Identification of Immunogenic Salmonella enterica Serotype Typhi Antigens Expressed in Chronic Biliary Carriers of S. Typhi in Kathmandu,Nepal

Background

Salmonella enterica serotype Typhi can colonize and persist in the biliary tract of infected individuals, resulting in a state of asymptomatic chronic carriage. Chronic carriers may act as persistent reservoirs of infection within a community and may introduce infection to susceptible individuals and new communities. Little is known about the interaction between the host and pathogen in the biliary tract of chronic carriers, and there is currently no reliable diagnostic assay to identify asymptomatic S. Typhi carriage.

Methodology/Principal Findings

To study host-pathogen interactions in the biliary tract during S. Typhi carriage, we applied an immunoscreening technique called in vivo-induced antigen technology (IVIAT), to identify potential biomarkers unique to carriers. IVIAT identifies humorally immunogenic bacterial antigens expressed uniquely in the in vivo environment, and we hypothesized that S. Typhi surviving in the biliary tract of humans may express a distinct antigenic profile. Thirteen S. Typhi antigens that were immunoreactive in carriers, but not in healthy individuals from a typhoid endemic area, were identified. The identified antigens included a number of putative membrane proteins, lipoproteins, and hemolysin-related proteins. YncE (STY1479), an uncharacterized protein with an ATP-binding motif, gave prominent responses in our screen. The response to YncE in patients whose biliary tract contained S. Typhi was compared to responses in patients whose biliary tract did not contain S. Typhi, patients with acute typhoid fever, and healthy controls residing in a typhoid endemic area. Seven of 10 (70%) chronic carriers, 0 of 8 bile culture-negative controls (0%), 0 of 8 healthy Bangladeshis (0%), and 1 of 8 (12.5%) Bangladeshis with acute typhoid fever had detectable anti-YncE IgG in blood. IgA responses were also present.

Conclusions/Significance

Further evaluation of YncE and other antigens identified by IVIAT could lead to the development of improved diagnostic assays to identify asymptomatic S. Typhi carriers.     

Oxidative stress-induced modifications of histidyl-tRNA synthetase affect its tRNA aminoacylation activity but not its immunoreactivity

The aminoacyl-tRNA synthetases are ubiquitously expressed enzymes that catalyze the esterification of amino acids to their cognate tRNAs. Autoantibodies against several aminoacyl-tRNA synthetases are found in autoimmune polymyositis and dermatomyositis patients. Because necrosis is often found in skeletal muscle biopsies of these patients, we hypothesized that cell-death-induced protein modifications may help in breaking immunological tolerance. Since cell death is associated with oxidative stress, the effect of oxidative stress on the main myositis-specific autoantibody target Jo-1 (histidyl-tRNA synthetase; HisRS) was studied in detail. The exposure of Jurkat cells to hydrogen peroxide resulted in the detection of several oxidized methionines and one oxidized tryptophan residue in the HisRS protein, as demonstrated by mass spectrometry. Unexpectedly, the tRNA aminoacylation activity of HisRS appeared to be increased upon oxidative modification. The analysis of myositis patient sera did not lead to the detection of autoantibodies that are specifically reactive with the modified HisRS protein. The results of this study demonstrate that the Jo-1/HisRS autoantigen is modified under oxidative stress conditions. The consequences of these modifications for the function of HisRS and its autoantigenicity are discussed.     

Association of anti-oxidized LDL and candidate genes with severity of coronary stenosis in the Women's Ischemia Syndrome Evaluation study

Atherosclerosis is the major cause of coronary artery disease (CAD), and oxidized LDL (oxLDL) is believed to play a key role in the initiation of the atherosclerotic process. Recent studies show that inflammation and autoimmune reactions are also relevant in atherosclerosis. In this study, we examined the association of antibodies against oxLDL (anti-oxLDL) with the severity of CAD in 558 Women's Ischemia Syndrome Evaluation (WISE) study samples (465 whites; 93 blacks) determined by coronary stenosis (< 20%, 20%-49%, > 50% stenosis). We also examined the relationship of anti-oxLDL with serum lipid levels and nine candidate genes including APOE, APOH, APOA5, LPL, LRP1, HL, CETP, PON1, and OLR1. IgM anti-oxLDL levels were significantly higher in the >20% stenosis group than in the ≥ 20% stenosis group in whites (0.69 ± 0.02 vs. 0.64 ± 0.01, respectively; P = 0.02). IgM anti-oxLDL levels correlated significantly with total cholesterol (r2 = 0.01; P = 0.03) and LDL cholesterol (r2 = 0.017; P = 0.004) in whites. Multiple regression analysis revealed a suggestive association of LPL/S447X single-nucleotide polymorphism (SNP) with both IgG anti-oxLDL (P = 0.02) and IgM anti-oxLDL (P = 0.07), as well as between IgM anti-oxLDL and the OLR1/3'UTR SNP (P = 0.020). Our data suggest that higher IgM anti-oxLDL levels may provide protection against coronary stenosis and that genetic variation in some candidate genes are determinants of anti-oxLDL levels.     

Mapping of cytochrome P450 2B4 substrate binding sites by photolabile probe 3-azidiamantane: identification of putative substrate access regions

To investigate structure-function relationships of cytochromes P450 (CYP), 3-azidiamantane was employed for photoaffinity labeling of rabbit microsomal CYP2B4. Four diamantane labeled tryptic fragments were identified by mass spectrometry and sequencing: peptide I (Leu³⁵⁹-Lys³⁷³), peptide II (Leu³⁰-Arg⁴⁸), peptide III (Phe¹²⁷-Arg¹⁴⁰), and peptide IV (Arg⁴³⁴-Arg⁴⁴³). Their positions were projected into CYP2B4 model structures and compared with substrate binding sites, proposed by docking of diamantane. We identified novel binding regions outside the active site of CYP2B4. One of them, defined with diamantane modified Arg¹³³, marks a possible entrance to the active site from the heme proximal face. In addition to crystal structures of CYP2B4 chimeras and molecular dynamics simulations, our data of photoaffinity labeling of the full CYP2B4 molecule provide further insight into functional and structural aspects of substrate binding.     

The most frequent chromosomal abnormalities in karyotypes of patients with reproductive problems

Results of cytogenetic and molecular-cytogenetic inspection of 210 matrimonial pairs with the problems of reproduction are presented. Different types of chromosomal aberrations have been detected in the karyotypes of the patients in 46 (10.95%) cases. Such structural chromosomal aberrations as pericentric inversions, Robertsonian translocations, balanced reciprocal translocations, and marker chromosomes as well prevailed the numerical chromosomal aberrations (89.13% and 10.87% cases accordingly). In the general group of the inspected patients there were 19 cases (4.52%) characterized by the low level of X and Y chromosome mosaicism. The authors suppose that the patients with the exposed chromosomal abnormalities need the differentiated approach at their treatment.     

Immunogenicity of a Killed Bivalent (O1 and O139) Whole Cell Oral Cholera Vaccine,Shanchol, in Haiti

Background

Studies of the immunogenicity of the killed bivalent whole cell oral cholera vaccine, Shanchol, have been performed in historically cholera-endemic areas of Asia. There is a need to assess the immunogenicity of the vaccine in Haiti and other populations without historical exposure to Vibrio cholerae.

Methodology/Principal Findings

We measured immune responses after administration of Shanchol, in 25 adults, 51 older children (6–17 years), and 47 younger children (1–5 years) in Haiti, where cholera was introduced in 2010. A≥4-fold increase in vibriocidal antibody titer against V. cholerae O1 Ogawa was observed in 91% of adults, 74% of older children, and 73% of younger children after two doses of Shanchol; similar responses were observed against the Inaba serotype. A≥2-fold increase in serum O-antigen specific polysaccharide IgA antibody levels against V. cholerae O1 Ogawa was observed in 59% of adults, 45% of older children, and 61% of younger children; similar responses were observed against the Inaba serotype. We compared immune responses in Haitian individuals with age- and blood group-matched individuals from Bangladesh, a historically cholera-endemic area. The geometric mean vibriocidal titers after the first dose of vaccine were lower in Haitian than in Bangladeshi vaccinees. However, the mean vibriocidal titers did not differ between the two groups after the second dose of the vaccine.

Conclusions/Significance

A killed bivalent whole cell oral cholera vaccine, Shanchol, is highly immunogenic in Haitian adults and children. A two-dose regimen may be important in Haiti, and other populations lacking previous repeated exposures to V. cholerae.     

Cardiac pressure overload initiates a systemic stem cell response

Background aims. Acute cardiac injury results in the activation and recruitment of resident and non-cardiac stem cells. In this study we sought to define the pattern of peripheral stem cells and resident cardiac stem cell (CSC) activation induced acutely by cardiac pressure overload (PO). Methods. PO was induced in mice by transaortic constriction (TAC). CSC, endothelial progenitor cells (EPC), hematopoietic stem cells (HSC) and stage-specific embryonic antigen (SSEA)-1(+) cells were profiled in the heart, spleen and bone marrow after TAC by flow cytometry. Results. The combination of a systemic and local stem cell response resulted in increases in SSEA-1 (+) cells and EPC in the heart 7 and 14 days post-TAC, respectively. Locally, modest SSEA-1(+) proliferation at 4 days preceded the elevated myocardial stem cell number. We observed no significant proliferation of EPC and CSC in the heart. The systemic stem cell response was characterized by a biphasic loss of splenic SSEA-1(+) cells at 2 and 7 days post-TAC and loss of bone marrow and spleen EPC at 4 and 7 days, respectively. Spleen size changed dynamically after TAC. A negligible response of HSC to TAC was observed. Significant EPC and SSEA-1(+) proliferation in the bone marrow and spleen occurred only after their local levels were decreased. Conclusions. Our results demonstrate that an orchestrated systemic stem cell response (EPC and SSEA-1 (+) ) takes place in response to TAC. The increase of SSEA-1(+) cells and EPC in the heart in response to pressure is likely to be because of a combination of local proliferation and stem cell recruitment.     

The Squash Aspartic Proteinase Inhibitor SQAPI Is Widely Present in the Cucurbitales, Comprises a Small Multigene Family, and Is a Member of the Phytocystatin Family

The squash (Cucurbita maxima) phloem exudate-expressed aspartic proteinase inhibitor (SQAPI) is a novel aspartic acid proteinase inhibitor, constituting a fifth family of aspartic proteinase inhibitors. However, a comparison of the SQAPI sequence to the phytocystatin (a cysteine proteinase inhibitor) family sequences showed ∼30% identity. Modeling SQAPI onto the structure of oryzacystatin gave an excellent fit; regions identified as proteinase binding loops in cystatin coincided with regions of SQAPI identified as hypervariable, and tryptophan fluorescence changes were also consistent with a cystatin structure. We show that SQAPI exists as a small gene family. Characterization of mRNA and clone walking of genomic DNA (gDNA) produced 10 different but highly homologous SQAPI genes from Cucurbita maxima and the small family size was confirmed by Southern blotting, where evidence for at least five loci was obtained. Using primers designed from squash sequences, PCR of gDNA showed the presence of SQAPI genes in other members of the Cucurbitaceae and in representative members of Coriariaceae, Corynocarpaceae, and Begoniaceae. Thus, at least four of seven families of the order Cucurbitales possess member species with SQAPI genes, covering ∼99% of the species in this order. A phylogenetic analysis of these Cucurbitales SQAPI genes indicated not only that SQAPI was present in the Cucurbitales ancestor but also that gene duplication has occurred during evolution of the order. Phytocystatins are widespread throughout the plant kingdom, suggesting that SQAPI has evolved recently from a phytocystatin ancestor. This appears to be the first instance of a cystatin being recruited as a proteinase inhibitor of another proteinase family. [Reviewing Editor: Dr. Antony Dean]     

Exploring the mode-of-action of bioactive compounds by chemical-genetic profiling in yeast

Discovering target and off-target effects of specific compounds is critical to drug discovery and development. We generated a compendium of "chemical-genetic interaction" profiles by testing the collection of viable yeast haploid deletion mutants for hypersensitivity to 82 compounds and natural product extracts. To cluster compounds with a similar mode-of-action and to reveal insights into the cellular pathways and proteins affected, we applied both a hierarchical clustering and a factorgram method, which allows a gene or compound to be associated with more than one group. In particular, tamoxifen, a breast cancer therapeutic, was found to disrupt calcium homeostasis and phosphatidylserine (PS) was recognized as a target for papuamide B, a cytotoxic lipopeptide with anti-HIV activity. Further, the profile of crude extracts resembled that of its constituent purified natural product, enabling detailed classification of extract activity prior to purification. This compendium should serve as a valuable key for interpreting cellular effects of novel compounds with similar activities.