Efficient inhibition of both syncytium-inducing and non-syncytium-inducing wild-type HIV-1 by lamivudine in vivo.

OBJECTIVE: To determine the effect of lamivudine (3TC) on syncytium-inducing (SI) and non-SI (NSI) HIV-1 populations in vivo. DESIGN: Responses in virus load and 3TC resistance in 40 3TC-treated subjects were analysed in relation to the presence or absence of SI HIV-1 variants. METHODS: Peripheral blood samples were collected at regular intervals from 40 HIV-1-infected subjects during 3TC treatment. Virus isolates obtained at the start of treatment were typed for SI-capacity by coculture with MT-2 cells. Changes in levels of viral RNA in plasma were determined by quantitative reverse transcriptase PCR. The relative amount of wild-type and mutant virus at codon 184, associated with HIV resistance to 3TC, was determined using a primer-guided nucleotide incorporation assay after amplification of part of the reverse transcriptase gene. In five subjects the frequency of productively infected CD4+ cells with SI or NSI variants was determined in relation to codon 184 genotype. RESULTS: Twenty-six subjects harboured only NSI variants at baseline, whereas 14 subjects also harboured SI variants. Although baseline plasma viral RNA load and CD4 cell counts were different between the two groups, no differences in the response to 3TC therapy were observed for these parameters. In-depth analysis of five subjects showed that the kinetics of virus load changes and emergence of 3TC resistance mutations were similar in plasma and cells, and comparable for the SI and NSI populations present in one individual. CONCLUSIONS: These data show that, in contrast to didanosine and zidovudine, the pressure exerted by 3TC is similar for SI and NSI M184 populations.

Zidovudine treatment results in the selection of human immunodeficiency virus type 1 variants whose genotypes confer increasing levels of drug resistance.

High level resistance to 3'-azido-3'-deoxythymidine (AZT, zidovudine or Retrovir) is conferred by the presence of four or five mutations (Met-41-->Leu; Asp-67-->Asn; Lys-70-->Arg; Thr-215-->Tyr or Phe; Lys-219-->Gln) in the human immunodeficiency virus (HIV) reverse transcriptase. The order of appearance of these five mutations in asymptomatic patients during therapy has been studied. This has enabled us to propose a model for the acquisition of zidovudine resistance mutations during the treatment of high-risk asymptomatic HIV-infected individuals. A consistent acquisition pattern of mutations at codons 41, 70 and 215 was observed in 17 individuals. Complex mixtures of HIV species containing different combinations of single and linked double resistance mutations were present early in zidovudine therapy in isolates from two patients studied in detail. From these mixtures the linked Leu-41/Tyr-215 genotype outgrew all others initially. The development of each new virus population is likely to be mediated primarily by the increase in the level of drug resistance rather than changes in the growth kinetics of the virus. This leads us to conclude that one major driving force in the outgrowth of different mutant viruses is the selective advantage conferred by higher levels of drug resistance.

Effects of Trichobilharzia ocellata on hemocytes of Lymnaea stagnalis.

We analyzed the effects of infection with Trichobilharzia ocellata on hemocytes of its snail host, Lymnaea stagnalis, and correlated them with successive stages of parasite development. Circulating hemocytes were studied at 0, 2, 4, 6, and 8 weeks post exposure (p.e.) with respect to cell number, distribution of subpopulations (as characterized by morphology, determinants recognized by either of two lectins and a monoclonal antibody) and to proliferative, phagocytic and endogenous peroxidase activity. Infection results in a net elevated level of activity of circulating hemocytes at 2 weeks p.e., when mother sporocysts are present in the head-foot-mantle region, as well as at 4 weeks p.e., when daughter sporocysts are migrating to and growing in the digestive gland region. A lower level of activity was observed at 6 weeks p.e., when cercariae are differentiating within daughter sporocysts. A net activation was again found at 8 weeks p.e., when cercariae are escaping. So, infection with T. ocellata results in a net general activation of the internal defense system of L. stagnalis, during several stages of development of the parasite.

Evolution of sequences encoding the principal neutralization epitope of human immunodeficiency virus 1 is host dependent, rapid, and continuous.

The principal neutralization epitope of human immunodeficiency virus 1 is localized in the third variable (V3) domain of the external envelope and has been shown to bind isolate-specific antibodies. Therefore, the extent of variation within the nucleic acid sequence encoding this epitope was studied in DNA directly obtained from peripheral blood mononuclear cells of six children and their plasma donor. This revealed that the quasi-species distribution of sequences obtained after cloning varied from recipient to recipient and that the distance from the donor sequences increased over time. V3 nucleotide evolution rates averaged 9.5 x 10(-3) per site per year for silent sites and 11.4 x 10(-3) per site per year for nonsilent sites (vs. 9.7 and 9.8 x 10(-3) per site per year for a control region 5' adjacent to the V3 region) and, although individual differences were observed, did not correlate with the serum antigen levels or disease progression. Sequences of both the epitope coding region itself (V3) and the control region upstream diverted more from the donor sequence among children not progressing to AIDS than among children progressing to AIDS. The evolution of V3 sequences is apparently host dependent, rapid, and independent of the level of antigen expression.

Possible role of reactive forms of oxygen in in vitro killing of Schistosoma mansoni sporocysts by hemocytes of Lymnaea stagnalis.

The interactions between sporocysts of Schistosoma mansoni and hemocytes of the pond snail Lymnaea stagnalis were studied in an in vitro system. All sporocysts were encapsulated and killed in the hemolymph of juvenile or adult snails within 24 h; the killing patterns of both age groups were not very different. The separation of hemolymph into hemocytes and plasma and subsequent incubation of sporocysts showed that the cells, not the plasma, were responsible for parasite killing. The possible involvement of reactive oxygen intermediates and the enzyme peroxidase in the cytotoxic mechanism was studied. Chemiluminescence (CL) could not be detected during the hemocyte-parasite encounter. Histochemical staining with nitroblue tetrazolium (indicative for superoxide) and diaminobenzidine (indicative for hydrogen peroxide) showed that oxygen intermediates were produced by snail hemocytes that encapsulated sporocysts. Histochemistry also suggested the presence of peroxidase activity at the hemocyte-sporocyst interface. The cytotoxic mechanism underlying parasite killing by hemocytes and the role of reactive forms of oxygen and peroxidase are discussed.

Monoclonal antibody recognized hemocyte subpopulations in juvenile and adult Lymnaea stagnalis: functional characteristics and lectin binding.

The mouse monoclonal antibody LS1 recognizes a membrane epitope present on circulating hemocytes of the gastropod mollusc Lymnaea stagnalis. In both juvenile and adult pond snails, LS1+ (LS1 positive) hemocytes have the morphology of immature cells. The percentage of LS1+ hemocytes is higher in juveniles (ca. 39%) than it is in adults (ca. 14%). Functional characteristics of LS1+ hemocytes and lectin binding to these cells were studied. In both age groups, the proliferative activity, as measured by the incorporation of deoxybromouridine, is much higher for LS1+ hemocytes than it is for LS1- (LS1 negative) cells. LS1+ hemocytes are phagocytically less active and have a lower lysosomal enzyme (peroxidase) content as compared to hemocytes that lack the epitope. Histochemical staining of the total population of circulating hemocytes shows that the lectins DBA, BS-l-A4 and BS-l-B4, PNA, SBA and ECA do not react with the hemocytes. LTA, APA, WGA, Con A and LCA bind to all hemocytes. RCA and STA recognize surface carbohydrate moieties present on subpopulations of hemocytes only. The LS1+ hemocyte population virtually lacks the carbohydrate residues recognized by STA, whereas the LS1- population never shows binding of RCA. Our results support the findings that the LS1 epitope is a membrane marker of less differentiated hemocytes in both juvenile and adult L. stagnalis. Furthermore, they suggest a correlation between the presence of the LS1 epitope and the absence of STA binding, whereas absence of the LS1 marker may correlate with the presence of a sugar recognized by RCA.

The production of toxic oxygen metabolites by hemocytes of different snail species.

The phagocytic hemocytes of four snail species were investigated for their ability to generate reactive oxygen metabolites upon stimulation by foreign material. Hemocytes of the pond snail Lymnaea stagnalis and of the garden snail Helix aspersa showed a luminol-dependent chemiluminescence (CL) when they phagocytosed zymosan particles. This CL was inhibited by superoxide dismutase (SOD) and sodium azide, indicating the involvement of oxygen intermediates. Hemocytes of the planorbid snails Planorbarius corneus and Biomphalaria glabrata did not give a detectable CL response. This is probably due to the presence of hemoglobin in the hemolymph; after isolation of the cells and subsequent stimulation, however, still no CL could be measured. Hemocytes of all four snail species showed a SOD-sensitive nitroblue tetrazolium (NBT) reduction, indicating the generation of superoxide anions. Regarding the NBT reaction, no differences were observed between strains of B. glabrata that were susceptible or resistant to PR-1 Schistosoma mansoni; neither did exposure to the parasite have an effect on the ability of the hemocytes to reduce NBT. Also, hemocytes from Trichobilharzia ocellata-infected L. stagnalis did not react differently from hemocytes of uninfected snails. It is now clear that phagocytically stimulated hemocytes of several molluscan species can generate reactive forms of oxygen; the relevance of this fact for the phylogeny of killing systems operative in leukocytes is discussed.