Intracellular Killing Activity Research Articles

Macrophage targeted polymeric curcumin nanoparticles limit intracellular survival of Mycobacterium tuberculosis through induction of autophagy and augment anti-TB activity of isoniazid in RAW 264.7 macrophages.

Rapid emergence of antibiotic resistance in tuberculosis has left us with limited resources to treat and manage multi drug resistant (MDR) cases of tuberculosis, prompting the development of novel therapeutics. Mycobacterium tuberculosis (MTB) perturbs the host protective pathways for its survival, therefore host directed therapeutic (HDT) interventions offer an attractive alternative strategy. Curcumin (CMN), the principle curcuminoid from Curcuma longa is known to have anti-TB activity against MDR strains of MTB in macrophages. We discovered that treatment of CMN induced autophagy in uninfected and MTB infected macrophages which was evident by conversion of LC3-I to LC3-II and degradation of p62. Inhibition of autophagy by a pharmacological inhibitor 3-MA resulted in significant inhibition of intracellular killing activity of CMN, suggesting the involvement of autophagy in intracellular clearance of MTB. Moreover, annexin v-FITC/PI staining data suggested induction of apoptosis in uninfected and MTB infected macrophages post CMN treatment. This finding was further corroborated by up-regulated expression of pro-apoptotic proteins, Bax, cleaved caspase-3 and PARP and diminished expression of anti-apoptotic protein Bcl-2 as evaluated by immunoblotting. Using GFP-MTB H37Rv and Lysotracker Red staining we demonstrated co-localization of GFP-MTB H37Rv containing phagosome to lysosome after CMN treatment, indicating enhanced phagosome lysosome fusion. Due to poor bioavailability of CMN, its clinical use is limited, therefore to overcome this issue, CMN was encapsulated in Poly(lactic-co-glycolic) acid (PLGA) shell, resulting in polymeric CMN nano particles (ISCurNP). Flow cytometric evaluation suggested >99% uptake of ISCurNP after 3h of treatment. In BALB/c mice, oral dose of ISCurNP resulted in 6.7-fold increase in the bioavailability compared to free CMN. Moreover, ISCurNP treatment resulted in significant decrease in the intracellular survival of MTB H37Rv through induction of autophagy. Adjunct action of ISCurNP and CMN in combination with isoniazid (INH) revealed >99% decrease in intracellular survival of MTB in macrophage as compared to ISCurNP, CMN or INH alone. In conclusion, our findings suggest the role of ISCurNP as novel host directed formulation to combat both sensitive and MDR strains of MTB by induction of autophagy.

Heavy metal bioaccumulation triggers oxystress, genotoxicity and immunomodulation in head kidney macrophages of Channa punctatus Bloch.

Studies on heavy metal induced toxicity have been conducted in many water bodies across the globe and such effects have been evaluated in various fish species. The present study was designed to determine the load of some heavy metals in select sites in Southern Assam, India, along with estimating their concentration in tissues of Channa punctatus Bloch. inhabiting those niches. The effect of heavy metals in oxystress generation, genotoxicity and subsequent immune response in fish was also evaluated. In all of these sites, the concentration of Hg, Cd, Pb and Cr were above the permissible ranges while their concentrations were several folds higher in the piscine tissues due to bioaccumulation and possible biomagnification. Kidney showed the highest metal pollution index followed by liver and gills. Generation of ROS was significantly elevated and that in turn triggered oxystress, as is evident from enhanced lipid peroxidation, protein carbonylation and respiratory burst activity. These were in association with the compromised antioxidant enzyme levels with concomitant damage to DNA as evident from Comet parameters. The innate immune potential was significantly impaired as evident from the compromised cell adhesion, phagocytosis, intracellular killing activity in head kidney macrophages (HKM) along with decreased release of nitric oxide (NO) and myeloperoxidase (MPO). Immunosuppression was further validated at protein levels where compromised release of cytokines viz. TNF-α, IL-1β, IL-6, IL-10 and IL-12 and cell signaling molecules iNOS and NF-κβ were noted. Thus the present study indicates genotoxicity along with a compromise in immune status of Channa punctatus Bloch. living in a habitat laden with heavy metals.

Novel oxazolidinones harbor potent in vitro activity against the clinical isolates of multidrug-resistant Mycobacterium tuberculosis in China.

To investigate the in vitro activities of five oxazolidinones in parallel against the reference strains of different mycobacterial species and clinical isolates of Mycobacterium tuberculosis (Mtb), and shed light on the differences in the efficacy of these homolog drugs. The minimum inhibitory concentrations (MICs) of linezolid, tedizolid, sutezolid, delpazolid, and contezolid against 16 mycobacterial reference strains and 69 M. tuberculosis clinical isolates, including 17 drug-susceptible isolates and 52 multidrug-resistant (MDR) isolates, were determined by microplate alamarBlue assay (MABA). The intracellular killing activities of contezolid and linezolid against Mtb H37Rv were compared. In addition, mutations in the linezolid resistance-related genes (rplC, rplD, and 23S rRNA) of the Mtb clinical isolates were also analyzed. Tedizolid exhibited the strongest inhibitory activities against the reference strains of both rapidly growing mycobacteria (RGM) and slowly growing mycobacteria (SGM), among the tested oxazolidinones. In contrast, sutezolid only manifested potent activity against reference strains of SGM. Linezolid, delpazolid, and contezolid were less active against the non-tuberculous mycobacterial references. For the Mtb clinical isolates, the antimicrobial action was ranked as: sutezolid > tedizolid > contezolid and linezolid > delpazolid, whereas no difference between drug-sensitive and multiple drug-resistant isolates was observed. Notably, contezolid demonstrated obviously superior intracellular antimicrobial activity than linezolid. Few strains harbored mutations in rrl gene or rplD genes, although these strains had drug susceptible profiles to linezolid. Different oxazolidinones can have discrepant antimicrobial activity against different mycobacterial species, or have different manifestations out of cell or in cell. Understanding these differences would be helpful in choosing the appropriate drug in clinical practice.

MEGAMOX MEDIATES ECO-FRIENDLY SYNTHESIS OF SELENIUM NANOPARTICLES AS IMMUNOMODULATORY, ANTIOXIDANT AND ANTIMICROBIAL AGENTS

The research herein includes methodology for eco-friendly preparation of selenium nanoparticles (SeNPs) using the broad-spectrum antibiotic, Megamox (Mega.). Characterization of SeNPs was done using UV-Visible spectroscopy, FTIR, XRD, DLS and TEM imaging. Additionally, the immunomodulatory, antioxidant and antimicrobial activities of both SeNPs and Mega. were checked against different strains of bacteria and fungi. TEM and DLS images showed that SeNPs are polydisperse spheres with mean diameter of 22.4 nm. FTIR analysis indicated that the hydroxyl and nitrogen moieties in Mega. were effective for reduction plus binding manner. According to the results of the nitro blue tetrazolium reduction test, both SeNPs and Mega. presented high intracellular killing activity, which confirmed their immunostimulatory effect. Antioxidant activity of SeNPs and Mega. were 90 and 82 %, respectively. SeNPs presented great activity facing multi-drug resistant bacteria and TB. SeNPs are considered promising cost-effective and eco-friendly anti-bacterial and anti-fungal agents that can represent a new potential nano-platform in both medical and infectious diseases control.

Osteocytes Serve as a Reservoir for Intracellular Persisting Staphylococcus aureus Due to the Lack of Defense Mechanisms.

Chronic staphylococcal osteomyelitis can persist for long time periods causing bone destruction. The ability of Staphylococcus aureus to develop chronic infections is linked to its capacity to invade and replicate within osteoblasts and osteocytes and to switch to a dormant phenotype called small colony variants. Recently, osteocytes were described as a main reservoir for this pathogen in bone tissue. However, the mechanisms involved in the persistence of S. aureus within these cells are still unknown. Here, we investigated the interaction between S. aureus and osteoblasts or osteocytes during infection. While osteoblasts are able to induce a strong antimicrobial response and eliminate intracellular S. aureus, osteocytes trigger signals to recruit immune cells and enhance inflammation but fail an efficient antimicrobial activity to clear the bacterial infection. Moreover, we found that extracellular signals from osteocytes enhance intracellular bacterial clearance by osteoblasts. Even though both cell types express Toll-like receptor (TLR) 2, the main TLR responsible for S. aureus detection, only osteoblasts were able to increase TLR2 expression after infection. Additionally, proteomic analysis indicates that reduced intracellular bacterial killing activity in osteocytes is related to low antimicrobial peptide expression. Nevertheless, high levels of lipid mediators and cytokines were secreted by osteocytes, suggesting that they can contribute to inflammation. Taken together, our results demonstrate that osteocytes contribute to severe inflammation observed in osteomyelitis and represent the main niche for S. aureus persistence due to their poor capacity for intracellular antimicrobial response.

Novel Assay Platform to Evaluate Intracellular Killing of Mycobacterium tuberculosis: In Vitro and In Vivo Validation.

One of the main hallmarks of tuberculosis (TB) is the ability of the causative agent to transform into a stage of dormancy and the capability of long persistence in the host phagocytes. It is believed that approximately one-third of the population of the world is latently infected with Mycobacterium tuberculosis (Mtb), and 5%–10% of these individuals can develop clinical manifestations of active TB even decades after the initial infection. In this latent, intracellular form, the bacillus is shielded by an extremely robust cell wall and becomes phenotypically resistant to most antituberculars. Therefore, there is a clear rationale to develop novel compounds or carrier-conjugated constructs of existing drugs that are effective against the intracellular form of the bacilli. In this paper, we describe an experimental road map to define optimal candidates against intracellular Mtb and potential compounds effective in the therapy of latent TB. To validate our approach, isoniazid, a first-line antitubercular drug was employed, which is active against extracellular Mtb in the submicromolar range, but ineffective against the intracellular form of the bacteria. Cationic peptide conjugates of isoniazid were synthesized and employed to study the host-directed drug delivery. To measure the intracellular killing activity of the compounds, Mtb-infected MonoMac-6 human monocytic cells were utilized. We have assessed the antitubercular activity, cytotoxicity, membrane interactions in combination with internalization efficacy, localization, and penetration ability on interface and tissue-mimicking 3D models. Based on these in vitro data, most active compounds were further evaluated in vivo in a murine model of TB. Intraperitoneal infectious route was employed to induce a course of slowly progressive and systemic disease. The well-being of the animals, monitored by the body weight, allows a prolonged experimental setup and provides a great opportunity to test the long-term activity of the drug candidates. Having shown the great potency of this simple and suitable experimental design for antimicrobial research, the proposed novel assay platform could be used in the future to develop further innovative and highly effective antituberculars.

Antibacterial Activity of Ikarugamycin against Intracellular Staphylococcus aureus in Bovine Mammary Epithelial Cells In Vitro Infection Model.

Simple SummaryAntibiotics are widely used for the treatment and control of bovine mastitis. However, the treatment has only been partially effective, as the cure percentage only ranging from 10–30%. Infection by Staphylococcus aureus (S. aureus) is particularly difficult to treat due to the bacteria’s ability to enter and resides inside the host cells. Most antibiotics are ineffective against intracellular bacterial due to the poor penetration into host cells to achieve optimal intracellular bactericidal bioavailability levels. There is therefore, an increasing need to evaluate candidate active substances and develop novel antibiotics effective against intracellular persistence infection. In this study, we examine the potential antibacterial properties of ikarugamycin compound as an alternative drug candidate to be explored for treating persistent bovine mastitis caused by intracellular S. aureus using bovine mammary cell line as an in vitro infection model. We also assessed the potential cytotoxicity effect of ikarugamycin in the infection model. We found that, the ikarugamycin possessed intracellular killing activity against S. aureus within the mammary epithelial cell. This finding highlights the potential application of ikarugamycin as a novel antimicrobial for the treatment of S. aureus mastitis.Staphylococcus aureus is an ubiquitous and versatile pathogen associated with a wide range of diseases. In animals, this bacterium is one of the causative agents of bovine mastitis, responsible for huge economic losses in the dairy industry. Besides the development of antibiotic resistance, the intracellular survival of S. aureus within udder cells has rendered many antibiotics ineffective, leading to therapeutic failure. Our study therefore aims to investigate the in vitro bactericidal activity of ikarugamycin (IKA) against intracellular S. aureus using a bovine mammary epithelial cells (Mac-T cells) infection model and determine the cytotoxic effect. Minimum inhibitory concentration (MIC) was used to determine the antibacterial activity of IKA, and Mac-T cells were infected with S. aureus using gentamicin protection assay. IKA intracellular antibacterial activity assays were used to determine the bactericidal activity of IKA against intracellular S. aureus. The cytotoxicity of IKA against Mac-T cells was evaluated using the resazurin assay. We showed that, S. aureus is susceptible to IKA with a MIC value of 0.6 μg/mL. IKA at 4 × MIC and 8 × MIC have bactericidal activity by reducing 3 and 5 logs10 CFU/mL of S. aureus in the first six-hour of treatment respectively. In addition, IKA demonstrated intracellular killing activity by killing 90% of intracellular S. aureus at 5 μg/mL. This level is comparatively lower than 9.2 μg/mL determined as the half-maximal inhibitory concentration (IC50) of IKA required to kill 50% of Mac-T cells, highlighting a lower concentration required for bactericidal effect compared to the cytotoxic effect. The study highlighted that importance of IKA as a potential antibiotic candidate to be explored for the in vivo efficacy in treating S. aureus mastitis.

A Clofazimine-Containing Regimen Confers Improved Treatment Outcomes in Macrophages and in a Murine Model of Chronic Progressive Pulmonary Infection Caused by the Mycobacterium avium Complex.

Treatment outcomes using the standard regimen (a macrolide, ethambutol, and rifampicin) for Mycobacterium avium complex-pulmonary disease (MAC-PD) remain unsatisfactory. Thus, improved treatment regimens for MAC-PD are required. Clofazimine has recently been revisited as an effective drug against mycobacterial infection. We performed a comparison between the standard regimen and an alternative regimen (replacing the rifampicin of the standard regimen with clofazimine) based on the intracellular anti-MAC activities of the individual drugs in a murine model of chronic progressive MAC-pulmonary infection (MAC-PI). The intracellular anti-MAC activities of the individual drugs and their combinations in murine bone marrow-derived macrophages (BMDMs) were determined. The treatment efficacies of the standard and clofazimine-containing regimens were evaluated in mice chronically infected with M. avium by initiating 2- and 4-week treatment at 8 weeks post-infection. Bacterial loads in the lung, spleen, and liver were assessed along with lung inflammation. Insufficient intracellular anti-MAC activity of rifampicin in BMDMs was recorded despite its low in vitro minimum inhibitory concentrations (MICs), whereas optimal intracellular killing activity against all tested MAC strains was achieved with clofazimine. Compared to the standard regimen, the clofazimine-containing regimen significantly reduced CFUs in all organs and achieved marked reductions in lung inflammation. The replacement of rifampicin with clofazimine in the treatment regimen resulted in more favorable outcomes in an animal model of chronic progressive MAC-PI. Intriguingly, 2 weeks of treatment with the clofazimine-containing regimen reduced bacterial loads more effectively than 4 weeks of treatment with the standard regimen in M. avium-infected mice. Thus, the clofazimine-containing regimen also had a treatment-shortening effect.

Short-Chain Fatty Acids Promote Intracellular Bactericidal Activity in Head Kidney Macrophages From Turbot (Scophthalmus maximus L.) via Hypoxia Inducible Factor-1α.

Short-chain fatty acids (SCFAs) are mainly produced by microbiota through the fermentation of carbohydrates in the intestine. Acetate, propionate, and butyrate are the most abundant SCFA metabolites and have been shown to be important in the maintenance of host health. In this study, head kidney macrophages (HKMs) were isolated and cultured from turbots. We found that the antibacterial activity of HKMs was increased after these cells were incubated with sodium butyrate, sodium propionate or sodium acetate. Interestingly, our results showed that all three SCFAs enhanced the expression of hypoxia inducible factor-1 α (HIF-1α) in HKMs, and further study confirmed that butyrate augmented the oxygen consumption of these cells. Moreover, HIF-1α inhibition diminished the butyrate-promoted intracellular bacterial killing activity of macrophages, and SCFAs also raised the gene expression and activity of lysozymes in HKMs via HIF-1α signaling. In addition, our results suggested that butyrate induced HIF-1α expression and the bactericidal activity of HKMs through histone deacetylase inhibition, while G protein-coupled receptors did not contribute to this effect. Finally, we demonstrated that butyrate induced a similar response in the murine macrophage cell line RAW264.7. In conclusion, our results demonstrated that SCFAs promoted HIF-1α expression via histone deacetylase inhibition, leading to the enhanced production of antibacterial effectors and increased bacterial killing of macrophages.

Mechanism of Microvascular Endothelial Cells Protected by Pulsatillae Decoction Activated Neutrophils to Kill Bacteria

Abstract Neutrophils play critical a role in bacterial infection and tumor immunity. The migration of neutrophils across microvascular endothelial cells has been a focus of increased interest within the past decade. A traditional Chinese herbal formula, Pulsatillae Decoction (PD), has the effects of improving the permeability of MVECs, inhibiting apoptosis and enhancing cell activity. But there are very few studies linking the three, and the effect of PD on the bactericidal function of PMNs transendothelial migration is unclear. To investigate the protective effect of PD on the damage of Rat microvascular endothelial cells (RIMVECs) induced by LPS and it’s mechanism of activating neutrophils (PMNs) to kill bacteria by protecting RIMVECs, we established a model of LPS-induced RIMVECs injury and to evaluate the protection of PD against LPS-induced RIMVECs injury. We observed that LPS-injured RIMVECs inhibited the extracellular and intracellular bactericidal activity of transendothelial neutrophils, PD-protected RIMVECs promoted the extracellular and intracellular killing activity of transendothelial neutrophils and significantly increased NE and CG concentration in transendothelial neutrophils. In addition, SWATH/DIA proteome results showed that 11 consensus proteins were down-regulated in the model group and up-regulated in the treatment group, 23 consensus proteins were up-regulated in the model group and down-regulated in the treatment group. PD was likely to protect RIMVECs and activate the sterilization of PMNs by regulating these differential proteins.

Aspirin enhances regulatory functional activities of monocytes and downregulates CD16 and CD40 expression in myocardial infarction autoinflammatory disease

BackgroundExacerbation of CD16 as molecule marker of both intermediate and non-classical monocytes (MOs) has been shown to be involved in the pathogenesis of myocardial infarction (MI). In this study, we have tried to evaluate the aspirin (acetylsalicylic acid, ASA) treatment effect on the CD16-expressed MOs and activation-associated CD40 in MI. MethodsMOs were isolated from the whole blood of healthy controls and patients with MI. The cells were stimulated and treated with different doses of ASA. ResultsASA significantly decreased nitric oxide (NO) production and inducible NO synthase (iNOS) activity, but significantly increased arginase activity. Levels of interleukin (IL)-1β, IL-6 and interferon-γ (IFN-γ) were downregulated, whereas those of IL-10 were upregulated. Additionally, ASA induced a markedly increase in both phagocytosis and intracellular pathogen killing activities. Moreover, ASA treatment induced significantly upregulation of intracellular levels of glucose (iGlu), and free calcium ions (ifCa2+), and, covertly, significantly downregulation of total cellular cholesterol content (tccCHOL). Furthermore, the expression levels of CD16 and CD40 were significantly downregulated in ASA-treated MOs. ConclusionsWe show for the first time that ASA immunomodulates the functional activities of MOs during MI and promotes their switching toward a classical phenotype, exhibiting low CD16 expression levels and thereby anti-inflammatory properties.

Effect of dietary resveratrol on cell-mediated immunity and hepatocyte morphometry in the model organism medaka (Oryzias latipes Temminck & Schlegel)

Abstract The effect of dietary resveratrol (40 µg g−1 BW day−1) on cell-mediated immunity (the activity of spleen phagocytes and the proliferative response of lymphocytes) and liver histology (hepatocyte morphometry, lipid vacuoles, and glycogen granules) in adult medaka (Oryzias latipes) (aged two years, body weight BW = 0.49 g) were tested after ten days of feeding with experimental diets. The fish were fed either a commercial diet (control group C) or this same diet supplemented with resveratrol (group R) three times daily at 3% of body weight (BW). Fish fed feed with resveratrol had significantly higher intracellular phagocyte killing activity than did those in the control group (P < 0.05). No differences in the sizes of hepatocytes or their nuclei were observed in the fish from groups C and R. The nucleo-cytoplasmic ratio ranged from 0.39 (± 0.03; group R) to 0.42 (± 0.03, group C) in adult medaka exposed to the two dietary treatments (P > 0.05). The results indicated that the diet supplemented with resveratrol at a dose of 40 µg g−1 BW day−1 had no impact on the liver tissues. Typical hepatocytes with lipids and glycogen were observed to the same extent in the vacuoles. Moreover, no mortality or pathological changes were noted in the fish studied. The diet containing resveratrol helped to maintain disease resistance without affecting the liver tissue after ten days of feeding.

One-pot synthesis and molecular docking of some new spiropyranindol-2-one derivatives as immunomodulatory agents and in vitro antimicrobial potential with DNA gyrase inhibitor

a series of 2-oxospiro[indoline-3,4′-pyran]derivatives 4 and 7 were obtained in good yield under mild conditions from the one-pot reaction of indole-2,3-dione derivatives 1, appropriate methylene active nitriles 2 and β-dicarbonyl compound 3 or 6. The newly synthesized compounds were characterized and evaluated for their in vitro antibacterial, antifungal as well as immunomodulatory activity. According to MIC values, the most potent compounds 4f, 4h, 7a, 7c, 7e, 7f, 7g, 8a, and 8c were evaluated for MBC and displayed high activity to killing pathogens with a good MBC value against norfloxacin as well as investigated against an extended panel of multidrug resistance bacteria (MDRB) and exhibited promising to moderate multidrug resistance activities, compounds 7f showed the much better than norfloxacin with higher potency results. Furthermore, the most potent compounds showed an increase in the intracellular killing activity of neutrophils which confirmed the immunostimulatory power. Eight of the nine active compounds exhibited inhibitory activities with IC50 ranged between (18.07 ± 0.18) to (27.03 ± 0.24) μM stronger than ciprofloxacin (26.43 ± 0.64 μM) for S. aureus DNA gyrase. Molecular docking was performed inside the active site of S. aureus DNA gyrase to predict the binding mode.

DNA Sensor IFI204 Contributes to Host Defense Against Staphylococcus aureus Infection in Mice.

Interferon-inducible protein (IFI204) (p204, the murine homolog of human IFI16) is known as a cytosolic DNA sensor to recognize DNA viruses and intracellular bacteria. However, little is known about its role during extracellular bacterial infection. Here we show that IFI204 is required for host defense against the infection of Staphylococcus aureus, an extracellular bacterial pathogen. IFI204 deficiency results in decreased survival, increased bacterial loads, severe organs damage, and decreased recruitment of neutrophils and macrophages. Production of several inflammatory cytokines/chemokines including IFN-β and KC is markedly decreased, as well as the related STING-IRF3 and NF-κB pathways are impaired. However, exogenous administration of recombinant KC or IFN-β is unable to rescue the susceptibility of IFI204-deficient mice, suggesting that other mechanisms rather than KC and IFN-β account for IFI204-mediated host defense. IFI204 deficiency leads to a defect in extracellular bacterial killing in macrophages and neutrophils, although bacterial engulf, and intracellular killing activity are normal. Moreover, the defect of bactericidal activity is mediated by decreased extracellular trap formation in the absence of IFI204. Adoptively transferred WT bone marrow cells significantly protect WT and IFI204-deficient recipients against Staphylococcus infection compared with transferred IFI204-deficient bone marrow cells. Hence, this study suggests that IFI204 is essential for the host defense against Staphylococcus infection.

Biomimetic hydroxyapatite nanocrystals are an active carrier for Salmonella bacteriophages.

PurposeThe use of bacteriophages represents a valid alternative to conventional antimicrobial treatments, overcoming the widespread bacterial antibiotic resistance phenomenon. In this work, we evaluated whether biomimetic hydroxyapatite (HA) nanocrystals are able to enhance some properties of bacteriophages. The final goal of this study was to demonstrate that biomimetic HA nanocrystals can be used for bacteriophage delivery in the context of bacterial infections, and contribute – at the same time – to enhance some of the biological properties of the same bacteriophages such as stability, preservation, antimicrobial activity, and so on.Materials and methodsPhage isolation and characterization were carried out by using Mitomycin C and following double-layer agar technique. The biomimetic HA water suspension was synthesized in order to obtain nanocrystals with plate-like morphology and nanometric dimensions. The interaction of phages with the HA was investigated by dynamic light scattering and Zeta potential analyses. The cytotoxicity and intracellular killing activities of the phage–HA complex were evaluated in human hepatocellular carcinoma HepG2 cells. The bacterial inhibition capacity of the complex was assessed on chicken minced meat samples infected with Salmonella Rissen.ResultsOur data highlighted that the biomimetic HA nanocrystal–bacteriophage complex was more stable and more effective than phages alone in all tested experimental conditions.ConclusionOur results evidenced the important contribution of biomimetic HA nanocrystals: they act as an excellent carrier for bacteriophage delivery and enhance its biological characteristics. This study confirmed the significant role of the mineral HA when it is complexed with biological entities like bacteriophages, as it has been shown for molecules such as lactoferrin.

Potential immunomodulatory effects of non-dialyzable materials of cranberry extract in poultry production

Studies on effects of cranberry products in animals, especially in chickens, are very scarce or even lacking. This study investigated the immunomodulatory effect of high molecular weight non-dialyzable materials (NDMs) of cranberry extract. The in vitro antioxidant and anti-inflammatory activities were investigated. The ability of NDMs (0, 1, 2, or 4 mg/mL) to enhance phagocytic activities was also evaluated using chicken heterophils (CHEs) against Staphylococcus aureus. Furthermore, a broiler model was used to determine the effect of NDMs on the humoral immune response. Seven-d-old chicks were vaccinated with the infectious bursal disease virus (IBDV) vaccine S-706, and treated orally with 0, 2, 4, or 8 mg/mL/bird NDMs for five consecutive days. Serum immunoglobulin level (Ig), and antibody concentration against IBDV, infectious bronchitis virus (IBV), Newcastle disease virus (NDV), and avian reovirus (ARV) were measured weekly by enzyme-linked immunosorbent assay (ELISA). NDMs showed >five-fold higher antioxidant activity (oxygen radical absorbance capacity = 222.7 mg trolox/g) than the commercial raw cranberry juice from which it derived (oxygen radical absorbance capacity = 39.6 mg trolox/g). Likewise, NDMs demonstrated anti-inflammatory activities comparable to Naproxen but better than those of Ibuprofen. The susceptibility of S. aureus to phagocytosis by CHEs increased significantly (P < 0.05) at 4 mg/mL NDMs in the medium. While no intracellular bacteria were counted in CHEs after phagocytosis in the presence of 2 and 4 mg/mL NDMs, 1 mg/mL NDMs demonstrated a significant (P < 0.05) intracellular killing activity in CHEs against S. aureus compared to the untreated CHEs. Results from the in vivo studies indicated that birds receiving 2 and 4 mg/mL/bird NDMs had a higher serum IgM level (P < 0.05), and their antibody titers against IBDV tended to increase with NDMs administration (P = 0.06) on d 35. These results suggest that NDMs enhances bacterial susceptibility to immuno-defense mechanisms, and may be useful as immunomodulators against infections.

PD-1/PD-L pathway inhibits M.tb-specific CD4+ T-cell functions and phagocytosis of macrophages in active tuberculosis.

The role of the PD-1/PD-L pathway in a murine model of tuberculosis remains controversial regarding viral infections and clinical tuberculosis. We conducted a case-control study to investigate the modulating role and mechanism of the PD-1/PD-L pathway in patients with active tuberculosis. Fifty-nine participants, including 43 active tuberculosis (ATB) patients and 16 healthy controls (HC), were enrolled. Cell surface staining and flow cytometry were used to detect the expressions of PD-1 and its ligands on T cells and monocytes. Intracellular cytokine staining was used to determine the PPD-specific IFN-γ-secreting T-cell proportion. CD4+ T-cell proliferation and macrophage functions were investigated in the presence or absence of PD-1/PD-L pathway blockade. Proportions of both PD-1+CD4+ and PD-L1+CD4+ T cells in ATB patients were more significantly increased than in the HC group (P = 0.0112 and P = 0.0141, respectively). The expressions of PD-1, PD-L1, and PD-L2 on CD14+ monocytes in ATB patients were much higher than those in the HC group (P = 0.0016, P = 0.0001, and P = 0.0088, respectively). Blockade of PD-1 could significantly enhance CD4+ T-cell proliferation (P = 0.0433). Phagocytosis and intracellular killing activity of macrophages increased significantly with PD-1/PD-L pathway blockade. In conclusion, the PD-1/PD-L pathway inhibits not only M.tb-specific CD4+ T-cell-mediated immunity but also innate immunity.

Intracellular, biofilm-inhibitory and membrane-damaging activities of nimbolide isolated from Azadirachta indica A. Juss (Meliaceae) against meticillin-resistant Staphylococcus aureus.

Staphylococcus aureus is a leading aetiologic agent of nosocomial- and community-acquired infectious diseases worldwide. The public health concern regarding staphylococcal infections is inflated by the increasing occurrence of multidrug-resistant strains, e.g. multidrug- and meticillin-resistant S.aureus (MDR MRSA). This study was designed to evaluate the intracellular killing, membrane-damaging and biofilm-inhibitory activities of nimbolide isolated from Azadirachta indica against MDR MRSA. In vitro antibacterial activity of nimbolide was determined by performing MIC, minimal bactericidal concentration (MBC) and time-kill kinetic studies. Bacterial membrane-damaging activity was determined by membrane perturbation and scanning electron microscopy (SEM) examination. Biofilm-inhibitory activities were determined by SEM. Cellular drug accumulation and assessments of intracellular activities were performed using Vero cell culture. SEM revealed that nimbolide caused significant membrane damage and lysis of the S. aureus cells. The biofilm structure was disrupted, and the biofilm formation was greatly reduced in the presence of nimbolide as examined by SEM. The level of accumulation of nimbolide in Vero cells incubated for 24 h is relatively higher than that of ciprofloxacin and nalidixic acid (Cc/Ce for nimbolide > ciprofloxacin and nalidixic acid). The viable number of intracellular S. aureus was decreased [reduction of ~2 log10 c.f.u. (mg Vero cell protein)-1] in a time-dependent manner in the presence of nimbolide (4× MBC) that was comparable to that of tetracycline and nalidixic acid. The significant intracellular, biofilm-inhibitory and bacterial membrane-damaging activities of nimbolide demonstrated here suggested that it has potential as an effective antibacterial agent for the treatment of severe infections caused by MDR MRSA.

5-Aminoimidazole-4-carboxamide ribonucleoside-mediated adenosine monophosphate-activated protein kinase activation induces protective innate responses in bacterial endophthalmitis.

The retina is considered to be the most metabolically active tissue in the body. However, the link between energy metabolism and retinal inflammation, as incited by microbial infection such as endophthalmitis, remains unexplored. In this study, using a mouse model of Staphylococcus aureus (SA) endophthalmitis, we demonstrate that the activity (phosphorylation) of 5' adenosine monophosphate-activated protein kinase alpha (AMPKα), a cellular energy sensor and its endogenous substrate; acetyl-CoA carboxylase is down-regulated in the SA-infected retina. Intravitreal administration of an AMPK activator, 5-aminoimidazole-4-carboxamide ribonucleoside (AICAR), restored AMPKα and acetyl-CoA carboxylase phosphorylation. AICAR treatment reduced both the bacterial burden and intraocular inflammation in SA-infected eyes by inhibiting NF-kB and MAP kinases (p38 and JNK) signalling. The anti-inflammatory effects of AICAR were diminished in eyes pretreated with AMPK inhibitor, Compound C. The bioenergetics (Seahorse) analysis of SA-infected microglia and bone marrow-derived macrophages revealed an increase in glycolysis, which was reinstated by AICAR treatment. AICAR also reduced the expression of SA-induced glycolytic genes, including hexokinase 2 and glucose transporter 1 in microglia, bone marrow-derived macrophages and the mouse retina. Interestingly, AICAR treatment enhanced the bacterial phagocytic and intracellular killing activities of cultured microglia, macrophages and neutrophils. Furthermore, AMPKα1 global knockout mice exhibited increased susceptibility towards SA endophthalmitis, as evidenced by increased inflammatory mediators and bacterial burden and reduced retinal function. Together, these findings provide the first evidence that AMPK activation promotes retinal innate defence in endophthalmitis by modulating energy metabolism and that it can be targeted therapeutically to treat ocular infections.

Dietary resveratrol improves immunity but reduces reproduction of broodstock medaka Oryzias latipes (Temminck & Schlegel)

Here, we investigated the effect of dietary resveratrol (20, 40, and 80 µg/g BW/day) on cell-mediated immunity (activity of spleen phagocytes and proliferative response of lymphocytes) and reproductive parameters (egg and sperm quality, i.e. fecundity—total number of eggs produced by individual fish, fertility, embryo survival, and hatching rate) in medaka. Fish fed feed with resveratrol at 40 and 80 µg/g BW/day had significantly higher metabolic activity and intracellular phagocyte killing activity than control. The proliferative lymphocyte activity of the fish from R80 group was greater by more than 20 % in comparison with the control group (P < 0.05). The percentage of macrophages (MO) and their mean fluorescence intensities (MFI) in R40 and R80 groups were significantly higher compared to C and R20 groups (P < 0.05). The differences in MO and MFI values ranged from 52.5 % (±1.5; R0 group) to 65.8 % (±1.6; R80 group) and from 23.2 (±1.4; R0 group) to 38.2 (±2.4; R80 group), respectively. Moreover, resveratrol at 80 µg/g BW/day decreased liver COX activity, i.e. 5.4 in R80 group and 7.9 in R0 group (P < 0.05). The motility parameters of the sperm obtained from the males fed feed supplemented with resveratrol at 80 µg/g BW/day exhibited the highest values except the linearity, which was lower as compared to the control (P < 0.05). The results indicate that diet supplemented with resveratrol at a dosage of 40 µg/g BW/day improves phagocyte killing ability and lymphocyte proliferation in broodstock and accelerates offspring hatch. Also, the results suggest that COX activity influences sperm and oocyte quality in fish; the presence of a COX inhibitor in the dose of 40 µg/g BW/day decreased the embryo survival.