Stany Depraetere

Hepatitis C Virus Targets DC-SIGN and L-SIGN To Escape Lysosomal Degradation

ABSTRACT Hepatitis C virus (HCV) is a major health problem. However, the mechanism of hepatocyte infection is largely unknown. We demonstrate that the dendritic cell (DC)-specific C-type lectin DC-SIGN and its liver-expressed homologue L-SIGN/DC-SIGNR are important receptors for HCV envelope glycoproteins E1 and E2. Mutagenesis analyses demonstrated that both HCV E1 and E2 bind the same binding site on DC-SIGN as the pathogens human immunodeficiency virus type 1 (HIV-1) and mycobacteria, which is distinct from the cellular ligand ICAM-3. HCV virus-like particles are efficiently captured and internalized by DCs through binding of DC-SIGN. Antibodies against DC-SIGN specifically block HCV capture by both immature and mature DCs, demonstrating that DC-SIGN is the major receptor on DCs. Interestingly, internalized HCV virus-like particles were targeted to nonlysosomal compartments within immature DCs, where they are protected from lysosomal degradation in a manner similar to that demonstrated for HIV-1. Lewis X antigen, another ligand of DC-SIGN, was internalized to lysosomes, demonstrating that the internalization pathway of DC-SIGN-captured ligands may depend on the structure of the ligand. Our results suggest that HCV may target DC-SIGN to “hide” within DCs and facilitate viral dissemination. L-SIGN, expressed by THP-1 cells, internalized HCV particles into similar nonlysosomal compartments, suggesting that L-SIGN on liver sinusoidal endothelial cells may capture HCV from blood and transmit it to hepatocytes, the primary target for HCV. We therefore conclude that both DCs and liver sinusoidal endothelial cells may act as reservoirs for HCV and that the C-type lectins DC-SIGN and L-SIGN, as important HCV receptors, may represent a molecular target for clinical intervention in HCV infection.

Enhanced soluble interleukin‐5 receptor alpha expression in nasal polyposis

Background: Alternative splicing of the interleukin‐5 receptor alpha (IL‐5Rα)‐subunit leads to the generation of a signalling, membrane‐anchored (TM) isoform, or a secreted [soluble (SOL)], antagonistic variant. Given the key role of IL‐5 in eosinophil function, we investigated SOL IL‐5Rα expression pattern in an eosinophil‐associated disease such as nasal polyposis (NP).

Mouse strain and conditioning regimen determine survival and function of human leucocytes in immunodeficient mice

The innate immune system of severe combined immunodeficient (SCID) mice represents an important barrier to the successful engraftment of human cells. Different genetic and pharmacological strategies improve the graft survival. Non‐obese diabetic (NOD)‐SCID mice are better hosts for reconstitution with human peripheral blood leucocytes (Hu‐PBL) because of their reduced natural killer cell and macrophage activity next to defective T and B cell functions. We investigated effects of TM‐β1, a rat monoclonal antibody recognizing the mouse IL‐2 receptor β‐chain, on Hu‐PBL survival and function in NOD‐SCID and SCID mice. Relative to untreated littermates, TM‐β1 improved Hu‐PBL survival in SCID and NOD‐SCID mice. Moreover, TM‐β1‐pretreated NOD‐SCID mice displayed significantly better Hu‐PBL survival and tissue distribution than TM‐β1‐pretreated SCID mice. Irradiation of NOD‐SCID mice further enhanced the effects of TM‐β1. However, these animals died within 3 weeks post‐grafting due to graft‐versus‐host disease. Secondary immune responses were evaluated with Hu‐PBL from a donor immune to hepatitis B surface antigen (HBsAg). In TM‐β1‐pretreated NOD‐SCID mice, human HBsAg‐specific memory B cells produced high titres of anti‐HBsAg immunoglobulin irrespective of the administration of a secondary antigen booster dose. This contrasts with secondary immune responses in TM‐β1‐pretreated SCID mice where high titred antigen‐specific immunoglobulins were produced when the appropriate antigen booster was given. In conclusion, reducing the function of the innate immune system in immunodeficient mice improves survival of the human graft and can result in an activation of the memory B cells without the need for recall antigen exposure.

Human B Cell Growth and Differentiation in the Spleen of Immunodeficient Mice

Human mAbs (HumAbs) have therapeutic potential against infectious diseases and cancer. Heretofore, their production has been hampered by ethical constraints preventing the isolation of Ag-specific activated B cells by in vivo immunization. Alternatively, severe combined immune deficient (SCID) mice, transplanted i.p. with human (Hu)-PBLs, allow the in vivo stimulation of human Ab responses without the usual constraints. Unfortunately, human B cells only represent a minor fraction of the surviving graft, they are scattered all over the animal body, and thus are hard to isolate for subsequent immortalization procedures. To prevent this dispersion and to provide the human B cells with a niche for expansion and maturation, SCID mice were engrafted with Hu-PBL directly into the spleen. Simultaneously endogenous murine NK cell activity was depleted by treatment with an anti-mouse IL-2 receptor β-chain Ab. During engraftment, human B lymphocytes became activated, divided intensely, and differentiated into plasmacytoid cells. In vivo exposure to a recall Ag after cell transfer induced expansion of Ag-specific B cell clones. One week after inoculation, human B cells were abundant in the spleen and could easily be recovered for fusion with a heteromyeloma line. This resulted in the formation of stable hybridoma cell lines that secreted Ag-specific HumAbs. Thus transplantation of human lymphoid cells in the spleens of immune deficient mice represents a model for the study of human T cell-dependent B cell activation and proves to be an excellent tool for the successful production of HumAbs.

Murine IL-2 receptor beta chain blockade improves human leukocyte engraftment in SCID mice

Severe combined immunodeficient (SCID) mice accept human xenografts and can act as a model for human immune functions. Murine natural killer cells (NK), however, represent an important barrier for the reconstitution of SCID mice with human peripheral blood leukocytes (Hu‐PBL). We investigated the effect on Hu‐PBL survival of pretreatment with TM‐β1, a rat monoclonal antibody for the mouse IL‐2 receptor beta chain. TM‐β1 greatly improved the survival of Hu‐PBL. Human lymphocytes, predominantly T cells, survived in the peritoneum and infiltrated spleen and lungs already 1 week after engraftment and liver and thymus from 2 weeks on. Secondary humoral responses were evaluated with Hu‐PBL from a donor immune to hepatitis‐B surface Ag (HBsAg) and tetanus toxoid (TT). TM‐β1 pretreatment enhanced the recall Ig response to HBsAg and did not affect the baseline anti‐TT Ig production. In conclusion, TM‐β1 pretreatment of SCID mice significantly improves the survival and functionality of the Hu‐PBL graft.

Long term response to interferon treatment in chronic hepatitis C patients is associated with a significant reduction in anti-E1 envelope antibody titers.

Interferon (IFN) alfa has been used widely for the treatment of chronic hepatitis C virus (HCV) infections but only a small number of patients treated have shown a sustained biochemical and virological response. Anti‐envelope E1 and E2 antibody titers were assessed retrospectively before, during, and after treatment with IFN in order to evaluate their usefulness for the prediction and monitoring of therapy outcome in 115 patients infected chronically with HCV genotype 1b. At baseline, E2 induced more frequent and stronger immunogenic responses than E1, irrespective of patient response to therapy. E1 and E2 antibodies also tended to be higher in patients with a long‐term or a transient response to IFN treatment than in patients who were absolute non‐responders. In most patients, E1 and E2 antibody levels tended to be lower after treatment. This reduction was most pronounced and occurred most frequently in long‐term responders to therapy. In this patient group, the reduction of E1 antibodies was more pronounced than that of E2 antibodies. In contrast to E2 antibodies, the decrease of E1 antibodies could already be observed at the end of therapy (week 24) and was significantly larger (p<0.05) than that observed in relapsers and non‐responders. Thus, a sustained elevation of E1 antibodies seems to be associated with ongoing infection even when HCV RNA levels were undetectable in serum. Monitoring of E1 antibody titers may represent a useful additional marker to discriminate sustained responders from those who relapse in patients receiving interferon therapy. J. Med. Virol. 60:126–132, 2000.