Patricia Keating

Semaphorin7A promotes tumor growth and exerts a pro-angiogenic effect in macrophages of mammary tumor-bearing mice

Semaphorins are a large family of molecules involved in axonal guidance during the development of the nervous system and have been recently shown to have both angiogenic and anti-angiogenic properties. Specifically, semaphorin 7A (SEMA7A) has been reported to have a chemotactic activity in neurogenesis and to be an immune modulator through α1β1integrins. SEMA7A has been shown to promote monocyte chemotaxis and induce them to produce proinflammatory mediators. In this study we explored the role of SEMA7A in a murine model of breast cancer. We show that SEMA7A is highly expressed by DA-3 murine mammary tumor cells in comparison to normal mammary cells (EpH4), and that peritoneal elicited macrophages from mammary tumor-bearing mice also express SEMA7A at higher levels compared to those derived from normal mice. We also show that murine macrophages treated with recombinant murine SEMA7A significantly increased their expression of proangiogenic molecule CXCL2/MIP-2. Gene silencing of SEMA7A in peritoneal elicited macrophages from DA-3 tumor-bearing mice resulted in decreased CXCL2/MIP-2 expression. Mice implanted with SEMA7A silenced tumor cells showed decreased angiogenesis in the tumors compared to the wild type tumors. Furthermore, peritoneal elicited macrophages from mice bearing SEMA7A-silenced tumors produce significantly (p < 0.01) lower levels of angiogenic proteins, such as CXCL2/MIP-2, CXCL1, and MMP-9, compared to those from control DA-3 mammary tumors. We postulate that SEMA7A in mammary carcinomas may skew monocytes into a pro-tumorigenic phenotype to support tumor growth. SEMA7A could prove to be valuable in establishing new research avenues toward unraveling important tumor-host immune interactions in breast cancer patients.

Effect of vitamin D on T-helper type 9 polarized human memory cells in chronic persistent asthma

BACKGROUND Vitamin D suppresses inflammation and vitamin D deficiency is linked to the severity of asthma symptoms. T-helper type 9 (TH9) cells are important in the pathogenesis, yet the effects of vitamin D on this subset of inflammatory T-helper cells from patients with chronic asthma is unknown. OBJECTIVE To determine the effects of vitamin D and dexamethasone on TH9 memory cells from adults with chronic persistent asthma and on a recall response to dust mite allergen. METHODS T-helper memory cells were cultured with cytokines that drive TH9 polarization with vitamin D and/or dexamethasone. Peripheral blood mononuclear cells (PBMCs) from patients with radioallergosorbent test results for house dust mite were stimulated with allergen in the presence or absence of vitamin D. Intracellular cytokines, transcription factors, and identification of cell surface phenotypic markers were determined by flow cytometry. RESULTS Vitamin D decreased interleukin (IL)-9, IL-5, and IL-8 but increased IL-13(+) cells in TH9 cultures. Transcription factors PU.1 and interferon regulatory factor 4 were downregulated by vitamin D but not GATA3 and c-MAF. When PBMCs from patients with positive radioallergosorbent test results were stimulated with dust mite allergen, vitamin D decreased IL-9, IL-5, and IL-13 in T-helper cells (CD4(+)). TH9 cells present in a recall response were classically TH2 (CD294(+)), and polarization by transforming growth factor-β and IL-4 altered that phenotype. CONCLUSION Vitamin D decreased inflammatory cytokine profiles in TH9 memory cells and CD4(+) cells stimulated with dust mite allergen. Vitamin D is additive with dexamethasone in decreasing inflammatory cytokine production from T-cell subsets implicated in asthma.

Exploring the role of CHI3L1 in “pre-metastatic” lungs of mammary tumor-bearing mice

Elevated levels of chitinase-3-like-1 (CHI3L1) are associated with poor prognosis, shorter recurrence-free intervals and low survival in breast cancer patients. Breast cancer often metastasizes to the lung. We hypothesized that molecules expressed in the “pre-metastatic” lung microenvironment could support the newly immigrant tumor cells by providing growth and angiogenic factors. Macrophages are known to play an important role in tumor growth by releasing pro-angiogenic molecules. Using mouse mammary tumor models, we have previously shown that during neoplastic progression both the mammary tumor cells and splenic macrophages from tumor-bearing mice express higher levels of CHI3L1 compared to normal control mice. However, the role of CHI3L1 in inducing angiogenesis by macrophages at the pulmonary microenvironment to support newly arriving breast cancer cells is not yet known. In this study, we determined the expression of CHI3L1 in bronchoalveolar lavage macrophages and interstitial macrophages in regulating angiogenesis that could support the growth of newly immigrant mammary tumor cells into the lung. Here we show that in vitro treatment of pulmonary macrophages with recombinant murine CHI3L1 resulted in enhanced expression of pro-angiogenic molecules including CCL2, CXCL2, and MMP-9. We and others have previously shown that inhibition of CHI3L1 decreases the production of angiogenic molecules. In this study, we explored if in vivo administration of chitin microparticles has an effect on the expression of CHI3L1 and pro-angiogenic molecules in the lungs of mammary tumor-bearing mice. We show that treatment with chitin microparticles decreases the expression of CHI3L1 and pro-angiogenic molecules in the “metastatic” lung. These studies suggest that targeting CHI3L1 may serve as a potential therapeutic agent to inhibit angiogenesis and thus possibly tumor growth and metastasis.

Highly Specific Novel Method for Isolation and Purification of Lupus Anti‐DNA Antibody via Oligo‐(dT) Magnetic Beads

Abstract:  A novel method for isolation and purification of anti‐ssDNA antibodies from human sera is developed. The process involves: antibody purification based on their affinity for single‐stranded sequence of thymidines and removal of remaining components via protein G coated magnetic beads, with high affinity for only IgG subclass. The high degree of purity and molecular weights of healthy versus lupus anti‐ssDNA antibodies were confirmed by SDS‐PAGE and silver staining. Western blot confirmed IgG isotype. This novel technique allows for diagnostic purposes, structural and functional analysis of anti‐DNA antibodies, and studies of their role in autoimmune diseases.

Production of functional native human interleukin-2 in tobacco chloroplasts.

Interleukin-2 (IL-2) is an important T lymphocyte-derived cytokine in the mammalian immune system. Non-native, recombinant IL-2 derived from Escherichiacoli is used widely in both medical research and treatment of diseases. Recombinant human IL-2 gene has been expressed in plant nuclear genomes, therefore it can be spread to the environment through pollen. Furthermore, all the plant-produced IL-2 reported thus far had been attached with artificial tags or fusion proteins, which may trigger unintended immunological responses and therefore compromise its full utility as a medicine. To expand the potential of using plant chloroplasts to produce functional native human therapeutic proteins, we inserted an engineered human interleukin-2 (hIL-2)-coding gene, without any tags, into the chloroplast genome of tobacco (Nicotiana tabacum L.). Partially purified hIL-2 protein from the leaves of the transplastomic plants induced in vitro proliferation of IL-2-dependent murine T lymphocytes. Our study demonstrates that plant chloroplasts can serve as a bio-factory for production of an active native human interleukin in a self-contained and therefore environmentally safe manner.

Allergen induced pulmonary inflammation enhances mammary tumor growth and metastasis: Role of CHI3L1

Metastasis is the primary cause of mortality in women with breast cancer. Metastasis to the lungs is greater in patients with pulmonary inflammatory illnesses. It is unknown how pre‐existing pulmonary inflammation affects mammary tumor progression. We developed a novel breast cancer model in which pulmonary inflammation is induced in mice prior to tumor cell implantation. In the present study, we determined how pre‐existing allergen‐induced inflammation changes the pulmonary microenvironment to exacerbate tumor metastasis. We showed that pre‐existing pulmonary inflammation in mammary tumor bearers is associated with: 1) an increase in growth of the primary tumor and metastasis; 2) an increase in the expression of a glycoprotein known as CHI3L1; and 3) increase in the levels of myeloid populations in their lungs. We also showed that myeloid derived cells from the lungs of allergic tumor bearers produce higher amounts of CHI3L1 than the saline controls. We previously showed that CHI3L1 induces the expression of proinflammatory and protumorigenic molecules. In this study, we show that CHI3L1 knockout tumor bearers with pre‐existing allergic pulmonary inflammation had decreased levels of myeloid‐derived cells, decreased levels of proinflammatory mediators, and a significant reduction in tumor volume and metastasis compared with the wild‐type controls. Pre‐existing inflammation and CHI3L1 might be driving the establishment of a premetastatic milieu in the lungs and aiding in the support of metastatic foci. Understanding the role of allergen‐induced CHI3L1 and inflammation in tumor bearers and its effects on the pulmonary microenvironment could result in targeted therapies for breast cancer.

Effects of α-conotoxin ImI on TNF-α, IL-8 and TGF-β expression by human macrophage-like cells derived from THP-1 pre-monocytic leukemic cells

Abstractα7 nicotinic acetylcholine receptors (nAChRs) are ubiquitous in the nervous system and ensure important neurophysiological functionality for many processes. However, they are also found in cells of the immune system, where their role has been less studied. Here we report the pro-inflammatory effect of ImI, a well characterized conotoxin that inhibits α7 nAChRs, on differentiated THP-1 pre-monocyte macrophages (MDM) obtained by phorbol 12-myristate 13 acetate (PMA) treatment. Enzyme-linked immunosorbent assay (ELISA) performed on supernatant fluids of LPS challenged MDM showed ImI-mediated upregulation of pro-inflammatory cytokine TNF-α in an ImI concentration-dependent manner from 0.5 to 5.0 µmol/L and for IL-8 up to 1.0 µmol/L. Levels of anti-inflammatory cytokine TGF-β remained practically unaffected in ImI treated MDMs. Nicotine at 10 µmol/L significantly downregulated the release of TNF-α, but showed a lesser effect on IL-8 secretion and no effect on TGF-β. Fluorescent competitive assays involving ImI, α-bungarotoxin and nicotine using MDM and the murine macrophage RAW 264.7 suggest a common binding site in the α7 receptor. This work extends the application of conotoxins as molecular probes to non-excitatory cells, such as macrophages and supports the involvement of the α7 nAChR in regulating the inflammatory response via the cholinergic anti-inflammatory pathway (CAP).

Isolation and Purification of Th9 Cells for the Study of Inflammatory Diseases in Research and Clinical Settings

Th9 cells are associated with atopic and inflammatory diseases, and their increased levels and function correlate with the severity of symptoms in various inflammatory disorders including asthma, food allergy, atopic dermatitis, ulcerative colitis, and psoriatic arthritis. Thus, clinical trials are warranted to evaluate the role of Th9 cells in allergic diseases with the goal of controlling these ailments.Circulating T cells (naïve or memory CD4+ T cells) purified from human blood and expanded using anti-CD3 and anti-CD28 antibodies can be treated with appropriate cytokines in order to polarize them to the Th9 phenotype as evidenced by their production of IL-9. When treated in vitro with cholecalciferol or 1,25(OH)2 vitamin D3, cells polarized under Th9 conditions significantly downregulate production of IL-9. The percentage of polarized Th9 memory cells from patients treated with steroids or other modalities can be monitored during clinical trials and compared to control populations.

Suppression of tumor-derived Semaphorin 7A and genetic ablation of host-derived Semaphorin 7A impairs tumor progression in a murine model of advanced breast carcinoma

Solid tumors can generate a plethora of neurogenesis-related molecules that enhance their growth and metastasis. Among them, we have identified axonal guidance molecule Semaphorin 7A (SEMA7A) in breast cancer. The goal of this study was to determine the therapeutic effect of suppressing SEMA7A levels in the 4T1 murine model of advanced breast carcinoma. We used anti-SEMA7A short hairpin RNA (shRNA) to gene silence SEMA7A in 4T1 mammary tumor cells. When implanted into the mammary fat pads of syngeneic mice, SEMA7A shRNA-expressing 4T1 tumors exhibited decreased growth rates, deferred metastasis and reduced mortality. In vitro, SEMA7A shRNA-expressing 4T1 cells had weakened proliferative, migratory and invasive abilities, and decreased levels of mesenchymal factors. Atomic force microscopy studies showed that SEMA7A shRNA-expressing 4T1 cells had an increase in cell stiffness that corresponded with their decreased malignant potential. Genetic ablation of host-derived SEMA7A further enhanced the antitumor effects of SEMA7A shRNA gene silencing in 4T1 cells. Our preclinical findings demonstrate a critical role for SEMA7A in mediating mammary tumor progression.