From 1995 to 1997 several defined species like V. alginolyticus, V. anguillarum, V. cholerae (non O1 and non O139), V. mimicus, V. parahaemolyticus and V. vulnificus were isolated during a survey to ...determine the presence of V. vulnificus in the brackish water of the Baltic Sea in Germany. Moreover atypical Vibrio isolates were detected. Four isolates belonging to a group of atypical Vibrio and possibly representing a new species in the genus Vibrio were characterized in detail. All four strains were isolated from surface costal waters. Based on 16S rDNA sequence analysis they showed the highest relatedness to the species V. navarrensis and V. vulnificus. The strains did not harbor the species specific hemolysin gene vvhA from V. vulnificus as shown by PCR and hybridization experiments. Moreover, they differed in at least two biochemical parameters tested from the hitherto described Vibrio species. All these strains induced hemolysis on washed blood agar dishes and showed phase variations on Luria Bertani agar dishes. Because of the similarity to the eel pathogen V. vulnificus, we infected eels with one of the four atypical strains (CH-291), but no pathogenicity for eels could be detected. Furthermore, Vero cell tests with supernatants of bacterial cultures did not reveal secreted Vero cell cytotoxic compounds. This indicates a nonpathogenic nature of these strains.
In recent years, epitopes of various origin have been inserted into the core protein of hepatitis B virus (HBc), allowing the formation of chimeric HBc particles. Although the C-terminus of a ...C-terminally truncated HBc (HBc) tolerates the insertion of extended foreign sequences, the insertion capacity is still a limiting factor for the construction of multivalent vaccines. Previously, we described a new system to generate HBc mosaic particles based on a read-through mechanism in an Escherichia coli suppressor strain J Gen Virol 1997;78:2049-2053. Those mosaic particles allowed the insertion of a 114-amino acid (aa)-long segment of a Puumala hantavirus (PUUV) nucleocapsid (N) protein. To study the value and the potential limitations of the mosaic approach in more detail, we investigated the assembly capacity of 'non-mosaic' HBc fusion proteins and the corresponding mosaic constructs carrying 94, 213 and 433 aa of the hantaviral N protein. Whereas the fusion proteins carrying 94, 114, 213 or 433 aa were not assembled into HBc particles, or only at a low yield, the insertion of a stop codon-bearing linker restored the ability to form particles with 94, 114 and 213 foreign aa. The mosaic particles formed exhibited PUUV-N protein antigenicity. Immunization of BALB/c mice with these mosaic particles carrying PUUV-N protein aa 1-114, aa 1-213 and aa 340-433, respectively, induced HBc-specific antibodies, whereas PUUV-N protein-specific antibodies were detected only in mice immunized with particles carrying N-terminal aa 1-114 or aa 1-213 of the N protein. Both the anti-HBc and anti-PUUV antibody responses were IgG1 dominated. In conclusion, stop codon suppression allows the formation of mosaic core particles carrying large-sized and 'problematic', e.g. hydrophobic, hantavirus sequences.
Summary Background Pazopanib, a multitargeted tyrosine kinase inhibitor, has single-agent activity in patients with advanced non-adipocytic soft-tissue sarcoma. We investigated the effect of ...pazopanib on progression-free survival in patients with metastatic non-adipocytic soft-tissue sarcoma after failure of standard chemotherapy. Methods This phase 3 study was done in 72 institutions, across 13 countries. Patients with angiogenesis inhibitor-naive, metastatic soft-tissue sarcoma, progressing despite previous standard chemotherapy, were randomly assigned by an interactive voice randomisation system in a 2:1 ratio in permuted blocks (with block sizes of six) to receive either pazopanib 800 mg once daily or placebo, with no subsequent cross-over. Patients, investigators who gave the treatment, those assessing outcomes, and those who did the analysis were masked to the allocation. The primary endpoint was progression-free survival. Efficacy analysis was by intention to treat. The trial is registered with ClinicalTrials.gov , number NCT00753688. Findings 372 patients were registered and 369 were randomly assigned to receive pazopanib (n=246) or placebo (n=123). Median progression-free survival was 4·6 months (95% CI 3·7–4·8) for pazopanib compared with 1·6 months (0·9–1·8) for placebo (hazard ratio HR 0·31, 95% CI 0·24–0·40; p<0·0001). Overall survival was 12·5 months (10·6–14·8) with pazopanib versus 10·7 months (8·7–12·8) with placebo (HR 0·86, 0·67–1·11; p=0·25). The most common adverse events were fatigue (60 in the placebo group 49% vs 155 in the pazopanib group 65%), diarrhoea (20 16% vs 138 58%), nausea (34 28% vs 129 54%), weight loss (25 20% vs 115 48%), and hypertension (8 7% vs 99 41%). The median relative dose intensity was 100% for placebo and 96% for pazopanib. Interpretation Pazopanib is a new treatment option for patients with metastatic non-adipocytic soft-tissue sarcoma after previous chemotherapy. Funding GlaxoSmithKline.
Oral anticancer drugs show a high interpatient variability in pharmacokinetics (PK), leading to large differences in drug exposure. For many of these drugs, exposure has been linked to efficacy and ...toxicity. Despite this knowledge, these drugs are still administered in a one-size-fits-all approach. Consequently, individual patients have a high probability to be either underdosed, which can lead to decreased antitumor efficacy, or overdosed, which could potentially result in increased toxicity. Therapeutic drug monitoring (TDM), personalized dosing based on measured drug levels, could be used to circumvent underdosing and overdosing and thereby optimize treatment outcomes.
In this prospective clinical study (www.trialregister.nl; NL6695), the feasibility, tolerability, and efficacy of TDM of oral anticancer drugs will be evaluated. In total, at least 600 patients will be included for (at least) 23 different compounds. Patients starting regular treatment with one of these compounds at the approved standard dose can be included. PK sampling will be performed at 4, 8, and 12 weeks after the start of treatment and every 12 weeks thereafter. Drug concentrations will be measured, and trough concentrations (Cmin) will be calculated. In cases where Cmin falls below the predefined target and acceptable toxicity, a PK-guided intervention will be recommended. This could include emphasizing compliance, adapting concomitant medication (due to drug-drug interactions), instructing to take the drug concomitant with food, splitting intake moments, or recommending a dose increase.
Despite a strong rationale for the use of TDM for oral anticancer drugs, this is currently not yet widely adopted in routine patient care. This prospective study will be a valuable contribution to demonstrate the additional value of dose optimization on treatment outcome for these drugs.
Tamoxifen is widely prescribed as adjuvant therapy in patients with early-stage breast cancer. It has been postulated that concentrations of endoxifen, the active metabolite of tamoxifen, are a ...better predictor of tamoxifen efficacy than CYP2D6 genotypes. Although in a retrospective study, an endoxifen threshold of 5.9 ng/mL for efficacy was described, confirmation based on prospective studies is lacking. The objective of the prospective CYPTAM (The Netherlands National Trial Register: NTR1509) study was to associate endoxifen concentrations and CYP2D6 genotypes with clinical outcome in patients with early-stage breast cancer receiving tamoxifen.
From February 2008 to December 2010, patients with breast cancer treated with adjuvant tamoxifen were included. Patients could be enrolled up to a maximum of 12 months after tamoxifen initiation. Blood samples were retrieved for CYP2D6 genotyping and endoxifen measurements by Amplichip (Roche Diagnostics, Indianapolis, IN) and high-performance liquid chromatography-tandem mass spectrometry, respectively. Endoxifen concentrations were analyzed as a continuous variable, classifying patients into quartiles and using an endoxifen threshold of 5.9 ng/mL. Endoxifen concentrations and CYP2D6 genotypes were associated with relapse-free survival (censored at the time of tamoxifen discontinuation; RFSt) by Cox regression analysis.
A total of 667 pre- and postmenopausal patients were enrolled and had received tamoxifen for a median time of 0.37 years (range, 0.23 to 0.6 years) before study entry. No association was found between endoxifen concentrations and RFSt (adjusted hazard ratio, 0.991; 95% CI, 0.946 to 1.038; P = .691). Also, neither categorizing endoxifen concentrations into quartiles nor using 5.9 ng/mL as threshold altered these results. In addition, no association was found between CYP2D6 genotype and RFSt (adjusted hazard ratio, 0.929; 95% CI, 0.525 to 1.642; P = .799).
This prospective clinical study shows no association between endoxifen concentrations or CYP2D6 genotypes and clinical outcome in patients with early-stage breast cancer receiving adjuvant tamoxifen.
Ultrathin section and surface replica electron microscopy were applied in combination with immunoelectron microscopy to elucidate the fine structure of HIV. The shell of the tubular core shows p24 ...antigenicity, while p17 is located at the inner leaflet of the lipid membrane. The virus particle is studded with 70–80 protrusions. These knobs have a diameter of 15 nm, a height of 9 nm, and are probably arranged in a
T = 7 I symmetry. The major envelope protein gpl20 is spontaneously shed from the viral surface. A possible role of released gpl20 in pathogenesis is discussed.
Hepatitis B virus (HBV) core-derived chimeric particles carrying a Puumala (PUU) hantavirus (strain Vranica/Hällnäs) nucleocapsid (N) protein sequence (aa 1–45), alternatively inserted at three ...distinct positions (N-, C-terminus, or the internal region), and mosaic particles consisting of HBV core as well as core/PUU (Vranica/Hällnäs) N (aa 1–45) readthrough protein were generated. Chimeric particles carrying the insert at the N-terminus or the internal region of core induced some protective immune response in bank voles (Clethrionomys glareolus) against a subsequent PUU virus (strain Kazan) challenge; 40–50% of the animals showed markers of protection. In contrast, internal insertion of PUU strain CG18–20 N (aa 1–45) into the HBV core caused a highly protective immune response in the bank vole model. Immunizations with particles carrying aa 75–119 of PUU (CG18–20) N at the C-terminus of core verified the presence of a second, minor protective region in the N protein. A strong PUU N-specific antibody response was detected not only in bank voles immunized with chimeric particles containing internal and N-terminal fusions of PUU N protein but also in animals immunized with the corresponding mosaic particles. Except for the exclusive occurrence of antibodies directed against aa 231–240 of N in non-protected animals post virus challenge, there was no additional obvious difference in the epitope-specificity of N-specific antibodies from immunized animals prior and post virus challenge.
Segments of the human immunodeficiency virus (HIV) type 1
gag and
pol genes and mutants thereof were transiently expressed in mammalian cells. Expression was dependent on the presence of the rev ...responsive element in
cis and the rev protein in
trans and was readily detected by indirect immunofluorescence or Western blotting. Transfection of constructs encoding the entire
gag and
pol open reading frames yielded efficient release of particles banding at a density of 1.16 g of sucrose per milliliter and consisting mainly of processed gag proteins. In addition, these particles contained the p66/p51 heterodimer of reverse transcriptase (RT), had associated RT activity, and contained RNA. Electron micrographs revealed immature retrovirus-like particles budding primarily from the plasma membrane and extracellular particles with morphological characteristics of HIV. Particle production was independent of the
pol open reading frame or an active HIV proteinase (PR) but without active PR, cell-associated and particle-associated proteins remained completely uncleaved and budding occurred primarily into intracellular vacuoles. A mutation preventing myristoylation of the viral polyproteins abolished particle release but did not interfere with polyprotein synthesis and did not prevent processing. Expression of
gag and PR in the same reading frame yielded complete processing of polyproteins but no budding and led to increased cell toxicity. A mutation of the PR active site in this construct prevented cytotoxicity and restored particle release indicating that the observed phenotype was caused by the overexpression of PR. These particles were aberrant in size and morphology when analyzed on sucrose density gradients and by electron microscopy. Budding was arrested at an early stage and extracellular particles appeared to be released by a different mechanism. Only short C-terminal extensions were compatible with this release mechanism since expression of a similar mutant construct encoding the entire
gag-pol open reading frame did not yield particles.
Diagnostic electron microscopy has two advantages over enzyme-linked immunosorbent assay and nucleic acid amplification tests. After a simple and fast negative stain preparation, the undirected, ...“open view” of electron microscopy allows rapid morphologic identification and differential diagnosis of different agents contained in the specimen. Details for efficient sample collection, preparation, and particle enrichment are given. Applications of diagnostic electron microscopy in clinically or epidemiologically critical situations as well as in bioterrorist events are discussed. Electron microscopy can be applied to many body samples and can also hasten routine cell culture diagnosis. To exploit the potential of diagnostic electron microscopy fully, it should be quality controlled, applied as a frontline method, and be coordinated and run in parallel with other diagnostic techniques.
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Dostopno za:
DOBA, IZUM, KILJ, NUK, ODKLJ, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
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