OBJECTIVES:This study investigated the impact of Janus kinase inhibitors (JAKi) on NK cell activity and phenotype in patients with RA. While JAKi are effective in achieving low disease activity (LDA), preclinical and pharmacokinetic studies suggest potential effects on NK cells, raising concerns about infection and malignancy risks. However, the extent of these effects in RA patients remains unclear. METHODS:We assessed the cytotoxic activity of peripheral blood NK cells in RA patients with LDA who had been treated with JAKi (n = 85) or TNF inhibitors (TNFi) (n = 131) for more than three months using a 51Cr-release assay. Propensity score matching was used to adjust for confounders, and multivariable logistic regression identified risk factors for low NK cell activity. NK cell maturation and differentiation were evaluated via flow cytometry. RESULTS:NK cell activity was significantly lower in the JAKi group compared with the TNFi group (median 50% vs 36%, P = 0.002). The proportion of NK and CD56dim cells was reduced, while CD56bright cells were more abundant in the JAKi group. In the JAKi group, glucocorticoid use (odds ratio [OR] 5.36, 95% CI 1.10-26.07, P = 0.04) and younger age (<50 years; OR 10.56, 95% CI 2.69-41.45, P < 0.01) were independent risk factors for reduced NK cell activity. Notably, herpes zoster incidence was significantly higher in the low NK cell activity group among RA patients younger than 50 years. CONCLUSION:JAKi treatment significantly impaired NK cell activity and differentiation in RA patients, with age influencing these effects, underscoring important safety concerns.

Differentiation of the human monocytic leukemia cell line THP-1 is widely used to analyze the function of monocyte/macrophage-like cells in vitro. Although chemotaxis, a critical function of monocytes/macrophages enabling tissue accumulation, has been extensively studied, methods to evaluate sustained, long-distance chemotaxis remain underexplored. Therefore, we aimed to evaluate macrophage-like cells in vitro by differentiating THP-1 cells into monocyte/macrophage-like cells exhibiting sustained, strong chemotaxis over long distances (up to 260 μm). Using various reagents, we identified the combination of vitamin D, panobinostat, and granulocyte–macrophage-colony-stimulating factor as optimal for achieving high directionality and velocity in cell migration, as analyzed using the TAXIScan cell dynamics assay device. The differentiated cells matured into M1 macrophage-like cells and displayed reduced migratory capacity post-maturation, along with enhanced phagocytosis and reactive oxygen species production. Collectively, our differentiation and analysis methods provide a reliable platform for basic research into cellular maturation processes and drug development targeting the regulation of monocyte/macrophage dynamics.

BACKGROUND:According to the gene-environment interactions concept, the mechanism of health impairment can be explained by genetic factors, environmental factors, or their interaction. Physical and mental health effects resulting from environmental exposure may be classified either as toxicity, immune response, and allergic reaction. Moreover, despite the already established therapeutic approaches to bronchial asthma and decreasing mortality due to bronchial asthma, patients with difficult and severe asthma are increasing in number. This review outlines recent topics in the field of allergies, focusing on asthma. RESULTS AND DISCUSSION:Living environment-derived pollutants and their involvement in the pathogenesis of asthma and its exacerbation, referred to here as an exposome concept, comprises the three domains of internal, specific external, and general external. Living environment-derived pollutants include exposure to pollutants in workplaces, climate change, air pollution, microplastics, tobacco smoke, biodiversity change and loss, changing dietary habits, and the microbiome. These are associated with the modernization, urbanization, and globalization of human society. Although many novel compounds are currently available, their harmful health effects, such as allergy, are not thoroughly understood. Hence, the means to mitigate these are unknown. Dietary changes from a traditional diet rich in fish to a Western-style diet are considered critical environmental factors and therefore, associated with an increased prevalence of allergies. Cytokines, including thymic stromal lymphopoietin, IL-25, and IL-33, released from the airway epithelium in response to various triggers (exposure to diverse environmental factors) are known as alarmins. Anti-alarmin antibodies are a promising therapeutic approach against severe and difficult allergic disorders. Collaboration between hospitals and clinics and occupational and clinical medicine is imperative for treating and managing severe asthma. In addition to avoiding environmental exposure, understanding the pathogenesis and exacerbation of asthma is essential for future research in the field of allergy and immunotoxicology.

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.

Genetic and environmental factors and their interactions cause diseases and deteriorate health (Genetic and Environmental Interaction). Exposure to environmental factors plays a major role in the deterioration of health in the workplace.Occupational asthma (OA) is a common disorder in the workplace. Approaches to OA are well described and discussed in "Japanese Guideline for Diagnosis and Management of Occupational Allergic Diseases" by the Japanese Society of Occupational and Environmental Allergy. According to the guideline, OA and work-aggravated asthma comprise work-related asthma, and OA can be further divided into two disease entities: sensitizer-induced OA and irritant-induced OA. The guidelines also describe diagnostic and therapeutic strategies for OA. Since a definitive diagnosis of OA requires a comprehensive decision based on a detailed interview on clinical symptoms related to employment status and clinical tests, including inhalation tests of suspected substances as needed, the possibility of OA should be considered as the first step toward diagnosis of the patient. Otherwise, OA may not be diagnosed. Therapeutic strategies include exposure avoidance, environmental arrangements in the workplace, utilization of social resources for workers, and conventional pharmacotherapy for asthma.Artificially synthesized small compounds are used in various industries and can cause allergies. For example, isocyanates are small compounds in the -NCO group, which have been toxicologically studied. It was later shown that isocyanate could cause various nontoxic adverse health effects, including allergic reactions. Since small agents with low molecular weights bind to proteins, detecting their specific immunoglobulin E (IgE) antibodies targeting small compounds is generally difficult. In contrast, isocyanate-specific IgE antibodies are detectable in individuals with isocyanate allergies.Suspecting OA is essential in cases exposed to newly synthesized compounds, or to those that are already known but applied to new uses, which can be better understood and predicted by studying the health effects of isocyanates.Academic interest in various issues related to allergies, immunology, and toxicology in the workplace includes clinical medicine, epidemiology, and epigenetics related to environmental exposure. Further advanced research in these areas is necessary and promising.

Asbestos is naturally occurring mineral fiber and one of representative harmful and tumorigenic materials. Occupational or environmental exposure to asbestos is still a global issue to cause asbestos-related diseases (ARD), in particular malignant mesothelioma is a serious disease with poor prognosis. It takes a long period of about 40 years to develop mesothelioma after inhalation exposure to asbestos, which is known to accumulate in regional lymph nodes. Those clues motivated us to examine immunological effects of asbestos exposure by cull culture experiments, omics data and broad analyses for peripheral blood of patients with mesothelioma. Our studies clarified several key events of asbestos-caused functional impairment in NK, helper T (Th) and cytotoxic T cells (CTL) as well as augmentation in regulatory T cells, associated with decrease in NKp46, IFN-gamma, CXCR3 and perforin and increase in CTLA-4, which were also observed in patients with malignant mesothelioma. In addition, it was also found that diffuse pleural thickening, a benign type of ARD, showed better characteristics of anti-tumor immunity compared with malignant mesothelioma, including high expression of perforin in CTL and low expression of GITR on cell surface of Treg. Those findings indicate that asbestos exposure has a potential to cause suppressed immune functions, which might contribute to the pathogenesis of malignant mesothelioma together with directly tumorigenic effect of asbestos on mesothelial cells. On the basis of our obtained knowledge, immunological scoring devices with mathematical formulae were also developed, in which M-score successfully distinguished patients with mesothelioma from other people. Moreover, our recent study about transcriptome of human cytotoxic T cell line demonstrated that continuous exposure to asbestos causes gradual decline in IFN-gamma expression and shared alteration in expression of transcripts among the types of asbestos fiber. All of our obtained findings together with more those in future will contribute to early diagnosis of and therapeutical strategy for mesothelioma in people exposed to asbestos.

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.

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.

The COVID-19 pandemic caused by the SARS-CoV-2 epidemic now requires the deployment of fast, sensitive and inexpensive diagnostic methods to facilitate disease management and containment worldwide. The real-time reverse transcription-polymerase chain reaction (rRT-PCR) assay is currently the standard method for detecting SARS-CoV-2 virus. However, lack of access to laboratory resources (including rRT-PCR instrumentation, reagents, or even trained laboratory technicians) limits rRT-PCR assays from being a field-deployable and rapid diagnostic tool, especially in resource-constrained pandemic zones. Given this limitation and the necessity of expediting the diagnostic procedures, there is a strong need to develop an accurate point-of-care (POC) testing platform for COVID-19 with quick turnaround times. A POC community-level quick test (used as an initial screening test before subsequent confirmation by rRT-PCR in a central laboratory) can significantly improve diagnostic utility and ultimately reduce the burden on medical resources. CRISPR-Cas technology has now surfaced as a promising diagnostic tool with rewarding prospects of rapid detection and low cost, thereby serving as a POC one-stop platform to detect COVID 19 infection and loads. Herein, we review various hitherto CRISPR-Cas-based methodologies for the detection of SARS-CoV-2 viral RNA.

Indoor air environments may affect humans in various ways such as their immune, neurological and psychological states. We have investigated and developed two methods to improve indoor air environments that result in enhancement of immune power and improvement of outcomes associated with pollen allergy that affects immune cells. One method involves the use of a negatively charged particle-dominant indoor air conditioner (NCPDIAC), while the other method involves use of a cloth containing a special natural ore powder (CCSNOP). We briefly explain these two devices and discuss their use in a world where viruses are widespread.

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.