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In the bloodstream of mammalian hosts, African trypanosomes face the challenge of protecting their invariant surface receptors from immune detection. This crucial role is fulfilled by a dense, glycosylated protein layer composed of variant surface glycoproteins (VSGs), which undergo antigenic variation and provide a physical barrier that shields the underlying invariant surface glycoproteins (ISGs). The protective shield's limited permeability comes at the cost of restricted access to the extracellular host environment, raising questions regarding the specific function of the ISG repertoire. In this study, we employ an integrative structural biology approach to show that intrinsically disordered membrane-proximal regions are a common feature of members of the ISG super-family, conferring the ability to switch between compact and elongated conformers. While the folded, membrane-distal ectodomain is buried within the VSG layer for compact conformers, their elongated counterparts would enable the extension beyond it. This dynamic behavior enables ISGs to maintain a low immunogenic footprint while still allowing them to engage with the host environment when necessary. Our findings add further evidence to a dynamic molecular organization of trypanosome surface antigens wherein intrinsic disorder underpins the characteristics of a highly flexible ISG proteome to circumvent the constraints imposed by the VSG coat. Author summary: In the blood of their human and animal hosts, single-celled parasites of the species Trypanosoma brucei need to hide from the body's defense system to survive. These parasites have a special armor made of proteins (known as VSGs) that changes regularly, helping them stay undetected by the immune system. This armor also covers other important proteins (ISGs) that the parasites need in order to interact with their host, but must hide to avoid being attacked. However, this protective layer makes it hard for the parasites to reach outside their shield. In our study, we looked at these hidden proteins in detail and discovered that they can change shape. Some can stretch out beyond their protective layer when necessary, while others stay concealed inside. This ability to change shape lets these otherwise hidden proteins interact with their surroundings without getting caught by the immune system. Our findings provide further evidence for a multi-layered defense strategy in these parasites. [ABSTRACT FROM AUTHOR]

Pharmacophores such as hydroxyethylamine (HEA) and phthalimide (PHT) have been identified as potential synthons for the development of compounds against various parasitic infections. In order to further advance our progress, we conducted an experiment utilising a collection of PHT and HEA derivatives through phenotypic screening against a diverse set of protist parasites. This approach led to the identification of a number of compounds that exhibited significant effects on the survival of Entamoeba histolytica, Trypanosoma brucei, and multiple life-cycle stages of Leishmania spp. The Leishmania hits were pursued due to the pressing necessity to expand our repertoire of reliable, cost-effective, and efficient medications for the treatment of leishmaniases. Antileishmanials must possess the essential capability to efficiently penetrate the host cells and their compartments in the disease context, to effectively eliminate the intracellular parasite. Hence, we performed a study to assess the effectiveness of eradicating L. infantum intracellular amastigotes in a model of macrophage infection. Among eleven L. infantum growth inhibitors with low-micromolar potency, PHT-39, which carries a trifluoromethyl substitution, demonstrated the highest efficacy in the intramacrophage assay, with an EC50 of 1.2 +/- 3.2 μM. Cytotoxicity testing of PHT-39 in HepG2 cells indicated a promising selectivity of over 90-fold. A chemogenomic profiling approach was conducted using an orthology-based method to elucidate the mode of action of PHT-39. This genome-wide RNA interference library of T. brucei identified sensitivity determinants for PHT-39, which included a P-type ATPase that is crucial for the uptake of miltefosine and amphotericin, strongly indicating a shared route for cellular entry. Notwithstanding the favourable properties and demonstrated efficacy in the Plasmodium berghei infection model, PHT-39 was unable to eradicate L. major infection in a murine infection model of cutaneous leishmaniasis. Currently, PHT-39 is undergoing derivatization to optimize its pharmacological characteristics. Author summary: Two novel compound series, based on hydroxyethylamine (HEA) and phthalimide (PHT) pharmacophores, previously led to the discovery of antiplasmodial lead compounds, both with therapeutic efficacy in a murine Plasmodium infection model. Here we employed these compound libraries, supplemented with newly designed HEA derivatives, for phenotypic screening against a wide range of protist parasites associated with unmet medical needs. We report potent hit compounds for Entamoeba histolytica, Trypanosoma brucei and Leishmania spp., with limited overlap in their structure–activity relation. PHT-39 efficiently penetrated host macrophages to effectively eliminate Leishmania intracellular amastigotes, while displaying high selectivity. Leveraging an orthology-based genome-wide RNAi library screen, we propose an uptake route for the PHT-39 that relies on a transmembrane flippase, known as crucial uptake transporter for the two frontline antileishmanials miltefosine and amphotericin. Despite favourable drug properties of PHT-39, experimental PHT-39 treatment failed to eliminate the Leishmania infection in a cutaneous leishmaniasis animal model. Considering PHT-39 high selectivity and the ability of the drug to effectively reach the intracellular stage of the parasite, we suggest PHT-39 for further optimization. [ABSTRACT FROM AUTHOR]

Leishmaniasis, as a major health problem in tropical and sub-tropical areas in the world, needs novel, safe, nontoxic and plausible therapeutic solutions for its control. As a part of innate immune system, natural antimicrobial peptides have a potential to be used as new generation of antibiotics especially after persistent resistance of conventional antimicrobial agents. Brevinin 2R, a member of Defensin families of host defense peptides, showed promising effects against bacterial and fungal infections as well as cancerous cell lines. In the current research, the anti-leishmanial effect of Brevinin 2R and its lauric acid conjugate was investigated against Leishmania major (L. major) parasite. The data revealed that, conjugation of fatty acid to Brevinin 2R, strengthen its effect on L. major promastigotes in respect to toxicity and hemolytic effect. These peptides showed anitleishmanial activity through cell membrane disruption and changes in the electrical and mitochondrial membrane potential. No signs of apoptosis induction or caspase activation were detected. Despite its hemolytic and cytotoxic effect in in vitro conditions, lauric acid- Brevinin 2R (L- Brevinin 2R) did not show site specific adverse reactions in animal model. Treatment course with L- Brevinin 2R in the L. major infected mice exhibited decreased parasite load in the lymph nodes adjacent to the infected site despite cytokine production profile and footpad swelling data. [ABSTRACT FROM AUTHOR]

Human Neutrophil Peptide-1 (HNP1) produced by neutrophils, is a well-known antimicrobial peptide which plays a role both in innate as well as in adaptive immunity and is under intensive investigation as a potential therapeutic agent. Previous in vitro experiments have indicated the leishmaniacidal effect of recombinant HNP1 on Leishmania major (L. major) promastigotes and amastigotes. In the current study, we further extended the idea to explore the remedial effect of HNP1 in the two modalities of peptide therapy (folded HNP1) and gene therapy in L. major infected BALB/c mice. To this end, mice in five different groups received synthetic folded HNP1 (G1), pcDNA-HNP1-EGFP (G2), pcDNA-EGFP (G3), Amphotericin B (G4) and PBS (G5), which was started three weeks after infection for three consecutive weeks. Footpad swelling was monitored weekly and a day after the therapy ended, IFN-γ, IL-4, IL-10, IL-6 and nitric oxide produced by splenocytes were analyzed together with the parasite load in draining lymph nodes. Arginase activity and dermal histopathological changes were also analyzed in the infected footpads. We demonstrated that both therapeutic approaches effectively induced Th1 polarization and restricted parasite burden. It can control disease progression in contrast to non-treated groups. However, pcDNA-HNP1-EGFP is more promising in respect to parasite control than folded HNP1, but less effective than AmB treatment. We concluded with the call for a future approach, that is, a DNA-based expression of HNP1 combined with AmB as it can improve the leishmaniacidal efficacy. [ABSTRACT FROM AUTHOR]

Canine Visceral Leishmaniasis (CVL) is a major veterinary and public health problem caused by Leishmania infantum (L. infantum) in many endemic countries. It is a severe chronic disease with generalized parasite spread to the reticuloendothelial system, such as spleen, liver and bone marrow and is often fatal when left untreated. Control of VL in dogs would dramatically decrease infection pressure of L. infantum for humans, since dogs are the main domestic reservoir. In the past decade, various subunits and DNA antigens have been identified as potential vaccine candidates in experimental animal models, but none has been approved for human use so far. In this study, we vaccinated outbreed dogs with a prime-boost regimen based on recombinant L. tarentolae expressing the L. donovani A2 antigen along with cysteine proteinase genes (CPA and CPB without its unusual C-terminal extension (CPB-CTE) and evaluated its immunogenicity and protective immunity against L. infantum infectious challenge. We showed that vaccinated animals produced significantly higher levels of IgG2, but not IgG1, and also IFN-γ and TNF-α, but low IL-10 levels, before and after challenge as compared to control animals. Protection in dogs was also correlated with a strong DTH response and low parasite burden in the vaccinated group. Altogether, immunization with recombinant L. tarentolae A2-CPA-CPB-CTE was proven to be immunogenic and induced partial protection in dogs, hence representing a promising live vaccine candidate against CVL. [ABSTRACT FROM AUTHOR]

Background: Novel vaccination approaches are needed to prevent leishmaniasis. Live attenuated vaccines are the gold standard for protection against intracellular pathogens such as Leishmania and there have been new developments in this field. The nonpathogenic to humans lizard protozoan parasite, Leishmania (L) tarentolae, has been used effectively as a vaccine platform against visceral leishmaniasis in experimental animal models. Correspondingly, pre-exposure to sand fly saliva or immunization with a salivary protein has been shown to protect mice against cutaneous leishmaniasis. Methodology/Principal Findings: Here, we tested the efficacy of a novel combination of established protective parasite antigens expressed by L. tarentolae together with a sand fly salivary antigen as a vaccine strategy against L. major infection. The immunogenicity and protective efficacy of different DNA/Live and Live/Live prime-boost vaccination modalities with live recombinant L. tarentolae stably expressing cysteine proteinases (type I and II, CPA/CPB) and PpSP15, an immunogenic salivary protein from Phlebotomus papatasi, a natural vector of L. major, were tested both in susceptible BALB/c and resistant C57BL/6 mice. Both humoral and cellular immune responses were assessed before challenge and at 3 and 10 weeks after Leishmania infection. In both strains of mice, the strongest protective effect was observed when priming with PpSP15 DNA and boosting with PpSP15 DNA and live recombinant L. tarentolae stably expressing cysteine proteinase genes. Conclusion/Significance: The present study is the first to use a combination of recombinant L. tarentolae with a sand fly salivary antigen (PpSP15) and represents a novel promising vaccination approach against leishmaniasis. Author Summary: More than 98 countries are reported as endemic for leishmaniasis, a vector-borne disease transmitted by sand flies. Drug-resistant forms have emerged and there is an increased need to develop advanced preventive strategies. Live attenuated vaccines are the gold standard for protection against intracellular pathogens such as Leishmania and there have been new developments in this field. The lizard protozoan parasite, L. tarentolae, is nonpathogenic to humans and has been used effectively as a vaccine platform against visceral leishmaniasis in experimental animal models. Correspondingly, pre-exposure to sand fly saliva or immunization with salivary proteins has been shown to protect mice against cutaneous leishmaniasis. Herein, we used DNA/Live and Live/Live prime-boost vaccination strategies against cutaneous leishmaniasis based on recombinant L. tarentolae stably expressing CPA/CPB genes with and without the sand fly salivary antigen PpSP15 in both resistant and susceptible mice models. Assessment of the immune response and parasite burden in vaccinated mice at different time intervals post-challenge demonstrated that combination of recombinant L. tarentolae CPA/CPB with PpSP15 DNA elicits an enhanced protective immune response against cutaneous leishmaniasis in mice. This parasite- and insect vector-derived antigen combination represents an important step forward in the development of new vaccine strategies against Leishmania infections. [ABSTRACT FROM AUTHOR]

Background: Novel vaccination approaches are needed to prevent leishmaniasis. Live attenuated vaccines are the gold standard for protection against intracellular pathogens such as Leishmania and there have been new developments in this field. The nonpathogenic to humans lizard protozoan parasite, Leishmania (L) tarentolae, has been used effectively as a vaccine platform against visceral leishmaniasis in experimental animal models. Correspondingly, pre-exposure to sand fly saliva or immunization with a salivary protein has been shown to protect mice against cutaneous leishmaniasis. Methodology/Principal Findings: Here, we tested the efficacy of a novel combination of established protective parasite antigens expressed by L. tarentolae together with a sand fly salivary antigen as a vaccine strategy against L. major infection. The immunogenicity and protective efficacy of different DNA/Live and Live/Live prime-boost vaccination modalities with live recombinant L. tarentolae stably expressing cysteine proteinases (type I and II, CPA/CPB) and PpSP15, an immunogenic salivary protein from Phlebotomus papatasi, a natural vector of L. major, were tested both in susceptible BALB/c and resistant C57BL/6 mice. Both humoral and cellular immune responses were assessed before challenge and at 3 and 10 weeks after Leishmania infection. In both strains of mice, the strongest protective effect was observed when priming with PpSP15 DNA and boosting with PpSP15 DNA and live recombinant L. tarentolae stably expressing cysteine proteinase genes. Conclusion/Significance: The present study is the first to use a combination of recombinant L. tarentolae with a sand fly salivary antigen (PpSP15) and represents a novel promising vaccination approach against leishmaniasis. [ABSTRACT FROM AUTHOR]

The toxicity of available drugs for treatment of leishmaniasis, coupled with emerging drug resistance, make it urgent to find new therapies. Antimicrobial peptides (AMPs) have a strong broad-spectrum antimicrobial activity with distinctive modes of action and are considered as promising therapeutic agents. The defensins, members of the large family of AMPs, are immunomodulatory molecules and important components of innate immune system. Human neutrophil peptide-1 (HNP-1), which is produced by neutrophils, is one of the most potent defensins. In this study, we described anti-parasitic activity of recombinant HNP-1 (rHNP-1) against Leishmania major promastigotes and amastigotes. Furthermore, we evaluated the immunomodulatory effect of rHNP-1 on parasite-infected neutrophils and how neutrophil apoptosis was affected. Our result showed that neutrophils isolated from healthy individuals were significantly delayed in the onset of apoptosis following rHNP-1 treatment. Moreover, there was a noteworthy increase in dying cells in rHNP-1- and/or CpG–treated neutrophils in comparison with untreated cells. There is a considerable increase in TNF-α production from rHNP-1-treated neutrophils and decreased level of TGF-β concentration, a response that should potentiate the immune system against parasite invasion. In addition, by using real-time polymerase chain reaction (real-time PCR), we showed that in vitro infectivity of Leishmania into neutrophils is significantly reduced following rHNP-1 treatment compared to untreated cells. Author Summary: In Iran, cutaneous leishmaniasis (CL) is a widespread and highly endemic disease in young individuals. To date, treatment strategy is based on chemotherapy accompanied with high incidence of toxicity and drug resistance. Distinctive mode of action of defensins (members of antimicrobial peptides) with low susceptibility to resistance and low toxicity to mammalian cells makes them suitable candidates for anti-leishmanial agents. The most active human defensin is human neutrophil peptide-1 (HNP-1) produced by neutrophils; the first effector cells during Leishmania infection. In this work, we used recombinant HNP-1 (rHNP-1) against both the promastigote and amastigote forms of Leishmania (L.) major. Furthermore, immunomodulatory effect of rHNP-1 on Leishmania-infected neutrophils was investigated. Our result showed that rHNP-1 has anti-parasitic effect against L. major promastigotes and amastigotes and also reduces infectivity rate of Leishmania-infected neutrophils. Moreover, assessment of cytokine production from Leishmania-infected neutrophils reveals an increase in TNF-α and a decrease in TGF-β production after rHNP-1 treatment; a cytokine pattern anticipated to facilitate control of parasites. The immunomodulatory effect of rHNP-1 on cytokine production from parasite-infected neutrophils besides its direct effect on free parasites is considered as promising step towards developing new anti-leishmanial agents. [ABSTRACT FROM AUTHOR]

Visceral leishmaniasis (VL) is a vector-borne disease affecting humans and domestic animals that constitutes a serious public health problem in many countries. Although many antigens have been examined so far as protein- or DNA-based vaccines, none of them conferred complete long-term protection. The use of the lizard non-pathogenic to humans Leishmania (L.) tarentolae species as a live vaccine vector to deliver specific Leishmania antigens is a recent approach that needs to be explored further. In this study, we evaluated the effectiveness of live vaccination in protecting BALB/c mice against L. infantum infection using prime-boost regimens, namely Live/Live and DNA/Live. As a live vaccine, we used recombinant L. tarentolae expressing the L. donovani A2 antigen along with cysteine proteinases (CPA and CPB without its unusual C-terminal extension (CPB-CTE)) as a tri-fusion gene. For DNA priming, the tri-fusion gene was encoded in pcDNA formulated with cationic solid lipid nanoparticles (cSLN) acting as an adjuvant. At different time points post-challenge, parasite burden and histopathological changes as well as humoral and cellular immune responses were assessed. Our results showed that immunization with both prime-boost A2-CPA-CPB-CTE-recombinant L. tarentolae protects BALB/c mice against L. infantum challenge. This protective immunity is associated with a Th1-type immune response due to high levels of IFN-γ production prior and after challenge and with lower levels of IL-10 production after challenge, leading to a significantly higher IFN-γ/IL-10 ratio compared to the control groups. Moreover, this immunization elicited high IgG1 and IgG2a humoral immune responses. Protection in mice was also correlated with a high nitric oxide production and low parasite burden. Altogether, these results indicate the promise of the A2-CPA-CPB-CTE-recombinant L. tarentolae as a safe live vaccine candidate against VL. Author Summary: Visceral leishmaniasis (VL) is the most severe form of leishmaniasis and has emerged as an opportunistic infection in HIV-1 infected patients in many parts of the world. Drug-resistant forms have developed so emergence and increased the need for advanced preventive strategies. Using live avirulent organisms as a vaccine has been proven to be more effective than other regimens. The lizard protozoan parasite Leishmania tarentolae is considered as nonpathogenic to humans. In our previous work, a recombinant L. tarentolae strain expressing the amastigote-specific L. donovani A2 antigen as a vaccine candidate elicited protection against L. infantum challenge in mice. Furthermore, combinations of CPA/CPB cysteine proteinases were more protective against visceral and cutaneous Leishmania infections than the individual forms. Herein, we used DNA/Live and Live/Live prime-boost vaccination strategies against visceral leishmaniasis in BALB/c mice consisting of the A2-CPA-CPB-CTE tri-fusion genes formulated with cationic solid lipid nanoparticles and a recombinant L. tarentolae expressing the tri-fusion. Assessments of cytokine production, humoral responses, parasite burden and histopathological studies support that the recombinant L. tarentolae A2-CPA-CPB-CTE candidate vaccine elicits a protective response against visceral leishmaniasis in mice and represents an important step forward in the development of new vaccine combinations against Leishmania infections. [ABSTRACT FROM AUTHOR]

Background: As a potent CD8+ T cell activator, peptide vaccine has found its way in vaccine development against intracellular infections and cancer, but not against leishmaniasis. The first step toward a peptide vaccine is epitope mapping of different proteins according to the most frequent HLA types in a population. Methods and Findings: Six Leishmania (L.) major-related candidate antigens (CPB,CPC,LmsTI-1,TSA,LeIF and LPG-3) were screened for potential CD8+ T cell activating 9-mer epitopes presented by HLA-A*0201 (the most frequent HLA-A allele). Online software including SYFPEITHI, BIMAS, EpiJen, Rankpep, nHLApred, NetCTL and Multipred were used. Peptides were selected only if predicted by almost all programs, according to their predictive scores. Pan-A2 presentation of selected peptides was confirmed by NetMHCPan1.1. Selected peptides were pooled in four peptide groups and the immunogenicity was evaluated by in vitro stimulation and intracellular cytokine assay of PBMCs from HLA-A2+ individuals recovered from L. major. HLA-A2− individuals recovered from L. major and HLA-A2+ healthy donors were included as control groups. Individual response of HLA-A2+ recovered volunteers as percent of CD8+/IFN-γ+ T cells after in vitro stimulation against peptide pools II and IV was notably higher than that of HLA-A2− recovered individuals. Based on cutoff scores calculated from the response of HLA-A2− recovered individuals, 31.6% and 13.3% of HLA-A2+ recovered persons responded above cutoff in pools II and IV, respectively. ELISpot and ELISA results confirmed flow cytometry analysis. The response of HLA-A2− recovered individuals against peptide pools I and III was detected similar and even higher than HLA-A2+ recovered individuals. Conclusion: Using in silico prediction we demonstrated specific response to LmsTI-1 (pool II) and LPG-3- (pool IV) related peptides specifically presented in HLA-A*0201 context. This is among the very few reports mapping L. major epitopes for human HLA types. Studies like this will speed up polytope vaccine idea towards leishmaniasis. Author Summary: Leishmaniasis is currently a serious health as well as economic problem in underdeveloped and developing countries in Africa, Asia, the Near and Middle East, Central and South America and the Mediterranean region. Cutaneous leishmaniasis is highly endemic in Iran, remarkably in Isfahan, Fars, Khorasan, Khozestan and Kerman provinces. Since effective prevention is not available and current curative therapy is expensive, often poorly tolerated and not always effective, alternative therapies including vaccination against leishmaniasis are of priority to overcome the problem. Although Th1 dominant response is so far considered as a pre-requisite for the immune system to overcome the infection, CD8+ T cell response could also be considered as a potent arm of immune system fighting against intracellular Leishmania. Polytope vaccine strategy may open up a new way in vaccine design against leishmaniasis, since they act as a potent tool to stimulate multi-CD8 T cell responses. Clearly there is a substantial need to evaluate the promising epitopes from different proteins of Leishmania parasite species. Some new immunoinformatic tools are now available to speed up this process, and we have shown here that in silico prediction can effectively evaluate HLA class I-restricted epitopes out of Leishmania proteins. [ABSTRACT FROM AUTHOR]

Background: We have demonstrated that vaccination with pDNA encoding cysteine proteinase Type II (CPA) and Type I (CPB) with its unusual C-terminal extension (CTE) can partially protect BALB/c mice against cutaneous leishmanial infection. Unfortunately, this protection is insufficient to completely control infection without booster injection. Furthermore, in developing vaccines for leishmaniasis, it is necessary to consider a proper adjuvant and/or delivery system to promote an antigen specific immune response. Solid lipid nanoparticles have found their way in drug delivery system development against intracellular infections and cancer, but not Leishmania DNA vaccination. Therefore, undefined effect of cationic solid lipid nanoparticles (cSLN) as an adjuvant in enhancing the immune response toward leishmanial antigens led us to refocus our vaccine development projects. Methodology/Principal Findings: Three pDNAs encoding L. major cysteine proteinase type I and II (with or without CTE) were formulated by cSLN. BALB/c mice were immunized twice by 3-week interval, with cSLN-pcDNA-cpa/b, pcDNA-cpa/b, cSLN-pcDNA-cpa/b-CTE, pcDNA-cpa/b-CTE, cSLN, cSLN-pcDNA and PBS. Mice vaccinated with cSLN-pcDNA-cpa/b-CTE showed significantly higher levels of parasite inhibition related to protection with specific Th1 immune response development, compared to other groups. Parasite inhibition was determined by different techniques currently available in exploration vacciation efficacy, i.e., flowcytometry on footpad and lymph node, footpad caliper based measurements and imaging as well as lymph node microtitration assay. Among these techniques, lymph node flowcytometry was found to be the most rapid, sensitive and easily reproducible method for discrimination between the efficacy of vaccination strategies. Conclusions/Significance: This report demonstrates cSLN's ability to boost immune response magnitude of cpa/cpb-CTE cocktail vaccination against leishmaniasis so that the average parasite inhibition percent could be increased significantly. Hence, cSLNs can be considered as suitable adjuvant and/or delivery systems for designing third generation cocktail vaccines. Author Summary: Cutaneous leishmaniasis (CL) is the most common form of leishmaniasis with an annual incidence of approximately 2 million cases and is endemic in 88 countries, including Iran. CL's continued spread, along with rather ineffectual treatments and drug-resistant variants emergence has increased the need for advanced preventive strategies. We studied Type II cysteine proteinase (CPA) and Type I (CPB) with its C-terminal extension (CTE) as cocktail DNA vaccine against murine and canine leishmaniasis. However, adjuvants' success in enhancing immune responses to selected antigens led us to refocus our vaccine development programs. Herein, we discuss cationic solid lipid nanoparticles' (cSLN) ability to improve vaccine-induced protective efficacy against CL and subsequent lesion size and parasite load reduction in BALB/c mice. For this work, we evaluated five different conventional as well as novel parasite detection techniques, i.e., footpad imaging, footpad flowcytometry and lymph node flowcytometry for disease progression assessments. Vaccination with cSLN-cpa/cpb-CTE formulation showed highest parasite inhibition at 3-month post vaccination. Immunized mice showed reduced IL-5 level and significant IFN-ã increase, compared to control groups. We think our study represents a potential future and a major step forward in vaccine development against leishmaniasis. [ABSTRACT FROM AUTHOR]