Synovial fibroblast gene expression is associated with sensory nerve growth and pain in rheumatoid arthritis.

It has been presumed that rheumatoid arthritis (RA) joint pain is related to inflammation in the synovium; however, recent studies reveal that pain scores in patients do not correlate with synovial inflammation. We developed a machine-learning approach (graph-based gene expression module identification or GbGMI) to identify an 815-gene expression module associated with pain in synovial biopsy samples from patients with established RA who had limited synovial inflammation at arthroplasty. We then validated this finding in an independent cohort of synovial biopsy samples from patients who had early untreated RA with little inflammation. Single-cell RNA sequencing analyses indicated that most of these 815 genes were most robustly expressed by lining layer synovial fibroblasts. Receptor-ligand interaction analysis predicted cross-talk between human lining layer fibroblasts and human dorsal root ganglion neurons expressing calcitonin gene-related peptide (CGRP+). Both RA synovial fibroblast culture supernatant and netrin-4, which is abundantly expressed by lining fibroblasts and was within the GbGMI-identified pain-associated gene module, increased the branching of pain-sensitive murine CGRP+ dorsal root ganglion neurons in vitro. Imaging of solvent-cleared synovial tissue with little inflammation from humans with RA revealed CGRP+ pain-sensing neurons encasing blood vessels growing into synovial hypertrophic papilla. Together, these findings support a model whereby synovial lining fibroblasts express genes associated with pain that enhance the growth of pain-sensing neurons into regions of synovial hypertrophy in RA.

Pilot Scale Assessment of High-Pressure Processing (HPP) to Enhance Microbiological Quality and Shelf Life of Fresh Ready-to-Eat (RTE) Blue Crab Meat.

Blue crab (Callinectes sapidus) is a highly valuable wild fishery species of crab native to the waters of the western Atlantic Ocean and the Gulf of Mexico. The annual commercial production of live blue crabs is approximately 50,000 metric tons with a dockside value of USD 200 million. Presently the US blue crab processing industry sells crab meat in three basic forms: fresh crab meat, pasteurized crab meat, and frozen crab meat. By far "Fresh" is the most desirable form of crab meat. However, fresh crab meat has a limited shelf life. This study evaluated the effects of high-pressure processing (HPP) on enhancing the microbiological quality and shelf life of blue crab meat. Live blue crabs were pressure-cooked in a retort (≥115 °C for 4-6 min). The crab meat was handpicked, packed in plastic containers with seals, subjected to HPP treatment, and stored at 4 °C. Container integrity and water leakage issues were examined by observation in addition to weight comparison before and after HPP treatment; the shelf life of crab meat with and without HPP treatments was examined via microbiological tests and sensory evaluations. Results show that polypropylene containers sealed with 10K OTR (oxygen transmission rate) film could withstand high pressure without water leakage issues; HPP treatment at 600 MPa for 3 min could extend the shelf life of fresh, cooked, and handpicked crab meat from 6 days to 18 days based on the strictest APC (aerobic plate account) limit (APC ≤ 100,000 CFU/g). The sensory quality of the HPP-treated crab meat was well accepted throughout the 3-week storage period. The results support the use of HPP as an effective non-thermal processing technology to enhance the microbiological quality and extend the shelf life of fresh RTE blue crab meat.

Microbiome Analysis of Organic and Conventional Chickens Processed Using Whole Carcass Enrichment and Rinse Methods.

Investigating the chicken microbiome is important to establish control measures for pathogens to protect consumers. This study aimed at evaluating the comparative efficiency of human pathogen detection through 16S rRNA sequencing of organic and conventional chickens processed using whole carcass enrichment (WCE) and rinse (WCR) methods. Organic and conventional whole broiler carcasses (n = 31) were vigorously shaken with 500 mL buffered peptone water (BPW). For the rinse method, a 30 mL aliquot was mixed with 30 mL of BPW. The rest of the sample, including the carcass, was used for the enrichment method. All samples were incubated at 37°C for 24 h. The samples were divided into five groups [Negative Control: only BPW without chicken (n = 5), Organic-Rinsed (n = 7), -Enriched (n = 8), Conventional-Rinsed (n = 7), and -Enriched (n = 9)]. Fifty milliliters of each sample were subjected to DNA extraction followed by 16S rRNA sequencing. Proteobacteria and Firmicutes predominated the microbiota of both conventional and organic chickens, followed by low abundances of Bacteroidetes and Fusobacterium. While the abundance of Proteobacteria and Firmicutes remained unchanged in organic chicken irrespective of the methods used, a noticeable shift in the Proteobacteria and Firmicutes ratio (59%:39% in rinsed to 38%:60% in enriched) was observed in conventional chicken. Furthermore, the choice of method did not yield any differences in Abundance-Based Coverage Estimator, and Jackknife, among conventional and organic chickens but resulted in a statistically significant difference in the Shannon, Simpson, Chao1, and phylogenetic diversity indices (p < 0.05). The relative abundance of Salmonella and Campylobacter was less than 0.1%. The results suggested the WCE method provides a broad range of information on the chicken microbiome.

Machine Learning Reveals Synovial Fibroblast Genes Associated with Pain Affect Sensory Nerve Growth in Rheumatoid Arthritis.

It has been presumed that rheumatoid arthritis (RA) joint pain is related to inflammation in the synovium; however, recent studies reveal that pain scores in patients do not correlate with synovial inflammation. We identified a module of 815 genes associated with pain, using a novel machine learning approach, Graph-based Gene expression Module Identification (GbGMI), in samples from patients with longstanding RA, but limited synovial inflammation at arthroplasty, and validated this finding in an independent cohort of synovial biopsy samples from early, untreated RA patients. Single-cell RNA-seq analyses indicated these genes were most robustly expressed by lining layer fibroblasts and receptor-ligand interaction analysis predicted robust lining layer fibroblast crosstalk with pain sensitive CGRP+ dorsal root ganglion sensory neurons. Netrin-4, which is abundantly expressed by lining fibroblasts and associated with pain, significantly increased the branching of pain-sensitive CGRP+ neurons in vitro . We conclude GbGMI is a useful method for identifying a module of genes that associate with a clinical feature of interest. Using this approach, we find that Netrin-4 is produced by synovial fibroblasts in the absence of inflammation and can enhance the outgrowth of CGRP+ pain sensitive nerve fibers. One Sentence Summary: Machine Learning reveals synovial fibroblast genes related to pain affect sensory nerve growth in Rheumatoid Arthritis addresses unmet clinical need.

RNA Identification of PRIME Cells Predicting Rheumatoid Arthritis Flares.

BACKGROUND: Rheumatoid arthritis, like many inflammatory diseases, is characterized by episodes of quiescence and exacerbation (flares). The molecular events leading to flares are unknown. METHODS: We established a clinical and technical protocol for repeated home collection of blood in patients with rheumatoid arthritis to allow for longitudinal RNA sequencing (RNA-seq). Specimens were obtained from 364 time points during eight flares over a period of 4 years in our index patient, as well as from 235 time points during flares in three additional patients. We identified transcripts that were differentially expressed before flares and compared these with data from synovial single-cell RNA-seq. Flow cytometry and sorted-blood-cell RNA-seq in additional patients were used to validate the findings. RESULTS: Consistent changes were observed in blood transcriptional profiles 1 to 2 weeks before a rheumatoid arthritis flare. B-cell activation was followed by expansion of circulating CD45-CD31-PDPN+ preinflammatory mesenchymal, or PRIME, cells in the blood from patients with rheumatoid arthritis; these cells shared features of inflammatory synovial fibroblasts. Levels of circulating PRIME cells decreased during flares in all 4 patients, and flow cytometry and sorted-cell RNA-seq confirmed the presence of PRIME cells in 19 additional patients with rheumatoid arthritis. CONCLUSIONS: Longitudinal genomic analysis of rheumatoid arthritis flares revealed PRIME cells in the blood during the period before a flare and suggested a model in which these cells become activated by B cells in the weeks before a flare and subsequently migrate out of the blood into the synovium. (Funded by the National Institutes of Health and others.).

ZFP36 RNA-binding proteins restrain T cell activation and anti-viral immunity.

Dynamic post-transcriptional control of RNA expression by RNA-binding proteins (RBPs) is critical during immune response. ZFP36 RBPs are prominent inflammatory regulators linked to autoimmunity and cancer, but functions in adaptive immunity are less clear. We used HITS-CLIP to define ZFP36 targets in mouse T cells, revealing unanticipated actions in regulating T-cell activation, proliferation, and effector functions. Transcriptome and ribosome profiling showed that ZFP36 represses mRNA target abundance and translation, notably through novel AU-rich sites in coding sequence. Functional studies revealed that ZFP36 regulates early T-cell activation kinetics cell autonomously, by attenuating activation marker expression, limiting T cell expansion, and promoting apoptosis. Strikingly, loss of ZFP36 in vivo accelerated T cell responses to acute viral infection and enhanced anti-viral immunity. These findings uncover a critical role for ZFP36 RBPs in restraining T cell expansion and effector functions, and suggest ZFP36 inhibition as a strategy to enhance immune-based therapies.

T cells presenting viral antigens or autoantigens induce cytotoxic T cell anergy.

In the course of modeling the naturally occurring tumor immunity seen in patients with paraneoplastic cerebellar degeneration (PCD), we discovered an unexpectedly high threshold for breaking CD8+ cytotoxic T cell (CTL) tolerance to the PCD autoantigen, CDR2. While CDR2 expression was previously found to be strictly restricted to immune-privileged cells (cerebellum, testes, and tumors), unexpectedly we have found that T cells also express CDR2. This expression underlies inhibition of CTL activation; CTLs that respond to epithelial cells expressing CDR2 fail to respond to T cells expressing CDR2. This was a general phenomenon, as T cells presenting influenza (flu) antigen also fail to activate otherwise potent flu-specific CTLs either in vitro or in vivo. Moreover, transfer of flu peptide-pulsed T cells into flu-infected mice inhibits endogenous flu-specific CTLs. Our finding that T cells serve as a site of immune privilege, inhibiting effector CTL function, uncovers an autorepressive loop with general biologic and clinical relevance.

Dendritic cell vaccines containing lymphocytes produce improved immunogenicity in patients with cancer.

BACKGROUND: Dendritic cells are currently under investigation for their ability to generate anti-cancer immune responses. No consensus has been reached as to the optimal method of dendritic cell vaccine preparation and is a barrier to success in the field. METHODS: Over a course of three separate dendritic cell vaccine studies to treat cancer, we tested two different methods for preparing dendritic cells from peripheral blood mononuclear cells: adherence and antibody-selected CD14+ cells. RESULTS: Surprisingly, we found that patients who received dendritic cell vaccines generated by the adherence method mounted increased T cell proliferation in response to vaccination. This difference could not be accounted for by dendritic cell vaccine dose, cell surface phenotype or dendritic cell function in vitro. One notable difference between the two vaccine preparation methods was that the dendritic cell vaccine cultures generated by the adherence method contained up to 10% lymphocytes, and these lymphocytes were proliferating and producing IFNγ in response to antigen in vitro at the time of administration. CONCLUSIONS: Enhanced immunogenicity of adherence dendritic cell vaccinations may be due to the presence of lymphocytes during dendritic cell culture. TRIAL REGISTRATION: Clinicaltrials.gov identifiers: NCT00289341, NCT00345293, and NCT00893945.

Cellular immune suppression in paraneoplastic neurologic syndromes targeting intracellular antigens.

BACKGROUND Tumor treatment is the mainstay of therapy for paraneoplastic neurologic disorders (PNDs), but it is only effective in some cases and other treatment options are limited. OBJECTIVE To evaluate the short-term use of a combination of prednisone and tacrolimus for acute neurologic worsening in PND in which intracellular antigens are targeted. DESIGN Retrospective single-center case series of patients with PND treated with tacrolimus. SETTING The Rockefeller University Hospital, a research hospital in New York, New York. PATIENTS Twenty-six patients with PND with high titer (≥1:1000) anti-HuD, anti-Yo, or anti-CRMP5 autoantibodies were enrolled. Patients were referred from Memorial Sloan Kettering Cancer Center or self-referred. Two patients discontinued intervention owing to adverse events. INTERVENTIONS Patients were treated with tacrolimus, 0.15-0.30 mg/kg per day, in 2 divided oral doses with 60 mg per day of oral prednisone, tapered off during 1 to 4 weeks. MAIN OUTCOME MEASURES The primary outcome measure was median survival. Neurologic examinations before and after treatment as well as adverse events are described. RESULTS Median survival time was 52 months from time of diagnosis. Some patients experienced neurologic improvement that was functionally meaningful. The incidence of adverse events was similar to that generally reported with tacrolimus. CONCLUSIONS A short course of prednisone and tacrolimus to target central nervous system T cells in patients with PND with acute neurologic decline in which intracellular antigens are targeted was well tolerated and warrants further study. TRIAL REGISTRATION clinicaltrials.gov Identifier: NCT00378326.

Harnessing naturally occurring tumor immunity: a clinical vaccine trial in prostate cancer.

BACKGROUND: Studies of patients with paraneoplastic neurologic disorders (PND) have revealed that apoptotic tumor serves as a potential potent trigger for the initiation of naturally occurring tumor immunity. The purpose of this study was to assess the feasibility, safety, and immunogenicity of an apoptotic tumor-autologous dendritic cell (DC) vaccine. METHODS AND FINDINGS: We have modeled PND tumor immunity in a clinical trial in which apoptotic allogeneic prostate tumor cells were used to generate an apoptotic tumor-autologous dendritic cell vaccine. Twenty-four prostate cancer patients were immunized in a Phase I, randomized, single-blind, placebo-controlled study to assess the safety and immunogenicity of this vaccine. Vaccinations were safe and well tolerated. Importantly, we also found that the vaccine was immunogenic, inducing delayed type hypersensitivity (DTH) responses and CD4+ and CD8+ T cell proliferation, with no effect on FoxP3+ regulatory T cells. A statistically significant increase in T cell proliferation responses to prostate tumor cells in vitro (p = 0.002), decrease in prostate specific antigen (PSA) slope (p = 0.016), and a two-fold increase in PSA doubling time (p = 0.003) were identified when we compared data before and after vaccination. CONCLUSIONS: An apoptotic cancer cell vaccine modeled on naturally occurring tumor immune responses in PND patients provides a safe and immunogenic tumor vaccine. TRIAL REGISTRATION: ClinicalTrials.gov NCT00289341.

Microinfusion of antineuronal antibodies into rodent striatum: failure to differentiate between elevated and low titers.

An autoimmune-mediated mechanism has been proposed for several pediatric movement disorders. In a three-center (Brown, Yale, and Johns Hopkins) collaborative effort, serum antineuronal antibodies (ANAb) were measured by use of ELISA or immunohistochemical techniques on 35 children (mean age 11.4 years) with Tourette syndrome, attention deficit hyperactivity disorder, and/or obsessive compulsive disorder. Eight sera, 4 containing the highest and 4 the lowest levels of ANAb, were identified at each institution. Selected sera (total of 9 with elevated and 7 with low ANAb) were re-encoded and sent to each center for infusion into the ventrolateral striatum of 16 male Sprague-Dawley rats. Animals were observed for behavioral abnormalities for 3 days before the start of infusion, during infusion on days 2-4, and for 2 days after infusion. Combined stereotypy scores increased after antibody infusion, but there was no significant effect based on serum titer (p=0.85). Scores differed among centers, but analyses based on individual institutional data again failed to show an effect based on elevated or low ANAb values (Brown, p=0.95; Yale and Johns Hopkins, p=0.81). Post hoc studies with sham surgery and infusion of phosphate-buffered saline support suggestions of nonspecific behavioral effects unrelated to antibody titer. This report emphasizes that any conclusions about antibody-mediated movement disorders that are based upon results from the rodent infusion model must be considered with caution.

A survey for phosphoglucose isomerase with lysyl aminopeptidase activity in Vibrionaceae and non-Vibrio pathogens.

Phosphoglucose isomerase (PGI) with a novel lysyl aminopeptidase (LysAP) activity was recently purified and characterized from Vibrio vulnificus. We showed that it cleaves the amino-terminal lysyl residue from des-Arg(10)-kallidin to produce des-Arg(9)-bradykinin, suggesting that it plays a role in virulence. A survey was conducted to determine the presence of this potential virulence-enhancing enzyme among twenty-three halotolerant human and fish pathogens from eleven species within the Vibrionaceae family, including V. vulnificus, V. parahaemolyticus, V. cholerae, Aeromonas hydrophila, and Plesiomonas shigelloides. In addition, fourteen species of non-Vibrionaceae pathogens were screened for LysAP activity. Cell lysates were partially purified by anion exchange chromatography and fractions were screened for LysAP and isomerase activities. PGI-LysAP activity was detected in chromatographic fractions from all the Vibrio species tested, but was not detected in any of the non-Vibrionaceae pathogens. Levels of isomerase and LysAP activity correlated (R(2)=0.92) for nine strains of V. vulnificus. Since the Vibrionaceae represent an important family of human and fish pathogens, our identification of PGI-LysAP activity in a broad array of vibrios may lead to the development of improved analytical methods for their identification as well as interventions to reduce the high morbidity and mortality associated with some Vibrionaceae infections in clinical, veterinary, and aquaculture settings.

Increased serum levels of interleukin-12 and tumor necrosis factor-alpha in Tourette's syndrome.

BACKGROUND: The hypothesis that common infections can modulate the onset and course of tic disorders and early-onset obsessive-compulsive disorder (OCD) in pediatric populations is longstanding. To date, most investigations have focused on the hypothesis of molecular mimicry and humoral immune responses. This study was carried out to investigate whether cytokines associated with the innate immune response or T cell activation were altered under baseline conditions and during periods of symptom exacerbation. METHODS: Forty-six patients with Tourette's syndrome and/or early-onset OCD, aged 7-17 years, and 31 age-matched control subjects participated in a prospective longitudinal study. Ratings of clinical severity and serum were collected at regular intervals, and serum concentrations of 10 cytokines were measured repeatedly. RESULTS: Interleukin-12 and tumor necrosis factor alpha concentrations at baseline were elevated in patients compared with control subjects. Both of these markers were further increased during periods of symptom exacerbation. CONCLUSIONS: These findings suggest that symptom exacerbations are associated with an inflammatory process propagated by systemic and local cytokine synthesis that might involve the central nervous system. We conclude that, in the future, longitudinal studies of children with neuropsychiatric disorders should examine the involvement of innate and T cell immunity.

Characterization of a lysyl aminopeptidase activity associated with phosphoglucose isomerase of Vibrio vulnificus.

Phosphoglucose isomerase (PGI) is a multifunctional enzyme involved in glycolysis and gluconeogenesis and, in mammalian cells, functions as neuroleukin, autocrine motility factor (AMF), and differentiation and maturation factor (MF). We isolated and characterized PGI with a novel lysyl aminopeptidase (LysAP) activity (PGI-LysAP) from Vibrio vulnificus. Mass spectrometry revealed that PGI-LysAP is a heterodimer consisting of 23.4- and 60.8-kDa subunits. Only the heterodimer displayed LysAP activity. PGI-LysAP has a pI around 6.0 and high specificity toward the synthetic, fluorogenic substrate l-lysyl-7-amino-4-methylcoumarin. LysAP activity is optimal at pH 8.0, is 64% higher at 37 degrees C than at 21 degrees C, does not directly correlate with virulence, and is strongly inhibited by serine protease and metalloprotease inhibitors. PGI-LysAP was also identified in Vibrio parahaemolyticus and V. cholerae, but was absent from non-Vibrio human pathogens. Sequencing of the pgi gene revealed 1653 bp coding for a 550-amino-acid protein. Cloned and expressed PGI formed a homodimer with isomerase activity, but not LysAP activity. The finding of LysAP activity associated with heterodimeric PGI should foster a broad search for putative substrates in an effort to elucidate the role of PGI-LysAP in bacteria and its roles in the pathophysiology of diseases.