Effects of Bovine Lactoferrin on the Maintenance of Respiratory and Systemic Physical Conditions in Healthy Adults-A Randomized, Double-Blind, Placebo-Controlled Trial.

OBJECTIVES: We investigated the effects of bovine lactoferrin (LF) on the maintenance of the respiratory and systemic physical conditions. METHODS: A randomized, double-blind, placebo-controlled trial was conducted. Healthy adults at Kyushu University of Health and Welfare ingested a placebo or bovine LF (200 mg/day) for 12 weeks. The primary endpoints were the total respiratory and systemic symptom scores. The secondary endpoint was the activity of plasmacytoid dendritic cells (pDCs) in peripheral blood. RESULTS: A total of 157 subjects were randomized (placebo, n = 79; LF, n = 78), of whom, 12 dropped out. The remaining 145 participants were included in the full analysis set (placebo group, n = 77; LF group, n = 68). The total scores for respiratory and systemic symptoms during the intervention were significantly lower in the LF group than in the placebo group. The expression of CD86 and HLA-DR on pDCs was significantly higher in the LF group than in the placebo group at week 12. Adverse events were comparable between the groups, and no adverse drug reactions were observed. CONCLUSIONS: These results suggest that orally ingested LF supports the normal immune system via maintaining pDC activity, and maintains respiratory and systemic physical conditions in healthy adults.

Nanopore Filter: A Method for Counting and Extracting Single DNA Molecules Using a Biological Nanopore.

This paper describes a method for the real-time counting and extraction of DNA molecules at the single-molecule level by nanopore technology. As a powerful tool for electrochemical single-molecule detection, nanopore technology eliminates the need for labeling or partitioning sample solutions at the femtoliter level. Here, we attempt to develop a DNA filtering system utilizing an α-hemolysin (αHL) nanopore. This system comprises two droplets, one filling with and one emptying DNA molecules, separated by a planar lipid bilayer containing αHL nanopores. The translocation of DNA through the nanopores is observed by measuring the channel current, and the number of translocated molecules can also be verified by quantitative polymerase chain reaction (qPCR). However, we found that the issue of contamination seems to be an almost insolvable problem in single-molecule counting. To tackle this problem, we tried to optimize the experimental environment, reduce the volume of solution containing the target molecule, and use the PCR clamp method. Although further efforts are still needed to achieve a single-molecule filter with electrical counting, our proposed method shows a linear relationship between the electrical counting and qPCR estimation of the number of DNA molecules.

Lactoferrin and its digestive peptides induce interferon-α production and activate plasmacytoid dendritic cells ex vivo.

Plasmacytoid dendritic cells (pDCs) recognise viral single-stranded RNA (ssRNA) or CpG DNA via Toll-like receptor (TLR)-7 and TLR9, and produce interferon (IFN)-α. Activated pDCs upregulate human leukocyte antigen (HLA)-DR and CD86 expression levels. Ingestion of bovine lactoferrin (LF) activates pDCs, but little is known about its effects. In this study, the effects of LF and its pepsin hydrolysate (LFH) on the production of IFN-α from peripheral blood mononuclear cells (PBMCs) and pDCs were examined. PBMCs were prepared from peripheral blood of healthy adults and incubated with LF, LFH, or lactoferricin (LFcin) in the absence or presence of ssRNA derived from human immunodeficiency virus. The concentration of IFN-α in the supernatant and the expression levels of IFN-α, HLA-DR, and CD86 in pDCs were quantified by enzyme-linked immunosorbent assay and flow cytometry. In the absence of ssRNA, the concentration of IFN-α was negligible and LF had no effect on it. In the presence of ssRNA, IFN-α was detected at a certain level, and LF and LFH significantly increased its concentration. The increase caused by LFH and LFcin were comparable. In addition, LF significantly upregulated the expression levels of IFN-α, HLA-DR, and CD86 in pDCs. LF and its digestive peptides induced IFN-α production and activated pDCs in the presence of ssRNA, suggesting that LF modulates the immune system by promoting pDC activation upon viral recognition.

Pharmaceutical Potential of Casein-Derived Tripeptide Met-Lys-Pro: Improvement in Cognitive Impairments and Suppression of Inflammation in APP/PS1 Mice.

BACKGROUND: Tripeptide Met-Lys-Pro (MKP), a component of casein hydrolysates, has effective angiotensin-converting enzyme (ACE) inhibitory activity. Brain angiotensin II enzyme activates the NADPH oxidase complex via angiotensin II receptor type 1 (AT1) and enhances oxidative stress injury. ACE inhibitors improved cognitive function in Alzheimer's disease (AD) mouse models and previous clinical trials. Thus, although undetermined, MKP may be effective against pathological amyloid-ß (Aß) accumulation-induced cognitive impairment. OBJECTIVE: The current study aimed to investigate the potential of MKP as a pharmaceutical against AD by examining MKP's effect on cognitive function and molecular changes in the brain using double transgenic (APP/PS1) mice. METHODS: Experimental procedures were conducted in APP/PS1 mice (n = 38) with a C57BL/6 background. A novel object recognition test was used to evaluate recognition memory. ELISA was used to measure insoluble Aß40, Aß42, and TNF-α levels in brain tissue. Immunohistochemical analysis allowed the assessment of glial cell activation in MKP-treated APP/PS1 mice. RESULTS: The novel object recognition test revealed that MKP-treated APP/PS1 mice showed significant improvement in recognition memory. ELISA of brain tissue showed that MKP significantly reduced insoluble Aß40, Aß42, and TNF-α levels. Immunohistochemical analysis indicated the suppression of the marker for microglia and reactive astrocytes in MKP-treated APP/PS1 mice. CONCLUSION: Based on these results, we consider that MKP could ameliorate pathological Aß accumulation-induced cognitive impairment in APP/PS1 mice. Furthermore, our findings suggest that MKP potentially contributes to preventing cognitive decline in AD.

Neuroprotective Effects of Casein-Derived Peptide Met-Lys-Pro (MKP) in a Hypertensive Model.

We have previously reported that casein hydrolysate, CH-3, from bovine milk and casein-derived tripeptide Met-Lys-Pro (MKP) has ACE inhibitory activity and reduces blood pressure. In this study, we investigated the therapeutic effects of MKP in a hypertensive rat model (7-week-old male SHRSP/Izm rats). For long term evaluation, rats were fed either a diet containing CH-3 or normal diet. The survival rate of SHRSP rats was significantly improved by intake of CH-3 for 181 days. For short term evaluation, rats were orally administered synthetic tripeptide MKP or distilled water for 4 weeks. MRI study demonstrated that hemorrhagic lesions were observed in two of five rats in the control group, while no hemorrhagic lesions were observed in the MKP group. Volumetric analysis using MRI revealed that MKP administration inhibited atrophy of diencephalic regions. Histological examinations revealed that hemorrhage areas and astrogliosis in the hippocampus and cerebral cortex were lower in the MKP group than in the control group. Gene expression analysis indicated that MKP administration reduced expression of genes related to cerebral circulation insufficiency such as immune responses (Cd74 and Prkcd), response to hypoxia (Ddit4, Apold1, and Prkcd), reactive oxygen species metabolic process (Ddit4 and Pdk4), and apoptotic process (Ddit4, Prkcd, and Sgk1), suggesting that MKP administration prevented cerebral ischemia associated with hypertension. In addition, some genes encoding responses to hormone stimulus (Fos, Dusp1, and Sik1) were also downregulated. Serum aldosterone and corticosterone levels were also significantly decreased following MKP administration. The present study indicates that MKP shows neuroprotective effects in SHRSP rats by regulating cerebral circulation insufficiency and corticoid levels. MKP administration may therefore be a potential therapeutic strategy for hypertensive brain diseases such as cerebrovascular disease.

Evaluation of Fat Accumulation and Adipokine Production during the Long-Term Adipogenic Differentiation of Porcine Intramuscular Preadipocytes and Study of the Influence of Immunobiotics.

The degree of fat accumulation and adipokine production are two major indicators of obesity that are correlated with increased adipose tissue mass and chronic inflammatory responses. Adipocytes have been considered effector cells for the inflammatory responses due to their capacity to express Toll-like receptors (TLRs). In this study, we evaluated the degree of fat accumulation and adipokine production in porcine intramuscular preadipocyte (PIP) cells maintained for in vitro differentiation over a long period without or with stimulation of either TNF-α or TLR2-, TLR3-, or TLR4-ligands. The cytosolic fat accumulation was measured by liquid chromatography and the expression of adipokines (CCL2, IL-6, IL-8 and IL-10) were quantified by RT-qPCR and ELISA at several time points (0 to 20 days) of PIP cells differentiation. Long-term adipogenic differentiation (LTAD) induced a progressive fat accumulation in the adipocytes over time. Activation of TLR3 and TLR4 resulted in an increased rate of fat accumulation into the adipocytes over the LTAD. The production of CCL2, IL-8 and IL-6 were significantly increased in unstimulated adipocytes during the LTAD, while IL-10 expression remained stable over the studied period. An increasing trend of adiponectin and leptin production was also observed during the LTAD. On the other hand, the stimulation of adipocytes with TLRs agonists or TNF-α resulted in an increasing trend of CCL2, IL-6 and IL-8 production while IL-10 remained stable in all four treatments during the LTAD. We also examined the influences of several immunoregulatory probiotic strains (immunobiotics) on the modulation of the fat accumulation and adipokine production using supernatants of immunobiotic-treated intestinal immune cells and the LTAD of PIP cells. Immunobiotics have shown a strain-specific ability to modulate the fat accumulation and adipokine production, and differentiation of adipocytes. Here, we expanded the utility and potential application of our in vitro PIP cells model by evaluating an LTAD period (20 days) in order to elucidate further insights of chronic inflammatory pathobiology of adipocytes associated with obesity as well as to explore the prospects of immunomodulatory intervention for obesity such as immunobiotics.

Investigating Anti-Obesity Effects by Oral Administration of Aloe vera Gel Extract (AVGE): Possible Involvement in Activation of Brown Adipose Tissue (BAT).

The aim of this study is to investigate the mechanism of anti-obesity effects of Aloe vera gel extract (AVGE) containing Aloe sterols. Previously, we reported that oral intake of Aloe vera components has an anti-diabetic and anti-obesity effect. This study was designed to assess the role of brown adipose tissue (BAT) in the anti-obesity effect of AVGE. Six-week-old male mice were divided into three groups; STD (standard diet), HFD (60% high fat diet) and AVGE (60% high fat diet with AVGE treatment). During 11 wk of AVGE administration, body weight has been monitored. Tissue samples were obtained to be measured the weight and evaluated the gene expressions. Mice treated with AVGE had suppressed body weight, and liver and fat weight gain. To investigate BAT activation, we measured the expression of mRNA related to BAT thermogenesis. Mice in the AVGE group had higher expression of Ucp1, Adrb3, and Cidea in BAT compared to HFD. Next, to investigate the possibility that AVGE induced hepatic FGF21, which is an important factor for nutrient and energy homeostasis including BAT regulation, in vitro study was conducted. HepG2 cell stimulated by AVGE were highly expressed FGF21. These results suggested that BAT activation partially contributes to mechanism of anti-obesity effect of Aloe sterols in diet-induced obesity (DIO) models. However, further study is needed to determine the predominant mechanism.

Transcriptome Modifications in the Porcine Intramuscular Adipocytes during Differentiation and Exogenous Stimulation with TNF-α and Serotonin.

Adipocytes are dynamic cells that have critical functions to maintain body energy homeostasis. Adipocyte physiology is affected by the adipogenic differentiation, cell program, as well as by the exogenous stimulation of biochemical factors, such as serotonin and TNF-α. In this work, we investigated the global transcriptome modifications when porcine intramuscular preadipocyte (PIP) was differentiated into porcine mature adipocyte (pMA). Moreover, we studied transcriptome changes in pMA after stimulation with serotonin or TNF-α by using a microarray approach. Transcriptome analysis revealed that the expression of 270, 261, and 249 genes were modified after differentiation, or after serotonin and TNF-α stimulation, respectively. Expression changes in APP, HNF4A, ESR1, EGR1, SRC, HNF1A, FN1, ALB, STAT3, CBL, CEBPB, AR, FOS, CFTR, PAN2, PTPN6, VDR, PPARG, STAT5A and NCOA3 genes which are enriched in the 'PPAR signaling' and 'insulin resistance' pathways were found in adipocytes during the differentiation process. Dose-dependent serotonin stimulation resulted in a decreased fat accumulation in pMAs. Serotonin-induced differentially expressed genes in pMAs were found to be involved in the significant enrichment of 'GPCR ligand-binding', 'cell chemotaxis', 'blood coagulation and complement', 'metabolism of lipid and lipoproteins', 'regulation of lipid metabolism by PPARA', and 'lipid digestion, mobilization and transport' pathways. TNF-α stimulation also resulted in transcriptome modifications linked with proinflammatory responses in the pMA of intramuscular origin. Our results provide a landscape of transcriptome modifications and their linked-biological pathways in response to adipogenesis, and exogenous stimulation of serotonin- and TNF-α to the pMA of intramuscular origin.

Transcriptome Modifications in Porcine Adipocytes via Toll-Like Receptors Activation.

Adipocytes are the most important cell type in adipose tissue playing key roles in immunometabolism. We previously reported that nine members of the Toll-like receptor (TLR) family are expressed in an originally established porcine intramuscular pre-adipocyte (PPI) cell line. However, the ability of TLR ligands to modulate immunometabolic transcriptome modifications in porcine adipocytes has not been elucidated. Herein, we characterized the global transcriptome modifications in porcine intramuscular mature adipocytes (pMA), differentiated from PPI, following stimulation with Pam3csk4, Poly(I:C) or LPS which are ligands for TLR2, TLR3, and TLR4, respectively. Analysis of microarray data identified 530 (218 up, 312 down), 520 (245 up, 275 down), and 525 (239 up, 286 down) differentially expressed genes (DEGs) in pMA following the stimulation with Pam3csk4, Poly(I:C), and LPS, respectively. Gene ontology classification revealed that DEGs are involved in several biological processes including those belonging to immune response and lipid metabolism pathways. Functionally annotated genes were organized into two groups for downstream analysis: immune response related genes (cytokines, chemokines, complement factors, adhesion molecules, and signal transduction), and genes involved with metabolic and endocrine functions (hormones and receptors, growth factors, and lipid biosynthesis). Differential expression analysis revealed that EGR1, NOTCH1, NOS2, TNFAIP3, TRAF3IP1, INSR, CXCR4, PPARA, MAPK10, and C3 are the top 10 commonly altered genes of TLRs induced transcriptional modification of pMA. However, the protein-protein interaction network of DEGs identified EPOR, C3, STAR, CCL2, and SAA2 as the major hub genes, which were also exhibited higher centrality estimates in the Gene-Transcription factor interaction network. Our results provide new insights of transcriptome modifications associated with TLRs activation in porcine adipocytes and identified key regulatory genes that could be used as biomarkers for the evaluation of treatments having immunomodularoty and/or metabolic functional beneficial effects in porcine adipocytes.

Therapeutic effect of cisplatin given with a lymphatic drug delivery system on false-negative metastatic lymph nodes.

Systemic administration of drugs into the blood circulation is standard treatment for prevention of metastasis. However, systemic delivery cannot maintain sufficiently high concentrations of anticancer drugs in lymph nodes (LN). Here, we show that giving cisplatin (CDDP) using a lymphatic drug delivery system (LDDS) has the potential to treat false-negative metastatic LN. We found that in MXH10/Mo-lpr/lpr mice, which develop systemic swelling of LN up to 10 mm in diameter, accumulation of indocyanine green (ICG), which has a similar molecular weight to CDDP, in a target LN was greater for lymphatic delivery of ICG than for systemic (i.v.) administration. Furthermore, CDDP administration with a LDDS inhibited tumor growth in false-negative metastatic LN and produced fewer adverse effects than systemically given CDDP. We anticipate that drug delivery using a LDDS will, in time, replace systemic chemotherapy for the treatment of false-negative metastatic LN.

Peptidoglycan from Immunobiotic Lactobacillus rhamnosus Improves Resistance of Infant Mice to Respiratory Syncytial Viral Infection and Secondary Pneumococcal Pneumonia.

Several research works have demonstrated that beneficial microbes with the capacity to modulate the mucosal immune system (immunobiotics) are an interesting alternative to improve the outcome of bacterial and viral respiratory infections. Among the immunobiotic strains with the capacity to beneficially modulate respiratory immunity, Lactobacillus rhamnosus CRL1505 has outstanding properties. Although we have significantly advanced in demonstrating the capacity of L. rhamnosus CRL1505 to improve resistance against respiratory infections as well as in the cellular and molecular mechanisms involved in its beneficial activities, the potential protective ability of this strain or its immunomodulatory cellular fractions in the context of a secondary bacterial pneumonia has not been addressed before. In this work, we demonstrated that the nasal priming with non-viable L. rhamnosus CRL1505 or its purified peptidoglycan differentially modulated the respiratory innate antiviral immune response triggered by toll-like receptor 3 activation in infant mice, improving the resistance to primary respiratory syncytial virus (RSV) infection, and secondary pneumococcal pneumonia. In association with the protection against RSV-pneumococcal superinfection, we found that peptidoglycan from L. rhamnosus CRL1505 significantly improved lung CD3+CD4+IFN-γ+, and CD3+CD4+IL-10+ T cells as well as CD11c+SiglecF+IFN-ß+ alveolar macrophages with the consequent increases of IFN-γ, IL-10, and IFN-ß in the respiratory tract. Our results also showed that the increase of these three cytokines is necessary to achieve protection against respiratory superinfection since each of them are involved in different aspect of the secondary pneumococcal pneumonia that have to be controlled in order to reduce the severity of the infectious disease: lung pneumococcal colonization, bacteremia, and inflammatory-mediated lung tissue injury.

New concept for the prevention and treatment of metastatic lymph nodes using chemotherapy administered via the lymphatic network.

Intravenous chemotherapy has poor access to metastatic lymph nodes (LNs) and is limited by short-lived drug concentrations. Here, we describe the administration of chemotherapy via the lymphatic network as a new concept for the prevention and treatment of metastatic LNs. A metastatic LN can be treated by the injection of drugs into an upstream LN, either the sentinel LN (SLN) or another upstream LN. In a mouse model, tumor cells were inoculated into the subiliac LN (SiLN) to induce metastasis to the proper axillary LN (PALN). Two routes were used for drug delivery to the PALN, namely from the SiLN and from the accessory axillary LN (AALN). We found that tumor masses were formed in lymphatic vessels between the SiLN and PALN. The flow of fluorescent solution injected into the SiLN towards the PALN decreased with tumor mass formation. Delivery from the AALN (free of metastatic tumor cells) to the PALN was identified as an alternative route. Intranodal injection can deliver high concentrations of drugs to secondary metastatic LNs. The study advocates a new concept for the prevention and treatment of metastatic lymph nodes whereby drugs injected into upstream lymph nodes can reach metastatic lymph nodes via the lymphatic network.

Immunobiotic Lactobacillus strains reduce small intestinal injury induced by intraepithelial lymphocytes after Toll-like receptor 3 activation.

OBJECTIVE: Intestinal intraepithelial lymphocytes (IELs) play critical roles in disrupting epithelial homeostasis after Toll-like receptor (TLR)-3 activation with genomic rotavirus dsRNA or the synthetic dsRNA analog poly(I:C). The capacity of immunobiotic Lactobacillus rhamnosus CRL1505 (Lr1505) or Lactobacillus plantarum CRL1506 (Lp1506) to beneficially modulate IELs response after TLR3 activation was investigated in vivo using a mice model. RESULTS: Intraperitoneal administration of poly(I:C) induced inflammatory-mediated intestinal tissue damage through the increase of inflammatory cells (CD3(+)NK1.1(+), CD3(+)CD8αα(+), CD8αα(+)NKG2D(+)) and pro-inflammatory mediators (TNF-α, IL-1ß, IFN-γ, IL-15, RAE1, IL-8). Increased expression of intestinal TLR3, MDA5, and RIG-I was also observed after poly(I:C) challenge. Treatment with Lr1505 or Lp1506 prior to TLR3 activation significantly reduced the levels of TNF-α, IL-15, RAE1, and increased serum and intestinal IL-10. Moreover, CD3(+)NK1.1(+), CD3(+)CD8αα(+), and CD8αα(+)NKG2D(+) cells were lower in lactobacilli-treated mice when compared to controls. The immunomodulatory capacities of lactobacilli allowed a significant reduction of intestinal tissue damage. CONCLUSIONS: This work demonstrates the reduction of TLR3-mediated intestinal tissue injury by immunobiotic lactobacilli through the modulation of intraepithelial lymphocytes response. It is a step forward in the understanding of the cellular mechanisms involved in the antiviral capabilities of immunobiotic strains.

Nasal priming with immunobiotic Lactobacillus rhamnosus modulates inflammation-coagulation interactions and reduces influenza virus-associated pulmonary damage.

OBJECTIVE: To evaluate the effect of the nasal administration of live and heat-killed Lactobacillus rhamnosus CRL1505 (Lr1505) on immune-coagulative response during influenza virus (IFV) infection to improve survival and reduce lung injury. METHODS: Six-week-old BALB/c mice were treated with live or heat-killed Lr1505 by the nasal route during two consecutive days. Treated and untreated control mice were then nasally challenged with IFV. RESULTS: Both viable and non-viable Lr1505 protected infected mice by reducing pulmonary injury and lung viral loads trough several mechanisms: (a) Inflammatory cytokines were efficiently regulated allowing higher clearance of virus and reduction of inflammatory lung tissue damage, associated to higher levels of the regulatory cytokine IL-10. (b) The antiviral immune response was enhanced with improved levels of type I interferons, CD4(+)IFN-γ(+) lymphocytes, and lung CD11c(+)CD11b(low)CD103(+) and CD11c(+)CD11b(high)CD103(-) dendritic cells. (c) The procoagulant state was reversed mainly by down-regulating tissue factor expression and restoring thrombomodulin levels in lung. The capacity of Lr1505 to improve the outcome of IFV infection would be related to its ability to beneficially modulate lung TLR3-triggered immune response. CONCLUSIONS: Our work is the first to demonstrate the ability of an immunobiotic strain to beneficially modulate inflammation-coagulation interactions during IFV infection. Interestingly, non-viable L. rhamnosus CRL1505 was as effective as the viable strain to beneficially modulate respiratory antiviral immune response.

Advanced application of porcine intramuscular adipocytes for evaluating anti-adipogenic and anti-inflammatory activities of immunobiotics.

We previously established a clonal porcine intramuscular preadipocyte (PIP) line and we were able to establish a protocol to obtain functional mature adipocytes from PIP cells. We hypothesized that both PIP cells and mature adipocytes are likely to be useful in vitro tools for increasing our understanding of immunobiology of adipose tissue, and for the selection and study of immunoregulatory probiotics (immunobiotics) able to modulate adipocytes immune responses. In this study, we investigated the immunobiology of PIP cells and mature adipocytes in relation to their response to TNF-α stimulation. In addition, we evaluated the possibility that immunobiotic microorganisms modify adipogenesis and immune functions of porcine adipose tissue through Peyer's patches (PPs) immune-competent cells. We treated the porcine PPs immune cells with different probiotic strains; and we evaluated the effect of conditioned media from probiotic-stimulated immune cells in PIP cells and mature adipocytes. The Lactobacillus GG and L. gasseri TMC0356 showed remarkable effects, and were able to significantly reduce the expression of pro-inflammatory factors and negative regulators (A20, Bcl-3, and MKP-1) in adipocytes challenged with TNF-α. The results of this study demonstrated that the evaluation of IL-6, and MCP-1 production, and A20 and Bcl-3 down-regulation in TNF-α-challenged adipocytes could function as biomarkers to screen and select potential immunobiotic strains. Taking into consideration that several in vivo and in vitro studies clearly demonstrated the beneficial effects of Lactobacillus GG and L. gasseri TMC0356 in adipose inflammation, the results presented in this work indicate that the PIP cells and porcine adipocytes could be used for the screening and the selection of new immunobiotic strains with the potential to functionally modulate adipose inflammation when orally administered.