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. Following IL-4/CD40L activation and RCI treatment we found up-regulation of 115 unique mRNA transcripts and down-regulation of 80 unique mRNAs. The effect on these RNA levels was dose-dependent for RCI and was distinct from changes in mRNA expression induced by treatment with a potent synthetic glucocorticoid. RCI down-regulated mRNAs were observed to include a significant over-representation of genes critical for B cell proliferation under activating conditions. These data confirm that RCI exerts direct effects on human B cells to modulate mRNA expression in specific pathways of importance to B cell function and that, at the molecular level, the effects of RCI are distinct from those exerted by glucocorticoids.

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. Baseline clinical, radiographic, molecular (n=16) and quantitative MRI-based measures of the meniscus and cartilage, including delayed gadolinium-enhanced MRI (dGEMRIC) and T2, were obtained. Differences in these baseline measures between radiographic osteoarthritic knees with longitudinal cartilage thinning (or thickening) and those with no significant change were evaluated by receiver operator characteristic analyses and Wilcoxon rank sum tests. RESULTS: The relatively strongest predictors of longitudinal cartilage thinning were reduced baseline cartilage thickness in the medial femur (area under the curve (AUC)=0.81), varus malalignment (AUC=0.77), reduced minimum joint space width and a greater radiographic joint space narrowing (JSN) score (both AUC=0.74). These remained significant after adjusting for multiple comparisons using false discovery rates. Reduced bone resorption (C-terminal telopeptide of type I collagen; AUC=0.65) and a low dGEMRIC index (reflecting low proteoglycan content) in the medial tibia (AUC=0.68) were associated with longitudinal cartilage thinning, but failed to reach statistical significance after correction for multiple testing in this (small) sample. CONCLUSIONS: This exploratory study indicates that baseline molecular or MRI cartilage compositional markers may not provide better discrimination between knees with cartilage thinning and those without longitudinal change than simple radiographic measures, such as greater JSN score.

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. The ability to quantify the procollagen peptides in biological fluids would enable a better understanding of OA disease pathology and provide means for assessing the proof of mechanism of anabolic disease modifying OA drugs (DMOADs). METHODS: A polyclonal antibody that recognizes the sequence GPKGQKGEPGDIKDI in the propeptide region of rat, dog, and human type II collagen was raised in chicken and peptide-affinity purified. The immunoaffinity liquid chromatography mass spectrometry (LC-MS/MS) was used to extensively characterize N-terminal procollagen type II (NPII) peptides found in biological fluids. The novel competition enzyme-linked immunosorbent assay (ELISA) assay was developed to quantitatively measure the NPII peptides. RESULTS: Several peptides ranging from 17 to 41 amino acids with various modifications including hydroxylations on proline and lysine residues, oxidation of lysines to allysines, and attachments of glucose and galactose moieties to hydroxylysines were identified in a simple system such as ex vivo cultures of human articular cartilage (HAC) explants as well as in more complex biological fluids such as human urine and plasma. A competitive ELISA assay has been developed and applied to urine, plasma, and synovial fluid matrices in human, rat and dog samples. CONCLUSION: A novel NPII assay has been developed and applied to OA and normal human subjects to understand the changes in collagen type II synthesis related to the pathology of OA.

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. Isolated human OCs and primary bone marrow-derived OC-like cells expressed both the signaling (IL-1RI) and decoy (IL-1RII) IL-1 receptors, whereas only IL-1RI was detected in osteoblasts. IL-1RII/IL-1RI mRNA ratios and release of soluble IL-1RII (sIL-1RII) were lower in OC-like cells derived from women in the late postmenopausal period compared with younger women, but were unrelated to male donor age, suggesting that estrogen might play a role in regulating IL-1 receptor levels in vivo. Estrogen directly reduced in vitro OC-like cell IL-1RI mRNA levels while increasing IL-1RII mRNA levels and sIL-1RII release. These estrogenic events were associated with inhibited IL-1-mediated cytokine (IL-8) mRNA induction and cell survival, i.e., increased apoptosis. In contrast, estrogen did not alter IL-1R levels or IL-1 responsiveness in primary human osteoblasts or bone marrow stromal cells. We conclude that one novel mechanism by which estrogen exerts bone-protective effects may include a selective modulation of IL-1R isoform levels in OC or OC-like cells, thereby reducing their IL-1 responsiveness and cell survival. Conversely, this restraint on IL-1 actions may be lost as estrogen levels decline in aging women, contributing to an enhanced OC-mediated postmenopausal bone loss.

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. This suggested that the block lies in the transition of inactive GTP-Gi to active GTP-Gi (G to G* transition). Experiments with purified Gi in solution and after incorporation into phospholipid vesicles showed that PT does not increase or decrease the intrinsic GTPase activity of Gi. Experiments in which Gi was incorporated into phospholipid vesicles with rhodopsin, a receptor that interacts with Gi to stimulate the rate of guanosine 5'-O-(3-thio)triphosphate binding and GTP hydrolysis, indicated that PT does not affect the basal GTPase activity of Gi, but blocks its activation by the photoreceptor. Taken together the results indicate that PT-mediated ADP ribosylation has two separate effects, one to block the interaction of receptor with Gi and another to impede the GTP-induced activation reaction from occurring, or that PT has only one effect, that of blocking interaction with receptors. In this latter case the present results add to a mounting series of data that are consistent with the hypothesis that unoccupied receptors are not inactive, but exhibit a basal agonist-independent activity responsible for the various effects of GTP observed on G protein-coupled effector functions in intact membranes.

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. This premise was investigated using isolated bone-resorptive avian osteoclasts and focusing on the inducible isoform of NO synthase (iNOS) responsible for inflammatory stimulated high-level NO synthesis in other cells. Avian osteoclasts expressed both iNOS messenger RNA (mRNA) and protein. However, inflammatory cytokines that induce iNOS mRNA, protein, and NO in other cells did not do so in avian osteoclasts, consistent with the known role of inflammatory stimuli in promoting osteoclast resorption and localized bone loss. In searching for potential modulators of osteoclast iNOS, protein kinase C activation [by phorbol 12-myristate 13-acetate (PMA)] and intracellular Ca2+ rises (A23187) were each found to elevate osteoclast iNOS mRNA and protein levels, while increasing NO release and reducing osteoclast bone resorption. The iNOS selective inhibitor aminoguanidine suppressed stimulated osteoclast NO production elicited by either signal, but reversed only the resorption inhibition due to raised Ca2+. Thus, whereas additional inhibitory signals are presumably coproduced in osteoclasts treated with PMA, osteoclast iNOS-derived NO may act as an autocrine signal to mediate Ca2+-inhibited bone resorption. These findings document for the first time an iNOS whose mRNA levels are regulated by Ca2+ or PMA, but not inflammatory stimuli, and the autocrine production of NO as a Ca2+ sensing signal to suppress osteoclast bone resorption. The unusual regulation of osteoclast iNOS makes it a potentially attractive target for designing novel therapeutic agents to alleviate excessive bone loss.

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. Although macrophages, neutrophils, and endothelial cells are considered the primary sources of inflammation-induced IL-8 increases, we report here for the first time that human bone marrow-derived osteoclast-like cells (hOCL) as well as authentic bone-resorbing human osteoclasts (hOC) isolated from osteoporotic femoral heads express messenger RNA (mRNA) for IL-8 and secrete high levels of IL-8 during culture. Basal IL-8 release by cultured hOC or hOCL was orders of magnitude greater than the release of the proinflammatory cytokines IL-1beta, IL-6, and tumor necrosis factor-alpha. At a cellular level, in situ hybridization analysis revealed that IL-8 mRNA was expressed in resorbing hOC of rheumatoid arthritic pannus and was substantially greater than that expressed in hOC of noninflammatory giant cell tumor of bone tissue. Therefore, the potential inflammation-mediated induction of IL-8 was directly assessed using cultured hOCL. IL-8 release was stimulated by proinflammatory signals (IL-1alpha, tumor necrosis factor-alpha, lipopolysaccharide, or phorbol 12-myristate 13-acetate), unaffected by various other osteotropic modulators (transforming growth factor-beta1 and -beta3, IL-6, 17beta-estradiol, or calcitonin) and was decreased by interferon-gamma, vitamin D3, and the antiinflammatory glucocorticoid dexamethasone. Changes in IL-8 secretion were paralleled by corresponding changes in IL-8 mRNA steady state levels. We conclude that hOC and hOCL synthesize and secrete high constitutive and inflammation-stimulated levels of the chemokine IL-8. Consequently, hOC-derived IL-8 could act as an important regulatory signal for bone, vascular, and immune cell recruitment and activation during normal and pathological bone remodeling.

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. Liquid chromatography-tandem mass spectrometry (LC-MS/MS) was used to identify peptides generated by MMP-driven degradation of human articular cartilage (HAC) in vivo. It was shown that a 45-mer peptide fragment of collagen type II with five hydroxyprolines (OH) can be selectively produced by the activity of collagenase, an enzyme purported to be involved in the pathology of OA. This 45-mer is the most abundant neoepitope peptide found in biological fluids such as urine and synovial fluid. An immunoaffinity LC-MS/MS assay has been developed to quantify collagen type II neoepitope peptides as biomarkers of collagenase modulation. The lower limit of quantification for this assay was established to be 0.035 nM. The assay was used to measure the levels of collagen type II peptides in the urine of both clinical (healthy human subjects) and preclinical species. The urinary levels of the most abundant peptides are reported for rat, rabbit, guinea pig, dog, and healthy human adult subjects. The utility of this peptide to monitor collagenase activity in vivo has been demonstrated through its detailed characterization in HAC explants as well as in the urine of human and other preclinical species.

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. Each group contained 30 subjects who exhibited clinically significant increases in knee pain over 30 months and 30 who did not. Urine samples were obtained every 6 months for determination of the creatinine (Cr)-adjusted uTIINE concentration. RESULTS: Baseline uTIINE levels were unrelated to JSN in the placebo group. However, among subjects in the active treatment group, a 1-standard deviation increment in baseline uTIINE (68 ng/mM Cr) was associated with a marginally significant, two-fold increase in the odds of progression of JSN (odds ratio 2.04, 95% confidence interval 0.98-4.28). The within-subject mean of uTIINE values at baseline, 6 months and 12 months was associated with concurrent JSN measured at 16 months (0.10mm of JSN per 69 ng/mM Cr, P=0.008). Similar results were seen in the interval between months 16 and 30 and in analyses using the maximum of intercurrent uTIINE levels. CONCLUSION: Baseline uTIINE was not a consistent predictor of JSN in subjects with knee OA. However, serial measurements of uTIINE reflect concurrent JSN.

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. Moreover, the sequence CAACGG was found 85 and 146 bases upstream from this site, indicating presumptive binding sites for the Myb protein. Gel-mobility shift assays revealed that a 120-base-pair fragment, which included the G + C-rich, TATA, and CAACGG sequences bound nuclear proteins specifically. DNA-protein complexes with similar gel mobilities were obtained with nuclear extracts from rat forebrain or cerebellum and from neonatal or adult brains. Extracts from rat liver, kidney, and spleen generated specific DNA-protein complexes with different electrophoretic mobilities, suggesting the occurrence of different nuclear proteins that bind to 5' regulatory elements of the Ca2+/calmodulin-dependent kinase II alpha-subunit gene.

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. This activity was independent of a myogenic factor putative binding site (E-box). However, transactivation experiments with recombinant myoD, myogenin, or MRF4 showed that the E-box was functional and that MRF4 preferentially transactivates the epsilon-promoter. Thus, like other AChR promoters, the proximal region of the epsilon-promoter contains information for cell type-specific and developmental regulation of CAT and can be transactivated by myogenic factors in cultured cell lines. Unlike the other AChR promoters characterized to date, epsilon-promoter function can be partially independent of myogenic factors of the helix-loop-helix class.

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. Preparations of Ns, free of detectable Ni, exhibited less than 10% the capacity to hydrolyze GTP, as compared to Ni on an equal protein basis. The basic properties of the GTP-hydrolyzing activity of Ni were determined. The activity is dependent on Mg ion (apparent Km = 5 to 15 nM), and is rapidly lost upon incubation with Mg2+ in the absence of GTP. MgGTP and free GTP serve equally well as substrate (apparent Km about 40 nM). Isotopic dilution studies indicate that the GTP binding site has a relative affinity for guanine nucleotides in the order GTP = GTP gamma S greater than GDP = GMP-P(NH)P greater than GDP beta S with the highest difference (GTP versus GDP beta S) being about 10-fold. NaF inhibited GTP hydrolysis by Ni at concentrations at which it activates Ni in intact membranes.

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. The possibility that osteoclasts, like macrophages, express a regulated iNOS and produce NO as a potential autocrine signal following inflammatory stimulation was investigated in well-characterized avian marrow-derived osteoclast-like cells. NO production (reflected by medium nitrite levels) was markedly elevated in these cells by the proinflammatory agents lipopolysaccharide (LPS) and the synergistic action of IL-1 alpha, TNF alpha, and IFN gama. inhibitors of NOS activity (aminoguanidine, L-NAME) or iNOS induction (dexamethasone, TGF beta) reduced LPS-stimulated nitrite production. LPS also increased the NOS-associated diaphorase activity of these cells and their reactivity with anti-iNOS antibodies. RT-PCR cloning, using avian osteoclast-like cell RNA and human iNOS primers, yielded a novel 900 bp cDNA with high sequence homology (76%) to human, rat, and mouse iNOS genes. In probing osteoclast-like cell RNA with the PCR-derived iNOS cDNA, a 4.8 kb mRNA species was detected whose levels were greatly increased by LPS. Induction of iNOS mRNA by LPS, or by proinflammatory cytokines, occurred prior to the rise of medium nitrite in time course studies and was diminished by dexamethasone. Moreover, osteoclast-like cells demonstrated an upregulation of NO production and iNOS mRNA by IL-8 and IL-10, regulatory mechanism's not previously described. It is concluded that osteoclast-like cells express a novel iNOS that is upregulated by inflammatory mediators, leading to NO production. Therefore, NO may serve as both a paracrine and autocrine signal for modulating osteoclast bone resorption.

Simultaneous human immunodeficiency virus and Hepatitis C infection following a needlestick injury.

Needlestick injuries to health professionals at the Hospital del Mar, Barcelona since 1987 have been prospectively studied; a total of 296 such accidents in 286 subjects have been registered. We report the first case to our knowledge of simultaneous human immunodeficiency virus (HIV) and hepatitis C (HCV) infection in a nurse who suffered a needlestick injury after a blood sampling. Forty-four days after the accident she had symptoms and laboratory findings of acute hepatitis. Subsequent laboratory tests showed elevation in the aminotransferases and antibodies against HIV. The seroconversion to HCV was not detected until 109 days after the injury. The precise sequence of clinical and biological events of this case of simultaneous HIV and HCV infection is reported.