Distinct MicroRNAs Identified in Rabbit Blood Arising from Induced Diabetes and a Surgically Simulated Diabetic Ischemia Complication.

BACKGROUND: Diabetic complications have been studied extensively in recent years. There are very few biomarkers in body fluids that can pinpoint a distinct diabetic complication due to insufficient known specific biomarkers for ischemia. OBJECTIVE: Identifying microRNA in animal models for each complication could enable early diagnosis of a given complication if verified in humans. MicroRNA (miRNA) profiling has been done in rodent models for a number of diabetic complications, like diabetic glomerular injury, atherosclerosis, cognitive impairment, diabetic wound healing, angiopathy and other complications. Due to multiple differences between rodents and humans, the changes in rabbit skin, considered closer to humans than even pigs, may better simulate human diabetic complications of ischemia. METHODS: To study the miRNA profile of rabbits in which diabetes was induced or ischemia was surgically generated, we studied whether diabetes or ischemia-induced specific miRNA could be detected. MicroRNA from the blood of diabetic rabbits and rabbits with local ischemia was collected in PAXgene Blood RNA tubes specifically designed for miRNA isolation and extracted using the PAX gene miRNA extraction kit. The isolated RNA was quality controlled using an RNA analyzer, and further, using RNA seq technology, it was analyzed for distinct miRNAs that were detected in diabetic and non-diabetic rabbits induced with ischemia. RESULTS: A miRNA that was found to be expressed in diabetic rabbits and ischemic rabbits but not in untreated rabbits was miRNA-183. Several miRNAs were differentially expressed across comparison groups, and several upregulated miRNAs were identified being unique to each comparison. In rabbits with a potential diabetic complication of a long-term ischemic model, there was one distinct microRNA, which was highly significantly upregulated in ischemia rabbit (miRNA-133-3p). One miRNA that was highly significantly upregulated in diabetic rabbit but not in ischemic rabbits was miRNA-3074-5p. Only statistically significant results have been considered and analyzed. CONCLUSION: These findings could lead to a precise and timely diagnosis of a potential single diabetic complication without invasive tissue biopsies and could be a novel tool in the management of diabetic patients developing complications due to the progression of diabetes.

Intracellular ATP Delivery Causes Rapid Tissue Regeneration via Upregulation of Cytokines, Chemokines, and Stem Cells.

We have reported accelerated wound healing induced by intracellular ATP delivery in rabbits, through early massive accumulation, in situ proliferation, and M2 polarization of macrophages. Granulation tissue started to grow within first 24 h of treatment and continued the growth till the wound cavity is completely covered. However, the mechanisms underlying this macrophage response are totally unclear because no one has ever reported this before. In this study, we performed a preliminary exploration of the possible mechanisms by focusing on the roles of cytokines, growth factors, and stem cells in this process. Among the 33 adult rabbits, 18 were used for cytokine measurements and the remaining were used for histological and immunohistochemical studies. Four wounds were created on the ventral side of each ear. Two wounds on one side were treated with ATP-vesicles (10 mM ATP), and the other two were treated with controls (normal saline or Regranex). Dressing changes were made daily and the rabbits were sacrificed at 5 h, 12 h, and 1, 2, 3, 4, 6, 9, 15, and 26 days after wounding. Tissue samples were analyzed for cytokines and growth factors using real-time PCR and immunohistochemical staining. The control wounds showed an immediate increase in proinflammatory cytokines after wound creation but no further increase after this initial spike. The growth factor levels in the control wounds remained unchanged throughout the study. Conversely, the wounds treated with ATP-vesicles showed significantly higher expression of MCP-1 and stem cell markers (CD44, CD106, CD146, and CD34) at day 1, significantly higher IL-1ß and TNF-α expression from day 1-4, and significantly higher VEGF-A, VEGF-D, and VEGFR-2 expression from day 4-6 when compared to the controls. The significant upregulation of these factors corresponded to the very early and rapid macrophage accumulation, in situ proliferation, and M2 polarization, resulting in unprecedented rapid granulation tissue generation due to direct macrophage collagen production and neovascularization.

Significant upregulation of U1 and U4 spliceosomal snRNAs by ATP nanoliposomes explains acceleration of wound healing, due to increased pre-mRNA processing to functional mRNA.

Delayed wound healing is one of the hallmarks of diabetic complications and certain autoimmune inflammatory diseases. Extensive wound healing studies in rabbits have indicated that the delivery of ATP encapsulated in unilamellar nanoliposomes causes rapid cell proliferation and fast tracks the wound healing process. In the current study, we explored the possible molecular mechanism underlying this response by comparing gene expression in cultured rabbit kidney cells treated with either ATP nanoliposomes (containing 1 mg Mg-ATP/ml formulation) or control nanoliposomes (containing 1 mg/ml unmetabolisable gamma-thio-ATP/ml formulation). High-quality total RNA was isolated 24 h from the cells and subjected to RNA seq technology, which revealed significant overexpression of specific noncoding RNAs. The U1 spliceosomal RNA, U1 snRNA, was upregulated more than 250-fold following treatment with ATP nanoliposomes. This multifunctional U1 spliceosomal RNA may function in transcription by speeding up the critical splicing step, thereby facilitating faster processing of pre-mRNA to translatable mRNA.

Macrophage Differentiation in Normal and Accelerated Wound Healing.

Chronic wounds pose considerable public health challenges and burden. Wound healing is known to require the participation of macrophages, but mechanisms remain unclear. The M1 phenotype macrophages have a known scavenger function, but they also play multiple roles in tissue repair and regeneration when they transition to an M2 phenotype. Macrophage precursors (mononuclear cells/monocytes) follow the influx of PMN neutrophils into a wound during the natural wound-healing process, to become the major cells in the wound. Natural wound-healing process is a four-phase progression consisting of hemostasis, inflammation, proliferation, and remodeling. A lag phase of 3-6 days precedes the remodeling phase, which is characterized by fibroblast activation and finally collagen production. This normal wound-healing process can be accelerated by the intracellular delivery of ATP to wound tissue. This novel ATP-mediated acceleration arises due to an alternative activation of the M1 to M2 transition (macrophage polarization), a central and critical feature of the wound-healing process. This response is also characterized by an early increased release of pro-inflammatory cytokines (TNF, IL-1 beta, IL-6), a chemokine (MCP-1), an activation of purinergic receptors (a family of plasma membrane receptors found in almost all mammalian cells), and an increased production of platelets and platelet microparticles. These factors trigger a massive influx of macrophages, as well as in situ proliferation of the resident macrophages and increased synthesis of VEGFs. These responses are followed, in turn, by rapid neovascularization and collagen production by the macrophages, resulting in wound covering with granulation tissue within 24 h.

Pivotal role of ATP in macrophages fast tracking wound repair and regeneration.

Chronic wounds occurring during aging or diabetes pose a significant burden to patients. The classical four-phase wound healing process has a 3-6 day lag before granulation starts to appear and it requires an intermediate step of activation of resident fibroblasts during the remodeling phase for production of collagen. This brief communication discusses published articles that demonstrate how the entire wound healing process can be fast tracked by intracellular ATP delivery, which triggers a novel pathway where alternatively activated macrophages play absolutely critical and central roles. This novel pathway involves an increase in proinflammatory cytokines (TNF, IL-1ß, IL-6) and a chemokine (MCP-1) release. This is followed by activation of purinergic receptor (a family of plasma membrane receptors found in almost all mammalian cells), production of platelets and platelet microparticles, and activation of ATP-dependent chromatin remodeling enzymes. The end result is a massive influx and in situ proliferation of macrophages, increases in vascular endothelial growth factors that promote neovascularization, and most prominently, the direct production of collagen.

Exploring the potential benefits of vaccinia virus complement control protein in controlling complement activation in pathogenesis of the central nervous system diseases.

Aging is a major risk factor for the development of diseases related to the central nervous system (CNS), such as Alzheimer's disease (AD) and age-related macular degeneration (AMD). In both cases, linkage studies and genome-wide association studies found strong links with complement regulatory genes and disease risk. In AD, both CLU and CR1 genes were implicated in the late-onset form of the disease. In AMD, polymorphisms in CFH, CFB and C2 were similarly implicated. The cost of caring for patients with AD or AMD is approaching billions of dollars, and with the baby boomers reaching their 60's, this amount is likely to increase further. Intervention using complement inhibitors for individuals in their early 50s who are at a higher risk of disease development, (testing positive for genetic risk factors), could slow the progression of AD or AMD and possibly prevent the severity of late stage symptoms. Although we have used the vaccinia virus complement control protein (VCP) to elucidate the role of complement in CNS diseases, it has merely been an investigational tool but not the only possible potential therapeutic agent.

Viral infection: an evolving insight into the signal transduction pathways responsible for the innate immune response.

The innate immune response is initiated by the interaction of stereotypical pathogen components with genetically conserved receptors for extracytosolic pathogen-associated molecular patterns (PAMPs) or intracytosolic nucleic acids. In multicellular organisms, this interaction typically clusters signal transduction molecules and leads to their activations, thereby initiating signals that activate innate immune effector mechanisms to protect the host. In some cases programmed cell death-a fundamental form of innate immunity-is initiated in response to genotoxic or biochemical stress that is associated with viral infection. In this paper we will summarize innate immune mechanisms that are relevant to viral pathogenesis and outline the continuing evolution of viral mechanisms that suppress the innate immunity in mammalian hosts. These mechanisms of viral innate immune evasion provide significant insight into the pathways of the antiviral innate immune response of many organisms. Examples of relevant mammalian innate immune defenses host defenses include signaling to interferon and cytokine response pathways as well as signaling to the inflammasome. Understanding which viral innate immune evasion mechanisms are linked to pathogenesis may translate into therapies and vaccines that are truly effective in eliminating the morbidity and mortality associated with viral infections in individuals.

Application of quartz crystal microbalance with dissipation monitoring technology for studying interactions of poxviral proteins with their ligands.

Poxviruses are one of the most complex of animal viruses and encode for over 150 proteins. The interactions of many of the poxviral-encoded proteins with host proteins, as well as with other proteins, such as transcription complexes, have been well characterized at the qualitative level. Some have also been characterized quantitatively by two hybrid systems and surface plasmon resonance approaches. Presented here is an alternative approach that can enable the understanding of complex interactions with multiple ligands. The example given is that of vaccinia virus complement control protein (VCP). The complement system forms the first line of defense against microorganisms and a failure to appropriately regulate it is implicated in many inflammatory disorders, such as traumatic brain injury, Alzheimer's disease (AD), and rheumatoid arthritis. The complement component C3 is central to the complement activation. Complement regulatory proteins, capable of binding to the central complement component C3, may therefore effectively be employed for the treatment and prevention of these disorders. There are many biochemical and/or immunoassays available to study the interaction of proteins with complement components. However, protocols for many of them are time consuming, and not all assays are useful for multiple screening. In addition, most of these assays may not give information regarding the nature of binding, the number of molecules interacting with the complement component C3, as well as kinetics of binding. Some of the assays may require labeling which may induce changes in protein confirmation. We report a protocol for an assay based on quartz crystal microbalance with dissipation monitoring (QCM-D) technology, which can effectively be employed to study poxviral proteins for their ability to interact with their ligand. A protocol was developed in our laboratories to study the interaction of VCP with the complement component C3 using Q-sense (D-300), equipment based on QCM-D technology. The protocol can also be used as a prototype for studying both proteins and small-sized compounds (for use as anti-poxvirals) for their ability to interact with and/or inhibit the activity of their ligands.

Characterization of poxvirus-encoded proteins that regulate innate immune signaling pathways.

Innate immune recognition of pathogens is critical to the prompt control of infections, permitting the host to survive to develop long-term immunity via an adaptive immune response. Poxviruses encode a family of proteins that inhibit signaling by Toll-like receptors to their downstream signaling components, severely limiting nuclear translocation of transcription factors such as IRF3 and NF-κB and thereby decreasing production of host interferons and cytokines. We describe bioinformatics techniques for identifying candidate poxviral inhibitors of the innate immune response based on similarity to the family of proteins that includes A52, A46, and N1. Robust luciferase assays can determine whether a given poxviral gene affects innate immune signaling, and in combination with other approaches can identify the cellular targets of poxviral innate immune evasion genes. Because apoptosis is an innate immune response of the cell to viral infection, assays for identifying poxviral genes that inhibit apoptosis can also be employed. Novel poxviral innate immune inhibitors are being identified via several approaches and these techniques promise to identify further complexities in the way that poxviruses interact with the host innate immune system.

Central nervous system distribution of the poxviral proteins after intranasal administration of proteins and titering of vaccinia virus in the brain after intracranial administration.

Poxviral proteins are known to interact with the immune system of the host. Some of them interact with the transcription factors of the host, whereas others interact with the components of the immune system. Vaccinia virus secretes a 28.8-kDa complement control protein (VCP), which is known to regulate the complement system. This protein helps the virus to evade the immune response of the host. Such viral proteins might also prove beneficial in the treatment and prevention of the progression of the disorders, where up-regulation of the complement system is evident. VCP has been shown experimentally to be effective in protecting tissues from inflammatory damage in the rodent models of Alzheimer's diseases (AD), spinal cord injury, traumatic brain injury, and rheumatoid arthritis. Not only VCP, but also other poxviral proteins could be used therapeutically to treat or prevent the progression of the brain disorders, where the immune system is inadequately controlled. However, being a protein that cannot traverse the brain barrier because of its size, delivery of such proteins to the central nervous system (CNS) could be a limiting factor in their usefulness as CNS therapeutics. In this chapter, we show methods for the intranasal route of administration of a protein and show ways to detect its distribution in the cerebrospinal fluid (CSF) and to the different parts of the brain. These protocols can be extended to examine the distribution of viral antigens in the brain. A protocol is also included to quantitate vaccinia virus in different segments of the brain after intracranial administration of the virus.

Influenza virus variation in susceptibility to inactivation by pomegranate polyphenols is determined by envelope glycoproteins.

Pomegranates have high levels of polyphenols (PPs) and may be a rich source of compounds with antiviral activity. We evaluated the direct anti-influenza activity of three commercially available pomegranate extracts: pomegranate juice (PJ), a concentrated liquid extract (POMxl), and a 93% PP powder extract (POMxp). The acidity of PJ and POMxl solutions contributed to rapid anti-influenza activity, but this was not a factor with POMxp. Studies using POMxp showed that 5min treatment at room temperature with 800µg/ml PPs resulted in at least a 3log reduction in the titers of influenza viruses PR8 (H1N1), X31 (H3N2), and a reassortant H5N1 virus derived from a human isolate. However, the antiviral activity was less against a coronavirus and reassortant H5N1 influenza viruses derived from avian isolates. The loss of influenza infectivity was frequently accompanied by loss of hemagglutinating activity. PP treatment decreased Ab binding to viral surface molecules, suggesting some coating of particles, but this did not always correlate with loss of infectivity. Electron microscopic analysis indicated that viral inactivation by PPs was primarily a consequence of virion structural damage. Our findings demonstrate that the direct anti-influenza activity of pomegranate PPs is substantially modulated by small changes in envelope glycoproteins.

Investigation of interaction of vaccinia virus complement control protein and curcumin with complement components c3 and c3b using quartz crystal microbalance with dissipation monitoring technology.

C3 and C3b, the components central to the complement activation, also play a damaging role in several inflammatory disorders. Vaccinia virus complement control protein (VCP) and curcumin (Cur) are natural compounds with different biological origins reported to regulate complement activation. However, both VCP and Cur have not been investigated for their interaction with the third component (C3) prior to it being converted to its activated form (C3b). These two compounds have also not been compared to each other with respect to their interactions with C3 and C3b. Quartz crystal microbalance with dissipation monitoring (QCM-D) is a novel technology used to study the interaction of biomolecules. This technology was applied to characterize the interactions of VCP, Cur and appropriate controls with the key complement components. Cur as well as VCP showed binding to both C3 and to C3b, Cur however bound to C3b to a lesser extent.

Influence of glycosylation and oligomerization of vaccinia virus complement control protein on level and pattern of functional activity and immunogenicity.

Vaccinia virus complement control protein (VCP) is one of the proteins encoded by vaccinia virus to modulate the host inflammatory response. VCP modulates the inflammatory response and protects viral habitat by inhibiting the classical and the alternative pathways of complement activation. The extended structure of VCP, mobility between its sequential domains, charge distribution and type of residues at the binding regions are factors that have been identified to influence its ability to bind to complement proteins. We report that a Lister strain of vaccinia virus encodes a VCP homolog (Lis VCP) that is functional, glycosylated, has two amino acids less than the well-characterized VCP from vaccinia virus WR strain (WR VCP), and the human smallpox inhibitor of complement enzymes (SPICE) from variola virus. The glycosylated VCP of Lister is immunogenic in contrast to the weak immunogenicity of the nonglycosylated VCP. Lis VCP is the only orthopoxviral VCP homolog found to be glycosylated, and we speculate that glycosylation influences its pattern of complement inhibition. We also correlate dimerization of VCP observed only in mammalian and baculovirus expression systems to higher levels of activity than monomers, observed in the yeast expression system.

Vaccinia virus complement control protein (VCP) improves kidney structure and function following ischemia/reperfusion injury in rats.

Renal transplantation is often confronted with ischemia reperfusion (I/R) injury that accounts for a delayed recovery of the graft. This surgically and biologically induced injury often results in activation of the complement system. The vaccinia virus complement control protein (VCP) down-regulates both the classical and alternative complement pathways by preventing the formation of C3b, a component where both pathways converge. The aim of the study was to investigate the effect of VCP on renal I/R injury. Long Evans rats were subjected to laparotomy, mobilization of the right kidney in unilateral ischemia, and both kidneys in bilateral ischemia. The renal arteries were clamped for 60 min followed by 24 h reperfusion time. The animals were randomly allocated to receive recombinant VCP (rVCP), natural VCP, and humanized recombinant VCP (hrVCP) combination, vehicle (PBS), or sham group. Blood samples were collected for biochemical studies, and the kidneys were removed for histopathologic and immunohistochemical studies. The biochemical studies in the bilateral ischemia showed that the PBS group displayed 1.5-fold and 5-fold increases in the urea and creatinine concentrations, respectively, compared with the VCP/hrVCP groups. In both models, the histopathologic study revealed focal necrosis of the tubular epithelial cells in the rVCP or VCP/hrVCP treated animals compared with the diffuse and markedly elevated field scores in the PBS controls. The immunohistochemical study showed significant C3 deposition in the renal tubules of the PBS controls compared with the rVCP or VCP/hrVCP groups, suggesting that rVCP, VCP/hrVCP reduced I/R injury by inhibiting the biosynthesis of C3.

Inhibition of complement and CD14 attenuates the Escherichia coli-induced inflammatory response in porcine whole blood.

The innate immune response is a double-edged sword in systemic inflammation and sepsis. Uncontrolled or inappropriate activation can damage and be lethal to the host. Several studies have investigated inhibition of downstream mediators, including tumor necrosis factor alpha (TNF-alpha) and interleukin-1beta (IL-1beta). Emerging evidence indicates that upstream inhibition is a better therapeutic approach for attenuating damaging immune activation. Therefore, we investigated inhibition of two central innate immune pathways, those of complement and CD14/Toll-like receptor 4 (TLR4)/myeloid differentiation protein 2 (MD-2), in a porcine in vitro model of Escherichia coli-induced inflammation. Porcine whole blood anticoagulated with lepuridin, which did not interfere with the complement system, was incubated with E. coli lipopolysaccharide (LPS) or whole bacteria. Inhibitors of complement and CD14 and thus the LPS CD14/TLR4/MD-2 receptor complex were tested to investigate the effect on the inflammatory response. A broad range of inflammatory readouts were used to monitor the effect. Anti-CD14 was found to saturate the CD14 molecule on granulocytes and completely inhibited LPS-induced proinflammatory cytokines in a dose-dependent manner. Anti-CD14 significantly reduced the levels of the E. coli-induced proinflammatory cytokines TNF-alpha and IL-1beta, but not IL-8, in a dose-dependent manner. No effect on bacterial clearance was seen. Vaccinia complement control protein and smallpox inhibitor of complement enzymes, two Orthopoxvirus-encoded complement inhibitors, completely inhibited complement activation. Furthermore, these agents almost completely inhibited the expression of wCD11R3, which is associated with CD18 as a beta2 integrin, on porcine granulocytes and decreased IL-8 levels significantly in a dose-dependent manner. As expected, complement inhibition reduced bacterial clearance. We conclude that inhibition of complement and CD14 attenuates E. coli-induced inflammation and might be used as a therapeutic regimen in gram-negative sepsis along with appropriate treatment with antibiotics.

Robust intrapulmonary CD8 T cell responses and protection with an attenuated N1L deleted vaccinia virus.

BACKGROUND: Vaccinia viruses have been used as a model for viral disease and as a protective live vaccine. METHODOLOGY AND PRINCIPAL FINDINGS: We investigated the immunogenicity of an attenuated strain of vaccinia virus engineered to inactivate the N1L gene (vGK5). Using the intranasal route, this recombinant virus was 2 logs less virulent compared to the wildtype VACV-WR. Infection by the intranasal, intraperitoneal, and tail scarification routes resulted in the robust induction of cytolytic virus-specific CD8 T cells in the spleens and the lungs. VACV-specific antibodies were also detected in the sera of mice infected 3-5 months prior with the attenuated vGK5 virus. Finally, mice immunized with vGK5 were significantly protected when challenged with a lethal dose of VACV-WR. CONCLUSIONS: These results indicate that the attenuated vGK5 virus protects against subsequent infection and suggest that the N1L protein limits the strength of the early antiviral CD8 T cell response following respiratory infection.

Intracranial administration of vaccinia virus complement control protein in Mo/Hu APPswe PS1dE9 transgenic mice at an early age shows enhanced performance at a later age using a cheese board maze test.

One of the key pro-inflammatory mediators activated by amyloid protein in neurodegenerative disorders of the brain, such as Alzheimer's disease is the complement system. Vaccinia virus complement control protein secreted by vaccinia virus, commonly known as VCP, was found to inhibit amyloid protein mediated up-regulation of complement system in vitro. In the current research investigation, VCP was administered twice (First dose at 3 weeks and the second dose at 6-7 months) intracranially into the parietal cortical area of Mo/Hu APPswe transgenic mice. At the age of 2 years or more, the same mice were subjected to cued-learning, spatial learning, probe and reverse probe trial paradigms of cheese board maze tasks for cognitive assessment. A significant difference was observed between VCP treated mice and the transgenic controls on days two and three of the cued trials and probe trials. The VCP treated group showed a similar trend as revealed during the spatial learning trial and reverse probe trial. A differential pattern of thioflavine S staining was observed in the VCP treated group. These results suggest that administration of VCP at an early age in transgenic mice may be effective in regulating the progression to the familial form of Alzheimer's disease at a later age.

Early detection of memory deficits and memory improvement with vaccinia virus complement control protein in an Alzheimer's disease model.

Vaccinia virus complement control protein (VCP) inhibits both the classical and alternate complement pathways. In diseases such as traumatic brain injury (TBI) and Alzheimer's disease (AD), pathological inflammation is caused by amongst several factors, prolonged or inappropriate activation of the complement system and is a significant cause of neurodegeneration. This study investigates for the first time the use of a cheeseboard maze to evaluate cognitive deficits and the effect of VCP on memory processes in 2- and 3-month-old mice that express mutant amyloid precursor protein (APPswe) and mutant presenilin 1 (Ps1dE9) that correspond to a form of early onset AD. A four-phase training schedule was carried out on the cheeseboard maze before intracranial injections of 5 microl of VCP (1.7 microg/microl) or 5 microl saline. Two weeks later the effect of VCP on memory was evaluated. A statistically significant decrease in goal latency in VCP-treated mice than saline-treated transgenic mice in both the first probe and reverse tasks was observed. Similarly, after a second intracranial VCP or saline injection performed 2 months later, the 6.5- and 7.5-month aged VCP-injected mice performed significantly better in goal latency in both second probe and reverse tasks than saline-treated mice. These data also demonstrated that the use of a dry maze is a sensitive technique for distinguishing cognitive measures between non-transgenic and APPswe/PS1De9 transgenic mice at a much earlier stage.