Saurabh Dixit

Live Borrelia burgdorferi spirochetes elicit inflammatory mediators from human monocytes via the Toll-like receptor signaling pathway.

ABSTRACT We investigated the mechanisms that lead to the production of proinflammatory mediators by human monocytes when these cells are exposed in vitro to live Borrelia burgdorferi spirochetes. We first focused on myeloid differentiation primary response protein 88 (MyD88), an adapter molecule that is essential in the Toll-like receptor (TLR) pathway. Real-time PCR, flow cytometry, and confocal microscopy experiments revealed that MyD88 was maximally expressed in THP-1 cells after 24-h stimulation of these cells with live B. burgdorferi. Silencing of the MYD88 gene by using small interfering RNA resulted in 24%, 35%, and 84% down-modulation of the production of tumor necrosis factor alpha (TNF-α), interleukin-8 (IL-8), and IL-6, respectively, in THP-1 cells stimulated with live B. burgdorferi. Specific silencing of the TLR1, TLR2, or TLR5 gene by RNA interference further revealed that silencing of the TLR1 and TLR2 genes alone or combined, but not the TLR5 gene, caused a downregulation of IL-6, IL-8, and TNF-α in live B. burgdorferi-stimulated THP-1 cells. Overall, similar results were obtained for THP-1 cells stimulated with purified lipoproteins. Our results indicate that the TLR pathway mediates, at least in part, the release of inflammatory mediators in human monocytes stimulated with live B. burgdorferi spirochetes and furthermore suggest that the TLR-dependent interaction between these cells and live spirochetes is mediated by spirochetal lipoproteins but not by flagellin.

Future Prospects for Scaffolding Methods and Biomaterials in Skin Tissue Engineering: A Review

Over centuries, the field of regenerative skin tissue engineering has had several advancements to facilitate faster wound healing and thereby restoration of skin. Skin tissue regeneration is mainly based on the use of suitable scaffold matrices. There are several scaffold types, such as porous, fibrous, microsphere, hydrogel, composite and acellular, etc., with discrete advantages and disadvantages. These scaffolds are either made up of highly biocompatible natural biomaterials, such as collagen, chitosan, etc., or synthetic materials, such as polycaprolactone (PCL), and poly-ethylene-glycol (PEG), etc. Composite scaffolds, which are a combination of natural or synthetic biomaterials, are highly biocompatible with improved tensile strength for effective skin tissue regeneration. Appropriate knowledge of the properties, advantages and disadvantages of various biomaterials and scaffolds will accelerate the production of suitable scaffolds for skin tissue regeneration applications. At the same time, emphasis on some of the leading challenges in the field of skin tissue engineering, such as cell interaction with scaffolds, faster cellular proliferation/differentiation, and vascularization of engineered tissues, is inevitable. In this review, we discuss various types of scaffolding approaches and biomaterials used in the field of skin tissue engineering and more importantly their future prospects in skin tissue regeneration efforts.

Anti-inflammatory effects of silver-polyvinyl pyrrolidone (Ag-PVP) nanoparticles in mouse macrophages infected with live Chlamydia trachomatis

Chlamydia trachomatis is a very common sexually transmissible infection in both developing and developed countries. A hallmark of C. trachomatis infection is the induction of severe inflammatory responses which play critical roles in its pathogenesis. Antibiotics are the only treatment option currently available for controlling C. trachomatis infection; however, they are efficacious only when administered early after an infection. The objectives of this study are to explore alternative strategies in the control and regulation of inflammatory responses triggered by a C. trachomatis infection. We employed silver-polyvinyl pyrrolidone (Ag-PVP) nanoparticles, which have been shown to possess anti-inflammatory properties, as our target and the in vitro mouse J774 macrophage model of C. trachomatis infection. Our hypothesis is that small sizes of Ag-PVP nanoparticles will control inflammatory mediators triggered by a C. trachomatis infection. Cytotoxicity studies using Ag-PVP nanoparticles of 10, 20, and 80 nm sizes revealed >80% macrophage viability up to a concentration of 6.25 μg/mL, with the 10 nm size being the least toxic. All sizes of Ag-PVP nanoparticles, especially the 10 nm size, reduced the levels of the prototypic cytokines, tumor necrosis factor (TNF) and interleukin (IL)-6, as elicited from C. trachomatis infected macrophages. Additionally, Ag-PVP nanoparticles (10 nm) selectively inhibited a broad spectrum of other cytokines and chemokines produced by infected macrophages. Of significance, Ag-PVP nanoparticles (10 nm) caused perturbations in a variety of upstream (toll like receptor 2 [TLR2], nucleotide-binding oligomerization-protein 2 [NOD2], cluster of differentiation [CD]40, CD80, and CD86) and downstream (IL-1 receptor-associated kinase 3 [IRAK3] and matrix metallopeptidase 9 [MMP9]) inflammatory signaling pathways by downregulating their messenger ribonucleic acid (mRNA) gene transcript expressions as induced by C. trachomatis in macrophages. Collectively, our data provides further evidence for the anti-inflammatory properties of Ag-PVP nanoparticles, and opens new possibilities for smaller sizes of Ag-PVP nanoparticles to be employed as regulators of inflammatory responses induced by C. trachomatis.

Interleukin-10 Alters Effector Functions of Multiple Genes Induced by Borrelia burgdorferi in Macrophages To Regulate Lyme Disease Inflammation

ABSTRACT Interleukin-10 (IL-10) modulates inflammatory responses elicited in vitro and in vivo by Borrelia burgdorferi, the Lyme disease spirochete. How IL-10 modulates these inflammatory responses still remains elusive. We hypothesize that IL-10 inhibits effector functions of multiple genes induced by B. burgdorferi in macrophages to control concomitantly elicited inflammation. Because macrophages are essential in the initiation of inflammation, we used mouse J774 macrophages and live B. burgdorferi spirochetes as the model target cell and stimulant, respectively. First, we employed transcriptome profiling to identify genes that were induced by stimulation of cells with live spirochetes and that were perturbed by addition of IL-10 to spirochete cultures. Spirochetes significantly induced upregulation of 347 genes at both the 4-h and 24-h time points. IL-10 inhibited the expression levels, respectively, of 53 and 65 of the 4-h and 24-h genes, and potentiated, respectively, at 4 h and 24 h, 65 and 50 genes. Prominent among the novel identified IL-10-inhibited genes also validated by quantitative real-time PCR (qRT-PCR) were Toll-like receptor 1 (TLR1), TLR2, IRAK3, TRAF1, IRG1, PTGS2, MMP9, IFI44, IFIT1, and CD40. Proteome analysis using a multiplex enzyme-linked immunosorbent assay (ELISA) revealed the IL-10 modulation/and or potentiation of RANTES/CCL5, macrophage inflammatory protein 2 (MIP-2)/CXCL2, IP-10/CXCL10, MIP-1α/CCL3, granulocyte colony-stimulating factor (G-CSF)/CSF3, CXCL1, CXCL5, CCL2, CCL4, IL-6, tumor necrosis factor alpha (TNF-α), IL-1α, IL-1β, gamma interferon (IFN-γ), and IL-9. Similar results were obtained using sonicated spirochetes or lipoprotein as stimulants. Our data show that IL-10 alters effectors induced by B. burgdorferi in macrophages to control concomitantly elicited inflammatory responses. Moreover, for the first time, this study provides global insight into potential mechanisms used by IL-10 to control Lyme disease inflammation.

Advances in Skin Regeneration Using Tissue Engineering

Tissue engineered skin substitutes for wound healing have evolved tremendously over the last couple of years. New advances have been made toward developing skin substitutes made up of artificial and natural materials. Engineered skin substitutes are developed from acellular materials or can be synthesized from autologous, allograft, xenogenic, or synthetic sources. Each of these engineered skin substitutes has their advantages and disadvantages. However, to this date, a complete functional skin substitute is not available, and research is continuing to develop a competent full thickness skin substitute product that can vascularize rapidly. There is also a need to redesign the currently available substitutes to make them user friendly, commercially affordable, and viable with longer shelf life. The present review focuses on providing an overview of advances in the field of tissue engineered skin substitute development, the availability of various types, and their application.

A Class of Tricyclic Compounds Blocking Malaria Parasite Oocyst Development and Transmission

ABSTRACT Malaria is a deadly infectious disease in many tropical and subtropical countries. Previous efforts to eradicate malaria have failed, largely due to the emergence of drug-resistant parasites, insecticide-resistant mosquitoes and, in particular, the lack of drugs or vaccines to block parasite transmission. ATP-binding cassette (ABC) transporters are known to play a role in drug transport, metabolism, and resistance in many organisms, including malaria parasites. To investigate whether a Plasmodium falciparum ABC transporter (Pf14_0244 or PfABCG2) modulates parasite susceptibility to chemical compounds or plays a role in drug resistance, we disrupted the gene encoding PfABCG2, screened the recombinant and the wild-type 3D7 parasites against a library containing 2,816 drugs approved for human or animal use, and identified an antihistamine (ketotifen) that became less active against the PfABCG2-disrupted parasite in culture. In addition to some activity against asexual stages and gametocytes, ketotifen was highly potent in blocking oocyst development of P. falciparum and the rodent parasite Plasmodium yoelii in mosquitoes. Tests of structurally related tricyclic compounds identified additional compounds with similar activities in inhibiting transmission. Additionally, ketotifen appeared to have some activity against relapse of Plasmodium cynomolgi infection in rhesus monkeys. Further clinical evaluation of ketotifen and related compounds, including synthetic new derivatives, in blocking malaria transmission may provide new weapons for the current effort of malaria eradication.

Chlamydia trachomatis recombinant MOMP encapsulated in PLGA nanoparticles triggers primarily T helper 1 cellular and antibody immune responses in mice: a desirable candidate nanovaccine

We recently demonstrated by in vitro experiments that PLGA (poly D, L-lactide-co-glycolide) potentiates T helper 1 (Th1) immune responses induced by a peptide derived from the recombinant major outer membrane protein (rMOMP) of Chlamydia trachomatis, and may be a promising vaccine delivery system. Herein we evaluated the immune-potentiating potential of PLGA by encapsulating the full-length rMOMP (PLGA-rMOMP), characterizing it in vitro, and investigating its immunogenicity in vivo. Our hypothesis was that PLGA-rMOMP triggers Th1 immune responses in mice, which are desirable prerequisites for a C. trachomatis candidate nanovaccine. Physical-structural characterizations of PLGA-rMOMP revealed its size (approximately 272 nm), zeta potential (−14.30 mV), apparent spherical smooth morphology, and continuous slow release pattern. PLGA potentiated the ability of encapsulated rMOMP to trigger production of cytokines and chemokines by mouse J774 macrophages. Flow cytometric analyses revealed that spleen cells from BALB/c mice immunized with PLGA-rMOMP had elevated numbers of CD4+ and CD8+ T cell subsets, and secreted more rMOMP-specific interferon-gamma (Th1) and interleukin (IL)-12p40 (Th1/Th17) than IL-4 and IL-10 (Th2) cytokines. PLGA-rMOMP-immunized mice produced higher serum immunoglobulin (Ig)G and IgG2a (Th1) than IgG1 (Th2) rMOMP-specific antibodies. Notably, sera from PLGA-rMOMP-immunized mice had a 64-fold higher Th1 than Th2 antibody titer, whereas mice immunized with rMOMP in Freund’s adjuvant had only a four-fold higher Th1 than Th2 antibody titer, suggesting primarily induction of a Th1 antibody response in PLGA-rMOMP-immunized mice. Our data underscore PLGA as an effective delivery system for a C. trachomatis vaccine. The capacity of PLGA-rMOMP to trigger primarily Th1 immune responses in mice promotes it as a highly desirable candidate nanovaccine against C. trachomatis.

Different Patterns of Expression and of IL-10 Modulation of Inflammatory Mediators from Macrophages of Lyme Disease-Resistant and -Susceptible Mice

C57BL/6J (C57) mice develop mild arthritis (Lyme disease-resistant) whereas C3H/HeN (C3H) mice develop severe arthritis (Lyme disease-susceptible) after infection with the spirochete Borrelia burgdorferi. We hypothesized that susceptibility and resistance to Lyme disease, as modeled in mice, is associated with early induction and regulation of inflammatory mediators by innate immune cells after their exposure to live B. burgdorferi spirochetes. Here, we employed multiplex ELISA and qRT-PCR to investigate quantitative differences in the levels of cytokines and chemokines produced by bone marrow-derived macrophages from C57 and C3H mice after these cells were exposed ex vivo to live spirochetes or spirochetal lipoprotein. Upon stimulation, the production of both cytokines and chemokines was up-regulated in macrophages from both mouse strains. Interestingly, however, our results uncovered two distinct patterns of spirochete- and lipoprotein-inducible inflammatory mediators displayed by mouse macrophages, such that the magnitude of the chemokine up-regulation was larger in C57 cells than it was in C3H cells, for most chemokines. Conversely, cytokine up-regulation was more intense in C3H cells. Gene transcript analyses showed that the displayed patterns of inflammatory mediators were associated with a TLR2/TLR1 transcript imbalance: C3H macrophages expressed higher TLR2 transcript levels as compared to those expressed by C57 macrophages. Exogenous IL-10 dampened production of inflammatory mediators, especially those elicited by lipoprotein stimulation. Neutralization of endogenously produced IL-10 increased production of inflammatory mediators, notably by macrophages of C57 mice, which also displayed more IL-10 than C3H macrophages. The distinct patterns of pro-inflammatory mediator production, along with TLR2/TLR1 expression, and regulation in macrophages from Lyme disease-resistant and -susceptible mice suggests itself as a blueprint to further investigate differential pathogenesis of Lyme disease.

Anti-Pfs25 Human Plasma Reduces Transmission of Plasmodium falciparum Isolates That Have Diverse Genetic Backgrounds

ABSTRACT Pfs25 is a leading candidate for a malaria transmission-blocking vaccine whose potential has been demonstrated in a phase 1 trial with recombinant Pfs25 formulated with Montanide ISA51. Because of limited sequence polymorphism, the anti-Pfs25 antibodies induced by this vaccine are likely to have transmission-blocking or -reducing activity against most, if not all, field isolates. To test this hypothesis, we evaluated transmission-blocking activities by membrane feeding assay of anti-Pfs25 plasma from the Pfs25/ISA51 phase 1 trial against Plasmodium falciparum parasites from patients in two different geographical regions of the world, Thailand and Burkina Faso. In parallel, parasite isolates from these patients were sequenced for the Pfs25 gene and genotyped for seven microsatellites. The results indicate that despite different genetic backgrounds among parasite isolates, the Pfs25 sequences are highly conserved, with a single nonsynonymous nucleotide polymorphism detected in 1 of 41 patients in Thailand and Burkina Faso. The anti-Pfs25 immune plasma had significantly higher transmission-reducing activity against parasite isolates from the two geographical regions than the nonimmune controls (P < 0.0001).

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.