Visualizing CD4 T-cell migration into inflamed skin and its inhibition by CCR4/CCR10 blockades using in vivo imaging model.

BACKGROUND: Chemokines are critical mediators of T-cell homing into inflamed skin. The complex nature of this multicellular response makes it difficult to analyse mechanisms mediating the early responses in vivo. OBJECTIVES: To visualize directly T-cell homing into inflamed skin and its inhibition by blockades using a unique noninvasive confocal microscopy. MATERIALS AND METHODS: A mouse model of allergic contact dermatitis was used. T cells from oxazolone-sensitized and -challenged Balb/c mice were first analysed phenotypically in vitro. CD4 T cells were then labelled with a tracker dye and transferred into Balb/c-SCID mice. The recipient mice were challenged with oxazolone and CD4 T-cell homing into inflamed skin was visualized. RESULTS: T cells with the skin homing receptors CCR4 and CCR10 were increased in the affected skin and draining lymph nodes, and effectively attracted by their specific chemokines CCL17, CCL22 and CCL27 in vitro. Using in vivo imaging, T-cell migration into the inflamed skin was observed at 2 h after application, peaking at 12 h and continuing for 48 h. Simultaneous systemic administration of neutralizing antibodies against CCR4 ligands (CCL17 and CCL22) and CCR10 ligand (CCL27) led to a significant suppression of T-cell migration and skin inflammation. CONCLUSIONS: Our data indicate that these tissue-selective adhesion molecules and chemokine/receptor pathways act in concert to attract specialized T-cell populations to mediate cutaneous inflammation. The in vivo imaging technique can be applicable to other models of cutaneous diseases to help with better understanding of the pathogenesis and monitoring the therapeutic effects.

Comprehensive Imaging of Sensory-Evoked Activity of Entire Neurons Within the Awake Developing Brain Using Ultrafast AOD-Based Random-Access Two-Photon Microscopy.

Determining how neurons transform synaptic input and encode information in action potential (AP) firing output is required for understanding dendritic integration, neural transforms and encoding. Limitations in the speed of imaging 3D volumes of brain encompassing complex dendritic arbors in vivo using conventional galvanometer mirror-based laser-scanning microscopy has hampered fully capturing fluorescent sensors of activity throughout an individual neuron's entire complement of synaptic inputs and somatic APs. To address this problem, we have developed a two-photon microscope that achieves high-speed scanning by employing inertia-free acousto-optic deflectors (AODs) for laser beam positioning, enabling random-access sampling of hundreds to thousands of points-of-interest restricted to a predetermined neuronal structure, avoiding wasted scanning of surrounding extracellular tissue. This system is capable of comprehensive imaging of the activity of single neurons within the intact and awake vertebrate brain. Here, we demonstrate imaging of tectal neurons within the brains of albino Xenopus laevis tadpoles labeled using single-cell electroporation for expression of a red space-filling fluorophore to determine dendritic arbor morphology, and either the calcium sensor jGCaMP7s or the glutamate sensor iGluSnFR as indicators of neural activity. Using discrete, point-of-interest scanning we achieve sampling rates of 3 Hz for saturation sampling of entire arbors at 2 µm resolution, 6 Hz for sequentially sampling 3 volumes encompassing the dendritic arbor and soma, and 200-250 Hz for scanning individual planes through the dendritic arbor. This system allows investigations of sensory-evoked information input-output relationships of neurons within the intact and awake brain.

Automating Event-detection of Brain Neuron Synaptic Activity and Action Potential Firing in vivo using a Random-access Multiphoton Laser Scanning Microscope for Real-time Analysis.

Determining how a neuron computes requires an understanding of the complex spatiotemporal relationship between its input (e.g. synaptic input as a result of external stimuli) and action potential output. Recent advances in in vivo, laser-scanning multiphoton technology, known as random-access microscopy (RAM), can capture this relationship by imaging fluorescent light, emitted from calcium-sensitive biosensors responding to synaptic and action potential firing in a neuron's full dendritic arbor and cell body. Ideally, a continuous output of fluorescent intensities from the neuron would be converted to a binary output (`event', 'or no-event'). These binary events can be used to correlate temporal and spatial associations between the input and output. However, neurons contain hundreds-to-thousands of synapses on the dendritic arbors generating an enormous quantity of data composed of physiological signals, which vary greatly in shape and size. Thus, automating data-processing tasks is essential to support high-throughput analysis for real-time/post-processing operations and to improve operators' comprehension of the data used to decipher neuron computations. Here, we describe an automated software algorithm to detect brain neuron events in real-time using an acousto-optic, multiphoton, laser scanning RAM developed in our laboratory. The fluorescent light intensities, from a genetically encoded, calcium biosensor (GCAMP 6m), are measured by our RAM system and are input to our 'event-detector', which converts them to a binary output meant for real-time applications. We evaluate three algorithms for this purpose: exponentially weighted moving average, cumulative sum, and template matching; present each algorithm's performance; and discuss user-feasibility of each. We validated our system in vivo, using the visual circuit of the Xenopus laevis.

An unreported variation of the cervical vagus nerve: anatomical and histological observations.

Variations involving the cervical portion of the vagus nerve are seemingly very rare. We report an adult male found to harbour a right cervical vagus nerve that crossed anterior to the right common carotid artery to terminate in the lateral aspect of the thyroid gland. A very small continuation of this nerve was found to continue distally into the thorax. Histologically, this part of the vagus nerve did not contain ganglion or other cell bodies. There were no heterologous inclusions (thyroid, parathyroid, thymus, salivary gland or branchial cleft remnants) present. Although grossly there was a connection into the thyroid gland, this was not observed histologically. No signs of trauma were found to the ipsilateral neck region. We hypothesise that this variation is due to entanglement between the thyroid gland and cervical vagus nerve during development. This rare variation might be considered by the clinician who operates in the cervical region or interprets imaging of the neck. To our knowledge, a vagus nerve with the above described morphology has not been described.

Synchronous bifocal alveolar rhabdomyosarcoma: a case report.

We report a case of bifocal rhabdomyosarcoma involving the hand and thigh in an 11-year-old female. We highlight the importance of a thorough clinical examination and an aggressive surgical approach in which each lesion is treated as a separate primary.

When is a butterfly like an elephant?

The behaviours of organisms as diverse as elephants and butterflies are affected by pheromones with identical or similar structures. Recent developments in the molecular biology of pheromone detection suggest why.

Desmin positivity in primitive neuroectodermal tumors of childhood.

In this report, we describe two rosette-forming primitive neuroectodermal tumors that were found to contain desmin by both immunohistochemistry and Western blotting. Electron microscopy on both cases was consistent with primitive neuroectodermal tumors and revealed that the tumor cells contained cytoplasmic bundles of intermediate filaments. In both cases, studies for MyoD1 protein using immunohistochemistry and Western blotting were negative. Thus, the detection of desmin in a pediatric neoplasm does not absolutely exclude the diagnosis of primitive neuroectodermal tumor and should not be considered as prima facie evidence that a small-cell tumor is a rhabdomyosarcoma.

Effects of cholinergic blockade on hemodynamic disturbances and intestinal lesions in endotoxic shock in newborn piglets.

The parasympathetic nervous system actively participates in the regulation of pathophysiologic responses in circulatory shock. To determine the effects of cholinergic blockade in endotoxic shock in newborn piglets, 16 chronically instrumented newborn piglets were infused with 10 mg/kg of endotoxin over 10 min. Eight animals were injected intravenously with 10 mg/kg of anisodamine, an anticholinergic drug, 10 min before endotoxin and then with escalating doses of 2, 5, 10, and 20 mg/kg every 10 min, beginning 60 min after endotoxin. The other eight animals were given saline as a control. Endotoxin infusion caused elevations in mean pulmonary artery pressure and vascular resistance index and an initial increase in systemic artery pressure followed by hypotension. Heart rate was stable for 45 min and then increased. Cardiac index fell from a baseline of 173 +/- 20 (mean +/- S.E.) to 136 +/- 23 mL.min-1.kg-1 60 min after endotoxin. Pretreatment with anisodamine increased heart rate from 163 +/- 15 to 289 +/- 10 beats.min-1 and cardiac index from 195 +/- 15 to 238 +/- 14 mL.min-1.kg-1 before endotoxin infusion. These variables remained at higher levels than in the control group until 60 min after endotoxin infusion; thereafter, the two groups were similar. The changes in pulmonary and systemic artery pressures were not significantly altered by anisodamine. After 60 min, additional doses of anisodamine caused no significant hemodynamic responses, and the differences between the two groups were not significant. Arterial plasma thromboxane B2 levels rose immediately and tumor necrosis factor-alpha levels increased 60 min after endotoxin infusion; no significant differences were noted between groups at any time.(ABSTRACT TRUNCATED AT 250 WORDS)

Primary rhabdomyosarcoma of the iris.

We describe herein a 4-year-old girl with primary rhabdomyosarcoma of the right iris. Malignant cells were aspirated from the aqueous humor, and the eye was enucleated 7 months after the tumor appeared. To our knowledge, only two cases of primary rhabdomyosarcoma of the iris have been reported. Both of these patients received radiation therapy prior to enucleation.

Megalocytic interstitial nephritis, xanthogranulomatous pyelonephritis, and malakoplakia. An ultrastructural comparison.

Light-microscopic and ultrastructural examinations of megalocytic interstitial nephritis, xanthogranulomatous pyelonephritis, and malakoplakia of the kidney were compared. The cases of megalocytic interstitial nephritis and xanthogranulomatous pyelonephritis represent the first reported electron-microscopic studies on human kidney of these diseases. The study confirmed the presence of a polymorphous cellular infiltrate with predominate histiocytes containing crystalloid material in the case of megalocytic interstitial nephritis; a polymorphous cellular infiltrate with histiocytes predominating in the case of xanthogranulomatous pyelonephritis; and macrophages containing Michaelis-Gutmann bodies in the case of malakoplakia. The characteristic sites of involvement within the kidney by each of the three lesions are discussed. It is believed that the lesions are distinct entities, but related to one another, and represent varied and unusual host responses to inflammation.

Partial liquid breathing with perflubron improves arterial oxygenation in acute canine lung injury.

Tidal liquid breathing with perfluorocarbon liquid improves lung mechanics and gas exchange in surfactant-deficient lambs. We asked whether an intratracheal dose of perflubron equal to liquid functional residual capacity (FRC) or less (partial liquid breathing) would alter gas exchange in gas-ventilated, anesthetized, and oleic acid (OA)-injured dogs. We gave eight dogs perflubron in six aliquots, each equal to one-sixth of liquid FRC [sequential dose (SEQ) group], starting 90 min after OA. Two other groups of eight dogs each received a liquid FRC dose of perflubron (60-70 ml/kg) either just before (P-Pre) or 90 min after (P-Post) OA, and eight control (CON) dogs received OA but no perflubron. In CON, SEQ, and P-Post dogs, OA caused a significant decrease in static respiratory system compliance (Crs) (1.50 +/- 0.05 to 1.15 +/- 0.06 ml.cmH2O-1.kg-1) and arterial PO2 (508 +/- 10 to 113 +/- 16 Torr) by minute 90. In P-Pre, P-Post, and after the third dose of perflubron (50% FRC) in SEQ dogs, arterial PO2 was significantly higher than that in the CON dogs but was similar to that in the CON dogs after perflubron removal. Perflubron doses less than two-thirds of liquid FRC were associated with increased Crs compared with CON dogs, but further lung filling caused a decrease in Crs. Lung histology showed widespread edema and hemorrhage and was similar in perflubron and CON dogs. O2 delivery and uptake were well maintained in all four groups.(ABSTRACT TRUNCATED AT 250 WORDS)

Analysis of disability resulting from treatment including radical neck dissection or modified neck dissection.

A multiinstitutional study to define the impact of total treatment programs involving radical neck dissection (RND) and modified neck dissection (MND) on patients' permanent disability was undertaken. A total of 243 patient responses were included in the study. Comparative analyses between the treatment groups show no advantage of one surgical operation over the other in returning patients to their pretreatment employment status. Radiation therapy was identified as adding significantly to the patient's permanent disability.

Successful medical treatment of a spinal histoplasmoma.

A woman with previously treated disseminated histoplasmosis was admitted with progressive paraparesis. Magnetic resonance imaging showed multifocal cerebritis and spinal histoplasmoma. She recovered following a second treatment with amphotericin B. This is the fourth reported patient with intramedullary spinal histoplasmoma, and the first demonstrating successful treatment of the lesion with medical therapy alone.

Short bowel syndrome.

Improvements in parenteral nutrition and supportive therapy have led to a growing population of patients who survive for prolonged periods with short bowel syndrome. Definitive treatment for these patients requires innovative therapy based on a sound knowledge of small intestinal physiology and adaptation. Current understanding of short bowel pathophysiology and of intestinal adaptation are reviewed. Medical and surgical therapeutic options are described, highlighting the promotion of small bowel adaptation and methods to increase the small intestinal mucosal mass.

Angiolipoma of the parotid: case report and review.

An angiolipoma is a benign adipose tissue tumor that can occur as either a circumscribed or an infiltrating lesion and represents up to 17% of all lipomas. Most angiolipomas occur in teenagers and young adults, and are rarely described in younger children. Angiolipoma generally arises on the extremities or trunk, and it is unusual when found in the head and neck. An angiolipoma of the parotid region was suspected in an infant girl after contrast-enhanced computerized tomographic scan, and was confirmed when the mass was surgically excised. The clinical characteristics, radiological appearance, and histopathology of angiolipoma are discussed.

Pyruvate carboxylase deficiency: acute exacerbation after ACTH treatment of infantile spasms.

Pyruvate carboxylase deficiency results in congenital lactic acidosis. We report the significant finding in a child with infantile spasms controlled with adrenocorticotrophin hormone (ACTH) but who then developed severe lactic acidosis; pyruvate carboxylase deficiency was subsequently diagnosed. Blood lactate, pyruvate, and alanine levels were elevated, as well as cerebrospinal fluid alanine. Plasma alanine concentration was doubled by ACTH therapy. Fibroblasts contained extremely low pyruvate carboxylase activity. The patient died at 12 weeks of age after recurrent episodes of profound acidosis. At autopsy, the brain manifested cystic degeneration and demyelination. Pyruvate carboxylase deficiency is associated with neonatal onset of acidosis, delayed development, seizures, hypotonia, recurrent profound acidosis, and early death. The dramatic rise in plasma alanine content coincident with ACTH therapy suggest that ACTH played a role in precipitating the catastrophic metabolic acidosis.

Twenty years of vocational training in the west of Scotland: the practice component.

A retrospective analysis of the experience and opinions of doctors receiving vocational training in general practice was obtained by postal questionnaire. Questionnaires were received by 974 doctors who had been vocationally trained in the west of Scotland between 1968 and 1987. The response rate was 64%. It was found that 94% of the respondents had enjoyed their trainee period, 82% had been given a choice of training practice and 86% had spent 12 months in a training practice. Only 81 respondents had trained in two practices. The most common method of monitoring the trainee's consultation was the trainer sitting in on the consultation; half of the doctors had experience of this (51%). For the majority regular tutorials were commonplace, but for 41% of respondents this was not so. However, those training after 1979 were significantly less likely to have never had tutorials than those training earlier. Nearly half of the doctors (49%) felt that certain aspects had been poorly covered or omitted from their training, notably practice management and finance. Again, this was significantly less likely among those training after 1979. When asked to give a rating of the training they had received 21% of the respondents rated it as excellent, 37% as very good, 30% as fairly good and 12% as poor/fairly poor or very poor. Notably, significantly fewer respondents training after 1979 rated their training as poor/fairly poor or very poor. Very few respondents had participated in a practice exchange but virtually all of those who had felt it had been beneficial.(ABSTRACT TRUNCATED AT 250 WORDS)

Second generation alpha-enones from a pyranosidic alpha-enone.

The Diels-Alder product from the reaction of methyl 2,3,6-trideoxy-alpha-D-glycero-hex-2-enopyranosid-4-ulose (1b) with trans-1-methoxy-3-tert-butyl-dimethylsilyloxy-1,3-butadiene is 3b (93%). Reaction of 3b wih sodium borohydride causes reduction of the C-4 carbonyl group only, but, with lithium aluminum hydride, further reactions occur which can be rationalized by fragmentation brought about by hydride cleavage on the silicon-oxygen bond, with simultaneous ejection of the beta-methoxyl group complexed to a trivalent aluminum species. The enone resulting from this fragmentation also reacts further with lithium aluminum hydride, and several products result. The behavior of postulated intermediates, which have been prepared separately and subjected to the reaction conditions, supports the proposed reaction mechanisms. The "second generation" enone (methyl 2,3,6-trideoxy-alpha-D-talopyranosido)-[3,2-d]-2-cyclohexenone+ ++ (10a), arising from the first generation precursors 1b, has been prepare by two routes.

Peripheral primitive neuroectodermal tumors. A flow cytometric analysis with immunohistochemical and ultrastructural observations.

Flow cytometry of classical neuroblastoma has provided provocative evidence that cell cycle and ploidy analysis generate prognostically useful information. To determine whether such analyses of peripheral primitive neuroectodermal tumors might yield similar results, formalin-fixed, paraffin-embedded tissue specimens from 19 peripheral primitive neuroectodermal tumors, each previously characterized by immunohistochemical or ultrastructural study, were assessed. An acceptable histogram was obtained in 16 cases. Of these, nine neoplasms were diploid and seven contained aneuploid DNA. Among patients with diploid lesions, four were free of disease, whereas three had persistent or recurrent disease, and two had died of tumor. Among patients with aneuploid neoplasms, four were free of disease, one had recurrence, and two had died. There was no apparent correlation between immunophenotype and proliferative activity with the clinical outcome. Among aneuploid peripheral primitive neuroectodermal tumors, DNA index did not predict survival. Hence, cell cycle and DNA ploidy analyses do not appear to contribute to the prognostic assessment of peripheral primitive neuroectodermal tumors, as they do to presumably related neoplasms of the central and peripheral nervous system.

Urinalysis: current status and prospects for the future.

More than 300 million routine clinical analyses are performed annually in the United States. Methods for routine clinical urine examination, including detection of bacteriuria, are briefly reviewed. Prospects of some newer, better techniques to carry out such analyses are introduced. A preliminary report is presented on the use of supravital microscopic fluorescence technique (SMFT), employing acridine orange as a non-specific staining fluorochrome. Results of examining 218 unspun urine specimens by SMFT are compared to traditional bacteriologic culture at a large pediatric hospital reference laboratory.