Tin K. Mao

Primary biliary cirrhosis is associated with altered hepatic microRNA expression

MicroRNAs (miRNAs) are small RNA molecules that negatively regulate protein coding gene expression and are thought to play a critical role in many biological processes. Aberrant levels of miRNAs have been associated with numerous diseases and cancers, and as such, miRNAs have gain much interests as diagnostic biomarkers, and as therapeutic targets. However, their role in autoimmunity is largely unknown. The aims of this study are to: (1) identify differentially expressed miRNAs in human primary biliary cirrhosis (PBC); (2) validate these independently; and (3) identify potential targets of differentially expressed miRNAs. We compared the expression of 377 miRNAs in explanted livers form subjects with PBC versus controls with normal liver histology. A total of 35 independent miRNAs were found to be differentially expressed in PBC (p < 0.001). Quantitative PCR was employed to validate down-regulation of microRNA-122a (miR-122a) and miR-26a and the increased expression of miR-328 and miR-299-5p. The predicted targets of these miRNAs are known to affect cell proliferation, apoptosis, inflammation, oxidative stress, and metabolism. Our data are the first to demonstrate that PBC is characterized by altered expression of hepatic miRNA; however additional studies are required to demonstrate a causal link between those miRNA and the development of PBC.

Altered monocyte responses to defined TLR ligands in patients with primary biliary cirrhosis

The role of the adaptive immune response, with regard to the development of autoantibodies, has been extensively studied in primary biliary cirrhosis (PBC). However, the importance of innate immunity has been noted only recently. Based on the proposed role of microorganisms in the pathogenesis of the disease, we hypothesize that patients with PBC possess a hyper‐responsive innate immune system to pathogen‐associated stimuli that may facilitate the loss of tolerance. To address this issue, we isolated peripheral blood monocytes from 33 patients with PBC and 26 age‐matched healthy controls and stimulated such cells in vitro with defined ligands for toll‐like receptor (TLR) 2 (lipoteichoic acid; LTA), TLR3 (polyIC), TLR4 (lipopolysaccharide; LPS), TLR5 (flagellin), and TLR9 (CpG‐B). Supernatant fluids from the cultures were analyzed for levels of 5 different pro‐inflammatory cytokines, interleukin (IL)‐1β, IL‐6, IL‐8, IL‐12p70, and TNF‐α. After in vitro challenge with TLR ligands, PBC monocytes produced higher relative levels of pro‐inflammatory cytokines, particularly IL‐1β, IL‐6, IL‐8, and TNF‐α, compared with controls. In conclusion, monocytes from patients with PBC appear more sensitive to signaling via select TLRs, resulting in secretion of selective pro‐inflammatory cytokines integral to the inflammatory response that may be critical in the breakdown of self‐tolerance. (HEPATOLOGY 2005;42:802–808.)

The effect of cocoa procyanidins on the transcription and secretion of interleukin 1β in peripheral blood mononuclear cells.

Recent data has demonstrated that cacao liquor polyphenols (procyanidins) have antioxidant activity, inhibit mRNA expression of interleukin-2 and are potent inhibitors of acute inflammation. Given the widespread ingestion of cocoa in many cultures, we investigated whether cocoa, in its isolated procyanidin fractions (monomer through decamer), would modulate synthesis of the pro-inflammatory cytokine, interleukin-1 beta. Both resting and phytohemagglutinin (PHA)-stimulated peripheral blood mononuclear cells (PBMC) were investigated at the levels of transcription and protein secretion. Individual cocoa fractions were shown to augment constitutive IL-1 beta gene expression, although values varied between subjects. Interestingly, the smaller fractions of cocoa (monomer-tetramer) consistently reduced IL-1 beta expression of PHA-stimulated cells by 1-15%, while the larger oligomers (pentamer-decamer) increased expression by 4-52%. These data, observed at the transcription level, were reflected in protein levels in PHA-induced PBMC. The presence or absence of PHA did not alter the effects of the cocoa procyanidins with the exception of the pentamer. This study offers additional data for the consideration of the health-benefits of dietary polyphenols from a wide variety of foods, including those benefits associated specifically with cocoa and chocolate consumption.

The anti-inflammatory properties of cocoa flavanols.

Signs of chronic or acute inflammation have been demonstrated in most cardiovascular diseases of multifactorial pathogenesis, including atherosclerosis and chronic heart failure. The triggers and mechanisms leading to inflammation may vary between clinical conditions but they share many common mediators, including specific patterns of eicosanoid and cytokine production. Certain cocoa-based products can be rich in a subclass of flavonoids known as flavanols, some of which have been found in model systems to possess potential anti-inflammatory activity relevant to cardiovascular health. Indeed, experimental evidence demonstrates that some cocoa-derived flavanols can reduce the production and effect of pro-inflammatory mediators either directly or by acting on signaling pathways. However, it should be noted that the evidence for any beneficial effects of cocoa flavanols in providing a meaningful anti-inflammatory action has been gathered predominantly from in vitro experiments. Therefore, additional research in well-designed human clinical experiments, using cocoa properly characterized in terms of flavanol content, would be a welcome addition to the evidence base to determine unambiguously if this benefit does indeed exist. If so, then flavanol-rich cocoa could be a potential candidate for the treatment, or possibly prevention, of the broad array of chronic diseases that are linked to dysfunctional inflammatory responses.

Cocoa Procyanidins and Human Cytokine Transcription and Secretion

We examined whether cocoa, in its isolated procyanidin fractions (monomer through decamer), would modulate cytokine production at the levels of transcription and protein secretion in both resting and phytohemagglutinin (PHA)-stimulated peripheral blood mononuclear cells (PBMC). In resting cells, interleukin (IL)-1beta and IL-4 gene expression from cocoa-treated cells varied markedly among the subjects tested. However, at the protein level, the larger fractions (pentamer through decamer) stimulated a dramatic increase in IL-1beta concentration (up to ninefold) with increasing degree of polymerization. Similarly, these larger fractions augmented IL-4 concentration by as much as 2 pg/ml, whereas the control displayed levels nearly undetectable. In the presence of PHA, gene expression also seemed to be most affected by the larger procyanidin fractions. The pentameric through decameric fractions increased IL-1 beta expression by 7-19% compared with PHA control, whereas the hexameric through decameric fractions significantly inhibited PHA-induced IL-4 transcription in the range of 71-86%. This observation at the transcription level for IL-1 beta was reflected at the protein level in PHA-stimulated PBMC. Significant reductions in mitogen-induced IL-4 production were also seen at the protein level with the hexamer, heptamer and octamer. Individual oligomeric cocoa fractions were unstimulatory for IL-2 in resting PBMC. However, when induced with PHA, the pentamer, hexamer and heptamer fractions caused a 61-73% inhibition in IL-2 gene expression. This study offers additional data for the consideration of the health benefits of dietary polyphenols from a wide variety of foods, including those benefits associated specifically with cocoa and chocolate consumption.

Modulation of TNF-α Secretion in Peripheral Blood Mononuclear Cells by Cocoa Flavanols and Procyanidins

Epidemiological reports have suggested that the consumption of foods rich in flavonoids is associated with a lower incidence of certain degenerative diseases, including cardiovascular disease. Flavanols and their related oligomers, the procyanidins CFP, isolated from cocoa can modulate the production and level of several signaling molecules associated with immune function and inflammation in vitro, including several cytokines and eicosanoids. To further elucidate the potential immuno-modulatory functions of flavanol-rich cocoa, the present investigation examined whether isolated CFP fractions (monomers through decamers) influence the secretion of tumor necrosis factor-α (TNF-α) from resting and phytohemagluttinin (PHA)-stimulated human peripheral blood mononuclear cells (PBMC). We used an in vitro culture system where PBMC from 14 healthy subjects were introduced to individual CFP fractions for 72 h prior to measuring the levels of TNF-α released. The intermediate-sized CFP fractions (tetramers through octamers) were the most active on resting cells, causing a 3–4 fold increase in TNF-α relative to media baseline. The monomers and dimers were the least stimulatory of the fractions tested, displaying a 42 and 31% increase, respectively, over media control, whereas the trimers, nonamers and decamers showed an intermediate stimulation of this cytokine. In the presence of PHA, the intermediate-sized CFP fractions again were the most active, enhancing TNF-α secretion in the range of 48–128% relative to the PHA control. The monomers and dimers were slightly inhibitory (–1.5 and –15%, respectively), while trimers, nonamers and decamers stimulated moderate increases in TNF-α levels (13, 19 and 15%, respectively). The above results lend support to the concept that CFP can be immunomodulatory. The stimulation of TNF-α secretion may contribute to the putative beneficial effects of dietary flavanoids against microbial infection and tumorigenesis.

Effect of Cocoa Flavanols and Their Related Oligomers on the Secretion of Interleukin-5 in Peripheral Blood Mononuclear Cells

We previously showed that flavanols and their related oligomers (FLO) isolated from cocoa can have immunomodulatory effects on production of the cytokines interleukin-1beta (IL-1beta), IL-2, and IL-4. In the present study, we examined whether selected FLO fractions isolated from cocoa (monomer through decamer) modulate IL-5 protein secretion from resting and phytohemagglutinin (PHA)-stimulated peripheral blood mononuclear cells (PBMC). Although FLO fractions were unstimulatory for IL-5 secretion in resting cells, PHA-induced IL-5 release from PBMC was markedly affected by certain FLO fractions. The monomeric and small oligomeric (dimer and trimer) fractions enhanced PHA stimulation by 50%, 54%, and 43%, respectively. In contrast, the larger oligomeric fractions (hexamer through decamer) inhibited IL-5 release in the range of 18% to 39%; the tetramer and pentamer showed intermediate effects. The increment in IL-5 suggests that FLO may preferentially stimulate immunoglobulin A. We suggest that in the oral cavity this could result in reduction in the risk for dental caries and periodontal disease. This work offers additional data for consideration of the health benefits of dietary FLO from a variety of foods, including those benefits associated specifically with consumption of some cocoas and chocolates.

Cocoa Flavonols and Procyanidins Promote Transforming Growth Factor-β1 Homeostasis in Peripheral Blood Mononuclear Cells

Evidence suggests that certain flavan-3-ols and procyanidins (FP) can have a positive influence on cardiovascular health. It has been previously reported that FP isolated from cocoa can potentially modulate the level and production of several signaling molecules associated with immune function and inflammation, including several cytokines and eicosanoids. In the present study, we examined whether FP fractions monomers through decamers modulate secretion of the cytokine transforming growth factor (TGF)-β1 from resting human peripheral blood mononuclear cells (PBMC). A total of 13 healthy subjects were studied and grouped according to their baseline production of TGF-β1. When cells from individuals with low baseline levels of TGF-β1 (n = 7) were stimulated by individual FP fractions (25 μg/ml), TGF-β1 release was enhanced in the range of 15%–66% over baseline (P < 0.05; monomer, dimer, and tetramer). The low-molecular-weight FP fractions (≤pentamer) were more effective at augmenting TGF-β1 secretion than their larger counterparts (≥hexamer), with the monomer and dimer inducing the greatest increases (66% and 68%, respectively). In contrast to the above, TGF-β1 secretion from high TGF-β1 baseline subjects (n = 6) was inhibited by individual FP fractions (P < 0.05; trimer through decamer). The inhibition was most pronounced with trimeric through decameric fractions (28%–42%), and monomers and dimers moderately inhibited TGF-β1 release (17% and 23%, respectively). Given the vascular actions associated with TGF-β1, we suggest that in healthy individuals, homeostatic modulation of its production by FP offers an additional mechanism by which FP-rich foods can potentially benefit cardiovascular health.

Sidechain biology and the immunogenicity of PDC‐E2, the major autoantigen of primary biliary cirrhosis

The E2 component of mitochondrial pyruvate dehydrogenase complex (PDC‐E2) is the immunodominant autoantigen of primary biliary cirrhosis. Whereas lipoylation of PDC‐E2 is essential for enzymatic activity and predominates under normal conditions, other biochemical systems exist that also target the lysine residue, including acylation of fatty acids or xenobiotics and ubiquitinylation. More importantly, the immunogenicity can be affected by derivatization of the lysine residue, as the recognition of lipoylated PDC‐E2 by patient autoantibodies is enhanced compared with octanoylated PDC‐E2. Furthermore, our laboratory has shown that various xenobiotic modifications of a peptide representing the immunodominant region of PDC‐E2 are immunoreactive against patient sera. The only purported regulatory system that prevents the accumulation of potentially autoreactive PDC‐E2 is glutathionylation, in which the lysine‐lipoic acid moiety is further modified with glutathione during apoptosis. Interestingly, this system is found in several cell lines, including HeLa, Jurkat, and Caco‐2 cells, but not in cholangiocytes and salivary gland epithelial cells, both of which are targets for destruction in primary biliary cirrhosis. Hence, the failure of this or other regulatory system(s) may overwhelm the immune system with immunogenic PDC‐E2 that can initiate the breakdown of tolerance in a genetically susceptible individual. In this review the authors survey the data available on the biochemical life of PDC‐E2, with particular emphasis on the lysine residue and its known interactions with machinery involved in various posttranslational modifications. (HEPATOLOGY 2004;40:1241–1248.)

Genetic polymorphisms influencing xenobiotic metabolism and transport in patients with primary biliary cirrhosis

Epidemiological data suggest that environmental factors may trigger autoimmunity in genetically susceptible individuals. In primary biliary cirrhosis (PBC), it has been postulated that halogenated xenobiotics can modify self‐molecules, facilitating the breakdown of tolerance to mitochondrial antigens. The transport and metabolism of xenobiotics is highly dependent on key genetic polymorphisms that alter enzymatic phenotype. We analyzed genomic DNA from 169 patients with PBC and 225 geographically and sex‐matched healthy subjects for polymorphisms of genes coding for cytochromes P450 (CYPs) 2D6 (CYP2D6*4, CYP2D6*3, CYP2D6*5, and CYP2D6*6) and 2E1 (c1/c2), multidrug resistance 1 (MDR1 C3435T) P‐glycoprotein, and pregnane X receptor (PXR C‐25385T, C8055T, and A7635G). We compared the genotype frequencies in patients and controls and also correlated polymorphisms with PBC severity. The distributions of the studied genotypes did not significantly differ between patients and controls. However, when clinical characteristics of patients with PBC were compared according to genotype, the CYP2E1 c2 allele was associated with signs of more severe disease. In conclusion, genetic polymorphisms of CYP 2D6 and 2E1, PXR, and MDR1 do not appear to play a role in the onset of PBC. (HEPATOLOGY 2005;41:55–63.)