CD169+ macrophages mediate pathological formation of woven bone in skeletal lesions of prostate cancer

Skeletal metastases present a major clinical challenge for prostate cancer patient care, inflicting distinctive mixed osteoblastic and osteolytic lesions that cause morbidity and refractory skeletal complications. Macrophages are abundant in bone and bone marrow and can influence both osteoblast and osteoclast function in physiology and pathology. Herein, we examined the role of macrophages in prostate cancer bone lesions, particularly the osteoblastic response. First, macrophage and lymphocyte distributions were qualitatively assessed in patient's prostate cancer skeletal lesions by immunohistochemistry. Second, macrophage functional contributions to prostate tumour growth in bone were explored using an immune‐competent mouse model combined with two independent approaches to achieve in vivo macrophage depletion: liposome encapsulated clodronate that depletes phagocytic cells (including macrophages and osteoclasts); and targeted depletion of CD169⁺ macrophages using a suicide gene knock‐in model. Immunohistochemistry and histomorphometric analysis were performed to quantitatively assess cancer‐induced bone changes. In human bone metastasis specimens, CD68⁺ macrophages were consistently located within the tumour mass. Osteal macrophages (osteomacs) were associated with pathological woven bone within the metastatic lesions. In contrast, lymphocytes were inconsistently present in prostate cancer skeletal lesions and when detected, had varied distributions. In the immune‐competent mouse model, CD169⁺ macrophage ablation significantly inhibited prostate cancer‐induced woven bone formation, suggesting that CD169⁺ macrophages within pathological woven bone are integral to tumour‐induced bone formation. In contrast, pan‐phagocytic cell, but not targeted CD169⁺ macrophage depletion resulted in increased tumour mass, indicating that CD169^? macrophage subset(s) and/or osteoclasts influenced tumour growth. In summary, these observations indicate a prominent role for macrophages in prostate cancer bone metastasis that may be therapeutically targetable to reduce the negative skeletal impacts of this malignancy, including tumour‐induced bone modelling. Copyright © 2016 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.     

m6A modifications of circular RNAs in ischemia-induced retinal neovascularization

Ischemia-induced pathological neovascularization in the retina is a leading cause of blindness in various age groups. The purpose of the current study was to identify the involvement of circular RNAs (circRNAs) methylated by N⁶-methyladenosine (m⁶A), and predict their potential roles in oxygen-induced retinopathy (OIR) in mice. Methylation assessment via microarray analysis indicated that 88 circRNAs were differentially modified by m⁶A methylation, including 56 hyper-methylated circRNAs and 32 hypo-methylated circRNAs. Gene ontology enrichment analysis predicted that the enriched host genes of the hyper-methylated circRNAs were involved in cellular process, cellular anatomical entity, and protein binding. Host genes of the hypo-methylated circRNAs were enriched in the regulation of cellular biosynthetic process, the nucleus, and binding. According to the Kyoto Encyclopedia of Genes and Genomes analysis, those host genes were involved in the pathways of selenocompound metabolism, salivary secretion, and lysine degradation. MeRIP-qPCR verified significant alterations in m⁶A methylation levels of mmu_circRNA_33363, mmu_circRNA_002816, and mmu_circRNA_009692. In conclusion, the study revealed the m⁶A modification alterations in OIR retinas, and the findings above shed light on the potential roles of m⁶A methylation in circRNA regulatory functions in the pathogenesis of ischemia-induced pathological retinal neovascularization.     

Parakeratosis in skin is associated with loss of inhibitor of differentiation 4 via promoter methylation

Parakeratosis refers to incomplete maturation of epidermal keratinocytes, resulting in abnormal retention of nuclei in the stratum corneum. It occurs in many diseases of the skin, particularly in psoriasis. Down-regulation of inhibitor of differentiation 4 messenger RNA has been demonstrated in psoriatic skin, but the specificity and mechanism for this finding are unknown. In this study, we addressed specificity by immunohistochemical staining for inhibitor of differentiation 4 protein in skin disorders showing parakeratosis, including: psoriasis (n = 9), chronic eczema (n = 6), and squamous cell carcinoma (n = 7). In these conditions, parakeratotic keratinocytes in the upper layers of the skin lacked inhibitor of differentiation 4 protein expression, whereas keratinocytes in the lower layers were densely stained, in contrast to diffuse expression in normal skin. Because promoter hypermethylation of inhibitor of differentiation 4 has been described in several cancers, we determined the methylation pattern of the inhibitor of differentiation 4 promoter in psoriasis and compared this with squamous cell carcinoma. We found a novel methylation pattern of the inhibitor of differentiation 4 promoter in both conditions. Inhibitor of differentiation 4 promoter methylation was significantly increased in psoriasis (34.8%) and squamous cell carcinoma (21.8%), compared with normal skin (0%). Moreover, cells in the upper and lower parts of psoriatic epidermis were, respectively, hypermethylated and nonmethylated, at the inhibitor of differentiation 4 promoter. Comparable studies in several cell lines confirmed that hypermethylation of the promoter was associated with loss of inhibitor of differentiation 4 messenger RNA and protein expression. Our study demonstrates a previously unreported link between gene-specific promoter hypermethylation and abnormal cellular differentiation in several skin diseases. This mechanism might provide clues for novel therapies for skin disorders characterized by parakeratosis.     

The Wistar Bonn Kobori rat, a unique animal model for autoimmune pancreatitis with extrapancreatic exocrinopathy

The male Wistar Bonn/Kobori (WBN/Kob) rat is known to be a unique animal model for chronic pancreatitis with widely distributed fibrosis and degeneration of parenchyma because of the infiltration of lymphocytes. In this report, we show that female (but not male) rats develop dacryoadenitis at 3 months of age, and that both male and female WBN/Kob rats develop sialoadenitis, thyroiditis, sclerotic cholangitis and tubulointerstitial nephritis over 18 months of age. The infiltration of CD8⁺ cells and the deposits of tissue-specific IgG2b were observed in the injured pancreas and lachrymal glands. Furthermore, the number of regulatory T cells (defined as CD4⁺ Forkhead box P3⁺ cells) decreased in the periphery of both male and female WBN/Kob rats, suggesting that the onset of these diseases is attributable, at least, to the failure in the maintenance of peripheral immune tolerance. These features show clearly that WBN/Kob rats are a useful animal model for autoimmune pancreatitis and Sjøgren-like syndrome or multi-focal fibrosclerosis in humans. We also show that these autoimmune diseases can be prevented by a newly devised strategy of bone marrow transplantation (BMT) in which bone marrow cells are injected directly into the bone marrow cavity: intrabone marrow–BMT.     

Unusual Endosteally Formed Bone Tissue in a Patagonian Basal Sauropodomorph Dinosaur

Mussaurus patagonicus (Dinosauria: Sauropodomorpha) is a basal sauropodomorph from the Late Triassic of southern Argentina that is known from a large number of individuals, including juveniles, subadults, and adults. Here, we report on the occurrence of an unusual bone tissue in an individual of M. patagonicus. The rather atypical bone tissue is located within the femoral medullary cavity and also occurs within several erosion cavities of the midinner part of the cortex. This tissue is well vascularized and is composed of a matrix that consists of abundant and densely packed osteocyte lacunae. Although some features of this tissue resembles avian medullary bone, the histological features are distinctive and share more features with the pathological, reactive bone produced in extant birds in response to a retrovirus‐induced disease (avian osteopetrosis). Here, we also discuss and provide histological features to effectively differentiate endosteally formed medullary bone from pathological avian osteopetrosis. Anat Rec, 297:1385–1391, 2014. © 2014 Wiley Periodicals, Inc.     

A Quick and Easy Approach to Prepare Ground Bone Slides from Undecalcified Skeleton

Ground bone slides are not only necessary for teaching histology of Haversian system to undergraduate and postgraduate students, but also essential for rapid pathological diagnosis in related diseases as well as to be used in polarizing microscopy and studying bone architectures from fossils. This technique is also applicable for hard tissues like teeth. Present histology manual-cum-practical literatures though prefer bone-slide preparation from decalcified skeleton, but Haversian architectures with osteons are vivid in ground bone slides. Here a technique is briefed; which have practiced well to prepare the ground bone slides with very simple, cheap and easily available equipments in a very short time, which can be adopted by anatomists, odontologists and geologists for quick making of compact bone slides without hampering the bone continuity avoiding hazardous and time consuming methods or costly equipments like freezing microtome.     

多发性骨髓瘤中p15基因高甲基化的研究

目的：探索p15（MST INK4b）基因在多发性骨髓瘤中的作用特点和甲基化发生的规律。方法：用甲基化特异的PCR的方法-MSP（methylation-specific PCR）,检测23例多发性骨髓瘤（multiple myeloma, MM）p15基因甲基化的发生率。结果：MM中甲基化的发生率为73.5%(17/23),扩增产物为148 bp片段。其中4例Ⅰ期,骨髓象为浆细胞型的MM p15基因未发生甲基化;1例Ⅱ期和1例Ⅲ期,骨髓象为分化成熟的浆细胞的MM中p15基因未发生甲基化;在Ⅰ、Ⅱ和Ⅲ期中多数为浆细胞型或混合型中的幼稚浆细胞的病例易发生甲基化。Ⅰ、Ⅱ期和Ⅲ期中甲基化的发生率分别为55%(5/9)、100%(7/7)和71.4%(5/7)。结论：p15基因甲基化可能是MM发病原因之一,与病程的进展和预后有一定的关系。     

Expression and functions of cellular prion proteins in immunocytes

Prion diseases are fatal neurodegenerative processes caused by the accumulation of the pathological prion protein, PrP^Sc. While pathological lesions are limited to the central nervous system (CNS), disease-specific proteins accumulate and replicate in secondary lymphoid organs prior to neuroinvasion, and their replication there depends on the abundance of cellular prion protein (PrP^C). PrP^C is expressed in both central and peripheral lymphoid tissues, and up- or downregulates innate and adaptive immune responses. In addition to prion diseases, PrP^C is also immunologically involved in other neurological disorders and infectious diseases, including Alzheimer's disease and human immunodeficiency virus infection. Herein, we summarize the expression and functions of PrP^C in various immunocytes, as well as its immunological and pathological roles in neurodegeneration and infection.     

The macroscopic and histomorphological properties of periosteal rib lesions and its relation with disease duration: evidence from the Luis Lopes Skeletal Collection (Lisbon,Portugal)

Periosteal new bone formation (PNBF) is a common finding in a large spectrum of diseases. In clinical practice, the morphology and location of periosteal lesions are frequently used to assist in the differential diagnosis of distinct bone conditions. Less commonly reported is the presence of PNBF on the ribs. This contrasts with the data retrieved from the study of skeletonized human remains that shows a high frequency of cases and a strong, albeit not specific, association between periosteal rib lesions and pulmonary conditions (e.g. tuberculosis). Despite that, an overall disagreement regarding the specificity and non‐specificity of periosteal reactions exists in the study of dry bone remains. The insufficient number of clinical models exploring the morphology and the pathophysiology of PNBF's and the lack of systematic studies of pathological samples with a known diagnosis are claimed as major reasons for the disagreements. This study aimed to describe and compare the macroscopic and the histomorphologic appearance of periosteal rib lesions and to discuss their usefulness as diagnostic indicators. To pursue this goal, an assemblage of 13 rib samples (males = 11, females = 2, mean age‐at‐death = 36.6 years old) was collected from the Luis Lopes Skeletal Collection (Museu Nacional de História Natural e da Ciência, Universidade de Lisboa, Portugal). The assemblage belongs to individuals who died from pulmonary‐TB (group 1), non‐TB pulmonary infections (group 2) and other conditions (group 3). Prior to sample preparation, the ribs were visually inspected and the PNBF described according to its thickness, the degree of cortical integration and the type of new bone formed (e.g. woven, lamellar or both). After sampling, each bone sample was prepared for histological analysis under plane and polarized light microscopy. Macroscopically, the results showed no differences in the new bone composition between cause‐of‐death groups. Only slight differences in the degree of cortical integration, which was most frequently classified as mild to high in the pulmonary‐TB group, were observed. Histologically, no distinguishing features were identified by pathological group. However, new bone microarchitectures were observed compatible with (1) acute, fast‐growing processes (e.g. spiculated reactions), (2) long‐standing processes with a rapid bone formation (e.g. appositional layering of bone) and/or (3) chronic, slow‐growing processes (e.g. layers of compact lamellae). To some extent, these distinct rates of disease progression resonate with the cause‐of‐death listed for some individuals. Despite the small sample size, the results of this investigation are in agreement with previous studies, according to which the macroscopic and histological appearance of periosteal formations are not specific for a particular pathological conditions. Nevertheless, the results support the conclusion that the morphology of periosteal lesions is a good biological indicator for inferring the rate of progression and duration of pathological processes. This study provides important reference data regarding the histomorphology of periosteal lesions that can be used for comparative purposes, as well as to narrow down the differential diagnosis in unidentified skeletal remains.     

Isolation of complementary DNA unique to the genome of avian myeloblastosis virus (AMV)

Avian myeloblastosis virus (AMV) can transform avian cells of hemopoietic origin, such as bone marrow, embryonic yolk sac, and circulating macrophages. The AMV is defective in its replication and can only replicate in the presence of helper viruses. This defectiveness in replication is probably due to a deletion or substitution of nucleotide sequences in its genome. The AMV genome contains no sequences homologous to the src gene, which is responsible for the transforming function of the sarcoma viruses. We attempted to identify the AMV sequences that may contain sequences which are responsible for its transforming function. We isolated a complementary DNA (cDNA_AMV) that hybridized preferentially to the RNA of the transforming AMV but not to the RNA of the helper virus. Using this cDNA_AMV as a probe, we determined the size of the AMV genome to be 33–34 S with a molecular weight of 2.6 × 10⁶. A similar molecular weight estimation of the AMV genome size was obtained by methylmercury-agarose gel electrophoresis of AMV RNA. In AMV-producer myeloblasts we can detect about 6000–7000 copies per cell of AMV-specific RNA, whereas fewer than 2 copies per cell of AMV RNA are found in helper virus-infected cells. In AMV nonproducer myeloblasts, about 2000 copies of AMV-specific RNA are detected. Furthermore, we find that RNA of AMV NP myeloblasts can only hybridize to 55% of cDNA complementary to helper virus genome. In uninfected hemopoietic cells, e.g., bone marrow cells, about 20 copies per cell of AMV-specific RNA are present, whereas in uninfected chick embryo fibroblasts less than 1 copy per cell is found.     

Hyperlipidemia compromises homing efficiency of systemically transplanted BMSCs and inhibits bone regeneration

Purpose: Mesenchymal stem cells (MSCs) can selectively home to bone defects and play an essential role in promoting bone regeneration. As an adverse effect factor for bone metabolism, hyperlipidemia significantly impairs bone regeneration. In this study, bone marrow stromal cells (BMSCs) were systemically transplanted into a hyperlipidemic mouse model to explore the effect of hyperlipidemia on stem cell recruitment and bone regeneration. Methods: Hyperlipidemia was established in ApoE^-/- mice (on C57BL/6J background) fed with a high fat diet (HFD) for five weeks. C57BL/6 mice fed with the same diet served as controls. BMSCs labeled with the green fluorescent protein (GFP) were then injected via the tail vein and bone defects were created in the mandibles. The animals were sacrificed at weeks 1, 2 and 4 after surgery, and the fate of the transplanted BMSCs was monitored with a fluorescence microscope and immunohistochemical analysis. After hematoxylin and eosin (HE) staining and Masson’s Trichrome (MT) staining, histomorphometric analysis was performed to evaluate bone regeneration. Results: In both groups transplanted with BMSCs, the number of GFP-positive BMSCs detected in the bone defects reached its peak at 1 week after surgery and was decreased thereafter. However, at all time points, less GFP⁺ cells were detected in the ApoE^-/- mice than in the corresponding control mice. BMSCs transplantation significantly enhanced new bone formation, but to a lesser degree in the ApoE^-/- mice when compared with the control mice. Conclusions: Hyperlipidemia compromises homing efficiency of systemically transplanted BMSCs and inhibits bone regeneration.     

Human osteoclast-like cells in primary culture

We describe a method for the isolation and culture of osteoclast-like cells from cancellous bone chips of iliac crests from patients undergoing reconstructive maxillofacial surgery. Under aseptic conditions, bone chips were cut into small pieces, incubated briefly with collagenase, and the isolated bone cells were separated from the bone chips by filtration using a nylon mesh. Bone cells were then cultured on a variety of surfaces for up to 10 days. Cell motility and fusion, together with the development of tartrate-resistant acid phosphatase activity, were seen in many cells soon after culture. The large osteoclast-like cells adhered to human cortical bone slices and produced resorption pits. These morphological and functional characteristics suggest that the cells we isolated and cultured were human osteoclasts and their precursors. Thus this method may provide a reliable means of obtaining human osteoclasts from normal tissue for short-term studies of their metabolism or from various skeletal diseases to study pathological aberrations and mechanisms. © 1996 Wiley-Liss, Inc.     

Keratan sulfate as a marker for medullary bone in fossil vertebrates

The ability to determine the sex of extinct dinosaurs by examining the bones they leave behind would revolutionize our understanding of their paleobiology; however, to date, definitive sex-specific skeletal traits remain elusive or controversial. Although living dinosaurs (i.e., extant birds) exhibit a sex-specific tissue called medullary bone that is unique to females, the confident identification of this tissue in non-avian archosaurs has proven a challenge. Tracing the evolution of medullary bone is complicated by existing variation of medullary bone tissues in living species; hypotheses that medullary bone structure or chemistry varied during its evolution; and a lack of studies aimed at distinguishing medullary bone from other types of endosteal tissues with which it shares microstructural and developmental characteristics, such as pathological tissues. A recent study attempted to capitalize on the molecular signature of medullary bone, which, in living birds, contains specific markers such as the sulfated glycosaminoglycan keratan sulfate, to support the proposed identification of medullary bone of a non-avian dinosaur specimen (Tyrannosaurus rex MOR 1125). Purported medullary bone samples of MOR 1125 reacted positively to histochemical analyses and the single pathological control tested (avian osteopetrosis) did not, suggesting the presence of keratan sulfate might serve to definitively discriminate these tissues for future studies. To further test these results, we sampled 20 avian bone pathologies of various etiologies (18 species), and several MB samples. Our new data universally support keratan sulfate as a reliable marker of medullary bone in birds. However, we also find that reactivity varies among pathological bone tissues, with reactivity in some pathologies indistinguishable from MB. In the current sample, some pathologies comprised of chondroid bone (often a major constituent of skeletal pathologies and developing fracture calluses in vertebrates) contain keratan sulfate. We note that beyond chemistry, chondroid bone shares many characteristics with medullary bone (fibrous matrix, numerous and large cell lacunae, potential endosteal origin, trabecular architecture) and medullary bone has even been considered by some to be a type of chondroid bone. Our results suggest that the presence of keratan sulfate is not exclusive evidence for MB, but rather must be used as one in a suite of criteria available for identifying medullary bone (and thus gravid females) in non-avian dinosaur specimens. Future studies should investigate whether there are definite chemical or microstructural differences between medullary bone and reactive chondroid bone that can discriminate these tissues.     

Poor osteoinductive potential of subcutaneous bone cement-induced membranes for tissue engineered bone

Abstract

Background: Large segmental bone defects remain a challenge for reconstructive surgeons. A two-stage repair strategy may offer a potential solution. Here, we sought to evaluate the osteoinductive potential of bone cement-induced membranes in an ectopic site. Methods: First, bone cements were inserted into the subcutaneous tissues of 16 rabbits to induce membrane formation. After 2, 4, 6 and 8 weeks, the induced membranes were harvested to assess their vascularization and osteoinductive potential. Next, bone cements were subcutaneously inserted into 12 rabbits for 4 weeks. These bone cements were then harvested from the newly formed membranes and replaced with granular porous β-TCP, with or without bone mesenchymal stem cells. New bone formation was then evaluated after 3, 6 and 9 weeks. Results: The highest level of blood vessel formation and bone morphogenetic protein-2 expression in the membranes were found at 4 weeks (p?<?0.05). In addition, vascular endothelial growth factor concentration was highest after 2 weeks (p?<?0.001), persisting until 8 weeks. However, the results showed little ectopic bone formation at these time points. Conclusion: While bone cement-induced membranes appear to provide a suitable environment for bone formation, they fail to drive osteoinduction in non-osseous sites for the purposes of bone tissue engineering.     

LncRNA在骨重建及骨相关疾病中的作用

骨重建可保持骨生物力学特性稳定,对维持骨强度具有重要意义。正常骨骼的生长和发育需要转录调控网络、信号传导通路、力学生物学及生物力学因素的紧密协调,而力学环境的改变,多种信号途径失调,影响骨的发育。长链非编码RNA(long non-coding RNA, lncRNA)是一类长度大于200 nt、生物来源广泛、2级及3级结构高度保守的RNA分子。研究显示,许多lncRNA参与骨骼系统的正常发育或平衡,调控成骨细胞的分化,以及参与骨肉瘤的发生。LncRNA表达失调与关节炎、骨质疏松症和骨癌等多种骨疾病密切相关,有望作为预测骨疾病的生物标志物。综述lncRNA的特征、参与骨重塑的lncRNA及其可能的作用机理,并讨论lncRNA作为生物标志物应用于治疗包括骨关节炎、骨质疏松症、骨癌等骨骼系统疾病的可能性,旨在为更好地理解和研究lncRNA在生物体内的作用提供参考。