Extraction of redox extracellular vesicles using exclusion-based sample preparation.

Studying specific subpopulations of cancer-derived extracellular vesicles (EVs) could help reveal their role in cancer progression. In cancer, an increase in reactive oxygen species (ROS) happens which results in lipid peroxidation with a major product of 4-hydroxynonenal (HNE). Adduction by HNE causes alteration to the structure of proteins, leading to loss of function. Blebbing of EVs carrying these HNE-adducted proteins as a cargo or carrying HNE-adducted on EV membrane are methods for clearing these molecules by the cells. We have referred to these EVs as Redox EVs. Here, we utilize a surface tension-mediated extraction process, termed exclusion-based sample preparation (ESP), for the rapid and efficient isolation of intact Redox EVs, from a mixed population of EVs derived from human glioblastoma cell line LN18. After optimizing different parameters, two populations of EVs were analyzed, those isolated from the sample (Redox EVs) and those remaining in the original sample (Remaining EVs). Electron microscopic imaging was used to confirm the presence of HNE adducts on the outer leaflet of Redox EVs. Moreover, the population of HNE-adducted Redox EVs shows significantly different characteristics to those of Remaining EVs including smaller size EVs and a more negative zeta potential EVs. We further treated glioblastoma cells (LN18), radiation-resistant glioblastoma cells (RR-LN18), and normal human astrocytes (NHA) with both Remaining and Redox EV populations. Our results indicate that Redox EVs promote the growth of glioblastoma cells, likely through the production of H2O2, and cause injury to normal astrocytes. In contrast, Remaining EVs have minimal impact on the viability of both glioblastoma cells and NHA cells. Thus, isolating a subpopulation of EVs employing ESP-based immunoaffinity could pave the way for a deeper mechanistic understanding of how subtypes of EVs, such as those containing HNE-adducted proteins, induce biological changes in the cells that take up these EVs.

Label-free detection of the cytotoxicity effect of cisplatin in human leukemic cells using Raman spectroscopy in conjunction with multivariate analysis.

The single-cell Raman spectra of human leukemic Jurkat cells can be obtained by confocal microscopy Raman spectroscopy, including cell groups treated with different doses of cisplatin (0, 3.5, 7, 10.5 and 14 µmol L-1) for 24 hours and those treated with 10.5 µmol L-1 cisplatin for different times (0, 6, 12, 24 and 36 hours). The structure and amount of protein, nucleic acid and other major molecules from different cell groups show special changes in the percentage of biochemical constituents. Compared with the control group, the two protein Raman bands (1449 and 1659 cm-1) and two DNA bands (1303 and 1338 cm-1) in the treatment groups decrease in intensity with the increase of the drug dose and treatment time of cisplatin. Partial least squares combined with support vector machines was used to develop diagnostic algorithms for distinguishing between control and treatment groups. The support vector machines for classification between the control group (0 µmol L-1) and cell groups treated with 10.5 and 14 µmol L-1 cisplatin for 24 hours have achieved good diagnostic results with a high sensitivity of 100%, specificity of 100% and accuracy of 100%, respectively, indicating that 10.5 µmol L-1 can be used as an appropriate therapeutic dose. Using the same method, the diagnostic sensitivity, specificity and accuracy between the control group (0 hours) and cell groups treated with 10.5 µmol L-1 cisplatin for 24 and 36 hours are all 100%, showing that 24 hours can be used as an appropriate therapeutic time. These results showed that Raman spectroscopy in conjunction with multivariate statistical analysis could be a useful tool for evaluating the cytotoxicity induced by cisplatin in human leukemic cells.

Role of vesicoureteral reflux on pediatric kidney allograft function.

BACKGROUND: Vesicoureteral reflux (VUR) is a common urologic complication of pediatric kidney transplant, though there is little data on the effect of VUR on histologic graft changes or graft survival. METHODS: All pediatric patients who received a kidney transplant from 2007 to 2020 were selected for retrospective chart review. All participants underwent a voiding cystourethrogram (VCUG) at a 6-month post-transplant. Patients were then categorized into two groups based on vesicoureteral reflux grade: no/low-grade VUR (grades 0-2) and high-grade VUR (grades 3-5). Outcomes collected included graft failure rates, graft function, urinary tract infections (UTIs), proteinuria, and Banff scores at 3- and 12-month post-transplant surveillance kidney biopsies. RESULTS: There were 74 pediatric patients who received a kidney transplant in the designated time-period, and of those 39 had no/low-grade VUR and 35 had high-grade VUR. There was no difference in graft failure among the two groups over time when stratified for age (p = 0.389, CI 0.53-5.08). Patients with high grade VUR had a higher risk of UTI development overall (RR 1.89, 95%CI 1-3.6, p = 0.041), mostly accounted for from increased development of febrile UTI (RR 1.66, 95%CI 1.1-2.6, p = 0.038). CONCLUSIONS: Unselected pediatric kidney transplant recipients with high-grade vesicoureteral reflux on VCUG at a 6-month post-kidney transplant are more likely to have febrile UTI compared to those in the low-grade VUR group. There is no difference in graft survival among the two groups.

BK polyomavirus infection: more than 50 years and still a threat to kidney transplant recipients.

BK polyomavirus (BKPyV) is a ubiquitous human polyomavirus and a major infection after kidney transplantation, primarily due to immunosuppression. BKPyV reactivation can manifest as viruria in 30%-40%, viremia in 10%-20%, and BK polyomavirus-associated nephropathy (BKPyVAN) in 1%-10% of recipients. BKPyVAN is an important cause of kidney graft failure. Although the first case of BKPyV was identified in 1971, progress in its management has been limited. Specifically, there is no safe and effective antiviral agent or vaccine to treat or prevent the infection. Even in the current era, the mainstay approach to BKPyV is a reduction in immunosuppression, which is also limited by safety (risk of de novo donor specific antibody and rejection) and efficacy (graft failure). However, recently BKPyV has been getting more attention in the field, and some new treatment strategies including the utilization of viral-specific T-cell therapy are emerging. Given all these challenges, the primary focus of this article is complications associated with BKPyV, as well as strategies to mitigate negative outcomes.

Rapid intracellular pH measurement based on electroporation- surface-enhanced Raman scattering.

In this study, electroporation-surface-enhanced Raman scattering (SERS) was applied to rapidly measure intracellular pH. The generation of a sensitive SERS probe for measuring pH in the range of 6.0-8.0 was accomplished through the conjugation of the pH-sensitive molecule 4-mercaptobenzoic acid (4-MBA) to the surface of gold nanoparticles (Au NPs) through its thiol functional group. This bioprobe was then rapidly introduced into nasopharyngeal carcinoma CNE-1 cells by electroporation, followed by SERS scanning and the fitting of intensity ratios of each detection point's Raman peaks at 1423 cm-1 and 1072 cm-1, to create the pH distribution map of CNE-1 cells. The electroporation-SERS assay introduces pH bioprobes into a living cell in a very short time and disperses the nanoprobe throughout the cytoplasm, ultimately enabling rapid and comprehensive pH analysis of the entire cell. Our work demonstrates the potential of electroporation-SERS for the biochemical analysis of live cells.

Oxalate Nephropathy After Kidney Transplantation: Risk Factors and Outcomes of Two Phenotypes.

Describing risk factors and outcomes in kidney transplant recipients with oxalate nephropathy (ON) may help elucidate the pathogenesis and guide treatment strategies. We used a large single-center database to identify patients with ON and categorized them into delayed graft function with ON (DGF-ON) and late ON. Incidence density sampling was used to select controls. A total of 37 ON cases were diagnosed between 1/2011 and 1/2021. DGF-ON (n = 13) was diagnosed in 1.05% of the DGF population. Pancreatic atrophy on imaging (36.4% vs. 2.9%, p = 0.002) and gastric bypass history (7.7% vs. 0%; p = 0.06) were more common in DGF-ON than with controls with DGF requiring biopsy but without evidence of ON. DGF-ON was not associated with worse graft survival (p = 0.98) or death-censored graft survival (p = 0.48). Late ON (n = 24) was diagnosed after a mean of 78.2 months. Late ON patients were older (mean age 55.1 vs. 48.4 years; p = 0.02), more likely to be women (61.7% vs. 37.5%; p = 0.03), have gastric bypass history (8.3% vs. 0.8%; p = 0.02) and pancreatic atrophy on imaging (38.9% vs. 13.3%; p = 0.02). Late ON was associated with an increased risk of graft failure (HR 2.0; p = 0.07) and death-censored graft loss (HR 2.5; p = 0.10). We describe two phenotypes of ON after kidney transplantation: DGF-ON and late ON. Our study is the first to our knowledge to evaluate DGF-ON with DGF controls without ON. Although limited by small sample size, DGF-ON was not associated with adverse outcomes when compared with controls. Late ON predicted worse allograft outcomes.

Histopathological Features and Role of Allograft Kidney Biopsy Among Recipients With Prolonged Delayed Graft Function: A Review.

Delayed graft function (DGF) is an early posttransplant complication predictive of adverse outcomes. This "acute kidney injury of transplantation" is often defined as allograft dysfunction requiring renal replacement within 7 d posttransplantation. DGF is an important area of study because it is emerging with efforts to expand the donor pool and address the supply-demand gap in kidney transplantation. DGF is often caused by severe kidney injury mechanisms because of multiple donors, recipients, and immunologic factors. The role of kidney biopsy, particularly in prolonged DGF, is an ongoing area of research and inquiry for clinicians and researchers alike to better define, manage, and predict outcomes of this early posttransplant event. This review aims to provide an in-depth, comprehensive summary of the literature to date on the histopathology of DGF and the role of kidney transplant biopsies in prolonged DGF.

Discriminative fusion of moments-aligned latent representation of multimodality medical data.

Fusion of multimodal medical data provides multifaceted, disease-relevant information for diagnosis or prognosis prediction modeling. Traditional fusion strategies such as feature concatenation often fail to learn hidden complementary and discriminative manifestations from high-dimensional multimodal data. To this end, we proposed a methodology for the integration of multimodality medical data by matching their moments in a latent space, where the hidden, shared information of multimodal data is gradually learned by optimization with multiple feature collinearity and correlation constrains. We first obtained the multimodal hidden representations by learning mappings between the original domain and shared latent space. Within this shared space, we utilized several relational regularizations, including data attribute preservation, feature collinearity and feature-task correlation, to encourage learning of the underlying associations inherent in multimodal data. The fused multimodal latent features were finally fed to a logistic regression classifier for diagnostic prediction. Extensive evaluations on three independent clinical datasets have demonstrated the effectiveness of the proposed method in fusing multimodal data for medical prediction modeling.

Helical TomoTherapy Total Lymphoid Irradiation and Hematopoietic Cell Transplantation for Kidney Transplant Tolerance in Rhesus Macaques.

Development of a post-transplant kidney transplant tolerance induction protocol involving a novel total lymphoid irradiation (TLI) conditioning method in a rhesus macaque model is described. We examined the feasibility of acheiving tolerance to MHC 1-haplotype matched kidney transplants by establishing a mixed chimeric state with infusion of donor hematopoietic cells (HC) using TomoTherapy TLI. The chimeric state was hypothesized to permit the elimination of all immunosuppressive (IS) medications while preserving allograft function long-term without development of graft-versus-host-disease (GVHD) or rejection. An experimental group of 11 renal transplant recipients received the tolerance induction protocol and outcomes were compared to a control group (n = 7) that received the same conditioning but without donor HC infusion. Development of mixed chimerism and operational tolerance was accomplished in two recipients in the experimental group. Both recipients were withdrawn from all IS and continued to maintain normal renal allograft function for 4 years without rejection or GVHD. None of the animals in the control group achieved tolerance when IS was eliminated. This novel experimental model demonstrated the feasibility for inducing of long-term operational tolerance when mixed chimerism is achieved using a TLI post-transplant conditioning protocol in 1-haplotype matched non-human primate recipients of combined kidney and HC transplantation.

The Trend of Serum Creatinine Does Not Predict Follow-Up Biopsy Findings Among Kidney Transplant Recipients With Antibody-Mediated Rejection.

Traditionally, antibody-mediated rejection (AMR) has been suspected mainly by a rise in serum creatinine (Scr) and confirmed by allograft biopsy. There is limited literature describing the trend of Scr after treatment, and how that trend might differ between patients with histological response and with no response to treatment. Methods: We included all cases of AMR at our program between March 2016 and July 2020 who had a follow-up biopsy after the index biopsy, with initial diagnosis of AMR. We trended the Scr and change in Scr (delta Scr) and its association with being a responder (microvascular inflammation, MVI ≤1) or nonresponder (MVI >1), as well as graft failure. Results: A total of 183 kidney transplant recipients were included, 66 in the responder group and 177 in the nonresponder group. The MVI scores and sum chronicity scores, along with transplant glomerulopathy scores, were higher in the nonresponder group. However, Scr at index biopsy was similar in responders (1.74 ± 0.70) versus nonresponders (1.83 ± 0.65; P = 0.39), as were the delta Scr at various time points. After adjustment for multiple variables, delta Scr was not associated with being a nonresponder. Also, delta Scr value at follow-up biopsy compared with index biopsy among responders was 0 ± 0.67 (P = 0.99) and among nonresponders was -0.01 ± 0.61 (P = 0.89). Being a nonresponder was significantly associated with an increased risk of graft failure at the last follow-up in univariate analysis but was not in multivariate analysis (hazard ratio 1.35; 95% confidence interval, 0.58-3.17; P = 0.49). Conclusions: We found that Scr is not a good predictor of the resolution of MVI, supporting the utility of follow-up biopsies after treatment of AMR.

An automated histological classification system for precision diagnostics of kidney allografts.

For three decades, the international Banff classification has been the gold standard for kidney allograft rejection diagnosis, but this system has become complex over time with the integration of multimodal data and rules, leading to misclassifications that can have deleterious therapeutic consequences for patients. To improve diagnosis, we developed a decision-support system, based on an algorithm covering all classification rules and diagnostic scenarios, that automatically assigns kidney allograft diagnoses. We then tested its ability to reclassify rejection diagnoses for adult and pediatric kidney transplant recipients in three international multicentric cohorts and two large prospective clinical trials, including 4,409 biopsies from 3,054 patients (62.05% male and 37.95% female) followed in 20 transplant referral centers in Europe and North America. In the adult kidney transplant population, the Banff Automation System reclassified 83 out of 279 (29.75%) antibody-mediated rejection cases and 57 out of 105 (54.29%) T cell-mediated rejection cases, whereas 237 out of 3,239 (7.32%) biopsies diagnosed as non-rejection by pathologists were reclassified as rejection. In the pediatric population, the reclassification rates were 8 out of 26 (30.77%) for antibody-mediated rejection and 12 out of 39 (30.77%) for T cell-mediated rejection. Finally, we found that reclassification of the initial diagnoses by the Banff Automation System was associated with an improved risk stratification of long-term allograft outcomes. This study demonstrates the potential of an automated histological classification to improve transplant patient care by correcting diagnostic errors and standardizing allograft rejection diagnoses.ClinicalTrials.gov registration: NCT05306795 .

Blockade of Indoleamine 2,3-Dioxygenase attenuates lipopolysaccharide-induced kidney injury by inhibiting TLR4/NF-κB signaling.

Blockade of indoleamine 2,3-dioxygenase (IDO) has been shown to alleviate lipopolysaccharide (LPS)-induced endotoxic shock and reduce sepsis mortality, but its effect on LPS-induced kidney damage has not been reported. Herein, we established a mouse kidney injury model by intraperitoneal injection of 10 mg/kg LPS and established an in vitro renal tubular epithelial cell injury model by stimulating TCMK-1 cells with 10 mg/L LPS. We found that pretreatment with 1-methyl tryptophan (1-MT), an IDO inhibitor, significantly improved LPS-induced mouse survival, and IDO knockout (KO) mice also had higher survival rates after LPS exposure than wild-type mice. At the same time, IDO KO or pretreatment with 1-MT not only reduced serum creatinine, blood urea nitrogen, renal tubular injury pathological score, but also inflammatory factors and oxidative stress status in serum or kidney of LPS-exposed mice. In vitro, blockade of IDO with 1-MT significantly inhibited LPS-induced apoptosis, inflammation and oxidative stress in TCMK-1 cells. In addition, blockade of IDO significantly inhibited LPS-activated TLR4/NF-κB signaling pathway in kidney of mice or in TCMK-1 cells. In conclusion, our results suggested that blockade of IDO attenuated kidney inflammation, apoptosis and oxidative stress to protect against LPS-induced septic kidney injury via inhibiting the TLR4/NF-κB signaling pathway.

What lies in-between: C3 glomerulopathy with non-hemolytic renal microangiopathy and an ultra-rare C3 variant.

We report a 36-year-old female with mixed nephritic-nephrotic syndrome and recurrent pancreatitis. Kidney biopsy showed a crescentic membranoproliferative glomerulonephritis with dominant C3 staining on immunofluorescence (IF) but only scant deposits on electron microscopy (EM) and instead, evidence of severe acute and chronic microangiopathy - endothelial swelling, sub-endothelial fluff, and segmental basement membrane remodeling. Her serum C3 was normal, Factor Ba, and serum Membrane attack complex (sMAC) levels were elevated, and Properdin was low. Genetic testing revealed a heterozygous ultra rare C3 variant of unknown significance (c.4838G>T, p.Gly1613Val) as well as a heterozygous deletion of CFHR3-CFHR1. She showed an initial response to terminal complement blockade with eculizumab, but her renal disease progressed in the next year. Notably, our patient never demonstrated microangiopathic hemolysis, yet pancreatitis of unclear etiology recurred periodically. Our case suggests the existence of a "C3G/aHUS overlap" clinicopathologic syndrome and highlights the challenges of treating complement-mediated kidney disease.

Post-transplant idiopathic immune complex membrano-proliferative glomerulonephritis: Characteristics and outcomes.

BACKGROUND: There is little published information on the natural history and the treatment of immune complex membrano-proliferative glomerulonephritis (IC-MPGN) of unknown cause in the transplanted kidney. MATERIALS AND METHODS: From 01/2004 to 12/2018, 41 patients had the diagnosis of post-transplant idiopathic IC-MPGN and were included in the study. RESULTS: The mean age of the cohort at the time of transplant was 50 ± 13 years. The most common presentation was increased proteinuria, followed by kidney dysfunction. Fewer than 50% of patients had hematuria at presentation. 25 patients (61%) had no change in their baseline immunosuppression after the diagnosis of idiopathic IC-MPGN. Eight patients (19.5%) received steroids alone, and 8 patients (19.5%) received rituximab with (7) or without (1) steroids. The patients who received rituximab had better uncensored graft survival than the patients who received no treatment (p = 0.02), but the benefit of steroids compared to no treatment did not reach statistical significance (p = 0.05). The multivariate analysis retained eGFR < 30 mL/min/1.73m2 at time of diagnosis (HR = 3.30, p = 0.02; 95% Cl 1.15 - 9.46) as a significant predictor of graft loss. In this analysis, treatment of idiopathic IC-MPGN was associated with lower graft loss (HR = 0.22, p = 0.02; 95% Cl 0.06 - 0.78). CONCLUSION: To the best of our knowledge, this is the largest clinic-pathological series of post-transplant idiopathic IC-MPGN. Treatment of idiopathic IC-MPGN may be associated with better graft outcomes.

Hydrogen Peroxide Promotes the Production of Radiation-Derived EVs Containing Mitochondrial Proteins.

In spite of extensive successes, cancer recurrence after radiation treatment (RT) remains one of the significant challenges in the cure of localized prostate cancer (PCa). This study focuses on elucidating a novel adaptive response to RT that could contribute to cancer recurrence. Here, we used PC3 cell line, an adenocarcinoma from a bone metastasis and radio-resistant clone 695 cell line, which survived after total radiation dose of 66 Gy (2 Gy × 33) and subsequently regrew in nude mice after exposure to fractionated radiation at 10 Gy (2 Gy × 5). Clone 695 cells not only showed an increase in surviving fraction post-radiation but also an increase in hydrogen peroxide (H2O2) production when compared to PC3 cells. At the single cell level, confocal microscope images coupled with IMARIS rendering software demonstrate an increase in mitochondrial mass and membrane potential in clone 695 cells. Utilizing the Seahorse XF96 instrument to investigate mitochondrial respiration, clone 695 cells demonstrated a higher basal Oxygen Consumption Rate (OCR), ATP-linked OCR, and proton leak compared to PC3 cells. The elevation of mitochondrial function in clone 695 cells is accompanied by an increase in mitochondrial H2O2 production. These data suggest that H2O2 could reprogram PCa's mitochondrial homeostasis, which allows the cancer to survive and regrow after RT. Upon exposure to RT, in addition to ROS production, we found that RT induces the release of extracellular vesicles (EVs) from PC3 cells (p < 0.05). Importantly, adding H2O2 to PC3 cells promotes EVs production in a dose-dependent manner and pre-treatment with polyethylene glycol-Catalase mitigates H2O2-mediated EV production. Both RT-derived EVs and H2O2-derived EVs carried higher levels of mitochondrial antioxidant proteins including, Peroxiredoxin 3, Glutathione Peroxidase 4 as well as mitochondrial-associated oxidative phosphorylation proteins. Significantly, adding isolated functional mitochondria 24 h prior to RT shows a significant increase in surviving fractions of PC3 cells (p < 0.05). Together, our findings reveal that H2O2 promotes the production of EVs carrying mitochondrial proteins and that functional mitochondria enhance cancer survival after RT.

APRIL/BLyS deficient rats prevent donor specific antibody (DSA) production and cell proliferation in rodent kidney transplant model.

APRIL (A proliferation inducing ligand) and BLyS (B Lymphocyte Stimulator) are two critical survival factors for B lymphocytes and plasma cells, the main source of alloantibody. We sought to characterize the specific effects of these cytokines in a kidney transplant model of antibody mediated rejection (AMR). We engineered APRIL-/- and BLyS-/- Lewis rats using CRISPR/Cas9. APRIL-/- and BLyS-/- rats were sensitized with Brown Norway (BN) blood (complete MHC mismatch). Twenty-one days following sensitization, animals were harvested and collected tissues were analyzed using flow cytometry, ELISPOT, and immunohistochemistry. Flow cross match and a 3 day mixed lymphocyte reaction (MLR) was performed to assess donor specific antibody (DSA) production and T-cell proliferation, respectively. Sensitized dual knock out Lewis rats (APRIL-/-/BLyS-/-) underwent kidney transplantation and were sacrificed on day 7 post-transplant. Sensitized BLyS-/- had significant decreases in DSA and cell proliferation compared to WT and APRIL-/- (p<0.02). Additionally, BLyS-/- rats had a significant reduction in IgG secreting cells in splenic marginal zone B lymphocytes, and in cell proliferation when challenged with alloantigen compared to WT and APRIL-/-. Transplanted APRIL-/-/BLyS-/- rodents had significantly less DSA and antibody secreting cells compared to WT (p<0.05); however, this did not translate into a significant difference in AMR seen between groups. In summary, our studies suggest that APRIL and BLyS play a greater role in DSA generation rather than AMR, highlighting the role of cellular pathways that regulate AMR.

Chronic Active Antibody-mediated Rejection in Kidney Transplant Recipients: Treatment Response Rates and Value of Early Surveillance Biopsies.

There is limited information on the value of short-term invasive and noninvasive monitoring in kidney transplant recipients (KTR) undergoing therapy for chronic active antibody-mediated rejection (cAMR). Methods: We describe response rates in patients with cAMR receiving pulse steroids/IVIG ± rituximab 3-mo after index biopsy. Results: The study included 82 consecutive KTR. Mean time from transplant to cAMR was 10 y. Mean peritubular capillaritis (ptc), glomerulitis (g), microvascular inflammation (MVI), C4d, and cg Banff scores were 1.1, 2.1, 3.2, 0.2, and 2, respectively. Mean estimated glomerular filtration rate (eGFR) and urine protein creatinine (UPC) ratio were 38 mL/min and 1.6 g/g, respectively. Thirty (37%) patients lost their allograft during the mean follow-up of 2.4 y. In patients treated with pulse steroids/IVIG (n = 41), response rates for eGFR, UPC, donor-specific antibodies (DSAs), and MVI were 27%, 49%, 7%, and 19%, respectively. In the pulse steroids/IVIG/rituximab group, response rates were 66%, 61%, 20%, and 69%, respectively. Univariate analysis identified response in eGFR (HR = 0.03; P = 0.001; 95% CI, 0.004-0.26), UPC (HR = 0.38; P = 0.01; 95% CI, 0.18-0.82), and DSA (HR = 0.11; P = 0.004; 95% CI, 0.02-0.49) as predictors of graft survival. Multivariate analysis only retained eGFR response (HR = 0.12; P = 0.01; 95% CI, 0.02-0.64). Conclusions: In cAMR, short-term response to treatment for kidney function and DSA was associated with graft survival, but the role of early surveillance biopsies needs further evaluation.

Evaluation of teaching effect of first-aid comprehensive simulation-based education in clinical medical students.

Background: Although students mastered the composition skills, they lack of the ability to effectively integrate these composition skills in real clinical situations. To address the problem, we set up different levels of situational simulation training for medical students in grades 2-4, and evaluate the teaching effect of first-aid situation comprehensive simulation-based education (SBE) on clinical medical students. Methods: The medical students in Grade 2, 3, and 4 received different situational SBE, respectively. The 2nd-year medical students received a single skill module which included cardiopulmonary resuscitation, endotracheal intubation, and electric defibrillation training. The 3rd-year medical students received a single subject module which included cardiovascular and respiratory system training. The 4th-year medical students received the integrated multidisciplinary module which combined first-aid skills, clinical thinking, and teamwork training. The primary outcome was the expert evaluation and peer evaluation. The secondary outcome was students' satisfaction questionnaire response. In our training, we arranged an adequate teaching staff for intensive training and timely feedback (the student-teacher ratio of 5:1), adequate time for repetitive practice (Each SBE was carried out within 4 h), curriculum design, and integration from real cases by clinicians, realistic computer-driven mannequins to ensure simulation fidelity, providing a different difficult level of SBE to different grades of students, and pre- and post-tests for outcome measurement. Results: In all of the single skill module, single subject module or comprehensive disciplines module, the scores in the expert evaluation and peer assessment after the training were significantly higher than before the training, and the differences were statistically significant (p < 0.05). The integrated subject training, although having the lowest pre-and post-test marks, had the largest increase in score. Conclusion: The first aid comprehensive simulation-based education in grade 2-4 clinical medical students, basing on timely feedback, repetitive practice, curriculum integration, simulation fidelity, and outcome measurement are effective in improving the students' proficiency in managing the real emergencies.

The RelB-BLNK Axis Determines Cellular Response to a Novel Redox-Active Agent Betamethasone during Radiation Therapy in Prostate Cancer.

Aberrant levels of reactive oxygen species (ROS) are potential mechanisms that contribute to both cancer therapy efficacy and the side effects of cancer treatment. Upregulation of the non-canonical redox-sensitive NF-kB family member, RelB, confers radioresistance in prostate cancer (PCa). We screened FDA-approved compounds and identified betamethasone (BET) as a drug that increases hydrogen peroxide levels in vitro and protects non-PCa tissues/cells while also enhancing radiation killing of PCa tissues/cells, both in vitro and in vivo. Significantly, BET increases ROS levels and exerts different effects on RelB expression in normal cells and PCa cells. BET induces protein expression of RelB and RelB target genes, including the primary antioxidant enzyme, manganese superoxide dismutase (MnSOD), in normal cells, while it suppresses protein expression of RelB and MnSOD in LNCaP cells and PC3 cells. RNA sequencing analysis identifies B-cell linker protein (BLNK) as a novel RelB complementary partner that BET differentially regulates in normal cells and PCa cells. RelB and BLNK are upregulated and correlate with the aggressiveness of PCa in human samples. The RelB-BLNK axis translocates to the nuclear compartment to activate MnSOD protein expression. BET promotes the RelB-BLNK axis in normal cells but suppresses the RelB-BLNK axis in PCa cells. Targeted disruptions of RelB-BLNK expressions mitigate the radioprotective effect of BET on normal cells and the radiosensitizing effect of BET on PCa cells. Our study identified a novel RelB complementary partner and reveals a complex redox-mediated mechanism showing that the RelB-BLNK axis, at least in part, triggers differential responses to the redox-active agent BET by stimulating adaptive responses in normal cells but pushing PCa cells into oxidative stress overload.

How Should Acute T-cell Mediated Rejection of Kidney Transplants Be Treated: Importance of Follow-up Biopsy.

Background: Limited published data exist to guide patient monitoring after the treatment of T-cell mediated rejection (TCMR) of kidney allografts. Methods: We reviewed the kidney function and histological outcomes after treatment of 163 first episodes of biopsy-proven TCMR between January 1' 2015' and July 31' 2020. Results: Of the 146 patients treated with steroid pulse alone, complete histological response was seen in 83% of patients with borderline rejection, 82.5% with grade 1A, 67% with grade 1B, and 50% with grade IIA. Of the 17 patients treated with steroids plus antithymocyte globulin, the complete histological response rate was 100% with grade 1A, 75% with grade 1B, 100% with grade IIA, and 57% with grade IIB. Among the patients with complete response as assessed by kidney function, 14% only had a partial or no response histologically. Among patients with no kidney function response, 68% had a complete response histologically. Conclusion: We thus find that responses based on kidney function alone do not correlate well with histological responses. If further treatment had been based solely on changes in estimated glomerular filtration rate, a significant number of patients would have been subsequently undertreated or overtreated. These results support the use of protocol follow-up biopsies after the treatment of TCMR.