Alleviation of the chronic stress response attributed to the antioxidant and anti-inflammatory effects of electrolyzed hydrogen water.

Reactive oxygen species (ROS) are highly reactive and directly attack surrounding biomolecules to deteriorate cellular and tissue functions. Meanwhile, ROS also serve as signaling mediators to upregulate pro-inflammatory cytokine expression via activation of the nuclear factor kappa B signaling pathway, and the increased pro-inflammatory cytokines trigger respiratory burst of inflammatory cells that further accelerates ROS production in the inflamed tissue. Such crosstalk between ROS and inflammatory responses leads to a chain reaction of negativity, and cause progression of several chronic pathologies. Since molecular hydrogen is known to preferentially remove cytotoxic hydroxyl radicals and peroxynitrites, and to prevent cell and tissue damage, we here examined whether electrolyzed hydrogen water (EHW) enriched with molecular hydrogen and reactive hydrogen storing platinum nanoparticles dissolved from an electrode could alleviate oxidative stress and inflammation induced by continuous stress challenges. Five-day continuous stress loading to rats elevated reactive oxygen metabolites-derived compounds (d-ROMs), interleukin (IL)-1ß, and adrenocorticotropic hormone (ACTH) levels and decreased the biological antioxidant potential (BAP) level. Drinking EHW during 5-day continuous stress loading significantly alleviated all of these changes. The results suggest that EHW could suppress stress-response-associated oxidative stress and IL-1ß level elevation in vivo, and that drinking of EHW is effective for controlling stress responses via its antioxidant potential.

Dissolved molecular hydrogen (H2) in Peritoneal Dialysis (PD) solutions preserves mesothelial cells and peritoneal membrane integrity.

BACKGROUND: Peritoneal dialysis (PD) is used as renal replacement therapy in patients with end-stage kidney disease. However, peritoneal membrane failure remains problematic and constitutes a critical cause of PD discontinuation. Recent studies have revealed the unique biological action of molecular hydrogen (H2) as an anti-oxidant, which ameliorates tissue injury. In the present study, we aimed to examine the effects of H2 on the peritoneal membrane of experimental PD rats. METHOD: Eight-week-old male Sprague-Dawley rats were divided into the following groups (n = 8-11 each) receiving different test solutions: control group (no treatment), PD group (commercially available lactate-based neutral 2.5% glucose PD solution), and H2PD group (PD solution with dissolved H2 at 400 ppb). Furthermore, the influence of iron (FeCl3: 5 µM: inducer of oxidative cellular injury) in the respective PD solutions was also examined (Fe-PD and Fe-H2PD groups). The H2PD solution was manufactured by bathing a PD bag in H2-oversaturated water created by electrolysis of the water. Twenty mL of the test solutions were intraperitoneally injected once a day for 10 days. Parietal peritoneum samples and cells collected from the peritoneal surface following treatment with trypsin were subjected to analysis. RESULTS: In the PD group as compared to controls, a mild but significant sub-mesothelial thickening was observed, with increase in the number of cells in the peritoneal surface tissue that were positive for apoptosis, proliferation and vimentin, as seen by immunostaining. There were significantly fewer of such changes in the H2PD group, in which there was a dominant presence of M2 (CD163+) macrophages in the peritoneum. The Fe-PD group showed a significant loss of mesothelial cells with sub-mesothelial thickening, these changes being ameliorated in the Fe-H2PD group. CONCLUSION: H2-dissolved PD solutions could preserve mesothelial cells and peritoneal membrane integrity in PD rats. Clinical application of H2 in PD could be a novel strategy for protection of peritoneal tissue during PD treatment.

Elucidation of the etiology and characteristics of pink urine in young healthy subjects.

BACKGROUND: Pink urine syndrome (PUS) is attributed to the precipitation of uric acid caused by low urinary pH (U-pH). However, the reasons for the lower U-pH are unclear. OBJECTIVES: To investigate the occurrence of PUS and verified the cause of U-pH reduction. METHODS: Participants comprised 4,940 students who had undergone a physical examination. Data on the presence [PUS (+)] or absence [PUS (-)] of PUS, as well as age, gender, body mass index (BMI), blood pressure (BP), heart rate (HR), and U-pH were collected. Of these participants, 300 randomly selected individuals were evaluated for their waist circumference, as well as their levels of urinary C-peptide, angiotensinogen, methylglyoxal, thiobarbituric acid-reactive substances (TBARS), and Na(+) excretion. Independent risk factors of lower U-pH were decided by a multiple-regression analysis. RESULTS: PUS was observed in 216 students (4.4 %). A greater number of men comprised the PUS (+) group compared with the PUS (-) group, and subjects in this group had high BMI, BP, and HR values, as well as low U-pH. A logistic regression analysis revealed that the BMI and U-pH were independent risk factors for PUS (+). The decrease of U-pH was closely related to the progress of chronic kidney disease (CKD). BMI value was related to PUS (+) in the CKD (-) subjects. On the other hand, low U-pH was related to PUS (+) in the CKD (+) subjects. All factors other than HR showed a significant negative correlation with U-pH. However, multiple-regression analysis revealed that TBARS and angiotensinogen were independent risk factors. CONCLUSION: Obesity and lower U-pH were each independently related to PUS, whereas increased intrarenal oxidative stress and exacerbation of the renin-angiotensin system activation were associated with the lowering of U-pH. U-pH low value is related to potential CKD.

Application of Electrolyzed Hydrogen Water for Management of Chronic Kidney Disease and Dialysis Treatment-Perspective View.

Chronic kidney disease (CKD), which is globally on the rise, has become an urgent challenge from the perspective of public health, given its risk factors such as end-stage renal failure, cardiovascular diseases, and infections. The pathophysiology of CKD, including dialysis patients, is deeply associated with enhanced oxidative stress in both the kidneys and the entire body. Therefore, the introduction of a safe and widely applicable antioxidant therapy is expected as a measure against CKD. Electrolyzed hydrogen water (EHW) generated through the electrolysis of water has been confirmed to possess chemical antioxidant capabilities. In Japan, devices producing this water have become popular for household drinking water. In CKD model experiments conducted to date, drinking EHW has been shown to suppress the progression of kidney damage related to hypertension. Furthermore, clinical studies have reported that systemic oxidative stress in patients undergoing dialysis treatment using EHW is suppressed, leading to a reduction in the incidence of cardiovascular complications. In the future, considering EHW as one of the comprehensive measures against CKD holds significant importance. The medical utility of EHW is believed to be substantial, and further investigation is warranted.

Health Benefits of Electrolyzed Hydrogen Water: Antioxidant and Anti-Inflammatory Effects in Living Organisms.

Molecular hydrogen, the smallest and lightest molecule, serves as an intense reducing agent. Its distinct characteristics, including minimal size and neutral charge, enhance bioavailability and facilitate significant biological effects. Previously considered physiologically inert, hydrogen has gained recognition as a powerful therapeutic agent, known for its antioxidative and anti-inflammatory properties. Electrolyzed hydrogen water (EHW), enriched with molecular hydrogen, demonstrates remarkable antioxidative capabilities, indicating potential benefits for various diseases. Inflammation-induced reactive oxygen species (ROS) amplify inflammation, leading to secondary oxidative stress and creating a crosstalk between ROS and inflammatory responses. This crosstalk contributes to the pathogenesis and progression of chronic diseases. EHW interrupts this crosstalk, reducing inflammatory cytokines and oxidative stress across various disease models, suggesting therapeutic potential. EHW is also known for its anti-inflammatory effects, extending to pain management, as evidenced in models like sciatic nerve ligation and inflammatory pain. In an inflammatory bowel disease (IBD) model, EHW effectively alleviates abdominal pain, mitigating 2,4,6-trinitrobenzene sulfonic acid (TNBS)-induced inflammation and oxidative stress, offering insights for clinical applications. Additionally, hydrogen selectively targets harmful radicals, and EHW intake helps balance stress-induced hormonal dysregulation, potentially easing disorders associated with chronic stress.

Health Effects of Electrolyzed Hydrogen Water for the Metabolic Syndrome and Pre-Metabolic Syndrome: A 3-Month Randomized Controlled Trial and Subsequent Analyses.

We studied the effect of three months' use of electrolyzed hydrogen water (EHW, Electrolyzed Hydrogen Water conditioner produced by Nihon Trim Co., Ltd.) on metabolic and pre-metabolic syndrome groups. This research was carried out jointly by Susaki City; Nihon Trim Co., Ltd.; and Kochi University as part of a local revitalization project with health as a keyword. A randomized, placebo-controlled, double-blind, parallel-group trial was conducted to evaluate the clinical impact of EHW on participants who suffered from metabolic syndrome or pre-metabolic syndrome. EHW was produced via electrolysis using a commercially available apparatus (Nihon Trim Co., Ltd., Osaka, Japan). During exercise, oxidative stress increases, and active oxygen species increase. In this study, we examined 181 subjects with metabolic syndrome or pre-metabolic syndrome. Among the group that drank EHW for 3 months, those who also engaged in a high level of physical activity showed a significant difference in waist circumference reduction. Although no significant difference was observed, several positive results were found in the participants who engaged in a high level of physical activity. Urinary 8-OHdG, urinary nitrotyrosine, HbA1c, and blood glucose levels increased in the filtered water (FW) group but decreased in the EHW group. High-sensitivity CRP increased less in the EHW group. 8-Isoprostane decreased more in the EHW group. In subgroup analysis, the EHW group showed a significantly greater reduction in waist circumference than the FW group only when controlled for high physical activity. Based on the result, we suggest that, among participants in the study who suffered from metabolic syndrome and pre-metabolic syndrome in which the level of active oxygen species is said to be higher than in healthy subjects, the group that consumed EHW and also engaged in a high level of physical activity experienced a suppressed or reduced increase in active oxygen species.

A rapid screening and production method using a novel mammalian cell display to isolate human monoclonal antibodies.

Antibody display methods are increasingly being used to produce human monoclonal antibodies for disease therapy. Rapid screening and isolation of specific human antibody genes are valuable for producing human monoclonal antibodies showing high specificity and affinity. In this report, we describe a novel mammalian cell display method in which whole human IgG is displayed on the cell surface of CHO cells. Cells expressing antigen-specific human monoclonal IgGs with high affinity on the cell surface after normal folding and posttranscriptional modification were screened using a cell sorter. The membrane-type IgG-expressing CHO cells were then converted to IgG-secreting cells by transfection with a plasmid coding Cre recombinase. This mammalian cell display method was applied to in vitro affinity maturation of monoclonal C9 IgG specific to the human high-affinity IgE receptor (FcεRIα). The CDR3 of the C9 heavy chain variable region gene was randomly mutated and inserted into pcDNA5FRT/IgG. A C9 IgG (CDRH3r)-expressing CHO cell display library consisting of 1.1×10(6) independent clones was constructed. IgG-displaying cells showing high reactivity to FcεRIα antigen were screened by the cell sorter, resulting in the establishment of a CHO cell line producing with higher reactivity than the parent C9 IgG.

A new colorimetric method for determining antioxidant levels using 3,5-dibromo-4-nitrosobenzene sulfonate (DBNBS).

We describe here a novel assay that determines the total a+ntioxidative activities of known antioxidants and antioxidants in beverages. The method employs the substrate 3,5-dibromo-4-nitrosobenzene sulfonate (DBNBS) that yields the colored product 3,5,3',5'-tetrabromoazobenzene sulfate sodium salt (azo-TBBS). The amounts of azo-TBBS are measured using HPLC and then used to calculate total antioxidative capacity (TAC) values. We first show that the TAC values measured using the new DBNBS system were significantly higher compared with the control. The assay was validated through further analysis of 56 compounds, including previously characterized antioxidants. The data are consistent with published values. Here we describe in detail the application of the DBNBS method to the measurement of the TAC values of eight beverages, including wines and fruit juices. The DBNBS assay employs a readily applicable protocol that sensitively determines the levels of antioxidants in foodstuffs. - A new DBNBS-mediated antioxidant assay system is compared with standard DPPH and ORAC assays - DBNBS traps hydrogen radicals to generate a readily measured colored reduction product that quantifies antioxidant levels.

Electrolyzed Hydrogen Water Alleviates Abdominal Pain through Suppression of Colonic Tissue Inflammation in a Rat Model of Inflammatory Bowel Disease.

Inflammatory bowel disease (IBD) is characterized by chronic inflammation of the digestive tract and is typically accompanied by characteristic symptoms, such as abdominal pain, diarrhea, and bloody stool, severely deteriorating the quality of the patient's life. Electrolyzed hydrogen water (EHW) has been shown to alleviate inflammation in several diseases, such as renal disease and polymyositis/dermatomyositis. To investigate whether and how daily EHW consumption alleviates abdominal pain, the most common symptom of IBD, we examined the antioxidative and anti-inflammatory effects of EHW in an IBD rat model, wherein colonic inflammation was induced by colorectal administration of 2,4,6-trinitrobenzene sulfonic acid (TNBS). We found that EHW significantly alleviated TNBS-induced abdominal pain and tissue inflammation. Moreover, the production of proinflammatory cytokines in inflamed colon tissue was also decreased significantly. Meanwhile, the overproduction of reactive oxygen species (ROS), which is intricately involved in intestinal inflammation, was significantly suppressed by EHW. Additionally, expression of S100A9, an inflammatory biomarker of IBD, was significantly suppressed by EHW. These results suggest that the EHW prevented the overproduction of ROS due to its powerful free-radical scavenging ability and blocked the crosstalk between oxidative stress and inflammation, thereby suppressing colonic inflammation and alleviating abdominal pain.

Electrolyzed hydrogen-rich water for oxidative stress suppression and improvement of insulin resistance: a multicenter prospective double-blind randomized control trial.

INTRODUCTION: Electrolyzed hydrogen-rich water (EHW) is known to have suppressive effects on oxidative stress (OS). However, its benefit in type 2 diabetes mellitus (T2DM) remains unclear. This study aimed to investigate the effect of EHW on T2DM. METHODS: This was a multicenter, prospective, double-blind, randomized controlled trial of 50 patients with T2DM who were assigned to the EHW or filtered water (FW) groups. The primary endpoint was changes in insulin resistance (IR) evaluated using the homeostasis model assessment of insulin resistance (HOMA-IR). OS markers such as urinary 8-hydroxy-2'-deoxyguanosine excretion (8-OHdG), plasma diacron-reactive oxygen metabolites (d-ROM), and plasma biological antioxidant potential (BAP) and other clinical data, including serum lactate concentration (lactate), were evaluated. RESULTS: There were no significant differences in the changes in HOMA-IR between the EHW and FW groups. However, lactate levels decreased significantly in the EHW group, and this decrease was significantly correlated with a reduction in HOMA-IR, fasting plasma glucose, and fasting plasma insulin level. Serum lactate level also significantly correlated to decreased insulin bolus secretion after 90 min with glucose loading in the EHW subjects with HOMA-IR > 1.73. No EHW treatment-related adverse effects were observed. CONCLUSION: There were no significant effect of EHW in the change in HOMA-IR in this study; larger-scale and longer-term study are needed to verify the effects of EHW in T2DM patients. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1007/s13340-021-00524-3.

Antioxidant effects of continuous intake of electrolyzed hydrogen water in healthy adults.

Chronic oxidative stress induces deterioration of health and a risk for the onset of various diseases. Previous clinical studies revealed that electrolyzed hydrogen water (EHW) is effective to reduce oxidative stress during hemodialysis in patients with chronic dialysis. In the present observational study, we investigated the antioxidant effects of a daily continuous intake of EHW in healthy adults. The concentrations of serum reactive oxygen metabolites-derived compounds (d-ROMs) and blood urea nitrogen in healthy volunteers (n = 64) who had a habit of intake over 500 mL/day of EHW at least 5 days a week for longer than 6 months were lower than those of age- and sex-matched controls (n = 470) without the habit of EHW intake. Oxidation stress index which the ratio between concentrations in d-ROMs and biological antioxidant potential was correlated with the serum concentration of high-sensitivity C-reactive protein or low-density lipoprotein cholesterol in the EHW group. These results suggest that the continuous intake of EHW induces antioxidant effects and may contribute to alleviate the risk of various oxidative stress-related dysfunctions and diseases in healthy adults.

Intake of water with high levels of dissolved hydrogen (H2) suppresses ischemia-induced cardio-renal injury in Dahl salt-sensitive rats.

BACKGROUND: Hydrogen (H(2)) reportedly produces an antioxidative effect by quenching cytotoxic oxygen radicals. We studied the biological effects of water with dissolved H(2) on ischemia-induced cardio-renal injury in a rat model of chronic kidney disease (CKD). METHODS: Dahl salt-sensitive rats (7 weeks old) were allowed ad libitum drinking of filtered water (FW: dissolved H(2), 0.00 ± 0.00 mg/L) or water with dissolved H(2) produced by electrolysis (EW: dissolved H(2), 0.35 ± 0.03 mg/L) for up to 6 weeks on a 0.5% salt diet. The rats then underwent ischemic reperfusion (I/R) of one kidney and were killed a week later for investigation of the contralateral kidney and the heart. RESULTS: In the rats given FW, unilateral kidney I/R induced significant increases in plasma monocyte chemoattractant protein-1, methylglyoxal and blood urea nitrogen. Histologically, significant increases were found in glomerular adhesion, cardiac fibrosis, number of ED-1 (CD68)-positive cells and nitrotyrosine staining in the contralateral kidney and the heart. In rats given EW, those findings were significantly ameliorated and there were significant histological differences between rats given FW and those given EW. CONCLUSION: Consumption of EW by ad libitum drinking has the potential to ameliorate ischemia-induced cardio-renal injury in CKD model rats. This indicates a novel strategy of applying H(2) produced by water electrolysis technology for the prevention of CKD cardio-renal syndrome.

Mechanism of the lifespan extension of Caenorhabditis elegans by electrolyzed reduced water--participation of Pt nanoparticles.

Electrolyzed reduced water (ERW) contains a large amount of molecular hydrogen and a small amount of Pt nanoparticles (Pt NPs). We have found that ERW significantly extended the lifespan of Caenorhabditis elegans in a novel culture medium designated Water Medium. In this study, we found that synthetic Pt NPs at ppb levels significantly extended the nematode lifespan and scavenged reactive oxygen species (ROS) in the nematode induced by paraquat treatment. In contrast, a high concentration of dissolved molecular hydrogen had no significant effect on the lifespan of the nematode. These findings suggest that the Pt NPs in ERW, rather than the molecular hydrogen, extend the longevity of the nematode, at least partly by scavenging ROS.

Electrolyzed Hydrogen Water Protects against Ethanol-Induced Cytotoxicity by Regulating Aldehyde Metabolism-Associated Enzymes in the Hepatic Cell Line HepG2.

Excessive alcohol consumption can cause multi-systemic diseases. Among them, alcoholic liver disease is the most frequent and serious disease. Electrolytic hydrogen water (EHW) is produced at the cathode during electrolysis of water and contains a large amount of molecular hydrogen and a low content of platinum nanoparticles with alkaline properties. In this study, we found that EHW inhibits ethanol-induced cytotoxicity by decreasing the intracellular acetaldehyde, a toxic substance produced by ethanol degradation, in hepatocyte cell lines HepG2. Analysis of the mechanism of action revealed that EHW inhibits the metabolism of ethanol to acetaldehyde by suppressing alcohol dehydrogenase. EHW also promotes the metabolism of acetaldehyde to acetic acid by activating aldehyde dehydrogenase, which plays to reduce aldehyde toxicity and intracellular reactive oxygen species in HepG2 cells. These functions were correlated with the concentration of molecular hydrogen in EHW, and were abolished by degassing treatment, suggesting that molecular hydrogen may contribute as a functional factor in the suppression of ethanol-induced hepatocellular damage. Furthermore, hydrogen water with high dissolved hydrogen molecule showed the same hepatocellular protective effect against ethanol as the EHW. These results suggest that EHW may be useful in the prevention of alcoholic liver disease.

Extension of the lifespan of Caenorhabditis elegans by the use of electrolyzed reduced water.

Electrolyzed reduced water (ERW) has attracted much attention because of its therapeutic effects. In the present study, a new culture medium, which we designated Water medium, was developed to elucidate the effects of ERW on the lifespan of Caenorhabditis elegans. Wild-type C. elegans had a significantly shorter lifespan in Water medium than in conventional S medium. However, worms cultured in ERW-Water medium exhibited a significantly extended lifespan (from 11% to 41%) compared with worms cultured in ultrapure water-Water medium. There was no difference between the lifespans of worms cultured in ERW-S medium and ultrapure water-S medium. Nematodes cultured in ultrapure water-Water medium showed significantly higher levels of reactive oxygen species than those cultured in ultrapure water-S medium. Moreover, ERW-Water medium significantly reduced the ROS accumulation induced in the worms by paraquat, suggesting that ERW-Water medium extends the longevity of nematodes at least partly by scavenging ROS.

Monolayered Platinum Nanoparticles as Efficient Electrocatalysts for the Mass Production of Electrolyzed Hydrogen Water.

High-performance/low-cost platinum (Pt)-based electrocatalysts have been established by top-coating both sides of a titanium plate with Pt nanoparticles. The average diameter of the Pt nanoparticles used in this study is approximately 100 nm. Three types of Pt top-coated Pt/Ti electrocatalysts, each having different top-coated Pt layer thicknesses, are prepared. Type I is a monolayered Pt top-coated type, in which the thickness of the top-coated Pt layer is approximately 100 nm; Type II is a few-layered type with a top-coated Pt layer thickness of approximately 200 nm, and Type III is a multilayered type with a top-coated Pt layer thickness of approximately 750 nm. The mass loading of Pt is 0.0215 mg cm-2 for Type I, 0.043 mg cm-2 for Type II, and 0.161 mg cm-2 for Type III. The electrocatalytic activities of each type of Pt/Ti electrocatalyst are evaluated through the electrolysis of acidic water and tap water. Type I gives the highest electrocatalytic efficiencies, which are comparable or even better than the electrocatalytic efficiencies of the state-of-the-art commercially available Pt/C electrode and other metal-/carbon-based HER catalysts. For example, in the case of the electrolysis of acidic water at an overpotential of 0.15 V, Type I shows a Tafel slope of 29 mV dec-1 and a current density of 27.5 mA cm-2. Even in the case of the electrolysis of tap water, Type I gives an HER Faradaic efficiency of 92%. A model of water (H2O), hydronium ions (H3O+), and hydroxyl ions (OH-) properly adsorbing on the Pt (111) facet is proposed to explain the electrocatalytic mechanism. New insights into the distinguishing properties of the resultant electrolyzed hydrogen water (EHW), namely, the healthy beneficial effects of EHW, are also described, and a new concept of storing and carrying reductive hydrogen (H*) by free Pt nanoparticles is proposed.

Biological effects of electrolyzed water in hemodialysis.

BACKGROUND/AIMS: The application of electrolyzed water (EW) at the cathode side to manufacture reverse osmosis (RO) water and hemodialysis (HD) solution can actually lead to less oxidative capacity in chemical terms. The present study examined the biological actions of this water on human polymorphonuclear leukocytes (PMNs), and the clinical feasibility of applying this technology to HD treatment. METHODS: RO water using EW (e-RO) exhibited less chemiluminescence in luminol-hydrogen peroxide and higher dissolved hydrogen levels (-99.0 ppb) compared with control RO water. The effects of e-RO on PMN viability were tested. HD using e-RO was performed for 12 consecutive sessions in 8 patients for the feasibility test. RESULTS: Basal cellular viability and function to generate superoxide radicals of PMNs were better preserved by e-RO application. In the clinical trial, reductions of blood pressure were noted, but no adverse events were observed. There were no changes in the blood dialysis parameters, although methylguanidine levels were significantly decreased at the end of study. CONCLUSION: The present study demonstrated the capacity of e-RO to preserve the viability of PMNs, and the clinical feasibility of applying this water for HD treatment. The clinical application of this technology may improve the bio-compatibility of HD treatment.

Production of human monoclonal antibodies against Fc(epsilon)RI(alpha) by a method combining in vitro immunization with phage display.

An in vitro immunization protocol using human peripheral blood mononuclear cells (PBMC) was developed to generate human antigen-specific antibodies. Monoclonal antibodies have great potential, and in particular, efficient acquirement of monoclonal antibodies against membrane proteins provides advantages. In this study, we tried to generate a human monoclonal antibody against the high affinity IgE receptor, Fc(epsilon)RI(alpha), using a method combining in vitro immunization and phage display. Heavy and light chain variable region genes were obtained from PBMC immunized in vitro with Fc(epsilon)RI(alpha)-expressed KU812F cells. Subsequently a combined phage antibody library 6 x 10(3) in the size was generated. Antigen-specific phage antibody clones were selected by panning with recombinant Fc(epsilon)RI(alpha) and recombined to produce human IgG format antibodies using CHO cells. The antibodies exhibited specific binding against Fc(epsilon)RI(alpha). These results suggest that one can obtain membrane protein-specific human monoclonal antibodies from a relatively small phage antibody library using in vitro immunized PBMCs.

A rapid and efficient strategy to generate antigen-specific human monoclonal antibody by in vitro immunization and the phage display method.

An in vitro immunization (IVI) protocol was developed for inducing antigen-specific immune responses in human peripheral blood mononuclear cells (PBMCs). After antigen sensitization of PBMCs by IVI, B cells producing antigen-specific antibody can be propagated within a week. Here, we attempted to establish a rapid, efficient strategy to obtain antigen-specific antibody by the phage display method using in vitro immunized PBMCs. Heavy and light chain variable region genes were easily amplified from these PBMCs immunized with mite extract (ME). After generating a combinatorial phage library (1.6 x 10(5) members), 4 antigen-specific clones were selected by 5 panning rounds using biotinylated antigen and streptavidin magnetic beads. Next, we combined variable region genes of these selected clones with human IgG constant region genes and produced human IgG-type antibody. Direct and competitive enzyme-linked immunosorbent assays demonstrated that the mAb 1C11 clone bound specifically to ME. We thus established a rapid, efficient method to obtain antigen-specific human antibody genes and produce human monoclonal IgG antibody using the phage antibody library generated from in vitro immunized PBMCs.

Translational Research of Peritoneal Dialysis Solution with Dissolved Molecular Hydrogen.

BACKGROUND: Improved biocompatibility of peritoneal dialysis solution (PDS) is crucial for peritoneal membrane preservation, thereby ensuring long-term peritoneal dialysis (PD) and preventing encapsulating peritoneal sclerosis. We previously reported the protective effect of molecular hydrogen (H2) on mesothelial cells from PDS in nonuremic rats. SUMMARY: In the present study, we examined the effect of H2-containing PDS (commercially available neutral pH type) regarding the protection of peritoneal tissue in experimental chronic kidney disease rats. Furthermore, we conducted a 2-week clinical trial in which H2-containing PDS was used in place of standard PDS and its feasibility was examined. In the experimental study, test solutions were injected through the subcutaneous port into the abdomen for 3 weeks. Histological study revealed a significant increase in the number of mesothelial cells and a significant decrease in peritoneal thickness in the H2-PD group as compared to the control and PD groups. Also, results of immunostaining analysis revealed increased vimentin and apoptotic cells in the membrane of the PD group, indicating that H2 may play a role in ameliorating PDS-induced peritoneal injury and preserving peritoneal integrity. In the clinical trial with 6 prevalent PD patients, all subjects completed the study with no adverse effects. Moreover, there were substantial changes in surrogate markers, such as increased CA125 and mesothelin, in the effluent in selected cases, suggesting enhanced mesothelial regeneration by H2. Key Message: H2-enriched PDS is a candidate novel PDS with improved biocompatibility. Further, our results support the significance of H2-PD clinical trials in the future.