Morphological Changes in Blood Cells in a Rat Model of Heatstroke: A Pilot Study.

Despite the increasing threat of heatstroke with global warming, pathophysiologic injury continues to be defined. In addition, morphological changes of the peripheral blood cells in heatstroke have not been well characterized. We evaluated pathophysiologic changes in bone marrow and blood cells in a rat heatstroke model using a 39.5 °C climate chamber. After three hours of incubation, blood and bone marrow samples were collected for morphology, and the direct effects of heat on leukocytes in vitro were evaluated using time-lapse observation. The blood cell count and peripheral/bone marrow smear were examined either in a lethal model (core body temperature exceeded 42.5 °C) or in a sublethal model (<41.5 °C). Significant decreases in platelet and white blood counts occurred in the lethal model (>35% and >20% decreases, respectively) and changes were less in the sublethal model. Platelet clumping with the appearance of large platelets was observed. The neutrophils often demonstrated hyper-segmented nuclei, and lymphocytes showed reactive or blast-like changes. Further, the direct effect of heat on leukocytes noted apoptotic cell death at 41.5 °C, but subsequent necrosis at 43 °C. In summary, our rodent model showed that heatstroke causes platelet aggregation, leukocyte injury, and aponecrotic cell death. Such changes were milder and reversible in sublethal heatstroke. The appearance of immature cells may result from damage to the bone marrow microenvironment. These findings may provide useful information for potential diagnostic and therapeutic considerations.

Isolation of TCR genes with tumor-killing activity from tumor-infiltrating and circulating lymphocytes in a tumor rejection cynomolgus macaque model.

To develop effective adoptive cell transfer therapy using T cell receptor (TCR)-engineered T cells, it is critical to isolate tumor-reactive TCRs that have potent anti-tumor activity. In humans, tumor-infiltrating lymphocytes (TILs) have been reported to contain CD8+PD-1+ T cells that express tumor-reactive TCRs. Characterization of tumor reactivity of TILs from non-human primate tumors could improve anti-tumor activity of TCR-engineered T cells in preclinical research. In this study, we sought to isolate TCR genes from CD8+PD-1+ T cells among TILs in a cynomolgus macaque model of tumor transplantation in which the tumors were infiltrated with CD8+ T cells and were eventually rejected. We analyzed the repertoire of TCRα and ß pairs obtained from single CD8+PD-1+ T cells in TILs and circulating lymphocytes and identified multiple TCR pairs with high frequency, suggesting that T cells expressing these recurrent TCRs were clonally expanded in response to tumor cells. We further showed that the recurrent TCRs exhibited cytotoxic activity to tumor cells in vitro and potent anti-tumor activity in mice transplanted with tumor cells. These results imply that this tumor transplantation macaque model recapitulates key features of human TILs and can serve as a platform toward preclinical studies of non-human primate tumor models.

Recombinant Human Soluble Thrombomodulin Suppresses Arteritis in a Mouse Model of Kawasaki Disease.

INTRODUCTION AND OBJECTIVE: Kawasaki disease (KD) is associated with diffuse and systemic vasculitis of unknown aetiology and primarily affects infants and children. Intravenous immunoglobulin (IVIG) treatment reduces the risk of developing coronary aneurysms, but some children have IVIG-resistant KD, which increases their risk of developing coronary artery injury. Here, we investigated the effect of recombinant human soluble thrombomodulin (rTM), which has anticoagulant, anti-inflammatory, and cytoprotective properties on the development of coronary arteritis in a mouse model of vasculitis. METHODS: An animal model of KD-like vasculitis was created by injecting mice with Candida albicans water-soluble fraction (CAWS). This model was used to investigate the mRNA expression of interleukin (IL)-10, tumour necrosis factor alpha (TNF-α), and tissue factor (TF), in addition to histopathology of heart tissues. RESULTS: rTM treatment significantly reduces cardiac vascular endothelium hypertrophy by 34 days after CAWS treatment. In addition, mRNA expression analysis revealed that rTM administration increased cardiac IL-10 expression until day 27, whereas expression of TNF-α was unaffected. Moreover, in the spleen, rTM treatment restores IL-10 and TF expression to normal levels. CONCLUSION: These findings suggest that rTM suppresses CAWS-induced vasculitis by upregulating IL-10. Therefore, rTM may be an effective treatment for KD.

A new SOFA score calculation to improve the predictive performance for mortality in sepsis-associated disseminated intravascular coagulopathy patients.

PURPOSE: The change in the sequential organ failure assessment (SOFA) score from the entry day, a delta-SOFA (SOFAΔ), has been proposed as a better indicator for predicting mortality, and potentially as an endpoint in clinical trials. However, there are some concerns that the value of the absolute SOFA score has not been considered. The purpose of the study is to examine whether the addition of an absolute SOFA score can increase the predictive performance of SOFAΔ. MATERIALS AND METHODS: Data obtained from 297 patients with sepsis-associated disseminated intravascular coagulopathy (DIC) in multiinstitutional post-marketing surveys were analyzed retrospectively. The SOFAComb (SOFAΔ score + absolute SOFA score) and SOFAΔ were calculated, and the performance of each indicator was analyzed in terms of predictive ability for 28-day mortality. RESULTS: The area under the receiver operating curve (AUC) for the mortality of SOFAComb on day 2, 4, 7 were significantly greater compared to those of SOFAΔ (P <0.001, =0.002, <0.001, respectively). In addition, the accuracy [(True positive + True negative) / total number at the best cutoff points] of SOFAComb was better than that of SOFAΔ. CONCLUSIONS: SOFAComb is simple to calculate and provides better predictive performance compared to SOFAΔ for predicting mortality.

PiTMaP: A New Analytical Platform for High-Throughput Direct Metabolome Analysis by Probe Electrospray Ionization/Tandem Mass Spectrometry Using an R Software-Based Data Pipeline.

A new analytical platform called PiTMaP was developed for high-throughput direct metabolome analysis by probe electrospray ionization/tandem mass spectrometry (PESI/MS/MS) using an R software-based data pipeline. PESI/MS/MS was used as the data acquisition technique, applying a scheduled-selected reaction monitoring method to expand the targeted metabolites. Seventy-two metabolites mainly related to the central energy metabolism were selected; data acquisition time was optimized using mouse liver and brain samples, indicating that the 2.4 min data acquisition method had a higher repeatability than the 1.2 and 4.8 min methods. A data pipeline was constructed using the R software, and it was proven that it can (i) automatically generate box-and-whisker plots for all metabolites, (ii) perform multivariate analyses such as principal component analysis (PCA) and projection to latent structures-discriminant analysis (PLS-DA), (iii) generate score and loading plots of PCA and PLS-DA, (iv) calculate variable importance of projection (VIP) values, (v) determine a statistical family by VIP value criterion, (vi) perform tests of significance with the false discovery rate (FDR) correction method, and (vii) draw box-and-whisker plots only for significantly changed metabolites. These tasks could be completed within ca. 1 min. Finally, PiTMaP was applied to two cases: (1) an acetaminophen-induced acute liver injury model and control mice and (2) human meningioma samples with different grades (G1-G3), demonstrating the feasibility of PiTMaP. PiTMaP was found to perform data acquisition without tedious sample preparation and a posthoc data analysis within ca. 1 min. Thus, it would be a universal platform to perform rapid metabolic profiling of biological samples.

Long non-coding RNA MANCR is a target of BET bromodomain protein BRD4 and plays a critical role in cellular migration and invasion abilities of prostate cancer.

Androgen receptor (AR)-negative castration-resistant prostate cancer (CRPC) is highly aggressive and is resistant to most of the current therapies. Bromodomain and extra terminal domain (BET) protein BRD4 binds to super-enhancers (SEs) that drive high expression of oncogenes in many cancers. A BET inhibitor, JQ1, has been found to suppress the malignant phenotypes of prostate cancer cells, however, the target genes of JQ1 remain largely unknown. Here we show that SE-associated genes specific for AR-negative CRPC PC3 cells include genes involved in migration and invasion, and that JQ1 impairs migration and invasion of PC3 cells. We identified a long non-coding RNA, MANCR, which was markedly down-regulated by JQ1, and found that BRD4 binds to the MANCR locus. MANCR knockdown led to a significant decrease in migration and invasion of PC3 cells. Furthermore, RNA sequencing analysis revealed that expression of the genes involved in migration and invasion was altered by MANCR knockdown. In summary, our data demonstrate that MANCR plays a critical role in migration and invasion of PC3 cells.

Trans-omics Impact of Thymoproteasome in Cortical Thymic Epithelial Cells.

The thymic function to produce self-protective and self-tolerant T cells is chiefly mediated by cortical thymic epithelial cells (cTECs) and medullary TECs (mTECs). Recent studies including single-cell transcriptomic analyses have highlighted a rich diversity in functional mTEC subpopulations. Because of their limited cellularity, however, the biochemical characterization of TECs, including the proteomic profiling of cTECs and mTECs, has remained unestablished. Utilizing genetically modified mice that carry enlarged but functional thymuses, here we show a combination of proteomic and transcriptomic profiles for cTECs and mTECs, which identified signature molecules that characterize a developmental and functional contrast between cTECs and mTECs. Our results reveal a highly specific impact of the thymoproteasome on proteasome subunit composition in cTECs and provide an integrated trans-omics platform for further exploration of thymus biology.

TCR Affinity for In Vivo Peptide-Induced Thymic Positive Selection Fine-Tunes TCR Responsiveness of Peripheral CD8+ T Cells.

The affinity for TCR interactions with self-peptide/MHC complexes (pMHC) in the thymus critically affects immature thymocytes that newly express TCRs. Previous fetal thymus organ culture experiments have indicated that difference in the affinity for thymic TCR/pMHC interactions not only determines thymocyte fate between positive and negative selection, but also affects Ag responsiveness of positively selected thymocytes. In the current study, we examined whether TCR/pMHC affinity during positive selection in the thymus would further affect Ag responsiveness of mature T cells in the periphery. To do so, OVA peptide variants were in vivo administered to TAP1-deficient OT-I/TCR-transgenic mice in which T cell development was otherwise arrested at CD4+CD8+ thymocytes because of the lack of self-pMHC presentation in thymic APCs. We found that a group of peptide variants induced the transient generation of OT-I CD8+ T cells in the thymus and the periphery. We also noticed that the affinity threshold for positive and negative selection detected in adult mice in vivo was higher than that measured in fetal thymus organ culture experiments in vitro. Interestingly, we further found that the affinity for positively selecting peptides proportionally affected TCR responsiveness of peripheral naive CD8+ T cells. These results indicate that in vivo administration of a peptide can promote T cell selection in the thymus and the affinity for TCR/pMHC interaction during positive selection fine-tunes Ag responsiveness of peripheral T cells.

Thymus machinery for T-cell selection.

An immunocompetent and self-tolerant pool of naive T cells is formed in the thymus through the process of repertoire selection. T cells that are potentially capable of responding to foreign antigens are positively selected in the thymic cortex and are further selected in the thymic medulla to help prevent self-reactivity. The affinity between T-cell antigen receptors expressed by newly generated T cells and self-peptide-major histocompatibility complexes displayed in the thymic microenvironments plays a key role in determining the fate of developing T cells during thymic selection. Recent advances in our knowledge of the biology of thymic epithelial cells have revealed unique machinery that contributes to positive and negative selection in the thymus. In this article, we summarize recent findings on thymic T-cell selection, focusing on the machinery unique to thymic epithelial cells.

Establishment of a novel platform cell line for efficient and precise evaluation of T cell receptor functional avidity.

Adoptive T-cell therapy with T cell receptor (TCR) -engineered T cells is an attractive strategy for cancer treatment and the success in this therapy is dependent on the functional avidity of the transduced TCRs against targeted tumor antigens. Therefore, the establishment of the methodology of the efficient and precise evaluation of TCR functional avidity has been awaited. Here, we show a novel platform cell line, named 2D3, which enables the functional avidity of transduced TCRs to be evaluated efficiently and precisely. In the 2D3, the precise TCR functional avidity of transduced TCRs is easily evaluable by the expression of green fluorescent protein (GFP) reporter gene driven by nuclear factor of activated T cells (NFAT) activation via TCR signaling. Four different TCRs of HLA-A*24:02-restricted Wilms' tumor gene 1 (WT1)-specific CD8+ cytotoxic T lymphocytes (CTLs) were transduced into 2D3 cells and the functional avidities of these four TCRs were evaluated. The evaluated functional avidity of these TCRs positively correlated with cell proliferation, cytokine production, and WT1-specific cytotoxicity of the TCR-transduced CD8+ T cells in response to WT1 antigen. These results showed that 2D3 cell line was a novel and stable tool useful for the efficient and precise evaluation of the functional avidity of isolated and transduced TCRs in developing TCR-based immunotherapy.

Antigen processing and presentation in the thymus: implications for T cell repertoire selection.

The processing and presentation of major histocompatibility complex (MHC)-associated antigens depend on the intracellular digestion of self- and nonself-proteins, the loading of digested peptides onto MHC molecules, and the traffic of peptide-MHC complexes to plasma membrane surface for display to interacting T cells. Recent studies have revealed unique machineries for antigen processing and presentation in thymic antigen-presenting cells that display self-antigens to developing thymocytes for the formation of functionally competent yet self-tolerant T cell repertoire. Here, we briefly summarize those machineries, focusing on the biology of cortical and medullary thymic epithelial cells.

Characteristics and components of poly-aluminum chloride coagulants that enhance arsenate removal by coagulation: Detailed analysis of aluminum species.

We evaluated 51 poly-aluminum chloride (PACl) coagulants to determine the coagulant characteristics that were responsible for effective arsenate removal from contaminated river water by means of experiments involving coagulation, settling, and microfiltration. Some of the high-basicity PACls exhibited high arsenate removal percentages, particularly under alkaline conditions, and we investigated various relevant properties and characteristics of these high-basicity PACls. Effective arsenate removal was correlated with the content of polymeric and colloidal aluminum species (Alb and Alc) in the PACls but was not well correlated with colloid charge or zeta potential. Multiple regression analysis revealed that a portion of Alb and Alc, which reacted with the ferron reagent during the period from 30 min to 3 h, that is, the (Al30min-3h) fraction, had the highest arsenate sorption capacity, followed by a colloidal aluminum fraction (Al>3h, which reacted with ferron at a time of >3 h). The Al30min-3h fraction was stable, and its arsenate sorption capacity did not decrease markedly with increasing pH. The Al30min-3h fraction did not correspond to the Keggin-type e-Al13 polycation or the δ-Al30 polycation; it is likely to be an aluminum polymer that is unobservable by 27Al NMR spectroscopy. Our results suggest that PACls with a high proportion of the Al30min-3h fraction should be used for enhanced arsenate removal by coagulation. A high content of the e-Al13 polycation or the δ-Al30 polycation was not indispensable for effective arsenate removal.

Generation of Peptides That Promote Positive Selection in the Thymus.

To establish an immunocompetent TCR repertoire that is useful yet harmless to the body, a de novo thymocyte repertoire generated through the rearrangement of genes that encode TCR is shaped in the thymus through positive and negative selection. The affinity between TCRs and self-peptides associated with MHC molecules determines the fate of developing thymocytes. Low-affinity TCR engagement with self-peptide-MHC complexes mediates positive selection, a process that primarily occurs in the thymic cortex. Massive efforts exerted by many laboratories have led to the characterization of peptides that can induce positive selection. Moreover, it is now evident that protein degradation machineries unique to cortical thymic epithelial cells play a crucial role in the production of MHC-associated self-peptides for inducing positive selection. This review summarizes current knowledge on positive selection-inducing self-peptides and Ag processing machineries in cortical thymic epithelial cells. Recent studies on the role of positive selection in the functional tuning of T cells are also discussed.

An Essential Role of the Avidity of T-Cell Receptor in Differentiation of Self-Antigen-reactive CD8+ T Cells.

Many studies demonstrated crucial roles of avidity of T-cell receptor (TCR) in T-cell fate. However, majority of these findings resulted from analysis of non-self-antigen-specific CD8 T cells, and little is known about roles of TCR avidity in the fate of self-antigen-specific CD8 T cells. Wilms tumor gene 1 (WT1) protein is a self-antigen most suitable for addressing this issue because WT1 protein is a highly immunogenic, typical self-antigen. Here, we isolated 2 distinct and functional TCRs, TCR1 and TCR2, from murine WT1 peptide (RMFPNAPYL)-specific cytotoxic T lymphocytes (WT1-CTLs) and generated TCR1-retrogenic (Rg) and TCR2-Rg mice under T and B-cell-deficient and -reconstituted conditions. TCR1-transduced CD8 T (TCR1-T) cells had approximately 2-fold higher avidity to WT1 peptide than TCR2-transduced CD8 T (TCR2-T) cells. Cytokine production profiles and cell surface phenotypes showed that TCR1-T cells were more differentiated than TCR2-T cells under both conditions. Therefore, TCR1-T cells with TCR avidity higher than that of TCR2-T cells are more differentiated compared with TCR2-T cells. Furthermore, TCR1-T cells that developed under T and B-cell-reconstituted conditions displayed cytotoxicity against endogenously WT1-expressing tumor cells, whereas TCR2 T cells that developed under the same conditions did not. Thus, it was demonstrated, for the first time, that TCR avidity played an essential role in differentiation of self-antigen-reactive T cells, through the success of establishment of two distinct WT1-CTLs with a difference in only TCR avidity under the identical genetic background. Present results should provide us with an insight for elucidation of the differentiation mechanisms of self-antigen-reactive T cells, including tumor antigen-reactive T cells.

Transduction of a novel HLA-DRB1*04:05-restricted, WT1-specific TCR gene into human CD4+ T cells confers killing activity against human leukemia cells.

BACKGROUND/AIM: Wilms' tumor gene 1 (WT1) product is a pan-tumor-associated antigen. We previously identified WT1 protein-derived promiscuous helper peptide, WT1332. Therefore, isolation and characterization of the WT1332-specific T-cell receptors (TCRs) are useful to develop broadly applicable TCR gene-based adoptive immunotherapy. MATERIALS AND METHODS: A novel HLA-DRB1*04:05-restricted WT1332-specific TCR gene was cloned and transduced into human CD4+ T-cells by using a lentiviral vector. RESULTS: The WT1332-specific TCR-transduced CD4+ T-cells showed strong proliferation and Th1-cytokine production in an HLA-DRB1*04:05-restricted, WT1332-specific manner. Furthermore, the WT1332-specific TCR-transduced CD4+ T-cells could lyse HLA-DRB1*04:05-positive, WT1-expressing leukemia cells in vitro. CONCLUSION: The novel TCR gene cloned here should be a promising tool to develop adoptive immunotherapy of WT1332-specific TCR-transduced CD4+ T-cells for the treatment of WT1-expressing cancer, such as leukemia.

Rigidity of a spherical capsule switches the localization of encapsulated particles between inner and peripheral regions under crowding condition: simple model on cellular architecture.

We have investigated the inhomogeneous interior of confined spherical cavities as capsules containing encapsulated binary hard sphere mixtures for different compositions and cavity wall rigidity. Such a greatly simplified model manifests the effects of macromolecular crowding arising from excluded volume interactions in a tiny cell or a cellular nucleus. By fixing the number of large particles, the level of crowding is adjusted by changing the amount of small hard spheres in the cavity. For a rigid cavity, large spheres tend to pack in liquid-like order apart from the surface to the center of the cavity as the crowding level is increased. Whereas, for a soft cavity, larger spheres tend to blend with small spheres in the peripheral region at near the boundary of the cavity, and are susceptible to be depleted from the interior of the cavity as the cavity becomes more crowded. These results may help future elucidation of the thermodynamic pathways to stabilize the inhomogeneous structure of mixtures confined in cavities, such as the derepression of genome materials around the interior rim of the nucleus in a cancerous cell.

Minimizing residual aluminum concentration in treated water by tailoring properties of polyaluminum coagulants.

Aluminum coagulants are widely used in water treatment plants to remove turbidity and dissolved substances. However, because high aluminum concentrations in treated water are associated with increased turbidity and because aluminum exerts undeniable human health effects, its concentration should be controlled in water treatment plants, especially in plants that use aluminum coagulants. In this study, the effect of polyaluminum chloride (PACl) coagulant characteristics on dissolved residual aluminum concentrations after coagulation and filtration was investigated. The dissolved residual aluminum concentrations at a given coagulation pH differed among the PACls tested. Very-high-basicity PACl yielded low dissolved residual aluminum concentrations and higher natural organic matter (NOM) removal. The low residual aluminum concentrations were related to the low content of monomeric aluminum (Ala) in the PACl. Polymeric (Alb)/colloidal (Alc) ratio in PACl did not greatly influence residual aluminum concentration. The presence of sulfate in PACl contributed to lower residual aluminum concentration only when coagulation was performed at around pH 6.5 or lower. At a wide pH range (6.5-8.5), residual aluminum concentrations <0.02 mg/L were attained by tailoring PACl properties (Ala percentage ≤0.5%, basicity ≥85%). The dissolved residual aluminum concentrations did not increase with increasing the dosage of high-basicity PACl, but did increase with increasing the dosage of normal-basicity PACl. We inferred that increasing the basicity of PACl afforded lower dissolved residual aluminum concentrations partly because the high-basicity PACls could have a small percentage of Ala, which tends to form soluble aluminum-NOM complexes with molecular weights of 100 kDa-0.45 µm.

Functional human Th17 clones with WT1-specific helper activity.

Th17 plays important roles in the pathogenesis of various inflammatory and autoimmune diseases. Although the importance of Th17 in tumor immunity has also been suggested, precise roles of tumor-associated antigen-specific Th17 still remain poorly understood, especially in humans. We previously identified WT1332, a 16-mer helper epitope derived from tumor-associated antigen Wilms' tumor gene 1 (WT1) product, and WT1332-specific Th1 clones were established. In the present study, WT1-specific Th17 clones were established by the stimulation of peripheral blood mononuclear cells with the WT1332 helper peptide under human Th17-polarizing conditions. The WT1-specific Th17 clone exhibited the helper function for proliferation of conventional CD4(+) T cells in the antigenic stimulation-specific manner. This is the first report of establishment of functional Th17 clones with both antigen (WT1332) specificity and antigen-specific helper activity. Th17 clones established here and the method to establish antigen-specific Th17 clones should be a useful tool to further analyze the roles of human Th17 in tumor immunity.

Controlled release using a polymer stereocomplex capsule through the selective extraction and incorporation of one capsule shell component.

Isotactic poly(methyl methacrylate) (it-PMMA)/syndiotactic poly(methacrylic acid) (st-PMAA) stereocomplex hollow capsules were fabricated by the deposition of stereocomplex films of it-PMMA and st-PMAA on silica particles by alternate layer-by-layer assembly and the subsequent removal of the silica particles with aqueous HF. The selective extraction of st-PMAA from the it-PMMA/st-PMAA stereocomplex capsule shells was successfully carried out by immersion in a pH 6-9 aqueous solution. The incorporation of st-PMAA into the resulting porous capsule shells was performed by immersion in an acetonitrile/water (1/1) solution of st-PMAA. The controlled release of an encapsulated dye from the it-PMMA/st-PMAA hollow capsules was achieved by combining the selective extraction of st-PMAA from the capsule shells and the incorporation of st-PMAA into the resulting porous shells.

Fabrication of nanofibers through a unique morphological transformation of poly(lactic acid) particles in water.

Poly(lactic acid) (PLA) particles dispersed in water were transformed into nanofibers by simply heating above the glass transition temperatures of the hydrated PLAs.