Antiplatelet-derived growth factor (PDGF) activity in the saliva of ixodid ticks is linked with their long mouthparts.

The saliva of blood-feeding arthropods modulates their vertebrate hosts' haemostatic, inflammatory and immune responses to facilitate blood feeding. In a previous study, we showed that salivary gland products from ixodid tick species also manipulate the wound-healing response by targeting at least four different mammalian growth factors: transforming growth factor ß1, hepatocyte growth factor, fibroblast growth factor 2 and platelet-derived growth factor (PDGF). In addition, species that showed PDGF-binding activity also inhibited cell proliferation in vitro and induced changes in cell morphology accompanied by disruption of the actin cytoskeleton. Here, we show a correlation between the length of the tick hypostome, the sclerotized feeding tube of the mouthparts inserted into the host's skin and anti-PDGF activity. This apparent link between hypostome length, and hence the potential depth of skin damage, and PDGF-binding activity was not apparent for the other growth factors or for other cytokines important in wound healing (keratinocyte growth factor, interleukin 6 and stromal cell-derived factor 1). However, PDGF-binding activity was no longer correlated with anti-cell activities, indicating that an additional as yet unidentified activity in tick saliva may affect cellular changes in wound repair.

Evasin-3-like anti-chemokine activity in salivary gland extracts of ixodid ticks during blood-feeding: a new target for tick control.

Ticks exploit many evasion mechanisms to circumvent the immune control of their hosts including subversion of the communication language between cells of the immune system provided by chemokines and other cytokines. One subversive molecule secreted in the saliva of Rhipicephalus sanguineus is Evasin-3, a structurally unique 7 kDa protein that selectively binds the neutrophil chemoattractants, CXCL8 and (with lower affinity) CXCL1. We compared anti-human CXCL8 and anti-mouse CXCL1/KC activities in salivary gland extracts prepared from adult Amblyomma variegatum, Rhipicephalus appendiculatus and Dermacentor reticulatus ticks during blood-feeding. Both anti-CXCL8 activity and anti-CXCL1 activity were detected in all species and in both adult females and males, with consistently higher activity levels against CXCL8. These results suggest that Evasin-3-like activity is common amongst metastriate ixodid tick species, and provide further evidence of the importance to ticks in controlling neutrophils during blood-feeding. As such, Evasin-3 offers a new target for anti-tick vaccine development.

Chemokine-binding activities of M3 protein encoded by Murine gammaherpesvirus 72.

Murine gammaherpesvirus 68 (MHV-68) contains gene-encoding M3 protein expressed during the acute and persistent phase of infection. This protein features a chemokine-binding activities (Parry et al., 2000; van Berkel et al., 2000). In this study, we demonstrated that the Murine gammaherpesvirus 72 (MHV-72) also contained M3 gene with the codon-changing mutation at the position 920 nt converting amino acid (aa) 307 Asp (GAC) to Gly (GGC). The mutation in the M3 protein was localized near chemokine-binding domain and was able to change the secondary structure of M3 protein. We examined the binding activities of M3 proteins of MHV-72 and MHV-68 to five human chemokines (CCL3, CCL5, CCL11, CCL2, and CXCL8). Binding activity of MHV-72 M3 protein to CCL5 as well as to CXCL8 reached only 11.1% (day 3 p.i.) to 20% (day 4 p.i.) of the activity detected for MHV-68 M3 protein. On the other hand, MHV-72 M3 protein bound to human cytokines CCL11 and CCL2 reached about 90% of the binding detected for MHV-68 M3 protein. The binding activity of both M3 proteins to human CCL3 was similar. These data implied that mutation identified in MHV-72 M3 protein might be involved in attenuation of immune response to infection with MHV-72.

Immunomodulatory arsenal of nymphal ticks.

Ticks have developed their own immunomodulatory mechanisms to inhibit the host inflammatory response. One of them involves the ability to subvert the cytokine network at the site of tick feeding by secreting cytokine binding molecules. Most studies have focused on the immunomodulatory prowess of adult female ticks. Here we describe anti-cytokine activity in salivary gland extracts (SGEs) prepared from 2-day-fed nymphs of Dermacentor reticulatus Fabricius, Ixodes ricinus L., Rhipicephalus appendiculatus Neumann and Amblyomma variegatum Fabricius. Anti-CXCL8 activity was detected in nymphs of all species. Relatively high activity against CCL2, CCL3 and CCL11 was observed in SGEs of R. appendiculatus and A. variegatum nymphs, whereas SGEs of I. ricinus nymphs showed comparatively high anti-interleukin-2 (-IL-2) and anti-IL-4 activities. These data show that nymphs, which epidemiologically are usually more important than adults as disease vectors, possess a range of anti-cytokine activities that may facilitate pathogen transmission.

Differential anti-chemokine activity of Amblyomma variegatum adult ticks during blood-feeding.

Ticks secrete a cocktail of immunomodulatory molecules in their saliva during blood-feeding, including chemokine-binding factors that help control the activity of host immunocompetent cells. Here we demonstrate differential dynamics of anti IL-8 (CXCL8), MCP-1 (CCL2), MIP-1 (CCL3), RANTES (CCL5) and eotaxin (CCL11) activities in salivary gland extracts of adult Amblyomma variegatum. Unfed male and female ticks showed activity against all the chemokines except CCL5; anti-CCL11 activity was particularly high. However, during feeding the dynamics of anti-chemokine activity differed significantly between males and females, and varied between chemokines. In males, anti-chemokine activities increased, whereas in females they declined or increased slightly as feeding progressed. The exception was anti-CCL11 activity, which declined and then increased in both males and females. Comparison of salivary gland equivalents of individual ticks prepared at various feeding intervals revealed some differences that were most pronounced between individual females fed for 8 days. These observations reflect the feeding behaviour of male and female A. variegatum. They support the concept of 'mate guarding', in which males help their mates to engorge by controlling their host's immune response, and the possibility that ticks benefit from feeding together by exploiting molecular individuality.

Manipulation of host cytokine network by ticks: a potential gateway for pathogen transmission.

Ticks are obligatory blood-feeding arthropods that secrete various immunomodulatory molecules to antagonize host inflammatory and immune responses. Cytokines play an important role in regulating these responses. We investigated the extent to which ticks interact with the sophisticated cytokine network by comparing the effect of salivary gland extracts (SGE) of 3 ixodid tick species, Dermacentor reticulatus, Amblyomma variegatum and Ixodes ricinus, all of which are important vectors of tick-borne pathogens. Using specific ELISAs, anti-cytokine activity was demonstrated with 7 cytokines: IL-8, MCP-1, MIP-1alpha, RANTES, eotaxin, IL-2 and IL-4. The results varied between species, and between adult males and females of the same species. Relatively high activity levels were detected in saliva of female D. reticulatus, confirming that the observed anti-cytokine activities are an integral part of tick saliva secreted into the host. Results with fractionated SGE indicated that from 2 to 6 putative cytokine binding molecules are produced, depending on species and sex. Binding ability of SGE molecules was verified by cross-linking with radio-isotope labelled MIP-1alpha. By targeting different cytokines, ixodid ticks can manipulate the cytokine network, which will greatly facilitate blood-feeding and provide a gateway for tick-borne pathogens that helps explain why ticks are such efficient and effective disease vectors.

Vesicular stomatitis virus nucleocapsid protein production in cells treated with selected fast protein liquid chromatography fractions of tick salivary gland extracts.

A salivary gland extract (SGE) prepared from 5-days-fed Dermacentor reticulatus female ticks was fractionated by fast protein liquid chromatography (FPLC). The effect of three FPLC fractions selected on the basis of anti-interleukin 8 (anti-IL-8) activity on vesicular stomatitis virus (VSV) nucleocapsid (N) protein formation in mouse L-cells was determined. Infected 14C-labeled cells treated with the FPLC fractions were analyzed by two-dimensional (2D) electrophoresis. The yields of VSV N protein were evaluated by Imagemaster software analysis. Most noticeable was an increase in the N protein production after treatment with the fraction 39 corresponding to the major peak of the anti-IL-8 activity. The nature of the substance in SGE that was responsible for this effect remains unclear.

Effect of Mahonia aquifolium stem bark crude extract and one of its polysaccharide components on production of IL-8.

The crude hydroalcoholic extract of Mahonia aquifolium stem bark and a polysaccharide isolated from the extract were tested for their activity on interleukin-8 (IL-8) production by human monocytic cell line THP-1. The crude extract partly inhibited the IL-8 spontaneous production after 48-h treatment of the cells, while the polysaccharide was found to be a potent inducer of IL-8 production.

Anti-interleukin-8 activity of tick salivary gland extracts.

Interleukin-8 (IL-8) is one of many mammalian chemokines (chemotactic cytokines) that direct mammalian inflammatory and immune cells to sites of injury and infection. Chemokines are produced locally and act on leucocytes through selective receptors. The principal role of IL-8 is to control the movement and activity of neutrophils. To date, several tick species have been shown to modulate the production or activity of certain cytokines but none of these are chemokines. Using an IL-8 specific ELISA, we showed that salivary gland extracts (SGE) from several ixodid tick species (Dermacentor reticulatus, Amblyomma variegatum, Rhipicephalus appendiculatus, Haemaphysalis inermis and Ixodes ricinus) reduced the level of detectable IL-8. Analyses of fractionated SGE revealed one similar peak of activity for D. reticulatus, A. variegatum and R. appendiculatus; a second peak, observed for D. reticulatus and A. variegatum, differed between the two species. Using radiolabelled IL-8, SGE and peak activity fractions of D. reticulatus were shown to bind the chemokine, and to inhibit binding of IL-8 to its receptors on human granuolocytes enriched for neutrophils. The biological significance of these observations was demonstrated by the ability of SGE to inhibit IL-8 induced chemotaxis of human blood granulocytes. Future isolation and characterization of the active molecules will enable determination of their functional roles in bloodfeeding and effect on tick-borne pathogen transmission.

Significance of anti-interferon-alpha2 and sICAM-1 activities in the sera of viral hepatitis B and C patients treated with human recombinant interferon-alpha2.

In this study the presence of an IFN-binding activity in the sera of patients with chronic viral hepatitis B or C treated with rIFN-alpha2 was screened by a radioimmune assay (RIA) using radiolabeled rIFN-alpha2. Incidence of an anti-IFN activitywas compared with hepatitis B virus (HBV) or hepatitis C virus (HCV) serum markers as hepatitis B s antigen (HBsAg), hepatitis B e antigen (HBeAg), antibodies to HBsAg (anti-HBsAg), antibodies to HBeAg (anti-HBeAg), seroconversion, HBV DNA, HCV RNA, and serum soluble intracellular adhesion molecule I (sICAM). Injections (intramuscular) of rIFN-alpha2 caused an anti-rIFN activity formation in 8 (27.6%) of 29 patients with chronic active hepatitis B (CAH-B) and in 8 (30.8%) of 26 patients with chronic active hepatitis C (CAH-C). The presence of the anti-rIFN activity in CAH-B patients correlated frequently with the persistence of HBsAg, HBeAg and HBV-DNA, while its absence was often accompanied by the anti-HBeAg and anti-HBsAg seroconversion, respectively, and HBV-DNA negativity. In two CAH-C patients who became HCV RNA-negative no anti-IFN activity was found. Levels of serum sICAM-1 in CAH-B patients responding to the IFN treatment were higher than those in non-responders or in which the anti-IFN activity was present. The anti-IFN activity may negatively influence the effect of the IFN therapy of CAH-B or CAH-C patients at early stages of the therapy. The appearance of the anti-IFN activity at the end of a long-term IFN therapy does not seem to influence the outcome of the therapy. sICAM-1 may be involved in the process of CAH-B reactivation and IFN-triggered cytotoxicity during the IFN therapy.

Comparison of the protein profiles of salivary gland extracts derived from three species of unfed and partially fed ixodid ticks analysed by SDS-PAGE.

Salivary gland extracts (SGE) from unfed and 5 days fed adult female Ixodes ricinus (Linnaeus, 1758); Haemaphysalis inermis (Birula, 1895) and Dermacentor reticulatus (Fabricius, 1794) ticks were prepared. The protein content after feeding increased by 10.6, 8.7 and 6.8 times, respectively. Extracts were equilibrated to the same protein content and submitted to SDS-polyacrylamide gel electrophoresis followed by computer analysis of the scanned gels. Relative differences in protein profiles of extracts obtained from unfed and partially fed ticks were found in all species and some of them were similar in all three species used in the study. Results demonstrate that the increase of the protein content in salivary glands during the feeding does not occur proportionally. Some proteins are synthesised preferentially (67.1 kDa, 13.5 kDa) but other bands (in range of 15-16 kDa) present in the SGE derived from unfed ticks are less discernible in that of fed ticks.

Inhibition of the antiviral action of interferon by tick salivary gland extract.

The saliva of haematophagous arthropods (e.g. mosquitoes, sandflies and ticks) contains potent immunomodulatory activities that counter their hosts' haemostatic, inflammatory and immune responses to facilitate blood-feeding. Such effects are exploited by arthropod-transmitted pathogens to promote their transmission. We investigated the ability of tick saliva to enhance arthropod-borne virus (arbovirus) transmission by determining its effect on the antiviral action of murine interferon (IFN alpha/beta). Salivary gland extract (SGE) was prepared from partially fed adult female Dermacentor reticulatus ticks that had been feeding on mice for either 3 or 5 days (SGED3 and SGED5, respectively). We demonstrated that SGE inhibits the antiviral effect of IFN as measured by a biological assay using vesicular stomatitis virus (VSV), and by two-dimensional electrophoretic analysis of the appearance of selected VSV proteins. The most pronounced effect was observed when mouse L cells were treated with SGE prior to IFN treatment. Following pretreatment with SGE, virus multiplication (which was fully blocked by IFN treatment alone) achieved yields similar to those obtained from infected cells not treated with IFN. Contemporaneous treatment, or treatment with SGE after IFN, was less effective. In parallel with these findings, formation of early viral proteins, N (nucleocapsid protein) and P (phosphoprotein), which was blocked by IFN, was detectable following pretreatment with SGE. The ability to inhibit the antiviral action of IFN was higher for SGED3 compared to SGED5. Demonstration that tick SGE can promote virus replication by suppressing the action of IFN helps explain why ticks are such efficient vectors of arboviruses.

Comparison of manganese superoxide dismutase precursor induction ability in human hepatoma cells with or without hepatitis B virus DNA insertion.

In a previous study (Hajnická et al., Acta virol. 38, 55-57 (1994)), we described synthesis of a 23 K protein in high amounts in the PLC/PRF/5 human hepatoma cell line after stimulation with sera of patients suffering from liver cirrhosis. In this study we identified this protein as manganense superoxide dismutase (Mn-SOD). When PLC/PRF/5 cells stimulated by various cytokines (interleukin-1 alpha (IL-1 alpha), IL-1 beta, tumor necrosis factor-alpha (TNF-alpha), TNF-beta, IL-6, tumor growth factor-alpha (TGF-alpha), TGF-beta, and interferon-gamma (IFN-gamma) were compared, the most effective was IL-1, followed by TNF-alpha and IL-6. Other cytokines had no effect on the stimulation of Mn-SOD. IL-1 alpha was selected for stimulation of Mn-SOD production in four human hepatoma cell lines (PLC/PRF/5, Hep-3B, Hep-G2 and Sk-Hep 1). Maximum Mn-SOD production occurred in PLC/PRF/5 cells. In other cell lines, Mn-SOD production was lower, reaching 35.7% and 31.5% in Hep-3B and Sk-Hep-1 cells, respectively, while it was only 4.3% in Hep-G2 cells.

Promotion of vesicular stomatitis virus nucleocapsid protein production by arthopod saliva.

In a previous study (Hajnicka, V. et al., Parasitology 116, 533-538, 1998), the infectivity titer of vesicular stomatitis virus (VSV) was shown to increase up to 10,000-fold when mouse L cells were treated with tick salivary gland extract (SGE) prior to infection. To examine this effect at the level of viral protein production, radiolabeled VSV-infected cells were analyzed by double-dimensional gel electrophoresis. A pre-treatment of cells with SGE from partially fed ticks in amounts corresponding to 1 or 3 salivary glands increased the level of both viral nucleocapsid (N) protein and phosphoprotein (P) in a dose-dependent manner. The effect was more pronounced for N protein and could account for the dramatic increase in infectious virus yield. Promotion of viral infectivity by arthropod saliva may support the arthropode-borne transmission cycle of VSV.

Tick salivary gland extracts promote virus growth in vitro.

Saliva of blood-feeding arthropods promotes infection by the vector-borne pathogens they transmit. To investigate this phenomenon in vitro, cultures of mouse L cells were treated with a salivary gland extract (SGE) prepared from feeding ticks and then infected with vesicular stomatitis virus (VSV). At low input doses of VSV, viral yield was increased 100-fold to 10,000-fold by 16-23 h post-infection compared with untreated cultures, and depending on the SGE concentration. SGE-mediated acceleration of viral yield corresponded with the earlier appearance of VSV nucleocapsid protein as detected by 2-dimensional electrophoresis of infected cells. The observation that physiological doses of virus (i.e. doses likely to be inoculated by an infected arthropod vector into its vertebrate host during blood-feeding) respond to SGE treatment in vitro provides a new opportunity for identifying the factors in tick saliva that promote virus transmission in vivo.

Therapy-induced antibodies to interferon-alpha 2a recognise its receptor-binding site.

Fifty-eight patients with chronic hepatitis B (HB) or C (HC) were treated with recombinant human interferon (rIFN)-alpha 2 and their sera were assayed for antibodies to rIFN-alpha 2c. Twelve of these patients produced low titres and two high titres of the antibodies. We localized the region which was recognised by the high-titre therapy-induced antibodies on the IFN molecule by testing the antibodies with a set of murine monoclonal antibodies (MoAbs) to IFN-alpha 2 in a competitive radioimmune assay (RIA). Only MoAbs with epitopes located in the amino-terminal portion of IFN-alpha 2 could inhibit the binding of radiolabelled IFN-alpha 2 by patients' sera. Our data indicate that the therapy-induced antibodies were directed to the receptor-binding domain of IFN-alpha 2 formed by amino acids (aa) 30-53. In accordance with this observation, human anti-IFN sera inhibited the binding of rIFN-alpha 2 to human cells.

Interferon-omega suppresses hepatitis B surface antigen production in human hepatoma cell line.

Biological activities of human interferon (IFN) omega are less well characterized than those of other type I human IFNs. We compared the ability of recombinant IFN-omega, IFN-alpha 2 and IFN-gamma to inhibit the production of viral hepatitis B surface antigen (HBsAg) in the human hepatoma cell line PLC/PRF/5. The results demonstrated that the capacity of IFN-omega to suppress the HBsAg synthesis was similar to that of IFN-alpha 2. The kinetics of the inhibitory effect of IFN-gamma differed from those of the two other IFNs.

Ixodid tick salivary gland extracts inhibit production of lipopolysaccharide-induced mRNA of several different human cytokines.

Extracts prepared from the salivary glands (SGE) of partially fed adult female Rhipicephalus appendiculatus ticks reduced the expression by human peripheral blood leukocytes 9PBLs) of lipopolysaccharide (LPS)-stimulated cytokine mRNA. Treatment with SGE had no obvious effect on cytokine mRNA production when compared with untreated PBLs. LPS treatment induced or increased mRNA production for IFN alpha, TNF-alpha, IL-1 alpha, IL-1 beta, IL-5, IL-6, IL-7 and IL-8. All the LPS-stimulated cytokine mRNAs were reduced when treated with a mixture of LPS and SGE. The results indicate the potential of ticks in modulating the cytokine network of their vertebrate hosts, possibly to facilitate blood feeding.

Priming with interferon-alpha 1 or interferon-alpha 2 enhances the production of both subtypes simultaneously.

A short period of incubation with interferon-alpha ("priming") increases the amounts of IFN-alpha formed by human peripheral blood leukocytes when subsequently induced with a virus. We investigated specifically the effect of priming on the production of two individual subtypes, IFN-alpha 1 and IFN-alpha 2. The rate of interferon synthesis and the amounts formed were equally potentiated in leukocytes primed with either IFN-alpha 1 or IFN-alpha 2. Whichever of these was used for priming had no selective effect on the relative increased production of IFN-alpha 1 or IFN-alpha 2.

Effect of sera of cirrhotic patients with or without hepatitis B virus infection on protein synthesis in hepatoma cells.

The in vitro effects of sera of 11 patients with liver cirrhosis on protein synthesis in PLC/PRF/5 cells were studied. Hepatitis B virus (HBV) infection was documented in 7 patients. Increased random production of several cell proteins of M(r) of approximately 25, 65, 90 and 130 K was shown by SDS-polyacrylamide gel electrophoresis (SDS-PAGE). There was no correlation between HBV-positive and HBV-negative cirrhosis and the induced proteins. One of them was identified as alpha-1 foetoprotein by immunoblot analysis. C-reactive protein (CRP) was determined only in one case; production of interleukin-6 (IL-6) was not detected.