Tacrolimus treatment of plasmacytoid dendritic cells inhibits dinucleotide (CpG-)-induced tumour necrosis factor-alpha secretion.

Tacrolimus is a widely used immunosuppressive agent. Although T cells are the main targets of these pharmacological drugs, antigen presentation may also be affected. Among antigen-presenting cells, plasmacytoid dendritic cells (PDCs) are the main source of type I interferons upon microbial challenge, and are involved in several diseases and autoimmune disorders. The aim of this study was to evaluate whether tacrolimus can modulate the function of PDCs in vitro. Maturation and function of PDCs was determined using flow cytometry, enzyme-linked immunosorbent assay and cytometry bead arrays. The effect of tacrolimus on PDCs was observed mainly when the cells were pretreated with the immunosuppressive agent before activation. Upon dinucleotide-oligodeoxynucleotide (CpG-ODN) activation, tacrolimus pretreated PDCs showed a significant reduction in the surface expression of co-stimulatory molecules and human leucocyte antigen D-related (HLA-DR) and secreted reduced levels of tumour necrosis factor (TNF)-alpha. These results show that tacrolimus treatment of PDCs impairs CpG-induced activation, which could affect the outcome of the immune response.

Effect of bottlenecking on evolution of the nonstructural protein 3 gene of hepatitis C virus during sexually transmitted acute resolving infection.

Sexual partners of patients infected with the hepatitis C virus (HCV) often have detectable HCV-specific T-cell responses in the absence of seroconversion, suggesting unapparent, spontaneously resolving infection. To determine whether differences in the evolutionary potential of bottlenecked inoculum may explain the low rate of HCV persistence after sexual exposure, we have investigated changes in the entire HCV nonstructural 3 (NS3) gene over time in a chronic carrier and compared his viral quasispecies with that of the acute-phase isolate of his sexual partner, who developed acute resolving hepatitis C. The overall rate of accumulation of mutations, estimated by regression analysis of six consecutive consensus NS3 sequences over 8 years, was 1.5 x 10(-3) mutations per site per year, with small intersample fluctuations related to changes in environmental conditions. Comparison of quasispecies parameters in one isolate of the chronic carrier with those of the acute-phase isolate of the infected partner revealed a higher heterogeneity and lower proportion of nonsynonymous mutations in the former. All NS3 sequences from the acute-phase isolate clustered with a single sequence from the chronic isolate, despite complete HLA mismatch between the patients, suggesting bottlenecking during transmission. The low risk of viral persistence after sexual exposure to HCV may be related to the selection of a limited number of viral particles carrying a particular combination of mutations which may further limit the potential of a relatively homogeneous quasispecies to rapidly diversify and overcome the immune response of the exposed host.

Improved attachment of natural HCV isolate to Daudi cells upon elimination of immune complexes and close pH control.

OBJECTIVES: To study the viral requirements for attachment, entry and infection using natural isolates of HCV (infectious plasma of a patient infected with HCV from genotype 1b) and a cell line which has previously been shown to support HCV replication (Daudi cells). METHODS: We studied attachment of HCV to Daudi cells, a human B-cell line. Quantification was done by real-time RT-PCR. RESULTS: The best attachment levels were obtained using plasma depleted of immunocomplexes. Results of kinetics of HCV attachment to Daudi cells show that attachment is maximum at pH 7.0, and that it decreased drastically at any other pH studied (5.5, 6.0, 6.5, 7.5 and 8.0). CONCLUSIONS: Depletion of immunocomplexes and pH control during infection are important parameters to improve attachment of HCV to Daudi cells using plasma as a natural source of virus.

FK506 in the maturation of dendritic cells.

BACKGROUND AND OBJECTIVES: FK506 (tacrolimus) is a potent immunosuppressive agent that inhibits interleukin-2 (IL-2) and interferon-g production by CD4+ cells. The effect of this agent on dendritic cells (DCs), the highly professional antigen-presenting cells for T-cells, has not been completely defined. We investigated the effect of FK506 on DC differentiation from monocytes, and on the shift from immature to mature immunophenotypes. DESIGN AND METHODS: DCs were generated in vitro from monocytes of healthy donors. Cells were exposed to lipopolysaccharide (LPS) and two doses of FK506, with variations in time of exposure and sequence of FK506 and LPS addition. Immunophenotype analysis in immature and mature DCs under FK506 treatment was performed by flow cytometry at the end of cell culture. The Student's t-test was used for statistical analyses. RESULTS: FK506 did not affect dendritic cell generation or viability. There were no changes in cell surface markers with addition of FK506 at physiologic concentrations (10 ng/mL). We found a decrease in CD1a median fluorescence intensity (MFI) and an increase in percentage of CD86-positive cells with lengthy exposure (6 days) to FK506 at 5000 ng/mL. In the sequential study, 5000 ng/mL FK506 before LPS addition resulted in a significant decrease in CD1a MFI and in the percentage of cells co-expressing CD83 and CD86. INTERPRETATION AND CONCLUSIONS: Our results indicate that lengthy exposure to 5000 ng/mL FK506 modified the expression of some DC-cell surface markers, maintaining DCs in a low maturity stage.