Equine and human mutual welfare: a whole subject? Critical aspects and possible strategies in equine-assisted activities and therapies

General aim of the study is equine welfare, particularly concerning different husbandry methodic and inter-specific relational factors. Specific aim is the evaluation of possible mutual (to humans and to equines) benefits and the analysis of critical factors/strength points, of human-horse relationship within Therapeutic Riding context (TR). The peculiarities of human-horse relationship (compared to the bond with “Pet”) are analyzed, concerning their socio-anthropological, psychological, psycho-dynamic distinctive characteristics. 8 European representative therapeutic riding centers (TRC) were therefore selected (on the basis of their different animals’ husbandry criteria, and of the different rehabilitative methodologies adopted). TRC were investigated through 2 different questionnaires, specifically settled to access objective/subjective animal welfare parameters; the quality of human-horse relationship; technicians’ emotional experienced. 3 Centers were further selected, and behavioral (145 hours of behavioral recording) and physiological parameters (heart rate and heart rate variability) were evaluated, aimed to access equine welfare and horses’ adaptive responses/coping (towards general environment and towards TR job). Moreover a specific “handling-task” was ideated and experimented, aimed to measure the quality of TR technicians-horses relationship. We did therefore evaluate both the individual horses’ responses and the possible differences among Centers. Data collected highlight the lack of univocal standardized methodic, concerning the general animals’ management and the specific methodologies (aimed to improve animal welfare and to empower TR efficacy). Some positive and some critical aspects were detected concerning TR personnel-horse relationship. Another experimental approach did evaluate the efficacy (concerning the mutual benefits’ empowerment) of an “ethologically-fitted” TR intervention, aimed to educate children to and through the relationship with horses. Our data evidenced that the improvement of human horse relationship, through structured educational programs for TR personnel might have important consequences both to human and equine welfare.

Generierung und Prozessierung oxidativer DNA-Schäden

Generierung und Prozessierung oxidativer DNA Schäden --- Ziel dieser Arbeit war es, adaptive Antworten der Zellen auf einen DNA Schädigung zu untersuchen. Hierzu wurden Experimente zur Reparatur oxidierter Basen (Substrate der Basen Exzisions Reparatur (BER)) oder von Pyrimidindimeren (Substrate der Nukleotid Exzisions Reparatur (NER)) nach einer Vorbehandlung mit DNA-schädigender Agenzien durchgeführt. Die Ergebnisse zeigten, dass sowohl eine Vorbehandlung mit einer alkylierenden als auch mit einer oxidierenden Substanz zu einer adaptiven Erhöhung des zellulären Glutathionspiegels führte, die 16 h nach der Schädigung ihr Maximum erreichte. Jedoch waren die 8-oxoG Glykosylaseaktivitäten über einen Zeitraum von 18 h konstant. Diese Effekte waren unabhängig davon, ob Maus Embryofibroblasten, primäre oder p53 profiziente menschliche Zellen verwendet wurden. Die BER war ebenfalls in keiner der verschiedenen Zelllinien signifikant verbessert. Die adaptive Antwort bezüglich der Glutathionspiegel war also nicht mit einer entsprechenden Veränderung bei der DNA-Reparatur verbunden. Folglich ist die Reparatur von oxidativen DNA-Schäden durch eine vorausgehende Schädigung nicht induzierbar. Der zweite Teil der Untersuchungen zu der Reparatur beschäftigte sich mit der NER. Hierzu wurde die Reaktivierung eines mit UVB-Strahlung geschädigten Plasmids untersucht. Als Wirtszellen fungierten primäre menschliche Fibroblasten und Keratinozyten, die entweder mit UVB vorbehandelt oder ungeschädigt waren. Auch für die NER konnte keine signifikante Beschleunigung der Reparatur von Pyrimidindimeren durch eine Vorbehandlung festgestellt werden. Die Reaktivierung erfolgte ferner unabhängig vom p53-Status der Zellen, wie Versuche mit p53-siRNA zeigten. Neben der Prozessierung war die Generierung oxidativer DNA Schäden Gegenstand der Arbeit. Die verwendete Substanz Tirapazamin (TPZ) ist ein für hypoxische Zellen selektives, neues Zytostatikum und befindet sich momentan in Phase 2/3 der klinischen Prüfung. Ziel war es die von TPZ verursachten DNA Modifikationen zu charakterisieren, sowie die Toxizität und Genotoxizität zu untersuchen. Da es Hinweise auf eine Aktivierung von TPZ über eine Oxidoreduktase (OR) gab, wurden die Experimente in Wildtyp und hOR überexprimierenden Zellen durchgeführt. Die Quantifizierung der verursachten DNA-Modifikationen zeigte, dass der von TPZ verursachte Schaden in Zellen mit hOR erhöht war. Das erhaltene Schadensprofil der durch TPZ verursachten DNA-Modifikationen war dem Schadensprofil von durch Gamma-Strahlung intrazellulär verursachten Hydroxylradikalen sehr ähnlich. Da es nach der Aktivierung von TPZ durch eine OR zu einer Abspaltung von Hydroxylradikalen kommt, bestätigte dies den vermuteten Mechanismus. Weitere Untersuchungen mit t-Butanol, einem Hydroxylradikal Fänger, ergaben eine verminderte DNA-Schädigung, was ebenfalls für eine DNA-Schädigung durch Hydroxylradikale spricht. Untersuchungen zur Mutagenität zeigten das die Mutationsrate in Zellen mit hOR um das 4 fache erhöht ist. Erstaunlich war jedoch, dass der im gleichen Ausmaß von Gamma-Strahlung verursachte DNA-Schaden für die beobachtete Toxizität dieser verantwortlich war, während bei TPZ unter den gleichen Bedingungen keine Toxizität vorlag. Erklärt werden könnte die erhöhte Toxizität und Mutagenität durch so genannte geclusterte DNA-Schäden, die von Gamma-Strahlen, nicht jedoch von TPZ gebildet werden. Nach einer verlängerten Inkubation wurde sowohl für die Toxizität als auch für die Genotoxizität erneut ein verstärkender Effekt durch die OR bestätigt. Überraschend war weiterhin die von der OR unabhängige Generierung von Doppelstrangbrüchen, für die demnach ein grundsätzlich anderer Mechanismus, wie zum Beispiel eine direkte Interaktion mit der Topoisomerase II, angenommen werden muss.

In remembrance of commensal intestinal microbes

Mammals contain an enormous load of commensal microbes in the lower intestine, which induce adaptive responses in the host immune system that ensure mutual coexistence of the host and its microbial passengers. The main way of studying how the host responds to commensal colonization has been to compare animals kept in entirely germ-free conditions and their colonized counterparts. We present an overview of our development of a reversible colonization system, whereby germ free animals can be treated with live commensal bacteria that do not persist in the host, so it becomes germ free again. We describe how this system has been used to demonstrate that there is little or no immune memory for specific IgA induction in the intestinal mucosal immune system by commensal intestinal bacteria.

A Measurement Model of Cortisol Reactivity of Healthy Older Adults

Research on the physiological adaptation process has found that stress is associated with the rate of cortisol secretion, the main hormone that reflects stress. However, considerable variation among subjects has been reported. Using a sample of older adults (N=46), we tested the hypothesis that cortisol reactivity is composed of (1) a situation-related component representing hypothalamic influence on cortisol secretion observed on three different occasions, and (2) a stable component representing a general trait responsible for cortisol responses observed from occasion to occasion. LISREL VIII was used to test this hypothesis. Results indicated that a homogeneous reliability model was not supported by the data. A congeneric measurement model represented a better fit to the data. Results suggest that subjects have consistent patterns of response during separate experimental occasions. However, results do not suggest a consistent pattern of response over time. The main implication of these results is that salivary cortisol measures are sensitive to experimental stress situations. As such, this noninvasive method may be useful in examining adaptive responses to stress.

Anti-HIV state but not apoptosis depends on IFN signature in CD4+ T cells

To gain insights into the molecular mechanisms underlying early host responses to HIV in the CD4(+) T cell target population, we examined gene expression in CD4(+) T cells isolated 24 h after ex vivo HIV infection of lymphocyte aggregate cultures derived from human tonsils. Gene profiling showed a distinct up-regulation of genes related to immune response and response to virus, notably of IFN-stimulated genes (ISGs), irrespective of the coreceptor tropism of the virus. This mostly IFN-alpha-dependent gene signature suggested the involvement of plasmacytoid dendritic cells, a principal component of the antiviral immune response. Indeed, depletion of plasmacytoid dendritic cells before HIV inoculation abrogated transcriptional up-regulation of several ISGs and resulted in increased levels of HIV replication. Treatment with a blocking anti-IFN-alphaR Ab yielded increased HIV replication; conversely, HIV replication was decreased in pDC-depleted cultures treated with IFN-alpha. Among up-regulated ISGs was also TRAIL, indicating a potential role of the IFN signature in apoptosis. However, a blocking anti-TRAIL Ab did not abrogate apoptosis of CD4(+) T cells in CXCR4-tropic HIV-infected cultures, suggesting the involvement of pathways other than TRAIL mediated. We conclude that acute HIV infection of lymphoid tissue results in up-regulation of ISGs in CD4(+) T cells, which induces an anti-HIV state but not apoptosis.

Rising sound intensity: an intrinsic warning cue activating the amygdala

Human subjects overestimate the change of rising intensity sounds compared with falling intensity sounds. Rising sound intensity has therefore been proposed to be an intrinsic warning cue. In order to test this hypothesis, we presented rising, falling, and constant intensity sounds to healthy humans and gathered psychophysiological and behavioral responses. Brain activity was measured using event-related functional magnetic resonance imaging. We found that rising compared with falling sound intensity facilitates autonomic orienting reflex and phasic alertness to auditory targets. Rising intensity sounds produced neural activity in the amygdala, which was accompanied by activity in intraparietal sulcus, superior temporal sulcus, and temporal plane. Our results indicate that rising sound intensity is an elementary warning cue eliciting adaptive responses by recruiting attentional and physiological resources. Regions involved in cross-modal integration were activated by rising sound intensity, while the right-hemisphere phasic alertness network could not be supported by this study.

Maternal tobacco smoking and decreased leukocytes, including dendritic cells, in neonates

Maternal smoking in pregnancy is associated with respiratory diseases in the offspring, possibly due to prenatal influences on the developing immune system. We investigated whether maternal smoking in pregnancy was associated with cord blood leukocyte numbers, including precursor dendritic cells, adjusting for concomitant factors. In a prospective healthy birth cohort study, total leukocyte counts were reduced in neonates of smoking mothers [10.7 (8.4-13.0), n=14] compared with nonexposed infants [14.7 (13.7-15.7), n=74, p=0.002] [geometric mean cells x 10(3)/microL (95% confidence interval)]. All leukocyte subsets were decreased, most prominently segmented neutrophils [4.3 (2.8-5.7) versus 6.2 (5.5-6.8), p=0.021], lymphocytes [3.8 (2.9-4.8) versus 5.0 (4.5-5.6), p=0.036], and myeloid precursor dendritic cells [12.7 cells/microL (9.1-17.8) versus 18.3 (15.8-21.2), p=0.055]. These differences persisted after adjustment for possible confounders. Predictors of myeloid precursor dendritic cell numbers in multivariable models were maternal smoking (-5.1 cells/microL, p=0.042), age (-0.5 cells/microL/y, p=0.035), and, marginally, asthma (+8.1 cells/microL, p=0.075). The decrease of all leukocytes in neonates of smoking mothers could be clinically significant and suggests a decreased cell production, increased peripheral recruitment, or retention in bone marrow. Given the importance of dendritic cells in early immune responses, their decrease might reflect an impact of maternal smoking on the developing fetal immune system.

Up-regulation of the peroxiredoxin-6 related metabolism of reactive oxygen species in skeletal muscle of mice lacking neuronal nitric oxide synthase

Although neuronal nitric oxide synthase (nNOS) plays a substantial role in skeletal muscle physiology, nNOS-knockout mice manifest an only mild phenotypic malfunction in this tissue. To identify proteins that might be involved in adaptive responses in skeletal muscle of knockout mice lacking nNOS, 2D-PAGE with silver-staining and subsequent tandem mass spectrometry (LC-MS/MS) was performed using extracts of extensor digitorum longus muscle (EDL) derived from nNOS-knockout mice in comparison to C57Bl/6 control mice. Six proteins were significantly (P < or = 0.05) more highly expressed in EDL of nNOS-knockout mice than in that of C57 control mice, all of which are involved in the metabolism of reactive oxygen species (ROS). These included prohibitin (2.0-fold increase), peroxiredoxin-3 (1.9-fold increase), Cu(2+)/Zn(2+)-dependent superoxide dismutase (SOD; 1.9-fold increase), heat shock protein beta-1 (HSP25; 1.7-fold increase) and nucleoside diphosphate kinase B (2.6-fold increase). A significantly higher expression (4.1-fold increase) and a pI shift from 6.5 to 5.9 of peroxiredoxin-6 in the EDL of nNOS-knockout mice were confirmed by quantitative immunoblotting. The concentrations of the mRNA encoding five of these proteins (the exception being prohibitin) were likewise significantly (P < or = 0.05) higher in the EDL of nNOS-knockout mice. A higher intrinsic hydrogen peroxidase activity (P < or = 0.05) was demonstrated in EDL of nNOS-knockout mice than C57 control mice, which was related to the presence of peroxiredoxin-6. The treatment of mice with the chemical NOS inhibitor L-NAME for 3 days induced a significant 3.4-fold up-regulation of peroxiredoxin-6 in the EDL of C57 control mice (P < or = 0.05), but did not alter its expression in EDL of nNOS-knockout mice. ESR spectrometry demonstrated the levels of superoxide to be 2.5-times higher (P < or = 0.05) in EDL of nNOS-knockout mice than in C57 control mice while an in vitro assay based on the emission of 2,7-dichlorofluorescein fluorescence disclosed the concentration of ROS to be similar in both strains of mice. We suggest that the up-regulation of proteins that are implicated in the metabolism of ROS, particularly of peroxiredoxin-6, within skeletal muscles of nNOS-knockout mice functionally compensates for the absence of nNOS in scavenging of superoxide.

The cannabinoid CB2 receptor-selective phytocannabinoid beta-caryophyllene exerts analgesic effects in mouse models of inflammatory and neuropathic pain

The widespread plant volatile beta-caryophyllene (BCP) was recently identified as a natural selective agonist of the peripherally expressed cannabinoid receptor 2 (CB2). It is found in relatively high concentrations in many spices and food plants. A number of studies have shown that CB2 is critically involved in the modulation of inflammatory and neuropathic pain responses. In this study, we have investigated the analgesic effects of BCP in animal models of inflammatory and neuropathic pain. We demonstrate that orally administered BCP reduced inflammatory (late phase) pain responses in the formalin test in a CB2 receptor-dependent manner, while it had no effect on acute (early phase) responses. In a neuropathic pain model the chronic oral administration of BCP attenuated thermal hyperalgesia and mechanical allodynia, and reduced spinal neuroinflammation. Importantly, we found no signs of tolerance to the anti-hyperalgesic effects of BCP after prolonged treatment. Oral BCP was more effective than the subcutaneously injected synthetic CB2 agonist JWH-133. Thus, the natural plant product BCP may be highly effective in the treatment of long lasting, debilitating pain states. Our results have important implications for the role of dietary factors in the development and modulation of chronic pain conditions.

Diversity versus disparity and the role of ecological opportunity in a continental bird radiation

Aim The Neotropical parrots (Arini) are an unusually diverse group which colonized South America in the Oligocene. The newly invaded Neotropics may have functioned as an underused adaptive zone and provided novel ecological opportunities that facilitated diversification. Alternatively, diversification may have been driven by ecological changes caused by Andean uplift and/or climate change from the Miocene onwards. Our aim was to find out whether Arini diversified in a classical adaptive radiation after their colonization of South America, or whether their diversification occurred later and was influenced by more recent environmental change. Location Neotropics. Methods We generated a time-calibrated phylogeny of more than 80% of all Arini species in order to analyse lineage diversification. This chronogram was also used as the basis for the reconstruction of morphological evolution within Arini using a multivariate ratio analysis of three size measurements. Results We found a concentration of size evolution and partitioning of size niches in the early history of Arini consistent with the process of adaptive radia- tion, but there were no signs of an early burst of speciation or a decrease in speci- ation rates through time. Although we detected no overall temporal shifts in diversification rates, we discovered two young, unexpectedly species-rich clades. Main conclusions Arini show signs of an early adaptive radiation, but we found no evidence of the slowdown in speciation rate generally considered a feature of island or lake radiations. Historical processes and environmental change from the Miocene onwards may have kept diversification rates roughly constant ever since the colonization of the Neotropics. Thus, Arini may not yet have reached equilibrium diversity. The lack of diversity-dependent speciation might be a general feature of adaptive radiations on a continental scale, and diversification processes on continents might therefore not be as ecologically limited as in isolated lakes or on oceanic islands.

Increased myocardial susceptibility to repetitive ischemia with high-fat diet-induced obesity.

Obesity and diabetes are frequently associated with cardiovascular disease. When a normal heart is subjected to brief/sublethal repetitive ischemia and reperfusion (I/R), adaptive responses are activated to preserve cardiac structure and function. These responses include but are not limited to alterations in cardiac metabolism, reduced calcium responsiveness, and induction of antioxidant enzymes. In a model of ischemic cardiomyopathy inducible by brief repetitive I/R, we hypothesized that dysregulation of these adaptive responses in diet-induced obese (DIO) mice would contribute to enhanced myocardial injury. DIO C57BL/6J mice were subjected to 15 min of daily repetitive I/R while under short-acting anesthesia, a protocol that results in the development of fibrotic cardiomyopathy. Cardiac lipids and candidate gene expression were analyzed at 3 days, and histology at 5 days of repetitive I/R. Total free fatty acids (FFAs) in the cardiac extracts of DIO mice were significantly elevated, reflecting primarily the dietary fatty acid (FA) composition. Compared with lean controls, cardiac FA oxidation (FAO) capacity of DIO mice was significantly higher, concurrent with increased expression of FA metabolism gene transcripts. Following 15 min of daily repetitive I/R for 3 or 5 days, DIO mice exhibited increased susceptibility to I/R and, in contrast to lean mice, developed microinfarction, which was associated with an exaggerated inflammatory response. Repetitive I/R in DIO mice was associated with more profound significant downregulation of FA metabolism gene transcripts and elevated FFAs and triglycerides. Maladaptive metabolic changes of FA metabolism contribute to enhanced myocardial injury in diet-induced obesity.

Molluscan memory of injury: evolutionary insights into chronic pain and neurological disorders.

Molluscan preparations have yielded seminal discoveries in neuroscience, but the experimental advantages of this group have not, until now, been complemented by adequate molecular or genomic information for comparisons to genetically defined model organisms in other phyla. The recent sequencing of the transcriptome and genome of Aplysia californica, however, will enable extensive comparative studies at the molecular level. Among other benefits, this will bring the power of individually identifiable and manipulable neurons to bear upon questions of cellular function for evolutionarily conserved genes associated with clinically important neural dysfunction. Because of the slower rate of gene evolution in this molluscan lineage, more homologs of genes associated with human disease are present in Aplysia than in leading model organisms from Arthropoda (Drosophila) or Nematoda (Caenorhabditis elegans). Research has hardly begun in molluscs on the cellular functions of gene products that in humans are associated with neurological diseases. On the other hand, much is known about molecular and cellular mechanisms of long-term neuronal plasticity. Persistent nociceptive sensitization of nociceptors in Aplysia displays many functional similarities to alterations in mammalian nociceptors associated with the clinical problem of chronic pain. Moreover, in Aplysia and mammals the same cell signaling pathways trigger persistent enhancement of excitability and synaptic transmission following noxious stimulation, and these highly conserved pathways are also used to induce memory traces in neural circuits of diverse species. This functional and molecular overlap in distantly related lineages and neuronal types supports the proposal that fundamental plasticity mechanisms important for memory, chronic pain, and other lasting alterations evolved from adaptive responses to peripheral injury in the earliest neurons. Molluscan preparations should become increasingly useful for comparative studies across phyla that can provide insight into cellular functions of clinically important genes.

Long-lasting hyperexcitability induced by depolarization in the absence of detectable Ca2+ signals.

Learning and memory depend on neuronal alterations induced by electrical activity. Most examples of activity-dependent plasticity, as well as adaptive responses to neuronal injury, have been linked explicitly or implicitly to induction by Ca(2+) signals produced by depolarization. Indeed, transient Ca(2+) signals are commonly assumed to be the only effective transducers of depolarization into adaptive neuronal responses. Nevertheless, Ca(2+)-independent depolarization-induced signals might also trigger plastic changes. Establishing the existence of such signals is a challenge because procedures that eliminate Ca(2+) transients also impair neuronal viability and tolerance to cellular stress. We have taken advantage of nociceptive sensory neurons in the marine snail Aplysia, which exhibit unusual tolerance to extreme reduction of extracellular and intracellular free Ca(2+) levels. The axons of these neurons exhibit a depolarization-induced memory-like hyperexcitability that lasts a day or longer and depends on local protein synthesis for induction. Here we show that transient localized depolarization of these axons in an excised nerve-ganglion preparation or in dissociated cell culture can induce short- and intermediate-term axonal hyperexcitability as well as long-term protein synthesis-dependent hyperexcitability under conditions in which Ca(2+) entry is prevented (by bathing in nominally Ca(2+) -free solutions containing EGTA) and detectable Ca(2+) transients are eliminated (by adding BAPTA-AM). Disruption of Ca(2+) release from intracellular stores by pretreatment with thapsigargin also failed to affect induction of axonal hyperexcitability. These findings suggest that unrecognized Ca(2+)-independent signals exist that can transduce intense depolarization into adaptive cellular responses during neuronal injury, prolonged high-frequency activity, or other sustained depolarizing events.

C9ORF72 Regulates Stress Granule Formation and Its Deficiency Impairs Stress Granule Assembly, Hypersensitizing Cells to Stress

Hexanucleotide repeat expansions in the C9ORF72 gene are causally associated with frontotemporal lobar dementia (FTLD) and/or amyotrophic lateral sclerosis (ALS). The physiological function of the normal C9ORF72 protein remains unclear. In this study, we characterized the subcellular localization of C9ORF72 to processing bodies (P-bodies) and its recruitment to stress granules (SGs) upon stress-related stimuli. Gain of function and loss of function experiments revealed that the long isoform of C9ORF72 protein regulates SG assembly. CRISPR/Cas9-mediated knockdown of C9ORF72 completely abolished SG formation, negatively impacted the expression of SG-associated proteins such as TIA-1 and HuR, and accelerated cell death. Loss of C9ORF72 expression further compromised cellular recovery responses after the removal of stress. Additionally, mimicking the pathogenic condition via the expression of hexanucleotide expansion upstream of C9ORF72 impaired the expression of the C9ORF72 protein, caused an abnormal accumulation of RNA foci, and led to the spontaneous formation of SGs. Our study identifies a novel function for normal C9ORF72 in SG assembly and sheds light into how the mutant expansions might impair SG formation and cellular-stress-related adaptive responses.

Predator-Induced Plasticity in Spotted Salamanders (Ambystoma maculatum)

The ability to respond plastically to the environment has allowed amphibians to evolve adaptive responses to spatial and temporal variation in predation threat. However, animals exposed to predators may also show costs of plasticity or tradeoffs. This study examines predator-induced plasticity in larval development, behavior, and metamorphosis in the spotted salamander, Ambystoma maculatum. Salamanders were raised in two treatments: with predator cues (a fish predator, genus Lepomis, on the other side of a divided tank), or without predator cues. During the larval stage the predator treatment group experienced higher mortality rates than the no-predator treatment group. Behavioral trials revealed that predator treatment animals ate less than those not exposed, and that this feeding response was immediately inducible and had lasting effects. Animals in the predator treatment group had smaller tail areas during the mid-larval period. Feeding and body size effects may have contributed to increased mortality in the predator-treatment animals. The timing of metamorphic onset was not affected by the presence of predators, but predator-treatment salamanders had shorter snout/vent lengths at metamorphosis. The duration of metamorphosis showed a potentially adaptive plastic response to the presence of predator cues: metamorphosis was longest in the no-predator treatment group, reduced in the predator treatment group, and even further reduced for animals exposed to predator cues only during metamorphosis. Overall, we found a mix of potentially adaptive and costly plastic responses in spotted salamanders.