creases in soluble immune factors, such as serum An imbalance between T helper cell (Th)1 and Th2-interleukin-2 (IL-2) receptor have provided peripheral evilike cytokines has been described in several chronic dence that significant intrahepatic anti-HCV immune proinfectious diseases. We therefore analyzed the intrahecesses may be occurring. 4 However, there is a paucity of patic messenger RNA (mRNA) expression of Th1-like (indata relating to the intrahepatic expression of cytokines that terleukin [IL]-2, interferon [IFN]-g) and Th2-like (IL-4, may be expected to mediate any immune response in the IL-10) cytokines in chronic hepatitis C patients (n Å 17) liver in chronic HCV infection. and controls (n Å 6) and correlated the results with liver Cytokines are regulatory molecules that play an important histology and intrahepatic viral load. Intrahepatic cytorole in many physiological and pathological processes. CD4/ kine mRNA and hepatitis C virus (HCV) RNA were quan-T cells, which are central to the induction of anti-viral retitatively assessed by polymerase chain reaction (PCR) sponses, have been subdivided according to two predominant using a dot-blot hybridization technique. Liver biopsy cytokine secretion profiles originally described in mouse Tspecimens were histologically graded using the Scheuer cell clones. 5 T helper (Th) 1 cells produce cytokines such as Score. IFN-g and IL-2 mRNA expression were signifi-IL-2 and interferon-g (IFN-g) which are important factors cantly upregulated in chronic HCV vs. controls (P õ .002, responsible for promoting the cell-mediated immune re-P õ .04, respectively). Both correlated significantly with sponse. In contrast Th2 cells produce cytokines such as IL-4 histological fibrosis and portal tract inflammation. In contrast, the expression of IL-10 mRNA was decreased and IL-10, which mediate the humoral response. However, in cirrhosis and chronic HCV compared with controls (P it is now recognized that these cytokines can be produced õ .02, P õ .0001, respectively). IL-4 mRNA was detected by cells other than CD4/ T cells and therefore it has been inconsistently at low levels in all groups. Intrahepatic suggested that these polarized cytokine responses be referred viral load did not correlate with either cytokine expres-to as Th1-like or type 1 and Th2-like or type 2 responses. 6 sion or tissue injury. In conclusion, the progressive liver Recently the relationship of Th1 versus Th2-like cytokines injury seen in chronic HCV is associated with the upreg-in chronic infections such as human immunodeficiency virus, ulation of intrahepatic Th1-like cytokines and the down-leprosy, and leishmaniasis has been explored.6-10 A decrease regulation of IL-10, a Th2-like cytokine. These results in cell-mediated immunity, i.e., a Th2-type profile, has been suggest a role for delayed-type hypersensitivity immune suggested to be associated with increased pathogen load and reactions in HCV related liver injury. (HEPATOLOGY progressive disease. It is unknown whether chronic HCV in-1996;24:759-765....

Summary Real-time quantitative reverse transcriptase-polymerase chain reaction (RT-PCR) is the method of choice for rapid and reproducible measurements of cytokine or growth factor expression in small samples. Fluorescence detection methods for monitoring real-time PCR include fluorogenic probes labelled with reporter and quencher dyes, such as Taqman probes or Molecular Beacons and the dsDNA-binding dye SYBR Green I. Fluorogenic (Taqman) probes for a range of human and rat cytokines and growth factors were tested for sensitivity and compared with an assay for SYBR Green I quantification using real-time fluorescence monitoring (PE Applied Biosystems Model 7700 sequence detector). SYBR Green I detection involved analysis of the melting temperature of the PCR product and measurement of fluorescence at the optimum temperature. Fluorogenic probes provided sensitive and reproducible detection of targets that ranged from low (<10 copies/reaction) to high (>10 7 copies/ reaction) expression. SYBR Green I gave reproducible quantification when the target gene was expressed at moderate to high levels (≥1000 copies/reaction), but did not give consistently reproducible quantification when the target gene was expressed at low levels. Although optimization of melting temperature improved the specificity of SYBR Green I detection, in our hands it did not equal the reproducible sensitivity and specificity of fluorogenic probes. The latter method is the first choice for measurement of low-level gene expression, although SYBR Green I is a simple and reproducible means to quantify genes that are expressed at moderate to high levels.

Liver transplants are not often rejected in patients weaned from immunosuppression and are spontaneously accepted in some animal models. We review past and recent findings of liver transplantation and propose a unified model in which several mechanisms act in concert to induce and maintain tolerance in both naïve and effector T cell compartments. First, passenger leukocytes migrate to lymphoid tissues and induce apoptosis of alloreactive naïve T cells. Second, antigen-specific activation and subsequent deletion of naïve and effector cells within the liver itself purge the repertoire of alloreactive T cells. Other mechanisms such as microchimerism and migration of donor dendritic cells to the thymus may play a predominant role in maintaining tolerance, and soluble major histocompatibility complex molecules, donor peptides, and regulatory T cells may participate in the induction and maintenance phases. Thus, the major challenge in liver transplantation will be to favor these tolerogenic processes while developing strategies that specifically inhibit alloreactive memory T cells.