Myeloid Nuclear Differentiation Antigen Research Articles

MNDA expression and its value in differential diagnosis of B-cell non-Hodgkin lymphomas: a comprehensive analysis of a large series of 1293 cases.

MNDA (myeloid nuclear differentiation antigen) has been considered as a potential diagnostic marker for marginal zone lymphoma (MZL), but its utility in distinguishing MZL from other B-cell non-Hodgkin lymphomas (B-NHLs) and its clinicopathologic relevance in diffuse large B-cell lymphoma (DLBCL) are ambiguous. We comprehensively investigated MNDA expression in a large series of B-NHLs and evaluated its diagnostic value. MNDA expression in a cohort of 1293 cases of B-NHLs and 338 cases of reactive lymphoid hyperplasia (RLH) was determined using immunohistochemistry and compared among different types of B-NHL. The clinicopathologic relevance of MNDA in DLBCL was investigated. MNDA was highly expressed in MZLs (437/663, 65.9%), compared with the confined staining in marginal zone B-cells in RLH; whereas neoplastic cells with plasmacytic differentiation lost MNDA expression. MNDA expression was significantly higher in mantle cell lymphoma (MCL, 79.6%, p = 0.006), whereas lower in chronic lymphocytic leukemia/small lymphocytic lymphoma (CLL/SLL, 44.8%, p = 0.001) and lymphoplasmacytic lymphoma (LPL, 25%, p = 0.016), and dramatically lower in follicular lymphoma (FL, 5.2%, p < 0.001), compared with MZL. 29.6% (63/213) of DLBCLs were positive for MNDA. The cases in non-GCB group exhibited a higher rate of MNDA positivity (39.8%) compared to those in GCB group (16.3%) (p < 0.001), and MNDA staining was more frequently observed in DLBCLs with BCL2/MYC double-expression (50%) than those without BCL2/MYC double-expression (24.8%) (p = 0.001). Furthermore, there was a significant correlation between MNDA and CD5 expression in DLBCL (p = 0.036). MNDA was highly expressed in MZL with a potential utility in differential diagnosis between MZL and RLH as well as FL, whereas its value in distinguishing MZL from MCL, CLL/SLL is limited. In addition, MNDA expression in DLBCL was more frequently seen in the non-GCB group and the BCL2/MYC double-expression group, and demonstrated a correlation with CD5, which deserves further investigation. The clinical relevance of MNDA and its correlation with the prognosis of these lymphomas also warrant to be fully elucidated.

MNDA, a PYHIN factor involved in transcriptional regulation and apoptosis control in leukocytes.

Inflammation control is critical during the innate immune response. Such response is triggered by the detection of molecules originating from pathogens or damaged host cells by pattern-recognition receptors (PRRs). PRRs subsequently initiate intra-cellular signalling through different pathways, resulting in i) the production of inflammatory cytokines, including type I interferon (IFN), and ii) the initiation of a cascade of events that promote both immediate host responses as well as adaptive immune responses. All human PYRIN and HIN-200 domains (PYHIN) protein family members were initially proposed to be PRRs, although this view has been challenged by reports that revealed their impact on other cellular mechanisms. Of relevance here, the human PYHIN factor myeloid nuclear differentiation antigen (MNDA) has recently been shown to directly control the transcription of genes encoding factors that regulate programmed cell death and inflammation. While MNDA is mainly found in the nucleus of leukocytes of both myeloid (neutrophils and monocytes) and lymphoid (B-cell) origin, its subcellular localization has been shown to be modulated in response to genotoxic agents that induce apoptosis and by bacterial constituents, mediators of inflammation. Prior studies have noted the importance of MNDA as a marker for certain forms of lymphoma, and as a clinical prognostic factor for hematopoietic diseases characterized by defective regulation of apoptosis. Abnormal expression of MNDA has also been associated with altered levels of cytokines and other inflammatory mediators. Refining our comprehension of the regulatory mechanisms governing the expression of MNDA and other PYHIN proteins, as well as enhancing our definition of their molecular functions, could significantly influence the management and treatment strategies of numerous human diseases. Here, we review the current state of knowledge regarding PYHIN proteins and their role in innate and adaptive immune responses. Emphasis will be placed on the regulation, function, and relevance of MNDA expression in the control of gene transcription and RNA stability during cell death and inflammation.

Myeloid nuclear differentiation antigen: an aid in differentiating lymphoplasmacytic lymphoma and splenic marginal zone lymphoma in bone marrow biopsies at presentation

The differential diagnosis between lymphoplasmacytic lymphoma (LPL) and marginal zone B-cell lymphoma, particularly splenic type (SMZL), can be challenging on onset of bone marrow biopsy (BMB) since morphology and phenotype are not specific and clinical features can overlap or be mildly developed at diagnosis. The LPL-specific L265P mutation in the MYD88 gene is not available in all laboratories, and genetic aberrancies identified in SMZL (del7q, mutations of NOTCH2 and KLF2) are seldom searched in routine practice. The study aim is to investigate the potential role of myeloid nuclear differentiation antigen (MNDA) expression in this specific differential diagnosis. We report MNDA reactivity in 559 patients with small B-cell lymphoma including bone marrow biopsies from 90 LPL and 91 SMZL cases. MYD88 p.Leu265Pro mutation status was assessed and confirmed as positive in 24 of 90 LPL cases, which served as the test set. MNDA staining was negative in 23 of 24 LPL cases in the test set (96%). In the 157 remaining cases (66 LPL, 91 SMZL), which served as the validation set, the MYD88 p.Leu265Pro mutation was unavailable and MNDA was more frequently expressed in SMZL (p<0.00001). In addition, immunohistochemical features more consistent with SMZL (i.e., presence of CD23+ follicular dendritic cell meshworks, polytypic plasma cells, DBA44 reactivity) were more often present in MNDA-positive cases (statistically significant for 2 such parameters). On the widest case series so far published focusing on LPL and SMZL immunohistochemical diagnosis at onset of BMB, we demonstrated that MNDA expression significantly support the diagnosis of SMZL. This observation may be of particular help in cases where the MYD88 p.Leu265Pro mutational status and/or SMZL-related genetic aberrations are unavailable.

Detecting Periprosthetic Joint Infection by Using Mass Spectrometry.

Novel methods for diagnosing periprosthetic joint infection (PJI) are currently being explored. Mass spectrometry (MS) is an approach that can detect whole-protein changes in synovial fluid and may represent a promising method. Between March 2017 and July 2018, we successively collected synovial fluid samples from patients who were undergoing diagnostic hip or knee aspiration because PJI was suspected. A PJI diagnosis was based on the modified Musculoskeletal Infection Society (MSIS) criteria. Cluster analysis and receiver operating characteristic (ROC) curves were used to evaluate the results, which were quantitatively confirmed with parallel reaction monitoring in another patient group who underwent aspiration between August 2018 and January 2019. A total of 117 synovial samples, including 51 PJI and 66 non-PJI samples, were analyzed with liquid chromatography-tandem MS (LC-MS/MS). The cluster analysis sensitivity and specificity based on differentially expressed proteins were 0.961 (95% confidence interval [CI], 0.854 to 0.993) and 0.924 (95% CI, 0.825 to 0.972), respectively. Myeloid nuclear differentiation antigen (MNDA) and polymorphonuclear leukocyte serine protease 3 (PRTN3) were the 2 most important markers for detecting PJI. The areas under the curves (AUCs) of MNDA and PRTN3 were 0.969 (95% CI, 0.936 to 1.000) and 0.900 (95% CI, 0.844 to 0.956), respectively. When MNDA and PRTN3 were combined as variables of a predictive model to diagnose PJI, the AUC reached 0.975 (95% CI, 0.943 to 1.000). Our parallel reaction monitoring-based quantitative analysis of another 40 synovial samples confirmed this result. MS could be a powerful tool for diagnosing PJI using proteome information or 2 specific markers, MNDA and PRTN3. The parallel reaction monitoring strategy simplified the PJI detection process and provided quantitative results with similar conclusions. The clinical application of MS adds a new powerful tool for the diagnosis of PJI, and the parallel reaction monitoring strategy lays a foundation for the clinical application of MS. Diagnostic Level II. See Instructions for Authors for a complete description of levels of evidence.

MNDA controls the expression of MCL-1 and BCL-2 in chronic lymphocytic leukemia cells

The myeloid nuclear differentiation antigen (MNDA) is a stress-induced protein that promotes degradation of the anti-apoptotic factor MCL-1 and apoptosis in myeloid cells. MNDA is also expressed in normal lymphoid cells and in B-cell clones isolated from individuals with chronic lymphocytic leukemia (CLL), a disease characterized by abnormal apoptosis control. We found that MNDA expression levels inversely correlate with the amount of the anti-apoptotic proteins MCL-1 and BCL-2 in human CLL samples. We report that in response to chemotherapeutic agents that induce genotoxic stress, MNDA exits its typical nucleolar localization and accumulates in the nucleoplasm of CLL and lymphoid cells. Then, MNDA binds chromatin at Mcl1 and Bcl2 genes and affects the transcriptional competence of RNA polymerase II. Our data also reveal that MNDA specifically associates with Mcl1 and Bcl2 (pre-) mRNAs and favors their rapid turnover as a prompt response to genotoxic stress. We propose that this rapid dynamic tuning of RNA levels, which leads to the destabilization of Mcl1 and Bcl2 transcripts, represents a post-transcriptional mechanism of apoptosis control in CLL cells. These results provide an explanation of previous clinical data and corroborate the finding that higher MNDA expression levels in CLL are associated with a better clinical course.

LTF, PRTN3, and MNDA in Synovial Fluid as Promising Biomarkers for Periprosthetic Joint Infection: Identification by Quadrupole Orbital-Trap Mass Spectrometry.

Diagnosing periprosthetic joint infection (PJI) requires various laboratory and clinical criteria. The purpose of this study was to explore novel biomarkers that could rapidly diagnose PJI with high accuracy. In this retrospective study of prospectively collected samples, 50 synovial fluid aspirates, 20 from the hip and 30 from the knee, were collected before surgery; 25 of the patients were diagnosed as having aseptic loosening (non-PJI) and 25, as having PJI according to the Musculoskeletal Infection Society criteria. A quadrupole orbital-trap mass spectrometry (MS) instrument was used to compare expression of proteins in patients with and without PJI. Proteins that were most efficacious for diagnosis of PJI were then determined using prediction analysis of microarray software and a random forest model. The most promising proteins were selected, and altered expression of these selected proteins was verified by ELISA (enzyme-linked immunosorbent assay) in an extended sample cohort. A total of 256 proteins were significantly upregulated (≥3.0-fold) and 14 proteins were downregulated in synovial fluid of patients with PJI compared with patients without PJI. The 3 most promising proteins were lactoferrin (LTF), polymorphonuclear leukocyte serine protease 3 (PRTN3), and myeloid nuclear differentiation antigen (MNDA). When MS was used for diagnosis of PJI, the area under the curve was 0.9888 for LTF, 0.9488 for PRTN3, and 0.9632 for MNDA. ELISA results verified that LTF, MNDA, and PRTN3 were sensitive, while LTF and MNDA were specific, for diagnosis of PJI. This proteomic study identified a previously noted protein and 2 novel candidate proteins as promising synovial fluid biomarkers for PJI diagnosis, and they should be further validated in future clinical trials. Diagnostic Level III. See Instructions for Authors for a complete description of levels of evidence.

IRTA1 and MNDA Expression in Marginal Zone Lymphoma: Utility in Differential Diagnosis and Implications for Classification.

To evaluate the clinical utility of immune receptor translocation-associated protein 1 (IRTA1) and myeloid nuclear differentiation antigen (MNDA) expression in the diagnosis and classification of marginal zone lymphomas (MZLs). IRTA1 was examined using a novel RNA in situ hybridization assay and MNDA expression determined by immunohistochemistry in 127 small B-cell neoplasms, including 80 cases of MZL. IRTA1 expression was detected in 31 (42%) of 74 MZLs vs one (2%) of 43 other small B-cell neoplasms (P < .001). MNDA staining was positive in 51 (64%) of 79 MZLs vs 21 (45%) of 46 non-MZLs (P = .06). MNDA expression was particularly uncommon in follicular lymphoma (3/14, 21%; P = .003 vs MZL). There was no association between MNDA and IRTA1 expression and the presence of monocytoid cytology. IRTA1 expression was less frequent in cases with a diffuse growth pattern. IRTA1 and MNDA are useful markers in the differential diagnosis of MZLs.

Value of Quantitative assessment of Myeloid Nuclear Differentiation Antigen expression and other flow cytometric parameters in the diagnosis of Myelodysplastic syndrome.

The diagnosis of myelodysplastic syndrome (MDS) based on morphology is particularly difficult in low-grade MDS. Thus, the role of myeloid nuclear differentiation antigen (MNDA) and other flow cytometric (FCM) parameters in MDS was evaluated. Bone marrow aspirates (BMA) collected from 52 patients with unexplained persistent cytopenias were divided into three groups: (i) proven MDS (n = 12) based on morphology and/or cytogenetics; (ii) suspected MDS (n = 6), noncontributory morphology, and cytogenetics; and (iii) non-MDS (n = 34). Sixteen control BMA were studied. Cases were analyzed for MNDA expression (on granulocytes, blasts, monocytes, and lymphocytes) and for seven quantitative parameters: CD34(+) myeloblasts % in nucleated cells, CD34(+) B-cell progenitor% in CD34(+) cells, lymphocyte/myeloblast CD45 MFI ratio, granulocyte/lymphocyte SSC peak channel ratio and the proportion of CD34(+) myeloblasts expressing CD15, CD11b, and CD56. A score of 1 was given to each parameter beyond the cutoff, and score ≥3 was considered FCM positive. MNDA expression on granulocytes and blasts was significantly lower in proven MDS and suspected MDS vs. non-MDS. Quantitative FCM parameters successfully distinguished MDS and suspected MDS from non-MDS. MNDA expression is an independent marker for the evaluation of dyspoiesis and may be added to the standard panel for quantitative assessment by FCM.

Myeloid Nuclear Differentiation Antigen (MNDA) Expression: A Useful Marker in the Diagnosis of Myelodysplastic Syndromes By Flow Cytometry

Background: Diagnostic workup for suspected myelodysplastic syndromes (MDS) is increasingly done with the aid of multiparamter flow cytometry (MFC) detecting aberrant antigen expression. Myeloid nuclear differentiation antigen (MNDA) is a hematopoietic protein expressed strongly in mature myeloid cells, but only weakly in myeloid progenitor cells. Recently, reduced MNDA expression was detected by MFC in monocytes (M), granulocytes (G) and myeloid progenitor cells (MP) in patients (pts) with MDS and MNDA as additional marker for MFC was proposed to improve diagnostic capabilities of MFC.Aim: To verify differences in MNDA expression in M, G and MP of pts with MDS vs. those without MDS and to test its value as additional MFC parameter in a cohort with suspected MDS.Patients and methods: We analysed bone marrow from 131 pts (median age 74, range 17-93 years) with suspected MDS by cytomorphology (CM), standard 10-color-MFC and cytogenetics (CG) in parallel. For detection of MNDA expression we applied a readily available five color intracellular staining assay using monoclonal antibodies against MNDA, CD45, CD64, CD15 and myeloperoxidase (MDS-Quant, Trillium Diagnostics, Bangor, ME). Different gating strategies to best define M [SSC/CD45 plot (A) vs CD16/CD15 plot (B)], G (A vs B) and MP (A) were used. MNDA expression in pts diagnosed with no MDS (n=35), possible MDS (n=40; based on morphologic dysplastic changes insufficient for diagnosis of MDS) and MDS (n=56) by CM was compared. We also correlated results to findings in standard MDS-MFC (n=128) and CG (n=120).Results: Based on CM diagnosis, pts with MDS showed higher percentages of G and M with weak expression of MNDA (%dimG and %dimM) than no-MDS pts irrespective of the gating strategy [mean±SD, %dimG: 16±17 vs 6±7, p<0.001 (A) and 3±3 vs 1±2, p=0.011 (B); %dimM: 22±21 vs 15±12, p=0.054 (A) and 14±25 vs 4±4, p=0.004 (B)]. Diagnostically challenging cases with "possible MDS" by CM also displayed significantly higher %dimG and %dimM than no-MDS cases [%dimG 13±14 vs 6±7, p=0.006 (A), %dimM: 23±20 vs 15±12, p=0.027 (A) and 17±24 vs 4±4, p=0.002 (B)]. Differences between MDS and possible MDS existed only in higher %dimGra in MDS gated in plot B (2.7±3.3 vs 1.6±1.9, p=0.036). Conversely, MDS pts and pts with possible MDS had higher percentages of MP with high MNDA expression (%hiMP) than no-MDS pts (17±16 vs 9±8, p=0.005 and 14±13 vs 9±8, p=0.041, respectively). MNDA expression levels measured by mean fluorescence intensity (MFI) were diminished in M of pts with MDS vs no-MDS [26±12 vs 33±9, p=0.006 (A) and 30±14 vs 39±10, p=0.002 (B), respectively] and in pts with possible MDS vs no-MDS pts [26±12 vs 33±9, p=0.004 (A) and 30±2 vs 39±10, p=0.005, respectively]. Looking at the low level MNDA MP (dimMP), higher MFIs were found in MDS compared to no-MDS (1±0.8 vs 0.7±0.4, p=0.002) and in possible MDS vs no-MDS (1.0±0.7 vs 0.7±0.4, p=0.011). Comparing results of MNDA MFC and standard MFC we also found higher MFI in dimMP (1.0±0.8 vs 0.7±0.3, p=0.039) in pts diagnosed MDS by MFC (n=63) vs those without signs of MDS (n=14). In the inexplicit cases diagnosed MDS possible by MFC (aberrant antigen expressions not sufficient for MDS diagnosis, n=51) higher percentages of dimG [16±18 vs 9±10, p=0.015 (A) and 3±3 vs 1±2, p=0.005 (B)] and dimM [16±25 vs 7±13, p=0.02 (B)] were seen as compared to no-MDS, in line with CM results. Moreover, a lower MFI in M [26±12 vs 31±11, p=0.024 (A) and 29±14 vs 36±13, p=0.02 (B)] and a higher MFI in dimMP (1.0±0.8 vs 0.9±0.6, p=0.021) could be detected. Considering CG, cases with an aberrant karyotype (n=41) had higher values for %dimM (27±23 vs 18±16, p=0.035) as compared to those with a normal karyotype (n=79). Including the most significant markers [%dimG>12 (A), %dimM>13 (B), %hiMP>15 (A), 1 point each], we created a new MNDA score with a score of ≥2 indicating MDS. Of 14 pts with a score of ≥2, 13 pts were concordantly diagnosed MDS using CM for validation; 1 pt had no MDS by CM but was classified MDS by standard MDS-MFC.Conclusions: Reduced MNDA levels and higher percentages of M and G with low MNDA expression as well as a higher percentage of MP with high MNDA expression could be confirmed in pts with MDS. Applying our newly defined MNDA score for MFC, identification of a subset of pts with MDS was possible at high specificity. Further analyses will have to evaluate this MNDA score incorporated into standard MDS-MFC panels to improve MFC-based diagnostic approaches for MDS. DisclosuresBellos:MLL Munich Leukemia Laboratory: Employment. Haferlach:MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Davis:Trillium Diagnostics: Employment, Equity Ownership. Haferlach:MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Schnittger:MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Kern:MLL Munich Leukemia Laboratory: Employment, Equity Ownership.

Purification, characterization and docking studies of the HIN domain of human myeloid nuclear differentiation antigen (MNDA)

The HIN domain of myeloid nuclear differentiation antigen (MNDA) was expressed and purified as a monomer using E. coli JM109 as host. The protein interacted with double-stranded DNA at a Kd of 3.15 μM and did not recognize the termini of double-stranded DNA. Isothermal titration calorimetry indicated that the interaction between the protein and double-stranded DNA is mainly mediated by electrostatic attractions and hydrogen bonding. We developed a model to analyze the potential DNA binding site of the MNDA HIN domain. Based on the model, molecular docking and mutation studies suggest that the double-stranded DNA binding site of the protein is different from other HIN-DNA structures. This work facilitates the design of specific drugs against pathogens detected by human MNDA.

THU0100 Characterization of Citrullinated Proteins in Complex Samples

BackgroundRheumatoid arthritis (RA) patients frequently produce autoantibodies against citrullinated proteins, proteins in which an arginine residue is converted into a citrulline by peptidylarginine deiminase (PAD). In view of the putative...

Resolution of Inflammation: Leukocytes and Molecular Pathways as Potential Therapeutic Targets

Inflammation is the principal defense mechanism against invading pathogens and host injury. Neutralization and elimination of the offending insult ideally prompts resolution of inflammation and restoration of tissue homeostasis. Excessive or dysregulated inflammatory responses together with impaired repair processes results in non-resolving inflammation and persisting tissue damage, which is now considered as a critical component of many chronic human diseases. Ground-breaking work during the past decade has led to the recognition that resolution of inflammation is a tightly controlled active process that involves generation of a new class of lipid mediators, proteins, and autacoids. These endogenous molecules possess overlapping but not fully identical biological properties and may act simultaneously or sequentially to regulate processes that are essential for timely and complete resolution of inflammation. Among these mechanisms are sequestering and suppression of generation of pro-inflammatory cytokines, inhibition of inflammatory cell trafficking into inflamed sites, promotion of neutrophil apoptosis, and macrophage polarization into M2 (alternatively activated or pro-resolution) phenotype, and facilitation of removal of apoptotic neutrophils and other cell types via efferocytosis and tissue repair. During the past years much progress has been made in the identification and characterization of pro-resolution mediators and signaling circuits. Of these, protein mediators, such as annexin A1, and novel classes of lipid mediators, including arachidonic acid-derived lipoxins, and omega-3 polyunsaturated fatty acid-derived resolvins, protectins, and maresins exhibit dual anti-inflammatory and pro-resolution properties. Extensive research has characterized biosynthetic pathways, G-protein-coupled receptors, downstream intracellular signaling pathways, and identified several microRNAs that are involved in the regulation of pro-resolving molecular and cellular circuits. While in inflamed sites neutrophils interact with other cells in their immediate vicinity to produce these lipid mediators, eosinophil granulocytes can also generate pro-resolving mediators from omega-3 polyunsaturated fatty acids, indicating an important role for this type of leukocytes in inflammatory resolution. Resolvins facilitate macrophage polarization into the M2 phenotype. Lipoxins and resolvins interfere with β2 integrin-mediated outside in signaling that in addition to governing leukocyte trafficking also modulates neutrophil apoptosis, another control point in resolving inflammation. Data emerging from a variety of experimental models demonstrated that resolvins and protectins limit adaptive immunity in asthma, attenuate adipose tissue inflammation and metabolic dysfunctions, including type-2 diabetes mellitus-associated insulin resistance and non-alcoholic fatty acid liver disease, and protect the kidney against diabetic nephropathy. The glucocorticoid-regulated protein annexin A1 may act in concert with lipoxins to limit inflammation underlying myocardial ischemia/reperfusion, arthritis, multiple sclerosis, and sepsis. Altering local adenosine signaling affects leukocyte activation and trafficking, and may restore tissue homeostasis in liver injury and rheumatoid arthritis. The nuclear proteins proliferating cell nuclear antigen (PCNA) and myeloid nuclear differentiation antigen (MNDA) play opposing roles in modulating neutrophil apoptosis in patient with sepsis. Targeting the complement C5a receptors C5Ra and C5L2 may attenuate excessive inflammation in sepsis and autoimmune diseases. Intracellular molecular mechanisms have also been implicated in dampening inflammation. The NOD-like receptors NOD1 and NOD2 recognize intracellular pathogens and activate transcriptional responses that would lead to restriction of infection. Suppression of IFNγ production in T lymphocytes by allopurinol may attenuate cell-mediated inflammatory diseases. This Research Topic focuses on articles that discuss the molecular mechanisms governing the function and fate of leukocytes during inflammation and identify potential targets for development of innovative therapeutic approaches for the treatment of inflammatory pathologies based on mimicking and/or activating endogenous pro-resolution mechanisms.

The rheumatoid arthritis synovial fluid citrullinome reveals novel citrullinated epitopes in apolipoprotein E, myeloid nuclear differentiation antigen, and β‐actin

To generate a catalog of citrullinated proteins that are present in the synovia of patients with rheumatoid arthritis (RA) and to elucidate their relevance for the anti-citrullinated protein antibody response in RA. Polypeptides isolated from the synovial fluid of patients with RA were identified by mass spectrometry. Three proteins (apolipoprotein E [Apo E], myeloid nuclear differentiation antigen [MNDA], and β-actin) were studied in more detail, using immunoprecipitation and Western blotting. The presence of autoantibodies to synthetic peptides derived from these proteins in sera from patients with RA, sera from patients with other diseases, and sera from healthy control subjects was studied by enzyme-linked immunosorbent assay (ELISA). RA synovial fluid samples displayed several distinct patterns of citrullinated proteins. Using mass spectrometry, (fragments of) 192 proteins were identified, including 53 citrullinated proteins, some of which contained multiple citrullinated residues. In addition to previously reported citrullinated proteins in RA synovia (e.g., vimentin and fibrinogen), a series of novel citrullinated proteins, including Apo E, MNDA, β-actin, and cyclophilin A, was identified. Immunoprecipitation experiments confirmed the citrullination of Apo E and MNDA. ELISAs demonstrated the presence of autoreactive citrullinated epitopes in Apo E, MNDA, and β-actin. Synovial fluid samples from the inflamed joints of patients with RA contain many citrullinated proteins. Citrullinated Apo E, MNDA, and β-actin are novel antigens identified in RA synovial fluid, and only a limited number of their citrullinated epitopes are targeted by the immune system in RA.

Evaluation of flow cytometric assessment of myeloid nuclear differentiation antigen expression as a diagnostic marker for myelodysplastic syndromes in a series of 269 patients

Myeloid nuclear differentiation antigen (MNDA) is expressed in myelomonocytic cells with highest levels in mature granulocytes and monocytes. It is suggested to be expressed more weakly in patients with myelodysplastic syndromes (MDS). The analysis of MNDA therefore may improve diagnostic capabilities of multiparameter flow cytometry (MFC) in MDS. We used MFC for detection of MNDA expression in 269 patients with suspected or known MDS, acute myeloid leukemia (AML) or chronic myelomonocytic leukemia (CMML), cytopenia of unknown cause or without malignancy (negative controls). Results were compared with the diagnoses revealed by cytomorphology (CM) and cytogenetics (CG). Percentages of granulocytes and monocytes with diminished MNDA expression (dimG and dimM) were higher in patients with MDS (mean ± SD, 20% ± 20%, P < 0.001 and 31% ± 24%, P < 0.001) and AML (27% ± 27%, P = 0.007 and 45% ± 31%, P = 0.001) diagnosed by CM, vs. patients without MDS (8% ± 10% and 16% ± 11%), respectively. Significant differences were also found for mean fluorescence intensity (MFI) of MNDA in monocytes which was lower in MDS (mean ± SD, 71 ± 36, P = 0.004) and AML (55 ± 39, P < 0.001) vs. no MDS samples (85 ± 28), respectively. Within patients with MDS, cases with cytogenetic aberrations showed a trend to higher %dimG (24% ± 18%, P = 0.083) compared with those without (16% ± 21%). Cut-off values for %dimG (12%) and %dimM (22%) as well as for MFI in monocytes (72) were defined capable of discriminating between MDS and non-MDS. MNDA expression in bone marrow cells can be assessed reliably by MFC and may facilitate evaluation of dyspoiesis when added to a standard MDS MFC panel.

Myeloid nuclear differentiation antigen, neutrophil apoptosis and sepsis

Sepsis and septic shock are characterized by prolonged inflammation and delayed resolution, which are associated with suppression of neutrophil apoptosis. The role of the intrinsic apoptotic pathway and intracellular factors in regulation of neutrophil apoptosis remain incompletely understood. We previously reported that the nuclear factor MNDA (myeloid nuclear differentiation antigen) is fundamental to execution of the constitutive neutrophil death program. During neutrophil apoptosis MNDA is cleaved by caspases and relocated to the cytoplasm. However, when challenged with known mediators of sepsis, human neutrophils of healthy donors or neutrophils from patients with sepsis exhibited impaired MNDA relocation/cleavage parallel with myeloid cell leukemia-1 (MCL-1) accumulation and suppression of apoptosis. MNDA knockdown in a model cell line indicated that upon induction of apoptosis, MNDA promotes proteasomal degradation of MCL-1, thereby aggravating mitochondrial dysfunction. Thus, MNDA is central to a novel nucleus-mitochondrion circuit that promotes progression of apoptosis. Disruption of this circuit contributes to neutrophil longevity, thereby identifying MNDA as a potential therapeutic target in sepsis and other inflammatory pathologies.

Flow Cytometric Assessment of Myeloid Nuclear Differentiation Antigen (MNDA) Expression in Myelodysplastic Syndromes As a Diagnostic Marker,

Abstract 3805 Background:Detection of aberrant antigen expression by multiparameter flow cytometry (MFC) is increasingly used in the diagnostic work-up of myelodysplastic syndromes (MDS). Myeloid nuclear differentiation antigen (MNDA) has been described to be strongly expressed in mature myeloid cells while myeloid progenitor cells show a weak expression only. MNDA has been suggested to be aberrantly expressed in MDS and therefore may improve the diagnostic capabilities of MFC. Aim: To analyze differences in the expression of MNDA between non-MDS cytopenias, MDS, and AML. Patients and methods: Bone marrow samples from a total of 269 patients with cytopenias and suspected MDS were analyzed by cytomorphology and MFC in parallel for diagnostic purposes. MNDA expression was determined by MFC in granulocytes, monocytes, and myeloid progenitor cells using the antibody clone 3C1 (provided by Trillium Diagnostics, Bangor, ME). MNDA expression was compared between patients grouped according to cytomorphology as: no MDS (n=103), MDS (n=85), CMML (n=9), AML (n=19), and possible MDS (n=53, based on cytomorphologic features of dysplasia but not sufficient to diagnose MDS). In addition, MNDA expression was compared between patients with aberrant and normal karyotype (cytogenetics was available in 238/269 patients, an aberrant karyotype was present in 59 patients). Results: A higher percentage of granulocytes (G) and monocytes (M) with weak expression of MNDA (%dimG and %dimM) was consistently found in MDS/AML samples as compared to samples without MDS. In detail, %dimG (mean±SD, 20±20% vs. 8±10%, p<0.001) and %dimM (31±24% vs. 16±11%, p<0.001) were significantly higher in cases with MDS as compared to those without. Furthermore, in patients with MDS, as compared to those without, the mean fluorescence intensity (MFI) of MNDA expression was found to be lower in monocytes (mean MFI, 71±36 vs. 85±27, p=0.004) as well as in myeloid progenitor cells with bright (67±24 vs. 86±66, p=0.007) or dim (2.5±0.9 vs. 2.8±1.2, p=0.037) expression of MNDA. In AML cases, as compared to cases with no MDS, similar figures were observed with higher values for %dimG (27±27% vs. 8±10%, p=0.007) and for %dimM (45±31% vs. 16±11%, p=0.001). In addition, in AML cases MFI of MNDA expression was lower in monocytes (55±39 vs. 85±27, p<0.001). Considering the diagnostically challenging cases with dysplastic features by cytomorphology but not sufficient to diagnose MDS similar although smaller differences were seen as compared to cases with no MDS with higher values for both %dimG (15±22% vs. 8±10%, p=0.026) and %dimM (25±19% vs. 16±11%, p=0.002) as well as a lower MFI of MNDA expression in monocytes (73±26 vs. 85±27, p=0.010). Interestingly, in CMML cases no reduced expression of MNDA could be found in any of the cell lines analyzed which might argue in favour of regarding CMML as myeloproliferative rather than myelodysplastic disease. From the cytogenetic point of view cases with an aberrant karyotype had higher values for both %dimG (22±20% vs. 12±18%, p=0.001) and %dimM (33±24% vs. 23±21%, p=0.004) as compared to those with a normal karyotype. Conclusions: These data suggests that MDS, as compared to non-MDS bone marrow, is associated with a reduced expression of MNDA in different cell lines which in part also applies to AML. Further analyses should define the potential of MNDA assessment to improve the MFC-based approach to diagnose MDS. Disclosures:Kern:MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Bellos:MLL Munich Leukemia Laboratory: Employment. Alpermann:MLL Munich Leukemia Laboratory: Employment. Haferlach:MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Schnittger:MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Haferlach:MLL Munich Leukemia Laboratory: Employment, Equity Ownership.

Quantitative Assessment of Myeloid Nuclear Differentiation Antigen Distinguishes Myelodysplastic Syndrome From Normal Bone Marrow

By using flow cytometry, we analyzed myeloid nuclear differentiation antigen (MNDA) expression in myeloid precursors in bone marrow from patients with myelodysplastic syndrome (MDS) and control samples from patients undergoing orthopedic procedures. The median percentage of MNDA-dim myeloid precursors in MDS cases was 67.4% (range, 0.7%-97.5%; interquartile range, 44.9%-82.7%) of myeloid cells, with bimodal MNDA expression in most MDS samples. Control samples demonstrated a median MNDA-dim percentage in myeloid precursors of 1.2% (range, 0.2%-13.7%; interquartile range, 0.6%-2.7%), with no bimodal pattern in most samples. The area under the receiver operating characteristic curve for MNDA-dim percentage in myeloid precursors was 0.96 (P = 9 × 10(-7)). Correlation of MNDA-dim levels with World Health Organization 2008 morphologic diagnoses was not significant (P = .21), but correlation with patient International Prognostic Scoring System scores suggested a trend (P = .07). Flow cytometric assessment of MNDA in myeloid precursors in bone marrow may be useful for the diagnosis of MDS.

Association of IFI200 Gene Polymorphisms with Susceptibility to Systemic Lupus Erythematosus

To the Editor: The MNDA (myeloid nuclear differentiation antigen), IFIX (interferon-inducible protein X), IFI16 (interferon-inducible protein 16), and AIM2 (absent in melanoma 2) are a group of interferon (IFN)-inducible genes whose products belong to the gene family denoted hematopoietic interferon-inducible protein with 200 amino acid repeat (IFI200). These genes map on chromosome 1q21–23, which is the major susceptibility locus of systemic lupus erythematosus (SLE). They are proposed as new candidate genes for SLE susceptibility for several reasons: (1) genetic mapping from a murine model of lupus1,2; (2) upregulation of all 4 genes in patients with SLE3; (3) the role of IFI16 as autoantigen in patients with SLE4,5; (4) the ability of IFI16 to bind single-strand DNA in a process of DNA repair6; and (5) the discovery of AIM2 as an intracellular DNA sensor leading to inflammation and apoptosis7. Therefore, IFI200 might be associated with abnormal inflammation and loss of tolerance to dsDNA observed in patients with SLE. We recruited 200 SLE patients (194 women, 6 men; mean age 36.21 ± SD 10.76 yrs) from King Chulalongkorn Memorial Hospital, Bangkok, each having at least 4 of the American College of Rheumatology revised criteria for SLE8, and 200 ethnically matched healthy volunteer blood donors from the Thai Red Cross Society (147 women, 53 men; mean age 23 ± SD 12.3 yrs)9. The study was approved by the ethics committee of the Faculty of Medicine, Chulalongkorn University, and all subjects gave their informed consent. Within 60 minutes after blood draw, peripheral blood mononuclear cells (PBMC) from 15 healthy donors containing different IFI16 genotypes were isolated by Ficoll-Hypaque gradient (Robbins Scientific, Sunnyvale, CA, USA) and resuspended in RPMI-1640 medium (Sigma, New York, NY, USA) with 10% … Address correspondence to Dr. Hirankarn; E-mail: fmednpt{at}md.chula.ac.th

Role for Myeloid Nuclear Differentiation Antigen in the Regulation of Neutrophil Apoptosis during Sepsis

Suppressed neutrophil apoptosis, a hallmark of sepsis, perpetuates inflammation and delays resolution. Myeloid nuclear differentiation antigen (MNDA) is expressed only in myeloid cells and has been implicated in cell differentiation; however, its function in mature neutrophils is not known. We studied whether MNDA could contribute to regulation of apoptosis of neutrophils from healthy subjects and patients with sepsis, and investigated the impact of MNDA knockdown on apoptosis. Human neutrophils were challenged with mediators of sepsis and neutrophils from patients with sepsis were cultured to investigate cleavage and cytoplasmic accumulation of MNDA. MNDA was knocked down in myeloid HL-60 cells to investigate development of apoptosis. During constitutive apoptosis of human neutrophils, MNDA is cleaved by caspases and accumulated in the cytoplasm, where it promotes degradation of the antiapoptotic protein Mcl-1, thereby accelerating collapse of mitochondrial transmembrane potential. Culture of neutrophils with LPS, bacterial DNA, or platelet-activating factor prevented MNDA cleavage and cytoplasmic accumulation. MNDA knockdown with short hairpin RNA markedly attenuated Mcl-1 turnover and conferred resistance to stress-induced apoptosis in HL-60 cells. Neutrophils from patients with severe sepsis exhibited markedly suppressed apoptosis that was associated with impaired cytoplasmic MNDA accumulation, preservation of Mcl-1 expression, and mitochondrial transmembrane potential. Culture of neutrophils of healthy subjects with septic plasma delayed apoptosis and cytoplasmic MNDA accumulation. These results indicate that cytoplasmic accumulation of MNDA facilitates progression of apoptosis and suggest that impaired cytoplasmic MNDA accumulation contributes to delayed neutrophil apoptosis in patients with severe sepsis.

MNDA regulates a novel nuclear‐mitochondrial circuit critical for progression of apoptosis in human neutrophils

Delayed neutrophil apoptosis perpetuates inflammation and delays the resolution of inflammation. We investigated the role of the nuclear protein MNDA (myeloid nuclear differentiation antigen) in progression of apoptosis of human neutrophils. We found that during constitutive apoptosis, MNDA is cleaved by caspase‐3 and relocated from the nucleus to the cytoplasm. MNDA co‐immunoprecipitates with the anti‐apoptotic protein Mcl‐1, accelerates proteasomal degradation of Mcl‐1, thereby contributing to collapse of mitochondrial transmembrane potential. MNDA knockdown with short hairpin RNA attenuates Mcl‐1 turnover, preserve mitochondrial function and confers resistance to apoptosis in myeloid HL‐60 cells. Culture of neutrophils with inflammatory mediators, including LPS, bacterial DNA, PAF or the acute phase reactants modified C‐reactive protein and serum amyloid A, prevents MNDA cleavage and translocation parallel with suppression of neutrophil apoptosis. Our results identify MNDA as a central mediator of a novel nucleus‐mitochondrion circuit that regulates progression of neutrophil apoptosis. (Grant support: LLSC and CIHR).