IL-18BP mediates the balance between protective and pathological immune responses to Toxoplasma gondii

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Back to basics: an evaluation of NaOH and alternative rapid DNA extraction protocols for DNA barcoding,genotyping, and disease diagnostics from fungal and oomycete samples

The ubiquity, high diversity and often‐cryptic manifestations of fungi and oomycetes frequently necessitate molecular tools for detecting and identifying them in the environment. In applications including DNA barcoding, pathogen detection from plant samples, and genotyping for population genetics and epidemiology, rapid and dependable DNA extraction methods scalable from one to hundreds of samples are desirable. We evaluated several rapid extraction methods (NaOH, Rapid one‐step extraction (ROSE), Chelex 100, proteinase K) for their ability to obtain DNA of quantity and quality suitable for the following applications: PCR amplification of the multicopy barcoding locus ITS1/5.8S/ITS2 from various fungal cultures and sporocarps; single‐copy microsatellite amplification from cultures of the phytopathogenic oomycete Phytophthora ramorum; probe‐based P. ramorum detection from leaves. Several methods were effective for most of the applications, with NaOH extraction favored in terms of success rate, cost, speed and simplicity. Frozen dilutions of ROSE and NaOH extracts maintained PCR viability for over 32 months. DNA from rapid extractions performed poorly compared to CTAB/phenol‐chloroform extracts for TaqMan diagnostics from tanoak leaves, suggesting that incomplete removal of PCR inhibitors is an issue for sensitive diagnostic procedures, especially from plants with recalcitrant leaf chemistry. NaOH extracts exhibited lower yield and size than CTAB/phenol‐chloroform extracts; however, NaOH extraction facilitated obtaining clean sequence data from sporocarps contaminated by other fungi, perhaps due to dilution resulting from low DNA yield. We conclude that conventional extractions are often unnecessary for routine DNA sequencing or genotyping of fungi and oomycetes, and recommend simpler strategies where source materials and intended applications warrant such use.     

Reliability of a computational platform as a surrogate for manually interpreted immunohistochemical markers in breast tumor tissue microarrays

BackgroundPathologist and computational assessments have been used to evaluate immunohistochemistry (IHC) in epidemiologic studies. We compared Definiens Tissue Studio® to pathologist scores for 17 markers measured in breast tumor tissue microarrays (TMAs) [AR, CD20, CD4, CD8, CD163, EPRS, ER, FASN, H3K27, IGF1R, IR, Ki67, phospho-mTOR, PR, PTEN, RXR, and VDR].Methods5 914 Nurses’ Health Study participants, diagnosed 1976–2006 (NHS) and 1989–2006 (NHS-II), were included. IHC was conducted by the Dana-Farber/Harvard Cancer Center Specialized Histopathology Laboratory. The percent of cells staining positive was assessed by breast pathologists. Definiens output was used to calculate a weighted average of percent of cells staining positive across TMA cores for each marker. Correlations between pathologist and computational scores were evaluated with Spearman correlation coefficients. Receiver-operator characteristic curves were constructed, using pathologist scores as comparison.ResultsSpearman correlations between pathologist and Definiens assessments ranged from weak (RXR, rho=-0.05; CD163, rho = 0.10) to strong (Ki67, rho = 0.79; pmTOR, rho = 0.77). The area under the curve was >0.70 for all markers except RXR.ConclusionOur data indicate that computational assessments exhibit variable correlations with interpretations made by an expert pathologist, depending on the marker evaluated. This study provides evidence supporting the use of computational platforms for IHC evaluation in large-scale epidemiologic studies, with the caveat that pilot studies are necessary to investigate agreement with expert assessments. In sum, computational platforms may provide greater efficiency and facilitate high-throughput epidemiologic analyses.     

Image Analysis Algorithms for Immunohistochemical Assessment of Cell Death Events and Fibrosis in Tissue Sections

Cell death is of broad physiological and pathological importance, making quantification of biochemical events associated with cell demise a high priority for experimental pathology. Fibrosis is a common consequence of tissue injury involving necrotic cell death. Using tissue specimens from experimental mouse models of traumatic brain injury, cardiac fibrosis, and cancer, as well as human tumor specimens assembled in tissue microarray (TMA) format, we undertook computer-assisted quantification of specific immunohistochemical and histological parameters that characterize processes associated with cell death. In this study, we demonstrated the utility of image analysis algorithms for color deconvolution, colocalization, and nuclear morphometry to characterize cell death events in tissue specimens: (a) subjected to immunostaining for detecting cleaved caspase-3, cleaved poly(ADP-ribose)-polymerase, cleaved lamin-A, phosphorylated histone H2AX, and Bcl-2; (b) analyzed by terminal deoxyribonucleotidyl transferase–mediated dUTP nick end labeling assay to detect DNA fragmentation; and (c) evaluated with Masson''s trichrome staining. We developed novel algorithm-based scoring methods and validated them using TMAs as a high-throughput format. The proposed computer-assisted scoring methods for digital images by brightfield microscopy permit linear quantification of immunohistochemical and histochemical stainings. Examples are provided of digital image analysis performed in automated or semiautomated fashion for successful quantification of molecular events associated with cell death in tissue sections. (J Histochem Cytochem 57:649–663, 2009)     

Hypoxia-inducible factor 1-alpha and vascular endothelial growth factor in cartilage tumors

Neoangiogenesis has been demonstrated in chondrosarcoma. Anti-angiogenic therapies are being tested in clinical trials for chondrosarcomas. Studies of the underlying mechanisms have been performed almost exclusively in cell lines. We immunostained 20 samples of chondrosarcoma and 20 samples of enchondromas with antibodies against hypoxia-inducible factor 1-alpha (HIF-1-alpha) and vascular endothelial growth factor (VEGF). The immunohistochemical staining of HIF-1-alpha and VEGF were highly correlated. Enchondromas were HIF-1-alpha and VEGF negative, whereas all chondrosarcoma exhibited HIF-1-alpha and VEGF immunostaining. HIF-1-alpha/VEGF double positive cases were almost exclusively chondrosarcomas with a high tumor grade. We suggest that HIF-1-alpha is a marker of malignancy in chondrosarcomas that correlates with tumor neo-angiogenesis. Our findings also suggest that a HIF-1-alpha/VEGF angiogenic pathway may exist in chondrosarcoma in vivo as in other malignant tumors. The inclusion of novel inhibitors to HIF-1-alpha and other factors may optimize anti-angiogenic interventions in chondrosarcoma.     

Proteomic tools against the neglected pathology of snake bite envenoming

This article covers the application of proteomic tools (‘venomics’, ‘antivenomics’ and ‘venom phenotyping’) to study the composition and natural history of snake venoms, and the cross-reactivity of antivenoms with homologous and heterologous venoms, to help address the neglected pathology of snake bite envenoming. The identification of evolutionary and immunological trends may help to replace the traditional geographic- and phylogenetic-driven hypotheses for antivenom production strategies with a more rational approach based on proteome phenotype and immunological profile similarities. Antivenomics and venom phenotyping may also contribute to expand the clinical range of currently existing antidotes.     

The effect on Orchestia hurleyi (Amphipoda: Talitridae) of a whitey disease caused by Bacillus subtilis

Abstract

Field observations made over 10 years suggested that a bacterial disease of adults of the terrestrial amphipod Orchestia hurleyi Duncan, caused by Bacillus subtilis, is progressing southwards down the eastern side of New Zealand's South Island. As the disease spread, amphipod density appeared to decline and population age structure became truncated. In the vicinity of Dunedin and further south the amphipods are still disease-free. Signs of the disease are a progressive weakening and wasting. The animal cannot jump, and its speed of walking is reduced. Its body becomes opaque white instead of the normal translucent reddish-brown. Diseased females do not brood. There is no evidence that diseased animals moult. Death is caused by general wasting or by predators. The disease-causing organism was isolated, and healthy amphipods were re-infected from the isolate. Signs of the disease were apparent within 7 days of inoculation. The presence of the disease-causing organism in the haemocoel causes host defences to be mobilised, as shown by elevated haemocyte counts (4512 mm^?3, cf. 300 mm^?3 in healthy, disease-free adults), but as the disease progresses the animal's defences are overcome, and haemocyte counts fall to an average of 784 mm^?3 during the later stages of disease. The blood of terminally diseased amphipods is thick and creamy white, packed with motile bacterial cells, and few (if any) haemocytes are present in the circulation. Two populations were studied, one disease-free (at Dunedin) and the other heavily diseased (at Christchurch). The incidence of disease (as measured by a performance test) was about 30%r in Christchurch adults. The disease-causing strain of B. subtilis was found on the body surface of almost all adults in the diseased population. It is possible that the bacterium gains entry to the haemocoel through wounds suffered during ecdysis, conflict, or predator attack. The main differences shown by the diseased population relative to the disease-free population were: lower average density (992 m^?2, cf. 1677 m^?2); lower maximum density (3104 m^?2, cf. 9971 m^?2); smaller average size, with fewer adult instars; a smaller proportion of females brooding in each instar; and much lower egg production. The brood size/mother age relationship was the same for both populations — number of eggs in brood = -4.9 + 0.64(instar number of mother)—because in the diseased population only healthy females breed. Lower egg production in the diseased population reflects the smaller proportion of healthy females, and the number of broods per female is lower since life expectancy is much less. A computer model based on Leslie matrices was used to simulate the ecological effects of the disease. It gave predictions which conformed with the observed population features with respect to age structure and density.     

Differentiation of ovarian and testicular interstitial cells during embryonic and post-embryonic development in mice

Summary Different steps in mouse ovarian and testicular development have been studied in order to compare the time sequences during the in vivo differentiation of steroidogenic cell populations growing in contact with male and female gonocytes. These time sequences indicated a basic common developmental pattern: early signs of steroid synthesis in the male gonad, but late entering into meiotic prophase of XY germ cells; early meiosis but late steroidogenic activity in the ovary. In both male and female interstitial tissues, signs of involution were found following a period of exponential development.Dedicated to Prof. Dr. med. H. Leonhardt on the occasion of his 60th birthdaySupported by the Deutsche Forschungsgemeinschaft (Pe 104/8)We wish to thank Dr. B. Nabarra, M.-F. Rousseau-Merck and D. Sandoz for helpful advices throughout this study, as well as Mrs. L. Andrianarison and Mrs. R. Sprang for skilful assistance     

Long-lasting sonographic and histopathological findings in cured clonorchiasis of rabbits.

To ascertain residual sonographic and histopathological findings of clonorchiasis after treatment, the present study evaluated sonographic findings in rabbits which were infected with 500 metacercariae of C. sinensis every 6 months for 18 months after treatment with praziquantel. The sonographic findings were analyzed in terms of intrahepatic bile duct dilatation and periductal echogenicity, and histopathological findings were observed after the last sonographic examination. Compared with the sonographic findings before treatment, dilatation of the intrahepatic bile ducts became mild to some degree in four of the seven cases and increased periductal echogenicity resolved in four of them. The histopathological specimens after 18 months showed that periductal inflammation has almost resolved but moderate dilatation of the intrahepatic ducts and mucosal hyperplasia persisted. The periductal fibrosis minimally resolved. The long-lasting sonographic findings in cured clonorchiasis make sonography less specific.