Tumoricidal activity of a novel anti-human DR5 monoclonal antibody without hepatocyte cytotoxicity.

A novel anti-human DR5 monoclonal antibody, TRA-8, induces apoptosis of most tumor necrosis factor-related apoptosis-inducing ligand (TRAIL)-sensitive tumor cells both in vitro and in vivo. In contrast to both the membrane-bound form of human TRAIL, which induced severe hepatitis in mice, and the soluble form of human TRAIL, which induced apoptosis of normal human hepatocytes in vitro, TRA-8 did not induce significant cell death of normal human hepatocytes. However, both primary hepatocellular carcinoma cells and an established liver cancer cell line were highly susceptible to the killing mediated by TRA-8. We show here that elevated levels of cell-surface expression of DR5 and increased susceptibility to DR5-mediated apoptosis are characteristics of malignant tumor cells. In contrast, DR5 alone is not sufficient to trigger apoptosis of normal hepatocytes. Therefore, selective, specific targeting of DR5 with an agonistic antibody might be a safe and effective strategy for cancer therapy.

Prevention of age-related T cell apoptosis defect in CD2-fas-transgenic mice.

T cell dysfunction and thymic involution are major immunologic abnormalities associated with aging. Fas (CD95) is a bifunctional molecule that is critical for apoptosis and stimulation during T cell development, but the role of Fas during aging has not been determined. Fas expression and function on T cells from old (22-26-mo-old) mice was compared with young (2-mo-old) mice and old CD2-fas-transgenic mice. Fas expression and ligand-induced apoptosis were decreased on T cells from old mice compared with young mice. This correlated with an age-related increase in CD44+Fas- T cells. There was a marked decrease in the proliferation of T cells from old mice after anti-CD3 stimulation compared with young mice. Anti-CD3-stimulated T cells from young mice exhibited increased production of interleukin (IL)-2 and decreased production of interferon-gamma and IL-10 compared with old mice. There was an age-related decrease in the total thymocyte count from 127 +/- 10 cells in young mice compared with 26 +/- 8 x 10(6) in old mice. In 26-mo-old CD2-fas-transgenic mice, Fas and CD44 expression, Fas-induced apoptosis, T cell proliferation, and cytokine production were comparable to that of the young mice. These results suggest that T cell senescence with age is associated with defective apoptosis, and that the CD2-fas transgene allows maintenance of Fas apoptosis function and T cell function in aged mice comparable to that of young mice.

Autoimmune disease in mice due to integration of an endogenous retrovirus in an apoptosis gene.

The MRL-lpr/lpr mouse strain is a model of systemic autoimmune disease. In this model, intrinsic defects of intrathymic T cell development include defective deletion of self-reactive T cells and expression of endogenous retroviruses. Defective deletion of self-reactive T cells in the thymus has been proposed to be due to germline mutation in the Fas apoptosis gene. Using different fragments of a Fas cDNA probe, we determined that the lpr/lpr mutation is a 5.3-kb insertion of DNA within the second intron of the Fas gene. cDNA corresponding to this region was then derived from thymic RNA from MRL-lpr/lpr and MRL- +/+ mice using the polymerase chain reaction. All thymic RNA samples from MRL-lpr/lpr mice yielded a unique product that was 168 bp larger than that of MRL- +/+ mice. Complete sequence analysis indicated that this inserted sequence had 98% homology with a sequence from the 3' long terminal repeat of the early transposon (ETn). RNA analysis indicated higher expression of ETn RNA in the thymus of MRL-lpr/lpr than MRL- +/+ mice. The interdependence of ETn expression and abnormal Fas expression was then analyzed in a CD2-Fas transgenic mouse model in which a full-length murine Fas cDNA under the regulation of the CD2 promoter and enhancer was used to correct defective Fas expression in T cells of MRL-lpr/lpr mice. In these mice, reduced thymic ETn expression was observed, confirming that high ETn expression is related to abnormal Fas expression. These results establish a link between endogenous retrovirus expression, abnormal Fas expression, and autoimmune disease.

Transgenic rearranged T cell receptor gene inhibits lymphadenopathy and accumulation of CD4-CD8-B220+ T cells in lpr/lpr mice.

The lpr gene in homozygous form induces development of CD4-CD8-B220+ T cells and lymphadenopathy in MRL and C57BL/6 mice. Although the propensity for excessive production of T cells is related to an intrinsic T cell defect, a thymus is also required because neonatal thymectomy eliminates lymphadenopathy. Recent evidence suggests that excessive production and release of autoreactive T cells from the thymus of lpr/lpr mice might lead to downregulation of CD4 and CD8 as a "fail safe" tolerance mechanism that occurs during late thymic or post-thymic development. To test this hypothesis, T cell receptor (TCR) transgenic mice that produce large numbers of immature thymocytes recognizing the H-2Db and male H-Y antigens were backcrossed with C57BL/6-lpr/lpr mice and MRL-lpr/lpr mice. It was predicted that Db male lpr/lpr mice would produce large numbers of autoreactive T cells during early thymic development that would lead to an accelerated lymphoproliferative disease. In contrast, Db female lpr/lpr mice would produce large numbers of Db H-Y-reactive T cells, but might not develop lymphadenopathy because the male H-Y antigen would not be present. Unexpectedly, there was complete elimination of lymphadenopathy in both male and female TCR transgenic lpr/lpr mice. The elimination of lymphadenopathy was not due to a failure of thymic maturation since the thymus of H-2Db female lpr/lpr mice contained nearly normal numbers of mature thymocytes. Elimination of lymphadenopathy was also not due to a lack of autoreactive T cells in the peripheral lymph nodes (LN) since there was an increased syngeneic mixed lymphocyte proliferative response of LNT cells from transgenic lpr/lpr compared with +/+ mice in vitro. Hypergammaglobulinemia and autoantibody production in the transgenic lpr/lpr was present at levels comparable with or higher than control nontransgenic lpr/lpr mice, suggesting a dissociation of autoantibody production from the lymphoproliferative disease in the TCR transgenic mice. Conversely, the development of lymphadenopathy and production of CD4-CD8-B220+ T cells appear to be intimately linked, as both were completely eliminated in T cells expressing the transgenic TCR. We propose that lymphoproliferation and production of CD4-CD8-6B2+ T cells in lpr/lpr mice is related to decreased expression of the TCR, and providing the T cells with a rearranged TCR transgene overcomes this defect.

Defective maintenance of T cell tolerance to a superantigen in MRL-lpr/lpr mice.

In normal mice neonatal injection of staphylococcal enterotoxin B (SEB) induces tolerance in T cells that express reactive T cell receptor (TCR) V beta regions. To determine if a T cell neonatal defect was present in MRL-lpr/lpr mice, 20 micrograms of SEB was injected intraperitoneally every other day into V beta 8.2 TCR transgenic and nontransgenic MRL(-)+/+ and MRL-lpr/lpr mice from birth to 2 wk of age. At 2 wk of age, V beta 8+ T cells were depleted, and SEB reactivity was lost, in spleen, lymph node, and thymus. These effects were equivalent in +/+ and lpr/lpr SEB-tolerized mice. However, MRL-lpr/lpr mice failed to maintain neonatal tolerance. By 4 wk of age, there was a dramatic increase in T cells expressing V beta 8.2 in the peripheral lymph nodes of MRL-lpr/lpr mice but not MRL(-)+/+ mice. In vitro stimulation with SEB or TCR crosslinking revealed a total loss of neonatal tolerance 2 wk after cessation of SEB treatment in lpr/lpr mice, but not +/+ mice. The time-course of recovery of V beta 8+ T cells and reactivity to SEB and TCR crosslinking in the thymus of MRL-lpr/lpr mice was similar to that in the lymph node. Thymectomy at 2 wk of age eliminated tolerance loss in lymph nodes of MRL-lpr/lpr mice at 4 wk of age, indicating that loss of peripheral tolerance was due to the emigration of untolerized T cells from the thymus. Challenge of neonatally tolerized MRL-lpr/lpr mice with SEB (100 micrograms, i.p.) at 8 wk of age resulted in a dramatic onset of T cell-mediated autoimmune disease characterized by 30% weight loss and 60% morality. This indicated that loss of tolerance to SEB also occurred in vivo. In contrast, neonatally tolerized MRL(-)+/+ mice remained totally unresponsive to SEB challenge and did not undergo any detectable weight loss. These results suggest that there is normal induction of neonatal tolerance to SEB in lpr/lpr mice, but that tolerance is not maintained after the tolerizing antigen is removed. This loss of neonatal tolerance can lead to severe weight loss and death on exposure to the tolerizing antigen later in life.

Oncogene expression in autoimmune and normal peripheral blood mononuclear cells.

PBMC from patients with autoimmune diseases and from normal controls were studied for the expression of several cellular oncogenes. Gene expression was assessed by Northern blot analysis of poly(A)+ RNA obtained from leukapheresis samples. Patients with SLE expressed significantly more c-myc protooncogene RNA than did normal controls. Increased expression of the N-ras protooncogene was found in that subset of patients whose autoimmune disease was very active. Cells from individuals with SLE, but not from those with other autoimmune illnesses, showed significantly decreased levels of the c-myb and c-fos protooncogenes. To examine the implications of these findings, B and T cells were purified from apheresis samples donated by normal volunteers. When mitogen was used to activate the B cells in vitro, their pattern of protooncogene expression changed to resemble that found in freshly isolated cells from lupus patients. These results suggest that the differences detected in the expression of protooncogenes by patients with SLE may be due to the abnormal activation of their B cells in vivo. The pattern of protooncogene expression found in patients with other autoimmune illnesses is consistent with the activation of additional cell types in those diseases.

Inhibition of Nur77/Nurr1 leads to inefficient clonal deletion of self-reactive T cells.

The Nur77/Nurr1 family of DNA binding proteins has been reported to be required for the signal transduction of CD3/T cell receptor (TCR)-mediated apoptosis in T cell hybridomas. To determine the role of this family of DNA-binding proteins in thymic clonal deletion, transgenic (Tg) mice bearing a dominant negative mutation were produced. The transgene consisted of a truncated Nur77 (deltaNur77) gene encoding the DNA-binding domain of Nur77 ligated to a TCR-beta enhancer resulting in early expression in thymocytes. Apoptosis of CD4+CD8+ thymocytes mediated by CD3/TCR signaling was greatly inhibited in the deltaNur77 Tg mice, compared with non-Tg littermates, after treatment with anti-CD3 or anti-TCR antibody in vivo and in vitro. Clonal deletion of self-reactive T cells was investigated in deltaNur77-Db/HY TCR-alpha/beta double Tg mice. There was a five-fold increase in the total number of thymocytes expressing self-reactive Db/HY TCR-alpha/beta in the deltaNur77-TCR-alpha/beta double Tg male mice. Deficient clonal deletion of self-reactive thymocytes was demonstrated by a 10-fold increase in the CD4+CD8+ thymocytes that expressed Tg TCR-alpha/beta. There was an eightfold increase in the CD8+, Db/HY TCR-alpha/beta T cells in the lymph nodes (LN) of delta Nur77-Db/HY TCR-alpha/beta double Tg compared with Db/HY TCR-alpha/beta Tg male mice. In spite of defective clonal deletion, the T cells expressing the Tg TCR were functionally anergic. In vivo analysis revealed increased activation and apoptosis of T cells associated with increased expression of Fas and Fas ligand in LN of deltaNur77-Db/HY TCR-alpha/beta double male mice. These results indicate that inhibition of Nur77/Nurr1 DNA binding in T cells leads to inefficient thymic clonal deletion, but T cell tolerance is maintained by Fas-dependent clonal deletion in LN and spleen.

Visualizing CD4 T-cell migration into inflamed skin and its inhibition by CCR4/CCR10 blockades using in vivo imaging model.

BACKGROUND: Chemokines are critical mediators of T-cell homing into inflamed skin. The complex nature of this multicellular response makes it difficult to analyse mechanisms mediating the early responses in vivo. OBJECTIVES: To visualize directly T-cell homing into inflamed skin and its inhibition by blockades using a unique noninvasive confocal microscopy. MATERIALS AND METHODS: A mouse model of allergic contact dermatitis was used. T cells from oxazolone-sensitized and -challenged Balb/c mice were first analysed phenotypically in vitro. CD4 T cells were then labelled with a tracker dye and transferred into Balb/c-SCID mice. The recipient mice were challenged with oxazolone and CD4 T-cell homing into inflamed skin was visualized. RESULTS: T cells with the skin homing receptors CCR4 and CCR10 were increased in the affected skin and draining lymph nodes, and effectively attracted by their specific chemokines CCL17, CCL22 and CCL27 in vitro. Using in vivo imaging, T-cell migration into the inflamed skin was observed at 2 h after application, peaking at 12 h and continuing for 48 h. Simultaneous systemic administration of neutralizing antibodies against CCR4 ligands (CCL17 and CCL22) and CCR10 ligand (CCL27) led to a significant suppression of T-cell migration and skin inflammation. CONCLUSIONS: Our data indicate that these tissue-selective adhesion molecules and chemokine/receptor pathways act in concert to attract specialized T-cell populations to mediate cutaneous inflammation. The in vivo imaging technique can be applicable to other models of cutaneous diseases to help with better understanding of the pathogenesis and monitoring the therapeutic effects.

Increased expression of activation-induced cytidine deaminase is associated with anti-CCP and rheumatoid factor in rheumatoid arthritis.

Rheumatoid arthritis (RA) is associated with higher levels of autoantibodies and IL-17. Here, we investigated if ectopic lymphoid follicles and peripheral blood mononuclear cells (PBMCs) from RA patients exhibit increased activation-induced cytidine deaminase (AID), and if increased AID is correlated with serum levels of autoantibodies and IL-17. The results of immunohistochemical staining showed that organized AID(+) germinal centres were observed in six of the 12 RA synovial samples, and AID(+) cells were found almost exclusively in the B-cell areas of these follicles. Aggregated but not organized lymphoid follicles were found in only one OA synovial sample without AID(+) cells. Significantly higher levels of AID mRNA (Aicda) detected by RT-PCR were found in the PBMCs from RA patients than PBMCs from normal controls (P < 0.01). In the PBMCs from RA patients, AID was expressed predominately by the CD10(+)IgM(+)CD20(+) B-cell population and the percentage of these cells that expressed AID was significantly higher than in normal controls (P < 0.01). AID expression in the PBMCs correlated significantly and positively with the serum levels of rheumatoid factor (RF) (P

Autoimmunity and increased c-myb transcription.

A single recessive gene, lpr, induces an autoimmune-lymphoproliferative syndrome in several strains of mice. The lymphoid organs of lpr/lpr mice contained cells with increased amounts of myb RNA, which codes for a protein found in the nucleus. A similar human lymphoproliferative disorder also had an increase in c-myb expression. Mouse T cells induced by mitogens to proliferate did not express large amounts of myb RNA, indicating that marked myb expression is not a general feature of lymphocyte activation and proliferation.

Protection from Fas-mediated apoptosis by a soluble form of the Fas molecule.

Fas is an apoptosis-signaling receptor molecule on the surface of a number of cell types. Molecular cloning and nucleotide sequence analysis revealed a human Fas messenger RNA variant capable of encoding a soluble Fas molecule lacking the transmembrane domain because of the deletion of an exon encoding this region. The expression of soluble Fas was confirmed by flow cytometry and immunocytochemical analysis. Supernatants from cells transfected with the variant messenger RNA blocked apoptosis induced by the antibody to Fas. Levels of soluble Fas were elevated in patients with systemic lupus erythematosus, and mice injected with soluble Fas displayed autoimmune features.

Cancer gene therapy using mesenchymal stem cells expressing interferon-beta in a mouse prostate cancer lung metastasis model.

Cell-based therapy for cancer is a promising new field. Among cell types that can be used for this purpose, mesenchymal stem cells (MSCs) appear to hold great advantage for reasons including easier propagation in culture, possible genetic modification to express therapeutic proteins and preferential homing to sites of cancer growth upon in vivo transfer. The present study evaluated the potential of genetically modified MSC, constitutively expressing interferon (IFN)-beta, in an immunocompetent mouse model of prostate cancer lung metastasis. A recombinant adeno-associated virus (rAAV) encoding mouse IFN-beta was constructed and initially tested in vitro for high-level expression and bioactivity of the transgenic protein. MSCs were transduced by the rAAV-IFN-beta or green fluorescent protein ex vivo and used as cellular vehicles to target lung metastasis of TRAMP-C2 prostate cancer cells in a therapy model. Cohorts of mice were killed on days 30 and 75 to determine the effect of therapy by measurement of tumor volume, histology, immunohistochemistry, enzyme-linked immunosorbent assay and flow cytometry. Results indicated a significant reduction in tumor volume in lungs following IFN-beta-expressing MSC therapy. Immunohistochemistry of the lung demonstrated increased tumor cell apoptosis and decreased tumor cell proliferation and blood vessel counts. A significant increase in the natural kill cell activity was observed following IFN-beta therapy correlating the antitumor effect. Systemic level of IFN-beta was not significantly elevated from this targeted cell therapy. These data demonstrate the potential of MSC-based IFN-beta therapy for prostate cancer lung metastasis.

Fas ligand mutation in a patient with systemic lupus erythematosus and lymphoproliferative disease.

The pathogenesis of systemic lupus erythematosus (SLE) is multifactorial and multigenetic. The apoptosis genes, fas and fas ligand (fasL), are candidate contributory genes in human SLE, as mutations of these genes result in autoimmunity in several murine models of this disease. In humans, fas mutations result in a familial autoimmune lymphoproliferative syndrome, but defects in FasL have not yet been identified. In this study, DNA from 75 patients with SLE was screened by single-stranded conformational polymorphism analysis for potential mutations of the extracellular domain of FasL. A heterozygous single-stranded conformational polymorphism for FasL, was identified in one SLE patient, who exhibited lymphadenopathy. Molecular cloning and sequencing indicated that the genomic DNA of this patient contained an 84-bp deletion within exon 4 of the fasL gene, resulting in a predicted 28 amino acid in-frame deletion. Analysis of PBMC from this patient revealed decreased FasL activity, decreased activation-induced cell death, and increased T cell proliferation after activation. This is the first report of defective FasL-mediated apoptosis related to a mutation of the human Fasl, gene in a patient with SLE and suggests that fasL mutations are an uncommon cause of the disease.

Apoptosis mediated by Fas but not tumor necrosis factor receptor 1 prevents chronic disease in mice infected with murine cytomegalovirus.

The role of Fas- and TNF-receptor 1 (TNF-R1)-mediated apoptosis in the clearance of virally infected cells and in the regulation of the immune response was analyzed after murine cytomegalovirus (MCMV) infection of C57BL/6 (B6)-+/+ mice, Fas-mutant B6-lpr/lpr mice, TNF-R1 knockout B6-tnfr0/0 mice, and double-deficient B6-tnfr0/0 lpr/lpr mice. There was approximately equivalent clearance of MCMV in B6-+/+, B6-tnfr0/0, and B6-lpr/lpr mice, and by day 28 no infectious virus could be detected in the liver, kidney, lung, or peritoneal exudate. However, delayed virus clearance was observed in B6-tnfr0/0 lpr/lpr mice. An acute inflammatory response occurred in the liver, lung, and kidney of all mice, which was most severe 7 d after MCMV infection, but resolved by day 28 in B6-+/+ and B6-tnfr0/0 mice, but not in B6-lpr/lpr or B6-tnfr0/0 lpr/lpr mice. These results indicate that apoptosis mediated by either Fas or TNF-R1 is sufficient for rapid clearance of the virus. However, apoptosis induced by Fas, but not TNF-R1, is required for the downmodulation of the immune response to the virus and prevention of a chronic inflammatory reaction.

Antigen presenting cells expressing Fas ligand down-modulate chronic inflammatory disease in Fas ligand-deficient mice.

We assessed the effect of modified antigen presenting cells (APCs) expressing high levels of Fas ligand (APC-FasL) on post-viral chronic inflammatory disease. FasL-deficient B6-gld/gld mice infected with murine cytomegalovirus (MCMV) cleared the virus from their lungs, kidneys, and livers within 2 weeks of infection. However, inflammation persisted in these organs for more than 8 weeks, with a chronically increased T-cell response to MCMV-infected APCs and production of autoantibodies. Administration of APC-AdFasL at 4 weeks suppressed this inflammation and diminished the T-cell response and autoantibody production. APC-AdFasL that had been transfected with ultraviolet-irradiated MCMV were more effective than uninfected APC-AdFasL in ameliorating the chronic inflammation. APC-AdFasL migrated preferentially to the spleen, where they triggered apoptosis of lymphocytes in the marginal zone of the spleen. These results confirm that Fas-mediated apoptosis is not required for clearance of virus, but is required for down-modulation of the virally induced chronic inflammatory response. This organwide effect of APC-AdFasL appears to be mediated by elimination of activated T lymphocytes in the spleen before their emigration to the target organs.

Nonhomologous recombination at sites within the mouse JH-C delta locus accompanies C mu deletion and switch to immunoglobulin D secretion.

Plasma cells secrete immunoglobulins other than immunoglobulin M (IgM) after a deletion and recombination in which a portion of the immunoglobulin heavy-chain locus (IgH), from the 5'-flanking region of the mu constant-region gene (C mu) to the 5'-flanking region of the secreted heavy-chain constant-region gene (CH), is deleted. The recombination step is believed to be targeted via switch regions, stretches of repetitive DNA which lie in the 5' flank of all CH genes except delta. Although serum levels of IgD are very low, particularly in the mouse, IgD-secreting plasmacytomas of BALB/c and C57BL/6 mice are known. In an earlier study of two BALB/c IgD-secreting hybridomas, we reported that both had deleted the C mu gene, and we concluded that this deletion was common in the normal generation of IgD-secreting cells. To learn how such switch recombinations occur in the absence of a switch region upstream of the C delta 1 exon, we isolated seven more BALB/c and two C57BL/6 IgD-secreting hybridomas. We determined the DNA sequences of the switch recombination junctions in eight of these hybridomas as well as that of the C57BL/6 hybridoma B1-8. delta 1 and of the BALB/c, IgD-secreting plasmacytoma TEPC 1033. All of the lines had deleted the C mu gene, and three had deleted the C delta 1 exon in the switch recombination event. The delta switch recombination junction sequences were similar to those of published productive switch recombinations occurring 5' to other heavy-chain genes, suggesting that nonhomologous, illegitimate recombination is utilized whenever the heavy-chain switch region is involved in recombination.

Induction of specific T-cell tolerance by adenovirus-transfected, Fas ligand-producing antigen presenting cells.

A major problem associated with adenovirus gene therapy is the T cell-mediated immune response, which is elicited by inoculation of the adenovirus vector and leads to rapid clearance of the virus and loss of transgene expression. In this study, the immune response to adenovirus was prevented by induction of specific T-cell tolerance by pretreatment with adenovirus-infected antigen-presenting cells (APC) that express Fas ligand. Compared with control-treated mice, the tolerized mice showed prolonged expression of lacZ upon administration of AdCMVlacZ 1 week after tolerance induction. In contrast to the control mice, the tolerized mice did not display proliferation of CD3+ T cells in the spleen in response to AdCMVlacZ. Tolerance induction also was indicated by the lower production of interferon-gamma and interleukin-2 by peripheral T cells isolated from AdCMVlacZ-challenged tolerized mice than by AdCMVlacZ-challenged control-treated mice. The T-cell tolerance was specific for the adenovirus as the T-cell responses to irrelative murine cytomegalovirus remained unimpaired. Our results indicate that adenovirus-specific T-cell tolerance can be induced by APCs that coexpress Fas ligand and adenovirus antigens. We propose that this new strategy can be used to induce tolerance to adenovirus vector gene therapy with resultant prolonged expression of the transgene.

Soluble Fas gene therapy protects against Fas-mediated apoptosis of hepatocytes but not the lethal effects of Fas-induced TNF-alpha production by Kupffer cells.

The elevation of soluble Fas (sFas) in the sera of patients with liver disease suggests a role for sFas in the disease process; whether it is protective or not is controversial. To determine the effects of sFas on Fas-induced liver apoptosis, we manipulated mice to produce sFas by transfecting them in vivo with different amounts of an adenovirus that produces mouse sFas driven by the CMV promoter (AdsFas). Fas-mediated apoptosis was induced by administration of anti-mouse Fas (Jo2; 10 microg/mouse) one week later. The administration of AdsFas (10(3), 10(7), or 10(9) pfu/mouse), which was associated with only minimal side-effects, resulted in a significant reduction in the liver transaminase levels and mortality of the mice on challenge with Jo2, as compared to control mice treated with AdLacZ. However, the protective effect of AdsFas was not complete. The possibility that Jo2-induction of TNF-alpha in the Kupffer cells of the liver contributes to the pathology was therefore tested. Although administration of soluble TNF receptor (sTNFRI) alone did not protect the mice from the lethal effects of Jo2, administration of sTNFRI (200 microg/mouse) after infection with AdsFas (10(9) pfu/mouse) resulted in 100% survival of the mice on challenge with Jo2. To confirm that the production of TNF-alpha by Kupffer cells produce the lethal effects of Jo2 that remained after treatment with AdsFas, these cells were selectively ablated by treatment of the mice with gadolinium chloride prior to challenge with Jo2. This treatment greatly reduced early mortality and hepatocellular damage as well as TNF-alpha production 6 h after injection of Jo2. These results indicate that: (1) AdsFas prevents Jo2-induced apoptosis of hepatocytes; (2) In addition to mediating Fas-mediated apoptosis of hepatocytes, Jo2 can separately induce TNF-alpha production by Kupffer cells resulting in early mortality, and (3) Optimal protection from Jo2-induced mortality can be achieved by protection of liver cells by pretreatment with both AdsFas and sTNFRI.

Regulation of apoptosis of synovial fibroblasts.

In 1992, the first gene to cause systemic autoimmune disease in mice was identified as the Fas gene that is mutated in lymphoproliferative (lpr mice). These mice exhibited a defect in activation-induced cell death of T cells and B cells in vivo. This leads to the failure of proper clearance and removal of immune cells and defective downmodulation of an immune response. This then leads to the speculation that apoptosis defects, including defects in Fas, Fas ligand and Fas apoptosis signaling, may play a role in defective downmodulation of the hyperimmune response observed in human autoimmune diseases. Over the past 7 years, many scientists have analyzed different proapoptotic genes such as Fas, Fas ligand, Bcl-X, caspases as well as antiapoptosis pathways including defects in Fas and Fas ligand, Bcl-2 and caspase inhibitors. Potential genetic defects have been analyzed at the RNA, protein and functional level in humans with autoimmune disease. Somewhat surprisingly, most studies indicate that there is excessive apoptosis of PBMCs in autoimmune disease and human autoimmune disease suggesting that human autoimmune disease is not due to defective apoptosis of immune cells. Some studies indicate that there is decreased apoptosis of parenchymal cells such as RASF that undergo hyperplasia. Gene therapy and other modulators of apoptosis, such as wortmannin, can be used to faciliate apoptosis of RASF.

Ethambutol-induced toxic epidermal necrolysis.

Toxic epidermal necrolysis (TEN) is a severe cutaneous reaction that most commonly is related to drug exposure and that clinically can be confused with other bullous dermatoses, particularly staphylococcal scalded skin syndrome (SSSS) and erythema multiforme major (the Stevens-Johnson syndrome). We report the first case, to our knowledge, of TEN associated with ethambutol hydrochloride administration. Toxic epidermal necrolysis can be partially differentiated from other bullous dermatoses by history and clinical presentation. Microbiological results (eg, the isolation of Staphylococcus aureus in SSSS) and immunological studies (eg, the demonstration of immune complexes in the Stevens-Johnson syndrome) may aid in differentiation, but ultimately the diagnosis depends on histopathological examination of involved skin.