Characterization of pulmonary responses in mice to asbestos/asbestiform fibers using gene expression profiles.

Humans exposed to asbestos and/or asbestiform fibers are at high risk of developing many lung diseases including asbestosis, lung cancer, and malignant mesothelioma. However, the disease-causing potential and specific metabolic mechanisms and pathways associated with various asbestos/asbestiform fiber exposures triggering different carcinogenic and non-carcinogenic outcomes are still largely unknown. The aim of this this study was to investigate gene expression profiles and inflammatory responses to different asbestos/asbestiform fibers at the acute/sub-acute phase that may be related to delayed pathological outcomes observed at later time points. Mice were exposed to asbestos (crocidolite, tremolite asbestos), asbestiform fibers (erionite), and a low pathogenicity mineral fiber (wollastonite) using oropharyngeal aspiration. Similarities in inflammatory and tissue damage responses, albeit with quantitative differences, were observed at day 1 and 7 post treatment. Exposure to different fibers induced significant changes in regulation and release of a number of inflammatory cytokines/chemokines. Comparative analysis of changes in gene regulation in the lung on day 7 post exposure were interpretable in the context of differential biological responses that were consistent with histopathological findings at days 7 and 56 post treatment. Our results noted differences in the magnitudes of pulmonary responses and gene regulation consistent with pathological alterations induced by exposures to four asbestos/asbestiform fibers examined. Further comparative mechanistic studies linking early responses with the long-term endpoints may be instrumental to understanding triggering mechanisms underlying pulmonary carcinogenesis, that is lung cancer versus mesothelioma.

Mediation of the single-walled carbon nanotubes induced pulmonary fibrogenic response by osteopontin and TGF-ß1.

PURPOSE OF THE STUDY: A number of in vivo studies have shown that pulmonary exposure to carbon nanotubes (CNTs) may lead to an acute local inflammatory response, pulmonary fibrosis, and granulomatous lesions. Among the factors that play direct roles in initiation and progression of fibrotic processes are epithelial-mesenchymal transition and myofibroblasts recruitment/differentiation, both mediated by transforming growth factor-ß1 (TGF-ß1). Yet, other contributors to TGF-ß1 associated signaling, such as osteopontin (OPN) has not been fully investigated. MATERIALS AND METHODS: OPN-knockout female mice (OPN-KO) along with their wild-type (WT) counterparts were exposed to single-walled carbon nanotubes (SWCNT) (40 µg/mouse) via pharyngeal aspiration and fibrotic response was assessed 1, 7, and 28 days post-exposure. Simultaneously, RAW 264.7 and MLE-15 cells were treated with SWCNT (24 hours, 6 µg/cm2 to 48 µg/cm2) or bleomycin (0.1 µg/ml) in the presence of OPN-blocking antibody or isotype control, and TGF-ß1 was measured in supernatants. RESULTS AND CONCLUSIONS: Diminished lactate dehydrogenase activity at all time points, along with less pronounced neutrophil influx 24 h post-exposure, were measured in broncho-alveolar lavage (BAL) of OPN-KO mice compared to WT. Pro-inflammatory cytokine release (IL-6, TNF-α, MCP-1) was reduced. A significant two-fold increase of TGF-ß1 was found in BAL of WT mice at 7 days, while TGF-ß1 levels in OPN-KO animals remained unaltered. Histological examination revealed marked decrease in granuloma formation and less collagen deposition in the lungs of OPN-KO mice compared to WT. RAW 264.7 but not MLE-15 cells exposed to SWCNT and bleomycin had significantly less TGF-ß1 released in the presence of OPN-blocking antibody. We believe that OPN is important in initiating the cellular mechanisms that produce an overall pathological response to SWCNT and it may act upstream of TGF-ß1. Further investigation to understand the mechanistic details of such interactions is critical to predict outcomes of pulmonary exposure to CNT.

Immunobiology and immunosurveillance in patients with intraductal papillary mucinous neoplasms (IPMNs), premalignant precursors of pancreatic adenocarcinomas.

Premalignant lesions for many cancers have been identified, and efforts are currently directed toward identification of antigens expressed on these lesions that would provide suitable targets for vaccines for cancer prevention. Intraductal papillary mucinous neoplasms (IPMNs) are premalignant pancreatic cysts of which a subset has the potential to progress to cancer. Currently, there are no validated predictive markers for progression to malignancy. We hypothesized that the presence or absence of immune surveillance of these lesions would be one such factor. Here we show that the tumor antigen MUC1, which is abnormally expressed on pancreatic cancer and is a target for cancer immunosurveillance, is also abnormally expressed on premalignant IPMN. We show that some IPMN patients make MUC1-specific IgG. Moreover, we show evidence of CD4 and CD8 T cell infiltration into IPMN areas of high dysplasia suggesting an ongoing immune response within the lesions. We also found, however, increased levels of circulating myeloid-derived suppressor cells (MDSCs) and regulatory T cells (Tregs) in some IPMN patients as well as evidence of T cell exhaustion. Further studies correlating immunosurveillance or immunosuppression with IPMN progression to malignancy will help define the immune response as a biomarker of risk, leading potentially to a vaccine to boost spontaneous immunity and prevent progression to cancer.

Gender differences in murine pulmonary responses elicited by cellulose nanocrystals.

BACKGROUND: Cellulose-based materials have been used for centuries to manufacture different goods derived from forestry and agricultural sources. In the growing field of nanocellulose applications, its uniquely engineered properties are instrumental for inventive products coming to competitive markets. Due to their high aspect ratio and stiffness, it is speculated that cellulose nanocrystals (CNC) may cause similar pulmonary toxicity as carbon nanotubes and asbestos, thus posing a potential negative impact on public health and the environment. METHODS: The present study was undertaken to investigate the pulmonary outcomes induced by repeated exposure to respirable CNC. C57BL/6 female and male mice were exposed by pharyngeal aspiration to CNC (40 µg/mouse) 2 times a week for 3 weeks. Several biochemical endpoints and pathophysiological outcomes along with gene expression changes were evaluated and compared in the lungs of male and female mice. RESULTS: Exposure to respirable CNC caused pulmonary inflammation and damage, induced oxidative stress, elevated TGF-ß and collagen levels in lung, and impaired pulmonary functions. Notably, these effects were markedly more pronounced in females compared to male mice. Moreover, sex differences in responses to pulmonary exposure to CNC were also detected at the level of global mRNA expression as well as in inflammatory cytokine/chemokine activity. CONCLUSIONS: Overall, our results indicate that there are considerable differences in responses to respirable CNC based on gender with a higher pulmonary toxicity observed in female mice.

Pulmonary exposure to cellulose nanocrystals caused deleterious effects to reproductive system in male mice.

Over the past several years there has been an increased number of applications of cellulosic materials in many sectors, including the food industry, cosmetics, and pharmaceuticals. However, to date, there are few studies investigating the potential adverse effects of cellulose nanocrystals (CNC). The objective of this study was to determine long-term outcomes on the male reproductive system of mice upon repeated pharyngeal aspiration exposure to CNC. To achieve this, cauda epididymal sperm samples were analyzed for sperm concentration, motility, morphological abnormalities, and DNA damage. Testicular and epididymal oxidative damage was evaluated, as well as histopathology examination of testes. In addition, changes in levels of testosterone in testes and serum and of luteinizing hormone (LH) in serum were determined. Three months after the last administration, CNC exposure significantly altered sperm concentration, motility, cell morphology, and sperm DNA integrity. These parameters correlated with elevated proinflammatory cytokines levels and myeloperoxidase (MPO) activity in testes, as well as oxidative stress in both testes and epididymis. Exposure to CNC also produced damage to testicular structure, as evidenced by presence of interstitial edema, frequent dystrophic seminiferous tubules with arrested spermatogenesis and degenerating spermatocytes, and imbalance in levels of testosterone and LH. Taken together, these results demonstrate that pulmonary exposure to CNC induces sustained adverse effects in spermatocytes/spermatozoa, suggesting male reproductive toxicity.

Origin and pharmacological modulation of tumor-associated regulatory dendritic cells.

Protumorigenic activity of immune regulatory cells has been proven to play a major role in precluding immunosurveillance and limiting the efficacy of anticancer therapies. Although several approaches have been offered to deplete myeloid-derived suppressor cells (MDSC) and regulatory T cells, there are no data on how to control suppressive dendritic cell (DC) accumulation or function in the tumor environment. Although immunosuppressive function of DC in cancer was implicated to immature and plasmacytoid DC, details of how conventional DC (cDC) develop immunosuppressive properties remain less understood. Here, we show that the development of lung cancer in mice was associated with fast accumulation of regulatory DC (regDC) prior to the appearance of MDSC. Using the in vitro and in vivo approaches, we demonstrated that (i)both cDC and MDSC could be polarized into protumor regDC in the lung cancer environment; (ii) cDC → regDC polarization was mediated by the small Rho GTPase signaling, which could be controlled by noncytotoxic doses of paclitaxel; and (iii) prevention of regDC appearance increased the antitumor potential of DC vaccine in lung cancer. These findings not only bring new players to the family of myeloid regulatory cells and provide new targets for cancer therapy, but offer novel insights into the immunomodulatory capacity of chemotherapeutic agents used in low, noncytotoxic doses.

Clinical evaluation of systemic and local immune responses in cancer: time for integration.

The immune system has a dual role in cancer development and progression. On the one hand, it can eradicate emerging malignant cells, but on the other hand, it can actively promote growth of malignant cells, their invasive capacities and their ability to metastasize. Immune cells with predominantly anti-tumor functionality include cells of the innate immune system, such as natural killer cells, and cells of adaptive immunity, such as conventional dendritic cells and cytotoxic T lymphocytes. Immune cells with predominantly pro-tumor functionality include a broad spectrum of cells of the innate and adaptive immune system, such as type 2 neutrophils and macrophages, plasmacytoid DC, myeloid-derived suppressor cells and regulatory T lymphocytes. The presence of immune cells with tumor-suppressive and tumor-promoting activity in the cancer microenvironment and in peripheral blood is usually associated with good clinical outcomes and poor clinical outcomes, respectively. Significant advances in experimental and clinical oncoimmunology achieved in the last decade open an opportunity for the use of modern morphologic, flow cytometric and functional tests in clinical practice. In this review, we describe an integrated approach to clinical evaluation of the immune status of cancer patients for diagnostic purposes, prognostic/predictive purposes (evaluation of patient prognosis and response to treatment) and for therapeutic purposes.

Tumor associated regulatory dendritic cells.

Immune effector and regulatory cells in the tumor microenvironment are key factors in tumor development and progression as the pathogenesis of cancer vitally depends on the multifaceted interactions between various microenvironmental stimuli provided by tumor-associated immune cells. Immune regulatory cells participate in all stages of cancer development from the induction of genomic instability to the maintenance of intratumoral angiogenesis, proliferation and spreading of malignant cells, and formation of premetastatic niches in distal tissues. Dendritic cells in the tumor microenvironment serve as a double-edged sword and, in addition to initiating potent anti-tumor immune responses, may mediate genomic damage, support neovascularization, block anti-tumor immunity and stimulate cancerous cell growth and spreading. Regulatory dendritic cells in cancer may directly and indirectly maintain antigen-specific and non-specific T cell unresponsiveness by controlling T cell polarization, MDSC and Treg differentiation and activity, and affecting specific microenvironmental conditions in premalignant niches. Understanding the mechanisms involved in regulatory dendritic cell polarization and operation and revealing pharmacological means for harnessing these pathways will provide additional opportunities for modifying the tumor microenvironment and improving the efficacy of different therapeutic approaches to cancer.

Carbon nanotubes enhance metastatic growth of lung carcinoma via up-regulation of myeloid-derived suppressor cells.

Metastatic establishment and growth of Lewis lung carcinoma is promoted by single-walled carbon nanotubes (SWCNT) in C57BL6/J mice. The effect is mediated by increased local and systemic accumulation of myeloid-derived suppressor cells (MDSC), as their depletion abrogated pro-tumor activity in vivo. These data are important for the design of novel theranostics platforms with modules capable of depleting or functionally suppressing MDSC to ensure effective immunosurveillance in the tumor microenvironment.

Biodiesel versus diesel exposure: enhanced pulmonary inflammation, oxidative stress, and differential morphological changes in the mouse lung.

The use of biodiesel (BD) or its blends with petroleum diesel (D) is considered to be a viable approach to reduce occupational and environmental exposures to particulate matter (PM). Due to its lower particulate mass emissions compared to D, use of BD is thought to alleviate adverse health effects. Considering BD fuel is mainly composed of unsaturated fatty acids, we hypothesize that BD exhaust particles could induce pronounced adverse outcomes, due to their ability to readily oxidize. The main objective of this study was to compare the effects of particles generated by engine fueled with neat BD and neat petroleum-based D. Biomarkers of tissue damage and inflammation were significantly elevated in lungs of mice exposed to BD particulates. Additionally, BD particulates caused a significant accumulation of oxidatively modified proteins and an increase in 4-hydroxynonenal. The up-regulation of inflammatory cytokines/chemokines/growth factors was higher in lungs upon BD particulate exposure. Histological evaluation of lung sections indicated presence of lymphocytic infiltrate and impaired clearance with prolonged retention of BD particulate in pigment laden macrophages. Taken together, these results clearly indicate that BD exhaust particles could exert more toxic effects compared to D.

Novel protein and immune response markers of human serous tubal intraepithelial carcinoma of the ovary.

Ovarian cancer is the leading cause of death among gynecologic diseases in the USA and Europe. High-grade serous carcinoma (HGSC) of the ovary, the most aggressive type of ovarian cancer, is typically diagnosed at advanced stages when the 5-year survival is dismal. Since the cure rate for stage I HGSC is high, early detection of localized initial disease may improve patient outcomes. Serous tubal intraepithelial carcinoma (STIC) is considered to be a precursor lesion of HGSC. Discovery of biomarkers associated with STIC could aid in the development of an HGSC screening algorithm. Using immunohistochemical staining, we have demonstrated overexpression of UCHL1, ADAMTS13, and GAPDH in patients' STIC lesions, but not in cancer-free fallopian tubes. We additionally demonstrated a marked increase of T cells in perineoplastic stroma surrounding STIC lesions (largely CD4 + cells), but not in normal fallopian tubes and HGSC. FOXP3 + T regulatory cells are absent in STIC lesions but are present in HGSC. These observations indicate the microenvironment surrounding a STIC lesion may be immune promoting in contrast to the immune suppressive microenvironment of invasive carcinoma. In summary, we have identified UCHL1, ADAMTS13, and GAPDH as novel potentially useful markers associated with early stages of HGSC tumorigenesis and possibly contribute to STIC immunogenicity. The lack of immune suppression in the STIC microenvironment indicates that the immune system can still recognize and keep STIC controlled at this stage of the tumor development.

TGF-ß-mediated enhancement of TH17 cell generation is inhibited by bone morphogenetic protein receptor 1α signaling.

The cytokines of the transforming growth factor-ß (TGF-ß) family promote the growth and differentiation of multiple tissues, but the role of only the founding member, TGF-ß, in regulating the immune responses has been extensively studied. TGF-ß is critical to prevent the spontaneous activation of self-reactive T cells and sustain immune homeostasis. In contrast, in the presence of proinflammatory cytokines, TGF-ß promotes the differentiation of effector T helper 17 (TH17) cells. Abrogating TGF-ß receptor signaling prevents the development of interleukin-17 (IL-17)-secreting cells and protects mice from TH17 cell-mediated autoimmunity. We found that the receptor of another member of TGF-ß family, bone morphogenetic protein receptor 1α (BMPR1α), regulates T helper cell activation. We found that the differentiation of TH17 cells from naive CD4+ T cells was inhibited in the presence of BMPs. Abrogation of BMPR1α signaling during CD4+ T cell activation induced a developmental program that led to the generation of inflammatory effector cells expressing large amounts of IL-17, IFN-γ, and TNF family cytokines and transcription factors defining the TH17 cell lineage. We found that TGF-ß and BMPs cooperated to establish effector cell functions and the cytokine profile of activated CD4+ T cells. Together, our data provide insight into the immunoregulatory function of BMPs.

VHA Practice Guideline Recommendations for Diffuse Gliomas.

Although histology still plays a critical role in diagnosing diffuse gliomas, additional ancillary testing is an essential tool for VA pathology laboratories.

Complex Malignancies: A Diagnostic and Therapeutic Trilemma.

A patient presented with esophageal adenocarcinoma, pulmonary small-cell carcinoma, and high-grade carcinoma, which is a rare occurrence that required a staged multidisciplinary approach to treatment.

Abnormalities in the male reproductive system after exposure to diesel and biodiesel blend.

Altering the fuel source from petroleum-based ultralow sulfur diesel to biodiesel and its blends is considered by many to be a sustainable choice for controlling exposures to particulate material. As the exhaust of biodiesel/diesel blends is composed of a combination of combustion products of polycyclic aromatic hydrocarbons and fatty acid methyl esters, we hypothesize that 50% biodiesel/diesel blend (BD50) exposure could induce harmful outcomes because of its ability to trigger oxidative damage. Here, adverse effects were compared in murine male reproductive organs after pharyngeal aspiration with particles generated by engine fueled with BD50 or neat petroleum diesel (D100). When compared with D100, exposure to BD50 significantly altered sperm integrity, including concentration, motility, and morphological abnormalities, as well as increasing testosterone levels in testes during the time course postexposure. Serum level of luteinizing hormone was significantly depleted only after BD50 exposure. Moreover, we observed that exposure to BD50 significantly increased sperm DNA fragmentation and the upregulation of inflammatory cytokines in the serum and testes on Day 7 postexposure when compared with D100. Histological evaluation of testes sections from BD50 exposure indicated more noticeable interstitial edema, degenerating spermatocytes, and dystrophic seminiferous tubules with arrested spermatogenesis. Significant differences in the level of oxidative stress assessed by accumulation of lipid peroxidation products and depletion of glutathione were detected on exposure to respirable BD50 and D100. Taken together, these results indicate that exposure of mice to inhalable BD50 caused more pronounced adverse effects on male reproductive function than diesel.

MDSC and TGFß Are Required for Facilitation of Tumor Growth in the Lungs of Mice Exposed to Carbon Nanotubes.

During the last decades, changes have been observed in the frequency of different histologic subtypes of lung cancer, one of the most common causes of morbidity and mortality, with a declining proportion of squamous cell carcinomas and an increasing proportion of adenocarcinomas, particularly in developed countries. This suggests the emergence of new etiologic factors and mechanisms, including those defining the lung microenvironment, promoting tumor growth. Assuming that the lung is the main portal of entry for broadly used nanomaterials and their established proinflammatory propensities, we hypothesized that nanomaterials may contribute to changes facilitating tumor growth. Here, we report that an acute exposure to single-walled carbon nanotubes (SWCNT) induces recruitment and accumulation of lung-associated myeloid-derived suppressor cells (MDSC) and MDSC-derived production of TGFß, resulting in upregulated tumor burden in the lung. The production of TGFß by MDSC requires their interaction with both SWCNT and tumor cells. We conclude that pulmonary exposure to SWCNT favors the formation of a niche that supports ingrowth of lung carcinoma in vivo via activation of TGFß production by SWCNT-attracted and -presensitized MDSC.

The role of TLR4 in the paclitaxel effects on neuronal growth in vitro.

Paclitaxel (Pac) is an antitumor agent that is widely used for treatment of solid cancers. While being effective as a chemotherapeutic agent, Pac in high doses is neurotoxic, specifically targeting sensory innervations. In view of these toxic effects associated with conventional chemotherapy, decreasing the dose of Pac has been recently suggested as an alternative approach, which might limit neurotoxicity and immunosuppression. However, it remains unclear if low doses of Pac retain its neurotoxic properties or might exhibit unusual effects on neuronal cells. The goal of this study was to analyze the concentration-dependent effect of Pac on isolated and cultured DRG neuronal cells from wild-type and TLR4 knockout mice. Three different morphological parameters were analyzed: the number of neurons which developed neurites, the number of neurites per cell and the total length of neurites per cell. Our data demonstrate that low concentrations of Pac (0.1 nM and 0.5 nM) do not influence the neuronal growth in cultures in both wild type and TLR4 knockout mice. Higher concentrations of Pac (1-100 nM) had a significant effect on DRG neurons from wild type mice, affecting the number of neurons which developed neurites, number of neurites per cell, and the length of neurites. In DRG from TLR4 knockout mice high concentrations of Pac showed a similar effect on the number of neurons which developed neurites and the length of neurites. At the same time, the number of neurites per cell, indicating the process of growth cone initiation, was not affected by high concentrations of Pac. Thus, our data showed that Pac in high concentrations has a significant damaging effect on axonal growth and that this effect is partially mediated through TLR4 pathways. Low doses of Pac are devoid of neuronal toxicity and thus can be safely used in a chemomodulation mode.

Epigenetic mechanisms of promigratory chemokine CXCL14 regulation in human prostate cancer cells.

Chemokines play the key role in initiating immune responses by regulating the attraction and homing of immune cells to the lymphoid and nonlymphoid tissues. CXCL14 is a chemokine that in tumors may act as chemoattractant for monocytes and dendritic cells (DC), which may modulate antitumor immune responses in certain cancers. In this study, we investigated the mechanisms of loss of CXCL14 in prostate cancer cells. Cell treatment with the demethylating agent 5-aza-2-deoxycytidine resulted in the recovery of CXCL14 mRNA and protein expression. Hypermethylated CpG island sequences encompassing the CXCL14 gene promoter were identified. The restoration of CXCL14 by 5-aza-2-deoxycytidine treatment had functional impact, based on the DC chemoattractant activity of conditioned medium from drug-treated cells. Conversely, CXCL14 removal from conditioned media by affinity chromatography abolished its chemotactic properties, confirming that functionally active CXCL14 was generated in prostate cancer cells by relieving its transcriptional silencing with 5-aza-2-deoxycytidine. Our findings offer the first direct evidence for epigenetic regulation of chemokine expression in tumor cells.

Sensitization of pelvic nerve afferents and mast cell infiltration in the urinary bladder following chronic colonic irritation is mediated by neuropeptides.

Irritable bowel syndrome and interstitial cystitis frequently overlap. We have shown that acute colitis sensitizes urinary bladder afferents to both mechanical and chemical stimuli and that chronic colitis similarly produces neurogenic cystitis. We hypothesize that chronic irritation of the colon releases neuropeptides from bladder afferents, leading to receptor sensitization and neurogenic inflammation. Female Sprague-Dawley rats received intrarectal trinitrobenzenesulfonic acid (TNBS) or vehicle 3 days following either systemic capsaicin (CP) pretreatment or vehicle. Ten days later, action potentials of single-unit pelvic C-fiber afferents with receptive fields in the bladder were recorded under urethane anesthesia during graded bladder distensions (UBD) or intravesical capsaicin (vCP) administration. In controls, UBD increased bladder afferent firing in proportion to intravesical pressure. At intravesical pressures of 30 mmHg and above, the percent increase in afferent firing was significantly accentuated following TNBS compared with controls (1,222 +/- 176 vs. 624 +/- 54%, P < 0.01). The response to vCP was also enhanced (4,126 +/- 775 vs. 1,979 +/- 438%, P < 0.01). Systemic depletion of neuropeptides from sensory nerves abolished these effects. Histological examination of the bladders revealed an increase in mast cell density in TNBS-treated animals compared with controls (18.02 +/- 1.25 vs. 3.11 +/- 0.27 mast cells/x100 field, P < 0.01). This effect was significantly ameliorated with CP (10.25 +/- 0.95, P < 0.5 vs. TNBS-treated animals). In summary, chronic colonic irritation in the rat sensitizes urinary bladder afferents to noxious stimuli and causes mast cell infiltration in the bladder. Depletion of neuropeptides from sensory afferents diminishes these effects, suggesting they play an important role.