In breast cancer, several chromosomal sites frequently undergo amplification, implicating the location of genes important for tumor development and progression. Here we cloned two novel genes, breast carcinoma amplified sequence 3 (BCAS3) and 4 (BCAS4), from the two most common amplification sites in breast cancer, 17q23 and 20q13. The BCAS3 gene at 17q23 spans more than 600 kb at the genomic level and was predicted to encode a 913 amino acid nuclear protein. The BCAS4 gene at 20q13.2 encodes a 211 amino acid cytoplasmic protein. Both BCAS3 and BCAS4 represent novel genes with no homologies to any other known gene or protein. In the MCF7 breast cancer cell line, the BCAS3 and BCAS4 genes were co-amplified, and cloning of a highly overexpressed 1.3-kb transcript revealed a rearrangement fusing the last two exons of BCAS3 with BCAS4. The fusion led to a novel message in which only the first exon of BCAS4 and part of exon 23 of BCAS3 were transcribed. The BCAS4-BCAS3 fusion transcript was detected only in MCF7 cells, but the BCAS4 gene was also overexpressed in nine of 13 breast cancer cell lines. In conclusion, our results indicate that these novel genes, BCAS3 at 17q23 and BCAS4 at 20q13.2, undergo amplification, overexpression, and fusion in breast cancer and therefore may have a role in the frequent chromosomal alterations affecting these two loci.

Poliovirus proteins 3A and 3AB are small, membrane-binding proteins that play multiple roles in viral RNA replication complex formation and function. In the infected cell, these proteins associate with other viral and cellular proteins as part of a supramolecular complex whose structure and composition are unknown. We isolated viable viruses with three different epitope tags (FLAG, hemagglutinin [HA], and c-myc) inserted into the N-terminal region of protein 3A. These viruses exhibited growth properties and characteristics very similar to those of the wild-type, untagged virus. Extracts prepared from the infected cells were subjected to immunoaffinity purification of the tagged proteins by adsorption to commercial antibody-linked beads and examined after elution for cellular and other viral proteins that remained bound to 3A sequences during purification. Viral proteins 2C, 2BC, 3D, and 3CD were detected in all three immunopurified 3A samples. Among the cellular proteins previously reported to interact with 3A either directly or indirectly, neither LIS1 nor phosphoinositol-4 kinase (PI4K) were detected in any of the purified tagged 3A samples. However, the guanine nucleotide exchange factor GBF1, which is a key regulator of membrane trafficking in the cellular protein secretory pathway and which has been shown previously to bind enteroviral protein 3A and to be required for viral RNA replication, was readily recovered along with immunoaffinity-purified 3A-FLAG. Surprisingly, we failed to cocapture GBF1 with 3A-HA or 3A-myc proteins. A model for variable binding of these 3A mutant proteins to GBF1 based on amino acid sequence motifs and the resulting practical and functional consequences thereof are discussed.Poliovirus (PV) is a member of the human enterovirus C cluster in the Enterovirus genus of the virus family Picornaviridae. The PV genome encodes a single polyprotein that is proteolytically processed to generate a set of intermediate precursors and final cleavage products that are all required for virus replication. The N-terminal region of the polyprotein (P1) forms the viral capsid proteins, which are dispensable for viral RNA translation and replication but are needed for encapsidation and assembly of infectious particles. The remainder of the polyprotein (P2 and P3 regions) generates proteins that contribute catalytic and structural functions for viral RNA translation and replication, as well as for disruption and/or reorganization of numerous cellular processes and activities that could restrict or combat virus replication. All of the PV noncapsid proteins are essential for viral RNA replication; most manifest multiple activities during the virus replication cycle, and all at least partially localize to large, membraneassociated replication complexes that form from preexisting subcellular organelles after infection. Viral proteins 2B, 2C, and 3A contain hydrophobic trans-membrane regions or amphipathic helices, and these proteins as well as their larger precursor proteins bind membranes directly (7,12,...

dInfectious agents are often considered potential triggers of chronic inflammatory disease, including autoimmunity; however, direct evidence is usually lacking. Here we show that following control of acute infection of mice with the myotropic Colombiana strain of Trypanosoma cruzi, parasites persisted in tissue at low levels associated with development of systemic necrotizing vasculitis. Lesions occurred in many but not all organs and tissues, with skeletal muscle arteries being the most severely affected, and were associated with myositis, atrophy, paresis/paralysis, and death. Histopathology showed fibrinoid vascular necrosis, rare amastigote nests within skeletal muscle myocytes, and massive leukocyte infiltrates composed mainly of inflammatory monocytes, F4/80 ؉ macrophages, and T. cruzi tetramer-specific CD8 ؉ T lymphocytes capable of producing gamma interferon (IFN-␥) and tumor necrosis factor alpha (TNF-␣) but not interleukin-17 (IL-17). T. cruzi-specific IgG was detected in sera from infected mice, but antibody deposits and neutrophilic inflammation were not features of the lesions. Thus, T. cruzi infection of mice may be a specific infectious trigger of paralyzing systemic necrotizing vasculitis most severely affecting skeletal muscle, driven by pathogen-specific type I immune responses. Vasculitis is a general term for a spectrum of diseases involving leukocyte infiltration of the blood vessel wall. Many subtypes of vasculitis are recognized based on the types of blood vessels involved and the nature of the underlying immunopathology. In some cases, an association of vasculitis with specific infectious agents has been reported; for example, polyarteritis nodosa (PAN) has been associated with hepatitis B virus (HBV), hepatitis C virus (HCV), and HIV infections (1). However, it has been difficult to establish either causality or pathogenetic mechanisms, in part because of the low prevalence of vasculitis and the lack of good animal models (2). In this regard, while studying the chronic phase of C57BL/6J mice infected with the myotropic Colombiana strain of Trypanosoma cruzi, the infectious cause of Chagas' disease in humans, we noticed that the majority of animals developed hind limb paralysis associated with severe systemic necrotizing vasculitis affecting arteries and arterioles in skeletal muscle. Lesions were also present in many other tissues. A review of the literature identified several studies from 1970 to 2000 that described perivascular inflammation and frank arteritis in chronically T. cruzi-infected mouse skeletal muscle, aorta, nerves, and heart (3-9); however, the underlying immunologic mechanisms of disease were not defined. Interestingly, Okumura et al. reported necrotizing arteritis in small arteries beneath the peritoneal lining of the bowel and, occasionally, of the aorta and coronary arteries of T. cruzi-infected mice, which was at that time interpreted as "allergic necrotizing angiitis" (10). Dias et al. reported obliterative changes of the small and medium-sized branches of coronary...

In chronic Trypanosoma cruzi infection, the cause of Chagas disease, life-threatening inflammatory diseases develop over time in the heart, esophagus, and colon of some patients. C57BL/6 mice infected with the myotropic Colombiana strain of T. cruzi model many of the immunological and parasitological features of human infection but succumb to chronic paralyzing myositis and skeletal muscle vasculitis, not cardiomyopathy or gastrointestinal disease. Here we show that T cell depletion in the chronic phase of this model increased tissue parasitism to acutephase levels and induced neutrophilic skeletal muscle inflammation. Conversely, after daily treatment with the trypanocide benznidazole for 8 weeks during the chronic phase, viable parasites were no longer detectable, myositis completely resolved, vasculitis was ϳ80% reduced, fibrosis was reduced, and myofiber morphology normalized. After the drug was discontinued, parasitism rebounded, and immunopathology recurred. The parasite load was statistically strongly correlated with the severity of inflammation. Thus, both T cell immunity and trypanocidal pharmacotherapy suppress to very low levels, but do not cure, T. cruzi infection, which is necessary and possibly sufficient to induce crippling chronic skeletal muscle myositis and vasculitis in the model.

No abstract

During experiments concerning the introduction of oncogenes into normal human keratinocytes, we observed long-lived colonies arising spontaneously at the same low frequency in control cultures as in those transfected with Ha-rasEJ or activated c-myc or both. Two of these were karyotyped early in their life span and showed additional chromosomal material on the short arm of chromosome 9 in one case and of chromosome 18 in the other, whereas the parental cells had a normal karyotype. This indicates the presence of a partial trisomy in each line, although the origin of the extra chromosomal material is not known. A similarly long-lived human keratinocyte line containing an isochromosome of the long arm of chromosome 8 has been described elsewhere. Together these results suggest that the spontaneous occurrence of long-lived lines is more common in human keratinocytes than in fibroblasts and that a triple dose of one or more genes may be the initial event in this process.

High pro-inflammatory chemokine levels have been reported in blood and lung in patients with COVID-19. To investigate specific roles in pathogenesis, we studied the regulation of chemokine ligands and receptors in the lungs of 4-6-month-old wild type C57BL/6 mice infected with the MA10 mouse-adapted strain of SARS-CoV-2. We found that atypical chemokine receptor 1 (Ackr1, also known as Duffy antigen receptor for chemokines/DARC) was the most highly upregulated chemokine receptor in infected lung, where it localized to endothelial cells of veins and arterioles. In a screen of 7 leukocyte chemoattractant or chemoattractant receptor knockout mouse lines, Ackr1-/- mice were unique in having lower mortality after SARS-CoV-2 infection, particularly in males. ACKR1 is a non-signaling chemokine receptor that in addition to endothelium is also expressed on erythrocytes and Purkinje cells of the cerebellum. It binds promiscuously to both inflammatory CC and CXC chemokines and has been reported to control chemokine availability which may influence the shape of chemotactic gradients and the ability of leukocytes to extravasate and produce immunopathology. Of note, erythrocyte ACKR1 deficiency is fixed in sub-Saharan African populations where COVID-19 has been reported to result in low mortality compared to worldwide data. Our data suggest the possibility of a causal contribution of ACKR1 deficiency to low sub-Saharan COVID-19 mortality and identify ACKR1 as a possible drug target in the disease.

Cataracts are a common consequence of aging; however, pathogenesis remains poorly understood. Here, we observed that after 3 months of age mice lacking the G protein-coupled leukocyte chemotactic receptor Fpr1 (N-formyl peptide receptor 1) began to develop bilateral posterior subcapsular cataracts that progressed to lens rupture and severe degeneration, without evidence of either systemic or local ocular infection or inflammation. Consistent with this, Fpr1 was detected in both mouse and human lens in primary lens epithelial cells (LECs), the only cell type present in the lens; however, expression was confined to subcapsular LECs located along the anterior hemispheric surface. To maximize translucency, LECs at the equator proliferate and migrate posteriorly, then differentiate into lens fiber cells by nonclassical apoptotic signaling, which results in loss of nuclei and other organelles, including mitochondria which are a rich source of endogenous N-formyl peptides. In this regard, denucleation and posterior migration of LECs were abnormal in lenses from Fpr1 −/− mice, and direct stimulation of LECs with the prototypic N-formyl peptide agonist fMLF promoted apoptosis. Thus, Fpr1 is repurposed beyond its immunoregulatory role in leukocytes to protect against cataract formation and lens degeneration during aging.