Adam C. Seegmiller

Molecular Identification of the Sterol-Regulated Luminal Protease that Cleaves SREBPs and Controls Lipid Composition of Animal Cells

The lipid composition of animal cells is controlled by SREBPs, transcription factors released from membranes by sterol-regulated proteolysis. Release is initiated by Site-1 protease (S1P), which cleaves SREBPs in the ER luminal loop between two membrane-spanning regions. To clone S1P, we prepared pCMV-PLAP-BP2, which encodes a fusion protein that contains placental alkaline phosphatase (PLAP) in the ER lumen flanked by cleavage sites for signal peptidase and S1P. In sterol-deprived cells, cleavage by both proteases leads to PLAP secretion. PLAP is not secreted by SRD-12B cells, cholesterol auxotrophs that lack S1P. We transfected SRD-12B cells with pCMV-PLAP-BP2 plus pools of CHO cDNAs and identified a cDNA that restores Site-1 cleavage and PLAP secretion. The cDNA encodes S1P, an intraluminal 1052-amino-acid membrane-bound subtilisin-like protease. We propose that S1P is the sterol-regulated protease that controls lipid metabolism in animal cells.

The SREBP Pathway in Drosophila: Regulation by Palmitate, Not Sterols

In mammals, synthesis of cholesterol and unsaturated fatty acids is controlled by SREBPs, a family of membrane-bound transcription factors. Here, we show that the Drosophila genome encodes all components of the SREBP pathway, including a single SREBP (dSREBP), SREBP cleavage-activating protein (dSCAP), and the two proteases that process SREBP at sites 1 and 2 to release the nuclear fragment. In cultured Drosophila S2 cells, dSREBP is processed at sites 1 and 2, and the liberated fragment increases mRNAs encoding enzymes of fatty acid biosynthesis, but not sterol or isoprenoid biosynthesis. Processing requires dSCAP, but is not inhibited by sterols as in mammals. Instead, dSREBP processing is blocked by palmitic acid. These findings suggest that the ancestral SREBP pathway functions to maintain membrane integrity rather than to control cholesterol homeostasis.

Elucidation of seventeen human peripheral blood B-cell subsets and quantification of the tetanus response using a density-based method for the automated identification of cell populations in multidimensional flow cytometry data

Advances in multiparameter flow cytometry (FCM) now allow for the independent detection of larger numbers of fluorochromes on individual cells, generating data with increasingly higher dimensionality. The increased complexity of these data has made it difficult to identify cell populations from high‐dimensional FCM data using traditional manual gating strategies based on single‐color or two‐color displays.

Human immunodeficiency virus-associated plasmablastic lymphoma: Poor prognosis in the era of highly active antiretroviral therapy

Plasmablastic lymphoma (PBL) is a rare and aggressive B‐cell lymphoma strongly associated with human immunodeficiency virus (HIV) infection. The authors conducted a multi‐institutional, retrospective study to describe characteristics and determine prognostic factors in HIV‐associated PBL.

Immunophenotypic Differentiation Between Neoplastic Plasma Cells in Mature B-Cell Lymphoma vs Plasma Cell Myeloma

Some non-Hodgkin lymphomas show marked plasmacytic differentiation. In such cases, it may be difficult to differentiate these lymphoma from plasmacytoma or myeloma, especially with limited diagnostic material. However, there may be immunophenotypic differences in the plasma cells in these disorders that distinguish them. This study characterizes the immunophenotypes of neoplastic plasma cells in 41 cases of B-lineage non-Hodgkin lymphoma and compares them with those in plasma cell myeloma. We found that plasma cells in lymphoma were significantly more likely to express CD19, CD45, and surface immunoglobulin and less likely to express CD56 than those in myeloma. We further show that CD 19 and CD56 expression can be used reliably to distinguish these entities. Myeloma-associated osseous lesions and solitary plasmacytoma of bone showed myeloma-like immunophenotypes. However, some extramedullary plasmacytomas showed lymphoma-like phenotypes, suggesting that, in reality, they may represent non-Hodgkin lymphomas with extensive plasmacytic differentiation.

Characterization of Immunophenotypic Aberrancies in 200 Cases of B Acute Lymphoblastic Leukemia

Morphologic distinction of leukemic lymphoblasts in B acute lymphoblastic leukemia (B-ALL) from their nonneoplastic counterparts in bone marrow (hematogones) can be difficult. Thus, the presence of aberrant antigen expression detectable by flow cytometry may be critical for diagnosis of B-ALL and detection of minimal residual disease. The current study examined the immunophenotype of B-lineage leukemic lymphoblasts in 200 consecutive, unique, pretreatment patient specimens. We found that all cases of B-ALL exhibited multiple immunophenotypic aberrancies by which they can be distinguished from hematogones. The most frequent aberrancies were uniform or a spectrum of expression of terminal deoxynucleotidyl transferase and CD34, underexpression of CD45, overexpression of CD10, underexpression of CD38, and underexpression of CD20. Asynchronous coexpression of CD34 and CD20 was also frequently observed. Of the 200 cases, 86.5% expressed myeloid-associated antigens, and 19.0% expressed 3 or more. Of 200 cases, 9.0% aberrantly expressed T cell-associated antigens. There were significant differences in antigen-expression patterns between adult and pediatric B-ALL. Specific aberrancies correlate with recurrent cytogenetic abnormalities in B-ALL.

Plasmablastic lymphomas with MYC/IgH rearrangement: Report of three cases and review of the literature

We report detailed clinicopathologic features of 3 cases of plasmablastic lymphoma (PBL) with MYC/IgH rearrangement, representing one third of PBL cases diagnosed at our institution. This study brings the total number of reported cases in the literature to 6. All patients were HIV+ with very low CD4 counts at diagnosis. The involved locations were mediastinum, anus, and bone marrow. Tumors exhibited predominantly immunoblastic/plasmablastic morphologic features and had a plasma cell-like immunophenotype. Bright CD38 expression by flow cytometry had a tendency to be more common in these cases compared with PBL without MYC rearrangement. All cases were positive for Epstein-Barr virus-encoded RNA but lacked human herpesvirus-8 latent nuclear antigen. The 2 patients with follow-up died within 3 months. These findings show that PBL is often associated with MYC/IgH rearrangements and that this finding may portend an aggressive clinical course, suggesting that cytogenetic studies should be routinely applied in cases of PBL.

Simple karyotype and bcl-6 expression predict a diagnosis of Burkitt lymphoma and better survival in IG-MYC rearranged high-grade B-cell lymphomas

Rearrangement of MYC with immunoglobulin genes is a hallmark of Burkitt lymphoma. However, this rearrangement is not entirely specific and is often accompanied by varying numbers of additional cytogenetic abnormalities. This study aimed to assess the impact of karyotypic complexity, in correlation with comprehensive immunophenotypic analyses on the diagnosis and clinical outcomes of 34 cases of MYC-IG rearranged lymphomas that included Burkitt lymphoma (twenty-two cases), diffuse large B-cell lymphoma (three cases), unclassifiable B-cell lymphoma with features intermediate between diffuse large B-cell lymphoma and Burkitt lymphoma (six cases), and plasmablastic lymphoma (three cases). Additional cytogenetic abnormalities were observed in 26 of 34 cases (76%), including four cases (12%) that harbored dual translocations involving BCL-2 or BCl-6. Burkitt lymphoma cases had a significantly lower number of additional abnormalities (mean of 1.7), compared with unclassified B-cell lymphoma (3.3), diffuse large B-cell lymphoma (21.7), and plasmablastic lymphoma (6.7). Cases with simple karyotype (⩽2 additional abnormalities) were more likely to have a diagnosis of Burkitt lymphoma (89 versus 33% in patients with >2 additional abnormalities, P<0.01) and express bcl-6 (95 versus 47%, P<0.01). In addition, Burkitt lymphoma, bcl-6 expression, and simple karyotype were individual predictors of better overall survival. However, in multivariate analyses, only bcl-6 expression remained an independent predictor, although survival could be further stratified by karyotypic complexity in bcl-6(+) patients. We conclude that simple karyotype and bcl-6 expression suggest a diagnosis of Burkitt lymphoma and may portend better overall survival. These results may be very useful in the diagnosis and stratification of MYC-IG rearranged high-grade B-cell lymphomas.

DHA and EPA reverse cystic fibrosis-related FA abnormalities by suppressing FA desaturase expression and activity.

Patients and models of cystic fibrosis (CF) exhibit consistent abnormalities of polyunsaturated fatty acid composition, including decreased linoleate (LA) and docosahexaenoate (DHA) and variably increased arachidonate (AA), related in part to increased expression and activity of fatty acid desaturases. These abnormalities and the consequent CF-related pathologic manifestations can be reversed in CF mouse models by dietary supplementation with DHA. However, the mechanism is unknown. This study investigates this mechanism by measuring the effect of exogenous DHA and eicosapentaenoate (EPA) supplementation on fatty acid composition and metabolism, as well as on metabolic enzyme expression, in a cell culture model of CF. We found that both DHA and EPA suppress the expression and activity of Δ5- and Δ6-desaturases, leading to decreased flux through the n-3 and n-6 PUFA metabolic pathways and decreased production of AA. The findings also uncover other metabolic abnormalities, including increased fatty acid uptake and markedly increased retroconversion of DHA to EPA, in CF cells. These results indicate that the fatty acid abnormalities of CF are related to intrinsic alterations of PUFA metabolism and that they may be reversed by supplementation with DHA and EPA.

B-cell lymphomas with concurrent MYC and BCL2 abnormalities other than translocations behave similarly to MYC/BCL2 double-hit lymphomas

Large B-cell lymphomas with IGH@BCL2 and MYC rearrangement, known as double-hit lymphoma (DHL), are clinically aggressive neoplasms with a poor prognosis. Some large B-cell lymphomas have concurrent abnormalities of MYC and BCL2 other than coexistent translocations. Little is known about patients with these lymphomas designated here as atypical DHL. We studied 40 patients of atypical DHL including 21 men and 19 women, with a median age of 60 years. Nine (23%) patients had a history of B-cell non-Hodgkin lymphoma. There were 30 diffuse large B-cell lymphoma (DLBCL), 7 B-cell lymphoma, unclassifiable, with features intermediate between DLBCL and Burkitt lymphoma, and 3 DLBCL with coexistent follicular lymphoma. CD10, BCL2, and MYC were expressed in 28/39 (72%), 33/35 (94%), and 14/20 (70%) cases, respectively. Patients were treated with standard (n=14) or more aggressive chemotherapy regimens (n=17). We compared the atypical DHL group with 76 patients with DHLand 35 patients with DLBCL lacking MYC and BCL2 abnormalities. The clinicopathologic features and therapies were similar between patients with atypical and typical DHL. The overall survival of patients with atypical double-hit lymphoma was similar to that of patients with double-hit lymphoma (P=0.47) and significantly worse than that of patients with DLBCL with normal MYC and BCL2 (P=0.02). There were some minor differences. Cases of atypical double-hit lymphoma more often have DLBCL morphology (P<0.01), less frequently expressed CD10 (P<0.01), and patients less often had an elevated serum lactate dehydrogenase level (P=0.01). In aggregate, these results support expanding the category of MYC/BCL2 DHL to include large B-cell lymphomas with coexistent MYC and BCL2 abnormalities other than concurrent translocations.