Division of Biological Sciences
https://etd.iisc.ac.in/handle/2005/1
2024-03-28T21:15:27ZAcoustic Signals, Mate Choice And Mate Sampling Strategies in a Field Cricket
https://etd.iisc.ac.in/handle/2005/2697
Acoustic Signals, Mate Choice And Mate Sampling Strategies in a Field Cricket
Nandi, Diptarup
Acoustic communication in orthopterans and anurans provides a suitable model system for studying the evolutionary mechanisms of sexual selection mainly because males use acoustic signals to attract females over long distances for pair formation. Females use these signals not only to localize conspecific males but also to discriminate between potential mates. Investigations on the effect of sexual selection on acoustic signals requires an understanding of how female preferences for different features of the acoustic signal affect male mating success under ecological constraints in wild populations. The effect of female preferences on male mating success depends on the mate sampling strategy that females employ to search for potential mates. Despite its relevance, female mate sampling strategies based on male acoustic signals have rarely investigated in orthopterans and anurans, especially in the field. Considering the elaborate knowledge of the role of sensory physiology in female phonotaxis behaviour and characterization of the male acoustic signal, I used the field cricket species Plebeiogryllus guttiventris as a model system in this study. In this thesis, I first investigated the ecology of callers in wild populations. I then investigated female mate sampling strategies by incorporating relevant information on the ecology of signalers and the sensory physiology of receivers.
Amount of calling activity is a strong determinant of male mating success in acoustically communicating species such as orthopterans and anurans. While many studies in crickets have investigated the determinants of calling effort, patterns of variability in male calling effort in natural choruses remain largely unexplored. I therefore investigated the spatio-temporal dynamics of acoustic chorusing behaviour in a wild population. I first studied the consistency of calling activity by quantifying variation in male calling effort across multiple nights of calling using repeatability analysis. Callers were inconsistent in their calling effort across nights and did not optimize nightly calling effort to increase their total number of nights spent calling. Next, I investigated calling site fidelity of males across multiple nights by quantifying movement of callers. Callers frequently changed their calling site across calling nights with substantial displacement but without any significant directionality. Finally, I investigated trade-offs between within-night calling effort and energetically expensive calling song features such as call intensity and chirp rate. Calling effort was not correlated with any of the calling song features, suggesting that energetically expensive song features do not constrain male calling effort. The two key features of signaling behaviour, calling effort and call intensity, which determine the duration and spatial coverage of the sexual signal, are uncorrelated and function independently
Acoustic signal variation and female preference for different signal components constitute the prerequisite framework to study the mechanisms of sexual selection that shape acoustic communication. Despite several studies of acoustic communication in crickets, information on both male calling song variation in the field and female preference in the same system is lacking for most species. First, I quantified variation in the spectral, temporal and amplitudinal characteristics of the male calling song in a wild population, at two temporal scales, within and across nights, using repeatability analysis. Carrier frequency (CF) was the most repeatable call trait across nights, whereas chirp period (CP) had low repeatability. I further investigated female preferences based on song features with high and low repeatability (CF and CP respectively). Females showed no consistent preferences for CF but were more attracted towards calls with higher rates (shorter CP). I also examined the effect of signal intensity, which is known to play a critical role in female phonotaxis behaviour, on female preferences for faster calls. Females preferred louder calls over faster ones, implying a dominant role for signal intensity in female evaluation of potential mates based on acoustic signals. Call intensity was also the only signal feature that was positively correlated with male size.
In the final chapter, I investigated female mate sampling strategies based on acoustic signals using both theoretical and empirical approaches. Analytical models of mate sampling have demonstrated significant differences in individual fitness returns for different sampling strategies. However these models have rarely incorporated relevant information on the ecology of signalers and the sensory physiology of receivers. I used simulation models to compare the costs and benefits of different mate sampling strategies by incorporating information on relative spacing of callers in natural choruses and the effect of signal intensity on female phonotaxis behaviour. The strategy of mating with males that were louder at the female position emerged as the optimal sampling rule in the simulations. When tested empirically in the field using callers in natural choruses, females seemed to follow the optimal strategy of mating with males that were perceived as louder at their position.
2017-10-05T00:00:00ZActivation Of Glycoprotein Hormone Receptors : Role Of Different Receptor Domains In Hormone Binding And Signaling
https://etd.iisc.ac.in/handle/2005/2344
Activation Of Glycoprotein Hormone Receptors : Role Of Different Receptor Domains In Hormone Binding And Signaling
Majumdar, Ritankar
The glycoprotein hormones, Luteinizing Hormone (LH), human Chorionic Gonadotropin (hCG), Follicle Stimulating Hormone (FSH) and Thyroid Stimulating Hormone (TSH) are heterodimeric proteins with an identical α-subunit associated non-covalently with the hormone specific β-subunit and play important roles in reproduction and overall physiology of the organism [1]. The receptors of these hormones belong to the family of G-protein coupled receptors (GPCR) and have a large extracellular domain (ECD) comprising of 9-10 leucine rich repeats (LRR) followed by a flexible hinge region, a seven helical transmembrane domain (TMD) and a C terminal cytoplasmic tail [2]. Despite significant sequence and structural homologies observed between the ECDs of the receptors and the specific β-subunits of the hormones, the hormone-receptor pairs exhibit exquisite specificity with very low cross-reactivity with other members of the family. The TSH receptor (TSHR) is an especially interesting member of this family as it not only recognizes is cognate ligand, i.e. TSH, but also binds to the non-cognate ligands such as autoantibodies. TSHR autoantibodies come in different flavors; inhibitory antibodies that compete with the hormone for receptor binding and block its action, stimulatory antibodies that activate the receptor in a hormone independent manner and neutral antibodies that bind to the receptor but do not directly influence its functions. The inhibitory autoantibodies cause hypothyroidism and are responsible for Hashimoto’s Thyroiditis, whereas the stimulatory autoantibodies cause Graves’ thyrotoxicosis characterized by hyperthyroid condition [3]. The exact epitopes of these autoantibodies are not well delineated although it has been hypothesized that the blocking type- and the stimulatory type- autoantibodies have predominant epitopes in the TSHR ECD that overlap with hormone binding regions [4]. Insights into the mode of hormone or autoantibody binding to the receptor was primarily derived from the crystal structure of FSHR leucine rich repeat domain (LRRD) bound to single chain analog of FSH, and the crystal structures of TSHR LRRD bound to the stimulatory type human monoclonal antibody M22 [5] and the inhibitory type- monoclonal antibody K1-70 [6]. Both these crystal structures propose LRRDs as the primary ligand binding site which interacts with the hormone through its determinant loop in a hand-clasp fashion [7] while the autoantibodies mimics the hormone binding to a large extent [8] . These structures, while providing detailed understanding of the molecular interactions of the LRRs with the hormone, shed little light on the mechanism by which the signal generated at the LRRD are transduced to the downstream effector regions at the distally situated TMD. Hence, while one understands the ligand binding to a large extent, the activation process is not well understood, one of the central objective of the present study.
Ligand-receptor interactions are typically studied by perturbing ligand/receptor structure by mutagenesis or by mapping conformational changes by biophysical or computational approaches. In addition to the above-mentioned approaches, the present work also uses highly specific antibodies against different domains of the receptor as molecular probes due to the ability of antibodies to distinguish between conformations likely to arise during the activation process. Use of antibodies to
understand the receptor activation process is especially apt for TSHR due to the presence of physiologically relevant TSHR autoantibodies and their ability to influence hormone binding and receptor activation [9, 10]. Chapter 2 attempts to provide a comparison between the interactions of the hormone and the autoantibodies with TSHR. For this purpose, two assays were developed for identification of TSHR autoantibodies in the sera of patients suffering from autoimmune thyroid diseases (AITD), the first assay is based on the ability of TSHR autoantibodies to compete for radiolabeled hormone (The TSH binding inhibition (TBI), assay) and the second based on the capability of stimulatory antibody to produce cAMP in cells expressing TSHR (TSHR stimulatory immunoglobin (TSI) assay). A stable cell line expressing TSHR capable of recognizing both TSH and TSHR autoantibodies was thus created and used for prospective and retrospective analysis of AITD patients. Based on the TBI and TSI profiles of IgGs, purified from AITD patient's sera, it was recognized that TSHR stimulatory and TSH binding inhibitory effects of these antibodies correlated well, indicating overlap between hormone binding and IgG binding epitopes. It was also recognized that stimulatory IgGs are not affected by negative regulatory mechanism that governs TSH secretion substantiating the persistence of these antibodies in circulation. Kinetics of cAMP production by Graves’ stimulatory IgG was found to be fundamentally distinct, where the autoantibodies displayed pronounce hysteresis during the onset of the activation process when compared to the hormone. This could possibly be explained by the oligoclonality of the autoantibody population, a different mechanism of receptor activation or dissimilarity in autoantibody and hormone epitopes. To gain additional insights into the epitopes of TSHR autoantibodies and the regions that might be critical in the activation process, different overlapping fragments encompassing the entire TSH receptor ECD were cloned, expressed in E.coli as GST fusion proteins and purified: 1] the first three LRRs (TLRR 1-3, amino acid (aa) 21-127), 2] the first six LRRs (TLRR 1-6, aa 21-200), 3] the putative major hormone binding domain (TLRR 4-6, aa 128-200), and 4] the hinge region of TSH receptor along with LRR 7 to 9, (TLRR 7-HinR, aa 201-413). The receptor fragment TLRR 7-HinR was further subdivided into LRR 7-9 (TLRR 7-9, aa 201-161) and the hinge region (TSHR HinR, aa 261-413), expressed as N-terminal His-Tagged protein and purified using IMAC chromatography. Simultaneously, the full-length TSHR ECD was cloned, expressed and purified using the Pichia pastoris expression system. ELISA or immunoblot analysis of autoantibodies with the TSHR exodomain fragments suggested that Graves’ stimulatory antibody epitopes were distributed throughout the ECD with LRR 4-9 being the predominant site of binding. Interestingly, experiments involving neutralization of Graves’ IgG stimulated cAMP response by different receptor fragment indicated that fragments corresponding to the TSHR hinge region were better inhibitors of autoantibody stimulated receptor response than corresponding LRR fragments, suggesting that the hinge region might be an important component of the receptor activation process.
This was in contrast to prevalent beliefs that considered the hinge region to be an inert linker connecting the LRRs to the TMD, a structural entity without any known functional significance.
Mutagenesis in TSHR hinge region and agonistic antibodies against FSHR and LHR hinge regions, reported by the laboratory, recognized the importance of the hinge regions as critical for receptor activation and may not simply be a scaffold [11-13]. Unfortunately, the mechanism by which the hinge region regulates binding or response or both have not been well understood partially due to unavailability of structural information about this region. In addition poor sequence similarity within the GpHR family and within proteins of known structure, make this region difficult to model structurally. In chapter 3, effort is made to model the hinge regions of the three GpHR based on the knowledge driven and Ab initio protocols. An assembled structure comprising of the LRR domain (derived from the known structures of FSHR and TSHR LRR domains) and the modeled hinge region and transmembrane domain presents interesting differences between the three receptors, especially in the manner the hormone bound LRRD is oriented towards the TMD. These models also suggested that the α-subunit interactions in these three receptors are fundamentally different and this was verified by investigating the effects of two α-subunit specific MAbs C10/2A6 on hCG-LHR and hTSH-TSHR interactions. These two α-subunit MAbs had inverse effects on binding of hormone to the receptor. MAb C10 inhibited TSH binding to TSHR but not that of hCG, whereas MAb 2A6 inhibited binding of hCG to LHR but not of hTSH. Investigation into the accessibility of their epitopes in a preformed hormone receptor complex indicated that the α-subunit may become buried or undergo conformational change during the activation process and interaction may be different for LHR and TSHR.
Fundamental differences in TSHR and LHR were further investigated in the next chapter (Chapter 4), especially with regards to the ligand independent receptor activation. Polyclonal antibodies were developed against LRR 1-6, TLRR 7-HinR and the TSHR HinR receptor fragments. The LRR 1-6 antibodies were potent inhibitor of receptor binding as well as response, similar to that observed with antibodies against the corresponding regions of LHR. Interestingly, the antibodies against the hinge region of TSHR were unable to inhibit hTSH binding, but were effective inhibitors of cAMP production suggesting that this region may be involved in a later stage of a multi-step activation process. This was also verified by studying the mechanism of inhibition of receptor response and their effect on ligand-receptor association and dissociation kinetics. Hinge region-specific antibodies immunopurified from TLRR 7-HinR antibodies behaved akin to those of the pure hinge region antibodies providing independent validation of the above results. This result was, however, in contrast to those observed with a similar antibody against LHR hinge region. As compared to the TSHR antibody, the LHR antibody inhibited both hormone binding and response. In addition, this antibody could dissociate a preformed hormone-receptor complex which was not observed for TSHR hinge region antibodies. Although unable to dissociate preformed hormone-receptor complex by itself, the TSHR HinR antibodies augmented hormone induced dissociation of the hormone-receptor complex suggesting that this region may be involved in modulation of negative cooperativity associated with TSHR.
Molecular dissection of the role of hinge region of TSHR was further carried out by using monoclonal antibodies against LRR 1-3 (MAb 413.1.F7), LRR 7-9 (MAb 311.87), TSHR hinge region (MAb 311.62 and MAb PD1.37). MAb 311.62 which identifies the LRR/Cb-2 junction (aa 265-275), increased the affinity of TSHR for the hormone while concomitantly decreasing its efficacy, whereas MAb 311.87 recognizing LRR 7-9 (aa 201-259) acted as a non-competitive inhibitor of TSH binding. MAb 413.1.F7 did not affect hormone binding or response and was used as the control antibody for different experiments. Binding of MAbs was sensitive to the conformational changes caused by the activating and inactivating mutations and exhibited differential effects on hormone binding and response of these mutants. By studying the effects of these MAbs on truncation and chimeric mutants of thyroid stimulating hormone receptor (TSHR), this study confirms the tethered inverse agonistic role played by the hinge region and maps the interactions between TSHR hinge region [14] and exoloops responsible for maintenance of the receptor in its basal state. Mechanistic studies on the antibody-receptor interactions suggest that MAb 311.87 is an allosteric insurmountable antagonist and inhibits initiation of the hormone induced conformational changes in the hinge region, whereas MAb 311.62 acts as a partial agonist that recognizes a conformational epitope critical for coupling of hormone binding to receptor activation. Estimation of apparent affinities of the antibody to the receptor and the cooperativity factor suggests that epitope of MAb 311.87 (LRR 7-9) may act as a pivot involved in the initial events immediate to hormone binding at the LRRs. The anatgonsitic effect of MAB 311.62 on binding and response also suggested that binding of hormone is conformationally selective rather than an induced event. The hinge region, probably in close proximity with the α-subunit in the hormone-receptor complex, acts as a tunable switch between hormone binding and receptor activation.
In contrast to the stimulatory nature of Cb-2 antibody such as MAb 311.62, MAb PD1.37, which identified residues aa 366–384 near Cb-3, was found to be inverse agonistic. Unlike other known inverse agonistic MAbs such as CS-17 [15] and 5C9 [16], MAb PD1.37 did not compete for TSH binding to TSHR, although it could inhibit hormone stimulated response. Moreover, unlike CS-17, MAb PD1.37 was able to decrease elevated basal cAMP of hinge region constitutively activated mutations only but not those in the extracellular loops. This is particularly important as interaction of hinge region residues with those of ECLs had been thought to be critical in maintenance of the basal level of receptor activation and are responsible for attenuating the constitutive basal activity of the mutant and wild-type receptors in the absence of the hormone. This was demonstrated by a marked increase in the basal constitutive activity of the receptor upon the complete removal of its extracellular domain, which returned to the wild-type levels upon reintroduction of the hinge region. However, careful comparison of the activities of the mutants (receptors harboring deletions and gain-of-function mutations) with maximally stimulated wild-type TSHR indicated that these mutations of the receptor resulted primarily in partial activation of the serpentine domain suggesting that only the ECD in complex with the hormone is the full agonist of the receptor.
Confirmation of the above proposition has been difficult to verify primarily due to a highly transient conformational change in the tripartite interaction of the hinge region/hormone and the ECLs. The current approaches of using antibodies to probe the ECLs are difficult due to the conformational nature of the antigen as well as difficulty in obtaining a soluble protein. In chapter 5, the ligand induced conformational alterations in the hinge regions and inter-helical loops of LHR/FSHR/TSHR were mapped using the exoloop specific antibodies generated against a mini-Transmembrane domain (mini-TMD) protein. This mini-TMD protein, designed to mimic the native exoloop conformations, was created by joining the TSHR exoloops, constrained through the helical tethers and library derived linkers. The antibody against mini-TMD specifically recognized all three GpHRs and inhibited the basal and hormone stimulated cAMP production without affecting hormone binding. Interestingly, binding of the antibody to all three receptors was abolished by prior incubation of the receptors with the respective hormones suggesting that the exoloops are buried in the hormone-receptor complexes. The antibody also suppressed the high basal activities of gain-of-function mutations in the hinge regions, exoloops and TMDs such as those involved precocious puberty and thyroid toxic adenomas. Using the antibody and point/deletion/chimeric receptor mutants, dynamic changes in hinge region-exoloop interactions were mapped. The computational analysis suggests that mini-TMD antibodies act by conformationally locking the transmembrane helices by restraining the exoloops and juxta-membrane regions. This computational approach of generating synthetic TMDs bears promise in development of interesting antibodies with therapeutic potential, as well as, explains the role of exoloops during receptor activation.
In conclusion (Chapter 6), the study provides a comprehensive outlook on the highly dynamic interaction of ligand and different subdomains of the TSHR (and to a certain extent of LHR and FSHR) and proposes a model of receptor activation where the receptor is in a dynamic equilibrium between the low affinities constrained state and the high affinity unconstrained state and bind to the hormone through the LRR 4-6. Upon binding the βL2 loop of the hormone contact LRR 8-10 that triggers a conformational change in the hinge region driving the α-subunit to contact the ECLs. Upon contact, the ECLs cooperatively causes helix movement in the TMH and ultimately in ICLs causing the inbuilt GTP-exchange function of a GPCR.
2014-07-16T00:00:00ZActivin and TGF-β signaling: Differential role of a Serine/Threonine phosphate in the regulation of SMAD2 activity
https://etd.iisc.ac.in/handle/2005/5695
Activin and TGF-β signaling: Differential role of a Serine/Threonine phosphate in the regulation of SMAD2 activity
Gautam, Srishti
Activins are the most prominent members of the Activin/Inhibin branch of the TGF-β superfamily. Similar to TGF-β, they play an essential role in regulating multiple physiological processes such as development, reproductive physiology, and immune responses. Besides, Activins demonstrate a vital role in wound healing and diseases like fibrosis and cancer. It contributes to various cancer processes, such as cell migration, metastasis, immune evasion, angiogenesis, drug resistance, and cancer cachexia.
In recent studies, several differences have been reported between Activin and TGF-β mediated signaling and response. For example, Activins have been shown to function as a morphogen gradient in early embryonic development, unlike TGF-and are crucial for maintaining pluripotency in embryonic stem cells Despite differences, the primary notion in the field is that Activin signaling mirrors TGF-β signaling, where both the ligands activate the same SMAD2 and SMAD3 proteins and induce similar cellular responses.
However, if we look closely at the available literature, it is intriguing that all known TGF-β superfamily ligands (over 40) signal through merely seven type I and five type II receptors and a set of 8 SMAD proteins. This undoubtedly results in a high degree of context-dependent promiscuity, where ligands of one subfamily could signal through receptors of other subfamilies. This could potentially result from additional complexity at the level of signaling, which is poorly understood and represent some of the main standing questions in the field.
One of the major non-canonical pathways induced by Activin and TGF-β is ERK/MAPK pathway. The ERK/MAPK pathway forms an essential component of tumorigenesis and is active in most cancers. In addition to being Serine/Threonine kinases, the two TGF-β receptors, type I and type II, also function as tyrosine kinases and undergo auto-phosphorylation at tyrosine residues. These dual-specificity kinases employ various mechanisms to initiate the non-canonical ERK/MAPK pathway; many of them have been extensively studied. However, the regulation of the ERK/MAPK pathway by Activin and its role in Activin-mediated signaling is not well studied.
We demonstrate that the non-canonical ERK/MAPK pathway is indispensable for Activin-induced EMT markers and cell migration. Next, we investigated the effect of ERK inhibition on Activin-mediated SMAD signaling. We found that the non-canonical ERK/MAPK pathway is crucial for Activin-mediated phosphorylation of SMAD2 and had no effect on phosphorylation of SMAD3.
Furthermore, we demonstrate that the active ERK/MAPK pathway is essential for the nuclear translocation of SMAD2 and transcription of specific gene targets. In addition to this, the activity of GSK3β was identified to be critical for the regulation of receptor-mediated phosphorylation of SMAD2. A Ser/Thr phosphatase, PHLPP1, was determined as the common target of both ERK/MAPK and GSK3β kinases, which differentially targets the phosphorylation of SMAD2 upon Activin stimulation. ERK/MAPK and GSK3β kinases inhibit the phosphatase activity of PHLPP1, thereby facilitating SMAD2 phosphorylation. This signaling interaction was recorded only upon stimulation with Activin and was absent in TGF-β signaling. Therefore, through this study, we present novel differences between Activin and TGF-β-mediated signal transduction.
Next, we attempted to understand how the non-canonical ERK/MAPK pathway is induced in response to the two ligands. We found that Activin and TGF-β differ in the mechanisms that they employ for ERK activation. We demonstrate that Activin signaling requires ectodomain shedding and EGFR transactivation to induce ERK/MAPK pathway; however, TGF-β promotes ligand-independent EGFR transactivation.
Besides ERK/MAPK and GSK3β, we identified a unique regulatory role for PI3K, and observed that the phosphatase activity of PHLPP1, is regulated by all three kinases independently. We identified a positive regulation of PHLPP1 by PI3K, whereas both ERK/MAPK and GSK3β were found to regulate it negatively. Therefore, we propose that an “incoherent feedforward loop” is functioning between these kinases that regulate the PHLPP1 activity, and its outcome governs the eventual phosphorylation of Activin-mediated SMAD2.
To understand the effect of this regulation on Activin response, we performed a stable knockdown of PHLPP1 in cells and examined their EMT phenotype and migratory ability. Weobserved that upon knockdown of PHLPP1 expression, the migratory ability of cells was retained even in the presence of ERK inhibition. Therefore, we demonstrate a unique role for PHLPP1 in Activin-mediated signaling and response.
In conclusion, this study provides evidence of some fundamental differences in the signal transduction pathways initiated by Activin and TGF-β. We demonstrate that the two ligands employ different mechanisms to induce the non-canonical ERK/MAPK pathway. ERK/MAPK pathway plays a unique regulatory role in Activin-mediated SMAD2 and not SMAD3. We also identified a novel regulation of PHLPP1 by ERK/MAPK, along with GSK3β and PI3K kinases, and found this crosstalk indispensable for Activin-mediated response.
Adjunct Therapy with Curcumin for the Treatment of Malaria : Studies in a Murine Model
https://etd.iisc.ac.in/handle/2005/3553
Adjunct Therapy with Curcumin for the Treatment of Malaria : Studies in a Murine Model
Dende, Chaitanya
Malaria accounts for 198 million cases worldwide; with a high mortality rate. 584000 deaths were reported in 2013. Malaria is a re-emerging disease globally due to drug resistance, parasite recrudescence and non-availability of a vaccine. Chloroquine, quinine and antifolates served as frontline antimalarial drugs for decades. Development of resistance to chloroquine and antifolates, and the decreased efficacy of mefloquine, and even quinine, in malaria-endemic regions, has led to artemisinin derivatives evolving as frontline drugs. Artemisinin is a potent antimalarial compound and clears around 104 parasites per cycle. Despite being a potent antimalarial, artemisinin derivatives suffer from poor pharmacokinetic properties and short half lives. This has led to the development of artemisinin-based combination therapies (ACTs) using a partner drug with a longer half-life. However, resistance to ACTs has been reported in the last few years, perhaps due to lack of adherence to prescribed regimens or suboptimal treatment and the use of counterfeit drugs. Therefore there is an urgent need to develop an alternative ACT which overcomes these limitations.
This thesis entitled “Adjunct therapy with curcumin for the treatment of malaria: studies in a murine model” describes the antimalarial activity of curcumin and artemisinin and the adjunct role of curcumin in the prevention of parasite recrudescence and cerebral malaria. The thesis is divided into three chapters:
The first chapter entitled “Introduction: Malaria and anti-malarial drugs” consists of a brief introduction of malaria, the parasite life cycle and currently known antimalarial drugs. During the course of infection, the Plasmodium undergoes sporogony in the mosquito, and merogony and schizogony in the human host. All these life cycle stages are briefly described with depictions. A major part of this chapter is dedicated to describe antimalarial compounds under the following headings 1. Quinoline derivatives 2. 4-aminoquinolines 3. Antifolates 4. Artemisinin derivatives 5. Antibiotics and 6. Curcumin.
The second chapter is aimed at examining the ability of curcumin-arteether (a synthetic derivative of artemisinin) combination therapy in preventing parasite recrudescence in a murine model through immunomodulation employing various immunological, molecular biological, and biochemical techniques. The use of suboptimal doses of antimalarial drugs leads to recrudescence or relapse of malaria (reappearance of the parasite in blood after antimalarial regimen). In the present study we have addressed this issue by the use of curcumin as an adjunct molecule with α,β arteether (a synthetic derivative of artemisinin). We have studied recrudescence in a Swiss mice model. A suboptimal dose was standardized by the use of different doses of α,β arteether (AE) ranging from 250µg to 1500 µg. We found 750 µg to be a suboptimal dose and studied the adjunct nature of curcumin when animals were treated with AE suboptimal dose or AE+curcumin (AC) combination treatment and monitored the survival of animals. Our results clearly demonstrate that ~95% of animals treated with the suboptimal AE dose died of recrudescent malaria but there was almost 100% survival of AC-treated animals; these animals were under observation for at least 3 months. We have studied the effect of curcumin in a recrudescence model at the molecular level. Curcumin by itself has antimalarial activity, but only in combination with α,β arteether prevented recrudescence. Our results indicate that curcumin has immunomodulatory activity. Serum cytokine analysis and spleen mRNA analysis for proinflammatory and anti-inflammatory mediators indicate that AC treatment effectively reduced both mRNA and serum cytokine levels of IFNγ, TNFα, IL-12 and effectively increased both mRNA and serum levels IL-10 and antibodies of the IgG subclass. Using TLR2 and IL-10 knockout animals, we have conclusively demonstrated that TLR2 is involved in the production of IL-10, and IL-10 is required for the AC-mediated protection of animals during the recrudescence period. We conclude that curcumin is able to prevent parasite recrudescence essentially by switching the Th1 response to a Th2 response.
The third chapter deals with the study the effect of areether-curcumin (AC) combination therapy in the prevention of Experimental Cerebral Malaria. Although malaria mortality rates have decreased by an impressive 47% between 2000 and 2013, it is still a major affliction of mankind (WHO 2014). Plasmodium falciparum infection causes human cerebral malaria (HCM). The mortality rate in HCM is unacceptably high (15–20%), despite the availability of artemisinin-based therapy. HCM is characterized by a rapid progression from headache, general malaise, and prostration to hemiparesis, ataxia, unrousable coma, and death. Paediatric HCM deaths are mostly due to respiratory arrest. Alternatively, death may be due to parasite-mediated injury to a sensitive location; a small lesion due to parasite in brain stem can cause sudden respiratory arrest. In HCM, cytoadherence of pRBCs in brain microvasculature has been implicated as a major contributing factor for CM pathology. The failure of a large number of adjunct therapies in HCM demands the development of new intervention strategies. An effective adjunct therapy is urgently needed. Experimental Cerebral Malaria (ECM) in mice manifests many of the neurological features of HCM. In this study, we have demonstrated the efficacy of curcumin and PLGA nanocurcumin in the treatment of Experimental Cerebral Malaria (ECM), using the Plasmodium berghei ANKA-infected mouse model (C57BL/6). Curcumin/PLGA nanocurcumin alone can prevent the onset of ECM. We have shown that curcumin/PLGA nanocurcumin can prevent CD8+ T cell, CXCR3+ CD8 T cell and parasite-infected RBC (pRBC) sequestration in the brain. These are also the essential parameters underlying HCM. We have also demonstrated that curcumin effectively inhibits T cell proliferation in spleen. We have explained the anti-inflammatory effects of curcumin by showing the inhibition of NF-B in both brain and spleen, which is a plausible explanation. But, curcumin/PLGA nanocurcumin treated animals died later due to build up of parasitemia in blood and subsequent anemia.
Moreover, a combination therapy with arteether and curcumin given even after the onset of neurological symptoms can completely cure and protect the animals against mortality. We have tested AC-combination after the onset of symptoms to mimic patient conditions in HCM, since the murine regimens reported were not successful in the treatment of HCM. Our results clearly demonstrate that AC treatment even after the onset of symptoms ensures 100% survival. Since the bioavailability of curcumin is reported to be poor, we have also tested the efficacy of PLGA nanocurcumin and find that it is superior to native curcumin in terms of therapeutic effects. It is concluded that curcumin would be an ideal adjunct drug to be used with the artemisinin derivatives to treat malaria, including cerebral malaria.
2018-05-16T00:00:00Z