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.

Bovine Meat and Milk Factors (BMMFs): Their Proposed Role in Common Human Cancers and Type 2 Diabetes Mellitus.

Exemplified by infections with bovine meat and milk factors (BMMFs), this manuscript emphasizes the different mechanistic aspects of infectious agents contributing to human cancers by "direct" or "indirect" interactions. The epidemiology of cancers linked to direct carcinogens (e.g., response to immunosuppression) differs from those cancers linked with indirect infectious interactions. Cancers induced by direct infectious carcinogens commonly increase under immunosuppression, whereas the cancer risk by indirect carcinogens is reduced. This influences their responses to preventive and therapeutic interferences. In addition, we discuss their role in colon, breast and prostate cancers and type II diabetes mellitus. A brief discussion covers the potential role of BMMF infections in acute myeloid leukemia.

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.

Isolation of Two Virus-Like Circular DNAs from Commercially Available Milk Samples.

Epidemiological data indicate a potential relationship between milk and dairy product consumption and the incidence of breast cancer, as well as neurodegenerative diseases. We report the isolation of two novel circular DNA molecules isolated from commercially available milk.

Infectious Agents in Bovine Red Meat and Milk and Their Potential Role in Cancer and Other Chronic Diseases.

Red meat and dairy products have frequently been suggested to represent risk factors for certain cancers, chronic neurodegenerative diseases, and autoimmune and cardiovascular disorders. This review summarizes the evidence and investigates the possible involvement of infectious factors in these diseases. The isolation of small circular single-stranded DNA molecules from serum and dairy products of Eurasian Aurochs (Bos taurus)-derived cattle, obviously persisting as episomes in infected cells, provides the basis for further investigations. Gene expression of these agents in human cells has been demonstrated, and frequent infection of humans is implicated by the detection of antibodies in a high percentage of healthy individuals. Epidemiological observations suggest their relationship to the development multiple sclerosis, to heterophile antibodies, and to N-glycolylneuraminic acid (Neu5Gc) containing cell surface receptors.

Viremia during pregnancy and risk of childhood leukemia and lymphomas in the offspring: Nested case-control study.

A possible role for infections of the pregnant mother in the development of childhood acute leukemias and lymphomas has been suggested. However, no specific infectious agent has been identified. Offspring of 74,000 mothers who had serum samples taken during pregnancy and stored in a large-scale biobank were followed up to the age of 15 years (750,000 person years) through over-generation linkages between the biobank files, the Swedish national population and cancer registers to identify incident leukemia/lymphoma cases in the offspring. First-trimester sera from mothers of 47 cases and 47 matched controls were retrieved and analyzed using next generation sequencing. Anelloviruses were the most common viruses detected, found in 37/47 cases and in 40/47 controls, respectively (OR: 0.6, 95% CI: 0.2-1.9). None of the detected viruses was associated with leukemia/lymphoma in the offspring. Viremia during pregnancy was common, but no association with leukemia/lymphoma risk in the offspring was found.

Reprint of: cancer "causation" by infections--individual contributions and synergistic networks.

The search for infectious agents playing a role in human carcinogenesis and their identification remain important issues. This could provide clues for a broader spectrum of cancers preventable by vaccination and accessible to specific therapeutic regimens. Yet, the various ways of interacting among different factors functioning synergistically and their different modes of affecting individual cells should bring to question the validity of the term "causation". It also should put a word of caution into all attempts to summarize criteria for "causality" of infectious agents in cancer development. At least in the opinion of these authors, we would be much better off avoiding these terms, replacing "causal factor" by "risk factor" and grading them according to their contribution to an individual's cancer risk.

Dairy cattle serum and milk factors contributing to the risk of colon and breast cancers.

The analysis of published epidemiological data on colon and breast cancer reveals a remarkable concordance for most regions of the world. A low incidence for both cancers has been recorded in Mongolia and Bolivia. Discrepant data, however, have been reported for India, Japan and Korea. In India, the incidence of breast cancer is significantly higher than for colon cancer, in Japan and Korea colon cancer exceeds by far the rate of breast cancer. Here, studies are summarized pointing to a species-specific risk for colon cancer after consumption of beef originating from dairy cattle. Uptake of dairy products of Bos taurus-derived milk cattle, particularly consumed at early age, is suggested to represent one of the main risk factors for the development of breast cancer. A recent demonstration of reduced breast cancer rates in individuals with lactose intolerance (Ji et al., Br J Cancer 2014; 112:149-52) seems to be in line with this interpretation. Species-specific risk factors for these cancers are compatible with the transmission of different infectious factors transferred via meat or dairy products. Countries with discordant rates of colon and breast cancer reveal a similar discordance between meat and milk product consumption of dairy cattle. The recent isolation of a larger number of novel presumably viral DNAs from serum, meat and dairy products of healthy dairy cows, at least part of them infectious for human cells, deserves further investigation. Systemic infections early in life, resulting in latency and prevention of subsequent infections with the same agent by neutralizing antibodies, would require reconsideration of ongoing prospective studies conducted in the adult population.

Cancer "causation" by infections--individual contributions and synergistic networks.

The search for infectious agents playing a role in human carcinogenesis and their identification remain important issues. This could provide clues for a broader spectrum of cancers preventable by vaccination and accessible to specific therapeutic regimens. Yet, the various ways of interacting among different factors functioning synergistically and their different modes of affecting individual cells should bring to question the validity of the term "causation". It also should put a word of caution into all attempts to summarize criteria for "causality" of infectious agents in cancer development. At least in the opinion of these authors, we would be much better off avoiding these terms, replacing "causal factor" by "risk factor" and grading them according to their contribution to an individual's cancer risk.

Isolation of protein-associated circular DNA from healthy cattle serum.

Three replication-competent single-stranded DNA molecules sharing nucleotide similarity to transmissible spongiform encephalopathy (TSE)-associated isolate Sphinx 2.36 were isolated from healthy bovine serum.

Isolation of bacterial plasmid-related replication-associated circular DNA from a serum sample of a multiple sclerosis patient.

Psychrobacter species are considered to be opportunistic human pathogens. We report here the isolation of a circular DNA molecule, MSSI1.162, from a serum sample taken from a multiple sclerosis patient during relapse. This isolate is distantly related to known Psychrobacter species and their plasmids.

Mycovirus-like DNA virus sequences from cattle serum and human brain and serum samples from multiple sclerosis patients.

Myco-like viruses have been isolated from fungi, feces of various animals, and plant leaves. We report here the isolation of 3 complete genome sequences of gemycircularvirus-related viruses from healthy bovine serum and human brain and serum samples from patients with multiple sclerosis (MS). Their putative capsid proteins share similarity to Torque teno virus (TTV) open reading frame 1 (ORF1) proteins.

Novel replication-competent circular DNA molecules from healthy cattle serum and milk and multiple sclerosis-affected human brain tissue.

Epidemiological data point to the involvement of a cow milk factor in the etiology of multiple sclerosis (MS). Eleven circular DNA molecules closely related to transmissible spongiform encephalopathy (TSE)-associated isolate Sphinx 1.76 were isolated from healthy cattle serum, cow milk, and serum and brain tissue from MS patients.

A human torque teno virus encodes a microRNA that inhibits interferon signaling.

Torque teno viruses (TTVs) are a group of viruses with small, circular DNA genomes. Members of this family are thought to ubiquitously infect humans, although causal disease associations are currently lacking. At present, there is no understanding of how infection with this diverse group of viruses is so prevalent. Using a combined computational and synthetic approach, we predict and identify miRNA-coding regions in diverse human TTVs and provide evidence for TTV miRNA production in vivo. The TTV miRNAs are transcribed by RNA polymerase II, processed by Drosha and Dicer, and are active in RISC. A TTV mutant defective for miRNA production replicates as well as wild type virus genome; demonstrating that the TTV miRNA is dispensable for genome replication in a cell culture model. We demonstrate that a recombinant TTV genome is capable of expressing an exogenous miRNA, indicating the potential utility of TTV as a small RNA vector. Gene expression profiling of host cells identifies N-myc (and STAT) interactor (NMI) as a target of a TTV miRNA. NMI transcripts are directly regulated through a binding site in the 3'UTR. SiRNA knockdown of NMI contributes to a decreased response to interferon signaling. Consistent with this, we show that a TTV miRNA mediates a decreased response to IFN and increased cellular proliferation in the presence of IFN. Thus, we add Annelloviridae to the growing list of virus families that encode miRNAs, and suggest that miRNA-mediated immune evasion can contribute to the pervasiveness associated with some of these viruses.

Cross-roads in the classification of papillomaviruses.

Acceptance of an official classification for the family Papillomaviridae based purely on DNA sequence relatedness, was achieved as late as 2003. The rate of isolation and characterization of new papillomavirus types has greatly depended on and subjected to the development of new laboratory techniques. Introduction of every new technique led to a temporarily burst in the number of new isolates. In the following, the bumpy road towards achieving a classification system combined with the controversies of implementing and accepting new techniques will be summarized. An update of the classification of the 170 human papillomavirus (HPV) types presently known is presented. Arguments towards the implementation of metagenomic sequencing for this rapidly growing family will be presented.

The transcriptional regulator gene E2 of the Human Papillomavirus (HPV) 16 influences the radiosensitivity of cervical keratinocytes.

BACKGROUND: Clinical studies have demonstrated that HPV induced tumors constitute a specific subclass of cancer with a better response to radiation treatment. The purpose of this study was to investigate meaning of viral E2-gene for radiosensitivity. METHODS: W12 cells contain episomal HPV 16 genomes, whereas S12 cells, which derive from the W12 line, contain HPV DNA as integrated copies. Clonogenic survival was analyzed using 96-well in vitro test. Using flow cytometry cell cycle analyses were performed. Expression of pRb and p53 were analyzed using intracellular staining. RESULTS: W12 cells (intact E2 gene) showed a lower survival fraction than S12 cells. W12 cells developed a G2/M block 24 h after irradiation with 2 Gy whereas S12 showed no G2/M bloc. After irradiation S12 cells developed polyploidy and pRb-positive cells decreased. W12 cells showed no change of pRb-positive cells. CONCLUSIONS: Depending on E2 gene status differences in cell cycle regulation might cause radioresistance. The E2/E7/pRb pathway seems to influence HPV-induced radiosensitivity. Our experiments demonstrated an effect of HPV on radiosensitivity of cervical keratinocytes via viral transcription regulator E2 pathway.

Phylogenetically diverse TT virus viremia among pregnant women.

Infections during pregnancy have been suggested to be involved in childhood leukemias. We used high-throughput sequencing to describe the viruses most readily detectable in serum samples of pregnant women. Serum DNA of 112 mothers to leukemic children was amplified using whole genome amplification. Sequencing identified one TT virus (TTV) isolate belonging to a known type and two putatively new TTVs. For 22 mothers, we also performed TTV amplification by general primer PCR before sequencing. This detected 39 TTVs, two of which were identical to the TTVs found after whole genome amplification. Altogether, we found 40 TTV isolates, 29 of which were putatively new types (similarities ranging from 89% to 69%). In conclusion, high throughput sequencing is useful to describe the known or unknown viruses that are present in serum samples of pregnant women.

Analysis of Tp53 codon 72 polymorphisms, Tp53 mutations, and HPV infection in cutaneous squamous cell carcinomas.

BACKGROUND: Non-melanoma skin cancers are one of the most common human malignancies accounting for 2-3% of tumors in the US and represent a significant health burden. Epidemiology studies have implicated Tp53 mutations triggered by UV exposure, and human papilloma virus (HPV) infection to be significant causes of non-melanoma skin cancer. However, the relationship between Tp53 and cutaneous HPV infection is not well understood in skin cancers. In this study we assessed the association of HPV infection and Tp53 polymorphisms and mutations in lesional specimens with squamous cell carcinomas. METHODS: We studied 55 cases of histologically confirmed cutaneous squamous cell carcinoma and 41 controls for the presence of HPV infection and Tp53 genotype (mutations and polymorphism). RESULTS: We found an increased number of Tp53 mutations in the squamous cell carcinoma samples compared with perilesional or control samples. There was increased frequency of homozygous Tp53-72R polymorphism in cases with squamous cell carcinomas, while the Tp53-72P allele (Tp53-72R/P and Tp53-72P/P) was more frequent in normal control samples. Carcinoma samples positive for HPV showed a decreased frequency of Tp53 mutations compared to those without HPV infection. In addition, carcinoma samples with a Tp53-72P allele showed an increased incidence of Tp53 mutations in comparison carcinomas samples homozygous for Tp53-72R. CONCLUSIONS: These studies suggest there are two separate pathways (HPV infection and Tp53 mutation) leading to cutaneous squamous cell carcinomas stratified by the Tp53 codon-72 polymorphism. The presence of a Tp53-72P allele is protective against cutaneous squamous cell carcinoma, and carcinoma specimens with Tp53-72P are more likely to have Tp53 mutations. In contrast Tp53-72R is a significant risk factor for cutaneous squamous cell carcinoma and is frequently associated with HPV infection instead of Tp53 mutations. Heterozygosity for Tp53-72R/P is protective against squamous cell carcinomas, possibly reflecting a requirement for both HPV infection and Tp53 mutations.