Genomic Evolution of Multiple Myeloma In Vivo over Time.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 3400-3400
Author(s):  
Yu-Tzu Tai ◽  
Benjamin King ◽  
Cheng Li ◽  
XianFeng Li ◽  
Peter Burger ◽  
...  
Keyword(s):  
Drug Resistance ◽  
Mononuclear Cells ◽  
Base Line ◽  
Genomic Evolution ◽  
Peripheral Blood Mononuclear ◽  
Myeloma Cells ◽  
Time Points ◽  
Over Time

Abstract Genetic instability is an important feature of malignant cells, specifically in multiple myeloma (MM). This instability, by activation of oncogene or deletion of tumor suppressor genes, plays an important role in oncogenesis. The ongoing genetic instability is responsible for tumor progression, acquisition of invasiveness, and development of drug resistance, as well as eventual mortality. We have previously demonstrated that MM cells have elevated homologous recombination activity that leads to acquisition of new genomic changes over time and is associated with development of drug resistance (Blood2004; 104: 3409). However, such genomic evolution in patient samples has not been documented. Here we have performed genome wide loss of heterozygosity (LOH) assay, using high-density oligonucleotide arrays capable of measuring up to 250K single nucleotide polymorphisms (SNP) loci and able to analyze small areas of gains or losses as an indicator of genomic instability to determine ongoing development of new changes that may reflect instability. We have evaluated nine MM patients with purified myeloma cells obtained at two time points, atleast six months apart. CD138-expressing myeloma cells from these patients were purified and their peripheral blood mononuclear cells were also obtained. Genomic DNA from these cells was digested with StyI, PCR amplified and hybridized to 250K SNP array. Results from CD138+ myeloma cells from two time points were compared with each other as well as with sample from normal peripheral blood mononuclear cells using the dChip software for LOH and copy number analysis and areas of deletions and amplifications. The changes at the first time point were considered as base line, and the subsequent sample was considered as test sample where acquisition of new changes was evaluated compared to base line. In two patients we had three samples available on a serial basis. We have observed that myeloma cells from the second time point demonstrated significant new areas of genomic change with acquisition of mean 4467 (range 79 to 18998) new LOH loci from base line sample. Of theses new areas of change, 41 SNP loci were found to be present in more than three patient samples indicating recurrent loci of interest in regards to progression and/or development of drug resistance. In 2 patients with 3 serial samples, we observed serial increase in acquisition of new SNPs, 4947 and 18998 at first follow up and 531 and 5275 at second follow up suggesting cumulative accumulation of new genetic change. The reproducible area of new acquisition interestingly involves the area with chromosome 1 and 8 that may have significant role in the pathogenesis and progression of the disease. We are currently obtaining the gene expression profile from these time points to identify expression changes correlating with the observed genomic changes on follow up samples. In summary our results confirm genomic evolution of myeloma in vivo over time and provide means to identify molecular targets associated with progression of MM that can be utilized to inhibit progression of the disease and possibly development of drug resistance.

Acquisition of New In Vivo Genomic Change over Time in Multiple Myeloma.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 2481-2481
Author(s):  
Yu-Tzu Tai ◽  
Leutz Buon ◽  
Benjamin King ◽  
Cheng Li Cheng Li ◽  
Peter Burger Peter Burger ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
Drug Resistance ◽  
Snp Array ◽  
Genomic Change ◽  
Myeloma Cells ◽  
Genomic Changes ◽  
Time Points ◽  
A Genome ◽  
Over Time

Abstract Multiple myeloma (MM) cells present with significant genetic abnormality. This genetic instability is considered responsible for not only development of malignant phenotype but also progression of myeloma as well as development of drug resistance. We have previously demonstrated that MM cell lines have elevated homologous recombination activity that leads to acquisition of new genomic changes over time and is associated with development of drug resistance (Blood2004; 104: 3409). However, such genomic evolution in patient samples has not been documented. Here, we have performed a genome wide loss of heterozygosity (LOH) assay, using high-density oligonucleotide arrays capable of measuring up to 500K single nucleotide polymorphisms (SNP) loci, and identify areas of amplification and losses to determine ongoing development of new changes that may reflect instability. We have evaluated 17 MM patients with purified myeloma cells obtained at two time points, at least six months apart. CD138–expressing myeloma cells from these patients were purified and their peripheral blood mononuclear cells were also obtained. Genomic DNA from these cells was digested with StyI, PCR amplified and hybridized to 250K SNP array. Results from CD138+ myeloma cells from two time points or more were compared with each other using the dChip software for LOH and copy number analysis. During the LOH analysis, we have adapted a tumor vs. tumor comparison to track all the new SNPs affected. We have observed that myeloma cells acquired new LOH loci in the subsequent samples. These new areas of genetic changes were recurrent; for example on chromosome 13, an average area of recurrent LOH of 87 Mb was found to be present among three patients. We were able to observe recurrence of significant copy changes on several chromosomes with/without LOH. The reproducible area of new acquisition of LOH and/or significant copy changes interestingly involved areas on chromosomes 1p, 1q, 8p, 9q, 13q, 15, 16q, 20p, 21q, and X that may have significant role in the pathogenesis and progression of the disease. We are currently analyzing the gene expression profile from these time points to identify expression changes correlating with the observed genomic changes on follow up samples. In summary we demonstrate continued acquisition of new genomic changes in vivo in MM over time and provide a molecular basis for evolution of more aggressive disease able to escape therapeutic interventions. The mechanism governing such evolution is an important target to not only inhibit progression of the disease but also development of drug resistance.

Inhibition of Notch signaling induces apoptosis of myeloma cells and enhances sensitivity to chemotherapy

Blood ◽  
2008 ◽  
Vol 111 (4) ◽  
pp. 2220-2229 ◽  
Author(s):  
Yulia Nefedova ◽  
Daniel M. Sullivan ◽  
Sophia C. Bolick ◽  
William S. Dalton ◽  
Dmitry I. Gabrilovich
Keyword(s):  
Multiple Myeloma ◽  
Drug Resistance ◽  
Notch Signaling ◽  
Mononuclear Cells ◽  
De Novo ◽  
Drug Induced ◽  
Peripheral Blood Mononuclear ◽  
Myeloma Cells ◽  
Induced Apoptosis

Drug resistance remains a critical problem in the treatment of patients with multiple myeloma. Recent studies have de-termined that Notch signaling plays a major role in bone marrow (BM) stroma-mediated protection of myeloma cells from de novo drug-induced apoptosis. Here, we investigated whether pharmacologic inhibition of Notch signaling could affect the viability of myeloma cells and their sensitivity to chemotherapy. Treatment with a γ-secretase inhibitor (GSI) alone induced apoptosis of myeloma cells via specific inhibition of Notch signaling. At concentrations toxic for myeloma cell lines and primary myeloma cells, GSI did not affect normal BM or peripheral blood mononuclear cells. Treatment with GSI prevented BM stroma-mediated protection of myeloma cells from drug-induced apoptosis. The cytotoxic effect of GSI was mediated via Hes-1 and up-regulation of the proapoptotic protein Noxa. In vivo experiments using xenograft and SCID-hu models of multiple myeloma demonstrated substantial antitumor effect of GSI. In addition, GSI significantly improved the cytotoxicity of the chemotherapeutic drugs doxorubicin and melphalan. Thus, this study demonstrates that inhibition of Notch signaling prevents BM-mediated drug resistance and sensitizes myeloma cells to chemotherapy. This may represent a promising approach for therapeutic intervention in multiple myeloma.

Perturbation of Genomic Instability by Wortmannin in Myeloma.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 1105-1105
Author(s):  
Jagannath Pal ◽  
Dheeraj Pelluru ◽  
Mariateresa Fulciniti ◽  
Samir B Amin ◽  
Leutz Buon ◽  
...  
Keyword(s):  
Drug Resistance ◽  
Tumor Growth ◽  
Board Of Directors ◽  
Gene Product ◽  
Genomic Instability ◽  
Research Funding ◽  
Advisory Committees ◽  
Myeloma Cells ◽  
Over Time

Abstract Abstract 1105 Poster Board I-127 Genetic recombination plays a critical role in telomere maintenance, chromosomal translocation, and gene amplification, and may therefore underlie the chromosomal aberrations observed with high frequency in number of malignancies. The molecular mechanism/s inducing genomic instability remains ill-defined and their elucidation may provide methods to prevent tumor progression and development of drug resistance. Our earlier work has demonstrated that homologous recombination (HR) activity is elevated in multiple myeloma (MM) cells and leads to increased rate of mutation and progressive accumulation of genetic variation over time. We have further demonstrated that the inhibition of HR activity in MM cells by siRNAs targeting recombinase leads to significant reduction in the acquisition of new genetic changes in the genome; and conversely, induction of HR activity leads to significant elevation in the number of new mutations over time, and development of drug resistance in MM cells. Here we have evaluated a PI3K inhibitor Wortmaninin which has significant inhibitory activity against both HR and non-HR (nHR) pathways. Exposure of MM cells (OPM1, ARP and RPMI 8226) to wortmannin (WM) led to reduced expression of recombinase (hsRAD51) and nearly complete inhibition of HR activity, within 24 hrs as determined by a plasmid based assay in which generation of active gene product by recombination is measured. Similarly nHR was evaluated by measuring generation of intact gene product from a linearized plasmid. We evaluated effect of WM on nHR by 3 hours preincubation before transfecting the plasmid followed by cell culture for 72 hrs at 37° C. Cells were harvested and analyzed for nHR as previously described. Treatment with WM led to >40% reduction in nHR, indicating that WM affects both HR and NHR pathways. Downregulation of these pathways by wortmannin was also associated with a reduced growth rate of myeloma cells in culture by 20-25% at 48 hours. Importantly, WM treatment markedly decreased the acquisition of new genomic changes in MM as measured by genome-wide loss of heterozygosity assay as an indicator of genomic stability. To evaluate the impact of WM on in vivo tumor growth, OPM1 cells were injected subcutaneously in SCID mice and following appearance of palpable tumors, mice were treated with WM at 0.75 mg/kg, injecting daily intraperitoneally. Treatment with WM was associated with almost complete inhibition of tumor growth in vivo. Long term exposure of myeloma cells to WM was consistently associated with reduced telomere length, probably by blocking HR dependent ALT pathway. These data identifies dysregulated recombination activity as a key mediator of DNA instability and progression of MM, and WM as a potential therapeutic agent for prevention of myeloma progression and possibly drug resistance. Disclosures Anderson: Millenium: Consultancy, Honoraria, Research Funding; Celgene: Consultancy, Honoraria, Research Funding; Novartis: Consultancy, Honoraria, Research Funding. Munshi:Celgene: Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Millennium: Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Novartis : Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau.

scholarly journals JaponiconeA Induces Apoptosis of Bortezomib-Sensitive and –Resistant Myeloma Cells In Vitro and In Vivo through Targeting IKKβ

2020 ◽  
Author(s):  
Zilu Zhang ◽  
Chenjing Ye ◽  
Jia Liu ◽  
Wenbin Xu ◽  
Chao Wu ◽  
...  
Keyword(s):  
Drug Resistance ◽  
Cell Lines ◽  
Mononuclear Cells ◽  
Plasma Cells ◽  
Synergetic Effect ◽  
Rna Seq ◽  
Mice Model ◽  
Myeloma Cells

Abstract Backgrounds: Multiple myeloma (MM) is a clonal proliferative disease of abnormal plasma cells. Relapse and drug resistance still remain to be solved, so new therapeutic drugs are needed to be adopted to further improve the prognosis of MM. JaponiconeA (JA) is a natural product isolated from Inula japonica Thunb, the anti-tumor effect and mechanism in MM has not been studied.Methods: CCK8 and flow cytometry were used to detect the proliferation, apoptosis and cell cycle of MM cell lines with JA treatment. And the in vivo effects of JA were verified in the subcutaneous xenograft mice model of MM. In addition, we analyzed the possible targets and mechanism of JA through RNA-seq and c-Map databases, and we verified the specific target of JA in MM cell lines and bortezomib-resistant MM cell line through CETSA and rescue experiments. JA and bortezomib were used separately or together to treat MM cell lines to explore the synergetic effect. Results: In vitro experiments, JA inhibited proliferation, induced apoptosis and G2/M phase arrest of MM cell lines, and JA selectively killed primary CD138+ MM cells but spared normal human mononuclear cells. In vivo experiments, JA also showed good anti-tumor effect with no observable toxicity. In addition, JA achieved good synergetic effect in combination with bortezomib, and enhanced the anti-tumor effect of bortezomib in bortezomib-resistant cells. According to RNA-seq and c-Map data, the target protein of JA might be IKKβ. CETSA experiment confirmed that JA can bind IKKβ directly in vitro, and overexpression of IKKβ could partly rescue the apoptosis induced by JA.Conclusion: JA exhibited strong anti-tumor effects in MM. It sensitized myeloma cells to bortezomib and overcame NF-κB induced drug resistance through inhibiting IKKβ, which providing a new treatment strategy for MM patients.

Preliminary Report of In vitro and In vivo Effectiveness of Dornase Alfa on SARS-CoV-2 Infection (Preprint)

2020 ◽  
Author(s):  
Hacer Kuzu Okur ◽  
Koray Yalcin ◽  
Cihan Tastan ◽  
Sevda Demir ◽  
Bulut Yurtsever ◽  
...  
Keyword(s):  
Cystic Fibrosis ◽  
Respiratory Tract ◽  
Mononuclear Cells ◽  
Multiple Organ ◽  
Peripheral Blood Mononuclear ◽  
Dornase Alfa ◽  
Tract Infections ◽  
Adult Peripheral Blood

UNSTRUCTURED Dornase alfa, the recombinant form of the human DNase I enzyme, breaks down neutrophil extracellular traps (NET) that include a vast amount of DNA fragments, histones, microbicidal proteins and oxidant enzymes released from necrotic neutrophils in the highly viscous mucus of cystic fibrosis patients. Dornase alfa has been used for decades in patients with cystic fibrosis to reduce the viscoelasticity of respiratory tract secretions, to decrease the severity of respiratory tract infections, and to improve lung function. Previous studies have linked abnormal NET formations to lung diseases, especially to acute respiratory distress syndrome (ARDS). Coronavirus disease 2019 (COVID-19) pandemic affected more than two million people over the world, resulting in unprecedented health, social and economic crises. The COVID-19, viral pneumonia that progresses to ARDS and even multiple organ failure, is caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). High blood neutrophil levels are an early indicator of SARS-CoV-2 infection and predict severe respiratory diseases. A similar mucus structure is detected in COVID-19 patients due to the accumulation of excessive NET in the lungs. Here, we show our preliminary results with dornase alfa that may have an in-vitro anti-viral effect against SARS-CoV-2 infection in a bovine kidney cell line, MDBK without drug toxicity on healthy adult peripheral blood mononuclear cells. In this preliminary study, we also showed that dornase alfa can promote clearance of NET formation in both an in-vitro and three COVID-19 cases who showed clinical improvement in radiological analysis (2-of-3 cases), oxygen saturation (SpO2), respiratory rate, disappearing of dyspnea and coughing.

scholarly journals Autologous Peripheral Blood Mononuclear Cells for Limb Salvage in Diabetic Foot Patients with No-Option Critical Limb Ischemia

2021 ◽  
Vol 10 (10) ◽  
pp. 2213
Author(s):  
Alessia Scatena ◽  
Pasquale Petruzzi ◽  
Filippo Maioli ◽  
Francesca Lucaroni ◽  
Cristina Ambrosone ◽  
...  
Keyword(s):  
Peripheral Blood Mononuclear Cells ◽  
Critical Limb Ischemia ◽  
Diabetic Foot ◽  
Peripheral Blood ◽  
Mononuclear Cells ◽  
Limb Ischemia ◽  
Control Group ◽  
Peripheral Blood Mononuclear ◽  
Blood Mononuclear Cells

Peripheral blood mononuclear cells (PBMNCs) are reported to prevent major amputation and healing in no-option critical limb ischemia (NO-CLI). The aim of this study is to evaluate PBMNC treatment in comparison to standard treatment in NO-CLI patients with diabetic foot ulcers (DFUs). The study included 76 NO-CLI patients admitted to our centers because of CLI with DFUs. All patients were treated with the same standard care (control group), but 38 patients were also treated with autologous PBMNC implants. Major amputations, overall mortality, and number of healed patients were evaluated as the primary endpoint. Only 4 out 38 amputations (10.5%) were observed in the PBMNC group, while 15 out of 38 amputations (39.5%) were recorded in the control group (p = 0.0037). The Kaplan–Meier curves and the log-rank test results showed a significantly lower amputation rate in the PBMNCs group vs. the control group (p = 0.000). At two years follow-up, nearly 80% of the PBMNCs group was still alive vs. only 20% of the control group (p = 0.000). In the PBMNC group, 33 patients healed (86.6%) while only one patient healed in the control group (p = 0.000). PBMNCs showed a positive clinical outcome at two years follow-up in patients with DFUs and NO-CLI, significantly reducing the amputation rate and improving survival and wound healing. According to our study results, intramuscular and peri-lesional injection of autologous PBMNCs could prevent amputations in NO-CLI diabetic patients.

T3 Are Burns a Chronic Condition: Examining Physical and Mental Functioning up to 20 Years After Injury

2021 ◽  
Vol 42 (Supplement_1) ◽  
pp. S2-S3
Author(s):  
Callie Abouzeid ◽  
Audrey E Wolfe ◽  
Gretchen J Carrougher ◽  
Nicole S Gibran ◽  
Radha K Holavanahalli ◽  
...  
Keyword(s):  
Mental Health ◽  
Hospital Stay ◽  
National Database ◽  
Time Points ◽  
Patient Reported ◽  
Burn Survivors ◽  
Population Demographic ◽  
The Impact ◽  
Over Time

Abstract Introduction Burn survivors often face many long-term physical and psychological symptoms associated with their injury. To date, however, few studies have examined the impact of burn injuries on quality of life beyond 2 years post-injury. The purpose of this study is to examine the physical and mental well-being of burn survivors up to 20 years after injury. Methods Data from the Burn Model System National Database (1997–2020) were analyzed. Patient-reported outcome measures were collected at discharge with a recall of preinjury status, and then at 5, 10, 15, and 20 years after injury. Outcomes examined were the Physical Component Summary (PCS) and Mental Component Summary (MCS) of the Short Form-12. Trajectories were developed using linear mixed methods model with repeated measures of PCS and MCS scores over time and controlling for demographic and clinical variables. The model fitted score trajectory was generated with 95% confidence intervals to demonstrate score changes over time and associations with covariates. Results The study population included 420 adult burn survivors with a mean age of 42.4 years. The population was mainly male (66%) and white (76.4%) with a mean burn size of 21.5% and length of hospital stay of 31.3 days. Higher PCS scores were associated with follow-up time points closer to injury, shorter hospital stay, and younger age. Similarly, higher MCS scores were associated with earlier follow-up time points, shorter hospital stay, female gender, and non-perineal burns. MCS trajectories are demonstrated in the Figure. Conclusions Burn survivors’ physical and mental health worsened over time. Such a trend is different from previous reported results for mental health in the general population. Demographic and clinical predictors of recovery over time are identified.

scholarly journals A non-human primate in vitro functional assay for the early evaluation of TB vaccine candidates

npj Vaccines ◽  
2021 ◽  
Vol 6 (1) ◽  
Author(s):  
Rachel Tanner ◽  
Andrew D. White ◽  
Charelle Boot ◽  
Claudia C. Sombroek ◽  
Matthew K. O’Shea ◽  
...  
Keyword(s):  
Growth Inhibition ◽  
Mononuclear Cells ◽  
Functional Assay ◽  
Intermediate Precision ◽  
Peripheral Blood Mononuclear ◽  
Growth Inhibition Assay ◽  
Early Evaluation ◽  
Human Primate

AbstractWe present a non-human primate mycobacterial growth inhibition assay (MGIA) using in vitro blood or cell co-culture with the aim of refining and expediting early tuberculosis vaccine testing. We have taken steps to optimise the assay using cryopreserved peripheral blood mononuclear cells, transfer it to end-user institutes, and assess technical and biological validity. Increasing cell concentration or mycobacterial input and co-culturing in static 48-well plates compared with rotating tubes improved intra-assay repeatability and sensitivity. Standardisation and harmonisation efforts resulted in high consistency agreements, with repeatability and intermediate precision <10% coefficient of variation (CV) and inter-site reproducibility <20% CV; although some systematic differences were observed. As proof-of-concept, we demonstrated ability to detect a BCG vaccine-induced improvement in growth inhibition in macaque samples, and a correlation between MGIA outcome and measures of protection from in vivo disease development following challenge with either intradermal BCG or aerosol/endobronchial Mycobacterium tuberculosis (M.tb) at a group and individual animal level.

Cortisol-induced CXCR4 augmentation mobilizes T lymphocytes after acute physical stress

2005 ◽  
Vol 288 (3) ◽  
pp. R591-R599 ◽  
Author(s):  
Mitsuharu Okutsu ◽  
Kenji Ishii ◽  
Kai Jun Niu ◽  
Ryoichi Nagatomi
Keyword(s):  
T Lymphocytes ◽  
Mononuclear Cells ◽  
Cxcr4 Expression ◽  
Peripheral Blood Mononuclear ◽  
Cycling Exercise ◽  
Ru 486 ◽  
Before And After ◽  
Stromal Cell Derived Factor ◽  
Chemokine Receptor 4

The aim of this study was to elucidate the mechanism responsible for lymphopenia after exercise. Seven young healthy men volunteered for this study. Peripheral blood mononuclear cells (PBMC) were cultured with cortisol and analyzed for C-X-C motif chemokine receptor 4 (CXCR4) expression by flow cytometry. To determine the effects of exercise, subjects performed exhaustive cycling exercise. PBMC were cultured with plasma obtained before and after the cycling exercise. Alternatively, PBMC obtained before and after exercise were cultured without plasma or glucocorticoid to examine whether PBMC were primed in vivo for CXCR4 expression. We analyzed cortisol- or plasma-treated PBMC to determine their ability to migrate through membrane filters in response to stromal cell-derived factor 1α/CXCL12. Cortisol dose- and time-dependently augmented CXCR4 expression on T lymphocytes, with <6 h of treatment sufficient to augment CXCR4 on T lymphocytes. Postexercise plasma also augmented CXCR4 expression. Cortisol or postexercise plasma treatment markedly enhanced migration of T lymphocytes toward CXCL12. Augmentation of CXCR4 on T lymphocytes by cortisol or plasma was effectively blocked by the glucocorticoid receptor antagonist RU-486. Thus exercise-elicited endogenous cortisol effectively augments CXCR4 expression on T lymphocytes, which may account for lymphopenia after exercise.

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