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ObjectiveOur objective was to determine the effects of prolonged exposure to tumor necrosis factor-alpha (TNF-alpha) on leptin secretion from and leptin (OB) gene expression in isolated adipocytes. Because glucose uptake and the metabolism of glucose beyond lactate are important determinants of leptin production in adipocytes, we examined the effects of TNF-alpha on glucose uptake and lactate production and their relationship to leptin secretion.Design and methodsIsolated rat adipocytes were anchored in a defined matrix of basement membrane components and cultured with media containing 5 mM glucose, 0.16 nM insulin and several concentrations of TNF-alpha. Leptin secretion, steady-state levels of leptin mRNA levels, glucose uptake, and lactate production were assessed over 96 h.ResultsTNF-alpha at concentrations of 0.024, 0.24, 2.4 and 24 ng/ml did not affect leptin secretion over 24 h. TNF-alpha at concentrations of 0.24 to 24 ng/ml significantly inhibited leptin secretion over 96 h by 19-60%. TNF-alpha at concentrations of 0.024 to 24 ng/ml significantly decreased steady-state levels of leptin mRNA after 96 h by 32-95%. In addition, TNF-alpha at concentrations of 2.4 and 24 ng/ml significantly increased glucose uptake and lactate production over 96 h by 30-57%. TNF-alpha at a concentration of 0.024 ng/ml did not affect leptin secretion, glucose uptake or lactate production. Overall, for the TNF-alpha concentrations tested, leptin secretion was inversely related to the percent of glucose carbon released as lactate; however, TNF-alpha did not induce a proportional increase of lactate production from glucose.ConclusionShort-term (24 h) exposure of isolated adipocytes to TNF-alpha does not affect leptin secretion. Prolonged exposure to TNF-alpha produces a concentration-dependent inhibition of leptin secretion and gene expression. This suggests that the acute effect of TNF-alpha to increase circulating leptin levels in vivo may be indirect. TNF-alpha at higher concentrations increases glucose uptake, but does not increase the conversion of glucose to lactate. Therefore, TNF-alpha appears to induce a dissociation between adipocyte glucose metabolism and leptin production.

HER2-positive breast cancers are among the most heterogeneous breast cancer subtypes. The early amplification of HER2 and its known oncogenic isoforms provide a plausible mechanism in which distinct programs of tumor heterogeneity could be traced to the initial oncogenic event. Here a Cancer rainbow mouse simultaneously expressing fluorescently barcoded wildtype ( WT HER2), exon-16 null ( d16 HER2), and N-terminally truncated ( p95 HER2) HER2 isoforms is used to trace tumorigenesis from initiation to invasion. Tumorigenesis was visualized using whole-gland fluorescent lineage tracing and single-cell molecular pathology. We demonstrate that within weeks of expression, morphologic aberrations were already present and unique to each HER2 isoform. Although WT HER2 cells were abundant throughout the mammary ducts, detectable lesions were exceptionally rare. In contrast, d16 HER2 and p95 HER2 induced rapid tumor development. d16 HER2 incited homogenous and proliferative luminal-like lesions which infrequently progressed to invasive phenotypes whereas p95 HER2 lesions were heterogenous and invasive at the smallest detectable stage. Distinct cancer trajectories were observed for d16 HER2 and p95 HER2 tumors as evidenced by oncogene-dependent changes in epithelial specification and the tumor microenvironment. These data provide direct experimental evidence that intratumor heterogeneity programs begin very early and well in advance of screen or clinically detectable breast cancer. IMPLICATIONS: Although all HER2 breast cancers are treated equally, we show a mechanism by which clinically undetected HER2 isoforms program heterogenous cancer phenotypes through biased epithelial specification and adaptations within the tumor microenvironment., (©2021 American Association for Cancer Research.)

Benign mammary tumours are among the most common tumours of companion rats (Rattus norvegicus domestica), as well as a major animal welfare concern and euthanasia. The first objective of this study was to evaluate the expression of oestrogen, progesterone, androgen, and prolactin receptors in neoplastic and normal mammary gland tissues and compare the expression of these receptors between groups. The second objective was to determine if the expression of these receptors in neoplastic mammary gland tissue correlates with overall survival and occurrence of an additional mass after initial mammary mass excision. The third objective was to determine if the expression of oestrogen, progesterone, androgen and prolactin receptors was associated with mammary tumor clinical parameters or with the age of the animals. Thirty-two benign mammary tumours were collected from companion rats and submitted for immunohistochemistry staining of prolactin receptor, oestrogen receptor alpha (ERa), progesterone and androgen receptors (AR). Allred score were obtained for mammary tumours (n = 32) and surrounding normal mammary tissue (n = 20) when present. Prolactin receptor expression increased significantly with mammary gland tumorigenesis (P < .0001), while AR expression decreased with tumorigenesis (P < .0001). Lower expression of ERa in tumor stroma was associated with shorter survival (P = .02). Hormonal receptor expression was not significantly associated with age, mass diameter, location nor likelihood of additional mass development. Further studies should investigate the effects of prolactin antagonists in a prospective study involving companion rats with benign mammary tumours., (© 2020 John Wiley & Sons Ltd.)

Gemcitabine delivery to pancreatic ductal adenocarcinoma is limited by poor pharmacokinetics, dense fibrosis and hypo-vascularization. Activatable liposomes, with drug release resulting from local heating, enhance serum stability and circulation, and the released drug retains the ability to diffuse within the tumor. A limitation of liposomal gemcitabine has been the low loading efficiency. To address this limitation, we used the superior solubilizing potential of copper (II) gluconate to form a complex with gemcitabine at copper:gemcitabine (1:4). Thermosensitive liposomes composed of DPPC:DSPC:DSPE-PEG2k (80:15:5, mole%) then reached 12 wt% loading, 4-fold greater than previously reported values. Cryo transmission electron microscopy confirmed the presence of a liquid crystalline gemcitabine‑copper mixture. The optimized gemcitabine liposomes released 60% and 80% of the gemcitabine within 1 and 5 min, respectively, at 42 °C. Liposomal encapsulation resulted in a circulation half-life of ~2 h in vivo (compared to reported circulation of 16 min for free gemcitabine in mice), and free drug was not detected within the plasma. The resulting gemcitabine liposomes were efficacious against both murine breast cancer and pancreatic cancer in vitro. Three repeated treatments of activatable gemcitabine liposomes plus ultrasound hyperthermia regressed or eliminated tumors in the neu deletion model of murine breast cancer with limited toxicity, enhancing survival when compared to treatment with gemcitabine alone. With 5% of the free gemcitabine dose (5 rather than 100 mg/kg), tumor growth was suppressed to the same degree as gemcitabine. Additionally, in a more aggressive tumor model of murine pancreatic cancer, liposomal gemcitabine combined with local hyperthermia induced cell death and regions of apoptosis and necrosis., (Copyright © 2019 Elsevier B.V. All rights reserved.)

A successful chemotherapy-immunotherapy solid-tumor protocol should accomplish the following goals: debulk large tumors, release tumor antigen for cross-presentation and cross-priming, release cancer-suppressive cytokines and enhance anti-tumor immune cell populations. Thermally-activated drug delivery particles have the potential to synergize with immunotherapeutics to accomplish these goals; activation can release chemotherapy within bulky solid tumors and can enhance response when combined with immunotherapy. We set out to determine whether a single protocol, combining locally-activated chemotherapy and agonist immunotherapy, could accomplish these goals and yield a potentially translational therapy. For effective delivery of free doxorubicin to tumors with minimal toxicity, we stabilized doxorubicin with copper in temperature-sensitive liposomes that rapidly release free drug in the vasculature of cancer lesions upon exposure to ultrasound-mediated hyperthermia. We found that in vitro exposure of tumor cells to hyperthermia and doxorubicin resulted in immunogenic cell death and the local release of type I interferons across murine cancer cell lines. Following intravenous injection, local activation of the liposomes within a single tumor released doxorubicin and enhanced cross-presentation of a model antigen at distant tumor sites. While a variety of protocols achieved a complete response in >50% of treated mice, the complete response rate was greatest (90%) when 1 week of immunotherapy priming preceded a single activatable chemotherapeutic administration. While repeated chemotherapeutic delivery reduced local viable tumor, the complete response rate and a subset of tumor immune cells were also reduced. Taken together, the results suggest that activatable chemotherapy can enhance adjuvant immunotherapy; however, in a murine model the systemic adaptive immune response was greatest with a single administration of chemotherapy., (Copyright © 2019 Elsevier B.V. All rights reserved.)

Both adjuvants and focal ablation can alter the local innate immune system and trigger a highly effective systemic response. Our goal is to determine the impact of these treatments on directly treated and distant disease and the mechanisms for the enhanced response obtained by combinatorial treatments. Methods: We combined RNA-sequencing, flow cytometry and TCR-sequencing to dissect the impact of immunotherapy and of immunotherapy combined with ablation on local and systemic immune components. Results: With administration of a toll-like receptor agonist agonist (CpG) alone or CpG combined with same-site ablation, we found dramatic differences between the local and distant tumor environments, where the directly treated tumors were skewed to high expression of F4/80 , Cd11b and Tnf and the distant tumors to enhanced Cd11c , Cd3 and Ifng . When ablation was added to immunotherapy, 100% (n=20/20) of directly treated tumors and 90% (n=18/20) of distant tumors were responsive. Comparing the combined ablation-immunotherapy treatment to immunotherapy alone, we find three major mechanistic differences. First, while ablation alone enhanced intratumoral antigen cross-presentation (up to ~8% of CD45 + cells), systemic cross-presentation of tumor antigen remained low. Combining same-site ablation with CpG amplified cross-presentation in the draining lymph node (~16% of CD45 + cells) compared to the ablation-only (~0.1% of CD45 + cells) and immunotherapy-only cohorts (~10% of CD45 + cells). Macrophages and DCs process and present this antigen to CD8 + T-cells, increasing the number of unique T-cell receptor rearrangements in distant tumors. Second, type I interferon (IFN) release from tumor cells increased with the ablation-immunotherapy treatment as compared with ablation or immunotherapy alone. Type I IFN release is synergistic with toll-like receptor activation in enhancing cytokine and chemokine expression. Expression of genes associated with T-cell activation and stimulation (Eomes, Prf1 and Icos) was 27, 56 and 89-fold higher with ablation-immunotherapy treatment as compared to the no-treatment controls (and 12, 32 and 60-fold higher for immunotherapy-only treatment as compared to the no-treatment controls). Third, we found that the ablation-immunotherapy treatment polarized macrophages and dendritic cells towards a CD169 subset systemically, where CD169 + macrophages are an IFN-enhanced subpopulation associated with dead-cell antigen presentation. Conclusion: While the local and distant responses are distinct, CpG combined with ablative focal therapy drives a highly effective systemic immune response., Competing Interests: Competing Interests: The authors have declared that no competing interest exists.

Goal: The aim of this study was to investigate gene expression levels of proteins involved in sphingosine-1-phosphate (S1P) metabolism and signaling in a pediatric inflammatory bowel disease (IBD) patient population., Background: IBD is a debilitating disease affecting 0.4% of the US population. The incidence of IBD in childhood is rising. Identifying effective targeted therapies that can be used safely in young patients and developing tools for selecting specific candidates for targeted therapies are important goals. Clinical IBD trials now underway target S1PR1, a receptor for the pro-inflammatory sphingolipid S1P. However, circulating and tissue sphingolipid levels and S1P-related gene expression have not been characterized in pediatric IBD., Methods: Pediatric IBD patients and controls were recruited in a four-site study. Patients received a clinical score using PUCAI or PCDAI evaluation. Colon biopsies were collected during endoscopy. Gene expression was measured by qRT-PCR. Plasma and gut tissue sphingolipids were measured by LC-MS/MS., Results: Genes of S1P synthesis (SPHK1, SPHK2), degradation (SGPL1), and signaling (S1PR1, S1PR2, and S1PR4) were significantly upregulated in colon biopsies of IBD patients with moderate/severe symptoms compared with controls or patients in remission. Tissue ceramide, dihydroceramide, and ceramide-1-phosphate (C1P) levels were significantly elevated in IBD patients compared with controls., Conclusions: A signature of elevated S1P-related gene expression in colon tissues of pediatric IBD patients correlates with active disease and normalizes in remission. Biopsied gut tissue from symptomatic IBD patients contains high levels of pro-apoptotic and pro-inflammatory sphingolipids. A combined analysis of gut tissue sphingolipid profiles with this S1P-related gene signature may be useful for monitoring response to conventional therapy.

We have performed an in-depth single-cell phenotypic characterization of high-grade serous ovarian cancer (HGSOC) by multiparametric mass cytometry (CyTOF). Using a CyTOF antibody panel to interrogate features of HGSOC biology, combined with unsupervised computational analysis, we identified noteworthy cell types co-occurring across the tumors. In addition to a dominant cell subset, each tumor harbored rarer cell phenotypes. One such group co-expressed E-cadherin and vimentin (EV), suggesting their potential role in epithelial mesenchymal transition, which was substantiated by pairwise correlation analyses. Furthermore, tumors from patients with poorer outcome had an increased frequency of another rare cell type that co-expressed vimentin, HE4, and cMyc. These poorer-outcome tumors also populated more cell phenotypes, as quantified by Simpson's diversity index. Thus, despite the recognized genomic complexity of the disease, the specific cell phenotypes uncovered here offer a focus for therapeutic intervention and disease monitoring., (Copyright © 2018 The Author(s). Published by Elsevier Inc. All rights reserved.)

Background: Stat1 gene-targeted knockout mice (129S6/SvEvTac-Stat1 tm1Rds ) develop estrogen receptor-positive (ER + ), luminal-type mammary carcinomas at an advanced age. There is evidence for both host environment as well as tumor cell-intrinsic mechanisms to initiate tumorigenesis in this model. In this report, we summarize details of the systemic and mammary pathology at preneoplastic and tumor-bearing time points. In addition, we investigate tumor progression in the 129:Stat1 -/- host compared with wild-type 129/SvEv, and we describe the immune cell reaction to the tumors., Methods: Mice housed and treated according to National Institutes of Health guidelines and Institutional Animal Care and Use Committee-approved methods were evaluated by histopathology, and their tissues were subjected to immunohistochemistry with computer-assisted quantitative image analysis. Tumor cell culture and conditioned media from cell culture were used to perform macrophage (RAW264.7) cell migration assays, including the 129:Stat1 -/- -derived SSM2 cells as well as control Met1 and NDL tumor cells and EpH4 normal cells., Results: Tumorigenesis in 129:Stat1 -/- originates from a population of FoxA1 + large oval pale cells that initially appear and accumulate along the mammary ducts in segments or regions of the gland prior to giving rise to mammary intraepithelial neoplasias. Progression to invasive carcinoma is accompanied by a marked local stromal and immune cell response composed predominantly of T cells and macrophages. In conditioned media experiments, cells derived from 129:Stat1 -/- tumors secrete both chemoattractant and chemoinhibitory factors, with greater attraction in the extracellular vesicular fraction and inhibition in the soluble fraction. The result appears to be recruitment of the immune reaction to the periphery of the tumor, with exclusion of immune cell infiltration into the tumor., Conclusions: 129:Stat1 -/- is a unique model for studying the critical origins and risk reduction strategies in age-related ER + breast cancer. In addition, it can be used in preclinical trials of hormonal and targeted therapies as well as immunotherapies.

Focal therapies play an important role in the treatment of cancers where palliation is desired, local control is needed, or surgical resection is not feasible. Pairing immunotherapy with such focal treatments is particularly attractive; however, there is emerging evidence that focal therapy can have a positive or negative impact on the efficacy of immunotherapy. Thermal ablation is an appealing modality to pair with such protocols, as tumors can be rapidly debulked (cell death occurring within minutes to hours), tumor antigens can be released locally, and treatment can be conducted and repeated without the concerns of radiation-based therapies. In a syngeneic model of epithelial cancer, we found that 7 days of immunotherapy (TLR9 agonist and checkpoint blockade), prior to thermal ablation, reduced macrophages and myeloid-derived suppressor cells and enhanced IFN-γ-producing CD8 + T cells, the M1 macrophage fraction, and PD-L1 expression on CD45 + cells. Continued treatment with immunotherapy alone or with immunotherapy combined with ablation (primed ablation) then resulted in a complete response in 80% of treated mice at day 90, and primed ablation expanded CD8 + T cells as compared with all control groups. When the tumor burden was increased by implantation of 3 orthotopic tumors, successive primed ablation of 2 discrete lesions resulted in survival of 60% of treated mice as compared with 25% of mice treated with immunotherapy alone. Alternatively, when immunotherapy was begun immediately after thermal ablation, the abscopal effect was diminished and none of the mice within the cohort exhibited a complete response. In summary, we found that immunotherapy begun before ablation can be curative and can enhance efficacy in the presence of a high tumor burden. Two mechanisms have potential to impact the efficacy of immunotherapy when begun immediately after thermal ablation: mechanical changes in the tumor microenvironment and inflammatory-mediated changes in immune phenotype., Competing Interests: Conflict of interest: The authors have declared that no conflict of interest exists.

Differential diagnosis and therapy of heterogeneous breast tumors poses a major clinical challenge. To address the need for a comprehensive, noninvasive strategy to define the molecular and functional profiles of tumors in vivo, we investigated a novel combination of metabolic PET and diffusion-weighted (DW)-MRI in the polyoma virus middle T antigen transgenic mouse model of breast cancer. The implementation of a voxelwise analysis for the clustering of intra- and intertumoral heterogeneity in this model resulted in a multiparametric profile based on [(18)F]Fluorodeoxyglucose ([(18)F]FDG)-PET and DW-MRI, which identified three distinct tumor phenotypes in vivo, including solid acinar, and solid nodular malignancies as well as cystic hyperplasia. To evaluate the feasibility of this approach for clinical use, we examined estrogen receptor-positive and progesterone receptor-positive breast tumors from five patient cases using DW-MRI and [(18)F]FDG-PET in a simultaneous PET/MRI system. The postsurgical in vivo PET/MRI data were correlated to whole-slide histology using the latter traditional diagnostic standard to define phenotype. By this approach, we showed how molecular, structural (microscopic, anatomic), and functional information could be simultaneously obtained noninvasively to identify precancerous and malignant subtypes within heterogeneous tumors. Combined with an automatized analysis, our results suggest that multiparametric molecular and functional imaging may be capable of providing comprehensive tumor profiling for noninvasive cancer diagnostics. Cancer Res; 76(18); 5512-22. ©2016 AACR., (©2016 American Association for Cancer Research.)

Mechanisms for the progression of ductal carcinoma in situ (DCIS) to invasive breast carcinoma remain unclear. Previously we showed that the transition to invasiveness in the mammary intraepithelial neoplastic outgrowth (MINO) model of DCIS does not correlate with its serial acquisition of genetic mutations. We hypothesized instead that progression to invasiveness depends on a change in the microenvironment and that precancer cells might create a more tumor-permissive microenvironment secondary to changes in glucose uptake and metabolism. Immunostaining for glucose transporter 1 (GLUT1) and the hypoxia marker carbonic anhydrase 9 (CAIX) in tumor, normal mammary gland and MINO (precancer) tissue showed differences in expression. The uptake of the fluorescent glucose analog dye, 2-[N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl) amino]-2-deoxy-D-glucose (2-NBDG), reflected differences in the cellular distributions of glucose uptake in normal mammary epithelial cells (nMEC), MINO, and Met1 cancer cells, with a broad distribution in the MINO population. The intracellular pH (pHi) measured using the fluorescent ratio dye 2',7'-bis(2-carboxyethyl)-5(6)-155 carboxyfluorescein (BCECF) revealed expected differences between normal and cancer cells (low and high, respectively), and a mixed distribution in the MINO cells, with a subset of cells in the MINO having an increased rate of acidification when proton efflux was inhibited. Invasive tumor cells had a more alkaline baseline pHi with high rates of proton production coupled with higher rates of proton export, compared with nMEC. MINO cells displayed considerable variation in baseline pHi that separated into two distinct populations: MINO high and MINO low. MINO high had a noticeably higher mean acidification rate compared with nMEC, but relatively high baseline pHi similar to tumor cells. MINO low cells also had an increased acidification rate compared with nMEC, but with a more acidic pHi similar to nMEC. These findings demonstrate that MINO is heterogeneous with respect to intracellular pH regulation which may be associated with an acidified regional microenvironment. A change in the pH of the microenvironment might contribute to a tumor-permissive or tumor-promoting progression. We are not aware of any previous work showing that a sub-population of cells in in situ precancer exhibits a higher than normal proton production and export rate.

Ultrasonic activation of nanoparticles provides the opportunity to deliver a large fraction of the injected dose to insonified tumors and produce a complete local response. Here, we evaluate whether the local and systemic response to chemotherapy can be enhanced by combining such a therapy with locally-administered CpG as an immune adjuvant. In order to create stable, activatable particles, a complex between copper and doxorubicin (CuDox) was created within temperature-sensitive liposomes. Whereas insonation of the CuDox liposomes alone has been shown to produce a complete response in murine breast cancer after 8 treatments of 6 mg/kg delivered over 4 weeks, combining this treatment with CpG resolved local cancers within 3 treatments delivered over 7 days. Further, contralateral tumors regressed as a result of the combined treatment, and survival was extended in systemic disease. In both the treated and contralateral tumor site, the combined treatment increased leukocytes and CD4+ and CD8+ T-effector cells and reduced myeloid-derived suppressor cells (MDSCs). Taken together, the results suggest that this combinatorial treatment significantly enhances the systemic efficacy of locally-activated nanotherapy., (Copyright © 2015 Elsevier B.V. All rights reserved.)

Tissue localization of immune cells is critical to the study of disease processes in mouse models of human diseases. However, immunohistochemistry (IHC) for immune cell phenotyping in mouse tissue sections presents specific technical challenges. For example, CD4 and CD8 have been difficult to detect using IHC on formalin-fixed and paraffin-embedded mouse tissue, prompting alternative methods. We investigated the use of formalin-free zinc-salt fixation (ZN) and optimized IHC protocols for detecting a panel of immune cell-related markers (CD3, CD4, CD8, Foxp3, B220, F4/80, CD68, and major histocompatibility complex [MHC] class-I, MHC class-II, and Gr-1). The IHC results for these markers were compared on mouse spleen tissue treated with neutral buffered formalin (NBF) or ZN with or ZN without antigen retrieval (AR). Whereas CD4 and CD8 were not detected in NBF-treated tissue, all markers were detected in ZN-treated tissue without AR. Thus, the use of ZN treatment for IHC staining can be a good tool for studying immunoreactive lesions in tissues., (© 2015 by The Author(s).)

Ultrasound molecular imaging has great potential to impact early disease diagnosis, evaluation of disease progression and the development of target-specific therapy. In this paper, two neuropilin-1 (NRP) targeted peptides, CRPPR and ATWLPPR, were conjugated onto the surface of lipid microbubbles (MBs) to evaluate molecular imaging of tumor angiogenesis in a breast cancer model. Development of a molecular imaging agent using CRPPR has particular importance due to the previously demonstrated internalizing capability of this and similar ligands. In vitro, CRPPR MBs bound to an NRP-expressing cell line 2.6 and 15.6 times more than ATWLPPR MBs and non-targeted (NT) MBs, respectively, and the binding was inhibited by pretreating the cells with an NRP antibody. In vivo, the backscattered intensity within the tumor, relative to nearby vasculature, increased over time during the ∼6 min circulation of the CRPPR-targeted contrast agents providing high contrast images of angiogenic tumors. Approximately 67% of the initial signal from CRPPR MBs remained bound after the majority of circulating MBs had cleared (8 min), 8 and 4.5 times greater than ATWLPPR and NT MBs, respectively. Finally, at 7-21 days after the first injection, we found that CRPPR MBs cleared faster from circulation and tumor accumulation was reduced likely due to a complement-mediated recognition of the targeted microbubble and a decrease in angiogenic vasculature, respectively. In summary, we find that CRPPR MBs specifically bind to NRP-expressing cells and provide an effective new agent for molecular imaging of angiogenesis., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

Female 129:Stat1-null mice (129S6/SvEvTac-Stat1(tm1Rds) homozygous) uniquely develop estrogen-receptor (ER)-positive mammary tumors. Herein we report that the mammary glands (MG) of these mice have altered growth and development with abnormal terminal end buds alongside defective branching morphogenesis and ductal elongation. We also find that the 129:Stat1-null mammary fat pad (MFP) fails to sustain the growth of 129S6/SvEv wild-type and Stat1-null epithelium. These abnormalities are partially reversed by elevated serum progesterone and prolactin whereas transplantation of wild-type bone marrow into 129:Stat1-null mice does not reverse the MG developmental defects. Medium conditioned by 129:Stat1-null epithelium-cleared MFP does not stimulate epithelial proliferation, whereas it is stimulated by medium conditioned by epithelium-cleared MFP from either wild-type or 129:Stat1-null females having elevated progesterone and prolactin. Microarrays and multiplexed cytokine assays reveal that the MG of 129:Stat1-null mice has lower levels of growth factors that have been implicated in normal MG growth and development. Transplanted 129:Stat1-null tumors and their isolated cells also grow slower in 129:Stat1-null MG compared to wild-type recipient MG. These studies demonstrate that growth of normal and neoplastic 129:Stat1-null epithelium is dependent on the hormonal milieu and on factors from the mammary stroma such as cytokines. While the individual or combined effects of these factors remains to be resolved, our data supports the role of STAT1 in maintaining a tumor-suppressive MG microenvironment.

Under magnetic resonance (MR) guidance, high intensity focused ultrasound (HIFU) is capable of precise and accurate delivery of thermal dose to tissues. Given the excellent soft tissue imaging capabilities of MRI, but the lack of data on the correlation of MRI findings to histology following HIFU, we sought to examine tumor response to HIFU ablation to determine whether there was a correlation between histological findings and common MR imaging protocols in the assessment of the extent of thermal damage. Female FVB mice (n = 34), bearing bilateral neu deletion tumors, were unilaterally insonated under MR guidance, with the contralateral tumor as a control. Between one and five spots (focal size 0.5 × 0.5 × 2.5 mm3) were insonated per tumor with each spot receiving approximately 74.2 J of acoustic energy over a period of 7 seconds. Animals were then imaged on a 7T MR scanner with several protocols. T1 weighted images (with and without gadolinium contrast) were collected in addition to a series of T2 weighted and diffusion weighted images (for later reconstruction into T2 and apparent diffusion coefficient maps), immediately following ablation and at 6, 24, and 48 hours post treatment. Animals were sacrificed at each time point and both insonated/treated and contralateral tumors removed and stained for NADH-diaphorase, caspase 3, or with hematoxylin and eosin (H&E). We found the area of non-enhancement on contrast enhanced T1 weighted imaging immediately post ablation correlated with the region of tissue receiving a thermal dose CEM43 ≥ 240 min. Moreover, while both tumor T2 and apparent diffusion coefficient values changed from pre-ablation values, contrast enhanced T1 weighted images appeared to be more senstive to changes in tissue viability following HIFU ablation.

Microwave methods of fixation can dramatically shorten fixation times while preserving tissue structure; however, it remains unclear if adequate tissue antigenicity is preserved. To assess and validate antigenicity, robust quantitative methods and animal disease models are needed. We used two mouse mammary models of human breast cancer to evaluate microwave-assisted and standard 24-hr formalin fixation. The mouse models expressed four antigens prognostic for breast cancer outcome: estrogen receptor, progesterone receptor, Ki67, and human epidermal growth factor receptor 2. Using pathologist evaluation and novel methods of quantitative image analysis, we measured and compared the quality of antigen preservation, percentage of positive cells, and line plots of cell intensity. Visual evaluations by pathologists established that the amounts and patterns of staining were similar in tissues fixed by the different methods. The results of the quantitative image analysis provided a fine-grained evaluation, demonstrating that tissue antigenicity is preserved in tissues fixed using microwave methods. Evaluation of the results demonstrated that a 1-hr, 150-W fixation is better than a 45-min, 150-W fixation followed by a 15-min, 650-W fixation. The results demonstrated that microwave-assisted formalin fixation can standardize fixation times to 1 hr and produce immunohistochemistry that is in every way commensurate with longer conventional fixation methods.

Osteopontin (OPN) Spp1 is involved in differentiation of the mammary gland. We engineered mice to overexpress OPN in mammary epithelium and describe an altered mammary phenotype. Three transgenic (Tg) founder lines FVB/N Tg(MMTV-Opn)((1-3BOR)) were propagated after FVB/NJ pronuclear injections. Mammary glands from Tg-OPN mice compared to littermate controls showed, at 4 weeks of age, exaggerated terminal end buds; at 8 and 12 weeks, more numerous and complex ducts with increased luminal protein; and at 16 weeks, increased lobulogenesis. Lactational Tg-OPN mammary glands showed more rapid lobulogenesis and lactational changes with slower gland involution and regression following weaning. Ex vivo lobulogenesis was noticeably increased from organoids of Tg-OPN mice. Immunohistochemistry revealed cytoplasmic OPN accumulation and increased Ki-67 positive mammary epithelial cells in Tg-OPN mammary glands. OPN appears to convey a proliferative stimulus for mammary epithelial cells and alters development and differentiation. These OPN mammary overexpressing mice provide a means to study the role of OPN in cancer progression.

Quantitative Image Analysis (QIA) of digitized whole slide images for morphometric parameters and immunohistochemistry of breast cancer antigens was used to evaluate the technical reproducibility, biological variability, and intratumoral heterogeneity in three transplantable mouse mammary tumor models of human breast cancer. The relative preservation of structure and immunogenicity of the three mouse models and three human breast cancers was also compared when fixed with representatives of four distinct classes of fixatives. The three mouse mammary tumor cell models were an ER+/PR+ model (SSM2), a Her2+ model (NDL), and a triple negative model (MET1). The four breast cancer antigens were ER, PR, Her2, and Ki67. The fixatives included examples of (1) strong cross-linkers, (2) weak cross-linkers, (3) coagulants, and (4) combination fixatives. Each parameter was quantitatively analyzed using modified Aperio Technologies ImageScope algorithms. Careful pre-analytical adjustments to the algorithms were required to provide accurate results. The QIA permitted rigorous statistical analysis of results and grading by rank order. The analyses suggested excellent technical reproducibility and confirmed biological heterogeneity within each tumor. The strong cross-linker fixatives, such as formalin, consistently ranked higher than weak cross-linker, coagulant and combination fixatives in both the morphometric and immunohistochemical parameters.

Tp53 mutations are common in human prostate cancer (CaP), occurring with a frequency of ∼30% and ∼70% in localized and metastatic disease, respectively. In vitro studies have determined several common mutations of Tp53 that have specific gain-of-function properties in addition to loss of function, including the ability to promote castration-resistant (CR) growth of CaP cells in some contexts. To date, a lack of suitable mouse models has prohibited investigation of the role played by Tp53 mutations in mediating CaP progression in vivo. Here, we describe the effects of conditional expression of a mutant Tp53 (Tp53(R270H); equivalent to the human hotspot mutant R273H) in the prostate epithelium of mice. Heterozygous "Tp53(LSL-R270H/+)" [129S4(Trp53(tm3Tyj))] and "Nkx3.1-Cre" [129S(Nkx3-1(tm3(cre)Mms))] mice with prostate-specific expression of the Tp53(R270H) mutation (p53(R270H/+) Nkx3.1-Cre mice) were bred onto an FVB/N background via speed congenesis to produce strain FVB.129S4(Trp53(tm3Tyj/wt)); FVB.129S(Nkx3-1(tm3(cre)Mms/wt)) and littermate genotype negative control mice. These mutant mice had significantly increased incidences of prostatic intraepithelial neoplasia (PIN) lesions, and these appeared earlier, compared with the Nkx3.1 haploinsufficient (Nkx3.1-Cre het) littermate mice, which did not express the Tp53 mutation. PIN lesions in these mice showed consistent progression and some developed into invasive adenocarcinoma with a high grade, sarcomatoid or epithelial-mesenchymal transition (EMT) phenotype. PIN lesions were similar to those seen in PTEN conditional knockout mice, with evidence of AKT activation concomitant with neoplastic proliferation. However, the invasive tumor phenotype is rarely seen in previously described mouse models of prostatic neoplasia. These data indicate that the Tp53(R270H) mutation plays a role in CaP initiation. This finding has not previously been reported. Further characterization of this model, particularly in a setting of androgen deprivation, should allow further insight into the mechanisms by which the Tp53(R270H) mutation mediates CaP progression.

Studies with animal models in vivo as well as with animal and human tumor cells in vitro suggest that specific fatty acids could reduce breast tumorigenesis. The most striking dietary fatty acid studies in animal models that show promise for reduction of breast cancer risk in humans are with conjugated linoleic acids (CLA) and n-3 fatty acids. Although a number of mechanisms have been proposed, the specific target of those fatty acids is not yet known. We sought to determine whether the effects of those fatty acids on terminally differentiated tumor cell seen could be due to alteration of breast cancer stem cells. The isomers, cis9, trans11-CLA and trans10, cis12-CLA, and the n-3 fatty acids, docosahexaenoic and eicosapentaenoic, reduced the proliferation of, and had increased toxicity towards, mammary tumor initiating cells. One mechanism involved in the effect of n-3 fatty acids may be due to alteration of the profile of prostaglandins. These results indicate that select fatty acids may be useful for preventing or reducing the risk of breast cancer as they may target the tumor initiating cell., (Copyright 2010 Elsevier Ltd. All rights reserved.)

A healthy mucosal immune system prevents numerous diseases whether they are caused by pathogens or faulty tolerance to non-pathogenic antigens. Some methods for assessing immune responses have not changed for decades but have been applied in conjunction with new strategies. New methods have been developed recently that improve on existing mouse models and allow for assessment of cellular and molecular pathways that are involved in mucosal immune responses. Reviewed here are components of the mucosal immune system with attention paid to the gut-associated lymphoid tissue and some of the new methods for assessing immune responses.

We reviewed the literature regarding the effects of conjugated linoleic acid (CLA) preparations enriched in specific isomers, cis9, trans11-CLA (c9, t11-CLA) or trans10, cis12-CLA (t10, c12-CLA), on tumorigenesis in vivo and growth of tumor cell lines in vitro. We also examined the potential mechanisms by which CLA isomers may alter the incidence of cancer. We found no published reports that examined the effects of purified CLA isomers on human cancer in vivo. Incidence of rat mammary tumors induced by methylnitrosourea was decreased by c9, t11-CLA in all studies and by t10, c12-CLA in just a few that included it. Those 2 isomers decreased the incidence of forestomach tumors induced by benzo (a) pyrene in mice. Both isomers reduced breast and forestomach tumorigenesis. The c9, t11-CLA isomer did not affect the development of spontaneous tumors of the intestine or mammary gland, whereas t10, c12-CLA increased development of genetically induced mammary and intestinal tumors. In vitro, t10, c12-CLA inhibited the growth of mammary, colon, colorectal, gastric, prostate, and hepatoma cell lines. These 2 CLA isomers may regulate tumor growth through different mechanisms, because they have markedly different effects on lipid metabolism and regulation of oncogenes. In addition, c9, t11-CLA inhibited the cyclooxygenase-2 pathway and t10, c12-CLA inhibited the lipooxygenase pathway. The t10, c12-CLA isomer induced the expression of apoptotic genes, whereas c9, t11-CLA did not increase apoptosis in most of the studies that assessed it. Several minor isomers including t9, t11-CLA; c11, t13-CLA; c9, c11-CLA; and t7, c11-CLA were more effective than c9, t11-CLA or t10, c12-CLA in inhibiting cell growth in vitro. Additional studies with purified isomers are needed to establish the health benefit and risk ratios of each isomer in humans.

Conjugated linoleic acid (CLA) is a group of linoleic acid derivatives that has been implicated in animal studies to reduce a number of components of mammary tumorigenesis. Previously, we showed that CLA could alter the latency and metastasis of the highly metastatic transplantable line 4526 mouse mammary tumor. Several possible mechanisms have been proposed for the actions of CLA, but here we assessed how CLA may act to alter the expression and activity of matrix-modifying proteins within tumors from line 4526. In vitro, highly metastatic mouse mammary tumor cells had significantly decreased invasiveness after treatment with CLA, an indication that matrix-modifying proteins may have been altered. Using these same highly metastatic cells, primary tumors were grown in mice of separate groups fed 0, 0.1, 0.5, and 1% CLA (wt:wt) and evaluated for their levels and activities of matrix-modifying enzymes, enzyme inhibitors, and enzyme activators. The addition of CLA to the diet increased steady-state levels of messenger RNA (mRNA) of the matrix metalloproteinases (MMP) -2 and -9 in primary tumors removed from mice. However, western analysis revealed that although relative levels of the proform of MMP-9 were consistent with the mRNA observations, MMP-2 proform levels were actually decreased by dietary CLA. The activity of MMP-2 was barely detectable, but gelatin zymography and an in vitro activity assay showed that MMP-9 activity was significantly decreased by CLA. The steady-state mRNA and protein levels of tissue inhibitors of metalloproteinase-1 (TIMP-1) and TIMP-2, natural inhibitors of MMP, were increased at higher dietary CLA levels relative to low or no CLA. Suppression of MMP activity, therefore, may be 1 pathway through which CLA reduces tumor invasion and spread.

Background: Chemotactic cytokines, referred to as chemokines, play an important role in leukocyte trafficking. The circulating levels of chemokines have been shown to increase in inflammatory processes including obesity-related pathologies (e.g. atherosclerosis and diabetes). However, little is currently known about the relationship between chemokines and human obesity. In the present study, we investigated the circulating levels of selected chemokines (monocyte chemoattractant protein-1 (MCP-1), macrophage inflammatory protein-1alpha (MIP-1alpha), leukotactin-1, interleukin-8 (IL-8)) and the association between the chemokine levels and obesity-related parameters: body mass index (BMI), waist circumference, fasting glucose and insulin levels, lipids profile, and the level of C-reactive protein (CRP)., Methods: A total of 100 subjects, 50 obese (BMI>or=25 kg/m2) and 50 who were not obese (BMI<25 kg/m2) participated in the present study. The levels of chemokines and CRP were measured in a fasting state serum by sandwich enzyme-linked immunosorbent assay. Total cholesterol, high-density lipoprotein (HDL)-cholesterol, triglyceride, glucose, and insulin levels were measured by enzymatic analysis and immunoassay., Results: The circulating levels of MCP-1 and IL-8 in the serum were significantly (P<0.05) higher in obese subjects (BMI>30 kg/m2) compared with those of nonobese controls (BMI<25 kg/m2). The levels of CRP were positively correlated with BMI (P<0.001) or waist circumference (P<0.0001). The levels of MCP-1 and IL-8 were positively related to BMI (MCP-1, P<0.02; IL-8, P<0.01) and/or waist circumference (MCP-1, P<0.009; IL-8, P<0.03). The levels of MCP-1 were positively related to the levels of CRP (P<0.007) or interleukin-6 (IL-6) (P<0.0001), and negatively related to the levels of HDL-cholesterol (P<0.01). Homeostasis model assessment (HOMA) score was positively related to the levels of MCP-1 (P<0.02) or IL-8 (P<0.03) in obese subject., Discussion: Our data demonstrated that the circulating levels of MCP-1 and IL-8 are related to obesity-related parameters such as BMI, waist circumference, CRP, IL-6, HOMA and HDL-cholesterol. These findings suggest that the circulating MCP-1 and/or IL-8 may be a potential candidate linking obesity with obesity-related metabolic complications such as atherosclerosis and diabetes.

Animal studies consistently show that dietary conjugated linoleic acid (CLA) reduces mammary tumorigenesis including metastasis. Relatively low concentrations of CLA are required for those effects, and a threshold level exists above which there is no added reduction. We reasoned that the concentration of CLA required to effectively alter mammary tumor metastasis may be dependent on the type of dietary fat because select fatty acids can enhance or suppress normal or malignant cell growth and metastasis. For this study, the diets (a total of 12 different groups) differed in fatty acid composition but not in energy from fat (40%). In experiments involving spontaneous metastasis, mice were fed for 11 wk; in experiments in which mice were injected i.v. with tumor cells, they were fed for 7 wk. Mice were then assessed for the effect of CLA concentration on mammary tumorigenesis. Mammary tumor growth was not altered, but metastasis was significantly decreased when beef tallow (BT) replaced half of a defined vegetable fat blend (VFB). That blend reflects the typical fat content of a Western diet. In addition, that same VFB:BT diet lowered the concentration of CLA required to significantly decrease mammary tumor metastasis from 0.1% of the diet to 0.05%. A diet in which corn oil replaced half of the VFB did not lower the threshold from 0.1 to 0.05%. In vitro, the main fatty acid in vegetable oil, linoleic acid, reduced the efficacy of CLA toxicity on mammary tumor cells in culture. Alternatively, fatty acids normally found in BT, such as oleic, stearic, and palmitic acids, either did not change or enhanced the cytolytic effects of CLA isomers on mouse mammary tumor cells in culture. These data provide evidence that dietary BT, itself with negligible levels of CLA, may increase the efficacy of dietary CLA in reducing mammary tumorigenesis.

Conjugated linoleic acid (CLA) consists of a group of linoleic acid geometric isomers that have been shown to reduce tumor growth and metastasis in animal models of breast, prostate and colon cancer. To delineate a possible mechanism of action for CLA, we have recently shown that the 5-lipoxygenase product, 5-hydroxyeicosatetraenoic acid (5-HETE), could play a role in CLA alteration of mammary tumorigenesis. In this study, we determined how CLA could modulate 5-lipoxygenase activity. The t10, c12-CLA isomer reduced production of 5-HETE but not 12- and 15-HETE in MDA-MB-231 human breast tumor cells. That isomer and the c9, t11-CLA isomer decreased 5-HETE production by competition with the lipoxygenase substrate, arachidonic acid (AA). Interestingly, t10, c12-CLA reduced the expression of five-lipoxygenase activating protein (FLAP) but not the 5-lipoxygenase enzyme. Over-expression of FLAP abrogated t10, c12-CLA-reduced viability of MDA-MB-231 cells. These data suggest that the reduction of 5-HETE by t10, c12-CLA was due to competition with AA and the reduction of FLAP expression.

Our understanding of angiogenesis has increased significantly in the past few years with the discovery of angiopoietins (Ang). Specifically, Ang2 has been associated with pathologic as well as normal vascularization. While previous studies have shown that a major source of Ang2 has been endothelial cells and tumor cells, we reasoned that macrophages would also have the ability to express angiopoietins, specifically Ang2, due to that cell's role in wound healing, tumor angiogenesis, and a number of non-oncological diseases, such as rheumatoid arthritis and psoriasis. In this study, murine macrophages constitutively expressed both transcripts and protein for Ang2 but not Ang1 or Ang3. The secretion of Ang2 was enhanced by treatment with lipopolysaccharide, interferon-gamma, prostaglandin E2 and other cyclic AMP-elevating agents, as well as vascular endothelial growth factor (VEGF). Cyclic AMP-dependent protein kinase (PKA) played a major role in this enhancement since the PKA inhibitor, H89, blocked secretion of Ang2. Since stimulation of the PKA pathway can lead to macrophage production of VEGF, it is possible that enhancement of Ang2 production by macrophages may be due to autocrine responsiveness to VEGF. Adding anti-VEGF antibodies to the supernatants of stimulated macrophages blocked secretion of Ang2. This study is the first to show murine macrophage production of Ang2 and to provide evidence that it can be regulated. Understanding the regulation of macrophage Ang2 production is especially important in an effort to target the pathologic role of macrophages while preserving their role in immunity and homeostasis.

Conjugated linoleic acid (CLA) is a dietary fatty acid that has been shown to reduce tumorigenesis and metastasis in breast, prostate and colon cancer in animals. However, the mechanism of its action has not been clarified. The goal of this study was to determine whether CLA altered mouse mammary tumor cell growth and whether specific metabolites of the lipoxygenase pathway were involved in CLA action. Both t10, c12-CLA and a lipoxygenase inhibitor, but not c9, t11-CLA or linoleic acid (LA), reduced mouse mammary tumor cell viability and growth by inducing apoptosis and reducing cell proliferation. t10, c12-CLA reduced the production of the 5-lipoxygenase metabolite, 5-hydroxyeicosatetraenoic acid (5-HETE). That effect was not seen with c9, t11-CLA or LA. Adding 5-HETE back to tumor cells reduced the t10, c12-CLA effect on both apoptosis and cell proliferation. These data suggest that t10, c12-CLA reduction of tumor cell growth may involve the suppression of the 5-lipoxygenase metabolite, 5-HETE, with subsequent effects on apoptosis and cell proliferation.

Introduction: Women who smoke have a higher rate of fatal breast cancer than nonsmoking women. An association between smoking and pulmonary metastases from breast cancer has been suggested by epidemiologic studies., Study Objectives: To examine the relationship between exposure to cigarette smoke and pulmonary metastasis in a murine model of metastatic mammary cancer., Study Design: Prospective, randomized study., Setting: Animal research laboratory., Experimental Subjects: Female sexually mature BALB/cAnN mice., Interventions: Mice were randomly divided into experimental and control groups. Experimental animals were exposed to cigarette smoke in specialized exposure chambers, at concentrations chosen to approximate active cigarette smoking. Control animals were exposed to filtered air. One week after the initiation of exposures, mouse mammary tumor cells (tumor cell line 4526) were injected into the tail veins of experimental animals at one of three concentrations (50,000, 100,000, or 150,000 cells per 100 micro L). Three weeks later, the mice were killed, and pulmonary metastases were counted and measured., Results: The mean metastatic burden in the lungs was consistently greater for smoke-exposed animals at each concentration of cells injected (at 50,000 cells per 100 micro L, 9.8 vs 4.8 micro m(3), respectively [p < 0.01]; at 100,000 cells per 100 micro L, 34.5 vs 17.4 micro m(3), respectively [p < 0.10]; and at 150,000 cells per 100 micro L, 54.0 vs 31.5 micro m(3), respectively [p < 0.05]). This was largely attributable to a significant increase in the number of metastatic nodules per animal (at 50,000 cells per 100 micro L, 8.7 vs 4.8, respectively [p < 0.001]; at 100,000 cells per 100 micro L, 24.3 vs 14.0, respectively [p > 0.10]; and at 150,000 cells per 100 micro L, 42.0 vs 20.1, respectively [p < 0.02]) rather than to a change in nodule size., Conclusions: Cigarette smoke exposure is associated with an increase in the total pulmonary metastatic burden in this murine model of metastatic mammary cell cancer. This study provides experimental support for an adverse effect of smoking on the metastatic process and suggests a possible mechanism for smokers' increased breast cancer mortality.

Recent studies have linked conjugated linoleic acid (CLA) to altered tumorigenesis of several sites. We showed recently that a mixture of CLA isomers was able to significantly decrease mammary tumor metastasis in mice. That effect was seen with as little as 0.1% CLA in the diet. Other studies with dietary CLA have shown that various isomers may have differential effects. The purpose of this work was to assess which individual CLA isomers had similar effects in alteration of mouse mammary tumor metastasis. For that, we fed six 20% (w/w) total fat diets which contained either no CLA, low (0.1%, w/w) or high (0.25%, w/w) levels of cis9,trans11-CLA (c9,t11), trans10,cis12-CLA (t10,c12) or a mixture of the 2 isomers (0.125% of each, w/w) as free fatty acids. Neither the separate isomers nor the mixture had an effect on the latency or growth of primary line 4526 tumors when compared to the group without CLA. However, all diets containing CLA significantly decreased the total tumor burden (volume of tumor, mm(3)) in lungs of mice from both spontaneous metastasis (reduced by 42-73%) as well as implantation and survival of the metastatic cell (reduced by 46-61%) when compared with diets containing no CLA. Diets containing a greater concentration of either c9,t11 or t10,c12 had a significantly greater effect compared to the lower concentrations of the respective isomers when metastatic nodule size and total tumor burden were assessed. The diet containing both isomers decreased total tumor burden similarly to the diets containing the lower concentration of each of the isomers. Thus, the effects of c9,t11 and t10,c12 may not be additive and possibly share similar mechanisms for decreasing metastatic tumor burden in mice transplanted with mammary tumor cells.

Assessing the regulation of macrophage receptors for prostaglandin (PGE2) is essential to understanding the control which that potent lipid mediator has in modulating macrophage activities. The purpose of this study was to assess the differential mRNA expression of PGE2 receptor subtypes (EP) during macrophage exposure to activating and transducing agents. RAW 264.7 macrophages constitutively expressed mRNA for EP2,EP3 and EP4 receptor subtypes. Messenger RNA for EP4 was expressed at a much higher level when compared to EP2 in unstimulated macrophages as assessed by kinetic quantitative RT-PCR. When macrophages were stimulated with LPS, EP2 m RNA levels were 12-fold higher when compared to unstimulated macrophages, while EP4 m RNA remained unchanged. Conversely, mRNA levels of both EP2 and EP4 receptors were lower after macrophages were treated with IFN-gamma. Messenger RNA levels of both receptors were lower in macrophages after treatment with PGE2 or dibutyryl (db) cAMP Addition of the PKA inhibitor H89 reversed the effects of PGE2 and dbcAMP to varying degrees. Proteosome and p38 MAP kinase inhibitors blocked the LPS-stimulated increase in EP2 mRNA levels. Those inhibitors had no effect on EP4 mRNA.Thus, activating agents such as LPS and IFN-gamma may differentially regulate mRNAfor PGE2 receptor types in macrophages but the ligand and its associated signal transducing factors probably have similar regulatory effects.

We have previously shown that dietary (n-3) fatty acids decrease mammary tumor vascularization and PGE(2) production. One possible mechanism may be the modulation of vascular endothelial growth factor (VEGF) production by PGE(2). Macrophages are major producers of VEGF, and thus we assessed the role of PGE(2) in vitro and in vivo on their VEGF production. When added to macrophages, pharmacological (10(-7) M) but not physiological (10(-9) to 10(-11) M) concentrations of PGE(2) increased VEGF mRNA and protein levels. That increased expression was relatively rapid and sustained up to 8 hrs, but declined by 24 hrs. Similarly, dibutryl cAMP increased production of VEGF protein which was completely inhibited by H89. Addition of cAMP-elevating agents further potentiated the production of VEGF by PGE(2). Next, (n-3) and (n-6) fatty acids were added to macrophages in vitro or provided in the diet. Macrophages of mice fed safflower oil (n-6) had 2- to 4-fold greater copy number of VEGF transcripts after lipopolysaccarhide (LPS) stimulation compared to fish oil (n-3). A decreasing trend was seen in LPS-induced VEGF secretion from macrophages in vitro after docosahexaenoic acid or eicosapentaenoic acid incubation compared to arachidonic acid. While pharmacological concentrations of PGE(2) modulate VEGF expression, physiological alterations did not alter VEGF protein production by macrophages., (Copyright 2001 Harcourt Publishers Ltd.)

Micronutrients such as zinc, selenium, iron, copper, beta-carotene, vitamins A, C, and E, and folic acid can influence several components of innate immunity. Select micronutrients play an important role in alteration of oxidant-mediated tissue injury, and phagocytic cells produce reactive oxidants as part of the defense against infectious agents. Thus, adequate micronutrients are required to prevent damage of cells participating in innate immunity. Deficiencies in zinc and vitamins A and D may reduce natural killer cell function, whereas supplemental zinc or vitamin C may enhance their activity. The specific effects of micronutrients on neutrophil functions are not clear. Select micronutrients may play a role in innate immunity associated with some disease processes. Future studies should focus on issues such as age-related micronutrient status and innate immunity, alterations of micronutrients in disease states and their effect on innate immunity, and the mechanisms by which micronutrients alter innate immunity.

Recent studies have shown that conjugated linoleic acid (CLA) can inhibit the initiation and thus, incidence of mammary tumors in rodents. The concentration of CLA required for these effects was as low as 0.1% of the diet, with no increased effects above 1%. To date, there is little evidence that CLA has any effect on growth or metastasis of mammary tumors. In this report, we demonstrate that CLA, at the concentrations used in previous studies, had a significant effect on the latency, metastasis, and pulmonary tumor burden of transplantable murine mammary tumors grown in mice fed 20% fat diets. The latency of tumors from mice fed CLA was significantly increased when compared with the 0% CLA control diet. The volume of pulmonary tumor burden, as a result of spontaneous metastasis, decreased proportionately with increasing concentrations of dietary CLA. With 0.5 and 1% CLA, pulmonary tumor burden was significantly decreased compared to mice treated with the eicosanoid inhibitor, indomethacin and fed diets containing no CLA. Tumors of mice fed as little as 0.1% CLA and as much as 1% had significantly decreased numbers of pulmonary nodules when compared with diets containing no CLA. The decrease in the number of pulmonary nodules by CLA was nearly as effective as indomethacin, a known suppressor of tumor growth and metastasis in this malignant model. These data suggest that effects of CLA on mammary tumorigenesis may go beyond the reported alterations in tumor incidence and effect later stages, especially metastasis.

We have previously shown that mice fed a high (n-3) fatty acid-containing diet with 20% (w/w) total fat had significantly slower mammary tumor growth, decreased numbers of metastatic pulmonary nodules, and decreased total metastatic load. In this study we sought to determine whether tumor vascularization was altered in mice fed diets varying in concentrations of (n-3) and (n-6) fatty acids. Several direct or indirect parameters of vascularization were tested. With 20% dietary fat, fish oil (FO) or a mixture of FO and safflower oil (FS) significantly reduced blood vascular area, mast cell number and macrophage infiltration in solid mammary tumors compared to tumors grown in mice fed safflower oil (SO). A decreasing trend was seen in the percent area of vessels positive for CD31 and vascular endothelial growth factor (VEGF) in the 20% FO and 20% FS compared to the 20% SO dietary groups. VEGF concentrations were twice as high in smaller tumors (100 mm3) from all dietary groups as compared to larger tumors (500 mm3). A two-fold increase in VEGF levels was found in the 20% SO dietary group compared to the 20% FO group in 100-mm3 but not larger tumors. We conclude that at 20% total fat, the n-3 fatty acids found in fish oil may inhibit primary mammary tumor growth through modulation of select determinants of vascularization.

Probiotics, microorganisms that have a favorable influence on physiologic and pathological processes of the host by their effect on the intestinal flora, may play a role in improving human health. One of the putative effects is the modulation of immune function. Thus, the mucosal immune system and methods to assess its function are reviewed briefly. Probiotic modulation of humoral, cellular and nonspecific immunity is reviewed, with emphasis placed on immune response in disease models. There are very few reports of human intervention studies with probiotics. However, some of the possible future directions for research with respect to probiotics, immunity, and human health are discussed. Although the application of probiotics has demonstrated trends with respect to altered aspects of immune response, the underlying mechanisms by which that occurs are unclear.

Background: Depression of myocardial contractility occurs in septic shock., Methods: Fourteen pigs were instrumented to measure cardiopulmonary dynamics after a challenge of Escherichia coli endotoxin (lipopolysaccharide endotoxin, LPS). A volumetric Swan-Ganz catheter was placed via the jugular vein, and a carotid arterial line was placed into the aortic root. Eight pigs received LPS alone and six pigs received tumor necrosis factor monoclonal antibody (TNF MAb) 15 minutes before the administration of LPS. Pulmonary artery and aortic root blood were sampled for amounts of TNF. Ninety minutes after LPS administration, thoracotomy was performed to biopsy the right and left ventricles for TNF levels. Contractility was determined from the end systolic pressure-volume relationships of pressure-volume diagrams., Results: Right ventricular end diastolic volume index nearly doubled and myocardial contractility decreased by 40% from baseline in the pigs receiving only LPS. Pigs that received TNF MAb had no change in myocardial contractility or right ventricular end diastolic volume index from baseline. There was a higher level of TNF in the aortic sample than in the pulmonary samples at 60 minutes. Right ventricular tissue TNF levels were significantly higher in the LPS-alone group. There was no such difference in left ventricular tissue., Conclusion: The left and right ventricles have different susceptibilities to TNF MAb. TNF may decrease myocardial contractility in sepsis. Blockade of TNF with TNF MAb reverses the depression of myocardial contractility and the right ventricular dilatation associated with septic shock.

The immunosuppressive effect of portal venous blood transfusions in organ transplantation has been well established and may be mediated by increased Kupffer cell production of the immunosuppressive arachidonic acid metabolite prostaglandin E2 (PGE2). In this study, butyrate, a short-chain fatty acid known to enhance gene transcription, is hypothesized to enhance Kupffer cell PGE2 production by altering cyclooxygenase or phospholipase A2 (PLA2) activity, thus augmenting the immunosuppressive effect of portal venous transfusion. Lewis rats were given a portal venous transfusion of Wistar-Firth blood or saline 1 h prior to Kupffer cell harvest. The in vitro effects of butyrate on Kupffer cell PGE2 production, cyclooxygenase, and PLA2 activity were assessed. Kupffer cell tumor necrosis factor-alpha (TNFalpha) production was also assessed due to its sensitivity to PGE2 and its proinflamatory effects. Kupffer cells from portally transfused animals produced significantly more PGE2 than saline-transfused controls. Addition of butyrate to the culture medium further increased PGE2 production by as much as sevenfold in Kupffer cells of portally transfused animals. Other short-chain fatty acids, propionate and hexanoate, did not increase PGE2 production. Butyrate added to Kupffer cells from transfused animals slightly upregulated inducible cyclooxygenase (COX-2) mRNA levels as measured by both Northern blot and reverse-transcriptase polymerase chain reaction and increased PLA2 activity fivefold as measured by Western blot. Kupffer cell immune function was also affected by in vitro butyrate treatment with a significant decrease in the production of TNFalpha. Thus, butyrate may be a useful immunoregulatory agent in organ transplantation protocols which seek to enhance transcription of immunosuppressive molecules., (Copyright 1998 Academic Press.)

Background: Enhanced Kupffer cell production of the immunosuppressive arachidonic acid metabolite prostaglandin E2 (PGE2) has been shown to be a mechanism of the immunosuppressive effect of portal venous transfusions (PVT). Butyrate, a four-carbon short-chain fatty acid, has received increased attention because of its ability to enhance gene transcription. This study tested the hypothesis that the intrahepatic delivery of butyrate enhances Kupffer cell PGE2 production and thus augments the immunosuppressive effect of PVT., Methods: Butyrate was incorporated into liposomes and administered intravenously to Lewis rats. Control rats were administered liposomes without butyrate. Twenty-four hours after liposome injection, rats were administered a PVT of 1 ml of Wistar-Furth blood. Kupffer cells were isolated, and PGE2 and tumor necrosis factor-alpha levels were measured in the culture medium after 24 hr. Additionally, Kupffer cells from butyrate-treated and control animals were added to one-way mixed lymphocyte reaction cultures., Results: Intrahepatic delivery of butyrate via liposomes increased Kupffer cell PGE2 (3800+/-1220 vs. 1010+/-119 pg/ml, P<0.05) and decreased tumor necrosis factor-alpha (1670+/-81 vs. 3360+/-415 pg/ml, P<0.01) production as compared with controls. Butyrate also augmented the Kupffer cell-mediated immunosuppression as demonstrated by significant depression of the mixed lymphocyte reaction (690+/-119 vs. 3850+/-148 cpm, P<0.01)., Conclusion: The results support the hypothesis that intrahepatic delivery of butyrate enhances Kupffer cell PGE2 production, and specific targeting of Kupffer cells with liposomes containing immunomodulating agents such as butyrate may be a useful means of augmenting immunosuppression protocols in organ transplantation.

We and others have previously shown that dietary fat can alter the growth and metastasis of rodent mammary tumors. Few transplantable tumor models have been used to study the effects of dietary n-6 versus n-3 fatty acids on mammary tumorigenesis. Here we study the effects of fish oil and safflower oil on the growth and metastasis of an animal model that in several ways parallels the human disease. Tumor latency, growth and metastasis were studied in mice fed diets that contained either 10 or 20% total fat which was varied in the type of fat with either menhaden fish oil (FO), safflower oil (SO) or a 50/50 mixture of the two. Tumor latency was significantly longer and tumor growth was significantly slower in mice fed the 20% FO diet. When spontaneous metastasis was assessed, mice fed diets containing FO had significantly decreased numbers of pulmonary nodules and total metastatic load. Likewise, mice fed FO diets had a lower level of implantation and survival of pulmonary metastases. Thus, in our animal model, diets containing n-3 fatty acids in fish oil significantly decrease primary breast tumor growth and its metastasis.

Expression of major histocompatibility complex class II molecules, Ia, can be significantly augmented by interferon-gamma (IFN-gamma) in macrophages. In this study we demonstrate that platelet-activating factor (PAF) was also a potent inducer of Ia antigen expression on macrophages. PAF-induced Ia expression was both time- and dose-dependent. Maximal Ia expression was induced with 25 nM PAF after 3-h exposure to PAF. Ia expression in macrophages stimulated with PAF for 24 h was not significantly greater than unstimulated macrophages. Treatment of macrophages with IFN-gamma and PAF did not affect either the kinetics or concentration required for maximal Ia expression induced by either IFN-gamma or PAF. PAF-induced Ia expression was inhibited by the specific PAF receptor antagonists, WEB 2086, Ro 24-0238, and Ro 24-4637, indicating a receptor-mediated event. Like IFN-gamma-induced Ia expression, PAF activity was inhibited by prostaglandin E2 (PGE2). However, that expression was only inhibited after 24 h when macrophages were treated with the PGE2 synthesis inhibitors, flurbiprofen and indomethacin. These findings demonstrate that PAF, along with its role as a potent proinflammatory mediator, was also capable of inducing Ia expression on macrophages through the PAF receptor and that expression was altered by PGE2.

Portal venous transfusions (PVTs) of blood have been shown to induce significant immunosuppression in animal models of organ transplantation. A proposed mechanism of PVT-induced immunosuppression is via alteration of Kupffer cell arachidonic acid metabolism with increased secretion of the suppressive metabolite prostaglandin E2 (PGE2). This study assessed the hypothesis that PVT increases Kupffer cell PGE2 production via up-regulation of Kupffer cell phospholipase A2 (PLA2) as well as constitutive (COX1) and inducible (COX2) cyclooxygenase. Kupffer cells from Lewis rats were harvested 1 hr after PVT with either 1 ml of Wistar-Furth blood, systemic transfusion (SVT), or saline via portal vein (PVSal). After lipopolysaccharide stimulation, 24-hr Kupffer cell supernatant fractions were assayed for PGE2. PGE2 was increased after SVT (1465+/-234 pg/ml) compared with PVSal (597+/-99; P<0.01). PVT increased Kupffer cell PGE2 (5370+/-533; P<0.001 vs. SVT and vs. PVSal) even more substantially. Kupffer cells from PVT-treated rats were then cultured in the presence of inhibitors of PLA2, COX1, or COX2. When Kupffer cells were treated with mepacrine to inhibit PLA2 (5575+/-453 pg/ml), PGE2 production was not different from that by PVSal-treated controls (6467+/-614 pg/ml), but when Kupffer cells were incubated in the presence of the COX1 inhibitor flurbiprofen (3512+/-407 pg/ml) or the COX2 inhibitor nimesulide (2800+/-830 pg/ml), production was decreased 46.7% and 56.7%, respectively, over control activity without added inhibitor. PVT also increased Kupffer cells COX1 and COX2 mRNA as measured by Northern blot. Heart transplants were then performed from Wistar-Furth donors into Lewis recipients at the time of PVT, SVT, PVSal, or PVT + indomethacin (COX1/2 inhibitor). PVT prolonged allograft survival (12.0+/-0.9 days) compared with PVSal (6.3+/-0.3; P<0.01) or SVT (6.3+/-0.3; P<0.04). Indomethacin shortened graft survival when given with PVT (6.5+/-0.3 days). In summary, PVT increased Kupffer cell PGE2 production, up-regulated transcription of Kupffer cells COX1 and COX2 mRNA, and prolonged cardiac allograft survival. COX1/2 inhibition abrogated the effect of PVT. The results indicated that the immunosuppressive effect of PVT may be mediated by up-regulation of Kupffer cell COX1 and COX2. Manipulation of Kupffer cell arachidonic acid metabolism may be useful in augmentation of PVT-induced immunosuppression.

Diets rich in polyunsaturated n-3 fatty acids can alter various macrophage functions. One possible mechanism by which this occurs is through modulation of the physicochemical properties of the cell membrane and the signal transduction pathways associated with macrophage activation. In this study, we investigated how n-3 fatty acids altered the signaling pathway of the lipid-based mediator platelet-activating factor (PAF). Macrophages from mice fed a diet containing n-3 fatty acids showed a greater increase in PAF-induced intracellular Ca2+ mobilization than macrophages from mice fed an n-6 fatty acid-rich diet. Macrophages treated in vitro with the n-3 fatty acids docosahexaenoic and eicosapentaenoic also showed higher intracellular Ca2+ mobilization than untreated or n-6 fatty acid-treated macrophages. Scatchard analysis of PAF binding showed the presence of one type of PAF receptor; their number and affinities were not altered by dietary fat. Mastoparan, which can activate G-protein-linked phosphoinositide (PI)-signaling pathway through the activation of G proteins, stimulated a higher Ca2+ mobilization in macrophages from mice fed n-3 compared to n-6 fatty acids. In addition, the response of macrophages from n-3-fed mice to PAF was less sensitive to phospholipase C inhibition than that of macrophages from those fed n-6 diets. The activity of phospholipase C in macrophages from mice fed n-3 diets was significantly higher than that of macrophages from mice fed diets containing n-6 fatty acids. Collectively, these results showed that n-3 fatty acids can enhance the PAF-signaling pathway in macrophages by increasing the activation potential of phospholipase C, without affecting PAF receptor number and affinity.

After stimulation with select activating agents such as lipopolysaccharide (LPS) or recombinant interferon-gamma (rIFNgamma), several macrophage proteins may be induced, acylated with myristic acid, or both. Our goal in this study was to determine whether altering the levels of myristic acid in the diet would modulate the levels of a specific acylated macrophage protein, MacMARCKS (myristoylated, alanine-rich C kinase substrate), because that fatty acid can be found in substantial quantities in some foods. Thioglycollate-elicited peritoneal macrophages from groups of mice fed diets with various levels of myristic acid (from 0.2 to 99 g/100 g fatty acids) were treated with LPS, phorbol myristate acetate (PMA), or rIFNgamma plus LPS, which are well-established macrophage activating agents. Levels of MacMARCKS were measured by enzyme-linked immunosorbent assay using a rabbit anti-mouse polyclonal antibody against the first 10 amino acids of murine MacMARCKS. A 42-kDa protein with the same molecular weight as MacMARCKS was identified in macrophage lysates by Western analysis using the antibody. Lipopolysaccharide- and PMA-activated macrophages from mice fed the trimyristin diet had significantly greater levels of MacMARCKS than LPS- and PMA-activated macrophages of mice fed the safflower oil-containing diet. The levels of MacMARCKS were also greater in lysates of LPS plus rIFNgamma-stimulated macrophages from mice fed the trimyristin diet and mice fed a diet containing a moderate level of myristic acid (12 g/100 g fatty acids) compared with the lysates of macrophages from mice fed the safflower oil diet. These results indicate that altering the level of myristic acid in the diet may alter the production of specific proteins that may be involved in macrophage activation.