Renal carcinoma CD105-/CD44- cells display stem-like properties in vitro and form aggressive tumors in vivo.

Clear cell renal cell carcinoma (ccRCC) is the most common kidney cancer. Prognosis for ccRCC is generally poor since it is largely resistant to chemo- and radiotherapy. Many studies suggested that cancer stem cells/tumor initiating cells (CSCs/TICs) are responsible for development of tumor, disease progression, aggressiveness, metastasis and drug resistance. However, tumorigenic potential of CSCs/TICs isolated from established RCC cell lines - basic ccRCC research model - has never been investigated in vivo. CD105+, CD105-, CD44+ and CD44- as well as CD44-/CD105- CD44+/CD105+ and CD44-/CD105+ cells were isolated from Caki-1 RCC cell line, confirming coexistence of multiple subpopulations of stem-related phenotype in stable cell line. Sorted cells were injected subcutaneously into NOD SCID mice and tumor growth was monitored with MRI and PET/CT. Tumor growth was observed after implantation of CD105+, CD44+, CD44-, CD44-/CD105+ and CD44-/CD105- but not CD105- or CD44+/CD105+. Implantation of CD44-/CD105- cells induced tumors that were characterized by longer T1 and distinct metabolic pattern than other tumors. All the tumors were characterized by low uptake of [18F]FDG. CD105+ and CD44- tumors expresses Nanog and Oct-4, while CD44- tumors additionally expressed endothelial cell marker - CD31.

SOD1/Rag2 Mice with Low Copy Number of SOD1 Gene as a New Long-Living Immunodeficient Model of ALS.

The most recent research concerning amyotrophic lateral sclerosis (ALS) emphasizes the role of glia in disease development. Thus, one can suspect that the effective therapeutic strategy in treatment of ALS would be replacement of defective glia. One of the basic problems with human glial progenitors (hGRPs) replacement strategies is the time needed for the cells to become fully functional in vivo. The lifespan of most popular high copy number SOD1 mutant mice might be too short to acknowledge benefits of transplanted cells. We focused on developing immunodeficient rag2-/- model of ALS with lower number of transgene copies and longer lifespan. The obtained hSOD1/rag2 double mutant mice have been characterized. QPCR analysis revealed that copy number of hSOD1 transgene varied in our colony (4-8 copies). The difference in transgene copy number may be translated to significant impact on the lifespan. The death of long- and short-living hSOD1/rag2 mice is preceded by muscular weakness as early as one month before death. Importantly, based on magnetic resonance imaging we identified that mutant mice demonstrated abnormalities within the medullar motor nuclei. To conclude, we developed long-living double mutant hSOD1/rag2 mice, which could be a promising model for testing therapeutic utility of human stem cells.

An in vivo evaluation of Brilliant Blue G in animals and humans.

BACKGROUND/AIMS: To evaluate the retinal toxicity of Brilliant Blue G (BBG) following intravitreal injection in rat eyes and examine the biocompatibility and the staining properties in humans. METHODS: BBG was injected into the 11 rat eyes to evaluate toxic effects with balanced salt solution (BSS) serving as control. Retinal toxicity was assessed by retinal ganglion cell (RGC) counts and by light microscopy 7 days later. In addition, BBG was applied during vitrectomy for macular hole (MH) (n = 15) or epiretinal membranes (ERM) (n = 3) in a prospective, non-comparative consecutive series of patients. Before and after surgery, all patients underwent a complete clinical examination including measurement of best corrected visual acuity (VA) and intraocular pressure, perimetry, fundus photography and optical coherence tomography. Patients were seen 1 day before surgery and then in approximately four weeks intervals. RESULTS: No significant reduction in RGC numbers and no morphological alterations were noted. A sufficient staining of the internal limiting membrane (ILM) was seen in patients with MH, while the staining pattern in ERM cases was patchy, indicating that parts of the ILM were peeled off along with the ERM in a variable extent. All MHs could be closed successfully. VA improved in 10 eyes (56%; 8/15 MH patients, 2/3 ERM patients), was unchanged in four eyes (22%; all MH patients) and was reduced in four eyes (22%; 3/15 MH, 1/3 ERM). No toxic effects attributable to the dye were noted during patient follow-up. The ultrastructure of tissue harvested during surgery was unremarkable. CONCLUSION: Brilliant Blue provides a sufficient and selective staining of the ILM. No retinal toxicity or adverse effects related to the dye were observed in animal and human studies. The long-term safety of this novel dye will have to be evaluated in larger patient series and a longer follow-up.

The photodynamic effect of Victoria blue BO on peripheral blood mononuclear and leukemic cells.

The photodynamic effect of Victoria blue BO (VB-BO) and photoirradiation on peripheral blood mononuclear cells was studied. The cells were preincubated with VB-BO followed by photoirradiation and overnight culture. The highest percentage of dead cells (propidium iodide assay in flow cytometry) was seen in the monocyte population. The lymphocytes showed a lower sensitivity to VB-BO photodynamic action than the monocytes (12% vs 80% of PI-positive cells). The effect of VB-BO and phototreatment on lymphocyte function was studied using a mitogen-induced proliferation assay. A decrease of mitogen response was observed. The VB-BO and photoirradiation were also used on leukemic cells. The leukemic cells from acute myeloid leukemia and B precursors leukemia were sensitive to VB-BO photodynamic action. The high VB-BO sensitivity of monocytes and leukemic cells (myeloid and lymphoid B derived) suggests possible application of VB-BO for selective depletion of monocytes or sensitive leukemic cells.

Efficient photodynamic action of Victoria blue BO against the human leukemic cell lines K-562 and TF-1.

Photodynamic induced cytotoxicity by Victoria blue BO (VB-BO), merocyanine 540 (MC540), Nile blue A (NB) and 4-tetrasulfonatophenyl-porphyrin (4-TSPP) has been studied on two human leukemic cell lines: K-562 and TF-1. Cells were incubated with dyes and irradiated with different doses of white light. Cell survival was assessed by propidium iodide (PI) staining using flow cytometry analysis. Concentrations of 5 x 10(-8) M VB-BO were found to kill 75% of cells, and a concentration of 1 x 10(-7) M induced more than 99% of cell killing. To obtain the same cytotoxic level, the presence of 2.6 x 10(-5) M of MC540 during irradiation was needed. Under the conditions used, NB was ineffective as a photosensitizer, although uptake studies showed that this dye was taken by the cells in much greater amounts than any other studied dye. Cell cycle distribution of TF-1 cells, surviving MC540 or VB-BO photosensitization has been studied by flow cytometry analysis after staining with Hoechst 33342 and PI. It was found that cells in G1 phase were slightly more resistant toward MC540- and VB-BO-mediated photosensitization than cells in other phases of the cell cycle.