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.

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.

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.

Chromosomes and causation of human cancer and leukemia. XX. Banding patterns of primary tumors.

Banding techniques were used in the study of the chromosomes of primary tumors from 16 patients with various types of cancer. The initial analysis with conventional Giemsa staining revealed chromosome abnormalities in 13 of the 16 tumors. Eleven of these 13 tumors and 2 of the 3 with normal karyotypes were reexamined with Q-, G-, and C-banding techniques: The 2 tumors were conventionally stained normal karyotypes were found to have no abnormalities. Nine of the tumors wre characterized by numerical changes only and 4 by both numerical and structural abnormalities. In 11 tumors, excessive chromosomes, identified with banding techniques, were usually found in the following groups (number of tumors involved is shown in parentheses): No. 5 (5), No. 8 (6), No. 11 (5), no. 13 (5), and No. 21 (5). The primary tumors examined had hyperdiploid modes; only 4 of these tumors contained marker chromosomes, as opposed to the high frequency of markers in metastatic cancer cells and the presence, usually, of high polidy (near-triploidy or near-tetraploidy). The data suggested that the karyotypic changes in primary cancers consist primarily of numerical changes (hyperploidy), rather infrequent appearance of marker chromosomes, and, when present, only 1 or 2 markers.

Effects of 1,2-symmetrical dimethylhydrazine on jejunocolic transposition in Sprague-Dawley rats.

In this experiment, a segment of the left colon including the upper part of the rectum was transposed to the upper jejunum, and a segment of upper jejunum was transposed to the left colon of the same animal. In another group, the same colon and jejunum segments were transsected and reanastomosed in place. A third group served as a normal control. After a recovery period, weekly s.c. 1,2-symmetrical dimethylhydrazine injections were begun. Each animal received a total of 20 injections at a dose of 20 mg/kg. Five weeks after the last 1,2-symmetrical dimethylhydrazine injection, 15 of 19 (79%) of the animals had one or more tumor(s) in the transposed colon segment, while none had tumor in the transposed jejunal segment. Transsected and reanastomosed animals showed the same distribution of tumors as did the normal control animals. All three groups had tumors at other sites in the colon and rectum. In addition, about 20% had tumors of the duodenojejunal area. These data indicate that the colonic mucosa is the primary target for the carcinogenic effect of 1,2-symmetrical dimethylhydrazine, independent of other variables such as the fecal stream.

Genetics of colon carcinogenesis in mice treated with 1,2-dimethylhydrazine.

Genetic analysis of colon tumor induction by symmetrical 1,2-dimethylhydrazine (DMH) was undertaken in F1, F2, and reciprocal backcross hybrids derived from a cross between two inbred mouse strains, the 100% susceptible ICR/Ha and completely resistant C57BL/Ha. Mice, 12 to 14 weeks old, received 22 successive weekly s.c. injections of 0.35% aqueous solution of DMH buffered to pH 6.5. A dose of 15 mg/kg/mouse/week produced invasive colon adenocarcinomas in all ICR/Ha males and females (60 of 60) within 22 weeks. None of the 90 C57BL/Ha mice developed DMH tumors during 44 weeks of observation. Susceptibility to the carcinogen was dominant, as indicated by 100% colon tumor incidence in reciprocal ICR/Ha X C57BL/HaF1 hybrids (68 of 68) and in the susceptible backcross ICR/Ha X F1 (42 of 42). Tumor yield in F2 hybrids (94 of 120) was 78%, which is in close agreement with the 3:1 ratio expected if a single dominant DMH susceptibility gene is inherited via the F1 from the ICR/Ha grandparent. Likewise, tumor yield in resistant backcross mice of genotype C57BL/Ha X F1 (46 of 117) is not out of line with the anticipated 1:1 ratio in the latter type of test hybrids. Tests with five isozyme markers and two coat color genes have tentatively ruled out linkage of DMH susceptibility on seven autosomes. The 47% tumor incidence among 57 male resistant backcross hybrids, regardless of whether their single X chromosome was inherited from the ICR/Ha or C57BL/Ha strain, provides evidence against sex linkage.

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.

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.

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.

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.

Efficient transduction of human lymphocytes and CD34+ cells via human immunodeficiency virus-based gene transfer vectors.

The development of gene transfer systems for the efficient transduction of human primary cells including lymphocytes and CD34+ cells is a significant step in the advancement of gene therapy and cell marking protocols. Efficient gene transfer systems also represent useful tools for basic research. Here we show that human primary lymphocytes and CD34+ cells can be efficiently transduced using a VSV-G pseudotyped HIV-1-based gene transfer system. The enhanced green fluorescent protein (EGFP) was chosen as the marker transgene, because it can be easily visualized and quantitated using fluorescence microscopy and flow cytometry, thus eliminating the need for selection or PCR to score transduction. Vectors produced with this system did not generate replication-competent retroviruses (RCRs) and efficiently transduced human cell lines (40-90%), PBMCs (60%), mobilized CD34+ cells (39%), and CD34+ cells from umbilical cord blood (60%) as measured by flow cytometry. Cells treated with AZT prior to infection did not express EGFP, ruling out passive protein or plasmid DNA transfer. This was further confirmed in methylcellulose cultures, where expression in myeloid and erythroid colonies was maintained for at least 3 weeks. In addition, this HIV-based vector was able to efficiently transduce freshly isolated, not-prestimulated CD34+ cells (70% EGFP positive) in serum-free medium. Under these same conditions, a Moloney murine leukemia virus-based vector failed to transduce not-prestimulated CD34+ cells. These characteristics make this gene transfer system an excellent choice for both basic science and possible gene therapy applications.

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.

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.

Quality assurance of clinical data: internal monitoring: patient and study management at the clinic.

Adquate methods to assure the quality of data collected at the clinic need to be developed. A full understanding of the limitations of physicians as information processors and reasonable performance expectations for physicians during peak information periods will result in concentrated planning for patient visits and will limit the data that must be collected at the clinic. It is mandatory for each clinical research project that protocol treatment take into account the question of variable provider follow-up versus constant provider follow-up. It is also imperative that all clinical research providers receive special training, testing, and follow-up evaluation. The prime responsibility for the overall conduct of clinical research rests with the principal investigator. A monitoring tool that should be more fully used is the informed patient.

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.

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.

Efficacy of an intraperitoneal antibiotic to reduce the incidence of infection in the trauma patient: a prospective, randomized study.

BACKGROUND: Antibiotic therapy in patients with blunt trauma remains an area of investigation. This study was undertaken in trauma patients evaluated with diagnostic peritoneal lavage to determine the effect of an intraperitoneal antibiotic on the following factors: infectious complications, length of hospital stay, and mortality. METHODS: A prospective, randomized double-blinded study compared using either 500 mg of intraperitoneal kanamycin or a saline control in 69 adult trauma patients requiring diagnostic peritoneal lavage was conducted over a 24-month period. Advanced trauma life support indications for performing diagnostic peritoneal lavage were used. Patients were randomized to receive 50 mL of solution intraperitoneally through a lavage catheter and were evaluated for all septic complications, length of hospital stay, and outcome. RESULTS: Over a 24-month period, 40 patients received kanamycin, and 29 patients received a placebo. Of patients receiving kanamycin, 27.5 percent experienced infectious complications compared to 65.5 percent of the control patients (p = 0.001, chi-square analysis). The average length of stay in the intensive care unit was 4.18 days in the kanamycin group and 6.96 days in the control group (p = 0.04, chi-square analysis). The average length of stay was 12.32 days for patients receiving kanamycin and 17.36 days for the control group (p = 0.03, chi-square analysis). The mortality rate for each group was 13 percent. CONCLUSIONS: Intraperitoneal kanamycin given to trauma patients requiring diagnostic peritoneal lavage within the first three hours following injury reduces the incidence of infectious complications and shortens intensive care unit and hospital stay.