p62 Is a Common Component of Cytoplasmic Inclusions in Protein Aggregation Diseases

Exposure of cells to stress, particularly oxidative stress, leads to misfolding of proteins and, if they are not refolded or degraded, to cytoplasmic protein aggregates. Protein aggregates are characteristic features of a variety of chronic toxic and degenerative diseases, such as Mallory bodies (MBs) in hepatocytes in alcoholic and non-alcoholic steatohepatitis, neurofibrillary tangles in neurons in Alzheimer's, and Lewy bodies in Parkinson's disease. Using 2D gel electrophoresis and mass spectrometry, we identified p62 as a novel MB component. p62 and cytokeratins (CKs) are major MB constituents; HSP 70, HSP 25, and ubiquitinated CKs are also present. These proteins characterize MBs as a prototype of disease-associated cytoplasmic inclusions generated by stress-induced protein misfolding. As revealed by transfection of tissue culture cells overexpressed p62 did not induce aggregation of regular CK filaments but selectively bound to misfolded and ubiquitinated CKs. The general role of p62 in the cellular response to misfolded proteins was substantiated by detection of p62 in other cytoplasmic inclusions, such as neurofibrillary tangles, Lewy bodies, Rosenthal fibers, intracytoplasmic hyaline bodies in hepatocellular carcinoma, and alpha1-antitrypsin aggregates. The presence of p62 along with other stress proteins and ubiquitin in cytoplasmic inclusions indicates deposition as aggregates as a third line of defense against misfolded proteins in addition to refolding and degradation.     

Rheumatoid arthritis

Summary: Therapeutic efficacy of depleting B cells or blocking T‐cell costimulation in rheumatoid arthritis (RA) has confirmed the critical pathogenic role of adaptive immune responses. Yet, RA preferentially affects elderly individuals, in whom adaptive immunity to exogenous antigens begins to fail. Here, we propose that senescence of the immune system is a risk factor for RA, with chronic inflammation resulting from the accumulation of degenerate T cells that have a low threshold for activation and utilize a spectrum of novel receptors to respond to microenvironmental cues. The process of immunosenescence is accelerated in RA and precedes the onset of disease, the acceleration, in part, being conferred by the HLA‐DR4 haplotype. Naive CD4⁺ T cells in RA are contracted in diversity and restricted in clonal burst. Senescence of effector CD4⁺ T cells is associated with the loss of CD28 and the de novo expression of KIR2DS2, NKG2D, and CX₃CR1, all of which function as costimulatory molecules and reduce the threshold for T‐cell activation. The synovial microenvironment promotes chronic persistent immune responses by facilitating ectopic lymphoid neogenesis, such as the formation of aberrant germinal centers. With the propensity to develop complex lymphoid architectures and to provide optimal activation conditions for senescent CD4⁺ T cells, the synovium becomes a natural target for pathogenic immune responses in prematurely aged individuals.     

Production of prostaglandin E2 in monocytes stimulated in vitro by Chlamydia trachomatis, Chlamydophila pneumoniae, and Mycoplasma fermentans

Chlamydia trachomatis (CT) as well as Chlamydophila pneumoniae (CP) cause chronic inflammatory diseases in humans. Persistently infected monocytes are involved in the pathogenesis by inducing mediators of inflammation. An in vitro system of chlamydial persistence in human peripheral blood monocytes (HPBM) was used to investigate prostaglandin E(2) (PGE(2)) production and the expression of the key enzyme for prostaglandin production, cyclooxygenase-2 (COX-2). PGE(2) production was determined by PGE(2)-ELISA of HPBM-culture supernatants. Cox-2 mRNA expression was measured by real-time RT-PCR of total RNA isolated from HPBM. Both, CT and CP, stimulated PGE(2) production of HPBM in vitro. Equivalent numbers of CT per host cell induced a higher PGE(2)-response compared to CP. The amount of synthesized PGE(2) depended on the chlamydial multiplicity of infection (MOI). Even at an MOI of 10 the amount of CT- and CP-induced prostaglandin, respectively, was lower than the amount of prostaglandin induced by E. coli lipopolysaccharide (LPS) at a concentration of 10microg/ml. In contrast to stimulation with LPS, Chlamydia-induced PGE(2) production as well as cox-2 mRNA decreased after day 1 post infection (p.i.). These data indicate that Chlamydia stimulate PGE(2) production in human monocytes. Since Chlamydia are often contaminated by mycoplasma, the influence of mycoplasma on the prostaglandin production was investigated additionally. Mycoplasma fermentans (MF) also stimulated PGE(2) production. The co-infection of mycoplasma and Chlamydia resulted in an additive effect in the production of PGE(2). Thus it is important to use host cells and Chlamydia free of mycoplasma contamination for the analysis of Chlamydia-induced prostaglandin production.     

Vegan Diet and Bone Health—Results from the Cross-Sectional RBVD Study

Scientific evidence suggests that a vegan diet might be associated with impaired bone health. Therefore, a cross-sectional study (n = 36 vegans, n = 36 omnivores) was used to investigate the associations of veganism with calcaneal quantitative ultrasound (QUS) measurements, along with the investigation of differences in the concentrations of nutrition- and bone-related biomarkers between vegans and omnivores. This study revealed lower levels in the QUS parameters in vegans compared to omnivores, e.g., broadband ultrasound attenuation (vegans: 111.8 ± 10.7 dB/MHz, omnivores: 118.0 ± 10.8 dB/MHz, p = 0.02). Vegans had lower levels of vitamin A, B2, lysine, zinc, selenoprotein P, n-3 fatty acids, urinary iodine, and calcium levels, while the concentrations of vitamin K1, folate, and glutamine were higher in vegans compared to omnivores. Applying a reduced rank regression, 12 out of the 28 biomarkers were identified to contribute most to bone health, i.e., lysine, urinary iodine, thyroid-stimulating hormone, selenoprotein P, vitamin A, leucine, α-klotho, n-3 fatty acids, urinary calcium/magnesium, vitamin B6, and FGF23. All QUS parameters increased across the tertiles of the pattern score. The study provides evidence of lower bone health in vegans compared to omnivores, additionally revealing a combination of nutrition-related biomarkers, which may contribute to bone health. Further studies are needed to confirm these findings.     

Meta-analysis of epigenome-wide association studies of carotid intima-media thickness

Common carotid intima-media thickness (cIMT) is an index of subclinical atherosclerosis that is associated with ischemic stroke and coronary artery disease (CAD). We undertook a cross-sectional epigenome-wide association study (EWAS) of measures of cIMT in 6400 individuals. Mendelian randomization analysis was applied to investigate the potential causal role of DNA methylation in the link between atherosclerotic cardiovascular risk factors and cIMT or clinical cardiovascular disease. The CpG site cg05575921 was associated with cIMT (beta?=??0.0264, p value?=?3.5?×?10^–8) in the discovery panel and was replicated in replication panel (beta?=??0.07, p value?=?0.005). This CpG is located at chr5:81649347 in the intron 3 of the aryl hydrocarbon receptor repressor gene (AHRR). Our results indicate that DNA methylation at cg05575921 might be in the pathway between smoking, cIMT and stroke. Moreover, in a region-based analysis, 34 differentially methylated regions (DMRs) were identified of which a DMR upstream of ALOX12 showed the strongest association with cIMT (p value?=?1.4?×?10^–13). In conclusion, our study suggests that DNA methylation may play a role in the link between cardiovascular risk factors, cIMT and clinical cardiovascular disease.

     

Ba2+ differentially inhibits the Rb+ efflux promoting and the vasorelaxant effects of levcromakalim and minoxidil sulfate in rat isolated aorta

The K⁺ channel openers activate ATP-sensitive K⁺ channels (K_ATP) in vascular smooth muscle and induce relaxation. In this study, the relationship between these two effects was examined in rings of rat aorta using levcromakalim and minoxidil sulfate as the openers and Ba²⁺ as the K⁺ channel blocker; K⁺ channel opening was assessed by determining the rate constant of ⁸⁶Rb⁺ efflux from the preparation.Ba²⁺ inhibited the ⁸⁶Rb⁺ efflux stimulated by levcromakalim in a noncompetitive manner with an IC₅₀ value of 29 M and a Hill-coefficient of 1.2. At concentrations > 300 M, Ba²⁺ increased the tension of rat aortic rings concentration-dependently. Levcromakalim relaxed contractions to Ba²⁺ (0.5 and 1 mM) with potencies similar to those determined against KCl (25 mM) or noradrenaline as spasmogens (EC₅₀ values 15–40 nM). The vasorelaxant effect against Ba²⁺ was inhibited by the K_ATP channel blockers, glibenclamide and tedisamil, and abolished in depolarizing medium (55 mM KCl). At 3 mM Ba²⁺, levcromakalim was still able to transiently induce complete relaxation; however, within 1 h oscillations in tension developed, leading to a stable level of only 15% relaxation. A similar level of relaxation was achieved against 10 mM Ba²⁺ whereas the combination of 0.5 mM Ba²⁺ and 3 M tedisamil blocked the relaxant effect of levcromakalim completely. With minoxidil sulfate as the K_ATP channel opener the results of the ⁸⁶Rb⁺ efflux and tension experiments were similar to those obtained with levcromakalim.It is concluded that Ba²⁺ is more potent in inhibiting the K⁺ channel opening than the vasorelaxant effects of the openers. On the basis of the ⁸⁶Rb⁺ efflux experiments it is estimated that at least 97% of the channels opened by the activators can be blocked without major effects on vasorelaxation suggesting a dissociation between the two effects. However, if the block is pushed to extremes ( 99.95%) the vasorelaxant effect of the openers is also abolished suggesting a link between both effects. This paradoxon remains to be solved.     

Growth Regulation of Thyroid and Thyroid Tumors in Humans

In a study of growth regulation of the human thyroid gland and thyroid tumors we investigated the impact of iodine and that of the thyroid-specific growth-stimulating hormone TSH. Further studies included locally active growth factors such as the epidermal growth factor, insulin-like growth factor, and tissue transforming growth factors alpha and beta. In addition to studies of growth regulation by the various growth factors in mostly normal thyrocytes, the impact of tumor-specific mutations in oncogenes and tumor-suppressor genes was investigated. The results demonstrated distinct changes in tissue specificity and sensitivity to external stimuli. This rather complex view on thyrocyte growth regulation may be confusing, but it describes the biologic reality more precisely. Increased knowledge of the regulatory processes may lead to the development of new tumor- and patient-specific therapeutic approaches, especially for preventing benign goiter recurrence and for treating follicular and papillary thyroid cancers.     

MTHFR C677T and A1298C polymorphisms: diet, estrogen, and risk of colon cancer.

5,10-methylenetetrahydrofolate reductase (MTHFR) is a key enzyme in folate metabolism, diverting metabolites toward methylation reactions or nucleotide synthesis. Using data from an incident case-control study (1608 cases and 1972 controls) we investigated two polymorphisms in the MTHFR gene, C677T and A1298C, and their associations with risk of colon cancer. All of the combined genotypes were evaluated separately, and the 1298AA/677CC (wild-type/wild-type) group was considered the reference group. Among both men and women, the 677TT/1298AA (variant/wild-type) genotype was associated with a small reduction in risk [men: odds ratio (OR), 0.7, 95% confidence interval (CI), 0.5-1.0; women: OR, 0.8, 95% CI, 0.5-1.2]. However, the 677CC/1298CC (wild-type/variant) genotype was associated with a statistically significant lower risk among women (OR, 0.6; 95% CI, 0.4-0.9) but not men. When the polymorphisms were considered individually, for A1298C a significant risk reduction associated with the homozygous variant CC genotype was seen among women only (OR, 0.6; 95% CI, 0.5-0.9), and nonstatistically significant reduced risks were observed for the variant 677 TT genotypes among both men and women. Stratification by nutrient intakes showed inverse associations with higher intakes of folate, vitamin B(2), B(6), B(12), and methionine among women with the MTHFR 677CC/1298AA genotypes, but not those with 677TT/1298AA. We observed opposite risk trends for both MTHFR variants, depending on whether women used hormone-replacement therapy or not (P for interaction = <.01). In summary, this study supports recent findings that the MTHFR A1298C polymorphism may be a predictor of colon cancer risk and have functional relevance. The possible interaction with hormone-replacement therapy warrants additional investigation.     

Expression of extracellular matrix metalloproteases inducer on micrometastatic and primary mammary carcinoma cells.

PURPOSE: EMMPRIN (extracellular matrix metalloprotease inducer) is a glycosylated member of the immunoglobulin superfamily known to stimulate the production of matrix metalloproteases (MMPs) 1, 2, and 3 and MT1-MMP in peritumoral fibroblasts. We here evaluated whether EMMPRIN expression is related to tumor progression in human breast cancer. EXPERIMENTAL DESIGN: An immunohistochemical study using high-density tissue microarrays (n = 2222 breast cancer samples) and EMMPRIN-specific antibodies HIM6 and MEM-M6/1 was performed, and staining results were statistically correlated with various clinicopathological parameters. To analyze the putative association between EMMPRIN expression and bone marrow (BM) micrometastasis, an additional set of 55 breast tumors from patients with or without micrometastatic cells as determined with anti-cytokeratin antibody A45-B/B3 were included in our study. Cytokeratin-positive cells in BM were costained with EMMPRIN-specific antibody 1G6.2. RESULTS: Positive EMMPRIN staining correlated significantly with various histopathological risk factors (higher tumor grade, increased tumor size, negative estrogen receptor status and progesterone receptor status, and higher mitotic index) as well as decreased tumor-specific survival (log-rank, P = 0.0027). In particular, in patients > 50 years (i.e., postmenopausal women), EMMPRIN expression was an independent prognosticator as shown by Cox regression analysis (relative risk = 1.7, 95% confidence interval 1.4-4.3, P = 0.036). An involvement of EMMPRIN in tumor progression was also supported by the fact that it was expressed on approximately 90% of micrometastatic cells in BM. CONCLUSIONS: EMMPRIN expression in primary tumor predicts an unfavorable prognosis in breast cancer, suggesting a crucial role of EMMPRIN in progression of human mammary carcinomas.