Purification and Characterization of a Methyl-DNA Binding Protein Complex from Primary Erythroid Cells

The chicken embryonic β-type globin gene, ρ, is silenced on day five of embryogenesis. Concomitant with this silencing is methylation of cytosine residues in the promoter and proximal transcribed region of the gene, which is first detected on day seven and is complete in adult cells. Once methylated, expression of the gene cannot be induced unless the methylation is removed by treatment of cells with Sazacytidine. Therefore ρ-globin is a member of a small group of genes whose normal developmentally regulated expression is mediated at least in part by DNA methylation.A methyl-DNA binding complex, termed the MeCPC (Erythroid Methyl Cytosine-binding Protein Complex), has been found to bind to the methylated, but not unmethylated, ρ-globin promoter and proximal transcribed region in nuclear extracts from definitive erythrocytes. This complex has a stronger binding affinity for its cognate binding sequence, the methylated ρ-globin proximal transcribed region (M-ρ248), than for an artificial 5-methylcytosine-rich sequence (M-CG11).To define the components of the MeCPC, we developed two chromatographic procedures to purify the complex from adult chicken red blood cell nuclear extracts (Purification Strategies I and II). Mass spectrometry was performed on the MeCPC obtained by Purification Strategy I and proteins were identified by a novel application of peptide mass fingerprint data fitting. Four components of the previously-purified MeCPl transcriptional repression complex were identified in the sample: MBD2, RbAp48, HDAC2 and MTA1. Another identified protein, MENT, is a factor expressed only in chicken hematopoietic cells. These five proteins, as well as the MeCPl component Mi2, were found to tightly coelute by Western blotting of gel-filtration fractions from Purification Strategy II. Therefore, we conclude that these five proteins are components of the MeCPC.To confirm that MBD2 is associated with the ρ-globin gene in vivo, we perfomed the chromatin immunoprecipitation assay using anti-MBD2 antibodies. In adult erythrocytes, significant enrichment for MBD2 is seen at the transcriptionally inactive ρ-globin gene, but no enrichment is observed at the transcriptionally active βA globin gene. These experiments confirm that MBD2 binds to the methylated p-globin gene in adult chicken erythroid cells

Report from the American Society of Transplantation on frailty in solid organ transplantation

Peer Reviewedhttps://deepblue.lib.umich.edu/bitstream/2027.42/148387/1/ajt15198_am.pdfhttps://deepblue.lib.umich.edu/bitstream/2027.42/148387/2/ajt15198.pd

Paradoxical coronary artery embolism - A rare cause of myocardial infarction

Paradoxical coronary artery embolism is a rare, but often an underdiagnosed cause of acute myocardial infarction. It should be considered in patient who presents with chest pain and otherwise having a low risk profile for atherosclerosis coronary artery disease. We describe a case of paradoxical coronary artery embolism causing ST segment elevation myocardial infarction in a patient with upper extremity venous thrombosis. Echocardiography demonstrated a patent foramen ovale (PFO) with bidirectional shunt. In addition to treatment of acute coronary event closure of the PFO should be considered to prevent a recurrence

Dapsone-Associated Anemia in Heart Transplant Recipients with Normal Glucose-6-Phosphate Dehydrogenase Activity

Dapsone is considered an alternative for pneumocystis jirovecii pneumonia (PJP) prophylaxis in sulfa-allergic or -intolerant transplant patients with normal glucose-6-phosphate dehydrogenase (G6PD) activity. Despite normal G6PD activity, anemia can still occur while on dapsone therapy. We retrospectively reviewed heart transplant patients transplanted at our center between January 2016 and June 2018 and identified those taking dapsone prophylaxis. There were 252 heart transplant recipients at our center between January 2016 and June 2018. 36 patients received dapsone prophylaxis. All had normal G6PD activity assessed prior to dapsone initiation. 8 (22%) patients developed significant anemia attributed to dapsone: 2 were hospitalized for anemia, 1 of whom required blood transfusion. These patients had a median reduction in hemoglobin of 2.1 g/dL from baseline prior to dapsone initiation. Overt evidence of hemolysis was present in six patients. Once dapsone was discontinued, Hgb increased by at least 2 g/dL in a median of 30 days. Anemia from dapsone may occur in a significant proportion of patients despite normal G6PD activity and resulting in significant morbidity. Careful monitoring of transplant recipients on dapsone prophylaxis is warranted, as well as consideration of alternative agents

MBD2 is a critical component of a methyl cytosine-binding protein complex isolated from primary erythroid cells

The chicken embryonic β-type globin gene, ρ, is a member of a small group of vertebrate genes whose developmentally regulated expression is mediated by DNA methylation. Previously, we have shown that a methyl cytosine-binding complex binds to the methylated ρ-globin gene in vitro. We have now chromatographically purified and characterized this complex from adult chicken primary erythroid cells. Four components of the MeCP1 transcriptional repression complex were identified: MBD2, RBAP48, HDAC2, and MTA1. These 4 proteins, as well as the zinc-finger protein p66 and the chromatin remodeling factor Mi2, were found to coelute by gel-filtration analysis and pull-down assays. We conclude that these 6 proteins are components of the MeCPC. In adult erythrocytes, significant enrichment for MBD2 is seen at the inactive ρ-globin gene by chromatin immunoprecipitation assay, whereas no enrichment is observed at the active βA-globin gene, demonstrating MBD2 binds to the methylated and transcriptionally silent ρ-globin gene in vivo. Knock-down of MBD2 resulted in up-regulation of a methylated ρ-gene construct in mouse erythroleukemic (MEL)-ρ cells. These results represent the first purification of a MeCP1-like complex from a primary cell source and provide support for a role for MBD2 in developmental gene regulation

IgM Marks Persistent IgG Anti-Human Leukocyte Antigen Antibodies in Highly Sensitized Heart Transplant Patients

BACKGROUND Sensitization to human leukocyte antigens (HLA) is a persistent problem in heart transplant (HT) candidates. We sought to characterize the anti-HLA antibody and circulating B cell repertoire in a cohort of highly sensitized HT candidates. METHODS We assessed IgG and IgM anti-HLA antibodies using luminex single antigen bead assays in a cohort of 11 highly sensitized (HS; calculated panel reactive antibody ≥ 90%) and 3 mildly sensitized (MS) candidates. We also performed B cell receptor repertoire sequencing (BCRseq) in HS candidates and 33 non-candidate controls. HLA antibody strength was measured by mean fluorescence intensity (MFI). RESULTS We found that IgM anti-HLA antibodies were present in all HS candidates, but with a lower breadth and strength as compared to IgG. When anti-HLA IgG specificities intersected with IgM, binding strength was higher. In contrast, there were IgM but no intersecting IgG specificities for the MS group. In four candidates in the HS group, IgG anti-HLA antibodies decreased in both breadth and strength after HT, but the decrease in strength was smaller if the IgG possessed a specificity that intersected with pre-transplant IgM. BCRseq revealed larger B cell clonotypes in HS candidates but similar diversity as compared to controls. CONCLUSIONS IgM marks IgG anti-HLA antibodies with higher strength before HT and persistence after HT. The presence of IgM intersecting IgG for an anti-HLA specificity may be useful approach to determine which donor HLA should be avoided for a sensitized candidate

Frailty in heart transplantation: Report from the heart workgroup of a consensus conference on frailty

A consensus conference on frailty in solid organ transplantation took place on February 11, 2018, to discuss the latest developments in frailty, adopt a standardized approach to assessment, and generate ideas for future research. The findings and consensus of the Frailty Heart Workgroup (American Society of Transplantation\u27s Thoracic and Critical Care Community of Practice) are presented here. Frailty is defined as a clinically recognizable state of increased vulnerability resulting from aging-associated decline in reserve and function across multiple physiologic systems such that the ability to cope with every day or acute stressors is compromised. Frailty is increasingly recognized as a distinct biologic entity that can adversely affect outcomes before and after heart transplantation. A greater proportion of patients referred for heart transplantation are older and have more complex comorbidities. However, outcomes data in the pretransplant setting, particularly for younger patients, are limited. Therefore, there is a need to develop objective frailty assessment tools for risk stratification in patients with advanced heart disease. These tools will help to determine appropriate recipient selection for advanced heart disease therapies including heart transplantation and mechanical circulatory support, improve overall outcomes, and help distinguish frailty phenotypes amenable to intervention

Mapping molecular HLA typing data to UNOS antigen equivalents.

BACKGROUND: Histocompatibility labs must convert molecular HLA typing data to antigen equivalencies for entry into the United Network for Organ Sharing (UNOS) UNet system. While an Organ Procurement and Transplantation Network (OPTN) policy document provides general guidelines for conversion, the process is complex because no antigen mapping table is available. We present a UNOS antigen equivalency table for all IPD-IMGT/HLA alleles at the A, B, C, DRB1, DRB3/4/5, DQA1, and DQB1 loci. METHODS: An automated script was developed to generate a UNOS antigen equivalency table. Data sources used in the conversion algorithm included the World Marrow Donor Association (WMDA) antigen table, the HLA Dictionary, and UNOS-provided tables. To validate antigen mappings, we converted National Marrow Donor Program (NMDP) high resolution allele frequencies to antigen equivalents and compared with the UNOS Calculated Panel Reactive Antibodies (CPRA) reference panel. RESULTS: Normalized frequency similarity scores between independent NMDP and UNOS panels for 4 US population categories (Caucasian, Hispanic, African American and Asian/Pacific Islander) ranged from 0.85 to 0.97, indicating correct antigen mapping. An open source web application (ALLele to ANtigen ( ALLAN )) and web services were also developed to map unambiguous and ambiguous HLA typing data to UNOS antigen equivalents based on NMDP population-specific allele frequencies (http://www.transplanttoolbox.org). CONCLUSIONS: Computer-assisted interpretation of molecular HLA data may aid in reducing typing discrepancies in UNet. This work also sets a foundation for molecular typing data to be utilized directly in the UNet match run as well as the virtual crossmatch process at transplant centers

HLA Homozygosity and Likelihood of Sensitization in Kidney Transplant Candidates.

BACKGROUND: Homozygosity for HLAs has been associated with adverse outcomes after viral infection as well as pregnancy-induced HLA sensitization. We sought to assess the relationship between HLA locus homozygosity and the level of HLA antibody sensitization. METHODS: We measured sensitization using the calculated panel reactive antibody value for a large cohort of 147 461 patients added to the US OPTN/United Network for Organ Sharing kidney transplant waitlist between December 2014 and December 2019. We used multinomial logistic modeling to compare 62 510 sensitized patients to 84 955 unsensitized controls. RESULTS: We found that the number of homozygous HLA loci was strongly associated with the level of sensitization. Within mildly, highly, or extremely sensitized candidates, women displayed a higher relative abundance of HLA homozygosity at multiple HLA loci as compared with men, with attenuation of this effect in Black candidates. In a multivariable logistic model, the number of homozygous HLA loci interacted with female sex but not with other factors associated with sensitization, including recipient ethnicity and a history of prior kidney transplant. CONCLUSIONS: This study shows that HLA homozygosity is an innate genetic factor that affects the likelihood of HLA sensitization. Further research is needed to identify the immunologic mechanisms that underlie this observation