Alterations in specific signal transduction pathways may explain the increased expression of proinflammatory cytokines seen in inflammatory diseases such as psoriasis. We reveal increased TNF-α protein expression, but similar TNF-α mRNA levels, in lesional compared with nonlesional psoriatic skin, demonstrating for the first time that TNF-α expression in lesional psoriatic skin is regulated posttranscriptionally. Increased levels of activated MAPK-activated protein kinase 2 (MK2) together with increased MK2 kinase activity were found in lesional compared with nonlesional psoriatic skin. Immunohistochemical analysis showed that activated MK2 was located in the basal layers of the psoriatic epidermis, whereas no positive staining was seen in nonlesional psoriatic skin. In vitro experiments demonstrated that both anisomycin and IL-1β caused a significant activation of p38 MAPK and MK2 in cultured normal human keratinocytes. In addition, TNF-α protein levels were significantly up-regulated in keratinocytes stimulated with anisomycin or IL-1β. This increase in TNF-α protein expression was completely blocked by the p38 inhibitor, SB202190. Transfection of cultured keratinocytes with MK2-specific small interfering RNA led to a significant decrease in MK2 expression and a subsequent significant reduction in the protein expression of the proinflammatory cytokines TNF-α, IL-6, and IL-8, whereas no change in the expression of the anti-inflammatory cytokine IL-10 was seen. This is the first time that MK2 expression and activity have been investigated in an inflammatory disease such as psoriasis. The results strongly suggest that increased activation of MK2 is responsible for the elevated and posttranscriptionally regulated TNF-α protein expression in psoriatic skin, making MK2 a potential target in the treatment of psoriasis.

The 2015 9th Workshop on Recent Issues in Bioanalysis (9th WRIB) took place in Miami, Florida with participation of 600 professionals from pharmaceutical and biopharmaceutical companies, biotechnology companies, contract research organizations and regulatory agencies worldwide. WRIB was once again a 5 day, week-long event - A Full Immersion Bioanalytical Week - specifically designed to facilitate sharing, reviewing, discussing and agreeing on approaches to address the most current issues of interest in bioanalysis. The topics covered included both small and large molecules, and involved LCMS, hybrid LBA/LCMS and LBA approaches, including the focus on biomarkers and immunogenicity. This 2015 White Paper encompasses recommendations emerging from the extensive discussions held during the workshop, and is aimed to provide the bioanalytical community with key information and practical solutions on topics and issues addressed, in an effort to enable advances in scientific excellence, improved quality and better regulatory compliance. Due to its length, the 2015 edition of this comprehensive White Paper has been divided into three parts. Part 3 discusses the recommendations for large molecule bioanalysis using LBA, biomarkers and immunogenicity. Part 1 (small molecule bioanalysis using LCMS) and Part 2 (hybrid LBA/LCMS and regulatory inputs from major global health authorities) have been published in volume 7, issues 22 and 23 of Bioanalysis, respectively.

Mitogen- and stress-activated protein kinase 1 (MSK1) is a downstream target of both the p38 and extracellular signal-regulated kinase 1/2 (ERK1/2) mitogen-activated protein kinases (MAPKs). MSK1 stimulates transcription of different pro-inflammatory genes through activation of transcription factors. The purpose of this study was to investigate the expression and activation of MSK1 in lesional psoriatic skin and its role in cytokine production in cultured normal human keratinocytes. Western blotting revealed a consistent and significant increase in phosphorylated (activated) MSK1(Ser376) in lesional psoriatic skin. Immunofluorescence staining revealed the phosphorylated MSK1(Thr581) to be localized in the basal layers of the epidermis in lesional psoriatic skin. No staining was found in non-lesional psoriatic skin. Cultured human keratinocytes incubated with anisomycin or IL-1beta resulted in the phosphorylation of the p38 MAPK and MSK1(Ser376). MSK1(Ser376) phosphorylation was inhibited by pre-incubation with the p38 inhibitor SB 202190. Transfection of the keratinocytes with specific MSK1 small interfering RNA resulted in 80% reduction of MSK1 expression and 51, 40, and 31% decrease in IL-6, IL-8, and tumor necrosis factor-alpha protein production, respectively. This study demonstrates for the first time the expression of MSK1 in epidermal keratinocytes and increased activation focally in psoriatic epidermis. As MSK1 regulates the production of pro-inflammatory cytokines, it may play a role in the pathogenesis of psoriasis.

We wish to report the synthesis and structure-activity relationship (SAR) of a series of 4-aminobenzophenones, as a novel compound class with high antiinflammatory activity. Our initial lead, (4-[(2-aminophenyl)amino]phenyl)(phenyl)methanone (3), was systematically optimized and resulted in compounds that potently inhibited the release of the proinflammatory cytokines IL-1beta and TNF-alpha in human peripheral blood mononuclear cells stimulated by LPS. One of the most potent compounds, among others, was (4-[(2-aminophenyl)amino]-2-chlorophenyl)(2-methylphenyl)methanone (45) with IC(50) values of 14 and 6 nM for the inhibition of IL-1beta and TNF-alpha, respectively. Furthermore, we found these types of compounds to be potent and selective p38 MAP kinase inhibitors, e.g. 45 had an IC(50) value of 10 nM. Molecular modeling was used to rationalize our SAR data and to propose a model for the interaction of compound 45 with the p38 MAP kinase. The model involved a favorable hydrogen bond between the carbonyl group of the benzophenone and the NH of Met-109, positioning ring A in the hydrophobic pocket I of the enzyme. Good antiinflammatory effects were demonstrated in two murine models of dermatitis after topical application (oxazolone and TPA model).

Transforming growth factor-␤ (TGF-␤) is a powerful modulator of bone metabolism, and both its anabolic and catabolic effects on bone have been described. Here we have tested the hypothesis that TGF-␤-induced changes in osteoblast shape promote bone resorption by increasing the surface area of bone that is accessible to osteoclasts. The addition of TGF-␤1 to MC3T3-E1 cells resulted in cytoskeletal reorganization, augmented expression of focal adhesion kinase, and cell elongation, accompanied by an increase in the area of cell-free substratum. TGF-␤1 also triggered activation of Erk1/2 and p38 mitogen-activated protein (MAP) kinase. The p38 MAP kinase inhibitor PD169316, but not an inhibitor of the Erk1/2 pathway, abrogated the effect of TGF-␤1 on cell shape. The matrix metalloproteinase inhibitor GM6001 also interfered with osteoblast elongation. Treatment of MC3T3-E1 cells seeded at confluence onto bone slices to mimic a bone lining cell layer with TGF-␤1 also induced cell elongation and increased pit formation by subsequently added osteoclasts. These effects were again blocked by PD169316 and GM6001. We propose that this novel pathway regulating osteoblast morphology plays an important role in the catabolic effects of TGF-␤ on bone metabolism.The skeletons of developing and adult mammals undergo constant remodeling, i.e. old bone is regularly removed and new bone is regularly laid down (1, 2). The major players in the remodeling of bone are two specialized and functionally coupled cell types: osteoblasts, which deposit organic and inorganic matrix, and osteoclasts, which remove bone matrix. Osteoclast function is controlled by both systemic and local factors, most of which act through or are produced by osteoblasts. Similarly, the ability of osteoblasts to deposit bone matrix is stimulated by factors that are produced by osteoclasts or released from bone in the wake of matrix dissolution. Thus, in living bone, osteoblasts play a paramount role in determining the functional state of osteoclasts, and osteoclasts are of supreme importance for osteoblast function.Most of the arguments concerned with the quest for identification of key mechanisms that produce bone resorbing osteoclasts, i.e. those mechanisms involved in osteoclast precursor proliferation and commitment, and osteoclast migration, fusion, and resorption focus on the direct effect of molecules found in the extracellular space or on the cell surface of osteoblasts or other stromal cells on osteoclast precursors and maturing osteoclasts (3). However, based on the observation that cells of the osteoblast lineage, the bone lining cells, display a cobblestone morphology and cover the bone surface in an epithelium-like manner, an additional mode of how osteoblasts may modulate the ability of osteoclasts to resorb bone has been proposed (4, 5). In this model, bone lining cells mechanically hinder the access of osteoclasts to the bone surface. Consequently, in order to allow osteoclasts to degrade bone matrix, bone lining cells must retreat from part of the bo...