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Current understanding of collective behaviour in nature is based largely on models that assume that identical agents obey the same interaction rules, but in reality interactions may be influenced by social relationships among group members. Here, we show that social relationships transform local interactions and collective dynamics. We tracked individuals' three-dimensional trajectories within flocks of jackdaws, a species that forms lifelong pair-bonds. Reflecting this social system, we find that flocks contain internal sub-structure, with discrete pairs of individuals tied together by spring-like effective forces. Within flocks, paired birds interacted with fewer neighbours than unpaired birds and flapped their wings more slowly, which may result in energy savings. However, flocks with more paired birds had shorter correlation lengths, which is likely to inhibit efficient information transfer through the flock. Similar changes to group properties emerge naturally from a generic self-propelled particle model. These results reveal a critical tension between individual- and group-level benefits during collective behaviour in species with differentiated social relationships, and have major evolutionary and cognitive implications.

Collective behaviour is typically thought to arise from individuals following fixed interaction rules. The possibility that interaction rules may change under different circumstances has thus only rarely been investigated. Here we show that local interactions in flocks of wild jackdaws (Corvus monedula) vary drastically in different contexts, leading to distinct group-level properties. Jackdaws interact with a fixed number of neighbours (topological interactions) when traveling to roosts, but coordinate with neighbours based on spatial distance (metric interactions) during collective anti-predator mobbing events. Consequently, mobbing flocks exhibit a dramatic transition from disordered aggregations to ordered motion as group density increases, unlike transit flocks where order is independent of density. The relationship between group density and group order during this transition agrees well with a generic self-propelled particle model. Our results demonstrate plasticity in local interaction rules and have implications for both natural and artificial collective systems., Modelling collective behaviour in different circumstances remains a challenge because of uncertainty related to interaction rule changes. Here, the authors report plasticity in local interaction rules in flocks of wild jackdaws with implications for both natural and artificial collective systems.

As one of nature's most striking examples of collective behaviour, bird flocks have attracted extensive research. However, we still lack an understanding of the attractive and repulsive forces that govern interactions between individuals within flocks and how these forces influence neighbours' relative positions and ultimately determine the shape of flocks. We address these issues by analysing the three-dimensional movements of wild jackdaws ( Corvus monedula ) in flocks containing 2–338 individuals. We quantify the social interaction forces in large, airborne flocks and find that these forces are highly anisotropic. The long-range attraction in the direction perpendicular to the movement direction is stronger than that along it, and the short-range repulsion is generated mainly by turning rather than changing speed. We explain this phenomenon by considering wingbeat frequency and the change in kinetic and gravitational potential energy during flight, and find that changing the direction of movement is less energetically costly than adjusting speed for birds. Furthermore, our data show that collision avoidance by turning can alter local neighbour distributions and ultimately change the group shape. Our results illustrate the macroscopic consequences of anisotropic interaction forces in bird flocks, and help to draw links between group structure, local interactions and the biophysics of animal locomotion.

Tracking the movements of birds in three dimensions is integral to a wide range of problems in animal ecology, behaviour and cognition. Multi-camera stereo-imaging has been used to track the three-dimensional (3D) motion of birds in dense flocks, but precise localization of birds remains a challenge due to imaging resolution in the depth direction and optical occlusion. This paper introduces a portable stereo-imaging system with improved accuracy and a simple stereo-matching algorithm that can resolve optical occlusion. This system allows us to decouple body and wing motion, and thus measure not only velocities and accelerations but also wingbeat frequencies along the 3D trajectories of birds. We demonstrate these new methods by analysing six flocking events consisting of 50 to 360 jackdaws (Corvus monedula) and rooks (Corvus frugilegus) as well as 32 jackdaws and 6 rooks flying in isolated pairs or alone. Our method allows us to (i) measure flight speed and wingbeat frequency in different flying modes; (ii) characterize the U-shaped flight performance curve of birds in the wild, showing that wingbeat frequency reaches its minimum at moderate flight speeds; (iii) examine group effects on individual flight performance, showing that birds have a higher wingbeat frequency when flying in a group than when flying alone and when flying in dense regions than when flying in sparse regions; and (iv) provide a potential avenue for automated discrimination of bird species. We argue that the experimental method developed in this paper opens new opportunities for understanding flight kinematics and collective behaviour in natural environments.

The rapid, cohesive turns of bird flocks are one of the most vivid examples of collective behaviour in nature, and have attracted much research. Three-dimensional imaging techniques now allow us to characterize the kinematics of turning and their group-level consequences in precise detail. We measured the kinematics of flocks of wild jackdaws executing collective turns in two contexts: during transit to roosts and anti-predator mobbing. All flocks reduced their speed during turns, probably because of constraints on individual flight capability. Turn rates increased with the angle of the turn so that the time to complete turns remained constant. We also find that context may alter where turns are initiated in the flocks: for transit flocks in the absence of predators, initiators were located throughout the flocks, but for mobbing flocks with a fixed ground-based predator, they were always located at the front. Moreover, in some transit flocks, initiators were far apart from each other, potentially because of the existence of subgroups and variation in individual interaction ranges. Finally, we find that as the group size increased the information transfer speed initially increased, but rapidly saturated to a constant value. Our results highlight previously unrecognized complexity in turning kinematics and information transfer in social animals.

Hurler disease or mucopolysaccharidosis type I (MPS-I) is an autosomal-recessive lysosomal storage disease caused by a defect in α-L-iduronidase (IDUA).1,2 It is the most common of the mucopolysaccharidoses, with an incidence of 1 per 75 000 live births.3 Deficiency of the IDUA enzyme leads to a pathological accumulation of glycosaminoglycans (GAG) in the brain and other organs. Typical central nervous system (CNS) features are severe mental retardation, leptomeningeal fibrosis with obstructive hydrocephalus, arachnoid cysts, and sensorineural deafness. Although systemic features of MPS-I can be treated with enzyme replacement therapy or hematopoietic stem cell transplantation, there is currently no accepted treatment for the CNS features. A caveat is that intervention in the neonatal period with bone marrow or cord blood transplantation has shown limited improvement in cognitive performance, likely through cross-correction by stem cells that migrate to the perivascular space.4,5 However, even in the best-case scenario, the existing treatment options do not reverse abnormal cognitive development, are associated with numerous long-term complications, and are prohibitively expensive for many patients. The pathological hallmark of MPS-I in the brain is the accumulation of GAG inside neurons, where they are increased up to 5-fold, as well as in mesodermally derived tissues of the perivascular space, leptomeninges, and choroid plexus, where they are often increased 10-fold and form so-called clear-cell periadventitial inclusions.6 As a result of pathological accumulation of GAG, there is a secondary perturbation of β-galactosidase (originally thought to be the main defect in Hurler disease),7 and consequently, the neuronal inclusions in Hurler disease are made up primarily of gangliosides (GM2, GM3) which are not usually present in substantial amounts in normal brain.8,9 Another downstream enzyme, β-hexosaminidase (defective in Tay-Sachs disease), which breaks down GM2, is upregulated in MPS-I, and normalization of this enzyme was used as a secondary outcome measure in canine gene therapy studies for Hurler disease.10 Our inability to achieve high-level neuronal gene expression throughout the large volume of the human brain is currently an impediment to more effective gene therapy for many neurodegenerative diseases. The purpose of this study was to test the feasibility of minimally invasive viral vector-based CNS gene therapy in Hurler disease and, more specifically, to compare endovascular with intracerebral ventricular (ICV) delivery. Recombinant adeno-associated viral vector (rAAV) was chosen because it is nonpathogenic and is able to express genes in the CNS at very high levels.11 It exists in more than 10 serotypes, most of which specifically transduce neurons when injected intraparenchymally, but some are capable of transducing astrocytes, microglia, ependyma, and other cells to various degrees. To maximize the unit dose per animal, we used a transgenic mouse model12 rather than one of the larger animal models,13,14 with an aggressive endovascular dosing regimen of 1 trillion genomic particles (g.p.) per mouse. We chose rAAV5 vector, which has an advantage of high-level neuronal expression and cellular internalization by the platelet-derived growth factor receptor,15 which also targets ependymal, choroid plexus, and perivascular cells affected in Hurler disease. Another theoretical benefit of rAAV5 is that neutralizing antibody titers are the lowest among AAV serotypes.16 Endovascular gene therapy offers widespread access to the brain but generally requires blood-brain barrier disruption, and until recently, results in animal models were disappointing. Rapoport and colleagues17,18 were the first to show reversible osmotic opening of the blood-brain barrier, and their technique of hyperosmolar disruption remains in widespread use. This method was adapted for clinical use in intra-arterial (IA) chemotherapy for brain tumors19 but has never been used clinically for gene therapy. Endovascular techniques have been used in rats20–22 or mice23 to deliver viral vectors to the brain, but gene expression was significantly less than with intraparenchymal delivery, and the microsurgical techniques are demanding. As a result, this approach was abandoned by the majority of gene therapists, even as microcatheter techniques and vector technology advanced. ICV gene therapy is appealing because the ependymal and meningeal surface area is large and may be particularly suited to Hurler disease, which involves these structures. The original clinical application of ICV gene therapy in 1996 used a nonviral vector with Ommaya reservoirs in patients with Canavan disease together with intravenous mannitol.24 The benefit of mannitol for this route of delivery is the greater bulk flow through the interstitial space and increased permeability of the ependyma, rather than effects on the blood-brain barrier. Systemic mannitol was shown in animal models to increase spread of vectors with ICV or intraparenchymal delivery.24–26 However, as a result of perceived limitations of the ICV route with viral vectors, an intraparenchymal approach with multiple rAAV2 injections and systemic mannitol was subsequently adopted for human use.27,28 At this time, the ICV approach deserves a closer look with new vector technology and techniques to optimize transependymal flow. Our study used rAAV5 vectors in adult animals to deliver foreign genes to the brain via IA or ICV delivery, with mannitol treatment used in both cases. In the first case, mannitol is administered directly to arterial vessels to transiently disrupt the endothelial tight junctions and to allow penetration of vector, whereas in the latter case, mannitol is used to improve bulk flow of rAAV5 through ependyma and interstitial space. We initially tested dose-response and biodistribution using rAAV5–green fluorescent protein (GFP) and then assessed the tolerability and efficacy of a very high unit dose (5 × 1013 viral particles per kg) of the therapeutic vector rAAV5-IDUA. With IA delivery, blood-brain barrier disruption was performed with 25% or 29.1% mannitol, and with ICV delivery, we used 25% mannitol injected systemically or directly into the cerebral ventricle. Our goal with ICV delivery was not merely to assess the use of mannitol, because this is already known to increase vector spread,24,29 but rather to compare the endovascular and ICV delivery routes using the best available techniques. Using either delivery paradigm, we significantly reversed brain pathology in animals with a Hurler phenotype.

The transfer and expression of cytokine genes into malignant cells to provide a more effective tumor response has shown promise. The majority of murine models in which tumor vaccination strategies have been tested have utilized selected and expanded clones of tumor cells, which is impractical clinically. In a model of murine B lineage lymphoma (BDL-2), we compared the effectiveness of tumor vaccines composed of a) a BDL-2 clone established by G-418 resistance following transduction with the LIL2SN retrovirus and screened for maximal IL-2 secretion, b) a syngeneic fibroblast line transduced with LIL2SN and screened for G-418 resistance and IL-2 expression, which was co-injected with the parental line, and c) a heterogeneous (unselected) population of BDL-2 cells transduced with the MFG/IL2 virus, reported to provide enhanced expression of cytokine genes and minimize the need for selection. Testing of splenocytes derived from vaccinated animals reveals that injections of BDL-2 expressing IL-2 results in an increased capacity of splenocytes to kill BDL-2 in vitro, compared to vaccination of BDL-2 alone or in combination with IL-2 secreting fibroblasts. We show that a vaccine composed of MFG/IL2 transduced, unselected BDL-2 cells is equivalent or superior to a clone derived from LIL2SN transduction in prolonging survival of animals with previously established tumor. These studies provide evidence that transduction of tumor with MFG based vectors without in vitro selection leads to expression of high levels of IL-2 and can impact the survival of animals with disseminated tumor.

In previous studies characterizing intron-dependent expression of the human purine nucleoside phosphorylase-encoding gene (PNP), we identified a putative enhancer sequence in the first intron which was capable of mediating increased cat reporter gene expression in transfected murine NIH 3T3 cells in a position- and orientation-independent manner. In order to further characterize this enhancer activity, the nucleotide sequence was determined for the region of intron 1 to which this activity was originally ascribed. The sequence was analyzed for the presence of binding sites for known transcription factors, but none were identified. A 444-bp downstream portion of the intron-1 sequence enhanced cat expression either in conjunction with a human PNP promoter sequence or with a 105-bp heterologous herpes simplex virus thymidine kinase (TK) promoter. Nested deletions of the downstream intron-1 sequence fused to a TK::cat fusion gene localized the enhancer activity to a 170-bp sequence in intron 1. A 154-bp HgiAI fragment (bp 424 to 577 of intron 1) excised from this region contained enhancer activity which varied directly with the number of fragments inserted upstream from the TK::cat fusion gene. However, inversion of the HgiAI fragment in a PNP abbreviated gene, or relocation of the HgiAI fragment from intron 1 to a position upstream from the PNP promoter, reduced or eliminated PNP expression. The effect of the intron-1 enhancer element on PNP expression is thus maximized in a position- and orientation-dependent manner.

Abbreviated purine nucleoside phosphorylase (PNP) genes were engineered to determine the effect of introns on human PNP gene expression. PNP minigenes containing the first intron (complete or shortened from 2.9 kb down to 855 bp), the first two introns or all five PNP introns resulted in substantial human PNP isozyme expression after transient transfection of murine NIH 3T3 cells. Low level human PNP activity was observed after transfection with a PNP minigene containing the last three introns. An intronless PNP minigene construct containing the PNP cDNA fused to genomic flanking sequences resulted in undetectable human PNP activity. Heterogeneous, stable NIH 3T3 transfectants of intron-containing PNP minigenes (verified by Southern analysis), expressed high levels of PNP activity and contained appropriately processed 1.7 kb message visualized by northern analysis. Stable transfectants of the intronless PNP minigene (40-45 copies per haploid genome) contained no detectable human PNP isozyme or mRNA. Insertion of the 855 bp shortened intron 1 sequence in either orientation upstream or downstream of a chimeric PNP promoter-bacterial chloramphenicol acetyltransferase (CAT) gene resulted in a several-fold increase in CAT expression in comparison with the parental PNP-CAT construct. We conclude that human PNP gene expression at the mRNA and protein level is dependent on the presence of intronic sequences and that the level of PNP expression varies directly with the number of introns included. The disproportionately greatest effect of intron 1 can be explained by the presence of an enhancer-like element retained in the shortened 855 bp intron 1 sequence.

The immune response to a murine myeloid leukemia (cell line C1498) was studied in vitro and in vivo. Natural killer (NK) cells and CD8+ cytotoxic T lymphocytes (CTL) were shown to lyse C1498 in vitro through the binding of leukocyte function antigen-1 (LFA-1) on effectors and intercellular adhesion molecule-1 (ICAM-1) and ICAM-2 on C1498 target cells. However, the ability of nonimmunized mice to resist an in vivo challenge of a low dose (10(4)) of C1498 was NK-cell, but not T-cell dependent. The failure of T cells to participate in the immune surveillance of a low leukemia burden appeared, in part, because of a lack of expansion of leukemia reactive CTL precursors (CTLp). Leukemia reactive CTLp frequency estimations in naive and leukemia bearing mice were not significantly different (range, 1:20,600 to 1:74,000) in contrast to immunized mice (range, 1:1,400 to 1:4,400). Leukemia reactive CTLp could be expanded to a level that could apparently mediate in vivo immune surveillance of 10(5) leukemia cells by injection of irradiated leukemia cells intraperitoneally (IP) or subcutaneously (SC), but not intravenously (IV). However, IV injection of 10(5) live leukemia cells engineered to secrete interleukin-2 (IL-2) resulted in systemic immunity mediated primarily by CD8+ T cells. We conclude that NK cells can mediate immune surveillance of a low leukemia burden. CD8+ CTL-mediated immune surveillance can eliminate a higher leukemia burden than NK cells, but requires T-cell help, which can be delivered by local IL-2. Both NK and CTL-mediated immune surveillance of C1498 murine myeloid leukemia is dependent on recognition through the LFA-1:ICAM adhesion pathway.

We have induced acute pulmonary oedema in upright anaesthetized dogs by increasing pulmonary microvascular permeability or by extracellular fluid volume overload in order to determine the sensitivity and specificity of the radiograph to the presence of abnormal lung water. Radiographs were taken before and after development of oedema. At the end of the experiment we removed the inflated lungs and froze them in liquid nitrogen and subsequently examined them macroscopically in the frozen state. The extravascular water/dry lung weight ratio was measured gravimetrically on seven portions of each lung. Finally without the base-line films for comparison. We directly measured the change in opacity of the films with a radiographic densitometer. When a dog's mean extravascular water/g dry lung was increased by more than 35% it was invariably recognized, in one or more zones, as definite oedema. Control dogs were reliably recognized as normal when base-line films were used but the distinction between normal and minor degrees of oedema could not be made without the base-line films. There was a positive correlation between radiological grade and lung water, but a great deal of overlap between grades. Densitometry was not a sensitive or reliable method for diagnosing or quantifying oedema. Oedema was usually associated with a decrease in volume of the lower lung zones.

The mechanism of sudden cardiac death following acute fluoride intoxication has been thought to result from profound hypocalcemia produced by the precipitation of calcium fluoride salts. In studies of a canine model, the onset of lethal ventricular arrhythmias was temporally more associated with an elevation of serum potassium than with a drop in serum calcium. Fluoride-induced hyperkalemia could not be prevented with glucose, insulin, or bicarbonate. In the erythrocytes, a five-minute exposure to 10 mM NaF caused a 50% increase in extracellular potassium concentrations after 12 hours compared to control erythrocyte suspensions (P less than .001). The total potassium efflux after 12 hours of incubation was linearly related to the log of fluoride contact time (r, 0.886; P less than .001). The treatment of fluoride-induced hyperkalemia may depend on removal of fluoride and potassium.