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Kulkarni A.P.,University of Cape Town | Kellaway L.A.,University of Cape Town | Kotwal G.J.,Kotwal Bioconsulting LLC | Kotwal G.J.,Inflamed Inc. | Kotwal G.J.,Health Science University
Methods in Molecular Biology | Year: 2012

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. © 2012 Springer Science+Business Media, LLC. Source


Kulkarni A.P.,University of Cape Town | Govender D.,University of Cape Town | Kellaway L.A.,University of Cape Town | Kotwal G.J.,Kotwal Bioconsulting LLC | And 2 more authors.
Methods in Molecular Biology | Year: 2012

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. © 2012 Springer Science+Business Media, LLC. Source


Kulkarni A.P.,University of Cape Town | Govender D.A.,University of Cape Town | Kotwal G.J.,Inflamed Inc. | Kotwal G.J.,Kotwal Bioconsulting LLC | And 2 more authors.
Current Alzheimer Research | Year: 2011

Widespread neuroinflammation in the central nervous system (CNS) of Alzheimer's disease (AD) patients, involving pro-inflammatory mediators such as complement components, might be responsible for AD associated behavioral symptoms such as anxiety. Vaccinia virus complement control protein (VCP) and curcumin (Cur) are the bioactive compounds of natural origin shown to inhibit the in-vitro complement activation. In order to develop complement regulatory compounds which could be delivered to the CNS by a non-invasive route, VCP, its truncated version (tVCP), and Cur were administered to Wistar rats intranasally. The distribution of these compounds in cerebrospinal fluid (CSF) was studied using an enzyme linked immunosorbent assay (ELISA), using VCP and tVCP as antigens and a modified fluorimetric method (Cur). VCP and tVCP were also detected in the olfactory lobes of the rat brain using immunohistochemical analysis. These compounds were then compared for their ability to attenuate the anxiety levels in APPswePS1δE9 mice using an elevated plus maze (EPM) apparatus. VCP treatment significantly improved the exploratory behavior and reduced the anxiety behavior in APPswePS1δE9 mice. tVCP however showed an opposite effect to VCP, whereas Cur showed no effect on the anxiety behavior of these mice. When these mice were subsequently tested for their cognitive performance in the Morris water maze (MWM), they showed tendencies to collide with the periphery of the walls of MWM. This unusual activity was termed "kissperi" behavior. This newly defined index of anxiety was comparable to the anxiety profile of the VCP and tVCP treated groups on EPM. VCP can thus be delivered to the CNS effectively via intranasal route of administration to attenuate anxiety associated with AD. © 2011 Bentham Science Publishers Ltd. Source


Rus F.,University of Massachusetts Medical School | Morlock K.,University of Massachusetts Medical School | Silverman N.,University of Massachusetts Medical School | Pham N.,University of Massachusetts Medical School | And 4 more authors.
Methods in Molecular Biology | Year: 2012

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. © 2012 Springer Science+Business Media, LLC. Source


Ghebremariam Y.T.,University of Cape Town | Engelbrecht G.,University of Cape Town | Tyler M.,University of Cape Town | Lotz Z.,University of Cape Town | And 4 more authors.
Journal of Surgical Research | Year: 2010

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. © 2010 Elsevier Inc. All rights reserved. Source

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