Chaperonin containing TCP1 as a marker for identification of circulating tumor cells in blood.

Herein we report the use of Chaperonin-Containing TCP-1 (CCT or TRiC) as a marker to detect circulating tumor cells (CTCs) that are shed from tumors during oncogenesis. Most detection methods used in liquid biopsy approaches for enumeration of CTCs from blood, employ epithelial markers like cytokeratin (CK). However, such markers provide little information on the potential of these shed tumor cells, which are normally short-lived, to seed metastatic sites. To identify a marker that could go beyond enumeration and provide actionable data on CTCs, we evaluated CCT. CCT is a protein-folding complex composed of eight subunits. Previously, we found that expression of the second subunit (CCT2 or CCTß) inversely correlated with cancer patient survival and was essential for tumorigenesis in mice, driving tumor-promoting processes like proliferation and anchorage-independent growth. In this study, we examined CCT2 expression in cancer compared to normal tissues and found statistically significant increases in tumors. Because not all blood samples from cancer patients contain detectable CTCs, we used the approach of spiking a known number of cancer cells into blood from healthy donors to test a liquid biopsy approach using CCT2 to distinguish rare cancer cells from the large number of non-cancer cells in blood. Using a clinically validated method for capturing CTCs, we evaluated detection of intracellular CCT2 staining for visualization of breast cancer and small cell lung (SCLC) cancer cells. We demonstrated that CCT2 staining could be incorporated into a CTC capture and staining protocol, providing biologically relevant information to improve detection of cancer cells shed in blood. These results were confirmed with a pilot study of blood from SCLC patients. Our studies demonstrate that detection of CCT2 could identify rare cancer cells in blood and has application in liquid biopsy approaches to enhance the use of minimally invasive methods for cancer diagnosis.

Voluntary exercise prevents the obese and diabetic metabolic syndrome of the melanocortin-4 receptor knockout mouse.

Exercise is a mechanism for maintenance of body weight in humans. Morbidly obese human patients have been shown to possess single nucleotide polymorphisms in the melanocortin-4 receptor (MC4R). MC4R knockout mice have been well characterized as a genetic model that possesses phenotypic metabolic disorders, including obesity, hyperphagia, hyperinsulinemia, and hyperleptinemia, similar to those observed in humans possessing dysfunctional hMC4Rs. Using this model, we examined the effect of voluntary exercise of MC4R knockout mice that were allowed access to a running wheel for a duration of 8 wk. Physiological parameters that were measured included body weight, body composition of fat and lean mass, food consumption, body length, and blood levels of cholesterol and nonfasted glucose, insulin, and leptin. At the termination of the experiment, hypothalamic mRNA expression levels of neuropeptide Y (NPY), agouti-related protein (AGRP), proopiomelanocortin (POMC), cocaine- and amphetamine-regulated transcript (CART), orexin, brain-derived neurotropic factor (BDNF), phosphatase with tensin homology (Pten), melanocortin-3 receptor (MC3R), and NPY-Y1R were determined. In addition, islet cell distribution and function in the pancreas were examined. In the exercising MC4R knockout mice, the pancreatic islet cell morphology and other physiological parameters resembled those observed in the wild-type littermate controls. Gene expression profiles identified exercise as having a significant effect on hypothalamic POMC, orexin, and MC3R levels. Genotype had a significant effect on AGRP, POMC, CART, and NPY-Y1R, with an exercise and genotype interaction effect on NPY gene expression. These data support the hypothesis that voluntary exercise can prevent the genetic predisposition of melanocortin-4 receptor-associated obesity and diabetes.

M-CSF and GM-CSF regulation of STAT5 activation and DNA binding in myeloid cell differentiation is disrupted in nonobese diabetic mice.

Defects in macrophage colony-stimulating factor (M-CSF) signaling disrupt myeloid cell differentiation in nonobese diabetic (NOD) mice, blocking myeloid maturation into tolerogenic antigen-presenting cells (APCs). In the absence of M-CSF signaling, NOD myeloid cells have abnormally high granulocyte macrophage colony-stimulating factor (GM-CSF) expression, and as a result, persistent activation of signal transducer/activator of transcription 5 (STAT5). Persistent STAT5 phosphorylation found in NOD macrophages is not affected by inhibiting GM-CSF. However, STAT5 phosphorylation in NOD bone marrow cells is diminished if GM-CSF signaling is blocked. Moreover, if M-CSF signaling is inhibited, GM-CSF stimulation in vitro can promote STAT5 phosphorylation in nonautoimmune C57BL/6 mouse bone marrow cultures to levels seen in the NOD. These findings suggest that excessive GM-CSF production in the NOD bone marrow may interfere with the temporal sequence of GM-CSF and M-CSF signaling needed to mediate normal STAT5 function in myeloid cell differentiation gene regulation.

Aberrant prostaglandin synthase 2 expression defines an antigen-presenting cell defect for insulin-dependent diabetes mellitus.

Prostaglandins (PGs) are lipid molecules that profoundly affect cellular processes including inflammation and immune response. Pathways contributing to PG output are highly regulated in antigen-presenting cells such as macrophages and monocytes, which produce large quantities of these molecules upon activation. In this report, we demonstrate aberrant constitutive expression of the normally inducible cyclooxygenase PG synthase 2 (PGS(2)/ COX-2) in nonactivated monocytes of humans with insulin-dependent diabetes mellitus (IDDM) and those with islet autoantibodies at increased risk of developing this disease. Constitutive PGS(2) appears to characterize a high risk for diabetes as it correlates with and predicts a low first-phase insulin response in autoantibody-positive subjects. Abnormal PGS(2) expression in at-risk subjects affected immune response in vitro, as the presence of a specific PGS(2) inhibitor, NS398, significantly increased IL-2 receptor alpha-chain (CD25) expression on phytohemagglutinin-stimulated T cells. The effect of PGS(2) on CD25 expression was most profound in subjects expressing both DR04 and DQbeta0302 high-risk alleles, suggesting that this cyclooxygenase interacts with diabetes-associated MHC class II antigens to limit T-cell activation. These results indicate that constitutive PGS(2) expression in monocytes defines an antigen-presenting cell defect affecting immune response, and that this expression is a novel cell-associated risk marker for IDDM.

GM-CSF induces STAT5 binding at epigenetic regulatory sites within the Csf2 promoter of non-obese diabetic (NOD) mouse myeloid cells.

Myeloid cells from non-obese diabetic (NOD) mouse and human type 1 diabetic (T1D) patients overexpress granulocyte-macrophage colony stimulation factor (GM-CSF). This overproduction prolongs the activation of signal transduction and activator of transcription 5 (STAT5) proteins, involved in GM-CSF-induced control of myeloid cell gene expression. We found that GM-CSF can regulate the binding of STAT5 on the promoter of its own gene, Csf2, within regions previously identified as sites of chromatin epigenetic modification important to the regulation of GM-CSF during myeloid differentiation and inflammation. We found multiple sequence polymorphisms within NOD mouse chromosome 11 Idd4.3 diabetes susceptibility region that alter STAT5 GAS binding sequences within the Csf2 promoter. STAT5 binding at these sites in vivo is increased significantly in GM-CSF-stimulated-bone marrow cells and in unactivated, high GM-CSF-producing macrophages from NOD mice as compared to non-autoimmune C57BL/6 mouse myeloid cells. Thus, GM-CSF overproduction by NOD myeloid cells may be perpetuating a positive epigenetic regulatory feedback on its own gene expression through its induction of STAT5 binding to its promoter. These findings suggest that aberrant STAT5 binding at epigenetic regulatory sites may contribute directly to immunopathology through cytokine-induced gene expression dysregulation that can derail myeloid differentiation and increase inflammatory responsiveness.

Characterization of HKE2: an ancient antigen encoded in the major histocompatibility complex.

Genes at the centromeric end of the human leukocyte antigen region influence adaptive autoimmune diseases and cancer. In this study, we characterized protein expression of HKE2, a gene located in the centromeric portion of the class II region of the major histocompatibility complex encoding subunit 6 of prefoldin. Immunohistochemical analysis using an anti-HKE2 antibody indicated that HKE2 protein expression is dramatically upregulated as a consequence of activation. In a tissue microarray and in several tumors, HKE2 was overexpressed in certain cancers compared with normal counterparts. The localization of the HKE2 gene to the class II region, its cytoplasmic expression and putative protein-binding domain suggest that HKE2 may function in adaptive immunity and cancer.

Signal transduction activator of transcription 5 (STAT5) dysfunction in autoimmune monocytes and macrophages.

Autocrine granulocyte macrophage-colony stimulating factor (GM-CSF) sequentially activates intracellular components in monocyte/macrophage production of the pro-inflammatory and immunoregulatory prostanoid, prostaglandin E2 (PGE2). GM-CSF first induces STAT5 signaling protein phosphorylation, then prostaglandin synthase 2 (COX2/PGS2) gene expression, and finally IL-10 production, to downregulate the cascade. Without activation, monocytes of at-risk, type 1 diabetic (T1D), and autoimmune thyroid disease (AITD) humans, and macrophages of nonobese diabetic (NOD) mice have aberrantly high GM-CSF, PGS2, and PGE2 expression, but normal levels of IL-10. After GM-CSF stimulation, repressor STAT5A and B isoforms (80-77kDa) in autoimmune human and NOD monocytes and activator STAT5A (96-94kDa) and B (94-92kDa) isoforms in NOD macrophages stay persistently tyrosine phosphorylated. This STAT5 phosphorylation persisted despite treatment in vitro with IL-10, anti-GM-CSF antibody, or the JAK2/3 inhibitor, AG490. Phosphorylated STAT5 repressor isoforms in autoimmune monocytes had diminished DNA binding capacity on GAS sequences found in the PGS2 gene enhancer. In contrast, STAT5 activator isoforms in NOD macrophages retained their DNA binding capacity on these sites much longer than in healthy control strain macrophages. These findings suggest that STAT5 dysfunction may contribute to dysregulation of GM-CSF signaling and gene activation, including PGS2, in autoimmune monocytes and macrophages.

Efficacy of the rat bioassay for the determination of biologically available vitamin B-6.

Research was conducted to evaluate the merits of rat bioassays in studies concerning the bioavailability of vitamin B-6. A protocol was devised, which included pair-feeding and prevention of coprophagy. Plasma pyridoxal 5'-phosphate (PLP) was measured by a rapid chromatographic method as an indicator of vitamin B-6 status. Pyridoxamine and pyridoxal exhibited 70% molar activity relative to pyridoxine at suboptimal dietary levels. Selected foods (spinach, cornmeal and potato) were evaluated at three dietary levels with this protocol. Analysis of dose-response curves (dietary vitamin B-6 vs. plasma PLP) by slope-ratio methods yielded imprecise estimates of the relative vitamin B-6 bioavailability. This low precision, in addition to the potential for interference by direct absorption of B-6 vitamers synthesized by intestinal microflora, indicates the need for improved methods for measurement of biologically available vitamin B-6. It was concluded that rat bioassay methods, even as modified here, may be frequently unsuitable for use in studies of vitamin B-6 bioavailability. Results presented provide further support for the use of plasma PLP as an indicator of vitamin B-6 nutriture.

IL10 resistant PGS2 expression in at-risk/Type 1 diabetic human monocytes.

Aberrant prostaglandin synthase 2 (PGS2/COX2) expression constitutes an antigen presenting cell (APC) dysfunction seen in monocytes of humans at risk for or with Type 1 diabetes. During endotoxin activation of PGS2 expression in healthy monocytes, granulocyte-monocyte colony stimulating factor (GM-CSF) is activated and, in turn, promotes PGS2 gene activation. GM-CSF is considered a major target the action for IL10 in its suppression of PGS2. We found that the PGS2 expression in monocytes from 47% of at-risk and diabetic humans tested were highly resistant to suppression by IL10 (maintaining > or =50% of their untreated expression), and had significantly increased GM-CSF production in vitro (1043+/-SD2798 pg/10(6)cells, subject n=35, vs 29.7+/-SD91 pg/10(6)cells, control n=20; P=0.0165). The PGS2 insensitivity to IL10 of these cells was not due to a lack of IL10 functionality or its suppression of GM-CSF. In contrast to its effects on PGS2, IL10 regulation of GM-CSF and other monocyte factors (i.e., DR, IL1beta, TNFalpha, IL12, CD54, and CD64) remained intact. These findings suggest that the inability of IL10 to properly downregulate PGS2 gene expression may contribute to its dysregulation in Type 1 diabetes.

Aberrant monocyte prostaglandin synthase 2 (PGS2) expression in type 1 diabetes before and after disease onset.

METHODS: We examined monocyte prostaglandin synthase 2 (PGS2/COX2) expression in individuals at risk for or with type 1 diabetes including: (i) 58 established type 1 and 2 diabetic patients; (ii) 34 autoantibody positive (AA+) children and adults; (iii) 164 infants and young children with insulin-dependent diabetes mellitus (IDDM) susceptibility human leukocyte antigen (HLA) alleles; and (iv) 37 healthy control individuals, over a 5-yr period. RESULTS: Established type 1 diabetic patients (1 month to 30+ yr post-disease onset) had significantly higher PGS2 expression than healthy controls; by contrast, insulin-treated type 2 diabetic patients had significantly lower PGS2 expression than healthy controls. Longitudinal studies of AA+ subjects at risk for type 1 diabetes indicated that 73% (11/15) of individuals who developed this disease during the study period expressed high levels of PGS2 prior to or after onset. We also found high level PGS2 expression in genetically at-risk infants and young children that correlated with having a first-degree relative with type 1 diabetes, but not with age, gender, or HLA genotype. In this population, high level PGS2 expression coincided with or preceded autoantibody detection in 30% (3/10) of subjects. CONCLUSIONS: These findings suggest that high level monocyte PGS2 expression, although subject to fluctuation, is present in at-risk subjects at an early age and is maintained during progression to and after type 1 diabetes onset.