Untersuchungen zur Standardisierung der Messung mikroskopischer Strukturen hinsichtlich Fluoreszenzlebensdauer und bildgebender Lumineszenz zur endoskopischen Anwendung

Tumour cells usually have an altered metabolism compared to healthy cells, since irrespective of the availability of oxygen, they prefer to cover their energy requirements by glycolysis instead of the more efficient oxidative phosphorylation (OXPHOS). The transition from OXPHOS to glycolysis leads to a change in the ratio of free and protein-bound cofactors involved in complex I and II of the respiratory chain, namely nicotinamide adenine dinucleotide (NADH) and flavin adenine dinucleotide (FAD). These endogenous fluorophores are accessible by fluorescence imaging in their reduced (NADH) and oxidized (FAD) forms. Due to the significant change in fluorescence lifetimes of the free and protein-bound forms of NADH and FAD, they are, in principle, suitable for obtaining information about the metabolic state of cells in vivo by means of fluorescence lifetime imaging microscopy (FLIM). However, clinical applications of FLIM are rare and only known in the field of dermatology, but they are frequently used in research. Two-photon excitation is preferable for this purpose, due to the longer excitation wavelength and thus increased optical penetration depth. The implementation of this technology in a clinical application requires both a validation of the measurement procedure and suitable artificial tissue phantoms as reproducible measurement objects. The diagnosis of squamous cell carcinomas, which are predominant in the ear, nose, and throat (ENT) area, requires knowledge of the differentiation of the tumour from normal cells, as well as about the depth of infiltration. Furthermore the differentiation from inflammatory processes is essential. Current diagnostics are done by white light endoscopy and partly by narrow band imaging (NBI), less frequently by optical coherence tomography (OCT) or by fluorescence techniques. The latter three methods enable diagnosis via improved visualisation of structural tissue features compared to white light endoscopy. However, these methods do not provide information about the metabolic state. In the clinic, in addition to endoscopy, a biopsy and time-consuming tissue pathology are required for a systematic diagnosis. By evaluating the fluorescence lifetimes of free and protein-bound NADH and FAD, a distinction between high-grade and low-grade tumours or different cell types is possible. This evaluation procedure is called metabolic imaging. Since tumours differ from normal tissue in their metabolism, it is possible to differentiate between the aforementioned tissue states by measuring the metabolic state of cells or tissue. The transfer of metabolic imaging to clinical diagnostics would offer immediate differentiation from inflammation sites, between high-grade and low-grade tumours or the infiltration of the basal membrane. This could be done during the clinical examination and would offer the possibility of a more exact determination of tumour margins. The analysis of the relative proportions of free and protein-bound NADH and FAD in the examined cell area are suitable for this. Four calculation methods of metabolic indices were compared to each other. The published NADH metabolic index (ratio of free to protein-bound NADH) and Fluorescence Lifetime Induced Redox Ratio (FLIRR, hereinafter referred to as FLIRR1, compares protein-bound and free NADH and FAD) were compared with two modifications of the FLIRR1 index, hereinafter referred to as FLIRR2 and FLIRR3, which take free FAD and additionally protein-bound flavin mononucleotide (FMN) into account. By comparing the metabolic indices, a conclusion can be drawn about the basic suitability of a calculation method for assessing the condition of cells or tissue. However, for development, characterisation and validation of metabolic imaging devices, artificial tissue phantoms showing on one hand side the optical properties of the target fluorophores as well as the optical properties of the target tissue on the other hand side are recommended including reproducible and controllable conditions. Since the optical parameters of tissue samples differ even within the same tissue type of the same species, controllable and reproducible optical properties are needed. This technical need can be provided by the development and subsequent use of artificial optical tissue phantoms. In the course of the investigations, two artificial phantoms were developed serving two different requests: one to simulate the fluorescence lifetimes of free and protein-bound NADH and FAD, and one that allows the determination of the imaging depth and optical distortions or resolution of a device without z-stage by the use of fluorescent objects of known size. Due to their chemical properties the substances NADH and FAD are only suitable as reference standards to a limited extent. This can be deduced also from the varying lifetime data available in literature and their dependence on the concentration and the pH value. Interestingly coumarin 1 and coumarin 6 have been identified as fluorophores with spectral properties sufficiently similar to those of NADH and FAD, thus their quenching properties have been investigated. Based on a modified Stern-Volmer equation, the required quencher concentrations to represent the fluorescence lifetimes of free and protein-bound NADH and FAD were calculated and experimentally tested in the case of coumarin 1. The stability of the coumarin 1 solution was tested over a period of 41 days. Fluorophore solutions based on coumarin 6 as a substitute for FAD were investigated in a cooperation with the Multiphoton Imaging Laboratory of the Munich University of Applied Sciences (AG Hellerer). In contrast to research microscopes, endoscopic systems in medicine are usually lacking the capability to precisely adjust the position of the object plane on the scale of the anatomical structures of the mucous membranes. Automatic focusing to a certain tissue depth is therefore not possible with a commercially available endoscope. For the application to metabolic imaging, an optical resolution in x, y, and z in the range of the size of mitochondria (approximately 1 µm diameter) was aimed for. The challenge is that the substances usually involved in optical phantoms are of similar size. The solution was a staircase-like structure, carved out of a glass body by laser microstructuring, beginning from the surface into the glass volume. With the stair-like structure, a depth range up to approximately 250 µm in 5 µm steps can be covered. The staircase structure was written into glass slides (n=10) by laser microstructuring and characterised in terms of step height, step length, surface texture and total depth. The tread surfaces where coated with fluorescent polystyrene microspheres of 1 µm diameter. The carved out volume was filled with a substance with known reduced scattering coefficient for the wavelengths 780 nm and 880 nm, according to literature values for the mucosa of human maxillary sinuses. For this cover layer, the concentration ratios of a hydrogel solution mixed with a suspension of non-fluorescent polystyrene microspheres as scattering centres were determined via an optimisation program, based on a published code for Mie scattering calculations. The performances of the artificial phantoms were characterised with a multiphoton FLIM microscope and a confocal microscope with regard to the achievable imaging depth, distinguishability of the stairs and their long-time stability. Furthermore, images of the structured phantoms created with all used devices were compared. The investigations carried out within the framework of the present dissertation showed the challenges as well as the limitations of phantoms for simulating the free and protein-bound states of the endogenous fluorophores NADH and FAD with a longterm-stability of more than 40 days. In addition, the manufacturing challenges as well as the limitations of a tissue mimicking phantom with the required x, y, z-resolution were investigated. By means of such a phantom, the maximum imaging depth as well as optical distortion effects could be comparatively examined on two different microscopy systems and an endoscopic system. Based on these developments, protocols for the validation or comparison of dedicated microscope and endoscopic devices can be defined, in order to achieve comparability of reported results and device characteristics. Consequently, with regard to clinical application, the results of metabolic imaging may become more reliable, so that the value of this technique as a diagnostic tool can be further improved.Tumorzellen weisen in der Regel einen veränderten Stoffwechsel im Vergleich zu gesunden Zellen auf. Unabhängig von der Verfügbarkeit von Sauerstoff decken sie ihren Energiebedarf bevorzugt über Glykolyse und nicht über die effektivere oxidative Phosphorellierung (OXPHOS). Dieser Übergang von OXPHOS zu Glykolyse geht mit einer Änderung des Anteils der freien und proteingebundenen, am Komplex I und II der Atmungskette beteiligten, Kofaktoren Nicotinamidadenindinukleotid (NADH) und Flavin-Adenin-Dinukleotid (FAD) einher. Als endogene Fluorophore sind diese in ihrer reduzierten, NADH, und oxidierten, FAD, Form einer Fluoreszenzbildgebung zugänglich. Da sich die Fluoreszenzlebensdauern der freien und proteingebundenen Formen von NADH und FAD signifikant ändern, ist deren Bestimmung über Fluoreszenzlebensdauer-Mikroskopie (Fluorescence Lifetime Imaging Microscopy, FLIM) grundsätzlich geeignet, um in vivo Informationen über den Stoffwechselzustand von Zellen zu erlangen. Jedoch findet FLIM gegenwärtig fast ausschließlich in der Forschung Anwendung, eine klinische Anwendung in der Diagnostik ist selten und nur in der Dermatologie bekannt. Aufgrund der höheren Eindringtiefe und Anregungswellenlängen im infraroten Spektralbereich ist eine Zwei-Photonen-Anregung hierfür vorzuziehen. Die Translation dieser Technologie in eine klinische Anwendung erfordert sowohl eine Validierung des Messverfahrens als auch die Bereitstellung von geeigneten künstlichen Gewebephantomen als reproduzierbare Messobjekte. In der vorliegenden Arbeit erfolgt dies mit dem Ziel der endoskopischen Anwendung im HNO-Bereich, ist aber prinzipiell auf andere Gebiete übertragbar. Bei der Diagnose der im HNO-Bereich dominierenden Plattenepithelkarzinome spielt die Kenntnis der Differenzierung der Tumorzellen sowie der Infiltrationstiefe und die Abgrenzung zu entzündlichen Prozessen eine entscheidende Rolle. Die gegenwärtige Diagnostik erfolgt primär über Weißlicht-Endoskopie und teilweise über Narrow-Band-Immaging (NBI), selten über Optical Coherence Tomography (OCT) oder über Fluoreszenzverfahren. Die letzteren drei Verfahren ermöglichen eine Diagnose über eine verbesserte Darstellung von Gewebestrukturen im Vergleich zur Weißlicht-Endoskopie. Diese Verfahren geben jedoch keinen Aufschluss über den Stoffwechselzustand einer Zelle. In der Klinik ist zusätzlich zur Endoskopie eine Biopsie und zeitaufwändige Gewebepathologie für eine fundierte Diagnose erforderlich. Durch die Auswertung der Fluoreszenzlebensdauern von freiem und proteingebundenem NADH und FAD konnte gezeigt werden, dass eine Unterscheidung sowohl zwischen verschiedenen Zelltypen als auch hochgradigen und niedriggradigen Tumoren prinzipiell möglich ist. Dieses Auswerteverfahren wird als metabolische Bildgebung bezeichnet. Der Vorteil einer Übertragung der metabolischen Bildgebung in die klinische Diagnostik läge darin, noch während der Untersuchung, eine Unterscheidung zwischen Normalgewebe, einer Entzündung und hochgradigem oder niedriggradigem Tumor zur ermöglichen, beziehungsweise Aussagen über eine Infiltration der Basalmembran treffen zu können. Zusätzlich würde sich die Möglichkeit der exakteren Bestimmung von Tumorrändern als es über eine Biopsie oder gegebenenfalls anderen optische Methoden möglich ist eröffnen. Da sich Tumore in ihrem Metabolismus von normalem Gewebe unterscheiden, ist über die Messung des metabolischen Zustands von Zellen beziehungsweise Gewebe grundsätzlich eine Unterscheidung zwischen den vorgenannten Gewebezuständen möglich. Hierfür eignet sich die Bestimmung der relativen Anteile von freiem und proteingebundenem NADH und FAD im untersuchten Zellgebiet. Es wurden vier Berechnungsverfahren von metabolischen Indizes untereinander verglichen. Die publizierten, als NADH Metabolic Index (setzt den Anteil von freiem zu proteingebundenem NADH ins Verhältnis) und Fluorescence Lifetime Induced Redox Ratio (FLIRR; im Folgenden FLIRR1 genannt; setzt proteingebundenes und freies NADH und FAD ins Verhältnis) bekannten Berechnungsverfahren wurden mit zwei Abwandlungen des FLIRR1-Indices, im folgenden FLIRR2 und FLIRR3 genannt, welche freies FAD und zusätzlich proteingebundenes Flavinmononukleotid (FMN) berücksichtigen, verglichen. Durch den Vergleich der metabolischen Indices kann eine Aussage über die grundsätzliche Eignung eines Berechnungsverfahrens zur Bewertung des Zustandes von Zellen oder Gewebe getroffen werden. Diese Erkenntnis alleine ist jedoch nicht für die Entwicklung, Charakterisierung und Validierung eines Gerätes ausreichend. Da sich die optischen Parameter von Probengeweben, auch innerhalb desselben Gewebetyps und der selben Spezies, unterscheiden, werden kontrollierbare und wiederholbare Eigenschaften, wie sie artifizielle Gewebephantomen zur Verfügung stellen, benötigt. Dies wird in der Regel durch die Nutzung von künstlichen (optischen) Gewebephantomen sichergestellt. Im Rahmen der Untersuchungen wurden zwei, unterschiedliche Zwecke erfüllende, künstliche Phantome entwickelt: Eines zur Simulation der Fluoreszenzlebensdauern von freiem und proteingebundenem NADH und FAD und eines, welches über fluoreszierende Objekte bekannter Größe, die Bestimmung der Bildgebungstiefe und gegebenenfalls optischer Verzerrungen oder der Auflösung eines Gerätes ohne z-Stage erlaubt. Die Substanzen NADH und FAD sind nur bedingt als Referenzstandard geeignet, da die in der Literatur angegebenen Werte zur Fluoreszenzlebensdauer zum Teil erheblich schwanken. Außerdem besteht eine Abhängigkeit der Fluoreszenzlebensdauer von der Konzentration und dem pH-Wert. Coumarin 1 und Coumarin 6 wurden als Fluorophore mit monoexponentiellem Abklingverhalten und hinreichend ähnlichen spektralen Eigenschaften zu NADH und FAD identifiziert und ihre Quencheigenschaften untersucht. Auf der Basis einer modifizierten Stern-Volmer-Gleichung wurden die notwendigen Quencherkonzentrationen, um die Fluoreszenzlebensdauern von freiem und proteingebundenem NADH und FAD nachzustellen, ermittelt und im Falle von Coumarin 1 als Substituat für NADH getestet. Weiterhin wurde die Stabilität der Coumarin 1 Lösungen für einen Zeitraum von 41 Tagen verifiziert. Die auf Coumarin 6, als Substituat für FAD, basierenden Fluorophorlösungen wurden im Rahmen einer Kooperation mit dem Multiphoton Imaging Labor der Hochschule München (AG Hellerer) für angewandte Wissenschaften untersucht. Im Gegensatz zu Forschungsmikroskopen verfügen endoskopische Systeme in der Medizin in der Regel nicht über die Möglichkeit zur präzisen Verstellung der Position der Objektebene in der Größenordnung der anatomischen Strukturen der Schleimhäute. Eine automatische Fokussierung in eine gewisse Gewebetiefe ist mit einem handelsüblichen Endoskop nicht möglich. Für die Anwendung zur metabolischen Bildgebung wurde ein Auflösungsvermögen im Bereich der Größe von Mitochondrien (1 µm Durchmesser) angestrebt. Damit ist die optische Auflösung des zu untersuchenden Gerätes in der Größenordnung der, in der Regel in optischen Phantomen eingesetzten Substanzen, um Streuung kontrolliert zu erzeugen. Mit dem Ziel ein Phantom zu entwickeln, welches sowohl mit einem Forschungsmikroskop als auch mit einem Endoskopsystem verwendet werden kann, wurde in einen Glaskörper eine treppenartige Struktur von der Oberfläche aus in das Glasvolumen hineingearbeitet. Mit dieser treppenartigen Struktur kann ein Tiefenbereich bis ca. 250 µm in 5 µm Schritten abgedeckt werden. Die Treppenstruktur wurde mittels Laser-Mikrostrukturierung in Glas-Objektträger (n=10) geschrieben und hinsichtlich der Stufenhöhe, Stufenlänge, Oberflächenbeschaffenheit und Gesamttiefe charakterisiert. Fluoreszierende Strukturen wurden durch eine Beschichtung der Treppenstufenflächen mit fluoreszierenden Polystyrene Microspheres von 1 µm Durchmesser erzeugt. Das herausgearbeitete Volumen wurde später mit einer Substanz mit bekanntem reduziertem Streukoeffizienenten für die Wellenlängen 780 nm und 880 nm aufgefüllt. Für diese Deckschicht wurden die Konzentrationsverhältnisse einer Hydrogellösung mit einer Suspension nicht fluoreszierenden Polystyrene Microspheres als Streuzentren über ein Optimierungsprogramm basierend auf einem publizierten Code zur Berechnung von Mie-Streuung nach Literaturwerten für die Schleimhaut der humanen Kieferhöhlen bestimmt. Die künstlichen Phantome wurden mit einem Multiphotonen-FLIM-Mikroskop und einem Konfokalkmikroskop hinsichtlich der erreichbaren Bildgebungstiefe, Unterscheidbarkeit der Stufen und ihrer Alterungsstabilität charakterisiert. Weiterhin wurden Aufnahmen eines der Phantome mit einem modifizierten FLIM-Tomographen (MPTFlex, Jenlab GmbH, Berlin), an welchen ein Mikro-Endoskop adaptiert wurde, mit den Aufnahmen der beiden Mikroskopsysteme verglichen. Die im Rahmen der vorliegenden Dissertation durchgeführten Untersuchungen zeigten die Fertigungsmöglichkeiten und Limitationen von Phantomen zur Simulation der freien und proteingebundenen Zustände der endogenen Fluorophore NADH und FAD. Die Phantome blieben, im Gegensatz zu NADH und FAD Lösungen, über einen Zeitraum von mehr als 40 Tagen stabil. Mittels eines Zweiten, durch Laser-Mikrostrukturierung erzeugten künstlichen Gewebephantoms, konnten Verzeichnungseffekte, sowie maximale Bildgebungstiefe, auch im Vergleich zwischen zwei unterschiedlichen Mikroskopiesystemen und einem endoskopischen System, analysiert werden. Zusammenfassend konnte mit den durchgeführten Untersuchungen und Entwicklungen ein wesentlicher Beitrag für Vergleichs- und Validierungsverfahren von bildgebenden Systemen in der Biophotonik geleistet werden

EVALUATION OF THE ULTRASTRUCTURE OF THE SMALL INTESTINE OF HIV INFECTED CHILDREN BY TRANSMISSION AND SCANNING ELECTRONIC MICROSCOPY

Objectives To describe HIV children's small intestinal ultrastructural findings. Methods Descriptive, observational study of small intestine biopsies performed between August 1994 and May 1995 at São Paulo, SP, Brazil. This material pertained to 11 HIV infected children and was stored in a laboratory in paraffin blocks. Scanning and transmission electronic microscopy were used to view those intestine samples and ultrastructural findings were described by analyzing digitalized photos of this material. Ethical Committee approval was obtained. Results In most samples scanning microscopy showed various degrees of shortening and decreasing number of microvilli and also completes effacements in some areas. Derangement of the enterocytes was seen frequently and sometimes cells well defined borders limits seemed to be loosened. In some areas a mucous-fibrin like membrane with variable thickness and extension appeared to partially or totally coat the epithelial surface. Fat drops were present in the intestinal lumen in various samples and a bacterium morphologically resembling bacilli was seen in two occasions. Scanning microscopy confirmed transmission microscopy microvilli findings and also showed little “tufts” of those structures. In addition, it showed an increased number of vacuoles and multivesicular bodies inside various enterocytes, an increased presence of intraepithelial lymphocytes, mitochondrial vacuolization and basement membrane enlargement in the majority of samples analyzed. However, some samples exhibited normal aspect. Conclusions Our study showed the common occurrence of various important intestinal ultrastructural alterations with variable degrees among HIV infected children, some of them in our knowledge not described before.Objetivos Descrever achados ultra-estruturais do intestino delgado de crianças infectadas pelo HIV. Métodos Estudo descritivo, observacional de biopsias do intestino delgado, realizada entre agosto de 1994 e maio de 1995 em São Paulo - Brasil. Este material pertencia a 11 crianças infectadas pelo HIV e foi armazenado em um laboratório em blocos de parafina. As amostras de intestino delgado foram analisadas por microscopia eletrônica de transmissão e de varredura e achados os achados ultra-estruturais foram descritos por meio da análise de fotos digitalizadas desse material. Foi obtida aprovação pelo Comitê de Ética. Resultados Na maioria das amostras a microscopia de varredura mostrou vários graus de encurtamento e diminuição do número das microvilosidades e até o completo apagamento dessas estruturas em algumas áreas. O desarranjo dos enterócitos foi visto com freqüência e, por vezes, os limites celulares estavam imprecisos. Em algumas áreas uma membrana fibrino-mucosa com espessura e extensão variáveis aparentava revestir parcial ou totalmente a superfície epitelial. Gotas de gordura no lúmen intestinal estavam presentes em várias amostras e bactérias morfologicamente semelhantes a bacilos foram observadas em duas amostras. A microscopia eletrônica de varredura confirmou as observações constatadas nas microvilosidades através da microscopia de transmissão e também mostrou pequenos “tufos” dessas estruturas. Além disso, mostrou aumento do número de vacúolos e de formações multivesiculares dentro de vários enterócitos, aumento da presença de linfócitos intraepiteliais, vacuolização mitocondrial e alargamento da membrana basal na maioria das amostras analisadas. No entanto, algumas amostras apresentavam-se com aspecto normal. Conclusões Nosso estudo mostrou a ocorrência comum de várias alterações ultra-estruturais intestinais importantes e de intensidade variável entre crianças infectadas pelo HIV, algumas delas, ao nosso conhecimento, não descritas anteriormente.Universidade Federal do Ceara PediatricsUniversidade Federal de São Paulo (UNIFESP) Escola Paulista de MedicinaUniversidade Federal de São Paulo (UNIFESP) Escola Paulista de Medicina Centro de Microscopia EletronicaUNIFESP, EPM, Centro de Microscopia EletronicaSciEL

Estudo ultraestrutural de degeneração transneuronal dos elementos axonais do núcleo paratrigeminal de ratos com desnervação sino-aórtica

OBJECTIVE: Morphological study that searched to authenticate the presence of sinoaortic baroreceptor inputs within the dorsolateral medullary nucleus under electron microscopy analysis. METHODS: After a 5-day survival period, 9 baroreceptor-denervated rats deeply anaesthetized with equithesin were transcardially perfused and their brains were histologically processed. RESULTS: The neuronal cytoarchitecture of the paratrigeminal nucleus comprehends afferent projections from other nuclei that have a distributive character regarding visceral and nociceptive functions in the cardiovascular reflex integration response. CONCLUSION: The medial portion of the nucleus receives afferent projections of the rostral ventrolateral medulla, as shown by retrograde neurotracing studies. The present results show that the medial extent of the paratrigeminal nucleus contains degenerated axoplasmic cellular components in sinoaortic deafferented rats. The number of degenerated axonal fibers was also larger in this area of the nucleus.OBJETIVO: Estudo morfológico que buscou verificar, por meio de microscopia eletrônica, a presença de aferências de receptores sino-aórticos em núcleo localizado na região dorso-lateral bulbar. MÉTODOS: Após 5 dias de sobrevida, 9 ratos com desnervação sino-aórtica anestesiados com equitesina foram submetidos à perfusão transcardíaca, e o encéfalo de cada um deles foi processado histologicamente. RESULTADOS: A citoarquitetura neuronal do núcleo paratrigeminal compreende projeções aferentes de outros núcleos que apresentam uma característica distributiva em relação às funções viscerais e nociceptivas na integração do reflexo cardiovascular. CONCLUSÃO: A porção medial do núcleo recebe projeções aferentes da região rostro-ventrolateral do troncoencefálico, confirmadas por meio de estudos com rastreadores neuronais. Os resultados indicam que a região medial do núcleo paratrigeminal contém o maior número de fibras axonais degeneradas.Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)Hospital Israelita Albert Einstein Instituto do CérebroUniversidade Federal de São Paulo (UNIFESP)Universidade Federal de São Paulo (UNIFESP) Centro de Microscopia EletrônicaUNIFESP, Centro de Microscopia EletrônicaSciEL

Escherichia coli enteroagregativa como agente provocador de diarrea persistente: modelo experimental utilizando microscopia óptica de luz

OBJECTIVE: To examine the interactions of Enteroaggregative Escherichia coli strains with small and large intestinal mucosa, in order to detect potential alterations in both regions of the digestive tract. METHODS: Enteroaggregative Escherichia coli strains, isolated from stools of infants with persistent diarrhea and the prototype strain 042 (O44:H18), isolated from a child with diarrhea in Lima, Peru (positive control), were analised by light microscopy after in vitro organ culture assay of ileal and colonic mucosa. The interactions between the different enteroaggregative Escherichia coli strains and the ileal and colonic mucosa were analysed. RESULTS: Light microscopy analysis suggested an association of enteroaggregative Escherichia coli strains with the epithelium, inducing alterations. These bacteria adhered to both small and large bowel mucosa. The enteroaggregative Escherichia coli strains induced alterations in those areas where they were directly interacting with the epithelium. In the ileum, some areas showed a secondary internalization. CONCLUSIONS: The enteroaggregative Escherichia coli strains could cause persistent diarrhea inducing alterations in the small intestinal structures, where the digestive-absorptive functions take place. Inflammatory lesions observed in colons could justify the colitis described in some children infected by enteroaggregative Escherichia coli.OBJETIVO: Evaluar interacciones de muestras de Escherichia coli enteroagregativa (EAEC) con tejido intestinal humano, a fin de documentar potenciales alteraciones en distintas regiones del tracto digestivo (intestino delgado distal e intestino grueso) y definir, con eso, su rol en la persistencia del proceso diarreico. MÉTODOS: Muestras de EAEC aislada de las heces de niños con diarrea persistente y la muestra prototipo 042, aislada de un niño con diarrea en Lima, Perú (control positivo) fueron analizadas por microscopía óptica de luz (ML) después de siembra en cultura de órgano in vitro de fragmentos de mucosa ileal y del colon. Fueron analizadas las interacciones entra las distintas cepas de EAEC y las mucosas ileal y del colon. RESULTADOS: El análisis por ML indicó asociación de estos microorganismos con el epitelio, provocando alteraciones. Las cepas estudiadas adhirieron a ambas regiones evaluadas: intestino delgado distal y grueso y causaron alteraciones, especialmente en aquellas áreas donde interactuaron directamente con el epitelio. En el íleo, algunas regiones mostraron internalización secundaria. CONCLUSIÓN: Estos agentes pueden causar diarrea persistente mediante alteraciones en el intestino delgado, donde ocurren las funciones digestivo-absortibas. Las lesiones inflamatorias descritas en la mucosa del colon podrían explicar la colitis descrita en algunos niños infectados por EAEC.OBJETIVO: Avaliar interações de amostras de Escherichia coli enteroagregativa com tecido intestinal humano, a fim de documentar potenciais alterações em diferentes regiões do trato digestivo. MÉTODOS: Amostras de Escherichia coli enteroagregativa isoladas das fezes de crianças com diarreia persistente e a amostra protótipo 042, isolada de uma criança com diarreia em Lima, no Peru (controle positivo), foram analisadas por microscopia óptica de luz após semeadura em cultura de orgão in vitro de fragmentos de mucosa ileal e colônica. Foram analisadas as interações entre as diferentes cepas de Escherichia coli enteroagregativa e as mucosas ileal e colônica. RESULTADOS: A análise por microscopia óptica de luz indicou associação destes micro-organismos com o epitélio, provocando alterações. As cepas estudadas aderiram a ambas as regiões avaliadas (intestino delgado distal e grosso) e causaram alterações, especialmente naquelas áreas onde interagiram diretamente com o epitélio. No íleo, algumas regiões mostraram internalização secundária. CONCLUSÕES: Esses agentes podem causar diarreia persistente por meio de alterações no intestino delgado, no qual ocorrem as funções digestivo-absortivas. As lesões inflamatórias descritas na mucosa colônica poderiam explicar a colite mostrada em algumas crianças infectadas por Escherichia coli enteroagregativa.Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)UNIFESP Centro de Microscopia EletrônicaUNIFESPUNIFESP, Centro de Microscopia EletrônicaUNIFESPSciEL

Quantifying intra- and inter-annual dynamics of river-floodplain connectivity and wetland inundation with remote sensing and wavelet analysis

Peer reviewe

Hairy cell leukemia: a histo-cytochemical and ultra-structural study

We studied five patients with hairy cell leukemia (HCL) diagnosed within the last ten years at the Department of Hematology of Universidade Federal de São Paulo (UNIFESP) - Escola Paulista de Medicina. Our purpose was to analyze the value of transmission electron microscopy (TEM) by comparing this method with the conventional ones. At diagnosis, patients presented weight loss, spleen enlargement and hairy cells (HC) in peripheral blood and bone marrow slides. HC was characterized by morphology and tartrate test resistance in the acid phosphatase reaction (TRAP). At the evaluation time, the amount of HC ranged from 1% to 85% of WBC count. All patients, except two, had phenotype B. In these last two, TRAP as well as phenotype B could not be documented due to low HC numbers in their exams. Cytoplasmatic projections and the absence of lamellar ribosomic complex were the most frequent ultrastructural findings, even in those patients with the lowest HC numbers. Based on these features, TEM is an efficient method for searching for HC at HCL diagnosis and during the course of the disease.Estudamos cinco pacientes com leucemia de células pilosas (LCP), diagnosticada nos últimos dez anos na Disciplina de Hematologia da Escola Paulista de Medicina. O principal objetivo foi analisar o valor da microscopia eletrônica de transmissão (MET) pela comparação deste método com os convencionais. Pacientes apresentavam no diagnóstico perda de peso, esplenomegalia e células pilosas (CP) no sangue periférico e medula óssea. As CP foram caracterizadas pela morfologia e resistência ao tartarato na reação de fosfatase ácida (FATR). Na época em que foram avaliados, a quantidade de CP variou de 1 a 85% da leucometria. Todos os pacientes tinham fenótipo B, excetuando-se dois nos quais FATR e imunofenótipo não puderam ser documentados devido ao baixo número de CP no sangue periférico e medula óssea. Projeções citoplasmáticas e ausência de complexo ribosômico lamelar foram os mais freqüentes achados na MET, mesmo nos pacientes com baixa porcentagem de CP. Baseado nestes achados concluímos que MET é um método eficiente no diagnóstico e evolução da LCP.Universidade Federal de São Paulo (UNIFESP)UNIFESPSciEL

Comparative investigation of reusable and single-use flexible endoscopes for urological interventions

In order to evaluate the technical adaptability of a type of disposable endoscope compared to reusable flexible endoscopes, in vitro and in vivo studies were conducted. A disposable digital ureteroscope ("chip on tip") and two reusable endoscopes were investigated with respect to spatial resolution, geometric distortion in air and water the maximum. Additionally, the clinical performance of the disposable device was tested during clinical procedures (n = 20). The disposable endoscope showed an optical resolution of 6.72 lines/mm at 10 mm distance, similar to the other devices. In comparison, the disposable endoscope showed a barrel-shaped image distortion in air of -24.2%, which is in the middle range, but was best under water (-8.6%). The bendability of 297 degrees (275 mu m fiber) and 316 degrees (empty channel, 1.5 F basket) and the maximum irrigation (1 m: 58.1 ml/min, 2 m: 91.9 ml/min) were convincing. Clinically the maneuverability was very good in (13/20), good or satisfactory in (7/20). Visibility was evaluated as very good in (11/20), just in (1/20) either satisfactory or sufficient. The consistency of visibility was not affected in (19/20). In all cases there were no adverse events. The technical examination and clinical application of the disposable endoscope are of equal quality compared to reusable devices. Disposable endoscopes can be an alternative to reusable devices, but economic aspects such as reduction of repair costs, sterilization effort and additional waste must be taken into account

Quantifying intra‐ and inter‐annual dynamics of river‐floodplain connectivity and wetland inundation with remote sensing and wavelet analysis

We used imagery from remote sensing (FORCE Time Series Analysis submodule (combining Landsat and Sentinel-2 imagery) to derive spatially distributed times series (8 years) of NDWI data to infer patterns of floodplain inundation and river-floodplain connectivity in two contrasting polders in the Lower Oder Valley National Park. The upstream Polder A (14.4 km2) was extensively flooded for prolonged periods most winters. Wavelet analysis showed that this strong seasonality was primarily driven by winter water levels in the river Oder that could enter and leave the polder through two opened floodgates. Subsequent drainage was slow and aided by a pumping station. Inundation of the downstream Polder 10 (17.7km2) was lower and had less marked seasonality. This reflected the impact of flood attenuation by storage in Polder A upstream, but also the greater connectivity (via 10 floodgates) to the Oder and a functional network of channels, which facilitated rapid drainage after flood peaks. In Polder A, secondary periods of transient inundation could also occur in response to local intense summer rainfall. Wavelet analysis also showed that groundwater recharge in and around Polder A is primarily induced by floodwater, whilst Polder 10 also reflects the influence of local rainfall-driven recharge. The flood regimes of the two polders showed marked inter-annual variation, largely dependent on flows from the upper Oder catchment. Understanding these patterns and processes of inundation is important for both managing flows and sustaining valuable wetland habitats within the National Park. Given projected climate change in eastern Europe and possible management alterations to the flow regime of the Oder, the potential implications for these habitats needs urgent attention.Leibnitz Association GermanyChina Scholarship Council http://dx.doi.org/10.13039/501100004543Einstein Stiftung Berlin http://dx.doi.org/10.13039/501100006188Berlin University Alliance http://dx.doi.org/10.13039/501100021727Peer Reviewe