Pavel Svoboda

University of Central Florida, Orlando, FL, United States

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Publications (11)54.65 Total impact

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    ABSTRACT: A pneumococcal serotyping/genotyping system (PSGS) was developed based upon targeted PCR, followed by electrospray ion-ization mass spectrometry and amplicon base composition analysis. Eight multiplex PCRs, 32 targeting serotype-determining capsular biosynthetic loci, and 8 targeting multilocus sequence typing (MLST) loci were employed for each of 229 highly diverse Streptococcus pneumoniae isolates. The most powerful aspect of the PSGS system was the identification of capsular serotypes accounting for the majority of invasive and carried pneumococcal strains. Altogether, 45 different serotypes or serogroups were correctly predicted among the 196 resolvable isolates, with only 2 unexpected negative results. All 33 isolates that represented 23 serotypes not included in the PSGS yielded negative serotyping results. A genotyping database was constructed using the base compositions of 65-to 100-bp sections of MLST alleles compiled within http://www.mlst.net. From this database, one or more MLST sequence types (STs) that comprised a PSGS genotype were identified. The end result of more PSGS genotypes (163) than conventional STs actually tested (155) was primarily due to amplification failures of 1 to 3 targets. In many instances, the PSGS genotype could provide resolution of single-and double-locus variants. This molecular serotyping/genotyping scheme is well suited to rapid characterization of large sets of pneumococcal isolates.
    Journal of Clinical Microbiology 06/2012; · 4.07 Impact Factor
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    ABSTRACT: The extensive world-wide morbidity and mortality caused by influenza A viruses highlights the need for new insights into the host immune response and novel treatment approaches. Cationic Host Defense Peptides (CHDP, also known as antimicrobial peptides), which include cathelicidins and defensins, are key components of the innate immune system that are upregulated during infection and inflammation. Cathelicidins have immunomodulatory and anti-viral effects, but their impact on influenza virus infection has not been previously assessed. We therefore evaluated the effect of cathelicidin peptides on disease caused by influenza A virus in mice. The human cathelicidin, LL-37, and the murine cathelicidin, mCRAMP, demonstrated significant anti-viral activity in vivo, reducing disease severity and viral replication in infected mice to a similar extent as the well-characterized influenza virus-specific antiviral drug zanamivir. In vitro and in vivo experiments suggested that the peptides may act directly on the influenza virion rather than via receptor-based mechanisms. Influenza virus-infected mice treated with LL-37 had lower concentrations of pro-inflammatory cytokines in the lung than did infected animals that had not been treated with cathelicidin peptides. These data suggest that treatment of influenza-infected individuals with cathelicidin-derived therapeutics, or modulation of endogenous cathelicidin production may provide significant protection against disease.
    PLoS ONE 01/2011; 6(10):e25333. · 3.73 Impact Factor
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    ABSTRACT: We recently reported that HIV-1 infection can be inhibited by innate antimicrobial components of human seminal plasma (SP). Conversely, naturally occurring peptidic fragments from the SP-derived prostatic acid phosphatase (PAP) have been reported to form amyloid fibrils called "SEVI" and enhance HIV-1 infection in vitro. In order to understand the biological consequence of this proviral effect, we extended these studies in the presence of human SP. PAP-derived peptides were agitated to form SEVI and incubated in the presence or absence of SP. While PAP-derived peptides and SEVI alone were proviral, the presence of 1% SP ablated their proviral activity in several different anti-HIV-1 assays. The anti-HIV-1 activity of SP was concentration dependent and was reduced following filtration. Supraphysiological concentrations of PAP peptides and SEVI incubated with diluted SP were degraded within hours, with SP exhibiting proteolytic activity at dilutions as high as 1:200. Sub-physiological concentrations of two prominent proteases of SP, prostate-specific antigen (PSA) and matriptase, could degrade physiological and supraphysiological concentrations of PAP peptides and SEVI. While human SP is a complex biological fluid, containing both antiviral and proviral factors, our results suggest that PAP peptides and SEVI may be subject to naturally occurring proteolytic components capable of reducing their proviral activity.
    PLoS ONE 01/2011; 6(1):e16285. · 3.73 Impact Factor
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    ABSTRACT: The emergence and rapid spread of the 2009 H1N1 pandemic influenza A virus (H1N1pdm) in humans highlights the importance of enhancing the capability of existing influenza surveillance systems with tools for rapid identification of emerging and re-emerging viruses. One of the new approaches is the RT-PCR electrospray ionization mass spectrometry (RT-PCR/ESI-MS) technology, which is based on analysis of base composition (BC) of RT-PCR amplicons from influenza "core" genes. Combination of the BC signatures represents a "genomic print" of an influenza A virus. Here, 757 samples collected between 2006 and 2009 were tested, including 302 seasonal H1N1, 171 H3N2, 7 swine triple reassortants, and 277 H1N1pdm viruses. Of the 277 H1N1pdm samples, 209 were clinical specimens (throat, nasal and nasopharyngeal swabs, nasal washes, blood and sputum). BC signatures for the clinical specimen from one of the first cases of the 2009 pandemic, A/California/04/2009, confirmed it as an unusual, previously unrecognized influenza A virus, with "core" genes related to viruses of avian, human and swine origins. Subsequent analysis of additional 276 H1N1pdm samples revealed that they shared the genomic print of A/California/04/2009, which differed from those of North American swine triple reassortant viruses, seasonal H1N1 and H3N2 and other viruses tested. Moreover, this assay allowed distinction between "core" genes of co-circulating groups of seasonal H1N1, such as clades 2B, 2C, and their reassortants with dual antiviral resistance to adamantanes and oseltamivir. The RT-PCR/ESI-MS assay is a broad range influenza identification tool that can be used directly on clinical specimens for rapid and accurate detection of influenza virus genes. The assay differentiates the H1N1pdm from seasonal and other nonhuman hosts viruses. Although not a diagnostic tool, this assay demonstrates its usefulness and robustness in influenza virus surveillance and detection of novel and unusual viruses with previously unseen genomic prints.
    PLoS ONE 01/2010; 5(10):e13293. · 3.73 Impact Factor
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    ABSTRACT: Capsular polysaccharides (CPS) are a major virulence factor in meningococcal infections and form the basis for serogroup designation and protective vaccines. Our work has identified meningococcal CPS as a pro-inflammatory ligand that functions through TLR2 and TLR4-MD2-dependent activation. We hypothesized that human cationic host defense peptides interact with CPS and influence its biologic activity. Accordingly, the interaction of meningococcal CPS with the human-derived cationic peptide LL-37, which is expressed by phagocytic and epithelial cells that interface with meningococci during infection, was investigated. LL-37 neutralized the pro-inflammatory activity of endotoxin-free CPS as assessed by TLR2 and TLR4-MD-2-dependent release of TNFα, IL-6 and IL-8 from human and murine macrophages. The cationic and hydrophobic properties of LL-37 were crucial for this inhibition, which was due to binding of LL-37 to CPS. LL-37 also inhibited the ability of meningococcal CPS to induce nitric oxide release, as well as TNFα and CXCL10 (IP-10) release from TLR4-sufficient and TLR4-deficient murine macrophages. Truncated LL-37 analogs, especially those that retained the antibacterial domain, inhibited vaccine grade CPS and meningococcal CPS prepared from the major serogroups (A, B C, Y and W135). Thus, LL-37 interaction with CPS was independent of specific glucan structure. We conclude that the capacity of meningococcal CPS to activate macrophages via TLR2 and TLR4-MD-2 can be inhibited by the human cationic host defense peptide LL-37 and propose that this impacts CPS-based vaccine responses.
    PLoS ONE 01/2010; 5(10):e13627. · 3.73 Impact Factor
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    ABSTRACT: Rabies prevention and appropriate population management of free-ranging animals have an important role to play in the eventual elimination of rabies in dogs. An effective sterilant based on rabies vaccines has the potential to create a supportive measure of public acceptability and to reduce associated clinic visit costs. We inserted the coding sequence of gonadotropin-releasing hormone (GnRH) into different locations within the rabies virus ERA glycoprotein (G) gene, and demonstrated that the amino terminus (N), antigenic site IIa, and the junction between the ecto- and cytoplasmic domains (C) of the G were suitable sites for GnRH insertion. The rescued recombinant rabies viruses ERA-N-GnRH and ERA-C-GnRH grew as well as the parental ERA virus, reaching 1x10(9)ffu/ml in cell culture. Insertion and expression of the GnRH were stable in the viruses after multiple passages in vitro. To increase immunogenicity of the GnRH peptide, two copies of GnRH, aligned in tandem, were fused to the N terminus of the G. The recombinant rabies virus ERA-N-2GnRH was recovered and grown to high titers in cell culture. All GnRH-carrying rabies viruses induced antibodies against GnRH in immunized mice and protected 100% of the animals after rabies virus challenge. The recombinant viruses reacted strongly with the serum from a GonaCon-immunized animal. The GnRH-carrying rabies viruses have significant potential in rabies and animal population control.
    Vaccine 11/2009; 27(51):7202-9. · 3.77 Impact Factor
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    ABSTRACT: Mucosal surfaces of the reproductive tract as well as their secretions have important roles in preventing sexual transmission of HIV-1. In the current study, the majority of the intrinsic anti-HIV-1 activity of human seminal plasma (SP) was determined to reside in the cationic polypeptide fraction. Antiviral assays utilizing luciferase reporter cells and lymphocytic cells revealed the ability of whole SP to prevent HIV-1 infection, even when SP was diluted 3200-fold. Subsequent fractionation by continuous flow acid-urea (AU)-PAGE and antiviral testing revealed that cationic polypeptides within SP were responsible for the majority of anti-HIV-1 activity. A proteomic approach was utilized to resolve and identify 52 individual cationic polypeptides that contribute to the aggregate anti-HIV-1 activity of SP. One peptide fragment of semenogelin I, termed SG-1, was purified from SP by a multistep chromatographic approach, protein sequenced, and determined to exhibit anti-HIV-1 activity against HIV-1. Anti-HIV-1 activity was transient, as whole SP incubated for prolonged time intervals exhibited a proportional decrease in anti-HIV-1 activity that was directly attributed to the degradation of semenogelin I peptides. Collectively, these results indicate that the cationic polypeptide fraction of SP is active against HIV-1, and that semenogelin-derived peptides contribute to the intrinsic anti-HIV-1 activity of SP.
    The FASEB Journal 07/2009; 23(10):3609-18. · 5.70 Impact Factor
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    ABSTRACT: Tumor-stroma interactions play a major role in tumor development, maintenance and progression. Yet little is known on how the genetic alterations that underlie cell transformation elicit cell extrinsic changes modulating heterotypic cell interactions. We hypothesized that these events involve a modification in the complement of secreted proteins by the cell, acting as mediators of intercellular communication. To test this hypothesis, we examined the role of wt-p53, a major tumor suppressor, on the tumor microenvironment through its regulation of secreted factors. Using a combination of 2-DE and cICAT proteomic techniques, we found a total of 111 secreted proteins, 39 of which showed enhanced and 21 inhibited secretion in response to wt-p53 expression. The majority of these were not direct targets of p53 transcription factor activity, suggesting a novel role for wt-p53 in the control of intracellular protein trafficking and/or secreted protein stability. Evidence for p53-controlled post-translational modifications on nine secreted proteins was also found. These findings will enhance our understanding of wt-p53 modulated interactions of the tumor with its environment.
    Oncogene 01/2007; 25(58):7650-61. · 7.36 Impact Factor
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    ABSTRACT: Human beta-defensin-1 (hBD-1) is a candidate tumor suppressor gene located on chromosome 8p23. Previously, we showed that cancer-specific loss of hBD-1 was found in 90% of renal clear cell carcinomas and in 82% of prostate cancers. To investigate the possible mechanisms of decreased gene expression and determine the function of hBD-1 protein in urological cancers, we sequenced hBD-1 gene coding regions in prostatic and renal cancer samples. We then analyzed the frequency distribution of promoter polymorphisms and determined the effect of these base changes on transcriptional activity of the hBD-1 promoter. A polymorphism at -688 bases upstream of the ATG start codon affects hBD-1 promoter activity, leading to a rate of reporter gene transcription that is 40% to 50% lower than the wild-type sequence when tested in either DU145 or TSU-Pr1 cell lines. In addition, a polymorphism at -44 bases was shown to enhance transcription up to 2.3 times more than the wild-type sequence in the same cell lines. In addition, three novel hBD-1 promoter mutations were found in renal and prostate cancer clinical samples. An iso-5-aza-2'-deoxycytidine treatment was effective in transcription up-regulation in DU145, suggesting a possible upstream methylation-dependent effect. Synthetic hBD-1 peptide inhibited bladder cancer cell TSU-Pr1 proliferation. Overexpression of the hBD-1 gene in renal cancer cells SW156 resulted in caspase-3-mediated apoptosis. These data support the hypothesis that hBD-1 is a potential tumor suppressor gene for urological cancers. Promoter point mutations may be responsible for cancer-specific loss of hDB-1 expression.
    Cancer Research 10/2006; 66(17):8542-9. · 8.65 Impact Factor
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    ABSTRACT: Mucosal surfaces of the vagina are the portals for heterosexual transmission of HIV-1 and therefore play a fundamental role in the pathogenesis of primary infection. In the search for direct biological evidence for the role of human vaginal fluid in innate host defense, we characterized the anti-HIV-1 function of cationic polypeptides within minimally manipulated vaginal fluid. In the current study we revealed that vaginal fluid confers intrinsic anti-HIV-1 properties against both X4 and R5 strains of HIV-1 and could protect against HIV-1 infection and reduce proviral genome integration in organotypic cultures of human cervicovaginal tissue. The majority of this activity was contained in the cationic polypeptide fraction, and the depletion of cationic polypeptides using a selective cation exchange resin ablated most of the intrinsic activity against HIV-1. By adding the cationic polypeptide fraction to depleted vaginal fluid, we were able to restore activity against HIV-1. Using a proteomic approach, we identified 18 cationic polypeptides within vaginal fluid, nearly all of which are either known antimicrobials or have other purported roles in host defense. Interestingly, physiologic concentrations of 13 of the cationic polypeptides were not active alone against HIV-1, yet in concert they partially restored the anti-HIV-1 activity of cation-depleted vaginal fluid. These results suggest that synergism between cationic polypeptides is complex, and full anti-HIV-1 activity probably involves the aggregate of the cationic peptides and proteins in vaginal fluid.
    The Journal of Immunology 01/2006; 175(11):7560-7. · 5.52 Impact Factor
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    ABSTRACT: Two extracellular endo-beta-1,4-mannanases, MAN I (major form) and MAN II (minor form), were purified to electrophoretic homogeneity from a locust bean gum-spent culture fluid of Aspergillus fumigatus IMI 385708 (formerly Thermomyces lanuginosus IMI 158749). Molecular weights of MAN I and MAN II estimated by SDS-PAGE were 60 and 63 kDa, respectively. IEF afforded several glycoprotein bands with pI values in the range of 4.9-5.2 for MAN I and 4.75-4.9 for MAN II, each exhibiting enzyme activity. MAN I as well as MAN II showed highest activity at pH 4.5 and 60 degrees C and were stable in the pH range 4.5-8.5 and up to 55 degrees C. In accordance with the ability of the enzymes to catalyze transglycosylation reactions, 1H NMR spectroscopy of reaction products generated from mannopentaitol confirmed the retaining character of both enzymes. Both MAN I and MAN II exhibited essentially identical kinetic parameters for polysaccharides and a similar hydrolysis pattern of various oligomeric and polymeric substrates. Both beta-mannanases contained identical internal amino acid sequence corresponding to glycoside hydrolase family 5 and also a cellulose-binding module. These data suggested that both MAN I and MAN II are products of the same gene differing in posttranslational modification. Indeed, the corresponding gene was identified within the recently sequenced Aspergillus fumigatus genome (http://sanger.ac.uk/Projects/A_fumigatus/).
    Biochimica et Biophysica Acta 11/2004; 1674(3):239-50. · 4.66 Impact Factor

Publication Stats

244 Citations
307 Downloads
957 Views
54.65 Total Impact Points

Institutions

  • 2006–2009
    • University of Central Florida
      • • Graduate Program in Molecular Biology and Microbiology
      • • Burnett School of Biomedical Sciences
      Orlando, FL, United States
  • 2004
    • Emory University
      • School of Medicine
      Atlanta, Georgia, United States