Honggang Liu

Date of Award

1997

Degree Name

Biomedical Sciences

College

Joan C. Edwards School of Medicine

Type of Degree

Ph.D.

Document Type

Dissertation

First Advisor

Susan H. Jackman

Second Advisor

Bryan Larsen

Third Advisor

Donald A. Primerano

Fourth Advisor

Vernon E. Reichenbecher

Fifth Advisor

John L. Szarek

Sixth Advisor

Leonard J. Deutsch

Abstract

Various fungal products have immunomodulating activity and some have been studied regarding prevention of transplantation rejection. Prior to this investigation, the mycotoxin, gliotoxin (GT), has never been investigated as an immunotherapeutic drug for autoimmune disease. GT is a fungal secondary metabolite and a member of the epipolythiodioxopiperazine (ETP) family which has been shown to inhibit phagocytosis, induction of cytolytic T cells and the proliferation of T cells following mitogen stimulation. GT also induces in vitro apoptosis in certain immune cell types. More importantly, GT exhibits selective activity towards cells of hemopoietic origin.

Autoimmune diseases are disorders caused by immune responses to self antigens. Insulin dependent diabetes mellitus (IDDM) is an organ-specific autoimmune disease in which insulin secreting pancreatic islet β-cells are destroyed leading to hyperglycemia, ketoacidosis and various systemic complications. Because of its potential effects on the immune system, we evaluated GT for its ability to prevent IDDM. This study is the first to successfully use GT to prevent an autoimmune process.

GT prevented IDDM in spontaneously diabetic DP/BB rats without causing significant adverse effects among the treated animals. GT treated rats developed diabetes at a rate of 55% by 120 days of age compared to 90% for control rats. GT treatment also significantly decreased serum glucose levels from an average 278 mg/dl to 185.67 mg/dl among non-diabetic/pre-diabetic animals.

A series of studies was conducted on 65 days old DP/BB rats, prior to development of diabetes to phenotypically characterize the splenic lymphocytes recovered from animals chronically treated with GT. A parallel study examined the direct effects of GT on splenocyte preparations incubated with this mycotoxin.

This study found that GT selectively affects certain lymphocyte subsets. Animals treated with GT showed involution of splenic follicles and several effects on lymphocyte subpopulations were found. In vitro treatment of splenocytes with GT revealed decreased CD4+ and increased CD8+ T cell subsets. CD8+ T cells function as an important regulator of autoimmunity, especially influencing the activity of CD4+ T cells. GT effects on CD4+ and CD8+ T cells are consistent with changes anticipated to inhibit IDDM pathogenesis. In vivo treatment with GT did not result in detectable alterations in relative CD4+ and CD8+ cell subsets, although this may have been more related to pharmacologic reasons than the physiological effects of GT.

Importantly, this study found that both in vitro and in vivo GT treatments significantly enhanced the detectable RT6 surface marker. The RT6+ T cell subset is a key regulatory element in IDDM pathogenesis. Increased numbers of RT6 surface markers may be involved with IDDM prevention or may be a result of it.

GT induced lymphocyte apoptosis among spleen cells from DP/BB rats was altered in vitro. The average increase in apoptotic cells due to GT treatment was nearly four fold. Results from this study suggested that the mechanism whereby GT prevents IDDM in DP/BB rats is through apoptosis. Coupled with the finding of altered lymphocyte populations, it may be suggested that apoptosis of regulatory cells, or effector cells is involved in diabetes prevention in this system. The finding that CD8+ cells and NK cells which include cytotoxic effectors that can promote pancreatic damage, were not decreased by GT treatment suggests that the effects may reside with regulatory cells rather than with effectors, although additional study is warranted to fully understand this process.

This research is the first to show that GT has a protective effect against an autoimmune disease. We also found that GT is a selective immunomodulator altering the ratio of CD4+ and CD8+ lymphocytes and causing increased RT6+ surface marker to appear as an important subset of lymphocytes. This study is also the first to demonstrate that apoptosis due to GT treatment occurs in intact animals.

Because indicators of systemic toxicity showed that GT is relatively benign in experimental animals, as evidenced by lack of irreversible histopathology, normal weight gain and normal leukocyte counts, and it has a beneficial effect on IDDM development, GT should be considered for continued evaluation as a potential IDDM preventive drug.

Subject(s)

Mycotoxins – Physiological effect.

Diabetes – Prevention –Research.

Autoimmune diseases – Research.

Immune response – Regulation.

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