should preferably comprise cells from the target (human) species. The whole suite of immune responses to vaccination that occur in vivo in humans cannot be tested in vitro using a single cell type. Even so, dendritic cells (DC) form an important candidate cell type since they are pivotal in inducing and orchestrating immune responses. Cell lines are preferred over ex vivo cells for reasons of safety, accessibility, and reproducibility. In this first feasibility study we used the human cell line MUTZ-3, because it most closely resembles ex vivo human DC, and compared its response to monocyte-derived DC (moDC).Haemophilus influenzae type B (HiB) vaccine was chosen because its components exert different effects in vivo: while the HiB antigen, polyribosyl ribitol phosphate (PRP) fails to induce sufficient protection in children below 2 years of age, conjugation of this polysaccharide antigen to outer membrane protein (OMP) of Neisseria meningitides, results in sufficient protection. Effects of PRP, OMP, conjugated PRP-OMP, and adjuvanted vaccine (PedVax HiB), on cytokine production and surface marker expression were established. PRP induced no effects on cytokine production and the effect on surface marker expression was limited to a minor decrease in CD209 (DC-SIGN). In both MUTZ-3 and moDC, OMP induced the strongest response both in cytokine production and surface marker expression. Compared to OMP alone conjugated PRP-OMP generally induced a weaker response in cytokine production and surface marker expression. The effects of PedVax HiB were comparable to conjugated PRP-OMP. While moDC showed a larger dynamic range than MUTZ-3 DC, these cells also showed considerable variability between donors, with MUTZ-3 DC showing a consistent response between the replicate assays. In our view, this makes MUTZ-3 DC the cells of choice. In conclusion, our results demonstrate that the MUTZ-3 DC assay allows discrimination between compounds with different immunogenicity. The potential of this cell line as (part of) an in vitro immunogenicity assay should be further explored.     

Uptake of Vitamins D2, D3, D4, D5, D6, and D7 Solubilized in Mixed Micelles by Human Intestinal Cells,Caco-2, an Enhancing Effect of Lysophosphatidylcholine on the Cellular Uptake,and Estimation of Vitamins D’ Biological Activities

Vitamins D have various biological activities, as well as intestinal calcium absorption. There has been recent concern about insufficient vitamin D intake. In addition to vitamins D₂ and D₃, there are lesser-known vitamins D₄–D₇. We synthesized vitamins D₅–D₇, which are not commercially available, and then evaluated and compared the mixed micelles-solubilized vitamins D uptake by Caco-2 cells. Except for vitamin D₅, the uptake amounts of vitamins D₄–D₇ by differentiated Caco-2 cells were similar to those of vitamins D₂ and D₃. The facilitative diffusion rate in the ezetimibe inhibited pathway was approximately 20% for each vitamin D type, suggesting that they would pass through the pathway at a similar rate. Lysophosphatidylcholine enhanced each vitamin D uptake by approximately 2.5-fold. Lysophosphatidylcholine showed an enhancing effect on vitamin D uptake by reducing the intercellular barrier formation of Caco-2 cells by reducing cellular cholesterol, suggesting that increasing the uptakes of vitamins D and/or co-ingesting them with lysophosphatidylcholine, would improve vitamin D insufficiency. The various biological activities in the activated form of vitamins D₄–D₇ were estimated by Prediction of Activity Spectra for Substances (PASS) online simulation. These may have some biological activities, supporting the potential as nutritional components.     

A single dose of inactivated hepatitis A vaccine promotes HAV-specific memory cellular response similar to that induced by a natural infection

Based on current studies on the effects of single dose vaccines on antibody production, Latin American countries have adopted a single dose vaccine program. However, no data are available on the activation of cellular response to a single dose of hepatitis A. Our study investigated the functional reactivity of the memory cell phenotype after hepatitis A virus (HAV) stimulation through administration of the first or second dose of HAV vaccine and compared the response to that of a baseline group to an initial natural infection. Proliferation assays showed that the first vaccine dose induced HAV-specific cellular response; this response was similar to that induced by a second dose or an initial natural infection. Thus, from the first dose to the second dose, increase in the frequencies of classical memory B cells, TCD8 cells, and central memory TCD4 and TCD8 cells were observed. Regarding cytokine production, increased IL-6, IL-10, TNF, and IFNγ levels were observed after vaccination. Our findings suggest that a single dose of HAV vaccine promotes HAV-specific memory cell response similar to that induced by a natural infection. The HAV-specific T cell immunity induced by primary vaccination persisted independently of the protective plasma antibody level. In addition, our results suggest that a single dose immunization system could serve as an alternative strategy for the prevention of hepatitis A in developing countries.