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Immune-mediated rippling muscle disease (iRMD) is a rare myopathy characterized by wavelike muscle contractions (rippling) and percussion- or stretch-induced muscle mounding. A serological biomarker of this disease is lacking.To describe a novel autoantibody biomarker of iRMD and report associated clinicopathological characteristics.This retrospective cohort study evaluated archived sera from 10 adult patients at tertiary care centers at the Mayo Clinic, Rochester, Minnesota, and BrighamWomen's Hospital, Boston, Massachusetts, who were diagnosed with iRMD by neuromuscular specialists in 2000 and 2021, based on the presence of electrically silent percussion- or stretch-induced muscle rippling and percussion-induced rapid muscle contraction with or without muscle mounding and an autoimmune basis. Sera were evaluated for a common biomarker using phage immunoprecipitation sequencing. Myopathology consistent with iRMD was documented in most patients. The median (range) follow-up was 18 (1-30) months.Diagnosis of iRMD.Detection of a common autoantibody in serum of patients sharing similar clinical and myopathological features.Seven male individuals and 3 female individuals with iRMD were identified (median [range] age at onset, 60 [18-76] years). An IgG autoantibody specific for caveolae-associated protein 4 (cavin-4) was identified in serum of patients with iRMD using human proteome phage immunoprecipitation sequencing. Immunoassays using recombinant cavin-4 confirmed cavin-4 IgG seropositivity in 8 of 10 patients with iRMD. Results for healthy and disease-control individuals (n = 241, including myasthenia gravis and immune-mediated myopathies) were cavin-4 IgG seronegative. Six of the 8 individuals with cavin-4 IgG were male, and the median (range) age was 60 (18-76) years. Initial symptoms included rippling of lower limb muscles in 5 of 8 individuals or all limb muscles in 2 of 8 sparing bulbar muscles, fatigue in 9 of 10, mild proximal weakness in 3 of 8, and isolated myalgia in 1 of 8, followed by development of diffuse rippling. All patients had percussion-induced muscle rippling and half had percussion- or stretch-induced muscle mounding. Four of the 10 patients had proximal weakness. Plasma creatine kinase was elevated in all but 1 patient. Six of the 10 patients underwent malignancy screening; cancer was detected prospectively in only 1. Muscle biopsy was performed in 7 of the 8 patients with cavin-4 IgG; 6 of 6 specimens analyzed immunohistochemically revealed a mosaic pattern of sarcolemmal cavin-4 immunoreactivity. Three of 6 patients whose results were seropositive and who received immunotherapy had complete resolution of symptoms, 1 had mild improvement, and 2 had no change.The findings indicate that cavin-4 IgG may be the first specific serological autoantibody biomarker identified in iRMD. Depletion of cavin-4 expression in muscle biopsies of patients with iRMD suggests the potential role of this autoantigen in disease pathogenesis.

Longitudinal studies assessing durability of the anti-severe acute respiratory syndrome coronavirus 2 (anti-SARS-CoV-2) humoral immune response have generated conflicting results. This has been proposed to be due to differences in patient populations, the lack of standardized methodologies, and the use of assays that measure distinct aspects of the humoral response. SARS-CoV-2 antibodies were serially measured in sera from a cohort of 44 well-characterized convalescent plasma donors over 120 days post-COVID-19 symptom onset, utilizing eight assays, which varied according to antigen source, the detected antibody isotype, and the activity measured (i.e., binding, blocking, or neutralizing). While the majority of assays demonstrated a gradual decline in antibody titers over the course of 120 days, the two electrochemiluminescence immunoassay Roche assays (Roche Diagnostics Elecsys anti-SARS-CoV-2 [qualitative, nucleocapsid based] and Roche Diagnostics Elecsys anti-SARS-CoV-2 S [semiquantitative, spike based]), which utilize dual-antigen binding for antibody detection, demonstrated stable and/or increasing antibody titers over the study period. This study is among the first to assess longitudinal, rather than cross-sectional, SARS-CoV-2 antibody profiles among convalescent COVID-19 patients, primarily using commercially available serologic assays with Food and Drug Administration emergency use authorization. We show that SARS-CoV-2 antibody detection is dependent on the serologic method used, which has implications for future assay utilization and clinical value.

Islet autoantibodies are predominantly measured by radioassay to facilitate risk assessment and diagnosis of type 1 diabetes. However, the reliance on radioactive components, large sample volumes and limited throughput renders radioassay testing costly and challenging. We developed a multiplex analysis platform based on antibody detection by agglutination-PCR (ADAP) for the sample-sparing measurement of GAD, IA-2 and insulin autoantibodies/antibodies in 1 μL serum. The assay was developed and validated in 7 distinct cohorts (n = 858) with the majority of the cohorts blinded prior to analysis. Measurements from the ADAP assay were compared to radioassay to determine correlation, concordance, agreement, clinical sensitivity and specificity. The average overall agreement between ADAP and radioassay was above 91%. The average clinical sensitivity and specificity were 96% and 97%. In the IASP 2018 workshop, ADAP achieved the highest sensitivity of all assays tested at 95% specificity (AS95) rating for GAD and IA-2 autoantibodies and top-tier performance for insulin autoantibodies. Furthermore, ADAP correctly identified 95% high-risk individuals with two or more autoantibodies by radioassay amongst 39 relatives of T1D patients tested. In conclusion, the new ADAP assay can reliably detect the three cardinal islet autoantibodies/antibodies in 1μL serum with high sensitivity. This novel assay may improve pediatric testing compliance and facilitate easier community-wide screening for islet autoantibodies.

Neutralizing antibodies are key determinants of protection from future infection, yet well-validated high-throughput assays for measuring titers of SARS-CoV-2-neutralizing antibodies are not generally available. Here, we describe the development and validation of IMMUNO-COV v2.0, a scalable surrogate virus assay, which titrates antibodies that block infection of Vero-ACE2 cells by a luciferase-encoding vesicular stomatitis virus displaying SARS-CoV-2 spike glycoproteins (VSV-SARS2-Fluc). Antibody titers, calculated using a standard curve consisting of stepped concentrations of SARS-CoV-2 spike monoclonal antibody, correlated closely (P

Introduction Multifocal motor neuropathy (MMN) is a motor only, asymmetric onset neuropathy that is relatively treatment-refractory compared with classic chronic inflammatory demyelinating polyradiculoneuropathy (CIDP) and multifocal acquired demyelinating sensory and motor (MADSAM) neuropathy. Methods We reviewed 35 patients seropositive for GM1 (monosialo-asialo [immunoglobulin M, IgM; immunoglobulin G, IgG]) and/or GD1b (disialo [IgG, IgM]) autoantibodies having MMN, classic CIDP, or MADSAM. Immune-treatment responsiveness and clinical course was compared with antibody negative disease controls. Results Seventy-nine percent of seropositives with an initial diagnosis of MMN were immunotherapy responsive compared with 46% of seronegatives (P = 0.045). Eight ganglioside antibody positive MMN patients of 19 (42%) developed sensory findings consistent with MADSAM compared with 3 of 41 (7%) seronegative MMN patients (P = 0.003). MMN and MADSAM patients with ganglioside antibody positivity had more sustained treatment responses (P = 0.03). Discussion Patients initially diagnosed with MMN seropositive for diverse GM1 autoantibodies appear more likely to have sustained treatment response and evolution to MADSAM. Muscle Nerve 57: 1000-1005, 2018.

Polyneuropathies co-occurring with multiple sclerosis (MS) may be underdiagnosed while causing additional disability burden.To determine polyneuropathy presence and type in MS and compare MS with chronic inflammatory demyelinating polyradiculoneuropathy (MS-CIDP) versus MS with other non-inflammatory polyneuropathies.Retrospective chart review of Mayo Clinic cases diagnosed with MS and polyneuropathy. Serum from MS-CIDP for pan-IgG autoantibodies to neurofascin-155 were tested when available.From 1980-2013, 133 co-existing MS/ polyneuropathy cases were identified. Twenty-eight MS patients had inflammatory neuropathy (11 CIDP, 5 plexopathy, 2 vasculitis, 4 monoclonal gammopathy-associated, 6 other), 15 inherited neuropathy (8 axonal, 7 demyelinating), 32 diabetic sensorimotor polyneuropathy, and 58 other. 109 had neuropathy beginning simultaneous to or after MS diagnosis (82%). Compared to MS cases with other polyneuropathy subtypes, MS-CIDP cases had absent or reduced ankle reflexes (100 vs. 70%, p = 0.04), earlier age of neuropathy recognition (52 vs. 58 years, p = 0.048), worse impairment (NIS 27 vs. 22 points, p 0.03), and more acquired demyelinating electrophysiology features (46% vs. 9%, p 0.003). Of MS-CIDP cases with available serum, 1-in-3 had IgG4 autoantibodies to neurofascin-155.(1) Polyneuropathies occurring in MS contribute to neurological disability. (2) Diagnosing polyneuropathies in people with MS is challenging and, likely, under-diagnosed. Recognition is important as some polyneuropathies (e.g., CIDP) are treatable. (3) The probable over-representation of inflammatory neuropathy (especially CIDP) in MS suggests a shared dysimmune pathogenesis, supported by autoantibodies to neurofascin-155.

Age-related bone loss in humans is associated with a decrease in bone formation relative to bone resorption, although the mechanisms for this impairment in bone formation with aging are not well understood. It is known that the precursors for the bone-forming osteoblasts reside in the mesenchymal cell population in bone marrow. Thus, in an effort to identify relevant genetic pathways that are altered with aging, we examined the gene expression and DNA methylation patterns from a highly enriched bone marrow mesenchymal cell population from young (mean age, 28.7 years) versus old (mean age, 73.3 years) women. Bone marrow mononuclear cells from these women were depleted of hematopoietic lineage (lin) and endothelial cells using a combination of magnetic- and fluorescence-activated cell sorting, yielding a previously characterized mesenchymal cell population (lin−/CD34−/CD31− cells) that is capable of osteoblast differentiation. Whole transcriptome RNA sequencing (RNAseq) of freshly isolated cells (without in vitro culture) identified 279 differentially expressed genes (p < 0.05, false discovery rate [q] < 0.10) between the young and old subjects. Pathway analysis revealed statistically significant (all p < 0.05) alterations in protein synthesis and degradation pathways, as well as mTOR, gap junction, calcium, melatonin and NFAT signaling pathways. Further, Reduced Representational Bisulphite sequencing (RRBS DNA methylation sequencing) revealed significant differences in methylation between the young and old subjects surrounding the promoters of 1528 target genes that also exhibited significant differences in gene expression by RNAseq. In summary, these studies provide novel insights into potential pathways affected by aging in a highly enriched human mesenchymal cell population analyzed without the confounding effects of in vitro culture. Specifically, our finding of alterations in several genes and pathways leading to impaired protein synthesis and turnover with aging in bone marrow mesenchymal cells points to the need for further studies examining how these changes, as well as the other alterations with aging that we identified, may contribute to the age-related impairment in osteoblast formation and/or function.

Multifocal motor neuropathy (MMN) is a motor only, asymmetric onset neuropathy that is relatively treatment-refractory compared with classic chronic inflammatory demyelinating polyradiculoneuropathy (CIDP) and multifocal acquired demyelinating sensory and motor (MADSAM) neuropathy.We reviewed 35 patients seropositive for GM1 (monosialo-asialo [immunoglobulin M, IgM; immunoglobulin G, IgG]) and/or GD1b (disialo [IgG, IgM]) autoantibodies having MMN, classic CIDP, or MADSAM. Immune-treatment responsiveness and clinical course was compared with antibody negative disease controls.Seventy-nine percent of seropositives with an initial diagnosis of MMN were immunotherapy responsive compared with 46% of seronegatives (P = 0.045). Eight ganglioside antibody positive MMN patients of 19 (42%) developed sensory findings consistent with MADSAM compared with 3 of 41 (7%) seronegative MMN patients (P = 0.003). MMN and MADSAM patients with ganglioside antibody positivity had more sustained treatment responses (P = 0.03).Patients initially diagnosed with MMN seropositive for diverse GM1 autoantibodies appear more likely to have sustained treatment response and evolution to MADSAM. Muscle Nerve 57: 1000-1005, 2018.

Hematopoietic stem cells (HSCs) are maintained in specialized microenvironments, termed niches, in which supporting cells that promote their proliferation and differentiation are located. Several studies have documented the regulatory role of osteoblasts in the fate of HSCs [Calvi et al., 2003; Zhang et al., 2003; Arai et al., 2004; Mayack and Wagers, 2008], while other studies have shown that HSCs can be observed adjacent to the vasculature in bone marrow [Kiel et al., 2005]. Indeed, recent data have indicated an important role of the endothelial and perivascular stromal cells in supporting HSC cells, with these cells being characterized by the production of high levels of CXCL12, alkaline phosphatase (AP), vascular cell adhesion molecule 1 (Vcam1), platelet-derived growth factor receptor α and β and stem cell factor (SCF; also known as KITL) which are necessary to maintain HSCs [Ding et al., 2012]. Nevertheless, at present the true identity of the supporting cells and the nature of the supporting factors remain uncertain. Previous studies demonstrated that conditional ablation of osteoblasts in transgenic mice expressing thymidine kinase (Tk) under the control of the 2.3 kb rat collagen 1 alpha 1 promoter (2.3Col1α1) resulted in a marked decrease in bone marrow cells (BMCs). In these mice, the depletion of BMCs was parallel to that of osteoblasts, suggesting a supportive role of osteoblasts in hematopoiesis [Visnjic et al., 2004]. Ganciclovir (GCV) treatment induced ablation of replicating osteoblast progenitors in mice expressing Tk under the control of the 3.6 kb rat collagen 1 alpha 1 promoter (3.6Col1α1) [Jilka et al., 2009]. GCV treatment induced depletion of hematopoietic cells which was evident after 2 weeks of treatment but only in 1–2-month-old animals, again suggesting that the self-renewal of HSCs, the cells that generate a lifelong supply of all blood cell types, might be partly regulated by osteoblastic cells within the hematopoietic niche. Bone marrow stromal cells (MSCs) have the capacity to differentiate into different mesodermal lineages, including osteoblasts and adipocytes, constitute an essential part of the bone marrow microenvironment, and have the ability to support hematopoiesis. Infusion of MSCs in humans has been associated with a rapid recovery of hematopoiesis after bone marrow transplantation for hematological diseases as well as after chemotherapy for breast cancer [Koc et al., 2000; Meuleman et al., 2009]. Recent experimental studies have shown a recovery of hematopoiesis after MSC infusion in mice exposed to severe radiation [Lange et al., 2011]. Other studies demonstrated the reconstitution of a functional hematopoietic microenvironment through the infusion of human MSCs in the murine bone marrow compartment [Muguruma et al., 2006]. One of the principal ways by which MSCs support hematopoiesis is by supplying HSCs and their progeny with signals for survival, proliferation and differentiation through direct cell-to-cell contact and/or production of cytokines, adhesion molecules, and extracellular matrix proteins. It has been reported that MSCs produce SCF, Flt-3 ligand, thrombopoietin, leukemia-inhibiting factor (LIF), TGF-β, interleukin (IL)-6, IL-7, IL-8, IL-11, IL-12, IL-14, IL-15, granulocyte-macrophage (GM) and macrophage colony stimulating factor (M-CSF) as well as intercellular adhesion molecule-1 and VCAM-1 [Prockop, 1997; Majumdar et al., 2000; Di Nicola et al., 2002]. Our group has previously identified circulating cells in humans expressing the osteoblast markers, osteocalcin (OCN), or AP. These cells have the ability to mineralize in vitro and to induce bone formation in vivo [Eghbali-Fatourechi et al., 2005]. In subsequent studies we further enriched cells expressing mesenchymal markers by first depleting hematopoietic cells, resulting in a hematopoietic negative fraction (lin−) which was then stained with antibodies to AP or Stro1. The gene expression data of these cells suggested that they were quiescent cells that could be involved in supporting hematopoiesis, with nearly 40% of these cells expressing the hematopoietic/endothelial marker CD34 [Eghbali-Fatourechi et al., 2007; Undale et al., 2010]. Recent work has further demonstrated that these cells also contained a significant population of CD31 cells (marker for mature endothelial cells) [Modder et al., 2012]. Although several lines of evidence have suggested a regulatory role for mesenchymal/osteoblastic cells in regulating hematopoiesis, at present the role of these circulating cells expressing osteoblastic and endothelial markers remains unknown. A significant proportion of HSCs are localized adjacent to sinusoidal blood vessels in the bone marrow [Kiel et al., 2005; Kiel et al., 2007]. Administration of antibodies against endothelial cells in vivo impairs HSC engraftment and transformed endothelial cells promote HSC expansion in culture [Butler et al., 2010]. Co-culture of progenitor cells with endothelial cells increased their capacity to repopulate the bone marrow of SCID mice [Chute et al., 2002]. In addition, the exposure of HSCs to endothelial cells isolated from different tissues resulted in varying HSC growth and repopulation ability [Li et al., 2004]. Endothelial cells play an important role in HSC maintenance; however, it is now also accepted that factors secreted by these cells may have a beneficial effect. Recently, Ding et al. (2012) demonstrated that endothelial and perivascular cells are major sources of SCF. In their study conditional deletion of Scf from endothelial cells resulted in a significant reduction of HSCs in the bone marrow of transgenic mice compared to controls. The authors showed that endothelial cells and especially perivascular stromal cells were the principal source of SCF for HSC maintenance. Nevertheless, perivascular stromal cells are probably heterogeneous and may include multiple cell types that contribute to HSC maintenance through additional mechanisms other than SCF secretion [Ding et al., 2012]. The aim of this study was to generate mice which express Tk under the control of the 3.6Col1α1 promoter in an immunocompromised (Rag) background in order to evaluate the ability of circulating human peripheral hematopoietic lineage negative/AP+ (lin−/AP+) cells to support hematopoiesis in vivo and to compare these results with the effect of human MSCs.

We isolate and characterize osteoblasts from humans without in vitro culture. These techniques should be broadly applicable to studying the pathogenesis of osteoporosis and other bone disorders.There is currently no data regarding the expression of specific genes or pathways in human osteoblasts that have not been subjected to extensive in vitro culture. Thus, we developed methods to rapidly isolate progressively enriched osteoblast populations from humans and characterized these cells.Needle bone biopsies of the posterior iliac crest were subjected to sequential collagenase digests. The cells from the second digest were stained with an alkaline phosphatase (AP) antibody, and the AP+ cells were isolated using magnetic cell sorting.Relative to AP- cells, the AP+ cells contained virtually all of the mineralizing cells and were enriched for key osteoblast marker genes. The AP+ cells were further purified by depletion of cells expressing CD45, CD34, or CD31 (AP+/CD45/34/31- cells), which represented a highly enriched human osteoblast population devoid of hematopoietic/endothelial cells. These cells expressed osteoblast marker genes but very low to undetectable levels of SOST. We next used high-throughput RNA sequencing to compare the transcriptome of the AP+/CD45/34/31- cells to human fibroblasts and identified genes and pathways expressed only in human osteoblasts in vivo, but not in fibroblasts, including 448 genes unique to human osteoblasts.We provide a detailed characterization of highly enriched human osteoblast populations without in vitro culture. These techniques should be broadly applicable to studying the pathogenesis of osteoporosis and other bone disorders.

Studies on the pathogenesis of osteoporosis and other metabolic bone diseases would be greatly facilitated by the development of approaches to assess changes in gene expression in osteoblast/osteoprogenitor populations in vivo without the potentially confounding effects of in vitro culture and expansion of the cells. While positive selection to identify a progenitor population in human marrow can be used to select for cells capable of osteoblast differentiation, each of the markers that have been used to identify marrow mesenchymal populations (alkaline phosphatase [AP], Stro-1, CD29, CD49a, CD73, CD90, CD105, CD166, CD44, CD146 and CD271) may be expressed on distinct subsets of marrow mesenchymal cells. Thus, positive selection with one or more of these markers could exclude a possibly relevant cell population that may undergo important changes in various clinical conditions. In the present report, we describe the isolation and characterization of human osteoprogenitor cells obtained by depletion of bone marrow cells of all hematopoietic lineage/hematopoietic stem cells and endothelial/endothelial precursor cells (lin−/CD34/CD31−). The yield of lin−/CD34/CD31− cells from ~10 mL of bone marrow (~ 80 million mononuclear cells) was ~80,000 cells (0.1% of mononuclear cells). While not selected on the basis of expression for the mesenchymal marker, Stro-1, 68% of these cells were Stro-1+. Using linear whole transcriptome amplification followed by quantitative polymerase chain reaction (QPCR) analysis, we also demonstrated that, compared to lin− cells (which are already depleted of hematopoietic cells), lin−/CD34/31− cells expressed markedly lower mRNA levels for the endothelial/hematopoietic markers, CD34, CD31, CD45, and CD133. Lin−/CD34/31− cells were also enriched for the expression of mesenchymal/osteoblastic markers, with a further increase in runx2, osterix, and AP mRNA expression following in vitro culture under osteogenic conditions. Importantly, lin−/CD34/31− cells contained virtually all of the mineralizing cells in human marrow: while these cells displayed robust calcium deposition in vitro, lin−/CD34/31+ cells demonstrated little or no mineralization when cultured under identical osteogenic conditions. Lin−/CD34/31− cells thus represent a human bone marrow population highly enriched for mesenchymal/osteoblast progenitor cells that can be analyzed without in vitro culture in various metabolic bone disorders, including osteoporosis and aging.

While female mice do not have the equivalent of a menopause, they do undergo reproductive senescence. Thus, to dissociate the effects of aging versus estrogen deficiency on age-related bone loss, we sham-operated, ovariectomized, or ovariectomized and estrogen-replaced female C57/BL6 mice at 6 months of age and followed them to age 18 to 22 months. Lumbar spines and femurs were excised for analysis, and bone marrow hematopoietic lineage negative (lin-) cells (enriched for osteoprogenitor cells) were isolated for gene expression studies. Six-month-old intact control mice were euthanized to define baseline parameters. Compared with young mice, aged/sham-operated mice had a 42% reduction in lumbar spine bone volume/total volume (BV/TV), and maintaining constant estrogen levels over life in ovariectomized/estrogen-treated mice did not prevent age-related trabecular bone loss at this site. By contrast, lifelong estrogen treatment of ovariectomized mice completely prevented the age-related reduction in cortical volumetric bone mineral density (vBMD) and thickness at the tibial diaphysis present in the aged/sham-operated mice. As compared with cells from young mice, lin- cells from aged/sham-operated mice expressed significantly higher mRNA levels for osteoblast differentiation and proliferation marker genes. These data thus demonstrate that, in mice, age-related loss of cortical bone in the appendicular skeleton, but not loss of trabecular bone in the spine, can be prevented by maintaining constant estrogen levels over life. The observed increase in osteoblastic differentiation and proliferation marker gene expression in progenitor bone marrow cells from aged versus young mice may represent a compensatory mechanism in response to ongoing bone loss.

Precise delineation of the specific genes and pathways altered with aging and estrogen (E) therapy may lead to new skeletal biomarkers and the development of novel bone therapeutics. Previous human bone studies, however, have been limited by only examining pre-specified genes and pathways. High-throughput RNA sequencing (RNAseq), on the other hand, offers an unbiased approach to examine the entire transcriptome. Here we present an RNAseq analysis of human bone samples, obtained from iliac crest needle biopsies, to yield the first in vivo interrogation of all genes and pathways that may be altered in bone with aging and E therapy in humans. 58 healthy women were studied, including 19 young women (mean age ± SD, 30.3 ± 5.4 years), 19 old women (73.1 ± 6.6 years), and 20 old women treated with 3 weeks of E therapy (70.5 ± 5.2 years). Using generally accepted criteria (false discovery rate [q] < 0.10), aging altered a total of 678 genes and 12 pathways, including a subset known to regulate bone metabolism (e.g., Notch). Interestingly, the LEF1 transcription factor, which is a classical downstream target of the Wnt/β-catenin signaling pathway, was significantly downregulated in the bones from the old versus young women; consistent with this, LEF1 binding sites were significantly enriched in the promoter regions of the differentially expressed genes in the old versus young women, suggesting that aging was associated with alterations in Wnt signaling in bone. Further, of the 21 unique genes altered in bone by E therapy, the expression of INHBB (encoding for the inhibin, beta B polypeptide), which decreased with aging (by 0.6-fold), was restored to young adult levels in response to E therapy. In conclusion, our data demonstrate that aging alters a substantial portion of the skeletal transcriptome, whereas E therapy appears to have significant, albeit less wide-ranging effects. These data provide a valuable resource for the potential identification of novel biomarkers associated with age-related bone loss and also highlight potential pathways that could be targeted to treat osteoporosis. Trial Registration ClinicalTrials.gov {"type":"clinical-trial","attrs":{"text":"NCT02349113","term_id":"NCT02349113"}}NCT02349113

Hematopoietic stem cell (HSC) self-renewal is regulated by osteoblast and/or endothelial cells within the hematopoietic niche. However, the true identity of the supporting cells and the nature of the secreted factors remain uncertain. We developed a novel mouse model and analyzed whether circulating human peripheral hematopoietic lineage negative/AP+ (lin−/AP+) cells support hematopoiesis in vivo. Thus, immunocompromised (Rag) mice expressing thymidine kinase (Tk) under the control of the 3.6Col1α1 promoter (Tk-Rag) were treated with ganciclovir, resulting in osteoblast progenitor cell ablation and subsequent loss of hematopoiesis (evaluated by measuring mouse Ter119+ erythroid cells). Following hematopoietic cell depletion, human bone marrow-derived marrow stromal cells (MSCs) or lin−/AP+ cells were infused into Tk-Rag mice and compared with saline infusions. Ganciclovir significantly reduced (7.4-fold) Ter119+cells in the bone marrow of Tk-Rag mice compared to saline injections. Infusion of either MSCs or lin−/AP+ cells into ganciclovir-treated mice resulted in a 3.3-fold and 2.7-fold increase (P

The effects of 17-β-estradiol in osteoblasts are primarily mediated by the nuclear transcription factors, estrogen receptor (ER)α and ERβ. ERs function through three general modes of action: DNA-binding dependent through estrogen response elements (EREs; designated nuclear ERE signaling); nuclear signaling via protein-protein interactions to other transcription factors (nuclear non-ERE signaling); and extra-nuclear signaling (membrane-bound functions of ERs). Identification of the specific transcriptional signatures regulated by each of these modes of action should contribute to an enhanced understanding of estrogen signaling in osteoblasts. To achieve this goal, we utilized specific mutations of ERa that eliminate the ability of the receptor to signal through a specific mode of action. The non-classical ERα knock-in (NERKI) mutation is incapable of signaling through direct DNA binding to EREs and the nuclear only ERα (NOER) mutation eliminates all membrane-localized signaling. Comparison of the gene expression patterns elicited by these mutations with the wild-type ERα (WT) pattern provides mode-specific data concerning transcriptional regulation by ERα. We expressed these constructs in the ER-negative osteoblastic cell line hFOB (-/+ estrogen) and performed global RNA-sequencing. Using a series of pairwise comparisons, we generated three lists of genes that were regulated either by the nuclear ERE-dependent, nuclear ERE-independent, or extra-nuclear actions of ERα. Pathway and gene ontology analyses revealed that genes regulated through the nuclear ERE and nuclear non-ERE pathways were largely involved in transcriptional regulation, whereas genes regulated through extra-nuclear mechanisms are involved in cytoplasmic signaling transduction pathways. We also intersected our data with genes linked to bone density and fractures from a recent genome-wide association study and found 25 of 72 genes (35%) regulated by estrogen. These data provide a comprehensive list of genes and pathways targeted by these specific modes of ERα action and suggest that "mode-specific" ligands could be developed to modulate specific ERα functionality in bone. © 2014 Roforth et al.

Carboranyl aldehydes react with alpha,beta-unsaturated esters, ketones, and nitriles in the presence of DABCO to provide functionalized carboranyl alcohols in good yields. Acetates of these alcohols undergo a facile isomerization with a variety of nucleophiles and afford structurally interesting carboranes. Biological evaluation of these molecules exhibited impressive antiproliferative activity for brain and breast cancer cells.

Context: Studies in postmenopausal women have shown that estrogen reduces circulating sclerostin levels, but effects of estrogen on skeletal sclerostin mRNA levels are unknown. Objective: The objective of the study was to evaluate the effects of short-term estrogen treatment on bone mRNA levels of sclerostin and other genes relevant to bone metabolism. Design, Setting, and Patients: Needle bone biopsies were obtained from 20 postmenopausal women treated with transdermal estrogen for 3 weeks and 20 untreated controls. Quantitative PCR analyses were used to examine the expression of sclerostin and other genes related to bone metabolism, including 71 additional genes linked to bone density/fracture from genome-wide association studies. Results: Estrogen treatment was associated with lower bone sclerostin mRNA levels (by 48%, P < .05) and with lower (by 54%, P < .01) mRNA levels of the sclerostin-related protein, sclerostin domain-containing protein 1 (SOSTDC1), which is also a Wnt/bone morphogenetic protein inhibitor. Consistent with studies in mice showing that ovariectomy increased nuclear factor-κB (NF-κB) activation, we found that estrogen treatment was associated with a significant reduction in inflammatory genes as a group (P = .028), with bone mRNA levels of NFKB2 and RELB (both encoding proteins in the NF-κB transcription factor complex) being significantly reduced individual genes. Eight of the 71 genome-wide association study-related genes examined were modulated by estrogen (P < .05, false discovery rate < 0.10). Conclusion: In humans, estrogen-induced decreases in two key inhibitors of Wnt/bone morphogenetic protein signaling, sclerostin and SOSTDC1, along with reductions in NF-κB signaling, may be responsible for at least part of the protective effects of estrogen on bone.

Control of osteoblastic bone formation involves the cumulative action of numerous transcription factors, including both activating and repressive functions that are important during specific stages of differentiation. The nuclear receptor retinoic acid receptor-related orphan receptor β (Rorβ) has been recently shown to suppress the osteogenic phenotype in cultured osteoblasts, and is highly upregulated in bone marrow-derived osteogenic precursors isolated from aged osteoporotic mice, suggesting Rorβ is an important regulator of osteoblast function. However the specific gene expression patterns elicited by Rorβ are unknown. Using microarray analysis, we identified 281 genes regulated by Rorβ in an MC3T3-E1 mouse osteoblast cell model (MC3T3-Rorβ-GFP). Pathway analysis revealed alterations in genes involved in MAPK signaling, genes involved in extracellular matrix (ECM) regulation, and cytokine-receptor interactions. Whereas the identified Rorβ-regulated ECM genes normally decline during osteoblastic differentiation, they were highly upregulated in this non-mineralizing MC3T3-Rorβ-GFP model system, suggesting that Rorβ may exert its anti-osteogenic effects through ECM disruption. Consistent with these in vitro findings, the expression of both RORβ and a subset of RORβ-regulated genes were increased in bone biopsies from postmenopausal women (73 ± 7 years old) compared to premenopausal women (30 ± 5 years old), suggesting a role for RORβ in human age-related bone loss. Collectively, these data demonstrate that Rorβ regulates known osteogenic pathways, and may represent a novel therapeutic target for age-associated bone loss.

Purkinje cell cytoplasmic antibody, type Tr (PCA-Tr) is typically encountered in patients with Hodgkin lymphoma–related paraneoplastic cerebellar ataxia.1 Immunohistochemical techniques, including indirect immunofluorescence assays (IFAs), employing rodent brain as substrate, reveal a specific immunoglobulin G (IgG) binding pattern on cerebellar Purkinje cell cytoplasm and molecular layer dendritic spines.2 PCA-Tr characterization as delta/notch-like epidermal growth factor–related receptor (DNER)–IgG has permitted antigen-specific assay development.3 With rare exception, the phenotype encountered is cerebellar ataxia among patients with Hodgkin lymphoma. Tr autoimmunity is occasionally reported in some patients with noncerebellar neurologic disorders, and in others with non-Hodgkin lymphoma.4 Acknowledgment: The authors thank John Schmeling for technical support and the Center for Individualized Medicine and Department of Laboratory Medicine and Pathology, Mayo Clinic, for funding.

The mechanisms of estrogen receptor (ER)-α activity can be categorized into those involving direct (classical) or indirect (nonclassical) DNA binding. Although various mouse models have demonstrated the importance of ERα in bone, the specific gene expression patterns affected by these modes of ERα action are unknown. In this report, the gene expression patterns of ERα-deficient (ERKO) mice and nonclassical ER knock-in (NERKI) mice, which can function only by nonclassical means, were analyzed. Three-month-old mice were ovariectomized and implanted with estrogen pellets for 1 month to normalize estrogen levels. Microarray analysis of flushed cortical bone revealed 28% (210 of 763) of the genes differentially expressed in ERKO mice were altered in NERKI mice, suggesting estrogen response element-dependent regulation of these genes in bone. Pathway analysis revealed alterations in genes involved in focal adhesion and extracellular matrix interactions. However, the majority of genes regulated in ERKO mice (72%) were unique (i.e. not altered in NERKI mice), suggesting these are regulated by nonclassical mechanisms. To further explore the pathways affected in ERKO mice, we performed focused quantitative PCR arrays for genes involved in various aspects of bone physiology. Genes involved in bone formation, senescence, apoptosis, and autophagy were significantly regulated. Overall, the majority of the genes regulated by ERα in bone are via nonclassical pathways. However, because NERKI mice display an osteoporotic phenotype, it can be deduced that the minority of the estrogen response element-dependent genes/pathways play critical roles in the regulation of bone physiology. These data demonstrate the importance of classical ERα signaling in regulating bone metabolism.

A complex network of transcription factors contributes to the establishment and maintenance of the osteoblastic phenotype. Although relatively few transcription factors, such as Runx2 and osterix, are essential to the process of osteoblastic differentiation, others serve the purpose of fine-tuning in response to various environmental and hormonal cues. The nuclear receptor (NR) superfamily of transcription factors are involved in numerous aspects of bone biology. In this study, we characterized the expression pattern of the entire NR superfamily in differentiating primary murine calvarial cells in order to identify novel NR regulatory patterns. Dynamic patterns of NR expression were observed throughout the differentiation process. Interestingly, retinoic acid receptor-related orphan receptor β (Rorβ) expression was markedly suppressed at later stages of differentiation. To gain further insight into the function of NRs in bone biology, the NR superfamily was also profiled in mouse bone marrow precursor cells isolated from either young (6-month) or aging, osteoporotic (18-22-month) mice. Of interest, Rorβ was potently overexpressed in the aged cohort. Collectively, these data provided evidence that Rorβ expression is inversely correlated with osteogenic potential, suggesting Rorβ may be an important and unexplored regulator of osteogenesis. To validate this hypothesis, a cell model stably expressing Rorβ in mouse osteoblastic MC3T3-E1 cells was produced (MC3T3-Rorβ). These cells displayed markedly suppressed bone nodule formation as well as reduced osteocalcin and osterix gene expression. Because these genes are Runx2 targets, we reasoned that Rorβ may interfere with Runx2 activity. Consistent with this, transient transfection analysis demonstrated that Rorβ inhibited Runx2-dependent activation of a Runx2-reporter construct. In summary, our data provide a comprehensive profile of NR expression during osteoblast differentiation and identify Rorβ as a novel regulator of osteogenesis and potentially of age-related bone loss through antagonism of Runx2 activity.

Intermittent parathyroid hormone (PTH) 1–34 treatment stimulates bone formation, but the molecular mechanisms mediating this effect have not been previously studied in humans. Thus, we used magnetic activated cell sorting to isolate hematopoietic lineage negative (lin−)/alkaline phosphatase positive (AP+) osteoprogenitor cells from bone marrow of 20 postmenopausal women treated with PTH (1–34) for 14 days and 19 control subjects. Serum PINP and CTX increased in PTH-treated subjects (by 97% and 30%, respectively, P < 0.001). Bone marrow lin−/AP+ cells from PTH-treated subjects showed an increase in the RANKL/OPG mRNA ratio (by 7.5-fold, P = 0.011) and in the mRNAs for c-fos (a known PTH-responsive gene, by 42%, P = 0.035) and VEGF-C (by 57%, P = 0.046). Gene Set Enrichment Analysis (GSEA, testing for changes in pre-specified pathways) demonstrated that PTH had no effect on osteoblast proliferation, apoptosis, or differentiation markers. However, PTH treatment resulted in a significant decrease (GSEA P-value, 0.005) in a panel of BMP target genes in the lin−/AP+ cells. Our findings thus identify several future directions for studying mechanisms of PTH action in humans. First, given the increasing evidence that PTH induces angiogenesis, the role of increased VEGF-C production by bone marrow osteoprogenitor cells in mediating this effect and the anabolic response to PTH warrants further study. Second, while the observed inhibition of BMP target gene expression by PTH is not consistent with the anabolic effects of PTH on bone and requires further validation, these data do generate the hypothesis that an inhibition of BMP signaling by PTH may, over time, limit the availability of mature osteoblasts on bone surfaces and thereby contribute to the observed waning of the anabolic response to PTH.

Increased Akt phosphorylation was reported in cancer cell lines and tumor tissues of patients exposed to rapamycin, a response likely contributing to the attenuated antitumor activity of rapamycin. It is, therefore, necessary to develop and validate combination strategies to reverse rapamycin-induced Akt signaling. We now report that Akt activation in response to rapamycin is abrogated by 17-allylamino-17-demethoxygeldanamycin (17-AAG), a heat shock protein 90 (HSP90) inhibitor. Rapamycin/17-AAG combination results in an enhanced antiproliferative activity in both MCF-7 and MDA-MB-231 breast cancer cells. In combination 17-AAG confers potent suppression of Raf-MEK-extracellular signal-regulated kinase signaling, a pathway that is otherwise not inhibited by rapamycin individually. Importantly, 17-AAG cooperates with rapamycin to block the phosphorylation of the mammalian target of rapamycin at Ser2448, as well as its downstream effectors ribosomal p70 S6 kinase and eukaryotic initiation factor 4E binding protein 1, which is accompanied by a substantial reduction in cyclins D1 and E. The potency of rapamycin/17-AAG combination is not affected by the activation of insulin-like growth factor 1 receptor signaling, which has been previously shown to diminish the antiproliferative activity of rapamycin. Rapamycin/17-AAG combination alleviates the induction of HSP90 protein, a heat shock response frequently associated with 17-AAG monotherapy. Our findings establish a mechanistic rationale for a combination approach using rapamycin and 17-AAG in the treatment of breast cancer.

Although allosensitization results in the priming of both cellular and humoral immunity, our data indicate that preformed antibody is the major barrier to successful bone marrow transplantation (BMT) in the sensitized recipient. A single priming event led to rapid, high, long-lived antibody levels that practically precluded waiting for sufficient waning of antibody titer for successful BMT. C57BL/6 (B6) mice, whether primed against BALB/c alloantigen 7 days or 4 months before BMT, died of graft failure by 2 wks after transplantation. Antibody-mediated BM rejection was not entirely antigen specific as B6 mice primed against BALB/c also rejected B10.BR BM albeit more slowly than BALB/c BM. However, B6 mice primed against B10.BR rejected B10.BR BM but not BALB/c BM. Examination of serum from primed B6 mice indicated that BALB/c priming resulted in a more vigorous and broadly cross-reactive antibody response than did B10.BR priming perhaps due to the multiple minor antigen differences (as well as MHC disparity) that exist between BALB/c and B6 mice but not between B10.BR and B6 mice. Moreover, BM incubated with serum from primed mice was rejected in non-primed recipients indicating that ex vivo antibody-coating of donor BM was sufficient for its elimination and that continual in vivo exposure to high titer antibody was not essential for rejection. Antibody-mediated rejection was formally shown to be dependent upon host Fc receptor+ cells. We hypothesized that if donor BM cells could be delivered directly to the bone marrow cavity they might bypass destruction by the RES system or a BM homing defect, but direct intra-BM infusion of donor BM did not abrogate rejection in primed recipients. Imaging studies using B6 GFP+ BM indicated that antibody-mediated BM rejection was complete by 18 hrs in B6-primed BALB/c mice. In contrast, T cell-mediated rejection in non-primed mice took >6 days with donor BM first eliminated from lymphoid organs and later, from the BM cavity and parenchymal organs. We hypothesized that mega-BM doses might overcome primed rejection however, increasing the BM cell dose 10-fold was sufficient to overcome T cell priming in B cell-deficient muMT mice but not antibody-mediated rejection in WT mice. Despite the formidable barrier to engraftment, high donor chimerism levels were achieved in a proportion of primed mice by the combination of high dose murine immunoglobulin (mIg) infusion pre-BMT, in vivo T cell-depletion peri-BMT and mega-BM dose. The addition of splenectomy pre-BMT to this protocol only modestly added to the efficacy of this combination strategy. Although mIg, especially in combination with splenectomy, reduced antibody levels, the reduction was unlikely to be of sufficient magnitude to account for the increased survival and engraftment seen in mice when used in conjunction with in vivo TCD. The efficacy of mIg was more likely due to inhibition of Fc receptor-mediated clearance of antibody-bound cells. The heretofore unappreciated rapidity of antibody-mediated BM rejection in allosensitized recipients indicates there is no window of opportunity for clinical intervention after BMT and dictates the need for greater patient evaluation and therapeutic strategies prior to BMT.