Exhaustion of bacteria-specific CD4 T cells and microbial translocation in common variable immunodeficiency disorders.

In the present study, we have investigated the functional profile of CD4 T cells from patients with common variable immunodeficiency (CVID), including production of cytokines and proliferation in response to bacteria and virus-derived antigens. We show that the functional impairment of CD4 T cells, including the reduced capacity to proliferate and to produce IFN-γ and IL-2, was restricted to bacteria-specific and not virus-specific CD4 T cells. High levels of endotoxins were found in the plasma of patients with CVID, suggesting that CD4 T cell dysfunction might be caused by bacterial translocation. Of note, endotoxemia was associated with significantly higher expression of programmed death 1 (PD-1) on CD4 T cells. The blockade of the PD-1-PD-L1/2 axis in vitro restored CD4 T cell proliferation capacity, thus indicating that PD-1 signaling negatively regulates CD4 T cell functions. Finally, we showed that intravenous immunoglobulin G (IVIG) treatment significantly reduced endotoxemia and the percentage of PD-1(+) CD4 T cells, and restored bacteria-specific CD4 T cell cytokine production and proliferation. In conclusion, the present study demonstrates that the CD4 T cell exhaustion and functional impairment observed in CVID patients is associated with bacterial translocation and that IVIG treatment resolves bacterial translocation and restores CD4 T cell functions.

Development of bacteria-based bioassays for arsenic detection in natural waters.

Arsenic contamination of natural waters is a worldwide concern, as the drinking water supplies for large populations can have high concentrations of arsenic. Traditional techniques to detect arsenic in natural water samples can be costly and time-consuming; therefore, robust and inexpensive methods to detect arsenic in water are highly desirable. Additionally, methods for detecting arsenic in the field have been greatly sought after. This article focuses on the use of bacteria-based assays as an emerging method that is both robust and inexpensive for the detection of arsenic in groundwater both in the field and in the laboratory. The arsenic detection elements in bacteria-based bioassays are biosensor-reporter strains; genetically modified strains of, e.g., Escherichia coli, Bacillus subtilis, Staphylococcus aureus, and Rhodopseudomonas palustris. In response to the presence of arsenic, such bacteria produce a reporter protein, the amount or activity of which is measured in the bioassay. Some of these bacterial biosensor-reporters have been successfully utilized for comparative in-field analyses through the use of simple solution-based assays, but future methods may concentrate on miniaturization using fiberoptics or microfluidics platforms. Additionally, there are other potential emerging bioassays for the detection of arsenic in natural waters including nematodes and clams.

ESCMID postgraduate technical workshop on intracellular bacteria: from biology to clinic.

Infection by intracellular bacteria can lead to several diseases in both veterinary and human medicine. Unfortunately, the biology of these intracellular bacteria is highly complex due to their interactions with their host cells. Thus, it is very important to develop several tools in order to better understand the complex intracellular life of these pathogens, so allowing to improve the diagnosis options and the treatments of infectious diseases that they are causing. The workshop organised in Villars-sur-Ollon (Switzerland) by the ESCMID Study group on intracellular bacteria was a good opportunity to enhance our knowledge on these fastidious pathogens. During 5 days, 15 speakers gave 41 talks, covering all fields, from biology to clinic of different intracellular bacteria such as Bartonella, Chlamydia, Coxiella, Ehrlichia, Listeria, Parachlamydia, Rickettsia, and Waddlia. The format of this postgraduate course, which took place in the Swiss mountains, allowed interactive sessions and living discussions between the participants coming from all around the world. One of the major strength was to gather epidemiologists, clinical microbiologists, infectious diseases specialists, entomologists, veterinarians as well as bioinformaticians, biochemists and biologists to deliver a unique "one-health science" on intracellular bacteria. Here, we summarise the main take-home messages delivered during this meeting.

Characterization of fecal indicator bacteria in sediments cores from the largest freshwater lake of Western Europe (Lake Geneva, Switzerland)

This study characterized the fecal indicator bacteria (FIB), including Escherichia coli (E. coli) and Enteroccocus (ENT), disseminated over time in the Bay of Vidy, which is the most contaminated area of Lake Geneva. Sediments were collected from a site located at similar to 500 m from the present waste water treatment plant (WWTP) outlet pipe, in front of the former WWTP outlet pipe, which was located at only 300 m from the coastal recreational area (before 2001). E. coil and ENT were enumerated in sediment suspension using the membrane filter method. The FIB characterization was performed for human Enterococcus faecalis (E. faecalis) and Enterococcus faecium (E. faecium) and human specific bacteroides by PCR using specific primers and a matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS). Bacterial cultures revealed that maximum values of 35.2 x 10(8) and 6.6 x 10(6) CFU g(-1) dry sediment for E. coil and ENT, respectively, were found in the sediments deposited following eutrophication of Lake Geneva in the 1970s. whereas the WWTP started operating in 1964. The same tendency was observed for the presence of human fecal pollution: the percentage of PCR amplification with primers ESP-1/ESP-2 for E. faecalis and E. faecium indicated that more than 90% of these bacteria were from human origin. Interestingly, the PCR assays for specific-human bacteroides HF183/HF134 were positive for DNA extracted from all isolated strains of sediment surrounding WWPT outlet pipe discharge. The MALDI-TOF MS confirmed the presence of general E. coli and predominance E. faecium in isolated strains. Our results demonstrated that human fecal bacteria highly increased in the sediments contaminated with WWTP effluent following the eutrophication of Lake Geneva. Additionally, other FIB cultivable strains from animals or adapted environmental strains were detected in the sediment of the bay. The approaches used in this research are valuable to assess the temporal distribution and the source of the human fecal pollution in aquatic environments. (C) 2011 Elsevier Inc. All rights reserved.

Basic calcium phosphate crystals induce monocyte/macrophage IL-1(beta) secretion through the NLRP3 inflammasome in vitro.

Basic calcium phosphate (BCP) crystals are associated with severe osteoarthritis and acute periarticular inflammation. Three main forms of BCP crystals have been identified from pathological tissues: octacalcium phosphate, carbonate-substituted apatite, and hydroxyapatite. We investigated the proinflammatory effects of these BCP crystals in vitro with special regard to the involvement of the NLRP3-inflammasome in THP-1 cells, primary human monocytes and macrophages, and mouse bone marrow-derived macrophages (BMDM). THP-1 cells stimulated with BCP crystals produced IL-1β in a dose-dependent manner. Similarly, primary human cells and BMDM from wild-type mice also produced high concentrations of IL-1β after crystal stimulation. THP-1 cells transfected with short hairpin RNA against the components of the NLRP3 inflammasome and mouse BMDM from mice deficient for NLRP3, apoptosis-associated speck-like protein, or caspase-1 did not produce IL-1β after BCP crystal stimulation. BCP crystals induced macrophage apoptosis/necrosis as demonstrated by MTT and flow cytometric analysis. Collectively, these results demonstrate that BCP crystals induce IL-1β secretion through activating the NLRP3 inflammasome. Furthermore, we speculate that IL-1 blockade could be a novel strategy to inhibit BCP-induced inflammation in human disease.

The role of potassium in inflammasome activation by bacteria.

Many Gram-negative bacteria possess a type III secretion system (TTSS( paragraph sign)) that can activate the NLRC4 inflammasome, process caspase-1 and lead to secretion of mature IL-1beta. This is dependent on the presence of intracellular flagellin. Previous reports have suggested that this activation is independent of extracellular K(+) and not accompanied by leakage of K(+) from the cell, in contrast to activation of the NLRP3 inflammasome. However, non-flagellated strains of Pseudomonas aeruginosa are able to activate NLRC4, suggesting that formation of a pore in the cell membrane by the TTSS apparatus may be sufficient for inflammasome activation. Thus, we set out to determine if extracellular K(+) influenced P. aeruginosa inflammasome activation. We found that raising extracellular K(+) prevented TTSS NLRC4 activation by the non-flagellated P. aeruginosa strain PA103DeltaUDeltaT at concentrations above 90 mm, higher than those reported to inhibit NLRP3 activation. Infection was accompanied by efflux of K(+) from a minority of cells as determined using the K(+)-sensitive fluorophore PBFI, but no formation of a leaky pore. We obtained exactly the same results following infection with Salmonella typhimurium, previously described as independent of extracellular K(+). The inhibitory effect of raised extracellular K(+) on NLRC4 activation thus reflects a requirement for a decrease in intracellular K(+) for this inflammasome component as well as that described for NLRP3.

Combination of fluorescence microscopy and nanomotion detection to characterize bacteria.

Antibiotic-resistant pathogens are a major health concern in everyday clinical practice. Because their detection by conventional microbial techniques requires minimally 24 h, some of us have recently introduced a nanomechanical sensor, which can reveal motion at the nanoscale. By monitoring the fluctuations of the sensor, this technique can evidence the presence of bacteria and their susceptibility to antibiotics in less than 1 h. Their amplitude correlates to the metabolism of the bacteria and is a powerful tool to characterize these microorganisms at low densities. This technique is new and calls for an effort to optimize its protocol and determine its limits. Indeed, many questions remain unanswered, such as the detection limits or the correlation between the bacterial distribution on the sensor and the detection's output. In this work, we couple fluorescence microscopy to the nanomotion investigation to determine the optimal experimental protocols and to highlight the effect of the different bacterial distributions on the sensor.

Health effects of occupational exposure in a dairy food industry, with a specific assessment of exposure to airborne lactic acid bacteria

OBJECTIVE:: Lactic acid bacteria (LAB) are used in food industries as probiotic agents. The aim of this study is to assess the potential health effects of airborne exposure to a mix of preblend (LAB and carbohydrate) and milk powder in workers. METHODS:: A medical questionnaire, lung function tests, and immunologic tests were carried out on 50 workers. Occupational exposure to inhalable dust and airborne LAB was measured. RESULTS:: Workers not using respiratory masks reported more symptoms of irritation than workers using protection. Workers from areas with higher levels of airborne LAB reported the most health symptoms and the immune responses of workers to LAB was higher than the immune responses of a control population. CONCLUSIONS:: Measures to reduce exposure to airborne LAB and milk powder in food industries are recommended.

Structure and expression profile of the Arabidopsis PHO1 gene family indicates a broad role in inorganic phosphate homeostasis.

PHO1 has been recently identified as a protein involved in the loading of inorganic phosphate into the xylem of roots in Arabidopsis. The genome of Arabidopsis contains 11 members of the PHO1 gene family. The cDNAs of all PHO1 homologs have been cloned and sequenced. All proteins have the same topology and harbor a SPX tripartite domain in the N-terminal hydrophilic portion and an EXS domain in the C-terminal hydrophobic portion. The SPX and EXS domains have been identified in yeast (Saccharomyces cerevisiae) proteins involved in either phosphate transport or sensing or in sorting proteins to endomembranes. The Arabidopsis genome contains additional proteins of unknown function containing either a SPX or an EXS domain. Phylogenetic analysis indicated that the PHO1 family is subdivided into at least three clusters. Reverse transcription-PCR revealed a broad pattern of expression in leaves, roots, stems, and flowers for most genes, although two genes are expressed exclusively in flowers. Analysis of the activity of the promoter of all PHO1 homologs using promoter-beta-glucuronidase fusions revealed a predominant expression in the vascular tissues of roots, leaves, stems, or flowers. beta-Glucuronidase expression is also detected for several promoters in nonvascular tissue, including hydathodes, trichomes, root tip, root cortical/epidermal cells, and pollen grains. The expression pattern of PHO1 homologs indicates a likely role of the PHO1 proteins not only in the transfer of phosphate to the vascular cylinder of various tissues but also in the acquisition of phosphate into cells, such as pollen or root epidermal/cortical cells.

Phosphate and zinc transport and signalling in plants: toward a better understanding of their homeostasis interaction.

Inorganic phosphate (Pi) and zinc (Zn) are two essential nutrients for plant growth. In soils, these two minerals are either present in low amounts or are poorly available to plants. Consequently, worldwide agriculture has become dependent on external sources of Pi and Zn fertilizers to increase crop yields. However, this strategy is neither economically nor ecologically sustainable in the long term, particularly for Pi, which is a non-renewable resource. To date, research has emphasized the analysis of mineral nutrition considering each nutrient individually, and showed that Pi and Zn homeostasis is highly regulated in a complex process. Interestingly, numerous observations point to an unexpected interconnection between the homeostasis of the two nutrients. Nevertheless, despite their fundamental importance, the molecular bases and biological significance of these interactions remain largely unknown. Such interconnections can account for shortcomings of current agronomic models that typically focus on improving the assimilation of individual elements. Here, current knowledge on the regulation of the transport and signalling of Pi and Zn individually is reviewed, and then insights are provided on the recent progress made towards a better understanding of the Zn-Pi homeostasis interaction in plants.

Identification and characterization of the Arabidopsis PHO1 gene involved in phosphate loading to the xylem.

The Arabidopsis mutant pho1 is deficient in the transfer of Pi from root epidermal and cortical cells to the xylem. The PHO1 gene was identified by a map-based cloning strategy. The N-terminal half of PHO1 is mainly hydrophilic, whereas the C-terminal half has six potential membrane-spanning domains. PHO1 shows no homology with any characterized solute transporter, including the family of H(+)-Pi cotransporters identified in plants and fungi. PHO1 shows highest homology with the Rcm1 mammalian receptor for xenotropic murine leukemia retroviruses and with the Saccharomyces cerevisiae Syg1 protein involved in the mating pheromone signal transduction pathway. PHO1 is expressed predominantly in the roots and is upregulated weakly under Pi stress. Studies with PHO1 promoter-beta-glucuronidase constructs reveal predominant expression of the PHO1 promoter in the stelar cells of the root and the lower part of the hypocotyl. There also is beta-glucuronidase staining of endodermal cells that are adjacent to the protoxylem vessels. The Arabidopsis genome contains 10 additional genes showing homology with PHO1. Thus, PHO1 defines a novel class of proteins involved in ion transport in plants.

Degradation of pathogen quorum-sensing molecules by soil bacteria: a preventive and curative biological control mechanism.

Abstract The plasmid pME6863, carrying the aiiA gene from the soil bacterium Bacillus sp. A24 that encodes a lactonase enzyme able to degrade N-acyl-homoserine lactones (AHLs), was introduced into the rhizosphere isolate Pseudomonas fluorescens P3. This strain is not an effective biological control agent against plant pathogens. The transformant P. fluorescens P3/pME6863 acquired the ability to degrade AHLs. In planta, P. fluorescens P3/pME6863 significantly reduced potato soft rot caused by Erwinia carotovora and crown gall of tomato caused by Agrobacterium tumefaciens to a similar level as Bacillus sp. A24. Little or no disease reduction was observed for the wild-type strain P3 carrying the vector plasmid without aiiA. Suppression of potato soft rot was observed even when the AHL-degrading P. fluorescens P3/pME6863 was applied to tubers 2 days after the pathogen, indicating that biocontrol was not only preventive but also curative. When antagonists were applied individually with the bacterial plant pathogens, biocontrol activity of the AHL degraders was greater than that observed with several Pseudomonas 2,4-diacetylphloroglucinol-producing strains and with Pseudomonas chlororaphis PCL1391, which relies on production of phenazine antibiotic for disease suppression. Phenazine production by this well characterized biological control strain P. chlororaphis PCL1391 is regulated by AHL-mediated quorum sensing. When P. chlororaphis PCL1391 was co-inoculated with P. fluorescens P3/pME6863 in a strain mixture, the AHL degrader interfered with the normally excellent ability of the antibiotic producer to suppress tomato vascular wilt caused by Fusarium oxysporum f. sp. lycopersici. Our results demonstrate AHL degradation as a novel biocontrol mechanism, but also demonstrate the potential for non-target interactions that can interfere with the biocontrol efficacy of other strains.

Staphylococcal biofilm formation on the surface of three different calcium phosphate bone grafts: a qualitative and quantitative in vivo analysis.

Differences in physico-chemical characteristics of bone grafts to fill bone defects have been demonstrated to influence in vitro bacterial biofilm formation. Aim of the study was to investigate in vivo staphylococcal biofilm formation on different calcium phosphate bone substitutes. A foreign-body guinea-pig infection model was used. Teflon cages prefilled with β-tricalcium phosphate, calcium-deficient hydroxyapatite, or dicalcium phosphate (DCP) scaffold were implanted subcutaneously. Scaffolds were infected with 2 × 10(3) colony-forming unit of Staphylococcus aureus (two strains) or S. epidermidis and explanted after 3, 24 or 72 h of biofilm formation. Quantitative and qualitative biofilm analysis was performed by sonication followed by viable counts, and microcalorimetry, respectively. Independently of the material, S. aureus formed increasing amounts of biofilm on the surface of all scaffolds over time as determined by both methods. For S. epidermidis, the biofilm amount decreased over time, and no biofilm was detected by microcalorimetry on the DCP scaffolds after 72 h of infection. However, when using a higher S. epidermidis inoculum, increasing amounts of biofilm were formed on all scaffolds as determined by microcalorimetry. No significant variation in staphylococcal in vivo biofilm formation was observed between the different materials tested. This study highlights the importance of in vivo studies, in addition to in vitro studies, when investigating biofilm formation of bone grafts.