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(1) Background: an increasing number of breast cancer patients develop lethal brain metastases (BM). The complete removal of these tumors by surgery becomes complicated when cells infiltrate into the brain parenchyma. However, little is known about the nature of these invading cells in breast cancer brain metastasis (BCBM). (2) Methods: we use intravital microscopy through a cranial window to study the behavior of invading cells in a mouse model of BCBM. (3) Results: we demonstrate that BCBM cells that escape from the metastatic mass and infiltrate into brain parenchyma undergo epithelial-to-mesenchymal transition (EMT). Moreover, cells undergoing EMT revert to an epithelial state when growing tumor masses in the brain. Lastly, through multiplex immunohistochemistry, we confirm the presence of these infiltrative cells in EMT in patient samples. (4) Conclusions: together, our data identify the critical role of EMT in the invasive behavior of BCBM, which warrants further consideration to target those cells when treating BCBM.

The basement membrane (BM) around tumor lobes forms a barrier to prevent cancer cells from invading the surrounding tissue. Although myoepithelial cells are key producers of the healthy mammary epithelium BM, they are nearly absent in mammary tumors. To study the origin and dynamics of the BM, we developed and imaged a laminin beta1-Dendra2 mouse model. We show that the turnover of laminin beta1 is faster in the BMs that surround the tumor lobes than in the BMs that surround the healthy epithelium. Moreover, we find that epithelial cancer cells and tumor-infiltrating endothelial cells synthesize laminin beta1 and that this production is temporarily and locally heterogeneous, leading to local discontinuity of the BM laminin beta1. Collectively, our data draw a new paradigm for tumor BM turnover in which the disassembly happens at a constant rate, and a local misbalance of compensating production leads to reduction or even complete disappearance of the BM.

Purpose: Extensive work in preclinical models has shown that microenvironmental cells influence many aspects of cancer cell behavior, including metastatic potential and their sensitivity to therapeutics. In the human setting, this behavior is mainly correlated with the presence of immune cells. Here, in addition to T cells, B cells, macrophages, and mast cells, we identified the relevance of nonimmune cell types for breast cancer survival and therapy benefit, including fibroblasts, myoepithelial cells, muscle cells, endothelial cells, and seven distinct epithelial cell types. Experimental Design: Using single-cell sequencing data, we generated reference profiles for all these cell types. We used these reference profiles in deconvolution algorithms to optimally detangle the cellular composition of more than 3,500 primary breast tumors of patients that were enrolled in the SCAN-B and MATADOR clinical trials, and for which bulk mRNA sequencing data were available. Results: This large data set enables us to identify and subsequently validate the cellular composition of microenvironments that distinguish differential survival and treatment benefit for different treatment regimens in patients with primary breast cancer. In addition to immune cells, we have identified that survival and therapy benefit are characterized by various contributions of distinct epithelial cell types. Conclusions: From our study, we conclude that differential survival and therapy benefit of patients with breast cancer are characterized by distinct microenvironments that include specific populations of immune and epithelial cells.

GM1-gangliosidosis is caused by a reduced activity of β-galactosidase (Glb1), resulting in intralysosomal accumulations of GM1. The aim of this study was to reveal the pathogenic mechanisms of GM1-gangliosidosis in a new Glb1 knockout mouse model. Glb1−/− mice were analyzed clinically, histologically, immunohistochemically, electrophysiologically and biochemically. Morphological lesions in the central nervous system were already observed in two-month-old mice, whereas functional deficits, including ataxia and tremor, did not start before 3.5-months of age. This was most likely due to a reduced membrane resistance as a compensatory mechanism. Swollen neurons exhibited intralysosomal storage of lipids extending into axons and amyloid precursor protein positive spheroids. Additionally, axons showed a higher kinesin and lower dynein immunoreactivity compared to wildtype controls. Glb1−/− mice also demonstrated loss of phosphorylated neurofilament positive axons and a mild increase in non-phosphorylated neurofilament positive axons. Moreover, marked astrogliosis and microgliosis were found, but no demyelination. In addition to the main storage material GM1, GA1, sphingomyelin, phosphatidylcholine and phosphatidylserine were elevated in the brain. In summary, the current Glb1−/− mice exhibit a so far undescribed axonopathy and a reduced membrane resistance to compensate the functional effects of structural changes. They can be used for detailed examinations of axon–glial interactions and therapy trials of lysosomal storage diseases.

As sufficient extracellular arginine is crucial for T cell function, depletion of extracellular arginine by elevated arginase 1 (Arg1) activity has emerged as a hallmark immunosuppressive mechanism. However, the potential cell-autonomous roles of arginases in T cells have remained unexplored. Here, we show that the arginase isoform expressed by T cells, the mitochondrial Arg2, is a cell-intrinsic regulator of CD8(+) T cell activity. Both germline Arg2 deletion and adoptive transfer of Arg2(–/–) CD8(+) T cells significantly reduced tumor growth in preclinical cancer models by enhancing CD8(+) T cell activation, effector function, and persistence. Transcriptomic, proteomic, and high-dimensional flow cytometry characterization revealed a CD8(+) T cell–intrinsic role of Arg2 in modulating T cell activation, antitumor cytoxicity, and memory formation, independently of extracellular arginine availability. Furthermore, specific deletion of Arg2 in CD8(+) T cells strongly synergized with PD-1 blockade for the control of tumor growth and animal survival. These observations, coupled with the finding that pharmacologic arginase inhibition accelerates activation of ex vivo human T cells, unveil Arg2 as a potentially new therapeutic target for T cell–based cancer immunotherapies.

To the Editor: In 2013, a 6-week-old female piglet kept in a flatdeck cage had coughing, growth retardation, and diarrhea and was taken to a local veterinarian in Hannover, Germany; the piglet was euthanized. After necropsy at the University of Veterinary Medicine in Hannover, histologic investigation found interstitial pneumonia; a mild, multifocal, lymphohistiocytic panencephalitis that affected the cerebrum and cerebellum, including brain stem and medulla oblongata; and a mild, multifocal, lymphohistiocytic panmyelitis. Results from screening for typical neurotropic viruses (classical swine fever virus, suid herpesvirus 1, rabies virus, teschovirus, porcine enterovirus 8, 9, and 10) were negative; Mycoplasma hyorhinis was detected by multiplex PCR (Institute of Virology, University of Veterinary Medicine Hannover) within the lung and pulmonary lymph nodes. Cerebral tissue from the pig was processed for viral metagenomics by random RNA and DNA virus screening and next-generation sequencing (NGS) with the 454 sequencing platform (GS Junior; Roche, Basel, Switzerland), as described (1), and 21,359 reads were obtained. Analysis by using blastn and blastx (2) showed 10 reads had >97% nt identity with porcine bocavirus (PBoV) KU14. No other viral sequences were detected. By using primers based on sequence data of the PBoV, partially overlapping PCR amplicons were obtained to confirm and extend the NGS data of the isolate, which was named PBoV S1142/13 (1; GenBank accession no. {"type":"entrez-nucleotide","attrs":{"text":"KU311698","term_id":"1040287233","term_text":"KU311698"}}KU311698). A total of 2,176 nt of PBoV S1142/13 were obtained, consisting of the partial nucleoprotein (NP) 1 and the nearly complete viral protein (VP) 1 gene. By using MAFFT version 7 (http://mafft.cbrc.jp/alignment/server/), we aligned the nearly complete VP1 gene of PBoV S1142/13 with various closely related members of the genus Bocaparvovirus and built a maximum-likelihood tree by using the general time reversible plus invariable sites plus gamma distribution method, as determined by jModelTest 2.0 (3) and default parameters in MEGA6.06 (4). Results confirmed that PBoV S1142/13 was most closely related to PBoV KU14 (Figure, panel A). The partial genome of PBoV S1142/13 differed at 8 nt positions from PBoV KU14, resulting in 99.6% nt identity. Of these nucleotide differences, 4 resulted in an amino acid difference, including position 2733 (T→C on the basis of PBoV KU14 as a reference genome), which is part of the NP1 stopcodon of PBoV KU14. These results indicate that the stopcodon was located 39 nt farther downstream than for PBoV KU14. The other 3 aa differences were present in the VP1 protein; each of these differences was within the same group of amino acids as those detected in PBoV KU14. For further substantiation of a potential cause-effect relationship of histologic (Figure, panel B) and NGS results, we performed fluorescent in situ hybridization (FISH) on formalin-fixed, paraffin-embedded central nervous system (CNS) sections of the diseased animal and of a control pig with no CNS lesions. We used an RNA probe specific for the obtained NP1 and VP1 sequences covering 1,153 nt (Affymetrix, Santa Clara, CA, USA) according to the manufacturer’s protocol, with minor variations (ViewRNA ISH Tissue 1-Plex Assay Kit and ViewRNA Chromogenic Signal Amplification Kit, Affymetrix). A probe specific for porcine ubiquitin (Sus scrofa ubiquitin C; GenBank accession no. {"type":"entrez-nucleotide","attrs":{"text":"XM_005657305","term_id":"545868586","term_text":"XM_005657305"}}XM_005657305; nt 2–890) served as a positive control. The spinal cord of the diseased pig showed diffuse intracytoplasmic and intranuclear PBoV-specific signals within scattered neurons adjacent to the histologically detected inflammatory lesions (Figure, panel C). The negative control and the nonprobe incubation lacked PBoV-specific signals. The porcine ubiquitin probe provided a strong intracellular and extracellular staining within the CNS of both pigs. Figure Phylogenetic analysis and staining of porcine bocavirus (PBoV) from the spinal cord of a diseased pig, Hannover, Germany. A) Phylogenetic relationship of PBoV isolate S1142/13 (bold) with other bocaviruses. The nucleotide sequence of the nearly complete ... PBoV (genus Bocaparvovirus, family Parvoviridae) was first described in 2009 as porcine boca-like virus in pigs in Sweden with postweaning multisystemic wasting syndrome (5). PBoV is usually involved in respiratory and intestinal diseases in pigs (5) but has not been detected in the CNS. In the pig in our study, the lack of detection of other viral sequences by using NGS indicates the potential role of PBoV as a pathogen that triggers encephalomyelitis. FISH substantiated the NGS results and revealed neuronal intracytoplasmic and intranuclear PBoV-specific signals adjacent to the lesion, indicating intraneuronal transcription and replication (6). Nevertheless, a potential synergistic effect of M. hyorhinis on the PBoV pathogenesis cannot be ruled out. Similarly, co-infection of M. hyorhinis and porcine circovirus type 2 has been associated with enhanced inflammatory lesions in the lungs of pigs (7). The CNS tropism of PBoV S1142/13 could result from various factors, including specific amino acid changes that enable the virus to pass the blood–brain barrier and infect neurons. Additional studies are necessary to elucidate a possible role of the amino acid differences between PBoV S1142/13 and PBoV KU14 in the tropism of these viruses. Human bocavirus has recently been found in the cerebrospinal fluid of patients having encephalitis (8), and related human parvovirus 4 (9) and human parvovirus B19 (10) have been reported in human encephalitis. The correlation of PBoV-specific signals by using FISH for histologic detection of encephalomyelitis assigns PBoV a potential role in provoking CNS lesions. PBoV should be considered as a cause of encephalomyelitis but needs further investigation.

Serial passage of viruses in cell culture has been traditionally used to attenuate virulence and identify determinants of viral pathogenesis. In a previous study, we found that a strain of Schmallenberg virus (SBV) serially passaged in tissue culture (termed SBVp32) unexpectedly displayed increased pathogenicity in suckling mice compared to wild-type SBV. In this study, we mapped the determinants of SBVp32 virulence to the viral genome M segment. SBVp32 virulence is associated with the capacity of this virus to reach high titers in the brains of experimentally infected suckling mice. We also found that the Gc glycoprotein, encoded by the M segment of SBVp32, facilitates host cell protein shutoff in vitro . Interestingly, while the M segment of SBVp32 is a virulence factor, we found that the S segment of the same virus confers by itself an attenuated phenotype to wild-type SBV, as it has lost the ability to block the innate immune system of the host. Single mutations present in the Gc glycoprotein of SBVp32 are sufficient to compensate for both the attenuated phenotype of the SBVp32 S segment and the attenuated phenotype of NSs deletion mutants. Our data also indicate that the SBVp32 M segment does not act as an interferon (IFN) antagonist. Therefore, SBV mutants can retain pathogenicity even when they are unable to fully control the production of IFN by infected cells. Overall, this study suggests that the viral glycoprotein of orthobunyaviruses can compensate, at least in part, for the function of NSs. In addition, we also provide evidence that the induction of total cellular protein shutoff by SBV is determined by multiple viral proteins, while the ability to control the production of IFN maps to the NSs protein. IMPORTANCE The identification of viral determinants of pathogenesis is key to the development of prophylactic and intervention measures. In this study, we found that the bunyavirus Gc glycoprotein is a virulence factor. Importantly, we show that mutations in the Gc glycoprotein can restore the pathogenicity of attenuated mutants resulting from deletions or mutations in the nonstructural protein NSs. Our findings highlight the fact that careful consideration should be taken when designing live attenuated vaccines based on deletions of nonstructural proteins since single mutations in the viral glycoproteins appear to revert attenuated mutants to virulent phenotypes.

Clinical, pathological and genetic examination revealed an as yet uncharacterized juvenile-onset neuroaxonal dystrophy (NAD) in Spanish water dogs. Affected dogs presented with various neurological deficits including gait abnormalities and behavioral deficits. Histopathology demonstrated spheroid formation accentuated in the grey matter of the cerebral hemispheres, the cerebellum, the brain stem and in the sensory pathways of the spinal cord. Iron accumulation was absent. Ultrastructurally spheroids contained predominantly closely packed vesicles with a double-layered membrane, which were characterized as autophagosomes using immunohistochemistry. The family history of the four affected dogs suggested an autosomal recessive inheritance. SNP genotyping showed a single genomic region of extended homozygosity of 4.5 Mb in the four cases on CFA 8. Linkage analysis revealed a maximal parametric LOD score of 2.5 at this region. By whole genome re-sequencing of one affected dog, a perfectly associated, single, non-synonymous coding variant in the canine tectonin beta-propeller repeat-containing protein 2 (TECPR2) gene affecting a highly conserved region was detected (c.4009C>T or p.R1337W). This canine NAD form displays etiologic parallels to an inherited TECPR2 associated type of human hereditary spastic paraparesis (HSP). In contrast to the canine NAD, the spinal cord lesions in most types of human HSP involve the sensory and the motor pathways. Furthermore, the canine NAD form reveals similarities to cases of human NAD defined by widespread spheroid formation without iron accumulation in the basal ganglia. Thus TECPR2 should also be considered as candidate gene for human NAD. Immunohistochemistry and the ultrastructural findings further support the assumption, that TECPR2 regulates autophagosome accumulation in the autophagic pathways. Consequently, this report provides the first genetic characterization of juvenile canine NAD, describes the histopathological features associated with the TECPR2 mutation and provides evidence to emphasize the association between failure of autophagy and neurodegeneration.

Schmallenberg virus (SBV) is an emerging orthobunyavirus of ruminants associated with outbreaks of congenital malformations in aborted and stillborn animals. Since its discovery in November 2011, SBV has spread very rapidly to many European countries. Here, we developed molecular and serological tools, and an experimental in vivo model as a platform to study SBV pathogenesis, tropism and virus-host cell interactions. Using a synthetic biology approach, we developed a reverse genetics system for the rapid rescue and genetic manipulation of SBV. We showed that SBV has a wide tropism in cell culture and “synthetic” SBV replicates in vitro as efficiently as wild type virus. We developed an experimental mouse model to study SBV infection and showed that this virus replicates abundantly in neurons where it causes cerebral malacia and vacuolation of the cerebral cortex. These virus-induced acute lesions are useful in understanding the progression from vacuolation to porencephaly and extensive tissue destruction, often observed in aborted lambs and calves in naturally occurring Schmallenberg cases. Indeed, we detected high levels of SBV antigens in the neurons of the gray matter of brain and spinal cord of naturally affected lambs and calves, suggesting that muscular hypoplasia observed in SBV-infected lambs is mostly secondary to central nervous system damage. Finally, we investigated the molecular determinants of SBV virulence. Interestingly, we found a biological SBV clone that after passage in cell culture displays increased virulence in mice. We also found that a SBV deletion mutant of the non-structural NSs protein (SBVΔNSs) is less virulent in mice than wild type SBV. Attenuation of SBV virulence depends on the inability of SBVΔNSs to block IFN synthesis in virus infected cells. In conclusion, this work provides a useful experimental framework to study the biology and pathogenesis of SBV., Author Summary Schmallenberg virus (SBV) was discovered in Germany (near the town of Schmallenberg) in November 2011 and since then has been found to be the cause of malformations and stillbirths in ruminants. SBV has spread very rapidly to many European countries including the Netherlands, Belgium, France and the United Kingdom. Very little is known about the biological properties of this virus and there is no vaccine available. In this study (i) we developed an approach (called reverse genetics) that allows the recovery of “synthetic” SBV under laboratory conditions; (ii) we developed a mouse model of infection for SBV; (iii) we showed that SBV replicates in neurons of experimentally infected mice similar to naturally infected lambs and calves; (iv) we developed viral mutants that are not as pathogenic as the original virus due to the inability to counteract the host cell defenses; and v) we identified mutations that are associated with increased virulence. This work provides the experimental tools to understand how this newly emerged virus causes disease in ruminants. In addition, it will now be possible to manipulate the SBV genome in order to develop highly effective vaccines.

To the Editor: In 2011, a new virus spread throughout ruminant populations in the Netherlands, Germany, and other European countries (1). Infected dairy cattle exhibited fever, reduced milk yield, loss of appetite, and diarrhea. Weeks after these signs appeared, epidemic abortions; births of malformed or stillborn animals; and perinatal deaths of calves, lambs, and goat kids were reported. Metagenomic analysis identified a novel orthobunyavirus (family Bunyaviridae), termed Schmallenberg virus (SBV), which is closely related to Akabane and Shamonda viruses (1). These arthropod-borne viruses are well-recognized ruminant pathogens in Africa, Asia, and Oceania; fetal infection is associated with an arthrogryposis–hydranencephaly syndrome. Previous reports about SBV have addressed clinical signs (2), serologic findings (3), pathologic findings (4), and SBV RNA distribution in organs of malformed calves (5) detected by real-time quantitative reverse transcription PCR (qRT-PCR). However, pathogenesis and viral cell tropism are largely unknown. Therefore, our aims were to establish an in situ hybridization method (ISH) to detect SBV mRNA, to evaluate the usefulness of this method as a complementary diagnostic tool, and to further analyze SBV pathogenesis. The in situ probe was generated according to established protocols (6). The SBV qRT-PCR product and primers used for diagnostics (5) were used to amplify an 88-bp segment of the nucleoprotein, encoded by the small segment of the SBV genome. The amplicon was cloned into a pCR4-TOPO vector (Invitrogen, Darmstadt, Germany) and sequenced. For generation of a digoxigenin-labeled antisense probe to detect viral mRNA, we used M13 reverse and SBV-S-469R primers (5). Specificity of the probe was ensured by distinct ISH signals in the brains of SBV-positive (as determined by qRT-PCR) animals (6). No reaction was detected in SBV-negative (as determined by qRT-PCR) goats, sheep, and calves; in ruminants with various non–SBV-associated nervous system lesions; or in the brain of a mouse that was experimentally infected with Akabane virus. Thereafter, we investigated SBV mRNA distribution in the central nervous system (CNS) of 82 naturally infected ruminants (46 lambs, 2 goat kids, and 34 calves), all of which had previously been found by qRT-PCR to be positive for SBV. In addition, ISH was used to examine the following tissues from 10 of these animals (4 lambs, 1 goat kid, and 5 calves): placenta, muscle, eye, heart, aorta, lung, trachea, liver, kidney, spleen, small and large intestine, mesenteric and pulmonary lymph nodes, thymus, adrenal gland, testis, and uterus. SBV mRNA was found in varying amounts, predominantly in neurons (Figure) of the cerebrum, cerebellum, brain stem, medulla oblongata, and spinal cord of 7 lambs, 1 goat kid, and 2 calves. Randomly distributed clusters of SBV-positive neurons were frequently found in small ruminants, whereas only single positive cells were found in both calves. SBV mRNA was not found in any peripheral organ. The Technical Appendix provides an overview of ISH results in relation to malformations and inflammatory CNS lesions. Histologic examination identified encephalitis, characterized by lymphohistiocytic, perivascular cuffs, in 11 (9 lambs, 1 goat kid, and 1 calf) of the 82 animals. Among animals that were positive according to ISH, all small ruminants showed inflammation, whereas both calves lacked inflammatory changes. Figure In situ detection by using Nomarski differential interference contrast microscopy of Schmallenberg virus mRNA in neurons in the medulla of a neonatal sheep for which Schmallenberg virus infection was confirmed by ... The most frequent macroscopic lesions or conditions in animals that were SBV positive by qRT-PCR, especially calves, were arthrogryposis, brachygnathia inferior, torticollis, kyphosis, lordosis, scoliosis, and muscle hypoplasia (especially in calves) (4). The predominant CNS lesions or conditions were cerebellar and cerebral hypoplasia, hydranencephaly, porencephaly, hydrocephalus, and micromyelia. Among the 8 small ruminants that were SBV positive by ISH, hydrocephalus was found in 5, cerebellar hypoplasia in 5, hydranencephaly in 1, and arthrogryposis in 6. For the 2 calves that were SBV positive, micromyelia and arthrogryposis were found in both and cerebellar hypoplasia in 1. This study indicates that neurons are the predominant target in SBV-infected neonates. Surprisingly, only 10 of 82 ruminants were positive for SBV by qRT-PCR and ISH. This discrepancy could result from low SBV mRNA copy numbers per cell, which are detectable by qRT-PCR but not ISH, and/or from reduced viral load in individual cells because of the long time between assumed infection in early gestation and time of examination after birth (≈17 weeks in sheep and 27 weeks in calves). The latter explanation is indicated by the high incidence of malformations (7) typical for teratogenic insults and might especially apply to calves. Detection of CNS inflammation in all small ruminants that were SBV positive according to ISH indicates a cause-and-effect association between high SBV mRNA copy numbers per cell and lymphohistiocytic immune response. Furthermore, low numbers of infected cells and/or reduced replication as assumed for animals that were positive by qRT-PCR but negative by ISH might explain the absence of inflammation in the calf brains. The 3 tested animals in which inflammation was found but that were SBV negative by ISH could represent an advanced resolving lesion after viral clearance or reduction. In situ detection of SBV mRNA represents a suitable way to study SBV pathogenesis, especially in the active phase of infection, and might enable identification of SBV as the causative agent in cases of CNS inflammation of unknown etiology. Technical Appendix: Absolute numbers and percentages of animals that were positive for Schmallenberg virus according to in situ hybridization in relation to central nervous system inflammation and occurrence of skeletal and brain malformations. Data refer to absolute numbers of 82 small and large ruminants that were positive for Schmallenberg virus by real-time quantitative reverse transcription PCR. Click here to view.(91K, pdf)