Anti-histone H1 Research Articles

Conventional (bone-type) giant cell tumor of the larynx: the first case with proven H3-3A: c.103G >T (p.Gly35Trp) mutation.

This report documents the first case of a conventional (bone-type) giant cell tumor of the larynx, in which the diagnosis was confirmed by molecular genetic analysis. A 50-year-old non-smoking man experienced progressive hoarseness lasting for 3 months. Imaging showed a 40-mm tumor arising from the right thyroid cartilage. The total laryngectomy was performed. Grossly, the tumor was solid and whitish, with areas of hemorrhage. Microscopically, the tumor consisted of a biphasic population with mononuclear cells with round to oval nuclei, small nucleoli, and pale eosinophilic cytoplasm admixed with evenly distributed dispersed osteoclast-like giant cells. Immunohistochemically, the neoplastic mononuclear cells expressed diffusely vimentin and p63 and focally SATB2. Admixed mononuclear histiocytes coexpressed CD68 and CD163, while the osteoclast-like giant cells showed only CD68 expression. Most importantly, all mononuclear tumor cells showed strong nuclear expression of anti-histone H3.3 G34W antibody. Subsequent next-generation sequencing confirmed the missense mutation of gene H3-3A: c.103G>T (p.Gly35Trp).

Immune Checkpoint Inhibitors (ICI) Promote Neutrophil-Platelet Aggregate and NET Formation in Tumor-Bearing Mice

Introduction: Immune checkpoint inhibitor (ICI) therapy has revolutionized cancer treatment. Despite remarkable clinical benefit, there are numerous immune-related adverse events (irAEs) associated with ICI therapy. Cancer-associated thrombosis (CAT) is a common complication of cancer, and ICI-associated thrombosis (ICI-CAT) is an underappreciated irAE. There is no data available concerning the mechanisms underlying ICI-CAT. To address this issue, we have established a murine model of ICI-CAT in which tumor-bearing mice treated with ICI develop larger thrombi than those treated with control IgG. We have observed that these ICI-treated mice display greater numbers of neutrophils in thrombi, and increased levels of circulating neutrophil extracellular traps (NETs). Methods: ICI (anti-PD-1 and anti-CTLA-4) was infused into Balb/c mice bearing subcutaneous CT26 colorectal tumors. Neutrophils (CD11b+ Ly6G+) as a percentage of total WBC was assessed using an automated hematology analyzer. Neutrophil-platelet doublets and aggregates were determined by flow cytometry analysis in using antibodies for CD41 (GPIIb) and CD62P (P-selectin). Circulating neutrophils were isolated from tumor-bearing mice treated with IgG and ICI 48h after IVC occlusion using an EasySep mouse neutrophil enrichment kit (STEMCELL technologies). For NET staining, isolated neutrophils (5x105 cells/well) were seeded on poly-L lysine coated coverslips, and treated with or without phorbol myristate acetate (PMA, 100 nM) for 4 h in 37°C. Neutrophils were then blocked using 5% FBS and stained, without permeabilization, using rabbit monoclonal anti-histone H3 (citrulline R2+R8+R17) antibody (ab5103, Abcam). Extracellular CitH3 was detected using anti-rabbit Alexa Fluor 488 secondary antibody. Results: Thrombi in ICI-treated mice were larger and contained greater numbers of neutrophils than thrombi from IgG-treated mice. Complete blood counts demonstrated that neutrophil numbers increased 3-fold in tumor bearing (38.2%) vs non-tumor bearing mice (12.8%) (Data not shown). However, neutrophils from ICI-treated tumor-bearing mice demonstrated significantly more spontaneous NET formation than those isolated from mice treated with control IgG (66.7% vs 18.2%). Smaller differences were also noted in response to PMA (Fig. A). Since binding of platelets to neutrophils is one cause of NET release, we also determined the frequency of neutrophil-platelet aggregates circulating in tumor-bearing mice treated with ICI or control IgG. Increased levels of circulating neutrophil-platelet aggregate in ICI-treated mice were present in a doublet CD41+Ly6G+ population (44.8% vs 22.3%); similar results were noted when platelets were stained with the activation marker CD62 (P-selectin) (8.5% vs 1%) (Fig. B, Left). The frequency of larger neutrophil-platelet aggregates followed a similar trend (ICI vs IgG; 75.8% vs 30% for CD41; 48.1% vs 7.7% for CD62P) (Fig. B, Right). Conclusions: Circulating neutrophils from ICI-treated, tumor bearing mice demonstrate increased spontaneous NET release. Moreover, elevated levels of circulating platelet-neutrophil doublets and aggregates are present in ICI-treated mice compared to those treated with control IgG. These results suggest a potential mechanism of ICI-induced thrombosis, and that platelet-neutrophil interactions may stimulate NET release in ICI-treated mice. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Anti-histone H3.3K36M Antibody is a Highly Sensitive and Specific Immunohistochemistry Marker for the Diagnosis of Chondroblastoma. A Validation Based on Study 136 Cases Comprising Chondroblastoma and its Mimics from Single a Centre in India.

Introduction. Chondroblastoma has a wide range of differential diagnosis encompassing various benign and malignant entities. The closest differential diagnosis is giant cell tumor of the bone due to overlapping radiological and histomorphological features. Extensive aneurysmal bone cyst like changes and lack of adequately sampled chondroid matrix often masquerades the primary bone lesion and amplifies the diagnostic difficulty in small biopsies with limited tissue. Immunohistochemistry is helpful in such instances to resolve the diagnostic dilemma. Objectives. To analyze the immunohistochemical expression of anti-histone H3F3K36M antibody inchondroblastoma and validate its utility in differentiating chondroblastoma from its histological mimics. Material and methods. Immunohistochemistry was performed using anti-histone antibody H3.3K36M in 44 histologically diagnosed chondroblastoma and 92 other histological mimickers. All chondroblastoma and giant cell tumor of the bone included in the study were also tested for anti-histone H3.3 G34W antibody. Of the 33 giant cell tumors of bone with classic morphology and imaging findings, 24 H3.3 G34W positive and 9 negative tumors were included intentionally to rule out the possibility of chondroblastoma. The sensitivity, specificity, positive and negative predictive value of marker with regard to chondroblastoma was calculated. Results. Immunohistochemistry revealed unequivocal nuclear positivity for H3.3K36M in the mononuclear cells in all the 44 chondroblastoma tested, denoting a sensitivity of 100% cases. Allthesetumors tested simultaneously for anti-histone H3.3G34W were negative. None of the histological mimickers were positive H3.3K36M indicating a specificity of 100%. The positive and negative predictive value was 100%. Conclusion. H3.3K36M mutant antibody is highly sensitive and specific IHC marker and can be used as a valuable adjunct to distinguish chondroblastoma from its histological mimics especially on small biopsies.

Association of Neutrophil Extracellular Traps with Plaque Instability in Patient with Carotid Artery Stenosis

Vulnerable carotid plaques are related to cerebral thromboembolic and ischemic events. Neutrophil extracellular traps (NETs) can induce endothelial dysfunction and induce inflammation and coagulation. The aim of the present study was to investigate NETs in patients with carotid artery plaques. Carotid plaques were collected by carotid endarterectomy (CEA) from 26 symptomatic and 8 asymptomatic patients between August 2017 and January 2021. The specimens were stained with hematoxylin-eosin and Elastica-van Gieson. Immunohistochemistry was performed staining by CD31 for identifying endothelial cells. NETs were detected by digoxigenin-labeled antihistone H3 (HH3) (citrulline R2+R8+R17). The relationships between the presence of NETs and patient profile and histopathological findings were assessed. HH3-positive cells were detected in 17 (asymptomatic=2 symptomatic=15) of 34 carotid plaques (median=9.7/mm). The number of NETs was correlated with the number of diffusion-weighted imaging high-intensity lesions [P=0.01], plaque rupture [P=0.001], intraplaque hemorrhage [P=0.02], intra luminal thrombus [P=0.001], and thin fibrous cap [P=0.001]. The presence of NETs was associated with the instability of carotid plaques, intraluminal thrombus, which may lead to subsequent cerebral infarction. Clarifying the roles of NETs in carotid plaques may improve the treatment of carotid artery disease.

Diagnostic Utility of Genetic and Immunohistochemical H3-3A Mutation Analysis in Giant Cell Tumour of Bone.

To validate the reliability and implementation of an objective diagnostic method for giant cell tumour of bone (GCTB). H3-3A gene mutation testing was performed using two different methods, Sanger sequencing and immunohistochemical (IHC) assays. A total of 214 patients, including 120 with GCTB and 94 with other giant cell-rich bone lesions, participated in the study. Sanger sequencing and IHC with anti-histone H3.3 G34W and G34V antibodies were performed on formalin-fixed, paraffin-embedded tissues, which were previously decalcified in EDTA if needed. The sensitivity and specificity of the molecular method was 100% (95% CI: 96.97–100%) and 100% (95% CI: 96.15–100%), respectively. The sensitivity and specificity of IHC was 94.32% (95% CI: 87.24–98.13%) and 100% (95% CI: 93.94–100.0%), respectively. P.G35 mutations were discovered in 2/9 (22.2%) secondary malignant GCTBs and 9/13 (69.2%) GCTB after denosumab treatment. We confirmed in a large series of patients that evaluation of H3-3A mutational status using direct sequencing is a reliable tool for diagnosing GCTB, and it should be incorporated into the diagnostic algorithm. Additionally, we discovered IHC can be used as a screening tool. Proper tissue processing and decalcification are necessary. The presence of the H3-3A mutation did not exclude malignant GCTB. Denosumab did not eradicate the neoplastic cell population of GCTB.

Anti-Histone H3.3 G34W antibody is a sensitive and highly specific immunohistochemistry marker for the diagnosis of Giant cell tumor of bone. A validation based on analysis of 198 cases from a single centre in India.

The diagnosis of giant cell tumor of bone (GCTB) is difficult in small biopsies with unusual age of presentation, location, and extensive secondary changes. Most of the GCTBs harbor H3F3A G34W mutations with a subset of cases showing alternate G34V, G34R, and G34L mutations. To analyze the expression of anti-histone H3.3G34W antibody in different cellular components of GCTB across different locations and presentations (including the unusual ones) and validate the utility of this antibody in the diagnosis of GCTB and differentiate it from the other osteoclast-like giant-cell-rich lesions. Immunohistochemistry was performed using anti-histone H3.3G34W antibody in the diagnosed cases of GCTB (136 cases of GCTB from 133 patients, including two malignant GCTBs) and other giant cell-containing lesions (62 cases). The presence of unequivocal crisp nuclear staining was considered positive. Immunohistochemistry revealed unequivocal nuclear positivity in the mononuclear cells in 87.3% of the cases of GCTB. Of these, most showed diffuse expression with moderate to strong intensity staining. The positive staining was restricted to the nuclei of mononuclear cells with the nuclei of osteoclastic giant cells being distinctly negative. In addition to conventional GCTBs, two cases each of multicentric and malignant GCTB showed positive staining. The other giant-cell containing lesions were distinctly negative. The present study showed a sensitivity of 87.3% with specificity and positive predictive value of 100%. The anti-histone G34W antibody is a highly sensitive and specific marker for the diagnosis of GCTB and differentiating it from its mimics. The positive staining is restricted to the mononuclear cell component of GCTB with sparing the osteoclastic giant cells further reiterating the fact that the mononuclear stromal cells are the true neoplastic component of GCTB.

Post-Denosumab-Treated Giant Cell Tumor of Bone: A Retrospective Histomorphological and Immunohistochemical Study

Abstract Introduction Giant cell tumors of bone (GCTBs) are treated with surgery with or without local adjuvants. Denosumab is a human monoclonal antibody that has recently emerged to be effective in treating unresectable and recurrent GCTBs. Objective In this study, we analyzed the histomorphological changes in GCTB following treatment with denosumab. The expression of histone mutation H3.3G34W by immunohistochemistry (IHC) using mutant specific antibody was also determined. Materials and Methods Of the total 109 GCTBs encountered during the study period, 14 cases with neoadjuvant denosumab therapy were analyzed retrospectively. The post-treatment changes on histopathology were examined on routine hematoxylin and eosin-stained sections. IHC was done using antihistone H3.3G34 antibodies. Statistical analysis was limited to descriptive statistics. No hypothesis testing was performed. Results All these cases except three showed fibrosis with areas of hyalinization, prominent newly formed woven bone along with spindle cells in short fascicles and storiform pattern. There was complete absence and marked reduction in osteoclast-like giant cells in six and five patients, respectively. Only three patients showed a substantial amount of residual osteoclast-like giant cells. IHC with antihistone H3.3G34W antibody showed unequivocal nuclear positivity in the mononuclear cells in nine cases. The mononuclear cells rimming and entrapped within the woven bone were also positive on IHC. The spindle cells in the benign fibrous histiocytoma-like areas and septa of aneurysmal bone cyst-like areas also retained nuclear staining. Conclusion Awareness of post-denosumab-related histopathological changes are necessary to avoid misdiagnosis as fibroosseous lesion and low-grade central osteosarcoma. Expression of mutant-specific H3.3 G34W antibody suggests that the neoplastic stromal cells are largely retained after denosumab therapy. The positive staining of cells both within and those rimming the newly formed woven bone point toward osteoblastic phenotype of the neoplastic stromal cells.

Differential Regulation of Damage-Associated Molecular Pattern Release in a Mouse Model of Skeletal Muscle Ischemia/Reperfusion Injury

BackgroundSkeletal muscle ischemia/reperfusion (I/R) injury is an important clinical issue that can cause remote organ injury. Although its pathogenesis has not been fully elucidated, recent studies have suggested that damage-associated molecular patterns (DAMPs) are mediators of remote organ injury in sterile inflammation. The purpose of this study was to investigate the possible involvement of DAMPs, including the nuclear proteins high-mobility group box 1 (HMGB1) and histone H3, in the pathogenesis of skeletal muscle I/R injury in mice.MethodsHindlimb ischemia was induced in mice through bilateral ligation of inguinal regions using rubber grommets. Reperfusion was induced by cutting the rubber grommets after 2–12 h of ischemic period. Survival rates, localization of HMGB1 and histone H3 in the gastrocnemius muscle, and circulating HMGB1 and histone H3 levels were analyzed. The effect of anti-HMGB1 and anti-histone H3 antibodies on survival was analyzed in mice with I/R injury.ResultsAll mice with hindlimb ischemia survived for at least 36 h, while all mice died within 24 h if the hindlimbs were reperfused after ischemia for 4–12 h. Immunohistochemical analysis revealed that HMGB1 translocated from the nucleus to the cytoplasm in the ischemic gastrocnemius muscle, while histone H3 was confined to the nucleus. Accordingly, serum HMGB1 levels were significantly elevated in mice with hindlimb I/R compared with normal mice or mice with hindlimb ischemia (P < 0.05). Serum histone H3 levels were not elevated after I/R. Treatment with anti-HMGB1 antibodies significantly improved survival of mice with hindlimb I/R injury compared with control antibodies (P < 0.05).ConclusionsHMGB1, but not histone H3, translocated to the cytoplasm during skeletal muscle ischemia, and was released into the systemic circulation after reperfusion in mice with I/R injury. Treatment with anti-HMGB1 antibodies partially improved survival.

Abstract 2514: Cell-free chromatin particles released into the microenvironment from dying tumor cells activate cancer hallmarks in surviving cells: a study in advanced squamous cell carcinoma of oral cavity with therapeutic implications

Abstract Background: We have reported that combination of the nutraceuticals Resveratrol (R) and Copper(Cu) results in generation of free radicals which can degrade cell-free chromatin (cfCh) particles and have potential therapeutic effects (Mittra et al., Ann. Oncol. 2017 &amp; Mittra et al., PLos One 2020). We have also reported that cfCh particles that are released from dying tumor cells can integrate into genomes of healthy bystander cells to activate two critical hallmarks of cancer namely, genome instability and inflammation, which could lead to their oncogenic transformation (Mittra et al., Cell Death Discovery 2017). Here we show that cfCh particles released into tumor microenvironment (TME) from dying tumor cells of advanced squamous carcinoma of oral cavity activate all ten hallmarks of cancer defined by Hanahan and Weinberg, and that oral administration of R-Cu for two weeks prevents this potentially life-threatening manifestation. Patients and Methods: The study was approved by Institutional Ethics Committee, and a written consent was obtained from participants. In this exploratory study, R-Cu was administered in four escalating doses orally for two weeks, to 25 patients divided into 5 groups with 5 patients each, as follows: 1) Control: R 0, Cu 0; 2) Level I: R 5.6 mg, Cu 560 ng; 3) Level II: R 50 mg, Cu 5µg; 4) Level III: R 500 mg, Cu 50 µg; 5) Level IV: R 500 mg, Cu 5mg. A punch biopsy was obtained on the day 0 and day 14 post R-Cu treatment. FFPE histological sections were examined by 1) Fluorescence immuno-staining (FI) using anti-DNA and anti-histone H4 antibodies and confocal microscopy to detect presence of cfCh in TME; 2) Immuno-fluorescence (IF) analysis of intracellular antioxidant enzymes; and 3) IF analysis of 10 hallmarks of cancer represented by 23 biomarkers. Results: 1) FI and Confocal microscopy detected extensive presence of dually fluorescently labelled cfCh particles in TME, which were dramatically reduced following R-Cu treatment. 2) IF analysis detected significantly elevated levels of three intra-cellular anti-oxidant enzymes in post R-Cu samples, suggesting that cells had mounted a defense mechanism to counteract free radicals that had entered into them. No difference between day 0 and day 14 samples was seen in the untreated control group. 3) IF analysis of 23 cancer hallmark biomarkers showed significant down-regulation in the post R-Cu samples, which were most marked in dose levels I and II. No difference between day 0 and day 14 samples was seen in the control group. Conclusion: We show in a human tumor model that cfCh particles released into TME from dying tumor cells activate cancer hallmarks in surviving cells, and that the cfCh inactivating agent R-Cu can significantly down-regulate cancer hallmark levels suggesting its therapeutic potential. Citation Format: Indraneel Mittra, Hitesh Singhavi, Aishwarya Pilankar, Venkat Raghuram Gorantla, Sophiya Siddiqui, Naveen Kumar Khare, Vishalkumar Jadhav, Kavita Pal, Pankaj Chaturvedi. Cell-free chromatin particles released into the microenvironment from dying tumor cells activate cancer hallmarks in surviving cells: a study in advanced squamous cell carcinoma of oral cavity with therapeutic implications [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2514.

The Role of Kdm6a in Malignant Hematopoiesis

Our preliminary studies identified recurrent, somatic mutations in Kdm6a in a mouse model of acute promyelocytic leukemia (APL). Putative inactivating mutations of Kdm6a (primarily small deletions) occurred in approximately 50% of mouse APL samples, making these the most common acquired progression events in this model. We found somatic mutations of KDM6A in 4 of 200 acute myeloid leukemia (AML) samples. However, in a cohort of 42 patients with APL, we identified no somatic alterations in KDM6A . To better understand the role of Kdm6a in malignant hematopoiesis, we intercrossed our Kdm6a conditional knockout (KO) x Vav1 - Cre mice with a “knock-in” mouse model of APL (Ctsg-PML-RARA mice). To determine whether Kdm6a inactivation cooperates with PML - RARA, we performed a tumor watch that included all genotypes of Kdm6a conditional KO female and male triple cross mice. After 18 months of observation, we found that the latency of APL was significantly decreased in all three cohorts of Kdm6a conditional KO x Vav1 - Cre x Ctsg-PML-RARA mice (Figure 1). This finding was most pronounced in female homozygous Kdm6a KO triple cross mice, which had a median survival of 161 days versus 365 days in the control cohort (P < 0.01 by log-rank test). The Kdm6a -null mice developed the typical phenotype of mouse APL, including splenomegaly, myeloproliferation and leukemic cells that coexpressed Gr-1 and Kit. The triple cross APL cells remained sensitive to all- trans retinoic acid (ATRA) and arsenic trioxide ex vivo . Compared with 56% penetrance in Ctsg-PML-RARA mice, female homozygous and male hemizygous Kdm6a KO x Vav1 - Cre x Ctsg-PML-RARA mice developed APL with 100% penetrance. We fully characterized pre-leukemic hematopoiesis in young (6 to 8-weeks-old) Kdm6a conditional KO x Vav1 - Cre x Ctsg-PML-RARA mice (compared to appropriate littermate controls) to define the underlying mechanism(s) by which Kdm6a inactivation contributes to leukemogenesis. Like our double cross Kdm6a conditional KO x Vav1 - Cre mice, the loss of Kdm6a expression in young female homozygous triple cross mice caused mild thrombocytopenia and splenomegaly. These young mice had neutrophilia and lymphopenia, which the female Kdm6a -null double cross mice only acquired with age. We observed a trend for increased numbers of colonies in a myeloid serial replating assay that was also specific to this cohort. Next, we demonstrated that this cohort had significant decreases in the Kit+Lin-Sca-1+ (KLS) cell, short-term hematopoietic stem cell (ST-HSC), and multipotent progenitor (MPP) hematopoietic stem and precursor compartments. This recapitulated the findings from the young, double cross Kdm6a -null female mice but was more pronounced. We found a concomitant increase in granulocyte-macrophage progenitors (GMPs) and decrease in megakaryocyte-erythroid progenitors (MEPs) that was also specific for the female Kdm6a -null triple cross cohort. Finally, we performed gene expression profiling via exon arrays on flow-sorted SLAM cells from aged (1-year-old) female Kdm6a -null double cross mice and aged controls. Unsupervised hierarchical clustering revealed a clear distinction between cohorts. We identified 1520 differentially expressed genes by an ANOVA analysis (fold change > 2, P < 0.05) between the Kdm6a -null versus WT aged SLAM cells. The list of differentially expressed genes included Pml (downregulated in Kdm6a -null SLAM cells with fold change of 2.35) and Flt3 (upregulated in Kdm6a -null SLAM cells with fold change of 2.32). Expression changes of these two genes may be associated with the mechanism of how inactivation of Kdm6a cooperates with PML-RARA in our mouse model. Pml haploinsuffiency has been show to modestly cooperate with PML-RARA in mouse models of APL, and FLT3 mutations are the most common secondary mutations in APL. In an effort to further elucidate the mechanism by which loss of Kdm6a contributes to malignant hematopoiesis, we have completed RNA-Seq, ATAC-Seq, anti-histone H3 (monomethyl K4) ChIP-Seq, and anti-histone H3 (trimethyl K27) ChIP-Seq studies on mouse APL samples derived from Ctsg-PML-RARA mice that developed spontaneous leukemias (n = 3 Kdm6a -WT samples versus n = 3 Kdm6a -mutant samples). Analysis of this data set is now underway. In sum, our data have demonstrated a functional role of Kdm6a in malignant hematopoiesis and have improved our understanding of its subsequent connection to leukemogenesis. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Associations of NETs with inflammatory risk and atherosclerotic severity in ST-segment elevation myocardial infarction

AimTo investigate the association between neutrophil extracellular traps (NETs) in coronary thrombus and risk profile of patients with ST-segment elevation myocardial infarction (STEMI). MethodsAll participants enrolled in the Optical Coherence Tomography Examination in Acute Myocardial Infarction (NCT03593928) registry from March 2017 to November 2019 were screened and 104 patients were qualified for the current analysis. Coronary aspirated materials were collected and culprit lesion characteristics were examined by optical coherence tomography. NETs in thrombus were identified by immunofluorescence as the co-localization of anti-histone H3 and myeloperoxidase, and NETs level was assessed using a semi-quantitative score. Patient-oriented composite endpoint included all-cause death, myocardial infarction, stroke, any revascularization, and re-admission for heart failure. ResultsAccording to NETs burden, 44 patients were in the low NETs group and 60 in the high NETs group. The two groups were comparable in age, sex, and comorbidities but the high NETs group presented with significantly higher level of high-sensitivity C reactive protein than the low NETs group (median 9.3 mg/L vs 5.2 mg/L, p = 0.036). Although culprit plaque morphology did not differ between groups, the lipid arc was smaller (maximal lipid arc 320° vs 360°, p = 0.012) and the flow area was larger (median 1.85mm2 vs 1.65mm2, p = 0.041) in the high NETs group. No significant difference was observed between groups in patient-oriented composite endpoint. ConclusionIncreased NETs in aspirated coronary thrombus appear to be associated with more elevated inflammation but less advanced atherosclerosis.Trial registration number: ClinicalTrials.gov: NCT03593928

Autoantibodies targeting components of neutrophil extracellular traps are elevated in cystic fibrosis

Abstract Cystic fibrosis (CF) lung disease is characterized by the presence of neutrophils and released neutrophil extracellular traps (NETs). NETs are composed of a genomic DNA scaffold decorated with histones and neutrophil granule proteins. The long-term presence of NETs could lead to an autoimmune response in CF. This is supported by our published results showing elevated levels of anti-PAD4 autoantibodies in a U.S. CF cohort. To confirm and expand this observation, here we hypothesized that anti-PAD4 autoantibodies are also elevated in another CF cohort from Hungary, and additional, NET-targeting autoantibodies can be identified in CF. Fifty-seven CF patients (class I–III CFTR mutations, stable lung function, 26±−5.4 years of age (mean±SD), 54% female) and forty healthy subjects (26.2±3.6 years of age, 57% female) were recruited into this study in Hungary. Anti-PAD4 and anti-dsDNA autoantibodies were measured by ELISA while additional autoantibodies were determined by autoantigen microarray. Increased levels of anti-PAD4 autoantibodies were confirmed in this CF cohort. Serum levels of anti-dsDNA IgA autoantibodies were significantly higher in CF patients compared to control subjects. The autoantigen microarray analysis revealed significantly elevated levels of several, novel, NET-targeting autoantibodies in CF: anti-chromatin IgA, anti-core Histone IgA and IgE, anti-Histone H1 IgA, anti-Histone H2A IgA and IgM. No significant correlations of these autoantibodies with lung function (FEV1%pred), age or inflammatory markers (human epididymis protein 4, C-reactive protein) were found. Overall, these results indicate that chronic presence of NETs leads to a systemic autoimmune response in CF.

Observation of intracellular protein localization area in a single neuron using gold nanoparticles with a scanning electron microscope

The localization areas of intracellular proteins in rat cortical neurons were visualized using a scanning electron microscope (SEM) coupled with a focused ion beam (FIB) system. To obtain a clear contrast in the SEM images, gold nanoparticles (GNPs) were bound to specific intracellular proteins by antigen-antibody reactions. By obtaining a cross section of the desired location of the neurons by FIB milling under the SEM imaging condition, it was possible to observe the proteins inside the cells as clear bright spots. When a neuron was stained with anti-tau and anti-histone H1 antibodies, the bright spots were localized in the cross section of the axon and the nucleus, respectively. It was confirmed that targeted proteins in a single neuron on a substrate could be successfully identified. The development of FIB/SEM observation with immunological GNP staining will offer important information for the stable growth of neurons on various substrate structures, since the elongation and turning of axons on the substrates are activated by the redistribution of intracellular proteins.

Abstract 5183: ONC201 induces acetylation of histone binding within the p21 (CDKN1A) gene promoter

Abstract ONC201/TIC10 is a promising anticancer agent that upregulates cytotoxic TRAIL pathway signaling in cancer cells. Preliminary clinical data indicates ONC201 induces clinical benefit in a subset of patients with histone H3 K27M glioma, among other tumors. Since H3 K27M mutation reduces levels of H3K27 di- and tri-methylation and in turn alters the expression of genes by epigenetic modulation, we investigated ONC201 effects on epigenetic regulation in cancer. We treated the colorectal cancer cell line HCT116 with the histone deacetylase inhibitor entinostat or ONC201. Chromatin immunoprecipitation (ChIP) was performed with anti-histone H3 (acetyl K9) antibody using lysates from drug-treated tumor cells. Immunoprecipitated DNA was probed by PCR using primers across the promoter of p21 (WAF1; CDKN1A). The results indicate that both ONC201 and entinostat induce the acetylation of histone H3 binding within the p21 promoter. We further observed that ONC201 plus entinostat have synergistic effects in this p21 promoter acetylation activity. Activation of p21 gene expression by enhancing histone acetylation may be relevant in cancer suppression by ONC201. Citation Format: Yiqun Zhang, Lanlan Zhou, Shengliang Zhang, Marie D. Ralff, Shuai Zhao, Philip H. Abbosh, Rahmat Sidker, Wafik S. El-Deiry. ONC201 induces acetylation of histone binding within the p21 (CDKN1A) gene promoter [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 5183.

Abstract WP281: Histologic Features of Thrombi Retrieved From Ischemic Stroke Patients With Cancer

Background: Systemic cancer is a cause of stroke. However, histologic features of thrombus in stroke patients with cancer are not well-known. We investigated histologic features of intracranial thrombi in acute stroke patients with cancer. Methods: We prospectively collected thrombi that were retrieved during mechanical thrombectomy. This study included 32 consecutive patients with any history of systemic cancer (16 patients with active cancer and 16 patients with inactive cancer.) All patients underwent continuous electrocardiographic monitoring for at least 24 h. Echocardiography was performed in all but three patients. Immunohistochemical staining of thrombus was done using anti-CD42b antibody for platelets, anti-glycophorin antibody for erythrocytes, anti-fibrinogen antibody for fibrin, anti-myeloperoxidase antibody for leukocytes, and anti-Histone H3 antibody for neutrophil extracellular traps. After acquiring images of entire thrombus area using the stereo investigator imaging system, quantitative fraction (%) of different thrombus composition was semi-automatically assessed using ImageJ software. We compared the thrombus composition between the active cancer group and the inactive cancer group. Results: Comparing with the inactive cancer group, the active cancer group showed higher platelet fraction (median [interquartile range], 20.6 [7.3-27.6] vs 6.1 [2.5-9.9], p=0.002) and lower erythrocyte fraction (1.6 [0.8-11.1] vs 20.7 [13.3-23.7], p=0.001). Fractions for fibrin, leukocytes, and neutrophil extracellular traps were not different between the active and inactive cancer groups. Four patients had vegetation on echocardiography and showed very high platelet fraction (29.5 [26.1-33.9]) and very low erythrocyte fraction (0.8 [0.7-0.9]). Of the active cancer group, 6 patients with negative evaluation also showed high platelet fraction and low erythrocyte fraction, but the thrombus composition in 4 patients with determined etiology (3 atrial fibrillation, 1 &gt;50% stenosis) was similar to that in inactive cancer. Conclusion: Thrombi in stroke patients with active cancer were platelet-rich and erythrocyte-poor. These findings may aid to determine the treatment strategy for stroke patients with cancer.

Neutrophil Extracellular Traps and Fibrin in Otitis Media

Bacterial resistance in acute otitis can result in bacterial persistence and biofilm formation, triggering chronic and recurrent infections. To investigate the middle ear inflammatory response to bacterial infection in human and chinchilla temporal bones. Six chinchillas underwent intrabullar inoculations with 0.5 mL of 106 colony-forming units (CFUs) of Streptococcus pneumoniae, serotype 2. Two days later, we counted bacteria in middle ear effusions postmortem. One ear from each chinchilla was processed in paraffin and sectioned at 5 µm. The opposite ear was embedded in epoxy resin, sectioned at a thickness of 1 µm, and stained with toluidine blue. In addition, we examined human temporal bones from 2 deceased donors with clinical histories of otitis media (1 with acute onset otitis media, 1 with recurrent infection). Temporal bones had been previously removed at autopsy, processed, embedded in celloidin, and cut at a thickness of 20 µm. Sections of temporal bones from both chinchillas and humans were stained with hematoxylin-eosin and immunolabeled with antifibrin and antihistone H4 antibodies. Histopatological and imminohistochemical changes owing to otitis media. Bacterial counts in chinchilla middle ear effusions 2 days after inoculation were approximately 2 logs above initial inoculum counts. Both human and chinchilla middle ear effusions contained bacteria embedded in a fibrous matrix. Some fibers in the matrix showed positive staining with antifibrin antibody, others with antihistone H4 antibody. In acute and recurrent otitis media, fibrin and neutrophil extracellular traps (NETs) are part of the host inflammatory response to bacterial infection. In the early stages of otitis media the host defense system uses fibrin to entrap bacteria, and NETs function to eliminate bacteria. In chronic otitis media, fibrin and NETs appear to persist.

H3F3A (Histone 3.3) G34W Immunohistochemistry: A Reliable Marker Defining Benign and Malignant Giant Cell Tumor of Bone.

Giant cell tumor of bone (GCTB) is a locally aggressive subarticular tumor. Having recently reported that H3.3 G34W mutations are characteristic of this tumor type, we have now investigated the sensitivity and specificity of the anti-histone H3.3 G34W rabbit monoclonal antibody in a wide variety of tumors including histologic mimics of GCTB to assess its value as a diagnostic marker. We also determined the incidence of H3.3 G34 mutations in primary malignant bone tumors as assessed by genotype and H3.3 G34W immunostaining. A total of 3163 tumors were tested. Totally, 213/235 GCTB (90.6%) showed nuclear H3.3 p.G34W immunoreactivity. This was not the case for the rare variants, p.G34L, M, and V, which occurred most commonly in the small bones of the hands, patella, and the axial skeleton. If these sites were excluded from the analysis, H3.3 G34W expression was found in 97.8% of GCTB. Malignant bone tumors initially classified as osteosarcomas were the only other lesions (n=11) that showed G34W expression. Notably an additional 2 previously reported osteosarcomas with a p.G34R mutation were not immunoreactive for the antibody. A total of 11/13 of these malignant H3.3-mutant tumors exhibited an osteoclast-rich component: when imaging was available all but one presented at a subarticular site. We propose that subarticular primary malignant bone sarcoma with H3.3 mutations represent true malignant GCTB, even in the absence of a benign GCTB component.

ANTI-HISTONE H1 IgGs POSSESS PROLIFERATIVE ACTIVITY TOWARDS HUMAN T-LEUKAEMIA CEM CELLS

Anti-histone H1 IgGs were purified from blood serum of systemic lupus erythematosus patients by precipitation of serum proteins with 50% ammonium sulfate followed by a sequential affinity chromatography on Protein G-Sepharose and histone H1-Sepharose columns. To avoid contamination with other proteins, anti-histone H1 IgGs were subjected to strongly acidic pH 2.0 during gel filtration through HPLC column. The effects of the anti-histone H1 IgGs on cell viability and cell cycle were tested by MTS-assay and flow cytometry, correspondingly. The cross-reactivity of the anti-histone H1 antibodies towards heterogenetic and cellular antigens was evaluated by Western-blot analysis. It was found that incubation of CEM cells with the HPLC-purified anti-histone H1 IgGs resulted in significant stimulation of cell growth by 46% after 48 h of incubation. These IgGs possess an antigenic poly-specificity to positively charged heterogenetic antigens and different cellular antigens. FITC-labeled and biotinylated anti-histone H1 IgGs are internalized by CEM cells and preferentially accumulated in the cytoplasm. The anti-histone H1 IgGs are shown to internalize human T-leukemia CEM and stimulate their proliferation. These IgGs are polyspecific toward cellular antigens.

Abstract TMP26: Existence of Neutrophil Extracellular Traps in Arterial Thrombi from Patients with Acute Ischemic Stroke

Introduction: Neutrophil extracellular traps (NETs) are composed mainly of strands of filamentous DNA and nuclear proteins, such as chromatin or isoforms of histone and secretable neutrophil granular proteins. Recent studies have shown that NETs existed in the venous thrombi and might function as a scaffold and had implication for thrombolytic treatments. Hypothesis: NETs also exists in cerebral arterial thrombus collected from patients with acute ischemic stroke(AIS). Methods: Ten arterial thrombi were collected from 10 AIS patients (33-78 years old, 4 females) who were treated by endovascular therapy (eight of them accepted alteplase thrombolysis firstly)within 8 hours after symptoms onset. Hematoxylin-eosin (HE) staining, immunofluorescence staining (including anti-histone H3 and Hoechst 33342 which label DNA) and electron microscopy were performed to identify the existence of fibrin and/or NETs in thrombus. Results: There were 4 thrombi from middle cerebral artery, 3 from internal carotid artery and 3 from basilar artery. Cardioembolism were considered as etiology for 5 patients and large artery atherosclerosis was the etiology for another 5 patients. HE staining showed that large amounts of fibrin and white blood cells in all ten cerebral arterial thrombi. NETs structures were also identified in 10/10 cerebral arterial thrombi by immunofluorescence staining. Furthermore, the existence of neutrophil-released DNA network together with fibrin was confirmed using electron microscopy in all 10 thrombi. Conclusion: Our results directly demonstrated that NETs do exist in the arterial thrombi from patients with AIS. they may serve as another structural component in addition to fibrin in both cardiogenic and artery atherosclerotic thrombi. NETs can be a new potential target for the recanalization treatment. Keyword: Neutrophil extracellular traps (NETs); arterial thrombus; Acute ischemic stroke

Pulmonary endothelial activation caused by extracellular histones contributes to neutrophil activation in acute respiratory distress syndrome.

BackgroundDuring the acute respiratory distress syndrome (ARDS), neutrophils play a central role in the pathogenesis, and their activation requires interaction with the endothelium. Extracellular histones have been recognized as pivotal inflammatory mediators. This study was to investigate the role of pulmonary endothelial activation during the extracellular histone-induced inflammatory response in ARDS.MethodsARDS was induced in male C57BL/6 mice by intravenous injection with lipopolysaccharide (LPS) or exogenous histones. Concurrent with LPS administration, anti-histone H4 antibody (anti-H4) or non-specific IgG was administered to study the role of extracellular histones. The circulating von Willebrand factor (vWF) and soluble thrombomodulin (sTM) were measured with ELISA kits at the preset time points. Myeloperoxidase (MPO) activity in lung tissue was measured with a MPO detection kit. The translocation of P-selectin and neutrophil infiltration were measured by immunohistochemical detection. For in vitro studies, histone H4 in the supernatant of mouse lung vascular endothelial cells (MLVECs) was measured by Western blot. The binding of extracellular histones with endothelial membrane was examined by confocal laser microscopy. Endothelial P-selectin translocation was measured by cell surface ELISA. Adhesion of neutrophils to MLVECs was assessed with a color video digital camera.ResultsThe results showed that during LPS-induced ARDS extracellular histones caused endothelial and neutrophil activation, as seen by P-selectin translocation, release of vWF, an increase of circulating sTM, lung neutrophil infiltration and increased MPO activity. Extracellular histones directly bound and activated MLVECs in a dose-dependent manner. On the contrary, the direct stimulatory effect of exogenous histones on neutrophils was very limited, as measured by neutrophil adhesion and MPO activity. With the contribution of activated endothelium, extracellular histones could effectively activating neutrophils. Both inhibiting the endothelial activation with an anti-toll like receptor (TLR) antibody and inhibiting the interaction of the endothelium with neutrophil using an anti-P-selectin antibody decreased the degree of neutrophil activation.ConclusionsExtracellular histones are pro-inflammatory mediators in LPS-induced ARDS in mice. In addition to direct action to neutrophils, extracellular histones promote neutrophil adhesion and subsequent activation by first activating the pulmonary endothelium via TLR signaling. Thus, endothelial activation is important for extracellular histone-induced inflammatory injury.