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The potential use of bone progenitors, multipotential stromal cells (MSCs) helping spine fusion is increasing, but convenient MSC sources and effective processing methods are critical factors yet to be optimised. The aim of this study was to test the effect of bone marrow processing on the MSC abundance and to compare the differentiation capabilities of vertebral body-bone marrow (VB-BM) MSCs versus iliac crest-bone marrow (IC-BM) MSCs. We assessed the effect of the red blood cell lysis (ammonium chloride, AC) and density-gradient centrifugation (Lymphoprep™, LMP), on the extracted VB-BM and IC-BM MSC numbers. The MSC abundance (indicated by colony counts and CD45lowCD271high cell numbers), phenotype, proliferation and tri-lineage differentiation of VB-BM MSCs were compared with donor-matched IC-BM MSCs. Importantly, the MSC attachment and osteogenesis were examined when VB-BM and IC-BM samples were loaded on a beta-tricalcium phosphate scaffold. In contrast to LMP, using AC yielded more colonies from IC-BM and VB-BM aspirates (p = 0.0019 & p = 0.0201 respectively). For IC-BM and VB-BM, the colony counts and CD45lowCD271high cell numbers were comparable (p = 0.5186, p = 0.2640 respectively). Furthermore, cultured VB-BM MSCs exhibited the same phenotype, proliferative and adipogenic potential, but a higher osteogenic and chondrogenic capabilities than IC-BM MSCs (p = 0.0010 and p = 0.0005 for calcium and glycosaminoglycan (GAG) levels, respectively). The gene expression data confirmed higher chondrogenesis for VB-BM MSCs than IC-BM MSCs, but osteogenic gene expression levels were comparable. When loaded on Vitoss™, both MSCs showed a similar degree of attachment and survival, but a better osteogenic ability was detected for VB-BM MSCs as measured by alkaline phosphatase activity (p = 0.0386). Collectively, the BM processing using AC had more MSC yield than using LMP. VB-BM MSCs have a comparable phenotype and proliferative capacity, but higher chondrogenesis and osteogenesis with or without using scaffold than donor-matched IC-BM MSCs. Given better accessibility, VB-BM could be an ideal MSC source for spinal bone fusion.

Bacterial and viral infectious triggers are linked to spondyloarthritis (SpA) including psoriatic arthritis (PsA) development, likely dendritic cell activation. We investigated spinal entheseal plasmacytoid dendritic cells (pDCs) toll-like receptor (TLR)-7 and 9 activation and therapeutic modulation, including JAK inhibition. We also investigated if COVID-19 infection, a potent TLR-7 stimulator triggered PsA flares. Normal entheseal pDCs were characterized and stimulated with imiquimod and CpG oligodeoxynucleotides (ODN) to evaluate TNF and IFNα production. NanoString gene expression assay of total pDCs RNA was performed pre- and post- ODN stimulation. Pharmacological inhibition of induced IFNα protein was performed with Tofacitinib and PDE4 inhibition. The impact of SARS-CoV2 viral infection on PsA flares was evaluated. CD45+HLA-DR+CD123+CD303+CD11c- entheseal pDCs were more numerous than blood pDCs (1.9 ± 0.8% vs 0.2 ± 0.07% of CD45+ cells, p=0.008) and showed inducible IFNα and TNF protein following ODN/imiquimod stimulation and were the sole entheseal IFNα producers. NanoString data identified 11 significantly upregulated differentially expressed genes (DEGs) including TNF in stimulated pDCs. Canonical pathway analysis revealed activation of dendritic cell maturation, NF-κB signaling, toll-like receptor signaling and JAK/STAT signaling pathways following ODN stimulation. Both tofacitinib and PDE4i strongly attenuated ODN induced IFNα. DAPSA scores elevations occurred in 18 PsA cases with SARS-CoV2 infection (9.7 ± 4 pre-infection and 35.3 ± 7.5 during infection). Entheseal pDCs link microbes to TNF/IFNα production. SARS-CoV-2 infection is associated with PsA Flares and JAK inhibition suppressed activated entheseal plasmacytoid dendritic Type-1 interferon responses as pointers towards a novel mechanism of PsA and SpA-related arthropathy.

Purpose To provide a 5-year national overview of corrective spinal deformity surgery in the United Kingdom. Methods Since 2008, the British Scoliosis Society has collected predefined data on spinal deformity surgeries carried out by its members. Participating units collect and submit annual anonymised data pertaining to the number of deformity surgeries performed, age groups, aetiology (idiopathic versus non-idiopathic), mortality, deep infections and neurological deficit (complete, incomplete without resolution and incomplete with resolution). Overall aetiology proportions and complication rates were calculated, as well as funnel plots with control limits of individual complication rates by cases performed. Results Between 2008 and 2012, 9,295 corrective spinal deformity procedures were performed. 4,445 (48 %) were recorded as idiopathic and 2,917 (31 %) as non-idiopathic. There were a total of 339 complications (3.6 %). Deep infections occurred in 222 (2.82 %), incomplete neurological deficit with resolution in 59 (0.65 %), incomplete neurological deficit without resolution in 29 (0.32 %), complete neurological deficit in 12 (0.13 %) and mortality in 17 (0.19 %). Conclusion The complication rates reported in this study compare well with previously published studies. These reported results will hopefully serve to provide a benchmark for units in the UK providing corrective spinal deformity surgery to allow individual units to compare their complication rates against national averages and to provide national complication figures to aid in the consenting process of patients. Use of a spinal deformity registry, such as the British Spine Registry, is required to ensure ongoing service development and optimal healthcare provision.

ObjectivesMurine models of interleukin (IL)-23-driven spondyloarthritis (SpA) have demonstrated entheseal accumulation of γδT-cells which were responsible for the majority of local IL-17A production. However, IL-23 blockers are ineffective in axial inflammation in man. This study investigated γδT-cell subsets in the normal human enthesis to explore the biology of the IL-23/17 axis.MethodsHuman spinous processes entheseal soft tissue (EST) and peri-entheseal bone (PEB) were harvested during elective orthopaedic procedures. Entheseal γδT-cells were evaluated using immunohistochemistry and isolated and characterised using flow cytometry. RNA was isolated from γδT-cell subsets and analysed by qPCR. Entheseal γδT-cells were stimulated with phorbol 12-myristate 13-acetate (PMA) and ionomycin, anti-CD3/28 or IL-23 and IL-17A production was measured by high-sensitivity ELISA and qPCR.ResultsEntheseal γδT-cells were confirmed immunohistochemically with Vδ1 and Vδ2 subsets that are cytometrically defined. Transcript profiles of both cell populations suggested tissue residency and immunomodulatory status. Entheseal Vδ2 cells expressed high relative abundance of IL-23/17-associated transcripts including IL-23R, RORC and CCR6, whereas the Vδ1 subset almost completely lacked detectable IL-23R transcript. Following PMA stimulation IL-17A was detectable in both Vδ1 and Vδ2 subsets, and following CD3/CD28 stimulation both subsets showed IL-17A and IL-17F transcripts with neither transcript being detectable in the Vδ1 subset following IL-23 stimulation.ConclusionSpinal entheseal Vδ1 and Vδ2 subsets are tissue resident cells with inducible IL-17A production with evidence that the Vδ1 subset does so independently of IL-23R expression.

ObjectiveWe investigated whether the normal human spinal enthesis contained resident myeloid cell populations, capable of producing pivotal proinflammatory cytokines including tumour necrosis factor (TNF) and interleukin (IL)-23 and determined whether these could be modified by PDE4 inhibition.MethodsNormal human enthesis soft tissue (ST) and adjacent perientheseal bone (PEB) (n=15) were evaluated using immunohistochemistry (IHC), digested for myeloid cell phenotyping, sorted and stimulated with different adjuvants (lipopolysaccharide and mannan). Stimulated enthesis fractions were analysed for inducible production of spondyloarthropathy disease-relevant mediators (IL-23 full protein, TNF, IL-1β and CCL20). Myeloid populations were also compared with matched blood populations for further mRNA analysis and the effect of PDE4 inhibition was assessed.ResultsA myeloid cell population (CD45+ HLADR+ CD14+ CD11c+) phenotype was isolated from both the ST and adjacent PEB and termed ‘CD14+ myeloid cells’ with tissue localisation confirmed by CD14+ IHC. The CD14− fraction contained a CD123+ HLADR+ CD11c− cell population (plasmacytoid dendritic cells). The CD14+ population was the dominant entheseal producer of IL-23, IL-1β, TNF and CCL20. IL-23 and TNF from the CD14+ population could be downregulated by a PDE4I and other agents (histamine and 8-Bromo-cAMP) which elevate cAMP. Entheseal CD14+ cells had a broadly similar gene expression profile to the corresponding CD14+ population from matched blood but showed significantly lower CCR2 gene expression.ConclusionsThe human enthesis contains a CD14+ myeloid population that produces most of the inducible IL-23, IL-1β, TNF and CCL20. This population has similar gene expression profile to the matched blood CD14+ population.

BackgroundThe human enthesis conventional T cells are poorly characterised.ObjectivesTo study the biology of the conventional T cells in human enthesis.MethodsCD4+ and CD8+ T cells were investigated in 25 enthesis samples using immunofluorescence, cytometrically, bulk RNAseq and quantitative real-time PCR following anti-CD3/CD28 bead stimulation to determine interleukin (IL)-17A and tumour necrosis factor (TNF) levels. T-cell receptor (TCR) repertoires were characterised and a search for putative T-cell reactivity was carried out using TCR3 database. The impact of pharmacological antagonism with retinoic acid receptor-related orphan nuclear receptor gamma t inhibitor (RORγti), methotrexate and phosphodiesterase type 4 inhibitor (PDE4i) was investigated.ResultsImmunofluorescence and cytometry suggested entheseal resident CD4+ and CD8+ T cells with a resident memory phenotype (CD69+/CD45RA-) and tissue residency gene transcripts (higher NR4A1/AhR and lower KLF2/T-bet transcripts). Both CD4+ and CD8+ T cells showed increased expression of immunomodulatory genes including IL-10 and TGF-β compared with peripheral blood T cells with entheseal CD8+ T cells having higher CD103, CD49a and lower SIPR1 transcript that matched CD4+ T cells. Following stimulation, CD4+ T cells produced more TNF than CD8+ T cells and IL-17A was produced exclusively by CD4+ T cells. RNAseq suggested both Cytomegalovirus and influenza A virus entheseal resident T-cell clonotype reactivity. TNF and IL-17A production from CD4+ T cells was effectively inhibited by PDE4i, while RORγti only reduced IL-17A secretion.ConclusionsHealthy human entheseal CD4+ and CD8+ T cells exhibit regulatory characteristics and are predicted to exhibit antiviral reactivity with CD8+ T cells expressing higher levels of transcripts suggestive of tissue residency. Inducible IL-17A and TNF production can be robustly inhibited in vitro.

Specialist registrars working in district general hospitals often travel considerable distances while tired Author(s): Townley S A Stephentownley@hotmail.com specialist registrar in anaesthetics Published on: May 1, 2002 EDITOR-The deleterious effects of sleep deprivation on driving performance have long been suspected, and experimental studies have supported this. 1 2 The duration of sleep deprivation required to cause an effect equivalent to that of a blood alcohol concentration of 80 mg/100 ml (the legal driving limit) has been estimated at 21 hours. 3 The imposition of a five year custodial sentence on the driver found guilty of causing death by dangerous driving in the Selby train disaster marks the law's recognition of this equivalence.

Aims This study investigated the influences of herbivore grazing intensity and grazing season on decomposition and nutrient release of dung and litter, which aimed to improve our understandings of grazing affecting nutrient cycling in alpine meadows on the Qinghai-Tibetan Platean. Methods A factorial design experiment comprising 3 grazing intensities (non-grazing, moderate grazing, and heavy grazing) and 2 grazing seasons (summer and winter), was applied to quantify the decomposition and chemistry of dung and litter in an alpine pasture using the litterbag technique. Litterbags were retrieved for analysis of mass loss and nutrient release with 180, 360, 540, and 720 days after placement. Results Grazing activity accelerated the decomposition of dung and litter and increased nutrient release from dung and litter by increasing soil temperature compared with non-grazing pastures, whereas grazing season had no effect on decomposition. The decomposition time was shorter for dung than that for litter. Conclusions Herbivores grazing benefited dung and litter decomposition and nutrient cycling directly by increasing soil temperature, which is likely to promote soil microbial activity due to low temperatures in alpine meadows, and indirectly through herbage ingestion and dung deposition which increase the organic debris concentration used for microorganisms growth and reproduction. This study provides insights into the mechanisms of grazing regulating nutrient cycling in alpine ecosystems.