The lymphocytic infiltration in calcific aortic stenosis predominantly consists of clonally expanded T cells

Valve lesions in degenerative calcific aortic stenosis (CAS), a disorder affecting 3% of those older than 75 years, are infiltrated by T lymphocytes. We sought to determine whether the alphabeta TCR repertoire of these valve-infiltrating lymphocytes exhibited features either of a polyclonal nonselective response to inflammation or contained expanded clones suggesting a more specific immune process. TCR beta-chain CDR3-length distribution analysis using PCR primers specific for 23 Vbeta families performed in eight individuals with CAS affecting tri- or bileaflet aortic valves revealed considerable oligoclonal T cell expansion. In five cases, beta-chain nucleotide sequencing in five selected Vbeta families showed that an average of 92% of the valve-infiltrating T cell repertoire consisted of expanded T cell clones, differing markedly in composition from the relatively more polyclonal peripheral CD8 or CD4 T cell subsets found even in this elderly population. Twenty-four of the valve-infiltrating T cell clones also had the same clone identified in blood, some of which were highly expanded. Interestingly, 22 of these 24 shared clones were CD8 in lineage (p = 1.5 x 10(-12)), suggesting a possible relationship to the expanded CD8(+)CD28(-) T cell clones frequently present in the elderly. Additionally, the sequences of several TCR beta-chain CDR3 regions were homologous to TCR beta-chains identified previously in allograft arteriosclerosis. We infer that these findings are inconsistent with a nonselective secondary response of T cells to inflammation and instead suggest that clonally expanded alphabeta T cells are implicated in mediating a component of the valvular injury responsible for CAS.     

Knotting the NETs: Analyzing histone modifications in neutrophil extracellular traps

Neutrophil extracellular chromatin traps (NETs) are a recently described mechanism of innate immune responses to bacteria and fungi. Evidence indicates that NETs are induced by inflammation, that they contribute to diverse disease pathologies, and that they associate with bactericidal substances. Genomic DNA is released in NETs, leading to a cell death that has been labeled NETosis. Although NETosis clearly differs from apoptosis, the classical form of cell death, recent experiments indicate a connection between NETosis and autophagy. The regulated deployment of NETs may require covalent modification of histones, the basic DNA-binding proteins that organize chromatin in the cell''s nucleus and within NETs. Histone modification by peptidylarginine deiminase 4 (PAD4) is necessary for NET release. The functions of additional histone modifications, however, remain to be tested.Less than a decade since their discovery, neutrophil extracellular traps (NETs) remain in the headlines. Initially, interest focused on the structure of extracellular NET chromatin and its capacity to capture and damage bacteria. Soon, however, researchers began to see the implications of extracellular chromatin for the development of autoimmune diseases. One quintessential autoimmune disease, systemic lupus erythematosus (SLE), is known to arise together with autoantibodies to DNA and chromatin, although the immediate trigger for the production of these autoantibodies is unclear. A connection between NETs and autoimmunity was made by discovering that histones, a set of proteins that act as a structural harness for DNA in chromatin, are modified by peptidylarginine deiminase 4 (PAD4), an enzyme that converts arginines to citrullines. Researchers had long suspected that autoantigen modifications could provide the initial stimuli in autoimmunity because subtle alterations in a protein''s primary sequence can break tolerance. PAD4 is implicated in the development of rheumatoid arthritis (RA) because the most reliable clinical test for RA uses the detection of anti-citrulline antibodies in the sera of patients.In a sophisticated set of experiments reported in the previous issue of Arthritis Research & Therapy, Liu and colleagues [1] accomplished an extensive inventory of post-translational modifications in NET histones. The researchers induced NETs from human neutrophils, as well as two cell lines that assume neutrophil-like characteristics, and used a panel of 40 commercially available antisera to identify histone modifications that arise in parallel with NETs. Stimuli that were used to elicit NET release also induced histone H3 and H4 citrullination in human neutrophils and the EPRO cell line. However, other modifications such as histone H4 lysine 20 methylation and H4 lysine 16 acetylation showed inconsistent results in neutrophils versus the EPRO cells. To survey histone modifications, Liu and colleagues [1] confronted technical difficulties in that histone amino terminal tails contain the highest concentration of histone modifications yet are also highly susceptible to proteases secreted by activated neutrophils [2,3]. The histone tails act as flexible tethers that organize chromatin into higher-order structures. Interestingly, purified NETs failed to induce an immune response in mice, although a subset of SLE sera reacted strongly with citrullinated histone H3 [1]. Therefore, mechanisms that regulate histone modification deserve further attention.Neeli and colleagues [4] were the first to identify citrullinated histone H3 in NETs, a discovery that was confirmed by others [5]. Neeli and colleagues [4] provided a second important insight, namely that PAD4-citrullinated histone H3 is a reliable marker of inflammation. Thus, it became clear that the release of NETs is not an ''accident'' caused by a barrage of proteases and reactive oxygen species unleashed from neutrophils. Instead, production of NETs requires enzymatic activity and input from neutrophil surface receptors and the cytoskeleton [6]. By analyzing PAD4-deficient mice, Li and colleagues [7] demonstrated that PAD4 is essential for the production of NETs in response to bacterial infections. The regulation of PAD4 activity thus moved to the forefront of the research on NETs.It is now clear that NET release takes advantage of NADPH (nicotinamide adenine dinucleotide phosphate) oxidase and the main granule proteases to trigger and construct the extended chromatin network [3,8]. In addition, myeloperoxidase is found in NETs after their release from the cells, and this enzyme and its products are the main components in NETs that kill bacteria [9]. In a notable study from the labs of Banchereau and Pascual [10], it was reported that SLE neutrophils are poised to undergo NETosis upon stimulation with anti-ribonucleo-protein autoantibodies and that NETs released by these neutrophils contain LL37 and HMGB-1, well-known stimulators of immune responses. In subsequent analyses using sera from patients with connective tissue disease, anti-citrullinated histone antibodies were observed in Felty''s syndrome, a rare disorder that shares serologic features with RA and SLE, whereas such autoantibodies were infrequent in SLE and RA [11]. These findings indicate that the process of NETosis is highly relevant to the development of human autoimmune responses, although a direct cause and effect may not connect the release of NETs to the production of autoantibodies.The detailed characterization of NET histone modifications, as accomplished by Liu and colleagues [1], invites speculations about the possible functions of these modifications. Several questions deserve further study: Will NET histone modifications, such as methylation, acetylation, and citrullination, be found to participate in gene regulation that sets the stage for NET release? Will the primary function of histone modifications turn out to be the decondensation of nuclear chromatin that is required for NETs expand to their optimal size and internal structure? Alternatively, NET histone modifications may serve non-traditional purposes. For example, certain modifications may anchor other NET components such as elastase, LL37, or myeloperoxidase to the chromatin meshwork. Unique modifications in NETs may attract phagocytes and stimulate them to ingest the trapped microorganisms. Other histone modifications may activate or dampen the inflammatory response by acting on innate pattern recognition receptors. The answers to these questions will, no doubt, keep research on NETs in leading immunology and microbiology journals for years to come.     

The crystal structure of the FAD/NADPH-binding domain of flavocytochrome P450 BM3

We report the crystal structure of the FAD/NADPH-binding domain (FAD domain) of the biotechnologically important Bacillus megaterium flavocytochrome P450 BM3, the last domain of the enzyme to be structurally resolved. The structure was solved in both the absence and presence of the ligand NADP(+), identifying important protein interactions with the NADPH 2'-phosphate that helps to dictate specificity for NADPH over NADH, and involving residues Tyr974, Arg966, Lys972 and Ser965. The Trp1046 side chain shields the FAD isoalloxazine ring from NADPH, and motion of this residue is required to enable NADPH-dependent FAD reduction. Multiple binding interactions stabilize the FAD cofactor, including aromatic stacking with the adenine group from the side chains of Tyr860 and Trp854, and several interactions with FAD pyrophosphate oxygens, including bonding to tyrosines 828, 829 and 860. Mutagenesis of C773 and C999 to alanine was required for successful crystallization, with C773A predicted to disfavour intramolecular and intermolecular disulfide bonding. Multiangle laser light scattering analysis showed wild-type FAD domain to be near-exclusively dimeric, with dimer disruption achieved on treatment with the reducing agent dithiothreitol. By contrast, light scattering showed that the C773A/C999A FAD domain was monomeric. The C773A/C999A FAD domain structure confirms that Ala773 is surface exposed and in close proximity to Cys810, with this region of the enzyme's connecting domain (that links the FAD domain to the FMN-binding domain in P450?BM3) located at a crystal contact interface between FAD domains. The FAD domain crystal structure enables molecular modelling of its interactions with its cognate FMN (flavodoxin-like) domain within the BM3 reductase module.     

Apo[a] size and PNR explain African American-Caucasian differences in allele-specific apo[a] levels for small but not large apo[a

Apolipoprotein [a] (apo[a]) gene size is a major predictor of lipoprotein [a] level. To determine genetic predictors of allele-specific apo[a] levels beyond gene size, we evaluated the upstream C/T and pentanucleotide repeat (PNR) polymorphisms. We determined apo[a] sizes, allele-specific apo[a] levels, and C/T and PNR in 215 Caucasians and 139 African Americans. For Caucasians, apo[a] size affected allele-specific levels substantially greater in subjects with apo[a] < 24 K4; for African Americans, the size effect was smaller than in Caucasians, <24 K4, but did not decrease at higher repeats. In both groups, the level decreased with increasing size of the other allele. Controlling for apo[a] sizes, PNR decreased allele-specific apo[a] levels in Caucasians with increasing PNR > 8. In a multiple regression model, apo[a] allele size and size and expression of the other apo[a] allele (and PNR > 8 for Caucasians) significantly predicted allele-specific apo[a] levels. For a common PNR 8 allele, predicted values were similar in the two ethnicities for small size apo[a]. Allele-specific apo[a] levels were influenced by the other allele size and expression. Observed differences between Caucasians and African Americans in allele-specific apo[a] levels were explained for small apo[a] sizes by the other allele size and PNR; the ethnicity differences remain unexplained for larger sizes.     

Conjunctival MicroRNA Expression in Inflammatory Trachomatous Scarring

Purpose

Trachoma is a fibrotic disease of the conjunctiva initiated by Chlamydia trachomatis infection. This blinding disease affects over 40 million people worldwide yet the mechanisms underlying its pathogenesis remain poorly understood. We have investigated host microRNA (miR) expression in health (N) and disease (conjunctival scarring with (TSI) and without (TS) inflammation) to determine if these epigenetic differences are associated with pathology.

Methods

We collected two independent samples of human conjunctival swab specimens from individuals living in The Gambia (n = 63 & 194). miR was extracted, and we investigated the expression of 754 miR in the first sample of 63 specimens (23 N, 17 TS, 23 TSI) using Taqman qPCR array human miRNA genecards. Network and pathway analysis was performed on this dataset. Seven miR that were significantly differentially expressed between different phenotypic groups were then selected for validation by qPCR in the second sample of 194 specimens (93 N, 74 TS, 22 TSI).

Results

Array screening revealed differential expression of 82 miR between N, TS and TSI phenotypes (fold change >3, p<0.05). Predicted mRNA targets of these miR were enriched in pathways involved in fibrosis and epithelial cell differentiation. Two miR were confirmed as being differentially expressed upon validation by qPCR. miR-147b is significantly up-regulated in TSI versus N (fold change = 2.3, p = 0.03) and miR-1285 is up-regulated in TSI versus TS (fold change = 4.6, p = 0.005), which was consistent with the results of the qPCR array.

Conclusions

miR-147b and miR-1285 are up-regulated in inflammatory trachomatous scarring. Further investigation of the function of these miR will aid our understanding of the pathogenesis of trachoma.     

Phytochrome Control of Acid and Alkaline Phosphatases in Etiolated Shoot Apices of Sorghum bicolor

A short pulse of red light or continuous far-red light enhancedthe activities of acid and alkaline phosphatases over the valuesof the dark controls in 5-day-old etiolated seedlings of Sorghumbicolor. For 30 min after the red light pulse 100% of the red/far-redphotoreversibilities was maintained for the acid and 80% forthe alkaline phosphatases. Thereafter, the "photoreversibilityescape reaction" was fast, being completed within 180 min. Cycloheximideas well as 6-methyl purine markedly inhibited red light enhancementof the activities of the phosphatases, but chloramphenicol,lincomiycin and rifamycin SV were ineffective. In spite of photoregulationof both the phosphatases at the time of de novo synthesis, itappears that control of the acid and alkaline phosphatases maybe affected by two independent initial actions of phytochrome. ¹ Present address: Biologisches Institut II, University of Freiburg,FRG. (Received August 4, 1984; Accepted April 3, 1985)     

Transplacental delivery of retinoid: the role of retinol-binding protein and lipoprotein retinyl ester

Retinoids are required for normal embryonic development. Both embryonic retinoid deficiency and excess result in congenital malformations. There is little understanding of the physiology underlying retinoid transfer from the maternal circulation to the embryo. We now report studies that explore this process using retinol-binding protein-deficient (RBP-/-) mice and mice that express human RBP on the RBP-/-) background. Our studies establish that dietary retinoid, bound to lipoproteins, can serve as an important source for meeting tissue retinoid requirements during embryogenesis. Indeed, retinyl ester concentrations in the circulations of pregnant RBP-/- mice are significantly elevated over those observed in wild-type mice, suggesting that lipoprotein retinyl esters may compensate for the absence of retinol-RBP during pregnancy. We also demonstrate, contrary to earlier proposals, that maternal RBP does not cross the placenta and cannot enter the fetal circulation. Overall, our data indicate that both retinol-RBP and retinyl esters bound to lipoproteins are able to provide sufficient retinoid to the embryo to allow for normal embryonic development.