Understanding the organ tropism of metastatic breast cancer through the combination of liquid biopsy tools.

BACKGROUND: Liquid biopsy provides real-time data about prognosis and actionable mutations in metastatic breast cancer (MBC). The aim of this study was to explore the combination of circulating tumour DNA (ctDNA) analysis and circulating tumour cells (CTCs) enumeration in estimating target organs more susceptible to MBC involvement. METHODS: This retrospective study analysed 88 MBC patients characterised for both CTCs and ctDNA at baseline. CTCs were isolated through the CellSearch kit, while ctDNA was analysed using the Guardant360 NGS-based assay. Sites of disease were collected on the basis of imaging. Associations were explored both through uni- and multivariate logistic regression and Fisher's exact test and the random forest machine learning algorithm. RESULTS: After multivariate logistic regression, ESR1 mutation was the only significant factor associated with liver metastases (OR 8.10), while PIK3CA was associated with lung localisations (OR 3.74). CTC enumeration was independently associated with bone metastases (OR 10.41) and TP53 was associated with lymph node localisations (OR 2.98). The metastatic behaviour was further investigated through a random forest machine learning algorithm. Bone involvement was described by CTC enumeration and alterations in ESR1, GATA3, KIT, CDK4 and ERBB2, while subtype, CTC enumeration, inflammatory BC diagnosis, ESR1 and KIT aberrations described liver metastases. PIK3CA, MET, AR, CTC enumeration and TP53 were associated with lung organotropism. The model, moreover, showed that AR, CCNE1, ESR1, MYC and CTC enumeration were the main drivers in HR positive MBC metastatic pattern. CONCLUSIONS: These results indicate that ctDNA and CTCs enumeration could provide useful insights regarding MBC organotropism, suggesting a possible role for future monitoring strategies that dynamically focus on high-risk organs defined by tumourbiology.

A New Gorilla Adenoviral Vector with Natural Lung Tropism Avoids Liver Toxicity and Is Amenable to Capsid Engineering and Vector Retargeting.

Human adenoviruses have many attractive features for gene therapy applications. However, the high prevalence of preexisting immunity against these viruses in general populations worldwide has greatly limited their clinical utility. In addition, the most commonly used human adenovirus, human adenovirus subgroup C serotype 5 (HAd5), when systemically administered, triggers systemic inflammation and toxicity, with the liver being the most severely affected organ. Here, we evaluated the utility and safety of a new low-seroprevalence gorilla adenovirus (GAd; GC46) as a gene transfer vector in mice. Biodistribution studies revealed that systemically administered GAd had a selective and robust lung endothelial cell (EC) tropism with minimal vector expression throughout many other organs and tissues. Administration of a high dose of GAd accomplished extensive transgene expression in the lung yet elicited no detectable inflammatory histopathology in this organ. Furthermore, GAd, unlike HAd5, did not exhibit hepatotropism or induce liver inflammatory toxicity in mice, demonstrating the exceptional safety profile of the vector vis-à-vis systemic utility. We further demonstrated that the GAd capsid fiber shared the flexibility of the HAd5 equivalent for permitting genetic modification; GAd with the pan-EC-targeting ligand myeloid cell-binding peptide (MBP) incorporated in the capsid displayed a reduced lung tropism and efficiently retargeted gene expression to vascular beds in other organs.IMPORTANCE In the aggregate, our mouse studies suggest that GAd is a promising gene therapy vector that utilizes lung ECs as a source of therapeutic payload production and a highly desirable toxicity profile. Further genetic engineering of the GAd capsid holds the promise of in vivo vector tropism modification and targeting.

Precision mouse models with expanded tropism for human pathogens.

A major limitation of current humanized mouse models is that they primarily enable the analysis of human-specific pathogens that infect hematopoietic cells. However, most human pathogens target other cell types, including epithelial, endothelial and mesenchymal cells. Here, we show that implantation of human lung tissue, which contains up to 40 cell types, including nonhematopoietic cells, into immunodeficient mice (lung-only mice) resulted in the development of a highly vascularized lung implant. We demonstrate that emerging and clinically relevant human pathogens such as Middle East respiratory syndrome coronavirus, Zika virus, respiratory syncytial virus and cytomegalovirus replicate in vivo in these lung implants. When incorporated into bone marrow/liver/thymus humanized mice, lung implants are repopulated with autologous human hematopoietic cells. We show robust antigen-specific humoral and T-cell responses following cytomegalovirus infection that control virus replication. Lung-only mice and bone marrow/liver/thymus-lung humanized mice substantially increase the number of human pathogens that can be studied in vivo, facilitating the in vivo testing of therapeutics.

LXR Alpha Restricts Gammaherpesvirus Reactivation from Latently Infected Peritoneal Cells.

Gammaherpesviruses are ubiquitous viruses that establish lifelong infections. Importantly, these viruses are associated with numerous cancers and lymphoproliferative diseases. While risk factors for developing gammaherpesvirus-driven cancers are poorly understood, it is clear that elevated viral reactivation from latency often precedes oncogenesis. Here, we demonstrate that the liver X receptor alpha isoform (LXRα) restricts gammaherpesvirus reactivation in an anatomic-site-specific manner. We have previously demonstrated that deficiency of both LXR isoforms (α and ß) leads to an increase in fatty acid and cholesterol synthesis in primary macrophage cultures, with a corresponding increase in gammaherpesvirus replication. Interestingly, expression of fatty acid synthesis genes was not derepressed in LXRα-deficient hosts, indicating that the antiviral effects of LXRα are independent of lipogenesis. Additionally, the critical host defenses against gammaherpesvirus reactivation, virus-specific CD8+ T cells and interferon (IFN) signaling, remained intact in the absence of LXRα. Remarkably, using a murine gammaherpesvirus 68 (MHV68) reporter virus, we discovered that LXRα expression dictates the cellular tropism of MHV68 in the peritoneal cavity. Specifically, LXRα-/- mice exhibit reduced latency within the peritoneal B cell compartment and elevated latency within F4/80+ cells. Thus, LXRα restricts gammaherpesvirus reactivation through a novel mechanism that is independent of the known CD8+ T cell-based antiviral responses or changes in lipid synthesis and likely involves changes in the tropism of MHV68 in the peritoneal cavity.IMPORTANCE Liver X receptors (LXRs) are nuclear receptors that mediate cholesterol and fatty acid homeostasis. Importantly, as ligand-activated transcription factors, LXRs represent potential targets for the treatment of hypercholesterolemia and atherosclerosis. Here, we demonstrate that LXRα, one of the two LXR isoforms, restricts reactivation of latent gammaherpesvirus from peritoneal cells. As gammaherpesviruses are ubiquitous oncogenic agents, LXRs may represent a targetable host factor for the treatment of poorly controlled gammaherpesvirus infection and associated lymphomagenesis.

The pentameric complex drives immunologically covert cell-cell transmission of wild-type human cytomegalovirus.

Human cytomegalovirus (HCMV) strains that have been passaged in vitro rapidly acquire mutations that impact viral growth. These laboratory-adapted strains of HCMV generally exhibit restricted tropism, produce high levels of cell-free virus, and develop susceptibility to natural killer cells. To permit experimentation with a virus that retained a clinically relevant phenotype, we reconstructed a wild-type (WT) HCMV genome using bacterial artificial chromosome technology. Like clinical virus, this genome proved to be unstable in cell culture; however, propagation of intact virus was achieved by placing the RL13 and UL128 genes under conditional expression. In this study, we show that WT-HCMV produces extremely low titers of cell-free virus but can efficiently infect fibroblasts, epithelial, monocyte-derived dendritic, and Langerhans cells via direct cell-cell transmission. This process of cell-cell transfer required the UL128 locus, but not the RL13 gene, and was significantly less vulnerable to the disruptive effects of IFN, cellular restriction factors, and neutralizing antibodies compared with cell-free entry. Resistance to neutralizing antibodies was dependent on high-level expression of the pentameric gH/gL/gpUL128-131A complex, a feature of WT but not passaged strains of HCMV.

[Embryo implantation: role of interleukin 1 family members]. / L'implantation embryonnaire - importance de la famille de l'interleukine 1.

Endometrial receptivity to embryo implantation is one of the fundamental features of reproduction. Success of natural or assisted embryo implantation is low (20-25%). Implantation remains the result of a successful collaboration, tightly regulated and closely coordinated, between maternal and embryonic tissues located at the crossroads of endocrinology and immunology. In scientific terms, this collaboration is a mystery of human reproduction. The implanted blastocyst within the endometrium is dependent on a fine-tuned synchronization. Therefore, an accurate dialogue between the mother and the embryo is timely required to orchestrate mutual and well-synchronized changes in the developing embryo and maternal responsiveness in order to achieve a successful implantation. Maternal-derived mediators, such as steroid hormones, matrix-degrading enzymes, integrins, cytokines, chemokines, and many embryonic growth factors could be involved in the feto-maternal dialogue. Therefore, what is the maternal molecular signature compatible with embryo implantation?

Depletion of host CCR7(+) dendritic cells prevented donor T cell tissue tropism in anti-CD3-conditioned recipients.

We reported previously that anti-CD3 mAb treatment before hematopoietic cell transplantation (HCT) prevented graft-versus-host disease (GVHD) and preserved graft-versus-leukemia (GVL) effects in mice. These effects were associated with downregulated donor T cell expression of tissue-specific homing and chemokine receptors, marked reduction of donor T cell migration into GVHD target tissues, and deletion of CD103(+) dendritic cells (DCs) in mesenteric lymph nodes (MLN). MLN CD103(+) DCs and peripheral lymph node (PLN) DCs include CCR7(+) and CCR7(-) subsets, but the role of these DC subsets in regulating donor T cell expression of homing and chemokine receptors remain unclear. Here, we show that recipient CCR7(+), but not CCR7(-), DCs in MLN induced donor T cell expression of gut-specific homing and chemokine receptors in a retinoid acid-dependent manner. CCR7 regulated activated DC migration from tissue to draining lymph node, but it was not required for the ability of DCs to induce donor T cell expression of tissue-specific homing and chemokine receptors. Finally, anti-CD3 treatment depleted CCR7(+) but not CCR7(-) DCs by inducing sequential expansion and apoptosis of CCR7(+) DCs in MLN and PLN. Apoptosis of CCR7(+) DCs was associated with DC upregulation of Fas expression and natural killer cell but not T, B, or dendritic cell upregulation of FasL expression in the lymph nodes. These results suggest that depletion of CCR7(+) host-type DCs, with subsequent inhibition of donor T cell migration into GVHD target tissues, can be an effective approach in prevention of acute GVHD and preservation of GVL effects.

Tropism and innate host responses of a novel avian influenza A H7N9 virus: an analysis of ex-vivo and in-vitro cultures of the human respiratory tract.

BACKGROUND: Since March, 2013, an avian-origin influenza A H7N9 virus has caused severe pneumonia in China. The aim of this study was to investigate the pathogenesis of this new virus in human beings. METHODS: We obtained ex-vivo cultures of the human bronchus, lung, nasopharynx, and tonsil and in-vitro cultures of primary human alveolar epithelial cells and peripheral blood monocyte-derived macrophages. We compared virus tropism and induction of proinflammatory cytokine responses of two human influenza A H7N9 virus isolates, A/Shanghai/1/2013 and A/Shanghai/2/2013; a highly pathogenic avian influenza H5N1 virus; the highly pathogenic avian influenza H7N7 virus that infected human beings in the Netherlands in 2003; the 2009 pandemic influenza H1N1 virus, and a low pathogenic duck H7N9 virus that was genetically different to the human disease causing A H7N9 viruses. FINDINGS: Both human H7N9 viruses replicated efficiently in human bronchus and lung ex-vivo cultures, whereas duck/H7N9 virus failed to replicate in either. Both human A H7N9 viruses infected both ciliated and non-ciliated human bronchial epithelial cells and replicated to higher titres than did H5N1 (p<0.0001 to 0.0046) and A/Shanghai/1/2013 replicated to higher titres than did H7N7 (p=0.0002-0.01). Both human A H7N9 viruses predominantly infected type II alveolar epithelial cells and alveolar macrophages in the human lung and replicated to higher titres than did H5N1 (p<0.0001 to 0.0078); A/Shanghai/1/2013 replicated to higher titres than did H1N1 (p=0.0052-0.05) and H7N7 (p=0.0031-0.0151). Human H7N9 viruses were less potent inducers of proinflammatory cytokines compared with H5N1 virus. INTERPRETATION: Collectively, the results suggest that the novel H7N9 viruses are better adapted to infect and replicate in the human conducting and lower airways than are other avian influenza viruses, including H5N1, and pose an important pandemic threat. FUNDING: Area of Excellence Scheme of the University Grants Committee (AoE/M-12/96), Hong Kong Special Administrative Region.

EBV-gp350 confers B-cell tropism to tailored exosomes and is a neo-antigen in normal and malignant B cells--a new option for the treatment of B-CLL.

gp350, the major envelope protein of Epstein-Barr-Virus, confers B-cell tropism to the virus by interacting with the B lineage marker CD21. Here we utilize gp350 to generate tailored exosomes with an identical tropism. These exosomes can be used for the targeted co-transfer of functional proteins to normal and malignant human B cells. We demonstrate here the co-transfer of functional CD154 protein on tailored gp350+ exosomes to malignant B blasts from patients with B chronic lymphocytic leukemia (B-CLL), rendering B blasts immunogenic to tumor-reactive autologous T cells. Intriguingly, engulfment of gp350+ exosomes by B-CLL cells and presentation of gp350-derived peptides also re-stimulated EBV-specific T cells and redirected the strong antiviral cellular immune response in patients to leukemic B cells. In essence, we show that gp350 alone confers B-cell tropism to exosomes and that these exosomes can be further engineered to simultaneously trigger virus- and tumor-specific immune responses. The simultaneous exploitation of gp350 as a tropism molecule for tailored exosomes and as a neo-antigen in malignant B cells provides a novel attractive strategy for immunotherapy of B-CLL and other B-cell malignancies.

Cytokine determinants of viral tropism.

The specificity of a given virus for a cell type, tissue or species - collectively known as viral tropism - is an important factor in determining the outcome of viral infection in any particular host. Owing to the increased prevalence of zoonotic infections and the threat of emerging and re-emerging pathogens, gaining a better understanding of the factors that determine viral tropism has become particularly important. In this Review, we summarize our current understanding of the central role of antiviral and pro-inflammatory cytokines, particularly the interferons and tumour necrosis factor, in dictating viral tropism and how these cytokine pathways can be exploited therapeutically for cancer treatment and to better counter future threats from emerging zoonotic pathogens.

Variation of macrophage tropism among HIV-1 R5 envelopes in brain and other tissues.

Human immunodeficiency virus (HIV)-positive individuals frequently suffer from progressive encephelopathy, which is characterized by sensory neuropathy, sensory myelopathy, and dementia. Our group and others have reported the presence of highly macrophage-tropic R5 variants of HIV-1 in brain tissue of patients with neurological complications. These variants are able to exploit low amounts of CD4 and/or CCR5 for infection and potentially confer an expanded tropism for any cell types that express low CD4 and/or CCR5. In contrast to the brain-derived envelopes, we found that envelopes from lymph node tissue, blood, or semen were predominantly non-macrophage-tropic and required high amounts of CD4 for infection. Nevertheless, where tested, the non-macrophage-tropic envelopes conferred efficient replication in primary CD4(+) T-cell cultures. Determinants of R5 macrophage tropism appear to involve changes in the CD4 binding site, although further unknown determinants are also involved. The variation of R5 envelopes also affects their sensitivity to inhibition by ligands and entry inhibitors that target CD4 and CCR5. In summary, HIV-1 R5 viruses vary extensively in macrophage tropism. In the brain, highly macrophage-tropic variants may represent neurotropic or neurovirulent viruses. In addition, variation in R5 macrophage tropism may also have implications (1) for transmission, depending on what role macrophages or cells that express low CD4 and/or CCR5 play in the establishment of infection in a new host, and (2) for pathogenesis and depletion of CD4(+) T cells (i.e., do highly macrophage-tropic variants confer a broader tropism among CD4(+) T-cell populations late in disease and contribute to their depletion?).

Replication of infectious bursal disease virus in macrophages and altered tropism of progeny virus.

We serially passaged classical infectious bursal disease virus (cIBDV) and antigenic variant IBDV (vIBDV) in an avian macrophage cell line, NCSU cells, referred as mcIBDV and mvIBDV respectively and examined the in vitro and in vivo characteristics of the macrophage-adapted viruses. NCSU adapted viruses caused earlier destruction of NCSU cells than the unadapted viruses. Nitric oxide (NO) was detected earlier in cultures infected with mcIBDV and mvIBDV than in cultures infected with cIBDV and vIBDV. cIBDV and vIBDV were able to infect DF-1 cells, a chicken embryo fibroblast cell line, only after one replication cycle in NCSU cells. The genetic basis of altered tropism of progeny virus from NCSU cells infected cultures was not identified. No aa substitutions were observed in hypervariable region of VP2 of cIBDV and vIBDV passaged 1 time in NCSU cells whereas both mcIBDV and mvIBDV had multiple aa substitutions. To assess protective efficacy of mcIBDV and mvIBDV, embryonated chicken eggs were inoculated with mcIBDV and mvIBDV at embryonation day 18 (ED 18) and challenged with a virulent cIBDV at 3 weeks of age. mcIBDV and mvIBDV were immunogenic and generated antibody responses and provided 100% protection against cIBDV.

Retinoic acid imprints gut-homing specificity on T cells.

For a preferential homing of T cells to the gut, expression of the integrin alpha4beta7 and the chemokine receptor CCR9 is essential and is induced by antigenic stimulation with dendritic cells from the gut-associated lymphoid organs. Here, we show that the vitamin A (retinol) metabolite, retinoic acid, enhances the expression of alpha4beta7 and CCR9 on T cells upon activation and imprints them with the gut tropism. Dendritic cells from the gut-associated lymphoid organs produced retinoic acid from retinol. The enhanced alpha4beta7 expression on T cells by antigenic stimulation with these dendritic cells was suppressed by the retinal dehydrogenase inhibitor citral and the retinoic acid receptor antagonist LE135. Accordingly, vitamin A deficiency caused a reduction in alpha4beta7(+) memory/activated T cells in lymphoid organs and a depletion of T cells from the intestinal lamina propria. These findings revealed a novel role for retinoic acid in the imprinting of gut-homing specificity on T cells.

Recombinant feline leukemia virus (FeLV) variants establish a limited infection with altered cell tropism in specific-pathogen-free cats in the absence of FeLV subgroup A helper virus.

Feline leukemia virus subgroup B (FeLV-B) is commonly associated with feline lymphosarcoma and arises through recombination between endogenous retroviral elements inherited in the cat genome and corresponding regions of the envelope (env) gene from FeLV subgroup A (FeLV-A). In vivo infectivity for FeLV-B is thought to be inefficient in the absence of FeLV-A. Proposed FeLV-A helper functions include enhanced replication efficiency, immune evasion, and replication rescue for defective FeLV-B virions. In vitro analysis of the recombinant FeLV-B-like viruses (rFeLVs) employed in this study confirmed these viruses were replication competent prior to their use in an in vivo study without FeLV-A helper virus. Eight specific-pathogen-free kittens were inoculated with the rFeLVs alone. Subsequent hematology and histology results were within normal limits, however, in the absence of detectable viremia, virus expression, or significant seroconversion, rFeLV proviral DNA was detected in bone marrow tissue of 4/4 (100%) cats at 45 weeks postinoculation (pi), indicating these rFeLVs established a limited but persistent infection in the absence of FeLV-A. Altered cell tropism was also noted. Focal infection was seen in T-cell areas of the splenic follicles in 3/4 (75%) rFeLV-infected cats analyzed, while an FeLV-A-infected cat showed focal infection in B-cell areas of the splenic follicles. Nucleotide sequence analysis of the surface glycoprotein portion of the rFeLV env gene amplified from bone marrow tissue collected at 45 weeks pi showed no sequence alterations from the original rFeLV inocula.

The V1/V2 region of human immunodeficiency virus type 1 modulates the sensitivity to neutralization by soluble CD4 and cellular tropism.

A primary isolate (KMT) of human immunodeficiency virus type 1 (HIV-1) resistant to recombinant soluble CD4 (rsCD4) was isolated from an HIV-1-infected individual and grown in a T lymphoid cell line. KMT isolate passaged on CEM cells (KMT/CEM) was still resistant to rsCD4. The V1/V2 and V3 regions of the viral envelope glycoprotein are thought to be involved in various biological phenotypes. To determine the exact envelope region of the KMT isolate responsible for sensitivity to rsCD4 and cellular tropism, we performed sequence analysis of KMT and KMT/CEM isolates. Sequence analysis of the KMT isolate showed that the sequence of the V3 region was relatively homogeneous, whereas a considerable heterogeneity of the V1/V2 region was noted. In contrast, the sequences of the V1 to V3 regions were homogeneous in KMT/CEM isolates. Analysis of NL4-3-based recombinant viruses with amplified sequences of the V1 to V3 regions from KMT and KMT/CEM isolates showed that the V1/V2 region modulated the sensitivity to rsCD4. A change in resistance to rsCD4 by the V1/V2 region was associated with the ability of the isolate to replicate in macrophages and efficiently replicate in T lymphoid cell lines. A change to an isolate sensitive to rsCD4 was associated with reduced replication efficiency in T lymphoid cell lines. Our results suggest that the V1/V2 region is involved in modulating the sensitivity to rsCD4, macrophage tropism, and replication efficiency in T lymphoid cell lines.

The role of alpha E beta 7 integrin (CD103) and E-cadherin in epidermotropism in cutaneous T-cell lymphoma.

Adhesion molecules such as integrins and cadherins are thought to play a critical role in T-cell migration and localization within the epidermis (epidermotropism). The purpose of this study was to correlate T-cell expression of the integrin CD103 and E-cadherin in cutaneous T-cell lymphoma (CTCL). Serial sections of skin biopsies from 22 patients with CTCL and 13 with benign reactive dermatitis were stained with antibodies to CD4, CD103, and E-cadherin by the avidin-biotin peroxidase technique. CD103 was expressed on single epidermotropic CD4+ T-cells in 9/9 early stage (patch/plaque) CTCL and 6/10 reactive dermatitis biopsies. Less than 30% of dermal T-cells expressed CD103. All 4/4 late stage (tumor) CTCL were CD103-. Epidermal aggregates of CD4+ T-cells (Pautrier's microabscesses) were CD103-. E-cadherin was expressed on epidermal keratinocytes and follicular and sweat gland epithelia but not on T-cells. We conclude that CD103 expression on cutaneous T-cells parallels the degree of epidermotropism exhibited in both neoplastic and inflammatory disorders of the skin. E-cadherin is not expressed on T-cells infiltrating the skin. Further investigation is necessary to further elucidate the interaction between CD103 and E-cadherin in facilitating trafficking of T-cells into the epidermis.

Redefinition of tropism of common macrophage-tropic human immunodeficiency virus type 1.

Previous studies have suggested that the abilities of human immunodeficiency virus type 1 (HIV-1) to infect primary macrophages and transformed T cell lines are mutually exclusive and define an important biological distinction among HIV-1 strains. In a survey of eight macrophage-tropic HIV-1 strains and nine T cell lines, all frequently used in studies of tropism, we have found that six virus strains replicate in one or more T cell lines and that four T cell lines are highly susceptible to macrophage-tropic HIV-1. Passage through T cell lines did not affect the tropism or the env V3 sequence of monocytotropic HIV-1 strains. We conclude that HIV-1 replication in transformed T cells and primary macrophages are not mutually exclusive, and that as such, these definitions of tropism per se are not generally useful markers for other biological properties of HIV-1.