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Vaccine,
1999]
The parasitic nematode, Onchocerca volvulus is a major cause of blindness and dermal pathology in tropical regions. A vaccine directed to infective larvae would provide a valuable control tool alongside the current methods of chemotherapy and vector control. Previously we have described the identification of a chitinase cDNA that is expressed in a stage specific manner by O. volvulus infective third stage (L3) larvae. To evaluate its host protective potential, the complete open reading frame was cloned into the eukaryotic expression plasmid pJW4303 and used to vaccinate mice by DNA immunisation with the Accell GeneGun. The survival of challenge infective larvae was monitored using implanted micropore chambers. In the first trial, mice immunised 3 times over 4 months with 1 microg O. volvulus chitinase DNA responded with modest antibody responses dominated by IgG2a and exhibited a 36% (p=0.189, NS) reduction in parasite survival compared with challenge controls. In the second trial, an increased dose of DNA (5 microg) and more frequent immunisations (5 times over 6 months) stimulated an IgG1 dominant response and a 53% reduction in parasite survival (p=0.042). Antibodies from the vaccinated mice reacted with the cuticle of post-infective L3 larvae, implying that this may be the site of immune attack following secretion of chitinase.
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[
Ecotoxicol Environ Saf,
2013]
Heavy metals are ubiquitous environmental pollutants, and their toxic effects have been widely studied. However, their transgenerational effects between parent and progeny at environmental relevant concentrations need further investigations. Currently, L3 stage of Caenorhabditis elegans was exposed to aqueous metals (Cd, Cu, Pb and Zn) at environmentally realistic concentrations for 96 h. The whole exposure time covered the formation of sperm, ovum and eggs. Subsequently the behavior and growth indicators were measured. The parent nematodes were then bleached to gain synchronized eggs, which were cultured under non-toxic conditions to L3 stage when the same indicators were measured in the progeny. The parent suffered concentration-dependent inhibitions on behavior and growth. Based on the median effective concentration (EC(50)) values, body bending frequency showed relatively higher sensitivity than other behavior indicators. The inhibitions on growth and behavior of progeny were more severe than those of the parent, based on their respective EC(50) values. Interestingly, Cd was not the most toxic metal in either parent or progeny according to EC(50) values, but its EC(50) ratios between parent and progeny (EC(50, parent)/EC(50, progeny)) were the most significant, indicating its greatest transgenerational effects. The results demonstrated the higher sensitivity of L3 larva stage of C. elegans in the transgenerational effect studies than other life stages used before. Our findings suggested that parental exposure to heavy metals can multiply their harmful effects in following generations.
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J Gerontol A Biol Sci Med Sci,
1997]
Mutant alleles of the genes
age-1 and
daf-2 that lengthen life span (Age phenotype) of Caenorhabditis elegans cause higher protein kinase (PKA, PKC, PTK) activity levels in senescing worms relative to wild-type. Elevated levels of PKA and PTK were also present in dauer larvae, developmentally arrested juveniles specialized for long-term survival, relative to L3 larvae, the alternative developmental stage. PKC activity was downregulated in dauers of a non-Age control strain and in
age-1 mutant dauers, compared to L3 larvae, but similar activities were measured in dauers and L3 larvae of a
daf-2 mutant strain. Thus,
age-1 and
daf-2 mutant worms may express distinct elements of a dauer-specific survival program during adult life.
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J Biol Chem,
2001]
A recently reported chitinase gene, expressed in the infective, third-stage (L3) larvae of the human filarial parasite Onchocerca volvulus, belongs to the family 18 glycosyl hydrolases and has been designated
Ov-chi-1. The gene product of
Ov-chi-1 is chitinolytic. Allosamidin ablates activity of the native enzyme in a dose-dependent manner but did not significantly inhibit the moulting of L3 larvae. Mono-specific antibodies were used to characterize Ov-CHI-1 as a 60-kDa protein expressed almost exclusively in L3 stages. Immunoelectron microscopy showed that Ov-CHI-1 expression is initiated in late L2 larvae and increases markedly in infective, L3 larvae. It is synthesized exclusively in the glandular esophagus and stored within discrete secretory granules. Secretion occurs through de-granulation during post-infective development, and the primary route of transport appears to be via the pseudo-coelom. An orthologue of
Ov-chi-1 was detected in Caenorhabditis elegans by BLAST analysis. It is constitutively expressed at a low level and is overexpressed in dauer larvae and embryonated eggs. It is chitinolytic. We conclude that Ov-CHI-1 is a highly stage-specific enzyme that may have a role in infectivity of the parasite, aiding escape from the vector or participating in early post-infective migration and/or development. The identification of an orthologue in C. elegans opens the way for further studies into the biological function(s) of this intriguing parasite product.
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Biochem Biophys Res Commun,
2002]
Growth and development rely on the mitochondrial respiratory chain (MRC) as the major source of ATP. We measured the mitochondrial DNA (mtDNA) copy number of each of the Caenorhabditis elegans developmental stages. Embryos, L1, L2, and L3 larvae all have approximately 25,000 copies of maternally derived mtDNA. The copy number increases fivefold in L4 larvae and a further sixfold in adult hermaphrodites, but only twofold in adult males. The majority of mtDNA in adult worms is germline associated, and germline-deficient mutants show markedly reduced mtDNA contents. With sperm-deficient or oocyte-deficient mutants, we confirm that mtDNA amplification is primarily associated with oocyte production. When mtDNA replication is inhibited, a quantitative and homogeneous arrest as L3 larvae occurs. Thus, mtDNA amplification is a necessary component of normal development and its regulation may involve an energy-sensing decision or checkpoint that can be invoked when mitochondrial energy generation is impaired.
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Parasite Immunol,
2001]
Onchocerciasis is caused by the filarial nematode Onchocerca volvulus and is a major public health problem in West and Central Africa. With only partial and long-term treatment currently available, there is a need to develop a suitable vaccine. We analysed the antibody response to infective L3 larvae because this stage is thought to be associated with host protective immunity. In addition, we have related our findings to the age, gender and current infection intensity of our participants: variables that may significantly influence antibody production. Interestingly, whilst 90% of our study group were seropositive for adult specific immunoglobulin (Ig)E, only 23% produced L3 specific IgE. This is in contrast to IgG4 where seropositivity was comparable at 96% and 92%, respectively. Furthermore, IgG levels were significantly affected by age and the intensity of infection but unaffected by host gender. This finding is independent for the IgG subclass (IgG1, IgG2, IgG3 and IgG4) and its specificity (L3 versus adult antigen). In summary, we show that L3 larvae induce little specific IgE and the antibody response shows a different isotype balance than that against adult antigens. Both host and parasite variables can influence antibody production in this disease.
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J Infect Dis,
2015]
BACKGROUND: Elimination of onchocerciasis and lymphatic filariasis is targeted for 2020. Given the coincident Loa loa infections in Central Africa and the potential for drug resistance development, the need for new microfilaricides and macrofilaricides has never been greater. With the genomes of L. loa, Onchocerca volvulus, Wuchereria bancrofti, and Brugia malayi available, new drug targets have been identified. METHODS: The effects of the tyrosine kinase inhibitors imatinib, nilotinib, and dasatinib on B. malayi adult males, adult females, L3 larvae, and microfilariae were assessed using a wide dose range (0-100 M) in vitro. RESULTS: For microfilariae, median inhibitory concentrations (IC50 values) on day 6 were 6.06 M for imatinib, 3.72 M for dasatinib, and 81.35 M for nilotinib; for L3 larvae, 11.27 M, 13.64 M, and 70.98 M, respectively; for adult males, 41.6 M, 3.87 M, and 68.22 M, respectively; and for adult females, 42.89 M, 9.8 M, and >100 M, respectively. Three-dimensional modeling suggests how these tyrosine kinase inhibitors bind and inhibit filarial protein activity. CONCLUSIONS: Given the safety of imatinib in humans, plans are underway for pilot clinical trials to assess its efficacy in patients with filarial infections.
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Exp Parasitol,
2000]
Over the past several years, numerous attempts have been made to culture the infective-stage (L3) larvae of the human filarial parasite Brugia malayi in an in vitro system that promotes molting to the fourth larval stage (L4). Although there have been reports in the literature of successful L3 to L4 development in vitro, all of these systems have required serum supplementation. The complexity of serum as a culture supplement has made reproducibility of results and identification of specific factors necessary for L3 development problematic. We have developed a serum-free in vitro system consisting of RPMI 1640 supplemented with one of three fatty acids (arachidonic, linoleic, or linolenic) that supports consistent and reproducible molting to the fourth larval stage in the presence of a basidiomycetous yeast, Rhodotorula minuta. Coculture with this yeast, initially isolated as a culture contaminant, is required for successful molting. In serum-free cultures lacking R. minuta, L3 larvae survive for upward of 2 weeks, but do not molt successfully. The L4 larvae generated in cultures containing R. minuta are well formed, as seen by light and electron microscopy, and are capable of further development upon transfer to a permissive host. This culture system is inexpensive and easily reproducible, thus making it a useful tool for studying the requirements for the development of B. malayi L3.
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J Parasitol,
1994]
Membranes from both ivermectin-sensitive and -resistant Haemonchus contortus L3 larvae were examined for the presence of high affinity [3H]ivermectin binding sites. Both tissue preparations displayed high affinity drug binding sites (Kd = 0.13 nM). Receptor density (Bmax = 0.4 pmol/mg) was the same in both the sensitive and resistant nematodes suggesting that target site modification was not involved in the development of drug resistance in this particular strain of H. contortus. The H. contortus ivermectin binding site appeared to be similar to the well characterized Caenorhabditis elegans ivermectin binding site with respect to affinity for ivermectin and receptor density.
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Bull Environ Contam Toxicol,
2009]
Lethality changes were investigated during development in 4 h metal exposed Caenorhabditis elegans. Exposure to examined metals caused severe lethality toxicities in L1- and L2-larvae, in L3-larvae exposed to examined metals at concentrations of 50 and 100 microM and to Pb, Hg, and Cr at the concentration of 2.5 microM, in L4-larvae exposed to examined metals at concentrations of 50 and 100 microM, and in adults exposed to Pb, Hg, and Cr at the concentration of 100 microM. Moreover, the lethality toxicities induced by Pb and Hg in L1 larvae for 4 h could be largely comparable to those in young adults for 24 h.