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OBJECTIVE: To investigate the mechanism underlying neutrophil migration into the articular cavity in experimental arthritis and, by extension, human inflammatory synovitis. METHODS: Antigen-induced arthritis (AIA) was generated in mice with methylated bovine serum albumin (mBSA). Migration assays and histologic analysis were used to evaluate neutrophil recruitment to knee joints. Levels of inflammatory mediators were measured by enzyme-linked immunosorbent assay. Antibodies and pharmacologic inhibitors were used in vivo to determine the role of specific disease mediators. Samples of synovial tissue and synovial fluid from rheumatoid arthritis (RA) or osteoarthritis patients were evaluated for CXCL1 and CXCL5 expression. RESULTS: High levels of CXCL1, CXCL5, and leukotriene B4 (LTB4) were expressed in the joints of arthritic mice. Confirming their respective functional roles, repertaxin (a CXCR1/CXCR2 receptor antagonist), anti-CXCL1 antibody, anti-CXCL5 antibody, and MK886 (a leukotriene synthesis inhibitor) reduced mBSA-induced neutrophil migration to knee joints. Repertaxin reduced LTB4 production in joint tissue, and neutrophil recruitment induced by CXCL1 or CXCL5 was inhibited by MK886, suggesting a sequential mechanism. Levels of both CXCL1 and CXCL5 were elevated in synovial fluid and were released in vitro by RA synovial tissues. Moreover, RA synovial fluid neutrophils stimulated with CXCL1 or CXCL5 released significant amounts of LTB4. CONCLUSION: Our data implicate CXCL1, CXCL5, and LTB4, acting sequentially, in neutrophil migration in AIA. Elevated levels of CXCL1 and CXCL5 in the synovial compartment of RA patients provide robust comparative data indicating that this mechanism plays a role in inflammatory joint disease. Together, these results suggest that inhibition of CXCL1, CXCL5, or LTB4 may represent a potential therapeutic strategy in RA. [ABSTRACT FROM AUTHOR]

Biohydrogen is a potentially useful product of microbial energy metabolism. One approach to engineering biohydrogen production in bacteria is the production of non-native hydrogenase activity in a host cell, for example Escherichia coli. In some microbes, hydrogenase enzymes are linked directly to central metabolism via diaphorase enzymes that utilise NAD+/NADH cofactors. In this work, it was hypothesised that heterologous production of an NAD+/NADH-linked hydrogenase could connect hydrogen production in an E. coli host directly to its central metabolism. To test this, a synthetic operon was designed and characterised encoding an apparently NADH-dependent, hydrogen-evolving [FeFe]-hydrogenase from Caldanaerobacter subterranus. The synthetic operon was stably integrated into the E. coli chromosome and shown to produce an active hydrogenase, however no H2 production was observed. Subsequently, it was found that heterologous co-production of a pyruvate::ferredoxin oxidoreductase and ferredoxin from Thermotoga maritima was found to be essential to drive H2 production by this system. This work provides genetic evidence that the Ca.subterranus [FeFe]-hydrogenase could be operating in vivo as an electron-confurcating enzyme.

Objectives: To describe the MRI findings in a UK referral population of dogs with steroid-responsive meningitis-arteritis and to determine if they were associated with any specific clinical features or outcomes., Materials and Methods: We performed a multi-centre retrospective case series of dogs diagnosed with steroid-responsive meningitis-arteritis in the UK that underwent MRI. Blinded consensus review of the MRI studies was performed and the findings described. The presence or absence of specific MRI abnormalities were analysed for significant associations with presenting signs, results of investigations or case outcomes., Results: Fifty-three dogs were included. The most common MRI findings were paravertebral muscle changes (30/53; 56.6%), meningeal contrast enhancement (13/41; 31.7%) and spinal cord parenchymal T2-W hyperintensity (15/53; 28.3%). Haemorrhage was observed in five of 53 (9.4%) cases - three intradural-extramedullary, one intramedullary and one extradural. Following binary logistic regressions, T2-W spinal cord parenchymal hyperintensity had a significant positive association with paresis/paralysis (odds ratio 14.86, 95% confidence interval 1.42 to 154.99) as did haemorrhage (odds ratio 16.12, confidence interval 2.05 to 126.73). Fifty-two (98.1%) dogs survived to discharge. Relapse occurred in nine of 29 (31.0%) dogs with sufficient follow-up, and no MRI finding had a significant relationship with its occurrence., Clinical Significance: Magnetic resonance imaging findings for steroid-responsive meningitis-arteritis can be severe and extensive, as can the clinical presentation. The presence of paresis/paralysis should raise concern for haemorrhage, though most dogs still have a good prognosis., (© 2024 Linnaeus, Pride Veterinary Referrals and The Author(s). Journal of Small Animal Practice published by John Wiley & Sons Ltd on behalf of British Small Animal Veterinary Association.)

Competing Interests: Author AF was employed by Swarm Oncology Ltd. Author SC was employed by Adthera Bio Ltd. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. The author(s) declared that they were an editorial board member of Frontiers, at the time of submission. This had no impact on the peer review process and the final decision.

Adoptive immunotherapy with Epstein-Barr virus (EBV)-specific T cells is an effective treatment for relapsed or refractory EBV-induced post-transplant lymphoproliferative disorders (PTLD) with overall survival rates of up to 69%. EBV-specific T cells have been conventionally made by repeated stimulation with EBV-transformed lymphoblastoid cell lines (LCL), which act as antigen-presenting cells. However, this process is expensive, takes many months, and has practical risks associated with live virus. We have developed a peptide-based, virus-free, serum-free closed system to manufacture a bank of virus-specific T cells (VST) for clinical use. We compared these with standard LCL-derived VST using comprehensive characterization and potency assays to determine differences that might influence clinical benefits. Multi-parameter flow cytometry revealed that peptide-derived VST had an expanded central memory population and less exhaustion marker expression than LCL-derived VST. A quantitative HLA-matched allogeneic cytotoxicity assay demonstrated similar specific killing of EBV-infected targets, though peptide-derived EBV T cells had a significantly higher expression of antiviral cytokines and degranulation markers after antigen recall. High-throughput T cell receptor-beta (TCRβ) sequencing demonstrated oligoclonal repertoires, with more matches to known EBV-binding complementary determining region 3 (CDR3) sequences in peptide-derived EBV T cells. Peptide-derived products showed broader and enhanced specificities to EBV nuclear antigens (EBNAs) in both CD8 and CD4 compartments, which may improve the targeting of highly expressed latency antigens in PTLD. Importantly, peptide-based isolation and expansion allows rapid manufacture and significantly increased product yield over conventional LCL-based approaches., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. The author(s) declared that they were an editorial board member of Frontiers, at the time of submission. This had no impact on the peer review process and the final decision., (Copyright © 2024 Cooper, Sutherland, Smith, Cowan, Barnett, Mitchell, McLean, Imlach, Hayes, Zahra, Manchanayake, Vickers, Graham, McGowan, Turner, Campbell and Fraser.)

The manufacturing of clinical cellular therapies is a complex process frequently requiring manipulation of cells, exchange of buffers and volume reduction. Current manufacturing processes rely on either low throughput open centrifugation-based devices, or expensive closed-process alternatives. Inertial focusing (IF) microfluidic devices offer the potential for high-throughput, inexpensive equipment which can be integrated into a closed system, but to date no IF devices have been approved for use in cell therapy manufacturing, and there is limited evidence for the effects that IF processing has on human cells. The IF device described in this study was designed to simultaneously separate leucocytes, perform buffer exchange and provide a volume reduction to the cell suspension, using high flow rates with high Reynolds numbers. The performance and effects of the IF device were characterized using peripheral blood mononuclear cells and isolated monocytes. Post-processing cell effects were investigated using multi-parameter flow cytometry to track cell viability, functional changes and fate. The IF device was highly efficient at separating CD14+ monocytes (approx. 97% to one outlet, approx. 60% buffer exchange, 15 ml min -1 ) and leucocyte processing was well tolerated with no significant differences in downstream viability, immunophenotype or metabolic activity when compared with leucocytes processed with conventional processing techniques. This detailed approach provides robust evidence that IF devices could offer significant benefits to clinical cell therapy manufacture.

Objective: Nutcracker syndrome (NCS) is an increasingly recognized venous compressive disease. Although people with NCS can experience debilitating symptoms, making an accurate diagnosis can often be challenging owing to the broad spectrum of clinical presentations and radiologic findings. In the present systematic review, we assessed the most frequent clinical and radiologic criteria used in establishing a diagnosis of NCS and have proposed a comprehensive framework for clinical decision-making., Methods: We performed a systematic review to identify all observational studies or case series conducted within the previous 10 years that had studied patients with a clinical and radiologic diagnosis of NCS. The extracted data included details related to the study design, participant demographics, presenting clinical features, and radiologic criteria. These details were compared between studies and synthesized to establish a comprehensive diagnostic framework that would be applicable to clinical practice., Results: In the present review, we included 14 studies with a total of 384 patients with NCS. The most common clinical features of NCS were hematuria (69.5%), left flank or abdominal pain (48.4%), pelvic pain (23.1%), and varicocele (15.8%). Computed tomography and ultrasound were the most commonly used imaging modalities, with a threshold for left renal vein stenosis of >80% the most frequently used diagnostic parameter. Eight studies had used venography, with the renocaval pressure gradient the most commonly measured parameter. Two studies had reported using intravascular ultrasound. The findings from our review have shown that a thorough clinical workup of NCS should include critical evaluation of the presenting clinical features and exclusion of alternative diagnoses. All patients should undergo duplex ultrasound with or without the addition of computed tomography or magnetic resonance imaging. Any patient considered for therapeutic intervention should also undergo diagnostic venography with measurement of the renocaval pressure gradient and, if available, intravascular ultrasound with measurement of the left renal vein diameters., Conclusions: NCS is a challenging condition in terms of the diagnosis and management both. We have proposed a comprehensive diagnostic framework based on the currently available literature to aid in clinical decision-making., (Copyright © 2022 Society for Vascular Surgery. Published by Elsevier Inc. All rights reserved.)

Objectives: To assess the recovery of urinary continence, faecal continence and tail function in ambulatory dogs with caudal lumbar intervertebral disc extrusion and to explore clinical factors that may be associated with recovery., Materials and Methods: Medical records from January 2010 to December 2020 were searched to identify ambulatory dogs undergoing surgical treatment for a caudal lumbar intervertebral disc extrusion causing urinary incontinence, faecal incontinence and/or tail dysfunction. Signalment, history, presenting clinical signs, neurological examination findings, diagnostic test results, treatment and outcome were recorded for all dogs., Results: Eighteen dogs with caudal lumbar intervertebral disc extrusion causing tail dysfunction, urinary and/or faecal incontinence were included. Urinary continence was recovered in 12 (86%) of 14 affected dogs, faecal continence recovered in nine (90%) of 10 affected dogs and tail function recovered in 13 (87%) of 15 affected dogs. Loss of tail nociception was recorded in three dogs on presentation; two made a full recovery and one showed mild persistent tail paresis., Clinical Significance: The prognosis for functional recovery of urinary continence, faecal continence and tail function in ambulatory dogs with caudal lumbar intervertebral disc extrusion following surgical treatment is good. Larger studies are needed to identify prognostic factors associated with failure of recovery., (© 2022 The Authors. Journal of Small Animal Practice published by John Wiley & Sons Ltd on behalf of British Small Animal Veterinary Association.)

Biliary diseases can cause inflammation, fibrosis, bile duct destruction, and eventually liver failure. There are no curative treatments for biliary disease except for liver transplantation. New therapies are urgently required. We have therefore purified human biliary epithelial cells (hBECs) from human livers that were not used for liver transplantation. hBECs were tested as a cell therapy in a mouse model of biliary disease in which the conditional deletion of Mdm2 in cholangiocytes causes senescence, biliary strictures, and fibrosis. hBECs are expandable and phenotypically stable and help restore biliary structure and function, highlighting their regenerative capacity and a potential alternative to liver transplantation for biliary disease., Competing Interests: Declaration of interests S.J.F. and J.D.M.C. are founders and scientific advisors of Resolution Therapeutics Ltd (not related to this study). D.C.H. is a founder, director, and shareholder at Stemnovate Limited and Stimuliver ApS (not related to this study)., (Copyright © 2022 The Authors. Published by Elsevier Inc. All rights reserved.)

Introduction: Liver cirrhosis is a growing global healthcare challenge. Cirrhosis is characterised by severe liver fibrosis, organ dysfunction and complications related to portal hypertension. There are no licensed antifibrotic or proregenerative medicines and liver transplantation is a scarce resource. Hepatic macrophages can promote both liver fibrogenesis and fibrosis regression. The safety and feasibility of peripheral infusion of ex vivo matured autologous monocyte-derived macrophages in patients with compensated cirrhosis has been demonstrated., Methods and Analysis: The efficacy of autologous macrophage therapy, compared with standard medical care, will be investigated in a cohort of adult patients with compensated cirrhosis in a multicentre, open-label, parallel-group, phase 2, randomised controlled trial. The primary outcome is the change in Model for End-Stage Liver Disease score at 90 days. The trial will provide the first high-quality examination of the efficacy of autologous macrophage therapy in improving liver function, non-invasive fibrosis markers and other clinical outcomes in patients with compensated cirrhosis., Ethics and Dissemination: The trial will be conducted according to the ethical principles of the Declaration of Helsinki 2013 and has been approved by Scotland A Research Ethics Committee (reference 15/SS/0121), National Health Service Lothian Research and Development department and the Medicine and Health Care Regulatory Agency-UK. Final results will be presented in peer-reviewed journals and at relevant conferences., Trial Registration Numbers: ISRCTN10368050 and EudraCT; reference 2015-000963-15., Competing Interests: Competing interests: PNB has received honoraria from Takeda. JAF has received consultancy fees for Ferring Pharmaceuticals, Macrophage Pharma, Aquilla BioMedical, Caldan Therapeutics, Cypralis, Third Rock Ventures, Rallybio, Narrow River Management, Gilde Healthcare, Guidepoint, Techspert.io and acted as advisory board member for: Novartis, Galecto Biotech, Tectonic Therapeutic and received research grant funding from Novartis and Intercept Pharmaceuticals. JFD has received honoraria and research grants from Gilead, AbbVie and MSD. JDMC and SJF are founders and scientific advisers to Resolution Therapeutics., (© Author(s) (or their employer(s)) 2021. Re-use permitted under CC BY. Published by BMJ.)

Adoptive immunotherapy using Epstein-Barr Virus (EBV)-specific T cells is a potentially curative treatment for patients with EBV-related malignancies where other clinical options have proved ineffective. We describe improved good manufacturing practice (GMP)-compliant culture and analysis processes for conventional lymphoblastoid cell line (LCL)-driven EBV-specific T cell manufacture, and describe an improved phenotyping approach for analysing T cell products. We optimized the current LCL-mediated clinical manufacture of EBV-specific T cells to establish an improved process using xenoprotein-free GMP-compliant reagents throughout, and compared resulting products with our previous banked T cell clinical therapy. We assessed effects of changes to LCL:T cell ratio in T cell expansion, and developed a robust flow cytometric marker panel covering T cell memory, activation, differentiation and intracellular cytokine release to characterize T cells more effectively. These data were analysed using a t-stochastic neighbour embedding (t-SNE) algorithm. The optimized GMP-compliant process resulted in reduced cell processing time and improved retention and expansion of central memory T cells. Multi-parameter flow cytometry determined the optimal protocol for LCL stimulation and expansion of T cells and demonstrated that cytokine profiling using interleukin (IL)-2, tumour necrosis factor (TNF)-α and interferon (IFN)-γ was able to determine the differentiation status of T cells throughout culture and in the final product. We show that fully GMP-compliant closed-process culture of LCL-mediated EBV-specific T cells is feasible, and profiling of T cells through cytokine expression gives improved characterization of start material, in-process culture conditions and final product. Visualization of the complex multi-parameter flow cytometric data can be simplified using t-SNE analysis., (© 2021 The Authors. Clinical & Experimental Immunology published by John Wiley & Sons Ltd on behalf of British Society for Immunology.)

COVID-19 disease caused by the SARS-CoV-2 virus is characterized by dysregulation of effector T cells and accumulation of exhausted T cells. T cell responses to viruses can be corrected by adoptive cellular therapy using donor-derived virus-specific T cells. One approach is the establishment of banks of HLA-typed virus-specific T cells for rapid deployment to patients. Here we show that SARS-CoV-2-exposed blood donations contain CD4 and CD8 memory T cells which recognize SARS-CoV-2 spike, nucleocapsid and membrane antigens. Peptides of these antigens can be used to isolate virus-specific T cells in a GMP-compliant process. The isolated T cells can be rapidly expanded using GMP-compliant reagents for use as an allogeneic therapy. Memory and effector phenotypes are present in the selected virus-specific T cells, but our method rapidly expands the desirable central memory phenotype. A manufacturing yield ranging from 10 10 to 10 11 T cells can be obtained within 21 days culture. Thus, multiple therapeutic doses of virus-specific T cells can be rapidly generated from convalescent donors for potential treatment of COVID-19 patients., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Cooper, Fraser, Smith, Burgoyne, Imlach, Jarvis, Turner, Zahra, Turner and Campbell.)

Background: Cryopreserved platelets are generally produced and stored in plasma. With the advent of additive solutions being routinely used it would be prudent to examine producing and storing these units in a platelet additive solution (SSP+)., Study Design and Methods: Platelet concentrates were prepared from twenty overnight held whole blood units with 10 being re-suspended in 100% plasma and 10 in approximately 70% SSP + and 30% plasma. All had 6% v/v DMSO added prior to storage at -80°C. On thawing plasma stored platelets were re-constituted in fresh plasma with additive prepared platelets being subsequently suspended in 100% SSP+. Sample analysis was assessed pre cryopreservation, post thaw and 4 h., Results: We noted a significant increase in our annexin V levels along with a decrease in GP1bα Von Willebrand binding sites post thaw. The platelets ability to change shape was also significantly reduced as observed with our HSR and ESC values. However, despite this there was still sufficient material within the platelet to allow them to be viable as observed with our thromboelasticity results which, were still observed to be within normal parameters post thaw We also observed an increase in Extracellular vesicles post thaw, suggesting platelet damage which was supported by the reduction in platelet counts. Although there were still sufficient numbers to meet the minimum requirements of the UK guidelines., (© 2020 International Society of Blood Transfusion.)

Objectives: To assess the utility of the Idexx ProCyte Dx® haematology analyser for assessing total nucleated cell count and differential cell counts in canine cerebrospinal fluid., Materials and Methods: Seventy-three client-owned dogs undergoing investigations for pyrexia and/or neurological signs were prospectively included. Cerebrospinal fluid samples were assessed using an Idexx ProCyte Dx® analyser and the results were compared to those obtained with the external laboratory reference standard., Results: The Idexx ProCyte Dx® performed with good sensitivity (92.6%) and moderate specificity (67.4%) for total nucleated cell count when compared to the reference standard. Qualitative assessment of the Idexx ProCyte Dx® scatter plots, and quantitative assessment of differential cell counts where available, appeared to correlate well with the external laboratory manual differential cell counts, with a good-to-high agreement in 25 of 26 samples (96.2%)., Clinical Significance: The Idexx ProCyte Dx® analyser performed well in determining the total nucleated cell count and differential cell counts in canine cerebrospinal fluid when compared to a reference standard of external laboratory analysis, except for cell counts higher than ~1000/μL. As the Idexx ProCyte Dx® currently only provides a cell count in 10 cells/μL increments, software modification may improve agreement between the two methods. As in human medicine, automated methods may prove useful in the future for cerebrospinal fluid analysis in addition to manual assessment, particularly in an emergency setting., (© 2019 British Small Animal Veterinary Association.)

Islet transplantation is an efficacious therapy for type 1 diabetes; however, islets from multiple donor pancreata are required, and a gradual attrition in transplant function is seen. Here, we manufactured human umbilical cord perivascular mesenchymal stromal cells (HUCPVCs) to Good Manufacturing Practice (GMP) standards. HUCPVCs showed a stable phenotype while undergoing rapid ex vivo expansion at passage 2 (p2) to passage 4 (p4) and produced proregenerative factors, strongly suppressing T cell responses in the resting state and in response to inflammation. Transplanting an islet equivalent (IEQ):HUCPVC ratio of 1:30 under the kidney capsule in diabetic NSG mice demonstrated the fastest return to normoglycemia by 3 days after transplant: Superior glycemic control was seen at both early (2.7 weeks) and later stages (7, 12, and 16 weeks) versus ratios of 1:0, 1:10, and 1:50, respectively. Syngeneic islet transplantation in immunocompetent mice using the clinically relevant hepatic portal route with a marginal islet mass showed that mice transplanted with an IEQ:HUCPVC ratio of 1:150 had superior glycemic control versus ratios of 1:0, 1:90, and 1:210 up to 6 weeks after transplant. Immunodeficient mice transplanted with human islets (IEQ:HUCPVC ratio of 1:150) exhibited better glycemic control for 7 weeks after transplant versus islet transplant alone, and islets transplanted via the hepatic portal vein in an allogeneic mouse model using a curative islet mass demonstrated delayed rejection of islets when cotransplanted with HUCPVCs (IEQ:HUCPVC ratio of 1:150). The immunosuppressive and proregenerative properties of HUCPVCs demonstrated long-term positive effects on graft function in vivo, indicating that they may improve long-term human islet allotransplantation outcomes., (Copyright © 2020 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works.)

Therapies to reduce liver fibrosis and stimulate organ regeneration are urgently needed. We conducted a first-in-human, phase 1 dose-escalation trial of autologous macrophage therapy in nine adults with cirrhosis and a Model for End-Stage Liver Disease (MELD) score of 10-16 (ISRCTN 10368050). Groups of three participants received a single peripheral infusion of 10 7 , 10 8 or up to 10 9 cells. Leukapheresis and macrophage infusion were well tolerated with no transfusion reactions, dose-limiting toxicities or macrophage activation syndrome. All participants were alive and transplant-free at one year, with only one clinical event recorded, the occurrence of minimal ascites. The primary outcomes of safety and feasibility were met. This study informs and provides a rationale for efficacy studies in cirrhosis and other fibrotic diseases.

Limbal stem cell deficiency (LSCD) is a disease resulting from the loss or dysfunction of epithelial stem cells, which seriously impairs sight. Autologous limbal stem cell transplantation is effective in unilateral or partial bilateral disease but not applicable in total bilateral disease. An allogeneic source of transplantable cells for use in total bilateral disease can be obtained from culture of donated cadaveric corneal tissue. We performed a controlled multicenter study to examine the feasibility, safety, and efficacy of allogeneic corneal epithelial stem cells in the treatment of bilateral LSCD. Patients were randomized to receive corneal epithelial stem cells cultured on amniotic membrane (AM): investigational medicinal product (IMP) or control AM only. Patients received systemic immunosuppression. Primary endpoints were safety and visual acuity, secondary endpoint was change in composite ocular surface score (OSS). Sixteen patients were treated and 13 patients completed all assessments. Safety was demonstrated and 9/13 patients had improved visual acuity scores at the end of the trial, with no significant differences between IMP and control groups. Patients in the IMP arm demonstrated significant, sustained improvement in OSS, whereas those in the control arm did not. Serum cytokine levels were measured during and after the period of immune suppression and we identified strongly elevated levels of CXCL8 in the serum of patients with aniridia, which persisted throughout the trial. This first randomized control trial of allogeneic corneal epithelial stem cells in severe bilateral LSCD demonstrates the feasibility and safety of this approach. Stem Cells Translational Medicine 2019;8:323-331., (© 2019 The Authors Stem Cells Translational Medicine published by Wiley Periodicals, Inc. on behalf of AlphaMed Press.)

Mutation of the isocitrate dehydrogenase 1 (IDH1) gene at codon 132 has been identified in approximately 70% of low-grade (II and III) human gliomas and secondary glioblastomas, with the IDH1 R132H point mutation representing 92.7% of these mutations. In people, the presence of an IDH1 gene mutation is associated with a better prognosis (both progression-free survival time and overall survival time) and a better response to therapy, including chemotherapy and radiation therapy. Furthermore, IDH1 mutations are included in diagnostic panels to improve diagnosis and molecular classification. Canine gliomas resemble their human counterpart both morphologically and immunohistochemically, therefore they are likely to share similar genetic abnormalities. The IDH1 gene is also comparable between man and dogs. If the IDH1 R132H point mutation is demonstrated in canine gliomas, the prognostic significance of this mutation in people may be transferable to the dog. The objective of this study was to investigate canine gliomas for the IDH1 R132H point mutation using immunohistochemistry. Thirty-one formalin-fixed and paraffin wax-embedded canine gliomas were examined for both IDH1 R132H expression and pan-IDH1 (IDH1 wild-type and point mutated IDH1). Glial tumour specimens were recorded to be either positive or negative for expression. Pan-IDH1 expression was identified in all 31 tumours (100%), while the IDH1 R132H point mutation was identified in none of the tumours (0%). Therefore, the IDH1 R132H point mutation was not identified in this population of canine gliomas and may not be a suitable biomarker or treatment target in canine gliomas. Further investigation is required to determine if other point mutations occur in the IDH1 gene of canine gliomas., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

Chemokines have been shown to be essential players in a range of cancer contexts. In this study, we demonstrate that mice deficient in the atypical chemokine receptor Ackr2 display impaired development of metastasis in vivo in both cell line and spontaneous models. Further analysis reveals that this relates to increased expression of the chemokine receptor CCR2, specifically by KLRG1 + NK cells from the Ackr2 -/- mice. This leads to increased recruitment of KLRG1 + NK cells to CCL2-expressing tumors and enhanced tumor killing. Together, these data indicate that Ackr2 limits the expression of CCR2 on NK cells and restricts their tumoricidal activity. Our data have important implications for our understanding of the roles for chemokines in the metastatic process and highlight Ackr2 and CCR2 as potentially manipulable therapeutic targets in metastasis., (Copyright © 2018 The Authors.)

Cellular therapeutics are a fast-growing, highly innovative area of medicine. This field encompasses well-established immune therapies for infection and cancer, as well as newer cell therapies aimed at regenerating diseased tissue. Flow cytometry is arguably the most important tool in the development of advanced cellular therapeutics and plays a role in many aspects of manufacturing. Quality control of raw materials, assessing rates of cellular growth during complex in vitro culture processes, differentiation status, as well as final product characterization, viability, and product stability are all essential data to be collected and documented. This requires robust and validated flow cytometric analysis. In this review, we discuss flow cytometer set up; design and use of assays to maintain quality control in cell processing, and the building of a robust set of release criteria. The use of flow cytometric assays as surrogates of functional assays to allow comparison and release of cellular products for clinical use are reviewed with reference to monocyte-derived and T cell products., (© 2018 International Clinical Cytometry Society.)

Immune checkpoint inhibitors (ICIs) targeting cytotoxic T lymphocyte-associated protein-4 (CTLA-4) or programmed cell death protein 1 (PD-1) receptors have demonstrated remarkable efficacy in subsets of patients with malignant disease. This emerging treatment modality holds great promise for future cancer treatment and has engaged pharmaceutical research interests in tumour immunology. While ICIs can induce rapid and durable responses in some patients, identifying predictive factors for effective clinical responses has proved challenging. This review summarizes the mechanisms of action of ICIs and outlines important preclinical work that contributed to their development. We explore clinical data that has led to disease-specific drug licensing, and highlight key clinical trials that have revealed ICI efficacy across a range of malignancies. We describe how ICIs have been used as part of combination therapies, and explore their future prospects in this area. We conclude by discussing the incorporation of these new immunotherapeutics into precision approaches to cancer therapy., (© 2017 British Society for Immunology.)

Background Aims: Autologous macrophage therapy represents a potentially significant therapeutic advance for the treatment of severe progressive liver cirrhosis. Administration of macrophages has been shown to reduce inflammation and drive fibrotic scar breakdown and tissue repair in relevant models. This therapeutic approach is being assessed for safety and feasibility in a first-in-human trial (MAcrophages Therapy for liver CirrHosis [MATCH] trial)., Methods: We outline the development and validation phases of GMP production. This includes use of the CliniMACS Prodigy cell sorting system to isolate CD14 + cells; optimizing macrophage culture conditions, assessing cellular identity, product purity, functional capability and determining the stability of the final cell product., Results: The GMP-compliant macrophage products have a high level of purity and viability, and have a consistent phenotypic profile, expressing high levels of mature macrophage markers 25F9 and CD206 and low levels of CCR2. The macrophages demonstrate effective phagocytic capacity, are constitutively oriented to an anti-inflammatory profile and remain responsive to cytokine and TLR stimulation. The process validation shows that the cell product in excipient is remarkably robust, consistently passing the viability and phenotypic release criteria up to 48 hours after harvest., Conclusions: This is the first report of validation of a large-scale, fully Good Manufacturing Practice-compliant, autologous macrophage cell therapy product for the potential treatment of cirrhosis. Phenotypic and functional assays confirm that these cells remain functionally viable for up to 48 h, allowing significant flexibility in administration to patients., (Copyright © 2017 International Society for Cellular Therapy. All rights reserved.)

Background: Feline infectious peritonitis (FIP) is the most common infectious central nervous system (CNS) disease in the cat and is invariably fatal. Improved means of antemortem diagnosis is required to facilitate clinical decision making. Information regarding the magnetic resonance imaging (MRI) findings of neurologic FIP currently is limited, resulting in the need for better descriptions to optimize its use as a diagnostic tool., Objective: To describe the clinicopathologic features and MRI findings in cases of confirmed neurologic FIP., Animals: Twenty-four client-owned cats with histopathologic confirmation of neurologic FIP., Methods: Archived records from 5 institutions were retrospectively reviewed to identify cases with confirmed neurologic FIP that had undergone antemortem MRI of the CNS. Signalment, clinicopathologic, MRI, and histopathologic findings were evaluated., Results: Three distinct clinical syndromes were identified: T3-L3 myelopathy (3), central vestibular syndrome (7), and multifocal CNS disease (14). Magnetic resonance imaging abnormalities were detected in all cases, including meningeal contrast enhancement (22), ependymal contrast enhancement (20), ventriculomegaly (20), syringomyelia (17), and foramen magnum herniation (14). Cerebrospinal fluid was analysed in 11 cases; all demonstrated a marked increase in total protein concentration and total nucleated cell count. All 24 cats were euthanized with a median survival time of 14 days (range, 2-115) from onset of clinical signs. Histopathologic analysis identified perivascular pyogranulomatous infiltrates, lymphoplasmacytic infiltrates, or both affecting the leptomeninges (16), choroid plexuses (16), and periventricular parenchyma (13)., Conclusions and Clinical Importance: Magnetic resonance imaging is a sensitive means of detecting neurologic FIP, particularly in combination with a compatible signalment, clinical presentation, and CSF analysis., (Copyright © 2017 The Authors. Journal of Veterinary Internal Medicine published by Wiley Periodicals, Inc. on behalf of the American College of Veterinary Internal Medicine.)

Objective: To test the hypothesis that miR-155 regulates monocyte migratory potential via modulation of chemokine and chemokine receptor expression in RA, and thereby is associated with disease activity., Methods: The miR-155 copy-numbers in monocytes from peripheral blood (PB) of healthy (n = 22), RA (n = 24) and RA SF (n = 11) were assessed by real time-PCR using synthetic miR-155 as a quantitative standard. To evaluate the functional impact of miR-155, human monocytes were transfected with control or miR-155 mimic, and the effect on transcript levels, and production of chemokines was evaluated by Taqman low-density arrays and multiplex assays. A comparative study evaluated constitutive chemokine receptor expression in miR-155 -/- and wild-type murine (CD115  +  Ly6C  +  Ly6G - ) monocytes., Results: Compared with healthy monocytes, the miR-155 copy-number was higher in RA, peripheral blood (PB) and SF monocytes (PB P < 0.01, and SF P < 0.0001). The miR-155 copy-number in RA PB monocytes was higher in ACPA-positive compared with ACPA-negative patients (P = 0.033) and correlated (95% CI) with DAS28 (ESR), R = 0.728 (0.460, 0.874), and with tender, R = 0.631 (0.306, 0.824) and swollen, R = 0.503 (0.125, 0.753) joint counts. Enforced-expression of miR-155 in RA monocytes stimulated the production of CCL3, CCL4, CCL5 and CCL8; upregulated CCR7 expression; and downregulated CCR2. Conversely, miR155 -/- monocytes showed downregulated CCR7 and upregulated CCR2 expression., Conclusion: Given the observed correlations with disease activity, these data provide strong evidence that miR-155 can contribute to RA pathogenesis by regulating chemokine production and pro-inflammatory chemokine receptor expression, thereby promoting inflammatory cell recruitment and retention in the RA synovium., (© The Author 2016. Published by Oxford University Press on behalf of the British Society for Rheumatology.)

Novel therapies, including molecular targeted therapies, are being developed for the treatment of human gliomas. To use such therapies for canine gliomas, more complete characterization of molecular targets is required. Epidermal growth factor receptor (EGFR) is one such therapeutic target used in human glioma trials, and the Ki-67 labeling index (LI) is a marker of proliferation and a prognostic indicator. The objectives of this cross-sectional study were to evaluate the expression of EGFR and Ki-67 in canine gliomas and to determine if immunopositivity is associated with tumor type and histologic grade. Thirty-one formalin-fixed, paraffin-embedded canine gliomas were evaluated for EGFR and Ki-67 expression by immunohistochemistry. EGFR immunopositivity was evaluated using a semi-quantitative score and the Ki-67 LI calculated based on the percentage of positive cells. EGFR and Ki-67 expression were identified in 16 of 31 (52%) and 28 of 31 (90%) tumors, respectively. EGFR expression was significantly greater in high-grade tumors compared with low-grade tumors (P = .04) and was significantly greater in gliomatosis cerebri compared with oligodendroglioma (P = .002), astrocytoma (P = .01), and oligoastrocytoma (P = .04). The Ki-67 LI was significantly greater in high-grade tumors compared with low grade tumors (P = .02); the median Ki-67 LI was 2.3% (range, 0%-17.6%) for low-grade tumors and 9.3% (range, 1.7%-41.0%) for high-grade tumors. A significant moderate correlation was identified between EGFR immunopositivity and Ki-67 LI (r = 0.47, P = .007). Overall, EGFR may be a suitable therapeutic target for some canine gliomas, particularly gliomatosis cerebri., (© The Author(s) 2016.)

Mesenchymal stromal cells (MSCs), multipotent cells present in tissues throughout the body, can reconstitute adipogenic, osteogenic and chondrogenic tissues, but are also of great interest as mediators of immune modulation and suppression. MSCs are able to improve transplant engraftment, treat graft versus host disease and suppress T cell responses and therefore have great potential as therapeutic agents. Their immune modulatory capacity is mediated through both cell-to-cell contact and cytokine secretion, but it is becoming clear that extracellular vesicles (EV) produced by MSC also possess immunomodulatory properties. These vesicles are easy to prepare and store, do not carry nuclear material and cannot form tumours, and therefore also represent a highly desirable therapeutic agent. This review outlines the formation and characterisation of extracellular vesicles, the reported function of MSC-EVs in vitro and in vivo, and addresses some of the emerging issues with nomenclature, EV therapeutic dose and tissue source. The development of GMP-grade production protocols and effective characterisation of MSC extracellular vesicles is essential to their successful use as immune modulating therapeutic agents, and this review outlines the current status of the research in this area., (Crown Copyright © 2016. Published by Elsevier Ltd. All rights reserved.)

Background Aims: Macrophages have complex roles in the liver. The aim of this study was to compare profiles of human monocyte-derived macrophages between controls and cirrhotic patients, to determine whether chronic inflammation affects precursor number or the phenotype, with the eventual aim to develop a cell therapy for cirrhosis., Methods: Infusion of human macrophages in a murine liver fibrosis model demonstrated a decrease in markers of liver injury (alanine transaminase, bilirubin, aspartate transaminase) and fibrosis (transforming growth factor-β, α-smooth muscle actin, phosphatidylserine receptor) and an increase in markers of liver regeneration (matrix metalloproteinases [MMP]-9, MMP-12 and TNF-related weak inducer of apoptosis). CD14+ monocytes were then isolated from controls. Monocytes were matured into macrophages for 7 days using a Good Manufacturing Practice-compatible technique., Results: There was no significant difference between the mean number of CD14+ monocytes isolated from cirrhotic patients (n = 9) and controls (n = 10); 2.8 ± SEM 0.54 × 10(8) and 2.5 ± 0.56 × 10(8), respectively. The mean yield of mature macrophages cultured was also not significantly different between cirrhotic patients and controls (0.9 × 10(8) ± 0.38 × 10(8), with more than 90% viability and 0.65 × 10(8) ± 0.16 × 10(8), respectively. Maturation to macrophages resulted in up-regulation of a number of genes (MMP-9, CCL2, interleukin [IL]-10 and TNF-related weak inducer of apoptosis). A cytokine and chemokine polymerase chain reaction array, comparing the control and cirrhotic macrophages, revealed no statistically significant differences., Conclusions: Macrophages can be differentiated from cirrhotic patients' apheresis-derived CD14 monocytes and develop the same pro-resolution phenotype as control macrophages, indicating their suitability for clinical therapy., (Copyright © 2015 International Society for Cellular Therapy. Published by Elsevier Inc. All rights reserved.)

Cell therapy regimens are frequently compromised by low-efficiency cell homing to therapeutic niches. Improvements in this regard would enhance effectiveness of clinically applicable cell therapy. The major regulators of tissue-specific cellular migration are chemokines, and therefore selection of therapeutic cellular populations for appropriate chemokine receptor expression would enhance tissue-homing competence. A number of practical considerations preclude the use of Abs in this context, and alternative approaches are required. In this study, we demonstrate that appropriately labeled chemokines are at least as effective in detecting their cognate receptors as commercially available Abs. We also demonstrate the utility of biotinylated chemokines as cell-sorting reagents. Specifically, we demonstrate, in the context of CCR7 (essential for lymph node homing of leukocytes), the ability of biotinylated CCL19 with magnetic bead sorting to enrich for CCR7-expressing cells. The sorted cells demonstrate improved CCR7 responsiveness and lymph node-homing capability, and the sorting is effective for both T cells and dendritic cells. Importantly, the ability of chemokines to detect CCR7, and sort for CCR7 positivity, crosses species being effective on murine and human cells. This novel approach to cell sorting is therefore inexpensive, versatile, and applicable to numerous cell therapy contexts. We propose that this represents a significant technological advance with important therapeutic implications., (Copyright © 2014 by The American Association of Immunologists, Inc.)

Aims: Percutaneous revascularisation of chronic total occlusions (CTO) is limited by failure of guidewire crossing. Neovascularisation within the proximal CTO segment may be important for guidewire crossing and dramatically declines in CTO beyond six weeks of age. The aims of the current study were to determine whether local delivery of a pro-angiogenic growth factor increases neovascularisation in mature CTO and facilitates guidewire crossings., Methods and Results: CTO (n=51) were created in the femoral arteries of 44 New Zealand white rabbits using the thrombin injection model. At 12 weeks, CTO were treated with poly-lactic-glycolic-acid (PLGA) microspheres containing either bovine serum albumin (BSA) (n=15) or recombinant mouse VEGF164 (n=14), or received no intervention (controls, n=12). Contrast-enhanced magnetic resonance angiography (CEMRA) was performed prior to treatment and at three weeks post treatment. Animals were sacrificed at three weeks post treatment and arterial samples were excised for micro-computed tomography imaging (µCT) and histologic morphometric analysis. Guidewire crossing was assessed at three weeks post treatment in an additional 10 VEGF164-treated CTO. In comparison to BSA-treated and control non-intervened CTO, VEGF164-treated CTO showed a significant increase in relative blood volume index in the proximal segment of the CTO lesion as determined by CEMRA and by µCT. Histologic measurements of microvessel area were also higher in VEGF164-treated CTO. Guidewire crossing across the proximal fibrous cap was successful in eight out of 10 VEGF164-treated CTO., Conclusions: Angiogenic therapy appears to be a promising strategy to improve neovascularisation and guidewire crossing rates in CTO.

Regulatory T cells (T(regs) ) are crucial in mediating immune homeostasis and promoting the establishment and maintenance of peripheral tolerance. However, in the context of cancer their role is more complex, and they are thought to contribute to the progress of many tumours. As cancer cells express both self- and tumour-associated antigens, T(regs) are key to dampening effector cell responses, and therefore represent one of the main obstacles to effective anti-tumour responses. Suppression mechanisms employed by T(regs) are thought to contribute significantly to the failure of current therapies that rely on induction or potentiation of anti-tumour responses. This review will focus on the current evidence supporting the central role of T(regs) in establishing tumour-specific tolerance and promoting cancer escape. We outline the mechanisms underlying their suppressive function and discuss the potential routes of T(regs) accumulation within the tumour, including enhanced recruitment, in-situ or local proliferation, and de-novo differentiation. In addition, we review some of the cancer treatment strategies that act, at least in part, to eliminate or interfere with the function of T(regs) . The role of T(regs) is being recognized increasingly in cancer, and controlling the function of these suppressive cells in the tumour microenvironment without compromising peripheral tolerance represents a significant challenge for cancer therapies., (© 2012 British Society for Immunology.)

This study used a rodent air-pouch model to assess the acute inflammatory response to cobalt-chromium (CoCr) alloy wear debris from a metal-on-metal hip resurfacing implant that may contribute to joint failure. Air-pouches were injected with either sterile phosphate-buffered saline, 1 μg lipopolysaccharide (LPS) or 2.5 mg CoCr wear debris. The in situ inflammatory response was monitored 4, 24, 48 and 72 h and 7 days later. A flow cytometric analysis of the inflammatory exudates showed that CoCr wear debris induced a different inflammatory pattern compared with LPS. LPS induced a strong early (4 h) neutrophil influx, with monocyte/macrophage influx peaking at 24 h, whereas CoCr wear debris initiated almost equal numbers of early monocyte/macrophage and neutrophil recruitment. Histological analyses also showed CoCr debris accumulated in the pouch wall and this was accompanied by vast cellular infiltration and fibrosis around the debris throughout the duration of the experiment. Assessment of inflammatory gene transcripts from air-pouch tissue showed that CoCr wear debris increased the expression of cytokines involved in promoting inflammation and fibrosis (IL-1β, TGF-β) and chemokines that promote the recruitment of neutrophils and monocytes/macrophages (CXCL2 and CCL2). The data suggest that inflammatory responses to CoCr debris induce a specific acute process in which the recruitment of monocytes/macrophages is key.

Cancer is one of the most important pathological conditions facing mankind in the 21st century, and is likely to become the most important cause of death as improvements continue in health, diet and life expectancy. The immune response is responsible for controlling nascent cancer through immunosurveillance. If tumours escape this control, they can develop into clinical cancer. Although surgery and chemo- or radiotherapy have improved survival rates significantly, there is a drive to reharness immune responses to treat disease. As T cells are one of the key immune cells in controlling cancer, research is under way to enhance their function and improve tumour targeting. This can be achieved by transduction with tumour-specific T cell receptor (TCR) or chimaeric antigen receptors (CAR) to generate redirected T cells. Virus-specific cells can also be transduced with TCR or CAR to create bi-functional T cells with specificity for both virus and tumour. In this review we outline the development and optimization of redirected and bi-functional T cells, and outline the results from current clinical trials using these cells. From this we discuss the challenges involved in generating effective anti-tumour responses while avoiding concomitant damage to normal tissues and organs., (© 2012 The Authors. Clinical and Experimental Immunology © 2012 British Society for Immunology.)

MicroRNA (miRNA) species (miR) regulate mRNA translation and are implicated as mediators of disease pathology via coordinated regulation of molecular effector pathways. Unraveling miR disease-related activities will facilitate future therapeutic interventions. miR-155 recently has been identified with critical immune regulatory functions. Although detected in articular tissues, the functional role of miR-155 in inflammatory arthritis has not been defined. We report here that miR-155 is up-regulated in synovial membrane and synovial fluid (SF) macrophages from patients with rheumatoid arthritis (RA). The increased expression of miR-155 in SF CD14(+) cells was associated with lower expression of the miR-155 target, Src homology 2-containing inositol phosphatase-1 (SHIP-1), an inhibitor of inflammation. Similarly, SHIP-1 expression was decreased in CD68(+) cells in the synovial lining layer in RA patients as compared with osteoarthritis patients. Overexpression of miR-155 in PB CD14(+) cells led to down-regulation of SHIP-1 and an increase in the production of proinflammatory cytokines. Conversely, inhibition of miR-155 in RA synovial CD14(+) cells reduced TNF-α production. Finally, miR-155-deficient mice are resistant to collagen-induced arthritis, with profound suppression of antigen-specific Th17 cell and autoantibody responses and markedly reduced articular inflammation. Our data therefore identify a role of miR-155 in clinical and experimental arthritis and suggest that miR-155 may be an intriguing therapeutic target.

Objectives: Systemic sclerosis (SSc) is characterised by vasculopathy, an aberrantly activated immune system and excessive extracellular matrix deposition. Inflammatory chemokines control migration of cells to sites of tissue damage; their removal from inflamed sites is essential for resolution of the inflammatory response. The atypical chemokine receptor D6 has a critical role in this physiological balance. To explore potential deregulation of this system in SSc, inflammatory chemokine and D6 expression were compared with that in healthy controls (HC)., Methods: Serum levels of inflammatory mediators were assessed by luminex analysis. Peripheral blood mononuclear cells (PBMCs) were used in molecular and immunocytochemical analysis. Platelet-rich plasma was collected and assessed by western blotting for D6 expression levels. Sex-matched HC were used for comparison., Results: 72 patients with SSc and 30 HC were enrolled in the study. The chemokines MCP-1/CCL2, MIP-1α/CCL3, MIP-1β/CCL4 and IL-8/CXCL8 were significantly increased in patients with SSc, regardless of disease subtype and phase. Quantitative PCR analysis revealed a significant 10-fold upregulation of D6 transcripts in patients with SSc compared with controls, and this was paralleled by increased D6 protein expression in the PBMCs of patients with SSc. Platelet lysates also showed strong D6 expression in patients with SSc but not in controls. Importantly, high levels of D6 expression correlated with reduced levels of its ligands in serum., Conclusions: Inflammatory chemokines and the regulatory receptor D6 are significantly upregulated in SSc and high D6 levels are associated with lower systemic chemokine levels, indicating that some patients control systemic chemokine levels using D6. These results suggest that chemokines may represent a therapeutic target in SSc.

We evaluated the role of p15(Ink4), a member of the INK4 family of CDK inhibitors on vascular smooth muscle cells (VSMCs) proliferation, cell cycle progression and intimal hyperplasia after stenting. Aortic VSMCs transduced with either adenovirus encoding for p15(Ink4) or β-galactosidase were assessed for DNA synthesis, cell cycle progression, and pRb phosphorylation. Rabbit carotid arteries were stented and treated with peri-adventitial delivery of saline or adenovirus encoding for p15(Ink4) or β-galactosidase. p15(Ink4) transgene and protein expression were evaluated at 24 h and 72 h, respectively. In-stent cell proliferation was evaluated by BrdU at day 7. Histomorphometric analysis of in-stent intimal hyperplasia was performed at 10 weeks. Human p15(Ink4) DNA was detected in transduced VSMCs at 24h. p15(Ink4) over-expression reduced VSMCs DNA synthesis by 60%. Cell cycle progression was inhibited, with a 30% increase in G1 population accompanied by inhibition of pRb phosphorylation. Human p15(Ink4) transgene was identified in transduced stented arteries but not in control arteries. p15(Ink4) immunostaining was increased and cell proliferation significantly reduced by 50% in p15(Ink4) transduced arteries. Intimal cross-sectional area (CSA) of p15(Ink4)-treated group was significantly lower than the β-gal treated and non-transduced groups (p=0.008). There were no differences in the intimal or medial inflammatory response between groups. p15(Ink4) over-expression blocks cell cycle progression leading to inhibition of VSMCs proliferation. Peri-adventitial delivery of p15(Ink4) significantly inhibits in-stent intimal hyperplasia., (Copyright © 2010 Elsevier Ltd. All rights reserved.)

Toll-like receptors orchestrate rapid local protective innate-immune responses to invading pathogens and optimize leukocyte priming of subsequent adaptive responses. Paradoxically, systemic excess of the TLR2 ligand, bacterial lipoprotein (BLP), suppresses peripheral inflammatory responses. Here, we demonstrate that this phenomenon is regulated via the TLR2-dependent, cell-autonomous down-regulation of inflammatory chemokine receptor expression on a variety of leukocyte subsets. Remarkably, BLP mediated no effect on constitutive chemokine receptor expression. By tracking adoptively transferred wild-type and TLR2(-/-) leukocytes in vivo, we observed that BLP mediated chemokine receptor switching directed leukocytes away from inflamed sites toward secondary lymphoid organs. These data highlight a novel role for TLR ligands, such as BLP, in regulating leukocyte retention and migration away from innate immune lesions via discrete constitutive and inflammatory chemokine receptor regulation.

D6 scavenges inflammatory chemokines and is essential for the regulation of inflammatory and immune responses. Mechanisms explaining the cellular basis for D6 function have been based on D6 expression by lymphatic endothelial cells. In this study, we demonstrate that functional D6 is also expressed by murine and human hemopoietic cells and that this expression can be regulated by pro- and anti-inflammatory agents. D6 expression was highest in B cells and dendritic cells (DCs). In myeloid cells, LPS down-regulated expression, while TGF-beta up-regulated expression. Activation of T cells with anti-CD3 and soluble CD28 up-regulated mRNA expression 20-fold, while maturation of human macrophage and megakaryocyte precursors also up-regulated D6 expression. Competition assays demonstrated that chemokine uptake was D6 dependent in human leukocytes, whereas mouse D6-null cells failed to uptake and clear inflammatory chemokines. Furthermore, we present evidence indicating that D6 expression is GATA1 dependent, thus explaining D6 expression in myeloid progenitor cells, mast cells, megakaryocytes, and DCs. We propose a model for D6 function in which leukocytes, within inflamed sites, activate D6 expression and thus trigger resolution of inflammatory responses. Our data on D6 expression by circulating DCs and B cells also suggest alternative roles for D6, perhaps in the coordination of innate and adaptive immune responses. These data therefore alter our models of in vivo D6 function and suggest possible discrete, and novel, roles for D6 on lymphatic endothelial cells and leukocytes.

IL-33, a cytokine of the IL-1 family, is closely associated with type II T cell responses. Here, we report an unexpected proinflammatory role of IL-33 in inflammatory arthritis. IL-33 was expressed in synovial fibroblasts from patients with rheumatoid arthritis (RA). Expression was markedly elevated in vitro by inflammatory cytokines. Mice lacking ST2, the IL-33 receptor alpha-chain, developed attenuated collagen-induced arthritis (CIA) and reduced ex vivo collagen-specific induction of proinflammatory cytokines (IL-17, TNFalpha, and IFNgamma), and antibody production. Conversely, treatment of wild-type (WT) but not ST2(-/-) mice with IL-33 exacerbated CIA and elevated production of both proinflammatory cytokines and anti-collagen antibodies. Mast cells expressed high levels of ST2 and responded directly to IL-33 to produce a spectrum of inflammatory cytokines and chemokines in vitro. In vivo, IL-33 treatment exacerbated CIA in ST2(-/-) mice engrafted with mast cells from WT but not from ST2(-/-) mice. Disease exacerbation was accompanied by elevated expression levels of proinflammatory cytokines. Our results demonstrate that IL-33 is a critical proinflammatory cytokine for inflammatory joint disease that integrates fibroblast activation with downstream immune activation mainly via an IL-33-driven, mast-cell-dependent pathway. Thus, this IL-1 superfamily member represents a therapeutic target for RA.

Production of the anti-inflammatory cytokine IL-10 by monocytes has been implicated as a probable negative regulator of graft-versus-host disease (GvHD) in patients undergoing allogeneic stem cell transplants (SCT). Monocytes from G-CSF-mobilized peripheral blood stem cell (gmPBSC) collections have been reported to produce more IL-10 than unmobilized monocytes in response to proinflammatory factors such as LPS. Why this should occur is unclear. In this study, monocyte phenotype and IL-10 localization and release were investigated in PB mononuclear cells (MNC) from 27 healthy donors mobilized for allogeneic SCT and from 13 patients with hematological malignancies mobilized for autologous SCT. All isolates contained elevated total percentages of monocytes in comparison with unmobilized PB, a high proportion of which displayed an immature phenotype. Stimulation of gmPB MNC with an inflammatory stimulus [fixed Staphylococcus aureus cells (SAC)] induced rapid up-regulation of CD14, indicating conversion to mature status. Localization studies indicated that IL-10 was predominantly present, bound on the surface of CD64(+)/CD14(low/neg) immature monocytes. Inflammatory stimuli (LPS, polyinosinic:polycytidylic acid, or SAC) induced release of variable quantities of IL-10 from the cell surface. MNC, separated into surface IL-10-positive or -negative fractions, differed in their ability to stimulate alloreactivity in MLR, and IL-10(+) MNC induced significantly lower levels of proliferation than IL-10(-) MNC. Thus, the subset of immature monocytes carrying surface-bound IL-10 in gmPB has the potential to modulate alloreactivity and GvHD after allogeneic SCT through cell-to-cell contact and released IL-10.

Dendritic cells (DCs) comprise heterogeneous subsets of professional antigen-presenting cells, linking innate and adaptive immunity. Analysis of DC subsets has been hampered by a lack of specific DC markers and reliable quantitation assays. We characterised the immunophenotype and functional characteristics of psoriatic arthritis (PsA)-derived and rheumatoid arthritis (RA)-derived myeloid DCs (mDCs) and plasmacytoid DCs (pDCs) to evaluate their potential role in arthritis. Circulating peripheral blood (PB) pDC numbers were significantly reduced in PsA patients (P = 0.0098) and RA patients (P = 0.0194), and mDCs were significantly reduced in RA patients (P = 0.0086) compared with healthy controls. The number of circulating mDCs in RA PB was significantly inversely correlated to C-reactive protein (P = 0.021). The phenotype of both DC subsets in PsA PB and RA PB was immature as compared with healthy controls. Moreover, CD62L expression was significantly decreased on both mDCs (PsA, P = 0.0122; RA, P = 0.0371) and pDCs (PsA, P = 0.0373; RA, P = 0.0367) in PB. Both mDCs and pDCs were present in PsA synovial fluid (SF) and RA SF, with the mDC:pDC ratio significantly exceeding that in matched PB (PsA SF, P = 0.0453; RA SF, P = 0.0082). pDCs isolated from RA SF and PsA SF displayed an immature phenotype comparable with PB pDCs. RA and PsA SF mDCs, however, displayed a more mature phenotype (increased expression of CD80, CD83 and CD86) compared with PB mDCs. Functional analysis revealed that both SF DC subsets matured following toll-like receptor stimulation. pDCs from PB and SF produced interferon alpha and tumour necrosis factor alpha on TLR9 stimulation, but only SF pDCs produced IL-10. Similarly, mDCs from PB and SF produced similar tumour necrosis factor alpha levels to TLR2 agonism, whereas SF mDCs produced more IL-10 than PB controls. Circulating DC subset numbers are reduced in RA PB and PsA PB with reduced CD62L expression. Maturation is incomplete in the inflamed synovial compartment. Immature DCs in SF may contribute to the perpetuation of inflammation via sampling of the inflamed synovial environment, and in situ presentation of arthritogenic antigen.

Infrared spectroscopy was used to corroborate predictions made by newly developed heterogeneous site binding models for humic substances. Experimental conditions to acquire the spectra of soil humic substances (humic and fulvic acid and a polysaccharide fraction) in an aqueous state using horizontal attenuated total reflectance Fourier transform infrared spectroscopy (HATR-FTIR) were established. Elimination of the water spectrum from that of the sample was achieved by spectral subtraction of the water peak at 2020 cm(-1). A KSCN internal standard with an absorption band at 2067 cm(-1) was used to verify the efficacy of the subtraction procedure. Spectral artifacts produced by the water spectrum subtraction and from contaminants within the humic materials have been identified. Three fulvic and one humic acid solution were examined in solutions of varying pH. Results show that the observed proportion of ionized carboxylate in relation to pH is consistent with models that assume electrostatic effects and a continuous distribution of proton association constants (log KH). The spectroscopic data were in accordance with calculations made using the generic humic and fulvic acid NICA-Donnan model parameters.