Clinical Trial Protocol for Porcine Islet Xenotransplantation in South Korea.

Background: Islet transplantation holds promise for treating selected type 1 diabetes mellitus patients, yet the scarcity of human donor organs impedes widespread adoption. Porcine islets, deemed a viable alternative, recently demonstrated successful longterm survival without zoonotic risks in a clinically relevant pig-to-non-human primate islet transplantation model. This success prompted the development of a clinical trial protocol for porcine islet xenotransplantation in humans. Methods: A single-center, open-label clinical trial initiated by the sponsor will assess the safety and efficacy of porcine islet transplantation for diabetes patients at Gachon Hospital. The protocol received approval from the Gachon Hospital Institutional Review Board (IRB) and the Korean Ministry of Food and Drug Safety (MFDS) under the Investigational New Drug (IND) process. Two diabetic patients, experiencing inadequate glycemic control despite intensive insulin treatment and frequent hypoglycemic unawareness, will be enrolled. Participants and their family members will engage in deliberation before xenotransplantation during the screening period. Each patient will receive islets isolated from designated pathogen-free pigs. Immunosuppressants and systemic infection prophylaxis will follow the program schedule. The primary endpoint is to confirm the safety of porcine islets in patients, and the secondary endpoint is to assess whether porcine islets can reduce insulin dose and the frequency of hypoglycemic unawareness. Conclusion: A clinical trial protocol adhering to global consensus guidelines for porcine islet xenotransplantation is presented, facilitating streamlined implementation of comparable human trials worldwide.

Association between heat and hospital admissions in people with disabilities in South Korea: a nationwide, case-crossover study.

BACKGROUND: Despite extensive findings on the hazardous impacts of environmental heat exposure, little is known about the effect on people with disabilities. This study aimed to estimate the association between environmental heat exposure and emergency department admissions for people with disabilities compared with people without disabilities. METHODS: In this nationwide, case-crossover study, we linked data on emergency department admissions (cases) for any cause in the warm season in South Korea from the Korean National Health Insurance Service (NHIS)-National Sample Cohort database (a nationally representative database of 1 million systematically sampled beneficiaries covering all ages) from Jan 1, 2002, to Dec 31, 2019, and short-term daily mean temperature exposure (measured via Google Earth Engine at a 9 km spatial grid, aggregated to district). We defined beneficiaries with disabilities as those who were registered as disabled in the NHIS; disabilities included in our study were physical disability, brain lesion disorders, blindness or vision loss, and deafness or hearing loss. Other types of disability were not included for confidentiality reasons. A time-stratified case-crossover design, in which participants served as their own control, was used with conditional logistic regression to estimate the association between heat and emergency department admissions in people with and without disabilities. FINDINGS: 23 792 emergency department admissions were recorded for 59 527 people with disabilities. Of these 23 792 admissions, 10 234 (43·0%) individuals were female and 13 558 (57·0%) were male. The odds ratio (OR) of emergency department admissions associated with heat (99th temperature percentile vs 75th percentile) was 1·15 (95% CI 1·07-1·24) in people with disabilities and 1·06 (1·04-1·09) in people without disabilities. The annual excess number of emergency department admissions attributable to heat per 100 000 persons-years was 27·81 admissions (95% CI 9·20-45·69) and excess medical costs were US$638 739·47 (95% CI 201 900·12-1 059 641·87) in people with disabilities; these values were more than four times that of the non-disabled population. People with brain lesion disorders, people with severe physical disabilities, female individuals, and those aged 65 years or older showed higher heat risks. The risks of emergency department admissions due to mental disorder (1·89, 95% CI 1·18-3·00) and respiratory diseases (1·34, 1·06-1·70) also showed higher heat risks than for the other two analysed causes of admission (cardiovascular and genitourinary diseases). INTERPRETATION: Heat was associated with increased risk of emergency department admissions for people with and without disabilities, but the risk appeared to be higher for those with disabilities. These results can inform policy makers when establishing action plans for people with disabilities. FUNDING: National Research Foundation of Korea, the South Korean Ministry of Environment, and the South Korean Ministry of Education.

A Comparison Between GalT-/-;hCD39;hCD55 and GalT-/-;hCD39;hCD46;hCD55;TBM Pig Kidneys Transplanted in Nonhuman Primates.

Because there is a shortage of donor kidneys, researchers are exploring the possibility of using genetically modified pig kidneys for transplantation. Approaches involving knockout of carbohydrate genes or knockin of protective proteins have been attempted to determine the best gene modifications. In this study, we utilized GalT-/-;hCD39;hCD55 and GalT-/-;hCD39;hCD46;hCD55;thrombomodulin (TBM) pigs for transplantation in nonhuman primates (NHPs). The NHPs survived for 4 weeks after kidney transplantation (4 WAT) from the GalT-/-;hCD39;hCD55 pig and for 6 WAT from the GalT-/-;hCD39;hCD46;hCD55;TBM pig. However, messenger RNA (mRNA) sequencing and immunohistochemistry analysis revealed that the 6 WAT kidney exhibited more severe apoptosis, inflammation, loss of renal function, and renal fibrosis than the 4 WAT kidney. These results indicate that additional knockin of complement regulator (hCD46) and coagulation regulator (TBM) is not enough to prevent renal damage, suggesting that improved immune suppression is needed for more prolonged survival.

Initial investigation on the feasibility of porcine red blood cells from genetically modified pigs as an alternative to human red blood cells for transfusion.

The decline in blood donation rates and the ongoing shortage of blood products pose significant challenges to medical societies. One potential solution is to use porcine red blood cells (pRBCs) from genetically modified pigs as an alternative to human red blood cells (hRBCs). However, adverse immunological reactions remain a significant obstacle to their use. This study aimed to evaluate the compatibility of diverse genetically modified pRBCs with human serum. We acquired human complement-competent serum, complement 7 (C7)-deficient serum, and hRBCs from all ABO blood types. Additionally, we used leftover clinical samples from health checkups for further evaluation. pRBCs were collected from wild-type (WT) and genetically modified pigs: triple knockout (TKO), quadruple KO (QKO), and TKO/hCD55.hCD39 knockin (hCD55.hCD39KI). The extent of C3 deposition on RBCs was measured using flow cytometry after incubation in C7-deficient serum diluted in Ca++-enriched or Ca++-depleted and Mg++-enriched buffers. The binding of immunoglobulin (Ig) M/IgG antibody to RBCs after incubation in ABO-type human serum was evaluated using flow cytometry. Naïve human serum- or sensitized monkey serum-mediated hemolysis was also evaluated. Phagocytosis was assessed by incubating labeled RBCs with the human monocytic cell line THP-1 and measurement by flow cytometry. All three genetic modifications significantly improved the compatibility of pRBCs with human serum relative to that of WT pRBCs. The extent of IgM/IgG binding to genetically modified pRBCs was lower than that of WT pRBCs and similar to that of O-type hRBCs. Total and alternative pathway complement activation in all three genetically modified pRBCs was significantly weaker than that in WT pRBCs and did not differ from that in O-type hRBCs. The extent of serum-mediated hemolysis and phagocytosis of these genetically modified pRBCs was low and similar to that of O-type hRBCs. Sensitized monkey serum-mediated hemolysis in QKO and TKO/hCD55.hCD39KI pRBCs was higher than in O-type hRBCs but lower than in TKO pRBCs. The elimination of porcine carbohydrate antigens in genetically modified pigs significantly enhanced pRBC compatibility with naïve human sera, which was comparable to that of O-type hRBCs. These findings provide valuable insights into the development of pRBCs as potential alternatives to hRBCs.

Comparative transcriptome analysis between long- and short-term survival after pig-to-monkey cardiac xenotransplantation reveals differential heart failure development.

Cardiac xenotransplantation is the potential treatment for end-stage heart failure, but the allogenic organ supply needs to catch up to clinical demand. Therefore, genetically-modified porcine heart xenotransplantation could be a potential alternative. So far, pig-to-monkey heart xenografts have been studied using multi-transgenic pigs, indicating various survival periods. However, functional mechanisms based on survival period-related gene expression are unclear. This study aimed to identify the differential mechanisms between pig-to-monkey post-xenotransplantation long- and short-term survivals. Heterotopic abdominal transplantation was performed using a donor CD46-expressing GTKO pig and a recipient cynomolgus monkey. RNA-seq was performed using samples from POD60 XH from monkey and NH from age-matched pigs, D35 and D95. Gene-annotated DEGs for POD60 XH were compared with those for POD9 XH (Park et al. 2021). DEGs were identified by comparing gene expression levels in POD60 XH versus either D35 or D95 NH. 1,804 and 1,655 DEGs were identified in POD60 XH versus D35 NH and POD60 XH versus D95 NH, respectively. Overlapped 1,148 DEGs were annotated and compared with 1,348 DEGs for POD9 XH. Transcriptomic features for heart failure and inhibition of T cell activation were observed in both long (POD60)- and short (POD9)-term survived monkeys. Only short-term survived monkey showed heart remodeling and regeneration features, while long-term survived monkey indicated multi-organ failure by neural and hormonal signaling as well as suppression of B cell activation. Our results reveal differential heart failure development and survival at the transcriptome level and suggest candidate genes for specific signals to control adverse cardiac xenotransplantation effects.

Recent trends in perioperative blood transfusion during elective kidney transplantation.

Background: Accurately predicting the demand for blood transfusions is crucial for blood banks. Given the potential for emergency situations, it is imperative that blood banks maintain a sufficient inventory of blood products. In this study, we examined the use of perioperative transfusions in patients undergoing elective kidney transplants. Methods: Data on all complement-dependent cytotoxicity-crossmatched assays between 2013 and 2022 were collected. We excluded repeated assays and patients who did not undergo kidney transplantation. Transfusion records and transfusion adverse reactions were reviewed retrospectively. Results: In total, 30 patients underwent elective kidney transplantation from 2013 to 2022. The mean age of the patients was 48.1±9.7 years. The male-to-female ratio was 1.5:1. Four patients received transfusions intraoperatively, whereas eight patients were transfused postoperatively. The postoperative hemoglobin level of the transfusion group (n=9, 8.9±1.3) was significantly lower than that of the nontransfusion group (n=21, 10.4±1.2). The most commonly transfused blood product intraoperatively was leuko-reduced filtered red blood cells, followed by fresh frozen plasma. When the study period was divided into two halves based on the time of operation, the first half showed a higher number of significant transfusions. Conclusions: In most elective kidney transplant cases, surgery was conducted without the need for blood transfusion. The timing of transfusion, when necessary, shifted from during the operation to after the operation. The implementation of patient blood management, coupled with advancements in surgical techniques, appears to have impacted the pattern of perioperative transfusion.

Comparison of Graft Survival Between Full-Thickness and Lamellar Pig-to-Monkey Corneal Xenotransplantation from the Same Genetically Engineered Pig Model with Minimal Immunosuppression.

BACKGROUND: The graft survival rate of full-thickness corneal xenotransplantation (XTP) with minimal immunosuppression in genetically engineered pigs is unknown, whereas lamellar corneal XTP shows satisfactory results. We compared graft survival between full-thickness and lamellar transplantations in the same genetically engineered pig. METHODS: Six pig-to-monkey corneal transplantations were performed on 3 transgenic pigs. Two corneas harvested from 1 pig were transplanted into 2 monkeys using full-thickness and lamellar corneal xenotransplantation. The transgenic donor pigs used were α1,3-galactosyltransferase gene-knockout + membrane cofactor protein (GTKO+CD46) in one recipient and GTKO+CD46+ thrombomodulin (TBM) in the other. RESULTS: The graft survival time for GTKO+CD46 XTP was 28 days. With the addition of TBM, the survival differences between lamellar and full-thickness XTP were 98 days versus 14 days and >463 days (ongoing) versus 21 days, respectively. An excessive number of inflammatory cells was observed in failed grafts, but none were in the recipient's stromal bed. CONCLUSIONS: Unlike full-thickness corneal XTP, lamellar xenocorneal transplantation does not exhibit surgical complications, such as retrocorneal membrane or anterior synechia. The graft survival of lamellar XTP in this study was not as good as in our previous experiments, although the survival period was superior to that of full-thickness XTP. The difference in graft survival based on transgenic type is not definitive. Further studies using transgenic pigs and minimal immunosuppression need to focus on improving graft survival of lamellar XTP and using a larger sample size to determine the potential of full-thickness corneal XTP.

Clinical Applicability of Automated Hematology Analyzer Research Parameters for Disease Diagnosis: a Reference Interval Study.

BACKGROUND: We established reference intervals for research parameters of complete blood cell count and examined their usefulness for diagnosing certain diseases. METHODS: Reference intervals for 26 basic and 38 research parameters were established for 3,457 and 1,325 men and 2,742 and 830 women aged 20 - 59 and ≥ 60 years, respectively. Research parameter values for patients with iron deficiency anemia (IDA), appendicitis, sepsis, and myelodysplastic syndromes (MDS) were compared against gender- and age-matched reference values. RESULTS: Seven basic and 10 research parameters among men and one research parameter among women required partitioning by age. No partitioning by gender was required. Further, 67% patients with IDA showed micro red blood cell ratio values above the upper reference limits of their corresponding age and gender subgroups; 3% and 5% patients with appendicitis showed immature granulocyte percentages and counts above the upper reference limits, respectively; 12% - 42% of patients with sepsis showed numerous values exceeded their reference limits, and 67% and 100% patients with MDS showed neutrophil cell complexity and structural dispersion values outside their reference ranges, respectively. CONCLUSIONS: Overall, < 60% of research parameter values were outside their reference ranges among most patients, indicating their limited diagnostic usefulness.

Impact of the COVID-19 Pandemic on the Usage of Blood for Transfusions: A 2-Year Experience from a Tertiary Center in Korea.

The coronavirus disease (COVID-19) outbreak affected the utilization and management of blood products in hospitals. Blood shortages occurred owing to social distancing policies and reduction in blood donors. However, only a few studies examined whether these changes affected blood usage and transfusion patterns. We retrospectively reviewed blood component usage according to hospital departments and phases of surgery in transfused patients admitted between 1 March 2019 and 28 February 2021, in a single center in Anyang, Korea. We also analyzed the length of hospital stay and mortality to determine prognosis. In 2020, 32,050 blood components were transfused to 2877 patients, corresponding to 15.8% and 11.8% less than the rates in 2019, respectively. Postoperative usage of blood products significantly decreased in 2020 (3.87 ± 6.50) compared to 2019 (7.12 ± 21.71) (p = 0.047). The length of hospital stay of the patients who underwent postoperative transfusion in 2019 (n = 197) was 13.97 ± 11.95 days, which was not significantly different from that in 2020 (n = 167), i.e., 16.44 ± 17.90 days (p = 0.118). Further, 9 of 197 postoperative transfusion patients died in 2019, while 8 of 167 patients died in 2020 (p = 0.920). The COVID-19 pandemic resulted in limited blood supply and reduced postoperative transfusions; however, patient prognosis was not affected.

Biological response of nonhuman primates to controlled levels of acute blood loss.

Introduction: The global shortage of human blood for medical use has prompted the development of alternative blood sources. Nonhuman primates (NHPs) are commonly used owing to their physiological similarities to humans. The objective of the current study was to establish a controlled-blood-loss model in NHPs to explore their clinical and biological responses. Methods: Blood was sequentially withdrawn from 10 cynomolgus monkeys (10, 14, 18, 22, and 25% of the total blood volume); their vital signs were monitored, and blood parameters were serially analyzed. Humoral mediators in the blood were measured using flow cytometry and enzyme-linked immunosorbent assays. Results: In NHPs subjects to 25% blood loss and presenting with related clinical symptoms, the systolic blood pressure ratio on day 0 after bleeding was significantly lower than that of the animals from the other groups (median: 0.65 vs. 0.88, P = 0.0444). Red blood cell counts from day 0-14 and hematocrit levels from day 0-7 were markedly decreased relative to the baseline (P < 0.01). These parameters showed a direct correlation with the extent of blood loss. The levels of creatine phosphokinase, aspartate aminotransferase, and alanine aminotransferase exhibited increases in response to blood loss and had a stronger correlation with the hemoglobin ratio than the volume of blood loss. The levels of C3a and C4a, as well as interleukin (IL)-1α and IL-15, displayed a strong correlation, with no apparent association with blood loss. Conclusion: The findings of the present study showed that only NHPs with 25% blood loss exhibited clinical decompensation and significant systolic blood pressure reduction without fatalities, suggesting that this level of blood loss is suitable for evaluating blood transfusion efficacy or other treatments in NHP models. In addition, the ratio of hemoglobin may serve as a more dependable marker for predicting clinical status than the actual volume of blood loss. Thus, our study could serve as a basis for future xenotransfusion research and to predict biological responses to massive blood loss in humans where controlled experiments cannot be ethically performed.

Treatment strategies for Lennox-Gastaut syndrome: outcomes of multimodal treatment approaches.

Background: Multimodal treatment approaches are often considered for patients with Lennox-Gastaut syndrome (LGS). Creating an algorithm that can guide healthcare providers in selecting treatment options for patients with LGS remains a challenge. Herein, we assessed the long-term seizure-free and neurodevelopmental outcomes of stepwise multimodal treatment in patients with LGS. Objective: Herein, we assess the long-term seizure-free and neurodevelopmental outcomes of stepwise multimodal treatment in patients with LGS. Methods: We retrospectively examined the data of 371 patients with LGS who underwent stepwise multimodal treatment, including antiseizure medication (ASM) therapy, dietary therapy (DT), resective epilepsy surgery (R-ES), and palliative epilepsy surgery (P-ES). The seizure-free outcome was considered to be the effect of the final treatment according to the treatment algorithm, and the percentage of patients who remained seizure-free in each treatment group was calculated. ASM treatment, DT, R-ES, and P-ES were applied to 371 (100%), 201 (54.2%), 112 (30.2%), and 115 (31.0%) patients with LGS, respectively. We evaluated the stepwise multimodal treatment outcomes in these patients. Results: One hundred sixty-eight patients (45.3%) remained seizure-free for at least 1 year (seizure-free-for-1-year group), 61 of whom (16.5%) remained seizure-free for more than 5 years (remained-seizure-free group). Among the patients treated with ASM therapy, DT, R-ES, and P-ES, 41 (11.1%), 53 (14.3%), 56 (15.1%), and 29 (7.8%), respectively, remained seizure-free for 1 year. In addition, 15 (4.1%), 15 (4.1%), 19 (5.1%), and 12 (3.2%) patients in the ASM, DT, R-ES, and P-ES treatment groups, respectively, remained seizure-free for more than 5 years. Both the seizure-free-for-1-year and remained-seizure-free groups showed significant improvement in electroencephalography findings and neurodevelopmental status following treatment. Conclusion: This study provides an update on the long-term seizure outcomes and neurodevelopmental improvements in a large cohort of patients with LGS following comprehensive multimodal treatment. We emphasize that the active combination of multiple ASMs, DT, and surgical treatment could provide long-term seizure-free outcomes and significant neurological benefits to patients with LGS.

Calidad de la formación en el postgrado de especialización en pediatría clínica de la FCM-UNA: un estudio de percepciones de sus estudiantes, actuales y graduados / Quality of training in the postgraduate specialization in clinical pediatrics of the FCM-UNA: a study of perceptions of its current students and graduates

Introducción: Los procesos de acreditación de la calidad de los programas de formación de médicos tienen importancia en cuanto al impulso que pueden dar a la formación de mejores profesionales. El reconocimiento de la calidad educativa de un determinado programa o carrera por parte de los organismos facultados, como la Agencia Nacional de Evaluación y Acreditación de la Educación Superior (ANEAES) es una garantía del cumplimiento de los criterios mínimos de calidad establecidos. Sin embargo, la calidad de un postgrado no solo es valorada por el cumplimiento de los criterios de calidad mínimos establecidos por los organismos encargados, sino por otros criterios como serían la percepción de los estudiantes, actuales y graduados sobre la calidad de la formación ofrecida por el mismo. Objetivos: El presente trabajo tiene como objetivo general estudiar la calidad de la formación en el Postgrado de Especialización en Pediatría Clínica de la FCM-UNA, a partir de las percepciones de sus estudiantes, actuales y graduados. Materiales y métodos: La metodología se enmarca en la tradición cualitativa de investigación social y en su instrumentación se incluye la realización de entrevistas semiestructuradas aplicadas a los estudiantes actuales (residentes) y graduados (egresados) del programa, así como el desarrollo, implementación y análisis de matrices, con la discusión de los resultados correspondientes a cada una de ellas. Resultados: Los resultados obtenidos muestran que la mayor parte de los estudiantes, actuales y egresados, del Postgrado de Especialización en Pediatría Clínica de la FCM UNA, perciben que la formación en este es de buena a mediana calidad. Los factores que inciden positivamente en la percepción de la calidad de la formación son: el ámbito de desarrollo del programa, el plan de estudios, las actividades académicas, la calidad de la composición y el desempeño del plantel docente, así como las relaciones interpersonales, tanto con los docentes como con sus pares; con algunas excepciones puntuales. La inclusión en el currículum de los aspectos relativos a la responsabilidad social universitaria también tuvo una incidencia positiva. Los factores que incidieron negativamente en la percepción de la calidad de la formación fueron: la carga horaria excesiva, el sistema de evaluación exigente, las falencias en la infraestructura y las tareas de asistencia social, en desmedro de la optimización del tiempo para la formación. En lo referente a la incidencia de la pandemia de COVID 19 en la calidad de la formación, las percepciones fueron dispares al interior de los casos de estudio que cursaron el postgrado durante la misma, con elementos negativos y llamativamente algunos elementos positivos. A la hora de sopesar en qué medida los factores positivos y negativos incidieron globalmente en la percepción de la calidad de la formación, se evidenció que, a pesar del reconocimiento de la existencia de varios factores negativos, los primeros primaron sobre los segundos. Esto hace que la mayoría de los estudiantes actuales y egresados manifiesten su satisfacción con el postgrado y los graduados con su desempeño autónomo al cumplir con sus expectativas. Conclusión: La mayor parte de los estudiantes, actuales y graduados del Postgrado de especialización en Pediatría Clínica de la FCM UNA, perciben que la formación en el mismo es de buena a mediana calidad, con aspectos a mejorar que son mencionados en las recomendaciones.

Introduction: The quality accreditation processes of medical training programs are important in terms of the boost they can give to the training of better professionals. Recognition of the educational quality of a certain program or career by authorized bodies, such as the National Agency for the Evaluation and Accreditation of Higher Education (ANEAES) is a guarantee of compliance with the minimum established quality criteria. However, the quality of a postgraduate course is not only assessed by compliance with the minimum quality criteria established by the bodies in charge, but by other criteria such as the perception of current and graduate students about the quality of the training offered by the same. Objectives: The general objective is to study the quality of training in the Postgraduate Specialization in Clinical Pediatrics of the FCM-UNA, based on the perceptions of its current and graduate students. Materials and methods: The methodology is part of the qualitative tradition of social research and its instrumentation includes semi-structured interviews applied to current students (residents) and graduates of the program, as well as the development, implementation and analysis of matrices, with the discussion of the results corresponding to each of them. Results: The results obtained show that most of the students, current and graduates, of the Postgraduate Specialization in Clinical Pediatrics of the FCM UNA, perceive that the training is of good to medium quality. Factors that positively affect the perception of the quality of training are: the scope of development of the program, the study plan, academic activities, quality of the composition and performance of the teaching staff, as well as interpersonal relationships, both with teachers and with their peers; with some specific exceptions. The inclusion in the curriculum of aspects related to university social responsibility also had a positive impact. Factors that negatively affected perception of the quality of training were: excessive workload, the demanding evaluation system, shortcomings in the infrastructure and the tasks of social service, to the detriment of time dedicated to optimizing training. Regarding the incidence of the COVID 19 pandemic on the quality of training, perceptions were uneven within the case studies that took the postgraduate course during it, with negative elements and strikingly some positive elements. When weighing up the extent to which the positive and negative factors had a global impact on the perception of the quality of training, it was shown that, despite the recognition of the existence of several negative factors, the former prevailed over the latter. This makes the majority of current students and graduates express their satisfaction with the postgraduate course and graduates with their autonomous performance in meeting their expectations. Conclusion: Most of the students, current and graduates of the Postgraduate specialization in Clinical Pediatrics of the FCM UNA, perceive that the training in it is of good to medium quality, with aspects to improve that are mentioned in the recommendations.

Effect of Factor H on Complement Alternative Pathway Activation in Human Serum Remains on Porcine Cells Lacking N-Glycolylneuraminic Acid.

Background: Triple knockout (TKO) donor pigs lacking alpha-1,3-galactose (Gal), N-glycolylneuraminic acid (Neu5Gc), and Sd(a) expressions were developed to improve the clinical success of xenotransplantation. Neu5Gc, a sialic acid expressed on cell surfaces, recruits factor H to protect cells from attack by the complement system. Lack of Neu5Gc expression may cause unwanted complement activation, abrogating the potential benefit of gene-modified donor pigs. To investigate whether TKO porcine cells display increased susceptibility to complement activation in human serum, pathway-specific complement activation, apoptosis, and human platelet aggregation by porcine cells were compared between alpha-1,3-galactosyltransferase gene-knockout (GTKO) and TKO porcine cells. Methods: Primary porcine peripheral blood mononuclear cells (pPBMCs) and endothelial cells (pECs) from GTKO and TKO pigs were used. Cells were incubated in human serum diluted in gelatin veronal buffer (GVB++) or Mg++-EGTA GVB, and C3 deposition and apoptotic changes in these cells were measured by flow cytometry. C3 deposition levels were also measured after incubating these cells in 10% human serum supplemented with human factor H. Platelet aggregation in human platelet-rich plasma containing GTKO or TKO pECs was analyzed. Results: The C3 deposition level in GTKO pPBMCs or pECs in GVB++ was significantly higher than that of TKO pPBMCs or pECs, respectively, but C3 deposition levels in Mg++-EGTA-GVB were comparable between them. The addition of factor H into the porcine cell suspension in 10% serum in Mg++ -EGTA-GVB inhibited C3 deposition in a dose-dependent manner, and the extent of inhibition by factor H was similar between GTKO and TKO porcine cells. The percentage of late apoptotic cells in porcine cell suspension in GVB++ increased with the addition of human serum, of which the net increase was significantly less in TKO pPBMCs than in GTKO pPBMCs. Finally, the lag time of platelet aggregation in recalcified human plasma was significantly prolonged in the presence of TKO pECs compared to that in the presence of GTKO pECs. Conclusion: TKO genetic modification protects porcine cells from serum-induced complement activation and apoptotic changes, and delays recalcification-induced human platelet aggregation. It does not hamper factor H recruitment on cell surfaces, allowing the suppression of alternative complement pathway activation.

Early Interferon-Gamma Response in Nonhuman Primate Recipients of Solid-Organ Xenotransplantation.

BACKGROUND: To understand changes in biological responses in nonhuman primate (NHP) recipients of xenotransplantation (XTP), we retrospectively investigated chronological changes in cytokine profiles of NHP recipients after solid-organ XTP. METHODS: Plasma samples were collected from 7 NHP recipients of pig heart or kidney XTP with α-1,3-galactosyltransferase gene knockout (GTKO) under anti-CD154-based immune suppression at the following time points: immediately before; 2 hours, 3 days, and 7 days after XTP; and weekly thereafter until the graft failed. The plasma levels of the following cytokines were measured: interleukin (IL)-1α, IL-1ß, IL-6, IL-12p70, IL-8, IL-10, IL-15, tumor necrosis factor, interferon gamma (IFN-γ), D-dimer, C3a, and histone-complexed DNA fragments. For in vitro experiments, human natural killer (NK) cells were cocultured with wild-type porcine endothelial cells (PECs), GTKO-PECs, and human umbilical vein endothelial cells, with or without anti-CD154 antibody. IFN-γ levels in the culture supernatants were compared. RESULTS: IFN-γ levels peaked on day 7 or 10 of XTP and then decreased to basal levels, whereas proinflammatory cytokine levels increased along with the elevation of histone-complexed DNA fragments and were sustained until xenograft failure. In vitro, human NK cells produced more IFN-γ when in contact with wild-type PECs than with human umbilical vein endothelial cells, which was not reduced by the use of GTKO-PECs or addition of anti-CD154 antibody to the mixture. CONCLUSIONS: In NHP recipients of XTP, the early peak of IFN-γ priming subsequent inflammatory responses may be attributed to NK cell activation in response to xenografts.

Immunoglobulin gene rearrangement in Koreans with multiple myeloma: Clonality assessment and repertoire analysis using next-generation sequencing.

INTRODUCTION: We assessed the applicability of next-generation sequencing (NGS)-based IGH/IGK clonality testing and analyzed the repertoire of immunoglobulin heavy chain (IGH) or immunoglobulin kappa light chain (IGK) gene usage in Korean patients with multiple myeloma (MM) for the first time. METHODS: Fifty-nine bone marrow samples from 57 Korean patients with MM were analyzed, and NGS-based clonality testing that targeted the IGH and IGK genes was performed using IGH FR1 and IGK primer sets. RESULTS: Clonal IGH and IGK rearrangements were observed in 74.2% and 67.7% of samples from Korean patients with kappa-restricted MM, respectively (90.3% had one or both), and in 60.7% and 95.5% of samples from those with lambda-restricted MM, respectively (85.7% had one or both). In total, 88.1% of samples from Koreans with MM had clonal IGH and/or IGK rearrangement. Clonal rearrangement was not significantly associated with the bone marrow plasma cells as a proportion of all BM lymphoid cells. IGHV3-9 (11.63%) and IGHV4-31 (9.30%) were the most frequently reported IGHV genes and were more common in Koreans with MM than in Western counterparts. IGHD3-10 and IGHD3-3 (13.95% each) were the most frequent IGHD genes; IGHD3-3 was more common in Koreans with MM. No IGK rearrangement was particularly prevalent, but single IGKV-J rearrangements were less common in Koreans with kappa-restricted MM than in Western counterparts. IGKV4-1 was less frequent in Koreans regardless of light chain type. Otherwise, the usages of the IGH V, D, and J genes and of the IGK gene were like those observed in previous Western studies. CONCLUSION: NGS-based IGH/IGK clonality testing ought to be applicable to most Koreans with MM. The overrepresentation of IGHV3-9, IGHV4-31, and IGHD3-3 along with the underrepresentation of IGKV4-1 and the differences in IGK gene rearrangement types suggest the existence of ethnicity-specific variations in this disease.

Long-term control of diabetes in a nonhuman primate by two separate transplantations of porcine adult islets under immunosuppression.

Porcine islet transplantation is an alternative to allo-islet transplantation. Retransplantation of islets is a routine clinical practice in islet allotransplantation in immunosuppressed recipients and will most likely be required in islet xenotransplantation in immunosuppressed recipients. We examined whether a second infusion of porcine islets could restore normoglycemia and further evaluated the efficacy of a clinically available immunosuppression regimen including anti-thymocyte globulin for induction; belimumab, sirolimus, and tofacitinib for maintenance and adalimumab, anakinra, IVIg, and tocilizumab for inflammation control in a pig to nonhuman primate transplantation setting. Of note, all nonhuman primates were normoglycemic after the retransplantation of porcine islets without induction therapy. Graft survival was >100 days for all 3 recipients, and 1 of the 3 monkeys showed insulin independence for >237 days. Serious lymphodepletion was not observed, and rhesus cytomegalovirus reactivation was controlled without any serious adverse effects throughout the observation period in all recipients. These results support the clinical applicability of additional infusions of porcine islets. The maintenance immunosuppression regimen we used could protect the reinfused islets from acute rejection.

A comparison of the analysis of 3 types of body fluids using the XN-350 hematology analyzer versus light microscopy assessment.

ABSTRACT: We evaluated the capacity of the XN-350 instrument to analyze 3 different types of body fluid samples under "body fluid mode."The performance of XN-350 was evaluated in terms of precision, carryover, limit of blank, limit of detection, limit of quantification, and linearity. Cell enumeration and differential data produced by the XN-350 were compared to manual chamber counting results in 63 cerebrospinal fluid (CSF), 51 ascitic fluid, and 51 pleural fluid (PF) samples. Comparisons between XN-350 versus Cytospin data were also performed in PF samples.The precision, carry-over, limit of blank, and linearity of the XN-350 were acceptable. The limits of detection for white blood cells (WBCs) and red blood cells were 1.0/µL, and 1,000.0/µL, respectively; the corresponding limits of quantitation (LOQs) were 5.0/µL and 2,000.0/µL, respectively. The XN-350's cell enumeration and differential counting correlated well with those of manual chamber counting for all 3 sample types (except for differential counting in CSF samples), particularly parameters involving monocytes (r = 0.33) and mononuclear cells (MO- body fluid [BF]; r = 0.26), as well as total cell (TC-BF) enumeration (r = 0.50) and WBC-BF (r = 0.50) in PF samples. The MO-BF in CSF samples differed significantly from manual chamber counting results, but neither TC-BF nor WBC-BF in PF samples did. The XN-350 also showed good correlations with Cytospin analyses for differential counting of neutrophils, lymphocytes, and monocytes in PF samples. The differential counting of eosinophils via the XN-350 and Cytospin were not significantly correlated, but the difference between them was not significant.The XN-350 is an acceptable alternative to manual fluid analysis. Samples with low cellularity around the LOQ should be checked manually. Moreover, manual differential counting should be performed on CSF samples, particularity those with low cell numbers.

Immunoglobulin M and Immunoglobulin G Subclass Distribution of Anti-galactose-Alpha-1,3-Galactose and Anti-N-Glycolylneuraminic Acid Antibodies in Healthy Korean Adults.

BACKGROUND: Human preformed antibodies (Abs), anti-galactose-alpha-1,3-galactose (Gal) and anti-N-glycolylneuraminic acid (Neu5Gc), can react with porcine antigens of wild-type pigs. To provide basic population data of the Abs for potential application in clinical xenotransplantation, we developed enzyme-linked immunosorbent assay methods and investigated the serum titers of anti-Gal and anti-Neu5Gc Abs, including immunoglobulin (Ig) M and IgG along with its subclasses, in humans. METHODS: Anti-Gal and anti-Neu5Gc Abs serum titers were measured in 380 healthy Korean adults using the in-house enzyme-linked immunosorbent assays. The frequency and median values of anti-Gal and anti-Neu5Gc were measured, and their class and subclass distribution were evaluated. RESULTS: The detection frequencies of anti-Gal were 99.2%, 95.0%, 23.2%, 94.5%, 12.4%, and 3.4% for IgM, IgG, IgG1, IgG2, IgG3, and IgG4, respectively. The detection frequencies of anti-Neu5Gc Abs were 87.4%, 96.6%, 1.6%, 46.3%, 0.0%, and 0.0% for IgM, IgG, IgG1, IgG2, IgG3, and IgG4, respectively. The median values of anti-Gal IgM (1001.6 ng/mL) and IgG (1198.3 ng/mL) were significantly higher than those of anti-Neu5Gc Abs (IgM, 328.4 ng/mL; IgG, 194.7 ng/mL; P < .001). IgG2 titers of both anti-Gal and anti-Neu5Gc Abs correlated better with the IgG class than the titers of other IgG subclasses. CONCLUSIONS: The titers of anti-Gal Abs were higher than those of anti-Neu5Gc Abs. IgG2 was the main IgG subclass in both anti-Gal and anti-Neu5Gc Abs. Variation in the titers of anti-Gal or anti-Neu5Gc Abs may partly explain the biological and immunologic changes that occur in recipients of xenotransplants.

Comprehensive Analysis of Cardiac Xeno-Graft Unveils Rejection Mechanisms.

Porcine heart xenotransplantation is a potential treatment for patients with end-stage heart failure. To understand molecular mechanisms of graft rejection after heart transplantation, we transplanted a 31-day-old alpha-1,3-galactosyltransferase knockout (GTKO) porcine heart to a five-year-old cynomolgus monkey. Histological and transcriptome analyses were conducted on xenografted cardiac tissue at rejection (nine days after transplantation). The recipient monkey's blood parameters were analyzed on days -7, -3, 1, 4, and 7. Validation was conducted by quantitative real-time PCR (qPCR) with selected genes. A non-transplanted GTKO porcine heart from an age-matched litter was used as a control. The recipient monkey showed systemic inflammatory responses, and the rejected cardiac graft indicated myocardial infarction and cardiac fibrosis. The transplanted heart exhibited a total of 3748 differentially expressed genes compared to the non-transplanted heart transcriptome, with 2443 upregulated and 1305 downregulated genes. Key biological pathways involved at the terminal stage of graft rejection were cardiomyopathies, extracellular interactions, and ion channel activities. The results of qPCR evaluation were in agreement with the transcriptome data. Transcriptome analysis of porcine cardiac tissue at graft rejection reveals dysregulation of the key molecules and signaling pathways, which play relevant roles on structural and functional integrities of the heart.

Long-term porcine islet graft survival in diabetic non-human primates treated with clinically available immunosuppressants.

BACKGROUND: Although pancreatic islet transplantation is becoming an effective therapeutic option for patients with type 1 diabetes (T1D) who suffer from a substantially impaired awareness of hypoglycemia, its application is limited due to the lack of donors. Thus, pig-to-human islet xenotransplantation has been regarded as a promising alternative due to the unlimited number of "donor organs." Long-term xenogeneic islet graft survival in pig-to-non-human primate (NHP) models has mainly been achieved by administering the anti-CD154 mAb-based immunosuppressant regimen. Since the anti-CD154 mAb treatment has been associated with unexpected fatal thromboembolic complications in clinical trials, the establishment of a new immunosuppressant regimen that is able to be directly applied in clinical trials is an urgent need. METHODS: We assessed an immunosuppressant regimen composed of clinically available agents at porcine islet transplantation in consecutive diabetic NHPs. RESULTS: Porcine islet graft survival in consecutive diabetic NHPs (n = 7; >222, >200, 181, 89, 62, 55, and 34 days) without severe adverse events. CONCLUSION: We believe that our study could contribute greatly to the initiation of islet xenotransplantation clinical trials.