Ultra-performance liquid chromatography-Quadrupole time-of-flight tandem mass spectrometry-based metabolite profiling, quality evaluation, and marker analysis of Trachyspermum ammi (L.) Sprague by high-performance thin-layer chromatography.

Trachyspermum ammi (L.) Sprague (Apiaceae), commonly known as "Ajwain" is distributed throughout India. Ajwain fruits contain fiber, carbohydrates, phenolic acids, flavonoids, and tannins. The fruits also yield a small amount of essential oil, with Thymol as the principal constituent. Ajwain has various pharmacological activities like anti-leishmanial, antimicrobial, cytotoxic, antispasmodic, nematocidal, and anthelmintic. The fruits are of high therapeutic value; thus, it becomes quite essential to evaluate the quality of Trachyspermum ammi (L.) Sprague to authenticate and ensure its therapeutic and nutritional properties. The ethyl acetate fraction of Trachyspermum ammi (L.) Sprague fruits exhibited the highest total phenolic and flavonoid content values of 149.55 ± 1.19 mg rutin equivalent and 682.85 ± 3.68 mg gallic acid equivalent, respectively. Metabolite profiling of the ethyl acetate fraction using ultra-performance liquid chromatography-quadrupole time-of-flight tandem mass spectrometry analysis resulted in identifying 19 phytomolecules. A validated high-performance thin-layer chromatography method was developed to quantify standard phytomolecules in the ethyl acetate fraction. The highest and lowest percentages of phytomarker were found to be caffeic acid (5.51% ± 0.16% w/w) and gallic acid (1.29% ± 0.09% w/w), respectively. This validated rapid, accurate, and precise method for standardization of Trachyspermum ammi (L.) Sprague will be beneficial for its quality evaluation as well as the derived products.

Preventive as well as therapeutic significances of linoleic acid in the containment of Leishmania donovani infection.

People suffering from malnutrition show compromised levels of ω-6 fatty acid and malnutrition is frequently observed among visceral leishmaniasis (VL) patients as disease inflicts primarily the socioeconomic destitute communities. Dietary linoleic acid (LA, 18:2; ω-6 fatty acid) is the principal source of essential fatty acid and its derivatives i.e. eicosanoids possess immune-modulatory activities. However, its role in VL is not yet established. LA was measured in VL human subjects (serum) as well as in Leishmania(L.)donovani infected hamsters (serum and visceral organs). Organ-specific mRNA expressions of various enzymes of the LA metabolic pathway were measured in visceral organs of infected hamsters. Our findings showed a decrease in the concentrations of LA in the serum samples of VL patients, suggesting malnutrition among these patients. However, in L. donovani infected hamsters, its level was not altered in the early infection (15 days) and then increased at late infection (60 days). Importantly, the supplementation of LA restored the Th-1 type of immune response and significantly reduced the parasite load within infected macrophages in vitro. This protective response of LA was mediated through 5-lipoxygenase pathway not via the cyclooxygenase pathway. Preventive usage of LA to mϕ followed by L. donovani infection also showed the strengthening of Th-1 immune response and significantly fewer parasite loads. Our findings demonstrate the protective role of LA in the containment of the parasite load. Incorporating LA rich oils in daily food habits across highly inflicted regions may be a significant advancement towards the eradication of the disease.

Comparison Between Immuno-Clinicopathological Features of Experimental and Human Visceral Leishmaniasis by Leishmania donovani.

BACKGROUND: Current understanding of visceral leishmaniasis (VL) depends upon the experimental model. Different species of mouse and hamster have been used as model for VL. It is already evident that the mouse model of VL is not a true reflection of the pathology of human visceral leishmaniasis (HuVL). On the other hand, hamster is reported to be a better model of VL to study the progressive as well as chronic pathology of the disease. OBJECTIVE: To compare immuno-clinicopathological features of experimental VL (ExVL) and HuVL by Leishmania donovani. METHODS: Hamsters were infected (15 and 60 days) and their immunological, clinical and biochemical parameters were compared with the cases of HuVL. RESULTS: Splenomegaly and hepatomegaly were observed in infected hamster post-infection, which are hallmarks of symptomatic HuVL cases. Clinical, biochemical and pathological manifestations of infected hamsters were consistent with that of HuVL cases, except parameters such as body weight, uric acid, alkaline phosphatase and random glucose. The absence of clear dichotomy between pro- and anti-inflammatory cytokines was also observed after infection at different sites of infection. CONCLUSION: Our results suggest that the golden hamster (Mesocricetus auratus), infected via the intracardiac route, constitutes a very good model for the study of experimental Leishmania donovani infections. However, certain differences in clinical presentations of infected hamsters (via intracardiac route) with HuVL suggest further optimization of this animal model like route of infection such as intradermal, which is more close to natural infection.

Leishmania donovani reduces the levels of retinoic acid-synthesizing enzymes in infected macrophages and favoring its own survival.

People suffering from malnutrition become susceptible to the infection like Leishmania sp., as it results in a compromised immune response. Retinoic acid (RA), an important constituent of nutrition, shows an immune-modulatory activity. However, its role in the containment of infection is not yet ascertained, particularly in case of visceral leishmaniasis (VL). VL patients (n = 10) and healthy endemic controls (n = 9) were recruited to measure the serum levels of RA. An in vitro model of Leishmania infection using the murine mφ cell line J774.1 was used to investigate the RA-synthesizing enzymes (RALDH-1 and RALDH-2). Parasite loads among infected mφ were measured by quantitative expression of kDNA in the presence of an inhibitor of the RALDH-2 enzyme. We found a significant decrease in the serum levels of RA in VL cases. Importantly, we observed decreased levels of RALDH-1 and RALDH-2 among L. donovani-infected mφ along with simultaneous decrease as well as increase in the Th-1 and Th-2-associated factors, respectively. Furthermore, the pretreatment of mφ with an RALDH-2 inhibitor improved parasite in vitro infection. Our findings show impaired RA pathway among infected mφ and indicate that an intact RA pathway is critical for anti-Leishmania immune response. Graphical abstract ᅟ.

Decrease in the Frequency of Circulating CD56+CD161+ NK Cells in Human Visceral Leishmaniasis.

BACKGROUND: Natural Killer (NK) cell plays an important role in the innate immune system and is known to produce IFN-γ at an early stage of infection that is essential to eliminate intracellular infection like Leishmania spp. It is already established that Leishmania parasite inhibits the activity of NK cells, avoiding the encounter with the early innate immune response. This, in turn, favors establishment and further dissemination of the infection. METHODS: In the present study, we have tried to measure the frequency of different phenotypic subsets of NK cells among visceral leishmaniasis (VL) patients. RESULTS: We have phenotyped three distinct three distinct subsets (CD56-CD161+, CD56+CD161-, and CD56+CD161+) of NK (CD3-) cell using their specific markers CD161 and CD56. CONCLUSION: Interestingly, we observed selective loss of CD56+CD161+ subset of circulating NK (CD3-) cells. Importantly, the other subsets (i.e., CD56-CD161+ and CD56+CD161-) of circulating NK cells remain unaffected as compared with healthy subjects.

Cedrus deodara: In vitro antileishmanial efficacy & immumomodulatory activity.

BACKGROUND & OBJECTIVES: The existing antileishmanial drugs for complete cure of visceral leishmaniasis (kala-azar) are limited. The available drugs are either toxic or less effective leading to disease relapse or conversion to post-kala-azar dermal leishmaniasis. Several herbal extracts have been shown to have antileishmanial activity, but a herbal drug may not always be safe. In the present study, the extract of Cedrus deodara leaves has been standardized and tested for immunomodulatory antileishmanial activities. METHODS: The extracts of C. deodara leaves with different solvents such as benzene, chloroform, ethyl acetate and methanol were made by soxhlation process. Solvents were removed under reduced pressure and temperature using rotary evaporator. The antileishmanial bioassay test was performed with in vitro maintained parasites. Immunomodulatory activity of different extracts was tested by flow cytometry. Standardization of the effective fraction was performed with Linalool as a marker compound through reverse-phase high-performance liquid chromatography. RESULTS: The extract with the use of benzene solvent showed strong antileishmanial activities within a dose 25-200 µg/ml culture with non-significant haemolytic activities and significant immunomodulant activities against the host cells. Linalool was found to be 1.29 per cent in the effective extract of C. deodara. INTERPRETATION & CONCLUSIONS: The antileishmanial activity of C. deodara, as assessed by bioassay testing on. LEISHMANIA DONOVANI: parasites and immunomodulatory effect of benzene extract of leaves on host cells indicated that it might be a potential new safe therapeutic target to cure the visceral leishmaniasis.

Efficacy and safety of amphotericin B emulsion versus liposomal formulation in Indian patients with visceral leishmaniasis: a randomized, open-label study.

BACKGROUND: India is home to 60% of the total global visceral leishmaniasis (VL) population. Use of long-term oral (e.g. miltefosine) and parenteral drugs, considered the mainstay for treatment of VL, is now faced with increased resistance, decreased efficacy, low compliance and safety issues. The authors evaluated the efficacy and safety of an alternate treatment option, i.e. single infusion of preformed amphotericin B (AmB) lipid emulsion (ABLE) in comparison with that of liposomal formulation (LAmB). METHODS: In this multicentric, open-label study, 500 patients with VL were randomly assigned in a 3:1 ratio to receive 15 mg/kg single infusion of either ABLE (N = 376) or LAmB (N = 124). Initial cure (Day 30/45), clinical improvement (Day 30) and long term definitive cure (Day 180) were assessed. FINDINGS: A total of 326 (86.7%) patients in the ABLE group and 122 (98.4%) patients in the LAmB group completed the study. Initial cure was achieved by 95.9% of patients in the ABLE group compared to 100% in the LAmB group (p = 0.028; 95% CI: -0.0663, -0.0150). Clinical improvement was comparable between treatments (ABLE: 98.9% vs. LAmB: 98.4%). Definitive cure was achieved in 85.9% with ABLE compared to 98.4% with LAmB. Infusion-related pyrexia (37.2% vs. 32.3%) and chills (18.4% vs. 18.5%) were comparable between ABLE and LAmB, respectively. Treatment-related serious adverse events were fewer in ABLE (0.3%) compared to LAmB (1.6%). Two deaths occurred in the ABLE group, of which one was probably related to the study drug. Nephrotoxicity and hepatotoxicity was not observed in either group. CONCLUSIONS: ABLE 15 mg/kg single infusion had favorable efficacy and was well tolerated. Considering the demographic profile of the population in this region, a single dose treatment offers advantages in terms of compliance, cost and applicability. TRIAL REGISTRATION: www.clinicaltrials.gov NCT00876824.

Early activated Th-1 type and dominantly diverse natural killer T (CD3⁺CD161⁺Vα24⁻) cells in bone marrow among visceral leishmaniasis patients.

Lipid antigens of Leishmania donovani-like lipophosphoglycans (LPG) are demonstrated to be a potent ligand for natural killer T (NKT) cell activation. Little is known about the phenotype or function of these cells and their trafficking pattern to the bone marrow (BM) of visceral leishmaniasis (VL) patients. Their precise role in humans still requires pathological validation. The study included 42 parasitologically confirmed patients (mean age 24.80±16.26 years; range 3-70 years; 25 males and 17 females), 33 healthy contact subjects (family/non-family members) and normal BM specimens (NBM; n=9). Enumeration of NKT cells and quantification of parasites (before and after therapy) were performed for the recruited patients. Results established that non-CD1d restricted, diverse cells are the dominant population among resident but not enriched NKT (CD3⁺CD161⁺) cells at the disease site (BM). Expression profiles for various markers are indicative of their early activated (CD69⁺, CD62L(low), CD11a(high)) CCR5⁺ phenotype at the BM. Functionally, BM-derived NKT cells were dominantly producing IFN-γ in response to L. donovani antigen in vitro. Given these observations, these data indicate that CD3⁺CD161⁺ diverse NKT cells are heterogeneous in function and of the dominant Th-1 phenotype at the disease site.

Effectiveness and feasibility of active and passive case detection in the visceral leishmaniasis elimination initiative in India, Bangladesh, and Nepal.

This study analyzed the effectiveness of active case detection (ACD) for new visceral leishmaniasis (VL) cases. ACD detection was carried out using house to house screening in Bangladesh and India and by neighborhood screening around index cases in Nepal. The percent increase of new VL cases through ACD compared to PCD was 6.7-17.1% in India; 38.8% in Nepal; and 60% in Bangladesh. The screening effort was high in India and Bangladesh (house to house screening) compared to Nepal (index case screening). The additional cost per new VL case detected varied: $50 to $106 in India; $172 in Bangladesh; $262 in Nepal depending on the type of screening staff, transport and training costs. The estimated annual VL incidence in the ACD arm ranged from 315-383 in India; 109 in Bangladesh, and 43 per 100,000 in Nepal. The additional effort and cost rises as disease incidence declines or PCD improves.