Alcohol and e-cigarette damage alveolar-epithelial barrier by activation of P2X7r and provoke brain endothelial injury via extracellular vesicles.

BACKGROUND: Use of nicotine containing products like electronic cigarettes (e-Cig) and alcohol are associated with mitochondrial membrane depolarization, resulting in the extracellular release of ATP, and mitochondrial DNA (mtDNA), mediating inflammatory responses. While nicotine effects on lungs is well-known, chronic alcohol (ETH) exposure also weakens lung immune responses and cause inflammation. Extracellular ATP (eATP) released by inflammatory/stressed cells stimulate purinergic P2X7 receptors (P2X7r) activation in adjacent cells. We hypothesized that injury caused by alcohol and e-Cig to pulmonary alveolar epithelial cells (hPAEpiC) promote the release of eATP, mtDNA and P2X7r in circulation. This induces a paracrine signaling communication either directly or via EVs to affect brain cells (human brain endothelial cells - hBMVEC). METHODS: We used a model of primary human pulmonary alveolar epithelial cells (hPAEpiC) and exposed the cells to 100 mM ethanol (ETH), 100 µM acetaldehyde (ALD), or e-Cig (1.75 µg/mL of 1.8% or 0% nicotine) conditioned media, and measured the mitochondrial efficiency using Agilent Seahorse machine. Gene expression was measured by Taqman RT-qPCR and digital PCR. hPAEpiC-EVs were extracted from culture supernatant and characterized by flow cytometric analysis. Calcium (Ca2+) and eATP levels were quantified using commercial kits. To study intercellular communication via paracrine signaling or by EVs, we stimulated hBMVECs with hPAEpiC cell culture medium conditioned with ETH, ALD or e-cig or hPAEpiC-EVs and measured Ca2+ levels. RESULTS: ETH, ALD, or e-Cig (1.8% nicotine) stimulation depleted the mitochondrial spare respiration capacity in hPAEpiC. We observed increased expression of P2X7r and TRPV1 genes (3-6-fold) and increased intracellular Ca2+ accumulation (20-30-fold increase) in hPAEpiC, resulting in greater expression of endoplasmic reticulum (ER) stress markers. hPAEpiC stimulated by ETH, ALD, and e-Cig conditioned media shed more EVs with larger particle sizes, carrying higher amounts of eATP and mtDNA. ETH, ALD and e-Cig (1.8% nicotine) exposure also increased the P2X7r shedding in media and via EVs. hPAEpiC-EVs carrying P2X7r and eATP cargo triggered paracrine signaling in human brain microvascular endothelial cells (BMVECs) and increased Ca2+ levels. P2X7r inhibition by A804598 compound normalized mitochondrial spare respiration, reduced ER stress and diminished EV release, thus protecting the BBB function. CONCLUSION: Abusive drugs like ETH and e-Cig promote mitochondrial and endoplasmic reticulum stress in hPAEpiC and disrupts the cell functions via P2X7 receptor signaling. EVs released by lung epithelial cells against ETH/e-cig insults, carry a cargo of secondary messengers that stimulate brain cells via paracrine signals.

Alcohol and e-cigarette damage alveolar-epithelial barrier by activation of P2X7r and provoke brain endothelial injury via extracellular vesicles.

Background: Use of nicotine containing products like electronic cigarettes (e-Cig) and alcohol are associated with mitochondrial membrane depolarization, resulting in the extracellular release of ATP, and mitochondrial DNA (mtDNA), mediating inflammatory responses. While nicotine effects on lungs is well-known, chronic alcohol (ETH) exposure also weakens lung immune responses and cause inflammation. Extracellular ATP (eATP) released by inflammatory/stressed cells stimulate purinergic P2X7 receptors (P2X7r) activation in adjacent cells. We hypothesized that injury caused by alcohol and e-Cig to pulmonary alveolar epithelial cells (hPAEpiC) promote the release of eATP, mtDNA and P2X7r in circulation. This induces a paracrine signaling communication either directly or via EVs to affect brain cells (human brain endothelial cells - hBMVEC). Methods: We used a model of primary human pulmonary alveolar epithelial cells (hPAEpiC) and exposed the cells to 100 mM ethanol (ETH), 100 µM acetaldehyde (ALD), or e-Cig (1.75µg/mL of 1.8% or 0% nicotine) conditioned media, and measured the mitochondrial efficiency using Agilent Seahorse machine. Gene expression was measured by Taqman RT-qPCR and digital PCR. hPAEpiC-EVs were extracted from culture supernatant and characterized by flow cytometric analysis. Calcium (Ca2+) and eATP levels were quantified using commercial kits. To study intercellular communication via paracrine signaling or by EVs, we stimulated hBMVECs with hPAEpiC cell culture medium conditioned with ETH, ALD or e-cig or hPAEpiC-EVs and measured Ca2+ levels. Results: ETH, ALD, or e-Cig (1.8% nicotine) stimulation depleted the mitochondrial spare respiration capacity in hPAEpiC. We observed increased expression of P2X7r and TRPV1 genes (3-6-fold) and increased intracellular Ca2+ accumulation (20-30-fold increase) in hPAEpiC, resulting in greater expression of endoplasmic reticulum (ER) stress markers. hPAEpiC stimulated by ETH, ALD, and e-Cig conditioned media shed more EVs with larger particle sizes, carrying higher amounts of eATP and mtDNA. ETH, ALD and e-Cig (1.8% nicotine) exposure also increased the P2X7r shedding in media and via EVs. hPAEpiC-EVs carrying P2X7r and eATP cargo triggered paracrine signaling in human brain microvascular endothelial cells (BMVECs) and increased Ca2+ levels. P2X7r inhibition by A804598 compound normalized mitochondrial spare respiration, reduced ER stress and diminished EV release, thus protecting the BBB function. Conclusion: Abusive drugs like ETH and e-Cig promote mitochondrial and endoplasmic reticulum stress in hPAEpiC and disrupts the cell functions via P2X7 receptor signaling. EVs released by lung epithelial cells against ETH/e-cig insults, carry a cargo of secondary messengers that stimulate brain cells via paracrine signals.

Michael Adduct of Sulfonamide Chalcone Targets Folate Metabolism in Brugia Malayi Parasite.

A series of Michael adducts of malononitrile and sulfonamide chalcones were synthesized, characterized, and evaluated for their antifilarial activity. Out of 14 compounds, N-(4-(4,4-dicyano-3-p-tolylbutanoyl)phenyl)benzenesulfonamide showed favorable drug-likeness properties with marked antifilarial effects at micro-molar dosages. Apoptosis in Brugia malayi microfilariae was confirmed by EB/AO staining, MTT assay, and cytoplasmic cytochrome c ELISA. Since chalcone and folate synthesis pathways share the same substrate, we hypothesize a structural analogy-based inhibition of folate metabolism by this compound. Molecular docking against a pre-validated BmDHFR protein showed more favorable thermodynamic parameters than a positive control, epicatechin-3-gallate. The compound significantly suppressed the DHFR activity in a parasite extract in vitro. Our hypothesis is also supported by a significant reversal of DHFR inhibition by folate addition, which indicated a plausible mechanism of competitive inhibition. These results demonstrate that targeting filarial folate metabolism through DHFR with consequent apoptosis induction might be rewarding for therapeutic intervention. This study reveals a novel rationale of the structural analogy-based competitive inhibition of DHFR by Michael adducts of sulfonamide chalcones.

Harnessing Immune Evasion Strategy of Lymphatic Filariae: A Therapeutic Approach against Inflammatory and Infective Pathology.

Human lymphatic filariae have evolved numerous immune evasion strategies to secure their long-term survival in a host. These strategies include regulation of pattern recognition receptors, mimicry with host glycans and immune molecules, manipulation of innate and adaptive immune cells, induction of apoptosis in effector immune cells, and neutralization of free radicals. This creates an anti-inflammatory and immunoregulatory milieu in the host: a modified Th2 immune response. Therefore, targeting filarial immunomodulators and manipulating the filariae-driven immune system against the filariae can be a potential therapeutic and prophylactic strategy. Filariae-derived immunosuppression can also be exploited to treat other inflammatory diseases and immunopathologic states of parasitic diseases, such as cerebral malaria, and to prevent leishmaniasis. This paper reviews immunomodulatory mechanisms acquired by these filariae for their own survival and their potential application in the development of novel therapeutic approaches against parasitic and inflammatory diseases. Insight into the intricate network of host immune-parasite interactions would aid in the development of effective immune-therapeutic options for both infectious and immune-pathological diseases.

The Role of Oxidative and Nitrosative Stress of Silver Nanoparticles in Human Parasitic Helminth Brugia malayi: A Mechanistic Insight.

PURPOSE: Silver nanoparticles (AgNPs) mediated apoptosis is well-known but its rationale is yet to be elucidated. This study explored the mechanistic underpinning of the apoptosis in the Brugia malayi parasitic model. METHOD: Silver nanoparticles were synthesized and tested against B. malayi microfilariae (Mf) to explore the role of oxidative and nitrosative stress in its apoptotic effect. RESULTS: AgNPs caused significant decrease in reduced glutathione (GSH) level and increase in both protein carbonylation and nitric oxide (NO) level indicating oxidative as well as nitrosative stress. Both GSH and nitric oxide synthase (NOS) inhibitors exhibited marked reversal. Nanoparticles and NO-donor in combination but not the NO-donor alone showed significant antiparasitic effect implying the requisite of combined oxidative and nitrosative stress to induce apoptosis. Synthetically prepared peroxynitrite from NaNO2 to H2O2 showed marked antiparasitic effect in very low dose which could be achieved neither by NaNO2 or H2O2 alone. GSH reversed the effect of peroxynitrite similar to its specific inhibitor, acetaminophen. GSH also reversed the plummet in mitochondrial oxygen consumption by AgNPs. CONCLUSION: We conclude that apoptosis by AgNPs is possibly mediated through peroxynitrite dependent depletion of GSH; this provides a significant insight into the pharmacological as well as toxicological impact of AgNPs.

In vitro apoptotic effect on human lymphatic filarial parasite by piperidine derivatives and thymidine reversal study.

A novel library of synthetic piperidine derivatives was used to screen against human lymphatic filarial parasite Brugia malayi. Piperidine has earlier been reported to have effect against parasites including rodent filarial nematodes. Compounds with hydroxyl substitutions (4Q and 4H) showed marked antifilarial effect. Molecular docking of 4H derivative showed more favorable thermodynamic parameters against thymidylate synthase of B. malayi than human counterpart. A wide difference between IC50 and LD50 ensured the therapeutic safety of the candidates against the filarial parasites. Addition of thymidine to the treatment regimen led to a significant reversal of antifilarial effect of 4H that confirmed inhibition of thymidylate synthase as pharmacological rationale. Apoptosis induced in the parasite as a consequence of probable inhibition of thymidylate synthase was studied by acridine orange/ethidium bromide fluorescent staining and poly (ADP-ribose) polymerase activity inhibition. Involvement of mitochondria was confirmed by decreased 3-[4,5-dimethylthiazol-2-yl]-2,5 diphenyl tetrazolium bromide (MTT) conversion and increased cytosolic cytochrome c level in 4H treated microfilariae, compared with the untreated microfilariae. Moreover, Michael adduct of chalcone targeting dihydrofolate reductase and piperidine targeting thymidylate synthase demonstrated synergistic effect on the parasite, indicating the importance of inhibition of DNA synthesis by combined effect. In conclusion, piperidine derivatives with hydroxyl substitution have a great therapeutic potential with an apoptotic rationale involving mitochondrial pathway, due to possible inhibition of parasitic thymidylate synthase.

Immunomodulatory Activity of Sulfonamide Chalcone Compounds in Mice Infected with Filarial Parasite, Brugia malayi.

Diethyl carbamazine (DEC) is being used as a sole drug to treat the lymphatic filariasis, although encountered with many limitations. Importantly, DEC works with putative host immunomodulating activities without any direct antifilarial effect. This study aimed to assess the possible modulatory effect on host immune system by sulfonamide chalcone compound, having direct antifilarial activity. The immunomodulatory activity of DEC and/or chalcone compound, 4t on mice peritoneal exudate cells (PECs) was analyzed initially in vitro. This was followed by the study of in vivo effect of these test agents in the parasitaemic BALB/c mice induced by Brugia malayi microfilariae. Cytokine profile and iNOS induction were measured from PECs of mice. 4t compound showed anti-inflammatory activity in vivo in contrast to DEC. Further 4t was found to increase anti-inflammatory and regulatory cytokines, IL-10 and TGF-ß gene expression with down regulation of pro-inflammatory cytokines TNF-α and IFN-γ and iNOS in mice PECs in in vitro. In conclusion, chalcones having direct antifilarial effect also upsurges anti-inflammatory host immune response. Therefore, the results might be envisaged as 4t to be a better alternative to DEC in the chronic case of lymphatic filariasis.

Glutathione S-transferase omega 1 inhibition activates JNK-mediated apoptotic response in breast cancer stem cells.

Glutathione S-transferase omega 1 (GSTO1) contributes to the inactivation of a wide range of drug compounds via conjugation to glutathione during phase reactions. Chemotherapy-induced GSTO1 expression in breast cancer cells leads to chemoresistance and promotes metastasis. In search of novel GSTO1 inhibitors, we identified S2E, a thia-Michael adduct of sulfonamide chalcone with low LC50 (3.75 ± 0.73 µm) that binds to the active site of GSTO1, as revealed by molecular docking (glide score: -8.1), cellular thermal shift assay and fluorescence quenching assay (Kb  ≈ 10 × 105  mol·L-1 ). Docking studies confirmed molecular interactions between GSTO1 and S2E, and identified the hydrogen bond donor Val-72 (2.14 Å) and hydrogen bond acceptor Ser-86 (2.77 Å). Best pharmacophore hypotheses could effectively map S2E and identified the 4-methyl group of the benzene sulfonamide ring as crucial to its anti-cancer activity. Lack of a thiophenyl group in another analog, 2e, reduced its efficacy as observed by cytotoxicity and pharmacophore matching. Furthermore, GSTO1 inhibition by S2E, along with tamoxifen, led to a significant increase in apoptosis and decreased migration of aggressive MDA-MB-231 cells, as well as significantly decreased migration, invasion and mammosphere formation in sorted breast cancer stem cells (CSCs, CD24- /CD44+ ). GSTO1 silencing in breast CSCs also significantly increased apoptosis and decreased migration. Mechanistically, GSTO1 inhibition activated the c-Jun N-terminal kinase stress kinase, inducing a mitochondrial apoptosis signaling pathway in breast CSCs via the pro-apoptotic proteins BAX, cytochrome c and cleaved caspase 3. Our study elucidated the role of the GSTO1 inhibitor S2E as a potential therapeutic strategy for preventing chemotherapy-induced breast CSC-mediated cancer metastasis and recurrence.

SXP-RAL Family Filarial Protein, rWbL2, Prevents Development of DSS-Induced Acute Ulcerative Colitis.

Helminthic infections lead to the release of various molecules which play an important role in modulation of the host immune system. Such filarial proteins with immunomodulatory potential can be used for therapeutic purpose in inflammatory and immune mediated diseases. In the present study, we have explored the prophylactic effect of filarial SXP-RAL family protein of Wuchereria bancrofti i.e. rWbL2 protein in DSS induced inflammatory ulcerative colitis in a mouse model. Prior treatment of rWbL2, followed by induction of colitis, showed significantly reduced disease severity as indicated by the decreased disease manifestations and improved macroscopic and microscopic inflammation. This preventive effect was found to be associated with increased release of anti-inflammatory cytokine IL-10 and decreased release of proinflammatory cytokines IFN-γ, TNF-α, IL-6 and IL-17 by the splenocytes of treated mice. From this study, it can be envisaged that pretreatment with filarial protein, rWbL2, can prevent the establishment of ulcerative colitis in BALB/c mice. The underlying immunological mechanism may involve the up-regulation of Th2 immune response with down-regulation of Th1 response.

Apoptotic impact on Brugia malayi by sulphonamido-quinoxaline: search for a novel therapeutic rationale.

Human lymphatic filariasis although not fatal but poses serious socioeconomic burden due to associated disability. This is reflected by the huge magnitude of the estimated disability-adjusted life years of about 5.09 million. Therefore, following WHO mandate, our earlier studies on antifilarial drug development revealed the significance of apoptosis. Apoptotic impact has been implicated in anticancer rationale of several drugs. In this study, we explored the antifilarial potential of sulphonamido-quinoxaline compounds, shown to be specific inhibitor for c-Met kinase in human cancer cells. Out of studied compounds, Q4, showing favorable drug-likeness and medicinal chemistry properties on bioinformatics platform along with subsequently recorded lowest IC100 value, was considered as a suitable antifilarial candidate. Significant apoptosis due to mitochondrial involvement was recorded in drug-treated parasite unlike untreated control. In spite of homology between human c-Met kinase and Brugia malayi counterpart, comparative docking result of this compound showed more favorable binding parameters with the parasitic target. The wide gap between IC100 and LD50 values further confirmed the therapeutic safety. We propose sulphonamido-quinoxaline derivative as a lead candidate for antifilarial drug development. Further study is warranted to authenticate parasitic c-Met kinase as a novel therapeutic target reminiscent of anticancer rationale implicating inhibition of proliferation.

Immunomodulatory potential of recombinant filarial protein, rWbL2, and its therapeutic implication in experimental ulcerative colitis in mouse.

OBJECTIVE: Immunomodulation by helminth proteins has potential therapeutic implications in inflammatory bowel disease. In the present study, we have explored the therapeutic effect of a RAL family protein of filarial parasite Wuchereria bancrofti i.e., rWbL2 protein against DSS induced colitis in a mouse model. MATERIALS AND METHODS: Anti-inflammatory activity of rWbL2 on mice peritoneal exudate cells was analyzed under in vitro condition. The colitis mice were treated intraperitoneally (i.p.) with rWbL2 in increasing doses (10 µg, 25 µg, and 50 µg) on days 4, 5, and 6. Disease severity was assessed by disease activity index (DAI), macroscopic and histopathological scores, and enzyme myeloperoxidase activity (MPO) in the colon. The response of the cultured splenocytes from treated mice to Con-A stimulation, in terms of ELISA-based assessment of the protein followed by the assessment of mRNA expression of cytokines, was measured by real-time PCR analysis. RESULT: rWbL2 protein showed anti-inflammatory activity in vitro. Treatment with rWbL2 (at 25 µg/dose) effectively attenuated disease severity by reducing weight loss, DAI, mucosal edema, colon damage, and MPO activity. This therapeutic effect was found to be associated with increased release of anti-inflammatory cytokine IL-10 and decreased release of pro-inflammatory cytokine IFN-γ and TNF-α by the splenocytes of treated mice followed by stimulation with Con-A. CONCLUSIONS: These results provide evidence of the strong immunomodulatory potential of rWbL2 protein implicating its therapeutic application against ulcerative colitis.

Sulfonamide chalcones: Synthesis and in vitro exploration for therapeutic potential against Brugia malayi.

Keeping in mind the immense biological potential of chalcones and sulfonamide scaffolds, a library of sulfonamide chalcones has been synthesized and evaluated for in vitro antifilarial assay against human lymphatic filarial parasite Brugia malayi. Experimental evidence showcased for the first time the potential of some sulfonamide chalcones as effective and safe antifilarial lead molecules against human lymphatic filarial parasite B. malayi. Sulfonamide chalcones 4d, 4p, 4q, 4t and 4aa displayed the significantly wide therapeutic window. Particularly chalcones with halogen substitution in aromatic ring proved to be potent antifilarial agents against Brugia malayi. Sulphonamide chalcones with lipophilic methyl moiety (4q and 4aa) at para position of terminal phenyl rings of compounds were found to have remarkable antifilarial activities with therapeutic efficacy. Observed preliminary evidence of apoptosis by effective chalcone derivatives envisaged its fair possibility to inhibit folate pathway with consequent defect in DNA synthesis.

Targeting folate metabolism for therapeutic option: A bioinformatics approach.

Lymphatic filariasis, commonly called elephantiasis, poses a burden of estimated level of 5.09 million disability adjusted life year. Limitations of its sole drug, diethylcarbamazine (DEC) drive exploration of effective filarial target. A few plant extracts having polyphenolic ingredients and some synthetic compounds possess potential dihydrofolate reductase (DHFR) inhibitory effect. Here, we postulated a plausible link between folates and polyphenolics based on their common precursor in shikimate metabolism. Considering its implication in structural resemblance based antagonism, we have attempted to validate parasitic DHFR protein as a target. The bioinformatics approach, in the absence of crystal structure of the proposed target, used to authenticate and for virtual docking with suitable tested compounds, showed remarkably lower thermodynamic parameters as opposed to the positive control. A comparative docking analysis between human and Brugia malayi DHFR also showed effective binding parameters with lower inhibition constants of these ligands with parasitic target, but not with human counterpart highlighting safety and efficacy. This study suggests that DHFR could be a valid drug target for lymphatic filariasis, and further reveal that bioinformatics may be an effective tool in reverse pharmacological approach for drug design.