Resistance to Fas-mediated apoptosis in human hepatoma cells.

CTLs- and lymphokine-induced apoptosis of infected hepatocytes during the course of chronic viral hepatitis is thought to be important for both disease termination and prevention of hepatocellular transformation. We therefore studied apoptosis induced by Fas (APO-1 or CD95)-a widely expressed cell surface receptor whose ligand is involved in lymphocyte cytotoxicity-in a set of human hepatoma cell lines. As normal hepatocytes, all of the human hepatoma cell lines tested do express detectable amounts of Fas on their surface. Nevertheless, only PLC/PRF/5 cells undergo apoptosis following treatment with anti-Fas. Systematic cloning and sequence analysis of the Fas cDNA did not show mutations in the Fas gene in any of the cells lines tested. However, due to alternative splicing, 5 to 10% of the Fas cDNAs are deleted of 63 internal nucleotides corresponding to the transmembrane domain, thus encoding for a soluble and secreted form of Fas (Fas delta TM), potentially able to neutralize anti-Fas or Fas-Ligand. Although we could not demonstrate a direct correlation between resistance of different hepatoma cell lines to Fas mediated death and endogenous expression of this transcript, we show that PLC/PRF 5 stable transfectants overexpressing Fas delta TM are less sensitive to anti-Fas than control cells. In three different cell lines, resistance to anti-Fas was overcome by treatment with the protein synthesis inhibitor cycloheximide. Although this could suggest the existence of short-lived repressors of the Fas-activated apoptotic signalling pathway(s), we show that translational inhibition is not required for the synergistic effect of cycloheximide to take place, and that resistant hepatoma cells can be sensitized to anti-Fas by subinhibitory concentrations of this protein synthesis inhibitor. Since cycloheximide is able to activate intracellular signalling independently on its effects on protein synthesis, we suggest that it might provide a costimulatory signal that cooperates with Fas in the induction of cell death and that, at least in the cells we tested, resistance to Fas is not an active process involving gene transcription and translation but only the consequence of an inadequate apoptotic stimulation.

Normalization of depressed natural killer activity after interferon-alpha therapy is associated with a low frequency of relapse in patients with chronic hepatitis C.

In the present study, we found that human recombinant interferon-alpha (rIFN-alpha) given at a dose of 3 x 10(6) units thrice weekly for three months, and 1.5 x 10(6) units thrice weekly for the next three months, was able to restore depressed natural-killer (NK) activity to normal values in 12 out of 21 chronic hepatitis C patients positive for anti-HCV antibodies. In all of these patients, NK normalization was still sustained after three months from suspension of therapy. Eighteen patients also showed a normalization of the alanine aminotransferase (ALT) level by the end of treatment (responder patients), independently of changes in NK activity. No significant improvement in either NK activity or aminotransferase levels was seen among 20 untreated patients. In 8 responder patients (1 with normalized and 7 with low NK activity), ALT levels returned to pre-therapy values within three months after suspension of rIFN-alpha administration (relapse). We found that patients who normalized NK activity had a lower frequency of relapse as compared to patients with low NK activity by the end of treatment (p > 0.01). Immunofluorescence analysis of biopsy-derived liver tissue revealed that rIFN-alpha was able to induce strong MHC class I antigen expression on hepatocytes of treated patients, but this was not related to the clinical course.

Prevalence of chronic fatigue syndrome in Italian patients with persistent fatigue.

Our study was carried out to determine the prevalence of chronic fatigue syndrome (CFS) within a selected population of patients suffering from persistent fatigue. We studied subjects with recurrent or persistent fatigue lasting 6 months and fulfilling at least four minor Center for Disease Control (CDC) criteria for the diagnosis of CFS. Evaluation included both clinical examination and laboratory testing. All subjects filled out a questionnaire specifically designed to gain information about the length and severity of symptoms, and patients with a previously diagnosed illness associated with fatigue were excluded. The study was carried out at the Fatigue Clinic of an internal medicine unit (Clinica Medica I) of the University of Rome "La Sapienza". Sixty-three subjects, residents of the Lazio region (central Italy), completed the diagnostic assessment. Alternative diagnoses were established in 37 (59%) of the 63 patients. A diagnosis of CFS based on the CDC criteria was established in only 6 cases. In 2 subjects, CFS had appeared following infectious mononucleosis, and no definitive diagnosis could be formulated for 18 patients. In Italy, CFS seems to be an infrequent cause of severe and persistent fatigue in a selected population. Numerous morbid conditions may be responsible for a clinical picture closely resembling CFS. We recommend that patients suffering from fatigue be thoroughly evaluated.

Human hepatoma cells expressing HLA class I molecules stimulate primary responses of purified CD8+ T lymphocytes.

In the present study, we investigated the ability of major histocompatibility complex (MHC) class-I+ human hepatoma cell lines to induce primary proliferative responses of purified allogeneic CD8+ T lymphocytes. We found that HA22T/VGH and Li7A, but not HepG2 cells induced significant proliferation of CD8+ T cells and that these responses were dependent on class I molecule expression. In blocking experiments carried out to identify the costimulatory signals involved, we found that anti-ICAM1 monoclonal antibodies drastically inhibited CD8+ T-cell proliferative responses. These findings suggest that transformed hepatocytes expressing HLA class I molecules may participate in anti-tumour immunosurveillance by the direct induction of cytotoxic T-cell responses through ICAM1-mediated adhesive interaction.

Interleukin-6 production by human hepatoma lines is related to a low degree of cell differentiation.

In this study, we showed by Northern blot analysis and bioassays that scarcely differentiated human hepatoma cell line HA22T/VGH constitutively produces interleukin-6 (IL6). This cytokine was produced neither by moderately differentiated Li7A nor by well-differentiated HepG2 human hepatoma cell lines. The finding that transcripts for IL6 were not present in 2.2.15 cells, a HepG2-derived clone transfected with the intracellular replicative form of hepatitis B virus (HBV) DNA, suggests that production of this cytokine is not affected by HBV-encoded gene products, but is more likely related to the dedifferentiated state of hepatoma cells.

Flow cytometric evaluation of nitro blue tetrazolium (NBT) reduction in human polymorphonuclear leukocytes.

Oxidative metabolic burst of activated human polymorphonuclear leukocytes (PMN) is most commonly investigated in clinical practice by evaluating nitroblue tetrazolium (NBT) reduction at the single cell level. Reduced NBT precipitates where the redox reaction has taken place and can be visualized as PMN-associated dark blue granules of formazan in light microscopy. Although widely used and not technically demanding, this method remains subjective and labor intensive, especially when large numbers of samples need to be investigated. We developed a new flow cytometry technique in which PMN membrane was rendered fluorescent by a short incubation with fluorescein-conjugated Concanavalin A. PMN were then incubated with NBT and increasing doses of a suitable stimulus, such as phorbol myristate acetate (PMA). Formazan has a distinct peak of absorption at 520 nm that represents the peak of emission of fluorescein. As a consequence, formazan quenches the PMN-associated fluorescence. Data show that a dose-dependent reduction of fluorescence can be obtained using graded amounts of PMA in normal PMN cultures. PMN-associated fluorescence remains unchanged in control patients with chronic granulomatous (CGD) disease, a disorder characterized by a selective impairment of PMN oxidative metabolism. Electronic cell size increases upon PMA incubation in normal PMN, irrespective of the presence of NBT. Conversely, forward light scatter intensity decreases in the presence, but not in the absence, of NBT indicating that the phenomenon is due to the capacity of formazan to absorb/scatter the incident light. The present method for easily detecting NBT reducing activity at single cell level by flow cytometry makes use of commonly available, inexpensive reagents and standard instrumentation. It could become a useful test for clinical purposes.

Antigen targeting to antigen-presenting cells enhances presentation to class II-restricted T lymphocytes.

Receptor-mediated uptake increases by several orders of magnitude the efficiency of APC to internalize Ag, and is stringently required for the Ag-presenting function of T lymphocytes due to their inability to take up Ag non-specifically. We have previously reported that hepatitis B envelope antigen (HBenvAg) can be internalized by T cells via transferrin receptor (TfR). To evaluate if Ag targeting to receptors expressed on APC could be an effective tool for promoting Ag uptake and presentation, we tested the capacity of activated T cells not expressing TfR to induce HBenvAg-specific T-cell responses when pulsed with a hybrid particle containing HBenvAg coupled to gp120 of human immunodeficiency virus (HIV), exploiting the ability of gp120 to bind to CD4 receptor. We found that CD4+/TfR- T cells pulsed either with the hybrid particle or peptide (S193-207) but not with S, L Ag, a recombinant form of HBenvAg, induced a specific proliferative response of a T-cell clone recognizing peptide (S193-207) of HBenvAg. The finding that the addition of anti-CD4 monoclonal antibody (mAb) before the pulsing of CD4+/TfR- T cells with the hybrid particle drastically blocked the specific T-cell response, together with the finding that CD8+/TfR- T cells were unable to serve as APC even if pulsed with this molecule, demonstrated that CD4 receptor was crucial for the HBenvAg internalization. On the other hand, HBenvAg presentation by CD4+/TfR+ T cells pulsed with the hybrid particle was inhibited only when both anti-CD4 and anti-TfR were added before the pulsing. These results suggest that Ag targeting to APC receptors may be usefully exploited to improve Ag-presentation efficiency in potential immunotherapeutic approaches.

Human hepatoma cells expressing MHC antigens display accessory cell function: dependence on LFA-1/ICAM-1 interaction.

Malignant transformation of human hepatocytes is often accompanied by an increased expression of major histocompatibility complex (MHC) molecules, but whether this phenomenon is related to an enhanced immunogenicity remains unknown. In this study, we tested the capacity of a series of human hepatoma cell lines to induce proliferation of allogeneic T cells in primary mixed lymphocyte tumour cultures (MLTC). These cell lines were positive for class I molecules, whereas class II molecule expression was either constitutive or inducible by treatment with interferon-gamma (IFN-gamma). We found that HA22T/VGH cells expressing class II molecules constitutively stimulated high proliferative responses of purified CD4+ T lymphocytes, whereas class II-negative Li7A cells stimulated CD4+ T-cell responses only when induced by treatment with IFN-gamma. HA22T/VGH and Li7A cells also exerted a significant stimulatory activity for purified CD8+ T cells whereas HepG2 cells, in which MHC class II molecules are neither constitutive IFN-gamma-inducible, were unable to induce CD4+ and CD8+ T-cell proliferative responses. Phenotypical analysis revealed that HA22T/VGH and Li7A expressed high levels of intracellular adhesion molecule-1 (ICAM-1) and experiments with blocking monoclonal antibodies (mAb) demonstrated that this molecule played a key role in mediating the co-stimulatory function of hepatoma cells. In addition, HA22T/VGH cells were found to produce mRNA for interleukin-1 (IL-1) beta and IL-6, while Li7a only produced IL-1 beta, yet both these cytokines were found to play a small part, if any, in T-cell co-activation. On the whole, these results show tht hepatoma cells expression MHC antigens and ICAM-1 are able to deliver signals necessary for activation of resting CD4+ and CD8+ T cells and suggest that they may actively participate in the anti-tumour immune response.

Allergy and asthma: distinguishing the causes from the triggers. An eye model for the study of inflammatory events following allergen challenge.

The study of the late-phase reaction to allergen challenge has greatly contributed to increasing understanding of the relationship existing between immunoglobulin E (IgE) triggering, allergic inflammation and bronchial hyperreactivity, as well as to changing our concept from "causes" to "triggers" of asthma. This paper reviews personal studies on a model of conjunctival provocation in allergic subjects. These studies represent the first evidence of a late-phase reaction in the human eye and contribute to a better knowledge of the network of cells and mediators involved in allergic inflammation which are responsible for heightened bronchial reactivity in allergic asthma.

Soluble transferrin mediates targeting of hepatitis B envelope antigen to transferrin receptor and its presentation by activated T cells.

In a previous study, we identified that transferrin receptor (TfR) is the receptor utilized by hepatitis B virus (HBV) to enter T cells. We demonstrated that hepatitis B envelope antigen (HBenvAg) is taken up by activated T cells via TfR, processed in endosomal compartments, and presented on class II molecules to specific CD4+ T cell clones. Herein, we report that binding to soluble ferric Tf by HBenvAg is needed in TfR-mediated endocytosis. Accordingly, presentation of HBenvAg by activated T cells is not observed in serum-free medium and is restored by addition of soluble Tf. Moreover, we provide evidence that pre-S2 and S regions of HBenvAg contain the critical residues for the interaction with soluble Tf. Our data not only explain HBV entry into a variety of host activated cells, but may also help in developing strategies to alter the course of chronic HBV infection.

Serum levels of eosinophil cationic protein in allergic diseases and natural allergen exposure.

BACKGROUND: Eosinophil cationic protein (ECP) is a cytotoxic performed mediator stored in eosinophil granules and released under various in vitro and in vivo conditions. OBJECTIVE: This study was carried out to evaluate the clinical value of ECP as a marker of allergic inflammation. METHODS: ECP was measured by a competitive radioimmunoassay in serum samples from 265 patients and 45 matched control subjects and related to the type of allergic disease (asthma, rhinitis, conjunctivitis) and to the type of allergic sensitization. RESULTS: All the patient groups studied showed significantly higher levels of serum ECP than control groups (p < 0.001). The type of sensitization was shown to be the only variable influencing ECP serum levels. In fact, subjects sensitized to perennial allergens had significantly higher ECP values than subjects with seasonal allergy (p < 0.001), whereas in patients with seasonal allergy ECP levels were significantly increased only during the pollen season. Differences in ECP values between various allergic diseases or age groups were only due to a nonhomogeneous distribution of the type of sensitization or to time of sera collection. CONCLUSIONS: Results obtained indicate that persistent natural exposure to a sensitizing allergen is responsible for a measurable increase in serum ECP levels in patients with allergy.

Selective expansion of cytotoxic T lymphocytes with a CD4+CD56+ surface phenotype and a T helper type 1 profile of cytokine secretion in the liver of patients chronically infected with Hepatitis B virus.

Highly purified CD4+ T cells isolated from liver biopsies of patients with hepatitis B virus-induced CAH had a strong cytotoxic activity and were comprised of a substantial number of cells (25%-40%) expressing CD56 surface marker. These cells were absent in CD4+ T cells from the peripheral blood of CAH patients or normal controls and these suspensions did not have cytotoxic activity. CD4+CD56+ T cells were further characterized by studies at the clonal level. A total of 71 hepatitis B envelope antigen-specific CD4+ T cell clones was investigated (23 from liver biopsies, 48 from peripheral blood of patients or normal vaccinated individuals). A total of 16 out of 23 (69.5%) of the clones from liver biopsies, but only 4.1% (2 out of 48) of those from PBLs, expressed CD56. A clone was defined as CD56+ when 40% or more of the cells expressed the marker. Production of TNF-alpha, IL-4, IL-5, IL-2, and IFN-gamma was investigated in 15 CD4+CD56+ and in 18 CD4+CD56- T cell clones, which shared the same HLA restriction element (DR2w15) and the same fine specificity (peptide 193-207 of the S region). All of the clones from the two groups released TNF-alpha and IL-2. However, all of the CD4+CD56+ T cell clones produced IFN-gamma but not IL-4 and IL-5 (Th1-like cell clones). Fourteen of the CD4+CD56- clones released IFN-gamma, IL-4, and IL-5 (Th0-like cell clones); three produced IL-4 and IL-5 but not IFN-gamma (Th2-like cell clones); and only one had a Th1 cytokine secretion profile. Cell fractionating studies within single CD4+CD56+ T cell clones showed that cells expressing high density CD56 had a stronger cytotoxic activity and produced higher levels of IFN-gamma than cells with low density CD56, thus further supporting a correlation between CD56 expression and cell functions. The results indicate that: 1) in CAH patients, cytotoxic CD4+ T cells with a Th1 cytokine secretion profile are compartmentalized in the liver, 2) these cells may be identified by the expression of CD56, 3) the expansion of these cells may be facilitated by antigenic stimulation within the inflammatory environment of the liver, and 4) CD4+CD56+ cells may play a pathogenetic role in hepatitis B virus infection.

Presentation of peptides by cultured monocytes or activated T cells allows specific priming of human cytotoxic T lymphocytes in vitro.

The conditions favouring effective specific cytotoxic T lymphocyte (CTL) priming have been exploited to set up a simple and reproducible method to induce a primary CTL response in vitro. We report that cultured monocytes, as well as activated T cells, pulsed with exogenous HLA-A2 binding immunogenic peptides, can induce primary peptide-specific CTL responses in vitro in a Th-independent manner. Primary viral peptide-induced CTL were HLA-A2 restricted, and recognized both peptide-pulsed target cells and targets infected with recombinant vaccinia virus expressing viral endogenous antigens. In addition, both cultured monocytes and activated T cells primed peptide-specific CD8+ T cells depleted from the CD45RO+ memory cell fraction. The efficiency of CTL priming by monocytes was dependent upon the strong up-regulation of class I, adhesion and co-stimulatory molecules occurring spontaneously upon in vitro culture. The inability of unseparated peripheral blood mononuclear cells to mount a peptide-specific CTL response could be reverted by direct co-stimulation of responding CD8+ T cells by soluble B7.1 or a stimulatory anti-CD28 antibody, that allowed a specific response to take place. Although co-stimulation via the B7-CD28 interaction appeared sufficient to trigger CTL responses, it was not essential for CTL priming, since neither anti-B7.1 mAb nor soluble CTLA-4 inhibited induction of primary CTL response. This new method for induction of specific CD8+ T cell response in vitro may be exploited in adoptive immunotherapy in cancer or in HIV-infected patients.