Risks, Benefits, and Therapeutic Potential of Hematopoietic Stem Cell Transplantation for Autoimmune Diabetes

Type 1 diabetes mellitus is a chronic disease that results from the autoimmune response against pancreatic insulin producing beta cells. Apart of several insulin regimens, since the decade of 80s various immunomodulatory regimens were tested aiming at blocking some steps of the autoimmune process against beta cell mass and at promoting beta cell preservation. In the last years, some independent research groups tried to cure type 1 diabetes with an "immunologic reset" provided by autologous hematopoietic stem cell transplantation in newly diagnosed patients, and the majority of patients became free form insulin with increasing levels of C-peptide along the time. In this review, we discuss the biology of hematopoietic stem cells and the possible advantages and disadvantages related to the high dose immunosuppression followed by autologous hematopoietic stem cell transplantation.

Healthcare-associated infection in hematopoietic stem cell transplantation patients: risk factors and impact on outcome

Objective: The objective of this study was to analyze the incidence of and risk factors for healthcare-associated infections (HAI) among hematopoietic stem cell transplantation (HSCT) patients, and the impact of such infections on mortality during hospitalization. Methods: We conducted a 9-year (2001-2009) retrospective cohort study including patients submitted to HSCT at a reference center in Sao Paulo, Brazil. The incidence of HAI was calculated using days of neutropenia as the denominator. Data were analyzed using EpiInfo 3.5.1. Results: Over the 9-year period there were 429 neutropenic HSCT patients, with a total of 6816 days of neutropenia. Bloodstream infections (BSI) were the most frequent infection, presenting in 80 (18.6%) patients, with an incidence of 11.7 per 1000 days of neutropenia. Most bacteremia was due to Gram-negative bacteria: 43 (53.8%) cases were caused by Gram-negative species, while 33 (41.2%) were caused by Gram-positive species, and four (5%) by fungal species. Independent risk factors associated with HAI were prolonged neutropenia (odds ratio (OR) 1.07, 95% confidence interval (CI) 1.04-1.10) and duration of fever (OR 1.20, 95% CI 1.12-1.30). Risk factors associated with death in multivariate analyses were age (OR 1.02, 95% CI 1.01-1.43), being submitted to an allogeneic transplant (OR 3.08, 95% CI 1.68-5.56), a microbiologically documented infection (OR 2.96, 95% CI 1.87-4.6), invasive aspergillosis disease (OR 2.21, 95% CI 1.1-4.3), and acute leukemias (OR 2.24, 95% CI 1.3-3.6). Conclusions: BSI was the most frequent HAI, and there was a predominance of Gram-negative microorganisms. Independent risk factors associated with HAI were duration of neutropenia and fever, and the risk factors for a poor outcome were older age, type of transplant (allogeneic), the presence of a microbiologically documented infection, invasive aspergillosis, and acute leukemia. Further prospective studies with larger numbers of patients may confirm the role of these risk factors for a poor clinical outcome and death in this transplant population. (C) 2012 Published by Elsevier Ltd on behalf of International Society for Infectious Diseases.

Expression analysis of stem cell-related genes reveal OCT4 as a predictor of poor clinical outcome in medulloblastoma

Aberrant expression of stem cell-related genes in tumors may confer more primitive and aggressive traits affecting clinical outcome. Here, we investigated expression and prognostic value of the neural stem cell marker CD133, as well as of the pluripotency genes LIN28 and OCT4 in 37 samples of pediatric medulloblastoma, the most common and challenging type of embryonal tumor. While most medulloblastoma samples expressed CD133 and LIN28, OCT4 expression was found to be more sporadic, with detectable levels occurring in 48% of tumors. Expression levels of OCT4, but not CD133 or LIN28, were significantly correlated with shorter survival (P <= 0.0001). Median survival time of patients with tumors hyperexpressing OCT4 and tumors displaying low/undetectable OCT4 expression were 6 and 153 months, respectively. More importantly, when patients were clinically stratified according to their risk of tumor recurrence, positive OCT4 expression in primary tumor specimens could discriminate patients classified as average risk but which further deceased within 5 years of diagnosis (median survival time of 28 months), a poor clinical outcome typical of high risk patients. Our findings reveal a previously unknown prognostic value for OCT4 expression status in medulloblastoma, which might be used as a further indicator of poor survival and aid postoperative treatment selection, with a particular potential benefit for clinically average risk patients.

Age-adjusted recipient pretransplantation telomere length and treatment-related mortality after hematopoietic stem cell transplantation

Telomere attrition induces cell senescence and apoptosis. We hypothesized that age-adjusted pretransplantation telomere length might predict treatment-related mortality (TRM) after hematopoietic stem cell transplantation (HSCT). Between 2000 and 2005, 178 consecutive patients underwent HSCT from HLA-identical sibling donors after myeloablative conditioning regimens, mainly for hematologic malignancies (n = 153). Blood lymphocytes' telomere length was measured by real-time quantitative PCR before HSCT. Age-adjusted pretransplantation telomere lengths were analyzed for correlation with clinical outcomes. After age adjustment, patients' telomere-length distribution was similar among all 4 quartiles except for disease stage. There was no correlation between telomere length and engraftment, GVHD, or relapse. The overall survival was 62% at 5 years (95% confidence interval [CI], 54-70). After a median follow-up of 51 months (range, 1-121 months), 43 patients died because of TRM. The TRM rate inversely correlated with telomere length. TRM in patients in the first (lowest telomere length) quartile was significantly higher than in patients with longer telomeres (P = .017). In multivariate analysis, recipients' age (hazard ratio, 1.1; 95% CI, .0-1.1; P = .0001) and age-adjusted telomere length (hazard ratio, 0.4; 95% CI; 0.2-0.8; P = .01) were independently associated with TRM. In conclusion, age-adjusted recipients' telomere length is an independent biologic marker of TRM after HSCT. (Blood. 2012;120(16):3353-3359)

Stem cell therapy for type 1 diabetes mellitus: a review of recent clinical trials

Stem cell therapy is one of the most promising treatments for the near future. It is expected that this kind of therapy can ameliorate or even reverse some diseases. With regard to type 1 diabetes, studies analyzing the therapeutic effects of stem cells in humans began in 2003 in the Hospital das Clínicas of the Faculty of Medicine of Ribeirão Preto - SP USP, Brazil, and since then other centers in different countries started to randomize patients in their clinical trials. Herein we summarize recent data about beta cell regeneration, different ways of immune intervention and what is being employed in type 1 diabetic patients with regard to stem cell repertoire to promote regeneration and/or preservation of beta cell mass.

Stem cell transplantation for type 1 diabetes mellitus

Abstract Background The use of stem cells to treat type 1 diabetes mellitus has been proposed for many years, both to downregulate the immune system and to provide β cell regeneration. Conclusion High dose immunosuppression followed by autologous hematopoietic stem cell transplantation is able to induce complete remission (insulin independence) in most patients with early onset type 1 diabetes mellitus.

Thrombotic obstruction of the central venous catheter in patients undergoing hematopoietic stem cell transplantation

This is an integrative literature review with the aim of summarizing the prevention measures and treatment of thrombotic obstruction of long-term semi-implanted central venous catheters, in patients undergoing hematopoietic stem cell transplantation. The sample consisted of seven studies, being two randomized controlled clinical trials, three cohort studies and two case series. Regarding the prevention measures, one single study demonstrated effectiveness, which was a cohort study on the oral use of warfarin. In relation to the treatment measures, three studies evidenced effectiveness, one highlighted the efficacy of streptokinase or urokinase, one demonstrated the benefit of using low-molecular-weight heparin and the other treated the obstruction with heparin or urokinase. Catheter patency research shows a restricted evolution that does not follow the evolution of transplantations, mainly regarding nursing care.

In search of mechanisms associated with mesenchymal stem cell-based therapies for acute kidney injury

Acute kidney injury (AKI) is classically described as a rapid loss of kidney function. AKI affects more than 15% of all hospital admissions and is associated with elevated mortality rates. Although many advances have occurred, intermittent or continuous renal replacement therapies are still considered the best options for reversing mild and severe AKI syndrome. For this reason, it is essential that innovative and effective therapies, without side effects and complications, be developed to treat AKI and the end-stages of renal disease. Mesenchymal stem cell (MSC) based therapies have numerous advantages in helping to repair inflamed and damaged tissues and are being considered as a new alternative for treating kidney injuries. Numerous experimental models have shown that MSCs can act via differentiation-independent mechanisms to help renal recovery. Essentially, MSCs can secrete a pool of cytokines, growth factors and chemokines, express enzymes, interact via cell-to-cell contacts and release bioagents such as microvesicles to orchestrate renal protection. In this review, we propose seven distinct properties of MSCs which explain how renoprotection may be conferred: 1) anti-inflammatory; 2) pro-angiogenic; 3) stimulation of endogenous progenitor cells; 4) anti-apoptotic; 5) anti-fibrotic; 6) anti-oxidant; and 7) promotion of cellular reprogramming. In this context, these mechanisms, either individually or synergically, could induce renal protection and functional recovery. This review summarises the most important effects and benefits associated with MSC-based therapies in experimental renal disease models and attempts to clarify the mechanisms behind the MSC-related renoprotection. MSCs may prove to be an effective, innovative and affordable treatment for moderate and severe AKI. However, more studies need to be performed to provide a more comprehensive global understanding of MSC-related therapies and to ensure their safety for future clinical applications.

Computational modelling of human stem cell-derived cardiomyocytes - Texture descriptors for biological image processing

This thesis investigates two distinct research topics. The main topic (Part I) is the computational modelling of cardiomyocytes derived from human stem cells, both embryonic (hESC-CM) and induced-pluripotent (hiPSC-CM). The aim of this research line lies in developing models of the electrophysiology of hESC-CM and hiPSC-CM in order to integrate the available experimental data and getting in-silico models to be used for studying/making new hypotheses/planning experiments on aspects not fully understood yet, such as the maturation process, the functionality of the Ca2+ hangling or why the hESC-CM/hiPSC-CM action potentials (APs) show some differences with respect to APs from adult cardiomyocytes. Chapter I.1 introduces the main concepts about hESC-CMs/hiPSC-CMs, the cardiac AP, and computational modelling. Chapter I.2 presents the hESC-CM AP model, able to simulate the maturation process through two developmental stages, Early and Late, based on experimental and literature data. Chapter I.3 describes the hiPSC-CM AP model, able to simulate the ventricular-like and atrial-like phenotypes. This model was used to assess which currents are responsible for the differences between the ventricular-like AP and the adult ventricular AP. The secondary topic (Part II) consists in the study of texture descriptors for biological image processing. Chapter II.1 provides an overview on important texture descriptors such as Local Binary Pattern or Local Phase Quantization. Moreover the non-binary coding and the multi-threshold approach are here introduced. Chapter II.2 shows that the non-binary coding and the multi-threshold approach improve the classification performance of cellular/sub-cellular part images, taken from six datasets. Chapter II.3 describes the case study of the classification of indirect immunofluorescence images of HEp2 cells, used for the antinuclear antibody clinical test. Finally the general conclusions are reported.

Pre-clinical and clinical development of leukemia stem cell inhibitors in acute leukemias

In Leukemias, recent developments have demonstrated that the Hedgehog pathway plays a key-role in the peculiar ability of self renewal of leukemia stem cells. The aim of this research activity was to investigate, through a first in man, Phase I, open label, clinical trial, the role and the impact, mainly in terms of safety profile, adverse events and pharmacokinetics, of a Sonic Hedgehog inhibitor compound on a population of heavely pretreated patients affected by AML, CML, MF, or MDS, resistant or refractory to standard chemotherapy. Thirty-five patients have been enrolled. The drug was administered orally, in 28 days cycles, without rest periods. The compound showed a good safety profile. The half life was of 17-35 hours, justifying the daily administration. Significant signs of activity, in terms of reduction of bone marrow blast cell amount were seen in most of the patients enrolled. Interestingly, correlative biological studies demonstrated that, comparing the gene expression profyiling signature of separated CD34+ cells before and after one cycle of treatment, the most variably expressed genes were involved in the Hh pathway. Moreover, we observed that many genes involved in MDR (multidrug resistance)were significantly down regulated after treatment. These data might lead to future clinical trials based on combinatory approaches, including, for instance, Hh inhibitors and conventional chemotherapy.

Interactions between the gut microbiota, short-chain fatty acids and the immune system in pediatric patients undergoing hematopoietic stem cell transplantation

The gut microbiota (GM) is essential for human health and contributes to several diseases; indeed it can be considered an extension of the self and, together with the genetic makeup, determines the physiology of an organism. In this thesis has been studied the peripheral immune system reconstitution in pediatric patients undergoing allogeneic hematopoietic stem cell transplantation (aHSCT) in the early phase; in parallel, have been also explored the gut microbiota variations as one of the of primary factors in governing the fate of the immunological recovery, predisposing or protecting from complications such as the onset of acute graft-versus-host disease (GvHD). Has been demonstrated, to our knowledge for the first time, that aHSCT in pediatric patients is associated to a profound modification of the GM ecosystem with a disruption of its mutualistic asset. aGvHD and non-aGvHD subjects showed differences in the process of GM recovery, in members abundance of the phylum Bacteroidetes, and in propionate fecal concentration; the latter are higher in the pre-HSCT composition of non-GvHD subjects than GvHD ones. Short-chain fatty acids (SCFAs), such as acetate, butyrate and propionate, are end-products of microbial fermentation of macronutrients and distribute systemically from the gut to blood. For this reason, has been studied their effect in vitro on human DCs, the key regulators of our immune system and the main player of aGvHD onset. Has been observed that propionate and, particularly, butyrate show a strong and direct immunomodulatory activity on DCs reducing inflammatory markers such as chemokines and interleukins. This study, with the needed caution, suggests that the pre-existing GM structure can be protective against aGvHD onset, exerting its protective role through SCFAs. They, indeed, may regulate cell traffic within secondary lymphoid tissues, influence T cell development during antigen recognition, and, thus, directly shape the immune system.

In vitro characterisation and expansion of human regulatory T cells for their in vivo application in the induction of tolerance in haematopoietic stem cell and solid organ transplantation

Solid organ transplantation (SOT) is considered the treatment of choice for many end-stage organ diseases. Thus far, short term results are excellent, with patient survival rates greater than 90% one year post-surgery, but there are several problems with the long term acceptance and use of immunosuppressive drugs. Hematopoietic Stem Cells Transplantation (HSCT) concerns the infusion of haematopoietic stem cells to re-establish acquired and congenital disorders of the hematopoietic system. The main side effect is the Graft versus Host Disease (GvHD) where donor T cells can cause pathology involving the damage of host tissues. Patients undergoing acute or chronic GvHD receive immunosuppressive regimen that is responsible for several side effects. The use of immunosuppressive drugs in the setting of SOT and GvHD has markedly reduced the incidence of acute rejection and the tissue damage in GvHD however, the numerous adverse side effects observed boost the development of alternative strategies to improve the long-term outcome. To this effect, the use of CD4+CD25+FOXP3+ regulatory T cells (Treg) as a cellular therapy is an attractive approach for autoimmunity disease, GvHD and limiting immune responses to allograft after transplantation. Treg have a pivotal role in maintaining peripheral immunological tolerance, by preventing autoimmunity and chronic inflammation. Results of my thesis provide the characterization and cell processing of Tregs from healthy controls and patients in waiting list for liver transplantation, followed by the development of an efficient expansion-protocol and the investigation of the impact of the main immunosuppressive drugs on viability, proliferative capacity and function of expanded cells after expansion. The conclusion is that ex vivo expansion is necessary to infuse a high Treg dose and although many other factors in vivo can contribute to the success of Treg therapy, the infusion of Tregs during the administration of the highest dose of immunosuppressants should be carefully considered.

CD137-selektierte leukämiereaktive T-Zellen gesunder Spender zur adoptiven Immuntherapie stammzelltransplantierter Leukämiepatienten = CD137-selected leukaemia-reactive T Cells from healthy donors for adoptive immunotherapy in stem-cell-transplanted leukaemia-patients

Bei stammzelltransplantierten Patienten, die ein Rezidiv ihrer Leukämie erleiden, kann eine Donor-Lymphozyten-Infusion (DLI) dauerhafte vollständige Leukämieremissionen induzieren. T-Zellen in der DLI vermitteln sowohl den potentiell kurativen Graft-versus-Leukaemia (GVL) Effekt, als auch die potentiell lebensbedrohliche Graft-versus-Host Disease (GVHD). Hingegen könnte die Infusion von leukämiereaktiven T-Zellen einen selektiven GVL Effekt und einen Langzeitschutz vor Rezidiven durch eine spezifisch gegen die Leukämie gerichtete Immunantwort und Immunität vermitteln. Unsere Arbeitsgruppe hat Protokolle zur in vitro Generierung leukämiereaktiver T-Zellen entwickelt, die hohe zytotoxische Aktivität gegen akute myeloische Leukämie-Blasten (AML) bei minimaler Reaktion auf mögliche GVHD Zielstrukturen zeigen. Für die klinische Anwendung sind diese Protokolle jedoch zu aufwändig, wobei vor allem eine erhebliche Verkürzung der Kulturzeit auf wenige Wochen erforderlich ist. Diese Verkürzung der in vitro Kulturzeit könnte das Wachstum von T-Zellen vom central memory oder frühen effector memory Phänotyp fördern, für die eine bessere in vivo Effektorfunktion und längere Persistenz im Rezipienten verglichen mit T-Zellen aus Langzeitkultur gezeigt werden konnte. Der Aktivierungsmarker und Kostimulations-Rezeptor CD137 kann zur Erkennung und Isolation antigenspezifischer T-Zellen genutzt werden, ohne dass dafür das von den T-Zellen erkannte Peptidepitop bekannt sein muss. Eine CD137-vermittelte Anreicherung mit Hilfe von clinical grade Materialien könnte verwendet werden, um DLI-Produkte mit leukämiespezifischen T-Zellen herzustellen, die sich sowohl durch eine effizientere T-Zell Generierung durch in vitro Selektion und Kostimulation, als auch durch eine verbesserte Spezifität des T-Zell-Produkts auszeichnen. Lymphozyten-Leukämie Cokulturen (mixed lymphocyte leukaemia cultures) wurden mit CD8 T-Zellen gesunder Spender und HLA-identischen oder einzel-HLA-mismatch AML-Blasten angesetzt und wöchentlich restimuliert. Nach zwei Wochen wurden die T-Zellen 12 Stunden nach Restimulation über den Marker CD137 positiv isoliert und anschließend separat weiterkultiviert. Die isolierten Fraktionen und unseparierten Kontrollen wurden im ELISPOT-Assay und im Chrom-Freisetzungstest an Tag 5 nach der Restimulation getestet. Es wurden keine konsistent nachweisbaren Vorteile im Hinblick auf Wachstum und Funktion der isolierten CD137-positiv Fraktion im Vergleich zur unseparierten Kontrolle gefunden. Verschiedene Isolationsmethoden, Patient-Spender-Systeme, Methoden zur Restimulation, Temperaturbedingungen, Zytokinkombinationen und Methoden der Zytokinzugabe sowie zusätzliche Feeder-Zellen oder AML-Blasten konnten Wachstum, funktionelle Daten und die deutlichen Zellverluste während der Isolation nicht entscheidend beeinflussen. Vitalfärbungen zeigten, dass aktivierungsinduzierter Zelltod CD137-positiver Zellen zu diesen Ergebnissen beitragen könnte. Im Gegensatz zur Stimulation mit AML-Blasten wurden erfolgreiche CD137-Anreicherungen für peptidstimulierte T-Zellen publiziert. Unterschiedliche CD137-Expressionskinetiken, aktivierungsinduzierter Zelltod und regulatorische T-Zellen sind mögliche Faktoren aufgrund derer die CD137-Anreicherung in diesem spezifischen Kontext ungeeinet sein könnte. Der stimulatorische Effekt eines CD137-Signals auf AML-reaktive CD8 T-Zellen wurde mit Hilfe von CD3/CD28 und CD3/CD28/CD137 Antikörper-beschichteten magnetischen beads untersucht. Für Nierenzellkarzinom-reaktive T-Zellen war die Stimulation mit CD3/CD28/CD137 beads genauso effektiv wie mit Tumorzellen und effektiver als mit CD3/CD28 beads. Beide Arten von beads waren für eine Stimulation während der ersten Wochen der Zellkultur geeignet, sodass ein zusätzliches CD137-Signal für die länger anhaltende Expansion tumorreaktiver T-Zellen zur klinischen Anwendung nützlich sein könnte. Die bead-Expansion veränderte die IFN-Sekretion im ELISPOT nicht, aber verursachte eine mäßige Verschlechterung der Zytotoxizität im Chrom-Freisetzungstest. Im Gegensatz dazu zeigten bei AML-reaktiven T-Zellen beide Arten von beads einen nicht apoptosevermittelten, dosisabhängigen zellschädigenden Effekt, der zu einer raschen Abnahme der Zellzahl in Kulturen mit beads führte. Unerwünschte Effekte auf die T-Zell-Funktionalität durch bead-Stimulation sind in der Literatur beschrieben, dennoch gibt es aktuell keine Veröffentlichungen, die eine fundierte Erklärung für den Effekt auf AML-reaktive T-Zellen bieten könnten. Abgesehen von Literaturdaten, die darauf hindeuten, dass CD137 ein vielversprechendes Kandidatenmolekül für die Anreicherung und Expansion von AML-reaktiven T-Zellen sein könnte, zeigen die eigenen Daten sowohl zur CD137-Isolation als auch zur bead-Stimulation, dass für diese spezielle Anwendung CD137 ein ungeeigneter Aktivierungsmarker und Kostimulations-Ligand ist.