Divergent modes of clonal spread and intraperitoneal mixing in high-grade serous ovarian cancer.

We performed phylogenetic analysis of high-grade serous ovarian cancers (68 samples from seven patients), identifying constituent clones and quantifying their relative abundances at multiple intraperitoneal sites. Through whole-genome and single-nucleus sequencing, we identified evolutionary features including mutation loss, convergence of the structural genome and temporal activation of mutational processes that patterned clonal progression. We then determined the precise clonal mixtures comprising each tumor sample. The majority of sites were clonally pure or composed of clones from a single phylogenetic clade. However, each patient contained at least one site composed of polyphyletic clones. Five patients exhibited monoclonal and unidirectional seeding from the ovary to intraperitoneal sites, and two patients demonstrated polyclonal spread and reseeding. Our findings indicate that at least two distinct modes of intraperitoneal spread operate in clonal dissemination and highlight the distribution of migratory potential over clonal populations comprising high-grade serous ovarian cancers.

Systematic analysis of somatic mutations impacting gene expression in 12 tumour types.

We present a novel hierarchical Bayes statistical model, xseq, to systematically quantify the impact of somatic mutations on expression profiles. We establish the theoretical framework and robust inference characteristics of the method using computational benchmarking. We then use xseq to analyse thousands of tumour data sets available through The Cancer Genome Atlas, to systematically quantify somatic mutations impacting expression profiles. We identify 30 novel cis-effect tumour suppressor gene candidates, enriched in loss-of-function mutations and biallelic inactivation. Analysis of trans-effects of mutations and copy number alterations with xseq identifies mutations in 150 genes impacting expression networks, with 89 novel predictions. We reveal two important novel characteristics of mutation impact on expression: (1) patients harbouring known driver mutations exhibit different downstream gene expression consequences; (2) expression patterns for some mutations are stable across tumour types. These results have critical implications for identification and interpretation of mutations with consequent impact on transcription in cancer.

The omics of triple-negative breast cancers.

BACKGROUND: Triple-negative breast cancers (TNBC) do not represent a single disease subgroup and are often aggressive breast cancers with poor prognoses. Unlike estrogen/progesterone receptor and HER2 (human epidermal growth factor receptor 2) breast cancers, which are responsive to targeted treatments, there is no effective targeted therapy for TNBC, although approximately 50% of patients respond to conventional chemotherapies, including taxanes, anthracyclines, cyclophosphamide, and platinum salts. CONTENT: Genomic studies have helped clarify some of the possible disease groupings that make up TNBC. We discuss the findings, including copy number-transcriptome analysis, whole genome sequencing, and exome sequencing, in terms of the biological properties and phenotypes that make up the constellation of TNBC. The relationships between subgroups defined by transcriptome and genome analysis are discussed. SUMMARY: TNBC is not a uniform molecular or disease entity but a constellation of variably well-defined biological properties whose relationship to each other is not understood. There is good support for the existence of a basal expression subtype, p53 mutated, high-genomic instability subtype of TNBC. This should be considered a distinct TNBC subtype. Other subtypes with variable degrees of supporting evidence exist within the nonbasal/p53wt (wild-type p53) TNBC, including a group of TNBC with PI3K (phosphoinositide 3-kinase) pathway activation that have better overall prognosis than the basal TNBC. Consistent molecular phenotyping of TNBC by whole genome sequencing, transcriptomics, and functional studies with patient-derived tumor xenograft models will be essential components in clinical and biological studies as means of resolving this heterogeneity.

Requirement for core 2 O-glycans for optimal resistance to helminth infection.

The migration of lymphocytes to the small intestine is controlled by expression of the integrin α4ß7 and the chemokine receptor CCR9. However, the molecules that specifically regulate migration to the large intestine remain unclear. Immunity to infection with the large intestinal helminth parasite Trichuris muris is dependent upon CD4(+) T cells that migrate to the large intestine. We examine the role of specific chemokine receptors, adhesion molecules and glycosyltransferases in the development of protective immunity to Trichuris. Mice deficient in expression of the chemokine receptors CCR2 or CCR6 were resistant to infection with Trichuris. Similarly, loss of CD34, CD43, CD44 or PSGL-1 had no effect on resistance to infection. In contrast, simultaneous deletion of the Core2 ß1,6-N-acetylglucosaminyltransferase (C2GnT) enzymes C2GnT1 and C2Gnt2 resulted in delayed expulsion of worms. These results suggest that C2GnT-dependent modifications may play a role in migration of protective immune cells to the large intestine.

Trichuris muris infection: a model of type 2 immunity and inflammation in the gut.

Trichuris muris is a natural pathogen of mice and is biologically and antigenically similar to species of Trichuris that infect humans and livestock. Infective eggs are given by oral gavage, hatch in the distal small intestine, invade the intestinal epithelial cells (IECs) that line the crypts of the cecum and proximal colon and upon maturation the worms release eggs into the environment. This model is a powerful tool to examine factors that control CD4(+) T helper (Th) cell activation as well as changes in the intestinal epithelium. The immune response that occurs in resistant inbred strains, such as C57BL/6 and BALB/c, is characterized by Th2 polarized cytokines (IL-4, IL-5 and IL-13) and expulsion of worms while Th1-associated cytokines (IL-12, IL-18, IFN-γ) promote chronic infections in genetically susceptible AKR/J mice. Th2 cytokines promote physiological changes in the intestinal microenvironment including rapid turnover of IECs, goblet cell differentiation, recruitment and changes in epithelial permeability and smooth muscle contraction, all of which have been implicated in worm expulsion. Here we detail a protocol for propagating Trichuris muris eggs which can be used in subsequent experiments. We also provide a sample experimental harvest with suggestions for post-infection analysis. Overall, this protocol will provide researchers with the basic tools to perform a Trichuris muris mouse infection model which can be used to address questions pertaining to Th proclivity in the gastrointestinal tract as well as immune effector functions of IECs.

Assessing the role of CD103 in immunity to an intestinal helminth parasite.

BACKGROUND: In the intestine, the integrin CD103 is expressed on a subset of T regulatory (T(reg)) cells and a population of dendritic cells (DCs) that produce retinoic acid and promote immune homeostasis. However, the role of CD103 during intestinal helminth infection has not been tested. METHODOLOGY/PRINCIPAL FINDINGS: We demonstrate that CD103 is dispensable for the development of protective immunity to the helminth parasite Trichuris muris. While we observed an increase in the frequency of CD103(+) DCs in the lamina propria (LP) following acute high-dose infection with Trichuris, lack of CD103 had no effect on the frequency of CD11c(+) DCs in the LP or mesenteric lymph nodes (mLN). CD103-deficient (CD103(-/-)) mice develop a slightly increased and earlier T cell response but resolve infection with similar kinetics to control mice. Similarly, low-dose chronic infection of CD103(-/-) mice with Trichuris resulted in no significant difference in immunity or parasite burden. Absence of CD103 also had no effect on the frequency of CD4(+)CD25(+)Foxp3(+) T(reg) cells in the mLN or LP. CONCLUSIONS/SIGNIFICANCE: These results suggest that CD103 is dispensable for intestinal immunity during helminth infection. Furthermore, lack of CD103 had no effect on DC or T(reg) recruitment or retention within the large intestine.

Activating and inhibitory functions for the histone lysine methyltransferase G9a in T helper cell differentiation and function.

Accumulating evidence suggests that the regulation of gene expression by histone lysine methylation is crucial for several biological processes. The histone lysine methyltransferase G9a is responsible for the majority of dimethylation of histone H3 at lysine 9 (H3K9me2) and is required for the efficient repression of developmentally regulated genes during embryonic stem cell differentiation. However, whether G9a plays a similar role in adult cells is still unclear. We identify a critical role for G9a in CD4(+) T helper (Th) cell differentiation and function. G9a-deficient Th cells are specifically impaired in their induction of Th2 lineage-specific cytokines IL-4, IL-5, and IL-13 and fail to protect against infection with the intestinal helminth Trichuris muris. Furthermore, G9a-deficient Th cells are characterised by the increased expression of IL-17A, which is associated with a loss of H3K9me2 at the Il17a locus. Collectively, our results establish unpredicted and complex roles for G9a in regulating gene expression during lineage commitment in adult CD4(+) T cells.

Synthetic cationic peptide IDR-1002 provides protection against bacterial infections through chemokine induction and enhanced leukocyte recruitment.

With the rapid rise in the incidence of multidrug resistant infections, there is substantial interest in host defense peptides as templates for production of new antimicrobial therapeutics. Natural peptides are multifunctional mediators of the innate immune response, with some direct antimicrobial activity and diverse immunomodulatory properties. We have previously developed an innate defense regulator (IDR) 1, with protective activity against bacterial infection mediated entirely through its effects on the immunity of the host, as a novel approach to anti-infective therapy. In this study, an immunomodulatory peptide IDR-1002 was selected from a library of bactenecin derivatives based on its substantially more potent ability to induce chemokines in human PBMCs. The enhanced chemokine induction activity of the peptide in vitro correlated with stronger protective activity in vivo in the Staphylococcus aureus-invasive infection model, with a >5-fold reduction in the protective dose in direct comparison with IDR-1. IDR-1002 also afforded protection against the Gram-negative bacterial pathogen Escherichia coli. Chemokine induction by IDR-1002 was found to be mediated through a Gi-coupled receptor and the PI3K, NF-kappaB, and MAPK signaling pathways. The protective activity of the peptide was associated with in vivo augmentation of chemokine production and recruitment of neutrophils and monocytes to the site of infection. These results highlight the importance of the chemokine induction activity of host defense peptides and demonstrate that the optimization of the ex vivo chemokine-induction properties of peptides is a promising method for the rational development of immunomodulatory IDR peptides with enhanced anti-infective activity.

Mice that exclusively express TLR4 on endothelial cells can efficiently clear a lethal systemic Gram-negative bacterial infection.

Recognition of LPS by TLR4 on immune sentinel cells such as macrophages is thought to be key to the recruitment of neutrophils to sites of infection with Gram-negative bacteria. To explore whether endothelial TLR4 plays a role in this process, we engineered and imaged mice that expressed TLR4 exclusively on endothelium (known herein as EndotheliumTLR4 mice). Local administration of LPS into tissue induced comparable neutrophil recruitment in EndotheliumTLR4 and wild-type mice. Following systemic LPS or intraperitoneal E. coli administration, most neutrophils were sequestered in the lungs of wild-type mice and did not accumulate at primary sites of infection. In contrast, EndotheliumTLR4 mice showed reduced pulmonary capillary neutrophil sequestration over the first 24 hours; as a result, they mobilized neutrophils to primary sites of infection, cleared bacteria, and resisted a dose of E. coli that killed 50% of wild-type mice in the first 48 hours. In fact, the only defect we detected in EndotheliumTLR4 mice was a failure to accumulate neutrophils in the lungs following intratracheal administration of LPS; this response required TLR4 on bone marrow-derived immune cells. Therefore, endothelial TLR4 functions as the primary intravascular sentinel system for detection of bacteria, whereas bone marrow-derived immune cells are critical for pathogen detection at barrier sites. Nonendothelial TLR4 contributes to failure to accumulate neutrophils at primary infection sites in a disseminated systemic infection.

Vav1 is essential for mechanotactic crawling and migration of neutrophils out of the inflamed microvasculature.

Mac-1-dependent crawling is a new step in the leukocyte recruitment cascade that follows LFA-1-dependent adhesion and precedes emigration. Neutrophil adhesion via LFA-1 has been shown to induce cytoskeletal reorganization through Vav1-dependent signaling, and the current study investigates the role of Vav1 in the leukocyte recruitment process in vivo with particular attention to the events immediately downstream of LFA-1-dependent adhesion. Intravital and spinning-disk-confocal microscopy was used to investigate intravascular crawling in relation to endothelial junctions in vivo in wild-type and Vav1(-/-) mice. Adherent wild-type neutrophils almost immediately began crawling perpendicular to blood flow via Mac-1 until they reached an endothelial junction where they often changed direction. This pattern of perpendicular, mechanotactic crawling was recapitulated in vitro when shear was applied. In sharp contrast, the movement of Vav1(-/-) neutrophils was always in the direction of flow and appeared more passive as if the cells were dragged in the direction of flow in vivo and in vitro. More than 80% of Vav1(-/-) neutrophils moved independent of Mac-1 and could be detached with LFA-1 Abs. An inability to release the uropod was frequently noted for Vav1(-/-) neutrophils, leading to greatly elongated tails. The Vav1(-/-) neutrophils failed to stop or follow junctions and ultimately detached, leading to fewer emigrated neutrophils. The Vav1(-/-) phenotype resulted in fewer neutrophils recruited in a relevant model of infectious peritonitis. Clearly, Vav1 is critical for the complex interplay between LFA-1 and Mac-1 that underlies the programmed intravascular crawling of neutrophils.

Use of artificial intelligence in the design of small peptide antibiotics effective against a broad spectrum of highly antibiotic-resistant superbugs.

Increased multiple antibiotic resistance in the face of declining antibiotic discovery is one of society's most pressing health issues. Antimicrobial peptides represent a promising new class of antibiotics. Here we ask whether it is possible to make small broad spectrum peptides employing minimal assumptions, by capitalizing on accumulating chemical biology information. Using peptide array technology, two large random 9-amino-acid peptide libraries were iteratively created using the amino acid composition of the most active peptides. The resultant data was used together with Artificial Neural Networks, a powerful machine learning technique, to create quantitative in silico models of antibiotic activity. On the basis of random testing, these models proved remarkably effective in predicting the activity of 100,000 virtual peptides. The best peptides, representing the top quartile of predicted activities, were effective against a broad array of multidrug-resistant "Superbugs" with activities that were equal to or better than four highly used conventional antibiotics, more effective than the most advanced clinical candidate antimicrobial peptide, and protective against Staphylococcus aureus infections in animal models.

Mast cell-expressed complement receptor, not TLR2, is the main detector of zymosan in peritonitis.

The in vitro macrophage response to zymosan has been attributed to Toll-like receptor 2 (TLR2). Whether TLR2 is obligatory for the zymosan-induced in vivo response has not been assessed. The importance of this question is underscored by the fact that zymosan activates complement in a cell-independent manner. We have investigated whether the in vitro observation of TLR2 as the dominant zymosan receptor on macrophages would translate to an experimental peritonitis model in vivo. We have treated mice with zymosan, resulting in significant leukocyte (primarily neutrophil) accumulation in the peritoneum at 4 h. Zymosan-mediated leukocyte recruitment was TLR2 independent, but was predominantly dependent on the complement components, C3 and C5a with a minor contribution from LTB4. Peritoneal neutrophilia was 50% mast cell dependent and this defect was reproduced using C5a receptor (C5aR)-deficient mast cells in mast cell-deficient mice, suggesting that C5aR is responsible for mast cell activation following zymosan challenge. By 24 h, the response to zymosan involved primarily monocyte recruitment and was C3 and C5aR independent. Taken together, these studies indicate that the in vivo inflammatory response to zymosan does not necessarily mimic the TLR2 dependence observed in vitro, and that complement plays a dominant role in early, but not late, zymosan-mediated peritonitis.

The role of TLR2 in vivo following challenge with Staphylococcus aureus and prototypic ligands.

Based on a wealth of in vitro macrophage studies, immunity to Staphylococcus aureus cell wall-derived peptidoglycan (PGN) and lipoteichoic acid has been attributed to TLR2. We investigated whether the in vitro paradigm of TLR2 dominance would hold true in vivo. Using an experimental peritonitis model, we challenged mice with PGN or lipoteichoic acid and found that only PGN resulted in significant leukocyte (primarily neutrophil) accumulation in the peritoneum at 4 h. PGN-mediated leukocyte recruitment was P-/E-selectin dependent but only partially TLR2 dependent, and also involved the C5aR. Concomitant inhibition of TLR2 and C5aR resulted in a further reduction in PGN-induced peritonitis. Peritoneal neutrophilia was partially mast cell dependent; however, the defect could not be reconstituted with TLR2(-/-) or C5aR(-/-) mast cells. Interestingly, macrophage-deficient mice did not have defective neutrophil recruitment. By 24 h, the response to PGN involved primarily monocytes and was TLR2 and C5aR independent. Finally, we challenged mice with live S. aureus and found a similar degree of TLR2 involvement in leukocyte recruitment to that observed with PGN. Most importantly, bacterial clearance from the spleen and peritoneum was not altered in TLR2(-/-) mice vs wild-type mice. Morbidity was only significantly increased in S. aureus-infected mice treated with a blocking Fab against C5aR. Taken together, these studies indicate that in vivo responses to prototypic TLR2 ligands do not necessarily recapitulate the absolute necessity for TLR2 observed in vitro, and additional receptors contribute, in a significant manner, to PGN and S. aureus-mediated immune responses.

LSP1 is an endothelial gatekeeper of leukocyte transendothelial migration.

Leukocyte-specific protein 1 (LSP1), an F-actin binding protein and a major downstream substrate of p38 mitogen-activated protein kinase as well as protein kinase C, has been reported to be important in leukocyte chemotaxis. Although its distribution has been thought to be restricted to leukocytes, herein we report that LSP1 is expressed in endothelium and is essential to permit neutrophil emigration. Using intravital microscopy to directly visualize leukocyte rolling, adhesion, and emigration in postcapillary venules in LSP1-deficient (Lsp1-/-) mice, we found that LSP1 deficiency inhibits neutrophil extravasation in response to various cytokines (tumor necrosis factor-alpha and interleukin-1beta) and to neutrophil chemokine keratinocyte-derived chemokine in vivo. LSP1 deficiency did not affect leukocyte rolling or adhesion. Generation of Lsp1-/- chimeric mice using bone marrow transplantation revealed that in mice with Lsp1-/- endothelial cells and wild-type leukocytes, neutrophil transendothelial migration out of postcapillary venules is markedly restricted. In contrast, Lsp1-/- neutrophils in wild-type mice were able to extravasate normally. Consistent with altered endothelial function was a reduction in vascular permeability to histamine in Lsp1-/- animals. Western blot analysis and immunofluorescence microscopy examination confirmed the presence of LSP1 in wild-type but not in Lsp1-/- mouse microvascular endothelial cells. Cultured human endothelial cells also stained positive for LSP1. Our results suggest that LSP1 expressed in endothelium regulates neutrophil transendothelial migration.

Endothelium-derived Toll-like receptor-4 is the key molecule in LPS-induced neutrophil sequestration into lungs.

The rapid and selective accumulation of neutrophils into the lungs is thought to underlie the pulmonary failure that leads to sepsis-related death. In this study we investigated whether neutrophil TLR4 is important in LPS-induced pulmonary neutrophil recruitment by creating chimeric mice (transferring bone marrow between TLR4(+/+) and TLR4(-/-) mice). In TLR4(+/+) mice receiving TLR4(-/-) bone marrow, 6 weeks after transplant TLR4 was absent in all circulating leukocytes as well as in resident macrophages (these mice were termed LeukocyteTLR4(-/-)), and these cells were completely nonresponsive to LPS. In TLR4(-/-) mice receiving TLR4(+/+) bone marrow, endothelial cells but not leukocytes were deficient in TLR4 (EndotheliumTLR4(-/-)). Surprisingly, systemic LPS (0.5 mg/kg) induced a dramatic increase in neutrophil sequestration into the lungs of LeukocyteTLR4(-/-) mice over the first 4 hours. Concomitantly, numbers of circulating leukocytes decreased by 90%. By contrast, EndotheliumTLR4(-/-) mice showed very little increase in neutrophil sequestration in the lungs, suggesting that endothelium rather than leukocyte TLR4 was important. Intravital microscopy of peripheral microcirculation in the cremaster muscle revealed about 30-fold more leukocyte-endothelial cell interactions in LPS-treated EndotheliumTLR4(-/-) mice than in LPS-treated LeukocyteTLR4(-/-) mice. This is consistent with less sequestration of leukocytes into the lungs of EndotheliumTLR4(-/-) mice. In conclusion, our data challenge the view that LPS directly activates neutrophils to trap in lungs and suggest a far more important role than previously appreciated for the endothelial cells.

Stable lymphocyte contact induces remodeling of endothelial cell matrix receptor complexes.

Endothelial cells (EC) actively participate in lymphocyte transendothelial migration by remodeling their actin cytoskeleton. We studied the endothelial cell abluminal matrix receptor (focal adhesion, FA) complexes to determine if these structures were remodeled following lymphocyte adhesion. Lymphocytes (PBL) were isolated from whole blood and added to cultured EC. Lectin-stimulated PBL adhered to EC spontaneously, whereas adhesion of freshly isolated lymphocytes to EC was induced by pre-treatment with MCP-1 or activating anti-CD11a mAb. Sustained adhesion between lymphocytes and EC resulted in a significant, contact-dependent decrease in paxillin incorporation into the FA following 15, but not 5, min of contact. EC FA remodeling was associated with increased phosphorylation of pp125 FA kinase. Pretreatment of the EC with an activating beta1 integrin monoclonal antibody, TS2/16, prevented lymphocyte-stimulated FA remodeling. Further, TS2/16 pretreatment inhibited transendothelial migration of lymphocytes and beta1 integrin-deficient JY lymphoblasts. These data demonstrate that sustained lymphocyte adhesion induces remodeling of EC FA structures and that this remodeling event is required for efficient lymphocyte transendothelial migration in vitro.