The pan-ErbB receptor tyrosine kinase inhibitor canertinib promotes apoptosis of malignant melanoma in vitro and displays anti-tumor activity in vivo

The ErbB receptor family has been suggested to constitute a therapeutic target for tumor-specific treatment of malignant melanoma. Here we investigate the effect of the pan-ErbB tyrosine kinase inhibitor canertinib on cell growth and survival in human melanoma cells in vitro and in vivo. Canertinib significantly inhibited growth of cultured melanoma cells, RaH3 and RaH5, in a dose-dependent manner as determined by cell counting. Half-maximum growth inhibitory dose (IC₅₀) was approximately 0.8 μM and by 5 μM both cell lines were completely growth-arrested within 72 h of treatment. Incubation of exponentially growing RaH3 and RaH5 with 1 μM canertinib accumulated the cells in the G₁-phase of the cell cycle within 24 h of treatment without induction of apoptosis as determined by flow cytometry. Immunoblot analysis showed that 1 μM canertinib inhibited ErbB1-3 receptor phosphorylation with a concomitant decrease of Akt-, Erk1/2- and Stat3 activity in both cell lines. In contrast to the cytostatic effect observed at doses ?5 μM canertinib, higher concentrations induced apoptosis as demonstrated by the Annexin V method and Western blot analysis of PARP cleavage. Furthermore, canertinib significantly inhibited growth of RaH3 and RaH5 melanoma xenografts in nude mice. Pharmacological targeting of the ErbB receptors may prove successful in the treatment of patients with metastatic melanoma.     

Validation of the IHE Cohort Model of Type 2 Diabetes and the Impact of Choice of Macrovascular Risk Equations

Background

Health-economic models of diabetes are complex since the disease is chronic, progressive and there are many diabetic complications. External validation of these models helps building trust and satisfies demands from decision makers. We evaluated the external validity of the IHE Cohort Model of Type 2 Diabetes; the impact of using alternative macrovascular risk equations; and compared the results to those from microsimulation models.

Methods

The external validity of the model was analysed from 12 clinical trials and observational studies by comparing 167 predicted microvascular, macrovascular and mortality outcomes to the observed study outcomes. Concordance was examined using visual inspection of scatterplots and regression-based analysis, where an intercept of 0 and a slope of 1 indicate perfect concordance. Additional subgroup analyses were conducted on ‘dependent’ vs. ‘independent’ endpoints and microvascular vs. macrovascular vs. mortality endpoints.

Results

Visual inspection indicates that the model predicts outcomes well. The UKPDS-OM1 equations showed almost perfect concordance with observed values (slope 0.996), whereas Swedish NDR (0.952) and UKPDS-OM2 (0.899) had a slight tendency to underestimate. The R ² values were uniformly high (>0.96). There were no major differences between ‘dependent’ and ‘independent’ outcomes, nor for microvascular and mortality outcomes. Macrovascular outcomes tended to be underestimated, most so for UKPDS-OM2 and least so for NDR risk equations.

Conclusions

External validation indicates that the IHE Cohort Model of Type 2 Diabetes has predictive accuracy in line with microsimulation models, indicating that the trade-off in accuracy using cohort simulation might not be that large. While the choice of risk equations was seen to matter, each were associated with generally reasonable results, indicating that the choice must reflect the specifics of the application. The largest variation was observed for macrovascular outcomes. There, NDR performed best for relatively recent and well-treated patients, while UKPDS-OM1 performed best for the older UKPDS cohort.     

Survival possibilities of the dragonfly <Emphasis Type="Italic">Aeshna viridis</Emphasis> (Insecta,Odonata) in southern Sweden predicted from dispersal possibilities

We use public records from 1980 to 2014 to analyse survival of the EU Annex IV species Aeshna viridis in Sweden, a dragonfly strongly associated with the plant Stratiotes aloides. We clustered localities with S. aloides based on assumed dispersal abilities of A. viridis, using a dispersing radius of 2–100 km, calculating the proportion of sites with S. aloides that A. viridis is able to reach. If mean dispersal capability is high (40 km or above) 92.6 % or more of the localities are connected. For a good disperser, the probability of long-time survival is good. We further analysed the species richness of other Odonata and aquatic plants at 98 localities from the dataset. A. viridis co-occurred with more Odonata in the presence of S. aloides and running water but not in lakes. S. aloides sites had a higher number of other aquatic plants. Area had no impact on the occurrence of the species. For the present situation we surveyed 32 localities with known occurrence of the species. Only half of the sites for S. aloides contained any specimens while A. viridis occurred in the same number of sites. The species co-occurred in only 8 of 32 sites. In four sites A. viridis larvae appeared among Menyanthes trifoliata, Phragmites australis, Potamogeton natans and Sphagnum spp., indicating that at high latitudes A. viridis breeds among other species. Indirect monitoring based only on S. aloides would underestimate the number of populations of the dragonfly.     

Expression and Prognostic Significance of Human Epidermal Growth Factor Receptors 1 and 3 in Gastric and Esophageal Adenocarcinoma

Background

Gastric and esophageal adenocarcinomas are major global cancer burdens. These cancer forms are characterized by a poor prognosis and a modest response to chemo- radio- and targeted treatment. Hence there is an obvious need for further enhanced diagnostic and treatment strategies. The aim of this study was to examine the expression and prognostic impact of human epidermal growth factor receptor 1 (HER1/EGFR) and 3 (HER3), as well as the occurrence of EGFR and KRAS mutations in gastric and esophageal adenocarcinoma.

Methods

Immunohistochemical expression of EGFR and HER3 was analysed in all primary tumours and a subset of lymph node metastases in a consecutive cohort of 174 patients with adenocarcinoma of the stomach, cardia and esophagus. The anti-HER3 antibody used was validated by siRNA-mediated knockdown, immunohistochemistry and quantitative real-time PCR. EGFR and KRAS mutation status was analysed by pyrosequencing tecchnology.

Results and Discussion

High EGFR expression was an independent risk factor for shorter overall survival (OS), whereas high HER3 expression was associated with a borderline significant trend towards a longer OS. KRAS mutations were present in only 4% of the tumours and had no prognostic impact. All tumours were EGFR wild-type. These findings contribute to the ongoing efforts to decide on the potential clinical value of different HERs and druggable mutations in gastric and esophageal adenocarcinomas, and attention is drawn to the need for more standardised investigational methods.     

Heavy chains of inter alpha inhibitor (IαI) inhibit the human complement system at early stages of the cascade

Inter alpha inhibitor (IαI) is an abundant serum protein consisting of three polypeptides: two heavy chains (HC1 and HC2) and bikunin, a broad-specificity Kunitz-type proteinase inhibitor. The complex is covalently held together by chondroitin sulfate but during inflammation IαI may interact with TNF-stimulated gene 6 protein (TSG-6), which supports transesterification of heavy chains to hyaluronan. Recently, IαI was shown to inhibit mouse complement in vivo and to protect from complement-mediated lung injury but the mechanism of such activity was not elucidated. Using human serum depleted from IαI, we found that IαI is not an essential human complement inhibitor as was reported for mice and that such serum has unaltered hemolytic activity. However, purified human IαI inhibited classical, lectin and alternative complement pathways in vitro when added in excess to human serum. The inhibitory activity was dependent on heavy chains but not bikunin and detected at the level of initiating molecules (MBL, properdin) in the lectin/alternative pathways or C4b in the classical pathway. Furthermore, IαI affected formation and assembly of the C1 complex and prevented assembly of the classical pathway C3-convertase. Presence and putative interactions with TSG-6 did not affect the ability of IαI to inhibit complement thus implicating IαI as a potentially important complement inhibitor once enriched onto hyaluronan moieties in the course of local inflammatory processes. In support of this, we found a correlation between IαI/HC-containing proteins and hemolytic activity of synovial fluid from patients suffering from rheumatoid arthritis.     

Direct repeat-mediated deletion of a type IV pilin gene results in major virulence attenuation of Francisella tularensis

Francisella tularensis, the causative agent of tularaemia, is a highly infectious and virulent intracellular pathogen. There are two main human pathogenic subspecies, Francisella tularensis ssp. tularensis (type A), and Francisella tularensis ssp. holarctica (type B). So far, knowledge regarding key virulence determinants is limited but it is clear that intracellular survival and multiplication is one major virulence strategy of Francisella. In addition, genome sequencing has revealed the presence of genes encoding type IV pili (Tfp). One genomic region encoding three proteins with signatures typical for type IV pilins contained two 120 bp direct repeats. Here we establish that repeat-mediated loss of one of the putative pilin genes in a type B strain results in severe virulence attenuation in mice infected by subcutaneous route. Complementation of the mutant by introduction of the pilin gene in cis resulted in complete restoration of virulence. The level of attenuation was similar to that of the live vaccine strain and this strain was also found to lack the pilin gene as result of a similar deletion event mediated by the direct repeats. Presence of the pilin had no major effect on the ability to interact, survive and multiply inside macrophage-like cell lines. Importantly, the pilin-negative strain was impaired in its ability to spread from the initial site of infection to the spleen. Our findings indicate that this putative pilin is critical for Francisella infections that occur via peripheral routes.     

Patterns of between-farm contacts via professionals in Sweden

Background

Infectious diseases of livestock have negative consequences for animal production as well as animal health and welfare and can be transmitted between farms via direct (live animal movements) as well as indirect (via physical vectors such as, people, transport vehicles and fomites) contacts. The objective of the study was to examine the travel patterns of professionals visiting Swedish farms (veterinarians, milk tanker drivers, artificial inseminators, maintenance technicians and livestock hauliers). This was done by obtaining records of the farms visited by a sample of professionals in the above categories in one week in January, one week in April, one week in July and one week in October in the Swedish counties Västerbotten, Södermanland, Västergötland and Skåne.

Results

There were twelve participating organisations, and data was provided for one to three individuals/vehicles/veterinary practices per professional category and per geographic region (except for dairy service technicians and livestock hauliers who did not provide data from all regions). There was a trend towards larger areas covered and smaller number of farms visited per week in the north, but exceptions occurred and there were regional variations. Generally, the greatest areas were travelled by milk tankers and livestock hauliers, and the profession travelling over the smallest areas tended to be the veterinarians. Milk tankers visited most farms per week, one milk tanker could visit between 23 and 90 farms per week and travel over areas between 717 km² and 23,512 km² per week.

Conclusions

Valuable insight into the travel patterns of Swedish professionals has emerged although the implications of the study largely concern highly infectious diseases. Movement of live animals pose the greatest risk for the spread of infectious animal diseases; however indirect contacts are important for many diseases. The results of this study indicate that in Sweden a highly contagious disease might spread over a large area in the time span of one incubation period, which ought to be kept in mind in case of an outbreak and in outbreak investigations. The difficulties in contacting some professionals visiting farms could be a problem in an outbreak situation.