Pig Kidney Research Articles

Features of accumulation and variability of stable strontium in some organs and tissues of landrace pigs

The authors in the article demonstrated the results of assessing the level of stable strontium in the skeletal muscles, liver, kidneys and spleen of Landrace pigs at the end of the technological cycle. The studies were performed on clinically healthy animals raised in a large pig farm in the Altai Territory. The conditions of keeping the animals corresponded to those typical for meat fattening up to 100 kg. Feeding was carried out with complete feed. Elemental analysis of samples of parenchymal organs and muscle tissue was performed using inductively coupled plasma atomic emission spectral analysis. Data processing was performed using Microsoft Office Excel and the R programming language in the data analysis environment RStudio version 2022.07.2+576 (RStudio, PBC). The distribution did not correspond to normal in all cases. The dispersions are not homogeneous. Based on the mean value and median, an increasing ranked series of strontium content in organs and tissues were established: liver = skeletal muscles < spleen < kidneys, in numerical terms: 1: 1: 3.7: 4.7 and 1: 1: 3.9: 5.7 respectively. Medians for stable strontium in the liver, kidney, spleen, and skeletal muscle were 0.024; 0.130; 0.089 and 0.023 mg/kg, respectively. Reference intervals for the liver are 0.004-0.043 and for the spleen - 0.030-0.145 mg/kg. The most significant range of variability is characteristic of the kidneys of pigs. A considerable uniformity is typical of the liver and skeletal muscles. Using the Kruskal-Wallis test, it was established that the accumulation of stable strontium significantly differs in the examined organs and tissues of pigs (H = 68.9, df = 3, p < 0.0001). Pairwise comparison showed significant differences for the kidney-liver, spleen-liver, skeletal muscle-kidney, and skeletal muscle-spleen pairs. Two clusters were identified according to the similarity of strontium accumulation: liver and skeletal muscles, kidneys and spleen. The calcium-strontium ratio, taking into account medians, for skeletal muscles was 1:1833, and for the liver - 1:1870. The established results can serve as an approximate physiological norm for the concentration of stable strontium in individual structures of the organism of Landrace pigs under the conditions of Western Siberia.

Molecular hydrogen: prospective treatment strategy of kidney damage after cardiac surgery.

Cardiac surgery-associated acute kidney injury is a common post-operative complication, mostly due to increasing oxidative stress. Recently, molecular hydrogen (H2 gas) has also been applied to cardiac surgery due to its ability to reduce oxidative stress. We evaluated the potential effect of H2 application on the kidney in an in vivo model of simulated heart transplantation. Pigs underwent cardiac surgery within 3 h while connected to extracorporeal circulation (ECC) and subsequent 60 min of spontaneous reperfusion of the heart. We used two experimental groups: T-pigs after transplantation and TH-pigs after transplantation treated with 4% H2 mixed with air during inhalation of anesthesia and throughout oxygenation of blood in ECC. The levels of creatinine, urea and phosphorus were measured in plasma. Renal tissue samples were analyzed by Western blot method for protein levels of nuclear factor erythroid 2-related factor 2 (Nrf2), Kelch-like ECH-associated protein 1 (Keap-1), and superoxide dismutase (SOD1). After cardiac surgery, selected plasma biomarkers were elevated. However, H2 therapy was followed by the normalization of all these parameters. Our results suggest activation of Nrf2/Keap1 pathway as well as increased SOD1 protein expression in the group treated with H2. The administration of H2 had a protective effect on the kidneys of pigs after cardiac surgery, especially in terms of normalization of plasma biomarkers to control levels.

Profiling of Chromatin Accessibility in Pigs across Multiple Tissues and Developmental Stages

The study of chromatin accessibility across tissues and developmental stages is essential for elucidating the transcriptional regulation of various phenotypes and biological processes. However, the chromatin accessibility profiles of multiple tissues in newborn pigs and across porcine liver development remain poorly investigated. Here, we used ATAC-seq and rRNA-depleted RNA-seq to profile open chromatin maps and transcriptional features of heart, kidney, liver, lung, skeletal muscle, and spleen in newborn pigs and porcine liver tissue in the suckling and adult stages, respectively. Specifically, by analyzing a union set of protein-coding genes (PCGs) and two types of transcripts (lncRNAs and TUCPs), we obtained a comprehensive annotation of consensus ATAC-seq peaks for each tissue and developmental stage. As expected, the PCGs with tissue-specific accessible promoters had active transcription and were relevant to tissue-specific functions. In addition, other non-coding tissue-specific peaks were involved in both physical activity and the morphogenesis of neonatal tissues. We also characterized stage-specific peaks and observed a close association between dynamic chromatin accessibility and hepatic function transition during liver postnatal development. Overall, this study expands our current understanding of epigenetic regulation in mammalian tissues and organ development, which can benefit both economic trait improvement and improve the biomedical usage of pigs.

#6465 MIR-451B AND CHRONIC KIDNEY DISEASE: A NOVEL TARGET TO MODULATE RENAL VEGF SIGNALING

Abstract Background and Aims Chronic kidney disease (CKD) is a progressive disorder that affects over 15% of adults worldwide. A major component of CKD pathophysiology is renal microvascular rarefaction and loss that pairs the progression of the disease and complications. We developed a translational swine model of CKD and showed that loss of renal function associates with a significant decrease in renal vascular endothelial growth factor (VEGF) signaling and progressive microvascular (MV) rarefaction. VEGF is a key endogenous cytokine for MV proliferation and repair, but underlying mechanisms of renal VEGF downregulation in CKD are unknown. We hypothesized that micro-RNA (miRNA)-mediated processes contribute to the downregulation of renal VEGF signaling in CKD. Method Unbiased renal micro-RNA (miRNA)-seq was performed in a swine model of CKD stage 3 (14 weeks of bilateral renal artery stenosis + atherogenic diet) and normal controls (n = 5/group). Micro-RNAs up- (>1.4 FC, p<0.05) and down-regulated (<0.7 FC, p<0.05) were identified, followed by target prediction analysis (miRwalk 3.0, Target Scan 7.2). Renal gene and protein expression of VEGF was measured by qPCR and western blot, respectively, and renal MV architecture was studied by 3D micro-CT. Results Unbiased miRNA-seq and target prediction analysis identified four miRs upregulated in the kidney of CKD pigs vs. controls, of which miR-451b (validated by qPCR), broadly conserved among species, was capable of targeting VEGFA (FC = 4.46, p = 0.04). Renal gene and protein expression of VEGF was downregulated in CKD vs. controls, associated with significant cortical and medullary microvascular rarefaction (Figure 1). Conclusion Our data suggest that post-transcriptional modulation of renal VEGF signaling may serve as a prominent mechanism of VEGF downregulation, which leads to MV rarefaction and disease progression in CKD. Furthermore, this work in progress identified a novel candidate (miR-451b) and may set the stage for its targeted modulation to preserve VEGF signaling as a potential strategy to protect the renal microvasculature in CKD.

Intermittent Surface Oxygenation Results in Similar Mitochondrial Protection and Maintenance of Aerobic Metabolism as Compared to Continuous Oxygenation during Hypothermic Machine Kidney Machine Perfusion.

Short bubble and subsequent surface oxygenation is an innovative oxygenation technique and alternative for membrane oxygenation during hypothermic machine perfusion (HMP). The metabolic effect of the interruption of surface oxygenation for 4 h (mimicking organ transport) during HMP was compared to continuous surface and membrane oxygenation in a pig kidney ex situ preservation model. After 30 min of warm ischemia by vascular clamping, a kidney of a ±40 kg pig was procured and subsequently preserved according to one of the following groups: (1) 22-h HMP + intermittent surface oxygenation (n = 12); (2) 22-h HMP + continuous membrane oxygenation (n = 6); and (3) 22-h HMP + continuous surface oxygenation (n = 7). Brief perfusate O2 uploading before kidney perfusion was either obtained by direct bubble (groups 1, 3) or by membrane (group 2) oxygenation. Bubble oxygenation during minimum 15 min was as efficient as membrane oxygenation in achieving supraphysiological perfusate pO2 levels before kidney perfusion. Metabolic tissue analysis (i.e., lactate, succinate, ATP, NADH, and FMN) during and at the end of the preservation period demonstrated similar mitochondrial protection between all study groups. Short bubble and subsequent intermittent surface oxygenation of the perfusate of an HMP-kidney might be an effective and cheap preservation strategy to protect mitochondria, eliminating the need/costs of a membrane oxygenator and oxygen source during transport.

Zinc trafficking: 1,10-phenanthroline, glutathione, and other metal binding ligands form adducts with proteomic Zn2+

Hypotheses were tested that the proteome of pig kidney LLC-PK1 cells (i) contains Zn-proteins that react with a diversity of native and pharmacologically active metal-binding ligands to form ternary complexes and (ii) includes proteins that bind Zn2+ nonspecifically and together form ternary adducts with a variety of metal-binding agents. The method to observe ternary complex formation with Zn-proteins and proteome•Zn involved preformation of fluorescent TSQ [6-Methoxy-(8-p-toluenesulfonamido)quinoline]-Zn-proteins and/or proteome•Zn-TSQ adducts followed by competitive reaction with selected ligands. The loss of TSQ-dependent fluorescence signaled the replacement of TSQ by the competing ligand in the starting adducts. In vitro, 1,10-phenanthroline competed effectively with TSQ for binding to Zn-proteins in the proteome. The successful competition of 1,10-phenanthroline with TSQ-Zn-proteins was also observed in cells. Similarly, 1,10-phenanthroline was shown to bind to a sizable fraction of Zn2+ associated adventitiously with proteome (proteome•Zn). Other synthetic ligands that bind to Zn-proteins and proteome•Zn include 2,2-bipyridyl, 8-hydroxyquinoline, 2,2'-dicarboxypyridine, and pyrithione. Such results suggest that ligand binding to such sites may play a role in the observed biological effects of these and other metal-binding molecules. Although cysteine does not significantly compete with TSQ, glutathione displaces TSQ from Zn-proteins and proteome•Zn at concentrations well below those found in cells, implying that ternary complex formation involving glutathione may be physiologically significant.

Assessing Kidney Injury Induced by Mercuric Chloride in Guinea Pigs with In Vivo and In Vitro Experiments.

Acute kidney injury, which is associated with high levels of morbidity and mortality, affects a significant number of individuals, and can be triggered by multiple factors, such as medications, exposure to toxic chemicals or other substances, disease, and trauma. Because the kidney is a critical organ, understanding and identifying early cellular or gene-level changes can provide a foundation for designing medical interventions. In our earlier work, we identified gene modules anchored to histopathology phenotypes associated with toxicant-induced liver and kidney injuries. Here, using in vivo and in vitro experiments, we assessed and validated these kidney injury-associated modules by analyzing gene expression data from the kidneys of male Hartley guinea pigs exposed to mercuric chloride. Using plasma creatinine levels and cell-viability assays as measures of the extent of renal dysfunction under in vivo and in vitro conditions, we performed an initial range-finding study to identify the appropriate doses and exposure times associated with mild and severe kidney injuries. We then monitored changes in kidney gene expression at the selected doses and time points post-toxicant exposure to characterize the mechanisms of kidney injury. Our injury module-based analysis revealed a dose-dependent activation of several phenotypic cellular processes associated with dilatation, necrosis, and fibrogenesis that were common across the experimental platforms and indicative of processes that initiate kidney damage. Furthermore, a comparison of activated injury modules between guinea pigs and rats indicated a strong correlation between the modules, highlighting their potential for cross-species translational studies.

First transplantation of a pig heart from a multiple gene-modified donor, porcine cytomegalovirus/roseolovirus, and antiviral drugs.

First transplantation of a pig heart from a multiple gene-modified donor, porcine cytomegalovirus/roseolovirus, and antiviral drugs.

PD28-03 EFFICACY AND SAFETY OF A MAGNETIC HYDROGEL FOR STONE FRAGMENT REMOVAL: AN IN VITRO AND IN VIVO STUDY

You have accessJournal of UrologyCME1 Apr 2023PD28-03 EFFICACY AND SAFETY OF A MAGNETIC HYDROGEL FOR STONE FRAGMENT REMOVAL: AN IN VITRO AND IN VIVO STUDY Daniel Massana Roquero, T. Jessie Ge, Grace H. Holton, Kathleen E. Mach, Zachary Kornberg, Ryan Sun, Simon Conti, Shan X. Wang, and joseph C. Liao Daniel Massana RoqueroDaniel Massana Roquero More articles by this author , T. Jessie GeT. Jessie Ge More articles by this author , Grace H. HoltonGrace H. Holton More articles by this author , Kathleen E. MachKathleen E. Mach More articles by this author , Zachary KornbergZachary Kornberg More articles by this author , Ryan SunRyan Sun More articles by this author , Simon ContiSimon Conti More articles by this author , Shan X. WangShan X. Wang More articles by this author , and joseph C. Liao joseph C. Liao More articles by this author View All Author Informationhttps://doi.org/10.1097/JU.0000000000003313.03AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookLinked InTwitterEmail Abstract INTRODUCTION AND OBJECTIVE: Ureteroscopic laser lithotripsy is the standard treatment for kidney stones<2 cm. Following lithotripsy, fragments<3 mm are often left behind due to limitations of standard stone retrieval tools and time. Residual stone fragments that do not pass contribute to recurrence. We have developed MagSToNE, an integrated stone fragment removal system consisting of a magnetic hydrogel to coat and magnetize stone fragments and a magnetic wire for extraction. Here we compare MagSToNE with standard nitinol basket extraction in an in vitro model and assess the safety of the approach in vivo in a porcine model. METHODS: Stone fragments (15-30, < 3 mm) were loaded in an ex vivo kidney model to compare ureteroscopic removal time and efficiency using MagSToNE and a conventional basket. With APLAC approval MagSToNE was assessed in vivo in porcine models. Fragments were introduced endoscopically to the kidneys of female pigs under general anesthesia. Magnetic hydrogel components were injected via flexible ureteroscopy, and magnetized stones were captured with a magnetic wire inserted through the working channel of the ureteroscope. Pigs were immediately euthanized or monitored for up to 72 hours post-procedure. Blood was collected at regular intervals, starting at hydrogel injection, for complete blood count (CBC) and metabolic panels. After nephrectomy, kidneys were bivalved for gross examination and processed for histology. RESULTS: In the in vitro model, we found that MagSToNE provided a faster (up to 57 %) and more efficient approach for extraction of kidney stone fragments than conventional basketing. Further, we demonstrated the feasibility of MagSToNE in in vivo porcine models. The injection catheter and magnetic wire (1 mm diameter) showed excellent compatibility with a disposable clinical ureteroscope, and magnetized fragments were removed through the ureteral access sheath. Initial results from in vivo MagSToNE application indicate that the hydrogel and extraction method are safe as no significant changes in vitals, blood count, or metabolic serum chemistries were observed in the porcine models. Visual inspection of the bivalved kidneys post procedure confirmed the absence of visible hydrogel. Survival studies in the porcine model are ongoing to further assess the safety of MagSToNE. CONCLUSIONS: MagSToNE is a promising approach to render patients stone free during ureteroscopy. We demonstrated that MagSToNE is superior to basketing in model systems, and we applied our technology in vivo to demonstrate the safe magnetic extraction of kidney stone fragments. Source of Funding: NIH R21DK131776 © 2023 by American Urological Association Education and Research, Inc.FiguresReferencesRelatedDetails Volume 209Issue Supplement 4April 2023Page: e819 Advertisement Copyright & Permissions© 2023 by American Urological Association Education and Research, Inc.MetricsAuthor Information Daniel Massana Roquero More articles by this author T. Jessie Ge More articles by this author Grace H. Holton More articles by this author Kathleen E. Mach More articles by this author Zachary Kornberg More articles by this author Ryan Sun More articles by this author Simon Conti More articles by this author Shan X. Wang More articles by this author joseph C. Liao More articles by this author Expand All Advertisement PDF downloadLoading ...

Androgen-Dependent Expression of CUX2 mRNA in the Pig Liver Is Associated with That of Drug Metabolizing Enzymes and Drug Transporters.

We previously identified androgen-dependent sex differences in the mRNA expression of drug metabolizing enzymes (DMEs), including CYPs, sulfotransferases and uridine 5'-diphospho-glucuronosyltransferases, and drug transporters in the pig liver and kidney. To elucidate the mechanism for such sex differences in pigs, we herein focused on the key regulators cut-like homeobox 2 (Cux2), B-cell lymphoma 6 (Bcl6), and signal transducer and activator of transcription 5b (Stat5b), which are reported to be responsible for the sex-biased gene expression of Cyps in the mouse liver. We used real-time RT-PCR to examine androgen-dependent sex differences in the mRNA levels of these regulators in the liver and kidney basically using Meishan and Landrace pigs. Significant sex differences (male > female) in the level of CUX2 mRNA were detected in the liver of both breeds, and levels were significantly decreased in males by castration and increased in castrated males and intact females by administering testosterone propionate. No such clear androgen-dependent sex differences in hepatic BCL6 or STAT5B mRNA expression were observed in either breed. In the kidney, androgen-dependent gene expression of these regulators was not observed. In the liver, CUX2 mRNA expression closely correlated with that of DMEs and drug transporters, which were previously shown to have androgen-dependent expression. Together, these findings demonstrate that hepatic CUX2 mRNA is expressed in an androgen-dependent manner, and strongly suggest that CUX2 plays a key role in the androgen-dependent gene expression of hepatic DMEs and drug transporters.

A A Novel Computed Tomography-Ultrasound Image Fusion Technique for Guiding the Percutaneous Kidney Access.

To describe the feasibility of computed tomography (CT)-ultrasound image fusion technique on guiding percutaneous kidney access in vitro and vivo. we compare CT-ultrasound image fusion technique and ultrasound for percutaneous kidney puncture guidance by using an in vitro pig kidney model. The fusion method, fusion time, ultrasound screening time, and success rate of puncture were compared between the groups. Next, patients with kidney stones in our hospital were randomized in the study of simulated puncture guidance. The general condition of patients, fusion method, fusion time, and ultrasound screening time were compared between the groups. A total of 45 pig models were established, including 23 in CT-ultrasound group and 22 in ultrasound group. The ultrasound screening time in CT-ultrasound group was significantly shorter than that in ultrasound group (P < .001). In addition, the success rate of puncture in CT-ultrasound group was significantly higher than that in ultrasound group (P =.015). Furthermore, in simulated PCNL puncture study, baseline data including age, BMI, and S.T.O.N.E score between the two groups showed no statistical difference. The ultrasound screening time of the two groups was (2.60 ± 0.33) min and (3.37 ± 0.51) min respectively, and the difference was statistically significant (P < .001). Our research revealed that CT-ultrasound image fusion technique was a feasible and safe method to guide PCNL puncture. Compared with traditional ultrasound guidance, CT-ultrasound image fusion technique can shorten the learning curve of PCNL puncture, improve the success rate of puncture, and shorten the ultrasound screening time.

Assessment of glomerular filtration and tubular secretion in baboons with life-supporting pig kidney grafts.

With pig kidney xenotransplantation nearing clinical reality, it is imperative to measure pig kidney function in the graft recipients. Our aims were (i) to compare inulin clearance after a short intravenous (IV) bolus with steady-state inulin IV infusion, (ii) to use this method to measure the glomerular filtration rate (GFR), and (iii) to determine the tubular secretory function using cefoxitin in a pig-to-baboon renal transplant model. A short IV infusion of inulin and cefoxitin were followed by a maintenance IV infusion of inulin over 5 h in seven healthy baboons, three healthy pigs, and five baboons after bilateral native nephrectomy and intra-abdominal pig renal transplantation. Blood and urine samples were collected. Serum and urinary inulin and serum cefoxitin concentrations measured by validated assays were used to calculate GFR and renal secretion. GFR calculatedwere similar by both methods. The body weight normalized total body clearance of inulin was similar in pigs and baboons despite differences in absolute clearances. Pig kidney transplanted into baboons provided similar clearance in baboons when normalized to baboon body weight and sustained filtration and secretory functions. The study documented that pig kidneys support the physiologic needs of baboons and are likely to support human recipients as well.

The impact of oxygen supply and erythrocytes during normothermic kidney perfusion

The influence of erythrocytes and oxygen concentration on kidneys during long-term normothermic kidney perfusion is under debate. This study compares acellular and erythrocyte-based NMP with focus on oxygen delivery to the tissue as well as the effects of high oxygenation on tissue integrity. Pig kidneys were connected to NMP for six hours. The first group (n = 6; AC500) was perfused without addition of oxygen carriers, arterial perfusate pO2 was maintained at 500 mmHg. In the second group (n = 6; RBC500) washed erythrocytes were added to the perfusate at pO2 of 500 mmHg. Third group (n = 6; RBC200) was perfused with erythrocyte containing perfusate at more physiological pO2 of 200 mmHg. Addition of RBC did not relevantly increase oxygen consumption of the kidneys during perfusion. Likewise, there were no differences in kidney functional and injury parameters between AC500 and RBC500 group. Expression of erythropoietin as indicator of tissue hypoxia was comparable in all three groups. Cell free NMP at supraphysiological oxygen partial pressure seems to be a safe alternative to erythrocyte based perfusion without adverse effect on kidney integrity and provides a less cumbersome application of NMP in clinical practice.

Renin-angiotensin-aldosterone system function in the pig-to-baboon kidney xenotransplantation model

After pig-to-baboon kidney transplantation, episodes of hypovolemia and hypotension from an unexplained mechanism have been reported. This study evaluated the renin-angiotensin-aldosterone system post–kidney xenotransplantation. Kidneys from genetically-engineered pigs were transplanted into 5 immunosuppressed baboons after the excision of the native kidneys. Immunosuppressive therapy was based on the blockade of the CD40/CD154 costimulation pathway. Plasma renin, angiotensinogen (AGT), angiotensin II (Ang II), aldosterone levels, and urine osmolality and electrolytes were measured in healthy pigs, healthy nonimmunosuppressed baboons, and immunosuppressed baboons with life-supporting pig kidney grafts. After pig kidney transplantation, plasma renin and Ang II levels were not significantly different, although Ang II trended lower, even though plasma AGT and potassium were increased. Plasma aldosterone levels were unchanged. Urine osmolality and sodium concentration were decreased. Even in the presence of increasing AGT and potassium levels, lower plasma Ang II concentrations may be because of reduced, albeit not absent, the reactivity of pig renin to cleave baboon AGT, suggesting an impaired response of the renin-angiotensin-aldosterone system to hypovolemic and hypotensive episodes. The maintenance of aldosterone may be protective. The reduced urine osmolality and sodium concentration reflect the decreased ability of the pig kidney to concentrate urine. These considerations should not prohibit successful clinical pig kidney xenotransplantation.

High-power laser lithotripsy - do we treat or harm? Histological evaluation of temperature effects in an in vivo study with thulium fiber laser.

The aim of this study was to evaluate the possible histopathological alterations that occur in the kidneys due to a continuous temperature increase above 43°C for one hour of lithotripsy using a newly introduced thulium fiber laser (TFL). Two female pigs were used. After the insertion of a 9.5/11.5 ureteral access sheath, flexible ureteroscopy and laser lithotripsy for one hour were conducted. A TFL laser with a 200-μm fiber was used. The power setting used was 8 W (0.5 J × 16 Hz). A K-type thermocouple was inserted and fixed in the upper calyx of the right porcine kidney to record the temperature in the pelvicalyceal system during the laser activation. Second-look flexible nephroscopy followed by nephrectomy and pathohistological evaluation of the operated kidney was performed one week after the procedure in the first pig and 2 weeks after the surgery in the second pig. Flexible nephroscopy did not reveal significant differences between the 2 porcine kidneys. Nevertheless, the histopathological report demonstrated severe alterations in the kidney of the first pig. Mild changes were reported in the kidney of the second pig. A significant improvement in inflammation and haemorrhagic lesions was demonstrated when comparing the 2 kidneys. The difference demonstrated between the 2 kidneys based on the histopathological report shows that the healing process is capable of improving severe to mild alterations within a one-week time frame. Two weeks after the surgery, only minor changes were observed, suggesting that even temperature increases above the threshold can be tolerated regarding renal damage.

A photoelectrochemical aptasensor for tetracycline based on the self-assembly of 2D MoS2 on a 3D ZnO/Au/ITO electrode.

Two-dimensional (2D) layered MoS2 has good dispersion and adsorption properties, but being a narrow bandgap semiconductor limits its application in photoelectric sensing. In this study, a homogeneous photoelectrochemical sensor based on three-dimensional (3D) ZnO/Au/2D MoS2 is proposed for the ultrasensitive detection of tetracycline (TET). MoS2 is uniformly embedded on the 3D ZnO/Au surface by ordered self-assembly. The physical method of π-π interaction of MoS2 replaces the conventional use of chemically modifying aptamers on the electrode material surface. Under optimal conditions, this method has been successfully applied to the detection of TET in milk, honey, pig kidney and pork samples with reliable results. We believe that this study presents a method for the preparation of sensing carriers and target detection with great potential for application.

Physiological and Pathological Evaluation of Mechanically Ventilated Anesthetized Pigs

Prolonged assisted ventilation during anesthesia and critical care is associated with increased morbidity and mortality in both humans and animals. However, no study of mechanically ventilated patients has examined the mortality-related physiology and pathology. Our study investigated physiological, hematological and pathological changes in pigs that died during anesthesia. Six pigs were placed under ventilation-assisted anesthesia for up to 48h. The heart rate (HR), respiratory rate (RR), mean arterial blood (MAP), end-tidal carbon dioxide (ETCO2), and oxygen saturation (SpO2) were monitored every 15min until the pigs dies. Blood glucose, serum lactate, pH, and serum bicarbonate (HCO3-) were measured every 12h until death. Tissues were harvested from lung, liver, heart, and kidney of pigs immediately after death during anesthesia. All pigs died during anesthesia. Times of death varied. One pig died at approximately 18h (Group I), two pigs at 24h (Group II), one pig at 36h (Group III), and two pigs at 48h (Group IV). Decreased MAP was reported in all groups throughout anesthesia. Each group showed different changes before death; Group I and III showed decreased blood glucose (45mg/dL) and (17mg/dL), respectively; Group III showed elevated serum lactate (9.76 mmol/l) and reduced pH (7.13) that suggested metabolic acidosis; Group IV showed increased ETCO2 (64.35±5.79 mmHg). Pigs that survived longer showed a higher level of cellular injury to the liver and respiratory system. Blood glucose, MAP, and ETCO2 should be carefully monitored during anesthesia for better patient outcomes and reduced mortality.

Establishment of guinea pig kidney cell lines with potential application in the production of a classical swine fever live GPE- vaccine.

Classical swine fever (CSF) live vaccine used in Japan, GPE- strain, is produced using guinea pig kidney (GPK)-derived primary culture cells. This means that a large number of guinea pigs are used to generate the primary GPK cells needed to produce the CSF live vaccine, and alternative solution is desired. Hence, we established two GPK cell lines capable of culturing the GPE- strain: spontaneously immortalized GPK (GPK-SI) cells were generated by repeated passaging of primary GPK cells, and the other cell line, artificially immortalized GPK (GPK-AI) cells, were obtained by introducing the SV40 large T antigen gene into primary GPK cells. Both cell lines were susceptible to the GPE- virus, and the virus grew more efficiently in GPK-SI cells at 37°C. When the culture temperature was set to 30°C, the virus titer reached 104.8 50% Tissue Culture Infectious Dose (TCID50)/mL in GPK-SI cells 7 days after virus inoculation at a multiplicity of infection (MOI) of 1, which was equivalent to that in cells cultured at 37°C. When the virus was inoculated at MOI <1, the virus titer 7 days after inoculation was higher when cultured at 30°C than when cultured at 37°C in both cell lines, reaching 105.63 TCID50/mL in GPK-SI cells. These results indicate that GPK-SI and GPK-AI cells can potentially replace primary GPK cells for the production of CSF live vaccines. This could also contribute to stable CSF vaccine production and animal welfare.

How, where and when to screen for porcine cytomegalovirus (PCMV) in donor pigs for xenotransplantation

Porcine cytomegalovirus (PCMV), that is actually a porcine roseolovirus (PRV), is a common herpesvirus in domestic pigs and wild boars. In xenotransplantation, PCMV/PRV has been shown to significantly reduce the survival time of pig kidneys and hearts in preclinical trials with different non-human primates. Furthermore, PCMV/PRV has been transmitted in the first pig to human heart xenotransplantation and contributed to the death of the patient. Although transmitted to the recipient, there is no evidence that PCMV/PRV can infect primate cells including human cells. PCMV/PRV is closely related to the human herpesviruses 6 and 7, and only distantly related to the human CMV (HCMV). Antiviral drugs used for the treatment of HCMV are less effective against PCMV/PRV. However, there are well described strategies to eliminate the virus from pig facilities. In order to detect the virus and to eliminate it, highly sensitive detection methods and the knowledge of how, where and when to screen the donor pigs is required. Here, a comparative testing of organs from pigs of different ages using polymerase chain reaction (PCR)-based and immunological methods was performed. Testing young piglets, PCMV/PRV was detected effectively by PCR in blood, bronchoalveolar lavage fluid, tonsils and heart. In adult animals, detection by PCR was not successful in most cases, because the virus load was below the detection limit or the virus was in its latent stage. Therefore, detection of antibodies against selected recombinant proteins corresponding to epitopes detected by nearly all infected animals in a Western blot assay is advantageous. By contrast, immunological testing is not beneficial in young animals as piglets might have PCMV/PRV-specific antibodies obtained from their infected mother via the colostrum. Using a thoughtful combination of PCR-based and immunological methods, detection of PCMV/PRV in donor pigs for xenotransplantation is feasible and a controlled elimination of the virus by early weaning or other methods is possible.

Patient informed consent for a clinical trial of gene-edited pig kidney transplantation: A representative consent form.

When clinical trials of gene-edited pig organ transplantation are initiated, the consent form that the patient is requested to sign will be an important document. Consent to receive a pig xenograft will have significant differences when compared with the requirements of most experimental clinical procedures. We here suggest a consent form for pig kidney transplantation that addresses the major points that will be required and hope it will provide a basis for discussion and future modification, if necessary. There is purposely some repetition in the document, but we believe this is necessary to ensure that the patient has a clear understanding of what he/she is consenting to.