Molecular Hydrogen as a Novel Protective Agent against Pre-Symptomatic Diseases.

Mibyou, or pre-symptomatic diseases, refers to state of health in which a disease is slowly developing within the body yet the symptoms are not apparent. Common examples of mibyou in modern medicine include inflammatory diseases that are caused by chronic inflammation. It is known that chronic inflammation is triggered by the uncontrolled release of proinflammatory cytokines by neutrophils and macrophages in the innate immune system. In a recent study, it was shown that molecular hydrogen (H2) has the ability to treat chronic inflammation by eliminating hydroxyl radicals (·OH), a mitochondrial reactive oxygen species (ROS). In doing so, H2 suppresses oxidative stress, which is implicated in several mechanisms at the root of chronic inflammation, including the activation of NLRP3 inflammasomes. This review explains these mechanisms by which H2 can suppress chronic inflammation and studies its applications as a protective agent against different inflammatory diseases in their pre-symptomatic state. While mibyou cannot be detected nor treated by modern medicine, H2 is able to suppress the pathogenesis of pre-symptomatic diseases, and thus exhibits prospects as a novel protective agent.

Potential Therapeutic Applications of Hydrogen in Chronic Inflammatory Diseases: Possible Inhibiting Role on Mitochondrial Stress.

Mitochondria are the largest source of reactive oxygen species (ROS) and are intracellular organelles that produce large amounts of the most potent hydroxyl radical (·OH). Molecular hydrogen (H2) can selectively eliminate ·OH generated inside of the mitochondria. Inflammation is induced by the release of proinflammatory cytokines produced by macrophages and neutrophils. However, an uncontrolled or exaggerated response often occurs, resulting in severe inflammation that can lead to acute or chronic inflammatory diseases. Recent studies have reported that ROS activate NLRP3 inflammasomes, and that this stimulation triggers the production of proinflammatory cytokines. It has been shown in literature that H2 can be based on the mechanisms that inhibit mitochondrial ROS. However, the ability for H2 to inhibit NLRP3 inflammasome activation via mitochondrial oxidation is poorly understood. In this review, we hypothesize a possible mechanism by which H2 inhibits mitochondrial oxidation. Medical applications of H2 may solve the problem of many chronic inflammation-based diseases, including coronavirus disease 2019 (COVID-19).

Molecular Hydrogen as a Potential Clinically Applicable Radioprotective Agent.

Although ionizing radiation (radiation) is commonly used for medical diagnosis and cancer treatment, radiation-induced damages cannot be avoided. Such damages can be classified into direct and indirect damages, caused by the direct absorption of radiation energy into DNA and by free radicals, such as hydroxyl radicals (•OH), generated in the process of water radiolysis. More specifically, radiation damage concerns not only direct damages to DNA, but also secondary damages to non-DNA targets, because low-dose radiation damage is mainly caused by these indirect effects. Molecular hydrogen (H2) has the potential to be a radioprotective agent because it can selectively scavenge •OH, a reactive oxygen species with strong oxidizing power. Animal experiments and clinical trials have reported that H2 exhibits a highly safe radioprotective effect. This paper reviews previously reported radioprotective effects of H2 and discusses the mechanisms of H2, not only as an antioxidant, but also in intracellular responses including anti-inflammation, anti-apoptosis, and the regulation of gene expression. In doing so, we demonstrate the prospects of H2 as a novel and clinically applicable radioprotective agent.

Molecular Hydrogen as a Novel Antitumor Agent: Possible Mechanisms Underlying Gene Expression.

While many antitumor drugs have yielded unsatisfactory therapeutic results, drugs are one of the most prevalent therapeutic measures for the treatment of cancer. The development of cancer largely results from mutations in nuclear DNA, as well as from those in mitochondrial DNA (mtDNA). Molecular hydrogen (H2), an inert molecule, can scavenge hydroxyl radicals (·OH), which are known to be the strongest oxidizing reactive oxygen species (ROS) in the body that causes these DNA mutations. It has been reported that H2 has no side effects, unlike conventional antitumor drugs, and that it is effective against many diseases caused by oxidative stress and chronic inflammation. Recently, there has been an increasing number of papers on the efficacy of H2 against cancer and its effects in mitigating the side effects of cancer treatment. In this review, we demonstrate the efficacy and safety of H2 as a novel antitumor agent and show that its mechanisms may not only involve the direct scavenging of ·OH, but also other indirect biological defense mechanisms via the regulation of gene expression.

A 45-year-old man with shortness of breath and eosinophilia.

A 45-year-old man who presented with dyspnea and chest tightness was found to have obstructive lung disease and eosinophilia of 10,300 eosinophils/µL. The differential diagnosis encompassed causes of primary eosinophilia and secondary eosinophilia associated with pulmonary disease, including asthma, environmental allergic reaction, eosinophilic granulomatosis with polyangiitis, allergic bronchopulmonary aspergillosis, acute eosinophilic pneumonia, chronic eosinophilic pneumonia, parasitic infections, tuberculosis, fungal infection, sarcoidosis, mastocytosis, drug reaction with eosinophilia and systemic symptoms, lymphoproliferative hypereosinophilic syndrome, and myeloproliferative hypereosinophilic syndrome. Infectious workup, fiberoptic bronchoscopy with biopsy, and tests for myeloproliferative mutations help differentiate among these causes. Identifying the underlying cause of eosinophilia is imperative in guiding treatment.

Salinomycin induces apoptosis and potentiates the antitumor effect of doxorubicin against feline mammary tumor 2.5D organoids.

Feline mammary tumors (FMT) are the third most common form of neoplasm in cats. The prognosis of FMT is poor due to its high malignancy and metastatic potential. The outcomes of treatment using the common anticancer drug doxorubicin (DOX) are unsatisfactory, with resistance inevitably leading to treatment failure and disease recurrence. Salinomycin (SAL), an antibiotic, has been reported to exert anticancer effects on both human and canine mammary tumors. To recapitulate the genetic and molecular imprints of the original tumor sample, we generated four strains of patient-derived FMT 2.5D organoids (FMTO) to examine the anti-tumor potential of SAL. Our results revealed that SAL decreased cell viability in a dose-dependent manner. Treatment of FMTO with SAL-induced cell apoptosis, represented by an upregulation of P21, Caspase-8, and Caspase-9, and increased activity of Caspase-3/7. The combination of low-dose SAL with DOX (SD) potentiated the cytotoxicity of the latter in both DOX-resistant and DOX-sensitive strains, promoting cell apoptosis and cell-cycle arrest. In vivo, experiments using FMTO-derived xenografts engrafted into mice revealed decreased tumor growth following SAL administration. In conclusion, SAL showed anticancer activity against FMTO and potentiated the anticancer effect of DOX by inhibiting cell proliferation and inducing apoptosis and cell cycle arrest. These results suggest that SAL may represent a new adjuvant treatment option for patients with FMT.

Comparative pharmacokinetics of tyrosine kinase inhibitor, lapatinib, in dogs and cats following single oral administration.

Lapatinib is an orally administered tyrosine kinase inhibitor used to treat human epidermal growth factor receptor 2 (HER2) -overexpressing breast cancers in humans. Recently, the potential of lapatinib treatment against canine urothelial carcinoma or feline mammary tumor was investigated. However, the pharmacokinetic studies of lapatinib in dogs and cats are not well-defined. In the present study, the pharmacokinetic characteristics of lapatinib in both cats and dogs after a single oral administration at a dose of 25 mg/kg were compared with each other. Lapatinib was administered orally to four female laboratory cats and four female beagle dogs. Blood samples were collected over time, and the plasma lapatinib concentrations were analyzed by HPLC. Following a single dose of 25 mg/kg, the averaged maximum plasma concentration (Cmax) of lapatinib in cats was 0.47 µg/mL and achieved at 7.1 hr post-administration, while the Cmax in dogs was 1.63 µg/mL and achieved at 9.5 hr post-administration. The mean elimination half-life was 6.5 hr in cats and 7.8 hr in dogs. The average area under the plasma concentration-time curve of dogs (37.2 hr·µg/mL) was significantly higher than that of cats (7.97 hr·µg/mL). These results exhibited slow absorptions of lapatinib in both animals after oral administration. The Cmax observed in cats was significantly lower and the half-life was shorter than those observed in dogs. Based on these results, a larger dose or shorter dosing intervals might be recommended in cats to achieve similar plasma concentration as dogs.

The overlooked benefits of hydrogen-producing bacteria.

Intestinal bacteria can be classified into "beneficial bacteria" and "harmful bacteria." However, it is difficult to explain the mechanisms that make "beneficial bacteria" truly beneficial to human health. This issue can be addressed by focusing on hydrogen-producing bacteria in the intestines. Although it is widely known that molecular hydrogen can react with hydroxyl radicals, generated in the mitochondria, to protect cells from oxidative stress, the beneficial effects of hydrogen are not fully pervasive because it is not generally thought to be metabolized in vivo. In recent years, it has become clear that there is a close relationship between the amount of hydrogen produced by intestinal bacteria and various diseases, and this report discusses this relationship.

Guidelines for the selection of hydrogen gas inhalers based on hydrogen explosion accidents.

Despite the fact that we have reported on the dangers of the explosion of hydrogen gas inhalers, hydrogen gas inhalers with explosive hazards are, as a matter of fact, still being sold today. In this study, we investigated past reports of hydrogen gas inhaler explosion accidents to clarify the causes of these explosion incidents. As a result of this investigation, we found that the central cause was the leakage of hydrogen gas inside the hydrogen gas inhaler. Although it is said that the explosive concentration of hydrogen is between 10% and 75%, and that the gas does not explode above 75% due to the lack of oxygen, we confirmed through a series of ignition experiments that explosions can occur even in hydrogen gas inhalers that produce 100% hydrogen gas. Some manufacturers of such highly concentrated hydrogen gas inhalers claim that the high concentration and purity of hydrogen is safe and that there is no risk of explosion. We believe that manufacturing or selling such products that pose a risk of explosion or detonation is a violation of social justice. This paper presents ideas for selecting safe hydrogen gas inhalers based on a survey of past accident cases.

Evaluation of the efficacy of mitochondrial fission inhibitor (Mdivi-1) using non-alcoholic steatohepatitis (NASH) liver organoids.

Non-alcoholic steatohepatitis (NASH) is known to progress to cirrhosis and hepatocellular carcinoma in some patients. Although NASH is associated with abnormal mitochondrial function related to lipid metabolism, mechanisms for the development and effective treatments are still unclear. Therefore, new approaches to elucidate the pathophysiology are needed. In the previous study, we generated liver organoids from different stages of NASH model mice that could recapitulate the part of NASH pathology. In the present study, we investigated the relationship between mitochondrial function and NASH disease by comparing NASH liver organoids (NLO) and control liver organoids (CLO). Compared with CLO, mitochondrial and organoid morphology was abnormal in NLO, with increased expression of mitochondrial mitogen protein, DRP1, and mitochondria-derived reactive oxygen species (ROS) production. Treatment of NLO with a DPR1 inhibitor, Mdivi-1 resulted in the improvement of morphology and the decreased expression of fibrosis-related markers, Col1a1 and Acta2. In addition, treatment of NASH model mice with Mdivi-1 showed a decrease in fatty liver. Mdivi-1 treatment also prevented fibrosis and ROS production in the liver. These results indicate that NLO undergoes enhanced metabolism and abnormal mitochondrial morphology compared with CLO. It was also suggested that Mdivi-1 may be useful as a therapeutic agent to ameliorate NASH pathology.

Anti-cancer activity of Chaga mushroom (Inonotus obliquus) against dog bladder cancer organoids.

Despite its disadvantages, chemotherapy is still commonly used for the treatment of bladder cancer (BC). Developing natural supplements that can target cancer stem cells (CSCs) which cause drug resistance and distant metastasis is necessary. Chaga mushrooms are popular to have several health-promoting and anti-cancer potentials. Organoid culture can recapitulate tumor heterogeneity, epithelial environment, and genetic and molecular imprints of the original tissues. In the previous study, we generated dog bladder cancer organoids (DBCO) as a novel experimental model of muscle-invasive BCO. Therefore, the present study aimed to examine the anti-tumor potentials of Chaga mushroom extract (Chaga) against DBCO. Four strains of DBCO were used in the present study. Treatment with Chaga inhibited the cell viability of DBCO in a concentration-dependent way. Treatment of DBCO with Chaga has significantly arrested its cell cycle and induced apoptosis. Expression of bladder CSC markers, CD44, C-MYC, SOX2, and YAP1, declined in the Chaga-treated DBCO. Also, Chaga inhibited the phosphorylation of ERK in DBCO. Expression of downstream signals of ERK, C-MYC, and Cyclins (Cyclin-A2, Cyclin-D1, Cyclin-E1, and CDK4) was also inhibited by Chaga in DBCO. Interestingly, the combinational treatment of DBCO with Chaga and anti-cancer drugs, vinblastine, mitoxantrone, or carboplatin, showed a potentiating activity. In vivo, Chaga administration decreased tumor growth and weight of DBCO-derived xenograft in mice with the induction of necrotic lesions. In conclusion, Chaga diminished the cell viability of DBCO by inhibiting proliferation-related signals and stemness conditions as well as by arresting the cell cycle. Collectively, these data suggest the value of Chaga as a promising natural supplement that could potentiate the effect of adjuvant chemotherapy, lower its adverse effects, and thus, limit the recurrence and metastasis of BC.

Oral pharmacokinetics of sulfadiazine and sulfamonomethoxine in female Holstein milking cows.

The efficacy of orally administered drugs in cattle is thought to be slow because of the anatomical and physiological features of their forestomach. Thus, parenteral routes are mainly preferred to administer drugs. However, the effect of some drugs with unique physicochemical properties was promptly obtained even after oral administration in clinically ill cattle. Therefore, the present study aimed to investigate pharmacokinetically the usefulness of the oral route in cattle by comparing the oral pharmacokinetic properties of two sulfonamides with different physicochemical properties. Sulfadiazine (SDZ) and sulfamonomethoxine (SMM) were administered by intravenous and oral route to four female Holstein cows with a 4-weeks washout period. Blood samples were collected over time, and SDZ and SMM concentrations in plasma were analyzed by HPLC. Data obtained from the same animal after intravenous and oral administration were simultaneously analyzed with the one compartment model, and kinetic parameters were calculated. The Tmax (mean ± SD) of SMM (2.75 ± 0.96 hr) was significantly achieved earlier than that of SDZ (5.00 ± 1.15 hr). Further, the mean absorption time of SMM (5.24 ± 0.69 hr) was significantly shorter than that of SDZ (5.92 ± 1.11 hr). Also, the half-life of absorption of SMM (3.91 ± 0.51 hr) was significantly shorter than that of SDZ (4.51 ± 0.82 hr). These data suggest that the absorption rates of highly unionized drugs (such as SMM) from the forestomach of cattle may be markedly higher than less unionized ones (such as SDZ).

Establishment of an experimental model of canine malignant mesothelioma organoid culture using a three-dimensional culture method.

Canine malignant mesothelioma (cMM) is a rare and drug-resistant malignant tumor. Due to few patients and experimental models, there have not been enough studies to demonstrate the pathogenesis of the disease and novel effective treatment for cMM. Since cMM resembles human MM (hMM) in histopathological characteristics, it is also considered a promising research model of hMM. Compared with conventional 2-dimensional (2D) culture methods, 3-dimensional (3D) organoid culture can recapitulate the properties of original tumor tissues. However, cMM organoids have never been developed. In the present study, we for the first time generated cMM organoids using the pleural effusion samples. Organoids from individual MM dogs were successfully generated. They exhibited the characteristics of MM and expressed mesothelial cell markers, such as WT-1 and mesothelin. The sensitivity to anti-cancer drugs was different in each strain of cMM organoids. RNA sequencing analysis showed cell adhesion molecule pathways were specifically upregulated in cMM organoids compared with their corresponding 2D cultured cells. Among these genes, the expression level of E-cadherin was drastically higher in the organoids than that in the 2D cells. In conclusion, our established cMM organoids might become a new experimental tool to provide new insights into canine and human MM therapy.

Derivation of a new model of lung adenocarcinoma using canine lung cancer organoids for translational research in pulmonary medicine.

Canine primary lung cancer (cPLC) is a rare malignant tumor in dogs, and exhibits poor prognosis. Effective therapeutic drugs against cPLC have not been established yet. Also, cPLC resembles human lung cancer in histopathological characteristics and gene expression profiles and thus could be an important research model for this disease. Three-dimensional organoid culture is known to recapitulate the tissue dynamics in vivo. We, therefore, tried to generate cPLC organoids (cPLCO) for analyzing the profiles of cPLC. After samples from cPLC and the corresponding normal lung tissue were collected, cPLCO were successfully generated, which recapitulated the tissue architecture of cPLC, expressed lung adenocarcinoma marker (TTF1), and exhibited tumorigenesis in vivo. The sensitivity of cPLCO to anti-cancer drugs was different among strains. RNA-sequencing analysis showed significantly upregulated 11 genes in cPLCO compared with canine normal lung organoids (cNLO). Moreover, cPLCO were enriched with the MEK-signaling pathway compared with cNLO. The MEK inhibitor, trametinib decreased the viability of several strains of cPLCO and inhibited the growth of cPLC xenografts. Collectively, our established cPLCO model might be a useful tool for identifying novel biomarkers for cPLC and a new research model for dog and human lung cancer.

Effect of the liquid form of traditional Chinese medicine, Hozen-S, on gastric motility in dogs.

Juzen-taiho-to, a traditional Chinese herbal medicine, is used for patients with anorexia and fatigue in human medicine. In our previous study, granulated Juzen-taiho-to improved vincristine-induced gastrointestinal adverse effects through increasing gastric motility in dogs. As the effect of Hozen-S, the sweet liquid form of Juzen-taiho-to, on dog gastric motility has not been investigated, we examined the effect of administration of Hozen-S on gastric motility. Furthermore, we assessed dog plasma ghrelin level to further elucidate the mechanism of the effect of Hozen-S on gastric contraction. Finally, we assessed the palatability of Hozen-S compared to granulated Juzen-taiho-to and its effect on body weight in dogs. Administration of Hozen-S significantly increased gastric motility, plasma ghrelin concentration, and body weight. A palatability evaluation revealed that the dogs preferred Hozen-S to granulated Juzen-taiho-to. In conclusion, Hozen-S administration to dogs promoted gastric motility by raising plasma ghrelin levels. Considering these functional and palatability data, Hozen-S may replace granulated type Juzen-taiho-to and become a prominent traditional Chinese veterinary medicament.

Establishment of a direct 2.5D organoid culture model using companion animal cancer tissues.

Like humans, cancer affects companion animals with similar genetic risks and incident rates. To improve treatment strategies for pet cancers, new research models are necessary. Patient-derived 3D organoid culture models are valuable and ensure the development of new effective therapies. In the previous study, we established a 3D organoid-derived 2.5D organoid culture model that recapitulated some characteristics of their parental 3D organoids. In the present study, we aimed to generate a 2.5D organoid culture model directly from cancer-diseased dogs and cats using special 2.5D media. The primary cultured cells in 2.5D media (direct 2.5D organoids) showed better attachment, growth, marker expression, and faster proliferation speed than those cultured in normal Dulbecco's Modified Eagle Medium media. The direct 2.5D organoids showed expression of each specific marker to their original cancer tissues and exhibited tumorigenesis in vivo. Moreover, the direct 2.5D organoids exhibited concentration-dependent responses to anti-cancer drugs, and different sensitivity profiles were shown among the strains. Our data suggest that the direct 2.5D organoid culture model might become a useful tool beyond 2D cell lines to study cancer biology in companion animals and could provide new platforms for screening the anti-cancer drugs.

Establishment of an experimental model of normal dog bladder organoid using a three-dimensional culture method.

Dog bladder cancer (BC) is mostly muscle-invasive (MI) with poor prognosis, and its pathogenesis is close to human MIBC. Three-dimensional (3D) organoid culture ensures novel knowledge on cancer diseases including BC. Recently, we have established dog BC organoids (BCO) using their urine samples. BCO recapitulated the epithelial structures, characteristics, and drug sensitivity of BC-diseased dogs. However, organoids from dog normal bladder epithelium are not established yet. Therefore, the present study aimed to establish dog normal bladder organoids (NBO) for further understanding the pathogenesis of dog BC and human MIBC. The established NBO underwent various analyzes including cell marker expressions, histopathological structures, cancer-related gene expression patterns, and drug sensitivity. NBO could be produced non-invasively with a continuous culturing and recapitulated the structures and characteristics of the dog's normal bladder mucosal tissues. Different drug sensitivities were observed in each NBO. The analysis of RNA sequencing revealed that several novel genes were changed in NBO compared with BCO. NBO showed a higher expression of p53 and E-cadherin, but a lower expression of MDM2 and Twist1 compared with BCO. These results suggest that NBO could be a promising experimental 3D model for studying the developmental mechanisms of dog BC and human MIBC.

[Development of liver organoids from NASH model mice and their application to drug discovery].

Nonalcoholic steatohepatitis (NASH) is one of the most common causes of chronic liver disease, with the increased prevalence of obesity, type 2 diabetes, and metabolic disorders in recent years. As the disease progresses, it leads to hepatic fibrosis, which may progress to hepatocellular carcinoma, but there is still no cure for severe hepatic fibrosis. Currently, in order to develop drugs for the treatment of NASH, the effects of candidate drugs are evaluated by a long-term administration to mice and rats that are fed a high-fat or methionine-deficient diet to reproduce the pathology of fatty liver and liver fibrosis. Since drug development using these experimental animals is time-consuming and costly, in vitro models that reproduce the pathology of NASH have recently been developing. In this review, we will outline the current issues in the diagnosis and treatment of NASH, and introduce our research for the discovery of early diagnostic markers and the development of new therapeutic agents using liver organoid cultures derived from mouse models of NASH.

Evaluation of the Safety and Feasibility of Apheresis in Dogs: For Application in Metastatic Cancer Research.

In patients with solid tumors, circulating tumor cells (CTCs) spread in their blood and function as a seed for metastases. However, the study of CTCs has been limited by their rarity, low frequency, and heterogeneity. The efficient collection of CTCs will contribute to further research of metastatic cancers. Apheresis is a process in which the whole blood of an individual is passed through a machine that isolates a particular constituent and returns the remainder to the circulation. In the present study, we investigated the safety and feasibility of apheresis to separate peripheral blood monocytes (PBMCs), whose density is closely similar to that of CTCs, and to capture intravenously administered human breast cancer cells, MCF7s, from the dogs. No life-threatening events were observed in dogs during the apheresis process. The changes in the hemogram were transient and recovered gradually within a few days after apheresis. During apheresis, 50 mL of PBMCs could be collected from each dog. Notably, a thrombus was formed along the circuit wall during apheresis, which decreased the blood collection pressure. MCF7 cells were successfully captured by the apheresis machine. The captured cells were regrown in vitro and characterized compared with the original cells. In conclusion, apheresis could be safely performed in dogs to isolate CTCs with precautions to maintain hemodynamic stability.

Discontinuing VEGF-targeted Therapy for Progression Versus Toxicity Affects Outcomes of Second-line Therapies in Metastatic Renal Cell Carcinoma.

BACKGROUND: A significant subgroup of metastatic renal cell carcinoma (mRCC) patients discontinue vascular endothelial growth factor-targeted therapies (VEGF-TT) because of toxicity. Whether clinical outcomes differ in patients who receive second-line (2L) targeted therapy on the basis of reason for discontinuation of first-line (1L) therapy is unknown. PATIENTS AND METHODS: Patients from 15 International Metastatic Renal Cell Carcinoma Database Consortium (IMDC) centers who started 2L targeted therapy were included and the reason for discontinuation of 1L therapy retrospectively collected. Treatment outcomes of 2L, including response, time to treatment failure, and overall survival (OS) were assessed. RESULTS: In total, 1124 patients were identified: 866 patients (77%) discontinued 1L VEGF-TT because of disease progression, and 208 patients (19%) because of toxicity. The reason for discontinuation of 1L therapy did not differ according to IMDC risk group. Compared with patients who stopped 1L VEGF-TT because of disease progression, patients who stopped because of toxicity had greater clinical benefit (nonprogressive disease as best response) in 2L treatment (68% vs. 56%; adjusted odds ratio, 1.58; 95% confidence interval [CI], 1.07-2.35; P = .023) and longer OS (17.4 vs. 11.2 months; adjusted hazard ratio, 0.69; 95% CI, 0.56-0.84; P = .0002) adjusted for type of therapy, time to initiation of 2L treatment, IMDC risk group, and number of metastases at initiation of 2L treatment. CONCLUSION: mRCC patients who discontinue 1L VEGF-TT because of toxicity have better outcomes with 2L therapy than patients who stop therapy because of disease progression. These findings should be taken into consideration when designing clinical trials for 2L therapies in mRCC.