Leishmaniasis Disease Research Articles

Meroterpenoids from Gongolaria abies-marina against Kinetoplastids: In Vitro Activity and Programmed Cell Death Study.

Leishmaniasis and Chagas disease affect millions of people worldwide. The available treatments against these parasitic diseases are limited and display multiple undesired effects. The brown alga belonging to the genus Gongolaria has been previously reported as a source of compounds with different biological activities. In a recent study from our group, Gongolaria abies-marine was proven to present antiamebic activity. Hence, this brown alga could be a promising source of interesting molecules for the development of new antiprotozoal drugs. In this study, four meroterpenoids were isolated and purified from a dichloromethane/ethyl acetate crude extract through a bioguided fractionation process targeting kinetoplastids. Moreover, the in vitro activity and toxicity were evaluated, and the induction of programmed cell death was checked in the most active and less toxic compounds, namely gongolarone B (2), 6Z-1'-methoxyamentadione (3) and 1'-methoxyamentadione (4). These meroterpenoids triggered mitochondrial malfunction, oxidative stress, chromatin condensation and alterations of the tubulin network. Furthermore, a transmission electron microscopy (TEM) image analysis showed that meroterpenoids (2-4) induced the formation of autophagy vacuoles and ER and Golgi complex disorganization. The obtained results demonstrated that the mechanisms of action at the cellular level of these compounds were able to induce autophagy as well as an apoptosis-like process in the treated parasites.

Pathogens, reservoirs, and vectors involved in the transmission of vector-borne and zoonotic diseases in a Colombian region

The ecology of vector-borne diseases (VBDs) is an important system of great complexity, which involves the knowledge about the pathogens and animal species entailed in maintaining transmission cycles in a given locality, including those that act as vectors and reservoirs for the transmitted pathogens. To understand the ecology of some VBDs, we studied vectors, reservoirs, and pathogens of different VBDs, including dengue, leishmaniasis, Chagas disease, malaria, Zika, and chikungunya in the municipality of La Mesa, Cundinamarca, Colombia, a locality close to the capital, Bogotá. Vectors and mammals were sampled in urban and rural areas between May and August 2019. Molecular analyses were performed for the detection of pathogens in mammals and vectors, and of blood-meal sources in insects. Several vectors and mammals collected in this study have been involved in pathogen transmission cycles or may have a potential role in them. The findings of this study suggest that in the municipality of La Mesa, there are both vector and potential reservoir species, which are or could be implicated in the maintenance of the cycles of vector-borne diseases such as leishmaniasis and Chagas disease. Although arbovirus infections, such as dengue, are reported in the municipality, arbovirus presence was not detected. These findings highlight the importance of ongoing surveillance of vectors and associated control operations in La Mesa, of relevance to other locations where vectors and animal hosts also occur.

Flavonoid Derivatives as New Potent Inhibitors of Cysteine Proteases: An Important Step toward the Design of New Compounds for the Treatment of Leishmaniasis.

Leishmaniasis is a neglected tropical disease, affecting more than 350 million people globally. However, there is currently no vaccine available against human leishmaniasis, and current treatment is hampered by high cost, side-effects, and painful administration routes. It has become a United Nations goal to end leishmaniasis epidemics by 2030, and multitarget drug strategy emerges as a promising alternative. Among the multitarget compounds, flavonoids are a renowned class of natural products, and a structurally diverse library can be prepared through organic synthesis, which can be tested for biological effectiveness. In this study, we synthesised 17 flavonoid analogues using a scalable, easy-to-reproduce, and inexpensive method. All synthesised compounds presented an impressive inhibition capacity against rCPB2.8, rCPB3, and rH84Y enzymes, which are highly expressed in the amastigote stage, the target form of the parasite. Compounds 3c, f12a, and f12b were found to be effective against all isoforms. Furthermore, their intermolecular interactions were also investigated through a molecular modelling study. These compounds were highly potent against the parasite and demonstrated low cytotoxic action against mammalian cells. These results are pioneering, representing an advance in the investigation of the mechanisms behind the antileishmanial action of flavonoid derivatives. Moreover, compounds have been shown to be promising leads for the design of other cysteine protease inhibitors for the treatment of leishmaniasis diseases.

The comparison of potential key genes on rat uterus and mammary gland regulated by estradiol

Context Although numerous studies have investigated the regulation of estrogen (E2) on the female reproductive system, there is still a lack of understanding regarding the specific genes and pathways involved in E2 regulation of the uterus and breast. Aims The aim of this study was to explore the shared genes and pathways involved in estrogen regulation of the uterus and mammary gland, which could provide a theoretical basis for disease treatment. Methods Bioinformatics analysis was employed to identify potential genes and pathways associated with E2 regulation of the uterus and breast. Key results The analysis revealed 233 differentially expressed genes (DEGs) in datasets GSE89321 and GSE95783 (adjusted-P < 0.05). Additionally, five KEGG (Kyoto Encyclopedia of Genes and Genomes) pathways were identified: Glutathione Metabolism Pathway, Chagas Disease Pathway, Leishmaniasis Pathway, Complement and Coagulation Cascades Pathway, and p53 Signalling Pathway. A protein interaction network was constructed, and 10 hub genes (Alb, Il1a, Serpine1, Timp1, Ccl11, Fos, Krt19, Krt7, Lcn2, and C3) were selected, with Alb being the highest scoring hub gene. The study also predicted the association of Mo-mir-200a-5p and Mo-Mir-200a-3p with Alb and Krt19. Furthermore, 28 DEGs were identified in E2 regulation of the mammary gland, encompassing pathways such as Acute Myeloid Leukemia, ErbB Signalling Pathway, Th1 and Th2 Cell Differentiation, Th17 Cell Differentiation, and Transcriptional Misregulation in Cancer. Hub genes in the mammary gland included Pgr, Gata3, Areg, Prom1, Stat5a, Cldn4, Greb1, Tfap2c, Pdk4, and Mb. Prom1, Prom2, Fam84a, and Padi2 were found to be common DEGs in E2 regulation of both the uterus and mammary gland. Conclusions The findings of this study, together with functional annotation and pathway analysis, suggest that Alb serves as a marker protein in E2 regulation of uterus development. Additionally, Prom1, Prom2, Fam84a, and Padi2 are identified as common DEGs involved in E2 regulation of both the uterus and mammary gland. Implications This study provides comprehensive omics data to elucidate the mechanisms underlying estrogen regulation of the uterus and mammary gland, opening up new research directions for disease treatment.

Detection of Leishmania DNA in Phlebotomine Sand Flies in Tsatee, a Community in the Volta Region, Ghana.

Leishmania parasites, which are spread by infected female sand flies, are the cause of the disease leishmaniasis. Although cutaneous leishmaniasis has been found to occur in the Volta Region, there is limited data on vector species and reservoirs. This study focused on the Tsatee community, in the South Dayi District of the Volta Region, and is aimed at identifying the sand fly fauna and detecting the presence of Leishmania DNA by the use of primers that target the conserved region of Leishmania spp. minicircle DNA of the parasite kinetoplast. The miniature light traps and hand aspirators provided by the Centers for Disease Control and Prevention (CDC) were used to collect outdoor and indoor sand flies for five months in a guinea woodland and semideciduous forest area. From the collections, 4,580 phlebotomine sand flies were obtained and identified, and females were examined for Leishmania DNA using PCR. The male flies were 1,202 (26.24%), non-blood-fed females were 3,321 (72.51%), and 57 (1.25%) were blood-fed females. It was observed that Sergentomyia species constituted 99.91% of the total collected sand flies with S. africana (76.77%) as the predominant species. Phlebotomus rodhaini (0.09%) was the only Phlebotomus species identified from the study area. From 283 non-blood-fed sand fly pools and 57 individual blood-fed species screened, Leishmania DNA was detected in 12 (4.24%) pools and 8 (14.04%) individuals, respectively. It was observed that Leishmania DNA was detected in all the sand fly species identified except S. collarti. This study reports the first detection of Leishmania DNA in P. rodhaini in Ghana, with an infection rate of 33.33% (95% CI, 1.23-88.32). The findings suggest that the role of Phlebotomus in disease transmission in the study area cannot be discounted. Future studies should include continuous surveillance, blood meal preferences, and vector competence of the various infected phlebotomine sand flies to create effective control measures.

Models of the putative antimony(V)-diolate motifs in antileishmanial pentavalent antimonial drugs.

The structures of the pentavalent antimonials, small-molecule Sb-containing drugs used to treat the neglected tropical disease leishmaniasis, remain unknown despite their widespread use for over half a century. These drugs are prepared by combination of an Sb(V) precursor and a sugar derivative and proposed structures frequently invoke a cyclic stiborane motif in which a vicinal diolate ligand chelates an Sb(V) center. As a step towards better understanding the structures of the pentavalent antimonial drugs, a series of cyclic organostiboranes spanning the stereochemical space afforded by a vicinal diolate motif has been synthesized and characterized. X-ray crystallography and NMR spectroscopy provide unambiguous characterization of the structures of these model compounds and of the interaction of the diolate with the Sb(V) center. Particularly notable are the systematic trends observed in the NMR spectroscopic signals as a function of the stereochemistry of the diolate. The spectroscopic signatures identified with these model compounds will provide a framework for elucidating the structures of the pentavalent antimonial drugs.

GIS-Based Situational Analysis of Cutaneous Leishmaniasis Disease (CLD) in Sri Lanka

Cutaneous Leishmaniasis (CL) is a severe health problem and a parasitic disease on human dermal and widely pervades tropical and subtropical developing counties. The study is mainly focused on Geographic Information System (GIS) based Situational Analysis (SA). The clinically recorded 394 CL patients’ information was obtained from the District General Hospital of Polonnaruwa (DGHP) for 2017 and 2018. The spatial distribution of these patients was collected using Global Positing System (GPS). Moran’s I Index spatial autocorrelation technique and Getis-Ord Gi were used to identify the study site’s hot spot and cold spot areas. More than 75% of the CL patients’ population were highly involved with agricultural activities, and they are the highly exposed group of the CL in the study area. Also, 75% of the CL population were men, and the highly vulnerable age group was 35 - 39 men and 40 - 44 women. The generated Moran’s I Index indicates 0.0321, representing a randomly distributed pattern of CL patients over the District, and the Getis-Ord Gi Z Score value was >1.96 (p Hence to prevent the disease spread, it is essential to implement an awareness program regarding sandflies’ behaviour and CLD.

Lethal action of Licarin A derivatives in Leishmania (L.) infantum: Imbalance of calcium and bioenergetic metabolism

Natural metabolites present an extraordinary chemo-diversity and have been used as the inspiration for new drugs. Considering the need for new treatments against the neglected parasitic disease leishmaniasis, three semi-synthetic derivatives of natural neolignane licarin A were prepared: O-acetyl (1a), O-allyl (1b), and 5-allyl (1c). Using an ex vivo assay, compounds 1a, 1b, and 1c showed activity against the intracellular amastigotes of Leishmania (L.) infantum, with IC50 values of 9, 13, and 10 μM, respectively. Despite no induction of hemolytic activity, only compound 1b resulted in mammalian cytotoxicity (CC50 = 64 μM). The most potent compounds (1a and 1c) resulted in selectivity indexes >18. The mechanism of action of compound 1c was evaluated by fluorescent/luminescent based techniques and MALDI-TOF/MS. After a short incubation period, increased levels of the cytosolic calcium were observed in the parasites, with alkalinization of the acidocalcisomes. Compound 1c also induced mitochondrial hyperpolarization, resulting in decreased levels of ATP without altering the reactive oxygen species (ROS). Neither plasma membrane damages nor DNA fragmentation were observed after the treatment, but a reduction in the cellular proliferation was detected. Using MALDI-TOF/MS, mass spectral alterations of promastigote proteins were observed when compared to untreated and miltefosine-treated groups. This chemically modified neolignan induced lethal alterations of the bioenergetic and protein metabolism of Leishmania. Future PKPD and animal efficacy studies are needed to optimize this promising natural-derived compound.

Natural Products and Synthetic Derivatives as Promising Candidates Against Neglected Tropical Diseases

Neglected Tropical Diseases (NTD) are a group of endemic diseases that cause up to one million deaths annually. Leishmaniasis and Chagas Disease are classified as NTD whose current treatments are of limited access. There is an urgent need to develop new agents to combat or alleviate the symptoms of these pathologies. The present work highlights the activities of several natural products and their derivatives which have been tested against the causative parasites of both diseases. The activities of natural products illustrate their potential to assist in the combat against NTD. Research in this area could provide an opportunity to advance medical research and to provide treatments to low- and middle-income countries.

Unraveling the role of Breg cells in digestive tract cancer and infectious immunity.

Over the past two decades, regulatory B cells (Breg cells or Bregs) have emerged as an immunosuppressive subset of B lymphocytes playing a key role in inflammation, infection, allergy, transplantation, and cancer. However, the involvement of Bregs in various pathological conditions of the gastrointestinal tract is not fully understood and is the subject of much recent research. In this review, we aimed to summarize the current state of knowledge about the origin, phenotype, and suppressive mechanisms of Bregs. The relationship between the host gut microbiota and the function of Bregs in the context of the disturbance of mucosal immune homeostasis is also discussed. Moreover, we focused our attention on the role of Bregs in certain diseases and pathological conditions related to the digestive tract, especially Helicobacter pylori infection, parasitic diseases (leishmaniasis and schistosomiasis), and gastrointestinal neoplasms. Increasing evidence points to a relationship between the presence and number of Bregs and the severity and progression of these pathologies. As the number of cases is increasing year by year, also among young people, it is extremely important to understand the role of these cells in the digestive tract.

Limitations of current chemotherapy and future of nanoformulation-based AmB delivery for visceral leishmaniasis-An updated review.

Visceral leishmaniasis (VL) is the most lethal of all leishmaniasis diseasesand the second most common parasiticdisease after malaria and,still, categorized as a neglected tropical disease (NTD). According to the latest WHO study, >20 Leishmania species spread 0.7-1.0 million new cases of leishmaniasis each year. VL is caused by the genus, Leishmania donovani (LD), which affects between 50,000 and 90,000 people worldwide each year. Lack of new drug development, increasing drug resistance, toxicity and high cost even with the first line of treatmentof Amphotericin B (AmB), demands new formulation for treatment of VLFurther the lack of a vaccine, allowedthe researchers to develop nanofomulation-based AmB for improved delivery. The limitation of AmB is its kidney and liver toxicity which forced the development of costly liposomal AmB (AmBisome) nanoformulation. Success of AmBisome have inspired and attracted a wide range of AmB nanoformulations ranging from polymeric, solid lipid, liposomal/micellar, metallic, macrophage receptor-targetednanoparticles (NP) and even with sophisticated carbon/quantum dot-based AmBnano delivery systems. Notably, NP-based AmB delivery has shown increased efficacy due to increased uptake, on-target delivery and synergistic impact of NP and AmB. In this review, we have discussed the different forms of leishmaniasis disease and their current treatment options with limitations. The discovery, mechanism of action of AmB, clinical status of AmB and improvement with AmBisome over fungizone (AmB-deoxycholate)for VL treatment was further discussed. At last, the development of various AmB nanoformulation was discussed along with its adavantages over traditional chemotherapy-based delivery.

Kidney Pathology of Tropical and Nontropical Infectious Diseases in the Pediatric Population.

To review kidney pathology of tropical and nontropical infectious diseases in the pediatric population. We review 4 tropical and 2 nontropical infectious diseases that affect the kidneys of children in terms of their direct and indirect pathogenetic mechanism in inducing kidney damage. We demonstrate clinical manifestations, pathogenesis, kidney pathology, and laboratory diagnostic methods for (1) renal cryptococcosis, which represents involvement of a pure direct pathway; (2) schistosomiasis and dengue fever as examples of dual direct and indirect pathways; and (3) congenital syphilis, visceral leishmaniasis, and Chagas disease, which represent indirect pathways. Infective agents affect the kidneys of children mainly through indirect mechanisms, such as through immunological mechanisms as part of an antigenic response. A direct mechanism of kidney injury, however, is less known within the medical community simply because the direct mechanism is rarely encountered in nontropical countries. In some infectious diseases, both indirect and direct pathways are responsible in inducing 2 sets of morphologically separate kidney lesions.

A Novel Protocol for the Synthesis of 1,2,4-Oxadiazoles Active against Trypanosomatids and Drug-Resistant Leukemia Cell Lines.

Cancer and parasitic diseases, such as leishmaniasis and Chagas disease, share similarities that allow the co-development of new antiproliferative agents as a strategy to quickly track the discovery of new drugs. This strategy is especially interesting regarding tropical neglected diseases, for which chemotherapeutic alternatives are extremely outdated. We designed a series of (E)-3-aryl-5-(2-aryl-vinyl)-1,2,4-oxadiazoles based on the reported antiparasitic and anticancer activities of structurally related compounds. The synthesis of such compounds led to the development of a new, fast, and efficient strategy for the construction of a 1,2,4-oxadiazole ring on a silica-supported system under microwave irradiation. One hit compound (23) was identified during the in vitro evaluation against drug-sensitive and drug-resistant chronic myeloid leukemia cell lines (EC50 values ranging from 5.5 to 13.2 µM), Trypanosoma cruzi amastigotes (EC50 = 2.9 µM) and Leishmania amazonensis promastigotes (EC50 = 12.2 µM) and amastigotes (EC50 = 13.5 µM). In silico studies indicate a correlation between the in vitro activity and the interaction with tubulin at the colchicine binding site. Furthermore, ADMET in silico predictions indicate that the compounds possess a high druggability potential due to their physicochemical, pharmacokinetic, and toxicity profiles, and for hit 23, it was identified by multiple spectroscopic approaches that this compound binds with human serum albumin (HSA) via a spontaneous ground-state association with a moderate affinity driven by entropically and enthalpically energies into subdomain IIA (site I) without significantly perturbing the secondary content of the protein.

Withaferin A-silyl ether analogs as potential anti-kinetoplastid agents targeting the programmed cell death

Current therapies of leishmaniasis and Chagas disease, two of the most widespread neglected tropical diseases, have limited efficacy and toxic side effects. In this regard, natural products play an important role in overcoming the current need for new antiparasitic agents. The present study reports the leishmanicidal and trypanocidal activities of twenty-four known silyl-ether derivatives of withaferin A. Eleven compounds from this series (4, 7, 8, 10, 12, 15, 17, 18, 20, 22 and 25) showed a potent dose-dependent inhibitory effect on the proliferation of Leishmania amazonensis promastigotes and Trypanosoma cruzi epimastigotes respectively, even higher than the references drugs, miltefosine and benznidazole. Among them, the most promising compound, derivative 10, exhibited approximately 34-fold higher leishmanicidal activity and 49-fold higher trypanocidal activity compared to the reference drugs, as well as lower cytotoxicity. Moreover, compounds 4, 7, 10, 12 and 15 were more active than the reference drugs against the amastigote forms of L. amazonensis, presenting a high selectivity index. Assays performed to study the ATP levels, mitochondrial membrane potential, plasma membrane permeability, chromatin condensation, reactive oxygen species and autophagy indicated that these withaferin A-silyl analogs appear to induce events characteristic of apoptosis-like and also autophagy leading to programmed cell death. These findings support the therapeutic potential of withaferin A-related steroids as anti-Leishmania and Trypanosoma agents.

Biological activities of 1,4-naphthoquinones derivatives against T. cruzi and L. amazonensis

Introduction: Chagas disease and Leishmaniasis are neglected diseases caused bythe Trypanosoma cruzi and kentoplastid parasites Leishmania spp. Parasitic diseasescause great impact on social and economic, affecting millions of people in the worldand represent a major global health problem. In the search for new alternatives for thetreatment of Leishmaniasis and Chagas disease, strategies have been used to discovernew active molecules, because there is an urgent need for the development of newdrugs. In this scenario, 1,4-naphthoquinones have shown notable activity in thecontext of neglected diseases. Aim: To synthesis of 1,4-naphthoquinones derivativesand evaluated these compounds against Trypanosoma cruzi epimastigotes, Leishmaniapromastigotes (Leishmania amazonensis) and cytotoxicity to LLCMK2 cells.Results: Nine 1,4-naphthoquinones derivatives were synthesized using 2-Bromo-1,4-naphthoquinone (1), 1,4-Naphthoquinone (5) and 2-Hydroxi-1,4-naphthoquinone(9) as starting material. Derivative 6a exhibited excellent trypanocidal activity,IC50 of 0.25 ± 0.02 μM, superior potency compared with the reference drug Benznidazol.Besides, these compounds displayed low activity against promastigote from L.amazonensis. Conclusion: The results indicate that compound 6a may have potentialfor agent against Chagas disease.

Chemical Constituents from Uapaca guineensis (Phyllanthaceae), and the Computational Validation of Their Antileishmanial and Anti-inflammatory Potencies

From the chemical investigations of the root bark of Uapaca guineensis, nine distinct compounds (1–9) have been isolated and characterized as lupeol, betulin, betulinic acid, β-amyryl acetate, physcion, quercetin, rutin, β-sitosterol, and β-sitosterol-3-O-β-D-glucopyranoside, respectively. The structures of all the isolated compounds have been established using their NMR data as well as the comparison of those data with the ones reported in the literature. Interestingly, to the best of our knowledge, except for the lupane-type triterpenoids (1–3) and compounds 4 and 9, all the other compounds are reported for the first time from this genus. Since the plant is widely used for the treatment of skin diseases, leishmaniasis and inflammatory diseases, the antileishmanial and anti-inflammatory potencies of all the isolated compounds have been computationally validated through their ability to inhibit the receptors 1QCC and 2XOX (for the antileishmanial studies) and 6Y3C and 1CX2 (for the anti-inflammatory studies). Furthermore, the ADMET studies of compounds have been done to evaluate their drug-likeness. Results demonstrate that all the isolated compounds showed a better affinity for both receptors’ binding sites than the standard drugs miltefosine and aspirin. Moreover, the compounds would not cause addiction when used as lead molecules whereas, aspirin is predicted to violate the BBB over a long term of usage as a drug. This study gives additional information on the chemistry of U. guineensis and its classification as a potential source of good leads for the development of potent antileishmanial and anti-inflammatory drugs.

Development and validation of an ultra-high performance liquid chromatography coupled to tandem mass spectrometry method for the quantification of the antileishmanial drug paromomycin in human skin tissue

Bioanalytical assay development and validation procedures were performed to quantify antiprotozoal drug paromomycin in human skin tissue by ultra-high performance liquid chromatography coupled to tandem mass spectrometry. Paromomycin, an aminoglycoside drug, is administered intra-muscularly and used in the treatment of multiple clinical presentations of the neglected tropical disease leishmaniasis. It is currently studied in the treatment of post-kala-azar dermal leishmaniasis, a disease where the Leishmania parasites divide and reside in the skin. We present a target-site bioanalytical method to accurately quantify paromomycin in human skin tissue, with the clinical purpose of quantifying paromomycin in skin biopsies from post-kala-azar dermal leishmaniasis patients originating from Sudan. Enzymatic digestion using collagenase A incubated at 37 °C overnight was employed as homogenization method to produce skin tissue homogenates. Further sample preparation was performed by protein precipitation using trichloroacetic acid and a dilution step. Final extracts were injected onto a C18 analytical column and isocratic heptafluorobutyric acid ion-pair separation and elution were employed. The chromatography system was coupled to a triple quadrupole mass spectrometer for detection. The method was validated in digestion solution over a linear range from 5 to 1000 ng/mL (r2 ≥ 0.9967) with the assay performance of accuracy and precision within acceptable criteria values as stated by the EMA guidelines. Furthermore, matrix effects were observed in human skin tissue and were corrected by the multiple deuterated paromomycin internal standard. No substantial IS-normalized matrix effect was detected along with relatively high sample preparation recovery. Consequently, digestion solution matrix serving as the preparation of calibration standards can be used as surrogate matrix for human skin tissue, which is convenient given the limited availability of control matrix. Finally, paromomycin was accurately quantified in skin of post-kala-azar dermal leishmaniasis patients originating from clinical trials in Sudan.

Selective Anti-Leishmanial Strathclyde Minor Groove Binders Using an N-Oxide Tail-Group Modification.

The neglected tropical disease leishmaniasis, caused by Leishmania spp., is becoming more problematic due to the emergence of drug-resistant strains. Therefore, new drugs to treat leishmaniasis, with novel mechanisms of action, are urgently required. Strathclyde minor groove binders (S-MGBs) are an emerging class of anti-infective agent that have been shown to have potent activity against various bacteria, viruses, fungi and parasites. Herein, it is shown that S-MGBs have potent activity against L. donovani, and that an N-oxide derivation of the tertiary amine tail of typical S-MGBs leads to selective anti-leishmanial activity. Additionally, using S-MGB-219, the N-oxide derivation is shown to retain strong binding to DNA as a 2:1 dimer. These findings support the further study of anti-leishmanial S-MGBs as novel therapeutics.

Development and Evaluation of Wild-Derived Rats as Potential Experimental Models for Research

Wild rodents have been used as animal models in experimental studies of cellular aging, cerebral malaria, schistosomiasis, and leishmaniasis. Since they are the reservoirs of these diseases, recent studies have shown that research involving wild rodents is more efficient in giving a mirror image of the disease progression outside the laboratory setting. The study design was an analytic experimental study where observations were made between two groups of animals i.e. the wild-derived and lab-bred rats. The outcome of the intervention was obtained by comparing the two groups. This study sought to determine the usability of wild-derived rats as potential experimental animal models for biomedical research. Wild rats were randomly captured in areas with high transmission of Schistosoma mansoni and Leishmania donovani in Kisumu and Kitui counties, in Kenya and maintained in the animal house at Kenya Medical Research Institute (KEMRI) in Nairobi. Breeding was carried out, and the subsequent generations were used to assess schistosomiasis and leishmaniasis disease progression between the wild-derived, and laboratory-bred rats. We further monitored behavior patterns and food consumption rate for 3 months in a total of 6 experimental trials. The results on feed consumption capacity indicated that consumption was significantly higher in laboratory-bred groups (p=0.001) compared to wild-derived rats. Overall, laboratory-bred rats were significantly heavier than wild rats. In the use of rats to assess schistosomiasis and leishmaniasis disease progression, the wild-derived rats were more susceptible to leishmaniasis and may be considered for S. mansoni compared to wild (worms recovered p=0.031). In conclusion, the study indicated that wild rats are potential reservoir hosts for both schistosomiasis and leishmaniasis and have the potential to maintain cycles of infection until after successful chemotherapeutic intervention. Therefore, wild rats may provide natural means for parasites like S. mansoni and L. donovani to re-infect humans in endemic areas.

Genome deletions to overcome the directed loss of gene function in Leishmania.

With the global reach of the Neglected Tropical Disease leishmaniasis increasing, coupled with a tiny armory of therapeutics which all have problems with resistance, cost, toxicity and/or administration, the validation of new drug targets in the causative insect vector borne protozoa Leishmania spp is more important than ever. Before the introduction of CRISPR Cas9 technology in 2015 genetic validation of new targets was carried out largely by targeted gene knockout through homologous recombination, with the majority of genes targeted (~70%) deemed non-essential. In this study we exploit the ready availability of whole genome sequencing technology to reanalyze one of these historic cell lines, a L. major knockout in the catalytic subunit of serine palmitoyltransferase (LCB2), which causes a complete loss of sphingolipid biosynthesis but remains viable and infective. This revealed a number of Single Nucleotide Polymorphisms, but also the complete loss of several coding regions including a gene encoding a putative ABC3A orthologue, a putative sterol transporter. Hypothesizing that the loss of such a transporter may have facilitated the directed knockout of the catalytic subunit of LCB2 and the complete loss of de novo sphingolipid biosynthesis, we re-examined LCB2 in a L. mexicana line engineered for straightforward CRISPR Cas9 directed manipulation. Strikingly, LCB2 could not be knocked out indicating essentiality. However, simultaneous deletion of LCB2 and the putative ABC3A was possible. This indicated that the loss of the putative ABC3A facilitated the loss of sphingolipid biosynthesis in Leishmania, and suggested that we should re-examine the many other Leishmania knockout lines where genes were deemed non-essential.