A novel haemodilution chip mounted on the total thrombus-formation analysis system, a flow chamber system, enables stable analysis of platelet products under low platelet concentration/high shear conditions.

BACKGROUND AND OBJECTIVES: The total thrombus-formation analysis system (T-TAS) can quantitatively analyse the contribution of platelets to haemostasis using reconstituted blood samples. However, it is unsuitable in cases with low platelet counts. We introduced a haemodilution (HD) chip with a shallow chamber depth, adapted to low platelet counts and high shear conditions (1500 s-1). MATERIALS AND METHODS: Blood samples were prepared by mixing red blood cell products, standard human plasma and platelet products; the final platelet count was 50 × 103/µL. Aggregation tests were performed by using the aggregation inducers collagen, adenosine diphosphate (ADP) and ristocetin. Samples with 2-, 4- and 9-day-old platelet products (N = 10) were evaluated. RESULTS: The HD chip enabled the stable analysis of the haemostatic function of all samples at a platelet count of 50 × 103/µL. Haemostatic function was correlated with ADP aggregation (time to 10 kPa [T10]: r = -0.53; area under the curve for 30 min: r = 0.40) and storage period (T10: r = 0.44). CONCLUSION: The HD chip-mounted T-TAS can stably analyse haemostatic function under low platelet counts and high shear conditions; this approach is expected to serve as a bridge to in vivo haemostatic tests with experimental animals.

5,6-dimethylxanthenone-4-acetic acid (DMXAA), a partial STING agonist, competes for human STING activation.

5,6-dimethylxanthenone-4-acetic acid (DMXAA) is a mouse-selective stimulator of interferon gene (STING) agonist exerting STING-dependent anti-tumor activity. Although DMXAA cannot fully activate human STING, DMXAA reached phase III in lung cancer clinical trials. How DMXAA is effective against human lung cancer is completely unknown. Here, we show that DMXAA is a partial STING agonist interfering with agonistic STING activation, which may explain its partial anti-tumor effect observed in humans, as STING was reported to be pro-tumorigenic for lung cancer cells with low antigenicity. Furthermore, we developed a DMXAA derivative-3-hydroxy-5-(4-hydroxybenzyl)-4-methyl-9H-xanthen-9-one (HHMX)-that can potently antagonize STING-mediated immune responses both in humans and mice. Notably, HHMX suppressed aberrant responses induced by STING gain-of-function mutations causing STING-associated vasculopathy with onset in infancy (SAVI) in in vitro experiments. Furthermore, HHMX treatment suppressed aberrant STING pathway activity in peripheral blood mononuclear cells from SAVI patients. Lastly, HHMX showed a potent therapeutic effect in SAVI mouse model by mitigating disease progression. Thus, HHMX offers therapeutic potential for STING-associated autoinflammatory diseases.

Reproducibility of subscapularis muscle thickness measurement using ultrasound imaging. -Relationship between subscapularis muscle thickness and internal rotation torque of the shoulder joint.

BACKGROUND: Few studies have reported on the morphometry of the subscapularis muscle using ultrasound imaging (USI); and their reproducibility has not been verified. OBJECTIVES: This study aimed to clarify the relative and absolute reproducibility of USI measurements of subscapularis muscle thickness at rest and during isometric contraction as well as the degree of change in muscle thickness caused by the amount of internal rotational torque in the shoulder joint. DESIGN: Two-group repeated-measures study. METHODS: The subjects were the inferior fibers of the subscapularis muscle of 40 healthy adult males. Muscle thickness was measured at rest and at 10%-30% of the maximum isometric internal rotation torque. Intraclass correlation coefficients (ICC) and Brand Altman analysis were used for reproducibility measurement. The degree of change in muscle thickness at each torque was also calculated. RESULTS: Intra- and inter-rater ICCs (ranged from 0.69 to 0.91) were good. A proportional error was observed in intra-rater measurements. Both minimal detectable change 95 (ranged from 2.33 to 6.47) were high. The subscapularis muscle thickness was significantly increased at 10% torque (25.49 ± 3.80 mm), 20% torque (26.07 ± 3.90 mm), and 30% torque (25.96 ± 3.82 mm) as compared to that in resting conditions (24.53 ± 4.46 mm) (p < 0.05). CONCLUSION: The reproducibility and error of the subscapularis muscle thickness measurement using USI used in this study were clarified when repeated measurements were made in the same limb position and under the same probe installation conditions, suggesting that the contraction of the subscapularis muscle can be estimated by muscle thickness measurement.

Identification of a Self-Assembling Small-Molecule Cancer Vaccine Adjuvant with an Improved Toxicity Profile.

Protein or peptide cancer vaccines usually include immune potentiators, so-called adjuvants. However, it remains challenging to identify structurally simple, chemically accessible synthetic molecules that are effective and safe as vaccine adjuvant. Here, we present cholicamideß (6), a self-assembling small-molecule vaccine adjuvant with an improved toxicity profile and proven efficacy in vivo. We demonstrate that cholicamideß (6), which is less cytotoxic than its parent compound, forms virus-like particles to potently activate dendritic cells with the concomitant secretion of cytokines. When combined with a peptide antigen, cholicamideß (6) potentiated the antigen presentation on dendritic cells to induce antigen-specific T cells. As a therapeutic cancer vaccine adjuvant in mice, a mixture of cholicamideß (6) and a peptide antigen protected mice from the challenges of malignant cancer cells without overt toxicity. Cholicamideß (6) may offer a translational opportunity as an unprecedented class of small-molecule cancer vaccine adjuvants.

Activity of deep trunk muscles for postural control to predictable and unpredictable sagittal plane perturbations: Electromyographic analysis.

BACKGROUND: The activity of deep trunk muscles (psoas major; PM, quadratus lumborum; QL, transverse abdominis; TrA, and lumbar multifidus; MF) in response to external perturbation is not clearly known. OBJECTIVE: This study aimed to record the onset and amount of activity of the deep trunk muscles during sagittal plane perturbations. METHODS: Fourteen healthy males participated in this study. The activity of the right deep trunk muscles was recorded using wire electrodes. In standing, the participants performed three tasks: a pendulum impacted from anterior with predictable and unpredictable and posterior with unpredictable. RESULTS: In predictable anterior perturbation, the TrA and PM demonstrated feedforward activation, while all deep trunk muscles demonstrated feedback activation in unpredictable anterior and posterior perturbations. In the anticipatory postural adjustment phase, the activity of the TrA was large in predictable anterior perturbation, while that of all deep trunk muscles was slight in other perturbations. In the compensatory postural adjustment phase, the activity of the PM, QL, and TrA in unpredictable anterior perturbation and those of the PM, QL, and MF in unpredictable posterior perturbation were large. CONCLUSIONS: These results showed that the onset and magnitude of deep trunk muscle activity changed depending on both predictable or unpredictable perturbation and the direction of perturbation.

Intratumoural Delivery of mRNA Loaded on a Cationic Hyper-Branched Cyclodextrin-Based Polymer Induced an Anti-Tumour Immunological Response in Melanoma.

mRNA technology has demonstrated potential for use as an effective cancer immunotherapy. However, inefficient in vivo mRNA delivery and the requirements for immune co-stimulation present major hurdles to achieving anti-tumour therapeutic efficacy. Therefore, we used a cationic hyper-branched cyclodextrin-based polymer to increase mRNA delivery in both in vitro and in vivo melanoma cancer. We found that the transfection efficacy of the mRNA-EGFP-loaded Ppoly system was significantly higher than that of lipofectamine and free mRNA in both 2D and 3D melanoma cancer cells; also, this delivery system did not show cytotoxicity. In addition, the biodistribution results revealed time-dependent and significantly higher mEGFP expression in complexes with Ppoly compared to free mRNA. We then checked the anti-tumour effect of intratumourally injected free mRNA-OVA, a foreign antigen, and loaded Ppoly; the results showed a considerable decrease in both tumour size and weight in the group treated with OVA-mRNA in loaded Ppoly compared to other formulations with an efficient adaptive immune response by dramatically increasing most leukocyte subtypes and OVA-specific CD8+ T cells in both the spleen and tumour tissues. Collectively, our findings suggest that the local delivery of cationic cyclodextrin-based polymer complexes containing foreign mRNA antigens might be a good and reliable concept for cancer immunotherapy.

A novel quantitative method to evaluate the contribution of platelet products to white thrombus formation in reconstituted blood under flow conditions.

BACKGROUND AND OBJECTIVES: Currently, the quality of platelet (PLT) products is evaluated using a series of in vitro tests, which only analyse PLTs as an inspection material. However, it would be ideal to assess the physiological functions of PLTs under conditions similar to the sequential blood haemostatic process. In this study, we attempted to establish an in vitro system where the thrombogenicity of PLT products was evaluated in the presence of red blood cells (RBCs) and plasma using a microchamber under constant shear stress (600/s). MATERIALS AND METHODS: Blood samples were reconstituted by mixing PLT products, standard human plasma (SHP) and standard RBCs. Each component was serially diluted keeping the other two components fixed. The samples were applied onto a flow chamber system (Total Thrombus-formation Analysis System [T-TAS]), and white thrombus formation (WTF) was assessed under large arterial shear conditions. RESULTS: We observed a good correlation between the PLT numbers in the test samples and WTF. The WTF of samples containing ≦10% SHP was significantly lower than those containing ≧40% SHP, and no difference was observed in WTF among samples containing 40%-100% SHP. WTF significantly declined in the absence of RBCs, whereas no change in WTF was observed in the presence of RBCs, over haematocrit range of 12.5%-50%. CONCLUSION: The WTF assessed on the T-TAS using reconstituted blood may serve as a new physiological blood thrombus test to quantitatively determine the quality of PLT products.

A-910823, a squalene-based emulsion adjuvant, induces T follicular helper cells and humoral immune responses via α-tocopherol component.

Background: Adjuvants are chemical or biological materials that enhance the efficacy of vaccines. A-910823 is a squalene-based emulsion adjuvant used for S-268019-b, a novel vaccine against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) that is currently in clinical development. Published evidence has demonstrated that A-910823 can enhance the induction of neutralizing antibodies against SARS-CoV-2 in humans and animal models. However, the characteristics and mechanisms of the immune responses induced by A-910823 are not yet known. Methods and Results: To characterize A-910823, we compared the adaptive immune response profile enhanced by A-910823 with that of other adjuvants (AddaVax, QS21, aluminum salt-based adjuvants, and empty lipid nanoparticle [eLNP]) in a murine model. Compared with other adjuvants, A-910823 enhanced humoral immune responses to an equal or greater extent following potent T follicular helper (Tfh) and germinal center B (GCB) cell induction, without inducing a strong systemic inflammatory cytokine response. Furthermore, S-268019-b containing A-910823 adjuvant produced similar results even when given as a booster dose following primary administration of a lipid nanoparticle-encapsulated messenger RNA (mRNA-LNP) vaccine. Preparation of modified A-910823 adjuvants to identify which components of A-910823 play a role in driving the adjuvant effect and detailed evaluation of the immunological characteristics induced by each adjuvant showed that the induction of humoral immunity and Tfh and GCB cell induction in A-910823 were dependent on α-tocopherol. Finally, we revealed that the recruitment of inflammatory cells to the draining lymph nodes and induction of serum cytokines and chemokines by A-910823 were also dependent on the α-tocopherol component. Conclusions: This study demonstrates that the novel adjuvant A-910823 is capable of robust Tfh cell induction and humoral immune responses, even when given as a booster dose. The findings also emphasize that α-tocopherol drives the potent Tfh-inducing adjuvant function of A-910823. Overall, our data provide key information that may inform the future production of improved adjuvants.

Antidepressant effects of acupuncture in a murine model: regulation of neurotrophic factors.

BACKGROUND: GV20 and Yintang are important targets in acupuncture treatment for depression. In this study, we examined the antidepressant effects of simultaneous acupuncture stimulation at GV20 and Yintang. METHODS: We compared the antidepressant effects of manual acupuncture (MA) stimulation at GV20 and Yintang, compared to acupuncture stimulation at two control point locations on the back of the mice (overlying the spinal column) and imipramine administration in a forced swimming (FS)-induced mouse model of depression, and examined the mRNA and protein expression of neurotrophic factors, including nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF), neurotrophin (NT)-3, and NT-4/5 in the brains by real-time polymerase chain reaction in two different experimental schedules - preventive (MA given alongside FS modelling) and therapeutic (MA given after FS-induced depression was already established). RESULTS: MA at GV20 and Yintang significantly reduced the immobility time of mice with FS-induced depression in both preventive and therapeutic experimental designs, with effects that were comparable to those of imipramine administration. Immobility time following simultaneous acupuncture stimulation of the two control point locations overlying the spinal column was significantly suppressed only 2 weeks after the start of FS in the preventive effect experiment, and the suppressive effect was significantly lower than that of simultaneous acupuncture stimulation at GV20 and Yintang. In the therapeutic effect experiment, there was no change in the increase in immobility time after the end of FS. MA at GV20 and Yintang significantly increased the expression of BDNF and NT-3 in the preventive evaluation and NGF, BDNF, NT-3, and NT-4/5 in the therapeutic effect evaluation. CONCLUSION: Our findings suggest that simultaneous acupuncture stimulation at GV20 and Yintang is effective for the prevention and treatment of depression, and the effect likely involves modulation of the expression of multiple neurotrophic factors.

Antigen stabilizing hydrogels based on cyclodextrins and polyethylene glycol act as type-2 adjuvants with suppressed local irritation.

Recent viral pandemics have increased global demand for vaccines. However, the supply of effective and safe vaccine not only to developed countries but also developing countries with inadequate storage equipment is still challenging due to the lack of robust systems which improve the efficacy and the stability of vaccines with few side effects. In our previous study, polypseudorotaxane (PPRX) hydrogels based on cyclodextrin (CyD) and polyethylene glycol (PEG) significantly improved the stability of antibody preparations and showed no serious adverse effects after subcutaneous injection, suggesting the possibility as safe vaccine formulations to stabilize an antigen protein. Moreover, recent studies have reported that one of the CyD derivatives, hydroxypropyl-ß-CyD (HP-ß-CyD), acts as an adjuvant to enhance protective type-2 immune responses. However, it is still unknown that CyD PPRX hydrogels enhance not only the stability of an antigen protein but also its immunogenicity with tolerable side effects. Here, we demonstrate that α- and γ-CyD PPRX hydrogels containing an antigen protein significantly induce antigen-specific type-2 immune responses. Moreover, α- and γ-CyD PPRX hydrogels showed negligible local irritation at the injection site, although subcutaneous injection of α-CyD alone induced skin lesion. Finally, shaking stability of the antigen protein at room temperature was significantly improved by being included in α- and γ-CyD PPRX hydrogels. These results propose the possibility of α- and γ-CyD PPRX hydrogels as novel vaccine formulations which improve both the immunogenicity and stability of an antigen protein with suppressed local irritation.

Hot Electron Extraction in SWCNT/TiO2 for Photocatalytic H2 Evolution from Water.

Single-walled carbon nanotube (SWCNT)/TiO2 hybrids were synthesized using 1,10-bis(decyloxy)decane-core PAMAM dendrimer as a molecular glue. Upon photoirradiation of a water dispersion of SWCNT/TiO2 hybrids with visible light (λ > 422 nm), the hydrogen evolution reaction proceeded at a rate of 0.95 mmol/h·g in the presence of a sacrificial agent (1-benzyl-1,4-dihydronicotinamide, BNAH). External quantum yields (EQYs) of the hydrogen production reaction photosensitized by (6,5), (7,5), and (8,3) tubes were estimated to be 5.5%, 3.6%, and 2.2%, respectively, using monochromatic lights corresponding to their E22 absorptions (570 nm, 650 nm, and 680 nm). This order of EQYs (i.e., (6,5) > (7,5) > (8,3)SWCNTs) exhibited the dependence on the C2 energy level of SWCNT for EQY and proved the hot electron extraction pathway.

Automated preparation of washed platelet concentrates through spinning-membrane filtration.

BACKGROUND: Washed platelet concentrates (WPC), prepared with an automated system cell processor (ACP), have recently been approved to be manufactured and marketed in Japan. From the perspective of risk management, it is preferable to secure alternative technologies for ACP. Here, we conducted a study to evaluate the quality of WPC prepared using an automated membrane filtration-based system, Lovo. STUDY DESIGN AND METHODS: Replaced PCs prepared from apheresis PCs were equally divided into control and test units, and subsequently washed using ACP and Lovo respectively. Work and operational efficiencies were evaluated by in vitro analyses, including total handling time, platelet recovery, and plasma protein removal rate. Product quality, including a set of biochemical and physiological indicators of platelets and supernatants, were assessed before and 3 days after washing. RESULTS: In vitro platelet recovery rates and plasma protein removal rates were >85% and >95%, respectively, in both groups. The pH values on day 0 were significantly high (6.97 vs. 6.86) due to low pCO2 in the test group, while no significant differences in glucose consumption and lactate production were observed between the two groups. The levels of hypotonic shock responses, aggregation response, platelet shape, CD62P expression, and sCD62P concentration were similar in both groups during the 3-day storage period. CONCLUSION: Platelet washing with Lovo provides platelet quality equivalent to, or better than, conventional washing with ACP. Thus, the new automated system, Lovo, can be considered as an alternative to ACP for WPC preparation.

Production and nonclinical evaluation of an autologous iPSC-derived platelet product for the iPLAT1 clinical trial.

Donor-derived platelets are used to treat or prevent hemorrhage in patients with thrombocytopenia. However, ∼5% or more of these patients are complicated with alloimmune platelet transfusion refractoriness (allo-PTR) due to alloantibodies against HLA-I or human platelet antigens (HPA). In these cases, platelets from compatible donors are necessary, but it is difficult to find such donors for patients with rare HLA-I or HPA. To produce platelet products for patients with aplastic anemia with allo-PTR due to rare HPA-1 mismatch in Japan, we developed an ex vivo good manufacturing process (GMP)-based production system for an induced pluripotent stem cell-derived platelet product (iPSC-PLTs). Immortalized megakaryocyte progenitor cell lines (imMKCLs) were established from patient iPSCs, and a competent imMKCL clone was selected for the master cell bank (MCB) and confirmed for safety, including negativity of pathogens. From this MCB, iPSC-PLTs were produced using turbulent flow bioreactors and new drugs. In extensive nonclinical studies, iPSC-PLTs were confirmed for quality, safety, and efficacy, including hemostasis in a rabbit model. This report presents a complete system for the GMP-based production of iPSC-PLTs and the required nonclinical studies and thus supports the iPLAT1 study, the first-in-human clinical trial of iPSC-PLTs in a patient with allo-PTR and no compatible donor using the autologous product. It also serves as a comprehensive reference for the development of widely applicable allogeneic iPSC-PLTs and other cell products that use iPSC-derived progenitor cells as MCB.

Feedforward coactivation of trunk muscles during rapid shoulder movements.

Background: Shoulder movements that involve unilateral and bilateral flexion, extension, abduction, and asymmetrical flexion-extension cause the activity of trunk muscles. There has not been a fixed consensus on the onset of deep trunk muscle activities including the psoas major (PM), quadratus lumborum (QL), transversus abdominis (TrA), and lumbar multifidus (MF) during shoulder movements. The purpose of this study was to measure the onset of electromyographic activity of the deep trunk muscles during rapid shoulder movements and clarify the coordinated activity pattern of the deep trunk muscles during 11 shoulder movements. Methods: Thirteen men participated in this study. The onset of activity of the right deep trunk muscles (PM, QL, TrA, and MF) were measured using fine-wire electrodes, and those of the right and left deltoid (anterior, middle, and posterior) and right superficial trunk muscles (rectus abdominis, external oblique [EO], and internal oblique [IO]) were measured using surface electrodes as participants performed 6 types of unilateral, 3 types of bilateral, and 2 types of asymmetrical rapid shoulder movements. We defined feedforward activation as the onset of activity of trunk muscle before or within +50 ms onset of the deltoid muscle and feedback activation as that after +50 ms. A 1-way analysis of variance was performed to compare the onset of activity of each muscle during each shoulder movement. Results: The mean onset of activity of the PM (26.0 ms), QL (13.1 ms), TrA (-19.7 ms), and MF (20.4 ms) muscles demonstrated feedforward activation during left shoulder flexion. The onset of activity of the TrA (1.6-48.7 ms), rectus abdominis (-1.7 to 17.3 ms), and EO (5.6-40.8 ms) muscles demonstrated feedforward activation during left, right, and bilateral shoulder extension. The onset of activity of the PM (22.9 ms), QL (23.0 ms), TrA (18.9 ms), and EO (15.4 ms) demonstrated feedforward activation during left shoulder abduction, while that of the IO (4.4-10.9 ms) only demonstrated feedforward activation during right and bilateral shoulder abduction. The onset of activity of the TrA (-27.6 ms) and IO (-23.9 ms) demonstrated feedforward activation during left shoulder flexion-right shoulder extension, and that of the MF (33.4 ms) and EO (-17.2 ms), during left shoulder extension-right shoulder flexion. Conclusion: Rapid shoulder movements occur with coordinated muscle activation of the deep trunk muscles depending on the direction of shoulder movements. Feedforward activation of single or combined deep trunk muscles may facilitate rapid shoulder movements.

Machine Learning-Assisted Screening of Herbal Medicine Extracts as Vaccine Adjuvants.

Adjuvants are important vaccine components, composed of a variety of chemical and biological materials that enhance the vaccine antigen-specific immune responses by stimulating the innate immune cells in both direct and indirect manners to produce a variety cytokines, chemokines, and growth factors. It has been developed by empirical methods for decades and considered difficult to choose a single screening method for an ideal vaccine adjuvant, due to their diverse biochemical characteristics, complex mechanisms of, and species specificity for their adjuvanticity. We therefore established a robust adjuvant screening strategy by combining multiparametric analysis of adjuvanticity in vivo and immunological profiles in vitro (such as cytokines, chemokines, and growth factor secretion) of various library compounds derived from hot-water extracts of herbal medicines, together with their diverse distribution of nano-sized physical particle properties with a machine learning algorithm. By combining multiparametric analysis with a machine learning algorithm such as rCCA, sparse-PLS, and DIABLO, we identified that human G-CSF and mouse RANTES, produced upon adjuvant stimulation in vitro, are the most robust biological parameters that can predict the adjuvanticity of various library compounds. Notably, we revealed a certain nano-sized particle population that functioned as an independent negative parameter to adjuvanticity. Finally, we proved that the two-step strategy pairing the negative and positive parameters significantly improved the efficacy of screening and a screening strategy applying principal component analysis using the identified parameters. These novel parameters we identified for adjuvant screening by machine learning with multiple biological and physical parameters may provide new insights into the future development of effective and safe adjuvants for human use.

Specific acupuncture stimulation of Shenshu (BL23) affects sympathetic nervous activity-associated plasma renin concentration changes.

OBJECTIVE: To examine whether specific stimulation of Shenshu (BL23) affects sympathetic nervous activity (SNA)-associated plasma renin concentration (PRC). METHODS: Eight healthy volunteers participated in three pattern conditions in random order: control (Cont), stimulation of Shenshu (BL23), and stimulation of sham point (Sham). All participants were initially in the supine position for > 60 min, and then remained in the standing position during the experimental procedure to increase SNA. An electrocardiogram was used to calculate low frequency/high frequency (LF/HF) ratio; blood was collected to analyze PRC. RESULTS: The LF/HF ratio was significantly increased in the standing position when compared with the supine position ( 0.01). There was no difference in LF/HF ratio during or after stimulation of Shenshu (BL23) in the standing position when compared with before the stimulation in the supine position; however, the LF/HF ratio was significantly increased in Cont and Sham conditions ( 0.01). There was no difference in PRC after stimulation of Shenshu (BL23) in the standing position when compared with before the stimulation in the supine position; however, there was a significant increase in PRC in the Cont and Sham conditions (Cont 0.05, Sham 0.01). CONCLUSION: Our results demonstrated that specific acupuncture stimulation of Shenshu (BL23) in the standing position decreased SNA-associated PRC, which was not observed during acupuncture stimulation of the sham point.

Dual preparation of plasma and platelet concentrates in platelet additive solution from platelet concentrates in plasma using a novel filtration system.

BACKGROUND AND OBJECTIVES: Platelet concentrates suspended in a platelet additive solution (PAS-PC) are associated with a reduction in allergic response and are suitable for preparing pathogen-inactivated PC. We aimed to develop an efficient platform for the dual preparation of PAS-PC and platelet-poor plasma. MATERIALS AND METHODS: PAS-PC was prepared in six steps by using a hollow-fibre system based on cross-flow filtration: priming, loading PC, loading PAS, collection of filtered liquid (flow-through) and collection of platelets by washing with PAS followed by washing with air. In this study, the efficacy of platelet and plasma protein recovery and characteristics of recovered PAS-PC and flow-through plasma were analysed in detail. RESULTS: Recoveries of platelet in PAS-PC and plasma protein in the flow-through were 95.4% ± 3.7% and 61.6% ± 5.0%, respectively. The residual plasma protein in PAS-PC was 34.1% ± 2.8%. Although the expression level of CD62P, a platelet activation marker, in recovered platelets was approximately 1.2-fold of that in original platelets, swirling patterns were well retained, and aggregation in PAS-PC was not visible. Agonist-induced aggregabilities, platelet morphology and hypotonic shock recovery were conserved. The patterns of plasma protein and lipoprotein in the flow-through were comparable with those in the original PCs. The multimeric pattern analysis of VWF remained unaltered. CONCLUSION: We propose a highly efficient preparation system that enables the simultaneous production of PAS-PC and platelet-poor plasma. It also achieves a high recovery of functionally well-retained platelets with very low activation.

B cell-intrinsic TBK1 is essential for germinal center formation during infection and vaccination in mice.

The germinal center (GC) is a site where somatic hypermutation and clonal selection are coupled for antibody affinity maturation against infections. However, how GCs are formed and regulated is incompletely understood. Here, we identified an unexpected role of Tank-binding kinase-1 (TBK1) as a crucial B cell-intrinsic factor for GC formation. Using immunization and malaria infection models, we show that TBK1-deficient B cells failed to form GC despite normal Tfh cell differentiation, although some malaria-infected B cell-specific TBK1-deficient mice could survive by GC-independent mechanisms. Mechanistically, TBK1 phosphorylation elevates in B cells during GC differentiation and regulates the balance of IRF4/BCL6 expression by limiting CD40 and BCR activation through noncanonical NF-κB and AKTT308 signaling. In the absence of TBK1, CD40 and BCR signaling synergistically enhanced IRF4 expression in Pre-GC, leading to BCL6 suppression, and therefore failed to form GCs. As a result, memory B cells generated from TBK1-deficient B cells fail to confer sterile immunity upon reinfection, suggesting that TBK1 determines B cell fate to promote long-lasting humoral immunity.

Fasting impairs type 2 helper T cell infiltration in the lung of an eosinophilic asthma mouse model.

Eosinophilic asthma is a form of bronchial asthma that is caused by the pulmonary infiltration of eosinophils and accounts for approximately half of the patients with severe asthma. Several cell types of the immune system in synergy with the epithelial cells of the lung provoke an inflammatory response in patients with asthma. Recently, the effect of fasting on immune cells and inflammation has attracted considerable attention. Therefore, we examined whether fasting may serve as novel preventive strategy in patients with asthma. In our study, we employed a previously established mouse model of eosinophilic asthma. C57BL/6 mice were inoculated intranasally with interleukin-33 and ovalbumin (OVA) in order to induce eosinophil infiltration in the lung and subjected to a 48-h long fasting period directly after or 7 days postinoculation. We used flow cytometry to characterise infiltrated immune cells in the lung and measured the quantity of inflammatory cytokines as well as antigen-specific immunoglobins (Ig) by ELISA. Our results indicated that fasting lowered the number of eosinophilic pulmonary infiltrates in the eosinophilic asthma model mice. Furthermore, fasting suppressed anti-OVA IgG1 production. Fasting suppressed Th2 cytokine production by impairing Th2 accumulation in the lung. The findings suggest that fasting may be a novel preventive strategy for eosinophilic asthma.