Reticulin Fibers Research Articles

Mode of Ossification and Extracellular Fiber Characterization of Osteoderm Matrix in the American Alligator (Alligator mississippiensis) and Comparisons to Inherited and Acquired Heterotopic Ossification Disorders

Osteoderms are bones that form in the dermis of several reptilian species and have been a source of scientific curiosity since at least the 1800s. Though there are several hypotheses for their function, a consensus has not yet emerged. The function of osteoderms is further obscured, since there is little histomorphological information on these structures. Indeed, understanding the cellular and molecular events that initiate and drive osteoderm development may clarify the functional significance of these structures. Dubansky & Dubansky (2018) conducted the first histological description of the development of the osteoderm in the American alligator, and identified discrete developmental stages based on the organization of collagen fibers and contributing cell mediators, as well as the degree of mineralization of the matrix. However, questions remain regarding the type of collagen and the identity of the cell precursors that are involved at different stages, as well as the existence of a hyaline cartilage model. In this study, differential staining techniques characterized changes in the fibrous component of the developing matrix of the osteoderm, and investigated whether ossification proceeds via an endochondral mechanism. Early stage osteoderms exhibit an extensive reticulin (type III collagen) fiber scaffold, which is replaced by disorganized, thick type I collagen fibers in mineralizing maturing matrix, and highly organized, thin type I collagen at the proliferating edge of the matrix. Despite morphological similarities to cartilage in some areas, staining showed that hyaline cartilage was absent and, therefore, the mode of ossification more resembled intramembranous ossification, than endochondral ossification. Osteoderm development resembles Heterotopic Ossification (HO) disorders in humans and is being used as a model to study several forms of this pathology. Based on this study, osteoderm histomorphology most closely resembles the inherited disorder Progressive Osseous Heteroplasia, in which pathological bone formation is restricted to the dermis and superficial fascia and proceeds via intramembranous ossification. Documenting similarities and differences among the various types of HO lesions and osteoderms may provide insights into the disease progression and future therapeutic strategies.Support or Funding InformationThis research was supported by a Faculty‐Student Research and Creative Activity Internal Grant from Tarleton State University, Office of Research and Innovation.

Association of mast cells and bone marrow reticulin fibrosis in patients with bcr-abl negative chronic myeloproliferative neoplasms

BackgroundWe aimed to investigate the association of bone marrow mast cell numbers (MCN) and the degree of reticulin fibrosis in patients with chronic myeloproliferative neoplasms (MPN). MethodsThis was a case-control study that recruited 47 patients who were diagnosed with bcr-abl negative MPN. Thirty patients with lymphoma served as controls. JAK2 mutation was studied and all subjects underwent bone marrow biopsy at the time of diagnosis. Mast and CD34+ cells were counted. Marrow reticulin fiber was graded. ResultsThirty-four patients had essential thrombocythemia (ET), 8 patients had primary myelofibrosis (PMF) and 5 patients had polycythemia vera (PV). Fourteen MPN patients had JAK2, whereas the controls had not. MCN was higher in patients than controls (p = 0.001). There was no significant difference regarding CD34. Reticulin fibrosis was present in 57.4% of MPN patients, whereas there was any in controls. PMF patients had more CD34 + cells than PV and ET. PMF patients had more reticulin fibers compared with other subgroups (p < 0.001). MCN, but not CD34+ cell counts, was significantly higher in JAK2(+) patients than JAK2(−) patients. ConclusionMCN and reticulin fibrosis were significantly increased in MPN patients. JAK2 positivity had significantly increased MCN compared to patients without JAK2. JAK2 was associated with increased reticulin fibrosis.

Depletion of Neoplastic CD11b Positive Cells in Jak2V617F Mutant Mice Reduced Fibrocytes in Bone Marrow and Improved Myelofibrosis

Myelofibrosis (MF) associated with myeloproliferative neoplasms (MPN) has been considered to be a reactive phenomenon caused by mesenchymal stromal cells (MSCs) stimulated by cytokines such as TGFb-1 overproduced by neoplastic megakaryocytes (MKs) and platelets. TGFb-1 stimulates non-neoplastic mesenchymal cells to produce collagen and fibronectin and to induces bone marrow (BM) fibrosis. However, the involvement of neoplastic fibrocyte in MF has recently been reported (Verstovsek et al. JEM 2016), and among blood cells, monocytes in particular are considered to be the main source of neoplastic fibrocytes. In this study, we assesed the role of neoplastic fibrocytes using a mouse model of MPN induced by Jak2V617F (Shide et al. Leukemia 2008). First, the distribution of neoplastic fibrocyte in the BM of Jak2V617F transgenic (TG) mice was examined. We transplanted wild-type (WT) or Jak2V617F TG cells (B6-CD45.2), together with WT BM cells (B6-CD45.1) into irradiated WT recipient mice (B6-CD45.1). Only recipient mice transplanted with a mixture of Jak2V617F cells and WT cells developed BM fibrosis. In immunofluorescent staining of fibrotic BM, cells expressing the fibrocyte marker CD45/Collagen-1(Col-1) were observed much more than cells expressing the fibroblast marker CD90(usually positive for MSCs)/Col-1. As for CD45/Col-1 positive cells, cells expressing CD45.2/Col-1 were much more than cells expressing CD45.1/Col-1, clearly indicating that these cells were derived from Jak2V617F mutant blood cells. On the other hand, in the BM of recipient mice transplanted with control WT cells, few cells expressing CD45/Col-1 or CD90/Col-1 were present. To examine the differentiation ability of Jak2V617F blood cells to fibrocytes directly, peripheral blood (PB) mononuclear cells (MNC) of Jak2V617F mice or WT mice were cultured in vitro. After 5 days of culture, PB MNCs from Jak2V617F mice differentiated into mature fibrocytes exhibiting a long spindle shape with Col-1 expression. On the other hand, there were very few fibrocytes differentiated from PB MNC from WT mice. Next, we depleted monocytes, the main source of fibrocytes, and observed its effects on BM fibrosis in vivo. Jak2V617F TG mice were mated with CD11b-diphtheria toxin receptor (DTR) TG mice (Duffield et al. JCI 2005) to obtain Jak2V617F/CD11b-DTR double TG mice. Mice transplanted with BM cells from Jak2V617F/CD11b-DTR double TG mice (hereinafter called Jak2V617F/CD11b-DTR mice) exhibit leukocytosis, thrombocytosis, anemia, splenomegaly, and BM fibrosis with increased megakaryocytes. Jak2V617F/CD11b-DTR mice was administered diphtheria toxin (DT) intraperitoneally to deplete monocytes. One day after DT administration, the number of PB monocytes (CD11b+/F4/80+) drastically decreased in Jak2V617F/CD11b-DTR mice, and the reduction of monocyte was maintained by every-other-day DT administration. After 8 weeks DT treatment, mice were sacrificed and analyzed. As a control group, Jak2V617F/CD11b-DTR mice treated with PBS were examined. DT treatment drastically decreased the number of neoplastic fibrocytes expressing CD45.2/Col-1 in BM and spleen of Jak2V617F/CD11b-DTR mice compared with control mice treated with PBS. Consistently, reticulin fibers were eliminated almost completely and collagen fibers almost fully disappeared in BM, which led to a reversal of the decrease in BM cellularity, although the number of MKs was not affected. Similar findings were observed in the spleen, although not completely. Plasma TGF-b1 level were about 2-fold higher in Jak2V617F/CD11b-DTR mice than in WT mice. Neoplastic monocyte depletion significantly decreased TGF-b1 level. Since MK numbers did not change, this indicates that fibrocytes are one of the main sources of TGF-b1. In other features of MF in Jak2V617F/CD11b-DTR mice, splenomegaly was ameliorated by DT treatment. Microscopic analysis revealed an improvement in the damaged spleen architecture and the disappearance of splenic fibrosis. In summary, most collagen-producing cells in BM were neoplastic fibrocytes in Jak2V617F-induced MPN, indicating that neoplastic fibrocytes played an essential role and mesenchymal fibroblasts had a minor contribution in fibrosis in MPN. Depletion of neoplastic monocytes also improved splenomegaly as well as BM fibrosis in mice, and this cell fraction could be a promising therapeutic target. Disclosures No relevant conflicts of interest to declare.

Disease Phenotype and Clonal Amplification in Calreticulin del52 and ins5 Knock-in Mice Are Dependent on the Type of Mutations and Gene Dosage

Introduction BCR-ABL-negative myeloproliferative neoplasms (MPNs) result from the transformation of a hematopoietic stem cell (HSC). Somatic mutations in the calreticulin (CALR) gene are associated with approximately 30% of essential thrombocythemia (ET) and primary myelofibrosis (PMF). All CALR mutations induce a frameshift to the same alternative reading frame generating a new C-terminal tail. The two most frequent CALR mutations are a 52 bp deletion (del52) or type 1 and a 5 bp insertion (ins5) or type 2. In patients, del52 and ins5 are equally found in ET but del52 is more frequent in PMF. In mouse retroviral model, del52 mice progress from ET to myelofibrosis (MF) while ins5 mice remain mostly with an ET. Methods In order to study the effect of endogenous levels of del52 and ins5 in hematopoiesis, we generated conditional knock-in (KI) mice expressing the murine CALR del52 or ins5 with the human mutated C-terminal tail under the control of a Scl-driven tamoxifen-inducible Cre recombinase (Scl-CreERT). We have also used Ubi-GFP transgenic mice to perform competitive engraftments. Results After tamoxifen-induction, both del52 and ins5 KI mice developed a rapid thrombocytosis, more severe in the homozygous than the heterozygous setting. In contrast, leukocytosis was observed only in homozygous setting. At similar zygosity, del52 induced a higher thrombocytosis compared to ins5. After 10 months of induction, both the bone marrow (BM) and the spleen of homozygous del52 KI mice and, to a much lower extent of homozygous ins5 KI mice, presented a significant increase in megakaryocytes (MKs) and in MK progenitors by flow cytometry. Von Willebrand factor staining showed that both del52 and ins5 homozygous mice displayed giant polylobulated MKs, associated with a similar increase in ploidy (mean ploidy 32N-33N). Heterozygous del52 presented also an increase ploidy of MK (mean of 25N) compared to controls (mean of 17N), whereas the MK ploidy of heterozygous ins5 mice was similar to control mice. The increase in number and size of MKs in homozygous del52 mice partially explained the significant decrease in BM cellularity and the splenomegaly. Moreover, we observed a decrease in BM erythroblasts and, in spleen, an increase in both erythroblast and granulocytic precursors together with a decrease in lymphocytes associated with a major disorganization of white pulp territories. Thus, the del52 homozygous KI mice developed features of a MF-like disease further illustrated by the presence of reticulin fibers stained with silver, mainly in spleen. Presence of fibrosis was not as pronounced in heterozygous del52 mice and more rarely observed in spleen of homozygous ins5 KI mice. In homozygous del52 KI mice, there was a significant amplification of the HSC compartment in both BM and spleen that was stronger than in homozygous ins5 mice. To study whether del52 and ins5 could provide a competitive advantage to HSCs, we performed BM transplantation with increasing percentages of non-induced homozygous del52 or ins5 cells with wild-type GFP+ cells into lethally-irradiated recipient mice. The homozygous del52 BM cells strongly competed wild-type hematopoiesis from an initial engraftment as low as 10% of mutated clones, reaching 100% in both blood myeloid cells and BM HSC compartments at 4 months. In contrast, out-competition of wild-type hematopoiesis by homozygous ins5 cells was slower, especially when less than 50% of mutated cells were initially engrafted suggesting that del52 provides a stronger advantage to the HSCs than ins5. Conclusion In conclusion, these results demonstrate that modeling CALRdel52 and ins5 mutations in mice can successfully recapitulate the differences in phenotype observed in patients, i.e del52 KI mice recapitulate an ET progressing to MF while ins5 KI mice only mimic an ET. This might be explained by a more profound effect of del52 than ins5 at the level of HSC. These KI mice offer solid in vivo models to investigate the mechanism of action of both types of mutations on HSCs and MKs and will be used to test new therapeutic approaches. Disclosures: No relevant conflicts of interest to declare. Disclosures Constantinescu: AlsaTech: Other: Co-Founde; Wiley &amp; Sons: Other: Editor in Chief, Journal of Cellular and Molecular Medicine; Novartis: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; AgenDix GmbH: Other: Co-Founder, MyeloPro Research and Diagnostics.

Chitinase 3-like 1 Produced By Fibrocytes Promotes Myelofibrosis

Introduction Monocyte-derived fibrocytes are spindle-shaped fibroblast-like blood cells derived from a sub-population of CD14+ monocytes. Recent studies have shown that fibrocytes play a key role in the pathogenesis of primary myelofibrosis (MF), and these cells have attracted attention as novel therapeutic targets for MF treatment. Using a Romiplostim (Rom)-induced murine MF model, we have previously reported that fibrocyte differentiation, which is directly induced by activating the thorombopoietin receptor signaling pathway, can potentially trigger the progression of MF (Leukemia 2017; 31: 2709). However, the precise molecular mechanism of fibrocyte activity in MF have not been elucidated. To address this question, we compared the RNA sequencing-based gene expression profiles of monocytes and fibrocytes and detected that fibrocytes exhibited a higher level of Chitinase 3-like 1 (CHI3L1) expression than monocytes. CHI3L1, an 18-glycosyl hydrolase-related molecule, is a member of the enzymatically inactive chitinase-like protein family. These proteins are associated with inflammation, tissue remodeling, and organ fibrosis. Therefore, we hypothesized that CHI3L1 produced by fibrocytes is potentially involved in the progression of MF and could be a therapeutic target. In the present study, we evaluated CHI3L1 using serum samples obtained from patients with myeloproliferative neoplasms (MPN) and a Rom-induced murine MF model. Methods Monocytes and fibrocytes (Day 9 and Day 12) were prepared using peripheral blood samples from healthy volunteers. Following total RNA extraction, RNA qualification, and library preparation, a Hiseq4000 (Illumina) system was used for RNA sequencing. ELISA-based analyses were performed using the supernatant of the blood cells to confirm the presence of the CHI3L1 protein. Furthermore, we determined the serum CHI3L1 levels of MPN patients with or without MF using ELISA-based analyses. Overall, 21 patients without MF and 31 patients with MF (MF-1 to MF-3) were enrolled after obtaining written informed consent. Using the Rom-induced murine MF model, we performed in vivo analyses of Chi3l1. Female C57BL/6J mice (8-week-old) were administered with 1 mg/kg Rom once a week for 2 or 3 weeks, following which serum samples, spleens, and bone marrow samples were examined. To eliminate fibrocytes, clodronate liposomes were administered to mice. Chi3l1-deficient (Chi3l1−/−) mice were administered with Rom in the same manner. Following these treatments, RT-PCR and histopathological analyses of bone marrow were conducted. Non-contact co-culture assays of human fibrocytes and fibroblasts were performed using the human fibroblast cell line HS-5 to evaluate the interaction between CHI3L1 and fibroblasts. HS-5 cells were cultured using the supernatant of fibrocytes with or without the neutralizing antibody for CHI3L1 and mRNA expression of HS-5 cells was examined. Results Comparing the RNA sequencing gene expression profiles of monocytes and fibrocytes revealed a significant increase in CHI3L1 expression associated with fibrocyte differentiation. ELISA-based analyses of human monocyte and fibrocyte supernatants confirmed that the secretion of CHI3l1 protein concurred with the amount of mRNA expression (P &lt; 0.01) (a). We demonstrated that CHI3L1 levels were elevated in in serum samples obtained from MPN patients with MF (N = 31) compared with those obtained from patients without MF (N = 21; P = 0.027) (b). High levels of Chi3l1 mRNA expression were observed in the bone marrow of the Rom-induced murine MF model (P &lt; 0.01), which was abrogated by clodronate treatment. Furthermore, compared with wild-type mice, Chi3l1−/− mice showed fewer reticulin fibers in silver-stained bone marrow sections (c) and significantly decreased Col3A1 and Acta2 mRNA expression in the bone marrow (P &lt; 0.05). In addition, co-culture assays demonstrated that HS-5 cells cultured with the supernatant of fibrocytes showed significantly higher levels of COL1A1 and COL3A1 expression (P &lt; 0.05) than that of monocytes and the addition of CHI3L1 neutralizing antibody abrogated these increases (d). Conclusion CHI3L1 is involved in the interaction between fibrocytes and fibroblasts and this molecule exacerbates the progression of bone marrow fibrosis by promoting the production of the extracellular matrix. Therefore, CHI3L1 could be a potential therapeutic target for MF treatment. Figure Disclosures Makishima: Bristol-Myers Squibb: Research Funding. Komatsu:Wako Pure Chemical Industries, Ltd.: Research Funding; Fuso Pharmaceutical Industries, Ltd.: Research Funding; Takeda Pharmaceutical Company Limited: Research Funding, Speakers Bureau; Pharma Essentia: Research Funding, Speakers Bureau; Novartis K.K: Speakers Bureau. Kimura:JSPS KAKENHI: Research Funding.

Progressive Diagnosis of Chronic Myeloproliferative Neoplasms: A Case Study

Chronic myeloproliferative neoplasms are hematologic diseases characterized by the clonal proliferation of peripheral blood components. A series of laboratory tests are required for diagnosis. This patient is a 74-year-old male incidentally noted to have a leukocytosis, monocytosis, and rare blasts in his peripheral blood. He presented with a CBC showing 6.6 g/dL Hgb, 114fL MCV, 15.3x10(9)/L, WBC, 304.10(9)/L Platelet. WBC differential consisting of 21% neutrophils, 21% lymphocytes, 45% monocytes, 6% eosinophils, 4% basophils and 3% blasts. The peripheral blood smear showed hypochromic microcytic red cells with marked macrocytosis and slight polychromasia. In addition, atypical hypogranular neutrophils, and hypogranular platelets were revealed. A pathology consultation reported a bone marrow aspirate of 22% neutrophils, 6% metamyelocytes, 11% myelocytes, 1% promyelocytes, 12% eosinophils, 8% blasts, 18% normoblasts, 15% monocytes, and 7% lymphocytes. The M:E ratio was increased, 4:1, with 90% hypercellularity. There was a decreased quantity of erythroid precursors and an increased quantity of myeloid precursors. Blasts contained intermediate-sized nuclei, reticulated chromatin and variably granulated cytoplasm and megakaryocytes were markedly increased. Ancillary tests included both iron and reticulin staining of the bone marrow. Storage iron was decreased and reticulin fibers increased with a myelofibrosis grade 1 of 3. Flow cytometry immunophenotyping of the bone marrow revealed mildly increased myeloid blasts, increased mature monocytes, increased monoblasts/promonocytes with other abnormal findings consistent with a myeloid neoplasm with high grade features. In summary, the pertinent and unusual findings were the rare blasts, increased monocytes and basophils, and dysplastic neutrophils, and leukocytosis seen in the peripheral blood along with slightly increased blasts in the marrow. Along with additional cytogenetic and molecular testing results, the above laboratory finds were essential to the diagnosis of an unusual myelodysplastic/myeloproliferative neoplasm, Chronic Myelomonocytic Leukemia (CMML). The sequence of testing lead to the proper diagnosis and will aid in treatment options.

Evaluation of antifibrotic and antifungal combined therapies in experimental pulmonary paracoccidioidomycosis.

Paracoccidioidomycosis (PCM) is a systemic mycosis caused by the Paracoccidioides genus. Most of the patients with chronic form present sequelae, like pulmonary fibrosis, with no effective treatment, leading to impaired lung functions. In the present study, we aimed to investigate the antifibrotic activity of three compounds: pentoxifylline (PTX), azithromycin (AZT), and thalidomide (Thal) in a murine model of pulmonary PCM treated with itraconazole (ITC) or cotrimoxazole (CMX). BALB/c mice were inoculated with P. brasiliensis (Pb) by the intratracheal route and after 8 weeks, they were submitted to one of the following six treatments: PTX/ITC, PTX/CMX, AZT/ITC, AZT/CMX, Thal/ITC, and Thal/CMX. After 8 weeks of treatment, the lungs were collected for determination of fungal burden, production of OH-proline, deposition of reticulin fibers, and pulmonary concentrations of cytokines and growth factors. Pb-infected mice treated with PTX/ITC presented a reduction in the pulmonary concentrations of OH-proline, associated with lower concentrations of interleukin (IL)-6, IL-17, and transforming growth factor (TGF)-β1 and higher concentrations of IL-10 compared to the controls. The Pb-infected mice treated with AZT/CMX exhibited decreased pulmonary concentrations of OH-proline associated with lower levels of TGF-β1, and higher levels of IL-10 compared controls. The mice treated with ITC/Thal and CMX/Thal showed intense weight loss, increased deposition of reticulin fibers, high pulmonary concentrations of CCL3, IFN-γ and VEGF, and decreased concentrations of IL-6, IL-1β, IL-17, and TGF-β1. In conclusion, our findings reinforce the antifibrotic role of PTX only when associated with ITC, and AZT only when associated with CMX, but Thal did not show any action upon addition.

PS1439 ENHANCED SUPPORT OF MYELOFIBROSIS STEM CELLS IN NEXT GENERATION HUMANIZED MICE

Background:Myelofibrosis (MF) is a hematopoietic stem cell (HSC) neoplasm characterized by an expansion of myeloid cells followed by deposition of fibers in the bone marrow (BM) and leukemic transformation depending on the clonal evolution of MF stem cells (MF SCs). Mutations in the receptor tyrosine kinase JAK2, the endoplasmic reticulum chaperone CALR or the thrombopoietin receptor (MPL) drive the disease phenotype through cytokine‐independent activation of the JAK‐STAT signaling pathway. Patient‐derived xenograft (PDX) models have emerged as powerful tools for investigating aggressive malignancies, such as acute leukemia's. However, the development of less‐aggressive malignancies, like MF, is often limited in PDX models. We hypothesized that the constitutive expression of human cytokines and growth factors in immunocompromised mice (i.e. MISTRG mice) may provide a supportive microenvironment for MF SC development.Aims:To develop a pre‐clinical MF PDX model that faithfully recapitulates the disease phenotype and the genetic heterogeneity observed in patients.Methods:Purified peripheral blood (PB) stem and progenitor (CD34+) cells from 14 MF patients were transplanted intra‐hepatically into sub‐lethally irradiated newborn MISTRG mice and NSG mice (controls). For secondary transplantations, human CD45+ cells purified from primary animals were transplanted intra‐hepatically. Mice were sacrificed 5–26 weeks (median: 9.8 weeks) post‐transplantation and characterized by flow cytometry, immunohistochemistry, and mutational profiling.Results:MISTRG mice supported significantly higher human MF engraftment in the BM (29.20% vs 4.175%, p &lt; 0.0001), the PB (48.70% vs. 0.73%, p &lt; 0.0001) and the spleen (8.19% vs. 0.33%, p &lt; 0.0001) compared to NSG mice. All 14 investigated patient samples engrafted in MISTRG mice, while 9/14 patient samples engrafted in NSG mice. MISTRG mice exhibited superior engraftment independent of risk categories (DIPSS, MIPSS70, and MYSEC), disease stage (chronic, accelerated) and diagnoses (primary, secondary MF). Both NSG and MISTRG mice supported robust monocytic and granulocytic engraftment in the BM. Furthermore, immunohistochemistry revealed human megakaryocytes in both strains. An increase in reticulin fibers was detected in MISTRG mice transplanted with 1 patient sample that was analyzed after 18 weeks. To determine whether the engrafted human cells were derived from the MF clone, NSG and MISTRG mice that showed &gt;10% human engraftment were analyzed for driver mutations (JAK2 or CALR). JAK2 or CALR mutations were preserved in all NSG (7/7) and MISTRG (39/39) mice transplanted with the respective sample. Next, we performed targeted sequencing of 54 myeloid genes frequently mutated in myeloid neoplasms on 7 NSG mice transplanted with 4 patient samples and 22 MISTRG mice transplanted with 8 patient samples to determine whether the mutational profile of the primary sample was preserved in the corresponding engrafted xenografts. This data showed maintenance of the primary patient sample clonal composition in both NSG and MISTRG mice. Finally, purified human MF cells isolated from primary mice showed myeloid reconstitution in secondary recipients.Summary/Conclusion:These results show that MISTRG mice support robust engraftment of MF SCs across all disease stages and categories and faithfully reproduce the genetic complexity observed in patients. This novel MISTRG MF PDX model is currently being used to assess the effect of the JAK inhibitor (JAKi) Ruxolitinib on engrafted human MF cells.

Fibrosis driving myofibroblast precursors in MPN and new therapeutic pathways.

Fibrosis driving myofibroblast precursors in MPN and new therapeutic pathways.

PB2109 NICHEFORMING STRUCTURES OF BONE MARROW OF PATIENTS WITH MULTIPLE MYELOMA BEFORE AND AFTER AUTOHSCT

Background: Autologous transplantation of hematopoietic stem cells (AutoHSCT) is one of the most effective methods of treatment in patients with MM, which can significantly reduce the volume of cells of the pathological clone and improve the quality of the response. However, there remain groups of patients with ineffective AutoHSCT: patients with unsuccessful mobilization and with early relapse after AutoHSCT. AutoHSCT success depends on many factors, including the state of the niche of HSC, which has been subjected to toxic effects of aggressive treatment and damaged by the underlying disease. Aims: To investigate the morphological characteristics of the main stromal components of the niche HSC and cultural characteristics of MSC patients with multiple myeloma (MM) before and after treatment, including AutoHSCT. Methods 32 trepanobiopsy of bone marrow from patients diagnosed with MM the ages of 48 and 68 (median age 56) before and after AutoHSCT were used in the study. The study applied histological, immunohistochemical and cultural methods. 20 patients diagnosed with primary MM included in the study. 12 patients diagnosed with progressive MM: inefficient mobilization of HSC was noted for 5 patients and early relapse for seven. Results: Changes of stromal microenvironment of bone marrow (BM) showed of all patients with MM: increased microvascular density and the number of endosteal stromal cells, strengthening of reticulin fiber in subendosteal and perivascular spaces. Increased angiogenesis correlates with the number of plasma cells in the myelogram (r = 0.58; p < 0.05) and with the type of infiltration of BM (r = 0.85; P < 0,05), as well as with osteodestructive changes in the patient's history (r = 0.65; p < 0.05). Shown significant changes in cultures of mesenchymal stromal cells (MSC), both before and after therapy, including AutoHSCT. In cultural studies of MSC BM MM patients compared to healthy donors showed a decrease in the speed of proliferation (2.4 times on average), a decrease in the total time of passionation from 7 to 5 passages. A marked increase expression of markers associated with myofibroblastic phenotype and aging (smooth-muscle actin, β-galactosidase) - 45-80% of cells in the culture were positively stained with these markers. In MSC cultures of patients after AutoHSCT β-galactosidase were synthesized weaker than before treatment, but nevertheless higher than in the control group. MSCS of healthy donors BM of the enzyme is almost not synthesized. Fibrils smooth-muscle actin was detected in MSCS to AutoHSCT, after AutoHSCT was practically absent. Summary/Conclusion: Thus, the cultures of patients with MSC and BM nicheforming elements of patients with MM possess the features of tumor-associated microenvironment, despite the treatment of the underlying disease.

Delayed Contrast Enhancement in Magnetic Resonance Imaging and Vascular Morphology of Primary Diffuse Large B-Cell Lymphoma (DLBCL) of the Central Nervous System (CNS): A Retrospective Study.

BackgroundThis study aimed to compare the magnetic resonance imaging (MRI) findings of primary diffuse large B-cell lymphoma (DLBCL) of the central nervous system (CNS) with delayed contrast enhancement and histological microvessel density (MVD). T1-weighted and T2-weighted contrast-enhanced and non-enhanced brain imaging were used. CNS lymphoma tissue was evaluated using primary antibodies to endothelial cells and smooth muscle cells, and histochemical staining for reticulin fibers and basement membrane, which allowed quantification of the MVD.Material/MethodsTwenty-one patients with histologically confirmed primary DLBCL of the CNS underwent pre-contrast-enhanced and postcontrast-enhanced MRI. Histology of the CNS lymphoma tissue included immunohistochemical staining with antibodies to CD34 for vascular endothelial cells and alpha smooth muscle actin (ASMA) for vascular smooth muscle cells, and histochemical staining included periodic acid-Schiff (PAS) and silver staining for reticulin fibers to evaluate microvessel density (MVD).ResultsIn primary DLBCL of the CNS, a positive correlation was found between the degree of necrosis and the size of the lymphoma (r=0.546, P=0.01). Delayed imaging enhancement was significantly correlated with the number of mature vessels, MVD, basement membrane, and reticulin fibers (r=0.593, 0.466, 0.446 and 0.497, respectively). Standardized β regression coefficient analysis showed that the MVD, PAS-positive structures, the number of mature vessels, and reticulin fibers, were significantly associated with delayed enhancement on MRI (β values, 0.425, 0.409, 0.295, and 0.188, respectively).ConclusionsIn primary DLBCL of the CNS, delayed imaging enhancement on MRI may be due to reduced neovascularization and vascular infiltration by lymphoma cells.

Human Atrial Natriuretic Peptide in Cold Storage of Donation After Circulatory Death Rat Livers: An Old but New Agent for Protecting Vascular Endothelia?

Current critical shortage of donor organs has increased the use of donation after circulatory death (DCD) livers for transplantation, despite higher risk for primary nonfunction or ischemic cholangiopathy. Human atrial natriuretic peptide (hANP) is a cardiovascular hormone that possesses protective action to vascular endothelia. We aimed to clarify the therapeutic potential of hANP in cold storage of DCD livers. Male Wistar rats were exposed to 30-minute warm ischemia in situ. Livers were then retrieved and cold-preserved for 6 hours with or without hANP supplementation. Functional and morphological integrity of the livers was evaluated by oxygenated ex vivo reperfusion at 37°C. hANP supplementation resulted in significant reduction of portal venous pressure (12.2 ± 0.5 versus 22.5 ± 3.5 mm Hg, P < 0.001). As underlying mechanisms, hANP supplementation significantly increased tissue adenosine concentration (P = 0.008), resulting in significant upregulation of endothelial nitric oxide synthase and significant downregulation of endothelin-1 (P = 0.01 and P = 0.004 vs. the controls, respectively). Consequently, hANP significantly decreased transaminase release (P < 0.001) and increased bile production (96.2 ± 18.2 versus 36.2 ± 15.2 μL/g-liver/h, P < 0.001). Morphologically, hepatocytes and sinusoidal endothelia were both better maintained by hANP (P = 0.021). Electron microscopy also revealed that sinusoidal ultrastructures and microvilli formation in bile canaliculi were both better preserved by hANP supplementation. Silver staining also demonstrated that hANP significantly preserved reticulin fibers in Disse space (P = 0.017), representing significant protection of sinusoidal frameworks/architectures. Supplementation of hANP during cold storage significantly attenuated cold ischemia/warm reperfusion injury of DCD livers.

In-vivo assessment of a case of cutaneous sarcoidosis using reflectance confocal microscopy

Reflectance confocal microscopy (RCM) is a noninvasive imaging technique that allows visualization of the epidermis and papillary dermis with cellular-level resolution. Granulomatous reactions such as sarcoidosis could be assessed using RCM. The identification of bright beaded-like structures that could correspond to reticulin fibers overlying granulomas, in association with dermoscopy, may be a very useful approach in the diagnosis of sarcoidosis and for the differentiation of this granulomatous entity with superficial cutaneous metastasis.

CHARACTERISTICS OF BONE MARROW MICROENVIRONMENT IN MULTIPLE MYELOMA BEFORE AND AFTER TREATMENT

The purpose of the study is to evaluate the morphological and functional features of the tumor microenvironment and the cultural characteristics of mesenchymal stromal cells (MSC) of patients with multiple myeloma (MM) before and after treatment. Material and methods. Parenchymal and stromal bone marrow tissue in iliac bone trepanobiopsy of 32 patients with MM aged from 48 to 78 years (median age 56 years) before and after treatment was investigated. The study applied histological, immunohistochemical and cultural methods. Results and discussion. Сhanges of stromal microenvironment of bone marrow in all patients with MM have been revealed: increased microvascular density and the number of endosteal stromal cells, strengthening of reticulin fiber in subendosteal and perivascular spaces. Increased angiogenesis correlates with the number of plasma cells in the myelogram ( r = 0.58; p < 0.05) and with the type of bone marrow infiltration ( r = 0.85; р < 0.05), as well as with osteodestructive changes in the patient’s history ( r = 0.65; p < 0.05). Significant changes in MSC cultures, both before and after therapy, including autologous hematopoietic stem-cell transplantation, were shown. Bone marrow MSC of patients with MM had decreased speed of proliferation (2.4 times on average), total time of passaging from 7 to 5 passages, increased expression of markers associated with myofibroblastic phenotype and aging (smooth muscle actin, β-galactosidase) compared to healthy donors. Conclusion. MSC cultures and BM niche forming elements of patients with MM have signs of tumor-associated microenvironment, despite the treatment of the underlying disease.

Recent Advancement in the Diagnosis and Treatment of Leprosy.

Many of the tropical diseases are neglected by the researchers and medicinal companies due to lack of profit and other interests. The Drugs for Neglected Diseases initiative (DNDi) is established to overcome the problems associated with these neglected diseases. According to a report published by the WHO, leprosy (Hansen's disease) is also a neglected infectious disease. A negligible amount of advancements has been made in last few decades which includes the tools of diagnosis, causes, treatment, and genetic studies of the bacterium (Mycobacterium leprae) that causes leprosy. The diagnosis of leprosy at earlier stages is important for its effective treatment. Recent studies on vitamin D and its receptors make leprosy diagnosis easier at earlier stages. Skin biopsies and qPCR are the other tools to identify the disease at its initial stages. Until now a specific drug for the treatment of leprosy is not available, therefore, Multi-Drug Therapy (MDT) is used, which is hazardous to health. Besides Mycobacterium leprae, recently a new bacterium Mycobacterium lepromatosis was also identified as a cause of leprosy. During the last few years the genetic studies of Mycobacterium leprae, the role of vitamin D and vitamin D receptors (VDR), and the skin biopsies made the treatment and diagnosis of leprosy easier at early stages. The studies of micro RNAs (miRNAs) made it easy to differentiate leprosy from other diseases especially from tuberculosis. Leprosy can be distinguished from sarcoidosis by quantitative study of reticulin fibers present in skin. The treatment used until now for leprosy is multi-drug treatment. The complete genome identification of Mycobacterium leprae makes the research easy to develop target specified drugs for leprosy. Rifampicin, identified as a potent drug, along with other drugs in uniform multi-drug treatment, has a significant effect when given to leprosy patients at initial stages. These are effective treatments but a specific drug for leprosy is still needed to be identified. The current review highlights the use of modern methods for the identification of leprosy at its earlier stages and the effective use of drugs alone as well as in combination.

The Hypomorphic Gata1low Mutation Induces Fibrosis in Multiple Organs

Mice carrying the hypomorphic mutation which reduces the transcription factor GATA1 in megakaryocytes (Gata1low mice), develop by 8-months myelofibrosis a phenotype resembling primary myelofibrosis, the most severe of myeloproliferative neoplasms (Vannucchi et al Blood 2002;100:1123). The high levels of TGF-β expressed by the abnormal Gata1low megakaryocytes that drive myelofibrosis in these mice alter the bone marrow (BM) expression profiling up-regulating expression of the transcription factor c-Jun (Ciaffoni et al BCD 2015; 54:234). Recently, Dr. Weissman reported that c-Jun over-expression in response to TGF-β activation induces in mice fibrosis in multiple organs (Werning et al PNAS 2017, 114, 4757), suggesting that also Gata1low mice may develop multi-organ fibrosis. To test this hypothesis, we compared morphology (by haematoxylin-eosin staining) and fibrosis (reticulin fibres by trichrome Mallory and collagen fibres by Gomory or Sirius Red Picrate staining) of organs from Gata 1low and wild-type (WT) mice at 1-, 8- and 15-months of age (3 mice/group).With age, the organs from WT mice presented histological abnormalities consistent with the mild one expected for being associated with aging and were seldom positive for fibrosis. By contrast, all the organs from Gata1low mice analysed had profound abnormal morphologies with reticulin or collagen fibers detectable, in addition to BM and spleen, in skin, lung, and kidney in an age-specific fashion (Fig 1). Gata1lowskin was thicker than normal and the connective layer presented numerous reticulin strikes already by 1-month and heavily dense connective regions strongly positive for collagen fibres by 8-15 months, resembling the skin from scleroderma patients. In lung, the alveoli had thickened walls with reticulin fibers detectable by 8-months and collagen bundles by 15-months near the bronchus walls. The abnormal morphology of kidney included reduced numbers of glomeruli and poorly organized cortical parenchyma. Reticulin and collagen fibres were observed in the medullary and nephron region by 8- and 15-months, respectively. In heart, cardiomyocytes presented a strong reduction of intercalary disks by 8-months and reticulin fibers were detectable at 15-months, suggesting that this mild fibrosis is driven by lung insufficiency. The liver presented abnormal localization and morphology of hepatocytes and presence of extramedullary hematopoiesis in perisinusoid areas. Sirius Red Picrate staining revealed few reticulin fibres mostly within erythroid islands.To clarify the mechanisms leading to multi-organ fibrosis in Gata1low mice, the transcription signature of 8-months Gata1low BM was compared with that published for murine liver, lung and kidney fibrosis (https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi? token = ijgpwgsyjhwpdoj&acc = GSE89630). Gata1low BM presented abnormal expression of 1724 genes (821 up- and 903 down). Pathway analyses indicated that most of the down genes were in the Gata1 pathway while the most prominent up genes were in the c-Jun, EZH2, SCL and p53 pathways. Some of the gene abnormal in Gata1low BM were also abnormal in liver (64), lung (29) and kidney (432) fibrosis. Only 28 genes were abnormal in more than one organ (25 liver and kidney, 7 lung). These common genes were obvious markers for fibrosis (5 collagen genes, fibronectin, TGF-β) and did not include c-Jun. Among genes up in Gata1low BM, kidney and liver there was lipocalin 2 (LCN2), a growth factor overexpressed in primary myelofibrosis patients (Lu et al Blood 2015;126:972) that exerts positive and negative effects, respectively, on fibrosis in BM and liver. By comparing the plasma levels of LCN2 in Gata1low and WT mice at 8-9 -and 15-17-months (8-29 mice/group), we determined that levels of LNC2 do not change with age in WT mice but increase by 3-fold in old Gata1low mice (p=0.028), explaining why liver was not one of the organs in which the mutants develop fibrosis.In conclusion, in addition to myelofibrosis, Gata1low mice develop fibrosis in skin, lung and kidney but not in heart and liver and represent genetic models for studies on the pathogenesis of fibrosis in multiple organs. Moreover, these results suggest that although the initiation factor(s) for fibrosis in the various organs are likely different, most of them are expressed by Gata1low megakaryocytes highlighting the importance for studies on the secretome profile of these cells. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

The Role of Nestin+ Mesenchymal Stem Cells (MSCs) in Bone Marrow Chronic Graft-Versus-Host Disease

Chronic graft-versus-host disease (cGVHD) is a major cause of morbidity and mortality after allogeneic hematopoietic stem cell transplantation (allo-HSCT). cGVHD is a syndrome of variable clinical features resembling autoimmune, involving not only the epithelial target tissues (skin, lung, gastrointestinal tract and liver) but also any other organ system (musculoskeletal, joint, ocular and genital tissues). Bone marrow (BM) suppression without infection is often observed in patients undergoing allo-HSCT as GVHD symptoms appear, suggesting that BM could be a target of GVHD (Yusuke S. et. al. Blood 2010). However, the BM cGVHD is rarely reported and the mechanism for BM cGVHD is still unclear. Nestin+ mesenchymal stem cells (MSCs) are capable of differentiating into cells of multiple cell lineages and involved in the formation of fibrotic lesions (Miaohua M. et. al. Cell Mol Life Sci 2016). To determine whether Nestin+ MSCs play an important role in the development of BM cGVHD. We used two murine models of cGVHD, including MHC-matched BALB/c (H-2d) recipients given B10D2 (H-2d) grafts, and MHC-mismatched BALB/c (H-2d) recipients given C57BL/6 (H-2b) grafts. We observed that BM displayed a significant increase in collagen and reticulin fibers deposition around blood vessels in cGVHD group compared to non-cGVHD group. The decreased absolute numbers of monocytes and neutrophils in the BM indicated the broad BM suppression in the cGVHD group. We further demonstrated that Nestin+MSCs located in perivascular BM niche underwent tremendous expansion in cGVHD group (p<0.001). Nestin+ MSCs in cGVHD group acquired expression of alpha smooth muscle actin (a-SMA), indicating myofibroblast differentiation. Moreover, transforming growth factor- beta1 (TGF-β1) concentrations also increased in cGVHD BM microenvironment (p<0.0001). High concentrations of TGF-β1 induced formation of Nestin+ MSC clusters and more transition from Nestin+ MSCs to a-SMA+ cells (p<0.05). Besides, expanded Nestin+ MSCs were surrounded by many macrophages and expressed high level of TGF-β1 receptor (p<0.001 and p<0.005). Taken together, these results indicate that Nestin+ MSCs participate BM cGVHD development through transition to a-SMA+ cells induced by high TGF-β1 in cGVHD microenvironment. DisclosuresZhang:National Natural Science Foundation of China (No. 81600141, No. 81770190) and Natural Science Foundation of Guangdong Province (No. 2016A030310390): Research Funding. Yang:National Natural Science Foundation of China (No. 81600141, No. 81770190) and Natural Science Foundation of Guangdong Province (No. 2016A030310390): Research Funding. Chen:National Natural Science Foundation of China (No. 81600141, No. 81770190) and Natural Science Foundation of Guangdong Province (No. 2016A030310390): Research Funding.

Safety and Efficacy of Romiplostim in over 200 Children with Immune Thrombocytopenia (ITP): Results of an Integrated Database of 5 Clinical Trials

Safety and Efficacy of Romiplostim in over 200 Children with Immune Thrombocytopenia (ITP): Results of an Integrated Database of 5 Clinical Trials

Captopril mitigates splenomegaly and myelofibrosis in the Gata1low murine model of myelofibrosis.

Allogeneic stem cell transplantation is currently the only curative therapy for primary myelofibrosis (MF), while the JAK2 inhibitor, ruxolitinib. Has been approved only for palliation. Other therapies are desperately needed to reverse life‐threatening MF. However, the cell(s) and cytokine(s) that promote MF remain unclear. Several reports have demonstrated that captopril, an inhibitor of angiotensin‐converting enzyme that blocks the production of angiotensin II (Ang II), mitigates fibrosis in heart, lung, skin and kidney. Here, we show that captopril can mitigate the development of MF in the Gata1 low mouse model of primary MF. Gata1 low mice were treated with 79 mg/kg/d captopril in the drinking water from 10 to 12 months of age. At 13 months of age, bone marrows were examined for fibrosis, megakaryocytosis and collagen expression; spleens were examined for megakaryocytosis, splenomegaly and collagen expression. Treatment of Gata1 low mice with captopril in the drinking water was associated with normalization of the bone marrow cellularity; reduced reticulin fibres, splenomegaly and megakaryocytosis; and decreased collagen expression. Our findings suggest that treating with the ACE inhibitors captopril has a significant benefit in overcoming pathological changes associated with MF.

Abnormal dartos fascia in buried penis and hypospadias: Evidence from histopathology

The importance of the pathology of the dartos fascia in hypospadias and buried penis is still debatable. Understanding the properties of connective tissue of dartos fascia in hypospadias and buried penis may give a clue to the underlying mechanism. This study aimed to compare connective tissue and vascularization of dartos fascia between normal penis, buried penis, and hypospadias. We conducted this prospective study from May 2013 to November 2016. We collected dartos fascia specimens from three groups: buried penis, hypospadias, and normal penis as control. All of the patients underwent primary surgery in all groups. Patients with penile abnormalities, such as phimosis or Balanitis Xerotica Obliterans (BXO) were excluded from the normal penis group. We compared the fibers between these groups using Masson trichrome histochemical staining, Gomori's silver impregnation staining, Weigert resorcin-fuchsin staining, and CD31 immunohistochemistry staining for evaluation of collagen fibers, reticulin fibers, elastin fibers, and endothelial cells of blood vessels, respectively. The collagen fibers, reticular fibers, elastic fibers, and vascular vessels were counted with ImageJ and manually calibrated and counted and were analyzed using the one-way ANOVA test. The assessment conducted by two pathologists was blinded, without knowing the clinical diagnosis of patients. There was a total of 60 patients with 20 patients in each group. Collagen fibers for most cases of buried penis and hypospadias showed thicker but fewer collagen fibers than the normal penis. There was a reduction of total collagen and elastin of dartos fascia in hypospadias and buried penis cases. On the other hand, the ratio of reticulin fibers, which represents collagen type III to total collagen, was increased compared to normal penis. Although the dartos fascia in buried penis and hypospadias is thick and inelastic when palpated or during traction/counter traction, it is well-vascularized tissue. This inelastic dartos fascia tissue is an abnormal tissue, but its characteristics are not similar to fibrotic tissue. However, further study with a larger sample is warranted and should differentiate the degree of chordee in patients with hypospadias and buried penis. There was a difference between connective the tissue of dartos fascia in buried penis and patients with hypospadias compared with normal penis. Inelastic dartos fascia tissue in patients diagnosed with buried penis and hypospadias is an abnormal tissue. Therefore, it is suggested that this tissue is excised during reconstructive surgery. Further research is needed to unveil the pathophysiology of the condition.