Optoacoustic real-time dosimetry for selective retina treatment

The selective retina treatment (SRT) targets retinal diseases associated with disorders in the retinal pigment epithelium (RPE). Due to the ophthalmoscopic invisibility of the laser-induced RPE effects, we investigate a noninvasive optoacoustic real-time dosimetry system. In vitro porcine RPE is irradiated with a Nd:YLF laser (527 nm, 1.7-micros pulse duration, 5 to 40 microJ, 30 pulses, 100-Hz repetition rate). Generated acoustic transients are measured with a piezoelectric transducer. During 27 patient treatments, the acoustic transients are measured with a transducer embedded in an ophthalmic contact lens. After treatment, RPE damage is visualized by fluorescein angiographic leakage. Below the RPE damage threshold, the optoacoustic transients show no pulse-to-pulse fluctuations within a laser pulse train. Above threshold, fluctuations of the individual transients among each other are observed. If optoacoustic pulse-to-pulse fluctuations are present, RPE leakage is observed in fluorescein angiography. In 96% of the irradiated areas, RPE leakage correlated with the optoacoustic defined threshold value. A noninvasive optoacoustic real-time dosimetry for SRT is developed and proved in vitro and during patient treatment. It detects the ophthalmoscopically invisible laser-induced damage of RPE cells and overcomes practical limitations of SRT for use in private practice.     

Receptor-mediated endocytosis of albumin in cultured opossum kidney cells: a model for proximal tubular protein reabsorption

The accumulation of fluorescein(FITC)-labelled bovine albumin was measured against the extracellular-fluid-phase marker FITC-inulin within confluent monolayers of the opossum kidney cell line OK. Fluorescence and electron microscopic pictures show that FITC-albumin is taken up by endocytosis and appears in a vesicular intracellular distribution. The uptake of FITC-albumin was quantified by measuring the cell-adherent fluorescence fluorimetrically. FITC-albumin uptake shows a time- and concentration-dependent saturation kinetics in contrast to the non-saturable FITC-inulin uptake, and exceeds the latter more than tenfold at low concentrations. Half-maximum saturation occurs at 20–30mg/l. Initial FITC-albumin uptake/mg protein is stimulated by cell maturation, being six-to sevenfold higher in the confluent than in the subconfluent state, while FITC-inulin uptake is unchanged. Both an elevation of ambient osmolality to 600–750 mOsm/kg and disruption of the cytoskeleton by cytochalasin B (0.1 mmol/l) reduce initial FITC-albumin uptake by 50%–60% in a non-additive fashion. Albumin endocytosis is reduced both in acidic (pH 5.4) and alkaline (pH 8.4) medium, but does not depend on extracellular sodium, calcium or chloride. High concentrations of fetal calf serum or unlabelled albumin reduce FITC-albumin endocytosis dose-dependently. The present study is the first to investigate both the protein uptake and the fluid-phase endocytosis in a cultured proximal tubular cell line, using these cells as a model systemfor proximal tubular protein reabsorption.Parts of this study have been reported in abstract form (Nieren Hochdruckkr 9:416; J Am Soc Nephrol 1:706)     

Common genetic variants associated with open-angle glaucoma

Open-angle glaucoma (glaucoma) is a major eye disorder characterized by optic disc pathology. Recent genome-wide association studies identified new loci associated with clinically relevant optic disc parameters, such as the optic disc area and vertical cup-disc ratio (VCDR). We examined to what extent these loci are involved in glaucoma. The loci studied include ATOH7, CDC7/TGFBR3 and SALL1 for optic disc area, and CDKN2B, SIX1, SCYL1/LTBP3, CHEK2, ATOH7 and DCLK1 for VCDR. We performed a meta-analysis using data from six independent studies including: the Rotterdam Study (n= 5736), Genetic Research in Isolated Populations combined with Erasmus Rucphen Family study (n= 1750), Amsterdam Glaucoma Study (n= 296) and cohorts from Erlangen and Tübingen (n= 1363), Southampton (n= 702) and deCODE (n= 36 151) resulting in a total of 3161 glaucoma cases and 42 837 controls. Of the eight loci, we found significant evidence (P= 1.41 × 10(-8)) for the association of CDKN2B with glaucoma [odds ratio (OR) for those homozygous for the risk allele: 0.76; 95% confidence interval (CI): 0.70-0.84], for the role of ATOH7 (OR: 1.28; 95% CI: 1.12-1.47) and for SIX1 (OR: 1.20; 95% CI: 1.10-1.31) when adjusting for the number of tested loci. Furthermore, there was a borderline significant association of CDC7/TGFBR3 and SALL1 (both P= 0.04) with glaucoma. In conclusion, we found consistent evidence for three common variants (CDKN2B, ATOH7 and SIX1) significantly associated with glaucoma. These findings may shed new light on the pathophysiological protein pathways leading to glaucoma, and point to pathways involved in the growth and development of the optic nerve.     

Enhanced endothelial cell retention on shear-stressed synthetic vascular grafts precoated with RGD-cross-linked fibrin

Clinical in vitro endothelialization has been shown to increase the patency of synthetic vascular grafts. The shear stress resistance of the cultured autologous endothelium represents a crucial cornerstone of the concept. We investigated whether an enrichment of the precoating matrix with adhesion sites can augment endothelial cell attachment. Adult human saphenous vein endothelial cells (AHSVECs) were seeded confluently ([58 +/- 11] x 10(3) AHSVECs/cm2) onto 10-cm-long ePTFE (expanded polytetrafluorethylene) vascular grafts (n = 24) precoated with commercial clinically approved fibrin gel (Tisseal) containing various concentrations of cross-linked RGD peptide (0.0, 4.0, 8.0, or 16.0 mg of RGD per milliliter of Tisseal fibrinogen component). Endothelialized grafts were postcultivated for 9 days before they were exposed to a pulsatile circulation model mimicking peak physiological shear stress conditions of the femoral artery (12 dyn/cm2; min/max, -60/+28 dyn/cm2). Cell loss after 24 h was quantitatively determined by image analysis of vital stains. Initial 24-h cell loss was 27.2 +/- 1.7% in grafts precoated with the non-RGD-enriched fibrin matrix. In contrast, cell loss was significantly less on fibrin containing 4.0 mg of RGD peptide per milliliter of Tisseal fibrinogen component (13.3 +/- 7.9%; p < 0.05). Cell loss on fibrin containing 8 and 16 mg of RGD per milliliter of Tisseal fibrinogen component was 41.0 +/- 27.4 and 43.0 +/- 23.2% (p > 0.05), respectively. We conclude that low concentrations of RGD peptide cross-linked into commercial fibrin matrices used for clinical in vitro lining of vascular grafts led to significantly increased endothelial cell retention. The failure of higher RGD concentrations to enhance endothelial cell attachment may be explained by competitive binding of endothelial cells to non-cross-linked RGD.     

Hypersusceptibility to vesicular stomatitis virus infection in Dicer1-deficient mice is due to impaired miR24 and miR93 expression

Dicer is essential for plant, Caenorhabditis elegans, and Drosophila antiviral responses because of its role in generating small interfering RNA (siRNA) from viral genomes. We show that because of impaired miRNA production, mice with a variant Dicer1 allele (Dicer1(d/d)) were more susceptible to vesicular stomatitis virus (VSV) infection. We did not detect VSV genome-derived siRNA in wild-type cells or any alteration of interferon-mediated antiviral responses by Dicer1 deficiency. Rather, we found that host miR24 and miR93 could target viral large protein (L protein) and phosphoprotein (P protein) genes, and a lack of miR24 and miR93 was responsible for increased VSV replication in Dicer1(d/d) cells. Our data suggest that host miRNA can play a role in host interactions with viruses.     

Time of transplantation and cell preparation determine neural stem cell survival in a mouse model of Huntington’s disease

Cell replacement therapies for neurodegenerative diseases, using multipotent neural stem cells (NSCs), require above all, a good survival of the graft. In this study, we unilaterally injected quinolinic acid (QA) into the striatum of adult mice and transplanted syngeneic NSCs of enhanced green fluorescent protein-transgenic mice into the lesioned striatum. The injection of QA leads to an excitotoxic lesion with selective cell death of the medium sized spiny neurons, the same cells that are affected in Huntington’s disease. In order to investigate the best timing of transplantation for the survival of donor cells, we transplanted the stem cells at 2, 7 and 14 days after injury. In addition, the influence of graft preparation prior to transplantation, i.e., intact neurospheres versus dissociated cell suspension on graft survival was investigated. By far the best survival was found with the combination of early transplantation (i.e., 2 days after QA-lesion) with the use of neurospheres instead of dissociated cell suspension. This might be due to the different states of host’s astrocytic and microglia activation which we found to be moderate at 2, but pronounced at 7 and 14 days after QA-lesion. We also investigated brain derived neurotrophic factor (BDNF)-expression in the striatum after QA-lesion and found no significant change in BDNF protein-level. We conclude that already the method of graft preparation of NSCs for transplantation, as well as the timing of the transplantation procedure strongly affects the survival of the donor cells when grafted into the QA-lesioned striatum of adult mice.     

Time-course and regional analyses of the physiopathological changes induced after cerebral injection of an amyloid β fragment in rats

Alzheimer's disease (AD) is a neurodegenerative pathology characterized by the presence of senile plaques and neurofibrillary tangles, accompanied by synaptic and neuronal loss. The major component of senile plaques is an amyloid β protein (Aβ) formed by pathological processing of the Aβ precursor protein. We assessed the time-course and regional effects of a single intracerebroventricular injection of aggregated Aβ fragment 25-35 (Aβ(25-35)) in rats. Using a combined biochemical, behavioral, and morphological approach, we analyzed the peptide effects after 1, 2, and 3 weeks in the hippocampus, cortex, amygdala, and hypothalamus. The scrambled Aβ(25-35) peptide was used as negative control. The aggregated forms of Aβ peptides were first characterized using electron microscopy, infrared spectroscopy, and Congo Red staining. Intracerebroventricular injection of Aβ(25-35) decreased body weight, induced short- and long-term memory impairments, increased endocrine stress, cerebral oxidative and cellular stress, neuroinflammation, and neuroprotective reactions, and modified endogenous amyloid processing, with specific time-course and regional responses. Moreover, Aβ(25-35), the presence of which was shown in the different brain structures and over 3 weeks, provoked a rapid glial activation, acetylcholine homeostasis perturbation, and hippocampal morphological alterations. In conclusion, the acute intracerebroventricular Aβ(25-35) injection induced substantial central modifications in rats, highly reminiscent of the human physiopathology, that could contribute to physiological and cognitive deficits observed in AD.     

Biochemical and functional characterization of histone H1-like proteins in procyclicTrypanosoma brucei brucei

Four variants and/or posttranslational modifications of histone H1-like proteins ofTrypanosoma brucei brucei procyclic culture forms were extracted with 0.25N HCl from isolated nuclei and analyzed by two-dimensional gel electrophoresis. The amino acid composition of these proteins, their ability to space nucleosomes regularly and to induce salt-dependent condensation of the chromatin indicated their histone H1 nature. On the other hand, the histone H1-like proteins clearly differed from their higher-eukaryote counterparts by their weak interaction with DNA under low-salt conditions. As a consequence, intact nucleosome filaments were prepared according to a new preparation protocol especially adapted to the unstable chromatin ofT. b. brucei. Our results indicate that the biochemical properties of the histone H1-like proteins contribute to the structural and functional differences between the chromatin of procyclicT. b. brucei and that of higher eukaryotes.