Ruth A. Gault

Protection from direct cerebral Cryptococcus infection by interferon-γ-dependent activation of microglial cells

The brain represents a significant barrier for protective immune responses in both infectious disease and cancer. We have recently demonstrated that immunotherapy with anti-CD40 and IL-2 can protect mice against disseminated Cryptococcus infection. We now applied this immunotherapy using a direct cerebral cryptococcosis model to study direct effects in the brain. Administration of anti-CD40 and IL-2 significantly prolonged the survival time of mice infected intracerebrally with Cryptococcus neoformans. The protection was correlated with activation of microglial cells indicated by the up-regulation of MHC II expression on brain CD45lowCD11b+ cells. CD4+ T cells were not required for either the microglial cell activation or anticryptococcal efficacy induced by this immunotherapy. Experiments with IFN-γ knockout mice and IFN-γR knockout mice demonstrated that IFN-γ was critical for both microglial cell activation and the anticryptococcal efficacy induced by anti-CD40/IL-2. Interestingly, while peripheral IFN-γ production and microglial cell activation were observed early after treatment, negligible IFN-γ was detected locally in the brain. These studies indicate that immunotherapy using anti-CD40 and IL-2 can augment host immunity directly in the brain against C. neoformans infection and that IFN-γ is essential for this effect.

Use of Systemic Proteasome Inhibition as an Immune-Modulating Agent in Disease

Bortezomib is the first proteasome inhibitor to be used clinically for the treatment of multiple myeloma and has been suggested as a possible treatment for a wide variety of hematologic and solid malignancies. Recent data suggests that potent immunomodulatory effects can also occur with systemic proteasome inhibition. This has been recently shown to occur in a graft-versus host disease model following bone marrow transplantation in mice. The suggested direct immunological effects of bortezomib treatment to include a decrease in anti-apoptotic protein levels, an increase in expression of TNF-family receptors (specifically Apo2L/TRAIL), induction of apoptosis, and inhibition of the transcription factor NF-kappaB. The NF-kappaB pathway has been associated with the regulation of numerous immune and inflammatory response mediators. In this review, we will present recent information concerning the potential therapeutic implications of bortezomib for a range of immune disorders. These findings would suggest that bortezomib treatment may be of clinical significance to suppress solid organ transplant rejection, autoreactive T cell responses, pro-inflammatory cytokine production, and consequently disease progression and pathology in autoimmunity.

Synthesis of cetyl myristoleate and evaluation of its therapeutic efficacy in a murine model of collagen-induced arthritis

Cetyl myristoleate (CM) was reported by Diehl and May [J Pharm Sci 83 (1994) 296] to block inflammation and prevent adjuvant-induced arthritis in rats. To verify this earlier work, we have synthesized pure CM and tested its anti-arthritic properties in a collagen-induced arthritis model in DBA/1LacJ mice. Multiple intraperitoneal injections of CM in 450 and 900 mg kg(-1) doses resulted in a significantly lower incidence of disease and caused a modest but significant diminution in clinical signs in those mice that developed arthritis. CM administered in daily oral doses of 20 mg kg(-1) also reduced the incidence of arthritis and caused a small reduction in the clinical signs in mice that developed arthritis. Although the protective effect of CM in collagen-induced arthritis observed in the present study was less dramatic than that reported earlier, our results confirm the anti-arthritic properties of pure CM.

Immunomodulation with CD40 Stimulation and Interleukin-2 Protects Mice from Disseminated Cryptococcosis

ABSTRACT Cryptococcus neoformans is a ubiquitous fungus that can cause life-threatening infections during immunosuppressive states such as AIDS and after bone marrow transplantation. In this study we investigated the antifungal efficacy of an agonist antibody to CD40, an important costimulator of immune function, in combination with interleukin 2 (IL-2) in a murine model of disseminated cryptococcosis. Only the combination of anti-CD40 and IL-2 significantly prolonged the survival time of infected mice. This protection was correlated with decreased yeast burdens in the brain and kidney. Increased immune cell populations in the spleens, as well as increased serum gamma interferon (IFN-γ) and tumor necrosis factor alpha levels were observed in infected mice treated with anti-CD40 and IL-2. Further experiments with IFN-γ knockout mice demonstrated that the protection induced by anti-CD40 and IL-2 treatment was dependent on IFN-γ. Depletion of CD4+ T cells did not affect the increased serum IFN-γ levels induced by anti-CD40 and IL-2 treatment and, importantly, did not affect the antifungal effect of combination therapy. These studies indicate that immunotherapy using anti-CD40 and IL-2 has therapeutic potential in augmenting host resistance to disseminated cryptococcosis and that IFN-γ is essential for efficacy.

Effects of housing on the thymic deficiency in dwarf mice and its reversal by growth hormone administration.

Initial studies on T cell development in the Snell Dwarf (dw/dw) strain of mice, which are deficient in the production of anterior pituitary hormones, have been interpreted to indicate a clear dependence of T cell development on endocrine system-derived factors. However, normal thymopoiesis in this strain has also been reported. The aim of the present study was to reconcile these contradictory data in order to define the role of anterior pituitary hormones in the thymus. The results indicated that if female dw/dw mice are housed together with their normal-sized littermates, thymic cellularity and the frequency of CD4(+)CD8(+) thymocytes are markedly reduced. However, administration of growth hormone could reverse these decreases seen in the double-positive T progenitor cells. Taken together, the data indicate that stress is the unifying parameter that can explain the disparate dw/dw mouse literature and suggest that endocrine effects on the T cell development can best be understood by interpreting the literature in this context.

Immunologic and hematopoietic effects of recombinant human prolactin after syngeneic bone marrow transplantation in mice

The period of immune deficiency following bone marrow transplantation (BMT) results in a susceptibility to opportunistic infections and remains a growing obstacle in improving the efficacy of BMT. Neuroendocrine hormones have been shown to affect numerous immunologic and hematologic responses after in vivo administration. We investigated whether neuroendocrine hormones, notably prolactin (PRL), could be administered after BMT and result in improved immunologic recovery. Mice were given lethal total body irradiation followed with a congeneic or a syngeneic BMT. Some groups then received recombinant human PRL (rhPRL) daily for 3 weeks. Effects on immune reconstitution and function were then monitored. The results show that PRL could increase thymic cellularity and donor T-cell reconstitution after congeneic BMT. Increases in B cells and myeloid progenitors were also observed. Mitogenic responses by both T and B cells were observed after PRL treatment. These results suggest that PRL may be of use to promote immune and myeloid reconstitution after BMT.

Characterization, application and potential uses of biotin-tagged inhibitors for lymphocyte serine proteases (granzymes)

Cytotoxic lymphocytes and natural killer cells are able to kill their target cells in minutes. The death of the target cell occurs after the release of cytoplasmic granules from the effector cell. These granules contain the pore-forming protein perforin and serine proteases (granzymes). To date 10 genes encoding lymphocyte granzymes have been discovered; of these only four have been purified and characterized for their substrate specificity. Several are predicted to have a common chymase, like specificity which is found in the granule extracts. Others may need to be enriched as active enzymes before they can be evaluated for substrate hydrolysis. Due to the limitations of detection by substrate hydrolysis, a more sensitive method for the detection of dilute granules was needed. We report the differing reactivities of seven biotin (Bi)-tagged isocoumarin (IC) inhibitors for Asp-ase, chymase, tryptase and Met-ase granzymes. The inhibitors contained different substituents at their no. 3 position: methoxy (OMe), ethoxy (OEt), propoxy (OPr) or 2-phenylethoxy (OEtPh) groups. The OMe group conferred general reactivity, whereas the OEtPh group conferred selective reactivity with chymase granzymes. The inhibitors that contained the longest aminocaproyl (Aca) spacers between the biotin-tag and the isocoumarin ring mediated the most stable granzyme inactivation. These inhibitors were the most effective at blocking lysis of red blood cells by the granule extracts. The inhibitors were used in protein blotting experiments where the biotin was detected with an avidin-enzyme complex. Over 10 granzymes were labelled by the inhibitor Bi-Aca-Aca-IC-OMe. The inhibitors detected granzymes when they were not readily detected by substrate hydrolysis.

Serine Protease Control of Lymphocyte-Mediated Cytolysis

Serine proteases initiate several complex biochemical responses to sudden, life-threatening situations. The immediate and localized activation of coagulant serine proteases prevents mammals from bleeding to death. The immediate activation of complement serine proteases kills common bacteria and prevents bacterial sepsis. The granules of cytotoxic lymphocytes (CTL) are packages which contain a unique subfamily of serine proteases and the pore-forming protein, perforin. These sequestered serine proteases are induced during the differentiation of lymphocytes to become competent killer cells (Lobe et al., 1986; Gershenfeld and Weisman, 1986). In vivo, lymphocytes laden with toxic granules accumulate at sites of graft rejection (Mueller et al., 1988). Exocytosis exposes the granule contents to high extracellular concentrations of calcium, the cation required for cell lysis. In vitro, freshly isolated natural killer (NK) lymphocytes and lymphokine-activated T lymphocytes release these protease-packed granules during killing. Proteases alone do not mediate lysis. Perform is necessary for cell lysis. The proteases undoubtably perform significant functions immediately associated with cytolysis.