Filariasis in Africa--treatment challenges and prospects.

Lymphatic filariasis (LF) and onchocerciasis are parasitic nematode infections that are responsible for a major disease burden in the African continent. Disease symptoms are induced by the immune reactions of the host, with lymphoedema and hydrocoele in LF, and dermatitis and ocular inflammation in onchocerciasis. Wuchereria bancrofti and Onchocerca volvulus, the species causing LF and onchocerciasis in Africa, live in mutual symbiosis with Wolbachia endobacteria, which cause a major part of the inflammation leading to symptoms and are antibiotic targets for treatment. The standard microfilaricidal drugs ivermectin and albendazole are used in mass drug administration programmes, with the aim of interrupting transmission, with a consequent reduction in the burden of infection and, in some situations, leading to regional elimination of LF and onchocerciasis. Co-endemicity of Loa loa with W. bancrofti or O. volvulus is an impediment to mass drug administration with ivermectin and albendazole, owing to the risk of encephalopathy being encountered upon administration of ivermectin. Research into new treatment options is exploring several improved delivery strategies for the classic drugs or new antibiotic treatment regimens for anti-wolbachial chemotherapy.

Filariasis and lymphoedema.

Among the causes of lymphoedema (LE), secondary LE due to filariasis is the most prevalent. It affects only a minority of the 120 million people infected with the causative organisms of lymphatic filariasis (LF), Wuchereria bancrofti and Brugia malayi/timori, but is clustered in families, indicating a genetic basis for development of this pathology. The majority of infected individuals develop filarial-specific immunosuppression that starts even before birth in cases where mothers are infected and is characterized by regulatory T-cell responses and high levels of IgG4, thus tolerating high parasite loads and microfilaraemia. In contrast, individuals with this pathology show stronger immune reactions biased towards Th1, Th2 and probably also Th17. Importantly, as for the aberrant lymph vessel development, innate immune responses that are triggered by the filarial antigen ultimately result in the activation of vascular endothelial growth factors (VEGF), thus promoting lymph vessel hyperplasia as a first step to lymphoedema development. Wolbachia endosymbionts are major inducers of these responses in vitro, and their depletion by doxycycline in LF patients reduces plasma VEGF and soluble VEGF-receptor-3 levels to those seen in endemic normals preceding pathology improvement. The search for the immunogenetic basis for LE could lead to the identification of risk factors and thus, to prevention; and has so far led to the identification of single-nucleotide polymorphisms (SNP) with potential functional relevance to VEGF, cytokine and toll-like receptor (TLR) genes. Hydrocele, a pathology with some similarity to LE in which both lymph vessel dilation and lymph extravasation are shared sequelae, has been found to be strongly associated with a VEGF-A SNP known for upregulation of this (lymph-)angiogenesis factor.

The mitochondrial heat shock protein 60 (HSP60) is up-regulated in Onchocerca volvulus after the depletion of Wolbachia.

Wolbachia, a genus of endosymbiotic bacteria of filarial worms, represent novel targets for anti-filarial therapy. The efficacy of compounds against Wolbachia has been evaluated using antiserum raised against the 60 kDa heat shock protein (HSP60) which binds specifically to this protein in both Wolbachia and mitochondria. It has been shown that Wolbachia stains (using such specific probes) stronger than the mitochondria in untreated Onchocerca volvulus, whereas after the depletion of Wolbachia (with drugs) staining of the mitochondria is increased. Herein, immunogold electron microscopy showed that specific anti-HSP60 serum specifically labelled Wolbachia and filarial mitochondria, and that both have distinct localization patterns, thus allowing them to be differentiated. Immunohistochemistry of O. volvulus showed that HSP60 staining is increased in the mitochondria after Wolbachia depletion in the hypodermis, epithelia, muscles, oocytes, embryos, and developing spermatozoa. This could have been the result of the antiserum preferentially binding to the Wolbachia when they are present or due to increased expression of the protein in the absence of the bacteria. To address this, mRNA levels of filarial hsp60 in O. volvulus were measured. After the depletion of Wolbachia, the transcription of hsp60 was significantly greater (7.7 fold) compared with untreated worms. We hypothesize that the increased expression of HSP60 in the absence of Wolbachia is due to a disruption of the homeostasis of the endosymbiosis.

Antibiotics which target the Wolbachia endosymbionts of filarial parasites: a new strategy for control of filariasis and amelioration of pathology.

Wolbachia endosymbionts of filariae are targets for the development of new antifilarial chemotherapy. Doxycycline to deplete Wolbachia from the worm has demonstrated the feasibility of this strategy and has provided a new chemotherapeutic tool. Recent research shows that depleting Wolbachia will also lessen pathology, and lessen adverse reactions to traditional antifilarial drugs.

Nitric oxide synthase in filariae: demonstration of nitric oxide production by embryos in Brugia malayi and Acanthocheilonema viteae.

The radical gas nitric oxide (NO) is synthesized by nitric oxide synthase (NOS) from l-arginine and molecular oxygen. Nitric oxide is an important signaling molecule in invertebrate and vertebrate systems. Previously we have shown that NOS is localized to more tissues in Brugia malayi than has been reported in Ascaris suum. In this paper, we analyze the distribution of NOS in Acanthocheilonema viteae, a filarial nematode that differs from B. malayi in that A. viteae females release microfilariae without a sheath. A. viteae is also one of a few filarial parasites without the Wolbachia intracellular endosymbiont. By use of a specific antibody, NOS was demonstrated in extracts of A. viteae and Dirofilaria immitis. The localization pattern of NOS in A. viteae was similar to that seen in B. malayi, with the enzyme localized to the body wall muscles of both sexes, developing spermatozoa, intrauterine sperm, and early embryos. By use of DAF-2, a fluorescent indicator specific for nitric oxide, the embryos of B. malayi and A. viteae were demonstrated to produce NO ex utero. The near identical staining patterns seen in A. viteae and B. malayi argue that NO is not produced by Wolbachia, nor is it produced by the nematodes in response to the infection. Localization of NOS to the sperm of filarial nematodes suggests a role for NO during fertilization as has been described for sea urchin and ascidian fertilization. Demonstration of the activity of embryonic NOS supports our earlier hypothesis that NO is a signaling molecule during embryogenesis in filarial nematodes.

Brugia malayi: localization of nitric oxide synthase in a lymphatic filariid.

Nitric oxide synthase converts L-arginine to citrulline and nitric oxide, a gaseous signaling molecule critical to multiple physiological responses. Nitric oxide synthase was detected by Western blot analysis of Brugia malayi extracts using an antibody raised against a peptide from murine brain nitric oxide synthase. Using NADPH diaphorase staining and immunohistochemistry, nitric oxide synthase was localized in the parasitic nematode B. malayi. As in Ascaris suum, nitric oxide synthase was detected in the body wall muscles of adult B. malayi. This localization pattern is in agreement with the role of nitric oxide in the control of muscle tone in other invertebrates and in vertebrates. A novel finding was the localization of nitric oxide synthase in the oocytes, in developing embryos, and in spermatozoa. B. malayi nitric oxide synthase may play a role in developmental signaling, as has been suggested for Drosophila and Ilyanassa, a marine mud snail.

Endo16, a large multidomain protein found on the surface and ECM of endodermal cells during sea urchin gastrulation, binds calcium.

Endo16 encodes a developmentally regulated protein restricted to cells participating in the formation of the archenteron during sea urchin gastrulation and to the stomach of the pluteus. The 4680-nt coding region of the Endo16 gene has been sequenced from overlapping cDNAs. Sequence analysis revealed that Endo16 is a large multidomain protein starting with a putative signal sequence at its amino terminus which is followed by a cysteine-rich region, two potential heparin-binding regions, an acidic domain of 5 clustered repeats, an RGD cell binding motif, and a group of 12 additional acidic repeats. Immunolocalization by confocal and electron microscopy demonstrate that the Endo16 protein is in the extracellular matrix and associated with the surface of endodermal cells in the mid and hindgut of the archenteron. The two distinct acidic repeat regions are similar to known calcium-binding sequences. A recombinant Endo16 protein containing both putative calcium-binding repeat regions has been shown to bind radioactive calcium. Tryptic digests of gastrula stage protein extracts in the presence and the absence of calcium have established that calcium stabilizes Endo16 protein against proteolytic degradation.