In vitro and in vivo immunomodulatory properties of octyl-β-d-galactofuranoside during Leishmania donovani infection

Abstract Background The chemotherapeutic arsenal available to treat visceral leishmaniasis is currently limited, in view of many drawbacks such as high cost, toxicity or emerging resistance. New therapeutic strategies are particularly needed to improve the management and the outcome in immunosuppressed patients. The combination of an immunomodulatory drug to a conventional anti-Leishmania treatment is an emerging concept to reverse the immune bias from Th2 to Th1 response to boost healing and prevent relapses. Methods Here, immunostimulating and leishmanicidal properties of octyl-β-d-galactofuranose (Galf) were assessed in human monocyte-derived macrophages (HM) and in a murine model, after challenge with Leishmania donovani promastigotes. We recorded parasite loads and expression of various cytokines and immune effectors in HM and mouse organs (liver, spleen, bone marrow), following treatment with free (Galf) and liposomal (L-Galf) formulations. Results Both treatments significantly reduced parasite proliferation in HM, as well as liver parasite burden in vivo (Galf, P < 0.05). Consistent with in vitro results, we showed that Galf- and L-Galf-treated mice displayed an enhanced Th1 immune response, particularly in the spleen where pro-inflammatory cytokines TNF-α, IL-1β and IL-12 were significantly overexpressed compared to control group. The hepatic recruitment of myeloid cells was also favored by L-Galf treatment as evidenced by the five-fold increase of myeloperoxidase (MPO) induction, which was associated with a higher number of MPO-positive cells within granulomas. By contrast, the systemic level of various cytokines such as IL-1β, IL-6, IL-17A or IL-27 was drastically reduced at the end of treatment. Conclusions Overall, these results suggest that Galf could be tested as an adjuvant in combination with current anti-parasitic drugs, to restore an efficient immune response against infection in a model of immunosuppressed mice.

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Hydroxytyrosol Plays Antiatherosclerotic Effects through Regulating Lipid Metabolism via Inhibiting the p38 Signal Pathway

BioMed Research International ◽

10.1155/2020/5036572 ◽

2020 ◽

Vol 2020 ◽

pp. 1-12

Author(s):

Xinxin Zhang ◽

Yating Qin ◽

Xiaoning Wan ◽

Hao Liu ◽

Chao Iv ◽

...

Keyword(s):

Lipid Metabolism ◽

Molecular Mechanisms ◽

Cholesterol Metabolism ◽

Fold Increase ◽

Heart Tissue ◽

Control Group ◽

Atherosclerotic Lesions ◽

Serum Crp

Purpose. Hydroxytyrosol (HT) processes multiaspect pharmacological properties such as antithrombosis and antidiabetes. The aim of this study was to explore the antistherosclerotic roles and relevant mechanisms of HT. Methods. Male apoE-/- mice were randomly divided into 2 groups: the control group and the HT group (10 mg/kg/day orally). After 16 weeks, blood tissue, heart tissue, and liver tissue were obtained to detect the atherosclerotic lesions, histological analysis, lipid parameters, and inflammation. And the underlying molecular mechanisms of HT were also studied in vivo and in vitro. Results. HT administration significantly reduced the extent of atherosclerotic lesions in the aorta of apoE-/- mice. We found that HT markedly lowered the levels of serum TG, TC, and LDL-C approximately by 17.4% (p=0.004), 15.2% (p=0.003), and 17.9% (p=0.009), respectively, as well as hepatic TG and TC by 15.0% (p<0.001) and 12.3% (p=0.003), respectively, while inducing a 26.9% (p=0.033) increase in serum HDL-C. Besides, HT improved hepatic steatosis and lipid deposition. Then, we discovered that HT could regulate the signal flow of AMPK/SREBP2 and increase the expression of ABCA1, apoAI, and SRBI. In addition, HT reduced the levels of serum CRP, TNF-α, IL-1β, and IL-6 approximately by 23.5% (p<0.001), 27.8% (p<0.001), 18.4% (p<0.001), and 19.1% (p<0.001), respectively, and induced a 1.4-fold increase in IL-10 level (p=0.014). Further, we found that HT might regulate cholesterol metabolism via decreasing phosphorylation of p38, followed by activation of AMPK and inactivation of NF-κB, which in turn triggered the blockade of SREBP2/PCSK9 and upregulation of LDLR, apoAI, and ABCA1, finally leading to a reduction of LDL-C and increase of HDL-C in the circulation. Conclusion. Our results provide the first evidence that HT displays antiatherosclerotic actions via mediating lipid metabolism-related pathways through regulating the activities of inflammatory signaling molecules.

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335 CYTOPLASMIC MICROINJECTION OF EXOGENOUS DNA IN IN VITRO AND IN VIVO DERIVED SHEEP EMBRYOS

Reproduction Fertility and Development ◽

10.1071/rdv23n1ab335 ◽

2011 ◽

Vol 23 (1) ◽

pp. 263

Author(s):

F. Pereyra-Bonnet ◽

A. Gibbons ◽

M. Cueto ◽

R. Bevacqua ◽

L. Escobar ◽

...

Keyword(s):

Fluorescent Protein ◽

Genetically Modified ◽

Fold Increase ◽

Egfp Expression ◽

Control Group ◽

Corpora Lutea ◽

Exogenous Dna ◽

Frozen Semen

Microinjection of DNA into the male pronucleus is a commonly used method to generate transgenic animals. However, it is only moderately efficient in several species because it requires proper male pronuclear visualisation, which occurs only in a narrow window of time in mice. The cytoplasmic microinjection of exogenous DNA (eDNA) is an alternative method that has not been fully investigated. Our objective was to evaluate if cytoplasmic microinjection of eDNA is capable of producing genetically modified embryos. In vitro and in vivo derived sheep embryos were cytoplasmically microinjected with pCX-EGFP previously incubated (5 min in a PVP droplet) with oolemma-cytoplasm fragments obtained from donor oocytes by microsurgery. A control group using microinjected plasmid alone was included in the in vivo procedure. For in vitro microinjection, IVF embryos were microinjected with circular plasmid with promoter (50 or 500 ng μL–1) or without promoter (50 ng μL–1) at 6 h after fertilization. The IVF was performed following (Brackett and Olliphant 1975 Biol. Reprod. 12, 260–274) with 15 × 106 spermatozoa mL–1, and presumptive zygotes were cultured in SOF. The expression of enhance green fluorescent protein (EGFP) was determined under blue light. For in vivo microinjection, embryos from superovulated sheep (by standard procedures) were recovered and microinjected with 50 ng μL–1 of linearized plasmid without promoter at 12 h after laparoscopic insemination with frozen semen (100 × 106 spermatozoa per sheep). Plasmid without promoter was used to avoid any possible cytotoxic effect produced by EGFP expression. The microinjection of IVF embryos with 50 ng μL–1 of plasmid was the best condition to produce embryos expressing eDNA (n = 96; 46.9% cleaved; 12.2% blastocysts; 53.0 and 4.1% of green embryos and blastocysts, respectively). Variables between the groups with or without promoter IVF were not statistically different (Fisher test: P < 0.05); however, when 500 ng μL–1 was microinjected, no blastocysts were obtained. In the in vivo embryo production group, 111 presumptive zygotes were microinjected (n = 37; with plasmid alone) from 16 donor sheep (11.5 ± 4.0 corpora lutea; 8.4 ± 4.8 presumptive zygotes recovered; 74.3% recovery rate). The mean time from injection to cleavage was 18.0 ± 4.5 h, and the percentage of cleavage and damage (due to the embryo injection) were >70% and <10%, respectively. Fifty-eight good quality embryos were transferred into the oviducts of 19 surrogate ewes; 12 of them are pregnant (63.1%). The presence of green IVF embryos demonstrates that eDNA was transported to the nucleus after cytoplasmic injection. We believe that the multi-fold increase (50- to 100-fold) in plasmid concentration compared with that used by others was the key step to our successful cytoplasmic microinjection. Accordingly, the new/old methodology described in this study provides an easy DNA construct delivery system of interest for the implementation of early reprogramming events. In addition, results obtained in the near future using in vivo cytoplasmic microinjection with high concentrations of eDNA could revalidate this technique for producing genetically modified large animals.

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The Diverse Potential of Gluten from Different Durum Wheat Varieties in Triggering Celiac Disease: A Multilevel In Vitro, Ex Vivo and In Vivo Approach

Nutrients ◽

10.3390/nu12113566 ◽

2020 ◽

Vol 12 (11) ◽

pp. 3566

Author(s):

Federica Gaiani ◽

Sara Graziano ◽

Fatma Boukid ◽

Barbara Prandi ◽

Lorena Bottarelli ◽

...

Keyword(s):

Immune Response ◽

Celiac Disease ◽

Durum Wheat ◽

Ex Vivo ◽

Diet Group ◽

Control Group ◽

Wheat Varieties ◽

Before And After

The reasons behind the increasing prevalence of celiac disease (CD) worldwide are still not fully understood. This study adopted a multilevel approach (in vitro, ex vivo, in vivo) to assess the potential of gluten from different wheat varieties in triggering CD. Peptides triggering CD were identified and quantified in mixtures generated from simulated gastrointestinal digestion of wheat varieties (n = 82). Multivariate statistics enabled the discrimination of varieties generating low impact on CD (e.g., Saragolla) and high impact (e.g., Cappelli). Enrolled subjects (n = 46) were: 19 healthy subjects included in the control group; 27 celiac patients enrolled for the in vivo phase. Celiacs were divided into a gluten-free diet group (CD-GFD), and a GFD with Saragolla-based pasta group (CD-Sar). The diet was followed for 3 months. Data were compared between CD-Sar and CD-GFD before and after the experimental diet, demonstrating a limited ability of Saragolla to trigger immunity, although not comparable to a GFD. Ex vivo studies showed that Saragolla and Cappelli activated immune responses, although with great variability among patients. The diverse potential of durum wheat varieties in triggering CD immune response was demonstrated. Saragolla is not indicated for celiacs, yet it has a limited potential to trigger adverse immune response.

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In vitro and in vivo antileishmanial efficacy of a combination therapy of diminazene and artesunate against Leishmania donovani in BALB/c mice

Revista do Instituto de Medicina Tropical de São Paulo ◽

10.1590/s0036-46652011000300003 ◽

2011 ◽

Vol 53 (3) ◽

pp. 129-132 ◽

Cited By ~ 12

Author(s):

Joshua Muli Mutiso ◽

John Chege Macharia ◽

Mustafa Barasa ◽

Evans Taracha ◽

Alain J. Bourdichon ◽

...

Keyword(s):

Leishmania Donovani ◽

Combined Therapy ◽

Parasite Burden ◽

Mice Model ◽

In Vivo Evaluation ◽

Reference Drug ◽

Drug Treatments ◽

Potential Use

The in vitro and in vivo activity of diminazene (Dim), artesunate (Art) and combination of Dim and Art (Dim-Art) against Leishmania donovani was compared to reference drug; amphotericin B. IC50 of Dim-Art was found to be 2.28 ± 0.24 µg/mL while those of Dim and Art were 9.16 ± 0.3 µg/mL and 4.64 ± 0.48 µg/mL respectively. The IC50 for Amphot B was 0.16 ± 0.32 µg/mL against stationary-phase promastigotes. In vivo evaluation in the L. donovani BALB/c mice model indicated that treatments with the combined drug therapy at doses of 12.5 mg/kg for 28 consecutive days significantly (p < 0.001) reduced parasite burden in the spleen as compared to the single drug treatments given at the same dosages. Although parasite burden was slightly lower (p < 0.05) in the Amphot B group than in the Dim-Art treatment group, the present study demonstrates the positive advantage and the potential use of the combined therapy of Dim-Art over the constituent drugs, Dim or Art when used alone. Further evaluation is recommended to determine the most efficacious combination ratio of the two compounds.

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Antileishmanial Efficacy and Pharmacokinetics of DB766-Azole Combinations

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.01129-17 ◽

2017 ◽

Vol 62 (1) ◽

Cited By ~ 4

Author(s):

April C. Joice ◽

Sihyung Yang ◽

Abdelbasset A. Farahat ◽

Heidi Meeds ◽

Mei Feng ◽

...

Keyword(s):

Visceral Leishmaniasis ◽

Leishmania Donovani ◽

Treatment Strategies ◽

Parasite Burden ◽

Pharmacokinetic Analysis ◽

Content Type ◽

New Treatment ◽

Almost All

ABSTRACT Given the limitations of current antileishmanial drugs and the utility of oral combination therapy for other infections, developing an oral combination against visceral leishmaniasis should be a high priority. In vitro combination studies with DB766 and antifungal azoles against intracellular Leishmania donovani showed that posaconazole and ketoconazole, but not fluconazole, enhanced DB766 potency. Pharmacokinetic analysis of DB766-azole combinations in uninfected Swiss Webster mice revealed that DB766 exposure was increased by higher posaconazole and ketoconazole doses, while DB766 decreased ketoconazole exposure. In L. donovani-infected BALB/c mice, DB766-posaconazole combinations given orally for 5 days were more effective than DB766 or posaconazole alone. For example, 81% ± 1% (means ± standard errors) inhibition of liver parasite burden was observed for 37.5 mg/kg of body weight DB766 plus 15 mg/kg posaconazole, while 37.5 mg/kg DB766 and 15 mg/kg posaconazole administered as monotherapy gave 40% ± 5% and 21% ± 3% inhibition, respectively. Combination index (CI) analysis indicated that synergy or moderate synergy was observed in six of nine combined dose groups, while the other three were nearly additive. Liver concentrations of DB766 and posaconazole increased in almost all combination groups compared to monotherapy groups, although many increases were not statistically significant. For DB766-ketoconazole combinations evaluated in this model, two were antagonistic, one displayed synergy, and one was nearly additive. These data indicate that the efficacy of DB766-posaconazole and DB766-ketoconazole combinations in vivo is influenced in part by the pharmacokinetics of the combination, and that the former combination deserves further consideration in developing new treatment strategies against visceral leishmaniasis.

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LmjF.22.0810 from Leishmania major Modulates the Th2-Type Immune Response and Is Involved in Leishmaniasis Outcome

Biomedicines ◽

10.3390/biomedicines8110452 ◽

2020 ◽

Vol 8 (11) ◽

pp. 452

Author(s):

Andrés Vacas ◽

Celia Fernández-Rubio ◽

Esther Larrea ◽

José Peña-Guerrero ◽

Paul A. Nguewa

Keyword(s):

Immune Response ◽

Protein Kinase ◽

Leishmania Major ◽

Parasite Burden ◽

Mrna Levels ◽

Expression Change ◽

Arginase 1 ◽

Th2 Immune Response

A novel serine/threonine protein kinase, LmjF.22.0810, was recently described in Leishmania major. After generating an L. major cell line overexpressing LmjF.22.0810 (named LmJ3OE), the ability of this novel protein to modulate the Th2-type immune response was analyzed. Our results suggest that the protein kinase LmjF.22.0810 might be involved in leishmaniasis outcomes. Indeed, our study outlined the LmJ3OE parasites infectivity in vitro and in vivo. Transgenic parasites displayed lower phagocytosis rates in vitro, and their promastigote forms exhibited lower expression levels of virulence factors compared to their counterparts in control parasites. In addition, LmJ3OE parasites developed significantly smaller footpad swelling in susceptible BALB/c mice. Hematoxylin–eosin staining allowed the observation of a lower inflammatory infiltrate in the footpad from LmJ3OE-infected mice compared to animals inoculated with control parasites. Gene expression of Th2-associated cytokines and effectors revealed a dramatically lower induction in interleukin (IL)-4, IL-10, and arginase 1 (ARG1) mRNA levels at the beginning of the swelling; no expression change was found in Th1-associated cytokines except for IL-12. Accordingly, such results were validated by immunohistochemistry studies, illustrating a weaker expression of ARG1 and a similar induction for inducible NO synthase (iNOS) in footpads from LmJ3OE-infected mice compared to control L. major infected animals. Furthermore, the parasite burden was lower in footpads from LmJ3OE-infected mice. Our analysis indicated that such significant smaller footpad swellings might be due to an impairment of the Th2 immune response that subsequently benefits Th1 prevalence. Altogether, these studies depict LmjF.22.0810 as a potential modulator of host immune responses to Leishmania. Finally, this promising target might be involved in the modulation of infection outcome.

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HIV-Infected Patients Developing Tuberculosis Disease Show Early Changes in the Immune Response to Novel Mycobacterium tuberculosis Antigens

Frontiers in Immunology ◽

10.3389/fimmu.2021.620622 ◽

2021 ◽

Vol 12 ◽

Author(s):

Noemi Rebecca Meier ◽

Manuel Battegay ◽

Tom H. M. Ottenhoff ◽

Hansjakob Furrer ◽

Johannes Nemeth ◽

...

Keyword(s):

Immune Response ◽

Mycobacterium Tuberculosis ◽

Early Diagnosis ◽

Early Stage ◽

Control Group ◽

Rapid Progression ◽

Operating Characteristics ◽

Tnf Α

Background: In individuals living with HIV infection the development of tuberculosis (TB) is associated with rapid progression from asymptomatic TB infection to active TB disease. Sputum-based diagnostic tests for TB have low sensitivity in minimal and subclinical TB precluding early diagnosis. The immune response to novel Mycobacterium tuberculosis in-vivo expressed and latency associated antigens may help to measure the early stages of infection and disease progression and thereby improve early diagnosis of active TB disease.Methods: Serial prospectively sampled cryopreserved lymphocytes from patients of the Swiss HIV Cohort Study developing TB disease (“cases”) and matched patients with no TB disease (“controls”) were stimulated with 10 novel Mycobacterium tuberculosis antigens. Cytokine concentrations were measured in cases and controls at four time points prior to diagnosis of TB: T1-T4 with T4 being the closest time point to diagnosis.Results: 50 samples from nine cases and nine controls were included. Median CD4 cell count at T4 was 289/ul for the TB-group and 456/ul for the control group. Viral loads were suppressed in both groups. At T4 Rv2431c-induced and Rv3614/15c-induced interferon gamma-induced protein (IP)-10 responses and Rv2031c-induced and Rv2346/Rv2347c-induced tumor necrosis factor (TNF)-α responses were significantly higher in cases compared to controls (p < 0.004). At T3 - being up to 2 years prior to TB diagnosis - Rv2031c-induced TNF-α was significantly higher in cases compared to controls (p < 0.004). Area under the receiver operating characteristics (AUROC) curves resulted in an AUC > 0.92 for all four antigen-cytokine pairs.Conclusion: The in vitro Mycobacterium tuberculosis-specific immune response in HIV-infected individuals that progress toward developing TB disease is different from those in HIV-infected individuals that do not progress to developing TB. These differences precede the clinical diagnosis of active TB up to 2 years, paving the way for the development of immune based diagnostics to predict TB disease at an early stage.

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Antileishmanial Activities of Stearylamine-Bearing Liposomes

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.44.6.1739-1742.2000 ◽

2000 ◽

Vol 44 (6) ◽

pp. 1739-1742 ◽

Cited By ~ 37

Author(s):

Tuhina Dey ◽

Khairul Anam ◽

Farhat Afrin ◽

Nahid Ali

Keyword(s):

Single Dose ◽

Visceral Leishmaniasis ◽

Leishmania Donovani ◽

Therapeutic Potential ◽

Parasite Burden ◽

Intracellular Amastigotes ◽

Antileishmanial Activities

ABSTRACT Here we report the activity of liposomes comprising egg phosphatidylcholine (PC) and stearylamine (SA) against Leishmania donovani parasites. Both promastigotes and intracellular amastigotes in vitro and in vivo were susceptible to SA-PC liposomes. A single dose of 55 mg of SA-PC liposomes/animal could significantly reduce the hepatic parasite burden by 85 and 68% against recent and established experimental visceral leishmaniasis, respectively, suggesting their strong therapeutic potential.

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PREPARATION AND CHARACTERIZATION OF ANDROGRAPHOLIDE NANOPARTICLES FOR VISCERAL LEISHMANIASIS CHEMOTHERAPY: IN VITRO AND IN VIVO EVALUATIONS

International Journal of Pharmacy and Pharmaceutical Sciences ◽

10.22159/ijpps.2016v8i12.14773 ◽

2016 ◽

Vol 8 (12) ◽

pp. 102

Author(s):

Pallab Ghosh ◽

Subhasish Mondal ◽

Tanmoy Bera

Keyword(s):

Visceral Leishmaniasis ◽

Leishmania Donovani ◽

Parasite Burden ◽

Plga Nanoparticles ◽

Antileishmanial Activity ◽

Infection Model ◽

Vitamin E Tpgs ◽

Polyethylene Glycol 1000

Objective: To overcome low physiological solubility, poor bioavailability, the short plasma half-life of andrographolide (AG), a delivery system based on poly (D, L-lactide-co-glycolide) (PLGA) nanoparticles (NPs) were developed to increase the efficiency of AG against visceral leishmaniasis (VL). Methods: Andrographolide-PLGA nanoparticles (AGnp) were prepared with Pgp efflux inhibitor vitamin E TPGS (D-α-tocopheryl polyethylene glycol 1000 succinate) by emulsion solvent evaporation method and characterized. Antileishmanial activity was evaluated using in vitro and in vivo VL infection model. Results: The particle size of AGnp was found to be171.4±11.5 nm with an encapsulation efficiency of 81%. The AGnp reduced AG cellular toxicity, retained it's in vitro antileishmanial activity and lead to a reduction (99.9%) of parasite burden in the Leishmania donovani infected spleen and liver. AGnp was more active in infected mice liver at low dose than in spleen. Therapeutic indexes (TI) were 6.9-fold greater in AG and 68-fold in AGnp compared to amphotericin B (AmB) when evaluated in L. donovani infected spleen. Conclusion: Incorporation of AG in PLGA nanoparticles, provided controlled and improved in vivo performance against VL

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TheREnantiomer of the Antitubercular Drug PA-824 as a Potential Oral Treatment for Visceral Leishmaniasis

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.00722-13 ◽

2013 ◽

Vol 57 (10) ◽

pp. 4699-4706 ◽

Cited By ~ 38

Author(s):

Stephen Patterson ◽

Susan Wyllie ◽

Laste Stojanovski ◽

Meghan R. Perry ◽

Frederick R. C. Simeons ◽

...

Keyword(s):

Clinical Trials ◽

Visceral Leishmaniasis ◽

Phase Ii ◽

Leishmania Donovani ◽

Oral Treatment ◽

Parasite Burden ◽

Content Type ◽

Phase Ii Clinical Trials

ABSTRACTThe novel nitroimidazopyran agent (S)-PA-824 has potent antibacterial activity againstMycobacterium tuberculosisin vitroandin vivoand is currently in phase II clinical trials for tuberculosis (TB). In contrast toM. tuberculosis, where (R)-PA-824 is inactive, we report here that both enantiomers of PA-824 show potent parasiticidal activity againstLeishmania donovani, the causative agent of visceral leishmaniasis (VL). In leishmania-infected macrophages, (R)-PA-824 is 6-fold more active than (S)-PA-824. Both des-nitro analogues are inactive, underlining the importance of the nitro group in the mechanism of action. Although thein vitroandin vivopharmacological profiles of the two enantiomers are similar, (R)-PA-824 is more efficacious in the murine model of VL, with >99% suppression of parasite burden when administered orally at 100 mg kg of body weight−1, twice daily for 5 days. InM. tuberculosis, (S)-PA-824 is a prodrug that is activated by a deazaflavin-dependent nitroreductase (Ddn), an enzyme which is absent inLeishmaniaspp. Unlike the case with nifurtimox and fexinidazole, transgenic parasites overexpressing the leishmania nitroreductase are not hypersensitive to either (R)-PA-824 or (S)-PA-824, indicating that this enzyme is not the primary target of these compounds. Drug combination studiesin vitroindicate that fexinidazole and (R)-PA-824 are additive whereas (S)-PA-824 and (R)-PA-824 show mild antagonistic behavior. Thus, (R)-PA-824 is a promising candidate for late lead optimization for VL and may have potential for future use in combination therapy with fexinidazole, currently in phase II clinical trials against VL.

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