Quantitative proteome analysis in cardiovascular physiology and pathology. I. Data processing

Methodological evaluation of the proteomic analysis of cardiovascular-tissue material has been performed with a special emphasis on establishing examinations that allow reliable quantitative analysis of silver-stained readouts. Reliability, reproducibility, robustness and linearity were addressed and clarified. In addition, several types of normalization procedures were evaluated and new approaches are proposed. It has been found that the silver-stained readout offers a convenient approach for quantitation if a linear range for gel loading is defined. In addition, a broad range of a 10-fold input (loading 20-200 microg per gel) fulfills the linearity criteria, although at the lowest input (20 microg) a portion of protein species will remain undetected. The method is reliable and reproducible within a range of 65-200 microg input. The normalization procedure using the sum of all spot intensities from a silver-stained 2D pattern has been shown to be less reliable than other approaches, namely, normalization through median or through involvement of interquartile range. A special refinement of the normalization through virtual segmentation of pattern, and calculation of normalization factor for each stratum provides highly satisfactory results. The presented results not only provide evidence for the usefulness of silver-stained gels for quantitative evaluation, but they are directly applicable to the research endeavor of monitoring alterations in cardiovascular pathophysiology.

[Implantation: physiology, pathology and therapeutic options in disorders of implantation]

So far, implantation is a poorly understood process, which involves several paradoxical cell-biological mechanisms. First, 50% of the embryo is paternal and immunologically foreign material, and second, both the endometrium and embryo are covered by epithelial tissue to prevent cellular fusion. The adhesion and invasion of the blastocyst require an accurate coordination of embryonic and endometrial physiology and the modulation of maternal immune tolerance. Endometrial function plays an important role in assisted reproduction. Pathologies such as fibroids, hydrosalpinges, endometriosis and the polycystic ovary syndrome have a significant negative impact on implantation but can be treated in most cases. Therapeutic strategies to improve endometrial and embryonic function in recurrent implantation disorders are however still controversially discussed.

Proliferative kidney disease in rainbow trout: time- and temperature-related renal pathology and parasite distribution

Proliferative kidney disease is a parasitic infection of salmonid fishes caused by Tetracapsuloides bryosalmonae. The main target organ of the parasite in the fish is the kidney. To investigate the influence of water temperature on the disease in fish, rainbow trout Oncorhynchus mykiss infected with T bryosalmonae were kept at 12 degrees C and 18 degrees C. The number of parasites, the type and degree of lesions in the kidney and the mortality rate was evaluated from infection until full development of disease. While mortality stayed low at 12 degrees C, it reached 77% at 18 degrees C. At 12 degrees C, pathological lesions were dominated by a multifocal proliferative and granulomatous interstitial nephritis. This was accompanied by low numbers of T. bryosalmonae, mainly located in the interstitial lesions. With progression of the disease, small numbers of parasites appeared in the excretory tubuli, and parasite DNA was detected in the urine. Parasite degeneration in the interstitium was observed at late stages of the disease. At 18 degrees C, pathological lesions in kidneys were more severe and more widely distributed, and accompanied by significantly higher parasite numbers. Distribution of parasites in the renal compartments, onset of parasite degeneration and time course of appearance of parasite DNA in urine were not clearly different from the 12 degrees C group. These findings indicate that higher mortality at 18 degrees C compared to 12 degrees C is associated with an enhanced severity of renal pathology and increased parasite numbers.

Background pathology of the ovary in a laboratory population of zebrafish Danio rerio

Adult zebrafish Danio rerio originating from one stock used as control animals in a toxicological study were examined histopathologically for the occurrence of spontaneous lesions in the gonads. While no histopathological changes were seen in the testes, the ovaries showed lesions consisting mainly of acute granulomatous inflammation with increased atresia and the presence of egg debris in the ovarian parenchyma and in the oviduct. Since infectious agents could not be detected and the fish were not exposed to toxicants, we consider these lesions as spontaneous alterations of the ovaries.

Foundations of stochastic geometry and theory of random sets

The first section of this chapter starts with the Buffon problem, which is one of the oldest in stochastic geometry, and then continues with the definition of measures on the space of lines. The second section defines random closed sets and related measurability issues, explains how to characterize distributions of random closed sets by means of capacity functionals and introduces the concept of a selection. Based on this concept, the third section starts with the definition of the expectation and proves its convexifying effect that is related to the Lyapunov theorem for ranges of vector-valued measures. Finally, the strong law of large numbers for Minkowski sums of random sets is proved and the corresponding limit theorem is formulated. The chapter is concluded by a discussion of the union-scheme for random closed sets and a characterization of the corresponding stable laws.

Pathology of sarcoptic mange in red foxes (Vulpes vulpes): macroscopic and histologic characterization of three disease stages

Sarcoptic mange is a highly contagious skin disease that can have a devastating impact on affected wild mammal populations. There are notable variations in the clinical and pathologic picture of sarcoptic mange among species and among conspecifics. However, the origin of these variations is unclear. We propose a classification scheme for skin lesions associated with Sarcoptes scabiei infestation to provide a basis for a subsequent risk factor analysis. We conducted a case-control study focused on macroscopic and histologic examination of the skin, using 279 red foxes (Vulpes vulpes) found dead or shot in Switzerland between November 2004 and February 2006. All animals were submitted to gross necropsy following a detailed protocol. Selection criteria for cases (n=147) vs. controls (n=111) were the presence or absence of mange-like lesions, mite detection by isolation or histologic examination, and serologic testing for S. scabiei antibodies. Characteristic features of mange lesions were scored macroscopically in all foxes and histologically in 67 cases and 15 controls. We classified skin lesions and associated necropsy findings into three types of mange: A) early stage (n=45): focal-extensive skin lesions, thin crusts, mild to moderate alopecia, few mites, numerous eosinophils, and mild lymph node enlargement; B) hyperkeratotic, fatal form (n=86): generalized skin lesions, thick crusts with or without alopecia, foul odor, abundance of mites, numerous bacteria and yeasts, numerous lymphocytes and mast cells, severe lymph node enlargement, and emaciation; C) alopecic, healing form (n=16): focal lesions, no crusts, severe alopecia, hyperpigmentation and lichenification, absence of mites, mixed cell infiltration, and rare mild lymph node enlargement. We hypothesize that after stage A, the animal either enters stage B and dies, or stage C and survives, depending on largely unknown extrinsic or intrinsic factors affecting the host ability to control mite infestation.

Pathology working group review of histopathologic specimens from three laboratory studies of diclofenac in trout.

While the pathology peer review/pathology working group (PWG) model has long been used in mammalian toxicologic pathology to ensure the accuracy, consistency, and objectivity of histopathology data, application of this paradigm to ecotoxicological studies has thus far been limited. In the current project, the PWG approach was used to evaluate histopathologic sections of gills, liver, kidney, and/or intestines from three previously published studies of diclofenac in trout, among which there was substantial variation in the reported histopathologic findings. The main objectives of this review process were to investigate and potentially reconcile these interstudy differences, and based on the results, to establish an appropriate no observed effect concentration (NOEC). Following a complete examination of all histologic sections and original diagnoses by a single experienced fish pathologist (pathology peer review), a two-day PWG session was conducted to allow members of a four-person expert panel to determine the extent of treatment-related findings in each of the three trout studies. The PWG was performed according to the United States Environmental Protection Agency (US EPA) Pesticide Regulation (PR) 94-5 (EPA Pesticide Regulation, 1994). In accordance with standard procedures, the PWG review was conducted by the non-voting chairperson in a manner intended to minimize bias, and thus during the evaluation, the four voting panelists were unaware of the treatment group status of individual fish and the original diagnoses associated with the histologic sections. Based on the results of this review, findings related to diclofenac exposure included minimal to slightly increased thickening of the gill filament tips in fish exposed to the highest concentration tested (1,000 μg/L), plus a previously undiagnosed finding, decreased hepatic glycogen, which also occurred at the 1,000 μg/L dose level. The panel found little evidence to support other reported effects of diclofenac in trout, and thus the overall NOEC was determined to be >320 μg/L. By consensus, the PWG panel was able to identify diagnostic inconsistencies among and within the three prior studies; therefore this exercise demonstrated the value of the pathology peer review/PWG approach for assessing the reliability of histopathology results that may be used by regulatory agencies for risk assessment.