Comparison of polymerase chain reaction with light microscopy for detection of microsporidia in clinical specimens

The detection of microsporidial DNA by the polymerase chain reaction (PCR) has been suggested as an alternative or supplement to conventional microscopic methods. However, the relative merits of these techniques remain uncertain. In the present study, clinical specimens of different origin (stool, urine, sputum, nasal discharge, and cerebrospinal fluid) containing four different microsporidial species were blinded after microscopic examination and analyzed by PCR and subsequent restriction fragment length polymorphism (RFLP) to determine the respective species. Thirty-four specimens from 31 patients were evaluated, 16 of which were positive and 18 negative by microscopic examination; PCR detection of microsporidia produced identical results in 82% (28/34) of these specimens. Four samples were microscopically negative, PCR-positive, and two were microscopically positive, PCR-negative. Species determination by RFLP analysis of the amplified product was accurate for all isolates containingEnterocytozoon bieneusi, Encephalitozoon intestinalis, Encephalitozoon hellem, andEncephalitozoon cuniculi compared with microscopic identification of the genusEnterocytozoon or molecular analysis ofEncephalitozoon species after in vitro culture. Therefore, PCR-RFLP is useful for the rapid detection and differentiation of microsporidian spores in clinical specimens.     

Regulation of cytokine synthesis in cardiac surgery: Role of extracorporeal circuit and humoral mediators <Emphasis Type="Italic">in vivo</Emphasis> and <Emphasis Type="Italic">in vitro</Emphasis>

Objective and design: Cardiopulmonary bypass (CPB) impairs monocyte and neutrophil proliferation, cytokine synthesis, and antigen presentation. This study compares in vivo data with results from an extracorporeal circulation (ECC) model, distinguishing direct effects on cytokine synthesis from regulatory mechanisms. Patients and methods: Whole blood from 18 patients prior to, during and after CPB was stimulated with lipopolysaccharide (LPS). Tumor necrosis factor (TNF)-α, interleukin (IL)-6, and IL-8 levels were measured. Additionally, blood from 4 volunteers was circulated in an ECC model. Cytokine levels were measured before and during mock ECC. Results: LPS-induced cytokine synthesis was reduced after CPB (TNF-α: 11 %; IL-6: 29 %; IL-8: 48 % of preoperative values, all p < 0.001). In mock ECC, cytokine production (except IL-8) was suppressed: TNF-α production was lowest 60 min after starting ECC, IL-6 synthesis was lowest at 90 min (33 % and 15 % vs. pre-ECC levels; both p < 0.001). Patient sera contained cytokine-inhibitory activity after CPB, an activity not found in mock ECC. Conclusions: (1) In patients, CPB induces early transient LPS hyporesponsiveness; (2) blood contact with foreign surfaces induces LPS hyporesponsiveness; (3) serum cytokineinhibitory activities are released after CPB, but not in mock ECC. Impaired leukocyte function may explain increased susceptibility to infections after CPB. Received 16 September 2006; accepted without revision by K. Visvanathan 18 October 2006     

Percepciones estudiantiles sobre las propiedades del examen clínico objetivo estructurado en Uruguay. Disponiendo un cuestionario válido para analizar la factibilidad del nuevo formato durante la transición curricular

Introduction

Although an objective structured clinical examination (OSCE) format has been applied in Uruguay since 2004, and providing reliable performance measures, perceptions of it properties and level of student satisfaction have not been determined.

Resorptions- und Sekretionsprüfungen bei Perfusion des Rattendarms mit hypo- und hypertonischen Glucoselösungen

Ohne Zusammenfassung     

The osmotic behaviour of toad skin epithelium (Bufo viridis)

The effect of saline adaptation on the intracellular Na, K, Cl, P concentrations and dry weight content of the toad skin epithelium (Bufo viridis) was studied using the technique of electron microprobe analysis. The measurements were performed on isolated abdominal skins either directly after dissection or after additional incubation in Ussing-type chambers.Adaptations of the toads to increasing NaCl concentrations for 7 days resulted in increased blood plasma osmolarity and a parallel increase in the cellular electrolyte, P and dry weight concentrations of the epithelium, the K increase representing the most significant fraction of the intracellular osmolarity increase. No evidence was obtained to show that the nucleus and cytoplasm reacted differently from each other and all living epithelial cell types basically showed the same response.Incubation of the isolated skins under control conditions showed a drastic inhibition of the transepithelial Na transport after adaptation to high salinities. In spite of the large variations in the transport rate almost identical intracellular electrolyte concentrations were observed. In tap water adapted toads the average cellular concentrations were 8.8 mmole/kg wet weight for Na, 109.6 for K, 41.5 for Cl, and 135.3 for P, respectively. Incubation of the skin with Ringer's solution of different osmolarities demonstrated that the epithelial cells are in osmotic equilibrium with the inner bathing solution. The results are consistent with the view that the osmotic adaptation is mainly accomplished by the movement of water.This work was supported by grants from the Deutsche Forschungsgemeinschaft and the Stiftung Volkswagenwerk     

Membranes of the adrenal medulla: A comparison of membranes of chromaffin granules with those of the endoplasmic reticulum

The protein and lipid composition of the membrane of the chromaffin granule has been compared with that of an endoplasmic reticulum fraction isolated from bovine adrenal medulla. The endoplasmic reticulum was purified by centrifugation of a microsomal fraction in sucrose and Ficoll density gradients. Only insignificant amounts of chromomembrin B, dopamine β-hydroxylase and cytochrome b₅₅₉, which are characteristic protein components of the chromaffin granule membrane, were present in the endoplasmic reticulum fraction; whereas glucose-6-phosphatase and galactosyltransferase, marker enzymes of membranes of endoplasmic reticulum and Golgi complex respectively, were absent from the membranes of chromaffin granules. The lipid composition of the two types of membranes varies in the relative amounts of lysolecithin, lecithin and sphingomyelin and in the molar ratio of cholesterol to phospholipid. Lysolecithin, which is a major phospholipid of the chromaffin granule membrane, is virtually absent from membranes of the endoplasmic reticulum. Different protein patterns were obtained when these two membrane fractions were solubilized with detergent and subjected to polyacrylamide gel electrophoresis.It is concluded that membranes of the endoplasmic reticulum and those of the chromaffin granules are basically different and therefore that the transport of secretory proteins from their site of synthesis to their site of storage occurs, in the chromaffin cell, by a mechanism that does not involve a mixing of the participating membranes.     

Elektronenmikroskopische Untersuchungen am embryonalen Mycetom der Kleinzikade Euscelis plebejus Fall. (Homoptera,Cicadina)

Zusammenfassung Die a-Symbionten von Euscelis plebejus treten während der Embryonalentwicklung ihres Wirts in zwei morphologischen Typen auf, die als Infektionsstadium und als vegetative Form beschrieben werden. Die Infektionsstadien teilen sich durch septenartiges Einwachsen der Symbiontenmembranen. Infektionsstadien von Zikadensymbionten waren bisher nur während der Ovarialinfektion aus den Mycetomen weiblicher Imagines bekannt. Die a-Infektionsformen sind gekennzeichnet durch ihr elektronendichtes Cytoplasma, eine ungleichmäßige Verteilung der Ribosomen mit einer ribosomenfreien Randzone und durch Membrankörper. Das Cytoplasma der vegetativen Symbionten erscheint elektronendurch lässiger. Die Ribosomen liegen homogen in der Cytoplasmagrundsubstanz verteilt.Die a-Symbionten liegen vom Zeitpunkt der Eiablage bis zum Eindringen in die a₁-Mycetocyten als Infektionsstadien vor. Nach der Aufnahme in die a₁-Mycetocyten nehmen sie das Aussehen vegetativer Formen an. Im transitorischen Mycetom erscheinen erneut Infektionsstadien während der Übersiedelung in die zweikernigen a₂-Mycetocyten.Jeder Symbiont wird von zwei eigenen Membranen umgeben. Beim Eindringen von Ooplasma in den Symbiontenball wird jeder Symbiont von einer Wirtsmembran umhüllt, die er bis zur Infektion der Wirtszelle behält. In den Mycetocyten umgibt eine offensichtlich vom Wirtscytoplasma erneut gebildete Hüllmembran die vegetativen Symbionten. In den vegetativen a-Symbionten tritt häufig ein mikrofibrillärer kristallartiger Einschlußkörper auf. — Kernäquivalentstrukturen waren in keinem Symbiontenstadium cytologisch mit Sicherheit nachzuweisen.

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Electron microscopic studies on the embryonic mycetome of the leafhopper Euscelis plebejus fall. (Homoptera, cicadina)

Summary During the embryonic development of their host the a-symbiotes of the leafhopper Euscelis plebejus appear in two different morphological types: infective form and vegetative form. The infective forms are able to multiply by ingrowth of their membranes. Until now infective forms of leafhopper symbiotes were only known from the ovarial infection by adult female mycetomes. The infective form of the a-symbiote is characterized by its electron dense cytoplasm, an asymmetric distribution of the ribosomes, a ribosome-free border, and by membraneous bodies. The cytoplasm of the vegetative form is less electron dense and the ribosomes are scattered throughout the cytoplasm.From the time of oviposition until they enter the a₁-mycetocytes, the a-symbiotes are found as the infective form. After entering the a₁-mycetocytes they take on the appearance of the vegetative form. In the transitory mycetome the infective forms appear again during the transition into the binucleate a₂-mycetocytes.Each symbiote is surrounded by two unit membranes of its own. As the ooplasm penetrates the symbiote mass, each symbiote is also surrounded by a host membrane, which remains until the infection of the host cells. In the mycetocytes the vegetative symbiotes have a new membrane around them, which is obviously developed from the cytoplasm of the host cell. In the vegetative a-symbiotes there is frequently a paracrystalline inclusion. — It was impossible to demonstrate nucleoid structure with certainty in any symbiote form.

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Abkürzungen a a-Symbionten - a_i a-Infektionsstadien - a_v vegetative a-Symbionten - a-Mc a-Mycetocyt - a-Z a-Zelle - eK elektronendichter Körper - ER endoplasmat. Retikulum - M₁ innere Symbiontenmembran - M₂ äußere Symbiontenmembran - M₃ Hüllmembran - Mf Mikrofibrillen - Mi Mitochondrien - Mk Membrankörper - Mt Mikrotubuli - Nu Zellkern - Py Kernpyknosen - Ri Ribosomen - Sb Symbiontenball - Sm Symbiontenmembranen - t t-Symbionten - t_v vegetative t-Symbionten - t-Mc t-Mycetocyt - t-Z t-Zelle - Zm Zellmembran Mit Unterstützung durch die Deutsche Forschungsgemeinschaft.Herrn Prof. Dr. K. Sander danke ich für stete Förderung meiner Arbeit und Frau A. Kaufmann für die Einführung in die elektronenmikroskopische Technik.     

Soft-tissue angiomatosis in adulthood: a case in the forearm showing a prominent myxoid adipose tissue component mimicking liposarcoma

Soft-tissue angiomatosis is a rare condition characterized by diffuse proliferation of benign vascular structures, accompanied by mature adipose tissue, which affect a large segment of the body in a contiguous fashion. Most patients present in childhood or adolescence. We report a case of large soft-tissue angiomatosis, located in the left forearm of a 65-year-old man, which showed extensive intramuscular and intermuscular infiltration, and embracement of the radius and the main vascular and neural bundles of the forearm. Due to the deep location, large size, ill-defined limits and non-homogeneous composition with areas suggestive of an adipose component in the radiological examination, the lesion raised the possibility clinically of a liposarcoma. The myxoid adipose tissue component of the lesion, a feature previously undescribed, and the predominance of small, capillary-sized vessels were considered concordant with the clinical diagnosis. This presentation is rare. The features of the lesion made complete surgical excision impossible and amputation of the extremity was considered the best treatment. The main differential diagnoses include infiltrating lipoma, myxolipoma, angiomyxolipoma, angiolipoma, intramuscular angioma, liposarcoma and low-grade myxofibrosarcoma. In conclusion, angiomatosis might simulate liposarcoma, particularly if the mass is large, heterogeneous, myxoid and appears later in life.