Phagocytosis is coupled to the formation of phagosome-associated podosomes and a transient disruption of podosomes in human macrophages

Phagocytosis consists in ingestion and digestion of large particles, a process strictly dependent on actin re-organization. Using synchronized phagocytosis of IgG-coated latex beads (IgG-LB), zymosan or serum opsonized-zymosan, we report the formation of actin structures on both phagocytic cups and closed phagosomes in human macrophages. Their lifespan, size, protein composition and organization are similar to podosomes. Thus, we called these actin structures phagosome-associated podosomes (PAPs). Concomitantly to the formation of PAPs, a transient disruption of podosomes occurred at the ventral face of macrophages. Similarly to podosomes, which are targeted by vesicles containing proteases, the presence of PAPs correlated with the maturation of phagosomes into phagolysosomes. The ingestion of LB without IgG did not trigger PAPs formation, did not lead to podosome disruption and maturation to phagolysosomes, suggesting that these events are linked together. Although similar to podosomes, we found that PAPs differed by being resistant to the Arp2/3 inhibitor CK666. Thus, we describe a podosome subtype which forms on phagosomes where it probably serves several tasks of this multifunctional structure.     

Continuous and Discontinuous Cigarette Smoke Exposure Differentially Affects Protective Th1 Immunity against Pulmonary Tuberculosis

Pulmonary tuberculosis (TB), caused by Mycobacterium tuberculosis, is the leading cause of death due to a bacterial pathogen. Emerging epidemiologic evidence suggests that the leading risk factor associated with TB mortality is cigarette smoke exposure. Despite this, it remains poorly understood what is the effect of cigarette smoke exposure on anti-TB immunity and whether its potential detrimental effect can be reversed by cigarette smoking cessation. In our current study, we have investigated the impact of both continuous and discontinuous cigarette smoke exposure on the development of anti-mycobacterial type 1 immunity in murine models. We find that while continuous cigarette smoke exposure severely impairs type 1 immunity in the lung, a short-term smoking cessation allows rapid restoration of anti-mycobacterial immunity. The ability of continuous cigarette smoke exposure to dampen type 1 protective immunity is attributed locally to its affects on innate immune cells in the lung. Continuous cigarette smoke exposure locally, by not systemically, impairs APC accumulation and their production of TNF, IL-12, and RANTES, blunts the recruitment of CD4+IFN-γ+ T cells to the lung, and weakens the formation of granuloma. On the other hand, smoking cessation was found to help restore type 1 immunity by rapidly improving the functionality of lung APCs, enhancing the recruitment of CD4+IFN-γ+ T cells to the lung, and promoting the formation of granuloma. Our study for the first time demonstrates that continuous, but not discontinuous, cigarette smoke exposure severely impedes the lung expression of anti-TB Th1 immunity via inhibiting innate immune activation and lung T cell recruitment. Our findings thus suggest cigarette smoking cessation to be beneficial to the control of pulmonary TB.     

A Novel Flow Cytometric Hemozoin Detection Assay for Real-Time Sensitivity Testing of Plasmodium falciparum

Resistance of Plasmodium falciparum to almost all antimalarial drugs, including the first-line treatment with artemisinins, has been described, representing an obvious threat to malaria control. In vitro antimalarial sensitivity testing is crucial to detect and monitor drug resistance. Current assays have been successfully used to detect drug effects on parasites. However, they have some limitations, such as the use of radioactive or expensive reagents or long incubation times. Here we describe a novel assay to detect antimalarial drug effects, based on flow cytometric detection of hemozoin (Hz), which is rapid and does not require any additional reagents. Hz is an optimal parasite maturation indicator since its amount increases as the parasite matures. Due to its physical property of birefringence, Hz depolarizes light, hence it can be detected using optical methods such as flow cytometry. A common flow cytometer was adapted to detect light depolarization caused by Hz. Synchronized in vitro cultures of P. falciparum were incubated for 48 hours with several antimalarial drugs. Analysis of depolarizing events, corresponding to parasitized red blood cells containing Hz, allowed the detection of parasite maturation. Moreover, chloroquine resistance and the inhibitory effect of all antimalarial drugs tested, except for pyrimethamine, could be determined as early as 18 to 24 hours of incubation. At 24 hours incubation, 50% inhibitory concentrations (IC50) were comparable to previously reported values. These results indicate that the reagent-free, real-time Hz detection assay could become a novel assay for the detection of drug effects on Plasmodium falciparum.     

Cardiac Fibroblasts Contribute to Myocardial Dysfunction in Mice with Sepsis: The Role of NLRP3 Inflammasome Activation

Myocardial contractile dysfunction in sepsis is associated with the increased morbidity and mortality. Although the underlying mechanisms of the cardiac depression have not been fully elucidated, an exaggerated inflammatory response is believed to be responsible. Nucleotide-binding oligomerization domain-like receptor containing pyrin domain 3 (NLRP3) inflammasome is an intracellular platform that is involved in the maturation and release of interleukin (IL)-1β. The aim of the present study is to evaluate whether sepsis activates NLRP3 inflammasome/caspase-1/IL-1β pathway in cardiac fibroblasts (CFs) and whether this cytokine can subsequently impact the function of cardiomyocytes (cardiac fibroblast-myocyte cross-talk). We show that treatment of CFs with lipopolysaccharide (LPS) induces upregulation of NLRP3, activation of caspase-1, as well as the maturation (activation) and release of IL-1β. In addition, the genetic (small interfering ribonucleic acid [siRNA]) and pharmacological (glyburide) inhibition of the NLRP3 inflammasome in CFs can block this signaling pathway. Furthermore, the inhibition of the NLRP3 inflammasome in cardiac fibroblasts ameliorated the ability of LPS-chalenged CFs to impact cardiomyocyte function as assessed by intracellular cyclic adenosine monophosphate (cAMP) responses in cardiomyocytes. Salient features of this the NLP3 inflammasome/ caspase-1 pathway were confirmed in in vivo models of endotoxemia/sepsis. We found that inhibition of the NLRP3 inflammasome attenuated myocardial dysfunction in mice with LPS and increased the survival rate in mice with feces-induced peritonitis. Our results indicate that the activation of the NLRP3 inflammasome in cardiac fibroblasts is pivotal in the induction of myocardial dysfunction in sepsis.     

Adjusting for reporting delay in cancer incidence when combining different sets of cancer registries

Cancer registries collect cancer incidence data that can be used to calculate incidence rates in a population and track changes over time. For incidence rates to be accurate, it is critical that diagnosed cases be reported in a timely manner. Registries typically allow a fixed amount of time (e.g. two years) for diagnosed cases to be reported before releasing the initial case counts for a particular diagnosis year. Inevitably, however, additional cases are reported after the initial counts are released; these extra cases are included in subsequent releases that become more complete over time, while incidence rates based on earlier releases will underestimate the true rates. Statistical methods have been developed to estimate the distribution of reporting delay (the amount of time until a diagnosed case is reported) and to correct incidence rates for underestimation due to reporting delay. Since the observed reporting delays must be less than the length of time the registry has been collecting data, most methods estimate a truncated delay distribution. These methods can be applied to a group of registries that began collecting data in the same diagnosis year. In this paper, we extend the methods to two groups of registries that began collecting data in two different diagnosis years (so that the delay distributions are truncated at different times). We apply the proposed method to data from the National Cancer Institute's Surveillance Epidemiology and End Results (SEER) program, a consortium of U.S. cancer registries that includes nine registries with data collection beginning in 1981 and four registries with data collection beginning in 1992. We use the method to obtain delay‐adjusted incidence rates for melanoma, liver cancer, and Hodgkin lymphoma.     

Inhibition of Aspartate Aminotransferase by Glycation In Vitro Under Various Conditions

Incubation of 50 mM d -glucose with aspartate aminotransferase (AST, EC 2.6.1.1) preparations (purified pig heart enzyme or a rat liver 20,000 × g supernatant) at 25°C had no effect on enzyme activity. 50 mM d -fructose or d -ribose gradually inhibited pig heart AST under the same conditions to zero activity after 14 days. 50 mM dl -glyceraldehyde decreased enzyme activity to zero after 6 days of incubation. The inhibition of pig heart AST by 50 mM d -fructose or d -ribose was marked even at a temperature of 4°C but it was less pronounced than at 25°C. There was no effect of 0.5 mM 2-oxoglutarate on AST activity during incubation, while the presence of 25 mM l -aspartate decreased it rapidly. 0.5 mM 2-oxoglutarate partly prevented inhibition of AST by d -ribose or d -fructose, while an analogous experiment with 25 mM aspartate resulted in a rapid decline similar to that in the absence of sugars.     

Osteoderm histology of the Pampatheriidae (Cingulata,Xenarthra, Mammalia): Implications for systematics,osteoderm growth,and biomechanical adaptation

Pampatheres are extinct, large‐bodied cingulates, which share morphological characters with both armadillos and glyptodonts but are considered to be more closely related to the latter. The osteoderm histology of six pampathere taxa was examined and compared to the histology of other cingulate osteoderms. This study investigates the development and functional adaptation of pampathere osteoderms as well as the phylogenetic relationships of the Pampatheriidae within the Cingulata. We found that pampathere osteoderms share a uniform histological organization based on a basic diploe‐like structure. After initial stages of intramembranous growth, metaplastic ossification, that is, the direct incorporation and mineralization of pre‐existing protein fibers, plays an important role in osteoderm development and provides information on various kinds of soft tissue otherwise not preserved. The latest stages of osteoderm growth are dominated by periosteal bone formation especially in the superficial cortex. Movable band osteoderms show regular arrangements of incorporated fibers that may increase the resistance of particularly weak areas against strain. The histological composition of pampathere osteoderms is plesiomorphic in its basic structure but shows a number of derived features. A unique array of Sharpey's fibers that are incorporated into the bone matrix at sutured osteoderm margins is interpreted as a synapomorphy of pampatheres. The arrangement of dermal fibers in the deep and superficial cortexes supports the close relationship between pampatheres and glyptodonts. J. Morphol., 2012. © 2011 Wiley Periodicals, Inc.     

Pathogenicity for Chickens of a Paramyxovirus Type 1, Isolated from Racing Pigeons in Sweden

Strains of paramyxovirus type 1 (PMV-1) have been isolated from diseased racing pigeons in Sweden. One of these isolates was selected for studies of the pathogenicity and contagiousness in chickens. The same isolate was previously found to have a high intravenous pathogenicity index (IVPI) in 6 weeks old chickens. In three experiments it was found that the PMV-1 isolate was very pathogenic for 1 week old chickens but not pathogenic for 120 day old pullets inoculated intranasally and ocularly. Symptoms in the young chickens were similar to those seen in the neurotropic form of Newcastle disease. The mortality was high and the incubation period 5–11 days. The disease easily spread to young chickens kept in contact with diseased birds. The microscopic examination revealed an interstitial nonpurulent pneumonia and a nonpurulent encephalitis in the young chickens. In the pullets the only finding was a mild encephalitis. PMV-1 was recovered from all young chickens but not from the pullets. Both the chickens and the inoculated pullets developed antibodies to PMV-1.     

Volatiles from Merino fleece evoke antennal and behavioural responses in the Australian sheep blow fly Lucilia cuprina

To identify flystrike‐related volatile compounds in wool from Merino sheep, the attractiveness of wool to Lucilia cuprina Wiedmann (Diptera: Calliphoridae) was examined. First, a selection of wool samples guided by previous knowledge of sheep lines, predicted to be more susceptible or more resistant to flystrike, was tested. The attractiveness of the 10 samples selected was not associated with field susceptibility: two samples from the more resistant line were identified as most attractive and two samples from the more susceptible line were identified as least attractive, based on the behavioural assays with gravid flies. Comparison of the headspace volatiles of these samples, using solid phase microextraction and gas chromatography‐mass spectrometry‐electroantennographic detection, revealed octanal and nonanal to be present in the attractive wool samples that elicited responses from the fly antenna. Furthermore, the two compounds were not present in wool that was least attractive to L. cuprina. In laboratory bioassays, octanal and nonanal evoked antennal and behavioural responses in gravid L. cuprina, thus confirming their potential role as semiochemicals responsible for attracting L. cuprina to Merino sheep.