Th17 Cell and Inflammatory Infiltrate Interactions in Cutaneous Leishmaniasis: Unraveling Immunopathogenic Mechanisms.

The inflammatory response during cutaneous leishmaniasis (CL) involves immune and non-immune cell cooperation to contain and eliminate Leishmania parasites. The orchestration of these responses is coordinated primarily by CD4+ T cells; however, the disease outcome depends on the Th cell predominant phenotype. Although Th1 and Th2 phenotypes are the most addressed as steers for the resolution or perpetuation of the disease, Th17 cell activities, especially IL-17 release, are recognized to be vital during CL development. Th17 cells perform vital functions during both acute and chronic phases of CL. Overall, Th17 cells induce the migration of phagocytes (neutrophils, macrophages) to the infection site and CD8+ T cells and NK cell activation. They also provoke granzyme and perforin secretion from CD8+ T cells, macrophage differentiation towards an M2 phenotype, and expansion of B and Treg cells. Likewise, immune cells from the inflammatory infiltrate have modulatory activities over Th17 cells involving their differentiation from naive CD4+ T cells and further expansion by generating a microenvironment rich in optimal cytokines such as IL-1ß, TGF-ß, IL-6, and IL-21. Th17 cell activities and synergies are crucial for the resistance of the infection during the early and acute stages; however, if unchecked, Th17 cells might lead to a chronic stage. This review discusses the synergies between Th17 cells and the inflammatory infiltrate and how these interactions might destine the course of CL.

Artificial Intelligence in Cutaneous Leishmaniasis Diagnosis: Current Developments and Future Perspectives.

Cutaneous Leishmaniasis (CL) is a major global health problem requiring appropriate diagnosis methods. Its diagnosis is challenging, particularly in resource-limited settings. The integration of Artificial Intelligence (AI) into medical diagnostics has shown promising results in various fields, including dermatology. In this systematic review, we aim to highlight the value of using AI for CL diagnosis and the AI-based algorithms that are employed in this process, and to identify gaps that need to be addressed. Our work highlights that only a limited number of studies are related to using AI algorithms for CL diagnosis. Among these studies, seven gaps were identified for future research. Addressing these considerations will pave the way for the development of robust AI systems and encourage more research in CL detection by AI. This could contribute to improving CL diagnosis and, ultimately, healthcare outcomes in CL-endemic regions.

Attractiveness of golden hamster infected with Leishmania amazonensis (Kinetoplastida: Trypanosomatidae) to laboratory-reared Lutzomyia longipalpis (Diptera: Psychodidae).

Lutzomyia longipalpis is the primary vector of Leishmania infantum in the Americas and a permissive vector for Leishmania amazonensis. Previous studies showed that Leishmania infantum-infected hosts can release different volatile organic compounds (VOCs) compared with uninfected hosts, presenting a higher attractiveness to vectors. In this study, we aimed to evaluate a possible effect of L. amazonensis infection of golden hamsters in three parameters: attractiveness to Lu. longipalpis females; blood volume ingested by sand fly females; and VOCs released by the animals.. Attractiveness was measured indirectly by the number of Lu. longipalpis females that blood fed in each L. (L.) amazonensis-infected and uninfected animal. For VOCs extraction, solid phase micro extraction fibers were used, which were analyzed by gas chromatography-mass spectrometry. Behavioral trials did not show any effect of L. amazonensis infection on the attraction of sand flies nor difference on blood meal rates of Lu. longipalpis fed in both goups of hamsters. Additionally, there was no difference between the VOCs profiles of L. (L.) amazonensis-infected or uninfected hamsters.

Preparation and characterization of artemether-loaded niosomes in Leishmania major-induced cutaneous leishmaniasis.

Cutaneous leishmaniasis is the most prevalent form of leishmaniasis worldwide. Although various anti-leishmanial regimens have been considered, due to the lack of efficacy or occurrence of adverse reactions, design and development of novel topical delivery systems would be essential. This study aimed to prepare artemether (ART)-loaded niosomes and evaluate their anti-leishmanial effects against Leishmania major. ART-loaded niosomes were prepared through the thin-film hydration technique and characterized in terms of particle size, zeta potential, morphology, differential scanning calorimetry, drug loading, and drug release. Furthermore, anti-leishmanial effect of the preparation was assessed in vitro and in vivo. The prepared ART-loaded niosomes were spherical with an average diameter of about 100 and 300 nm with high encapsulation efficiencies of > 99%. The results of in vitro cytotoxicity revealed that ART-loaded niosomes had significantly higher anti-leishmanial activity, lower general toxicity, and higher selectivity index (SI). Half-maximal inhibitory concentration (IC50) values of ART, ART-loaded niosomes, and liposomal amphotericin B were 39.09, 15.12, and 20 µg/mL, respectively. Also, according to the in vivo study results, ART-loaded niosomes with an average size of 300 nm showed the highest anti-leishmanial effects in animal studies. ART-loaded niosomes would be promising topical drug delivery system for the management of cutaneous leishmaniasis.

NKG2C+CD57+ natural killer with senescent features cells are induced in cutaneous leishmaniasis and accumulate in patients with lesional healing impairment.

Natural killer (NK) cells include different subsets with diverse effector capacities that are poorly understood in the context of parasitic diseases. Here, we investigated inhibitory and activating receptor expression on NK cells in patients with cutaneous leishmaniasis (CL) and explored their phenotypic and functional heterogeneity based on CD57 and NKG2C expression. The expression of CD57 identified NK cells that accumulated in CL patients and exhibited features of senescence. The CD57+ cells exhibited heightened levels of the activating receptor NKG2C and diminished expression of the inhibitory receptor NKG2A. RNA sequencing analyses based on NKG2C transcriptome have revealed two distinct profiles among CL patients associated with cytotoxic and functional genes. The CD57+NKG2C+ subset accumulated in the blood of patients and presented conspicuous features of senescence, including the expression of markers such as p16, yH2ax, and p38, as well as reduced proliferative capacity. In addition, they positively correlated with the number of days until lesion resolution. This study provides a broad understanding of the NK cell biology during Leishmania infection and reinforces the role of senescent cells in the adverse clinical outcomes of cutaneous leishmaniasis.

Epidemiology of cutaneous leishmaniasis in children of Khyber Pakhtunkhwa, Pakistan.

OBJECTIVES: In Pakistan, cutaneous leishmaniasis is an emerging tropical disease and a very high number (>70%) of children are afflicted by this marring infection. This study aimed to scrutinise the prevalence, spatial distribution and socio-demographic and behavioural risk factors associated with cutaneous leishmaniasis in children aged <5-15 years in Khyber Pakhtunkhwa. METHODS: A total of 1, 559 clinically confirmed records of children diagnosed with cutaneous leishmaniasis (January-December) from 2020 and 2022 were obtained from selected district hospitals. In addition, a risk factors-related questionnaire was administered to 1, 011 households (400 in 2020 and 611 in 2022) in nine districts during a household survey. RESULTS: The maximum number of cutaneous leishmaniasis cases was recorded in 2022 (n = 877, 56.25%) as compared to 2020 (n = 682, 43.75%). The hospital records showed a greater number of male patients in the 2022 cohort (n = 603, 68.76%). The highest number of cases were observed in children aged 5-9 years in 2022 (n = 282, 32.16%) and 2020 (n = 255, 37.39%). In 2020 and 2022, cutaneous leishmaniasis cases showed peak aggregation in March (n = 118, 17.3%) and January (n = 322, 36.72%). From a spatial analysis, the maximum number of cutaneous leishmaniasis cases was recorded at 59-1700 m elevation in various land-use/land-cover and climatic regions with quaternary alluvium rock formations. A multivariate logistic regression model analysis of risk factors from the households survey suggested that age group, socio-economic status, construction materials of the house, use of insect repellents, Afghan refugee camps in the village/district, knowledge and biting times of sand flies, frequent use of mosquito bed nets, presence of domestic animals in the house, knowledge of the transmission period and peak month of leishmaniasis infection increased the risk of acquiring cutaneous leishmaniasis (p value < 0.05). CONCLUSION: Our analysis demonstrated that cutaneous leishmaniasis in children is influenced by a variety of environmental, socio-demographic and behavioural risk factors in Khyber Pakhtunkhwa. The increase in recorded cases of cutaneous leishmaniasis in children in 2022 compared to 2020 suggests that the infection likely extended to new foci in the province.

Pentavalent Antimony Associated with G-CSF in the Treatment of Cutaneous Leishmaniasis Caused by Leishmania (Viannia) braziliensis.

Cutaneous leishmaniasis (CL), caused by Leishmania braziliensis, in recent decades has shown decreasing cure rates after treatment with meglumine antimoniate (MA). Granulocyte colony-stimulating factor (G-CSF) is a cytokine associated with epithelialization and healing processes. METHODS: This study compares the effectiveness of G-CSF associated with MA in the treatment of CL. A total of 32 patients aged between 18 and 50 years with CL confirmed for L. braziliensis were included in this study. G-CSF or placebo (0.9% saline) was applied by intralesional infiltration at four equidistant points on the edges of the largest ulcer on days 0 and 15 of treatment associated with intravenous MA. RESULTS: Males predominated in the G-CSF group (59%), while females predominated in the control group (53%). Injuries to the lower limbs predominated in both study groups. The cure rate in the G-CSF group was 65% and in the control group it was 47%, 90 days after initiation of therapy. CONCLUSIONS: Our data indicate that the association of G-CSF with MA is not superior to MA monotherapy. Although not significant, the potential benefit of this combination deserves further investigation. The use of higher doses or other routes of application of G-CSF in a greater number of patients should contribute to a definitive response.

A comparative genomics approach reveals a local genetic signature of Leishmania tropica in Morocco.

In Morocco, cutaneous leishmaniasis (CL) caused by Leishmania (L.) tropica is an important health problem. Despite the high incidence of CL in the country, the genomic heterogeneity of these parasites is still incompletely understood. In this study, we sequenced the genomes of 14 Moroccan isolates of L. tropica collected from confirmed cases of CL to investigate their genomic heterogeneity. Comparative genomics analyses were conducted by applying the recently established Genome Instability Pipeline (GIP), which allowed us to conduct phylogenomic and principal components analyses (PCA), and to assess genomic variations at the levels of the karyotype, gene copy number, single nucleotide polymorphisms (SNPs) and small insertions/deletions (INDELs) variants. Read-depth analyses revealed a mostly disomic karyotype, with the exception of the stable tetrasomy of chromosome 31. In contrast, we identified important gene copy number variations across all isolates, which affect known virulence genes and thus were probably selected in the field. SNP-based cluster analysis of the 14 isolates revealed a core group of 12 strains that formed a tight cluster and shared 45.1 % (87 751) of SNPs, as well as two strains (M3015, Ltr_16) that clustered separately from each other and the core group, suggesting the circulation of genetically highly diverse strains in Morocco. Phylogenetic analysis, which compared our 14 L. tropica isolates against 40 published genomes of L. tropica from a diverse array of locations, confirmed the genetic difference of our Moroccan isolates from all other isolates examined. In conclusion, our results indicate potential regional variations in SNP profiles that may differentiate Moroccan L. tropica from other L. tropica strains circulating in endemic countries in the Middle East. Our report paves the way for future research with a larger number of strains that will allow correlation of diverse phenotypes (resistance to treatments, virulence) and origins (geography, host species, year of isolation) to defined genomic signals such as gene copy number variations or SNP profiles that may represent interesting biomarker candidates.

A Rare Case of Cutaneous Leishmaniasis Presenting to the Emergency Department of a Large Community Hospital.

Cutaneous leishmaniasis should be considered a possible cause of skin ulcers in a patient who has traveled abroad recently and comes to the emergency department (ED) for an assessment. Before getting an accurate diagnosis, ED assessment, and proper treatment with intravenous amphotericin B, the patient presented to several other healthcare providers. This case displays the importance of a multidisciplinary approach with consultation from infectious diseases to determine an accurate diagnosis and effective treatment plan for patients with cutaneous leishmaniasis.

Comparative Efficacy of Intralesional Chloroquine With Intralesional Meglumine Antimoniate in the Treatment of Cutaneous Leishmaniasis.

BACKGROUND AND AIM: This comparative prospective study was conducted at the Department of Dermatology, Pak Emirates Military Hospital, Rawalpindi, from August 1, 2018, to January 31, 2019 (six months). This study aimed to compare the efficacy of intralesional chloroquine with intralesional meglumine antimoniate in the treatment of cutaneous leishmaniasis. MATERIALS AND METHODS: A total of 64 patients fulfilling the inclusion criteria reporting to the Department of Dermatology, Pak Emirates Military Hospital were included in this study. Informed consent was taken and demographic data including patients' hospital registration number, age, gender, and number of lesions were noted. The subjects were randomly assigned into two groups. In group A, intralesional chloroquine was injected two times per week, and in group B, intralesional meglumine antimoniate was injected two times per week. The efficacy of both treatments was noted after eight weeks of treatment. Frequency and percentages were computed for qualitative variables like gender and number of lesions. Mean±standard deviation was presented for quantitative variables like age. Analysis was done to compare the proportion of both groups. Chi-square test was applied to compare the efficacy of both groups, p≤0.05 was taken as significant. RESULTS: In this study, the mean age of patients was 29.69±08.95 years. There were 63 (98.44%) males and one (1.56%) female. In this study, efficacy was achieved in six (18.8%) patients in group A, while in 17 (53.1%) patients in group B. This difference was statistically significant, i.e., p=0.004. CONCLUSION: This study concluded that intralesional meglumine antimoniate is more effective in treating cutaneous leishmaniasis than intralesional chloroquine.

Complex cutaneous leishmaniasis in a pediatric patient.

Cutaneous leishmaniasis (CL), a parasitic infection caused by Leishmania protozoa and transmitted by sandfly bites, can be classified into Old World and New World subtypes. We report a case of a 2-year-old female who developed complex CL after travel to Panama. Ultimately, successful treatment required two rounds of liposomal amphotericin B. We report this case for its challenging clinical course and management.

Surge in Imported Cutaneous Leishmaniasis Cases from Mexico in 2023: A Case Series.

In Europe, American cutaneous leishmaniasis caused by Leishmania mexicana is a rare imported disease. A series of six cases in 2023 is a noteworthy escalation at our institutions compared to the past two decades. This surge is likely linked to an increase of cases and environmental changes in South-Eastern Mexico.

Environmental and socio-demographic factors associated with cutaneous leishmaniasis in district Khyber, Pakistan; alarming spread of the disease to new foci.

Cutaneous leishmaniasis (CL) is a sand fly-borne infection of significant public health concern in Pakistan (endemic for CL). This study aimed to scrutinize the environmental and socio-economic risk factors for CL in district Khyber (located on Pak-Afghan border), Pakistan. Clinically confirmed 2881 CL case records for (January-December) 2017 and 2020 were obtained from district hospital. In addition, a questionnaire for CL risk factors assessment was administered to 525 households (175 in 2017 and 350 in 2020) in 40 villages throughout the district in a household survey. Higher number of CL cases were recorded in 2020 (N = 1824 belonging to 90 villages) compared to 2017 (N = 1057 from 42 villages). Highest number of CL patient cases were recorded in tehsil Jamrud (N = 2248, 39.01 %), followed by Landi Kotal (N = 398, 6.91 %) and Bara (N = 235, 4.08 %). Records showed higher number of CL cases in males (N = 1,659, 57.58 %) compared to females (N = 1,222, 42.41 %). In 2017 and 2020 the disease burden peaked in January. GIS-based spatial analyses of hospital records revealed that CL cases were abundant at 294-1,916 m elevation, in agriculture and range lands. Univariate logistic regression model analysis of risk factors suggested that higher education, family size >15, knowledge of CL, having family member/s that suffered from CL in the past, knowledge about biting time of sand flies, use of mosquito spray, presence of Afghan refugees in the village/s and living in mud-made houses increased the risk of acquiring CL. The multivariable logistic regression model showed none of the risk factors to be statistically significant. Findings of the study are crucial towards effective and targeted control of CL in district Khyber.

Combination of the Topical Photodynamic Therapy of Chloroaluminum Phthalocyanine Liposomes with Fexinidazole Oral Self-Emulsifying System as a New Strategy for Cutaneous Leishmaniasis Treatment.

Cutaneous leishmaniasis (CL) is a neglected tropical disease. The treatment is restricted to drugs, such as meglumine antimoniate and amphotericin B, that exhibit toxic effects, high cost, long-term treatment, and limited efficacy. The development of new alternative therapies, including the identification of effective drugs for the topical and oral treatment of CL, is of great interest. In this sense, a combination of topical photodynamic therapy (PDT) with chloroaluminum phthalocyanine liposomes (Lip-ClAlPc) and the oral administration of a self-emulsifying drug delivery system containing fexinidazole (SEDDS-FEX) emerges as a new strategy. The aim of the present study was to prepare, characterize, and evaluate the efficacy of combined therapy with Lip-ClAlPc and SEDDS-FEX in the experimental treatment of Leishmania (Leishmania) major. Lip-ClAlPc and SEDDS-FEX were prepared, and the antileishmanial efficacy study was conducted with the following groups: 1. Lip-ClAlPc (0.05 mL); 2. SEDDS-FEX (50 mg/kg/day); 3. Lip-ClAlPc (0.05 mL)+SEDDS-FEX (50 mg/kg/day) combination; 4. FEX suspension (50 mg/kg/day); and 5. control (untreated). BALB/c mice received 10 sessions of topical Lip-ClAlPc on alternate days and 20 consecutive days of SEDDS-FEX or FEX oral suspension. Therapeutical efficacy was evaluated via the parasite burden (limiting-dilution assay), lesion size (mm), healing of the lesion, and histological analyses. Lip-ClAlPc and SEDDS-FEX presented physicochemical characteristics that are compatible with the administration routes used in the treatments. Lip-ClAlPc+SEDDS-FEX led to a significant reduction in the parasitic burden in the lesion and spleen when compared to the control group (p < 0.05) and the complete healing of the lesion in 43% of animals. The Lip-ClAlPc+SEDDS-FEX combination may be promising for the treatment of CL caused by L. major.

Geriatric cutaneous leishmaniasis: a retrospective analysis of 622 cases.

BACKGROUND: Cutaneous leishmaniasis (CL) is most common in childhood because children are exposed to the parasite early and, unlike adults, do not have immunity to CL. Since CL is less common in geriatric patients, clinical and epidemiological data in this age group are limited. This study aims to compare the clinical and demographic characteristics of geriatric patients diagnosed with CL with young patients. METHODS: In this retrospective study, 622 patients aged 65 and over and 6350 patients aged 19-64, who applied to Sanliurfa Oriental Boil Diagnosis and Treatment Center between January 2013 and February 2024 and were diagnosed with CL by parasitological examination, were included. Clinical and demographic characteristics of patients diagnosed with CL, such as age, gender, location of the lesion, lesion size, duration of the lesion, and treatments applied due to the diagnosis of CL, were recorded. Clinical and demographic characteristics of geriatric and young patients were compared. RESULTS: The mean age of elderly CL cases was 72.95 ± 6.54 years, and 65.2% were female. The most common clinical forms were ulcers (51.9%) and plaques (41%), respectively, in young and elderly patients. The most common locations of the lesions were upper limbs (54.8%) in all patients. The most preferred treatment method was intralesional (IL) meglumine antimoniate (MA) treatment (98.3%) in all patients. There were no difference between young and elderly CL cases in terms of mean number of lesions, average lesion duration, average lesion size, lesion location, clinical forms of lesions, and treatments options (P > 0.05). CONCLUSIONS: Based on the results of our study, it can be said that the clinical and demographic characteristics of CL are similar in young and old patients and systemic MA treatment shows similar clinical benefit in both age groups. In addition, it can be said that systemic MA therapy can be used safely in young patients and elderly patients without contraindications. IL MA therapy can be used in elderly patients where systemic MA therapy is contraindicated.

Ecology and Infection Status of Sand Flies in Rural and Urban Cutaneous Leishmaniasis Endemic Areas in Northwest Ethiopia.

Cutaneous leishmaniasis (CL) caused by Leishmania aethiopica is transmitted by Phlebotomus longipes in northern Ethiopia. No studies have been conducted to investigate the transmission dynamics of CL, despite its high endemicity in both rural and urban settings. Evidence on the ecology and behavior of the vector from this area are required to develop integrated disease control strategies. Sand flies were collected in the dry and wet seasons in 2021 in CL-endemic rural Gindmeteaye and urban Addis-Alem in northwest Ethiopia. Trapping was performed with sticky and Centers for Disease Control and Prevention (CDC) light traps in three habitats, including inside patients' houses, peridomestic areasand in caves/rocky areas. Sand flies were morphologically identified to species level. Female Phlebotomus species were categorized according to blood feeding status and tested by spliced-leader (SL-) ribonucleic acid (RNA) polymerase chain reaction (PCR) to screen for Leishmania infection. Of 1161 sand flies, the majority (77%) were P. longipes, six (0.5%) were P. orientalis and the remaining were Sergentomyia. The abundance of the 430 female P. longipes was significantly linked to seasonality (p < 0.001), with the majority in the dry season occurring in the outdoor rocky (37%) and peridomestic (34%) sites, while, in the wet season, most (62%) were captured indoors. This seasonality was more pronounced in rural Gindmeteaye, where housing construction is poor. The number of blood-fed and gravid P. longipes was significantly higher in the wet (31%; 22%), compared to the dry season (13%; 8%), and their proportion was highest indoors. Eighteen (4%) female P. longipes were Leishmania positive, with highest infection prevalence in caves (7% compared to 3% indoors, p = 0.022), and in the dry season (6%, p < 0.001). Phlebotomus orientalis specimens were all captured in May in rural Gindmeteaye, five indoors and one in a peridomestic site. Further research should be conducted to investigate the absolute contribution of humans and indoor transmission to the transmission cycle of CL. Inhabitants of endemic villages should be made aware that evening outdoor activities near caves may increase their exposure to infectious sand flies. Whether P. orientalis can breed and become infected at high altitudes should be further studied.

Immunization with centrin-Deficient Leishmania braziliensis Does Not Protect against Homologous Challenge.

Immunization with various Leishmania species lacking centrin induces robust immunity against a homologous and heterologous virulent challenge, making centrin mutants a putative candidate for a leishmaniasis vaccine. Centrin is a calcium-binding cytoskeletal protein involved in centrosome duplication in higher eukaryotes and Leishmania spp. lacking centrin are unable to replicate in vivo and are non-pathogenic. We developed a centrin-deficient Leishmania braziliensis (LbCen-/-) cell line and confirmed its impaired survival following phagocytosis by macrophages. Upon experimental inoculation into BALB/c mice, LbCen-/- failed to induce lesions and parasites were rapidly eliminated. The immune response following inoculation with LbCen-/- was characterized by a mixed IFN-γ, IL-4, and IL-10 response and did not confer protection against L. braziliensis infection, distinct from L. major, L. donovani, and L mexicana centrin-deficient mutants. A prime-boost strategy also did not lead to a protective immune response against homologous challenge. On the contrary, immunization with centrin-deficient L. donovani (LdonCen-/-) cross-protected against L. braziliensis challenge, illustrating the ability of LdonCen-/- to induce the Th1-dominant protective immunity needed for leishmaniasis control. In conclusion, while centrin deficiency in L. braziliensis causes attenuation of virulence, and disrupts the ability to cause disease, it fails to stimulate a protective immune response.

In Situ versus Systemic Immune Response in the Pathogenesis of Cutaneous Leishmaniasis.

The role of the immune response in the pathogenesis of cutaneous leishmaniasis (CL) due to Leishmania (Viannia) braziliensis is predominantly carried out via blood cells. Here, we evaluate whether cytokine production by peripheral blood mononuclear cells (PBMCs) reflects what has been documented at the lesion site. The participants included 22 CL patients diagnosed with a positive PCR. PBMCs were stimulated for 72 h with a soluble leishmania antigen (SLA). Biopsies obtained from the edge of the ulcers were incubated for the same period. Cytokines in supernatants were assessed via ELISA. TNF, IL-1ß, IL-6, IL-17, and granzyme B (GzmB) were higher in the supernatants of biopsies than in PBMCs, but IFN-γ was higher in the supernatants of PBMCs than in biopsies. There was a positive correlation between IFN-γ and TNF in PBMCs, and an inverse correlation between TNF and IL-10 in the cells from the lesion site. A strong correlation between IL-1ß, IL-17, and GzmB was observed in the biopsies, and a positive correlation was detected between these cytokines and the lesion size. Our results indicate that the immune response in L. braziliensis lesions is different from that observed in peripheral blood, and our data suggest that in addition to IL-1ß and GzmB, IL-17 participates in the pathology of CL.

In silico prediction of CD8+ and CD4+ T cell epitopes in Leishmania major proteome: Using immunoinformatics.

The leishmaniases are NDTs (neglected tropical diseases) that affect people all over the world. They are brought on by protozoans from the genus Leishmania and disseminated by phlebotomine flies that are afflicted with the disease. The best option to manage and lower the incidence of these diseases has been thought by the creation of a safe and effective vaccination. This research used an in silico based mining approach to look for high potential epitopes that might bind to MHC Class I and MHC Class II molecules (mainly; HLA-A*02:01 & HLA-DRB1*03:01) from human population in order to promote vaccine development. Based on the presence of signal peptides, GPI anchors, antigenicity predictions, and a subtractive proteomic technique, we have screened 17 putative antigenic proteins from the 8083 total proteins of L. major. After that thorough immunogenic epitope prediction were done using IEDB-AR tools. We isolated five immunogenic epitopes (three 9-mer & two 15-mer) from five antigenic proteins through docking and MD simulation analysis. Finally, these five anticipated epitopes, viz., TLPEIPVNV, ELMAPVFGL, TLAAAVALL, NSINIRLDGVTSAGF and NVPLVVDASSLFRVA have considerably stronger binding potential with their respective alleles and may trigger immunological responses. The goal of this work was to identify MHC restricted epitopes for CD8+ and CD4+ T cells activation using immunoinformatics in order to identify potential vaccine candidates against L. major parasites.

Assessment of Autophagy in Leishmania Parasites.

Leishmaniasis is a neglected tropical disease caused by numerous species of Leishmania parasites, including Leishmania major. The parasite is transmitted by several species of sandfly vectors and infects myeloid cells leading to a myriad of inflammatory responses, immune dysregulations, and disease manifestations. Every cell undergoes autophagy, a self-regulated degradative process that permits the cells to recycle damaged or worn-out organelles in order to maintain cellular health and homeostasis. Studies have shown that Leishmania modulates their host cell autophagic machinery and there are indications that the parasite-specific autophagic processes may be valuable for parasite virulence and survival. However, the role of autophagy in Leishmania is inconclusive because of the limited tools available to study the Leishmania-specific autophagic machinery. Here, we describe methods to study and definitively confirm autophagy in Leishmania major. Transmission electron microscopy (TEM) allowed us to visualize Leishmania autophagosomes, especially those containing damaged mitochondrial content, as well as dividing mitochondria with ongoing fusion/fission processes. Flow cytometry enabled us to identify the amount of acridine orange dye accumulating in the acidic vacuolar compartments in Leishmania major by detecting fluorescence in the red laser when autophagic inhibitors or enhancers were included. These methods will advance studies that aim to understand autophagic regulation in Leishmania parasites that could provide insights into developing improved therapeutic targets against leishmaniasis.