Development of non-antibiotic-resistant, chromosomally based, constitutive and inducible expression systems for aroA-attenuated Salmonella enterica serovar typhimurium

Live-vaccine delivery systems expressing two model antigens from Mycoplasma hyopneumoniae, F2(P97) (Adh) and NrdF, were constructed using Salmonella enterica serovar Typhimurium aroA (STM-1), and immunogenicity in mice was evaluated. Recombinant plasmid-based expression (PBE) and chromosomally based expression (CBE) systems were constructed. The PBE system was formed by cloning both antigen genes into pJLA507 to create an operon downstream of temperature-inducible promoters. Constitutive CBE was achieved using a promoter-trapping technique whereby the promoterless operon was stably integrated into the chromosome of STM-1, and the expression of antigens was assessed. The chromosomal position of the operon was mapped in four clones. Inducible CBE was obtained by using the in vivo-induced sspA promoter and recombining the expression construct into aroD. Dual expression of the antigens was detected in all systems, with PBE producing much larger quantities of both antigens. The stability of antigen expression after in vivo passage was 100% for all CBE strains recovered. PBE and CBE strains were selected for comparison in a vaccination trial. The vaccine strains were delivered orally into mice, and significant systemic immunoglobulin M(IgM) and IgG responses against both antigens were detected among all CBE groups. No significant immune response was detected using PBE strains. Expression of recombinant antigens in S. enterica serovar Typhimurium aroA from chromosomally located strong promoters without the use of antibiotic resistance markers is a reliable and effective method of inducing a significant immune response

Circulating gluten-specific FOXP3 + CD39 + regulatory T cells have impaired suppressive function in patients with celiac disease

Background Celiac disease is a chronic immune-mediated inflammatory disorder of the gut triggered by dietary gluten. Although the effector T-cell response in patients with celiac disease has been well characterized, the role of regulatory T (Treg) cells in the loss of tolerance to gluten remains poorly understood. Objective We sought to define whether patients with celiac disease have a dysfunction or lack of gluten-specific forkhead box protein 3 (FOXP3)+ Treg cells. Methods Treated patients with celiac disease underwent oral wheat challenge to stimulate recirculation of gluten-specific T cells. Peripheral blood was collected before and after challenge. To comprehensively measure the gluten-specific CD4+ T-cell response, we paired traditional IFN-γ ELISpot with an assay to detect antigen-specific CD4+ T cells that does not rely on tetramers, antigen-stimulated cytokine production, or proliferation but rather on antigen-induced coexpression of CD25 and OX40 (CD134). Results Numbers of circulating gluten-specific Treg cells and effector T cells both increased significantly after oral wheat challenge, peaking at day 6. Surprisingly, we found that approximately 80% of the ex vivo circulating gluten-specific CD4+ T cells were FOXP3+CD39+ Treg cells, which reside within the pool of memory CD4+CD25+CD127lowCD45RO+ Treg cells. Although we observed normal suppressive function in peripheral polyclonal Treg cells from patients with celiac disease, after a short in vitro expansion, the gluten-specific FOXP3+CD39+ Treg cells exhibited significantly reduced suppressive function compared with polyclonal Treg cells. Conclusion This study provides the first estimation of FOXP3+CD39+ Treg cell frequency within circulating gluten-specific CD4+ T cells after oral gluten challenge of patients with celiac disease. FOXP3+CD39+ Treg cells comprised a major proportion of all circulating gluten-specific CD4+ T cells but had impaired suppressive function, indicating that Treg cell dysfunction might be a key contributor to disease pathogenesis

Qualitative analysis of T-cell repertoire for relevance to non-progressive HIV infection

Cytotoxic T-lymphocytes are important for the control of viral replication during HIV infection, however the magnitude and breadth of HIV-specific CD8+ T-cell response does not correlate well. The purpose for this study was the examination of the HLA-B*2705-specific CD8+ T-cell response to the KRWIILGLNK (KK10) epitope as a definitive model of immune control over HIV replication. The breadth of the T-cell receptor (TCR) repertoire was determined for an association between the qualitative nature of this response and immune escape and therefore, disease progression. Methodology was developed and validated for TCR repertoire analysis in formaldehyde fixed antigen-specific CD8+ T-cells. The TCR repertoire for the KK10-specific CD8+ T-cell response was defined in cross-section and longitudinally for 6 HLA-B*2705+ patients. Comparison was made to cognate HLA-A*0201 CMV NV9 and HLA-B*2705 EBV RL9-specific CD8+ T-cell populations using the Simpsons diversity index and the Morisita-Horn similarity index for standardized repertoire analysis. HLA-B*2705 KK10-specific TCR repertoire was not found to be a determinant of control. Greater clonotype variation was found within CMV-specific CD8+ T-cell populations, suggesting an association with reactivation of CMV and disease state. An association was found between KK10-specific population diversity and the prevalence of cognate KK10 epitope in vivo. Cross-reactivity observed for dominant KK10-specific clonotypes suggested that avidity of CD8+ T-cells was important for in vivo survival. Phenotype and function was tested through multiparameter analysis of HIV and CMV-specific CD8+ T-cells. Increased frequency of CD127 (IL-7R) and Bcl-2 expression within dominant populations was suggestive of selective advantage. Division of dominant and sub-dominant CMV-specific CD8+ T-cell populations into early and late differentiation phenotypes indicated virus-specific mechanisms of clonotype turn over. No simple association of TCR expression was found for HIV and CMV-specific CD8+ T-cells with published examples of definitive TCR bias. Over-represented TCR ß-chain families of patients were found in association with public clonotypes. Convergent recombination of TCR genes was demonstrated as a mechanism for the prevalence of shared clonotypes. Standardized assessment of T-cell repertoire successfully identified mechanisms of antigen-specific CD8+ T-cell recruitment. A substantial increase in sample numbers is required before this methodology can be used to accurately demonstrate the importance of TCR repertoire usage in the control of human viral infection

Education in vascular surgery: Critical issues around the globe—training and qualification in vascular surgery in Europe

In 1958, the Union Européene des Médecins Spécialistes (UEMS), or European Union (EU) of Medical Specialists the European Union, was founded by the professional organizations of medical specialists in Europe. Among the objectives of the UEMS are to promote the highest level of patient care in the EU and to promote the harmonization of high-quality training programs within the various specialities throughout the EU. Within the 38 Specialist Sections of the UEMS are the European Boards, which are the working groups of the Specialist Sections. In 2005 Vascular Surgery was recognized as a separate and independent Section, a monospecialty, within the UEMS. The efforts of the UEMS are directed at facilitating the free exchange of training and work of trainees and medical specialists between EU countries. This situation, in combination with large differences in requirements and length of training in vascular surgery within the EU, stresses the importance of harmonization in training and certification in vascular surgery within the EU. For that reason, the European Board of Vascular Surgery has organized voluntary examinations yearly since 1996. The candidates who pass qualify as “Fellow of the European Board of Vascular Surgery” (FEBVS) since 2005. The first part of the examination evaluates the eligibility of the candidate (Certificate of Completion of Specialist Training, training center, logbook). The second part is a viva voce assessment that includes (1) case analyses, (2) a review of a scientific article, (3) an overall assessment, (4) a technical skills, and (5) an endovascular skills assessment. To pass the examination, the candidates must achieve a 67% success rate in each part of the examination. During the last 10 years, approximately 75% of the candidates have successfully taken the examination. In the near future the Section and Board, in close collaboration with the vascular societies in the EU, will develop a European vascular surgical syllabus and curriculum that will further harmonize and professionalize the training and certification of vascular surgery in Europe

Validation of RNA-based molecular clonotype analysis for virus-specific CD8+ T-cells in formaldehyde-fixed specimens isolated from peripheral blood

Recent advances in the field of molecular clonotype analysis have enabled detailed repertoire characterization of viably isolated antigen-specific T cell populations directly ex vivo. However, in the absence of a biologically contained FACS facility, peripheral blood mononuclear cell (PBMC) preparations derived from patients infected with agents such as HIV must be formaldehyde fixed to inactivate the pathogen; this procedure adversely affects nucleic acid template quality. Here, we developed and validated a method to amplify and sequence mRNA species derived from formaldehyde fixed PBMC specimens. Antigen-specific CD8+ cytotoxic T-lymphocyte populations were identified with standard fluorochrome-conjugated peptide-major histocompatibility complex class I tetramers refolded around synthetic peptides representing immunodominant epitopes from HIV p24 Gag (KRWII[M/L]GLNK/HLA B*2705) and CMV pp65 (NLVPMVATV/HLA A*0201 and TPRVTGGGAM/HLA B*0702), and acquired in separate laboratories with or without fixation. In the presence of proteinase K pre-treatment, the observed antigen-specific CD8+ T-cell repertoire determined by molecular clonotype analysis was statistically no different whether derived from fixed or unfixed PBMC. However, oligo-dT recovery methods were not suitable for use with fixed tissue as significant skewing of clonotypic representation was observed. Thus, we have developed a reliable RNA-based method for molecular clonotype analysis that is compatible with formaldehyde fixation and therefore suitable for use with primary human samples isolated by FACS outside the context of a biological safety level 3 containment facility

Education in vascular surgery: Critical issues around the globe-training and qualification in vascular surgery in Europe

In 1958, the Union Europeene des Medecins Specialistes (UEMS), or European Union (EU) of Medical Specialists the European Union, was founded by the professional organizations of medical specialists in Europe. Among the objectives of the U EMS arc to promote the highest level of patient care in the EU and to promote the harmonization of high-quality training programs within the various specialities throughout the EU. Within the 38 Specialist Sections of the UEMS are the European Boards, which are the working groups of the Specialist Sections. In 2005 Vascular Surgery was recognized as a separate and independent Section, a monospecialty, within the UEMS. The efforts of the UEMS are directed at facilitating the free exchange of training and work of trainees and medical specialists between EU countries. This situation, in combination with large differences in requirements and length of training in vascular surgery within the EU, stresses the importance of harmonization in training and certification in vascular surgery within the EU. For that reason, the European Board of Vascular Surgery has organized voluntary examinations yearly since 1996. The candidates who pass qualify as “Fellow of the European Board of Vascular Surgery” (FEBVS) since 2005. The first part of the examination evaluates the eligibility of the candidate (Certificate of Completion of Specialist Training, training center, logbook). The second part is a viva voce assessment that includes (1) case analyses, (2) a review of a scientific article, (3) an overall assessment, (4) a technical skills, and (5) an endovascular skills assessment. To pass the examination, the candidates must achieve a 67% success rate in each part of the examination. During the last 10 years, approximately 75% of the candidates have successfully taken the examination. In the near future the Section and Board, in close collaboration with the vascular societies in the EU, will develop a European vascular surgical syllabus and curriculum that will further harmonize and professionalize the training and certification of vascular surgery in Europe. (J Vasc Surg 2008;48:69S-75S.

TCR β-Chain sharing in human CD8+ T cell responses to cytomegalovirus and EBV

The CD8+ TCR repertoires specific for many immunogenic epitopes of CMV and EBV are dominated by a few TCR clonotypes and involve public TCRs that are shared between many MHC-matched individuals. In previous studies, we demonstrated that the observed sharing of epitope-specific TCRβ chains between individuals is strongly associated with TCRβ production frequency, and that a process of convergent recombination facilitates the more efficient production of some TCRβ sequences. In this study, we analyzed a total of 2836 TCRβ sequences from 23 CMV-infected and 10 EBV-infected individuals to investigate the factors that influence the sharing of TCRβ sequences in the CD8+ T cell responses to two immunodominant HLA-A*0201-restricted epitopes from these viruses. The most shared TCRβ amino acid sequences were found to have two features that indicate efficient TCRβ production, as follows: 1) they required fewer nucleotide additions, and 2) they were encoded by a greater variety of nucleotide sequences. We used simulations of random V(D)J recombination to demonstrate that the in silico TCRβ production frequency was predictive of the extent to which both TCRβ nucleotide and amino acid sequences were shared in vivo. These results suggest that TCRβ production frequency plays an important role in the interindividual sharing of TCRβ sequences within CD8+ T cell responses specific for CMV and EBV

Persistent survival of prevalent clonotypes within an immunodominant HIV gag-specific CD8+ T cell response

CD8+ T cells play a significant role in the control of HIV replication, yet the associated qualitative and quantitative factors that determine the outcome of infection remain obscure. In this study, we examined Ag-specific CD8+ TCR repertoires longitudinally in a cohort of HLA-B*2705+ long-term nonprogressors with chronic HIV-1 infection using a combination of molecular clonotype analysis and polychromatic flow cytometry. In each case, CD8+ T cell populations specific for the immunodominant p24 Gag epitope KRWIILGLNK (KK10; residues 263–272) and naturally occurring variants thereof, restricted by HLA-B*2705, were studied at multiple time points; in addition, comparative data were collected for CD8+ T cell populations specific for the CMV pp65 epitope NLVPMVATV (NV9; residues 495–503), restricted by HLA-A*0201. Dominant KK10-specific clonotypes persisted for several years and exhibited greater stability than their contemporaneous NV9-specific counterparts. Furthermore, these dominant KK10-specific clonotypes exhibited cross-reactivity with antigenic variants and expressed significantly higher levels of CD127 (IL-7Rα) and Bcl-2. Of note, we also found evidence that promiscuous TCR α-chain pairing associated with alterations in fine specificity for KK10 variants could contribute to TCR β-chain prevalence. Taken together, these data suggest that an antiapoptotic phenotype and the ability to cross-recognize variant epitopes contribute to clonotype longevity and selection within the peripheral memory T cell pool in the presence of persistent infection with a genetically unstable virus