Chemotactic activity of serum obtained from patients with idiopathic dilated cardiomyopathy.

Elevated circulating levels of alpha- and beta-chemokines in heart failure have been reported. The objective of this study was to investigate the interrelation of chemotactic activity of serum and circulating chemokine levels in patients suffering from idiopathic dilated cardiomyopathy (IDCM). Chemokine serum levels (MCP-1, MIP1-alpha, RANTES, IL-8 and TNF-alpha) were determined in patients with IDCM (n = 10), patients with coronary artery disease with normal (CAD-1; n = 10) or depressed (CAD-2; n = 10) left ventricular function and healthy controls (n = 10). The chemotactic effect of sera obtained from these groups was measured using an in vitro chemotaxis assay. Sera obtained from IDCM (5475 +/- 681 cells) showed the highest chemotactic activity when compared to controls (1850 +/- 215 cells), CAD-1 (3325 +/- 275 cells) and CAD-2 (2800 +/- 275 cells, P < 0.05) associated with significantly higher circulating MCP-1 levels. Sera obtained from IDCM patients show a high chemotactic activity associated with significantly elevated circulating MCP-1.

IL-2 and IL-3 production in high and low IgG-responding strains of mice.

The antibody response of mouse strain C57Bl/10ScSn (B10) is characterized by a low IgG responsiveness to a number of different antigens. Aberrant function of antigen-presenting cells and/or low activity of the Th cell population have been suggested as the cause of the defect. We studied the production of IL-2 and IL-3 in vitro by unstimulated and ConA-stimulated spleen cells. Unstimulated spleen cells of low-responding B10 mice produce significantly less IL-2 compared with the high-responding A/J mice in both intervals tested, i.e. after 24 and 48 h of in vitro incubation. IL-3 production is low but almost comparable in unstimulated cells of both strains. Stimulation of spleen cells by 5 micrograms/ml of ConA leads to considerably higher production of IL-2 in A/J spleen cells. IL-3 production by ConA-stimulated spleen cells showed the same pattern of activity. This low IL-3 production by B10 cells is most likely due to the low production of IL-2 during Th cell activation and to the limited proliferation of these cells. The low IgG production of B10 spleen cells during the secondary response to SRBC in vitro could be restored by IL-2 added to the medium. 50 U/ml of IL-2 increased the number of anti-SRBC IgG-producing cells 40 times in B10 cells, but only 4 times in A/J cells, so that the IgG production in B10 cells reached the same level as that in the A/J cells without exogenous IL-2. We suppose that the limited IL-2 production in the low-responding strain B10 is the cause of the low IgG responsiveness of these mice.

Intracellular processing of hapten-modified protein for MHC class I presentation: cytoplasmic delivery by pH-sensitive liposomes.

Phagocytic or endocytic uptake of pH-sensitive liposomes has been shown to result in the release of entrapped material into the cytosol. This system can therefore be applied to the targeted delivery of protein antigens into the MHC class I presentation pathway of antigen-processing cells. We have used trinitrophenyl (TNP)-modified chicken ovalbumin encapsulated in liposomes to examine the intracellular processing of haptenated proteins and the presentation of TNP-modified peptides to MHC class I-restricted hapten-specific CTL. Here, we demonstrate for the first time that hapten-modified proteins can undergo intracellular processing by macrophages, that similar peptides are produced in the form of unmodified or haptenated derivatives, and that TNP-peptides are transported to the cell surface and presented to class I-restricted CTL via the ER/Golgi pathway. This system can now be used to study T-cell responses to naturally processed hapten-conjugated peptides in vitro and in vivo.

Coxsackievirus B3-induced myocarditis in MHC class II-deficient mice.

OBJECTIVES: The pathogenesis of coxsackievirus B3 (CVB3)-induced myocarditis was investigated in immunocompetent C57BL/6 and MHC class II knockout mice with identical genetic backgrounds. STUDY DESIGN/METHODS: We analyzed the histology and immunohistology of myocardial injury, the replicating virus titer, and antibody response in the early and late phase of disease. RESULTS: CVB3-infected C57BL/6 mice showed acute myocarditis, with spontaneous healing, virus elimination, anti-CVB3 IgM/IgG production, and neutralizing antibody response. In contrast, MHC class II knockout mice developed less severe acute myocarditis, persistence of infiltrations and strong fibrosis, virus persistence, and weak IgG response, with absence of virus neutralizing antibodies. CONCLUSIONS: Immunodeficient organisms are more susceptible to long-term heart muscle injuries after infection with CVB3. The presence of CD4+ T cells are necessary to prevent the development of chronic disease.

Selective activation of CD8 T cell effector functions by epitope variants of lymphocytic choriomeningitis virus glycoprotein.

We provide evidence for selective activation of different effector functions of CD8+ T lymphocytes by altered peptide ligands. A T cell epitope from the glycoprotein of lymphocytic choriomeningitis virus (p33-41) and single amino acid variants thereof were used for primary in vitro induction of CTL clones. When the CTL were analyzed for cytotoxicity, proliferation, IFN-gamma production, and Ca2+ mobilization, we found that some of the clones showed activation of only their cytotoxic effector function when stimulated with variants of their inducing peptides. For one clone, cytotoxic reactivity was readily detected to the inducing peptide and three of four variants, but only the former was also able to trigger proliferation, IFN-gamma production, and Ca2+ mobilization. Another clone also revealed this dichotomy, but in this case some of the altered peptide ligands in addition to the inducing peptide were able to stimulate the full spectrum of effector functions, whereas others only stimulated cytotoxicity. A third clone revealed inefficient triggering of some effector functions by the peptide variants. Our data suggest that, as described for CD4 T cells, altered peptide ligands may lead to partial activation of effector functions of CD8 T cells. In addition, ligands with glycine substitutions in potential TCR contact positions induced CTL, which were able to recognize peptides with a variety of amino acids in the former glycine position.

The outcome of coxsackievirus B3-(CVB3-) induced myocarditis is influenced by the cellular immune status.

Mice develop a marked age-related susceptibility to myocardial coxsackievirus B3 (CVB3) infections. The lesions observed in mice resemble closely those seen in the human disease. Experimental murine models of CVB3-induced myocarditis have shown that both, host and viral genetic factors, can influence susceptibility to the infection as well as the persistence and progression of the disease. Recently, we have shown that CD4 T cell-deficient MHC Class II knockout mice develop a strong fibrosis with virus persistence in the heart tissue and without production of neutralizing antibodies. To examine the role of CD4+ T cells and especially the role of the T helper 1 cell response for the outcome and pathogenesis of CVB3-induced myocarditis in more detail, 2 different mouse strains with identical genetic background (H-2b) were infected with CVB3-Mü/J (Nancy strain). Immunocompetent C57BL/6 mice and mice with targeted disruption of interleukin (IL-)4 gene (IL-4-/- mice) developed a severe acute myocarditis on day 7 post infection (p.i.). The CVB3-induced inflammation was cured until the 21st day p.i. in hearts of C57BL/6 mice. IL-4-/- mice with insufficient T helper-2 cell immune response developed a severe myocardial damage between day 7 and 21 p.i. with prolonged virus persistence in the heart tissue. Therefore, we suggest that despite an obvious normal T helper-1 cell cytokine pattern, IL-4-/- mice are more susceptible to long-term heart muscle injuries after infection with CVB3.

Human immune response to HIV-1-Nef. I. CD45RO- T lymphocytes of non-infected donors contain cytotoxic T lymphocyte precursors at high frequency.

The immune response of peripheral blood lymphocytes (PBL) of non-exposed human individuals to the Nef protein of HIV-1 was studied. Nef is a regulatory protein of HIV which is immediately expressed after infection and which seems to be important in the pathogenicity of HIV. Nef may therefore serve as a potential target for effective immunity against HIV infection. Epstein-Barr (EBV)-transformed lymphoblastoid B cell lines (LCL) were established from four healthy young seronegative adults and transfected with the Nef gene. These cells served as stimulator cells for autologous PBL in vitro and as target cells for CTL. CTL responses were readily generated against Nef-transfected LCL, consisting of Nef-specific and putative EBV-specific CTL. Nef-specific CTL were generated exclusively from CD8+ cells and were MHC class I restricted. Since a vigorous Nef-specific CTL response in non-infected individuals was unexpected, CTL precursor frequencies were determined by limiting dilution analyses in non-fractionated PBL and in PBL separated into the CD45RO- (naive) and CD45RO+ (memory) T cell populations. As expected, the putative EBV-specific CTL precursors were predominantly found in the CD45RO+ subset at frequencies typical for memory T cells. Nef-specific CTL precursors, in contrast, were found predominantly in the CD45RO- population, at even higher frequencies of approximately 1/1000-1/3000. Nef may thus display either an unusually high number of immunogenic peptides or a limited number of peptides presented in a very efficient way, so that many T cells including low affinity cells, would be triggered.