Publications by authors named "Sara Deola"

25 Publications

  • Page 1 of 1

A modular framework for the development of targeted Covid-19 blood transcript profiling panels.

J Transl Med 2020 07 31;18(1):291. Epub 2020 Jul 31.

Sidra Medicine, Doha, Qatar.

Background: Covid-19 morbidity and mortality are associated with a dysregulated immune response. Tools are needed to enhance existing immune profiling capabilities in affected patients. Here we aimed to develop an approach to support the design of targeted blood transcriptome panels for profiling the immune response to SARS-CoV-2 infection.

Methods: We designed a pool of candidates based on a pre-existing and well-characterized repertoire of blood transcriptional modules. Available Covid-19 blood transcriptome data was also used to guide this process. Further selection steps relied on expert curation. Additionally, we developed several custom web applications to support the evaluation of candidates.

Results: As a proof of principle, we designed three targeted blood transcript panels, each with a different translational connotation: immunological relevance, therapeutic development relevance and SARS biology relevance.

Conclusion: Altogether the work presented here may contribute to the future expansion of immune profiling capabilities via targeted profiling of blood transcript abundance in Covid-19 patients.
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http://dx.doi.org/10.1186/s12967-020-02456-zDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7393249PMC
July 2020

Influence of storage conditions of small volumes of blood on immune transcriptomic profiles.

BMC Res Notes 2020 Mar 13;13(1):150. Epub 2020 Mar 13.

Omics Core, Research Branch, Out Patient Clinic, Sidra Medicine, PO 26999, Doha, Qatar.

Objective: Transcriptome analysis of human whole blood is used to discover biomarkers of diseases and to assess phenotypic traits. Here we have collected small volumes of blood in Tempus solution and tested whether different storage conditions have an impact on transcriptomic profiling. Fifty µl of blood were collected in 100µl of Tempus solutions, freezed at - 20 °C for 1 day and eventually thawed, stored and processed under five different conditions: (i) - 20 °C for 1 week; (ii) +4 °C for 1 week; (iii) room temperature for 1 week; (iv) room temperature for 1 day, - 20 °C for 1 day, room temperature until testing at day 7, (v) - 20 °C for 1 week, RNA was isolated and stored in GenTegra solution. We used 272 immune transcript specific assays to test the expression profiling using qPCR based Fluidigm BioMark HD dynamic array.

Results: RNA yield ranged between 0.17 and 1.39µg. Except for one sample, RIN values were > 7. Using Principal Component Analysis, we saw that the storage conditions did not drive sample distribution. The condition that showed larger variability was the RT-FR-RT (room temperature-freezing-room temperature), suggesting that freezing-thawing cycles may have a worse effect on data reproducibility than keeping the samples at room temperature.
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http://dx.doi.org/10.1186/s13104-020-04980-zDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7069204PMC
March 2020

Flow-Cytometry Platform for Intracellular Detection of FVIII in Blood Cells: A New Tool to Assess Gene Therapy Efficiency for Hemophilia A.

Mol Ther Methods Clin Dev 2020 Jun 15;17:1-12. Epub 2019 Nov 15.

Research Department, Sidra Medicine, PO Box 26999, Doha, Qatar.

Detection of factor VIII (FVIII) in cells by flow cytometry is controversial, and no monoclonal fluorescent antibody is commercially available. In this study, we optimized such an assay and successfully used it as a platform to study the functional properties of phosphoglycerate kinase (PGK)-FVIII lentiviral vector-transduced cells by directly visualizing FVIII in cells after different gene transfer conditions. We could measure cellular stress parameters after transduction by correlating gene expression and protein accumulation data. Flow cytometry performed on transduced cell lines showed that increasing MOI rates resulted in increased protein levels, plateauing after an MOI of 30. We speculated that, at higher MOI, FVIII production could be impaired by a limiting factor required for proper folding. To test this hypothesis, we interfered with the unfolded protein response by blocking proteasomal degradation and measured the accumulation of intracellular misfolded protein. Interestingly, at higher MOIs the cells displayed signs of toxicity with reactive oxygen species accumulation. This suggests the need for identifying a safe window of transduction dose to avoid consequent cell toxicity. Herein, we show that our flow cytometry platform for intracytoplasmic FVIII protein detection is a reliable method for optimizing gene therapy protocols in hemophilia A by shedding light on the functional status of cells after gene transfer.
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http://dx.doi.org/10.1016/j.omtm.2019.11.003DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6920166PMC
June 2020

Clofarabine and Treosulfan as Conditioning for Matched Related and Unrelated Hematopoietic Stem Cell Transplantation: Results from the Clo3o Phase II Trial.

Biol Blood Marrow Transplant 2020 02 9;26(2):316-322. Epub 2019 Oct 9.

Hematology and Bone Marrow Transplant Unit, IRCCS San Raffaele Scientific Institute, Milan, Italy. Electronic address:

Allogeneic hematopoietic stem cell transplantation (allo-HSCT) can be curative for patients with hematologic malignancies. The ideal conditioning regimen before allo-HSCT has not been established. We conducted a Phase II study to evaluate the tolerability and efficacy of clofarabine and treosulfan as conditioning regimen before allo-HSCT. The primary objective was to evaluate the cumulative incidence of nonrelapse mortality (NRM) on day +100. Forty-four patients (36 with acute myelogenous leukemia, 5 with acute lymphoblastic leukemia, 3 with myelodysplastic syndromes) were enrolled. The median patient age was 47 years, and the median duration of follow-up was 27 months. The conditioning regimen was based on clofarabine 40 mg/m (days -6 to -2) and treosulfan 14 g/m (days -6 to -4). Allogeneic hematopoietic stem cells were derived from a sibling (n = 22) or a well-matched unrelated donor (n = 22). Graft-versus-host disease (GVHD) prophylaxis consisted of antithymocyte globulin, rituximab, cyclosporine, and a short-course of methotrexate. The regimen allowed for rapid engraftment and a 100-day NRM of 18%, due mainly to bacterial infections. The incidences of grade II-IV acute GVHD and chronic GVHD were 16% and 19%, respectively. The rates of overall survival (OS), progression-free survival, and relapse at 2 years were 51%, 31%, and 50%, respectively. Significantly different outcomes were observed between patients with low-intermediate and patients with high-very high Disease Risk Index (DRI) scores (1-year OS, 78% and 24%, respectively). Our findings show that the use of treosulfan and clofarabine as a conditioning regimen for allo-HSCT is feasible, with a 78% 1-year OS in patients with a low-intermediate DRI score. However, 1-year NRM was 18%, and despite the intensified conditioning regimen, relapse incidence remains a major issue in patients with poor prognostic risk factors.
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http://dx.doi.org/10.1016/j.bbmt.2019.09.032DOI Listing
February 2020

Tailoring cells for clinical needs: Meeting report from the Advanced Therapy in Healthcare symposium (October 28-29 2017, Doha, Qatar).

J Transl Med 2018 10 10;16(1):276. Epub 2018 Oct 10.

Research Department, Clinical Research Center, Sidra Medicine, Doha, Qatar.

New technologies and therapies designed to facilitate development of personalized treatments are rapidly emerging in the field of biomedicine. Strikingly, the goal of personalized medicine refined the concept of therapy by developing cell-based therapies, the so-called "living drugs". Breakthrough advancements were achieved in this regard in the fields of gene therapy, cell therapy, tissue-engineered products and advanced therapeutic techniques. The Advanced Therapies in Healthcare symposium, organized by the Clinical Research Center Department of Sidra Medicine, in Doha, Qatar (October 2017), brought together world-renowned experts from the fields of oncology, hematology, immunology, inflammation, autoimmune disorders, and stem cells to offer a comprehensive picture of the status of worldwide advanced therapies in both pre-clinical and clinical development, providing insights to the research phase, clinical data and regulatory aspects of these therapies. Highlights of the meeting are provided in this meeting report.
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http://dx.doi.org/10.1186/s12967-018-1652-yDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6180452PMC
October 2018

Phase II Study of Sequential Infusion of Donor Lymphocyte Infusion and Cytokine-Induced Killer Cells for Patients Relapsed after Allogeneic Hematopoietic Stem Cell Transplantation.

Biol Blood Marrow Transplant 2017 Dec 13;23(12):2070-2078. Epub 2017 Jul 13.

USC Hematology and Bone Marrow Transplant Unit ASST Papa Giovanni XXIII Bergamo, Bergamo, Italy; Università degli Studi di Milano, Milan Italy.

Seventy-four patients who relapsed after allogeneic stem cell transplantation were enrolled in a phase IIA study and treated with the sequential infusion of donor lymphocyte infusion (DLI) followed by cytokine-induced killer (CIK) cells. Seventy-three patients were available for the intention to treat analysis. At least 1 infusion of CIK cells was given to 59 patients, whereas 43 patients received the complete cell therapy planned (58%). Overall, 12 patients (16%) developed acute graft-versus-host disease (aGVHD) of grades I to II in 7 cases and grades III to IV in 5). In 8 of 12 cases, aGVHD developed during DLI treatment, leading to interruption of the cellular program in 3 patients, whereas in the remaining 5 cases aGVHD was controlled by steroids treatment, thus allowing the subsequent planned administration of CIK cells. Chronic GVHD (cGVHD) was observed in 11 patients (15%). A complete response was observed in 19 (26%), partial response in 3 (4%), stable disease in 8 (11%), early death in 2 (3%), and disease progression in 41 (56%). At 1 and 3 years, rates of progression-free survival were 31% and 29%, whereas rates of overall survival were 51% and 40%, respectively. By multivariate analysis, the type of relapse, the presence of cGVHD, and a short (<6 months) time from allogeneic hematopoietic stem cell transplantation to relapse were the significant predictors of survival. In conclusion, a low incidence of GVHD is observed after the sequential administration of DLI and CIK cells, and disease control can be achieved mostly after a cytogenetic or molecular relapse.
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http://dx.doi.org/10.1016/j.bbmt.2017.07.005DOI Listing
December 2017

Two hits in one: whole genome sequencing unveils LIG4 syndrome and urofacial syndrome in a case report of a child with complex phenotype.

BMC Med Genet 2016 Nov 17;17(1):84. Epub 2016 Nov 17.

Division of Translational Medicine, Research Branch, Sidra Medical and Research Center, Doha, Qatar.

Background: Ligase IV syndrome, a hereditary disease associated with compromised DNA damage response mechanisms, and Urofacial syndrome, caused by an impairment of neural cell signaling, are both rare genetic disorders, whose reports in literature are limited. We describe the first case combining both disorders in a specific phenotype.

Case Presentation: We report a case of a 7-year old girl presenting with a complex phenotype characterized by multiple congenital abnormalities and dysmorphic features, microcephaly, short stature, combined immunodeficiency and severe vesicoureteral reflux. Whole Genome Sequencing was performed and a novel ligase IV homozygous missense c.T1312C/p.Y438H mutation was detected, and is believed to be responsible for most of the clinical features of the child, except vesicoureteral reflux which has not been previously described for ligase IV deficiency. However, we observed a second rare damaging (nonsense) homozygous mutation (c.C2125T/p.R709X) in the leucine-rich repeats and immunoglobulin-like domains 2 gene that encodes a protein implicated in neural cell signaling and oncogenesis. Interestingly, this mutation has recently been reported as pathogenic and causing urofacial syndrome, typically displaying vesicoureteral reflux. Thus, this second mutation completes the missing genetic explanation for this intriguing clinical puzzle. We verified that both mutations fit an autosomal recessive inheritance model due to extensive consanguinity.

Conclusions: We successfully identified a novel ligase IV mutation, causing ligase IV syndrome, and an additional rare leucine-rich repeats and immunoglobulin-like domains 2 gene nonsense mutation, in the context of multiple autosomal recessive conditions due to extensive consanguinity. This work demonstrates the utility of Whole Genome Sequencing data in clinical diagnosis in such cases where the combination of multiple rare phenotypes results in very intricate clinical pictures. It also reports a novel causative mutation and a clinical phenotype, which will help in better defining the essential features of both ligase IV and leucine-rich repeats and immunoglobulin-like domains 2 deficiency syndromes.
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http://dx.doi.org/10.1186/s12881-016-0346-7DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5114772PMC
November 2016

Granulocyte transfusions in children and adults with hematological malignancies: benefits and controversies.

J Transl Med 2015 Nov 16;13:362. Epub 2015 Nov 16.

Division of Translational Medicine, Clinical Research Center, Sidra Medical and Research Center, Out-Patient Clinic, Al Luqta Street, Education City North Campus, P.O. Box 26999, Doha, Qatar.

Bacterial and fungal infections continue to pose a major clinical challenge in patients with prolonged severe neutropenia after chemotherapy or hematopoietic stem cell transplantation (HSCT). With the advent of granulocyte colony-stimulating factor (G-CSF) to mobilize neutrophils in healthy donors, granulocyte transfusions have been broadly used to prevent and/or treat life-threatening infections in patients with severe febrile neutropenia and/or neutrophil dysfunction. Although the results of randomized controlled trials are inconclusive, there are suggestions from pilot and retrospective studies that granulocyte transfusions may benefit selected categories of patients. We will critically appraise the evidence related to the use of therapeutic granulocyte transfusions in children and adults, highlighting current controversies in the field and discussing complementary approaches to modulate phagocyte function in the host.
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http://dx.doi.org/10.1186/s12967-015-0724-5DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4647505PMC
November 2015

Abnormal cervical cytology after allogeneic bone marrow transplantation.

Am J Clin Pathol 2014 Aug;142(2):222-6

Explora Research and Statistical Analysis, Padova, Italy.

Objectives: Allogeneic bone marrow transplantation (BMT) is a procedure mostly used for high-risk hematologic malignances. In women, follow-up protocols after BMT include gynecologic checkups with Papanicolaou (Pap) smears.

Methods: We evaluated 117 Pap smears in 54 women who underwent allogeneic BMT and correlated the smear morphology with the BMT-related medical treatment.

Results: Abnormal Pap smears after BMT were found in 13 (24.1%) women. Four (7.4%) women had at least one smear with atypical squamous cells of unknown significance, six (11.1%) had a low-grade squamous intraepithelial lesion, and three (5.6%) had atypical squamous cells/high-grade lesion cannot be excluded (ASC-H). The three patients with ASC-H showed high-grade atypia mimicking cancer but had a negative follow-up. Nine women, including the three with ASC-H, had undergone a conditioning therapy for BMT that included busulfan. No association between other drugs and therapy-related atypia was found.

Conclusions: Pap smears after BMT show a high incidence of dysplastic lesions. Moreover, conditioning including busulfan is often associated with therapy-related cytologic atypia, which may lead to unnecessary colposcopies and biopsies. Knowledge of the patient's history and a careful evaluation of the smears are mandatory in these cases.
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http://dx.doi.org/10.1309/AJCP4SKAUS9TOTJXDOI Listing
August 2014

Next generation sequencing: new tools in immunology and hematology.

Blood Res 2013 Dec 24;48(4):242-9. Epub 2013 Dec 24.

Department of Pathology and Diagnostics, University of Verona, Verona, Italy.

One of the hallmarks of the adaptive immune system is the specificity of B and T cell receptors. Thanks to somatic recombination, a large repertoire of receptors can be generated within an individual that guarantee the recognition of a vast number of antigens. Monoclonal antibodies have limited applicability, given the high degree of diversity among these receptors, in BCR and TCR monitoring. Furthermore, with regard to cancer, better characterization of complex genomes and the ability to monitor tumor-specific cryptic mutations or translocations are needed to develop better tailored therapies. Novel technologies, by enhancing the ability of BCR and TCR monitoring, can help in the search for minimal residual disease during hematological malignancy diagnosis and follow-up, and can aid in improving bone marrow transplantation techniques. Recently, a novel technology known as next generation sequencing has been developed; this allows the recognition of unique sequences and provides depth of coverage, heterogeneity, and accuracy of sequencing. This provides a powerful tool that, along with microarray analysis for gene expression, may become integral in resolving the remaining key problems in hematology. This review describes the state of the art of this novel technology, its application in the immunological and hematological fields, and the possible benefits it will provide for the hematology and immunology community.
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http://dx.doi.org/10.5045/br.2013.48.4.242DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3894381PMC
December 2013

Treatment of graft versus host disease with mesenchymal stromal cells: a phase I study on 40 adult and pediatric patients.

Biol Blood Marrow Transplant 2014 Mar 7;20(3):375-81. Epub 2013 Dec 7.

HSCT Pediatric Unit and Laboratory of Cell Therapy "S. Verri", San Gerardo Hospital, Monza, Italy.

This phase I multicenter study was aimed at assessing the feasibility and safety of intravenous administration of third party bone marrow-derived mesenchymal stromal cells (MSC) expanded in platelet lysate in 40 patients (15 children and 25 adults), experiencing steroid-resistant grade II to IV graft-versus-host disease (GVHD). Patients received a median of 3 MSC infusions after having failed conventional immunosuppressive therapy. A median cell dose of 1.5 × 10(6)/kg per infusion was administered. No acute toxicity was reported. Overall, 86 adverse events and serious adverse events were reported in the study, most of which (72.1%) were of infectious nature. Overall response rate, measured at 28 days after the last MSC injection, was 67.5%, with 27.5% complete response. The latter was significantly more frequent in patients exhibiting grade II GVHD as compared with higher grades (61.5% versus 11.1%, P = .002) and was borderline significant in children as compared with adults (46.7 versus 16.0%, P = .065). Overall survival at 1 and 2 years from the first MSC administration was 50.0% and 38.6%, with a median survival time of 1.1 years. In conclusion, MSC can be safely administered on top of conventional immunosuppression for steroid resistant GVHD treatment. Eudract Number 2008-007869-23, NCT01764100.
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http://dx.doi.org/10.1016/j.bbmt.2013.11.033DOI Listing
March 2014

Increased effector-target cell conjugate formation due to HLA restricted specific antigen recognition.

Immunol Res 2009 ;45(1):13-24

Department of Laboratory Medicine, Clinical Center, National Institutes of Health, Bethesda, MD, USA.

The T cell receptor (TCR) orchestrates T cell mediated-cytotoxicity through a complex interaction that results in an antigen-specific effector-target cell conjugate formation. While it is well recognized that specific TCR/antigen interactions generate the immunological synapse, their direct contribution to the effector-target cell conjugate has not been conclusively demonstrated. Moreover, since human cytotoxic T lymphocyte (CTL) clones are also susceptible to antigen-independent adhesion to target cells, it remains unclear whether effector-target cell conjugate formation can serve as an indicator of specific antigen recognition by the TCR. To address this question, a well-characterized epitope-specific CTL clone recognizing the melanoma-associated antigen epitope gp100:209-217 in association with HLA-A*0201 was tested against melanoma cell lines lacking or expressing the HLA-A*0201 allele and/or gp100. In this model, TCR/HLA/antigen interactions cooperated with accessory/adhesion molecules to facilitate effector-target cell conjugate formation. HLA-restricted antigen recognition played a dominant role resulting in up to 2-fold increases in conjugate frequency, and a 50% increase of CTL binding to tumor cells over background. The increased number of CTL contained in conjugates correlated with the number of IFN-gamma producing CTL. These results warrant further investigation to evaluate conjugate assays as a potential tool to detect and isolate viable and functionally active CTL. Since conjugate formation analysis does not require knowledge of the target antigen, this assay could potentially be used for enrichment of CTL directed against novel antigens.
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http://dx.doi.org/10.1007/s12026-008-8041-1DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3506644PMC
May 2010

Induction of pro-inflammatory programs in enteroendocrine cells by the Toll-like receptor agonists flagellin and bacterial LPS.

Int Immunol 2008 Aug 10;20(8):961-70. Epub 2008 Jun 10.

Mucosal Immunity Laboratory, Department of Human Morphology, Università degli Studi di Milano, via Mangiagalli 31, 20133 Milano, Italy.

Enteroendocrine cells are hormone-secreting cells spread along the intestinal epithelium. Their principal function is to promote the digestion of food. However, little is known about other functions that these cells may play, since they are difficult to study as a whole endocrine organ due to their diffuse localization. It is known that the intestinal epithelial barrier is actively involved in the host defense against pathogen invasion. Here we applied gene expression profiling to characterize the response of the human LCC-18 enteroendocrine cell line to physiological and pathological stimuli mimicked by fatty acids (FAs), flagellin and LPS exposure. We observed that these cells participate in an innate immune reaction to pathogens through the expression of pro-inflammatory factors (i.e. CXCL1 and 3 and IL-32) that we could validate by molecular and proteomic approach. Interestingly, IL-32 has been recently found over-expressed in the inflamed mucosa of patients affected by inflammatory bowel disease. This is very important because modifications of enteroendocrine cells during intestinal inflammation have been so far considered as secondary effects of the inflammatory status rather than due to direct pathogen/enteroendocrine cell interaction. As expected, FAs exposure up-regulates pro-differentiative genes and the production of cholecystokinin but it does not enhance the expression of pro-inflammatory genes. The present observations enlighten a new aspect of the cross talk between immune and endocrine system and suggest enteroendocrine cells as important contributors of inflammatory processes occurring in the gut in response to pathogen exposure and direct enhancers of the inflammatory status associated with human inflammatory bowel disease.
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http://dx.doi.org/10.1093/intimm/dxn055DOI Listing
August 2008

GM-CSF/IL-3/IL-5 receptor common beta chain (CD131) expression as a biomarker of antigen-stimulated CD8+ T cells.

J Transl Med 2008 Apr 15;6:17. Epub 2008 Apr 15.

Infectious Disease and Immunogenetics Section (IDIS), Department of Transfusion Medicine, Clinical Center, National Institutes of Health, Bethesda, MD, USA.

Background: Upon Ag-activation cytotoxic T cells (CTLs) produce IFN-gamma GM-CSF and TNF-alpha, which deliver simultaneously pro-apoptotic and pro-inflammatory signals to the surrounding microenvironment. Whether this secretion affects in an autocrine loop the CTLs themselves is unknown.

Methods: Here, we compared the transcriptional profile of Ag-activated, Flu-specific CTL stimulated with the FLU M1:58-66 peptide to that of convivial CTLs expanded in vitro in the same culture. PBMCs from 6 HLA-A*0201 expressing donors were expanded for 7 days in culture following Flu M1:58-66 stimulation in the presence of 300 IU/ml of interleukin-2 and than sorted by high speed sorting to high purity CD8+ expressing T cells gated according to FluM1:58-66 tetrameric human leukocyte antigen complexes expression.

Results: Ag-activated CTLs displayed higher levels of IFN-gamma, GM-CSF (CSF2) and GM-CSF/IL-3/IL-5 receptor common beta- chain (CD131) but lacked completely expression of IFN-gamma receptor-II and IFN-stimulated genes (ISGs). This observation suggested that Ag-activated CTLs in preparation for the release of IFN-gamma and GM-CSF shield themselves from the potentially apoptotic effects of the former entrusting their survival to GM-SCF. In vitro phenotyping confirmed the selective surface expression of CD131 by Ag-activated CTLs and their increased proliferation upon exogenous administration of GM-CSF.

Conclusion: The selective responsiveness of Ag-activated CTLs to GM-CSF may provide an alternative explanation to the usefulness of this chemokine as an adjuvant for T cell aimed vaccines. Moreover, the selective expression of CD131 by Ag-activated CTLs proposes CD131 as a novel biomarker of Ag-dependent CTL activation.
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http://dx.doi.org/10.1186/1479-5876-6-17DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2330025PMC
April 2008

Helper B cells promote cytotoxic T cell survival and proliferation independently of antigen presentation through CD27/CD70 interactions.

J Immunol 2008 Feb;180(3):1362-72

Infectious Disease and Immunogenetics Section, Department of Transfusion Medicine, Clinical Center, National Institute of Neurological Disorders and Stroke, National Institutes of Health, 9000 Rockville Pike, Bethesda, MD 20892, USA.

CD8-expressing cytotoxic T cell (CTL) interactions with APCs and helper T cells determine their function and ability to survive. In this study, we describe a novel interaction independent of Ag presentation between activated CTLs and bystander CD19-expressing B lymphocytes. Ag-stimulated CTLs serially engage autologous B lymphocytes through CD27/CD70 contact that promotes their survival and proliferation. Moreover, these interactions induce the release of proinflammatory cytokines that follows two general patterns: 1) an epitope-dependent enhancement of cytokine release, and 2) a previously undiscovered coordinate release of cytokines independent of epitope exposure. The latter includes chemoattractants targeting activated T cells. As a result, activated T cells are attracted to B cells, which exert a "helper" role in lymphatic organs or in areas of inflammation. This observation provides a mechanistic explanation to previously reported experimental observations suggesting that B cells are required for T cell priming in vivo.
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http://dx.doi.org/10.4049/jimmunol.180.3.1362DOI Listing
February 2008

Molecular purging of multiple myeloma cells by ex-vivo culture and retroviral transduction of mobilized-blood CD34+ cells.

J Transl Med 2007 Jul 12;5:35. Epub 2007 Jul 12.

San Raffaele Telethon Institute for Gene Therapy (HSR-TIGET), Scientific Institute HS Raffaele, Milan, Italy.

Background: Tumor cell contamination of the apheresis in multiple myeloma is likely to affect disease-free and overall survival after autografting.

Objective: To purge myeloma aphereses from tumor contaminants with a novel culture-based purging method.

Methods: We cultured myeloma-positive CD34+ PB samples in conditions that retained multipotency of hematopoietic stem cells, but were unfavourable to survival of plasma cells. Moreover, we exploited the resistance of myeloma plasma cells to retroviral transduction by targeting the hematopoietic CD34+ cell population with a retroviral vector carrying a selectable marker (the truncated form of the human receptor for nerve growth factor, DeltaNGFR). We performed therefore a further myeloma purging step by selecting the transduced cells at the end of the culture.

Results: Overall recovery of CD34+ cells after culture was 128.5%; DeltaNGFR transduction rate was 28.8% for CD34+ cells and 0% for CD138-selected primary myeloma cells, respectively. Recovery of CD34+ cells after DeltaNGFR selection was 22.3%. By patient-specific Ig-gene rearrangements, we assessed a decrease of 0.7-1.4 logs in tumor load after the CD34+ cell selection, and up to 2.3 logs after culture and DeltaNGFR selection.

Conclusion: We conclude that ex-vivo culture and retroviral-mediated transduction of myeloma leukaphereses provide an efficient tumor cell purging.
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http://dx.doi.org/10.1186/1479-5876-5-35DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1948885PMC
July 2007

Molecular signatures induced by interleukin-2 on peripheral blood mononuclear cells and T cell subsets.

J Transl Med 2006 Jun 28;4:26. Epub 2006 Jun 28.

Immunogenetics Section, Department of Transfusion Medicine, Clinical Center, National Institutes of Health, Bethesda, Maryland 20892, USA.

Experimentally, interleukin-2 (IL-2) exerts complex immunological functions promoting the proliferation, survival and activation of T cells on one hand and inducing immune regulatory mechanisms on the other. This complexity results from a cross talk among immune cells which sways the effects of IL-2 according to the experimental or clinical condition tested. Recombinant IL-2 (rIL-2) stimulation of peripheral blood mononuclear cells (PBMC) from 47 donors of different genetic background induced generalized T cell activation and anti-apoptotic effects. Most effects were dependent upon interactions among immune cells. Specialized functions of CD4 and CD8 T cells were less dependent upon and often dampened by the presence of other PBMC populations. In particular, cytotoxic T cell effector function was variably affected with a component strictly dependent upon the direct stimulation of CD8 T cells in the absence of other PBMC. This observation may provide a roadmap for the interpretation of the discrepant biological activities of rIL-2 observed in distinct pathological conditions or treatment modalities.
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http://dx.doi.org/10.1186/1479-5876-4-26DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1557669PMC
June 2006

Delayed polarization of mononuclear phagocyte transcriptional program by type I interferon isoforms.

J Transl Med 2005 Jun 13;3:24. Epub 2005 Jun 13.

Department of Transfusion Medicine, Warren G. Magnuson Clinical Center, National Institutes of Health, Bethesda, Maryland, USA.

Background: Interferon (IFN)-alpha is considered a key modulator of immunopathological processes through a signature-specific activation of mononuclear phagocytes (MPs). This study utilized global transcript analysis to characterize the effects of the entire type I IFN family in comparison to a broad panel of other cytokines on MP previously exposed to Lipopolysaccharide (LPS) stimulation in vitro.

Results: Immature peripheral blood CD14+ MPs were stimulated with LPS and 1 hour later with 42 separate soluble factors including cytokines, chemokines, interleukins, growth factors and IFNs. Gene expression profiling of MPs was analyzed 4 and 9 hours after cytokine stimulation. Four hours after stimulation, the transcriptional analysis of MPs revealed two main classes of cytokines: one associated with the alternative and the other with the classical pathway of MP activation without a clear polarization of type I IFNs effects. In contrast, after 9 hours of stimulation most type I IFN isoforms induced a characteristic and unique transcriptional pattern separate from other cytokines. These "signature" IFNs included; IFN-beta, IFN-alpha2b/alpha2, IFN-alphaI, IFN-alpha2, IFN-alphaC, IFN-alphaJ1, IFN-alphaH2, and INF-alpha4B and induced the over-expression of 44 genes, all of which had known functional relationships with IFN such as myxovirus resistance (Mx)-1, Mx-2, and interferon-induced hepatitis C-associated microtubular aggregation protein. A second group of type I IFNs segregated separately and in closer association with the type II IFN-gamma. The phylogenetic relationship of amino acid sequences among type I IFNs did not explain their sub-classification, although differences at positions 94 through 109 and 175 through 189 were present between the signature and other IFNs.

Conclusion: Seven IFN-alpha isoforms and IFN-beta participate in the late phase polarization of MPs conditioned by LPS. This information broadens the previous view of the central role played by IFN-alpha in autoimmunity and tumor rejection by including and/or excluding an array of related factors likely to be heterogeneously expressed by distinct sub-populations of individuals in sickness or in response to biological therapy.
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http://dx.doi.org/10.1186/1479-5876-3-24DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1187927PMC
June 2005

Polarized monocyte response to cytokine stimulation.

Genome Biol 2005 21;6(2):R15. Epub 2005 Jan 21.

Immunogenetics Section, Department of Transfusion Medicine, Clinical Center, National Institutes of Health, Bethesda, MD 20892-1502, USA.

Background: Mononuclear phagocytes (MPs) stand at the crossroads between the induction of acute inflammation to recruit and activate immune effector cells and the downmodulation of the inflammatory process to contain collateral damage. This decision is extensively modulated by the cytokine microenvironment, which includes a broad array of cytokines whose direct effect on MPs remains largely unexplored. Therefore, we tested whether polarized responses of MPs to pathogens are related to the influence of selected cytokines or represent a mandatory molecular switch through which most cytokines operate.

Results: Circulating CD14+ MPs were exposed to bacterial lipopolysaccharide (LPS) followed by exposure to an array of cytokines, chemokines and soluble factors involved in the immune response. Gene expression was studied by global transcript analysis. Two main classes of cytokines were identified that induced a classical or an alternative pathway of MP activation. Expression of genes affected by NFkappaB activation was most predictive of the two main classes, suggesting that this pathway is a fundamental target of cytokine regulation. As LPS itself induces a classical type of activation, the most dramatic modulation was observed toward the alternative pathway, suggesting that a broad array of cytokines may counteract the pro-inflammatory effects of bacterial components.

Conclusions: This analysis is directly informative of the primary effect of individual cytokines on the early stages of LPS stimulation and, therefore, may be most informative of the way MP maturation may be polarized at the early stages of the immune response.
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http://dx.doi.org/10.1186/gb-2005-6-2-r15DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC551535PMC
March 2006

IL-3 or IL-7 increases ex vivo gene transfer efficiency in ADA-SCID BM CD34+ cells while maintaining in vivo lymphoid potential.

Mol Ther 2004 Dec;10(6):1096-108

San Raffaele Telethon Institute for Gene Therapy, 20132 Milan, Italy.

To improve maintenance and gene transfer of human lymphoid progenitors for clinical use in gene therapy of adenosine deaminase (ADA)-deficient SCID we investigated several gene transfer protocols using various stem cell-enriched sources. The lymphoid differentiation potential was measured by an in vitro clonal assay for B/NK cells and in the in vivo SCID-hu mouse model. Ex vivo culture with the cytokines TPO, FLT3-ligand, and SCF (T/F/S) plus IL-3 or IL-7 substantially increased the yield of transduced bone marrow (BM) CD34(+) cells purified from ADA-SCID patients or healthy donors, compared to T/F/S alone. Moreover, the use of IL-3 or IL-7 significantly improved the maintenance of in vitro B cell progenitors from ADA-SCID BM cells and allowed the efficient transduction of B and NK cell progenitors. Under these optimized conditions transduced CD34(+) cells were efficiently engrafted into SCID-hu mice and gave rise to B and T cell progeny, demonstrating the maintenance of in vivo lymphoid reconstitution capacity. The protocol based on the T/F/S + IL-3 combination was included in a gene therapy clinical trial for ADA-SCID, resulting in long-term engraftment of stem/progenitor cells. Remarkably, gene-corrected BM CD34(+) cells obtained from one patient 4 and 11 months after gene therapy were capable of repopulating the lymphoid compartment of SCID-hu hosts.
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http://dx.doi.org/10.1016/j.ymthe.2004.08.014DOI Listing
December 2004

Efficient gene transfer into primitive hematopoietic progenitors using a bone marrow microenvironment cell line engineered to produce retroviral vectors.

Haematologica 2004 Apr;89(4):462-70

San Raffaele Telethon Institute for Gene Therapy (HSR-TIGET), Via Olgettina 58, Milan 20132, Italy.

Background And Objectives: Effective gene transfer into human hematopoietic stem/progenitor cells is a compromise between achieving high transduction efficiency and maintaining the desired biological characteristics of the target cell. The aim of our work was to exploit the stromal microenvironment to increase gene transfer and maintenance of hematopoietic progenitors.

Design And Methods: The murine bone marrow stromal cell line MS-5, known to support primitive human progenitors, was modified into an amphotropic packaging cell, by the stable introduction of DNA coding for retroviral structural proteins, and a viral vector encoding a marker gene. The gene transfer efficiency of the recombinant virus was evaluated by flow cytometry, in vitro assays for committed (CFC) and primitive (LTC-CFC) progenitors, as well as a clonal assay for B and NK lymphoid progenitors.

Results: The new packaging cell line (NEXUS) produced equivalent levels of virus as did the established GP+Am12 system, also under serum-free conditions. On average 30% of human mobilized peripheral blood CD34(+) cells were transduced by a single exposure to NEXUS supernatant, representing a three-fold increase over GP+Am12-based technology. Gene transfer into both committed and primitive progenitors increased on average two-fold using NEXUS retroviral supernatant. Furthermore, CD34(+) CD38(low) early progenitor cells purified from umbilical cord blood were efficiently transduced with NEXUS retroviral vector and gave rise to a high frequency of marked B and NK lymphocytes.

Interpretation And Conclusions: Our data show that that an established bone marrow stromal cell can be engineered to enhance the genetic modification of primitive hematopoietic and lymphoid progenitors using a clinically relevant method.
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April 2004

Mobilized blood CD34+ cells transduced and selected with a clinically applicable protocol reconstitute lymphopoiesis in SCID-Hu mice.

Hum Gene Ther 2004 Mar;15(3):305-11

San Raffaele Telethon Institute for Gene Therapy (HSR-TIGET), 20132 Milan, Italy.

We developed a clinically applicable gene transfer procedure into mobilized peripheral blood (MPB) CD34(+) hematopoietic progenitor cells, based on single viral exposure and selection of engineered cells. CD34(+) cells were transduced with a retroviral vector carrying the truncated form of the nerve growth factor receptor (Delta NGFR) marker gene, and immunoselected for Delta NGFR expression. Optimal time and procedure for viral exposure, length of culture, and transgene expression of MPB CD34(+) cells were determined using in vitro assays. The multipotent capacity of MPB CD34(+)-transduced cells was demonstrated in the SCID-hu bone/liver/thymus mouse model. Transduced Delta NGFR(+) cells retained 50% of long-term culture-colony forming cells (LTC-CFC) compared to unmanipulated CD34(+) cells. In SCID-hu mice, 52% of CD45(+) cells, 27% of CD34(+) cells, 49% of B cells, and more than 50% of T cells were derived from transplanted CD34(+)/Delta NGFR(+) cells. Furthermore, transplantation of purified transduced cells greatly reduced the competition with untransduced progenitors occurring in unselected grafts. These data demonstrate that MPB CD34(+) cells, transduced with a single viral exposure and selected by transgene expression, retain multilineage reconstitution capacity and remarkable transgene expression.
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http://dx.doi.org/10.1089/104303404322886156DOI Listing
March 2004

Correction of ADA-SCID by stem cell gene therapy combined with nonmyeloablative conditioning.

Science 2002 Jun;296(5577):2410-3

San Raffaele Telethon Institute for Gene Therapy (HSR-TIGET), Milan, Italy.

Hematopoietic stem cell (HSC) gene therapy for adenosine deaminase (ADA)-deficient severe combined immunodeficiency (SCID) has shown limited clinical efficacy because of the small proportion of engrafted genetically corrected HSCs. We describe an improved protocol for gene transfer into HSCs associated with nonmyeloablative conditioning. This protocol was used in two patients for whom enzyme replacement therapy was not available, which allowed the effect of gene therapy alone to be evaluated. Sustained engraftment of engineered HSCs with differentiation into multiple lineages resulted in increased lymphocyte counts, improved immune functions (including antigen-specific responses), and lower toxic metabolites. Both patients are currently at home and clinically well, with normal growth and development. These results indicate the safety and efficacy of HSC gene therapy combined with nonmyeloablative conditioning for the treatment of SCID.
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http://dx.doi.org/10.1126/science.1070104DOI Listing
June 2002