Growth pattern of experimental glioblastoma.

Glioblastoma multiforme (GBM) is an aggressive primary brain malignancy with a very poor prognosis. Researchers employ animal models to develop potential therapies. It is important that these models have clinical relevance. This means that old models, propagated for decades in cultures, should be questioned. Parameters to be evaluated include whether animals are immune competent or not, the infiltrative growth pattern of the tumor, tumor volume resulting in symptoms and growth rate. We here describe the growth pattern of an experimental glioblastoma model in detail with GFP positive glioblastoma cells in fully immune competent animals and study tumor growth rate and tumor mass as a function of time from inoculation. We were able to correlate findings made with classical immunohistochemistry and MR findings. The tumor growth rate was fitted by a Gompertz function. The model predicted the time until onset of symptoms for 5000 inoculated cells to 18.7±0.4 days, and the tumor mass at days 10 and 14, which are commonly used as the start of treatment in therapeutic studies, were 5.97±0.62 mg and 29.1±3.0 mg, respectively. We want to raise the question regarding the clinical relevance of the outline of glioblastoma experiments, where treatment is often initiated at a very early stage. The approach presented here could potentially be modified to gain information also from other tumor models.

Zebularine induces long-term survival of pancreatic islet allotransplants in streptozotocin treated diabetic rats.

BACKGROUND: Coping with the immune rejection of allotransplants or autologous cells in patients with an active sensitization towards their autoantigens and autoimmunity presently necessitates life-long immune suppressive therapy acting on the immune system as a whole, which makes the patients vulnerable to infections and increases their risk of developing cancer. New technologies to induce antigen selective long-lasting immunosuppression or immune tolerance are therefore much needed. METHODOLOGY/PRINCIPAL FINDINGS: The DNA demethylating agent Zebularine, previously demonstrated to induce expression of the genes for the immunosuppressive enzymes indolamine-2,3-deoxygenase-1 (IDO1) and kynureninase of the kynurenine pathway, is tested for capacity to suppress rejection of allotransplants. Allogeneic pancreatic islets from Lewis rats were transplanted under the kidney capsule of Fischer rats previously made diabetic by a streptozotocin injection (40 mg/kg). One group was treated with Zebularine (225 mg/kg) daily for 14 days from day 6 or 8 after transplantation, and a control group received no further treatment. Survival of the transplants was monitored by blood sugar measurements. Rats, normoglycemic for 90 days after allografting, were subjected to transplant removal by nephrectomy to confirm whether normoglycemia was indeed due to a surviving insulin producing transplant, or alternatively was a result of recovery of pancreatic insulin production in some toxin-treated rats. Of 9 Zebularine treated rats, 4 were still normoglycemic after 90 days and became hyperglycemic after nephrectomy. The mean length of normoglycemia in the Zebularine group was 67±8 days as compared to 14±3 days in 9 controls. Seven rats (2 controls and 5 Zebularine treated) were normoglycemic at 90 days due to pancreatic recovery as demonstrated by failure of nephrectomy to induce hyperglycemia. CONCLUSIONS/SIGNIFICANCE: Zebularine treatment in vivo induces a long-lasting suppression of the immune destruction of allogeneic pancreatic islets resulting in protection of allograft function for more than 10 weeks after end of treatment.

Detection of cell cycle- and differentiation stage-dependent human telomerase reverse transcriptase expression in single living cancer cells.

Elevated telomerase activity is an important molecular signature of cancer cells and primitive cells in regenerative tissues. However, isolation of single living cells with endogenous telomerase activity has not yet been possible. Here, we developed adenovirus serotype 35 tropism-based vectors encoding destabilized enhanced green fluorescence protein with a half-life of 2 h (d2EGFP) driven by the human telomerase reverse transcriptase (hTERT) promoter. As assessed in telomerase-positive or -negative cell lines, the d2EGFP expression positively correlated with hTERT transcript content and telomerase activity. In retinoic acid-induced differentiating HL-60 cells, the d2EGFP expression is diminished in the same manner as the hTERT expression. Individual cells from HeLa and HL-60 cell lines exhibited heterogeneous d2EGFP expression, which was cell cycle dependent, as the sorted d2EGFP+ HL-60 cells contained twice as many cells in S/G2/M phase of the cell cycle compared with the d2EGFP- HL-60 cells. However, both cell populations exhibited the same proliferation and regeneration capacities. Heterogeneous d2EGFP expression was also detected in xenograft glioblastoma multiforme cells with tumor formation capacity. Thus, d2EGFP expression reported cell cycle- and differentiation stage-dependent hTERT expression. Our study facilitates isolation and characterization of single living cells with telomerase activity.

A sequential fluorescence method for neurotransmitter-specific retrograde tracing in the central nervous system of the rat; utilizing True Blue and immunohistochemistry in combination with computer-assisted photography.

Aiming to map the distribution of spinally projecting, hypothalamic neurons containing neuronal nitric oxide synthase (nNOS), True Blue (TB) is injected into the rat spinal cord. After survival times of 7-14 days the animals are anaesthetized and perfused transcardially with a solution containing paraformaldehyde and sucrose. After dissection, the injection site is further fixed for 4-8 h, cut in a cryostat, and documented by computer-assisted digital photography. The brain region of interest is fixed for 4 h, rinsed in phosphate buffer for 48 h, sectioned, and photographically documented utilizing filter settings for visualization of TB. The brain sections are then immunohistochemically processed using a primary antibody against nNOS and a Texas Red (TR)-labelled secondary antibody and once again photographically documented, now using filter settings for visualization of TB and TR, respectively. Utilizing the Photoshop program, the TB containing cells can then be exactly aligned and the presence of TB and/or TR fluorescence in the same cell bodies are evaluated. This method for neurotransmitter-specific retrograde tracing derives its high sensitivity from the optimization of fixation/rinsing parameters, the use of appropriate fluorophores, and sequential digital microphotography.