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International audience; One of the main functions of Intestinal Alkaline Phosphatase (FAI) is to detoxify bacterial lipopolysaccharides (LPS) to control intestinal inflammation. Recent data indicate that FAI participates in the detoxification of other bacterial compounds (flagellin and DNA CpG motifs) and many free nucleotides (ATP, UDP). FAI is directly involved in the resolution of tissue inflammation mediated by Resolvin E1. The anti-inflammatory action of FAI indirectly improves the intestinal barrier function and affects the diversity of microbiota. Various intestinal diseases, including necrotizing enterocolitis, celiac disease and chronic intestinal inflammation (inflammatory bowel disease) are related to a decrease in the expression and activity of FAI. Furthermore, a high FAI activity in the colon is related with inflammatory processes due to high concentration of tissue nonspecific alkaline phosphatase isoform (FANE) and tissue infiltration by neutrophils, which also contain FANE. Exogenous administration of FAI reduces intestinal and/or systemic inflammation (depending on the route of administration). In conclusion, intestinal homeostasis and health largely depend on the capacity of FAI to detoxify LPS and remove LPS-induced metabolic inflammation. However, how our diets can actually limit gut pools of pro-inflammatory bacterial compounds and maximize IAP activity needs more in-depth investigations.; Una de las principales funciones de la Fosfatasa Alcalina Intestinal (FAI) es la detoxificación de los lipopolisacáridos (LPS) bacterianos para controlar la inflamación intestinal. Recientes publicaciones indican que FAI participa en la detoxificación de otros compuestos bacterianos (flagelina y motivos CpG de DNA) y de muchos nucleótidos libres (ATP, UDP). FAI está involucrada de manera directa en la recuperación tisular de la inflamación por la Resolvina E1. La acción antiinflamatoria de FAI mejora indirectamente la función de la barrera intestinal e impacta la diversidad y la composición de la microbiota. Diversas enfermedades intestinales, incluyendo enterocolitis necrótica, enfermedad celíaca y la inflamación crónica intestinal (o inflammatory bowel disease, IBD) están relacionadas con disminuciones en la expresión y actividad de FAI. Por otro lado, una elevada actividad de FAI en colon es sinónimo de procesos inflamatorios, debido a la elevada concentración de la isoforma tisular de Fosfatasa Alcalina no específica (FANE), y a la infiltración tisular por los neutrófilos (que también contienen FANE). En algunos ensayos en humanos se ha observado que la administración exógena de FAI reduce la inflamación intestinal/sistémica (dependiendo de la vía de administración). En conclusión, la homeóstasis intestinal y la preservación de la salud dependen en gran medida de la capacidad de FAI para detoxificar los LPS y suprimir la inflamación metabólica inducida por estos. Sin embargo, es necesario realizar investigaciones a fondo sobre como los hábitos alimenticios pueden modificar la detoxificación de los diferentes compuestos proinflamatorios bacterianos y maximizar la actividad de FAI.

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Maternal environment during pregnancy and lactation influences health of the offspring and its microbiota.Immune tolerance to dietary and commensal microbiota antigens develops early in life. Our aim was toinvestigate intestinal barrier and local immune response to LPS in piglets deriving from sows whosemicrobiota was manipulated by antibiotics. Long-term consequences on gut adaptation to a high fat diet wereexamined in growing offspring. Sows were given amoxicillin (AMX; 40 mg/kg/d) per os (n=11 AMX) ornot (n=12 CTL) from 10 d before to 21 d after parturition. One piglet per litter was sacrificed at post-natalday (PND) 14, 21 and after weaning at PND42. The remaining ones were given either a low fat (LF) or ahigh fat (HF) diet (to enhance LPS epithelial passage) from PND140 to 169. At PND14, ileal permeabilitywas greater in AMX than CTL piglets (P

Gut permeability is a key function often incriminated in the development of local and systemic inflammation. Chronic consumption of high-fat diets leads to obesity and inflammation. Data in mice revealed a causal increase in intestinal permeability to bacterial lipopolysaccharide (LPS) that may be responsible for adipose tissue development and inflammation (Cani et al, 2009). Whether intestinal or colonic barrier can be modulated by omega-6 (O6) or omega-3 (O3) PUFA in the context of high-fat diet-induced obesity is poorly understood. Intestinal epithelial barrier in vitro is strengthened by both O6 and O3 fatty acids. In vivo, rats chronically fed high fat diets enriched in PUFA did not show different paracellular permeabilities in the jejunum, compared to rats fed a diet rich in saturated fat. Whether colonic permeability is altered has not been reported. We hypothesized that PUFA-enriched diets modulate gut permeability differentially according to permeability pathway and gut site. This hypothesis was tested in 16 obese adult minipigs fed for 2 months with iso-protein and iso-calorie high-fat (12%) diets based on lard (saturated fat, S), sunflower oil (rich in O6), or fish oil (rich in O3). Ileal and colonic mucosa were collected and both para- and trans-cellular permeability were investigated with fluorescein isothiocyanate-dextran 4 and horseradish peroxidase in Ussing chambers. Gut permeability was assessed without and with an inflammatory challenge (E. coli LPS added at 0.1 and 10 μg/ml). After two months of regimen, body weight was not different between diet groups. Ileal mucosa density was not affected by the diet. Ileal permeability to FD4 was not influenced by the diet in the basal state but it tended to be higher with O3 compared to O6 diet after LPS challenge (e.g. with LPS 0.1 μg/ml: 1272 ± 389, 775 ± 355 and 1723 ± 389) ng/cm2/h; P = 0.095). Interestingly, basal colonic permeability to FD4 was higher with O3 compared to O6 diet (692 ± 165, 509 ± 150 and 1032 ± 165 ng/cm2/h for diets based on saturated, O6 and O3 FA, respectively; P,0.05). Dietary treatments did not affect basal ileal or colonic permeability to HRP. However, both ileal and colonic permeability to HRP tended to be higher with O3 than O6 diet after LPS challenge. Our data indicate that gut permeability is modulated by dietary FA source regionally and according to permeability pathway. Para-cellular permeability in the colon (but not in the ileum) was affected in the basal state by diet FA composition. Surprisingly, it was higher with the O3 as compared to O6-enriched diet. The same trends for differences between O3 and O6 diets on para- or trans-cellular permeability were also revealed under inflammatory condition.

Background: Peripartum antibiotics are thought to have long-lasting negative consequences on intestinal barrier function, immune system development and long-term health, e.g. allergy. Various kinds of stress (e.g. nutrition, environment) can be deleterious to gut barrier function. We have shown that broad spectrum antibiotic administration to sows around parturition alters various facets of gut barrier in their offspring during development and in adulthood. We tested with this model the hypothesis that early administration of a probiotic to offspring can modulate gut barrier function in young adults depending on diet and stressor. Methods: Sows (n=20) received amoxicillin orally (40mg/kgBW/d) around parturition (day-10 to day+21) in order to induce gut barrier disturbances in offspring. Piglets from half of the litters received orally a probiotic suspension (Lactobacillus sobrius, 108/ml, 1 ml/pig and intervention, PROB) at birth and then three times a week until postnatal day PND21. Offspring were reared under the sow until weaning at PND28. They were reared with a similar diet until PND141. Then, each group was split into two halves. One half in each group remained on the control (low fat, LF 2%) diet while the other halves were fed a high fat (HF, 11%) diet. Pigs were sacrificed at PND169 and pieces of ileum and colon were collected and mounted in Ussing chambers (UC). UC treatment included control, oxidative stress (monochloramine) and mast cell degranulation stress (48/80). Mucosal para- and trans-cellular permeabilities (PCP, TCP) were measured using FD4 and HRP, respectively. Results: Early PROB supplementation had no effect on offspring basal ileal and colonic permeabilities. Ileum from PROB pigs fed LF diet displayed reduced PCP in oxidative (P,0.05) and degranulation (P,0.10) conditions. No differences between PROB and CTL were seen for colonic PCP. Contrasting with this, ileal TCP was much higher (x 2 to 3) in PROB pigs with both LF and HF diets (P ,0.01) after degranulation stress. An interaction (P,0.10) between early PROB treatment and adult diet was observed for TCP after oxidative stress (decrease with LF, increase with HF). Colonic TCP was increased after both oxidative and degranulation stress in PROB pigs fed LF (but not HF) diet (P ,0.05). Conclusion: Our data in the swine model suggest that probiotics provided early to gut barrier-disturbed offspring born to mothers treated with broad-spectrum antibiotic around parturition can contribute to long-term modulation of gut barrier function following stress. However, the responses are complex and depend on gut site, and diet composition and type of stressor in adluthood. Work is in progress to investigate long-term changes in gut microbiota composition induced by early probiotic administration.

Background: Peripartum antibiotics are thought to have negative consequences on intestinal barrier function, immune system development and long-term health. These effects might be related to disturbances in neonatal bacterial colonization. Protective heat shock proteins 25 and 72 are induced physiologically by luminal bacteria in cultured intestinal epithelial cell (IEC) and in mouse intestine. HSP60 has been recently shown to contribute to IEC protection in vitro but data in vivo are lacking. We aimed at testing the hypothesis that early alterations in gut microbial colonization impact intestinal cytoprotective HSPs. Methods: Sows received amoxicillin orally (40mg/kgBW/d, ATBQ n=11; vs. untreated CTL, n=12) around parturition (d-10 till d+21). Offspring (1/litter) were sacrificed during the suckling period (d14, d28) and after weaning (d42), and ileal tissues were collected. HSP27, HSP60, HSP70 and cognate HSC70, as well as the transcription factor HSF1 were determined by western blotting and tissue levels expressed relative to β-actin. Enzyme activity of intestinal alkaline phosphatase (IAP), an HSP-like protein crucial in the detoxification of pro-inflammatory bacterial components (e.g. LPS) was also investigated in comparison. Results: Ileal tissue levels of HSP27 in offspring were unaffected by antibiotic treatment of mothers. HSP60 tended to be (P=0.07) and HSC70 was (P,0.05) higher in offspring born to ATBQ sows than in CTL (+40 and +24%, respectively). By contrast, HSP70 was much lower in offspring born to ATBQ sows than in CTL (P,0.01) and this difference was significant (P,0.01) at day 28 (-69%) and day 42 (-59%) (treatment by age interaction, P ,0.05). Ileal protein expression of HSF1 in offspring was unaffected by ATBQ treatment of sows or offspring post-natal age. In comparison to HSPs, IAP activity displayed a treatment by time interaction: it was twice lower at day 14 in offspring born to ATBQ sows than in CTL (P ,0.01) with no differences at days 28 and 42. Conclusion: Maternal antibiotic treatment peripartum had differential consequences on protein expression of intestinal heat shock proteins in her offspring in the swine. This did not involve the modulation of HSP-gene transcription factor HSF1, suggesting alternate regulatory mechanisms. Collectively, the data suggest complex, time-dependent interactions between colonizing maternal fecal microbiota and offspring intestinal HSP expression. Changes in offspring intestinal microbiota and long-term effects of perinatal antibiotic treatment on offspring intestinal HSP family are being investigated.

High-fat diets consumption induces low-grade obesity-related inflammation (Clark et al, 2011). The blood-brain barrier (BBB) can be damaged by immune cells invasion facilitated by pro-inflammatory cytokines. Interestingly, omega 3 fatty acids inhibit the production of pro-inflammatory cytokines, and omega 3 fatty acids supplementation (DHA) can increase the activity of the prefrontal cortex, which is one of the brain areas that was found deactivated in obese humans and minipigs (Val-Laillet et al, 2011). We hypothesized that a diet enriched with omega 3 might prevent the onset of central inflammation and protect some brain areas that are important for food intake control. We decided to investigate this question in 15 obese minipigs fed with high-fat diets of which the lipids were provided by either fish oil rich in omega 3 (O3), sunflower oil rich in omega 6 (O6), or lard rich in saturated fat (S). Various measures were performed on these animals, including adiposity estimation by CTscan, BBB permeability evaluation by dynamic injected CT-scan, and brain metabolism exploration by Positronic Emission Tomography (PET). After five weeks of treatment, there was no significant difference between groups for the body weight, adiposity accumulation and distribution (P.0.10). The BBB was the most permeable in the O3 group, intermediary in the S group, and the least permeable in the O6 group for the cortical areas (P=0.0326 for O3 vs O6 - Fig. 1). Basal metabolism differences were found in several brain regions involved in food intake control. For example, the anterior prefrontal cortex metabolism was lesser for O3, intermediary for S, and higher for O6 (Punc ,0.0001 - Fig. 2). There was also a decreased activity in the nucleus accumbens (Punc=0.038) for O3 compared to O6. There was a significant correlation between the BBB permeability and the prefrontal cortex activity (R=0.55, P=0.041 - Fig. 3). Contrary to what was expected, these results demonstrate that a diet enriched with O3 did not protect the BBB nor the basal metabolism of brain areas (e.g. prefrontal cortex and basal nuclei) usually altered by obesity and high-fat diets. Surprisingly, the O6 diet induced the least damage to the BBB and deactivation of the prefrontal cortex and nucleus accumbens. Further research is needed to elucidate whether a causal relationship between these two phenomena exists, and why the O6 diet, unlike O3, had this protective effect.

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Neonatal microbial colonization of the gut participates in anatomical and functional development of this organ. Perinatal use of antibiotics (AB) is suspected to contribute to later development of diet-related diseases in human, through early alterations in microbial colonization impacting gut and distant-organ function. In that respect, gut barrier function, including permeability, detoxification, immune and cytoprotection systems are important aspects. AB treatment around farrowing also happens in pig production. However, the consequences on gut barrier throughout life in offspring are unknown. During the Interplay EU project, we developed a pig model of AB-induced early microbiota disturbances for investigating short- and longerterm consequences on offspring gut barrier function. Sows were given orally a broad spectrum AB around farrowing (day -10 to +21). Offspring were sacrificed during suckling, after weaning and at 6 months of age. In the long-term protocol, offspring were fed a high fat (vs. low fat) diet for 4 weeks as a model of diet-induced low grade inflammation. AB treatment of sows affected their faecal microbiota and that of their offspring. We observed age-dependent alterations in offspring ileal and colonic permeability, ileal epithelial transcriptome, mucosal inflammation traits, digestive enzymes, ileal and colonic protective heat shock proteins and in digesta concentrations of pro-inflammatory bacterial components. Interactions between perinatal antibiotic treatment of mothers and offspring adult diet was often significant for gut parameters. Data indicate that both early gut microbial colonization disturbances and late nutritional environment condition gut function in adulthood according to specific spatio-temporal patterns. Underlying molecular mechanisms and correlations between physiological and microbial traits are under investigation.

Effets d'une supplémentation orale en butyrate sur la physiologie intestinale chez le porc en croissance. 10. Journées Francophones de Nutrition (JFN)

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International audience; Early disturbance by antibiotic treatment on the microbial colonization process can have drastic consequences for gastrointestinal tract development. This study aimed at evaluating the effects of amoxicillin treatment (AT) of sows before and after farrowing on the intestinal microbiota in their offspring during the suckling and the post-weaning period. Two groups of 4 sows each were defined as control and treatment. In the treatment, sows were treated orally with amoxicillin (40 mg/kg BW/d) during 10 d before and 21 d after parturition. Feces of sows were collected at the beginning and the end of the AT. Offsprings (1/sow/time) were sacrificed at d 14, 21, 28 and 42 after birth, to collect ileal digesta. The microbial composition of feces and ileal digesta was analyzed by the Pig Gastrointestinal Tract Chip. Cluster analysis of microbiota profiles of sows showed separate grouping of samples from treated sows at the end of AT. This was further confirmed by principal response curves (PRC) analysis which showed a large deviation of the treatment from the control at the end of AT and indicated a significant effect (P = 0.045) of AT. PRC analysis revealed a reduction of abundance of Mycoplasmalike, L. gasseri-like, Porphyromonas asaccharolytica-like and L. delbrueckii-like organisms of treated sows. Cluster analysis of ileal microbiota of piglets showed samples from d 42 grouped in a distinct assembly, while all other samples formed, with few exceptions, 2 clusters according to treatment, indicating AT of sows influenced the microbiota of piglets. Microbial diversity of piglets derived from treated sows was lower than that of controls at all time points, and was significantly different at d 42(P = 0.018). The PRC analysis showed deviations of the treatment from the control at each sampling point and confirmed the sustained AT effect on the ileal microbiota. Reduction of L. gasserilike and Mycoplasma-like organisms of sows caused by AT also appeared in digesta of piglets. In conclusion, pre and postpartum AT had a significant effect on intestinal microbiota of sows, which in turn influenced the microbial colonization of their offspring.

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International audience; The Developmental Origins of Health and Disease hypothesis formulated in the early 1990s has stimulated research on long-term effects of early nutrition and environment over the last decades. Longterm is understood in this review as physiologically relevant periods such as after weaning, around sexual maturity, and in adulthood, as opposed to early developmental periods. The small and large intestines as targets for the study of long-term effects have received little attention until recent years and the stomach has been considered very rarely. Data have accumulated for laboratory animal models but they are still scarce in the swine species. Following the epidemics of metabolic diseases and obesity in western countries, experimental evidence has been published showing that nutritional factors, including energy, fat and fatty acids, protein, and micronutrients impact various facets of gut function. These include alterations in intestinal digestive, absorptive, secretory, barrier, and defense systems, often in a way potentially detrimental to the host. Environmental factors with long-term influence include stress (e. g., maternal deprivation, neonatal gut irritation), chemical pollutants (e. g., bisphenol A), and gut microbiota disturbances (e. g., by antibiotics). Examples of such long-term effects on the gut are provided in both laboratory animals and pigs together with underlying physiological mechanisms whenever available. Experimental evidence for the involvement of underlying epigenetic modifications (e. g., genomic DNA methylation) in long-term studies has just started to emerge with regard to the gastrointestinal tract. Also, interactions between the microbiota and the host are being considered pivotal in the early programming of gut functions. Finally, suggestions for future research are provided in order to better understand and then control early programming as an attempt to optimize vital functions of the gastrointestinal tract throughout adult life.

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International audience; Gut maturation during suckling and after weaning is influenced by maternal environment. Scarce is the knowledge on the presence and the regulation of taste receptors in the pig gastro-intestinal tract. The same is for the machinery that processes the taste signals, including α-transducin. Our main goal was to assess if the age of offspring and maternal environment, as influenced by a maternal antibiotic treatment, could affect the expression of the receptors for umami and sweet taste in offspring stomach. Eighty 4 pigs reared from sows fed a diet with or without Amoxicillin (40 mg/kg BW/d, on −10 d to +21 d from farrowing, ATB) were sacrificed at 14 d, 21 d, 28 d (weaning) or 42 d, and sampled for oxyntic (OXY) and pyloric mucosa (PY). In PY, gene expression (RT Real-Time PCR, corrected for 2 housekeeping genes) for TAS1R3 (necessary for sweet and umami taste) was stable in time and not affected by ATB. TAS1R1 (complementary for umami) increased from suckling to post-weaning period (P = 0.001), and was lower in ATB offspring (P = 0.013). TAS1R2 (complementary for sweet) gene expression was not detected. TAS1R1 gene expression in OXY, increased with age in suckling period (P = 0.013), but not later. Transducin gene expression in PY was higher in ATB offspring (P = 0.043); the number of cells staining for transducin per gland in PY decreased during the suckling and post-weaning periods (P < 0.001), while cell counts tended to be lower in ATB offspring (P =0.092). TAS1R1 and TAS1R3 gene expression in PY was also tested in littermate offspring that were reared up to the age of 5 mo, and then kept on the same diet (low fat, 2%) or switched to a high fat (11%) diet for 4 weeks (n = 10 pigs per group). Neither perinatal ATB treatment nor later fat diet content affected the studied variables. Data show that sensing for umami taste has relevance for 2 different gastric mucosae. It can change with age and be potentially affected by maternal environment. For α-transducin, divergent observations on gene expression and positive cell counts require further evaluation.

International audience

International audience; The gastrointestinal tract (GIT) of young mammals is submitted to aggressions early in life and GIT stress proteins are up-regulated in pigs following weaning. We hypothesized that transient food deprivation may contribute to these changes. Therefore, the effects of fasting and refeeding on GIT stress proteins in weaned pigs were investigated. A complete block experimental design with three groups of five pigs each was set up with the following treatments: A - food offered, B - fasted for 1.5 days, C - fasted for 1.5 days and then re-fed for 2.5 days. After slaughter, the GIT was removed, weighed and sampled. Intestinal villi and crypts were measured and alkaline phosphatase activity was determined. GIT tissue stress protein concentrations were measured by Western blotting. Fasting led to intestinal mucosa and villous-crypt atrophy (p < 0.01) and reduced mucosal alkaline phosphatase total activity in the proximal small intestine (p < 0.05). Heat shock proteins HSP 27 and HSP 90 (but not HSP 70) and neuronal NO synthase (nNOS) increased (p < 0.01) in the stomach, mid-intestine and proximal colon with fasting. Inducible NOS (iNOS) did so in the stomach (p < 0.001). Refeeding partially or totally restored GIT characteristics and stress protein concentrations, except for gastric HSP 90 and iNOS. Significant correlations (p < 0.05 to p < 0.0001) were found among stress proteins, between nNOS and digesta weight, between HSP 27 or HSP 90 and intestinal mucosa weight, and between intestinal or colonic HSP or nNOS and alkaline phosphatase. In conclusion, fasting and refeeding modulate GIT HSP proteins and nNOS in pigs following weaning. Changes in digesta and intestinal mucosa weights and alkaline phosphatase activity may be involved in the modulation of stress proteins along the GIT.

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National audience; The diverse roles of intestinal alkaline phosphatase (IAP) have long remained elusive. However, four novel functions have been reported recently. It was shown that IAP participates in the regulations of lipid absorption across enterocyte’s apical membrane, and of bicarbonate secretion and extracellular duodenal pH. IAP is responsible for bacterial lipopolysaccharide (LPS) detoxification and for controlling LPS-induced inflammation. IAP regulates bacterial translocation across intestinal epithelium. A retrospective analysis of the literature was then conducted for analysing the potential of the diet to modulate IAP. This analysis revealed that many dietary components including fat, protein and carbohydrate are important IAP modulators. In conclusion, IAP plays key roles in the control of intestinal homeostasis and its activity could be increased through the diet, especially in pathological situations (e.g. inflammatory bowel diseases) where the involvement of commensal bacteria is suspected, and when intestinal AP is too low to detoxify enough of bacterial LPS.; Les rôles exacts de la phosphatase alcaline intestinale (PAI) sont restés mal connus pendant longtemps. C’est seulement récemment que quatre nouvelles fonctions ont été démontrées pour la PAI. Elle joue un rôle crucial dans la régulation de l’absorption intestinale des lipides, de la sécrétion duodénale de bicarbonate et du maintien du pH extracellulaire local, dans la détoxification des endotoxines et le contrôle de l’inflammation induite par celles-ci, et enfin dans la prévention de la translocation bactérienne intestinale. Une analyse rétrospective de la littérature a été conduite pour évaluer la capacité de l’alimentation à moduler l’activité intestinale de la PAI. Il en ressort que plusieurs constituants, incluant les lipides, les protéines et les glucides, modulent l’expression et/ou l’activité de la PAI. En conclusion, la PAI joue un rôle clé dans le contrôle de l’homéostasie intestinale et son activité peut être augmentée par voie nutritionnelle, en particulier dans les situations pathologiques (par exemple les maladies inflammatoires chroniques de l’intestin) dans lesquelles l’implication des bactéries commensales est suspectée, et quand l’activité de la PAI est trop faible pour détoxifier les endotoxines.

The weight-of-evidence, case-by-case approach is considered the most appropriate way of assessing the allergenicity of genetically modified (GM) food and feed. This scientific opinion discusses various aspects to increase the strength and accuracy of this approach, including the latest developments pertaining to clinical aspects of allergic reactions, structural aspects of GM food and feed and in silico approaches, as well as IgE binding studies and cell-based methods, profiling techniques and animal models. In this context, conclusions and recommendations are provided to update and complement current risk assessment strategies for the allergenicity assessment of newly expressed protein(s) and whole GM food and feed. In summary, it is recommended that with regard to the search for sequence homology and structural similarities, the local alignment method with a known allergen with a threshold of 35% sequence identity over a window of at least 80 amino acids is considered a minimal requirement. When IgE binding tests are considered necessary, e.g. when there is sequence homology and/or structure similarity with known allergens, the use of individual sera from allergic individuals rather than pooled sera is recommended. In addition to the pepsin resistance test, it is recommended that the resistance to digestion of the newly expressed proteins is evaluated using other in vitro digestibility tests mimicking physiological conditions of humans. Finally, when the recipient of the introduced gene is allergenic, in order to compare the allergenicty of the whole GM plant with that of its appropriate comparator(s), it is recommended that relevant characterised endogenous allergens are included in the comparative compositional analysis of the GM plant and its appropriate comparator(s). Proposals for the use of additional testing that may improve the weight-of-evidence approach and suggestions for further evaluation of new promising methods that are as yet in an early phase of development are also addressed.

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