The structure of poly(L-lysine) (PLL)/hyaluronan (HA) polyelectrolyte multilayers formed by electrostatic self-assembly is studied by using confocal laser scanning microscopy, quartz crystal ...microbalance, and optical waveguide lightmode spectroscopy. These films exhibit an exponential growth regime where the thickness increases exponentially with the number of deposited layers, leading to micrometer thick films. Previously such a growth regime was suggested to result from an "in" and "out" diffusion of the PLL chains through the film during buildup, but direct evidence was lacking. The use of dye-conjugated polyelectrolytes now allows a direct three-dimensional visualization of the film construction by introducing fluorescent polyelectrolytes at different steps during the film buildup. We find that, as postulated, PLL diffuses throughout the film down into the substrate after each new PLL injection and out of the film after each PLL rinsing and further after each HA injection. As PLL reaches the outer layer of the film it interacts with the incoming HA, forming the new HA/PLL layer. The thickness of this new layer is thus proportional to the amount of PLL that diffuses out of the film during the buildup step, which explains the exponential growth regime. HA layers are also visualized but no diffusion is observed, leading to a stratified film structure. We believe that such a diffusion-based buildup mechanism explains most of the exponential-like growth processes of polyelectrolyte multilayers reported in the literature.
In this placebo-controlled phase II randomized clinical trial, 103 human immunodeficiency virus type 1 (HIV-1)-infected patients under cART (combined antiretroviral treatment) were randomized 2:1 to ...receive either 3 doses of DNA GTU-MultiHIV B (coding for Rev, Nef, Tat, Gag, and gp160) at week 0 (W0), W4, and W12, followed by 2 doses of LIPO-5 vaccine containing long peptides from Gag, Pol, and Nef at W20 and W24, or placebo. Analytical treatment interruption (ATI) was performed between W36 to W48. At W28, vaccinees experienced an increase in functional CD4
T-cell responses (
< 0.001 for each cytokine compared to W0) measured, predominantly against Gag and Pol/Env, and an increase in HIV-specific CD8
T cells producing interleukin 2 (IL-2) and tumor necrosis factor alpha (TNF-α) (
= 0.001 and 0.013, respectively), predominantly against Pol/Env and Nef. However, analysis of T-cell subsets by mass cytometry in a subpopulation showed an increase in the W28/W0 ratio for memory CD8
T cells coexpressing exhaustion and senescence markers such as PD-1/TIGIT (
= 0.004) and CD27/CD57 (
= 0.044) in vaccinees compared to the placebo group. During ATI, all patients experienced viral rebound, with the maximum observed HIV RNA level at W42 (median, 4.63 log
copies cp/ml; interquartile range IQR, 4.00 to 5.09), without any difference between arms. No patient resumed cART for CD4 cell count drop. Globally, the vaccine strategy was safe. However, a secondary HIV transmission during ATI was observed. These data show that the prime-boost combination of DNA and LIPO-5 vaccines elicited broad and polyfunctional T cells. The contrast between the quality of immune responses and the lack of potent viral control underscores the need for combined immunomodulatory strategies. (This study has been registered at ClinicalTrials.gov under registration no. NCT01492985.)
In this placebo-controlled phase II randomized clinical trial, we evaluated the safety and immunogenicity of a therapeutic prime-boost vaccine strategy using a recombinant DNA vaccine (GTU-MultiHIV B clade) followed by a boost vaccination with a lipopeptide vaccine (HIV-LIPO-5) in HIV-infected patients on combined antiretroviral therapy. We show here that this prime-boost strategy is well tolerated, consistently with previous studies in HIV-1-infected individuals and healthy volunteers who received each vaccine component individually. Compared to the placebo group, vaccinees elicited strong and polyfunctional HIV-specific CD4
and CD8
T-cell responses. However, these immune responses presented some qualitative defects and were not able to control viremia following antiretroviral treatment interruption, as no difference in HIV viral rebound was observed in the vaccine and placebo groups. Several lessons were learned from these results, pointing out the urgent need to combine vaccine strategies with other immune-based interventions.
Polyelectrolyte multilayers (PEMs) are now widely used for biomedical applications. In this work, we investigated the primary osteoblast adhesion properties of PEMs of poly(L‐lysine) (PLL), ...poly(L‐glutamic acid) (PGA), poly(alginic acid) (Palg), and poly(galacturonic acid) (Pgal). In order to compensate for the poor adhesion of the as‐synthesized films, two kinds of film modifications were achieved: a purely physical modification by film crosslinking, and a chemical modification by grafting a arginine–glycine–aspartic acid (RGD) peptide to PGA. Crosslinking was performed using a water‐soluble carbodiimide in combination with N‐hydroxysulfosuccinimide (sulfo‐NHS) to induce amide formation. This reaction was followed by Fourier‐transform IR spectroscopy. For film functionalization, a 15‐amino‐acid peptide was grafted to PGA and deposited as the top layer of the film. PLL/PGA, PLL/Palg, and PLL/Pgal films were crosslinked or functionalized. The films were tested for both short‐term adhesion properties and long‐term proliferation of primary osteoblasts. Whereas the effect of film crosslinking on short‐term adhesion was moderate, it was much more important for the RGD‐functionalized films. On the other hand, the long‐term proliferation was the same or even higher for the crosslinked films as compared with the functionalized films. This effect was particularly enhanced for the PLL/Palg and PLL/Pgal films. Finally, we functionalized PLL/PGA that had been crosslinked prior to PGA‐RGD deposition. These architectures exhibited even higher short‐term adhesion and proliferation. These results clearly show the important role of the physical properties of the films, besides their chemical properties, for the modulation of primary cell‐adhesion behavior.
Adhesion and proliferation of osteoblasts on polyelectrolyte multilayer (PEM) films (see Figure) is enhanced by crosslinking, even for films functionalized with the RGD cell‐binding peptide sequence. Cell attachment after short times was enhanced by the RGD sequence, but long‐term proliferation (after 8 days) was greatly improved by crosslinking, demonstrating the influence of both the chemical and the mechanical properties of the films on cell adhesion.
HIV-1-infected individuals with protective HLA class I alleles exhibit better control of viremia and slower disease progression. Virus control in these individuals has been associated with strong and ...potent HIV-1-specific cytotoxic-T-lymphocyte (CTL) responses restricted by protective HLA alleles, but control of viremia also occurs in the presence of selected CTL escape mutations. CTL escape mutations restricted by protective HLA class I molecules are frequently located in the conserved p24 Gag sequence of HIV-1 that encodes the conical capsid core and have been suggested to reduce viral replication capacity. In this study, the consequences of well-described CTL-associated p24 Gag sequence mutations for HIV-1 capsid stability were assessed using a cyclosporine (CsA) washout assay. The frequently occurring HLA-B57- and HLA-B27-associated CTL escape mutations T242N and R264K resulted in delayed capsid uncoating, suggesting modulation of capsid stability. The described compensatory mutations L268M and S173A observed in R264K viruses reconstituted the capsid-uncoating half-time. Interestingly, capsid stability was correlated with infectivity. Taken together, these data demonstrate that CTL-driven escape mutations within p24 Gag restricted by protective HLA class I alleles have a significant impact on capsid stability that might contribute to the persistent control of viral replication observed despite viral escape from CTL responses.
Sequence mutations within p24 Gag selected by CTL responses restricted by protective HLA class I alleles have been associated with reduced viral fitness. However, the precise mechanisms underlying the reduced viral replication capacity and lower viral loads associated with these mutations remain unclear. Here, we demonstrate that dominant HLA-B27-associated CTL escape mutations within HIV-1 capsid lead to enhanced capsid rigidity, providing a possible mechanism for the reduced viral fitness of these variants.
The inhibitory effect of
Andrographis paniculata extract (APE) and andrographolide (AND), the most medicinally active phytochemical in the extract, on hepatic cytochrome P450s (CYPs) activities was ...examined using rat and human liver microsomes. For this purpose, CYP1A2-dependent ethoxyresorufin-
O-deethylation, CYP2B1-dependent benzyloxyresorufin-
O-dealkylation, CYP2B6-dependent bupropion hydroxylation, CYP2C-dependent tolbutamide hydroxylation, CYP2E1-dependent
p-nitrophenol hydroxylation and CYP3A-dependent testosterone 6β-hydroxylation activities, were determined in the presence and absence of APE or AND (0–200
μM). APE inhibited ethoxyresorufin-
O-deethylation activity in rat and human liver microsomes, with apparent
K
i
values of 8.85 and 24.46
μM, respectively. In each case, the mode of inhibition was noncompetitive. APE also inhibited tolbutamide hydroxylation both in rat and human microsomes with apparent
K
i
values of 8.21 and 7.51
μM, respectively and the mode of inhibition was mixed type. In addition, APE showed a competitive inhibition only on CYP3A4 in human microsomes with
K
i
of 25.43
μM. AND was found to be a weak inhibitor of rat CYP2E1 with a
K
i
of 61.1
μM but did not affect human CYP2E1. In conclusion, it cannot be excluded from the present study that APE could cause drug–drug interactions in humans through CYP3A and 2C9 inhibition.
•Kinetics of cyclosporine A were analyzed in 3 liver cell models over a 14-day period.•Sandwich primary rat and human hepatocytes and HepaRG cells were compared.•The cyclophilin B was produced and ...CsA-induced secreted in all cell models.•CsA biotransformation was higher in human liver cells than in rat hepatocytes.•Metabolic clearances were 12–14-fold higher in HepaRG cells than in primary hepatocytes.
In vitro experiments have a high potential to improve current chemical safety assessment and reduce the number of animals used. However, most studies conduct hazard assessment alone, largely ignoring exposure and kinetic parameters. Therefore, in this study the kinetics of cyclosporine A (CsA) and the dynamics of CsA-induced cyclophilin B (Cyp-B) secretion were investigated in three widely used hepatic in vitro models: primary rat hepatocytes (PRH), primary human hepatocytes (PHH) and HepaRG cells. Cells were exposed daily to CsA for up to 14days. CsA in cells and culture media was quantified by LC-MS/MS and used for pharmacokinetic modeling. Cyp-B was quantified by western blot analysis in cells and media. All cell systems took up CsA rapidly from the medium after initial exposure and all showed a time- and concentration-dependent Cyp-B cellular depletion and extracellular secretion. Only in PRH an accumulation of CsA over 14days repeated exposure was observed. Donor-specific effects in CsA clearance were observed in the PHH model and both PHH and HepaRG cells significantly metabolized CsA, with no bioaccumulation being observed after repeated exposure. The developed kinetic models are described in detail and show that all models under-predict the in vivo hepatic clearance of CsA, but to different extents with 27-, 24- and 2-fold for PRH, PHH and HepaRG cells, respectively. This study highlights the need for more attention to kinetics in in vitro studies.
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