INTRODUCTION: Chimeric antigen receptor (CAR) T-cell therapy is a new treatment for hematologic malignancies including aggressive B-cell non-Hodgkin’s lymphoma (NHL). Although it has provided an effective treatment option for patients who have few options, CAR T-cell therapy does have many associated toxicities. Prolonged cytopenias are one of the lesser understood toxicities that can affect upwards of 40% of patients. METHODS: In this retrospective study, we reviewed 106 patients who received commercial CAR T-cell therapy between November 2017 and September 2019. Prolonged cytopenias were defined as having absolute neutrophil count (ANC) <1000/mm3, platelets (PLT) <50,000/mm3, and/or hemoglobin (Hgb) <10 g/dL at least once after 30 days post-CAR T-cell infusion. Furthermore, if only one incidence of cytopenia was recorded 30 days post infusion, we required that the patient had to have received either a transfusion or granulocyte-colony stimulating factor (GCSF) after the date of the recorded cytopenic value to be considered a part of the cytopenic cohort. RESULTS: 22 patients met the criteria of having prolonged cytopenias. 64% of the cytopenic cohort had >1 type of prolonged cytopenias. Anemia was the most prevalent affecting 72% of cytopenic patients. The length of time from diagnosis of aggressive B-cell NHL to date of CAR T-cell infusion was found to be positively correlated with an increased risk of developing prolonged cytopenias following CAR T-cell therapy. Additional risk factors associated with an increased risk of delayed cytopenias by univariate analysis included neutropenia on the day of infusion (day 0), a high C-reactive protein (CRP) before lymphodepletion and on day 0, day 0 PLT count, and Hgb before lymphodepletion and on day 0. On multivariate analysis, only high CRP before lymphodepletion was associated with an increased risk of prolonged cytopenias while high ferritin and PLT values on day 0 were associated with not developing prolonged cytopenias. There was no statistical difference between the cytopenic and non-cytopenic cohorts in rates of progression free survival (PFS) and overall survival (OS). Also, no difference was seen in rates or severity of other toxicities between cohorts. 41% of the cytopenic cohort experienced infectious complications post-infusion with one patient dying from their infectious complications. However, there was no association with incidence of infection and prolonged cytopenias when compared to the incidence of infection in the non-cytopenic cohort. CONCLUSIONS: A longer time from diagnosis of aggressive B-cell NHL to time of CAR T-cell infusion was associated with prolonged cytopenias while the number of lines of prior chemotherapy and rate of prior high dose chemotherapy with an autologous stem cell transplant (HD-ASCT) were not associated. It would be valuable to confirm this association and why it is associated since the other two factors were not. We lacked bone marrow biopsies before CAR T-cell infusion and did not have bone marrow biopsies for many patients after CAR T-cell infusion. It would be beneficial to collect data regarding bone marrow biopsies from these time points to highlight any changes that could be related to CAR T-cell therapy. Cytogenetic information of individual patient’s diseases would be worth analyzing to help determine if there are biological factors associated with prolonged cytopenias in response to CAR T-cell therapy. Additional studies should investigate the laboratory values we found to have associations with either cohort to help identify possible predictive values providers could use to identify patients at higher risk of having prolonged cytopenias. There is also a need to see if specific prior chemotherapy regimens increase a patient’s risk of having prolonged cytopenias. Overall, since prolonged cytopenias after CAR T-cell infusions have not been heavily investigated, further investigation is needed to better understand the predictive factors and identify possible mechanisms of prolonged cytopenias seen in CAR T-cell patients.

Background: Large-scale expansion of myogenic progenitors is necessary to support the development of high-throughput cellular assays in vitro and to advance genetic engineering approaches necessary to develop cellular therapies for rare muscle diseases. However, optimization has not been performed in order to maintain the differentiation capacity of myogenic cells undergoing long-term cell culture. Multiple extracellular matrices have been utilized for myogenic cell studies, but it remains unclear how different matrices influence long-term myogenic activity in culture. To address this challenge, we have evaluated multiple extracellular matrices in myogenic studies over long-term expansion. Methods: We evaluated the consequence of propagating mouse and human myogenic stem cell progenitors on various extracellular matrices to determine if they could enhance long-term myogenic potential. For the first time reported, we comprehensively examine the effect of physiologically relevant laminins, laminin 211 and laminin 521, compared to traditionally utilized ECMs (e.g., laminin 111, gelatin, and Matrigel) to assess their capacity to preserve myogenic differentiation potential. Results: Laminin 521 supported increased proliferation in early phases of expansion and was the only substrate facilitating high-level fusion following eight passages in mouse myoblast cell cultures. In human myoblast cell cultures, laminin 521 supported increased proliferation during expansion and superior differentiation with myotube hypertrophy. Counterintuitively however, laminin 211, the native laminin isoform in resting skeletal muscle, resulted in low proliferation and poor differentiation in mouse and human cultures. Matrigel performed excellent in short-term mouse studies but showed high amounts of variability following long-term expansion. Conclusions: These results demonstrate laminin 521 is a superior substrate for both short-term and long-term myogenic cell culture applications compared to other commonly utilized substrates. Since Matrigel cannot be used for clinical applications, we propose that laminin 521 could possibly be employed in the future to provide myoblasts for cellular therapy directed clinical studies.

The burden of sickle cell disease lies in Africa where resources are limited . Hydroxyurea may be an affordable treatmentoption for these patients. Purpose To asses whether hydroxyurea has any effect in reducing vaso-occlusive crises, hospitalization and transfusion requirements in children with homozygous sickle cell a n a e m i a . To e v a l u a t e t h e t o x i c i t y o f h y d r o x y u r e a . M e t h o d s T h i s i s a r e t r o s p e c t i v e , d e s c r i p t i v e s t u d y o f c l i n i c a l and h a e m a t o l o g i c a l o u t c o m e s i n c h i l d r e n w i t h s i c k l e c e l l a n a e m i a t r e a t e d w i t h h y d r o x y u r e a . R e s u 1t s Ten p a t i e n t s w e r e e v a l u a t e d . H y d r o x y u r e a d e c r e a s e d t h e r a t e o f v a s o - o c c 1 u s i o n and d e c r e a s e d t h e t r a n s f u s i o n r e q u i r e m e n t s . The c l i n i c a l and h a e m a t o l o g i c a l b e n e f i t s w e r e g r e a t e s t when f o e t a l h a e m o b l o b i n was m a x i m a l . T h e r e was no s h o r t - t e r m t o x i c i t y . C o n c l u s i o n H y d r o x y u r e a a m e l i o r a t e s symptoms in sickle cell disease. On going studies are n e e d e d t o a s s e s s l o n g - t e r m e f f e c t s .

Stem cells have yet to contribute to their full potential in the field of regenerative medicine and further understanding of the underlying kinetics of cell differentiation could be the step forward. Various methods have been used to characterise stem cell lineage commitment. However, most of these techniques are end-point assays and provide very little information about the changes occurring in the early stages of the differentiation process. This project aims to explore if the structural and geometrical specificity of the cytoskeletal components (actin in particular) encode information regarding cell lineage. Adipogenic and osteogenic differentiation lineages were selected, as they have been extensively studied over the past few decades. We have developed a novel approach to describe cells by defining their cytoskeletal and nuclear morphology in terms of 19 geometric measurements. This set of parameters has a range of complexity, extending from one dimensional (e.g. fibre length, fibre thickness) to compound geometrical readings (e.g. chirality and fibre alignment), while some estimate morphological and mechanical properties of the nucleus i.e. Poisson ratio and chromatin condensation. A proprietary image analysis algorithm is used to analyse fluorescent images of cells biochemically and mechanically stimulated to differentiate for a period of up to 10 days. Our analysis pipeline is currently optimised for images acquired at x20 magnification using epi-fluorescence but can be further adapted for high throughput live cell imaging. Factorial analysis of the measured features showed that some parameters change markedly in the early stages of differentiation. More interestingly we observed these changes to be non-linear and non-monotonic. This analysis, in light with previously published literature on the subject has allowed us to more intricately hypothesise probable mechanisms involved with mechanotransduction which direct the lineage commitments. As our technique quantifies the morphology of individual cells, we used our extracted feature data to characterise each cell using a multivariate predictive model (LDA).

Cartilage lesion is a fairly common problem in orthopaedic practise. It is often a consequence of traumas, inflammatory conditions, and biomechanics alterations. However, as an avascular and aneural tissue, articular cartilage has minimal healing ability. Over the past decades, surgeons and scientists have proposed a nubmer of treatment strategies to promote restoration of articular cartilage, like arthroscopic lavage, microfracture surgery, osteochoncral autografts and allografts, autologous chondrocyte implantation, and other cell-based repairs. Nevertheless, these solutions often result in fibrocartilage, which has inferior mechanical and biochemical properties, with increased susceptibility to injury, which usually ultimately leads to osteoarthritis (OA). Stem cell therapy techniques are widely applied in treating disease or injury. Many medical researchers have proposed stem cell transplantation treatment for enhancing cartilage repair by using mesenchymal stem cells (MSCs) along with biocompatible scaffolds. In addition to that, chondrogenic progenitor cells (CPCs) have also been discovered in OA patients and healthy articular cartilage. However, neither the method for isolating CPCs is not well established, nor the origin and function is not fully understood. Stem cells may be measured in CFUs (Colony-forming units). Ideally, adult stem cells should be clonogenic. In other words, a single adult stem cell should be able to generate a line of genetically identical cells. Fully characteraization of stem/progenitor cell potential requires purified population. Single-cell cloned population maybe serve as a convincing source for study of stem/progenitor cells. Therefore, a single cell clonogenecity screening system was developed to identify and isolate putative stem/progenitor cells from cartilage based on fluorescence-activated cell sorting (FACS). Also, genetical and functional characterization of isolated cells was taken.

Magnetic and plasmonic properties combined in a single nanostructure provide a synergy that is advantageous in a number of biomedical applications, such as contrast enhancement in multimodal imaging, simultaneous capture and detection of circulating tumor cells, and photothermal therapy of cancer. These applications have stimulated significant interest in development of magneto-plasmonic nanoparticles with optical absorbance in the near-infrared region and a strong magnetic moment. In this dissertation, we addressed this need to create a novel immunotargeted magneto-plasmonic nanoparticle platform. The nanostructures were synthetized by self-assembly of primary 6 nm iron oxide core-gold shell particles, resulting in densely packed spherical nanoclusters. The close proximity of the primary particles in the nanoclusters generates a greatly improved response to an external magnetic field and strong near-infrared plasmon resonances. A procedure for antibody conjugation and PEGylation to the hybrid nanoparticles was developed for biomedical applications which require molecular and biocompatible targeting. Furthermore, we presented two biomedical applications based on the immunotargeted hybrid nanoparticle platform, including circulating tumor cell (CTC) detection and cell-based immunotherapy of cancer. In the CTC detection assays, rare cancer cells were specifically targeted by antibody-conjugated nanoparticles and efficiently separated from normal blood cells by a magnetic force in a microfluidic chamber. The experiments in whole blood showed capture efficiency greater than 90% for a variety of cancers. We also explored photoacoustic imaging to detect nanoparticle-labeled CTCs in whole blood. The results showed excellent sensitivity to delineate the distribution of hybrid nanoparticles on the cancer cells. Thus, these works paves the way for a novel CTC detection approach which utilizes immunotargeted magneto-plasmonic nanoclusters for a simultaneous magnetic capture and photoacoustic detection of CTCs. In another application, we introduced a novel approach to label cytotoxic T cells using the magnetic nanoparticles with an expectation to enhance T cell recruitment in tumor under external magnetic stimulus. A series of in vitro experiments demonstrated highly controllable manipulation of labeled T cells. Thus, these results highlight the promise of using our nanoparticle platform as a multifunctional probe to manipulate and track immune cells in vivo and further improve the efficacy of cell-based cancer immunotherapy. / text

No description available.

Der Schlaganfall stellt die häufigste Ursache für dauerhafte körperliche Behinderungen im Erwachsenenalter dar. Derzeit steht aufgrund des engen therapeutischen Zeitfensters lediglich für 10% aller Schlaganfallpatienten die kausale Thrombolysetherapie zur Eröffnung des verschlossenen Blutgefäßes zur Verfügung. Eine vielversprechende Therapieoption stellen humane Nabelschnurblutzellen dar, deren Effektivität bisher ausschließlich bei jungen und meist gesunden Tieren nachgewiesen wurde. Die vorliegende Arbeit untersuchte die Wirksamkeit intravenös applizierter, kryokonservierter mononukleärer Zellen des humanen Nabelschnurblutes auf die Entwicklung eines experimentellen Schlaganfalles in jungen und alten hypertensiven Ratten. Die sensomotorische Regeneration der Tiere wurde anhand der BeamWalk- und mNSS-Tests untersucht. Im Rahmen dieser Arbeit wurde außerdem der LadderRung Walking-Test etabliert. Parallel durchgeführte MRT-Untersuchungen dienten zur Analyse des Infarktvolumens. Nach Ablauf des 64-tägigen Beobachtungszeitraumes wurde die Proliferationsaktivität von Astrozyten im Randbereich des Infarktes überprüft. Die Auswertungen sensomotorischer Fähigkeiten, der Infarktvolumina und der Zellproliferation ergaben keine signifikanten Unterschiede zwischen Therapie- und Kontrollgruppe beider Altersstufen. Junge und alte Ratten entwickelten vergleichbare Infarktvolumina und wiesen ähnliche motorische Fähigkeiten auf. Ein erhöhtes Alter der Versuchstiere war darüber hinaus mit methodischen Problemen behaftet, was die sensomotorische Funktionalitätsprüfung alter Tiere eingeschränkte und eine histopathologische Auswertung ausschloss.

<p>The ends of long bones that articulate with respect to one another are lined with a crucial connective tissue called articular cartilage. This tissue plays an essential biomechanical function in synovial joints, as it serves to both dissipate load and lubricate articulating surfaces. Osteoarthritis is a painful and debilitating disease that drives the deterioration of articular cartilage. Like many chronic diseases, pro-inflammatory cytokines feature prominently in the onset and progression of osteoarthritis. Because cartilage lacks physiologic features critical for regeneration and self-repair, the development of effective strategies to create functional cartilage tissue substitutes remains a priority for the fields of tissue engineering and regenerative medicine. The overall objectives of this dissertation are to (1) develop a bioactive scaffold capable of mediating cell differentiation and formation of extracellular matrix that recapitulates native cartilage tissue and (2) to produce stem cells specifically tailored at the scale of the genome with the ability to resist inflammatory cues that normally lead to degeneration and pain. </p><p>Engineered replacements for musculoskeletal tissues generally require extensive ex vivo manipulation of stem cells to achieve controlled differentiation and phenotypic stability. By immobilizing lentivirus driving the expression of transforming growth factor-β3 to a highly structured, three dimensionally woven tissue engineering scaffold, we developed a technique for producing cell-instructive scaffolds that control human mesenchymal stem cell differentiation and possess biomechanical properties approximating those of native tissues. This work represents an important advance, as it establishes a method for generating constructs capable of restoring biological and mechanical function that may circumvent the need for ex vivo conditioning of engineered tissue substitutes.</p><p>Any functional cartilage tissue substitute must tolerate the inflammation intrinsic to an arthritic joint. Recently emerging tools from synthetic biology and genome engineering facilitate an unprecedented ability to modify how cells respond to their microenvironments. We exploited these developments to engineer cells that can evade signaling of the pro-inflammatory cytokine interleukin-1 (IL-1). Our study provides proof-of-principle evidence that cartilage derived from such engineered stem cells are resistant to IL-1-mediated degradation. </p><p>Extending on this work, we developed a synthetic biology strategy to further customize stem cells to combat inflammatory cues. We commandeered the highly responsive endogenous locus of the chemokine (C-C motif) ligand 2 gene in pluripotent stem cells to impart self-regulated, feedback-controlled production of biologic therapy. We demonstrated that repurposing of degradative signaling pathways induced by IL-1 and tumor necrosis factor toward transient production of cytokine antagonists enabled engineered cartilage tissue to withstand the action of inflammatory cytokines and to serve as a cell-based, auto-regulated drug delivery system.</p><p>In this work, we combine principles from synthetic biology, gene therapy, and functional tissue engineering to develop methods for generating constructs with biomimetic molecular and mechanical features of articular cartilage while precisely defining how cells respond to dysfunction in the body’s finely-tuned inflammatory systems. Moreover, our strategy for customizing intrinsic cellular signaling pathways in therapeutic stem cell populations opens innovative possibilities for controlled drug delivery to native tissues, which may provide safer and more effective treatments applicable to a wide variety of chronic diseases and may transform the landscape of regenerative medicine.</p> / Dissertation

Cell based therapy using stem cell derived cardiomyocytes, has emerged as a potential therapeutic approach for cardiac diseases such as myocardial infarction and heart failure. Induced pluripotent stem cells (iPS cells) could be an ideal source of cardiomyocytes (iPS-CM). Challenges facing cell therapy include the high number of viable cells needed to survive in pathological conditions. The Hippo signalling pathway has been described as a key pathway involved in regulating cardiomyocyte proliferation and survival in both embryonic and adult hearts. We hypothesise that modification of the Hippo pathway will enhance the efficiency of iPS-CM generation and will increase iPS-CM survival and viability in pathological conditions. Skin fibroblasts were reprogrammed to iPS cells and then differentiated to cardiomyocytes. The Hippo signalling pathway was modified by genetic ablation of MST1, a major upstream regulator of the Hippo pathway, or by overexpressing YAP, the main downstream effector of the pathway. Cell proliferation was analysed using an EdU incorporation assay and staining for cytokinesis markers Ki67 and phospho-histone H3. Cell death and viability were analysed by measuring caspase 3/7 and MTT activity and by trypan blue staining in both normal and hypoxic conditions (CoCl2 treatment). Analysis of cell proliferation shows that genetic ablation of Mst1 leads to significantly increased proliferation (+12±1.5% P < 0.001), survival and viability (+20±4.3% P < 0.001) of iPS cells in both normal and hypoxic (CoCl2 treatment) conditions compared to controls. In addition, overexpression of YAP, which is normally inhibited by upstream Hippo pathway components, and overexpression of mutated constitutively active form of YAP (S127A) increases cell proliferation in iPS-CM compared to control iPS-CM as shown with EdU assay (46±2.60% P < 0.01) and Ki67 staining (4.9±0.9% P < 0.001). Overexpression of YAP leads to up regulation of genes associated with inhibition of apoptosis and promotion of cell proliferation. Preliminary studies show mouse iPS-CM are retained in the myocardium following intra-cardiac injection and do not cause any adverse effects confirmed with histological, echocardiography and electrocardiogram analysis. In conclusion targeting the Hippo pathway in iPS cells and iPS-CM significantly increases proliferation and survival in both normal and hypoxic conditions. Therefore, modulation of the Hippo pathway could become a new strategy to enhance the therapeutic potential of iPS-CM.