Adaptive radiotherapy for breast cancer.

Research in the field of local and locoregional breast cancer radiotherapy aims to maintain excellent oncological outcomes while reducing treatment-related toxicity. Adaptive radiotherapy (ART) considers variations in target and organs at risk (OARs) anatomy occurring during the treatment course and integrates these in re-optimized treatment plans. Exploiting ART routinely in clinic may result in smaller target volumes and better OAR sparing, which may lead to reduction of acute as well as late toxicities. In this review MR-guided and CT-guided ART for breast cancer patients according to different clinical scenarios (neoadjuvant and adjuvant partial breast irradiation, whole breast, chest wall and regional nodal irradiation) are reviewed and their advantages as well as challenging aspects discussed.

Comparative phylogeography of African savannah ungulates.

The savannah biome of sub-Saharan Africa harbours the highest diversity of ungulates (hoofed mammals) on Earth. In this review, we compile population genetic data from 19 codistributed ungulate taxa of the savannah biome and find striking concordance in the phylogeographic structuring of species. Data from across taxa reveal distinct regional lineages, which reflect the survival and divergence of populations in isolated savannah refugia during the climatic oscillations of the Pleistocene. Data from taxa across trophic levels suggest distinct savannah refugia were present in West, East, Southern and South-West Africa. Furthermore, differing Pleistocene evolutionary biogeographic scenarios are proposed for East and Southern Africa, supported by palaeoclimatic data and the fossil record. Environmental instability in East Africa facilitated several spatial and temporal refugia and is reflected in the high inter- and intraspecific diversity of the region. In contrast, phylogeographic data suggest a stable, long-standing savannah refuge in the south.

Ocean-wide genomic variation in Gray's beaked whales, Mesoplodon grayi.

The deep oceans of the Southern Hemisphere are home to several elusive and poorly studied marine megafauna. In the absence of robust observational data for these species, genetic data can aid inferences on population connectivity, demography and ecology. A previous investigation of genetic diversity and population structure in Gray's beaked whale (Mesoplodon grayi) from Western Australia and New Zealand found high levels of mtDNA diversity, no geographic structure and stable demographic history. To further investigate phylogeographic and demographic patterns across their range, we generated complete mitochondrial and partial nuclear genomes of 16 of the individuals previously analysed and included additional samples from South Africa (n = 2) and South Australia (n = 4), greatly expanding the spatial range of genomic data for the species. Gray's beaked whales are highly elusive and rarely observed, and our data represents a unique and geographically broad dataset. We find relatively high levels of diversity in the mitochondrial genome, despite an absence of population structure at the mitochondrial and nuclear level. Demographic analyses suggest these whales existed at stable levels over at least the past 1.1 million years, with an approximately twofold increase in female effective population size approximately 250 thousand years ago, coinciding with a period of increased Southern Ocean productivity, sea surface temperature and a potential expansion of suitable habitat. Our results suggest that Gray's beaked whales are likely to be resilient to near-future ecosystem changes, facilitating their conservation. Our study demonstrates the utility of low-effort shotgun sequencing in providing ecological information on highly elusive species.

Postmastectomy Radiation Therapy Planning After Immediate Implant-based Reconstruction Using the European Society for Radiotherapy and Oncology-Advisory Committee in Radiation Oncology Practice Consensus Guidelines for Target Volume Delineation.

AIMS: To evaluate how common radiation therapy techniques perform in the setting of the new European Society for Radiotherapy and Oncology-Advisory Committee in Radiation Oncology Practice (ESTRO-ACROP) delineation recommendations for immediate breast reconstruction (IBR). MATERIALS AND METHODS: Seven Danish radiation therapy centres and six international European centres participated in this project. Two breast cancer cases (one left-sided and one right-sided) with a retropectoral implant were chosen for radiation therapy planning using deep-inspiration breath-hold. Target volumes were delineated according to ESTRO-ACROP delineation recommendations. The centres were asked to plan the cases using any radiation therapy technique according to the Danish Breast Cancer Group plan objectives. RESULTS: In total, 35 treatment plans were collected. Half of the submitted plans, for both the left-sided and the right-sided case, used the field-in-field (FiF) technique (nine for each), a quarter used volumetric arc radiation therapy (VMAT; five for right-sided, four for left-sided) and the remaining quarter was a mix of inverse intensity-modulated radiation therapy (IMRT), helicoidal therapy and hybrid (combined open fields and VMAT) techniques. Mean clinical target volume doses were in the range 99-102% of the prescribed dose. The median FiF mean heart dose (MHD) for right-sided radiation therapy was 1 Gy (range 0.8-3.7) and 5.2 Gy for left-sided radiation therapy (range 2.2-6.5). For right-sided radiation therapy, the median VMAT MHD was 3.42 Gy, for IMRT was 2.3 Gy and for helicoidal therapy was 5.1 Gy. For left-sided radiation therapy, the median VMAT MHD was 6.3 Gy, for IMRT was 7.8 Gy and for helicoidal therapy was 7.3 Gy. CONCLUSIONS: Different radiation therapy techniques could be used to plan radiation therapy in the setting of IBR. FiF provided good coverage with acceptable organ at risk doses. The best dose distribution results as a trade-off between the objectives of target volume coverage and high-dose organ at risk inclusion. The radiation therapy technique affects the interplay between these objectives.

Immersion exposure of rainbow trout (Oncorhynchus mykiss) fry to wildtype Flavobacterium psychrophilum induces no mortality, but protects against later intraperitoneal challenge.

Flavobacterium psychrophilum, the causative agent of RTFS or rainbow trout fry syndrome, causes high mortality among hatchery reared rainbow trout (Oncorhynchus mykiss) fry in Europe and the USA. Despite several attempts, no efficient vaccines have yet been developed, the main obstacle being that the fry have to be vaccinated very early, i.e. around 0.2-0.5 g, where RTFS usually starts to give problems in the fish farms. Consequently, only oral or bath vaccines are relevant. Immersion of fry in inactivated or attenuated bacteria has resulted in RPS values of less than 50%. However, the results are biased by the fact that the fish have been challenged by intraperitoneal (ip) or subcutaneous (sc) injection against which an immersion/oral vaccine may not protect. Therefore, the present study was undertaken in order to investigate whether the presumably most potent immersion immunization, i.e. bathing in high titres of non-attenuated isolates of F. psychrophilum, was able to induce immunity to a subsequent ip challenge. Immersion in live bacteria for 30 or 50 min caused no mortality and protected a major fraction of the fry against challenges 26 and 47 days later with RPS values of 88.2 and 60.3%, respectively. Increased specific antibody titres suggested that adaptive immune mechanisms were involved in the protection.

Mid-Holocene decline in African buffalos inferred from Bayesian coalescent-based analyses of microsatellites and mitochondrial DNA.

Genetic studies concerned with the demographic history of wildlife species can help elucidate the role of climate change and other forces such as human activity in shaping patterns of divergence and distribution. The African buffalo (Syncerus caffer) declined dramatically during the rinderpest pandemic in the late 1800s, but little is known about the earlier demographic history of the species. We analysed genetic variation at 17 microsatellite loci and a 302-bp fragment of the mitochondrial DNA control region to infer past demographic changes in buffalo populations from East Africa. Two Bayesian coalescent-based methods as well as traditional bottleneck tests were applied to infer detailed dynamics in buffalo demographic history. No clear genetic signature of population declines related to the rinderpest pandemic could be detected. However, Bayesian coalescent modelling detected a strong signal of African buffalo population declines in the order of 75-98%, starting in the mid-Holocene (approximately 3-7000 years ago). The signature of decline was remarkably consistent using two different coalescent-based methods and two types of molecular markers. Exploratory analyses involving various prior assumptions did not seriously affect the magnitude or timing of the inferred population decline. Climate data show that tropical Africa experienced a pronounced transition to a drier climate approximately 4500 years ago, concurrent with the buffalo decline. We therefore propose that the mid-Holocene aridification of East Africa caused a major decline in the effective population size of the buffalo, a species reliant on moist savannah habitat for its existence.

Immunoprophylaxis in fish by injection of mouse antibody genes.

Antibodies are a crucial part of the body's specific defense against infectious diseases and have considerable potential as therapeutic and prophylactic agents in humans and animals. The development of recombinant single-chain antibodies allows a genetic application strategy for prevention of infectious diseases. To test this in a fish model, a gene construct encoding a neutralizing single-chain antibody to the fish-pathogenic rhabdovirus VHSV (viral hemorrhagic septicemia virus) was administered to rainbow trout by intramuscular injection of plasmid DNA. Circulating recombinant antibodies could later be detected in the fish, and protective immunity to the viral disease was established.

Genetic vaccination of rainbow trout against viral haemorrhagic septicaemia virus: small amounts of plasmid DNA protect against a heterologous serotype.

Viral haemorrhagic septicaemia (VHS) is known as one of the most important diseases in cultured rainbow trout in Europe. An efficient vaccine is highly desirable, but so far only limited success has been obtained with traditional products based on killed or attenuated virus. Genetic immunization with a plasmid vector containing the VHS virus glycoprotein gene under the control of a cytomegalovirus promoter has recently been shown to induce high levels of protection against the homologous virus isolate. Expressed glycoprotein could be detected immunohistochemically in fish muscle and about 70% of the vaccinated animals had neutralizing antibodies in their serum. To further evaluate the potential of the DNA vaccine technology for prophylaxis of VHS, a vaccination trial including lower doses of DNA and different virus isolates was performed. Eight weeks after injection, rainbow trout were challenged by immersion with the homologous virus isolate or with a serologically different isolate. Cumulative mortalities demonstrated that even the lowest dose of DNA tested (0.1 microg per fish) induced protective immunity against both virus isolates. Virus neutralization tests in cell culture indicated that trout sera neutralized VHS virus isolates independently of serotypes defined with mammalian mono- and polyclonal antibodies. No protection was observed following vaccination with a plasmid construct carrying the VHS virus nucleocapsid-protein gene.

Inter-laboratory comparison of cell lines for susceptibility to three viruses: VHSV, IHNV and IPNV.

Eleven European National Reference Laboratories participated in an inter-laboratory comparison of the susceptibility of 5 selected cell lines to 3 fish pathogenic viruses. The test included viral hemorrhagic septicaemia virus (VHSV); infectious hematopoietic necrosis virus (IHNV) and infectious pancreatic necrosis virus (IPNV), and the cell lines derived from bluegill fry (BF-2), chinook salmon embryo (CHSE-214), epithelioma papulosum cyprini (EPC), fathead minnow (FHM) and rainbow trout gonad (RTG-2). The results showed that for isolation of VHSV, BF-2 and RTG-2 cells performed equally well and had higher sensitivity compared to the other cell lines. For IHNV, EPC and FHM cells gave the best results, and for IPNV it was BF-2 and CHSE-214 cells. FHM cells showed the largest variability among laboratories, whereas EPC was the cell line showing the smallest variability.

General and family-specific gene expression responses to viral hemorrhagic septicaemia virus infection in rainbow trout (Oncorhynchus mykiss).

The ability of rainbow trout (Oncorhynchus mykiss) to respond successfully to infection by viral hemorrhagic septicaemia virus (VHSV) is expected to involve a large number of biochemical processes. We hypothesized that this would be reflected at the gene expression level in infected fish, and we tested it by examining gene expression levels in the head kidney of trout at a genome-wide scale with a 16K cDNA microarray for salmonids. Expression levels were recorded during 16 days following bath challenge. The challenge experiment included a relatively low susceptibility (32% survival following challenge) and a relatively high susceptibility (18% survival following challenge) trout family that were both split into a group exposed to virus and a non-exposed control group. In total, 939 genes were differentially expressed between infected and non-infected fish (FDR p=0.05). Five groups of Gene Ontology categories were involved in immune-related processes and over-represented in infected fish: (i) stress and defense response, (ii) NFkappaB signal transduction, (iii) response to non-self, (iv) antigen processing and presentation, and (v) proteasome complexes. The first four categories were also over-represented among the 642 differentially expressed genes in the low-susceptibility trout family but not among the 556 differentially expressed genes in the high-susceptibility trout family. Expression profiles for most immune genes discussed showed increased transcription from day 3 post-challenge. The results suggest that the innate immune system may play an important role in the successful response to VHSV in rainbow trout. In addition, the results indicate that a superior regulation of the transcription of several key innate immune-related genes contribute to the increased survival in resistant fish.

The protective mechanisms induced by a fish rhabdovirus DNA vaccine depend on temperature.

DNA vaccines encoding the viral glycoproteins of viral haemorrhagic septicaemia virus (VHSV) and infectious haematopoietic necrosis virus (IHNV) have proved highly efficient in rainbow trout (Oncorhynchus mykiss) under experimental conditions. Non-specific as well as specific immune mechanisms seem to be activated. Temperature is an important external parameter affecting the immune response in fish. The present study aimed at determining the effectiveness of a DNA vaccine against VHS at different temperatures. Rainbow trout fingerlings acclimated at 5 degrees C, 10 degrees C or 15 degrees C, were given an intramuscular injection of 1 microg purified plasmid DNA and challenged with virulent VHSV 8 or 36-40 days later. The vaccine protected the fish well at all three temperatures, but the involvement of innate and adaptive mechanisms differed: at low temperature, non-specific protection lasted longer and at 36 dpv fish kept at 5 degrees C had no detectable response of neutralizing antibodies while 67% of the fish kept at 15 degrees C had seroconverted. Induction of Mx as measured in liver samples was delayed at 5 degrees C with no detectable response 7 dpv whereas fish maintained at 10 degrees C had significantly elevated levels of Mx3-transcripts at that time point. Immunohistochemical studies of the injection site of vaccinated fish also showed a clear effect of temperature: in fish maintained at 15 degrees C the vhsG-protein appeared earlier on the surface of transfected myocytes and the inflammatory response clearing away these myocytes arose earlier compared to fish kept at the lower temperatures of 5 and 10 degrees C.

Immunity to viral haemorrhagic septicaemia (VHS) following DNA vaccination of rainbow trout at an early life-stage.

Rainbow trout fry of average weight 0.5 g were vaccinated against viral haemorrhagic septicaemia (VHS) by intramuscular injection of 1 microg of plasmid DNA encoding the VHS virus glycoprotein gene. Challenge with a lethal dose of virus at two different time points, 9 and 71 days post-vaccination respectively, revealed that a highly protective and lasting immunity was established shortly after vaccination, in accordance with earlier experiments with larger fish. The defence mechanisms activated by the DNA vaccine are thus functional at an early life-stage in rainbow trout.

DNA vaccines as a tool for analysing the protective immune response against rhabdoviruses in rainbow trout.

DNA vaccines based on the glycoprotein genes of the salmonid rhabdoviruses VHSV and IHNV have been demonstrated to be very efficient in inducing a protective immune response against the respective diseases in rainbow trout. Nanogram doses of plasmid DNA delivered by intramuscular injection are sufficient to induce high levels of immunity in fingerling-size fish, whereas larger fish require more vaccine for protection. The protection is long lasting and, more surprisingly, is partly established already 4 days post vaccination. The early protection involves cross-protective anti-viral defence mechanisms, while the long duration immunity is highly specific. The nature of these immune response mechanisms is discussed and it is suggested that the efficacy of the vaccines is related to their ability to activate the innate immune system as it is activated by live virus.