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Introduction: Locally implanted antibiotic-eluting carriers may be a valuable adjuvant to the management of prosthetic joint infections. Aim: to assess local and plasma antibiotic concentrations as well as cumulative antibiotic urine excretion associated with clinical use of a gentamicin - or vancomycin-loaded mineral composite antibiotic carrier.Methods: 32 patients (male/female=19/13, mean age=56; 21-82 years) were prospectively followed after implantation of gentamicin (n=11), vancomycin (n=15), or a combination (n=7), using an antibiotic carrier (CERAMENT™|G or CERAMENT™|V, mean amount 11 (3-20) mL) during resection arthroplasty of the hip/knee. We measured antibiotic concentrations in plasma (1h, 3h, 24h, 48h and 72h post-implantation), urine (24h, 48h and 72h post-implantation) and in drain (n=15).Results: We observed low antibiotic concentrations in plasma (Gentamicin: 0.33 mg/L (95%-CI: 0.25-0.44) and vancomycin: 1.33 mg/L (95%-CI: 1.02-1.66)) and high concentrations in drain (Gentamicin: mean 57.8 mg/L (95%-CI: 45.8-69.7) and vancomycin: mean 234.4 mg/L (95%-CI: 198.9-269.7)). Use of a drain was associated with a statistically significant reduction in vancomycin urine excretion (55.6% (95% CI: 36.45-74.92) to 28.71% (95% CI: 13.07-44.35), p=0.042). A similar trend was observed for gentamicin (34.17% (95% CI: 24.62-43.72) to 16.22% (95% CI: 0-33.86), p=0.078).Conclusions: CERAMENT™G/V was associated with safe plasma concentrations and high local concentrations above minimum inhibitory concentration. Installation of a surgical drain results in removal of a substantial amount of antibiotics and reduces antibiotic urine excretion.

Objectives: The objective is to present the antibiotic elution from a locally implanted gentamicin containing hydroxyapatite and calcium sulphate bone substitute with an extended follow up of 30 days.We also compare the pharmacokinetics of the ceramic bone substitute with a published study on gentamicin containing poly (methyl methacrylate) (PMMA) bone cement used in primary total hip arthroplasty.Methods: Gentamicin release was measured in the urine for a month and the serum for 4 days in 10 patients operated for trochanteric hip fractures and 10 patients in uncemented hip revisions. 17 patients were followed up at one year and 3 patients at 6 months.Results and Discussion: The gentamicin concentrations measured in serum were low and approximately 100 times less than in urine during the first days, indicating high local concentrations at the implant site. The elution from the biphasic bone substitute showed a stronger burst and higher gentamicin concentrations for the first week compared to that reported for PMMA used in hip arthroplasty. Also, for the bone substitute a complete gentamicin elution was obtained after 30 days, while for the PMMA cement sub-inhibitory MIC levels of gentamicin were still present in urine 60 days past surgery. No infections were detected.Conclusions: A new biphasic bone substitute containing antibiotics could potentially be used to prevent infection in patients treated for trochanteric hip fractures or uncemented hip revisions. The gentamicin elution from the bone substitute is efficient with high initial local gentamicin concentrations and complete release at 30 days.

In a prospective study, we evaluated the adaptive bone remodelling pattern of the distal femur using dual energy X-ray absorptiometry (DEXA) after total knee arthroplasty (TKA). Eleven patients underwent TKA with insertion of an Interax total knee system (Howmedica) because of primary osteoarthrosis of the knee. All patients received an uncemented femoral component with an uncoated cast-mesh ingrowth surface. The components had a 20-mm long central peg placed anteriorly just behind the anterior flange. DEXA scans were performed postoperatively at 2 weeks and at 3, 6, 12 and 24 months in the mediolateral plane of the distal femur and in the anteroposterior plane of the distal tibia and fibula. Compared to the immediate 2-week postoperative bone mineral density (BMD) value, we found no significant changes in BMD in 4 regions of interest (ROI) in the distal femur during the 2-year follow-up at 3 months. A temporary but significant decrease in BMD of 11.2% (95% CL, -17.3% to -5.0%) was observed in the most distal ROI just posterior to the fixation peg. Bone mineral content (BMC) of the distal tibia and fibula showed no significant changes in the operated and contralateral legs during the follow-up. The adaptive bone remodelling of the distal femur after TKA using an uncemented femoral component not previously examined by DEXA induced only a very limited and temporary loss of bone mineral. [ABSTRACT FROM AUTHOR]

Background and Purpose: Revision total hip arthroplasty (rTHA) is a challenging procedure especially in the presence of severe bone loss where implant fixation is compromised. The aim of this study was to evaluate implant survival, clinical outcome, and midterm results in a group of complex patients after femoral revision using an uncemented modular implant design. Patients and Methods: We performed a retrospective study including 100 patients (101 hips) treated with revision THA using an uncemented modular implant design. We identified 51 hips as Paprosky types I-II and 50 hips as Paprosky III-IV bone defects. We reviewed operative reports and radiographs. Patients underwent a clinical examination to assess the Harris Hip Score (HHS) and completed patient-reported outcome measures (PROM), including the Oxford Hip Score (OHS) and the EQ-5D Visual Analog Scale (VAS). Minimum follow-up was 2 years (average, 5.8 years; range, 2.0-9.4 years). Results: Among the cases, 46 hips were revised for infection in a 2-stage procedure and 44 hips for aseptic loosening. 11 hips had periprosthetic fractures of Vancouver type B2 or B3. A total of 5 hips required revision with removal of the femoral implant and 11 patients experienced complications resulting in fracture ( n  = 1), dislocation ( n  = 10), and soft tissue revision ( n  = 1). The 5-year implant survival estimated by Kaplan-Meier survival analysis was 95% (95%-CI: 91%-99%). All hips had radiographic evidence of osseointegration and no one with subsidence greater than 5 mm. Additionally, 80% showed radiographic evidence of restoration of proximal femoral bone. Mean HHS was 78. Conclusion: In complex cases of revision THA, using a modular revision femoral system yielded promising results. The 5-year implant survival estimated by Kaplan-Meier survival analysis was 95% (95%-CI: 91%-99%), with all hips demonstrating radiographic evidence of osseointegration and no one with subsidence greater than 5 mm. Notably 80% showed radiographic evidence of restoration of proximal femoral bone., Competing Interests: The authors declare no conflicts of interest., (Copyright © 2024 Sebastian Winther et al.)

Background: Four large community-randomized trials examining universal testing and treatment (UTT) to reduce HIV transmission were conducted between 2012-2018 in Botswana, Kenya, Uganda, Zambia and South Africa. In 2014, the UNAIDS 90-90-90 targets were adopted as a useful metric to monitor coverage. We systematically review the approaches used by the trials to measure intervention delivery, and estimate coverage against the 90-90-90 targets. We aim to provide in-depth understanding of the background contexts and complexities that affect estimation of population-level coverage related to the 90-90-90 targets., Methods: Estimates were based predominantly on "process" data obtained during delivery of the interventions which included a combination of home-based and community-based services. Cascade coverage data included routine electronic health records, self-reported data, survey data, and active ascertainment of HIV viral load measurements in the field., Results: The estimated total adult populations of trial intervention communities included in this study ranged from 4,290 (TasP) to 142,250 (Zambian PopART Arm-B). The estimated total numbers of PLHIV ranged from 1,283 (TasP) to 20,541 (Zambian PopART Arm-B). By the end of intervention delivery, the first-90 target (knowledge of HIV status among all PLHIV) was met by all the trials (89.2%-94.0%). Three of the four trials also achieved the second- and third-90 targets, and viral suppression in BCPP and SEARCH exceeded the UNAIDS target of 73%, while viral suppression in the Zambian PopART Arm-A and B communities was within a small margin (~ 3%) of the target., Conclusions: All four UTT trials aimed to implement wide-scale testing and treatment for HIV prevention at population level and showed substantial increases in testing and treatment for HIV in the intervention communities. This study has not uncovered any one estimation approach which is superior, rather that several approaches are available and researchers or policy makers seeking to measure coverage should reflect on background contexts and complexities that affect estimation of population-level coverage in their specific settings. All four trials surpassed UNAIDS targets for universal testing in their intervention communities ahead of the 2020 milestone. All but one of the trials also achieved the 90-90 targets for treatment and viral suppression. UTT is a realistic option to achieve 95-95-95 by 2030 and fast-track the end of the HIV epidemic., (© 2022. The Author(s).)

Background: Haemophilic arthropathy is a chronic and debilitating joint disease caused by recurrent spontaneous joint bleeds in patients with haemophilia. Understanding how characteristics of individual joint bleeds relate to the subsequent development of arthropathy could improve management and prevention of this joint disease. Here, we aimed to explore relations between joint bleed characteristics and development of bone pathology in a mouse model of haemophilic arthropathy by using novel in vivo imaging methodology., Methods: We characterised induced knee bleeds in a murine model of haemophilic arthropathy by quantitative in vivo fluorescence molecular tomography (FMT) and by measurements of changes in the diameter of the injured knee. Wild-type mice and non-injured haemophilic mice acted as controls. Development of arthropathy was characterised by post mortem evaluation of bone pathology by micro-CT 14 days after bleed-induction. In an in vitro study, we assessed the effect of blood on the quantification of fluorescent signal with FMT., Results: In most injured haemophilic mice, we observed significant loss of trabecular bone, and half of the mice developed pathological bone remodelling. Development of pathological bone remodelling was associated with significantly increased fluorescent signal and diameter of the injured knee just 1 day after induction of the bleed. Further, a correlation between the fluorescent signal 1 day after induction of the bleed and loss of trabecular bone reached borderline significance. In the in vitro study, we found that high concentrations of blood significantly decreased the fluorescent signal., Conclusion: Our results add novel insights on the pathogenesis of haemophilic arthropathy and underline the importance of the acute phase of joint bleeds for the subsequent development of arthropathy.

Introduction: Alzheimer disease (AD) is a neurodegenerative disease characterised by progressive dementia associated with global cognitive dysfunction., Methods: We conducted a systematic review and meta-analysis of clinical trials evaluating omega-3 supplementation in patients with AD., Objective: To determine if there is scientific evidence of the effectiveness of omega-3 supplementation in improving cognitive function in patients with AD., Search Strategy: We included only randomised controlled trials (RCTs) from the following databases: Medline, Cochrane Central, Cinahl, and LILACS. An electronic search was also conducted using Google Scholar., Study Selection: Six articles met the eligibility criteria. The risk of bias was assessed following the Cochrane method., Conclusion: There is no consistent evidence to support the effectiveness of omega-3 supplementation in improving cognitive function in AD patients in the short and medium term., (Copyright © 2017 Sociedad Española de Neurología. Publicado por Elsevier España, S.L.U. All rights reserved.)

Background: Survival predictions before surgery for metastatic bone disease in the extremities (based on statistical models and data of previous patients) are important for choosing an implant that will function for the remainder of the patient's life. The 2008-SPRING model, presented in 2016, enables the clinician to predict expected survival before surgery for metastatic bone disease in the extremities. However, to maximize the model's accuracy, it is necessary to maintain and update the patient database to refit the prediction models achieving more accurate calibration., Questions/purposes: The purposes of this study were (1) to refit the 2008-SPRING model for prediction of survival before surgery for metastatic bone disease in the extremities with a more modern cohort; and (2) to evaluate the performance of the refitted SPRING model in a population-based cohort of patients having surgery for metastatic bone disease in the extremities., Methods: We produced the 2013-SPRING model by adding to the 2008-SPRING model (n = 130) a cohort of patients from a consecutive institutional database of patients who underwent surgery for bone metastases in the extremities with bone resection and reconstruction between 2009 and 2013 at a highly specialized surgical center in Denmark (n = 140). Currently the model is only available as the nomogram fully available in the current article, which is sufficient to use in daily clinical work, but we are working on making the tool available online. As such, the 2013-SPRING model was produced using a consecutive cohort of patients (n = 270) treated during an 11-year period (2003-2013) called the training cohort, all treated with bone resection and reconstruction. We externally validated the 2008-SPRING and the 2013-SPRING models in a prospective cohort (n = 164) of patients who underwent surgery for metastatic bone disease in the extremities from May 2014 to May 2016, called the validation cohort. The validation cohort was identified from a cross-section of the Danish population who were treated for metastatic lesions (using endoprostheses and internal fixation) in the extremities at five secondary surgical centers and one highly specialized surgical center. This cross-section is representative of the Danish population and no patients were treated outside the included centers as a result of public healthcare settings. The indications for surgery for training and the validation cohort were pathologic fracture, impending fracture, or intractable pain despite radiation. Exact date of death was known for all patients as a result of the Danish Civil Registration System and no loss to followup existed. In the training cohort, 150 patients (out of 270 [56%]) and in the validation cohort 97 patients (out of 164 [59%]) died of disease within 1 year postoperatively. The 2013 model did not differ from the 2008 model and included hemoglobin, complete fracture/impending fracture, visceral and multiple bone metastases, Karnofsky Performance Status, and the American Society of Anesthesiologists score and primary cancer. The models were evaluated by area under the receiver operating characteristic curve (AUC ROC) and Brier score (the lower the better)., Results: The 2013-SPRING model was successfully refitted with a cohort using more patients than the 2008-SPRING model. Comparison of performance in external validation between the 2008 and 2013-SPRING models showed the AUC ROC was increased by 3% (95% confidence interval [CI], 0%-5%; p = 0.027) and 2% (95% CI, 0%-4%; p = 0.013) at 3-month and 6-month survival predictions, respectively, but not at 12 months at 1% (95% CI, 0%-3%; p = 0.112). Brier score was improved by -0.018 (95% CI, -0.032 to -0.004; p = 0.011) for 3-month, -0.028 (95% CI, -0.043 to -0.0123; p < 0.001) for 6-month, and -0.014 (95% CI, -0.025 to -0.002; p = 0.017) for 12-month survival prediction., Conclusions: We improved the SPRING model's ability to predict survival after surgery for metastatic bone disease in the extremities. As such, the refitted 2013-SPRING model gives the surgeon a tool to assist in the decision-making of a surgical implant that will serve the patient for the remainder of their life. The 2013-SPRING model may provide increased quality of life for patients with bone metastasis because potential implant failures can be minimized by precise survival prediction preoperatively and the model is freely available and ready to use from the current article., Level of Evidence: Level I, diagnostic study.

Essentials N8-GP is an extended half-life recombinant factor VIII (FVIII) for the treatment of hemophilia A. Subcutaneous (SC) FVIII dosing might reduce the treatment burden of prophylaxis. SC N8-GP has a favorable PK profile in animal models and disappears from skin injection sites. Combined animal (SC) and clinical (IV) data suggest that daily SC dosing may provide prophylaxis., Summary: Background N8-GP is an extended half-life recombinant factor VIII (FVIII) for the treatment of hemophilia A. Subcutaneous administration of FVIII may reduce the treatment burden of prophylaxis; however, standard FVIII products have low bioavailability after subcutaneous dosing in animals. Objective To evaluate the pharmacokinetics, effectiveness and local distribution of subcutaneously administered N8-GP in preclinical models and predict the human pharmacokinetic (PK) profile. Methods The pharmacokinetics of subcutaneously administered N8-GP were evaluated in FVIII knockout (F8-KO) mice and cynomolgus monkeys; a human PK prediction model in hemophilia A patients was developed. The hemostatic effect was evaluated in a tail vein bleeding model in F8-KO mice. The injection-site distribution and absorption of subcutaneously administered N8-GP were assessed in F8-KO mice by the use of temporal fluorescence imaging and immunohistochemistry. Results Subcutaneously administered N8-GP had a bioavailability, a first-order absorption rate and a half-life, respectively, of 24%, 0.094 h -1 and 14 h in F8-KO mice, and 26%, 0.33 h -1 and 15 h in cynomolgus monkeys. A dose-dependent effect of subcutaneously administered N8-GP on blood loss was observed in mice. A minimal amount of N8-GP was detected at the injection site 48-72 h after single or multiple dose(s) in F8-KO mice. Subcutaneously administered N8-GP was localized to the skin around the injection site, with time-dependent disappearance from the depot. PK modeling predicted that subcutaneously administered N8-GP at a daily dose of 12.5 IU kg -1 will provide FVIII trough levels of 2.5-10% in 95% of patients with severe hemophilia A. Conclusions Subcutaneously administered N8-GP may provide effective hemophilia A prophylaxis. A phase I clinical trial is underway to investigate this possibility., (© 2018 Novo Nordisk A/S. Journal of Thombosis and Haemostasis published by Wiley Periodicals, Inc. on behalf of International Society of Thombosis and Haemostasis.)

Bretschneider (histidine-tryptophan-ketoglutarate) solution with its high histidine concentration (198 mM) is one of many cardioplegic solutions, which are routinely used for cardiac arrest. The aim of this study was to evaluate the physiological biochemical degradation of administered histidine to histamine and its major urinary metabolite N-methylimidazole acetic acid. A total number of thirteen consecutive patients scheduled for elective isolated coronary artery bypass grafting with cardiopulmonary bypass were enrolled in the prospective observational designed study at the Department of Thoracic and Cardiovascular Surgery between 04/2016 and 06/2016. Patients received 1.7 l Bretschneider solution on average. Before and at the end of operation as well as in the postoperative course, urine samples gathered from the urinary catheter bag were analyzed. During the operative period, urinary histidine concentration significantly increased from 29 micromol/mmol creatinine to 9,609 micromol/mmol creatinine. Postoperatively, histidine excretion reduced while histamine as well as N-methylimidazole acetic acid excretion rose significantly. Patients showed elevated levels of histidine, histamine as well as N-methylimidazole acetic acid in urine, but no unmanageable hemodynamic instability possibly arising from the histamine's biological properties. Chemically modified histidine might reduce uptake and metabolization while maintaining the advantages of buffer capacity.

Background: With the recognition of the need for research capacity strengthening for advancing health and development, this research capacity article explores the use of technology enhanced learning in the delivery of a collaborative postgraduate blended Master's degree in Malawi. Two research questions are addressed: (i) Can technology enhanced learning be used to develop health research capacity?, and: (ii) How can learning content be designed that is transferrable across different contexts?, Methods: An explanatory sequential mixed methods design was adopted for the evaluation of technology enhanced learning in the Masters programme. A number of online surveys were administered, student participation in online activities monitored and an independent evaluation of the programme conducted., Results: Remote collaboration and engagement are paramount in the design of a blended learning programme and support was needed for selecting the most appropriate technical tools. Internet access proved problematic despite developing the content around low bandwidth availability and training was required for students and teachers/trainers on the tools used. Varying degrees of engagement with the tools used was recorded, and the support of a learning technologist was needed to navigate through challenges faced., Conclusion: Capacity can be built in health research through blended learning programmes. In relation to transferability, the support required institutionally for technology enhanced learning needs to be conceptualised differently from support for face-to-face teaching. Additionally, differences in pedagogical approaches and styles between institutions, as well as existing social norms and values around communication, need to be embedded in the content development if the material is to be used beyond the pilot resource-intensive phase of a project.

The antibiotic trimethoprim acts as a folate antagonist. Since trophoblasts are very sensitive to drugs that interfere with the folic acid cycle and thereby inhibit DNA synthesis, use of trimethoprim during the first trimester could be associated with miscarriage. A nationwide cohort study including all women in Denmark with a registered pregnancy between 1997 and 2005 was conducted. We used nationwide registers to identify all women giving birth, having a record of miscarriage or induced abortion. Data on exposure to trimethoprim were obtained from the National Prescription Register. Cox proportional hazard regression analysis with exposure to trimethoprim as a time-dependent variable was used to estimate the risk of miscarriage. The adjusted hazard ratio of having a miscarriage after exposure to trimethoprim in the first trimester compared to non-exposure was 2∙04 (95% confidence interval 1∙43-2∙91). Our results indicate that trimethoprim exposure in the first trimester is associated with a doubling of the hazard of miscarriage.

Accumulating evidence suggests that a subpopulation of breast cancer cells, referred to as cancer stem cells (CSCs), have the ability to propagate a tumor and potentially seed new metastases. Furthermore, stimulation of an epithelial-to-mesenchymal transition by factors like transforming growth factor-β (TGFβ) is accompanied with the generation of breast CSCs. Previous observations indicated that bone morphogenetic protein-7 (BMP7) antagonizes the protumorigenic and prometastatic actions of TGFβ, but whether BMP7 action is mechanistically linked to breast CSCs has remained elusive. Here, we have studied the effects of BMP7, BMP2 and a BMP2/7 heterodimer on the formation of human breast CSCs (ALDH(hi)/CD44(hi)/CD24(-/low)) and bone metastases formation in a preclinical model of intra-cardiac injection of MDA-MB-231 cells in athymic nude (Balb/c nu/nu) mice. The BMP2/7 heterodimer was the most efficient stimulator of BMP signaling and very effectively reduced TGFβ-driven Smad signaling and cancer cell invasiveness. The tested BMPs-particularly the heterodimeric BMP2/7-strongly reduced the size of the ALDH(hi)/CD44(hi)/CD24(-/low) CSC subpopulation. In keeping with these in vitro observations, pretreatment of cancer cells with BMPs for 72 h prior to systemic inoculation of the cancer cells inhibited the formation of bone metastases. Collectively, our data support the notion that breast CSCs are involved in bone metastasis formation and describe heterodimeric BMP2/7 as a powerful TGFβ antagonist with anti-metastatic potency.

Ancient autophagy pathways are emerging as key defense modules in host eukaryotic cells against microbial pathogens. Apart from actively eliminating intracellular intruders, autophagy is also responsible for cell survival, for example by reducing the deleterious effects of endoplasmic reticulum stress. At the same time, autophagy can contribute to cellular suicide. The concurrent engagement of autophagy in these processes during infection may sometimes mask its contribution to differing pro-survival and pro-death decisions. The importance of autophagy in innate immunity in mammals is well documented, but how autophagy contributes to plant innate immunity and cell death is not that clear. A few research reports have appeared recently to shed light on the roles of autophagy in plant-pathogen interactions and in disease-associated host cell death. We present a first attempt to reconcile the results of this research.

Programmed cell death (PCD) is an integral part of plant development and of responses to abiotic stress or pathogens. Although the morphology of plant PCD is, in some cases, well characterised and molecular mechanisms controlling plant PCD are beginning to emerge, there is still confusion about the classification of PCD in plants. Here we suggest a classification based on morphological criteria. According to this classification, the use of the term 'apoptosis' is not justified in plants, but at least two classes of PCD can be distinguished: vacuolar cell death and necrosis. During vacuolar cell death, the cell contents are removed by a combination of autophagy-like process and release of hydrolases from collapsed lytic vacuoles. Necrosis is characterised by early rupture of the plasma membrane, shrinkage of the protoplast and absence of vacuolar cell death features. Vacuolar cell death is common during tissue and organ formation and elimination, whereas necrosis is typically found under abiotic stress. Some examples of plant PCD cannot be ascribed to either major class and are therefore classified as separate modalities. These are PCD associated with the hypersensitive response to biotrophic pathogens, which can express features of both necrosis and vacuolar cell death, PCD in starchy cereal endosperm and during self-incompatibility. The present classification is not static, but will be subject to further revision, especially when specific biochemical pathways are better defined.

Transforming growth factor (TGF)-beta can suppress and promote breast cancer progression. How TGF-beta elicits these dichotomous functions and which roles the principle intracellular effector proteins Smad2 and Smad3 have therein, is unclear. Here, we investigated the specific functions of Smad2 and Smad3 in TGF-beta-induced responses in breast cancer cells in vitro and in a mouse model for breast cancer metastasis. We stably knocked down Smad2 or Smad3 expression in MDA-MB-231 breast cancer cells. The TGF-beta-induced Smad3-mediated transcriptional response was mitigated and enhanced by Smad3 and Smad2 knockdown, respectively. This response was also seen for TGF-beta-induced vascular endothelial growth factor (VEGF) expression. TGF-beta induction of key target genes involved in bone metastasis, were found to be dependent on Smad3 but not Smad2. Strikingly, whereas knockdown of Smad3 in MDA-MB-231 resulted in prolonged latency and delayed growth of bone metastasis, Smad2 knockdown resulted in a more aggressive phenotype compared with control MDA-MB-231 cells. Consistent with differential effects of Smad knockdown on TGF-beta-induced VEGF expression, these opposing effects of Smad2 versus Smad3 could be directly correlated with divergence in the regulation of tumor angiogenesis in vivo. Thus, Smad2 and Smad3 differentially affect breast cancer bone metastasis formation in vivo.

Progressive kidney fibrosis precedes end-stage renal failure in up to a third of patients with diabetes mellitus. Elevated intra-renal transforming growth factor-beta (TGF-beta) is thought to underlie disease progression by promoting deposition of extracellular matrix and epithelial-mesenchymal transition. GW788388 is a new TGF-beta type I receptor inhibitor with a much improved pharmacokinetic profile compared with SB431542. We studied its effect in vitro and found that it inhibited both the TGF-beta type I and type II receptor kinase activities, but not that of the related bone morphogenic protein type II receptor. Further, it blocked TGF-beta-induced Smad activation and target gene expression, while decreasing epithelial-mesenchymal transitions and fibrogenesis. Using db/db mice, which develop diabetic nephropathy, we found that GW788388 given orally for 5 weeks significantly reduced renal fibrosis and decreased the mRNA levels of key mediators of extracellular matrix deposition in kidneys. Our study shows that GW788388 is a potent and selective inhibitor of TGF-beta signalling in vitro and renal fibrosis in vivo.

1-Bromopropane (1-BP) has been marketed as an alternative for ozone depleting solvents and suspect carcinogens and is in aerosol products, adhesives and solvents used for metal, precision and electronics cleaning. Toxicity of 1-BP is poorly understood, but it may be a neurologic, reproductive and hematologic toxin. Sparse exposure information prompted this exposure assessment study using air sampling, and measurement of urinary metabolites. Mercapturic acid conjugates are excreted in urine from 1-BP metabolism involving removal of bromide (Br) from the propyl group. One research objective was to evaluate the utility of urinary Br analysis for assessing 1-BP exposure using a relatively inexpensive, commercially available method. Complete 48 h urine specimens were obtained from 30 workers on two consecutive days at two facilities using 1-BP adhesives to construct polyurethane foam seat cushions and from seven unexposed control subjects. All of the workers' urine was collected into composite samples representing three daily time intervals (at work; after work but before bedtime; and upon wake-up) and analyzed for Br ion by inductively coupled plasma-mass spectrometry. Full-shift breathing zone samples were collected for 1-BP on Anasorb carbon molecular sieve sorbent tubes and analyzed by gas chromatography-flame ionization detection via NIOSH method 1025. Geometric mean (GM) breathing zone concentrations of 1-BP were 92 parts per million (p.p.m.) for adhesive sprayers and 11 p.p.m. for other jobs. For sprayers, urinary Br concentrations ranged from 77 to 542 milligrams per gram of creatinine [mg (g-cr)(-1)] at work; from 58 to 308 mg (g-cr)(-1) after work; and from 46 to 672 mg (g-cr)(-1) in wake-up samples. Pre-week urinary Br concentrations for sprayers were substantially higher than for the non-sprayers and controls, with GMs of 102, 31 and 3.8 mg (g-cr)(-1), respectively. An association of 48 h urinary Br concentration with 1-BP exposure was statistically significant (r(2) = 0.89) for all jobs combined. This study demonstrates that urinary elimination is an important excretion pathway for 1-BP metabolism, and Br may be a useful biomarker of exposure.

Objective: To investigate whether a hypo-energetic low-fat diet is superior to a hypo-energetic high-fat diet for the treatment of obesity., Design: Open-label, 10-week dietary intervention comparing two hypo-energetic (-600 kcal/day) diets with a fat energy percent of 20-25 or 40-45., Subjects: Obese (BMI >or=30 kg/m(2)) adult subjects (n = 771), from eight European centers., Measurements: Body weight loss, dropout rates, proportion of subjects who lost more than 10% of initial body weight, blood lipid profile, insulin and glucose., Results: The dietary fat energy percent was 25% in the low-fat group and 40% in the high-fat group (mean difference: 16 (95% confidence interval (CI) 15-17)%). Average weight loss was 6.9 kg in the low-fat group and 6.6 kg in the high-fat group (mean difference: 0.3 (95% CI -0.2 to 0.8) kg). Dropout was 13.6% (n = 53) in the low-fat group and 18.3% (n = 70) in the high-fat group (P=0.001). Among completers, more subjects lost >10% in the low-fat group than in the high-fat group ((20.8%, n = 70) versus (14.7%, n = 46), P = 0.02). Fasting plasma total, low-density lipoprotein- and high-density lipoprotein-cholesterol decreased in both groups, but more so in the low-fat group than in the high-fat group. Fasting plasma insulin and glucose were lowered equally by both diets., Conclusions: The low-fat diet produced similar mean weight loss as the high-fat diet, but resulted in more subjects losing >10% of initial body weight and fewer dropouts. Both diets produced favorable changes in fasting blood lipids, insulin and glucose.

Objective: Fish oil supplement has been proposed as a non-pharmacological strategy to correct the atherogenic lipid profile associated with type 2 diabetes mellitus. However, fish oil may have deleterious effects on lipid peroxidation and glycemic control., Design: In this study, 44 type 2 diabetic patients were randomized to vitamin E standardized (53.6 mg/day) supplementation (capsules) with 4 g daily of either fish oil (n=23) or corn oil (n=21) for 8 weeks preceded by a 4 week run-in period of corn oil supplementation. LDL was isolated by density gradient ultracentrifugation and oxidized in vitro with Cu(2+). As a marker of in vivo oxidation malondialdehyde concentration in LDL (LDL-MDA) was measured., Results: Fish oil reduced both mean lag time (before, 57.8; after, 48.8 min, P<0.001) and mean propagation rate (before, 0.018 DeltaOD/min; after, 0.015 DeltaOD/min, P<0.001), whereas corn oil had no influence on lag time and propagation rate. The changes in lag time and propagation rate differed significantly between fish oil and corn oil treatment. LDL-MDA changes differed borderline significantly between groups (FO, 110.4 pmol/mg protein; CO, 6.7 pmol/mg protein; P=0.057). Fish oil supplementation had no influence on glycemic control as assessed from HbA(1c) and fasting blood glucose., Conclusion: According to our findings, fish oil supplementation leads to increased in vivo oxidation and increased in vitro oxidation susceptibility of LDL particles. More studies are needed to clarify the clinical importance of this finding.

Cell-suspension cultures of Linum flavum L. (Linaceae) synthesize and accumulate aryltetrahydronaphthalene lignans with 6-methoxypodophyllotoxin as the main component. The experimental data indicate that the biosynthesis of 6-methoxypodophyllotoxin occurs via deoxypodophyllotoxin, beta-peltatin, and beta-peltatin-A methyl ether. The enzyme catalyzing the introduction of the hydroxyl group in position 6 is deoxypodophyllotoxin 6-hydroxylase (DOP6H). The enzyme was shown to be a cytochrome P450-dependent monooxygenase by blue-light reversion of carbon monoxide inhibition and inhibition by cytochrome c. DOP6H is a membrane-bound microsomal enzyme with a pH optimum of 7.6 and a temperature optimum of 26 degrees C. Deoxypodophyllotoxin is specifically accepted with an apparent Km of 20 microM and a saturation concentration of 200 microM; 4'-demethyldeoxypodophyllotoxin is the only other tested substrate accepted for hydroxylation. DOP6H predominantly accepts NADPH as electron donor; NADH can only sustain low hydroxylation activities. A synergistic effect of NADPH and NADH is not observed. The enzyme is saturated around 250 microM NADPH; the apparent Km for this substrate is 36 microM.

Hyperphenylalaninemia due to a deficiency of phenylalanine hydroxylase (PAH) is an autosomal recessive disorder caused by >400 mutations in the PAH gene. Recent work has suggested that the majority of PAH missense mutations impair enzyme activity by causing increased protein instability and aggregation. In this study, we describe an alternative mechanism by which some PAH mutations may render PAH defective. Database searches were used to identify regions in the N-terminal domain of PAH with homology to the regulatory domain of prephenate dehydratase (PDH), the rate-limiting enzyme in the bacterial phenylalanine biosynthesis pathway. Naturally occurring N-terminal PAH mutations are distributed in a nonrandom pattern and cluster within residues 46-48 (GAL) and 65-69 (IESRP), two motifs highly conserved in PDH. To examine whether N-terminal PAH mutations affect the ability of PAH to bind phenylalanine at the regulatory domain, wild-type and five mutant (G46S, A47V, T63P/H64N, I65T, and R68S) forms of the N-terminal domain (residues 2-120) of human PAH were expressed as fusion proteins in Escherichia coli. Binding studies showed that the wild-type form of this domain specifically binds phenylalanine, whereas all mutations abolished or significantly reduced this phenylalanine-binding capacity. Our data suggest that impairment of phenylalanine-mediated activation of PAH may be an important disease-causing mechanism of some N-terminal PAH mutations, which may explain some well-documented genotype-phenotype discrepancies in PAH deficiency.

Cytotoxic lignans derived from podophyllotoxin are currently used in cancer chemotherapy. Podophyllotoxin for semi-synthetic derivatization is isolated from the rhizomes of Podophyllum plants growing wild, some of which are counted as endangered species. An alternative source for podophyllotoxin or related lignans may in future be cell cultures derived from different plant species, such as Podophyllum spp or Linum spp. These cell cultures were shown to accumulate considerable amounts of podophyllotoxin or 5-methoxypodophyllotoxin. Optimization of the cell cultivation regime might lead to a renewable source of cytotoxic lignans for medicinal uses. This Mini-Review summarizes the attempts to establish plant cell cultures for the production of podophyllotoxin and related lignans and their optimization towards high levels of these target compounds. It also summarizes the results of studies on the biosynthesis of podophyllotoxin and 5-methoxypodophyllotoxin.

Transposon inactivation of Arabidopsis MAP kinase 4 produced the mpk4 mutant exhibiting constitutive systemic acquired resistance (SAR) including elevated salicylic acid (SA) levels, increased resistance to virulent pathogens, and constitutive pathogenesis-related gene expression shown by Northern and microarray hybridizations. MPK4 kinase activity is required to repress SAR, as an inactive MPK4 form failed to complement mpk4. Analysis of mpk4 expressing the SA hydroxylase NahG and of mpk4/npr1 double mutants indicated that SAR expression in mpk4 is dependent upon elevated SA levels but is independent of NPR1. PDF1.2 and THI2.1 gene induction by jasmonate was blocked in mpk4 expressing NahG, suggesting that MPK4 is required for jasmonic acid-responsive gene expression.

Congenital deafness accounts for about 1 in 1000 infants and approximately 80% of cases are inherited as an autosomal recessive trait. Recently, it has been demonstrated that connexin 26 (GJB2) gene is a major gene for congenital sensorineural deafness. A single mutation (named 35delG) was found in most recessive families and sporadic cases of congenital deafness, among Caucasoids, with relative frequencies ranging from 28% to 63%. We present here the analysis of the 35delG mutation in 3270 random controls from 17 European countries. We have detected a carrier frequency for 35delG of 1 in 35 in southern Europe and 1 in 79 in central and northern Europe. In addition, 35delG was detected in five out of 376 Jewish subjects of different origin, but was absent in other non-European populations. The study suggests either a single origin for 35delG somewhere in Europe or in the Middle East, and the possible presence of a carrier advantage together with a founder effect. The 35delG carrier frequency of 1 in 51 in the overall European population clearly indicates that this genetic alteration is a major mutation for autosomal recessive deafness in Caucasoids. This finding should facilitate diagnosis of congenital deafness and allow early treatment of the affected subjects.

1. A [3H]-resiniferatoxin (RTX) binding assay utilizing rat spinal cord membranes was employed to identify novel vanilloids in a collection of natural products of fungal origin. Of the five active compounds found (scutigeral, acetyl-scutigeral, ovinal, neogrifolin, and methyl-neogrifolin), scutigeral (Ki=19 microM), isolated from the edible mushroom Albatrellus ovinus, was selected for further characterization. 2. Scutigeral induced a dose-dependent 45Ca uptake by rat dorsal root ganglion neurons with an EC50 of 1.6 microM, which was fully inhibited by the competitive vanilloid receptor antagonist capsazepine (IC50=5.2 microM). 3. [3H]-RTX binding isotherms were shifted by scutigeral (10-80 microM) in a competitive manner. The Schild plot of the data had a slope of 0.8 and gave an apparent Kd estimate for scutigeral of 32 microM. 4. Although in the above assays scutigeral mimicked capsaicin, it was not pungent on the human tongue up to a dose of 100 nmol per tongue, nor did it provoke protective wiping movements in the rat (up to 100 microM) upon intraocular instillation. 5. In accord with being non-pungent, scutigeral (5 microM) did not elicit a measurable inward current in isolated rat dorsal root ganglion neurons under voltage-clamp conditions. It did, however, reduce the proportion of neurons (from 61 to 15%) that responded to a subsequent capsaicin (1 microM) challenge. In these neurons, scutigeral both delayed (from 27 to 72 s) and diminished (from 5.0 to 1.9 nA) the maximal current evoked by capsaicin. 6. In conclusion, scutigeral and its congeners form a new chemical class of vanilloids, the triprenyl phenols. Scutigeral promises to be a novel chemical lead for the development of orally active, non-pungent vanilloids.

Objectives: To examine the association of immune cell number and function with occupational exposure to substances contaminated with 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD)., Methods: A cross sectional medical survey. The exposed participants were employed at two chemical plants between 1951 and 1972 in the manufacture of 2,4,5-trichlorophenate and its derivatives. The reference group consisted of people with no occupational exposure to phenoxy herbicides who lived within the communities of the workers. Data from a total of 259 workers and 243 unexposed referents were included in the analysis of immune function. Laboratory tests for immune status included enumeration of circulating leukocyte and lymphocyte populations, proliferative responses of circulating lymphocytes to mitogens and antigens, and serum concentrations of the major immunoglobulins and complement factor C3., Results: The workers had substantial exposure to substances contaminated with TCDD, as indicated by a lipid adjusted mean serum TCDD concentration of 229 ppt compared with a mean of 6 ppt in the unexposed referents. Workers were divided into categories based on their serum TCDD concentration. For all categories except the lowest, with values of serum TCDD comparable with the unexposed referents, there were increased odds of having lower counts of CD26 cells (activated T cells) (odds ratio (OR) 1.0, 95% confidence interval (95% CI) 0.5 to 1.8 for TCDD < 20 ppt; OR 1.6, 95% CI 0.8 to 3.2 for TCDD 20-51 ppt; OR 2.7, 95% CI 1.4 to 5.1 for TCDD 52-125 ppt; OR 2.6, 95% CI 1.4 to 4.9 for TCDD 125-297 ppt; OR 2.4, 95% CI 1.3 to 4.6 for TCDD 298-3389 ppt). A less consistent finding was decreased spontaneous proliferation of cultured lymphocytes. However, increases were found in proliferation of lymphocytes in response to concanavalin and pokeweed in workers in the high TCDD category. Age, cigarette smoking, and alcohol were significant predictors of several immunological outcomes., Conclusions: Associations between serum TCDD concentration and both a decrease in circulating CD26 cells and decreased spontaneous background proliferation were the major findings of this study. These results are unlikely to be of clinical importance but may reflect limited evidence for an association between immunological changes in workers and high serum concentrations of TCDD, or chance findings resulting from the evaluation of multiple immunological variables.

Physical mapping across a duplication can be a tour de force if the region is larger than the size of a bacterial clone. This was the case of the 170- to 275-kb duplication present on the long arm of chromosome 21 in normal human at 21q11.1 (proximal region) and at 21q22.1 (distal region), which we described previously. We have constructed sequence-ready contigs of the two copies of the duplication of which all the clones are genuine representatives of one copy or the other. This required the identification of four duplicon polymorphisms that are copy-specific and nonallelic variations in the sequence of the STSs. Thirteen STSs were mapped inside the duplicated region and 5 outside but close to the boundaries. Among these STSs 10 were end clones from YACs, PACs, or cosmids, and the average interval between two markers in the duplicated region was 16 kb. Eight PACs and cosmids showing minimal overlaps were selected in both copies of the duplication. Comparative sequence analysis along the duplication showed three single-basepair changes between the two copies over 659 bp sequenced (4 STSs), suggesting that the duplication is recent (less than 4 mya). Two CpG islands were located in the duplication, but no genes were identified after a 36-kb cosmid from the proximal copy of the duplication was sequenced. The homology of this chromosome 21 duplicated region with the pericentromeric regions of chromosomes 13, 2, and 18 suggests that the mechanism involved is probably similar to pericentromeric-directed mechanisms described in interchromosomal duplications., (Copyright 1998 Academic Press.)

The vaccinia virus A39R protein is a member of the semaphorin family. A39R.Fc protein was used to affinity purify an A39R receptor from a human B cell line. Tandem mass spectrometry of receptor peptides yielded partial amino acid sequences that allowed the identification of corresponding cDNA clones. Sequence analysis of this receptor indicated that it is a novel member of the plexin family and identified a semaphorin-like domain within this family, thus suggesting an evolutionary relationship between receptor and ligand. A39R up-regulated ICAM-1 on, and induced cytokine production from, human monocytes. These data, then, describe a receptor for an immunologically active semaphorin and suggest that it may serve as a prototype for other plexin-semaphorin binding pairs.

Tumor necrosis factor-alpha (TNFalpha) is a cytokine that induces protective inflammatory reactions and kills tumor cells but also causes severe damage when produced in excess, as in rheumatoid arthritis and septic shock. Soluble TNFalpha is released from its membrane-bound precursor by a membrane-anchored proteinase, recently identified as a multidomain metalloproteinase called TNFalpha-converting enzyme or TACE. We have cocrystallized the catalytic domain of TACE with a hydroxamic acid inhibitor and have solved its 2.0 A crystal structure. This structure reveals a polypeptide fold and a catalytic zinc environment resembling that of the snake venom metalloproteinases, identifying TACE as a member of the adamalysin/ADAM family. However, a number of large insertion loops generate unique surface features. The pro-TNFalpha cleavage site fits to the active site of TACE but seems also to be determined by its position relative to the base of the compact trimeric TNFalpha cone. The active-site cleft of TACE shares properties with the matrix metalloproteinases but exhibits unique features such as a deep S3' pocket merging with the S1' specificity pocket below the surface. The structure thus opens a different approach toward the design of specific synthetic TACE inhibitors, which could act as effective therapeutic agents in vivo to modulate TNFalpha-induced pathophysiological effects, and might also help to control related shedding processes.

The diagnosis of cancer in a child leaves the parents and child faced with a life-threatening illness in addition to the need to learn about how the illness will impact their lives. Education about the disease process, treatment regimen, and possible side effects of therapy must occur. The educational process, often consisting of didactic teaching sessions, often does not lend itself well to the individual learning needs of the child. A more individualized and developmentally appropriate method of educating children was developed, consisting of the development of a computer-assisted instructional (CAI) program on blood counts.

Objectives: A cross-sectional study was conducted to assess the association of upper extremity musculoskeletal disorders and work-related factors among employees using video display terminals at a large metropolitan newspaper., Methods: The study included 1050 randomly selected workers from four departments. The workers were asked to complete questionnaires on symptoms, job tasks, and psychosocial and work organization conditions. Musculoskeletal disorders of the upper extremities were defined by frequency, duration, and intensity of symptoms not attributable to acute injury. Data were analyzed with the use of logistic regression., Results: A total of 973 workers completed the survey. The one-year period prevalence rate for any musculoskeletal disorder of the upper extremities was 41%. Neck symptoms (26%) were the most frequently reported, followed by hand or wrist (22%), shoulder (17%), and elbow (10%) symptoms. Greater time working at the video display station was associated with increased hand or wrist symptoms in a dose-response relationship. In addition, variables corresponding to increased work-load demands (eg, increased time working under deadline and increased job pressure) were associated with increased neck, shoulder, and hand or wrist disorders. Women were more likely to report symptoms in several areas, but this finding may reflect the concentration of women in jobs involving more risk factors., Conclusions: The results suggest a high prevalence of musculoskeletal disorders of the upper extremities among newspaper employees, and they provide additional evidence that increased work load, time pressure, and greater hours of computer use are related to the occurrence of work-related musculoskeletal disorders among these workers, particularly for disorders in the hand or wrist area.

Cross-linking of antibodies bound to CD11b or CD18 on the neutrophil surface produced a large Ca2+ transient (max. approximately 500 nM). The major source for this Ca2+ rise (more than 81%) was from an intracellular Ca2+ store. After integrin cross-linking, the Ca2+ transient induced by N-formyl-methionyl-leucyl-phenylalanine (FMLP) was decreased, suggesting that both stimuli acted on the same Ca2+ store. Release from the Ca2+ store, induced by integrin cross-linking, was visualized as a 'cloud' of elevated Ca2+, which remained localized even in the presence of extracellular Ca2+. The generation of localized cytosolic Ca2+ signals by cross-linking integrin may be important for triggering localized cellular responses.

A healthy elderly white man was admitted for right inguinal herniorrhaphy. Preoperative chest x-ray revealed a large mass in the lower lobe of the left lung. CT-directed needle biopsy was nondiagnostic. Technetium-99m methylene diphosphonate (99mTc-MDP) bone scan revealed intense soft-tissue activity in the lower left hemithorax with no evidence of abnormal activity in the osseous skeleton. Because of the degree of uptake in the lesion, a presumptive diagnosis of extraosseous osteogenic sarcoma was made. The patient had a left lower lobectomy and the lesion was diagnosed as an osteosarcoma of primary pulmonary origin.

Tumour necrosis factor (TNF) is a mediator of inflammation that has been shown to enhance neutrophil responses to soluble and particulate stimuli. The release of leukotriene B4 (LTB4) by human neutrophils stimulated by unopsonized zymosan was measured in the presence or absence of recombinant human TNF-alpha (rhTNF-alpha) preincubation. There was a threefold increase in the LTB4 response at an optimal TNF concentration of 10(-9) M and an optimal preincubation time of 10-20 min. A similar time and dose dependency was observed for CR3 receptor expression and for the release of the secondary lysosomal granule marker, vitamin B12-binding protein. In contrast, the chemotactic peptide formyl-methionyl-leucyl-phenylalanine (FMLP), although stimulating an increase in both CR3 receptor number and in particle phagocytosis, failed to induce an increase in LTB4 release in response to zymosan. In addition, the present study demonstrated that, unlike FMLP, the exocytotic mechanism for secondary granule release by rhTNF-alpha functioned in the absence of a rise in cytosolic free calcium. Furthermore, it was independent of changes in cyclic nucleotide concentrations and did not require an intact cytoskeleton. Thus the capacity of rhTNF-alpha to amplify the neutrophil response to zymosan through the CR3 receptor appears to be related to the amplification of post-membrane events as well as to an increase in the number of functionally active receptors.

The effects that Actinobacillus pleuropneumoniae bacterins containing paraffin (mineral) oil, Al(OH)3, or lecithin-base oil adjuvants had on antibody production and muscle irritation were studied. Four hundred and thirty-two pigs were vaccinated twice (864 injection sites) at a 30-day interval. To assess effects of skin surface contamination on tissue reaction, 247 of the injections were made through skin soiled with manure. Serum samples for antibody determination and tissue samples were obtained at slaughter (15 to 90 days after vaccination). Bacterins containing Al(OH)3 or 5% lecithin-base oil as an adjuvant resulted in minimal tissue irritation. Bacterins containing paraffin oil or greater than 20% lecithin-base oil were more irritating to muscle, often resulting in a granuloma or an abscess at the injection site. Pigs given bacterins containing Al(OH)3 adjuvant had lower antibody titers than did pigs given bacterins containing paraffin oil or lecithin-base oil adjuvants.