Current challenges in software solutions for mass spectrometry-based quantitative proteomics

This work was in part supported by the PRIME-XS project, grant agreement number 262067, funded by the European Union seventh Framework Programme; The Netherlands Proteomics Centre, embedded in The Netherlands Genomics Initiative; The Netherlands Bioinformatics Centre; and the Centre for Biomedical Genetics (to S.C., B.B. and A.J.R.H); by NIH grants NCRR RR001614 and RR019934 (to the UCSF Mass Spectrometry Facility, director: A.L. Burlingame, P.B.); and by grants from the MRC, CR-UK, BBSRC and Barts and the London Charity (to P.C.

Therapeutic DNA Vaccine Encoding Peptide P10 against Experimental Paracoccidioidomycosis

Paracoccidioidomycosis (PCM), caused by Paracoccidioides brasiliensis, is the most prevalent invasive fungal disease in South America. Systemic mycoses are the 10th most common cause of death among infectious diseases in Brazil and PCM is responsible for more than 50% of deaths due to fungal infections. PCM is typically treated with sulfonamides, amphotericin B or azoles, although complete eradication of the fungus may not occur and relapsing disease is frequently reported. A 15-mer peptide from the major diagnostic antigen gp43, named P10, can induce a strong T-CD4+ helper-1 immune response in mice. The TEPITOPE algorithm and experimental data have confirmed that most HLA-DR molecules can present P10, which suggests that P10 is a candidate antigen for a PCM vaccine. In the current work, the therapeutic efficacy of plasmid immunization with P10 and/or IL-12 inserts was tested in murine models of PCM. When given prior to or after infection with P. brasiliensis virulent Pb 18 isolate, plasmid-vaccination with P10 and/or IL-12 inserts successfully reduced the fungal burden in lungs of infected mice. In fact, intramuscular administration of a combination of plasmids expressing P10 and IL-12 given weekly for one month, followed by single injections every month for 3 months restored normal lung architecture and eradicated the fungus in mice that were infected one month prior to treatment. The data indicate that immunization with these plasmids is a powerful procedure for prevention and treatment of experimental PCM, with the perspective of being also effective in human patients

Evidence for Positive Selection in Putative Virulence Factors within the Paracoccidioides brasiliensis Species Complex

Paracoccidioides brasiliensis is a dimorphic fungus that is the causative agent of paracoccidioidomycosis, the most important prevalent systemic mycosis in Latin America. Recently, the existence of three genetically isolated groups in P. brasiliensis was demonstrated, enabling comparative studies of molecular evolution among P. brasiliensis lineages. Thirty-two gene sequences coding for putative virulence factors were analyzed to determine whether they were under positive selection. Our maximum likelihood–based approach yielded evidence for selection in 12 genes that are involved in different cellular processes. An in-depth analysis of four of these genes showed them to be either antigenic or involved in pathogenesis. Here, we present evidence indicating that several replacement mutations in gp43 are under positive balancing selection. The other three genes (fks, cdc42 and p27) show very little variation among the P. brasiliensis lineages and appear to be under positive directional selection. Our results are consistent with the more general observations that selective constraints are variable across the genome, and that even in the genes under positive selection, only a few sites are altered. We present our results within an evolutionary framework that may be applicable for studying adaptation and pathogenesis in P. brasiliensis and other pathogenic fungi

A DNA Vaccine Encoding Multiple HIV CD4 Epitopes Elicits Vigorous Polyfunctional, Long-Lived CD4+ and CD8+ T Cell Responses

T-cell based vaccines against HIV have the goal of limiting both transmission and disease progression by inducing broad and functionally relevant T cell responses. Moreover, polyfunctional and long-lived specific memory T cells have been associated to vaccine-induced protection. CD4+ T cells are important for the generation and maintenance of functional CD8+ cytotoxic T cells. We have recently developed a DNA vaccine encoding 18 conserved multiple HLA-DR-binding HIV-1 CD4 epitopes (HIVBr18), capable of eliciting broad CD4+ T cell responses in multiple HLA class II transgenic mice. Here, we evaluated the breadth and functional profile of HIVBr18-induced immune responses in BALB/c mice. Immunized mice displayed high-magnitude, broad CD4+/CD8+ T cell responses, and 8/18 vaccine-encoded peptides were recognized. In addition, HIVBr18 immunization was able to induce polyfunctional CD4+ and CD8+ T cells that proliferate and produce any two cytokines (IFNγ/TNFα, IFNγ/IL-2 or TNFα/IL-2) simultaneously in response to HIV-1 peptides. For CD4+ T cells exclusively, we also detected cells that proliferate and produce all three tested cytokines simultaneously (IFNγ/TNFα/IL-2). The vaccine also generated long-lived central and effector memory CD4+ T cells, a desirable feature for T-cell based vaccines. By virtue of inducing broad, polyfunctional and long-lived T cell responses against conserved CD4+ T cell epitopes, combined administration of this vaccine concept may provide sustained help for CD8+ T cells and antibody responses- elicited by other HIV immunogens

Usefulness of PCR-based assays to assess drug efficacy in Chagas disease chemotherapy: value and limitations

One major goal of research on Chagas disease is the development of effective chemotherapy to eliminate the infection from individuals who have not yet developed cardiac and/or digestive disease manifestations. Cure evaluation is the more complex aspect of its treatment, often leading to diverse and controversial results. The absence of reliable methods or a diagnostic gold standard to assess etiologic treatment efficacy still constitutes a major challenge. In an effort to develop more sensitive tools, polymerase chain reaction (PCR)-based assays were introduced to detect low amounts of Trypanosoma cruzi DNA in blood samples from chagasic patients, thus improving the diagnosis and follow-up evaluation after chemotherapy. In this article, I review the main problems concerning drug efficacy and criteria used for cure estimation in treated chagasic patients, and the work conducted by different groups on developing PCR methodologies to monitor treatment outcome of congenital infections as well as recent and late chronic T. cruzi infections

Mycobacterium tuberculosis epitope-specific interferon-g production in healthy Brazilians reactive and non-reactive to tuberculin skin test

The interferon (IFN)-gamma response to peptides can be a useful diagnostic marker of Mycobacterium tuberculosis (MTB) latent infection. We identified promiscuous and potentially protective CD4(+) T-cell epitopes from the most conserved regions of MTB antigenic proteins by scanning the MTB antigenic proteins GroEL2, phosphate-binding protein 1 precursor and 19 kDa antigen with the TEPITOPE algorithm. Seven peptide sequences predicted to bind to multiple human leukocyte antigen (HLA)-DR molecules were synthesised and tested with IFN-gamma enzyme-linked immunospot (ELISPOT) assays using peripheral blood mononuclear cells (PBMCs) from 16 Mantoux tuberculin skin test (TST)-positive and 16 TST-negative healthy donors. Eighty-eight percent of TST-positive donors responded to at least one of the peptides, compared to 25% of TST-negative donors. Each individual peptide induced IFN-gamma production by PBMCs from at least 31% of the TST-positive donors. the magnitude of the response against all peptides was 182 +/- 230 x 10(6) IFN-gamma spot forming cells (SFC) among TST-positive donors and 36 +/- 62 x 10(6) SFC among TST-negative donors (p = 0.007). the response to GroEL2 (463-477) was only observed in the TST-positive group. This combination of novel MTB CD4 T-cell epitopes should be tested in a larger cohort of individuals with latent tuberculosis (TB) to evaluate its potential to diagnose latent TB and it may be included in ELISPOT-based IFN-gamma assays to identify individuals with this condition.Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)Universidade Federal de São Paulo, Fac Med, Inst Coracao, Immunol Lab, São Paulo, BrazilUniversidade Federal de São Paulo, Fac Med, Dept Med, Div Imunol Clin & Alergia, São Paulo, BrazilFundacao Oswaldo Cruz, Lab Avancado Saude Publ, Salvador, BA, BrazilEscola Bahiana Med & Saude Publ, Salvador, BA, BrazilInst Invest Immunol, São Paulo, BrazilUniversidade Federal de São Paulo, Fac Med, Inst Coracao, Immunol Lab, São Paulo, BrazilUniversidade Federal de São Paulo, Fac Med, Dept Med, Div Imunol Clin & Alergia, São Paulo, BrazilWeb of Scienc