Calcium Concentration Effects on the Mechanical and Biochemical Properties of Chondrocyte-Alginate Constructs.

Alginate gel crosslinked by calcium ions (Ca(2+)) has been widely used in cartilage tissue engineering. However, most studies have been largely performed in vitro in medium with a calcium concentration ([Ca(2+)]) of 1.8mM, while the calcium level in the synovial fluid of the human knee joints, for example, has been reported to be 4mM or even higher. To simulate the synovial environment, the two studies in this paper were designed to investigate how the alginate scaffold alone, as well as the chondrocytes seeded alginate gel responds to variations in medium [Ca(2+)]. In Study A, the mechanical properties of 2% alginate hydrogel were tested in 0.15M NaCl and various [Ca(2+)] (1.0mM, 1.8mM, and 4mM). In Study B, primary bovine chondrocytes was seeded in alginate gel, and biochemical contents and mechanical properties were determined after incubation for 28 days in three [Ca(2+)] (1.8mM, 4mM, and 8mM). For both studies, it was found that the magnitude of the complex shear modulus (|G*|) at 1Hz doubled and the corresponding phase angle shift angle (δ) increased > 2° as a result of the approximate 4-fold change in [Ca(2+)]. At high [Ca(2+)], the chondrocyte glycosaminogylcan (GAG) production inside the chondrocyte-alginate constructs was suppressed significantly. This is likely due to a decrease in the porosity of the chondrocyte-alginate constructs as a result of compaction in structure caused by an increased crosslinking density with [Ca(2+)]. These may be important considerations in the eventual successful implementation of cartilage tissue-engineered constructs in the clinical setting.

Single nucleotide polymorphisms as a prerequisite for autoantigens.

It is still elusive why certain self proteins induce an autoimmune response. One immunological hypothesis is that only modified or altered self-proteins may become a target. Thus, we asked whether such alterations may actually be genetic polymorphisms that can be revealed by analyzing sequence variability in the known human autoantigens. Indeed, we found autoantigens to contain significantly more single nucleotide polymorphisms (SNP) than other human genes do. Our finding may offer an explanation for autoimmune responses through allogeneic exposure. Besides other contributing factors in autoimmunity, SNP may represent an essential prerequisite for the primary induction of an autoimmune response.

Sensitizing antigen-specific CD8+ T cells for accelerated suicide causes immune incompetence.

Death receptor-mediated activation-induced apoptosis of antigen-specific T cells is a major mechanism of peripheral tolerance induction and immune homeostasis. Failure to undergo activation-induced cell death (AICD) is an important underlying cause of many autoimmune diseases. Thus, enhancing the T cell's own suicide mechanism may provide an efficient therapy for the treatment of autoimmune diseases. Bisindolylmaleimide VIII (Bis VIII), a PKC inhibitor, can sensitize T cells for death receptor-induced apoptosis and thus can inhibit the development of T cell-mediated autoimmune disease in vivo. In this study, we have analyzed the functional consequences of accelerated suicide for a protective CD8+ T cell-mediated immune response. Our data indicate that CD8+ T cells are sensitized by Bis VIII to AICD, both in vitro and in vivo. The sensitizing effect of Bis VIII appears to be mediated by specific downmodulation of the antiapoptotic molecule cellular FLICE-like inhibitory protein (cFLIP(L)). Importantly, Bis VIII administration during an acute lymphocytic choriomeningitis virus (LCMV) infection causes the depletion of virus-specific CD8+ T cells and subsequently impaired cytotoxicity and virus clearance. We conclude that resistance to death receptor-induced apoptosis is crucial for the efficient induction of a protective immune response, and that Bis VIII-based immunotherapies have to be applied under well-controlled conditions to avoid the induction of immune incompetence and the inability to respond to pathogen infection.

IL-18-independent cytotoxic T lymphocyte activation and IFN-gamma production during experimental acute graft-versus-host disease.

Acute graft-versus-host disease (GvHD) is a serious complication after allogeneic bone marrow transplantation. Donor-derived T cells infiltrate recipient target organs and cause severe tissue damage, often leading to death of the affected patient. Tissue destruction is a direct result of donor CD8+ T cell activation and cell-mediated cytotoxicity. IL-18 is a novel pro-inflammatory cytokine with potent T(h)1 immune response-promoting and cytotoxic T lymphocyte (CTL)-inducing activity. IL-18 is strongly induced in experimental mouse models and human patients with acute GvHD. However, the precise role of IL-18 in the development of acute GvHD is still unknown. In this study, we have used IL-18-binding protein, a soluble IL-18 decoy receptor, to specifically neutralize IL-18 in vivo and in vitro. Our results demonstrate that IL-18 is induced during GvHD. However, its effect in the induction of GvHD appears to be redundant, since neutralization of IL-18 does not alter any disease parameter analyzed. Our study further shows that IFN-gamma production and CTL induction upon activation by T cell mitogens or by alloantigen does not involve IL-18-mediated amplification, in contrast to lipopolysaccharide-induced IFN-gamma production. We conclude that IL-18 expression correlates with the course of GvHD; however, its effect is dispensable for IFN-gamma and CTL induction for the initiation phase of this disease, most likely due to direct, IL-18-independent, CTL activation.