Gobierno universitario : entre la autogestión estamental y la responsabilidad social

<p>Low doses of the relatively neutralization resistant SHIV _SF162P3 isolate were incubated at 37⁰C for four hours with concentrations of the human monoclonal antibody IgG1 b12. The mixture was then added to GHOST cells and allowed to absorb for 24 hours. The cells were washed and cultured for a further 24 hours (4/24/2 assays). Four duplicate cultures were used for each point within a replicate. Data are fitted to a second-order (quadratic) equation. Dotted lines are extrapolations to the horizontal axis calculated from the quadratic plots. Axes are truncated and some symbols are excluded to improve clarity, especially around the origin. <b>A</b>. SHIV_SF162P3 exposed to GHOST cells from passage 7 (1 replicate) and 9 (2 replicates). Gray: control cultures where virus were incubated without monoclonal antibody: y = -0.00285 x² + 1.310 x -6.009; green: Virus pre-incubated with 0.625 µg/ml IgG1 b12: y = -0.00284 x² + 0.939 x -0.517. <b>B</b>. Gray same as for A. blue: Virus pre-incubated with 0.25 µg/ml IgG1 b12: y = -0.000606 x² + 0.870 x + 3.152. <b>C</b>. SHIV_SF162P3 exposed to GHOST cells from passages 15, 17 and 21. Gray: control cultures where virus were incubated without monoclonal antibody: y = 0.00182 x² + 0.665 x + 11.01; green: Virus pre-incubated with 0.625 µg/ml IgG1 b12: y = + 0.00135 x² + 0.487 x + 8.334. <b>D</b>. Gray same as for C. blue: where cultures are exposed to virus pre-incubated with 0.25 µg/ml IgG1 b12: y = 0.00140x² + 0.616x + 5.768. Interval between points where control and 0.25 µg/ml IgG1 b12 plots cut x-axis: 7.81 infectious virus.</p

<i>In Vitro</i> Neutralization of Low Dose Inocula at Physiological Concentrations of a Monoclonal Antibody Which Protects Macaques against SHIV Challenge

<div><p>Background</p><p>Passive transfer of antibodies can be protective in the simian human immunodeficiency virus (SHIV) – rhesus macaque challenge model. The human monoclonal antibody IgG1 b12 neutralizes human immunodeficiency type 1 (HIV-1) <i>in vitro</i> and protects against challenge by SHIV. Our hypothesis is that neutralizing antibodies can only completely inactivate a relatively small number of infectious virus.</p> <p>Methods And Findings</p><p>We have used GHOST cell assays to quantify individual infectious events with HIV-1_SF162 and its SHIV derivatives: the relatively neutralization sensitive SHIV_SF162P4 isolate and the more resistant SHIV_SF162P3. A plot of the number of fluorescent GHOST cells with increasing HIV-1_SF162 dose is not linear. It is likely that with high-dose inocula, infection with multiple virus produces additive fluorescence in individual cells. In studies of the neutralization kinetics of IgG1 b12 against these isolates, events during the absorption phase of the assay, as well as the incubation phase, determine the level of neutralization. It is possible that complete inactivation of a virus is limited to the time it is exposed on the cell surface. Assays can be modified so that neutralization of these very low doses of virus can be quantified. A higher concentration of antibody is required to neutralize the same dose of resistant SHIV_SF162P3 than the sensitive SHIV_SF162P4. In the absence of selection during passage, the density of the CCR5 co-receptor on the GHOST cell surface is reduced. Changes in the CD4 : CCR5 density ratio influence neutralization.</p> <p>Conclusions</p><p>Low concentrations of IgG1 b12 completely inactivate small doses of the neutralization resistant SHIV _SF162P3. Assays need to be modified to quantify this effect. Results from modified assays may predict protection following repeated low-dose shiv challenges in rhesus macaques. It should be possible to induce this level of antibody by vaccination so that modified assays could predict the outcome of human trials.</p> </div

Dose–response plots of HIV-1 _SF162 and SHIV variants on GHOST cells.

<p>GHOST cell cultures were exposed to different doses of virus, plotted on the x-axis (= horizontal). The number of cells which fluoresce after infection is plotted on the y-axis (= vertical). <b>A</b>. Linear regression of HIV-1 _SF162 - infected cultures: y = 1.457 ± 0.066 x -584.3 ± 89.86. <b>B</b>. Fitting of data to second-order (quadratic) equation: y = 4.31 x 10^-4 x² + 0.347 x + 26.63. <b>C</b>. Quadratic plot of SHIV_SF162P4 on GHOST cells: y = -1.03 x 10^-3 x² + 1.349 x + -4.17 x 10^-4. <b>D</b>. Quadratic plot of SHIV_SF162P3 on GHOST cells: y = -7.63 x 10^-5 x² + 0.963x + -4.077.</p

FACS analysis of Hi5 GHOST cells at different passage levels.

<p>Fluorescent intensities are plotted on the horizontal axis and the number of cells on the vertical. <b>A</b>. Passage 7; <b>B</b>. Passage 9; <b>C</b>. Passage 11; <b>D</b>. Passage 13; <b>E</b>. Passage 15; <b>F</b>. Passage 17; <b>G</b>. Passage 19; <b>H</b>. Passage 21; <b>I</b>. Passage 23.</p

Comparison of linear regression and fitted second-order plots of reductions in infectious virus in HIV-1 _SF162 - GHOST cell neutralization assays.

<p>Low doses of the relatively neutralization sensitive HIV-1 _SF162 isolate were incubated at 37⁰C for four hours with concentrations of the human monoclonal antibody IgG1 b12. The mixture was then added to GHOST cells and allowed to absorb for 24 hours. The cells were washed and cultured for a further 24 hours (= 4/24/2 assays). Four duplicate cultures were used for each point within a replicate. Parameters are given as means with their standard errors. Regression lines with the formula y = mx + c where y is the number of fluorescent cells plotted on the vertical axis and x is the dose of virus along the horizontal axis. <b>A</b>. Gray: three control replicates where cells were cultured without monoclonal antibody: m = 1.081 ± 0.063; c = -5.187 ± 4.375; green: three replicates where virus were incubated with 0.125 µg/ml IgG1 b12 : m = 0.545 ± 0.042; c = -1.731 ± 2.938. <b>B</b>. Gray: seven control replicates where cells were cultured without monoclonal antibody: m = 1.003 ± 0.037 ; c = -0.300 ± 3.086; blue: seven replicates where virus were incubated with 0.05 µg/ml IgG1 b12 : m = 0.737 ± 0.028; c = -0.409 ± 2.350. <b>C</b>. Gray: two control replicates where cells from early passages were cultured without monoclonal antibody: m = 0.973 ± 0.076; c = 2.409 ± 7.608; red: two replicates where virus were incubated with 0.02 µg/ml IgG1 b12: m = 0.815 ± 0.059; c = -4.545 ± 5.928; D. Gray: four control replicates where cells from later passages were cultured without monoclonal antibody: m = 1.003 ± 0.046; c = -0.301 ± 3.402; red: four replicates where virus were incubated with 0.02 µg/ml IgG1 b12: m = 1.013 ± 0.036; c = -7.313 ± 2.719. Dotted lines are extrapolations to the axes using the formula of the regression / fitted lines. Some data points have been excluded and axes truncated to improve clarity and magnify the situation around the origin.</p

Reductions in infectious titer following exposure of HIV-1_SF162 or SHIV_SF162P4 to monoclonal antibody IgG 1 b12.

<p>Reductions in infectious virus are calculated as the ratio of the titer (V_t) at time t for the virus exposed to antibody divided by the titer (V_c) at the same time for control cultures without antibody. The ratio is transformed to log₁₀ (V_t / V_c). Incubation and absorption phases are measured in hours. Data are displayed as means with standard errors. Plots are regression lines with their 95% confidence band. Horizontal broken line represents 50% neutralization. Green: 1 µg/ml IgG1 b12; Blue: 0.4 µg/ml IgG1 b12; Red: 0.2 µg/ml IgG1 b12. Expected ratio of neutralization rates is the ratio of the antibody concentrations within an individual assay. <b>A</b>. Incubation plots of IgG1 b12 against HIV-1 _<b>SF162</b> (Ratios: Expected 1 µg/ml : 0.4 µg/ml = 2.5; The observed ratio of the gradients of the regression lines = 3.14; p < 0.0001; Expected 1 µg/ml : 0.2 µg/ml = 5; Observed: 8.03p < 0.0001; Expected 0.4 µg/ml : 0.2 µg/ml = 2; Observed = 2.56; p = 0.0251); <b>B</b>. absorption plots of IgG1 b12 against HIV-1 _<b>SF162</b> (Ratios Expected = 2.5; Observed = 1.25; p = 0.4879); C. Incubation plots of IgG1 b12 against SHIV <b>_SF162P4</b> (Ratios: Expected = 2.50; Observed = 2.50; p < 0.0001); D. Absorption plots of IgG1 b12 against SHIV <b>_SF162P4</b> (Ratios: Expected = 2.50; Observed = 1.69; p < 0.02773).</p

Pandemic Swine-Origin H1N1 Influenza Virus Replicates to Higher Levels and Induces More Fever and Acute Inflammatory Cytokines in Cynomolgus versus Rhesus Monkeys and Can Replicate in Common Marmosets

<div><p>The close immunological and physiological resemblance with humans makes non-human primates a valuable model for studying influenza virus pathogenesis and immunity and vaccine efficacy against infection. Although both cynomolgus and rhesus macaques are frequently used in influenza virus research, a direct comparison of susceptibility to infection and disease has not yet been performed. In the current study a head-to-head comparison was made between these species, by using a recently described swine-origin pandemic H1N1 strain, A/Mexico/InDRE4487/2009. In comparison to rhesus macaques, cynomolgus macaques developed significantly higher levels of virus replication in the upper airways and in the lungs, involving both peak level and duration of virus production, as well as higher increases in body temperature. In contrast, clinical symptoms, including respiratory distress, were more easily observed in rhesus macaques. Expression of sialyl-α-2,6-Gal saccharides, the main receptor for human influenza A viruses, was 50 to 73 times more abundant in trachea and bronchus of cynomolgus macaques relative to rhesus macaques. The study also shows that common marmosets, a New World non-human primate species, are susceptible to infection with pandemic H1N1. The study results favor the cynomolgus macaque as model for pandemic H1N1 influenza virus research because of the more uniform and high levels of virus replication, as well as temperature increases, which may be due to a more abundant expression of the main human influenza virus receptor in the trachea and bronchi.</p></div

Quantification of intensity of 56 kD band in SNA staining on SDS-PAGE of trachea and bronchus tissue samples from cynomolgus and rhesus monkeys.

<p>Indicated is the intensity of signal as percentage of signal from 62kD band of Fetuin (positive control).</p><p>Quantification of intensity of 56 kD band in SNA staining on SDS-PAGE of trachea and bronchus tissue samples from cynomolgus and rhesus monkeys.</p

Body temperature of cynomolgus macaques, rhesus macaques and common marmosets before and after Mex4487 influenza virus infection.

<p>(A) Circadian temperature pattern, shown for two animals of each species (C5, C6, R5, R6, M5 and M6). The circadian pattern was calculated from the temperatures recorded during three weeks before infection. Grey areas represent the mean temperature with the 95% confidence interval. (B) Body temperature increase during infection. Shown is the net-increase in temperature, which was calculated by subtracting the individual circadian body temperature from the actually recorded temperature in time after infection. Data are depicted from the two animals of each species that were maintained in study for 14 days (C5, C6, R5, R6, M5 and M6). (C) Cumulative net temperature increase, calculated as area under the curve (AUC) from the net-increase data, either for the first 3.5 days or the first 6.5 days of infection. Colored dots represent individual animals (green: CRM1, orange: CRM2, red: CRM3, light blue: CRM4, purple: CRM5, dark blue: CRM6). C: cynomolgus, R: rhesus, M: marmoset. Statistical differences between groups were determined with Mann-Whitney test.</p

Radiographic score in cynomolgus macaques, rhesus monkeys and common marmosets in time.

<p>Both left and right lung lobes were scored from 0–3, giving a maximal radiographic score of 6 of the complete lung.</p