A One–Step, Operator–Independent Method for Isolating Islets of Langerhans from the Porcine Pancreas

Abstract: Large–scale isolation of islets of Langerhans is one of the major obstacles in islet transplantation. Until now, isolation methods relied on enzymatic digestion, the duration of which relies on a decision dictated by the operator's experience. This approach has always hindered development of an automated method. The aim of this study was to develop a one–step method based on complete digestion of the pancreas. The original aspect of the technique (derived from the Ricordi method) is use of the University of Wisconsin (UW) solution in the digestion medium and a continuous flow collagenase processing circuit with local cooling and rewarming to allow tissue digestion to proceed at 37°C while settling of the cell suspension takes place at 4°C. A stopcock system permits the alternate use of two settling chambers so that while one is in the circuit, the other can be removed for cen–trifugation, resuspension of the crude islet preparation in collagenase in free UW solution, and further purification in a density gradient system. Ten experiments were performed, and 545, 750 ± 48, 670 purified pig islets were obtained per totally digested pancreas. Histological studies showed cell integrity. Insulin secretion in response to double glucose stimulation under perfusion conditions demonstrated the functional viability of the isolated islets. In conclusion, this one–step method makes it possible to obtain a high number of viable islets of Langerhans in the absence of any decision by an operator, and it should therefore provide basis for an automated method.     

Überempfindlichkeitsversuche an Bakterien im Infizierten Organismus

Ohne ZusammenfassungSCHNABEL: Dtsch. med. Wochenschr. 1922, Nr. 20; Zentralbl. f. Bakteriol., Parasitenk. u. Infektionskrankh., Abt. I, Orig.,89, 111; Zeitschr. f. Hyg. u. Infektionskrankh.96. 1922.     

Binding of nucleosomes to a cell surface receptor: Redistribution and endocytosis in the presence of lupus antibodies

In the present study, we sought evidence for a surface nucleosome receptor in the fibroblastic cell line CV-1, and questioned whether anti-double-stranded (ds)DNA and/or anti-histone autoantibodies could recognized and influence the fate of cell surface-bound nucleosomes. ¹²⁵I-labeled mononucleosomes were shown to bind to the cell layer in a specific, concentration-dependent and a saturable manner. Scatchard analysis revealed the presence of two binding sites: a high-affinity site with a Kd of ～ 7nM and a low-affinity site (Kd ～ 400 nM) with a high capacity of 9 × 10⁷ sites. Visualization of bound mononucleosomes by fluorescence revealed staining on both the cell surface and the extracellular matrix (ECM). Purified mononucleosome-derived dsDNA (180–200 bp) was found to compete for binding of ¹²⁵I-mononucleosomes on the low-affinity site, to stain exclusively the ECM in immunofluorescence, and to precipitate three specific proteins of 43, 180 and 240 kDa from ¹²⁵-I-labeled cell lysates. Nucleosomes were found to precipitate not only the 180-kDa dsDNA-reactive component, but also a unique protein of 50 kDa, suggesting that this protein is a cell surface receptor for nucleosomes on these fibroblasts. Once bound on the cell surface, mononucleosomes were recognized and secondarily complexed by lupus anti-dsDNA or anti-histone antibodies (i.e. anti-nucleosome antibodies), thus forming immune complexes in situ. The presence of these complexing auto-antibodies was found dramatically to enhance the kinetics of mononucleosome internalization. Following the internalization of the nucleosome-anti-nucleosome complexes by immunofluorescence, we observed the formation of vesicles at the edge of the cells by 5–10 min which moved toward the perinuclear region by 20–30 min. By means of double-fluorescence labeling and proteolytic treatment, these fluorescent vesicles were shown to be in the cytoplasm, suggesting true endocytosis of nucleosome-anti-nucleosome immune complexes. As shown by confocal microscopy, at no stage of this endocytic process was there any indication that coated pits or coated vesicles participated. Co-distribution of the endocytic vesicles with regions rich in actin filaments and inhibition of endocytosis of nucleosome-anti-nucleosome complexes by disruption of the micro-filament network with cytochalasin D suggest a mechanism mediated by the cytoskeleton. Taken together, our data provide evidence for the presence of a surface nucleosome receptor. We also show that anti-dsDNA and anti-histone antibodies can form nucleosome-anti-nucleosome immune complexes in situ at the cell surface, and thus dramatically enhance the kinetics of nucleosome endocytosis.     

A mammary gland adenomyoepithelioma in a C57BL/6 mouse

Mammary gland adenomyoepitheliomas are benign complex mammary gland tumors composed of neoplastic cells of epithelial and myoepithelial origins, described in many species (humans, dogs, cats, rats) and rarely in mice. We report here an adenomyoepithelioma in a C57BL/6 female mouse. Histologically, tubes and cords formed by neoplastic epithelial cells were separated by bundles of neoplastic myoepithelial cells in a clear and partially mucinous matrix. The tumor displayed characteristics of a benign neoplastic proliferation with a compressive growth pattern, and moderate cellular pleomorphism and mitotic index. At immunohistochemistry, the epithelial cells were strongly cytokeratin positive; the myoepithelial cells were weakly cytokeratin positive and strongly smooth muscle actin positive. This is to our knowledge, the first report of a mammary gland adenomyoepithelioma in a C57BL/6 mouse.     

Radial gradient-based segmentation of mammographic microcalcifications: observer evaluation and effect on CAD performance

Precise segmentation of microcalcifications is essential in the development of accurate mammographic computer-aided diagnosis (CAD) schemes. We have designed a radial gradient-based segmentation method for microcalcifications, and compared it to both the region-growing segmentation method currently used in our CAD scheme and to the watershed segmentation method. Two observer studies were conducted to subjectively evaluate the proposed segmentation method. The first study (A) required observers to rate the segmentation accuracy on a 100-point scale. The second observer evaluation (B) was a preference study in which observers selected their preferred method from three displayed segmentation methods. In study A, the observers gave an average accuracy rating of 88 for the radial gradient-based and 50 for the region-growing segmentation method. In study B, the two observers selected the proposed method 56% and 62% of the time. We also investigated the effect of the proposed segmentation method on the performance of computerized classification scheme in differentiating malignant from benign clustered microcalcifications. The performances of the classification scheme using a linear discriminant analysis (LDA) or a Bayesian artificial neural network classifier both showed statistically significant improvements when using the proposed segmentation method. The areas under the receiver-operating characteristic curves for case-based performance when using the LDA classifier were 0.86 with the proposed segmentation method, 0.80 with the region-growing method, and 0.83 with the watershed method.     

Latent inhibition of conditioned odor potentiation of startle: a developmental analysis

We conducted a two-part study of age and latent inhibition in the rat. In the first part of the study, rats given odor-shock pairings at 23 or 75 days of age exhibited a potentiated startle response in the presence of the odor the following day. This effect did not occur in rats trained at 16 or 20 days of age. Odor pre-exposure on the day prior to conditioning markedly reduced the odor potentiation of startle effect in 23- and 75-day-old rats but had no effect in 16 and 20-day-olds. In the second part of the study, rats were pre-exposed to the odor at 16 or 20 days of age and then conditioned at 23 days of age. When tested the day after conditioning, these pre-exposed rats exhibited a disruption in the odor potentiation of startle effect. We compare our results with other studies of latent inhibition, and with recent studies on whether conditioned responses are appropriate to the animal's age at training or their age at test.     

Relationship among pulmonary function, bronchial reactivity, and exhaled nitric oxide in a large group of asthmatic patients.

BACKGROUND: Bronchial reactivity and exhaled nitric oxide (eNO) are not often used to monitor control and severity of asthma in clinical practice. OBJECTIVE: To evaluate the relationship among different physiologic measures (pulmonary function, nonspecific bronchial reactivity, and eNO) in asthmatic patients. METHODS: Cross-sectional, hospital-based study conducted in patients with varied asthma severity. RESULTS: A total of 392 patients participated in the study. There was no difference in eNO levels between patients taking inhaled corticosteroids (ICS group) and patients not receiving inhaled corticosteroids (NICS group). However, the percentage of predicted forced expiratory volume in 1 second (FEV1) and the provocative dose of methacholine causing a 20% decrease in FEV1 were significantly lower in the ICS group compared with the NICS group (mean, 83.2%; 95% confidence interval [CI], 80.4%-86.0%; vs mean, 94.1%; 95% CI, 91.1%-97.1%; P = .001; and geometric mean, 0.32 mg; 95% CI, 0.23-0.45 mg; vs geometric mean, 0.58 mg; 95% CI, 0.42-0.81 mg; P = .01; respectively). Patients with more severe bronchial hyperresponsiveness had a lower percentage of predicted FEV1 values (P < .001) and levels of eNO were significantly increased with increasing bronchial hyperresponsiveness (P < .001). There was no relationship between the percentage of predicted FEV1 and eNO. Atopic patients had significantly higher eNO levels than nonatopic patients (geometric mean, 11.21 ppb; 95% CI, 10.07-12.49 ppb; vs geometric mean, 7.76 ppb; 95% CI, 6.11-9.85 ppb; P = .006; respectively). CONCLUSIONS: eNO values are not related to the degree of airway obstruction but are related to airway reactivity and atopic status independent of inhaled corticosteroid use. Higher values of eNO are seen with increased airway reactivity.     

Specific pattern of ionic channel gene expression associated with pacemaker activity in the mouse heart

Even though sequencing of the mammalian genome has led to the discovery of a large number of ionic channel genes, identification of the molecular determinants of cellular electrical properties in different regions of the heart has been rarely obtained. We developed a high-throughput approach capable of simultaneously assessing the expression pattern of ionic channel repertoires from different regions of the mouse heart. By using large-scale real-time RT-PCR, we have profiled 71 channels and related genes in the sinoatrial node (SAN), atrioventricular node (AVN), the atria (A) and ventricles (V). Hearts from 30 adult male C57BL/6 mice were microdissected and RNA was isolated from six pools of five mice each. TaqMan data were analysed using the threshold cycle ( C _t) relative quantification method. Cross-contamination of each region was checked with expression of the atrial and ventricular myosin light chains. Two-way hierarchical clustering analysis of the 71 genes successfully classified the six pools from the four distinct regions. In comparison with the A, the SAN and AVN were characterized by higher expression of Navβ1, Navβ3, Cav1.3, Cav3.1 and Cavα2δ2, and lower expression of Kv4.2, Cx40, Cx43 and Kir3.1. In addition, the SAN was characterized by higher expression of HCN1 and HCN4, and lower expression of RYR2, Kir6.2, Cavβ2 and Cavγ4. The AVN was characterized by higher expression of Nav1.1, Nav1.7, Kv1.6, Kvβ1, MinK and Cavγ7. Other gene expression profiles discriminate between the ventricular and the atrial myocardium. The present study provides the first genome-scale regional ionic channel expression profile in the mouse heart.