A Vibrio cholerae ghost-based subunit vaccine induces cross-protective chlamydial immunity that is enhanced by CTA2B, the nontoxic derivative of cholera toxin

The Vibrio cholerae ghost (rVCG) platform is an effective carrier and delivery system for designing efficacious Chlamydia vaccines. We investigated whether CTA2B, the nontoxic derivative of cholera toxin, can augment protective immunity conferred by an rVCG-based chlamydial vaccine and enhance cross-protection against heterologous chlamydial strains. An rVCG vaccine coexpressing chlamydial major outer membrane protein and CTA2B was genetically constructed and antigens were targeted to the inner membrane of V. cholerae before ghost production by gene E -mediated lysis. Effective immunomodulation by CTA2B was demonstrated by the ability of the vaccine construct to enhance the activation and maturation of dendritic cells in vitro . Also, C57BL/6 mice immunized via mucosal and systemic routes showed increased specific mucosal and systemic antibody and T-helper type-1 (Th1) responses, irrespective of the route. The enhanced production of IFN-γ, but not IL-4 by genital mucosal and splenic T cells, indicated a predominantly Th1 response. Clearance of the Chlamydia muridarum vaginal infection was significantly enhanced by codelivery of the vaccine with CTA2B, with the intravaginal route showing a moderate advantage. These results indicate that the rVCG-based vaccine is capable of inducing cross-protection against heterologous chlamydial serovars and that incorporation of mucosal adjuvants, such as CTA2B in the rVCG delivery platform, may enhance protective immunity.     

5-Aminopyrimidin-2-ylnitriles as Cathepsin K inhibitors

A series of pyrimidine nitrile inhibitors of Cathepsin K with reduced glutathione reactivity has been identified and Molecular Core Matching (MoCoM) has been used to quantify the effect of an amino substituent at C5.     

Galactose metabolism in cell walls of opening and senescing petunia petals

Galactose was the major non-cellulosic neutral sugar present in the cell walls of ‘Mitchell’ petunia (Petunia axillaris × P. axillaris × P. hybrida) flower petals. Over the 24 h period associated with flower opening, there was a doubling of the galactose content of polymers strongly associated with cellulose and insoluble in strong alkali (‘residual’ fraction). By two days after flower opening, the galactose content of both the residual fraction and a Na₂CO₃-soluble pectin-rich cell wall fraction had sharply decreased, and continued to decline as flowers began to wilt. In contrast, amounts of other neutral sugars showed little change over this time, and depolymerisation of pectins and hemicelluloses was barely detectable throughout petal development. Size exclusion chromatography of Na₂CO₃-soluble pectins showed that there was a loss of neutral sugar relative to uronic acid content, consistent with a substantial loss of galactose from rhamnogalacturonan-I-type pectin. β-Galactosidase activity (EC 3.2.1.23) increased at bud opening, and remained high through to petal senescence. Two cDNAs encoding β-galactosidase were isolated from a mixed stage petal library. Both deduced proteins are β-galactosidases of Glycosyl Hydrolase Family 35, possessing lectin-like sugar-binding domains at their carboxyl terminus. PhBGAL1 was expressed at relatively high levels only during flower opening, while PhBGAL2 mRNA accumulation occurred at lower levels in mature and senescent petals. The data suggest that metabolism of cell wall-associated polymeric galactose is the major feature of both the opening and senescence of ‘Mitchell’ petunia flower petals.     

Viral Load Levels Measured at Set-Point Have Risen Over the Last Decade of the HIV Epidemic in the Netherlands

Background

HIV-1 RNA plasma concentration at viral set-point is associated not only with disease outcome but also with the transmission dynamics of HIV-1. We investigated whether plasma HIV-1 RNA concentration and CD4 cell count at viral set-point have changed over time in the HIV epidemic in the Netherlands.

Methodology/Principal Findings

We selected 906 therapy-naïve patients with at least one plasma HIV-1 RNA concentration measured 9 to 27 months after estimated seroconversion. Changes in HIV-1 RNA and CD4 cell count at viral set-point over time were analysed using linear regression models. The ATHENA national observational cohort contributed all patients who seroconverted in or after 1996; the Amsterdam Cohort Studies (ACS) contributed seroconverters before 1996. The mean of the first HIV-1 RNA concentration measured 9–27 months after seroconversion was 4.30 log₁₀ copies/ml (95% CI 4.17–4.42) for seroconverters from 1984 through 1995 (n = 163); 4.27 (4.16–4.37) for seroconverters 1996–2002 (n = 232), and 4.59 (4.52–4.66) for seroconverters 2003–2007 (n = 511). Compared to patients seroconverting between 2003–2007, the adjusted mean HIV-1 RNA concentration at set-point was 0.28 log₁₀ copies/ml (95% CI 0.16–0.40; p<0.0001) and 0.26 (0.11–0.41; p = 0.0006) lower for those seroconverting between 1996–2002 and 1984–1995, respectively. Results were robust regardless of type of HIV-1 RNA assay, HIV-1 subtype, and interval between measurement and seroconversion. CD4 cell count at viral set-point declined over calendar time at approximately 5 cells/mm³/year.

Conclusion

The HIV-1 RNA plasma concentration at viral set-point has increased over the last decade of the HIV epidemic in the Netherlands. This is accompanied by a decreasing CD4 cell count over the period 1984–2007 and may have implications for both the course of the HIV infection and the epidemic.     

The Severity of Pandemic H1N1 Influenza in the United States,from April to July 2009: A Bayesian Analysis

Background

Accurate measures of the severity of pandemic (H1N1) 2009 influenza (pH1N1) are needed to assess the likely impact of an anticipated resurgence in the autumn in the Northern Hemisphere. Severity has been difficult to measure because jurisdictions with large numbers of deaths and other severe outcomes have had too many cases to assess the total number with confidence. Also, detection of severe cases may be more likely, resulting in overestimation of the severity of an average case. We sought to estimate the probabilities that symptomatic infection would lead to hospitalization, ICU admission, and death by combining data from multiple sources.

Methods and Findings

We used complementary data from two US cities: Milwaukee attempted to identify cases of medically attended infection whether or not they required hospitalization, while New York City focused on the identification of hospitalizations, intensive care admission or mechanical ventilation (hereafter, ICU), and deaths. New York data were used to estimate numerators for ICU and death, and two sources of data—medically attended cases in Milwaukee or self-reported influenza-like illness (ILI) in New York—were used to estimate ratios of symptomatic cases to hospitalizations. Combining these data with estimates of the fraction detected for each level of severity, we estimated the proportion of symptomatic patients who died (symptomatic case-fatality ratio, sCFR), required ICU (sCIR), and required hospitalization (sCHR), overall and by age category. Evidence, prior information, and associated uncertainty were analyzed in a Bayesian evidence synthesis framework. Using medically attended cases and estimates of the proportion of symptomatic cases medically attended, we estimated an sCFR of 0.048% (95% credible interval [CI] 0.026%–0.096%), sCIR of 0.239% (0.134%–0.458%), and sCHR of 1.44% (0.83%–2.64%). Using self-reported ILI, we obtained estimates approximately 7–9× lower. sCFR and sCIR appear to be highest in persons aged 18 y and older, and lowest in children aged 5–17 y. sCHR appears to be lowest in persons aged 5–17; our data were too sparse to allow us to determine the group in which it was the highest.

Conclusions

These estimates suggest that an autumn–winter pandemic wave of pH1N1 with comparable severity per case could lead to a number of deaths in the range from considerably below that associated with seasonal influenza to slightly higher, but with the greatest impact in children aged 0–4 and adults 18–64. These estimates of impact depend on assumptions about total incidence of infection and would be larger if incidence of symptomatic infection were higher or shifted toward adults, if viral virulence increased, or if suboptimal treatment resulted from stress on the health care system; numbers would decrease if the total proportion of the population symptomatically infected were lower than assumed. Please see later in the article for the Editors'' Summary     

Gall-induction in insects: evolutionary dead-end or speciation driver?

Background

The tree of life is significantly asymmetrical - a result of differential speciation and extinction - but general causes of such asymmetry are unclear. Differences in niche partitioning are thought to be one possible general explanation. Ecological specialization might lead to increases in diversification rate or, alternatively, specialization might limit the evolutionary potential of specialist lineages and increase their extinction risk. Here we compare the diversification rates of gall-inducing and non-galling insect lineages. Compared with other insect herbivores feeding on the same host plant, gall-inducing insects feed on plant tissue that is more nutritious and less defended, and they do so in a favorable microhabitat that may also provide some protection from natural enemies. We use sister-taxon comparisons to test whether gall-inducing lineages are more host-specific than non-galling lineages, and more or less diverse than non-gallers. We evaluate the significance of diversity bipartitions under Equal Rates Markov models, and use maximum likelihood model-fitting to test for shifts in diversification rates.

Results

We find that, although gall-inducing insect groups are more host-specific than their non-galling relatives, there is no general significant increase in diversification rate in gallers. However, gallers are found at both extremes - two gall-inducing lineages are exceptionally diverse (Euurina sawflies on Salicaceae and Apiomorpha scale insects on Eucalytpus), and one gall-inducing lineage is exceptionally species-poor (Maskellia armored scales on Eucalyptus).

Conclusions

The effect of ecological specialization on diversification rates is complex in the case of gall-inducing insects, but host range may be an important factor. When a gall-inducing lineage has a host range approximate to that of its non-galling sister, the gallers are more diverse. When the non-galler clade has a much wider host range than the galler, the non-galler is also much more diverse. There are also lineage-specific effects, with gallers on the same host group exhibiting very different diversities. No single general model explains the observed pattern.     

Evaluation of allelic expression of imprinted genes in adult human blood

Background

Imprinted genes are expressed from only one allele in a parent-of-origin dependent manner. Loss of imprinted (LOI) expression can result in a variety of human disorders and is frequently reported in cancer. Biallelic expression of imprinted genes in adult blood has been suggested as a useful biomarker and is currently being investigated in colorectal cancer. In general, the expression profiles of imprinted genes are well characterised during human and mouse fetal development, but not in human adults.

Methodology/Principal Findings

We investigated quantitative expression of 36 imprinted genes in adult human peripheral blood leukocytes obtained from healthy individuals. Allelic expression was also investigated in B and T lymphocytes and myeloid cells. We found that 21 genes were essentially undetectable in adult blood. Only six genes were demonstrably monoallelic, and most importantly, we found that nine genes were either biallelic or showed variable expression in different individuals. Separated leukocyte populations showed the same expression patterns as whole blood. Differential methylation at each of the imprinting control loci analysed was maintained, including regions that contained biallelically expressed genes. This suggests in some cases methylation has become uncoupled from its role in regulating gene expression.

Conclusions/Significance

We conclude that only a limited set of imprinted genes, including IGF2 and SNRPN, may be useful for LOI cancer biomarker studies. In addition, blood is not a good tissue to use for the discovery of new imprinted genes. Finally, lymphocyte DNA methylation status in the adult may not always be a reliable indicator of monoallelic gene expression.     

Fibulin-1 Is Increased in Asthma – A Novel Mediator of Airway Remodeling?

Background

The extracellular matrix is a dynamic and complex network of macromolecules responsible for maintaining and influencing cellular functions of the airway. The role of fibronectin, an extracellular matrix protein, is well documented in asthma. However, the expression and function of fibulin-1, a secreted glycoprotein which interacts with fibronectin, has not been reported. Fibulin-1 is widely expressed in basement membranes in many organs including the lung. There are four isoforms in humans (A–D) of which fibulin-1C and 1D predominate. The objective of this study was to study the expression of fibulin-1 in volunteers with and without asthma, and to examine its function in vitro.

Methodology/Principal Findings

We used immunohistochemistry and dot-blots to examine fibulin-1 levels in bronchial biopsies, bronchoalveolar lavage fluid and serum. Real-time PCR for fibulin-1C and 1D, and ELISA and western blotting for fibulin-1 were used to study the levels in airway smooth muscle cells. The function of fibulin-1C was determined by assessing its role, using an antisense oligonucleotide, in cell proliferation, migration and wound healing. A murine model of airway hyperresponsiveness (AHR) was used to explore the biological significance of fibulin-1. Levels of fibulin-1 were significantly increased in the serum and bronchoalveolar lavage fluid of 21 asthmatics compared with 11 healthy volunteers. In addition fibulin-1 was increased in asthma derived airway smooth muscle cells and fibulin-1C contributed to the enhanced proliferation and wound repair in these cells. These features were reversed when fibulin-1C was suppressed using an antisense oligomer. In a mouse model of AHR, treatment with an AO inhibited the development of AHR to methacholine.

Conclusions

Our data collectively suggest fibulin-1C may be worthy of further investigation as a target for airway remodeling in asthma.