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Objectives: To determine the representation of particular HIV-1 genotypes during cultivation in different primary cell-culture systems compared with the spectrum of the quasispecies in vivo. Methods: Primary isolates of HIV-1 were recovered by isolation in cultures of lymphocytes, mixed mononuclear cells (MNC), and monocytes/macrophages. Nucleotide sequence determination of the C2-V3 region of gp120 of HIV was performed on 10-20 independently isolated clones derived by polymerase chain reaction from the culture systems, the uncultured peripheral blood MNC (PBMC) as well as plasma. Results: Several predominant HIV genotypes were found in the uncultured PBMC from each of the patients. The most frequent genotypes in PBMC were also the most frequent types in plasma. In addition, lymphocytes, macrophages or mixed MNC cultures allowed the outgrowth of variants that were underrepresented in uncultured PBMC. We showed that the virus cultivation systems used in this study selected differently for the genetic variants. Whereas some genotypes were present in all three culture systems, although at different frequencies, others were exclusively found in a specific culture system. Conclusions: These results demonstrate that monocyte/macrophage and mixed MNC culture systems complement the standard lymphocyte culture in terms of the spectrum of genotypically different virus variants obtained in vitro.

HIV-1 isolates were obtained from four countries within the framework of the WHO Network for HIV Isolation and Characterization. The use of standard HIV isolation procedures allowed us to compare the biological properties of 126 HIV-1 isolates spanning five genetic subtypes. In primary isolation cultures, viruses from Uganda and Brazil appeared early and replicated without delay, whereas the replication of Thai viruses was delayed by several weeks. Regardless of genetic subtype or country of origin, blood samples collected more than 2 years after seroconversion yielded virus that replicated efficiently in the primary isolation cultures. None of the isolates obtained from Thailand or Rwanda replicated in cell lines, whereas 5 of the 13 Brazilian isolates and 7 of the 11 Ugandan isolates replicated and induced syncytia in MT-2 cells. As expected for virus isolates obtained early in HIV-1 infection (within 2 years of seroconversion), all viruses from Brazil, Rwanda, and Thailand showed a slow/low replicative pattern. For the Ugandan samples, the time from seroconversion was known precisely for a few of the samples and only in one case was less than 2 years. This may explain why the five viruses that were able to replicate in all cell lines, and thus classified as rapid/high, were of Ugandan origin. Viruses able to induce syncytia in MT-2 cells, also induced syncytia in PBMC. However, 8 slow/low viruses (out of 27) gave discordant results, inducing syncytia in PBMC but not in MT-2 cells. Furthermore, using syncytium induction as a marker, changes in virus populations during early in vitro passage in PBMC could be observed

A high-risk population (patients of a sexually transmitted disease clinic and the GT hospital in Bombay) was tested for antibodies against HIV-1 and HIV-2. Among 405 serum samples, 226 had previously been classified HIV-positive in India using different locally available enzyme-linked immunosorbent assay (ELISA) tests. The serology of 179 samples was unknown. All 405 samples were tested at the Georg-Speyer-Haus (GSH) with the Pasteur HIV-1/2-Combi-ELISA. Positive samples were further analyzed with HIV-1 and HIV-2 Western blot kits from Dupont and Pasteur, respectively. A very high seroprevalence of HIV was found in this population. Among the 179 unscreened samples, 69 (38.5%) were positive in the ELISAs as well as the Western blots for HIV-1 or HIV-2. Among the prescreened samples, only 174 (77%) were confirmed HIV-positive. Altogether, 243 of 405 sera were HIV-positive. Of these, 184 (76%) were reactive with HIV-1, 10 (4%) were reactive with HIV-2, and 49 (20%) had dual reactivity to HIV-1 and HIV-2. Previous data from the Indian Council of Medical Research had already suggested a possible high prevalence of HIV-1 in India. Our results confirm this view. The finding of a substantial spread of HIV-2 infection was, however, totally unexpected in India, but confirms our previous study which had already demonstrated the existence of HIV-2 in this country. Asia can thus no longer be considered free of HIV-2, and testing for HIV-2 appears mandatory, at least in India.(ABSTRACT TRUNCATED AT 250 WORDS)

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.

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.