Analysis of KC-like as biomarker for sepsis in bitches with pyometra

Sepsis är ett komplext och livshotande tillstånd orsakat av en infektion som skapar en dysreglerad inflammation, vilket ger upphov till ett så kallat Systemic Inflammatory Response Syndrome (SIRS). Mortaliteten vid sepsis är hög hos hund och en tidig diagnos och insättande av behandling är därför mycket viktigt överlevnaden. Pyometra är en vanlig sjukdom hos tikar och i en studie av tikar med pyometra klassificerades över hälften som SIRS-positiva, enligt de kriterier som idag används för diagnostik av sepsis hos hund. Patogenesen till sepsis är ännu inte helt klarlagd och definitionen och kriterierna har därför ändrats under årens lopp. Senast år 2016 kom nya riktlinjer inom humanmedicinen som ändrade synen på syndromet till att ses som ett obalanserat hyper- och hypoinflammatoriskt tillstånd och även omfatta organsvikt. SIRS-kriterierna som tidigare varit rådande byttes till det så kallade Sepsis-related Organ Failure Assessment (SOFA), där fler organspecifika parametrar mäts. Detta system har dock ännu inte utvärderats i större omfattning hos hundar med sepsis. Biomarkörer som har studerats för identifiering av individer med hög sannolikhet för sepsis är bland annat akutfasproteinet C-reaktivt protein (CRP) och ett antal inflammatoriska cytokiner. Ingen av dessa har dock ännu setts på ett säkert sätt kunna identifiera hundar med sepsis. Studier har istället visat att en antikropp mot kemokinen keratinocyte chemoattractant (KC) hos mus kan känna igen förhöjda koncentrationer av ett protein (kallat KC-like) i blodet hos tikar med sepsis. Vid en gensekvensanalys hittades ingen motsvarande gen hos hund för KC, men ett antal C-X-C motif ligand (CXCL)-kemokiner sågs ha hög likhet. Studien syftade till att analysera vilken av dessa CXCL-kemokiner som KC-antikroppen binder starkast till samt om förhöjda koncentrationer av protein som KC-antikroppen binder till förekommer hos tikar med pyometra och SIRS jämfört med de utan SIRS. De utvalda CXCL-kemokinerna uttrycktes därför i Escherichia coli och analyserades med Sodium Dodecyl Sulfate-Polyacrylamid Gel Electrophoresis (SDS-PAGE) och western blot. Blodprover insamlade från tikar med pyometra med eller utan SIRS analyserades också med en Enzyme-linked Immunosorbent Assay (ELISA) med KC-antikropp hos mus. Resultatet från analyserna visar att KC-antikroppen binder framförallt en av de utvalda CXCL-kemokinerna och denna skulle därför kunna vara en potentiell biomarkör för sepsis hos hund. För att bekräfta detta fynd krävs dock ett förbättrat analysprotokoll. Signifikant skillnad i koncentration KC-like sågs mellan grupperna i studien där tikar med pyometra och SIRS sågs ha förhöjda nivåer. Emellertid krävs större och mer detaljerade studier på KC-like för att verifiera dessa resultat. De obalanserade immunologiska processer som sker vid sepsis gör att det i dagsläget är svårt att hitta en tillräckligt säker enskild biomarkör för sepsis. Utveckling av de nuvarande SIRS-kriterierna samt en panel av pro- och antiinflammatoriska biomarkörer, där KC-like skulle kunna ingå, skulle därför kunna vara det effektivaste sättet att identifiera detta allvarliga och komplexa syndrom hos hund.Sepsis is a complex and life-threatening condition caused by an infection creating a dysregulated inflammation, leading to a Systemic Inflammatory Response Syndrome (SIRS). Sepsis-related mortality is high for dogs and an early diagnosis and start of treatment is very important for survival. Pyometra is a common disease in bitches and in a Swedish study of bitches with pyometra over half were classified as SIRS-positive, according to the criterias of that today is used for diagnostics of sepsis for dogs. The pathogenesis of sepsis is not yet clear and the definition and the criterias has changed during the years. The latest guidelines in the human medicine came in 2016 and it changed the view of the syndrome to be seen as an unbalanced hyper- and hypoinflammatory condition and to also include organ failure. The SIRS-criteria which was used earlier were changed to the so called Sepsis-related Organ Failure Assessment (SOFA), where multiple organ-specific parameters are measured. This system has not yet been fully evaluated in dogs with sepsis. Biomarkers that have been studied to identify individuals with a high likelihood of sepsis is among others the acute-phase protein C-reactive protein (CRP) and a number of inflammatory cytokines. None of these have yet, in a safe way, been able to identify dogs with sepsis. Instead studies have shown that an antibody, directed against the chemokine keratinocyte chemoattractant (KC) in mice, can recognize elevated concentrations of a protein (called KC-like) in the blood of bitches with sepsis. A gene-sequence analysis found no corresponding gene in dogs for KC but a number of C-X-C motif ligand (CXCL)-chemokines was seen to have a high resemblance. This study aimed to analyse which of these CXCL-chemokines the KC-antibody binds the strongest to and if elevated concentrations of protein that the KC-antibody binds to occur in bitches with pyometra and SIRS compared to bitches without SIRS. The selected CXCL-chemokines was therefore expressed in Escherichia coli and were analysed with Sodium Dodecyl Sulfate-Polyacrylamid Gel Electrophoresis (SDS-PAGE) and western blot. Blood samples collected from bitches with pyometra with or without SIRS was analysed with an Enzyme-linked Immunosorbent Assay (ELISA) with an antibody against KC in mouse. Results from the analyses show that the KC-antibody especially bind one of the studied CXCL-chemokines, which therefore could be a potential biomarker for sepsis in dogs. To confirm this an improved protocol for analysis is needed. A significant difference in concentration of KC-like was seen between groups in the study where bitches with pyometra and SIRS had higher levels. Detailed studies including more dogs are needed on KC-like to verify these findings. The unbalanced immunologic processes that occur in sepsis make it hard to find a safe individual biomarker for sepsis today. Development of the current SIRS-criteria and a panel of pro- and anti-inflammatory biomarkers, where KC-like could be included, could therefore be the most efficient way to identify this severe and complex syndrome in dogs

Hormonal influence and receptor expression in canine mammary tumours

Juvertumörer är de vanligaste tumörerna hos tikar och det finns olika typer av både benigna och maligna juvertumörer. De hormonella fluktuationerna som sker under östralcykeln driver utvecklingen av juvervävnaden och behandling med progestiner (syntetiska ekvivalenter till progesteron) för uppskjutande av löp har kopplats samman med förekomst av juvertumörer, vilket har lett till ett intresse för den hormonella påverkan på juvertumörutvecklingen. Hormonerna är i sin tur beroende av uttrycket av receptorer för att kunna ha effekt i vävnaden. Syftet med den här litteraturstudien är att utreda uttrycket av hormoner och receptorer för progesteron, tillväxthormon (growth hormone, GH) och insulinliknande tillväxtfaktor (insulin-like growth factor, IGF) i normal juvervävnad och i benigna och maligna tumörer, såväl som deras påverkan på juvertumörutvecklingen. De intracellulära signalvägarna för progesteronreceptorer (PR), GH-receptorer (GHR) och IGF-I-receptorn (IGF-IR) är kopplade till cellproliferation och genreglering. De komplexa nätverken av signalvägar interagerar med varandra och en obalans i uttryck och reglering av hormoner, receptorer eller molekyler i cellen kan leda till att tumörer uppstår. Vid progestinbehandling har ökade nivåer av GH och IGF-I uppmätts och högst nivåer sågs i maligna tumörer. Uttrycket av PR visades vara högt i benigna juvertumörer i motsats till maligna tumörer där PR-uttrycket istället var lågt. Samtidigt har resultat visat på ett högre uttryck av GHR och IGF-IR i maligna tumörer. PR har också visats finnas i två isoformer, kallade PR-A och PR-B, och PR-A sågs dominera över PR-B i maligna tumörer. Nya studier på hundens PR-B tyder på att den kan ha en nedsatt funktion via mutationer i aktiveringsdomänen AF3 i PR-genen. Därför diskuteras vilken betydelse PR-B har hos hund och vikten framhävs av att i framtida studier analysera inte bara totalt PR-uttryck utan även skillnader mellan PR-A och PR-B. Varierande resultat från olika morfologiska juvertumörtyper har också påvisats och det anses därför viktigt att särskilja mellan slutsatser från tumörer med olika cellulärt ursprung. Avslutningsvis konstateras progesteron-GH-IGF-axeln ha en stimulerande påverkan på juvertillväxten och att det finns skillnader i uttryck av hormoner och receptorer i tumöromvandlad vävnad. Resultaten tyder på att förlust av PR leder till att uttrycket av GH-genen blir progestinoberoende efter transformering till maligna tumörer och genen regleras då inte längre av de normala hormonella fluktuationerna. Istället produceras GH och IGF-I lokalt av tumörerna och uttrycket av deras receptorer ökar. Sammantaget tyder det på att det skapats en oberoende parakrin och autokrin produktion hos tumörcellerna som driver tumörens tillväxt. Motstridiga resultat kring GH/GHR-systemets proliferativa påverkan i olika juvertumörtyper anses tyda på att det krävs ytterligare forskning kring kopplingen mellan olika signalvägar, hormoner och receptorer som är inblandade i utvecklingen av juvertumörer.Mammary tumours are the most common tumours seen in female dogs and there are many different types of benign and malignant mammary tumours. The hormonal fluctuations during the oestrus cycle is the driving force of development of the mammary tissue and treatment with progestins (synthetic progesterone equivalents) for the postponement of ovulation has been correlated to the incidence of canine mammary tumours, which has led to the interest of studying the hormonal influence on the development of mammary tumours. The effect of hormones is in turn dependent on the expression of receptors in tissues. The purpose of this literature study is to investigate the expression of hormones and receptors for progesterone, growth hormone (GH) and insulin-like growth factor (IGF) in normal mammary tissue and benign and malignant tumours as well as their influence on the development of mammary tumours. Intracellular signalling pathways for the progesterone receptor (PR), the GH-receptor (GHR) and the IGF-receptor (IGF-IR) are connected to cell proliferation and gene regulation. The complex networks of the signalling pathways interact with each other and an imbalance in the expression and regulation of hormones, receptors or molecules in the cell can lead to the development of tumours. During treatment with progestins, increasing levels of GH and IGF-I has been registered and the highest levels are seen in malignant tumours. The expression of PR was shown to be elevated in benign tumours. In contrast, malignant tumours showed a low expression of PR with a concurrent higher expression of GHR and IGF-IR. PR has also been shown to be expressed in two isoforms, PR-A and PR-B, of which the levels of PR-A are dominating over PR-B. Recent studies have revealed that the canine PR-B might have a diminished function caused by mutations in an activation domain in the PR-gene. Therefore the relevance of PR-B in dogs is discussed and the importance of analysing not only total expression of PR, but also the differences between PR-A and PR-B in future studies is emphasized. Varying results in different morphological types of mammary tumours has been shown and it is therefore considered important to distinguish between conclusions from tumours with different cellular origin. In conclusion, it is determined that the progesterone-GH-IGF-axis has a stimulating effect on the growth of mammary tissue and there are differences in expression of hormones and receptors in mammary tumours. Results indicate that the loss of PR leads to independence from progestins in the expression of the GH-gene after malignant transformation of tumours and it is no longer regulated by the normal hormonal fluctuations. Instead, GH and IGF-I is produced locally by the tumour and the expression of their receptors are increased, indicating that an independent paracrine and autocrine production by the tumour cells themselves is created which leads to tumour growth. Contradicting results of the proliferative influence of the GH/GHR-system is considered to indicate the need for additional research regarding the link between the different signalling pathways, hormones and receptors that are involved in the development of canine mammary tumours

Males respond to female begging signals of need: A handicapping experiment in the pied flycatcher, Ficedula hypoleuca

The 'female nutrition' hypothesis proposes that food provided by males during incubation is an important energy source for females in bird species in which females alone incubate. Females should be able to communicate their needs through begging signals to mates and males may compensate for the energetic limitations of females through their feeding visits, owing to their overlapping reproductive interests. To test whether female begging during incubation is an honest signal of energetic need and whether mates respond to it we experimentally handicapped female pied flycatchers at the beginning of incubation by clipping two primary flight feathers on each wing. Experimental manipulation led females to intensify begging displays arising from condition impairment and males accordingly increased their incubation feeding rates. Female begging intensity explained more than half of the variation in male incubation feeding rate, thereby showing that female nutrition is the main factor explaining male incubation feeding. Moreover, handicapped females consumed a higher proportion of male food deliveries during the first few days after hatching and weighed less at the end of the nestling period than control females. Handicapping had no influence on female incubation behaviour, hatching and breeding success, nestling and male condition or female nestling provisioning. The provisioning rates of males in the late nestling stage were higher in experimental nests. This is the first experimental study showing that males adjust incubation feeding rates to behavioural displays of need by their mates. The ability of females to modify their begging displays according to need may be an important adaptation that allows females to maintain a good energetic condition during incubation. © 2014 The Association for the Study of Animal Behaviour.This study was financed by project CGL2010-19233-C03-02 to J.M. from Spanish MICINN.Peer Reviewe

Oxidative Stress in Early Life: Associations with Sex, Rearing Conditions, and Parental Physiological Traits in Nestling Pied Flycatchers

The chapter IV of the dissertation: López-Arrabé, Jimena. Physiological adaptations in the pied flycatcher (Ficedula hypoleuca): oxidative stress, reproduction and development (2017), reproduces the manuscript of: López-Arrabé, Jimena ; Cantarero, Alejandro ; Pérez-Rodríguez, Lorenzo ; Palma, Antonio; Moreno, Juan. Oxidative Stress in Early Life: Associations with Sex, Rearing Conditions, and Parental Physiological Traits in Nestling Pied Flycatchers. Physiological and Biochemical Zoology, 89(2): 83-92(2016). http://dx.doi.org/10.1086/685476. http://hdl.handle.net/10261/130300El capítulo IV de la tesis doctoral: López-Arrabé, Jimena. Physiological adaptations in the pied flycatcher (Ficedula hypoleuca): oxidative stress, reproduction and development (2017), reproduce la información del artículo: López-Arrabé, Jimena ; Cantarero, Alejandro ; Pérez-Rodríguez, Lorenzo ; Palma, Antonio; Moreno, Juan. Oxidative Stress in Early Life: Associations with Sex, Rearing Conditions, and Parental Physiological Traits in Nestling Pied Flycatchers. Physiological and Biochemical Zoology, 89(2): 83-92(2016). http://dx.doi.org/10.1086/685476. http://hdl.handle.net/10261/130300Conditions experienced during juvenile development can affect the fitness of an organism. During early life, oxidative stress levels can be particularly high as a result of the increased metabolism and the relatively immature antioxidant system of the individual, and this may have medium- and long-term fitness consequences. Here we explore variation in levels of oxidative stress measured during early life in relation to sex, rearing conditions (hatching date and brood size), and parental condition and levels of oxidative markers in a wild population of the pied flycatcher (Ficedula hypoleuca) followed for 2 yr. A marker of total antioxidant status (TAS) in plasma and total levels of glutathione (GSH) in red blood cells, as well as a marker of oxidative damage in plasma lipids (malondialdehyde [MDA]), were assessed simultaneously. Our results show that nestling total GSH levels were associated with parental oxidative status, correlating negatively with maternal MDA and positively with total GSH levels of both parents, with a high estimated heritability. This suggests that parental physiology and genes could be determinants for endogenous components of the antioxidant system of the offspring. Moreover, we found that total GSH levels were higher in female than in male nestlings and that hatching date was positively associated with antioxidant defenses (higher TAS and total GSH levels). These results suggest that different components of oxidative balance are related to a variety of environmental and intrinsic—including parental—influencing factors. Future experimental studies must disentangle the relative contribution of each of these on nestling oxidative status and how the resulting oxidative stress at early phases shape adult phenotype and fitness.Peer reviewe

On the heritability of blue-green eggshell coloration

Avian blue-green eggshell coloration has been proposed as a female signal of genetic or phenotypic quality to males. However, little is known about the relative importance of additive genetic and environmental effects as sources of eggshell colour variation in natural populations. Using 5 years of data and animal models, we explored these effects in a free-living population of pied flycatchers. Permanent environmental and year effects were negligible, although year environmental variance (VYear) was significant for all but one of the traits. However, we found high–moderate narrow-sense heritabilities for some colour parameters. Within-clutch colour variability showed the highest coefficient of additive genetic variation (i.e. evolvability). Previous evidence suggests that eggshell colour is sexually selected in this species, males enhancing parental effort in clutches with higher colour variability and peak values. Eggshell colour could be driven by good-genes selection in pied flycatchers although further genetic studies should confirm this possibilityPeer reviewe

Immediate skin-to-skin contact after birth ensures stable thermoregulation in very preterm infants in high-resource settings

Aim To investigate the impact of immediate skin-to-skin contact with a parent after birth on thermal regulation in very preterm infants. Methods This clinical trial was conducted in three neonatal intensive care units in Scandinavia from 2018 to 2021. Infants born between 28 + 0 and 32 + 6 weeks and days of gestation were randomised to immediate skin-to-skin contact or conventional care in an incubator during the first 6 postnatal hours. We report on a secondary outcome: serial measurements of axillary temperature. Results Ninety-one infants were randomised to skin-to-skin contact or conventional care. Mean (range) gestational ages were 31 + 2 (28 + 6, 32 + 5) and 31 + 0 (28 + 4, 32 + 6) weeks and days, mean birth weights were 1572 (702, 2352) and 1495 (555, 2440) grams, respectively. Mean (95%CI, p-value) temperatures were within the normal range in both groups, 0.2°C (−0.29, −0.14, p < 0.001) lower in the skin-to-skin contact group. The skin-to-skin contact group had a lower relative risk (95%CI, p-value) of developing events of hyperthermia, RR = 0.70 (0.50, 0.99, p = 0.04). Conclusions Very preterm infants, irrespective of clinical stability, do not develop hypothermia during immediate skin-to-skin contact after birth. Immediate skin-to-skin contact did protect against events of hyperthermia. Concerns about thermal regulation should not limit implementation of immediate skin-to-skin contact in high-resource settings.publishedVersio

VI Curso de Ornitología

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La naturaleza no enseña nada: los errores del instruccionismo en el estudio de la conducta y de la adaptación

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La estructuración de las comunidades ecológicas por selección natural: una lección para la ecología de Wallace y Darwin

Estamos tan acostumbrados a relacionar a la selección natural con procesos evolutivos en el seno de poblaciones de organismos, que apenas paramos a pensar en otras consecuencias de las diferencias individuales en probabilidades de supervivencia y reproducción, es decir en eficacia biológica o “fitness” en inglés, que existen en todas las poblaciones naturales. Una de ellas es la composición de comunidades de organismos distintos que comparten un determinado hábitat. La ecología de comunidades ha sido tradicionalmente abordada desde un enfoque claramente taxonómico, intentando explicar las diferentes abundancias relativas de distintas especies que coexisten en diversos lugares. El ejemplo más claro son los estudios de la vegetación, su variación geográfica y sus cambios temporales, incluida la famosa sucesión. Los organismos coexistentes han sido considerados exclusivamente como miembros de una determinada especie, y los rasgos de estos individuos incluidos en los de la especie correspondiente. Distintas tradiciones dentro de la ecología han abordado el estudio de las comunidades como el de interacciones entre especies. Así la tradición basada en los nichos ecológicos, iniciada por Gause, Hutchinson y resumida por MacArthur (1972), ha intentado explorar las implicaciones de la coexistencia de organismos con nichos de especie más o menos similares. La competencia interespecífica basada en similitud de nichos ha sido valorada como una de los principales factores explicativos de la composición de comunidades por esta venerable tradición que tiene su origen, como no, en Darwin mismo. Otra tradición, iniciada por las críticas a los modelos de nichos de Simberloff y llevada a su apogeo por Bell y especialmente por Hubbell (2001), es la que postula que las comunidades son agregados al azar o ‘neutrales’ de especies provenientes de un “pool” disponible de especies y mezclados por contingentes procesos de dispersión.Peer Reviewe

Malentendidos sobre la Teoría de Darwin

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