第9回千葉県胆膵研究会 11.

Additional file 13: Table S5. Statistical analysis of all experiments separated for transmission pairs with high and low diversity transmitters and for transmission pairs where recipient is closer to ancestral genotype, respectively

V1V2 deletion impairs virus infectivity and is reflected by a high stoichiometry of entry.

<p>(A) Comparison of infectivity of pseudoviruses expressing wt and V1V2-deleted envs upon infection of TZM-bl cells. Data points depict mean values of luciferase reporter activity per µl virus stock measured in 3 independent experiments. The p-value was calculated by a paired t-test. (B and C) Relative infectivity of mixed trimer infection experiments of 10 wt envs and their V1V2 deleted variants using the R508S/R511S (B) and the V513E (C) dominant-negative mutations are shown. Infectivity of pseudotyped virus stocks expressing the indicated ratios of dominant-negative mutant envs was measured on TZM-bl cells. Infectivity of virus stocks containing solely functional envelope were set as 100%. Data depict mean and SD from 2 to 4 independent experiments. (D) Estimates of T for the wt and V1V2-deleted envs derived from mixed trimer experiments. Data points are the mean of the individual estimates of T obtained with the R508S/R511S and V513E dominant-negative mutations. The p-value was calculated by a paired t-test.</p

Loss of the N160 glycosylation site in gp120 steers the stoichiometry of entry and virus infectivity.

<p>(A) Titration of CAP88 wt (black) and CAP88 K160N (red) pseudoviruses on TZM-bl reporter cells. Data shown are mean and SD of luciferase reporter activity upon infection measured in 2 independent experiments. Inset: Infectivity comparison of CAP88 wt and K160N depicted as relative light units (RLU) of luciferase reporter activity per µl of pseudovirus stock upon infection of TZM-bl cells. The fold infectivity difference is indicated. (B) Estimates of T for the CAP88 wt and K160N variant. Mean and range of the individual estimates using the R508S/R511S and V513E dominant-negative mutations are shown.</p

Virus entry kinetics correlate with the stoichiometry of entry.

<p>(A) Entry kinetic curves for JR-FL wt and JR-FL ΔV1V2. Synchronized pseudovirus infection of TZM-bl cells following spinoculation was terminated by addition of T-20 at the indicated timepoints. Infectivity reached after 120 minutes was set as 100% and all data were normalized relative to this value. Data are mean and SD from 3 independent experiments. (B) Half maximal entry time for wt and V1V2-deleted envs was calculated from kinetic profiles shown in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1004595#ppat-1004595-g004" target="_blank">Fig. 4A</a> and <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1004595#ppat.1004595.s007" target="_blank">S7B Fig.</a> Time (in minutes) required to reach 50% entry into target cells is depicted. Data shown are means derived from 2 to 4 independent experiments. The p-value was calculated by a paired t-test. (C) Correlation analysis (Pearson) of wt (black symbols) and V1V2-deleted env (red symbols) half-maximal entry time and estimated T.</p

The entry stoichiometry governs virus population infectivity.

<p>(A) Scheme depicting the influence of the entry stoichiometry on virus population infectivity. Different Ts (exemplified here: T = 1 and T = 7) will determine the minimum number of trimers that a virion requires in order to be infectious. (B) Correlation analysis (Pearson) of virus strain infectivity (measured by infection of TZM-bl reporter cells and expressed in arbitrary relative light units (RLU) per µl of virus stock) and the estimated T (plotted as mean of the independent R508S/R511S and V513E estimates shown in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1004595#ppat-1004595-g001" target="_blank">Fig. 1C</a>). Virus infectivities are depicted as mean values derived from 3 independent experiments. (C) Mathematical modeling to investigate the influence of entry stoichiometry on virion population infectivity. The data depict how T = 2 and T = 7 translate into different fractions of a virion population being potentially infectious, in dependence on the trimer number distribution across the virion population. As shown in (D), the overall infectivity of a virus population decreases with increasing T. For (C) and (D) we assumed the trimer number distribution across virions to follow a discretized Beta distribution with constant mean 12.95 and variance 45 <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1004595#ppat.1004595-Magnus1" target="_blank">[15]</a>.</p

HIV-1 strains and mutants.

<p>Eleven HIV-1 strains from subtypes A, B and C were employed in this study. Mean virion trimer numbers and expression levels of the dominant-negative R508S/R511S and V513E mutants of the utilized virus preparations are shown.</p>a<p>Mean virion trimer numbers as estimated by gp120/p24 ELISA of purified HIV-1 pseudoparticle stocks. Data are means of 2 to 3 independent experiments.</p>b<p>The expression level of the dominant-negative mutants is recorded as percentage of the corresponding wt env based on gp120 quantification by ELISA of purified HIV-1 pseudoparticle stocks.</p><p>HIV-1 strains and mutants.</p

HIV-1 strains differ in the number of trimers required for entry.

<p>(A) Theoretical predictions of relative virus infectivity over the fraction of dominant-negative mutant env (f_m) according to our model. Curves for T ranging from 1 to 8 are shown assuming the trimer number distribution across virions to follow a discretized Beta distribution with constant mean 12.95 and variance 45 <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1004595#ppat.1004595-Magnus1" target="_blank">[15]</a>. (B) Relative infectivity of mixed trimer infection experiments with 11 HIV-1 strains using the R508S/R511S (left) and V513E (right) dominant-negative env mutants. Infectivity of pseudotyped virus stocks expressing the indicated ratios of wild type and dominant-negative mutant envs was measured on TZM-bl reporter cells. Infectivity of virus stocks containing solely wt envelope were set as 100%. Data depict mean and SD from 2 to 4 independent experiments. For each virus the individual curve fits resulting from the model to the obtained data were evaluated (<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1004595#ppat.1004595.s001" target="_blank">S1 Fig.</a>). (C) Mathematical estimates of T derived from the data shown in (B). The R508S/R511S (black circles) and V513E (open squares) mutations were analyzed individually. Bootstrap analyses demonstrating the robustness of the obtained estimates of T are shown in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1004595#ppat.1004595.s001" target="_blank">S1 Fig.</a> (D and E) Mixed trimer virus stocks for strain JR-FL (D) and SF-162 (E) carrying the V513E mutation were assayed on healthy donor PBMC and compared to data obtained with TZM-bl target cells. Data depict mean and SD from 2 independent experiments.</p

Point mutations in JR-FL dictate virus infection efficacy and entry stoichiometry.

<p>(A) Infectivities of JR-FL wt and indicated point mutant envs were determined by titration of virus stocks on TZM-bl reporter cells and are shown normalized to JR-FL wt. Data depict mean and SD from 4 independent experiments. (B) Relative infectivity of mixed trimer infection experiments with the specified JR-FL variants using the R508S/R511S and V513E dominant-negative mutations are shown. Infectivity of pseudotyped virus stocks expressing the indicated ratios of dominant-negative mutant envs was measured on TZM-bl cells. Infectivity of virus stocks containing solely functional envelope were set as 100%. Data depict mean and SD from 2 independent experiments. (C) Mathematical analyses of the data shown in (B) yielded estimates of T, shown here as mean and range of the individual T estimates obtained with the R508S/R511S and V513E dominant-negative mutations. (D) Analysis of virus entry kinetics for the four JR-FL variants were performed as shown <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1004595#ppat.1004595.s007" target="_blank">S7A Fig.</a></p

Local deposition of calcium pyrophosphate crystals in evolution of knee osteoarthritis.

The aim of the study was to investigate the frequency of development of local calcium pyrophosphate (CPPD) crystal deposition in patients with knee OA initially found negative for these crystals, as well as to discover whether prognostic indicators for this subset of patients can be found. A clinical follow-up of records of outpatients with idiopathic knee OA was established. An anteroposterior plain radiography of the knee joints was made initially and at the end of the observation period. The follow-up period needed to be more than 1 year. Patients were divided into two groups. The first included patients with knee OA who did not develop intra-articular CPPD crystal deposition during the observation period (OA group). The second included those patients whose X-rays or synovial fluid (SF) analysis in the follow-up showed these crystal deposits to be present (OA + CPPD group). There were 59 patients (42 women, 17 men) who met the selection criteria. During the observation period (8.1 + 7.4 years in the OA group, 10.4 +/- 6 years in the OA + CPPD group), intra-articular CPPD deposits were observed in 15 patients (25%): 10 on the X-rays, eight in the SF and three in both examinations. Age at diagnosis of OA and incidence of obesity were similar in both groups. There was a trend (P = 0.21) towards men developing intra-articular CPPD crystal deposits more frequently than women. OA in only one knee joint was significantly more frequent in the group with CPPD (P&amp;lt;0.01). Of those with CPPD deposits 40% required surgery at the end of the observation period, compared to 27.2% of those without deposits (P = 0.27). The waiting period before knee surgery was shorter in the OA + CPPD group but the difference was not statistically significant. In conclusion, local CPPD crystal deposition was observed in 25% of cases during the evolution of knee OA. No predictive factors were found. OA of the knee could, per se, favour the development of CPPD deposits. The occurrence of intra-articular CPPD deposits seemed to be related to a more rapid and severe evolution of OA of the knee

Unique properties of bnAbs CAP256-VRC26.08 and CAP256-VRC26.09.

<p><b>A: Comparable potency of bnAbs CAP256-VRC26.08 and CAP256-VRC26.09 during free virus and cell-cell neutralization.</b> Inhibition of free virus (black circles) and cell-cell (red circles) transmission of the indicated virus strains by bnAbs CAP256-VRC26.08 and CAP256-VRC26.09 is shown. The graphs depict means and standard error of means (SEM, error bars) of two to three independent experiments performed in duplicates and curve fits to sigmoid dose response curves (variable slope). <b>B-C: Comparison of inhibitory activity of CAP256-VRC26.08 and CAP256-VRC26.09</b>. <b>B:</b> Comparison of IC₅₀ values of free virus and cell-cell inhibition determined in <b>(A)</b> Subtype A and C viruses are denoted in blue and orange, respectively. <b>C:</b> Comparison of the change in inhibitory activity in cell-cell transmission relative to free virus inhibition (fold change IC₅₀). <b>D: Comparison of the capacity to neutralize post-CD4 receptor engagement.</b> Activity of bnAbs added after attachment of virions to A3.01-CCR5 target cells was assessed and compared to samples, where bnAbs were present before and after attachment (total activity, set to 100% inhibition). Post- attachment inhibition is displayed relative to the total inhibition activity. Subtype A and C virus are denoted in blue and orange, respectively. One representative of two independent experiments is shown. <b>E: Comparison of the capacity to neutralize pre-CD4 receptor engagement.</b> The relative activity of bnAbs in the pre-attachment phase compared to total activity (bnAbs present before and after attachment) was assessed. Data are means of two to three independent experiments.</p