Revision total knee replacement: A two-year review of complexity data and regional workload in South West England.

BACKGROUND: The GIRFT report (2012) sought to address the need for sustainable orthopaedic treatment delivered through regional "networks"; the aim being improved care, decreased cost and reduced revision rate. The aims of this study were to record the number and complexity of revision total knee replacements within a regional network using a validated classification over a two-year period and audit this against National Joint Registry (NJR) records. METHODS: A region-wide network model where revision TKR cases are assessed locally using the Revision Knee Complexity Classification (RKCC) and local multi-disciplinary team (MDT) was introduced. Data was collected from 8 revision centres over a two-year period using the RKCC. The case volume was audited against the NJR records. RESULTS: In year 1 (01/01/2018-31/12/2018) 237 RKCC forms were collected from eight centres. 46% of R2s and 63% of R3s were carried out at the higher volume centre. 211 K2 forms were received by the NJR. In year 2 (01/01/2019-31/12/2019) 252 RKCC forms were collected. 46% of R2s and 64% of R3s were carried out at the higher volume centre. 267 K2 forms were received by the NJR. CONCLUSION: This is the first published set of revision knee data showing complexity percentages across a region. The RKCC has been successfully introduced into the region and this has been sustained. The findings show that a successful network has been established and majority of complex revision knee surgery is occurring in the high-volume centre. NJR data suggests that the RKCC is capturing the complexity and volume of our work accurately.

A tractable covalent linker strategy for the production of immunogenic antigen-TLR7/8L bioconjugates.

Despite the ease of production and improved safety profiles of recombinant vaccines, the inherently low immunogenicity of unadjuvanted proteins remains an impediment to their widespread adoption. The covalent tethering of TLR agonists to antigenic proteins offers a unique approach to co-deliver both constituents to the same cell-enhancing vaccine efficacy while minimizing reactogenicity. However, the paucity of simple and effective linker chemistries continues to hamper progress. Here, we present a modular, PEG-based linker system compatible with even extremely lipophilic and challenging TLR7/8 agonists. To advance the field and address previous obstacles, we offer the most straightforward and antigen-preserving linker system to date. These antigen-adjuvant conjugates enhance antigen-specific immune responses in mice, demonstrating the power of our approach within the context of modern vaccinology.

Programmatic utility of tuberculosis cluster investigation using a social network approach in Birmingham, United Kingdom.

SETTING: Birmingham, United Kingdom, 2010-2014. OBJECTIVE: To investigate predictors for clustering of tuberculosis (TB) cases and cluster size and to evaluate the impact of cluster investigation using social network data. DESIGN: Retrospective observational cohort study. Prioritised cases linked using 24-locus mycobacterial interspersed repetitive units-variable number of tandem repeats (MIRU-VNTR) were interviewed using a social network approach to find epidemiological links. RESULTS: Of 2055 TB cases notified, 56% could be typed. Clustering was associated with younger age, UK birth, Black Caribbean ethnicity, social risk factors, pulmonary TB and negative human immunodeficiency virus status. Only UK birth and presence of more than one social risk factor were associated with larger cluster size, while drug resistance was associated with smaller cluster size. Social network data from 139/431 clustered cases found new epidemiological links in 11/19 clusters with ⩾5 members (undirected median network density 0.09, interquartile range 0.05-0.4). Ninety-eight additional contacts were assessed, with one case of active TB and 24 with latent tuberculous infection diagnosed. CONCLUSION: A social network approach increased knowledge of likely transmission events, but few additional TB cases were diagnosed. Obtaining social network data for all typed and untyped TB cases may improve contact tracing and reduce unexpected transmission detected from molecular data.

Complications associated with opening wedge high tibial osteotomy--A review of the literature and of 15 years of experience.

BACKGROUND: Complication rates following opening wedge high tibial osteotomy (OWHTO) is an issue that has not been comprehensively addressed in current literature. METHODS: We performed a retrospective study of local patients who underwent OWHTO for isolated medial compartment knee osteoarthritis from 1997 to 2013. We analysed survivorship and complication rates and compared this to a literature review of previously reported data. RESULTS: One hundred and fifteen patients met the inclusion criteria. Mean follow-up=8.4 years. Mean age=47 (range 32 to 62). Mean Body Mass Index (BMI)=29.1 (range 20.3 to 40.2). Devices used consisted of Tomofix (72%), Puddu plate (21%) and Orthofix (seven percent) (no significant differences in age/sex/BMI). Wedge defects were filled with autologous graft (30%), Chronos (35%) or left empty (35%). Five years survival rate (without requiring conversion to arthroplasty)=80%. Overall complication rate=31%. Twenty five percent of patients suffered 36 complications including minor wound infections (9.6%), major wound infections (3.5%), metalwork irritation necessitating plate removal (seven percent), non-union requiring revision (4.3%), vascular injury (1.7%), compartment syndrome (0.9%), and other minor complications (four percent). No thromboembolic complications were observed. CONCLUSION: No significant differences existed in complication rates following OWHTO relative to BMI, implant type, type of bone graft used or patient age at surgery. When the complications from OWHTO were analysed closely they appear higher than previously reported in the literature; however serious complications appear rare. LEVEL OF EVIDENCE 3: Retrospective cohort study.

Displaced femoral neck stress fractures in Royal Marine recruits--management and results of operative treatment.

Femoral neck stress fractures (FNSF) represent 3.5%-8% of stress fractures in military recruits; potentially resulting in medical discharge and/or complications. The incidence of displaced FNSF in the British Army has been reported as 1.8 in 10,000 recruits. We aimed to review the incidence and outcome of displaced FNSF in Royal Marine recruits. Retrospective review identified 6 recruits who sustained a displaced FNSF from 2001 to 2011 representing an incidence of 9.3 in 10,000 recruits. All were treated urgently by internal fixation. There were no cases of avascular necrosis, no surgical complications and no further procedures required. All united with a mean time to union of 11 months. 50% had a union time greater than 1 year. These fractures are slow to unite but with urgent surgical intervention and stable fixation 100% union was achieved. Awareness of this guides the management and rehabilitation whilst avoiding the risks of unnecessary secondary surgical interventions.

A tale of two STAT6 knock out mice in the induction of experimental autoimmune encephalomyelitis.

T helper 2 (Th2) cytokines are known to be important in protection against experimental autoimmune encephalomyelitis (EAE). To investigate the role of the signal transducer and activator of transcription factor 6 (STAT6) in EAE we used mice with two different targeted disruptions of the STAT6 gene. In this report, we show that mice with a targeted deletion of the first coding exon of the SH2 domain of STAT6 induce Th2 cell differentiation and are resistant to EAE induction. By contrast, STAT6(-/-) mice generated by deletion of amino acids 505 to 584 encoding the SH2 domain of STAT6 are defective in Th2 cell differentiation and develop very severe EAE. These results suggest that an altered STAT6 gene can be more efficient than wild type STAT6 in regulating the autoimmune response in EAE.

An IS6110-targeting fluorescent amplified fragment length polymorphism alternative to IS6110 restriction fragment length polymorphism analysis for Mycobacterium tuberculosis DNA fingerprinting.

A rapid, simple and highly discriminatory DNA fingerprinting methodology which produces data that can be easily interpreted, compared and transported is the ultimate goal for studying the epidemiology of Mycobacterium tuberculosis. A novel TaqI fluorescent amplified fragment length polymorphism (fAFLP) approach to M. tuberculosis DNA fingerprinting that targeted the variable IS6110 marker was developed in this study. The new method was tested for specificity and reproducibility, and compared with the standard reference IS6110 restriction fragment length polymorphism (RFLP) method for a panel of 78 isolates. Clustering conflicts between the two methods were resolved using mycobacterial interspersed repetitive unit-variable number tandem repeat (MIRU-VNTR) data. Comparison with an in-silico digestion of strain H37Rv showed that fAFLP-detected fragments were highly specific in vitro. The reproducibility of repeated digestions of strain H37Rv was 100%. Clustering results obtained by fAFLP and RFLP were highly congruent, with fAFLP allocating 97% of RFLP-clustered isolates to the same eight clusters as RFLP. Two single-copy isolates that had been clustered by RFLP were not clustered by fAFLP, but the MIRU-VNTR patterns of these isolates were different, indicating that the RFLP data had falsely clustered these isolates. Analysis by fAFLP will allow rapid screening of isolates to confirm or refute epidemiological links, and thereby provide insights into the frequency, conservation and consequences of specific transposition events.

Occurrence, prevalence and genetic environment of CTX-M beta-lactamases in Enterobacteriaceae from Indian hospitals.

OBJECTIVES: To determine occurrence, prevalence and CTX-M genotypes produced by Enterobacteriaceae from clinical samples from three geographically distant Indian hospitals and to detect linkage of IS26 with bla(CTX-M) and map its precise insertion position. METHODS: A total of 130, non-duplicate Escherichia coli and Klebsiella pneumoniae resistant to a third-generation cephalosporin (3GC) from three Indian centres were screened for extended-spectrum beta-lactamase (ESBL) production using phenotypic detection methods. All isolates were screened for bla(CTX-M) using multiplex PCR. Precise CTX-M genotype was identified using reverse-line hybridization. All CTX-M-producing isolates were screened for linkage of IS26 with bla(CTX-M). DNA sequencing was used to map the exact insertion position of this mobile element. RESULTS: Ninety-five of 130 3GC-resistant (73%) (73% of total E. coli, 72% of total K. pneumoniae) isolates were found to carry bla(CTX-M-15). No other CTX-M genotype was detected. IS26 linkage with bla(CTX-M-15) was detected in 31% of isolates carrying bla(CTX-M-15). DNA sequencing revealed variable insertion of this mobile element within tnpA of ISEcp1. RAPD-PCR typing demonstrated great diversity in isolates carrying bla(CTX-M-15); no predominant clone was identified. CONCLUSIONS: In contrast with other studies where greater diversity exists, CTX-M-15 was the only CTX-M ESBL produced in this Indian collection of unrelated E. coli and K. pneumoniae. This is the first systematic survey report from India detecting CTX-M-type beta-lactamases This is also the first report indicating such high mobility/diversity of insertion of IS26 in close association with bla(CTX-M) in a single bacterial collection.

Purification of native alpha-enolase from Streptococcus pneumoniae that binds plasminogen and is immunogenic.

Many pathogenic bacteria express plasminogen receptors on their surface, which may play a role in the dissemination of organisms by binding plasminogen that, when converted to plasmin, can digest extracellular matrix proteins. A 45-kDa protein was purified from Streptococcus pneumoniae and confirmed as an alpha-enolase by its ability to catalyse the dehydration of 2-phospho-D-glycerate to phosphoenolpyruvate and by N-terminal sequencing. The activity of alpha-enolase was found in the cytoplasm and in whole cells. Activity was also demonstrated in cell wall fractions, which confirmed that alpha-enolase is a cytoplasmic antigen also expressed on the surface of S. pneumoniae. The plasminogen-binding activity of alpha-enolase was examined by Western blot, which showed that purified alpha-enolase was able to bind human plasminogen. Immunoblots of the purified 45-kDa alpha-enolase with 22 sera from patients with pneumococcal disease showed binding in 15 cases, indicating that pneumococcal enolase is immunogenic.

Pre-clinical evaluation of an in vitro selection protocol for the enrichment of transduced CD34+ cell-derived human dendritic cells.

The efficient genetic modification of CD34+ cell-derived dendritic cells (DC) will provide a significant advancement towards the development of immunotherapy protocols for cancer, autoimmune disorders and infectious diseases. Recent reports have described the transduction of CD34+ cells via retrovirus- and lentivirus-based gene transfer vectors and subsequent differentiation into functional DC. Since there is significant apprehension regarding the clinical uses of HIV-based vectors, in this report, we compare a murine leukemia virus (MLV)- and a human immunodeficiency virus (HIV)-based bicistronic vector for gene transfer into human CD34+ cells and subsequent differentiation into mature DC. Each vector expressed both EGFP and the dominant selectable marker DHFR(L22Y) allowing for the enrichment of marked cells in the presence of the antifolate drug trimetrexate (TMTX). Both MLV-based and HIV-based vectors efficiently transduced cytokine mobilized human peripheral blood CD34+ cells. However, in vitro expansion and differentiation in the presence of GM-CSF, TNF-alpha, Flt-3L, SCF and IL-4 resulted in a reduction in the percentage of DC expressing the transgene. Selection with TMTX during differentiation increased the percentage of marked DC, resulting in up to 79% (MLV vector) and up to 94% (lentivirus-vector) transduced cells expressing EGFP without loss of DC phenotype. Thus, MLV-based vectors and in vitro selection of transduced human DC show great promise for immunotherapy protocols.

Differentiation and expansion of lentivirus vector-marked dendritic cells derived from human CD34(+) cells.

The in vitro genetic manipulation of dendritic cells (DCs) for the expression of foreign proteins or peptides will assist in the development of immunotherapeutic approaches to treat cancer, immunological disorders, and/or infectious diseases. Reports have shown the expansion and differentiation of CD34(+) progenitor cells into mature DCs. In this article we describe the differentiation and expansion of lentivirus vector-marked DCs from umbilical cord blood, bone marrow, and cytokine-mobilized peripheral blood CD34(+) cells in the presence of GM-CSF, TNF-alpha, SCF, Flt-3, and IL-4. Lentivirus-marked DCs expressed high levels of enhanced green fluorescent protein and the characteristic DC surface markers CD1a, CD83, HLA-DR, and CD80. Transduced DCs activated allogeneic CD3(+) T cells as efficiently as control (nontransduced) DCs in mixed lymphocyte reactions. These results demonstrate the potential utility of lentivirus-transduced DCs in future immunotherapy protocols.

Lentivirus vector mobilization and spread by human immunodeficiency virus.

The rapid advancement of lentivirus-based gene transfer systems and their demonstrated utility in a variety of in vitro and in vivo settings has heightened the need for assays to evaluate the safety of these vectors prior to human clinical trials. Two major concerns relating to the use of lentivirus-based vectors in a clinical setting are the presence of contaminating replication-competent retroviruses in vector preparations and the efficiency of vector mobilization and spread by wild-type helper virus (rescue). This article describes an in vitro system to study the rescue of lentivirus-based vectors by wild-type HIV. We show that lentivirus-based vectors can be readily rescued from T cell lines and to a lesser extent from primary human lymphocytes by wildtype HIV, resulting in the spread of mobilized vector particles to previously untransduced cells. Furthermore, we show that vector mobilization can be prevented by antiretroviral drugs such as AZT. In contrast to recently published reports by Bukovsky et al. and An et al., the lentivirus vectors used in these studies had little or no effect on the replication and spread of HIV in transduced cells [Bukovsky et al. (1999). J. Virol. 73, 7087-7092; An et al. (1999). J. Virol. 73, 7671-7677]. Whereas vector spread is a significant concern for most gene therapy applications, in the context of gene therapy for HIV infection it may have beneficial effects.

Thymocyte differentiation from lentivirus-marked CD34(+) cells in infant and adult human thymus.

Changes in thymic function and immune system homeostasis associated with HIV infection or chemotherapy have significant effects on the ability of patients to maintain a complete T cell receptor repertoire. Therefore, the development of in vitro systems to evaluate thymic function in children and adults may aid in the understanding of thymopoiesis and the development of new therapies to improve thymic output. Here we use a lentivirus-based gene transfer system to mark CD34(+) cells with EGFP and follow their differentiation into CD4(+) and CD8(+) single positive thymocytes in human thymic organ cultures. Lentivirus-marked cells entered the thymus and were detected in both the cortex and medulla. Pretreatment of the thymus with 2-deoxyguanosine depleted resident thymocytes and significantly increased the percentage of EGFP(+) thymocytes. High frequency gene transfer into CD34(+) cells and maintained expression throughout differentiation allows for the in vitro assessment of thymic function. In thymuses ranging in age from fetal to adult we observed EGFP(+) thymocytes at all stages of development suggesting that thymuses of all ages are capable of accepting new T cell progenitors and contributing to the maintenance of T cell homeostasis.

In vitro selection of lentivirus vector-transduced human CD34+ cells.

Human CD34(+) cells with in vivo repopulating potential hold much promise as a target for corrective gene transfer for numerous hematopoietic disorders. However, the efficient introduction of exogenous genes into this small, quiescent population of cells continues to present a significant challenge. To circumvent the need for high initial transduction efficiency of human hematopoietic cells, we investigated a dominant selection strategy using a variant of the DHFR gene (DHFR(L22Y)). For this purpose, we constructed a lentivirus-based bicistronic vector expressing EGFP and DHFR(L22Y). Here we demonstrate efficient in vitro selection and enrichment of lentivirus vector-transduced human CD34(+) hematopoietic cells from fetal liver, umbilical cord blood, bone marrow, and peripheral blood after cytokine mobilization. Growth of transduced human CD34(+) cells in semisolid culture under selective pressure resulted in enrichment of transduced progenitor cells to 99.5% (n = 14). Selection for DHFR(L22Y)(+) cells after expansion of transduced progenitors in liquid culture resulted in a 7- to 13-fold increase in the percentage of marked cells. Thus we have shown that transduced human hematopoietic cells may be effectively enriched in vitro by dominant selection, suggesting that development of such strategies holds promise for future in vivo application.

Role of platelet-derived growth factor in allograft vasculopathy.

OBJECTIVE: To test the hypothesis that platelet-derived growth factor (PDGF) accelerates the formation of allograft vascular disease. SUMMARY BACKGROUND DATA: Allograft vasculopathy, characterized by myointimal hyperplasia of the coronary arteries in the transplanted heart, is the most common cause of late graft failure and death in heart transplant recipients. The cause of the process is unclear, and no treatment exists. PDGF has been implicated in alterations in vascular endothelial biology and in vascular restenosis, but the role of PDGF in allograft vasculopathy has not been explored. METHODS: An orthotopic heart transplant model was established in the rat mismatched at one class II locus using the PVGR8 and PVGR23 strains. No immunosuppressive regimen was used. Six treatment groups (PDGF-A, PDGF-A antibody, and PDGF-A receptor antibody) using 10 rats per group were examined. An untreated group of 10 rats manifesting chronic rejection as well as the native hearts were used as controls. PDGF-A at 1 ng/dL (10 rats) or 10 ng/dL (10 rats) was administered intraperitoneally to each transplant group. Similar groups were treated with PDGF-A antibody and PDGF-A receptor antibody. The animals were killed after 50 days; transplanted and native hearts were removed and coronary arteries were examined morphometrically. Smooth muscle proliferation was confirmed by immunohistochemistry. Statistical analysis was performed using multivariate analysis of variance. RESULTS: Coronary myointimal hyperplasia was seen in the chronic rejection group. The PDGF-A groups showed significant myointimal hyperplasia. Administration of PDGF-A antibody did not attenuate the process. Administration of PDGF-A receptor antibody at 1 ng/dL resulted in reduction of the hyperplasia, and 10 ng/dL significantly attenuated the process. CONCLUSIONS: This study establishes a cause-and-effect relation between PDGF-A and coronary myointimal hyperplasia in the rat transplant model. Blockade of the PDGF-A receptor clearly attenuates the process, indicating a potential mode of therapy to be explored.

Anal duct carcinoma: case report and review of the literature.

This report details the clinical course of two patients with true anal duct carcinoma. The incidence of this malignancy is low. The tissues of origination are the glands of the anal duct. The features that differentiate this tumor from the usual rectal carcinoma are prominent ductal structures, abundant mucin production with organized mucinous pools, and infiltration into the perirectal soft tissue. The clinical management of anal duct carcinoma remains a surgical challenge. The extent of surgical resection must be radical because of the infiltrative nature of the tumor. This report describes treatment of two patients with anal duct carcinoma. The first patient was a black woman with no previous history of rectal disease. Her operative procedure was an abdominoperineal resection with posterior vaginectomy. Nine months after initial surgery a local recurrence was resected. The second patient was a white man with a previous history of hemorrhoidectomy and anal fissure. He underwent an abdominoperineal resection but had positive dermal skin margins on permanent sections despite wide perirectal soft tissue resection. A secondary resection with confirmed clear margins of the skin was performed 2 weeks postoperatively. One management aspect of anal duct carcinoma that needs emphasis is the need for wide local excision of the perirectal soft tissues.

Human cord blood CD34+CD38- cell transduction via lentivirus-based gene transfer vectors.

The efficient transfer and sustained expression of a transgene in human hematopoietic cells with in vivo repopulating potential would provide a significant advancement in the development of protocols for the treatment of hematopoietic diseases. Recent advances in the ability to purify and culture hematopoietic cells with the CD34+CD38- phenotype and with in vivo repopulating potential from human umbilical cord blood provide a direct means of testing the ability of transfer vectors to transduce these cells. Here we demonstrate the efficient transduction and expression of enhanced green fluorescent protein (EGFP) in human umbilical cord-derived CD34+CD38- cells, without prestimulation, using a lentivirus-based gene transfer system. Transduced CD34+CD38- cells cultured in serum-free medium supplemented with SCF, Flt-3, IL-3, and IL-6 maintained their surface phenotype for 5 days and expressed readily detectable levels of the transgene. The average transduction efficiency of the CD34+CD38- cells was 59 +/- 7% as determined by flow cytometry. Erythroid and myeloid colonies derived from transduced CD34+CD38- cells were EGFP positive at a high frequency (66 +/- 9%). In contrast, a murine leukemia virus-based vector transduced the CD34+CD38- cells at a low frequency (<4%). These results demonstrate the utility of lentiviral-based gene transfer vectors in the transduction of primitive human hematopoietic CD34+CD38- cells.