Potent lung tumor promotion by inhaled MWCNT.

In the lung, carcinogenesis is a multi-stage process that includes initiation by a genotoxic agent, promotion that expands the population of cells with damaged DNA to form a tumor, and progression from benign to malignant neoplasms. We have previously shown that Mitsui-7, a long and rigid multi-walled carbon nanotube (MWCNT), promotes pulmonary carcinogenesis in a mouse model. To investigate the potential exposure threshold and dose-response for tumor promotion by this MWCNT, 3-methylcholanthrene (MC) initiated (10 µg/g, i.p., once) or vehicle (corn oil) treated B6C3F1 mice were exposed by inhalation to filtered air or MWCNT (5 mg/m3) for 5 h/day for 0, 2, 5, or 10 days and were followed for 17 months post-exposure for evidence of lung tumors. Pulmonary neoplasia incidence in MC-initiated mice significantly increased with each MWCNT exposure duration. Exposure to either MC or MWCNT alone did not affect pulmonary neoplasia incidence compared with vehicle controls. Lung tumor multiplicity in MC-initiated mice also significantly increased with each MWCNT exposure duration. Thus, a significantly higher lung tumor multiplicity was observed after a 10-day MWCNT exposure than following a 2-day exposure. Both bronchioloalveolar adenoma and bronchioloalveolar adenocarcinoma multiplicity in MC-initiated mice were significantly increased following 5- and 10-day MWCNT exposure, while a 2-day MWCNT exposure in MC-initiated mice significantly increased the multiplicity of adenomas but not adenocarcinomas. In this study, even the lowest MWCNT exposure promoted lung tumors in MC-initiated mice. Our findings indicate that exposure to this MWCNT strongly promotes pulmonary carcinogenesis.

Historical shift in pathological type of progressive massive fibrosis among coal miners in the USA.

BACKGROUND: Pneumoconiosis among coal miners in the USA has been resurgent over the past two decades, despite modern dust controls and regulatory standards. Previously published studies have suggested that respirable crystalline silica (RCS) is a contributor to this disease resurgence. However, evidence has been primarily indirect, in the form of radiographic features. METHODS: We obtained lung tissue specimens and data from the National Coal Workers' Autopsy Study. We evaluated specimens for the presence of progressive massive fibrosis (PMF) and used histopathological classifications to type these specimens into coal-type, mixed-type and silica-type PMF. Rates of each were compared by birth cohort. Logistic regression was used to assess demographic and mining characteristics associated with silica-type PMF. RESULTS: Of 322 cases found to have PMF, study pathologists characterised 138 (43%) as coal-type, 129 (40%) as mixed-type and 55 (17%) as silica-type PMF. Among earlier birth cohorts, coal-type and mixed-type PMF were more common than silica-type PMF, but their rates declined in later birth cohorts. In contrast, the rate of silica-type PMF did not decline in cases from more recent birth cohorts. More recent year of birth was significantly associated with silica-type PMF. CONCLUSIONS: Our findings demonstrate a shift in PMF types among US coal miners, from a predominance of coal- and mixed-type PMF to a more commonly encountered silica-type PMF. These results are further evidence of the prominent role of RCS in the pathogenesis of pneumoconiosis among contemporary US coal miners.

Mining Tenure and Job Duties Differ Among Contemporary and Historic Underground Coal Miners With Progressive Massive Fibrosis.

OBJECTIVE: To characterize differences in mining jobs and tenure between contemporary (born 1930+, working primarily with modern mining technologies) and historic coal miners with progressive massive fibrosis (PMF). METHODS: We classified jobs as designated occupations (DOs) and non-DOs based on regulatory sampling requirements. Demographic, occupational characteristics, and histopathological PMF type were compared between groups. RESULTS: Contemporary miners ( n = 33) had significantly shorter mean total (30.4 years vs 37.1 years, P = 0.0006) and underground (28.8 years vs 35.8 years, P = 0.001) mining tenure compared with historic miners ( n = 289). Silica-type PMF was significantly more common among miners in non-DOs (30.1% vs 15.8%, P = 0.03) and contemporary miners (58.1% vs 15.2%, P < 0.0001). CONCLUSIONS: Primary jobs changed over time with the introduction of modern mining technologies and likely changed exposures for workers. Elevated crystalline silica exposures are likely in non-DOs and require attention.

Pulmonary toxicity and gene expression changes in response to whole-body inhalation exposure to multi-walled carbon nanotubes in rats.

Purpose: To investigate the molecular mechanisms underlying the pulmonary toxicity induced by exposure to one form of multi-walled carbon nanotubes (MWCNT-7).Materials and methods: Rats were exposed, by whole-body inhalation, to air or an aerosol containing MWCNT-7 particles at target cumulative doses (concentration x time) ranging from 22.5 to 180 (mg/m3)h over a three-day (6 hours/day) period and toxicity and global gene expression profiles were determined in the lungs.Results: MWCNT-7 particles, associated with alveolar macrophages (AMs), were detected in rat lungs following the exposure. Mild to moderate lung pathological changes consisting of increased cellularity, thickening of the alveolar wall, alveolitis, fibrosis, and granuloma formation were detected. Bronchoalveolar lavage (BAL) toxicity parameters such as lactate dehydrogenase activity, number of AMs and polymorphonuclear leukocytes (PMNs), intracellular oxidant generation by phagocytes, and levels of cytokines were significantly (p < 0.05) increased in response to exposure to MWCNT-7. Global gene expression profiling identified several significantly differentially expressed genes (fold change >1.5 and FDR p value <0.05) in all the MWCNT-7 exposed rats. Bioinformatic analysis of the gene expression data identified significant enrichment of several diseases/biological function categories (for example, cancer, leukocyte migration, inflammatory response, mitosis, and movement of phagocytes) and canonical pathways (for example, kinetochore metaphase signaling pathway, granulocyte and agranulocyte adhesion and diapedesis, acute phase response, and LXR/RXR activation). The alterations in the lung toxicity parameters and gene expression changes exhibited a dose-response to the MWCNT exposure.Conclusions: Taken together, the data provided insights into the molecular mechanisms underlying the pulmonary toxicity induced by inhalation exposure of rats to MWCNT-7.

Pathology and Mineralogy Demonstrate Respirable Crystalline Silica Is a Major Cause of Severe Pneumoconiosis in U.S. Coal Miners.

Rationale: The reasons for resurgent coal workers' pneumoconiosis and its most severe forms, rapidly progressive pneumoconiosis and progressive massive fibrosis (PMF), in the United States are not yet fully understood. Objectives: To compare the pathologic and mineralogic features of contemporary coal miners with severe pneumoconiosis with those of their historical counterparts. Methods: Lung pathology specimens from 85 coal miners with PMF were included for evaluation and analysis. We compared the proportion of cases with pathologic and mineralogic findings in miners born between 1910 and 1930 (historical) with those in miners born in or after 1930 (contemporary). Results: We found a significantly higher proportion of silica-type PMF (57% vs. 18%; P < 0.001) among contemporary miners compared with their historical counterparts. Mineral dust alveolar proteinosis was also more common in contemporary miners compared with their historical counterparts (70% vs. 37%; P < 0.01). In situ mineralogic analysis showed that the percentage (26.1% vs. 17.8%; P < 0.01) and concentration (47.3 × 108 vs. 25.8 × 108 particles/cm3; P = 0.036) of silica particles were significantly greater in specimens from contemporary miners compared with their historical counterparts. The concentration of silica particles was significantly greater when silica-type PMF, mineral dust alveolar proteinosis, silicotic nodules, or immature silicotic nodules were present (P < 0.05). Conclusions: Exposure to respirable crystalline silica appears causal in the unexpected surge of severe disease in contemporary miners. Our findings underscore the importance of controlling workplace silica exposure to prevent the disabling and untreatable adverse health effects afflicting U.S. coal miners.

Lung toxicity and gene expression changes in response to whole-body inhalation exposure to cellulose nanocrystal in rats.

OBJECTIVE: Human exposure to cellulose nanocrystal (CNC) is possible during the production and/or use of products containing CNC. The objectives of the current study were to determine the lung toxicity of CNC and the underlying molecular mechanisms of the toxicity. METHODS: Rats were exposed to air or CNC (20 mg/m3, six hours/day, 14 d) by whole-body inhalation and lung toxicity and global gene expression profile were determined. RESULTS: Significant increases in lactate dehydrogenase activity, pro-inflammatory cytokine levels, phagocyte oxidant production, and macrophage and neutrophil counts were detected in the bronchoalveolar lavage cells or fluid from the CNC exposed rats. Mild lung histological changes, such as the accumulation of macrophages and neutrophils, were detected in the CNC exposed rats. Gene expression profiling by next generation sequencing identified 531 genes whose expressions were significantly different in the lungs of the CNC exposed rats, compared with the controls. Bioinformatic analysis of the lung gene expression data identified significant enrichment in several biological functions and canonical pathways including those related to inflammation (cellular movement, immune cell trafficking, inflammatory diseases and response, respiratory disease, complement system, acute phase response, leukocyte extravasation signaling, granulocyte and agranulocyte adhesion and diapedesis, IL-10 signaling, and phagosome formation and maturation) and oxidative stress (NRF2-mediated oxidative stress response, production of nitric oxide and reactive oxygen species in macrophages, and free radical scavenging). CONCLUSION: Our data demonstrated that inhalation exposure of rats to CNC resulted in lung toxicity mediated mainly through the induction of inflammation and oxidative stress.

Resolution of Pulmonary Inflammation Induced by Carbon Nanotubes and Fullerenes in Mice: Role of Macrophage Polarization.

Pulmonary exposure to certain engineered nanomaterials (ENMs) causes chronic lesions like fibrosis and cancer in animal models as a result of unresolved inflammation. Resolution of inflammation involves the time-dependent biosynthesis of lipid mediators (LMs)-in particular, specialized pro-resolving mediators (SPMs). To understand how ENM-induced pulmonary inflammation is resolved, we analyzed the inflammatory and pro-resolving responses to fibrogenic multi-walled carbon nanotubes (MWCNTs, Mitsui-7) and low-toxicity fullerenes (fullerene C60, C60F). Pharyngeal aspiration of MWCNTs at 40 µg/mouse or C60F at a dose above 640 µg/mouse elicited pulmonary effects in B6C3F1 mice. Both ENMs stimulated acute inflammation, predominated by neutrophils, in the lung at day 1, which transitioned to histiocytic inflammation by day 7. By day 28, the lesion in MWCNT-exposed mice progressed to fibrotic granulomas, whereas it remained as alveolar histiocytosis in C60F-exposed mice. Flow cytometric profiling of whole lung lavage (WLL) cells revealed that neutrophil recruitment was the greatest at day 1 and declined to 36.6% of that level in MWCNT- and 16.8% in C60F-treated mice by day 7, and to basal levels by day 28, suggesting a rapid initiation phase and an extended resolution phase. Both ENMs induced high levels of proinflammatory leukotriene B4 (LTB4) and prostaglandin E2 (PGE2) with peaks at day 1, and high levels of SPMs resolvin D1 (RvD1) and E1 (RvE1) with peaks at day 7. MWCNTs and C60F induced time-dependent polarization of M1 macrophages with a peak at day 1 and subsequently of M2 macrophages with a peak at day 7 in the lung, accompanied by elevated levels of type 1 or type 2 cytokines, respectively. M1 macrophages exhibited preferential induction of arachidonate 5-lipoxygenase activating protein (ALOX5AP), whereas M2 macrophages had a high level expression of arachidonate 15-lipoxygenase (ALOX15). Polarization of macrophages in vitro differentially induced ALOX5AP in M1 macrophages or ALOX15 in M2 macrophages resulting in increased preferential biosynthesis of proinflammatory LMs or SPMs. MWCNTs increased the M1- or M2-specific production of LMs accordingly. These findings support a mechanism by which persistent ENM-induced neutrophilic inflammation is actively resolved through time-dependent polarization of macrophages and enhanced biosynthesis of specialized LMs via distinct ALOX pathways.

Inhalation of Stachybotrys chartarum Fragments Induces Pulmonary Arterial Remodeling.

Stachybotrys chartarum is a fungal contaminant within the built environment and a respiratory health concern in the United States. The objective of this study was to characterize the mechanisms influencing pulmonary immune responses to repeatedly inhaled S. chartarum. Groups of B6C3F1/N mice repeatedly inhaled viable trichothecene-producing S. chartarum conidia (strain A or strain B), heat-inactivated conidia, or high-efficiency particulate absolute-filtered air twice per week for 4 and 13 weeks. Strain A was found to produce higher amounts of respirable fragments than strain B. Lung tissue, serum, and BAL fluid were collected at 24 and 48 hours after final exposure and processed for histology, flow cytometry, and RNA and proteomic analyses. At 4 weeks after exposure, a T-helper cell type 2-mediated response was observed. After 13 weeks, a mixed T-cell response was observed after exposure to strain A compared with a T-helper cell type 2-mediated response after strain B exposure. After exposure, both strains induced pulmonary arterial remodeling at 13 weeks; however, strain A-exposed mice progressed more quickly than strain B-exposed mice. BAL fluid was composed primarily of eosinophils, neutrophils, and macrophages. Both the immune response and the observed pulmonary arterial remodeling were supported by specific cellular, molecular, and proteomic profiles. The immunopathological responses occurred earlier in mice exposed to high fragment-producing strain A. The rather striking induction of pulmonary remodeling by S. chartarum appears to be related to the presence of fungal fragments during exposure.

Flavorings-Related Lung Disease: A Brief Review and New Mechanistic Data.

Flavorings-related lung disease is a potentially disabling and sometimes fatal lung disease of workers making or using flavorings. First identified almost 20 years ago in microwave popcorn workers exposed to butter-flavoring vapors, flavorings-related lung disease remains a concern today. In some cases, workers develop bronchiolitis obliterans, a severe form of fixed airways disease. Affected workers have been reported in microwave popcorn, flavorings, and coffee production workplaces. Volatile α-dicarbonyl compounds, particularly diacetyl (2,3-butanedione) and 2,3-pentanedione, are implicated in the etiology. Published studies on diacetyl and 2,3-pentanedione document their ability to cause airway epithelial necrosis, damage biological molecules, and perturb protein homeostasis. With chronic exposure in rats, they produce airway fibrosis resembling bronchiolitis obliterans. To add to this knowledge, we recently evaluated airway toxicity of the 3-carbon α-dicarbonyl compound, methylglyoxal. Methylglyoxal inhalation causes epithelial necrosis at even lower concentrations than diacetyl. In addition, we investigated airway toxicity of mixtures of diacetyl, acetoin, and acetic acid, common volatiles in butter flavoring. At ratios comparable to workplace scenarios, the mixtures or diacetyl alone, but not acetic acid or acetoin, cause airway epithelial necrosis. These new findings add to existing data to implicate α-dicarbonyl compounds in airway injury and flavorings-related lung disease.

Mouse pulmonary response to dust from sawing Corian®, a solid-surface composite material.

Corian®, a solid-surface composite (SSC), is composed of alumina trihydrate and acrylic polymer. The aim of the present study was to examine the pulmonary toxicity attributed to exposure to SSC sawing dust. Male mice were exposed to either phosphate buffer saline (PBS, control), 62.5, 125, 250, 500, or 1000 µg of SSC dust, or 1000 µg silica (positive control) via oropharyngeal aspiration. Body weights were measured for the duration of the study. Bronchoalveolar lavage fluid (BALF) and tissues were collected for analysis at 1 and 14 days post-exposure. Enhanced-darkfield and histopathologic analysis was performed to assess particle distribution and inflammatory responses. BALF cells and inflammatory cytokines were measured. The geometric mean diameter of SSC sawing dust following suspension in PBS was 1.25 µm. BALF analysis indicated that lactate dehydrogenase (LDH) activity, inflammatory cells, and pro-inflammatory cytokines were significantly elevated in the 500 and 1000 µg SSC exposure groups at days 1 and 14, suggesting that exposure to these concentrations of SSC induced inflammatory responses, in some cases to a greater degree than the silica positive control. Histopathology indicated the presence of acute alveolitis at all doses at day 1, which was largely resolved by day 14. Alveolar particle deposition and granulomatous mass formation were observed in all exposure groups at day 14. The SSC particles were poorly cleared, with 81% remaining at the end of the observation period. These findings demonstrate that SSC sawing dust exposure induces pulmonary inflammation and damage that warrants further investigation. Abbreviations: ANOVA: Analysis of Variance; ATH: Alumina Trihydrate; BALF: Bronchoalveolar Lavage Fluid; Dpg: Geometric Mean Diameter; FE-SEM: Field Emission Scanning Electron Microscopy; IACUC: Institutional Animal Care and Use Committee; IFN-γ: Interferon Gamma; IL-1 Β: Interleukin-1 Beta; IL-10: Interleukin-10; IL-12: Interleukin-12; IL-2: Interleukin-2; IL-4: Interleukin-4; IL-5: Interleukin-5; IL-6: Interleukin-6; KC/GRO: Neutrophil-Activating Protein 3; MMAD: Mass Median Aerodynamic Diameter; PBS: Phosphate-Buffered Saline; PEL: Permissible Exposure Limit; PM: Polymorphonuclear Leukocytes; PNOR: Particles Not Otherwise Regulated; SEM/EDX: Scanning Electron Microscope/Energy-Dispersive X-Ray; SSA: Specific Surface Area; SSC: Solid Surface Composite; TNFα: Tumor Necrosis Factor-Alpha; VOC: Volatile Organic Compounds; σg: Geometric Standard Deviation.

Toxicological assessment of dust from sanding micronized copper-treated lumber in vivo.

Micronized copper azole (MCA) is a lumber treatment improve longevity. In this study, the in vivo response to PM2.5 sanding dust generated from MCA-treated lumber was compared to that of untreated yellow pine (UYP) or soluble copper azole-treated (CA-C) lumber to determine if the MCA was more bioactive than CA-C. Mice were exposed to doses (28, 140, or 280 µg/mouse) of UYP, MCA, or CA-C sanding dust using oropharyngeal aspiration. Bronchoalveolar lavage fluid (BALF) lactate dehydrogenase activity was increased at 1 day post-exposure to 280 µg/mouse of MCA and CA-C compared to UYP. BALF polymorphonuclear cells were increased by MCA and CA-C. There were increases in BALF cytokines in MCA and CA-C-exposed groups at 1 day post-exposure. Lung histopathology indicated inflammation with infiltration of neutrophils and macrophages. Pulmonary responses were more severe in MCA and CA-C-exposed groups at 1 day post-exposure. MCA caused more severe inflammatory responses than CA-C at 1 day post-exposure. These findings suggest that the MCA and CA-C sanding dusts are more bioactive than the UYP sanding dust, and, moreover, the MCA sanding dust is more bioactive in comparison to the CA-C sanding dust. No chronic toxic effects were observed among all observed sanding dusts.

Sparse Supervised Classification Methods Predict and Characterize Nanomaterial Exposures: Independent Markers of MWCNT Exposures.

Recent experimental evidence indicates significant pulmonary toxicity of multiwalled carbon nanotubes (MWCNTs), such as inflammation, interstitial fibrosis, granuloma formation, and carcinogenicity. Although numerous studies explored the adverse potential of various CNTs, their comparability is often limited. This is due to differences in administered dose, physicochemical characteristics, exposure methods, and end points monitored. Here, we addressed the problem through sparse classification method, a supervised machine learning approach that can reduce the noise contained in redundant variables for discriminating among MWCNT-exposed and MWCNT-unexposed groups. A panel of proteins measured from bronchoalveolar lavage fluid (BAL) samples was used to predict exposure to various MWCNT and determine markers that are attributable to MWCNT exposure and toxicity in mice. Using sparse support vector machine-based classification technique, we identified a small subset of proteins clearly distinguishing each exposure. Macrophage-derived chemokine (MDC/CCL22), in particular, was associated with various MWCNT exposures and was independent of exposure method employed, that is, oropharyngeal aspiration versus inhalation exposure. Sustained expression of some of the selected protein markers identified also suggests their potential role in MWCNT-induced toxicity and proposes hypotheses for future mechanistic studies. Such approaches can be used more broadly for nanomaterial risk profiling studies to evaluate decisions related to dose/time-response relationships that could delineate experimental variables from exposure markers.

Mechanism of splenic cell death and host mortality in a Plasmodium yoelii malaria model.

Malaria is a fatal disease that displays a spectrum of symptoms and severity, which are determined by complex host-parasite interactions. It has been difficult to study the effects of parasite strains on disease severity in human infections, but the mechanisms leading to specific disease phenotypes can be investigated using strains of rodent malaria parasites that cause different disease symptoms in inbred mice. Using a unique mouse malaria model, here we investigated the mechanisms of splenic cell death and their relationship to control of parasitemia and host mortality. C57BL/6 mice infected with Plasmodium yoelii nigeriensis N67C display high levels of pro-inflammatory cytokines and chemokines (IL-6, IFN-γ, TNF-α, CXCL1, and CCL2) and extensive splenic damage with dramatic reduction of splenic cell populations. These disease phenotypes were rescued in RAG2-/-, IFN-γ-/-, or T cell depleted mice, suggesting IFN-γ and T cell mediated disease mechanisms. Additionally, apoptosis was one of the major pathways involved in splenic cell death, which coincides with the peaks of pro-inflammatory cytokines. Our results demonstrate the critical roles of T cells and IFN-γ in mediating splenic cell apoptosis, parasitemia control, and host lethality and thus may provide important insights for preventing/reducing morbidity associated with severe malaria in humans.

A Plasmodium yoelii HECT-like E3 ubiquitin ligase regulates parasite growth and virulence.

Infection of mice with strains of Plasmodium yoelii parasites can result in different pathology, but molecular mechanisms to explain this variation are unclear. Here we show that a P. yoelii gene encoding a HECT-like E3 ubiquitin ligase (Pyheul) influences parasitemia and host mortality. We genetically cross two lethal parasites with distinct disease phenotypes, and identify 43 genetically diverse progeny by typing with microsatellites and 9230 single-nucleotide polymorphisms. A genome-wide quantitative trait loci scan links parasite growth and host mortality to two major loci on chromosomes 1 and 7 with LOD (logarithm of the odds) scores = 6.1 and 8.1, respectively. Allelic exchange of partial sequences of Pyheul in the chromosome 7 locus and modification of the gene expression alter parasite growth and host mortality. This study identifies a gene that may have a function in parasite growth, virulence, and host-parasite interaction, and therefore could be a target for drug or vaccine development.Many strains of Plasmodium differ in virulence, but factors that control these distinctions are not known. Here the authors comparatively map virulence loci using the offspring from a P. yoelii YM and N67 genetic cross, and identify a putative HECT E3 ubiquitin ligase that may explain the variance.

Pulmonary toxicity of indium-tin oxide production facility particles in rats.

Indium-tin oxide (ITO) is used to make transparent conductive coatings for touch-screen and liquid crystal display electronics. Occupational exposures to potentially toxic particles generated during ITO production have increased in recent years as the demand for consumer electronics continues to rise. Previous studies have demonstrated cytotoxicity in vitro and animal models have shown pulmonary inflammation and injury in response to various indium-containing particles. In humans, pulmonary alveolar proteinosis (PAP) and fibrotic interstitial lung disease have been observed in ITO facility workers. However, which indium materials or specific processes in the workplace may be the most toxic to workers is unknown. Here we examined the pulmonary toxicity of three different particle samples that represent real-life worker exposures, as they were collected at various production stages throughout an ITO facility. Indium oxide (In2O3), sintered ITO (SITO) and ventilation dust (VD) particles each caused pulmonary inflammation and damage in rats over a time course (1, 7 and 90 days post-intratracheal instillation), but SITO and VD appeared to induce greater toxicity in rat lungs than In2O3 at a dose of 1 mg per rat. Downstream pathological changes such as PAP and fibrosis were observed in response to all three particles 90 days after treatment, with a trend towards greatest severity in animals exposed to VD when comparing animals that received the same dose. These findings may inform workplace exposure reduction efforts and provide a better understanding of the pathogenesis of an emerging occupational health issue.

Strain-specific innate immune signaling pathways determine malaria parasitemia dynamics and host mortality.

Malaria infection triggers vigorous host immune responses; however, the parasite ligands, host receptors, and the signaling pathways responsible for these reactions remain unknown or controversial. Malaria parasites primarily reside within RBCs, thereby hiding themselves from direct contact and recognition by host immune cells. Host responses to malaria infection are very different from those elicited by bacterial and viral infections and the host receptors recognizing parasite ligands have been elusive. Here we investigated mouse genome-wide transcriptional responses to infections with two strains of Plasmodium yoelii (N67 and N67C) and discovered differences in innate response pathways corresponding to strain-specific disease phenotypes. Using in vitro RNAi-based gene knockdown and KO mice, we demonstrated that a strong type I IFN (IFN-I) response triggered by RNA polymerase III and melanoma differentiation-associated protein 5, not Toll-like receptors (TLRs), binding of parasite DNA/RNA contributed to a decline of parasitemia in N67-infected mice. We showed that conventional dendritic cells were the major sources of early IFN-I, and that surface expression of phosphatidylserine on infected RBCs might promote their phagocytic uptake, leading to the release of parasite ligands and the IFN-I response in N67 infection. In contrast, an elevated inflammatory response mediated by CD14/TLR and p38 signaling played a role in disease severity and early host death in N67C-infected mice. In addition to identifying cytosolic DNA/RNA sensors and signaling pathways previously unrecognized in malaria infection, our study demonstrates the importance of parasite genetic backgrounds in malaria pathology and provides important information for studying human malaria pathogenesis.

The D10 decapping enzyme of vaccinia virus contributes to decay of cellular and viral mRNAs and to virulence in mice.

Posttranscriptional mechanisms are important for regulation of cellular and viral gene expression. The presence of the 5' cap structure m(7)G(5')ppp(5')Nm is a general feature of mRNAs that provides protection from exoribonuclease digestion and enhances translation. Vaccinia virus and other poxviruses encode enzymes for both cap synthesis and decapping. Decapping is mediated by two related enzymes, D9 and D10, which are synthesized before and after viral DNA replication, respectively. The timing of D10 synthesis correlates better with the shutdown of host gene expression, and deletion of this gene has been shown to cause persistence of host and viral mRNAs in infected cells. Here, we constructed specific mutant viruses in which translation of D10 was prevented by stop codons or activity of D10 was abrogated by catalytic site mutations, without other genomic alterations. Both mutants formed plaques of normal size and replicated to similar extents as the parental virus in monkey epithelial cells and mouse embryonic fibroblasts. The synthesis of viral proteins was slightly delayed, and cellular and viral mRNAs persisted longer in cells infected with the mutants compared to either the parental virus or clonal revertant. Despite the mild effects in vitro, both mutants were more attenuated than the revertants in intranasal and intraperitoneal mouse models, and less infectious virus was recovered from organs. In addition, there was less lung histopathology following intranasal infection with mutant viruses. These data suggest that the D10 decapping enzyme may help restrict antiviral responses by accelerating host mRNA degradation during poxvirus infection.

p47(phox) directs murine macrophage cell fate decisions.

Macrophage differentiation and function are pivotal for cell survival from infection and involve the processing of microenvironmental signals that determine macrophage cell fate decisions to establish appropriate inflammatory balance. NADPH oxidase 2 (Nox2)-deficient chronic granulomatous disease (CGD) mice that lack the gp91(phox) (gp91(phox-/-)) catalytic subunit show high mortality rates compared with wild-type mice when challenged by infection with Listeria monocytogenes (Lm), whereas p47(phox)-deficient (p47(phox-/-)) CGD mice show survival rates that are similar to those of wild-type mice. We demonstrate that such survival results from a skewed macrophage differentiation program in p47(phox-/-) mice that favors the production of higher levels of alternatively activated macrophages (AAMacs) compared with levels of either wild-type or gp91(phox-/-) mice. Furthermore, the adoptive transfer of AAMacs from p47(phox-/-) mice can rescue gp91(phox-/-) mice during primary Lm infection. Key features of the protective function provided by p47(phox-/-) AAMacs against Lm infection are enhanced production of IL-1α and killing of Lm. Molecular analysis of this process indicates that p47(phox-/-) macrophages are hyperresponsive to IL-4 and show higher Stat6 phosphorylation levels and signaling coupled to downstream activation of AAMac transcripts in response to IL-4 stimulation. Notably, restoring p47(phox) protein expression levels reverts the p47(phox)-dependent AAMac phenotype. Our results indicate that p47(phox) is a previously unrecognized regulator for IL-4 signaling pathways that are important for macrophage cell fate choice.

Idiopathic solitary cutaneous xanthoma in a dog.

A 6-year-old female spayed Boxer mix dog was presented with multiple cutaneous masses, one of which was determined to be a xanthoma. Fine-needle aspirates of this mass revealed large round cells that were consistent with macrophages. These macrophages had lightly basophilic cytoplasm that was filled with many clear circular spaces that varied in size. The nuclei of these cells displayed mild anisokaryosis with condensed chromatin and lacked prominent nucleoli. The cytologic interpretation was lipid-laden histiocytic inflammation most consistent with a cutaneous xanthoma, which was confirmed histologically. Mild hypertriglyceridemia and persistent moderate hypercholesterolemia were present. After ruling out other causes of hyperlipidemia, we concluded that the dog likely had idiopathic hyperlipidemia with secondary xanthoma formation.