29 results on '"Emmons RV"'
Search Results
2. Lack of effect of donor-recipient Rh mismatch on outcomes after allogeneic hematopoietic stem cell transplantation.
- Author
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Wirk B, Klumpp TR, Ulicny J, Herman JH, Gajewski JL, Martin ME, Emmons RV, and Mangan KF
- Abstract
BACKGROUND: A recently published study has reported that donor-recipient Rhesus (Rh)-mismatched allogeneic hematopoietic stem cell transplantation independently led to significantly poorer survival. This suggests that donor-recipient Rh mismatching is a risk factor in allogeneic hematopoietic stem cell transplantation and should be a criterion for donor selection. STUDY DESIGN AND METHODS: To further evaluate this issue, 258 consecutive patients who underwent myeloablative or submyeloablative allogeneic hematopoietic stem cell transplantation at our institution were analyzed to determine the association between the Rh mismatch pattern and 5-year actuarial survival. Secondary endpoints analyzed were the association of donor-recipient Rh mismatch and event-free survival, transplant-related mortality, incidence of acute graft-versus-host disease (GVHD), and incidence of chronic GVHD. RESULTS: In our analysis, there were no significant associations between donor-recipient Rh mismatch pattern and overall survival, event-free survival, transplant-related mortality, incidence of acute GVHD, or incidence of chronic GVHD. On multivariate Cox proportional hazard analyses, the donor-recipient Rh mismatch pattern was not independently predictive of overall survival. CONCLUSION: Donor-recipient Rh mismatch is not a risk factor in allogeneic hematopoietic stem cell transplantation and does not affect transplant outcomes. [ABSTRACT FROM AUTHOR]
- Published
- 2008
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3. Matrix effects demystified: Strategies for resolving challenges in analytical separations of complex samples.
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Williams ML, Olomukoro AA, Emmons RV, Godage NH, and Gionfriddo E
- Abstract
Matrix effects can significantly impede the accuracy, sensitivity, and reliability of separation techniques presenting a formidable challenge to the analytical process. It is crucial to address matrix effects to achieve accurate and precise measurements in complex matrices. The multifaceted nature of matrix effects which can be influenced by factors such as target analyte, sample preparation protocol, composition, and choice of instrument necessitates a pragmatic approach when analyzing complex matrices. This review aims to highlight common challenges associated with matrix effects throughout the entire analytical process with emphasis on gas chromatography-mass spectrometry, liquid chromatography-mass spectrometry, and sample preparation techniques. These techniques are susceptible to matrix effects that could lead to ion suppression/enhancement or impact the analyte signal at various stages of the analytical workflow. The assessment, quantification, and mitigation of matrix effects are necessary in developing any analytical method. Strategies can be implemented to reduce or eliminate the matrix effect by changing the type of ionization, improving extraction and clean-up methods, optimization of chromatography conditions, and corrective calibration methods. While development of an effective strategy to completely mitigate matrix effects remains elusive, an integrated approach that combines sample preparation, analytical extraction, and effective instrumental analysis remains the most promising avenue for identifying and resolving matrix effects., (© 2023 The Authors. Journal of Separation Science published by Wiley-VCH GmbH.)
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- 2023
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4. Rapid Screening and Quantification of PFAS Enabled by SPME-DART-MS.
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Emmons RV, Fatigante W, Olomukoro AA, Musselman B, and Gionfriddo E
- Abstract
Per- and polyfluoroalkyl substances (PFAS), an emerging class of toxic anthropogenic chemicals persistent in the environment, are currently regulated at the low part-per-trillion level worldwide in drinking water. Quantification and screening of these compounds currently rely primarily on liquid chromatography hyphenated to mass spectrometry (LC-MS). The growing need for quicker and more robust analysis in routine monitoring has been, in many ways, spearheaded by the advent of direct ambient mass spectrometry (AMS) technologies. Direct analysis in real time (DART), a plasma-based ambient ionization technique that permits rapid automated analysis, effectively ionizes a broad range of compounds, including PFAS. This work evaluates the performance of DART-MS for the screening and quantification of PFAS of different chemical classes, employing a central composite design (CCD) to better understand the interactions of DART parameters on their ionization. Furthermore, in-source fragmentation of the model PFAS was investigated based on the DART parameters evaluated. Preconcentration of PFAS from water samples was achieved by solid phase microextraction (SPME), and extracts were analyzed using the optimized DART-MS conditions, which allowed obtaining linear dynamic ranges (LDRs) within 10 and 5000 ng/L and LOQs of 10, 25, and 50 ng/L for all analytes. Instrumental analysis was achieved in less than 20 s per sample.
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- 2023
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5. Leveraging multi-mode microextraction and liquid chromatography stationary phases for quantitative analysis of neurotoxin β-N-methylamino-L-alanine and other non-proteinogenic amino acids.
- Author
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Emmons RV, Karaj E, Cudjoe E, Bell DS, Tillekeratne LMV, and Gionfriddo E
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- Chromatography, Liquid, Amino Acids, Neurotoxins
- Abstract
Effective quantitative analysis of BMAA (β-N-methylamino-L-alanine) and its isomers without the need for derivatization has always been an analytical challenge due to their poor retention and separation on various liquid chromatography stationary phases. Previous studies that utilized conventional hydrophilic interaction chromatography (HILIC) demonstrate false negatives compared to reverse-phase workflows with derivatization. This work evaluates the chromatographic behavior of BMAA and its isomers, in their underivatized forms, on selected stationary phases, in particular fluorophenyl-based columns, to attain effective retention and separation. Detection and quantification were achieved with an ion-trap mass spectrometer. Extraction and preconcentration were achieved via solid phase microextraction (SPME) by assessing the effectiveness of multiple extraction phases, including hydrophilic-lipophilic balanced (HLB) and mixed-mode (MM). A MM extraction phase consisting of C
8 and benzene sulfonic acid moieties provided ideal extraction performance for BMAA and its isomers (2,4-diaminobutyric acid, DABA; N-(2-aminoethyl) glycine, AEG). Chromatographic separation was achieved within 8 min on a fluorophenyl stationary phase, ensuring high throughput without derivatization, and showing exceptional improvement from conventional HILIC methods. Limits of quantification in water for BMAA and AEG were 2.5 µg L-1 and DABA was 5 µg L-1 , with linear dynamic ranges from 2.5 µg L-1 - 200 µg L-1 for BMAA and AEG and 5 µg L-1 - 200 µg L-1 for DABA., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2022. Published by Elsevier B.V.)- Published
- 2022
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6. Unraveling the Complex Composition of Produced Water by Specialized Extraction Methodologies.
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Emmons RV, Shyam Sunder GS, Liden T, Schug KA, Asfaha TY, Lawrence JG, Kirchhoff JR, and Gionfriddo E
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- Solid Phase Microextraction, Wastewater chemistry, Water, Polycyclic Aromatic Hydrocarbons analysis, Water Pollutants, Chemical analysis
- Abstract
Produced water (PW), a waste byproduct of oil and gas extraction, is a complex mixture containing numerous organic solubles and elemental species; these constituents range from polycyclic aromatic hydrocarbons to naturally occurring radioactive materials. Identification of these compounds is critical in developing reuse and disposal protocols to minimize environmental contamination and health risks. In this study, versatile extraction methodologies were investigated for the untargeted analysis of PW. Thin-film solid-phase microextraction with hydrophilic-lipophilic balance particles was utilized for the extraction of organic solubles from eight PW samples from the Permian Basin and Eagle Ford formation in Texas. Gas chromatography-mass spectrometry analysis found a total of 266 different organic constituents including 1,4-dioxane, atrazine, pyridine, and PAHs. The elemental composition of PW was evaluated using dispersive solid-phase extraction followed by inductively coupled plasma-mass spectrometry, utilizing a new coordinating sorbent, poly(pyrrole-1-carboxylic acid). This confirmed the presence of 29 elements including rare earth elements, as well as hazardous metals such as Cr, Cd, Pb, and U. Utilizing chemometric analysis, both approaches facilitated the discrimination of each PW sample based on their geochemical origin with a prediction accuracy above 90% using partial least-squares-discriminant analysis, paving the way for PW origin tracing in the environment.
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- 2022
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7. Ion exchange solid phase microextraction coupled to liquid chromatography/laminar flow tandem mass spectrometry for the determination of perfluoroalkyl substances in water samples.
- Author
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Olomukoro AA, Emmons RV, Godage NH, Cudjoe E, and Gionfriddo E
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- Caprylates analysis, Fluorocarbons isolation & purification, Water Pollutants, Chemical isolation & purification, Chromatography, Liquid, Fluorocarbons analysis, Ion Exchange, Solid Phase Microextraction, Tandem Mass Spectrometry, Water Pollutants, Chemical analysis
- Abstract
Per- and polyfluoroalkyl substances (PFAS) are toxic and bioaccumulative compounds that are persistent in the environment due to their water and heat resistant properties. These compounds have been demonstrated to be ubiquitous in the environment, being found in water, soil, air and various biological matrices. The determination of PFAS at ultra-trace levels is thus critical to assess the extent of contamination in a particular matrix. In this work, solid phase microextraction (SPME) was evaluated as a pre-concentration technique to aid the quantitation of this class of pollutants below the EPA established advisory limits in drinking water at parts-per-trillion levels. Four model PFAS with varying physicochemical properties, namely hexafluoropropylene oxide dimer acid (GenX), perfluoro-1- butanesulfonate (PFBS), perfluoro-n-octanoic acid (PFOA) and perfluoro-1-octanesulfonate (PFOS) were studied as a proof of concept. Analysis was performed with the use of ultra-high pressure liquid chromatography-laminar flow tandem mass spectrometry (UHPLC-MS/MS). This study proposes the use of hydrophilic-lipophilic balance-weak anion-exchange/polyacrylonitrile (HLB-WAX/PAN) as a SPME coating, ideal for all model analytes. A sample volume of 1.5 mL was used for analysis, the optimized protocol including 20 min extraction, 20 min desorption and 6 min LC/MS analysis. This method achieved LOQs of 2.5 ng L
- 1 (PFOS) and 1 ng L- 1 (GenX, PFBS and PFOA) with satisfactory precision and accuracy values evaluated over a period of 5 days., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2021. Published by Elsevier B.V.)- Published
- 2021
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8. Minimizing transient microenvironment-associated variability for analysis of environmental anthropogenic contaminants via ambient ionization.
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Emmons RV and Gionfriddo E
- Abstract
The rapid and quantitative analysis of anthropogenic contaminants in environmental matrices is crucial for regulatory testing and to elucidate the environmental fate of these pollutants. Direct ambient mass spectrometry (AMS) methodologies greatly increase sample throughput, can be adapted for onsite analysis and are often regarded as semi-quantitative by most developed protocols. One of the limitations of AMS, especially for on site analysis applications, is the irreproducibility of the measurements related to the occurrence of transient microenvironments (TME) and variable background interferences. In this work we report an effective strategy to minimize these effects by hyphenating, for the first time, solid phase microextraction (SPME) arrow to mass spectrometry via a thermal desorption unit (TDU) and direct analysis in real time (DART) source. The developed method was optimized for the extraction and analysis of pesticides and pharmaceuticals from surface water. It was demonstrated that the hyphenation of the SPME and TDU-DART resulted in reduced background contamination, indicating the suitability of the method for onsite analysis even in variable and non-ideal environments. Model analytes were quantitated in the low μg/L range with a total analysis time of less than 5 min, linear dynamic ranges (LDR) and interday reproducibility for most compounds being 2.5-500 μg/L and lower than 10%, respectively. The developed approach provides an excellent analytical tool that can be applied for the onsite high-throughput analysis of water samples as well as air and aereosols. Considering the tunability of our extraction process, time-resolved environmental monitoring can be achieved onsite within minutes., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2021 Elsevier B.V. All rights reserved.)
- Published
- 2021
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9. Optimization of thin film solid phase microextraction and data deconvolution methods for accurate characterization of organic compounds in produced water.
- Author
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Emmons RV, Liden T, Schug KA, and Gionfriddo E
- Abstract
The continued rise in the extraction of unconventional oil and gas across the globe poses many questions about how to manage these relatively new waste-streams. Produced water, the primary waste by-product, contains a diverse number of anthropogenic additives together with the numerous hydrocarbons extracted from the well. Due to potential environmental hazards, it is critical to characterize the chemical composition of this type of waste before proper disposal or remediation/reuse. In this work, a thin film solid phase microextraction approach was developed and optimized to characterize produced water. The thin film device consisted of hydrophilic-lipophilic balance particles embedded in polydimethylsiloxane and immobilized on a carbon mesh surface. These devices were chosen to provide broad extraction coverage and high reusability. Various parameters were evaluated to ensure reproducible results while minimizing analyte loss. This optimized protocol, consisting of a 15 min extraction followed by a short (3 s) rinsing step, enabled the reproducible analysis of produced water without any sample pretreatment. Extraction efficiency was suitable for both produced water additives and hydrocarbons. The developed approach was able to tentatively identify a total of 201 compounds from produced water samples, by using one-dimensional gas chromatography hyphenated to mass spectrometry and data deconvolution., (© 2020 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)
- Published
- 2020
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10. Randomized Trial of Lenalidomide Versus Observation in Smoldering Multiple Myeloma.
- Author
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Lonial S, Jacobus S, Fonseca R, Weiss M, Kumar S, Orlowski RZ, Kaufman JL, Yacoub AM, Buadi FK, O'Brien T, Matous JV, Anderson DM, Emmons RV, Mahindra A, Wagner LI, Dhodapkar MV, and Rajkumar SV
- Subjects
- Adult, Aged, Aged, 80 and over, Female, Humans, Lenalidomide adverse effects, Male, Middle Aged, Quality of Life, Smoldering Multiple Myeloma mortality, Lenalidomide therapeutic use, Smoldering Multiple Myeloma drug therapy
- Abstract
Purpose: Observation is the current standard of care for smoldering multiple myeloma. We hypothesized that early intervention with lenalidomide could delay progression to symptomatic multiple myeloma., Methods: We conducted a randomized trial that assessed the efficacy of single-agent lenalidomide compared with observation in patients with intermediate- or high-risk smoldering multiple myeloma. Lenalidomide was administered orally at a dose of 25 mg on days 1 to 21 of a 28-day cycle. The primary end point was progression-free survival, with disease progression requiring the development of end-organ damage attributable to multiple myeloma and biochemical progression., Results: One hundred eighty-two patients were randomly assigned-92 patients to the lenalidomide arm and 90 to the observation arm. Median follow-up is 35 months. Response to therapy was observed in 50% (95% CI, 39% to 61%) of patients in the lenalidomide arm, with no responses in the observation arm. Progression-free survival was significantly longer with lenalidomide compared with observation (hazard ratio, 0.28; 95% CI, 0.12 to 0.62; P = .002). One-, 2-, and 3-year progression-free survival was 98%, 93%, and 91% for the lenalidomide arm versus 89%, 76%, and 66% for the observation arm, respectively. Only six deaths have been reported, two in the lenalidomide arm versus four in the observation arm (hazard ratio for death, 0.46; 95% CI, 0.08 to 2.53). Grade 3 or 4 nonhematologic adverse events occurred in 25 patients (28%) on lenalidomide., Conclusion: Early intervention with lenalidomide in smoldering multiple myeloma significantly delays progression to symptomatic multiple myeloma and the development of end-organ damage.
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- 2020
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11. Tackling mantle cell lymphoma (MCL): Potential benefit of allogeneic stem cell transplantation.
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Shanbhag S, Smith MR, and Emmons RV
- Abstract
Mantle cell lymphoma (MCL) is a type of non-Hodgkins lymphoma (NHL) associated with poor progression-free and overall survival. There is a high relapse rate with conventional cytotoxic chemotherapy. Intensive combination chemotherapy including rituximab, dose intense CHOP- (cyclophosphamide-doxorubicin-vincristine-prednisone) like regimens, high dose cytarabine, and/or consolidation with autologous stem cell transplant (autoSCT) have shown promise in significantly prolonging remissions. Data from phase II studies show that even in patients with chemotherapy refractory MCL, allogeneic stem cell transplant (alloSCT) can lead to long term disease control. Most patients with MCL are not candidates for myeloablative alloSCT due to their age, comorbidities, and performance status. The advent of less toxic reduced intensity conditioning (RIC) regimens, which rely more on the graft-versus-lymphoma (GVL) effect, have expanded the population of patients who would be eligible for alloSCT. RIC regimens alter the balance of toxicity and efficacy favoring its use. Treatment decisions are complicated by introduction of novel agents which are attractive options for older, frail patients. Further studies are needed to determine the role and timing of alloSCT in MCL. Currently, for selected fit patients with chemotherapy resistant MCL or those who progress after autoSCT, alloSCT may provide long term survival.
- Published
- 2010
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12. Increased natural killer cells and decreased regulatory T cells are seen in complex atypical endometrial hyperplasia and well-differentiated carcinoma treated with progestins.
- Author
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Witkiewicz AK, McConnell T, Potoczek M, Emmons RV, and Kurman RJ
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- Adult, Blood Cell Count, Carcinoma, Endometrioid immunology, Carcinoma, Endometrioid pathology, Endometrial Hyperplasia immunology, Endometrial Hyperplasia pathology, Endometrial Neoplasms immunology, Endometrial Neoplasms pathology, Endometrium drug effects, Endometrium pathology, Female, Humans, Antineoplastic Agents therapeutic use, Carcinoma, Endometrioid drug therapy, Endometrial Hyperplasia drug therapy, Endometrial Neoplasms drug therapy, Killer Cells, Natural immunology, Progestins therapeutic use, T-Lymphocytes, Regulatory immunology
- Abstract
Progestins are used to treat complex atypical hyperplasia and well-differentiated endometrial carcinoma in women who desire fertility preservation and those who are poor surgical candidates. Although sensitivity to progestins is thought to be associated with the presence of estrogen and progesterone receptors, it is known that receptor-negative tumors can also respond to the agent, suggesting that there is another direct antitumor action of progestin. Because tumor immune response is an additional predictor of survival in well-differentiated endometrial carcinoma, it is surprising that the role of progestins in tumor immunity has not been investigated. Regulatory T cells modulate the immune response, whereas cytotoxic T cells directly target tumor cells. In this study, we investigated the effect of progestins on regulatory T cells and cytotoxic T cells. The pre- and posttreatment endometrial samples of 15 progestin-treated patients with complex atypical hyperplasia or well-differentiated endometrial carcinoma were evaluated for therapeutic response and the presence of cytotoxic T cells and regulatory T cells. Immunohistochemical analysis was performed for FOXP3 to identify regulatory T cells and for granzyme B to identify activated cytotoxic T cells. To further characterize the cytotoxic T cell's subpopulations, we performed CD8 (cytotoxic T-cell marker) and CD56 (natural killer cells marker). Ten of 15 patients had normal morphology on follow-up endometrial samplings, and 4 patients had persistence or progression of the disease. Regulatory T-cell counts pretreatment were significantly higher in complex atypical hyperplasia and well-differentiated endometrial carcinoma than in posttreatment normal endometrium. Residual complex atypical hyperplasia and well-differentiated endometrial carcinoma present in posttreatment samples maintained high regulatory T cells and low number of cytotoxic T cells. Progestin treatment was associated with striking increase in cytotoxic T cells in areas with decidual reaction. Before treatment, most of the granzyme B+ cytotoxic T cells in complex atypical hyperplasia and well-differentiated endometrial carcinoma were CD8(+) T cells, whereas after treatment, up to 80% of cytotoxic T cells were natural killer cells. These results suggest that progestin treatment affects subpopulations of lymphocytes in the endometrium and may induce immune suppression of complex atypical hyperplasia and well-differentiated endometrial carcinoma.
- Published
- 2010
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13. Foxp3-expressing T regulatory cells and mast cells in acute graft-versus-host disease of the skin.
- Author
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Wu KN, Emmons RV, Lisanti MP, Farber JL, and Witkiewicz AK
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- Adolescent, Adult, Aged, Biopsy, Female, Forkhead Transcription Factors immunology, Forkhead Transcription Factors metabolism, Graft vs Host Disease metabolism, Graft vs Host Disease pathology, Hematopoietic Stem Cell Transplantation, Humans, Inflammation metabolism, Male, Mast Cells metabolism, Middle Aged, Proto-Oncogene Proteins c-kit immunology, Proto-Oncogene Proteins c-kit metabolism, Skin immunology, Skin pathology, T-Lymphocytes, Regulatory metabolism, Young Adult, Graft vs Host Disease immunology, Inflammation immunology, Mast Cells immunology, T-Lymphocytes, Regulatory immunology
- Abstract
Acute graft-versus-host disease (aGVHD) limits the effectiveness of allogeneic hematopoietic stem cell transplantation. Foxp3 is required for the development and function of CD4(+)/CD25(+) regulatory T cells (T-regs). Foxp3-expressing T-regs are thought to protect against GVHD. Mast cells are thought to be essential in CD4(+)/CD25(+) regulatory T cell-dependent peripheral tolerance. Twenty biopsies of skin with grades I-III aGVHD were stained for Foxp3 and CD117. Inflammation was quantified by a 4 point scale, 0 = no inflammation, 1 = <25% of 20x field, 2 = 25-50%, and 3 = >50%. T-regs and mast cells were quantified by a 4 point scale, 0 = no cells per 20x field, 1 = <5 cells per 20x field, 2 = 5-10 cells, and 3 = >10 cells. T-regs were positively correlated with both inflammation and aGVHD grade. Twelve cases with low T-regs had mild inflammation and lower grades of aGVHD and 6 cases with high T-regs had dense inflammatory infiltrate and higher grades of aGVHD. The number of T-regs, mast cells and density of the inflammatory infiltrate were positively correlated only in cases with mild inflammation. In aGVHD of the skin, T-regs increased with the degree of inflammation and GVHD grade. Mast cells were present at the same density whether aGVHD was of lower or higher grade.
- Published
- 2009
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14. Expression of indoleamine 2,3-dioxygenase in metastatic malignant melanoma recruits regulatory T cells to avoid immune detection and affects survival.
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Brody JR, Costantino CL, Berger AC, Sato T, Lisanti MP, Yeo CJ, Emmons RV, and Witkiewicz AK
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- Forkhead Transcription Factors immunology, Forkhead Transcription Factors metabolism, Humans, Lymphatic Metastasis, Melanoma immunology, Melanoma mortality, Melanoma pathology, Retrospective Studies, Skin Neoplasms immunology, Skin Neoplasms mortality, Skin Neoplasms pathology, Immunologic Surveillance, Indoleamine-Pyrrole 2,3,-Dioxygenase biosynthesis, Melanoma enzymology, Skin Neoplasms enzymology, T-Lymphocytes, Regulatory immunology
- Abstract
The mechanism by which malignant melanoma (MM) cells survive in lymph nodes is poorly understood. One possible mechanism by which MM cells can escape immune surveillance is through upregulation of immunomodulatory enzymes such as indoleamine 2,3-dioxygenase (IDO). In this study, 25 cases of MM lymph node metastases from patients with long and short survival were evaluated for expression of IDO and the number of Forkhead box p3 (FOXP3)-expressing regulatory T cells. Moderate to strong cytoplasmic IDO expression was present in all (15/15) MM lymph node metastases in patients with poor survival. Eight of 10 patients with metastatic MM and long survival were negative or only weakly positive for IDO. Upregulation of IDO in metastatic MM cells was associated with an increased number of regulatory T cells (Tregs). There was a statistically significant association between shorter survival and both a stronger IDO expression (p = 0.0019) and a higher number of FOXP3 expressing Tregs (p < 0.001). Using RT-PCR analysis, we showed that IDO expression in MM cells is induced by interferon-gamma. These data support the notion that metastatic MM cells select for expression of IDO to evade immunologic detection. Therefore, inhibition of IDO in MM patients may be a useful treatment strategy.
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- 2009
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15. Mixed chimerism and graft failure following conditioning with the fludarabine and cyclophosphamide nonablative regimen; conversion to full donor chimerism.
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Jillella AP, Shafer D, Klumpp TR, Emmons RV, and Mangan KF
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- Acute Disease, Adult, Aged, Antineoplastic Combined Chemotherapy Protocols therapeutic use, Chronic Disease, Cohort Studies, Disease Progression, Female, Follow-Up Studies, Graft Rejection etiology, Graft vs Host Disease etiology, Hematologic Neoplasms etiology, Humans, Injections, Intravenous, Lymphocyte Transfusion adverse effects, Male, Middle Aged, Retrospective Studies, Stem Cell Transplantation adverse effects, Survival Rate, Tissue Donors, Transplantation Conditioning, Treatment Outcome, Vidarabine administration & dosage, Antineoplastic Combined Chemotherapy Protocols adverse effects, Chimerism, Cyclophosphamide administration & dosage, Graft Rejection therapy, Graft vs Host Disease therapy, Hematologic Neoplasms therapy, Vidarabine analogs & derivatives
- Abstract
Twenty-one patients with hematologic malignancies were treated with the fludarabine (120-125 mg/m(2)) and cyclophosphamide (120 mg/kg) nonmyeloablative conditioning regimen. Graft versus host disease (GVHD) and graft rejection prophylaxis was with tacrolimus and mycophenolate mofetil. Thirteen of the 21 patients (62%) had mixed chimerism (< or = 90% donor cells) at day 60 and 11 (52%) of these patients had mixed chimerism which persisted until day 100. Immunosuppression was discontinued in 12 of 13 patients and two of them converted to full chimerism by day 100. Eight patients received a donor lymphocyte infusion (DLI) and five of them converted to full donor chimerism with DLI alone. Two patients were given GM-CSF in addition to a DLI with conversion to full donor chimerism. Three patients (14%) had graft failure requiring a second transplant using fludarabine (125 mg/m(2)) and melphalan (140 mg/m(2)). With a median followup of 2.8 years, 15 patients are alive - one with disease and 14 with no disease. Two patients died of acute GVHD, one of chronic GVHD, and three due to progressive disease. We conclude that the nonmyeloablative fludarabine/cyclophosphamide regimen results in a significant incidence of mixed chimerism and graft rejection but is well tolerated. We suggest a more intense regimen, such as fludarabine and melphalan, be used in patients with a high risk of early disease progression to establish early engraftment and graft versus tumor effect., ((c) 2007 Wiley-Liss, Inc.)
- Published
- 2007
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16. Lack of effect of donor-recipient ABO mismatching on outcome following allogeneic hematopoietic stem cell transplantation.
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Klumpp TR, Herman JH, Ulicny J, Emmons RV, Martin ME, and Mangan KF
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- Blood Grouping and Crossmatching methods, Disease-Free Survival, Female, Graft vs Host Disease mortality, Humans, Male, Neoplasms therapy, Recurrence, Remission Induction, Retrospective Studies, Time Factors, Transplantation, Homologous, ABO Blood-Group System, Donor Selection methods, Hematopoietic Stem Cell Transplantation mortality, Neoplasms mortality, Tissue Donors
- Abstract
Several recently published studies have suggested that patients who undergo ABO mismatched hematopoietic stem cell transplantation may be at increased risk for relapse, graft-versus-host disease, transplant-related mortality, and/or all-cause mortality. To investigate this issue further, we analyzed potential associations between the donor-recipient ABO mismatch pattern and the above outcome measures among 240 consecutive patients who underwent allogeneic hematopoietic stem cell transplantation at our institution. Our analyses uncovered no significant associations between donor-recipient ABO mismatch pattern and overall survival, event-free survival, transplant-related mortality, incidence of acute graft-versus-host disease (GVHD), or incidence of chronic GVHD. Our data do not support recent assertions that donor-recipient ABO mismatching is a major risk factor for patients undergoing allogeneic transplant, nor do they support recent assertions that ABO matching should be an important consideration in selecting allogeneic hematopoietic stem cell donors.
- Published
- 2006
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17. High-dose cyclophosphamide, BCNU, and VP-16 (CBV) conditioning before allogeneic stem cell transplantation for patients with non-Hodgkin's lymphoma.
- Author
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Rossi HA, Becker PS, Emmons RV, Westervelt P, Levy W, Liu Q, Clark Y, and Ballen K
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- Adolescent, Adult, Blood Platelets cytology, Disease-Free Survival, Female, Graft vs Host Disease mortality, Humans, Infections mortality, Liver Diseases mortality, Lung Diseases mortality, Lymphoma, Non-Hodgkin mortality, Male, Middle Aged, Neutrophils cytology, Recurrence, Transplantation Conditioning methods, Transplantation, Homologous, Treatment Outcome, Antineoplastic Agents, Alkylating administration & dosage, Antineoplastic Agents, Phytogenic administration & dosage, Carmustine administration & dosage, Cyclophosphamide administration & dosage, Etoposide administration & dosage, Hematopoietic Stem Cell Transplantation, Lymphoma, Non-Hodgkin drug therapy
- Abstract
Allogeneic stem cell transplantation (SCT) has been shown to be a curative therapy for some patients with non-Hodgkin's lymphoma (NHL). Total-body irradiation and high-dose cyclophosphamide combinations are the most established conditioning regimens used in this setting. We examined the efficacy and toxicity of cyclophosphamide, BCNU, and VP-16 (CBV) as a suitable chemotherapy-only regimen for NHL patients. In total, 18 patients, median age 42 years, with NHL were treated with CBV followed by allotransplant. Patients had received a median of two prior chemotherapy regimens. Median times to neutrophil and platelet recovery were 19 and 15 days, respectively. Interstitial pneumonitis occurred in one patient. There have been four relapses after a median follow-up of 39 months. Overall, there were four deaths, one because of relapse. The 2-year estimates of relapse-free and overall survival are 56 and 76%, respectively. CBV is a safe and an effective alternative to TBI-containing regimens before allogeneic SCT for NHL.
- Published
- 2003
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18. Low-dose total body irradiation followed by allogeneic lymphocyte infusion may induce remission in patients with refractory hematologic malignancy.
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Ballen KK, Becker PS, Emmons RV, Fitzgerald TJ, Hsieh CC, Liu Q, Heyes C, Clark Y, Levy W, Lambert JF, Chiafari F, Szymanski I, Rososhansky S, Popovsky MA, Stewart FM, and Quesenberry PJ
- Subjects
- Adult, Aged, Fetal Blood, Graft Survival, Hematologic Neoplasms complications, Hematologic Neoplasms mortality, Hematopoietic Stem Cell Transplantation adverse effects, Hematopoietic Stem Cell Transplantation mortality, Humans, Middle Aged, Radiation Dosage, Remission Induction methods, Salvage Therapy, Transplantation Chimera, Transplantation Conditioning adverse effects, Transplantation Conditioning methods, Transplantation Conditioning mortality, Treatment Outcome, Whole-Body Irradiation adverse effects, Hematologic Neoplasms therapy, Hematopoietic Stem Cell Transplantation methods, Lymphocyte Transfusion methods, Whole-Body Irradiation methods
- Abstract
Allogeneic stem cell transplantation is curative for certain cancers, but the high doses of chemotherapy/radiotherapy lead to toxicity. Here, we treat patients with refractory cancer with 100 cGy total body irradiation (TBI) followed by infusion of nonmobilized pheresed allogeneic peripheral blood cells. Twenty-five patients, with a median age of 47 years, with refractory cancers were enrolled. Eighteen patients received sibling and 7 received unrelated cord blood cells. Donor chimerism was assessed at weeks 1, 2, 3, 4, and 8 after transplantation. Seven patients with solid tumors received a sibling transplant and 6 received a cord blood transplant; none achieved donor chimerism, but 1 treated at the higher dose level of 1 x 10(8) CD3+ cells/kg had a transient nodal response. Twelve patients with hematologic malignancies were treated; 1 received a cord blood transplant and 11 received sibling donor cells. Nine of these 11 patients achieved donor chimerism, ranging from 5% to 100%. Four patients had sustained complete remission of their cancers, including one patient with transient 5% donor chimerism. The development of chimerism correlated with hematologic malignancy (P <.001), total previous myelotoxic chemotherapy (P <.001), T-cell dose (P =.03), and graft-versus-host disease (P =.01). Tumor response correlated with donor chimerism (P =.01). Engraftment was achieved in patients with hematologic malignancies who had been heavily pretreated, suggesting the degree of immunosuppression may be a determinant of engraftment. Low-dose TBI and allogeneic lymphocyte infusion may induce remission in patients with refractory hematologic malignancy.
- Published
- 2002
- Full Text
- View/download PDF
19. Prospective evaluation of antiemetic outcome following high-dose chemotherapy with hematopoietic stem cell support.
- Author
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Ballen KK, Hesketh AM, Heyes C, Becker PS, Emmons RV, Fogarty K, LaPointe J, Liu Q, Hsieh CC, and Hesketh PJ
- Subjects
- Adult, Aged, Female, Humans, Logistic Models, Male, Middle Aged, Nausea chemically induced, Nausea prevention & control, Prospective Studies, Transplantation Conditioning, Treatment Outcome, Vomiting chemically induced, Vomiting prevention & control, Antiemetics therapeutic use, Antineoplastic Agents adverse effects, Hematopoietic Stem Cell Transplantation
- Abstract
Considerable progress has been made in improving the control of chemotherapy-induced emesis. The impact of available antiemetic options for patients receiving stem cell transplants is unclear, as few prospective data have been collected. We prospectively evaluated antiemetic outcome in patients receiving stem cell transplantation over a 7-day period following the initiation of chemotherapy. The primary endpoints were the number of emetic episodes and the extent of nausea measured on a four-point scale. Eighty-two patients were evaluated. Ninety-five percent of patients had nausea during the first week of treatment; 80% had at least one emetic episode. The percentage of patients with emesis was as follows: day 1: 13%, day 2: 21%, day 3: 30%, day 4: 38%, day 5: 44%, day 6: 39%, day 7: 18%. In multivariate analysis, gender, emesis with prior chemotherapy, history of morning or motion sickness, type of transplant (auto vs allo), use of total body irradiation, or use of dexamethasone did not effect emesis control. Most patients receiving high-dose chemotherapy experience incompletely controlled emesis. Control of nausea and emesis progressively worsened with each subsequent day following initiation of chemotherapy, reaching a nadir on day 5. New treatment approaches are needed to improve emesis control in this patient population.
- Published
- 2001
- Full Text
- View/download PDF
20. Successful autologous bone marrow transplant without the use of blood product support.
- Author
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Ballen KK, Ford PA, Waitkus H, Emmons RV, Levy W, Doyle P, Stewart FM, Quesenberry PJ, and Becker PS
- Subjects
- Adult, Anemia drug therapy, Anemia economics, Anemia prevention & control, Blood Transfusion economics, Blood Transfusion psychology, Bone Marrow Transplantation standards, Christianity psychology, Humans, Lymphoma, Large B-Cell, Diffuse therapy, Male, Thrombocytopenia drug therapy, Thrombocytopenia economics, Thrombocytopenia prevention & control, Transplantation, Autologous methods, Transplantation, Autologous standards, Treatment Outcome, Bone Marrow Transplantation methods
- Abstract
We describe a successful autologous bone marrow transplant without the use of any blood products. The patient had relapsed large cell lymphoma. He was a Jehovah's Witness and would not accept transfusions of red blood cells or platelets. He enrolled in our Bloodless Medicine and Surgery Program and was maintained on a regimen of erythropoietin, iron, Amicar, and G-CSF throughout the transplant. He tolerated the transplant well and is alive with no evidence of disease 10 months after autografting.
- Published
- 2000
- Full Text
- View/download PDF
21. The administration of 10 microg/kg granulocyte colony-stimulating factor (G-CSF) alone results in a successful peripheral blood stem cell collection when previous mobilization with chemotherapy and hematopoietic growth factor failed.
- Author
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D'Hondt L, Emmons RV, André M, Guillaume T, Feyens AM, Canon JL, Humblet Y, Longueville J, and Symann M
- Subjects
- Adult, Breast Neoplasms blood, Combined Modality Therapy, Dose-Response Relationship, Drug, Drug Administration Schedule, Female, Granulocyte Colony-Stimulating Factor adverse effects, Hematopoietic Stem Cell Transplantation, Hodgkin Disease blood, Humans, Injections, Subcutaneous, Leukapheresis, Lymphoma, Non-Hodgkin blood, Male, Middle Aged, Antineoplastic Combined Chemotherapy Protocols therapeutic use, Breast Neoplasms drug therapy, Granulocyte Colony-Stimulating Factor administration & dosage, Hematopoietic Cell Growth Factors therapeutic use, Hematopoietic Stem Cell Mobilization methods, Hodgkin Disease drug therapy, Lymphoma, Non-Hodgkin drug therapy
- Abstract
Some heavily pretreated cancer patients fail to mobilize enough peripheral blood stem cells (PBSC) after stimulation with chemotherapy and hematopoietic growth factors. For these patients the best way to obtain an adequate PBSC collection is unknown. Here we report 6 heavily pretreated cancer patients who failed to mobilize sufficient PBSC after stimulation with chemotherapy and G-CSF 5 microg/kg/day. In these cases, we used G-CSF 10 microg/kg/day alone for six days at least 3 weeks after the last chemotherapy. After three consecutive leukaphereses starting on day 5, five patients had adequate PBSC collections. With 6 days of G-CSF 10 microg/kg/day alone, 2.8 x 10(6) (+/- 1) CD34+ cells/kg were collected. This was significantly higher than the number of CD34+ cells/kg collected after chemotherapy and G-CSF 5 microg/kg 0.3 x 10(6) (+/- 0.1) [P = 0.05]. Four patients received high-dose chemotherapy with PBSC support. Hematologic recovery observed in these patients was as expected. In conclusion, G-CSF 10 microg/kg alone can mobilize progenitor cells into peripheral blood when previous mobilization with chemotherapy and G-CSF 5 microg/kg fails.
- Published
- 1999
- Full Text
- View/download PDF
22. Expression of interferon-gamma by stromal cells inhibits murine long-term repopulating hematopoietic stem cell activity.
- Author
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Yu JM, Emmons RV, Hanazono Y, Sellers S, Young NS, and Dunbar CE
- Subjects
- Animals, Base Sequence, Coculture Techniques, DNA Primers, Female, Humans, Mice, Mice, Inbred C57BL, Recombinant Proteins genetics, Retroviridae genetics, Transduction, Genetic, Cell Division genetics, Hematopoietic Stem Cells cytology, Interferon-gamma genetics, Stromal Cells metabolism
- Abstract
Several lines of evidence suggest that overexpression of interferon gamma (IFN-gamma) in the marrow microenvironment may play a role in the pathogenesis of marrow suppression in aplastic anemia. We previously showed that overexpression of IFN-gamma by marrow stromal cells inhibits human long-term culture initiating cell activity assayed in vitro to a much greater degree than the addition of soluble IFN-gamma. The effect of IFN-gamma on true repopulating stem cells assayed in vivo has not been studied previously. We compared the effect of co-culture of murine marrow cells in the presence of stromal cells transduced with a retroviral vector expressing murine IFN-gamma vs stromal cells transduced with a control neo vector. Using a murine congenic competitive repopulation assay, there was significantly less long-term repopulating stem cell activity remaining after culture on mIFN-gamma-expressing stroma as compared to control stroma. We also investigated the effect of directly transducing murine bone marrow cells with the mIFN-gamma or control vector. Marrow cells transduced with either vector were transplanted into W/Wv recipient mice. The percentage of vector-containing cells in the mIFN-gamma mice was significantly lower than in the control mice, suggesting that mIFN-gamma-transduced primitive cells may not have survived culture, or that mIFN-gamma directly decreases gene transfer into repopulating cells. Despite no significant differences in white or red blood cells in the mice transplanted with the mIFN-gamma-transduced cells, the number of bone marrow colony-forming unit-C 16 weeks after transplantation was significantly lower in the IFN-gamma group. These data indicate that ectopic or overexpression of mIFN-gamma, especially by marrow microenvironmental elements, may have a marked effect on primitive hematopoiesis as assayed in vivo.
- Published
- 1999
- Full Text
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23. Retroviral transfer of the glucocerebrosidase gene into CD34+ cells from patients with Gaucher disease: in vivo detection of transduced cells without myeloablation.
- Author
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Dunbar CE, Kohn DB, Schiffmann R, Barton NW, Nolta JA, Esplin JA, Pensiero M, Long Z, Lockey C, Emmons RV, Csik S, Leitman S, Krebs CB, Carter C, Brady RO, and Karlsson S
- Subjects
- Adult, Antigens, CD34 analysis, Base Sequence, Bone Marrow Cells immunology, DNA Primers, Female, Gaucher Disease enzymology, Gaucher Disease genetics, Gene Transfer Techniques, Genetic Vectors, Glucosylceramidase blood, Granulocyte Colony-Stimulating Factor therapeutic use, Hematopoietic Stem Cell Mobilization, Humans, Male, Stromal Cells cytology, Bone Marrow Cells metabolism, Gaucher Disease therapy, Genetic Therapy, Glucosylceramidase genetics, Retroviridae genetics, Transduction, Genetic
- Abstract
Retroviral gene transfer of the glucocerebrosidase gene to hematopoietic progenitor and stem cells has shown promising results in animal models and corrected the enzyme deficiency in cells from Gaucher patients in vitro. Therefore, a clinical protocol was initiated to explore the safety and feasibility of retroviral transduction of peripheral blood (PB) or bone marrow (BM) CD34+ cells with the G1Gc vector. This vector uses the viral LTR promoter to express the human glucocerebrosidase cDNA. Three adult patients have been entered with follow-up of 6-15 months. Target cells were G-CSF-mobilized and CD34-enriched PB cells or CD34-enriched steady state BM cells, and were transduced ex vivo for 72 hr. Patient 1 had PB cells transduced in the presence of autologous stromal marrow cells. Patient 2 had PB cells transduced in the presence of autologous stroma, IL-3, IL-6, and SCF. Patient 3 had BM cells transduced in the presence of autologous stroma, IL-3, IL-6, and SCF. At the end of transduction, the cells were collected and infused immediately without any preparative treatment of the patients. The transduction efficiency of the CD34+ cells at the end of transduction was approximately 1, 10, and 1 for patients 1, 2, and 3, respectively, as estimated by semiquantitative PCR on bulk samples and PCR analysis of individual hematopoietic colonies. Gene marking in vivo was demonstrated in patients 2 and 3. Patient 2 had vector-positive PB granulocytes and mononuclear bone marrow cells at 1 month postinfusion and positive PB mononuclear cells at 2 and 3 months postinfusion. Patient 3 had a positive BM sample at 1 month postinfusion but was negative thereafter. These results indicate that gene-marked cells can engraft and persist for at least 3 months postinfusion, even without myeloablation. However, the level of corrected cells (<0.02%) is too low to result in any clinical benefit, and glucocerebrosidase enzyme activity did not increase in any patient following infusion of transduced cells. Modifications of vector systems and transduction conditions, along with partial myeloablation to allow higher levels of engraftment, may be necessary to achieve beneficial levels of correction in patients with Gaucher disease.
- Published
- 1998
- Full Text
- View/download PDF
24. Green fluorescent protein retroviral vectors: low titer and high recombination frequency suggest a selective disadvantage.
- Author
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Hanazono Y, Yu JM, Dunbar CE, and Emmons RV
- Subjects
- Animals, Blotting, Southern, Cell Line, Gene Transfer Techniques, Genes, Viral genetics, Genetic Markers, Genetic Vectors genetics, Green Fluorescent Proteins, Luminescent Proteins metabolism, Microscopy, Fluorescence, Plasmids, RNA, Viral analysis, Retroviridae physiology, Virus Replication, Genes, Reporter genetics, Genetic Vectors metabolism, Luminescent Proteins genetics, Retroviridae genetics
- Abstract
Green fluorescent protein (GFP) has been used as a reporter molecule for gene expression because it fluoresces green after blue-light excitation. Inclusion of this gene in a vector could allow rapid, nontoxic selection of successfully transduced cells. However, many attempts by our laboratory to isolate stable retroviral producer cell clones secreting biologically active vectors containing either the highly fluorescent S65T-GFP mutant or humanized GFP have failed. Vector plasmids containing various forms of GFP and the neomycin resistance gene were transfected into three different packaging cell lines and fluorescence was observed for several days, but stable clones selected with G418 no longer fluoresced. Using confocal microscopy, the brightest cells were observed to contract and die within a matter of days. RNA slot-blot analysis of retroviral producer supernatants showed no viral production from the GFP plasmid-transfected clones, although all clones derived after transfection with an identical retroviral construct not containing GFP produced virus. Genomic Southern analysis of the GFP-transduced clones showed a much higher probability of rearrangement of the priviral sequences than in the control non-GFP clones. Overall, 18/34 S65T-GFP clones and 17/33 humanized-GFP clones had rearrangements, whereas 2/15 control non-GFP clones had rearrangements. Hence, producer cells expressing high levels of these GFP genes seem to be selected against, with stable clones undergoing major rearrangements or other mutations that both abrogate GFP expression and prevent vector production. These observations indicate that GFP may not be an appropriate reporter gene for gene transfer applications in our vector/packaging system.
- Published
- 1997
- Full Text
- View/download PDF
25. Retroviral gene transduction of adult peripheral blood or marrow-derived CD34+ cells for six hours without growth factors or on autologous stroma does not improve marking efficiency assessed in vivo.
- Author
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Emmons RV, Doren S, Zujewski J, Cottler-Fox M, Carter CS, Hines K, O'Shaughnessy JA, Leitman SF, Greenblatt JJ, Cowan K, and Dunbar CE
- Subjects
- Adult, Bone Marrow drug effects, Drug Resistance, Microbial genetics, Female, Genetic Vectors, Hematopoietic Stem Cells drug effects, Humans, Male, Middle Aged, Neomycin pharmacology, Retroviridae genetics, Stromal Cells drug effects, Stromal Cells pathology, Stromal Cells transplantation, Transplantation, Autologous, Bone Marrow pathology, Gene Transfer Techniques, Hematopoietic Stem Cell Transplantation, Hematopoietic Stem Cells pathology
- Abstract
Our previous work in patients undergoing autologous transplant for multiple myeloma (MM) or breast cancer (BC) has shown that retroviral transduction of adult CD34+ cells for 72 hours in the presence of interleukin-3 (IL-3), IL-6, and stem cell factor (SCF) resulted in .01% to 1% long-term marking of peripheral blood and marrow cells (Blood 85:3948, 1995). In this study we compare these previous studies to transduction with no added growth factors, previously shown to result in higher levels of marking in children (Lancet 342:1134, 1993) or transduction in the presence of an autologous stromal layer. Peripheral blood (PB) mononuclear cells were collected via apheresis after high-dose cyclophosphamide and granulocyte colony-stimulating factor. Bone marrow (BM) was also harvested in all patients. One third of both BM and PB collections were enriched for CD34+ cells and transduced with one of two marking vectors containing the neomycin-resistance gene to distinguish cells originating from BM and PB posttransplantation. Cells from 3 MM and 2 BC patients were transduced without growth factors for 6 hours and cells from 2 MM and 2 BC patients were transduced in the presence of autologous marrow stroma. Immediately posttransduction, the percentage of Neo-resistant PB and BM progenitors (colony-forming units) were: 0% to 19% in the 6-hour no growth factor group and 0% to 36% in the autologous stroma group. After conditioning therapy, both transduced and untransduced PB and BM fractions were infused into the patients. Semi-quantitative nested DNA polymerase chain reaction was performed on total, mononuclear, and granulocyte fractions of PB and BM at 1, 3, 6, 9, 12, and 18 months. Poor marking has been observed in both groups, with no consistently positive patients. These results compare unfavorably with our prior experience using growth factors during transduction. Further optimization of transduction conditions and vectors needs to be developed to improve transduction efficiency of adult human repopulating hematopoietic cells.
- Published
- 1997
26. Bone formation in vivo: comparison of osteogenesis by transplanted mouse and human marrow stromal fibroblasts.
- Author
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Krebsbach PH, Kuznetsov SA, Satomura K, Emmons RV, Rowe DW, and Robey PG
- Subjects
- Animals, Bone Marrow Cells, Calcium Phosphates, Cell Transplantation methods, Cells, Cultured, Ceramics, Child, Child, Preschool, Collagen, Durapatite, Female, Fibroblasts transplantation, Humans, Male, Mice, Mice, Transgenic, Osteogenesis physiology, Polyvinyls, Porifera, Tissue Donors, Bone Development physiology
- Abstract
Background: Marrow stromal fibroblasts (MSFs) are known to contain bone precursor cells. However, the osteogenic potential of human MSFs has been poorly characterized. The aim of this study was to compare the osteogenic capacity of mouse and human MSFs after implantation in vivo., Methods: After in vitro expansion, MSFs were loaded into a number of different vehicles and transplanted subcutaneously into immunodeficient mice., Results: Mouse MSFs transplanted within gelatin, polyvinyl sponges, and collagen matrices all formed a capsule of cortical-like bone surrounding a cavity with active hematopoiesis. In transplants of MSFs from transgenic mice harboring type I procollagen-chloramphenicol acetyltransferase constructs, chloramphenicol acetyltransferase activity was maintained for up to 14 weeks, indicating prolonged bone formation by transplanted MSFs. New bone formation by human MSFs was more dependent on both the in vitro expansion conditions and transplantation vehicles. Within gelatin, woven bone was observed sporadically and only after culture in the presence of dexamethasone and L-ascorbic acid phosphate magnesium salt n-hydrate. Consistent bone formation by human MSFs was achieved only within vehicles containing hydroxyapatite/tricalcium phosphate ceramics (HA/TCP) in the form of blocks, powder, and HA/TCP powder-type I bovine fibrillar collagen strips, and bone was maintained for at least 19 weeks. Cells of the new bone were positive for human osteonectin showing their donor origin. HA/TCP powder, the HA/TCP powder-type I bovine fibrillar collagen strips, and HA/TCP powder held together with fibrin were easier to load and supported more extensive osteogenesis than HA/TCP blocks and thus may be more applicable for therapeutic use., Conclusions: In this article, we describe the differences in the requirements for mouse and human MSFs to form bone, and report the development of a methodology for the consistent in vivo generation of extensive bone from human MSFs.
- Published
- 1997
- Full Text
- View/download PDF
27. Transduction of CD34-enriched human peripheral and umbilical cord blood progenitors using a retroviral vector with the Fanconi anemia group C gene.
- Author
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Walsh CE, Mann MM, Emmons RV, Wang S, and Liu JM
- Subjects
- Base Sequence, Fetal Blood, Humans, Molecular Sequence Data, Antigens, CD34 genetics, Fanconi Anemia genetics, Gene Transfer Techniques, Genetic Vectors genetics, Hematopoietic Stem Cells immunology, Retroviridae, Transduction, Genetic
- Abstract
Background: Fanconi anemia (FA) is an autosomal recessive inherited form of bone marrow failure. FA cells are characterized by their extreme sensitivity to DNA cross-linking agents that cause DNA instability and cell death. Four genetic complementation groups for FA have been identified and the gene for the complementation C group (FACC) has been cloned. Genetic transfer of the FACC gene should provide a growth advantage in transduced hematopoietic cells. We have previously demonstrated efficient retroviral-mediated gene transduction and correction of FA(C) cell lines and peripheral blood-derived CD34+ progenitors from patients carrying mutant FACC alleles. In this report we sought to define the optimal conditions for transduction of CD34+ progenitors from mobilized peripheral blood and umbilical cord blood., Methods: Peripheral blood hematopoietic progenitors were obtained by G-CSF mobilization followed by apheresis. Human fetal cord blood cells were obtained from full-term gestation deliveries. Cells were immunoselected for CD34 antigen expression and then incubated with recombinant retroviruses containing a selectable marker gene (neomycin). Recombinant colony stimulating factors were added to facilitate viral transduction. Cells were plated in methylcellulose and resulting hematopoietic colonies were isolated and analyzed by PCR., Results: Transduction efficiency of peripheral blood progenitors (from normal individuals) using a retrovirus encoding the FACC cDNA was comparable to that of the retroviral producer G1Na.40 currently being used in clinical gene therapy marking studies. We extended our standard transduction protocol to analyze CD34+ and CD34+ CD38-subpopulations of progenitors derived from umbilical cord blood (from normal pregnancies). In addition, we tested whether FACC cDNA transduction could be improved by vector infection supported by autologous stroma. For FA(C) hematopoietic cell infection, vector supernatant transduction in the presence of recombinant human IL-3, IL-6, and SCF was found to be superior to transduction supported by autologous FA(C) patient stroma., Conclusions: We documented efficient retroviral transduction of umbilical cord blood and peripheral blood enriched for hematopoietic progenitor cells. These results suggest the feasibility of a clinical gene therapy protocol utilizing progenitor cells from both peripheral blood and umbilical cord blood.
- Published
- 1995
28. Gene transfer into hematopoietic progenitor and stem cells: progress and problems.
- Author
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Dunbar CE and Emmons RV
- Subjects
- Animals, Clinical Protocols, Dependovirus genetics, Genetic Engineering, Genetic Therapy adverse effects, Genetic Therapy trends, Genetic Vectors, Hematopoietic Stem Cell Transplantation adverse effects, Hematopoietic Stem Cell Transplantation trends, Humans, Models, Biological, Retroviridae genetics, Safety, Gene Transfer Techniques, Genetic Therapy methods, Hematopoietic Stem Cell Transplantation methods, Hematopoietic Stem Cells
- Abstract
Gene transfer to hematopoietic cells for the purpose of "gene therapy" is a new and rapidly developing field with clinical trials in progress. A fundamental goal of research in this field is the incorporation of exogenous genes into the chromosomes of the most primitive hematopoietic progenitor cells--stem cells. Recombinantly engineered retroviral vectors are the best characterized and are currently the only vector type in clinical trials directed at the hematopoietic system. High efficiency gene transfer and expression in murine stem cells and their progeny is now routine, but in larger animal models such as dogs or primates and preliminary clinical trials, gene transfer has been less successful. Problems such as retroviral efficiency, gene expression, insertional mutagenesis and helper virus contamination are being addressed. A promising new vector, the adeno-associated virus (AAV), has shown promise and may allow production of high titer, stable, recombinant virions without helper contamination and with potentially better safety parameters. However, the technology for AAV gene transfer is currently underdeveloped, and issues related to the reproducible production of vectors must be addressed. Other non-viral vector systems are being explored, but little data are available on applications to hematopoietic cells. Better preclinical models are needed to study gene targeting and expression in human cells. An overview of recombinant retroviral and adeno-associated viral vector production, preclinical data and preliminary clinical data will be given, and problems needing to be addressed at all stages of development before broad clinical utility can be achieved will be discussed.
- Published
- 1994
- Full Text
- View/download PDF
29. Role of proteoglycans and cytoskeleton in the effects of TGF-beta 1 on renal proximal tubule cells.
- Author
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Humes HD, Nakamura T, Cieslinski DA, Miller D, Emmons RV, and Border WA
- Subjects
- Actins metabolism, Animals, Cells, Cultured, Cytochalasin B pharmacology, DNA biosynthesis, Fibronectins biosynthesis, Glycosides pharmacology, Kidney Tubules, Proximal metabolism, Kidney Tubules, Proximal ultrastructure, Phenotype, Rabbits, Cytoskeleton metabolism, Kidney Tubules, Proximal drug effects, Proteoglycans biosynthesis, Transforming Growth Factor beta pharmacology
- Abstract
Transforming growth factor-beta (TGF-beta) is a critical cell regulatory protein which influences cell growth, cell differentiation and cell chemotaxis. TGF-beta 1 has been previously shown to promote a migratory and adherent transformation of monolayers of renal proximal tubule cells in primary culture to form solid clusters of cells. To better understand the cellular basis of this TGF-beta 1 effect, these studies evaluated the influence of TGF-beta 1 on the synthesis of proteoglycans and on cytoskeleton rearrangement in rabbit renal proximal tubule cells in primary culture, and their role in this transformation effect of TGF-beta 1. Biosynthetic labeling of proteoglycans with 35S sulfate and enzyme digestion studies demonstrated that TGF-beta 1 promoted the synthesis of heparan sulfate proteoglycans in these cells. The importance of proteoglycan synthesis induced by TGF-beta 1 in this migration and aggregation process was demonstrated with the use of two chemically-dissimilar proteoglycan synthesis inhibitors: xyloside and galactosamine. Both compounds inhibited TGF-beta 1 stimulation of proteoglycan synthesis and diminished TGF-beta 1 promoted transformation of proximal tubule cells as assessed by quantitative morphometry. Further experiments evaluated the influence of TGF-beta 1 on actin microfilaments with the use of rhodamine conjugated phalloidin staining and immunofluorescent microscopy, and demonstrated that TGF-beta 1 provoked a dramatic rearrangement of actin microfilaments into stress fibers. The use of actin microfilament disrupting agents, cytochalasin B and D, attenuated the stress fiber formation promoted by TGF-beta 1 and inhibited the TGF-beta 1-induced morphologic transformation of these cells. Further studies evaluated these effects on the rate of DNA synthesis in these cells, as assessed with 3H-thymidine incorporation. Proteoglycan synthesis inhibitors significantly diminished the maximal proliferative response of these epithelial cells to epidermal growth factor (EGF). In contrast, actin microfilament disaggregation with cytochalasin B or D did not change the rate of DNA synthesis in response to EGF but did attenuate the antiproliferative effect of TGF-beta 1 on EGF-induced DNA synthesis cells. These studies demonstrate that the TGF-beta 1 promoted synthesis cells. These studies demonstrate that the TGF-beta 1 promoted an increase in the production of proteoglycans and a higher ordered structure of the cytoskeleton. Both effects were instrumental in the adhesive migratory response of proximal tubule cells to TGF-beta 1 as well as the DNA synthesis rate response to both EGF and TGF-beta 1.
- Published
- 1993
- Full Text
- View/download PDF
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