Multinucleated Research Articles

Clinical and histopathological features of lentigo maligna and lentigo maligna melanoma: a retrospective analysis in Korea.

Lentigo maligna (LM) and lentigo maligna melanoma (LMM) are rare in Asian countries. The histopathological diagnosis of LM is often challenging, and misdiagnosis is common. Although histopathologic features of LM/LMM are known, statistical analysis of them were scarcely reported. In this study, we aimed to investigate the histopathological characteristics of LM/LMM in Korean patients and identify key histopathological clues distinguishing LM from benign lentigo. We performed a retrospective study of the clinical and histopathological features of patients diagnosed with LM/LMM at our center between 2011 and 2022. We assessed the histopathological features in each case based on 16 pathological criteria according to previous literature. Pathologically confirmed cases of benign lentigo were analyzed for comparison. Twenty-one patients (10 with LM and 11 with LMM) were analyzed. Several statistically significant difference existed between the features of LM and benign lentigo (N = 10), including asymmetry of overall structure (p < 0.001), cytologic atypia (p < 0.001), predominant single-cell proliferation (p < 0.001), melanocytic nests (p = 0.033), melanocytes forming rows (p = 0.003), pagetoid spread of melanocytes (p < 0.001), and hair follicle invasion by atypical melanocytes (p < 0.001). Degree of solar elastosis was more severe in group "Age ≥ 60" (p = 0.015), and group "Diameter ≥ 20 mm" (p = 0.043). Presence of elongated rete ridges were less common in the older than 60 age group (p = 0.015) and group "Diameter ≥ 20 mm." Invasion was associated with mitosis (p = 0.001, OR 49.285), multinucleated cells (p = 0.035, OR 17.769), and degree of lymphocyte infiltration (p = 0.004). This study investigated the clinical and histopathologic characteristics of LM and LMM in Koreans. Although histopathological diagnosis is challenging, especially in the early stages of LM, our data showed essential histopathological changes in architectural, cytological, and dermal patterns. Considering the potential aggressiveness of LM/LMM, it is essential to recognize its histopathological features and provide timely management.

A NOVEL NON-CODING RNA-BASED APPROACH TO MODULATE OSTEOCLAST BEHAVIOUR

Osteoclasts (OCs) are multinucleated cells that play a pivotal role in skeletal development and bone remodeling. Abnormal activation of OCs contributes to the development of bone-related diseases, such as osteoporosis, bone metastasis and osteoarthritis. Restoring the normal function of OCs is crucial for bone homeostasis. Recently, RNA therapeutics emerged as a new field of research for osteoarticular diseases.The aim of this study is to use non-coding RNAs (ncRNAs) to molecularly engineer OCs and modulate their function. Specifically, we investigated the role of the microRNAs (namely miR-16) and long ncRNAs (namely DLEU1) in OCs differentiation and fusion.DLEU1/DLEU2 region, located at chromosome 13q14, also encodes miR-15 and miR-16. Our results show that levels of these ncRNA transcripts are differently expressed at distinct stages of the OCs differentiation. Specifically, silencing of DLEU1 by small interfering RNAs (siDLEU1) and overexpression of miR-16 by synthetic miRNA mimics (miR-16-mimics) led to a significant reduction in the number of OCs formed per field (OC/field), both at day 5 and 9 of the differentiation stage. Importantly, time-lapse analysis, used to track OCs behavior, revealed a significant decrease in fusion events after transfection with siDLEU1 or miR-16-mimics and an alteration in the fusion mode and partners. Next, we investigated the migration profile of these OCs, and the results show that only miR-16-mimics-OCs, but not siDLEU-OCs, have a lower percentage of immobile cells and an increase in cells with mobile regime, compared with controls. No differences in cell shape were found. Moreover, mass-spectrometry quantitative proteomic analysis revealed independent effects of siDLEU1 and miR-16-mimics at the protein levels. Importantly, DLEU1 and miR-16 act by distinct processes and pathways.Collectively, our findings support the ncRNAs DLEU1 and miR-16 as therapeutic targets to modulate early stages of OCs differentiation and, consequently, to impair OC fusion, advancing ncRNA-therapeutics for bone-related diseases.Acknowledgements: Authors would like to thank to AO CMF / AO Foundation (AOCMFS-21-23A). SRM and MIA are supported by FCT (SFRH/BD/147229/2019 and BiotechHealth Program; CEECINST/00091/2018/CP1500/CT0011, respectively).

Somatic Cell Fusion in Host Defense and Adaptation.

Evidence of fusion of somatic cells has been noted in health and in disease for more than a century. The most obvious but uncertain hallmark has been the presence of multiple nuclei in cells. Although multinucleated cells are found in normal and diseased tissues, the benefit or harm of such cells can be difficult to elucidate. Still more difficult however is the identification of mononuclear cells previously formed by fusion of somatic cells with one or more nuclei disposed. The later process can introduce mutations that promote viral diversification, cancer, and tissue senescence. Less obvious the potential benefits of cell fusion. Recent work in cell biology, immunology, and genomic analysis however makes it possible to postulate benefits and potentially arrive at novel therapeutic agents and approaches that replicate or enhance these benefits.

Histological Evaluation of Internal Dental Resorption: An Analysis of a Cohort of 50 Cases.

This study aimed to perform a histological evaluation of teeth diagnosed with internal root resorption. A descriptive study involved the examination of 50 human teeth extracted due to an unfavorable prognosis for retention in the oral cavity. Teeth were preserved in 10% buffered formalin and subsequently subjected to the decalcification process. Masson-Goldner staining was applied for comprehensive histological assessment. In all the 50 teeth examined, resorption gaps within the dentin tissue were identified, accompanied by the presence of reparative cells in the vicinity of these cavities. Marked structural loss and dentin fragmentation were evident, with regions exhibiting fissures and an absence of dentinal tubules. The histological evaluation of 50 teeth diagnosed with internal dental resorption revealed significant structural alterations, including resorption lacunae, the presence of multinucleated osteoclast-like cells, and reparative connective tissue. These findings highlight the complex and multifaceted nature of internal dental resorption. These histological insights provide a deeper understanding of the pathological processes involved in internal dental resorption and underscore the necessity for early detection and intervention to mitigate tooth loss and preserve dental health.

Chemotaxis of Large Multinucleate Cells of Dictyostelium Produced by Electric-Pulse Induced Fusion.

Normal-sized cells of Dictyostelium build up a front-tail polarity when they respond to a gradient of chemoattractant. To challenge the polarity-generating system, cells are fused to study the chemotactic response of oversized cells that extend multiple fronts toward the source of attractant. An aspect that can be explored in these cells is the relationship of spontaneously generated actin waves to actin reorganization in response to chemoattractant.

Preclinical Evaluation of the ATR Inhibitor BAY 1895344 as a Radiosensitizer for Head and Neck Squamous Cell Carcinoma

Despite aggressive multimodal treatment that typically includes definitive or adjuvant radiation therapy (RT), locoregional recurrence rates approach 50% for patients with locally advanced human papillomavirus (HPV)-negative head and neck squamous cell carcinoma (HNSCC). Thus, more effective therapeutics are needed to improve patient outcomes. We evaluated the radiosensitizing effects of ataxia telangiectasia and RAD3-related (ATR) inhibitor (ATRi) BAY 1895344 in preclinical models of HNSCC. Murine and human HPV-negative HNSCC cells (MOC2, MOC1, JHU-012) were treated with vehicle or ATRi with or without 4 Gy. Checkpoint kinase 1 phosphorylation and DNA damage (γH2AX) were evaluated by Western blot, and ATRi half-maximal inhibitory concentration was determined by MTT assay for HNSCC cells and immortalized murine oral keratinocytes. In vitro radiosensitization was tested by clonogenic assay. Cell cycle distribution and mitotic catastrophe were evaluated by flow cytometry. Mitotic aberrations were quantified by fluorescent microscopy. Tumor growth delay and survival were assessed in mice bearing MOC2 or JHU-012 transplant tumors treated with vehicle, ATRi, RT (10 Gy × 1 or 8 Gy × 3), or combined ATRi + RT. ATRi caused dose-dependent reduction in checkpoint kinase 1 phosphorylation at 1 hour post-RT (4 Gy) and dose-dependent increase in γH2AX at 18 hours post-RT. Addition of RT to ATRi led to decreased BAY 1895344 half-maximal inhibitory concentration in HNSCC cell lines but not in normal tissue surrogate immortalized murine oral keratinocytes. Clonogenic assays demonstrated radiosensitization in the HNSCC cell lines. ATRi abrogated the RT-induced G2/M checkpoint, leading to mitosis with unrepaired DNA damage and increased mitotic aberrations (multinucleated cells, micronuclei, nuclear buds, nucleoplasmic bridges). ATRi and RT significantly delayed tumor growth in MOC2 and JHU-012 in vivo models, with improved overall survival in the MOC2 model. These findings demonstrated that BAY 1895344 increased in vitro and in vivo radiosensitivity in HPV-negative HNSCC preclinical models, suggesting therapeutic potential warranting evaluation in clinical trials for patients with locally advanced or recurrent HPV-negative HNSCC.

Uterine leiomyoma with bizarre nuclei – A series of four cases

Uterine leiomyoma is the most common benign mesenchymal tumor of the uterus occurring in females of reproductive age group and are derived from smooth muscle. Degenerative changes are usual in leiomyoma. There is a wide spectrum of morphological patterns in leiomyomas, among them 90% of leiomyomas are the conventional type or usual type. Leiomyoma with bizarre nuclei is an unusual variant of uterine leiomyoma with presence of marked nuclear atypia and pleomorphism. However, there is low mitotic activity (&amp;#60; 5 mitoses/10 high power fields), absence of tumor cell necrosis and intermixed normal spindled smooth muscle cells. The present study describes histopathological analysis of 4 cases of leiomyoma with bizarre nuclei. All these cases underwent abdominal hysterectomy for leiomyomas in the uterus. The age range was 47 to 52 years. On gross examination the cases had well-defined masses with grey white, whorled areas and microscopic impression was leiomyoma with bizarre nuclei. These cases had low mitotic activity ranging from 1 to 3/ 10 high power fields and absence of tumor necrosis. Ki 67 index was 0.5 -1.0%. Leiomyoma with bizarre nuclei can create a diagnostic dilemma due to marked nuclear atypia, multinucleated tumor cells and karyorrhectic cells resembling mitotic figures. Hence it is important to rule out more aggressive and malignant mesenchymal tumors.

Profilin1 is required for prevention of mitotic catastrophe in murine and human glomerular diseases.

The progression of proteinuric kidney diseases is associated with podocyte loss, but the mechanisms underlying this process remain unclear. Podocytes reenter the cell cycle to repair double-stranded DNA breaks. However, unsuccessful repair can result in podocytes crossing the G1/S checkpoint and undergoing abortive cytokinesis. In this study, we identified Pfn1 as indispensable in maintaining glomerular integrity - its tissue-specific loss in mouse podocytes resulted in severe proteinuria and kidney failure. Our results suggest that this phenotype is due to podocyte mitotic catastrophe (MC), characterized histologically and ultrastructurally by abundant multinucleated cells, irregular nuclei, and mitotic spindles. Podocyte cell cycle reentry was identified using FUCCI2aR mice, and we observed altered expression of cell-cycle associated proteins, such as p21, p53, cyclin B1, and cyclin D1. Podocyte-specific translating ribosome affinity purification and RNA-Seq revealed the downregulation of ribosomal RNA-processing 8 (Rrp8). Overexpression of Rrp8 in Pfn1-KO podocytes partially rescued the phenotype in vitro. Clinical and ultrastructural tomographic analysis of patients with diverse proteinuric kidney diseases further validated the presence of MC podocytes and reduction in podocyte PFN1 expression within kidney tissues. These results suggest that profilin1 is essential in regulating the podocyte cell cycle and its disruption leads to MC and subsequent podocyte loss.

Tissue-specific accumulation, depuration and histopathological effects of 3,6-dichlorocarbazole and 2,7-dibromocarbazole in adult zebrafish (Danio rerio)

Although polyhalogenated carbazoles have been detected with increasing frequency in aquatic ecosystems, their bioaccumulation in fish and corresponding pathological effects related to bioaccumulation are still unclear. Here, we investigated the tissue-specific accumulation, depuration, and histopathological effects of two typical PHCZs, 3,6-dichlorocarbazole (36-CCZ) and 2,7-dibromocarbazole (27-BCZ), in adult zebrafish at three levels (0, 0.15 μg/L (5 × environmentally relevant level), and 50 μg/L (1/10 LC50). The lowest concentrations of 36-CCZ (1.2 μg/g ww) and 27-BCZ (1.4 μg/g ww) were observed in muscle, and the greatest concentrations of 36-CCZ (3.6 μg/g ww) and 27-BCZ (4 μg/g ww) were detected in intestine among the tested tissues. BCFww of 36-CCZ and 27-BCZ in zebrafish ranged from 172.9 (muscle) to 606.6 (intestine) and 285.2 (muscle) to 987.5 (intestine), respectively, indicating that both 36-CCZ and 27-BCZ have high potential of bioaccumulation in aquatic system. The 0.15 μg/L level of 36-CCZ or 27-BCZ caused lipid accumulation in liver, while 50 μg/L of 36-CCZ or 27-BCZ induced liver lesions such as fibrous septa, cytolysis, and nuclear dissolution. Brain damage such as multinucleated cells and nuclear solidification were only observed at 50 μg/L of 27-BCZ. This study provided valuable information in assessing the health and ecological risks of 36-CCZ and 27-BCZ.

Bcl2l1 Deficiency in Osteoblasts Reduces the Trabecular Bone Due to Enhanced Osteoclastogenesis Likely through Osteoblast Apoptosis.

Bcl2l1 (Bcl-XL) belongs to the Bcl-2 family, Bcl2 and Bcl2-XL are major anti-apoptotic proteins, and the apoptosis of osteoblasts is a key event for bone homeostasis. As the functions of Bcl2l1 in osteoblasts and bone homeostasis remain unclear, we generated osteoblast-specific Bcl2l1-deficient (Bcl2l1fl/flCre) mice using 2.3-kb Col1a1 Cre. Trabecular bone volume and the trabecular number were lower in Bcl2l1fl/flCre mice of both sexes than in Bcl2l1fl/fl mice. In bone histomorphometric analysis, osteoclast parameters were increased in Bcl2l1fl/flCre mice, whereas osteoblast parameters and the bone formation rate were similar to those in Bcl2l1fl/fl mice. TUNEL-positive osteoblastic cells and serum TRAP5b levels were increased in Bcl2l1fl/flCre mice. The deletion of Bcl2l1 in osteoblasts induced Tnfsf11 expression, whereas the overexpression of Bcl-XL had no effect. In a co-culture of Bcl2l1-deficient primary osteoblasts and wild-type bone-marrow-derived monocyte/macrophage lineage cells, the numbers of multinucleated TRAP-positive cells and resorption pits increased. Furthermore, serum deprivation or the deletion of Bcl2l1 in primary osteoblasts increased apoptosis and ATP levels in the medium. Therefore, the reduction in trabecular bone in Bcl2l1fl/flCre mice may be due to enhanced bone resorption through osteoblast apoptosis and the release of ATP from apoptotic osteoblasts, and Bcl2l1 may inhibit bone resorption by preventing osteoblast apoptosis.

Bona fide dendritic cells are pivotal precursors for osteoclasts

ObjectivesOsteoclasts (OCs) are myeloid-derived multinucleated cells uniquely able to degrade bone. However, the exact nature of their myeloid precursors is not yet defined.MethodsCD11c-diphtheria toxin receptor (CD11cDTR) transgenic mice were treated...

“Keep on rolling”: circulating cells in a botryllid ascidian torpor

Hemocytes of the colonial tunicate Botrylloides leachii play important roles throughout the animal’s life span, including transport and storage of nutrients, respiration, regeneration, budding, sexual reproduction, defense responses, and tunic generation. Nevertheless, very little is known about the involvement of hemocytes or their functions in the remarkable torpor phenomenon characteristic of this species. Changes in water temperature result in rapid and dramatic morphological changes in which the entire colony degenerates, leaving remnants devoid of feeding and reproductive organs that form compacted and opaque masses of lacunae filled with hemocyte cells. Here, we study hemocyte populations in active and hibernating colonies at the morphological, cellular, and molecular levels using histological and transmission electron microscopy observations, primary cell culture observations, and single-cell transcriptomics. This study defines and captures the different cell types in torpor and control stages and further highlights torpor-associated cell types. Multinucleated cells (MNCs) appear only in torpor stages and under in vitro conditions and are most likely formed from cell fusions. Bacteria-carrying phagocytes are cells specific to a torpor state with yet unknown function. Single-cell sequencing analysis revealed 14 transcriptionally distinct cell clusters. The hibernating colonies had a low throughput of cells, yet all but two transcriptional clusters were present in hibernating colonies. A comparison of gene expressions in the same cell clusters revealed torpor-specific transcriptional modalities in seven of the cell types. Single-cell sequencing generates an enormous amount of valuable data that can serve researchers in future studies of ascidians and torpor phenomena and provide opportunities for future meta-analysis studies. However, the technical challenges create bottlenecks for the full exploitation of single-cell RNA sequencing (scRNA-seq) data. We developed an interactive, searchable, and intuitive cloud-based database where researchers can easily explore the single-cell transcriptomics data generated by us from active and torpid B. leachii colonies. The data can be interrogated and downloaded for further analysis and comparative studies. The Dashboard is available at: http://bleachii.tauberbioinformatics.org/seurat?id=Bleachii-v1. By elucidating hemocyte populations during hibernation, these results provide the basis for future studies of hibernation at the cellular and molecular levels in B. leachii and comparative studies of hibernation phenomena in other organisms.

Progeny counter mechanism in malaria parasites is linked to extracellular resources.

Malaria is caused by the rapid proliferation of Plasmodium parasites in patients and disease severity correlates with the number of infected red blood cells in circulation. Parasite multiplication within red blood cells is called schizogony and occurs through an atypical multinucleated cell division mode. The mechanisms regulating the number of daughter cells produced by a single progenitor are poorly understood. We investigated underlying regulatory principles by quantifying nuclear multiplication dynamics in Plasmodium falciparum and knowlesi using super-resolution time-lapse microscopy. This confirmed that the number of daughter cells was consistent with a model in which a counter mechanism regulates multiplication yet incompatible with a timer mechanism. P. falciparum cell volume at the start of nuclear division correlated with the final number of daughter cells. As schizogony progressed, the nucleocytoplasmic volume ratio, which has been found to be constant in all eukaryotes characterized so far, increased significantly, possibly to accommodate the exponentially multiplying nuclei. Depleting nutrients by dilution of culture medium caused parasites to produce fewer merozoites and reduced proliferation but did not affect cell volume or total nuclear volume at the end of schizogony. Our findings suggest that the counter mechanism implicated in malaria parasite proliferation integrates extracellular resource status to modify progeny number during blood stage infection.

Serum calprotectin correlates more strongly with inflammation and disease activity in ACPA positive than ACPA negative rheumatoid arthritis.

The aim of the present study was to evaluate performance of serum and synovial fluid levels of the granulocyte protein calprotectin as inflammatory biomarker in rheumatoid arthritis (RA) patients with knee synovitis. 76 RA patients with ongoing knee synovitis were included. Data on disease activity score with 28 joints and their subcomponents and radiological destruction of the affected knee were collected. White blood cell count, C-reactive protein, anti-citrullinated peptide antibodies (ACPA) against cyclic citrullinated peptide version 2 (anti-CCP2), IgM rheumatoid factor (RF) and calprotectin were analysed in parallel in circulation and in synovial fluid (SF). Counts of polynuclear and mononuclear cells were measured in SF. Serum (S) calprotectin correlated stronger than SF-calprotectin with inflammatory markers and disease activity. Instead, SF-calprotectin showed a strong correlation to SF counts of white blood cells, and especially to polymorphonuclear cell counts (Spearman's rho = 0.72, p< 0.001). S-calprotectin showed markedly stronger correlation with inflammatory markers and disease activity in ACPA positive as compared with ACPA negative RA patients; a similar difference was observed for patients with and without IgM RF. The particularly strong association between circulating calprotectin and inflammation in ACPA positive RA is a new argument for a specific role for polymorphonuclear granulocytes/neutrophils in this RA subset. Measurement of calprotectin in SF does not convey any additional benefit compared with measurement in the circulation in RA patients with knee synovitis.

Evaluating the antioxidant potential of resveratrol-gold nanoparticles in preventing oxidative stress in endothelium on a chip

Vascular endothelial cells play a vital role in the health and maintenance of vascular homeostasis, but hyperglycemia disrupts their function by increasing cellular oxidative stress. Resveratrol, a plant polyphenol, possesses antioxidant properties that can mitigate oxidative stress. Addressing the challenges of its limited solubility and stability, gold nanoparticles (GNps) were utilized as carriers. A microfluidic chip (MFC) with dynamic flow conditions was designed to simulate body vessels and to investigate the antioxidant properties of resveratrol gold nanoparticles (RGNps), citrate gold nanoparticles (CGNps), and free Resveratrol on human umbilical vein endothelial cells (HUVEC). The 2, 2-diphenyl-1-picrylhydrazyl (DPPH) assay was employed to measure the extracellular antioxidant potential, and cell viability was determined using the Alamar Blue test. For assessing intracellular oxidative stress, the 2′,7′-dichlorodihydrofluorescein diacetate (DCFH-DA) assay was conducted, and results from both the cell culture plate and MFC were compared. Free Resveratrol demonstrated peak DPPH scavenging activity but had a cell viability of about 24–35%. RGNPs, both 3.0 ± 0.5 nm and 20.2 ± 4.7 nm, consistently showed high cell viability (more than about 90%) across tested concentrations. Notably, RGNPs (20 nm) exhibited antioxidative properties through DPPH scavenging activity (%) in the range of approximately 38–86% which was greater than that of CGNps at about 21–32%. In the MFC,the DCFH-DA analysis indicated that RGNPs (20 nm) reduced cellular oxidative stress by 57–82%, surpassing both CGNps and free Resveratrol. Morphologically, cells in the MFC presented superior structure compared to those in traditional cell culture plates, and the induction of hyperglycemia successfully led to the formation of multinucleated variant endothelial cells (MVECs). The MFC provides a distinct advantage in observing cell morphology and inducing endothelial cell dysfunction. RGNps have demonstrated significant potential in alleviating oxidative stress and preventing endothelial cell disorders.

Possible mechanisms of phlebitis-like abnormal reaction (PLAR) after cyanoacrylate obliteration of varicose veins

Based on their own research and a review of the literature, the authors analyze the possible cellular mechanisms of the development of an inflammatory reaction after the obliteration of varicose veins with cyanoarylate adhesive compounds (CAO), which received the name phlebitis- Like abnormal Reaction (PLAR) in foreign sources. Despite the existing opinion about the “abnormal” nature of the inflammatory reaction, it is noted that the main stages of its development are fully consistent with the currently known molecular and cellular mechanisms of the response of biological tissues to contact with a foreign antigenic substance and are of a natural nature. The cause of the development of acute alterative inflammation in the vein wall is the direct contact of the endothelium with an aggressive environment, which is cyanoacrylate. A specific feature of the development of chronic inflammation in the vein wall is its productive interdaily character, which is replaced by proliferative processes. The main role in the development of successive stages of PLAR development is played by monocytic, mast and giant cells of foreign bodies, as well as the mechanisms underlying the regulation of the functional activity of these cells. During the period of cyanoacrylate biodegradation, its cellular environment corresponds to all morphological features of a phagocytoma, whose activity decreases with the biodegradation of cyanoacrylate with simultaneous connective tissue proliferation. The development of possible chronic granulomatous inflammation is based on a local autoimmune process associated with the formation of giant multinucleated epithelioid cells (Langerhans cells). In conclusion, it is emphasized that today, when using various cyanoacrylate compounds for the purpose of adhesive obliteration of veins, taking into account the accumulated clinical data and morphological studies, the final answers to the existing reasonable objections about the complete safety of the use of cyanoacrylates in clinical practice should be given by fundamental immunohistochemical and genetic studies.

Isolation and Maintenance in Culture of Primary Human Trophoblast from Term Placentae.

Trophoblasts are placenta-specific epithelial cells that play an essential role in conducting nutrient, gas, and waste exchange between the fetus and the mother. Primary culture of human trophoblasts from donated term placentae is an important tool to study placental functions. Currently, there is a lack of general consensus of the optimal culture conditions for maintaining term trophoblast cells in vitro. A key problem with culturing trophoblasts from term placentae is overgrowth of the trophoblasts by rapidly proliferating cellular contaminants. Recently we reported a system to culture trophoblasts from term placentae which differentiate into syncytiotrophoblast-like multinucleated cells that can be maintained in culture for at least 30days with minimal contamination. This chapter details our optimized approach for long-term, contaminant-free in vitro culture of primary trophoblasts from term placentae.

In Vitro Analysis of Bone Remodeling Alterations in Hemophilia: Influence of Coagulation Factors on Bone Cells

Background. Today, one of the main comorbidities affecting the quality of life of the haemophilia patient is bone pathology. Hemophilia A (HA) is associated with reduced bone mass and mineral density (BMD). Due to the rarity of the disease and the heterogeneity among the studies, pathogenesis of bone loss is still under investigation and both processes of bone resorption and bone formation could be hypothesized to be altered. FVIII deficiency could alter BMD directly affecting bone cells or indirectly by decreasing thrombin generation. The reduction of BMD seems due to perturbations of the Receptor Activator of Nuclear factor-B RANK Ligand (RANKL) and osteoprotegerin (OPG) pathways. In haemophilia alterations of bone remodeling, osteoclasts seem to play a major role. Osteoclasts are multinucleated cells, which derive from the CD14+ monocyte/macrophage lineage. Until recently, the identity of osteoclast progenitors has not been well defined, but evidences report that CD16−CD14+ rather than CD16+CD14+ monocytes were prone to differentiate into osteoclasts (Komano et al. Arthritis Res Ther. 2006). Objectives. To dissect the mechanism of bone loss in hemophilia, we studied the effects of coagulation factors, such as factor VIII, von Willebrand factor (VWF), activated factor X (FXa) and thrombin, on both osteoclasts and osteoblasts biology and we characterized the osteoclastogenic potential of HA patients' osteoclast precursors. Patients/Methods: Peripheral Blood Mononuclear Cells (PBMC) isolated from healthy donors were induced to differentiate into osteoclasts and treated with plasma derived VWF/FVIII complex, human rVWF, human full length rFVIII, activated FX and thrombin. Osteoclastogenesis and expression analysis of RANK, TRAF6 (TNF receptor-associated factor 6), TCIRG1 (T Cell Immune Regulator 1) and osteoclasts protease CTSK (cathepsin K) expression were assessed. Moreover, in vitro assays assessed the osteoclastogenic potential of PBMC isolated from different hemophilic A patients. FACS analyses of osteoclast precursors isolated from patients were performed. Osteoblasts differentiation, mineralization and genes expression (Alkaline Phosphatase and COL1A2) were performed in the presence of aforementioned coagulation factors. Results. We showed a significant reduction of mature osteoclasts after treatment of HD-PBMC with FVIII, VWF, FVIII/VWF, FXa and thrombin. VWF appears to play a major role to regulate osteoclast differentation from healthy donor-derived PBMC. Indeed, it inhibits by itself ~45% the osteoclastogenesis comparable to OPG, and even more if is complexed with FVIII (53% inhibition). About 50% and 70% reduced levels of osteoclast differentiation were also revealed following treatment with FXa and thrombin, respectively. Interestingly, PBMC from HA patients showed increased ability to form mature osteoclasts compared to those obtained from healthy controls. Osteoclast precursors (CD16−CD14+CD11b+) are significantly higher in HA patients than age and sex matched controls. Moreover, transcriptional analysis revealed increased RANK, TRAF6, CTSK and TCIRG1 genes expression in adult hemophilia patient's osteoclasts compared to matched controls. FVIII and VWF treatments led also to a statistically significant reduction of ALP positivity in control osteoblasts; opposite effect was shown following thrombin treatment. Conclusions. All these data support that bone loss observed in haemophilia patients could be related to increased osteoclast formation and activity and that coagulation factors directly impact on bone cells. These results may have important implications in the clinical management of hemophilic patients to prevent bleeding as well as to preserve bone health.

Sarcomatoid mesothelioma diagnosed in a patient with mesothelioma in situ: a case report on morphologic differences after 9-month interval with details analysis of cytology in early-stage mesothelioma

BackgroundOverlapping morphological features of mesothelial cells have been rendered it difficult to distinguish between reactive and malignant conditions. The development of methods based on detecting genomic abnormalities using immunohistochemistry and fluorescence in situ hybridization have contributed markedly to solving this problem. It is important to identify bland mesothelioma cells on cytological screening, perform efficient genomic-based testing, and diagnose mesothelioma, because the first clinical manifestation of pleural mesothelioma is pleural effusion, which is the first sample available for pathological diagnosis. However, certain diagnostic aspects remain challenging even for experts.Case presentationThis report describes a case of a 72-year-old man with a history of asbestos exposure who presented with pleural effusion as the first symptom and was eventually diagnosed as mesothelioma. Mesothelioma was suspected owing to prominent cell-in-cell engulfment in mesothelial cells on the first cytological sample, and the diagnosis of mesothelioma in situ was confirmed by histology. Unexpectedly, sarcomatoid morphology of mesothelioma was found in the second pathology samples 9 months after the first pathological examination. Both the mesothelioma in situ and invasive lesion showed immunohistochemical loss of methylthioadenosine phosphorylase (MTAP) and homozygous deletion of cyclin dependent kinase inhibitor 2A (CDKN2A) on fluorescence in situ hybridization. The patient received medication therapy but died of disease progression 12 months after the diagnosis of the sarcomatoid morphology of mesothelioma.ConclusionOur case suggests that cell-in-cell engulfment can be conspicuous in early-stage mesothelioma with inconspicuous nuclear atypia and few multinucleated cells. In addition, the presence of MTAP loss and CDKN2A homozygous deletion are suspected to be involved in early formation to invasive lesions and/or sarcomatoid morphology. We believe that it is important to consider genetic abnormalities when deciding on individual patient management. Furthermore, cases of mesothelioma, even those of an in situ lesion, with MTAP loss and/or CDKN2A deletion should be carefully followed up or subjected to early treatment.

Dysfunction of poly (ADP-ribose) glycohydrolase suppresses osteoclast differentiation in RANKL-stimulated RAW264 cells

Poly (ADP-ribose) glycohydrolase (PARG) is an enzyme that mainly degrades poly (ADP-ribose) (PAR) synthesized by poly (ADP-ribose) polymerase (PARP) family proteins. Although PARG is involved in many biological phenomena, including DNA repair, cell differentiation, and cell death, little is known about the relationship between osteoclast differentiation and PARG. It has also not been clarified whether PARG is a valuable target for therapeutic agents in the excessive activity of osteoclast-related bone diseases such as osteoporosis. In the present study, we examined the effects of PARG inhibitor PDD00017273 on osteoclast differentiation in RANKL-induced RAW264 cells. PDD00017273 induced the accumulation of intracellular PAR and suppressed the number of tartrate-resistant acid phosphatase (TRAP)-positive multinucleated cells. PDD00017273 also downregulated osteoclast differentiation marker genes such as Trap, cathepsin K (Ctsk), and dendrocyte expressed seven transmembrane protein (Dcstamp) and protein expression of nuclear factor of activated T cells 1 (NFATc1), a master regulator of osteoclast differentiation. Taken together, our findings suggest that dysfunction of PARG suppresses osteoclast differentiation via the PAR accumulation and partial inactivation of the NFATc1.