Pituitary neuropeptides and B lymphocyte function.

This review discusses the accumulated evidence that pro-opiomelanocortin (POMC) gene products as well as other pituitary neuropeptides derived from related genes (Proenkephalin, PENK; Prodynorphin, PDYN, and Pronociceptin, PNOC) can exert direct effects on B lymphocytes to modulate their functions. We also review the available data on receptor systems that might be involved in the transmission of such hormonal signals to B cells.

Individual pituitary neuropeptides do not recapitulate the effects of repository corticotropin (Acthar®) on human B cells in vitro.

Repository corticotropin injection (RCI), a complex mixture of adrenocorticotropic hormone (ACTH) analogs and other pituitary peptides, has been found to suppress key aspects of gene expression and cellular function in human B lymphocytes in vitro. The present studies reveal that neither individual POMC peptides (α-MSH, ACTH, ACTH, β-endorphin) nor other related pituitary neuropeptides are sufficient to elicit these effects, even though specific receptors capable of transmitting signals from these peptides are expressed by human B cells. RCI's direct effects on human B cells may require complementary signals from multiple components of the preparation.

Repository corticotropin injection reverses critical elements of the TLR9/B cell receptor activation response in human B cells in vitro.

We sought evidence for direct effects of repository corticotropin (RCI; an FDA-approved treatment for selected cases of SLE) on isolated human B lymphocytes activated by engagement of TLR9 and B cell receptors. ODN 2395/αIgM treatment was found to result in induction of 162 distinct mRNAs and suppression of 80 mRNAs at 24 h. RCI treatment resulted in suppression of 14 of the ODN 2395/αIgM -induced mRNAs (mean suppression to 23.

Repository corticotrophin injection exerts direct acute effects on human B cell gene expression distinct from the actions of glucocorticoids.

Repository corticotrophin injection (RCI, H.P Acthar gel) has been approved for use in the management of multiple autoimmune and inflammatory diseases for more than a half-century, but its mechanism of action is not well understood. We used RNA-Seq methods to define RCI-regulated mRNAs in cultured human B cells under conditions of activation by interleukin (IL)-4 and CD40 ligand.

Discovery of N-(4-Fluoro-3-methoxybenzyl)-6-(2-(((2S,5R)-5-(hydroxymethyl)-1,4-dioxan-2-yl)methyl)-2H-tetrazol-5-yl)-2-methylpyrimidine-4-carboxamide. A Highly Selective and Orally Bioavailable Matrix Metalloproteinase-13 Inhibitor for the Potential Treatment of Osteoarthritis.

Matrix metalloproteinase-13 (MMP-13) is a zinc-dependent protease responsible for the cleavage of type II collagen, the major structural protein of articular cartilage. Degradation of this cartilage matrix leads to the development of osteoarthritis. We previously have described highly potent and selective carboxylic acid containing MMP-13 inhibitors; however, nephrotoxicity in preclinical toxicology species precluded development.

Clinical, radiographic, molecular and MRI-based predictors of cartilage loss in knee osteoarthritis.

Objective: To examine the relationship of baseline clinical, radiographic, molecular and MRI measures with structural progression (subregional MRI-based femorotibial cartilage loss) in knee osteoarthritis (OA).

Methods: Single knees of 75 female participants with radiographic knee OA (and 77 healthy control participants) were examined over 24 months using MRI. Subregional femorotibial cartilage thickness was determined at baseline and follow-up.

Orally active MMP-1 sparing α-tetrahydropyranyl and α-piperidinyl sulfone matrix metalloproteinase (MMP) inhibitors with efficacy in cancer, arthritis, and cardiovascular disease.

α-Sulfone-α-piperidine and α-tetrahydropyranyl hydroxamates were explored that are potent inhibitors of MMP's-2, -9, and -13 that spare MMP-1, with oral efficacy in inhibiting tumor growth in mice and left-ventricular hypertrophy in rats and in the bovine cartilage degradation ex vivo explant system. α-Piperidine 19v (SC-78080/SD-2590) was selected for development toward the initial indication of cancer, while α-piperidine and α-tetrahydropyranyl hydroxamates 19w (SC-77964) and 9i (SC-77774), respectively, were identified as backup compounds.

Cartilage degradation biomarkers predict efficacy of a novel, highly selective matrix metalloproteinase 13 inhibitor in a dog model of osteoarthritis: confirmation by multivariate analysis that modulation of type II collagen and aggrecan degradation peptides parallels pathologic changes.

Objective: To demonstrate that the novel highly selective matrix metalloproteinase 13 (MMP-13) inhibitor PF152 reduces joint lesions in adult dogs with osteoarthritis (OA) and decreases biomarkers of cartilage degradation.

Methods: The potency and selectivity of PF152 were evaluated in vitro using 16 MMPs, TACE, and ADAMTS-4 and ADAMTS-5, as well as ex vivo in human cartilage explants. In vivo effects were evaluated at 3 concentrations in mature beagles with partial medial meniscectomy.

Pharmacokinetic/pharmacodynamic (PK/PD) differentiation of native and PEGylated recombinant human growth hormone (rhGH and PEG-rhGH) in the rat model of osteoarthritis.

Osteoarthritis (OA) is a degenerative joint disease that has no FDA-approved treatment. The current standard of care does not address the regeneration of the damaged cartilage. Human growth hormone (hGH) is part of the insulin-like growth factor (IGF)-1 axis.

Discovery of (pyridin-4-yl)-2H-tetrazole as a novel scaffold to identify highly selective matrix metalloproteinase-13 inhibitors for the treatment of osteoarthritis.

Potent, highly selective and orally-bioavailable MMP-13 inhibitors have been identified based upon a (pyridin-4-yl)-2H-tetrazole scaffold. Co-crystal structure analysis revealed that the inhibitors bind at the S(1)(') active site pocket and are not ligands for the catalytic zinc atom. Compound 29b demonstrated reduction of cartilage degradation biomarker (TIINE) levels associated with cartilage protection in a preclinical rat osteoarthritis model.

Effect of the oral application of a highly selective MMP-13 inhibitor in three different animal models of rheumatoid arthritis.

Objective: To evaluate the decrease of cartilage destruction by a novel orally active and specific matrix metalloproteinase 13 (MMP-13) inhibitor in three different animal models of rheumatoid arthritis (RA).

Materials And Methods: The SCID mouse co-implantation model of RA, the collagen-induced arthritis (CIA) model in mice and the antigen-induced arthritis model (AIA) in rabbits were used.

Results: In the SCID mouse co-implantation model, the MMP-13 inhibitor reduced cartilage destruction by 75%.

Plasma 3-nitrotyrosine is a biomarker in animal models of arthritis: Pharmacological dissection of iNOS' role in disease.

The contribution of inducible nitric oxide synthase (iNOS) to oxidative/nitrative stress is well-documented in inflammation, but difficult to quantify. Using a novel, recently developed assay for 3-nitrotyrosine (3-NT), we characterized iNOS activity and its inhibition in preclinical models of inflammation. In particular, we utilized the 3-NT assay to assess the role of iNOS in the disease pathology as well as for proof of pharmacology of iNOS inhibitors in an acute endotoxin challenge model, in models of rheumatoid arthritis (RA) such as rat adjuvant- and collagen-induced arthritis (AIA and CIA) and a model of osteoarthritis (OA) such as rat sodium monoiodoacetate-induced arthritis (MIA).

Discovery and development of the N-terminal procollagen type II (NPII) biomarker: a tool for measuring collagen type II synthesis.

Objective: Progression of joint damage in osteoarthritis (OA) is likely to result from an imbalance between cartilage degradation and synthesis processes. Markers reflecting these two components appear to be promising in predicting the rate of OA progression. Both N- and C-terminal propeptides of type II collagen reflect the rates of collagen type II synthesis.

Discovery and development of a type II collagen neoepitope (TIINE) biomarker for matrix metalloproteinase activity: from in vitro to in vivo.

Destruction of cartilage by matrix metalloproteinases (MMPs) plays a significant role in the pathology of osteoarthritis (OA). A translatable biomarker of MMP activity would enable development of MMP inhibitors for the treatment of OA and potentially the improved diagnosis of OA. A directed approach to identifying specific MMP cleavage products as potential biomarkers has been undertaken.

Association between concentrations of urinary type II collagen neoepitope (uTIINE) and joint space narrowing in patients with knee osteoarthritis.

Objective: To examine whether urine concentrations of type II collagen neoepitope (uTIINE) distinguish subjects with progressive radiographic and/or symptomatic knee osteoarthritis (OA) from those with stable disease.

Methods: Subjects were 120 obese middle-aged women with unilateral knee OA who participated in a 30-month randomized-controlled trial of structure modification with doxycycline, in which a standardized semiflexed anteroposterior view of the knee was obtained at baseline, 16 months and 30 months. Subjects were selected from a larger sample to permit a priori comparisons between 60 OA progressors and 60 nonprogressors, as defined by joint space narrowing (JSN) in the medial tibiofemoral compartment.

Estrogen's bone-protective effects may involve differential IL-1 receptor regulation in human osteoclast-like cells.

Declining estrogen levels during the first postmenopausal decade lead to rapid bone loss and increased fracture risk that can be reversed by estrogen replacement therapy. The bone-protective effects of estrogen may involve suppression of inflammatory cytokines that promote osteoclastogenesis and bone resorption, such as IL-1, TNF-alpha, and IL-6. We investigated whether estrogen modulates IL-1 actions on human osteoclasts (OCs) and other bone cell types.

Human osteoclasts and osteoclast-like cells synthesize and release high basal and inflammatory stimulated levels of the potent chemokine interleukin-8.

Chemokines, including interleukin-8 (IL-8), function as key mediators in diverse inflammatory disorders via promoting the recruitment, proliferation, and activation of vascular and immune cells. IL-8 levels are elevated in inflammatory diseases, such as rheumatoid arthritis, osteoarthritis, osteomyelitis, and periodontal disease, that also exhibit progressive bone loss. Therefore, it is possible that IL-8 contributes to the osteopenia associated with these pathological conditions.

Ca2+ or phorbol ester but not inflammatory stimuli elevate inducible nitric oxide synthase messenger ribonucleic acid and nitric oxide (NO) release in avian osteoclasts: autocrine NO mediates Ca2+-inhibited bone resorption.

Osteoclast bone resorption is essential for normal calcium homeostasis and is therefore tightly controlled by calciotropic hormones and local modulatory cytokines and factors. Among these is nitric oxide (NO), a multifunctional free radical that potently inhibits osteoclast bone resorption in vitro and in vivo. Previous findings led us to propose that NO might serve as an autocrine, as well as paracrine, regulator of osteoclast function.

Proinflammatory agents, IL-8 and IL-10, upregulate inducible nitric oxide synthase expression and nitric oxide production in avian osteoclast-like cells.

Nitric oxide synthase (NOS) isoenzymes generate nitric oxide (NO), a sensitive multifunctional intercellular signal molecule. High NO levels are produced by an inducible NOS (iNOS) in activated macrophages in response to proinflammatory agents, many of which also regulate local bone metabolism. NO is a potent inhibitor of osteoclast bone resorption, whereas inhibitors of NOS promote bone resorption both in vitro and in vivo.

Cell type- and differentiation-dependent expression from the mouse acetylcholine receptor epsilon-subunit promoter.

The nicotinic acetylcholine receptor (AChR) in adult skeletal muscle is composed of alpha-, beta-, epsilon-, and delta-subunits and is localized at the neuromuscular junction; in contrast, the more diffusely distributed fetal form is composed of alpha-, beta-, gamma-, and delta-subunits. To define sequences necessary for the transcriptional regulation of the mouse epsilon-subunit gene, we sequenced and analyzed 1036 bp upstream of the transcription start site. Using deletion analysis of the 5'-flanking region linked to the bacterial chloramphenicol acetyltransferase (CAT) gene and transfection of the resulting constructs into established cell lines, we demonstrate that a 151 bp fragment exhibits cell type- and differentiation-specific promoter activity.

Sequence analysis and DNA-protein interactions within the 5' flanking region of the Ca2+/calmodulin-dependent protein kinase II alpha-subunit gene.

The 5' flanking region of the brain Ca2+/calmodulin-dependent protein kinase II alpha-subunit gene was identified and characterized. A total of 430 bases was sequenced upstream from the translation initiation codon, and the site of transcription initiation was located at -149 or -147 bases as determined by primer extension and S1 nuclease protection analysis, respectively. TATA and CAAT boxes were absent from their standard positions; however, the 5' flanking region was rich in G + C and contained a GGGCG and a TATATAA sequence 76 and 160 bases upstream from the transcription initiation site, respectively.

Studies on nucleotide and receptor regulation of Gi proteins: effects of pertussis toxin.

In intact membranes as well as after reconstitution into phospholipid vesicles, pertussis toxin (PT)-mediated ADP-ribosylation of G proteins causes loss of receptor-mediated regulation of effectors and/or G protein-mediated regulation of receptor binding. Studies were carried out to test which of several discrete steps known to constitute the basal and receptor-stimulated regulatory cycles of Gi proteins are affected by PT. Experiments with the Gs-deficient Gi-regulated adenylyl cyclase of cyc- S49 cell membranes indicated that PT blocks Gi activation by GTP without affecting GDP dissociation or GTP binding to a major extent.

GTP hydrolysis by pure Ni, the inhibitory regulatory component of adenylyl cyclases.

The stimulatory and inhibitory regulatory components of adenylyl cyclase (Ns and Ni), purified to apparent homogeneity without the use of regulatory ligands such as Mg, NaF, and guanyl-5'-yl imidodiphosphate, were tested for GTPase activity by incubating them with [gamma-32P]GTP and measuring 32Pi liberation using a charcoal adsorption assay to separate hydrolyzed from nonhydrolyzed radioactivity. We found that Ni is capable of hydrolyzing GTP. The activity was shown to be due to Ni itself and not to presence of one of its minor contaminants by correlating activity with abundance of the 40,000 Da alpha i subunit throughout the last stages of purification and by showing co-migration on a sucrose density gradient of the GTP-hydrolyzing activity with the alpha i, beta, and gamma subunits of Ni and not with any one of three minor contaminants present in the preparation tested.