IFN-λ3 is induced by Leishmania donovani and can inhibit parasite growth in cell line models but not in the mouse model, while it shows a significant association with leishmaniasis in humans.

The intracellular protozoan parasite Leishmania donovani causes debilitating human diseases that involve visceral and dermal manifestations. Type 3 interferons (IFNs), also referred to as lambda IFNs (IFNL, IFN-L, or IFN-λ), are known to play protective roles against intracellular pathogens at the epithelial surfaces. Herein, we show that L. donovani induces IFN-λ3 in human as well as mouse cell line-derived macrophages. Interestingly, IFN-λ3 treatment significantly decreased parasite load in infected cells, mainly by increasing reactive oxygen species production. Microscopic examination showed that IFN-λ3 inhibited uptake but not replication, while the phagocytic ability of the cells was not affected. This was confirmed by experiments that showed that IFN-λ3 could decrease parasite load only when added to the medium at earlier time points, either during or soon after parasite uptake, but had no effect on parasite load when added at 24 h post-infection, suggesting that an early event during parasite uptake was targeted. Furthermore, the parasites could overcome the inhibitory effect of IFN-λ3, which was added at earlier time points, within 2-3 days post-infection. BALB/c mice treated with IFN-λ3 before infection led to a significant increase in expression of IL-4 and ARG1 post-infection in the spleen and liver, respectively, and to different pathological changes, especially in the liver, but not to changes in parasite load. Treatment with IFN-λ3 during infection did not decrease the parasite load in the spleen either. However, IFN-λ3 was significantly increased in the sera of visceral leishmaniasis patients, and the IFNL genetic variant rs12979860 was significantly associated with susceptibility to leishmaniasis.

Status of B-Lymphocyte Subsets and Their Homing Markers in Patients With Post-Kala-Azar Dermal Leishmaniasis.

In visceral leishmaniasis, the Type II helper T cell predominance results in B cell modulation and enhancement of anti-leishmanial IgG. However, information regarding its dermal sequel, post-kala-azar dermal leishmaniasis (PKDL), remains limited. Accordingly, this study aimed to elucidate the B cell-mediated antibody-dependent/independent immune profiles of PKDL patients. In the peripheral blood of PKDL patients, immunophenotyping of B cell subsets was performed by flow cytometry and by immunohistochemistry at lesional sites. The functionality of B cells was assessed in terms of skin IgG by immunofluorescence, while the circulating levels of B cell chemoattractants (CCL20, CXCL13, CCL17, CCL22, CCL19, CCL27, CXCL9, CXCL10 and CXCL11) were evaluated by a multiplex assay. In patients with PKDL as compared with healthy controls, there was a significant decrease in pan CD19+ B cells. However, within the CD19+ B cell population, there was a significantly raised proportion of switched memory B cells (CD19+IgD-CD27+) and plasma cells (CD19+IgD-CD38+CD27+). This was corroborated at lesional sites where a higher expression of CD20+ B cells and CD138+ plasma cells was evident; they were Ki67 negative and demonstrated a raised IgG. The circulating levels of B cell chemoattractants were raised and correlated positively with lesional CD20+ B cells. The increased levels of B cell homing markers possibly accounted for their enhanced presence at the lesional sites. There was a high proportion of plasma cells, which accounted for the increased presence of IgG that possibly facilitated parasite persistence and disease progression.

Portable smartphone-based molecular test for rapid detection of Leishmania spp.

PURPOSE: Leishmaniasis, caused by the parasite of the genus Leishmania, is a neglected tropical disease which is endemic in more than 60 countries. In South-East Asia, Brazil, and East Africa, it mainly occurs as kala-azar (visceral leishmaniasis, VL), and subsequently as post kala-azar dermal leishmaniasis (PKDL) in a smaller portion of cases. As stated per WHO roadmap, accessibility to accurate diagnostic methods is an essential step to achieve elimination. This study aimed to test the accuracy of a portable minoo device, a small battery-driven, multi-use fluorimeter operating with isothermal technology for molecular diagnosis of VL and PKDL. METHODS: Fluorescence data measured by the device within 20 min are reported back to the mobile application (or app) via Bluetooth and onward via the internet to a backend. This allows anonymous analysis and storage of the test data. The test result is immediately returned to the app displaying it to the user. RESULTS: The limit of detection was 11.2 genome copies (95% CI) as determined by screening a tenfold dilution range of whole Leishmania donovani genomes using isothermal recombinase polymerase amplification (RPA). Pathogens considered for differential diagnosis were tested and no cross-reactivity was observed. For its diagnostic performance, DNA extracted from 170 VL and PKDL cases, comprising peripheral blood samples (VL, n = 96) and skin biopsies (PKDL, n = 74) from India (n = 108) and Bangladesh (n = 62), was screened. Clinical sensitivity and specificity were 88% and 91%, respectively. CONCLUSION: Minoo devices can offer a convenient, cheaper alternative to other molecular diagnostics. Its easy handling makes it ideal for use in low-resource settings to identify parasite burden.

Dermal microdialysis: A method to determine drug levels in the skin of patients with Post kala-azar dermal leishmaniasis (PKDL).

OBJECTIVES: Post-kala-azar-dermal leishmaniasis (PKDL) is an infectious skin disease that occurs as sequela of visceral leishmaniasis (VL) and causes cutaneous lesions on the face and other exposed body parts. While the first-line drug miltefosine is typically used for 28 days to treat VL, 12 weeks of therapy is required for PKDL, highlighting the need to evaluate the extent of drug penetration at the dermal site of infection. In this proof-of-concept study, we demonstrate the use of a minimally invasive sampling technique called microdialysis to measure dermal drug exposure in a PKDL patient, providing a tool for the optimization of treatment regimens. METHODS AND MATERIALS: One PKDL patient receiving treatment with miltefosine (50 mg twice daily for 12 weeks) was recruited to this proof-of-concept study and consented to undergo dermal microdialysis. Briefly, a µDialysis Linear Catheter 66 for skin and muscle, a probe with a semi-permeable membrane, was inserted in the dermis. A perfusate (a drug-free physiological solution) was pumped through the probe at a low flow rate, allowing miltefosine present in the dermis to cross the membrane and be collected in the dialysates over time. Protein-free (dialysates) and total (blood and skin biopsies) drug concentrations were analysed using LC-MS/MS. RESULTS: and conclusions: Using microdialysis, protein-free miltefosine drug concentrations could be detected in the infected dermis over time (Cmax ≈ 450 ng/ml). This clinical proof-of-concept study thus illustrates the potential of dermal microdialysis as a minimally invasive alternative to invasive skin biopsies to quantify drug concentrations directly at the pharmacological site of action in PKDL.

Does immune dysregulation contribute towards development of hypopigmentation in Indian post kala-azar dermal leishmaniasis?

Post kala-azar dermal leishmaniasis (PKDL), a sequel of apparently cured visceral leishmaniasis (VL) presents with papulonodular (polymorphic) or hypopigmented lesions (macular) and is the proposed disease reservoir. As hypopigmentation appears consistently in PKDL, especially the macular form, this study aimed to delineate immune factors that singly or in combination could contribute towards this hypopigmentation. At lesional sites, the presence of melanocytes and CD8+ T-cells was assessed by immunohistochemistry and mRNA expression of melanogenic markers (tyrosinase, tyrosinase-related protein-1 and MITF) by droplet digital PCR, while plasma levels of cytokines and chemokines were measured by a multiplex assay. In comparison with skin from healthy individuals, macular PKDL demonstrated a near total absence of Melan-A+ cells at dermal sites, while the polymorphic cases demonstrated a 3.2-fold decrease, along with a dramatic reduction in the expression of key enzymes related to the melanogenesis signalling pathway in both forms. The levels of circulating IFN-γ, IL-6, IL-2, IL-1ß, TNF-α and IFN-γ-inducible chemokines (CXCL9/10/11) were elevated and was accompanied by an increased lesional infiltration of CD8+ T-cells. The proportion of CD8+ T-cells correlated strongly with plasma levels of IFN-γ (r = 0.8), IL-6 (r = 0.9, p < 0.05), IL-2 (r = 0.7), TNF-α (r = 0.9, p < 0.05) and IL-1ß (r = 0.7), as also with CXCL9 (r = 0.5) and CXCL10 (r = 0.6). Taken together, the absence/reduction in Melan-A suggested hypopigmentation in PKDL was associated with the destruction of melanocytes, following the impairment of the melanogenesis pathway. Furthermore, the presence of CD8+ T-cells and an enhanced IFN-γ-associated immune milieu suggested the generation of a pro-inflammatory landscape that facilitated melanocyte dysfunction/destruction.

Attenuation of methotrexate induced hepatotoxicity by epigallocatechin 3-gallate.

Methotrexate (MTX) is currently used as first-line therapy for autoimmune diseases like rheumatoid arthritis, psoriasis, and systemic lupus erythematous. However, its use is limited by its hepatotoxic potential. Epigallocatechin-3-gallate (EGCG), an abundant catechin present in tea possesses potent antioxidant activity and effectively ameliorates oxidative stress-related disorders. This study aimed to investigate the hepatoprotective influence of EGCG in a MTX-induced rat model of hepatotoxicity. Sprague Dawley rats pretreated with EGCG (40 mg kg-1 b.w., p.o.) were administered a single dose of MTX (20 mg kg-1 b.w., i.p.) and its hepatoprotective efficacy compared with folic acid (1 mg kg-1 b.w., i.p.). On day 10, blood samples were collected to determine plasma levels of aspartate aminotransferase (AST), alanine transaminase (ALT), alkaline phosphatase (ALP), and lactate dehydrogenase (LDH), while the livers were examined for histopathogical changes along with levels of oxidative stress measured in terms of myeloperoxidase (MPO) activity, protein carbonylation (PCO), lipid peroxidation (LPO), and activities of cellular enzymatic antioxidants - superoxide dismutase (SOD), catalase (CAT), and glutathione peroxidase (GPx). MTX significantly increased the plasma levels of AST, ALT, ALP, and LDH, which were prevented by pretreatment with EGCG, and was corroborated by histopathology. Additionally, MTX-induced hepatic oxidative stress as measured by increased generation of MPO, enhanced PCO, LPO, and decreased activities of antioxidant enzymes was mitigated by pretreatment with EGCG. The amelioration of MTX-induced hepatotoxicity by EGCG endorsed the inclusion of an anti-oxidant during chronic administration of MTX.

Quantitative monitoring of experimental and human leishmaniasis employing amastigote-specific genes.

The gold standard for diagnosis of leishmaniasis is the microscopic detection of amastigotes/Leishman Donovan (LD) bodies, but its moderate sensitivity necessitates the development of molecular approaches. This study aimed to quantify in experimental animal models and human leishmaniasis the expression of amastigote-specific virulence genes, A2 and amastin by droplet digital polymerase chain reaction (ddPCR). Total RNA was isolated from L. donovani-infected hamsters or murine peritoneal macrophages and lesional biopsies from patients with post kala-azar dermal leishmaniasis (PKDL). Following cDNA conversion, EvaGreen-based ddPCR was performed using specific primers for A2 or amastin and parasite load expressed in copies per µL. Assay was optimized and the specificity of amastigote-specific A2 and amastin was confirmed. In hepatic and splenic tissues of L. donovani-infected hamsters and peritoneal macrophages, ddPCR demonstrated a greater abundance of A2 than amastin. Treatment of L. donovani-infected peritoneal macrophages with conventional anti-leishmanials, miltefosine and amphotericin B translated into a dose-dependent reduction in copies per µL of A2 and amastin, and the extrapolated IC50 was comparable with results obtained by counting LD bodies in Giemsa-stained macrophages. Similarly, in dermal biopsies of patients with PKDL, A2 and amastin were detected. Overall, monitoring of A2 by ddPCR can be an objective measure of parasite burden and potentially adaptable into a high throughput approach necessary for drug development and monitoring disease progression when the causative species is L. donovani.

First-in-class pyrido[2,3-d]pyrimidine-2,4(1H,3H)-diones against leishmaniasis and tuberculosis: Rationale, in vitro, ex vivo studies and mechanistic insights.

Pyrido[2,3-d]pyrimidine-2,4(1H,3H)-diones were synthesized, for the first time, from indole chalcones and 6-aminouracil, and their ability to inhibit leishmaniasis and tuberculosis (Tb) infections was evaluated. The in vitro antileishmanial activity against promastigotes of Leishmania donovani revealed exceptional activities of compounds 3, 12 and 13, with IC50 values ranging from 10.23 ± 1.50 to 15.58 ± 1.67 µg/ml, which is better than the IC50 value of the standard drug pentostam of 500 µg/ml. The selectivity of the compounds towards Leishmania parasites was evaluated via ex vivo studies in Swiss albino mice. The efficiency of these compounds against Tb infection was then evaluated using the in vitro anti-Tb microplate Alamar Blue assay. Five compounds, 3, 7, 8, 9 and 12, showed MIC100 values against the Mycobacterium tuberculosis H37 Rv strain at 25 µg/ml, and compound 20 yielded an MIC100 value of 50 µg/ml. Molecular modelling of these compounds highlighted interactions with binding sites of dihydrofolate reductase, pteridine reductase and thymidylate kinase, thus establishing the rationale of their pharmacological activity against both pathogens, which is consistent with the in vitro results. From the above results, it is clear that compounds 3 and 12 are promising lead candidates for Leishmania and Mycobacterium infections and may be promising for coinfections.

Status of IL-4 and IL-10 driven markers in experimental models of Visceral Leishmaniasis.

AIM: Leishmania donovani, the causative agent for visceral leishmaniasis (VL), modulates host monocytes/macrophages to ensure its survival. However, knowledge regarding the host-parasite interactions underpinning the disease remains limited. As disease progression is associated with polarization of monocytes/macrophages towards M2, which is regulated by cytokines IL-4/IL-13 and IL-10, this study evaluated the status of key IL-4- and IL-10 driven markers in experimental models of VL, as also evaluated their correlation, if any, with parasite load. METHODS: In liver and splenic tissues from L donovani-infected hamsters and BALB/c mice, the parasite burden was determined along with mRNA expression of IL-4-driven markers, that is CD206, Arginase-I, CCL17, CCL22, PPAR-γ, STAT6, KLF4, FIZZ1 and YM1 along with IL-10-driven markers, CXCL13, IL-10, TGF-ß, VDR, CCR2 and CYP27A1. RESULTS: The mRNA expression of IL-4- and IL-10-driven markers was enhanced in both models, but only in the hamster model, the splenic tissues demonstrated a positive correlation between all the IL-10-driven markers and parasite load. CONCLUSIONS: Contrary to human VL, both models demonstrated an increased expression of IL-4- and IL-10-driven markers.

Methotrexate and theaflavin-3, 3'-digallate synergistically restore the balance between apoptosis and autophagy in synovial fibroblast of RA: an ex vivo approach with cultured human RA FLS.

BACKGROUND: Imbalance between apoptosis and autophagy in fibroblast-like synoviocytes (FLS) is one of the pathogenic mechanisms responsible for their abnormal proliferation in rheumatoid arthritis (RA). Methotrexate (MTX) demonstrated limited efficacy in amending this imbalance in fluid-derived (fd)-FLS. The active compound of black tea Theaflavin 3,3'-digallate (TF3) may be effective in restoring apoptosis-autophagy imbalance in (fd)-FLS. The combined effect of MTX + TF3 upon the same is yet to be elucidated. OBJECTIVE: To evaluate the effect of MTX + TF3 on fd-FLS to induce apoptosis and inhibit autophagy through Endoplasmic Reticulum (ER) stress-mediated pathways. METHODS: FLS from synovial fluid of 11 RA and 10 osteoarthritis patients were cultured after treatment with MTX/TF3 or a combination of MTX (125 nM) and TF3(10 µM) and the following parameters were evaluated. C-reactive protein, cytokines (TNF-α, IL-6), angiogenic markers were quantified by ELISA. fd-FLS viability was determined by MTT assay and apoptosis by flow cytometry. ER stress markers were estimated by RT-PCR (IRE1A, spliced-XBP-1) and immunoblotting (Grp78, Hsp70, CHOP, HIF-1α). Immunoblot studies were done to evaluate apoptotic (Bcl-2, Bax, Caspases) and autophagic (Beclin1, LC3b, p62) proteins. RESULTS: MTX (IC25) and TF3 (IC50) both in single doses could down-regulate the levels of pro-inflammatory and angiogenic markers. Combinatorial treatment modulated autophagosomal proteins in fd-FLS and induced apoptosis by regulating ER stress response. CONCLUSION: Disruption in homeostasis between apoptosis and autophagy in fd-FLS might be an underlying phenomenon in the progression of pathophysiology in RA. Co-administration of MTX + TF3 successfully restored the homeostasis by inducing apoptosis.

The leishmanicidal activity of artemisinin is mediated by cleavage of the endoperoxide bridge and mitochondrial dysfunction.

Endoperoxides kill malaria parasites via cleavage of their endoperoxide bridge by haem or iron, leading to generation of cytotoxic oxygen-centred radicals. In view of the Leishmania parasites having a relatively compromised anti-oxidant defense and high iron content, this study aims to establish the underlying mechanism(s) accounting for the apoptotic-like death of Leishmania promastigotes by artemisinin, an endoperoxide. The formation of reactive oxygen species was confirmed by flow cytometry and was accompanied by inhibition of mitochondrial complexes I-III and II-III. However, this did not translate into a generation of mitochondrial superoxide or decrease in oxygen consumption, indicating minimal impairment of the electron transport chain. Artemisinin caused depolarization of the mitochondrial membrane along with a substantial depletion of adenosine triphosphatase (ATP), but it was not accompanied by enhancement of ATP hydrolysis. Collectively, the endoperoxide-mediated radical formation by artemisinin in Leishmania promastigotes was the key step for triggering its antileishmanial activity, leading secondarily to mitochondrial dysfunction indicating that endoperoxides represent a promising therapeutic strategy against Leishmania worthy of pharmacological consideration.

Berberine chloride mediates its antileishmanial activity by inhibiting Leishmania mitochondria.

Berberine chloride, a plant-derived isoquinoline alkaloid, has been demonstrated to have leishmanicidal activity, which is mediated by generation of a redox imbalance and depolarization of the mitochondrial membrane, resulting in a caspase-independent apoptotic-like cell death. However, its impact on mitochondrial function remains to be delineated and is the focus of this study. In UR6 promastigotes, berberine chloride demonstrated a dose-dependent increase in generation of reactive oxygen species and mitochondrial superoxide, depolarization of the mitochondrial membrane potential, a dose-dependent inhibition of mitochondrial complexes I-III and II-III, along with a substantial depletion of ATP, collectively suggesting inhibition of parasite mitochondria. Accordingly, the oxidative stress induced by berberine chloride resulting in an apoptotic-like cell death in Leishmania can be exploited as a potent chemotherapeutic strategy, mitochondria being a prime contributor.

Oncogenic potential of hepatitis B virus subgenotype D1 surpasses D3: significance in the development of hepatocellular carcinoma.

Despite widespread distribution of hepatitis B virus (HBV)-genotype D, the clinical implications of its ten subgenotypes (D1-D10) have not been well documented. Here, we have investigated the impact of two major circulating HBV/D subgenotypes, D1 and D3 in Eastern India towards pathogenesis of liver disease progression to hepatocellular carcinoma (HCC). HBV subgenotypes were determined using full-length genome sequences of HBV isolates from patients with chronic hepatitis B (CHB), liver cirrhosis (LC) and HCC. Impact of D1 and D3 on viral lifecycle and disease progression was assessed by several in vitro assays. Phylogenetic tree analysis revealed that HBV/D1 and HBV/D3 were the two predominating HBV subgenotypes circulating in Eastern India. Interestingly, the frequency of patients infected with HBV/D1 was noticed progressively rising from CHB to HCC through LC while the increasing frequency of HBV/D3 declined suddenly in HCC implicating HBV/D1 might have greater oncogenic potential than HBV/D3. Similar to higher viral load noted in HCC patients infected with HBV/D1 than HBV/D3, the larger amount of intracellular/extracellular viral DNA and secreted HBsAg levels in transfected cell lines also implicated that HBV/D1 might replicate faster than HBV/D3. Again, higher expression of marker genes related to endoplasmic reticulum stress, epithelial-mesenchymal transition, DNA double strand breaks, angiogenesis etc. and faster rate of cellular migration and anchorage independent growth cumulatively suggested that compared to HBV/D3, HBV/D1 generates more liver injuries which eventually culminates into HCC. Therefore, our results highlight the importance of determination of subgenotypes of HBV in CHB patients, so that high-risk individual can be monitor periodically that may help to detect HCC at early stages.

Monitoring of Parasite Kinetics in Indian Post-Kala-azar Dermal Leishmaniasis.

Background: The potential reservoirs of leishmaniasis in South Asia include relapsed cases of visceral leishmaniasis (VL), patients with post-kala-azar dermal leishmaniasis (PKDL), and an asymptomatically infected population. Therefore, assessment of cure in terms of parasite clearance, early detection of PKDL, and asymptomatic VL are pivotal for ensuring elimination. This study aimed to monitor the efficacy of miltefosine and liposomal amphotericin B (LAmB) in PKDL based on parasite load. Methods: Patients with PKDL were recruited from the dermatology outpatient departments or during active field surveys. Skin biopsies were collected at disease presentation, immediately at the end of treatment, and 6 months later. The presence of parasite DNA was assessed by internal transcribed spacer-1 polymerase chain reaction, and quantified by amplification of parasite kinetoplastid DNA. Results: At disease presentation (n = 184), the median parasite load was 5229 (interquartile range [IQR], 896-50898)/µg genomic DNA (gDNA). Miltefosine cleared the parasites to <10 in the macular (n = 17) and polymorphic (n = 21) variants, and remained so up to 6 months later (<10 parasites). LAmB reduced the parasite burden substantially in macular (n = 34; 2128 [IQR, 544-5763]/µg gDNA) and polymorphic PKDL (n = 36; 2541 [IQR, 650-9073]/µg gDNA). Importantly, in patients who returned 6 months later (n = 38), a resurgence of parasites was evident, as the parasites increased to 5665 (IQR, 1840-17067)/µg gDNA. Conclusions: This study established that quantifying parasite load is an effective approach for monitoring patients with PKDL, wherein miltefosine demonstrated near-total parasite clearance and resolution of symptoms. However, in cases treated with LAmB, the persistence of parasites suggested treatment inadequacy. This needs immediate redressal in view of the leishmaniasis elimination program targeted for 2020.

Progression of histopathological and behavioral abnormalities following mild traumatic brain injury in the male ferret.

White matter damage is an important consequence of traumatic brain injury (TBI) in humans. Unlike rodents, ferrets have a substantial amount of white matter and a gyrencephalic brain; therefore, they may represent an ideal small mammal model to study human-pertinent consequences of TBI. Here we report immunohistochemical and behavioral results after a controlled cortical impact (CCI) injury to the sensorimotor cortex of adult male ferrets. We assessed inflammation in the neocortex and white matter, and behavior at 1 day post injury and 1, 4, and 16 weeks post injury (WPI). CCI in the ferret produced inflammation that originated in the neocortex near the site of the injury and progressed deep into the white matter with time. The density of microglia and astrocytes increased in the neocortex near the injury, peaking at 4WPI and remaining elevated at 16WPI. Microglial morphology in the neocortex was significantly altered in the first 4 weeks, but showed a return toward normal at 16 weeks. Clusters of microglial cells in the white matter persisted until 16WPI. We assessed motor and cognitive behavior using the open field, novel object recognition, T-maze, and gait tests. A transient deficit in memory occurred at 4WPI, with a reduction of rearing and motor ability at 12 and 16WPI. Behavioral impairments coincide with features of the inflammatory changes in the neocortex revealed by immunohistochemistry. The ferret represents an important animal model to explore ongoing damage in the white matter and cerebral cortex after TBI.

Interaction of ascaridole, carvacrol, and caryophyllene oxide from essential oil of Chenopodium ambrosioides L. with mitochondria in Leishmania and other eukaryotes.

The antileishmanial activity of the essential oil (EO) from Chenopodium ambrosioides L. has been demonstrated in vitro and in animal models, attributed to the major components of the EO. This study focused on the effects of the three major EO compounds carvacrol, caryophyllene oxide (Caryo), and the antileishmanial endoperoxide ascaridole (Asc) on mitochondrial functions in Leishmania tarentolae promastigotes (LtP). EO and Caryo were able to partially inhibit the leishmanial electron transport chain, whereas other components failed to demonstrate a direct immediate effect. Caryo demonstrated inhibition of complex III activity in LtP and in isolated complex III from other species. The formation of superoxide radicals was studied in Leishmania by electron spin resonance spectroscopy in the presence of iron chelators wherein selected compounds failed to trigger a significant immediate additional superoxide production in LtP. However, upon prolonged incubation of Leishmania with Asc and especially in the absence of iron chelators (allowing the activation of Asc), an increased superoxide radical production and significant impairment of mitochondrial coupling in Leishmania was observed. Prolonged incubation with all EO components resulted in thiol depletion. Taken together, the major components of EO mediate their leishmanicidal activity via different mitochondrial targets and time profiles. Further studies are required to elucidate possible synergistic effects of carvacrol and Asc and the influence of minor compounds.

Activation of Anthracene Endoperoxides in Leishmania and Impairment of Mitochondrial Functions.

Leishmaniasis is a vector-borne disease caused by protozoal Leishmania. Because of resistance development against current drugs, new antileishmanial compounds are urgently needed. Endoperoxides (EPs) are successfully used in malaria therapy, and experimental evidence of their potential against leishmaniasis exists. Anthracene endoperoxides (AcEPs) have so far been only technically used and not explored for their leishmanicidal potential. This study verified the in vitro efficiency and mechanism of AcEPs against both Leishmania promastigotes and axenic amastigotes (L. tarentolae and L. donovani) as well as their toxicity in J774 macrophages. Additionally, the kinetics and radical products of AcEPs' reaction with iron, the formation of radicals by AcEPs in Leishmania, as well as the resulting impairment of parasite mitochondrial functions were studied. Using electron paramagnetic resonance combined with spin trapping, photometry, and fluorescence-based oximetry, AcEPs were demonstrated to (i) show antileishmanial activity in vitro at IC50 values in a low micromolar range, (ii) exhibit host cell toxicity in J774 macrophages, (iii) react rapidly with iron (II) resulting in the formation of oxygen- and carbon-centered radicals, (iv) produce carbon-centered radicals which could secondarily trigger superoxide radical formation in Leishmania, and (v) impair mitochondrial functions in Leishmania during parasite killing. Overall, the data of different AcEPs demonstrate that their structures besides the peroxo bridge strongly influence their activity and mechanism of their antileishmanial action.

Allylpyrocatechol Attenuates Collagen-Induced Arthritis via Attenuation of Oxidative Stress Secondary to Modulation of the MAPK, JAK/STAT, and Nrf2/HO-1 Pathways.

Rheumatoid arthritis (RA), an inflammatory autoimmune disorder, is characterized by synovial hyperplasia and bony destruction. The pathogenesis of RA includes redox dysregulation, concomitant with increased levels of proinflammatory mediators. As the ability of allylpyrocatechol (APC), a phytoconstituent of Piper betle leaves, to alleviate oxidative stress has been demonstrated in patients with RA, its antiarthritic activity was evaluated in an animal model of arthritis, and the underlying mechanism(s) of action clarified. The animal model was established by immunizing rats with bovine collagen type II (CII) followed by lipopolysaccharide, along with a booster dose of CII on day 15. Rats were treated with APC or methotrexate (MTX) from days 11 to 27, when paw edema, radiography, histopathology, and markers of inflammation were evaluated. The pro/antiinflammatory signaling pathways were studied in a RAW264.7 macrophage cell line. Allylpyrocatechol (APC) prevented the progression of arthritis as was evident from the reduction in paw edema, and attenuation of damage to bones and cartilage shown by radiography and histopathology. Additionally, there was reduction in the levels of proinflammatory cytokines [tumor necrosis factor-α (TNF-α) and interleukin-6 (IL-6)] and restoration of the redox balance. Importantly, MTX ameliorated the features of arthritis but not the associated oxidative stress. In RAW264.7, APC inhibited generation of nitric oxide and proinflammatory cytokines (TNF-α, IL-6, and IL-12p40), and modulated the phosphorylation of proinflammatory (extracellular signal-regulated kinase 1/2, stress-activated protein kinase/c-Jun N-terminal protein kinase, and Janus kinase/signal transducers and activators of transcription) and cytoprotective (nuclear factor erythroid 2-related factor 2, heme oxygenase-1) signaling pathways. Taken together, APC controlled the development of arthritis, possibly via modulation of signaling pathways, and deserves further consideration as a therapy for RA.

Decreased Frequency and Secretion of CD26 Promotes Disease Progression in Indian Post Kala-azar Dermal Leishmaniasis.

PURPOSE: Leishmania, the causative organisms for leishmaniasis, reside in host macrophages and survive by modulating the microbicidal pathways via attenuation of the oxidative burst and/or suppression of cell-mediated immunity. As post kala-azar dermal leishmaniasis (PKDL), the dermal sequela of visceral leishmaniasis, has no animal model, the underlying mechanism(s) that nullify the microbicidal effector mechanisms remain poorly understood. This study was aimed at assessing the status of dipeptidyl peptidase CD26, a co-stimulatory molecule that is essential for T-cell signal activation. METHODS: The frequency/expression of CD26 and CD45RO/RA was evaluated by flow cytometry, while levels of soluble CD26 (sCD26), CXCL-10, RANTES, IL-10 and TGF-ß along with adenosine deaminase (ADA) activity were measured using ELISA. RESULTS: In patients with PKDL vis-à-vis healthy individuals, there was a significant decrease in the frequency and expression of CD26 on CD3(+)CD8(+) T-cells, which was accompanied by a significant lowering of plasma levels of sCD26. Furthermore, these patients showed a significant decrease in the frequency of CD45RO(+)/CD8(+) T-cells, concomitant with a significant increase in the proportion of CD45RA(+)/CD8(+) T-cells. This could collectively translate into reduced formation of the immunological synapse of CD26, CD45RO, and ADA, and lead to an attenuation of the Th1 responses. The decreased levels of CD26 and sCD26 correlated negatively with raised levels of Th2 cytokines, IL-10, and TGF-ß along with the lesional parasite load, indicating disease specificity. CONCLUSIONS: Taken together, the decreased expression and secretion of CD26 in patients with PKDL resulted in impairment of the CD26-ADA interaction, and thereby possibly contributed to T-cell unresponsiveness, emphasizing the need to develop immunomodulatory therapies against PKDL and by extension, the leishmaniases.