Effects of Bifidobacterium longum BB536 and Bifidobacterium breve MCC1274 on Body Composition in Normal and Overweight Adults in Randomized Placebo-Controlled Study.

Visceral fat accumulation is considered to be associated with a higher risk of chronic diseases. We investigated the effects of Bifidobacterium longum subsp. longum (B. longum) BB536 and Bifidobacterium breve (B. breve) MCC1274 on body composition, including visceral fat, in a randomized, parallel-group, placebo-controlled study. Participants were between 29 and 64 years of age and had a body mass index (BMI) of greater than 23 and less than 30. One hundred participants were randomly assigned to the probiotics group or placebo group. Participants were administered probiotic capsules containing 1 × 1010 colony-forming units (CFUs) of B. longum BB536 and 5 × 109 CFU of B. breve MCC1274 or placebo capsules without bifidobacteria for 16 weeks. In the probiotics group, abdominal visceral fat area, total abdominal fat area, and serum triglyceride levels were significantly decreased compared to those in the placebo group. Additionally, the increase in BMI observed in the placebo group was significantly suppressed in the probiotics group. This study showed that B. longum BB536 and B. breve MCC1274 reduced abdominal visceral fat and total fat levels in healthy normal and overweight adults, suggesting their beneficial effects on body composition.

Effects of Heat-Killed Lacticaseibacillus paracasei MCC1849 on Immune Parameters in Healthy Adults-A Randomized, Double-Blind, Placebo-Controlled, Parallel-Group Study.

Previous clinical studies have shown that heat-killed Lacticaseibacillus paracasei MCC1849 suppresses subjective symptoms among healthy adults. However, the mechanism underlying this beneficial effect remains unclear. This clinical study aimed to investigate the effects of MCC1849 on immune functions in humans. In this randomized, double-blind, placebo-controlled, parallel-group study, 100 healthy adults were randomly divided into MCC1849 or placebo groups. Participants ingested test powder with 5 × 1010 MCC1849 cells or placebo powder for 4 weeks. Immune functions were evaluated using expression levels of CD86 and HLA-DR on dendritic cells (DCs), neutrophils, and natural killer cells. The expression levels of interferon (IFN)-α, -ß, and -γ in peripheral blood mononuclear cells incubated with Cpg2216 in vitro were quantified. Efficacy analysis was performed on participants in the per-protocol set (placebo group; n = 47, MCC1849 group; n = 49). The expression level of CD86 on pDCs and the gene expression levels of IFN-α, -ß, and -γ upon TLR9 agonist stimulation were significantly higher in the MCC1849 group at 4 weeks. No side effects were observed. This is the first report to show the positive effects of MCC1849 on human immune cells. These findings reveal one possible mechanism of how MCC1849 suppresses subjective symptoms.

Screening of novel DPP-IV inhibitory peptides derived from bovine milk proteins using a peptide array platform.

Dipeptidyl peptidase IV (DPP-IV) has become an important target in the prevention and treatment of diabetes. Although many DPP-IV inhibitory peptides have been identified by a general approach involving the repeated fractionation of food protein hydrolysates, the obtained results have been dependent on the content of each peptide and fractionation conditions. In the present study, a peptide array that provides comprehensive assays of peptide sequences was used to identify novel DPP-IV inhibitory peptides derived from bovine milk proteins; these peptides were then compared with those identified using the general approach. While the general approach identified only known peptides that were abundant in the hydrolysate, the peptide array-based approach identified 10 novel DPP-IV inhibitory peptides, all of which had proline at the second residue from the N-terminus. The proper or combined use of these two approaches, which have different advantages, will enable the efficient development of novel bioactive foods and drugs.

Bifidobacterium breve M-16V regulates the autonomic nervous system via the intestinal environment: A double-blind, placebo-controlled study.

We conducted a randomized controlled trial to investigate the potential of Bifidobacterium breve M-16 V to improve mood in humans. In this evaluation, we incorporated the use of near-infrared spectroscopy (NIRS), which has been used to evaluate mood states in studies with small sample sizes. Participants were given B. breve M-16 V (20 billion cells/day) for 6 weeks, and their mood state was assessed before and after ingestion. NIRS data were collected at rest and during a mental arithmetic task (under stress). Intake of B. breve M-16 V decreased the heart rate under stress and increased levels of the GABA-like substance pipecolic acid in stool samples. In addition, B. breve M-16 V improved mood and sleep scores in participants with high anxiety levels. These results suggest that B. breve M-16 V affects the metabolites of the gut microbiota and has the potential to modulate the autonomic nervous system and to improve mood and sleep.

Erratum: Ligand-based, piggyBac-engineered CAR-T cells targeting EGFR are safe and effective against non-small cell lung cancers.

[This corrects the article DOI: 10.1016/j.omto.2023.100728.].

Ligand-based, piggyBac-engineered CAR-T cells targeting EGFR are safe and effective against non-small cell lung cancers.

Epidermal growth factor receptor (EGFR) is overexpressed in various cancers, including non-small cell lung cancer (NSCLC), and in some somatic cells at a limited level, rendering it an attractive antitumor target. In this study, we engineered chimeric antigen receptor (CAR)-T cells using the piggyBac transposon system, autologous artificial antigen-presenting cells, and natural ligands of EGFR. We showed that this approach yielded CAR-T cells with favorable phenotypes and CAR positivity. They exhibited potent antitumor activity against NSCLC both in vitro and in vivo. When administered to tumor-bearing mice and non-tumor-bearing cynomolgus macaques, they did not elicit toxicity despite their cross-reactivity to both murine and simian EGFRs. In total we tested three ligands and found that the CAR candidate with the highest affinity consistently displayed greater potency without adverse events. Taken together, our results demonstrate the feasibility and safety of targeting EGFR-expressing NSCLCs using ligand-based, piggyBac-engineered CAR-T cells. Our data also show that lowering the affinity of CAR molecules is not always beneficial.

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.

Effects of Lactoferrin on Oral and Throat Conditions under Low Humidity Environments: A Randomized, Double-Blind, and Placebo-Controlled Crossover Trial.

To evaluate the effects of a single ingestion of bovine lactoferrin (bLF) on oral and throat conditions under a low-humidity environment. A randomized, double-blind, 2-sequence, 2-treatment, and 2-period placebo-controlled crossover trial was conducted. Healthy adult subjects orally ingested bLF dissolved in water, or placebo water, followed by exposure to low humidity (20 °C, 20% relative humidity (RH)) for 2 h. The primary endpoint was subjective oral and throat discomfort assessed by a visual analog scale (VAS), which positively correlated with the discomfort. Secondary endpoints were unstimulated whole salivary flow rate (UWSFR) and salivary immunoglobulin A (IgA) secretion rate. Overall, 40 subjects were randomly assigned to two sequences (20 each) and 34 were analyzed. The VAS values for oral and throat discomfort in the bLF treatment were significantly lower than in the placebo treatment, whereas UWSFR and IgA secretion rates were comparable between the two treatments. Adverse drug reactions were not observed. Subjective oral and throat discomfort associated with low humidity is suppressed by a single ingestion of bLF. Our findings demonstrate the novel use of bLF in a clinical situation that leverages its unique characteristics.

Effects of Heat-Killed Lacticaseibacillus paracasei MCC1849 on the Maintenance of Physical Condition in Healthy Adults: A Randomized, Double-Blind, Placebo-Controlled, Parallel-Group Study.

We previously reported that the intake of heat-killed Lacticaseibacillus paracasei MCC1849 suppressed the onset of cold-like symptoms in healthy young women who were susceptible to colds. This study aimed to investigate the effects of MCC1849 on subjective symptoms of physical condition in healthy adults of a wide age range. In this randomized, double-blind, placebo-controlled, parallel-group study, 200 healthy adults were randomly divided into the MCC1849 group or placebo group. The participants received test powder with 50 billion MCC1849 cells or placebo powder without MCC1849 for 24 weeks. Subjective symptoms were assessed by diary scores. Analysis was performed on 183 participants (MCC1849 group; n = 91, placebo group; n = 92) in the per-protocol set. The number of days of stuffy nose and cold-like symptoms was significantly reduced in the MCC1849 group compared with the placebo group. In addition, the duration of stuffy nose, sore throat and cold-like symptoms was significantly lower in the MCC1849 group. No side effects were observed. Therefore, oral intake of MCC1849 suppressed subjective symptoms in healthy adults of a wide age range. These data suggest that MCC1849 may help maintain physical condition.

Comprehensive genetic analyses of childhood acute leukemia in Iraq using next-generation sequencing.

Background: Molecular analyses in hematological malignancies provide insights about genetic makeup. Probable etiological factors in leukemogenesis could also be disclosed. Since genetic analyses are still primitive in Iraq, a country of repeated wars, we conceived of performing next-generation sequencing (NGS), to disclose the genomic landscape of acute lymphoblastic leukemia (ALL) and acute myeloid leukemia (AML) among a cohort of Iraqi children. Methods: Dried blood samples were collected from Iraqi children with ALL (n=55), or AML (n=11), and transferred to Japan where NGS was done. Whole-exome, whole-genome, and targeted gene sequencings were performed. Results: Somatic point mutations and the copy number variations among Iraqi children with acute leukemia were comparable with those in other countries, and cytosine-to-thymine nucleotide alterations were dominant. Strikingly, TCF3-PBX1 was the most recurrent fusion gene (22.4%) in B-cell precursor ALL (B-ALL), and acute promyelocytic leukemia (AML-M3) was subtyped in 5 AML cases. Additionally, a high frequency of RAS signaling pathway mutations was detected in children with B-ALL (38.8%), along with 3 AML cases that carried oncogenic RAS. Conclusions: Apart from disclosing the high frequency of TCF3-PBX1, NGS confirmed our previous finding of recurrent RAS mutations in Iraqi childhood acute leukemia. Our results suggest that the biology of Iraqi childhood acute leukemia is in part characteristic, where the war-aftermath environment or geography might play a role.

Heat-Killed L. helveticus Enhances Positive Mood States: A Randomized, Double-Blind, Placebo-Controlled Study.

When mood states are impaired, daily life is severely disrupted. To maintain a specific mood state, both positive and negative moods must be controlled; however, methods to maintain a positive mood have not been fully established. Previous studies have suggested that heat-killed L. helveticus MCC1848 has the potential to improve positive moods. This study aimed to test the efficacy of heat-killed L. helveticus MCC1848 in maintaining and improving a positive mood with PANAS, a questionnaire specifically designed to assess positive and negative mood, as the primary endpoint. Healthy Japanese nursing students (n = 46) were randomized to receive heat-killed L. helveticus MCC1848 (5 billion/day) or placebo powder for four weeks. Mood state was assessed before and two and four weeks after the intervention began; ingestion of heat-killed L. helveticus MCC1848 significantly improved PANAS 'Positive Affect' compared to the placebo. These results indicate that heat-killed L. helveticus MCC1848 is effective in enhancing positive mood.

Early Probiotic Supplementation of Healthy Term Infants with Bifidobacterium longum subsp. infantis M-63 Is Safe and Leads to the Development of Bifidobacterium-Predominant Gut Microbiota: A Double-Blind, Placebo-Controlled Trial.

Bifidobacteria are important intestinal bacteria that provide a variety of health benefits in infants. We investigated the efficacy and safety of Bifidobacterium longum subsp. infantis (B. infantis) M-63 in healthy infants in a double-blind, randomized, placebo-controlled trial. Healthy term infants were given B. infantis M-63 (n = 56; 1 × 109 CFU/day) or placebo (n = 54) from postnatal age ≤ 7 days to 3 months. Fecal samples were collected, and fecal microbiota, stool pH, short-chain fatty acids, and immune substances were analyzed. Supplementation with B. infantis M-63 significantly increased the relative abundance of Bifidobacterium compared with the placebo group, with a positive correlation with the frequency of breastfeeding. Supplementation with B. infantis M-63 led to decreased stool pH and increased levels of acetic acid and IgA in the stool at 1 month of age compared with the placebo group. There was a decreased frequency of defecation and watery stools in the probiotic group. No adverse events related to test foods were observed. These results indicate that early supplementation with B. infantis M-63 is well tolerated and contributes to the development of Bifidobacterium-predominant gut microbiota during a critical developmental phase in term infants.

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.

Clinical research review: usefulness of bovine lactoferrin in child health.

Lactoferrin (LF) is abundant in human milk and plays an important role in the health of children. Bovine LF (bLF) has high homology with human LF and has been reported to have multiple biological functions. Several clinical studies have been conducted considering these properties, which reported the usefulness of bLF. This review was aimed to provide an overview of the clinical evidence in children. We searched clinical reports investigating the effects of bLF in children and identified 36 studies on the role of bLF in infections, iron metabolism, body growth, cerebral development, and fecal microbiome. Considering the accumulated evidence, bLF may contribute to the child health, particularly by suppressing or alleviating gastrointestinal and respiratory symptoms, and improving the iron status of children with anemia or those at high risk of anemia. The dose of bLF varies depending on the expected effect and target age, but may not necessarily have to be as high as human LF in human milk. Some of the beneficial effects of bLF have not been fully validated due to limited clinical evidence or being observed in the secondary analysis of some studies. Further clinical evidence would add significant value to the use of bLF in child health.

Basophil activation test for allergic and febrile non-haemolytic transfusion reactions among paediatric patients with haematological or oncological disease.

BACKGROUND AND OBJECTIVES: Allergic transfusion reactions (ATRs) and febrile non-haemolytic transfusion reactions (FNHTRs) are common, although their mechanisms remain unclear. Immunoglobulin E (IgE)-mediated type I hypersensitivity may be involved in the pathogenesis of ATR. A basophil activation test (BAT) may help elucidate this process. MATERIALS AND METHODS: The BAT was based on peripheral blood samples from paediatric patients with a haematological or oncological disease and on samples of residual blood products transfused in each case. Dasatinib was used to evaluate whether basophil activation was mediated by an IgE-dependent pathway. RESULTS: Twenty-seven patients with and 19 patients without ATR/FNHTR were included in this study, respectively. The median BAT values associated with ATR- (n = 41) and FNHTR-causing (n = 5) blood products were 22.1% (range = 6.1%-77.0%) and 27.8% (range = 15.2%-47.8%), respectively, which were higher than the median value of 8.5% (range = 1.1%-40.9%) observed in blood products without a transfusion reaction. Dasatinib suppressed basophil activity. BAT values were comparable in patients with ATR regardless of severity. Meanwhile, BAT values analysed with blood products non-causal for ATR/FNHTR were higher in patients with ATR/FNHTR than in those without. CONCLUSION: The IgE-mediated type I hypersensitivity may be involved in the pathogenesis of ATR and FNHTR. BAT analyses may help elucidate the underlying mechanisms and identify patients at risk.

The Potential Immunomodulatory Effect of Bifidobacterium longum subsp. longum BB536 on Healthy Adults through Plasmacytoid Dendritic Cell Activation in the Peripheral Blood.

The interaction between the gut microbiota and the host can influence the host's immune system. Bifidobacterium, a commensal genus of gut bacteria, seems to have positive effects on host health. Our previous clinical research showed that B. longum subsp. longum BB536 enhanced innate and adaptive immune responses in elderly individuals with a lower grade of immunity, but the immunomodulatory mechanism is still unclear. In this study, dendritic cell (DC) surface markers in peripheral blood mononuclear cells isolated from healthy individuals were evaluated through coculture with heat-killed BB536. DC markers, innate immune activity and cytokine levels in plasma were also evaluated by a randomized, double-blind, placebo-controlled, parallel-group study (UMIN000045564) with 4 weeks of continuous live BB536 intake. BB536 significantly increased the expression of CD86 and HLA-DR on plasmacytoid DCs (pDCs) in vitro. Compared to placebo (n = 48), a significant increase in the expression of CD86 on peripheral pDCs was detected at week 4 of live BB536 intake (n = 49; 1 × 1010 CFU/day). Furthermore, coculture with hk-BB536 significantly increased the IFNγ expression level and demonstrated trends of increased IFNα1 and IFNß expression. These findings suggest that consumption of BB536 has potential immunomodulatory effects on healthy individuals through the activation of peripheral pDCs.

Efficient differentiation of human neutrophils with recapitulation of emergency granulopoiesis in human G-CSF knockin humanized mice.

Neutrophils are critical mediators during the early stages of innate inflammation in response to bacterial or fungal infections. A human hematopoietic system reconstituted in humanized mice aids in the study of human hematology and immunology. However, the poor development of human neutrophils is a well-known limitation of humanized mice. Here, we generate a human granulocyte colony-stimulating factor (hG-CSF) knockin (KI) NOD/Shi-scid-IL2rgnull (NOG) mouse in which hG-CSF is systemically expressed while the mouse G-CSF receptor is disrupted. These mice generate high numbers of mature human neutrophils, which can be readily mobilized into the periphery, compared with conventional NOG mice. Moreover, these neutrophils exhibit infection-mediated emergency granulopoiesis and are capable of efficient phagocytosis and reactive oxygen species production. Thus, hG-CSF KI mice provide a useful model for studying the development of human neutrophils, emergency granulopoiesis, and a potential therapeutic model for sepsis.

Lactulose Ingestion Induces a Rapid Increase in Genus Bifidobacterium in Healthy Japanese: A Randomised, Double-Blind, Placebo-Controlled Crossover Trial.

Oral administration of a low dose of lactulose increases the abundance of genus Bifidobacterium in the large intestine; however, the details of the daily variation in Bifidobacterium have not been researched. To observe how the intestinal microbiota, including Bifidobacterium, change, especially immediately after the initiation of ingestion, we conducted a randomised, placebo-controlled, double-blind crossover study of ingestion of 4 g lactulose/day for 2 weeks in 36 healthy Japanese (including males and females). The primary outcome was the percentage of Bifidobacterium in the faecal bacteria. In the lactulose-treatment group, the percentage of Bifidobacterium was already significantly higher 2 days after starting lactulose ingestion than in the placebo group (20.5 ± 1.2% vs. 17.1 ± 1.2%, p = 0.021). Significant differences were maintained, gradually widening, until the end of the 2-week intervention period. There were significant increases in the percentage and the number of Bifidobacterium with ingestion of 4 g lactulose/day for 2 weeks, but no significant changes in the beta diversity of the intestinal microbiota between lactulose and placebo ingestion. The percentage of Bifidobacterium in the faecal bacteria returned to its original level within a week of the end of intervention with lactulose.

Humanized liver TK-NOG mice with functional deletion of hepatic murine cytochrome P450s as a model for studying human drug metabolism.

Chimeric TK-NOG mice with a humanized liver (normal Hu-liver) are a unique animal model for predicting drug metabolism in humans. However, residual mouse hepatocytes occasionally prevent the precise evaluation of human drug metabolism. Herein, we developed a novel humanized liver TK-NOG mouse with a conditional knockout of liver-specific cytochrome P450 oxidoreductase (POR cKO Hu-liver). Immunohistochemical analysis revealed only a few POR-expressing cells around the portal vein in POR cKO mouse livers. NADPH-cytochrome c reductase and cytochrome P450 (P450)-mediated drug oxidation activity in liver microsomes from POR cKO mice was negligible. After the intravenous administration of S-warfarin, high circulating and urinary levels of S-7-hydroxywarfarin (a major human metabolite) were observed in POR cKO Hu-liver mice. Notably, the circulating and urinary levels of S-4'-hydroxywarfarin (a major warfarin metabolite in mice) were much lower in POR cKO Hu-liver mice than in normal Hu-liver mice. POR cKO Hu-liver mice with minimal interference from mouse hepatic P450 oxidation activity are a valuable model for predicting human drug metabolism.