Minimizing inequality in access to precision medicine in breast cancer by real-time population-based molecular analysis in the SCAN-B initiative.

BACKGROUND: Selection of systemic therapy for primary breast cancer is currently based on clinical biomarkers along with stage. Novel genomic tests are continuously being introduced as more precise tools for guidance of therapy, although they are often developed for specific patient subgroups. The Sweden Cancerome Analysis Network - Breast (SCAN-B) initiative aims to include all patients with breast cancer for tumour genomic analysis, and to deliver molecular subtype and mutational data back to the treating physician. METHODS: An infrastructure for collection of blood and fresh tumour tissue from all patients newly diagnosed with breast cancer was set up in 2010, initially including seven hospitals within the southern Sweden regional catchment area, which has 1.8 million inhabitants. Inclusion of patients was implemented into routine clinical care, with collection of tumour tissue at local pathology departments for transport to the central laboratory, where routines for rapid sample processing, RNA sequencing and biomarker reporting were developed. RESULTS: More than 10 000 patients from nine hospitals have currently consented to inclusion in SCAN-B with high (90 per cent) inclusion rates from both university and secondary hospitals. Tumour samples and successful RNA sequencing are being obtained from more than 70 per cent of patients, showing excellent representation compared with the national quality registry as a truly population-based cohort. Molecular biomarker reports can be delivered to multidisciplinary conferences within 1 week. CONCLUSION: Population-based collection of fresh tumour tissue is feasible given a decisive joint effort between academia and collaborative healthcare groups, and with governmental support. An infrastructure for genomic analysis and prompt data output paves the way for novel systemic therapy for patients from all hospitals, irrespective of size and location.

G protein-coupled estrogen receptor 1 (GPER, GPR 30) in normal human endometrium and early pregnancy decidua.

The recently identified trans-membrane G protein-coupled estrogen receptor 1 (GPER, GPR30) has been implicated in rapid non-genomic effects of estrogens. This focuses on expression and localization of GPER mRNA and protein in normal cyclic endometrium and early pregnancy decidua. Real-time PCR, western blotting, in situ hybridization and immuno-histochemistry were used. Endometrial expression of GPER mRNA was lower in the secretory phase than in the proliferative phase, and even lower in the decidua. The expression pattern was similar to that of ERα mRNA, but different from that of ERß mRNA. Western blot detected GPER protein as a 54 kDa band in all endometrial and decidual samples. In contrast to the mRNA, GPER protein did not show cyclic variations. Apparently, a lower amount of mRNA is sufficient to maintain protein levels in the secretory phase. GPER mRNA was predominantly localized in the epithelium of mid- and late-proliferative phase endometrium, whereas expression in early proliferative and secretory glands could not be distinguished from the diffuse stromal signal, which was present throughout the cycle. Immuno-staining for GPER was stronger in glandular and luminal epithelium than in the stroma throughout the cycle. The cyclic variations of GPER mRNA obviously relate to strong epithelial expression in the proliferative phase, and the expression pattern suggests regulation by ovarian steroids. GPER protein is present in endometrial tissue throughout the cycle, and the epithelial localization suggests potential functions during sperm migration at mid-cycle, as well as decidualization and blastocyst implantation in the mid-secretory phase.

Tissue distribution of cefquinome after intramammary and "systemic" administration in the isolated perfused bovine udder.

Mammary glands taken at slaughter from healthy lactating cows were perfused in vitro with warmed and gassed Tyrode solution. Cefquinome (88.8mg cefquinome sulphate per 8mL) was administered by the intramammary route to all quarters and/or "systemically" via the perfusion fluid at concentrations similar to those measured in plasma following intramuscular administration of 1mg cefquinome per kg body weight. Samples of the perfusate were taken over a 6-h period and from the regional lymph nodes after 6h. Using a scalpel, sections of glandular tissue - at different distances from and vertical to the teat right up to the udder base - were gathered from four quarters each per route of administration at 2, 4 and 6h. The cefquinome content of the tissue samples was analysed by high performance liquid chromatography with diode array detection and of the perfusate samples by bioassay. After intramammary administration, the concentration of cefquinome in the glandular tissue decreased exponentially with increasing distance from the teat. The addition of cefquinome to the perfusion fluid produced a mean concentration of 0.2-0.5microg/g at all glandular tissue sites. Combined intramammary and systemic treatment ensured that concentrations exceeded the MIC(90) values of the most common mastitis pathogens in all areas of the udder by 2h post-administration. There was considerable variability in the tissue concentrations of cefquinome, particularly after intramammary administration. These results suggest that for the treatment of acute mastitis a combination of both intramammary and systemic administration is likely to be advantageous in order to rapidly produce maximum cefquinome concentrations in all regions of the udder.

Differential localization and expression of urokinase plasminogen activator (uPA), its receptor (uPAR), and its inhibitor (PAI-1) mRNA and protein in endometrial tissue during the menstrual cycle.

Normal endometrium is a highly dynamic tissue, which responds to ovarian steroids with cyclic proliferation, differentiation (secretion), and degradation (menstruation). The urokinase plasminogen activator (uPA)-dependent proteolytic cascade as well as ligand activation of the uPA receptor (uPAR) is critically involved in physiological as well as pathophysiological aspects of tissue expansion and remodelling. Cyclic variation and distribution of uPA, uPAR and plasminogen activator inhibitor 1 (PAI-1) mRNA were examined by in situ hybridization, real-time PCR and northern blot in normal endometrium. Their corresponding proteins were localized with immunohistochemistry. uPA mRNA is exclusively expressed by stromal cells, whereas uPA protein is present in both epithelial and stromal cells. Immunostaining for uPA protein is reduced or undetectable at midcycle, thus coinciding with peak concentration of uPA in the uterine fluid. uPAR mRNA is expressed by epithelial cells in the proliferative phase and by stromal cells in the secretory phase. However, epithelial cells stain for uPAR protein throughout the cycle, suggesting that uPAR may detach from stromal cells and then bind to epithelial cells in the secretory phase. PAI-1 mRNA is located in vessel walls. The late secretory phase has greatly increased expression of all three mRNA and their proteins, mainly in pre-decidual cells in the superficial stroma. Discordant localization of the mRNA and proteins suggest that uPA is produced by stromal cells, released and bound to epithelial cells in both the proliferative and secretory phases, whereas uPAR is released from the stroma and bound to epithelial cells in the secretory phase. Also, the present data together with earlier reports suggest that uPA is released from the epithelial cells to the uterine fluid.

Concentration of enrofloxacin and its metabolite ciprofloxacin in canine matrices of the locomotor system.

Due to its pharmacodynamic and pharmacokinetic properties, the use of enrofloxacin may be indicated in canine osteomyelitis, but there is insufficient data on its distribution within the musculoskeletal tissues. The dogs used in this study were 31 regular veterinary orthopaedic patients. Four hours after their oral or subcutaneous treatment with 10 mg/kg enrofloxacin (Baytril; Bayer, Leverkusen, Germany) once daily for 1 or 3 days, the concentration of enrofloxacin and its main metabolite ciprofloxacin was quantified in plasma, bone, musculature and other matrices of the locomotor system by high pressure liquid chromatography with fluorescence-detection after homogenization and solid phase extraction of the samples. By oral or subcutaneous administration of enrofloxacin once daily for 3 days, higher concentrations of the active constituents in the samples were achieved than by single treatment. Nevertheless, even after single injection, minimal inhibitory enrofloxacin concentrations of up to 0.5 microg ml or microg/g sample against most pathogens of osteomyelitis were exceeded. In the musculature, on average, higher concentrations of active constituents were detected than in less perfused matrices (bones and synovial membranes) at sampling time. The enrofloxacin diffusion into inflamed bone was higher compared with mechanically damaged bone, whereas for ciprofloxacin it was lower. In conclusion, a dosage of 10 mg/kg enrofloxacin is sufficient to exceed the minimal inhibitory concentrations in osteomyelitic bone against most pathogens that are sensitive in vitro, but clinical efficacy remains to be evaluated.

[Pharmacokinetics of cephalexin from two oral formulations in dogs]. / Pharmakokinetik von Cephalexin aus zwei oralen Formulierungen bei Hunden.

Six beagle dogs were treated with cephalexin-monohydrate from 2 oral formulations (Rilexine tablets and Cefaseptin dragees, respectively) in a dosage of 25 mg/kg and plasma concentrations of cephalexin were measured over 8 hours. After solid phase extraction of the samples, cephalexin was determined by high pressure liquid chromatography with UV detection. After administration, Cephalexin was absorbed rapidly and mean maximum plasma concentrations of 30.9 and 27.9 micrograms/ml, respectively, were acquired after approximately 1.6 hours. Minimal inhibitory concentrations of < or = 6.25 micrograms/ml for in vitro sensitive bacteria were maintained for about 5 hours. Cephalexin from the tested preparations reached a mean area under the plasma concentration-time curve of 115.3 and 102.4 micrograms.h/ml, respectively. The plasma concentration decreased rapidly with a mean half life period of 1.4 hours in average. The other calculated pharmacokinetic parameters were also in the area of the data for dogs stated in the literature. There was no clear difference in the pharmacokinetics of both products, especially the bioavailability. Furthermore, both formulations were well tolerated clinically.

[The tissue distribution of antibiotics in the udder--comparison of the situation in vivo with the isolated perfused bovine udder]. / Untersuchung der Gewebeverteilung von Antibiotika im Euter--Vergleich der In-vivo-Situation zum isoliert perfundierten Rindereuter.

Especially for animal protection reasons, tissue concentrations of intracisternally administered antibiotics in the mammary gland hardly can be determined in the live cow. Therefore, this paper assessed the use of the isolated perfused bovine udder to study the distribution of penicillins in glandular tissue. With this intention, the in vitro results acquired with this model were compared to tissue concentrations as well as absorption data from the few in vivo studies in the literature and differences were interpreted. This approach must consider inevitable deviations of experimental materials and methods. Furthermore, in vivo the udder is included in a closed circulatory system with other metabolism and excretion feasibilities than the isolated model. Moreover, the lower flow rate in the vessels in vitro has to be taken into account with respect to absorption capacities. Nevertheless, the tissue concentrations and the distribution equilibrium across the blood-udder-barrier in both experimental concepts corresponded with each other, if the deviating conditions are considered by calculating correction factors. Advantages and disadvantages of the isolated udder are discussed critically. In conclusion, this method is a useful completion of pharmacokinetic in vivo studies that are supposed to compare intracisternally administered formulations.

Paracrine stimulation of capillary endothelial cell migration by endometrial tissue involves epidermal growth factor and is mediated via up-regulation of the urokinase plasminogen activator receptor.

Endometrial angiogenesis is not well studied, but has potential as a model for studies of physiological angiogenesis. Migration as well as proliferation of vascular endothelial cells are modulated by other endometrial cells. This study analyzes the chemotactic signal released from endometrial tissue in a wound assay using human microvascular endothelial cells. Endometrial tissue explants stimulate migration, and this effect is significantly weaker with explants taken at midcycle than those obtained earlier or later in the cycle. Migration is inhibited more than 50% by either blocking antibodies to the urokinase plasminogen activator receptor (uPAR) or enzymatic removal of uPAR from the cell surface. Also, migration is inhibited more than 50% by antibodies to epidermal growth factor (EGF), but not by antibodies to vascular endothelial growth factor or basic fibroblast growth factor. The combination of anti-EGF and anti-uPAR antibodies does not further reduce the response, suggesting that these antibodies target a common pathway. Conditioned medium from endometrial explants contains EGF, and EGF stimulates the migration of endothelial cells in a dose-dependent way. This effect is completely blocked by antibodies to uPAR. These data suggest up-regulation of the uPA system by EGF. Conditioned medium from EGF-treated cells contains less uPA than medium from control cells. In contrast, binding of radiolabeled uPA reveals an increased number of uPA-binding sites in EGF-treated cells. Increased expression of uPAR potentially increases the activation and assembly of focal adhesion sites, a prerequisite for cell migration. We conclude that the endometrial migratory signal has two components. The major part of the signal is blocked by antibodies to EGF, and the response is mediated via up-regulation of uPAR in the endothelial cells. The other part of the signal is unknown, and the response does not involve uPAR. Decreased endometrial chemotactic signal at midcycle is probably related to decreased release of EGF, which is secondary to increased binding to endometrial cell membranes.

[Pharmacokinetics of kanamycin after subcutaneous and intravenous administration in dogs]. / Pharmakokinetik von Kanamycin nach subkutaner und intravenöser Applikation beim Hund.

Six beagle dogs were treated with kanamycin subcutaneously or intravenously in a dosage of 5 mg/kg. The plasma kanamycin concentration was measured over 24 hours by high pressure liquid chromatography with UV detection after derivatization and solid phase extraction. After subcutaneous application, kanamycin was absorbed quickly, and maximum plasma levels of 18.9 micrograms/ml in average after ca. 1 hour were measured. With complete systemic availability, the minimal inhibitory concentration of 4 micrograms/ml was maintained for 4 hours. After subcutaneous administration, kanamycin was terminally eliminated with a mean half life period of 2 hours.

Tissue distribution of benzylpenicillin after intramammary administration in the isolated perfused bovine udder.

Udders from previously healthy lactating cows were perfused with warmed and gassed Tyrode solution in vitro. Benzylpenicillin was administered in three formulations: an oily suspension with micronized particles of <10 microm diameter, an oily suspension with average particle size of 40 microm and an aqueous solution (3 million IU benzylpenicillin-potassium, volume 15 mL). The antibiotics were administered intracisternally to six front and six rear quarters each. Moreover, a dry-off-ointment (100 000 IU benzylpenicillin-potassium and 100 000 IU benzylpenicillin-benzathine, volume 7.5 mL) was tested in four udder halves. Perfusate samples were collected over 3 h. Furthermore, glandular tissue at different vertical distances from the teat base and the regional lymph node were sampled after 3 h. The determination of benzylpenicillin was performed by high pressure liquid chromatography with UV detection. With increasing distance from the teat base, the concentration of benzylpenicillin in tissue exponentially decreased. Using the aqueous solution or oily suspension that contained micronized active principle, higher concentrations were reached compared to the formulation with particle sizes of 40 microm. In udder lymph nodes, the concentration was highest after treatment with the coarse suspension. The transfer from the dry-off-ointment with benzathine-salt into perfusate was very low. These results suggest that it is possible to study tissue distribution of antibiotics in the isolated perfused bovine udder.

Tissue distribution of oxacillin and ampicillin in the isolated perfused bovine udder.

In vivo, tissue distribution of intra-mammarily administered antibiotics is mostly only assessed by sampling milk and blood. Therefore, the described study analysed whether measurement of tissue concentrations makes sense in vitro instead. Isolated bovine udders were perfused with gassed and warmed Tyrode solution. To four front and rear quarters each, 1000 mg oxacillin in 7.5 ml vehicle was administered intracisternally, completely formulated as sodium monohydrate in two lactation ointments (with or without sodium dodecylsulphate) or 80% as benzathine salt in a dry-off ointment. Over 3 h, perfusate and glandular tissue from different locations were sampled and analysed by high pressure liquid chromatography. With increasing vertical distance to the teat base, the tissue concentration of antibiotics decreased. With the lactation ointment containing sodium dodecylsulphate, lower oxacillin concentrations were reached in glandular tissue and lymph nodes compared to those without. The ointments led to a higher recovery of oxacillin in glandular tissue than in perfusate. Aluminium monostearate in the dry-off ointment caused an even poorer absorption of oxacillin into perfusate. The isolated perfused bovine udder is suitable to study the tissue distribution of antibiotics, since the results were mainly comparable with the few existing in vivo studies and show the influence of different formulations.

Effects of the phosphodiesterase 4 inhibitor RPR 73401 in a model of immunological inflammation.

The study was performed to investigate effects of the phosphodiesterase 4 inhibitor RPR 73401 [N-(3, 5-dichloropyrid-4-yl)-3-cyclopentyl-oxy-4-methoxybenzamid] on an allergic skin reaction. To simulate an immunological inflammation, BALB/c mice were sensitized to dinitrochlorobenzene or toluenediisocyanate. At first, the abdominal skin was shaved and 50 microliter Freund's adjuvant were injected intracutaneously once. Then, the horny layer was removed by adhesive tape stripping and 100 microliter 0.5% dinitrochlorobenzene or 5% toluenediisocyanate were administered on the epidermis for 4 days. After repeated local treatment of the ear skin with 20 microliter 3% RPR 73401 or intraperitoneal administration of 1 and 5 mg/kg RPR 73401, 20 microliter 1% dinitrochlorobenzene or 0.5% toluenediisocyanate were given topically as a challenge. The vehicle controls showed a high increase in ear thickness over 48 h after challenge, whereas RPR 73401 administered on either route reduced this increase significantly. Nevertheless after topical administration, RPR 73401 had a longer lasting effect. These and other results may point to an indication for RPR 73401 in immunological dermatitis.

[Pharmacokinetic aspects of mastitis therapy]. / Pharmakokinetische Aspekte der Mastitistherapie.

Treating mastitis in dairy cattle, most often antibiotics are used systemically or intracisternally, supported by influencing the inflammatory process with other measures. The success of systemic treatment is influenced by the bioavailability of the chemotherapeutic agent. Here, apart from the molecular size and protein binding, the fat-solubility of the active principle as well as its degree of ionisation play a major role. The degree of ionisation is determined by the isoelectric point (pKa) of the substance and by the pH-value of the surrounding medium. Thus, weak acids are mainly ionised in blood and cannot be transferred into udder tissue and milk. Weak bases react in the opposite way. Applied locally, the drug must not irritate the udder to secure deep diffusion into the glandular tissue. Therefore, topically administered drugs are formulated on an oily basis, also for longer prophylaxis during the dry-off-period. The pharmaceutic and pharmacokinetic relevance of the chemical and physical factors as well as the influence of the particle size of the active principle were demonstrated with benzylpenicillin potassium in the in vitro model of the isolated perfused bovine udder.

Sequence, organization and expression of the core histone genes of Aspergillus nidulans.

The core histone gene family of Aspergillus nidulans was characterized. The H2A, H2B and H3 genes are unique in the A. nidulans genome. In contrast there are two H4 genes, H4.1 and H4.2. As previously reported for the H2A gene (May and Morris 1987) introns also interrupt the other core histone genes. The H2B gene, like the H2A gene, is interrupted by three introns, the H3 and H4.1 gene are each interrupted by two introns and the H4.2 gene contains one intron. The position of the single intron in H4.2 is the same as that the first intron of the H4.1 gene. The H2A and H2B genes are arranged as a gene pair separated by approximately 600 bp and are divergently transcribed. The H3 and H4.1 genes are similarly arranged and are separated by approximately 800 bp. The H4.2 gene is not closely linked to either the H2A-H2B or H3-H4.1 gene pairs. Using pulse field gel electrophoresis an electrophoretic karyotype was established for A. nidulans. This karyotype was used to assign the H3-H4.1 gene pair and the H4.2 gene to linkage group VIII and the H2A-H2B gene pair to either linkage group III or VI. The abundance of each of the histone messenger RNAs was determined to be cell cycle regulated but the abundance of the H4.2 mRNA appears to be regulated differently from the others.