Bone Blood Research Articles

Role of Dashmool Siddha Majjabasti in the Management of Avascular Necrosis of the Femoral Head: A successful case study

Avascular necrosis is a condition brought on by a temporary or irreversible reduction in the bone's blood supply. Bone tissue dies and crumbles when blood supply is interrupted. The joint surface may collapse if there is avascular necrosis close to joint. Young individuals with a 60% bilateral incidence rate have a condition known as avascular necrosis (AVN), which is also known as osteochondritis dissecans or Chandler's disease and is characterized by dead bone. Complete hip replacement surgery is the only option for treating AVN, but it has disadvantages of a lengthy recovery period and a short lifespan for the hip. Contrary to popular belief, ayurvedic medicine offers long-term cure and halts the course of disease without any negative effects. Avascular necrosis and Asthi-Majja GataVata are not directly related, but their clinical manifestations point to Vata Pradhana Tridoshaja. Vyadhi and Asthi-Majja Dhatu's primary Vikruti. The first portion of the Acharayas' Vata-dominant illness therapy is called Basti Chikitsa. It is regarded as Param Aushadh for illnesses of Vata. To evaluate Dashmool Siddha Majjabasti's effectiveness in the treatment of AVN. The goal was to prevent future hip joint degeneration and lessen the likelihood that surgical intervention would be necessary to treat avascular necrosis. A patient with stage 4 AVN in the right hip joint and stage 2 AVN in left hip joint who had not undergone surgery and who presented to the hospital's outpatient department with limited mobility and a limping gait was treated using panchakarma treatment and shaman yoga. Significant improvement was seen following the therapy. Range of motion was improved while pain levels were significantly lowered. Following the procedure, patient was pain- and stiffness-free when walking and climbing stairs. Ayurvedic principles are used in the conservative therapy of AVN, which significantly reduces signs and symptoms and enhances quality of life.

Cytogenetic abnormalities in patients with hematological malignancies in Lahore city, Pakistan.

Hematological and hematopoietic cells malignancies of the genes and hematopoietic cells are associated with the genetic mutation, often at the chromosomal level. The standard cytogenetic study is widely accepted as one of the main diagnostics and prognostic determinants in patients. Therefore, the current descriptive and cross-sectional study sought to determine the cytogenetic analysis of frequent hematological malignancies in Pakistan. A total of 202 peripheral bone marrow or blood samples from patients with benign and malignant hematological malignancy were taken using a conventional G-banding technique. Among enrolled patients, the mean age was 21.5 years ± 23.4, and gender-wise distribution showed a marked predominance of the male 147 (73%) population compared to the female 55 (27%). Patients in the age group (2-10 years) had the highest frequency, 48 (24%), of hematological neoplasms, followed by age (11-20 years) with 40 (20%). Normal karyotypes (46, XX/46, XY) was found in 51% (n=103) patients. Furthermore, the frequency of complex karyotype was 30 (15%), while normal was seen in 171 (85%) patients. Pre-B Acute Lymphoblastic Leukemia (Pre-B ALL) was the most prevalent malignancy of 66 (33%), followed by Chronic Myelogenous Leukemia (CML) of 41 (20%) and Acute Lymphocytic Leukemia of 29 (14%). Translocation was the most prevalent 50 (25%), followed by hypotriploidy 14 (7%) and monosomy 8 (4%) on chromosome aberration analysis. In addition, t(9:22) translocation was found to be 20 (10%) in CML, with the majority in the age group (31-40 years). This study recommends that karyotyping should be tested frequently in hematological conditions because it may provide insight into the relative chromosomal changes associated with particular malignancies.

Abstract 6251: Evaluation of a dual-targeting BCMA-CS1 HLE BiTE® molecule for multiple myeloma

Abstract Multiple myeloma (MM) is a cancer of the antibody-producing plasma cells (PC). MM is invariably fatal due to frequent disease relapse and/or treatment refractoriness, and therapies that provide deeper and more durable responses are needed. BiTE® (Bispecific T Cell Engager) molecules are immunotherapy agents that redirect a patient’s T cells to lyse tumor cells by simultaneously engaging a tumor associated antigen (TAA) on cancer cells and CD3ε on T cells. Clinical activity has been observed in MM using BiTE® molecules and other T cell engagers that target B-cell maturation antigen (BCMA). BCMA is an attractive MM TAA due to its broad prevalence and restricted normal tissue profile, mainly PCs and plasmablasts. However, heterogenous expression in MM cancer cells, potential antigen loss, and the presence of high levels of soluble BCMA in MM patient sera present challenges that may prevent BCMA-only-targeted therapies from achieving their full potential. To address these issues, we generated a BiTE® molecule capable of engaging BCMA and a second MM TAA, CS1 (also known as SLAMF7). This BiTE® molecule also contains an Fc-based domain to provide half-life extension (HLE). Here, we evaluated the in vitro and in vivo properties of this BCMA-CS1 HLE BiTE® molecule. The BCMA-CS1 HLE BiTE® molecule had nanomolar binding affinity for human BCMA, CS1, and CD3ε and the non-human primate (NHP) orthologues. In vitro, we observed picomolar activity against human MM cell lines with a range of BCMA and CS1 expression in a T cell dependent cellular cytotoxicity (TDCC) assay using human T cells or NHP peripheral blood mononuclear cells. The BCMA-CS1 HLE BiTE® molecule retained TDCC activity in the presence of soluble BCMA up to 2500 ng/mL as well as against human MM cells engineered to express only BCMA or CS1. In vivo, this BiTE® molecule inhibited tumor growth in a MM xenograft model. We also evaluated the BCMA-CS1 HLE BiTE® molecule in NHP over 15 days (IV dosing; intra-animal dose escalation from 60→240 μg/kg or 180→540 μg/kg on days 1 and 8). We observed hallmarks of BiTE® molecule activity in all groups, including transient decreases in circulating lymphocytes and moderate increases in cytokines like MCP-1. We measured the PC-specific transcripts BCMA and J-chain in NHP bone marrow and blood as surrogates for PC levels using ddPCR. These transcripts were reduced in both treatment groups (≥90%) with the strongest effects occurring in the 180→540 μg/kg group. Lastly, we showed that CS1 is highly and broadly expressed in MM patient samples and is restricted to a few normal hematopoietic cell types including PCs, NK cells, T cells, and some monocytes. These data suggest that the BCMA-CS1 HLE BiTE® molecule has potent in vitro and in vivo activity and may provide therapeutic benefit for MM patients by expanding the population of MM cancer cells that can be eliminated by a BiTE® molecule while overcoming common mechanisms that can impair BCMA-only-targeted MM therapies. Citation Format: Elizabeth T. Andrews, Stephanie C. Casey, Mohammad Farhad Amani, Grit Lorenczewski, Mozhgan Farshbaf, Lisa Winkel, Matthias Klinger, John M. Harrold, Famke Aeffner, Ana Goyos, Matthias Friedrich, Tara Arvedson, Matthew G. Chun. Evaluation of a dual-targeting BCMA-CS1 HLE BiTE® molecule for multiple myeloma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 6251.

Abstract 2909: CD123-CODV-TCE bispecific T-cell engager for acute myeloid leukemia (AML): Activity on primary AML and safety in non-human primates

Abstract Acute myeloid leukemia (AML) is characterized by accumulation of abnormal blast cells in the bone marrow and blood. Although treatments like chemotherapy, allogeneic stem cell transplantation and new biotherapies improve the survival of patients, relapse is common and may be due to residual leukemic stem cells (LSCs). Since LSCs express high levels of CD123 (interleukin-3 receptor), CD123 represents an attractive target for relapsed/refractory (R/R) AML patients. Here, the expression of CD123 on leukemic cells and LSCs from fresh AML patient samples was confirmed, with a similar receptor density ranging from 350 to 12,500 and from 1,200 to 29,000 sites per cell, respectively. Also, the cytotoxic activity of the bispecific T-cell engager (TCE) CD123-CODV-TCE (that binds to CD3 on T cells and CD123 on blast cells) against primary blast cells isolated from the blood of 30 AML patients was evaluated. CD123-CODV-TCE induced high killing of AML blast cells with both high and low CD123 antigen density (from 298 to 18,719 sites per cell). The Effector: Target (E:T) ratio was highly variable among all patients, ranging from less than 1 to more than 100 blast cells for one T cell, and did not impact the activity of CD123-CODV-TCE. Since cytokine release syndrome is associated with TCE therapies, the release of proinflammatory cytokines was monitored in parallel to cytotoxicity. CD123-CODV-TCE induced the release of all tested cytokines (IL-6, IFNγ, TNFα and IL-8) without clear correlation with blast killing efficacy level. Since a similar CD123 expression pattern in human and cynomolgus monkey tissues was found, and CD123-CODV-TCE binds to human and cynomolgus CD3ε and CD123, non-human primates were used for the evaluation of pharmacodynamic effects and safety. A repeat-dose monkey study was performed using a progressive, weekly (X4) intra-monkey dose escalation scheme. The depletion of CD123 positive cells in blood was evidenced from the lowest dose tested (0.1 μg/kg) to the highest (5 µg/kg) with no adverse effects observed at 0.1 µg/kg. The main adverse effect consisted of a marked increase of the level of cytokines at 5 hours following compound administration at 1 μg/kg and above. Importantly, following the dose-escalation scheme 0.1/1/3/3 µg/kg, only limited cytokine release after the 4th administration was observed, suggesting that mitigation of cytokine release can be obtained by progressive intra-monkey dose escalation. Taken together, these results indicate that CD123-CODV-TCE leads to potent and specific leukemic cancer cell killing independently of CD123 receptor density and E:T ratio, and that studies monitoring cytokine release in monkeys can be useful for future clinical development of TCEs in the setting of R/R AML. Citation Format: Helene Bonnevaux, Jacqueline Courta, Wilfried Passe-Coutrin, Anne-Laure Bauchet, Annelies Roobrouck, Celine Amara, Eric Beys, Marielle Balzano, Maxime Moulard, Marco Meloni, Melissa Dullaers, Marielle Chiron, Angela Virone-Oddos. CD123-CODV-TCE bispecific T-cell engager for acute myeloid leukemia (AML): Activity on primary AML and safety in non-human primates [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2909.

Abstract 2649: Orally bioavailable small molecule CXCR4 antagonists with enhanced efficacy in mouse models of genitourinary cancers

Abstract Purpose: Pro-angiogenic and immune cells expressing chemokine receptor CXCR4 traffic along concentration gradients of the chemokine ligand CXCL12, which disseminates from stromal niches. The CXCR4/CXCL12 axis is hijacked by various cancer types characterized by dramatic CXCR4 and/or CXCL12 upregulation. This chemokine network misregulation causes hyperactivation of CXCR4-mediated processes, leading to (1) excessive stimulation of protective tumor-stromal interactions, (2) metastatic expansion of CXCR4+ cancer cells to CXCL12-rich sites, and (3) intratumoral infiltration of CXCR4+ immunosuppressive cells. Accordingly, CXCR4 antagonists have significant therapeutic potential against cancer. While clinical CXCR4 inhibitors are severely limited by suboptimal pharmacokinetic (PK) properties and off-target activities, several of our tetrahydroisoquinoline-based CXCR4 antagonists exhibit superior selectivity and PK profiles to these clinical comparators (e.g., X4P-001). Methods: To test the hypothesis that improved PK profiles would translate to enhanced efficacy, our lead CXCR4 antagonist EMU-116 was compared to X4P-001 head-to-head in three mouse models of genitourinary cancers. First, human 786.0 renal cell carcinoma (RCC) xenograft-bearing female nude mice were treated p.o., q.d. with vehicle, axitinib (30 mg/kg), X4P-001 (100 mg/kg), EMU-116 (3, 10, or 30 mg/kg), X4P + axitinib, or EMU-116 + axitinib. Subcutaneous tumor volume was monitored using calipers. Second, nude male mice bearing intratibial, luciferase-expressing human PC-3 prostate cancer xenografts were treated with vehicle (p.o., q.d.), docetaxel (10 mg/kg i.p. weekly), X4P-001 (10 or 30 mg/kg p.o., q.d.), EMU-116 (10 or 30 mg/kg p.o., q.d.), X4P + axitinib, or EMU + axitinib. Intratibial tumor volume was monitored via luminescence imaging. Third, syngeneic RENCA RCC tumor-bearing female Balb/c mice were treated p.o., q.d. with vehicle, X4P-001 (30 mg/kg), or EMU-116 (30 mg/kg). Subcutaneous tumor volume was monitored using calipers, and immune cell subsets in bone marrow, blood, tumor, and tumor-draining lymph nodes were quantified via flow cytometry. Results: In the RCC xenograft model in combination with axitinib, EMU-116 (3 or 10 mg/kg) was equally effective at decreasing tumor burden compared to X4P-001 (100 mg/kg), whereas EMU-116 (30 mg/kg) was more effective. In the bone metastatic prostate cancer xenograft model, EMU-116 was as or more effective than X4P-001 when paired with docetaxel. In the syngeneic RCC model, EMU-116 mobilized T cells more effectively than X4P-001. Conclusion: EMU-116, at the same or lower dose, was more efficacious than X4P-001 in these models. These results highlight EMU-116 as a clinical candidate with significant therapeutic potential to synergize with chemotherapeutics, targeted therapies, and immuno-oncology agents. Citation Format: Eric J. Miller, Carrie Q. Sun, Petra Gregorova, Edgars Jecs, Yesim Altas Tahirovic, Robert J. Wilson, Huy H. Nguyen, Savita K. Sharma, Perry Bartsch, Zachary Sticher, Levi Moellering, Priscilla Davidson, Ryan Jajosky, Michael D'Erasmo, Manohar Saindane, Zafer Sahin, Nicholas S. Akins, Alexander A. Kolykhalov, Lawrence Wilson, Rebecca S. Arnold, John A. Petros, Haydn Kissick, Lingjie Xu, Yi Jiang, Dennis C. Liotta. Orally bioavailable small molecule CXCR4 antagonists with enhanced efficacy in mouse models of genitourinary cancers [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2649.

Hairy cell leukemia: a specific 17-gene expression signature points to new targets for therapy

BackgroundHairy cell leukemia (HCL) is a rare chronic B cell malignancy, characterized by infiltration of bone marrow, blood and spleen by typical “hairy cells” that bear the BRAFV600E mutation. However, in addition to the intrinsic activation of the MAP kinase pathway as a consequence of the BRAFV600E mutation, the potential participation of other signaling pathways to the pathophysiology of the disease remains unclear as the precise origin of the malignant hairy B cells.Materials and methodsUsing mRNA gene expression profiling based on the Nanostring technology and the analysis of 290 genes with crucial roles in B cell lymphomas, we defined a 17 gene expression signature specific for HCL.ResultsSeparate analysis of samples from classical and variant forms of hairy cell leukemia showed almost similar mRNA expression profiles apart from overexpression in vHCL of the immune checkpoints CD274 and PDCD1LG2 and underexpression of FAS. Our results point to a post-germinal memory B cell origin and in some samples to the activation of the non-canonical NF-κB pathway.ConclusionsThis study provides a better understanding of the pathogenesis of HCL and describes new and potential targets for treatment approaches and guidance for studies in the molecular mechanisms of HCL.

Prediction of Femoral Strength Based on Bone Density and Biochemical Markers in Elderly Men With Type 2 Diabetes Mellitus.

Purpose: Effects of bone density, bone turnover and advanced glycation end products (AGEs) on femoral strength (FS) are still unclear in patients with type 2 diabetes mellitus (T2DM). This study aims to assess and predict femoral strength and its influencing factors in elderly men with T2DM. Methods: T2DM patients (n = 10, mean age, 66.98 years) and age-matched controls (n = 8, mean age, 60.38 years) were recruited. Femoral bone mineral density (BMD) and serum biochemical indices of all subjects were measured. FS was evaluated through finite element analysis based on quantitative computed tomography. Multiple linear regression was performed to obtain the best predictive models of FS and to analyze the ability of predictors of FS in both groups. Results: FS (p = 0.034), HbA1c (p = 0.000) and fasting blood glucose (p = 0.000) levels of T2DM group were significantly higher than those of control group; however, the P1NP level (p = 0.034) was significantly lower. FS was positively correlated with femoral neck T score (FNTS) (r = 0.794, p < 0.01; r = 0.881, p < 0.01) in both groups. FS was correlated with age (r = -0.750, p < 0.05) and pentosidine (r = -0.673, p < 0.05) in T2DM group. According to multiple linear regression, FNTS and P1NP both contributed to FS in two groups. P1NP significantly improved the prediction of FS in both groups, but significant effect of FNTS on predicting FS was only presented in control group. Furthermore, pentosidine, age and HbA1c all played significant roles in predicting FS of T2DM. Conclusion: Femoral strength was higher in elderly men with T2DM, which might be caused by higher BMD and lower bone turnover rate. Moreover, besides BMD and bone formation level, AGEs, blood glucose and age might significantly impact the prediction of femoral strength in T2DM.

Abstract P2-14-07: Marrow-infiltrating lymphocytes as adoptive immunotherapy for breast cancer

Abstract Background: The bone marrow harbors tumor-antigen specific T cells in patients with breast cancer (1). Marrow-infiltrating lymphocytes (MILs®) are the product of activating and expanding bone-marrow T cells (2). In multiple myeloma, transfer of MILs as adoptive cell therapy has demonstrated anti-tumor activity (3). The use of adoptive transfer of autologous tumor-infiltrating lymphocytes (TILs) has been used successfully for hormone-receptor positive (HR+) metastatic breast cancer (4). Expansion of MILs was previously shown to be feasible in breast cancer, non-small cell lung cancer (NSCLC), prostate, head and neck, glioblastoma and multiple myeloma (5). Using an expanded cohort, we demonstrate that tumor-specific MILs can be expanded from bone marrow in patients with breast cancer. Methods: Bone marrow aspiration and blood samples were collected from 8 patients with metastatic and high-risk early-stage breast cancer, including 3 patients with HR+/HER2-, 2 patients with HER2+, and 3 patients with triple negative subtypes, at Providence Cancer Institute (Portland, OR). MILs and peripheral blood lymphocytes were activated and expanded from patient samples during a 10-day proprietary process. T cell lineage-specific markers CD3, CD4 and CD8 were characterized by flow cytometry pre- and post- expansion. Tumor-specific T cells were quantitated in expanded MILs and peripheral blood lymphocytes using a previously described cytokine-secretion assay (3). Briefly, they were defined as the IFNy-producing population by flow cytometry. Autologous antigen-presenting cells (APCs) were pulsed with lysates from allogeneic cancer cell lines and co-cultured with activated MILs or peripheral blood lymphocytes. APCs pulsed with irrelevant mis-matched cancer cell line lysates or media alone were used as negative controls. Results: In all harvested samples, MILs expansion successfully resulted in a selective increase in CD3+ T cells. Cytokine-producing CD4+ and CD8+ T cells were detected in all expanded MILs samples, but not in any of the matched activated and expanded peripheral blood lymphocytes. MILs were successfully expanded in HR+/HER2-, HER2+, and triple negative subtypes without apparent differences in activity as measured by cytokine production. Conclusion: MILs isolated from this expanded cohort of metastatic and high-risk early stage breast cancer patients demonstrated cytokine production in response to pulsed cancer cell line lysates, consistent with our experience with other solid (5) and hematological (3) malignancies. A phase II trial to evaluate MILs in combination with a checkpoint inhibitor is underway in patients with anti-PD1/PDL1-refractory NSCLC (NCT04069936). These preclinical data demonstrate that expanding MILs is feasible and could be evaluated therapeutically in breast cancer.

A single session of antimicrobial photodynamic therapy does not influence the alveolar repair process in rats.

The aim of this study was to use microscopic and molecular techniques to evaluate the effects of a single session of antimicrobial photodynamic therapy (aPDT) on the alveolar repair process after tooth extraction in rats. The study sample included 84 rats divided into four groups, as follows: a) Control - untreated socket; b) Laser - socket treated using photobiomodulation; c) TBO - socket treated with topic application of the photosensitizer agent, toluidine blue O (TBO); and d) aPDT - socket treated with TBO and laser irradiation. An additional rat was used for thermal mapping during socket irradiation. The animals were euthanatized at 6, 15, and 28 days after unilateral extraction of the upper incisor. Quantitative and qualitative analyses of the connective and bone tissues, blood clot, blood vessel, and inflammatory infiltrate were performed, and real-time polymerase chain reaction was used to study the expression of genes (collagen type I, osteocalcin, alkaline phosphatase [ALP], runt-related transcription factor 2 [RUNX2], and vascular endothelial growth factor [VEGF]) involved in the bone healing process. No statistically significant differences in microscopic and molecular outcomes were observed between the groups (p > 0.05). A positive correlation was seen to exist between blood clot and VEGF (p = 0.000), and a negative correlation was observed between bone tissue and ALP (p = 0.028) and blood vessel and VEGF (p = 0.018). A single session of aPDT in the dental extraction site did not influence the alveolar repair process in rats.

Analysis of Peripheral Blood Mononuclear Cells Gene Expression Highlights the Role of Extracellular Vesicles in the Immune Response following Hematopoietic Stem Cell Transplantation in Children

Hematopoietic stem cell transplantation (HSCT) is an effective treatment method used in many neoplastic and non-neoplastic diseases that affect the bone marrow, blood cells, and immune system. The procedure is associated with a risk of adverse events, mostly related to the immune response after transplantation. The aim of our research was to identify genes, processes and cellular entities involved in the variety of changes occurring after allogeneic HSCT in children by performing a whole genome expression assessment together with pathway enrichment analysis. We conducted a prospective study of 27 patients (aged 1.5–18 years) qualified for allogenic HSCT. Blood samples were obtained before HSCT and 6 months after the procedure. Microarrays were used to analyze gene expressions in peripheral blood mononuclear cells. This was followed by Gene Ontology (GO) functional enrichment analysis, Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis, and protein–protein interaction (PPI) analysis using bioinformatic tools. We found 139 differentially expressed genes (DEGs) of which 91 were upregulated and 48 were downregulated. “Blood microparticle”, “extracellular exosome”, “B-cell receptor signaling pathway”, “complement activation” and “antigen binding” were among GO terms found to be significantly enriched. The PPI analysis identified 16 hub genes. Our results provide insight into a broad spectrum of epigenetic changes that occur after HSCT. In particular, they further highlight the importance of extracellular vesicles (exosomes and microparticles) in the post-HSCT immune response.

MYD88 Gene Polimorphism in Patients With Chronic Lenfoid Leukemia

Leukemia is a type of cancer that manifests itself with the excessive proliferation of blood cells, especially white blood cells, and affects the blood production system in the body. Leukemias are classified as acute or chronic and according to the extent and development of the tumor. In chronic lymphocyte leukemia (CLL), cancer cells are found in the bone marrow, blood, and lymph nodes. Therefore, in this study, the effects of polymorphisms in the MYD88 gene of CLL patients were investigated. For this purpose, 100 patients diagnosed with CLL and 70 healthy individuals without cancer history were included in the study as the control group. DNA was isolated from CLL patients and control groups. Polymorphisms in the MYD88 (L265p T/C) promoter regions were studied by RT-PCR. Genotype and allele frequencies were calculated directly. Statistical significance of genotypic distributions between CLL patients and control groups was evaluated with Z Ratios test and Fisher's Exact test.For the MYD88 L265p T/C region in 100 CLL patients, 95 wild type TT, 3 TC and 5 mutant type CC genotypes were detected. No statistically significant difference was found between CLL patients and control groups in the MYD88 L265p T/C region (p 0.05). According to the allele frequencies of the region, it was determined as 0.965 and 0.065 for T and C, respectively. According to Fisher's Exact results, it was calculated as 0.078 for TT, 0.078 for TC and 0.269 for Mutant, respectively. The MYD88 genotype frequencies of the patients included in the study do not appear to be statistically correlated with CLL patients. The research we have done as a result of our study will shed light on the studies with different SNPs to be made with the increase in the number of CLL patients..

The Diagnostic Usefulness of Bone Marrow Examination and Bone Marrow Culture in Patients with Pyrexia of Unknown Origin

Introduction: Bone marrow cultures (BMCs) are usually obtained in the diagnosis of pyrexia of unknown origin (PUO). In the effort to identify the cause of systemic infection, BMCs are often performed in addition with blood cultures (BCs) and cultures of other body fluids and tissues. The practice of performing BMC and Blood cultures is long-standing, but the medical importance is questionable. Given the importance of cost effectiveness, such practices must be assessed on the basis of their cost-benefit to patient outcomes. In this study we compared the value of BMC with BC and studied the usefulness of the histopathological features of bone marrow trephine biopsy specimens in detecting the cause of systemic infections in selected patient population with PUO.&#x0D; Objective: To determine the usefulness of bone marrow culture for the diagnosis of different causes of pyrexia of unknown origin.&#x0D; Study Design: Descriptive Cross-sectional study&#x0D; Place and Duration of Study: This study was conducted at Peoples University of Medical and Health Sciences for Women Nawabshah Pakistan from June January 2020 to January 2021.&#x0D; Materials and Methods: A total 150 patients were included in this study who fulfilled the criteria of pyrexia of unknown origin. The bone marrow aspiration, biopsy, and blood cultures specimens were obtained using standard techniques and bone marrow culture send for microbial analysis.&#x0D; Results: 1.5% of patients had hematomas, one diagnosed with Behcet disease, other patients with acute leukemia. The bone marrow aspiration results exhibited that 23 patients (15.3%) diagnose with a hemophagocytic syndrome where 3 cases of infection, 3 cases with hematological malignant disorder, and 7 cases with hemophagocytosis, and repercussion of bone marrow aspiration diagnosed 5 acute leukemia and 2 with visceral leishmaniasis. The usefulness of BMB was determined through an odd ratio of few parameters including thrombocytopenia with odd ratio 4.59 and 95% CI (1.07-8.4), anemia odd ratio 3.17, and CI (1.09-12.92) were considered most associated factors for the usefulness of BMB. BMC in 13 (8.66%) patients out of 150 were found with bacterial growth and 2 (1.33%) cases with bacterial growth were observed in blood culture. There was no fungal and mycobacterial growth observed. In the BMC analysis 2 cases of E. coli, 3 cases with Staphylococcus aureus, 2 cases of Enterococcus sps. while 4 cases of Salmonella typhi and 2 cases of Salmonella paratyphi-A were diagnosed in blood culture analysis.&#x0D; Conclusion: The obtained data indicate that blood culture did not divulge bacterial growth without the bone marrow culture. Therefore bone marrow culture was found to be an important diagnostic modality and correlation was found for thrombocytopenia and anemia. Bone marrow biopsy is deemed a useful method to diagnose pyrexia of unknown origin.

Tofacitinib Blocks Entheseal Lymphocyte Activation and Modulates MSC Adipogenesis, but Does Not Directly Affect Chondro- and Osteogenesis

Entheseal spinal inflammation and new bone formation with progressive ankylosis may occur in ankylosing spondylitis (AS) and psoriatic arthritis (PsA). This study evaluated whether JAK inhibition with tofacitinib modulated the key disease associated cytokines, TNF and IL-17A, and whether tofacitinib also modulated bone marrow stromal cell-derived mesenchymal stem cell (MSCs) function, including osteogenesis, since post inflammation new bone formation occurs under these conditions. Methods: Conventional entheseal derived αβ CD4+ and CD8+ T-cells were investigated following anti-CD3/CD28 bead stimulation to determine IL-17A and TNF levels in tofacitinib treated (1000 nM) peri-entheseal bone (PEB) and peripheral blood mononuclear cells (PBMC) using ELISA. Bone marrow stromal cell-derived mesenchymal stem cell (MSC) colony forming units (CFU-F) and multi-lineage potential were evaluated using tofacitinib (dosages ranging between 100, 500, 1000 and 10,000 nM). Results: Induced IL-17A and TNF cytokine production from both entheseal CD4+ T-cells and CD8+ T-cells was effectively inhibited by tofacitinib. Tofacitinib treatment did not impact on CFU-F potential or in vitro chondro- and osteogenesis. However, tofacitinib stimulation increased MSC adipogenic potential with greater Oil Red O stained areas. Conclusion: Inducible IL-17A and TNF production by healthy human entheseal CD4+ and CD8+ T-cells was robustly inhibited in vitro by tofacitinib. However, tofacitinib did not impact MSC osteogenesis, but stimulated in vitro MSC adipogenesis, the relevance of which needs further evaluation given that the adipocytes are associated with new bone formation in SpA.

Quantitative and qualitative composition of the innate immune response are equally important in wound healing after myocardial infarction

Abstract Background Emergency hematopoiesis (EH) serves as the foundation of monocyte-derived and macrophage (Mφ) driven efferocytosis and ventricular remodeling after myocardial infarction (MI). Excessive myelopoiesis, however, can stipulate maladaptive wound healing and its therapeutic reduction may be a novel approach to preserve cardiac function. All-trans retinoic acid (ATRA) is a pleiotropic modulator of EH and innate immunity shielding hematopoietic stem cells from activation and driving survival and differentiation of myeloid cells. Purpose This study aimed to investigate this intriguing interplay of ATRA in wound healing after MI. Methods MI was induced by permanent coronary ligation in C57BL/6 mice and treated with daily injections of either ATRA (30mg/kg) or DMSO (vehicle) up to five days, starting 24h after ligation. Flow cytometry (FACS) was used for cell cycle analysis and immunophenotyping of leukocytes in bone marrow (BM), blood and heart. Immunohistochemistry (IH), masson trichrome (MT) staining and echocardiography evaluated inflammatory-fibrotic and functional development. Cytokine expression was analyzed by qPCR in bulk infarct and isolated, polarized Mφ-populations of BM-derived and cardiac resident origin. Results On day 2 after MI, EH was significantly reduced in ATRA-treated mice as compared to vehicle controls by means of cell cycle activity (n=6–13 per group; p&amp;lt;0,01) and myeloid cells in BM, blood and infarct tissue (n=5–13; p&amp;lt;0,05). Consequently, mRNA-expression of key inflammatory cytokines, IL-1β and TNFα, was diminished in the infarct tissue in this early phase (n=5–12; p&amp;lt;0,05). These changes, however, failed to preserve cardiac function and ventricular remodeling, 21 days after MI (n=10–11; not significant). By qPCR, non-canonical activation of recruited ATRA-primed monocyte-derived Mφ, was found to propagate a pro-inflammatory phenotype with higher expression of MMP2 and MMP9 in sorted cardiac Mφ (n=4–5; p&amp;lt;0,001). Furthermore, prominent IL-1β-expression in M2-polarized BM-derived Mφ indicated an impaired anti-inflammatory phenotype after ATRA treatment (n=4–6; p&amp;lt;0,05). Strikingly, these changes also occurred in remote myocardium where IH revealed a 2-fold increase of CD11b - positive myeloid cells accompanied by increased expression of TNFα and TGFβ (n=9; p&amp;lt;0,001). MT-staining, performed 21 days after MI, demonstrated an almost 3-fold increase in collagen deposition in remote myocardium of ATRA treated mice in contrast to vehicle controls (n=4–6; p&amp;lt;0,0001). Conclusion Despite a beneficial reduction of EH after MI, short-term treatment with ATRA induced profound and persisting changes in the cytokine expression of monocyte-derived Mφ, which significantly altered their function and thus prevented improvements in cardiac function. Our data provide evidence that quantitative and qualitative changes in innate immunity are equally important for cardiac remodeling after MI. Funding Acknowledgement Type of funding sources: Public grant(s) – National budget only. Main funding source(s): Deutsche Forschungsgemeinschaft

Assessment of Knowledge and Attitude Among College Students Toward Umbilical Cord Blood and its Banking

Introduction: Umbilical cord blood (UCB) is a good source of haematopoietic stem cells and progenitor cells. UCB stem cells are used in treatment of a variety of blood and bone marrow diseases, blood cancers, metabolic disorders and immune deficiencies. Objective: This study aimed in investigating college students’ knowledge and attitude about UCB and UCB banking. Methods: It is a descriptive cross-sectional study. The study was conducted in East West College of Management, Bangalore, India from September 2018 to August 2019. A total 80 college students were selected by random sampling techniques. There were self-structured questionnaires used to assess the knowledge and attitude regarding UCB and UCB banking. The data analysis was done using descriptive and inferential statistics in SPSS version 21. Results: The maximum number of participants were from age group 19-21 years followed by age group 16-18 years. The overall knowledge score was 21.05±5.11 with mean percentage score of 60.14. The 71.25% (57) of subjects had moderately adequate knowledge; whereas, 25% (20) of subjects showed adequate knowledge. Similarly, the overall attitude score of subjects was 61.5±5.17 with a mean percentage score of 51.25%. Out of the total participants, 90% (72) had neutral attitude, 10% (8) exhibited positive attitude and none of the subjects exhibited negative attitude towards UCB banking. There was no association found between knowledge and sociodemographic variables and between attitude and sociodemographic variables. Conclusion: The majority of the respondents had moderate knowledge and maximum had neutral atti­tude on umbilical cord blood and umbilical cord blood banking.

Роль молекулярно-генетичних транслокацій у первинній відповіді на лікування дітей, хворих на гостру лімфобластну лейкемію, за програмою ALLIC BFM 2009

The correlation analysis between the number of blast cells in bone marrow and peripheral blood was performed, and the dependence of blast percentage on the presence of molecular genetic translocations (AF4/MLL, BCR/ABL1, TEL/AML, E2A/PBX1) in patients with acute lymphoblastic leukemia (ALL) under the conditions of ALLIC-BFM 2009 cytostatic therapy was researched. The purpose of the study was to establish a relationship between the number of blast cells in bone marrow and peripheral blood depending on the presence of molecular genetic translocations for early detection of induction treatment by ALLIC BFM 2009. Materials and methods. The survey group consisted of 105 children aged 12 months to 16 years (median age was 6 years). Among those surveyed were 62 boys (59.0%) and 43 girls (41.0%). All patients were diagnosed with acute lymphoblastic leukemia. Results and discussion. Correlation analysis revealed a high degree of correlation between the number of blast cells in the bone marrow and peripheral blood, as the correlation coefficient (r) is 0.87. It is shown that the increase in the number of blast cells depends on the presence of chromosomal translocations. The highest number of blasts was observed in patients with BCR/ABL1 and E2A/PBX1 translocations, in whom the content of blasts in bone marrow was 97 and 96%, respectively, and in peripheral blood - 67 and 73%, respectively. It was found that treatment under the ALLIC BFM 2009 program leads to a decrease in the number of blast cells in the bone marrow and blood with minimal values on the 33rd day of treatment. It has been shown that the highest levels of blast cells during chemotherapy are observed in patients with chromosomal translocations BCR/ABL1 and E2A/PBX1. In patients with AF4/MLL translocation, the efficacy of therapy was the highest because no blast cells in the bone marrow were visualized on day 33 of treatment. The study of the primary response of patients with acute lymphoblastic leukemia to induction treatment according to the ALLIC BFM 2009 program revealed the dependence of the level of blast cells of bone marrow and blood on the type of chromosomal aberration. Patients with BCR/ABL1 and E2A/PBX1 have the highest resistance to chemotherapy with molecular genetic translocations, and patients with AF4/MLL and TEL/AML have the lowest resistance, as evidenced by the presence and absence of blast cells in the peripheral blood, respectively. Conclusion. Establishing the relationship between cytogenetic and molecular genetic features of the tumour clone will help determine the degree of malignancy of the process, as well as the risk group for the course of the disease. Determining the dependence of acute leukemia on molecular genetic translocations will make it possible to further modify the treatment program

Novel methods to assess cell-free circulating tumor DNA in acute lymphoblastic leukemia.

10034 Background: Acute lymphoblastic leukemia (ALL) is the most common pediatric cancer, with a relatively high relapse rate, which is associated with poor prognosis. Currently, minimal residual disease (MRD) at the end of induction and consolidation therapy is the best predictor of patient relapse, however obtaining bone marrow aspirate is invasive and not always accurate. Another major concern in ALL is the presence of central nervous system (CNS) disease, which is often present long before clinical diagnosis can be made by flow cytometry. To circumvent these clinical challenges, we developed a new assay quantifying cell-free, circulating tumor (cfDNA) as a biomarker of disease progression, which can be correlated with MRD status as a predictor of relapse. cfDNA is frequently used to monitor progression of solid tumors, but pediatric leukemias lack common mutations that can be used to distinguish leukemic cfDNA from normal cfDNA. Methods: We examined two possible methods for using ctDNA as a biomarker: leukemia cell clonality and DNA methylation profiling. We developed a novel workflow for identifying VDJ rearrangements in leukemia cells and tracking their presence in cfDNA. We collected bone marrow, blood, and CSF samples from newly diagnosed patients, and cfDNA was isolated from blood and CSF samples throughout treatment. Invivoscribe Lymphotrack PCR assays combined with MinION (Oxford Nanopore Technologies) sequencing were used to identify the VDJ sequence of the immunoglobulin (B-ALL) or T-cell receptor (T-ALL) rearrangements of leukemic clones in genomic DNA. The MinION assay relies on patient-specific sequencing. We are also in the process of developing a universal assay that utilizes recurrent methylation changes in ALL to identify leukemic cfDNA in patient samples. Results: The MinION workflow was used to follow leukemic cfDNA throughout the course of treatment, and accurately identified MRD and CNS disease in patients. This workflow performed equivalent or better at detecting leukemic clones compared to MiSeq and droplet digital polymerase chain reaction (ddPCR), and is faster and less expensive than traditional Illumina sequencing. Methylation analysis of 865 ALL and 79 healthy samples yielded 55 regions and 19 specific methylation sites that were uniquely present in ALL samples. We are validating these sites by ddPCR to establish a panel of biomarkers to track ALL over time via cfDNA. Conclusions: The end goal of our study is provide a more sensitive and less invasive method for tracking MRD and CNS disease than current approaches. Results will ultimately be correlated with patient response to therapy, the presence of relapse or CNS disease, and overall outcomes determined by standard clinical diagnostic procedures.

Influence of calcium ion-modified implant surfaces in protein adsorption and implant integration

BackgroundCalcium (Ca) is a well-known element in bone metabolism and blood coagulation. Here, we investigate the link between the protein adsorption pattern and the in vivo responses of surfaces modified with calcium ions (Ca-ion) as compared to standard titanium implant surfaces (control). We used LC–MS/MS to identify the proteins adhered to the surfaces after incubation with human serum and performed bilateral surgeries in the medial section of the femoral condyles of 18 New Zealand white rabbits to test osseointegration at 2 and 8 weeks post-implantation (n=9).ResultsCa-ion surfaces adsorbed 181.42 times more FA10 and 3.85 times less FA12 (p<0.001), which are factors of the common and the intrinsic coagulation pathways respectively. We also detected differences in A1AT, PLMN, FA12, KNG1, HEP2, LYSC, PIP, SAMP, VTNC, SAA4, and CFAH (p<0.01). At 2 and 8 weeks post-implantation, the mean bone implant contact (BIC) with Ca-ion surfaces was respectively 1.52 and 1.25 times higher, and the mean bone volume density (BVD) was respectively 1.35 and 1.13 times higher. Differences were statistically significant for BIC at 2 and 8 weeks and for BVD at 2 weeks (p<0.05).ConclusionsThe strong thrombogenic protein adsorption pattern at Ca-ion surfaces correlated with significantly higher levels of implant osseointegration. More effective implant surfaces combined with smaller implants enable less invasive surgeries, shorter healing times, and overall lower intervention costs, especially in cases of low quantity or quality of bone.

129 Stimulating the Cholinergic Anti-inflammatory Pathway Alters Inflammatory Cell Mobilization after Burn Injury

Abstract Introduction Severe burn injury causes a systemic inflammatory response (SIRS) that is characterized by mobilization of inflammatory cells into the circulation and is associated with distant organ injury that can lead to significant morbidity and mortality. The cholinergic anti-inflammatory pathway, mediated by the vagus nerve, regulates the anti-inflammatory response to injury and infection. We have previously shown in models of burn injury that stimulating the vagus nerve may be a potential therapy aimed at limiting SIRS. Here, we hypothesized that stimulating the vagus nerve (VNS) would limit the SIRS response by altering the mobilization and trafficking of inflammatory cells after burn injury. Methods Wild type 10–12-week-old C57BL/6 mice were injured with a 30% total body surface area steam burn. A separate cohort of animals was treated with electrical stimulation of the cervical vagus nerve for 10 minutes immediately post-burn. Bone marrow, blood and lung tissue were collected 24 hours after burn injury. Flow cytometry of bone marrow was performed to measure Lineage- c-kit± Sca-1+ (LSK) hematopoietic stem cells (HSC), then further analyzed to quantify changes in Long-term (LT) HSC, short-term (ST) HSC, and Multipotential Progenitor (MPP) compartments. Bone marrow, blood and perfused lung tissue were analyzed by flow cytometry using a panel of myeloid cell markers. Results Severe burn injury decreased bone marrow LSK expression by 50% compared to sham, with LT-HSC and MPP expression decreasing to a greater degree than ST-HSCs. VNS did not alter burn-induced changes in any bone marrow HSC cell type. Burn injury was associated with increased mobilization of CD45+CD11b+ monocytes and CD11b+Ly6Chi inflammatory monocytes into the peripheral blood and lung, while increased CD11b+Ly6Clo patrolling monocytes and Gr1+Ly6C- neutrophils was seen in the lung only. VNS significantly prevented the burn-induced increase in CD45+ inflammatory cells, CD11b+Ly6Clo patrolling monocytes and Gr1+Ly6C- neutrophils in the lung (see Figure), reducing their expression to sham levels, despite only modest changes to myeloid cell expression in the blood. Conclusions VNS attenuates myeloid cell cell trafficking to the lung after severe burn injury despite having no effect on emergency myelopoiesis in the bone marrow. Further studies are needed to define the mechanism by which the cholinergic anti-inflammatory pathway attenuates the SIRS response to burn.

Leptin Receptor q223r Polymorphism Influences Clostridioides difficile Infection-Induced Neutrophil CXCR2 Expression in an Interleukin-1β Dependent Manner.

Neutrophils are key first-responders in the innate immune response to C. difficile infection (CDI) and play a central role in disease pathogenesis. Studies have clearly shown that tissue neutrophil numbers need to be tightly regulated for optimal CDI outcomes: while excessive colonic neutrophilia is associated with severe CDI, neutrophil depletion also results in worse outcomes. However, the biological mechanisms that control CDI-induced neutrophilia remain poorly defined. C-X-C chemokine receptor 2 (CXCR2) is a chemotactic receptor that is critical in neutrophil mobilization from bone marrow to blood and tissue sites. We have previously reported that a single nucleotide polymorphism (SNP) in leptin receptor (LEPR), present in up to 50% of people, influenced CDI-induced neutrophil CXCR2 expression and tissue neutrophilia. Homozygosity for mutant LEPR (i.e. RR genotype) was associated with higher CXCR2 expression and more tissue neutrophils. Here, we investigated the biological mechanisms that regulate neutrophil CXCR2 expression after CDI, and the influence of host genetics on this process. Our data reveal that: a) CXCR2 plays a key role in CDI-induced neutrophil extravasation from blood to colonic tissue; b) plasma from C. difficile-infected mice upregulated CXCR2 on bone marrow neutrophils; c) plasma from C. difficile-infected RR mice induced a higher magnitude of CXCR2 upregulation and had more IL-1β; and d) IL-1β neutralization reduced CXCR2 expression on bone marrow and blood neutrophils and their subsequent accrual to colonic tissue. In sum, our data indicate that IL-1β is a key molecular mediator that communicates between gastro-intestinal tract (i.e. site of CDI) and bone marrow (i.e. primary neutrophil reservoir) and regulates the intensity of CDI-induced tissue neutrophilia by modulating CXCR2 expression. Further, our studies highlight the importance of host genetics in affecting these innate immune responses and provide novel insights into the mechanisms by which a common SNP influences CDI-induced neutrophilia.