Anterior cruciate ligament (ACL) rupture occurs most commonly in young and active individuals and can have negative long-term physical and psychological impacts. The diagnosis is made with a combination of patient's history, clinical examination, and, if appropriate, magnetic resonance imaging. The objectives of management are to restore knee function, address psychological barriers to activity participation, prevent further injury and osteoarthritis, and optimize long-term quality of life. The three main treatment options for ACL rupture are (1) rehabilitation as first-line treatment (followed by ACL reconstruction (ACLR) in patients, who develop functional instability), (2) ACLR and post-operative rehabilitation as the first-line treatment, and (3) pre-operative rehabilitation followed by ACLR and post-operative rehabilitation. We provide practical recommendations for informing and discussing management options with patients, and describe patient-related factors associated with a worse ACL-rupture outcome. Finally, we define evidence-based rehabilitation and present phase-specific rehabilitation recommendations and criteria to inform return to sport decisions. In the USA alone, 250,000 individuals suffer an anterior cruciate ligament (ACL) rupture per year To accurately diagnose an ACL rupture, the clinician will combine information from a patient history, clinical examination and, if appropriate, imaging. ACL ruptures often occur with concomitant injury to menisci, cartilage, or other knee ligaments. Special consideration should be taken to diagnose substantial concomitant injuries accurately.An ACL rupture should always be suspected if the patient reports (1) an injury mechanism that involves deceleration/acceleration in combination with a knee valgus load, (2) hearing or feeling a “pop” at the time of injury, or (3) hemarthrosis within 2 h of injury Several clinical tests can be used to detect an ACL rupture. The Lachman test is the most accurate clinical diagnostic test, with a pooled reported sensitivity of 85% and specificity of 94% In experienced assessors, the combination of patient history and clinical examination will often be sufficient to diagnose an ACL rupture. However, pain and effusion in an acute setting may make it difficult to detect the injury during clinical examination. Misdiagnosis is common; half of patients with an acute ACL rupture were misdiagnosed as an uncomplicated knee sprain in an orthopedic emergency unit Following substantial acute knee trauma, the Ottawa Knee Rule should be used to rule out fracture ACL ruptures often occur with concomitant ligament sprains, meniscus tears, bone marrow lesions, articular cartilage injuries, and intra-articular fractures Once the diagnosis is clear, the clinician should inform the patient of the injury and known consequences. For many individuals, their ACL-injured knee will never feel as it did before the injury. More than five years after ACL rupture, knee pain, symptoms, recreational limitations, and impaired QOL are common The objectives of management for an individual with ACL rupture are to (1) restore knee function, (2) address psychological barriers to resuming activity participation, (3) prevent further knee injury and reduce the risk of knee osteoarthritis, and (4) optimize long-term QOL.Treatments to achieve this objective are targeted toward the specific impairments of the patient. Establishing realistic and specific goals, and accurate assessment of current impairments that need to be addressed to reach these goals, are therefore key. Typical impairments after injury include varying degrees of muscle strength deficits, altered movement patterns, decreased knee joint proprioception, and increased passive knee laxity. Collectively, these impairments contribute to the varying degrees of functional knee instability that patients with ACL rupture experience.Although the functional status of the knee is associated with whether the patient returns to sport The main threats to future knee health are knee re-injury and the development of posttraumatic knee osteoarthritis. To date, no randomized trials exist to support interventions that can eliminate these risks. The risk for knee re-injury is lower in those who (1) do not participate in sports with frequent pivoting and cutting, (2) complete rehabilitation to the point where they pass functional return to sport criteria before returning to pivoting sports, and (3) return to pivoting sports later than 9 months after an ACLR The risk for knee osteoarthritis is higher in those with higher BMI, those who are physically inactive, and those with quadriceps muscle weakness Maintaining participation in a satisfying form of physical activity is a key determinant of long-term QOL following ACLR The treatment for patients with ACL rupture needs to be individualized and several options are currently in use in clinical practice. In this chapter, we present three main options for the reader . ConsideThere are a number of literature reviews comparing outcomes between individuals that are ACL-deficient or have had an ACLR There is a notable shortage of high quality research comparing outcomes following the management of patients with an ACL rupture with high-quality rehabilitation compared to ACLR. A recent review of all randomized controlled trials (RCTs) for ACL injury identified only 1  out of 412 trials that compared outcomes following ACL management with rehabilitation plus optional delayed ACLR vs. ACLR and postoperative rehabilitation Several relevant recent studies did not feature in the previous reviews. A recent cohort study compared outcomes between patients with an ACL rupture managed with strength and neuromuscular training or ACLR followed by postoperative rehabilitation. The treatment groups had similar five-year functional and radiographic outcomes, but the ACLR group had more joint effusion, better self-reported knee function, and lower self-reported fear laxity refers to the passive intra-articular translation available in the tibiofemoral joint  whereas functional instability refers to the patient's perception of an “unstable” knee functional stability can be achieved through neuromuscular training Knee Meniscus or cartilage damage at the time of ACLR is associated with worse outcomes and higher rates of osteoarthritis Subsequent knee injury is common following ACLR; one third of young individuals who undergo ACLR experience a second ACL tear does not indicate that an individual is at greater risk of subsequent injury if they are managed with rehabilitation as the first-line treatment as opposed to ACLR.A common belief is that early ACLR prevents additional meniscus and cartilage injury. However, studies referenced to support this belief are typically retrospective reviews of surgical records, and show more severe joint injury in patients presenting for ACLR months or years after ACL rupture, following unknown/no rehabilitation Understanding patient-related factors that are associated with outcomes after ACL rupture can enable the clinician to better tailor the information and management strategy to the individual patient. In those who are treated with ACLR, younger athletes, men, and elite athletes are more likely to return to sport Patients who are better prepared, both psychologically and functionally, prior to ACLR also have better outcomes after an ACLR. Preoperative factors associated with better postoperative outcomes include optimism, self-efficacy, greater quadriceps muscle strength, and passive knee extension range of motion Fewer studies are available on factors that are associated with outcomes following rehabilitation alone, and it is largely unknown how and if these factors interact with treatment choice. An exploratory analysis of the KANON trial showed that undergoing repeat knee surgery was associated with worse five-year pain, symptoms, function in sport, and QOL, and regardless of treatment and had self-reported knee function within the normative range of individuals with no history of knee injury. Athletes who were older, women, and individuals with better knee function early after ACL rupture were more likely to have successful two-year outcomes Recently, Grindem et al. ACL ruptures can have serious long-term consequences, optimal management requires substantial investment from the patient, and the patient's short- and long-term priorities may be conflicting . The treatment choice should therefore be a shared decision between the individual patient and the treating health-care professionals. To ensure that the patient makes an informed commitment to a treatment plan, the first step of this process is to provide high-quality information about the injury, short- and long-term consequences, what the different treatment options are, and the patient's likely prognosis with the different treatment options.Patients tend to have very high expectations prior to ACLR, which do not match average outcomes. Of 181 patients who awaited ACLR, all patients expected to have almost normal or normal knee function within 12 months of surgery, 91% expected to return to sport within one year of surgery and 98% expected no or only a slight increased risk of knee osteoarthritis after ACLR Discussing the pros and cons of different management options will allow the patient to make an informed decision and select the approach that is best suited to him or her. To give the patient the greatest chance of success, it is critical that the initial advice is based on best-evidence recommendations and not biased toward a particular treatment strategy. This requires the clinician to synthesize the scientific evidence and their clinical experience to make specific recommendations for the individual patient. The rationale behind these recommendations should be as transparent as possible.As the only randomized trial on the topic did not support a superior outcome with early ACLR Clinical practice patterns also show that many nonprofessional athletes choose to have an ACLR to enable return to pivoting sports Other factors such as concomitant injuries, access to treatment, patient preferences, and life plans may also influence the final decision. For example, external pressure will often give a professional athlete limited time for the decision-making process. It may therefore not be feasible to delay a surgical decision to see if they can return to sport with rehabilitation alone. On the other hand, if the athlete sustains an ACL rupture close to an important event and has no signs of functional instability, having an early ACLR would mean they could not compete in this event. He or she might therefore choose to participate in the event before considering whether surgery is likely to be of additional benefit to rehabilitation. The key to the treatment decision is to agree on a realistic treatment plan that sets the patient up with the best opportunity to achieve his or her goals.Evidence-based ACL rupture rehabilitation recommendations are presented in Over the last decades, rehabilitation has moved from time-based protocols to individualized and criterion-based content and progression. There are five distinct phases of rehabilitation after ACL rupture . ProgresThe principles of rehabilitation are similar for those who are treated with rehabilitation alone and those who elect to undergo ACLR. However, a shorter time-frame should be expected for those who do not have ACLR. After both ACL rupture and ACLR, concomitant injury and/or surgery may also require adjustments to the rehabilitation program. There are multiple combinations of concomitant injury presentations, and an injured structure may be nonoperatively managed or surgically treated using a variety of methods. For example, meniscus injuries may be surgically treated with a small resection, repair, or meniscus transplantation. These surgical methods require very different adaptations to the rehabilitation program and impairments from graft harvesting. ACLR with a patellar tendon autograft is associated with donor-site pain and quadriceps weakness In patients, who undergo ACLR, the graft choice should be considered in postoperative rehabilitation. An ACLR can be performed with allograft (a donor graft) or autograft . Rehabilitation after ACLR should address impairments from the ACL rupture As outlined in the previous section, a gradual return to activity/sport is an integrated part of the rehabilitation progress. Different sports, and even different aspects within one sport, pose different demands to physical and psychological readiness. As the patient improves with rehabilitation, a gradual increase in sports participation is recommended to ensure the best transition back to full participation There are no high-level interventional studies that compare different criteria for return to sport. Current guidelines in this area are therefore informed by observational studies and expert opinion. Regardless of which activity/sport the patient is aiming to return to, there are three key considerations for the medical decision-making team: (1) is the athlete physically ready to participate in the activity/sport? (2) is the athlete mentally ready to perform in the activity/sport? (3) have we allowed enough time from injury/surgery for sufficient biological healing to occur?Assessment of the patient's knee function is a key aspect in the clinical decision of whether they should return to activity/sports. Pivoting sport athletes who pass specific criteria prior to return to sport have 4–6 times lower risk for re-injury Performance-based tests of muscle strength and single-legged hop ability have traditionally been the cornerstone of functional return to sport criteria Psychological factors are highly associated with not returning to sport after ACLR. These factors can be related to the patient's competence, autonomy, and relatedness  Over the last decades, clinical practice patterns have shifted toward earlier return to activity. Recent research has now highlighted that important biological healing processes are still ongoing at the time when athletes traditionally resume sports activities. Free tendon autografts are commonly used for ACLR. After systematically reviewing the literature, Claes et al. ∗The objective of ACL-injury management is to restore knee function, address psychological impacts, prevent further knee injury and osteoarthritis, and optimize long-term QOL;∗Current evidence suggests average long-term outcomes are similar following management of ACL rupture with ACLR and rehabilitation or with rehabilitation alone;∗Rehabilitation should start as early as possible after injury, and progress in rehabilitation is performed when the patient meets specific clinical or functional milestones;∗For some patients, not returning to sport can negatively impact long-term QOL, but returning to pivoting sport increases the risk of subsequent knee injury;∗Subsequent knee injury after ACL-injury is associated with poor long-term outcome, and reducing risk of subsequent knee injury should be a key priority when managing ACL-injured individuals;∗To reduce the risk for subsequent injury, pivoting sport athletes with ACLR should pass functional criteria for return to sport and delay full participation for at least nine months after surgery∗Research to determine optimal strategies for preventing subsequent knee injury and osteoarthritis after ACL rupture is required;∗Research is needed to establish return to sport guidelines for ACL-injured people who are treated with rehabilitation alone;∗Further research on late-phase rehabilitation and the efficacy of return to sport criteria is required;∗There is a need for research on interventions to improve psychological readiness to return to sport, and for research to evaluate the implications of such interventions on re-injury rates and long-term QOL;∗There is a need for further research to identify which patients will benefit most from management with or without ACLRFollowing an ACL rupture the clinician and patient should, together, devise a treatment plan that addresses (1) rehabilitation, (2) appropriateness of ACLR, and (3) return to sport. Average long-term outcomes are similar following management of ACL rupture with rehabilitation alone or with ACLR and rehabilitation. Irrespective of treatment strategy, ACL rupture management should aim to restore knee function, address psychological barriers to sport/activity participation, reduce the risk of further injury and knee osteoarthritis, and optimize long-term QOL. Rehabilitation after an ACL rupture and/or ACLR should be individualized and criterion-based, with gradual return to sport/activity as an integrated part of rehabilitation progression. The three main factors in deciding readiness to participate in sport are (1) physical readiness, (2) psychological readiness, and (3) biological healing. Athletes who return to pivoting sport after ACLR can reduce their risk for re-injury by passing time-based and functional criteria before returning to sport.None.


Elevated intracranial pressure (ICP) is observed in association with a range of brain disorders. One of these challenging disorders is idiopathic intracranial hypertension (IIH), characterized by raised ICP of unknown cause with significant morbidity and limited therapeutic options. In this review, special focus is put on the preclinical research performed in order to understand the pathophysiology behind ICP regulation and IIH. This includes cerebrospinal fluid dynamics, molecular mechanisms underlying disturbances in brain fluids leading to elevated ICP, role of obesity in IIH, development of an IIH model and ICP measurements in rodents. The review also discusses existing and new drug targets for IIH that have been evaluated in vivo.ICP monitoring in rodents is challenging and different methods have been applied. Some of these methods are invasive, depend on use of anesthesia and only allow short-term monitoring. Long-term ICP recordings are needed to study IIH but existing methods are hampered by several limitations. As obesity is one of the most common risk factors for IIH, a rodent obese model has been developed that mimics some key aspects of IIH. The most commonly used drugs for IIH have been evaluated in vivo for their efficacy at lowering ICP in the existing animal models. These studies suggest these drugs, including acetazolamide, might have limited or no reducing effect on ICP. Two drug targets that can impact ICP in healthy rodents are topiramate and a glucagon-like peptide-1 receptor (GLP-1R) agonist. However, it remains to evaluate their effect in an IIH model with more precise and valid ICP monitoring system. Therefore, continued evaluation in the preclinical research with refined tools is of great importance to further understand the pathophysiology behind disorders with raised ICP and to explore new drug targets. Elevated intracranial pressure (ICP) is observed in association with a range of brain disorders and their underlying pathophysiology is widely unknown. Actually, there is limited insight into the regulatory mechanisms of this essential balance under normal conditions and consequently also a significant lack of specific treatments. In this review, we aim to focus on the preclinical research behind ICP regulation mainly with Idiopathic Intracranial Hypertension (IIH) as a disease model. Further, we aim to discuss the existing and novel drug targets for IIH that have been evaluated in preclinical models.The regulation of ICP is fundamental for providing a stable environment to enable normal brain function. According to the anatomists Monro and Kellie, ICP is determined by three parameters; the volume of the brain, cerebral blood volume and cerebrospinal fluid (CSF) volume, where cerebral blood volume and CSF are homeostatically controlled and compensate for acute changes in ICP . Several+/K+ ATPase and the Na+-K+-2Cl−-1 cotransporter (NKCC1) are fundamental in CSF secretion at the CP, and are targeted clinically to reduce ICP , organs that reside in the cerebral ventricles and display the characteristic asymmetrical distribution of ion transporters of fluid transporting epithelia  of the superior sagittal sinus are thought to drain CSF directly into the venous system . The preIt has been hypothesised that CSF can be drained via perivascular spaces that penetrate the parenchyma, termed glymphatics, in an Aquaporin-4 (AQP4) dependent manner . This glThere are currently a limited number of studies that assess CSF dynamics in IIH. Several studies utilizing small cohorts of IIH patients identified an increased CSF flow rate through the cerebral aqueduct, indicating increased CSF secretion –23. StudHistological data from cortical biopsies of IIH patients showed increased AQP4 expression at astrocyte end feet, a constituent of the glymphatic drainage. This is suggestive of an altered state of CSF clearance via the glymphatic system . FurtherAlthough it is apparent that ICP and in particular CSF dynamics are altered in IIH, the current literature fails to elucidate conclusively whether IIH patients have CSF hypersecretion, reduced CSF drainage or a combination of these factors. Furthermore the studies do not address the etiology of the alterations they describe, consequently further studies are required.The gold standard for measuring ICP in patients is by using an intracranial probe however, this requires neuro-surgical intervention with risk of infections. Lumbar puncture (LP) opening pressure measured during an LP is less invasive however, the obtained ICP values are less accurate than the gold standard method, and can only provide a short-term pressure reading during the procedure. There are some LP measurement methods that have been shown to be comparable to intraparenchymal ICP recordings –32. The In rodents, ICP values have been reported to vary between 2 and 8 mmHg with a mean value around 6 mmHg , 40. In As mentioned earlier, IIH is a challenging disease with increased ICP of unknown aetiology and was previously called benign intracranial hypertension or pseudotumor cerebri. The most disabling symptoms are chronic headache, impaired vision due to optic nerve compression and cognitive impairment –45. IIH As the molecular regulation of CSF is not yet fully understood, there is a lack of understanding for many pathological conditions associated with abnormal CSF dynamics. Hence, diseases involving elevated ICP are generally poorly understood and managed, due to the lack of specific targeted treatment options. Medical interventions for IIH range from dietary therapy (weight loss) to diuretics and surgical CSF diversion . In IIH IIH has a prominent association with both the female gender and obesity as 90% of patients with IIH are obese , 48, 59.There is also a strong association between abdominal fat deposition and health outcomes consequently adipose distribution in patients with IIH may be of relevance . It has +/K+-ATPase at the CP, suggesting that testosterone may modulate CSF secretion [Currently there are very few animal models with elevated ICP and they are associated with the induction of severe cerebral traumas such as subarachnoid hemorrhage, TBI, brain tumors, kaolin induced hydrocephalus and cerebral ischemia , 70–73. ecretion . It remaAnother fundamental factor in the development of an IIH animal model is the ability to perform long-term ICP measurements in rodents. ICP measurements are widely used for diagnostic purposes and to inform treatment options for conditions with raised ICP such as IIH. However, the invasive nature of performing ICP monitoring in humans limits clinical research. Thereby, there is a great need of consistent and precise ICP recording in animal models, where ICP can be measured across the day, in response to a drug and during disease development. However, it has been very challenging to measure ICP accurately in animal models due to the lack of refined equipment designed specifically to measure ICP in rodents. Various locations and techniques for measuring ICP have been investigated such as epidural, subdural, ventricular cisterna magna placement using a cannula, fluid-filled catheter or fiber-optic pressure transducers –86, summAlthough the implementation of epidural ICP recording using fluid-filled systems has been a major improvement for measuring ICP in rodents, this method has some limitations. In this method the transducer is not permanently implanted, which prevents the possibility of continuous ICP recordings over larger time intervals. Furthermore, this ICP method as the others depends on repeated use of anesthesia/sedation before each recording to immobilize the animals which are additional cofounders affecting ICP. Additionally, the fluid-filled systems can develop air bubbles, resulting in false ICP values. Fiber-optic pressure transducers have been used to avoid these problems, however, it has only been developed for animals under anesthesia . TelemetRecently, telemetric probes with specific ICP transducers have been developed to measure ICP continuously in freely moving rats (Kaha Sciences) . This haIn the management of IIH, the focus is on reducing the elevated ICP by pharmacological therapy. Given that IIH patients often suffer from visual disturbances and headache, management of IIH has been focused on preserving vision and reducing headache morbidity . In thisTopiramate was originally used for epilepsy treatment and as a migraine prophylactic drug , 102. To+/H+ exchanger and/or Na+ channels on the CP epithelial cells [Diuretic agents have long been known to reduce CSF secretion and have therefore been regarded as a treatment option for IIH Fig. . One of al cells , 117. Onal cells . Howeveral cells . ClinicaOctreotide is a synthetic somatostatin analogue and a potent inhibitor of growth hormone. Two uncontrolled clinical studies have shown its efficacy for IIH treatment , 120. In+- and K+-dependent adenosine triphosphatase activity in cell cultures. Subcutaneous injections with exendin-4 showed a reduction in ICP in both healthy and hydrocephalic rats [Glucagon-like peptide-1 receptor (GLP-1R) agonists are used to treat diabetes and promote weight loss. Due to its ability to affect fluid homeostasis in the kidney, we investigated if the GLP-1R agonist, exendin-4, could modulate CSF secretion and reduce ICP in rats . This stlic rats . Importalic rats . Taken tThe role of obesity on ICP regulation has been evaluated in vivo and lead to the first animal model mimicking some aspects of IIH. However, a refined model is needed to further investigate the mechanism behind IIH and also to provide valuable new knowledge regarding both regulation and dysregulation of CSF and ICP dynamics. The most used drugs in the management of IIH have been evaluated in rodents and although with some limitations, some of these are not efficient for lowering ICP. However, their effect on IIH-related symptoms such as visual outcome and headache has not been evaluated in animal models. Preclinical research suggests a GLP-1R agonist as a novel drug target for elevated ICP which needs to be further evaluated. Furthermore, a novel ICP monitoring system has been developed for long-term ICP monitoring in freely moving animals, which is a prerequisite to resolve the molecular origin of IIH-elevated ICP and to evaluate drug targets. In conclusion, preclinical investigations including the development of adequate in vivo models are warranted in order to increase our understanding in IIH pathophysiology and to explore novel drug targets able to reduce ICP.


Our skin and mucosal surfaces are colonized by diverse microbial communities, collectively known as the microbiota . The micColonization resistance is an acquired trait, because the microbiota is assembled after birth by attaining maternal and environmental microbes . To coexHost genetic variation explains only a small fraction of taxonomic microbiota variation between individuals, whereas environmental influences dominate this trait . An impoEnterobacteriaceae (phylum Proteobacteria) within the microbiota of the large intestine is the availability of respiratory electron acceptors, such as oxygen [Escherichia coli (family Enterobacteriaceae) has access to oxygen in the ceca of neonate chicks when it is inoculated one day prior to challenge with Salmonella enterica (family Enterobacteriaceae) but not when neonate chicks receive both species at the same time [The colonization order influences both the outcome of microbial community assembly and the ecological success of individual microbes , 9. Thess oxygen . Escheriame time , suggestame time .The composition and function of mature microbial communities within an individual host is relatively stable against perturbations, a phenomenon known as microbiota resistance . For insA correlate of the nutrient-niche hypothesis is that an antibiotic-mediated extinction of strains from the gut microbiota should be followed by an acquisition of new microbes, each encoding a metabolic pathway needed to fill one of the nutrient-niches vacated by the elimination of an antibiotic-sensitive microbe. The founder hypothesis predicts that these newly acquired microbes will be steadily maintained subsequently through priority effects, thereby resulting in a permanent shift in the microbiota composition. Consistent with these projections, daily gut microbiota profiling over the course of one year shows that the gut microbiota composition in one individual was stable until an episode of gastroenteritis required antibiotic therapy. Antibiotic treatment was associated with a permanent decline of most gut bacterial taxa, which were replaced subsequently with new species that were genetically similar and were afterward maintained steadily for the remainder of the study . TherefoA mature microbiota confers colonization resistance, because each of its members occupies one of the available nutrient-niches (nutrient-niche hypothesis) and holds a competitive advantage over newly arriving microbes through priority effects (founder hypothesis). As a result, it is difficult for newly arriving microbes to establish permanent residency because the best seats in the house are already taken , which eBifidobacterium infantis (phylum Actinobacteria) in breast-fed infants, as human milk oligosaccharides are a growth-limiting nutrient for this microbe [B. infantis, which results in a loss of the microbe from the human gut microbiota.An alternate strategy for overcoming colonization resistance is to generate an additional nutrient-niche that allows a new microbe to be added to the microbiota . In the  microbe . WeaningMucosal pathogens can establish residency in the microbiota of apparently healthy individuals using a strategy that involves neither antibiotics nor dietary supplements. To solve the challenge that all available nutrient-niches are occupied and the occupants are difficult to remove, mucosal pathogens use their virulence factors to create a new nutrient-niche for themselves by eliciting host responses that alter the environment at the mucosal surface . Upon enAn unexplained diversity in taxa carriage across individuals and the poorly understood phenomenon of microbiota resistance support the idea that a theoretical framework for hypothesis-driven research is largely still lacking in microbiome studies . Here, w


Animals harbor an extensive, dynamic microbial ecosystem in their gut. Gut microbiota (GM) supposedly modulate various host functions including fecundity, metabolism, immunity, cognition and behavior. Starting by analyzing the concept of the holobiont as a unit of selection, we highlight recent findings suggesting an intimate link between GM and animal social behavior. We consider two reciprocal emerging themes: (i) that GM influence host social behavior; and (ii) that social behavior and social structure shape the composition of the GM across individuals. We propose that, throughout a long history of coevolution, GM may have become involved in the modulation of their host’s sociality to foster their own transmission, while in turn social organization may have fine-tuned the transmission of beneficial endosymbionts and prevented pathogen infection. We suggest that investigating these reciprocal interactions can advance our understanding of sociality, from healthy and impaired social cognition to the evolution of specific social behaviors and societal structure. Firmicutes and Bacteroidetes as the dominant phyla  interact symbiotically  that ferment ingested food and proliferate, causing periodic, vagus nerve-mediated increases in host thermogenesis aimed at curtailing the microbial expansion. The “insurance policy hypothesis” proposes that biodiversity insures ecosystems against declines in their functioning because many species provide greater guarantees that some will maintain functioning even if others fail , which supposedly regulates emotional behavior in healthy mice via the vagus nerve  that may give extreme elders (>104 years old) some kind of “longevity adaptation”  suggest that the integrity of the GM communities and particularly that of the Lactobacillus population was decreased due to the disruption of the mother-infant bond, leaving those infants with the highest responses to separation as the most susceptible to bacterial infection. The abundance of Prevotella was in turn associated to the stress physiology of the period of peer group-formation in young monkeys  axis, controlling the “fight or flight” response to acute threats, but also allowing for the inadequate or chronic activation of adrenocortical function, resulting in deleterious effects in terms of health and survival McEwen, . RecentlWhile laboratory studies are essential for understanding cause and effect relationships, studies in wild animals should be crucial for understanding host-microbe coevolution Amato, . If we cPantoea agglomerans, Klebsiella pneumoniae pneumoniae; Enterobacter cloacae) inhabiting locusts’ guts produce components of the locust cohesion pheromone involved in their ability to change from its solitary to its swarming gregarious form  locustae can inhibit aggregation of locusts (Locusta migratoria manilensis) by acidifying the gut and thus suppressing the growth of hindgut bacteria producing aggregation pheromones  locustae, or one with similar capacities, could be present in the gut of patients suffering from psychiatric disorders related to alterations in the production of serotonin or dopamine. This may actually be true across a variety of taxa, since other species of bees, Apis mellifera and Bombus sp., harbor GM profiles different to those found in solitary species  shows a more homogenous microbial diversity, one showing fluid fission-fusion dynamics has higher microbial dissimilarities between individuals , which harbor specialized GM that are absent in solitary bee species  can gauge the parasitic status of group members by sniffing their feces and avoid long grooming sessions with parasitized individuals  from two different social groups differed, although both groups exploited adjacent habitats and ate similar diets. Furthermore, within social groups, closer grooming partners had more similar GM, and such relationship extended to the functional similarity of the gut microbiomes , primates that live in small social groups with relatively little social interaction, adult female-female dyads who spent more time in contact or in close proximity had more similar GM than other group members  from the Kasekala community (Tanzania) were more similar throughout a season during which chimpanzees were more sociable than during a season when they were less sociable, and sociability was positively associated with GM species richness  were significantly higher than in monandrous females, suggesting that a larger number of sexual partners increased bacterial diversity in females’ cloaca. Likewise, White et al. (Rissa tridactyla) by first allowing and then impeding insemination, finding that: (a) cloacal diversity decreased; and (b) the microbial communities of mates became more dissimilar after the experimental blocking took place  interacting primarily with males had a lower microbial diversity than females interacting with various males  White et al., Rissa tridactyla) White et al., Hirundo rustica erythrogaster) Levin et al., Suricata suricatta) Leclaire et al., Alouatta pigra) Amato et al., Desmodus rotudus), a species that maintains the rabies virus in the wilderness (Benavides et al., This allows considering a possible association between the direct transmission of microbial life (i.e., via physical contact between infected and susceptible conspecifics; Cortez and Weitz, e et al.  experimeLactobacillus (which modulated social behavior in mouse models: Buffington et al., Bifidobacterium (which were socially structured in baboons; Tung et al., In humans, the GM are increasingly recognized as important modulators of autoimmune diseases (Kosiewicz et al., Evidence emerging from biomedical and ecological studies suggests that GM plays an important role in shaping host sociality and that, vice versa, social organization and behavior influence the GM associated with individuals. The two processes appear to be linked, as microbes acquired through horizontal transmission by mothers can be transmitted vertically to the next generation. Gestation and infancy appear to be critical periods for the GM to influence the infant’s brain development, and may be critical in determining its future degree of sociability Figure .These findings reinforce earlier hypotheses proposing that gregariousness, social structures, and social behaviors might in part have evolved because they enhance or fine-tune the beneficial transmission of endosymbionts Troyer, . SimultaBeyond shedding light on the ecology and evolution of species, such findings may have major implications for human health. The fact that specific GM may shape social behavior could eventually translate into GM-based therapies for mental disorders associated with social deficits, i.e., psychobiotics (Vétizou et al., Overall, it appears that the microbes, in particular GM, and host social behavior have coevolved to become virtually inseparable. The GM appear to shape the social behavior and structure of their hosts, and depend on them for transmission. In turn, GM offer benefits by protecting from several diseases. Further investigation of these fascinating reciprocal interactions may open avenues for the treatment of neurological disorders or the management of health and diseases with an evolutionary perspective. Moreover, investigating these reciprocal interactions may advance our understanding of sociality, from social cognition to the basis of society structures.EM and GP-L initiated this work as part of the ETH Zurich course 701-1701-00L “Human Health, Nutrition, and Environment: Term Paper” during the autumn semester 2016. All authors listed, have made substantial, direct and intellectual contribution to the work, and approved it for publication. In particular: EM, AM-C and GP-L performed the literature review. EM, AM-C, WL and GP-L wrote the manuscript. EM designed the figures.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


While rodent cancer models are essential for early proof-of-concept and mechanistic studies for immune therapies, these models have limitations with regards to predicting the ultimate effectiveness of new immunotherapies in humans. As a unique spontaneous, large animal model of cancer, the value of conducting studies in pet dogs with cancer has been increasingly recognized by the research community. This review will therefore summarize key aspects of the dog cancer immunotherapy model and the role that these studies may play in the overall immunotherapy drug research effort. We will focus on cancer types and settings in which the dog model is most likely to impact clinical immuno-oncology research and drug development. Immunological reagent availability is discussed, along with some unique opportunities and challenges associated with the dog immunotherapy model. Overall it is hoped that this review will increase awareness of the dog cancer immunotherapy model and stimulate additional collaborative studies to benefit both man and man's best friend. Cancer immunotherapy continues to make remarkable strides in just the few years since the first checkpoint molecule targeted therapeutic antibodies were evaluated in trials and approved by the FDA. Indeed, there is the sense by the author and colleagues in the veterinary immune-oncology community  that the field of human immune-oncology is advancing so rapidly that new immunotherapy combinations are being evaluated before there is time to determine whether the combinations are truly effective, as judged by evidence of synergistic or additive antitumor activity in realistic animal models –5.Increasingly the NIH and pharmaceutical and biotechnology companies are looking to alternative animal models with which to screen immunotherapeutic drugs. The dog spontaneous cancer model has received considerable attention recently , 6–13. STherefore, this review will summarize key aspects of the dog cancer model that make it particularly well-suited to evaluating cancer immunotherapies and drug combinations. This will not however be a comprehensive review of all dog cancer immunotherapy studies, which have been reviewed elsewhere and are beyond the scope of this work , 14–20. The uniquely valuable aspects of the canine cancer model have been well-covered in recent reviews , 7, 13. Another relevant aspect of the dog model is that the immune system of dogs in cancer immunotherapy studies is typically already very immunologically experienced, with animals having experienced exposure to multiple immunizations during their early years, and to multiple different viral and bacterial infections prior to development of cancer. These immunological events all shape the immune repertoire of dogs and consequently render the dog much more immunologically experienced than rodents raised in sterile cages and fed sterilized water and chow. Dogs also share the same environment of their human companions, and are therefore exposed to many of the same allergens, food antigens, and environmental chemicals , 8. ThusDogs also develop tumors spontaneously, which means that the immune system has typically had weeks to months to recognize the tumor and mount immune responses prior to the appearance of a tumor large enough to diagnose. This long-term exposure to tumor antigens and secreted factors thus educates and conditions the canine immune system in a way that cannot be recapitulated in rodent implanted or induced tumor models , 5, 27. From the standpoint of dosing immunotherapy drugs (other than vaccines), dogs with their similar body sizes and metabolic pathways also fill a gap not currently addressed by rodent studies. Drugs dosed based on weight or body surface area in dogs are much more likely to predict drug activity and toxicity than drugs dosed in mice frequently treated at much higher drug concentrations than can be tolerated by human patients . This feThe canine immune system and immune responses in general are very similar to those of humans, with a few notable differences. In broad terms, numbers and proportions of T cells (CD4 and CD8) and B cells in blood of adult dogs closely resemble those of humans . Moreover, the ratio of CD4 to CD8 T cells (~2:1) in blood and lymph nodes is similar in dogs and humans. Numbers and percentages of neutrophils and monocytes in blood of both species are also very similar. However, recent reagent development for canine NK cells may improve our ability to quantitate dog NK cell responses , 29. DogCirculating concentrations of immunoglobulins in adult dogs are approximately the same as those of adult humans, though much less is known about normal immunoglobulin concentrations in young dogs and when final adult IgG concentrations are attained . Canine IgG molecules can be classified into four functional subclasses (A–D), similar to the human IgG subclasses, with two subclasses capable of binding Fc receptors and two subclasses being Fc functionally negative .T cells in dogs express many of the same co-stimulatory or co-inhibitory molecules as present in humans, including CD28, PD-1, OX40, TIGIT, TIM-3, and Lag3. Like human T cells, canine T cells constitutively express low levels of MHCII, which can be upregulated following T cell activation. In addition, canine effector T cells upregulate production of granzyme B, in addition to CD25 and MHCII . AntigenAn unusual feature of dog neutrophils, which differs from neutrophils in humans, is their expression of CD4 . The function of CD4 molecule expression by canine neutrophils is unclear, and CD4 is not expressed by other myeloid lineage cells such as monocytes in dogs. Dogs also appear to have a greater abundance of mast cells than humans, especially in mucosal sites such as the skin and airways, and mast cell tumors are much more common in dogs than in humans. Dogs also develop malignancies of cells of the DC and macrophage lineage  at a much higher rate than in humans  –44.Dogs will never replace rodent cancer models for cancer immunotherapy drug research and development, since early drug screening and mechanism of action studies can realistically only be done in rodent models. However, there are several tumor models where the dog may offer clear advantages, particularly for assessment of new immunotherapies and their potential efficacy against metastatic disease , 9, 45. A key question related to the new generation of cancer vaccines currently under development, which is difficult to fully address in rodent models, is whether they can effectively prevent metastatic disease, or control metastases once they develop. As noted above, dogs develop several highly metastatic cancers closely related to human cancers, including in particular osteosarcoma and melanoma , 20, 57.Adoptive cell therapy (ACT) with CAR T cells has transformed the treatment of certain leukemias and several other hematopoietic cancers in humans. However, progress in using CAR T cells to treat solid tumors in humans has been more fraught with difficulty, including serious and occasionally fatal toxicities, as well as less overall anti-tumor activity. The adverse events associated with CAR T cell treatment of solid tumors were unfortunately not predicted by rodent cancer models. Given the strong similarities between canine and human immune responses, dogs with solid tumors offer a unique opportunity to evaluate the safety and potential efficacy of new ACTs such as CAR T cell therapy before initiating human clinical trials. Indeed, ACT with CAR T cells has been evaluated in small scale studies in dogs, including CAR T cell studies with a CD20 targeted CAR T cells in dogs with B cell lymphoma . Other oIncreasingly studies point to the essential role of the tumor microenvironment (TME) in regulating overall anti-tumor immune responses. Thus, a new wave of therapeutics that target the TME are under development and evaluation in clinical trials. Our studies have identified a wide spectrum of immune responses in tumor tissues of dogs, ranging from highly inflammatory tumors  to tumors that are immunologically “cold”  . Each of these tumor models in dogs therefore offers the opportunity for evaluation of agents that target the TME, particularly for those designed to remove immune suppressive cells to help activate immunologically “cold” tumors. For example, depleting target tumor-associated macrophages by administration of agents such as liposomal clodronate that deplete tumor macrophages outright has been evaluated in dogs , 61. We A second strategy to eliminate the immune suppressive tumor macrophage population is to prevent their recruitment to tumor tissues by administering agents that block signaling by the chemokine receptor CCR2. Given that there are no currently approved (or affordable) pure CCR2 antagonists available for evaluation in dogs, our group has identified several drugs, most notably angiotensin receptor antagonists (ARBs), that can be repurposed as monocyte migration inhibitors. For example, we reported recently that the ARB losartan exerts potent antitumor activity by blocking signaling via the CCR2 chemokine receptor, thereby inhibiting the recruitment of inflammatory monocytes into tumor tissues, leading to overall tumor macrophage depletion . A recenAs another strategy to modify the TME, it was recently reported that blockade of CCR4 signaling with humanized antibodies could significantly deplete Tregs in a canine model of invasive bladder cancer . In thatper se.Other groups have investigated indoleamine deoxygenase inhibitors, which target an immune suppressive metabolic pathway in the TME , 63. TheCheckpoint targeted therapeutics are far advanced in development and approval for treatment of multiple cancers in humans. As new checkpoint molecule targeted drugs become available in dogs, opportunities exist where the dog model may provide important new information, particularly with respect rational combination therapies of immune targeted drugs given with checkpoint inhibitors. A fully canine PD-1 antibody is currently nearing phase I trial completion in dogs with a variety of different cancers and a product launch is possible in 2020 . Other cThus, the anticipated availability of new checkpoint immunotherapy reagents will make it possible to conduct creative trials in dogs. For example, a number of questions could be addressed, including: Are checkpoint molecule therapeutics effective when administered in an adjuvant setting in dogs with highly metastatic disease such as osteosarcoma or hemangiosarcoma? Or can checkpoint inhibitors be effectively combined with cytotoxic drugs such as CHOP for treatment of lymphoma? Or does co-administration of a checkpoint inhibitor with a tumor vaccine such as the Her2/neu vaccine improve vaccine efficacy in the setting of advanced, bulky tumors? As these examples suggest, a number of important questions related to adjuvant therapy and checkpoint therapy combinations can be addressed in targeted populations of dogs with relevant cancers.Salmonella in soft tissue sarcoma), regulatory T cell depletion with metronomically delivered chemotherapeutics , adoptive transfer of non-specifically activated T cells and IL-15 activated NK cells (osteosarcoma), along with a variety of different cancer vaccines , systemic administration of IL-12 nanoparticles (soft tissue sarcoma), bacterial delivered therapeutics  required to support such studies. However, all of these challenges are surmountable, given sufficient support from funding agencies, including more recently the NIH. The setting of realistic expectations at the outset of studies also helps minimize the impacts of these challenges.The era of effective cancer immunotherapy represents a major change in how cancer is treated, and the dog cancer model undoubtedly has an opportunity to play an important role in advancing this field. The value of the dog cancer model for immunotherapy has been demonstrated previously, with the best example being the essential role played by dogs with osteosarcoma development of the non-specific immunotherapeutic L-MTP  as an approved immunotherapy for pediatric osteosarcoma , 68. TheThe author confirms being the sole contributor of this work and has approved it for publication.The author declares that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Bufo bufo) captured in natural, agricultural and urban habitats, and in their offspring raised in a common-garden experiment. We found that, compared to toads captured from natural habitats, adults from both types of anthropogenic habitats had larger toxin glands (parotoids) and their toxin secretion contained higher concentrations of bufagenins, the more potent class of bufadienolide toxins. Furthermore, urban toads had lower concentrations of bufotoxins, the compounds with lower toxicity. None of these differences were present in the captive-raised juveniles; instead, toadlets originating from agricultural habitats had smaller parotoids and lower bufotoxin concentrations. These results suggest that toads’ chemical defences respond to the challenges of anthropogenic environments via phenotypic plasticity. These responses may constitute non-adaptive consequences of pollution by endocrine-disrupting chemicals as well as adaptive adjustments to the altered predator assemblages of urban and agricultural habitats.Despite the well-documented effects of human-induced environmental changes on the morphology, physiology, behaviour and life history of wild animals, next to nothing is known about how anthropogenic habitats influence anti-predatory chemical defence, a crucial fitness component of many species. We investigated the amount and composition of defensive toxins in adult common toads ( The abiotic and biotic changes induced by both forms of anthropogenic land use include habitat fragmentation, various kinds of pollution, and altered flora and fauna with lower diversity and more invasive species5. Over the past two decades, a surge of studies documented wide-ranging effects of these environmental changes on the morphology, physiology, behaviour and life history of wild organisms8. Some of these phenotypic changes are maladaptive and contribute to population declines, while some are adaptive and help living in anthropogenic habitats4. The adaptive responses can result from phenotypic plasticity of individuals or from genetic divergence between populations5. In most cases, there is yet insufficient knowledge to identify which of these two mechanisms are responsible, although such knowledge would be crucial for predicting and dealing with the eco-evolutionary consequences of anthropogenic environmental change5.Natural habitats worldwide are increasingly modified by human-induced environmental change; for example, agricultural and urban areas have taken up about 40% and 5% of the Earth’s land surface, respectively9. Chemical defence may have important consequences for life-history evolution and ecology, as chemically protected animals can live longer10 and occupy a larger niche space11. Changes in toxicity can affect not only the defended animal’s survival12 but also other species’; for example, predators can suffer serious mortality when consuming unusually toxic prey which can lead to predator population declines13. Some predators can learn to avoid toxic prey and switch to other species which then may alter trophic interactions and community structure15; whereas other predators adapt to consuming toxic prey by evolving toxin resistance, leading to co-evolutionary arms races between the defended organisms and their enemies16. Despite this potential of defensive toxins to impact multiple populations across wildlife communities and thereby biodiversity conservation, we have very little understanding of how environmental changes influence chemical defences in animals17, and we know virtually nothing about whether and how their toxicity is altered by anthropogenic habitats.Animals’ chemical defences are especially under-studied in this context. Like plants, many animal species rely on defensive chemicals or toxins for protection from their natural enemies such as predators, parasites and competitorsBufo bufo) in natural and anthropogenically influenced environments. Bufonid toads synthesize toxic steroids called bufadienolides, which they store in their skin glands including their main toxin depot, the pair of parotoid glands13  and we stored it in 1 mL HPLC-grade absolute methanol. We used somewhat different methods for the juveniles than for the adults because the toadlets were raised, photographed and euthanized as part of another experiment, and we decided to study their chemical defences only after finishing the analyses of adult data, when the toadlets had already been sacrificed. For the purposes of the present study, we used one offspring of each pair; 73 juveniles in total .From the eggs laid by the toads in our lab, we raised toadlets as described earlier32, with slight modification as detailed in the Supplementary Material  was a significant random effect, because toads from one site may be non-independent from each other36. For all variables we found that site of origin as a random effect did not improve model fit significantly , although there was no sexual dimorphism in juveniles’ SVL . As we had two alternative measurements for parotoid size in juveniles, we present the results on calliper measurements (i.e. the method that was used in adults) in the main text and photo measurements in the Supplementary Material . Hedges’ d is a standardized difference in means 38. For the adults, we calculated the effect sizes for each compound for the two sexes separately, because 6 out of 31 compounds were not detected in the majority (86–100%) of males 37. We conducted separate meta-analyses for adults and juveniles. In each meta-analysis, compound was included as a random intercept to take into account the non-independence of effect sizes comparing the data of urban and agricultural animals to the same animals of natural origin for any given compound. For both age groups, we ran 3 meta-analyses. The first was a simple meta-analytical model (an intercept-only model without any moderators) on all available data to examine the overall effect of anthropogenic habitats on the concentrations of bufadienolides. In the second model we added habitat type as a moderator to test if urban and agricultural habitats had different effects. In the third model we added the interaction of habitat type and toxin type to test if the effects of each habitat type differed between bufotoxins and bufagenins. Point estimates from statistical models were considered significantly different from zero when their 95% confidence intervals (CI) did not overlap zero.In the second step, we analysed the effect size estimates  in meta-analysis models, which are similar to weighted regression analyses, i.e. they account for the uncertainty of estimatesIn adults, parotoid area was significantly larger in animals captured from urban as well as agricultural habitats compared to toads from natural habitats Table , Fig. 2.For adults, the meta-analytic mean effect of anthropogenic habitats on the concentrations of bufadienolide compounds did not differ from zero Table , but theSimilarly to the adults, the juveniles’ meta-analytic mean effect of anthropogenic habitats on the concentrations of bufadienolide compounds did not differ from zero Table , but the18. Thus, our finding that adult toads had larger parotoids in urban and agricultural habitats than in natural habitats suggests that the total amount of stored toxins was higher in the animals captured from anthropogenic habitats. Furthermore, the overall amount of bufadienolides per unit dry mass of parotoid secretion was higher in adult toads from agricultural habitats, suggesting that their toxin secretion was more potent. These results parallel the earlier experimental findings on toad tadpoles that herbicide exposure increases their bufadienolide content26. Altogether, one potential explanation for these results is that chemical pollutants in anthropogenic habitats may enhance the production of bufadienolides, which may be either a non-adaptive constraint or an adaptive response. On the one hand, bufadienolides are synthesized from the same precursor as glucocorticoid stress hormones and steroid sex hormones, starting with the same initial steps20. Thus, elevated bufadienolide levels may be by-products of physiological stress and/or the endocrine-disrupting effects known to be elicited by many anthropogenic pollutants26. Notably, elevated levels of stress hormones have been found in aquatic amphibians in urban areas39. On the other hand, increasing the levels of bufadienolides might also be an adaptive response to chemical stressors, because bufadienolides may act as regulators of the amphibian skin’s ion transport40 which can be altered by several pollutants43. Both of these potential mechanisms can apply to the adult toads in our present study, because in a joint study we found that our ponds contained larger numbers and higher concentrations of endocrine-disrupting chemicals in anthropogenic habitats than in natural habitats27. Experimental studies are needed to test whether these chemicals or other pollutants affect bufadienolide levels by any of the two mechanisms proposed above.The parotoid is by far the largest toxin gland in toads, and larger parotoids contain more toxins17. Although we have no data on predator densities at our study sites, ample literature shows that generally both urban and agricultural environments differ from natural habitats in the composition of predator fauna45. While larger, specialist predators typically avoid anthropogenic habitats, avian and mammalian generalist mesopredators like corvids, badgers, martens, racoons, skunks and foxes  are often more abundant in urban habitats, as are domestic cats and dogs46. Similarly, some corvids47 and herons48 occur at higher densities in agricultural habitats, and some toad-eating snakes can spend up to 80% of their time in crop fields49. Therefore, toads living in anthropogenic habitats may perceive higher overall predation risk and may adjust their toxin levels accordingly. This scenario is supported by our results that bufagenins, the typically more toxic compounds23, were present in higher concentrations in the parotoid secretion of adult toads from urban as well as agricultural habitats, compared to their counterparts from natural habitats. This difference was particularly prominent for urban habitats , where the increase in bufagenins seems to have come at the expense of bufotoxins. It is thus possible that urban toads invest more heavily into producing and storing higher amounts of the more potent compounds even at the price of having to deal with higher autotoxicity18. Alternatively, toads might not store the bufagenins but rather produce them upon gland discharge by enzymatically cutting the side chain off of the bufotoxins19. In this case, our urban toads may have had a faster machinery for this process, allowing them to respond more rapidly to a predator attack (i.e. a human squeezing their parotoids). Clarifying the avenues by which anthropogenic environments affect toad chemical defences will take more detailed studies on their toxin physiology.However, pollution and physiological stress are not the only features of anthropogenic habitats that may contribute to the higher levels of toad toxins. Deterrence of predators is considered the main function of amphibian toxins, and experiments with newts showed that individuals can upregulate their chemical defences in response to higher predation risk perceived because of predator presence or repeated predatory attacks28 and little if any seasonal variation has been found in parotoid size and toxin composition50. Also, we cannot rule out that some similarity in habitat effects between the two age groups might have been masked by the minor differences in the methods applied to adults and juveniles to measure parotoids and sample toxins. However, calliper and photo measurements yielded qualitatively identical results for juvenile parotoid size (Table 27). Therefore, our findings suggest that phenotypic plasticity at the level of individuals may play an important role in the enhanced chemical defences of toads living in anthropogenic habitats. This is intriguing, because out of the few studies that have so far investigated the mechanisms by which anthropogenic environments influence the animals’ physiological and behavioural responses to risk53 or chemical stress55, the majority suggested microevolution or other transgenerational effects. It is possible that anthropogenic environments exert complex selection forces on toads’ chemical defences because of spatio-temporal heterogeneity in pollution27 and predation risk44, which should then favour the evolution and maintenance of phenotypic plasticity9. Also, the potential for microevolution may be constrained if heritability is low. Further studies are thus needed to quantify heritability and plasticity of chemical defence in toads, as has been done with other adaptive inducible defences56.Our common garden experiment showed that the differences found in adult toads were not retained in their offspring when the latter were raised in the lab under identical environmental conditions. When we compared the animals originating from anthropogenic and natural habitats, the significant differences seen in adults were either not present in the juveniles or even showed the opposite direction. In contrast with the larger parotoids and higher bufadienolide concentrations in adults from agricultural habitats, their offspring had smaller parotoids and lower bufotoxin concentrations. Similarly, the juveniles originating from urban habitats showed neither the larger parotoids nor the higher concentration of bufagenins and lower concentration of bufotoxins that we found in their parents. These results indicate that genetic or epigenetic changes are not likely to be responsible for the differences we observed in the adult toads’ chemical defences between anthropogenic and natural habitats. Cautionarily, we cannot exclude the possibility that the differences seen in the adults are genetically determined but are only expressed after maturity and/or during the breeding season. However, the juveniles had well developed gonads by the time of sampling and, in other toad species, parotoids acquire the morphological and histochemical characteristics of adults before sexual maturityze Table , and cheze Table  despite ze Table . Thus, h57, the adults in our present study might have traded-off some resources for increased toxin synthesis. For example, if toads from anthropogenic habitats converted the common steroid precursor into bufadienolides at the expense of sex hormones20, they might have suffered reduced reproductive success. In line with this idea, we found significantly reduced offspring performance in our toads from anthropogenic habitats27, which might also explain the lower bufotoxin levels of juveniles from agricultural habitats. Furthermore, higher toxicity of toads may negatively affect other species too. For example, the invasive cane toad (Rhinella marina) has caused drastic mortality among Australian native predators that are highly sensitive to bufadienolides13. Thus, understanding how widespread the anthropogenic effects on toad toxicity are, and what mechanisms govern them, may prove important for the conservation of toads and their predators alike.A further important question is whether the elevated levels of chemical defence are costly in terms of fitness. Although experiments with common toad tadpoles did not reveal any cost of bufadienolide productionSupplementary InformationSupplementary Dataset


Although many individuals who experience a trauma go on to develop post-traumatic stress disorder (PTSD), the rate of PTSD following trauma is only about 15–24%. There must be some pre-existing conditions that impart increased vulnerability to some individuals and not others. Diathesis models of PTSD theorize that pre-existing vulnerabilities interact with traumatic experiences to produce psychopathology. Recent work has indicated that personality factors such as behavioral inhibition (BI), harm avoidance (HA), and distressed (Type D) personality are vulnerability factors for the development of PTSD and anxiety disorders. These personality temperaments produce enhanced acquisition or maintenance of associations, especially avoidance, which is a criterion symptom of PTSD. In this review, we highlight the evidence for a relationship between these personality types and enhanced avoidance and associative learning, which may increase risk for the development of PTSD. First, we provide the evidence confirming a relationship among BI, HA, distressed (Type D) personality, and PTSD. Second, we present recent findings that BI is associated with enhanced avoidance learning in both humans and animal models. Third, we will review evidence that BI is also associated with enhanced eyeblink conditioning in both humans and animal models. Overall, data from both humans and animals suggest that these personality traits promote enhanced avoidance and associative learning, as well as slowing of extinction in some training protocols, which all support the learning diathesis model. These findings of enhanced learning in vulnerable individuals can be used to develop objective behavioral measures to pre-identify individuals who are more at risk for development of PTSD following traumatic events, allowing for early (possibly preventative) intervention, as well as suggesting possible therapies for PTSD targeted on remediating avoidance or associative learning. Future work should explore the neural substrates of enhanced avoidance and associative learning for behaviorally inhibited individuals in both the animal model and human participants. Post-traumatic stress disorder (PTSD) is a devastating mental illness that can develop following exposure to a traumatic event. However, it is unclear why only a percentage (15–24%) of individuals who experience trauma go on to develop PTSD . For exaDiagnostic and Statistical Manual  or trauma-related thoughts or feelings , or both. Avoidance of traumatic or stressful situations is usually adaptive in that it protects from possible harm. However, avoidance can become maladaptive, limit social and non-social interactions, and disrupt normal life functioning as in the case of PTSD. Moreover, avoidance contributes to the chronicity of disorder as it prevents the individual from learning that certain situations or stimuli are not or no longer dangerous.One behavioral tendency, which has received much attention, is avoidance. Although disorders such as generalized anxiety disorder (GAD), obsessive-compulsive disorder (OCD), panic disorder, social anxiety disorder (SAD), social phobia, and PTSD have distinguishing symptoms, excessive or maladaptive avoidance is a common symptom in all these disorders. To satisfy Avoidant tendencies may be particularly important in differentiating individuals likely to develop PTSD following exposure to a traumatic event from those who display resilience in the face of trauma. Avoidance or escape behaviors have been identified as a significant predictor of PTSD  as well DSM-5). One expression of this altered arousal and reactivity is hypervigilance. Hypervigilance can be expressed in several ways. An individual may express general hypervigilance by over-attending to possible aversive stimuli by searching for possible threats , which is defined as a temperamental tendency to avoid novel individuals and situations . BI can Recent work has demonstrated a relationship between BI and PTSD in a variety of samples. AMBI, but not RMBI, scores were associated with scores on the PTSD checklist (PCL-M) administered to Veterans ,b. This Other personality factors found to be strongly related to BI are harm avoidance or HA  and distType D personality is measured with the Type D scale DS-14,  and invoTaken together, these findings have supported the relationship between PTSD and a variety of constructs describing anxious and inhibited personality temperaments; however, the degree to which trait anxiety, BI, HA, and Type D are distinct constructs has not been adequately addressed. Recent work has revealed that these temperaments have some degree of relationship varying from weak to moderate. Future work needs to examine how much overlap exists between these various measures of inhibited temperament.In addition to personality temperaments like BI, HA, and Type D, females express greater lifetime prevalence for PTSD than males . Partly,Overall, several personality temperaments related to avoidance including trait anxiety, behavioral inhibition, harm avoidance, and distressed (Type D) personality have been identified, along with female gender, as vulnerability factors for the development of PTSD. Inhibitory temperaments and a tendency to avoid can possibly identify which trauma-exposed individuals are more at risk to develop PTSD or display resilience, allowing optimal targeting of therapeutic resources.Besides the strong clinical evidence for a relationship between inhibited temperaments and PTSD, inhibited temperament has been investigated in an animal model, the inbred Wistar Kyoto (WKY) rat strain. WKY rats express many of the behavioral tendencies associated with BI and PTSD in humans. For a detailed review of the WKY rat as an animal model in which pathological avoidance is an endophenotype for PTSD, see WKY rats also exhibit inhibited temperament in a variety of behavioral tasks. One such task is the open field test that measures locomotion and exploration of a novel environment and has been used to test anxiety-like behaviors in rats and mice. Anxiety-related behaviors are thought to come about due to social separation from cage mates and the stress of the novel open field apparatus . There iDSM-5 Criterion E  for PTSD. A common measure of hypervigilance is the acoustic startle response (ASR), which tests arousal and vigilance to an unexpected sound. ASR is typically measured by full body movement in rats as compared to an eyeblink response in humans. WKY rats exhibit an enhanced ASR compared to outbred SD rats  or active avoidance (performing an arbitrary response to prevent an aversive stimulus). Passive avoidance involves inhibiting innate or species-specific defense responses (SSDRs) such as a preference for a dark area. WKY rats acquire passive avoidance faster than SD rats , 1996, wActive avoidance in rodents occurs when a voluntary behavior is made in response to a warning signal such as a light or tone in order to avoid an impending aversive event such as an electric foot shock. A variety of behaviors have been tested in recent active avoidance studies in rats including shuttlebox activity  and stepWKY rats are faster to acquire the lever-press avoidance response and exhibit higher levels of avoidance than SD rats  as shownWKY rats exhibit almost perfect avoidance, eventually making an avoidance response on almost every trial. A well-trained WKY rat will then begin each subsequent session with an avoidance response on the first trial and continuing to avoid on all or most trials thereafter. However, SD rats display a different pattern. Even after extensive training, SD rats will typically begin each session with a trial on which they fail to make the avoidance response, experience the shock, and perform an escape response – even though avoidance responses were expressed at high levels at the end of the previous session. This effect is termed warm up ,b and haThe near perfect avoidance behavior of well-trained WKY rats also insulates them from experiencing alterations in shock contingency. For example, when a rat consistently avoids the shock, it cannot become aware of a shift from acquisition trials in which the warning signal is followed by the shock, to extinction training, where warning signal remains but the shock has been removed. Accordingly, WKY rats continue to perform avoidance responses during extinction training, even though the shock is no longer delivered  as shownImportantly, these findings of resistance to avoidance extinction fit with the current reports of impaired inhibition of conditioned fear responses in PTSD patients e.g., . One of Avoidance in humans has not been as widely investigated as in animal models. Some recent human avoidance studies have utilized various aversive events including mild electric shocks e.g.,  and an uSome studies of human avoidance have employed computer-based avoidance tasks involving a loss of points or loss of monetary reward as the aversive event. For example, A computer-based avoidance task that may be comparable to escape and avoidance learning in rats was developed by More recently, Overall, humans expressing inhibited temperament and/or PTSD symptoms exhibit enhanced avoidance learning much like WKY rats. In addition, females exhibited greater avoidance learning than males in both WKY rats and humans. Taken together, the findings from these avoidance studies with WKY rats and humans support the idea that inhibited temperament and female gender are associated with enhanced avoidance and slowed extinction, which may be indicative of PTSD.In addition to avoidance, several theories of PTSD  suggest Continued over-prediction of aversive stimuli related to a traumatic event can come about through two mechanisms. First, the formation of associations between a cue (the CS) and an aversive event (the US) may be enhanced. For example, a soldier may experience a seemingly neutral event such as a crowd of people (CS) immediately before an aversive event such as an explosion (US). The degree of predictiveness between the neutral and aversive events may be low in which most crowds do not predict upcoming explosions; however, some individuals exposed to one instance of the crowd-explosion association may form stronger than normal associations between the events, such that a future exposure to a crowd may trigger strong memories or feelings associated with the traumatic event (CR). Individuals with PTSD might then avoid similar situations in order to avoid triggering the CR.Second, the associations formed between a cue and an undesirable stimulus may persist longer than normal. Normally, when cues no longer predict an undesirable event, the association weakens and the behavior is reduced. A real-world example would be a soldier who learned an association between crowds and explosions during periods of combat exposure and then returns home to experience many instances of crowds  with no explosion. The repeated lack of pairing between crowd CS and explosion US would normally lead to extinction of the CS-US association. However, an individual with PTSD might experience crowds many times without the outcome of an explosion and yet continue to experience stress and fear responses related to the traumatic event due to the initial strength of the association between crowds and the traumatic event or an inability to associate crowds with alternative responses, or lack of extinction.Individual differences in personality temperament  may produce differences in either the acquisition of conditioned responses through CS-US paired training or the reduction of previously trained responses through CS-alone extinction training, or both. Behaviorally inhibited individuals would form strong associations between traumatic events and trauma-related cues resulting in fear, anxiety, and avoidance in the presence of these cues. These learned responses contribute to dysfunction when expressed pathologically in the absence of danger.Consistent with this view, some studies utilizing associative learning paradigms with aversive stimuli, such as shock, have reported facilitated acquisition of autonomic  conditioned responses in humans expressing PTSD symptoms . HoweverThe most well-studied form of classical conditioning other than fear conditioning involves the conditioning of an eyeblink response. Classical eyeblink conditioning involves the pairing of an initially neutral CS  with an aversive US  that elicits a reflexive eyeblink termed the unconditioned response or UR. Initially, the tone is neutral in which it does not produce an eyeblink response. However, following paired presentations of the tone followed by the air puff, a conditioned eyeblink or CR is exhibited prior to the onset of the air puff.There is a long history of studying anxiety in classical eyeblink conditioning going back to the 1950s. Janet Taylor Spence  developeRecent work has attempted to determine if the enhanced avoidance in WKY rats and inhibited humans carried over to eyeblink classical conditioning. As expected, WKY rats acquired conditioned eyeblinks faster and to a greater degree than control SD rats  as shownIn addition to the enhanced CR acquisition, More recent work with eyeblink classical conditioning and inhibited personality has extended beyond BI to test the effects of distressed (Type D) personality in non-clinical populations . As descGiven the findings of enhanced avoidance and associative learning  in WKY rats and humans with inhibited temperament, a form of eyeblink conditioning that involves an instrumental avoidance response was also tested. The avoidance version of eyeblink conditioning involves the omission of the US on trials in which a CR is performed to the CS. Early studies of omission training with human eyeblink conditioning revealed that omission training resulted in reduced acquisition compared to 100% CS-US paired trials . FurtherIn a rat version of this eyeblink conditioning omission task, the US (eyelid stimulation) was omitted on trials in which the rat performed a CR to the tone . TherefoThe effect of omission training on eyeblink conditioning was also tested with healthy young adults assessed for BI . While bEven though humans did not exhibit avoidance learning, the eyeblink conditioning omission study in humans did lead to a novel finding that the enhanced acquisition of conditioned eyeblinks was more evident in the omission and yoked conditions than in the 100% CS-US paired trials. The finding of enhanced learning in non-optimal learning conditions was theorized to be due to the effects of schedules of partial reinforcement or situations in which some of the trials were tone alone rather than CS-US paired presentations.Within the context of eyeblink classical conditioning, the US air puff has been interpreted by The partial reinforcement effect was subsequently tested in humans with BI, using specific schedules of partial reinforcement in which the CS or US were presented alone on 50% of training trials . Both paWe hypothesized that the enhancement observed in inhibited individuals under partial reinforcement may also have been due in part to the fact that introducing CS- and US-alone trails caused variations in the timing between CS-US paired training trials, compared to the fairly regular presentations of CS-US trails under 100% CS-US training. This hypothesis was tested by extending and varying the inter-trial interval (ITI) between CS-US paired training trials under an 100% CS-US training schedule . Simply Another variation of eyeblink conditioning that has been tested with WKY rats and inhibited humans involves the proactive interference effects of stimulus pre-exposure prior to conditioning. Normally, repeated exposures of the CS alone , US alone, or uncorrelated presentations of the CS or US  retard subsequent acquisition of conditioned responses. LI is not observed in stressed humans  or thoseHowever, when In addition to studies of behaviorally inhibited individuals, eyeblink conditioning has also been tested with individuals diagnosed or identified with PTSD symptoms. More recently, Overall, several measures of anxiety or inhibited temperament including manifest anxiety, trait anxiety, behavioral inhibition, and distressed (Type D) personality have been associated with enhanced associative learning in humans. In addition, individuals with PTSD symptoms also show enhanced associative learning. These findings generally agree with the findings from the behaviorally inhibited WKY rat strain. In addition, these findings support the idea that parallel rodent and human studies provide an opportunity to explore the mechanisms for the development of PTSD through altered associative learning. The work reviewed from WKY rats and inhibited individuals as well as individuals with symptoms of PTSD support for the learning diathesis model of PTSD as represented in In addition to eyeblink conditioning studies with rats and humans, a separate animal model of PTSD utilizing eyeblink conditioning involves modulation of the eyeblink reflex in rabbits , the cerebellum is the substrate of CS-US association (for a detailed review, see There is also some limited evidence that the cerebellum is involved in avoidance learning. Removal of the cerebellum in rats impaired passive avoidance learning in a shuttle box task . More spThe possible role of cerebellar learning in PTSD is supported by recent imaging studies that have identified a role for the cerebellum in PTSD . CerebelBeyond the cerebellum, the hippocampus is known to modulate eyeblink conditioning and be involved in avoidance learning as well as PTSD. Although the hippocampus is not required for simple delay eyeblink conditioning, when present it normally contributes to acquisition of the CS-US association (for reviews, see These findings of hippocampal abnormalities in WKY rats fit with hippocampal abnormalities observed in humans with PTSD. For example, reduced hippocampal volume is observed in patients with PTSD , along wThe amygdala also modulates eyeblink conditioning, is involved in avoidance learning, and has been found to be related to PTSD. Specifically, lesions to the amygdala retard acquisition of classically conditioned eyeblinks, disrupt hippocampal activity in rats , and redOverall, there is strong evidence that neural substrates including the cerebellum, hippocampus, amygdala, and medial prefrontal cortex are involved in avoidance learning and eyeblink conditioning as well as PTSD. These brain structures are part of the circuit identified as the cerebellar-limbic-thalamo-cortical network  that serFuture work should focus on further aligning the findings between human studies and animal models of PTSD. Specifically, there is a need for a human avoidance task that more closely matches the trial and error learning aspect as well as the aversive nature of the rat lever-press avoidance paradigm. In addition, studies in WKY rats are needed to replicate recent human findings with schedules of partial reinforcement in both eyeblink conditioning and avoidance learning. To date, eyeblink classical conditioning studies in behaviorally inhibited humans and individuals with PTSD symptoms have been tested exclusively with delay eyeblink conditioning. Future work should also target trace conditioning, which involves the hippocampus and has been found to produce stronger gender differences than delay conditioning . Future via a series of small challenges that can be overcome (e.g., Future work can also have potential clinical applications. Avoidance and classical conditioning tasks could be used as pre-tests for PTSD vulnerability in trauma-exposed individuals. These objective measures may be better than standard paper and pencil self-report inventories, which are susceptible to biased responding as well as to individual variations in the ability to introspect and describe one’s own feelings and behaviors in comparison to accepted social norms. If successful, such testing would offer an opportunity for identifying high-risk individuals for a pre-emptive intervention to possibly reduce or avert PTSD before it develops. If the mechanisms through which BI interacts with stimulus pre-exposure could be fully delineated, possible therapies could be developed in which individuals are “inoculated” for PTSD prior to traumatic experiences – similar to the concept of “inoculating” an individual against learned helplessness by training me e.g., .The work with extinction in avoidance learning and eyeblink conditioning is applicable to both identifying individuals at risk for PTSD as well as therapies to reduce PTSD symptoms. Prior work with fear conditioning has indicated that pre-trauma differences in classical conditioning  and extiThe exact mechanisms through which a traumatic event leads a limited number of individuals to develop of PTSD, while others develop resilience, remain elusive. What is abundantly clear is that PTSD is best explained with diathesis models  that addMA wrote the original manuscript based on the prior publications of MA, CM, KB, KP, and RS. CM, KB, KP, and RS revised the manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.The contents do not necessarily reflect the views of the U.S. Department of Veterans Affairs or the United States Government.