Autoreactive monoclonal antibodies from patients with primary biliary cholangitis recognize environmental xenobiotics.

A major problem in autoimmunity has been identification of the earliest events that lead to breach of tolerance. Although there have been major advances in dissecting effector pathways and the multilineage immune responses to mitochondrial self-antigens in primary biliary cholangitis, the critical links between environmental factors and tolerance remain elusive. We hypothesized that environmental xenobiotic modification of the E2 subunit of the pyruvate dehydrogenase (PDC-E2) inner lipoyl domain can lead to loss of tolerance to genetically susceptible hosts. Previously we demonstrated that serum anti-PDC-E2 autoantibodies cross-react with the chemical xenobiotics 2-octynoic acid and 6,8-bis (acetylthio) octanoic acid and further that there is a high frequency of PDC-E2-specific peripheral plasmablasts. Herein we generated 104 recombinant monoclonal antibodies (mAbs) based on paired heavy-chain and light-chain variable regions of individual plasmablasts derived from primary biliary cholangitis patients. We identified 32 mAbs reactive with native PDC-E2, including 20 specific for PDC-E2 and 12 cross-reactive with both PDC-E2 and 2-octynoic acid and 6,8-bis (acetylthio) octanoic acid. A lower frequency of replacement somatic hypermutations, indicating a lower level of affinity maturation, was observed in the complementarity-determining regions of the cross-reactive mAbs in comparison to mAbs exclusively recognizing PDC-E2 or those for irrelevant antigens. In particular, when the highly mutated heavy-chain gene of a cross-reactive mAb was reverted to the germline sequence, the PDC-E2 reactivity was reduced dramatically, whereas the xenobiotic reactivity was retained. Importantly, cross-reactive mAbs also recognized lipoic acid, a mitochondrial fatty acid that is covalently bound to PDC-E2. CONCLUSION: Our data reflect that chemically modified lipoic acid or lipoic acid itself, through molecular mimicry, is the initial target that leads to the development of primary biliary cholangitis. (Hepatology 2017;66:885-895).

The fingerprint of antimitochondrial antibodies and the etiology of primary biliary cholangitis.

The identification of environmental factors that lead to loss of tolerance has been coined the holy grail of autoimmunity. Our work has focused on the reactivity of antimitochondrial autoantibodies (AMA) to chemical xenobiotics and has hypothesized that a modified peptide within PDC-E2, the major mitochondrial autoantigen, will have been immunologically recognized at the time of loss of tolerance. Herein, we successfully applied intein technology to construct a PDC-E2 protein fragment containing amino acid residues 177-314 of PDC-E2 by joining a recombinant peptide spanning residues 177-252 (PDC-228) with a 62-residue synthetic peptide from 253 to 314 (PP), which encompasses PDC-E2 inner lipoyl domain (ILD). We named this intein-constructed fragment PPL. Importantly, PPL, as well as lipoic acid conjugated PPL (LA-PPL) and xenobiotic 2-octynoic acid conjugated PPL (2OA-PPL), are recognized by AMA. Of great importance, AMA has specificity for the 2OA-modified PDC-E2 ILD peptide backbone distinct from antibodies that react with native lipoylated PDC-E2 peptide. Interestingly, this unique AMA subfraction is of the immunoglobulin M isotype and more dominant in early-stage primary biliary cholangitis (PBC), suggesting that exposure to 2OA-PPL-like compounds occurs early in the generation of AMA. To understand the structural basis of this differential recognition, we analyzed PPL, LA-PPL, and 2OA-PPL using electron paramagnetic resonance spectroscopy, with confirmations by enzyme-linked immunosorbent assay, immunoblotting, and affinity antibody analysis. We demonstrate that the conformation of PDC-E2 ILD is altered when conjugated with 2OA, compared to conjugation with lipoic acid. CONCLUSION: A molecular understanding of the conformation of xenobiotic-modified PDC-E2 is critical for understanding xenobiotic modification and loss of tolerance in PBC with widespread implications for a role of environmental chemicals in the induction of autoimmunity. (Hepatology 2017;65:1670-1682).

Ongoing activation of autoantigen-specific B cells in primary biliary cirrhosis.

UNLABELLED: The serologic hallmark of primary biliary cirrhosis (PBC), the antimitochondrial response to the E2 component of the pyruvate dehydrogenase complex (PDC-E2), has unique features, including continuous high titers of immunoglobulin M (IgM) and IgG reactivity throughout all stages of disease, capable not only of target enzyme inhibition, but also crossreactive with chemical xenobiotics that share molecular homology with the inner lipoyl domain of PDC-E2; such chemicals have been proposed as potential etiological agents. We used flow cytometry and enzyme-linked immunospot assay (ELISPOT) to examine B-cell subsets in 59 subjects, including 28 with PBC, 13 with primary sclerosing cholangitis (PSC), and 18 healthy controls. Strikingly, in PBC, although there were no significant differences in B-cell phenotype subpopulations, 10% of the total IgG and IgA plasmablast population and 23% of the IgM plasmablast population were uniquely reactive with PDC-E2, detected in the CXCR7+ CCR10low plasmablast population. In contrast, plasmablast reactivity to a control antigen, tetanus toxoid, was minimal and similar in all groups. Additionally, we isolated plasmablast-derived polyclonal antibodies and compared reactivity with plasma-derived antibodies and noted a distinct noncirculating tissue source of xenobiotic crossreacting antibodies. The high levels of autoantigen specific peripheral plasmablasts indicate recent activation of naive or memory B cells and a continuous and robust activation. The presence of CXCR7+ CCR10low PDC-E2-specific ASCs suggests a mechanistic basis for the migration of circulating antigen specific plasmablasts to the mucosal epithelial ligands CXCL12 and CCL28. CONCLUSION: Our findings suggest a sustained rigorous B-cell response in PBC, likely activated and perpetuated by cognate autoantigen.

Antimitochondrial antibody recognition and structural integrity of the inner lipoyl domain of the E2 subunit of pyruvate dehydrogenase complex.

Antimitochondrial autoantibodies (AMAs), the serological hallmark of primary biliary cirrhosis, are directed against the lipoyl domain of the E2 subunit of pyruvate dehydrogenase (PDC-E2). However, comprehensive analysis of the amino acid residues of PDC-E2 lipoyl ß-sheet with AMA specificity is lacking. In this study, we postulated that specific residues within the lipoyl domain are critical to AMA recognition by maintaining conformational integrity. We systematically replaced each of 19 residue peptides of the inner lipoyl domain with alanine and analyzed these mutants for reactivities against 60 primary biliary cirrhosis and 103 control sera. Based on these data, we then constructed mutants with two, three, or four replacements and, in addition, probed the structure of the substituted domains using thiol-specific spin labeling and electron paramagnetic resonance (EPR) of a (5)Ile→Ala and (12)Ile→Ala double mutant. Single alanine replacement at (5)Ile, (12)Ile, and (15)Glu significantly reduced AMA recognition. In addition, mutants with two, three, or four replacements at (5)Ile, (12)Ile, and (15)Glu reduced AMA reactivity even further. Indeed, EPR reveals a highly flexible structure within the (5)Ile and (12)Ile double-alanine mutant. Autoreactivity is largely focused on specific residues in the PDC-E2 lipoyl domain critical in maintaining the lipoyl loop conformation necessary for AMA recognition. Collectively, the AMA binding studies and EPR analysis demonstrate the necessity of the lipoyl ß-sheet structural conformation in anti-PDC-E2 recognition.

Antimitochondrial antibody heterogeneity and the xenobiotic etiology of primary biliary cirrhosis.

UNLABELLED: Antimitochondrial antibodies (AMAs) directed against the lipoyl domain of the E2 subunit of pyruvate dehydrogenase (PDC-E2) are detected in 95% of patients with primary biliary cirrhosis (PBC) and are present before the onset of clinical disease. The recent demonstration that AMAs recognize xenobiotic modified PDC-E2 with higher titers than native PDC-E2 raises the possibility that the earliest events involved in loss of tolerance are related to xenobiotic modification. We hypothesized that reactivity to such xenobiotics would be predominantly immunoglobulin M (IgM) and using sera from a large cohort of PBC patients and controls (n = 516), we examined in detail sera reactivity against either 6,8-bis(acetylthio) octanoic acid (SAc)-conjugated bovine serum albumin (BSA), recombinant PDC-E2 (rPDC-E2) or BSA alone. Further, we also defined the relative specificity to the SAc moiety using inhibition enzyme-linked immunosorbent assay (ELISA); SAc conjugate and rPDC-E2-specific affinity-purified antibodies were also examined for antigen specificity, isotype, and crossreactivity. Reactivity to SAc conjugates is predominantly IgM; such reactivity reflects a footprint of previous xenobiotic exposure. Indeed, this observation is supported by both direct binding, crossreactivity, and inhibition studies. In both early and late-stage PBC, the predominant Ig isotype to SAc is IgM, with titers higher with advanced stage disease. We also note that there was a higher level of IgM reactivity to SAc than to rPDC-E2 in early-stage versus late-stage PBC. Interestingly, this finding is particularly significant in light of the structural similarity between SAc and the reduced form of lipoic acid, a step which is similar to the normal physiological oxidation of lipoic acid. CONCLUSION: Specific modifications of the disulfide bond within the lipoic-acid-conjugated PDC-E2 moiety, i.e., by an electrophilic agent renders PDC-E2 immunogenic in a genetically susceptible host.

Environment and primary biliary cirrhosis: electrophilic drugs and the induction of AMA.

Environmental stimulation is a major factor in the initiation and perpetuation of autoimmune diseases. We have addressed this issue and focused on primary biliary cirrhosis (PBC), an autoimmune disease of the liver. Immunologically, PBC is distinguished by immune mediated destruction of the intra hepatic bile ducts and the presence of high titer antimitochondrial autoantibodies (AMA) directed against a highly specific epitope within the lipoic acid binding domain of the pyruvate dehydrogenase E2 subunit (PDC-E2). We submit that the uniqueness of AMA epitope specificity and the conformational changes of the PDC-E2 lipoyl domain during physiological acyl transfer could be the lynchpin to the etiology of PBC and postulate that chemical xenobiotics modification of the lipoyl domain of PDC-E2 is sufficient to break self-tolerance, with subsequent production of AMA in patients with PBC. Indeed, using quantitative structure activity relationship (QSAR) analysis on a peptide-xenobiotic conjugate microarray platform, we have demonstrated that when the lipoyl domain of PDC-E2 was modified with specific synthetic small molecule lipoyl mimics, the ensuing structures displayed highly specific reactivity to PBC sera, at levels often higher than the native PDC-E2 molecule. Hereby, we discuss our recent QSAR analysis data on specific AMA reactivity against a focused panel of lipoic acid mimic in which the lipoyl di-sulfide bond are modified. Furthermore, data on the immunological characterization of antigen and Ig isotype specificities against one such lipoic acid mimic; 6,8-bis(acetylthio)octanoic acid (SAc), when compared with rPDC-E2, strongly support a xenobiotic etiology in PBC. This observation is of particular significance in that approximately one third of patients who have taken excessive acetaminophen (APAP) developed AMA with same specificity as patients with PBC, suggesting that the lipoic domain are a target of APAP electrophilic metabolites such as NAPQI. We submit that in genetically susceptible hosts, electrophilic modification of lipoic acid in PDC-E2 by acetaminophen or similar drugs can facilitate loss of tolerance and lead to the development of PBC.

Overexpression of microRNA-21 is associated with elevated pro-inflammatory cytokines in dominant-negative TGF-ß receptor type II mouse.

Dominant-negative TGF-ß receptor II (dnTGF-ßRII) mice spontaneously develop an autoimmune cholangitis resembling human primary biliary cirrhosis (PBC). Interestingly, the dominant-negative TGF-ß receptor is expressed by both CD4(+) and CD8(+) T cells and leads to greatly reduced (but not absent) TGF-ß signaling resulting in T cell intrinsic cell mediated autoimmunity. However, the mechanisms of the T cell dysregulation remain unclear. Recently it has been shown that TGF-ß signaling is intimately involved with miRNA biogenesis and control. Herein we show that lack of T cell TGF-ß signaling leads to down regulation of T cell miRNAs but up-regulation of the key inflammatory miRNA 21. Furthermore, the expression of miR-21 from hepatic effector CD8(+) T cells is significantly higher than in the same subsets isolated from spleen and mesenteric lymph nodes of the dnTGF-ßRII mice. Previous studies indicate that miR-21 increases the synthesis of IFN-γ and IL-17A by T cells and suppresses apoptosis via programmed cell death protein 4 (PDCD4). Data presented herein demonstrate that transfecting w.t. B6 T cell subsets with miR-21 resulted in up-regulation of the inflammatory cytokines TNF-α and IFN-γ, thus partly replicating the dnTGF-ßRII T cell phenotype. In conclusion, these data suggest miR-21 plays a critical role in the production of pro-inflammatory cytokines in dnTGF-ßRII mice, which could be a contributing factor for the development of the organ-specific autoimmune cholangitis and colitis in this murine model of human PBC.

Mincle and human B cell function.

C-type lectin receptors are pattern recognition receptors that are critical for autoimmunity and the immune response. Mincle is a C-type lectin receptor expressed by a variety of antigen presenting cells including macrophages, neutrophils, dendritic cells and B cells; a variety of stimuli including stress are known to induce the expression of Mincle. Mincle is an FcRγ-associated activation receptor that senses damaged cells and upon ligation induces activated macrophages to produce inflammatory cytokines. Recently, while several studies have reported that Mincle plays an important role in macrophage responses to fungal infection its function on B cells remains to be defined. In efforts to elucidate the function of Mincle expressed by B cells, we studied the expression of Mincle on subsets of B cells and analyzed cytokines and synthesized immunoglobulin upon ligation of Mincle. The expression of Mincle on CD27-CD19(+) naïve B cells is significantly higher than CD27 + CD19(+) memory B cells. The stimulation of TLR9 ligand induced Mincle expression on B cells. Furthermore, co-stimulation of TLR9 and Mincle ligand reduced IgG and IgA production from B cells without a significant change in the inflammatory cytokines TNF-α, IL-6, IL-8 and IL-10. Our data identifies Mincle as a potentially critical player in human B cell responses.

Electrophile-modified lipoic derivatives of PDC-E2 elicits anti-mitochondrial antibody reactivity.

Our laboratory has hypothesized that xenobiotic modification of the native lipoyl moiety of the major mitochondrial autoantigen, the E2 subunit of the pyruvate dehydrogenase complex (PDC-E2), may lead to loss of self-tolerance in primary biliary cirrhosis (PBC). This thesis is based on the finding of readily detectable levels of immunoreactivity of PBC sera against extensive panels of protein microarrays containing mimics of the inner lipoyl domain of PDC-E2 and subsequent quantitative structure-activity relationships (QSARs). Importantly, we have demonstrated that murine immunization with one such mimic, 2-octynoic acid coupled to bovine serum albumin (BSA), induces anti-mitochondrial antibodies (AMAs) and cholangitis. Based upon these data, we have focused on covalent modifications of the lipoic acid disulfide ring and subsequent analysis of such xenobiotics coupled to a 15mer of PDC-E2 for immunoreactivity against a broad panel of sera from patients with PBC and controls. Our results demonstrate that AMA-positive PBC sera demonstrate marked reactivity against 6,8-bis(acetylthio)octanoic acid, implying that chemical modification of the lipoyl ring, i.e. disruption of the S-S disulfide, renders lipoic acid to its reduced form that will promote xenobiotic modification. This observation is particularly significant in light of the function of the lipoyl moiety in electron transport of which the catalytic disulfide constantly opens and closes and, thus, raises the intriguing thesis that common electrophilic agents, i.e. acetaminophen or non-steroidal anti-inflammatory drugs (NSAIDs), may lead to xenobiotic modification in genetically susceptible individuals that results in the generation of AMAs and ultimately clinical PBC.

Assessment of IgE response and cytokine gene expression in pulmonary efferent lymph collected after ovalbumin inhalation during experimental infection of calves with bovine respiratory syncytial virus.

OBJECTIVE: To assess IgE response and cytokine gene expressions in pulmonary lymph collected from bovine respiratory syncytial virus (BRSV)-infected calves after ovalbumin inhalation. ANIMALS: Thirteen 7- to 8-week-old calves. PROCEDURES: The efferent lymphatic duct of the caudal mediastinal lymph node of each calf was cannulated 3 or 4 days before experiment commencement. Calves were inoculated (day 0) with BRSV (n = 7) or BRSV-free tissue culture medium (mock exposure; 6) via aerosolization and exposed to aerosolized ovalbumin on days 1 through 6 and day 15. An efferent lymph sample was collected daily from each calf on days -1 through 16; CD4+ and CD8+ T lymphocyte subsets in lymph samples were enumerated with a fluorescence-activated cell scanner. Expressions of several cytokines by efferent lymphocytes and lymph ovalbumin-specific IgE concentration were measured. Each calf was euthanized on day 16 and then necropsied for evaluation of lungs. RESULTS: Mean fold increase in ovalbumin-specific IgE concentration was greater in BRSV-infected calves than in mock-infected calves. At various time points from days 4 through 10, percentages of T lymphocyte subsets and CD4+:CD8+ T lymphocyte ratios differed between BRSV-infected calves and day -1 values or from values in mock-infected calves. On days 3 through 5, IL-4 and IL-13 gene expressions in BRSV-infected calves were increased, compared with expressions in mock-infected calves. Lung lesions were consistent with antigen exposure. CONCLUSIONS AND CLINICAL RELEVANCE: In response to the inhalation of aerosolized ovalbumin, BRSV infection in calves appeared to facilitate induction of a T helper 2 cell response and ovalbumin-specific IgE production.

Immune effects of cocoa procyanidin oligomers on peripheral blood mononuclear cells.

There has been considerable work on the relationships between nutrition and the immune response, particularly on studies that have focused on adaptive responses. There is increasing recognition of the importance of innate immunity in host protection and initiation of cytokine networks. In this study, we examined the effect of select cocoa flavanols and procyanidins on innate responses in vitro. Peripheral blood mono-nuclear cells (PBMCs), as well as purified monocytes and CD4 and CD8 T cells, were isolated from healthy volunteers and cultured in the presence of cocoa flavanol fractions that differ from another by the degree of flavanol polymerization: short-chain flavanol fraction (SCFF), monomers to pentamers; and long-chain flavanol fraction (LCFF), hexamers to decamers. Parallel investigations were also done with highly purified flavanol monomers and procyanidin dimers. The isolated cells were then challenged with lipopolysaccharide (LPS) with quantitation of activation using CD69 and CD83 expression and analysis of secreted tumor necrosis factor (TNF)-alpha, interleukin (IL)-1beta, IL-6, IL-10, and granulocyte macrophage colony-stimulating factor (GM-CSF). The chain length of flavanol fractions had a significant effect on cytokine release from both unstimulated and LPS-stimulated PBMCs. For example, there was a striking increase of LPS-induced synthesis of IL-1beta, IL-6, IL-10, and TNF-alpha in the presence of LCFF. LCFF and SCFF, in the absence of LPS, stimulated the production of GM-CSF. In addition, LCFF and SCFF increased expression of the B cell markers CD69 and CD83. There were also unique differential responses in the mononuclear cell populations studied. We conclude that the oligomers are potent stimulators of both the innate immune system and early events in adaptive immunity.

A contemporary perspective on the molecular characteristics of mitochondrial autoantigens and diagnosis in primary biliary cholangitis.

Primary biliary cholangitis (PBC) is an autoimmune hepatobiliary disease characterized by immune mediated destruction of the intrahepatic small bile ducts and the presence of antimitochondrial antibodies (AMAs). The mitochondrial autoantigens have been identified as the E2 subunits of the 2-oxo-acid dehydrogenase complex, including the E2 subunits of pyruvate dehydrogenase, branched-chain 2-oxo acid dehydrogenase complex, oxoglutarate dehydrogenase complex, E3 binding protein and PDC E1 alpha subunit. The AMA epitope is mapped within the E2 lipoic acid binding domain, which is particularly important for oxidative phosphorylation. In addition, lipoic acid, which serves as a swinging arm to capture electrons, is particularly susceptible to an electrophilic attack and may provide clues to the etiology of PBC. This review emphasizes the molecular characteristics of AMAs, including detection, immunochemistry and the putative role in disease. These data have significance not only specifically for PBC, but generically for autoimmunity.

Chemokine and chemokine receptors in autoimmunity: the case of primary biliary cholangitis.

Chemokines represent a major mediator of innate immunity and play a key role in the selective recruitment of cells during localized inflammatory responses. Beyond critical extracellular mediators of leukocyte trafficking, chemokines and their cognate receptors are expressed by a variety of resident and infiltrating cells (monocytes, lymphocytes, NK cells, mast cells, and NKT cells). Chemokines represent ideal candidates for mechanistic studies (particularly in murine models) to better understand the pathogenesis of chronic inflammation and possibly become biomarkers of disease. Nonetheless, therapeutic approaches targeting chemokines have led to unsatisfactory results in rheumatoid arthritis, while biologics against pro-inflammatory cytokines are being used worldwide with success. In this comprehensive review we will discuss the evidence supporting the involvement of chemokines and their specific receptors in mediating the effector cell response, utilizing the autoimmune/primary biliary cholangitis setting as a paradigm.

Elevated expression of tyrosine kinase DDR2 in primary biliary cirrhosis.

BACKGROUND: Primary biliary cirrhosis (PBC) is characterized by chronic progressive destruction of small intrahepatic bile ducts with portal inflammation and cholestasis, leading to fibrosis. METHODS: We utilized a novel restriction analysis system to profile the expression of tyrosine kinases (TKs). This methodology targets a conserved sequence present in the majority of human TKs, and exploits the known restriction map of the TK cDNA sequences. We isolated mRNA from biliary epithelial cell (BEC)-enriched cell fractions, amplified the TK transcripts using degenerative primers, and identified specific TKs by restriction enzyme digest analysis and then performed in situ hybridization. RESULTS: BEC-enriched samples from PBC livers displayed marked expression of discoidin domain receptor-2 (DDR2), whereas, non-diseased livers showed no detectable DDR2. Furthermore, in situ hybridization of PBC livers revealed that DDR2 is expressed in the small bile duct epithelial regions as well as in fibroblasts/stromal cells of fibrotic regions. A similar pattern was observed in livers of primary sclerosing cholangitis (PSC), although the amount of small ducts that were positively stained was lower than in PBC. Furthermore, cirrhotic livers of patients with other diseases, including alcoholic liver disease and chronic hepatitis C, DDR2 transcripts were noted only within fibrotic lesions and the degree of intensity was much lower than in PBC and PSC. CONCLUSIONS: DDR2, a TK that is stimulated by fibrillar collagens that accumulate in cirrhotic livers, is present at elevated levels in the small bile ducts of PBC patients. DDR2 is part of a positive feedback loop in which its enhanced expression leads to enhanced deposition of fibrillar collagens (types I andIII). These fibrillar collagens can also provide binding sites for immune mediators, such as cytokines and chemokines. Therefore, unusually high DDR2 expression in the bile ducts of PBC patients could contribute to duct injury by altering local cytokine levels and thereby increasing immune-mediated damage.

Cocoa procyanidins inhibit proliferation and angiogenic signals in human dermal microvascular endothelial cells following stimulation by low-level H2O2.

Procyanidins extracted from cocoa play a role in the defense against oxidative stress, as well as in vascular and immune functions. We previously reported that pentameric procyanidins isolated from cocoa inhibit the expression of the tyrosine kinase ErbB2 gene, thus slowing the growth of cultured human aortic endothelial cells. We herein investigate the further consequences of such inhibition by cocoa procyanidins, particularly regarding the protein level in phosphorylation patterns and the effects on the proliferation of human dermal microvascular endothelial cells (HDMECs) following angiogenic stimulation with low-level H2O2. We report herein that both the pentameric and octameric procyanidin fractions of cocoa inhibit the proliferation of HDMECs, whereas the pentameric fraction modulates the activity of several crucial proteins in angiogenic signaling by altering their tyrosine phosphorylation. Similar to aortic endothelial cells, the pentameric procyanidin fraction down-regulates the expression of ErbB2 tyrosine kinase in HDMECs. In conclusion, we report evidence suggesting that polyphenols may influence endothelial growth signaling, thus affecting angiogenesis in vitro. If these observations are applicable in vivo, they suggest a beneficial effect for cells overexpressing ErbB2, such as in specific neoplasias

Pentameric procyanidins isolated from Theobroma cacao seeds selectively downregulate ErbB2 in human aortic endothelial cells.

Flavonoids isolated from cocoa have biological activities relevant to oxidant defenses, vascular health, tumor suppression, and immune function. The intake of certain dietary flavonoids, along with other dietary substances such as tocopherols, ascorbate, and carotenoids, is epidemiologically associated with a reduced risk of cardiovascular disease. Flavonoids have also been shown to modulate tumor pathology in vitro and in animal models. We took advantage of the conserved sequences found in tyrosine kinases to study the influence of cocoa fractions and controls on gene expression. We report that the pentameric procyanidin (molecular weight of 1442 daltons) fraction isolated from cocoa was a potent inhibitor of tyrosine kinase ErbB2 expression, a receptor important in angiogenesis regulation. Consistent with this primary observation, the cocoa flavonoid fraction also suppressed human aortic endothelial cell (HAEC) growth and decreased expression of two tyrosine kinases responsive to ErbB2 modulation, namely VEGFR-2/KDR and MapK 11/p38beta2. These inhibitory effects were observed when HAECs were treated with the flavonol fraction (molecular weight 280 daltons) isolated from cocoa, which comprise the structural subunits from which the procyanidin flavonoid subclass is biosynthetically constructed. Down-regulation of ErbB2 and inhibition of HAEC growth by cocoa procyanidins may have several downstream implications, including reduced vascular endothelial growth factor (VEGF) activity and angiogenic activity associated with tumor pathology. These results suggest specific dietary flavonoids are capable of selectively inhibiting ErbB2 and therefore may offer important insight into the design of therapeutic agents that target tumors overexpressing ErbB2.

Stem cell therapy in autoimmune rheumatic diseases: a comprehensive review.

The clinical management of autoimmune rheumatic diseases (ARD) has undergone significant changes in the last few decades, leading to remarkable improvements in clinical outcomes of many patients with mild to moderate ARD. On the other hand, severe refractory ARD patients often have high morbidity and mortality. Extensive basic research and clinical evidence has opened the door to new encouraging perspectives, such as the establishment of a role of stem cell transplantation (SCT) in the strategic management of ARD. Given the great heterogeneity of ARD, it is difficult to assign an optimal SCT regimen to all ARD patients. SCT remains a challenging mode of therapy in ARD patients from the standpoints of both efficacy and safety. As the clinical data of SCT in ARD increases and as we improve our understanding of stem cell biology and the downstream effects on the immune system, the future is promising for the development of optimal personalized SCT regimens in ARD.

Current immunological and molecular biological perspectives on seafood allergy: a comprehensive review.

Seafood is an important component in human diet and nutrition worldwide. However, seafood also constitutes one of the most important groups of foods in the induction of immediate (type I) food hypersensitivity, which significantly impacts the quality of life and healthcare cost. Extensive efforts within the past two decades have revealed the molecular identities and immunological properties of the major fish and shellfish allergens. The major allergen involved in allergy and cross-reactivity among different fish species was identified as parvalbumin while that responsible for shellfish (crustaceans and mollusks) allergy was identified as tropomyosin. The cloning and expression of the recombinant forms of these seafood allergens facilitate the investigation on the detailed mechanisms leading to seafood allergies, mapping of IgE-binding epitopes, and assessment of their allergenicity and stability. Future research focusing on the immunological cross-reactivity and discovery of novel allergens will greatly facilitate the management of seafood allergies and the design of effective and life-long allergen-specific immunotherapies.

The effects of Spirulina on anemia and immune function in senior citizens.

Anemia and immunological dysfunction (i.e. immunosenescence) are commonly found in older subjects and nutritional approaches are sought to counteract these phenomena. Spirulina is a filamentous and multicellular bule-green alga capable of reducing inflammation and also manifesting antioxidant effects. We hypothesized that Spirulina may ameliorate anemia and immunosenescence in senior citizens with a history of anemia. We enrolled 40 volunteers of both sexes with an age of 50 years or older who had no history of major chronic diseases. Participants took a Spirulina supplementation for 12 weeks and were administered comprehensive dietary questionnaires to determine their nutritional regimen during the study. Complete cell count (CCC) and indoleamine 2,3-dioxygenase (IDO) enzyme activity, as a sign of immune function, were determined at baseline and weeks 6 and 12 of supplementation. Thirty study participants completed the entire study and the data obtained were analyzed. Over the 12-week study period, there was a steady increase in average values of mean corpuscular hemoglobin in subjects of both sexes. In addition, mean corpuscular volume and mean corpuscular hemoglobin concentration also increased in male participants. Older women appeared to benefit more rapidly from Spirulina supplements. Similarly, the majority of subjects manifested increased IDO activity and white blood cell count at 6 and 12 weeks of Spirulina supplementation. Spirulina may ameliorate anemia and immunosenescence in older subjects. We encourage large human studies to determine whether this safe supplement could prove beneficial in randomized clinical trials.

Development and validation of gene therapies in autoimmune diseases: Epidemiology to animal models.

Recent advancement in immunology, molecular biology, and bioinformatics has yielded extensive information on the pathophysiological mechanisms of autoimmunity, which has greatly facilitated the identification of potential therapeutic targets and the development of gene therapy in the treatment of autoimmune disease. Preclinical studies were carried out in animal models. This phenomenon is well illustrated in two prototypic animal models of autoimmune disease: the autoimmune encephalomyelitis (EAE) model of multiple sclerosis (MS) and collagen-induced arthritis (CIA) model in rheumatoid arthritis (RA). Here we discuss the current data on the development and validation of gene therapy in autoimmunity in these two models. The success in preclinical animal model studies provides the proof-of-concept of gene therapy for potential future applications in the treatment of autoimmune diseases. Furthermore, the identification of risk factors from epidemiological studies reveals further potential therapeutic targets to be examined in animal models.