An integrative correlation of myopathology, phenotype and genotype in late onset Pompe disease.

AIMS: Pompe disease is caused by pathogenic mutations in the alpha 1,4-glucosidase (GAA) gene and in patients with late onset Pome disease (LOPD), genotype-phenotype correlations are unpredictable. Skeletal muscle pathology includes glycogen accumulation and altered autophagy of various degrees. A correlation of the muscle morphology with clinical features and the genetic background in GAA may contribute to the understanding of the phenotypic variability. METHODS: Muscle biopsies taken before enzyme replacement therapy were analysed from 53 patients with LOPD. On resin sections, glycogen accumulation, fibrosis, autophagic vacuoles and the degree of muscle damage (morphology-score) were analysed and the results were compared with clinical findings. Additional autophagy markers microtubule-associated protein 1A/1B-light chain 3, p62 and Bcl2-associated athanogene 3 were analysed on cryosections from 22 LOPD biopsies. RESULTS: The myopathology showed a high variability with, in most patients, a moderate glycogen accumulation and a low morphology-score. High morphology-scores were associated with increased fibrosis and autophagy highlighting the role of autophagy in severe stages of skeletal muscle damage. The morphology-score did not correlate with the patient's age at biopsy, disease duration, nor with the residual GAA enzyme activity or creatine-kinase levels. In 37 patients with LOPD, genetic analysis identified the most frequent mutation, c.-32-13T>G, in 95%, most commonly in combination with c.525delT (19%). No significant correlation was found between the different GAA genotypes and muscle morphology type. CONCLUSIONS: Muscle morphology in LOPD patients shows a high variability with, in most cases, moderate pathology. Increased pathology is associated with more fibrosis and autophagy.

Spinocerebellar ataxia type 17 associated with an expansion of 42 glutamine residues in TATA-box binding protein gene.

BACKGROUND: Spinocerebellar ataxia type 17 (SCA17) is caused by abnormal expansions of CAG/CAA trinucleotides within the TATA-box binding protein gene (TBP). The currently accepted critical threshold of abnormal expansions is ≥43. OBJECTIVE: To investigate the minimal CAG/CAA expansion within the TBP in SCA17. RESULTS: 285 patients with autosomal-dominant ataxia were examined, and abnormal or borderline expansions of CAG/CAA within TBP in eight cases were found. Of those, four patients from three families had exactly 42 CAG/CAA trinucleotides, that is, one codon less than the currently accepted critical threshold of 43. The four patients presented with a relatively benign phenotype. All had dysdiadochokinesia and dysarthria. Mild gait ataxia was observed in three of the four patients. CONCLUSION: The reference definition of at least 43 CAG/CAA codons for pathological SCA17 alleles should be lowered to 42.

Three-dimensional holographic imaging of living tissue using a highly sensitive photorefractive polymer device.

Photorefractive materials are dynamic holographic storage media that are highly sensitive to coherent light fields and relatively insensitive to a uniform light background. This can be exploited to effectively separate ballistic light from multiply-scattered light when imaging through turbid media. We developed a highly sensitive photorefractive polymer composite and incorporated it into a holographic optical coherence imaging system. This approach combines the advantages of coherence-domain imaging with the benefits of holography to form a high-speed wide-field imaging technique. By using coherence-gated holography, image-bearing ballistic light can be captured in real-time without computed tomography. We analyzed the implications of Fourier-domain and image-domain holography on the field of view and image resolution for a transmission recording geometry, and demonstrate holographic depth-resolved imaging of tumor spheroids with 12 microm axial and 10 microm lateral resolution, achieving a data acquisition speed of 8 x 10(5) voxels/s.

The critical size of a defect in the glenoid causing anterior instability of the shoulder after a Bankart repair, under physiological joint loading.

AIMS: Patients with recurrent anterior dislocation of the shoulder commonly have an anterior osseous defect of the glenoid. Once the defect reaches a critical size, stability may be restored by bone grafting. The critical size of this defect under non-physiological loading conditions has previously been identified as 20% of the length of the glenoid. As the stability of the shoulder is load-dependent, with higher joint forces leading to a loss of stability, the aim of this study was to determine the critical size of an osseous defect that leads to further anterior instability of the shoulder under physiological loading despite a Bankart repair. PATIENTS AND METHODS: Two finite element (FE) models were used to determine the risk of dislocation of the shoulder during 30 activities of daily living (ADLs) for the intact glenoid and after creating anterior osseous defects of increasing magnitudes. A Bankart repair was simulated for each size of defect, and the shoulder was tested under loading conditions that replicate in vivo forces during these ADLs. The critical size of a defect was defined as the smallest osseous defect that leads to dislocation. RESULTS: The FE models showed a high risk of dislocation during ADLs after a Bankart repair for anterior defects corresponding to 16% of the length of the glenoid. CONCLUSION: This computational study suggests that bone grafting should be undertaken for an anterior osseous defect in the glenoid of more than 16% of its length rather than a solely soft-tissue procedure, in order to optimize stability by restoring the concavity of the glenoid.

Leukotrienes as mediators in ischemia-reperfusion injury in a microcirculation model in the hamster.

Leukotriene (LT)B4 promotes leukocyte chemotaxis and adhesion to the endothelium of postcapillary venules. The cysteinyl leukotrienes, LTC4, LTD4, and LTE4, elicit macromolecular leakage from this vessel segment. Both leukocyte adhesion to the endothelium and macromolecular leakage from postcapillary venules hallmark the microcirculatory failure after ischemia-reperfusion, suggesting a role of leukotrienes as mediators of ischemia-reperfusion injury. Using the dorsal skinfold chamber model for intravital fluorescence microscopy of the microcirculation in striated muscle in awake hamsters and sequential RP-HPLC and RIA for leukotrienes, we demonstrate in this study that (a) the leukotrienes (LT)B4 and LTD4 elicit leukocyte/endothelium interaction and macromolecular leakage from postcapillary venules, respectively, that (b) leukotrienes accumulate in the tissue after ischemia and reperfusion, and that (c) selective inhibition of leukotriene biosynthesis (by MK-886) prevents both postischemic leukotriene accumulation and the microcirculatory changes after ischemia-reperfusion, while blocking of LTD4/E4 receptors (by MK-571) inhibits postischemic macromolecular leakage. These results demonstrate a key role of leukotrienes in ischemia-reperfusion injury in striated muscle in vivo.

Role of leukotrienes in leukocyte adhesion following systemic administration of oxidatively modified human low density lipoprotein in hamsters.

In vitro studies indicate that oxidatively modified low density lipoprotein (oxLDL) promotes leukocyte adhesion to the vascular endothelium, a constant feature of early atherogenesis. Using intravital fluorescence microscopy in the dorsal skinfold chamber model in awake Syrian golden hamsters, we studied whether (a) oxLDL elicits leukocyte/endothelium interaction in vivo, and whether (b) leukotrienes play a mediator role in this event. Leukocyte/endothelium interaction was assessed in the time course after intravenous injection of native human LDL (4 mg/kg body wt) and of oxLDL (7.5 microM Cu++, 6 h, 37 degrees C) into control hamsters and into hamsters, pretreated with the selective leukotriene biosynthesis inhibitor MK-886 (20 mumol/kg, i.v.). While no effect was seen after injection of native LDL, oxLDL elicited an immediate induction of leukocyte adhesion to the endothelium of arterioles and postcapillary venules. Total and differential leukocyte counts suggest that all leukocyte subsets were likewise affected by oxLDL with no specific preference for monocytes. Stimulation of leukocyte adhesion was entirely prevented in inhibitor-treated animals, suggesting the important mediator role of leukotrienes in oxLDL-induced leukocyte/endothelium interaction.

Reversible ischaemia after raising a radial forearm flap with ulceration of three fingers in a cigarette smoker.

The radial forearm flap is a standard method for the reconstruction of intraoral defects of soft tissues. We report the case of a middle-aged man who developed ischaemia in three fingers after a fasciocutaneous radial flap had been raised. The preoperative Allen test to diagnose occlusion of radial or ulnar artery was satisfactory. Soon after the operation the patient resumed smoking and four weeks later he developed ulcers on the thumb, index, and middle fingers. Only after he had stopped smoking and been given acetylsalicylic acid and heparin did blood flow and capillary hemoglobin oxygenation increase. As a result, his radial fingers recovered completely.

Protoporphyrin IX occurs naturally in colorectal cancers and their metastases.

Colorectal cancers exhibit a red fluorescence. The nature of the responsible fluorophore and its eventual diagnostic potential were investigated. Thirty-three consecutive colorectal resection specimen, 32 of which with histologically confirmed cancer, and a total of 1053 palpable mesenteric nodes were fluorimetrically characterized ex vivo. Furthermore, frozen material from 28 patients was analyzed, selected for the availability of primary tumor material and metastatic tissue, e.g., lymphatic and liver metastases from the same patient. Biochemical characterization was carried out through chemical extraction and reversed phase high-performance liquid chromatography. The fluorescence spectra of tissues, tissue extracts, and standard solutions of porphyrins were determined using a pulsed solid-state laser system for excitation and an imaging polychromator, together with an intensified CCD camera for time-delayed observation. Protoporphyrin IX (PpIX) was identified as the predominant fluorophore in primary tumors and their metastases. The fluorophore occurred in the absence of necrosis and in sterile locations. In untreated cases (n = 24), PpIX fluorescence discriminates metastatically involved lymph nodes from all other palpable nodes with a sensitivity of 62% at a specificity of 78% (P < 0.0001). After neoadjuvant treatment of rectal cancer, the PpIX fluorescence level of the primary tumors was reduced and a discrimination of lymph nodes based on PpIX-fluorescence was impossible. We conclude that colorectal cancer metastases accumulate diagnostic levels of endogenous PpIX as a result of a tumor-specific metabolic alteration.

Predictive value of ex vivo biodynamic imaging in determining response to chemotherapy in dogs with spontaneous non-Hodgkin's lymphomas: a preliminary study.

Biodynamic imaging (BDI) is a novel phenotypic cancer profiling technology which optically characterizes changes in subcellular motion within living tumor tissue samples in response to ex vivo treatment with cancer chemotherapy drugs. The purpose of this preliminary study was to assess the ability of ex vivo BDI to predict in vivo clinical response to chemotherapy in ten dogs with naturally-occurring non-Hodgkin's lymphomas. Pre-treatment tumor biopsy samples were obtained from all dogs and treated ex vivo with doxorubicin (10 µM). BDI measured six dynamic biomarkers of subcellular motion from all biopsy samples at baseline and at regular intervals for 9 h following drug application. All dogs subsequently received doxorubicin to treat their lymphomas. Best overall response to and progression-free survival time following chemotherapy were recorded for all dogs. Receiver operating characteristic (ROC) curves were used to determine accuracy and identify possible cut-off values for the BDI-measured biomarkers which could accurately predict those dogs' cancers that would and would not respond to doxorubicin chemotherapy. One biomarker (designated 'MEM') showed 100% discriminative capability for predicting clinical response to doxorubicin (area under the ROC curve = 1.00, 95% CI 0.692-1.000), while other biomarkers also showed promising predictive capability. These preliminary findings suggest that ex vivo BDI can accurately predict treatment outcome following doxorubicin chemotherapy in a spontaneous animal cancer model, and is worthy of further investigation as a technology for personalized cancer medicine.

Point-of-Care Clinical Ultrasound for Medical Students.

PURPOSE: Our institution has recently implemented a point-of-care (POC) ultrasound training program, consisting of an e-learning course and systematic practical hands-on training. The aim of this prospective study was to evaluate the learning outcome of this curriculum. MATERIALS AND METHODS: 16 medical students with no previous ultrasound experience comprised the study group. The program covered a combination of 4 well-described point-of-care (POC) ultrasound protocols (focus assessed transthoracic echocardiography, focused assessment with sonography in trauma, lung ultrasound, and dynamic needle tip positioning for ultrasound-guided vascular access) and it consisted of an e-learning course followed by 4 h of practical hands-on training. Practical skills and image quality were tested 3 times during the study: at baseline, after e-learning, and after hands-on training. RESULTS: Practical skills improved for all 4 protocols; after e-learning as well as after hands-on training. The number of students who were able to perform at least one interpretable image of the heart increased from 7 at baseline to 12 after e-learning, p<0.01, and to all 16 students after hands-on-training, p<0.01. The number of students able to cannulate an artificial vessel increased from 3 to 8 after e-learning and to 15 after hands-on training. CONCLUSION: Medical students with no previous ultrasound experience demonstrated a considerable improvement in practical skill after interactive e-learning and 4 h of hands-on training.

The human S3a ribosomal protein: sequence, location and cell-free transcription of the functional gene.

The intron-containing gene encoding human ribosomal protein S3a (hRPS3a) was isolated by utilizing a PCR-based strategy to detect a gene-specific intron which was subsequently used as a probe for cloning of the entire gene. The hRPS3a gene is composed of six exons and five introns spanning 5013 bp. As described for other hRP-encoding genes, the promoter lacks a canonical TATA sequence and a defined CAAT box. Primer extension experiments, as well as cell-free transcription, revealed that a cytosine functions as the major transcription start point in a polypyrimidine region, but a guanosine at position -1 was also able to initiate transcription. Hybridization analysis of chromosomal DNA from a panel of human-rodent somatic cell hybrids revealed that hRPS3a is encoded by a single locus in the human genome, present on chromosome 4.

DNA analysis of Huntington's disease: five years of experience in Germany, Austria, and Switzerland.

OBJECTIVE: To review the direct DNA testing for Huntington's disease (HD) in Germany, Switzerland, and Austria from 1993 to 1997, and to analyze the population with regard to age structure, gender, and family history. METHODS: Twelve laboratories (nine in Germany, two in Austria, and one in Switzerland) recorded data pertaining to repeat number, gender, age at molecular diagnosis, and family history of probands. The molecular test was categorized as either diagnostic (for symptomatic individuals), presymptomatic (for individuals at risk), and prenatal (for pregnancies at risk). RESULTS: A total of 3,090 HD patients, 992 individuals at risk, and 24 fetuses were investigated using DNA analysis. The clinical diagnosis was confirmed in 65.6% of patients. A total of 38.5% of individuals at risk inherited an expanded CAG repeat. The female-to-male ratio showed a distinct predominance of women both in the diagnostic and presymptomatic groups. Of the fetuses tested, six were carriers of an expanded CAG repeat. Two pregnancies were interrupted; one pregnancy was not. No information about the parents' decision was obtained for the remaining three pregnancies. CONCLUSIONS: Approximately 20% of the estimated 10,000 HD patients living in Germany, Switzerland, and Austria have been identified by DNA analysis (total population, approximately 100 million; incidence of HD, 1:10,000). Assuming a ratio of HD patients to individuals at risk of 1:3, approximately 30,000 individuals are, in principle, eligible for a presymptomatic test. Less than 3 to 4% of individuals at risk have requested a presymptomatic test. This shows that the assumed enormous request of predictive testing has not occurred. More surprisingly, prenatal diagnoses were found to be rare.

A Plasmodium falciparum blood stage antigen highly homologous to the glycophorin binding protein GBP.

We have isolated a gene coding for a protein highly homologous to an antigen known as the glycophorin binding protein (GBP) which was therefore called GBPH. The gene consists of 2 exons interrupted by an intron located at a position corresponding to that of the GBP gene. The deduced amino acid sequence of GBPH comprises 427 residues and is characterized by a signal sequence and by an extended repeat region consisting of 8 units of 40 amino acid residues. The comparison of the amino acid sequences of GBPH and GBP reveals an identity of 69%. Antisera raised against a GBPH fragment that carries part of the repetitive region cross-react with GBP (105 kDa) and additionally detect some bands between 40 and 70 kDa, one of which may correspond to GBPH. The genes coding for GBP and GBPH are located on chromosomes 10 and 14, respectively. The GBP gene is transcribed as a highly abundant 6.5 kb mRNA in the blood-stage form, whereas Northern blot analysis using a GBPH specific probe detects 2 less abundant mRNAs of 2.3 kb and 2.7 kb. Southern blot analysis of P. falciparum DNA identifies a third member of the GBP gene family.

Transient dynamics during two-wave mixing in photorefractive quantum well diodes using moving gratings.

The temporal dynamics of photorefractive and absorptive gains during two-wave mixing in Stark geometry photorefractive quantum wells are investigated using moving gratings to break the symmetry of the photorefractive diodes to achieve nonreciprocal energy transfer between two coherent laser beams.

Direct-to-video holographic 3-D imaging using photorefractive multiple quantum well devices.

Customised photorefractive quantum well devices have been developed for real-time video acquisition of coherence-gated, three-dimensional images. Holographic imaging with direct video capture has been demonstrated. The technique has been applied to 3-D imaging through turbid media with 50 mm transverse and 60 mm depth resolution being achieved using near infrared light through a phantom of 13 mean free paths scattering depth. Spectrally-resolved holographic imaging has also been demonstrated.

Direct-to-video holographic 3-D imaging using photorefractive multiple quantum well devices: errata.

Due to an oversight during the revision process, one of the authors was not mentioned in this paper. The author list should read: R. Jones, M. Tziraki, D. Parsons Karavassilis, P. M. W. French, K. M. Kwolek, D. D. Nolte and M. R. Melloch.

Reduction of postischemic leukocyte-endothelium interaction by adenosine via A2 receptor.

The adhesion of leukocytes to the endothelium of postcapillary venules hallmarks a key event in ischemia-reperfusion injury. Adenosine has been shown to protect from postischemic reperfusion injury, presumably through inhibition of postischemic leukocyte-endothelial interaction. This study was performed to investigate in vivo by which receptors the effect of adenosine on postischemic leukocyte-endothelium interaction is mediated. The hamster dorsal skinfold model and fluorescence microscopy were used for intravital investigation of red cell velocity, vessel diameter, and leukocyte-endothelium interaction in postcapillary venules of a thin striated skin muscle. Leukocytes were stained in vivo with acridine orange (0.5 mg kg-1 min-1 i.v.). Parameters were assessed prior to induction of 4 h ischemia to the muscle tissue and 0.5 h, 2 h, and 24 h after reperfusion. Adenosine, the adenosine A1-selective agonist 2-chloro-N6-cyclopentyladenosine (CCPA), the A2-selective agonist CGS 21,680, the non-selective adenosine receptor antagonist xanthine amine congener (XAC), and the adenosine uptake blocker S-(p-nitrobenzyl)-6-thioinosine (NBTI) were infused via jugular vein starting 15 min prior to release of ischemia until 0.5 h after reperfusion. Adenosine and CGS 21,680 significantly reduced postischemic leukocyte-endothelium interaction 0.5 h after reperfusion (p less than 0.01), while no inhibitory effect was observed with CCPA. Coadministration of XAC blocked the inhibitory effects of adenosine. Infusion of NBTI alone effectively decreased postischemic leukocyte-endothelium interaction. These findings indicate that adenosine reduces post-ischemic leukocyte-endothelium interaction via A2 receptor and suggest a protective role of endogenous adenosine during ischemia-reperfusion.

Maternal ingestion of ortho-aminoacetophenone during gestation affects intake by offspring.

Ingested flavor chemicals cross the placental barrier and occur in the fetal blood and amniotic fluid. This occurrence is detectable by the fetus, and can influence post parturition feeding. In the present experiment, pregnant mice were offered either 0.1% ortho-aminoacetophenone emulsions (OAP) or water throughout gestation. OAP is normally avoided by mice, apparently on the basis of chemosensory characteristics. Subsequently, offspring were offered 0.5%, 0.25%, or 0.1% OAP in one-bottle tests at 26 or 88 days of age. Offspring of mothers given OAP drank greater amounts of OAP than did offspring of mothers given water. Enhanced acceptance of OAP was not detected in mice exposed to 0.1% OAP as adults for a duration similar to that given during gestation. We conclude that fetal experiences with OAP lowered sensitivity and/or raised tolerance for the compound.

Garlic in the ovine fetal environment.

We investigated whether the odor of garlic can cross the placental barrier from the mother to the fetal environment. Samples of amniotic fluid, allantoic fluid, fetal blood, and maternal blood were collected 0, 50, 100, and 150 min after a pregnant ewe (approximately day 110 of gestation) was gavaged with 6 ml of Egyptian garlic oil. A panel of judges detected (p < 0.05) garlic odor in samples of allantoic fluid, fetal blood, and maternal blood collected 50, 100, and 150 min after the ewe was given garlic and in samples of amniotic fluid collected 100 min after treatment.

Effectiveness of thirteen vertebrate repellents as rodent trigeminal stimulants.

Repellent chemicals are presumed to activate trigeminal neurons, including polymodal nociceptors, but few data are available that bear on this notion. In the present experiment, we assessed multi-unit and single-unit responses of neurons in the rat lingual trigeminal nerve to 13 candidate repellents and a thermal stimulus. All of the chemicals evoked trigeminal responses, and neural activity was predictable from available behavioral data. These results are consistent with the view that repellents are irritants. The results also suggest that electrophysiological methods may represent a useful method for screening candidate repellent compounds.