The interaction between silver nanoparticles and herpesviruses is attracting great interest due to their antiviral activity and possibility to use as microbicides for oral and anogenital herpes. In this work, we demonstrate that tannic acid modified silver nanoparticles sized 13 nm, 33 nm and 46 nm are capable of reducing HSV-2 infectivity both in vitro and in vivo. The antiviral activity of tannic acid modified silver nanoparticles was size-related, required direct interaction and blocked virus attachment, penetration and further spread. All tested tannic acid modified silver nanoparticles reduced both infection and inflammatory reaction in the mouse model of HSV-2 infection when used at infection or for a post-infection treatment. Smaller-sized nanoparticles induced production of cytokines and chemokines important for anti-viral response. The corresponding control buffers with tannic acid showed inferior antiviral effects in vitro and were ineffective in blocking in vivo infection. Our results show that tannic acid modified silver nanoparticles are good candidates for microbicides used in treatment of herpesvirus infections.
Fas receptor-Fas ligand (FasL) signaling is involved in apoptosis of virus-infected cells but increasing evidence accumulates on Fas receptor as a mediator of apoptosis-independent processes such as induction of activating and pro-inflammatory signals. In this study, we examined the role of Fas/FasL pathway in regulation of anti-viral response to genital HSV-2 infection using a murine model of HSV-2 infection applied to C57BL6/J, B6. MRL-Faslpr/J and B6Smn.C3-Faslgld/J mice. HSV-2 infection of Fas- and FasL-deficient mice led to decreased migration of IFN-? expressing NK cells and CD4+ T cells, but not of ?? T cells, into the vaginal tissue. The vaginal tissues of HSV-2 infected Fas- and FasL-deficient mice showed increased production of IL-10, followed by low expression of the early CD69 activation marker on CD4+ and CD8+ T cells and increased numbers of regulatory T cells (Tregs). Experiments in co-cultures of CD4+ T cells and bone marrow derived dendritic cells showed that lack of bilateral Fas-FasL signaling led to expansion of Tregs and increased production of IL-10 and TGF-?1. Our results demonstrate that Fas/FasL can regulate development of tolerogenic dendritic cells and expansion of Tregs early during HSV-2 infection, which further influences effective anti-viral response.
Glutamate-cysteine ligase (GCL) is a heterodimer enzyme composed of a catalytic subunit (GCLC) and a modifier subunit (GCLM). This enzyme catalyses the synthesis of ?-glutamylcysteine, a precursor of glutathione. cDNAs of the putative glutamate-cysteine ligase catalytic (Ace-GCLC) and modifier subunits (Ace-GCLM) of Ancylostoma ceylanicum were cloned using the RACE-PCR amplification method. The Ace-gclc and Ace-gclm cDNAs encode proteins with 655 and 254 amino acids and calculated molecular masses of 74.76 and 28.51kDa, respectively. The Ace-GCLC amino acid sequence shares about 70% identity and 80% sequence similarity with orthologs in Loa loa, Onchocerca volvulus, Brugia malayi, and Ascaris suum, whereas the Ace-GCLM amino acid sequence has only about 30% sequence identity and 50% similarity to homologous proteins in those species. Real-time PCR analysis of mRNA expression in L3, serum stimulated L3 and adult stages of A. ceylanicum showed the highest level of Ace-GCLC and Ace-GCLM expression occurred in adult worms. No differences were detected among adult hookworms harvested 21 and 35dpi indicating expression of Ace-gclc and Ace-gclm in adult worms is constant during the course of infection. Positive interaction between two subunits of glutamate-cysteine ligase was detected using the yeast two-hybrid system, and by specific enzymatic reaction. Ace-GCL is an intracellular enzyme and is not exposed to the host immune system. Thus, as expected, we did not detect IgG antibodies against Ace-GCLC or Ace-GCLM on days 21, 60 and 120 of A. ceylanicum infection in hamsters. Furthermore, vaccination with one or both antigens did not reduce worm burdens, and resulted in no improvement of clinical parameters (hematocrit and hemoglobin) of infected hamsters. Therefore, due to the significant role of the enzyme in parasite metabolism, our analyses raises hope for the development of a successful new drug against ancylostomiasis based on the specific GCL inhibitor.
Canine babesiosis is a tick-borne disease caused by parasites of the genus Babesia. Tumour necrosis factor alpha (TNF-?) is a cytokine that plays a role in the pathogenesis of canine babesiosis. In this study, the authors determined the concentration of serum TNF-? in 11 dogs infected with Babesia canis and calculated Spearman's rank correlations between the concentration of TNF-? and blood pressure, and between TNF-? and indices of renal damage such as: fractional excretion of sodium (FE(Na(+))), urinary creatinine to serum creatinine ratio (UCr/SCr), renal failure index (RFI), urine specific gravity (USG) and urinary protein to urinary creatinine ratio (UPC). The results demonstrated statistically significant strong negative correlations between TNF-? and systolic arterial pressure (r = -0.7246), diastolic arterial pressure (r = -0.6642) and mean arterial pressure (r = -0.7151). Serum TNF-? concentration was also statistically significantly correlated with FE(Na(+)) (r = 0.7056), UCr/SCr (r = -0.8199), USG (r = -0.8075) and duration of the disease (r = 0.6767). The results of this study show there is an increase of serum TNF-? concentration during canine babesiosis, and the increased TNF-? concentration has an influence on the development of hypotension and renal failure in canine babesiosis. This probably results from the fact that TNF-? is involved in the production of nitric oxide and induction of vasodilation and hypotension, which may cause renal ischaemia and hypoxia, and finally acute tubular necrosis and renal failure.
Fasciola hepatica is a liver fluke that infects 2.4 million of people and causes great economical loss in animal production. To date a 100% effective vaccine has not been developed and the disease is controlled by drug therapy. Great efforts are put into development of effective vaccine against parasite what is difficult since Fasciola spp. (like other helmints) during evolutionary process has developed sophisticated and efficient methods to evade immune response. During preliminary experiments it is convenient to use cell lines which are relatively cheap and allow for reproducible comparison of results between laboratories. We stimulated BOMA (bovine monocyte/macrophage cell line) and BOMAC (bovine macrophage cell line) with native or recombinant antigens of Fasciola hepatica and assessed IFN-?, IL-4 and TNF-? level upon stimulation. We observed diminished secretion of proinflammatory TNF-? in LPS activated BOMA cells stimulated with Excretory/Secretory products of adult fluke (Fh-ES). We also observed greater changes in gene expression in LPS activated BOMA cells than in non activated BOMA cells upon stimulation using Fh-ES. The results show possibility of using cell lines for in vitro research of bovine immune response against liver fluke, although this model still requires validation and further characterization.
Hookworms are blood feeding intestinal nematodes that infect more than 500 million people and cause iron deficiency anemia. Infected children suffer from physical and cognitive growth retardation. Because of potential anthelminthic drug resistance, the need for vaccine development is urgent. Numerous antigens have been tested in animal models as vaccines against hookworm infection, but there is no effective human vaccine. We cloned a cDNA encoding Ancylostoma ceylanicum metalloprotease 6 (Acemep-6). Ace-MEP-6 is a protein with a predicted molecular mass of 101.87 kDa and based on computational analysis it is very likely to be engaged in food processing via hemoglobin digestion. Groups of hamsters were immunized with an Ace-mep-6 cDNA vaccine, either once or three times. Animals that were administered one dose developed high resistance (80%, p < 0.01) against challenge infection, whereas triple immunization resulted in no worm burden reduction. These results suggest that DNA vaccines can be powerful tools in ancylostomiasis control, although the mechanisms through which protection is conferred remain unclear.
Uncinaria stenocephala belongs to Ancylostomatidae family. Members of this family - hookworms - infect millions of people and animals worldwide. U. stenocephala is most pathogenic in dogs and other Canidae, which are the main hosts, and infection causes anemia or even death. So far no effective hookworm vaccine has been developed that is economically viable. Attempts to identify vaccine antigens have led to a group of aspartic proteases, which play a key role in parasite feeding, migration through host tissues and immune evasion. The cDNA of an aspartic protease from U. stenocephala was cloned using the RACE-PCR method. Computational analysis showed that the cDNA encodes a 447 amino acid protein with a molecular mass of 52kDa that shows high homology to aspartic proteases from related hookworms. Analysis identified 1 potential N-glycosylation site, 3 potential disulfide bonds and no O-glycosylation sites. The recombinant protein was expressed in Escherichia coli followed by purification and mouse immunization. Using raised anti-Us-APR-1(2) (Uncinaria stenocephala Aspartic protease-1) serum the presence of Us-APR-1 in the adult stage of U. stenocephala and the expression of homologous protease in L3 and adult stages of A. ceylanicum was confirmed. This analysis is the first phase of work exploring the biological role of Us-APR-1 in parasite-host interactions and raises hope for successful vaccine development against Uncinaria sp. and possibly Ancylostoma sp.
Ancylostoma ceylanicum belongs to a group of soil-transmitted helminths, which infect almost 576 mln people worldwide and are a major cause of anaemia and malnutrition. Upon contact with a permissive host, third-stage larvae (L3) residing in the environment become activated larvae (ssL3), a process associated with changes in the profile of gene expression. Ancylostoma secreted proteins (ASPs) are the major proteins secreted during larvae activation and play a crucial role in hookworm adaptation to parasitism. Here we report the cloning using RACE-PCR technique of three novel ASPs from the hookworm A. ceylanicum (Ace-asp-3, Ace-asp-4, and Ace-asp-5) and computational analysis of the protein sequences. All three proteins contain SCP (Sperm Coating Protein) domain characteristic for previously described ASP proteins. Real-time PCR analysis shows significant up-regulation of Ace-asp-3 and Ace-asp-5 expression in adult worms and correlated down-regulation in ssL3 larvae. On the other hand, expression of Ace-asp-4 was increased in ssL3 stages and decreased in adult parasites.
Apoptotic cell death is critical for maintaining integrity of the epithelia as well as for removal of the virus infected cells. We assessed the role of Fas/FasL-dependent pathway in apoptosis of genital epithelium during HSV-2 infection using a murine model of HSV-2 infection applied to C57BL6, MRL-Fas(lpr)/J (Fas-/-) and C3-Fasl(gld)/J (FasL-/-) mice and an in vitro model of HSV-2 infection in monocyte RAW 264.7 and keratinocyte 291.03C cell cultures and peritoneal macrophages. In contrast to keratinocyte in vitro cultures, HSV-2 infection of the monocytic cell cultures led to early induction of apoptosis. HSV-2 infection of peritoneal macrophages isolated from Fas- and FasL-deficient mice showed decreased activation of apoptosis, which could be further blocked by caspase-9 inhibitor. Infection of Fas and FasL-deficient mice increased the percentage of apoptotic cells and activation of caspase-9 in the vaginal tissue in comparison to C57BL6 wild type strain. Furthermore, Fas and FasL-deficient mice showed increased infiltration of neutrophiles in the vaginal mucosal epithelium at 3 and 7 day of infection in contrast to HSV-2 infected wild-type mice. Our results show that while the Fas/FasL pathway during HSV-2 infection of the vaginal epithelium plays an important role in controlling early local inflammatory response, mitochondrial apoptotic pathway also becomes activated by the inflammatory reaction.
Ancylostoma ceylanicum belongs to the group of parasites commonly known as hookworms, blood-sucking nematodes which infect around 576 million people and hundreds of millions of animals. The interactions between these parasites and host immune systems are complicated and yet to be determined. Hookworm infections are usually long lasting and recurrent, due in part to their ability to synthesize macromolecules capable of modulating the host immune response. The interaction of parasite proteins with host immune systems has been proven, but so far there is no data describing the influence of astacin-like metalloproteases (expressed among different parasitic nematodes) on the human immune system. The cDNA encoding A. ceylanicum metalloprotease 2 (Ace-mtp-2) was cloned using RACE-PCR. Computational analysis was used to examine the immunogenicity and recombinant Ace-MTP-2 was used to investigate its influence on human THP-1 monocytes and macrophages. The Ace-mtp-2 gene encodes an astascin-like metalloprotease, with a theoretical molecular mass of 26.7 kDa. The protease has a putative signal peptide, 11 potential phosphorylation sites, and two disulfide bridges revealed by computational analysis. Maximal expression of Ace-mtp-2 by A. ceylanicum occurs in the adult stage of the parasite, and Western blot indicates the secretory nature of the protease. This suggests the protease is working at the host-parasite interface and would likely be exposed to the hosts immune response. Recombinant protein were expressed in Escherichia coli and Pichia pastoris. Recombinant Ace-MTP-2 amplified the in vitro release of TNF? and induced release of IFN? by lipopolysaccharide activated THP-1 macrophages. The presence of Ace-MTP-2 in secretory products of the adult parasite and the induction of IFN? release may suggest an important role for Ace-MTP-2 in host-parasite interactions since IFN? is suggested to be responsible for the protective immune response against adult hookworms.
The liver fluke Fasciola hepatica infects a wide range of hosts, and has a considerable impact on the agriculture industry, mainly through infections of sheep and cattle. Further, human infection is now considered of public health importance and is hyperendemic in some regions. The fluke infection causes considerable damage to the hosts liver. However, the mechanisms of liver destruction have not yet been completely elucidated. In the present report we incubated a human liver cell line in the presence of either F. hepatica excretory/secretory material (FhES) or recombinant phosphoglycerate kinase (FhPGK). Dosedependent cytotoxicity in the presence of FhES was observed, indicating that FhES is capable of killing human hepatocytes, supporting a role for FhES in damaging host liver cells during infection; while treatment with a recombinant intracellular protein - FhPGK, had no impact on cell survival.
Monocytic cells represent important cellular elements of the innate and adaptive immune responses in viral infections. We assessed the role of Fas/FasL in promoting monocyte apoptosis during HSV-2 infection by using an in vitro model based on the murine RAW 264.7 monocytic cell line and an in vivo murine model of HSV-2 infection applied to C57BL6, MRL-Fas(lpr)/J (Fas-/-) and C3-Fasl(gld)/J (FasL-/-) mice. HSV-2 infection of the monocytic cell line led to early induction of apoptosis, with no protective expression of anti-apoptotic Bcl-2. HSV-2 infected monocytes up-regulated Fas and FasL expression early during in vitro infection but were susceptible to Fas induced apoptosis. The vaginal monocytes in the HSV-2 murine model of infection up-regulated FasL expression and were susceptible to Fas induced apoptosis. HSV-2 infection of Fas and FasL- deficient mice led to decreased apoptosis of monocytes and impaired recruitment of NK, CD4+ and CD8+ T cells within the infection sites. The vaginal lavages of HSV-2 infected Fas and FasL- deficient showed decreased production of CXCL9, CXCL10 and TNF-? in comparison to HSV-2 infected wild-type mice strain. The decreased recruitment of immune competent cells was accompanied by delayed virus clearance from the infected tissue. Triggering of the Fas receptor on HSV-2 infected monocytes in vitro up-regulated the expression of CXCL9 chemokines and the cytokine TNF-?. Our study provides novel insights on the role of Fas/FasL pathway not only in apoptosis of monocytes but also in regulating local immune response by monocytes during HSV-2 infection.
The purpose of this study was to specify the occurrence and prevalence of Babesia microti in hard ticks removed from dogs in Warsaw (central Poland). Among 590 collected ticks, 209 were identified as Ixodes ricinus, and 381 as Dermacentor reticulatus. B. microti DNA was detected in 11 out of 590 (1.86%) samples of ticks. The DNA of the parasite was detected only in lysates from female I. ricinus ticks (11 out of 193; 5.7%). The result of this study is the first evidence of B. microti in I. ricinus ticks in Warsaw.
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