Search

T cell alloreactivity against minor histocompatibility antigens (mHAgs)-polymorphic peptides resulting from donor-recipient (D-R) disparity at sites of genetic polymorphisms-is at the core of the therapeutic effect of allogeneic hematopoietic cell transplantation (allo-HCT). Despite the crucial role of mHAgs in graft-versus-leukemia (GvL) and graft-versus-host disease (GvHD) reactions, it remains challenging to consistently link patient-specific mHAg repertoires to clinical outcomes. Here we devise an analytic framework to systematically identify mHAgs, including their detection on HLA class I ligandomes and functional verification of their immunogenicity. The method relies on the integration of polymorphism detection by whole-exome sequencing of germline DNA from D-R pairs with organ-specific transcriptional- and proteome-level expression. Application of this pipeline to 220 HLA-matched allo-HCT D-R pairs demonstrated that total and organ-specific mHAg load could independently predict the occurrence of acute GvHD and chronic pulmonary GvHD, respectively, and defined promising GvL targets, confirmed in a validation cohort of 58 D-R pairs, for the prevention or treatment of post-transplant disease recurrence., (© 2024. The Author(s), under exclusive licence to Springer Nature America, Inc.)

Background: The Er blood group system was recently shown to be defined by PIEZO1. The system consists of high prevalence antigens Er a , Er3, ERSA, and ERAMA; and low prevalence antigen Er b . Er a /Er b are antithetical with Er(a-b+) defined by the ER*B allele [c.7180G>A p.(Gly2394Ser)]. A nonsense variant c.5289C>G p.(Tyr1763*) is associated with a predicted Er null phenotype, and a missense variant c.7174G>A p.(Glu2392Lys) in close proximity to p.2394 causes loss of both Er a and Er b expression., Study Design and Methods: We investigated PIEZO1 in four Er(a-) individuals who presented with anti-Er a . Whole genome sequencing (WGS) and Sanger sequencing were performed. The location and structural differences of predicted protein changes were visualized using the predicted 3-D structure of Piezo1 created using AlphaFold2., Results: One individual was homozygous for the reported ER*B. A second had a novel heterozygous nonsense variant c.3331C>T p.(Gln1111*), but a second allelic variant was not found. In the remaining two individuals, two different heterozygous novel missense variants, c.7184C>T p.(Ala2395Val) or c.7195G>A p.(Gly2399Ser), were in trans to the reported c.7180G>A variant, ER*B. AlphaFold2 protein modeling showed that each of the missense variants is predicted to encode an altered structural conformation near Er a and Er b ., Conclusions: Investigation of archived samples resulted in the identification of three novel PIEZO1 alleles including a predicted Er null and two missense variants. Structural modeling suggests that the missense changes potentially alter Er a /Er b epitope expression with p.2399Ser resulting in a small increase in the negative electrostatic potential., (© 2024 AABB.)

Although immune tolerance evolved to reduce reactivity with self, it creates a gap in the adaptive immune response against microbes that decorate themselves in self-like antigens. This is particularly apparent with carbohydrate-based blood group antigens, wherein microbes can envelope themselves in blood group structures similar to human cells. In this study, we demonstrate that the innate immune lectin, galectin-4 (Gal-4), exhibits strain-specific binding and killing behavior towards microbes that display blood group-like antigens. Examination of binding preferences using a combination of microarrays populated with ABO(H) glycans and a variety of microbial strains, including those that express blood group-like antigens, demonstrated that Gal-4 binds mammalian and microbial antigens that have features of blood group and mammalian-like structures. Although Gal-4 was thought to exist as a monomer that achieves functional bivalency through its two linked carbohydrate recognition domains, our data demonstrate that Gal-4 forms dimers and that differences in the intrinsic ability of each domain to dimerize likely influences binding affinity. While each Gal-4 domain exhibited blood group-binding activity, the C-terminal domain (Gal-4C) exhibited dimeric properties, while the N-terminal domain (Gal-4N) failed to similarly display dimeric activity. Gal-4C not only exhibited the ability to dimerize but also possessed higher affinity toward ABO(H) blood group antigens and microbes expressing glycans with blood group-like features. Furthermore, when compared to Gal-4N, Gal-4C exhibited more potent antimicrobial activity. Even in the context of the full-length protein, where Gal-4N is functionally bivalent by virtue of Gal-4C dimerization, Gal-4C continued to display higher antimicrobial activity. These results demonstrate that Gal-4 exists as a dimer and exhibits its antimicrobial activity primarily through its C-terminal domain. In doing so, these data provide important insight into key features of Gal-4 responsible for its innate immune activity against molecular mimicry., Competing Interests: Conflict of interest The authors declare no competing interests., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

Introduction: Severe respiratory illness is the most prominent manifestation of patients infected with SARS-CoV-2, and yet the molecular mechanisms underlying severe lung disease in COVID-19 affected patients still require elucidation. Human leukocyte antigen class I (HLA-I) expression is crucial for antigen presentation and the host's response to SARS-CoV-2., Methods: To gain insights into the immune response and molecular pathways involved in severe lung disease, we performed immunopeptidomic and proteomic analyses of lung tissues recovered at four COVID-19 autopsy and six non-COVID-19 transplants., Results: We found signals of tissue injury and regeneration in lung fibroblast and alveolar type I/II cells, resulting in the production of highly immunogenic self-antigens within the lungs of COVID-19 patients. We also identified immune activation of the M2c macrophage as the primary source of HLA-I presentation and immunogenicity in this context. Additionally, we identified 28 lung signatures that can serve as early plasma markers for predicting infection and severe COVID-19 disease. These protein signatures were predominantly expressed in macrophages and epithelial cells and were associated with complement and coagulation cascades., Discussion: Our findings emphasize the significant role of macrophage-mediated immunity in the development of severe lung disease in COVID-19 patients., Competing Interests: DK is a scientific advisor for Immunitrack and Breakbio. DK owns equity in Affimed N.V., Agenus, Armata Pharmaceuticals, Breakbio, BioMarin Pharmaceutical, Celldex Therapeutics, Editas Medicine, Gilead Sciences, Immunitybio, ImmunoGen, IMV, Lexicon Pharmaceuticals, Neoleukin Therapeutics. BeiGene, a Chinese biotech company, supported unrelated SARS COV-2 research at TIGL. EK has an unrelated financial interest in Novartis AG. EK receives unrelated research funding from Bayer AG and 10x Genomics, Inc. CW receives funding support from Pharmacyclics and holds equity in BioNTech, Inc. SY holds equity in Yihui Bio, Inc. RK receives grant support from Boehringer Ingelheim and Bayer, LH receives grants from Boehringer Ingelheim and has received personal consulting fees from Boehringer Ingelheim, Pliant Therapeutics, Bioclinica, Abbvie and Biogen Idec. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be constructed as a potential conflict of interest. The author(s) declared that they were an editorial board member of Frontiers, at the time of submission. This had no impact on the peer review process and the final decision., (Copyright © 2023 Yin, Klaeger, Chea, Carulli, Rachimi, Black, Filbin, Hariri, Knipe, Padera, Stevens, Lane, Carr, Wu, Kim and Keskin.)

Use of data-driven methodologies in enhancing blood transfusion practices is rising, leveraging big data, machine learning, and optimization techniques to improve demand forecasting and supply chain management. This review used a narrative approach to identify, evaluate, and synthesize key studies that considered novel computational techniques for blood demand forecasting and inventory management through a search of PubMed and Web of Sciences databases for studies published from January 01, 2016, to March 30, 2023. The studies were analyzed for their utilization of various techniques, and their strengths, limitations, and areas for improvement. Seven key studies were identified. The studies focused on different blood components using various computational methods, such as regression, machine learning, hybrid models, and time series models, across different locations and time periods. Key variables used for demand forecasting were largely derived from electronic health record data, including clinical related predictors such as laboratory test results and hospital census by location. Each study offered unique strengths and valuable insights into the use of data-driven methods in blood bank management. Common limitations were unknown generalizability to other healthcare settings or blood components, need for field-specific performance measures, lack of ABO compatibility consideration, and ethical challenges in resource allocation. While data-driven research in blood demand forecasting and management has progressed, limitations persist and further exploration is needed. Understanding these innovative, interdisciplinary methods and their complexities can help refine inventory strategies and address healthcare challenges more effectively, leading to more robust, accurate models to enhance blood management across diverse healthcare scenarios., Competing Interests: Declaration of Competing Interest The authors report no relevant conflicts of interest., (Copyright © 2023. Published by Elsevier Inc.)

Technological advances in the field of histocompatibility have allowed us to define anti-human leukocyte antigen (HLA) antibody specificity at the allelic level. However, how allele-specific antibodies affect organ allocation is poorly studied. We examined allelic specificities of class I HLA antibodies in 6726 consecutive serum samples from 2953 transplant candidates and evaluated their impact on the corresponding crossmatch and organ allocation. Out of 17 class I HLA antigens represented by >1 allele in the LABScreen single antigen bead assay, 12 had potential allele-specific reactivity. Taking advantage of our unbiased cohort of deceased donor-candidate testing (123,135 complement-dependent cytotoxicity crossmatches between 2014 and 2017), we estimated that the presence of allele-specific antibody detected using a single antigen bead assay (median fluorescence intensity, >3000) against only the rare allele was a poor predictor of a positive complement-dependent cytotoxicity crossmatch, with a positive predictive value of 0% to 7%, compared with 52.5% in allele-concordant class I HLA antibodies against A or B locus antigens. Further, we confirmed allele-specific reactivity using flow crossmatch in 3 scenarios: A11:01/A11:02, A68:01/A68:02, and B44:02/B44:03. Our results suggest that allele-specific antibodies may unnecessarily exclude transplant candidates (up to 10%) from organ offers by overcalling unacceptable antigens; incorporation of selective reactivity pattern in allocation may promote precision matching and more equitable allocation., Competing Interests: Declaration of Competing Interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests:Melissa Yeung reports a relationship with One Lambda Inc that includes: consulting or advisory. Mike Chen reports a relationship with One Lambda Inc that includes: employment. Jar-How Lee reports a relationship with One Lambda Inc that includes: employment., (Copyright © 2023 American Society of Transplantation & American Society of Transplant Surgeons. Published by Elsevier Inc. All rights reserved.)

ABO compatibility is important for kidney transplantation, with longer waitlist times for blood group B kidney transplant candidates. However, kidneys from non-A 1 (eg, A 2 ) subtype donors, which express less A antigen, can be safely transplanted into group B recipients. ABO subtyping is routinely performed using anti-A 1 lectin, but DNA-based genotyping is also possible. Here, we compare lectin and genotyping testing. Lectin and genotype subtyping was performed on 554 group A deceased donor samples at 2 transplant laboratories. The findings were supported by 2 additional data sets of 210 group A living kidney donors and 124 samples with unclear lectin testing sent to a reference laboratory. In deceased donors, genotyping found 65% more A 2 donors than lectin testing, most with weak lectin reactivity, a finding supported in living donors and samples sent for reference testing. DNA sequencing and flow cytometry showed that the discordances were because of several factors, including transfusion, small variability in A antigen levels, and rare ABO∗A2.06 and ABO∗A2.16 sequences. Although lectin testing is the current standard for transplantation subtyping, genotyping is accurate and could increase A 2 kidney transplant opportunities for group B candidates, a difference that should reduce group B wait times and improve transplant equity., (Copyright © 2022 American Society of Transplantation & American Society of Transplant Surgeons. Published by Elsevier Inc. All rights reserved.)

Competing Interests: Disclosure W.J. Lane reports receiving personal consulting fees from CareDx, Inc, and his institution is a founding member of the Blood transfusion Genomics Consortium (BGC) that has received fees from Thermo Fisher Scientific Inc. to help codevelop a high-density DNA genotyping array. J.K. Cone Sullivan has nothing to disclose. N. Gleadall’s institution is a founding member of the Blood transfusion Genomics Consortium (BGC) that has received fees from Thermo Fisher Scientific Inc. to help codevelop a high-density DNA genotyping array.

Background and Objectives: Under the ISBT, the Working Party (WP) for Red Cell Immunogenetics and Blood Group Terminology is charged with ratifying blood group systems, antigens and alleles. This report presents the outcomes from four WP business meetings, one located in Basel in 2019 and three held as virtual meetings during the COVID-19 pandemic in 2020 and 2021., Materials and Methods: As in previous meetings, matters pertaining to blood group antigen nomenclature were discussed. New blood group systems and antigens were approved and named according to the serologic, genetic, biochemical and cell biological evidence presented., Results: Seven new blood group systems, KANNO (defined numerically as ISBT 037), SID (038), CTL2 (039), PEL (040), MAM (041), EMM (042) and ABCC1 (043) were ratified. Two (039 and 043) were de novo discoveries, and the remainder comprised reported antigens where the causal genes were previously unknown. A further 15 blood group antigens were added to the existing blood group systems: MNS (002), RH (004), LU (005), DI (010), SC (013), GE (020), KN (022), JMH (026) and RHAG (030)., Conclusion: The ISBT now recognizes 378 antigens, of which 345 are clustered within 43 blood group systems while 33 still have an unknown genetic basis. The ongoing discovery of new blood group systems and antigens underscores the diverse and complex biology of the red cell membrane. The WP continues to update the blood group antigen tables and the allele nomenclature tables. These can be found on the ISBT website (http://www.isbtweb.org/working-parties/red-cell-immunogenetics-and-blood-group-terminology/)., (© 2022 International Society of Blood Transfusion.)

Genome sequencing holds the promise for great public health benefits. It is currently being used in the context of rare disease diagnosis and novel gene identification, but also has the potential to identify genetic disease risk factors in healthy individuals. Genome sequencing technologies are currently being used to identify genetic factors that may influence variability in symptom severity and immune response among patients infected by SARS-CoV-2. The GENCOV study aims to look at the relationship between genetic, serological, and biochemical factors and variability of SARS-CoV-2 symptom severity, and to evaluate the utility of returning genome screening results to study participants. Study participants select which results they wish to receive with a decision aid. Medically actionable information for diagnosis, disease risk estimation, disease prevention, and patient management are provided in a comprehensive genome report. Using a combination of bioinformatics software and custom tools, this article describes a pipeline for the analysis and reporting of genetic results to individuals with COVID-19, including HLA genotyping, large-scale continental ancestry estimation, and pharmacogenomic analysis to determine metabolizer status and drug response. In addition, this pipeline includes reporting of medically actionable conditions from comprehensive gene panels for Cardiology, Neurology, Metabolism, Hereditary Cancer, and Hereditary Kidney, and carrier screening for reproductive planning. Incorporated into the genome report are polygenic risk scores for six diseases-coronary artery disease; atrial fibrillation; type-2 diabetes; and breast, prostate, and colon cancer-as well as blood group genotyping analysis for ABO and Rh blood types and genotyping for other antigens of clinical relevance. The genome report summarizes the findings of these analyses in a way that extensively communicates clinically relevant results to patients and their physicians. © 2022 Wiley Periodicals LLC. Basic Protocol 1: HLA genotyping and disease association Basic Protocol 2: Large-scale continental ancestry estimation Basic Protocol 3: Dosage recommendations for pharmacogenomic gene variants associated with drug response Support Protocol: System setup., (© 2022 Wiley Periodicals LLC.)

Cancers avoid immune surveillance through an array of mechanisms, including perturbation of HLA class I antigen presentation. Merkel cell carcinoma (MCC) is an aggressive, HLA-I-low, neuroendocrine carcinoma of the skin often caused by the Merkel cell polyomavirus (MCPyV). Through the characterization of 11 newly generated MCC patient-derived cell lines, we identified transcriptional suppression of several class I antigen presentation genes. To systematically identify regulators of HLA-I loss in MCC, we performed parallel, genome-scale, gain- and loss-of-function screens in a patient-derived MCPyV-positive cell line and identified MYCL and the non-canonical Polycomb repressive complex 1.1 (PRC1.1) as HLA-I repressors. We observed physical interaction of MYCL with the MCPyV small T viral antigen, supporting a mechanism of virally mediated HLA-I suppression. We further identify the PRC1.1 component USP7 as a pharmacologic target to restore HLA-I expression in MCC.

Adaptive immunity can target a nearly infinite range of antigens, yet it is tempered by tolerogenic mechanisms that limit autoimmunity. Such immunological tolerance, however, creates a gap in adaptive immunity against microbes decorated with self-like antigens as a form of molecular mimicry. Our results demonstrate that the innate immune lectin galectin-7 (Gal-7) binds a variety of distinct microbes, all of which share features of blood group-like antigens. Gal-7 binding to each blood group expressing microbe, including strains of Escherichia coli , Klebsiella pneumoniae , Providencia alcalifaciens, and Streptococcus pneumoniae , results in loss of microbial viability. Although Gal-7 also binds red blood cells (RBCs), this interaction does not alter RBC membrane integrity. These results demonstrate that Gal-7 recognizes a diverse range of microbes, each of which use molecular mimicry while failing to induce host cell injury, and thus may provide an innate form of immunity against molecular mimicry., Competing Interests: The authors declare no competing interests., (© 2022 The Author(s).)

Background and Objectives: Non-invasive assays for predicting foetal blood group status in pregnancy serve as valuable clinical tools in the management of pregnancies at risk of detrimental consequences due to blood group antigen incompatibility. To secure clinical applicability, assays for non-invasive prenatal testing of foetal blood groups need to follow strict rules for validation and quality assurance. Here, we present a multi-national position paper with specific recommendations for validation and quality assurance for such assays and discuss their risk classification according to EU regulations., Materials and Methods: We reviewed the literature covering validation for in-vitro diagnostic (IVD) assays in general and for non-invasive foetal RHD genotyping in particular. Recommendations were based on the result of discussions between co-authors., Results: In relation to Annex VIII of the In-Vitro-Diagnostic Medical Device Regulation 2017/746 of the European Parliament and the Council, assays for non-invasive prenatal testing of foetal blood groups are risk class D devices. In our opinion, screening for targeted anti-D prophylaxis for non-immunized RhD negative women should be placed under risk class C. To ensure high quality of non-invasive foetal blood group assays within and beyond the European Union, we present specific recommendations for validation and quality assurance in terms of analytical detection limit, range and linearity, precision, robustness, pre-analytics and use of controls in routine testing. With respect to immunized women, different requirements for validation and IVD risk classification are discussed., Conclusion: These recommendations should be followed to ensure appropriate assay performance and applicability for clinical use of both commercial and in-house assays., (© 2021 International Society of Blood Transfusion.)

Emm is a high incidence red cell antigen with eight previously reported Emm- probands. Anti-Emm appears to be naturally occurring yet responsible for a clinically significant acute hemolytic transfusion reaction. Previous work suggests that Emm is located on a GPI-anchored protein, but the antigenic epitope and genetic basis have been elusive. We investigated samples from a South Asian Indian family with two Emm- brothers by whole genome sequencing (WGS). Additionally, samples from four unrelated Emm- individuals were investigated for variants in the candidate gene. Filtering for homozygous variants found in the Emm- brothers and by gnomAD frequency of < 0.001 resulted in 1818 variants with one of high impact; a 2-bp deletion causing a frameshift and premature stop codon in PIGG [NM&#95;001127178.3:c.2624&#95;2625delTA, p.(Leu875*), rs771819481]. PIGG encodes for a transferase, GPI-ethanolaminephosphate transferase II, which adds ethanolamine phosphate (EtNP) to the second mannose in a GPI-anchor. The four additional unrelated Emm- individuals had various PIGG mutations; deletion of Exons 2-3, deletion of Exons 7-9, insertion/deletion (indel) in Exon 3, and new stop codon in Exon 5. The Emm- phenotype is associated with a rare deficiency of PIGG, potentially defining a new Emm blood group system composed of EtNP bound to mannose, part of the GPI-anchor. The results are consistent with the known PI-linked association of the Emm antigen, and may explain the production of the antibody in the absence of RBC transfusion. Any association with neurologic phenotypes requires further research., (© 2021. The Author(s).)

Background and Objectives: Rh is one of the most diverse and complex blood group systems. Recently, next generation sequencing (NGS) has proven to be a viable option for RH genotyping. We have developed automated software (bloodTyper) for determining alleles encoding RBC antigens from NGS-based whole genome sequencing (WGS). The bloodTyper algorithm has not yet been optimized and evaluated for complex and uncommon RH alleles., Materials and Methods: Twenty-two samples with previous polymerase chain reaction (PCR) and Sanger sequencing-based RH genotyping underwent WGS. bloodTyper was used to detect RH alleles including those defined by structural variation (SV) using a combination of three independent strategies: sequence read depth of coverage, split reads and paired reads., Results: bloodTyper was programmed to identify D negative and positive phenotypes as well as the presence of alleles encoding weak D, partial D and variant RHCE. Sequence read depth of coverage calculation accurately determined RHD zygosity and detected the presence of RHD/RHCE hybrids. RHCE*C was determined by sequence read depth of coverage and by split read methods. RHD hybrid alleles and RHCE*C were confirmed by using a paired read approach. Small SVs present in RHCE*CeRN and RHCE*ceHAR were detected by a combined read depth of coverage and paired read approach., Conclusions: The combination of several different interpretive approaches allowed for automated software based-RH genotyping of WGS data including RHD zygosity and complex compound RHD and RHCE heterozygotes. The scalable nature of this automated analysis will enable RH genotyping in large genomic sequencing projects., (© 2020 International Society of Blood Transfusion.)

Each year, blood transfusions save millions of lives. However, under current blood-matching practices, sensitization to non-self-antigens is an unavoidable adverse side effect of transfusion. We describe a universal donor typing platform that could be adopted by blood services worldwide to facilitate a universal extended blood-matching policy and reduce sensitization rates. This DNA-based test is capable of simultaneously typing most clinically relevant red blood cell (RBC), human platelet (HPA), and human leukocyte (HLA) antigens. Validation was performed, using samples from 7927 European, 27 South Asian, 21 East Asian, and 9 African blood donors enrolled in 2 national biobanks. We illustrated the usefulness of the platform by analyzing antibody data from patients sensitized with multiple RBC alloantibodies. Genotyping results demonstrated concordance of 99.91%, 99.97%, and 99.03% with RBC, HPA, and HLA clinically validated typing results in 89 371, 3016, and 9289 comparisons, respectively. Genotyping increased the total number of antigen typing results available from 110 980 to >1 200 000. Dense donor typing allowed identification of 2 to 6 times more compatible donors to serve 3146 patients with multiple RBC alloantibodies, providing at least 1 match for 176 individuals for whom previously no blood could be found among the same donors. This genotyping technology is already being used to type thousands of donors taking part in national genotyping studies. Extraction of dense antigen-typing data from these cohorts provides blood supply organizations with the opportunity to implement a policy of genomics-based precision matching of blood.

Background: The MNS blood group system is defined by three homologous genes: GYPA, GYPB, and GYPE. GYPB encodes for glycophorin B (GPB) carrying S/s and the "universal" antigen U. RBCs of approximately 1% of individuals of African ancestry are U- due to absence of GPB. The U- phenotype has long been attributed to a deletion encompassing GYPB exons 2 to 5 and GYPE exon 1 (GYPB*01N)., Study Design and Methods: Samples from two U-individuals underwent Illumina short read whole genome sequencing (WGS) and Nanopore long read WGS. In addition, two existing WGS datasets, MedSeq (n = 110) and 1000 Genomes (1000G, n = 2535), were analyzed for GYPB deletions. Deletions were confirmed by Sanger sequencing. Twenty known U- donor samples were tested by a PCR assay to determine the specific deletion alleles present in African Americans., Results: Two large GYPB deletions in U- samples of African ancestry were identified: a 110 kb deletion extending left of GYPB (DEL&#95;B&#95;LEFT) and a 103 kb deletion extending right (DEL&#95;B&#95;RIGHT). DEL&#95;B&#95;LEFT and DEL&#95;B&#95;RIGHT were the most common GYPB deletions in the 1000 Genomes Project 669 African genomes (allele frequencies 0.04 and 0.02). Seven additional deletions involving GYPB were seen in African, Admixed American, and South Asian samples. No samples analyzed had GYPB*01N., Conclusions: The U- phenotype in those of African ancestry is primarily associated with two different complete deletions of GYPB (with intact GYPE). Seven additional less common GYPB deletion backgrounds were found. GYPB*01N, long assumed to be the allele commonly encoding U- phenotypes, appears to be rare., (© 2020 AABB.)

Prediction of HLA epitopes is important for the development of cancer immunotherapies and vaccines. However, current prediction algorithms have limited predictive power, in part because they were not trained on high-quality epitope datasets covering a broad range of HLA alleles. To enable prediction of endogenous HLA class I-associated peptides across a large fraction of the human population, we used mass spectrometry to profile >185,000 peptides eluted from 95 HLA-A, -B, -C and -G mono-allelic cell lines. We identified canonical peptide motifs per HLA allele, unique and shared binding submotifs across alleles and distinct motifs associated with different peptide lengths. By integrating these data with transcript abundance and peptide processing, we developed HLAthena, providing allele-and-length-specific and pan-allele-pan-length prediction models for endogenous peptide presentation. These models predicted endogenous HLA class I-associated ligands with 1.5-fold improvement in positive predictive value compared with existing tools and correctly identified >75% of HLA-bound peptides that were observed experimentally in 11 patient-derived tumor cell lines.

Background: Genotyping has expanded the number red blood cell (RBC) and platelet (PLT) antigens that can readily be typed, but often represents an additional testing cost. The analysis of existing genomic data offers a cost-effective approach. We recently developed automated software (bloodTyper) for determination of RBC and PLT antigens from whole genome sequencing. Here we extend the algorithm to whole exome sequencing (WES)., Study Design and Methods: Whole exome sequencing was performed on samples from 75 individuals. WES-based bloodTyper RBC and PLT typing was compared to conventional polymerase chain reaction (PCR) RHD zygosity testing and serologic and single-nucleotide polymorphism (SNP) typing for 38 RBC antigens in 12 systems (17 serologic and 35 SNPs) and 22 PLT antigens (22 SNPs). Samples from the first 20 individuals were used to modify bloodTyper to interpret WES followed by blinded typing of 55 samples., Results: Over the first 20 samples, discordances were noted for C, M, and N antigens, which were due to WES-specific biases. After modification, bloodTyper was 100% accurate on blinded evaluation of the last 55 samples and outperformed both serologic (99.67% accurate) and SNP typing (99.97% accurate) reflected by two Fy b and one N serologic typing errors and one undetected SNP encoding a Jk null phenotype. RHD zygosity testing by bloodTyper was 100% concordant with a combination of hybrid Rhesus box PCR and PCR-restriction fragment length polymorphism for all samples., Conclusion: The automated bloodTyper software was modified for WES biases to allow for accurate RBC and PLT antigen typing. Such analysis could become a routing part of future WES efforts., (© 2019 AABB.)

Background: Calculated panel reactive antibody (cPRA) scoring is used to assess whether platelet refractoriness is mediated by human leukocyte antigen (HLA) antibodies in the recipient. cPRA testing uses a national sample of US kidney donors to estimate the population frequency of HLA antigens, which may be different than HLA frequencies within local platelet inventories. We aimed to determine the impact on patient cPRA scores of using HLA frequencies derived from typing local platelet donations rather than national HLA frequencies., Methods: We built an open-source web service to calculate cPRA scores based on national frequencies or custom-derived frequencies. We calculated cPRA scores for every hematopoietic stem cell transplantation (HSCT) patient at our institution based on the United Network for Organ Sharing (UNOS) frequencies and local frequencies. We compared frequencies and correlations between the calculators, segmented by gender. Finally, we put all scores into three buckets (mild, moderate, and high sensitizations) and looked at intergroup movement., Results: 2531 patients that underwent HSCT at our institution had at least 1 antibody and were included in the analysis. Overall, the difference in medians between each group's UNOS cPRA and local cPRA was statistically significant, but highly correlated (UNOS vs. local total: 0.249 and 0.243, ρ = 0.994; UNOS vs. local female: 0.474 and 0.463, ρ = 0.987, UNOS vs. local male: 0.165 and 0.141, ρ = 0.996; P < 0.001 for all comparisons). The median difference between UNOS and cPRA scores for all patients was low (male: 0.014, interquartile range [IQR]: 0.004-0.029; female: 0.0013, IQR: 0.003-0.028). Placement of patients into three groups revealed little intergroup movement, with 2.96% (75/2531) of patients differentially classified., Conclusions: cPRA scores using local frequencies were modestly but significantly different than those obtained using national HLA frequencies. We released our software as open source, so other groups can calculate cPRA scores from national or custom-derived frequencies. Further investigation is needed to determine whether a local-HLA frequency approach can improve outcomes in patients who are immune-refractory to platelets., Competing Interests: There are no conflicts of interest.

Immune evasion is a hallmark of cancer and a central mechanism underlying acquired resistance to immune therapy. In allogeneic hematopoietic cell transplantation (alloHCT), late relapses can arise after prolonged alloreactive T-cell control, but the molecular mechanisms of immune escape remain unclear. To identify mechanisms of immune evasion, we performed a genetic analysis of serial samples from 25 patients with myeloid malignancies who relapsed ≥1 year after alloHCT. Using targeted sequencing and microarray analysis to determine HLA allele-specific copy number, we identified copy-neutral loss of heterozygosity events and focal deletions spanning class 1 HLA genes in 2 of 12 recipients of matched unrelated-donor HCT and in 1 of 4 recipients of mismatched unrelated-donor HCT. Relapsed clones, although highly related to their antecedent pretransplantation malignancies, frequently acquired additional mutations in transcription factors and mitogenic signaling genes. Previously, the study of relapse after haploidentical HCT established the paradigm of immune evasion via loss of mismatched HLA. Here, in the context of matched unrelated-donor HCT, HLA loss provides genetic evidence that allogeneic immune recognition may be mediated by minor histocompatibility antigens and suggests opportunities for novel immunologic approaches for relapse prevention., (© 2019 by The American Society of Hematology.)

Background: Although P1 and Xg a are known to be associated with the A4GALT and XG genes, respectively, the genetic basis of antigen expression has been elusive. Recent reports link both P1 and Xg a expression with nucleotide changes in the promotor regions and with antigen-negative phenotypes due to disruption of transcription factor binding., Study Design and Methods: Whole genome sequencing was performed on 113 individuals as part of the MedSeq Project with serologic RBC antigen typing for P1 (n = 77) and Xg a (n = 15). Genomic data were analyzed by two approaches, nucleotide frequency correlation and serologic correlation, to find A4GALT and XG changes associated with P1 and Xg a expression., Results: For P1, the frequency approach identified 29 possible associated nucleotide changes, and the serologic approach revealed four among them correlating with the P1+/P1- phenotype: chr22:43,115,523&#95;43,115,520AAAG/delAAAG (rs66781836); chr 22:43,114,551C/T (rs8138197); chr22:43,114,020 T/G (rs2143918); and chr22:43,113,793G/T (rs5751348). For Xg a , the frequency approach identified 82 possible associated nucleotide changes, and among these the serologic approach revealed one correlating with the Xg(a+)/Xg(a-) phenotype: chrX:2,666,384G/C (rs311103)., Conclusion: A bioinformatics analysis pipeline was created to identify genetic changes responsible for RBC antigen expression. This study, in progress before the recently published reports, independently confirms the basis for P1 and Xg a . Although this enabled molecular typing of these antigens, the Y chromosome PAR1 region interfered with Xg a typing in males. This approach could be used to identify and confirm the genetic basis of antigens, potentially replacing the historical approach using family pedigrees as genomic sequencing becomes commonplace., (© 2018 AABB.)

Neoantigens, which are derived from tumour-specific protein-coding mutations, are exempt from central tolerance, can generate robust immune responses 1,2 and can function as bona fide antigens that facilitate tumour rejection 3 . Here we demonstrate that a strategy that uses multi-epitope, personalized neoantigen vaccination, which has previously been tested in patients with high-risk melanoma 4-6 , is feasible for tumours such as glioblastoma, which typically have a relatively low mutation load 1,7 and an immunologically 'cold' tumour microenvironment 8 . We used personalized neoantigen-targeting vaccines to immunize patients newly diagnosed with glioblastoma following surgical resection and conventional radiotherapy in a phase I/Ib study. Patients who did not receive dexamethasone-a highly potent corticosteroid that is frequently prescribed to treat cerebral oedema in patients with glioblastoma-generated circulating polyfunctional neoantigen-specific CD4 + and CD8 + T cell responses that were enriched in a memory phenotype and showed an increase in the number of tumour-infiltrating T cells. Using single-cell T cell receptor analysis, we provide evidence that neoantigen-specific T cells from the peripheral blood can migrate into an intracranial glioblastoma tumour. Neoantigen-targeting vaccines thus have the potential to favourably alter the immune milieu of glioblastoma.

Recent studies have highlighted the promise of targeting tumor neoantigens to generate potent antitumor immune responses and provide strong motivation for improving our understanding of antigen-T-cell receptor (TCR) interactions. Advances in single-cell sequencing technologies have opened the door for detailed investigation of the TCR repertoire, providing paired information from TCRα and TCRβ, which together determine specificity. However, a need remains for efficient methods to assess the specificity of discovered TCRs. We developed a streamlined approach for matching TCR sequences with cognate antigen through on-demand cloning and expression of TCRs and screening against candidate antigens. Here, we first demonstrate the system's capacity to identify viral-antigen-specific TCRs and compare the functional avidity of TCRs specific for a given antigen target. We then apply this system to identify neoantigen-specific TCR sequences from patients with melanoma treated with personalized neoantigen vaccines and characterize functional avidity of neoantigen-specific TCRs. Furthermore, we use a neoantigen-prediction pipeline to show that an insertion-deletion mutation in a putative chronic lymphocytic leukemia (CLL) driver gives rise to an immunogenic neoantigen mut- MGA, and use this approach to identify the mut- MGA -specific TCR sequence. This approach provides a means to identify and express TCRs, and then rapidly assess antigen specificity and functional avidity of a reconstructed TCR, which can be applied for monitoring antigen-specific T-cell responses, and potentially for guiding the design of effective T-cell-based immunotherapies., (© 2018 by The American Society of Hematology.)

Background: There are more than 300 known red blood cell (RBC) antigens and 33 platelet antigens that differ between individuals. Sensitisation to antigens is a serious complication that can occur in prenatal medicine and after blood transfusion, particularly for patients who require multiple transfusions. Although pre-transfusion compatibility testing largely relies on serological methods, reagents are not available for many antigens. Methods based on single-nucleotide polymorphism (SNP) arrays have been used, but typing for ABO and Rh-the most important blood groups-cannot be done with SNP typing alone. We aimed to develop a novel method based on whole-genome sequencing to identify RBC and platelet antigens., Methods: This whole-genome sequencing study is a subanalysis of data from patients in the whole-genome sequencing arm of the MedSeq Project randomised controlled trial (NCT01736566) with no measured patient outcomes. We created a database of molecular changes in RBC and platelet antigens and developed an automated antigen-typing algorithm based on whole-genome sequencing (bloodTyper). This algorithm was iteratively improved to address cis-trans haplotype ambiguities and homologous gene alignments. Whole-genome sequencing data from 110 MedSeq participants (30 × depth) were used to initially validate bloodTyper through comparison with conventional serology and SNP methods for typing of 38 RBC antigens in 12 blood-group systems and 22 human platelet antigens. bloodTyper was further validated with whole-genome sequencing data from 200 INTERVAL trial participants (15 × depth) with serological comparisons., Findings: We iteratively improved bloodTyper by comparing its typing results with conventional serological and SNP typing in three rounds of testing. The initial whole-genome sequencing typing algorithm was 99·5% concordant across the first 20 MedSeq genomes. Addressing discordances led to development of an improved algorithm that was 99·8% concordant for the remaining 90 MedSeq genomes. Additional modifications led to the final algorithm, which was 99·2% concordant across 200 INTERVAL genomes (or 99·9% after adjustment for the lower depth of coverage)., Interpretation: By enabling more precise antigen-matching of patients with blood donors, antigen typing based on whole-genome sequencing provides a novel approach to improve transfusion outcomes with the potential to transform the practice of transfusion medicine., Funding: National Human Genome Research Institute, Doris Duke Charitable Foundation, National Health Service Blood and Transplant, National Institute for Health Research, and Wellcome Trust., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

This corrects the article DOI: 10.1038/nature22991.

Effective anti-tumour immunity in humans has been associated with the presence of T cells directed at cancer neoantigens, a class of HLA-bound peptides that arise from tumour-specific mutations. They are highly immunogenic because they are not present in normal tissues and hence bypass central thymic tolerance. Although neoantigens were long-envisioned as optimal targets for an anti-tumour immune response, their systematic discovery and evaluation only became feasible with the recent availability of massively parallel sequencing for detection of all coding mutations within tumours, and of machine learning approaches to reliably predict those mutated peptides with high-affinity binding of autologous human leukocyte antigen (HLA) molecules. We hypothesized that vaccination with neoantigens can both expand pre-existing neoantigen-specific T-cell populations and induce a broader repertoire of new T-cell specificities in cancer patients, tipping the intra-tumoural balance in favour of enhanced tumour control. Here we demonstrate the feasibility, safety, and immunogenicity of a vaccine that targets up to 20 predicted personal tumour neoantigens. Vaccine-induced polyfunctional CD4 + and CD8 + T cells targeted 58 (60%) and 15 (16%) of the 97 unique neoantigens used across patients, respectively. These T cells discriminated mutated from wild-type antigens, and in some cases directly recognized autologous tumour. Of six vaccinated patients, four had no recurrence at 25 months after vaccination, while two with recurrent disease were subsequently treated with anti-PD-1 (anti-programmed cell death-1) therapy and experienced complete tumour regression, with expansion of the repertoire of neoantigen-specific T cells. These data provide a strong rationale for further development of this approach, alone and in combination with checkpoint blockade or other immunotherapies.

Platelet transfusions are a cornerstone of therapy for patients who develop thrombocytopenia while undergoing Hematopoietic Stem Cell Transplantation (HSCT). Many patients who develop Platelet Transfusion Refractoriness (PTR) require HLA-matched platelets. Identifying these patients early could lead to better utilization of platelets as well as increased platelet counts. We built a SMART on FHIR visualization tool to aid the oncology, blood bank, and blood donor center teams in identifying these patients by showing trends in thrombocytopenia along with a computer generated calculated Panel Reactive Antibody (cPRA) level. To do this, we required a FHIR interface to our HLA database. We describe our methods and outcome for constructing this FHIR interface, as well as the architecture and data flow of HLA data from its proprietary database to the SMART on FHIR environment and application database along with RESTful cPRA web service calculator. Future work will evaluate the clinical impact of this platelet visualization tool and overall success of our FHIR implementation.

Background: Cold agglutinin disease (CAD) is a rare autoimmune hemolytic anemia mediated by autoantibodies that preferentially react at 4°C. Laboratory testing for cold-reactive autoantibodies is laborious and may not be ordered judiciously, particularly in patients with a negative direct antiglobulin test (DAT). We sought to determine whether a negative DAT using anti-human complement (anti-C3) rules out elevated cold agglutinin (CA) titers and the diagnosis of CAD., Study Design and Methods: We performed a retrospective study of patients with a CA test performed at three major academic medical centers: Barnes-Jewish Hospital (2003-2014), Vanderbilt University Medical Center (2007-2009), and Massachusetts General Hospital (2009-2014)., Results: This study included 801 patients, of whom 51% (n = 410) had a DAT within the 7 days before CA testing. A total of 98% of patients with a negative DAT using anti-C3 had a negative CA titer (<64). Only five subjects had a negative DAT using anti-C3 and an elevated CA titer., Conclusions: Overutilization of CA testing could be reduced by establishing laboratory acceptance criteria based on a positive DAT using anti-C3. Such acceptance criteria would have reduced CA testing by 68% for those with an available DAT result., (© 2017 AABB.)

Competing Interests: of conflicts of interest CAT holds a research grant from Terumo BCT. FFW and WAF are inventors of patents owned by the German Red Cross Blood Service Baden-Württemberg-Hessen. GAD is the inventor of patents on red cell genotyping owned by the Canadian Blood Services and on the speakers’ bureau of Grifols. GS receives research support from Immucor. LC is on the Transfusion Advisory Board of Grifols. MAK is on the speakers’ bureau of Agena Bioscience. WAF holds intellectual property rights for RHD genotyping applications. WALH is a consultant for Cerus Corporation and Seres Therapeutics. WJL receives support from Wiley Publishing for educational activities related to the Transfusion News Question of the Day. The remaining authors declared that they have no competing financial interests relevant to this article.

Background: There are 346 serologically defined red blood cell (RBC) antigens and 33 serologically defined platelet (PLT) antigens, most of which have known genetic changes in 45 RBC or six PLT genes that correlate with antigen expression. Polymorphic sites associated with antigen expression in the primary literature and reference databases are annotated according to nucleotide positions in cDNA. This makes antigen prediction from next-generation sequencing data challenging, since it uses genomic coordinates., Study Design and Methods: The conventional cDNA reference sequences for all known RBC and PLT genes that correlate with antigen expression were aligned to the human reference genome. The alignments allowed conversion of conventional cDNA nucleotide positions to the corresponding genomic coordinates. RBC and PLT antigen prediction was then performed using the human reference genome and whole genome sequencing (WGS) data with serologic confirmation., Results: Some major differences and alignment issues were found when attempting to convert the conventional cDNA to human reference genome sequences for the following genes: ABO, A4GALT, RHD, RHCE, FUT3, ACKR1 (previously DARC), ACHE, FUT2, CR1, GCNT2, and RHAG. However, it was possible to create usable alignments, which facilitated the prediction of all RBC and PLT antigens with a known molecular basis from WGS data. Traditional serologic typing for 18 RBC antigens were in agreement with the WGS-based antigen predictions, providing proof of principle for this approach., Conclusion: Detailed mapping of conventional cDNA annotated RBC and PLT alleles can enable accurate prediction of RBC and PLT antigens from whole genomic sequencing data., (© 2015 The Authors Transfusion published by Wiley Periodicals, Inc. on behalf of AABB.)

Detection of somatic mutations in human leukocyte antigen (HLA) genes using whole-exome sequencing (WES) is hampered by the high polymorphism of the HLA loci, which prevents alignment of sequencing reads to the human reference genome. We describe a computational pipeline that enables accurate inference of germline alleles of class I HLA-A, B and C genes and subsequent detection of mutations in these genes using the inferred alleles as a reference. Analysis of WES data from 7,930 pairs of tumor and healthy tissue from the same patient revealed 298 nonsilent HLA mutations in tumors from 266 patients. These 298 mutations are enriched for likely functional mutations, including putative loss-of-function events. Recurrence of mutations suggested that these 'hotspot' sites were positively selected. Cancers with recurrent somatic HLA mutations were associated with upregulation of signatures of cytolytic activity characteristic of tumor infiltration by effector lymphocytes, supporting immune evasion by altered HLA function as a contributory mechanism in cancer.

As genome sequencing technologies increasingly enter medical practice, genetics laboratories must communicate sequencing results effectively to nongeneticist physicians. We describe the design and delivery of a clinical genome sequencing report, including a one-page summary suitable for interpretation by primary care physicians. To illustrate our preliminary experience with this report, we summarize the genomic findings from 10 healthy participants in a study of genome sequencing in primary care., (© 2015 S. Karger AG, Basel.)

Background: The MedSeq Project is a randomized clinical trial developing approaches to assess the impact of integrating genome sequencing into clinical medicine. To facilitate the return of results of potential medical relevance to physicians and patients participating in the MedSeq Project, we sought to develop a reporting approach for the effective communication of such findings., Methods: Genome sequencing was performed on the Illumina HiSeq platform. Variants were filtered, interpreted, and validated according to methods developed by the Laboratory for Molecular Medicine and consistent with current professional guidelines. The GeneInsight software suite, which is integrated with the Partners HealthCare electronic health record, was used for variant curation, report drafting, and delivery., Results: We developed a concise 5-6 page Genome Report (GR) featuring a single-page summary of results of potential medical relevance with additional pages containing structured variant, gene, and disease information along with supporting evidence for reported variants and brief descriptions of associated diseases and clinical implications. The GR is formatted to provide a succinct summary of genomic findings, enabling physicians to take appropriate steps for disease diagnosis, prevention, and management in their patients., Conclusions: Our experience highlights important considerations for the reporting of results of potential medical relevance and provides a framework for interpretation and reporting practices in clinical genome sequencing.

Background: Pathology informatics is both emerging as a distinct subspecialty and simultaneously becoming deeply integrated within the breadth of pathology practice. As specialists, pathology informaticians need a broad skill set, including aptitude with information fundamentals, information systems, workflow and process, and governance and management. Currently, many of those seeking training in pathology informatics additionally choose training in a second subspecialty. Combining pathology informatics training with molecular pathology is a natural extension, as molecular pathology is a subspecialty with high potential for application of modern biomedical informatics techniques., Methods and Results: Pathology informatics and molecular pathology fellows and faculty evaluated the current fellowship program's core curriculum topics and subtopics for relevance to molecular pathology. By focusing on the overlap between the two disciplines, a structured curriculum consisting of didactics, operational rotations, and research projects was developed for those fellows interested in both pathology informatics and molecular pathology., Conclusions: The scope of molecular diagnostics is expanding dramatically as technology advances and our understanding of disease extends to the genetic level. Here, we highlight many of the informatics challenges facing molecular pathology today, and outline specific informatics principles necessary for the training of future molecular pathologists.

Background: In 2007, our healthcare system established a clinical fellowship program in Pathology Informatics. In 2010 a core didactic course was implemented to supplement the fellowship research and operational rotations. In 2011, the course was enhanced by a formal, structured core curriculum and reading list. We present and discuss our rationale and development process for the Core Curriculum and the role it plays in our Pathology Informatics Fellowship Training Program., Materials and Methods: The Core Curriculum for Pathology Informatics was developed, and is maintained, through the combined efforts of our Pathology Informatics Fellows and Faculty. The curriculum was created with a three-tiered structure, consisting of divisions, topics, and subtopics. Primary (required) and suggested readings were selected for each subtopic in the curriculum and incorporated into a curated reading list, which is reviewed and maintained on a regular basis., Results: Our Core Curriculum is composed of four major divisions, 22 topics, and 92 subtopics that cover the wide breadth of Pathology Informatics. The four major divisions include: (1) Information Fundamentals, (2) Information Systems, (3) Workflow and Process, and (4) Governance and Management. A detailed, comprehensive reading list for the curriculum is presented in the Appendix to the manuscript and contains 570 total readings (current as of March 2012)., Discussion: The adoption of a formal, core curriculum in a Pathology Informatics fellowship has significant impacts on both fellowship training and the general field of Pathology Informatics itself. For a fellowship, a core curriculum defines a basic, common scope of knowledge that the fellowship expects all of its graduates will know, while at the same time enhancing and broadening the traditional fellowship experience of research and operational rotations. For the field of Pathology Informatics itself, a core curriculum defines to the outside world, including departments, companies, and health systems considering hiring a pathology informatician, the core knowledge set expected of a person trained in the field and, more fundamentally, it helps to define the scope of the field within Pathology and healthcare in general.

Background: Pathology Informatics is a new field; a field that is still defining itself even as it begins the formalization, accreditation, and board certification process. At the same time, Pathology itself is changing in a variety of ways that impact informatics, including subspecialization and an increased use of data analysis. In this paper, we examine how these changes impact both the structure of Pathology Informatics fellowship programs and the fellows' goals within those programs., Materials and Methods: As part of our regular program review process, the fellows evaluated the value and effectiveness of our existing fellowship tracks (Research Informatics, Clinical Two-year Focused Informatics, Clinical One-year Focused Informatics, and Clinical 1 + 1 Subspecialty Pathology and Informatics). They compared their education, informatics background, and anticipated career paths and analyzed them for correlations between those parameters and the fellowship track chosen. All current and past fellows of the program were actively involved with the project., Results: Fellows' anticipated career paths correlated very well with the specific tracks in the program. A small set of fellows (Clinical - one or two year - Focused Informatics tracks) anticipated clinical careers primarily focused in informatics (Director of Informatics). The majority of the fellows, however, anticipated a career practicing in a Pathology subspecialty, using their informatics training to enhance that practice (Clinical 1 + 1 Subspecialty Pathology and Informatics Track). Significantly, all fellows on this track reported they would not have considered a Clinical Two-year Focused Informatics track if it was the only track offered. The Research and the Clinical One-year Focused Informatics tracks each displayed unique value for different situations., Conclusions: It seems a "one size fits all" fellowship structure does not fit the needs of the majority of potential Pathology Informatics candidates. Increasingly, these fellowships must be able to accommodate the needs of candidates anticipating a wide range of Pathology Informatics career paths, be able to accommodate Pathology's increasingly subspecialized structure, and do this in a way that respects the multiple fellowships needed to become a subspecialty pathologist and informatician. This is further complicated as Pathology Informatics begins to look outward and takes its place in the growing, and still ill-defined, field of Clinical Informatics, a field that is not confined to just one medical specialty, to one way of practicing medicine, or to one way of providing patient care.

Background: Last year, our pathology informatics fellowship added informatics-based interactive case studies to its existing educational platform of operational and research rotations, clinical conferences, a common core curriculum with an accompanying didactic course, and national meetings., Methods: The structure of the informatics case studies was based on the traditional business school case study format. Three different formats were used, varying in length from short, 15-minute scenarios to more formal multiple hour-long case studies. Case studies were presented over the course of three retreats (Fall 2011, Winter 2012, and Spring 2012) and involved both local and visiting faculty and fellows., Results: Both faculty and fellows found the case studies and the retreats educational, valuable, and enjoyable. From this positive feedback, we plan to incorporate the retreats in future academic years as an educational component of our fellowship program., Conclusions: Interactive case studies appear to be valuable in teaching several aspects of pathology informatics that are difficult to teach in more traditional venues (rotations and didactic class sessions). Case studies have become an important component of our fellowship's educational platform.

The Escherichia coli transcription system is the best characterized from a biochemical and genetic point of view and has served as a model system. Nevertheless, a molecular understanding of the details of E. coli transcription and its regulation, and therefore its full exploitation as a model system, has been hampered by the absence of high-resolution structural information on E. coli RNA polymerase (RNAP). We use a combination of approaches, including high-resolution X-ray crystallography, ab initio structural prediction, homology modeling, and single-particle cryo-electron microscopy, to generate complete atomic models of E. coli core RNAP and an E. coli RNAP ternary elongation complex. The detailed and comprehensive structural descriptions can be used to help interpret previous biochemical and genetic data in a new light and provide a structural framework for designing experiments to understand the function of the E. coli lineage-specific insertions and their role in the E. coli transcription program., Competing Interests: The authors have declared that no competing interests exist.

Comprehensive multiple sequence alignments of the multisubunit DNA-dependent RNA polymerase (RNAP) large subunits, including the bacterial beta and beta' subunits and their homologs from archaebacterial RNAPs, eukaryotic RNAPs I-III, nuclear-cytoplasmic large double-stranded DNA virus RNAPs, and plant plastid RNAPs, were created [Lane, W. J. and Darst, S. A. (2009). Molecular evolution of multisubunit RNA polymerases: sequence analysis. In press]. The alignments were used to delineate sequence regions shared among all classes of multisubunit RNAPs, defining common, fundamental RNAP features as well as identifying highly conserved positions. Here, we present a systematic, detailed structural analysis of these shared regions and highly conserved positions in terms of the RNAP structure, as well as the RNAP structure/function relationship, when known.

Transcription in all cellular organisms is performed by multisubunit, DNA-dependent RNA polymerases that synthesize RNA from DNA templates. Previous sequence and structural studies have elucidated the importance of shared regions common to all multisubunit RNA polymerases. In addition, RNA polymerases contain multiple lineage-specific domain insertions involved in protein-protein and protein-nucleic acid interactions. We have created comprehensive multiple sequence alignments using all available sequence data for the multisubunit RNA polymerase large subunits, including the bacterial beta and beta' subunits and their homologs from archaebacterial RNA polymerases, the eukaryotic RNA polymerases I, II, and III, the nuclear-cytoplasmic large double-stranded DNA virus RNA polymerases, and plant plastid RNA polymerases. To overcome technical difficulties inherent to the large-subunit sequences, including large sequence length, small and large lineage-specific insertions, split subunits, and fused proteins, we created an automated and customizable sequence retrieval and processing system. In addition, we used our alignments to create a more expansive set of shared sequence regions and bacterial lineage-specific domain insertions. We also analyzed the intergenic gap between the bacterial beta and beta' genes.

The Escherichia coli Rsd protein binds tightly and specifically to the RNA polymerase (RNAP) sigma(70) factor. Rsd plays a role in alternative sigma factor-dependent transcription by biasing the competition between sigma(70) and alternative sigma factors for the available core RNAP. Here, we determined the 2.6 A-resolution X-ray crystal structure of Rsd bound to sigma(70) domain 4 (sigma(70)(4)), the primary determinant for Rsd binding within sigma(70). The structure reveals that Rsd binding interferes with the two primary functions of sigma(70)(4), core RNAP binding and promoter -35 element binding. Interestingly, the most highly conserved Rsd residues form a network of interactions through the middle of the Rsd structure that connect the sigma(70)(4)-binding surface with three cavities exposed on distant surfaces of Rsd, suggesting functional coupling between sigma(70)(4) binding and other binding surfaces of Rsd, either for other proteins or for as yet unknown small molecule effectors. These results provide a structural basis for understanding the role of Rsd, as well as its ortholog, AlgQ, a positive regulator of Pseudomonas aeruginosa virulence, in transcription regulation.

The control of bacterial transcription initiation depends on a primary sigma factor for housekeeping functions, as well as alternative sigma factors that control regulons in response to environmental stresses. The largest and most diverse subgroup of alternative sigma factors, the group IV extracytoplasmic function sigma factors, directs the transcription of genes that regulate a wide variety of responses, including envelope stress and pathogenesis. We determined the 2.3-A resolution crystal structure of the -35 element recognition domain of a group IV sigma factor, Escherichia coli sigma(E)4, bound to its consensus -35 element, GGAACTT. Despite similar function and secondary structure, the primary and group IV sigma factors recognize their -35 elements using distinct mechanisms. Conserved sequence elements of the sigma(E) -35 element induce a DNA geometry characteristic of AA/TT-tract DNA, including a rigid, straight double-helical axis and a narrow minor groove. For this reason, the highly conserved AA in the middle of the GGAACTT motif is essential for -35 element recognition by sigma(E)4, despite the absence of direct protein-DNA interactions with these DNA bases. These principles of sigma(E)4/-35 element recognition can be applied to a wide range of other group IV sigma factors., Competing Interests: Competing interests. The authors have declared that no competing interests exist.

Background: CD133 (AC133) is a surface antigen that defines a broad population of stem cells, including myogenic and endothelial progenitors. CD133+ cells are rare in adult tissues, and the factors that support their differentiation into mature angiomyogenic cells are not known. These hurdles have hampered the use of CD133+ cells for therapeutic purposes. Because human fetal liver is a rich source of CD133+ cells, we sought to identify the growth factors that promote codifferentiation of these cells into angiogenic and myogenic cells., Methods and Results: Human fetal liver CD133+ and CD133- cell subpopulations were cultured with 5'-azacytidine or vascular endothelial growth factor (VEGF165) and/or brain-derived nerve growth factor (BDNF). CD133+ but not CD133- cells from human fetal liver codifferentiated into spindle-shaped cells, as well as flat adherent multinucleated cells capable of spontaneous contractions in culture. The resulting spindle-shaped cells were confirmed to be endothelial cells by immunohistochemistry analysis for von Willebrand factor and by acetylated LDL uptake. Multinucleated cells were characterized as striated muscles by electron microscopy and immunohistochemistry analysis for myosin heavy chain. Presence of VEGF165 and BDNF significantly enhanced angiomyogenesis in vitro. Inoculation of cells derived from CD133+ cells, but not CD133- cells, into the ear pinna of NOD/SCID mice resulted in the formation of cardiomyocytes, as identified by immunostaining with cardiac troponin-T antibody. These cells generated electrical action potentials, detectable by ECG tracing., Conclusions: CD133 defines a population of human fetal liver cells capable of differentiating into both angiogenic and myogenic cells. Preconditioning of these CD133+ cells with VEGF165 and BDNF enhances the angiomyogenesis. CD133+ fetal liver cells ultimately may be used for therapeutic angiomyogenesis.

Objective: The mechanism for anagrelide's potent platelet lowering activity in human subjects is not well defined. Studies related to anagrelide function have been hampered by its lack of activity in nonhuman primates and water insolubility. In an effort to define the mechanism whereby anagrelide exerts its therapeutic effect, we identified a water-soluble metabolite (anagrelide.met). The availability of anagrelide.met allowed, for the first time, parallel in vitro and in vivo animal studies centered on the mechanisms by which anagrelide lowers platelet levels., Materials and Methods: The effects of anagrelide.met on proliferation and maturation of mega-karyocytes (MKs) as well as platelet production were studied both in vitro and in vivo., Results: Anagrelide.met is capable of blocking in vitro MK migration by 20% to 40%. At 100 ng/mL, anagrelide.met selectively blocked in vitro MK maturation, resulting in a 50% decrease in the total number of CD41a(+) MKs, corresponding with a 30% decrease in MK ploidy by day 10 and a 60% decrease by day 20. Daily intraperitoneal injections of anagrelide.met 100 microg into BALB/c mice was sufficient to significantly decrease platelet counts within 24 to 48 hours, stabilizing to 40 to 50% of normal levels by day 5. This was associated with a 45% decrease in the number of developing MKs and an increase in thrombopoietin levels. Anagrelide.met did not alter WBC counts, hematocrit, or bleeding time, or lead to any apparent signs of toxicity. Furthermore, unlike the parent anagrelide compound, anagrelide.met did not inhibit ADP-induced platelet aggregation even at high concentrations (10 microg/mL)., Conclusions: We describe a cross-species reactive anagrelide metabolite that selectively inhibits MK maturation and migration, lowering platelet levels without influencing platelet aggregation.

Despite the discovery of thrombopoietin (TPO) and its contribution to megakaryocytopoiesis, the exact mechanisms and sites of platelet production are unknown. It has been shown that mature megakaryocytes (MKs) functionally express the stromal-derived factor 1 (SDF-1) receptor, CXCR4. SDF-1-induced migration of mature MKs through endothelial cell layers results in increased platelet production. Because the migration of polyploid MKs from the bone marrow microenvironment requires remodeling of the perivascular extracellular matrix, it was hypothesized that mature polyploid MKs may express matrix metalloproteinases (MMPs), facilitating their exit into the bone marrow extravascular space. In this report, it is demonstrated that SDF-1 induces the expression and release of gelatinase B (MMP-9) by purified mature polyploid human MKs and an adeno-CXCR4-infected megakaryocytic cell line. Neutralizing antibody to MMP-9, but not MMP-2, blocked SDF-1-induced migration of MKs through reconstituted basement membrane, suggesting that expression of MMP-9 is critical for MK migration. Incubation of mature MKs with a synthetic MMP inhibitor, 5-phenyl-1,10-phenanthrolene, resulted in the inhibition of platelet formation, suggesting that the expression of MMPs is not only critical for megakaryocyte migration but also for subsequent platelet release. Confirming these results, adeno-SDF-1 injection into normal mice resulted in increased platelet counts, a process that could be blocked by a synthetic MMP inhibitor. These results suggest mobilization of MKs involves sequential expression and activation of chemokine receptors such as CXCR4, MMP-9, followed by transendothelial migration. MMP inhibitors may have potential use in the treatment of thrombotic and myeloproliferative disorders. (Blood. 2000;96:4152-4159)