Background: Minimal residual disease (MRD) in B lymphoblastic leukemia (B-ALL) as measured by flow cytometry is well-established as an important prognostic factor; Its presence is used to adjust ...treatment in most therapeutic protocols in children , while the lack of a standardized assay has hampered the introduction of flow cytometric MRD in adult ALL trials. On the other hand, measuring MRD has become part of the standard of care even for patients not on clinical trials. Although flow cytometric analysis of MRD in B-ALL has been well standardized in clinical trials of the Children's Oncology Group (COG) in North America (Borowitz et al Blood 2015;126:964), there are no data on performance characteristics of this assay within routine clinical labs.
Methods: As part of an ongoing effort to standardize and decentralize ALL MRD measurement, list-mode data from post-induction marrows were distributed from one COG reference lab to 7 different clinical flow cytometry labs self-identified as having experience with ALL MRD. All labs were provided with the COG protocol used for MRD analysis along with a template illustrating recommended gating strategies, and formulas for calculating MRD burden. List-mode files of pre-treatment B-ALL samples analyzed with the standard COG B-ALL MRD antibody panel were distributed for comparison. In the first rounds, list-mode files from 15 samples were distributed to the 7 labs. Samples included those with and without MRD as assayed in the reference lab. Samples were selected to include normal B-cell precursors (hematogones) or MRD that had undergone phenotypic change with therapy. Some samples had artifacts that could potentially mimic small MRD populations. To improve performance, educational sessions were implemented, and 10 more list-mode file samples were distributed in a second round of challenges.
Results: There was considerable dispersion of MRD results among the 7 labs that analyzed the list-mode files (Fig 1A). Although high levels of MRD were uniformly recognized, several labs misclassified normal B-cell precursors and/or mischaracterized small artifacts as MRD. Moreover, among samples correctly identified as positive, quantitative differences in MRD levels from those reported by the reference lab were seen. Among 95 total challenges, the overall discordance rate was 24%. This included 11 false positives, 7 false negatives, and an additional 5 quantitatively discordant cases among positives (defined as outside +/- 0.5 log of the reference lab value). In the second round, positive and negative samples, as well as those with normal precursors were included, though these samples contained fewer artifacts than those of the first round. Performance improved considerably (Fig 1B); out of 70 challenges, there were 5 false positives and 1 false negative (8.6% discordance), and no cases were quantitatively discordant. Four of the 6 deviations occurred in a single lab. Three samples with hematogones were still misclassified as MRD.
Conclusions: Despite the provision of a standardized analysis protocol, even experienced laboratories have difficulty with B-ALL MRD analysis by flow cytometry. Some of these difficulties can be overcome with education, but even with education recognition of hematogones still remains a challenge for some labs. Extrapolating these results to other laboratories with less experience indicates the need for caution in migrating MRD testing from centralized reference laboratories, and suggests that implementation of MRD testing as part of routine clinical management of B-ALL patients in a manner similar to that of routine flow cytometric classification of leukemia may require additional training and resources.
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Wood:Pfizer: Honoraria, Other: Laboratory Services Agreement; Juno: Other: Laboratory Services Agreement; Amgen: Honoraria, Other: Laboratory Services Agreement; Seattle Genetics: Honoraria, Other: Laboratory Services Agreement. Lozanski:Stemline Therapeutics Inc.: Research Funding; Beckman Coulter: Research Funding; Boehringer Ingelheim: Research Funding; Genentech: Research Funding. Mukundan:CCS Associates: Employment. Higley:CCS Associates: Employment. Sigman:CCS Associates: Equity Ownership. Borowitz:BD Biosciences: Research Funding; Medimmune: Research Funding; Bristol Myers Squibb: Research Funding; HTG Molecular: Consultancy.
All homeotherms use thermogenesis to maintain their core body temperature, ensuring that cellular functions and physiological processes can continue in cold environments (1-3). In the prevailing ...model of thermogenesis, when the hypothalamus senses cold temperatures it triggers sympathetic discharge, resulting in the release of noradrenaline in brown adipose tissue and white adipose tissue (4,5). Acting via the β.sub.3-adrenergic receptors, noradrenaline induces lipolysis in white adipocytes (6), whereas it stimulates the expression of thermogenic genes, such as PPAR-γ coactivator 1a (Ppargc1a), uncoupling protein 1 (Ucp1) and acyl-CoA synthetase long-chain family member 1 (Acsll), in brown adipocytes (7-9). However, the precise nature of all the cell types involved in this efferent loop is not well established. Here we report in mice an unexpected requirement for the interleukin-4 (IL-4)-stimulated program of alternative macrophage activation in adaptive thermogenesis. Exposure to cold temperature rapidly promoted alternative activation of adipose tissue macrophages, which secrete catecholamines to induce thermogenic gene expression in brown adipose tissue and lipolysis in white adipose tissue. Absence of alternatively activated macrophages impaired metabolic adaptations to cold, whereas administration of IL-4 increased thermogenic gene expression, fatty acid mobilization and energy expenditure, all in a macrophage-dependent manner. Thus, we have discovered a role for alternatively activated macrophages in the orchestration of an important mammalian stress response, the response to cold.
The interaction between CD40 ligand (CD154) expressed on activated T cells and its receptor, CD40, has been shown to play a role in the onset and maintenance of autoimmune inflammation. Recent ...studies suggest that CD154+T cells also contribute to the regulation of atherogenesis due to their capacity to activate CD40+cells of the vasculature, including vascular smooth muscle cells (VSMC). The present study evaluated the signalling events initiated through CD40 ligation which culminate in VSMC chemokine production. CD40 ligation resulted in the phosphorylation/activation of mitogen-activated protein kinases (MAPKs), extracellular signal-regulated kinases 1 and 2 (ERK1/2), and p38, but not c-jun N-terminal kinase. Inhibition of both ERK1/2 and p38 activity abrogated CD40 stimulation of IL-8 and MCP-1 production. CD40-mediated induction of chemokines also showed dependence on the Src family kinase activity. The Src kinase inhibitor, PP2, was found to inhibit CD40-induced phosphorylation of ERK1/2 as well as activation of IκB kinase. An evaluation of Src kinases that may be important in CD40 signalling identified Lyn as a potential candidate. These data indicate that CD40 signalling in VSMC activates a Src family kinase-initiated pathway that results in the induction of MAPK activities required for successful induction of chemokine synthesis.
Macrophage infiltration and activation in metabolic tissues underlie obesity-induced insulin resistance and type 2 diabetes. While inflammatory activation of resident hepatic macrophages potentiates ...insulin resistance, the functions of alternatively activated Kupffer cells in metabolic disease remain unknown. Here we show that, in response to the Th2 cytokine interleukin-4 (IL-4), peroxisome proliferator activated receptor δ (PPARδ) directs expression of the alternative phenotype in Kupffer cells and adipose tissue macrophages of lean mice. However, adoptive transfer of PPARδ null bone marrow into wild type mice only diminishes alternative activation of hepatic macrophages, causing hepatic dysfunction and systemic insulin resistance. Suppression of hepatic oxidative metabolism is recapitulated by treatment of primary hepatocytes with conditioned media from PPARδ null macrophages, indicating direct involvement of Kupffer cells in liver lipid metabolism. Taken together, these data suggest an unexpected beneficial role for alternatively activated Kupffer cells in metabolic syndrome and type 2 diabetes.
Macrophages rapidly engulf apoptotic cells to limit the release of noxious cellular contents and to restrict autoimmune responses against self antigens. Although factors participating in recognition ...and engulfment of apoptotic cells have been identified, the transcriptional basis for the sensing and the silent disposal of apoptotic cells is unknown. Here we show that peroxisome proliferator-activated receptor-delta (PPAR-delta) is induced when macrophages engulf apoptotic cells and functions as a transcriptional sensor of dying cells. Genetic deletion of PPAR-delta decreases expression of opsonins such as complement component-1qb (C1qb), resulting in impairment of apoptotic cell clearance and reduction in anti-inflammatory cytokine production. This increases autoantibody production and predisposes global and macrophage-specific Ppard.sup.-/- mice to autoimmune kidney disease, a phenotype resembling the human disease systemic lupus erythematosus. Thus, PPAR-delta has a pivotal role in orchestrating the timely disposal of apoptotic cells by macrophages, ensuring that tolerance to self is maintained.
Obesity and insulin resistance, the cardinal features of metabolic syndrome, are closely associated with a state of low-grade inflammation. In adipose tissue chronic overnutrition leads to macrophage ...infiltration, resulting in local inflammation that potentiates insulin resistance. For instance, transgenic expression of Mcp1 (also known as chemokine ligand 2, Ccl2) in adipose tissue increases macrophage infiltration, inflammation and insulin resistance. Conversely, disruption of Mcp1 or its receptor Ccr2 impairs migration of macrophages into adipose tissue, thereby lowering adipose tissue inflammation and improving insulin sensitivity. These findings together suggest a correlation between macrophage content in adipose tissue and insulin resistance. However, resident macrophages in tissues display tremendous heterogeneity in their activities and functions, primarily reflecting their local metabolic and immune microenvironment. While Mcp1 directs recruitment of pro-inflammatory classically activated macrophages to sites of tissue damage, resident macrophages, such as those present in the adipose tissue of lean mice, display the alternatively activated phenotype. Despite their higher capacity to repair tissue, the precise role of alternatively activated macrophages in obesity-induced insulin resistance remains unknown. Using mice with macrophage-specific deletion of the peroxisome proliferator activated receptor-gamma (PPARgamma), we show here that PPARgamma is required for maturation of alternatively activated macrophages. Disruption of PPARgamma in myeloid cells impairs alternative macrophage activation, and predisposes these animals to development of diet-induced obesity, insulin resistance, and glucose intolerance. Furthermore, gene expression profiling revealed that downregulation of oxidative phosphorylation gene expression in skeletal muscle and liver leads to decreased insulin sensitivity in these tissues. Together, our findings suggest that resident alternatively activated macrophages have a beneficial role in regulating nutrient homeostasis and suggest that macrophage polarization towards the alternative state might be a useful strategy for treating type 2 diabetes.
Obesity and insulin resistance, the cardinal features of metabolic syndrome, are closely associated with a state of low-grade inflammation. In adipose tissue chronic overnutrition leads to macrophage ...infiltration, resulting in local inflammation that potentiates insulin resistance. For instance, transgenic expression of Mcp1 (also known as chemokine ligand 2, Ccl2) in adipose tissue increases macrophage infiltration, inflammation and insulin resistance. Conversely, disruption of Mcp1 or its receptor Ccr2 impairs migration of macrophages into adipose tissue, thereby lowering adipose tissue inflammation and improving insulin sensitivity. These findings together suggest a correlation between macrophage content in adipose tissue and insulin resistance. However, resident macrophages in tissues display tremendous heterogeneity in their activities and functions, primarily reflecting their local metabolic and immune microenvironment. While Mcp1 directs recruitment of pro-inflammatory classically activated macrophages to sites of tissue damage, resident macrophages, such as those present in the adipose tissue of lean mice, display the alternatively activated phenotype. Despite their higher capacity to repair tissue, the precise role of alternatively activated macrophages in obesity-induced insulin resistance remains unknown. Using mice with macrophage-specific deletion of the peroxisome proliferator activated receptor-gamma (PPARgamma), we show here that PPARgamma is required for maturation of alternatively activated macrophages. Disruption of PPARgamma in myeloid cells impairs alternative macrophage activation, and predisposes these animals to development of diet-induced obesity, insulin resistance, and glucose intolerance. Furthermore, gene expression profiling revealed that downregulation of oxidative phosphorylation gene expression in skeletal muscle and liver leads to decreased insulin sensitivity in these tissues. Together, our findings suggest that resident alternatively activated macrophages have a beneficial role in regulating nutrient homeostasis and suggest that macrophage polarization towards the alternative state might be a useful strategy for treating type 2 diabetes. PUBLICATION ABSTRACT
Effective aggregation of cell surface immune receptors with their ligands is critical in promoting humoral and cellular immune responses. Simulation of these interactions using soluble multimeric ...ligands having potent adjuvant effects may prove an effective alternative to agonistic antibodies as immunotherapeutics. Multimeric ligands may effectively engage their receptors, leading to aggregation and effective signal transduction. We exploited the structural characteristics of streptavidin (SA) for the generation of multimeric chimeric proteins. Streptavidin forms stable tetramers and oligomers under physiological conditions, and, as such, chimeric molecules with SA are expected to possess similar features. Two chimeric molecules consisting of the extracellular domains of human and mouse CD40L and a modified form of core streptavidin were generated. These proteins form stable oligomers that could only be dissociated into monomers by heating at 100°C, but not 60°C, under denaturing conditions. The chimeric proteins vigorously stimulated B cells, monocytes, and dendritic cells for the production of cytokines and chemokines and upregulation of immunostimulatory molecules. The use of SA as a chaperon presents a novel approach to generate multimeric immunological molecules with potent activities and their use as potential therapeutics for the treatment of cancer and other immune-based disorders.
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