A Human Betaretrovirus (HBRV) has been identified in humans, dating as far back as about 4500 years ago, with a high probability of it being acquired by our species around 10,000 years ago, following a species jump from mice to humans. HBRV is the human homolog of the MMTV (mouse mammary tumor virus), which is the etiological agent of murine mammary tumors. The hypothesis of a HMTV (human mammary tumor virus) was proposed about 50 years ago, and has acquired a solid scientific basis during the last 30 years, with the demonstration of a robust link with breast cancer and with PBC, primary biliary cholangitis. This article summarizes most of what is known about MMTV/HMTV/HBRV since the discovery of MMTV at the beginning of last century, to make evident both the quantity and the quality of the research supporting the existence of HBRV and its pathogenic role. Here, it is sufficient to mention that scientific evidence includes that viral sequences have been identified in breast-cancer samples in a worldwide distribution, that the complete proviral genome has been cloned from breast cancer and patients with PBC, and that saliva contains HBRV, as a possible route of inter-human infection. Controversies that have arisen concerning results obtained from human tissues, many of them outdated by new scientific evidence, are critically discussed and confuted. IndexPreamblePrefacePart I1. Introduction Cancer and Its Many Names1.1. Breast and Mammary Gland1.2. Basic Concepts of Breast Oncogenesis1.3. 1.3.1. The Mammary Gland Is a Dynamic Organ, with Consequence on Carcinogenesis1.3.2. The Terminal Duct Lobular Unit Is the Site of Origin of Breast Carcinoma1.3.3. Initiation, Promotion, Progression1.3.4. Preinvasive Lesions1.3.5. Risk FactorsSporadic and Hereditary Breast Carcinoma1.4. 1.4.1. The Knudson’s Model1.4.2. HaploinsufficiencyHuman Breast Cancer Etiology Is Unknown, So Far1.5. Part II: Mouse Mammary Tumor Virus—MMTV2. The Experimental Model of Breast Cancer: The Mouse Mammary Tumors and the Mouse Mammary Tumor Virus (MMTV), with Its Long List of Names3. The Mouse Mammary Tumor VirusClassification3.1. Viral Structure and Molecular Trafficking3.2. 3.2.1. Viral Packaging3.2.2. Viral BuddingTransmission by Nursing3.3. Intestinal Absorption3.4. Cell Infection3.5. Molecular Structure3.6. Oncogenesis3.7. 3.7.1. Insertional Mutagenesis, Int, Cis3.7.2. Oncogene3.7.3. TranscriptionMouse Strains and Their Tumors3.8. How Many Types of MMTV?3.9. MMTV Can Infect Adult Mice by an Alternative Way of Entry: The Nasal-Associated Lymphoid Tissue3.10. Not Only Mammary Tumors3.11. Endogenous MMTV3.12. ERVs3.12.1. Transposons, Retrotransposons, Part III: Human Mammary Tumor Virus—HMTV4. The Human Mammary Tumor VirusHMTV in Human Milk4.1. HMTV in Human Cancer4.2. 4.2.1. Molecular Biology and Immunohistochemistry4.2.2. The gp52 Glycoprotein5. The ENV Sequence, the HMTV, and the Breast Carcinoma: The New York LaboratoryEnv Sequences in Human Breast Carcinoma5.1. Env Sequence Expression5.2. MMTV Provirus5.3. MMTV-Like LTR and LTR-Env Gene Sequences5.4. The Superantigen (sag)5.5. The Interferon Signature5.6. MMTV Particles in Human Cells5.7. MMTV Proteins in Human Cells5.8. The HMTV—Human Mammary Tumor Virus5.9. 6. MMTV/HMTV Is Significantly Present All over the World7. HMTV Is Able to Infect Human Cells: The Vienna LaboratoryTfR1—The Transferrin Receptor 17.1. HMTV Can Infect Human Cells7.2. HMTV Does Not Use TfR1 to Infect Human Cells7.3. 8. HMTV Is Involved in Almost All Sporadic Breast Carcinomas: The Pisa LaboratoryStringent Laboratory Procedures Guarantee the Quality of the Result8.1. HMTV Is Present in the Vast Majority of Sporadic Breast Carcinomas8.2. Hereditary Breast Carcinomas Do Not Need a Viral Etiology8.3. HMTV in Saliva and the Paleoanthropological Study8.4. 9. The P14, a MMTV Signal Peptide—A Possible Tool for Vaccination Strategies: The Laboratory of Jerusalem10. The Point of View of an Epidemiologist: The Laboratory of Sidney11. HMTV in Human Neoplasia Other Than Breast CarcinomaNon-Hodgkin Lymphoma (NHL)11.1. Tumors Influenced by Hormones11.2. Lung Cancer11.3. 12. HMTV and the Human Primary Biliary Cholangitis: The Laboratory of Edmonton13. HERVs—Human Endogenous Retroviruses14. The Leitmotiv of Contamination: No EvidenceContamination14.1. HERVs14.2. Other Issues14.3. 15. Breast Cancer Incidence in Immunosuppressed Patients16. The Pisa Meetings2012 (2–4 April): HMTV—Viruses and Breast Cancer16.1. 2016 (22–23 September): HBRV—MMTV and Human Diseases16.2. Part IV: Human Betaretrovirus—HBRV17. The Human Species Has Its First Human Betaretrovirus 18. Cross-Species Transmission19. HBRV as the Product of a Cross-Species Transmission20. The Possibility of a Contemporary Zoonosis21. Inter-Human Transmission of HBRVMilk (Breast Feeding) Cannot Be the Answer21.1. Retroviruses (MMTV Included) Can Be Transmitted by Saliva in Animals21.2. MMTV Can Infect Adult Mice through the Nose21.3. HBRV Is Present in Human Saliva21.4. 22. HBRV Transmission by SalivaSaliva Contains a Little Universe22.1. Saliva Spreading22.2. Saliva Transmits Even Prions22.3. What Is in Saliva That Can Diffuse HBRV22.4. 23. Breast Cancer in the Same Family: A Transmissible Agent?24. The Abundance of BC Histotypes and Biological Types Can Find a Reason in a Viral Etiology25. Oncogenesis of HMTVProteins with the Characteristics of Oncogene25.1. 25.1.1. The Env Protein25.1.2. The Sag, Superantigen25.1.3. The Naf, Negative Acting Factor25.1.4. The REM, Regulator of Expression/Export of MMTV mRNACell Fusion25.2. Activation of a Second Oncogenic Virus25.3. HERVs25.4. 26. How Many HBRV?Part V27. Keynotes28. For the FutureTo Francesco Squartini, 1927–1992,pioneer of the mouse mammary tumor biologyPreambleThese notes synthesize most of what is known about the possible viral origin of human breast cancer and would, perhaps, be useful also to scientists who are not strictly involved in the field. For this reason, the paper recalls some basic concepts, of breast cancer biology and cancerology in particular.The whole of almost one century of study demonstrates the existence of HBRV, the human homologue of MMTV, endemic in the human species, with high probability as the etiologic agent of breast cancer (BC) and primary biliary cholangitis (PBC).PrefaceThis is the story of a virus grown in the mouse and passed to humans thousands of years ago, which has many names, causes mammary tumors in mice, and is strictly related to BC and to PBC.The story begins in the countryside of Maine almost a century ago, in a golden age for science: inbred mice are raised to study the genetic transmission of diseases, viruses are discovered, Oswald Theodore Avery, Colin Munro MacLeod, and Maclyn McCarty individuate DNA as the transforming principle able to modify the characteristics of bacteria (Pneumococcus type III) .In 1908 Ellerman and Bang  discoverPart I.The number of cells of an adult human organism has to remain constant; in the single tissue so many cells die, while so many cells are born. This balance is guaranteed by sophisticated biomolecular processes involved in controlling cell-cycle progression, apoptosis, DNA repair, degradation of proteins, assemblage of the mitotic spindle, etc. Some genes and their coded proteins (a) exert an inhibitory control on proliferation, whereas others (b) activate the process. The disarrangement of this machinery can lead to an uncontrolled cell proliferation, that is, a tumor. This result can be consequence of a damage in specific genes, with the consequent origin of mutated proteins. When the mutated gene/protein belongs to the above mentioned “a” group, they are called suppressor (or oncosuppressor) genes/proteins, whereas when they belong to the “b” group, they are named oncogenes/oncoproteins.tumor means swelling. Neoplasia derives from the ancient Greek words νέοσ  and πλάσσω  and means “new formation”. Cancer is the Latin word for crab. Carcinoma derives from the ancient Greek καρκίνος . Cancer, karkinos, and crab are all linked by the Indo-European root kar- that means “hard”. Actually, we might say “hardoma”(!). The strict link between hardness and disease probably derives from breast cancer, one of the most frequent and distinctly visible growths, because it occurs in a superficial organ, and often very hard as a consequence of its abundant stromal component. In fact, one of the common histotypes of breast cancer was called “scirrhous”, , with the same root.The Latin word σάρξ  and indicates cancer of mesenchymal tissues.For no evident reason, cancer indicates any malignant tumor, whereas carcinoma indicates cancers deriving from epithelial cells. The two words are often wrongly used as synonyms. Sarcoma derives from Breast is made by a more or less abundant quantity of adipose tissue hosting the mammary gland tree. From the nipple a dozen of galactoforous (lactiferous) ducts penetrates the subcutaneous fat giving origin, by a series of dichotomous divisions, to segmental and subsegmental ducts. Each of them ends with a short and thin peduncle that holds a bunch (lobule) of very small glandular acini: the terminal duct lobular unit (TDLU). Milk is produced in the lobule and then transported to the nipple by the duct system.labile, i.e., divide all the time, makes them highly susceptible to carcinogenetic agents, which more easily can reach the cell DNA during the mitotic division. Stable (quiescent) cells spend most of their life in G0, and then have a low incidence of cancer (hepatocytes). Permanent cells are considered terminally differentiated and, therefore, are non-proliferative; for instance, neurons only exceptionally participate in the formation of tumors of the nervous system. Labile cells are more prone to develop cancer, also because of their high proliferative rate facilitates the occurrence and accumulation of the gene mutations necessary for the neoplastic transformation and progression.The mammary gland is characterized by an amazing activity. During a woman’s reproductive life, each month there is an intense proliferation of the TDLUs, which prepare themselves to produce large quantities of milk. In contrast, after the menstruation, lobules disappear following an apoptotic program. The fact that mammary epithelial cells are TDLU is the dynamic part of the glandular tree, while ducts are stable. As expected, TDLU is the site of origin of a carcinoma, both in a woman and in the mouse.initiation, the moment of the transformation of normal cells in neoplastic cells by a carcinogenetic agent/process; (b) promotion, the stimulus to proliferation exerted not only on the transformed cells but on the entire cell compartment to which they belong; (c) progression, the whole of all the phases that characterize the entire life of the process. The concept of progression was created by Leslie Foulds in the ‘50s, mainly by the observation of mouse mammary neoplasia  .Tumors of the mouse mammary gland have been extensively studied and classified. Two main types are the “adenocarcinoma type A” typical of C3H mice and the “adenocarcinoma type B” typical of RIII mice. According to Thelma Brumfield Dunn, type A tumors have a “fine uniform acinar structure” with small uniform acini, whereas type B “represent a diversified, multiform group in which the tumor is clearly of glandular epithelial origin, but in which there are no predominant features”, with acinar structures, solid areas, irregular glands, cysts filled with blood or clear fluid, intracystic papillary projections etc. .The model of MMTV mouse mammary tumors was crucial for the development of the concept of tumor progression and preneoplastic/preinvasive lesions, as a mainstay of modern oncology . In factThe question, “How Many Types of MMTV are there?”, was asked several decades ago, but a complete answer is still missing. A difference of about 20% has been reported in the viral genome between high-incidence and low-incidence mouse strains. Conversely, no sound difference was demonstrated among the high tumor incidence strains .However, an experiment of 60 years ago unveiled the existence of types of MMTV with relevant biological dissimilarities .Francesco Squartini was intrigued by the different characteristics of C3H and RIII mammary tumors. The C3H tumors have HAN as preneoplastic lesions, are type A adenocarcinoma, are hormone independent, and are highly metastatic. The RIII tumors have plaques as precancerous lesions, are type B adenocarcinoma, are hormone dependent, and are poorly metastatic. The question asked was as follows: Are these morphological and biological differences, which are so evident in the mammary carcinogenesis between C3H and RII mice due to the different genetic constitution of the animals, or to relevant differences between MMTV/C3H and MMTV/RIII viruses?The answer was obtained by a “simple” experiment aimed to cancel the influence of the genetic characters of the host: to study the tumors induced by the two different virus strains when introduced in the same mouse strain. Two new mouse strains were created by fostering BALB/c mice by C3H or RIII mice; actually, the BALB/cfC3H had been already established, whereas the BALB/cfRIII was brand new. BALB/cfC3H animals developed mammary tumors identical to those of the C3H strain, whereas BALB/cfRIII animals developed tumors identical to those of the RIII strain. “These results indicate that the tumor characters may be transferred from one strain to another by means of foster-nursing”.The intentionally soft conclusion of Squartini means that different MMTV strains exist, able to drive different carcinogenetic pathways.As seen above, in the newborn mouse MMTV enters the small intestine at the level of Peyer’s plaques, where an intimate interaction with the immune system takes place.In 1997, Swiss scientists were successful in infecting adult mice with mouse milk containing the viral particles . They deHence MMTV can enter an organism even from sites different from the small intestine, provided that well represented lymphatic structures are present.These findings are quite relevant in the formulation of hypotheses for the inter-human transmission of HBRV.MMTV has a pleiotropic role, in that it is able to induce in mouse tumors other than the mammary ones.Int common to mammary and kidney tumors [The BALB/cf/Cd mouse has a 70% incidence of spontaneous kidney adenocarcinomas, which show viral particles, the presence of several copies of integrated exogenous MMTV, and an y tumors ,87.MMTV is able to induce also T-cell lymphoma/leukemia (MMTV variant type B leukemogenic virus—TBLV) and B-cell lymphoma ,89,90. TMtv indicates the endogenous virus. Mtv is an ERV (Endogenous Retrovirus). ERVs are retrotransposons.The previous pages dealt with the exogenous form of MMTV, a viral particle that buds on cell surface and is transmitted horizontally via milk. However, MMTV exists also as an endogenous form, a particle that usually does not bud and is transmitted vertically by germ cells according to the Mendelian inheritance. The exogenous virus is indicated with the acronym MMTV, while Transposons or transposable elements (TEs) are DNA sequences that are able to move within the genome. The 1983 Nobel prize for Physiology or Medicine was awarded to Barbara McClintock for her discovery of mobile genetic elements . TranspoRetrotransposons are subdivided according to the presence or absence of LTRs  flanking a central coding region. Non-LTR groups are LINEs (long-interspersed nucleotide elements) and SINEs (short-interspersed nucleotide elements). LTR-retrotransposons are the endogenous retroviruses (ERVs).Mtv, MusD/ETn (early transposon) family, and IAP  [ERVs are sequences of exogenous viruses inserted into the host genome millions of years ago that have remained with no functions (living fossils) or with functions mainly regulatory. Three classes of ERVs can be recognized on the base of their similarity with exogenous retroviruses: class I: resembles gamma and epsilon retroviruses; class II: alpha, beta, and delta retroviruses; class III: spumaviruses. Class II includes rticles) ,96,97,98env gene, in that they do not have the envelope.ERVs usually do not own the gag, pro, and pol genes, whereas env is missing; (b) the matrix domain (MA) of the Gag polyprotein is lacking in its myristoylation signal, necessary for anchoring the lipid molecules of the cell membrane, preventing Gag from exert its role in assembling the mature virions.In the previous chapters, it was clarified that MMTV is the B particle, formed by the budding at the cell membrane of an intracytoplasmic A particle, its immature form. However, A particles are the morphological expression also of the ERVs, IAP in particular, with the difference that they accumulate in the cytoplasmic cisternae and do not form infectious particles. Two facts explain why IAP are unable to become B particles and to infect: (a) IAP have a long LTR with env gene. No longer IAP, but IAPE, “intracisternal A-particle-related elements coding for envelope”. There are data in favor of the fact that IAPs descend from IAPEs [However, it is well known that mice hosting ERVs can form XRVs (exogenous retroviruses), as in the GR mouse strain, in which the virus can be transmitted by milk. When an endogenous virus becomes infectious and forms B particles, IAP are, however, present and indistinguishable from those of strictly non-infectious endogenous MMTV, but with a molecular difference that easily explains their new behavior: they have an om IAPEs ,100,101.ERVs can express an oncogenetic action through at least a couple of mechanisms, such as insertional mutagenesis or the production of proteins that play a role in carcinogenesis ,103,104.Part III: Human Mammary Tumor Virus—HMTVNews and Views [On February 1971, Nature titles in nd Views  “Human bnd Views  have detAt the same time (1970), the reverse transcriptase was discovered by David Baltimore  and HowaOther laboratories were not able to morphologically identify viral particles in human milk; however, they highlighted the relevance of the presence of reverse transcriptase . Quite iAs described above, the early 1970s were a period full of enthusiasm for the possibility of a viral etiology of human breast cancer and of discoveries in that direction. The driving idea was that the human model could copy the murine model, with MMTV present in milk and transmitted to newborns. Results of morphological and biological studies on milk, even if not unanimous, supported this idea. Unfortunately, epidemiology did not: “the weight of epidemiologic data has not been consistent with milk transmission of human breast cancer” and “no association between breast feeding and breast cancer risk in offspring was found”. Yet, “there is little doubt of a definite genetic component to human breast cancer. The increased familial risk to this disease seems to be associated with an altered state of hormone excretion or metabolism” . All theBecause of the discouraging results given by searching the virus in human milk, scientists began to focus on breast-cancer tissues.In 1972, Sol Spiegelman, using molecular hybridization, demonstrated the presence of RNA homologous to MMTV RNA in 66% of 29 infiltrating breast cancers, with normal breast tissues, benign breast lesions, and non-breast tumors all negative . In the Biochemical evidence is also provided for the presence of an RNA tumor virus in purified fractions of human malignant breast tumors; these particles are similar to the subviral particulate of MMTV isolated from murine mammary tumors; furthermore, particles exhibit features diagnostic of cores and ribonucleoproteins of oncornaviruses .Then, the same laboratory adopted the newly developed immunohistochemical approach to the study of proteins and identified, in paraffin sections of human breast cancer, an antigen, immunologically related to a group-specific antigen of the MMTV, the gp52, a 52,000-dalton glycoprotein.Env gene, which is a main gene of MMTV, encodes for a gp70 polyprotein that gives origin to a couple of subunits, SU (surface) and TM (transmembrane). The SU is a gp52 glycoprotein.As seen before, the For the above reasons, Spiegelman et al. decided to study the immunohistochemical expression of gp52 in human samples. The protein was demonstrated in 39% of 131 infiltrating breast cancers, whereas all 119 samples of normal breast and benign breast lesions were found negative; moreover, 99 non-breast tumors and eight cystosarcoma phyllodes of the breast were all negative; interestingly, some cases of negative benign lesions were coincidental with a positive breast carcinoma. In an extended series of 376 cases, the percentage of positive cases arose to 45.5% ,121. IntFinally, they demonstrated a positivity for gp52 of the particles released from the human T47D breast-carcinoma cell line, which have characteristics of retroviruses .Env gene is one of the main genes of MMTV, and is very important for its life cycle and, therefore, of high interest in the search for MMTV in human tissues. It has been well studied since the 1970s, and received more attention in the first decade of this century after the demonstration of its direct role in carcinogenesis. In fact, it encodes an ITAM (immunoreceptor tyrosine-based activation motif), which is able to trigger transformation in murine and human mammary epithelial cells and to function in non-hematopoietic cells as potent oncoproteins by scaffolding downstream mediators [As described in the first part of this article, the ediators ,124.env gene in human breast cancers. In 1995, they were able to detect a 660-bp MMTV env gene-like sequences in a large part (38.5%) of 314 breast cancer samples and in two human breast-cancer cell lines. The sequence was selected on the basis of its very high homology to MMTV (95–99%) and of its very low homology to human endogenous retroviruses (<15% when compared to the endogenous provirus HERV-K10) or to all known viral and human genes . The 660-bp sequence does not seem to belong to an endogenous retrovirus, as demonstrated also by the fact that normal organs and non-breast cancers were all negative, as well as blood lymphocytes from positive breast cancer patients and breast tissues homolateral to cancer. Additionally, a 250-bp sequence within the 660-bp stretch was used for the analysis of paraffin tissues and was found present in 40% of breast cancer samples. Two human breast-cancer cell lines (MCF-7 and ED) were shown to contain the 660-bp sequence, while four other cell lines were positive only for the 250-bp sequence  [env sequence to MMTV and its low homology to endogenous retroviruses were demonstrated further in a following paper [A renewed and strengthened interest towards a possible human MMTV as a cause of breast cancer appeared after about twenty years after the studies on gp52 protein, when in New York City, Beatriz Pogo et al. decided to focus on the presence of  MB 231) . The higng paper .env+ breast cancers and the 256-bp sequence in all env+ cancers, while they were absent in env− cases. The 660-bp sequence was 98% homologous to the env gene of the MMTV [The 660-bp sequence was found expressed in 66% of the MMTV .env positive was amplified, in particular LTR-gag, gag-pol, and pol-env segments. The 9.9-kb provirus is 95% homologous to MMTV but only 57% to human endogenous retrovirus K10 in 3.5 kb.Then, a whole proviral structure from each of two human breast carcinomas that were Gag and pol showed a higher homology to HERV-K10. PCR approaches appear never to have been confirmed using regions of low homology in the gag and pol genes. It can be hypothesized that there should be regions (such as the dUTPase or part of pol) that should be homologous enough to allow confirmation that human breast cancer contain HMTV .env-LTR sequence of an env-positive breast cancer cell line revealed that the sequence is inserted in several chromosomes but not in chromosomes from normal breast cells [The provirus displays typical features of a replication competent virus, plus the open reading frame for the superantigen and the glucocorticoid responsive element. Fluorescence in situ hybridization with a 2.7-kb st cells .env and LTR sequences was amplified, cloned and sequenced from breast-cancer DNA. The human LTRs were highly homologous to MMTV and contain enhancer and promoter elements, the glucocorticoid responsive element (GRE) and the super-antigen (Sag) sequences. The presence of functional sequences implies involvement in transcriptional regulation, whereas the presence of an env-LTR sequence indicates contiguity within the genome of a potential provirus. Their presence in breast-cancer DNA, but not in normal tissue, suggests an exogenous origin [At the same time, MMTV-like LTRs were detected in human breast cancer. A 630-bp LTR sequence, amplified by PCR using primers LTR5 and LTR3, was identified in 41.5% of 65 human breast cancers, with normal breast tissues negative. A larger 1.2 kb LTR fragment was also amplified with high homology to MMTV. Finally, a 1.6 kb fragment containing s origin .sag sequences were amplified and sequenced from 10 human breast cancers and found highly homologous to those of MMTV. Moreover, the LTR Sag was shown able to stimulate human T lymphocytes inducing them to proliferate and to secrete cytokines, obtaining the same response that occurs in the mouse [MMTV-like he mouse .env+ cells, 7 were interferon-related, 5 TNF-connected, and 2 TGFβ-related. The authors concluded that MMTV sequences stimulate a chronic inflammatory response, expected in a viral infection and in line with the intense relationship between MMTV and the immune system in the mouse [Stimulated by a paper of Uri Einav et al., reporting the expression of an interferon-related signature in breast and ovarian cancer , researche mouse . B and TAs already seen, in the previous years, many attempts were undertaken to identify MMTV particles in human cancer cells, without conclusive results. Pogo’s laboratory identified them in primary cultures of cells obtained by pleural effusions of patients with metastatic breast cancer. Particles showed ultrastructural characteristics compatible with MMTV. Env protein was expressed and reverse transcriptase activity was determined. cDNA sequences from virion DNA were synthesized, amplified, and sequenced; all the virion genes were detected, and 70% of the virion RNA was sequenced. The sequence homology with MMTV and human MTV proviruses were 85% and 95%, respectively. A 52 kDa protein was detected in tumor cells .Envelope and capsid viral proteins were detected in primary cultures of human breast cancer cells containing human MTV sequences by Western blot and FACS using a panel of antibodies .The expression of HMTV envelope (Env) and capsid (Ca) was detected in 10 primary cultures of human breast cancer containing HMTV sequences (MSSM) by Western blot and fluorescence activated cell sorting (FACS), using a panel of antibodies against HMTV Env, HBRV Env and Ca and the MMTV Env Gp36 and Ca P27 proteins. All the antibodies detected MMTV proteins. Approximately 13% of the MSSM cells showed HMTV protein expression by FACS analysis.On the basis of the results obtained in her laboratory, Beatriz Pogo introduced the term Human Mammary Tumor Virus (HMTV), to express in a synthetic but exhaustive way her convincement of the existence of a human homolog of MMTV .env sequences in human breast cancers. Most of the laboratories had positive results, while others could not find a correlation. Almost all studies analyzed the original env sequences. Data provided by Pogo’s laboratory motivated many other groups to investigate the presence of The table lists 46 articles, with a total of 51 patient populations. MMTV sequences are present in 42 populations (82%), while absent in 9 (18%). The total number of cases studied is 5015, with 1320 positive cases, accounting for a 26%. The percentage of positivity among North America, South America, Europe, and Asia is substantially the same . The percentage doubles in North Africa (40%) and Australia (40%).The studies were conducted on frozen material or on FFPE tissues, with the majority on the latter. The laboratory procedures seem substantially adequate; however, it is hard to go into details. It is impossible to obtain clear information about histotypes and hereditary tumors.Some papers report positive cases also in normal breast tissues, in benign breast lesions, in blood, and in human cancer cell cultures.There is one and only one message that is possible to get from the Some of the authors of the “negative” papers suggest that the “positive” data could be a consequence of a contamination with endogenous viral sequences or with plasmids or amplimers, actually denying the viral hypothesis for BC. Others suggest that the absence of viral sequence could be due to a concentration too low for being detected or to a different geographic and ethnic distribution of the virus. A possible explanation that can be considered “prophetical” (2003) is that “the agent may be a still unknown human retrovirus” . MoreoveThe 2011 article of Daniel Park et al. , with onTwenty years ago, the laboratory of Susan Ross showed that the mouse TfR1 is the entry receptor for MMTV . Then, tIn 2005, in Vienna, Stanislav Indik, Walter Günzburg, Brian Salmons, and Francoise Rouault demonstrated that MMTV is able to infect human normal (Hs578T) and cancerous (T47D and MCF7) cells . Then, tFinally, Indik et al., 7 years ago, already had demonstrated that MMTV is capable of infecting cells in the absence of TrF1. In fact, the viral Env G42E mutation enhances the interaction virus/receptor on the cell surface and confers to the virus a greater infectivity. The mutation consists in a substitution of glutamic acid in place of glycine at position 42 of SU. Authors concluded that the transferrin receptor 1 is not essential for infection and that a different cell surface molecule mediates the MMTV entry into human cells .In 2006, the author, stimulated from Pogo’s results on human carcinomas  and fromIn a previous chapter, the concepts of cancer progression and of preinvasive lesion were discussed.env sequences were investigated in the various types of preinvasive lesions. As expected, HMTV is well expressed since the beginning of the nuclear modifications, with a 27% of positive cases in ADH . Quite surprisingly and interestingly, DCIS  doubles the positivity of invasive tumors, 82% vs. 35%. The dramatic decrease in positive cases in invasive tumors is paralleled by the even dramatic decrease in the viral load analyzed by CISH (chromogenic in situ hybridization). The virus is present also in normal glandular structures proximal to invasive carcinomas (19%), while normal epithelia at distant sites are always negative [Applying the methodology illustrated above, negative .The interpretation of these results is made easier bearing in mind that gene mutations induced by MMTV integration are initiating events , not invThe halving of the positive cases in invasive lesions when compared to DCIS can be explained by the fact that the virus is no longer necessary and can be lost . In factAs detailed in the first part of this article, HBCs recognize a specific genetic etiology, based on the hereditary transmission of highly penetrant pathogenic mutations affecting a TSG. As a consequence, it was hypothesized that HBCs do not need an exogenous etiological agent, such as HMTV, for their development.A study on the presence of viral sequences in two groups of sporadic and hereditary breast tumors confirmed the hypothesis: HBCs have a very low percentage of cases positive for HMTV, only 4% (2/47), in comparison to a 30% (17/56) of sporadic carcinomas. All patients with HBCs hosted a BRCA germline mutation .Interestingly, this study well supports the reliability of the data obtained by FFPE tissues. In addition to the usual laboratory precautions, all the paraffin blocks (HBCs and SBCs) were from the same archive and were processed in the same laboratory, with the same equipment and reagents; moreover, the laboratory did not host mice nor murine cell cultures. Such a significant difference (30% vs. 4%) cannot be due to a contamination with murine material, unless of an improbable “selective” contamination.These data on HBC also allow researchers to view in a different light Daniel J. Park et al.’s 2011 paper  reportinThese studies will be described in Part IV.Env-derived peptide, has a nucleolar localization and interacts with the nucleolar protein B23 . B23 is associated with other retroviral proteins, such as Rev and Rex [Jacob Hochman et al. identified a MMTV p14 protein in tumorigenic and nontumorigenic cells of murine T-cell lymphomas . This pr and Rex .The authors developed antibodies against recombinant p14, which revealed it was useful to localize it in the nucleoli of murine mammary carcinomas and human BC . ActuallEnv, was shown to be a phosphoprotein with two phosphorylation sites, at serine 18 and 65. p14 behaves as a tumor modulator: a Ser65Ala mutation is associated with impaired tumorigenicity, whereas Ser18Ala mutation is associated with enhanced tumorigenicity [Notably, p14, identified as the signal peptide of MMTV genicity .Finally, Hochman et al. showed p14 expression on the cell surface of both murine and human cells containing MMTV. Since p14 is immunogenic, they propose it as target for vaccination strategies .In the last 20 years, James Sutherland Lawson (Jim to his friends) et al., put an amazing energy in the transmission to the scientific community of their conviction that viruses play a major role in human diseases: breast cancer, prostate cancer, atherosclerosis. MMTV occupies a major place in their interests and some of their many results deserve a special mention: the presence of MMTV in breast tissues years before the development of a carcinoma , the resDetails about Jim’s work are outlined in the article co-authored with Wendy K. Glenn published in this same issue of Viruses .Jim also wrote two delightful books, one on his adventurous life, in which MMTV plays an important role , and oneIn humans, an association has been found between MMTV and non-breast tumors: non-Hodgkin lymphoma (NHL), tumors influenced by hormones, and lung cancer.The association between BC and NHL has been described during the last twenty years ,198. QuiHMTV has been detected in carcinomas of endometrium (10%), ovary (16%), and prostate (36%) ,200. ThiHMTV has been detected in a few cases of lung cancer. Unfortunately, not sufficient information about the specimens is provided .Taken together, these observations (lung cancer apart) are a solid suggestion for a viral role of HMTV in human carcinogenesis, not limited to the breast, as is also observed for MMTV in mice.The story begins in 1998, when Andrew Mason et al. showed that many patients with primary biliary cirrhosis (PBC) had antibody reactivity to proteins that share antigenic determinants with HIV-1 p24 or HIAP  . Having Mason’s scientific work is of special relevance in the research of a human betaretrovirus. An important component of this work was to link human betaretrovirus with autoimmunity by detecting infection in cells displaying upregulated mitochondrial autoantigen expression . A new aUsing perihepatic lymph node DNA, in 2004, human betaretrovirus nucleic acid sequences were cloned from patients with PBC . They shThen, a correlation between MMTV infection and the production of AMA  was shown in many mouse models of primary biliary cirrhosis, where MMTV proteins were localized to cells displaying upregulated mitochondrial autoantigen expression . AntivirIn 2015, the human betaretrovirus was found frequently integrated in biliary epithelium of patients with PBC, autoimmune hepatitis and other idiopathic liver disease In 2020, human betaretrovirus was produced, purified and characterized and antibodies against the viral surface protein (Su) were detected in patients with breast cancer or liver disease .In the meantime, several papers on antiviral therapies in patients with primary biliary cholangitis were published reporting significant improvements in liver biochemistry and histology ,214,215.An important result is illustrated in an article published in this same issue of Viruses: this year they were able to isolate a human betaretrovirus from patients with primary biliary cholangitis .The search in humans for exogenous retroviruses must take into account the existence of HERVs. In fact, their presence can cause false positive results. For a better understanding of the issue, the following notes can be useful. Murine ERVs have been already discussed.env, gag, pol, pro) flanked by LTRs [Millions of years ago, unknown retroviruses used to infect the somatic cells of vertebrates. Sometimes, germ cells were infected and viruses could become stably integrated in their genome, acquiring the ability to be transmitted to the next generation (Mendelian inheritance). These retro-elements (or transposable elements) are called endogenous retroviruses (ERVs). In humans, they are called HERVs (human endogenous retroviruses) and comprises about the 8% of the genome. A complete HERV is made of four viral genes , II (beta-like), and III (spuma-like), on the basis of their similarity to exogenous retroviruses. The HML supergroup of class II (human MMTV-like) arouses a special interest, for its relationships with MMTV, and, as consequence, with a well-known murine model of oncogenesis .Quite interestingly, the production of MMTV B-like particles were described in human cells and considered of endogenous origin ; moreoveThe research of a viral etiology for human breast cancer has been accompanied since its beginning by a doubtful school of thought, according to which the presence of MMTV sequences in human breast carcinomas is due to contamination with murine material. It must be emphasized that some of these laboratories do not seem familiar with the biology of the MMTV and/or of the human breast carcinoma. Moreover, some of these papers were published even years before clarifying discoveries.Some of these papers ,226,227 Results showing a correlation between MMTV/HMTV/HBRV are false positive caused by contamination with murine DNA containing integrated MMTV DNA.Rodent DNA is present throughout building walls and ventilation systems.Negative data are consistent with the majority of papers on this topic, while only a few labs describe MMTV positive human breast-cancer specimens.The present article summarizes the numerous studies demonstrating that the viral sequences investigated are exogenous and human, using appropriate laboratory procedures. In particular, the use of frozen tissue, of the laser microdissection for the selection of the cell population and its enrichment, and of the fluorescent PCR represents a quality assurance .The studies carried out in Pisa were performed in laboratories that did not host animal facilities, nor viral cultures, and neither murine cell cultures; the author believes that at least the largest part of the laboratories in which similar studies were conducted was in the same condition.Statement n. 3 is not supported by the analysis made in this article, as it appears by the reading of the -In the same laboratory (Pisa), with the same equipment and reagents, the percentage of MMTV positive cases remains constant in the years ,149,150:-Again, in Pisa, the positivity was of 4% in hereditary tumors against 30% in sporadic ones; all the FFPE tissue came from the same archives and was processed in the same laboratory with the same equipment and reagents . The str-As detailed before, all over the world MMTV has been examined in 51 BC populations and found present in 82% of them. The percentage of positivity remains between 18% and 28%, with the only exception of North Africa and Australia, where it reaches 40%.Moreover, some facts cannot be interpreted due to contamination:One of the preferred claims against the existence of HMTV is that the presence in human tissues of HERVs similar to MMTV can induce false positive results.Some of the most relevant papers on HMTV solved the problem by a careful comparative analysis of the viral sequences used in their experiments with HERVs sequences ,126. The-MMTV tumors and human BC have different morphology.This statement has no scientific base. The reading of Thelma Dunn’s original article  and of a-If HMLV has a similar life-cycle to MMTV, it would be expected that transmission via breast milk should occur. Epidemiological studies failed to demonstrate that breast-feeding was a risk factor for human breast cancer.As detailed in -Traces of MMTV are detected in normal mouse breast tissues and epithelia.MMTV is well documented in human normal mammary tissues ,193, as -Pregnancy has a well-known protective effect against the risk of developing breast cancer in humans: the opposite is true for MMTV caused murine hyperplasia.As detailed in -Unlike all established human oncogenic viruses, chronic immuno-suppression does not predispose to human breast cancers.In the mouse, MMTV needs an efficient immune system, necessary to infect the host and for its distribution to the organs, and to mammary glands, in particular. Studies report that in immunosuppressed women the incidence of BCs either decreases or does not increase, in favor of the MMTV etiology ,232,233.-Human cells lack the receptor necessary for the viral entry of MMTV.As discussed in point 7.3, MMTV can infect human cells in absence of the TfR1 .-The presence of the viral marker should be more frequent in cases than in normal controls in the same geographic setting, as measured by the virus present in human mammary epithelial cells, and antibodies to the virus being present/elevated.Point 6 illustrates the large number of studies worldwide, with results in line with these requests.-Temporal relationship: exposure to the virus should precede disease outcome. This was demonstrated in 2017 .-The association of the virus with breast cancer should be replicated by multiple investigators”.The association MMTV/human BC was shown in 42 different studies against nine with negative results, with 1320 positive cases out of 5015 (26%); see point 6.-The geographical prevalence of the virus should be highest where the incidence of breast cancer is highest.This study is very difficult. The epidemiological data on cancer are not fully reliable due to differences among countries in terms of Cancer Registries, awareness campaigns, and early diagnosis (see point 20).-There should be a dose–response relationship between exposure level and the incidence of breast cancer.Not applicable.-Biological plausibility/coherence: viral causation should make sense in terms of mode of transmission, natural history/pathology, and oncogenic capacity .The answer is positive for all these points, as illustrated in the different sections of this article.-Negative data are consistent with the majority of papers on this topic, while only a few laboratories describe MMTV positive human breast-cancer specimens.This statement is not supported by the analysis carried out in this article and previously summarized.Some of the cited papers gave special attention to the issue of contamination by adopting very accurate laboratory methodologies ,193,228.Quite interestingly, in several studies the incidence of BC is described as unchanged or even reduced in immunosuppressed women, such as HIV or transplant patients ,233,235.As already discussed, MMTV has special relationships with the immune system of the mouse, which has to be working perfectly to allow the infection. Hence, the above epidemiological data are expected, following MMTV biology. Of course, these studies are observational and require a biological confirmation.Until ten years ago some of the few scientists interested in demonstrating the existence of a human MMTV had never met. As a remedy, the author took the initiative to organize scientific meetings in Pisa.It was the first international meeting on MMTV in humans, under the auspices of the University of Pisa, the European Society of Pathology, and the Tuscan Regional Government.The speakers were as follows: Gertrude Buehring, University of California, Berkley; Robert Callahan, NCI, NIH, Bethesda, MD; Jaquelin Dudley, University of Texas, Austin; Ezequiel Moises Fuentes Pananá, Hospital de Pediatriá, Mexico City; Walter Gunzburg, University of Veterinary Medicine, Vienna; Jacob Hochman, The Hebrew University of Jerusalem; James Lawson, University of New South Wales, Sydney; Andrew Mason, University of Alberta, Edmonton; Chiara Maria Mazzanti, University of Pisa; Stella Melana, Mount Sinai School of Medicine, New York; Mauro Pistello, University of Pisa; Beatriz Pogo, Mount Sinai School of Medicine, New York; Susan Ross, University of Pennsylvania, Philadelphia; Brian Salmons, Austrianova Singapore Pte Ltd., Singapore; Alexander Stewart, University of Ottawa; Harald zur Hausen, German Cancer Research Center, Heidelberg, and; the author.The three conference days were full of constructive discussion and stimulating ideas and useful for planning future research. Harald zur Hausen closed the meeting with a synthesis of the presentations. The conclusion was that the quality and quantity of the data justified pursuing the hypothesis of the MMTV etiology of human breast cancer.The following scientists took part in the event: Ori Braitbard, The Hebrew University of Jerusalem; Giulia Freer, University of Pisa; Eliyahu Golomb, The Hebrew University of Jerusalem; Nicole Grandi, University of Cagliari; Walter Gunzburg, University of Veterinary Medicine, Vienna; Jacob Hochman, The Hebrew University of Jerusalem; Stanislav Indik, University of Veterinary Medicine, Vienna; James Lawson, University of New South Wales, Sydney; Michael Lisanti, University of Salford, Manchester; Andrew Mason, University of Alberta, Edmonton; Chiara Maria Mazzanti, Fondazione Pisana per la Scienza; Stella Melana, Mount Sinai School of Medicine, New York; James Neil, University of Glasgow; Mauro Pistello, University of Pisa; Beatriz Pogo, Mount Sinai School of Medicine, New York; Brian Salmons, Austrianova Singapore Pte Ltd., Singapore; Enzo Tramontano, University of Cagliari, and; the author.The existence of a human betaretrovirus involved in breast cancer and primary biliary cholangitis was considered in detail. The first data on the presence of HBRV in ancient humans were presented and discussed, giving more determination to carry on.Part IV: Human Betaretrovirus—HBRVDuring this first part of the century, two concepts became more solid and finally accepted: (a) there is a virus (MMTV/HMTV) well established in humans, strictly connected to breast carcinoma; (b) the same virus (or highly homologous) is strictly connected with the primary biliary cholangitis (PBC), a chronic, inflammatory, autoimmune disease.As a consequence, the names used to date to indicate the virus were no longer appropriate, in that the virus was human and no longer identified only with BC. HBRV means Human BetaRetroVirus, and is well suited to the situation: a human virus, a betaretrovirus, a virus linked to different types of disease and potentially involved in other diseases.Two years ago, the author and his colleagues hypothesized that the finding of viral sequences in remnants of ancient individuals would have demonstrated the existence of a human MMTV/HMTV, in other words of the HBRV.The strategy consisted in the analysis of 36 ancient skulls dated from the Copper Age to the 17th century, all from Italian regions, namely, Sardinia, Tuscany and Liguria. The dental calculus (tartar) was chosen as the material to be investigated. The choice was suggested by the previous demonstration of MMTV/HMTV in human saliva and salivary glands in that the calculus originates from saliva, which provides many constituents of the dental plaque, a bacterial biofilm covering the surface of teeth. Over time, plaque undergoes a process of calcification, giving origin to the calculus through gradual calcium phosphate mineralization. Interestingly, calculus is an excellent material for the analysis of the paleomicrobiome, in that it provides an excellent preservation of the nucleic acids of the microorganism remained entombed in it during the life. Moreover, the calculus is not easily colonized by environmental bacteria, does not have points of entry for exogenous bacteria and lacks nutrient sources that are able to attract bacteria and support their growth ,237,238.Env sequences being investigated. Two of them were dated back to the Sardinian Copper Age, approximately 4500 years ago, while the other four were from the 6th–17th century [Six of the 36 individuals examined revealed the presence of the  century .Most importantly, an accurate bioinformatic analysis demonstrated that the amplified sequences do not derive from the amplification of HERV-K HML (human endogenous retrovirus-K human MMTV like). Moreover, the phylogenetic analysis of the amplified regions showed that all the amplicons grouped together with HMTV and MMTV C3H were phylogenetically well distinct from all the other exogenous and endogenous betaretroviruses.HBRV was finally born.zoonosis, from the ancient Greek ζῷον  and νόσος . A CST can occur today or it could have occurred in ancient times. In the latter, the new host is permanent and the disease becomes typical of the new species.Pathogens can change their host, moving from one species to another. This phenomenon is called cross-species transmission (CST) or interspecies transmission or host jump or spillover. When CST causes a disease, it is called Several modern diseases began in the past as a zoonosis, such as measles, small pox, influenza, diphtheria. As any pathogen can, retroviruses too can jump from animals to humans. An example is HIV, which became human at the beginning of the last century, moving from its original host, the chimpanzee .fertile crescent, which includes Western Asia, the Nile Valley and the Nile Delta, where and when agriculture arose. Humans began to cohabit with other species, some of which were then domesticated. Mice, having a guaranteed abundance of food and with a very high reproduction rate, became a very large population, beginning their long story of strict “friendship” with man. In this so promiscuous habitat, animal pathogens began to infect humans to definitively settle within them. The rise of agriculture is considered a key moment for CST [Many animal pathogens became human pathogens about 10,000 years ago, at the beginning of civilization, in the area named the  for CST .In the past millennia there was a rise in the frequency of house mice with the intensification of urbanization. Cats became domestic to control rodents and possibly participated in giving rise to the origin of zoonoses .The finding of HBRV in our ancestors from thousands of years ago is highly indicative that a mouse-human cross-species transmission occurred in the fertile crescent in prehistoric times. This process led to the humanization of MMTV, which finally became HBRV.Bearing in mind the murine model, the following hypothesis comes spontaneously: mice host the MMTV, mice are everywhere, ergo mice transmit viruses to humans.(a)Mus musculus, Mus m. musculus and Mus m. domesticus, each have a different geographic distribution: Mus m. musculus lives in eastern Europe and in northern Asia, while Mus M. domesticus lives in western Europe, in north Africa, and in the near East. A third subspecies, Mus m. castaneus, lives in southeast Asia, from the eastern part of the Indian subcontinent to all southeastern Asia.The two most numerous subspecies of (b)Mus m. domesticus than in Mus m. musculus and Mus m. castaneus; exogenous infectious particles can evolve from endogenous particles; as a consequence, Mus m. domesticus is more infectious for what concerns MMTV.The number of endogenous MMTV loci is higher in (c)The incidence of breast cancer is higher in western Europe in comparison to eastern Europe.(d)Mus m. domesticus.The higher number of breast cancer patients in western Europe can be a consequence of a zoonosis, with a direct transmission to humans of MMTV from In 2000, Thomas Stewart et al.,  publisheThe paper had a strong impact on the scientific community interested in the existence of a human MMTV and was an object of particular attention in the 2012 Pisa Meeting on Viruses and Breast Cancer. Very recently, Alexandre Stewart and Hsiao-Huei Chen revisited the MMTV zoonotic hypothesis, basically confirming their data of 2000 .(a)M. musculus, M. domesticus, M. castaneus [m. domesticus (50%) and M. musculus (43%) [Knowledge about the presence of MMTV in wild mice is quite limited, with studies usually conducted on exiguous numbers of animals. However, exogenous MMTV has been found in all the three Mus subspecies mentioned, astaneus ,242. In (b)According to Murray B. Gardner, “the prevalence of type B virus (mammary tumor virus) and of spontaneous breast tumors was uniformly low in all the population of wild mice” and “in wild mice MMTV is transmitted by milk and not genetically, exists in low prevalence, causes a few breast tumors, and plays an insignificant role in the overall biology or causes of death” .(c)In recent times, several papers indicate that BC epidemiological data are changing ,248,249.(d)Moreover, a classic assumption of BC epidemiology is now in question: the change in BC incidence in individuals moving from one country to another with different frequency of the disease is currently under debate ,251.(e)Finally, the paleoantrophological data cannot be ignored .The hypothesis that mice can transmit MMTV to humans makes sense, not least for the billions of mice living on the earth. There are, however, a few points that deserve attention:In any case, the two situations are not necessarily mutually exclusive and could coexist.Ascertaining the human nature of HBRV: how is the infection transmitted?-To infect the newborn mouse, a large quantity of virus is required. Because at any lactation, most of the particles are denatured in the stomach ,48,252, -12 virions/mL [To obtain a high incidence of mammary tumors by nursing, it is necessary to nurse for an appreciable period of time, despite that during the first few days, milk is not at all deficient in virus, containing about 10rions/mL .-Human milk has been shown to exert a destructive effect on MMTV particle ,253.-B particles are very difficult to find in human milk .As seen in the previous parts of this article, scientists for a long time, and in vain, have tried to demonstrate the presence in human milk of MMTV/HMTV in a quantity sufficient to infect the newborn. This result is in line with what is known about the biological basis of infection in mice:However, the presence in human milk of traces of MMTV in terms of viral particles, viral sequences, and reverse transcriptase ,254,255,In the mouse and in other animals, MMTV as well as exogenous retroviruses, in general, are present in salivary glands and saliva, and can be transmitted horizontally ,256,257.Waldeyer’s ring is, in humans, a major defensive system against microbiological agents  situated at the main entry of the body. It is made by several tonsils , lymphoid immunocompetent organs. The tonsils’ structure and function are quite similar to those of Peyer’s plaques, discussed previously. M cells uptake antigens and stimulate B and T cells . NALT  the absence of viral sequences in the saliva of newborns eliminates the possibility of a trans-placental transmission and strongly supports an inter-human transmission; (b) the saliva and the salivary glands of adults have a very similar percentage of positivity ; (c) the fact that the percentage of positive cases in pediatric ages is almost three times that of adults does not surprise, considering that children are much more prone than adults to infections; (d) the very high positivity in breast-cancer patients is a highly significant link between the virus and breast carcinoma.All the data here summarized make conceivable the possibility that HBRV can be spread into a population by the saliva of an infected person. The virus can easily reach the Waldeyer’s ring structures. The oro-pharingeal lymphatic structure can be an entry-side for HBRV, as seen before.Saliva and its droplets contain viruses, bacteria, epithelial cells, immunocompetent cells, molecules of different type, and ions .Saliva transmits its content by contact or by air  .Saliva transmits prions and the diseases they cause. This happens in chronic wasting disease (CWD) in cervids, a very interesting natural model of neurodegenerative disease quite similar to Alzheimers. Moreover, despite the very low salivary concentration of prions, a single exposure can initiate infection .This is a difficult question to answer. However, the betaretrovirus HBRV is a “slow” virus with a special link with the immunocompetent cells, which it uses as a “taxi”. So, a situation different from that of the more common viruses can be expected. Are there complete viral particles in a small but sufficient quantity? Do the lymphocytes contain viral nuclei acids? Do the epithelial cells contain viral nuclei acids? Are there free viral nucleic acids? Indeed, all these questions are waiting for definitive answers.The appearance of breast cancer in genetically unrelated individuals of the same family has been described several times. Most cases deal with wife and husband, but there is also a case with father, mother, and daughter. These reports ,265,266 The term “ductal” carcinoma has now been disproven and thus mothballed. In fact, the site of origin of BC is the TDLU and not the ducts, for reasons explained before.Up to 80% of invasive carcinoma are today called “no special type” (NST) or “not otherwise specified” (NOS); invasive lobular carcinoma represents about 10%; the others, about twenty histotypes, are about the remaining 10% under the definition of “special types” invasive carcinoma [Actually, NSTs, in turn, have a large number of architectures, difficult to catalog somehow, made of a variable quantity of acini, solid groups of cells, and stroma, often in large quantities. Moreover, the analysis of hormone receptors and of HER2 expression allows a further examination with prognostic/therapeutics purpose.(a)The morphology of murine mammary tumors is determined by the strain of virus and not from the genetics of the host.(b)MMTV exerts its oncogenic action in mice primarily by a random process of insertional mutagenesis, with certain trigger integrations leading to the activation of many pathogenetic molecular pathways.The question “why this richness of architectures?” is legitimate and might be explained by a viral etiology of the disease, for a couple of possible reasons by analogy to the situation in mice, which have been discussed before:In respect to the latter point, it is noteworthy that “MMTV displays the most random dispersion of integration sites among retroviruses determined so far” .”(1)Different strains of HRBV exist, and are able to activate oncogenesis in different ways.(2)HRBV adopts the mechanism of insertional mutagenesis, activating a various and wide number of pathways, with different morphological and biological consequences.Hence, two explanations are possible and deserve to be investigated:Preliminary data from the Pisa laboratory indicate the presence of HBRV in almost all the histotypes.On the basis of what is known about MMTV, it is possible that HBRV carries out its oncogenetic mechanism through a process of insertional mutagenesis. MMTV INT and CIS have been of great relevance in the studies on human BC, with the identification of genes and pathways related to BC and to general carcinogenesis ,282,283.In the meantime, mechanisms other than insertional mutagenesis could play a role, at least in theory, as discussed by Salmons and Gunzburg ,287:The surface unit (SU) of the Env protein hosts an ITAM sequence, which has tyrosine residues phosphorylated by Src kinases. Src recruits Syk kinases, able to phosphorylate substrates involved in cell proliferation and differentiation. ITAMs are encoded also by viruses as EBV and BLV. The ITAM associated to Env is able to initiate malignant transformation of mammary epithelial cells ,77. InteSag sequences isolated from human BC were shown to be able to perform functions similar to those in the mouse [Sag may act as an oncogene, stimulating the proliferation of epithelial cells and promoting tumorigenicity of hyperplastic mammary epithelial cells [MMTV has an open reading frame (ORF) in the 3′ LTR, encoding also for Sag, indispensable for the infection of lymphocytes and the diffusion of infection. he mouse . Then, ial cells .Naf is hosted by the same ORF. Actually, sag and naf share parts of their coding sequences. Naf is a transcriptional repressor of retroviral genome and is able to induce differential expression of several proteins and to reduce epithelial cell growth. These characteristics suggest a possible role in oncogenesis [ogenesis ,290,291.regulator of expression of virion proteins) present in the nucleus and involved in the export of unspliced, or incompletely spliced, mRNA. Rem is an alternative splice variant of the env mRNA localized to nucleoli. p14, a 14-kDa phosphoprotein, is the Rem-derived signal peptide. Of particular interest are two phosphorylation sites at Serines 18 and 65, phosphorylated by two different kinases of the PKC and CK2 types, respectively. Interestingly, p14 can function as either an oncogene, when phosphorylated by CK2, or as a tumor suppressor, when phosphorylated by PKC [Rem is a Rev-like protein  by regulating PI3K/PTEN/AKT/GSK-3b pathway .There may be an interaction between HBRV and HERVs  or even the production of pseudotyped HBRV and HERVs virions or retrotranspositions that may also play a role in the causation of breast cancer ,297.HBRV is linked to BC. At the same time, it is linked to PBC. Moreover, BC has a myriad of histotypes, possibly related to the virus.In the mouse, several types of MMTV exist, able to give origin to tumors different in terms of morphology and biology.The possibility that different strains of HBRV exist has to be taken into consideration.Part VA few million new cases of breast cancer are diagnosed worldwide every year.No cause is known for breast cancer. Estrogens are essential pathogenetic factors, but their etiological role has never been proven. No sound etiological hypothesis alternative to the viral one has ever been advanced.The betaretrovirus MMTV, the mammary tumor virus, is a potent oncogenic agent that in the mouse causes mammary tumors, and also lymphomas.The experimental model of MMTV-induced murine mammary tumors had a significant role in unveiling central aspects of the biology of cancer, in general, and of human breast cancer, in particular.Murine mammary tumors and human breast carcinomas have unquestionable biological and morphological similarities.MMTV needs an efficient immune system to infect the host and to spread to its organs.A vast and solid amount of data collected worldwide indicates the existence of MMTV in humans, therefore named HMTV, human mammary tumor virus. At the same time, data strongly indicate the involvement of HMTV in human breast cancer and in PBC, primary biliary cholangitis.HMTV is detectable in normal breasts years before the appearance of cancer; moreover, it is present in preinvasive breast lesions, with 80% positivity in ductal carcinoma in situ (DCIS), whereas it is about 40% in invasive tumors.A paleoanthropological study has demonstrated that HMTV is a human virus living with humans over thousands of years.This “new” virus, actually the first human Betaretrovirus, is named simply HBRV, human betaretrovirus, in that it is not any more related only to the mouse or to mammary tumors, but also to PBC, an inflammatory liver disease.HBRV could be spread by saliva and infect through the Waldeyer’s ring lymphatic structures.Human cancers with a viral etiology are numerous.The evidence for a causal role of HMTV/HBRV in human breast cancer has continued to increase over the last 25 years due to the advances in technology and methodology, on which we count for its conclusive demonstration.The hope is that a century of passionate and enthusiastic research can motivate the scientific community to invest in this goal.


Temporomandibular disorders (TMD) is an umbrella term that encompasses many musculoskeletal problems that include the masticatory muscles, the temporomandibular joint, and other associated structures. TMD can be divided into two large groups: those that affect the musculature and those that affect the joint. The treatment of TMD requires the combined skills of physiotherapists and dentists, as well as sometimes psychologists and other medical specialists. This study aims to examine the effectiveness of the interdisciplinary approach using physiotherapy and dental techniques on pain in patients with temporomandibular disorders (TMDs). This is a Scoping Review of studies investigating the effects of combined therapy on patients with TMD. PRISMA guidelines were followed during this review’s design, search, and reporting stages. The search was carried out in the MEDLINE, CINHAL, and EMBASE databases. A total of 1031 studies were detected and analyzed by performing the proposed searches in the detailed databases. After removing duplicates and analyzing the titles and abstracts of the remaining articles, six studies were ultimately selected for this review. All the included studies showed a positive effect on pain decreasing after a combined intervention. The interdisciplinary approach characterized by the combination of manual therapy and splint or electrotherapy can positively influence the perceived symptoms; positively decrease pain; and reduce disability, occlusal impairments, and perception of change. Temporomandibular disorders (TMDs) occur due to excessive and/or prolonged joint overload, influenced by biomechanical factors that may lead to excessive or unbalanced joint loading as well as reduced joint adaptability . InjurieThe treatment of TMD requires the combined skills of physiotherapists and dentists, as well as sometimes rheumatologists and other medical specialists . In certThe effectiveness of TMD treatments is controversial, although it is recognized that multidisciplinary treatment with education and counseling, exercise therapy, manual and invasive physiotherapy, and occlusal splint therapy, in addition to pharmacotherapy in moderate to severe pain, is effective in many patients ,8,9.As with other conditions with chronic pain, such as low back pain, in TMD, the psychological and physical factors that influence the disability of patients are of great importance and must be considered when treating them . Manual While it is true that some physiotherapists and dentists may claim that an altered occlusion may affect the spine, scientifically, there is no evidence of the beneficial effects of orthodontic treatment on spinal deformity . FurtherSince in the treatment of this region, physiotherapists and dentists collaborate, the purpose of this study is to examine the current evidence on the effectiveness of the interdisciplinary approach using physiotherapy and dental techniques on pain in patients with TMD.This is a Scoping Review of studies investigating the effects of combined therapy on patients with TMD. PRISMA guidelines were followed during this review’s design, search, and reporting stages.The literature search was conducted independently by two authors (N.B. and A.B.) on MEDLINE, EMBASE, and CINAHL, for articles published before 1 September 2022, using the keywords “temporomandibular joint disorders”, “physiotherapy”, “dentistry”, “interdisciplinary approach”, “manual therapy”, “exercise therapy”, and “occlusal splint”, combining with Boolean operators and MeSH terms and completing the searching operation with manual research by a search methodology expert. The search strategy was not language-restricted, and only human research was considered, including randomized controlled trials (RCTs) and observational studies (cohort studies and case-control studies).The selected studies’ participants had to be male or female adults with a diagnosis of TMD based on the homonymous diagnostic criteria (DC/TMD)  or any cAfter the independent titles and abstracts screening of the identified studies by two authors (N.B. and A.B.), full texts of the potentially relevant articles were retrieved. All disagreements between the reviewers were settled with another author (J.H.V.). The manual search of relevant studies’ references was applied to retrieve additional articles. Exclusion criteria based on study design were editorials, comments, case reports/series, letters to the editor, reviews, and meta-analyses. The studies that included subjects under 18 years of age and subjects with a history of TMD surgery or systemic pathologies were excluded.RCT methodological quality was evaluated using the PEDro scale. The PEDro scale is an 11-item scale designed to rate randomized clinical trials (RCTs) methodological quality. Each item that is satisfied on the scale contributes one point to the total possible score of 10 points . The metInitially, 1031 articles were found through the databases search on MEDLINE, CINHAL, and EMBASE. Once duplicates were eliminated, and the titles and abstracts of the remaining papers were examined, nine full-text articles were explored to verify their eligibility for inclusion in this review. Four of these articles were excluded, and lastly, one study was included from related research. Six studies ,20,21,22The risk of bias analysis of the four RCTs included in this review ,19,21,22The PEDro scale was used to assess the quality of the four RCTs included in the review. Three of the articles reviewed ,19,21 wep = 0.001), which was maintained at T2 (p = 0.001), with the treatment factor explaining 33.2% (R2) of pain-score improvement with a large effect size (f2: 0.50). About the distinctions between groups, statistical differences were showed at T1 and T2 for VAS, with a large effect . The results of the Helkimo Index revealed a significant reduction of 4.4 points, between T0 and T1, in the EG (p = 0.001). The CG, however, showed no statistical differences inside the group. Regarding pain pressure threshold (PPT) there was a significant improvement in the EG for all the TM muscles, whereas the CG remained at similar values for the entire experimentation time. The group variances were statistically significant for all the analyzed muscles at T1 and T2. About the effect of the intervention over the course of time: in the EG at T1, the algometry of the three muscle groups improved significantly, and the Helkimo and the VAS both reduced significantly. These improvements were maintained at T2. The Patient Global Impression of Change Scale (PGICS) showed that, at T2, the EG perceived a greater improvement after treatment , and this difference between groups was statistically significant . In conclusion, this study showed that MT combined with ST leads to a decrease in pain (3-point decrease), higher PPT (of at least 1.0 kg/cm2), improvement of disability caused by pain (4.4-point decrease), and positive perception of change (EG: 50% felt “much improvement”), compared to ST alone.The study realized by Espí-Lopez et al.  was condThe aim of the study realized by Gomes et al.  was to ap = 0.014). Statistical analysis of these comparisons, the Chi-square, showed that all physiotherapy modalities contributed to TMD pain reduction. The statistical analysis was realized by the Chi-square test, and results showed that physical therapy procedures performed without the presence of professionals for TMD treatment are very helpful in pain reduction (p = 0.002). In conclusion, the study realized by Toledo et al. [The study realized by Toledo et al.  analyzedo et al.  showed tp < 0.05). In contrast, the were no statistically significant difference between groups for the passive jaw opening. Analysis of subjective pain evaluation in group 1 showed an improvement in total pain intensity, pain intensity during mandibular movement, pain intensity without mandibular movement, and pain intensity after mandibular loading (p < 0.05). Similarly, pain intensity in group 2 reduced after intervention (p < 0.05). No significant differences were found between the two groups for the subjective parameters. In conclusion, the results of this study showed that physical therapy combined with temporomandibular spin seems to have a positive effect in patients with TMD. The work produced by Ismail et al.  was realp = 0.0001, effect size = 0.853) and control  groups. Results showed that pain-free opening and maximum assisted opening improved notably in the course of time only in group 1 . Using an occlusal splint alone did not lead to statistically significant improvements in the amount of mouth opening (p > 0.05). In conclusion, this study provided evidence that the combination of SS with PT was more effective than the use of SS alone in decreasing deviations and improving the range of mouth opening in a treatment period of 6 months. Moreover, both interventions reduced pain in subjects with anterior disc displacement.The study realized by Alajbeg et al.  was realp < 0.05). Results showed a significant decrease in the occurrence of muscle pain during free movement and TMJ pain on palpation with an average successful outcome of 81%. The change in joint clicking was nonsignificant. Results showed that the opening ROM significantly increased by 8 mm (19%). The accompanying deviations and the constituent values of this movement also increased. The protrusion movement showed no statistical difference from the healthy group, but the lateral movement range improved on both sides by an average of 2.1 mm (both sides 36%). In functional evaluations, a satisfactory result was registered by 86% of the participants. In conclusion, the results of this study showed how myorelaxation therapy was effective in the treatment of TMD. After six months of intervention, a significant improvement in opening/closing velocity, opening movement range, and lateral movement range was obtained.In the study realized by Gawriołek et al. , the jawThe main objective of this scoping review was to examine the current evidence on the effectiveness of the interdisciplinary approach using physiotherapy and dental techniques on pain in patients with TMDs. In terms of the effectiveness of the multidisciplinary approach, three of the RCTs ,19,21 inFor example, one RCT of “a fair quality”  found thIn the same way, one good quality observational study  evaluateFurthermore, the type of exercise may be very important for achieving successful outcomes, such as therapeutic exercise based on scientific evidence . BoucharIn the quasiexperimental study by Gawriolek et al. , the exaIn terms of the effectiveness of the interdisciplinary approach, two of the three RCTs ,19,21 weFinally, the study by Gomes et al.  did haveHowever, in only one  of the tIt should be noted that, although sleep in patients was not analyzed in the included studies, a recent systematic review found that there is insufficient evidence to suggest that patients with temporomandibular joint osteoarthritis are associated with increased sleep disorders or poorer sleep quality .It is recognized that the sample of studies included was low. Even so, in return, it was decided to analyze the studies that used physical therapy and dental techniques together rather than separately to analyze the pure relationship between this type of interdisciplinary approach. A meta-analysis could not be performed due to the considerable heterogeneity of the studies included, which must be viewed as a limitation of the study. However, the scoping review performed widely responds to the objectives established.The interdisciplinary approach characterized by the combination of manual therapy and splint or electrotherapy can influence the perceived symptoms positively, showing a positive effect on pain decreasing, reduction in disability, occlusal impairments, and perception of change. Future interdisciplinary research in physiotherapy and dentistry should include large sample sizes of experimental studies and further investigation of the development of physiotherapy and dentistry techniques used in clinical practice to treat patients with TMD.


Aspergillus sp., isolated from the Amazon biome, which is stored at the Amazon Fungi Collection-CFAM at ILMD/FIOCRUZ, was evaluated. The fungal culture was cultivated in yeast extract agar and sucrose (YES) for cold extraction of the biocompounds in ethyl acetate at 28 °C for 7 days in a BOD type incubator. The obtained extract was evaluated for its antimicrobial activity against Candida albicans and Gram-positive and negative bacteria by the “cup plate” method and the determination of the minimum inhibitory concentration (MIC) by the broth microdilution method. The extract was subjected to thin layer chromatography (TLC) and fractionated by open and semipreparative column chromatography. The fractions of interest had their chemical constituents elucidated by nuclear magnetic resonance and mass spectrometry. The elucidated molecule was evaluated for cytotoxicity against the human fibroblast strain (MRC5). The extract presented inhibitory activity against both Gram-positive and negative bacteria, with the range of inhibition halos from 5.3 to 14 mm in diameter and an MIC ranging from 500 to 15.6 μg/mL. Seventy-one fractions were collected and TLC analysis suggested the presence of substances with double bond groups: coumarins, flavonoids, phenolic, alkaloids, and terpenes. NMR and MS analyses demonstrated that the isolated molecule was kojic acid. The results of the cytotoxicity test showed that MRC5 cells presented viability at concentrations from 500 to 7.81 μg/mL. The kojic acid molecule of Aspergillus sp., with antibacterial activity and moderate toxicity at the concentrations tested, is a promising prototype of an alternative active principle of an antimicrobial drug.The antimicrobial potential of  Staphylococcus aureus (MRSA) and β-lactamase-producing Escherichia coli, which are no longer associated exclusively with hospitals, but are emerging in the community -4-pyrone is an organic acid that was discovered in Japan by Saito in 1907 from the fungus  on rice . Since t on rice –37.It has the capacity of acting as an ultraviolet protector, suppressing skin hyperpigmentation and restricting the formation of melanin through the inhibition of tyrosinase formation, an enzyme responsible for pigmentation . And witIn addition to having antioxidant activity and being used in cosmetics intended for skin lightening, kojic acid and its derivatives also have antibiotic properties against certain Gram-negative bacteria, as well as Gram-positive bacteria . Wei et L. amazonensis amastigotes. In addition, in in vivo experiments, it was possible to observe that the topical application of kojic acid promoted the production of type I collagen fibers in the infection sites, as well as a reduction in the size of the skin lesion.Studies also show the antiparasitic effect of this substance. Rodrigues et al. , for exaAspergillus stand out, such as A. oryzae, A. parasiticus, A. flavus, A. tamari, A. candidus, and A. pseudonomius [Kojic acid is biologically produced by several fungi during aerobic fermentation, using carbohydrates as a source of energy, and among them, some species of the genus donomius . Regardidonomius .Aspergillus species.Even with the valorization of kojic acid, there are no records of industrial production in Brazil. However, several research studies are being carried out for the production of kojic acid from Aspergillus sp. extract isolated from the soil of the Amazon biome showed antimicrobial potential against strains of S. aureus, S. aureus MRSA, and E. coli, presenting in its composition substances of various chemical classes. The product of this work culminated in the isolation of kojic acid, an unprecedented result for Amazonian fungi, according to the bibliographic survey carried out. This result reinforces the potential of fungi present in the region, demonstrating that they can be viable alternative sources for the production of strategic substances for the industrial sector.The


Dubin-Johnson syndrome (DJS) is an often-missed diagnosis of neonatal cholestasis. We report two patients with DJS, who presented with neonatal cholestasis. The first patient underwent extensive investigations for infantile cholestasis with no definitive etiology reached; the diagnosis of DJS was missed until the age of 14 years old. The diagnosis was confirmed genetically with c.2273G > T, p.G758V mutation in exon 18 of the ABCC2 gene. The 2nd patient is a 7-day-old baby, the son of the 1st patient who gave birth to him at the age of 21 years old. He was diagnosed with DJS at the age of 2 weeks based on normal clinical and laboratory workup apart from direct hyperbilirubinemia. He had the same mutation as his mother in homozygous status. The husband was heterozygous for the same mutation. DJS is one of the often-missed differential diagnoses of neonatal cholestasis. It should be suspected in patients of infantile cholestasis, who have an, otherwise, normal physical examination, and laboratory investigations to avoid unnecessary lengthy, invasive, and expensive workups. Dubin-Johnson syndrome (DJS) was first reported in 1954 by Dubin and Johnson . Most paThe syndrome occurs due to expression defects of the MRP2 gene, an ATP-dependent canalicular membrane transporter –5. The dA 14-year-old female child was born to consanguineous Saudi first-degree cousins who are descents from a tribe with highly consanguineous marriage. She was referred to a pediatric gastroenterology clinic as a case of persistent conjugated hyperbilirubinemia for investigations.Tracing her history revealed that jaundice started at the age of 4 days with elevated total bilirubin (350 μmol/L), mainly indirect. Her direct bilirubin was 30 μmol/L with normal alanine and aspartate transaminases (ALT and AST). Phototherapy was started, and the patient was discharged in good condition after 3 days.She returned to her primary physician at the age of 40 days with unresolved jaundice and mild abdominal distension with no organ enlargement. Her investigations revealed mild direct hyperbilirubinemia. Her total and direct bilirubins were 50 and 35 μmol/L, respectively. Her ALT, AST, gamma-glutamyl transpeptidase (GGT), prothrombin time/concentration, and abdominal ultrasonography were all normal. Extensive workups of cholestasis, including complete blood count, retics, coombs, hemoglobin electrophoresis, urine and blood cultures, TORCH screening, serum bile acids, thyroid profile, tandem metabolic screening, and non-glucose-reducing substances in the urine, were all normal. HIDA scan and MRCP were not available in that hospital and were not done. Liver biopsy was refused by the parents, and she was discharged against medical advice in good general condition without a definitive diagnosis. Her total and direct bilirubin at time of discharge was 48 and 40 μmol/L, respectively.Since that time and until her presentation to our care at the age of 14 years, the parents used to visit different health care facilities when their child’s jaundice deepened with different intercurrent illnesses. Laboratory workups, including liver function tests and hepatitis markers, were done many times with normal results apart from direct hyperbilirubinemia.m Tc-HIDA scan was requested. The HIDA scan serial images revealed rapid clearance of blood pool activity with a good hepatocyte function as evidenced by the adequate ascending limb of the dynamic curve. However, there was a slow excretion of radioactivity into the biliary radicles with retained activity in the liver up to 6 h. The gall bladder was seen at 1 h and the small intestine at 2 h. This good hepatocyte uptake function with impairment of excretory function in absence of obstruction was highly suggesting DJS. Urinary coproporphyrins were not done .On presentation to our hospital, she had tinge jaundice with stable vital signs, normal abdominal examination with no organomegaly, and normal assessment of different body systems. Her laboratory investigation showed high total bilirubin of 32 μmol/L, mainly in the form of direct bilirubin (31 μmol/L), with normal ALT, AST, GGT, complete blood picture, and a renal profile with normal abdominal US. The diagnosis of DJS was suspected, and a 99Molecular genetic testing for DJS, the ABCC2 gene, was requested to confirm the diagnosis.PCR amplification and direct sequencing of all coding exons and flanking intronic sequence  gene dosage analysis by quantitative real-time PCR (qPCR) with 5 amplifications  .Unclassified variant c.2273G⟩T, pG785V in Exon 18 of the gene ABCC2 gene in the homozygous state. By qPCR, no deletion or duplication was detected, Molecular analysis confirmed the clinical suspicion of DJS syndrome. The variant c.2273G⟩T, pG785V in Exon 18 of the gene ABCC2 gene was detected in homozygous state.The patient was diagnosed in 2014, and, at that time, this mutation was a novel mutation, which has not been described yet . “Polyphen2”  called tIn October 2021, the parents presented to us once again with the patient who was a 21-year young adult female. She got married to her cousin, and she experienced an intermittent deepening of her jaundice during pregnancy with no associated pruritus or dark urine. Her liver biochemistry was within normal values apart from direct hyperbilirubinemia.She gave birth to a 3.5 kg male baby by normal vertex delivery with uneventful antenatal and perinatal histories. Her baby developed jaundice at the age of 1 week with no history of pallor, blood transfusion, or medications intake.At the age of 4 weeks, she thought about our medical advice for her newly born jaundiced baby. On assessment, his physical examination was normal apart from mild jaundice with no organomegaly. His workup was assuring with normal abdominal ultrasound, ALT, AST, albumin, prothrombin time/concentration, and GGT with a high total bilirubin of 78 μmol/L and high direct bilirubin of 43 μmol/L, suggesting the diagnosis of DJS.Sanger sequencing of the p.G785V variant detected in his mother was performed for him, which came out to be positive. The husband was also tested and was heterozygous for the same mutation.In January 2022, we got the consent of the patient and her husband for publishing their family case series. The family pedigree is illustrated in Dubin-Johnson syndrome is a rare hereditary disease, inherited as autosomal recessive inheritance mainly, but some cases with an autosomal dominant mode of transmission had been reported , 11. TheMany cases were reported worldwide with the ABCC2 gene mutation . Our patTwo reports were released from Saudi Arabia, one with a large series of 28 genetically proven cases with DJS  and the Patients with DJS have metabolic defect since birth, but it rarely presents in infancy and usually becomes more apparent in the late teens. In our reported two patients and in the series of Al-Hussaini and his colleagues, jaundice started in early infancy.The first case was missed until the age of 14 years with extensive workup of cholestasis in infancy up to a liver biopsy with more than 20 physicians’ visits afterward, but the diagnosis of DJS was overlooked. This unfortunate course reflects a lack of knowledge about DJS, which should have been the first differential diagnosis of direct hyperbilirubinemia in otherwise normal patients with unremarkable clinical and laboratory workups.A HIDA scan is a good diagnostic modality for DJS , 21, andThe presence of cholestasis in well-appearing neonates and infants, who have an otherwise normal examination with normal liver biochemical functions and hepatocytes and biliary enzymes, should alert physicians to the possibility of the diagnosis of DJS. A high index of suspicion for DJS in those patients prevents unnecessary lengthy, costly, and invasive workup. Molecular genetic testing of the ABCC2 gene is confirmatory.The datasets for this article are not publicly available due to concerns regarding participant/patient anonymity. Requests to access the datasets should be directed to the corresponding author.The studies involving human participants were reviewed and approved by IRB Committee of Alhada Armed Forces Hospital, Taif, Saudi Arabia. Written informed consent was obtained from the individual(s), and minor(s)’ legal guardian/next of kin, for the publication of any potentially identifiable images or data included in this article.NK diagnosed the patient, set the idea of the study, and designed the study. NK, OS, SA, LS, ME, HA, and AA reviewed literature, drafted the manuscript, and critically analyzed the data. SA, NK, HA, and AA collected patient’s data. All authors reviewed and approved the manuscript for final publication.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.