Injectable Hydrogel To Deliver Bone Mesenchymal Stem Cells Preloaded with Azithromycin To Promote Spinal Cord Repair.

Spinal cord injury is a disease that causes severe damage to the central nervous system. Currently, there is no cure for spinal cord injury. Azithromycin is commonly used as an antibiotic, but it can also exert anti-inflammatory effects by down-regulating M1-type macrophage genes and up-regulating M2-type macrophage genes, which may make it effective for treating spinal cord injury. Bone mesenchymal stem cells possess tissue regenerative capabilities that may help promote the repair of the injured spinal cord. In this study, our objective was to explore the potential of promoting repair in the injured spinal cord by delivering bone mesenchymal stem cells that had internalized nanoparticles preloaded with azithromycin. To achieve this objective, we formulated azithromycin into nanoparticles along with a trans-activating transcriptional activator, which should enhance nanoparticle uptake by bone mesenchymal stem cells. These stem cells were then incorporated into an injectable hydrogel. The therapeutic effects of this formulation were analyzed in vitro using a mouse microglial cell line and a human neuroblastoma cell line, as well as in vivo using a rat model of spinal cord injury. The results showed that the formulation exhibited anti-inflammatory and neuroprotective effects in vitro as well as therapeutic effects in vivo. These results highlight the potential of a hydrogel containing bone mesenchymal stem cells preloaded with azithromycin and trans-activating transcriptional activator to mitigate spinal cord injury and promote tissue repair.

Clofazimine as a substitute for rifampicin improves efficacy of Mycobacterium avium pulmonary disease treatment in the hollow-fiber model.

Mycobacterium avium complex pulmonary disease is treated with an azithromycin, ethambutol, and rifampicin regimen, with limited efficacy. The role of rifampicin is controversial due to inactivity, adverse effects, and drug interactions. Here, we evaluated the efficacy of clofazimine as a substitute for rifampicin in an intracellular hollow-fiber infection model. THP-1 cells, which are monocytes isolated from peripheral blood from an acute monocytic leukemia patient, were infected with M. avium ATCC 700898 and exposed to a regimen of azithromycin and ethambutol with either rifampicin or clofazimine. Intrapulmonary pharmacokinetic profiles of azithromycin, ethambutol, and rifampicin were simulated. For clofazimine, a steady-state average concentration was targeted. Drug concentrations and bacterial densities were monitored over 21 days. Exposures to azithromycin and ethambutol were 20%-40% lower than targeted but within clinically observed ranges. Clofazimine exposures were 1.7 times higher than targeted. Until day 7, both regimens were able to maintain stasis. Thereafter, regrowth was observed for the rifampicin-containing regimen, while the clofazimine-containing regimen yielded a 2 Log10 colony forming unit (CFU) per mL decrease in bacterial load. The clofazimine regimen also successfully suppressed the emergence of macrolide tolerance. In summary, substitution of rifampicin with clofazimine in the hollow-fiber model improved the antimycobacterial activity of the regimen. Clofazimine-containing regimens merit investigation in clinical trials.

Azithromycin Augments Bacterial Uptake and Anti-Inflammatory Macrophage Polarization in Cystic Fibrosis.

BACKGROUND: Azithromycin (AZM) is widely being used for treating patients with cystic fibrosis (pwCF) following clinical trials demonstrating improved lung function and fewer incidents of pulmonary exacerba-tions. While the precise mechanisms remain elusive, immunomodulatory actions are thought to be involved. We previously reported impaired phagocytosis and defective anti-inflammatory M2 macrophage polarization in CF. This study systematically analyzed the effect of AZM on the functions of unpolarized and M1/M2 polarized macrophages in CF. METHODS: Monocytes, isolated from the venous blood of patients with CF (pwCF) and healthy controls (HCs), were differentiated into monocyte-derived macrophages (MDMs) and subsequently infected with P. aeruginosa. P. aeruginosa uptake and killing by MDMs in the presence or absence of AZM was studied. M1 and M2 macrophage polarizations were induced and their functions and cytokine release were analyzed. RESULTS: Following AZM treatment, both HC and CF MDMs exhibited a significant increase in P. aeruginosa uptake and killing, however, lysosomal acidification remained unchanged. AZM treatment led to higher activation of ERK1/2 in both HC and CF MDMs. Pharmacological inhibition of ERK1/2 using U0126 significantly reduced P. aeruginosa uptake in HC MDMs. M1 macrophage polarization remained unaffected; however, AZM treatment led to increased IL-6 and IL-10 release in both HC and CF M1 macrophages. AZM also significantly increased the phagocytic index for both pHrodo E. coli and S. aureus in CF M1 macrophages. In CF, AZM treatment promoted anti-inflammatory M2 macrophage polarization, with an increased percentage of CD209+ M2 macrophages, induction of the M2 gene CCL18, along with its secretion in the culture supernatant. However, AZM d'd not restore endocytosis in CF, another essential feature of M2 macrophages. CONCLUSIONS: This study highlights the cellular functions and molecular targets of AZM which may involve an improved uptake of both Gram-positive and Gram-negative bacteria, restored anti-inflammatory macrophage polarization in CF. This may in turn shape the reduced lung inflammation observed in clinical trials. In addition, we confirmed the role of ERK1/2 activation for bacterial uptake.

Azithromycin reduces hemoglobin-induced innate neuroimmune activation.

Neonatal intraventricular hemorrhage (IVH) releases blood products into the lateral ventricles and brain parenchyma. There are currently no medical treatments for IVH and surgery is used to treat a delayed effect of IVH, post-hemorrhagic hydrocephalus. However, surgery is not a cure for intrinsic brain injury from IVH, and is performed in a subacute time frame. Like many neurological diseases and injuries, innate immune activation is implicated in the pathogenesis of IVH. Innate immune activation is a pharmaceutically targetable mechanism to reduce brain injury and post-hemorrhagic hydrocephalus after IVH. Here, we tested the macrolide antibiotic azithromycin, which has immunomodulatory properties, to reduce innate immune activation in an in vitro model of microglial activation using the blood product hemoglobin (Hgb). We then utilized azithromycin in our in vivo model of IVH, using intraventricular blood injection into the lateral ventricle of post-natal day 5 rat pups. In both models, azithromycin modulated innate immune activation by several outcome measures including mitochondrial bioenergetic analysis, cytokine expression and flow cytometric analysis. This suggests that azithromycin, which is safe for neonates, could hold promise for modulating innate immune activation after IVH.

Preventive and therapeutic effects of azithromycin on acrylamide-induced neurotoxicity in rats.

BACKGROUND: Acrylamide (ACR) can induce neurotoxicity through different pathways, including oxidative stress and apoptosis. Azithromycin is well-known for its antioxidant and anti-apoptotic properties. OBJECTIVE: To evaluate the potential neuroprotective effect of azithromycin in an in vivo model of ACR-induced neurotoxicity, by investigating its impact on oxidative stress and apoptosis pathways. METHODS: Male rats were divided into eleven groups at random (n = 6). 1:control (vehicle), 2:ACR (50 mg/kg, 11 days, I.P.), 3-7:ACR+ azithromycin (3.1, 6.25, 12.5, 25, 50 mg/kg, 11 days, I.P.), 8-9:ACR+ azithromycin (3.1, 6.25 mg/kg, from day 3-11), 10: ACR+ vitamin E (200 mg/kg, every other day, I.P.), 11. Azithromycin (50 mg/kg). Following the treatment period, a gait score examination was performed, and malondialdehyde (MDA), glutathione (GSH), Bcl-2-associated X protein (Bax)/B-cell lymphoma 2 (Bcl-2) ratio and caspase-3 levels in the cerebral cortex were measured. RESULTS: Gait abnormality, a drop in GSH, and an increase in lipid peroxidation, Bax/Bcl-2 ratio, and caspase-3 levels were all significantly triggered by ACR in the cerebral cortex versus the control group. Azithromycin 3.1 and 6.25 mg/kg with ACR and azithromycin 6.25 mg/kg with ACR from day 3-11 ameliorated movement disorders caused by ACR. Azithromycin in all doses and both protocols along with ACR decreased the MDA level. Azithromycin (3.1, 6.25 mg/kg) along with ACR in both protocols increased the level of GSH, reduced the Bax/Bcl-2 ratio and caspase-3 amounts in the brain tissue versus the ACR group. CONCLUSIONS: Administration of azithromycin had both preventive and therapeutic effects on ACR-induced neurotoxicity through its antioxidant and antiapoptotic properties.

Anti-babesial activity of a series of 6,7-dimethoxyquinazoline-2,4-diamines (DMQDAs).

Diminazene aceturate (DA), imidocarb dipropionate (ID), atovaquone (ATO), azithromycin (AZI), clindamycin, and quinine have been used to treat animal and human babesiosis for many years, despite their negative effects and rising indications of resistance. Thus, finding anti-babesial compounds that can either treat the infection or lower the dose of drugs given has been a primary objective. Quinazolines are one of the most important nitrogen heterocycles, with a wide range of pharmacological activities including analgesic, anti-inflammatory, sedative-hypnotic, anti-histaminic, anti-cancer, and anti-protozoan properties. The present study investigated the anti-babesial activities of twenty 6,7-dimethoxyquinazoline-2,4-diamines on Babesia spp. One candidate, 6,7-dimethoxy-N4-ethylisopropyl-N2-ethyl(pyridin-4-yl)quinazoline-2,4-diamine (SHG02), showed potent inhibition on Babesia gibsoni in vitro, as well as on B. microti and B. rodhaini in mice. Our findings indicate that the candidate compound SHG02 is promising for further development of anti-babesial drugs and provides a new structure to be explored for developing anti-Babesia therapeutics.

Limited effects of azithromycin on the oropharyngeal microbiome in children with CF and early pseudomonas infection.

BACKGROUND: Tobramycin inhalation solution (TIS) and chronic azithromycin (AZ) have known clinical benefits for children with CF, likely due to antimicrobial and anti-inflammatory activity. The effects of chronic AZ in combination with TIS on the airway microbiome have not been extensively investigated. Oropharyngeal swab samples were collected in the OPTIMIZE multicenter, randomized, placebo-controlled trial examining the addition of AZ to TIS in 198 children with CF and early P. aeruginosa infection. Bacterial small subunit rRNA gene community profiles were determined. The effects of TIS and AZ were assessed on oropharyngeal microbial diversity and composition to uncover whether effects on the bacterial community may be a mechanism of action related to the observed changes in clinical outcomes. RESULTS: Substantial changes in bacterial communities (total bacterial load, diversity and relative abundance of specific taxa) were observed by week 3 of TIS treatment for both the AZ and placebo groups. On average, these shifts were due to changes in non-traditional CF taxa that were not sustained at the later study visits (weeks 13 and 26). Bacterial community measures did not differ between the AZ and placebo groups. CONCLUSIONS: This study provides further evidence that the mechanism for AZ's effect on clinical outcomes is not due solely to action on airway microbial composition.

Potential mitigating role of ivermectin on the spread of Chlamydia trachomatis by Musca sorbens.

Trachoma is the world's most frequent cause of blindness from an infectious agent. The disease caused by infection is associated with lack of access to sanitation and low hygiene standards. Trachoma is controlled through the Surgery, Antibiotics, Facial cleanliness, and Environmental improvement (SAFE) strategy, which delivers azithromycin (AZM) mass drug administration (MDA) in endemic areas. The putative vector Musca sorbens principally reproduce in human faecal matter left in the environment due to open defecation. Ivermectin (IVM) is on the WHO's essential medicines list and is administered as preventative chemotherapy against two neglected tropical diseases (NTDs)-onchocerciasis, as an annual or bi-annual treatment, and lymphatic filariasis, as an annual treatment in combination with albendazole. Ivermectin has a known inhibitive effect on insects that reproduce in dung. To assess if IVM could be a viable vector control tool against M. sorbens, this study evaluates existing data from trachoma, onchocerciasis and lymphatic filariasis mass drug administration (MDA) operations in Ethiopia. Persistent and recrudescent trachoma in evaluation units (EUs) were examined for whether AZM MDA in EUs was accompanied by IVM MDA, and whether co-administration was associated with greater likelihood of trachoma control. Results show an association suggesting that EUs that received both IVM and AZM MDA benefit from improved control of trachoma in persistent or recrudescent areas, when compared to EUs that received AZM MDA. This initial investigation supports the potential for ivermectin's use to support SAFE. Findings warrant further work to validate ivermectin's impact on M. sorbens reproduction through controlled lab and field-based studies.

Azithromycin promotes proliferation, and inhibits inflammation in nasal epithelial cells in primary ciliary dyskinesia.

Primary ciliary dyskinesia (PCD) is a genetic disorder associated with recurrent and chronic respiratory infections due to functional defects of motile cilia. In this study, we aimed to elucidate inflammatory and proliferative responses in PCD respiratory epithelium and evaluate the effect of Azithromycin (AZT) on these responses. Airway basal cells (BCs) were isolated from nasal samples of Wild-type (WT) epitope of healthy donors and PCD donors with bi-allelic mutations in DNAH5, DNAH11 and CCDC39. Cells were expanded in vitro and stimulated with either Lipopolysaccharide (LPS) or vehicle control. Post stimulation, cells were treated with either Azithromycin (AZT) or vehicle control. Cell proliferation was imaged in real-time. Separately, BCs from the same donors were expanded and grown at an air-liquid interface (ALI) to generate a multi-ciliated epithelium (MCE). Once fully mature, cells were stimulated with LPS, AZT, LPS + AZT or vehicle control. Inflammatory profiling was performed on collected media by cytokine Luminex assay. At baseline, there was a significantly higher mean production of pro-inflammatory cytokines by CCDC39 BCs and MCEs when compared to WT, DNAH11 and DNAH5 cells. AZT inhibited production of cytokines induced by LPS in PCD cells. Differences in cell proliferation were noted in PCD and this was also corrected with AZT treatment.

Quercetin inhibits Toxoplasma gondii tachyzoite proliferation and acts synergically with azithromycin.

Quercetin (QUE) is a natural polyphenol known to have numerous pharmacological properties against infectious and non-infectious diseases. Azithromycin (AZ) is an antibiotic that belongs to the azalide class of antimicrobials and an antiparasitic that is known to be effective in combination with clindamycin against pyrimethamine/sulfadiazine-resistant Toxoplasma gondii tachyzoites in clinical settings. Both compounds are known to target protein synthesis and have anti-inflammatory properties. However, little is known about QUE and AZ synergistic interaction against T. gondii growth. Here, we report for the first time the effects of the combination of QUE and AZ on T. gondii growth. The 50% inhibitory concentration (IC50) for QUE at 72 h of interaction was determined to be 0.50 µM, whereas AZ gave an IC50 value of 0.66 µM at 72 h of interaction with parasites. Combination testing of QUE and AZ in a ratio of 2:1 (QUE:AZ) showed an IC50 value of 0.081 µM. Interestingly, a fractional inhibitory index value of 0.28 was observed, indicating a strong synergy. QUE was also found to upregulate the generation of reactive oxygen species and cause dysfunction of the mitochondria membrane of both intracellular and extracellular T. gondii tachyzoites. Overall, the results indicate that QUE is a novel lead capable of synergizing with AZ for inhibiting T. gondii growth and may merit future investigation in vivo for possible combination drug development.

Azithromycin alters Colony Stimulating Factor-1R (CSF-1R) expression and functional output of murine bone marrow-derived macrophages: A novel report.

Antibiotic treatment may lead to side effects that require mechanistic explanation. We investigated the effect of azithromycin (AZM) treatment on bone marrow-derived macrophage (Mφ) generation, their functional output, and the subsequent effect on bacterial clearance in a mouse model of S. flexneri infection. To our fascination, AZM increased PU.1, C/EBPß, CSF-1R/pCSF-1R expressions leading to M2-skewed in vitro BMDM generation. Altered Mφ-functions like- phagocytosis, oxidative stress generation, inflammasome-activation, cytokine release, and phenotype (pro-inflammatory-M1, anti-inflammatory-M2) even in the presence of infection were observed with AZM treatment. AZM increased CD206, egr2, arg1 (M2-marker) expression and activity while reducing CD68, inducible nitric oxide (iNOS) expression, and activity (M1-marker) in Mφs during infection. Pro-inflammatory cytokines (TNF-α, IL-12, IL-1ß) were reduced and anti-inflammatory IL-10 release was augmented by AZM-treated-iMφs (aiMφs) along with decreased asc, nlrp3, aim2, nlrp1a, caspase1 expressions, and caspase3 activity signifying that aMφs/aiMφs were primed towards an anti-inflammatory phenotype. Interestingly, CSF-1R blockade increased NO, IL-12, TNF-α, IL-1ß, decreased TGF-ß release, and CD206 expression in aiMφs. T-cell co-stimulatory molecule cd40, cd86, and cd80 expressions were decreased in ai/aM1-Mφs and co-cultured CD8+, CD4+ T-cells had decreased proliferation, t-bet, IFN-γ, IL-17, IL-2 but increased foxp3, TGF-ß, IL-4 which were rescued with CSF-1R blockade. Thus AZM affected Mφ-functions and subsequent T-cell responses independent of its antibacterial actions. This was validated in the balb/c model of S. flexneri infection. We conclude that AZM skewed BMDM generation to anti-inflammatory M2-like via increased CSF-1R expression. This warrants further investigation of AZM-induced altered-Mφ-generation during intracellular infections.

Azithromycin inhibits glioblastoma angiogenesis in mice via inducing mitochondrial dysfunction and oxidative stress.

The poor outcomes in glioblastoma (GBM) necessitate new treatments. As GBM is highly vascularized and its growth is largely dependent on angiogenesis, angiogenesis inhibitors have been hotly evaluated in clinical trials for GBM treatment for the last decade. In line with these efforts, our work reveals that azithromycin, a clinically available antibiotic, is a novel angiogenesis inhibitor. Azithromycin inhibits vessel structure formation on Matrigel of GBM-derived endothelial cell (ECs) and other types of ECs. Time course analysis shows that azithromycin interferes with the early stage of angiogenesis. Azithromycin also inhibits GBM-derived EC adhesion, growth and survival but not migration. The transgenic zebrafish Tg (fli1a: EGFP) model clearly shows that azithromycin inhibits angiogenesis in vivo. Of note, azithromycin at non-toxic dose inhibits GBM growth in mice and increases overall survival, and furthermore, this is associated with angiogenesis inhibition. Mechanism studies show that azithromycin decreases mitochondrial respiration by suppressing the activity of multiple complexes, leading to ATP reduction, oxidative stress and damage. In addition, oxidative stress induced by azithromycin is through thiol redox-mediated pathways. Our work demonstrates the anti-angiogenic activity of azithromycin via inducing mitochondrial dysfunction and oxidative stress. Our pre-clinical evidence provides a rationale for initiating clinical trials using azithromycin in combination with standard-of-care drugs for GBM patients.

Poly(sebacic acid) microparticles loaded with azithromycin as potential pulmonary drug delivery system: Physicochemical properties, antibacterial behavior, and cytocompatibility studies.

Recurrent bacterial infections are a common cause of death for patients with cystic fibrosis and chronic obstructive pulmonary disease. Herein, we present the development of the degradable poly(sebacic acid) (PSA) microparticles loaded with different concentrations of azithromycin (AZ) as a potential powder formulation to deliver AZ locally to the lungs. We characterized microparticle size, morphology, zeta potential, encapsulation efficiency, interaction PSA with AZ and degradation profile in phosphate buffered saline (PBS). The antibacterial properties were evaluated using the Kirby-Bauer method against Staphylococcus aureus. Potential cytotoxicity was evaluated in BEAS-2B and A549 lung epithelial cells by the resazurin reduction assay and live/dead staining. The results show that microparticles are spherical and their size, being in the range of 1-5 µm, should be optimal for pulmonary delivery. The AZ encapsulation efficiency is nearly 100 % for all types of microparticles. The microparticles degradation rate is relatively fast - after 24 h their mass decreased by around 50 %. The antibacterial test showed that released AZ was able to successfully inhibit bacteria growth. The cytotoxicity test showed that the safe concentration of both unloaded and AZ-loaded microparticles was equal to 50 µg/ml. Thus, appropriate physicochemical properties, controlled degradation and drug release, cytocompatibility, and antibacterial behavior showed that our microparticles may be promising for the local treatment of lung infections.

Azithromycin induces read-through of the nonsense Apc allele and prevents intestinal tumorigenesis in C3B6F1 ApcMin/+ mice.

Therapeutic strategies that promote read-through of a mutant gene have proved effective for certain non-neoplastic diseases. However, the efficacy of this approach is unproven regarding neoplastic diseases with germline nonsense mutations, including familial adenomatous polyposis. Here we examined the cancer-preventive efficacy of the macrolide antibiotic azithromycin, with a reported read-through effect, on intestinal tumorigenesis in C3B6F1 ApcMin/+ mice harboring a nonsense Apc mutation resulting in a truncated Apc protein. Mice were given drinking water lacking azithromycin or containing 0.0125-0.2 mg/mL azithromycin from 3 weeks of age. The small intestine and cecum were analyzed for pathological changes and alterations of intestinal flora. Azithromycin suppressed the number of tumors and the proportion of adenocarcinomas, with the most effective drinking-water concentration being 0.0125 mg/mL. Furthermore, azithromycin recovered the cellular level of full-length Apc, resulting in downregulation of ß-catenin and cyclin D1. Conversely, the effect of azithromycin on the diversity of the intestinal microbiota depended on the drinking-water concentration. These results suggest that the balance between azithromycin-mediate read-through of mutant Apc mRNA and antibacterial effects influences intestinal tumorigenesis. Thus, azithromycin is a potential anticancer agent for familial adenomatous polyposis patients harboring nonsense mutations.

The immunomodulatory effects of probiotics and azithromycin in dextran sodium sulfate-induced ulcerative colitis in rats via TLR4-NF-κB and p38-MAPK pathway.

Ulcerative colitis (UC), a chronic autoimmune disease of the gut with a relapsing and remitting nature, considers a major health-care problem. DSS is a well-studied pharmacologically-induced model for UC. Toll-Like Receptor 4 (TLR4) and its close association with p-38-Mitogen-Activated Protein Kinase (p-38 MAPK) and nuclear factor kappa B (NF-κB) has important regulatory roles in inflammation and developing UC. Probiotics are gaining popularity for their potential in UC therapy. The immunomodulatory and anti-inflammatory role of azithromycin in UC remains a knowledge need. In the present rats-established UC, the therapeutic roles of oral probiotics (60 billion probiotic bacteria per kg per day) and azithromycin (40 mg per kg per day) regimens were evaluated by measuring changes in disease activity index, macroscopic damage index, oxidative stress markers, TLR4, p-38 MAPK, NF-κB signaling pathway in addition to their molecular downstream; tumor necrosis factor alpha (TNFα), interleukin (IL)1ß, IL6, IL10 and inducible nitric oxide synthase (iNOS). After individual and combination therapy with probiotics and azithromycin regimens, the histological architecture of the UC improved with restoration of intestinal tissue normal architecture. These findings were consistent with the histopathological score of colon tissues. Each separate regimen lowered the remarkable TLR4, p-38 MAPK, iNOS, NF-κB as well as TNFα, IL1ß, IL6 and MDA expressions and elevated the low IL10, glutathione and superoxide dismutase expressions in UC tissues. The combination regimen possesses the most synergistic beneficial effects in UC that, following thorough research, should be incorporated into the therapeutic approach in UC to boost the patients' quality of life.

Detection of bile acids in bronchoalveolar lavage fluid defines the inflammatory and microbial landscape of the lower airways in infants with cystic fibrosis.

BACKGROUND: Cystic Fibrosis (CF) is a genetic condition characterized by neutrophilic inflammation and recurrent infection of the airways. How these processes are initiated and perpetuated in CF remains largely unknown. We have demonstrated a link between the intestinal microbiota-related metabolites bile acids (BA) and inflammation in the bronchoalveolar lavage fluid (BALF) from children with stable CF lung disease. To establish if BA indicate early pathological processes in CF lung disease, we combined targeted mass spectrometry and amplicon sequencing-based microbial characterization of 121 BALF specimens collected from 12-month old infants with CF enrolled in the COMBAT-CF study, a multicentre randomized placebo-controlled clinical trial comparing azithromycin versus placebo. We evaluated whether detection of BA in BALF is associated with the establishment of the inflammatory and microbial landscape of early CF lung disease, and whether azithromycin, a motilin agonist that has been demonstrated to reduce aspiration of gastric contents, alters the odds of detecting BA in BALF. We also explored how different prophylactic antibiotics regimens impact the early life BALF microbiota. RESULTS: Detection of BA in BALF was strongly associated with biomarkers of airway inflammation, more exacerbation episodes during the first year of life, increased use of oral antibiotics with prolonged treatment periods, a higher degree of structural lung damage, and distinct microbial profiles. Treatment with azithromycin, a motilin agonist, which has been reported to reduce aspiration of gastric contents, did not reduce the odds of detecting BA in BALF. Culture and molecular methods showed that azithromycin does not alter bacterial load or diversity in BALF. Conversely, penicillin-type prophylaxis reduced the odds of detecting BAs in BALF, which was associated with elevated levels of circulating biomarkers of cholestasis. We also observed that environmental factors such as penicillin-type prophylaxis or BAs detection were linked to distinct early microbial communities of the CF airways, which were associated with different inflammatory landscapes but not with structural lung damage. CONCLUSIONS: Detection of BA in BALF portend early pathological events in CF lung disease. Benefits early in life associated with azithromycin are not linked to its antimicrobial properties. Video Abstract.

Azithromycin Mechanisms of Action in CRS Include Epithelial Barrier Restoration and Type 1 Inflammation Reduction.

OBJECTIVE: Previous in vitro transcriptomic profiling suggests azithromycin exerts its effects in patients with chronic rhinosinusitis (CRS) via modulation of type 1 inflammation and restoration of epithelial barrier function. We wished to verify these postulated effects using in vitro models of epithelial repair and in vivo transcriptional profiling. STUDY DESIGN: Functional effects of azithromycin in CRS were verified using in vitro models of wounding. The mechanism of the effect of azithromycin was assessed in vivo using transcriptomic profiling. SETTING: Academic medical center. METHODS: Effects of azithromycin on the speed of epithelial repair were verified in a wounding model using primary nasal epithelial cells (pNEC) from CRS patients. Nasal brushings collected pre-and posttreatment during a placebo-controlled trial of azithromycin for CRS patients unresponsive to surgery underwent transcriptomic profiling to identify implicated pathways. RESULTS: Administration of azithromycin improved the wound healing rates in CRS pNECs and prevented the negative effect of Staphylococcus aureus on epithelial repair. In vivo, response to azithromycin was associated with downregulation in pathways of type 1 inflammation, and upregulation of pathways implicated in the restoration of the cell cycle. CONCLUSION: Restoration of healthy epithelial function may represent a major mode of action of azithromycin in CRS. In vitro models show enhanced epithelial repair, while in vivo transcriptomics shows downregulation of pathways type 1 inflammation accompanied by upregulation of DNA repair and cell-cycle pathways. The maximal effect in patients with high levels of type 1-enhanced inflammation suggests that azithromycin may represent a novel therapeutic option for surgery-unresponsive CRS patients.

A mini-review on anticancer-related properties of azithromycin and its potential activities in overcoming the challenges of glioblastoma.

The resistance, plasticity and heterogeneity of cancer cells, including glioblastoma (GB) cells, have prompted the investigation of various agents for possible adjuncts and alternatives to existing therapies. This includes a macrolide antibiotic, azithromycin (AZI). It possesses intriguing anticancer properties in a range of cancer models in vitro, such as antiproliferative, pro-apoptotic, anti-autophagy and anti-angiogenic effects. In fact, AZI is renowned for its ability to eradicate cancer stem cells by inhibiting mitochondrial biogenesis and respiration. AZI-containing regimens in cancer patients for different purposes have shown favourable (i.e., attributed to its antibacterial activity) and unfavourable outcomes. Whilst its direct anticancer effects have yet to be clinically proven. To that end, this review provides a summary of AZI anticancer studies and delineates its potential activities in overcoming the challenges of GB.

Azithromycin ameliorated cigarette smoke-induced airway epithelial barrier dysfunction by activating Nrf2/GCL/GSH signaling pathway.

BACKGROUND: Airway epithelium is the first barrier against environmental insults, and epithelial barrier dysfunction caused by cigarette smoke (CS) is particularly relevant to chronic obstructive pulmonary disease (COPD) progression. Our study was to determine whether Azithromycin (AZI) ameliorates CS-induced airway epithelial barrier dysfunction and the underlying mechanisms. METHODS: Primary bronchial epithelial cells (PBECs), human bronchial epithelial cells (HBECs), Sprague Dawley rats and nuclear factor erythroid 2-related factor 2 (Nrf2)-/- mice were pretreated with AZI and subsequently exposed to CS. Transepithelial electronic resistance (TEER), junction proteins as well as pro-inflammatory cytokines and apoptosis markers were examined to assess epithelial barrier dysfunction. Metabolomics study was applied to explore the underlying mechanism of AZI. RESULTS: CS-induced TEER decline and intercellular junction destruction, accompanied with inflammatory response and cell apoptosis in PBECs were restored by AZI dose-dependently, which were also observed in CS-exposed rats. Mechanistically, GSH metabolism pathway was identified as the top differentially impacted pathway and AZI treatment upregulated the activities of glutamate cysteine ligase (GCL) and the contents of metabolites in GSH metabolic pathway. Furthermore, AZI apparently reversed CS-induced Nrf2 suppression, and similar effects on airway epithelial barrier dysfunction were also found for Nrf2 agonist tert-butylhydroquinone and vitamin C. Finally, deletion of Nrf2 in both HBECs and C57BL/6N mice aggravated CS-induced GSH metabolism imbalance to disrupt airway epithelial barrier and partially deprived the effects of AZI. CONCLUSION: These findings suggest that the clinical benefits of AZI for COPD management are related with the protection of CS-induced airway epithelial barrier dysfunction via activating Nrf2/GCL/GSH pathway, providing potential therapeutic strategies for COPD.

Azithromycin, a potent autophagy inhibitor for cancer therapy, perturbs cytoskeletal protein dynamics.

BACKGROUND: Autophagy plays an important role in tumour cell growth and survival and also promotes resistance to chemotherapy. Hence, autophagy has been targeted for cancer therapy. We previously reported that macrolide antibiotics including azithromycin (AZM) inhibit autophagy in various types of cancer cells in vitro. However, the underlying molecular mechanism for autophagy inhibition remains unclear. Here, we aimed to identify the molecular target of AZM for inhibiting autophagy. METHODS: We identified the AZM-binding proteins using AZM-conjugated magnetic nanobeads for high-throughput affinity purification. Autophagy inhibitory mechanism of AZM was analysed by confocal microscopic and transmission electron microscopic observation. The anti-tumour effect with autophagy inhibition by oral AZM administration was assessed in the xenografted mice model. RESULTS: We elucidated that keratin-18 (KRT18) and α/ß-tubulin specifically bind to AZM. Treatment of the cells with AZM disrupts intracellular KRT18 dynamics, and KRT18 knockdown resulted in autophagy inhibition. Additionally, AZM treatment suppresses intracellular lysosomal trafficking along the microtubules for blocking autophagic flux. Oral AZM administration suppressed tumour growth while inhibiting autophagy in tumour tissue. CONCLUSIONS: As drug-repurposing, our results indicate that AZM is a potent autophagy inhibitor for cancer treatment, which acts by directly interacting with cytoskeletal proteins and perturbing their dynamics.