scholarly journals 4053 A TL1 Team Approach to CNS-Localized Delivery of Glial Cell-Derived Neurotrophic Factor for Treatment of Parkinson’s Disease

2020 ◽  
Vol 4 (s1) ◽  
pp. 1-1
Author(s):  
Shaheen Farhadi ◽  
Adithya Gopinath ◽  
Wolfgang Streit ◽  
Gregory A Hudalla ◽  
Habibeh Khoshbouei
Keyword(s):  
Cell Surface ◽  
Dopaminergic Neurons ◽  
Fusion Proteins ◽  
Ex Vivo ◽  
Symptomatic Relief ◽  
Dopamine Neuron ◽  
Team Approach ◽  
Depth Of Penetration ◽  
Tissue Surfaces ◽  
Galectin 3

OBJECTIVES/GOALS: Develop a strategy to restrict GDNF diffusion at an injected CNS tissue site for dopamine neuron rescue by endowing it with binding affinity for carbohydrates that are abundant on the cell surface and extracellular matrix. METHODS/STUDY POPULATION: GDNF will be fused to galectin-3 (G3), a human protein that binds to β-galactoside residues of cell surface and matrix glycoproteins. We characterized the binding of G3 fusion proteins to various glycoproteins and primary human myeloid cells. We incubated G3 fusions with CNS tissue ex vivo to measure their binding and depth of penetration via diffusion. We next plan to administer GDNF-G3 via CNS intracranial infusion in a murine PD model and then conduct behavioral PD phenotype testing via rotarod and pole descent to compare to non-parkinsonian controls. We will further examine the effects of GDNF-G3 on degeneration using immunohistochemical examination of post-mortem brain tissue. RESULTS/ANTICIPATED RESULTS: Based on results from previous clinical trials of GDNF delivery, we anticipate that a successful intervention using GDNF-G3 will result in rescue of midbrain dopaminergic neurons in a murine PD model. In murine CNS tissue, we observed binding to glycans at the tissue surfaces when incubated with G3 fusion proteins ex vivo, suggesting GDNF-G3 will remain localized to the injection site. Next we will administer GDNF-G3 via CNS intracranial infusion in a murine PD model and assess efficacy by behavior and histopathology. GDNF-G3-mediated dopamine neuron rescue are expected to slow or reverse the progression of PD in these animal models. DISCUSSION/SIGNIFICANCE OF IMPACT: PD treatments focus on symptomatic relief. Standard therapies have not been efficacious in rescuing of dopaminergic neurons. GDNF-G3 administered at the site of neurodegeneration would represent a milestone on the path to treating PD pathology and address limitations of GDNF delivery.

scholarly journals 3475 A TL1 Team Approach to CNS-Localized Delivery of Neurotrophic Factors for Treatment of Parkinson’s Disease

2019 ◽  
Vol 3 (s1) ◽  
pp. 129-130
Author(s):  
Adithya Gopinath ◽  
Shaheen Farhadi
Keyword(s):  
Dopaminergic Neurons ◽  
Standard Of Care ◽  
Team Approach ◽  
Midbrain Dopaminergic Neurons ◽  
Post Mortem Brain ◽  
Galectin 3 ◽  
Study Population ◽  
Localized Delivery ◽  
Dopamine Signaling ◽  
Tissue Site

OBJECTIVES/SPECIFIC AIMS: We present an alternative strategy to retain NTFs at an injected CNS tissue site by endowing them with binding affinity for carbohydrates that are abundant on the cell surface and within extracellular matrices. METHODS/STUDY POPULATION: We are creating recombinant fusions in which glial cell-derived neurotrophic factor (GDNF) is linked to galectin-3 (G3), a human protein that binds to extracellular beta-galactoside glycans and glycosaminoglycans. GDNF-G3 fusion proteins will circumvent major therapeutic shortcomings of early GDNF human trials by anchoring GDNF to the midbrain in a preclinical animal model of PD over a therapeutically-relevant timescale in order to achieve DA neuron rescue. Further, in PD patients, we have detected significantly dysregulated dopamine signaling in peripheral, blood-derived monocytes, suggesting a systemic dopamine signaling change in PD. RESULTS/ANTICIPATED RESULTS: Based on results from published human NTF administration trials, we anticipate that a successful intervention using GDNF-G3 will result in rescue or delayed degeneration of midbrain dopaminergic neurons in a murine PD model. Outcome measures include behavioral PD phenotype testing via rotarod and pole descent compared to non-parkinsonian control animals, as well as corroborating immunohistological evidence from immunohistochemical examination of post-mortem brain tissue from the same animals to examine degree of degeneration. DISCUSSION/SIGNIFICANCE OF IMPACT: Current treatments for PD, whether pharmacological or surgical, center on alleviating movement symptoms that impair daily function - in other words, largely palliative care. Little has been accomplished by way of rescue of dopaminergic neurons or slowing disease progression using standard-of-care therapy. If successful, GDNF-G3 constructs administered intracranially at the site of degeneration would represent a milestone on the path to treating the basic pathology associated with PD, while addressing major shortcomings in earlier NTF-delivery attempts, namely NTF diffusion away from target site.

O-191 Assessing the use of tumour-specific DARPin-toxin fusion proteins for ex vivo purging of cancer metastases from human ovarian cortex tissue fragments before autotransplantation

2021 ◽  
Vol 36 (Supplement_1) ◽  
Author(s):  
L Eijkenboom ◽  
V Palacio-Castañeda ◽  
F A Groenman ◽  
D D M Braat ◽  
C C M Beerendonk ◽  
...  
Keyword(s):  
Breast Cancer ◽  
Cancer Cells ◽  
Fusion Proteins ◽  
Breast Cancer Cells ◽  
Ex Vivo ◽  
Ovarian Tissue ◽  
In Vitro Growth ◽  
Ovarian Cortex ◽  
Positive Breast Cancer

Abstract Study question Is it possible to eradicate cancer cells from ovarian cortex by using tumour-specific designed ankyrin repeat protein (DARPin)-toxin fusion proteins, without compromising the ovarian tissue? Summary answer Purging ovarian cortex ex vivo from experimentally induced breast cancer tumour foci is possible by tumour-targeted DARPin-toxin fusion proteins trough inhibition of protein synthesis. What is known already Ovarian tissue cryopreservation and autotransplantation is a successful technique for fertility restoration in cancer patients. The procedure is not without risk since malignant cells may still be present in the graft. Procedures to detect cancer cells render the tissue fragment useless for autotransplantation. Strategies to circumvent this problem such as in vitro maturation of follicles or the construction of artificial ovaries are pursued but are still experimental. Alternatively, we have shown ex vivo purging of ovarian cortex is possible by elimination of rhabdomyosarcoma after treatment with verteporfin. This allows treatment of cortex fragments before autotransplantation without compromising ovarian tissue integrity. Study design, size, duration Human ovarian cortex fragments harbouring breast cancer tumour foci were exposed for 24 h to DARPins fused to the translocation and catalytic domain of Pseudomonas aeruginosa exotoxin A (DARPin-toxin fusion proteins) targeting EpCAM or HER2. After treatment with the DARPin-toxin fusion proteins the tissue was cultured for an additional 6 days to allow any remaining tumour cells to form foci. In addition, the functional integrity of the ovarian tissue was analysed after purging. Participants/materials, setting, methods Breast cancer cell lines expressing different levels of EpCAM and HER2 were introduced in human ovarian tissue to form tumour foci. After purging with DARPin-toxin fusion proteins, the presence of any remaining cancer cells in the tissue was analysed with (immuno)histochemistry and RT-qPCR. Possible detrimental effects on the viability of ovarian cortex and follicles were determined by (immuno)histology, a follicular viability assay and an assay to determine the in vitro growth capacity of small follicles. Main results and the role of chance Ovarian cortex harbouring EpCAM-positive breast cancer cells showed a significant decrease in the number of tumour foci after treatment with the EpCAM-targeted DARPin-toxin fusion proteins. Although exposure to the EpCAM-specific DARPin had no effect on morphology or viability of follicles, a decrease in oocyte viability after in vitro growth experiments was observed, presumably due to low level expression of EpCAM on oocytes. In contrast to the EpCAM-specific DARPin-toxin fusion protein, the DARPin-toxin fusion protein targeting HER2 had no detrimental effects on morphology, viability or in vitro growth of follicles while foci of HER2-positive breast cancer cells were severely affected as indicated by the presence of apoptotic bodies, tumour cell remnants and the absence of viable tumour cells. The histological results after purging with the HER2-specific DARPin-toxin fusions proteins were confirmed by RT-qPCR, showing a decrease to basal levels of HER2 mRNA in the ovarian cortex tissue. Limitations, reasons for caution The effect of DARPin-toxin fusion proteins depends heavily on the expression of their target on the cancer cell. The target protein should not be expressed by ovarian cortex as this may lead to tissue damage. The functional integrity of ovarian cortex after the treatment requires further investigation in vivo. Wider implications of the findings Purging metastases from ovarian cortex without harming ovarian tissue is possible by targeting tumour specific surface expressed antigens with DARPin-toxin fusion proteins. Purging ovarian cortex tissue with DARPin-toxin fusion proteins provides a feasible therapeutic strategy to prevent reintroduction of cancer by autotransplantation in case of malignancies expressing tumour-specific surface markers. Trial registration number not applicable

The Role of Different Molecular Markers in Papillary Thyroid Cancer Patients with Acromegaly

2018 ◽  
Vol 127 (07) ◽  
pp. 437-444 ◽  
Author(s):  
Fatma Ela Keskin ◽  
Hande Mefkure Ozkaya ◽  
Sina Ferahman ◽  
Ozlem Haliloglu ◽  
Adem Karatas ◽  
...  
Keyword(s):  
Thyroid Cancer ◽  
Papillary Thyroid Cancer ◽  
Ex Vivo ◽  
Papillary Thyroid ◽  
Cerrahpasa Medical Faculty ◽  
Protein Expressions ◽  
Galectin 3 ◽  
Benign Nodules ◽  
Igf1 Expression

Abstract Purpose Prevalence of papillary thyroid cancer (PTC) is increased in patients with acromegaly. We aimed to determine the protein expression of BRAF, RAS, RET, insulin like growth factor 1(IGF1), Galectine 3, CD56 in patients with PTC related acromegaly and to compare the extensity of these expressions with normal PTC patients and benign thyroid nodules. Methods We studied 313 patients with acromegaly followed in Cerrahpasa Medical Faculty, Endocrinology and Metabolism Clinic between 1998 and 2015. On the basis of availability of pathological specimen of thyroid tissues, thyroid samples of 13 patients from 19 with acromegaly related PTC (APTC), 20 normal PTC and 20 patients with multinodulary goiter (MNG) were histopathologically evaluated. Protein expressions were determined via immunohistochemical staining in ex-vivo tumor samples and benign nodules. Results The incidence of PTC in acromegaly patients were 6% (n=19). Among patients with PTC, APTC and MNG, all the immunohistochemical protein expressions we have studied were higher in papillary thyroid cancer groups (p<0.01, for all). Between PTC group without acromegaly and APTC, galectin 3 and IGF1 expression was significantly higher in acromegalic patients (p<0.01 for all) while RAS was predominantly higher in PTC patients without acromegaly (p<0.01). Conclusion BRAF expression was not higher in PTC with acromegaly patients compared to PTC patients without acromegaly. Galectine 3 and IGF1 were expressed more intensively in APTC. These positive protein expressions may have more influence on determining malign nodules among acromegaly patients.

Abstract 158: A Tether Containing a UBX Domain (TUG) Mediates Glucose Transporter Translocation in the Ischemic Heart

2017 ◽  
Vol 121 (suppl_1) ◽  
Author(s):  
Ji Li ◽  
Yina Ma ◽  
Jonathan Bogan
Keyword(s):  
Cell Surface ◽  
Glucose Uptake ◽  
Glucose Transporter ◽  
Ex Vivo ◽  
Ischemia And Reperfusion ◽  
Ampk Activation ◽  
Glut4 Translocation ◽  
Heart Perfusion ◽  
Intracellular Vesicles

Introduction: The adaptive metabolic regulation of glucose and fatty acid in the heart plays a critical role in limiting cardiac damage caused by ischemia and reperfusion (I/R). TUG (tether containing a UBX domain, for GLUT4) can be cleaved to mobilize glucose transporter GLUT4 from intracellular vesicles to the cell surface in skeletal muscle and adipose in response to insulin stimulation. The energy sensor AMP-activated protein kinase (AMPK) plays an important cardioprotective role in response to ischemic insults by modulating GLUT4 translocation. Hypothesis: TUG is one of the downstream targets of AMPK in the heart. TUG could be phosphorylated by ischemic AMPK and cleaved to dissociate with GLUT4 and increase GLUT4 translocation in the ischemic heart. Methods: In vivo regional ischemia by ligation of left anterior coronary artery and ex vivo isolated mouse heart perfusion Langendorff system were used to test the hypothesis. Results: Antithrombin (AT) is an endogenous AMPK agonist in the heart and used to define the role of TUG in regulating GLUT4 trafficking during ischemia and reperfusion in the heart. AT showed its cardioprotective function through recovering cardiac pumping function and activating AMPK. The results showed that AMPK activation by AT treatment was through LKB1 and Sesn2 complex. Furthermore, the ex vivo heart perfusion data demonstrated that AT administration significantly increase GLUT4 translocation, glucose uptake, glycolysis and glucose oxidation during ischemia and reperfusion (p<0.05 vs . vehicle). Moreover, AT treatment increased abundance of a TUG cleavage product (42 KD) in response to I/R. The TUG protein was clearly phosphorylated by activated AMPK in HL-1 cardiomyocytes. The in vivo myocardial ischemia results demonstrated that ischemic AMPK activation triggers TUG cleavage and significantly increases GLUT4 translocation to the cell surface. Moreover, an augmented interaction between AMPK and TUG was observed during ischemia. Conclusions: Cardiac AMPK activation stimulates TUG cleavage and causes the dissociation between TUG and GLUT4 in the intracellular vesicles. TUG is a critical mediator that modulates cardiac GLUT4 translocation to cell surface and enhances glucose uptake by AMPK signaling pathway.

scholarly journals Earliest Mechanisms of Dopaminergic Neurons Sufferance in a Novel Slow Progressing Ex Vivo Model of Parkinson Disease in Rat Organotypic Cultures of Substantia Nigra

2019 ◽  
Vol 20 (9) ◽  
pp. 2224 ◽  
Author(s):  
Matteo Dal Ben ◽  
Rosario Bongiovanni ◽  
Simone Tuniz ◽  
Emanuela Fioriti ◽  
Claudio Tiribelli ◽  
...  
Keyword(s):  
Substantia Nigra ◽  
Parkinson Disease ◽  
Dopaminergic Neurons ◽  
Molecular Mechanisms ◽  
Ex Vivo ◽  
Ex Vivo Model ◽  
Redox Imbalance ◽  
Clinical Phase ◽  
Protein Clearance ◽  
Persistent Inflammation

The current treatments of Parkinson disease (PD) are ineffective mainly due to the poor understanding of the early events causing the decline of dopaminergic neurons (DOPAn). To overcome this problem, slow progressively degenerating models of PD allowing the study of the pre-clinical phase are crucial. We recreated in a short ex vivo time scale (96 h) all the features of human PD (needing dozens of years) by challenging organotypic culture of rat substantia nigra with low doses of rotenone. Thus, taking advantage of the existent knowledge, the model was used to perform a time-dependent comparative study of the principal possible causative molecular mechanisms undergoing DOPAn demise. Alteration in the redox state and inflammation started at 3 h, preceding the reduction in DOPAn number (pre-diagnosis phase). The number of DOPAn declined to levels compatible with diagnosis only at 12 h. The decline was accompanied by a persistent inflammation and redox imbalance. Significant microglia activation, apoptosis, a reduction in dopamine vesicle transporters, and the ubiquitination of misfolded protein clearance pathways were late (96 h, consequential) events. The work suggests inflammation and redox imbalance as simultaneous early mechanisms undergoing DOPAn sufferance, to be targeted for a causative treatment aimed to stop/delay PD.

scholarly journals MT1-MMP–dependent neovessel formation within the confines of the three-dimensional extracellular matrix

2004 ◽  
Vol 167 (4) ◽  
pp. 757-767 ◽  
Author(s):  
Tae-Hwa Chun ◽  
Farideh Sabeh ◽  
Ichiro Ota ◽  
Hedwig Murphy ◽  
Kevin T. McDonagh ◽  
...  
Keyword(s):  
Extracellular Matrix ◽  
Endothelial Cells ◽  
Cell Surface ◽  
Type I Collagen ◽  
Ex Vivo ◽  
Three Dimensional ◽  
Collagenolytic Activity ◽  
Type I ◽  
Ex Vivo Model

During angiogenesis, endothelial cells initiate a tissue-invasive program within an interstitial matrix comprised largely of type I collagen. Extracellular matrix–degradative enzymes, including the matrix metalloproteinases (MMPs) MMP-2 and MMP-9, are thought to play key roles in angiogenesis by binding to docking sites on the cell surface after activation by plasmin- and/or membrane-type (MT) 1-MMP–dependent processes. To identify proteinases critical to neovessel formation, an ex vivo model of angiogenesis has been established wherein tissue explants from gene-targeted mice are embedded within a three-dimensional, type I collagen matrix. Unexpectedly, neither MMP-2, MMP-9, their cognate cell-surface receptors (i.e., β3 integrin and CD44), nor plasminogen are essential for collagenolytic activity, endothelial cell invasion, or neovessel formation. Instead, the membrane-anchored MMP, MT1-MMP, confers endothelial cells with the ability to express invasive and tubulogenic activity in a collagen-rich milieu, in vitro or in vivo, where it plays an indispensable role in driving neovessel formation.

Assembly, organization and regulation of cell-surface receptors by lectin–glycan complexes

2015 ◽  
Vol 469 (1) ◽  
pp. 1-16 ◽  
Author(s):  
María T. Elola ◽  
Ada G. Blidner ◽  
Fátima Ferragut ◽  
Candelaria Bracalente ◽  
Gabriel A. Rabinovich
Keyword(s):  
Cell Surface ◽  
Protein Interactions ◽  
Consensus Sequence ◽  
Hierarchical Organization ◽  
Receptor Expression ◽  
Receptor Internalization ◽  
Local Concentration ◽  
Surface Receptors ◽  
Extracellular Milieu ◽  
Galectin 3

Galectins are a family of β-galactoside-binding lectins carrying at least one consensus sequence in the carbohydrate-recognition domain. Properties of glycosylated ligands, such as N- and O-glycan branching, LacNAc (N-acetyl-lactosamine) content and the balance of α2,3- and α2,6-linked sialic acid dramatically influence galectin binding to a preferential set of counter-receptors. The presentation of specific glycans in galectin-binding partners is also critical, as proper orientation and clustering of oligosaccharide ligands on multiple carbohydrate side chains increase the binding avidity of galectins for particular glycosylated receptors. When galectins are released from the cells, they typically concentrate on the cell surface and the local matrix, raising their local concentration. Thus galectins can form their own multimers in the extracellular milieu, which in turn cross-link glycoconjugates on the cell surface generating galectin–glycan complexes that modulate intracellular signalling pathways, thus regulating cellular processes such as apoptosis, proliferation, migration and angiogenesis. Subtle changes in receptor expression, rates of protein synthesis, activities of Golgi enzymes, metabolite concentrations supporting glycan biosynthesis, density of glycans, strength of protein–protein interactions at the plasma membrane and stoichiometry may modify galectin–glycan complexes. Although galectins are key contributors to the formation of these extended glycan complexes leading to promotion of receptor segregation/clustering, and inhibition of receptor internalization by surface retention, when these complexes are disrupted, some galectins, particularly galectin-3 and -4, showed the ability to drive clathrin-independent mechanisms of endocytosis. In the present review, we summarize the data available on the assembly, hierarchical organization and regulation of conspicuous galectin–glycan complexes, and their implications in health and disease.

A Non-Internalizing Anti-CD40 Antibody, CHIR-12.12, Blocks CD40L-Induced Cytokine Production and Mediates Greater ADCC Than Rituximab in Primary CLL Cells.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 2964-2964
Author(s):  
Xia Tong ◽  
Sharon Lea Aukerman ◽  
Karen Lin ◽  
Natasha Aziz ◽  
Cheryl Goldbeck ◽  
...  
Keyword(s):  
Cell Surface ◽  
B Cell ◽  
Cytokine Production ◽  
Ex Vivo ◽  
Lymphocytic Leukemia ◽  
Target Cells ◽  
Adcc Activity ◽  
Effector Cells ◽  
Cells Cultured

Abstract CD40 is expressed on chronic lymphocytic leukemia (CLL) cells, and CD40 activation leads to signaling critical for cell survival and proliferation. We have previously described a novel, fully human IgG1 anti-CD40 antagonistic monoclonal antibody, CHIR-12.12, generated in XenoMouse® mice (Abgenix, Inc.), and have demonstrated that it inhibits normal human B cell proliferation and survival and mediates potent antibody-dependent cellular cytotoxicity (ADCC) against primary CLL and non-Hodgkin’s lymphoma cells. In this study, we examined the ability of CHIR-12.12 to modulate cytokine production by primary CLL cells and compared the ADCC activity of CHIR-12.12 with rituximab against primary CLL cells. Primary CLL cells stimulated with CD40L produced a variety of cytokines, including IL-10, TNF-α , IL-8, GM-CSF, IL-6, MCP-1, and MIP-1β. Addition of CHIR-12.12 to primary CLL cells inhibited CD40L-mediated production of these cytokines. Cytokine production by primary CLL cells cultured with CHIR-12.12 alone in the absence of CD40L did not exceed levels produced by CLL cells cultured in medium. These data suggest that CHIR-12.12 is a potent antagonist for CD40L-mediated cytokine production by primary CLL cells and shows no agonistic activity by itself. We next compared the relative ADCC activity of CHIR-12.12 and rituximab against ex vivo primary CLL cells from 8 patients. CHIR-12.12 exhibited greater ADCC than rituximab against CLL cells from all patients. The average percent of maximum lysis by CHIR-12.12 and rituximab were 49 ± 16% and 31 ± 14%, respectively. CHIR-12.12 was greater than 10-fold more potent than rituximab, as measured by ED50 values (14.1 pM versus 155.5 pM, respectively). Quantitative CD20 and CD40 density on CLL cells and the degree of antibody internalization were investigated as potential reasons for the difference in ADCC activity. The greater ADCC potency and efficacy of CHIR-12.12 was not dependent on a higher density of cell surface CD40 molecules, as there were 1.3 to 14-fold higher numbers of CD20 than CD40 molecules on the cell surface. Antibody internalization studies using primary CLL cells conducted by flow cytometry and confocal microscopy show that upon binding to CD40 at 37°C, CHIR-12.12 remains uniformly distributed on the cell surface, even after 3 hours. In contrast, after binding at 37°C, rituximab is redistributed into caps and internalized. These data suggest that the potent ADCC activity of CHIR-12.12 may be partly related to its ability to remain on the surface of target cells uniformly, allowing optimal interaction with effector cells. Taken together, these results suggest that CHIR-12.12 may be effective at mediating potent ADCC against CLL cells in vivo. CHIR-12.12 is currently in Phase I trials for B-cell malignancies.

Sign in / Sign up

Export Citation Format

Share Document