Visceral leishmaniasis misdiagnosed as an upper respiratory infection and iron-deficiency anemia in a 20-month-old male patient: a case report.

BACKGROUND: Visceral Leishmaniasis should be suspected in every patient with a history of splenomegaly, fever, and pancytopenia. It is one of the most dangerous forms of infection and prompt recognition is the key to positive outcome. CASE PRESENTATION: A 20-month-old Caucasian male patient was brought to our hospital as an outpatient with the complaint of persistent fever, which did not improve with empiric antibiotic treatment (> 96 hour after the initial dose). The antibiotic treatment had been prescribed by primary care physician at polyclinic, who also referred the patient to hematologist due to anemia, who prescribed iron supplement. Despite multiple subspecialist visits, bicytopenia was, unfortunately, left unidentified. Upon physical examination no specific signs were detected, however, spleen seemed slightly enlarged. Patient was admitted to the hospital for further work-up, management and evaluation. Abdominal ultrasound, complete blood count and c-reactive protein had been ordered. Hematologist and infectionist were involved, both advised to run serology for Epstein-Barr Virus and Visceral Leishmaniasis. The latter was positive; therefore, patient was transferred to the specialized clinic for specific management. CONCLUSION: Both in endemic and non-endemic areas the awareness about VL should be increased among the medical professionals. We also recommend that our colleagues take the same approach when dealing with bicytopenia and fever, just as with pancytopenia and fever. The medical community should make sure that none of the cases of fever and pancytopenia are overlooked, especially if we have hepatomegaly and/or splenomegaly.

Prosopis juliflora (Sw.) DC.induces apoptotic-like programmed cell death in Leishmania donovani via over production of oxidative stress, mitochondrial dysfunction and ATP depletion.

Background: Leishmaniasis is endemic in more than 60 countries with a large number of mortality cases. The current chemotherapy approaches employed for managing the leishmaniasis is associated with severe side effects. Therefore there is a need to develop effective, safe, and cost affordable antileishmanial drug candidates. Purpose of the study: This study was designed to evaluate the in vitro antileishmanial activity of a Prosopis juliflora leaves extract (PJLME) towards the Leishmania donovani parasites. Material and methods: PJLME was evaluated for its cytotoxicity against the L. donovani parasites and the mouse macrophage cells. Further, various in vitro experiments like ROS assay, mitochondrial membrane potential assay, annexin v assay, cell cycle assay, and caspase 3/7 assay were performed to understand the mechanism of cell death. Phytochemical profiling of P. juliflorawas performed by utilizing HPTLC and GC-MS analysis. Results: PJLME demonstrated antileishmanial activity at a remarkably lower concentration of IC50 6.5 µg/mL. Of note, interestingly PJLME IC50 concentration has not demonstrated cytotoxicity against the mouse macrophage cell line. Performed experiments confirmed ROS inducing potential of PJLME which adversely affected the mitochondrial membrane potential and caused loss of mitochondrial membrane potential and thereby ATP levels. PJLME also arrested the cell cycle and induced apoptotic-like cell death in PJLME treated L. donovani promastigotes. Conclusion: The results clearly established the significance of Prosopis juliflora as an effective and safe natural resource for managing visceral leishmaniasis. The findings can be used as a baseline reference for developing novel leads/formulations for effective management of visceral leishmaniasis.

Computational evaluation of phytochemicals targeting DNA topoisomerase I in Leishmania donovani: molecular docking and molecular dynamics simulation studies.

DNA topoisomerase I (Topo I) is a ubiquitous enzyme that plays a crucial role in resolving the topological constraints of supercoiled DNA during various cellular activities, including repair, replication, recombination, transcription, and chromatin remodeling. Multiple studies have confirmed the essential role of Topo I in nucleic acid metabolism of Leishmania donovani, the kinetoplastid parasite responsible for visceral leishmaniasis or kala-azar. Inhibition of this enzyme has shown promise as a strategy for therapy against visceral leishmaniasis. However, current treatment options suffer from limitations related to effectiveness, cost, and side effects. To address these challenges, computational methods have been employed in this study to investigate the inhibition of Leishmania donovani DNA topoisomerase I (LdTopo I) by phytochemicals derived from Indian medicinal plants known for their anti-leishmanial activity. A library of phytochemicals and known inhibitors was assembled, and virtual screening based on docking binding affinities was conducted to identify potent phytochemical inhibitors. To assess the drug-likeness of the docked phytochemicals, their physicochemical properties were predicted. Additionally, molecular dynamics (MD) simulations were performed on the docked complexes for a duration of 100 ns to evaluate their stability, intermolecular interactions, and dynamic behavior. Among all the docked phytochemicals, three compounds, namely CID23266147 (withanolide N), CID5488537 (fagopyrine), and CID100947536 (isozeylanone), exhibited the highest inhibitory potential against LdTopo I. These findings hold promise for the development of novel inhibitors targeting LdTopo I, which could potentially lead to improved therapies for visceral leishmaniasis.Communicated by Ramaswamy H. Sarma.

Insights into Leishmania donovani potassium channel family and their biological functions.

Leishmania donovani is the causative organism for visceral leishmaniasis. Although this parasite was discovered over a century ago, nothing is known about role of potassium channels in L. donovani. Potassium channels are known for their crucial roles in cellular functions in other organisms. Recently the presence of a calcium-activated potassium channel in L. donovani was reported which prompted us to look for other proteins which could be potassium channels and to investigate their possible physiological roles. Twenty sequences were identified in L. donovani genome and subjected to estimation of physio-chemical properties, motif analysis, localization prediction and transmembrane domain analysis. Structural predictions were also done. The channels were majorly α-helical and predominantly localized in cell membrane and lysosomes. The signature selectivity filter of potassium channel was present in all the sequences. In addition to the conventional potassium channel activity, they were associated with gene ontology terms for mitotic cell cycle, cell death, modulation by virus of host process, cell motility etc. The entire study indicates the presence of potassium channel families in L. donovani which may have involvement in several cellular pathways. Further investigations on these putative potassium channels are needed to elucidate their roles in Leishmania. Supplementary Information: The online version contains supplementary material available at 10.1007/s13205-023-03692-y.

Andrographolide-Soya-L-α-Phosphatidyl Choline Complex Augmented Solubility and Drug Delivery in Leishmania donovani, a Causative Agent for Cutaneous and Visceral Leishmaniasis.

The utility of andrographolide (AN) in visceral leishmaniasis (VL) and cutaneous leishmaniasis (CL) is limited owing to poor solubility, hindered permeation, and unstable structure under physiological conditions. The present study mainly focuses on synthesizing of andrographolide-Soya-L-α-phosphatidyl choline (ANSPC) complex in ethanol and its characterization using various spectral and analytical techniques. Results from FT-IR, 1H NMR, ROSEY, and in silico docking techniques suggest ANSPC complex formation due to inter-molecular interaction between the hydrophilic head of SPC and hydroxyl group of AN present at 24th position. ANSPC complex demonstrated the solubility of 113.93 ± 6.66 µg/mL significantly (P < 0.05) greater than 6.39 ± 0.47 µg/mL of AN. The particle size of ANSPC complex was found to be 182.2 ± 2.69 nm. The IC50 value of AN suspension (PBS, pH ~ 7.4) at 24, 48, and 72 h against Leishmania donovani (L. donovani) was noticed to be 32.76 ± 4.53, 20.87 ± 2.37, and 17.71 ± 3.06 µM/mL, respectively. Moreover, augmented aqueous solubility of ANSPC complex led to significant (P < 0.05) reduction in IC50 value, i.e., 25.02 ± 4.35, 11.31 ± 0.60, and 8.33 ± 2.71 µM/mL at 24, 48, and 72 h, respectively. The IC50 values for miltefosine were noted to be 9.84 ± 2.65, 12.13 ± 7.26, and 6.56 ± 0.61 µM/mL at similar time periods. Moreover, ANSPC complex demonstrated augmented cellular uptake at 24 h as compared to 6 h in L. donovani. We suppose that submicron size and phospholipid-mediated complexation might have endorsed the permeation of ANSPC complex across the plasma membrane of L. donovani parasite by transport mechanisms such as P-type ATPase. ANSPC complex warrants further in-depth in vivo studies under a set of stringent parameters for translating the product into a clinically viable form.

Drug repurposing based novel anti-leishmanial drug screening using in-silico and in-vitro approaches.

Visceral leishmaniasis is a neglected tropical disease and is mainly caused by L. donovani in the Indian subcontinent. The mitochondria genome replication in Leishmania spp. is having a very specific mechanism, and it is initiated by a key enzyme called mitochondrial primase. This enzyme is essential for the onset of the replication process and growth of the parasite. Therefore, we focused on the primase protein as a potential therapeutic target for combating leishmaniasis diseases. We started our studies molecular modeling and followed by docking of the FDA-approved drug library into the binding site of the primase protein. The top 30 selected compounds were subjected for molecular dynamics studies. Also, the target protein was cloned, purified, and tested experimentally (primase activity assays and inhibition assays). Some compounds were very effective against the Leishmania cell culture. All these approaches helped us to identify few possible novel anti-leishmanial drugs such as Pioglitazone and Mupirocin. These drugs are effectively involved in inhibiting the promastigote of L. donovani, and it can be utilized in the next level of clinical trials. Communicated by Ramaswamy H. Sarma.

Use of N-acetylcysteine as treatment adjuvant regulates immune response in visceral leishmaniasis: Pilot clinical trial and in vitro experiments.

This investigation aimed to assess the effect of N-acetylcysteine (NAC) as an adjuvant treatment to alleviate visceral leishmaniasis (VL). The present work includes both blinded randomized clinical intervention and experimental in vitro studies. The clinical trial included 60 patients with VL randomly allocated into two groups: a test group (n = 30) treated with meglumine antimoniate plus NAC (SbV + NAC) and a control group (n = 30) treated with meglumine antimoniate only (SbV). The primary outcome was clinical cure (absence of fever, spleen and liver sizes reduction, and hematological improvement) in 180 days. The cure rate did not differ between the groups; both groups had similar results in all readout indices. The immunological parameters of the patients treated with SbV + NAC showed higher sCD40L in sera during treatment, and the levels of sCD40L were negatively correlated with Interleukin-10 (IL-10) serum levels. In addition, data estimation showed a negative correlation between the sCD40L levels and the spleen size in patients with VL. For the in vitro experiments, peripheral blood mononuclear cells (PBMCs) or PBMC-derived macrophages from healthy donors were exposed to soluble Leishmania antigen (SLA) or infected with stationary promastigotes of Leishmania infantum in the presence or absence of NAC. Results revealed that NAC treatment of SLA-stimulated PBMCs reduces the frequency of monocytes producing IL-10 and lowers the frequency of CD4+ and CD8+ T cells expressing (pro-)inflammatory cytokines. Together, these results suggest that NAC treatment may modulate the immune response in patients with VL, thus warranting additional investigations to support its case use as an adjuvant to antimony therapy for VL.

Thymus, undernutrition, and infection: Approaching cellular and molecular interactions.

Undernutrition remains a major issue in global health. Low protein-energy consumption, results in stunting, wasting and/or underweight, three deleterious forms of malnutrition that affect roughly 200 million children under the age of five years. Undernutrition compromises the immune system with the generation of various degrees of immunodeficiency, which in turn, renders undernourished individuals more sensitive to acute infections. The severity of various infectious diseases including visceral leishmaniasis (VL), influenza, and tuberculosis is associated with undernutrition. Immunosuppression resulting from protein-energy undernutrition severely impacts primary and secondary lymphoid organs involved in the response to related pathogens. The thymus-a primary lymphoid organ responsible for the generation of T lymphocytes-is particularly compromised by both undernutrition and infectious diseases. In this respect, we will discuss herein various intrathymic cellular and molecular interactions seen in undernutrition alone or in combination with acute infections. Many examples illustrated in studies on humans and experimental animals clearly revealed that protein-related undernutrition causes thymic atrophy, with cortical thymocyte depletion. Moreover, the non-lymphoid microenvironmental compartment of the organ undergoes important changes in thymic epithelial cells, including their secretory products such as hormones and extracellular matrix proteins. Of note, deficiencies in vitamins and trace elements also induce thymic atrophy. Interestingly, among the molecular interactions involved in the control of undernutrition-induced thymic atrophy is a hormonal imbalance with a rise in glucocorticoids and a decrease in leptin serum levels. Undernutrition also yields a negative impact of acute infections upon the thymus, frequently with the intrathymic detection of pathogens or their antigens. For instance, undernourished mice infected with Leishmania infantum (that causes VL) undergo drastic thymic atrophy, with significant reduction in thymocyte numbers, and decreased levels of intrathymic chemokines and cytokines, indicating that both lymphoid and microenvironmental compartments of the organ are affected. Lastly, recent data revealed that some probiotic bacteria or probiotic fermented milks improve the thymus status in a model of malnutrition, thus raising a new field for investigation, namely the thymus-gut connection, indicating that probiotics can be envisioned as a further adjuvant therapy in the control of thymic changes in undernutrition accompanied or not by infection.

Therapeutic Potential of Quercetin-Loaded Nanoemulsion against Experimental Visceral Leishmaniasis: In Vitro/Ex Vivo Studies and Mechanistic Insights.

Visceral leishmaniasis (VL) is one of the most fatal and neglected tropical diseases caused by Leishmania donovani (L. donovani). The applications of currently available chemotherapy (amphotericin B, miltefosine, and others) in VL treatment have been limited due to their poor bioavailability, unfavorable toxicity profile, and prolonged parenteral dosing. Quercetin (QT), a potent natural antioxidant, is a prominent target when conducting investigations on alternative therapies against L. donovani infections. However, the therapeutic applications of QT have been restricted due to its low solubility and bioavailability. In the present study, we developed and evaluated the antileishmanial activity (ALA) of quercetin-loaded nanoemulsion (QTNE) against L. donovani clinical strains. In vitro anti-promastigote assay results demonstrated that QTNE (IC50 6.6 µM, 48 h) significantly inhibited the growth of parasites more efficiently than the pure QT suspension in a dose- and time-dependent manner. Results of the anti-amastigote assay revealed that the infected macrophages (%) of QTNE were significantly more than those of the pure QT suspension at all concentrations (6.6, 26.4, and 52.8 µM; p < 0.05, p < 0.01 compared to the control). Moreover, the results of in vitro and ex vivo studies assisted in determining the mechanistic insights associated with the ALA of QTNE. The overall findings suggested that QTNE exhibited potential ALA by enhancing the intracellular ROS and nitric oxide levels, inducing distortion of membrane integrity and phosphatidylserine release (AV/PI), rupturing the parasite DNA (late apoptosis/necrosis process), and upregulating the immunomodulatory effects (IFN-γ and IL-10 levels). Additionally, QTNE showed superior biocompatibility against all of the treated healthy cells (PBMCs, PECs, and BMCs) as compared to the control. In conclusion, QTNE acts as a potential antileishmanial agent targeting both promastigote and intracellular amastigote forms of L. donovani, which thus opens a new avenue for the use of QTNE in VL therapy.

Activation of TLR-pathway to induce host Th1 immune response against visceral leishmaniasis: Involvement of galactosylated-flavonoids.

Immunotherapeutic strategies against visceral leishmaniasis (VL) are pertinent because of the emergence of resistance against existing chemotherapy, coupled with their toxicity and high costs. Various bioactive components with potential immunomodulatory activity, such as alkaloids, terpenes, saponins, flavonoids obtained primarily from medicinal plants, have been screened against different disease models. Reports suggested that glycans containing terminal ß-galactose can skew host immune response towards Th1 by engaging TLRs. In this study, two synthesized terminal galactose-containing flavones, Quercetin 3-d-galactoside (Q-gal) and Kaempferol 3-O-d-galactoside (K-gal), are profiled in terms of inducing host protective Th1 response in both in vitro & in vivo animal models of experimental VL individually against antimony-resistant & antimony-susceptible Leishmania donovani. Further, we explored that both Q-gal and K-gal induce TLR4 mediated Th1 response to encounter VL. Molecular docking analysis also suggested strong interaction with TLR4 for both the galactosides, with a slightly better binding potential towards Q-gal. Treatment with both Q-gal and K-gal showed significant antileishmanial efficacy. Each considerably diminished the liver and splenic parasite burden 60 days after post-infection (>90% in AG83 infected mice and >87% in GE1F8R infected mice) when administered at a 5 mg/kg/day body-weight dose for ten consecutive days. However, the treatments failed to clear the parasites in the TLR4 deficient C3H/HeJ mice. Treatment with these compounds favors the elevation of TLR4 dependent host protective Th1 cytokines and suppression of disease-promoting IL-10. Q-gal and K-gal also triggered sufficient ROS generation in macrophages to kill intracellular parasites directly.

Screening of medicinal plants unraveled the leishmanicidal credibility of Garcinia cowa; highlighting Norcowanin, a novel anti-leishmanial phytochemical through in-silico study.

Leishmaniasis, one of the most prevalent yet neglected parasitic causes of death, yearns for therapeutic control and treatment. Severely toxic and inefficient modern-day pentavalent antimonials, caters the search for naturally derived drugs, as efficient alternatives for disease treatment. The anti-promastigote activity of ten different plants selected for their ethnomedicinal properties revealed significant leishmanicidal capacity; the most potent being Garcinia cowa methanolic extract with an IC50 value of 21.4 µg/ml. Garcinia cowa, a plant endemic to North-Eastern India that is of the Clusiaceae family, is replete with such medicinal qualities as antimicrobial, antiviral, antiparasitic, and antiproliferative activities. Computational biology with its tools such as molecular docking has opened new horizons aimed at a better understanding of biological systems, complexes, and their interactions, and subsequently drug discovery via in silico techniques. Therefore, an in-silico study was designed to evaluate the binding capability of six phytochemicals- cowanin, cowanol, cowaxanthone, norcowanin, rubraxanthone, and a basic xanthone, found in Garcinia cowa against Pentamidine, a synthetic anti-leishmanial drug. The active sites of three characteristic enzymes belonging to the Leishmania donovani parasite: O-acetylserine sulfhydrylase (OASS), Trypanothione reductase (TryR), and N-Myristoyltransferase (NMT) were chosen as target proteins. Results revealed lower binding energies and higher affinities, of nearly all the phytochemicals with respect to Pentamidine, indicating their leishmanicidal potential. Norcowanin showed the lowest average binding of - 9.8 kcal/mol against all the three enzymes under study.

Retinoic acid restores the levels of cellular cholesterol in Leishmania donovani infected macrophages by increasing npc1 and npc2 expressions.

Visceral leishmaniasis (VL) is a fatal form among all forms of leishmaniasis and is caused by visceralization of the Leishmania donovani (Ld) parasite to the critical organs. Mild to severe malnutrition is common in VL patients and the deficiency of retinoic acid (RA), an important micronutrient, results in a compromised state of immune response in macrophages (mφ) leading to the increased parasite load. In the continuation of our earlier work, we observed loss of cellular cholesterol in infected mφ in the absence of RA i.e., upon inhibition of RALDH pathway. Moreover, the Leishmania utilizes host cholesterol for the establishment of infection and causes a decrease in the expressions of Niemann-Pick C2 (npc2) and Niemann-Pick C1 (npc1) genes involved in the uptake of extracellular cholesterol. This results in reduced levels of cellular cholesterol in infected mφ. Intrigued by this, as the first sign of our hypothesis, we investigated the presence of RA Response Element (RARE) sequences in the upstream of npc1 and npc2 genes. To functionally confirm this, we measured their expressions and the levels of cellular cholesterol in Ld infected mφ in the absence (i.e., using an inhibitor of RALDH pathway) and presence of RA. We found restoration of the levels of cellular cholesterol in infected mφ under the supplementation of RA resulting in the decreased parasite load. Hence, the supplementation of RA with the standard therapy and/or preventive use of RA could be potentially an advancement in the treatment and cure of VL patients.

Effects of terpenes in the treatment of visceral leishmaniasis: A systematic review of preclinical evidence.

Visceral leishmaniasis (VL) is a severe and potentially fatal neglected tropical disease, being considered a public health concern in many countries worldwide. There are still no vaccines against human VL, and the existing chemotherapy is often toxic. Thereby, alternative treatments have been investigated, and byproducts from plant metabolism have been a source of promising pharmacological compounds. Terpenes are secondary metabolites that exhibit a large spectrum of therapeutic activities. Herein, we conducted a systematic review to evaluate the effects of terpenes in the treatment of VL in rodents. After an extensive search using the PubMed/MEDLINE, Scopus, and Web of Science databases, we included 34 articles in this review. Our results revealed that triterpenes were the most used terpenes by the eligible studies. Overall, terpene treatment showed no or negligible toxicity, causing an increase in the Th1-type immune response profile and nitric oxide production. It also reduced the Th2 cytokines levels and parasite load (> 90% to > 99%). Moreover, terpenes induced apoptosis by damaging the plasma membrane and inhibiting DNA topoisomerases in the parasite. The use of terpene carriers increased the terpene bioavailability in the body, preventing their rapid excretion and promoting the drug delivery at the site of Leishmania infection. Terpene derivatives showed better pharmacokinetics than the original terpenes. Altogether, the benefits of VL treatment with terpenes in preclinical studies may open new directions for other preclinical and human trials.

GC-MS screening and antiparasitic action of Putranjiva roxburghii leaves against sensitive and resistant strains of Leishmania donovani.

Looming drug resistance cases of leishmaniasis infection are an undeniably serious danger to worldwide public health, also jeopardize the efficacy of available drugs. Besides this, no successful vaccine is available till date. Since the ancient era, many plants and their parts have been used as medicines against various ailments. Hence, the importance of drug development for new molecules against Leishmania infection is significant that is a cost-effective and safer drug preferably from the natural herbal resources. We evaluated the GC-MS screening and efficacy of Putranjiva roxburghii (PR) against the sensitive and resistant promastigotes of L. donovani. GC-MS profiling revealed that the extract was rich in myo-inositol-4-C-methyl, azulene and desulphosinigrin. Quantitative investigation of phytoconstituents confirmed that PR was rich in phenols, flavonoids and terpenoids. We found an IC50 25.61 ± 0.57 µg/mL and 29.02 ± 1.21 µg/mL of PR against sodium stibogluconate sensitive and resistant strain respectively. It was found to be safer in cytotoxicity assay and generated ROS mediated oxidative stress in the parasitic cells which was evidenced by the increased and decreased levels of superoxide radicals, lipid peroxidation products, lipid bodies and levels of thiol, plasma membrane integrity respectively. Therefore, our results support the importance of P. roxburghii as a medicinal plant against L. donovani and showed potential for exploration as an antileishmanial agent.

In vivo evaluation of anti-Leishmania activity of alkyltriazoles and alkylphosphocholines by oral route.

The failures in the treatment of leishmaniasis is an increasing problem around the world, especially related to resistance. Thus, we describe the synthesis and in vivo anti-Leishmania activity of alkylphosphocholine and alkyltriazoles; besides, their likely action mechanisms stem from some eventual inhibition of parasite enzymes using computational tools. These compounds were tested in an in vivo hamster model infected with Leishmania Leishmania infantum chagasi. Fifty days after parasite inoculation, the two compounds 12-azidedodecylphosphocholine (3) and 3-(1-(12-fluorododecyl)-1H-1,2,3-triazol-1-yl)propano-1-ol (9), were separately administered once a day as oral suspensions (25 and 12.5 mg/kg/day, respectively) during ten days, and their efficacy was compared to the reference compound pentavalent antimonial Glucantime (GLU). Compound 3 significantly reduced the number of parasites in the spleen (4.93 × 102 amastigotes/g) and liver (4.52 × 103 amastigotes/g). Compound 9 reduced the number of amastigotes in the spleen to 1.30 × 104 and 1.36 × 103 amastigotes/g in the liver. GLU was the most effective overall treatment (7.50 × 101 and 2.28 × 102 amastigotes/g in the spleen and liver, respectively). The high activity levels of these compounds in vivo may stem from their high in vitro leishmanicidal activity and lipophilicity. The in silico absorption, distribution, metabolism, and excretion studies also showed some anti-Leishmania potential. Compound 9 had more lipophilic characteristics than those of compound 3. In silico studies of the nine enzymes of compounds 3 and 9 showed significant evidence of interactions with nicotimidase and tyrosine aminotransferase, demonstrating possible inhibition enzymes present in L. (L.) infantum chagasi. These compounds could be a promising template for developing a new class of leishmanicidal agents, by oral route, and deserve further investigation to explore different therapeutic regimens.

Acarbose presents in vitro and in vivo antileishmanial activity against Leishmania infantum and is a promising therapeutic candidate against visceral leishmaniasis.

Treatment against visceral leishmaniasis (VL) is mainly hampered by drug toxicity, long treatment regimens and/or high costs. Thus, the identification of novel and low-cost antileishmanial agents is urgent. Acarbose (ACA) is a specific inhibitor of glucosidase-like proteins, which has been used for treating diabetes. In the present study, we show that this molecule also presents in vitro and in vivo specific antileishmanial activity against Leishmania infantum. Results showed an in vitro direct action against L. infantum promastigotes and amastigotes, and low toxicity to mammalian cells. In addition, in vivo experiments performed using free ACA or incorporated in a Pluronic® F127-based polymeric micelle system called ACA/Mic proved effective for the treatment of L. infantum-infected BALB/c mice. Treated animals presented significant reductions in the parasite load in their spleens, livers, bone marrows and draining lymph nodes when compared to the controls, as well as the development of antileishmanial Th1-type humoral and cellular responses based on high levels of IFN-γ, IL-12, TNF-α, GM-CSF, nitrite and IgG2a isotype antibodies. In addition, ACA or ACA-treated animals suffered from low organ toxicity. Treatment with ACA/Mic outperformed treatments using either Miltefosine or free ACA based on parasitological and immunological evaluations performed one and 15 days post-therapy. In conclusion, data suggest that the ACA/Mic is a potential therapeutic agent against L. infantum and merits further consideration for VL treatment.

A Systematic Review of Curcumin and its Derivatives as Valuable Sources of Antileishmanial Agents.

BACKGROUND: In recent years, antimonial agents and other synthetic antileishmanial drugs, such as amphotericin B, paromomycin, and many other drugs, have restrictions in use due to the toxicity risk, high cost, and emerging resistance to these drugs. The present study aimed to review the antileishmanial effects of curcumin, its derivatives, and other relevant pharmaceutical formulations on leishmaniasis. METHODS: The present study was carried out according to the 06-preferred reporting items for systematic reviews and meta-analyses (PRISMA) guideline and registered in the CAMARADES-NC3Rs Preclinical Systematic Review and Meta-Analysis Facility (SyRF) database. Some English-language databases including PubMed, Google Scholar, Web of Science, EBSCO, Science Direct, and Scopus were searched for publications worldwide related to antileishmanial effects of curcumin, its derivatives, and other relevant pharmaceutical formulations, without date limitation, to identify all the published articles (in vitro, in vivo, and clinical studies). Keywords included "curcumin", "Curcuma longa", "antileishmanial", "Leishmania", "leishmaniasis", "cutaneous leishmaniasis", "visceral leishmaniasis", "in vitro", and "in vivo". RESULTS: Out of 5492 papers, 29 papers including 20 in vitro (69.0%), 1 in vivo (3.4%), and 8 in vitro/in vivo (27.6%) studies conducted up to 2020, met the inclusion criteria for discussion in this systematic review. The most common species of the Leishmania parasite used in these studies were L. donovani (n = 13, 44.8%), L. major (n = 10, 34.5%), and L. amazonensis (n = 6, 20.7%), respectively. The most used derivatives in these studies were curcumin (n = 15, 33.3%) and curcuminoids (n = 5, 16.7%), respectively. CONCLUSION: In the present review, according to the studies in the literature, various forms of drugs based on curcumin and their derivatives exhibited significant in vitro and in vivo antileishmanial activity against different Leishmania spp. The results revealed that curcumin and its derivatives could be considered as an alternative and complementary source of valuable antileishmanial components against leishmaniasis, which had no significant toxicity. However, further studies are required to elucidate this concluding remark, especially in clinical settings.

Micronutrient levels and their effects on the prognosis of visceral leishmaniasis treatment, a prospective cohort study.

BACKGROUND: Micronutrients are minerals and vitamins and they are essential for normal physiological activities. The objectives of the study were to describe the progress and determinants of micronutrient levels and to assess the effects of micronutrients in the treatment outcome of kalazar. METHODS: A prospective cohort study design was used. The data were collected using patient interviews, measuring anthropometric indicators, and collecting laboratory samples. The blood samples were collected at five different periods during the leishmaniasis treatments: before starting anti-leishmaniasis treatments, in the first week, in the second week, in the third week, and in the 4th week of anti-leishmaniasis treatments. Descriptive statistics were used to describe the profile of patients and to compare the treatment success rate. The generalized estimating equation was used to identify the determinants of serum micronutrients. RESULTS: The mean age of the patients were 32.88 years [SD (standard deviation) ±15.95]. Male constitute 62.3% of the patients and problematic alcohol use was present in 11.5% of the patients. The serum zinc level of visceral leishmaniasis patients was affected by alcohol (B - 2.7 [95% CI: - 4.01 - -1.5]), DDS (B 9.75 [95% CI: 7.71-11.79]), family size (B -1.63 [95% CI: - 2.68 - -0.58]), HIV (B -2.95 [95% CI: - 4.97 - -0.92]), and sex (B - 1.28 [95% CI: - 2.5 - -0.07]). The serum iron level of visceral leishmaniasis patients was affected by alcohol (B 7.6 [95% CI: 5.86-9.35]), family size (B -5.14 [95% CI: - 7.01 - -3.28]), malaria (B -12.69 [95% CI: - 14.53 - -10.87]), Hookworm (- 4.48 [- 6.82 - -2.14]), chronic diseases (B -7.44 [95% CI: - 9.75 - -5.13]), and HIV (B -5.51 [95% CI: - 8.23 - -2.78]). The serum selenium level of visceral leishmaniasis patient was affected by HIV (B -18.1 [95% CI: - 20.63 - -15.58]) and family size (B -11.36 [95% CI: - 13.02 - -9.7]). The iodine level of visceral leishmaniasis patient was affected by HIV (B -38.02 [95% CI: - 41.98 - -34.06]), DDS (B 25 .84 [95% CI: 22.57-29.1]), smoking (B -12.34 [95% CI: - 15.98 - -8.7]), chronic illness (B -5.14 [95% CI: - 7.82 - -2.46]), and regular physical exercise (B 5.82 [95% CI: 0.39-11.26]). The serum vitamin D level of visceral leishmaniasis patient was affected by HIV (B -9.43 [95% CI: - 10.92 - -7.94]), DDS (B 16.24 [95% CI: 14.89-17.58]), malaria (B -0.61 [95% CI: - 3.37 - -3.37]), and family size (B -1.15 [95% CI: - 2.03 - -0.28]). The serum vitamin A level of visceral leishmaniasis patient was affected by residence (B 0.81 [95% CI: 0.08-1.54]), BMI (B 1.52 [95% CI: 0.42-2.6]), DDS (B 1.62 [95% CI: 0.36-2.88]), family size (B -5.03 [95% CI: - 5.83 - -4.22]), HIV (B -2.89 [95% CI: - 4.44 - -1.34]),MUAC (B 0.86 [95% CI: 0.52-1.21]), and age (B 0.09 [95% CI: 0.07-0.12]). CONCLUSION: The micronutrient levels of visceral leishmaniasis patients were significantly lower. The anti-leishmaniasis treatment did not increase the serum micronutrient level of the patients.

Berberine-Loaded Liposomes for the Treatment of Leishmania infantum-Infected BALB/c Mice.

Berberine (BER)-an anti-inflammatory quaternary isoquinoline alkaloid extracted from plants-has been reported to have a variety of biologic properties, including antileishmanial activity. This work addresses the preparation of BER-loaded liposomes with the aim to prevent its rapid liver metabolism and improve the drug selective delivery to the infected organs in visceral leishmaniasis (VL). BER liposomes (LP-BER) displayed a mean size of 120 nm, negative Z-potential of -38 mV and loaded 6 nmol/µmol lipid. In vitro, the loading of BER in liposomes enhanced its selectivity index more than 7-fold by decreasing its cytotoxicity to macrophages. In mice, LP-BER enhanced drug accumulation in the liver and the spleen. Consequently, the liposomal delivery of the drug reduced parasite burden in the liver and spleen by three and one logarithms (99.2 and 93.5%), whereas the free drug only decreased the infection in the liver by 1-log. The organ drug concentrations-far from IC50 values- indicate that BER immunomodulatory activity or drug metabolites also contribute to the efficacy. Although LP-BER decreased 10-fold-an extremely rapid clearance of the free drug in mice-the value remains very high. Moreover, LP-BER reduced plasma triglycerides levels.

Cytokine profile and nitric oxide levels in peritoneal macrophages of BALB/c mice exposed to the fucose-mannose ligand of Leishmania infantum combined with glycyrrhizin.

BACKGROUND: The fucose-mannose ligand (FML) of Leishmania infantum is a complex glycoprotein which does not elicit adequate immunogenicity in humans. In recent years, adjuvant compounds derived from plants have been used for improving the immunogenicity of vaccines. Glycyrrhizin (GL) is a natural triterpenoid saponin that has known immunomodulatory activities. In the present study, we investigated the effects of co-treatment with FML and GL on the production of cytokines and nitric oxide (NO) by macrophages, in vitro. METHODS: Lipopolysaccharide (LPS) stimulated murine peritoneal macrophages were treated with FML (5 µg/ml) of L. infantum and various concentrations of GL (1 µg/ml, 10 µg/ml and 20 µg/ml). After 48 h of treatment, cell culture supernatants were recovered and the levels of TNF-α, IL-10, IL-12p70 and IP-10 were measured by sandwich ELISA and NO concentration by Griess reaction. RESULTS: Our results indicate that the treatment of activated macrophages with FML plus GL leads to enhanced production of NO, TNF-α and IL-12p70, and reduction of IL-10 levels in comparison with FML treatment alone. CONCLUSIONS: Therefore, we concluded that GL can improve the immunostimulatory effect of FML on macrophages and leads to their polarization towards an M1-like phenotype.