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Mammary gland cancer is the most common cancer occurring in women globally. Incidences of this cancer in Japan are on the increase. Annually, more than 70,000 new cases are recorded in Japan and about 1.7 million in the world. Many cases are still difficult to cure completely, and animal models are required for the characterization of the biology, therapeutic strategy, and preventive measures for spontaneous mammary tumor. The mouse model used currently has some limitations owing to structural differences between mouse and human mammary glands. Tupaia belangeri (tree shrew), which belongs to the Tupaiidae family, shows relatively high genetic homology and structural similarity to human mammary glands. Here, we characterized the spontaneous mammary tumors in 61 female tree shrews of different ages. The incidence rate was 24.6% (15/61), and the rate of simultaneous or metachronous multiplex tumors was 60% (9/15). From the incidence pattern, some cases seemed to be of familial mammary gland tumor, as the offspring of female tree shrews No. 3 and 9 and male tree shrew No. 11 showed a high incidence rate, of 73.3% (11/15). Average incidence age for tumor development was 2 years and 3 months, and the earliest was 10 months. Histochemical analysis indicated that spontaneous mammary gland tumors in the tree shrew show the features of intraductal papillary adenomas (22 cases), except 2 tubulopapillary carcinoma cases (No. 75 and 131). All the cases were positive for the progesterone receptor, whereas 91.3% were positive for the estrogen receptor, and 4.3% were HER-2 positive. We have also confirmed the expression of nectin-4 in some mammary tumor cells. Additionally, we subjected tree shrews to cytodiagnosis or X-ray CT. Thus, the findings of this study highlight the potential of the tree shrew as a valuable new animal model for mammary gland tumor study.

The molecular mechanisms that allow pathogenic bacteria to infect animals have been intensively studied. On the other hand, the molecular mechanisms by which bacteria acquire virulence functions are not fully understood. In the present study, we experimentally evaluated the evolution of a non-pathogenic strain of Escherichia coli in a silkworm infection model and obtained pathogenic mutant strains. As one cause of the high virulence properties of E. coli mutants, we identified amino acid substitutions in LptD (G580S) and LptE (T95I) constituting the lipopolysaccharide (LPS) transporter, which translocates LPS from the inner to the outer membrane and is essential for E. coli growth. The growth of the LptD and LptE mutants obtained in this study was indistinguishable from that of the parent strain. The LptD and LptE mutants exhibited increased secretion of outer membrane vesicles containing LPS and resistance against various antibiotics, antimicrobial peptides, and host complement. In vivo cross-linking studies revealed that the conformation of the LptD-LptE complex was altered in the LptD and LptE mutants. Furthermore, several clinical isolates of E. coli carried amino acid substitutions of LptD and LptE that conferred resistance against antimicrobial substances. This study demonstrated an experimental evolution of bacterial virulence properties in an animal infection model and identified functional alterations of the growth-essential LPS transporter that led to high bacterial virulence by conferring resistance against antimicrobial substances. These findings suggest that non-pathogenic bacteria can gain virulence traits by changing the functions of essential genes, and provide new insight to bacterial evolution in a host environment., Author summary Pathogenic bacteria developed their virulence properties by changing the functions of various genes after the emergence of the host animals on earth. The types of gene function alterations that confer bacterial virulence properties, however, have remained unclear. We utilized a silkworm infection model to perform an experimental evolution of bacterial virulence activity. From a non-pathogenic strain of Escherichia coli, we obtained a mutant strain that exhibited 500-fold higher virulence than the original strain and identified mutations of the lipopolysaccharide (LPS) transporter, which translocates LPS onto the bacterial surface, as one cause of the high virulence. The mutations changed the structure of the LPS transporter, increased the secretion of outer membrane vesicles, and enabled bacterial survival in the presence of host antimicrobial substances. This mechanism to gain high virulence occurs naturally, as several E. coli clinical isolates carried mutations of the LPS transporter that confer resistance against antimicrobial substances. Our study unveiled a novel mechanism by which bacteria increase their virulence through modifying their gene function.

One of the most refractory breast cancer types is triple negative (TN) breast cancer, in which cells are resistant to both hormone and Herceptin treatments and, thus, often cause recurrence and metastasis. Effective treatments are needed to treat TN breast cancer. We previously demonstrated that rMV-SLAMblind, a recombinant measles virus, showed anti-tumor activity against breast cancer cells. Here, we examined whether rMV-SLAMblind is effective for treating TN breast cancer. Nectin-4, a receptor for rMV-SLAMblind, was expressed on the surface of 75% of the analyzed TN breast cancer cell lines. rMV-SLAMblind infected the nectin-4-expressing TN breast cancer cell lines, and significantly decreased the viability in half of the analyzed cell lines in vitro. Additionally, intratumoral injection of rMV-SLAMblind suppressed tumor growth in xenografts of MDA-MB-468 and HCC70 cells. To assess treatment for metastatic breast cancer, we performed intravenous administration of the luciferase-expressing-rMV-SLAMblind to MDA xenografted mice. Virus replicated in the tumor and resulted in significant suppression of the tumor growth. The safety of the virus was tested by its intravenous injection into healthy cynomolgus monkeys, which did not cause any measles-like symptoms. These results suggest that rMV-SLAMblind is a promising candidate as a therapeutic agent for treating metastatic and/or TN type breast cancer., Graphical Abstract, Triple negative breast cancer (TNBC) is one of the most refractory breast cancer types. A recombinant measles virus, rMV-SLAMblind, infected nectin-4-positive TNBC cells and significantly suppressed the tumor growth in xenograft models. Thus, rMV-SLAMblind is a promising candidate as a therapeutic agent for TNBC.

Highly pathogenic avian influenza virus (HPAIV) is a serious threat not only to domestic fowls but also to humans. Vaccines inducing long-lasting immunity against HPAIV are required. In the present study, we generated recombinant measles virus (MV) expressing the hemagglutinin protein of HPAIV without the multibasic site necessary for its pathogenicity in chickens using the backbone of an MV vaccine strain (rMV-Ed-H5HA) or a wild-type MV-derived mutant (rMV-HL-Vko-H5HA). We examined protective efficacy of the candidate vaccines in the monkey infection model by the challenge with a HPAIV (H5N1). Cynomolgus monkeys inoculated with the candidate vaccines produced both anti-H5 HA and anti-MV antibodies. They recovered earlier from influenza symptoms than unvaccinated monkeys after the challenge with the HPAIV strain. Chest radiography and histopathological analyses confirmed less severe pneumonia in the vaccinated monkeys. Vaccination tended to suppress viral shedding and reduced the interleukin-6 levels in the lungs. Furthermore, the vaccination with rMV-Ed-H5HA of monkeys with pre-existing anti-MV immunity induced the production of anti-H5 HA antibodies. These results suggest that both candidate vaccines effectively reduce disease severity in naïve hosts, and that rMV-Ed-H5HA is a particularly good candidate vaccine against HPAIV infection.

Summary Pancreatic cancer is one of the most intractable cancers and has a devastating prognosis over the past three decades the 5-year survival rate has been less than 10%. Therefore, development of a novel anticancer treatment for pancreatic cancer is a matter of urgency. We previously developed an oncolytic recombinant measles virus (MV), rMV-SLAMblind, which had lost the ability to bind to its principal receptor, signaling lymphocyte activity molecule (SLAM), but which selectively infected and efficiently killed nectin-4-expressing breast and lung cancer cells. In this study, we analyzed the antitumor effect of this virus against pancreatic cancer. Nectin-4 was expressed on the surface of 4/16 tested pancreatic cancer cell lines, which were efficiently infected and killed by rMV-SLAMblind in vitro. The intratumoral inoculation of rMV-SLAMblind suppressed the growth of KLM1 and Capan-2 cells xenografted in SCID mice. The sequence analysis of MV isolated from the tumor revealed that the designed mutation in the H protein of rMV-SLAMblind had been stably maintained for 47 days after the last inoculation. These results suggest that rMV-SLAMblind is a promising candidate for the novel treatment of pancreatic cancer. This article is protected by copyright. All rights reserved.

Oncolytic virotherapy is a distinctive antitumor therapy based on the cancer-cell-specific infectivity and killing activity of viruses, which exert a considerable antitumor effect with only a few treatments. Because colorectal cancer cells often acquire resistance to the molecular-targeted therapies and alternative treatments are called for, in this study, we evaluated the oncolytic activity against colorectal cancer cells of a recombinant measles virus (rMV-SLAMblind), which is blind to signaling lymphocytic activation molecule (SLAM) and infects target cells via nectin-4/poliovirus receptor-related 4 protein. We examined 10 cell lines including 8 cell lines that were resistant to epidermal-growth-factor-receptor (EGFR) targeted therapy. rMV-SLAMblind infected and lysed the nectin-4-positive cell lines dependently on nectin-4 expression, in spite of mutation in EGFR cascade. Tumour progression in xenograft models was also abrogated by the virus and the infection of cancer cells in vivo by the virus was demonstrated with both flow cytometry and a histological analysis. Therefore, rMV-SLAMblind is considered a novel therapeutic agent for colorectal cancers, including those resistant to molecular-targeted therapies.

Oncolytic virotherapy is a promising treatment strategy for cancer. We previously generated a recombinant measles virus (rMV-SLAMblind) that selectively uses a poliovirus receptor-related 4 (PVRL4/Nectin4) receptor, but not signaling lymphocyte activation molecule (SLAM). We demonstrated that the virus exerts therapeutic effects against human breast cancer cells. Here, we examined the applicability of rMV-SLAMblind to treating canine mammary cancers (CMCs). We found that the susceptibilities of host cells to rMV-SLAMblind were dependent on canine Nectin-4 expression. Nectin-4 was detected in four of nine CMC cell lines. The rMV-SLAMblind efficiently infected those four Nectin-4-positive cell lines and was cytotoxic for three of them (CF33, CHMm, and CTBm). In vivo experiment showed that the administration of rMV-SLAMblind greatly suppressed the progression of tumors in mice xenografted with a CMC cell line (CF33). Immunohistochemistry revealed that canine Nectin-4 was expressed in 45% of canine mammary tumors, and the tumor cells derived from one clinical specimen were efficiently infected with rMV-SLAMblind. These results suggest that rMV-SLAMblind infects CMC cells and displays antitumor activity in vitro, in xenografts, and ex vivo. Therefore, oncolytic virotherapy with rMV-SLAMblind can be a novel method for treating CMCs.

Community acquired-methicillin resistant Staphylococcus aureus (CA-MRSA) is a socially problematic pathogen that infects healthy individuals, causing severe disease. CA-MRSA is more virulent than hospital associated-MRSA (HA-MRSA). The underlying mechanism for the high virulence of CA-MRSA is not known. The transcription product of the psm-mec gene, located in the mobile genetic element SCCmec of HA-MRSA, but not CA-MRSA, suppresses the expression of phenol-soluble modulin α (PSMα), a cytolytic toxin of S. aureus. Here we report that psm-mec RNA inhibits translation of the agrA gene encoding a positive transcription factor for the PSMα gene via specific binding to agrA mRNA. Furthermore, 25% of 325 clinical MRSA isolates had a mutation in the psm-mec promoter that attenuated transcription, and 9% of the strains had no psm-mec. In most of these psm-mec-mutated or psm-mec-deleted HA-MRSAs, PSMα expression was increased compared with strains carrying intact psm-mec, and some mutated strains produced high amounts of PSMα comparable with that of CA-MRSA. Deletion of psm-mec from HA-MRSA strains carrying intact psm-mec increased the expression of AgrA protein and PSMα, and virulence in mice. Thus, psm-mec RNA suppresses MRSA virulence via inhibition of agrA translation and the absence of psm-mec function in CA-MRSA causes its high virulence property., Author Summary Methicillin-resistant Staphylococcus aureus (MRSA) is resistant to various antibiotics, including β-lactams, thus causing serious clinical problems. Hospital-associated (HA)-MRSA infects immunocompromised patients in hospitals. Community-acquired (CA)-MRSA causes serious diseases in healthy people who have not had contact with hospitals in the United States, Canada, or Europe. CA-MRSA produces higher amounts of extracellular toxins and has higher virulence than HA-MRSA, although the reason for this is unclear. SCCmec is a foreign DNA integrated into the MRSA chromosome that contains several genes including the mecA gene that confers resistance against methicillin. The SCCmec of CA-MRSA does not contain the psm-mec gene that exists in the HA-MRSA SCCmec. In the present study, we found that the transcription product of psm-mec inhibits translation of the agrA gene encoding a positive transcription factor for many extracellular toxins by direct binding to the agrA mRNA, resulting in decreased extracellular toxin production. Furthermore, some HA-MRSA strains carry mutated psm-mec or no psm-mec and produce higher amounts of extracellular toxins than HA-MRSA strains carrying intact psm-mec. These findings suggest that psm-mec RNA negatively regulates agrA and mutation or absence of psm-mec leads to a high virulence capacity of MRSA.

Highly pathogenic avian influenza virus (HPAIV) continues to threaten human health. Non-human primate infection models of human influenza are desired. To establish an animal infection model with more natural transmission and to determine the pathogenicity of HPAIV isolated from a wild water bird in primates, we administered a Japanese isolate of HPAIV (A/whooper swan/Hokkaido/1/2008, H5N1 clade 2.3.2.1) to rhesus and cynomolgus monkeys, in droplet form, via the intratracheal route. Infection of the lower and upper respiratory tracts and viral shedding were observed in both macaques. Inoculation of rhesus monkeys with higher doses of the isolate resulted in stronger clinical symptoms of influenza. Our results demonstrate that HPAIV isolated from a water bird in Japan is pathogenic in monkeys by experimental inoculation, and provide a new method for HPAIV infection of non-human primate hosts, a good animal model for investigation of HPAIV pathogenicity.

In silkworm larvae, the mature form of paralytic peptide (PP), an insect cytokine, is produced from pro-PP in association with activation of innate immune responses, resulting in slow muscle contraction. We utilized this reaction, muscle contraction in silkworms coupled with innate immunity stimulation, to quantitatively measure the innate immune stimulating activity of various natural polysaccharides. β-Glucan of Gyrophora esculenta (GE-3), fucoidan from sporophyll of Undaria pinnatifida, and curldan induced silkworm muscle contraction. We further demonstrated that GE-3 had therapeutic effects on silkworms infected by baculovirus. Based on these findings, we propose that the silkworm muscle contraction assay is useful for screening substances that stimulate innate immunity before evaluating therapeutic effectiveness in mammals.

Canine distemper virus (CDV) vaccination confers long-term protection against CDV reinfection. To investigate the utility of CDV as a polyvalent vaccine vector for Leishmania, we generated recombinant CDVs, based on an avirulent Yanaka strain, that expressed Leishmania antigens: LACK, TSA, or LmSTI1 (rCDV–LACK, rCDV–TSA, and rCDV–LmSTI1, respectively). Dogs immunized with rCDV-LACK were protected against challenge with lethal doses of virulent CDV, in the same way as the parental Yanaka strain. To evaluate the protective effects of the recombinant CDVs against cutaneous leishmaniasis in dogs, dogs were immunized with one recombinant CDV or a cocktail of three recombinant CDVs, before intradermal challenge (in the ears) with infective-stage promastigotes of Leishmania major. Unvaccinated dogs showed increased nodules with ulcer formation after 3 weeks, whereas dogs immunized with rCDV–LACK showed markedly smaller nodules without ulceration. Although the rCDV–TSA- and rCDV–LmSTI1-immunized dogs showed little protection against L. major, the cocktail of three recombinant CDVs more effectively suppressed the progression of nodule formation than immunization with rCDV–LACK alone. These results indicate that recombinant CDV is suitable for use as a polyvalent live attenuated vaccine for protection against both CDV and L. major infections in dogs., Author Summary More than 1 million new cases of leishmaniasis occur throughout the world every year. Leishmaniasis typically presents as one of two clinical forms, either cutaneous or visceral. Dogs harboring Leishmania act as reservoirs, and are closely associated with human infections in South America and southern Europe. Therefore, immunization of dogs with effective vaccines against Leishmania will also effectively prevent Leishmania infection in humans. In this study, we have evaluated the utility of recombinant canine distemper viruses (CDVs) that express Leishmania antigen as effective polyvalent candidate vaccines against CDV and cutaneous Leishmania infections. The results indicated that recombinant CDV completely protected against challenge with a virulent strain of CDV. Furthermore, mixed immunization with three recombinant CDVs that express different antigens that mediate distinct immune responses, significantly reduced the nodule size after Leishmania major challenge. These results strongly suggest that a cocktail of multiple antigens confers more effective immunity throughout the life cycle of Leishmania. We propose that a combination of recombinant CDV-based vaccines expressing different antigens has utility as a polyvalent vaccine for the prevention of leishmaniasis epidemics by inhibiting the transmission of the parasites through dogs.

Background The importance of visual sense in Hymenopteran social behavior is suggested by the existence of a Hymenopteran insect-specific neural circuit related to visual processing and the fact that worker honeybee brain changes morphologically according to its foraging experience. To analyze molecular and neural bases that underlie the visual abilities of the honeybees, we used a cDNA microarray to search for gene(s) expressed in a neural cell-type preferential manner in a visual center of the honeybee brain, the optic lobes (OLs). Methodology/Principal Findings Expression analysis of candidate genes using in situ hybridization revealed two genes expressed in a neural cell-type preferential manner in the OLs. One is a homologue of Drosophila futsch, which encodes a microtubule-associated protein and is preferentially expressed in the monopolar cells in the lamina of the OLs. The gene for another microtubule-associated protein, tau, which functionally overlaps with futsch, was also preferentially expressed in the monopolar cells, strongly suggesting the functional importance of these two microtubule-associated proteins in monopolar cells. The other gene encoded a homologue of Misexpression Suppressor of Dominant-negative Kinase Suppressor of Ras 2 (MESK2), which might activate Ras/MAPK-signaling in Drosophila. MESK2 was expressed preferentially in a subclass of neurons located in the ventral region between the lamina and medulla neuropil in the OLs, suggesting that this subclass is a novel OL neuron type characterized by MESK2-expression. These three genes exhibited similar expression patterns in the worker, drone, and queen brains, suggesting that they function similarly irrespective of the honeybee sex or caste. Conclusions Here we identified genes that are expressed in a monopolar cell (Amfutsch and Amtau) or ventral medulla-preferential manner (AmMESK2) in insect OLs. These genes may aid in visualizing neurites of monopolar cells and ventral medulla cells, as well as in analyzing the function of these neurons.