The Differential Anti-HIV Effect of a New Humic Substance-Derived Preparation in Diverse Cells of the Immune System.

The anti-HIV activity of a new humic substance-derived preparation has been studied in individual pools of immune cells (CD4+ T lymphocytes, macrophages, dendritic cells). Near-complete inhibition of the HIV infection (by more than 90%) was achieved by treating each of the abovementioned cell types with non-toxic concentrations of the preparation. The inhibitory effect demonstrates the possibility of preventing the depletion of a significant portion of functionally important immune cells. A comparative study of infection inhibition in individual cell pools has allowed us to reveal the differences in the preparation's effectiveness in each of the cell populations. A R5-tropic HIV-1 infection in macrophages exhibited maximum sensitivity to the preparation: 90% and 50% inhibition of the infection were observed in the presence of concentrations as low as 1.4 and 0.35 µg/ml, respectively. A 15- and 19-fold higher concentration was required to achieve the same extent of inhibition in dendritic cells infected with the same strain. The effectiveness of the drug in CD4 + T lymphocytes is quite comparable to its effectiveness in macrophages. The drug is universally effective for both the T- and M-tropic variants of HIV-1.

Enhancing the X-ray contrast of polymeric biochromatography particles for three-dimensional imaging.

A major limitation of the three-dimensional imaging of polymeric biochromatography particle packings using X-ray computed tomography is that the particles have a low density and a high porosity, making them almost undistinguishable from the surrounding liquid phase. Additionally, the employed media are typically composed of materials with low atomic numbers, which exhibit low X-ray absorption. We report an improvement of packed column reconstruction using micro X-ray computed tomography. A simple, inexpensive, and fast method to increase the contrast factor of highly porous polymer-based chromatographic particles was developed by applying a modified pore-blocking method. This approach relies on the selective filling of the porous chromatographic particles with a hydrophilic phase while a hydrophobic phase occupies the void spaces between the particles. The hydrophilic phase contains a dissolved X-ray absorbing radiocontrast agent. No chemical modifications of the chromatographic beads or columns were necessary. The developed method can be applied in-situ in a previously packed column and can be used for media with different organic backbones. We show the applicability of this method by carrying out the first 3D-reconstruction of packed micro columns with an inner diameter of 760 µm. The micro column contained agarose- and methacrylate-based particles commonly used in preparative biochromatography with mean diameters of 40 and 65 µm, respectively. Based on the obtained high-resolution 3D-reconstructions, we exemplarily computed packing properties such as global extraparticle porosity and radial porosity profiles, and visualized the presence of void spaces using 3D image analysis.

Towards a Full Automation of the ELISpot Assay for Safe and Parallelized Immunomonitoring.

The ELISpot assay, as a sensitive and specific method, enables the detection of cytokines for immunological purposes and in vaccine development. Here we describe the successful transfer of the manual procedure to a commercially available automated liquid handling platform, based on the work described in Neubauer et al. (Cytotechnology 69:57-73, 2017). Different kinds of technical issues (dead volume reduction, instrumental handling limitations, liquid class improvement) have been solved and biological effects (reagents concentration, selectivity tests, dispensing way, etc.) have been controlled during the implementation process. At the end a maximum of 6% mean delta difference and a lower mean dispersion than the manual assay were reached as well as a turnaround time of four to six times higher than the manual process.

Towards standardized automated immunomonitoring: an automated ELISpot assay for safe and parallelized functionality analysis of immune cells.

The ELISpot assay is used for the detection of T cell responses in clinical trials and vaccine evaluations. Standardization and reproducibility are necessary to compare the results worldwide, inter- and intra-assay variability being critical factors. To assure operator safety as well as high-quality experiment performance, the ELISpot assay was implemented on an automated liquid handling platform, a Tecan Freedom EVO. After validation of the liquid handling, automated loading of plates with cells and reagents was investigated. With step by step implementation of the manual procedure and liquid dispensing optimization on the robot platform, a fully automated ELISpot assay was accomplished with plates remaining in the system from the plate blocking step to spot development. The mean delta difference amounted to a maximum of 6%, and the mean dispersion was smaller than in the manual assay. Taken together, we achieved with this system not only a lower personnel attendance but also higher throughput and a more precise and parallelized analysis. This platform has the potential to guarantee validated, safe, fast, reproducible and cost-efficient immunological and toxicological assays in the future.

In vitro-to-in vivo correlation of the skin penetration, liver clearance and hepatotoxicity of caffeine.

This work illustrates the use of Physiologically-Based Toxicokinetic (PBTK) modelling for the healthy Caucasian population in in vitro-to-in vivo correlation of kinetic measures of caffeine skin penetration and liver clearance (based on literature experiments), as well as dose metrics of caffeine-induced measured HepaRG toxicity. We applied a simple correlation factor to quantify the in vitro and in vivo differences in the amount of caffeine permeated through the skin and concentration-time profiles of caffeine in the liver. We developed a multi-scale computational approach by linking the PBTK model with a Virtual Cell-Based Assay to relate an external oral and dermal dose with the measured in vitro HepaRG cell viability. The results revealed higher in vivo skin permeation profiles than those determined in vitro using identical exposure conditions. Liver clearance of caffeine derived from in vitro metabolism rates was found to be much slower than the optimised in vivo clearance with respect to caffeine plasma concentrations. Finally, HepaRG cell viability was shown to remain almost unchanged for external caffeine doses of 5-400 mg for both oral and dermal absorption routes. We modelled single exposure to caffeine only.

The acute effects of daily nicotine intake on heart rate--a toxicokinetic and toxicodynamic modelling study.

Joint physiologically-based toxicokinetic and toxicodynamic (PBTK/TD) modelling was applied to simulate concentration-time profiles of nicotine, a well-known stimulant, in the human body following single and repeated dosing. Both kinetic and dynamic models were first calibrated by using in vivo literature data for the Caucasian population. The models were then used to estimate the blood and liver concentrations of nicotine in terms of the Area Under Curve (AUC) and the peak concentration (Cmax) for selected exposure scenarios based on inhalation (cigarette smoking), oral intake (nicotine lozenges) and dermal absorption (nicotine patches). The model simulations indicated that whereas frequent cigarette smoking gives rise to high AUC and Cmax in blood, the use of nicotine-rich dermal patches leads to high AUC and Cmax in the liver. Venous blood concentrations were used to estimate one of the most common acute effects, mean heart rate, both at rest and during exercise. These estimations showed that cigarette smoking causes a high peak heart rate, whereas dermal absorption causes a high mean heart rate over 48h. This study illustrates the potential of using PBTK/TD modelling in the safety assessment of nicotine-containing products.

Application of physiologically-based toxicokinetic modelling in oral-to-dermal extrapolation of threshold doses of cosmetic ingredients.

The application of physiologically based toxicokinetic (PBTK) modelling in route-to-route (RtR) extrapolation of three cosmetic ingredients: coumarin, hydroquinone and caffeine is shown in this study. In particular, the oral no-observed-adverse-effect-level (NOAEL) doses of these chemicals are extrapolated to their corresponding dermal values by comparing the internal concentrations resulting from oral and dermal exposure scenarios. The PBTK model structure has been constructed to give a good simulation performance of biochemical processes within the human body. The model parameters are calibrated based on oral and dermal experimental data for the Caucasian population available in the literature. Particular attention is given to modelling the absorption stage (skin and gastrointestinal tract) in the form of several sub-compartments. This gives better model prediction results when compared to those of a PBTK model with a simpler structure of the absorption barrier. In addition, the role of quantitative structure-property relationships (QSPRs) in predicting skin penetration is evaluated for the three substances with a view to incorporating QSPR-predicted penetration parameters in the PBTK model when experimental values are lacking. Finally, PBTK modelling is used, first to extrapolate oral NOAEL doses derived from rat studies to humans, and then to simulate internal systemic/liver concentrations - Area Under Curve (AUC) and peak concentration - resulting from specified dermal and oral exposure conditions. Based on these simulations, AUC-based dermal thresholds for the three case study compounds are derived and compared with the experimentally obtained oral threshold (NOAEL) values.

The effect of EIF dynamics on the cryopreservation process of a size distributed cell population.

Typical mathematical modeling of cryopreservation of cell suspensions assumes a thermodynamic equilibrium between the ice and liquid water in the extracellular solution. This work investigates the validity of this assumption by introducing a population balance approach for dynamic extracellular ice formation (EIF) in the absence of any cryo-protectant agent (CPA). The population balance model reflects nucleation and diffusion-limited growth in the suspending solution whose driving forces are evaluated in the relevant phase diagram. This population balance description of the extracellular compartment has been coupled to a model recently proposed in the literature [Fadda et al., AIChE Journal, 56, 2173-2185, (2010)], which is capable of quantitatively describing and predicting internal ice formation (IIF) inside the cells. The cells are characterized by a size distribution (i.e. through another population balance), thus overcoming the classic view of a population of identically sized cells. From the comparison of the system behavior in terms of the dynamics of the cell size distribution it can be concluded that the assumption of a thermodynamic equilibrium in the extracellular compartment is not always justified. Depending on the cooling rate, the dynamics of EIF needs to be considered.

Is there a difference in the efficacy of peripartum antiretroviral regimens in reducing mother-to-child transmission of HIV in Africa?

BACKGROUND: Peripartum antiretroviral regimens have been shown to prevent mother-to-child transmission of HIV (MTCT) in randomized clinical trials; however, direct comparison of published results is impossible given methodological and population differences. OBJECTIVE: To directly compare the efficacy of different antiretroviral regimens in reducing the risk of 6-week MTCT rate in African breastfeeding populations. METHODS: Pooled analysis including all mother-infant pairs from any relevant trial: West African ZDV-placebo trials, Petra ZDV+3TC [two regimens A (pre/intra/post-partum) and B (intra/post-partum), placebo from Uganda and Tanzania], SAINT (NVP and Petra arm B), HIVNET012 (NVP, ultra short ZDV pp) and the Vitamin A trial (as placebo arm in South Africa). Peripartum HIV infection was any positive RNA or DNA polymerase chain reaction test < day 60. The MTCT risk was estimated at 6 weeks for each treatment arm and compared with placebo or single-dose NVP using logistic regression adjusting for maternal CD4 cell count, breastfeeding and birthweight. RESULTS: Overall, 4125 singleton live-births were included; 3629 (88%) were assessed for HIV status at 6 weeks of age. In comparison with placebo, zidovudine + lamivudine (ZDV+3TC) arm A [adjusted odds ratio (AOR), 0.23; P < 0.0001], ZDV+3TC arm B (AOR, 0.49; P < 0.001), antenatal ZDV short (AOR, 0.55; P = 0.006) and nevirapine (NVP) (AOR, 0.60; P = 0.0007) significantly reduced MTCT. In comparison with NVP, only the longest regimen of ZDV+3TC (AOR, 0.39, P < 0.0005) was significantly more effective. CONCLUSION: These results are in line with current World Health Organisation guidelines suggesting equivalence of choice between single-dose NVP and short-course ZDV, and confirm the greater efficacy of ZDV+3TC than with any single antiretroviral drug.

Physicochemical characterization of protamine-phosphorothioate nanoparticles.

Protamine-oligonucleotide nanoparticles represent effective colloidal drug carriers for antisense phosphorothioate oligonucleotides (PTO). This study describes improvements in particle preparation and the physicochemical properties of the complexes prepared. The influence of component concentrations, length of the PTO chain and the PTO/protamine weight ratio on particle formation and size, shape and surface charge of the particles were studied in detail. Nanoparticles with diameters of 90-200nm were obtained, using protamine free base (PFB) and phosphorothioate in water. The chemical composition of the nanoparticles was analysed. More than 90% of the PTO could be assembled in the particle matrix using a > or = 1:2 ratio (w/w) of PTO and PFB. About 53-68% of the PFB was incorporated in the particle matrix. The complexes had a zetapotential of -19 up to +32 mV, depending on the PTO/PFB ratio. The kinetics of the assembly of this binary system were observed by dynamic light scattering (DLS) measurements and by sedimentation velocity analysis in the analytical ultracentrifuge (AUC). In addition, scanning electron microscopy (SEM) and atomic force microscopy (AFM) were applied to verify the results of DLS and the ultracentrifuge measurements. According to sedimentation velocity analysis, the particles were only moderately stable in water and unstable in salt solutions. However, the colloidal solution in water could be stabilized by polyethylenglycol 20000 (PEG), which also led to an increase of stability in cell medium.

Optimization of the preparation process for human serum albumin (HSA) nanoparticles.

Nanoparticles prepared by desolvation and subsequent crosslinking of human serum albumin (HSA) represent promising carriers for drug delivery. Particle size is a crucial parameter, in particular for the in vivo behaviour of nanoparticles after intravenous injection. The objective of the present study is the development of a desolvation procedure for the preparation of HSA-based nanoparticles under the aspect of a controllable particle size between 100 and 300 nm in combination with a narrow size distribution. A pump-controlled preparation method was established which enabled particle preparation under defined conditions. Several factors of the preparation process, such as the rate of addition of the desolvating agent, the pH value and the ionic composition of the HSA solution, the protein concentration, and the conditions of particle purification were evaluated. The pH value of the HSA solution prior to the desolvation procedure was identified as the major factor determining particle size. Varying this parameter, (mean) particle diameters could be adjusted between 150 and 280 nm, higher pH values leading to smaller nanoparticles. Washing the particles by differential centrifugation led to significantly narrower size distributions. The reproducibility of the particle size and particle size distribution under the proposed preparation conditions was demonstrated by sedimentation velocity analysis in the analytical ultracentrifuge and the cellular uptake of those nanoparticles was studied by confocal microscope imaging and FACS analysis. The stability of the resulting nanoparticles was evaluated by pH and buffer titration experiments. Only pH values distinctly outside the isoelectric pH range of HSA and low salt concentrations were able to prevent nanoparticle agglomeration.

Analysis of cellular factors influencing the replication of human immunodeficiency virus type I in human macrophages derived from blood of different healthy donors.

We analyzed parameters influencing HIV-1 infectibility of cells of the monocyte/macrophage lineage (MO/MAC) isolated from different healthy donors. The proportion of in vitro-infected cells and replication kinetics in different donor MAC ranged from 0.03 to 99% p24 antigen-positive MAC and from undetectable RT activity up to 5 x 10(6) cpm/ml/90 min, respectively. As a quantitative measurement for HIV-1 susceptibility of donor MO/MAC, we determined TCID(50) values of defined virus stocks which varied up to 3000-fold depending on the donor MAC used for titration. As host factors which may influence the viral infection we determined the expression of virus receptors CD4, CCR5, CXCR4, and CCR3 as well as the secretion of the natural ligands of CCR5, which altogether showed no correlation with HIV-1 infectibility of the cells. Moreover, other MO-derived secretory factors which might affect viral infection of these cells could be excluded. Furthermore, expression of maturation-related antigens CD14, CD16, HLA-DR, and MAX.1/CPM was determined. Analysis of the reverse transcription process revealed that restricted HIV-1 infection was reflected by highly reduced or even undetectable full-length HIV-1 DNA formation, although early and intermediate transcripts appeared, suggesting that viral replication is blocked after entry at the level of early reverse transcription.

Characterization of a virtually full-length human immunodeficiency virus type 1 genome of a prevalent intersubtype (C/B') recombinant strain in China.

A molecular epidemiology study was conducted among more than 100 human immunodeficiency virus type 1 (HIV-1) subtype C seropositive intravenous drug users (IDUs) from China. Genotyping based on the envelope C2V3 coding region revealed the highest homology of the most prevalent virus strains circulating throughout China to subtype C sequences of Indian origin. Based on these results, a virtually full-length genome representing the most prevalent class of clade C strains circulating throughout China was directly amplified from peripheral blood mononuclear cells of a selected HIV-infected IDU and subcloned. Sequence analysis identified a mosaic structure, suggesting extensive intersubtype recombination events between genomes of the prevalent clade C and (B')-subtype Thai virus strains of that geographic region. Recombinant Identification Program analysis and phylogenetic bootstrapping suggested that there were 10 breakpoints (i) in the gag-pol coding region, (ii) in vpr and at the 3' end of the vpu gene, and (iii) in the nef open reading frame. (B')-sequences therefore include (i) several insertions in the gag-pol coding region; (ii) 3'-vpr, the complete vpu gene, and the first exons of tat and rev; and (iii) the 5' half of the nef gene. Breakpoints located in the vpr/vpu coding region as well as in the nef gene of 97cn54 were found at almost identical positions of all subtype C strains isolated from IDUs living in different areas of China, suggesting a common ancestor for the C/B' recombinant strains. More than 50% of well-defined subtype B-derived cytotoxic T-lymphocyte epitopes within Gag and Pol and 10% of the known epitopes in Env were found to exactly match sequences within in this clade C/B' chimeric reference strain. These results may substantially facilitate a biological comparison of clade C-derived reference strains as well as the generation of useful reagents supporting vaccine-related efforts in China.

Polysorbate-80 coating enhances uptake of polybutylcyanoacrylate (PBCA)-nanoparticles by human and bovine primary brain capillary endothelial cells.

Certain drugs such as dalargin, loperamide or tubocurarine are not transported across the blood-brain barrier (BBB) and therefore exhibit no effects on the central nervous system. However, effects on the central nervous system can be observed when these drugs are loaded onto polybutylcyanoacrylate (PBCA)-nanoparticles and coated with polysorbate 80. The mechanism by which these complexed nanoparticles cross the BBB and exhibit their effects has not been elucidated. Cultured microvessel brain endothelial cells of human and bovine origin were used as an in vitro model for the BBB to gain further insight into the mechanism of uptake of nanoparticles. With cells from these species we were able to show that polysorbate 80-coated nanoparticles were taken up by brain endothelial cells much more rapidly and in significantly higher amounts (20-fold) than uncoated nanoparticles. The process of uptake was followed by fluorescence and confocal laser scanning microscopy. The results demonstrate that the nanoparticles are taken up by cells and that this uptake occurs via an endocytotic mechanism.

Preparation of avidin-labeled protein nanoparticles as carriers for biotinylated peptide nucleic acid.

The possibility of preparing protein nanoparticles followed by covalent linkage of avidin was investigated. Free sulfhydryl groups were introduced onto the surface of protein nanoparticles either by aldehyde quenching with cysteine or reaction of free amino groups with 2-iminothiolane. The number of primary amino groups and sulfhydryl groups on the surface of the resulting particles was quantified with site-specific reagents. Avidin was attached to the surface of the thiolated nanoparticles via a bifunctional spacer which reacted in a first step with amino groups of avidin and in a second step with the sulfhydryl groups introduced onto the surface of the nanoparticles. Biotinylated peptide nucleic acid (PNA) as a model compound for biotinylated drugs was effectively coupled to the nanoparticles by complex formation with the covalently attached avidin. Since the formation of the interaction between biotin and avidin is very rapid and stable a highly effective drug carrier system for biotinylated compounds such as PNAs was achieved.

Gelatin nanoparticles by two step desolvation--a new preparation method, surface modifications and cell uptake.

A new two-step desolvation method for manufacturing gelatin nanoparticles was developed. After the first desolvation step, the low molecular gelatin fractions present in the supernatant were removed by decanting. The high molecular fractions present in the sediment were redesolved and then desolvated again at pH 2.5 in the second step. The resulting particles can then be easily purified by centrifugation and redispersion. The different fractions obtained during the process were analysed by gel permeation chromatography (GPC). Based on these results, it can be concluded that the molecular weight of gelatin has a decisive influence on the stability of the manufactured gelatin nanoparticles. In addition, two fluorescent dyes (Texasred and fluoresceinamine) were coupled to the nanoparticles for cell uptake studies. The fluorescent nanoparticles showed a high uptake into monocytes/macrophages.

Preparation of avidin-labelled gelatin nanoparticles as carriers for biotinylated peptide nucleic acid (PNA).

The possibility of preparing uniform nanoparticles consisting of proteins such as gelatin followed by covalent linkage of avidin was investigated. Gelatin nanoparticles were prepared by two step desolvation. Functional groups at the surface of the particulate system were quantified with site-specific reagents. The surface of the nanoparticles was thiolated and avidin was covalently attached to the nanoparticles via a bifunctional spacer at high levels. Biotinylated peptide nucleic acid (PNA) was effectively complexed by the avidin-conjugated nanoparticles. Avidin-conjugated protein nanoparticles should prove as potential carrier system for biotinylated drug derivatives in antisense therapy.

Epidemic transmission of human immunodeficiency virus in renal dialysis centers in Egypt.

In 1993 an epidemic of human immunodeficiency virus (HIV) infection occurred among 39 patients at 2 renal dialysis centers in Egypt. The centers, private center A (PCA) and university center A (UCA) were visited, HIV-infected patients were interviewed, seroconversion rates at UCA were calculated, and relatedness of HIV strains was determined by sequence analysis; 34 (62%) of 55 patients from UCA and 5 (42%) of 12 patients from PCA were HIV-infected. The HIV seroconversion risk at UCA varied significantly with day and shift of dialysis session. Practices that resulted in sharing of syringes among patients were observed at both centers. The analyzed V3 loop sequences of the HIV strain of 12 outbreak patients were >96% related to each other. V3 loop sequences from each of 8 HIV-infected Egyptians unrelated to the 1993 epidemic were only 76%-89% related to those from outbreak strains. Dialysis patients may be at risk for HIV infection if infection control guidelines are not followed.

Resistance mutations selected in vivo under therapy with anti-HIV drug HBY 097 differ from resistance pattern selected in vitro.

The quinoxaline derivative HBY 097, an orally active nonnucleoside inhibitor of HIV-1 reverse transcriptase (NNRTI), showed an efficient suppression of viral load in a dose-escalating phase I study with mean trough concentrations increasing from 137-1299 ug/l [Rübsamen-Waigmann et al., Lancet 349:1517]. Half-maximal inhibitory concentrations (IC50) for viruses grown from the patients at entry of the study were 0.1-3 nM, except for one patient who had a virus with reduced susceptibility to HBY 097 at entry (IC50: 160 nM). During therapy, only two patients developed a virus with a moderately increased IC50 (2.2 and 15 nM). This reduced susceptibility was associated with the known NNRTI-resistance mutation K ==> N at position 103, in contrast to resistance selection in vitro, which had yielded predominant mutations at positions 179 and 190. The Tyr mutation at position 181, inducing high resistance for other NNRTIs, was never observed. The resistant virus at study entry (IC50 = 160 nM) had a mutation at position 103 as well, combined with an AZT resistance mutation (K ==> R) at position 70, suggesting that nucleoside-resistance mutations may help increasing resistance to HBY 097. This is in line with our in vitro selection studies, where resistance mutations at the 'nucleoside sites' 74 and 75 increased the resistance phenotype of NNRTI mutations. Our findings highlight the crucial importance of IC50 determinations from cultured virus for determination of phenotypic resistance development during therapy and demonstrate that in vivo resistance development cannot be predicted from in vitro selection.