Publications by authors named "Eiji Kobayashi"

426 Publications

Techniques of orthotopic renal transplantation. II. Size-matched porcine grafts in monkey recipients.

Acta Cir Bras 2021 18;36(5):e360503. Epub 2021 Jun 18.

PhD. Department of Kidney Regenerative Medicine - The Jikei University School of Medicine - Tokyo, Japan.

Purpose: As a classical xenotransplantation model, porcine kidneys have been transplanted into the lower abdomen of non-human primates. However, we have improved upon this model by using size-matched grafting in the orthotopic position. The beneficial aspects and surgical details of our method are reported herein.

Methods: Donors were two newborn pigs (weighting 5 to 6 kg) and recipients were two cynomolgus monkeys (weighting, approximately, 7 kg). After bilateral nephrectomy, kidneys were cold-transported in Euro-Collins solution. The porcine kidney was transplanted to the site of a left nephrectomy and fixed to the peritoneum.

Results: Kidneys transplanted to the lower abdomen by the conventional method were more susceptible to torsion of the renal vein (two cases). In contrast, early-stage blood flow insufficiency did not occur in orthotopic transplants of theleft kidney.

Conclusions: Size-matched porcine-primate renal grafting using our method of transplanting tothe natural position of the kidneys contributes to stable post-transplant blood flow to the kidney.
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http://dx.doi.org/10.1590/ACB360503DOI Listing
June 2021

Rapid Metabolic Recovery of Donor Circulatory Death Liver Graft Using Whole Blood Perfusion: A Pig Study.

Transplant Direct 2021 Jul 10;7(7):e712. Epub 2021 Jun 10.

Department of Organ Fabrication, Keio University School of Medicine, Tokyo, Japan.

Ex vivo perfusion technology has been actively developed to solve the problem of severe donor shortage. In this study, the ex vivo metabolic characteristics of porcine donation after circulatory death (DCD) liver in short-term perfusion using whole or diluted blood were compared with those of the in vivo transplanted state to evaluate their initial response to resuscitation.

Methods: The porcine DCD model was constructed by clamping the thoracic aorta. After 60 min of blood flow cessation, retrieved livers were flushed with 500 mL of heparin saline (20 000 IU/L) followed by perfusion with 500 mL of cold histidine-tryptophan-ketoglutarate solution. The liver grafts were immersed in cold histidine-tryptophan-ketoglutarate solution for 60 min. Subsequently, normothermic ex vivo perfusion was performed with 20 000 IU/L of heparin added to the collected blood (whole blood group) or medium mixed with 10% whole blood (dilution group) for 3 h. Blood from the portal vein, the hepatic artery, and infra hepatica inferior vena cava was collected hourly and metabolomic analyses were performed. The other liver graft was heterotopically transplanted as a control (in vivo group). Each experiment was conducted once.

Results: The guanosine levels demonstrated similar fluctuating trends in the whole blood and in vivo groups. In contrast, the levels increased during the perfusion in the diluted blood group. Fluctuations in choline metabolism demonstrated similar trends in the whole blood and in vivo groups.

Conclusions: Ex vivo machine perfusion with whole blood over a short time resulted in a metabolic trend similar to that in the in vivo model. Further studies in this regard are warranted to progress in the utilization of DCD organs.
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http://dx.doi.org/10.1097/TXD.0000000000001170DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8196120PMC
July 2021

CD70 antibody-drug conjugate: A potential novel therapeutic agent for ovarian cancer.

Cancer Sci 2021 Jun 12. Epub 2021 Jun 12.

Department of Obstetrics and Gynecology, Osaka University, Osaka, Japan.

This study aimed to investigate the cytotoxicity of a CD70 antibody-drug conjugate (CD70-ADC) against ovarian cancer in-vitro and in-vivo xenograft models. CD70 expression was assessed in clinical samples by immunohistochemical analysis. Western blotting and fluorescence-activated cell sorting analyses were used to determine CD70 expression in the ovarian cancer cell lines, A2780 and SKOV3, and in the cisplatin-resistant ovarian cancer cell lines, A2780cisR and SKOV3cisR. CD70 expression after cisplatin exposure was determined in A2780 cells transfected with mock- or nuclear factor (NF)-κB-p65-small interfering RNA. We developed an ADC with an anti-CD70 monoclonal antibody linked to monomethyl auristatin F and investigated its cytotoxic effect. We examined 63 ovarian cancer clinical samples; 43 (68.3%) of them expressed CD70. Among patients with advanced stage disease (n=50), those who received neoadjuvant chemotherapy were more likely to exhibit high CD70 expression compared to those who did not (55.6% [15/27] versus 17.4% [4/23], P<0.01). CD70 expression was confirmed in A2780cisR, SKOV3, and SKOV3cisR cells. Notably, CD70 expression was induced after cisplatin treatment in A2780 mock cells but not in A2780-NF-κB-p65-silenced cells. CD70-ADC was cytotoxic to A2780cisR, SKOV3, and SKOV3cisR cells, with IC values ranging from 0.104 to 0.341 nM. In A2780cisR and SKOV3cisR xenograft models, tumor growth in CD70-ADC treated mice was significantly inhibited compared to that in the control-ADC treated mice (A2780cisR: 32.0 versus 1639.0 mm , P<0.01; SKOV3cisR: 232.2 versus 584.9 mm , P<0.01). Platinum treatment induced CD70 expression in ovarian cancer cells. CD70-ADC may have potential therapeutic implications in the treatment of CD70 expressing ovarian cancer.
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http://dx.doi.org/10.1111/cas.15027DOI Listing
June 2021

Micro- and macro-borderless surgery using a newly developed high-resolution (4K) three-dimensional video system.

PLoS One 2021 12;16(5):e0250559. Epub 2021 May 12.

Department of Organ Fabrication, Keio University School of Medicine, Tokyo, Japan.

Objective: Microsurgery using conventional optical microscopes or surgical loupes features a limited field of view and imposes a serious strain on surgeons especially during long surgeries. Here we advocate the micro- and macro-borderless surgery (MMBS) using a novel high-resolution (4K) three-dimensional (3D) video system. This study aimed to confirm the applicability of this concept in several surgical procedures.

Methods: We evaluated the possible use and efficacy of MMBS in the following experiments in porcine subjects. Experiment 1 (non-inferiority test) consisted of dissection and anastomosis of carotid artery, portal vein, proper hepatic artery, and pancreatoduodenectomy with surgical loupe versus MMBS. Experiment 2 (feasibility test) consisted of intra-abdominal and intra-thoracic smaller arteries anastomosed by MMBS as a pre-clinical setting. Experiment 3 (challenge on new surgery) consisted of orthotopic liver transplantation of the graft from a donor after circulatory death maintained by machine perfusion. Circulation of the cardiac sheet with a vascular bed in experiment 2 and liver graft during preservation in experiment 3 was evaluated with indocyanine green fluorescence imaging equipped with this system.

Results: Every procedure was completed by MMBS. The operator and assistants could share the same field of view in heads-up status. The focal depth was deep enough not to be disturbed by pulsing blood vessels or respiratory movement. The tissue circulation could be evaluated using fluorescence imaging of this system.

Conclusions: MMBS using the novel system is applicable to various surgeries and valuable for both fine surgical procedures and high-level surgical education.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0250559PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8115828PMC
May 2021

Effects of early high nutrition related to metabolic imprinting events on growth, carcass characteristics, and meat quality of grass-fed Wagyu (Japanese Black cattle).

J Anim Sci 2021 Jun;99(6)

Kuju Agriculture Research Center, Kyushu University, Taketa, Oita 878-0201, Japan.

The study was conducted to clarify how early high plane of nutrition related to metabolic imprinting affected growth, carcass characteristics, and meat quality of grass-fed Wagyu (Japanese Black cattle). Wagyu steers were allocated randomly into 2 dietary groups: (1) steers fed milk replacer (crude protein 26.0%, crude fat 25.5%; maximum intake 0.6 kg/d) until 3 mo of age and then fed roughage (orchard grass hay) ad libitum from 4 to 10 mo of age (roughage group, RG; n = 11); (2) steers fed milk replacer (maximum intake of 1.8 kg/d) until 3 mo of age and then fed a high-concentrate diet from 4 to 10 mo of age (early high nutrition, EHN; n = 12). After 11 mo of age, all steers were fed roughage ad libitum until 31 mo of age and then slaughtered. Growth performance, carcass traits, longissimus muscle (LM) meat quality and intramuscular fat (IMF) content, plasma insulin-like growth factor I (IGF-I) concentration, and bone mineral density were measured. Body weight was greater in EHN steers (571 kg) than RG steers (520 kg; P < 0.01). Plasma IGF-I levels were higher in EHN steers than in RG steers at 3, 10, and 14 mo of age (P < 0.01, P < 0.005, P < 0.001, respectively); however, plasma IGF-I levels were lower in EHN steers compared with RG steers at 30 mo of age (P < 0.01). The total weight of the muscles and bones of the left half of the carcass was not different between the 2 groups (P = 0.065). Five of the 19 muscles investigated (semimembranosus, P = 0.036; infraspinatus, P = 0.024; supraspinatus, P = 0.0019; serratus ventralis cervicis, P = 0.032; serratus ventralis thoracis, P = 0.027) were heavier in EHN steers. Total fat weight in the left half of the carcass was 30% greater (P = 0.025) in HNE carcasses. Subcutaneous and perirenal fat weights were 53% and 84% greater (P = 0.008, P = 0.002, respectively) in EHN carcasses. The LM IMF content was greater in EHN loins (13.2%) compared with RG loins (9.4%) at 31 mo of age (P = 0.038); however, no differences were found for shear force, tenderness, and cook loss. These results suggested early high-nutrition affected the growth and meat quality of livestock.
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http://dx.doi.org/10.1093/jas/skab123DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8212569PMC
June 2021

Testing of human equivalent dose of health food 5-aminolevulinic acid using the experimental pig.

Authors:
Eiji Kobayashi

Biomed Pharmacother 2021 07 24;139:111629. Epub 2021 Apr 24.

Department of Organ Fabrication, Keio University School of Medicine, 35 Shinanomachi, Shinjuku-ku, Tokyo 160-8582, Japan. Electronic address:

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http://dx.doi.org/10.1016/j.biopha.2021.111629DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8065248PMC
July 2021

Feasibility of large experimental animal models in testing novel therapeutic strategies for diabetes.

World J Diabetes 2021 Apr;12(4):306-330

Meiji University International Institute for Bio-Resource Research, Meiji University, Kawasaki 214-8571, Kanagawa, Japan.

Diabetes is among the top 10 causes of death in adults and caused approximately four million deaths worldwide in 2017. The incidence and prevalence of diabetes is predicted to increase. To alleviate this potentially severe situation, safer and more effective therapeutics are urgently required. Mice have long been the mainstay as preclinical models for basic research on diabetes, although they are not ideally suited for translating basic knowledge into clinical applications. To validate and optimize novel therapeutics for safe application in humans, an appropriate large animal model is needed. Large animals, especially pigs, are well suited for biomedical research and share many similarities with humans, including body size, anatomical features, physiology, and pathophysiology. Moreover, pigs already play an important role in translational studies, including clinical trials for xenotransplantation. Progress in genetic engineering over the past few decades has facilitated the development of transgenic animals, including porcine models of diabetes. This article discusses features that attest to the attractiveness of genetically modified porcine models of diabetes for testing novel treatment strategies using recent technical advances.
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http://dx.doi.org/10.4239/wjd.v12.i4.306DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8040081PMC
April 2021

A potential means to help the HPV vaccine penetrate the Japanese public while under the continued suspension of governmental recommendation.

Hum Vaccin Immunother 2021 Apr 20:1-6. Epub 2021 Apr 20.

Department of Obstetrics and Gynecology , Tsubaki Women's Clinic, Matsuyama, Japan.

In Japan, the government's recommendation for the HPV vaccine has been suspended for almost 8 years. A questionnaire survey was conducted in the Tsubaki Women's Clinic, Matsuyama, Japan, to examine responses of the mothers of girls eligible for HPV vaccine before and after their doctor provided them an informative leaflet explaining the need for cervical cancer prevention.Among the 53 mothers who admitted to imposing some preconditions before being willing to encourage their daughters' HPV vaccination, 21 (40%) mothers became more willing to vaccinate their daughters immediately after receiving the cervical cancer prevention linkage explanation provided by their doctor, and seven of the mothers (33%) even returned to the clinic to get their daughter vaccinated during our study period. Logistical regression analysis revealed that having initial preconditions required for their daughters' HPV vaccination was an independent variable influencing the mothers' change of willingness to get their daughters vaccinated immediately after receiving the explanation using the leaflet.We have found that to achieve maximum effectiveness, we can use an appropriate leaflet even under suspension of the governmental recommendation. Our future efforts should be focused on those mothers who are less likely to impose preconditions on their daughter's vaccination.
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http://dx.doi.org/10.1080/21645515.2021.1910001DOI Listing
April 2021

Dose rate in the highest irradiation area of the rectum correlates with late rectal complications in patients treated with high-dose-rate computed tomography-based image-guided brachytherapy for cervical cancer.

J Radiat Res 2021 May;62(3):494-501

Department of Radiation Oncology, Osaka University Graduate School of Medicine, Osaka, 565-0871, Japan.

The purpose of this study was to evaluate the effect of dose rate to the rectum on late rectal complications in patients treated with computed tomography (CT)-based image-guided brachytherapy (IGBT) for cervical cancer. The subjects were 142 patients with cervical cancer who underwent Ir-192 high-dose-rate (HDR)-IGBT between March 2012 and January 2018. The dose rate to the rectum was calculated using in-house software. The minimum, mean and maximum effective dose rate (EDR) was calculated for voxels of the rectal volume covered by cumulative doses >D0.1cc, >D2cc, and > D5cc. The average EDR of three to four brachytherapy sessions was calculated (EDR for patients; EDRp). The total dose of the rectum was calculated as the biologically equivalent dose in 2-Gy fractions (EQD2). The associations between EDRp for D0.1cc, D2cc, and D5cc and the respective rectal EQD2 values with late rectal complications were then analyzed. The median follow-up period was 40 months. Patients with rectal complications of ≥Grade 1 received a significantly higher mean EDRp for D0.1cc-5cc and had a greater EQD2 for D0.1cc-5cc. Multivariate analysis was performed using the mean EDRp for D2cc, EQD2 for D2cc, heavy smoking and BMI. Of these four variables, mean EDRp for D2cc (HR = 3.38, p = 0.004) and EQD2 for D2cc (HR = 2.59, p = 0.045) emerged as independent predictors for late rectal complications. In conclusion, mean EDRp and EQD2 were associated with late rectal complications in patients treated with HDR CT-based IGBT for cervical cancer.
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http://dx.doi.org/10.1093/jrr/rrab023DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8127676PMC
May 2021

Epstein-Barr Virus LMP1 Induces Soluble PD-L1 in Nasopharyngeal Carcinoma.

Microorganisms 2021 Mar 15;9(3). Epub 2021 Mar 15.

Division of Otolaryngology and Head and Neck Surgery, Graduate School of Medical Science, Kanazawa University, Kanazawa 920-8640, Japan.

Nasopharyngeal carcinoma (NPC) is an Epstein-Barr virus (EBV)-associated malignancy. The principal oncogene of EBV, latent membrane protein 1 (LMP1), induces the expression of programmed death-ligand 1 (PD-L1), which is an immunosuppressive transmembrane protein and a promising therapeutic target for various malignancies. Recent studies have revealed an association between the level of soluble PD-L1 (sPD-L1) and disease progression. However, the role of sPD-L1 in NPC or its relevance to LMP1 has not been elucidated. This study aimed to examine whether LMP1 induces sPD-L1 in vitro and analyze the clinical relevance of LMP1, PD-L1, and sPD-L1 in NPC patients. Analysis of nasopharyngeal cell lines revealed that LMP1 induces both cellular PD-L1 and sPD-L1. Analysis of biopsy specimens from 32 NPC patients revealed that LMP1 expression was significantly correlated with PD-L1 expression. Finally, the serum sPD-L1 level in NPC patients was higher than that in the controls. Moreover, the sPD-L1 level in the advanced stage was higher than that in the early stage. However, LMP1 expression, PD-L1 expression, and sPD-L1 levels were not associated with prognosis. These results suggest that LMP1 induces both sPD-L1 and PD-L1, which are associated with NPC progression.
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http://dx.doi.org/10.3390/microorganisms9030603DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7998736PMC
March 2021

Intramyocardial Transplantation of Human iPS Cell-Derived Cardiac Spheroids Improves Cardiac Function in Heart Failure Animals.

JACC Basic Transl Sci 2021 Mar 19;6(3):239-254. Epub 2021 Feb 19.

Department of Cardiology, Keio University School of Medicine, Tokyo, Japan.

The severe shortage of donor hearts hampered the cardiac transplantation to patients with advanced heart failure. Therefore, cardiac regenerative therapies are eagerly awaited as a substitution. Human induced pluripotent stem cells (hiPSCs) are realistic cell source for regenerative cardiomyocytes. The hiPSC-derived cardiomyocytes are highly expected to help the recovery of heart. Avoidance of teratoma formation and large-scale culture of cardiomyocytes are definitely necessary for clinical setting. The combination of pure cardiac spheroids and gelatin hydrogel succeeded to recover reduced ejection fraction. The feasible transplantation strategy including transplantation device for regenerative cardiomyocytes are established in this study.
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http://dx.doi.org/10.1016/j.jacbts.2020.11.017DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7987543PMC
March 2021

Rapid and efficient generation of T-cell receptor-like antibodies using chip-based single-cell analysis.

Eur J Immunol 2021 Mar 17. Epub 2021 Mar 17.

Department of Immunology, Faculty of Medicine, Academic Assembly, University of Toyama, Toyama, Japan.

Generation of TCR-like monoclonal antibodies using conventional methods is markedly laborious and inefficient. We have proposed improvements of ISAAC (chip-based Ab-secreting cell [ASC] screening method), allows comprehensive analysis of ASCs at the single-cell level to obtain TCR-like antibodies; blocking procedure enables us to avoid the detection of non-TCR-like antibodies.
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http://dx.doi.org/10.1002/eji.202049083DOI Listing
March 2021

Prevention of Chronic Rejection of Marginal Kidney Graft by Using a Hydrogen Gas-Containing Preservation Solution and Adequate Immunosuppression in a Miniature Pig Model.

Front Immunol 2020 17;11:626295. Epub 2021 Feb 17.

Department of Organ Fabrication, Keio University School of Medicine, Tokyo, Japan.

In clinical kidney transplantation, the marginal kidney donors are known to develop chronic allograft rejection more frequently than living kidney donors. In our previous study, we have reported that the hydrogen gas-containing organ preservation solution prevented the development of acute injuries in the kidney of the donor after cardiac death by using preclinical miniature pig model. In the present study, we verified the impact of hydrogen gas treatment in transplantation with the optimal immunosuppressive protocol based on human clinical setting by using the miniature pig model. Marginal kidney processed by hydrogen gas-containing preservation solution has been engrafted for long-term (longer than 100 days). A few cases showed chronic rejection reaction; however, most were found to be free of chronic rejection such as graft tissue fibrosis or renal vasculitis. We concluded that marginal kidney graft from donor after cardiac death is an acceptable model for chronic rejection and that if the transplantation is carried out using a strict immunosuppressive protocol, chronic rejection may be alleviated even with the marginal kidney.
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http://dx.doi.org/10.3389/fimmu.2020.626295DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7925892PMC
February 2021

Mechanism and design of a novel 8K ultra-high-definition video microscope for microsurgery.

Heliyon 2021 Feb 15;7(2):e06244. Epub 2021 Feb 15.

Department of Organ Fabrication, Keio University School of Medicine, Tokyo, Japan.

The practical application of microscopes using 8K ultra-high definition (UHD) technology is progressing. However, due to insufficiencies in factors such as luminous intensity and stereopsis, it has not been possible to achieve sufficient image quality for close observation of submillimeter order microlymphatic anastomosis using a combination of 8K-UHD cameras with a rigid endoscope. We have improved the quality of microsurgery by the introduction of a new heads up 8K-UHD surgical system. Herein, we show the mechanisms of this next-generation technology that makes optical improvements to the electronic image input data, resolving the initial drawback. We have developed a new 8K-UHD digital microscope system with digital zooming to enable maximum 300X magnification of the surgical field. This system has specific lighting settings for shadows dropped in surgical field to expand the three-dimensional effect while still being a monocular camera. The original mechanism and design enable the increase of the depth of field with optimal angles between the imaging direction and approaching direction towards the surgical field. Assessment during a pre-clinical trial using rats demonstrated that it is possible to perform microlymphatic anastomosis in a heads-up position with a 70-inch 8K-UHD monitor and the 8K-UHD monocular camera system. Performing supermicrosurgery is difficult with conventional surgical microscopes. Our results illustrate the application of this new 8K-UHD microscope system to this new field.
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http://dx.doi.org/10.1016/j.heliyon.2021.e06244DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7900699PMC
February 2021

An organoid-based organ-repurposing approach to treat short bowel syndrome.

Nature 2021 04 24;592(7852):99-104. Epub 2021 Feb 24.

Department of Organoid Medicine, Keio University School of Medicine, Tokyo, Japan.

The small intestine is the main organ for nutrient absorption, and its extensive resection leads to malabsorption and wasting conditions referred to as short bowel syndrome (SBS). Organoid technology enables an efficient expansion of intestinal epithelium tissue in vitro, but reconstruction of the whole small intestine, including the complex lymphovascular system, has remained challenging. Here we generate a functional small intestinalized colon (SIC) by replacing the native colonic epithelium with ileum-derived organoids. We first find that xenotransplanted human ileum organoids maintain their regional identity and form nascent villus structures in the mouse colon. In vitro culture of an organoid monolayer further reveals an essential role for luminal mechanistic flow in the formation of villi. We then develop a rat SIC model by repositioning the SIC at the ileocaecal junction, where the epithelium is exposed to a constant luminal stream of intestinal juice. This anatomical relocation provides the SIC with organ structures of the small intestine, including intact vasculature and innervation, villous structures, and the lacteal (a fat-absorbing lymphatic structure specific to the small intestine). The SIC has absorptive functions and markedly ameliorates intestinal failure in a rat model of SBS, whereas transplantation of colon organoids instead of ileum organoids invariably leads to mortality. These data provide a proof of principle for the use of intestinal organoids for regenerative purposes, and offer a feasible strategy for SBS treatment.
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http://dx.doi.org/10.1038/s41586-021-03247-2DOI Listing
April 2021

Techniques of orthotopic renal transplantation in pigs. One donor to two recipients via inverted grafting.

Acta Cir Bras 2021 22;36(2):e360208. Epub 2021 Feb 22.

MD, PhD. The Jikei University School of Medicine - Department of Kidney Regenerative Medicine; Keio University School of Medicine - Department of Organ Fabrication - Tokyo, Japan.

Purpose: Although transplanting two kidneys from a single donor to two recipients has some advantages, the right and left kidneys are not anatomically identical; thus, a surgical procedure considering the anatomical features of the donor kidneys is needed when transplanting them into the opposite renal fossae. Based on vast experience, the surgical details of pig orthotopic kidney transplantation from one donor to two recipients was reported.

Methods: When the right kidney was transplanted to the left renal fossa, the graft was inverted upside down, not backwards, thus ensuring that the anteroposterior relationship of the renal vessels was maintained and anatomically natural vascular anastomosis could be performed.

Results: Using this technique, we could have developed a pig experimental model that is safe and has a high success rate, even for researchers in the middle of their training. This technique of inverting the graft upside down was reported in human kidney transplantation to make vascular anastomosis easier.

Conclusions: In pig orthotopic kidney transplantation from one donor to two recipients, an anatomically natural vascular anastomosis could be performed via inverted grafting when the right kidney was transplanted into the left renal fossa.
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http://dx.doi.org/10.1590/ACB360208DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7902078PMC
February 2021

Tryptophan Metabolism Regulates Proliferative Capacity of Human Pluripotent Stem Cells.

iScience 2021 Feb 26;24(2):102090. Epub 2021 Jan 26.

Department of Cardiology, Keio University School of Medicine, Shinjuku, Tokyo 160-8582, Japan.

Human pluripotent stem cells (hPSCs) have a unique metabolic signature for maintenance of pluripotency, self-renewal, and survival. Although hPSCs could be potentially used in regenerative medicine, the prohibitive cost associated with large-scale cell culture presents a major barrier to the clinical application of hPSC. Moreover, without a fully characterized metabolic signature, hPSC culture conditions are not optimized. Here, we performed detailed amino acid profiling and found that tryptophan (TRP) plays a key role in the proliferation with maintenance of pluripotency. In addition, metabolome analyses revealed that intra- and extracellular kynurenine (KYN) is decreased under TRP-supplemented conditions, whereas N-formylkynurenine (NFK), the upstream metabolite of KYN, is increased thereby contributing to proliferation promotion. Taken together, we demonstrate that TRP is indispensable for survival and proliferation of hPSCs. A deeper understanding of TRP metabolism will enable cost-effective large-scale production of hPSCs, leading to advances in regenerative medicine.
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http://dx.doi.org/10.1016/j.isci.2021.102090DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7878994PMC
February 2021

Differentially methylated CpG sites related to fertility in Japanese Black bull spermatozoa: epigenetic biomarker candidates to predict sire conception rate.

J Reprod Dev 2021 Apr 14;67(2):99-107. Epub 2021 Jan 14.

Institute of Livestock and Grassland Science, NARO, Ibaraki 305-0901, Japan.

For semen suppliers, predicting the low fertility of service bull candidates before artificial insemination would help prevent economic loss; however, predicting bull fertility through in vitro assessment of semen is yet to be established. In the present study, we focused on the methylated CpG sites of sperm nuclear DNA and examined methylation levels to screen new biomarkers for predicting bull fertility. In frozen-thawed semen samples collected from Japanese Black bulls, for which the sire conception rate (SCR) was recorded, the methylation level of each CpG site was analyzed using human methylation microarray. According to regression analysis, 143 CpG sites related to SCR were significantly differentially methylated. Whole genome bisulfite sequence data were obtained from three semen samples and the differentially methylated regions (DMRs) that included the target CpG sites selected by human methylation microarray were confirmed. Using combined bisulfite restriction analysis, fertility-related methylation changes were detected in 10 DMRs. With the exception of one DMR, the methylation levels of these DMRs were significantly different between groups with high fertility (> 50%) and low fertility (< 40%). From multiple regression analysis of methylation levels and SCR, three DMRs were selected that could effectively predict bull fertility. We suggest that these fertility-related differences in spermatozoal methylation levels could be new epigenetic biomarkers for predicting bull fertility.
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http://dx.doi.org/10.1262/jrd.2020-137DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8075730PMC
April 2021

A retrospective assessment of the safety and efficacy of laparoscopic radical hysterectomy in Japan during the early years following its introduction: a Japanese Gynecologic Oncology Group study (JGOG1081S).

Int J Clin Oncol 2021 Feb 12;26(2):417-428. Epub 2021 Jan 12.

Department of Obstetrics and Gynecology, Tokai University School of Medicine, Kanagawa, Japan.

Objective: To evaluate the safety and efficacy of laparoscopic radical hysterectomy (LRH) for cervical cancer, in terms of morbidity and short-term oncologic outcome following LRH's introduction into Japan.

Methods: We conducted a retrospective analysis of patients with early-stage cervical cancer (FIGO staging IA2, IB1, and IIA1) who underwent LRH from Dec 2014 to Dec 2016. We assessed the morbidity, overall survival (OS) and recurrence-free survival (RFS), and prognostic factors for RFS.

Results: A total of 251 patients were included from 22 facilities across Japan. There were 8 cases of stage IA2 cervical cancer, 226 of IB1, and 17 of IIA1. The median operating time was 343 min and the median blood loss was 190 ml. Two patients (0.8%) had a postoperative complication with a Clavien-Dindo classification of grade 3 or higher. After a median follow-up time of 15.6 months, the 2-year RFS was 87.4%, and the 2-year OS was 97.8%. When the 2-year RFS rate was compared with whether the patient pathologically had tumors of less than 2 cm, versus 2 cm or more, the RFS was 95.8% and 80.4%, respectively. Multivariate analysis found that tumor size and the route of lymph node removal were independent prognostic factors for recurrence.

Conclusion: When LRH was first introduced into Japan, we found that the route of lymph node removal was an independent prognostic factor for recurrence in addition to large tumors (≥ 2 cm). Our results suggest that prognosis may be secured by paying attention to the lymph node removal route.
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http://dx.doi.org/10.1007/s10147-020-01799-3DOI Listing
February 2021

Genetically engineered pigs manifesting pancreatic agenesis with severe diabetes.

BMJ Open Diabetes Res Care 2020 11;8(2)

Meiji University International Institute for Bio-Resource Research, Meiji University - Ikuta Campus, Kawasaki, Japan

Introduction: Pancreatic duodenum homeobox 1 () expression is crucial for pancreatic organogenesis and is a key regulator of insulin gene expression. Hairy and enhancer of split 1 () controls tissue morphogenesis by maintaining undifferentiated cells. encodes a basic helix loop helix (bHLH) transcriptional repressor and functionally antagonizes positive bHLH genes, such as the endocrine determination gene neurogenin-3. Here, we generated a new pig model for diabetes by genetic engineering and genes.

Research Design And Methods: A transgenic (Tg) chimera pig with germ cells carrying a construct expressing under the control of the promoter was used to mate with wild-type gilts to obtain Tg piglets.

Results: The Tg pigs showed perinatal death; however, this phenotype could be rescued by insulin treatment. The duodenal and splenic lobes of the Tg pigs were slender and did not fully develop, whereas the connective lobe was absent. β cells were not detected, even in the adult pancreas, although other endocrine cells were detected, and exocrine cells functioned normally. The pigs showed no irregularities in any organs, except diabetes-associated pathological alterations, such as retinopathy and renal damage.

Conclusion: Tg pigs were an attractive model for the analysis of pancreatic development and testing of novel treatment strategies for diabetes.
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http://dx.doi.org/10.1136/bmjdrc-2020-001792DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7705540PMC
November 2020

Daily inhalation of hydrogen gas has a blood pressure-lowering effect in a rat model of hypertension.

Sci Rep 2020 11 26;10(1):20173. Epub 2020 Nov 26.

Department of Basic Science, Nippon Veterinary and Life Science University, School of Veterinary Nursing and Technology, Tokyo, 180-8602, Japan.

A recent clinical study demonstrated that haemodialysis with a dialysate containing hydrogen (H) improves blood pressure control in end-stage kidney disease. Herein, we examined whether H has a salutary effect on hypertension in animal models. We subjected 5/6 nephrectomised rats to inhalation of either H (1.3% H + 21% O + 77.7% N) or control (21% O + 79% N) gas mixture for 1 h per day. H significantly suppressed increases in blood pressure after 5/6 nephrectomy. The anti-hypertensive effect of H was also confirmed in rats in a stable hypertensive state 3 weeks after nephrectomy. To examine the detailed effects of H on hypertension, we used an implanted telemetry system to continuously monitor blood pressure. H exerted an anti-hypertensive effect not only during daytime rest, but also during night-time activities. Spectral analysis of blood pressure variability revealed that H improved autonomic imbalance, namely by suppressing the overly active sympathetic nervous system and augmenting parasympathetic nervous system activity; these effects co-occurred with the blood pressure-lowering effect. In conclusion, 1-h daily exposure to H exerts an anti-hypertensive effect in an animal model of hypertension.
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http://dx.doi.org/10.1038/s41598-020-77349-8DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7692487PMC
November 2020

A microsurgical technique for catheter insertion in the rat femoral artery.

Acta Cir Bras 2020 23;35(10):e202001004. Epub 2020 Nov 23.

MD, PhD, Department of Organ Fabrication, Keio University School of Medicine, Tokyo, Japan. Conception and design, technical procedures, manuscript preparation, final approval.

Purpose: To modify a surgical catheterization method using the bent needle introducer in small animals.

Methods: Eight-week-old male Lewis rats were used in the study. A needle introducer was created by bending a 21G injection needle at 45°. The bent needle introducer was used for catheter insertion into the left femoral artery of the rats under anesthesia. As a control, a catheter was directly inserted into the blood vessel without the introducer. The insertion time of each method was measured. Blood pressure and heart rate were measured 24 h after catheter insertion using the telemetry system.

Results: Using the introducer, the catheter was successfully inserted within a short time in all rats. Without the introducer, a longer duration was required for catheter insertion. The frequency of the insertion with no catheter-based errors with the introducer tended to be higher than that without the introducer. The mean arterial pressure and heart rate 24 h after catheter insertion in each group were almost the same.

Conclusions: We developed a surgical catheterization method using the introducer in small animals. This could potentially reduce the frequency of the insertion with catheter-based errors and insertion time.
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http://dx.doi.org/10.1590/s0102-865020200100000004DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7709899PMC
November 2020

Development of a microsurgery-assisted robot for high-precision thread traction and tension control, and confirmation of its applicability.

Int J Med Robot 2021 Apr 1;17(2):e2205. Epub 2020 Dec 1.

Department of Organ Fabrication, Keio University School of Medicine, Shinjuku-ku, Tokyo, Japan.

Background: Microsurgery requires high skills for suturing using fragile threads, often within narrow surgical fields. Precise tension is required for good healing and to avoid the risk of thread breakage.

Methods: To meet the demands, we developed a novel assist robot utilizing high-precision sensorless haptic technology. The robot adopts a cable-driven mechanism to maintain a distance from the surgical area and enhances compatibility with surgical equipment such as microscopes. The robot performance was verified through in vitro and in vivo experiments using a rat model.

Results: The realization of precise tension control was confirmed in both experiments. In particular, in the in vivo experiments, the developed robot succeeded to produce a knot with an accurate tension of 0.66% error.

Conclusions: The developed robot can realize to control traction force precisely. This technology might open up the window for a full assist robot for microsurgery with haptic feeling.
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http://dx.doi.org/10.1002/rcs.2205DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7988610PMC
April 2021

Low-Flow Nasal Cannula Hydrogen Therapy.

J Clin Med Res 2020 Oct 21;12(10):674-680. Epub 2020 Sep 21.

Department of Cardiology, Keio University School of Medicine, 35 Shinanomachi, Shinjuku-ku, Tokyo 160-8582, Japan.

Background: Molecular hydrogen (H) is a biologically active gas that is widely used in the healthcare sector. In recent years, on-site H gas generators, which produce high-purity H by water electrolysis, have begun to be introduced in hospitals, clinics, beauty salons, and fitness clubs because of their ease of use. In general, these generators produce H at a low-flow rate, so physicians are concerned that an effective blood concentration of H may not be ensured when the gas is delivered through a nasal cannula. Therefore, this study aimed to evaluate blood concentrations of H delivered from an H gas generator via a nasal cannula.

Methods: We administered 100% H, produced by an H gas generator, at a low-flow rate of 250 mL/min via a nasal cannula to three spontaneously breathing micro miniature pigs. An oxygen mask was placed over the nasal cannula to administer oxygen while minimizing H leakage, and a catheter was inserted into the carotid artery to monitor the arterial blood H concentration.

Results: During the first hour of H inhalation, the mean (standard error (SE)) H concentrations and saturations in the arterial blood of the three pigs were 1,560 (413) nL/mL and 8.85% (2.34%); 1,190 (102) nL/mL and 6.74% (0.58%); and 1,740 (181) nL/mL and 9.88% (1.03%), respectively. These values are comparable to the concentration one would expect if 100% of the H released from the H gas generator is taken up by the body.

Conclusions: Inhalation of 100% H produced by an H gas generator, even at low-flow rates, can increase blood H concentrations to levels that previous non-clinical and clinical studies demonstrated to be therapeutically effective. The combination of a nasal cannula and an oxygen mask is a convenient way to reduce H leakage while maintaining oxygenation.
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http://dx.doi.org/10.14740/jocmr4323DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7524558PMC
October 2020

Challenges of laparoscopic lymphadenectomy for cervical cancer in pregnant women: techniques and tips.

Int J Gynecol Cancer 2020 12 30;30(12):2017-2018. Epub 2020 Sep 30.

Department of Obstetrics and Gynecology, Osaka University, Suita, Osaka, Japan.

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http://dx.doi.org/10.1136/ijgc-2020-001703DOI Listing
December 2020

Oncological and Reproductive Outcomes of Abdominal Radical Trachelectomy.

Anticancer Res 2020 Oct;40(10):5939-5947

Department of Obstetrics and Gynecology, Osaka University Graduate School of Medicine, Osaka, Japan.

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http://dx.doi.org/10.21873/anticanres.14615DOI Listing
October 2020

Viral infections of pigs used for medical education. A Japanese experience.

Authors:
Eiji Kobayashi

Acta Cir Bras 2020 21;35(8):e202000808. Epub 2020 Sep 21.

MD, PhD, Department of Organ Fabrication, Keio University School of Medicine, Shinjuku-ku, Tokyo, Japan.

Infectious viruses pose a threat to all living organisms, including humans, and can cause significant morbidity. Previous experience with pigs in medical education and research, rather than in domestic control settings, has led to a unique perspective on viral infections in swine. In this article, common porcine infectious diseases have been listed, based mainly on the authors' experience thus far. For example, young domestic pigs that were used in surgical training and infected with hepatitis E were subjected to quarantine and isolation treatment, and attempts were made to develop a DNA vaccine for swine influenza arising from swine-to-human transmission. More recent research has focused on preventing infection by the African swine virus, a current threat. We hope that this article of porcine infectious diseases identified at the School of Medicine will help develop a breakthrough with regard to coronavirus disease.
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http://dx.doi.org/10.1590/s0102-865020200080000008DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7518223PMC
September 2020

CD70 antibody-drug conjugate as a potential therapeutic agent for uterine leiomyosarcoma.

Am J Obstet Gynecol 2021 02 19;224(2):197.e1-197.e23. Epub 2020 Aug 19.

Department of Obstetrics and Gynecology, Osaka University, Osaka, Japan.

Background: Uterine leiomyosarcoma is a rare and aggressive gynecologic malignancy originating in the myometrium of the uterine corpus that tends to recur even after complete surgical excision. Current therapeutic agents have only modest effects on uterine leiomyosarcoma. Although antibodies and antibody-drug conjugates have been recognized as useful targeted therapies for other cancers, no study has yet evaluated the effects of this approach on uterine leiomyosarcoma.

Objective: This study aimed to examine the activity of tumoral CD70 in uterine leiomyosarcoma and assess the antitumor activity of CD70-antibody-drug conjugate treatment in uterine leiomyosarcoma.

Study Design: Target membrane proteins were screened by profiling and comparing membrane protein expression in 3 uterine leiomyosarcoma cell lines (SK-UT-1, SK-LMS-1, and SKN) and normal uterine myometrium cells using the isobaric tags for relative and absolute quantitation labeling method. Western blotting, fluorescence-activated cell sorting analyses, and immunohistochemistry were used to examine CD70 expression in the membrane proteins in uterine leiomyosarcoma cell lines and clinical samples. We developed an antibody-drug conjugate with a monoclonal antibody of the target membrane protein linked to monomethyl auristatin F and investigated its antitumor effects against uterine leiomyosarcoma (in vitro, in vivo, and in patient-derived xenograft models).

Results: CD70 was identified as a specific antigen highly expressed in uterine leiomyosarcoma cell lines. Of the 3 uterine leiomyosarcoma cell lines, CD70 expression was confirmed in SK-LMS-1 cells by western blotting and fluorescence-activated cell sorting analysis. CD70 overexpression was observed in 19 of 21 (90.5%) tumor specimens from women with uterine leiomyosarcoma. To generate CD70-antibody-drug conjugate, anti-CD70 monoclonal antibody was conjugated with a novel derivative of monomethyl auristatin F. CD70-antibody-drug conjugate showed significant antitumor effects on SK-LMS-1 cells (half maximal inhibitory concentration, 0.120 nM) and no antitumor effects on CD70-negative uterine leiomyosarcoma cells. CD70-antibody-drug conjugate significantly inhibited tumor growth in the SK-LMS-1 xenograft mouse model (tumor volume, 129.8 vs 285.5 mm; relative reduction, 54.5%; P<.001) and patient-derived xenograft mouse model (tumor volume, 128.1 vs 837.7 mm; relative reduction, 84.7%; P<.001).

Conclusion: Uterine leiomyosarcoma tumors highly express CD70 and targeted therapy with CD70-antibody-drug conjugate may have a potential therapeutic implication in the treatment of uterine leiomyosarcoma.
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http://dx.doi.org/10.1016/j.ajog.2020.08.028DOI Listing
February 2021