Intestinal Organoids in Colitis Research: Focusing on Variability and Cryopreservation

In recent years, stem cell-derived organoids have become a cell culture standard that is widely used for studying various scientific issues that were previously investigated through animal experiments and using common tumor cell lines. After their initial hype, concerns regarding their standardization have been raised. Here, we aim to provide some insights into our experience in standardizing murine colonic epithelial organoids, which we use as a replacement method for research on inflammatory bowel disease. Considering good scientific practice, we examined various factors that might challenge the design and outcome of experiments using these organoids. First, to analyze the impact of antibiotics/antimycotics, we performed kinetic experiments using ZellShield® and measured the gene expression levels of the tight junction markers Ocln, Zo-1, and Cldn4, the proliferation marker Ki67, and the proinflammatory cytokine Tnfα. Because we found no differences between cultivations with and without ZellShield®, we then performed infection experiments using the probiotic Escherichia coli Nissle 1917 as an already established model setup to analyze the impact of technical, interexperimental, and biologic replicates. We demonstrate that interexperimental differences pose the greatest challenge for reproducibility and explain our strategies for addressing these differences. Additionally, we conducted infection experiments using freshly isolated and cryopreserved/thawed organoids and found that cryopreservation influenced the experimental outcome during early passages. Formerly cryopreserved colonoids exhibited a premature appearance and a higher proinflammatory response to bacterial stimulation. Therefore, we recommend analyzing the growth characteristics and reliability of cryopreserved organoids before to their use in experiments together with conducting several independent experiments under standardized conditions. Taken together, our findings demonstrate that organoid culture, if standardized, constitutes a good tool for reducing the need for animal experiments and might further improve our understanding of, for example, the role of epithelial cells in inflammatory bowel disease development

Distribution of immune cells in head and neck cancer: CD8+ T-cells and CD20+ B-cells in metastatic lymph nodes are associated with favourable outcome in patients with oro- and hypopharyngeal carcinoma

<p>Abstract</p> <p>Background</p> <p>Tumour infiltrating lymphocytes (TIL) are generally considered to represent a host immune response directed against tumour antigens. TIL are also increasingly recognised as possible prognostic parameters. However, the effects observed are variable indicating that results cannot be extrapolated from type of tumour to another. Moreover, it has been suggested that primary solid tumours may be ignored by the immune system and that a meaningful immune response is only mounted in regional lymph nodes.</p> <p>Methods</p> <p>We have examined the local distribution of immune cells in tumour-related compartments in head and neck squamous cell carcinomas (HNSCC). In a second step, the prognostic impact of these cells on disease-free survival (DFS) was analysed. A total of 198 tissue cores from 33 patients were evaluated using tissue mircroarray technique and immunohistochemistry. Tumour-infiltrating immune cells were identified using antibodies specific for CD3, CD8, GranzymeB, FoxP3, CD20 and CD68 and quantified using an image analysis system.</p> <p>Results</p> <p>We demonstrate a relative expansion of FoxP3⁺regulatory T-cells (Treg) and of cytotoxic T-cells among tumour infitrating T-cells. We also show that intratumoural CD20⁺B-cells are significantly more frequent in metastatic deposits than in primary tumours. Furthermore, we observed a reduced number of peritumoural CD8⁺T-cells in metastatic lymph nodes as compared to univolved regional nodes suggesting a local down-modulation of cellular immunity. All other immune cells did not show significant alterations in distribution. We did not observe an association of tumour infiltrating immune cells at the primary site with outcome. However, increased numbers of intraepithelial CD8⁺TIL in metastatic tumours as well as large numbers of peritumoural B-cells in lymph node metastases were associated with favourable outcome. Unexpectedly, no effect on patient outcome was observed for Treg in any compartment.</p> <p>Conclusion</p> <p>Our results suggest that alterations in lymphocyte distribution in regional lymph nodes rather than at the primary tumour site may be relevant for patient prognosis. Moreover, we demonstrate that in addition to cellular immunity humoral immune responses may be clinically relevant in anti-tumour immunity.</p

Non-small cell lung cancer-pathology and biology

BackgroundNon-small cell lung cancer (NSCLC) accounts for ca.75% of malignant epithelial neoplasms of the lungs. In recent years profound insight has been gained regarding the molecular mechanisms of lung carcinogenesis and subsequently new targeted therapies as well as immunotherapies have been developed. These advances have had asignificant impact on routine diagnostics in pathology.ObjectiveThe article aims to give an overview of the most common histological subtypes of NSCLC as well as the morphological, immunohistochemical and molecular characteristics.Material and methodsSelective search of the PubMed database.Results and discussionAdenocarcinomas, squamous cell carcinomas and large cell carcinomas are the most common histological subtypes. With the ancillary methods available in routine pathology even poorly differentiated tumors can be assigned to these entities. The NSCLC show numerous genetic changes of which alterations of EGFR, MET, ALK1 and ROS1 are target structures for personalized therapy

Genotype/Phenotype Correlations in Patients with Hereditary Breast Cancer

Of all breast cancer cases, 5-10% can be attributed to germline mutations, and the high-susceptibility genes BRCA1 and BRCA2 account for about 25-28% of these cases. For the remainder, several genes of moderate and low penetrance have been discovered. Histopathologic characteristics have been studied in small cohorts, but for most of the known non-BRCA1/2-associated hereditary breast cancers, the histologic and immunohistochemical phenotypes are not yet identified. Particularly BRCA1 tumors are associated with a distinct morphology and immunohistochemical characteristics that differ from sporadic breast cancer of age-matched controls. The recognition of features characteristic of these mutations can be helpful to identify patients likely to carry a germline mutation and to assess which gene should be screened for first, in families with a high occurrence of breast and ovarian cancer

First biodegradable metal stent in a child with congenital heart disease: Evaluation of macro and histopathology

New developments in stent technology led to the first biodegradable magnesium stents To overcome the fundamental restrictions of conventional stent implantation, these new stents may improve interventional therapy, also in small children. What remains after complete degradation of a magnesium stent is of particular interest and concern. At the autopsy, 2 months after the projected complete degradation time of the 3.0 x 10 mm(2) stent, no solid compounds were detected, and the vessel diameter had increased slightly to 3.7 mm. Histological preparation revealed an amorphous to jelly-like substitute of the magnesium struts mainly consisting of calcium phosphate covered by fibrotic tissue. Immunological staining revealed no relevant inflammatory reaction to the stent material. Neointima proliferation was detected around the struts with some cellular infiltration of the calcium-phosphate material. These pathological and histological findings show minimal alteration of the vessel wall and an increase of the arterial diameter after stent degradation. This is an important precondition for further use of biodegradable stents in small infants. Further observations have to prove whether these findings do reproduce in other settings also. (C) 2007 Wiley-Liss, Inc

Comparative expression profiling for human endoplasmic reticulum-resident aminopeptidases 1 and 2 in normal kidney versus distinct renal cell carcinoma subtypes

Altered expression of the ER-resident aminopeptidases ERAP1 and ERAP2 might play an important role in shaping the MHC class I-presented peptide repertoire, but their function in tumors has not been determined in detail. Thus, the expression of ERAP1, ERAP2 and HLA class I heavy chain (HC) was analysed in various renal tumor types and corresponding kidney parenchyma by immunohistochemistry. Additionally, comparative expression profilings of untreated versus interferon (IFN)-γ-treated RCC cell lines were performed applying qRT-PCR, Western blot and/or flow cytometry. Normal kidney tissues showed strong ERAP1 staining in the proximal tubules of 57.4 % of cases, in the distal tubules of 94.3 % of cases and in the medulla of 88.6 % of cases, whereas high ERAP2 levels were observed in the medulla of 77.1 % of cases and in both, proximal and distal tubules of about 88 % of cases. Imbalanced, downregulated and RCC subtype-specific ERAP1 or ERAP2 expression was detected in 12.7 % or 43.8 % of samples analyzed, respectively. A coordinated downregulation of ERAPs was found in 4.8 %, an upregulation of ERAP1 or ERAP2 in 22.8 % or 2.0 % of RCC lesions. No association exists between ERAP and HLA class I HC expression for any tissue type. A heterogeneous constitutive ERAP expression pattern was also detected in RCC cell lines with lower ERAP2 than ERAP1 expression levels, which was in 11/17 RCC cell lines inducible by IFN-γ. Conclusively, ERAP1 and ERAP2 might be involved in the development of immune escape mechanisms of RCC

Prediction of Radioresistant Prostate Cancer Based on Differentially Expressed Proteins

Introduction: Although relapses after radiotherapy are common in prostate cancer (PCA) patients, those with a high risk for radioresistance cannot be identified prior to treatment yet. Therefore, this proof-of-concept study was performed to compare protein expression profiles of patients with radio-recurrent PCA to patients treated with primary radical prostatectomy separated by Gleason risk groups. We hypothesized that radio-recurrent PCA have a similar protein expression as high-risk Gleason PCA. Methods: Patient cohorts consisted of (i) 31 patients treated with salvage prostatectomy for locally recurrent PCA after primary radiotherapy and (ii) 94 patients treated with primary prostatectomy split into a Gleason high-risk (>= 4 + 3; n = 42 [44.7%]) versus a low-risk group (<= 3 + 4; n = 52 [55.3%]). Immunohistochemistry was performed using 15 antibodies with known association to radioresistance in PCA in vitro. ELISA was used for validation of selected markers in serum. Results: Androgen receptor (AR) was overexpressed in most radio-recurrent PCA (89.7%) and in most primary high-risk Gleason PCA (87.8%; p = 0.851), while only 67.3% of the low-risk group showed an expression (p = 0.017). Considering the highest Gleason pattern in primary PCA, aldo-keto reductase family 1 member C3 (AKR1C3) was most similarly expressed by patients with radio-recurrent PCA and patients with Gleason patterns 4 and 5 (p = 0.827 and p = 0.893) compared to Gleason pattern 3 (p = 0.20). These findings were supported by ELISA. Conclusion: This is the first study to evaluate protein markers in order to predict radioresistance in PCA. Our results point to AR and AKR1C3 as the most promising markers that might help stratify patients for radiotherapy