Effects of prime-boost strategies on the protective efficacy and immunogenicity of a PLGA (85:15)-encapsulated Chlamydia recombinant MOMP nanovaccine.

To begin to optimize the immunization routes for our reported PLGA-rMOMP nanovaccine [PLGA-encapsulated Chlamydia muridarum (Cm) recombinant major outer membrane protein (rMOMP)], we compared two prime-boost immunization strategies [subcutaneous (SC) and intramuscular (IM-p) prime routes followed by two SC-boosts)] to evaluate the nanovaccine-induced protective efficacy and immunogenicity in female BALB/c mice. Our results showed that mice immunized via the SC and IM-p routes were protected against a Cm genital challenge by a reduction in bacterial burden and with fewer bacteria in the SC mice. Protection of mice correlated with rMOMP-specific Th1 (IL-2 and IFN-γ) and not Th2 (IL-4, IL-9, and IL-13) cytokines, and CD4+ memory (CD44highCD62Lhigh) T-cells, especially in the SC mice. We also observed higher levels of IL-1α, IL-6, IL-17, CCL-2, and G-CSF in SC-immunized mice. Notably, an increase of cytokines/chemokines was seen after the challenge in the SC, IM-p, and control mice (rMOMP and PBS), suggesting a Cm stimulation. In parallel, rMOMP-specific Th1 (IgG2a and IgG2b) and Th2 (IgG1) serum, mucosal, serum avidity, and neutralizing antibodies were more elevated in SC than in IM-p mice. Overall, the homologous SC prime-boost immunization of mice induced enhanced cellular and antibody responses with better protection against a genital challenge compared to the heterologous IM-p.

PLGA-Chitosan Encapsulated IL-10 Nanoparticles Modulate Chlamydia Inflammation in Mice.

Introduction: Interleukin-10 (IL-10) is a key anti-inflammatory mediator in protecting host from over-exuberant responses to pathogens and play important roles in wound healing, autoimmunity, cancer, and homeostasis. However, its application as a therapeutic agent for biomedical applications has been limited due to its short biological half-life. Therefore, it is important to prolong the half-life of IL-10 to replace the current therapeutic application, which relies on administering large and repeated dosages. Therefore, not a cost-effective approach. Thus, studies that aim to address this type of challenges are always in need. Methods: Recombinant IL-10 was encapsulated in biodegradable nanoparticles (Poly-(Lactic-co-Glycolic Acid) and Chitosan)) by the double emulsion method and then characterized for size, surface charge, thermal stability, cytotoxicity, in vitro release, UV-visible spectroscopy, and Fourier Transform-Infrared Spectroscopy as well as evaluated for its anti-inflammatory effects. Bioactivity of encapsulated IL-10 was evaluated in vitro using J774A.1 macrophage cell-line and in vivo using BALB/c mice. Inflammatory cytokines (IL-6 and TNF-α) were quantified from culture supernatants using specific enzyme-linked immunosorbent assay (ELISA), and significance was analyzed using ANOVA. Results: We obtained a high 96% encapsulation efficiency with smooth encapsulated IL-10 nanoparticles of ~100-150 nm size and release from nanoparticles as measurable to 22 days. Our result demonstrated that encapsulated IL-10 was biocompatible and functional by reducing the inflammatory responses induced by LPS in macrophages. Of significance, we also proved the functionality of encapsulated IL-10 by its capacity to reduce inflammation in BALB/c mice as provoked by Chlamydia trachomatis, an inflammatory sexually transmitted infectious bacterium. Discussion: Collectively, our results show the successful IL-10 encapsulation, slow release to prolong its biological half-life and reduce inflammatory cytokines IL-6 and TNF production in vitro and in mice. Our results serve as proof of concept to further explore the therapeutic prospective of encapsulated IL-10 for biomedical applications, including inflammatory diseases.

Bystander activation of microglia by Brucella abortus-infected astrocytes induces neuronal death via IL-6 trans-signaling.

Inflammation plays a key role in the pathogenesis of neurobrucellosis where glial cell interactions are at the root of this pathological condition. In this study, we present evidence indicating that soluble factors secreted by Brucella abortus-infected astrocytes activate microglia to induce neuronal death. Culture supernatants (SN) from B. abortus-infected astrocytes induce the release of pro-inflammatory mediators and the increase of the microglial phagocytic capacity, which are two key features in the execution of live neurons by primary phagocytosis, a recently described mechanism whereby B. abortus-activated microglia kills neurons by phagocytosing them. IL-6 neutralization completely abrogates neuronal loss. IL-6 is solely involved in increasing the phagocytic capacity of activated microglia as induced by SN from B. abortus-infected astrocytes and does not participate in their inflammatory activation. Both autocrine microglia-derived and paracrine astrocyte-secreted IL-6 endow microglial cells with up-regulated phagocytic capacity that allows them to phagocytose neurons. Blocking of IL-6 signaling by soluble gp130 abrogates microglial phagocytosis and concomitant neuronal death, indicating that IL-6 activates microglia via trans-signaling. Altogether, these results demonstrate that soluble factors secreted by B. abortus-infected astrocytes activate microglia to induce, via IL-6 trans-signaling, the death of neurons. IL-6 signaling inhibition may thus be considered a strategy to control inflammation and CNS damage in neurobrucellosis.

Pseudomonas aeruginosa reference strains PAO1 and PA14: A genomic, phenotypic, and therapeutic review.

Pseudomonas aeruginosa is a ubiquitous, motile, gram-negative bacterium that has been recently identified as a multi-drug resistant pathogen in critical need of novel therapeutics. Of the approximately 5,000 strains, PAO1 and PA14 are common laboratory reference strains, modeling moderately and hyper-virulent phenotypes, respectively. PAO1 and PA14 have been instrumental in facilitating the discovery of novel drug targets, testing novel therapeutics, and supplying critical genomic information on the bacterium. While the two strains have contributed to a wide breadth of knowledge on the natural behaviors and therapeutic susceptibilities of P. aeruginosa, they have demonstrated significant deviations from observations in human infections. Many of these deviations are related to experimental inconsistencies in laboratory strain environment that complicate and, at times, terminate translation from laboratory results to clinical applications. This review aims to provide a comparative analysis of the two strains and potential methods to improve their clinical relevance.

Encapsulation of Recombinant MOMP in Extended-Releasing PLGA 85:15 Nanoparticles Confer Protective Immunity Against a Chlamydia muridarum Genital Challenge and Re-Challenge.

Recently we reported the immune-potentiating capacity of a Chlamydia nanovaccine (PLGA-rMOMP) comprising rMOMP (recombinant major outer membrane protein) encapsulated in extended-releasing PLGA [poly (D, L-lactide-co-glycolide) (85:15)] nanoparticles. Here we hypothesized that PLGA-rMOMP would bolster immune-effector mechanisms to confer protective efficacy in mice against a Chlamydia muridarum genital challenge and re-challenge. Female BALB/c mice received three immunizations, either subcutaneously (SC) or intranasally (IN), before receiving an intravaginal challenge with C. muridarum on day 49 and a re-challenge on day 170. Both the SC and IN immunization routes protected mice against genital challenge with enhanced protection after a re-challenge, especially in the SC mice. The nanovaccine induced robust antigen-specific Th1 (IFN-γ, IL-2) and IL-17 cytokines plus CD4+ proliferating T-cells and memory (CD44high CD62Lhigh) and effector (CD44high CD62Llow) phenotypes in immunized mice. Parallel induction of antigen-specific systemic and mucosal Th1 (IgG2a, IgG2b), Th2 (IgG1), and IgA antibodies were also noted. Importantly, immunized mice produced highly functional Th1 avidity and serum antibodies that neutralized C. muridarum infectivity of McCoy fibroblasts in-vitro that correlated with their respective protection levels. The SC, rather than the IN immunization route, triggered higher cellular and humoral immune effectors that improved mice protection against genital C. muridarum. We report for the first time that the extended-releasing PLGA 85:15 encapsulated rMOMP nanovaccine confers protective immunity in mice against genital Chlamydia and advances the potential towards acquiring a nano-based Chlamydia vaccine.

Nanofiller-Enhanced Soft Non-Gelatin Alginate Capsules for Modified Drug Delivery.

Capsules are one of the major solid dosage forms available in a variety of compositions and shapes. Developments in this dosage form are not new, but the production of non-gelatin capsules is a recent trend. In pharmaceutical as well as other biomedical research, alginate has great versatility. On the other hand, the use of inorganic material to enhance material strength is a common research topic in tissue engineering. The research presented here is a combination of qualities of alginate and montmorillonite (MMT). These two materials were used in this research to produce a soft non-gelatin modified-release capsule. Moreover, the research describes a facile benchtop production of these capsules. The produced capsules were critically analyzed for their appearance confirming resemblance with marketed capsules, functionality in terms of drug encapsulation, as well as release and durability.

Suppressors of Cytokine Signaling (SOCS)1 and SOCS3 Proteins Are Mediators of Interleukin-10 Modulation of Inflammatory Responses Induced by Chlamydia muridarum and Its Major Outer Membrane Protein (MOMP) in Mouse J774 Macrophages.

The immunopathology of chlamydial diseases is exacerbated by a broad-spectrum of inflammatory mediators, which we reported are inhibited by IL-10 in macrophages. However, the chlamydial protein moiety that induces the inflammatory mediators and the mechanisms by which IL-10 inhibits them are unknown. We hypothesized that Chlamydia major outer membrane protein (MOMP) mediates its disease pathogenesis, and the suppressor of cytokine signaling (SOCS)1 and SOCS3 proteins are mediators of the IL-10 inhibitory actions. Our hypothesis was tested by exposing mouse J774 macrophages to chlamydial stimulants (live Chlamydia muridarum and MOMP) with and without IL-10. MOMP significantly induced several inflammatory mediators (IL-6, IL-12p40, CCL5, CXCL10), which were dose-dependently inhibited by IL-10. Chlamydial stimulants induced the mRNA gene transcripts and protein expression of SOCS1 and SOCS3, with more SOCS3 expression. Notably, IL-10 reciprocally regulated their expression by reducing SOCS1 and increasing SOCS3. Specific inhibitions of MAPK pathways revealed that p38, JNK, and MEK1/2 are required for inducing inflammatory mediators as well as SOCS1 and SOCS3. Chlamydial stimulants triggered an M1 pro-inflammatory phenotype evidently by an enhanced nos2 (M1 marker) expression, which was skewed by IL-10 towards a more M2 anti-inflammatory phenotype by the increased expression of mrc1 and arg1 (M2 markers) and the reduced SOCS1/SOCS3 ratios. Neutralization of endogenously produced IL-10 augmented the secretion of inflammatory mediators, reduced SOCS3 expression, and skewed the chlamydial M1 to an M2 phenotype. Inhibition of proteasome degradation increased TNF but decreased IL-10, CCL5, and CXCL10 secretion by suppressing SOCS1 and SOCS3 expressions and dysregulating their STAT1 and STAT3 transcription factors. Our data show that SOCS1 and SOCS3 are regulators of IL-10 inhibitory actions, and underscore SOCS proteins as therapeutic targets for IL-10 control of inflammation for Chlamydia and other bacterial inflammatory diseases.

A nanovaccine formulation of Chlamydia recombinant MOMP encapsulated in PLGA 85:15 nanoparticles augments CD4+ effector (CD44high CD62Llow) and memory (CD44high CD62Lhigh) T-cells in immunized mice.

Vaccine developmental strategies are utilizing antigens encapsulated in biodegradable polymeric nanoparticles. Here, we developed a Chlamydia nanovaccine (PLGA-rMOMP) by encapsulating its recombinant major outer membrane protein (rMOMP) in the extended-releasing and self-adjuvanting PLGA [poly (D, L-lactide-co-glycolide) (85:15)] nanoparticles. PLGA-rMOMP was small (nanometer size), round and smooth, thermally stable, and exhibited a sustained release of rMOMP. Stimulation of mouse primary dendritic cells (DCs) with PLGA-rMOMP augmented endosome processing, induced Th1 cytokines (IL-6 and IL-12p40), and expression of MHC-II and co-stimulatory (CD40, CD80, and CD86) molecules. BALB/c mice immunized with PLGA-rMOMP produced enhanced CD4+ T-cells-derived memory (CD44high CD62Lhigh), and effector (CD44high CD62Llow) phenotypes and functional antigen-specific serum IgG antibodies. In vivo biodistribution of PLGA-rMOMP revealed its localization within lymph nodes, suggesting migration from the injection site via DCs. Our data provide evidence that the PLGA (85:15) nanovaccine activates DCs and augments Chlamydia-specific rMOMP adaptive immune responses that are worthy of efficacy testing.

Antibiotic Minocycline Prevents Respiratory Syncytial Virus Infection.

Treatment drugs, besides their specific activity, often have multiple effects on the body. The undesired effect of the drug may be repurposed as therapeutics, saving significant investigative time and effort. Minocycline has anti-cancer, anti-oxidant, anti-inflammatory, and anti-apoptotic properties. Presently, minocycline is also known to show anti-viral activity against Influenza virus, Japanese encephalitis virus, Simian immunodeficiency virus, Human immunodeficiency virus and West Nile virus. Here, we investigate the effect of minocycline on Respiratory syncytial virus (RSV), a common respiratory virus that causes severe mortality and morbidity in infants, children, and older adult populations. Currently, there is no effective vaccine or treatment for RSV infection; hence, there is a critical need for alternative and effective drug choices. Our study shows that minocycline reduces the RSV-mediated cytopathic effect and prevents RSV infection. This is the first study demonstrating the anti-viral activity of minocycline against RSV.

Prolonged Release and Functionality of Interleukin-10 Encapsulated within PLA-PEG Nanoparticles.

Inflammation, as induced by the presence of cytokines and chemokines, is an integral part of chlamydial infections. The anti-inflammatory cytokine, interleukin (IL)-10, has been reported to efficiently suppress the secretion of inflammatory cytokines triggered by Chlamydia in mouse macrophages. Though IL-10 is employed in clinical applications, its therapeutic usage is limited due to its short half-life. Here, we document the successful encapsulation of IL-10 within the biodegradable polymeric nanoparticles of PLA-PEG (Poly (lactic acid)-Poly (ethylene glycol), to prolong its half-life. Our results show the encapsulated-IL-10 size (~238 nm), zeta potential (-14.2 mV), polydispersity index (0.256), encapsulation efficiency (~77%), and a prolonged slow release pattern up to 60 days. Temperature stability of encapsulated-IL-10 was favorable, demonstrating a heat capacity of up to 89 °C as shown by differential scanning calorimetry analysis. Encapsulated-IL-10 modulated the release of IL-6 and IL-12p40 in stimulated macrophages in a time- and concentration-dependent fashion, and differentially induced SOCS1 and SOCS3 as induced by chlamydial stimulants in macrophages. Our finding offers the tremendous potential for encapsulated-IL-10 not only for chlamydial inflammatory diseases but also biomedical therapeutic applications.

A three-dimensional human skin model to evaluate the inhibition of Staphylococcus aureus by antimicrobial peptide-functionalized silver carbon nanotubes.

OBJECTIVE: To investigate the toxicity and antibacterial application of antimicrobial peptide-functionalized silver-coated carbon nanotubes against Staphylococcus infection using a full thickness human three-dimensional skin model. MATERIALS AND METHODS: The three-dimensional skin formation on the scaffolds was characterized by electron microscopy and investigation of several skin cell markers by real time-reverse transcriptase polymerase chain reaction. Functionalized silver-coated carbon nanotubes were prepared using carboxylated silver-coated carbon nanotubes with antimicrobial peptides such as TP359, TP226 and TP557. Following the characterization and toxicity evaluation, the antibacterial activity of functionalized silver-coated carbon nanotubes against Staphylococcus aureus was investigated using a bacterial enumeration assay and scanning electron microscopy. For this purpose, a scar on the human three-dimensional skin grown on Alvetex scaffold using keratinocytes and fibroblasts cells was created by taking precaution not to break the scaffold beneath, followed by incubation with 5 µg/mL of functionalized silver-coated carbon nanotubes re-suspended in minimum essential medium for 2 h. Post 2-h incubation, 200 µL of minimum essential medium containing 1 × 104 colony forming units of Staphylococcus aureus were incubated for 2 h. After incubation with bacteria, the colony forming unit/gram (cfu/g) of skin tissue were counted using the plate count assay and the samples were processed for scanning electron microscopy analysis. RESULTS: MTT assay revealed no toxicity of functionalized silver-coated carbon nanotubes to the skin cells such as keratinocytes and fibroblasts at 5 µg/mL with 98% cell viability. The bacterial count increased from 104 to 108 cfu/g in the non-treated skin model, whereas skin treated with functionalized silver-coated carbon nanotubes showed only a small increase from 104 to 105 cfu/g (1000-fold viable cfu difference). Scanning electron microscopy analysis showed the presence of Staphylococcus aureus on the non-treated skin as opposed to the treated skin. CONCLUSION: Thus, our results showed that functionalized silver-coated carbon nanotubes are not only non-toxic, but also help reduce the infection due to their antibacterial activity. These findings will aid in the development of novel antibacterial skin substitutes.

The Chlamydia M278 Major Outer Membrane Peptide Encapsulated in the Poly(lactic acid)-Poly(ethylene glycol) Nanoparticulate Self-Adjuvanting Delivery System Protects Mice Against a Chlamydia muridarum Genital Tract Challenge by Stimulating Robust Systemic and Local Mucosal Immune Responses.

Recently, we reported that our PPM chlamydial nanovaccine [a biodegradable co-polymeric PLA-PEG (poly(lactic acid)-poly(ethylene glycol))-encapsulated M278 peptide (derived from the major outer membrane protein (MOMP) of Chlamydia)] exploits the caveolin-mediated endocytosis pathway for endosomal processing and MHC class II presentation to immune-potentiate Chlamydia-specific CD4+ T-cell immune effector responses. In the present study, we employed the Chlamydia muridarum mouse infection model to evaluate the protective efficacy of PPM against a genital tract challenge. Our results show that mice immunized with PPM were significantly protected against a homologous genital tract challenge evidently by reduced vaginal bacterial loads. Protection of mice correlated with enhanced Chlamydia-specific adaptive immune responses predominated by IFN-γ along with CD4+ T-cells proliferation and their differentiation to CD4+ memory (CD44high CD62Lhigh) and effector (CD44high CD62Llow) T-cell phenotypes. We observed the elevation of M278- and MOMP-specific serum antibodies with high avidity in the ascending order IgG1 > IgG2b > IgG2a. A key finding was the elevated mucosal IgG1 and IgA antibody titers followed by an increase in MOMP-specific IgA after the challenge. The Th1/Th2 antibody titer ratios (IgG2a/IgG1 and IgG2b/IgG1) revealed that PPM evoked a Th2-directed response, which skewed to a Th1-dominated antibody response after the bacterial challenge of mice. In addition, PPM immune sera neutralized the infectivity of C. muridarum in McCoy cells, suggesting the triggering of functional neutralizing antibodies. Herein, we reveal for the first time that subcutaneous immunization with the self-adjuvanting biodegradable co-polymeric PPM nanovaccine immune-potentiated robust CD4+ T cell-mediated immune effector responses; a mixed Th1 and Th2 antibody response and local mucosal IgA to protect mice against a chlamydial genital tract challenge.

Brucella abortus Traverses Brain Microvascular Endothelial Cells Using Infected Monocytes as a Trojan Horse.

Neurobrucellosis is an inflammatory disease caused by the invasion of Brucella spp. to the central nervous system (CNS). The pathogenesis of the disease is not well characterized; however, for Brucella to gain access to the brain parenchyma, traversing of the blood-brain barrier (BBB) must take place. To understand the CNS determinants of the pathogenesis of B. abortus, we have used the in vitro BBB model of human brain microvascular endothelial cells (HBMEC) to study the interactions between B. abortus and brain endothelial cells. In this study, we showed that B. abortus is able to adhere and invade HBMEC which was dependent on microtubules, microfilaments, endosome acidification and de novo protein synthesis. After infection, B. abortus rapidly escapes the endosomal compartment of HBMEC and forms a replicative Brucella-containing vacuole that involves interactions with the endoplasmic reticulum. Despite the ability of B. abortus to invade and replicate in HBMEC, the bacterium was unable by itself to traverse HBMEC, but could traverse polarized HBMEC monolayers within infected monocytes. Importantly, infected monocytes that traversed the HBMEC monolayer were a bacterial source for de novo infection of glial cells. This is the first demonstration of the mechanism whereby B. abortus is able to traverse the BBB and infect cells of the CNS. These results may have important implications in our understanding of the pathogenesis of neurobrucellosis.

Future of human Chlamydia vaccine: potential of self-adjuvanting biodegradable nanoparticles as safe vaccine delivery vehicles.

INTRODUCTION: There is a persisting global burden and considerable public health challenge by the plethora of ocular, genital and respiratory diseases caused by members of the Gram-negative bacteria of the genus Chlamydia. The major diseases are conjunctivitis and blinding trachoma, non-gonococcal urethritis, cervicitis, pelvic inflammatory disease, ectopic pregnancy, tubal factor infertility, and interstitial pneumonia. The failures in screening and other prevention programs led to the current medical opinion that an efficacious prophylactic vaccine is the best approach to protect humans from chlamydial infections. Unfortunately, there is no human Chlamydia vaccine despite successful veterinary vaccines. A major challenge has been the effective delivery of vaccine antigens to induce safe and effective immune effectors to confer long-term protective immunity. The dawn of the era of biodegradable polymeric nanoparticles and the adjuvanted derivatives may accelerate the realization of the dream of human vaccine in the foreseeable future. AREAS COVERED: This review focuses on the current status of human chlamydial vaccine research, specifically the potential of biodegradable polymeric nanovaccines to provide efficacious Chlamydia vaccines in the near future. EXPERT COMMENTARY: The safety of biodegradable polymeric nanoparticles-based experimental vaccines with or without adjuvants and the array of available chlamydial vaccine candidates would suggest that clinical trials in humans may be imminent. Also, the promising results from vaccine testing in animal models could lead to human vaccines against trachoma and reproductive diseases simultaneously.

Caveolin-mediated endocytosis of the Chlamydia M278 outer membrane peptide encapsulated in poly(lactic acid)-Poly(ethylene glycol) nanoparticles by mouse primary dendritic cells enhances specific immune effectors mediated by MHC class II and CD4+ T cells.

We previously developed a Chlamydia trachomatis nanovaccine (PPM) by encapsulating a chlamydial M278 peptide within poly(lactic acid)-poly(ethylene glycol) biodegradable nanoparticles that immunopotentiated Chlamydia-specific immune effector responses in mice. Herein, we investigated the mechanistic interactions of PPM with mouse bone marrow-derived dendritic cells (DCs) for its uptake, trafficking, and T cell activation. Our results reveal that PPM triggered enhanced expression of effector cytokines and chemokines, surface activation markers (Cd1d2, Fcgr1), pathogen-sensing receptors (TLR2, Nod1), co-stimulatory (CD40, CD80, CD86) and MHC class I and II molecules. Co-culturing of PPM-primed DCs with T cells from C. muridarum vaccinated mice yielded an increase in Chlamydia-specific immune effector responses including CD3+ lymphoproliferation, CD3+CD4+ IFN-γ-secreting cells along with CD3+CD4+ memory (CD44high and CD62Lhigh) and effector (CD44high and CD62Llow) phenotypes. Intracellular trafficking analyses revealed an intense expression and colocalization of PPM predominantly in endosomes. PPM also upregulated the transcriptional and protein expression of the endocytic mediator, caveolin-1 in DCs. More importantly, the specific inhibition of caveolin-1 led to decreased expression of PPM-induced cytokines and co-stimulatory molecules. Our investigation shows that PPM provided enhancement of uptake, probably by exploiting the caveolin-mediated endocytosis pathway, endosomal processing, and MHC II presentation to immunopotentiate Chlamydia-specific immune effector responses mediated by CD4+ T cells.

Brucella abortus Promotes a Fibrotic Phenotype in Hepatic Stellate Cells, with Concomitant Activation of the Autophagy Pathway.

The liver is frequently affected in patients with active brucellosis. The present study demonstrates that Brucella abortus infection induces the activation of the autophagic pathway in hepatic stellate cells to create a microenvironment that promotes a profibrogenic phenotype through the induction of transforming growth factor-ß1 (TGF-ß1), collagen deposition, and inhibition of matrix metalloproteinase-9 (MMP-9) secretion. Autophagy was revealed by upregulation of the LC3II/LC3I ratio and Beclin-1 expression as well as inhibition of p62 expression in infected cells. The above-described findings were dependent on the type IV secretion system (VirB) and the secreted BPE005 protein, which were partially corroborated using the pharmacological inhibitors wortmannin, a phosphatidyl inositol 3-kinase inhibitor, and leupeptin plus E64 (inhibitors of lysosomal proteases). Activation of the autophagic pathway in hepatic stellate cells during Brucella infection could have an important contribution to attenuating inflammatory hepatic injury by inducing fibrosis. However, with time, B. abortus infection induced Beclin-1 cleavage with concomitant cleavage of caspase-3, indicating the onset of apoptosis of LX-2 cells, as was confirmed by the terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling assay and Hoechst staining. These results demonstrate that the cross talk of LX-2 cells and B. abortus induces autophagy and fibrosis with concomitant apoptosis of LX-2 cells, which may explain some potential mechanisms of liver damage observed in human brucellosis.

Immunological challenges associated with artificial skin grafts: available solutions and stem cells in future design of synthetic skin.

The repair or replacement of damaged skins is still an important, challenging public health problem. Immune acceptance and long-term survival of skin grafts represent the major problem to overcome in grafting given that in most situations autografts cannot be used. The emergence of artificial skin substitutes provides alternative treatment with the capacity to reduce the dependency on the increasing demand of cadaver skin grafts. Over the years, considerable research efforts have focused on strategies for skin repair or permanent skin graft transplantations. Available skin substitutes include pre- or post-transplantation treatments of donor cells, stem cell-based therapies, and skin equivalents composed of bio-engineered acellular or cellular skin substitutes. However, skin substitutes are still prone to immunological rejection, and as such, there is currently no skin substitute available to overcome this phenomenon. This review focuses on the mechanisms of skin rejection and tolerance induction and outlines in detail current available strategies and alternatives that may allow achieving full-thickness skin replacement and repair.

Interleukin-10 Conjugation to Carboxylated PVP-Coated Silver Nanoparticles for Improved Stability and Therapeutic Efficacy.

Interleukin-10 (IL-10) is a key anti-inflammatory and immunosuppressive cytokine and therefore represents a potential therapeutic agent especially in inflammatory diseases. However, despite its proven therapeutic efficacy, its short half-life and proteolytic degradation in vivo combined with its low storage stability have limited its therapeutic use. Strategies have been developed to overcome most of these shortcomings, including in particular bioconjugation with stabilizing agents such as polyethylene glycol (PEG) and poly (vinylpyrolidone) (PVP), but so far these have had limited success. In this paper, we present an alternative method consisting of bioconjugating IL-10 to PVP-coated silver nanoparticles (Ag-PVPs) in order to achieve its storage stability by preventing denaturation and to improve its anti-inflammatory efficacy. Silver nanoparticles capped with a carboxylated PVP were produced and further covalently conjugated with IL-10 protein by carbodiimide crosslinker chemistry. The IL-10 conjugated Ag-PVPs exhibited increased stability and anti-inflammatory effectiveness in vitro. This study therefore provides a novel approach to bioconjugating PVP-coated silver nanoparticles with therapeutic proteins, which could be useful in drug delivery and anti-inflammatory therapies.

The anti-microbial peptide TP359 attenuates inflammation in human lung cells infected with Pseudomonas aeruginosa via TLR5 and MAPK pathways.

Pseudomonas aeruginosa infection induces vigorous inflammatory mediators secreted by epithelial cells, which do not necessarily eradicate the pathogen. Nonetheless, it reduces lung function due to significant airway damage, most importantly in cystic fibrosis patients. Recently, we published that TP359, a proprietary cationic peptide had potent bactericidal effects against P. aeruginosa, which were mediated by down-regulating its outer membrane biogenesis genes. Herein, we hypothesized that TP359 bactericidal effects could also serve to regulate P. aeruginosa-induced lung inflammation. We explored this hypothesis by infecting human A549 lung cells with live P. aeruginosa non-isogenic, mucoid and non-mucoid strains and assessed the capacity of TP359 to regulate the levels of elicited TNFα, IL-6 and IL-8 inflammatory cytokines. In all instances, the mucoid strain elicited higher concentrations of cytokines in comparison to the non-mucoid strain, and TP359 dose-dependently down-regulated their respective levels, suggesting its regulation of lung inflammation. Surprisingly, P. aeruginosa flagellin, and not its lipopolysaccharide moiety, was the primary inducer of inflammatory cytokines in lung cells, which were similarly down-regulated by TP359. Blocking of TLR5, the putative flagellin receptor, completely abrogated the capacity of infected lung cells to secrete cytokines, underscoring that TP359 regulates inflammation via the TLR5-dependent signaling pathway. Downstream pathway-specific inhibition studies further revealed that the MAPK pathway, essentially p38 and JNK are necessary for induction of P. aeruginosa elicited inflammatory cytokines and their down-regulation by TP359. Collectively, our data provides evidence to support exploring the relevancy of TP359 as an anti-microbial and anti-inflammatory agent against P. aeruginosa for clinical applications.

Brucella abortus-activated microglia induce neuronal death through primary phagocytosis.

Inflammation has long been implicated as a contributor to pathogenesis in neurobrucellosis. Many of the associated neurocognitive symptoms of neurobrucellosis may be the result of neuronal dysfunction resulting from the inflammatory response induced by Brucella abortus infection in the central nervous system. In this manuscript, we describe an immune mechanism for inflammatory activation of microglia that leads to neuronal death upon B. abortus infection. B. abortus was unable to infect or harm primary cultures of mouse neurons. However, when neurons were co-cultured with microglia and infected with B. abortus significant neuronal loss occurred. This phenomenon was dependent on TLR2 activation by Brucella lipoproteins. Neuronal death was not due to apoptosis, but it was dependent on the microglial release of nitric oxide (NO). B. abortus infection stimulated microglial proliferation, phagocytic activity and engulfment of neurons. NO secreted by B. abortus-activated microglia induced neuronal exposure of the "eat-me" signal phosphatidylserine (PS). Blocking of PS-binding to protein milk fat globule epidermal growth factor-8 (MFG-E8) or microglial vitronectin receptor-MFG-E8 interaction was sufficient to prevent neuronal loss by inhibiting microglial phagocytosis without affecting their activation. Taken together, our results indicate that B. abortus is not directly toxic to neurons; rather, these cells become distressed and are killed by phagocytosis in the inflammatory surroundings generated by infected microglia. Neuronal loss induced by B. abortus-activated microglia may explain, in part, the neurological deficits observed during neurobrucellosis.