Research:

Currently my research studies are divided in three distinct but complementary approaches. First the patient oriented study of redox regulation and lymphocytes subtypes, the complementary basic science and in vitro method development and the relationship between the redox parameters and B cell development. The unique 11 color- 13 parameter (Hi-D) FACS gives us the unparalleled opportunity to study the lymphocyte subtypes as well as the redox parameters that we are interested in.

A) Study of thioredoxin and thioredoxin reductase in healthy individuals and in HIV infected individuals with or without B cell lymphomas. In HIV patients, there is an imbalance of T-cell function favoring the presence of pro-inflammatory cytokines that exert continuous oxidative stress on cells. The Herzenberg laboratory and others have shown that reduced glutathione (GSH) levels decrease progressively as HIV disease progresses. In fact, the advanced stages of HIV infection may be considered a model dominated by excessive pro-inflammatory cytokines and low intracellular GSH levels. Importantly, for our interest here, elevated thioredoxin levels in HIV infected individuals correlate with decreased GSH levels and decreased numbers of CD4 T cells.
Placebo controlled trials in the Herzenberg laboratory have shown that intracellular GSH levels are restored in HIV infected individuals a by per oral administration with N-acetylcysteine (NAC), which supplies the cysteine needed to restore GSH. Consistent with this finding, a small substudy in the Herzenberg placebo-controlled trial showed that NAC treatment resulted in significantly decreases thioredoxin levels. These data are consistent with evidence showing that in vitro treatment of a thioredoxin-producing neoplastic cell line decreases thioredoxin release. Both findings thus implicate oxidative stress in increased production/release of thioredoxin.
The oxidative stress in HIV infection recently suggested a new study in which the Herzenberg and Nolan laboratories at Stanford collaboratively showed that motexafin gadolinium (Gd-Tex) selectively kills human T cells infected in vitro with HIV. GD-Tex, which induces intracellular oxidative stress, is in Phase III trials as a drug to increase sensitivity of neoplasms to irradiation. Our studies with Gd-Tex showed that it selectively induces apoptosis in HIV-1-infected CD4+ T cells in IL-2-stimulated cultures of peripheral blood mononuclear cells infected in vitro with HIV-1.
Continuing with studies in which the Herzenberg laboratory detected elevated thioredoxin levels in HIV infected people and showed that these elevated levels were associated with impaired survival, the studies proposed here will determine whether the frequency of thioredoxin elevation is decreased in the protease inhibitor (triple therapy) era. In addition, my studies will evaluate the potential relationship between thioredoxin elevations and the occurrence or progress of AIDS-related B cell neoplasms.

B) Investigation of redox status of the cell surface. Since the cell surface microenvironment contributes to the redox status of the thiol (-SH) groups on thiol-containing proteins on the cell surface, we are including surface thiol measurements as an additional index of redox changes that may influence T and B cell functions. In the studies I will use measurements of intracellular glutathione, intracellular thioredoxin and thioredoxin reductase, and surface thiols to determine whether the general redox imbalance that occurs in HIV infection predicts the neoplastic changes that lead to the development of B lymphomas in HIV disease. We have recently demonstrated that increased intracellular thioredoxin correlates with elevated surface thiols on T cells in HIV-infected individuals. We now plan to extend our studies to include these and the remaining markers to evaluate the relationship between oxidative stress and neoplastic transformation of B cells in HIV-infected and uninfected subjects. We also have developed methods to measure the glutathionylation of proteins using the HI-D FACS, which will also help us understand the redox changes on the cell surface proteins and the consequences for the cell function.

C) Relationship between the redox regulation (intracellular GSH, surface thiol and thioredoxin system) and B-1 cell development, clonal expansion and survival in mice. In studies conducted in parallel with the HIV studies, we will use mouse model systems to investigate the expansion of normal and neoplastic B cells (principally B-1 cells).
Based on the characteristic cell surface features, anatomical location and developmental history, B cells in adult mice are divided into two major subsets/lineage, termed B-1 and B-2. B-2 constitutes the majority of the B cells in mouse spleen and lymph nodes. Most of the B-2 cells are small, long lived resting cells that reside in B cell follicles and express low levels of surface IgM, high amounts of IgD, a variety of other surface markers, including CD23. However, they do not express the CD5 surface glycoprotein that is found on all T cells. B-1 cells, in contrast, comprise less than 5% of splenic B cells, are found at very low frequencies in lymph nodes but represent the dominant population in the peritoneal cavity. These cells express high levels of IgM, low levels of IgD and, for the most part, express low level of CD5.
B-1 cells produce most or all of the innate antibodies in serum. They undergo Ig class switching and represent roughly half of the IgA producing plasma cells in lamina propria of the gut. They respond to antigenic stimulation but are mainly (although not exclusively) involved in T-cell independent antibody responses. Reconstitution and other studies demonstrate that unlike B-2 cells, which develop de novo from precursors in the bone marrow throughout life, B-1 cells develop almost exclusively during the fetal/neonatal period and persist as a self-replenishing population thereafter.
B-1 cell neoplasms in the mouse have often been considered a model for human chronic lymphocytic leukemia (CLL) since CLL frequently express the CD5 surface marker. However, AIDS-related B cell lymphomas also often express CD5 and thus information derived from studies of B-1 normal and neoplastic cells may also shed light on the origins of the AIDS-related tumors and the mechanisms that control their growth.
In particular, since thioredoxin has been implicated in the regulation of B-1 neoplastic growth, and also redox (through hypoxia induced factor HIF-1 ) seems to regulate the B-1 cell proliferation, there is reason to suspect that the elevated thioredoxin levels in HIV disease may play a role in inducing or expanding B-1 neoplasms. Therefore, we have planned this dual-species project in which we will investigate the relationship between the thioredoxin system and B-1 cell development, clonal expansion and survival in mice and in HIV infected individuals.
Basically, we plan to study the effect of this increased thioredoxin levels on B-1 cell development, clonal expansion and survival in the mouse model and in HIV infected individuals (particularly those with AIDS-related B cell lymphomas), applying knowledge gained in each sphere to the other as our work progresses.

Publications:

Sahaf B, Heydari K, Holmgren A, Herzenberg LA and Herzennberg LA. Surface thiols and intracellular thioredoxin as sensors of redox imbalance in HIV infection. (in preparation)

Herzenberg LA, Parks D, Sahaf B, Perez O, Roederer M, and Herzenberg LA. The History and future of fluorescence activated cell sorter and flow cytometry: A view from Stanford, Clinical Chemistry, 48 (10) 2002 (In Press)

Sahaf B, Soderberg A, Ekerfelt C, Paulie S, Rosén A. Enzyme-linked immunospot assay for detection of thioredoxin and thioredoxin reductase secretion from cells. Methods Enzymol. 2002; (353): 22-35

Sahaf B. and Rosén A. Secretion of 10 kDa and 12 kDa thioredoxin species from blood monocytes and transformed leukocytes. Antioxid Redox Signal. 2000; 2(4) 717-26

Abdiu A., Nakamura H, Sahaf B, Yodoi J, Holmgren A, Rosén A. Thioredoxin blood levels increases after severe burn injury. Antioxid Redox Signal. 2000 (4) 707-16

Soderberg A, Sahaf B, Rosén A. Thioredoxin reductase, a redox-active selenoprotein, is secreted by normal and neoplastic cells: presence in human plasma. Cancer Res. 2000; 60 (8): 2281-9

Soderberg A , Sahaf B, Holmgren A, Rosén A. Monoclonal antibodies to human thioredoxin reductase. Biochem Biophys Res Commun. 1998; 249 (1):86-9

Sahaf B, Soderberg A, Spyrou G, Barral AM, Pekkari K, Holmgren A, and Rosén A. Thioredoxin expression and localization in human cell lines: detection of full-length and truncated species. Exp Cell Res 1997 236 (1): 181-92