Morphological diversity among closely related animals can be the result of differing growth patterns. The Australian radiation of agamid lizards (Amphibolurinae) exhibits great ecological and morphological diversity, which they have achieved on a continent-wide scale, in a relatively short period of time (30 million years). Amphibolurines therefore make an ideal study group for examining ontogenetic allometry. We used two-dimensional landmark-based geometric morphometric methods to characterise the postnatal growth patterns in cranial shape of 18 species of amphibolurine lizards and investigate the associations between cranial morphology, and life habit and phylogeny.Ctenophorus includes species that undergo small magnitudes of shape change during growth. They have dorsoventrally deep, blunt-snouted skulls , and also dorsoventrally shallow skulls (associated with saxicolous lifestyles). The sister clade to Ctenophorus, which includes the bearded dragon (Pogona), frill-neck lizard (Chlamydosaurus), and long-nosed dragon (Gowidon), exhibit broad and robust post-orbital regions and differing snout lengths .For most amphibolurine species, juveniles share a similar cranial phenotype, but by adulthood crania are more disparate in shape and occupy different sub-spaces of the total shape space. To achieve this disparity, crania do not follow a common post-natal growth pattern; there are differences among species in both the direction and magnitude of change in morphospace. We found that these growth patterns among the amphibolurines are significantly associated with ecological life habits. The clade Australian agamids show great variability in the timing of development and divergence of growth trajectories which results in a diversity of adult cranial shapes. Phylogenetic signal in cranial morphology appears to be largely overwritten by signals that reflect life habit. This knowledge about growth patterns and skull shape diversity in agamid lizards will be valuable for placing phylogenetic, functional and ecological studies in a morphological context.The online version of this article (10.1186/s12862-018-1335-6) contains supplementary material, which is available to authorized users. The role of natural selection in producing morphological variation is limited by the changes that can be generated by organism-specific processes of growth and development (ontogeny) –4. In eaThere are two ways in which changes to an ancestral post-natal growth pathway can generate morphological diversity. Firstly, changes in adult shape can occur simply by changing adult size, without altering the shape-size relationship. Modifications in timing or rate of the ancestral growth pathway (heterochrony) account for diversification of shape. Such heterochronic changes, “when a descendant retains the ancestral relationship between size and shape”  p. 87), 7, 8]. SeDifferences in ontogenetic patterns have often been associated with differences in ecology, in an interplay between selective forces and developmental processes. Evolutionary radiations consisting of many closely related species provide opportunities to examine how changes to an ancestral growth pathway may have contributed to morphological diversification within a particular clade. The Australian radiation of dragon lizards, the Amphibolurinae (Agamidae), includes iconic species such as the frill-neck lizard, bearded dragon, and thorny devil. They constitute a diverse component of Australia’s reptile fauna comprising around 108 species, and probably represent the descendants of a single continental colonisation from Southeast Asia approximately 30 million years ago (Ma) –27. TheyThis study aims to investigate whether the evolution of adult cranial shape diversity among 18 species of the Amphibolurinae is achieved through heterochrony alone, without changes to the ancestral growth pathway , or through heterochrony and modification of these pathways. We use two-dimensional landmark based geometric morphometrics to characterise cranial shape, and compare the direction and magnitude of postnatal growth trajectories using a phenotypic trajectory analysis . We alsoGowidon longirostris is one example of an extreme morphotype  which has a relatively long and pointed snout, long maxillary facial process, a wide postorbital bar , small orbit, and an overall dorsoventrally shallow skull. Chlamydosaurus kingii, Intellagama lesueurii, Pogona barbata and P. vitticeps have relatively short snouts (compared to the most long snouted forms) and large, robust post-orbit elements . Interestingly, these four species are also those with the largest adult size. Ch. kingii and both species of Pogona have relatively short but pointed snouts, whereas I. lesueurii has a more rounded snout. The third extreme form of shape variation has a short and blunt snout, robust post-orbit elements, and a dorsoventrally deep overall skull profile, e.g. Ctenophorus nuchalis, Ct. reticulatus and Moloch horridus. For any particular species, the intermediate and adult ontogenetic stages occupy morphospace between the large-orbit form associated with smaller individuals , and one of these three broad morphotypes.A principal component analysis (PCA) characterising overall cranial shape across the sampled specimens shows that most of the smaller individuals have high PC1 values and low PC2 values  greater among adult crania for each species  than among juvenile crania of each species . The disparity calculated for the smallest juvenile and largest adult representatives of each species show that different agamid species begin life with a similar cranial shape and later disperse towards more disparate adult forms.Morphological disparity (Procrustes variance) for Amphibolurinae was significantly . The variation detected in the shape data by a phenotypic trajectory analysis (PTA) revealed significant differences in growth trajectories: among directions (angles) of shape change, and also among magnitudes of shape change Fig. a. For PCP-values for direction (angle) differences in the PTA are reported in Additional file Ctenophorus isolepis and Ct. nuchalis). M. horridus has a significantly different direction of shape change than all other sampled species. I. lesueurii, Ch. kingii, P. barbata, and P. vitticeps have the largest adult size, and all have similar directions of ontogenetic shape change. The species with the smallest adult sizes are Ct. isolepis, Diporiphora winneckei, Rankinia diemensis, Tympanocryptis tetraporophora, and M. horridus, and they also have mostly similar directions of ontogenetic shape change, apart from M. horridus.The direction of ontogenetic shape change varies between different species Fig. a. PairwiCh. kingii, I. lesueurii, P. barbata, P. vitticeps) have greater magnitudes of ontogenetic shape changes than other sampled agamids. Ct. isolepis shows the smallest magnitude of shape change compared with all other sampled species. Ct. cristatus, Ct. decresii, Ct. nuchalis and Ct. reticulatus all have relatively small magnitudes of shape change. In Ctenophorus, only three significant pairwise differences in magnitude were detected (both involving Ct. isolepis). Within the sister clade to Ctenophorus we detected 21 pairwise differences, mostly involving Ch. kingii and the species of Pogona, which show the largest magnitude of shape change of any species  = 128.35, P = 0.001; habit F = 39.83, P = 0.001; size*habit F = 5.24, P = 0.001). Thus, there is significant allometry in cranial shape, and the allometric slopes differ among life habit categories .Cranial shape is influenced by cranial size, life habit and the interactions of the two . The amount of phylogenetic signal is less than 1, indicating that species resemble each other less than is expected under Brownian motion. This is reflected in the dispersion of the species in the two cranial phylomorphospaces .Tests for phylogenetic signal (relative to what is expected under a Brownian motion model of evolution) in cranial shape of the smallest juveniles and the largest adults revealed that both juveniles and adults show significant phylogenetic signal in their shape . However, heterochrony is insufficient to explain the entirety of high morphological diversity of the amphibolurines. More often than not, we observe differences in both direction and magnitude of growth pathways between pairs of species, which suggests ontogenetic divergence has been a major factor in the evolution of the disparity seen in adult amphibolurine lizards.Most of the disparity in the adult cranial form of the sampled amphibolurines develops during post-hatching ontogeny. For just under half of the sampled species, we observe shape changes along a shared allometric slope, with indicates paedomorphy as a particular heterochronic pattern in a number of species of The extent to which ontogenies are conserved during evolution has been a continued topic of controversy that has influenced the development of phylogenetic methods , 39, 40;In both juvenile and adult skull shapes, phylogenetic affinity is less strongly correlated with shape than are associations with ecological groups. Although we find strong associations with ecological life habit, this does not preclude the existence of other many important factors, such as diet or patterns of interspecific selection. The weak phylogenetic signal we observe agrees with other studies on the skulls of reptiles , 46, mamOur findings add to the growing body of evidence , 37, 56 Diversity in cranial shape of amphibolurine lizards seems to be the result of a combination of heterochrony and changes in growth patterns, which are related to phylogenetic affinity and adaptive evolution. The expectation of a conserved phylogenetic pattern, as predicted by the ontogenetic scaling hypothesis, does not fully explain the variation in skull shapes, and we find compelling evidence that there is an adaptive basis for much of the variation in ontogenetic allometry that we observe. Our study emphasises the power of growth pathways for facilitating the morphological variation that is characteristic of large and speciose evolutionary radiations. It also underlines the importance of using quantitative multivariate analyses to properly appreciate the role of developmental processes in shaping phenotypic diversity across species.Material comprised 2D lateral view images of 361 specimens representing 18 different species of amphibolurine lizards  and photographed using an Olympus TG-4 camera mounted on a large flexible tripod, with a ruler set to the sagittal (midline) axis of the skull as a reference for scale. We also used 2D images of 3D rendered surface models generated from micro-Computed Tomography (CT) reconstructions of specimens in alcohol from South Australian Museum. These specimens had been micro CT scanned at either ~ 18 or ~ 9 μm resolution (depending on the size of the specimen) using a Skyscan 1076 (Bruker micro-CT) at Adelaide Microscopy. Each CT scan was reconstructed using the NRecon software interface . We usedgeomorph [Lateral cranial shape was characterised using 2D landmark-based geometric morphometrics. Landmarks were digitised on the images of the crania using tpsDig v. 2.21 . We set geomorph . This efgeomorph . The resgeomorph, and a wireframe representation of the skull, to represent shape differences between corresponding landmarks of the mean shape and minimum and maximum values for PC1 and PC2.We performed a principal component analysis (PCA) on the Procrustes aligned shape coordinates to visualise the variation among sets of landmarks in the data set. To interpret the shape differences described by the major axes of variation identified by the PCA, we plotted a morphospace (PC1 versus PC2) with points identified by species and scaled to represent centroid size. To visualise the shape variation associated with the major axes of variation, we used thin-plate spline deformation grids , producegeomorph, which estimates Procrustes variance while accounting for group size, and uses absolute differences in variances to test for pairwise differences in morphological disparity between groups. The statistical significance between the juvenile and adult groups was assessed using a randomised residual permutation test with 1000 iterations.To examine whether the morphological disparity in cranial shape among species differs between juveniles and adults, we quantified the disparity for two separate groups: the smallest three juveniles of each species (start of growth trajectory); and the largest three adults of each species (end of growth trajectory). We calculated morphological disparity using the “morphol.disparity” function in ” function from the R package geomorph, which assesses significance via distributions generated with resampling permutations (we used 1000 iterations). If the association between size and shape for a particular species was significant, this result indicated that there was an ontogenetic allometric (and not isometric) pattern present for that particular species.We determined whether any species displayed isometric growth (no change in shape with a change in size), by fitting individual regression of log-transformed size on shape for each species, using the “procD.lmgeomorph. This procedure quantifies different attributes of a shape change trajectory between two or more points, in this case we measure the attributes of shape change between two groups: juveniles and adults. To circumvent issues with estimating nearness to adulthood in a clade with such broad variation in adult body sizes, we were able to categorise each specimen as either a juvenile or adult based on the number of acrodont teeth they had. If a specimen had more than 80% of the maximum number of acrodont teeth observed for that species, they were categorised as an adult. In some cases we altered the categorisation if a specimen was missing enough teeth to hinder obtaining a count, and were able to categorise these as either juveniles or adults based on the centroid sizes observed for other specimens of that particular species. We used species as groups, and juveniles and adults as the trajectory points. This analysis involved pairwise comparisons of two different attributes: the magnitudes of the trajectories among species, and also directions of the trajectories among species. Attribute differences were evaluated from sampling distributions generated from 1000 random permutations (based on a null model that lacked coefficients for a species-transect interaction) [To test whether ontogenetic trajectories differ among species we conducted a phenotypic trajectory analysis (PTA)  on the sraction) . To visugeomorph function “procD.allometry”, with log transformed centroid size, life habit, and their interaction as model effects. Life habit was split into three categories  *species), on log transformed centroid size, and identified points by life habit.We tested for differences in ontogenetic allometric patterns of skull shape among ecological life habit groups by running a multivariate analysis of covariance (MANCOVA) model using the mult [mult using phylogenetic permutation with 1000 iterations, which is calculated by permuting the multivariate shape data of the specimens among all tips of the phylogenetic tree. This was done using the “physignal” function in geomorph. To visualise how shape variation among species is associated with phylogeny we carried out separate PCAs on the landmark data for the mean shape of the three smallest juveniles and mean shape of the three largest adults, and used the phylomorphospace approach to project a phylogeny into the juvenile and adult PC biplots , implemented with the function “phylomorphospace” in the R package phytools [We inferred an evolutionary tree using Hugall et al., Melville et al., and Pyron et al. , 29, 68,mult , which imult . We detephytools .Additional file 1:Table S1. Pairwise results for angles in phenotypic trajectory analysis. Upper triangle shows pairwise p-values, while lower triangle shows pairwise angle differences (red highlighted cells indicate p < 0.05). Table S2. Pairwise results for magnitude in phenotypic trajectory analysis. Upper triangle shows pairwise p-values, while lower triangle shows pairwise magnitude differences (red highlighted cells indicate p < 0.05). (XLSX 19 kb)Additional file 2:Table S3. Examining allometry: MANCOVAs of cranial shape predicted by size and life habit (shape ~ log(size)*habit). Table S4. Examining allometry of life habit groups: pairwise angle and length differences. Upper triangle = p-values. Lower triangle = angles. (DOCX 15 kb)Additional file 3:Figure S1. Images of A. muricatus with landmark numbers which correspond to landmark definitions found in Additional file Additional file 4:Table S5. Landmark definitions used in geometric morphometric analysis . (DOCX 12 kb)


Aceria tosichella, a major pest of the world’s grain industry, to identify the factors behind its extensive diversification.Understanding the mechanisms that underlie the diversification of herbivores through interactions with their hosts is important for their diversity assessment and identification of expansion events, particularly in a human-altered world where evolutionary processes can be exacerbated. We studied patterns of host usage and genetic structure in the wheat curl mite complex (WCM), We expanded on previous phylogenetic research, demonstrating deep lineage diversification within the taxon, a complex of distinctive host specialist and generalist lineages more diverse than previously assumed. Time-calibrated phylogenetic reconstruction inferred from mitochondrial DNA sequence data suggests that lineage diversification pre-dates the influence of agricultural practices, and lineages started to radiate in the mid Miocene when major radiations of C4 grasses is known to have occurred. Furthermore, we demonstrated that host specificity is not phylogenetically constrained, while host generalization appears to be a more derived trait coinciding with the expansion of the world’s grasslands. Demographic history of specialist lineages have been more stable when compared to generalists, and their expansion pre-dated all generalist lineages. The lack of host-associated genetic structure of generalists indicates gene flow between mite populations from different hosts.Our analyses demonstrated that WCM is an unexpectedly diverse complex of genetic lineages and its differentiation is likely associated with the time of diversification and expansion of its hosts. Signatures of demographic histories and expansion of generalists are consistent with the observed proliferation of the globally most common lineages. The apparent lack of constrains on host use, coupled with a high colonization potential, hinders mite management, which may be further compromised by host range expansion. This study provides a significant contribution to the growing literature on host-association and diversification in herbivorous invertebrates.The online version of this article (10.1186/s12862-018-1234-x) contains supplementary material, which is available to authorized users. Niche breadth varies significantly among species, with life history traits, physical tolerances, and evolutionary adaptations directly influencing the range of habitats which species can successfully colonize and thrive within , 2. ConsRhagoletis pomonella being the most well-known example )  tulipae , and thiWe identified some discrepancies in lineage delineation between the mitochondrial and nuclear DNA datasets, a phenomenon frequently reported in many animal taxa –86. The Our research has characterized WCM as a complex of at least seven specialist, six generalist, and two semi-specialist taxa, with specificity levels yet to be assigned for several other distinct genetic lineages. We expect that semi-specialist lineages MT-12 and MT-13 are in fact host specialists, given they occur predominantly on single grass host species, and only sporadically on other hosts. This is in concordance with previous research on host-related variation in population density of WCM lineages, which has indicated that MT-12 and MT-13 exhibit preferences toward one host species , although the lineages have been found on the other hosts sporadically .Our phylogenetic reconstructions also indicate that neither host generalization nor specialization is a phylogenetically constrained trait, as the tree topology provides clear evidence of their convergent evolution in unrelated taxa. Shifting patterns of host generalization to specialization between closely-related lineages suggest that host range expansion and contraction is likely to be dynamic through time. The phenomenon has been well documented and can be attributed to environmental changes, including anthropogenic influence , 12. AltVanessa cardui [Related phytophagous insect species tend to feed on plants from similar taxonomic groupings –92. Howea cardui , leaf bea cardui  and monoa cardui . These fWCM is inherently associated with grass hosts which are intensively farmed across multiple continents. Therefore, the global distribution of WCM has been previously attributed to the extension of agriculture around the globe , and herOur analyses suggest that some generalist WCM lineages have undergone major historical demographic expansion, but again the timing of these events precedes the potential influence of agriculture. Generalist lineages associated with wild grasses appear to have underwent much earlier demographic expansions during the Pleistocene 160,000–290,000 years ago. In contrast, the most widespread generalists MT-1 and MT-8, which nowadays predominate on cereal hosts, appear to have undergone expansion events more recently, approximately 27,000 and 86,000 years ago, respectively. It is likely that during the time of recent expansion MT-1 and MT-8 lineages infested wild grasses, since many cereal hosts are no older than 11,000 years old . It is pIn contrast, the demographic history of specialists MT-7  and MT-9 (associated with smooth brome) appears to have been more stable with evidence of expansion events predating all generalist lineages . Expansions seem to be more likely in generalists rather than in specialists, probably given their enhanced dispersal capacities and abilities to capitalize on host availability on greater geographic scales , 101.High host-specificity is a prevalent feeding strategy in eriophyoid mites , therefoBemisia tabaci [Our research focuses on patterns of genetic diversity and host association in obligatory phytophagous wheat curl mite (WCM), which is a major agricultural pest in cropping regions of the world. We revealed that lineage diversity within the taxon is significantly greater than was assumed, and consists of at least 29 genetically divergent lineages with distinctive host-use patterns, ranges from host generalists to specialists. The timing of WCM diversification events pre-dates agriculture, instead appears to be associated with timing of global diversification events within Poaceae during the mid-Miocene and the Pliocene. These novel findings provide insight into co-evolutionary processes among herbivores and their hosts, and revoke hypothesis that WCM lineage diversity is primarily linked to human activity, similarly like it has been shown for whitefly a tabaci .Furthermore, our results shed new light on the mechanisms associated with specialization and generalization, each appearing to have evolved convergently in unrelated WCM lineages on numerous occasions. Such a pattern supports the notion that host use strategy can be highly dynamic in herbivores , 105, alAdditional file 1:Sequence and sampling information. (DOCX 99 kb)Additional file 2:Phylogenetic trees for wheat curl mite sequence datasets. (DOCX 776 kb)Additional file 3:Statistics for genetic diversity of WCM genetic lineages. (DOCX 738 kb)Additional file 4:Mismatch distributions of WCM genetic lineages. (DOCX 747 kb)Additional file 5:Haplotype networks for the mtDNA Cox1 wheat curl mite (WCM). (DOCX 950 kb)


Cranial nerve palsies are well-known complications of basal meningitis, especially in patients with tuberculous meningitis. However, a minority of bacterial meningitis gets complicated with cranial nerve palsies. Although cerebral infarctions are known to occur with acute bacterial meningitis, infarctions occurring in the brainstem are infrequently described.  We report a 46-year-old healthy female who presented with dysarthria with fever, headache, and vomiting and was diagnosed to have acute pyogenic meningitis complicated with a brainstem infarction resulting in bilateral hypoglossal palsy. Her MRI revealed an infarction in the lower part of the medulla oblongata, probably involving the bilateral hypoglossal nuclei. Isolated bilateral hypoglossal nerve palsy is an extremely rare cranial nerve palsy, secondary to pyogenic meningitis. To our knowledge, this should be the first reported case of isolated bilateral hypoglossal nerve palsy due to a brainstem infarct in the background of pyogenic meningitis. Despite the current advanced treatment, meningitis still carries a substantial morbidity and mortality . Fever, We herewith report a rare case of bilateral hypoglossal nerve palsy due to posteromedial infarction in the medulla oblongata secondary to acute bacterial meningitis. As far as we are aware, this is the first reported case of isolated bilateral hypoglossal nerve palsy, secondary to acute bacterial meningitis.A 46-year-old previously healthy female developed an insidious onset severe persistent headache, most prominent in the occipital region lasting for 10 days. Six days after the onset, she experienced dysarthria and a difficulty in moving her tongue within the mouth with a difficulty in eating and drinking. She did not complain of nasal regurgitation of food or nasal quality of speech. After admission, she was found to have a high-grade fever. She was otherwise healthy and denied symptoms of cough, decreased appetite, weight loss, or past history of tuberculosis. On admission, she was found to be ill with elicitable neck stiffness. Neurological examination revealed bilateral hypoglossal nerve palsy with marked tongue atrophy, more prominent in the left side  with tonµL; 92.2% of neutrophils) with elevated ESR (100 1st Hr) and CRP (195 u/L). Her blood cultures were negative. Noncontrast CT brain did not reveal any abnormality. Cerebrospinal fluid (CSF) biochemistry revealed significant elevation of protein (111 mg/dL) with 59 polymorphs and 8 lymphocytes per cubic millimetre with reduced CSF glucose (29 mg/dL). CSF for GeneXpert for tuberculosis and staining for acid-fast bacillus (AFB) and fungal and atypical cells were negative. Pyogenic, mycobacterial, and fungal CSF cultures were negative and CSF for Meningococcus, Haemophilus, and Pneumococcus antigens were also negative. Her chest radiograph did not reveal any changes suggestive of pulmonary tuberculosis or sarcoidosis. Syphilis (VDRL & THPA), HIV serology, and autoimmune markers for vasculitis , and p & c-ANCA) were negative.Her blood tests revealed a haemoglobin of 12.5g/dl with a neutrophil leukocytosis  is an uncommon cranial nerve palsy comparing to other commoner cranial nerves such as 3rd, 6th, and 7th cranial nerves . Kean et Neisseria Meningitidis complicated with obstructive hydrocephalus who had isolated unilateral HNP which resolved over 5 months [Rocholt et al. described a patient with meningitis due to5 months . AlthougA recent retrospective study was conducted in patients affected with acute bacterial meningitis (ABM) or tuberculous meningitis (TBM); the predictive value of each neurological sign on the diagnosis of above was analysed. They observed that the presence of cranial nerve palsies was the most important neurological predictor favouring TBM over ABM  . Since tWith treatment, our patient showed a significant clinical improvement and inflammatory markers became normal. In addition, repeat CSF analysis two weeks later showed complete resolution of CSF abnormalities. All these findings stabilized the diagnosis of acute pyogenic meningitis. We attributed the uncommon presentation of bilateral tongue involvement to a posteromedial medullary infarction which was observed in the MRI scan of the brain .For several decades, attention has been paid to the neurological complications of meningitis including cerebral infarctions. Diederik van de Beek et al. in a review described that 15-20% of community-acquired bacterial meningitis is complicated with infarctions of the brain matter .According to another Danish population-based cohort study, 14% infarctions were observed in the course of ABM . Many siApart from the involvement of bilateral HNP, our patient had other brainstem signs such as saccadic eye movements and cerebellar ataxia which got completely resolved with treatment. According to an animal experimental study carried out to explain the pathophysiology of eye saccades, it was suggested that the saccade palsy could have resulted from an insult to the lower pons . This isIsolated bilateral hypoglossal nerve palsy is extremely rare cranial nerve palsy secondary to pyogenic meningitis. Brainstem infarcts involving the medial medullary region of the medulla oblongata can lead to a presentation of bilateral hypoglossal nerve palsy. To our knowledge, this is the first reported case of bilateral hypoglossal nerve palsy due to a brainstem infarct in the background of pyogenic meningitis.


Evolution of indolent to aggressive lymphoma has been described in dogs but is difficult to distinguish from the de novo development of a second, clonally distinct lymphoma. Differentiation of these scenarios can be aided by next generation sequencing (NGS)-based assessment of clonality of lymphocyte antigen receptor genes.An 8-year-old male intact Mastiff presented with generalized lymphadenomegaly was diagnosed with nodal T zone lymphoma (TZL) based on cytology, histopathology, immunohistochemistry and flow cytometry. Thirteen months later, the dog re-presented with progressive lymphadenomegaly, and based on cytology and flow cytometry, a large B cell lymphoma (LBCL) was diagnosed. Sequencing-based clonality testing confirmed the de novo development of a LBCL and the persistence of a TZL.The occurrence of two distinct lymphoid neoplasms should be considered if patient features and tumor cytomorphology or immunophenotype differ among sequential samples. Sequencing-based clonality testing may provide conclusive evidence of two concurrent and distinct clonal lymphocyte populations, termed most appropriately “composite lymphoma”. Lymphoma is the most common hematopoietic neoplasm in dogs and is due to clonal proliferation of lymphocytes . The cliApproaches to diagnose and classify lymphoma include cytology, histopathology, immunohistochemistry, flow cytometry, and clonality testing. Although histopathology combined with immunohistochemistry is generally sufficient for diagnosis, obtaining a tissue biopsy requires sedation or anesthesia. Aspirating lymph nodes is less invasive, and since most lymphomas in dogs have diffuse architecture, representative samples for diagnosis are readily obtained. Aspirated samples are also suitable for flow cytometric characterization and, therefore, prognostication . In caseThis is the first report of a dog with concurrent TZL and LBCL diagnosed using multiple diagnostic approaches. Clinical features and tumor cytomorphology suggested emergence of a high-grade lymphoma in a patient with a pre-existing indolent TZL. Flow cytometry confirmed the initial subtype of TZL and subsequent LBCL, and NGS-based clonality testing identified two distinct clones within one affected lymph node.An 8-year-old male intact Mastiff presented to a veterinary specialty hospital with an acute onset of head shaking. Magnetic resonance imaging (MRI) of the head revealed an intra-axial lesion within the right pyriform lobe. The lesion was T2/FLAIR hyperintense, T1 hypointense, and non-contrast enhancing with a mild mass effect, consistent with a primary neoplasm such as glioma. The dog was subsequently referred to the Ontario Veterinary College Animal Cancer Centre, and definitive radiation therapy consisting of 18 fractions was administered over 4 weeks at a total dose of 45 Gray.At a re-check appointment 7 months after completion of radiation therapy, the dog was free of neurological signs while receiving solely oral phenobarbital  but physical examination revealed mild generalized lymphadenomegaly. Results of a complete blood cell count (CBC) and serum biochemistry analysis were unremarkable. Fine needle aspirates of three peripheral lymph nodes yielded slides with a relatively uniform population of medium size lymphocytes with pale cytoplasm, central or slightly eccentric nuclei and frequent cytoplasmic projections. The findings were interpreted as lymphoma, and the cytomorphology was consistent with TZL   was initiated with the intent to alleviate edema and hydrocephalus. The dog’s lethargy and dullness improved; however, reduction in prednisone dosage over the subsequent weeks resulted in recurrent malaise. Therefore, a maintenance prednisone dosage of 0.7 mg/kg q48h was prescribed.2 q24h) was added to prednisone. Three weeks later, the owners reported that an episode of pain and weakness had occurred at home, which prompted an increase in prednisone dosage to 1 mg/kg q24h. The owners noted no further episodes.Two months later, the owner noted progressive peripheral lymphadenomegaly, which was confirmed during physical examination at the teaching hospital. The marked increase in lymph node size was interpreted as progression of the TZL, and chemotherapy with chlorambucil  due to progressive lethargy, weakness, and lymphadenomegaly. An necropsy was not performed.At a recheck assessment 6 weeks following the start of chlorambucil therapy, lymphadenomegaly was unchanged. The dosage of chlorambucil was increased to 20 mg/mTo investigate a potential relationship between the initial TZL and subsequent LBCL, NGS-based clonality testing was performed after euthanasia using DNA extracted from lymph node samples collected at the time of diagnosis of each lymphoma Figs. c, and clThis report describes the emergence of LBCL in a dog with pre-existent and persistent TZL. In human oncology, presence of two distinct, clonally unrelated, lymphomas within the same organ is termed “composite lymphoma”. This entity comprises < 5% of all lymphomas in humans , but hasDifferentiating relapse from development of a de novo tumor is challenging for most cancer types. Lymphoid cancers differ in this regard because every lymphocyte clone carries a unique DNA sequence that can be used as a genetic fingerprint to track lymphocyte clones over time and across anatomic sites. This unique gene sequence is generated early in lymphocyte development by rearrangement of antigen receptor genes, and confers every lymphocyte clone with unique antigen specificity. Clonality testing assesses the diversity of lymphocyte antigen receptor genes in a given lymphocyte population. In the initial sample, clonality testing confirmed the diagnosis of TZL based on the presence of clonal TRB and TRG rearrangements. A single productive TRB rearrangement and two unproductive TRG rearrangements were consistent with an alpha/beta T cell lineage of the neoplastic clone, and usage of TRGV2/TRJ3–2 by both dominant clones was suggestive of a bi-allelic rearrangement rather than the rearrangement of two different cassettes on the same chromosome. The same rearrangements were found in the second sample at similar abundance, which suggests persistence of the neoplastic T cell clone in the face of treatment. In addition to the clonal TRB and TRG rearrangements, the second sample showed a dominant IGH clone that comprised about 88% of all rearrangements. This finding not only confirms the diagnosis of a B cell lymphoma but also suggests that the B cell lymphoma is a de novo tumor rather than a progression of the TZL with altered immunophenotype. In contrast to cell surface marker expression, which can be influenced by micro-environmental stimuli and a cell's developmental stage and viability, lymphocyte antigen receptor gene rearrangements are stable throughout the life of a lymphocyte . ConsequThe use of sequencing-based clonality testing provided a distinct advantage over electrophoresis-based methods. Traditionally, clonality testing utilizes gel electrophoresis to visualize the diversity of lymphocyte antigen receptor gene arrangements in a given sample. Since this method only distinguishes antigen receptor genes by size, it can result in equivocal results when the signal of a neoplastic clone is quenched by noise of non-neoplastic lymphocytes. Sequencing-based clonality testing yields a higher ‘clonal resolution’ because it can distinguish lymphocyte clones based on sequence on top of size , 19. In Although NGS was useful to identify the presence of two distinct clones in this case, other diagnostic approaches were also necessary. An entire affected lymph node was initially removed and assessed histopathologically and immunohistochemically to confirm the cytological diagnosis of TZL. Sections consisted of a homogeneous population of small lymphocytes with rare remnant follicles. Cytologic evaluation of an aspirate of the contralateral popliteal lymph node collected 1 year after the initial sample identified cells morphologically consistent with LBCL rather than TZL, which prompted immunophenotypic assessment. Flow cytometry confirmed LBCL, which in virtually all cases in dogs is a DLBCL . AlthougIt is possible that the dog in this report had an increased risk of developing secondary neoplasms following radiation of the brain tumor. Radiation therapy induces a multitude of adverse effects, including systemic effects from local therapy . Such efTreatment and prognosis of composite lymphoma in humans varies depending on histological subtype . Canine The patient had been diagnosed with an intracranial mass most consistent with glioma several months prior to the first diagnosis of lymphoma. Without histopathologic evaluation, non-neoplastic causes of brain masses such as vascular events or granulomatous inflammation cannot be entirely ruled out. However, the clinical features and MRI characteristics of intra-axial location, T2/FLAIR hyperintensity, T1 hypointensity, lack of contrast enhancement, and mass effect, were most suggestive of a neoplasm such as a low-grade glioma –29. DefiLimitations to this investigation were the unavailability of a biopsy from the lymph node with concurrent TZL/LBCL, and lack of post mortem assessment. Histopathology and immunohistochemistry of the second lymphoma would have allowed more definitive diagnosis of LBCL, and illustrated morphological findings associated with concurrent TZL and LBCL. Similarly, post mortem evaluation would have allowed conclusive identification of the brain lesion and the extent of the lymphoma. Nevertheless, evidence for a composite lymphoma in this case was considered very strong based on multiple sophisticated and complementary diagnostic approaches.In conclusion, this report of a composite lymphoma in a dog highlights the value of multiple diagnostic approaches to differentiate between two de novo lymphomas rather than transformation of a single clone.Additional file 1: Table S1. Results of complete blood count and serum biochemistry.Additional file 2: Table S2. Primer Sequences.Additional file 3. Materials and Methods.


Brezinacantha tolis gen. et sp. nov., preserved as articulated skeletons from an upper Campanian (Late Cretaceous) methane seep of South Dakota. It is uniquely the first fossil case of a seep-associated ophiuroid. The articulated skeletons overlie centimeter-thick accumulations of dissociated skeletal parts, suggesting lifetime densities of approximately 1000 individuals per m2, persisting at that particular location for several generations. The ophiuroid skeletons on top of the occurrence were preserved intact most probably because of increased methane seepage, killing the individuals and inducing rapid cementation, rather than due to storm-induced burial or slumping. The mass occurrence described herein is an unambiguous case of an autochthonous, dense ophiuroid community that persisted at a particular spot for some time. Thus, it represents a true fossil equivalent of a recent ophiuroid dense bed, unlike other cases that were used in the past to substantiate the claim of a mid-Mesozoic predation-induced decline of ophiuroid dense beds.Articulated brittle stars are rare fossils because the skeleton rapidly disintegrates after death and only fossilises intact under special conditions. Here, we describe an extraordinary mass occurrence of the ophiacanthid ophiuroid  Most of the time, however, ophiuroids tend to live a cryptic life hidden under rocks, inside sponges, epizoic on corals or buried in the mud (e.g.2) to such a point that their real abundance is rarely appreciated at first sight.Brittle stars, or ophiuroids, are among the most abundant and widespread components of the marine benthos, occurring at all depths and latitudes of the world oceans8). One of the most remarkable examples of a recent ophiuroid mass occurrence was discovered on the Macquarie Ridge chain of Seamounts in 2008 as part of the Census of Marine Life initiative: dubbed “Brittle star city”, the spectacular mass aggregation of ophiuroids was met with wide academic and public interest. That same year, the flanks of the Admiralty Seamount in Antarctica yielded a similarly impressive but smaller ophiuroid mass occurrence, consequently called “Brittle star village”9.As a result, openly visible mass occurrences of brittle stars raise attention and often incite scientific studies to explore the ecological factors explaining the alleged aberrant abundance (e.g.15) have shaped the hypothesis that dense ophiuroid populations were common in shallow waters until the Jurassic and then significantly declined by the end of the Mesozoic (e.g.16). In combination with an assumed lower predation pressure in the Jurassic16, ophiuroid mass aggregations gained key witness status for the so-called Marine Mesozoic Revolution: a hypothesised causal connection between the evolution of durophagous predators and the Mesozoic decline of epifaunal suspension-feeder communities18. Although recent studies suggest that ophiuroid mass aggregations in modern oceans are neither rare nor restricted to low-predation areas (e.g.19), such communities are still commonly considered archaic benthic assemblages20.Interest in dense ophiuroid aggregations has gone far beyond the mere ecological background of the occurrences. Assumed fossil equivalents (e.g.21) and the first from the fossil record. Second, it is an unambiguous case of an autochthonous, dense ophiuroid community that persisted at a particular spot during several generations, thus representing a true fossil equivalent of a recent ophiuroid dense bed. As such, it casts doubt on several other cases of assumed fossil ophiuroid dense beds used in the past to substantiate the predation-induced decline of ophiuroid dense beds during the mid-Mesozoic (e.g.22). Finally, it significantly adds to the very sparse Mesozoic ophiuroid fossil record of the United States and the Western Interior Seaway in particular, currently counting as few as 9 species in total29. Apart from the systematic assessment of the material, we elucidate its intriguing taphonomy and discuss its significance for the interpretation of other fossil ophiuroid mass aggregations.We here describe an extraordinary fossil ophiuroid occurrence from the Late Cretaceous of South Dakota. The material is outstanding in several aspects. First, it represents one of the very few seep-associated ophiuroids known to date  of the U.S. Western Interior since the late 1800 s and have been extensively studied over the last several decades37. The presence of methane was confirmed through isotopic analyses of the carbonate cements39. Methane gas probably migrated along faults and fractures that developed in the Late Cretaceous prior to the uplift of the Black Hills during the Laramide Orogeny35. Dewatering of the thick, black shales (hundreds of meters thick) due to the weight of the water and sediments also, most likely, contributed to this process35.Around the Black Hills, these deposits were formed from methane enriched fluids escaping from underlying layers of the Pierre, Niobrara, Belle Fourche, Mowery and Greenhorn Formations41. Seeps may have represented semi-permanent self-sustaining habitats that were inhabited by a unique group of animals that lived on the nutrients provided by symbiotic, sulfur-oxidizing chemosynthetic bacteria42.Carbonate by-products precipitated out of solution as a result of anaerobic oxidation of methane thus allowing carbonate cementation to occur below the sediment water interface and in and around the fauna at the seeps that might have formed small, elevated mounds on the seafloor, creating refuges in an otherwise less than friendly environmentDidymoceras cheyennense and Baculites compressus zones around the Black Hills but are still mostly rare at seeps. Both regular and irregular echinoids have been found along with stalked crinoids41, feather stars (in preparation), asteroids42 and ophiuroids (this paper). An abundant food source and a hard substrate in an otherwise soft, muddy ocean bottom were most likely the key factors for echinoderms to habitat the seeps.Echinoderms seem more common in the Didymoceras cheyennense Zone, upper Campanian, Late Cretaceous  , Hoploscaphites brevis , H. nodosus , Didymoceras cheyennense , Spiroxybeloceras meekanum  and Baculites corrugatus ), nautilus ), ghost shrimp , crabs , oysters, lucinids, inoceramids and other miscellaneous bivalves, gastropods, crinoids , echinoids (regular), ophiuroid (this paper), tube worms, radiolarians, foraminifera, dinoflagellates, a mosasaur vertebra (Plioplatecarpus) along with a few miscellaneous fish vertebrae.AMNH 3509A has a rich, varied fauna that contains: ammonites  and the Natural History Museum Luxembourg (MnhnL).Selected ophiuroid skeletal plates were mechanically removed from one slab, mounted on a stub and gold-coated for Scanning Electron Microscopy using a Jeol Neoscope JMC-5000. Terminology follows Stöhr http://zoobank.org/”. The LSID for this publication is: urn:lsid:zoobank.org:pub:3A98CFE8-7F81-4906-9D41-072CF02D61C5; for Brezinacantha: urn:lsid:zoobank.org:act:621CED39-46AF-4B98-B531-0CC362AAEE3E; for Brezinacantha tolis: urn:lsid:zoobank.org:act:BABFCFB5-F991-44EF-8DB6-A1A701187BCB. The electronic edition of this work was published in a journal with an ISSN, and has been archived and is available from the following digital repositories: PubMed Central, LOCKSS.The electronic edition of this article conforms to the requirements of the amended International Code of Zoological Nomenclature, and hence the new names contained herein are available under that Code from the electronic edition of this article. This published work and the nomenclatural acts it contains have been registered in ZooBank, the online registration system for the ICZN. The ZooBank LSIDs (Life Science Identifiers) can be resolved and the associated information viewed through any standard web browser by appending the LSID to the prefix “Class: Ophiuroidea Gray, 1840Subclass: Myophiuroidea Matsumoto, 1915Infraclass: Metophiurida Matsumoto, 1915et al., 2017Superorder: Ophintegrida O’Hara et al., 2017Order Ophiacanthida O’Hara et al., 2017Suborder: Ophiacanthina O’Hara Family: Ophiacanthidae Ljungman, 1867Brezinacantha gen. nov.Genus: Diagnosis: as for speciesBrezinacantha tolis™Type and only known species: Etymology: Genus named after Jamie Brezina  who discovered the ophiuroid specimens described herein, to honour his indefatigable commitment to the study of seep deposits and their fossil content in the Western Interior Seaway; suffix derived from ‘Acantha’, a Greek nymph whose name literally translates into “thorny”; gender feminine.Brezinacantha tolis sp. nov.Etymology: Species named after Sakis and Themis Tolis, founding members of a Greek Black Metal band, to honour their unorthodox yet powerful approach to music. The ophiuroids described herein merit in many ways a Black-Metal-referenced name: they gathered in great numbers in a spooky, toxic environment on top of their dead predecessors’ remains. The species name is entirely the responsibility of the lead author, Ben Thuy.Holotype: AMNH 113563Didymoceras cheyennense Zone, upper Campanian, Late Cretaceous, Pierre Shale, Pennington County, South Dakota.Type locality and stratum: AMNH locality 3495 (WPT 130a), Diagnosis: Ophiacanthid with very large triangular radial shields accounting for three quarters of the disc radius, interradially separated by a narrow band of tiny, radially elongate scales; interradii not indented; two to three apical oral papillae; small, narrow adoral shields; distal portion of lateral arm plates strongly elevated, separated from remaining outer surface by a poorly defined, wavy ridge; up to five arm spines as long as one and a half arm segments, round in cross section, devoid of thorns.Paratypes: AMNH 113564–113568, MnhnL OPH039 - 040Other material examined: AMNH 113569–113585, AMNH 82689–82691, MnhnL OPH041 - 043.Description of holotype: AMNH 113563 Fig.  is an arArms moderately wide, with first two segments covered by disc; first ventral arm plate wider than long, oval; following ventral arm plates nearly two times larger, nearly as wide as long, widest distally, with convex distal edge, deeply concave lateral edges and obtuse proximal angle; ventral arm plates separated by lateral arm plates from first arm segments onwards; no spurs or conspicuous ornamentation discernible. Lateral arm plates with conspicuously bulging distal portion sharply offset with respect to proximal portion of plate; elevated distal portion with at least four large, freestanding, ear-shaped spine articulations; stereom of lateral arm plate outer surface with slightly protruding trabecular intersections arranged in faint vertical rows; ventral portion of lateral arm plate not protruding ventro-proximalwards; spine articulations composed of narrow and strongly arched dorsal and ventral lobes proximally merged to form a circle around a large muscle opening, separated from much smaller nerve opening by sigmoidal fold; spine articulations increasing in size dorsalwards; large, conical, pointed arm spines at least as long as one arm segment. Tentacle openings small, covered by single oval tentacle scale.Paratypes: MnhnL OPH039 Fig.  is a fraAMNH 113564 Fig.  is a fraMnhnL OPH040 Fig.  is a disAMNH 113565 Fig.  is a disAMNH 113566 Fig.  is a disAMNH 113567 Fig.  is a disAMNH 113568 Fig.  are two Ophioplinthaca Verrill, 1899. In this genus, however, the interradii are strongly indented and the radial shields are interradially bordered by a series of large, crescent-shaped scales (e.g.45).Remarks: The shape of the spine articulations and their dorsalward increase in size clearly place the present specimens in the ophiacanthid suborder Ophiacanthina. Within this group, the single row of lateral oral papillae, the absence of a ventral tooth cluster, the non-fragmented dental plate, the ornamentation of the outer lateral arm plate surface and the non-protruding ventral portion of the lateral arm plates suggest closest affinities with the family Ophiacanthidae. The excessively large radial shields in combination with the strongly elevated distal portion of the lateral arm plates and the relatively long arm spines place the present ophiuroids close to extant Ophiosternle crinitum  from the Late Jurassic of Germany46, especially with respect to the size and position of the radial shields that are interradially separated by tiny scales. In the present specimens, however, the ventral tips of the jaws lack a well-defined cluster of apical papillae, the adoral shields are much narrower, the dorsal arm plates are smaller and not overlapping, and the arm spines are smaller and less numerous, thus precluding assignment to Ophiosternle. We thus suggest the new taxon Brezinacantha tolis to accommodate the ophiuroids described herein. With respect to the relevant morphological similarities, Brezinacantha seems the most closely related to Ophioplinthaca and Ophiosternle, and we anticipate close phylogenetic ties between the three.Remarkable similarities are shared with the genus 47). Yet, the site of discovery shows no sedimentary evidence for such an obrution event. Microbial and bacterial mats are a common feature of cold methane seeps and were highlighted as a possible factor in the preservation of partially articulated crinoids at neighboring seep locations41. Another very likely factor to explain the exceptional preservation of the ophiuroid skeletons is rapid carbonate cementation related to anaerobic oxidation of methane, known to enable exceptional fossil preservation in seeps (e.g.49). Considering the speed of post-mortem disarticulation of the ophiuroid skeleton, however, trapping and overgrowth by microbial mats and rapid cementation alone might not be sufficient to explain the here-described occurrence of fully articulated ophiuroid skeletons.The state of articulation of the ophiuroids mostly varies between the two extremes of the spectrum: fully dissociated skeletal plates on the one hand and fully articulated skeletons with no or little sign of post-mortem decay. The most likely mechanism to preserve an ophiuroid skeleton in its original anatomical disposition is rapid and effective burial either by storm-induced currents or by slumping (e.g.50). These occurrences suggest that the otherwise rapid post-mortem decay of the ophiuroid skeleton can be significantly delayed by a lack of oxygen, an assumption corroborated by yet unpublished experiments on decay of dead ophiuroids exposed to high concentrations of methane or hydrogen sulfide . We thus speculate that the ophiuroids described herein were killed by an increase in methane seepage, delaying skeletal decomposition and allowing microbial mats and rapid cementation to fossilise the skeletons intact.Interestingly, intact ophiuroid skeletons are known from sediments that lack obrution-related features but instead show signs of low oxygen levels at the time of deposition , Brezinacantha probably lifted its spinose arms in the water column to suspension feed, probably benefitting from a weak bottom water current (see above). The seep created a nutrient-rich environment with vast amounts of organic matter and thus provided abundant sources of food to sustain an extremely dense aggregation of suspension-feeding ophiuroids for several generations.Judging from the distribution of the ophiuroid fossils at the site of discovery, the dense ophiuroid aggregation covered an area of at least 3 to 4 m, representing a fossil case of a brittle-star mass aggregation. It was found in an area with bivalve and ammonite shell hash and numerous seep-associated microbialite nodules at approximately 10 m distance from the central core of the seep . In the same logic, a Mesozoic decline of brittle-star beds visible in the fossil record was postulated, in association with the diversification of durophagous predators22, except in areas with anomalously low predation (e.g.5), e.g. at high latitudes (e.g.15).Proponents of the Marine Mesozoic Revolution postulate that dominance within communities of suspension-feeding invertebrates was pushed to more cryptic, active, or heavily armoured species by increased predation pressure in the course of the Mesozoic . Others are the result of storm-induced obrution, implying that the ophiuroids might not be autochthonous but instead washed together before burial (Early and Mid-Triassic records). Finally, the Mid-Jurassic occurrence from La-Voulte-sur-Rhône, while probably representing a true fossil ophiuroid mass occurrence, originates from a bathyal rather than shallow-water setting51. Furthermore, from a methodological point of view, it should be noted that the Late Devonian records of Velbert and Angerbachtal and the Mid-Triassic records of Nordwürttemberg, Weimar and Mergentheim originate from the same depositional settings respectively, implying that they should possibly be considered as a single Late Devonian and a single Mid-Triassic occurrence.In an attempt to understand the ophiuroid mass occurrence described herein in a broader evolutionary context, we noticed that the compilation of fossil ophiuroid beds used to substantiate the assumed mid-Mesozoic decline of such occurrencesWith so many dubious, if not invalid, fossil occurrences in the original compilation, and with the number of true fossil ophiuroid dense beds described since including the present one, the hypothesis of the mid-Mesozoic decline of ophiuroid dense beds should be revisited.


We know little about the budget impact of integrating robotic exoskeleton over-ground training into therapy services for locomotor training. The purpose of this study was to estimate the budget impact of adding robotic exoskeleton over-ground training to existing locomotor training strategies in the rehabilitation of people with spinal cord injury.A Budget Impact Analysis (BIA) was conducted using data provided by four Spinal Cord Injury (SCI) Model Systems rehabilitation hospitals. Hospitals provided estimates of therapy utilization and costs about people with spinal cord injury who participated in locomotor training in the calendar year 2017. Interventions were standard of care walking training including body-weight supported treadmill training, overground training, stationary robotic systems , and overground robotic exoskeleton training. The main outcome measures included device costs, training costs for personnel to use the device, human capital costs of locomotor training, device demand, and the number of training sessions per person with SCI.Robotic exoskeletons for over-ground training decreased hospital costs associated with delivering locomotor training in the base case analysis. This analysis assumed no difference in intervention effectiveness across locomotor training strategies. Providing robotic exoskeleton overground training for 10% of locomotor training sessions over the course of the year (range 226–397 sessions) results in decreased annual locomotor training costs  between $1114 to $4784 per annum. The base case shows small savings that are sensitive to parameters of the BIA model which were tested in one-way sensitivity analyses, scenarios analyses, and probability sensitivity analyses. The base case scenario was more sensitive to clinical utilization parameters  than device-specific parameters . Probabilistic sensitivity analysis simultaneously considered human capital cost, device cost, and locomotor device substitution. With probabilistic sensitivity analysis, the introduction of a robotic exoskeleton only remained cost saving for one facility.Providing robotic exoskeleton for over-ground training was associated with lower costs for the locomotor training of people with SCI in the base case analyses. The analysis was sensitive to parameter assumptions. Between 249,000 to 363,000 persons live with disabilities due to spinal cord injuries (SCI) in the United States, and approximately 17,730 individuals experience new SCI each year [For most individuals, some form of functional limitation persists; however, improvement in mobility and function is possible following the injury. The preservation of volitional motor or sensory function below the lesion level provides an opportunity for motor recovery from intensive training during rehabilitation. This population has great potential for motor recovery and walking. Locomotor training is a standard of care for the rehabilitation of the individual with incomplete SCI. Likewise, walking is usually one of the primary goals of rehabilitation due to physiological and social benefits , 4.Conventional locomotor training strategies include those in which body-weight is supported while clinicians may manually facilitate stepping motions. This training requires multiple personnel to assist with support or lower extremity movement. Although body-weight supported treadmill training (BWSTT) and overground training (OGT) remain common locomotor training strategies, alternative strategies using robotic therapy (RT) devices require less human capital and allow greater repetition than typically is delivered in personnel-intensive approaches . RT may Robotic exoskeletons (RT-exo) are prescription devices comprising an external, motorized orthosis that is placed over paralyzed or weakened limbs for the purpose of facilitating standing, walking, climbing stairs, and performing activities of daily living , 12. ExoP < .00001) and endurance  in robot-assisted training groups [Multiple randomized trials have found outcomes associated with RT to be no different from conventional therapy when similar doses are compared –20;; howg groups . Additiog groups . Tefertig groups , 22. Wheg groups , 23. Salg groups .RT-exo shares many of the features of treadmill-based RT devices while allowing greater user autonomy and fewer environmental restrictions than treadmill-based RT devices . A recenOn average new technologies improve health outcomesMany new technologies do not improve health outcomes and it is not always easy to discriminate between effective and ineffective technologies.On average new technologies add to health care costsDiffusion of new technology often is inefficientDemand for new technology almost always is high.New technology in health care is a substantial contributor to rising costs,  creatingThe rapid growth and development of robotic devices is an excellent example of such a technology in rehabilitation. The costs of robotic exoskeletons include a high initial purchase price (relative to competing rehabilitation technology), annual maintenance costs, and training costs per physical therapist user. Whereas many new technologies can be cost-inefficient, others have been shown to be cost-neutral or cost-saving . It has We know little about the budget impact of integrating RT-exo devices into routine therapy services. Carpino et al. conducted a review and model-based cost-effectiveness analysis of lower extremity robotics, showing RT-exo to be cost-effective relative to conventional care and stationary robotics; however, model assumptions have been criticized . MorrisoEstimation of the budgetary impact of a new intervention involves a comparison of the “world without” against the “world with” an RT-exo locomotor strategy. The net difference in estimated health-care costs between these two scenarios constitutes the budgetary impact of the intervention. The BIA framework takes into consideration health system features, population eligibility, and resource use in order to understand what current and future costs are for the treatment of a given condition Fig. .Fig. 1BFigure The perspective of the analysis is from the rehabilitation facilities, specifically Inpatient Rehabilitation Facilities and Long Term Care Hospitals, as the purchasers of technology. A retrospective pre-post study design was implemented to estimate the treatment cost of locomotor strategies. The budget impact of RT-exo was evaluated over one year. The calculation was replicated in a Microsoft Excel-based spreadsheet. Probabilistic sensitivity analysis was conducted using R Statistical Software .Four SCI Model Systems agreed to collaborate on this project:  The Shirhttps://www.bls.gov/bls/blswage.htm) according to the model system facility location.The base case scenario represents the most likely or preferred set of assumptions, and sensitivity analyses explore deviations for this estimate . Our basTable Table Several parameters are likely to influence the RT-exo BIA, including device cost, the choice of locomotor strategy for substitution, and the efficiency of the use of RT-exo. One way sensitivity analyses will vary the following parameters: cost of robotic exoskeleton , adoption rate of robotic exoskeleton device (100–10%), training strategy substituted , and exoskeleton device life (8 year device life – 3 year device life). A probabilistic sensitivity analysis was performed where uncertainty across all parameters was considered simultaneously. For the probabilistic sensitivity analysis, we considered the uncertainty around human capital cost. Finally, we also tested the assumption of varying conventional locomotor training strategies by 10% (without the inclusion of RT-exo) to assess the influence of varying conventional locomotor strategies on budget impact as scenario analysis.In the base case scenario for all hospital systems, offering RT-exo for locomotor training decreased hospital costs associated with delivering locomotor training Tables  & 4. ProThe base case scenario was sensitive to several parameters, such as the cost of a robotic exoskeleton, efficiency of robotic exoskeleton use, training strategy substituted, and exoskeleton device life. Fig. We explored additional scenario analyses where, instead of choosing to introduce RT-exo, we shift market share to each of the conventional locomotor training strategies by 10%, e.g., in Facility A we assess how locomotor training costs change after we shift from 33 to 43% BWSTT. When stationary robotic and BWSTT are each increased by 10% locomotor training costs increase at all facilities. This is because of the higher personnel cost in BWSTT and higher device cost of stationary robotics relative to the overground training cost. When conventional over-ground training is increased by 10% it lowers costs at all facilities. Full scenario analyses are presented in The base case analysis shows small savings that are sensitive to cost or structural components of RT-exo. Despite the expense of RT-exo devices, savings related to a decrease in staffing needs and training for the therapeutic device exist. For hospital systems considering the addition of RT-exo as a locomotor training strategy, those with a higher percentage of human capital intensive strategies such as BWSTT may experience reduced costs if the introduction of RT-exo substitutes for these locomotor training interventions. However, the savings are very small with large degrees of uncertainty.In the base case scenario, we assumed the robotic overground exoskeleton adoption rate would be 50%. This estimate may be too generous given two factors, the population eligible for RT-exo and clinician acceptance of new technology. RT-exo is a new technology that clinicians could choose not to use for locomotor training despite hospital purchasing decisions. Interestingly, the base case scenario was more sensitive to assumptions on clinical utilization parameters than device-specific parameters. For example, limiting the RT-exo useful life to 3 years (versus 5) only resulted in an increase in locomotor training cost per annum of between $3847 and $7611 across facilities. However, if RT-exo was adopted at a rate of 10%, i.e., the device is idle 90% of the time (as opposed to the 50% used in our model), annual costs of locomotor training would increase between $32,096 and $52,934 across facilities. When considering all sensitivity and scenario analyses the least costly alternative is when over-ground training sessions are increased by 10%. This makes intuitive sense because device costs and personnel costs are lowest. Over-ground training is a well developed and clinically integrated technology, therefore few efficiencies can be expected from changes in technological adoption and further integration into clinical practice. Conversely, further technological development stands to improve the business case for RT-exo but barriers in adoption and integration can impair its efficiency. Issues of technological adoption warrant additional attention.Turchetti et al. provide a comprehensive overview of technology adoption with application to robotic devices in rehabilitation . The autAnother barrier to adoption is the different perspectives on the value added by the new technology suggesting the need for further economic studies that consider multiple perspectives in order to address the interests of all stakeholders . Swank aWe did not quantify several key considerations regarding RT-exo in this analysis. Hospital systems may invest in RT-exo as a means of building or maintaining their reputation as a leader in the use of cutting-edge technology. In this model, RT-exo is considered only as a substitute for locomotor training in the rehabilitation setting; however, there is potential for RT-exo to augment some community mobility. Devices such as RT-exo are likely to decrease in cost with greater competition in the market . NumerouClinicalTrials.gov Identifier: NCT03340792, NCT03477123, NCT02322125, NCT03443700). Therefore, the findings of this BIA must be interpreted with caution because we assume non-inferiority. In addition, there is limited data on the risks to users with SCI and personnel associated with locomotor training for RT-exo relative to other locomotor training strategies [This project did not take into account differences in locomotor training effectiveness between alternatives. There is no strong evidence showing the superiority or non-inferiority of RT-exo. The evidence for the comparative effectiveness of robotic overground exoskeleton for locomotor training in SCI is limited, but several pilot studies show promising results , 23, andrategies . We may The assessment of economic efficiency-with robotic exoskeleton is in its infancy. Our model provides a framework upon which economic studies can build. Utilizing RT-exo for locomotor training may be an efficient use of hospital resources if key assumptions concerning efficacy hold true and the technology is adopted within the health system; however, a large degree of uncertainty surrounds our estimates as evidence for differences in the effectiveness of among approaches is limited.


Fibrosis, cancer, and autoimmunity developing upon particle exposure have been exclusively linked with uncontrolled inflammatory processes. The critical role of inflammation is now challenged by several contradictory observations indicating that the emergence of these chronic disorders may result from non-inflammatory events. A growing number of studies reveals that micro- and nano-particles can cause exaggerated and persistent immunosuppression characterized by the release of potent anti-inflammatory cytokines (IL-10 and TGF-β), and the recruitment of major regulatory immune cells . This persistent immunosuppressive environment is initially established to limit early inflammation but contributes later to fibrosis, cancer, and infection. Immunosuppression promotes fibroblast proliferation and matrix element synthesis and subverts innate and adaptive immune surveillance against tumor cells and microorganisms. This review details the contribution of immunosuppressive cells and their derived immunoregulatory mediators and delineates the mutual role of inflammatory vs. immunosuppressive mechanisms in the pathogenesis of chronic diseases induced by particles. The consideration of these new results explains how particle-related diseases can develop independently of chronic inflammation, enriches current bioassays predicting particle toxicity and suggests new clinical strategies for treating patients affected by particle-associated diseases. The toxicity of inhaled particles and the subsequent development of health diseases including fibrosis, cancer and autoimmunity are recognized since several centuries . UnfortuThe fundamental concept, usually put forward to explain the pathogenesis of particle-induced fibrosis, cancer, and autoimmune diseases, is relatively simple. An unresolved and chronic inflammatory process, characterized by marked inflammatory cell accumulation and sustained release of inflammatory molecules, damages tissues, orchestrates accumulation of mesenchymal cells and their matrix protein products, transforms normal cells to tumor cells or activates adaptive immune responses to self-antigens .A precise cascade of cellular and molecular inflammatory events has been proposed from an extensive and growing number of data in human and animal exposed to particles Figure . First, A myriad of inflammatory mediators accompanying inflammation are linked to scar formation, cell transformation, and autoimmunity but only few of them are definitively identified as important in the pathological processes. Among those, the master pro-inflammatory cytokines TNF-α, IL-1 (mainly produced by M1 macrophages), type I and II IFN and IL-17  are essential in the pathogenesis of particle-induced diseases –20, 21. Current treatment modalities for treating patients developing particle-related diseases have been based on the assumption that these disorders are exclusively related to chronic inflammation diseases. However, it is becoming evident that most available anti-inflammatory drugs , 35 or cBesides the strong evidence of a major role of inflammation in particle-treated animals, several experimental studies did not find an evident association between inflammatory responses and experimental diseases and, thus, have also challenged this inflammatory dogma. Several studies, conducted in mice, reported that exacerbated lung inflammation is not always accompanied by a subsequent increase of fibrosis after silica or carbon nanotubes (CNT) treatment , 39. InvAltogether, these results strongly suggest that other pathophysiological mechanisms unrelated to inflammation are operational after particle exposure. There are now emerging studies from the literature that explain why diseases achieved by particles are not systematically related to inflammation. This review covers existing evidence supporting that immunosuppression represents an alternative pathological pathway in the development of particle-induced disorders. The most relevant studies delineating the immunosuppressive properties of (nano)particles are depicted in the following sections and commented in Table Indubitably, TGF-β is associated with the development of responses to particles in human and animal. Elevated TGF-β expression has been observed in patients with asbestosis or silicosis –71. In aBecause the anti-inflammatory cytokine IL-10 was effective in the regulation of inflammation, several authors have suggested that this cytokine could also be active in controlling inflammation in the early and late responses to mineral particles and thus plays a beneficial role based on the inflammatory paradigm –83. HoweIn vitro and in vivo studies additionally showed that mesotheliomagenic fibers such as asbestos directly enhance the immunosuppressive function of T regs and their capacity to release TGF-β and IL-10  in the development of lung responses to particles was investigated. This specialized thymus-derived sub-population of Th lymphocytes acts to suppress immune responses, thereby maintaining homeostasis and self-tolerance. They mediate immunosuppression by producing the immunoregulatory cytokines IL-10 and TGF-β . Recent nd IL-10 , 94. In nd IL-10 , 100. FrMore recently, Chen and colleagues have identified an additional immunosuppressive subpopulation of B lymphocytes recruited in response to silica in human and animal , 101 Fi. RegulatInfiltrated macrophages in cancer or asthma acquire a particular phenotype called M2, which provides an immunosuppressive microenvironment for tumor growth and allergic tolerance , 106. ReMyeloid-derived suppressor cells (MDSC) represent a heterogeneous population of immature neutrophilic and monocytic myeloid cells that displays potent immunosuppressive activities and inhibits immune effector cell functions in diverse chronic pathologies such as cancer . The accThe emerging concept that particles orchestrate the establishment of immunosuppressive responses is consolidated by several studies exploring the impact of nanoparticles on the immune system. As mentioned above for micrometric particles, the major concern in the field of nanotoxicology is historically related toward the capacity of nanoparticles to induce inflammation and immunostimulation . NeverthThe particles inducing immunosuppression mainly include metal  and carbon  nanoparticles . The immin vitro and in vivo  could promote chronic diseases after particle inhalation. The mutual roles of inflammation and immunosuppression well explain the diversity of particle-induced immune polarization and pathologies Figure . InteresIt remains, however, to learn how to extrapolate and how to benefit from these observations in predictive toxicology and translational medicine. Current bioassays testing particle reactivity are exclusively based on the appreciation of inflammatory components and suffer from a lack of specificity , 136. ThIn recent years, the increase of anti-tumor responses obtained in clinic trials by blocking the immunosuppressive environment of tumors have revolutionized cancer treatment. Monoclonal antibody-based immunotherapies targeting TGF-β and IL-10 or T cell immune checkpoint receptors (PD-1 or CTLA4) are now available and confer long-term benefit for patients –140. AddThe existence of at least two different pathogenic routes may explain why existing therapies are controversial and disappointing with a strong differential effect in specific subgroups of patients , 142. ThThe 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.


While neutralizing antibody titers measured by hemagglutination inhibition have been proposed as a correlate of protection following influenza vaccination, neutralization alone is a modest predictor of protection against seasonal influenza. Instead, emerging data point to a critical role for additional extra-neutralizing functions of antibodies in protection from infection. Specifically, beyond binding and neutralization, antibodies mediate a variety of additional immune functions via their ability to recruit and deploy innate immune effector function. Along these lines, antibody-dependent cellular cytotoxicity, antibody-mediated macrophage phagocytosis and activation, antibody-driven neutrophil activation, antibody-dependent complement deposition, and non-classical Fc-receptor antibody trafficking have all been implicated in protection from influenza infection. However, the precise mechanism(s) by which the immune system actively tunes antibody functionality to drive protective immunity has been poorly characterized. Here we review the data related to Fc-effector functional protection from influenza and discuss prospects to leverage this humoral immune activity for the development of a universal influenza vaccine. Influenza viruses are enveloped negative-strand RNA viruses with segmented genomes that can infect a variety of birds and mammals, including humans . Seasonade novo immunity to novel circulating strains. The development of yearly influenza vaccines relies on predictions published by the World Health Organization to determine the strain composition for a given year  has named the development of a universal influenza vaccine, defined as one that provides protection against symptomatic disease from ≥75% of influenza A strains, as one of its research priorities . While nven year . Due to ven year , leadingven year .Beyond efforts to match sequences to ensure seasonal immunity, the current surrogate of protection used to evaluate influenza vaccines is the hemagglutination inhibition (HAI) assay . HAI wasHumoral immune responses to influenza are largely directed toward the hemagglutinin molecule (HA). HA, the primary viral glycoprotein, exists as a trimer made of monomers composed of two subunits, HA1, roughly corresponding to the “head” and HA2, or “stem”  Figure . HeterosHeterosubtypic protective antibodies against influenza primarily target either the receptor binding site on the HA head or the more conserved HA stem . HoweverWhile the mechanism of protection mediated by neutralizing antibodies is simple to comprehend, the extra-neutralizing mechanisms of action of antibodies are less well-understood. Emerging evidence has suggested that mechanisms including the ability of antibodies to leverage the innate immune system may contribute to protection against influenza –31. CritAntibody-dependent cellular cytotoxicity (ADCC) is largely mediated by the interaction of pathogen or cell-surface bound antibodies with Fc gamma receptor IIIa (FcγRIIIa) on NK cells in humans  and FcγRIn vitro ADCC activity was linked to protection conferred by antibodies in a mouse model of influenza infection (in vitro (measured by CD107a expression) exhibited increased survival and decreased morbidity compared to mice that received either an antibody unable to bind to FcRs or a less potent ADCC-inducing antibody  activity  in healt+ T cells to the lung . Im. Im57). oduction . Influenoduction . Despiteing mAbs , arguinging mAbs , 64. Thuin vitro only when opsonized by antibodies . Th. Th33). tibodies . Howevertibodies . In thestibodies .Beyond antibody driven virion elimination, the complement protein C3 was also shown to promote higher titers of influenza-specific IgG antibodies. C3 also improved CD4+ and CD8+ T cell responses in the mouse models of influenza infection . VaccinaIn human serum, neutralization and complement-dependent lysis activities by mAbs have not always correlated, although neutralizing antibodies can induce complement-dependent lysis . Both IgThe neonatal Fc receptor, FcRn, is involved in transcytosing IgG across the placenta during fetal development, across the vascular endothelium to increase extravascular antibody levels, and across the mucosal epithelium to provide humoral defense within the mucosa . AdditioSystems level analyses aimed at defining biomarkers of productive immunity to flu vaccination identified pre-existing antibody titers as a negative predictor of response to vaccination , thoughtIn addition to trapping and delivery, these sialylated Fcs were also shown to increase B cell inhibitory FcR, FcγRIIb, expression, resulting in elevated thresholds required to activate B cells during development in the germinal center . With thTogether, these data suggest that the quality of the antibody response may not only influence direct antiviral activity, but may also be critical in influencing the response to vaccination. Seasonal influenza infection in humans has been shown to induce moderate antibody cross-reactivity , and theThe functional potency of an antibody is significantly affected by the antibody's subclass, which determines the binding affinity of the antibody for FcRs . Antibodex vivo with infectious influenza virus, suggesting that enhanced IgG3 responses were a marker of a more effective response to vaccination , MF59, AS03, and virosomes . In addiOther adjuvants are currently under investigation to specifically and selectively enhance influenza specific immunity. For example, liposomes provide unique scaffolds for antigen delivery , and werIn addition to efforts to promote more effective immune stimulation through adjuvants, intense investigation has focused on the development and design of unique antigens able to selectively direct the immune response away from strain-specific immunodominant sites to those that are more conserved . These iComputationally optimized broadly reactive antigens (COBRAs), designed based on computational modeling of influenza strains to create mosaic antigens aimed at focusing the immune response on the evolution of heterosubtypic responses, had some success in eliciting broadly reactive HAI titers that target both seasonal and pandemic strains of influenza –120. In Given the complexity of altering immunodominance using whole HA molecules, additional efforts have aimed to direct immunity against minimal antigenic regions associated with broadly protective responses including the stem , 122 andin vitro and in vivo , nucleoprotein (NP), and Matrix-2 (M2) . AntibodThe highly conserved internal viral proteins NP and M2 have been shown to induce an immune response that is also broadly reactive . NP-specMore than 4 decades of research has clearly illustrated the importance of both direct neutralization and non-neutralizing functional antibodies in protection against influenza infection and disease. Because neutralizing and non-neutralizing antibody activities are not induced in a mutually exclusive manner, vaccine strategies able to leverage both functions of antibodies are likely to confer the greatest level of protection. However, the precise innate immune effector functions to precise sites of viral vulnerability on HA or other target antigens remain to be determined. With emerging novel vaccine design strategies, coupled to emerging immune modulatory adjuvants, opportunities to drive universal protection are on the horizon.CB and GA wrote the review article, contributed to revision, and approved the submitted version.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.