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Migration independently evolved numerous times in animals, with a myriad of ecological and evolutionary implications. In fishes, perhaps the most extreme form of migration is diadromy, the migration between marine and freshwater environments. Key and long-standing questions are: how many times has diadromy evolved in fishes, how frequently do diadromous clades give rise to non-diadromous species, and does diadromy influence lineage diversification rates? Many diadromous fishes have large geographic ranges with constituent populations that use isolated freshwater habitats. This may limit gene flow between some populations, increasing the likelihood of speciation in diadromous lineages relative to nondiadromous lineages. Alternatively, diadromy may reduce lineage diversification rates if migration is associated with enhanced dispersal capacity that facilitates gene flow within and between populations. Clupeiformes (herrings, sardines, shads, and anchovies) is a model clade for testing hypotheses about the evolution of diadromy because it includes an exceptionally high proportion of diadromous species and several independent evolutionary origins of diadromy. However, relationships among major clupeiform lineages remain unresolved, and existing phylogenies sparsely sampled diadromous species, limiting the resolution of phylogenetically informed statistical analyses. We assembled a phylogenomic dataset and used multi-species coalescent and concatenation-based approaches to generate the most comprehensive, highly resolved clupeiform phylogeny to date, clarifying associations among several major clades and identifying recalcitrant relationships needing further examination. We determined that variation in rates of sequence evolution (heterotachy) and base-composition (nonstationarity) had little impact on our results. Using this phylogeny, we characterized evolutionary patterns of diadromy and tested for differences in lineage diversification rates between diadromous, marine, and freshwater lineages. We identified 13 transitions to diadromy, all during the Cenozoic Era (10 origins of anadromy, 2 origins of catadromy, and 1 origin of amphidromy), and 7 losses of diadromy. Two diadromous lineages rapidly generated nondiadromous species, demonstrating that diadromy is not an evolutionary dead end. We discovered considerably faster transition rates out of diadromy than to diadromy. The largest lineage diversification rate increase in Clupeiformes was associated with a transition to diadromy, but we uncovered little statistical support for categorically faster lineage diversification rates in diadromous versus nondiadromous fishes. We propose that diadromy may increase the potential for accelerated lineage diversification, particularly in species that migrate long distances. However, this potential may only be realized in certain biogeographic contexts, such as when diadromy allows access to ecosystems in which there is limited competition from incumbent species., (© The Author(s) 2024. Published by Oxford University Press, on behalf of the Society of Systematic Biologists. All rights reserved. For commercial re-use, please contact reprints@oup.com for reprints and translation rights for reprints. All other permissions can be obtained through our RightsLink service via the Permissions link on the article page on our site—for further information please contact journals.permissions@oup.com.)

AbstractMigration can have a profound influence on rates and patterns of phenotypic evolution. Diadromy is the migration between marine and freshwater habitats for feeding and reproduction that can require individuals to travel tens to thousands of kilometers. The high energetic demands of diadromy are predicted to select for ecomorphological traits that maximize swimming and locomotor efficiency. Intraspecific studies have shown repeated instances of divergence among diadromous and nondiadromous populations in locomotor and foraging traits, which suggests that at a macroevolutionary scale diadromous lineages may experience convergent evolution onto one or multiple adaptive optima. We tested for differences in rates and patterns of phenotypic evolution among diadromous and nondiadromous lineages in Clupeiformes, a clade that has evolved diadromy more than 10 times. Our results show that diadromous clupeiforms show convergent evolution for some locomotor traits and faster rates of evolution, which we propose are adaptive responses to the locomotor demands of migration. We also find evidence that diadromous lineages show convergence into multiple regions of multivariate trait space and suggest that these respective trait spaces are associated with differences in migration and trophic ecology. However, not all locomotor traits and no trophic traits show evidence of convergence or elevated rates of evolution associated with diadromy. Our results show that long-distance migration influences the tempo and patterns of phenotypic evolution at macroevolutionary scales, but there is not a single diadromous syndrome.

Evolutionary transitions between marine and freshwater ecosystems have occurred repeatedly throughout the phylogenetic history of fishes. The theory of ecological opportunity predicts that lineages that colonize species-poor regions will have greater potential for phenotypic diversification than lineages invading species-rich regions. Thus, transitions between marine and freshwaters may promote phenotypic diversification in trans-marine/freshwater fish clades. We used phylogenetic comparative methods to analyze body size data in nine major fish clades that have crossed the marine/freshwater boundary. We explored how habitat transitions, ecological opportunity, and community interactions influenced patterns of phenotypic diversity. Our analyses indicated that transitions between marine and freshwater habitats did not drive body size evolution, and there are few differences in body size between marine and freshwater lineages. We found that body size disparity in freshwater lineages is not correlated with the number of independent transitions to freshwaters. We found a positive correlation between body size disparity and overall species richness of a given area, and a negative correlation between body size disparity and diversity of closely related species. Our results indicate that the diversity of incumbent freshwater species does not restrict phenotypic diversification, but the diversity of closely related taxa can limit body size diversification. Ecological opportunity arising from colonization of novel habitats does not seem to have a major effect in the trajectory of body size evolution in trans-marine/freshwater clades. Moreover, competition with closely related taxa in freshwaters has a greater effect than competition with distantly related incumbent species., (© The Author(s) 2022. Published by Oxford University Press on behalf of the Society for Integrative and Comparative Biology.)

Species introductions provide opportunities to quantify rates and patterns of evolutionary change in response to novel environments. Alewives ( Alosa pseudoharengus ) are native to the East Coast of North America where they ascend coastal rivers to spawn in lakes and then return to the ocean. Some populations have become landlocked within the last 350 years and diverged phenotypically from their ancestral marine population. More recently, alewives were introduced to the Laurentian Great Lakes (~150 years ago), but these populations have not been compared to East Coast anadromous and landlocked populations. We quantified 95 years of evolution in foraging traits and overall body shape of Great Lakes alewives and compared patterns of phenotypic evolution of Great Lakes alewives to East Coast anadromous and landlocked populations. Our results suggest that gill raker spacing in Great Lakes alewives has evolved in a dynamic pattern that is consistent with responses to strong but intermittent eco-evolutionary feedbacks with zooplankton size. Following their initial colonization of Lakes Ontario and Michigan, dense alewife populations likely depleted large-bodied zooplankton, which drove a decrease in alewife gill raker spacing. However, the introduction of large, non-native zooplankton to the Great Lakes in later decades resulted in an increase in gill raker spacing, and present-day Great Lakes alewives have gill raker spacing patterns that are similar to the ancestral East Coast anadromous population. Conversely, contemporary Great Lakes alewife populations possess a gape width consistent with East Coast landlocked populations. Body shape showed remarkable parallel evolution with East Coast landlocked populations, likely due to a shared response to the loss of long-distance movement or migrations. Our results suggest the colonization of a new environment and cessation of migration can result in rapid parallel evolution in some traits, but contingency also plays a role, and a dynamic ecosystem can also yield novel trait combinations., (© 2020 The Authors. Evolutionary Applications published by John Wiley & Sons Ltd.)

Habitat occupancy can have a profound influence on macroevolutionary dynamics, and a switch in major habitat type may alter the evolutionary trajectory of a lineage. In this study, we investigate how evolutionary transitions between marine and freshwater habitats affect macroevolutionary adaptive landscapes, using needlefishes (Belonidae) as a model system. We examined the evolution of body shape and size in marine and freshwater needlefishes and tested for phenotypic change in response to transitions between habitats. Using micro-computed tomographic (µCT) scanning and geometric morphometrics, we quantified body shape, size, and vertebral counts of 31 belonid species. We then examined the pattern and tempo of body shape and size evolution using phylogenetic comparative methods. Our results show that transitions from marine to freshwater habitats have altered the adaptive landscape for needlefishes and expanded morphospace relative to marine taxa. We provide further evidence that freshwater taxa attain reduced sizes either through dwarfism (as inferred from axial skeletal reduction) or through developmental truncation (as inferred from axial skeletal loss). We propose that transitions to freshwater habitats produce morphological novelty in response to novel prey resources and changes in locomotor demands. We find that repeated invasions of different habitats have prompted predictable changes in morphology., Competing Interests: The authors declare no conflicts of interest., (© 2020 The Authors. Ecology and Evolution published by John Wiley & Sons Ltd.)

Migratory animals respond to environmental heterogeneity by predictably moving long distances in their lifetime. Migration has evolved repeatedly in animals, and many adaptations are found across the tree of life that increase migration efficiency. Life-history theory predicts that migratory species should evolve a larger body size than non-migratory species, and some empirical studies have shown this pattern. A recent study analysed the evolution of body size between diadromous and non-diadromous shads, herrings, anchovies and allies, finding that species evolved larger body sizes when adapting to a diadromous lifestyle. It remains unknown whether different fish clades adapt to migration similarly. We used an adaptive landscape framework to explore body size evolution for over 4500 migratory and non-migratory species of ray-finned fishes. By fitting models of macroevolution, we show that migratory species are evolving towards a body size that is larger than non-migratory species. Furthermore, we find that migratory lineages evolve towards their optimal body size more rapidly than non-migratory lineages, indicating body size is a key adaption for migratory fishes. Our results show, for the first time, that the largest vertebrate radiation on the planet exhibited strong evolutionary determinism when adapting to a migratory lifestyle.

We explored the macroevolutionary dynamics of miniaturisation in New World anchovies by integrating a time-calibrated phylogeny, geometric morphometrics and phylogenetic comparative methods. We found that the paedomorphic species Amazonsprattus scintilla occupies a novel region of shape space, while the dwarf species Anchoviella manamensis has an overall shape consistent with other anchovies. We found that miniaturisation did not increase overall clade disparity in size or shape beyond the expectations of Brownian motion, nor were there differences in rates of size or shape evolution among clades. Overall, our study shows that while the mode of miniaturisation influences shape evolution, the phenotypic novelty produced by the evolution of miniaturisation did not seem to alter macroevolutionary dynamics., (© 2019 The Fisheries Society of the British Isles.)

Biotic and abiotic forces govern the evolution of trophic niches, which profoundly impact ecological and evolutionary processes and aspects of species biology. Herbivory is a particularly interesting trophic niche because there are theorized trade-offs associated with diets comprised of low quality food that might prevent the evolution of herbivory in certain environments. Herbivory has also been identified as a potential evolutionary "dead-end" that hinders subsequent trophic diversification. For this study we investigated trophic niche evolution in Clupeoidei (anchovies, sardines, herrings, and their relatives) and tested the hypotheses that herbivory is negatively correlated with salinity and latitude using a novel, time-calibrated molecular phylogeny, trophic guilds delimited using diet data and cluster analysis, and standard and phylogenetically-informed statistical methods. We identified eight clupeoid trophic guilds: molluscivore, terrestrial invertivore, phytoplanktivore, macroalgivore, detritivore, piscivore, crustacivore, and zooplanktivore. Standard statistical methods found a significant negative correlation between latitude and the proportion of herbivorous clupeoids (herbivorous clupeoid species/total clupeoid species), but no significant difference in the proportion of herbivorous clupeoids between freshwater and marine environments. Phylogenetic least squares regression did not identify significant negative correlations between latitude and herbivory or salinity and herbivory. In clupeoids there were five evolutionary transitions from non-herbivore to herbivore guilds and no transitions from herbivore to non-herbivore guilds. There were no transitions to zooplanktivore, the most common guild, but it gave rise to all trophic guilds, except algivore, at least once. Transitions to herbivory comprised a significantly greater proportion of diet transitions in tropical and subtropical (<35°) relative to temperate areas (>35°). Our findings suggest cold temperatures may constrain the evolution of herbivory and that herbivory might act as an evolutionary "dead-end" that hinders subsequent trophic diversification, while zooplanktivory acts as an evolutionary "cradle" that facilitates trophic diversification., (Copyright © 2018 Elsevier Inc. All rights reserved.)

The BAFF receptor BR3 plays key roles in B-cell activation, maturation, and survival whereas the function of BR3 on T lymphocytes is less well characterized. Previous reports have demonstrated that BR3 costimulates human T-cell activation in vitro in the presence of high nonphysiological levels of plate-bound BAFF. Here, relying on the soluble and membrane-bound BAFF expressed by T cells themselves, we investigated the function of BR3 on activated primary CD4 and CD8 T lymphocytes using a BR3-specific neutralization antibody and shRNA gene down-modulation. Interestingly, the anti-BR3 blocking antibody resulted in significant augmentation of CD25 and IFN-γ expression by both subsets, as did shRNA-mediated down-modulation of BR3. In addition, granzyme B expression was substantially elevated in anti-BR3-treated and BR3-silenced T cells. Anti-BR3 blockade increased the expression of CD25 on cytolytic CRTAM T cells. Importantly, anti-BR3 significantly enhanced redirected killing of P-815 cells by both CD4 and CD8 cytotoxic T cells [cytotoxic T lymphocytes (CTLs)]. Furthermore, anti-BR3-augmented CD4 T-cell-mediated killing of class II melanoma cell line A375 and cervical cancer cell line HeLa in vitro, increasing the level of granzyme B activity as measured by PARP-1 cleavage and active caspase 3. Together, our data indicate that BR3 neutralization increases the activation and cytolytic function of CD4 and CD8 cytotoxic T lymphocytes. Our findings provide a novel strategy for ex vivo T-cell activation applicable to T-cell immunotherapy platforms such as TIL or CAR-T cell therapeutics.

Mesenchymal stem cells (MSCs) have immunosuppressive and tissue repair properties, but clinical trials using MSCs to prevent or treat graft-versus-host disease (GVHD) have shown mixed results. Macrophages (MØs) are important regulators of immunity and can promote tissue regeneration and remodeling. We have previously shown that MSCs can educate MØs toward a unique anti-inflammatory immunophenotype (MSC-educated MØs [MEMs]); however, their implications for in vivo models of inflammation have not been studied yet. We now show that in comparison with MØs, MEMs have increased expression of the inhibitory molecules PD-L1, PD-L2, in addition to markers of alternatively activated MØs: CD206 and CD163. RNA-Seq analysis of MEMs, as compared with MØs, show a distinct gene expression profile that positively correlates with multiple pathways important in tissue repair. MEMs also show increased expression of IL-6, transforming growth factor-β, arginase-1, CD73, and decreased expression of IL-12 and tumor necrosis factor-α. We show that IL-6 secretion is controlled in part by the cyclo-oxygenase-2, arginase, and JAK1/STAT1 pathway. When tested in vivo, we show that human MEMs significantly enhance survival from lethal GVHD and improve survival of mice from radiation injury. We show these effects could be mediated in part through suppression of human T cell proliferation and may have attenuated host tissue injury in part by enhancing murine fibroblast proliferation. MEMs are a unique MØ subset with therapeutic potential for the management of GVHD and/or protection from radiation-induced injury., (Copyright © 2017 The American Society for Blood and Marrow Transplantation. Published by Elsevier Inc. All rights reserved.)

Mid-substance rupture of the canine cranial cruciate ligament rupture (CR) and associated stifle osteoarthritis (OA) is an important veterinary health problem. CR causes stifle joint instability and contralateral CR often develops. The dog is an important model for human anterior cruciate ligament (ACL) rupture, where rupture of graft repair or the contralateral ACL is also common. This suggests that both genetic and environmental factors may increase ligament rupture risk. We investigated use of bone marrow-derived mesenchymal stem cells (BM-MSCs) to reduce systemic and stifle joint inflammatory responses in dogs with CR. Twelve dogs with unilateral CR and contralateral stable partial CR were enrolled prospectively. BM-MSCs were collected during surgical treatment of the unstable CR stifle and culture-expanded. BM-MSCs were subsequently injected at a dose of 2x106 BM-MSCs/kg intravenously and 5x106 BM-MSCs by intra-articular injection of the partial CR stifle. Blood (entry, 4 and 8 weeks) and stifle synovial fluid (entry and 8 weeks) were obtained after BM-MSC injection. No adverse events after BM-MSC treatment were detected. Circulating CD8+ T lymphocytes were lower after BM-MSC injection. Serum C-reactive protein (CRP) was decreased at 4 weeks and serum CXCL8 was increased at 8 weeks. Synovial CRP in the complete CR stifle was decreased at 8 weeks. Synovial IFNγ was also lower in both stifles after BM-MSC injection. Synovial/serum CRP ratio at diagnosis in the partial CR stifle was significantly correlated with development of a second CR. Systemic and intra-articular injection of autologous BM-MSCs in dogs with partial CR suppresses systemic and stifle joint inflammation, including CRP concentrations. Intra-articular injection of autologous BM-MSCs had profound effects on the correlation and conditional dependencies of cytokines using causal networks. Such treatment effects could ameliorate risk of a second CR by modifying the stifle joint inflammatory response associated with cranial cruciate ligament matrix degeneration or damage., Competing Interests: The authors have declared that no competing interests exist.

Background/aims: Human mesenchymal stromal/stem cells (MSCs), derived from many different tissues, are characterized by a fibroblast-like morphology, the expression of certain cell surface markers and their ability to differentiate into adipocytes, chondrocytes and osteoblasts. A number of studies have shown that MSCs share many characteristics with fibroblasts; however, there is no well-defined set of phenotypic characteristics that could distinguish between these 2 types of cells., Methods: We used 4 well-established human fibroblast strains from 3 different tissue sources and several human MSC strains from 2 different tissue sources to compare the phenotypic and immunological characteristics of these cells., Results: Fibroblast strains had a similar morphology to MSCs, expressed the same cell surface markers as MSCs and could also differentiate into adipocytes, chondrocytes and osteoblasts. Also, similar to MSCs, these fibroblasts were capable of suppressing T cell proliferation and modulating the immunophenotype of macrophages. We also show that MSCs deposit extracellular matrices of collagen type I and fibronectin, and express FSP1 in patterns similar to fibroblasts., Conclusions: Based on currently accepted definitions for cultured human MSCs and fibroblasts, we could not find any immunophenotypic property that could make a characteristic distinction between MSCs and fibroblasts., (© 2016 S. Karger AG, Basel.)

Phylogenetic relationships among families within the order Atheriniformes have been difficult to resolve on the basis of morphological evidence. Molecular studies so far have been fragmentary and based on a small number taxa and loci. In this study, we provide a new phylogenetic hypothesis based on sequence data collected for eight molecular markers for a representative sample of 103 atheriniform species, covering 2/3 of the genera in this order. The phylogeny is calibrated with six carefully chosen fossil taxa to provide an explicit timeframe for the diversification of this group. Our results support the subdivision of Atheriniformes into two suborders (Atherinopsoidei and Atherinoidei), the nesting of Notocheirinae within Atherinopsidae, and the monophyly of tribe Menidiini, among others. We propose taxonomic changes for Atherinopsoidei, but a few weakly supported nodes in our phylogeny suggests that further study is necessary to support a revised taxonomy of Atherinoidei. The time-calibrated phylogeny was used to infer ancestral habitat reconstructions to explain the current distribution of marine and freshwater taxa. Based on these results, the current distribution of Atheriniformes is likely due to widespread marine dispersal along the margins of continents, infrequent trans-oceanic dispersal, and repeated invasion of freshwater habitats. This conclusion is supported by post-Gondwanan divergence times among families within the order, and a high probability of a marine ancestral habitat., (Copyright © 2015 Elsevier Inc. All rights reserved.)

Background Aims: The T-cell suppressive property of bone marrow-derived mesenchymal stromal cells (MSCs) has been considered a major mode of action and basis for their utilization in a number of human clinical trials. However, there is no well-established reproducible assay to measure MSC-mediated T-cell suppression., Methods: At the University of Wisconsin-Madison Production Assistance for Cellular Therapy (PACT) Center, we developed an in vitro quality control T-cell suppression immunopotency assay (IPA) that uses anti-CD3 and anti-CD28 antibodies to stimulate T-cell proliferation. We measured MSC-induced suppression of CD4+ T-cell proliferation at various effector-to-target cell ratios with the use of defined peripheral blood mononuclear cells and in parallel compared with a reference standard MSC product. We calculated an IPA value for suppression of CD4+ T cells for each MSC product., Results: Eleven MSC products generated at three independent PACT centers were evaluated for cell surface phenotypic markers and T-cell suppressive properties. Flow cytometry results demonstrated typical MSC cell surface marker profiles. There was significant variability in the level of suppression of T-cell proliferation, with immunopotency assay values ranging from 27% to 88%. However, MSC suppression did not correlate with human leukocyte antigen-DR expression., Conclusions: We have developed a reproducible immunopotency assay to measure allogeneic MSC-mediated suppression of CD4+ T cells. Additional studies may be warranted to determine how these in vitro assay results may correlate with other immunomodulatory properties of MSCs, in addition to evaluating the ability of this assay to predict in vivo efficacy., (Copyright © 2015 International Society for Cellular Therapy. Published by Elsevier Inc. All rights reserved.)

Explaining the disparity of species richness across the tree of life is one of the great challenges in evolutionary biology. Some lineages are exceptionally species rich, while others are relatively species poor. One explanation for heterogeneity among clade richness is that older clades are more species rich because they have had more time to accrue diversity than younger clades. Alternatively, disparity in species richness may be due to among-lineage diversification rate variation. Here we investigate diversification in water scavenger beetles (Hydrophilidae), which vary in species richness among major lineages by as much as 20 fold. Using a time-calibrated phylogeny and comparative methods, we test for a relationship between clade age and species richness and for shifts in diversification rate in hydrophilids. We detected a single diversification rate increase in Megasternini, a relatively young and species rich clade whose diversity might be explained by the stunning diversity of ecological niches occupied by this clade. We find that Amphiopini, an old clade, is significantly more species poor than expected, possibly due to its restricted geographic range. The remaining lineages show a correlation between species richness and clade age, suggesting that both clade age and variation in diversification rates explain the disparity in species richness in hydrophilids. We find little evidence that transitions between aquatic, semiaquatic, and terrestrial habitats are linked to shifts in diversification rates.

One of the most remarkable types of migration found in animals is diadromy, a life-history behaviour in which individuals move between oceans and freshwater habitats for feeding and reproduction. Diadromous fishes include iconic species such as salmon, eels and shad, and have long fascinated biologists because they undergo extraordinary physiological and behavioural modifications to survive in very different habitats. However, the evolutionary origins of diadromy remain poorly understood. Here, we examine the widely accepted productivity hypothesis, which states that differences in productivity between marine and freshwater biomes determine the origins of the different modes of diadromy. Specifically, the productivity hypothesis predicts that anadromous lineages should evolve in temperate areas from freshwater ancestors and catadromous lineages should evolve in tropical areas from marine ancestors. To test this, we generated a time-calibrated phylogeny for Clupeiformes (herrings, anchovies, sardines and allies), an ecologically and economically important group that includes high diversity of diadromous species. Our results do not support the productivity hypothesis. Instead we find that the different modes of diadromy do not have predictable ancestry based on latitude, and that predation, competition and geological history may be at least as important as productivity in determining the origins of diadromy.

Freshwater habitats make up only ∼0.01% of available aquatic habitat and yet harbor 40% of all fish species, whereas marine habitats comprise >99% of available aquatic habitat and have only 60% of fish species. One possible explanation for this pattern is that diversification rates are higher in freshwater habitats than in marine habitats. We investigated diversification in marine and freshwater lineages in the New World silverside fish clade Menidiinae (Teleostei, Atherinopsidae). Using a time-calibrated phylogeny and a state-dependent speciation-extinction framework, we determined the frequency and timing of habitat transitions in Menidiinae and tested for differences in diversification parameters between marine and freshwater lineages. We found that Menidiinae is an ancestrally marine lineage that independently colonized freshwater habitats four times followed by three reversals to the marine environment. Our state-dependent diversification analyses showed that freshwater lineages have higher speciation and extinction rates than marine lineages. Net diversification rates were higher (but not significant) in freshwater than marine environments. The marine lineage-through time (LTT) plot shows constant accumulation, suggesting that ecological limits to clade growth have not slowed diversification in marine lineages. Freshwater lineages exhibited an upturn near the recent in their LTT plot, which is consistent with our estimates of high background extinction rates. All sequence data are currently being archived on Genbank and phylogenetic trees archived on Treebase., (© 2013 The Author(s). Evolution © 2013 The Society for the Study of Evolution.)

Background Aims: Mesenchymal stromal cells (MSC) have now been shown to reside in numerous tissues throughout the body, including the pancreas. Ex vivo culture-expanded MSC derived from many tissues display important interactions with different types of immune cells in vitro and potentially play a significant role in tissue homeostasis in vivo. In this study, we investigated the biologic and immunomodulatory properties of human pancreatic islet-derived MSC., Methods: We culture-expanded MSC from cadaveric human pancreatic islets and characterized them using flow cytometry, differentiation assays and nuclear magnetic resonance-based metabolomics. We also investigated the immunologic properties of pancreatic islet-derived MSC compared with bone marrow (BM) MSC., Results: Pancreatic islet and BM-derived MSC expressed the same cell-surface markers by flow cytometry, and both could differentiate into bone, fat and cartilage. Metabolomics analysis of MSC from BM and pancreatic islets also showed a similar set of metabolic markers but quantitative polymerase chain reactions showed that pancreatic islet MSC expressed more interleukin(IL)-1b, IL-6, STAT3 and FGF9 compared with BM MSC, and less IL-10. However, similar to BM MSC, pancreatic islet MSC were able to suppress proliferation of allogeneic T lymphocytes stimulated with anti-CD3 and anti-CD28 antibodies., Conclusions: Our in vitro analysis shows pancreatic islet-derived MSC have phenotypic, biologic and immunomodulatory characteristics similar, but not identical, to BM-derived MSC. We propose that pancreatic islet-derived MSC could potentially play an important role in improving the outcome of pancreatic islet transplantation by promoting engraftment and creating a favorable immune environment for long-term survival of islet allografts.

Evolutionary transitions between marine and freshwater biomes are relatively rare events, yielding a widespread pattern of biome conservatism among aquatic organisms. We investigated biome transitions in anchovies (Engraulidae), a globally distributed clade of economically important fishes. Most anchovy species are near-shore marine fishes, but several exclusively freshwater species are known from tropical rivers of South America and were previously thought to be the product of six or more independent freshwater invasions. We generated a comprehensive molecular phylogeny for Engraulidae, including representatives from 15 of 17 currently recognized genera. Our data support previous hypotheses of higher-level relationships within Engraulidae, but show that most New World genera are not monophyletic and in need of revision. Ancestral character reconstruction reveals that New World freshwater anchovies are the product of a single marine to freshwater transition, supporting a pattern of biome conservatism. We argue that competition is the principal mechanism that regulates aquatic biome transitions on a continental scale., (© 2012 The Authors. Journal of Evolutionary Biology © 2012 European Society For Evolutionary Biology.)

Phylogenetic relationships among families of Atheriniformes have long been problematic. The affinities of one of the most enigmatic lineages, surf silversides (Notocheiridae), have proven particularly elusive due to this taxon's unique morphology and rarity in museum collections. In this study, we use mitochondrial and nuclear sequence data to generate a phylogeny for seven of the eight families of Atheriniformes. Our results reveal that four families within Atheriniformes (Atherinopsidae, Notocheiridae, Atherinidae, Melanotaeniidae) are not monophyletic. Most notably, Notocheiridae is polyphyletic, with Notocheirus hubbsi nested within New World silversides (Atherinopsidae), while members of Iso are sister to all other Old World Atheriniforms. These data suggest that the unique morphology of Notocheirus and Iso is a result of adaptive convergent evolution to the high-energy surf habitat where these species live., (Copyright © 2011 Elsevier Inc. All rights reserved.)

Pediatric renal transplant recipients experience side effects of immunosuppression. Few immunoassays exist which can assess the adequacy of immunosuppression. We developed a CKT, whereby cytokine levels are measured in a five-day mixed lymphocyte reaction. We describe the in vitro cytokine responses to donor and third-party antigen in a pilot study of nine children after living-donor renal transplantation. The CKT identified five patterns of IFN-gamma secretion relative to donor and third-party alloantigen: no response to alloantigen (n = 2), hypo-response to donor (n = 3), equal response (n = 1), hyper-response to donor (n = 1), and intermediate response (n = 2). IL-2 and IL-13 patterning correlated with IFN-gamma expression. Two of nine subjects had acute rejection, which correlated with intermediate and hyper-responsive profiles. No rejection occurred during immunosuppression or donor-specific hypo-responsiveness. Significant immunosuppression was universal early after transplantation. Two of four children showed strong pretransplant responses to donor, which were regained three months post-transplant, and associated with rejection in one subject. The CKT reflects the level of immunosuppression and may offer a method to assess the adequacy of immunosuppression. A pattern of complete non-responsiveness or hypo-responsiveness correlated with lack of acute rejection. The CKT may prove useful in titrating immunosuppression and in improving live donor selection.

Alemtuzumab induction with 60 days of tacrolimus treatment and continuous sirolimus treatment prevented acute rejection in nine of 10 consecutive renal allograft recipients. All patients are alive with a functioning kidney graft at 27-39 months of follow-up. Extensive immune monitoring was performed in all patients. Alloantibody detection, cytokine kinetics assay (CKA), and trans vivo delayed-type hypersensitivity (DTH) assay were performed every 6 months showing correlation with clinical evolution. Despite alloantibody presence in five patients, eight patients remain without the need for specific treatment and only sirolimus monotherapy in decreasing dosage. Four patients take only 1 mg sirolimus daily with levels of 3-4 ng/mL. One patient showed clinical signs of rejection at month 9 post-transplant, with slow increase in serum creatinine and histological signs of mixed cellular (endarteritis) and humoral rejection (C4d positivity in peritubular capillaries and donor-specific antibody (DSA)). In summary, the addition of tacrolimus therapy for 2 months to a steroid-free, alemtuzumab induction and sirolimus maintenance protocol limited the previously shown acute rejection development. Nevertheless, alloantibody was present in serum and/or C4d present on 1-year biopsy in half the patients. The combination of CKA and DSA monitoring or the performance of transvivo DTH correlated with immune status of the patients.

Campath-1H (Alemtuzumab) is an effective immunodepletion agent used in renal transplantation. To evaluate its influence on T lymphocytes during repletion, we analyzed peripheral blood from Campath-1H-treated renal allograft recipients for the presence of FOXP3(+) regulatory T (Treg) cells. Flow cytometry demonstrated that CD4(+)CD25(+)FOXP3(+) lymphocytes increased significantly within the CD4(+) T-cell population, skewing Treg/Teff (T effector) ratios for up to several years. In contrast, Treg levels in patients treated with anti-CD25 (Basiliximab) and maintained on CsA demonstrated a sustained decrease. The increase in Tregs in Campath-1H treated patients developed independent of maintenance immunosuppression. Importantly, the increase in Tregs was not fully explained by their homeostatic proliferation, increased thymic output, or Treg sparing, suggesting de novo generation/expansion. Consistent with this, in vitro stimulation of PBMCs with Campath-1H, with or without anti-CD3, activation led to an increase in CD4(+)CD25(+)FOXP3(+) cells that had suppressive capabilities. Together, these data suggest that Campath-1H promotes an increase in peripheral Tregs and may act as an intrinsic generator of Tregs in vivo.

Background: Kidney transplant patients given Campath-1H (Alemtuzumab) immunodepletion therapy and long-term rapamycin monotherapy have excellent graft survival and function at three years. As an initial step in understanding the characteristics of repopulated T lymphocytes in these patients, we performed several assays to assess alloreactivity., Methods: We measured T-cell responses using CFSE-labeled recipient lymphocytes in a direct one-way MLR, and also analyzed the kinetics of expression of IFN-gamma. We examined the T-cell responses of Campath-treated transplant patients on monotherapy versus those treated with anti-CD25 (Basiliximab) induction therapy and maintenance immunosuppression consisting of cyclosporine A, mycophenolate mofetil, and steroids., Results: On average, proliferative responses to donor antigen were equal between Campath and control groups. However, the Campath group displayed a greater response to third party compared to donor antigen (CD3 P = 0.04, CD4 P = 0.07, CD8 P < 0.01), whereas the control group did not display a greater response to third party (CD3 P = 0.69, CD4 P = 0.72, CD8 P = 0.60). Interestingly, more Campath patients (4 of 15) than control patients (0 of 8) displayed donor specific unresponsiveness as gauged by IFN-gamma expression and T-cell proliferation (P = 0.15)., Conclusions: These studies suggest that Campath-1H in conjunction with rapamycin monotherapy retains intact immune responses to third party alloantigen, yet may promote hyporesponsiveness to donor antigen.

Background: In this study, we determined whether Campath-1H induction followed by sirolimus monotherapy inhibited alloantibody production in renal transplantation. Second, we evaluated the correlation between human leukocyte antigen (HLA) antibody production and serum creatinine levels., Methods: Sera were taken 1 to 24 months after transplantation from 24 patients treated with Campath-1H and sirolimus and tested for serum creatinine and HLA-specific antibody by using flow cytometry and enzyme-linked immunosorbent assay., Results: Ten (42%) of the 24 patients treated with Campath-1H and sirolimus produced HLA antibodies. Six of these 10 developed both donor-specific antibodies (DSAs) and non-donor-specific antibodies (NDSAs), whereas only NDSAs were detected in the other four patients. In patients with biopsy-diagnosed humoral rejection (C4d+), serum levels of both DSA and NDSA significantly correlated with patient serum creatinine levels. Rejection treatment successfully reduced both DSAs and NDSAs and reversed humoral rejection., Conclusions: The numeric relationship between serum creatinine and DSA levels suggests a causal relationship between alloantibody and transplant rejection.

Background: This study assesses the safety and efficacy of the novel human anti-human CD154 monoclonal antibody ABI793 in rhesus monkeys., Methods: Outbred rhesus monkeys were used for renal transplantation from major histocompatibility complex-mismatched donors. Seven recipients were treated with ABI793, and six untreated recipients were used as controls. Graft function was monitored by urine output, serum creatinine, and renal biopsy. Phenotypic analysis of peripheral blood lymphocytes and mixed lymphocyte reaction were performed before transplantation and periodically after transplantation. Anti-donor major histocompatibility complex class I antibody levels were measured at the time of sacrifice., Results: Monkeys in the treated group demonstrated prolonged graft survival compared with controls. One monkey was sacrificed because of a urine leak on postoperative day 13. Three monkeys were sacrificed because of acute rejection (days 44, 149, and 158). Two monkeys were sacrificed because of chronic active rejection (days 154 and 221). One monkey was sacrificed on day 139 without rejection to observe the effects of ABI793 in the absence of rejection. There were no obvious clinical side effects of ABI793, but microscopic thromboembolic changes were observed in two monkeys. Lymphocyte subsets remained unaltered in all monkeys. Mixed lymphocyte reaction showed nonspecific suppression 6 weeks after transplantation. The monkeys with chronic active rejection showed relatively strong alloantibody responses., Conclusions: ABI793 induces prolonged renal allograft survival in rhesus monkeys. Nevertheless, thromboembolic complications may occur and chronic allograft nephropathy may develop after anti-CD154 treatment is discontinued.

Campath-1H, an anti-CD52 monoclonal antibody, was used as induction therapy (40 mg i.v. total dose) in 29 primary human renal transplants, and the patients were maintained on rapamycin monotherapy (levels 8-15 ng/mL) post-transplant. Campath-1H profoundly depletes lymphocytes long-term and more transiently depletes B cells and monocytes. All patients are alive and well at 3-29 months of follow up. One graft was lost because of rejection. There have been no systemic infections and no malignancies. Eight of 29 patients have experienced rejection, which was successfully treated in seven of eight patients. Five of these patients had pathological evidence of a humoral component of their rejection. Seven of the 29 patients were converted to standard triple therapy on account of rejection. Rapamycin was generally well tolerated in that there were no significant wound-healing problems; two lymphoceles required surgical drainage; and most patients were treated with a lipid-lowering agent. Flow crossmatch testing post-transplant revealed evidence of alloantibody in two patients tested with previous combined cellular and humoral rejection. Biopsies have shown no chronic allograft nephropathy to date. In view of the relatively high incidence of early humoral rejection, we plan to modify the immunosuppressive regimen in subsequent pilot studies. This clinical trial provides insight into the use of Campath-1H induction in combination with rapamycin maintenance monotherapy.

Objective: To evaluate standardized developmental test performance of infants and children who as fetuses had mild isolated cerebral ventriculomegaly diagnosed by ultrasound., Methods: Ultrasound records from 1990 to 1996 were searched for cases of mild isolated ventriculomegaly, and standardized developmental testing of the children was offered to their parents. Each consented child was matched to a normal antepartum subject with respect to sex, race, indication for ultrasound, and gestational age (+/- 2 weeks) at the time of ultrasound. Tests of cognitive, motor, and adaptive behavior were then administered by examiners blinded to the subjects' case or comparison status., Results: Twenty-two cases and an equal number of matched comparison subjects completed the testing. The ventriculomegaly and comparison groups were similar with respect to parental age, maternal education, and household income. The ventriculomegaly subjects scored significantly lower than the comparison group on both the Bayley Scales of Infant Development: mental development index (88.95 versus 99.68, P = .017) and psychomotor development index (95.99 versus 103.95, P = .039). Eight of the 22 ventriculomegaly children were classified as developmentally delayed on the mental developmental index compared with one of 22 children in the comparison group (P = .021). Adaptive behavior skills, as measured by the Vineland Behavior Scales (99.64 versus 102.68), were not significantly different between the groups (P = .571)., Conclusion: Mild isolated ventriculomegaly detected on antepartum sonographic examination is associated with a significant risk for developmental delay. Insofar as these children were judged to be completely normal at birth, our findings represent an important application of antepartum sonography for identifying infants who could be targeted for early childhood intervention.

(B6 X A)F1 mice were immunized with sperm whale myoglobin, and T cell clones and hybridomas were generated. Hybridoma 74a.e9 was specific for the sperm whale myoglobin 67-79 peptide and could be partially activated by a peptide analogue, equine myoglobin with a natural 74G substitution. Using this hybridoma in T cell activation assays, we studied the effects of varying the avidity of the TCR for its ligand, the concentration of MHC:peptide complex on the APC, and the density of TCR on the surface. Varying ligand concentration on the surface of the APC, the TCR avidity, or the density of TCR on the T cell were equally important parameters in driving T cell activation. The mouse myoglobin (74T) analogue, however, acted as an antagonist to the T cell response. Its effectiveness was also partially determined by its ability to bind to MHC. By independently altering each of these variables and following T cell activation, we describe the interrelationships among these three components (MHC:peptide:TCR) that control the activation of the T cell.

Autoantibodies to ribonucleoproteins (RNP) occur prominently in human systemic lupus erythematosus and murine lupus models. In previous studies we demonstrated a relationship in MRL/Mp-lpr/lpr (MRL/lpr) mice between antibodies to Sm, an RNP autoantigen, and antibodies to DNA. Thus, many anti-Sm monoclonals bound DNA and expressed the same V region genes as anti-DNA. In addition, many had multiple VHCDR3 Arg residues suggestive of selection by DNA, and some had somatic mutations suggesting selection for mutant B cells by DNA. To determine whether autoantibodies to other RNP antigens are also associated with the anti-DNA response, we have analyzed the response to the La RNP. Six anti-La B cell hybridomas were generated from a single MRL/lpr mouse. Southern blot analysis of Ig V gene rearrangements and V gene sequences indicated two clonally related pairs, suggesting an oligoclonal response. Antibodies from all six hybridomas bound single-stranded DNA, while antibodies from five hybridomas bound double-stranded DNA. Two hybridomas expressed a VH7183 gene which is used by members of two previously reported anti-DNA clones and two anti-Sm/DNA clones of MRL/lpr origin. These data demonstrate an association between the anti-La and anti-DNA responses in MRL/lpr mice, suggesting that cross-reactive anti-RNP and anti-DNA responses are a general feature of autoimmunity in this lupus model.

Immunization of normal mice with bacterial DNA induces a significant anti-DNA response that includes antibodies resembling some lupus anti-DNA in their binding properties, although lacking specificity for mammalian dsDNA. To determine the structure of these induced antibodies and their relationship to anti-DNA from lupus mice, we have characterized the clonality and selected V-region sequences of a panel of 20 anti-DNA antibodies from 3 BALB/c mice immunized with ssDNA from Escherichia coli. Southern blot analysis of H and L chain rearrangements indicated that two of the animals expressed pairs of clonally related antibodies. Amino acid sequences of 10 of the induced antibodies demonstrated predominant utilization of J558 family VH genes and JH4 in association with various DH, J kappa and V kappa genes. Among the VH CDR3 of these 10 antibodies, 4 displayed arginine residues as a result of N region additions. None of these antibodies, however, had more than one arginine residue in VH CDR3 nor arginines at positions 100 or 100a, characteristic features of lupus antibodies to dsDNA. These results suggest that normal mice immunized with bacterial DNA display certain facets of DNA Ag drive, although lacking the mechanisms for the production of antibodies to mammalian dsDNA.

Anti-Sm autoantibodies are unique to SLE, but are present in only 25% of patients with this disease. This response also occurs at a similar frequency in mice of the autoimmune MRL strains. Previous analyses of the anti-Sm response in these mice indicate that its occurrence is controlled by stochastic events, and suggest that Sm is the driving Ag. To further elucidate the role of Ag in this response, and to test the hypothesis that the 25% incidence is due to a requirement for particular Ig gene rearrangements or somatic mutations, we have analyzed the specificity and V-region gene sequences of 41 anti-Sm B cell hybridomas derived from nine anti-Sm-positive MRL/Mp-lpr/lpr mice. The majority of hybridomas are specific for the D peptide of the Sm particle. Hybridomas of independent origin express unique VH/V kappa combinations with diverse junctional sequences and are variable in the extent of somatic mutation. Thus, the response does not appear to be dependent upon the occurrence of a rare Ig gene rearrangement or specific somatic mutation. The response exhibits restriction in JH and VH gene use, and in individual mice is oligoclonal, suggestive of Ag selection. In the few B cells for which mutations can be identified, the evidence for selection of mutant B lymphocytes, based on patterns of mutation, is ambiguous. However, there is remarkably little intraclonal diversity, suggesting that the overall mutation rates in these clones are low.

Autoantibodies specific for the Sm ribonucleoprotein are spontaneously produced in patients with SLE and in mice of the MRL mouse strains. We have previously reported the characterization of the clonality and V region gene use of 41 MRL/Mp-lpr/lpr (MRL/lpr)-derived B cell hybridomas selected for Sm binding. In this report, we show that many of the expressed V genes of these hybridomas are also expressed by anti-DNA hybridomas of MRL/lpr mice. Moreover, the anti-Sm hybridomas from nine clonal groups produce antibodies that bind ssDNA, and those of five clones produce antibodies that also bind dsDNA. Sm/DNA-specific hybridomas, but not Sm-only-specific hybridomas, have a higher than expected content of arginine residues in CDR3 of the H chain, similar to MRL/lpr hybridomas selected on the basis of DNA binding. One clone displays intraclonal differences in DNA binding, inasmuch as the most extensively mutated members produce antibodies that are able to bind dsDNA and have a higher affinity for ssDNA than the least mutated members of this clone. Thus, DNA appears to be a selecting Ag in this response. These data indicate an overlap in the anti-Sm and anti-DNA autoimmune responses in MRL mice that may have implications for the activation of anti-Sm B cells, and for defining the spectrum of Ag targeted in SLE.

We have quantified neurotransmitter changes in the rat jejunum in which the myenteric neurons were ablated by serosal application of benzalkonium chloride. Within 2 days after benzalkonium chloride treatment, there was a 40% reduction in the activity of choline acetyltransferase, a specific marker for cholinergic neurons, and a 25% reduction in the amount of vasoactive intestinal peptide per centimeter length of jejunum. By 15 days, levels were comparable to those in control segments, and by 45 days after myenteric neuronal ablation, levels in treated tissues were twice those in controls. In contrast, we observed no reduction in the amount of leucine-enkephalin per centimeter length of jejunum at early times after benzalkonium chloride treatment, although by 45 days, levels of this neurotransmitter in treated segments of jejunum were more than twice those in controls. Significant increases in muscle thickness and tissue weight were also observed at 15, 30, and 45 days after myenteric neuronal ablation. Thus we have observed that in response to chemical ablation of myenteric neurons in the rat jejunum, there is a thickening of the smooth muscle layers and a compensatory increase in the production of certain neurotransmitters by the surviving neuronal elements in the gut.