Impacts of Self-Administered 3,4-Methylenedioxypyrovalerone (MDPV) Alone, and in Combination with Caffeine, on Recognition Memory and Striatal Monoamine Neurochemistry in Male Sprague Dawley Rats: Comparisons with Methamphetamine and Cocaine.

Recent data suggest that 3,4-methylenedioxypyrovalerone (MDPV) has neurotoxic effects; however, the cognitive and neurochemical consequences of MDPV self-administration remain largely unexplored. Furthermore, despite the fact that drug preparations that contain MDPV often also contain caffeine, little is known regarding the toxic effects produced by the co-use of these two stimulants. The current study investigated the degree to which self-administered MDPV or a mixture of MDPV+caffeine can produce deficits in recognition memory and alter neurochemistry relative to prototypical stimulants. Male Sprague Dawley rats were provided 90 min or 12 h access to MDPV, MDPV+caffeine, methamphetamine, cocaine, or saline for 6 weeks. Novel object recognition (NOR) memory was evaluated prior to any drug self-administration history and 3 weeks after the final self-administration session. Rats that had 12 h access to methamphetamine and those that had 90 min or 12 h access to MDPV+caffeine exhibited significant deficits in NOR, whereas no significant deficits were observed in rats that self-administered cocaine or MDPV. Striatal monoamine levels were not systematically affected. These data demonstrate synergism between MDPV and caffeine with regard to producing recognition memory deficits, highlighting the importance of recapitulating the manner in which drugs are used (e.g., in mixtures containing multiple stimulants, binge-like patterns of intake).

Impacts of Self-Administered 3,4-Methylenedioxypyrovalerone (MDPV) Alone, and in Combination with Caffeine, on Recognition Memory and Striatal Monoamine Neurochemistry in Male Sprague-Dawley Rats: Comparisons with Methamphetamine and Cocaine.

Recent data suggest that 3,4-methylenedioxypyrovalerone (MDPV) has neurotoxic effects; however, the cognitive and neurochemical consequences of MDPV self-administration remain largely unexplored. Furthermore, despite the fact that drug preparations that contain MDPV often also contain caffeine, little is known regarding the toxic effects produced by the co-use of these two stimulants. The current study investigated the degree to which self-administered MDPV, or a mixture of MDPV+caffeine can produce deficits in recognition memory and alter neurochemistry relative to prototypical stimulants. Male Sprague-Dawley rats were provided 90-min or 12-h access to MDPV, MDPV+caffeine, methamphetamine, cocaine, or saline for 6 weeks. Novel object recognition (NOR) memory was evaluated prior to any drug self-administration history and 3 weeks after the final self-administration session. Rats that had 12-h access to methamphetamine and those that had 90-min or 12-h access to MDPV+caffeine exhibited significant deficits in NOR, whereas no significant deficits were observed in rats that self-administered cocaine or MDPV. Striatal mono-amine levels were not systematically affected. These data demonstrate synergism between MDPV and caffeine with regard to producing recognition memory deficits and lethality, highlighting the importance of recapitulating the manner in which drugs are used (e.g., in mixtures containing multiple stimulants, binge-like patterns of intake).

The intracellular growth of the vacuolar pathogen Legionella pneumophila is dependent on the acyl chain composition of host membranes.

Legionella pneumophila is an accidental human bacterial pathogen that infects and replicates within alveolar macrophages causing a severe atypical pneumonia known as Legionnaires' disease. As a prototypical vacuolar pathogen L. pneumophila establishes a unique endoplasmic reticulum (ER)-derived organelle within which bacterial replication takes place. Bacteria-derived proteins are deposited in the host cytosol and in the lumen of the pathogen-occupied vacuole via a type IVb (T4bSS) and a type II (T2SS) secretion system respectively. These secretion system effector proteins manipulate multiple host functions to facilitate intracellular survival of the bacteria. Subversion of host membrane glycerophospholipids (GPLs) by the internalized bacteria via distinct mechanisms feature prominently in trafficking and biogenesis of the Legionella -containing vacuole (LCV). Conventional GPLs composed of a glycerol backbone linked to a polar headgroup and esterified with two fatty acids constitute the bulk of membrane lipids in eukaryotic cells. The acyl chain composition of GPLs dictates phase separation of the lipid bilayer and therefore determines the physiochemical properties of biological membranes - such as membrane disorder, fluidity and permeability. In mammalian cells, fatty acids esterified in membrane GPLs are sourced endogenously from de novo synthesis or via internalization from the exogenous pool of lipids present in serum and other interstitial fluids. Here, we exploited the preferential utilization of exogenous fatty acids for GPL synthesis by macrophages to reprogram the acyl chain composition of host membranes and investigated its impact on LCV homeostasis and L. pneumophila intracellular replication. Using saturated fatty acids as well as cis - and trans - isomers of monounsaturated fatty acids we discovered that under conditions promoting lipid packing and membrane rigidification L. pneumophila intracellular replication was significantly reduced. Palmitoleic acid - a C16:1 monounsaturated fatty acid - that promotes membrane disorder when enriched in GPLs significantly increased bacterial replication within human and murine macrophages but not in axenic growth assays. Lipidome analysis of infected macrophages showed that treatment with exogenous palmitoleic acid resulted in membrane acyl chain reprogramming in a manner that promotes membrane disorder and live-cell imaging revealed that the consequences of increasing membrane disorder impinge on several LCV homeostasis parameters. Collectively, we provide experimental evidence that L. pneumophila replication within its intracellular niche is a function of the lipid bilayer disorder and hydrophobic thickness.

Cellular cholesterol licenses Legionella pneumophila intracellular replication in macrophages.

Host membranes are inherently critical for niche homeostasis of vacuolar pathogens. Thus, intracellular bacteria frequently encode the capacity to regulate host lipogenesis as well as to modulate the lipid composition of host membranes. One membrane component that is often subverted by vacuolar bacteria is cholesterol - an abundant lipid that mammalian cells produce de novo at the endoplasmic reticulum (ER) or acquire exogenously from serum-derived lipoprotein carriers. Legionella pneumophila is an accidental human bacterial pathogen that infects and replicates within alveolar macrophages causing a severe atypical pneumonia known as Legionnaires' disease. From within a unique ER-derived vacuole L. pneumophila promotes host lipogenesis and experimental evidence indicates that cholesterol production might be one facet of this response. Here we investigated the link between cellular cholesterol and L. pneumophila intracellular replication and discovered that disruption of cholesterol biosynthesis or cholesterol trafficking lowered bacterial replication in infected cells. These growth defects were rescued by addition of exogenous cholesterol. Conversely, bacterial growth within cholesterol-leaden macrophages was enhanced. Importantly, the growth benefit of cholesterol was observed strictly in cellular infections and L. pneumophila growth kinetics in axenic cultures did not change in the presence of cholesterol. Microscopy analyses indicate that cholesterol regulates a step in L. pneumophila intracellular lifecycle that occurs after bacteria begin to replicate within an established intracellular niche. Collectively, we provide experimental evidence that cellular cholesterol promotes L. pneumophila replication within a membrane bound organelle in infected macrophages.

Fomepizole dosing during continuous renal replacement therapy - an observational study.

BACKGROUND: Fomepizole is the preferred antidote for treatment of methanol and ethylene glycol poisoning, acting by inhibiting the formation of the toxic metabolites. Although very effective, the price is high and the availability is limited. Its availability is further challenged in situations with mass poisonings. Therefore, a 50% reduced maintenance dose for fomepizole during continuous renal replacement therapy (CRRT) was suggested in 2016, based on pharmacokinetic data only. Our aim was to study whether this new dosing for fomepizole during CRRT gave plasma concentrations above the required 10 µmol/L. Secondly, we wanted to study the elimination kinetics of fomepizole during CRRT, which has never been studied before. METHODS: Prospective observational study of adult patients treated with fomepizole and CRRT. We collected samples from arterial line (pre-filter) = plasma concentration, post-filter and dialysate for fomepizole measurements. Fomepizole was measured using high-pressure liquid chromatography with a reverse phase column. RESULTS: Ten patients were included in the study. Seven were treated with continuous veno-venous hemodialysis (CVVHD) and three with continuous veno-venous hemodiafiltration (CVVHDF). Ninety-eight percent of the plasma samples were above the minimum plasma concentration of 10 µmol/L. Fomepizole was removed during CRRT with a median saturation/sieving coefficient of 0.85 and dialysis clearance of 28 mL/min. CONCLUSION: Fomepizole was eliminated during CCRT. The new dosing recommendations for fomepizole and CRRT appeared safe, by maintaining the plasma concentration above the minimum value of 10 µmol/L. Based on these data, the fomepizole maintenance dose during CRRT could be reduced to half as compared to intermittent hemodialysis.

Diethylene glycol produces nephrotoxic and neurotoxic effects in female rats.

CONTEXT: Diethylene glycol (DEG) is an organic compound found in household products but also as a counterfeit solvent in medicines. DEG poisonings are characterized by acute kidney injury (AKI) and by neurological sequelae such as decreased reflexes or face and limb weakness. Previous studies in male rats have demonstrated that neurotoxic effects develop only with the establishment of AKI, but the dose sensitivity of females to DEG toxicity is unknown. OBJECTIVES: Assessing whether subacute administration of DEG in female rats would delineate any sex-differences in neuropathy or in kidney injury. METHODS: Female Wistar-Han rats were orally administered doses of 4 - 6 g/kg DEG every 12 h and monitored for 7 days. Urine was collected every 12 h and endpoint blood and cerebrospinal fluid (CSF) were collected for renal plasma parameters and total protein estimation, respectively. Motor function tests were conducted before and after treatment. Kidney and brain tissue were analyzed for metabolite content. RESULTS: Of 12 animals treated with DEG, 3 developed AKI as confirmed by increased BUN and creatinine concentrations. Renal and brain DGA contents were increased in animals that developed AKI compared to animals without AKI. Total CSF protein content in animals with AKI was markedly elevated compared to control and to treated animals without AKI. Decreases in forelimb grip strength and in locomotor and rearing activity were observed in animals with AKI compared to control and to animals without AKI. DISCUSSION: Repeated dosing with DEG in a female model produced nephrotoxic effects at a dose similar to that in males. The decrease in motor function and increase in CSF protein were only present in females that developed AKI. However, kidney and neurologic effects were assessed only at the end of the treatments, thus limiting determination of which effect occurs first. Limb function and coordination were measured globally and more sensitive tests such as nerve conduction studies might offer a detailed neurotoxicity assessment of the effects of DEG. CONCLUSIONS: These studies show that DEG toxicity does not appear to be sex-specific and that, in males and females, neurological symptoms are present only when DGA accumulation and kidney injury also occur.

Neurotoxic effects of nephrotoxic compound diethylene glycol.

CONTEXT: Diethylene glycol (DEG) is an organic compound found in household products but also as an adulterant in medicines by acting as a counterfeit solvent. DEG poisonings have been characterized predominately by acute kidney injury (AKI), but also by delayed neurological sequelae such as decreased reflexes or face and limb weakness. OBJECTIVES: Characterizing the neurological symptoms of DEG poisoning in a subacute animal model would create a clearer picture of overall toxicity and possibly make mechanistic connections between kidney injury and neuropathy. METHODS: Male Wistar-Han rats were orally administered doses of 4 - 6 g/kg DEG every 12 or 24 h and monitored for 7 days. Urine was collected every 12 h and endpoint blood and cerebrospinal fluid (CSF) were collected for a renal plasma panel and total protein estimation, respectively. Motor function tests were conducted before and after treatment. Kidney and brain tissue was harvested for metabolic analysis. RESULTS: Of the 43 animals treated with DEG, 11 developed AKI as confirmed by increased BUN and creatinine levels. Renal and brain DGA accumulation was markedly increased in animals that developed AKI compared to animals without AKI. The total protein content in CSF in animals with kidney injury was markedly elevated compared to control and to treated animals without AKI. Significant decreases in forelimb grip strength and decreases in locomotor and rearing activity were observed in animals with AKI compared to control and to animals without AKI. DISCUSSION: Repeated dosing with DEG in an animal model produced nephrotoxic effects like those in studies with acute DEG administration. The decrease in motor function and increase in CSF protein were only present in animals that developed AKI. CONCLUSIONS: These studies show development of neurotoxicity in this DEG animal model and suggest that neurological symptoms are observed only when DGA accumulation and kidney injury also occur.

Transcervical sonography and human papillomavirus 16 E6 antibodies are sensitive for the detection of oropharyngeal cancer.

BACKGROUND: Human papillomavirus 16 (HPV-16) E6 seropositivity is a promising early marker of human papillomavirus-driven oropharyngeal cancer (HPV-OPC), yet more sensitive imaging modalities are needed before screening is considered. The objective of this study was to determine the sensitivity of transcervical sonography (TCS) for detecting clinically apparent HPV-OPC in comparison with computed tomography (CT) and positron emission tomography (PET)/CT. METHODS: Fifty-one patients with known or suspected HPV-OPC without prior treatment underwent oropharyngeal TCS and blood collection (for HPV multiplex serology testing). Eight standard sonographic images were collected; primary-site tumors were measured in 3 dimensions if identified. Each patient underwent a full diagnostic workup as part of standard clinical care. The pathologic details, HPV status, final staging, and imaging findings were abstracted from the medical record. The sensitivity of each imaging modality was compared with the final clinical diagnosis (the gold standard). RESULTS: Twenty-four base of tongue cancers (47%), 22 tonsillar cancers (43%), and 2 unknown primary cancers (4%) were diagnosed; 3 patients (6%) had no tumors. All p16-tested patients were positive (n = 47). Primary-site tumors were correctly identified in 90.2% (95% confidence interval [CI], 78.6%-96.7%) with TCS, in 69.4% (95% CI, 54.6%-81.7%) with CT, and in 83.3% (95% CI, 68.6%-93.0%) with PET/CT. TCS identified tumors in 10 of 14 cases missed by CT and recognized the absence of tumors in 3 cases for which CT or PET/CT was falsely positive. The smallest sonographically identified primary-site tumor was 0.5 cm in its greatest dimension; the average size was 2.3 cm. Among p16-positive patients, 76.1% (95% CI, 61.2%-87.4%) were seropositive for HPV-16 E6. CONCLUSIONS: TCS and HPV-16 E6 antibodies are sensitive for the diagnosis of HPV-OPC.

Dosage sensitivity intolerance of VIPR2 microduplication is disease causative to manifest schizophrenia-like phenotypes in a novel BAC transgenic mouse model.

Recent genome-wide association studies (GWAS) have identified copy number variations (CNVs) at chromosomal locus 7q36.3 that significantly contribute to the risk of schizophrenia, with all of the microduplications occurring within a single gene: vasoactive intestinal peptide receptor 2 (VIPR2). To confirm disease causality and translate such a genetic vulnerability into mechanistic and pathophysiological insights, we have developed a series of conditional VIPR2 bacterial artificial chromosome (BAC) transgenic mouse models of VIPR2 CNV. VIPR2 CNV mouse model recapitulates gene expression and signaling deficits seen in human CNV carriers. VIPR2 microduplication in mice elicits prominent dorsal striatal dopamine dysfunction, cognitive, sensorimotor gating, and social behavioral deficits preceded by an increase of striatal cAMP/PKA signaling and the disrupted early postnatal striatal development. Genetic removal of VIPR2 transgene expression via crossing with Drd1a-Cre BAC transgenic mice rescued the dopamine D2 receptor abnormality and multiple behavioral deficits, implicating a pathogenic role of VIPR2 overexpression in dopaminoceptive neurons. Thus, our results provide further evidence to support the GWAS studies that the dosage sensitivity intolerance of VIPR2 is disease causative to manifest schizophrenia-like dopamine, cognitive, and social behavioral deficits in mice. The conditional BAC transgenesis offers a novel strategy to model CNVs with a gain-of -copies and facilitate the genetic dissection of when/where/how the genetic vulnerabilities affect development, structure, and function of neural circuits. Our findings have important implications for therapeutic development, and the etiology-relevant mouse model provides a useful preclinical platform for drug discovery.

Curcumin gum formulation for prevention of oral cavity head and neck squamous cell carcinoma.

OBJECTIVES/HYPOTHESIS: Head and neck squamous cell carcinoma represents the sixth most common cancer. As a result of field cancerization, second primaries and recurrences are high. Hence, research has focused on chemoprevention. Curcumin, a polyphenol compound with anticarcinogenic properties, is one such promising nutraceutical. As poor bioavailability limits curcumin's use, a novel gum formulation was tested allowing for direct mucosal absorption into the bloodstream. This preliminary study validates curcumin gum efficacy by assessing release and transmucosal absorption, along with measuring its effects on serum cytokine levels. STUDY DESIGN: Clinical trial. METHODS: Protocols consisting of initial chew (chewing gum for 30 minutes) and revised chew (alternating chewing and parking gum against buccal mucosa for 30 minutes) were tested in healthy volunteers. High-performance liquid chromatography measured remnant curcumin in chewed gum, serum, and saliva. Serum levels were assayed for 15 proinflammatory cytokines via multiplex analysis. RESULTS: Revised chew samples demonstrated significantly higher curcumin release and absorption (P = .0078). Curcumin serum levels were significantly higher at 4 hours in samples > 2.0 g of curcumin release (P = .01). As saliva levels decreased, a concurrent increase in serum levels was observed, with no significance in the inverse relationship (P = .1423). When evaluating differences between gender, race, and age, the Asian population showed significantly lower curcumin release and serum levels (P = .009). CXCL1 (GRO-α) and TNF-α were significantly decreased in serum after chewing the gum (P = .036, P < .001, respectively). CONCLUSIONS: Enhanced mucosal contact appears critical in improving curcumin release and absorption. CXCL1 and TNF-α both represent potential biomarkers for the future study of curcumin chemoprevention. LEVEL OF EVIDENCE: 2b Laryngoscope, 129:1597-1603, 2019.

Dissociation of Striatal Dopamine and Tyrosine Hydroxylase Expression from Aging-Related Motor Decline: Evidence from Calorie Restriction Intervention.

The escalating increase in retirees living beyond their eighth decade brings increased prevalence of aging-related impairments, including locomotor impairment (Parkinsonism) that may affect ~50% of those reaching age 80, but has no confirmed neurobiological mechanism. Lifestyle strategies that attenuate motor decline, and its allied mechanisms, must be identified. Aging studies report little to moderate loss of striatal dopamine (DA) or tyrosine hydroxylase (TH) in nigrostriatal terminals, in contrast to ~70%-80% loss associated with bradykinesia onset in Parkinson's disease. These studies evaluated the effect of ~6 months 30% calorie restriction (CR) on nigrostriatal DA regulation and aging-related locomotor decline initiated at 12 months of age in Brown-Norway Fischer F1 hybrid rats. The aging-related decline in locomotor activity was prevented by CR. However, striatal DA or TH expression was decreased in the CR group, but increased in substantia nigra versus the ad libitum group or 12-month-old cohort. In a 4- to 6-month-old cohort, pharmacological TH inhibition reduced striatal DA ~30%, comparable with decreases reported in aged rats and the CR group, without affecting locomotor activity. The dissociation of moderate striatal DA reduction from locomotor activity seen in both studies suggests that aging-related decreases in striatal DA are dissociated from locomotor decline.

Identification of small molecules that disrupt vacuolar function in the pathogen Candida albicans.

The fungal vacuole is a large acidified organelle that performs a variety of cellular functions. At least a sub-set of these functions are crucial for pathogenic species of fungi, such as Candida albicans, to survive within and invade mammalian tissue as mutants with severe defects in vacuolar biogenesis are avirulent. We therefore sought to identify chemical probes that disrupt the normal function and/or integrity of the fungal vacuole to provide tools for the functional analysis of this organelle as well as potential experimental therapeutics. A convenient indicator of vacuolar integrity based upon the intracellular accumulation of an endogenously produced pigment was adapted to identify Vacuole Disrupting chemical Agents (VDAs). Several chemical libraries were screened and a set of 29 compounds demonstrated to reproducibly cause loss of pigmentation, including 9 azole antifungals, a statin and 3 NSAIDs. Quantitative analysis of vacuolar morphology revealed that (excluding the azoles) a sub-set of 14 VDAs significantly alter vacuolar number, size and/or shape. Many C. albicans mutants with impaired vacuolar function are deficient in the formation of hyphal elements, a process essential for its pathogenicity. Accordingly, all 14 VDAs negatively impact C. albicans hyphal morphogenesis. Fungal selectivity was observed for approximately half of the VDA compounds identified, since they did not alter the morphology of the equivalent mammalian organelle, the lysosome. Collectively, these compounds comprise of a new collection of chemical probes that directly or indirectly perturb normal vacuolar function in C. albicans.

In-vivo evidence of nephrotoxicity and altered hepatic function in rats following administration of diglycolic acid, a metabolite of diethylene glycol.

CONTEXT: Diglycolic acid (DGA) is one of the two primary metabolites of diethylene glycol (DEG). DEG is an industrial solvent that has been implicated in mass poisonings resulting from product misuse in the United States and worldwide, with the hallmark toxicity being acute kidney injury, hepatotoxicity, encephalopathy and peripheral neuropathy. Our laboratory has generated in-vitro evidence suggesting that DGA is the metabolite responsible for the proximal tubule necrosis and decreased kidney function observed following DEG ingestion. Furthermore, we have shown that DGA specifically accumulates in kidney tissues (100× higher than peak blood concentrations) following DEG administration. OBJECTIVE: To examine renal and hepatic accumulation and dysfunction following direct administration of DGA in-vivo. We hypothesize that administration of DGA will result in renal and hepatic DGA accumulation, as well as proximal tubular necrosis and liver injury. MATERIALS AND METHODS: Adult male Wistar rats were divided into three groups dosed with 0, 100 or 300 mg/kg DGA via single oral gavage. Urine was collected every 6-12 h and blood, kidneys and liver were removed upon sacrifice at 48 h post-dosing for analysis. RESULTS: DGA accumulated significantly in both kidney and liver tissue only at 300 mg DGA/kg. DGA concentrations in the kidneys and liver correlated with renal and hepatic injury, respectively. Histopathological and clinical chemistry analysis revealed that DGA-treated animals exhibited moderate liver fatty accumulation and marked renal injury, again only at 300 mg/kg. DISCUSSION: DGA-induced kidney injury demonstrated a steep dose response threshold, where severe damage occurred only in animals given 300 mg/kg DGA, while no toxicity was observed at 100 mg/kg. CONCLUSION: These results provide evidence for in-vivo toxicity following direct administration of DGA, a metabolite of DEG. The steep dose-response threshold for toxicity suggests mechanistically that there is likely a saturable step that results in DGA accumulation in target organs.

Initiation of calorie restriction in middle-aged male rats attenuates aging-related motoric decline and bradykinesia without increased striatal dopamine.

Aging-related bradykinesia affects ∼ 15% of those reaching age 65 and 50% of those reaching their 80s. Given this high risk and lack of pharmacologic therapeutics, noninvasive lifestyle strategies should be identified to diminish its risk and identify the neurobiological targets to reduce aging-related bradykinesia. Early-life, long-term calorie restriction (CR) attenuates aging-related bradykinesia in rodents. Here, we addressed whether CR initiation at middle age could attenuate aging-related bradykinesia and motoric decline measured as rotarod performance. A 30% CR regimen was implemented for 6 months duration in 12-month-old male Brown-Norway Fischer 344 F1 hybrid rats after establishing individual baseline locomotor activities. Locomotor capacity was assessed every 6 weeks thereafter. The ad libitum group exhibited predictably decreased locomotor activity, except movement speed, out to 18 months of age. In contrast, in the CR group, movement number and horizontal activity did not decrease during the 6-month trial, and aging-related decline in rotarod performance was attenuated. The response to CR was influenced by baseline locomotor activity. The lower the locomotor activity level at baseline, the greater the response to CR. Rats in the lower 50th percentile surpassed their baseline level of activity, whereas rats in the top 50th percentile decreased at 6 weeks and then returned to baseline by 12 weeks of CR. We hypothesized that nigrostriatal dopamine tissue content would be greater in the CR group and observed a modest increase only in substantia nigra with no group differences in striatum, nucleus accumbens, or ventral tegmental area. These results indicate that initiation of CR at middle age may reduce aging-related bradykinesia, and, furthermore, subjects with below average locomotor activity may increase baseline activity. Sustaining nigral dopamine neurotransmission may be one component of preserving locomotor capabilities during aging.

Enhanced Systemic Bioavailability of Curcumin Through Transmucosal Administration of a Novel Microgranular Formulation.

BACKGROUND/AIM: Curcumin is a promising nutraceutical for chemoprevention of head and neck squamous cell carcinoma (HNSCC). Capsular formulations of curcumin demonstrate low systemic bioavailability. We aimed to determine if curcumin levels were higher in healthy volunteers and cancer patients with microgranular curcumin that allows for transmucosal absorption and identify a consistent biomarker. PATIENTS AND METHODS: Eight healthy volunteers and 15 HNSCC patients completed the trials. Serum levels of curcumin were measured by HPLC. Biological activity of curcumin was assessed with Multiplex Immunoassay and immunohistochemistry. RESULTS: We achieved higher serum levels of curcumin compared to trials using capsular formulation. In cancer patients a significant decrease in expression of fibroblast growth factor-2 (FGF-2) in post-biopsy samples and decreased serum levels of FGF-2, granulocyte macrophage colony-stimulating factor (GM-CSF) and interleukin-17 (IL-17) (p<0.05) was observed. CONCLUSION: Transmucosal administration of microgranular curcumin leads to enhanced curcumin bioavailability that is associated with significant biological effects.

Diethylene glycol-induced toxicities show marked threshold dose response in rats.

Diethylene glycol (DEG) exposure poses risks to human health because of widespread industrial use and accidental exposures from contaminated products. To enhance the understanding of the mechanistic role of metabolites in DEG toxicity, this study used a dose response paradigm to determine a rat model that would best mimic DEG exposure in humans. Wistar and Fischer-344 (F-344) rats were treated by oral gavage with 0, 2, 5, or 10g/kg DEG and blood, kidney and liver tissues were collected at 48h. Both rat strains treated with 10g/kg DEG had equivalent degrees of metabolic acidosis, renal toxicity (increased BUN and creatinine and cortical necrosis) and liver toxicity (increased serum enzyme levels, centrilobular necrosis and severe glycogen depletion). There was no liver or kidney toxicity at the lower DEG doses (2 and 5g/kg) regardless of strain, demonstrating a steep threshold dose response. Kidney diglycolic acid (DGA), the presumed nephrotoxic metabolite of DEG, was markedly elevated in both rat strains administered 10g/kg DEG, but no DGA was present at 2 or 5g/kg, asserting its necessary role in DEG-induced toxicity. These results indicate that mechanistically in order to produce toxicity, metabolism to and significant target organ accumulation of DGA are required and that both strains would be useful for DEG risk assessments.

Strong HCV NS3/4a, NS4b, NS5a, NS5b-specific cellular immune responses induced in Rhesus macaques by a novel HCV genotype 1a/1b consensus DNA vaccine.

Chronic HCV is a surreptitious disease currently affecting approximately 3% of the world's population that can lead to liver failure and cancer decades following initial infection. However, there are currently no vaccines available for the prevention of chronic HCV. From patients who acutely resolve HCV infection, it is apparent that a strong and broad cytotoxic T lymphocyte (CTL) response is important in HCV clearance. DNA vaccines are naked plasmid DNA molecules that encode pathogen antigens to induce a pathogen-specific immune response. They are inexpensive to produce and have an excellent safety profile in animals and humans. Additionally, DNA vaccines are able to induce strong CTL responses, making them well-suited for an HCV vaccine. We aimed to maximize vaccine recipients' opportunity to induce a broad T cell response with a novel antigenic sequence, multi-antigen vaccine strategy. We have generated DNA plasmids encoding consensus sequences of HCV genotypes 1a and 1b non-structural proteins NS3/4a, NS4b, NS5a, and NS5b. Rhesus macaques were used to study the immunogenicity of these constructs. Four animals were immunized 3 times, 6 weeks apart, at a dose of 1.0mg per antigen construct, as an intramuscular injection followed by in vivo electroporation, which greatly increases DNA uptake by local cells. Immune responses were measured 2 weeks post-immunization regimen (PIR) in immunized rhesus macaques and showed a broad response to multiple HCV nonstructural antigens, with up to 4680 spot-forming units per million peripheral blood mononuclear cells (PBMCs) as measured by Interferon-γ ELISpot. In addition, multiparametric flow cytometry detected HCV-specific CD4+ and CD8+ T cell responses by intracellular cytokine staining and detected HCV-specific CD107a+/GrzB+ CD8+ T cells indicating an antigen specific cytolytic response 2 weeks PIR compared with baseline measurements. At the final study time point, 6 weeks PIR, HCV-specific CD45RA- memory-like T cells remained detectable in peripheral blood. Data presented in this manuscript support the notion that vaccine immunogenicity studies using a macaque model can be used to depict key anti-HCV nonstructural antigenic cellular immune responses and support the development of DNA-based prophylactic HCV vaccines.

An optimized, synthetic DNA vaccine encoding the toxin A and toxin B receptor binding domains of Clostridium difficile induces protective antibody responses in vivo.

Clostridium difficile-associated disease (CDAD) constitutes a large majority of nosocomial diarrhea cases in industrialized nations and is mediated by the effects of two secreted toxins, toxin A (TcdA) and toxin B (TcdB). Patients who develop strong antitoxin antibody responses can clear C. difficile infection and remain disease free. Key toxin-neutralizing epitopes have been found within the carboxy-terminal receptor binding domains (RBDs) of TcdA and TcdB, which has generated interest in developing the RBD as a viable vaccine target. While numerous platforms have been studied, very little data describes the potential of DNA vaccination against CDAD. Therefore, we created highly optimized plasmids encoding the RBDs from TcdA and TcdB in which any putative N-linked glycosylation sites were altered. Mice and nonhuman primates were immunized intramuscularly, followed by in vivo electroporation, and in these animal models, vaccination induced significant levels of both anti-RBD antibodies (blood and stool) and RBD-specific antibody-secreting cells. Further characterization revealed that sera from immunized mice and nonhuman primates could detect RBD protein from transfected cells, as well as neutralize purified toxins in an in vitro cytotoxicity assay. Mice that were immunized with plasmids or given nonhuman-primate sera were protected from a lethal challenge with purified TcdA and/or TcdB. Moreover, immunized mice were significantly protected when challenged with C. difficile spores from homologous (VPI 10463) and heterologous, epidemic (UK1) strains. These data demonstrate the robust immunogenicity and efficacy of a TcdA/B RBD-based DNA vaccine in preclinical models of acute toxin-associated and intragastric, spore-induced colonic disease.

Model combustion-generated particulate matter containing persistent free radicals redox cycle to produce reactive oxygen species.

Particulate matter (PM) is emitted during thermal decomposition of waste. During this process, aromatic compounds chemisorb to the surface of metal-oxide-containing PM, forming a surface-stabilized environmentally persistent free radical (EPFR). We hypothesized that EPFR-containing PM redox cycle to produce ROS and that this redox cycle is maintained in biological environments. To test our hypothesis, we incubated model EPFRs with the fluorescent probe dihydrorhodamine (DHR). Marked increases in DHR fluorescence were observed. Using a more specific assay, hydroxyl radicals ((•)OH) were also detected, and their level was further increased by cotreatment with thiols or ascorbic acid (AA), known components of epithelial lining fluid. Next, we incubated our model EPFR in bronchoalveolar lavage fluid (BALF) or serum. Detection of EPFRs and (•)OH verified that PM generate ROS in biological fluids. Moreover, incubation of pulmonary epithelial cells with EPFR-containing PM increased (•)OH levels compared to those in PM lacking EPFRs. Finally, measurements of oxidant injury in neonatal rats exposed to EPFRs by inhalation suggested that EPFRs induce an oxidant injury within the lung lining fluid and that the lung responds by increasing antioxidant levels. In summary, our EPFR-containing PM redox cycle to produce ROS, and these ROS are maintained in biological fluids and environments. Moreover, these ROS may modulate toxic responses of PM in biological tissues such as the lung.

Kinetics of fomepizole in pregnant rats.

CONTEXT/OBJECTIVES: Fomepizole has been utilized with remarkable success for ethylene glycol and methanol poisonings in children and adults. However, very little information is available regarding the safe and effective use of fomepizole in pregnancy. The goal of this research was to utilize an animal model to investigate the kinetics of fomepizole in pregnancy. MATERIALS/METHODS: Male and pregnant Sprague-Dawley rats, which were obtained at 19 days gestation, were administered fomepizole 15 mg/kg intraperitoneally. The animals were anesthetized and blood, liver, kidney, and fetus samples were collected at 1-24 hours post administration. Tissue samples were homogenized, deproteinized and prepared by solid phase extraction. Fomepizole concentrations from tissue and serum samples were analyzed using high pressure liquid chromatography. RESULTS: Between males and pregnant females, tissue and serum fomepizole levels were similar. Fomepizole concentrations in whole fetal tissue were similar to those in the maternal liver and kidney tissue. Fetal fomepizole concentrations were fivefold higher than maternal serum concentrations. The zero order elimination rate of fomepizole from maternal serum was 7.6 mol/L/h, which was slightly slower than the elimination rate in male rats (12.9 mol/L/h). Elimination of fomepizole from the fetus followed a similar time course to that in the maternal tissues. DISCUSSION/CONCLUSION: Elevated concentrations of fomepizole were detected in the fetus following maternal administration. Although the levels of fomepizole in the fetal tissue would imply significant protection against fetal formation of toxic alcohol metabolites, further research is needed to explore the long-term effects of fomepizole on the fetus.