IFNγ production in peripheral blood of early Lyme disease patients to hLFAα(L) (aa326-345)

BACKGROUND: It has been proposed that outer surface protein A (OspA) of Borrelia burgdorferi sensu stricto contains a T helper 1 (Th1) cell epitope that could play a role in an autoimmune response to hLFA1. METHODS: We used two peptides, hLFAα(L) (aa326-345) and Borrelia burgdorferi OspAB31 (aa164-183), as stimulating antigens to measure Th1 proinflammatory IFNγ cytokine production in peripheral blood of Lyme disease patients presenting with EM without history of arthritis, as well as in peripheral blood of healthy individuals. RESULTS: IFNγ responses to hLFA1 peptide were observed in 11 of 19 Lyme disease patients and in 3 of 15 healthy controls. In contrast, only 2 of 19 of the Lyme disease patients and none of the controls responded to the homologous OspAB31 peptide. CONCLUSIONS: IFNγ was produced in response to stimulation with peptide hLFAα(L) (aa326-345) in peripheral blood of 58% of patients with early Lyme disease without signs of arthritis, as well as in peripheral blood of 20% of healthy individuals, but not in response to stimulation with the homologous OspAB31 (aa164-183) peptide (p < 0.05). Our results suggest that reactivity to the hLFA1 peptide in peripheral blood may be the result of T cell degeneracy

The microaerophilic microbiota of de-novo paediatric inflammatory bowel disease: the BISCUIT study

&lt;p&gt;Introduction: Children presenting for the first time with inflammatory bowel disease (IBD) offer a unique opportunity to study aetiological agents before the confounders of treatment. Microaerophilic bacteria can exploit the ecological niche of the intestinal epithelium; Helicobacter and Campylobacter are previously implicated in IBD pathogenesis. We set out to study these and other microaerophilic bacteria in de-novo paediatric IBD.&lt;/p&gt; &lt;p&gt;Patients and Methods: 100 children undergoing colonoscopy were recruited including 44 treatment naïve de-novo IBD patients and 42 with normal colons. Colonic biopsies were subjected to microaerophilic culture with Gram-negative isolates then identified by sequencing. Biopsies were also PCR screened for the specific microaerophilic bacterial groups: Helicobacteraceae, Campylobacteraceae and Sutterella wadsworthensis.&lt;/p&gt; &lt;p&gt;Results: 129 Gram-negative microaerophilic bacterial isolates were identified from 10 genera. The most frequently cultured was S. wadsworthensis (32 distinct isolates). Unusual Campylobacter were isolated from 8 subjects (including 3 C. concisus, 1 C. curvus, 1 C. lari, 1 C. rectus, 3 C. showae). No Helicobacter were cultured. When comparing IBD vs. normal colon control by PCR the prevalence figures were not significantly different (Helicobacter 11% vs. 12%, p = 1.00; Campylobacter 75% vs. 76%, p = 1.00; S. wadsworthensis 82% vs. 71%, p = 0.312).&lt;/p&gt; &lt;p&gt;Conclusions: This study offers a comprehensive overview of the microaerophilic microbiota of the paediatric colon including at IBD onset. Campylobacter appear to be surprisingly common, are not more strongly associated with IBD and can be isolated from around 8% of paediatric colonic biopsies. S. wadsworthensis appears to be a common commensal. Helicobacter species are relatively rare in the paediatric colon.&lt;/p&gt

Evidence That Two ATP-Dependent (Lon) Proteases in Borrelia burgdorferi Serve Different Functions

The canonical ATP-dependent protease Lon participates in an assortment of biological processes in bacteria, including the catalysis of damaged or senescent proteins and short-lived regulatory proteins. Borrelia spirochetes are unusual in that they code for two putative ATP-dependent Lon homologs, Lon-1 and Lon-2. Borrelia burgdorferi, the etiologic agent of Lyme disease, is transmitted through the blood feeding of Ixodes ticks. Previous work in our laboratory reported that B. burgdorferi lon-1 is upregulated transcriptionally by exposure to blood in vitro, while lon-2 is not. Because blood induction of Lon-1 may be of importance in the regulation of virulence factors critical for spirochete transmission, the clarification of functional roles for these two proteases in B. burgdorferi was the object of this study. On the chromosome, lon-2 is immediately downstream of ATP-dependent proteases clpP and clpX, an arrangement identical to that of lon of Escherichia coli. Phylogenetic analysis revealed that Lon-1 and Lon-2 cluster separately due to differences in the NH2-terminal substrate binding domains that may reflect differences in substrate specificity. Recombinant Lon-1 manifested properties of an ATP-dependent chaperone-protease in vitro but did not complement an E. coli Lon mutant, while Lon-2 corrected two characteristic Lon-mutant phenotypes. We conclude that B. burgdorferi Lons -1 and -2 have distinct functional roles. Lon-2 functions in a manner consistent with canonical Lon, engaged in cellular homeostasis. Lon-1, by virtue of its blood induction, and as a unique feature of the Borreliae, may be important in host adaptation from the arthropod to a warm-blooded host

Nonrandom Distribution of Vector Ticks (Dermacentor variabilis) Infected by Francisella tularensis

The island of Martha's Vineyard, Massachusetts, is the site of a sustained outbreak of tularemia due to Francisella tularensis tularensis. Dog ticks, Dermacentor variabilis, appear to be critical in the perpetuation of the agent there. Tularemia has long been characterized as an agent of natural focality, stably persisting in characteristic sites of transmission, but this suggestion has never been rigorously tested. Accordingly, we sought to identify a natural focus of transmission of the agent of tularemia by mapping the distribution of PCR-positive ticks. From 2004 to 2007, questing D. variabilis were collected from 85 individual waypoints along a 1.5 km transect in a field site on Martha's Vineyard. The positions of PCR-positive ticks were then mapped using ArcGIS. Cluster analysis identified an area approximately 290 meters in diameter, 9 waypoints, that was significantly more likely to yield PCR-positive ticks (relative risk 3.3, P = 0.001) than the rest of the field site. Genotyping of F. tularensis using variable number tandem repeat (VNTR) analysis on PCR-positive ticks yielded 13 different haplotypes, the vast majority of which was one dominant haplotype. Positive ticks collected in the cluster were 3.4 times (relative risk = 3.4, P<0.0001) more likely to have an uncommon haplotype than those collected elsewhere from the transect. We conclude that we have identified a microfocus where the agent of tularemia stably perpetuates and that this area is where genetic diversity is generated

Social factors related to the clinical severity of influenza cases in Spain during the A(H1N1)2009 virus pandemic

Background During the 2009 influenza pandemic, a change in the type of patients most often affected by influenza was observed. The objective of this study was to assess the role of individual and social determinants in hospitalizations due to influenza A (H1N1) 2009 infection. Methods We studied hospitalized patients (cases) and outpatients (controls) with confirmed influenza A (H1N1) 2009 infection. A standardized questionnaire was used to collect data. Variables that might be related to the hospitalization of influenza cases were compared by estimation of the odds ratio (OR) and 95% confidence intervals (CI) and the variables entered into binomial logistic regression models. Results Hospitalization due to pandemic A (H1N1) 2009 influenza virus infections was associated with non-Caucasian ethnicity (OR: 2.18, 95% CI 1.17 − 4.08), overcrowding (OR: 2.84, 95% CI 1.20 − 6.72), comorbidity and the lack of previous preventive information (OR: 2.69, 95% CI: 1.50 − 4.83). Secondary or higher education was associated with a lower risk of hospitalization (OR 0.56, 95% CI: 0.36 − 0.87) Conclusions In addition to individual factors such as comorbidity, other factors such as educational level, ethnicity or overcrowding were associated with hospitalization due to A (H1N1) 2009 influenza virus infections

An RND-Type Efflux System in Borrelia burgdorferi Is Involved in Virulence and Resistance to Antimicrobial Compounds

Borrelia burgdorferi is remarkable for its ability to thrive in widely different environments due to its ability to infect various organisms. In comparison to enteric Gram-negative bacteria, these spirochetes have only a few transmembrane proteins some of which are thought to play a role in solute and nutrient uptake and excretion of toxic substances. Here, we have identified an outer membrane protein, BesC, which is part of a putative export system comprising the components BesA, BesB and BesC. We show that BesC, a TolC homolog, forms channels in planar lipid bilayers and is involved in antibiotic resistance. A besC knockout was unable to establish infection in mice, signifying the importance of this outer membrane channel in the mammalian host. The biophysical properties of BesC could be explained by a model based on the channel-tunnel structure. We have also generated a structural model of the efflux apparatus showing the putative spatial orientation of BesC with respect to the AcrAB homologs BesAB. We believe that our findings will be helpful in unraveling the pathogenic mechanisms of borreliae as well as in developing novel therapeutic agents aiming to block the function of this secretion apparatus

A Novel Tetrameric PilZ Domain Structure from Xanthomonads

PilZ domain is one of the key receptors for the newly discovered secondary messenger molecule cyclic di-GMP (c-di-GMP). To date, several monomeric PilZ domain proteins have been identified. Some exhibit strong c-di-GMP binding activity, while others have barely detectable c-di-GMP binding activity and require an accessory protein such as FimX to indirectly respond to the c-di-GMP signal. We now report a novel tetrameric PilZ domain structure of XCC6012 from the plant pathogen Xanthomonas campestris pv. campestris (Xcc). It is one of the four PilZ domain proteins essential for Xcc pathogenicity. Although the monomer adopts a structure similar to those of the PilZ domains with very weak c-di-GMP binding activity, it is nevertheless interrupted in the middle by two extra long helices. Four XCC6012 proteins are thus self-assembled into a tetramer via the extra heptad repeat α3 helices to form a parallel four-stranded coiled-coil, which is further enclosed by two sets of inclined α2 and α4 helices. We further generated a series of XCC6012 variants and measured the unfolding temperatures and oligomeric states in order to investigate the nature of this novel tetramer. Discovery of this new PilZ domain architecture increases the complexity of c-di-GMP-mediated regulation

Enhancing Social-Emotional Outcomes in Early Years (E-SEE): Randomized Pilot Study of Incredible Years Infant and Toddler Programs

Abstract: Social emotional development in infancy is a predictor of outcomes in later life, yet there is little evidence of effectiveness for parenting interventions designed to enhance social emotional wellbeing in infancy. An 18-month two-arm randomized controlled pilot trial evaluated the feasibility of a definitive trial of Incredible Years (IY) Infant and Toddler parent programs delivered in a proportionate universal model, called Enhancing Social-Emotional Health and Wellbeing in the Early Years (E-SEE) Steps. Intervention families received an IY Babies book (universal dose), followed by the IY Infant and/or the Toddler group-based programs, based on parent depression (PHQ-9) and/or child social emotional development (ASQ:SE-2) scores. Control parents received services as usual. Parents from two English local authorities with a child eight-weeks-old or younger participated, and were block randomized using a web-based system. Primary endpoints for the study were feasibility parameters relating to recruitment, retention, intervention fidelity and appropriateness of measures. 205 participants were randomized (152:53, intervention:control). Our target was 288 parents. Trial retention rate was higher than expected, with a completion rate of 88% (n = 181, 137:44) at follow-up 3; equating to 94% of 192 expected participants. Intervention uptake was lower than expected. Fidelity of delivery was acceptable and measures were deemed appropriate. A definitive trial is feasible with design amendments to include: introduction of a child screener for intervention eligibility; enhanced intervention material; revised sample size and random allocation ratio. Our internal pilot became an external pilot due to these changes

MrkH, a Novel c-di-GMP-Dependent Transcriptional Activator, Controls Klebsiella pneumoniae Biofilm Formation by Regulating Type 3 Fimbriae Expression

Klebsiella pneumoniae causes significant morbidity and mortality worldwide, particularly amongst hospitalized individuals. The principle mechanism for pathogenesis in hospital environments involves the formation of biofilms, primarily on implanted medical devices. In this study, we constructed a transposon mutant library in a clinical isolate, K. pneumoniae AJ218, to identify the genes and pathways implicated in biofilm formation. Three mutants severely defective in biofilm formation contained insertions within the mrkABCDF genes encoding the main structural subunit and assembly machinery for type 3 fimbriae. Two other mutants carried insertions within the yfiN and mrkJ genes, which encode GGDEF domain- and EAL domain-containing c-di-GMP turnover enzymes, respectively. The remaining two isolates contained insertions that inactivated the mrkH and mrkI genes, which encode for novel proteins with a c-di-GMP-binding PilZ domain and a LuxR-type transcriptional regulator, respectively. Biochemical and functional assays indicated that the effects of these factors on biofilm formation accompany concomitant changes in type 3 fimbriae expression. We mapped the transcriptional start site of mrkA, demonstrated that MrkH directly activates transcription of the mrkA promoter and showed that MrkH binds strongly to the mrkA regulatory region only in the presence of c-di-GMP. Furthermore, a point mutation in the putative c-di-GMP-binding domain of MrkH completely abolished its function as a transcriptional activator. In vivo analysis of the yfiN and mrkJ genes strongly indicated their c-di-GMP-specific function as diguanylate cyclase and phosphodiesterase, respectively. In addition, in vitro assays showed that purified MrkJ protein has strong c-di-GMP phosphodiesterase activity. These results demonstrate for the first time that c-di-GMP can function as an effector to stimulate the activity of a transcriptional activator, and explain how type 3 fimbriae expression is coordinated with other gene expression programs in K. pneumoniae to promote biofilm formation to implanted medical devices