Toll-like receptor 2 regulates CXC chemokine production and neutrophil recruitment to the cornea in Onchocerca volvulus/Wolbachia-induced keratitis.

The filarial nematode Onchocerca volvulus is the causative organism of river blindness. Our previous studies demonstrated an essential role for endosymbiotic Wolbachia bacteria in corneal disease, which is characterized by neutrophil infiltration into the corneal stroma and the development of corneal haze. To determine the role of Toll-like receptors (TLRs) in neutrophil recruitment and activation, we injected a soluble extract of O. volvulus containing Wolbachia bacteria into the corneal stromata of C57BL/6, TLR2-/-, TLR4-/-, TLR2/4-/-, and TLR9-/- mice. We found an essential role for TLR2, but not TLR4 or TLR9, in neutrophil recruitment to the cornea and development of corneal haze. Furthermore, chimeric mouse bone marrow studies showed that resident bone marrow-derived cells in the cornea can initiate this response. TLR2 expression was also essential for CXC chemokine production by resident cells in the cornea, including corneal fibroblasts, and for neutrophil activation. Taken together, these findings indicate that Wolbachia activates TLR2 on resident bone marrow-derived cells in the corneal stroma to produce CXC chemokines, leading to neutrophil recruitment to the corneal stroma, and that TLR2 mediates O. volvulus/Wolbachia-induced neutrophil activation and development of corneal haze.

The murine local lymph node assay: regulatory and potency considerations under REACH.

From June 2007, new chemicals legislation on the registration, evaluation, authorization and restriction of chemicals (REACH) will come into force across the European Union. This will require the submission of data on human health effects of chemicals, including chemical safety assessments which will require measurements of potency. For skin sensitization hazard identification, REACH states that the first-choice in vivo assay is the local lymph node assay (LLNA). This test has also been the UK competent authority's preferred test for skin sensitization since 2002, and has now replaced guinea pig tests in dossiers submitted to it under the Notification of New Substances Regulations. Advantages of the LLNA over guinea pig tests include improvements in animal welfare, a more scientific approach to hazard identification, and the inclusion of a dose-response element in the endpoint, which enables an estimation of potency. However, notifiers to the UK competent authority have sometimes been reluctant to use the assay because of concerns over false-positive reactions. Across Europe, these concerns have been heightened in the lead-up to the introduction of REACH, since the use of in vivo alternatives to the LLNA will require scientific justification. This review will address some of these concerns from a regulatory perspective.

A randomized, double-blind clinical trial of a 3-week course of doxycycline plus albendazole and ivermectin for the treatment of Wuchereria bancrofti infection.

BACKGROUND: Eight- and 6-week courses of doxycycline are superior to standard treatment of bancroftian filariasis. Standard treatment (albendazole plus ivermectin) is associated with adverse reactions. We assessed whether a shorter (i.e, 3-week) course of doxycycline with standard treatment would show superior efficacy to standard treatment alone and reduce the incidence of adverse reactions. METHODS: A total of 44 adults from Ghana were recruited in January 2003: 20 received doxycycline (200 mg/day) for 3 weeks, and 24 received matching placebo. Participants received albendazole (400 mg) and ivermectin (150 microg/kg) at month 4, and adverse reactions were assessed 48 h later. Treatment efficacy was evaluated at months 4, 12, and 24. RESULTS: The microfilariae level was significantly reduced after receipt of doxycycline treatment at months 4 (P = .017), 12 (P = .001), and 24 (P = .005). The microfilariae level was only significantly reduced at month 12 in the placebo group (P = .041). At all follow-up points, the microfilariae level was significantly lower in the doxycycline group. Adverse reactions to standard antifilarial treatment were similar in frequency between the doxycycline group (in 7 of 11 subjects) and the placebo group (in 13 of 17 subjects). Moderate reactions only occurred in the placebo group (in 3 of 17 subjects). Severity of adverse reaction was associated with microfilaremia (P = .037), Wolbachia bacteria in plasma (P = .048), and proinflammatory cytokines in plasma (P = .019). Adult parasite viability was not significantly different between doxycycline and placebo groups at months 12 or 24. CONCLUSIONS: Treatment with doxycycline for 3 weeks is more effective in inducing a long-term amicrofilaremia than is standard treatment alone, but it is ineffective at inducing curative effects. Inflammatory reactions to antifilarial treatment are associated with levels of microfilariae and Wolbachia endosymbionts released into plasma.

Macrofilaricidal activity after doxycycline treatment of Wuchereria bancrofti: a double-blind, randomised placebo-controlled trial.

BACKGROUND: Wolbachia endosymbionts of filarial nematodes are vital for larval development and adult-worm fertility and viability. This essential dependency on the bacterium for survival of the parasites has provided a new approach to treat filariasis with antibiotics. We used this strategy to investigate the effects of doxycycline treatment on the major cause of lymphatic filariasis, Wuchereria bancrofti. METHODS: We undertook a double-blind, randomised, placebo-controlled field trial of doxycycline (200 mg per day) for 8 weeks in 72 individuals infected with W bancrofti from Kimang'a village, Pangani, Tanzania. Participants were randomly assigned by block randomisation to receive capsules of doxycycline (n=34) or placebo (n=38). We assessed treatment efficacy by monitoring microfilaraemia, antigenaemia, and ultrasound detection of adult worms. Follow-up assessments were done at 5, 8, 11, and 14 months after the start of treatment. Analysis was per protocol. FINDINGS: One person from the doxycycline group died from HIV infection. Five (doxycycline) and 11 (placebo) individuals were absent at the time of ultrasound analysis. Doxycycline treatment almost completely eliminated microfilaraemia at 8-14 months' follow-up (for all timepoints p<0.001). Ultrasonography detected adult worms in only six (22%) of 27 individuals treated with doxycycline compared with 24 (88%) of 27 with placebo at 14 months after the start of treatment (p<0.0001). At the same timepoint, filarial antigenaemia in the doxycycline group fell to about half of that before treatment (p=0.015). Adverse events were few and mild. INTERPRETATION: An 8-week course of doxycycline is a safe and well-tolerated treatment for lymphatic filariasis with significant activity against adult worms and microfilaraemia.

Onchocerca parasites and Wolbachia endosymbionts: evaluation of a spectrum of antibiotic types for activity against Onchocerca gutturosa in vitro.

BACKGROUND: The filarial parasites of major importance in humans contain the symbiotic bacterium Wolbachia and recent studies have shown that targeting of these bacteria with antibiotics results in a reduction in worm viability, development, embryogenesis, and survival. Doxycycline has been effective in human trials, but there is a need to develop drugs that can be given for shorter periods and to pregnant women and children. The World Health Organisation-approved assay to screen for anti-filarial activity in vitro uses male Onchocerca gutturosa, with effects being determined by worm motility and viability as measured by reduction of MTT to MTT formazan. Here we have used this system to screen antibiotics for anti-filarial activity. In addition we have determined the contribution of Wolbachia depletion to the MTT reduction assay. METHODS: Adult male O. gutturosa were cultured on a monkey kidney cell (LLCMK 2) feeder layer in 24-well plates with antibiotics and antibiotic combinations (6 to 10 worms per group). The macrofilaricide CGP 6140 (Amocarzine) was used as a positive control. Worm viability was assessed by two methods, (i) motility levels and (ii) MTT/formazan colorimetry. Worm motility was scored on a scale of 0 (immotile) to 10 (maximum) every 5 days up to 40 days. On day 40 worm viability was evaluated by MTT/formazan colorimetry, and results were expressed as a mean percentage reduction compared with untreated control values at day 40. To determine the contribution of Wolbachia to the MTT assay, the MTT formazan formation of an insect cell-line (C6/36) with or without insect Wolbachia infection and treated or untreated with tetracycline was compared. RESULTS: Antibiotics with known anti-Wolbachia activity were efficacious in this system. Rifampicin (5 x 10(-5) M) was the most effective anti-mycobacterial agent; clofazimine (1.25 x 10(-5) M and 3.13 x 10(-6) M) produced a gradual reduction in motility and by 40 days had reduced worm viability. The other anti-mycobacterial drugs tested had limited or no activity. Doxycycline (5 x 10(-5) M) was filaricidal, but minocycline was more effective and at a lower concentration (5 x 10(-5) M and 1.25 x 10(-5) M). Inactive compounds included erythromycin, oxytetracycline, trimethoprim and sulphamethoxazole. The MTT assay on the insect cell-line showed that Wolbachia made a significant contribution to the metabolic activity within the cells, which could be reduced when they were exposed to tetracycline. CONCLUSION: The O. gutturosa adult male screen for anti-filarial drug activity is also valid for the screening of antibiotics for anti-Wolbachia activity. In agreement with previous findings, rifampicin and doxycycline were effective; however, the most active antibiotic was minocycline. Wolbachia contributed to the formation of MTT formazan in the MTT assay of viability and is therefore not exclusively a measure of worm viability and indicates that Wolbachia contributes directly to the metabolic activity of the nematode.

Diethylcarbamazine activity against Brugia malayi microfilariae is dependent on inducible nitric-oxide synthase and the cyclooxygenase pathway.

BACKGROUND: Diethylcarbamazine (DEC) has been used for many years in the treatment of human lymphatic filariasis. Its mode of action is not well understood, but it is known to interact with the arachidonic acid pathway. Here we have investigated the contribution of the nitric oxide and cyclooxygenase (COX) pathways to the activity of DEC against B. malayi microfilariae in mice. METHODS: B. malayi microfilariae were injected intravenously into mice and parasitaemia was measured 24 hours later. DEC was then administered to BALB/c mice with and without pre-treatment with indomethacin or dexamethasone and the parasitaemia monitored. To investigate a role for inducible nitric oxide in DEC's activity, DEC and ivermectin were administered to microfilaraemic iNOS-/- mice and their background strain (129/SV). Western blot analysis was used to determine any effect of DEC on the production of COX and inducible nitric-oxide synthase (iNOS) proteins. RESULTS: DEC administered alone to BALB/c mice resulted in a rapid and profound reduction in circulating microfilariae within five minutes of treatment. Microfilarial levels began to recover after 24 hours and returned to near pre-treatment levels two weeks later, suggesting that the sequestration of microfilariae occurs independently of parasite killing. Pre-treatment of animals with dexamethasone or indomethacin reduced DEC's efficacy by almost 90% or 56%, respectively, supporting a role for the arachidonic acid and cyclooxygenase pathways in vivo. Furthermore, experiments showed that treatment with DEC results in a reduction in the amount of COX-1 protein in peritoneal exudate cells. Additionally, in iNOS-/- mice infected with B. malayi microfilariae, DEC showed no activity, whereas the efficacy of another antifilarial drug, ivermectin, was unaffected. CONCLUSION: These results confirm the important role of the arachidonic acid metabolic pathway in DEC's mechanism of action in vivo and show that in addition to its effects on the 5-lipoxygenase pathway, it targets the cyclooxygenase pathway and COX-1. Moreover, we show for the first time that inducible nitric oxide is essential for the rapid sequestration of microfilariae by DEC.

Population dynamics of Wolbachia bacterial endosymbionts in Brugia malayi.

The human filarial nematode Brugia malayi contains an endosymbiotic bacterium, Wolbachia. We used real-time quantitative polymerase chain reaction (QPCR) and microscopy to investigate the population dynamics of the bacterium-nematode association. Two Wolbachia (wsp and ftsZ) and one nematode (gst) genes were amplified from all life-cycle stages of B. malayi and results expressed as gene copies per worm and as Wolbachia/nematode ratios. Since the genes were single copy and there was one genome per Wolbachia, the gene copy numbers were equivalent to the numbers of bacteria. These were similar in microfilariae and the mosquito-borne larval stages (L2 and L3), with the lowest ratios of Wolbachia/nematode DNA. However, within 7 days of infection of the mammalian host, bacteria had increased 600-fold and the bacteria/worm ratio was the highest of all life-cycle stages. The rapid multiplication continued throughout L4 development, so that the major period of bacterial population growth occurred within 4 weeks of infection of the definitive host. Microscopy confirmed that there were few bacteria in mosquito-derived L3 but many, in large groups, in L4 collected 9 and 21 days after infection. In adult male worms up to 15 months of age, the bacterial populations were maintained, whilst in females, bacteria numbers increased as the worms matured and as the ovary and embryonic larval stages became infected. These results support the hypothesis that the bacteria are essential for larval development in the mammalian host and for the long-term survival of adult worms.

Evidence against Wolbachia symbiosis in Loa loa.

BACKGROUND: The majority of filarial nematode species are host to Wolbachia bacterial endosymbionts, although a few including Acanthocheilonema viteae, Onchocerca flexuosa and Setaria equina have been shown to be free of infection. Comparisons of species with and without symbionts can provide important information on the role of Wolbachia symbiosis in the biology of the nematode hosts and the contribution of the bacteria to the development of disease. Previous studies by electron microscopy and PCR have failed to detect intracellular bacterial infection in Loa loa. Here we use molecular and immunohistological techniques to confirm this finding. METHODS: We have used a combination of PCR amplification of bacterial genes (16S ribosomal DNA [rDNA], ftsZ and Wolbachia surface protein [WSP]) on samples of L. loa adults, third-stage larvae (L3) and microfilariae (mf) and immunohistology on L. loa adults and mf derived from human volunteers to determine the presence or absence of Wolbachia endosymbionts. Samples used in the PCR analysis included 5 adult female worms, 4 adult male worms, 5 mf samples and 2 samples of L3. The quality and purity of nematode DNA was tested by PCR amplification of nematode 5S rDNA and with diagnostic primers from the target species and used to confirm the absence of contamination from Onchocerca sp., Mansonella perstans, M. streptocerca and Wuchereria bancrofti. Immunohistology was carried out by light and electron microscopy on L. loa adults and mf and sections were probed with rabbit antibodies raised to recombinant Brugia malayi Wolbachia WSP. Samples from nematodes known to be infected with Wolbachia (O. volvulus, O. ochengi, Litomosoides sigmodontis and B. malayi) were used as positive controls and A. viteae as a negative control. RESULTS: Single PCR analysis using primer sets for the bacterial genes 16S rDNA, ftsZ, and WSP were negative for all DNA samples from L. loa. Positive PCR reactions were obtained from DNA samples derived from species known to be infected with Wolbachia, which confirmed the suitability of the primers and PCR conditions. The quality and purity of nematode DNA samples was verified by PCR amplification of 5S rDNA and with nematode diagnostic primers. Additional analysis by 'long PCR' failed to produce any further evidence for Wolbachia symbiosis. Immunohistology of L. loa adults and mf confirmed the results of the PCR with no evidence for Wolbachia symbiosis. CONCLUSION: DNA analysis and immunohistology provided no evidence for Wolbachia symbiosis in L. loa.

Innate immune responses to endosymbiotic Wolbachia bacteria in Brugia malayi and Onchocerca volvulus are dependent on TLR2, TLR6, MyD88, and Mal, but not TLR4, TRIF, or TRAM.

The discovery that endosymbiotic Wolbachia bacteria play an important role in the pathophysiology of diseases caused by filarial nematodes, including lymphatic filariasis and onchocerciasis (river blindness) has transformed our approach to these disabling diseases. Because these parasites infect hundreds of millions of individuals worldwide, understanding host factors involved in the pathogenesis of filarial-induced diseases is paramount. However, the role of early innate responses to filarial and Wolbachia ligands in the development of filarial diseases has not been fully elucidated. To determine the role of TLRs, we used cell lines transfected with human TLRs and macrophages from TLR and adaptor molecule-deficient mice and evaluated macrophage recruitment in vivo. Extracts of Brugia malayi and Onchocerca volvulus, which contain Wolbachia, directly stimulated human embryonic kidney cells expressing TLR2, but not TLR3 or TLR4. Wolbachia containing filarial extracts stimulated cytokine production in macrophages from C57BL/6 and TLR4(-/-) mice, but not from TLR2(-/-) or TLR6(-/-) mice. Similarly, macrophages from mice deficient in adaptor molecules Toll/IL-1R domain-containing adaptor-inducing IFN-beta and Toll/IL-1R domain-containing adaptor-inducing IFN-beta-related adaptor molecule produced equivalent cytokines as wild-type cells, whereas responses were absent in macrophages from MyD88(-/-) and Toll/IL-1R domain-containing adaptor protein (TIRAP)/MyD88 adaptor-like (Mal) deficient mice. Isolated Wolbachia bacteria demonstrated similar TLR and adaptor molecule requirements. In vivo, macrophage migration to the cornea in response to filarial extracts containing Wolbachia was dependent on TLR2 but not TLR4. These results establish that the innate inflammatory pathways activated by endosymbiotic Wolbachia in B. malayi and O. volvulus filaria are dependent on TLR2-TLR6 interactions and are mediated by adaptor molecules MyD88 and TIRAP/Mal.

Wolbachia- and Onchocerca volvulus-induced keratitis (river blindness) is dependent on myeloid differentiation factor 88.

Endosymbiotic Wolbachia bacteria that infect the filarial nematode Onchocerca volvulus were previously found to have an essential role in the pathogenesis of river blindness. The current study demonstrates that corneal inflammation induced by Wolbachia or O. volvulus antigens containing Wolbachia is completely dependent on expression of myeloid differentiation factor 88.

IL-4 dependent resistance to the tapeworm Mesocestoides corti (Cestoda) in mice.

Three-week Mesocestoides corti infections of C57BL/6 mice showed significantly raised parasite counts in animals with a targeted knockout of the IL-4 gene. By contrast, antibody neutralization of IL-5 and inhibition of eosinophilia had no effect on parasite numbers. In SV/129 mice, knockout of the interferon gamma receptor and inducible nitric oxide synthase genes had no significant effect on parasite counts. In IL-4(-/-)mice the dominant IgG1 antibody response was dramatically reduced, with a concomitant increase in IgG2a/b responses and a partial twofold reduction in IgM and IgE responses. We conclude that murine resistance to M. corti is dependent on IL-4, but occurs independently of IL-5 and eosinophils. This provides the first direct evidence for IL-4 mediated immunity of mice to infection with a tissue-dwelling platyhelminth.

Wolbachia-induced neutrophil activation in a mouse model of ocular onchocerciasis (river blindness).

Endosymbiotic Wolbachia bacteria are abundant in the filarial nematodes that cause onchocerciasis (river blindness), including the larvae (microfilariae) that migrate into the cornea. Using a mouse model of ocular onchocerciasis, we recently demonstrated that it is these endosymbiotic bacteria rather than the nematodes per se that induce neutrophil infiltration to the corneal stroma and loss of corneal clarity (Saint Andre et al., Science 295:1892-1895, 2002). To better understand the role of Wolbachia organisms in the pathogenesis of this disease, we examined the fate of these bacteria in the cornea by immunoelectron microscopy. Microfilariae harboring Wolbachia organisms were injected into mouse corneas, and bacteria were detected with antibody to Wolbachia surface protein. Within 18 h of injection, neutrophils completely surrounded the nematodes and were in close proximity to Wolbachia organisms. Wolbachia surface protein labeling was also prominent in neutrophil phagosomes, indicating neutrophil ingestion of Wolbachia organisms. Furthermore, the presence of numerous electron-dense granules around the phagosomes indicated that neutrophils were activated. To determine if Wolbachia organisms directly activate neutrophils, peritoneal neutrophils were incubated with either parasite extracts containing Wolbachia organisms, parasite extracts depleted of Wolbachia organisms (by antibiotic treatment of worms), or Wolbachia organisms isolated from filarial nematodes. After 18 h of incubation, we found that isolated Wolbachia organisms stimulated production of tumor necrosis factor alpha and CXC chemokines macrophage inflammatory protein 2 and KC by neutrophils in a dose-dependent manner. Similarly, these cytokines were induced by filarial extracts containing Wolbachia organisms but not by Wolbachia-depleted extracts. Taken together, these findings indicate that neutrophil activation is an important mechanism by which Wolbachia organisms contribute to the pathogenesis of ocular onchocerciasis.