Inheritable Silencing of Endogenous Genes by Hit-and-Run Targeted Epigenetic Editing.

Gene silencing is instrumental to interrogate gene function and holds promise for therapeutic applications. Here, we repurpose the endogenous retroviruses' silencing machinery of embryonic stem cells to stably silence three highly expressed genes in somatic cells by epigenetics. This was achieved by transiently expressing combinations of engineered transcriptional repressors that bind to and synergize at the target locus to instruct repressive histone marks and de novo DNA methylation, thus ensuring long-term memory of the repressive epigenetic state. Silencing was highly specific, as shown by genome-wide analyses, sharply confined to the targeted locus without spreading to nearby genes, resistant to activation induced by cytokine stimulation, and relieved only by targeted DNA demethylation. We demonstrate the portability of this technology by multiplex gene silencing, adopting different DNA binding platforms and interrogating thousands of genomic loci in different cell types, including primary T lymphocytes. Targeted epigenome editing might have broad application in research and medicine.

Targeting MHC-I molecules for cancer: function, mechanism, and therapeutic prospects.

The molecules of Major histocompatibility class I (MHC-I) load peptides and present them on the cell surface, which provided the immune system with the signal to detect and eliminate the infected or cancerous cells. In the context of cancer, owing to the crucial immune-regulatory roles played by MHC-I molecules, the abnormal modulation of MHC-I expression and function could be hijacked by tumor cells to escape the immune surveillance and attack, thereby promoting tumoral progression and impairing the efficacy of cancer immunotherapy. Here we reviewed and discussed the recent studies and discoveries related to the MHC-I molecules and their multidirectional functions in the development of cancer, mainly focusing on the interactions between MHC-I and the multiple participators in the tumor microenvironment and highlighting the significance of targeting MHC-I for optimizing the efficacy of cancer immunotherapy and a deeper understanding of the dynamic nature and functioning mechanism of MHC-I in cancer.

Prevalence and Associations of Beta2-Microglobulin Mutations in MSI-H/dMMR Cancers.

Microsatellite instability (MSI) has emerged as an important predictor of sensitivity for immunotherapy-based strategies. ß-2-Microglobulin (B2M) contains microsatellites within the coding regions and is prone to somatic changes in MSI/mismatch repair deficiency (MSI/dMMR) tumors. To delineate prevalence and associations of B2M mutations in MSI-H/dMMR cancers, we investigated the mutational profile of B2M and clinical and pathological features in gastric cancer (GC), colorectal cancer (CRC), and endometrial cancer (EC) with a high incidence of microsatellite instability-high (MSI-H)/dMMR. Formalin-fixed paraffin-embedded (FFPE) tumor tissues along with matched normal tissues were collected from 108 MSI/dMMR patients with GC, CRC, and EC. Genomic profiling of tissue and blood samples were assessed next-generation sequencing (NGS). Immunohistochemistry (IHC) was used to examine the presence or absence of B2M protein. Alternations in the exonic microsatellite regions of B2M were observed at various but high frequencies (57.5% in CRC, 23.9% in GC, and 13.6% in EC) and in different forms. NGS assay revealed that genes involved in chromatin regulation, the PI3K pathway, the WNT pathway, and mismatch repair were extensively altered in the MSI-H cohort. Signature 6 and 26, 2 of 4 mutational signatures associated with defective DNA mismatch repair, featured with high numbers of small insertion/deletions (INDEL) dominated in all 3 types of cancer. Alternations in the exonic microsatellite regions of B2M were observed at various but high frequencies (57.5% in CRC, 23.9% in GC, and 13.6% in EC) and in different forms. Tumor mutational burden (TMB) was significantly higher in the patients carrying MSI-H/dMMR tumors with B2M mutation than that in patients with wild-type B2M (P = .026).The frame shift alteration occurring at the exonic microsatellite sties caused loss of function of B2M gene. In addition, a case with CRC carrying indels in B2M gene resisted the ICI treatment was reported. In conclusion, patients carrying MSI-H/dMMR tumors with B2M mutation showed significantly higher TMB. Prescription of ICIs should be thoroughly evaluated for these patients.

Performance of Panel-Estimated GFR Among Hospitalized Older Adults.

RATIONALE & OBJECTIVE: Older adults represent nearly half of all hospitalized patients and are vulnerable to inappropriate dosing of medications eliminated through the kidneys. However, few studies in this population have evaluated the performance of equations for estimating the glomerular filtration rate (GFR)-particularly those that incorporate multiple filtration markers. STUDY DESIGN: Cross-sectional diagnostic test substudy of a randomized clinical trial. SETTING & PARTICIPANTS: Adults≥65 years of age presenting to the emergency department of Copenhagen University Hospital Amager and Hvidovre in Hvidovre, Denmark, between October 2018 and April 2021. TESTS COMPARED: Measured GFR (mGFR) determined using 99mTc-DTPA plasma clearance compared with estimated GFR (eGFR) calculated using 6 different equations based on creatinine; 3 based on creatinine and cystatin C combined; and 2 based on panels of markers including creatinine, cystatin C, ß-trace protein (BTP) and/or ß2-microglobulin (B2M). OUTCOME: The performance of each eGFR equation compared with mGFR with respect to bias, relative bias, inaccuracy (1-P30), and root mean squared error (RMSE). RESULTS: We assessed eGFR performance for 106 patients (58% female, median age 78.3 years, median mGFR 62.9mL/min/1.73m2). Among the creatinine-based equations, the 2009 CKD-EPIcr equation yielded the smallest relative bias (+4.2%). Among the creatinine-cystatin C combination equations, the 2021 CKD-EPIcomb equation yielded the smallest relative bias (-3.4%), inaccuracy (3.8%), and RMSE (0.139). Compared with the 2021 CKD-EPIcomb, the CKD-EPIpanel equation yielded a smaller RMSE (0.136) but larger relative bias (-4.0%) and inaccuracy (5.7%). LIMITATIONS: Only White patients were included; only a subset of patients from the original clinical trial underwent GFR measurement; and filtration marker concentration can be affected by subclinical changes in volume status. CONCLUSIONS: The 2009 CKD-EPIcr, 2021 CKD-EPIcomb, and CKD-EPIpanel equations performed best and notably outperformed their respective full-age spectrum equations. The addition of cystatin C to creatinine-based equations improved performance, while the addition of BTP and/or B2M yielded minimal improvement. FUNDING: Grants from public sector industry (Amgros I/S) and government (Capital Region of Denmark). TRIAL REGISTRATION: Registered at ClinicalTrials.gov with registration number NCT03741283. PLAIN-LANGUAGE SUMMARY: Inaccurate kidney function assessment can lead to medication errors, a common cause of hospitalization and early readmission among older adults. Several novel methods have been developed to estimate kidney function based on a panel of kidney function markers that can be measured from a single blood sample. We evaluated the accuracy of these new methods (relative to a gold standard method) among 106 hospitalized older adults. We found that kidney function estimates combining 2 markers (creatinine and cystatin C) were highly accurate and noticeably more accurate than estimates based on creatinine alone. Estimates incorporating additional markers such as ß-trace protein and ß2-microglobulin did not further improve accuracy.

Association of Kidney Function Measures With Signs of Neurodegeneration and Small Vessel Disease on Brain Magnetic Resonance Imaging: The Atherosclerosis Risk in Communities (ARIC) Study.

RATIONALE & OBJECTIVE: Chronic kidney disease (CKD) is a risk factor for cognitive decline, but evidence is limited on its etiology and morphological manifestation in the brain. We evaluated the association of estimated glomerular filtration rate (eGFR) and urinary albumin-creatinine ratio (UACR) with structural brain abnormalities visible on magnetic resonance imaging (MRI). We also assessed whether this association was altered when different filtration markers were used to estimate GFR. STUDY DESIGN: Cross-sectional study nested in a cohort study. SETTING & PARTICIPANTS: 1,527 participants in the Atherosclerosis Risk in Communities (ARIC) Study. PREDICTORS: Log(UACR) and eGFR based on cystatin C, creatinine, cystatin C and creatinine in combination, or ß2-microglobulin (B2M). OUTCOMES: Brain volume reduction, infarcts, microhemorrhages, white matter lesions. ANALYTICAL APPROACH: Multivariable linear and logistic regression models fit separately for each predictor based on a 1-IQR difference in the predictor value. RESULTS: Each 1-IQR lower eGFR was associated with reduced cortex volume (regression coefficient: -0.07 [95% CI, -0.12 to-0.02]), greater white matter hyperintensity volume (logarithmically transformed; regression coefficient: 0.07 [95% CI, 0.01-0.15]), and lower white matter fractional anisotropy (regression coefficient: -0.08 [95% CI, -0.17 to-0.01]). The results were similar when eGFR was estimated with different equations based on cystatin C, creatinine, a combination of cystatin C and creatinine, or B2M. Higher log(UACR) was similarly associated with these outcomes as well as brain infarcts and microhemorrhages (odds ratios per 1-IQR-fold greater UACR of 1.31 [95% CI, 1.13-1.52] and 1.30 [95% CI, 1.12-1.51], respectively). The degree to which brain volume was lower in regions usually susceptible to Alzheimer disease and LATE (limbic-predominant age-related TDP-43 [Tar DNA binding protein 43] encephalopathy) was similar to that seen in the rest of the cortex. LIMITATIONS: No inference about longitudinal effects due to cross-sectional design. CONCLUSIONS: We found eGFR and UACR are associated with structural brain damage across different domains of etiology, and eGFR- and UACR-related brain atrophy is not selective for regions typically affected by Alzheimer disease and LATE. Hence, Alzheimer disease or LATE may not be leading contributors to neurodegeneration associated with CKD.

Cell-free protein synthesis of CD1E and B2M protein and in vitro interaction.

CD1E, one of the most important glycolipid antigens on T cell membranes, is required for glycolipid antigen presentation on the cell surface. Cell-based recombinant expression systems have many limitations for synthesizing transmembrane proteins such as CD1E, including low protein yields and miss folding. To overcome these challenges, here we successfully synthesized high-quality soluble CD1E using an E.coli cell-free protein synthesis system (CFPS) with the aid of detergent. Following purification by Ni2+ affinity chromatography, we were able to obtain CD1E with ≥90% purity. Furthermore, we used the string website to predict the protein interaction network of CD1E and identified a potential binding partner━B2M. Similarly, we synthesized soluble B2M in the E.coli CFPS. Finally, we verified the interaction between CD1E and B2M by using Surface Plasmon Resonance (SPR). Taken together, the methods described here provide an alternative way to obtain active transmembrane protein and may facilitate future structural and functional studies on CD1E.

Efficacy and safety of PD-1 monoclonal antibody plus rituximab in relapsed/refractory diffuse large B cell lymphoma patients.

BACKGROUND: Patients with relapsed/refractory diffuse large B-cell lymphoma (r/r DLBCL) have poor outcomes and few treatment options. We report the preliminary results of the efficacy and safety of PD-1 monoclonal antibody (mab) plus Rituximab for r/r DLBCL. METHODS: In this single-center, single-arm phase 2 and retrospective study, r/r DLBCL patients received PD-1 mab and Rituximab every 3 weeks. Immunohistochemistry, fluorescence in situ hybridization, and probe capture-based high-resolution sequencing were performed. Efficacy, safety and prognostic factors were analyzed. RESULTS: Between October 16th, 2018, and July 10th, 2022, 36 patients (10 patients in retrospective study and 26 patients in phase 2 study) were enrolled and received at least one dose of PD-1 mab combined with Rituximab. The objective response rate was 52.8%. The median progression free survival (PFS) and overall survival was 2.8 and 19.6 months, respectively. The median duration of response was 18.7 months. Rare grade 3 or 4 treatment related adverse events were observed. B2M mutations correlated with a significantly poor PFS (p = .013) and OS (p = .009) in DLBCL patients treated with this regimen. CONCLUSION: PD-1 mab combined with Rituximab could be a potential treatment option for r/r DLBCL with manageable safety profile.

Circ_0058063 contributes to cisplatin-resistance of bladder cancer cells by upregulating B2M through acting as RNA sponges for miR-335-5p.

Bladder cancer (BC) is one of the most common malignant tumors of the urinary system, and cisplatin (CDDP) is a critical chemical drug for the treatment of BC. However, CDDP-resistance seriously limits the therapeutic efficacy of this drug for clinical utilization. Thus, identification of pivotal molecule targets that regulate CDDP-resistance in BC become urgent and necessary. In this study, we firstly identified a novel BC-associated circular RNA circ_0058063 that participates in the regulation of CDDP-resistance in BC. Specifically, circ_0058063 was significantly overexpressed in CDDP-resistant tissue and cells, in contrast with the corresponding CDDP-sensitive counterparts. Further loss-of-function experiments validated that downregulation of circ_0058063 suppressed cell proliferation and tumor growth, whereas induced cell apoptosis in the CDDP-resistant BC cells in vitro and in vivo. In addition, we disclosed that circ_0058063 acts as a sponge for miR-335-5p to positively regulate B2M expression, and further rescuing experiments verified that the enhancing effects of sh-circ_0058063 on CDDP-sensitivity in the CDDP-resistant BC cells were abrogated by silencing miR-335-5p. Taken together, our results demonstrated that circ_0058063 contributed to CDDP resistance of bladder cancer cells via sponging miR-335-5p, and B2M might be the downstream effector gene. This study firstly evidenced that targeting circ_0058063 might be an effective strategy to improve CDDP-sensitivity in BC.

Highly expressed genes in multiple myeloma cells - what can they tell us about the disease?

Cancer cells can convert proto-oncoproteins into oncoproteins by increasing the expression of genes that are oncogenic when expressed at high levels. Such genes can promote oncogenesis without being mutated. To find overexpressed genes in cancer cells from patients with multiple myeloma, we retrieved mRNA expression data from the CoMMpass database and ranked genes by their expression levels. We grouped the most highly expressed genes based on a set of criteria and we discuss the role a selection of them can play in the disease pathophysiology. The list was highly concordant with a similar list based on mRNA expression data from the PADIMAC study. Many well-known "myeloma genes" such as MCL1, CXCR4, TNFRSF17, SDC1, SLAMF7, PTP4A3, and XBP1 were identified as highly expressed, and we believe that hitherto unrecognized key players in myeloma pathogenesis are also enriched on the list. Highly expressed genes in malignant plasma cells that were absent or expressed at only a low level in healthy plasma cells included IFI6, IFITM1, PTP4A3, SIK1, ALDOA, ATP5MF, ATP5ME, and PSMB4. The ambition of this article is not to validate the role of each gene but to serve as a guide for studies aiming at identifying promising treatment targets.

B2M gene expression shapes the immune landscape of lung adenocarcinoma and determines the response to immunotherapy.

Loss of the B2M gene is associated with tumour immune escape and resistance to immunotherapy. However, genetic alterations of the B2M gene are rare. We performed an integrative analysis of the mutational and transcriptional profiles of large cohorts of non-small-cell lung cancer (NSCLC) patients and found that epigenetic downregulation of B2M is common. B2M-low tumours exhibit a suppressive immune microenvironment characterized by reduced infiltration of immune cells of various lineages; in B2M-high tumours, more T and natural killer cells are present, but their activities are constrained by immune checkpoint molecules, indicating the diverse mechanisms of immune evasion. High levels of B2M mRNA, but not PD-L1, are correlated with an enhanced response to PD-1-based immunotherapy, suggesting its value for immunotherapy response prediction in solid tumours. Notably, a high tumour mutation burden (TMB) is associated with low B2M expression, which may explain the poor predictive value of the TMB in some situations. In syngeneic mouse models, genetic ablation of B2M in tumour cells causes resistance to PD-1-based immunotherapy, and B2M knockdown also diminishes the therapeutic efficacy. Moreover, forced expression of B2M in tumour models improves the response to immunotherapy, suggesting that B2M levels have significant impacts on treatment outcomes. Finally, we provide insight into the roles of transcription factors and KRAS mutations in B2M expression and the anticancer immune response. In conclusion, genetic and epigenetic regulation of B2M fundamentally shapes the NSCLC immune microenvironment and may determine the response to checkpoint blockade-based immunotherapy.

Long-Term Longitudinal Stability of Kidney Filtration Marker Measurements: Implications for Epidemiological Studies and Clinical Care.

BACKGROUND: Establishment and improvement of glomerular filtration rate estimating equations requires accurate and precise laboratory measurement procedures (MPs) for filtration markers. The Advanced Research and Diagnostic Laboratory (ARDL) at the University of Minnesota, which has served as the central laboratory for the Chronic Kidney Disease Epidemiology Collaboration since 2009, has implemented several quality assurance measures to monitor the accuracy and stability of filtration marker assays over time. METHODS: To assess longitudinal stability for filtration marker assays, a 40-sample calibration panel was created using pooled serum, divided into multiple frozen aliquots stored at -80 °C. ARDL monitored 4 markers-creatinine, cystatin C, beta-2-microglobulin (B2M) and beta-trace protein-measuring 15 calibration panel aliquots from 2009 to 2019. Initial target values were established using the mean of the first 3 measurements performed in 2009-10, and differences from target were monitored over time. New MPs for cystatin C and B2M were added in 2012, with target values established using the first measurement. RESULTS: The mean percentage difference from mean target values across time was <2% for all original MPs (-0.59% for creatinine; -0.94% for cystatin C; -0.82% for B2M; 1.24% for beta-trace protein). CONCLUSIONS: Close monitoring of filtration marker trends with a calibration panel at ARDL demonstrates remarkable long-term stability of the MPs. Routine use of a calibration panel for both research studies and clinical care is recommended for filtration markers where longitudinal monitoring is important to detect analytical biases, which can mask or confound true clinical trends in patients.

A New Panel-Estimated GFR, Including ß2-Microglobulin and ß-Trace Protein and Not Including Race, Developed in a Diverse Population.

RATIONALE AND OBJECTIVE: Glomerular filtration rate (GFR) estimation based on creatinine and cystatin C (eGFRcr-cys) is more accurate than estimated GFR (eGFR) based on creatinine or cystatin C alone (eGFRcr or eGFRcys, respectively), but the inclusion of creatinine in eGFRcr-cys requires specification of a person's race. ß2-Microglobulin (B2M) and ß-trace protein (BTP) are alternative filtration markers that appear to be less influenced by race than creatinine is. STUDY DESIGN: Study of diagnostic test accuracy. SETTING AND PARTICIPANTS: Development in a pooled population of 7 studies with 5,017 participants with and without chronic kidney disease. External validation in a pooled population of 7 other studies with 2,245 participants. TESTS COMPARED: Panel eGFR using B2M and BTP in addition to cystatin C (3-marker panel) or creatinine and cystatin C (4-marker panel) with and without age and sex or race. OUTCOMES: GFR measured as the urinary clearance of iothalamate, plasma clearance of iohexol, or plasma clearance of [51Cr]EDTA. RESULTS: Mean measured GFRs were 58.1 and 83.2 mL/min/1.73 m2, and the proportions of Black participants were 38.6% and 24.0%, in the development and validation populations, respectively. In development, addition of age and sex improved the performance of all equations compared with equations without age and sex, but addition of race did not further improve the performance. In validation, the 4-marker panels were more accurate than the 3-marker panels (P < 0.001). The 3-marker panel without race was more accurate than eGFRcys (percentage of estimates greater than 30% different from measured GFR [1 - P30] of 15.6% vs 17.4%; P = 0.01), and the 4-marker panel without race was as accurate as eGFRcr-cys (1 - P30 of 8.6% vs 9.4%; P = 0.2). Results were generally consistent across subgroups. LIMITATIONS: No representation of participants with severe comorbid illness and from geographic areas outside of North America and Europe. CONCLUSIONS: The 4-marker panel eGFR is as accurate as eGFRcr-cys without requiring specification of race. A more accurate race-free eGFR could be an important advance.

Generation of hypoimmunogenic human pluripotent stem cells via expression of membrane-bound and secreted ß2m-HLA-G fusion proteins.

Allogeneic immune rejection is a major barrier for the application of human pluripotent stem cells (hPSCs) in regenerative medicine. A broad spectrum of immune cells, including T cells, natural killer (NK) cells, and antigen-presenting cells, which either cause direct cell killing or constitute an immunogenic environment, are involved in allograft immune rejection. A strategy to protect donor cells from cytotoxicity while decreasing the secretion of inflammatory cytokines of lymphocytes is still lacking. Here, we engineered hPSCs with no surface expression of classical human leukocyte antigen (HLA) class I proteins via beta-2 microglobulin (B2M) knockout or biallelic knockin of HLA-G1 within the frame of endogenous B2M loci. Elimination of the surface expression of HLA class I proteins protected the engineered hPSCs from cytotoxicity mediated by T cells. However, this lack of surface expression also resulted in missing-self response and NK cell activation, which were largely compromised by expression of ß2m-HLA-G1 fusion proteins. We also proved that the engineered ß2m-HLA-G5 fusion proteins were soluble, secretable, and capable of safeguarding low immunogenic environments by lowering inflammatory cytokines secretion in allografts. Our current study reveals a novel strategy that may offer unique advantages to construct hypoimmunogenic hPSCs via the expression of membrane-bound and secreted ß2m-HLA-G fusion proteins. These engineered hPSCs are expected to serve as an unlimited cell source for generating universally compatible "off-the-shelf" cell grafts in the future.

Albuminuria and Estimated GFR as Risk Factors for Dementia in Midlife and Older Age: Findings From the ARIC Study.

RATIONALE & OBJECTIVE: Evidence is limited on how estimated glomerular filtration rate (eGFR) and urinary albumin-creatinine ratio (UACR) relate to dementia at different ages. We evaluated eGFR and UACR in midlife and older age as risk factors for dementia. Additionally, we assessed whether the association between eGFR and dementia is altered when cystatin C and ß2-microglobulin (B2M) levels are used for GFR estimation. STUDY DESIGN: Prospective cohort study. SETTING & PARTICIPANTS: Two baselines from the Atherosclerosis Risk in Communities (ARIC) Study were used: visit 4 (1996-1998), including 9,967 participants 54 to 74 years old, and visit 5 (2011-2013), including 4,626 participants 70 to 90 years old. Participants were followed up until 2017. PREDICTORS: Log(UACR); eGFR based on creatinine, cystatin C, creatinine and cystatin C, or B2M levels (denoted as eGFRcr, eGFRcys, eGFRcr-cys, and eGFRB2M). OUTCOME: Incident dementia. ANALYTICAL APPROACH: Multivariable Cox proportional hazards regression models fit separately for each of the 5 predictors and based on a change in the predictor equivalent to the interquartile range for that predictor at visit 4 (IQRV4). eGFR models were adjusted for log(UACR) and log(UACR) models were adjusted for eGFRcys. RESULTS: We observed 1,821 dementia cases after visit 4 and 438 cases after visit 5. Dementia risk increased with higher albuminuria levels (HRs per IQRV4 [equivalent to 4.2-fold greater log albuminuria] of 1.15 [95% CI, 1.09-1.21] after visit 4 and 1.27 [95% CI, 1.13-1.42] after visit 5). An association with lower eGFR was seen for only eGFRcys (HRs per IQRV4 [equivalent to 24.3mL/min/1.73m2 lesser eGFRcys] of 1.12 [95% CI, 1.04-1.21] after visit 4 and 1.30 [95% CI, 1.12-1.52] after visit 5) and eGFRB2M (HRs per IQRV4 [equivalent to 18.3mL/min/1.73m2 lesser eGFRB2M] of 1.15 [95% CI, 1.07-1.23] after visit 4 and 1.34 [95% CI, 1.17-1.55] after visit 5). Differences between these associations in midlife and older age were not statistically significant. LIMITATIONS: Changes in potentially time-varying covariates were not measured. Dementia was not subclassified by cause. CONCLUSIONS: Albuminuria was consistently associated with dementia incidence. Lower eGFR based on cystatin C or B2M, but not creatinine, levels was also associated with dementia. Risk associations were similar when kidney measures were assessed at midlife and older age.

Viable pigs after simultaneous inactivation of porcine MHC class I and three xenoreactive antigen genes GGTA1, CMAH and B4GALNT2.

BACKGROUND: Cell surface carbohydrate antigens play a major role in the rejection of porcine xenografts. The most important for human recipients are α-1,3 Gal (Galactose-alpha-1,3-galactose) causing hyperacute rejection, also Neu5Gc (N-glycolylneuraminic acid) and Sd(a) blood group antigens both of which are likely to elicit acute vascular rejection given the known human immune status. Porcine cells with knockouts of the three genes responsible, GGTA1, CMAH and B4GALNT2, revealed minimal xenoreactive antibody binding after incubation with human serum. However, human leucocyte antigen (HLA) antibodies cross-reacted with swine leucocyte antigen class I (SLA-I). We previously demonstrated efficient generation of pigs with multiple xeno-transgenes placed at a single genomic locus. Here we wished to assess whether key xenoreactive antigen genes can be simultaneously inactivated and if combination with the multi-transgenic background further reduces antibody deposition and complement activation. METHODS: Multiplex CRISPR/Cas9 gene editing and somatic cell nuclear transfer were used to generate pigs carrying functional knockouts of GGTA1, CMAH, B4GALNT2 and SLA class I. Fibroblasts derived from one- to four-fold knockout animals, and from multi-transgenic cells (human CD46, CD55, CD59, HO1 and A20) with the four-fold knockout were used to examine the effects on human IgG and IgM binding or complement activation in vitro. RESULTS: Pigs were generated carrying four-fold knockouts of important xenoreactive genes. In vitro assays revealed that combination of all four gene knockouts reduced human IgG and IgM binding to porcine kidney cells more effectively than single or double knockouts. The multi-transgenic background combined with GGTA1 knockout alone reduced C3b/c and C4b/c complement activation to such an extent that further knockouts had no significant additional effect. CONCLUSION: We showed that pigs carrying several xenoprotective transgenes and knockouts of xenoreactive antigens can be readily generated and these modifications will have significant effects on xenograft survival.

Association of liver and kidney functions with Klotho gene methylation in a population environment exposed to cadmium in China.

Exposure to the heavy metal cadmium has adverse effects on human health, including DNA methylation. This study aimed to investigate the effects of cadmium on liver and kidney functions and Klotho gene methylation and to explore the relationship of methylation level with indicators of liver and kidney functions. Graphite furnace atomic absorption spectrometry was conducted to determine urinary cadmium, and an automatic biochemical analyzer was used to detect indices of liver and kidney functions. PCR pyrosequencing was performed to detect the methylation rate of Klotho. One-way ANOVA was adopted to compare the differences between groups, and the linear correlation to variables was analyzed. Cadmium exposure was negatively correlated with albumin level (r=-0.143, p=0.021) and positively correlated with urinary ß2-microglobulin level (r=0.229, p<0.001). However, the methylation levels of Klotho gene was decreased and increased by low and high doses of cadmium exposure, respectively. And Klothomethylation levels were negatively correlated with albumin levels and positively correlated with ß2-microglobulin levels.In this study, cadmium exposure affects liver and kidney functions as well as Klotho methylation levels, but the effect on Klotho methylation levels is not linear. Klotho methylation levels also influence liver and kidney functions.

Comprehensive Genomic Profiling of Hodgkin Lymphoma Reveals Recurrently Mutated Genes and Increased Mutation Burden.

BACKGROUND: The genomic landscape of Hodgkin lymphoma (HL) has been difficult to characterize due to the paucity of neoplastic cells and an abundant microenvironment. Such characterization is needed in order to improve treatment strategies. MATERIALS AND METHODS: We performed comprehensive genomic profiling (CGP) using targeted next-generation sequencing on archival formalin-fixed paraffin embedded tumor samples from 63 patients to analyze the landscape of HL. RESULTS: CGP was successful for 49/63 archival specimens (78%), and revealed aberrations impacting genes including B2M, TP53, and XPO1 (E571). Of the 34 patients for whom total mutation burden (TMB; mutations/megabase [Mb]) was assessed, 5 (15%) had high TMB (≥20 mutations/Mb), 18 (53%) had intermediate TMB (6-19 mutations/Mb), and 11 (32%) had low TMB (≤5 mutations/Mb). We next tested 13 patients' plasma cell-free DNA with droplet digital polymerase chain reaction for the presence of XPO1 E571 mutation, which was confirmed in the plasma of 31% of patients. In three patients with serially collected plasma samples, XPO1 E571K allelic frequency changes corresponded with changes in tumor size on conventional radiographic imaging. CONCLUSION: The study demonstrates that comprehensive genomic profiling of archival Hodgkin lymphoma tumor samples is feasible and leads to the identification of genes that are recurrently mutated and that Hodgkin lymphoma has increased mutation burden in the majority of samples analyzed. Furthermore, tracking of XPO1 E571 mutant allele frequency in a subset of patients may also represent a potential disease-monitoring strategy and warrants further investigation. IMPLICATIONS FOR PRACTICE: This study provides the first evidence that comprehensive genomic profiling can be performed to map the genomic landscape of Hodgkin lymphoma and that a subpopulation of patients has mutations in TP53, B2M, XPO1, and other genes. It was found that 15% of patients have high mutation burden, which, in cancers such as melanoma, may indicate sensitivity to immune checkpoint inhibitors, and may thus be explored for Hodgkin lymphoma. Lastly, this work demonstrates that changes in the mutant allele frequency of XPO1 in serially collected plasma cell-free DNA samples correspond with treatment outcomes measured with conventional radiographic imaging.

Confirmation that somatic mutations of beta-2 microglobulin correlate with a lack of recurrence in a subset of stage II mismatch repair deficient colorectal cancers from the QUASAR trial.

AIMS: Beta2-microglobulin (B2M) forms part of the HLA class I complex and plays a role in metastatic biology. B2M mutations occur frequently in mismatch repair-deficient colorectal cancer (dMMR CRC), with limited data suggesting they may protect against recurrence. Our experimental study tested this hypothesis by investigating B2M mutation status and B2M protein expression and recurrence in patients in the stage II QUASAR clinical trial. METHODS AND RESULTS: Sanger sequencing was performed for the three coding exons of B2M on 121 dMMR and a subsample of 108 pMMR tumours; 52 with recurrence and 56 without. B2M protein expression was assessed by immunohistochemistry. Mutation status and protein expression were correlated with recurrence and compared to proficient mismatch repair (pMMR) CRCs. Deleterious B2M mutations were detected in 39 of 121 (32%) dMMR tumours. Five contained missense B2M-variants of unknown significance, so were excluded from further analyses. With median follow-up of 7.4 years, none of the 39 B2M-mutant tumours recurred, compared with 14 of 77 (18%) B2M-wild-type tumours (P = 0.005); six at local and eight at distant sites. Sensitivity and specificity of IHC in detecting B2M mutations was 87 and 71%, respectively. Significantly (P < 0.0001) fewer (three of 104, 2.9%) of the 108 pMMR CRCs demonstrated deleterious B2M mutations. One pMMR tumour, containing a frameshift mutation, later recurred. CONCLUSION: B2M mutations were detected in nearly one-third of dMMR cancers, none of which recurred. B2M mutation status has potential clinical utility as a prognostic biomarker in stage II dMMR CRC. The mechanism of protection against recurrence and whether this protection extends to stage III disease remains unclear.

Possible detrimental effects of beta-2-microglobulin knockout in pigs.

BACKGROUND: Despite major improvements in pig-to-primate xenotransplantation, long-term survival of xenografts is still challenging. The major histocompatibility complex (MHC) class I, which is crucial in cellular immune response, is an important xenoantigen. Abrogating MHC class I expression on xenografts might be beneficial for extending graft survival beyond current limits. METHODS: In this study, we employed the CRISPR/Cas9 system to target exon 2 of the porcine beta-2-microglobulin (B2M) gene to abrogate SLA class I expression on porcine cells. B2M-KO cells served as donor cells for somatic cell nuclear transfer, and cloned embryos were transferred to three recipient sows. The offspring were genotyped for mutations at the B2M locus, and blood samples were analyzed via flow cytometry for the absence of SLA class I molecules. RESULTS: Pregnancies were successfully established and led to the birth of seven viable piglets. Genomic sequencing proved that all piglets carried biallelic modifications at the B2M locus leading to a frameshift, a premature stop codon, and ultimately a functional knockout. However, survival times of these animals did not exceed 4 weeks due to unexpected disease processes. CONCLUSION: Here, we demonstrate the feasibility of generating SLA class I knockout pigs by targeting the porcine beta-2-microglobulin gene using the CRISPR/Cas9 system. Additionally, our findings indicate for the first time that this genetic modification might have a negative impact on the viability of the animals. These issues need to be solved to unveil the real value for xenotransplantation in the future.

Nonstructural Protein 4 of Porcine Reproductive and Respiratory Syndrome Virus Modulates Cell Surface Swine Leukocyte Antigen Class I Expression by Downregulating ß2-Microglobulin Transcription.

Porcine reproductive and respiratory syndrome virus (PRRSV) is the causative agent of PRRS, which has important impacts on the pig industry. PRRSV infection results in disruption of the swine leukocyte antigen class I (SLA-I) antigen presentation pathway. In this study, highly pathogenic PRRSV (HP-PRRSV) infection inhibited transcription of the ß2-microglobulin (ß2M) gene (B2M) and reduced cellular levels of ß2M, which forms a heterotrimeric complex with the SLA-I heavy chain and a variable peptide and plays a critical role in SLA-I antigen presentation. HP-PRRSV nonstructural protein 4 (Nsp4) was involved in the downregulation of ß2M expression. Exogenous expression of Nsp4 downregulated ß2M expression at both the mRNA and the protein level and reduced SLA-I expression on the cell surface. Nsp4 bound to the porcine B2M promoter and inhibited its transcriptional activity. Domain III of Nsp4 and the enhancer PAM element of the porcine B2M promoter were identified as essential for the interaction between Nsp4 and B2M These findings demonstrate a novel mechanism whereby HP-PRRSV may modulate the SLA-I antigen presentation pathway and provide new insights into the functions of HP-PRRSV Nsp4. IMPORTANCE PRRSV modulates the host response by disrupting the SLA-I antigen presentation pathway. We show that HP-PRRSV downregulates SLA-I expression on the cell surface via transcriptional inhibition of B2M expression by viral Nsp4. The interaction between domain III of Nsp4 and the enhancer PAM element of the porcine B2M promoter is essential for inhibiting B2M transcription. These observations reveal a novel mechanism whereby HP-PRRSV may modulate SLA-I antigen presentation and provide new insights into the functions of viral Nsp4.