Standard Operating Procedures for Biospecimen Collection, Processing, and Storage: From the Type 1 Diabetes in Acute Pancreatitis Consortium.

ABSTRACT: Differences in methods for biospecimen collection, processing, and storage can yield considerable variability and error. Therefore, best practices for standard operating procedures are critical for successful discovery, development, and validation of disease biomarkers. Here, we describe standard operating procedures developed for biospecimen collection during the DREAM (Diabetes RElated to Acute pancreatitis and its Mechanisms) Study within the Type 1 Diabetes in Acute Pancreatitis Consortium. Notably, these protocols were developed using an integrative process based on prior consortium experience and with input from working groups with expertise in immunology, pancreatitis, and diabetes. Publication and adoption consistent biospecimen protocols will inform future studies and allow for better comparisons across different metabolic research efforts.

Rationale and Development of a Data Coordinating Center to Support the Type 1 Diabetes in Acute Pancreatitis Consortium.

ABSTRACT: A data coordinating center (DCC) is a critical member of any multicenter research undertaking, and that is especially true for the Type 1 Diabetes in Acute Pancreatitis Consortium (T1DAPC). We describe how the T1DAPC DCC supports the consortium via its experience and expertise in project management, administration, financial management, regulatory compliance, scientific coordination, data management, research computing, and biostatistics and in facilitating scientific publications. The DCC's matrix management system has been extremely effective in managing all of its responsibilities. The first 16 months in the life of the T1DAPC have been dedicated to the development of its first protocol, titled Diabetes RElated to Acute pancreatitis and its Mechanisms (DREAM), addressing the institutional review board and regulatory components, developing the T1DAPC data management system, and providing training and certification of clinical center staff. As a result of its efforts, the DCC was a major contributor to the T1DAPC being able to initiate recruitment for the DREAM study in January 2022.

C6-ceramide nanoliposomes target the Warburg effect in chronic lymphocytic leukemia.

Ceramide is a sphingolipid metabolite that induces cancer cell death. When C6-ceramide is encapsulated in a nanoliposome bilayer formulation, cell death is selectively induced in tumor models. However, the mechanism underlying this selectivity is unknown. As most tumors exhibit a preferential switch to glycolysis, as described in the "Warburg effect", we hypothesize that ceramide nanoliposomes selectively target this glycolytic pathway in cancer. We utilize chronic lymphocytic leukemia (CLL) as a cancer model, which has an increased dependency on glycolysis. In CLL cells, we demonstrate that C6-ceramide nanoliposomes, but not control nanoliposomes, induce caspase 3/7-independent necrotic cell death. Nanoliposomal ceramide inhibits both the RNA and protein expression of GAPDH, an enzyme in the glycolytic pathway, which is overexpressed in CLL. To confirm that ceramide targets GAPDH, we demonstrate that downregulation of GAPDH potentiates the decrease in ATP after ceramide treatment and exogenous pyruvate treatment as well as GAPDH overexpression partially rescues ceramide-induced necrosis. Finally, an in vivo murine model of CLL shows that nanoliposomal C6-ceramide treatment elicits tumor regression, concomitant with GAPDH downregulation. We conclude that selective inhibition of the glycolytic pathway in CLL cells with nanoliposomal C6-ceramide could potentially be an effective therapy for leukemia by targeting the Warburg effect.

Seroreactivity to LGL leukemia-specific epitopes in aplastic anemia, myelodysplastic syndrome and paroxysmal nocturnal hemoglobinuria: results of a bone marrow failure consortium study.

Large granular lymphocyte (LGL) leukemia is characterized by clonal expansion of antigen-activated cytotoxic T cells (CTL). Patients frequently exhibit seroreactivity against a human T-cell leukemia virus (HTLV) epitope, BA21. Aplastic anemia, paroxysmal nocturnal hemoglobinuria and myelodysplastic syndrome are bone marrow failure diseases that can also be associated with similar aberrant CTL activation (LGL-BMF). We identified a BA21 peptide that was specifically reactive with LGL leukemia sera and found significantly elevated antibody reactivity against the same peptide in LGL-BMF sera. This finding of shared seroreactivity in LGL-BMF conditions and LGL leukemia suggests that these diseases might share a common pathogenesis.

Therapeutic efficacy of FTY720 in a rat model of NK-cell leukemia.

NK-cell leukemia is a clonal expansion of NK cells. The illness can occur in an aggressive or chronic form. We studied cell lines from human and rat NK-cell leukemias (aggressive NK-cell leukemia) as well as samples from patients with chronic NK-cell leukemia to investigate pathogenic mechanisms. Here we report that Mcl-1 was overexpressed in leukemic NK cells and that knockdown of Mcl-1 induced apoptosis in these leukemic cells. In vitro treatment of human and rat NK leukemia cells with FTY720 led to caspase-dependent apoptosis and decreased Mcl-1 expression in a time- and-dose-dependent manner. These biologic effects could be inhibited by blockade of reactive oxygen species generation and the lysosomal degradation pathway. Lipidomic analyses after FTY720 treatment demonstrated elevated levels of sphingosine, which mediated apoptosis of leukemic NK cells in vitro. Importantly, systemic administration of FTY720 induced complete remission in the syngeneic Fischer rat model of NK-cell leukemia. Therapeutic efficacy was associated with decreased expression of Mcl-1 in vivo. These data demonstrate that therapeutic benefit of FTY720 may result from both altered sphingolipid metabolism as well as enhanced degradation of a key component of survival signaling.

Targeting of survivin by nanoliposomal ceramide induces complete remission in a rat model of NK-LGL leukemia.

The natural killer (NK) type of aggressive large granular lymphocytic (LGL) leukemia is a fatal illness that pursues a rapid clinical course. There are no effective therapies for this illness, and pathogenetic mechanisms remain undefined. Here we report that the survivin was highly expressed in both aggressive and chronic leukemic NK cells but not in normal NK cells. In vitro treatment of human and rat NK-LGL leukemia cells with cell-permeable, short-chain C6-ceramide (C6) in nanoliposomal formulation led to caspase-dependent apoptosis and diminished survivin protein expression, in a time- and dose-dependent manner. Importantly, systemic intravenous delivery of nanoliposomal ceramide induced complete remission in the syngeneic Fischer F344 rat model of aggressive NK-LGL leukemia. Therapeutic efficacy was associated with decreased expression of survivin in vivo. These data suggest that in vivo targeting of survivin through delivery of nanoliposomal C6-ceramide may be a promising therapeutic approach for a fatal leukemia.

Platelet-derived growth factor mediates survival of leukemic large granular lymphocytes via an autocrine regulatory pathway.

Large granular lymphocyte (LGL) leukemia results from chronic expansion of cytotoxic T cells or natural killer (NK) cells. Apoptotic resistance resulting from constitutive activation of survival signaling pathways is a fundamental pathogenic mechanism. Recent network modeling analyses identified platelet-derived growth factor (PDGF) as a key master switch in controlling these survival pathways in T-cell LGL leukemia. Here we show that an autocrine PDGF regulatory loop mediates survival of leukemic LGLs of both T- and NK-cell origin. We found high levels of circulating PDGF-BB in platelet-poor plasma samples from LGL leukemia patients. Production of PDGF-BB by leukemic LGLs was demonstrated by immunocytochemical staining. Leukemic cells expressed much higher levels of PDGFR-beta transcripts than purified normal CD8(+) T cells or NK cells. We observed that phosphatidylinositol-3-kinase (PI3 kinase), Src family kinase (SFK), and downstream protein kinase B (PKB)/AKT pathways were constitutively activated in both T- and NK-LGL leukemia. Pharmacologic blockade of these pathways led to apoptosis of leukemic LGLs. Neutralizing antibody to PDGF-BB inhibited PKB/AKT phosphorylation induced by LGL leukemia sera. These results suggest that targeting of PDGF-BB, a pivotal regulator for the long-term survival of leukemic LGLs, may be an important therapeutic strategy.