Bovine meat and milk factor protein expression in tumor-free mucosa of colorectal cancer patients coincides with macrophages and might interfere with patient survival.

Bovine milk and meat factors (BMMFs) are plasmid-like DNA molecules isolated from bovine milk and serum, as well as the peritumor of colorectal cancer (CRC) patients. BMMFs have been proposed as zoonotic infectious agents and drivers of indirect carcinogenesis of CRC, inducing chronic tissue inflammation, radical formation and increased levels of DNA damage. Data on expression of BMMFs in large clinical cohorts to test an association with co-markers and clinical parameters were not previously available and were therefore assessed in this study. Tissue sections with paired tumor-adjacent mucosa and tumor tissues of CRC patients [individual cohorts and tissue microarrays (TMAs) (n = 246)], low-/high-grade dysplasia (LGD/HGD) and mucosa of healthy donors were used for immunohistochemical quantification of the expression of BMMF replication protein (Rep) and CD68/CD163 (macrophages) by co-immunofluorescence microscopy and immunohistochemical scoring (TMA). Rep was expressed in the tumor-adjacent mucosa of 99% of CRC patients (TMA), was histologically associated with CD68+ /CD163+ macrophages and was increased in CRC patients when compared to healthy controls. Tumor tissues showed only low stromal Rep expression. Rep was expressed in LGD and less in HGD but was strongly expressed in LGD/HGD-adjacent tissues. Albeit not reaching statistical significance, incidence curves for CRC-specific death were increased for higher Rep expression (TMA), with high tumor-adjacent Rep expression being linked to the highest incidence of death. BMMF Rep expression might represent a marker and early risk factor for CRC. The correlation between Rep and CD68 expression supports a previous hypothesis that BMMF-specific inflammatory regulations, including macrophages, are involved in the pathogenesis of CRC.

Structural expression of bovine milk and meat factors in tissues of colorectal, lung and pancreatic cancer patients.

Chronic inflammation, linked to the presence of bovine milk and meat factors (BMMFs) and specific subsets of macrophages, results in oxygen radical synthesis and induction of mutations in DNA of actively replicating cells and replicating single stranded DNA. Cancers arising from this process have been characterized as indirect carcinogenesis by infectious agents (without persistence of genes of the agent in premalignant or cancers cells). Here, we investigate structural properties of pleomorphic vesicles, regularly identified by staining peritumor tissues of colorectal, lung and pancreatic cancer for expression of BMMF Rep. The latter represents a subgroup of BMMF1 proteins involved in replication of small single-stranded circular plasmids of BMMF, but most likely also contributing to pleomorphic vesicular structures found in the periphery of colorectal, lung and pancreatic cancers. Structurally dense regions are demonstrated in preselected areas of colorectal cancer, after staining with monoclonal antibodies against BMMF1 Rep. Similar structures were observed in human embryonic cells (HEK293TT) overexpressing Rep. These data suggest that Rep or Rep isoforms contribute to the structural formation of vesicles.

Analysis of chronic inflammatory lesions of the colon for BMMF Rep antigen expression and CD68 macrophage interactions.

Consumption of Eurasian bovine meat and milk has been associated with cancer development, in particular with colorectal cancer (CRC). In addition, zoonotic infectious agents from bovine products were proposed to cause colon cancer (zur Hausen et al., 2009). Bovine meat and milk factors (BMMF) are small episomal DNA molecules frequently isolated from bovine sera and milk products, and recently, also from colon cancer (de Villiers et al., 2019). BMMF are bioactive in human cells and were proposed to induce chronic inflammation in precancerous tissue leading to increased radical formation: for example, reactive oxygen and reactive nitrogen species and elevated levels of DNA mutations in replicating cells, such as cancer progenitor cells (zur Hausen et al., 2018). Mouse monoclonal antibodies against the replication (Rep) protein of H1MSB.1 (BMMF1) were used to analyze BMMF presence in different cohorts of CRC peritumor and tumor tissues and cancer-free individuals by immunohistochemistry and Western blot. BMMF DNA was isolated by laser microdissection from immunohistochemistry-positive tissue regions. We found BMMF Rep protein present specifically in close vicinity of CD68+ macrophages in the interstitial lamina propria adjacent to CRC tissues, suggesting the presence of local chronic inflammation. BMMF1 (modified H1MSB.1) DNA was isolated from the same tissue regions. Rep and CD68+ detection increased significantly in peritumor cancer tissues when compared to tissues of cancer-free individuals. This strengthens previous postulations that BMMF function as indirect carcinogens by inducing chronic inflammation and DNA damage in replicating cells, which represent progress to progenitor cells for adenoma (polyps) formation and cancer.

A specific class of infectious agents isolated from bovine serum and dairy products and peritumoral colon cancer tissue.

The in silico analyses of 109 replication-competent genomic DNA sequences isolated from cow milk and its products (97 in the bovine meat and milk factors 2 group - BMMF2, and additional 4 in BMMF1) seems to place these in a specific class of infectious agents spanning between bacterial plasmid and circular ssDNA viruses. Satellite-type small plasmids with partial homology to larger genomes, were also isolated in both groups. A member of the BMMF1 group H1MBS.1 was recovered in a distinctly modified form from colon tissue by laser microdissection. Although the evolutionary origin is unknown, it draws the attention to the existence of a hitherto unrecognized, broad spectrum of potential pathogens. Indirect hints to the origin and structure of our isolates, as well as to their replicative behaviour, result from parallels drawn to the Hepatitis deltavirus genome structure and replication.

Wound healing revised: a novel reepithelialization mechanism revealed by in vitro and in silico models.

Wound healing is a complex process in which a tissue's individual cells have to be orchestrated in an efficient and robust way. We integrated multiplex protein analysis, immunohistochemical analysis, and whole-slide imaging into a novel medium-throughput platform for quantitatively capturing proliferation, differentiation, and migration in large numbers of organotypic skin cultures comprising epidermis and dermis. Using fluorescent time-lag staining, we were able to infer source and final destination of keratinocytes in the healing epidermis. This resulted in a novel extending shield reepithelialization mechanism, which we confirmed by computational multicellular modeling and perturbation of tongue extension. This work provides a consistent experimental and theoretical model for epidermal wound closure in 3D, negating the previously proposed concepts of epidermal tongue extension and highlighting the so far underestimated role of the surrounding tissue. Based on our findings, epidermal wound closure is a process in which cell behavior is orchestrated by a higher level of tissue control that 2D monolayer assays are not able to capture.

Spatial quantification and classification of skin response following perturbation using organotypic skin cultures.

MOTIVATION: For a mechanistic understanding of skin and its response to an induced perturbation, systems biology is gaining increasing attention. Unfortunately, quantitative and spatial expression data for skin, like for most other tissues, are almost not available. RESULTS: Integrating organotypic skin cultures, whole-slide scanning and subsequent image processing provides bioinformatics with a novel source of spatial expression data. We here used this approach to quantitatively describe the effect of treating organotypic skin cultures with sodium dodecyl sulphate in a non-corrosive concentration. We first measured the differentiation-related spatial expression gradient of Heat-Shock-Protein 27 in a time series of up to 24 h. Secondly, a multi-dimensional tissue classifier for predicting skin irritation was developed based on abstract features of these profiles. We obtained a high specificity of 0.94 and a sensitivity of 0.92 compared with manual classification. Our results demonstrate that the integration of tissue cultures, whole-slide scanning and image processing is well suited for both the standardized data acquisition for systems biological tissue models and a highly robust classification of tissue responses.

Robust gridding of TMAs after whole-slide imaging using template matching.

Tissue microarrays (TMAs) represent an important approach for the high-throughput cellular analysis of large numbers of tissue samples on one single slide in research related to diagnostics and oncology. Whole-slide imaging now enables full scanning and subsequent image analysis of such TMAs. In contrast to automatically spotted RNA microarrays, TMAs are fabricated manually and mechanically by arranging hundreds of tissue cores in a single paraffin block. This procedure frequently results in quality problems severely hampering the later automatic image analysis of TMAs after whole-slide imaging. We therefore set out to (a) determine the extent of these quality issues in exemplary TMAs and (b) to develop a robust gridding method to identify the logical position coordinates of each TMA core on a virtual TMA slide. We present the first robust method identifying these coordinates by shifting a template grid over all cores of the TMA (template matching) and thereby measuring in how far the grid matches a predefined list of cores on the virtual TMA Slide. Analysis of 20 TMAs from Breast Cancer as well as 40 Head-and-Neck Cancer showed that frequent TMA layout issues comprise low staining, debris, core displacement, nonuniform background, missing cores, and rotated subarrays. On this highly demanding test comprising chromogen as well as fluorescence stained TMAs, our template matching method achieved an excellent position analysis. Of 8900 cores, 8864 (99.59%) were assigned properly. In all 60 slides of the test set, no localization error occurred. The automatic grid analysis of TMAs after whole-slide imaging is highly demanding and requires dedicated algorithms. We demonstrate such a method and evaluate its performance. © 2010 International Society for Advancement of Cytometry.