Inflammation and immunity in organ regeneration.

The ability of vertebrates to regenerate amputated appendages is increasingly well-understood at the cellular level. Cells mediating an innate immune response and inflammation in the injured tissues are a prominent feature of the limb prior to formation of a regeneration blastema, with macrophage activity necessary for blastema growth and successful development of the new limb. Studies involving either anti-inflammatory or pro-inflammatory agents suggest that the local inflammation produced by injury and its timely resolution are both important for regeneration, with blastema patterning inhibited in the presence of unresolved inflammation. Various experiments with Xenopus larvae at stages where regenerative competence is declining show improved digit formation after treatment with certain immunosuppressive, anti-inflammatory, or antioxidant agents. Similar work with the larval Xenopus tail has implicated adaptive immunity with regenerative competence and suggests a requirement for regulatory T cells in regeneration, which also occurs in many systems of tissue regeneration. Recent analyses of the human nail organ indicate a capacity for local immune tolerance, suggesting roles for adaptive immunity in the capacity for mammalian appendage regeneration. New information and better understanding regarding the neuroendocrine-immune axis in the response to stressors, including amputation, suggest additional approaches useful for investigating effects of the immune system during repair and regeneration.

Health Care Efficiencies: Consolidation and Alternative Models vs. Health Care and Antitrust Regulation - Irreconcilable Differences?

Despite the U.S. substantially outspending peer high income nations with almost 18% of GDP dedicated to health care, on any number of statistical measurements from life expectancy to birth rates to chronic disease, 1 the U.S. achieves inferior health outcomes. In short, Americans receive a very disappointing return on investment on their health care dollars, causing economic and social strain. 2 Accordingly, the debates rage on: what is the top driver of health care spending? Among the culprits: poor communication and coordination among disparate providers, paperwork required by payors and regulations, well-intentioned physicians overprescribing treatments, drugs and devices, outright fraud and abuse, and medical malpractice litigation. Fundamentally, what is the best way to reduce U.S. health care spending, while improving the patient experience of care in terms of quality and satisfaction, and driving better patient health outcomes? Mergers, partnerships, and consolidation in the health care industry, new care delivery models like Accountable Care Organizations and integrated care systems, bundled payments, information technology, innovation through new drugs and new medical devices, or some combination of the foregoing? More importantly, recent ambitious reform efforts fall short of a cohesive approach, leaving fundamental internal inconsistencies across divergent arms of the federal government, raising the issue of whether the U.S. health care system can drive sufficient efficiencies within the current health care and antitrust regulatory environments. While debate rages on Capitol Hill over "repeal and replace," only limited attention has been directed toward reforming the current "fee-for-service" model pursuant to which providers are paid for volume of care rather than quality or outcomes. Indeed, both the Patient Protection and Affordable Care Act ("ACA") 3 and proposals for its replacement focus primarily on the reach and cost of providing coverage for health care, rather than specifics for the delivery of health care. 4 With the U.S. expenditures on health care producing inferior results, experts see consolidation and alternatives to fee-for-service as fundamental to reducing costs. 5 Integrating care coordination and delivery and increasing scale to drive efficiencies allows organizations to benefit from shared savings and relationships with payors and vendors. 6 Deloitte forecasts that, by 2024, the current health system landscape-which includes roughly 80 national health systems, 275 regional systems, 130 academic medical centers, and 1,300 small community systems-will morph into just over 900 multi-hospital systems. 7 Even though health care market and payment reforms encourage organizations to consolidate and integrate, innovators must proceed with extreme caution. Health care organizations attempting to drive efficiencies and bring down costs through mergers may run afoul of numerous federal and state laws and regulations. 8 Calls for updates or leniency in these laws are growing, including the possible recognition of an "Obamacare defense" to antitrust restrictions 9 and speculation that laws restricting physicians from having financial relationships will be repealed, ostensibly to allow sharing of the rewards reaped from coordinated care. 10 In the meantime, however, absent specific waivers or exemptions, all the usual rules and regulations apply, including antitrust constraints, 11 physician self-referral 12 and anti-kickback laws and regulations, 13 state fraud and abuse restrictions, 14 and more. In short, a maelstrom of conflicting political prescriptions, health care regulations, and antitrust restrictions undermine the ability of innovators to achieve efficiencies through joint ventures, transactions, innovative models, and other structures. This article first considers the conflicting positions taken by the United States government with respect to achieving efficiencies in health care under the ACA and alternative delivery models, on the one hand, and health care regulatory enforcement and antitrust enforcement, on the other. At almost a fifth of the U.S. economy, 15 health care arguably has grown ungovernable, exceeding the ability of any one law or branch of government to create or implement coherent reform. Indeed, the article posits that although the ACA reformed and expanded access to health care, it failed to transform the way health care is delivered beyond limited "demonstration projects", leaving fee-for-service intact. Nonetheless, even with limited rather than revolutionary goals, the ACA still lacks sufficient authority across disparate branches of government to achieve its stated goals. The article then examines the conflicting positions of the various United States regulatory schemes and enforcement agencies governing health care, and whether they can be reconciled with the stated goal of the government, often referred to as the "Triple Aim": 16 improving quality of care, improving population health, and lowering health care costs. It examines fundamental, systemic challenges to achieving the "Triple Aim": longstanding health care regulatory laws that impede adoption of innovative delivery systems beyond their current "demonstration project" status, and antitrust enforcement that promotes waste and duplication in densely populated areas, while preventing necessary consolidation to more efficiently reach rural areas. The article concludes with recommendations for promoting efficiency through modest reconciliation of the conflicting goals and regulations in health care.

Neuropeptides as biologial system integrators – mini review.

This paper provides a review on the emerging role of neuropeptides for body systems integration. Neuropeptides are small protein-like substances that are released into the synaptic space and/or the blood, hence, acting as neurotransmitters and neurohormones. Classically, their role has been viewed as integrating the central nervous system and the endocrine system. More recently, however, there is an emerging understanding that neuropeptides integrate many more body systems into a larger cohesive body response that involves basic body functions, behavior, and mind. One indication is the discovery that neurohormones are also produced by many peripheral tissues and that non-neuronal peptides are released by neurons as well. Examples from the gastrointestinal and the immune system are presented. With the emerging role of neuropeptides for systems integration, immense opportunities arise for their use as pharmacological agents and biomarkers in the diagnosis and treatment of countless disorders such as chronic inflammatory diseases and obesity.

Changes in the inflammatory response to injury and its resolution during the loss of regenerative capacity in developing Xenopus limbs.

Tissue and organ regeneration, unlike development, involves an injury that in postembryonic animals triggers inflammation followed by resolution. How inflammation affects epimorphic regeneration is largely uninvestigated. Here we examine inflammation and its resolution in Xenopus laevis hindlimb regeneration, which declines during larval development. During the first 5 days postamputation, both regeneration-competent stage 53 and regeneration-deficient stage 57 hindlimbs showed very rapid accumulation of leukocytes and cells expressing interleukin-1ß and matrix metalloproteinase 9. Expression of genes for factors mediating inflammatory resolution appeared more persistent at stages 55 and 57 than at stage 53, suggesting changes in this process during development. FoxP3, a marker for regulatory T cells, was upregulated by amputation in limbs at all three stages but only persisted at stage 57, when it was also detected before amputation. Expression of genes for cellular reprogramming, such as SALL4, was upregulated in limbs at all 3 stages, but markers of limb patterning, such as Shh, were expressed later and less actively after amputation in regeneration-deficient limbs. Topical application of specific proinflammatory agents to freshly amputated limbs increased interleukin-1ß expression locally. With aqueous solutions of the proinflammatory metal beryllium sulfate, this effect persisted through 7 days postamputation and was accompanied by inhibition of regeneration. In BeSO4-treated limbs expression of markers for both inflammation and resolution, including FoxP3, was prolonged, while genes for cellular reprogramming were relatively unaffected and those for limb patterning failed to be expressed normally. These data imply that in Xenopus hindlimbs postamputation inflammation and its resolution change during development, with little effect on cellular dedifferentiation or reprogramming, but potentially interfering with the expression of genes required for blastema patterning. The results suggest that developmental changes in the larval anuran immune system may be involved in the ontogenetic loss of epimorphic regeneration in this system.

The developing Xenopus limb as a model for studies on the balance between inflammation and regeneration.

The roles of inflammation and immune cell reactivity triggered by amputation have only recently begun to be addressed in investigations of epimorphic regeneration, although studies of tissue repair in mammals clearly show the importance of the immune system in determining the quality of the repair process. Here, we first review inflammation-related work in non-mammalian systems of epimorphic regeneration which suggests that regeneration of an amputated appendage requires continuous modulation of the local immune response, from the first hours after amputation through the period of blastema patterning. We then present data on the effects of anti-inflammatory and proinflammatory agents on regeneration of larval Xenopus hindlimbs. Treatment with the glucocorticoid beclomethasone immediately after amputation inhibits regeneration in regeneration-complete stage 53 limbs. Other anti-inflammatory agents, including the inhibitors of cyclooxygenase-2 (COX-2) activity celecoxib and diclofenac, applied similarly to larvae amputated at stage 55, when the capacity for limb regeneration is normally being lost, restore regenerative capacity. This suggests that although injury-related events sensitive to glucocorticoids are necessary for regeneration, resolution of the inflammatory response may also be required to allow the complete regenerative response and normal blastema patterning. Conversely, if resolution of inflammation is prevented by local treatment of amputated limbs with beryllium, a strong immunoadjuvant, regeneration is inhibited, and gene expression data suggest that this inhibition results from a failure of normal blastema patterning. Both positive and negative effects of immune- or inflammation-related activities occur during anuran limb regeneration and this underscores the importance of considering immune cells in studies of epimorphic regeneration.

Dedifferentiation and the role of sall4 in reprogramming and patterning during amphibian limb regeneration.

A central feature of epimorphic regeneration during amphibian limb regeneration is cellular dedifferentiation. Two questions are discussed. First, what is the origin and nature of the soluble factors involved in triggering local cellular and tissue dedifferentiation? Secondly, what role does the key stem cell transcription factor Sall4 play in reprogramming gene expression during dedifferentiation? The pattern of Sall4 expression during Xenopus hindlimb regeneration is consistent with the hypothesis that Sall4 plays a role in dedifferentiation (reprogramming) and in maintaining limb blastema cells in an undifferentiated state. Sall4 is involved in maintenance of ESC pluripotency, is a major repressor of differentiation, plays a major role in reprogramming differentiated cells into iPSCs, and is a component of the stemness regulatory circuit of pluripotent ESCs and iPSCs. These functions suggest Sall4 as an excellent candidate to regulate reprogramming events that produce and maintain dedifferentiated blastema cells required for epimorphic regeneration.

RNA helicase Ddx39 is expressed in the developing central nervous system, limb, otic vesicle, branchial arches and facial mesenchyme of Xenopus laevis.

Ddx39, a DEAD-box RNA helicase, is a part of the homeostatic machinery that regulates the switch between cellular proliferation and differentiation. Ddx39 was shown to be differentially regulated in Xenopus laevis using a differential screen of mRNAs from regenerating limbs (King et al., 2003). Here, the expression patterns of Ddx39 in developing limb and nervous system are reported. Ddx39 was detected by RT-PCR in the Xenopus embryo, the earliest stage examined. Localization of the message by whole-mount in situ hybridization at stage 17 showed it to be localized primarily to the developing nervous system. Ddx39 was present in the ventricular region of the developing neural tube up to and including stage 48, and was also localized to the head mesenchyme, pharyngeal arches, and paraxial mesoderm. Strong label was also present in the developing limb buds at stages 48-55. Analysis of expression patterns in cryosections of the developing eye at stage 38 and 47 showed Ddx39 in the ciliary marginal zone (CMZ) adjacent to the neural retina and within the lens epithelium. Ddx39 was also present in the anterior eye during fibroblast growth factor 2 (FGF2)-mediated retinal regeneration. BrDU incorporation analyses and double-label studies with proliferating cell nuclear antigen showed that Ddx39 message was restricted to a subpopulation of proliferating cells in the developing and regenerating optic cup.

Proteomic analysis of blastema formation in regenerating axolotl limbs.

BACKGROUND: Following amputation, urodele salamander limbs reprogram somatic cells to form a blastema that self-organizes into the missing limb parts to restore the structure and function of the limb. To help understand the molecular basis of blastema formation, we used quantitative label-free liquid chromatography-mass spectrometry/mass spectrometry (LC-MS/MS)-based methods to analyze changes in the proteome that occurred 1, 4 and 7 days post amputation (dpa) through the mid-tibia/fibula of axolotl hind limbs. RESULTS: We identified 309 unique proteins with significant fold change relative to controls (0 dpa), representing 10 biological process categories: (1) signaling, (2) Ca2+ binding and translocation, (3) transcription, (4) translation, (5) cytoskeleton, (6) extracellular matrix (ECM), (7) metabolism, (8) cell protection, (9) degradation, and (10) cell cycle. In all, 43 proteins exhibited exceptionally high fold changes. Of these, the ecotropic viral integrative factor 5 (EVI5), a cell cycle-related oncoprotein that prevents cells from entering the mitotic phase of the cell cycle prematurely, was of special interest because its fold change was exceptionally high throughout blastema formation. CONCLUSION: Our data were consistent with previous studies indicating the importance of inositol triphosphate and Ca2+ signaling in initiating the ECM and cytoskeletal remodeling characteristic of histolysis and cell dedifferentiation. In addition, the data suggested that blastema formation requires several mechanisms to avoid apoptosis, including reduced metabolism, differential regulation of proapoptotic and antiapoptotic proteins, and initiation of an unfolded protein response (UPR). Since there is virtually no mitosis during blastema formation, we propose that high levels of EVI5 function to arrest dedifferentiated cells somewhere in the G1/S/G2 phases of the cell cycle until they have accumulated under the wound epidermis and enter mitosis in response to neural and epidermal factors. Our findings indicate the general value of quantitative proteomic analysis in understanding the regeneration of complex structures.

Gene expression profiles of lens regeneration and development in Xenopus laevis.

Seven hundred and thirty-four unique genes were recovered from a cDNA library enriched for genes up-regulated during the process of lens regeneration in the frog Xenopus laevis. The sequences represent transcription factors, proteins involved in RNA synthesis/processing, components of prominent cell signaling pathways, genes involved in protein processing, transport, and degradation (e.g., the ubiquitin/proteasome pathway), matrix metalloproteases (MMPs), as well as many other proteins. The findings implicate specific signal transduction pathways in the process of lens regeneration, including the FGF, TGF-beta, MAPK, Retinoic acid, Wnt, and hedgehog signaling pathways, which are known to play important roles in eye/lens development and regeneration in various systems. In situ hybridization revealed that the majority of genes recovered are expressed during embryogenesis, including in eye tissues. Several novel genes specifically expressed in lenses were identified. The suite of genes was compared to those up-regulated in other regenerating tissues/organisms, and a small degree of overlap was detected.

Proteomics analysis of regenerating amphibian limbs: changes during the onset of regeneration.

During amphibian epimorphic limb regeneration, local injury produces metabolic changes that lead to cellular dedifferentiation and formation of a blastema, but few details of these changes have been elucidated. Here we report the first global proteomic analysis of epimorphic regeneration comparing the profiles of abundant proteins in larval limbs of the anuran Xenopus laevis (stage 53) at the time of amputation (0dPA) and 3 days post-amputation when the regeneration blastema is developing (3dPA). We identified and quantified 1517 peptides, of which 1067 were identified with high peptide ID confidence. Of these 1067 proteins, 489 showed significant changes in quantity between the two groups. Taking into account identical peptides whose fold changes were within 20%, and not including peptides whose fold changes were below the observed fold changes of peptides for the internal standard (chicken lysozyme), we were able to identify 145 peptides elevated in 3dPA relative to 0dPA and 220 peptides in 0dPA relative to 3dPA. In this report, we focus on those proteins that were elevated in the 3dPA tissue relative to 0dPA. In this class were members of the annexin family (e.g. ANXA1, ANXA2, ANXA5) and the ANXA2-binding partner S100A10, which have important immunoregulatory roles in other systems and were also shown to be differentially expressed in stage 53 and 57 3dPA and 5dPA blastemas in our previous microarray studies. Besides elucidating the possible modulation of inflammation during amphibian limb regeneration, our proteomic study also provides insight into dedifferentiation by revealing up-regulation of proteins known to characterize many stem cells.

Loss of the protein NUPR1 (p8) leads to delayed LHB expression, delayed ovarian maturation, and testicular development of a sertoli-cell-only syndrome-like phenotype in mice.

The high mobility group factor NUPR1, also known as p8 and com1, plays a role in temporal expression of the beta subunit of luteinizing hormone, LHB, during gonadotroph development. At Embryonic Day (e) 16.5, LHB is detectable in wild-type (Nupr1(+/+)) but not Nupr1 knockout (Nupr1(-/-)) mice. LHB is initiated by e17.5 in Nupr1(-/-) mice, and expression is fully recovered by Postnatal Day (p) 2. Factors indicative of pituitary maturation, GATA2, CGA, and TSH, are not differentially expressed in Nupr1(-/-) and Nupr1(+/+) embryos at e17.5. Therefore, the delay in LHB expression does not appear to result from delayed pituitary development. In addition, the role of NUPR1 in gonadotropin expression appears specific for LHB, as no difference in FSHB is observed in Nupr1(-/-) and Nupr1(+/+) embryos. The gonads are also impacted by the absence of NUPR1. Ovaries of female Nupr1(-/-) mice lack corpora lutea (CL) at 8 wk, an age at which CL are present in all Nupr1(+/+) littermates. Sexual maturity is recovered by 11 wk in Nupr1(-/-) mice. Conversely, the testes of Nupr1(-/-) males appear normal through 8 mo of age. By 10 mo, however, these mice develop a condition in which a significant number of seminiferous tubules lack germ cells, an abnormality reminiscent of human Sertoli-cell-only syndrome. NUPR1 is undetectable in Nupr1(+/+) gonadotrophs by p2 and remains absent in adulthood, but quantitative PCR analysis indicates Nupr1(+/+) adult ovaries and testes express Nupr1 mRNA. Therefore, the ovarian and testicular phenotypes may be due to the loss of NUPR1 directly at the gonads.

Neural MMP-28 expression precedes myelination during development and peripheral nerve repair.

Mammalian matrix metalloproteinase 28 (MMP-28) is expressed in several normal adult tissues, and during cutaneous wound healing. We show that, in frog and mouse embryos, MMP-28 is expressed predominantly throughout the nervous system. Xenopus expression increases during neurulation and remains elevated through early limb development where it is expressed in nerves. In the mouse, neural expression peaks at embryonic day (E) 14 but remains detectable through E17. During frog hindlimb regeneration XMMP-28 is not initially expressed in the regenerating nerves but is detectable before myelination. Following hindlimb denervation, XMMP-28 expression is detectable along regenerating nerves before myelination. In embryonic rat neuron-glial co-cultures, MMP-28 decreases after the initiation of myelination. Incubation of embryonic brain tissue with purified MMP-28 leads to the degradation of multiple myelin proteins. These results suggest that MMP-28 plays an evolutionarily conserved role in neural development and is likely to modulate the axonal-glial extracellular microenvironment.

DP58, an inducible myeloid protein, is constitutively expressed in murine neuronal nuclei.

A novel cytosolic phosphoprotein, DP58 induced in bone marrow-derived dendritic progenitors was found in this study to be constitutively expressed at a very high level in neuronal nuclei. Amplified cDNA confirmed by sequencing to be DP58 was present only in brain tissue, and DP58-like protein was expressed in neurons as a 52 kDa nuclear protein, phosphorylated primarily at the serine residues. In contrast, its isoform in dendritic progenitors appeared as a 58 kDa inducible protein with phosphorylation at serine, threonine and tyrosine residues. Although protein markers common to brain and hematopoietic cells are known, no report was found on constitutive expression in neuronal nuclei of DP58, an inducible Pro-myloid marker. The sequence of DP58 reveals ankyrin repeats present in a wide spectrum of interacting proteins including NF-kappaB-binding BCL3, a predominantly nuclear protein of I-kappaB family. The contrasting phosphorylated forms of DP58 suggest a distinct physiological role in neuronal cells and early dendritic progenitors.

Global analysis of gene expression in Xenopus hindlimbs during stage-dependent complete and incomplete regeneration.

Xenopus laevis tadpoles are capable of limb regeneration after amputation, in a process that initially involves the formation of a blastema. However, Xenopus has full regenerative capacity only through premetamorphic stages. We have used the Affymetrix Xenopus laevis Genome Genechip microarray to perform a large-scale screen of gene expression in the regeneration-complete, stage 53 (st53), and regeneration-incomplete, stage 57 (st57), hindlimbs at 1 and 5 days postamputation. Through an exhaustive reannotation of the Genechip and a variety of comparative bioinformatic analyses, we have identified genes that are differentially expressed between the regeneration-complete and -incomplete stages, detected the transcriptional changes associated with the regenerating blastema, and compared these results with those of other regeneration researchers. We focus particular attention on striking transcriptional activity observed in genes associated with patterning, stress response, and inflammation. Overall, this work provides the most comprehensive views yet of a regenerating limb and different transcriptional compositions of regeneration-competent and deficient tissues.

Expression of Xenopus XlSALL4 during limb development and regeneration.

The multi-C2H2 zinc-finger domain containing transcriptional regulators of the spalt (SAL) family plays important developmental regulatory roles. In a competitive subtractive hybridization screen of genes expressed in Xenopus laevis hindlimb regeneration blastemas, we identified a SAL family member that, by phylogenetic analysis, falls in the same clade as human SALL4 and have designated it as XlSALL4. Mutations of human SALL4 have been linked to Okihiro syndrome, which includes preaxial (anterior) limb defects. The expression pattern of XlSALL4 transcripts during normal forelimb and hindlimb development and during hindlimb regeneration at the regeneration-competent and regeneration-incompetent stages is temporally and regionally dynamic. We show for the first time that a SAL family member (XlSALL4) is expressed at the right place and time to play a role regulating both digit identity along the anterior/posterior axis and epimorphic limb regeneration.

Regeneration or scarring: an immunologic perspective.

Complete regeneration of complex tissues and organs is usually precluded by fibrotic reactions that lead to scarring. Fish, salamanders, and larval anurans are among the few vertebrates capable of regenerating lost appendages, and this process seems to recapitulate ontogenic development of the structure in most respects. Recent work has revealed a capacity for excellent regeneration in certain mammalian tissues: embryonic or fetal skin and the ear of the MRL mouse. Analyses of these two systems suggest that processes of regenerative growth and patterning for the formation of new structures such as hair follicles may involve modulation of the inflammatory response to the injury in a way that reduces fibrosis and formation of scar tissue. We review evidence that this modulation includes changes in cytokine signaling and may involve properties of the extracellular matrix mediated by factors that include hyaluronic acid and "anti-adhesive substrates" such as tenascin-C. New studies and classic work on the capacity for limb regeneration in amphibians are then reviewed, focusing on the loss of this ability in prometamorphic anuran hindlimbs and the view that changing properties of the immune system may also underlie the declining regenerative potential in this system. Finally, we review recent work in comparative and developmental immunology, which raises the possibility that phylogenetic changes in regenerative capacity may be the result of evolutionary changes in cellular activities of the immune system.

Identification of genes expressed during Xenopus laevis limb regeneration by using subtractive hybridization.

Suppression polymerase chain reaction-based subtractive hybridization was used to identify genes that are expressed during Xenopus laevis hindlimb regeneration. Subtractions were done by using RNAs extracted from the regeneration-competent stage (stage 53) and regeneration-incompetent stage (stage 59) of limb development. Forward and reverse subtractions were done between stage 53 7-day blastema and stage 53 contralateral limb (competent stage), stage 59 7-day pseudoblastema and stage 59 contralateral limb (incompetent stage), and stage 53 7-day blastema and stage 59 7-day pseudoblastema. Several thousand clones were analyzed from the various subtracted libraries, either by random selection and sequencing (1,920) or by screening subtracted cDNA clones (6,150), arrayed on nylon membranes, with tissue-specific probes. Several hundred clones were identified from the array screens whose expression levels were at least twofold higher in experimental tissue vs. control tissue (e.g., blastema vs. limb) and selected for sequencing. In addition, primers were designed to assay several of the randomly selected clones and used to assess the level of expression of these genes during regeneration and normal limb development. Approximately half of the selected clones were differentially expressed, as expected, including several that demonstrate blastema-specific enhancement of expression. Three distinct categories of expression were identified in our screens: (1) clones that are expressed in both regeneration-competent blastemas and -incompetent pseudoblastemas, (2) clones that are expressed at highest levels in regeneration-competent blastemas, and (3) clones that are expressed at highest levels in regeneration-incompetent pseudoblastemas. Characterizing the role of each of these three categories of genes will be important in furthering our understanding of the process of tissue regeneration.