The ex vivo generation of platelets from human-induced pluripotent cells (hiPSCs) is expected to compensate donor-dependent transfusion systems. However, manufacturing the clinically required number of platelets remains unachieved due to the low platelet release from hiPSC-derived megakaryocytes (hiPSC-MKs). Here, we report turbulence as a physical regulator in thrombopoiesis in vivo and its application to turbulence-controllable bioreactors. The identification of turbulent energy as a determinant parameter allowed scale-up to 8 L for the generation of 100 billion-order platelets from hiPSC-MKs, which satisfies clinical requirements. Turbulent flow promoted the release from megakaryocytes of IGFBP2, MIF, and Nardilysin to facilitate platelet shedding. hiPSC-platelets showed properties of bona fide human platelets, including circulation and hemostasis capacities upon transfusion in two animal models. This study provides a concept in which a coordinated physico-chemical mechanism promotes platelet biogenesis and an innovative strategy for ex vivo platelet manufacturing.

The ex vivo production of platelets depleted of human leukocyte antigen class I (HLA-I) could serve as a universal measure to overcome platelet transfusion refractoriness caused by HLA-I incompatibility. Here, we developed human induced pluripotent cell-derived HLA-Ideficient platelets (HLA-KO iPLATs) in a clinically applicable imMKCL system by genetic manipulation and assessed their immunogenic properties including natural killer (NK) cells, which reject HLA-I downregulated cells. HLA-KO iPLATs were deficient for all HLA-I but did not elicit a cytotoxic response by NK cells in vitro and showed circulation equal to wild-type iPLATs upon transfusion in our newly established Hu-NK-MSTRG mice reconstituted with human NK cells. Additionally, HLA-KO iPLATs successfully circulated in an alloimmune platelet transfusion refractoriness model of Hu-NK-MISTRG mice. Mechanistically, the lack of NK cell-activating ligands on platelets may be responsible for evading the NK cell response. This study revealed the unique non-immunogenic property of platelets and provides a proof of concept for the clinical application of HLA-KO iPLATs.

ABSTRACT. Cell-mediated and humoral immune responses are attenuated with aging. Intracellular glutathione (GSH) levels also decrease with aging. Previously, we have reported that combined administration of L -cystine and L -theanine enhances antigen-specific IgG production, partly through augmentation of GSH levels and T helper 2-mediated responses in 12-week-old mice. These findings suggest that combined administration of L -cystine and L -theanine to aged mice improves immune responses via increase of GSH synthesis. Here, we examined the effects of combined administration of L -cystine and L -theanine on antigen-specific antibody production and influenza virus infection in aged mice. Combined administration of these amino acids for 14 days before primary immunization significantly enhanced the serum antigen-specific IgM and IgG levels in 24-month-old mice. Furthermore, 13-month-old mice co-treated with these amino acids orally for 10 days had significantly lower lung viral titers than controls at 6 days after influenza virus infection. In addition, this co-treatment also significantly prevented the weight loss associated with infection. Enhancement of anti-influenza-virus IgG antibodies by combined administration of L -cystine and L -theanine was seen 10 days after infection. The significantly elevated serum interleukin-10/interferon- ratio and -glutamylcysteine synthetase mRNA expression, which is the rate-limiting enzyme of GSH synthesis, in the spleen 3 days after infection may have contributed to the observed beneficial effects. These results suggest that combined administration of L -cystine and L -theanine enhances immune function and GSH synthesis which are compromised with advanced age, and may become a useful strategy in healthy aging. With aging, the human body's defense capability weakens, including production of antibodies and cytotoxic T lymphocytes (CTL) and cellular immune function [12,28]. These age-related alterations seem to result from oxidative stress [9]. Diet supplementation with antioxidants has been used to prevent or delay the onset of age-related immune impairment [7]. For example, a diet that contains the antioxidant 2-mercaptoethanol, which reduces cystine to cysteine and increases the intracellular concentration of glutathione (GSH; L --glutamyl-L -cysteinyl-glycine), improves several types of immune response in aged mice [17]. Dietary supplementation with GSH, another antioxidant, has also been shown to increase cell-mediated immunity in aged mice [11]. In addition, GSH levels and the gene expression of -glutamylcysteine synthetase (GCS), the rate-limiting enzyme in GSH synthesis, are known to decrease with aging [24]. These results suggest that GSH antioxidant therapy may aid in preventing or delaying the onset of age-related immune impairment.A recent study we performed indicated that combined administration of L -cystine and L -theanine (-glutamylethylamide, a specific amino acid found in green tea) to young mice increases the GSH level in the liver and also enhances the s...

Marked hyperglycaemia (30.9 mmol l(-1)) during interferon-gamma (IFN-gamma) therapy for asymptomatic recurrent renal cancer as multiple lung metastases in a 52-year-old man is described. Although the involvement of IFN-gamma has been reported in the development of autoimmune diabetes, in this case, antibodies against pancreatic beta-cells including anti-islet cell antibody (ICA) and anti-glutamic acid decarboxylase (GAD) antibody were negative. Moreover, serum level of immunoreactive insulin (IRI) (11 microU ml(-1) at fasting) and urinary excretion of C-peptide (108 microg day(-1), reference range: 20-130) suggested insulin resistance, supported by results of insulin tolerance tests. With insulin therapy and cessation of IFN-gamma, fasting blood glucose concentration returned to 6.2 mmol l(-1), and insulin therapy was discontinued. The injection of IFN-gamma may cause hyperglycaemia because of insulin resistance, rather than beta-cell injury.

The Chinese traditional medicine mao-bushi-saishin-to (MBST), which has anti-inflammatory effects and has been used to treat the common cold and nasal allergy in Japan, was examined for its effects on the therapeutic activity of a new benzoxazinorifamycin, KRM-1648 (KRM), against Mycobacterium avium complex (MAC) infection in mice. In addition, we examined the effects of MBST on the anti-MAC activity of murine peritoneal macrophages (Ms). First, MBST significantly increased the anti-MAC therapeutic activity of KRM when given to mice in combination with KRM, although MBST alone did not exhibit such effects. Second, MBST treatment of Ms significantly enhanced the KRM-mediated killing of MAC bacteria residing in Ms, although MBST alone did not potentiate the M anti-MAC activity. MBST-treated Ms showed decreased levels of reactive nitrogen intermediate (RNI) release, suggesting that RNIs are not decisive in the expression of the anti-MAC activity of such M populations. MBST partially blocked the interleukin-10 (IL-10) production of MAC-infected Ms without affecting their transforming growth factor ␤ (TGF-␤)-producing activity. Reverse transcription-PCR analysis of the lung tissues of MAC-infected mice at weeks 4 and 8 after infection revealed a marked increase in the levels of tumor necrosis factor alpha, gamma interferon (IFN-␥), IL-10, and TGF-␤ mRNAs. KRM treatment of infected mice tended to decrease the levels of the test cytokine mRNAs, except that it increased TGF-␤ mRNA expression at week 4. MBST treatment did not affect the levels of any cytokine mRNAs at week 8, while it down-regulated cytokine mRNA expression at week 4. At week 8, treatment of mice with a combination of KRM and MBST caused a marked decrease in the levels of the test cytokines mRNAs, especially IL-10 and IFN-␥ mRNAs, although such effects were obscure at week 4. These findings suggest that down-regulation of the expression of IL-10 and TGF-␤ is related to the combined therapeutic effects of KRM and MBST against MAC infection.on July 3, 2020 by guest http://aac.asm.org/ Downloaded from a Mice infected i.v. with MAC N-444 (10 7 CFU) were given or not given the indicated agents by gavage either once weekly (KRM) or once daily five times per week (MBST) from day 1 for up to 4 weeks. b There were six mice per regimen. c ND, not determined. d Significantly smaller than the value of untreated control mice (P Ͻ 0.05 by Bonferroni's multiple t test). e Significantly smaller than the value of mice given MBST alone (P Ͻ 0.05 by Bonferroni's multiple t test). f The difference from the values of mice given KRM alone was statistically significant (P Ͻ 0.05) when assessed by the Mann-Whitney test.