Semi-Quantitative MALDI Measurements of Blood-Based Samples for Molecular Diagnostics.

Accurate and precise measurement of the relative protein content of blood-based samples using mass spectrometry is challenging due to the large number of circulating proteins and the dynamic range of their abundances. Traditional spectral processing methods often struggle with accurately detecting overlapping peaks that are observed in these samples. In this work, we develop a novel spectral processing algorithm that effectively detects over 1650 peaks with over 3.5 orders of magnitude in intensity in the 3 to 30 kD m/z range. The algorithm utilizes a convolution of the peak shape to enhance peak detection, and accurate peak fitting to provide highly reproducible relative abundance estimates for both isolated peaks and overlapping peaks. We demonstrate a substantial increase in the reproducibility of the measurements of relative protein abundance when comparing this processing method to a traditional processing method for sample sets run on multiple matrix-assisted laser desorption/ionization-time of flight (MALDI-TOF) instruments. By utilizing protein set enrichment analysis, we find a sizable increase in the number of features associated with biological processes compared to previously reported results. The new processing method could be very beneficial when developing high-performance molecular diagnostic tests in disease indications.

Predicting prognosis in COVID-19 patients using machine learning and readily available clinical data.

RATIONALE: Prognostic tools for aiding in the treatment of hospitalized COVID-19 patients could help improve outcome by identifying patients at higher or lower risk of severe disease. The study objective was to develop models to stratify patients by risk of severe outcomes during COVID-19 hospitalization using readily available information at hospital admission. METHODS: Hierarchical ensemble classification models were trained on a set of 229 patients hospitalized with COVID-19 to predict severe outcomes, including ICU admission, development of acute respiratory distress syndrome, or intubation, using easily attainable attributes including basic patient characteristics, vital signs at admission, and basic lab results collected at time of presentation. Each test stratifies patients into groups of increasing risk. An additional cohort of 330 patients was used for blinded, independent validation. Shapley value analysis evaluated which attributes contributed most to the models' predictions of risk. MAIN RESULTS: Test performance was assessed using precision (positive predictive value) and recall (sensitivity) of the final risk groups. All test cut-offs were fixed prior to blinded validation. In development and validation, the tests achieved precision in the lowest risk groups near or above 0.9. The proportion of patients with severe outcomes significantly increased across increasing risk groups. While the importance of attributes varied by test and patient, C-reactive protein, lactate dehydrogenase, and D-dimer were often found to be important in the assignment of risk. CONCLUSIONS: Risk of severe outcomes for patients hospitalized with COVID-19 infection can be assessed using machine learning-based models based on attributes routinely collected at hospital admission.

A Genetic Variant in the BCL2 Gene Associates with Adalimumab Response in Hidradenitis Suppurativa Clinical Trials and Regulates Expression of BCL2.

Hidradenitis suppurativa is a chronic skin disease with a significant genetic component and prevalence from 0.5% to 4%. Adalimumab is the only treatment approved by either the European Medicines Agency or the U.S. Food and Drug Administration for the management of moderate to severe hidradenitis suppurativa. To identify genetic variants associated with adalimumab response, we performed a genome-wide association study (GWAS) from the most extensive two phase 3 hidradenitis suppurativa clinical trials (PIONEER I and II) to date. Through direct genotyping and imputation, we tested almost 7 million genetic variants with minor allele frequency > 5% and identified one single linkage disequilibrium block, located in the intron of the BCL2 gene, which reached genome-wide significance (lead single-nucleotide polymorphism, rs59532114; P = 2.35E-08). Bioinformatic analysis and functional genomics experiments suggested a correlation of the most strongly associated single-nucleotide polymorphism minor allele with increased BCL2 gene and protein expressions in hair follicle tissues. In reciprocal knockdown experiments, we found that BCL2 is down-regulated by TNF inhibition. These results highlight a pathway that involves BCL2 in response to adalimumab. Further work is required to determine how this pathway influences adalimumab effectiveness in patients with hidradenitis suppurativa.

Inhibition of myogenic microRNAs 1, 133, and 206 by inflammatory cytokines links inflammation and muscle degeneration in adult inflammatory myopathies.

OBJECTIVE: The molecular basis of inflammatory myopathies such as dermatomyositis (DM), polymyositis, and inclusion body myositis, which share the characteristics of chronic muscle inflammation and skeletal muscle wasting, are poorly understood. As such, effective targeted treatments for these diseases are lacking, resulting in critical unmet medical needs for these devastating diseases. The purpose of this study was to identify possible new targets for drug development by exploring the mechanism by which inflammation may play a role in the pathology of the inflammatory myopathies. METHODS: We compared expression levels of inflammatory cytokines and microRNAs (miRNAs) between muscle biopsy samples from patients with inflammatory myopathies and those from donors without myositis. In vitro human and mouse model systems were then used to characterize the role of these cytokines and microRNAs on myoblast-to-myocyte differentiation. RESULTS: We observed increased expression of inflammatory cytokines, including tumor necrosis factor α (TNFα), interferon-α (IFNα), IFNß, and interleukin-1ß, in different subtypes of inflammatory myopathies. We observed decreased expression of microRNA-1 (miR-1), miR-133a, and miR-133b in all of the inflammatory myopathy subtypes we evaluated, as well as decreased expression of miR-206 in DM; these miRNAs are essential for adult skeletal muscle differentiation and maintenance. TNFα was significantly inversely correlated with decreased myogenic miRNA expression in the inflammatory myopathy subtypes. In mechanistic studies, TNFα inhibited the expression of myogenic miRNAs and suppressed the differentiation of C2C12 myoblasts to myocytes/myotubes in an NF-κB-dependent manner. This block in differentiation by TNFα was relieved by overexpression of miR-1, miR-206, or miR-133a/b. CONCLUSION: Taken together, these results provide a new mechanistic link between the action of proinflammatory cytokines and the degenerative pathology of inflammatory myopathies, and suggest therapeutic approaches for these diseases.

MicroRNA-206 induces G1 arrest in melanoma by inhibition of CDK4 and Cyclin D.

Expression profiling of microRNAs in melanoma lesional skin biopsies compared with normal donor skin biopsies, as well as melanoma cell lines compared with normal melanocytes, revealed that hsa-miR-206 was down-regulated in melanoma (-75.4-fold, P = 1.7 × 10(-4)). MiR-206 has been implicated in a large number of cancers, including breast, lung, colorectal, ovarian, and prostate cancers; however, its role in tumor development remains largely unknown, its biologic function is poorly characterized, and its targets affecting cancer cells are largely unknown. MiR-206 reduced growth and migration/invasion of multiple melanoma cell lines. Bioinformatics identified cell cycle genes CDK2, CDK4, Cyclin C, and Cyclin D1 as strong candidate targets. Western blots and 3'UTR reporter gene assays revealed that miR-206 inhibited translation of CDK4, Cyclin D1, and Cyclin C. Additionally, hsa-miR-206 transfection induced G1 arrest in multiple melanoma cell lines. These observations support hsa-miR-206 as a tumor suppressor in melanoma and identify Cyclin C, Cyclin D1, and CDK4 as miR-206 targets.

Tumor recognition and self-recognition induce distinct transcriptional profiles in antigen-specific CD4 T cells.

Tumors express a wide variety of both mutated and nonmutated Ags. Whether these tumor Ags are broadly recognized as self or foreign by the immune system is currently unclear. Using an autochthonous prostate cancer model in which hemagglutinin (HA) is specifically expressed in the tumor (ProHA x TRAMP mice), as well as an analogous model wherein HA is expressed in normal tissues as a model self-Ag (C3HA(high)), we examined the transcriptional profile of CD4 T cells undergoing Ag-specific division. Consistent with our previous data, transfer of Ag-specific CD4 T cells into C3HA(high) resulted in a functionally inactivated CD4 T cell profile. Conversely, adoptive transfer of an identical CD4 T cell population into ProHA x TRAMP mice resulted in the induction of a regulatory phenotype of the T cell (Treg) both at the transcriptional and functional level. Interestingly, this Treg skewing was a property of even early-stage tumors, suggesting Treg induction as an important tolerance mechanism during tumor development.

Kruppel-like factor 4 is essential for inflammatory monocyte differentiation in vivo.

Several members of the Kruppel-like factor (KLF) family of transcription factors play important roles in differentiation, survival, and trafficking of blood and immune cell types. We demonstrate in this study that hematopoietic cells from KLF4(-/-) fetal livers (FL) contained normal numbers of functional hematopoietic progenitor cells, were radioprotective, and performed as well as KLF4(+/+) cells in competitive repopulation assays. However, hematopoietic "KLF4(-/-) chimeras" generated by transplantation of KLF4(-/-) fetal livers cells into lethally irradiated wild-type mice completely lacked circulating inflammatory (CD115(+)Gr1(+)) monocytes, and had reduced numbers of resident (CD115(+)Gr1(-)) monocytes. Although the numbers and function of peritoneal macrophages were normal in KLF4(-/-) chimeras, bone marrow monocytic cells from KLF4(-/-) chimeras expressed lower levels of key trafficking molecules and were more apoptotic. Thus, our in vivo loss-of-function studies demonstrate that KLF4, previously shown to mediate proinflammatory signaling in human macrophages in vitro, is essential for differentiation of mouse inflammatory monocytes, and is involved in the differentiation of resident monocytes. In addition, inducible expression of KLF4 in the HL60 human acute myeloid leukemia cell line stimulated monocytic differentiation and enhanced 12-O-tetradecanoylphorbol 13-acetate induced macrophage differentiation, but blocked all-trans-retinoic acid induced granulocytic differentiation of HL60 cells. The inflammation-selective effects of loss-of-KLF4 and the gain-of-KLF4-induced monocytic differentiation in HL60 cells identify KLF4 as a key regulator of monocytic differentiation and a potential target for translational immune modulation.

CD34+ hematopoietic stem-progenitor cell microRNA expression and function: a circuit diagram of differentiation control.

MicroRNAs (miRNAs) are a recently identified class of epigenetic elements consisting of small noncoding RNAs that bind to the 3' untranslated region of mRNAs and down-regulate their translation to protein. miRNAs play critical roles in many different cellular processes including metabolism, apoptosis, differentiation, and development. We found 33 miRNAs expressed in CD34+ hematopoietic stem-progenitor cells (HSPCs) from normal human bone marrow and mobilized human peripheral blood stem cell harvests. We then combined these data with human HSPC mRNA expression data and with miRNA-mRNA target predictions, into a previously undescribed miRNA:mRNA interaction database called the Transcriptome Interaction Database. The in silico predictions from the Transcriptome Interaction Database pointed to miRNA control of hematopoietic differentiation through translational control of mRNAs critical to hematopoiesis. From these predictions, we formulated a model for miRNA control of stages of hematopoiesis in which many of the genes specifying hematopoietic differentiation are expressed by HSPCs, but are held in check by miRNAs until differentiation occurs. We validated miRNA control of several of these target mRNAs by demonstrating that their translation in fact is decreased by miRNAs. Finally, we chose miRNA-155 for functional characterization in hematopoiesis, because we predicted that it would control both myelopoiesis and erythropoiesis. As predicted, miRNA-155 transduction greatly reduced both myeloid and erythroid colony formation of normal human HSPCs.

Ex vivo soluble fas ligand treatment of donor cells to selectively reduce murine acute graft versus host disease.

BACKGROUND: Allogeneic bone marrow transplantation (BMT) and donor lymphocyte infusion (DLI) provide valuable treatments for a range of diseases. However, the therapeutic utility of BMT and DLI is reduced by the high incidence of graft-versus-host disease (GvHD) mediated by activated donor T lymphocytes directed against recipient alloantigens. METHODS: Using mouse models, we developed and evaluated a strategy to selectively enhance activation-induced cell death (AICD) of anti-recipient T cells within transplant donor cell populations, with the goal of reducing GvHD. Responder T lymphocytes were incubated ex vivo with irradiated allogenic stimulator cells in a mixed lymphocyte reaction (MLR) in the presence of soluble Fas ligand (sFasL) to induce AICD in alloreactive cells. RESULTS: This ex vivo sFasL treatment reduced proliferation to the allogeneic stimulator cells in vitro and abrogated acute GvHD capacity in vivo. In contrast, the secondary immune responsiveness of the ex vivo sFasL-treated responder T cells to an unrelated model antigen was preserved. Furthermore, upon adoptive transfer in a DLI model, ex vivo sFasL-treated T cells were able to reject a model tumor. Finally, ex vivo sFasL treatment of bone marrow cells did not reduce their hematopoietic engraftment capacity. CONCLUSIONS: Thus, ex vivo treatment with sFasL appears to have potential for translation to clinical cell processing of BMT allografts and DLI infusions.

TAGmapper: a web-based tool for mapping SAGE tags.

Serial Analysis of Gene Expression (SAGE) is an important means of obtaining quantitative information about expression of genes in different samples. Short SAGE tags are 10 nucleotides long and often contain enough information to uniquely identify the gene(s) corresponding to the tag. We have observed, however, that the currently available resources are not adequate for accurate mapping of all SAGE tags to genes. Here, we describe development of a web-based tool called TAGmapper (http://tagmapper.ibioinformatics.org), which provides a comprehensive and accurate mapping of SAGE tags to genes. We were able to map SAGE tags accurately in several instances where two other popular resources, SAGEmap (http://www.ncbi.nlm.nih.gov/projects/SAGE/) and SAGE Genie (http://cgap.nci.nih.gov/SAGE), provided incorrect or no assignment of tags to genes. Finally, we experimentally determined the expression of a subset of genes assigned by TAGmapper using DNA microarrays and/or quantitative PCR to confirm the reliability of the gene mappings. We anticipate that TAGmapper will be a useful tool in functional genomic approaches by providing accurate identification of genes in SAGE experiments.

Microarray and serial analysis of gene expression analyses identify known and novel transcripts overexpressed in hematopoietic stem cells.

The human CD34(+)/CD38(-)/Lin(-) cell subset, comprising approximately 1-10% of the CD34(+) cell population, contains few of the less primitive hematopoietic (lineage-committed) progenitor cells (HPCs) but most of the primitive in vivo engrafting (lympho-)hematopoietic stem cells (HSCs). We analyzed gene expression in CD34(+)/CD38(-)/Lin(-) cell populations isolated from normal human adult donor bone marrow, neonatal placental/umbilical cord blood, and mobilized adult donor peripheral blood stem-progenitor cells. As measured by Affymetrix microarrays, 4746 genes were expressed in CD34(+)/CD38(-)/Lin(-) cells from all three tissues. We also determined the transcriptomes of the stem cell-depleted, HPC-enriched CD34(+)/[CD38/Lin](++) cell population from each tissue. Comparison of CD34(+)/CD38(-)/Lin(-) (HSC-enriched) versus CD34(+)/[CD38/Lin](++) (HPC-enriched, HSC-depleted) cells from each tissue yielded 81 genes overrepresented and 90 genes underrepresented, common to all three of the CD34(+)/CD38(-)/Lin(-) cell populations. These transcripts, which are selectively expressed in HSCs from all three tissues, include a number of known genes (e.g., transcription factors, receptors, and signaling molecules) that might play roles in key functions (e.g., survival, self-renewal, differentiation, and/or migration/adhesion) of human HSCs. Many genes/transcripts of unknown function were also detected by microarray analysis. Serial analysis of gene expression of the bone marrow HSC and HPC populations confirmed expression of most of the overrepresented transcripts for which reliable serial analysis of gene expression tags were detected and additionally suggested that current microarrays do not detect as many as 30% of the transcripts expressed in HSCs, including a number of previously unknown transcripts. This work is a step toward full definition of the transcriptome of normal human HSCs and may identify new genes involved in leukemogenesis and cancer stem cells.

Kinetics of CCR7 expression differ between primary activation and effector memory states of T(H)1 and T(H)2 cells.

We explored the kinetics of CCR7 expression on T(H)1 and T(H)2 polarized cells as well as on antigen-specific T cell lines at various stages of differentiation. A striking pattern of early (days 7-14) inducible CCR7 expression was seen preferentially on primary T(H)1 cell lines, as compared to T(H)2 cells, and was dependent on the strength and duration of the T cell receptor signal. Upon repeated restimulation (days 21-28) and differentiation, a switch occurred in which T(H)2 cells had high CCR7 expression, whereas T(H)1 cells lost CCR7 expression. Chronic (8 weeks and later) effector memory cell lines were 95% CCR7 negative. These data demonstrate an ordered pattern of CCR7 expression that suggest more rapid priming of T(H)1 cells in the lymph node, and delayed priming with prolonged CCR7 expression during T(H)2 responses, and may have implications for tracking T(H)1 effector T cells ex vivo in autoimmune diseases.

Human CD34+ hematopoietic stem/progenitor cells express high levels of FLIP and are resistant to Fas-mediated apoptosis.

We sought to determine whether lympho-hematopoietic stem-progenitor cells (HSC) from human placental/umbilical cord blood (CB) or adult mobilized blood (PBSC) are sensitive to Fas-induced apoptosis. Human CD34+ cells from CB or PBSC were cultured in serum-free medium, with or without hematopoietic growth factors (FKT: FLT-3 ligand [FL], KIT ligand [KL], and thrombopoietin [TPO]), and with or without soluble Fas ligand (sFasL) or agonistic anti-Fas antibody. After 5-48 hours of culture, cells were assessed for viability and stained with Annexin V and 7-Aminoactinomycin D for apoptosis analysis by fluorescence-activated cell sorting. Cultured cells were also assessed by in vitro hematopoietic colony-forming cell (CFC) and in vivo nonobese diabetic/severe combined immunodeficient mouse engraftment potential (SEP) assays. Levels of Fas, FLICE inhibitory protein (FLIP), and Caspase 8 mRNA in CD34+ cells were determined by real-time quantitative polymerase chain reaction. Expression of FLIP was confirmed by Western blotting. No decrease in viability, CFC, or SEP was observed in CB or PBSC CD34+ cells cultured in the presence of sFasL or agonistic anti-Fas antibody. Human CB and mobilized PBSC CD34+ cells expressed high levels of FLIP, low ratios of Caspase 8:FLIP, and low levels of Fas. Thus, human CB and PBSC CD34+ HSC were resistant to Fas pathway agonists. High-level expression of FLIP likely provides one level of protection of CD34+ cells from Fas-mediated apoptosis.