Drinking during marathon running in extreme heat: A video analysis study of the top finishers in the 2004 Athens Olympic marathons

Objective. To assess the drinking behaviours of top competitors during an Olympic marathon. Methods. Retrospective video analysis of the top four finishers in both the male and female 2004 Athens Olympic marathons plus the pre-race favourite in the female race in order to assess total time spent drinking. One male and female runner involved in a laboratory drinking simulation trial. Results. For the five female athletes, 37 of a possible 73 drinking episodes were captured. The female race winner was filmed at 11 of 15 drinking stations. Her total drinking time was 23.6 seconds; extrapolated over 15 seconds this would have increased to 32.2 seconds for a total of 27 sips of fluid during the race. Eighteen of a possible 60 drinking episodes for the top four male marathon finishers were filmed. The total drinking time for those 18 episodes was 11.4 seconds. A laboratory simulation found that a female athlete of approximately the same weight as the female Olympic winner might have been able to ingest a maximum of 810 ml (350 ml.h-1) from 27 sips whilst running at her best marathon pace whereas a male might have drunk a maximum of 720 ml (330 ml.h-1) from 9 sips under the same conditions. Conclusions. These data suggest that both the female and male 2004 Olympic Marathon winners drank minimal total amounts of fluid (30ºC) temperatures while completing the marathon with race times within 2.5% of the Olympic record

Patients with tuberculosis disease have Mycobacterium tuberculosis-specific CD8 T cells with a pro-apoptotic phenotype and impaired proliferative capacity, which is not restored following treatment

CD8 T cells play a critical role in control of chronic viral infections; however, the role of these cells in containing persistent bacterial infections, such as those caused by Mycobacterium tuberculosis (Mtb), is less clear. We assessed the phenotype and functional capacity of CD8 T cells specific for the immunodominant Mtb antigens CFP-10 and ESAT-6, in patients with pulmonary tuberculosis (TB) disease, before and after treatment, and in healthy persons with latent Mtb infection (LTBI). In patients with TB disease, CFP-10/ESAT-6-specific IFN-γ + CD8 T cells had an activated, pro-apoptotic phenotype, with lower Bcl-2 and CD127 expression, and higher Ki67, CD57, and CD95 expression, than in LTBI. When CFP-10/ESAT-6-specific IFN-γ + CD8 T cells were detectable, expression of distinct combinations of these markers was highly sensitive and specific for differentiating TB disease from LTBI. Successful treatment of disease resulted in changes of these markers, but not in restoration of CFP-10/ESAT-6-specific CD8 or CD4 memory T cell proliferative capacity. These data suggest that high mycobacterial load in active TB disease is associated with activated, short-lived CFP-10/ESAT-6-specific CD8 T cells with impaired functional capacity that is not restored following treatment. By contrast, LTBI is associated with preservation of long-lived CFP-10/ESAT-6-specific memory CD8 T cells that maintain high Bcl-2 expression and which may readily proliferate

Live-attenuated Mycobacterium tuberculosis vaccine MTBVAC versus BCG in adults and neonates: a randomised controlled, double-blind dose-escalation trial

Background: Infants are a key target population for new tuberculosis vaccines. We assessed the safety and immunogenicity of the live-attenuated Mycobacterium tuberculosis vaccine candidate MTBVAC in adults and infants in a region where transmission of tuberculosis is very high. Methods: We did a randomised, double-blind, BCG-controlled, dose-escalation trial at the South African Tuberculosis Vaccine Initiative site near Cape Town, South Africa. Healthy adult community volunteers who were aged 18–50 years, had received BCG vaccination as infants, were HIV negative, had negative interferon-¿ release assay (IGRA) results, and had no personal history of tuberculosis or current household contact with someone with tuberculosis were enrolled in a safety cohort. Infants born to HIV-negative women with no personal history of tuberculosis or current household contact with a person with tuberculosis and who were 96 h old or younger, generally healthy, and had not yet received routine BCG vaccination were enrolled in a separate infant cohort. Eligible adults were randomly assigned (1:1) to receive either BCG Vaccine SSI (5 × 105 colony forming units [CFU] of Danish strain 1331 in 0·1 mL diluent) or MTBVAC (5 × 105 CFU in 0·1 mL) intradermally in the deltoid region of the arm. After favourable review of 28-day reactogenicity and safety data in the adult cohort, infants were randomly assigned (1:3) to receive either BCG Vaccine SSI (2·5 × 105 CFU in 0·05 mL diluent) or MTBVAC in three sequential cohorts of increasing MTBVAC dose (2·5 × 103 CFU, 2·5 × 104 CFU, and 2·5 × 105 CFU in 0·05 mL) intradermally in the deltoid region of the arm. QuantiFERON-TB Gold In-Tube IGRA was done on days 180 and 360. For both randomisations, a pre-prepared block randomisation schedule was used. Participants (and their parents or guardians in the case of infant participants), investigators, and other clinical and laboratory staff were masked to intervention allocation. The primary outcomes, which were all measured in the infant cohort, were solicited and unsolicited local adverse events and serious adverse events until day 360; non-serious systemic adverse events until day 28 and vaccine-specific CD4 and CD8 T-cell responses on days 7, 28, 70, 180, and 360. Secondary outcomes measured in adults were local injection-site and systemic reactions and haematology and biochemistry at study day 7 and 28. Safety analyses and immunogenicity analyses were done in all participants who received a dose of vaccine. This trial is registered with ClinicalTrials.gov, number NCT02729571. Findings: Between Sept 29, 2015, and Nov 16, 2015, 62 adults were screened and 18 were enrolled and randomly assigned, nine each to the BCG and MTBVAC groups. Between Feb 12, 2016, and Sept 21, 2016, 36 infants were randomly assigned—eight to the BCG group, nine to the 2·5 × 103 CFU MTBVAC group, nine to the 2·5 × 104 CFU group, and ten to the 2·5 × 105 CFU group. Mild injection-site reactions occurred only in infants in the BCG and the 2·5 × 105 CFU MTBVAC group, with no evidence of local or regional injection-site complications. Systemic adverse events were evenly distributed across BCG and MTBVAC dose groups, and were mostly mild in severity. Eight serious adverse events were reported in seven vaccine recipients (one adult MTBVAC recipient, one infant BCG recipient, one infant in the 2·5 × 103 CFU MTBVAC group, two in the 2·5 × 104 CFU MTBVAC group, and two in the 2·5 × 105 CFU MTBVAC group), including one infant in the 2·5 × 103 CFU MTBVAC group treated for unconfirmed tuberculosis and one in the 2·5 × 105 CFU MTBVAC group treated for unlikely tuberculosis. One infant died as a result of possible viral pneumonia. Vaccination with all MTBVAC doses induced durable antigen-specific T-helper-1 cytokine-expressing CD4 cell responses in infants that peaked 70 days after vaccination and were detectable 360 days after vaccination. For the highest MTBVAC dose (ie, 2·5 × 105 CFU), these responses exceeded responses induced by an equivalent dose of the BCG vaccine up to 360 days after vaccination. Dose-related IGRA conversion was noted in three (38%) of eight infants in the 2·5 × 103 CFU MTBVAC group, six (75%) of eight in the 2·5 × 104 CFU MTBVAC group, and seven (78%) of nine in the 2·5 × 105 CFU MTBVAC group at day 180, compared with none of seven infants in the BCG group. By day 360, IGRA reversion had occurred in all three infants (100%) in the 2·5 × 103 CFU MTBVAC group, four (67%) of the six in the 2·5 × 104 CFU MTBVAC group, and three (43%) of the seven in the 2·5 × 105 CFU MTBVAC group. Interpretation: MTBVAC had acceptable reactogenicity, and induced a durable CD4 cell response in infants. The evidence of immunogenicity supports progression of MTBVAC into larger safety and efficacy trials, but also confounds interpretation of tests for M tuberculosis infection, highlighting the need for stringent endpoint definition. Funding: Norwegian Agency for Development Cooperation, TuBerculosis Vaccine Initiative, UK Department for International Development, and Biofabri

A quantitative analysis of complexity of human pathogen-specific CD4 T cell responses in healthy M. tuberculosis infected South Africans

Author Summary: Human pathogen-specific immune responses are tremendously complex and the techniques to study them ever expanding. There is an urgent need for a quantitative analysis and better understanding of pathogen-specific immune responses. Mycobacterium tuberculosis (Mtb) is one of the leading causes of mortality due to an infectious agent worldwide. Here, we were able to quantify the Mtb-specific response in healthy individuals with Mtb infection from South Africa. The response is highly diverse and 66 epitopes are required to capture 80% of the total reactivity. Our study also show that the majority of the identified epitopes are restricted by multiple HLA alleles. Thus, technical advances are required to capture and characterize the complete pathogen-specific response. This study demonstrates further that the approach combining identified epitopes into "megapools" allows capturing a large fraction of the total reactivity. This suggests that this technique is generally applicable to the characterization of immunity to other complex pathogens. Together, our data provide for the first time a quantitative analysis of the complex pathogen-specific T cell response and provide a new understanding of human infections in a natural infection setting

Differential co-expression patterns of Bcl-2, CD57 and CD95 by CFP-10/ESAT-6-specific CD8 T cells distinguish individuals with LTBI and patients with TB.

<p>Boolean analysis was performed to determine co-expression patterns of Bcl-2, CD57, and CD95 by CFP-10 and ESAT-6-specific CD8 T cells detected by ICS, as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0094949#pone-0094949-g001" target="_blank">Figure 1</a>. (A) Co-expression patterns of Bcl-2, CD57, and CD95 by specific IFN-γ⁺ CD8 T cells detectable in individuals with LTBI (open bars; n = 6) and patients with TB disease (grey bars; n = 13). Cells were gated on VIVID^l°CD3⁺CD8⁺IFN-γ⁺ cells. Differences were assessed using the Mann-Whitney test. § indicates p values that did not retain statistical significance after applying the Bonferroni correction for multiple comparisons. Pie graphs indicate the median proportion of each population contributing to the total specific IFN-γ⁺ CD8 T cell response. (B) Comparison of the proportion of Bcl-2⁺CD57⁻CD95⁺ cells contributing to the total CFP-10 and ESAT-6-specific CD8 T cell responses in individuals with LTBI and patients with TB disease. Horizontal lines represent the median. Differences were assessed using the Mann-Whitney test. The dotted line indicates the cut-off (18.7%) that distinguishes individuals with LTBI and TB disease, with 100% specificity and 100% sensitivity. (C) Receiver operator characteristic (ROC) curve indicating the sensitivity and specificity of the proportion of CFP-10/ESAT-6-specific CD8 T cells that are Bcl-2⁺CD57⁻CD95⁺ to distinguish individuals with LTBI and TB disease. An area under the ROC curve (AUC) analysis was performed to further evaluate the performance of this phenotypic expression profile in distinguishing individuals with LTBI and TB disease.</p

Lack of restoration of CFP-10/ESAT-6-specific CD4 and CD8 T cell proliferative capacity following completion of treatment.

<p>The proliferative capacity of CFP-10/ESAT-6-specific CD4 and CD8 T cells was measured following a 6-day stimulation of freshly isolated PBMCs with CFP-10 and ESAT-6 peptide pools. (A) Flow cytometry data of the proliferative capacity of antigen-specific CD8 T cells from a TB diseased patient analyzed at 4 time points: prior to treatment (Pre-TB Tx), 2 months of treatment (2 mo. TB Tx), 6 months of treatment (6 mo. TB Tx, corresponding to the end of the treatment period), and 6 months after completion of treatment (12 mo. TB Tx). The cells shown are gated on VIVID^l°CD3⁺CD8⁺ lymphocytes. The percentage on each plot indicates the percentage of proliferating CD8 T cells after subtraction of background proliferation in the negative control condition. (B, D) Longitudinal analysis of the proliferative capacity of CFP-10 and ESAT-6-specific CD8 (panel B) and CD4 (panel D) T cells in a subset of TB diseased patients who were followed for up to one year after TB diagnosis (n = 19 with at least 3 time points available for analysis). There were no significant differences in the frequency of proliferating antigen-specific CD8 or CD4 T cells over time (Kruskal-Wallis test). (C, E) Cross-sectional comparison of the CFP-10 and ESAT-6-specific CD8 (panel C) and CD4 (panel E) T cell proliferative capacity between individuals with LTBI (n = 34) and active TB disease at 4 time points (pre-TB tx: n = 34; 2 months of TB tx: n = 19; 6 months of TB Tx [corresponding to the end of the treatment period]: n = 25; 12 months post initiation of TB tx [corresponding to 6 months after the end of the treatment period]: n = 10). Data are shown after subtraction of background proliferation in the negative control condition. Differences in panels C and E were first assessed using a Kruskal-Wallis test, followed by a Dunn's post-test to correct for multiple comparisons. All comparisons remained significant following correction with the Dunn's post-test.</p

CFP-10/ESAT-6-specific CD8 T cells in patients with TB have a short-lived, pro-apoptotic phenotype.

<p>PBMCs from individuals with LTBI (n = 18) and patients with TB disease (n = 20) were stimulated for 6 hours with CFP-10 and ESAT-6 peptide pools; intracellular IFN-γ production was measured in CD8 T cells by flow cytometry. (<b>A</b>) Frequencies of CFP-10 and ESAT-6-specific IFN-γ⁺ CD8 T cells in persons with LTBI and patients with TB disease. Horizontal lines represent the median. Data are shown after subtraction of background cytokine production in the negative control condition. The dotted line separates individuals who met the criteria for a positive CD8 T cell response for further phenotypic analyses (above dotted line: n = 6 LTBI; n = 13 TB disease), and individuals in whom the frequency of IFN-γ⁺ CD8 T cells was too low to meet the criteria for further phenotypic analyses (below dotted line). (<b>B</b>) Representative flow cytometry data from an individual with LTBI and a patient with TB disease, following stimulation of PBMCs with an ESAT-6 peptide pool. Grey cells indicate the total CD8 T cell population (gated on VIVID^l°CD3⁺CD8⁺ cells); black cells indicate ESAT-6-specific CD8 T cells (gated on VIVID^l°CD3⁺CD8⁺IFN-γ⁺ cells). (<b>C</b>) Summary data of the percentage of specific IFN-γ⁺ CD8 T cells expressing Ki67, Bcl-2, CD127, CD57 and CD95 in individuals with LTBI and TB disease. Differences were assessed using the Mann-Whitney test. § indicates p values that did not remain significant after applying the Bonferroni correction for multiple comparisons. (<b>D</b>) Comparison of the expression of intracellular Ki67 and Bcl-2 between the total CD8 T cell population and specific IFN-γ⁺ CD8 T cells within the same individual. Only individuals meeting the criteria for a positive CD8 T cell response in the ICS assay were included in this paired analysis (n = 6 LTBI; n = 13 TB disease). Differences were assessed using the Wilcoxon matched-pairs test.</p

CFP-10/ESAT-6-specific CD8 T cell phenotype is associated with mycobacterial antigen load.

<p>Phenotypic analysis of CFP-10 and ESAT-6-specific IFN-γ⁺ CD8 T cells was performed as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0094949#pone-0094949-g001" target="_blank">Figure 1</a>. Cells were analyzed prior to initiating anti-TB treatment (Pre-TB Tx), and 2 months and 6 months after initiation of treatment. (<b>A</b>) Flow cytometry data indicating Ki67 and Bcl-2 expression by IFN-γ⁺ ESAT-6-specific CD8 T cells from a TB diseased patient at two time points: prior to treatment and 6 months after initiation of treatment, corresponding to the end of the treatment period. (<b>B</b>) Representative histogram overlays indicating expression of Bcl-2, CD127 and CD95 within CFP-10/ESAT-6-specific IFN-γ⁺ CD8 T cells. Black lines indicate expression prior to treatment; grey lines indicate expression 6 months after initiation of treatment. The numbers in the upper right corner of the histograms indicate the median fluorescence intensity (MFI) at each time point (black: pre-treatment MFI; grey: 6-month TB treatment MFI). Data shown in panels A and B are gated on VIVID^l°CD3⁺CD8⁺IFN-γ⁺ cells. (<b>C</b>) Summary data comparing the expression of Ki67, Bcl-2, CD127 and CD95 on CFP-10/ESAT-6-specific IFN-γ⁺ CD8 T cells from the same individual at two time points: prior to treatment (Pre-TB Tx) and at the end of the 6-month treatment period (6 mo. TB Tx). Data are shown from 7 individuals who maintained positive CFP-10/ESAT-6-specific CD8 T cell responses throughout the 6-month duration of treatment. Differences between time points were assessed using the Wilcoxon matched pairs test. § indicates p values that did not retain statistical significance after applying the Bonferroni correction for multiple comparisons. The proportion of CFP-10/ESAT-6-specific IFN-γ⁺ CD8 T cells that express Ki67 (<b>D</b>), CD95 (<b>E</b>), Bcl-2 (<b>F</b>), and CD127 (<b>G</b>) are shown for individuals with LTBI (n = 6), and TB diseased patients prior to treatment (n = 13; Pre-TB Tx), and 2 months (n = 10; 2 mo. TB Tx) and 6 months (n = 8; 6 mo. TB Tx) following initiation of treatment. Differences in panels D-G were first assessed using a Kruskal-Wallis test, followed by a Dunn's post-test to correct for multiple comparisons; § indicates p values that did not remain significant following correction with the Dunn's post-test.</p

Characteristics of study population.

a<p>The remaining 8 individuals with TB were sputum smear-negative, culture positive for <i>M. tuberculosis</i>.</p>b<p>Values denote median number of days on anti-TB treatment at first sample collection (range).</p>c<p>Values denote median (range).</p>d<p><i>p</i><0.05, compared with LTBI.</p><p>N/A, not applicable.</p