Advances in genetic technologies result in improved diagnosis of mismatch repair deficiency in colorectal and endometrial cancers.

BACKGROUND: Testing cancers for mismatch repair deficiency (dMMR) by immunohistochemistry (IHC) is a quick and inexpensive means of triaging individuals for germline Lynch syndrome testing. The aim of this study was to evaluate tumour dMMR and the prevalence of Lynch syndrome in patients referred to the Manchester Centre for Genomic Medicine, which serves a population of 5.6 million. METHODS: Tumour testing used IHC for MMR proteins with targeted BRAF and MLH1 promotor methylation testing followed by germline mutation and somatic testing as appropriate. RESULTS: In total, 3694 index tumours were tested by IHC (2204 colorectal cancers (CRCs), 739 endometrial cancers (ECs) and 761 other), of which 672/3694 (18.2%) had protein loss, including 348 (9.4%) with MLH1 loss. MLH1 loss was significantly higher for 739 ECs (15%) vs 2204 CRCs (10%) (p=0.0003) and was explained entirely by higher rates of somatic MLH1 promoter hypermethylation (87% vs 41%, p<0.0001). Overall, 65/134 (48.5%) patients with MLH1 loss and no MLH1 hypermethylation or BRAF c.1799T>A had constitutional MLH1 pathogenic variants. Of 456 patients with tumours showing loss of MSH2/MSH6, 216 (47.3%) had germline pathogenic variants in either gene. Isolated PMS2 loss was most suggestive of a germline MMR variant in 19/26 (73%). Of those with no germline pathogenic variant, somatic testing identified likely causal variants in 34/48 (71%) with MLH1 loss and in MSH2/MSH6 in 40/47 (85%) with MSH2/MSH6 loss. CONCLUSIONS: Reflex testing of EC/CRC leads to uncertain diagnoses in many individuals with dMMR following IHC but without germline pathogenic variants or MLH1 hypermethylation. Tumour mutation testing is effective at decreasing this by identifying somatic dMMR in >75% of cases.

Postoperative Packing of Perianal Abscess Cavities (PPAC2): randomized clinical trial.

BACKGROUND: Perianal abscess is common. Traditionally, postoperative perianal abscess cavities are managed with internal wound packing, a practice not supported by evidence. The aim of this randomized clinical trial (RCT) was to assess if non-packing is less painful and if it is associated with adverse outcomes. METHODS: The Postoperative Packing of Perianal Abscess Cavities (PPAC2) trial was a multicentre, RCT (two-group parallel design) of adult participants admitted to an NHS hospital for incision and drainage of a primary perianal abscess. Participants were randomized 1:1 (via an online system) to receive continued postoperative wound packing or non-packing. Blinded data were collected via symptom diaries, telephone, and clinics over 6 months. The objective was to determine whether non-packing of perianal abscess cavities is less painful than packing, without an increase in perianal fistula or abscess recurrence. The primary outcome was pain (mean maximum pain score on a 100-point visual analogue scale). RESULTS: Between February 2018 and March 2020, 433 participants (mean age 42 years) were randomized across 50 sites. Two hundred and thirteen participants allocated to packing reported higher pain scores than 220 allocated to non-packing (38.2 versus 28.2, mean difference 9.9; P < 0.0001). The occurrence of fistula-in-ano was low in both groups: 32/213 (15 per cent) in the packing group and 24/220 (11 per cent) in the non-packing group (OR 0.69, 95 per cent c.i. 0.39 to 1.22; P = 0.20). The proportion of patients with abscess recurrence was also low: 13/223 (6 per cent) in the non-packing group and 7/213 (3 per cent) in the packing group (OR 1.85, 95 per cent c.i. 0.72 to 4.73; P = 0.20). CONCLUSION: Avoiding abscess cavity packing is less painful without a negative morbidity risk. REGISTRATION NUMBER: ISRCTN93273484 (https://www.isrctn.com/ISRCTN93273484). REGISTRATION NUMBER: NCT03315169 (http://clinicaltrials.gov).

Perianal abscess is a common, painful condition due to infection and swelling around the anus caused by blockage of the anal glands. The treatment of perianal abscess has stayed the same for over 50 years. An operation is performed under general anaesthetic to cut the skin and drain the infection. This is followed by continued internal dressing (packing) of the remaining cavity (hole) until the skin has healed over. Packing changes are needed multiple times a week for several weeks. Packing is the accepted treatment as it is believed to reduce the chance of the abscess coming back, and also reduces the chance of perianal fistula forming. There are no medical studies to support this idea. Perianal fistula (an abnormal passage between the skin around the anus, and the inside of the anal canal or rectum) is a long-term condition, which causes pain, and pus (and sometimes faeces) discharge, and often needs another operation (or multiple operations) to fix it. This trial was performed to demonstrate if no packing of a perianal abscess would result in a reduction of pain, with no increase in unwanted abscess recurrences and fistulas, in comparison to the standard treatment of packing. The trial recruited 433 people, who were randomly chosen to enter one of two groups; one to have their wound packed and the other to have no packing of the wound. After being discharged from hospital following surgery, the patients attended or were visited by a community nurse for the dressing to be changed or wound packed. Each patient provided information on pain from their wound, including worst pain each day and pain before, during, and after the changing of their dressing or packing. This and other information was gathered for the first 10 days after surgery and periodically until 6 months after surgery. The no-packing group experienced much less pain than the packing group. There was no difference in abscess recurrence and fistula formation between the non-packing and packing groups. The findings demonstrate that no packing of perianal abscess wounds after drainage operation is the best treatment.

Implications for the colorectal surgeon following the 100 000 Genomes Project.

AIM: The 100 000 Genomes Project was completed in 2019 with the objective of integrating genomic medicine into routine National Health Service (NHS) clinical pathways. This project and genomic research will revolutionize the way we practice colorectal surgery in the 21st century. This paper aims to provide an overview of genomic medicine and its implications for the colorectal surgeon. RESULTS: Within NHS England, consolidation has created seven regional Genomic Laboratory Hubs. DNA from solid tumours, including colorectal cancers, will be assessed using 500-gene panels, results will be fed back to Genome Tumour Advisory Boards. Identifying variants from biopsies earlier in the clinical pathway may alter surgical and other treatment options for patients. However, there is an important distinction between somatic variants within a tumour biopsy and germline variants that may suggest a heritable condition such as Lynch syndrome. Novel drugs, for example immunotherapy, will increase treatment options including downstaging cancers and changing the surgical approach. The use of circulating tumour DNA (liquid biopsies) will have applications in diagnosis, treatment and surveillance of cancer. There are many exciting potential future applications of this technology for offering personalized medicine that will require multidisciplinary working and the colorectal community. CONCLUSION: There are many challenges but also exciting opportunities to embed new 'omic' technologies and innovation into 21st century colorectal surgery. The next phase for the colorectal community is how we engage with this change, with questions around training, identification of genomic multidisciplinary team (MDT) champions and how we collaborate with the core members of the MDT, clinical geneticists and national genomic testing.

Negative pressure wound therapy for open traumatic wounds.

BACKGROUND: Traumatic wounds (wounds caused by injury) range from abrasions and minor skin incisions or tears, to wounds with extensive tissue damage or loss as well as damage to bone and internal organs. Two key types of traumatic wounds considered in this review are those that damage soft tissue only and those that involve a broken bone, that is, open fractures. In some cases these wounds are left open and negative pressure wound therapy (NPWT) is used as a treatment. This medical device involves the application of a wound dressing through which negative pressure is applied and tissue fluid drawn away from the area. The treatment aims to support wound management, to prepare wounds for further surgery, to reduce the risk of infection and potentially to reduce time to healing (with or without surgical intervention). There are no systematic reviews assessing the effectiveness of NPWT for traumatic wounds. OBJECTIVES: To assess the effects of NPWT for treating open traumatic wounds in people managed in any care setting. SEARCH METHODS: In June 2018 we searched the Cochrane Wounds Specialised Register, the Cochrane Central Register of Controlled Trials (CENTRAL), Ovid MEDLINE (including In-Process & Other Non-Indexed Citations), Ovid Embase and EBSCO CINAHL Plus. We also searched clinical trials registries for ongoing and unpublished studies, and scanned reference lists of relevant included studies as well as reviews, meta-analyses and health technology reports to identify additional studies. There were no restrictions with respect to language, date of publication or study setting. SELECTION CRITERIA: Published and unpublished randomised controlled trials that used NPWT for open traumatic wounds involving either open fractures or soft tissue wounds. Wound healing, wound infection and adverse events were our primary outcomes. DATA COLLECTION AND ANALYSIS: Two review authors independently selected eligible studies, extracted data, carried out a 'Risk of bias' assessment and rated the certainty of the evidence. Data were presented and analysed separately for open fracture wounds and other open traumatic wounds (not involving a broken bone). MAIN RESULTS: Seven RCTs (1377 participants recruited) met the inclusion criteria of this review. Study sample sizes ranged from 40 to 586 participants. One study had three arms, which were all included in the review. Six studies compared NPWT at 125 mmHg with standard care: one of these studies did not report any relevant outcome data. One further study compared NPWT at 75 mmHg with standard care and NPWT 125mmHg with NPWT 75 mmHg.Open fracture wounds (four studies all comparing NPWT 125 mmHg with standard care)One study (460 participants) comparing NPWT 125 mmHg with standard care reported the proportions of wounds healed in each arm. At six weeks there was no clear difference between groups in the number of participants with a healed, open fracture wound: risk ratio (RR) 1.01 (95% confidence interval (CI) 0.81 to 1.27); moderate-certainty evidence, downgraded for imprecision.We pooled data on wound infection from four studies (596 participants). Follow-up varied between studies but was approximately 30 days. On average, it is uncertain whether NPWT at 125 mmHg reduces the risk of wound infection compared with standard care (RR 0.48, 95% CI 0.20 to 1.13; I2 = 56%); very low-certainty evidence downgraded for risk of bias, inconsistency and imprecision.Data from one study shows that there is probably no clear difference in health-related quality of life between participants treated with NPWT 125 mmHg and those treated with standard wound care (EQ-5D utility scores mean difference (MD) -0.01, 95% CI -0.08 to 0.06; 364 participants, moderate-certainty evidence; physical component summary score of the short-form 12 instrument MD -0.50, 95% CI -4.08 to 3.08; 329 participants; low-certainty evidence downgraded for imprecision).Moderate-certainty evidence from one trial (460 participants) suggests that NPWT is unlikely to be a cost-effective treatment for open fractures in the UK. On average, NPWT was more costly and conferred few additional quality-adjusted life years (QALYs) when compared with standard care. The incremental cost-effectiveness ratio was GBP 267,910 and NPWT was shown to be unlikely to be cost effective at a range of cost-per-QALYs thresholds. We downgraded the certainty of the evidence for imprecision.Other open traumatic wounds (two studies, one comparing NPWT 125 mmHg with standard care and a three-arm study comparing NPWT 125 mmHg, NPWT 75 mmHg and standard care)Pooled data from two studies (509 participants) suggests no clear difference in risk of wound infection between open traumatic wounds treated with NPWT at 125 mmHg or standard care (RR 0.61, 95% CI 0.31 to 1.18); low-certainty evidence downgraded for risk of bias and imprecision.One trial with 463 participants compared NPWT at 75 mmHg with standard care and with NPWT at 125 mmHg. Data on wound infection were reported for each comparison. It is uncertain if there is a difference in risk of wound infection between NPWT 75 mmHg and standard care (RR 0.44, 95% CI 0.17 to 1.10; 463 participants) and uncertain if there is a difference in risk of wound infection between NPWT 75 mmHg and 125 mmHg (RR 1.04, 95% CI 0.31 to 3.51; 251 participants. We downgraded the certainty of the evidence for risk of bias and imprecision. AUTHORS' CONCLUSIONS: There is moderate-certainty evidence for no clear difference between NPWT and standard care on the proportion of wounds healed at six weeks for open fracture wounds. There is moderate-certainty evidence that NPWT is not a cost-effective treatment for open fracture wounds. Moderate-certainty evidence means that the true effect is likely to be close to the estimate of the effect, but there is a possibility that it is substantially different. It is uncertain whether there is a difference in risk of wound infection, adverse events, time to closure or coverage surgery, pain or health-related quality of life between NPWT and standard care for any type of open traumatic wound.

Manufacture of GMP-compliant functional adenovirus-specific T-cell therapy for treatment of post-transplant infectious complications.

BACKGROUND AIMS: In pediatric patients, adenovirus (ADV) reactivation after allogeneic hematopoietic stem cell transplantation (allo HSCT) is a major cause of morbidity and mortality. For patients who do not respond to antiviral drug therapy, a new treatment approach using ADV-specific T cells can present a promising alternative. Here we describe the clinical scale Good Manufacturing Practice (GMP)-compliant manufacture and characterization of 40 ADV-specific T-cell products, Cytovir ADV, which are currently being tested in a multi-center phase I/IIa clinical trial. This process requires minimal intervention, is high yield, and results in a pure T-cell product that is functional. METHODS: Mononuclear cells (2 × 10(7)) were cultured in a closed system in the presence of GMP-grade ADV peptide pool and cytokines for 10 days. On day 10, the T-cell product was harvested, washed in a closed system, counted and assessed for purity and potency. Additional characterization was carried out where cell numbers allowed. RESULTS: Thirty-eight of 40 products (95%) met all release criteria. Median purity of the cell product was 88.3% CD3+ cells with a median yield of 2.9 × 10(7) CD3+ cells. Potency analyses showed a median ADV-specific interferon (IFN)γ response of 5.9% of CD3+ and 2345 IFNγ spot-forming cells/million. CD4 and CD8 T cells were capable of proliferating in response to ADV (63.3 and 56.3%, respectively). These virus-specific T cells (VST) were heterogenous, containing both effector memory and central memory T cells. In an exemplar patient with ADV viremia treated in the open ASPIRE trial, ADV-specific T-cell response was detected by IFNγ enzyme-linked immunospot from 13 days post-infusion. ADV DNA levels declined following cellular therapy and were below level of detection from day 64 post-infusion onward. CONCLUSIONS: The clinical-scale GMP-compliant One Touch manufacturing system is feasible and yields functional ADV-specific T cells at clinically relevant doses.

Internal dressings for healing perianal abscess cavities.

BACKGROUND: A perianal abscess is a collection of pus under the skin, around the anus. It usually occurs due to an infection of an anal gland. In the UK, the annual incidence is 40 per 100,000 of the adult population, and the standard treatment is admission to hospital for incision and drainage under general anaesthetic. Following drainage of the pus, an internal dressing (pack) is placed into the cavity to stop bleeding. Common practice is for community nursing teams to change the pack regularly until the cavity heals. Some practitioners in the USA and Australia make a small stab incision under local anaesthetic and place a catheter into the cavity which drains into an external dressing. It is removed when it stops draining. Elsewhere in the USA, simple drainage is performed in an outpatient setting under local anaesthetic. OBJECTIVES: To assess the effects of internal dressings in healing wound cavities resulting from drainage of perianal abscesses. SEARCH METHODS: In May 2016 we searched: The Cochrane Wounds Specialised Register; The Cochrane Central Register of Controlled Trials (CENTRAL) (The Cochrane Library); Ovid MEDLINE; Ovid MEDLINE (In-Process & Other Non-Indexed Citations); Ovid EMBASE and EBSCO CINAHL Plus. We also searched clinical trial registries to identify ongoing and unpublished studies, and searched reference lists of relevant reports to identify additional studies. We did not restrict studies with respect to language, date of publication, or study setting. SELECTION CRITERIA: Published or unpublished randomised controlled trials (RCTs) comparing any type of internal dressing (packing) used in the post-operative management of perianal abscess cavities with alternative treatments or different types of internal dressing. DATA COLLECTION AND ANALYSIS: Two review authors independently performed study selection, risk of bias assessment, and data extraction. MAIN RESULTS: We included two studies, with a total of 64 randomised participants (50 and 14 participants) aged 18 years or over, with a perianal abscess. In both studies, participants were enrolled on the first post-operative day and randomised to continued packing by community district nursing teams or to no packing. Participants in the non-packing group managed their own wounds in the community and used absorbant dressings to cover the area. Fortnightly follow-up was undertaken until the cavity closed and the skin re-epithelialised, which constituted healing. For non-attenders, telephone follow-up was conducted.Both studies were at high risk of bias due to risk of attrition, performance and detection bias.It was not possible to pool the two studies for the outcome of time to healing. It is unclear whether continued post-operative packing of the cavity of perianal abscesses affects time to complete healing. One study reported a mean time to wound healing of 26.8 days (95% confidence interval (CI) 22.7 to 30.7) in the packing group and 19.5 days (95% CI 13.6 to 25.4) in the non-packing group (it was not clear if all participants healed). We re-analysed the data and found no clear difference in the time to healing (7.30 days longer in the packing group, 95% CI -2.24 to 16.84; 14 participants). This was assessed as very low quality evidence (downgraded three levels for very serious imprecision and serious risk of bias). The second study reported a median time to complete wound healing of 24.5 days (range 10 to 150 days) in the packing group and 21 days (range 8 to 90 days) in the non-packed group. There was insufficient information to be able to recreate the analysis and the original analysis was inappropriate (did not account for censoring). This second study also provided very low quality evidence (downgraded four levels for serious risk of bias, serious indirectness and very serious imprecision).There was very low quality evidence (downgraded for risk of bias, indirectness and imprecision) of no difference in wound pain scores at the initial dressing change. Both studies also reported patients' retrospective judgement of wound pain over the preceding two weeks (visual analogue scale, VAS) as lower for the non-packed group (2; both studies) compared with the packed group (0; both studies); (very low quality evidence) but we have been unable to reproduce these analyses as no variance data were published.There was no clear evidence of a difference in the number of post-operative fistulae detected between the packed and non-packed groups (risk ratio (RR) 2.31, 95% CIs 0.56 to 9.45, I(2) = 0%) (very low quality evidence downgraded three levels for very serious imprecision and serious risk of bias).There was no clear evidence of a difference in the number of abscess recurrences between the packed and non-packed groups over the variable follow-up periods (RR 0.72, 95% CI 0.22 to 2.37, I(2) = 0%) (very low quality evidence downgraded three levels for serious risk of bias and very serious imprecision).No study reported participant health-related quality of life/health status, incontinence rates, time to return to work or normal function, resource use in terms of number of dressing changes or visits to a nurse, or change in wound size. AUTHORS' CONCLUSIONS: It is unclear whether using internal dressings (packing) for the healing of perianal abscess cavities influences time to healing, wound pain, development of fistulae, abscess recurrence or other outcomes. Despite this absence of evidence, the practice of packing abscess cavities is commonplace. Given the lack of high quality evidence, decisions to pack may be based on local practices or patient preferences. Further clinical research is needed to assess the effects and patient experience of packing.

Lynch syndrome caused by MLH1 mutations is associated with an increased risk of breast cancer: a cohort study.

INTRODUCTION: Lynch syndrome is known to cause an increased risk of malignancies, including bowel and endometrial cancers. However, the risk of breast cancer associated with mutations in the mismatch repair (MMR) genes that cause Lynch syndrome is still unclear. MATERIALS AND METHODS: This study assesses the cumulative risk of breast cancer in 106 MLH1 and 118 MSH2 families. Families were referred on the basis of clinical criteria. Pedigree information was obtained, and tumour immunohistochemistry and microsatellite testing performed. Appropriate patients underwent sequencing and multiple ligation dependent probe amplification of all relevant exons of the MMR genes. Kaplan-Meier analysis of cumulative lifetime risk of breast cancer was made combining proven mutation carriers and their first-degree female relatives. RESULTS: After allocation of mutation status, the cumulative risk of breast cancer to 70 years in MLH1 carriers was 18.6% (95% CI 11.3 to 25.9)). This is significantly higher than the cumulative risk for MSH2 which was 11.2% (95% CI 1.4 to 21.0) to age 70 years (p=0.014). The UK population risk is 7.5%-8% at the age of 70 years. Prospective analysis identified six breast cancers in 1120 years of follow-up with an OR of 3.41 (95% CI 1.53 to 7.59). DISCUSSIONS: Female MLH1 carriers would appear to be at moderate risk of breast cancer and should be considered for breast screening at ages earlier than national screening programmes.

Successful isolation and expansion of CMV-reactive T cells from G-CSF mobilized donors that retain a strong cytotoxic effector function.

Cytomegalovirus (CMV) infections post-haematopoietic stem cell transplantation (HSCT) can be effectively controlled through the adoptive transfer of donor-derived CMV-specific T cells (CMV-T). Current strategies involve a second leukapheresis collection from the original donor to manufacture CMV-T, which is often not possible in the unrelated donor setting. To overcome these limitations we have investigated the use of a small aliquot of the original granulocyte-colony stimulating factor (G-CSF) mobilized HSCT graft to manufacture CMV-T. We explored the T cell response to CMVpp65 peptide stimulation in G-CSF mobilized peripheral blood mononuclear cells (PBMC) and subsequently examined isolation of CMV-T based on the activation markers CD154 and CD25. CD25(+) enriched CMV-T from G-CSF mobilized PBMC contained a higher proportion of FoxP3 expression than non-mobilized PBMC and showed superior suppression of T cell proliferation. Expanded CMV-T enriched through CD154 were CD4(+) and CD8(+) , demonstrated a high specificity for CMV, secreted cytotoxic effector molecules and lysed CMVpp65 peptide-loaded phytohaemagglutinin-stimulated blasts. These data provide the first known evidence that CMV-T can be effectively manufactured from G-CSF mobilized PBMC and that they share the same characteristics as CMV-T isolated in an identical manner from conventional non-mobilized PBMC. This provides a novel strategy for adoptive immunotherapy that abrogates the need for successive donation.

Human dendritic cells infected with an adenoviral vector suppress proliferation of autologous and allogeneic T cells.

Dendritic cells (DCs) play a key role in the type and course of an immune response. The manipulation of human DCs to produce therapeutic agents by transduction with viral vectors is a growing area of research. We present an investigation into the effects of adenoviral vector infection on human DCs and other cell types, and on their subsequent ability to induce T-cell proliferation. We show that infection with replication-deficient adenovirus results in impaired proliferation of T cells in a mixed lymphocyte reaction (MLR). We show this to be an active suppression rather than a defect in the DCs as T cells also fail to proliferate in response to phytohaemagglutinin in the presence of adenoviral vector-infected DCs. This suppression is not attributable to phenotypic changes, death or inability of the DCs to produce cytokines on stimulation. By separation of DCs from T cells, and addition of conditioned supernatants, we show that suppression is mediated by a soluble factor. Blocking of interleukin (IL)-10 but not transforming growth factor (TGF)-beta could overcome the suppressive effect in some donors, and the source of the suppressive IL-10 was lymphocytes exposed to conditioned supernatant. Together our data suggest that infection of DCs by adenoviral vectors leads to suppression of the resulting immune response.

Isolation of highly suppressive CD25+FoxP3+ T regulatory cells from G-CSF-mobilized donors with retention of cytotoxic anti-viral CTLs: application for multi-functional immunotherapy post stem cell transplantation.

Previous studies have demonstrated the effective control of cytomegalovirus (CMV) infections post haematopoietic stem cell transplant through the adoptive transfer of donor derived CMV-specific T cells (CMV-T). Strategies for manufacturing CMV immunotherapies has involved a second leukapheresis or blood draw from the donor, which in the unrelated donor setting is not always possible. We have investigated the feasibility of using an aliquot of the original G-CSF-mobilized graft as a starting material for manufacture of CMV-T and examined the activation marker CD25 as a targeted approach for identification and isolation following CMVpp65 peptide stimulation. CD25+ cells isolated from G-CSF-mobilized apheresis revealed a significant increase in the proportion of FoxP3 expression when compared with conventional non-mobilized CD25+ cells and showed a superior suppressive capacity in a T cell proliferation assay, demonstrating the emergence of a population of Tregs not present in non-mobilized apheresis collections. The expansion of CD25+ CMV-T in short-term culture resulted in a mixed population of CD4+ and CD8+ T cells with CMV-specificity that secreted cytotoxic effector molecules and lysed CMVpp65 peptide-loaded phytohaemagglutinin-stimulated blasts. Furthermore CD25 expanded cells retained their suppressive capacity but did not maintain FoxP3 expression or secrete IL-10. In summary our data indicates that CD25 enrichment post CMV stimulation in G-CSF-mobilized PBMCs results in the simultaneous generation of both a functional population of anti-viral T cells and Tregs thus illustrating a potential single therapeutic strategy for the treatment of both GvHD and CMV reactivation following allogeneic haematopoietic stem cell transplantation. The use of G-CSF-mobilized cells as a starting material for cell therapy manufacture represents a feasible approach to alleviating the many problems incurred with successive donations and procurement of cells from unrelated donors. This approach may therefore simplify the clinical application of adoptive immunotherapy and broaden the approach for manufacturing multi-functional T cells.

Interleukin-17-producing T cells are enriched in the joints of children with arthritis, but have a reciprocal relationship to regulatory T cell numbers.

OBJECTIVE: To identify interleukin-17 (IL-17)-producing T cells from patients with juvenile idiopathic arthritis (JIA), and investigate their cytokine production, migratory capacity, and relationship to Treg cells at sites of inflammation, as well as to test the hypothesis that IL-17+ T cell numbers correlate with clinical phenotype in childhood arthritis. METHODS: Flow cytometry was used to analyze the phenotype, cytokine production, and chemokine receptor expression of IL-17-producing T cells in peripheral blood and synovial fluid mononuclear cells from 36 children with JIA, in parallel with analysis of forkhead box P3 (FoxP3)-positive Treg cells. Migration of IL-17+ T cells toward CCL20 was assessed by a Transwell assay. Synovial tissue was analyzed by immunohistochemistry for IL-17 and IL-22. RESULTS: IL-17+ T cells were enriched in the joints of children with JIA as compared with the blood of JIA patients (P = 0.0001) and controls (P = 0.018) and were demonstrated in synovial tissue. IL-17+ T cell numbers were higher in patients with extended oligoarthritis, the more severe subtype of JIA, as compared with patients with persistent oligoarthritis, the milder subtype (P = 0.046). Within the joint, there was an inverse relationship between IL-17+ T cells and FoxP3+ Treg cells (r = 0.61, P = 0.016). IL-17+,CD4+ T cells were uniformly CCR6+ and migrated toward CCL20, but synovial IL-17+ T cells had variable CCR4 expression. A proportion of IL-17+ synovial T cells produced IL-22 and interferon-gamma. CONCLUSION: This study is the first to define the frequency and characteristics of "Th17" cells in JIA. We suggest that these highly proinflammatory cells contribute to joint pathology, as indicated by relationships with clinical phenotypes, and that the balance between IL-17+ T cells and Treg cells may be critical to outcome.

Quantification of normal range of inflammatory changes in morphologically normal pediatric muscle.

The aim of this study was to define normal ranges of histological features in pediatric muscle in comparison with muscle demonstrating inflammatory changes. Sixteen pediatric muscle biopsy samples, considered normal by standard histology, were analyzed for the presence of inflammatory cells, and the expression of neonatal myosin and major histocompatibility complex (MHC) Class 1. Normal findings were defined for each feature. These data will facilitate quantitative analysis of inflammatory changes in pediatric muscle biopsy.

International consensus on a proposed score system for muscle biopsy evaluation in patients with juvenile dermatomyositis: a tool for potential use in clinical trials.

OBJECTIVE: To devise and test a system with which to evaluate abnormalities on muscle biopsy samples obtained from children diagnosed with juvenile dermatomyositis (DM). METHODS: We established an International Consensus Group on Juvenile DM Biopsy and carried out 2 phases of consensus process and scoring workshops. Biopsy sections (n = 33) were stained by standard methods. The scoring tool was based on 4 domains of change: inflammatory, vascular, muscle fiber, and connective tissue. Using a Latin square design, biopsy samples were scored by 11 experts for items in each domain, and for a global abnormality measure using a 10-cm visual analog score (VAS 0-10). The tool's reliability was assessed using an intraclass correlation coefficient (ICC) and scorer agreement (alpha) by determining variation in scorers' ratings. RESULTS: There was good agreement in many items of the tool, and several items refined between the meetings improved in reliability and/or agreement. The inflammatory and muscle fiber domains had the highest reliability and agreement. The overall VAS score for abnormality had high agreement and reliability, reaching an ICC of 0.863 at the second consensus meeting. CONCLUSION: We propose a provisional scoring system to measure abnormalities on muscle biopsy samples obtained from children with juvenile DM. This system needs to be validated, and then could be used in prospective studies to test which features of muscle pathology are prognostic of disease course or outcome. We suggest that the process we used could be a template for developing similar systems in other forms of myositis.

Expression of the inflammatory chemokines CCL5, CCL3 and CXCL10 in juvenile idiopathic arthritis, and demonstration of CCL5 production by an atypical subset of CD8+ T cells.

This study focuses upon three chemokines, namely CCL5, CXCL10 and CCL3, which are potential novel therapeutic targets in arthritis. The aim of the study was to analyse the expression and production of these three chemokines within the joints of children with juvenile idiopathic arthritis (JIA) of the oligoarticular and polyarticular subtypes. All three of these chemokines are highly expressed at the level of mRNA, with the most significant increase in mRNA levels being demonstrated for CCL5 when compared with matched peripheral blood samples and controls. We show that high levels of all three chemokines are present in synovial fluid of children with JIA. We investigate the major source of CCL5 from inflammatory synovial cells, which we show to be CD8+ T cells. This CD8+ synovial T cell population has an unexpected phenotype that has not been described previously, being CCR7- yet predominantly CD28+ and CD45RA-. These cells contain high levels of stored intracellular CCL5, and rapid release of CCL5 takes place on T cell stimulation, without requiring new protein synthesis. In addition, we demonstrate that CCL5 is present in synovial biopsies from these patients, in particular on the endothelium of small and medium sized vessels. We believe this to be the first in depth analysis of these mediators of inflammation in JIA.