Insights into the structure-function relationship of Brugia malayi thymidylate kinase (BmTMK).

Lymphatic filariasis is a debilitating disease caused by lymph dwelling nematodal parasites like Wuchereria bancrofti, Brugia malayi and Brugia timori. Thymidylate kinase of B. malayi is a key enzyme in the de novo and salvage pathways for thymidine 5'-triphosphate (dTTP) synthesis. Therefore, B. malayi thymidylate kinase (BmTMK) is an essential enzyme for DNA biosynthesis and an important drug target to rein in filariasis. In the present study, the structural and functional changes associated with recombinant BmTMK, in the presence of protein denaturant GdnHCl, urea and pH were studied. GdnHCl and urea induced unfolding of BmTMK is non-cooperative and influence the functional property of the enzyme much lower than their Cm values. The study delineate that BmTMK is more prone to ionic perturbation. The dimeric assembly of BmTMK is an absolute requirement for enzymatic acitivity and any subtle change in dimeric conformation due to denaturation leads to loss of enzymatic activity. The pH induced changes on structure and activity suggests that selective modification of active site microenvironment pertains to difference in activity profile. This study also envisages that chemical moieties which acts by modulating oligomeric assembly, could be used for better designing of inhibitors against BmTMK enzyme.

The secreted triose phosphate isomerase of Brugia malayi is required to sustain microfilaria production in vivo.

Human lymphatic filariasis is a major tropical disease transmitted through mosquito vectors which take up microfilarial larvae from the blood of infected subjects. Microfilariae are produced by long-lived adult parasites, which also release a suite of excretory-secretory products that have recently been subject to in-depth proteomic analysis. Surprisingly, the most abundant secreted protein of adult Brugia malayi is triose phosphate isomerase (TPI), a glycolytic enzyme usually associated with the cytosol. We now show that while TPI is a prominent target of the antibody response to infection, there is little antibody-mediated inhibition of catalytic activity by polyclonal sera. We generated a panel of twenty-three anti-TPI monoclonal antibodies and found only two were able to block TPI enzymatic activity. Immunisation of jirds with B. malayi TPI, or mice with the homologous protein from the rodent filaria Litomosoides sigmodontis, failed to induce neutralising antibodies or protective immunity. In contrast, passive transfer of neutralising monoclonal antibody to mice prior to implantation with adult B. malayi resulted in 60-70% reductions in microfilarial levels in vivo and both oocyte and microfilarial production by individual adult females. The loss of fecundity was accompanied by reduced IFNγ expression by CD4⁺ T cells and a higher proportion of macrophages at the site of infection. Thus, enzymatically active TPI plays an important role in the transmission cycle of B. malayi filarial parasites and is identified as a potential target for immunological and pharmacological intervention against filarial infections.

[Studies on allelic zymogram of periodic Brugia malayi from Zhejiang Province].

AIM: To study the allelic zymogram of periodic B. malayi from Zhejiang Province. METHODS: 180 adult worms(male 60, female 120), 32,000 microfilaria and 1,500 infective larvae of periodic B. malayi were examined for the isoenzymes of 14 enzymes by horizontal starch gel electrophoresis with 14 different isomyme. RESULTS: Twenty-seven allelic loci were found in three developing stages of B. malayi, most of them were homozygotes, however, two of them(MPI,MDH) were heterozygotes(7.4%). 17, 19 and 9 loci were presented in adult worms, microfilariae and infective larvae, respectively. At the same time, the allelic zymogram of B. malayi from Guizhou Province was also examined. CONCLUSION: The allelic zymogram of periodic B. malayi from Zhejiang Province was similar to that of periodic B. malayi from Guizhou Province but passaged in the laboratory.

Side reactions following ivermectin therapy in high density bancroftian microfilaraemics.

Side reactions following ivermectin treatment were evaluated in sixty males with high density bancroftian microfilaremia (GM 1388/ml). Following a single oral dose of ivermectin of different strengths (20, 50, 100 or 200 micrograms/kg), microfilariae clearance and side reactions were monitored in a double blind fashion. Microfilaria levels fell rapidly after ivermectin administration in all dosage groups and 98% of pretreatment microfilariae was cleared after 12 h of treatment. The rate of microfilaria (mf) clearance was slower with 20 micrograms/kg than with the highest dose (200 micrograms/kg) administered. Forty-six patients (77%) became amicrofilaraemic within 2 weeks of treatment. Side reactions were noted in 97% of cases. The most common reactions were fever, headache, weakness, myalgia and cough which appeared by 12 h and subsided by 72 h following treatment. The frequency and intensity of side reactions were related to pretreatment mf densities and were independent of the dose administered. Unusual side reactions were noted in a few patients with high density microfilaraemia. These included intense cough, shortness of breath, blood tinged mucoid expectoration associated with patchy pneumonitis of the lung. Itchy rashes, lymphatic nodules and raised alkaline phosphatase level were also observed in some patients. These side reactions were transient, self limiting and were not serious enough to warrant any treatment. These exaggerated unusual reactions were possibly due to allergic response of the susceptible host to rapid killing of large number of microfilariae.

Wuchereria bancrofti: identification of parasitic acetylcholinesterase in microfilariae infected human serum.

An antigen with cholinesterase activity was detected in the sera of patients infected with Wuchereria bancrofti. The asymptomatic microfilaremic sera showed 3 to 4 times more cholinesterase activity for acetylthiocholine (ATCh) as compared to sera of symptomatic amicrofilaremic, hookworm infected and endemic normals, whereas the activities for butyrylthiocholine (BTCh) did not significantly differ. The enzyme activities from both sources, namely from sera of microfilaremic cases and from endemic normals, were partially purified and according to substrate specificity for ATCh and BTCh as well as inhibition of the former activity by excess substrate classified as acetylcholinesterase (AChE; EC 3.1.1.7) and pseudocholinesterase (AChE; EC 3.1.1.8), respectively. The Km-value for ATCh of the cholinesterase from the microfilaremic sera was determined to be 0.87 mM. Eserine competitively inhibited the AChE activity; the inhibition constant was found to be 1.3 microM. The BChE from the normal sera had Km-values of 0.15 and 0.20 mM for BTCh and ATCh, respectively, and did not show significant inhibition by eserine. These and other dissimilarities suggest a difference in nature of the cholinesterases in microfilaremic and normal sera and propose that the former enzyme, a true acetylcholinesterase, originates from the parasite. Additional evidence for the origin of the AChE-activity from the parasite was provided by ELISA-studies; anti-Brugia malayi AChE antibodies confirmed antigenecity and cross reactivity of the AChE in infected sera, whereas the antibodies did not show any cross reactivity with the BChE in normal sera.

In vitro and in vivo effect of diethylcarbamazine on the activity of acetylcholinesterase from Wuchereria bancrofti infected human serum.

The acetylcholinesterase (AChE) activity was measured in human serum from persons infected with the filarial parasite Wuchereria bancrofti. The asymptomatic microfilaremic serum showed five times increase in AChE-activity as compared with normal serum, whereas only little difference was observed in serum from patients with elephantiasis. Similar results were obtained when the enzyme activity was measured in the immune complexes precipitated with polyethyleneglycol. Further, the effect of the antifilarial drug diethylcarbamazine (DEC), on the AChE activity of infected and normal serum was studied in in vivo and in vitro experiments. In vitro, DEC was found to be effective only with respect to AChE from asymptomatic microfilaremic serum where 75% decrease in enzyme activity was observed at 100 mumol. The oral administration of DEC (5 mg/kg of body weight/day) effected the activity of AChE from microfilaremic serum as shown after 1 hr, 1 and 3 weeks. A regular decrease in enzyme activity of asymptomatic microfilaremic serum was observed. By increasing time periods and after three weeks the level of AChE reaches the normal value. In vitro and in vivo the same concentration of DEC has negligible effect on the normal serum suggesting that in case of asymptomatic microfilaremic serum the increased activity of AChE is different in nature than the host acetylcho-[abstract incomplete in journal]

Isozymic pattern of lactate dehydrogenase in cases of bancroftian filariasis.

Isozymic patterns of lactate dehydrogenase (E.C. 1.1.1.27) by polyacrylamide gel electrophoresis (PAGE) were observed in various categories of filariasis and controls, i.e. asymptomatic microfilaraemia and symptomatic amicrofilaraemia, endemic normal and non-endemic normal. Lactate dehydrogenase (LDH) activity was also observed amongst the above categories of patients. An increase in enzyme activity and change in the isozymic pattern was observed in the above categories of filaria infected serum. LDH activity doubled in asymptomatic microfilaraemia whereas in symptomatic amicrofilaraemia the increase in LDH activity was thirtyfold. The isozymic pattern of microfilaraemic cases showed the presence of three bands B4, A1B3, A2B2, which are quite thick as compared to normal healthy subjects, whereas the patients with symptomatic amicrofilaraemia showed marked elevation of serum LDH-4 or A3B1. The LDH was partially purified by combined treatment of (NH4)2SO4 fractionation and gel filtration. The isozymic pattern of purified LDH showed a similar pattern.

Biochemical studies in Presbytis cristata infected with subperiodic Brugia malayi.

The Presbytis cristata--Brugia malayi model, now established as a reliable non-human primate model for the experimental screening of potential filaricides, was monitored at monthly intervals for changes in the liver and renal function tests and also for alkaline phosphatase levels during infection. Animals infected with 200-400 infective larvae became patient at 50-90 days post-infection and geometric mean microfilarial counts were above 1000 per ml from the fourth month onwards. There were no significant changes in the biochemical parameters monitored throughout the period of observation. This is an important observation as any changes seen in these parameters during experimental drug studies can be attributed to drug reaction or toxicity and this will be invaluable in decision making as to drug safety.