Immunohistological analysis of Tannerella forsythia-induced lesions in a murine model

Tannerella forsythia has been implicated as a defined periodontal pathogen. In the present study a mouse model was used to determine the phenotype of leukocytes in the lesions induced by subcutaneous injections of either live (group A) or nonviable (group B) T. forsythia. Control mice (group C) received the vehicle only. Lesions were excised at days 1, 2, 4, and 7. An avidin-biotin immunoperoxidase method was used to stain infiltrating CD4(+) and CD8(+) T cells, CD14(+) macrophages, CD19(+) B cells, and neutrophils. Hematoxylin and eosin sections demonstrated lesions with central necrotic cores surrounded by neutrophils, macrophages and lymphocytes in both group A and group B mice. Lesions from control mice exhibited no or only occasional solitary leukocytes. In both groups A and B, neutrophils were the dominant leukocyte in the lesion 1 day after injection, the numbers decreasing over the 7-day experimental period. There was a relatively low mean percent of CD4(+) and CD8(+) T cells in the lesions and, whereas the percent of CD8(+) T cells remained constant, there was a significant increase in the percent of CD4(+) T cells at day 7. This increase was more evident in group A mice. The mean percent of CD14(+) macrophages and CD19(+) B cells remained low over the experimental period, although there was a significantly higher mean percent of CD19(+) B cells at day 1. In conclusion, the results showed that immunization of mice with live T. forsythia induced a stronger immune response than nonviable organisms. The inflammatory response presented as a nonspecific immune response with evidence of an adaptive (T-cell) response by day 7. Unlike Porphyromonas gingivalis, there was no inhibition of neutrophil migration.

Enzymatic characterization and homology model of a catalytically active recombinant West Nile virus NS3 protease

West Nile Virus (WNV) is a mosquito-borne flavivirus with a rapidly expanding global distribution. Infection causes severe neurological disease and fatalities in both human and animal hosts. The West Nile viral protease (NS2B-NS3) is essential for post-translational processing in host-infected cells of a viral polypeptide precursor into structural and functional viral proteins, and its inhibition could represent a potential treatment for viral infections. This article describes the design, expression, and enzymatic characterization of a catalytically active recombinant WNV protease, consisting of a 40-residue component of cofactor NS2B tethered via a noncleavable nonapeptide (G(4)SG(4)) to the N-terminal 184 residues of NS3. A chromogenic assay using synthetic para-nitroanilide (pNA) hexapeptide substrates was used to identify optimal enzyme-processing conditions (pH 9.5, I < 0.1 M, 30% glycerol, 1 mM CHAPS), preferred substrate cleavage sites, and the first competitive inhibitor (Ac-FASGKR- H, IC50 &SIM; 1 μM). A putative three-dimensional structure of WNV protease, created through homology modeling based on the crystal structures of Dengue-2 and Hepatitis C NS3 viral proteases, provides some valuable insights for structure-based design of potent and selective inhibitors of WNV protease.

Optimal design for model discrimination and parameter estimation for itraconazole population pharmacokinetics in cystic fibrosis patients

Optimal sampling times are found for a study in which one of the primary purposes is to develop a model of the pharmacokinetics of itraconazole in patients with cystic fibrosis for both capsule and solution doses. The optimal design is expected to produce reliable estimates of population parameters for two different structural PK models. Data collected at these sampling times are also expected to provide the researchers with sufficient information to reasonably discriminate between the two competing structural models.

Cytotoxic T-lymphocyte epitope vaccination protects against human metapneumovirus infection and disease in mice

Human metapneumovirus (hMPV) has emerged as an important human respiratory pathogen causing upper and lower respiratory tract infections in young children and older adults. In addition, hMPV infection is associated with asthma exacerbation in young children. Recent epidemiological evidence indicates that hMPV may cocircullate with human respiratory syncytial virus (hRSV) and mediate clinical disease similar to that seen with hRSV. Therefore, a vaccine for hMPV is highly desirable. In the present study, we used predictive bioinformatics, peptide immunization, and functional T-cell assays to define hMPV cytotoxic T-lymphocyte (CTL) epitopes recognized by mouse T cells restricted through several major histocompatibility complex class I alleles, including HILA-A*0201. We demonstrate that peptide immunization with hMPV CTL epitopes reduces viral load and immunopathollogy in the lungs of hMPV-challenged mice and enhances the expression of Th1-type cytokines (gamma interferon and interleukin-12 [IL-12]) in lungs and regional lymph nodes. In addition, we show that levels of Th2-type cytolkines (IL-10 and IL-4) are significantly lower in hMPV CTL epitope-vaccinated mice challenged with hMPV. These results demonstrate for the first time the efficacy of an hMPV CTL epitope vaccine in the control of hMPV infection in a murine model.

Familial aggregation of human infection with Schistosoma japonicum in the Poyang Lake region, China

Despite the success of extensive control measures that have been implemented in China for over 50 years, the number of individuals infected with Schistosoma japonicum remains high in the existing endemic areas. A variance components analysis was undertaken to estimate the heritable and environmental components that contribute to S. japonicum infection in the Poyang Lake region of Jiangxi Province, PR China. The total target population was 3148 from four separate administrative villages. Two thousand seven hundred and five of these comprised 400 families ranging in size from 3 to 188. After adjustments were made for gender, water contact and past history of having had schistosomiasis, the heritable component was estimated to account for as much as 58% of the phenotype variation under the polygenic model. Household was not shown to be an important environmental factor. Incorporating village effects indicated that the results were valid for the total population. We conclude that genetic heritability in this region is high and plays an important role in determining risk of infection with S. japonicum. (c) 2005 Australian Society for Parasitology Inc. Published by Elsevier Ltd. All rights reserved.

Salicylic acid mediates resistance to the vascular wilt pathogen Fusarium oxysporum in the model host Arabidopsis thaliana

Fusarium oxysporum is a soilborne fungal pathogen that causes major economic losses by inducing necrosis and wilting symptoms in many crop plants. In this study, the interaction between F. oxysporum and the model plant Arabidopsis thaliana has been investigated to better understand the nature of host defences that are effective against the Fusarium wilt pathogen. The expression of salicylate- and jasmonate-responsive defence genes in F. oxysporum-challenged roots of A. thaliana plants as well as in the roots of plants whose leaves were treated with salicylate or jasmonate was analysed. Unexpectedly, genes (e.g. PR1, PDF1.2, and CHIB) encoding proteins with defensive functions or transcription factors (e.g. ERF1, AtERF2, AtERF4 and AtMYC2) known to positively or negatively regulate defences against F. oxysporum were not activated in F. oxysporum-inoculated roots. In contrast, the jasmonate-responsive defence gene PDF1.2 was induced in the leaves of plants whose roots were challenged with F. oxysporum, but the salicylate- responsive PR1 gene was not induced in the leaves of inoculated plants. Exogenous salicylic acid treatment prior to inoculation, however, activated PR1 and BGL2 defence gene expression in leaves and provided increased F. oxysporum resistance as evidenced by reduced foliar necrosis and plant death. Exogenous salicylic acid treatment of the foliar tissue did not activate defence gene expression in the roots of plants. This suggests that salicylate- dependent defences may function in foliar tissue to reduce the development of pathogen-induced wilting and necrosis. Despite the induction of defence gene expression in the leaves by jasmonate, this treatment did not lead to increased resistance to F. oxysporum. Overall, the results presented here suggest that the genetic manipulation of plant defence signalling pathways is a useful strategy to provide increased Fusarium wilt resistance.

The roles of exercise-induced immune system disturbances in the pathology of heat stroke - The dual pathway model of heat stroke

Heat stroke is a life-threatening condition that can be fatal if not appropriately managed. Although heat stroke has been recognised as a medical condition for centuries, a universally accepted definition of heat stroke is lacking and the pathology of heat stroke is not fully understood. Information derived from autopsy reports and the clinical presentation of patients with heat stroke indicates that hyperthermia, septicaemia, central nervous system impairment and cardiovascular failure play important roles in the pathology of heat stroke. The current models of heat stroke advocate that heat stroke is triggered by hyperthermia but is driven by endotoxaemia. Endotoxaemia triggers the systemic inflammatory response, which can lead to systemic coagulation and haemorrhage, necrosis, cell death and multi-organ failure. However, the current heat stroke models cannot fully explain the discrepancies in high core temperature (Tc) as a trigger of heat stroke within and between individuals. Research on the concept of critical Tc: as a limitation to endurance exercise implies that a high Tc may function as a signal to trigger the protective mechanisms against heat stroke. Athletes undergoing a period of intense training are subjected to a variety of immune and gastrointestinal (GI) disturbances. The immune disturbances include the suppression of immune cells and their functions, suppression of cell-mediated immunity, translocation of lipopolysaccharide (LPS), suppression of anti-LPS antibodies, increased macrophage activity due to muscle tissue damage, and increased concentration of circulating inflammatory and pyrogenic cytokines. Common symptoms of exercise-induced GI disturbances include diarrhoea, vomiting, gastrointestinal bleeding, and cramps, which may increase gut-related LPS translocation. This article discusses the current evidence that supports the argument that these exercise-induced immune and GI disturbances may contribute to the development of endotoxaemia and heat stroke. When endotoxaemia can be tolerated or prevented, continuing exercise and heat exposure will elevate Tc to a higher level (> 42 degrees C), where heat stroke may occur through the direct thermal effects of heat on organ tissues and cells. We also discuss the evidence suggesting that heat stroke may occur through endotoxaemia (heat sepsis), the primary pathway of heat stroke, or hyperthermia, the secondary pathway of heat stroke. The existence of these two pathways of heat stroke and the contribution of exercise-induced immune and GI disturbances in the primary pathway of heat stroke are illustrated in the dual pathway model of heat stroke. This model of heat stroke suggests that prolonged intense exercise suppresses anti-LPS mechanisms, and promotes inflammatory and pyrogenic activities in the pathway of heat stroke.

Non-curliation of Escherichia coli O78:K80 isolates associated with IS1 insertion in csgB and reduced persistence in poultry infection

The elaboration of curli fimbriae by Escherichia coli is associated with the development of a lacy colony morphology when grown on colonisation factor antigen agar at 25 degrees C. Avian colisepticaemia E. coli isolates screened for curliation by this culture technique showed lacy and smooth colonial morphologies and the genetic basis of the non-curliated smooth colonial phenotype was analysed. Two smooth E. coli O78:K80 isolates possessed about 40 copies of the IS1 element within their respective genomes of which one copy insertionally inactivated the csgB gene, the nucleator gene for curli fibril formation. One of these two isolates also possessed a defective rpoS gene which is a known regulator of curli expression. In the day-old chick model, both smooth isolates were as invasive as a known virulent O78:K80 isolate as determined by extent of liver and spleen colonisation post oral inoculation but were less persistent in terms of caecal colonisation.

The variant rpoS allele of S. enteritidis strain 27655R does not affect virulence in a chick model nor constitutive curliation but does generate a cold-sensitive phenotype

Adherence of pathogenic Escherichia coli and Salmonella spp. to host cells is in part mediated by curli fimbriae which, along with other virulence determinants, are positively regulated by RpoS. Interested in the role and regulation of curli (SEF17) fimbriae of Salmonella enteritidis in poultry infection, we tested the virulence of naturally occurring S. enteritidis PT4 strains 27655R and 27655S which displayed constitutive and null expression of curli (SEF17) fimbriae, respectively, in a chick invasion assay and analysed their rpoS alleles. Both strains were shown to be equally invasive and as invasive as a wild-type phage type 4 strain and an isogenic derivative defective for the elaboration of curli. We showed that the rpoS allele of 27655S was intact even though this strain was non-curliated and we confirmed that a S. enteritidis rpoS::strr null mutant was unable to express curli, as anticipated. Strain 27655R, constitutively curliated, possessed a frameshift mutation at position 697 of the rpoS coding sequence which resulted in a truncated product and remained curliated even when transduced to rpoS::strr. Additionally, rpoS mutants are known to be cold-sensitive, a phenotype confirmed for strain 27655R. Collectively, these data indicated that curliation was not a significant factor for pathogenesis of S. enteritidis in this model and that curliation of strains 27655R and 27655S was independent of RpoS. Significantly, strain 27655R possessed a defective rpoS allele and remained virulent. Here was evidence that supported the concept that different naturally occurring rpoS alleles may generate varying virulence phenotypic traits.

The surface of Pseudomonas aeruginosa in cystic fibrosis lung infection

Chronic experimental lung infection in rats was induced by intratracheal inoculation of agar beads containing Pseudomonas aeruginosa. Bacteria were recovered directly without subculture from the lungs of rats at 14 days post-infection and the outer membrane (OM) antigens were studied. The results indicated that bacteria grew under iron-restricted conditions as revealed by the expression of several iron-regulated membrane proteins (IRMPs) which could also be observed when the isolate was grown under iron-depleted conditions in laboratory media. The antibody response to P. aeruginosa OM protein antigens was investigated by immunoblotting with serum and lung fluid from infected rats. These fluids contained antibodies to all the major OM proteins, including the IRMPs, and protein H1. Results obtained using immunoblotting and enzyme-linked immunosorbent assay indicated that lipopolysaccharide (LPS) was the major antigen recognised by antibodies in sera from infected rats. The animal model was used to follow the development of the immune response to P. aeruginosa protein and LPS antigens. Immunoblotting was used to investigate the antigens recognised by antibodies in sequential serum samples. An antibody response to the IRMPs and OM proteins D, E, G and H1 and alao to rough LPS was detected as early as 4 days post-infection. Results obtained using immunoblotting and crossed immunoelectrophoresis techniques indicated that there was a progressive increase in the number of P. aeruginosa antigens recognised by antibodies in these sera. Both iron and magnesium depletion influenced protein H1 production. Antibodies in sera from patients with infections due to P. aeruginosa reacted with this antigen. Results obtained using quantitative gas-liquid chromatographic analysis indicated that growth phase and magnesium and iron depletion also affected the amount of LPS fatty acids, produced by P. aeruginosa. The silver stained SDS-polyacrylamide gels of proteinase K digested whole cell lysates of P. aeruginosa indicated that the O-antigen and core LPS were both affected by growth phase and specific nutrient depletion.

Development of a multicellular co-culture model of normal and cystic fibrosis human airways in vitro

Cystic fibrosis (CF) is the most common lethal inherited disease among Caucasians and arises due to mutations in a chloride channel, called cystic fibrosis transmembrane conductance regulator. A hallmark of this disease is the chronic bacterial infection of the airways, which is usually, associated with pathogens such as Pseudomonas aeruginosa, S. aureus and recently becoming more prominent, B. cepacia. The excessive inflammatory response, which leads to irreversible lung damage, will in the long term lead to mortality of the patient at around the age of 40 years. Understanding the pathogenesis of CF currently relies on animal models, such as those employing genetically-modified mice, and on single cell culture models, which are grown either as polarised or non-polarised epithelium in vitro. Whilst these approaches partially enable the study of disease progression in CF, both types of models have inherent limitations. The overall aim of this thesis was to establish a multicellular co-culture model of normal and CF human airways in vitro, which helps to partially overcome these limitations and permits analysis of cell-to-cell communication in the airways. These models could then be used to examine the co-ordinated response of the airways to infection with relevant pathogens in order to validate this approach over animals/single cell models. Therefore epithelial cell lines of non-CF and CF background were employed in a co-culture model together with human pulmonary fibroblasts. Co-cultures were grown on collagen-coated permeable supports at air-liquid interface to promote epithelial cell differentiation. The models were characterised and essential features for investigating CF infections and inflammatory responses were investigated and analysed. A pseudostratified like epithelial cell layer was established at air liquid interface (ALI) of mono-and co-cultures and cell layer integrity was verified by tight junction (TJ) staining and transepithelial resistance measurements (TER). Mono- and co-cultures were also found to secrete the airway mucin MUC5AC. Influence of bacterial infections was found to be most challenging when intact S. aureus, B. cepacia and P. aeruginosa were used. CF mono- and co-cultures were found to mimic the hyperinflammatory state found in CF, which was confirmed by analysing IL-8 secretions of these models. These co-culture models will help to elucidate the role fibroblasts play in the inflammatory response to bacteria and will provide a useful testing platform to further investigate the dysregulated airway responses seen in CF.

Statnote 30 : The one-way analysis of variance (ANOVA):fixed effects model

In Statnote 9, we described a one-way analysis of variance (ANOVA) ‘random effects’ model in which the objective was to estimate the degree of variation of a particular measurement and to compare different sources of variation in space and time. The illustrative scenario involved the role of computer keyboards in a University communal computer laboratory as a possible source of microbial contamination of the hands. The study estimated the aerobic colony count of ten selected keyboards with samples taken from two keys per keyboard determined at 9am and 5pm. This type of design is often referred to as a ‘nested’ or ‘hierarchical’ design and the ANOVA estimated the degree of variation: (1) between keyboards, (2) between keys within a keyboard, and (3) between sample times within a key. An alternative to this design is a 'fixed effects' model in which the objective is not to measure sources of variation per se but to estimate differences between specific groups or treatments, which are regarded as 'fixed' or discrete effects. This statnote describes two scenarios utilizing this type of analysis: (1) measuring the degree of bacterial contamination on 2p coins collected from three types of business property, viz., a butcher’s shop, a sandwich shop, and a newsagent and (2) the effectiveness of drugs in the treatment of a fungal eye infection.

Logistic Regression in Modelling Data for CVC - Related Infection

2000 Mathematics Subject Classification: 62J12, 62P10.