Multidisciplinary approach to diagnosis and management of osteosarcoma – a review of the St Vincent's Hospital experience

BACKGROUND: Osteosarcoma is the most common primary malignant bone tumour in children and young adults. Despite advances in the diagnosis and management of osteosarcoma, there have been few recent studies describing the experiences of tertiary referral centres. This paper aims to describe and discuss the clinical features, pre-operative work-up, management and outcomes of these patients at St Vincent's Hospital (Melbourne, Australia). METHODS: Retrospective study of fifty-nine consecutive patients managed for osteosarcoma at St Vincent's Hospital between 1995 and 2005. RESULTS: Median age at diagnosis was 21 (range, 11–84) years. Gender distribution was similar, with thirty-one male and twenty-eight female patients. Twenty-five patients had osteosarcoma in the femur, eleven each were located in the humerus and tibia, six were identified in the pelvis, and one each in the clavicle, maxilla, fibula, sacrum, ulna and radius. Pre-operative tissue diagnosis of osteosarcoma was obtained through computed tomography-guided percutaneous biopsy in over ninety percent of patients. Following initial therapy, over fifty percent of patients remained relapse-free during the follow-up period, with twelve percent and twenty-seven percent of patients documented as having local and distant disease recurrence, respectively. Of patients with recurrent disease, sixty-two percent remained disease-free following subsequent surgical intervention (most commonly, pulmonary metastatectomy). CONCLUSION: Patient outcomes can be optimised through a multidisciplinary approach in a tertiary referral centre. At St Vincent's Hospital, survival and relapse rates of patients managed for osteosarcoma compare favourably with the published literature

O6-methylguanine-DNA methyltransferase depletion and DNA damage in patients with melanoma treated with temozolomide alone or with lomeguatrib

We evaluated the pharmacodynamic effects of the O6-methylguanine-DNA methyltransferase (MGMT) inactivator lomeguatrib (LM) on patients with melanoma in two clinical trials. Patients received temozolomide (TMZ) for 5 days either alone or with LM for 5, 10 or 14 days. Peripheral blood mononuclear cells (PBMCs) were isolated before treatment and during cycle 1. Where available, tumour biopsies were obtained after the last drug dose in cycle 1. Samples were assayed for MGMT activity, total MGMT protein, and O6-methylguanine (O6-meG) and N7-methylguanine levels in DNA. MGMT was completely inactivated in PBMC from patients receiving LM, but detectable in those on TMZ alone. Tumours biopsied on the last day of treatment showed complete inactivation of MGMT but there was recovery of activity in tumours sampled later. Significantly more O6-meG was present in the PBMC DNA of LM/TMZ patients than those on TMZ alone. LM/TMZ leads to greater MGMT inactivation, and higher levels of O6-meG than TMZ alone. Early recovery of MGMT activity in tumours suggested that more protracted dosing with LM is required. Extended dosing of LM completely inactivated PBMC MGMT, and resulted in persistent levels of O6-meG in PBMC DNA during treatment

Affordable flow cytometry for enumeration of absolute CD4(+ )T-lymphocytes to identify subtype C HIV-1 infected adults requiring antiretroviral therapy (ART) and monitoring response to ART in a resource-limited setting

BACKGROUND: The World Health Organization (WHO)'s "3 × 5 program" has spurred efforts to place 3 million people on combination antiretroviral therapy (ART) for treatment of AIDS in resource-limited countries. Paradoxically, the cost of CD4(+ )T-lymphocyte count essential for decision-making to commence HIV positive adults on ART as well as for monitoring responses to ART remains unaffordable in most resource-limited countries. Thus, low-cost methods for enumerating CD4(+ )T-lymphocyte are urgently needed. OBJECTIVE: To evaluate Cyflow cytometry (Cyflow SL, Partec, Munster, Germany) for enumeration of absolute CD4(+ )T-lymphocyte in subtype C HIV-1 seropositive subjects using FACSCount (Becton and Dickinson, Immunocytometry Systems, San Jose, CA, USA) as the "predicate method". METHODS: A total of 150 HIV-1 seropositive subjects were included in the evaluation exercise. Fifty-eight specimens were collected from pregnant HIV-1 seropositive women (subtype C drug resistance study). Twenty-seven specimens were collected from women and their spouses with AIDS followed in a Duke ART study to assess the immunologic and virologic responses to generic ART, comprising Stavudine, Lamivudine and Nevirapine (Stalanev, Varichem Labs, Harare, Zimbabwe). Sixty-five specimens were collected from AIDS patients enrolled in an ongoing Kaposi Sarcoma (KS) study to investigate impact of ART on KS progression. Enumeration of CD4(+ )T-lymphocytes using FACSCount is routinely conducted for all the three studies. The Medical Research Council of Zimbabwe and Medicines Control Authority of Zimbabwe approved the studies. Whole blood was collected in EDTA vacutainer tubes and aliquoted into two tubes (200 μL in each). CD4(+ )T-lymphocyte counts were enumerated using a Cyflow counter, in the Department of Immunology and a FACSCount in the Department of Obstetrics and Gynaecology within 6 hours of phlebotomy following manufacturers' instructions. RESULTS: Using linear regression analysis, there was a very strong correlation (R = 0.991) between the overall CD4(+ )T-lymphocyte counts obtained by FACSCount and those obtained by Cyflow. When data analysis was stratified by study groups, there was a strong correlation between the FACSCount and Cyflow CD4(+ )T-lymphocyte counts from subjects in the three independent studies; Subtype C resistance (R(2 )= 0.987), Duke ART (R(2 )= 0.980) and KS (R(2 )= 0.994), Table 1. Using Bland-Altman plots, the overall, absolute CD4(+ )T lymphocytes obtained by the two methods were in excellent agreement (mean difference 1.21, 95% Confidence Interval {CI): -2.1 to 3.3). For the 0–250 CD4(+ )T-lymphocytes range, the CD4 counts obtained using FACSCount were also in good agreement with those obtained using Cyflow counter (mean difference = 2.6 cells/μL, 95% CI: -1.1 to 6.3). Similarly, in the 251–500 (mean difference 1.0, cells/μL, 95% CI: -3.7 to 5.6) and the 501–1200 (mean difference = 0.29 cells/μL, 95% CI: -8.1 to 8.7) CD4 T-lymphocytes range, good agreement was observed. CONCLUSION: The Cyflow counter is as accurate as the FACSCount in enumerating absolute CD4(+ )T-lymphocytes in the range 1–1200 cells/μL. Cyflow cytometry is relatively affordable, easy to use technology that is useful not only in identifying HIV seropositive individuals who require ART but also for monitoring immunologic responses to ART

A Novel Campylobacter jejuni Sequence Type from a Culture-Negative Patient in The Gambia

The introduction of molecular diagnostic methods is crucial for improved understanding of the aetiology and epidemiology of bacterial infections in communities in resource poor settings. A blood sample from a 7 month old patient diagnosed with malaria in 2001 in a Gambian outpatient clinic was reported as culture negative after it was subjected to traditional bacterial culture protocols. We re-addressed the analysis of the blood sample from this case more recently (after 6.5 years in archival storage) in pilot work establishing 16S rRNA PCR in our molecular laboratory. Initial 16S rRNA PCR results confirmed the presence of bacterial DNA in the sample. 16S rRNA sequence analysis identified the organism as Campylobacter spp. In light of the molecular evidence we successfully grew the organism using appropriate culture conditions and subsequently biochemically confirmed that the isolate was Campylobacter jejuni. PCR and DNA sequencing of a set of seven C. jejuni housekeeping genes and in silico Multilocus Sequence Typing (MLST) analysis revealed that the isolate exhibits a novel sequence type (ST) of C. jejuni (ST 2928) and belongs to ST-443 clonal complex. This study demonstrates the potential for molecular tools to enhance the diagnosis of bacterial infections, which remain a major killer globally, not least in children in the developing world. Improvements in diagnostics are needed, and will be important not only for sick individuals but also for populations, where better measures of disease burden will contribute significantly to the improvement of public health policy

Lipoxin A4 Stimulates Calcium-Activated Chloride Currents and Increases Airway Surface Liquid Height in Normal and Cystic Fibrosis Airway Epithelia

Cystic Fibrosis (CF) is a genetic disease characterised by a deficit in epithelial Cl− secretion which in the lung leads to airway dehydration and a reduced Airway Surface Liquid (ASL) height. The endogenous lipoxin LXA4 is a member of the newly identified eicosanoids playing a key role in ending the inflammatory process. Levels of LXA4 are reported to be decreased in the airways of patients with CF. We have previously shown that in normal human bronchial epithelial cells, LXA4 produced a rapid and transient increase in intracellular Ca2+. We have investigated, the effect of LXA4 on Cl− secretion and the functional consequences on ASL generation in bronchial epithelial cells obtained from CF and non-CF patient biopsies and in bronchial epithelial cell lines. We found that LXA4 stimulated a rapid intracellular Ca2+ increase in all of the different CF bronchial epithelial cells tested. In non-CF and CF bronchial epithelia, LXA4 stimulated whole-cell Cl− currents which were inhibited by NPPB (calcium-activated Cl− channel inhibitor), BAPTA-AM (chelator of intracellular Ca2+) but not by CFTRinh-172 (CFTR inhibitor). We found, using confocal imaging, that LXA4 increased the ASL height in non-CF and in CF airway bronchial epithelia. The LXA4 effect on ASL height was sensitive to bumetanide, an inhibitor of transepithelial Cl− secretion. The LXA4 stimulation of intracellular Ca2+, whole-cell Cl− currents, conductances and ASL height were inhibited by Boc-2, a specific antagonist of the ALX/FPR2 receptor. Our results provide, for the first time, evidence for a novel role of LXA4 in the stimulation of intracellular Ca2+ signalling leading to Ca2+-activated Cl− secretion and enhanced ASL height in non-CF and CF bronchial epithelia

Incidence, risk factors and treatment of diarrhoea among Dutch travellers: reasons not to routinely prescribe antibiotics

<p>Abstract</p> <p>Background</p> <p>Travellers' diarrhoea (TD) is the most common infectious disease among travellers. In the Netherlands, stand-by or prophylactic antibiotics are not routinely prescribed to travellers. This study prospectively assessed the incidence rate, risk factors, and treatment of TD among immunocompetent travellers.</p> <p>Methods</p> <p>Persons who attended the travel clinic of the Public Health Service Amsterdam in 2006-2007 before short-term travel to tropical and subtropical countries were invited to answer a questionnaire regarding sociodemographics and travel purpose; they were also asked to keep a daily structured travel diary, recording their itinerary, symptoms, and self-medication or consultation with a doctor. Diarrhoea episodes containing blood or mucous were considered severe.</p> <p>Results</p> <p>Of 1202 travellers, the median age was 38 years, and the median travel duration 3 weeks. Of all episodes, 96% were mild. The median duration of TD was 2 days and significantly shorter in subsequent episodes compared to first episodes (p < 0.0005). Of first episodes 38% started in the first travel week. The incidence rate (IR) for first episodes was 2.49 (95% confidence interval [CI], 2.30-2.70) per 100 travel days, with the highest IR among travellers to South-Central and West Asia. The IR for first and subsequent episodes was comparable. Risk factors for first episodes included female sex, a Western country of birth, and tourism as the purpose of travel. The lowest risk was in travellers to South America. An independent risk factor for subsequent episodes was female sex. In total, 5% of travellers used antibiotics; of those, 92% had mild diarrhoea, and 53% received antibiotics over the counter.</p> <p>Conclusions</p> <p>TD is common among travellers, but the overall course is mild, not requiring treatment. The incidence rates for first and second episodes are comparable. Female sex is a risk factor for the first episode, as well as subsequent ones. Prescription antibiotics are not needed in short-term healthy travellers.</p

BRCA1 and BRCA2 germline mutation analysis in the Indonesian population

Specific mutations in BRCA1 and BRCA2 genes have been identified in specific populations and ethnic groups. However, little is known about the contribution of BRCA1 and BRCA2 mutations to breast cancers in the Indonesian population. One hundred-twenty moderate to high risk breast cancer patients were tested using PCR-DGGE, and any aberrant band was sequenced. Multiplex ligation-dependent probe amplification (MLPA) was performed on all samples to detect large deletions in the two genes. Twenty-three different mutations were detected in 30 individuals, ten were deleterious mutations and 20 were “unclassified variants” with uncertain clinical consequences. Three of seven (c.2784_2875insT, p.Leu1415X and del exon 13–15) and two of four (p.Glu2183X and p.Gln2894X) deleterious mutations that were found in BRCA1 and BRCA2 respectively, are novel. Several novel, pathogenic BRCA1 and BRCA2 germline mutations are found in early onset Indonesian breast cancer patients, these may therefore be specific for the Indonesian population

A histological and micro-CT investigation in to the effect of NGF and EGF on the periodontal, alveolar bone, root and pulpal healing of replanted molars in a rat model - a pilot study

Background: This study aims to investigate, utilising micro-computed tomography (micro-CT) and histology, whether the topical application of nerve growth factor (NGF) and/or epidermal growth factor (EGF) can enhance periodontal, alveolar bone, root and pulpal tissue regeneration while minimising the risk of pulpal necrosis, root resorption and ankylosis of replanted molars in a rat model. Methods: Twelve four-week-old male Sprague-Dawley rats were divided into four groups: sham, collagen, EGF and NGF. The maxillary right first molar was elevated and replanted with or without a collagen membrane impregnated with either the growth factors EGF or NGF, or a saline solution. Four weeks after replantation, the animals were sacrificed and the posterior maxilla was assessed using histological and micro-CT analysis. The maxillary left first molar served as the control for the corresponding right first molar. Results: Micro-CT analysis revealed a tendency for all replanted molars to have reduced root length, root volume, alveolar bone height and inter-radicular alveolar bone volume. It appears that the use of the collagen membrane had a negative effect while no positive effect was noted with the incorporation of EGF or NGF. Histologically, the incorporation of the collagen membrane was found to negatively affect pulpal, root, periodontal and alveolar bone healing with pulpal inflammation and hard tissue formation, extensive root resorption and alveolar bone fragmentation. The incorporation of EGF and NGF did not improve root, periodontal or alveolar bone healing. However, EGF was found to improve pulp vascularisation while NGF improved pulpal architecture and cell organisation, although not to the level of the control group.Conclusions: Results indicate a possible benefit on pulpal vascularisation and pulpal cell organisation following the incorporation of EGF and NGF, respectively, into the alveolar socket of replanted molars in the rat model. No potential benefit of EGF and NGF was detected in periodontal or root healing, while the use of a collagen membrane carrier was found to have a negative effect on the healing response

Detection of green line emission in the dayside atmosphere of Mars from NOMAD-TGO observations

The oxygen emission at 557.7 nm is a ubiquitous component of the spectrum of the terrestrial polar aurora and the reason for its usual green colour1. It is also observed as a thin layer of glow surrounding the Earth near 90 km altitude in the dayside atmosphere2,3 but it has so far eluded detection in other planets. Here we report dayglow observations of the green line outside the Earth. They have been performed with the Nadir and Occultation for Mars Discovery ultraviolet and visible spectrometer instrument on board the European Space Agency’s ExoMars Trace Gas Orbiter. Using a special observation mode, scans of the dayside limb provide the altitude distribution of the intensity of the 557.7 nm line and its variability. Two intensity peaks are observed near 80 and 120 km altitude, corresponding to photodissociation of CO2 by solar Lyman α and extreme ultraviolet radiation, respectively. A weaker emission, originating from the same upper level of the oxygen atom, is observed in the near ultraviolet at 297.2 nm. These simultaneous measurements of both oxygen lines make it possible to directly derive a ratio of 16.5 between the visible and ultraviolet emissions, and thereby clarify a controversy between discordant ab initio calculations and atmospheric measurements that has persisted despite multiple efforts. This ratio is considered a standard for measurements connecting the ultraviolet and visible spectral regions. This result has consequences for the study of auroral and airglow processes and for spectral calibration

Diabetes and hypertension increase the placental and transcellular permeation of the lipophilic drug diazepam in pregnant women

Background: Previous studies carried out in our laboratories have demonstrated impaired drug permeation in diabetic animals. In this study the permeation of diazepam (after a single dose of 5 mg/day, administered intramuscularly) will be investigated in diabetic and hypertensive pregnant women.Methods: A total 75 pregnant women were divided into three groups: group 1 (healthy control, n = 31), group 2 (diabetic, n = 14) and group 3 (hypertensive, n = 30). Two sets of diazepam plasma concentrations were collected and measured (after the administration of the same dose of diazepam), before, during and after delivery. The first set of blood samples was taken from the mother (maternal venous plasma). The second set of samples was taken from the fetus (fetal umbilical venous and arterial plasma). In order to assess the effect of diabetes and hypertension on diazepam placental-permeation, the ratios of fetal to maternal blood concentrations were determined. Differences were considered statistically significant if p=0.05.Results: The diabetes and hypertension groups have 2-fold increase in the fetal umbilical-venous concentrations, compared to the maternal venous concentrations. Feto: maternal plasma-concentrations ratios were higher in diabetes (2.01 ± 1.10) and hypertension (2.26 ± 1.23) groups compared with control (1.30 ± 0.48) while, there was no difference in ratios between the diabetes and hypertension groups. Umbilical-cord arterial: venous ratios (within each group) were similar among all groups (control: 0.97 ± 0.32; hypertension: 1.08 ± 0.60 and diabetes: 1.02 ± 0.77).Conclusions: On line with our previous findings which demonstrate disturbed transcellular trafficking of lipophilic drugs in diabetes, this study shows significant increase in diazepam placental-permeation in diabetic and hypertensive pregnant women suggesting poor transcellular control of drug permeation and flux, and bigger exposure of the fetus to drug-placental transport