Partial characterization of purified glycoprotein from nutshell of Arachis hypogea L. towards macrophage activation and leishmaniacidal activity.

Arachis hypogea L. protein fraction-2 (AHP-F2) from the Peanut shell was extracted and characterized and its potent immunomodulatory and anti-leishmanial role was determined in this present study. AHP-F2 was found to be a glycoprotein as the presence of carbohydrates were confirmed by the analysis of high-performance liquid chromatography (HPLC) yielded glucose, galactose, mannose, and xylose. AHP-F2 molecular mass was found to be ∼28 kDa as indicated in MALDI-TOF and peptide mass fingerprinting analysis followed by Mascot search. The peptide matches revealed the similarity of the mannose/glucose binding lectin with 71.07% in the BLAST analysis. After that, the 3D structure of the AHP-F2 model was designed and validated by the Ramachandran plot. The immunomodulatory role of AHP-F2 was established in murine peritoneal macrophages as induction of nitric oxide (NO), and stimulation of proinflammatory cytokines (IL-12 and IFN-γ) in a dose-dependent manner was observed. Interestingly, it was also found that AHP-F2 has interacted with the innate immune receptor, toll-like receptors (TLRs) as established in molecular docking as well as mRNA expression. The anti-leishmanial potential of AHP-F2 was revealed with a prominent inhibition of amastigote growth within the murine macrophages with prompt induction of nitrite release. Altogether, the isolated AHP-F2 from Arachis hypogea L. has strong immunomodulatory and anti-leishmanial potential which may disclose a new path to treat leishmaniasis.

Oceanimonas sp. BPMS22-derived protein protease inhibitor induces anti-leishmanial immune responses through macrophage M2 to M1 repolarization.

The present study aimed to validate the potential of a novel serine protein protease inhibitor (PPI), purified from marine Oceanimonas sp. BPMS22, induced M2 to M1 repolarization of the macrophages to treat visceral leishmaniasis (VL). Peptide mass fingerprint of the purified trypsin digested PPI peptide was obtained using matrix-assisted laser desorption ionization-time of flight combined with tandem mass spectrometry (MALDI-TOF MS/MS) and the sequence was used to construct a 3D protein model by homology modelling. The IC50 of PPI were 25.28 ± 1.675 µg/mL and 0.415 ± 0.015 µg/mL against promastigotes and intracellular amastigotes, respectively, indicating the host-directed therapy using PPI. The PPI enhanced the effector molecule i.e., nitric oxide (NO), and dampened the arginase activity in a dose-dependent manner. In vitro studies revealed that the BPMS22-derived PPI significantly (p < 0.05) decreased the mRNA expressions of M2 markers (FIZZ-1, YM-1, CD206, Arg-1) and increased the mRNA expressions of M1 markers (iNOS, IL-1ß, IL-12) in rIL-4 + rIL-10 induced M2 macrophages. Interestingly, the BPMS22-derived PPI also significantly (p < 0.05) decreased the FIZZ-1, YM-1, CD206, and Arg-1; significantly (p < 0.05) increased iNOS, IL-12, and IFN-γ mRNA expression in L. donovani -infected murine macrophages, alongside the decreased parasite load in it. Hence, PPI has the potential to repolarize the cytokines (rIL-4 + rIL-10) pre-stimulated and L. donovani-infected M2 macrophages to M1 phenotype in vitro. A decrease in parasite burden after treatment with PPI indicated the acceleration of the parasite killing by enhancing the macrophage effector functions. Further, in vivo PPI treatment reduced hepatic and splenic Leishman donovan units (LDU) up to 93.34 % and 87.63 %, respectively. This was followed by a surge in pro-inflammatory cytokines and dampening anti-inflammatory cytokines (p < 0.01), which exhibited anti-VL immunity. These observations might open new perspectives on PPI in macrophage repolarization to treat VL.

Oral combination of eugenol oleate and miltefosine induce immune response during experimental visceral leishmaniasis through nitric oxide generation with advanced cytokine demand.

Conventional therapy of visceral leishmaniasis (VL) remains challenging with the pitfall of toxicity, drug resistance, and expensive. Hence, urgent need for an alternative approach is essential. In this study, we evaluated the potential of combination therapy with eugenol oleate and miltefosine in Leishmania donovani infected macrophages and in the BALB/c mouse model. The interactions between eugenol oleate and miltefosine were found to be additive against promastigotes and amastigotes with xΣFIC 1.13 and 0.68, respectively. Significantly (p < 0.001) decreased arginase activity, increased nitrite generation, improved pro-inflammatory cytokines, and phosphorylated p38MAPK were observed after combination therapy with eugenol oleate and miltefosine. >80% parasite clearance in splenic and hepatic tissue with concomitant nitrite generation, and anti-VL cytokines productions were observed after orally administered miltefosine (5 mg/kg body weight) and eugenol oleate (15 mg/kg body weight) in L. donovani-infected BALB/c mice. Altogether, this study suggested the possibility of an oral combination of miltefosine with eugenol oleate against visceral leishmaniasis.

Oral administration of eugenol oleate cures experimental visceral leishmaniasis through cytokines abundance.

Visceral leishmaniasis (VL) is an endemic fatal infectious disease in tropical and subtropical nations. The limited treatment options, long treatment regimens, invasive mode of administration of drugs, and lack of effective vaccination are the main reasons for the search of new alternative therapeutics against VL. On this quest, from a series of eugenol derivatives, we had demonstrated eugenol oleate as a lead immunomodulatory anti-VL molecule earlier. In this report, the oral efficacy and mechanism of eugenol oleate in inducing immunomodulatory anti-VL activity has been studied in BALB/c mice model. The plasma pharmacokinetic and acute toxicity studies suggested that the eugenol oleate is safe with an appreciable pharmacokinetic profile. Eugenol oleate (30 mg/kg B.W.) showed 86.5% of hepatic and 84.1% of splenic parasite clearance. The increased Th1 cytokine profile and decreased Th2 cytokine profile observed from ELISA and qRTPCR suggested that the eugenol oleate induced the parasite clearance through the activation of the host immune system. Subsequently, the mechanistic insights behind the anti-leishmanial activity of eugenol oleate were studied in peritoneal macrophages in vitro by inhibitor response study and immunoblotting. The results inferred that eugenol oleate activated the PKC-ßII-p38 MAPK and produced IL-12 and IFN-γ which intern activated the iNOS2 to produce NO free radicals that cleared the intracellular parasite.

Synergic effect of eugenol oleate with amphotericin B augments anti-leishmanial immune response in experimental visceral leishmaniasis in vitro and in vivo.

Present treatment regimen on visceral leishmaniasis has multiple limitations including severe side effects, toxicity, and resistance of Leishmania strains. Amphotericin B is a well-established pharmacologically approved drug; however, mainly toxicity is a foremost issue with that drug. Recently, our group identified eugenol oleate as an anti-leishmanial immunomodulatory compound. The important objectives of this present study was to evaluate the possible synergistic effect of eugenol oleate with amphotericin B to reduce the toxicity of this approved drug. Results obtained from this study signified that combination of eugenol oleate and amphotericin B showed indifferent combinatorial effect against promastigotes with xΣFIC 1.015, while, moderate synergistic activity with xΣFIC 0.456 against amastigotes. It was also notable that eugenol oleate (2.5 µM) with low concentrations of amphotericin B (0.3125 µM) showed 96.45% parasite reduction within L. donovani-infected murine macrophages. Furthermore, eugenol oleate and amphotericin B significantly (p < 0.01) enhanced the nitrite generation, and pro-inflammatory cytokines (IL-12, IFN-γ and TNF-α) in infected macrophages in vitro and in BALB/c mice in vivo. Eugenol oleate (10 mg/Kg b. wt.) with amphotericin B (1 mg/Kg b.wt.) significantly (p < 0.01) controlled the parasite burden in liver by 96.2% and in spleen by 93.12%. Hence, this study strongly suggested the synergic potential of eugenol oleate with low concentration of amphotericin B in experimental visceral leishmaniasis through anti-leishmanial immune response.

Genome-wide analysis reveals miR-3184-5p and miR-181c-3p as a critical regulator for adipocytes-associated breast cancer.

Obesity is considered as an independent risk factor for breast cancer (BCa) and plays a major role in the breast tumor microenvironment. The etiology and mechanisms by which obesity contributes to BCa development is not yet understood. Herein, we show that in vitro coculture of BCa cells with mature adipocytes (MA-BCa) increased proliferation, migration, and invasive phenotype of BCa cells. MA-BCa coculture led to increased production of proinflammatory cytokines and chemokines. To identify microRNAs (miRNAs) in BCa cells that are modulated by the presence of adipocytes, we used small RNA sequencing analysis. Sequencing data revealed that 98 miRNAs were differentially expressed in MA-BCa. Among them, miR-3184-5p and miR-181c-3p were found to be the most upregulated and downregulated miRNAs, and direct targets are FOXP4 and PPARα, respectively. In vitro functional assays using a combination of miR-3184-5p inhibitor and miR-181c-3p mimic synergistically decreased adipocytes-induced cell proliferation and invasive capacity of BCa cells. Gene Set Enrichment analysis indicated that transcription factors were highly enriched followed by protein kinases, oncogene, and protein regulators in MA-BCa. GeneGo Metacore pathway analysis uncovered "NOTCH-induced EMT pathway" was found to be the most abundant in MA-BCa. Consistently, epithelial-mesenchymal transition-associated markers were also increased in MA-BCa. The disease enrichment analysis of the predict target genes revealed that diabetes mellitus was significantly affected disease in MA-BCa. Taken together, our data suggest that miRNA-based regulatory mechanism associated with deregulation of pathways and biological functions orchestrated by adipocytes-secreted factors might drive the BCa progression and metastasis in obese patients.

Potential anti-proliferative activity of AgNPs synthesized using M. longifolia in 4T1 cell line through ROS generation and cell membrane damage.

To overcome the problem of breast cancer, silver nanoparticles (AgNPs) synthesized using Indian medicinal plant Madhuca longifolia could be explored as an alternative anticancer medicine. Synthesized AgNPs were studied their characteristics and their anti-proliferative property was investigated in breast cancer cell line (4T1). Based on zeta sizer analysis, the size of the AgNPs was 103.5 nm and potential -9.57 eV. Fe-SEM results showed particle size of 69.4-99.4 nm while TEM images indicated the particle size of 18-24 nm. In dose-dependent study, AgNPs showed 93% of anti-proliferative activity at 50 µg/ml whereas the methanolic extract of M. longifolia showed 80% activity only at 10-fold increased concentration (500 µg/ml). AgNPs exhibited higher level of cytotoxicity in breast cancer cell line than extract through cell wall degradation and ROS generation. Such effective AgNPs could be investigated further through in vivo models with a view to develop anticancer drug.

Eugenol derived immunomodulatory molecules against visceral leishmaniasis.

Visceral leishmaniasis (VL) is a life threatening infectious disease caused by Leishmania donovani. It leads to the severe immune suppression in the host defense system. Higher cytotoxicity, rigorous side effects and lower therapeutic indexes (TI) of current antileishmanial drugs have created a necessity to develop new molecules with better antileishmanial activity and high TI value. In this study, we have synthesized 36 derivatives of eugenol and screened them for their activity against promastigote and amastigote forms of L. donovani. Among the synthesized derivatives, comp.35 showed better antileishmanial activity against extra cellular promastigotes (IC50- 20.13 ± 0.91 µM) and intracellular amastigotes (EC50-4.25 ± 0.26 µM). The TI value (82.24 ± 3.77) was found to improve by 10-13 fold compared to Amphotericin B and Miltefosine respectively. Treatment with comp.35 (5 µg/ml) enhanced the nitric oxide (NO) generation, iNOS2 mRNA expression (∼8 folds increase) and decreased the arginase-1 activity (∼4 folds) in L. donovani infected peritoneal macrophages. Comp.35 had also increased the IL-12 (∼6 folds) and decreased the IL-10 (∼3 folds) mRNA expression and release in vitro. Results of in vivo studies revealed that comp.35 treatment at 25 mg/kg body weight efficiently cleared the hepatic and splenic parasite burden with enhanced Th1 response in L. donovani infected BALB/c mice. Hence, this study clearly represents comp.35, as an immunomodulatory molecule, can induce host protective immune response against visceral leishmaniasis through enhanced NO generation and Th1 response, which are essentials against this deadly disease.

Attenuation of Mycobacterium species through direct and macrophage mediated pathway by unsymmetrical diaryl urea.

Tuberculosis is a major threat for mankind and the emergence of resistance strain of Mycobacterium tuberculosis (Mtb) against first line antibiotics makes it lethal for human civilization. In this study, we have synthesized different diaryl urea derivatives targeting the inhibition of mycolic acid biosynthesis. Among the 39 synthesized molecules, compounds 46, 57, 58 and 86 showed MIC values ≤ 10 µg/ml against H37Rv and mc26030 strains. The best molecule with a methyl at ortho position of the first aromatic ring and prenyl group at the meta position of the second aromatic ring showed the MIC value of 5.2 µg/ml and 1 µg/ml against H37Rv and mc26030 respectively, with mammalian cytotoxicity of 163.4 µg/ml. The effective compounds showed selective inhibitory effect on mycolic acid (epoxy mycolate) biosynthesis in 14C-radiolabelled assay. At the same time these molecules also executed their potent immunomodulatory activity by up-regulation of IFN-γ and IL-12 and down-regulation of IL-10.