Inflammatory Bone Marrow Research Articles

INASL Guidelines on Management of Hepatitis B Virus Infection in Patients receiving Chemotherapy, Biologicals, Immunosupressants, or Corticosteroids

Hepatitis B Virus (HBV) reactivation in patients receiving chemotherapy, biologicals, immunosupressants, or corticosteroids is emerging to be an important cause of morbidity and mortality in patients with current or prior exposure to HBV infection. These patients suffer a dual onslaught of illness: one from the primary disease for which they are receiving the culprit drug that led to HBV reactivation, and the other from HBV reactivation itself. The HBV reactivation not only leads to a compromised liver function, which may culminate into hepatic failure; it also adversely impacts the treatment outcome of the primary illness. Hence, identification of patients at risk of reactivation before starting these drugs, and starting treatment aimed at prevention of HBV reactivation is the best strategy of managing these patients. There are no Indian guidelines on management of HBV infection in patients receiving chemotherapy, biologicals, immunosupressants, or corticosteroids for the treatment of rheumatologic conditions, malignancies, inflammatory bowel disease, dermatologic conditions, or solid-organ or bone marrow transplantation. The Indian National Association for Study of the Liver (INASL) had set up a taskforce on HBV in 2016, with a mandate to develop consensus guidelines for management of various aspects of HBV infection, relevant to India. In 2017 the taskforce had published the first INASL guidelines on management of HBV infection in India. In the present guidelines, which are in continuation with the previous guidelines, the issues on management of HBV infection in patients receiving chemotherapy, biologicals, immunosupressants, or corticosteroids are addressed.

Osteomyelitis in adults: A prospective study of 60 cases

Introduction: The exact definition of osteomyelitis is inflammation of bone or bone marrow or both, but for all practical purposes this inflammation invariably is the result of infection. Various factors responsible for osteomyelitis include vascular insufficiency due to any cause, hematogenous spread of infection from a distant focus and surgery or trauma. Acute Osteomyelitis usually presents with fever, chills, pain or irritability. The classic signs of inflammation, including swelling, or redness and limited joint movement may also occur. Chronic osteomyelitis may present with bone destruction and sequestrum formation and is a difficult form of osteomyelitis to treat. Aims and Objectives: To study demographic details, organisms involved and outcome of treatment in patients with osteomyelitis. Materials and Methods: After obtaining approval from institutional ethical committee we conducted a prospective study of 60 patients diagnosed with either acute, subacute or chronic osteomyelitis on the basis of imaging and culture and sensitivity. Patients were included in this study on the basis of predefined inclusion criteria and were treated by antibiotics and when necessary surgical intervention was done. In cases of skeletal tuberculosis appropriate antikochs treatment was given. Outcome of treatment was studied over a follow up period of 1 year. The data was tabulated and analyzed using SPSS 16.0 version software. Results: Out of 60 cases of either acute, subacute or chronic osteomyelitis there were 38 males (63.33%) and 22 females (36.66%) with a M:F ratio of 1:0.57. Most common age group affected was found to be between 51-60 (36.66%) years. Acute, subacute and chronic osteomyelitis was seen in 71.66%, 11.66% and 16.66% patients respectively. Most common bones involved were Tibia (20%) and Femur (18.33%) followed by iliac bones (15%) fibula (11.66%) and vertebrae (11.66%). In most of the cases (36/60) contagious spread or trauma was the mechanism of infection. Comorbidities like hypertension, diabetes, chemotherapeutic agents or steroid intake and immunosuppression was present in 32 (53.33%) patients. S. Aureus (25/60) followed by Pseudomonas (7/60) and enterococci (7/60) were commonly isolated organisms. Atypical mycobacterial infection was seen in 1 patient who was immunocompromised. 40 patients were completely cured while remaining 10 patients had some or the other problem associated with chronic osteomyelitis. Amputations was done in 4 cases and septic arthritis developed in other 2 cases. 4 patients died during study period due to causes unrelated to osteomyelitis. Conclusion: Diagnosis as well as management of osteomyelitis is a challenge for treating orthopaedician. Knowledge of predisposing factors, presenting complaints, possible complications and proper management is essential for successful management of acute as well as chronic osteomyelitis.

Feasibility and reliability of the Spondyloarthritis Research Consortium of Canada sacroiliac joint inflammation score in children

BackgroundPublished methods for quantification of magnetic resonance imaging (MRI) evidence of inflammation in the sacroiliac joint lack validation in pediatric populations. We evaluated the reliability and construct validity of the Spondyloarthritis Research Consortium of Canada (SPARCC) sacroiliac joint inflammation score (SIS) in children with suspected or confirmed juvenile spondyloarthritis (JSpA).MethodsThe SPARCC SIS measures the presence, depth, and intensity of bone marrow inflammation on MRI through the cartilaginous part of the joint. Six readers blinded to clinical details except age, participated in two reading exercises, each preceded by a calibration exercise. Inter-observer reliability was assessed using intraclass correlation coefficients (ICCs) and for pre-specified acceptable reliability the inraclass correlation coefficient (ICC) was > 0.8.ResultsThe SPARCC SIS had face validity and was feasible to score in pediatric cases in both reading exercises. Cases were mostly male (64%) and the median age at the time of imaging was 14.9 years. After calibration, the median ICC across all readers for the SIS total score was 0.81 (IQR 0.71–0.89). SPARCC SIS had weak correlation with disease activity (DA) as measured by the JSpADA (r = − 0.12) but discriminated significantly between those with and without elevated C-reactive protein (p = 0.03).ConclusionThe SPARCC SIS was feasible to score and had acceptable reliability in children. The ICC improved with additional calibration and reading exercises, for both experienced and inexperienced readers.

Non-parenchymal TREM-2 protects the liver from immune-mediated hepatocellular damage

ObjectiveLiver injury impacts hepatic inflammation in part via Toll-like receptor (TLR) signalling. Triggering receptor expressed on myeloid cells 2 (TREM-2) modulates TLR4-mediated inflammation in bone marrow (BM)-derived macrophages but its...

Modic type 1 change is an autoimmune response that requires a proinflammatory milieu provided by the ‘Modic disc’

Background ContextModic changes (MCs) are magnetic resonance imaging (MRI) evidence of inflammatory and fibrotic vertebral bone marrow lesions that associate with adjacent disc degeneration and end plate damage. Although MC etiology is uncertain, historical data suggest a linkage to an autoimmune response of bone marrow triggered by the nucleus pulposus (NP). PurposeThe aim of this study was to test whether bone marrow has an autoimmune response to NP cells that is amplified by an inflammatory milieu and ultimately leads to MC development in vivo. We hypothesized that an inflammatory co-stimulus is required for bone marrow/NP crosstalk to stimulate MC. Study DesignThis is an in-vitro cell co-culture study plus in-vivo experiments in rat caudal vertebrae. MethodsIn in-vitro study, bone marrow mononuclear cells (BMNCs) and NP cells (NPCs) from rats were co-cultured with and without interleukin (IL)-1α stimulation. Cell viability (n=3) of BMNCs and NPCs and gene expression (n=7) were analyzed. In in-vivo study, proinflammatory lipopolysaccharide (LPS) and control disc nucleus surrogates (NP micromass pellets) were generated in vitro from rat NPCs and implanted into rat tail vertebrae, and the response was compared with sham surgery (n=12 each). Tissue changes were investigated with T1w and T2w MRI (7T), histology, and immunohistochemistry (tumor necrosis factor, CD3) 1 (n=6) and 2 weeks (n=6) after implantation. ResultsBMNC/NPC co-culture significantly increased lymphocyte viability (42%–69%, p<.05) and reduced NPC viability (96%–88%, p<.001), indicating immunogenicity of NPC. However, IL-1α was required to cause significant transcriptional upregulation of IL-1, IL-6, IL-10, and tropomyosin receptor kinase A. Therefore, an inflammatory activation is required to amplify the immune response. Immunogenicity of the NP was corroborated in vivo by CD3 cell accumulation around LPS and control disc surrogates at Day 7. However, only the LPS disc surrogate group demonstrated infiltration of CD3 cells at Day 14. Furthermore, end plate defects (p<.05, LPS: n=4/6, Ctrl: n=0/6, sham: n=0/6) and MC1-like MRI changes (T2w hyperintensity, p<.05) were only seen with LPS disc surrogates. ConclusionsNPCs are immunogenic but cannot trigger MC without an additional proinflammatory stimulus. Our data suggest that MC requires end plate defects that allow marrow/NPC co-mingling plus an adjacent inflammatory “MC disc” that can amplify the immune response.

Preliminary Data Show Heterogenous Patterns of 18f-FDG PET/CT When Assessing the Inflammatory Component of Primary or Secondary Myelofibrosis

Abstract Background. Recent studies have highlighted the interest of non-invasive procedures to assess extramedullary hematopoiesis in patients with overt myelofibrosis, primary (PMF) or secondary (sMF) to essential thrombocytemia (ET) or to polycythemia vera (PV) (1-4). Several nuclear medicine imaging techniques have been employed without clear consensus about the predominant value of one over other in these mostly preliminary works (2-4). Population and objectives. Because the pathophysiology of PMF or sMF involves some inflammatory pathways we studied the interest of 18F-FDG (18F-fluorodeoxyglucose) PET/CT in patients with PMF (5). The main objective was to know whether the inflammatory component of PMF could be well detected by this technique. Secondly, we looked for some kind of correlation between the FDG uptake on medullary and extramedullary sites of hematopoiesis and the severity of the disease. Five patients with diagnosis of MF (3 PMF and 2 sMF) were assessed (see Table 1 for baseline characteristics and results) Methods.18F-FDG PET/CT imaging was performed following a 60 min. uptake after intravenous injection of a weight-adapted FDG activity (2,5 MBq/kg) on a Siemens Biograph mCT. Images were reviewed by board certified nuclear medicine physicians. Spleen size was measured on axial slices. SUVmax were assessed in a 5 mm diameter spheric volume of interest in homogenous parts of liver and spleen, and in the L2 vertebral body for axial skeleton assessment. Spleen uptake was considered as abnormal in case of a diffuse uptake exceeding hepatic baseline uptake. Skeleton uptake was considered as abnormal in case of diffuse increased uptake. Results. UPN01LP: slight higher metabolic activity in the axial skeleton and diffuse spleen uptake ; UPN02AP: no hypermetabolic activity in the spleen and slightly higher uptake in the axial skeleton ; UPN03AA: diffuse bone hypermetabolic activity and high metabolic activity in the spleen ; UPN05HL: diffuse bone hypermetabolic activity and higher metabolic activity in the spleen ; UPN06DF: no diffuse hypermetabolic activity in the spleen, slight higher uptake in the axial skeleton. In summary, those patients with overt myelofibrosis and constitutional signs tend to show a diffuse higher activity both in the axial skeleton and the spleen whereas those without overt myelofibrosis and without constitutional signs tend to present a normal metabolic activity in the axial skeleton and in the spleen. These patterns are in accordance with the progression and severity of the disease. The higher uptake of 18FDG in the axial skeleton in patients with overt MF contrasts with what is observed with some hybrid imaging techniques that show a lower radionuclide (111In/SPECT/CT) uptake at this level translating a lower hematopoietic activity (3). Conclusions. Based on these preliminary results we consider 18FDG-PET/CT procedures are able to detect the inflammatory component of PMF or sMF and the intensity of FDG uptake appears to be correlated to the severity of the disease. Those patients with a high metabolic activity in the axial skeleton and the spleen might be candidates for specific therapies, although these statements need to be confirmed in a larger series of patients. Bibliography. 1 Derlin T et al. Assessment of bone marrow inflammation in patients with myelofibrosis: an 18F-fluorodeoxyglucose PET/CT study. Eur J. of Nucl Med Mol Imaging 2015 2 Agool A et al. Radionuclide imaging of bone marrow disorders. Eur J Nucl Med Mol Imaging. 2011. 3 Ojeda-Uribe M et al. Assessment of sites of marrow and extramedullary hematopoiesis by hybrid imaging in primary myelofibrosis patients. Cancer Medicine 2016. 4 Vercellino L et al. Assessing bone marrow activity in patients with myelofibrosis: results of a pilot study of 18F-FLT PET. J. of Nucl Med 2017. 5 Desterke C et al. Inflammation as a Keystone of Bone Marrow Stroma Alterations in Primary Myelofibrosis. Mediators Inflamm. 2015 Download : Download high-res image (107KB) Download : Download full-size image Table 1 . Disclosures No relevant conflicts of interest to declare.

Bone Marrow Characterization in Sickle Cell Disease: Inflammation and Stress Erythropoiesis Lead to Suboptimal CD34 Recovery Compared to Normal Volunteer Bone Marrow

IntroductionGene therapy for sickle cell disease (SCD) requires modification of a high number of long term engrafting hematopoietic stem cells (LT-HSCs) sufficient to sustain production of the gene of interest at levels capable of overcoming the pathogenic HbSS phenotype. Unlike β-Thalassemia, the inflammatory bone marrow (BM) environment and stress erythropoiesis associated with SCD may have significant impacts on HSC quality and yield necessary for disease amelioration. Important work to optimize gene therapy through improvement in gene transfer efficiency, editing strategies, or transplant conditioning can only improve gene therapy in SCD if enough autologous HSCs are LT-HSCs, thus characterization of SCD BM and CD34+ HSCs is required. Collection type, storage, and delays in processing may further impact CD34+ recovery and should be investigated as a strategy to maximize LT-HSC recovery.MethodsTwenty milliliters of BM from subjects with SCD (HbSS genotype) and normal volunteers was collected in different anticoagulants (Heparin, ACD-A) and processed immediately(day 0) or stored at 40C and processed the following day(day 1). After isolation via Ficoll density gradient centrifugation, the mononuclear (MN) layer was stained with antibodies against inflammatory markers (CD36, CD35, CD11b, CD62L, CD62P), non-MN cells (GPA, CD66b, CD41/61), or processed for CD34+ selection using a magnetic microbead CD34+ selection kit and stained for CD34, CD45, and GPA expression. Data were analyzed by conventional and imaging flow cytometry, the latter confirming post-CD34+ selection flow data and demonstrating antibody intensity as a characterization of HSC heterogeneity and progenitor lineage. Complete blood count and hemoglobin (Hb) electrophoresis were obtained at the time of BM collection. Statistical analyses were performed using unpaired t-tests.ResultsBM was collected from 18 subjects (16 with SCD; 11M; age 21-41 years). Median Hb (8.6 vs. 13.5 gm/dL, p<0.01) and white blood cell count (8.8 vs. 4.2 K/mcL, p<0.05) differed significantly between SCD and non-SCD subjects. Median percent sickle Hb in SCD subjects was 62%. Inflammatory markers and contamination with red cell and platelet markers in the post-Ficoll MN layer were higher in SCD vs. non-SCD BM regardless of anticoagulant (CD35 24% vs. 13%, p<0.05; CD36 22% vs. 11%, p<0.05; CD62P 16% vs. 3%, p<0.05; GPA 16% vs. 4%, p<0.05; CD41/61 19% vs. 3%, p<0.05), and trended higher on day 1 in SCD BM in both anticoagulants, significantly in Heparin (GPA 23% vs. 33% on day 1, p<0.05). Total CD45 expression was lower in SCD vs. non-SCD BM in both anticoagulants (p<0.05) and on day 0 (p<0.05) and 1 (p<0.01), with Amnis data confirming a higher CD34+CD45- population in SCD BM (4 ± 2% vs. 0.5 ± 0.3%, p<0.05). While there was no significant difference in total CD34+ cell count between SCD and non-SCD BM after selection post-Ficoll, there was a trend for lower CD34+ count in SCD in both anticoagulants (2.6x10^5 vs. 4.7x10^5, p=0.1). SCD CD34+ cells were characterized by higher GPA expression (28 ± 5% vs. 13 ± 3% in non-SCD BM, p<0.01) that worsened in Heparin on day 1 (22 ± 6.3% vs. 35 ± 12.4%, p<0.05). Image cytometry confirmed a majority of GPA expression in SCD BM is from single cell CD34+CD45+GPA+ and CD34+CD45-GPA+ HSCs in addition to red cell aggregates, with an increase in CD34+CD45-GPA+ HSCs on day 1 (10 ± 5% vs. 0.6 ± 0.2 % on day 0, p<0.05). Furthermore, the percentage of CD34hi HSCs was lower in SCD vs. non-SCD BM, with >50% SCD HSCs characterized as CD34dim (56% vs. 4% in non-SCD BM, p<0.001). Lastly, the purity of CD34+ selection worsened from day 0 to day 1 in SCD BM in heparin (94% vs. 68 ± 8%, p<0.05) and ACD-A (88% vs. 68 ± 0.7%, p<0.05).ConclusionsSCD BM is characterized by increased inflammation and cell contamination in the MN layer regardless of anticoagulant that worsens over time in Heparin more significantly than in ACD-A. Compared to non-SCD BM, CD34+ HSC yield post-Ficoll is lower in SCD subjects, and is characterized by a larger proportion of CD34+CD45+GPA+ and CD34+CD45-GPA+ HSCs that rise with delays in processing. This indication of early differentiation along the erythroid lineage, with more than 50% of HSCs losing CD34+ intensity suggesting they are not LT-HSCs, suggests suppression of inflammation and stress erythropoiesis, combined with early cell processing may be critical for maximal HSC recovery necessary for successful gene therapy in SCD. [Display omitted] DisclosuresLuo:bluebird bio Inc.: Employment. Pierciey:bluebird bio: Employment.

Quantifying bone marrow inflammatory edema in the spine and sacroiliac joints with thresholding

BackgroundPsoriatic Arthritis (PsA) is a chronic inflammatory arthritis that develops in patients with psoriasis. Inflammatory edema in the spine may reflect subclinical disease activity and be a predictor of radiographic progression. A semi-quantitative method established by the spondyloarthritis research consortium of Canada (SPARCC) is commonly used to assess the disease activity in MR images of the spine. This study aims to evaluate thresholding for quantification of subtle bone marrow inflammation in the spine and the sacroiliac (SI) joints of patients with PsA and compare it with the SPARCC scoring system.MethodsShort tau inversion recovery (STIR) MR images of the spine (N = 85) and the SI joints (N = 95) of patients with PsA (N = 41) were analyzed. A threshold was applied to visible bone marrow in order to mask areas with higher signal intensity, which are consistent with inflammation. These areas were considered as inflammatory lesions. The volume and relative signal intensity of the lesions were calculated. Results from thresholding were compared to SPARCC scores using linear mixed-effects models. The specificity and sensitivity of thresholding were also calculated.ResultsA significant positive correlation between the volumes and mean relative signal intensities, which were calculated by thresholding analysis, and the SPARCC scores was detected for both spine (p < 0.001) and SI joints (p < 0.001). For the spine, thresholding had sensitivity and specificity of 83% and 76% respectively, while for the SI joints the values were 51% and 88% respectively.ConclusionsThresholding allows quantification of subtle bone marrow inflammatory edema in patients with psoriatic arthritis, and could support SPARCC scoring of the spine. Improved image processing and inclusion of automatic segmentation are required for thresholding of STIR images to become a rapid and reliable method for quantitative measures of inflammation.Trial registrationNCT02995460 (December 14, 2016) – Retrospectively registered.

Age-related inflammatory bone marrow microenvironment induces ineffective erythropoiesis mimicking del(5q) MDS

Anemia is characteristic of myelodysplastic syndromes (MDS). The mechanisms of anemia in MDS are unclear. Using a mouse genetic approach, here we show that dual deficiency of mDia1 and miR-146a, encoded on chromosome 5q and commonly deleted in MDS (del(5q) MDS), causes an age-related anemia and ineffective erythropoiesis mimicking human MDS. We demonstrate that the ageing bone marrow microenvironment is important for the development of ineffective erythropoiesis in these mice. Damage-associated molecular pattern molecules (DAMPs), whose levels increase in ageing bone marrow, induced TNFα and IL-6 upregulation in myeloid-derived suppressor cells (MDSCs) in mDia1/miR-146a double knockout mice. Mechanistically, we reveal that pathologic levels of TNFα and IL-6 inhibit erythroid colony formation and differentially affect terminal erythropoiesis through reactive oxygen species-induced caspase-3 activation and apoptosis. Treatment of the mDia1/miR-146a double knockout mice with all-trans retinoic acid, which promoted the differentiation of MDSCs and ameliorated the inflammatory bone marrow microenvironment, significantly rescued anemia and ineffective erythropoiesis. Our study underscores the dual roles of the ageing microenvironment and genetic abnormalities in the pathogenesis of ineffective erythropoiesis in del(5q) MDS.

Muscle paralysis induces bone marrow inflammation and predisposition to formation of giant osteoclasts.

Transient muscle paralysis engendered by a single injection of botulinum toxin A (BTxA) rapidly induces profound focal bone resorption within the medullary cavity of adjacent bones. While initially conceived as a model of mechanical disuse, osteoclastic resorption in this model is disproportionately severe compared with the modest gait defect that is created. Preliminary studies of bone marrow following muscle paralysis suggested acute upregulation of inflammatory cytokines, including TNF-α and IL-1. We therefore hypothesized that BTxA-induced muscle paralysis would rapidly alter the inflammatory microenvironment and the osteoclastic potential of bone marrow. We tested this hypothesis by defining the time course of inflammatory cell infiltration, osteoinflammatory cytokine expression, and alteration in osteoclastogenic potential in the tibia bone marrow following transient muscle paralysis of the calf muscles. Our findings identified inflammatory cell infiltration within 24 h of muscle paralysis. By 72 h, osteoclast fusion and pro-osteoclastic inflammatory gene expression were upregulated in tibia bone marrow. These alterations coincided with bone marrow becoming permissive to the formation of osteoclasts of greater size and greater nuclei numbers. Taken together, our data are consistent with the thesis that transient calf muscle paralysis induces acute inflammation within the marrow of the adjacent tibia and that these alterations are temporally consistent with a role in mediating muscle paralysis-induced bone resorption.

Amygdala Activity May Predict Future Cardiovascular Events

Back to table of contents Previous article Next article Clinical and Research NewsFull AccessAmygdala Activity May Predict Future Cardiovascular EventsAaron LevinAaron LevinSearch for more papers by this authorPublished Online:20 Apr 2017https://doi.org/10.1176/appi.pn.2017.3b8AbstractResearchers find a biological link between effects of stress on the amygdala and later cardiovascular events.Higher emotional stress as measured by resting metabolic activity of the amygdala is associated with greater risk of subsequent cardiovascular disease events, according to a study published online January 11 in The Lancet. The findings “provide novel insights into the mechanism through which emotional stressors can lead to cardiovascular disease,” wrote researchers led by Ahmed Tawakol, M.D., an associate professor of medicine at Massachusetts General Hospital and Harvard Medical School.Tawakol and colleagues hypothesized that activity in the amygdala in response to stress increases arterial inflammation both directly and by way of activity in bone marrow.“What is novel here is the bridging of multiple areas of research,” said Peter Gianaros, Ph.D., a professor of psychology and psychiatry and director of the Behavioral Neurophysiology Lab at the University of Pittsburgh. Gianaros has also studied the relationship between the amygdala and cardiovascular disease but was not involved with Tawakol’s study.“It builds on threads of prior research that pointed in this direction and makes strong connections with research on the neurobiology of stress as well as with clinical cardiology,” said Gianaros. “Now we have the proof of concept and a vision of the future.”The amygdala is known to be a key component of the brain’s stress network. Past studies in mice suggested that amygdala activity in response to stress accelerates cytokine production and atherosclerosis, but whether the same was true of humans was unknown.Tawakol and colleagues examined data originally derived from cancer screening imaging studies of 293 patients. Using positron emission tomography/computed tomography (PET/CT) scans, they analyzed activity in the amygdala and in bone marrow, as well as arterial inflammation.During the median follow-up of 3.7 years, 22 patients experienced a cardiovascular disease event—including heart attacks, strokes, angina, or other events. Each increase of one standard deviation in resting amygdala activity predicted a 1.6 risk of a cardiovascular event. The results were significant even after adjusting for established cardiovascular risk factors.Greater amygdala activity was also associated with increased bone marrow activity, which the authors said led to increased arterial inflammation followed by cardiovascular disease events.“[T]he brain’s salience network, bone marrow, and arterial inflammation together form an axis that could accelerate the development of cardiovascular disease,” concluded Tawakol and colleagues. “Furthermore, our findings raise the possibility that efforts to attenuate psychosocial stress could produce benefits that extend beyond an improved sense of psychological well-being and could beneficially impact the atherosclerotic milieu.”Clinicians might eventually screen for and treat chronic stress just as they do for other cardiovascular risk factors, they suggested.Realistically, clinical applications are still well down the road, and much needs to be done to move the field forward, said Gianaros.“We’ll need to set norms and thresholds for amygdala activity and see cost reductions in the methods used to assess it,” he said. For instance, it would be helpful to know more about what factors contribute to elevated amygdala activity, how people vary in their appraisal of stressors, and whether the amygdala sensitizes individuals to stressors in the environment or if the increased activity is a result of repeated stressors, he said.“This rigorous study is a good example of how multidisciplinary work can lead to potential breakthroughs or make connections that don’t happen when working in silos,” said Gianaros. ■An abstract of “Relation between resting amygdalar activity and cardiovascular events: a longitudinal and cohort study” can be accessed here. ISSUES NewArchived

Administration of an activin receptor IIB ligand trap protects male juvenile rhesus macaques from simian immunodeficiency virus-associated bone loss

HIV-infected individuals are at an increased risk of osteoporosis despite effective viral suppression. Observations that myostatin null mice have increased bone mass led us to hypothesize that simian immunodeficiency virus (SIV)-associated bone loss may be attenuated by blocking myostatin/TGFβ signaling. In this proof-of-concept study, pair-housed juvenile male rhesus macaques were inoculated with SIVmac239. Four weeks later, animals were treated with vehicle or Fc-conjugated soluble activin receptor IIB (ActR2B·Fc, iv. 10mg∗kg−1∗week−1) – an antagonist of myostatin and related members of TGFβ superfamily. Limb and trunk bone mineral content (BMC) and density (BMD) using dual-energy X-Ray absorptiometry, circulating markers of bone growth and turnover, and serum testosterone levels were measured at baseline and during the 12-week intervention period. The increase in BMC was significantly greater in the ActRIIB.Fc-treated group (+8g) than in the placebo group (−4g) (p<0.05). BMD also increased significantly more in the ActRIIB.Fc-treated macaques (+0.03g/cm2) than in the placebo-treated animals (+0g/cm2) (p<0.005). Serum osteocalcin was about two-fold higher in the ActRIIB.Fc-treated group than in the placebo group (p<0.05), but serum C-terminal telopeptide and testosterone levels did not differ significantly between groups. The expression levels of TNFalpha (p<0.05), GADD45 (p<0.005), and sclerostin (p<0.038) in the bone-marrow were significantly lower in the ActRIIB.Fc-treated group than in the placebo group. ConclusionThe administration of ActRIIB.FC in SIV-infected juvenile macaques significantly increases BMC and BMD in association with reduced expression levels of markers of bone marrow inflammation.

IL-32-induced Inflammatory Cytokines Are Selectively Suppressed by α1-antitrypsin in Mouse Bone Marrow Cells

The induction of interleukin (IL)-32 in bone marrow (BM) inflammation is crucial in graft versus host disease (GvHD) that is a common side effect of allogeneic BM transplantation. Clinical trials on α-1 antitrypsin (AAT) in patients with GvHD are based on the preliminary human and mouse studies on AAT reducing the severity of GvHD. Proteinase 3 (PR3) is an IL-32-binding protein that was isolated from human urine. IL-32 primarily induces inflammatory cytokines in myeloid cells, probably due to PR3 expression on the membrane of the myeloid lineage cells. The inhibitory activity of AAT on serine proteinases may explain the anti-inflammatory effect of AAT on GvHD. However, the anti-inflammatory activity of AAT on BM cells remains unclear. Mouse BM cells were treated with IL-32γ and different inflammatory stimuli to investigate the anti-inflammatory activity of AAT. Recombinant AAT-Fc fusion protein inhibited IL-32γ-induced IL-6 expression in BM cells, but failed to suppress that induced by other stimuli. In addition, the binding of IL-32γ to PR3 was abrogated by AAT-Fc. The data suggest that the specific anti-inflammatory effect of AAT in mouse BM cells is due to the blocking of IL-32 binding to membrane PR3.

Selected issues in diagnostic imaging of spondyloarthritides: psoriatic arthritis and juvenile spondyloarthritis.

Spondyloarthritides (also known as spondyloarthropathies) are a group of rheumatic diseases that consists of diversified entities, i.e. ankylosing spondylitis, reactive arthritis, psoriatic arthritis, arthritis in the course of Crohn’s disease and ulcerative colitis, and juvenile spondyloarthropathies. In the diagnostics of spondyloarthritides, plain radiography has played a crucial role for years due to its undisputed ability to show distinctive bony changes. Yet as those diseases often manifest themselves by soft tissue pathology and bone marrow inflammation, ultrasonography and magnetic resonance imaging are currently a subject of numerous studies in the quest for setting up diagnostic criteria, especially at early stages of inflammatory processes. In our review, we present an up-to-date insight into classifications, etiopathogenesis and imaging of psoriatic arthritis and juvenile spondyloarthritis.

Interleukin-32 Induce Production of IL-6 in Multiple Myeloma Bone Marrow Stromal Cells

Background: Multiple myeloma (MM) is closely associated with inflammation. Patients with auto-immune disease history of infection and other inflammatory disease have higher incidence of MM. IL-6 is the most important inflammatory factor in MM which plays a key role in the proliferation and progression. We previously demonstrated that MM cells were modified by bone marrow stromal cells (BMSCs) that formulate a inflammatory microenvironment in bone marrow (BM) and secret IL-6. How the inflammation makes BMSCs secret IL-6, however, remained undocumented. Our subsequent study compared the differential secretion of peripheral blood (PB) between MM patients and normal people by cytokine array, and showed that interleukin 32(IL-32) is highly expressed in MM patients. IL-32, also named natural killer 4(NK-4), is a newly found inflammatory factor. It was reported in solid tumors IL-32 is a pro-inflammatory factor which triggers a massive amplification of inflammatory process resulting in the change of other inflammatory factor including IL-6,IL-10,TNF-α. In this study, we examined BMSCs cytokines in MM BM and found that IL-32 was a functional factor in the process of inflammation in MM BM microenvironment.Results: First, to test our previous study, we detected IL-32 in BM supernatant and PB supernatant in both MM patients (n=45) and healthy controls (n=13) by ELISA. Result showed that in both BM and PB, MM patients have higher expression of IL-32 compared to healthy controls (P<0.05). Next, total BM cells(both CD138+ and CD138- cells) from MM patients were assayed by qRT-PCR for gene expression analysis.IL-32 were highly expressed in MM BM cells and the CD138+ cells (P<0.05). We also detected IL-32 in MM cell lines (RPMI 8226,OPM-2) and BMSCs isolated from MM patients by qRT-PCR, Western blot, and ELISA, and found that IL-32 was highly expressed in MM cell lines than BMSCs. In contrast, proteinase 3(PR3, receptor of IL-32) was highly expressed in BMSCs compared to MM cell lines.Second, we stimulated the MM BMSCs with recombinant IL-32α, and found that the secretion of IL-6,CCL3 (MIP1-α), CCL4(MIP-1β) were significantly increased and CCL-5(RANTES)and IL-10 were decreased (P<0.05). Further, Western blot was applied to detect the inflammation molecular pathway in BMSCs. JAK-STAT pathway and NF-κB pathway were activated, and the phosphorylation of STAT3 was increased. After we knock down the PR3 in BMSCs, these changes were reduced. We repeated these experiments in BMSCs isolated from 15 different MM patients, the phenomenon mentioned above showed in 11 patients. The recombinant IL-32α was also used to stimulate 8226 and OPM-2 cells, but these two kinds of MM cells didn’t secret IL-6, and no significant change in cell proliferation or cell apoptosis.Finally, our group co-cultured the MM BMSCs with 8226 and OPM-2 cells. The secretion of IL-6 and the phosphorylation of JAK-STAT pathway in BMSCs were also increased. Knockdown of IL-32 in 8226 and OPM-2 cells weakened these changes.MM Cell proliferation and cell cycles after co-culture with MM BMSCs are under investigation.Conclusion: our findings suggest that IL-32 is mainly secreted by MM cells. It may not directly promote the MM cells to grow. However, IL-32 promote the MM BMSCs to secret more cytokines including IL-6,CCL3,CCL4 by activating the JAK-STAT3 pathway, which lead to a amplification of inflammation in BM environment, resulting in the cell proliferation . DisclosuresNo relevant conflicts of interest to declare.

Single Cell Transcriptional Profiling Reveals Activation of TNF-Alpha Signaling in Hematopoietic Stem and Progenitor Cells from Shwachman-Diamond Syndrome Patients

Shwachman-Diamond Syndrome (SDS) is an inherited bone marrow failure caused by mutations in SBDS, which encodes a conserved ribosome assembly factor. Despite simple genetic underpinnings, SDS is surprisingly complex. Patients suffer varying degrees of neutropenia, thrombocytopenia and anemia, and are predisposed to myelodysplasia and acute myeloid leukemia. The only curative treatment is stem cell transplant, but patients are unusually susceptible to toxic side effects. Mapping molecular pathways in affected lineages is a critical step towards developing safer, targeted therapies.The affected cell type(s) and genetic networks in SDS have remained elusive, but bone marrow hypocellularity and the involvement of multiple lineages points to a defect in the CD34+ hematopoietic stem and progenitor cell (HSPC) population. To identify the molecular basis of SDS, we set out to transcriptionally profile HSPC from SDS patients. However, these cells are rare and heterogeneous, even in normal donors. To overcome this challenge, we developed a pipeline to define the transcriptional architecture of early hematopoiesis at single cell resolution.Key features include rapid conversion of fresh cells to stable cDNA libraries which preserves their natural biology and unbiased sampling to capture all aspects of HSPC heterogeneity. To date, we have sequenced full-length cDNA libraries from 92 normal donor and 176 SDS patient cells.To deconvolute developmental heterogeneity among HSPC, we clustered cells based on the expression of empirically-determined lineage signature genes. Normal and SDS cells were similarly distributed along the developmental continuum, though there was an increased proportion of SDS cells in the stem and multipotent progenitor stage (HSC/MPP). This analysis produced the first single cell roadmap of human hematopoiesis, which illustrates that hematopoiesis is a continuous process rather than a series of discrete steps.To identify genes that contribute to impaired hematopoiesis in SDS, we used the MAST statistical framework for single cell expression analysis. The top upregulated pathway in SDS was TNF-alpha signaling via NF-kB. Interestingly, when we mapped this pathway back to our single cell roadmap we found that it was activated to varying degrees in cells at the HSC/MPP stage, but not in more committed progenitors. This finding is intriguing given that TNF-alpha has suppressive effects on HSC growth and long-term engraftment in mice. Moreover, HSC from patients with Fanconi Anemia, a related bone marrow failure, are hypersensitive to TNF-alpha-mediated suppression.Our study establishes the first link between an inflammatory pathway, TNF-alpha, and bone marrow failure in SDS. We are currently investigating the mechanistic basis for this link using patient-derived induced pluripotent stem cells. In the future, we will examine whether TNF-alpha inhibition is a viable therapy to counteract bone marrow failure in SDS. DisclosuresNo relevant conflicts of interest to declare.

Nilotinib Plus Ruxolitinib Produces Anti-Fibrotic Effect

Myelofibrosis (MP) is characterized by stem cell-derived clonal myeloproliferation and a reactive cytokine-driven inflammatory bone marrow fibrosis (BMF). BMF plays an important role in MF symptom such us extramedullary hematopoiesis that implies hepatosplenomegaly and bone marrow failure with anemia and thrombocytopenia.Ruxolitinib is the only drug approved for MF therapy. It was associated with significant reduction in symptomatic splenomegaly and improved constitutional symptoms (Harrison et al. N Engl J Med. 2012 Mar 1;366(9):787-98; Verstovsek et al. Haematologica2015 Apr;100(4):479-88). Recently, Masarova and co-workers reported a case a MF patient who achieved a complete remission, including the complete disappearance of BMF (Masarova et al. Blood. 2016 Jun 20).In a previous work (Arenas et al. Blood Volume 122, Issue 21 (ASH Annual Meeting Abstrat)), we tried to identify the most promising synergistic drugs combinations for a Ruxolitinib. Nilotinib was selected between a set of synergistic drug. It is a BCR-ABL inhibitor but also is a potent PDGF-R inhibitor. Interestingly, different studies reported an anti-fibrotic activity in liver (Liu et al. J Hepatol. 2011 Sep;55(3):612-25.) and skin (Akhmetshina et al. FASEB J. 2008;22(7):2214-22). For these reasons we decided study the effect of the combination of Ruxolitinib and Nilotinib in BMF.To determine the ideal concentration of TGF-β to induce collagen mRNA expression in HS27a, we treated the culture of HSP27a with increasing concentration of TGF-β (0.625-10 ng/mL) during 24h. The best TGF-β concentration was 2ng/mL, with a fold change of 2.74 (SD=0.89) (p<0.05) relative to control without TGF (figure 1).Figure 1: Fold change in COL1 mRNA expression in HSP27a cell line relative to control without TGF-β. Two-sample Wilcoxon rank-sum (Mann-Whitney) test was performed. * p<0.05. R: Ruxolitinib. N: Nilotinib.To assess the anti-fibrotic activity of Ruxolitinib and Nilotinib individually, we preincubated the HSP27a cultures with 0.1 - 0.5 µM of Ruxolitinib or 0.5 - 1 µM of Nilotinib during 1 h. After that, we added TGF-β during 24h. Both 0.5 - 1 µM of Nilotinib reduced the mRNA expression of COL1 with a fold change of 0.75 (SD = 17.6) (p<0.05) and 0.78 (SD = 11.5) (p<0.05) respectively, relative to control with TGF-β but without drug (figure 2).Figure 2: Fold change in COL1 mRNA expression in HSP27a cell line relative to control with TGF-β but without drugs. Two-sample Wilcoxon rank-sum (Mann-Whitney) test was performed. * p<0.05. R: Ruxolitinib. N: Nilotinib.Finally, to verify if the combination of both drug had the capacity of reducing COL1 expression, we pretreated the HS27a cultures with 0.1 µM of Ruxolitinib and 1 µM of Nilotinib simultaneously. The expression of COL1 were 74 % (SD = 27.32%) (p<0.05) relatively to control with TGF-β but without drug (figure 2).In conclusion, Nilotinib have synergistic and anti-fibrotic activity individually, but also in combination with Ruxolitinib. For these reasons, the combined therapy Ruxolitinib-Nilotinib could be a promising therapy to MF and these results support a clinical trial of this combination in MF patients. [Display omitted] [Display omitted] DisclosuresMartínez-López:Novartis: Honoraria, Speakers Bureau.

Dual Deficiency of mDia1 and Mir-146a in an Age-Related Inflammatory Bone Marrow Microenvironment Induces Ineffective Erythropoiesis in Del(5q) MDS

Myelodysplastic syndromes (MDS) are a group of age-related clonal hematologic diseases characterized by anemia, neutropenia, and thromobocytopenia. The mechanisms of anemia in MDS are unclear, which is partially due to the heterogeneity of MDS involving many cytogenetic and molecular abnormalities. Using a mouse genetic approach, here we show that dual deficiency of mDia1 and miR-146a, two genes located at chromosome 5q that is commonly deleted in MDS, causes an aged-related anemia and ineffective erythropoiesis that closely mimics human MDS. Bone marrow erythropoiesis at various stages was dramatically affected in mDia1/miR-146a double knockout mice, which induced a massive splenomegaly with potent extramedullary erythropoiesis. Old (> 1 year of age), but not young (2-4 months of age), wild type recipient mice that were transplanted with bone marrow cells from mDia1/miR-146a double knockout mice, exhibited severe anemia and rapid lethality, which indicates that the aged microenvironment is important for the development of ineffective erythropoiesis. Consistent with the roles of mDia1 and miR-146a in the innate immune response, the serum levels of TNFα and IL-6 were significantly elevated in mDia1/miR-146a double knockout mice. Pathogen-associated molecular pattern proteins (PAMPs), or damage-associated molecular pattern proteins (DAMPs), whose levels increase in aged microenvironment, both induced TNFα and IL-6 upregulation in mDia1/miR-146a double knockout granulocytes and T cells. Mechanistically, we demonstrated that the anemia and ineffective erythropoiesis was independent of hepcidin expression in mDia1/miR-146a double knockout mice. Instead, pathologic levels of TNFα and IL-6 inhibit erythroid colony formation and differentially affect terminal erythropoiesis through reactive oxygen species-induced caspase-3 activation and cell apoptosis. Our study highlights the dual roles of age-related microenvironment and cytogenetic abnormalities in the pathogenesis of ineffective erythropoiesis in MDS. DisclosuresNo relevant conflicts of interest to declare.

Pre-Transplantation Blockade of TNF-α-Mediated Oxygen Species Accumulation Protects Hematopoietic Stem Cells.

Hematopoietic stem cell (HSC) transplantation (HSCT) for malignancy requires toxic pre-conditioning to maximize anti-tumor effects and donor-HSC engraftment. While this induces bone marrow (BM)-localized inflammation, how this BM environmental change affects transplanted HSCs in vivo remains largely unknown. We here report that, depending on interval between irradiation and HSCT, residence within lethally irradiated recipient BM compromises donor-HSC reconstitution ability. Both in vivo and in vitro we demonstrate that, among inflammatory cytokines, TNF-α plays a role in HSC damage: TNF-α stimulation leads to accumulation of reactive oxygen species (ROS) in highly purified hematopoietic stem/progenitor cells (HSCs/HSPCs). Transplantation of flow-cytometry-sorted murine HSCs reveals damaging effects of accumulated ROS on HSCs. Short-term incubation either with an specific inhibitor of tumor necrosis factor receptor 1 signaling or an antioxidant N-acetyl-L-cysteine (NAC) prevents TNF-α-mediated ROS accumulation in HSCs. Importantly, pre-transplantation exposure to NAC successfully demonstrats protective effects in inflammatory BM on graft-HSCs, exhibiting better reconstitution capability than that of nonprotected control grafts. We thus suggest that in vivo protection of graft-HSCs from BM inflammation is a feasible and attractive approach, which may lead to improved hematopoietic reconstitution kinetics in transplantation with myeloablative conditioning that inevitably causes inflammation in recipient BM. Stem Cells 2017;35:989-1002.

Abstract 13040: Repeated Social Defeat Stress Enhances Neutrophil Extracellular Traps Formation in vivo and in vitro: Implication in the Exaggerated Atherosclerosis in Chronic Social Stress-exposed Apoe -/- Mice

Introduction: Neutrophil extracellular traps (NETs) has been reported to promote atherosclerosis through induction of NETosis, a new type of neutrophil cell death; however, the role of NETosis in psychosocial stress-induced atherosclerosis remains undefined. Methods and Results: Eight-week-old male apoE -/- mice were exposed to the repeated social defeat (RSD) by housing with a larger CD-1 mice in a shared home cage with a clear perforated divider. They were repeatedly subjected to vigorous physical contact for 5-10 min each day on 10 consecutive days. Control mice were housed in the same gage without physical contact. After 6 weeks of high-cholesterol diet feeding, atherosclerotic lesion area of the aortic root was significantly increased in RSD mice compared with control mice (19.4% vs 13.9%, P &lt; 0.05). There was no significant difference in body weight, food intake, mean blood pressure, and lipid profile between the 2 groups. Although both of Ly6G-positive and Mac3-positive area in immunofluorescent staining were comparable between the 2 groups, Ly6G-positive area in RSD mice was much strongly co-localized with MPO (16.4% vs. 6.6%; P &lt; 0.05) and H3Cit (15.1% vs. 9.0%; P &lt; 0.05), suggesting the augmented NETosis in RSD mice. Consistently, serum IL-1β level was markedly higher in RSD mice (2.9-fold; P &lt; 0.05). The percentages of circulating neutrophils (Ly6G + CD11b + F4/80 - CD11c - ) were significantly increased in RSD mice compared with control mice (27.1% vs. 13.0%; P &lt; 0.05), whereas the fractions of circulating inflammatory monocytes (Lin - CD11b + Ly6C + ) and bone marrow (BM) monocyte-lineage progenitor cells were equivalent between the 2 groups. To further examine the NETs formation of individual neutrophils in vitro, BM neutrophils (Ly6G + ) were isolated by flow cytometry and stimulated by PMA for 4 hr. The percentages of MPO- and H3Cit-positive cells were significantly increased by 2.5-fold in RSD mice compared with control mice ( P &lt; 0.01). Conclusions: Our findings demonstrated for the first time that RSD promotes NETs formation in BM neutrophils as well as in atherosclerotic lesions, which precede high-cholesterol diet-induced monocytosis, suggesting that NETosis could be a new therapeutic target in psychosocial stress-induced atherosclerosis.