Clinical manifestations of invasive pneumococcal disease by vaccine and non-vaccine types.

Pneumococcal conjugated vaccines (PCVs) have shown protection against invasive pneumococcal disease by vaccine serotypes, but an increase in non-vaccine serotype disease has been observed. Type-specific effects on clinical manifestation need to be explored. Clinical data from 2096 adults and 192 children with invasive pneumococcal disease were correlated to pneumococcal molecular serotypes. Invasive disease potential for pneumococcal serotypes were calculated using 165 invasive and 550 carriage isolates from children. The invasive disease potential was lower for non-PCV13 compared to vaccine-type strains. Patients infected with non-PCV13 strains had more underlying diseases, were less likely to have pneumonia and, in adults, tended to have a higher mortality. Furthermore, patients infected with pneumococci belonging to clonal serotypes only expressing non-PCV13 capsules had a higher risk for septicaemia and mortality. PCV vaccination will probably lead to a decrease in invasive pneumococcal disease but an alteration in the clinical manifestation of invasive pneumococcal disease. Genetic lineages causing invasive pneumococcal disease in adults often express non-vaccine serotypes, which can expand after vaccination with an increased risk of infection in patients with underlying diseases.

Exposing kinetic disparities between inflammasome readouts using time-resolved analysis.

The NLRP3 inflammasome is an intracellular multiprotein complex described to be involved in both an effective host response to infectious agents and various diseases. Investigation into the NLRP3 inflammasome has been extensive in the past two decades, and often revolves around the analysis of a few specific readouts, including ASC-speck formation, caspase-1 cleavage or activation, and cleavage and release of IL-1ß and/or IL-18. Quantification of these readouts is commonly undertaken as an endpoint analysis, where the presence of each positive outcome is assessed independently of the others. In this study, we apply time-resolved analysis of a human macrophage model (differentiated THP-1-ASC-GFP cells) to commonly accessible methods. This approach yields the additional quantifiable metrics time-resolved absolute change and acceleration, allowing comparisons between readouts. Using this methodological approach, we reveal (potential) discrepancies between inflammasome-related readouts that otherwise might go undiscovered. The study highlights the importance of time-resolved data in general and may be further extended as well as incorporated into other areas of research.

Two adult siblings with atypical cryopyrin-associated periodic syndrome due to a novel M299V mutation in NLRP3.

OBJECTIVE: The NALP3 inflammasome is a multiprotein complex that triggers caspase 1-mediated interleukin-1beta (IL-1beta) release. Mutations in the gene encoding NALP3 (NLRP3) underlie the cryopyrin-associated periodic syndrome (CAPS). The aim of this study was to report a novel NLRP3 mutation in 2 siblings of Swedish descent in whom symptoms first presented in adulthood. METHODS: Mutation analysis of NLRP3 was performed on DNA from patients with CAPS and 100 control subjects. For assessment of caspase 1 and IL-1beta, blood was collected from patients and age- and sex-matched healthy control subjects. Genetic constructs containing mutant or wild-type NLRP3 were transduced into THP-1 cells, followed by assessment of IL-1beta levels in cell supernatant. RESULTS: Both siblings carried a novel M299V mutation in NLRP3, which was not present in the control population. The samples obtained from the patients displayed increased caspase 1 activity and elevated IL-1beta levels at basal conditions as compared with healthy control subjects. THP-1 cells expressing mutated M299V revealed almost 10-fold higher IL-1beta production compared with the wild-type construct. CONCLUSION: M299V is an activating mutation in NLRP3 resulting in elevated spontaneous caspase 1 activity and IL-1beta levels. The classic CAPS phenotype was lacking in these adult siblings. Whereas one sibling displayed a milder phenotype that has so far responded satisfactorily to oral nonsteroidal antiinflammatory drugs in combination with low-dose corticosteroids, the inflammatory symptoms in the sibling with the more severe case responded well to IL-1beta blockade. Understanding the pathogenic mechanism underlying such disorders can be helpful for the physician. Our study reinforces the importance of genetic testing and laboratory investigations in combination with careful phenotypic evaluation for the diagnosis of such patients.

The crystal structure of the spinach plastocyanin double mutant G8D/L12E gives insight into its low reactivity towards photosystem 1 and cytochrome f.

Plastocyanin (Pc) is a copper-containing protein, which functions as an electron carrier between the cytochrome b(6)f and photosystem 1 (PS1) complexes in the photosynthetic electron transfer (ET) chain. The ET is mediated by His87 situated in the hydrophobic surface in the north region of Pc. Also situated in this region is Leu12, which mutated to other amino acids severely disturbs the ET from cytochrome f and to PS1, indicating the importance of the hydrophobic surface. The crystal structure of the Pc double mutant G8D/L12E has been determined to 2.0 A resolution, with a crystallographic R-factor of 18.3% (R(free)=23.2%). A comparison with the wild-type structure reveals that structural differences are limited to the sites of the mutations. In particular, there is a small but significant change in the hydrophobic surface close to His87. Evidently, this leads to a mismatch in the reactive complex with the redox partners. For PS1 this results in a 20 times weaker binding and an eightfold slower ET as determined by kinetic measurements. The mutations that have been introduced do not affect the optical absorption spectrum. However, there is a small change in the EPR spectrum, which can be related to changes in the copper coordination geometry.

Effects of sucking and skin-to-skin contact on maternal ACTH and cortisol levels during the second day postpartum-influence of epidural analgesia and oxytocin in the perinatal period.

BACKGROUND AND AIMS: In this study we made a detailed analysis of the mothers' release pattern of adrenocorticotropic hormone (ACTH) and cortisol during a breastfeeding session during the second day postpartum and related these patterns to maternal oxytocin levels as well to the duration of sucking and the duration of skin-to-skin contact before sucking the breast. Furthermore, we investigated if epidural analgesia and oxytocin administration during and after labor influenced the release pattern of ACTH and cortisol. METHODS: Sixty-three primiparae were included in the study. Fourteen received oxytocin intramuscularly postpartum, nine received oxytocin infusion, 14 received epidural analgesia combined with oxytocin infusion, and six received epidural analgesia alone. Twenty mothers did not receive any of these medical interventions. Blood samples were analyzed for ACTH and cortisol by enzyme-linked immunoassay. RESULTS: Both ACTH and cortisol levels fell significantly during the breastfeeding session. A significant negative relationship was found between oxytocin and ACTH levels, but not between oxytocin and cortisol levels. A positive and significant relationship was found between ACTH and cortisol levels. The duration of skin-to-skin contact before onset of sucking was significantly and negatively associated with lower cortisol levels, but not with ACTH levels. Cortisol levels differed significantly between mothers having received epidural analgesia with and without oxytocin. CONCLUSIONS: Breastfeeding is associated with a decrease of ACTH and cortisol levels. Skin-to-skin contact contributes to this effect. ACTH correlated negatively with the duration of sucking and with median oxytocin levels, whereas cortisol levels correlated inversely with the duration of skin-to-skin contact preceding sucking, suggesting a partial dissociation between the mechanisms regulating ACTH and cortisol release. In addition, medical interventions in connection with birth influence the activity of the hypothalamic-pituitary-adrenal axis 2 days after birth.

Functional prediction of a T-DNA tagged gene of Arabidopsis thaliana by in silico analysis.

We have employed a gene-knockout approach using T-DNA tagging and in vivo gene fusion in Arabidopsis thaliana for identification and isolation of specific plant genes. Screening of about 3,000 T-DNA tagged lines resulted in identification of a mutant line (no. 197) exhibiting a significant delay in flowering. From this line a 600-bp plant DNA fragment downstream of the left T-DNA junction was cloned by inverse PCR. BLAST searching in the A. thaliana genomic database indicated a putative gene, frf (flowering regulating factor), with unknown function downstream of the T-DNA insert. Bioinformatic tools were used to predict possible protein structure and function. The protein structure predicted by fold recognition indicates that frf is a transcriptional regulator, a ligand-binding receptor responsive to steroids and hormones. Analyzing the predicted results and the phenotype of the T-DNA tagged plant we hypothesized that FRF might be involved in hormone response in A. thaliana. For verification of this hypothesis we exposed the plants of line no. 197 to gibberellic acid (GA3), a potential growth regulator in higher plants. This treatment resulted in an earlier onset of flowering, almost similar to that in wild type control plants.

Characterization of the type I interferon locus and identification of novel genes.

The human type I interferon (IFN) genes are clustered on human chromosome 9p21 and the mouse genes are located in the region of conserved synteny on mouse chromosome 4. We have identified two novel mouse Ifna genes (Ifna12, Ifna13) and Ifnl2 (IFN-like 2, a homologue of Limitin/IFN-like 1). Another type I IFN gene was designated Ifne1. Mouse Ifne1 was expressed in ovaries and uterus but not in tissues of hematopoietic origin. IFN-epsilon1 has general structural characteristics of a type I IFN. These studies represent the first detailed annotation of the mouse type I IFN locus, and the products of these novel genes may have important functions in reproduction and host defense.

Structural complementarity of Toll/interleukin-1 receptor domains in Toll-like receptors and the adaptors Mal and MyD88.

The Toll/interleukin 1 receptor (TIR) domain is a region found in the cytoplasmic tails of members of the Toll-like receptor/interleukin-1 receptor superfamily. The domain is essential for signaling and is also found in the adaptor proteins Mal (MyD88 adaptor-like) and MyD88, which function to couple activation of the receptor to downstream signaling components. Experimental structures of two Toll/interleukin 1 receptor domains reveal a alpha-beta-fold similar to that of the bacterial chemotaxis protein CheY, and other evidence suggests that the adaptors can make heterotypic interactions with both the receptors and themselves. Here we show that the purified TIR domains of Mal and MyD88 can form stable heterodimers and also that Mal homodimers and oligomers are dissociated in the presence of ATP. To identify structural features that may contribute to the formation of signaling complexes, we produced models of the TIR domains from human Toll-like receptor 4 (TLR4), Mal, and MyD88. We found that although the overall fold is conserved the electrostatic surface potentials are quite distinct. Docking studies of the models suggest that Mal and MyD88 bind to different regions in TLRs 2 and 4, a finding consistent with a cooperative role of the two adaptors in signaling. Mal and MyD88 are predicted to interact at a third non-overlapping site, suggesting that the receptor and adaptors may form heterotetrameric complexes. The theoretical model of the interactions is supported by experimental data from glutathione S-transferase pull-downs and co-immunoprecipitations. Neither theoretical nor experimental data suggest a direct role for the conserved proline in the BB-loop in the association of TLR4, Mal, and MyD88. Finally we show a sequence relationship between the Drosophila protein Tube and Mal that may indicate a functional equivalence of these two adaptors in the Drosophila and vertebrate Toll pathways.