RESUMEN
PURPOSE: In search of a vaccine for the control of human lymphatic filariasis (LF) caused by Wuchereria bancrofti, Brugia malayi and B. timori, we identified three parasite-specific potential candidates: the disorganized muscle protein-1 (D), calponin (C) and troponin 1 (T) in B. malayi adult worm. In the present study, we investigated the immune response profile of the cocktails of the recombinant D, T and C proteins. METHODS: Groups of BALB/c mice were immunized with individual rproteins or their cocktails DT, TC, DC and DTC, and the immunogen-specific IgG and its subclasses and IgE were determined. Cells from the immunized animals were challenged in vitro with the respective rproteins and cocktails and the release of nitric oxide (NO) from macrophages and Th1 and Th2 cytokines from splenocytes were determined. RESULTS: Among the immunized groups, DTC elicited comparatively a stronger response which included augmented release of NO, Th1 (IL-1ß, IL-2, IFN-γ and TNF-α) and Th2 (IL-4, IL-6, IL-10 and TGF-ß) cytokines, and increased levels of immunogen-specific IgG, IgG1 and IgG2b and low levels of immunogen-specific IgG2a and IgE and the Th2 cytokine IL-13. CONCLUSION: Immune responses that play important role in host protection were elicited strongly by DTC cocktail compared to the individual rproteins or DT, TC and DC cocktails. The findings provide a sound rationale for further studies on DTC cocktail as a vaccine candidate for the control of LF.
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Brugia Malayi , Vacunas , Ratones , Animales , Humanos , Ratones Endogámicos BALB C , Troponina I , Citocinas , Inmunoglobulina G , Inmunidad , Inmunoglobulina E , CalponinasRESUMEN
Visceral leishmaniasis is neglected tropical protozoan disease caused by Leishmania donovani and are associated with high fatality rate in developing countries since prophylactic vaccines are not available. In the present study, we evaluated the immunomodulatory potential of L. donovani histidyl-tRNA synthetase (LdHisRS) and predicted the epitopes using immunoinformatic tools. Histidyl-tRNA synthetase (HisRS) is a class IIa aminoacyl t-RNA synthetase enzyme (aaRS) required for histidine incorporation into proteins during protein synthesis. The recombinant LdHisRS protein (rLdHisRS) was expressed in E coli BL-21cells, and its immunomodulatory role was assessed in J774A.1 murine macrophage and in BALB/c mice, respectively. LdHisRS specifically stimulated and triggered enhance cell proliferation, nitric oxide release and IFN-γ (70%; P < 0.001), and IL-12 (55.37%; P < 0.05) cytokine release in vitro, whereas BALB/c mice immunized with rLdHisRS show higher NO release (80.95%; P<0.001), higher levels of Th1 cytokines IFN-γ (14%; P < 0.05), TNF-α (34.93%; P < 0.001), and IL-12 (28.49%; P < 0.001) and robust IgG (p<0.001) and IgG2a (p<0.001) production. We also identified 20 Helper T-lymphocytes (HTLs), 30 cytotoxic T lymphocytes (CTLs), and 18 B-cell epitopes from HisRS protein of L. donovani. All these epitopes can be further used to make a multiepitope vaccine against L. donovani.
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Leishmania donovani , Vacunas contra la Leishmaniasis , Leishmaniasis Visceral , Vacunas , Animales , Ratones , Leishmania donovani/genética , Leishmaniasis Visceral/prevención & control , Histidina-ARNt Ligasa , Escherichia coli , Células TH1 , Citocinas , Interleucina-12 , Proteínas Recombinantes/genética , Epítopos , Ratones Endogámicos BALB C , Proteínas Protozoarias/genética , Antígenos de Protozoos/genéticaRESUMEN
TriTryp diseases (Leishmaniasis, Human African Trypanosomiasis (HAT), and Chagas disease) are devastating parasitic neglected tropical diseases (NTDs) that affect billions of people in developing countries, cause high mortality in humans, and impose a large socio-economic burden. The current treatment options against tritryp diseases are suboptimal and challenging due to the emergence of resistance against available tritryp drugs. Hence, designing and developing effective anti-tritryp drugs with novel targets are required. Aminoacyl-tRNA synthetases (AARSs) involved in specific aminoacylation of transfer RNAs (tRNAs), interrupt protein synthesis through inhibitors, and retard the parasite growth. AaRSs have long been studied as therapeutic targets in bacteria, and three aaRS inhibitors, mupirocin (against IleRS), tavaborole AN2690 (against LeuRS), and halofuginone (against ProRS), are already in clinical practice. The structural differences between tritryp and human aaRSs and the presence of unique sequences (N-terminal domain/C-terminal domain/catalytic domain) make them potential target for developing selective inhibitors. Drugs based on a single aaRS target developed by high-throughput screening (HTS) are less effective due to the emergence of resistance. However, designing multi-targeted drugs may be a better strategy for resistance development. In this perspective, we discuss the characteristics of tritryp aaRSs, sequence conservation in their orthologs and their peculiarities, recent advancements towards the single-target and multi-target aaRS inhibitors developed through rational design.
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Aminoacil-ARNt Sintetasas , Enfermedad de Chagas , Leishmaniasis , Tripanosomiasis Africana , Aminoacil-ARNt Sintetasas/química , Aminoacil-ARNt Sintetasas/genética , Aminoacil-ARNt Sintetasas/metabolismo , Aminoacilación , Animales , Humanos , Leishmaniasis/tratamiento farmacológico , Enfermedades Desatendidas/tratamiento farmacológico , Tripanosomiasis Africana/tratamiento farmacológicoRESUMEN
A cross-equatorial low-level wind, known as Findlater Jet (FJ), modulates the thermocline in the Arabian Sea (AS) during summer monsoon (June to September). By analysing ocean and atmospheric data, we show that the FJ signal gets 'trapped' in the AS in the form of upper ocean heat content till the following winter months (December to February). This memory is the consequence of the combined effect of FJ-induced wind stress curl and the annual downwelling Rossby waves in the AS. During the summer monsoon months, the strong low-level westerly winds cause a negative wind stress curl in the south of the FJ axis over the central AS, resulting in a deep thermocline and high magnitude of heat being trapped. In winter monsoon months, though the wind stress curl is positive over large parts of the AS and could potentially shoal the thermocline and reduce the upper ocean heat content in the central AS, this does not happen due to two reasons. Firstly, winds are weaker, and spread over a larger area over the AS making the magnitude of the wind stress curl low. Secondly, westward propagating downwelling Rossby wave radiated from the eastern AS deepens the thermocline and prevents ventilation of the trapped heat. During the following spring, the collapse of the Rossby waves leads to the shoaling and mixing of underlying waters with surface waters thereby resurfacing of the trapped heat. The resurfacing of the trapped heat makes the AS a memory bank of the FJ induced signal.
RESUMEN
Coinfection is less commonly observed in individuals around the world, yet it is more common than the single infection. Around 800 million people worldwide are infected with helminths as a result of various diseases. Lymphatic filariasis (LF) and visceral leishmaniasis (VL) are chronic, deadly, crippling, and debilitating neglected tropical diseases (NTDs) that are endemic in tropical and subtropical regions of the world. Due to poor hygienic conditions, poverty, and genetic predisposition, those living in endemic areas are more likely to develop both leishmaniasis and filariasis. One of the key challenges in the management of LF/VL coinfection is the development of an effective therapeutic strategy that not only treats the first episode of VL but also prevents LF. However, there is a scarcity of knowledge and data on the relationship between LF and VL coinfection. While reviewing it was apparent that only a few studies relevant to LF/VL coinfections have been reported from southeastern Spain, Sudan, and the Indian subcontinents, highlighting the need for greater research in the most affected areas. We also looked at LF and VL as a single disease and also as a coinfection. Some features of the immune response evolved in mammalian hosts against LF and VL alone or against coinfection are also discussed, including epidemiology, therapeutic regimens, and vaccines. In addition to being potentially useful in clinical research, our findings imply the need for improved diagnostic methodology and therapeutics, which could accelerate the deployment of more specific and effective diagnosis for treatments to lessen the impact of VL/LF coinfections in the population.
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Coinfección , Filariasis Linfática , Leishmania donovani , Leishmaniasis Visceral , Leishmaniasis , Animales , Coinfección/epidemiología , Filariasis Linfática/complicaciones , Filariasis Linfática/tratamiento farmacológico , Filariasis Linfática/epidemiología , Humanos , Inmunidad , Leishmaniasis Visceral/complicaciones , Leishmaniasis Visceral/tratamiento farmacológico , Leishmaniasis Visceral/epidemiologíaRESUMEN
Coinfection of Leishmania with bacteria, viruses, protozoans, and nematodes alter the immune system of the host, thereby influencing the disease outcomes. Here, we have determined the immunogenic property and protective efficacy of the cross-reactive molecule HSP60 of filarial parasite B. malayi against the L. donovani in BALB/c mice. Parasitological parameters results showed a significant decrease in the parasite burden (~59%; P < 0.001) and also a substantial increase in the delayed-type hypersensitivity (DTH) response (P < 0.001) in mice immunized with 10 µg of rBmHSP60. Protection against L. donovani in mice immunized with rBmHSP60 resulted from activation of the T cells, which is characterized by higher levels of nitric oxide (NO) production, enhanced cell proliferation, higher levels (expression and release) of IFN- γ, TNF- α, and IL-12, also, higher production of IgG and IgG2a antibodies. This strong Th1 immune response creates an inflammatory domain for L. donovani and protects the host from VL.
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Brugia Malayi , Chaperonina 60/inmunología , Protección Cruzada , Proteínas del Helminto/inmunología , Leishmaniasis Visceral , Animales , Citocinas/inmunología , Inmunidad Celular , Leishmania donovani , Leishmaniasis Visceral/prevención & control , Ratones , Ratones Endogámicos BALB C , Proteínas Recombinantes/inmunología , Células TH1/inmunologíaRESUMEN
PURPOSE: We recently identified disorganized muscle protein-1 of Brugia malayi (DIM-1bm) as a vaccine candidate for human lymphatic filariasis. The present study was aimed at investigating the localization of DIM-1bm in the life-stages of B. malayi to identify the tissue target of vaccine action. METHODS: Recombinant DIM-1bm (rDIM-1bm) was prepared and antibodies were raised in BALB/c mice. Immunoblots of SDS-PAGE resolved B. malayi infective 3rd stage larvae (L3) and adult worm antigens and rDIM-1bm were prepared and reacted with anti-rDIM-1bm sera. Sections of adult female worms and whole-mount preparations of L3 and microfilariae (mf) were stained by immunofluorescence using rDIM-1bm antibodies and Alexa Fluor 488 labeled secondary antibodies, and examined under a confocal microscope. RESULTS: Immunofluorescence staining showed that DIM-1bm is localized mainly in the subcuticular muscle layer in the L3 and the adult worms; no fluorescent signal could be detected in mf. CONCLUSION: The localization of DIM-1bm in the parasites' muscle layer suggests that the immunoprophylactic efficacy of DIM-1 is evidently due to immobilization of the parasite and its subsequent immune elimination.
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Brugia Malayi , Filariasis Linfática , Animales , Anticuerpos Antihelmínticos , Antígenos Helmínticos , Femenino , Humanos , Ratones , Ratones Endogámicos BALB C , Proteínas MuscularesRESUMEN
BACKGROUND: Lymphatic Filariasis (LF) is one of the incapacitating and mosquito-borne sicknesses that on progression may prompt a few recognizable types of clutters like extreme lymphedema, hydrocele, and elephantiasis. METHODS: Antigenic preparations of B. malayi adult (BmA), S. cervi adult parasites and microfilariae (mf) total parasite extract were used to analyze the serological reactivity profile with human infectious sera collected from endemic areas of Bancroftian filariasis by performing Western blot and ELISA analysis. Sera from healthy human subjects were also included in the study to determine the variation incurred in the reactivity due to the filariasis infection. Gelelectrophoresis analysis of the crude-extract of BmA revealed seven protein bands while more than ten bands were recognized in S. cervi. RESULTS: our results represent a clear variation in protein patterns among the crude-antigens. ELISA results showed highest prevalence of IgG, IgM and IgG4 antibodies against all antigen preparations when recorded among microfilaraemic chronic infected patients. In both the antigenic preparations, the positive reactions were in the order of microfilaraemic>endemic normal>chronic>acute>nonendemic normal subjects. All sera of Mf+ patients were uniformly positive, while sera of both chronic and endemic normal subjects showed less reactivity. CONCLUSION: In the present study, we endeavoured to establish the extent of cross-reactivity of antigens derived from animal filarial parasites such as B. malayi and S. cervi with W. bancrofti filariasis sera of human patients. Besides, we further analyzed antibody-isotype profile of IgG, IgG4 and IgM in various human infection sera of bancroftian filarial subjects reactive to heterologous parasite antigens derived from adult worms of S. cervi from bovine and B. malayi from bovine and jirds.
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Anticuerpos Antihelmínticos , Filariasis Linfática , Inmunoglobulina G , Inmunoglobulina M , Wuchereria bancrofti , Animales , Anticuerpos Antihelmínticos/sangre , Anticuerpos Antihelmínticos/inmunología , Antígenos Helmínticos/química , Antígenos Helmínticos/inmunología , Reacciones Cruzadas , Filariasis Linfática/sangre , Filariasis Linfática/inmunología , Humanos , Inmunoglobulina G/sangre , Inmunoglobulina G/inmunología , Inmunoglobulina M/sangre , Inmunoglobulina M/inmunología , Masculino , Wuchereria bancrofti/inmunología , Wuchereria bancrofti/metabolismoRESUMEN
BACKGROUND: Lymphatic filariasis (LF) is a parasitic disease that causes permanent disability (elephantiasis). Currently used antifilarial drugs are failing to control LF and there is resurgence in some areas. Looking for new antifilarial leads, we found that Calotropis procera plant parts have been used in traditional medicine for alleviating elephantiasis but the antifilarial activity is not known. OBJECTIVE: In the present study, the antifilarial activity of ethanolic extract (A001) and its hexane fraction (F001) of C. procera flowers was investigated using the human filarial parasite Brugia malayi. METHODS: A001 and F001 were tested for antifilarial activity using motility and 3-(4,5-dimethylthiazol-2- yl)-2,5 diphenyltetrazolium bromide (MTT) assays (in vitro) and in the rodent models B. malayi- Meriones unguiculatus and B. malayi-Mastomys coucha. In the rodent models, A001 and F001 were administered orally for 5 consecutive days, and the adult worm burden and course of microfilaraemia were determined. RESULTS: Both A001 and F001 showed microfilaricidal and macrofilaricidal activity in vitro. In animal models, A001 killed ~49-54% adult worms. In M. coucha model, F001 killed 12-60% adult worms in a dose (125-500 mg/kg) dependent manner; A001 and F001 suppressed microfilaraemia till days 91 and 35 post initiation of treatment, respectively. HPTLC revealed 0.61% lupeol, 0.50% ß-sitosterol and 1.50% triacontanol in F001. CONCLUSION: Flowers of C. procera have definite microfilaricidal and macrofilaricidal activities. Whether this activity is due to lupeol, ß-sitosterol and triacontanol found in the hexane fraction remains to be investigated. This is the first report on the antifilarial efficacy of flowers of the plant C. procera.
Asunto(s)
Brugia Malayi/efectos de los fármacos , Calotropis/química , Filaricidas/farmacología , Flores/química , Extractos Vegetales/farmacología , Animales , Filariasis Linfática/tratamiento farmacológico , Filaricidas/química , Filaricidas/aislamiento & purificación , Pruebas de Sensibilidad Parasitaria , Extractos Vegetales/química , Extractos Vegetales/aislamiento & purificaciónRESUMEN
In the search for immunoprophylactics for the control of human lymphatic filariasis, we recently identified troponin 1 (Tn1) in Brugia malayi adult worms. The present study reports the cloning and expression of the B. malayi Tn1 (Tn1bm), its immunoprophylactic efficacy against B. malayi infection, and the immunological responses of the host. The Tn1bm gene was cloned (Acc no. JF912447) and expressed, and the purified recombinant Tn1bm (rTn1bm) presented a single ~ 27 kDa band. Parasite load in rTn1bm-immunized BALB/c mice challenged with B. malayi infective larvae (L3) was assessed. In rTn1bm-immunized animals, IgE, IgG, and IgG subclasses in the serum, cell proliferative response, Th1 and Th2 cytokine secretion (from splenocytes), and NO release (from peritoneal macrophages) were determined. Antibody-dependent cell-mediated cytotoxicity (ADCC) to L3 was assayed using rTn1bm-immune serum. The innate immune response markers MHC class-I, MHC class-II, TLR2, TLR4, and TLR6 expression in peritoneal macrophages and CD3+, CD4+, CD8+, and CD19+ in the splenocyte population were determined in Tn1bm-exposed cells from naïve mice. rTn1bm-immunized L3-challenged animals showed a 60% reduction in parasite burden. Immunization upregulated cellular proliferation, cytokine (IFN-γ, TNF-α, IL-1ß, IL-4, IL-6, and IL-10) secretion, NO release, and antigen-specific IgG, IgG1, and IgG2b antibody levels. rTn1bm-immune serum killed > 65% of L3 in the ADCC assay. Increased MHC class-II, TLR2, and TLR6 expression and the relative CD4+ and CD19+ cell populations of naïve animal cells indicated the ability of rTn1bm to mobilize innate immune responses. This is the first report of the immunoprophylactic potential of rTn1bm against B. malayi.
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Anticuerpos Antiprotozoarios/sangre , Brugia Malayi/inmunología , Filariasis Linfática/inmunología , Filariasis Linfática/prevención & control , Troponina I/genética , Troponina I/inmunología , Animales , Citotoxicidad Celular Dependiente de Anticuerpos/inmunología , Brugia Malayi/genética , Clonación Molecular , Citocinas/sangre , Citocinas/inmunología , ADN Complementario/metabolismo , Humanos , Inmunoglobulina E/sangre , Inmunoglobulina G/sangre , Macrófagos Peritoneales/inmunología , Ratones , Ratones Endogámicos BALB C , Carga de Parásitos , Células TH1/inmunología , Células Th2/inmunología , VacunaciónRESUMEN
We earlier found that F6 fraction of human filaria Brugia malayi cross-reacted with sera of Leishmania donovani infected hamsters and immunization with F6 inhibited both filarial and leishmanial infections. In the present study, we identified a 52.9-93.6 kDa fraction (Ld1) of L. donovani that cross-reacted with sera of B. malayi infected animals and investigated effect of Ld1 on filarial infection. Immunization of BALB/c mice with Ld1 facilitated B. malayi infection with remarkable increase in parasite burden. Facilitation of filarial infection was associated with downregulated cell proliferation, IL-5, IL-13, IFN-γ, TNF-α, and IL-2 levels and upregulated IL-4 and TGF-ß. Ld1 exposure also suppressed MHC class-I, MHC class-II, and FcεR1 expression, and phagocytosis in naive mouse macrophages, and CD4+, CD8+, and CD19+ cell population in mouse spleen. Two-dimensional electrophoresis and matrix-assisted laser desorption ionization-time of flight-mass spectrometry revealed eight proteins in Ld1: putative heat shock protein (HSP) 70-related protein 1, HSP70 mitochondrial precursor, alanine aminotransferase, 2,3-bisphosphoglycerate-independent phosphoglycerate mutase, protein disulfide isomerase, putative ATPase beta subunit, trypanothione reductase, and a hypothetical protein. HSP70 protein mitochondrial precursor and trypanothione reductase showed homology with Trypanosoma cruzi and L. donovani, respectively, and the rest 6 proteins including hypothetical protein bear homology with L. infantum. In conclusion, the present study for the first time shows that immunization with filarial cross-reactive Ld1 fraction of L. donovani facilitates filarial infection by modulating Th1 and Th2 responses. Ld1 molecules may therefore facilitate filarial infection in filaria-leishmania co-infection.
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Brugia Malayi/inmunología , Reacciones Cruzadas/inmunología , Filariasis/inmunología , Leishmania donovani/inmunología , Leishmaniasis Visceral/inmunología , Leishmaniasis/inmunología , Animales , Proliferación Celular , Coinfección/inmunología , Coinfección/parasitología , Cricetinae , Filariasis/parasitología , Humanos , Leishmaniasis/parasitología , Leishmaniasis Visceral/parasitología , Ratones , Ratones Endogámicos BALB C , Células TH1/inmunología , Células Th2/inmunología , VacunaciónRESUMEN
BACKGROUND: Lymphatic filariasis caused by Wuchereria bancrofti, Brugia malayi and B. timori, is a debilitating disease with an adverse social and economic impact. The infection remains unabated in spite of treatment with existing antifilarial drugs diethylcarbamazine (DEC) and ivermectin which are chiefly microfilaricides. There is therefore, need for macrofilaricides, embryostatic agents and better microfilaricides. In the present study we explored the antifilarial potential of crude extract and its molecular fractions of the plant Taxodium distichum using in vitro assay systems and rodent models of B. malayi infection. METHODS: Ethanolic extract (A001) of aerial parts of T. distichum was solvent fractionated and sub-fractionated. Four molecules, 3-Acetoxylabda-8(20), 13-diene-15-oic acid (K001), Beta-sitosterol (K002), labda-8(20),13-diene-15-oic acid (K003) and Metasequoic acid A (K004) were isolated from the fractions and their structure determined by spectroscopic analysis. The extract, subfractions and molecules were evaluated for antifilarial activity against B. malayi by 3-(4,5-dimethylthiazol-2-yl)-2,5 diphenyltetrazolium bromide (MTT) reduction and motility assays in vitro and in two animal models, Meriones unguiculatus and Mastomys coucha, harbouring B. malayi infection. RESULTS: A001 was effective in killing microfilariae (mf) and adult worms in vitro. The diterpenoid K003 produced 100 % reduction in motility of both mf and adult worms and > 80 % inhibition in MTT reduction potential of adult female worms. In B. malayi-M. unguiculatus model, A001 killed all the adult worms in > 80 % of infected animals. K003 was embryostatic (> 95 %) in this model. In the B. malayi-M. coucha model, K003 killed ~54 % of adult worms (macrofilaricidal activity) and rendered > 36 % female worms sterile; it also stopped any further rise in microfilaraemia after day 42 post-initiation of treatment. CONCLUSION: Ethanolic extract of aerial parts of the plant T. distichum possesses potent antifilarial activity and the active principle was localised to K003 which showed significant macrofilaricidal activity and late suppression of peripheral microfilaraemia and some embryostatic activity. These findings indicate that labdane diterpenoid molecule(s) may provide valuable leads for design and development of new macrofilaricidal agent(s). To the best of our knowledge, this is the first report on antifilarial efficacy of products from the plant T. distichum.
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Brugia Malayi/efectos de los fármacos , Diterpenos/farmacología , Filariasis Linfática/tratamiento farmacológico , Filaricidas/farmacología , Extractos Vegetales/farmacología , Taxodium/química , Animales , Brugia Malayi/citología , Dietilcarbamazina/uso terapéutico , Modelos Animales de Enfermedad , Diterpenos/química , Diterpenos/aislamiento & purificación , Femenino , Filaricidas/química , Filaricidas/aislamiento & purificación , Gerbillinae , Humanos , Ivermectina/uso terapéutico , Masculino , Microfilarias , Murinae , Componentes Aéreos de las Plantas/química , Extractos Vegetales/químicaRESUMEN
Coinfections are common in natural populations and the outcome of their interactions depends on the immune responses of the host elicited by the parasites. Earlier we showed that immunization with BmAFII (Sephadex G-200 eluted) fraction of human lymphatic filaria Brugia malayi inhibited progression of Leishmania donovani infection in golden hamsters. In the present study we identified cross reactive molecules of B. malayi, and investigated their effect on L. donovani infection and associated immune responses in the host. The sequence alignment and sharing of linear T- and B-cell epitopes in protein molecules of B. malayi and L. donovani counterparts were studied in silico. Hamsters were immunized with robustly cross reactive SDS-PAGE resolved fractions F6 (54.2-67.8kDa) and F9 (41.3-45.0kDa) of B. malayi and subsequently inoculated with amastigotes of L. donovani intracardially. F6 inhibited (â¼72%) L. donovani infection and upregulated Th1 cytokine expression, lymphoproliferation, IgG2, IgG2/3 levels and NO production, and downregulated Th2 cytokine expression. Sequences in HSP60 and EF-2 of F6 and L. donovani counterparts were conserved and B- and T-cell epitopes in the proteins shared antigenic regions. In conclusion, leishmania-cross reactive molecules of filarial parasite considerably inhibited leishmanial infection via Th1-mediated immune responses and NO production. Common B- and T-cell epitope regions in HSP60 and EF-2 of the parasites might have contributed to the inhibitory effect on the L. donovani infection. Thus, leishmania-cross reactive filarial parasite molecules may help in designing prophylactic(s) against L. donovani.
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Brugia Malayi/inmunología , Leishmania donovani , Leishmaniasis Visceral/prevención & control , Animales , Cricetinae , Citocinas/sangre , Epítopos de Linfocito T , Humanos , Inmunización , Leishmania donovani/inmunología , MesocricetusRESUMEN
Nitric oxide (NO) mediated mechanisms have been implicated in killing of some life-stages of Brugia malayi/Wuchereria bancrofti and protect the host through type 1 responses and IFN-γ stimulated toxic mediators' release. However, the identity of NO stimulating molecules of the parasites is not known. Three predominantly NO-stimulating SDS-PAGE resolved fractions F8 (45.24-48.64 kDa), F11 (33.44-38.44 kDa) and F12 (28.44-33.44 kDa) from B. malayi were identified and their proteins were analyzed by 2-DE and MALDI-TOF/TOF. Tropomyosin, calponin and de novo peptides were identified by 2-DE and MALDI-TOF/TOF in F8 and immunization with F8 conferred most significant protection against L3-initiated infection in Mastomys coucha. Immunized animals showed upregulated F8-induced NO, IFN-γ, TNF-α, IL-1ß, IL-10, TGF-ß release, cellular proliferative responses and specific IgG and IgG1. Anti-IFN-γ, anti-TNF-α, and anti-IL-1ß significantly reduced F8-mediated NO generation and iNOS induction at protein levels. Anti-IFN-γ treated cells showed maximum reduction (>74%) in NO generation suggesting a predominant role of IFN-γ in iNOS induction. In conclusion, the findings suggest that F8 which contains tropomyosin, calponin and de novo peptides protects the host via IFN-γ mediated iNOS induction and may hold promise as vaccine candidate(s). This is also the first report of identification of tropomyosin and calponin in B. malayi.
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Antígenos Helmínticos/administración & dosificación , Brugia Malayi/inmunología , Activadores de Enzimas/administración & dosificación , Interferón gamma/metabolismo , Óxido Nítrico Sintasa de Tipo II/metabolismo , Animales , Anticuerpos Antihelmínticos/sangre , Antígenos Helmínticos/química , Antígenos Helmínticos/aislamiento & purificación , Brugia Malayi/química , Proliferación Celular , Electroforesis en Gel Bidimensional , Electroforesis en Gel de Poliacrilamida , Activadores de Enzimas/química , Activadores de Enzimas/aislamiento & purificación , Inmunoglobulina G/sangre , Interleucinas/metabolismo , Leucocitos Mononucleares/inmunología , Masculino , Peso Molecular , Murinae , Óxido Nítrico/metabolismo , Espectrometría de Masa por Láser de Matriz Asistida de Ionización Desorción , Factor de Crecimiento Transformador beta/metabolismo , Factor de Necrosis Tumoral alfa/metabolismo , Vacunación/métodosRESUMEN
As part of our drug discovery program for anti-filarial agents from Indian medicinal plants, leaves of Eucalyptus tereticornis were chemically investigated, which resulted in the isolation and characterization of an anti-filarial agent, ursolic acid (UA) as a major constituent. Antifilarial activity of UA against the human lymphatic filarial parasite Brugia malayi using in vitro and in vivo assays, and in silico docking search on glutathione-s-transferase (GST) parasitic enzyme were carried out. The UA was lethal to microfilariae (mf; LC100: 50; IC50: 8.84 µM) and female adult worms (LC100: 100; IC50: 35.36 µM) as observed by motility assay; it exerted 86% inhibition in MTT reduction potential of the adult parasites. The selectivity index (SI) of UA for the parasites was found safe. This was supported by the molecular docking studies, which showed adequate docking (LibDock) scores for UA (-8.6) with respect to the standard antifilarial drugs, ivermectin (IVM -8.4) and diethylcarbamazine (DEC-C -4.6) on glutathione-s-transferase enzyme. Further, in silico pharmacokinetic and drug-likeness studies showed that UA possesses drug-like properties. Furthermore, UA was evaluated in vivo in B. malayi-M. coucha model (natural infection), which showed 54% macrofilaricidal activity, 56% female worm sterility and almost unchanged microfilaraemia maintained throughout observation period with no adverse effect on the host. Thus, in conclusion in vitro, in silico and in vivo results indicate that UA is a promising, inexpensive, widely available natural lead, which can be designed and developed into a macrofilaricidal drug. To the best of our knowledge this is the first ever report on the anti-filarial potential of UA from E. tereticornis, which is in full agreement with the Thomson Reuter's 'Metadrug' tool screening predictions.
Asunto(s)
Brugia Malayi/efectos de los fármacos , Filariasis/tratamiento farmacológico , Filaricidas/uso terapéutico , Hojas de la Planta , Plantas Medicinales , Triterpenos/uso terapéutico , Animales , Simulación por Computador , Filaricidas/farmacología , Humanos , Técnicas In Vitro , Triterpenos/farmacología , Ácido UrsólicoRESUMEN
Here we report the synthesis of novel chalcone-thiazole compounds and their antifilarial activity. The antifilarial properties of these hybrids were assessed against microfilariae as well as adult worms of Brugia malayi. Among all the synthesized compounds, only two compounds, namely 4g and 4n were identified to be promising in vitro. These active compounds were tested in B. malayi-jird (Meriones unguiculatus) and B. malayi-Mastomys coucha models. Compound 4n showed 100% embryostatic effect and 49% macrofilaricidal in jirds and M. coucha models, respectively. This study provides a new structural clue for the development of novel antifilarial lead molecules.
Asunto(s)
Brugia Malayi/efectos de los fármacos , Chalcona/análogos & derivados , Chalcona/farmacología , Filariasis/tratamiento farmacológico , Filaricidas/síntesis química , Filaricidas/farmacología , Tiazoles/farmacología , Animales , Chalcona/química , Modelos Animales de Enfermedad , Relación Dosis-Respuesta a Droga , Femenino , Filariasis/parasitología , Filaricidas/química , Gerbillinae , Masculino , Estructura Molecular , Murinae , Pruebas de Sensibilidad Parasitaria , Relación Estructura-Actividad , Tiazoles/químicaRESUMEN
We have recently identified disorganized muscle protein-1 (DIM-1) in one of the proinflammatory fractions of the human filaria Brugia malayi adult worm. The present study was undertaken to characterize B. malayi DIM-1 (DIM-1bm) and explore its vaccine potential. In this study we cloned and expressed the DIM-1bm gene, investigated its sequence homology with other nematodes, constructed in silico structural model, purified the recombinant DIM-1bm (rDIM-1bm) protein, and studied the effect of immunization with rDIM-1bm on the establishment of B. malayi infection in Mastomys coucha. DIM-1bm showed similarity with DIM-1 of Caenorhabditis elegans, Ascaris suum and Loa loa. Structural modeling revealed three immunoglobulin domains in DIM-1bm indicating that it is a member of immunoglobulin superfamily (IgSF) and 'blastn' results showed that DIM-1bm coding sequence (CDS) have almost no homology with human and mouse nucleotide sequences. Immunization with rDIM-1bm partially protected M. coucha against establishment of infection as inferred by a low recovery of microfilariae (37-64%) and parasite burden (â¼50%). The enhanced activity of macrophages, and IFN-γ and NO responses, and elevated levels of specific IgG, IgG1, IgG2a and IgG2b correlated with parasitological findings. This is the first report on cloning, expression, structural modeling and purification of rDIM-1bm and its ability to partially prevent establishment of B. malayi infection. DIM-1bm's almost complete lack of homology with the human counterpart makes it an attractive protein for exploring its vaccine potential.