Effect of chlorpyrifos on VEGF gene expression.

Chlorpyrifos (CPF; 0,0-diethyl 0-(3,5,6-trichloro-2-pyridinyl)-phosphorothioate), a cholinesterase inhibitor, compromised the integrity of the blood-brain barrier (BBB) when used at low concentrations during our previous experiments in vitro. To determine if BBB leakage would also occur in vivo, we used FITC-dextrans to evaluate BBB permeability in CPF-dosed mice. Results indicated BBB leakages that were evident at 2 h after treatment with 70 mg/kg CPF ip. Since vascular endothelial growth factor (VEGF), a potent vasopermeability factor, is a signaling protein that promotes the growth of new blood vessels, we investigated the possible involvement of VEGF in BBB disruption by CPF. We found that VEGF serum concentration was significantly increased at 24 h after CPF exposure. To further explore VEGF involving BBB disruption by CPF treatment, the receptor antagonist for VEGF (sFlt-1) was used for pretreatment before CPF exposure. After sFlt-1 pretreatment, gene expressions of the tight junction (TJ) proteins claudin5 and occludin were significantly downregulated at 1, 2, and 3 h, but returned to control levels at 24 h after CPF treatment. These results suggest that VEGF is involved in BBB disruption by CPF through BBB-TJs regulation.

Formulation of Nanovaccines toward an Extended Immunity against Nicotine.

Nicotine vaccines have been investigated to assist with smoking cessation. Because smoking cessation is a long process, past nicotine vaccines required multiple injections to achieve long-term efficacy. It would be of great significance if extended efficacy can be achieved with fewer injections. Here, we report the assembly of lipid-polylactic acid (PLA) and lipid-poly(lactic-co-glycolic acid) (PLGA) hybrid nanoparticle (NP) based nicotine vaccines. Mice immunized with the lipid-PLGA vaccine produced higher titers of nicotine-specific antibodies than the lipid-PLA vaccine in short-term. However, the lipid-PLA vaccine was found to induce long-lasting antibodies. Three months after the immunization, only mice that received first two injections of the lipid-PLGA vaccine and a third injection of the lipid-PLA vaccine achieved a significantly lower brain nicotine concentration of 65.13 ± 20.59 ng/mg than 115.88 ± 37.62 ng/mg from the negative controls. The results indicate that not only the stability of the vaccines but also the combination of the vaccines impacted the long-term efficacy of the immunization. Lastly, both the body weight and the histopathology study suggest that the vaccines were safe to mice. These findings suggest that long-term immunity against nicotine can be realized by a rational administration of nanovaccines of different levels of stability.

The Safety, Tolerability, and Pharmacokinetics Profile of BT-11, an Oral, Gut-Restricted Lanthionine Synthetase C-Like 2 Agonist Investigational New Drug for Inflammatory Bowel Disease: A Randomized, Double-Blind, Placebo-Controlled Phase I Clinical Trial.

BT-11 is a new oral, gut-restricted, first-in-class investigational drug for Crohn disease (CD) and ulcerative colitis (UC) that targets the lanthionine synthetase C-like 2 (LANCL2) pathway and immunometabolic mechanisms. Oral BT-11 was assessed for safety, tolerability, and pharmacokinetics (PK) in normal healthy volunteers (n = 70) in a randomized, double-blind, placebo-controlled trial. Subjects (n = 70) were randomly assigned to one of five single ascending dose cohorts (up to 100 mg/kg, p.o.) and three multiple ascending dose cohorts [up to 100 mg/kg daily (QD) for seven days, orally]. Safety and tolerability were assessed by adverse event (AE) reporting, vital signs, electrocardiogram, hematology, and clinical chemistry. BT-11 did not increase total or gastrointestinal AE rates, as compared with placebo, and no serious adverse events were observed. Oral BT-11 dosing does not result in any clinically significant findings by biochemistry, coagulation, electrocardiogram, hematology, or urinalysis as compared with placebo. Mean fecal concentrations of BT-11 increased linearly with increasing oral doses, with 2.39 mg/g at 7.7 mg/kg on day 7 of the multiple ascending dose (MAD). Analysis of plasma pharmacokinetics indicates that maximum systemic concentrations are approximately 1/6000th of observed concentrations in feces and the distal gastrointestinal tract. Fecal calprotectin levels were lower in BT-11 treated groups as compared to placebo. BT-11 significantly decreases interferon gamma positive (IFNγ+) and tumor necrosis factor alpha positive (TNFα+) cluster of differentiation 4 positive (CD4+) T cells and increases forkhead box P3 positive (FOXP3+) CD4+ T cells in colonic lamina propria mononuclear cells from patients with CD and patients with UC at concentrations of 0.01 µM when treated ex vivo. BT-11 treatment is well-tolerated with no dose-limiting toxicities up to daily oral doses of 100 mg/kg (16 tablets); whereas the efficacious dose is a single tablet (8 mg/kg). Phase II studies in CD and UC patients are ongoing.

Alum as an adjuvant for nanoparticle based vaccines: A case study with a hybrid nanoparticle-based nicotine vaccine.

The treatment efficacy of a nicotine vaccine largely relies on its ability to induce high titers of nicotine-specific antibodies. Due to its strong immune-potentiating effects, aluminum salt (Alum) has been commonly used as an adjuvant in various nicotine vaccine formulations. In this study, we attempted to improve the immunological performance of a hybrid nanoparticle-based nicotine vaccine (NanoNicVac) by co-administering it with Alum. It was found that Alum severely restricted the release of NanoNicVac at the site of injection. Moreover, Alum damaged the hybrid structure of the vaccine. In the animal trial, mice immunized with NanoNicVac alone achieved an anti-nicotine IgG titer of 3.5 ±â€¯0.2 × 104 after three injections. Unexpectedly, Alum with quantities of 125, 250, 500, and 1000 µg did not enhance the immunogenicity of NanoNicVac. In addition, Alum did not improve the ability of the vaccine to reduce the entry of nicotine into the brain.

Effect of Adjuvant Release Rate on the Immunogenicity of Nanoparticle-Based Vaccines: A Case Study with a Nanoparticle-Based Nicotine Vaccine.

Adjuvants are a critical component for vaccines, especially for a poorly immunogenic antigen, such as nicotine. However, the impact of adjuvant release rate from a vaccine formulation on its immunogenicity has not been well illustrated. In this study, we fabricated a series of hybrid-nanoparticle-based nicotine vaccines to study the impact of adjuvant release rate on their immunological efficacy. It was found that the nanovaccine with a medium or slow adjuvant release rate induced a significantly higher anti-nicotine antibody titer than that with a fast release rate. Furthermore, the medium and slow adjuvant release rates resulted in a significantly lower brain nicotine concentration than the fast release rate after nicotine challenge. All findings suggest that adjuvant release rate affects the immunological efficacy of nanoparticle-based nicotine vaccines, providing a potential strategy to rationally designing vaccine formulations against psychoactive drugs or even other antigens. The hybrid-nanoparticle-based nicotine vaccine with an optimized adjuvant release rate can be a promising next-generation immunotherapeutic candidate against nicotine.

Paradox of PEGylation in fabricating hybrid nanoparticle-based nicotine vaccines.

Polyethylene glycol (PEG) has long been used in nanoparticle-based drug or vaccine delivery platforms. In this study, nano-nicotine vaccines (NanoNicVac) were PEGylated to different degrees to investigate the impact of PEG on the immunological efficacy of the vaccine. Hybrid nanoparticles with various degrees of PEGylation (2.5%-30%) were assembled. It was found that 30% PEGylation resulted in a hybrid nanoparticle of a compromised core-shell structure. A higher concentration of PEG also led to a slower cellular uptake of hybrid nanoparticles by dendritic cells. However, increasing the quantity of the PEG could effectively reduce nanoparticle aggregation during storage and improve the stability of the hybrid nanoparticles. Subsequently, nicotine vaccines were synthesized by conjugating nicotine haptens to the differently PEGylated hybrid nanoparticles. In both in vitro and in vivo studies, it was found that a nicotine vaccine with 20% PEGylation (NanoNicVac 20.0) was significantly more stable than the vaccines with lower PEGylation. In addition, NanoNicVac 20.0 induced a significantly higher anti-nicotine antibody titer of 3.7 ±â€¯0.6 × 104 in mice than the other NanoNicVacs with lower concentrations of PEG. In a subsequent pharmacokinetic study, the lowest brain nicotine concentration of 34 ±â€¯11 ng/g was detected in mice that were immunized with NanoNicVac 20.0. In addition, no apparent adverse events were observed in mice immunized with NanoNicVac. In summary, 20% PEGylation confers NanoNicVac with desirable safety, the highest stability, and the best immunological efficacy in mice.

High-throughput toxicity testing of chemicals and mixtures in organotypic multi-cellular cultures of primary human hepatic cells.

High-throughput screening (HTS) of liver toxicants can bridge the gap in understanding adverse effects of chemicals on humans. Toxicity testing of mixtures is time consuming and expensive, since the number of possible combinations increases exponentially with the number of chemicals. The combination of organotypic culture models (OCMs) and HTS assays can lead to the rapidly evaluation of chemical toxicity in a cost and time-effective manner while prioritizing chemicals that warrant additional investigation. We describe the design, assembly and toxicant response of multi-cellular hepatic organotypic culture models comprised of primary human or rat cells assembled in 96-well plates (denoted as µOCMs). These models were assembled using automated procedures that did not affect hepatocyte function or viability, rendering them ideal for large-scale toxicity evaluations. Rat µOCMs were assembled to obtain insights into deviations from human toxicity. Four test chemicals (acetaminophen, ethanol, isoniazid, and perfluorooctanoic acid) were added to the µOCMs individually or in mixtures. HTS assays were utilized to measure cell death, apoptosis, glutathione depletion, mitochondrial membrane damage, and cytochrome P450 2E1 activity. The µOCMs exhibited increased toxicant sensitivity compared to hepatocyte sandwich cultures. Synergistic and non-synergistic interactions were observed when the toxicants were added as mixtures. Specifically, chemical interactions in the µOCMs were manifested by changes in apoptosis and decreased glutathione. The µOCMs accurately predicted hepatotoxicity for individual and mixtures of toxicants, demonstrating their potential for large-scale toxicity evaluations in the future.

Hybrid nanoparticle-based nicotine nanovaccines: Boosting the immunological efficacy by conjugation of potent carrier proteins.

A series of hybrid nanoparticle-based nicotine nanovaccines (NanoNicVac) were engineered in this work by conjugating potent carrier protein candidates (Keyhole limpet hemocyanin (KLH) multimer, KLH subunit, cross-reactive material 197 (CRM197), or tetanus toxoid (TT)) for enhanced immunological efficacy. NanoNicVac with CRM197 or TT were processed by dendritic cells more efficiently than that with KLH multimer or subunit. NanoNicVac carrying CRM197 or TT exhibited a significantly higher immunogenicity against nicotine and a considerably lower immunogenicity against carrier proteins than NanoNicVac carrying KLH multimer or subunit in mice. The in vivo results revealed that NanoNicVac with CRM197 or TT resulted in lower levels of nicotine in the brain of mice after nicotine challenge. All findings suggest that an enhanced immunological efficacy of NanoNicVac can be achieved by using CRM197 or TT instead of KLH or KLH subunit as carrier proteins, making NanoNicVac a promising next-generation immunotherapeutic candidate against nicotine addiction.

Rational incorporation of molecular adjuvants into a hybrid nanoparticle-based nicotine vaccine for immunotherapy against nicotine addiction.

Current clinically-tested nicotine vaccines have yet shown enhanced smoking cessation efficacy due to their low immunogenicity. Achieving a sufficiently high immunogenicity is a necessity for establishing a clinically-viable nicotine vaccine. This study aims to facilitate the immunogenicity of a hybrid nanoparticle-based nicotine vaccine by rationally incorporating toll-like receptor (TLR)-based adjuvants, including monophosphoryl lipid A (MPLA), Resiquimod (R848), CpG oligodeoxynucleotide 1826 (CpG ODN 1826), and their combinations. The nanoparticle-delivered model adjuvant was found to be taken up more efficiently by dendritic cells than the free counterpart. Nanovaccine particles were transported to endosomal compartments upon cellular internalization. The incorporation of single or dual TLR adjuvants not only considerably increased total anti-nicotine IgG titers but also significantly affected IgG subtype distribution in mice. Particularly, the nanovaccines carrying MPLA+R848 or MPLA+ODN 1826 generated a much higher anti-nicotine antibody titer than those carrying none or one adjuvant. Meanwhile, the anti-nicotine antibody elicited by the nanovaccine adjuvanted with MPLA+R848 had a significantly higher affinity than that elicited by the nanovaccine carrying MPLA+ODN 1826. Moreover, the incorporation of all the selected TLR adjuvants (except MPLA) reduced the brain nicotine levels in mice after nicotine challenge. Particularly, the nanovaccine with MPLA+R848 exhibited the best ability to reduce the level of nicotine entering the brain. Collectively, rational incorporation of TLR adjuvants could enhance the immunological efficacy of the hybrid nanoparticle-based nicotine vaccine, making it a promising next-generation immunotherapeutic candidate for treating nicotine addiction.

Synthesis and Evaluation of Doxorubicin-Loaded Gold Nanoparticles for Tumor-Targeted Drug Delivery.

Doxorubicin is an effective and widely used cancer chemotherapeutic agent, but its application is greatly compromised by its cumulative dose-dependent side effect of cardiotoxicity. A gold nanoparticle-based drug delivery system has been designed to overcome this limitation. Five novel thiolated doxorubicin analogs were synthesized and their biological activities evaluated. Two of these analogs and PEG stabilizing ligands were then conjugated to gold nanoparticles, and the resulting Au-Dox constructs were evaluated. The results show that release of native drug can be achieved by the action of reducing agents such as glutathione or under acidic conditions, but reductive drug release gave the cleanest drug release. Gold nanoparticles (Au-Dox) were prepared with different loadings of PEG and doxorubicin, and one formulation was evaluated for mammalian stability and toxicity. Plasma levels of doxorubicin in mice treated with Au-Dox were significantly lower than in mice treated with the same amount of doxorubicin, indicating that the construct is stable under physiological conditions. Treatment of mice with Au-Dox gave no histopathologically observable differences from mice treated with saline, while mice treated with an equivalent dose of doxorubicin showed significant histopathologically observable lesions.

Rationalization of a nanoparticle-based nicotine nanovaccine as an effective next-generation nicotine vaccine: A focus on hapten localization.

A lipid-polymeric hybrid nanoparticle-based next-generation nicotine nanovaccine was rationalized in this study to combat nicotine addiction. A series of nanovaccines, which had nicotine-haptens localized on carrier protein (LPKN), nanoparticle surface (LPNK), or both (LPNKN), were designed to study the impact of hapten localization on their immunological efficacy. All three nanovaccines were efficiently taken up and processed by dendritic cells. LPNKN induced a significantly higher immunogenicity against nicotine and a significantly lower anti-carrier protein antibody level compared to LPKN and LPNK. Meanwhile, it was found that the anti-nicotine antibodies elicited by LPKN and LPNKN bind nicotine stronger than those elicited by LPKN, and LPNK and LPNKN resulted in a more balanced Th1-Th2 immunity than LPKN. Moreover, LPNKN exhibited the best ability to block nicotine from entering the brain of mice. Collectively, the results demonstrated that the immunological efficacy of the hybrid nanoparticle-based nicotine vaccine could be enhanced by modulating hapten localization, providing a promising strategy to combatting nicotine addiction.

Investigating acetaminophen hepatotoxicity in multi-cellular organotypic liver models.

In vivo studies clearly demonstrate the participation and subsequent death of non-parenchymal liver cells (NPCs) with corresponding hepatocyte effects. This results in a critical need to investigate how major liver cell types function cohesively during hepatotoxicity. However, virtually no studies replicate these phenomena in vitro. We report the design of multi-cellular three-dimensional (3D) organotypic liver models of primary rat hepatocytes, liver sinusoidal endothelial cells (LSECs) and Kupffer cells (KCs). LSECs and KCs were separated from hepatocytes by a detachable membrane that emulates the physical and chemical properties of the Space of Disse. Acetaminophen (APAP)-induced changes to cellular function and phenotype were investigated. LSECs exhibited approximately 40% cell death at 20mM APAP. KCs exhibited decreased interleukin-10 and increased tumor necrosis factor-alpha and interferon-gamma secretion. The secretion of these proteins altered hepatocyte function and signaling. Both LSECs and KCs maintained phenotypic markers. At 20mM APAP, the 3D models exhibited aspartate aminotransferase to alanine aminotransferase ratios from 2.1-2.5 and 45% glutathione depletion, corresponding to what is seen in vivo. At 10 and 20mM APAP, the 3D models exhibited cell death, primarily through necrosis. Therefore, the 3D cultures described in this report demonstrate significant potential as realistic models for hepatotoxicity studies.

The next-generation nicotine vaccine: a novel and potent hybrid nanoparticle-based nicotine vaccine.

Owing to the urgent need for more effective treatment against nicotine addiction, a hybrid nanoparticle-based nicotine vaccine (NanoNiccine) was developed in this study. NanoNiccine was composed of a poly(lactide-co-glycolide) acid (PLGA) core, keyhole limpet hemocyanin (KLH) as an adjuvant protein enclosed within the PLGA core, a lipid layer, and nicotine haptens conjugated to the outer surface of the lipid layer. In contrast to the traditional nicotine vaccine, NanoNiccine is not a nicotine-protein conjugate vaccine. Instead, the nicotine hapten and protein are separately located in the nanostructure to minimize antibody production towards KLH. The cellular uptake study demonstrated that NanoNiccine was ideal for internalization and processing by dendritic cells (DCs). Mice immunized with NanoNiccine produced much lower IgG level against KLH as compared to that immunized with the traditional nicotine-KLH (Nic-KLH) vaccine. In addition, NanoNiccine achieved up to a 400% higher titer of anti-nicotine IgG than the positive control, Nic-KLH. Additionally, the Th1/Th2 index of NanoNiccine suggested that the immune response induced by NanoNiccine was antibody response dominant. Furthermore, NanoNiccine was found to be safe in mice.

Exploratory Studies With BT-11: A Proposed Orally Active Therapeutic for Crohn's Disease.

Lanthionine synthetase cyclase-like receptor 2 (LANCL2) is a novel therapeutic target for Crohn's disease (CD). BT-11 is a small molecule that binds LANCL2, is orally active, and has demonstrated therapeutic efficacy in 3 validated mouse models of colitis at doses as low as 8 mg/kg/d. Exploratory experiments evaluated BT-11 in male Harlan Sprague Dawley rats with a single oral dose of 500 mg/kg and 80 mg/kg/d for 14 days (n = 10 rats dosed/group). Treated and control rats were observed for behavioral detriments, and blood and tissues were collected for clinical pathology and histopathological examination. A functional observational battery demonstrated no differences between treated and control groups over multiple times of observation for quantal, categorical, and continuous end points, including posture, in cage activity, approach, response to touch, weight, grip strength, body temperature, and time on a rotarod. Histopathological examination of the brain, kidney, liver, adrenal gland, testes, stomach, small and large intestines, duodenum, pancreas, heart, lungs, spleen, thymus, and rib found no significant differences between the groups. Plasma enzymes associated with liver function were transiently elevated 2 to 4 days after the 500 mg/kg single dose but returned to normal values by 8 days and were not observed at any time in rats given 80 mg/kg/d for 14 days. One hour after oral administration of a single dose of 80 mg/kg, BT-11 had a maximal concentration of 21 ng/mL; the half-life was 3 hours. These experimental results demonstrated that BT-11 is well tolerated in rats, and, with further testing, may hold promise as an orally active therapeutic for CD.

Negatively Charged Carbon Nanohorn Supported Cationic Liposome Nanoparticles: A Novel Delivery Vehicle for Anti-Nicotine Vaccine.

Tobacco addiction is the second-leading cause of death in the world. Due to the nature of nicotine (a small molecule), finding ways to combat nicotine's deleterious effects has been a constant challenge to the society and the medical field. In the present work, a novel anti-nicotine vaccine based on nanohorn supported liposome nanoparticles (NsL NPs) was developed. The nano-vaccine was constructed by using negatively charged carbon nanohorns as a scaffold for the assembly of cationic liposomes, which allow the conjugation of hapten conjugated carrier proteins. The assembled bio-nanoparticles are stable. Mice were immunized subcutaneously with the nano-vaccine, which induced high titer and high affinity of nicotine specific antibodies in mice. Furthermore, no evidence of clinical signs or systemic toxicity followed multiple administrations of NsL-based anti-nicotine vaccine. These results suggest that NsL-based anti-nicotine vaccine is a promising candidate in treating nicotine dependence and could have potential to significantly contribute to smoking cessation.

Engineering the lipid layer of lipid-PLGA hybrid nanoparticles for enhanced in vitro cellular uptake and improved stability.

Lipid-polymer hybrid nanoparticles (NPs), consisting of a polymeric core and a lipid shell, have been intensively examined as delivery systems for cancer drugs, imaging agents, and vaccines. For applications in vaccine particularly, the hybrid NPs need to be able to protect the enclosed antigens during circulation, easily be up-taken by dendritic cells, and possess good stability for prolonged storage. However, the influence of lipid composition on the performance of hybrid NPs has not been well studied. In this study, we demonstrate that higher concentrations of cholesterol in the lipid layer enable slower and more controlled antigen release from lipid-poly(lactide-co-glycolide) acid (lipid-PLGA) NPs in human serum and phosphate buffered saline (PBS). Higher concentrations of cholesterol also promoted in vitro cellular uptake of hybrid NPs, improved the stability of the lipid layer, and protected the integrity of the hybrid structure during long-term storage. However, stabilized hybrid structures of high cholesterol content tended to fuse with each other during storage, resulting in significant size increase and lowered cellular uptake. Additional experiments demonstrated that PEGylation of NPs could effectively minimize fusion-caused size increase after long term storage, leading to improved cellular uptake, although excessive PEGylation will not be beneficial and led to reduced improvement. STATEMENT OF SIGNIFICANCE: This paper reports the engineering of the lipid layer that encloses a polymeric nanoparticle, which can be used as a carrier for drug and vaccine molecules for targeted delivery. We demonstrated that the concentration of cholesterol is critical for the stability and uptake of the hybrid nanoparticles by dendritic cells, a targeted cell for the delivery of immune effector molecules. However, we found that hybrid nanoparticles with high cholesterol concentration tend to fuse during storage resulting in larger particles with decreased cellular uptake. This problem is subsequently solved by PEGylating the hybrid nanoparticles. With increased research and clinical applications of lipid-polymer hybrid nanoparticles in drug and vaccine delivery, this work will significantly impact the design of the hybrid nanoparticles for minimized molecule release during circulation and increased bioavailability of the target molecules.

Cefazolin concentration in surgically created wounds treated with negative pressure wound therapy compared to surgically created wounds treated with nonadherent wound dressings.

OBJECTIVE: To compare cefazolin concentrations in biopsied tissue samples collected from surgically created wounds treated with negative pressure wound therapy to those collected from surgically created wounds treated with nonadherent dressings. STUDY DESIGN: Prospective, controlled, experimental study. ANIMALS: Adult female spayed Beagles (n = 12). METHODS: Full thickness cutaneous wounds were created on each antebrachium (n = 24). Immediately after surgery, cefazolin (22 mg/kg intravenously [IV]) was administered to each dog and continued every 8 hours during the study. The right wound was randomly assigned to group I or group II whereas the wound on the contralateral antebrachium was assigned to the other group. Group I wounds were treated with negative pressure wound therapy (NPWT) and group II wounds were treated with nonadherent dressings for 3 days. Dressings were changed and tissue biopsies obtained from wound beds at 24 hours intervals for both groups. Cefazolin wound tissue and plasma concentrations were measured by liquid chromatography mass spectrometry (LC-MS/MS). Blood samples for measuring plasma cefazolin concentrations were collected before biopsy sampling. At the time of surgery and at each subsequent bandage change, wound beds were swabbed and submitted for aerobic and anaerobic culture. RESULTS: After initiating cefazolin treatment, wound tissue antibiotic concentrations between treatment groups were not significantly different at any sampling time. Similarly, after initiating cefazolin treatment, plasma cefazolin concentrations were not significantly different at any sampling time for individual dogs. CONCLUSIONS: Using a canine experimental model, NPWT treatment of surgically created wounds does not statistically impact cefazolin tissue concentrations when compared with conventional nonadherent bandage therapy.

In vitro performance of lipid-PLGA hybrid nanoparticles as an antigen delivery system: lipid composition matters.

Due to the many beneficial properties combined from both poly(lactic-co-glycolic acid) (PLGA) nanoparticles (NPs) and liposomes, lipid-PLGA hybrid NPs have been intensively studied as cancer drug delivery systems, bio-imaging agent carriers, as well as antigen delivery vehicles. However, the impact of lipid composition on the performance of lipid-PLGA hybrid NPs as a delivery system has not been well investigated. In this study, the influence of lipid composition on the stability of the hybrid NPs and in vitro antigen release from NPs under different conditions was examined. The uptake of hybrid NPs with various surface charges by dendritic cells (DCs) was carefully studied. The results showed that PLGA NPs enveloped by a lipid shell with more positive surface charges could improve the stability of the hybrid NPs, enable better controlled release of antigens encapsulated in PLGA NPs, as well as enhance uptake of NPs by DC.

Organophosphorus compound effects on neurotrophin receptors and intracellular signaling.

Neurite outgrowth of SH-SY5Y neuroblastoma cells following the addition of spinal cord extracts from chickens exposed to a neuropathic organophosphorus (OP) compound suggests the presence of a growth factor during OP neuropathy. However, exposure of SH-SY5Y cells directly to neuropathic OP compounds results in apoptosis and/or decreased neurite outgrowth. These cellular effects may follow OP-induced interference with neurotrophin-receptor binding and/or intracellular signaling resulting from receptor binding. We hypothesized that sub-lethal concentrations of a neuropathic OP compound interferes with neurotrophin-receptor binding as well as specific intracellular signaling pathways in neuroblastoma cells which would not occur with a non-neuropathic OP compound. SH-SY5Y cells were exposed to a neuropathic OP compound (PSP; 0.01, 0.1, 1.0µM), a neuropathic OP compound with nerve growth factor (1.0µM PSP+1ng/ml NGF), a non-neuropathic OP compound (paraoxon; 100µM), and medium only for 4, 8, 24, and 48h. Western blots indicate that cells exposed to a low dose of PSP or the high dose of PSP+NGF contained the phosphorylated form of a common neurotrophin receptor (pp75) that was four times greater than that of the phosphorylated form of the high-affinity NGF receptor (pTrkA) suggesting that p75 activation may contribute to early cell death after exposure to OP compounds. Furthermore, events in signaling pathways after exposure to PSP differed from those after exposure to paraoxon, with activation of the MEK1/2 protein increasing significantly only after exposure to paraoxon. Both types of OP compounds, however, caused significant activation of Akt in the PI-3K cell-survival pathway. These results suggest that exposure to a non-neuropathic OP compound causes increased activity of the MAPK pathway whereas exposure to neuropathic OP compounds prevented upregulation of the pathway. Since this pathway is integral to neurite outgrowth and cell survival, this study has revealed molecular mechanisms implicated in neuronal response after exposure to neuropathic OP compounds.

Comparative in vitro study of the inhibition of human and hen esterases by methamidophos enantiomers.

The current Organisation for Economic Co-operation and Development (OECD) guidelines for evaluating organophosphorus-induced delayed neuropathy (OPIDN) require the observation of dosed animals over several days and the sacrifice of 48 hens. Adhering to these protocols in tests with enantiomers is difficult because large quantities of the compound are needed and many animals must be utilized. Thus, developing an in vitro screening protocol to evaluate chiral organophosphorus pesticides (OPs) that can induce delayed neuropathy is important. This work aimed to evaluate, in blood and brain samples from hens, human blood, and human cell culture samples, the potential of the enantiomeric forms of methamidophos to induce acetylcholinesterase (AChE) inhibition and/or delayed neurotoxicity. Calpain activation was also evaluated in the hen brain and SH-SY5Y human neuroblastoma cells. The ratio between the inhibition of neuropathy target esterase (NTE) and AChE activities by the methamidophos enantiomers was evaluated as a possible indicator of the enantiomers' abilities to induce OPIDN. The (-)-methamidophos exhibited an IC(50) value approximately 6 times greater than that of the (+)-methamidophos for the lymphocyte NTE (LNTE) of hens, and (+)-methamidophos exhibited an IC(50) value approximately 7 times larger than that of the (-)-methamidophos for the hen brain AChE. The IC(50) values were 7 times higher for the human erythrocyte AChE and 5 times higher for AChE in the SH-SY5Y human neuroblastoma cells. Considering the esterases inhibition and calpain results, (+)-methamidophos would be expected to have a greater ability to induce OPIDN than the (-)-methamidophos in humans and in hens.