Unwinding the mechanism of macrophage repolarization potential of Oceanimonas sp. BPMS22-derived protein protease inhibitor through Toll-like receptor 4 against experimental visceral leishmaniasis.

The Oceanimonas sp. BPMS22-derived protein protease inhibitor (PPI) has been proven to shift macrophages towards an inflammatory state and reduce Leishmania donovani infection in vitro and in vivo. The current study explored and validated the mechanistic aspects of the PPI and Toll-like receptor (TLR) interaction. The PPI exhibited the upregulation of TLR2, TLR4, and TLR6 during treatment which was proven to orchestrate parasite clearance effectively. An in silico study confirmed the high interaction with TLR4 and PPI. Immune blotting confirmed the significant upregulation of TLR4 in macrophages irrespective of L. donovani infection. Pharmacological inhibition and immune blot study confirmed the involvement of the PPI in TLR4-mediated phosphorylation of p38 MAPK and dephosphorylation of ERK1/2, repolarizing to pro-inflammatory macrophage state against experimental visceral leishmaniasis. In addition, in TLR4 knockdown condition, PPI treatment failed to diminish M2 phenotypical markers (CD68, Fizz1, Ym1, CD206, and MSR-2) and anti-inflammatory cytokines (IL-4, IL-10, and TGF-ß). Simultaneously, the PPI failed to upregulate the M1 phenotypical markers and pro-inflammatory cytokines (IL-1ß, IL-6, IL-12, and IFN-γ) (p < 0.001) during the TLR4 knockdown condition. In the absence of TLR4, the PPI also failed to reduce the parasite load and T-cell proliferation and impaired the delayed-type hypersensitivity response. The absence of pro-inflammatory cytokines was observed during a co-culture study with PPI-treated macrophages (in the TLR4 knockdown condition) with day 10 T-cell obtained from L. donovani-infected mice. This study supports the immunotherapeutic potential of the PPI as it interacted with TLR4 and promoted macrophage repolarization (M2-M1) to restrict the L. donovani parasite burden and helps in the mounting immune response against experimental visceral leishmaniasis.

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.

Vitexin isolated from Prosopis cineraria leaves induce apoptosis in K-562 leukemia cells via inhibition of the BCR-ABL-Ras-Raf pathway.

OBJECTIVES: Leukemia is one of the severe cancer types all around the globe. Even though some chemotherapeutic drugs are available for treating leukemia, they have various side effects. As an alternative approach, herbal drugs are focused on current research to overcome leukemia. The present work was conducted to investigate the antileukemic mechanism of active phytochemical vitexin, which was isolated from ethno-medicine (Prosopis cineraria leaf) used by traditional healers of West Bengal, India. METHODS: Antiproliferative mechanisms of selected phyto-compound against K-562 cells were evaluated using cellular uptake, morphological changes, DNA fragmentation, mitochondrial membrane potential and signaling pathways analysis. KEY FINDINGS: Vitexin exhibited cytotoxicity by reducing mitochondrial membrane potential (32.40%) and causing DNA fragmentation (84.15%). The western blotting study indicated inhibition of cell survival proteins (BCR, ABL, H-RAS, N-RAS, K-RAS and RAF) and expression of apoptotic proteins (p38, BAX and caspase-9) in leukemia cells upon treatment with vitexin. CONCLUSIONS: Based on the results, presently investigated phyto-compound vitexin could be considered for developing safe and natural drugs to treat leukemia after conducting suitable preclinical and clinical trials.

Dynamicity in Host Metabolic Adaptation Is Influenced by the Synergistic Effect of Eugenol Oleate and Amphotericin B During Leishmania donovani Infection In Vitro.

Immune metabolic adaptation in macrophages by intracellular parasites is recognized to play a crucial role during Leishmania infection. However, there is little accessible information about changes in a metabolic switch in L. donovani infected macrophages. In previous studies, we have reported on the anti-leishmanial synergic effect of eugenol oleate with amphotericin B. In the present study, we demonstrated that glycolytic enzymes were highly expressed in infected macrophages during combinatorial treatment of eugenol oleate (2.5 µM) and amphotericin B (0.3125 µM). Additionally, we found that the biphasic role in arachidonic acid metabolite, PGE2, and LTB4, is released during this treatment. In vitro data showed that COX-2 mediated PGE2 synthesis increased significantly (p<0.01) in infected macrophages. Not only was the level of prostaglandin synthesis decreased 4.38 fold in infected macrophages after treatment with eugenol oleate with amphotericin B. The mRNA expression of PTGES, MPGES, and PTGER4 were also moderately expressed in infected macrophages, and found to be decreased in combinatorial treatment. In addition, NOS2 expression was activated by the phosphorylation of p38MAPK when combination-treated macrophages were promoted to kill intracellular parasites. The findings of the present study indicate that the synergism between eugenol oleate and amphotericin B could play an important role in immune metabolism adaptation with a concomitant increase in host immune response against the intracellular pathogen, L. donovani.

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.

Investigation of phytochemical constituents of anti-leukemic herbal drugs used by the traditional healers of Purulia, Birbhum and Bankura districts of West Bengal.

In the present work, 16 different plant drugs used by traditional healers from West Bengal were screened through in vitro cell line model. Herbal drugs used by traditional tribal healers in Purulia, Birbhum and Bankura districts of West Bengal were collected and screening against acute myeloid leukemia (AML) cell line (HL-60). Among 16 plant extracts, bark of Flacourtia indica (66.67%), leaf of Madhuca longifolia (69.17%), and leaf of Prosopis cineraria (68.08%) showed better cytotoxicity results than other herbals. Further, time-dependent study showed maximum cytotoxicity of the selected herbal extracts between 36 and 48 hours of treatment in both acute and chronic myeloid leukemia (CML) cell lines (HL-60 and K562). The LC-MS/MS analysis of the selected drugs revealed the presence of picrotoxinin and 10-deacetylbaccatin from F. indica, isoorientin and hirsutrin from M. longifolia, vitexin and rhoifolin in P. cineraria.

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.

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.