We herein elucidate the function of SARS-CoV-2derived 5'UTR in the human cells. 5'UTR bound host cellular RNAs were immunoprecipitated by gRNA-dCas13 (targeting luciferase RNA fused to SARS-CoV-2 5'UTR) in HEK293T and A549 cells. The 5'UTR bound RNA extractions were predominantly enriched for regulating lipid metabolism. Overexpression of SARS-CoV-2 5'UTR RNA altered the expression of factors involved in the process of the human Mevalonate pathway. In addition, we found that HMG-CoA reductase inhibitors were shown to suppress SARS-CoV-2 5'UTR-mediated translation activities. In conclusion, we deduce the array of host RNAs interacting with SARS-CoV-2 5'UTR that drives SARS-CoV-2 translation and influences host metabolic pathways.

Silicosis (SIL) is a well-known typical lung fibrosis categorized as pneumoconiosis. Patients suffer from dyspnea, shortness of breath, cough and sputum, which can develop into respiratory failure. The main complications of silicosis are pulmonary diseases. However, SIL is often complicated with various autoimmune diseases such as rheumatoid arthritis, systemic sclerosis, and anti-neutrophil cytoplasmic antibody (ANCA)-related vasculitis. We have been investigating the cellular and molecular alterations of immune cells derived from SIL as well as silica particles in vitro with particular emphasis on the effects on human immune cells. Results showed continuous activation and long survival of CD4 + responder T (Tresp) cells. Additionally, CD4 + 25 + regulatory T (Treg) cells were lost by earlier apoptosis since CD95/Fas death receptor was overexpressed as a result of chronic activation induced by silica particles. The imbalance between Tresp and Treg cells may be the basis for the alteration of dysregulation in autoimmunity. Then, we developed an autoimmune score (AIS) by multiple regression analysis using 19 immune factors in plasma of 19 healthy volunteers (HV) and 20 SIL patients who did not show any autoimmune clinical symptoms. The formula generated is [AIS = (0.119 × sFas) + (1.063 × IgG4) + (0.059 × IL-6) + 0.063]. Using this formula, all values from the 19 HV and 20 SIL patients ranged between 0.90 and 2.71. If we set the cut off value as 1.3935, HV and SIL patients were clearly divided, with only one false-negative SIL patient. We could use this formula for early preclinical detection of autoimmune diseases among various populations predominantly comprising relatively young and female subjects. There are presently some limitations to this formula, such as age differences between two groups and the use of limited factors. Thus, an altered version of this formula could be proposed to be used in the fields of preventive medicine.

Background Asbestos fibers possess tumorigenicity and are thought to cause mesothelioma. We have previously reported that exposure to asbestos fibers causes a reduction in antitumor immunity. Asbestos exposure in the mixed lymphocyte reaction (MLR) showed suppressed induction of cytotoxic T lymphocytes (CTLs), accompanied by a decrease in proliferation of CD8 + T cells. Recently, we reported that asbestos-induced suppression of CTL induction is not due to insufficient levels of interleukin-2 (IL-2). In this study, we continue to investigate the mechanism responsible for the effect of asbestos fibers on the differentiation of CTLs and focus on interleukin-15 (IL-15) which is known to be a regulator of T lymphocyte proliferation. Methods For MLR, human peripheral blood mononuclear cells (PBMCs) were cultured with irradiated allogenic PBMCs upon exposure to chrysotile B asbestos at 5 μg/ml for 7 days. After 2 days of culture, IL-15 was added at 1 ng/ml. After 7 days of MLR, PBMCs were collected and analyzed for phenotypic and functional markers of CD8 + T cells with fluorescence-labeled anti-CD3, anti-CD8, anti-CD45RA, anti-CD45RO, anti-CD25, and anti-granzyme B antibodies using flow cytometry. To examine the effect of IL-15 on the expression level of intracellular granzyme B in proliferating and non-proliferating CD8 + lymphocytes, PBMCs were stained using carboxyfluorescein diacetate succinimidyl ester (CFSE) and then washed and used for the MLR. Results IL-15 addition partially reversed the decrease in CD3 + CD8 + cell numbers and facilitated complete recovery of granzyme B + cell percentages. IL-15 completely reversed the asbestos-induced decrease in percentage of granzyme B + cells in both non-proliferating CFSE-positive and proliferating CFSE-negative CD8 + cells. The asbestos-induced decrease in the percentage of CD25 + and CD45RO + cells in CD8 + lymphocytes was not reversed by IL-15. Conclusion These findings indicate that CTLs induced upon exposure to asbestos possess dysfunctional machinery that can be partly compensated by IL-15 supplementation, and that IL-15 is more effective in the recovery of proliferation and granzyme B levels from asbestos-induced suppression of CTL induction compared with IL-2.

The effects of asbestos on immunocompetent cells have been investigated. In particular, attention was paid to regulatory T cell function, which was observed using the HTLV-1 immortalized human polyclonal T cell line MT-2. Exposure to asbestos (approximately more than 25 mu g/mL for 1-3 day) induced apoptosis, and we observed an increase in regulatory T cell function and acceleration of the cell cycle with continuous exposure to low concentrations of asbestos (5-10 mu g/mL for more than eight months). Furthermore, cDNA microarray analysis in this study revealed that expression of matrix metalloproteinase-7 (MMP-7) was markedly higher in exposed sublines compared to original MT-2 cells. It was determined that MMP-7 had no effect on Treg function, as determined by examination of sublines and by addition of recombinant MMP-7 and neutralizing antibodies or inhibitors of MMP-7. However, when examining melting of the extracellular matrix (an MMP-7-mediated event) or the extent to which the MT-2 parent strain or long-term exposed subline cells pass through a fibronectin-coated filter, more filter passes were observed for the subline. These results suggest that the effect of asbestos fibers on Treg cells results in excessive migration of the tumor microenvironment through hypersecretion of MMP-7 together with an increase in suppressive function and enhancement of cell cycle progression. Therefore, one possible way to prevent the development of asbestos-induced cancer is to reduce the function (including MMP-7 production) or amount of Treg cells by physiologically active substances or food ingredients. Alternatively, it may be possible to invoke immune checkpoint treatments when carcinogenesis occurs.

Pollen allergy to Japanese cedar and cypress is a serious illness that impairs daily life and sleep, especially during pollen season. We have reported that placing a cloth panel containing a specific natural ore powder (CCSNOP) in a room may alleviate the symptoms of hay fever and may also benefit the immune system. This ore is from the Aso mountain range, a volcano on Kyushu Island in the southwestern part of Japan. The purpose of this study was to verify the effect of CCSNOP on cypress pollen. Thirty-one double-blind tests, which investigated cedar pollen allergies, were conducted from February to March 2018 and have already been reported. After this, in early April, 10 of these cases were recruited and all had CCSNOP installed in their bedrooms. Before that, various symptoms and changes in medication were recorded in a "Symptom Diary" and included a mood survey by a questionnaire, stress test using saliva amylase, changes in cypress-specific immunoglobulins IgE and IgG4 by blood sampling, and eosinophil changes. In addition, changes in 29 types of cytokines were investigated. Exposure to CCSNOP relieved symptoms and subjects decreased their intake of medication. There was no change in mood or stress, but eosinophil levels tended to decrease. Although there were no statistical changes in cypress-specific IgE or IgG4, an increase in the former and a decrease in the latter were observed in some individuals during the period of pollen dispersal. Furthermore, levels of GM-CSF and IL8 decreased significantly after use of CCSNOP. The CCSNOP was shown to be effective against cypress pollen allergy, and future investigations will be necessary to observe the long-term effects of CCSNOP.

Exposure of human immune cells to asbestos causes a reduction in antitumor immunity. The present study aimed to investigate the recovery of reduced antitumor immunity by several ingredients taken as supplements or foods, including trehalose (Treh) and glycosylated hesperidin (gHesp). Peripheral blood CD4+ cells were stimulated with IL‑2, anti‑CD3 and anti‑CD28 antibodies for 3 days, followed by further stimulation with IL‑2 for 7 days. Subsequently, cells were stimulated with IL‑2 for an additional 28 days. During the 28 days, cells were cultured in the absence or presence of 50 µg/ml chrysotile asbestos fibers. In addition, cells were treated with 10 mM Treh or 10 µM gHesp. Following culture for 28 days, reverse transcription‑quantitative PCR was performed to assess the expression levels of transcription factors, cytokines and specific genes, including matrix metalloproteinase‑7 (MMP‑7), nicotinamide nucleotide transhydrogenase (NNT) and C‑X‑C motif chemokine receptor 3, in unstimulated cells (fresh) and cells stimulated with PMA and ionomycin (stimuli). The results demonstrated that compared with the control group, chrysotile‑exposure induced alterations in MMP‑7, NNT and IL‑17A expression levels were not observed in the ‘Treh’ and ‘gHesp’ groups in stimulated cells. The results suggested that Treh and gHesp may reverse asbestos exposure‑induced reduced antitumor immunity in T helper cells. However, further investigation is required to confirm the efficacy of future trials involving the use of these compounds with high‑risk human populations exposed to asbestos, such as workers involved in asbestos‑handling activities.

The immune system functions to eliminate abnormal cells that may arise for a variety of reasons. This process can help prevent tumor formation and is referred to as anti-tumor immunity. Inhaled asbestos fibers can accumulate in the non-lymphoid organ of the lungs as well as draining lymph nodes, exposing immune cells to the inhaled asbestos and thereby triggering potential immunological effects. On the basis of that idea, we have been investigating various kinds of asbestos exposure-mediated functional alterations in natural killer (NK), CD4+ T helper (Th), CD8+ cytotoxic T lymphocyte (CTL), and macrophage cells by in vitro experiments. The findings obtained indicate that exposure to asbestos causes decreased cytotoxicity of NK and CTL cells and decreased expression of cell surface activating receptors (NKp46, NKG2D), intracellular perforin levels, and IFN- γ production. Furthermore, an enhanced immune-suppressive role of Th (regulatory T (Treg)) cell function results, with increases in cell surface CTLA-4, and increased production of IL-10 and TGF-β cells as well as fibrogenic/immune-suppressive macrophages with high and lasting production of TGF-β. Interestingly, patients with malignant mesothelioma also show similar characteristics with respect to findings relating to peripheral blood mononuclear cells. Taken together, those data suggest that asbestos fibers elicit immune-suppressive potential, which might contribute to immune escape of abnormal mesothelial cells arising transiently, and promote the development of malignant mesothelioma in people exposed to asbestos. Continued investigations of these events may facilitate the development of early intervention strategies from an immunological perspective to mitigate the development of mesothelioma.

Asbestos exposure causes malignant tumors such as lung cancer and malignant mesothelioma. The effects of asbestos fibers on immunocompetent cells, however, have not been well studied. Asbestos physically comprises a fibrous substance, which differs from silica particles which are a particulate substance, although chemically it is a mineral silicate. Since silicosis patients previously exposed to silica particles often suffer from lung and autoimmune diseases, it is clear that silica exposure impairs immune tolerance. Similarly, asbestos may alter the immune system in asbestos-exposed individuals. Given that malignant tumors can result following exposure to asbestos, the attenuation of anti-tumor immunity in cases of asbestos exposure is an important area of investigation. We observed the effect of asbestos fibers on T lymphocytes, such as CD8+ cytotoxic T lymphocytes (CTLs), CD4+ helper T (Th), and regulatory T (Treg) cells, and showed that anti-tumor immunity was attenuated, as demonstrated in a system that stimulates fresh cells isolated from peripheral blood in vitro and a system that is continuously exposed to a cell line. In this manuscript, we introduce the experiments and results of studies on CTLs, as well as Th and Treg cells, and discuss how future changes in immunocompetent cells induced by asbestos fibers can be clinically linked.

Cas13 endonuclease activity depends on the RNA local secondary structure with strong preference for single-stranded (SS) regions. Hence, it becomes indispensable to identify the SS regions for effective Cas13 mediated RNA knockdown. We herein present rational gRNA design by integrating experimental structure-seq data and predicted structural models. Utilizing structure-seq data for XIST transcript, we observed that gRNAs targeting the SS regions significantly induce transcript knockdown and cleavage than those targeting double-stranded (DS) regions. Further, we identified the "central seed region" in the gRNA that upon targeting the SS regions efficiently facilitates Cas13 mediated cleavage. In our following pursuits, we considered the scenario wherein experimental structure-seq data is not available, hence we used SS18-SSX2 fusion transcript indicated in synovial sarcomas and computationally predicted its structure. We observed that gRNAs targeting the SS regions predicted from the structure, efficiently induced necrosis compared to gRNAs that target the DS regions. In conclusion, for the effective RNA knockdown, the Cas13 mediated targeting strategy presented herein emphasizes the designing of gRNAs specifically targeting SS regions by utilizing structural information. Further, this strategy, in turn, can be anticipated to narrow the search space for gRNA design (by exclusively targeting SS regions) especially when lncRNAs are the targets.

Asbestos fibers are known to cause not only benign pulmonary and pleural diseases such as asbestosis and pleural plaque, but also malignant tumors such as lung cancer and malignant mesothelioma. In addition to the carcinogenic activities possessed by the fibers themselves, it has been considered that asbestos fibers may affect the human immune system. In this review, a cell culture model using a human T cell line exposed to asbestos fibers continuously and at relatively low doses to mimic exposure of environmentally and occupationally exposed people to these fibers is introduced. Although transient and high-dose exposure caused cell apoptosis, the cell line employed acquired resistance to asbestos-induced apoptosis with continuous exposure as a result of various cellular and molecular changes such as changes in cytokine production and cytoskeletal molecules. On the other hand, changes in various immune cells such as cytotoxic T lymphocytes, natural killer cells, T helper cells, and regulatory T cells by in vitro exposure using certain cell lines as well as freshly isolated peripheral blood immune cells derived from healthy volunteers revealed impairment of antitumor immunity. Thereafter, the findings obtained were confirmed using peripheral blood immune cells derived from asbestos-exposed patients with pleural plaque or mesothelioma. The findings are also shown in this chapter. Further research should explore the effects of asbestos fibers on other immune cells such as Th17, investigate the development of diagnostic markers using altered immune cells, and pursue the identification of physiological substances from plants and other sources that can halt or recover the antitumor immunity caused by asbestos exposure.

Fibrous and particulate environmental substances such as asbestos fibers and silica particles cause not only lung fibrosis but also various health disturbances. Asbestos induce malignant tumors such as pleural mesothelioma and lung cancer. Silicosis patients exposed to silica particles show complications of various autoimmune diseases such as rheumatoid arthritis, systemic lupus erythematosus, systemic sclerosis, and antineutrophil cytoplasmic antibody (ANCA)-related vasculitis/nephritis. The causative alteration of immune cells exposed to these environmental substances may form baseline modification of human immune system not only localized pulmonary lesions, alteration of alveolar macrophages, and others but also general immune system and changes of function in effector, regulatory, and cytotoxic T cells and natural killer cells. In this review, both (localized and generalized) immune alterations caused by environmental fibrous and particulate substances are summarized and reported.

Mesothelioma is the most serious of the asbestos-related diseases. It is caused by exposure to relatively low doses of asbestos and takes a long period to develop, which suggests the enactment of gradual adverse effects other than cellular toxicity. The immune system, which can play a role in tumor prevention, is a presumable target of asbestos by accumulation in lymph nodes and then slowly affecting functions of immune cells. Here, we describe key findings obtained from our studies concerning the immune-suppressive effects of asbestos and functional alteration in immune cells of patients with mesothelioma as well as plaque-positive subjects. Asbestos exposure of cell cultures resulted in decreased natural and acquired cytotoxicity exerted by NK cells and CTLs and the ability of Th1 cells to activate and support antitumor immunity. In contrast, asbestos exposure augmented Treg cell function and generation of fibrogenic/suppressive macrophages. Mesothelioma patients also showed similar characteristics in certain alterations caused by asbestos exposure. Additionally, our recent study established immunological screening devices for mesothelioma and asbestos exposure on the basis of comprehensive analysis of peripheral blood. Those findings underscore the importance of the immunological effects of asbestos and should assist further understanding of the mechanism and early detection of mesothelioma.

The effects of asbestos fibers on human immune cells have not been well documented. We have developed a continuously exposed cell line model using the human T-lymphotropic virus 1 (HTLV-1)-immortalized human T cell line MT-2. Sublines continuously exposed to chrysotile (CH) or crocidolite (CR) showed acquired resistance to asbestos-induced apoptosis following transient and high-dose re-exposure with fibers. These sublines in addition to other immune cells such as natural killer cells or cytotoxic T lymphocytes exposed to asbestos showed a reduction in anti-tumor immunity. In this study, the expression of genes and molecules related to antioxidative stress was examined. Furthermore, complexes related to oxidative phosphorylation were investigated since the production of reactive oxygen species (ROS) is important when considering the effects of asbestos in carcinogenesis and the mechanisms involved in resistance to asbestos-induced apoptosis. In sublines continuously exposed to CH or CR, the expression of thioredoxin decreased. Interestingly, nicotinamide nucleotide transhydrogenase (NNT) expression was markedly enhanced. Thus, knockdown of NNT was then performed. Although the knockdown clones did not show any changes in proliferation or occurrence of apoptosis, these clones showed recovery of ROS production with returning NADPH/NADP+ ratio that increased with decreased production of ROS in continuously exposed sublines. These results indicated that NNT is a key factor in preventing ROS-induced cytotoxicity in T cells continuously exposed to asbestos. Considering that these sublines showed a reduction in anti-tumor immunity, modification of NNT may contribute to recovery of the anti-tumor effects in asbestos-exposed T cells.

Silicosis is a typical form of pneumoconiosis and is characterized as a type of lung fibrosis. Silica particles are captured and recognized upon by alveolar macrophages via the macrophage receptor with collagenous structure (MARCO) scavenger receptor, and thereafter the inflammasome is activated. Thereafter, various chemokines/cytokines play their roles to eventually form fibrosis. Additionally, silica particles chronically activate T helper cells which sets the background for the formation of silicosis-associated autoimmune disturbances. The occurrence and progression of lung fibrosis, the extracellular matrix-related molecules such as integrins and their ligands including fibronectin, vitronectin, laminin, and collagens, all play important roles. Here, the roles of these molecules in silicosis-related lung fibrosis are reviewed from the literature. Additionally, the measurement of serum nephronectin (Npnt), a new member of the integrin family of ligands, is discussed, together with investigations attempting to delineate the role of Npnt in silica-induced lung fibrosis. Serum Npnt was found to be higher in silicosis patients compared to healthy volunteers and seems to play a role in the progression of fibrosis with other cytokines. Therefore, serum Npnt levels may be employed as a suitable marker to monitor the progression of fibrosis in silicosis patients.

Pollen allergy remains a big problem in contemporary societies. We have shown in previous studies that a cloth containing a special natural ore powder (CCSNOP) is effective in relieving symptoms in patients with pollen allergies. However, in that study, subjects were exposed to CCSNOP for only one hour. In the present study, CCSNOP or control (non-woven cloth; NWC) panels were placed in the bedrooms of pollen allergy patients for two weeks during the pollen dispersal season in 2018, and the effects were investigated. Twenty-one subjects were exposed to CCSNOP panels and 10 subjects were exposed to NWC panels. Our investigations showed that use of CCSNOP resulted in relief of symptoms and reduced use of therapeutics. Moreover, the ratio of eosinophil count decrease during exposure was higher in the CCSNOP group. Furthermore, a formula for measuring various cytokines and other parameters was established and clearly showed a distinction between the CCSNOP and NWC groups. In this formula, Granulocyte Macrophage colony-stimulating Factor (GM-SCF), Interleukin (IL)-12p40, Immunoglobulin (Ig) G4 and eosinophil count were extracted. These results indicated that CCNSNOP has a beneficial effect on pollen allergy patients. Future studies shall engage in long-term monitoring of pollen allergy patients who will utilize this mineral powder for at least one year.

Various occupational and environmental substances alter the cellular and molecular function of the human lymphoid system. For example, silicosis patients who have been chronically exposed to silica particles often complicate with autoimmune diseases such as rheumatoid arthritis and systemic sclerosis. From our investigations, silica particles affect CD4+ responder T cells and regulatory T cells (Tregs), which results in the disrup - tion of autoimmunity. Asbestos fibers are a type of mineral silicate, and patients exposed to asbestos fibers revealed cancers such as mesothelioma and lung cancer. In these cases, asbestos fibers may reduce antitumor immunity. Our results investigating the effect of asbestos on cytotoxic T lymphocyte, natural killer (NK) cells, CD4+ cells, and Tregs revealed a reduction in antitumor immunity. To date, the effects of silica and asbestos on Th17 cells and antigen-presenting cells such as dendritic cells and macrophages remain unclear. Based on these findings, it will be possible to generate earlier detection methods to identify the occurrence of immune alterations in silicosis as well as the appearance of a decreased antitumor immunity in asbestos-exposed populations. Additionally, research efforts should also be directed at discovering and identifying physiological substances from foods, plants, and other sources that can restore the immune status in people exposed to particulate and fibrous substances.

Two attempts to address health issues by indoor environment modifications are introduced. One approach involves enhancement of natural killer cell activity by negatively charged particle dominant indoor air conditions (NCPDIAC) resulting from extra-porous charcoal paint and application of an electric voltage on the wall surface to adsorb positively charged small particles (approx. 20 nm in diameter). This apparatus creates relatively continuous NCPDIAC. The other approach involves prevention of pollen allergy symptoms by a cloth containing specific ore powder (CCSNOP). With the former approach, we engaged in short-term (2.5 hour), middle-term (2 W night stay), and long-term (3 M) stays under NCPDIAC and found that IL-2 levels increased under short-term stays and that natural killer cell activity was enhanced in middle- and long-term stay experiments. Implementation of this strategy may partially prevent the occurrence of cancers and viral-mediated diseases. With the latter approach, a 1-hour stay within a CCSNOP room resulted in improvement of symptoms in patients with pollen allergies in addition to a reduction in bad moods caused by any remaining symptoms. In both cases, longer-term experiments should be performed in an effort to confirm and delineate the biological effects of these indoor environment modifications on human health problems.