Modulation of anxiety behavior by intranasally administered vaccinia virus complement control protein and curcumin in a mouse model of Alzheimer's disease.

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.

Lack of N1L gene expression results in a significant decrease of vaccinia virus replication in mouse brain.

Vaccinia virus encodes secretory proteins termed virokines. One of the major virokines encoded by the N1L open reading frame is the 13.8 kDa protein. A recombinant virus, termed vGK5, lacking this protein when injected intracranially into mice, has one of the highest levels of in vivo attenuation achieved by deletion of any single open reading frame of vaccinia virus. Here we show that the 13.8 kDa protein significantly enhances viral replication within brain tissue; however, analysis of histology, neutrophil infiltrate, and nitric oxide synthase activity of brain tissue shows no significant differences between wild-type vaccinia virus and vGK5. Since there is poor growth of vGK5 virus in the brain, the possibility of postvaccinial encephalitis is significantly diminished. Mice injected with vGK5 became resistant to the lethal effects of vaccinia virus, indicating that vGK5 is immunogenic in the brain without being virulent and therefore is a vaccine candidate. This suggests that should vGK5 reach the brain it will not replicate efficiently but still serve as a live vaccine.

Prolonged retention of vaccinia virus complement control protein following IP injection: implications in blocking xenorejection.

The vaccinia virus complement control protein (VCP) blocks classic and alternate complement pathways by binding to the third and fourth complement components and by blocking the formation of the C3-convertase as well as by accelerating the decay of the C3 and C4 convertase. The therapeutic potential of VCP has been extensively studied for brain injury, xenotransplantation, Alzheimer's disease, and spinal cord injury. We investigated the pharmacokinetic behavior of rVCP in mice. Dosage of rVCP was studied by injecting different concentrations of rVCP. A 25 mg/kg or greater dose injected intraperitoneally was found to be adequate to suppress complement for more than 8 hours.

Dose-dependent inhibition of complement in baboons by vaccinia virus complement control protein: implications in xenotransplantation.

Vaccinia virus complement control protein (VCP) is a potent inhibitor of both the alternative and the classical complement pathways through its binding to activated third and fourth components. In addition to its complement inhibiting abilities, VCP can bind heparan sulfate on cell surfaces, resulting in further functional activities. Altogether, the multiple functions of VCP have been shown to reduce the inflammatory response of the host, helping the vaccinia virus to evade immune destruction. Recently, we reported that VCP is able to block hyperacute xenograft rejection, significantly prolonging graft survival in two separate in vivo heterotopic cervical cardiac xenograft models. Histopathological examination of the transplanted hearts receiving VCP revealed marked VCP deposition on the endothelium, a significant reduction in cardiac tissue damage, and significantly less C3, IgG and IgM deposition in the tissue. It is concluded that VCP may inhibit hyperacute xenorejection by binding to the endothelial surface, blocking complement fixation activation, thereby preventing xenoantibody attachment. In the current study, the level of serum complement inhibition was evaluated following different bolus dosages of VCP in baboons. The results indicated that to achieve a satisfactory level of complement inhibition higher doses of VCP are needed in baboons, than previously observed in rats. The current observations are critical for future assessment of the role of VCP to suppress hyperacute rejection following pig-to-baboon xenotransplantation.

Vaccinia virus complement control protein modulates inflammation following spinal cord injury.

The vaccinia virus complement control protein (VCP) possesses multiple modulatory functions. Functioning as a complement inhibitory protein, VCP reduces production of proinflammatory chemotactic factors produced during complement activation. Additionally, VCP binds heparin and heparan sulfate proteoglycans, resulting in added functions shown to block monocyte chemotaxis in vitro. Using an in vivo spinal cord contusive injury model in rats, the inflammation-modulating abilities of VCP were evaluated. The results of both myeloperoxidase assaying and H&E stained section counts of spinal tissue reveal that neutrophil infiltration to the area of the lesion was reduced in animals that received VCP as compared to saline-injected controls.

The role of the carboxyl-terminal fragments of amyloid precursor protein in Alzheimer's disease.

Two major pathological hallmarks of Alzheimer's disease (AD) are the senile plaques that are primarily composed of amyloid beta-peptide (Abeta) and neurofibrillary tangles consisting of tau aggregates. Abeta is generated proteolytically from a family of Abeta-containing precursor proteins (APP; 695-770 amino acid) by secretase enzymes to different specific carboxyl-terminal fragments (CTFs). Herein we examined APP and its products in autopsied brain sections from 10 AD and 10 non-AD control subjects immunochemically using an antibody that was raised against APP751-770 residue (O443). The O443 antibody was initially characterized by Western blot analysis and immunoprecipitation. In this study, we used this antibody for immunohistochemical analysis to determine the distribution of APP and its CTF species. In 10 brain regions showing different levels of plaques and tangles, antibody O443 stained the perinuclear region of the nucleus, plaques, and neurites. Tangle-bearing neurons also appeared to stain with the antibody, suggesting that these dysfunctional neurons continue to synthesize APP/CTF. Alternatively, the normally short-lived APP/CTF can be stabilized and persist in these neurons. Taken together, these results suggest that, in addition to the widely believed role of Abeta, CTFs may play a key role in the pathogenesis of AD. Studying their localization and biogenesis may reveal the biological activities of CTFs of APP. The present study may pave the way for possible antiamyloidogenic therapy in the treatment of AD.

Virokines: novel immunomodulatory agents.

Since the discovery of virokines in the 1980s, much time and research has been dedicated to exploring their potential use as therapeutic agents. Simply put, virokines are virally encoded proteins that are secreted from the infected host cell. Most of these proteins possess the ability to modulate different aspects of the host immune system, to better maintain a suitable habitat for viral replication. These proteins are often highly homologous to host immune proteins but are often smaller and more powerful. Examples of virokines include viral secreted proteins that: block components of the complement system, act as serine protease inhibitors, function as chemokine and cytokine agonists or antagonists and contribute to cell proliferation. Many of these proteins are currently being investigated for use as novel therapeutic immunomodulators to manage immune disorders, inflammation after trauma, graft rejection and autoimmune diseases.

Vaccinia virus complement control protein is capable of protecting xenoendothelial cells from antibody binding and killing by human complement and cytotoxic cells.

BACKGROUND: Vaccinia virus complement control protein (VCP) was the first secretory microbial protein shown to have structural similarity to the family of complement control proteins. VCP can block both the classical and alternate complement pathways. Recently, VCP has been shown to bind to heparin, and this property contributes to separate functions, making the molecule a multifunctional protein. METHODS: VCP prepared from a natural infection of RK-13 cells with vaccinia virus was purified to homogeneity. Cultured pig aortic endothelial cells (PAECs) were mixed with human serum, anti-Gal alpha1,3 Gal antibody, neutrophils, or natural killer (NK) cells in the presence or absence of VCP and either direct binding of FITC-labeled antibody or killing by cytotoxic cells was estimated. RESULTS: Our cell culture studies demonstrate that VCP blocks complement-mediated killing of PAECs by human serum in a dose-dependent manner. We also demonstrate that VCP is capable of blocking Gal alpha1,3 Gal binding sites on PAECS. Surprisingly, VCP effectively blocked interactions between PAECs and cytotoxic cells such as human naive neutrophils and NK cells. CONCLUSION: VCP is a novel protein amongst the complement control protein family and can, not only block xenorejection by inhibiting complement but also by blocking killing by cytotoxic cells.

RETRACTED: Crystal structure of a complement control protein that regulates both pathways of complement activation and binds heparan sulfate proteoglycans.

This article has been retracted: please see Elsevier Policy on Article Withdrawal (https://www.elsevier.com/about/our-business/policies/article-withdrawal). This article has been retracted at the request of request of the editors. Cell is retracting this paper reporting structures of a poxvirus protein, VCP, that inhibits the complement system. The paper presents a structural model derived from two crystal forms of the protein (PDB: 1G40 and 1G44) that defines an interaction surface implicated in inhibition of complement C3 proteins and visualizes heparin binding sites. We were contacted by the University of Alabama, Birmingham (UAB), the corresponding author's institution, with a report detailing concerns about the veracity of the structures and recommending that the structures be retracted from the Protein Data Bank. We then conducted an assessment with input from experts in the field who found that the structures as presented in the paper were not consistent with available data, including spatial packing and structure (B) factors. These findings were consistent with issues contained in the UAB report. A subsequent investigation by the Department of Health and Human Services Office of Research Integrity (https://www.federalregister.gov/documents/2018/04/16/2018-07782/findings-of-research-misconduct) has concluded that the corresponding author, Krishna H.M. Murthy, engaged in research misconduct and that the structures were falsified and/or fabricated. Given the results of our own assessment and the institutional investigations, the most appropriate course of action is to retract the paper. Co-authors Nick Mullin, Paul N. Barlow, and Craig M. Ogata support this retraction.

A novel approach using an attenuated recombinant vaccinia virus to test the antipoxviral effects of handsoaps.

Evidence indicates an increase in nosocomial and household infections due to viruses (Jeffries, D.J., 1995. Viral hazards to and from health care workers. J. Hosp. Infect. 30, 140-155). An antiviral assay was developed for evaluating efficiency of handsoaps at inactivating cell-free and cell-associated virus. A recombinant vaccinia virus, lacking a virulence factor (Isaacs, S.N., Kotwal, G.J., Moss, B., 1992. Vaccinia virus complement-control protein prevents antibody-dependent complement-enhanced neutralization of infectivity and contributes to virulence. Proc. Natl. Acad. Sci. USA 89, 628-632), whose construction was described earlier (Kotwal, G.J., Isaacs, S.N., McKenzie, R., Frank, M.M., Moss, B., 1990. Inhibition of the complement cascade by the major secretory protein of vaccinia virus. Science 250, 827-830), was used as the representative poxvirus. Two antibacterial handsoaps, one surgical handsoap, one moisturizing handsoap, and a sanitizing agent were tested. An aliquot of the virus was mixed and incubated with soap, then titrated onto BSC-1 cells for incubation at 37 degrees C for 48 h. The soaps' effect on cell-associated virus was tested similarly. The antibacterial soaps inactivated all cell-free virus in 1 min. The surgical soap was effective with a 5-min incubation. None of the soaps eliminated all of the cell-associated virus in 1 min. This safe and reproducible assay seems efficient to establish the comparative efficacy of household and surgical soaps.

Conserved surface-exposed K/R-X-K/R motifs and net positive charge on poxvirus complement control proteins serve as putative heparin binding sites and contribute to inhibition of molecular interactions with human endothelial cells: a novel mechanism for evasion of host defense.

Vaccinia virus complement control protein (VCP) has been shown to possess the ability to inhibit both classical and alternative complement pathway activation. The newly found ability of this protein to bind to heparin has been shown in previous studies to result in uptake by mast cells, possibly promoting tissue persistence. It has also been shown to reduce chemotactic migration of leukocytes by blocking chemokine binding. In addition, this study shows that VCP-through its ability to bind to glycosaminoglycans (heparin-like molecules) on the surface of human endothelial cells-is able to block antibody binding to surface major histocompatibility complex class I molecules. Since heparin binding is critical for many functions of this protein, we have attempted to characterize the molecular basis for this interaction. Segments of this protein, generated by genetic engineering of the DNA encoding VCP into the Pichia pastoris expression system, were used to localize the regions with heparin binding activity. These regions were then analyzed to more specifically define their properties for binding. It was found that the number of putative binding sites (K/R-X-K/R), the overall positive charge, and the percentage of positively charged amino acids within the protein were responsible for this interaction.

Poxviral mimicry of complement and chemokine system components: what's the end game?

Numerous viral proteins mimic host immunoregulatory proteins, both structurally and functionally. This phenomenon appears to underlie viral evasion of host defense. These viral immunomodulatory proteins block viral neutralization and destruction of infected cells, and are also able to influence their habitat, preserving habitats that favor their growth and that of their progeny. The end game seems to vary widely among viruses.

Local neutrophil influx following lateral fluid-percussion brain injury in rats is associated with accumulation of complement activation fragments of the third component (C3) of the complement system.

Traumatic brain injury can lead to locally destructive secondary events mediated by several inflammatory components. Following lateral fluid-percussion (FP) brain injury in rats, we examined cortical and hippocampal sections for neutrophil infiltration and accumulation of complement component C3. Neutrophil influx into the brain after injury was detected by an improved myeloperoxidase (MPO) microassay and manual cell counting, while C3 accumulation was detected using immunocytochemistry. MPO levels were elevated in the injured cortical tissue, whereas C3 immunoreactivity was increased in both injured cortical and ipsilateral hippocampal sections. These results show that the FP model of head injury leads to an intense local inflammatory reaction and subsequent tissue destruction.

Approaches of the diagnosis of hepatitis viruses.

Hepatitis virus infection occurs in close to a billion people worldwide at some point in their lifetimes. Hepatitis B and C viruses together account for infections in half a billion people and are considered the most carcinogenic of any known biological agent. The diagnostic approaches to detect hepatitis viruses are discussed.

Elucidation of the early events contributing to zymosan-induced multiple organ dysfunction syndrome using MIP-1alpha, C3 knockout, and C5-deficient mice.

Using a zymosan-induced mouse model of multiple organ dysfunction syndrome (MODS), it has been shown that the absence of MIP-1alpha increased mortality fourfold, whereas the absence of C5 decreased mortality fourfold. The purpose of the present study was to determine the early events following zymosan injection in MIP-1alpha knockout and C5-deficient mice. B10.D2/nSnJ (C5-sufficient) and B10.D2/0SnJ (C5-deficient) and genetically matched MIP-1alpha +/+ and MIP-1alpha -/- mice were divided into 3 groups: Group1 received no injection, Group 2 received intraperitoneal saline injection (1.0 mL), and Group 3 were given intraperitoneal zymosan (1 mg/gm, 1.0 mL). Two hours, 24 h, and 48 h after injection, peritoneal exudate leukocyte counts, total WBC count, lung MPO levels, and organ histology were examined for signs of changes in cellular infiltration. An acute local and systemic inflammatory response characterized by an increase in the peritoneal leukocyte count, total WBC counts, and circulating neutrophil levels was observed within 2-48 h of zymosan injection. Lack of MIP-1alpha attenuated local recruitment of phagocytes into the peritoneal cavity, and absence of MIP-1alpha or C5 caused a decrease in circulating neutrophil levels. The presence or absence of either C5 or MIP-1alpha did not affect early pulmonary neutrophil sequestration. Organ histopathology suggested early neutrophil infiltration in the lung and spleen within 48 h. These studies indicate that MIP-1alpha and C5 play a critical role in modulating cellular changes associated with lethality in a zymosan model of MODS.

Examination of the interactions of the amyloid precursor protein carboxyl terminus to intracellular protein: possible role in apoptosis.

1. The carboxyl terminus of the amyloid precursor protein (APP) has several identified regions that may potentially contribute to the pathogenic effects of Alzheimer's disease (AD). To examine these effects, the authors iodinated a short synthetic peptide corresponding to amino acids 679-687 of APP695. They also produced a [35S]-Methionine labeled peptide corresponding to the entire carboxyl 100 amino acids of APP695 via a reticulocyte lysate coupled in vitro transcription/translation system. 2. Human neuroblastoma cells (SK-N-SH) and non-neuronal epithelial cells (RK-13) were cultured, harvested, and lysed. The S1 cell extract fractions were combined with either of the labeled peptides and incubated at different temperatures to allow for interaction and binding of cellular proteins with the peptides. These interactions were identified as gel mobility shift patterns on native PAGE gels. The presence of distinct bands on the gels indicate that the APP C-terminus interacts with several intracellular proteins, some of which may be detrimental to the cell. The authors have tested the possibility that the accumulation of C-terminal proteins may result in apoptosis. 3. Apoptosis in neural cells is one detrimental effect that has been attributed to APP. The authors examined hippocampal tissue sections from Alzheimer's disease (AD) and age-matched normal control patients for a difference in the number of apoptotic nuclei present using an in situ apoptosis detection kit that labels the numerous free DNA ends present in apoptotic nuclei. The number of apoptotic nuclei found in AD neuronal tissue was significantly higher than in normal tissue.

Virokines: mediators of virus-host interaction and future immunomodulators in medicine.

A decade ago, after the discovery of the major secretory protein of vaccinia virus with structural similarity to complement control, the term virokine was coined. The term virokine, simply refers to a virally encoded secretory protein. During the past decade several virokines were discovered and most of them have been found to have immunomodulatory effects. A subset of virokines which resemble cytokine/chemokine receptors have been termed viroceptors. Examples of viroceptors include the TNF receptor homolog and the IL-1 beta receptor homolog. During the past several years animal models have been developed to try to understand the in vivo role of the virokines. It has become evident from at least two studies that quite a few of the virokines down-regulate the inflammatory response elicited during infection. This down-regulation at least in one model system seems to indicate that it ensures the preservation of the viral habitat (site of infection). One obvious spinoff of the research on virokines is a new class of immunomodulators has become available as therapeutics in alleviating the symptoms of inflammatory disease conditions.

Dextran sulfate inhibits IFN-gamma-induced Jak-Stat pathway in human vascular endothelial cells.

Human vascular endothelial cells can be induced by IFN-gamma to express class II MHC proteins. Previously, dextran sulfate was shown to selectively inhibit expression of class II MHC by preventing transcription of the gene encoding CIITA, a transactivator protein required for IFN-gamma-inducible expression of class II genes. In this study we characterized the effects of dextran sulfate on the intracellular events occurring prior to CIITA activation. Immunoprecipitation and Western blot analyses indicated that IFN-gamma-induced phosphorylation of Stat1 and Jak2 was blocked by dextran sulfate. In addition, electron micrographs showing the large accumulation of dextran sulfate particles in the cytoplasms of endothelial cells demonstrated that Stat and Jak proteins may directly interact with dextran sulfate. Binding of radiolabeled IFN-gamma to cells indicated that dextran sulfate may also modulate IFN-gamma interactions with the cell surface. Thus, dextran sulfate is capable of interfering with the IFN-gamma-induced expression of class II MHC genes at multiple sites.