Review: Embryonic stem cells as tools for in vitro gamete production in livestock.

The goal of in vitro gametogenesis is to reproduce the events of sperm and oocyte development in the laboratory. Significant advances have been made in the mouse in the last decade, but evolutionary divergence from the murine developmental program has prevented the replication of these advances in large mammals. In recent years, intensive work has been done in humans, non-human primates and livestock to elucidate species-specific differences that regulate germ cell development, due to the number of potential applications. One of the most promising applications is the use of in vitro gametes to optimize the spread of elite genetics in cattle. In this context, embryonic stem cells have been posed as excellent candidates for germ cell platforms. Here, we present the most relevant advances in in vitro gametogenesis of interest to livestock science, including new types of pluripotent stem cells with potential for germline derivation, characterization of the signaling environment in the gonadal niche, and experimental systems used to reproduce different stages of germ cell development in the laboratory.

bESC from cloned embryos do not retain transcriptomic or epigenetic memory from somatic donor cells.

In brief: Epigenetic reprogramming after mammalian somatic cell nuclear transfer is often incomplete, resulting in low efficiency of cloning. However, gene expression and histone modification analysis indicated high similarities in transcriptome and epigenomes of bovine embryonic stem cells from in vitro fertilized and somatic cell nuclear transfer embryos. Abstract: Embryonic stem cells (ESC) indefinitely maintain the pluripotent state of the blastocyst epiblast. Stem cells are invaluable for studying development and lineage commitment, and in livestock, they constitute a useful tool for genomic improvement and in vitro breeding programs. Although these cells have been recently derived from bovine blastocysts, a detailed characterization of their molecular state is lacking. Here, we apply cutting-edge technologies to analyze the transcriptomic and epigenomic landscape of bovine ESC (bESC) obtained from in vitro fertilized (IVF) and somatic cell nuclear transfer (SCNT) embryos. bESC were efficiently derived from SCNT and IVF embryos and expressed pluripotency markers while retaining genome stability. Transcriptome analysis revealed that only 46 genes were differentially expressed between IVF- and SCNT-derived bESC, which did not reflect significant deviation in cellular function. Interrogating histone 3 lysine 4 trimethylation, histone 3 lysine 9 trimethylation, and histone 3 lysine 27 trimethylation with cleavage under targets and tagmentation, we found that the epigenomes of both bESC groups were virtually indistinguishable. Minor epigenetic differences were randomly distributed throughout the genome and were not associated with differentially expressed or developmentally important genes. Finally, the categorization of genomic regions according to their combined histone mark signal demonstrated that all bESC shared the same epigenomic signatures, especially at gene promoters. Overall, we conclude that bESC derived from SCNT and IVF embryos are transcriptomically and epigenetically analogous, allowing for the production of an unlimited source of pluripotent cells from high genetic merit organisms without resorting to transgene-based techniques.

Genome activation in equine in vitro-produced embryos.

Embryonic genome activation is a critical event in embryo development, in which the transcriptional program of the embryo is initiated. The timing and regulation of this process are species-specific. In vitro embryo production is becoming an important clinical and research tool in the horse; however, very little is known about genome activation in this species. The objective of this work was to identify the timing of genome activation, and the transcriptional networks involved, in in vitro-produced horse embryos. RNA-Seq was performed on oocytes and embryos at eight stages of development (MII, zygote, 2-cell, 4-cell, 8-cell, 16-cell, morula, blastocyst; n = 6 per stage, 2 from each of 3 mares). Transcription of seven genes was initiated at the 2-cell stage. The first substantial increase in gene expression occurred at the 4-cell stage (minor activation), followed by massive gene upregulation and downregulation at the 8-cell stage (major activation). An increase in intronic nucleotides, indicative of transcription initiation, was also observed at the 4-cell stage. Co-expression network analyses identified groups of genes that appeared to be regulated by common mechanisms. Investigation of hub genes and binding motifs enriched in the promoters of co-expressed genes implicated several transcription factors. This work represents, to the best of our knowledge, the first genomic evaluation of embryonic genome activation in horse embryos.

Results of a single-arm pilot study of 32P microparticles in unresectable locally advanced pancreatic adenocarcinoma with gemcitabine/nab-paclitaxel or FOLFIRINOX chemotherapy.

BACKGROUND: Unresectable locally advanced pancreatic cancer (LAPC) is generally managed with chemotherapy or chemoradiotherapy, but prognosis is poor with a median survival of ∼13 months (or up to 19 months in some studies). We assessed a novel brachytherapy device, using phosphorous-32 (32P) microparticles, combined with standard-of-care chemotherapy. PATIENTS AND METHODS: In this international, multicentre, single-arm, open-label pilot study, adult patients with histologically or cytologically proven unresectable LAPC received 32P microparticles, via endoscopic ultrasound-guided fine-needle implantation, planned for week 4 of 5-fluorouracil, leucovorin, irinotecan and oxaliplatin (FOLFIRINOX) or gemcitabine/nab-paclitaxel chemotherapy, per investigator's choice. The primary endpoint was safety and tolerability measured using Common Terminology Criteria for Adverse Events version 4.0. The lead efficacy endpoint was local disease control rate at 16 weeks. RESULTS: Fifty patients were enrolled and received chemotherapy [intention-to-treat (ITT) population]. Forty-two patients received 32P microparticle implantation [per protocol (PP) population]. A total of 1102 treatment-emergent adverse events (TEAEs) were reported in the ITT/safety population (956 PP), of which 167 (139 PP) were grade ≥3. In the PP population, 41 TEAEs in 16 (38.1%) patients were possibly or probably related to 32P microparticles or implantation procedure, including 8 grade ≥3 in 3 (7.1%) patients, compared with 609 TEAEs in 42 (100%) patients attributed to chemotherapy, including 67 grade ≥3 in 28 patients (66.7%). The local disease control rate at 16 weeks was 82.0% (95% confidence interval: 68.6% to 90.9%) (ITT) and 90.5% (95% confidence interval: 77.4% to 97.3%) (PP). Tumour volume, carbohydrate antigen 19-9 levels, and metabolic tumour response at week 12 improved significantly. Ten patients (20.0% ITT; 23.8% PP) had surgical resection and median overall survival was 15.2 and 15.5 months for ITT and PP populations, respectively. CONCLUSIONS: Endoscopic ultrasound-guided 32P microparticle implantation has an acceptable safety profile. This study also suggests clinically relevant benefits of combining 32P microparticles with standard-of-care systemic chemotherapy for patients with unresectable LAPC.

Allele-specific expression analysis reveals conserved and unique features of preimplantation development in equine ICSI embryos†.

Embryonic genome activation and dosage compensation are major genetic events in early development. Combined analysis of single embryo RNA-seq data and parental genome sequencing was used to evaluate parental contributions to early development and investigate X-chromosome dynamics. In addition, we evaluated dimorphism in gene expression between male and female embryos. Evaluation of parent-specific gene expression revealed a minor increase in paternal expression at the 4-cell stage that increased at the 8-cell stage. We also detected eight genes with allelic expression bias that may have an important role in early development, notably NANOGNB. The main actor in X-chromosome inactivation, XIST, was significantly upregulated at the 8-cell, morula, and blastocyst stages in female embryos, with high expression at the latter. Sexual dimorphism in gene expression was identified at all stages, with strong representation of the X-chromosome in females from the 16-cell to the blastocyst stage. Female embryos showed biparental X-chromosome expression at all stages after the 4-cell stage, demonstrating the absence of imprinted X-inactivation at the embryo level. The analysis of gene dosage showed incomplete dosage compensation (0.5 < X:A < 1) in MII oocytes and embryos up to the 4-cell stage, an increase of the X:A ratio at the 16-cell and morula stages after genome activation, and a decrease of the X:A ratio at the blastocyst stage, which might be associated with the beginning of X-chromosome inactivation. This study represents the first critical analysis of parent- and sex-specific gene expression in early equine embryos produced in vitro.

Effects of sodium salicylate and time postpartum on mammary tissue proliferation, gene transcript profile, and DNA methylation.

Previous studies have demonstrated nonsteroidal antiinflammatory drug treatment in early lactation had a positive impact on whole-lactation milk production in older cows. The objective of this study was to evaluate proliferative, transcriptional, and epigenetic changes in the mammary gland that could explain increased production responses due to nonsteroidal antiinflammatory drug treatment. Sodium salicylate (SAL; 125 g/d) or water (CON) were administered via oral drench to multiparous Holstein cows (n = 8/treatment) once daily for 3 d beginning approximately 24 h after parturition, and mammary tissue was collected on d 1, 4, and 45 postpartum. Day 1 tissue was collected immediately preceding the initial drench, and d 4 tissue was collected 24 h following the final drench. Blood was collected twice weekly and analyzed for plasma glucose, insulin, ß-hydroxybutyrate, free fatty acids, and prolactin. Cows were milked twice daily until d 7 of lactation, and thrice daily for the remainder of the study. Total RNA extracted from tissue was deep-sequenced and analyzed for differential gene expression using DESeq2. We detected no treatment effect on milk yield or plasma metabolites through 45 d of lactation; additionally, no change in mammary epithelial cell proliferation was detected when assessed by Ki67 labeling. Comparison of SAL versus CON revealed that only 16 of 18,286 genes were differentially expressed (false discovery rate <0.1) in mammary tissue collected on d 45, whereas no differentially expressed genes due to treatment were detected on d 1 or 4. Analysis of transcriptional differences over time showed downregulation of pathways related to immune cell recruitment and differentiation, and extensive overlap with pathways related to cholesterol synthesis and liver X receptor signaling. Global DNA methylation of mammary tissue was decreased for CON compared with SAL. Transcriptome analysis emphasized extensive involvement of immune-related signaling pathways in the switch from lactogenesis to galactopoiesis, and changes in methylation with SAL treatment merit future investigation into epigenetic effects on milk production.

Systematic alteration of ATAC-seq for profiling open chromatin in cryopreserved nuclei preparations from livestock tissues.

The use of Assay for Transposase-Accessible Chromatin (ATAC-seq) to profile chromatin accessibility has surged over the past years, but its applicability to tissues has been very limited. With the intent of preserving nuclear architecture during long-term storage, cryopreserved nuclei preparations from chicken lung were used to optimize ATAC-seq. Sequencing data were compared with existing DNase-seq, ChIP-seq, and RNA-seq data to evaluate library quality, ultimately resulting in a modified ATAC-seq method capable of generating high quality chromatin accessibility data from cryopreserved nuclei preparations. Using this method, nucleosome-free regions (NFR) identified in chicken lung overlapped half of DNase-I hypersensitive sites, coincided with active histone modifications, and specifically marked actively expressed genes. Notably, sequencing only the subnucleosomal fraction dramatically improved signal, while separation of subnucleosomal reads post-sequencing did not improve signal or peak calling. The broader applicability of this modified ATAC-seq technique was tested using cryopreserved nuclei preparations from pig tissues, resulting in NFR that were highly consistent among biological replicates. Furthermore, tissue-specific NFR were enriched for binding motifs of transcription factors related to tissue-specific functions, and marked genes functionally enriched for tissue-specific processes. Overall, these results provide insights into the optimization of ATAC-seq and a platform for profiling open chromatin in animal tissues.

Review: Recent advances in bovine in vitro embryo production: reproductive biotechnology history and methods.

In vitro production (IVP) of embryos and associated technologies in cattle have shown significant progress in recent years, in part driven by a better understanding of the full potential of these tools by end users. The combination of IVP with sexed semen (SS) and genomic selection (GS) is being successfully and widely used in North America, South America and Europe. The main advantages offered by these technologies include a higher number of embryos and pregnancies per unit of time, and a wider range of potential female donors from which to retrieve oocytes (including open cyclic females and ones up to 3 months pregnant), including high index genomic calves, a reduced number of sperm required to produce embryos and increased chances of obtaining the desired sex of offspring. However, there are still unresolved aspects of IVP of embryos that limit a wider implementation of the technology, including potentially reduced fertility from the use of SS, reduced oocyte quality after in vitro oocyte maturation and lower embryo cryotolerance, resulting in reduced pregnancy rates compared to in vivo-produced embryos. Nevertheless, promising research results have been reported, and work is in progress to address current deficiencies. The combination of GS, IVP and SS has proven successful in the commercial field in several countries assisting practitioners and cattle producers to improve reproductive performance, efficiency and genetic gain.

Evaluation of protective and immune responses following vaccination with recombinant MIP and CPAF from Chlamydia abortus as novel vaccines for enzootic abortion of ewes.

MIP and CPAF from Chlamydia have been shown to be effective in inducing immune responses important in clearing chlamydial infections. This study evaluates the protection conferred by MIP and CPAF as novel vaccines in pregnant C. abortus challenged ewes. Fifty C. abortus sero-negative sheep were randomly allocated into 5 groups of 10 according to the treatment they were to receive (1) 100 µg of MBP-MIP (2) 100 µg CPAF (3) 50 µg MBP-MIP and 50 µg CPAF (4) Tris-buffer (negative control) (5) Enzovax (positive control). Booster inoculations were administered 3 weeks after primary inoculations. Blood samples were taken pre-vaccination and weekly for 5 weeks. Five months after vaccination the ewes were mated. Pregnant ewes were then challenged on day 90 of gestation. Blood samples taken at four time-points post challenge were analysed for IFNγ levels, TNFα and IL-10 expression and anti-chlamydial antibody levels. Vaginal swabs, placental and foetal tissue and bacterial shedding were analysed using qPCR to quantify levels of C. abortus. Enzovax was 100% effective with no abortions occurring. The MIP/CPAF combined vaccine offered the greatest protection of the novel vaccines with 67% of ewes giving birth to one or more live lambs equating to a 50% vaccine efficacy rate. MIP and CPAF administered singly did not confer protection. Enzovax and MIP/CPAF vaccinated ewes had longer gestations and lambs with higher birth weights than negative control ewes. Aborting ewes shed higher numbers of C. abortus than ewes that had live lambs, all vaccinated ewes demonstrated lower levels of bacterial shedding than negative control ewes with Enzovax ewes shedding significantly fewer bacteria. Ewes that went on to abort had significantly higher levels of IFNγ and IL-10 at day 35 post challenge and significantly higher levels of anti-chlamydial antibodies at 24 h post lambing compared to ewes that had live lambs.

Effect of different mini-volume colloid centrifugation configurations on flow cytometrically sorted sperm recovery efficiency and quality using a computer-assisted semen analyzer.

Straws of sex-sorted sperm are usually packaged at a low concentration (e.g., ~2.1 × 106  sperm/ml) and cost significantly more than unsorted conventional semen from the same sire. In order to maximize the efficiency of using sex-sorted sperm under in vitro fertilization conditions, the selection of an appropriate sperm separation technique is essential. In this study, the effect of using different silane-coated silica colloid dilutions and layering configurations during centrifugation of sex-sorted sperm was examined over an extended period of incubation time. Sperm recovery and viability after centrifugation using the colloid separation technique were measured along with several sperm motility parameters using CASA. For this purpose, frozen and thawed sex-sorted sperm samples were centrifuged using mini-volume single-layer (40%, 60% and 80%) and mini-volume two-layer (45%/90%, 40%/80% and 30%/60%) separation configurations using PureSperm® . A single layer of 40% PureSperm® recovered significantly more sex-sorted sperm (78.07% ± 2.28%) followed by a single layer of 80% PureSperm® (68.43% ± 2.33%). The lowest sperm recovery was obtained using a two-layer PureSperm® dilution of 45%/90% (47.57% ± 2.33%). Single-layer centrifugation recovered more sorted sperm (68.67% ± 1.74%) than two layer (53.74% ± 1.74%) (p < .0001). A single layer of 80% PureSperm® exhibited the highest sorted sperm viability (72.01% ± 2.90%) after centrifugation (p < .05). The mini-volume single layer of 80% PureSperm® was determined to be an effective alternative to a two-layer centrifugation configuration for sex-sorted sperm selection. In addition, single-layer colloid dilution of 80% performed either as well as or significantly outperformed the other treatments, as well as the control, with regard to motility (MOT) for all time periods of analysis.

Differing molecular response of young and advanced maternal age human oocytes to IVM.

STUDY QUESTION: What effect does maternal age have on the human oocyte's molecular response to in vitro oocyte maturation? SUMMARY ANSWER: Although polyadenylated transcript abundance is similar between young and advanced maternal age (AMA) germinal vesicle (GV) oocytes, metaphase II (MII) oocytes exhibit a divergent transcriptome resulting from a differential response to in vitro oocyte maturation. WHAT IS KNOWN ALREADY: Microarray studies considering maternal age or maturation stage have shown that either of these factors will affect oocyte polyadenylated transcript abundance in human oocytes. However, studies considering both human oocyte age and multiple stages simultaneously are limited to a single study that examined transcript levels for two genes by qPCR. Thus, polyadenylated RNA sequencing (RNA-Seq) could provide novel insight into age-associated aberrations in gene expression in GV and MII oocytes. STUDY DESIGN, SIZE, DURATION: The effect of maternal age (longitudinal analysis) on polyadenylated transcript abundance at different stages was analyzed by examining single GV and single in vitro matured MII oocytes derived from five young (YNG; < 30 years; average age 26.8; range 20-29) and five advanced maternal age (AMA; ≥40 years; average age 41.6 years; range 40-43 years) patients. Thus, a total of 10 YNG (5 GV and 5 MII) and 10 AMA (5 GV and 5 MII) oocytes were individually processed for RNA-Seq analysis. PARTICIPANTS/MATERIALS, SETTINGS, METHODS: Patients undergoing infertility treatment at the Colorado Center for Reproductive Medicine (Lone Tree, CO, USA) underwent ovarian stimulation with FSH and received hCG for final follicular maturation prior to ultrasound guided oocyte retrieval. Unused GV oocytes obtained at retrieval were donated for transcriptome analysis. Single oocytes were stored (at -80°C in PicoPure RNA Extraction Buffer; Thermo Fisher Scientific, USA) immediately upon verification of immaturity or after undergoing in vitro oocyte maturation (24 h incubation), representing GV and MII samples, respectively. After isolating RNA and generating single oocyte RNA-Seq libraries (SMARTer Ultra Low Input RNA HV kit; Clontech, USA), Illumina sequencing (100 bp paired-end reads on HiSeq 2500) and bioinformatics analysis (CLC Genomics Workbench, DESeq2, weighted gene correlation network analysis (WGCNA), Ingenuity Pathway Analysis) were performed. MAIN RESULTS AND THE ROLE OF CHANCE: A total of 12 770 genes were determined to be expressed in human oocytes (reads per kilobase per million mapped reads (RPKM) > 0.4 in at least three of five replicates for a minimum of one sample type). Differential gene expression analysis between YNG and AMA oocytes (within stage) identified 1 and 255 genes that significantly differed (adjusted P < 0.1 and log2 fold change >1) in polyadenylated transcript abundance for GV and MII oocytes, respectively. These genes included CDK1, NLRP5 and PRDX1, which have been reported to affect oocyte developmental potential. Despite the similarity in transcript abundance between GV oocytes irrespective of age, divergent expression patterns emerged during oocyte maturation. These age-specific differentially expressed genes were enriched (FDR < 0.05) for functions and pathways associated with mitochondria, cell cycle and cytoskeleton. Gene modules generated by WGCNA (based on gene expression) and patient traits related to oocyte quality (e.g. age and blastocyst development) were correlated (P < 0.05) and enriched (FDR < 0.05) for functions and pathways associated with oocyte maturation. LARGE SCALE DATA: Raw data from this study can be accessed through GSE95477. LIMITATIONS, REASONS FOR CAUTION: The human oocytes used in the current study were obtained from patients with varying causes of infertility (e.g. decreased oocyte quality and oocyte quality-independent factors), possibly affecting oocyte gene expression. Oocytes in this study were retrieved at the GV stage following hCG administration and the MII oocytes were derived by IVM of patient oocytes. Although the approach has the benefit of identifying intrinsic differences between samples, it may not be completely representative of in vivo matured oocytes. WIDER IMPLICATIONS OF THE FINDINGS: Transcriptome profiles of YNG and AMA oocytes, particularly at the MII stage, suggest that aberrant transcript abundance may contribute to the age-associated decline in fertility. STUDY FUNDING/COMPETING INTEREST(S): J.M.R. was supported by an Austin Eugene Lyons Fellowship awarded by the University of California, Davis. The Eunice Kennedy Shriver National Institute of Child Health and Human Development of the National Institutes of Health (awarded to P.J.R.; R01HD070044) and the Fertility Laboratories of Colorado partly supported the research presented in this manuscript.

Identification of long non-coding RNA in the horse transcriptome.

BACKGROUND: Efforts to resolve the transcribed sequences in the equine genome have focused on protein-coding RNA. The transcription of the intergenic regions, although detected via total RNA sequencing (RNA-seq), has yet to be characterized in the horse. The most recent equine transcriptome based on RNA-seq from several tissues was a prime opportunity to obtain a concurrent long non-coding RNA (lncRNA) database. RESULTS: This lncRNA database has a breadth of eight tissues and a depth of over 20 million reads for select tissues, providing the deepest and most expansive equine lncRNA database. Utilizing the intergenic reads and three categories of novel genes from a previously published equine transcriptome pipeline, we better describe these groups by annotating the lncRNA candidates. These lncRNA candidates were filtered using an approach adapted from human lncRNA annotation, which removes transcripts based on size, expression, protein-coding capability and distance to the start or stop of annotated protein-coding transcripts. CONCLUSION: Our equine lncRNA database has 20,800 transcripts that demonstrate characteristics unique to lncRNA including low expression, low exon diversity and low levels of sequence conservation. These candidate lncRNA will serve as a baseline lncRNA annotation and begin to describe the RNA-seq reads assigned to the intergenic space in the horse.

Tissue resolved, gene structure refined equine transcriptome.

BACKGROUND: Transcriptome interpretation relies on a good-quality reference transcriptome for accurate quantification of gene expression as well as functional analysis of genetic variants. The current annotation of the horse genome lacks the specificity and sensitivity necessary to assess gene expression especially at the isoform level, and suffers from insufficient annotation of untranslated regions (UTR) usage. We built an annotation pipeline for horse and used it to integrate 1.9 billion reads from multiple RNA-seq data sets into a new refined transcriptome. RESULTS: This equine transcriptome integrates eight different tissues from 59 individuals and improves gene structure and isoform resolution, while providing considerable tissue-specific information. We utilized four levels of transcript filtration in our pipeline, aimed at producing several transcriptome versions that are suitable for different downstream analyses. Our most refined transcriptome includes 36,876 genes and 76,125 isoforms, with 6474 candidate transcriptional loci novel to the equine transcriptome. CONCLUSIONS: We have employed a variety of descriptive statistics and figures that demonstrate the quality and content of the transcriptome. The equine transcriptomes that are provided by this pipeline show the best tissue-specific resolution of any equine transcriptome to date and are flexible for several downstream analyses. We encourage the integration of further equine transcriptomes with our annotation pipeline to continue and improve the equine transcriptome.

A comprehensive geriatric assessment screening questionnaire (CGA-GOLD) for older people undergoing treatment for cancer.

Oncology services do not routinely assess broader needs of older people with cancer. This study evaluates a comprehensive geriatric assessment and comorbidity screening questionnaire (CGA-GOLD) covering evidence-based domains and quality of life (EORTC-QLQ-C30). Patients aged 65+ attending oncology services were recruited into (1) Observational cohort (completed CGA-GOLD, received standard oncology care), (2) Intervention cohort (responses categorised 'low-risk', 'high-risk', 'possible need' by geriatricians). N = 417 observational patients (1002 invited by post, 418 consented, age 73.9 ± 5.4) completed CGA-GOLD in 11.7 ± 7.9 min, 86.3% required no assistance, 3.1% overall missing responses. Multiple problems reported: hypertension (18.1%), diabetes (16.9%), dyspnoea on flat surfaces (27.6%), polypharmacy (46%), difficulty walking (14.9%), fatigue (40.5%), living alone (30.9%), social isolation (11.2%), recent functional dependence (27.8%), urinary incontinence (21.4%), falls (13.3%). 237/239 intervention patients completed CGA-GOLD and consecutive subsets examined. The doctor and nurse specialist independently identified same need level in 87.3% (high inter-rater reliability kappa = 0.80), taking 1-2 min per questionnaire. Need level remained unchanged following hospital notes review against responses in 90% (75/83). 'Possible need' patients were telephoned with change in 29% (16/55) to low-risk and none to high-risk, confirming high need was not being missed. CGA-GOLD screening questionnaire was acceptable to older patients, feasibly administered in NHS cancer services, described comorbidities, CGA and QOL needs, and reliably identified higher risk patients requiring further input for optimal cancer treatment.

The impact of comprehensive geriatric assessment interventions on tolerance to chemotherapy in older people.

BACKGROUND: Although comorbidities are identified in routine oncology practice, intervention plans for the coexisting needs of older people receiving chemotherapy are rarely made. This study evaluates the impact of geriatrician-delivered comprehensive geriatric assessment (CGA) interventions on chemotherapy toxicity and tolerance for older people with cancer. METHODS: Comparative study of two cohorts of older patients (aged 70+ years) undergoing chemotherapy in a London Hospital. The observational control group (N=70, October 2010-July 2012) received standard oncology care. The intervention group (N=65, September 2011-February 2013) underwent risk stratification using a patient-completed screening questionnaire and high-risk patients received CGA. Impact of CGA interventions on chemotherapy tolerance outcomes and grade 3+ toxicity rate were evaluated. Outcomes were adjusted for age, comorbidity, metastatic disease and initial dose reductions. RESULTS: Intervention participants undergoing CGA received mean of 6.2±2.6 (range 0-15) CGA intervention plans each. They were more likely to complete cancer treatment as planned (odds ratio (OR) 4.14 (95% CI: 1.50-11.42), P=0.006) and fewer required treatment modifications (OR 0.34 (95% CI: 0.16-0.73), P=0.006). Overall grade 3+ toxicity rate was 43.8% in the intervention group and 52.9% in the control (P=0.292). CONCLUSIONS: Geriatrician-led CGA interventions were associated with improved chemotherapy tolerance. Standard oncology care should shift towards modifying coexisting conditions to optimise chemotherapy outcomes for older people.

Sorafenib for advanced hepatocellular carcinoma (HCC): impact of rationing in the United Kingdom.

BACKGROUND: The prognosis for hepatocellular carcinoma (HCC) is dependent upon tumour stage, performance status (PS), severity of underlying liver disease, and the availability of appropriate therapies. The unavailability of sorafenib may have a significantly adverse effect on the prognosis of UK patients with advanced HCC. During the study period, access to sorafenib was at the discretion of local health funding bodies, a process that may delay or deny access to the drug and that remains in place for Wales, Scotland, and Northern Ireland. Here, we attempt to address the impact of this system on patients with advanced HCC in the United Kingdom. METHODS: This is a retrospective study performed in the two largest specialist hepatobiliary oncology units in the United Kingdom. Funding applications were made to local funding bodies for patients with advanced HCC for whom sorafenib was considered appropriate (advanced HCC not suitable for loco-regional therapies, compensated chronic liver disease, PS 0-2). RESULTS: A total of 133 applications were made, of which 57 (43%) were approved and 76 (57%) declined. Demographics and prognostic factors were balanced between the two groups. This cohort had a number of adverse prognostic features: patients were predominantly PS 1-2; the majority had multifocal disease with the largest lesion being >5 cm; and macroscopic vascular invasion, metastases, and AFP >,000 ng ml(-1), were each present in one-third of cases. The median time from application to funding decision was 17 days (range 3-260 days). For the primary 'intention-to-treat' analysis, median overall survival was 4.1 months when funding was declined, and 9.5 months when funding was approved (hazard ratio (HR) 0.48; 95% CI 0.3186-0.7267; P=0.0005). CONCLUSION: These data support the use of sorafenib for patients with advanced HCC as an effective intervention. In the United Kingdom, this applies to a relatively small group of patients, estimated to total ∼800 per year who, unfortunately, do not survive long enough to themselves lobby for the availability of this drug. These data provide a comparison of sorafenib with supportive care and demonstrate the potential detrimental impact on patient outcomes of rationing health-care resources on the basis of cost.

Immunity to the respiratory pathogen Bordetella pertussis.

Bordetella pertussis causes whooping cough, a severe respiratory tract infection in infants and children, and also infects adults. Studies in murine models have shown that innate immune mechanisms involving dendritic cells, macrophages, neutrophils, natural killer cells, and antimicrobial peptides help to control the infection, while complete bacterial clearance requires cellular immunity mediated by T-helper type 1 (Th1) and Th17 cells. Whole cell pertussis vaccines (wP) are effective, but reactogenic, and have been replaced in most developed countries by acellular pertussis vaccines (aP). However, the incidence of pertussis is still high in many vaccinated populations; this may reflect sub-optimal, waning, or escape from immunity induced by current aP. Protective immunity generated by wP appears to be mediated largely by Th1 cells, whereas less efficacious alum-adjuvanted aP induce strong antibody Th2 and Th17 responses. New generation aP that induce Th1 rather than Th2 responses are required to improve vaccine efficacy and prevent further spread of B. pertussis.

Emerging strategies in the treatment of advanced hepatocellular carcinoma: the role of targeted therapies.

Hepatocellular cancer (HCC) is the fifth most common cause of cancer worldwide and its incidence is increasing as a result of the dissemination of hepatitis B and C virus infection. Surgical resection and liver transplantation are considered the only cures for HCC, but benefit approximately 10-15% of patients. In addition, radiofrequency ablation may is potentially curative for patients' with small HCC. Some patients with unresectable disease confined to the liver may benefit from embolisation or chemoembolisation. In the presence of disease not amenable to loco-regional therapy, median survival is only a few months. Current systemic therapy with cytotoxic chemotherapy induces relatively few responses and has no clear survival benefit. Current interest is focussed on the potential role of targeted therapies based on the key aspects of molecular pathogenesis of HCC, most notably sorafenib, an oral multikinase inhibitor. Recent developments discussed in this article demonstrate the potential benefits of this drug which seems destined to become first-line therapy for advanced HCC.

Epirubicin, cisplatin and protracted venous infusion 5-Fluorouracil chemotherapy for advanced salivary adenoid cystic carcinoma.

AIMS: Adenoid cystic carcinoma (ACC) is a rare tumour that usually arises in the salivary glands. Initial management is surgery often combined with adjuvant radiotherapy. Chemotherapy is reserved for treatment of symptomatic recurrence. We evaluated the combination of epirubicin, cisplatin and protracted venous infusion 5-fluorouracil (ECF) in the management of ACC. MATERIALS AND METHODS: Patients referred for treatment of advanced, symptomatic ACC were considered. The drugs given were epirubicin 50 mg/m(2) 3-weekly, cisplatin 60 mg/m(2) 3-weekly and protracted venous infusion 5-fluorouracil 200 mg/m(2)/day. RESULTS: Eight patients (median age 46 years) received a median of five cycles of chemotherapy. All patients had had previous surgery, seven had had previous radiotherapy and one had had previous chemotherapy. One patient showed a partial response (duration 34 months) and five showed stable disease (median duration 13.6 months [6.8-15.9+ months]). Median survival was 27 months (3.5-62.3 months). CONCLUSIONS: The activity of ECF in ACC of the head and neck seems to be similar to the combination of cisplatin and 5-fluorouracil and single-agent epirubicin.