Whole blood interferon-gamma responses to mycobacterium tuberculosis antigens in young household contacts of persons with tuberculosis in Uganda

Background

Due to immunologic immaturity, IFN-γ-producing T cell responses may be decreased in young children compared to adults, thus we hypothesized that IFN-γ responses to mycobacterial antigens in household contacts exposed to Mycobacterium tuberculosis (Mtb) would be impaired in young children relative to adults. The objective of this study was to compare whole blood IFN-γ production in response to mycobacterial antigens between children and adults in Uganda.

Methodology/Principal Findings

We studied household contacts of persons with culture-positive pulmonary tuberculosis (TB) enrolled in a cohort study conducted in Kampala, Uganda. Whole blood IFN-γ production in response to Mtb culture-filtrate antigens was measured by ELISA and compared between infants (<2 years old, n = 80), young children (2 <5 years old, n = 216), older children (5 <15 years old, n = 443) and adults (≥15 years old, n = 528). We evaluated the relationship between IFN-γ responses and the tuberculin skin test (TST), and between IFN-γ responses and epidemiologic factors that reflect exposure to Mtb, and the effect of prior BCG vaccination on IFN-γ responses. Young household contacts demonstrated robust IFN-γ responses comparable to those of adults that were associated with TST and known risk factors for infection. There was no effect of prior BCG immunization on the IFN-γ response.

Conclusions/Significance

Young children in a TB endemic setting can mount robust IFN-γ responses generally comparable to those of adults, and as in adults, these responses correlated with the TST and known epidemiologic risk factors for Mtb infection.     

Immunological relatedness of cytoplasmic and membrane-bound acid phosphatases from Dioscorea cayenensis rotundata

Antibodies were raised against one cytoplasmic and two membrane-bound acid phosphatases purified from yam tubers (Dioscorea cayenensis rotundata). Experiments of immunoinactivation and immunoelectrophoresis revealed cross-immunological reactions between the cytoplasmic enzyme (acid phosphatase A) and one of two membrane-bound counterparts (acid phosphatase B) suggesting that these molecules share common antigenic determinants. The antibodies raised against the other membrane-bound enzyme (acid phosphatase C) only inhibited and precipitated this enzyme.     

Structure and stability of an acidic fibroblast growth factor from Notophthalmus viridescens

The three-dimensional solution structure of an acidic fibroblast growth factor (nFGF-1) from the newt (Notophthalmus viridescens) is determined using multidimensional NMR techniques. Complete assignment of all the atoms ((1)H, (15)N, and (13)C) has been achieved using a variety of triple resonance experiments. 50 structures were calculated using hybrid distance geometry-dynamical simulated annealing technique with a total of 1359 constraints. The atomic root mean square distribution for the backbone atoms in the structured region is 0.60 A. The secondary structural elements include 12 beta-strands arranged antiparallely into a beta-barrel structure. The protein (nFGF-1) exists in a monomeric state upon binding to the ligand, sucrose octa sulfate (SOS), in a stoichiometric ratio of 1:1. The SOS binding site consists of a dense cluster of positively charged residues located at the C-terminal end of the molecule. The conformational stabilities of nFGF-1 and its structural and functional homologue from the human source (hFGF-1) are drastically different. The differential stabilities of nFGF-1 and hFGF-1 are attributed to the differences in the number of hydrogen bonds and the presence of solvent inaccessible cavities in the two proteins.     

New insights in protein phosphorylation: a signature for protein phosphatase 1 interacting proteins

Protein phosphatase 1 is regulated by the interaction between a catalytic subunit (PP1c) and multiple interacting proteins that allow the specific dephosphorylation of diverse cellular targets. This communication proposes to use the simultaneous presence of distinct consensus PP1c docking motifs R/K-x(0,1)-V-x-F and F-x-x-R/K-x-R/K as a signature to identify proteins putatively interacting with the PP1c. To develop this concept, we propose a new website, http://pp1 signature.pasteur.fr, which allows the identification of putative PP1-interacting proteins containing the two distinct PP1c docking consensus motifs represented in the Swissprot library. To validate the new concept of signature, we were able to characterise, by co-immunoprecipitation, four new PP1c interacting proteins randomly selected from the database in our website.     

Mevalonosomes: specific vacuoles containing the mevalonate pathway in Plocamium brasiliense cortical cells (Rhodophyta)

This paper has identified, for the first time in a member of the Rhodophyta, a vacuolar organelle containing enzymes that are involved in the mevalonate pathway—an important step in red algal isoprenoid biosynthesis. These organelles were named mevalonosomes (Mev) and were found in the cortical cells (CC) of Plocamium brasiliense, a marine macroalgae that synthesizes several halogenated monoterpenes. P. brasiliense specimens were submitted to a cytochemical analysis of the activity of the 3‐hydroxy‐3‐methylglutaryl‐CoA synthase (HMGS). Using transmission electron microscopy (TEM), we confirmed the presence of HMGS activity within the Mev. Because HMGS is necessary for the biosynthesis of halogenated monoterpenes, we isolated a hexanic fraction (HF) rich in halogenated monoterpenes from P. brasiliense that contained a pentachlorinated monoterpene as a major metabolite. Because terpenes are often related to chemical defense, the antifouling (AF) activity of pentachlorinated monoterpene was tested. We found that the settlement of the mussel Perna perna was reduced by HF treatment (2.25 times less than control; 40% and 90% of fouled surface, respectively; P = 0.001; F_9,9 = 1.13). The HF (at 10 μg · mL^?1) also inhibited three species of fouling microalgae (Chlorarachnion reptans, Cylindrotheca cloisterium, and Exanthemachrysis gayraliae), while at a higher concentration (50 μg · mL^?1), it inhibited the bacteria Halomonas marina, Polaribacter irgensii, Pseudoalteromonas elyakovii, Shewanella putrefaciens, and Vibrio aestuarianus. The AF activity of P. brasiliense halogenated monoterpenes and the localization of HMGS activity inside Mev suggest that this cellular structure found in CC may play a role in thallus protection against biofouling.     

Cough Aerosol Cultures of Mycobacterium tuberculosis: Insights on TST / IGRA Discordance and Transmission Dynamics

Rationale

The diagnosis of latent tuberculosis (TB) infection (LTBI) is complicated by the absence of a gold standard. Discordance between tuberculin skin tests (TST) and interferon gamma release assays (IGRA) occurs in 10–20% of individuals, but the underlying mechanisms are poorly understood.

Methods

We analyzed data from a prospective household contact study that included cough aerosol culture results from index cases, environmental and contact factors. We assessed contacts for LTBI using TST and IGRA at baseline and six weeks. We examined TST/IGRA discordance in qualitative and quantitative analyses, and used multivariable logistic regression analysis with generalized estimating equations to analyze predictors of discordance.

Measurements and Results

We included 96 TB patients and 384 contacts. Discordance decreased from 15% at baseline to 8% by six weeks. In adjusted analyses, discordance was related to less crowding (p = 0.004), non-cavitary disease (OR 1.41, 95% CI: 1.02–1.96; p = 0.03), and marginally with BCG vaccination in contacts (OR 1.40, 95% CI: 0.99–1.98, p = 0.06).

Conclusions

We observed significant individual variability and temporal dynamism in TST and IGRA results in household contacts of pulmonary TB cases. Discordance was associated with a less intense infectious exposure, and marginally associated with a BCG-mediated delay in IGRA conversion. Cough aerosols provide an additional dimension to the assessment of infectiousness and risk of infection in contacts.     

Functional interaction of Aurora-A and PP2A during mitosis

Entry into mitosis is a highly regulated process, promoted by the activated Cyclin B1/Cdk1 complex. Activation of this complex is controlled, in part, by the protein kinase Aurora-A, which is a member of a multigenic serine/threonine kinase family. In normal cells, Aurora-A activity is regulated, at least in part, by degradation through the APC-ubiquitin-proteasome pathway. It has recently been proposed that, in Xenopus, Aurora-A degradation can be inhibited by phosphorylation. It would thus be expected that a phosphatase activity would release this blockade at the end of mitosis. Here, we have shown that the protein phosphatase PP2A and Aurora-A are colocalized at the cell poles during mitosis in human cells and interact within the same complex. Using the PP2A inhibitor okadaic acid and an RNAi approach, we have shown that this interaction is functional within the cell. PP2A/Aurora-A interaction is promoted by an S51D mutation in Aurora-A and inhibited by a phosphomimetic peptide centered around Aurora-A S51, thereby strongly suggesting that PP2A controls Aurora-A degradation by dephosphorylating serine 51 in the A box of the human enzyme.     

Limited Breadth of the Protective Immunity Elicited by Simian Immunodeficiency Virus SIVmne gp160 Vaccines in a Combination Immunization Regimen

We previously reported that immunization with recombinant simian immunodeficiency virus SIVmne envelope (gp160) vaccines protected macaques against an intravenous challenge by the cloned homologous virus, E11S. In this study, we confirmed this observation and found that the vaccines were effective not only against virus grown on human T-cell lines but also against virus grown on macaque peripheral blood mononuclear cells (PBMC). The breadth of protection, however, was limited. In three experiments, 3 of 10 animals challenged with the parental uncloned SIVmne were completely protected. Of the remaining animals, three were transiently virus positive and four were persistently positive after challenge, as were 10 nonimmunized control animals. Protection was not correlated with levels of serum-neutralizing antibodies against the homologous SIVmne or a related virus, SIVmac251. To gain further insight into the protective mechanism, we analyzed nucleotide sequences in the envelope region of the uncloned challenge virus and compared them with those present in the PBMC of infected animals. The majority (85%) of the uncloned challenge virus was homologous to the molecular clone from which the vaccines were made (E11S type). The remaining 15% contained conserved changes in the V1 region (variant types). Control animals infected with this uncloned virus had different proportions of the two genotypes, whereas three of four immunized but persistently infected animals had >99% of the variant types early after infection. These results indicate that the protective immunity elicited by recombinant gp160 vaccines is restricted primarily to the homologous virus and suggest the possibility that immune responses directed to the V1 region of the envelope protein play a role in protection.     

Expression of Sonic hedgehog-Fc fusion protein in Pichia pastoris. Identification and control of post-translational,chemical, and proteolytic modifications

We have investigated the suitability of Pichia pastoris as an expression system for the candidate therapeutic protein, Sonic hedgehog fused to an immunoglobulin Fc domain (Shh-Fc). Sonic hedgehog is a morphogen protein involved in the patterning of a wide range of tissues during animal embryogenesis. The presence of Sonic hedgehog and its receptor, Patched, in adult nervous tissue suggests possible applications for the protein in the treatment of neurodegenerative disease and injury. We have engineered the Shh-Fc fusion protein in order to improve binding affinity and increase systemic exposure in animals. N-terminal sequencing, peptide mapping, mass spectrometry, and other biochemical and biological methods were used to characterize the purified protein. These analyses revealed several unanticipated problems, including thiaproline modification of the N-terminal cysteine, cleavage by a Kex2-like protease at a site near the N-terminus, proteolysis at sites near the hinge, addition of a hexose in the CH3 domain of the Fc region, and several sites of methionine oxidation. Sequence modifications to the protein and changes in fermentation conditions resulted in increased potency and greater consistency of the product. The final product was shown to be biologically active in animal studies.