Activation of the inflammasome upon Francisella tularensis infection: interplay of innate immune pathways and virulence factors

Tularaemia is a zoonotic disease caused by the facultative intracellular bacterium Francisella tularensis. The virulence of this pathogen depends on its ability to escape into the cytosol of host cells. Pathogens are detected by the innate immune system's pattern recognition receptors which are activated in response to conserved microbial molecules (pathogen-associated molecular patterns). Cytosolic bacteria are sensed intracellularly, often leading to activation of the cysteine protease caspase-1 within a multimolecular complex called the inflammasome. Caspase-1 activation leads to both host cell death and release of pro-inflammatory cytokines in a process called pyroptosis. Here we review the pathway leading to, and the consequences of, inflammasome activation upon F. tularensis infection both in vitro and in vivo. Finally, we discuss recent data on how other innate immune pathways and F. tularensis virulence factors control the activation of the inflammasome during infection.     

Inflammasome adaptors and sensors: intracellular regulators of infection and inflammation

The NOD-like receptors have important roles in innate immunity as intracellular sensors of microbial components and cell injury. It has been proposed that these cytosolic proteins regulate the cysteine protease caspase-1 within a multiprotein complex known as the 'inflammasome'. Activation of caspase-1 leads to the cleavage and activation of pro-inflammatory cytokines such as interleukin-1beta (IL-1beta) and IL-18, as well as host-cell death. The analysis of mice that are deficient in various inflammasome components has revealed that the inflammasome is a dynamic entity that is assembled from different adaptors in a stimulus-dependent manner. Here we review recent work on the activation of the inflammasome in response to various bacterial pathogens and tissue damage.     

Preoperative bi-fractionated accelerated radiation therapy for combined treatment of locally advanced rectal cancer in a consectutive series of unselected patients

Background

although preoperative RT (Radiation Therapy) is becoming the preferred approach for combined treatment of locally advanced rectal adenocarcinoma, no regimen can be now considered as a standard. Since the toxicity of preoperative RT isn't yet completely known, and the advantages of preoperative RT could be counterbalanced by increased postoperative morbidity and mortality, a monocentre series of preoperative bifractionated accelerated RT was retrospectively reviewed to clarify toxicity and outcomes after a prolonged follow up.

Methods

patients were screened following these eligibility criteria: histology-proven adenocarcinoma of the rectum; distal tumour extent at 12 cm or less from the anal verge; clinical stage T3–4/anyN, or anyT/N1–2; ECOG Performance Status 0–2. A total dose of 41.6 Gy (26 twice daily fractions of 1.6 Gy) was delivered. Surgery was carried out 17 ± 2 days after RT completion, adopting the total mesorectal excision technique.

Results

24 men and 23 women were enrolled; median age was 55 years (r.: 39–77). Twenty-eight patients were stage II and 19 stage III. 9 patients suffered from a recurrent tumour. 2 patients experienced a severe grade 4 gastrointestinal toxicity (a colo-vaginal fistula and an intestinal obstruction, both successfully treated). Operative mortality was nil; postoperative early complications occurred in 13 cases; mean length of hospital stay was 15 days. After a mean follow up of 44 months (r.: 18–84) 8 patients had deceased for recurrent disease, 15 were alive with a disease progression (2 pelvic recurrences and 13 pure distant deposits) and 24 were alive, without disease. The 5-year actuarial overall survival was 74.2%, the disease-free survival 62.9% and the regional control rate 84.7%. Long-term complications included 1 case of radiation enteritis requiring surgery, 2 cases of anastomotic stricture and 3 cases of bladder incontinence.

Conclusion

bifractionated accelerated RT administered in the preoperative setting to patients bearing locally advanced rectal cancer is reliable and safe, as its immediate and late toxicity (mainly infectious) is acceptably low and long-term survivals are achievable. These findings support the increasing use of preoperative RT for treatment of this malignancy in experienced centres. Ongoing multicentric trials are expected to address still unsolved issues, including the benefit of CT adjunct to preoperative RT.

     

Genetic characterization of Cryptosporidium species from humans in Spain

Several species of Cryptosporidium have been associated with infection. Cryptosporidium parvum and Cryptosporidium hominis are the main agents of cryptosporidiosis in humans. Stool samples from 108 Cryptosporidium-infected patients were submitted to PCR-RFLP analysis for a 553-bp fragment of Cryptosporidium oocyst wall protein (COWP) gene and an 826-864 bp fragment of the small-subunit ribosomal RNA (SSU-rRNA) gene. Ninety-two patients were immunocompetent children and 16 were HIV-infected adults. C. hominis was detected in 69 patients (59 immunocompetent and 10 HIV-infected); C. parvum, in 34 patients (28 immunocompetent and 6 HIV-infected); and C. meleagridis and C. felis in one patient each (both immunocompetent children). Three samples yielded negative results. C. parvum was significantly more frequent in children from rural areas than in those of urban residence (p=0.010). As far as we know, this is the first surveillance study about the molecular characterization of Cryptosporidium in humans performed in Spain. The finding of zoonotic species infecting humans calls for further research on this subject.     

Effects of food limitation and temperature on cladocerans from a tropical Brazilian lake

Food limitation was tested in the laboratory by individual growth and reproduction of two cladoceran species, Ceriodaphnia richardi and Daphnia gessneri, from the shallow tropical Brazilian Lake Monte Alegre. The cladocerans were fed cultivated green alga Scenedesmus spinosus in concentrations of 0.20, 0.10, 0.05, and 0.025 mg C l⁻¹. Higher biomass and growth rates occurred in the two highest-food concentrations; the two lowest ones negatively affected clutch size and first reproduction. The threshold food concentration is lower than 0.025 mg C l⁻¹ and the incipient limiting level is a value between 0.10 and 0.20 mg C l⁻¹. The largest species, D. gessneri, was more sensitive to low food concentrations. The effects of low and high temperatures (19 and 27°C) were evaluated by life table experiments with three cladocerans from the lake—Daphnia ambigua, D. gessneri, and Moina micrura—with no food limitation (1 mg C l⁻¹ of S. spinosus). Higher population growth rates for the three species were found at 27°C; better performance in most life table parameters was observed for the former two species at the highest temperature, D. gessneri being the most sensitive to the lowest temperature. There are indications that temperature is an important abiotic factor that constrains populations of cladocerans for a short period in winter in the lake, when temperature decreases to 18–19°C. However, its influence cannot be separated from a biotic factor such as food, whose effect is stronger in the cool season, when concentrations are lower and contribution of inedible algae is relatively higher.     

Correlated three-dimensional light and electron microscopy reveals transformation of mitochondria during apoptosis

In addition to their role in cellular bioenergetics, mitochondria also initiate common forms of programmed cell death (apoptosis) through the release of proteins such as cytochrome c from the intermembrane and intracristal spaces. The release of these proteins is studied in populations of cells by western blotting mitochondrial and cytoplasmic fractions of cellular extracts, and in single cells by fluorescence microscopy using fluorescent indicators and fusion proteins. However, studying the changes in ultrastructure associated with release of proteins requires the higher resolution provided by transmission electron microscopy. Here, we have used fluorescence microscopy to characterize the state of apoptosis in HeLa cells treated with etoposide followed by electron microscopy and three-dimensional electron microscope tomography of the identical cells to study the sequence of structural changes. We have identified a remodelling of the inner mitochondrial membrane into many separate vesicular matrix compartments that accompanies release of proteins; however, this remodelling is not required for efficient release of cytochrome c. Swelling occurs only late in apoptosis after release of cytochrome c and loss of the mitochondrial membrane potential.     

Fibroblast-led collective invasion of carcinoma cells with differing roles for RhoGTPases in leading and following cells

Imaging of collectively invading cocultures of carcinoma cells and stromal fibroblasts reveals that the leading cell is always a fibroblast and that carcinoma cells move within tracks in the extracellular matrix behind the fibroblast. The generation of these tracks by fibroblasts is sufficient to enable the collective invasion of the squamous cell carcinoma (SCC) cells and requires both protease- and force-mediated matrix remodelling. Force-mediated matrix remodelling depends on integrins alpha3 and alpha5, and Rho-mediated regulation of myosin light chain (MLC) activity in fibroblasts, but these factors are not required in carcinoma cells. Instead, carcinoma cells use Cdc42 and MRCK (myotonic dystrophy kinase-related CDC42-binding protein kinases) mediated regulation of MLC to follow the tracks generated by fibroblasts.     

Lactobacillli expressing llama VHH fragments neutralise <Emphasis Type="Italic">Lactococcus</Emphasis>phages

Background  

Bacteriophages infecting lactic acid bacteria (LAB) are widely acknowledged as the main cause of milk fermentation failures. In this study, we describe the surface-expression as well as the secretion of two functional llama heavy-chain antibody fragments, one binding to the major capsid protein (MCP) and the other to the receptor-binding proteins (RBP) of the lactococcal bacteriophage p2, by lactobacilli in order to neutralise lactococcal phages.