Functional characterization of equine dendritic cells propagated ex vivo using recombinant human GM-CSF and recombinant equine IL-4

AbstractVolume accommodation occurs via a novel mechanism involving interstitial cells in detrusor muscles. The interstitial cells in the bladder are PDGFRα+, and they restrain the excitability of smooth muscle at low levels and prevents the development of transient contractions (TCs). A common clinical manifestation of spinal cord injury (SCI)-induced bladder dysfunction is detrusor overactivity (DO). Although a myogenic origin of DO after SCI has been suggested, a mechanism for development of SCI-induced DO has not been determined. In this study we hypothesized that SCI-induced DO is related to loss of function in the regulatory mechanism provided by PDGFRα+ cells. Our results showed that transcriptional expression of Pdgfra and Kcnn3 was decreased after SCI. Proteins encoded by these genes also decreased after SCI, and a reduction in PDGFRα+ cell density was also documented. Loss of PDGFRα+ cells was due to apoptosis. TCs in ex vivo bladders during filling increased dramatically after SCI, and this was related to the loss of regulation provided by SK channels, as we observed decreased sensitivity to apamin. These findings show that damage to the mechanism restraining muscle contraction during bladder filling that is provided by PDGFRα+ cells is causative in the development of DO after SCI.

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Functional Characterization of Lamina X Neurons in ex-Vivo Spinal Cord Preparation

Frontiers in Cellular Neuroscience ◽

10.3389/fncel.2017.00342 ◽

2017 ◽

Vol 11 ◽

Cited By ~ 5

Author(s):

Volodymyr Krotov ◽

Anastasia Tokhtamysh ◽

Olga Kopach ◽

Andrew Dromaretsky ◽

Yevhenii Sheremet ◽

...

Keyword(s):

Spinal Cord ◽

Ex Vivo ◽

Functional Characterization

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Immunological Responses in CLL Patients Vaccinated with Autologous Dendritic Cells Loaded with Apoptotic Bodies (Apo-DC).

Blood ◽

10.1182/blood.v110.11.2061.2061 ◽

2007 ◽

Vol 110 (11) ◽

pp. 2061-2061

Author(s):

Aniruddha Choudhury ◽

Marzia Palma ◽

Lotta Hansson ◽

Karin Widen ◽

Ingrid Eriksson ◽

...

Keyword(s):

Clinical Trial ◽

Dendritic Cells ◽

T Cell ◽

Clinical Efficacy ◽

Ex Vivo ◽

Vaccine Dose ◽

Low Grade ◽

Apoptotic Bodies ◽

High Count ◽

Gm Csf

Abstract We have previously established the suitability of dendritic cells that have endocytosed apoptotic B-CLL cells (Apo-DC) as an antigen presentation platform to stimulate autologous antileukemic immune responses. The safety as well as immunological and clinical efficacy of Apo-DC vaccination was tested in a phase I/II clinical trial. CLL patients (Rai stage 0–2) who did not require concurrent therapy were leukapheresed and CD14+ monocytes as well as CD19+ B-CLL cells were purified immunomagnetically. Dendritic cells were generated ex vivo with GM-CSF and IL-4. Apoptotic bodies were obtained by irradiating B-CLL cells and overnight culture. DC were allowed to endocytose the apoptotic bodies and further matured with TNFα. The first cohort of 5 patients received 4 doses of the vaccine 2 weeks apart and a 5th dose at week 14. Each vaccine dose comprised of 107 autologous Apo-DC. The second cohort of 5 patients received the vaccine in a similar schedule with the addition of 75 μg of GM-CSF (Leukine™, Berlex Laboratories, Richmond VA) with every vaccine administration. Patients are scheduled to be monitored for a minimum of 52 weeks which has been completed for all of the patients in cohort 1 and is ongoing for cohort 2 The vaccine was well tolerated and occasional, low-grade toxicity was associated with the administration of GM-CSF. 4/5 patients in cohort 1 and 3/5 patients in cohort 2 responded immunologically in terms of increase in T cell proliferation or γ-IFN ELISPOT against autologous leukemic targets compared to prevaccination levels. Analysis of T cell responses at week 8 and week 16 following vaccination did not demonstrate an obvious augmentation of immunity by GM-CSF. One patient in cohort 1 demonstrated a transient decrease in WBC counts but no significant clinical responses have been noted thus far. Our results demonstrate that it is feasible to enrich monocyte precursors and generate Apo-DC for vaccinating patients with high count B-CLL. No significant toxicity was associated with this approach to vaccination therapy. The clinical efficacy of Apo-DC vaccination in CLL remains to be determined at the termination of the clinical trial.

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VHL-mutant renal cell carcinomas contain cancer cells with mesenchymal phenotypes.

Journal of Clinical Oncology ◽

10.1200/jco.2013.31.15_suppl.4568 ◽

2013 ◽

Vol 31 (15_suppl) ◽

pp. 4568-4568 ◽

Cited By ~ 2

Author(s):

Craig Gedye ◽

Danylo Sirskyj ◽

Nazleen Carol Lobo ◽

Andrew Evans ◽

Neil Eric Fleshner ◽

...

Keyword(s):

Stem Cells ◽

Cancer Stem Cells ◽

Cancer Cells ◽

Tumor Cells ◽

Renal Cell ◽

Clear Cell ◽

Ex Vivo ◽

Functional Characterization ◽

Mutant Cells

4568 Background: To study “cancer stem cells” it is imperative to account for all stromal cell populations within the tumour. The existence of “cancer stem cells” in clear cell renal cell carcinoma (ccRCC) has not been examined in ex vivo patient samples. Methods: We established a multiplex flow cytometry (FC) antibody panel in ccRCC, which reliably identified stromal lineages including CD45+ immune, CD31+/CD144+ endothelial and fibroblast-marker-positive subpopulations, thus allowing isolation of "lineage-negative" tumor cells. To verify the identity of tumour-derived populations as either cancer cells or normal stromal cells, we took advantage of the fact that mutations in VHL occur early during ccRCC tumorigenesis and are found in two-thirds of patients. Results: We sequenced 18 patient tumor samples, 12 of which had VHL exome mutations. Targeted re-sequencing of FC sorted subpopulations from these patients’ samples revealed that while CD45+ immune cells and CD31+/CD144+ endothelial cells were genetically normal, a population of VHL-mutant fibroblast-marker positive cells was consistently identified in every patient’s tumour. Immunohistochemistry showed that fibroblast marker-positive VHL-mutant cells do not have the large “clear cell” morphology typical of the majority of the cancer cells in these tumours. When purified and cultured, these fibroblast marker-positive VHL-mutant cells proliferate extensively under mesenchymal culture conditions, but displayed different morphologies to lineage-negative VHL-mutant tumor cells. Functional characterization of these FC sorted cell subpopulations is ongoing, including proliferation, migration, invasion, differentiation and treatment resistance. Conclusions: The phenotype and preliminary functional characterization of these VHL-mutant fibroblast-marker positive cells suggests a mesenchymal differentiation program in ccRCC, with implications for the ontogeny, biology and clinical management of VHL-mutant renal cancer.

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Identification, Structural, and Functional Characterization of a New Early Gene (6A3-5, 7 kb): Implication in the Proliferation and Differentiation of Smooth Muscle Cells

Journal of Biomedicine and Biotechnology ◽

10.1155/jbb.2005.254 ◽

2005 ◽

Vol 2005 (3) ◽

pp. 254-270 ◽

Cited By ~ 1

Author(s):

Kazem Zibara ◽

Gwenaële Garin ◽

John L. McGregor

Keyword(s):

Smooth Muscle ◽

Smooth Muscle Cells ◽

Ex Vivo ◽

Functional Characterization ◽

Muscle Cells ◽

Protein Band ◽

Early Gene ◽

New Family ◽

Transcription Profiles

Arterial smooth muscle cells (SMCs) play a major role in atherosclerosis and restenosis. Differential display was used to compare transcription profiles of synthetic SMCs to proliferating rat cultured SMC line. An isolated cDNA band (6A3-5) was shown by northern (7 kb) to be upregulated in the proliferating cell line. A rat tissue northern showed differential expression of this gene in different tissues. Using 5’ RACE and screening of a rat brain library, part of the cDNA was cloned and sequenced (5.4 kb). Sequence searches showed important similarities with a new family of transcription factors, bearing ARID motifs. A polyclonal antibody was raised and showed a protein band of 175 kd, which is localized intracellularly. We also showed that 6A3-5 is upregulated in dedifferentiated SMC (P9) in comparison to contractile SMC ex vivo (P0). This work describes cloning, structural, and functional characterization of a new early gene involved in SMC phenotype modulation.

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