Murine Borrelia arthritis is highly dependent on ASC and caspase-1, but independent of NLRP3

Introduction

The protein platform called the NOD-like-receptor -family member (NLRP)-3 inflammasome needs to be activated to process intracellular caspase-1. Active caspase-1 is able to cleave pro-Interleukin (IL)-1β, resulting in bioactive IL-1β. IL-1β is a potent proinflammatory cytokine, and thought to play a key role in the pathogenesis of Lyme arthritis, a common manifestation of Borrelia burgdorferi infection. The precise pathways through which B. burgdorferi recognition leads to inflammasome activation and processing of IL-1β in Lyme arthritis has not been elucidated. In the present study, we investigated the contribution of several pattern recognition receptors and inflammasome components in a novel murine model of Lyme arthritis.

Methods

Lyme arthritis was elicited by live B. burgdorferi, injected intra-articularly in knee joints of mice. To identify the relevant pathway components, the model was applied to wild-type, NLRP3-/-, ASC-/-, caspase-1-/-, NOD1-/-, NOD2-/-, and RICK-/- mice. As a control, TLR2-/-, Myd88-/- and IL-1R-/- mice were used. Peritoneal macrophages and bone marrow-derived macrophages were used for in vitro cytokine production and inflammasome activation studies. Joint inflammation was analyzed in synovial specimens and whole knee joints. Mann-Whitney U tests were used to detect statistical differences.

Results

We demonstrate that ASC/caspase-1-driven IL-1β is crucial for induction of B. burgdorferi-induced murine Lyme arthritis. In addition, we show that B. burgdorferi-induced murine Lyme arthritis is less dependent on NOD1/NOD2/RICK pathways while the TLR2-MyD88 pathway is crucial.

Conclusions

Murine Lyme arthritis is strongly dependent on IL-1 production, and B. burgdorferi induces inflammasome-mediated caspase-1 activation. Next to that, murine Lyme arthritis is ASC- and caspase-1-dependent, but NLRP3, NOD1, NOD2, and RICK independent. Also, caspase-1 activation by B. burgdorferi is dependent on TLR2 and MyD88. Based on present results indicating that IL-1 is one of the major mediators in Lyme arthritis, there is a rationale to propose that neutralizing IL-1 activity may also have beneficial effects in chronic Lyme arthritis.     

Nanocarrier for the Transdermal Delivery of an Antiparkinsonian Drug

The purpose of the present study was to investigate the potential of nanoemulsions as nanodrug carrier systems for the percutaneous delivery of ropinirole. Nanoemulsions comprised Capryol 90 as the oil phase, Tween 20 as the surfactant, Carbitol as the cosurfactant, and water as an external phase. The effects of composition of nanoemulsion, including the ratio of surfactant and cosurfactant (S _mix) and their concentration on skin permeation, were evaluated. All the prepared nanoemulsions showed a significant increase in permeation parameters such as steady state flux (J _ss) and permeability coefficient (K _p) when compared to the control (p < 0.01). Nanoemulsion composition (NEL3) comprising ropinirole (0.5% w/w), Capryol 90 (5% w/w), S _mix 2:1 (35% w/w), and water (59.5% w/w) showed the highest flux (51.81 ± 5.03 μg/cm²/h) and was selected for formulation into nanoemulsion gel. The gel was further optimized with respect to oil concentration (Capryol 90), polymer concentration (Carbopol), and drug content by employing the Box–Behnken design, which statistically evaluated the effects of these components on ropinirole permeation. Oil and polymer concentrations were found to have a negative influence on permeation, while the drug content had a positive effect. Nanoemulsion gel showed a 7.5-fold increase in skin permeation rate when compared to the conventional hydrogel. In conclusion, the results of the present investigation suggested a promising role of nanoemulsions in enhancing the transdermal permeation of ropinirole.     

Tyrosine phosphorylation of myosin heavy chain during skeletal muscle differentiation: an integrated bioinformatics approach

Background  

Previously it has been shown that insulin-mediated tyrosine phosphorylation of myosin heavy chain is concomitant with enhanced association of C-terminal SRC kinase during skeletal muscle differentiation. We sought to identify putative site(s) for this phosphorylation event.     

Exemestane Loaded Self-Microemulsifying Drug Delivery System (SMEDDS): Development and Optimization

The purpose of this research work was to formulate and characterize self-micro emulsifying drug delivery system containing exemestane. The solubility of exemestane was determined in various vehicles. Pseudo ternary phase diagram was used to evaluate the micro-emulsification existence area. SMEDDS formulations were tested for micro-emulsifying properties, and the resultant formulations loaded with exemestane (ME1, ME2, ME3, ME4 and ME5) were investigated for clarity, phase separation, globule size and shape, zeta potential, effect of various diluents and dilutions, thermodynamic and thermal stability. From the results it is concluded that increase in droplet size is proportional to the concentration of oil in SMEDDS formulation. Minor difference in the droplet size and zeta potential was observed by varying the diluents (deionized water and 0.1 N HCl) and dilutions (1:10, 1:50 and 1:100). Formulations, which were found to be thermodynamically stable (ME1, ME2, ME3 and ME4), were subjected to stability studies as per International Conference on Harmonization (ICH) guidelines. No significant variations were observed in the formulations over a period of 3 months at accelerated and long-term conditions. TEM photographs of microemulsions formulations further conformed the spherical shape of globules. Among the various SMEDDS formulations, ME4 offer the advantages of good clarity systems at high oil content and thus offer good solubilization of exemestane. Thus this study indicates that the SMEDDS can be used as a potential drug carrier for dissolution enhancement of exemestane and other lipophilic drug(s).     

Pre-clinical evidence for altered absorption and biliary excretion of irinotecan (CPT-11) in combination with quercetin: possible contribution of P-glycoprotein

P-glycoprotein (P-gp) is found to play a very significant role in intestinal and biliary transport of irinotecan and its active metabolite, SN-38. This makes P-gp inhibition a logical strategy for improving irinotecan's oral efficacy and reducing its toxicity. The objective of the present study was to identify the most suitable P-gp inhibitor, amongst various commonly used herbal components via in vitro screening; followed by determination of in vivo effects in rats. Caco-2 cell monolayers were used to investigate the influence of various components (quercetin, hesperitin, piperine, curcumin and naringenin) on the transport of irinotecan. The secretory transport (basolateral-to-apical) was significantly decreased by all components (p<0.05) except piperine. In the apical-to-basolateral transport, quercetin showed the highest absorptive permeability enhancement and P-gp interaction potential making it an appropriate candidate for further in vivo studies in female Wistar rats. Quercetin pre-treatment resulted in increased irinotecan C(max) and area under curve (AUC) with a concomitant decrease in t(max), plasma clearance and volume of distribution (p<0.05). The absolute bioavailability (F) of irinotecan control was 33%, which was increased to 43% (1.3 fold) by quercetin administration. The amounts of irinotecan and SN-38 eliminated in bile in control rats, is reduced to almost half when treated with quercetin. Our studies not only propose a safe approach for bioavailability enhancement and reducing toxicity of irinotecan by P-gp inhibition but in another way also reiterate the significance of elucidating herb-drug interactions for future insights.     

Activated macrophages migrate to the subcutaneous tumor site via the peritoneum: a novel route of cell trafficking

Spontaneous regression of AK-5 tumor in syngeneic hosts reported earlier involves the interplay of Th1-type cytokines and cell-mediated immunity. Upon subcutaneous transplantation of AK-5 cells, there was accumulation of immune cells in the peritoneum, of which macrophages were the predominant type and were found to be in a hyperactive state. They released macrophage-derived tumoricidal mediators like NO, O2(-), and ONOO(-) which exhibited potent cytotoxic activity against AK-5 cells in vitro. Interestingly, there was a dramatic disappearance of these hyperactive cells from the peritoneal cavity which correlated well with the onset of tumor regression at the subcutaneous site. Direct labeling of these cells in the peritoneum by the tracking dye PKH26 showed their migration to the tumor site. Similarly, frozen tumor sections when scanned under confocal microscope clearly exhibited fluorescent macrophages embedded into the tumor. Immunohistochemical sections of these intratumoral macrophages showed nitrotyrosine residues, indicating their contribution in the free-radical-mediated AK-5 cell death, thereby leading to successful tumor remission. These observations suggest a directional migration of the hyperactivated peritoneal population to the tumor site. We have also confirmed the influx of macrophages and other immune cells into the peritoneum after sc transplantation of Meth A tumor cells in Balb/c mice. Our studies suggest a role for the peritoneal compartment in imparting appropriate stimulus to the immune cells prior to their participation in the antitumor immune response. These studies suggest a novel route of macrophage trafficking via the peritoneum.     

Protection conferred by Bcl-2 expression involves reduced oxidative stress and increased glutathione production during hypothermia-induced apoptosis in AK-5 tumor cells

Hypothermia is known to retard mammalian cell growth, however, BC-8 cells, which have originated from AK-5 tumor after single cell cloning, were triggered into apoptotic pathway when grown at 30 degrees C. Cell death process showed typical apoptotic features like DNA fragmentation, cytochrome c release, etc. Introduction of Bcl-2 gene in BC-8 cells inhibited hypothermia-induced apoptotic process, which is ascribed to reduced ROS generation and higher glutathione production. Thus, Bcl-2 seems to control the apoptotic induction process at the level of redox regulation, in addition to its known effects at the mitochondrial dysregulation. These observations suggest that tumors, which are low in Bcl-2 expression, are sensitive to hypothermic shock and make hypothermia an interesting inducer of apoptosis in tumor cells.     

Regulation of CD40L expression on natural killer cells by interleukin-12 and interferon γ: its role in the elicitation of an effective antitumor immune response

Effector cell functions are regulated by a number of positive signals for the mediation of antitumor immunity. The CD40 and CD40 ligand (CD40L) interaction has been implicated in the generation of effective cell-mediated and humoral immune responses, where cytokines have been shown to play a significant role in the expression of these molecules. Our earlier studies have shown that spontaneous regression of a rat histiocytoma transplanted s.c. is mediated by CD8⁺ CD3⁻ NK cells. The CD40-CD40L mediation during tumor regression was of interest. Tumor-transplanted animals showed enhanced expression of CD40L on natural killer (NK) and T cells, when compared to cells from normal animals. CD40 expression on AK-5 tumor cells was also induced after s.c. transplantation. Administration of anti-(interleukin-12) (anti-IL-12) and anti-(interferon γ) (anti-IFNγ) antibodies in tumor-bearing animals showed down-regulation of the expression of CD40L on NK and T cells with simultaneous inhibition of cytotoxic acitivity of NK cells, cytokine release and the production of antitumor antibody. Naive NK cells, when co-cultured with fixed AK-5 cells, were induced to express CD40L. CD40L expression modulated the immune response exerted by NK cells, in part by the activation of nuclear factor kB (NF-kB). Furthermore, the signaling via CD40L through the use of anti-CD40L antibody promoted the in vitro activation of cytotoxic as well as NF-kB binding activity in NK cells from tumor-transplanted animals. These observations demonstrate that the expression of CD40L by the effector cells is regulated by IL-12 and IFNγ, and could effectively modulate the NK-cell-mediated immune response during the regression of AK-5 tumor. Received: 1 June 2000 / Accepted: 27 July 2000