Modulation of chemokine gene expression by Shiga-toxin producing Escherichia coli belonging to various origins and serotypes

Infection with Shiga-toxin producing Escherichia coli (STEC) may result in the development of the haemolytic-uremic syndrome (HUS), the main cause of acute renal failure in children. While O157:H7 STEC are associated with large outbreaks of HUS, it is difficult to predict whether a non-O157:H7 isolate can be pathogenic for humans. The mucosal innate immune response plays a central role in the pathogenesis of HUS; therefore, we compared the induction of IL-8 and CCL20 in human colon epithelial cells infected with strains belonging to different serotypes, isolated from cattle or from HUS patients. No correlation was observed between strain virulence and chemokine gene expression. Rather, the genetic background of the strains seems to determine the chemokine gene expression profile. Investigating the contribution of different bacterial factors in this process, we show that the type III secretion system of O157:H7 bacteria, but not the intimate adhesion, is required to stimulate the cells. In addition, H7, H10, and H21 flagellins are potent inducers of chemokine gene expression when synthesized in large amount.

AvxA, a composite serine-protease-RTX toxin of Avibacterium paragallinarum

Avibacterium paragallinarum, the etiological agent of infectious coryza in chicken, was found to encode a bivalent serine-protease - RTX-porin toxin named AvxA. This toxin is encoded on a classical RTX operon structure with the activator gene avxC, the structural serin-protease-RTX toxin gene avxA, and the genes for a proper type I secretion system avxBD. AvxA is activated by the product of the avxC gene, secreted by the avxBD specified type I secretion system and proteolytically processed leaving a 95 kDa RTX moiety that is found in culture supernatants of A. paragallinarum serovars A, B and C. The RTX moiety of AvxA (AvxA-RTX) is cytotoxic against the avian macrophage like cell line HD11 but not against bovine macrophage cell line BoMac. Purified IgG from hyper-immune rabbit anti-AvxA-RTX serum made by immunization with recombinant AvxA-RTX from a serotype A strain fully neutralizes the cytotoxic activity of recombinant active AvxA-RTX and of A. paragallinarum serotypes A, B and C. This indicates that AvxA is a common major virulence attribute of all A. paragallinarum serotypes.

Microbe sampling by mucosal dendritic cells is a discrete, MyD88-independent step in DeltainvG S. Typhimurium colitis.

Intestinal dendritic cells (DCs) are believed to sample and present commensal bacteria to the gut-associated immune system to maintain immune homeostasis. How antigen sampling pathways handle intestinal pathogens remains elusive. We present a murine colitogenic Salmonella infection model that is highly dependent on DCs. Conditional DC depletion experiments revealed that intestinal virulence of S. Typhimurium SL1344 DeltainvG mutant lacking a functional type 3 secretion system-1 (DeltainvG)critically required DCs for invasion across the epithelium. The DC-dependency was limited to the early phase of infection when bacteria colocalized with CD11c(+)CX3CR1(+) mucosal DCs. At later stages, the bacteria became associated with other (CD11c(-)CX3CR1(-)) lamina propria cells, DC depletion no longer attenuated the pathology, and a MyD88-dependent mucosal inflammation was initiated. Using bone marrow chimeric mice, we showed that the MyD88 signaling within hematopoietic cells, which are distinct from DCs, was required and sufficient for induction of the colitis. Moreover, MyD88-deficient DCs supported transepithelial uptake of the bacteria and the induction of MyD88-dependent colitis. These results establish that pathogen sampling by DCs is a discrete, and MyD88-independent, step during the initiation of a mucosal innate immune response to bacterial infection in vivo.

Comparison of Salmonella enterica serovar Typhimurium colitis in germfree mice and mice pretreated with streptomycin.

Salmonella enterica subspecies 1 serovar Typhimurium is a common cause of bacterial enterocolitis. Mice are generally protected from Salmonella serovar Typhimurium colonization and enterocolitis by their resident intestinal microflora. This phenomenon is called "colonization resistance" (CR). Two murine Salmonella serovar Typhimurium infection models are based on the neutralization of CR: (i) in specific-pathogen-free mice pretreated with streptomycin (StrSPF mice) antibiotics disrupt the intestinal microflora; and (ii) germfree (GF) mice are raised without any intestinal microflora, but their intestines show distinct physiologic and immunologic characteristics. It has been unclear whether the same pathogenetic mechanisms trigger Salmonella serovar Typhimurium colitis in GF and StrSPF mice. In this study, we compared the two colitis models. In both of the models Salmonella serovar Typhimurium efficiently colonized the large intestine and triggered cecum and colon inflammation starting 8 h postinfection. The type III secretion system encoded in Salmonella pathogenicity island 1 was essential in both disease models. Thus, Salmonella serovar Typhimurium colitis is triggered by similar pathogenetic mechanisms in StrSPF and GF mice. This is remarkable considering the distinct physiological properties of the GF mouse gut. One obvious difference was more pronounced damage and reduced regenerative response of the cecal epithelium in GF mice. Overall, StrSPF mice and GF mice provide similar but not identical models for Salmonella serovar Typhimurium colitis.

Flagella and chemotaxis are required for efficient induction of Salmonella enterica serovar Typhimurium colitis in streptomycin-pretreated mice.

Salmonella enterica subspecies 1 serovar Typhimurium is a common cause of gastrointestinal infections. The host's innate immune system and a complex set of Salmonella virulence factors are thought to contribute to enteric disease. The serovar Typhimurium virulence factors have been studied extensively by using tissue culture assays, and bovine infection models have been used to verify the role of these factors in enterocolitis. Streptomycin-pretreated mice provide an alternative animal model to study enteric salmonellosis. In this model, the Salmonella pathogenicity island 1 type III secretion system has a key virulence function. Nothing is known about the role of other virulence factors. We investigated the role of flagella in murine serovar Typhimurium colitis. A nonflagellated serovar Typhimurium mutant (fliGHI) efficiently colonized the intestine but caused little colitis during the early phase of infection (10 and 24 h postinfection). In competition assays with differentially labeled strains, the fliGHI mutant had a reduced capacity to get near the intestinal epithelium, as determined by fluorescence microscopy. A flagellated but nonchemotactic cheY mutant had the same virulence defects as the fliGHI mutant for causing colitis. In competitive infections, both mutants colonized the intestine of streptomycin-pretreated mice by day 1 postinfection but were outcompeted by the wild-type strain by day 3 postinfection. Together, these data demonstrate that flagella are required for efficient colonization and induction of colitis in streptomycin-pretreated mice. This effect is mostly attributable to chemotaxis. Recognition of flagellar subunits (i.e., flagellin) by innate immune receptors (i.e., Toll-like receptor 5) may be less important.

Role of the Salmonella pathogenicity island 1 effector proteins SipA, SopB, SopE, and SopE2 in Salmonella enterica subspecies 1 serovar Typhimurium colitis in streptomycin-pretreated mice.

Salmonella enterica subspecies 1 serovar Typhimurium (serovar Typhimurium) induces enterocolitis in humans and cattle. The mechanisms of enteric salmonellosis have been studied most extensively in calf infection models. The previous studies established that effector protein translocation into host cells via the Salmonella pathogenicity island 1 (SPI-1) type III secretion system (TTSS) is of central importance in serovar Typhimurium enterocolitis. We recently found that orally streptomycin-pretreated mice provide an alternative model for serovar Typhimurium colitis. In this model the SPI-1 TTSS also plays a key role in the elicitation of intestinal inflammation. However, whether intestinal inflammation in calves and intestinal inflammation in streptomycin-pretreated mice are induced by the same SPI-1 effector proteins is still unclear. Therefore, we analyzed the role of the SPI-1 effector proteins SopB/SigD, SopE, SopE2, and SipA/SspA in elicitation of intestinal inflammation in the murine model. We found that sipA, sopE, and, to a lesser degree, sopE2 contribute to murine colitis, but we could not assign an inflammation phenotype to sopB. These findings are in line with previous studies performed with orally infected calves. Extending these observations, we demonstrated that in addition to SipA, SopE and SopE2 can induce intestinal inflammation independent of each other and in the absence of SopB. In conclusion, our data corroborate the finding that streptomycin-pretreated mice provide a useful model for studying the molecular mechanisms of serovar Typhimurium colitis and are an important starting point for analysis of the molecular events triggered by SopE, SopE2, and SipA in vivo.

Pretreatment of mice with streptomycin provides a Salmonella enterica serovar Typhimurium colitis model that allows analysis of both pathogen and host.

Salmonella enterica subspecies 1 serovar Typhimurium is a principal cause of human enterocolitis. For unknown reasons, in mice serovar Typhimurium does not provoke intestinal inflammation but rather targets the gut-associated lymphatic tissues and causes a systemic typhoid-like infection. The lack of a suitable murine model has limited the analysis of the pathogenetic mechanisms of intestinal salmonellosis. We describe here how streptomycin-pretreated mice provide a mouse model for serovar Typhimurium colitis. Serovar Typhimurium colitis in streptomycin-pretreated mice resembles many aspects of the human infection, including epithelial ulceration, edema, induction of intercellular adhesion molecule 1, and massive infiltration of PMN/CD18(+) cells. This pathology is strongly dependent on protein translocation via the serovar Typhimurium SPI1 type III secretion system. Using a lymphotoxin beta-receptor knockout mouse strain that lacks all lymph nodes and organized gut-associated lymphatic tissues, we demonstrate that Peyer's patches and mesenteric lymph nodes are dispensable for the initiation of murine serovar Typhimurium colitis. Our results demonstrate that streptomycin-pretreated mice offer a unique infection model that allows for the first time to use mutants of both the pathogen and the host to study the molecular mechanisms of enteric salmonellosis.

Isolated GH deficiency type II: knockdown of the harmful Delta3GH recovers wt-GH secretion in rat tumor pituitary cells

Isolated GH deficiency type II (IGHD II) is the autosomal dominant form of GHD. In the majority of the cases, this disorder is due to specific GH-1 gene mutations that lead to mRNA missplicing and subsequent loss of exon 3 sequences. When misspliced RNA is translated, it produces a toxic 17.5-kDa GH (Delta3GH) isoform that reduces the accumulation and secretion of wild-type-GH. At present, patients suffering from this type of disease are treated with daily injections of recombinant human GH in order to maintain normal growth. However, this type of replacement therapy does not prevent toxic effects of the Delta3GH mutant on the pituitary gland, which can eventually lead to other hormonal deficiencies. We developed a strategy involving Delta3GH isoform knockdown mediated by expression of a microRNA-30-adapted short hairpin RNA (shRNA) specifically targeting the Delta3GH mRNA of human (shRNAmir-Delta3). Rat pituitary tumor GC cells expressing Delta3GH upon doxycycline induction were transduced with shRNAmir-Delta3 lentiviral vectors, which significantly reduced Delta3GH protein levels and improved human wild-type-GH secretion in comparison with a shRNAmir targeting a scrambled sequence. No toxicity due to shRNAmir expression could be observed in cell proliferation assays. Confocal microscopy strongly suggested that shRNAmir-Delta3 enabled the recovery of GH granule storage and secretory capacity. These viral vectors have shown their ability to stably integrate, express shRNAmir, and rescue IGHD II phenotype in rat pituitary tumor GC cells, a methodology that opens new perspectives for the development of gene therapy to treat IGHD patients.

Nucleic acid sensing by an orthogonal reporter system based on homo-DNA

We have developed an assay for single strand DNA or RNA detection which is based on the homo-DNA templated Staudinger reduction of the profluorophore rhodamine-azide. The assay is based on a three component system, consisting of a homo-DNA/DNA hybrid probe, a set of homo-DNA reporter strands and the target DNA or RNA. We present two different formats of the assay (Omega probe and linear probe) in which the linear probe was found to perform best with catalytic turnover of the reporter strands (TON: 8) and a match/mismatch discrimination of up to 19. The advantage of this system is that the reporting (homo-DNA) and sensing (DNA) domain are decoupled from each other since the two pairing systems are bioorthogonal. This allows independent optimization of either domain which may lead to higher selectivity in in vivo imaging.

Oral prednisolone induced acute generalized exanthematous pustulosis due to corticosteroids of group A confirmed by epicutaneous testing and lymphocyte transformation tests

BACKGROUND: Acute generalized exanthematous pustulosis (AGEP) is a rare cutaneous eruption which is often provoked by drugs. CASE REPORT: We report 2 cases of AGEP which showed rapidly spreading pustular eruptions accompanied by malaise, fever and neutrophilia after the administration of systemic prednisolone (corticosteroid of group A, hydrocortisone type). The histological examination showing neutrophilic subcorneal spongiform pustules was consistent with the diagnosis of AGEP. In both cases the rash cleared within a week upon treatment with topical steroids (corticosteroid of group D1, betamethasonedipropionate type and corticosteroid of group D2, hydrocortisone-17-butyrate type). Three months after recovery, the sensitization to corticosteroids of group A was confirmed by epicutaneous testing and positive lymphocyte transformation tests. CONCLUSION: These cases show that systemic corticosteroids can induce AGEP and demonstrate that epicutaneous testing and lymphocyte transformation tests may be helpful in identifying the causative drug. Our data support previous reports indicating an important role for drug-specific T cells in inducing neutrophil inflammation in this disease.

VirB6 is required for stabilization of VirB5 and VirB3 and formation of VirB7 homodimers in Agrobacterium tumefaciens.

VirB6 from Agrobacterium tumefaciens is an essential component of the type IV secretion machinery for T pilus formation and genetic transformation of plants. Due to its predicted topology as a polytopic inner membrane protein, it was proposed to form the transport pore for cell-to-cell transfer of genetic material and proteinaceous virulence factors. Here, we show that the absence of VirB6 leads to reduced cellular levels of VirB5 and VirB3, which were proposed to assist T pilus formation as minor component(s) or assembly factor(s), respectively. Overexpression of virB6 in trans restored levels of cell-bound and T pilus-associated VirB5 to wild type but did not restore VirB3 levels. Thus, VirB6 has a stabilizing effect on VirB5 accumulation, thereby regulating T pilus assembly. In the absence of VirB6, cell-bound VirB7 monomers and VirB7-VirB9 heterodimers were reduced and VirB7 homodimer formation was abolished. This effect could not be restored by expression of VirB6 in trans. Expression of TraD, a component of the transfer machinery of the IncN plasmid pKM101, with significant sequence similarity to VirB6, restored neither protein levels nor bacterial virulence but partly permitted T pilus formation in a virB6 deletion strain. VirB6 may therefore regulate T pilus formation by direct interaction with VirB5, and wild-type levels of VirB3 and VirB7 homodimers are not required.

Comparative carbon cycle dynamics of the present and last interglacial

Changes in temperature and carbon dioxide during glacial cycles recorded in Antarctic ice cores are tightly coupled. However, this relationship does not hold for interglacials. While climate cooled towards the end of both the last (Eemian) and present (Holocene) interglacials, CO₂ remained stable during the Eemian while rising in the Holocene. We identify and review twelve biogeochemical mechanisms of terrestrial (vegetation dynamics and CO₂ fertilization, land use, wild fire, accumulation of peat, changes in permafrost carbon, subaerial volcanic outgassing) and marine origin (changes in sea surface temperature, carbonate compensation to deglaciation and terrestrial biosphere regrowth, shallow-water carbonate sedimentation, changes in the soft tissue pump, and methane hydrates), which potentially may have contributed to the CO₂ dynamics during interglacials but which remain not well quantified. We use three Earth System Models (ESMs) of intermediate complexity to compare effects of selected mechanisms on the interglacial CO₂ and δ¹³ CO₂ changes, focusing on those with substantial potential impacts: namely carbonate sedimentation in shallow waters, peat growth, and (in the case of the Holocene) human land use. A set of specified carbon cycle forcings could qualitatively explain atmospheric CO₂ dynamics from 8ka BP to the pre-industrial. However, when applied to Eemian boundary conditions from 126 to 115 ka BP, the same set of forcings led to disagreement with the observed direction of CO₂ changes after 122 ka BP. This failure to simulate late-Eemian CO₂ dynamics could be a result of the imposed forcings such as prescribed CaCO₃ accumulation and/or an incorrect response of simulated terrestrial carbon to the surface cooling at the end of the interglacial. These experiments also reveal that key natural processes of interglacial CO₂ dynamics eshallow water CaCO₃ accumulation, peat and permafrost carbon dynamics are not well represented in the current ESMs. Global-scale modeling of these long-term carbon cycle components started only in the last decade, and uncertainty in parameterization of these mechanisms is a main limitation in the successful modeling of interglacial CO₂ dynamics.

Modularity and Integrity Issues in Instructional Engineering

Course materials for e-learning are a special type of information system (IS). Thus, in the development of educational material one may learn from principles, methods, and tools that originated in the Software Engineering (SE) discipline and that are relevant in similar ways in "Instructional Engineering". An important SE principle is mo dularization, which supports properties like reusability and adaptability of code. To foster the adaptability of courseware we present a concept in which learning material is organized as a library of modular course objects. A certain lecturer may customize the courseware according to his specific course requirements. He must consider logical dependencies of and relationship integrity between selected course objects. We discuss integrity issues that have to be regarded for the composition of consistent course materials.

Growth hormone (GH) deficiency type II: a novel GH-1 gene mutation (GH-R178H) affecting secretion and action

Context and Objective: Main features of the autosomal dominant form of GH deficiency (IGHD II) include markedly reduced secretion of GH combined with low concentrations of IGF-I leading to short stature. Design, Setting, and Patients: A female patient presented with short stature (height -6.0 sd score) and a delayed bone age of 2 yr at the chronological age of 5 yr. Later, at the age of 9 yr, GHD was confirmed by standard GH provocation test, which revealed subnormal concentrations of GH and a very low IGF-I. Genetic analysis of the GH-1 gene revealed the presence of a heterozygous R178H mutation. Interventions and Results: AtT-20 cells coexpressing both wt-GH and GH-R178H showed a reduced GH secretion after forskolin stimulation compared with the cells expressing only wt-GH, supporting the diagnosis of IGHD II. Because reduced GH concentrations found in the circulation of our untreated patient could not totally explain her severe short stature, functional characterization of the GH-R178H performed by studies of GH receptor binding and activation of the Janus kinase-2/signal transducer and activator of transcription-5 pathway revealed a reduced binding affinity of GH-R178H for GH receptor and signaling compared with the wt-GH. Conclusion: This is the first report of a patient suffering from short stature caused by a GH-1 gene alteration affecting not only GH secretion (IGHD II) but also GH binding and signaling, highlighting the necessity of functional analysis of any GH variant, even in the alleged situation of IGHD II.

Validity of an automatic measure protocol in distal femur for allograft selection from a three-dimensional virtual bone bank system

Osteoarticular allograft is one possible treatment in wide surgical resections with large defects. Performing best osteoarticular allograft selection is of great relevance for optimal exploitation of the bone databank, good surgery outcome and patient’s recovery. Current approaches are, however, very time consuming hindering these points in practice. We present a validation study of a software able to perform automatic bone measurements used to automatically assess the distal femur sizes across a databank. 170 distal femur surfaces were reconstructed from CT data and measured manually using a size measure protocol taking into account the transepicondyler distance (A), anterior-posterior distance in medial condyle (B) and anterior-posterior distance in lateral condyle (C). Intra- and inter-observer studies were conducted and regarded as ground truth measurements. Manual and automatic measures were compared. For the automatic measurements, the correlation coefficients between observer one and automatic method, were of 0.99 for A measure and 0.96 for B and C measures. The average time needed to perform the measurements was of 16 h for both manual measurements, and of 3 min for the automatic method. Results demonstrate the high reliability and, most importantly, high repeatability of the proposed approach, and considerable speed-up on the planning.