Astrocytes derived from fetal neural progenitor cells as a novel source for therapeutic adenosine delivery

PurposeIntracerebral delivery of anti-epileptic compounds represents a novel strategy for the treatment of refractory epilepsy. Adenosine is a possible candidate for local delivery based on its proven anti-epileptic effects. Neural stem cells constitute an ideal cell source for intracerebral transplantation and long-term drug delivery. In order to develop a cell-based system for the long-term delivery of adenosine, we isolated neural progenitor cells from adenosine kinase deficient mice (Adk^?/?) and compared their differentiation potential and adenosine release properties with corresponding wild-type cells.MethodsFetal neural progenitor cells were isolated from the brains of Adk^?/? and C57BL/6 mice fetuses and expanded in vitro. Before and after neural differentiation, supernatants were collected and assayed for adenosine release using liquid chromatography–tandem mass spectrometry (LC–MS/MS).ResultsAdk^?/? cells secreted significantly more adenosine compared to wild-type cells at any time point of differentiation. Undifferentiated Adk^?/? cells secreted 137 ± 5 ng adenosine per 10⁵ cells during 24 h in culture, compared to 11 ± 1 ng released from corresponding wild-type cells. Adenosine release was maintained after differentiation as differentiated Adk^?/? cells continued to release significantly more adenosine per 24 h (47 ± 1 ng per 10⁵ cells) compared to wild-type cells (3 ± 0.2 ng per 10⁵ cells).ConclusionsFetal neural progenitor cells isolated from Adk^?/? mice – but not those from C57BL/6 mice – release amounts of adenosine considered to be of therapeutic relevance.     

Ratiometric high-resolution imaging of JC-1 fluorescence reveals the subcellular heterogeneity of astrocytic mitochondria

Using the mitochondrial potential (ΔΨ_m) marker JC-1 (5,5′,6,6′-tetrachloro-1,1′,3,3′-tetraethylbenzimidazolylcarbocyanine iodide) and high-resolution imaging, we functionally analyzed mitochondria in cultured rat hippocampal astrocytes. Ratiometric detection of JC-1 fluorescence identified mitochondria with high and low ΔΨ_m. Mitochondrial density was highest in the perinuclear region, whereas ΔΨ_m tended to be higher in peripheral mitochondria. Spontaneous ΔΨ_m fluctuations, representing episodes of increased energization, appeared in individual mitochondria or synchronized in mitochondrial clusters. They continued upon withdrawal of extracellular Ca²⁺, but were antagonized by dantrolene or 2-aminoethoxydiphenylborate (2-APB). Fluo-3 imaging revealed local cytosolic Ca²⁺ transients with similar kinetics that also were depressed by dantrolene and 2-APB. Massive cellular Ca²⁺ load or metabolic impairment abolished ΔΨ_m fluctuations, occasionally evoking heterogeneous mitochondrial depolarizations. The detected diversity and ΔΨ_m heterogeneity of mitochondria confirms that even in less structurally polarized cells, such as astrocytes, specialized mitochondrial subpopulations coexist. We conclude that ΔΨ_m fluctuations are an indication of mitochondrial viability and are triggered by local Ca²⁺ release from the endoplasmic reticulum. This spatially confined organelle crosstalk contributes to the functional heterogeneity of mitochondria and may serve to adapt the metabolism of glial cells to the activity and metabolic demand of complex neuronal networks. The established ratiometric JC-1 imaging—especially combined with two-photon microscopy—enables quantitative functional analyses of individual mitochondria as well as the comparison of mitochondrial heterogeneity in different preparations and/or treatment conditions.     

Pancreatic Acinar Cell Carcinoma: A Multi-institutional Study

Introduction  The presentation and outcome of patients with acinar cell carcinoma (ACC) of the pancreas compared to the more common ductal cell adenocarcinoma (DCA) may be distinct. This study combines the experience with ACC from multiple academic institutions to better understand its natural history and outcomes. Methods  This study is a multi-institutional retrospective review of patients with ACC. Results  Between 1988 and 2008, 17 patients were identified with pathologically confirmed ACC. Median age at presentation was 59 years. Common presenting symptoms were abdominal pain (60%), back pain (50%), and weight loss (45%). Fifteen patients underwent 16 operations: pancreaticoduodenectomy (nine), distal pancreatectomy (four), and exploratory laparotomy (three). Mean tumor size was 5.3 cm. American Joint Commission on Cancer tumor stages were stage I (two), stage II (eight), stage III (four), and stage IV (three). Overall, 1- and 5-year survival rates were 88% and 50%, respectively. In resected cases (13), 1- and 5-year survival rates were 92% and 53%, respectively. Median survival in resected cases was 61 months. This is in contrast to 1,608 patients with ductal cell adenocarcinoma who underwent resection identified from recent literature reports where the average median survival was only 24 months. There was no discernable difference in the outcomes of patients with ACC between United States and Germany patients. Conclusion  Acinar cell carcinoma of the pancreas is rare and appears to have a presentation and outcome distinct from the more common pancreatic DCA. Based upon these data, the outcome of patients with ACC is superior to that of DCA. Jesus M. Matos and C. Max Schmidt contributed equally to this work. This paper was presented at the Society for Surgery of the Alimentary Tract (SSAT) in San Diego, California in May 2008.     

Polyarteritis nodosa and testicular pain: ultrasonography reveals vasculitis of the testicular artery

SIR, Polyarteritis nodosa (PAN) is a rare disease characterized bynecrotizing vasculitis that may present as an aggressive formassociated with multiorgan involvement [1]. Vast heterogeneityof initial clinical symptoms of PAN can occur and may lead todelayed diagnosis. We present a patient with testicular painas one of the initial clinical symptoms where ultrasonographyrevealed an impressive vasculitis of the testicular artery andreport the clinically protracted and complicated course of thedisease, which has been fully documented by laboratory parameters,photographs, videos and     

Lentiviral RNAi-induced downregulation of adenosine kinase in human mesenchymal stem cell grafts: a novel perspective for seizure control

Cell therapies based on focal delivery of the inhibitory neuromodulator adenosine were previously shown to provide potent seizure suppression in animal models of epilepsy. However, hitherto used therapeutic cells were derived from rodents and thus not suitable for clinical applications. Autologous patient-derived adenosine-releasing cell implants would constitute a major therapeutic advance to avoid both xenotransplantation and immunosuppression. Here we describe a novel approach based on lentiviral RNAi mediated downregulation of adenosine kinase (ADK), the major adenosine-removing enzyme, in human mesenchymal stem cells (hMSCs), which would be compatible with autologous cell grafting in patients. Following lentiviral transduction of hMSCs with anti-ADK miRNA expression cassettes we demonstrate up to 80% downregulation of ADK and a concentration of 8.5 ng adenosine per ml of medium after incubating 10(5) cells for 8 h. hMSCs with a knockdown of ADK or cells expressing a scrambled control sequence were transplanted into hippocampi of mice 1 week prior to the intraamygdaloid injection of kainic acid (KA). While mice with control implants expressing a scrambled miRNA sequence or sham treated control animals were characterized by KA-induced status epilepticus and subsequent CA3 neuronal cell loss, animals with therapeutic ADK knockdown implants displayed a 35% reduction in seizure duration and 65% reduction in CA3 neuronal cell loss, when analyzed 24 h after KA-injection. We conclude that lentiviral expression of anti-ADK miRNA constitutes a versatile tool to generate therapeutically effective adenosine releasing hMSCs, thus representing a model system to generate patient identical autologous adult stem cell grafts.     

Muller cells are living optical fibers in the vertebrate retina

Although biological cells are mostly transparent, they are phase objects that differ in shape and refractive index. Any image that is projected through layers of randomly oriented cells will normally be distorted by refraction, reflection, and scattering. Counterintuitively, the retina of the vertebrate eye is inverted with respect to its optical function and light must pass through several tissue layers before reaching the light-detecting photoreceptor cells. Here we report on the specific optical properties of glial cells present in the retina, which might contribute to optimize this apparently unfavorable situation. We investigated intact retinal tissue and individual Müller cells, which are radial glial cells spanning the entire retinal thickness. Müller cells have an extended funnel shape, a higher refractive index than their surrounding tissue, and are oriented along the direction of light propagation. Transmission and reflection confocal microscopy of retinal tissue in vitro and in vivo showed that these cells provide a low-scattering passage for light from the retinal surface to the photoreceptor cells. Using a modified dual-beam laser trap we could also demonstrate that individual Müller cells act as optical fibers. Furthermore, their parallel array in the retina is reminiscent of fiberoptic plates used for low-distortion image transfer. Thus, Müller cells seem to mediate the image transfer through the vertebrate retina with minimal distortion and low loss. This finding elucidates a fundamental feature of the inverted retina as an optical system and ascribes a new function to glial cells.