BackgroundEngineered carbon nanotubes are currently used in many consumer and industrial products such as paints, sunscreens, cosmetics, toiletries, electronic processes and industrial lubricants. Carbon nanotubes are among the more widely used nanoparticles and come in two major commercial forms, single-walled carbon nanotubes (SWCNT) and the more rigid, multi-walled carbon nanotubes (MWCNT). The low density and small size of these particles makes respiratory exposures likely. Many of the potential health hazards have not been investigated, including their potential for carcinogenicity. We, therefore, utilized a two stage initiation/promotion protocol to determine whether inhaled MWCNT act as a complete carcinogen and/or promote the growth of cells with existing DNA damage. Six week old, male, B6C3F1 mice received a single intraperitoneal (ip) injection of either the initiator methylcholanthrene(MCA, 10 μg/g BW, i.p.), or vehicle (corn oil). One week after i.p. injections, mice were exposed by inhalation to MWCNT (5 mg/m3, 5 hours/day, 5 days/week) or filtered air (controls) for a total of 15 days. At 17 months post-exposure, mice were euthanized and examined for lung tumor formation.ResultsTwenty-three percent of the filtered air controls, 26.5% of the MWCNT-exposed, and 51.9% of the MCA-exposed mice, had lung bronchiolo-alveolar adenomas and lung adenocarcinomas. The average number of tumors per mouse was 0.25, 0.81 and 0.38 respectively. By contrast, 90.5% of the mice which received MCA followed by MWCNT had bronchiolo-alveolar adenomas and adenocarcinomas with an average of 2.9 tumors per mouse 17months after exposure. Indeed, 62% of the mice exposed to MCA followed by MWCNT had bronchiolo-alveolar adenocarcinomas compared to 13% of the mice that received filtered air, 22% of the MCA-exposed, or 14% of the MWCNT-exposed. Mice with early morbidity resulting in euthanasia had the highest rate of metastatic disease. Three mice exposed to both MCA and MWCNT that were euthanized early had lung adenocarcinoma with evidence of metastasis (5.5%). Five mice (9%) exposed to MCA and MWCNT and 1 (1.6%) exposed to MCA developed serosal tumors morphologically consistent with sarcomatous mesotheliomas, whereas mice administered MWCNT or air alone did not develop similar neoplasms.ConclusionsThese data demonstrate that some MWCNT exposures promote the growth and neoplastic progression of initiated lung cells in B6C3F1 mice. In this study, the mouse MWCNT lung burden of 31.2 μg/mouse approximates feasible human occupational exposures. Therefore, the results of this study indicate that caution should be used to limit human exposures to MWCNT.

While several recent reports have described the toxicity of water-soluble C60 fullerene nanoparticles, none have reported the toxicity resulting from the inhalation exposures to C60 fullerene nanoparticles or microparticles. To address this knowledge gap, we exposed male rats to C60 fullerene nanoparticles (2.22 mg/m3, 55 nm diameter) and microparticles (2.35 mg/m3, 0.93 microm diameter) for 3 h a day, for 10 consecutive days using a nose-only exposure system. Nanoparticles were created utilizing an aerosol vaporization and condensation process. Nanoparticles and microparticles were subjected to high-pressure liquid chromatography (HPLC), XRD, and scanning laser Raman spectroscopy, which cumulatively indicated no chemical modification of the C60 fullerenes occurred during the aerosol generation. At necropsy, no gross or microscopic lesions were observed in either group of C60 fullerene exposures rats. Hematology and serum chemistry results found statistically significant differences, although small in magnitude, in both exposure groups. Comparisons of bronchoalveolar (BAL) lavage fluid parameters identified a significant increase in protein concentration in rats exposed to C60 fullerene nanoparticles. BAL fluid macrophages from both exposure groups contained brown pigments, consistent with C60 fullerenes. C60 lung particle burdens were greater in nanoparticle-exposed rats than in microparticle-exposed rats. The calculated lung deposition rate and deposition fraction were 41 and 50% greater, respectively, in C60 fullerene nanoparticle-exposed group than the C60 fullerene microparticle-exposed group. Lung half-lives for C60 fullerene nanoparticles and microparticles were 26 and 29 days, respectively. In summary, this first in vivo assessment of the toxicity resulting from inhalation exposures to C60 fullerene nanoparticles and microparticles found minimal changes in the toxicological endpoints examined. Additional toxicological assessments involving longer duration inhalation exposures are needed to develop a better and more conclusive understanding of the potential toxicity of inhaled C60 fullerenes whether in nanoparticle or microparticle form.

Cattle infected with Neospora caninum readily experience transplacental parasite transmission, presumably after maternal parasitemia, leading to abortion or birth of congenitally infected calves. Cytotoxic T lymphocytes (CTL) are important mediators of protective immunity against Toxoplasma gondii, an intracellular apicomplexan protozoan closely related to N. caninum. In this study, N. caninum-specific CTL expanded from peripheral blood mononuclear cells of two major histocompatibility complex-mismatched, experimentally infected cattle were identified by using a 51 Cr release cytotoxicity assay. Enrichment and blocking of CD4 ؉ -and CD8 ؉ -T-lymphocyte effector subsets indicated that CD4 ؉ CTL killed N. caninum-infected, autologous target cells and that killing was mediated through a perforin/granzyme pathway. Detection and characterization of CTL responses to N. caninum in the natural, outbred, bovine host will facilitate identification of immunogens and design of immunization strategies to induce parasite-specific CTL against transplacental N. caninum transmission in cattle.

Inhalation exposure to multi-walled carbon nanotubes (MWCNT) in mice results in inflammation, fibrosis, and the promotion of lung adenocarcinoma; however, the molecular basis behind these pathologies is unknown. This study determined global mRNA and miRNA profiles in whole blood from mice exposed by inhalation to MWCNT that correlated with the presence of lung hyperplasia, fibrosis, and bronchiolo-alveolar adenoma and adenocarcinoma. Six-week-old, male, B6C3F1 mice received a single intraperitoneal injection of either the DNA-damaging agent methylcholanthrene (MCA, 10 μg/g body weight) or vehicle (corn oil). One week after injections, mice were exposed by inhalation to MWCNT (5 mg/m³, 5 hours/day, 5 days/week) or filtered air (control) for a total of 15 days. At 17 months post-exposure, mice were euthanized and examined for the development of pathological changes in the lung, and whole blood was collected and analyzed using microarray analysis for global mRNA and miRNA expression. Numerous mRNAs and miRNAs in the blood were significantly up- or down-regulated in animals developing pathological changes in the lung after MCA/corn oil administration followed by MWCNT/air inhalation, including fcrl5 and miR-122-5p in the presence of hyperplasia, mthfd2 and miR-206-3p in the presence of fibrosis, fam178a and miR-130a-3p in the presence of bronchiolo-alveolar adenoma, and il7r and miR-210-3p in the presence of bronchiolo-alveolar adenocarcinoma, among others. The changes in miRNA and mRNA expression, and their respective regulatory networks, identified in this study may potentially serve as blood biomarkers for MWCNT-induced lung pathological changes.

Previously, our laboratory showed that Holstein cattle experimentally infected with Neospora caninum develop parasite-specific CD4 ؉ cytotoxic T lymphocytes (CTL) that lyse infected, autologous target cells through a perforin-granzyme pathway. To identify specific parasite antigens inducing bovine CTL and helper T-lymphocyte responses for vaccine development against bovine neosporosis, the tachyzoite major surface proteins NcSAG1 and NcSRS2 were targeted. In whole tachyzoite antigen-expanded bovine T-lymphocyte lines, recombinant NcSRS2 induced potent memory CD4؉ -and CD8 ؉ -T-lymphocyte activation, as indicated by proliferation and gamma interferon (IFN-␥) secretion, while recombinant NcSAG1 induced a minimal memory response. Subsequently, T-lymphocyte epitope-bearing peptides of NcSRS2 were mapped by using overlapping peptides covering the entire NcSRS2 sequence. Neospora caninum is an intracellular apicomplexan protozoan parasite that causes abortion and congenital infection in cattle (21). Transplacental parasite transmission is the major mode of infection (18, 45) which persists in the herd over successive generations, causing significant economic losses due to abortion, culling, and decreased milk production (13,20,25,52,53). Recrudescence of chronic, congenitally acquired infection due to downregulation of Th1 immune responses during pregnancy may be largely responsible for transplacental N. caninum transmission (10,22,27,42). A vaccine that could control tachyzoite parasitemia and decrease congenital infection or abortion rates is needed.Studies of Toxoplasma gondii, a protozoan parasite closely related to N. caninum in biology, phylogeny, and immunogenicity, unequivocally show the importance of T lymphocytes in resistance to infection by adoptive transfer, gene knockout, and depletion studies with mouse models (19). The presence of parasite-specific CD4 ϩ and CD8 ϩ cytotoxic T lymphocytes (CTL) is also associated with immunity to natural T. gondii infection in humans (14,41). In murine models of neosporosis, a T-lymphocyte-mediated immune response, characterized by increased levels of gamma interferon (IFN-␥) and interleukin 12 (IL-12), correlates with disease resistance (5,30,33), and IFN-␥ alone inhibits tachyzoite growth and cellular invasion (24,38,51,55). In pregnant mice, protection against vertical transmission correlates with induction of parasite-specific Tlymphocyte-mediated immunity (39). Cattle infected with N. caninum produce parasite-specific, major histocompatibility complex (MHC)-restricted, CD4 ϩ CTL (50) that could reduce N. caninum infection indirectly by IFN-␥ activation of mononuclear phagocytes and directly by killing N. caninum-infected cells.To identify vaccine candidate parasite antigens for induction of CTL and IFN-␥ secretion in cattle against neosporosis abortion, this study focused on major tachyzoite glycosylphosphatidylinositol-anchored surface proteins, N. caninum surface antigen 1 (NcSAG1) and N. caninum SAG1-related sequence 2 (NcSRS2) (26, 28). The T. gondii surface pr...