IL-8 as antibody therapeutic target in inflammatory diseases: reduction of clinical activity in palmoplantar pustulosis

IL-8 is a chemokine that has been implicated in a number of inflammatory diseases involving neutrophil activation. HuMab 10F8 is a novel fully human mAb against IL-8, which binds a discontinuous epitope on IL-8 overlapping the receptor binding site, and which effectively neutralizes IL-8-dependent human neutrophil activation and migration. We investigated whether interference in the cytokine network by HuMab 10F8 might benefit patients suffering from palmoplantar pustulosis, a chronic inflammatory skin disease. Treatment of patients with HuMab 10F8 was well tolerated and significantly reduced clinical disease activity at all five endpoints, which included a >or=50% reduction in the formation of fresh pustules. IL-8 neutralization was monitored at the site of inflammation by assessing exudates of palmoplantar pustulosis lesions. HuMab 10F8 sequestered IL-8 in situ, as observed by rapid dose-dependent decreases of IL-8 concentrations immediately following Ab infusion. These data demonstrate a critical role for IL-8 in the pathophysiology of palmoplantar pustulosis. HuMab 10F8 is capable of interrupting IL-8 activity in vivo and represents a candidate for treatment of inflammatory diseases and other pathological conditions associated with IL-8 overproduction.     

Differential expression of the human CD8beta splice variants and regulation of the M-2 isoform by ubiquitination

The CD8alphabeta heterodimer functions as a coreceptor with the TCR, influencing the outcome of CD8(+) T cell responses to pathogen-infected and tumor cells. In contrast to the murine CD8B gene, the human gene encodes alternatively spliced variants with different cytoplasmic tails (M-1, M-2, M-3, and M-4). At present, little is known about the expression patterns and functional significance of such variants. We used quantitative RT-PCR to demonstrate differential mRNA expression patterns of these splice variants in thymocytes and in resting, memory, and activated primary human CD8(+) T cells. In total CD8(+) T cells, mRNA levels of the M-1 variant were the most predominant and levels of M-3 were the least detected. The M-4 isoform was predominant in effector memory CD8(+) T cells. Upon stimulation of CD8(+) T cells, the M-2 variant mRNA levels were elevated 10-20-fold relative to resting cells in contrast to the other isoforms. Curiously, the M-2 isoform was not expressed on the cell surface in transfected cell lines. Using fluorescent chimeras of the extracellular domain of mouse CD8beta fused to the cytoplasmic tails of each isoform, the M-2 isoform was localized in a lysosomal compartment regulated by ubiquitination of a lysine residue (K215) in its cytoplasmic tail. In contrast, upon short-term stimulation, the M-2 protein localized to the cell surface with the TCR complex. The relatively recent evolution of CD8B gene splice variants in the chimpanzee/human lineage is most likely important for fine-tuning the CD8(+) T cell responses.     

Inhibition of arginase activity enhances inflammation in mice with allergic airway disease, in association with increases in protein S-nitrosylation and tyrosine nitration

Pulmonary inflammation in asthma is orchestrated by the activity of NF-kappaB. NO and NO synthase (NOS) activity are important modulators of inflammation. The availability of the NOS substrate, l-arginine, is one of the mechanisms that controls the activity of NOS. Arginase also uses l-arginine as its substrate, and arginase-1 expression is highly induced in a murine model of asthma. Because we have previously described that arginase affects NOx content and interferes with the activation of NF-kappaB in lung epithelial cells, the goal of this study was to investigate the impact of arginase inhibition on the bioavailability of NO and the implications for NF-kappaB activation and inflammation in a mouse model of allergic airway disease. Administration of the arginase inhibitor BEC (S-(2-boronoethyl)-l-cysteine) decreased arginase activity and caused alterations in NO homeostasis, which were reflected by increases in S-nitrosylated and nitrated proteins in the lungs from inflamed mice. In contrast to our expectations, BEC enhanced perivascular and peribronchiolar lung inflammation, mucus metaplasia, NF-kappaB DNA binding, and mRNA expression of the NF-kappaB-driven chemokine genes CCL20 and KC, and lead to further increases in airways hyperresponsiveness. These results suggest that inhibition of arginase activity enhanced a variety of parameters relevant to allergic airways disease, possibly by altering NO homeostasis.     

An endogenous prostaglandin enhances environmental phthalate-induced apoptosis in bone marrow B cells: activation of distinct but overlapping pathways

Phthalate esters are ubiquitous environmental contaminants that are produced for a variety of common industrial and commercial purposes. We have shown that mono-(2-ethylhexyl) phthalate (MEHP), the toxic metabolite of di-(2-ethylhexyl) phthalate, induces bone marrow B cell apoptosis that is enhanced in the presence of the endogenous prostaglandin 15-deoxy-Delta((12, 14))-PGJ(2) (15d-PGJ(2)). Here, studies were performed to determine whether 15d-PGJ(2)-mediated enhancement of MEHP-induced apoptosis represents activation of an overlapping or complementary apoptosis pathway. MEHP and 15d-PGJ(2) induced significant apoptosis within 8 and 5 h, respectively, in a pro/pre-B cell line and acted cooperatively to induce apoptosis in primary pro-B cells. Apoptosis induced with each chemical was accompanied by activation of a combination of initiator caspases (caspases-2, -8, and -9) and executed by caspase-3. Apoptosis induced with MEHP and 15d-PGJ(2) was reduced in APAF1 null primary pro-B cells and accompanied by alteration of mitochondrial membranes, albeit with different kinetics, indicating an intrinsically activated apoptosis pathway. Significant Bax translocation to the mitochondria supports its role in initiating release of cytochrome c. Both chemicals induced Bid cleavage, a result consistent with a truncated Bid-mediated release of cytochrome c in an apoptosis amplification feedback loop; however, significantly more Bid was cleaved following 15d-PGJ(2) treatment, potentially differentiating the two pathways. Indeed, Bid cleavage and cytochrome c release following 15d-PGJ(2) but not MEHP treatment was profoundly inhibited by Z-VAD-FMK, suggesting that 15d-PGJ(2) activates apoptosis via two pathways, Bax mobilization and protease-dependent Bid cleavage. Thus, endogenous 15d-PGJ(2)-mediated enhancement of environmental chemical-induced apoptosis represents activation of an overlapping but distinct signaling pathway.     

Identification of the proteins, including MAGEG1, that make up the human SMC5-6 protein complex

The SMC protein complexes play important roles in chromosome dynamics. The function of the SMC5-6 complex remains unclear, though it is involved in resolution of different DNA structures by recombination. We have now identified and characterized the four non-SMC components of the human complex and in particular demonstrated that the MAGEG1 protein is part of this complex. MAGE proteins play important but as yet undefined roles in carcinogenesis, apoptosis, and brain development. We show that, with the exception of the SUMO ligase hMMS21/hNSE2, depletion of any of the components results in degradation of all the other components. Depletion also confers sensitivity to methyl methanesulfonate. Several of the components are modified by sumoylation and ubiquitination.     

QTL analysis of early-season cold tolerance in sorghum

Cool temperatures during the early-growing season are a major limitation to growing sorghum [Sorghum bicolor (L.) Moench] in temperate areas. Several landraces from China have been found to exhibit higher emergence and greater seedling vigor under cool conditions than most breeding lines currently available, but tend to lack desirable agronomic characteristics. The introgression of desirable genes from Chinese landraces into elite lines could be expedited by marker-assisted selection. Using a population of 153 RI lines, developed from a cross between Chinese landrace ‘Shan Qui Red,’ (SQR, cold-tolerant) and SRN39 (cold-sensitive), QTL associated with early-season performance under both cold and optimal conditions were identified by single marker analysis, simple interval mapping (SIM), and composite interval mapping (CIM). Germination was observed under controlled conditions, and other traits were measured in field plantings. Two QTL for germination were identified: one on linkage group SBI-03a, derived from SRN39, was significant under cold and optimal temperatures. The other, on group SBI-07b, showed greater significance under cold temperatures and was contributed by SQR. A region of group SBI-01a, derived from SQR, showed strong associations with seedling emergence and seedling vigor scores under early and late field plantings. A QTL for both early and late emergence was identified by CIM on SBI-02 which favored the SRN39 allele. SIM identified a QTL for early vigor on SBI-04 favoring the SQR genotype. Further studies are needed to validate the effects of these QTL, but they represent the first step in development of a marker-assisted breeding effort to improve early-season performance in sorghum.     

Trypanosoma cruzi in non-human primates with a history of stillbirths: a retrospective study (Papio hamadryas spp.) and case report (Macaca fascicularis)

Background Congenital transmission of Trypanosoma cruzi has been described in humans and experimental work has been conducted with mice, but not with non‐human primates (NHPs). Methods We conducted a retrospective study of female baboons (Papio hamadryas spp.) naturally seropositive or seronegative for T. cruzi with history of fetal loss, and we report a stillbirth in a cynomolgus macaque (Macaca fascicularis) with placental T. cruzi amastigotes. Results There were no differences in menstrual cycle parameters and the number of fetal losses between seropositive and seronegative baboons with history of fetal loss. The amount of parasite DNA detected using quantitative polymerase chain reaction (Q‐PCR) in M. fascicularis placenta was within the range detected in infected baboon tissues. Conclusions There is no evidence that chronic maternal T. cruzi infection causes fetal loss in baboons. Q‐PCR is a useful diagnostic tool to study archived NHP placentas.     

Conformational stability and folding mechanisms of dimeric proteins

The folding of multisubunit proteins is of tremendous biological significance since the large majority of proteins exist as protein-protein complexes. Extensive experimental and computational studies have provided fundamental insights into the principles of folding of small monomeric proteins. Recently, important advances have been made in extending folding studies to multisubunit proteins, in particular homodimeric proteins. This review summarizes the equilibrium and kinetic theory and models underlying the quantitative analysis of dimeric protein folding using chemical denaturation, as well as the experimental results that have been obtained. Although various principles identified for monomer folding also apply to the folding of dimeric proteins, the effects of subunit association can manifest in complex ways, and are frequently overlooked. Changes in molecularity typically give rise to very different overall folding behaviour than is observed for monomeric proteins. The results obtained for dimers have provided key insights pertinent to understanding biological assembly and regulation of multisubunit proteins. These advances have set the stage for future advances in folding involving protein-protein interactions for natural multisubunit proteins and unnatural assemblies involved in disease.     

Implications for kinetochore-microtubule attachment from the structure of an engineered Ndc80 complex

Kinetochores are proteinaceous assemblies that mediate the interaction of chromosomes with the mitotic spindle. The 180 kDa Ndc80 complex is a direct point of contact between kinetochores and microtubules. Its four subunits contain coiled coils and form an elongated rod structure with functional globular domains at either end. We crystallized an engineered "bonsai" Ndc80 complex containing a shortened rod domain but retaining the globular domains required for kinetochore localization and microtubule binding. The structure reveals a microtubule-binding interface containing a pair of tightly interacting calponin-homology (CH) domains with a previously unknown arrangement. The interaction with microtubules is cooperative and predominantly electrostatic. It involves positive charges in the CH domains and in the N-terminal tail of the Ndc80 subunit and negative charges in tubulin C-terminal tails and is regulated by the Aurora B kinase. We discuss our results with reference to current models of kinetochore-microtubule attachment and centromere organization.     

Acetylated lysine 56 on histone H3 drives chromatin assembly after repair and signals for the completion of repair

DNA damage causes checkpoint activation leading to cell cycle arrest and repair, during which the chromatin structure is disrupted. The mechanisms whereby chromatin structure and cell cycle progression are restored after DNA repair are largely unknown. We show that chromatin reassembly following double-strand break (DSB) repair requires the histone chaperone Asf1 and that absence of Asf1 causes cell death, as cells are unable to recover from the DNA damage checkpoint. We find that Asf1 contributes toward chromatin assembly after DSB repair by promoting acetylation of free histone H3 on lysine 56 (K56) via the histone acetyl transferase Rtt109. Mimicking acetylation of K56 bypasses the requirement for Asf1 for chromatin reassembly and checkpoint recovery, whereas mutations that prevent K56 acetylation block chromatin reassembly after repair. These results indicate that restoration of the chromatin following DSB repair is driven by acetylated H3 K56 and that this is a signal for the completion of repair.