P J Quesenberry

Alpert Medical School - Brown University, Providence, Rhode Island, United States

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Publications (298)1478.08 Total impact

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    ABSTRACT: The mechanisms involved in renal repair by mesenchymal stromal cells (MSC) are not entirely elucidated. The paracrine secretion of bioactive molecules has been implicated in the protective effects. Besides soluble mediators, MSC have been shown to release extracellular vesicles (EVs), involved in renal repair process for different injury models. EVs have been shown to mediate communication between cells trough the transference of several molecules like protein, bioactive lipids, mRNA and miRNAs. The miRNAs are non-coding RNAs that posttranscriptionally modulate genes expression and are involved in the regulation of several cellular processes, including those related to repair. The aim of the present study was to investigate the role of MSC-EVs in the modulation of miRNAs inside renal proximal tubular epithelial cells (PTEC) in an in vitro model of ischemia-reperfusion injury induced by ATP depletion. In this model we evaluated whether changes in miRNA expression were dependent on direct miRNA transfer or on transcription induction by MSC-EVs. The obtained results showed an enhanced incorporation of MSC-EVs in injured PTEC with protection from cell death. This biological effect was associated with EV-mediated miRNA transfer and with transcriptional modulation of miRNAs expressed by injured PTEC. Prediction of miRNA targets showed that miRNAs modulated in PTEC are involved in process of renal recovery with down-regulation of coding-mRNAs associated with apoptosis, cytoskeleton reorganization and hypoxia such as caspase-3 and -7, SHC1, and SMAD4. In conclusion, these results indicate that MSC-EVs may transfer and modulate the expression of several miRNAs involved in the repair and recovery process in PTEC.
    Stem cells and development 03/2014; · 4.15 Impact Factor
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    ABSTRACT: Early work on platelet and erythrocyte vesicles interpreted the phenomena as a discard of material from cells. Subsequently vesicles were studied as possible vaccines and most recently there has been a focus on the effects of vesicles on cell fate. Recent studies have indicated that extracellular vesicles, previously referred to as microvesicles or exosomes, have the capacity to change the phenotype of neighboring cells. Extensive work has shown that vesicles derived from either lung or liver can enter bone marrow cells, (this is a prerequisite) and alter their fate towards that of the originating liver and lung tissue. Lung vesicles interacted with bone marrow cells result in the bone marrow cells expressing surfactants A B C D, Clara cell protein and aquaporin 5 mRNA. In a similar vein, liver derived vesicles induce albumin mRNA in target marrow cells. The vesicles contain protein, mRNA, microRNA, and non-coding RNA and variably some DNA. This genetic package is delivered to cells and alters phenotype. Further studies have shown that initially the altered phenotype is due to transfer of mRNA and a transcriptional modulator, but the long term epigenetic changes are induced via transfer of a transcriptional factor, the mRNA is rapidly degraded in the cell. Studies on the capacity of vesicles to restore injured tissue have been quite informative. Mesenchymal stem cell derived vesicles are able to reverse the injury to damaged liver and kidney. Other studies have shown that mesenchymal stem cell derived vesicles can reverse radiation toxicity of bone marrow stem cells. Extracellular vesicles offer an intriguing strategy for treating a number of diseases characterized by tissue injury.
    Stem cells and development 02/2014; · 4.15 Impact Factor
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    ABSTRACT: Mesenchymal stem cells (MSCs) contribute to the recovery of tissue injury, providing a paracrine support. Cell-derived extracellular vesicles (EVs), carrying membrane and cytoplasmatic constituents of the cell of origin, have been described as a fundamental mechanism of intercellular communication. We previously demonstrated that EVs derived from human MSCs accelerated recovery following acute kidney injury (AKI) in vivo. The aim of the present study was to investigate the biodistribution and the renal localization of EVs in AKI. For this purpose, two methods for EV labeling suitable for in vivo tracking with optical imaging (OI), were employed using near infrared (NIR) dye (DiD): i) labeled EVs were generated by MSCs pre-incubated with NIR dye and collected from cell supernatants; ii) purified EVs were directly labeled with NIR dye. EVs obtained with these two procedures were injected intravenously (i.v.) into mice with glycerol-induced AKI and into healthy mice to compare the efficacy of the two labeling methods for in vivo detection of EVs at the site of damage. We found that the labeled EVs accumulated specifically in the kidneys of the mice with AKI compared with the healthy controls. After 5 h, the EVs were detectable in whole body images and in dissected kidneys by OI with both types of labeling procedures. The directly labeled EVs showed a higher and brighter fluorescence compared with the labeled EVs produced by cells. The signal generated by the directly labeled EVs was maintained in time, but provided a higher background than that of the labeled EVs produced by cells. The comparison of the two methods indicated that the latter displayed a greater specificity for the injured kidney.
    International Journal of Molecular Medicine 02/2014; · 1.96 Impact Factor
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    ABSTRACT: Adoptive immunotherapy in the form of allogeneic hematopoietic stem cell transplantation (allo-HSCT) is a treatment modality for acute and chronic leukemias that has been in practice for several decades. Drawbacks to transplantation include toxicity from chemotherapy/radiation conditioning regimens, additional toxicity from graft versus host disease, and reliance on appropriate human leukocyte antigen matched donors. Newer modalities with increased specificity of donor cells to tumor cells in addition to therapies that do not require engraftment for anti-tumor effect reduce the risk of graft versus host disease and may create a more robust graft versus leukemia response. Without the need for engraftment, or at the very least in the absence of a 100% engraftment requirement, conditioning regimens may be minimized. Three methods of adoptive immunotherapy that may offer some of these advantages over traditional transplantation are donor lymphocyte infusions (DLI), chimeric antigen receptor modified T cells (CAR T cells), and cellular immunotherapy. DLIs and cellular therapy consist of transfusing T lymphocytes from the donor to recipient in an unmanipulated form. Alternatively, donor T lymphocytes can be modified with addition of chimeric antigen receptors for specific antigen directed killing of tumor cells. Significant responses and survival benefit have been reported with these modalities. Herein, we review the mechanisms for these newer adoptive immune therapies, clinical indications for their use, and potential future directions.
    Discovery medicine 01/2014; 17(91):15-24. · 2.97 Impact Factor
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    ABSTRACT: Extracellular vesicle (EV) trafficking is a fundamental cellular process that occurs in cells and is required for different aspects of pathophysiology. EV trafficking leads to changes in cellular function including apoptosis, angiogenesis and proliferation required for increased tumor formation. We report several phenotypic changes mediated by EVs isolated from non-malignant and malignant prostate cells as well as patient biopsied prostate tumor samples. EVs can reverse the resistance of prostate cancer cells to camptothecin EVs is olated from non-malignant PrECs (Prostate Epithelial Cells) can reverse soft agar colony formation of malignant DU145 cells, with the reciprocal effect observed. Isolation of EVs from 2 Gleason grade 8 prostate cancer patients significantly induced soft agar colony formation of non-malignant PrECs. We have identified proteins via antibody and Mass spectrometry analysis that may be responsible for the phenotypic changes. Mass spectrometry analysis of protein lysates using ProteoIQ revealed protein candidates associated with gene ontology annotations that may be responsible for this phenotypic change. Ingenuity Pathway Analysis was used to identify statistically relevant canonical pathways and functions associated the protein IDs and expression values obtained using ProteoIQ. Western blot analysis confirmed the increase of 14-3-3 zeta, pRKIP and prohibitin protein levels in PrEC cells co-cultured with patient EVs. 14-3-3 proteins were also found as common proteins of 3 other Gleason grade 8 patients. Our study provides a rational basis to further investigate putative proteins, such as 14-3-3 and prohibitin and genetic factors that may be responsible for phenotypic changes that are associated with prostate cancer progression.
    Molecular Cancer 10/2013; 12(1):118. · 5.13 Impact Factor
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    ABSTRACT: A major obstacle in treating colorectal cancer (CRC) is the acquired resistance to chemotherapeutic agents. An important protein in the regulation of cancer cell death and clinical outcome is Raf kinase inhibitor protein (RKIP). In contrast, activated signal transducer and activator of transcription 3 (STAT3) is a protein that promotes tumor cell survival by inhibiting apoptosis and has an important role in cancer progression in many of cancer types. The aim of this study was to evaluate the regulation of RKIP and STAT3 after treatment with clinically relevant chemotherapeutic agents (camptothecin (CPT) and oxaliplatin (OXP)) and the cytokine interleukin-6 (IL-6) in HCT116 colon cancer cells as well as evaluate the association between RKIP and STAT3 with clinical outcome of Stage II colon cancer patients. HCT-116 colon cancer cells were treated with CPT, OXP, and IL-6 separately or in combination in a time and dose-dependent manner and examined for phosphorylated and non-phosphorylated RKIP and STAT3 via Western blot analysis. STAT3 transcriptional activity was measured via a luciferase reporter assay in HCT116 cells treated with CPT, IL-6 or transfected with JAK 1, 2 separately or in combination. We extended these observations and determined STAT3 and RKIP/ pRKIP in tumor microarrays (TMA) in stage II colon cancer patients. We demonstrate IL-6-mediated activation of STAT3 occurs in conjunction with the phosphorylation of RKIP in vitro in human colon cancer cells. OXP and CPT block IL-6 mediated STAT3 activation and RKIP phosphorylation via the inhibition of the interaction of STAT3 with gp130. We determined that STAT3 and nuclear pRKIP are significantly associated with poor patient prognosis in stage II colon cancer patients. In the analysis of tumor samples from stage II colon cancer patients and the human colon carcinoma cell line HCT116, pRKIP and STAT3, 2 proteins potentially involved in the resistance to conventional treatments were detected. The phosphorylation of pRKIP and STAT3 are induced by the cytokine IL-6 and suppressed by the chemotherapeutic drugs CPT and OXP. Therefore, these results suggest that STAT3 and pRKIP may serve as prognostic biomarkers in stage II colon cancer patients and may improve chemotherapy.
    BMC Cancer 10/2013; 13(1):463. · 3.33 Impact Factor
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    ABSTRACT: Prevailing wisdom holds that hematopoietic stem cells (HSCs) are predominantly quiescent. Although HSC cycle status has long been the subject of scrutiny, virtually all marrow stem cell research has been based on studies of highly purified HSCs. Here we explored the cell cycle status of marrow stem cells in un-separated whole bone marrow (WBM). We show that a large number of long-term multi-lineage engraftable stem cells within WBM are in S/G2/M phase. Using bromodeoxyuridine, we show rapid transit through cell cycle of a previously defined relatively dormant purified stem cell, the long-term HSC (LT-HSC;Lineage(-)/c-kit(+)/Sca-1(+)/Flk-2(-)). Actively cycling marrow stem cells have continually changing phenotype with cell cycle transit, likely rendering them difficult to purify to homogeneity. Indeed, as WBM contains actively cycling stem cells, and highly purified stem cells engraft predominantly while quiescent, it follows that the population of cycling marrow stem cells within WBM are lost during purification. Our studies indicate that both the discarded lineage positive and lineage negative marrow cells in a stem cell separation contain cycling stem cells. We propose that future work should encompass this larger population of cycling stem cells that is poorly represented in current studies solely focused on purified stem cell populations.Leukemia accepted article preview online, 30 August 2013. doi:10.1038/leu.2013.252.
    Leukemia: official journal of the Leukemia Society of America, Leukemia Research Fund, U.K 08/2013; · 10.16 Impact Factor
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    ABSTRACT: Circulating endothelium-derived extracellular vesicles (EV) levels are altered in pulmonary arterial hypertension (PAH) but whether they are biomarkers of cellular injury or participants in disease pathogenesis is unknown. Previously, we found that lung-derived EV (LEV) induce bone marrow-derived progenitor cells to express lung-specific mRNA and protein. In this study, we sought to determine if LEV or plasma-derived EV (PEV) alter pulmonary vascular endothelial or marrow progenitor cell phenotype to induce pulmonary vascular remodeling.Methods and ResultsLEV, PEV isolated from monocrotaline (MCT-EV)- or vehicle-treated mice (vehicle-EV) were injected into healthy mice. Right ventricular (RV) hypertrophy and pulmonary vascular remodeling were assessed by RV-to-body weight (RV/BW) and blood vessel wall thickness-to-diameter (WT/D) ratios. RV/BW, WT/D ratios were elevated in MCT- vs. vehicle-injected mice (1.99+0.09vs.1.04+0.09 mg/g; 0.159+0.002vs.0.062+0.009%). RV/BW, WT/D ratios were higher in mice injected with MCT-EV vs. mice injected with vehicle-EV (1.63+0.09vs.1.08+0.09 mg/g; 0.113+0.02vs.0.056+0.01%). Lineage-depleted bone marrow cells incubated with MCT-EV and marrow cells isolated from mice infused with MCT-EV had greater expression of endothelial progenitor cell mRNAs and mRNAs abnormally expressed in PAH than cells incubated with vehicle-EV or isolated from vehicle-EV infused mice. MCT-EV induced an apoptosis-resistant phenotype in murine pulmonary endothelial cells and lineage-depleted bone marrow cells incubated with MCT-EV induced pulmonary hypertension when injected into healthy mice. EV from MCT-injured mice contribute to the development of MCT-induced pulmonary hypertension. This effect may be mediated directly by EV on the pulmonary vasculature or by differentiation of bone marrow cells to endothelial progenitor cells that induce pulmonary vascular remodeling.
    Cardiovascular research 07/2013; · 5.80 Impact Factor
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    ABSTRACT: The tyrosine phosphatase SHP2, encoded by PTPN11, is required for the survival, proliferation and differentiation of various cell types. Germline activating mutations in PTPN11 cause Noonan syndrome, whereas somatic PTPN11 mutations cause childhood myeloproliferative disease and contribute to some solid tumours. Recently, heterozygous inactivating mutations in PTPN11 were found in metachondromatosis, a rare inherited disorder featuring multiple exostoses, enchondromas, joint destruction and bony deformities. The detailed pathogenesis of this disorder has remained unclear. Here we use a conditional knockout (floxed) Ptpn11 allele (Ptpn11(fl)) and Cre recombinase transgenic mice to delete Ptpn11 specifically in monocytes, macrophages and osteoclasts (lysozyme M-Cre; LysMCre) or in cathepsin K (Ctsk)-expressing cells, previously thought to be osteoclasts. LysMCre;Ptpn11(fl/fl) mice had mild osteopetrosis. Notably, however, CtskCre;Ptpn11(fl/fl) mice developed features very similar to metachondromatosis. Lineage tracing revealed a novel population of CtskCre-expressing cells in the perichondrial groove of Ranvier that display markers and functional properties consistent with mesenchymal progenitors. Chondroid neoplasms arise from these cells and show decreased extracellular signal-regulated kinase (ERK) pathway activation, increased Indian hedgehog (Ihh) and parathyroid hormone-related protein (Pthrp, also known as Pthlh) expression and excessive proliferation. Shp2-deficient chondroprogenitors had decreased fibroblast growth factor-evoked ERK activation and enhanced Ihh and Pthrp expression, whereas fibroblast growth factor receptor (FGFR) or mitogen-activated protein kinase kinase (MEK) inhibitor treatment of chondroid cells increased Ihh and Pthrp expression. Importantly, smoothened inhibitor treatment ameliorated metachondromatosis features in CtskCre;Ptpn11(fl/fl) mice. Thus, in contrast to its pro-oncogenic role in haematopoietic and epithelial cells, Ptpn11 is a tumour suppressor in cartilage, acting through a FGFR/MEK/ERK-dependent pathway in a novel progenitor cell population to prevent excessive Ihh production.
    Nature 07/2013; · 38.60 Impact Factor
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    ABSTRACT: BACKGROUND: Acute myeloid leukemia (AML) and other aggressive refractory hematological malignancies unresponsive to upfront therapy remain difficult conditions to treat. Often, the focus of therapy is centered on achieving complete remission of disease in order to proceed with a consolidative stem cell transplant. At issue with this paradigm is the multitude of patients who are unable to achieve complete remission with standard chemotherapeutic options. A major benefit of transplantation is the graft versus tumor effect that follows successful engraftment. However, with this graft versus tumor effect comes the risk of graft versus host disease. Therefore, alternative treatment options that utilize immunotherapy while minimizing toxicity are warranted. Herein, we propose a novel treatment protocol in which haploidentical peripheral blood stem cells are infused into patients with refractory hematological malignancies. The end goal of cellular therapy is not engraftment but instead is the purposeful rejection of donor cells so as to elicit a potent immune reaction that appears to break host tumor tolerance.Methods/designThe trial is a FDA and institutional Rhode Island Hospital/The Miriam Hospital IRB approved Phase I/II study to determine the efficacy and safety of haploidentical peripheral blood cell infusions into patients with refractory hematological malignancies. The primary objective is the overall response rate while secondary objectives will assess the degree and duration of response as well as safety considerations. Patients with refractory acute leukemias and aggressive lymphomas over the age of 18 are eligible. Donors will be selected amongst family members. Full HLA typing of patients and donors will occur as will chimerism assessments. 1-2x108 CD3+ cells/kilogram will be infused on Day 0 without preconditioning. Patients will be monitored for their response to therapy, in particular for the development of a cytokine release syndrome (CRS) that has been previously described. Blood samples will be taken at the onset, during, and following the cessation of CRS so as to study effector cells, cytokine/chemokine release patterns, and extracellular vesicle populations. Initially, six patients will be enrolled on study to determine safety. Provided the treatment is deemed safe, a total of 25 patients will be enrolled to determine efficacy. DISCUSSION: Cellular Immunotherapy for Refractory Hematological Malignancies provides a novel treatment for patients with relapsed/refractory acute leukemia or aggressive lymphoma. We believe this therapy offers the immunological benefit of bone marrow transplantation without the deleterious effects of myeloablative conditioning regimens and minus the risk of GVHD. Laboratory correlative studies will be performed in conjunction with the clinical trial to determine the underlying mechanism of action. This provides a true bench to bedside approach that should serve to further enrich knowledge of host tumor tolerance and mechanisms by which this may be overcome.Trial registrationThe trial protocol is registered at http://www.clinicaltrials.gov with the trial number NCT01685606.
    Journal of Translational Medicine 06/2013; 11(1):150. · 3.46 Impact Factor
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    ABSTRACT: BACKGROUND: New drugs targeting specific genes required for unregulated growth and metastases have improved survival rates for patients with metastatic colorectal cancer. Resistance to monoclonal antibodies specific for the epidermal growth factor receptor (EGFR) has been attributed to the presence of activating point mutations in the proto-oncogene KRAS. The use of EGFR inhibitor monotherapy in patients that have KRAS wild type has produced response rates of only 10-20%. The molecular basis for clinical resistance remains poorly understood. We propose two possible explanations to explain these low response rates; 1) levels of resistant CRC cells carrying mutated KRAS are below the sensitivity of standard direct sequencing modalities (<5%) or 2) the standard practice of analyzing a single area within a heterogeneous tumor is a practice that can overlook areas with mutated KRAS. METHODS: In a collaborative effort with the surgical and molecular pathology departments, 3 formalin fixed paraffin embedded tissue blocks of human CRC were obtained from the human tissue bank maintained by Lifespan Pathology Department and/or the human tissue bank maintained by the Molecular Pathology Core of the COBRE for Cancer Research Development. The three specimens previously demonstrated KRAS mutations detected by the Applied Biosystems Kit. The Wave system 4500 (High performance ion-pairing liquid chromatography (IP-HPLC)) was utilized to evaluate tissue for presence of KRAS proto-oncogene mutations at codon 12 and 13. RESULTS: Initially, sensitivity of WAVE technology was compared with direct sequencing by evaluating a dilutional series. WAVE detected mutant alleles at levels of 2.5% compared to 20% performed with standard direct sequencing. Samples from three patients were evaluated by WAVE technology. Eight samples from patient 1 were analyzed. In two of eight samples, no mutations were detected at concentrations as low as 5%. In one sample a mutation was noted by WAVE and not by direct sequencing. All four samples from patient 2 tested positive for Exon 12/13 mutations. Of the seven samples from patient 3, five were positive for Exon 12/13 mutations and two were negative for Exon 12/13 mutations. CONCLUSION: In these studies the analysis of three patients' colorectal cancer tissue were analyzed utilizing the WAVE technology. Results demonstrated a greater degree of sensitivity in mutation detection when compared to standard sequencing. These studies also demonstrated heterogeneity of expression of KRAS mutations between areas of the tissue samples at a genomic level. The low clinical response rates to EGFR inhibition might be explained by the variation in mutation presence, which was dependent upon the region examined. The heterogeneity demonstrated in these studies provides another phenotypic variant that will impact clinical care.
    Experimental and Molecular Pathology 03/2013; · 2.13 Impact Factor
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    ABSTRACT: Abstract Spontaneous remission of chronic lymphocytic leukemia (CLL) is an unusual and poorly characterized event. We performed a search for spontaneous remission in CLL patients. Cases must have had a pathological diagnosis of CLL with disease duration >6 months. Spontaneous remission was defined as absence of lymphadenopathy or splenomegaly with lymphocyte counts <5 x 10(9)/L for >9 months without therapy. We identified 20 cases and included one additional case from our institution. Fourteen cases (67%) showed remission into monoclonal B-lymphocytosis (MBL) and seven (33%) into a normal phenotype. There was no difference in age distribution, lymphocyte counts or stage between groups. There was a significant difference in the median duration of CLL prior to remission, 13 years in the MBL versus 3 years in the normal phenotype group (p=0.03). This difference on the duration of CLL prior to remission could be due to a possible distinct pathophysiology for these events.
    Leukemia & lymphoma 11/2012; · 2.40 Impact Factor
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    Kenneth D Bishop, Meghana Rao, Peter J Quesenberry
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    ABSTRACT: Malignant epidural spinal cord compression is an oncologic emergency that often results in pain and neurologic dysfunction, which may be permanent. Few prospective studies have been performed to determine whether surgical intervention confers a significant benefit over radiation therapy. We believe the small amount of existing evidence to support surgical intervention or radiation therapy alone suffers from patient selection bias, and that such bias tends to bear out in practice as well. In addition to the objective benefits achieved by surgical decompression, practitioners need to consider the subjective benefits such as increased ambulatory ability, increased spine stability, and improved pain management provided by timely surgical decompression of metastatic spinal cord lesions. Malignant epidural spinal cord compression (MESCC) is an emergency that requires prompt evaluation and multi-disciplinary management. More than 20,000 new cases are reported each year [1]. Patients commonly present with pain and rapidly progressive neurologic dysfunction, and a high potential for permanent neurologic impairment exists if treatment is delayed. There is little prospective data to steer management regarding treatment modality. Conclusions from retrospective analysis are mixed. The benefits of surgical intervention alone were first called into question over thirty years ago when a small prospective study of patients with neurologically-significant compression could not demonstrate a difference in outcomes between radiation therapy and laminectomy plus radiation therapy [2]. Following this, an analysis suggested that laminectomy with or without radiation resulted in lower rates of restored neurologic function and had less benefit for pain relief than radiation alone [3]. In contrast, a later retrospective study showed a significant improvement in neurologic function in patients who received decompressive laminectomy followed by radiation therapy, compared to surgery or radiation alone [4]. Likely due to the move towards direct, selective surgical decompression as opposed to nonselective posterior laminectomy and the subsequent improvement of decompressive surgical techniques, later studies showed that surgery resulted in significant improvement in neurologic function (with
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    ABSTRACT: HIV-1 infection causes profound effects both inside and outside of cells through multiple mechanisms, including those mediated by exosomes. Using the technique of stable isotope labeling by amino acids in cell culture, we compared protein expression patterns in the exosomal compartment of HIV-1-infected and -uninfected lymphocytic H9 cells. Of 770 proteins identified in two independent sets of exosomal samples, 14 proteins were found to be differentially expressed in the exosomal fraction of HIV-1-infected cells versus -uninfected controls. Gene Ontology survey and DAVID analysis revealed that identified proteins were enriched for functional categories such as binding. Of these 14 proteins, three immunomodulatory molecules were reproducibly identified in both replicates and included ADP-ribosyl cyclase 1 (CD38), L-lactate dehydrogenase B chain (LDHB), and Annexin A5 (ANXA5). In addition to previously reported HIV-1 associations with CD38 and LDHB, new interactions were identified and validated for ANXA5, CD38, and LDHB, which were found to bind to HIV-1 p24 and Tat. In summary, our studies reveal that exosomes released from HIV-1-infected cells are composed of a unique and quantitatively different protein signature and harbor regulatory molecules that impact the processes of cellular apoptosis (ANXA5 and LDHB) and proliferation (CD38).
    Proteomics 07/2012; 12(13):2203-11. · 4.43 Impact Factor
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    ABSTRACT: We have shown that hematopoietic stem/progenitor cell phenotype and differentiative potential change throughout cell cycle. Lung-derived microvesicles (LDMVs) also change marrow cell phenotype by inducing them to express pulmonary epithelial cell-specific mRNA and protein. These changes are accentuated when microvesicles isolated from injured lung. We wish to determine if microvesicle-treated stem/progenitor cell phenotype is linked to cell cycle and to the injury status of the lung providing microvesicles. Lineage depleted, Sca-1+ (Lin-/Sca-1+) marrow isolated from mice were cultured with interleukin 3 (IL-3), IL-6, IL-11, and stem cell factor (cytokine-cultured cells), removed at hours zero (cell cycle phase G0/G1), 24 (late G1/early S), and 48 (late S/early G2/M), and cocultured with lung tissue, lung conditioned media (LCM), or LDMV from irradiated or nonirradiated mice. Alternatively, Lin-/Sca-1+ cells not exposed to exogenous cytokines were separated into G0/G1 and S/G2/M cell cycle phase populations by fluorescence-activated cell sorting (FACS) and used in coculture. Separately, LDMV from irradiated and nonirradiated mice were analyzed for the presence of adhesion proteins. Peak pulmonary epithelial cell-specific mRNA expression was seen in G0/G1 cytokine-cultured cells cocultured with irradiated lung and in late G1/early S cells cocultured with nonirradiated lung. The same pattern was seen in cytokine-cultured Lin-/Sca-1 cells cocultured with LCM and LDMV and when FACS-separated Lin-/Sca-1 cells unexposed to exogenous cytokines were used in coculture. Cells and LDMV expressed adhesion proteins whose levels differed based on cycle status (cells) or radiation injury (LDMV), suggesting a mechanism for microvesicle entry. These data demonstrate that microvesicle modification of progenitor/stem cells is influenced by cell cycle and the treatment of the originator lung tissue.
    Stem cells and development 01/2012; 21(10):1627-38. · 4.15 Impact Factor
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    ABSTRACT: Interest has been generated in the capacity of cellular-derived microvesicles to alter the fate of different target cells. Lung, liver, heart and brain-derived vesicles can alter the genetic phenotype of murine marrow cells; however, the stability of such changes and the mechanism of these changes remain unclear. In the present work, we show that lung-derived microvesicles (LDMV) alter the transcriptome and proteome of target marrow cells initially by mRNA and regulator(s) of transcription transfer, but that long term phenotype change is due solely to transfer of a transcriptional regulator with target cell. Whole bone marrow cells (WBM) were co-cultured with LDMV (both isolated from male C57BL/6 mice) or cultured alone (control). One week later, cultured WBM was transplanted into lethally-irradiated female C57BL/6 mice. Recipient mice were sacrificed 6 weeks later and WBM, spleens and livers were examined for the presence of lung-specific gene expression, including surfactants A, B, C and D, aquaporin-5, and clara cell specific protein, via real-time RT-PCR. Immunohistochemistry was also performed on lungs to determine the number of transplanted marrow-derived (Y chromosome+) type II pneumocytes (prosurfactant C+). Mice transplanted with LDMV co-cultured WBM expressed pulmonary epithelial cell genes in the cells of their bone marrow, livers and spleens and over fivefold more transplanted marrow-derived Y+/prosurfactant C+cells could be found in their lungs (vs. control mice). In vitro studies: WBM (from mice or rats) was cultured with or without LDMV (from mice or rats) for 1 week then washed and cultured alone. WBM was harvested at 2-week intervals for real-time RT-PCR analysis, using species-specific surfactant primers, and for Western Blot analysis. Proteomic and microRNA microarray analyses were also performed on cells. LDMV co-cultured WBM maintained expression of pulmonary epithelial cell genes and proteins for up to 12 weeks in culture. Surfactant produced at later time points was specific only to the species of the marrow cell in culture indicating de novo mRNA transcription. These findings, in addition to the altered protein and microRNA profiles of LDMV co-cultured WBM, support a stable transcriptional mechanism for these changes. These data indicate that microvesicle alteration of cell fate is robust and long-term and represents an important new aspect of cellular biology.
    Journal of extracellular vesicles. 01/2012; 1.
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    ABSTRACT: Long-term hematopoietic stem cells (LT-HSC) and short-term hematopoietic stem cells (ST-HSC) have been characterized as having markedly different in vivo repopulation, but similar in vitro growth in liquid culture. These differences could be due to differences in marrow homing. We evaluated this by comparing results when purified ST-HSC and LT-HSC were administered to irradiated mice by three different routes: intravenous, intraperitoneal, and directly into the femur. Purified stem cells derived from B6.SJL mice were competed with marrow cells from C57BL/6J mice into lethally irradiated C57BL/6J mice. Serial transplants into secondary recipients were also carried out. We found no advantage for ST-HSC engraftment when the cells were administered intraperitoneally or directly into femur. However, to our surprise, we found that the purified ST-HSC were not short-term in nature but rather gave long-term multilineage engraftment out to 387 days, albeit at a lower level than the LT-HSC. The ST-HSC also gave secondary engraftment. These observations challenge current models of the stem cell hierarchy and suggest that stem cells are in a continuum of change.
    PLoS ONE 01/2012; 7(2):e31300. · 3.73 Impact Factor
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    ABSTRACT: Much of the therapeutic benefit of allogeneic transplant is by a graft versus tumor effect. Further data shows that transplant engraftment is not dependant on myeloablation, instead relying on quantitative competition between donor and host cells. In the clinical setting, engraftment by competition alone is not feasible due to the need for large numbers of infused cells. Instead, low-level host irradiation has proven to be an effective engraftment strategy that is stem cell toxic but not myeloablative. The above observations served as the foundation for clinical trials utilizing allogeneic matched and haploidentical peripheral blood stem cell infusions with minimal conditioning in patients with refractory malignancies. Although engraftment was transient or not apparent, there were compelling responses in a heavily pretreated patient population that appear to result from the breaking of tumor immune tolerance by the host through the actions of IFNγ, invariant NK T cells, CD8 T cells, NK cells, or antigen presenting cells.
    Advances in Hematology 01/2012; 2012:784213.
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    ABSTRACT: The hierarchical models of stem cell biology have been based on work first demonstrating pluripotental spleen-colony-forming units, then showing progenitors with many differentiation fates assayed in in vitro culture; there followed the definition and separation of "stem cells" using monoclonal antibodies to surface epitopes and fluorescent-activated cell characterization and sorting (FACS). These studies led to an elegant model of stem cell biology in which primitive dormant G0 stem cells with tremendous proliferative and differentiative potential gave rise to progressively more restricted and differentiated classes of stem/progenitor cells, and finally differentiated marrow hematopoietic cells. The holy grail of hematopoietic stem cell biology became the purification of the stem cell and the clonal definition of this cell. Most recently, the long-term repopulating hematopoietic stem cell (LT-HSC) has been believed to be a lineage negative sca-1+C-kit+ Flk3- and CD150+ cell. However, a series of studies over the past 10 years has indicated that murine marrow stem cells continuously change phenotype with cell cycle passage. We present here studies using tritiated thymidine suicide and pyronin-Hoechst FACS separations indicating that the murine hematopoietic stem cell is a cycling cell. This would indicate that the hematopoietic stem cell must be continuously changing in phenotype and, thus, could not be purified. The extant data indicate that murine marrow stem cells are continually transiting cell cycle and that the purification has discarded these cycling cells. Further in vivo BrdU studies indicate that the "quiescent" LT-HSC in G0 rapidly transits cycle.Further complexity of the marrow stem cell system is indicated by studies on cell-derived microvesicles showing that they enter marrow cells and transcriptionally alter their cell fate and phenotype. Thus, the stem cell model is a model of continuing changing potential tied to cell cycle and microvesicle exposure. The challenge of the future is to define the stem cell population, not purify the stem cell. We are at the beginning of elucidation of quantum stemomics.
    Transactions of the American Clinical and Climatological Association 01/2012; 123:152-66.
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    ABSTRACT: JID: 101504271; OID: NLM: PMC3270515; 2011/09/14 [received]; 2011/11/24 [accepted]; 2012/01/18 [epublish]; ppublish
    Advances in hematology. 01/2012; 2012:784213.

Publication Stats

6k Citations
1,478.08 Total Impact Points

Institutions

  • 2008–2013
    • Alpert Medical School - Brown University
      • Department of Medicine
      Providence, Rhode Island, United States
  • 2012
    • Providence College
      Providence, Rhode Island, United States
  • 2007–2012
    • Rhode Island Hospital
      Providence, Rhode Island, United States
  • 2009–2010
    • Brown University
      Providence, Rhode Island, United States
  • 1994–2009
    • University of Massachusetts Medical School
      • • Department of Cancer Biology
      • • Department of Neurology
      • • Department of Medicine
      • • Cancer Center
      • • Department of Cell Biology
      Worcester, Massachusetts, United States
  • 1971–2008
    • St. Elizabeth's Medical Center
      Boston, Massachusetts, United States
  • 2005–2007
    • Lifespan
      Providence, Rhode Island, United States
  • 2002–2007
    • Roger Williams University
      Bristol, Rhode Island, United States
    • Yale-New Haven Hospital
      New Haven, Connecticut, United States
    • Henry Ford Health System
      Detroit, Michigan, United States
    • Partners HealthCare
      Boston, Massachusetts, United States
    • Worcester State University
      Worcester, Massachusetts, United States
    • Universität zu Lübeck
      • Institut für Medizinische Informatik
      Lübeck, Schleswig-Holstein, Germany
  • 2004–2006
    • Massachusetts General Hospital
      Boston, Massachusetts, United States
  • 1996–2003
    • University of Massachusetts Amherst
      Amherst Center, Massachusetts, United States
  • 2000
    • Children's Hospital Oakland Research Institute
      Oakland, California, United States
  • 1997
    • University of Innsbruck
      Innsbruck, Tyrol, Austria
  • 1985–1995
    • University of Virginia
      • • Department of Pediatrics
      • • Department of Medicine
      • • Division of Hematology and Oncology
      Charlottesville, VA, United States
  • 1986
    • Michiana Hematology Oncology
      Indiana, Pennsylvania, United States
  • 1977
    • Harvard Medical School
      • Department of Medicine
      Boston, Massachusetts, United States
  • 1972
    • University of Notre Dame
      South Bend, Indiana, United States