Rational thresholding of circulating tumor DNA concentration for improved surveillance of metastatic breast cancer.

BACKGROUND: The use of circulating tumor DNA (ctDNA) concentration for metastatic cancer surveillance is promising, but uncertainty remains about cut-offs with clinical validity. MATERIALS AND METHODS: This observational study recruited 136 subjects with advanced metastatic breast cancer (irrespective of ERBB2/hormone receptor status) for sequencing of their primary tumor in search for PIK3CA hotspot variants amenable for monitoring by droplet digital PCR (ddPCR). The study analyzed 341 on-treatment samples from 19 patients with PIK3CA variants H1047R or E545K enrolled for long-term (median 85 weeks, range 13-125 weeks), frequent (every 3-5 weeks, median of 14 time points per subject, range 2-29) blood sampling for ctDNA quantification by ddPCR, orthogonally validated by deep sequencing. The diagnostic accuracy of ctDNA versus cancer antigen 15-3 (CA15-3) concentrations to predict disease progression within 12 weeks was investigated using receiver operating characteristic (ROC) analysis. Likelihood ratios were used for rational selection of ctDNA result intervals. RESULTS: ctDNA [area under the ROC curve (AUC) 0.848, 95% confidence interval (CI) 0.791-0.895] showed superior diagnostic performance than CA15-3 (AUC 0.670, 95% CI 0.601-0.735, P < 0.001) to predict clinical progression within 12 weeks. ctDNA levels below 10 mutant allele copies/ml had high negative predictive value (88%), while levels above 100 copies/ml detected 64% of progressions 10 weeks earlier versus standard of care. Logistic regression analysis indicated complementary value of ctDNA and the presence of two consecutive CA15-3 rises, resulting in a model with 86% (95% CI 74% to 93%) positive predictive value and a clinically meaningful result in 89% of blood draws. CONCLUSIONS: Intensive ctDNA quantification improves metastatic breast cancer surveillance and enables individualized risk-based scheduling of clinical care.

MRI in hypertrophic mono- and polyneuropathies.

Different conditions that may lead to enlarged nerves or nerve roots include hereditary motor and sensory neuropathy (HMSN), neurofibromatosis (NF) type 1, chronic inflammatory demyelinating polyradiculoneuropathy (CIDP), and intraneural perineurioma. Differential diagnosis of hypertrophic mono- and polyradiculopathies remains challenging but is important because of different treatments and prognosis. Magnetic resonance imaging (MRI) can identify the hypertrophic nerve segments and guide a fascicular biopsy. A fascicular biopsy will often be necessary for precise diagnosis.

Pott's puffy tumour in a 5-year old boy: the role of ultrasound and contrast-enhanced CT imaging; surgical case report.

We report a case of Pott's puffy tumour, a subperiosteal abscess of the frontal bone associated with an underlying frontal osteomyelitis, in a 5-year-old boy. Ultrasonography played a crucial role in the diagnosis of our patient, suggesting the presence of a Pott's puffy tumour with epidural abscess by showing a subperiosteal abscess associated with erosion of the frontal bone. Subsequently, the diagnosis of Pott's puffy tumour with epidural abscess was confirmed by contrast-enhanced CT scanning. Prompt neurosurgical intervention with drainage of abscesses and debridement of bone sequestrate, together with prolonged antibiotic therapy, significantly contributes to a favorable outcome.

Spatiotemporal characteristics of spontaneous overground walk-to-run transition.

The purpose of the current study was to examine spontaneous overground walk-to-run transitions (WRT). For the first time, subjects' WRT was examined during an overground protocol that allowed them to accelerate freely. The overground speed profile prior to reaching the WRT was analysed together with the spatiotemporal characteristics of the actual transition. Nine women (height: 166.4+/-3.5 cm) performed five spontaneous WRT. Speed, step frequency (SF) and step length (SL) of the accelerating walking steps and the transition step were determined. By means of fourth degree polynomials, subjects' spatiotemporal profiles prior to reaching WRT were determined. A step length index (SLI) was used to calculate the contribution of SF and SL to the increase in walking speed. Subjects took on average 5.9+/-0.9 walking steps prior to reaching transition. When speeding up towards the transition to running, subjects chose to accelerate predominantly in the first half of the walking acceleration period, followed by smaller speed increments in the second half. The SLI values indicated that subjects tended to increase walking speed by increasing SL, more than SF, except during the first 20% of the acceleration period. WRT-speed was 2.664+/-0.230 m s(-1), which was higher than in former treadmill studies using a constant acceleration protocol (+/-2.1 m s(-1)). Subjects made a speed jump of 0.417 m s(-1) from the last walking step to the WRT-step. We can conclude that further transition studies studying the interaction between the acceleration and gait transition behaviour are necessary in order to complete the understanding of the transition phenomenon.

Metal ion levels in a triathlete with a metal-on-metal resurfacing arthroplasty of the hip.

A prospective study of serum and urinary ion levels was undertaken in a triathlete who had undergone a metal-on-metal resurfacing arthroplasty of the hip four years previously. The one month study period included the final two weeks of training, the day of the triathlon, and the two weeks immediately post-race. Serum cobalt and chromium levels did not vary significantly throughout this period, including levels recorded on the day after the 11-hour triathlon. Urinary excretion of chromium increased immediately after the race and had returned to pre-race levels six days later. The clinical implications are discussed.

Study of the environmental effect of a commercial wound cleanser used with different mechanical forces.

Important improvements have been made in wound care over the last decade. However, few data are available on the influence that these have outside their intended use. This study aimed to clarify the effects of the use of wound cleansers on bacterial contamination of the immediate surroundings. Little evidence was found from either laboratory or clinical settings that wound-derived micro-organisms become airborne during wound cleansing. Bacterial dispersion around wounds may be attributed to general activity rather than wound cleansing. If simple precautions are taken, risks for personnel and patients in hospitals and consultation rooms during wound cleansing can be minimized.

Tropist3: a cosmid vector for simplified mapping of both G+C-rich and A+T-rich genomic DNA.

We have constructed a cosmid vector, Tropist3, based on the lambda origin double-cos-site vector Lawrist4, which is designed for efficient cloning and mapping of genomic DNA. Tropist3 contains two cloning sites in addition to the HindIII and BamHI sites present in Lawrist4; a SalI site allows cloning of Sau3AI partial digests following partial filling-in of the ends, and a PmlI site is suitable for blunt-end cloning. Both these strategies reduce the chance of co-cloning two inserts. Tropist3 also contains NotI, PacI, SacII and KpnI sites flanking the cloning region; these allow most inserts to be excised cleanly and mapped by partial digestion followed by hybridization with short vector sequences which lie adjacent to the cloning sites. This will also be useful for recloning inserts into different vectors, or for cosmid sequencing projects.

Laboratory guidelines for the practical use of HIV drug resistance tests in patient follow-up.

HIV drug resistance is one of the major limitations in the successful treatment of HIV-infected patients using currently available antiretroviral combination therapies. When appropriate, drug susceptibility profiles should be taken into consideration in the choice of a specific combination therapy. Guidelines recommending resistance testing in certain circumstances have been issued. Many clinicians have access to resistance testing and will increasingly use these results in their treatment decisions. In this document, we comment on the different methods available, and the relevant issues relating to the clinical application of these tests. Specifically, the following recommendations can be made: (i) genotypic and phenotypic HIV-1 drug resistance analyses can yield complementary information for the clinician. However, insufficient information currently exists as to which approach is preferable in any particular clinical setting; (ii) when HIV-1 drug resistance testing is required, it is recommended that testing be performed on plasma samples obtained before starting, stopping or changing therapy, on samples that have a viral load above the detection limit of the resistance test; (iii) the panel recommends that genotypic and phenotypic HIV-1 drug resistance testing for clinical purposes be performed in a certified laboratory under strict quality control and quality assurance standards; and (iv) the panel recommends that resistance testing laboratories provide clinicians with resistance reports that include a list of drug-related resistance mutations (genotype) and/or a list of drug-related fold resistance values (phenotype), with interpretations of each by an experienced virologist. The interpretation of genotypic and phenotypic analysis is a complex and developing science, and in order to understand HIV-1 drug resistance reports, communication between the requesting clinician and the expert that interpreted the resistance report is recommended.

A versatile system for the production of recombinant chimeric peptides.

Production of chimeric and multimeric peptides is of interest for the analysis of topographic relationships between T and B cell stimulatory epitopes. Recombinant DNA technology has certain advantages over conventional chemical peptide synthesis for the production of peptide constructs of large size (more than 40 amino acid residues). We describe a methodology which is versatile and independent of the expression vector used because it only relies on the incorporation of appropriate restriction enzyme sites in oligonucleotides. The method was verified using two 20mer sequences from the 38 kDa antigen of M. tuberculosis. Peptide 201-220, containing an antibody binding linear epitope, has been made immunogenic in vivo when combined with T cell stimulatory peptide 350-369 in a chimeric peptide. The results demonstrate that a distinct orientation of the constituent peptides was essential for achieving optimal immunogenicity.

Effects of epidermal growth factor on CYP inducibility by xenobiotics, DNA replication, and caspase activations in collagen I gel sandwich cultures of rat hepatocytes.

In this study, we investigated the combined effects of EGF and collagen I gel on the phenotype of cultured rat hepatocytes and we focussed our investigations on the regulation of xenobiotic-mediated induction of CYP, cell cycle progression and activation of capases 8 and 3. We found that EGF, added to basal culture medium or phenobarbital (3.2 mM) containing medium, provoked a moderate decrease of CYP1A1 and CYP2B1/2 activities. However, EGF did not exert any inhibitory effect on 3-methylcholantrene (5 microM) and beta-naphtoflavone (25 microM) induction of CYP1A1 activities. In collagen gel sandwich cultures, hepatocytes remained arrested in mid-G1 phase of the cell cycle, even in the presence of EGF. In conventional primary cultures, caspases 8 and 3 were activated at 3 and 5 days after plating respectively. In collagen gel sandwich cultures, we found that neither collagen I nor EGF prevented activation of caspase 8 while collagen I gel inhibited activation of caspase 3, preventing spontaneous apoptosis of cultured rat hepatocytes. In contrast, EGF transiently increased caspase 3 activity at day 1 after plating. Altogether, our data demonstrate that collagen I gel triggers intracellular signals which strongly affect cultured hepatocyte phenotype, leading to a cell cycle arrest in G1 phase and long-term survival through the inhibition of caspase 3 activation and that EGF-free medium improves survival and liver-specific gene expression in hepatocytes maintained in collagen I gel sandwich cultures.

Correlation of pncA sequence with pyrazinamide resistance level in BACTEC for 21 mycobacterium tuberculosis clinical isolates.

Mutations in the pncA gene, encoding pyrazinamidase, are considered the major mechanism of pyrazinamide (PZA) resistance in Mycobacterium tuberculosis, but resistant strains containing the wild-type gene have been described. The correlation of pncA sequence with PZA resistance level was examined for 21 M. tuberculosis clinical isolates. Susceptibility patterns were determined for 100, 300, and 900 microg/ml concentrations of the drug in BACTEC. Insertions and deletions and a substitution in the putative promoter region led to high-level resistance, whereas substitutions within the open reading frame seemed to confer variable levels of resistance. Variable resistance levels and PZase activities were also observed among isolates lacking pncA mutations. The high-level resistance (900 microg/ml) in pncA wild-type isolates highlights the clinical significance of these isolates. These data also suggest that there may still be more than one alternative mechanism leading to PZA resistance in M. tuberculosis isolates.

Duplication of genes encoding the immunodominant 38 kDa antigen in Mycobacterium intracellulare.

Mycobacterium avium is a causative agent of mycobacterioses in systemically immunocompromised individuals, whereas Mycobacterium intracellulare is responsible for causing infections in relatively immunocompetent hosts. In an attempt to identify components that could be involved in virulence, we characterised the 38 kDa-encoding gene of M intracellulare that is absent in M. avium. This antigen cross reacts immunologically with a major 38 kDa antigen of M. tuberculosis, and both antigens are homologues of the phosphate transport subunit S (PstS) of the pst complex of Escherichia coli. Unlike the M. tuberculosis complex the M. intracellulare coding gene was found to be duplicated. We also identified and characterised other pst genes that may constitute an operon. Considering that multiple isoforms of PstS are present in mycobacteria the possible role of pstS1 genes for pathogenesis is discussed.

Fluorine-18 fluorodeoxyglucose-position emission tomography: a highly accurate imaging modality for the diagnosis of chronic musculoskeletal infections.

BACKGROUND: The noninvasive diagnosis of chronic musculoskeletal infections remains a challenge. Recent studies have indicated that fluorine-18 fluorodeoxyglucose-positron emission tomography is a highly accurate imaging technique and is significantly more accurate than the combination of a bone scan and a white blood-cell scan for the diagnosis of chronic infection in the central skeleton (p < 0.05). However, patients who had had surgery within the previous two years were excluded from study. It was our aim to evaluate the technique in an unselected, clinically representative population. METHODS: Sixty patients with a suspected chronic musculoskeletal infection involving the central skeleton (thirty-three patients) or the peripheral skeleton (twenty-seven patients) were studied with fluorine-18 fluorodeoxyglucose-positron emission tomography. Thirty-five patients had had surgery within the previous two years. The fluorine-18 fluorodeoxyglucose-positron emission tomography studies were read in a blinded, independent manner by two experienced readers. The final diagnosis was based on histopathological studies or microbiological culture (eighteen patients) or on clinical findings after at least six months of follow-up (forty-two patients). RESULTS: On the final composite assessment, twenty-five patients had infection and thirty-five did not. All twenty-five infections were correctly identified by both readers. There were four false-positive findings; in two of these cases, surgery had been performed less than six months prior to the study. The sensitivity, specificity, and accuracy were 100%, 88%, and 93% for the whole group; 100%, 90%, and 94% for the subgroup of patients with a suspected infection of the central skeleton; and 100%, 86%, and 93% for the subgroup of patients with a suspected infection of the peripheral skeleton. Interobserver agreement was excellent (kappa = 0.97). CONCLUSIONS: Fluorine-18 fluorodeoxyglucose-positron emission tomography is highly accurate as a single technique for the evaluation of chronic musculoskeletal infections. It is especially valuable in the evaluation of the central skeleton, where white blood-cell scans are less useful. Because of its simplicity and high degree of accuracy, it has the potential to become a standard technique for the diagnosis of chronic musculoskeletal infections. Further studies are needed to assess its ability to identify infections at the sites of total joint replacements and to distinguish infection from aseptic loosening of these prostheses.

Extent and duration of virulent virus excretion upon challenge of pigs vaccinated with different glycoprotein-deleted Aujeszky's disease vaccines.

Different deleted Aujeszky's disease vaccines were compared for their ability to induce an immunity which suppresses virus excretion optimally upon infection. Groups of pigs were vaccinated once with attenuated deleted Aujeszky's disease vaccine (gI, gX or gp63 negative), suspended in phosphate buffered saline. Two additional groups were vaccinated with a gI deleted vaccine virus suspended in an oil-in-water emulsion. Other groups were vaccinated twice with gI deleted inactivated vaccines. The three control groups included were: pigs immune after infection, unvaccinated pigs and pigs receiving vaccine without known deletion in the envelope. Experimental challenge took place 3 or 4 weeks after the only or the last vaccination. The number of excreting pigs, the duration of excretion and the virus titers excreted, were determined for all the groups. All the pigs vaccinated with glycoprotein deletion vaccines suspended in phosphate buffered saline, excreted virus for 2 to 6 days after challenge. A 100 to 1000 fold reduction in excreted virus titers was obtained in vaccinated pigs compared to unvaccinated ones. Some vaccines suppressed virus excretion better than others, but no correlation could be made between the type of deletion (gI, gX or gp63) and the degree of reduction in virus excretion. Similar results were obtained with two applications of inactivated vaccines. The lowest number of excreting pigs, the lowest duration of excretion and the lowest titers were obtained in groups vaccinated with the attenuated vaccine suspended in an oil-in-water emulsion. No vaccine suppressed virus excretion totally.

Effect of phenobarbital on 7-Ethoxycoumarin-O-deethylase and microsomal epoxide hydrase activities in collagen gel cultures of rat hepatocytes.

Ethoxycoumarin-O-deethylase (ECOD) and microsomal epoxide hydrase (mEH) activities have been studied in collagen gel cultures of rat hepatocytes under control conditions and after exposure to phénobarbital. The hepatocytes were either sandwiched between two layers of extracellular matrix (type I collagen) or directly mixed with the same type of collagen gel. In preliminary experiments it was first tested whether it was possible to measure ECOD activity directly in intact hepatocytes that were embedded in a collagen gel matrix. Probably by a delay of substrate penetration through the collagen gel, the latter had to be removed by a collagenase digestion before ECOD activity measurements took place. When cultured in control medium, the activity of both phase I enzymes studied decreased as a function of culture time in sandwich as well as in immobilization cultures. After 4 days, however, a steady-state situation was reached and this level of enzymatic activity was observed for at least 2 wk. When the cells were exposed to 3.2 mm phenobarbital, an increased ECOD activity remained detectable for at least 14 days. In sandwich cultures a two- to threefold increase of ECOD activity was observed, whereas in immobilization cultures, increases of only 30 to 46% of the values observed in the control medium were measured. After exposure of the collagen gel cultures to 3.2 mm phenobarbital, the mEH activity was increased to the same extent (factor 2 to 2.5) in both culture models.

Effect of epidermal growth factor in collagen gel cultures of rat hepatocytes.

Collagen gel cultures of hepatocytes represent a promising in vitro model in pharmaco-toxicology. Epidermal growth factor (EGF) is usually added to the culture medium, although one could question its value in a culture model aiming at maintaining a maximum of differentiated functional capacities. In this study, the effects of EGF (20 ng/ml) on albumin secretion, morphology and pentoxyresorufin O-depentylase (PROD), ethoxyresorufin O-deethylase (EROD) and glutathione S-transferase (GST) activities have been examined in both collagen gel sandwich and immobilization gel cultures of adult rat hepatocytes. Transmission electron microscopy did not show an obvious influence of epidermal growth factor (EGF) on the intracellular organization of organelles of the rat hepatocytes. It was found that EGF addition had no effect on albumin secretion in both culture models. On the contrary, the presence of EGF in the culture medium provoked in collagen gel sandwich cultures, after 7 days, significant decreases of 66% and 25% in EROD and PROD activities, respectively. On GST activities, no effect of EGF could be observed in both collagen gel cultures. Removal of EGF from the culture medium seemed to have a positive effect on the maintenance of the phase 1 biotransformation capacity of rat hepatocytes. Its addition should therefore be avoided in collagen gel cultures used in pharmaco-toxicology.

Effect of Extracellular Matrix Composition on the Expression of Glutathione S-transferase Isoenzymes in Organotypical Hepatocyte Cultures.

Collagen gel sandwich and immobilization cultures of hepatocytes, using hydrated collagen type I as extracellular matrix (ECM), have been proposed as long-term in vitro models in pharmaco-toxicology. The in vivo ECM composition in the space of Disse is, however, much more complex. As a differentiated hepatocyte phenotype is thought to be highly dependent on ECM composition and biophysical characteristics, we modulated the ECM to mimic the in vivo situation. Moreover, commercially available collagen type I (Boehringer-Ingelheim) was compared to the one prepared in the laboratory from rat tails. ECM composition had no effect on albumin secretion or hepatocyte morphology in both collagen gel sandwich and immobilization cultures. Total, Alpha and Mu class GST activities in organotypical cultures with a complex or a simple collagen type I ECM were similar. The Pi class GST activity increased as a function of culture time in all culture models. Thus, mimicking the in vivo composition of the ECM did not improve the changes in GST expression that were observed in simple collagen gel cultures. The collagen type I matrix is therefore assumed to confer sufficient protection to help the hepatocytes to maintain their differentiated phenotype to a certain extent. Moreover, we hypothesize that the collagen gel matrix may act as a scaffold to keep newly synthesized ECM components in the proximity of the basolateral surfaces of the hepatocytes.

Organochlorine contaminants and eggshell thinning in grebes from prairie Canada.

Eggs of five species of grebe were collected from Manitoba, Saskatchewan and Alberta during 1982-1987: red-necked (Podiceps grisegena), horned (P. auritus), eared (P. caspicus), western (Aechmophorus occidentalis) and pied-billed (Podilymbus podiceps). DDE and PCBs were present in all samples analyzed, whereas dieldrin, mirex and oxychlordane were occasionally present at low levels. Mercury was present at low levels in all samples for which it was analyzed. Red-necked grebes nesting in Manitoba had the highest contaminant levels and, assuming that contaminant burdens were accumulated principally on the wintering grounds, the mean PCB:DDE ratio (3.1) indicated that these birds and those from sites in central and eastern Saskatchewan probably wintered on the Atlantic coast. Overall low contaminant levels and a low mean PCB:DDE ratio (1.4) in the eggs of red-necked grebes breeding in Alberta and western Saskatchewan suggested that these birds wintered on the Pacific coast. A similar pattern was apparent in horned and eared grebes. Concentrations of DDE and PCB were both significantly correlated with Ratcliffe index (shell thickness), and were strongly correlated with each other. Ratcliffe indices were determined for historical collections of red-necked grebe eggs. Eggshell thickness of grebes nesting in Manitoba declined significantly during the years following the introduction of DDT (post-1947) and has only recovered partially since it was banned in 1972. The Alberta-breeding population did not appear to have undergone any significant decrease in shell thickness.

Influence of vaccine medium and vaccination schedules on the induction of active immunity against Aujeszky's disease in maternally immune pigs.

Active immunity against Aujeszky's disease virus (ADV) was compared at the end of the fattening period in pigs which had been vaccinated with the attenuated Bartha strain according to different schedules in the presence of different levels of maternal immunity. The percentage of seropositive pigs at the end of the fattening period varied from 21 to 94 per cent. The percentage was significantly higher when the vaccination schedules were applied to pigs from mothers vaccinated with an attenuated strain compared to pigs from mothers vaccinated with a subunit vaccine or from infected-immune mothers. Additionally, this percentage was two to three times lower when pigs were vaccinated once at 10 weeks old compared to pigs either revaccinated at 14 weeks or vaccinated once at 14 weeks old. When the virus strain used for vaccination had been suspended either in saline or in an oil-in-water emulsion, significant differences were not found in the serological response after vaccination and in the reduction of virus excretion upon subsequent challenge. In challenge experiments, a significantly longer duration of virus excretion was observed in vaccinated pigs which had not seroconverted than in vaccinated but seropositive pigs. The vaccination schedules for sows and fattening pigs in view of the eradication of ADV are discussed.

Field experiments to evaluate the feasibility of eradication of Aujeszky's disease virus by different vaccination schedules.

In the present report, the extent of the reduction in Aujeszky's disease virus (ADV) dissemination achieved when pigs were intensively vaccinated with gI-deleted vaccines under field circumstances, was examined. On widely dispersed breeding-fattening farms, a gI-negative status was most rapidly obtained and the rate of new waves of infections was lowest when the attenuated Bartha strain was administered to both the sows and the fatteners. It was more difficult not only to reach but also to keep a gI-negative status on farms on which the sows were vaccinated with an inactivated vaccine and the fatteners with the attenuated Bartha strain or when the fattening pigs were not vaccinated at all. In a densely populated area, 9 of the 17 farms had gI-positive fatteners at the start of the intensive vaccination programme in which the attenuated Bartha strain was given to both the sows and the fatteners. Antibodies were not detected in the sera of the fatteners of each farm at some time during the experiments, but the fatteners on 7 of the 18 farms still showed antibodies against gI after 20 months of vaccination. At the end of the experiment, the percentage of fatteners with antibodies on these farms was markedly reduced compared with the percentage at the start of the experiment. Therefore, elimination of field virus may be feasible if intensive vaccination is carried out over a sufficiently long period of time. However, the high rate of reinfections experienced either due to reintroduction of the virus or to recrudescence should be a warning against too much optimism, particularly in regions with a dense swine population.