A Bispecific Monoclonal Antibody Targeting Psl and PcrV Enhances Neutrophil-Mediated Killing of Pseudomonas aeruginosa in Patients with Bronchiectasis.

RATIONALE AND OBJECTIVES: Pseudomonas aeruginosa infection is associated with worse outcomes in bronchiectasis. Impaired neutrophil antimicrobial responses contribute to bacterial persistence. Gremubamab is a bivalent, bispecific monoclonal antibody targeting Psl exopolysaccharide and the type 3 secretion system component PcrV. This study evaluated the efficacy of gremubamab to enhance killing of P.aeruginosa by neutrophils from bronchiectasis patients and to prevent P.aeruginosa-associated cytotoxicity. METHODS: P.aeruginosa isolates from a global bronchiectasis cohort (n=100) underwent whole-genome sequencing to determine target prevalence. Functional activity of gremubamab against selected isolates was tested in-vitro and in-vivo. Patients with bronchiectasis (n=11) and controls (n=10) were enrolled and the effect of gremubamab in peripheral-blood neutrophil opsonophagocytic killing (OPK) assays against P.aeruginosa was evaluated. Serum antibody titers to Psl and PcrV were determined (n=30; 19: chronic P.aeruginosa infection, 11: no-known P.aeruginosa infection), as was the effect of gremubamab treatment in OPK and anti-cytotoxic activity assays. MEASUREMENTS AND RESULTS: Psl and PcrV were conserved in isolates from chronically-infected bronchiectasis patients. 73/100 isolates had a full psl locus and 99/100 contained the pcrV gene, with 20 distinct full-length PcrV protein subtypes identified. PcrV subtypes were successfully bound by gremubamab and the mAb mediated potent protective activity against tested isolates. Gremubamab increased bronchiectasis patient neutrophil-mediated OPK (+34.6±8.1%) and phagocytosis (+70.0±48.8%), similar to effects observed in neutrophils from controls (OPK:+30.1±7.6%). No evidence of competition between gremubamab and endogenous antibodies was found, with protection against P.aeruginosa-induced cytotoxicity and enhanced OPK demonstrated with and without addition of patient serum. CONCLUSION: Gremubamab enhanced bronchiectasis patient neutrophil phagocytosis and killing of P.aeruginosa and reduced virulence.

A microfluidic chip-based capillary zone electrophoresis-mass spectrometry method for measuring adenosine 5'-Triphosphate and its similar nucleotide analogues.

BACKGROUND: Extracellular ATP is involved in disorders that cause inflammation of the airways and cough, thus limiting its release has therapeutic benefits. Standard luminescence-based ATP assays measure levels indirectly through enzyme degradation and do not provide a simultaneous readout for other nucleotide analogues. Conversely, mass spectrometry can provide direct ATP measurements, however, common RPLC and HILIC methods face issues because these molecules are unstable, metal-sensitive analytes which are often poorly retained. These difficulties have traditionally been overcome using passivation or ion-pairing chromatography, but these approaches can be problematic for LC systems. As a result, more effective analytical methods are needed. RESULTS: Here, we introduce a new application that uses microfluidic chip-based capillary zone electrophoresis-mass spectrometry (µCZE-MS) to measure ATP and its analogues simultaneously in biofluids. The commercially available ZipChip Interface and a High-Resolution Bare-glass microchip (ZipChip, HRB, 908 Devices Inc.) coupled to a Thermo Scientific Tribrid Orbitrap, were successfully used to separate and detect various nucleotide standards, as well as ATP, ADP, AMP, and adenosine in plasma and BALF obtained from naïve Brown Norway rats. The findings demonstrate that this approach can rapidly and directly detect ATP and its related nucleotide analogues, while also highlighting the need to preserve these molecules in biofluids with chelators like EDTA. In addition, we demonstrate that this µCZE-MS method is also suitable for detecting a variety of metabolites, revealing additional potential future applications. SIGNIFICANCE: This innovative µCZE-MS approach provides a robust new tool to directly measure ATP and other nucleotide analogues in biofluids. This can enable the study of eATP in human disease and potentially contribute to the creation of ATP-targeting therapies for airway illnesses.

Serum-circulating His-tRNA synthetase inhibits organ-targeted immune responses.

His-tRNA synthetase (HARS) is targeted by autoantibodies in chronic and acute inflammatory anti-Jo-1-positive antisynthetase syndrome. The extensive activation and migration of immune cells into lung and muscle are associated with interstitial lung disease, myositis, and morbidity. It is unknown whether the sequestration of HARS is an epiphenomenon or plays a causal role in the disease. Here, we show that HARS circulates in healthy individuals, but it is largely undetectable in the serum of anti-Jo-1-positive antisynthetase syndrome patients. In cultured primary human skeletal muscle myoblasts (HSkMC), HARS is released in increasing amounts during their differentiation into myotubes. We further show that HARS regulates immune cell engagement and inhibits CD4+ and CD8+ T-cell activation. In mouse and rodent models of acute inflammatory diseases, HARS administration downregulates immune activation. In contrast, neutralization of extracellular HARS by high-titer antibody responses during tissue injury increases susceptibility to immune attack, similar to what is seen in humans with anti-Jo-1-positive disease. Collectively, these data suggest that extracellular HARS is homeostatic in normal subjects, and its sequestration contributes to the morbidity of the anti-Jo-1-positive antisynthetase syndrome.

The growth of a xenograft breast cancer tumor model with engineered hyaluronan-accumulating stroma is dependent on hyaluronan and independent of CD44.

Hyaluronan accumulation in the tumor microenvironment is associated with poor prognosis in several solid human cancers. To understand the role of stromal hyaluronan in tumor progression, we engineered 3T3HAS3, a hyaluronan-producing fibroblast cell line, by lentiviral transduction of Balb/c 3T3 cells with the human hyaluronan synthase 3 (HAS3) gene. 3T3HAS3 cells significantly enhanced tumor growth when co-grafted with MDA-MB-468 cells in nude mice. Immunohistochemical analysis of the xenograft tumors showed that MDA-MB-468 cells were surrounded by hyaluronan-accumulating stroma, closely resembling the morphology observed in human breast cancer specimens. Tumor growth of MDA-MB-468 + 3T3HAS3 co-grafts was greatly reduced upon hyaluronan degradation by lentiviral transduction of a human hyaluronidase gene in 3T3HAS3 cells, or by systemic administration of pegvorhyaluronidase alfa (PEGPH20). In contrast, the growth of the co-graft tumors was not inhibited when CD44 expression was reduced or ablated by small hairpin RNA-mediated CD44 knockdown in MDA-MB-468 cells, CD44 CRISPR knockout in 3T3HAS3 cells, or by grafting these cells in CD44 knockout nude mice. Collectively, these data demonstrate that tumor growth of an engineered xenograft breast cancer model with hyaluronan-accumulating stroma can be dependent on hyaluronan and independent of CD44.

Parallel Accumulation of Tumor Hyaluronan, Collagen, and Other Drivers of Tumor Progression.

Purpose: The tumor microenvironment (TME) evolves to support tumor progression. One marker of more aggressive malignancy is hyaluronan (HA) accumulation. Here, we characterize biological and physical changes associated with HA-accumulating (HA-high) tumors.Experimental Design: We used immunohistochemistry, in vivo imaging of tumor pH, and microdialysis to characterize the TME of HA-high tumors, including tumor vascular structure, hypoxia, tumor perfusion by doxorubicin, pH, content of collagen. and smooth muscle actin (α-SMA). A novel method was developed to measure real-time tumor-associated soluble cytokines and growth factors. We also evaluated biopsies of murine and pancreatic cancer patients to investigate HA and collagen content, important contributors to drug resistance.Results: In immunodeficient and immunocompetent mice, increasing tumor HA content is accompanied by increasing collagen content, vascular collapse, hypoxia, and increased metastatic potential, as reflected by increased α-SMA. In vivo treatment of HA-high tumors with PEGylated recombinant human hyaluronidase (PEGPH20) dramatically reversed these changes and depleted stores of VEGF-A165, suggesting that PEGPH20 may also diminish the angiogenic potential of the TME. Finally, we observed in xenografts and in pancreatic cancer patients a coordinated increase in HA and collagen tumor content.Conclusions: The accumulation of HA in tumors is associated with high tIP, vascular collapse, hypoxia, and drug resistance. These findings may partially explain why more aggressive malignancy is observed in the HA-high phenotype. We have shown that degradation of HA by PEGPH20 partially reverses this phenotype and leads to depletion of tumor-associated VEGF-A165. These results encourage further clinical investigation of PEGPH20. Clin Cancer Res; 24(19); 4798-807. ©2018 AACR.

Recombinant Human PH20: Baseline Analysis of the Reactive Antibody Prevalence in the General Population Using Healthy Subjects.

BACKGROUND: Recombinant human PH20 (rHuPH20) is used to depolymerize hyaluronan in the subcutaneous space, increasing the dispersion and absorption of co-administered drugs. While ~ 5 to 10% of rHuPH20 treatment-naïve healthy volunteers have demonstrated rHuPH20-reactive antibodies, associations with age, sex, fertility, and immune disorders remain unknown. OBJECTIVES: Using demographically diverse healthy volunteers, we assessed the prevalence of rHuPH20-reactive antibodies in the general population and potential associations with fertility and autoimmunity diseases. METHODS: In total, 896 subjects aged ≥ 12 years (767 adults; 129 children) without prior exposure to rHuPH20 were enrolled. A demographic and limited medical history review was performed, and K3-EDTA-anticoagulated plasma was analyzed for rHuPH20-reactive antibodies using a bridging immunoassay. RESULTS: Adult and pediatric positivity rates for rHuPH20-reactive antibodies were 5.2% (40/767) and 1.6% (2/129), respectively. Titers ranged from 5 to 2560 (median 30). In five antibody-positive subjects from whom repeated samples were available, antibody titers remained unchanged or decreased fourfold over periods up to 590 days. The prevalence of rHuPH20-reactive antibodies significantly increased with age (p = 0.0006) and was significantly higher in males than in females (p = 0.0010). Men who had fathered children had a significantly increased prevalence of rHuPH20-reactive antibodies than men who had not (p = 0.0036), whereas the rate of childbearing was not significantly different between rHuPH20 antibody-positive and -negative women. The prevalence between racial/ethnic groups was not significantly different, nor was the presence/absence of an autoimmune disorder. CONCLUSIONS: Approximately 1/20 of the adult population had rHuPH20-reactive antibodies. The reason remains unknown; however, no evidence for a negative effect on fertility or association with autoimmune disease was demonstrated.

Clinical Immunogenicity of rHuPH20, a Hyaluronidase Enabling Subcutaneous Drug Administration.

Recombinant human PH20 hyaluronidase (rHuPH20) is used to facilitate dispersion of subcutaneously delivered fluids and drugs. This report summarizes rHuPH20 immunogenicity findings from clinical trials where rHuPH20 was co-administered with SC human immunoglobulin, trastuzumab, rituximab, or insulin. Plasma samples were obtained from evaluable subjects participating in ten different clinical trials as well as from healthy plasma donors. A bridging immunoassay and a modified hyaluronidase activity assay were used to determine rHuPH20-reactive antibody titers and neutralizing antibodies, respectively. rHuPH20-binding antibody populations from selected subjects with positive titers were affinity-purified and subjected to further characterization such as cross-reactivity with endogenous PH20. Among individual trials, the prevalence of pre-existing rHuPH20-reactive antibodies varied between 3 and 12%, excepting the primary immunodeficiency (PID) studies. Incidence of treatment-induced rHuPH20 antibodies was 2 to 18%, with the highest titers (81,920) observed in PID. No neutralizing antibodies were observed. Within most trials, the kinetics of antibody responses were comparable between pre-existing and treatment-induced antibody responses, although responses classified as persistent were more common in subjects with pre-existing titers. There was no association between antibody positivity and either local or systemic adverse events. Pre-existing and treatment-induced antibody populations were of similar immunoglobulin isotypes and cross-reacted to endogenous PH20 to similar extents. No cross-reactivity to PH20 paralogs was detected. rHuPH20 induces only modest immunogenicity which has no association with adverse events. In addition, antibodies purified from baseline-positive individuals are qualitatively similar to those purified from individuals developing rHuPH20-reactive antibodies following exposure to the enzyme.

Serum C-X-C motif chemokine 13 is elevated in early and established rheumatoid arthritis and correlates with rheumatoid factor levels.

INTRODUCTION: We hypothesized that serum levels of C-X-C motif chemokine 13 (CXCL13), a B-cell chemokine, would delineate a subset of rheumatoid arthritis (RA) patients characterized by increased humoral immunity. METHODS: Serum from patients with established RA (the Dartmouth RA Cohort) was analyzed for CXCL13, rheumatoid factor (RF) levels, anticitrullinated peptide/protein antibody (ACPA) and total immunoglobulin G (IgG); other parameters were obtained by chart review. A confirmatory analysis was performed using samples from the Sherbrooke Early Undifferentiated PolyArthritis (EUPA) Cohort. The Wilcoxon rank-sum test, a t-test and Spearman's correlation analysis were utilized to determine relationships between variables. RESULTS: In both the Dartmouth and Sherbrooke cohorts, CXCL13 levels were selectively increased in seropositive relative to seronegative RA patients (P = 0.0002 and P < 0.0001 for the respective cohorts), with a strong correlation to both immunoglobulin M (IgM) and IgA RF levels (P < 0.0001). There was a weaker relationship to ACPA titers (P = 0.03 and P = 0.006, respectively) and total IgG (P = 0.02 and P = 0.14, respectively). No relationship was seen with regard to age, sex, shared epitope status or inclusion high-sensitivity C-reactive protein (hsCRP) in either cohort or regarding the presence of baseline erosions in the Sherbrooke Cohort, whereas a modest relationship with Disease Activity Score in 28 joints CRP (DAS28-CRP) was seen in the Dartmouth cohort but not the Sherbrooke cohort. CONCLUSION: Using both established and early RA cohorts, marked elevations of serum CXCL13 levels resided nearly completely within the seropositive population. CXCL13 levels exhibited a strong relationship with RF, whereas the association with clinical parameters (age, sex, DAS28-CRP and erosions) or other serologic markers (ACPA and IgG) was either much weaker or absent. Elevated serum CXCL13 levels may identify a subset of seropositive RA patients whose disease is shaped by or responsive to RF production.

Recombinant human hyaluronidase PH20 does not stimulate an acute inflammatory response and inhibits lipopolysaccharide-induced neutrophil recruitment in the air pouch model of inflammation.

Hyaluronidase (Hyal) and low m.w. hyaluronan (LMW HA) fragments have been widely reported to stimulate the innate immune response. However, most hyaluronidases used were purified from animal tissues (e.g., bovine testis Hyal [BTH]), and contain endotoxin and other unrelated proteins. We tested a highly purified recombinant human Hyal (rHuPH20) and endotoxin-free HA fragments from M(r) 5,000 to 1,500,000 in the rodent air pouch model of inflammation to determine their potential for stimulation of the innate immune response. Exogenous LMW HA fragments (average M(r) 200,000) failed to induce either cytokine/chemokine production or neutrophil infiltration into the air pouch. Challenging the air pouch with LPS or BTH stimulated production of cytokines and chemokines but rHuPH20 did not, suggesting that neither PH20 nor generation of LMW HA fragments in situ stimulates cytokine and chemokine production. LPS and BTH also induced neutrophil infiltration into the air pouch, which was not observed with rHuPH20 treatment. Endotoxin-depleted BTH had much reduced proinflammatory activity, suggesting that the difference in inflammatory responses between rHuPH20 and BTH is likely due to endotoxin contaminants in BTH. When rHuPH20 was dosed with LPS, the induction of cytokines and chemokines was the same as LPS alone, but neutrophil infiltration was inhibited, likely by interrupting HA-CD44 interaction. Our results indicate that neither rHuPH20 nor its directly generated HA catabolites have inflammatory properties in the air pouch model, and rHuPH20 can instead inhibit some aspects of inflammation, such as neutrophil infiltration into the air pouch.

Increased frequency of complement C4B deficiency in rheumatoid arthritis.

OBJECTIVE: To assess the copy number variation of complement C4A and C4B genes in patients with rheumatoid arthritis (RA). METHODS: DNA samples were obtained from 299 patients and controls and analyzed for copy number variation of total complement C4, C4A, and C4B genes. The results were compared by chi-square analysis, and odds ratios (ORs) and 95% confidence intervals (95% CIs) were calculated. RESULTS: Chi-square analysis revealed similar distribution patterns of total C4 alleles in RA patients (n = 160), non-RA patients (n = 88), and healthy controls (n = 51). There was no trend toward C4A deficiency as in lupus. Significant differences in C4B distribution were observed in RA patients, in whom an ∼2-fold increase in the frequency of homozygous and/or heterozygous C4B deficiency (0 or 1 allele) (40%) was present relative to non-RA patients or healthy controls (both 21.6%). C4B deficiency was more frequent in seropositive RA patients than in seronegative RA patients (44% versus 31%). The odds of C4B deficiency were 2.99 (95% CI 1.58-5.65) (P = 0.0006) in seropositive RA patients relative to non-RA controls. These findings were confirmed in a larger healthy control cohort, yielding an OR of 1.83 (95% CI 1.21-2.76) (P = 0.0056). The association of the shared epitope with C4B deficiency was significantly greater in seropositive RA patients than in non-seropositive RA controls (96% versus 54.5%) (P < 0.0001), suggesting that C4B deficiency interacts with the shared epitope in the development of seropositive RA. CONCLUSION: Our findings indicate a relationship between C4B copy number variation and RA that approximates that seen between C4A copy number variation and lupus. The concurrence of C4B deficiency and the shared epitope in seropositive RA may have broad implications for our understanding of RA pathogenesis.

The JAK inhibitor CP-690,550 (tofacitinib) inhibits TNF-induced chemokine expression in fibroblast-like synoviocytes: autocrine role of type I interferon.

OBJECTIVES: The objective of this study was to investigate the effect of the novel Janus kinase inhibitor CP-690,550 in fibroblast-like synoviocytes (FLSs) from patients with rheumatoid arthritis (RA). METHODS: RA FLSs were isolated from tissue obtained by arthroplasty, cultured and serum-starved 48 h prior to stimulation. Messenger RNA and protein levels were determined by quantitative PCR and ELISA or multiplex bead assay, respectively. Phosphorylation of STAT (signal transducers and activators of transcription) proteins was determined by western blot. RESULTS: Interleukin-6-induced phosphorylation of STAT1 and STAT3 was inhibited by CP-690,550 with IC(50) values of 23 and 77 nM, respectively. Unexpectedly, although tumour necrosis factor (TNF) did not induce immediate phosphorylation of either STAT, CP-690,550 inhibited TNF-induced expression of several chemokines (IP-10, RANTES and MCP1) at the messenger RNA and protein levels. Chemokine expression was inhibited by cycloheximide, implying a need for de novo protein synthesis, and cycloheximide abolished the effect of CP-690,550 (tofacitinib). TNF induced early interferon (IFN) ß expression and STAT1 phosphorylation beginning at 3 h, which was blocked by CP-690,550. The dependence of TNF-induced chemokine expression on type I IFN was confirmed in FLSs from mice lacking type I IFN receptors (IFNARs) and in RA FLSs using an IFNAR blocking antibody. CONCLUSIONS: The Janus kinase/STAT pathway in FLS is indirectly activated by TNF through autocrine expression of type I IFN, resulting in IFNAR engagement and production of T cell chemokines. These findings illuminate a novel role of CP-690,550 in the treatment of RA: the reduction of chemokine synthesis by FLS, thereby limiting recruitment of T cells and other infiltrating leucocytes.

CXCL13: a novel biomarker of B-cell return following rituximab treatment and synovitis in patients with rheumatoid arthritis.

OBJECTIVES: The B-cell chemokine, CXCL13, is a proposed serum biomarker of synovitis in RA. Its behaviour in the context of B-cell depletion therapy and reconstitution was studied during treatment of RA with rituximab. METHODS: Serum samples from 20 RA patients were analysed for CXCL13, RF-IgM and anti-CCP by ELISA before and 2 and 6 months following rituximab treatment. B cells were monitored by flow cytometry. Gene expression in blood and synovial biopsies was determined by qPCR. RESULTS: Patients with detectable B cells at 6 months had significantly higher levels of CXCL13 and RF-IgM and slightly higher levels of anti-CCP throughout the study, including at baseline, compared with patients with undetectable B cells at 6 months. Conversely, 10 of 12 patients with high baseline CXCL13 had detectable circulating B cells at 6 months, whereas no B cells could be detected at 6 months in patients with low baseline CXCL13. Synovial CXCL13 expression at baseline correlated significantly with serum CXCL13 levels, and the synovium of patients with high serum CXCL13 expressed elevated levels of IL-1ß, IL-8, MMP1 and MMP3. In addition, synovial CXCL13 expression correlated significantly with several synovial inflammatory markers. CONCLUSIONS: Serum CXCL13 is predictive of the rate of B-cell repopulation following a course of rituximab in RA. Serum CXCL13 correlates with synovial CXCL13 measured at a single joint, suggesting synovitis as an important source of circulating CXCL13. Within the synovium, CXCL13 expression is highly correlated with markers of synovitis. TRIAL REGISTRATION: ClinicalTrials.gov, http://clinicaltrials.gov/, NCT00147966.

Platelet-derived growth factor and transforming growth factor beta synergistically potentiate inflammatory mediator synthesis by fibroblast-like synoviocytes.

INTRODUCTION: The objective of this study was to model the effects of transforming growth factor beta (TGF-beta) and platelet-derived growth factor (PDGF), both present in rheumatoid arthritis (RA) synovia, on the behavior of fibroblast-like synoviocytes (FLS) in response to pro-inflammatory cytokine (interleukin (IL)1beta, tumor necrosis factor-alpha (TNFalpha)) challenge. METHODS: Gene and protein expression by fibroblast-like synoviocytes in vitro was studied by quantitative Polymerase Chain Reaction (qPCR), ELISA and multiplex bead cytokine assays. Intracellular signaling pathway activation was determined by Western blot for phospho-kinases and the use of specific inhibitors. RESULTS: In combination, TGF-beta and PDGF (2GF) synergistically augmented TNFalpha- or IL1beta-induced matrix metalloproteinase 3 (MMP3), IL6, IL8, and macrophage inflammatory protein 1 alpha (MIP1alpha) secretion by FLS. Other FLS-derived mediators remained unaffected. Individually, neither growth factor significantly potentiated TNFalpha or IL1beta-induced MMP3 secretion, and only slightly enhanced IL6. The effect of 2GF on TNFalpha-induced gene expression was transcriptionally mediated; blocked by imatinib mesylate; and occurred even if 2GF was added as much as four hours prior to TNFalpha. In addition, a 15-minute pulse of 2GF four hours prior to TNFalpha stimulation yielded a synergistic response. The extracellular-signal-regulated kinase (ERK) and phosphoinositide 3-kinase (PI3K) signaling pathways were induced for at least four hours by 2GF, as demonstrated by persistently upregulated levels of phospho-Akt and phospho-ERK. However, pharmacologic inhibitor studies demonstrated that the potentiating action of 2GF was dependent on PI3 kinase only, and not on ERK. CONCLUSIONS: The combination of PDGF and TGF-beta dramatically potentiates FLS response to cytokines in a receptor-mediated and PI3 kinase-dependent fashion. These data suggest that 2GF contribute to synovitis by directing synovial fibroblasts toward a more aggressive phenotype in response to TNFalpha. Therefore, inhibition of growth factor signaling may constitute a complementary therapeutic approach to cytokine-targeted treatments for RA.

Elevated autoantibody content in rheumatoid arthritis synovia with lymphoid aggregates and the effect of rituximab.

INTRODUCTION: The purpose of this study was to quantitatively evaluate the contribution of synovial lymphoid aggregates to autoantibody (rheumatoid factor [RF] and anti-cyclic citrullinated peptide [anti-CCP]) and total immunoglobulin (IgG and IgM) production in rheumatoid arthritis (RA) patients and the effect thereon of the B-cell-depleting antibody, rituximab, in the ARISE (Assessment of Rituximab's Immunomodulatory Synovial Effects) trial. METHODS: Autoantibodies as well as total IgM and IgG were quantified by enzyme-linked immunosorbent assay in extracts of synovial tissues and matched serum from patients with RA or osteoarthritis (OA). Synovial biopsies and serum were obtained at baseline and 8 weeks following rituximab therapy in 14 RA patients. A synovial/serum index (SSI) was calculated as the ratio of synovial to serum antibody/albumin, with values above 1 representing synovial enrichment. Lymphoid aggregates were evaluated histologically. RESULTS: Anti-CCP IgG, but not RF-IgM, was significantly enriched in RA synovia compared with serum. Total IgM and IgG were also enriched in RA, but not in OA. SSI correlated significantly with mRNA content for both IgM and IgG, demonstrating that it reflected synovial immunoglobulin production. RA synovia with lymphocyte aggregates contained significantly elevated RF-IgM and anti-CCP IgG compared with tissues with diffuse lymphoid infiltration. Rituximab treatment did not affect synovial autoantibody or total immunoglobulin SSI overall. However, in aggregate-containing tissues, rituximab significantly reduced total IgM and IgG SSI as well as IgM and IgG1 mRNA. Surprisingly, RF-IgM and anti-CCP IgG SSIs were unchanged by rituximab in aggregate-containing synovia. CONCLUSIONS: Combined with earlier observations that synovial lymphoid aggregates are unaltered by rituximab treatment, these data suggest that lymphoid aggregates may provide a protective niche for autoantibody-producing cells.

Modulation of melanocortin signaling ameliorates uremic cachexia.

Insulin-like growth factor (IGF)-I increases muscle mass while myostatin inhibits its development. Muscle wasting is common in patients with uremic cachexia and may be due to imbalance of this regulation. We had proposed a central mechanism involving leptin and melanocortin signaling in the pathogenesis of uremic cachexia since agouti-related peptide (AgRP), a melanocortin-4 receptor antagonist, reduced uremic cachexia. Here we found that injection of AgRP into the cerebral ventricles resulted in a gain of body mass and improved metabolic rate regulation in a mouse model of uremic cachexia. These salutary effects occurred independent of increased protein and calorie intake. Myostatin mRNA and protein concentrations were increased while those of IGF-I were decreased in the skeletal muscle of uremic mice. AgRP treatment partially corrected these uremia-induced changes. Suppressor of cytokine signaling-2 gene expression (SOCS2) was significantly increased in uremic animals and AgRP reduced this expression. We suggest that AgRP improves uremic cachexia and muscle wasting by a peripheral mechanism involving the balance between myostatin and IGF-I.

Acquisition, culture, and phenotyping of synovial fibroblasts.

The study of fibroblast-like synoviocytes (FLS) has yielded important insights into the pathogenic mechanisms of rheumatoid arthritis. FLS can be cultured from synovial tissue obtained at joint replacement surgery, synovectomy, or synovial biopsy. After collagenase digestion, adherent cells consist mainly of synovial fibroblasts and synovial macrophages. Proliferating FLS are enriched by repeated passage and comprise >95% of cells by passage 3. Because of cell senescence, use of FLS lines after passage 9 is generally not recommended. FLS in culture have a distinct phenotype with regard to morphology, ultrastructure, surface phenotype, and function. Surface markers that can be used to characterize FLS include positive staining for VCAM-1, CD44, CD55, CD90 (Thy-1), and cadherin-11, coupled with the absence of macrophage markers such as CD14 or CD68.

Monocytes from familial cold autoinflammatory syndrome patients are activated by mild hypothermia.

BACKGROUND: Familial cold autoinflammatory syndrome (FCAS) is characterized by rash, fever, and arthralgia in response to cold exposure. CIAS1, the gene that codes for cryopyrin, is mutated in FCAS. Treatment with anakinra (IL-1 receptor antagonist) prevents symptoms, indicating a crucial role for IL-1 in this disease. OBJECTIVE: To study cytokine responses to cold exposure in monocytes from subjects with FCAS. METHODS: Adherence-enriched monocytes were incubated at 32 degrees C or 37 degrees C. Transcription and release of IL-1beta, IL-6, and TNF-alpha were monitored by quantitative PCR and ELISA. RESULTS: The FCAS monocytes but not control cells responded to 4 h incubation at 32 degrees C with significant secretion of IL-1beta. At 16 h, IL-1beta, IL-6, and TNF-alpha were all significantly elevated in FCAS monocytes at 32 degrees C. Increased cytokine transcription was observed in all monocytes at 4 hours, but at 16 hours it was only seen in FCAS monocytes incubated at 32 degrees C. Incubation at 32 degrees C for as little as 1 hour sufficed to induce measurable IL-1beta release. Caspase-1 inhibitors prevented the cold-induced IL-1beta release, whereas a purinergic antagonist did not. Anakinra had no effect on the early IL-1beta release but significantly reduced the late-phase transcription and release of all cytokines. CONCLUSION: FCAS monocytes respond to mild hypothermia with IL-1beta release, which in turn induces autocrine transcription and secretion of IL-6 and TNF-alpha as well as stimulation of further IL-1beta production. CLINICAL IMPLICATIONS: These results confirm the central role of IL-1beta in FCAS and support the use of IL-1 targeted therapy in these patients.

Real-time quantitative PCR to detect changes in synovial gene expression in rheumatoid arthritis after corticosteroid treatment.

Synovial biomarkers are increasingly important in the development of novel therapeutic agents for the treatment of rheumatoid arthritis (RA). To identify biomarkers correlating with changes in clinical disease activity, real-time quantitative PCR (Q-PCR) was used to evaluate changes in synovial gene expression after treatment with corticosteroids. Patients with active RA received either oral prednisolone (n=10, 60 mg daily for the first week and 40 mg daily for the second week) or placebo (n=11) for 14 days. Real-time Q-PCR was used to quantify gene expression of tumour necrosis factor (TNF)alpha, IL1beta, IL8 and matrix metalloproteinase (MMP) 1 in synovial tissue samples obtained through an arthroscopic procedure before and after treatment. mRNA levels were reported as relative expression units compared with a cell-based standard. Statistical analysis was performed using an analysis of covariance model. Prednisolone markedly decreased IL8 and MMP1 expression compared with placebo, and the CIs excluded the likelihood of no effect. A trend towards reduction was seen in IL1beta and TNFalpha mRNA expression in the prednisolone group, although CIs included the value for no effect. These data suggest that Q-PCR can be used to measure synovial mRNA expression of mediators implicated in the pathogenesis of RA in small proof-of-concept trials.