Method development for the analysis of PBMC-mediated killing of K562 cells by bovine colostrum.

BACKGROUND: Colostrum is the first milk that supplies newborns with immune supporting peptides. Due to its heterogeneous and variable characteristics, standardized assays for assessment of its biological activities are a challenge. The current set of studies were aimed to investigate the immune activity of bovine colostrum blends as well as develop a method to assess variability across different lots. METHODS: Immune activity of a bovine colostrum blend was evaluated by their ability to enhance PBMC (peripheral blood mononuclear cell)-mediated killing of K562 cells. K562 cell killing was assessed by flow cytometry using DAPI. PBMCs derived from multiple healthy donors were initially investigated. Frozen PBMC aliquots from one of the highest responders were used for subsequent studies. Different doses and lots of product were assessed. Incubation time was also explored. RESULTS: Bovine colostrum blend similarly reduced K562 cells number and these effects were often greater than the IL-2 positive control. Despite consistent efficacy at enhancing PBMCs-mediated K562 killing, the degree of the effect was significantly variable across different lots. These biological effects were largely dependent on the solubility of the product. CONCLUSION: Assessment of PBMC-mediated killing of K562 cells by flow cytometry using DAPI can be a reliable method for measuring immune activity of bovine colostrum when the material is well-dissolved into solution and the same biological sample from a single donor is used.

Comparison of antimicrobial activities of natural essential oils and synthetic fragrances against selected environmental pathogens.

Plant essential oils (EOs) are known to inhibit the growth of bacteria and fungi. Whether these antimicrobial effects are comparable to synthetic household products is less clear. Furthermore, limited research is available on the potential additive effect of blending EOs. In this investigation, a new EO blend containing orange, patchouli, peppermint, and clary sage was compared to its individual single oils and to three household products-air freshener, liquid soap, and body spray-for their ability to inhibit the growth of Staphylococcus aureus, Streptococcus pneumoniae, Pseudonomas aeruginosa, and Aspergillus brasiliensis in the disc-diffusion assay. The new EO blend significantly inhibited the growth of the four microorganisms. The zones of inhibition of new EO blend were greater than the air freshener and similar to the liquid soap and body spray, with the exception of Str. pneumoniae in which the body spray provided greater inhibitory zone. The new EO blend and the single oils, with the exception of peppermint, equally inhibited the growth of S. aureus and Str. pneumoniae suggesting no additive effect. P. aeruginosa and A. brasiliensis showed variable susceptibility to all EOs except for no susceptibility to orange and limonene. No difference was found between (-) and (+)-limonene; whereas, (+)-menthol showed greater effect than (-)-menthol. In conclusion, blending the EO of orange, patchouli, peppermint, and clary sage was beneficial in inhibiting the growth of S. aureus, Str. pneumoniae, P. aeruginosa, and A. brasiliensis providing a natural antimicrobial fragrance option over synthetics fragrances used in soaps, body sprays, and air fresheners.

The Persistent Neurotoxic Effects of Methamphetamine on Dopaminergic and Serotonergic Markers in Male and Female Rats.

OBJECTIVE: Methamphetamine (METH) is a highly addictive substance abused world-wide in both males and females. Preclinical studies in male rodents suggest that large-dose exposure to METH can lead to persistent neurotoxic consequences to various brain regions. However, little research has focused on the potential role of sex in the neurotoxic consequences of METH exposure. METHODS: The current study exposed male and female rats to large-doses of METH (4 injections of 7.5 mg/kg) or saline. Hyperthermia was promoted in the females exposed to METH such that similar hyperthermia occurred in males and females. Rats were sacrificed 8 d later and neurochemical changes were assessed in the striatum, hippocampus, frontal cortex and olfactory bulbs. RESULTS: Results revealed that male and female rats exposed to METH had similar decreases in dopamine (DA) transporter (DAT) immunoreactivity in the striatum, serotonin (5-HT) content and 5-HT transporter (SERT) function in the hippocampus, and 5-HT content in the frontal cortex. However, female rats exposed to METH had greater decreases in 5-HT content in the olfactory bulbs compared to sex-matched controls while male rats exposed to METH did not significantly differ from sex-matched controls. CONCLUSIONS: These findings suggest that when similar hyperthermia is maintained between male and female rats exposed to METH, the neurotoxic effects of METH were similar in some, but not all brain regions.

Chronic Nicotine Exposure Attenuates Methamphetamine-Induced Dopaminergic Deficits.

Repeated methamphetamine (METH) administrations cause persistent dopaminergic deficits resembling aspects of Parkinson's disease. Many METH abusers smoke cigarettes and thus self-administer nicotine; yet few studies have investigated the effects of nicotine on METH-induced dopaminergic deficits. This interaction is of interest because preclinical studies demonstrate that nicotine can be neuroprotective, perhaps owing to effects involving α4ß2 and α6ß2 nicotinic acetylcholine receptors (nAChRs). This study revealed that oral nicotine exposure beginning in adolescence [postnatal day (PND) 40] through adulthood [PND 96] attenuated METH-induced striatal dopaminergic deficits when METH was administered at PND 89. This protection did not appear to be due to nicotine-induced alterations in METH pharmacokinetics. Short-term (i.e., 21-day) high-dose nicotine exposure also protected when administered from PND 40 to PND 61 (with METH at PND 54), but this protective effect did not persist. Short-term (i.e., 21-day) high-dose nicotine exposure did not protect when administered postadolescence (i.e., beginning at PND 61, with METH at PND 75). However, protection was engendered if the duration of nicotine exposure was extended to 39 days (with METH at PND 93). Autoradiographic analysis revealed that nicotine increased striatal α4ß2 expression, as assessed using [(125)I]epibatidine. Both METH and nicotine decreased striatal α6ß2 expression, as assessed using [(125)I]α-conotoxin MII. These findings indicate that nicotine protects against METH-induced striatal dopaminergic deficits, perhaps by affecting α4ß2 and/or α6ß2 expression, and that both age of onset and duration of nicotine exposure affect this protection.

Nicotine Administration Attenuates Methamphetamine-Induced Novel Object Recognition Deficits.

BACKGROUND: Previous studies have demonstrated that methamphetamine abuse leads to memory deficits and these are associated with relapse. Furthermore, extensive evidence indicates that nicotine prevents and/or improves memory deficits in different models of cognitive dysfunction and these nicotinic effects might be mediated by hippocampal or cortical nicotinic acetylcholine receptors. The present study investigated whether nicotine attenuates methamphetamine-induced novel object recognition deficits in rats and explored potential underlying mechanisms. METHODS: Adolescent or adult male Sprague-Dawley rats received either nicotine water (10-75 µg/mL) or tap water for several weeks. Methamphetamine (4 × 7.5mg/kg/injection) or saline was administered either before or after chronic nicotine exposure. Novel object recognition was evaluated 6 days after methamphetamine or saline. Serotonin transporter function and density and α4ß2 nicotinic acetylcholine receptor density were assessed on the following day. RESULTS: Chronic nicotine intake via drinking water beginning during either adolescence or adulthood attenuated the novel object recognition deficits caused by a high-dose methamphetamine administration. Similarly, nicotine attenuated methamphetamine-induced deficits in novel object recognition when administered after methamphetamine treatment. However, nicotine did not attenuate the serotonergic deficits caused by methamphetamine in adults. Conversely, nicotine attenuated methamphetamine-induced deficits in α4ß2 nicotinic acetylcholine receptor density in the hippocampal CA1 region. Furthermore, nicotine increased α4ß2 nicotinic acetylcholine receptor density in the hippocampal CA3, dentate gyrus and perirhinal cortex in both saline- and methamphetamine-treated rats. CONCLUSIONS: Overall, these findings suggest that nicotine-induced increases in α4ß2 nicotinic acetylcholine receptors in the hippocampus and perirhinal cortex might be one mechanism by which novel object recognition deficits are attenuated by nicotine in methamphetamine-treated rats.

Prior methamphetamine self-administration attenuates the dopaminergic deficits caused by a subsequent methamphetamine exposure.

Others and we have reported that prior methamphetamine (METH) exposure attenuates the persistent striatal dopaminergic deficits caused by a subsequent high-dose "binge" METH exposure. The current study investigated intermediate neurochemical changes that may contribute to, or serve to predict, this resistance. Rats self-administered METH or saline for 7 d. On the following day (specifically, 16 h after the conclusion of the final METH self-administration session), rats received a binge exposure of METH or saline (so as to assess the impact of prior METH self-administration), or were sacrificed without a subsequent METH exposure (i.e., to assess the status of the rats at what would have been the initiation of the binge METH treatment). Results revealed that METH self-administration per se decreased striatal dopamine (DA) transporter (DAT) function and DA content, as assessed 16 h after the last self-administration session. Exposure to a binge METH treatment beginning at this 16-h time point decreased DAT function and DA content as assessed 1 h after the binge METH exposure: this effect on DA content (but not DAT function) was attenuated if rats previously self-administered METH. In contrast, 24 h after the binge METH treatment prior METH self-administration: 1) attenuated deficits in DA content, DAT function and vesicular monoamine transporter-2 function; and 2) prevented increases in glial fibrillary acidic protein and DAT complex immunoreactivity. These data suggest that changes 24 h, but not 1 h, after binge METH exposure are predictive of tolerance against the persistence of neurotoxic changes following binge METH exposures.

Methamphetamine self-administration attenuates hippocampal serotonergic deficits: role of brain-derived neurotrophic factor.

Preclinical studies suggest that prior treatment with escalating doses of methamphetamine (METH) attenuates the persistent deficits in hippocampal serotonin (5-hydroxytryptamine; 5HT) transporter (SERT) function resulting from a subsequent 'binge' METH exposure. Previous work also demonstrates that brain-derived neurotrophic factor (BDNF) exposure increases SERT function. The current study investigated changes in hippocampal BDNF protein and SERT function in rats exposed to saline or METH self-administration prior to a binge exposure to METH or saline. Results revealed that METH self-administration increased hippocampal mature BDNF (mBDNF) immunoreactivity compared to saline-treated rats as assessed 24 h after the start of the last session. Further, mBDNF immunoreactivity was increased and SERT function was not altered in rats that self-administered METH prior to the binge METH exposure as assessed 24 h after the binge exposure. These results suggest that prior exposure to contingent METH increases hippocampal mBDNF, and this may contribute to attenuated deficits in SERT function.

Age-related differences in the disposition of nicotine and metabolites in rat brain and plasma.

INTRODUCTION: Studies have evaluated the behavioral and neurochemical impact of nicotine administration in rodents. However, the distribution of nicotine and metabolites in rat brain and plasma as a function of age has not been investigated. This is a significant issue because human adolescents have a greater risk for developing nicotine addiction than adults, and reasons underlying this observation have not been fully determined. Thus, in this present study, we evaluated the impact of the transition from adolescence (postnatal day [PND 40]) to adulthood (PND 90) on nicotine distribution in rats. METHODS: PND 40, 60, and 90 rats received a single injection of (-) nicotine (0.8 mg/kg, subcutaneously). Liquid chromatography tandem-mass spectrometry was used to measure concentration of nicotine and metabolites in selected biological matrices. RESULTS: Nicotine, cotinine, and nornicotine were detected in rat striata and frontal cortex 30 min, 1 hr, 2 hr, and 4 hr after a single administration. These and several additional metabolites (nicotine-1'-oxide, cotinine-N-oxide, norcotinine, and trans-3'-hydroxycotinine) were also detected in plasma at these same timepoints. The mean concentration of nicotine in brain and plasma was lower in PND 40 versus PND 90 rats. In contrast, the mean concentration of nornicotine was higher in the plasma and brain of PND 40 versus PND 90 rats. CONCLUSIONS: Nicotine and metabolite distribution differs between adolescent and adult rats. These data suggest that adolescent rats metabolize nicotine to some metabolites faster than adult rats. Further studies are needed to investigate the potential correlation between age, drug distribution, and nicotine addiction.

Prior methamphetamine self-administration attenuates serotonergic deficits induced by subsequent high-dose methamphetamine administrations.

BACKGROUND: Pre-clinical studies indicate that high-dose, non-contingent methamphetamine (METH) administration both rapidly and persistently decreases serotonergic neuronal function. Despite research indicating the hippocampus plays an important role in METH abuse and is affected by METH use, effects of METH self-administration on hippocampal serotonergic neurons are not well understood, and were thus an important focus of the current study. Because humans often administer METH in a binge-like pattern, effects of prior METH self-administration on a subsequent "binge-like" METH treatment were also examined. METHODS: Rats were treated as described above, and sacrificed 1 or 8d after self-administration or 1h or 7d after the final binge METH or saline exposure. Hippocampal serotonin (5-hydroxytryptamine; 5HT) content and transporter (SERT) function were assessed. RESULTS: METH self-administration per se had no persistent effect on hippocampal 5HT content or SERT function. However, this treatment attenuated the persistent, but not acute, hippocampal serotonergic deficits caused by a subsequent repeated, high-dose, non-continent METH treatment administered 1 d the last self-administration session. No attenuation in persistent deficits were seen when the high-dose administration of METH occurred 15d after the last self-administration session. CONCLUSIONS: The present findings demonstrate that METH self-administration alters serotonergic neurons so as to engender "tolerance" to the persistent serotonergic deficits caused by a subsequent METH exposure. However, this "tolerance" does not persist. These data provide a foundation to investigate complex questions including how the response of serotonergic neurons to METH may contribute to contingent-related disorders such as dependence and relapse.

Methamphetamine self-administration acutely decreases monoaminergic transporter function.

Numerous preclinical studies have demonstrated that noncontingent methamphetamine (METH) administration rapidly decreases both dopamine (DA) transporter (DAT) and vesicular monoamine-2 transporter (VMAT-2) function. Because of the importance of transporter function to the abuse and neurotoxic liabilities of METH, and previous research indicating that the effects of noncontingent METH treatment do not necessarily predict effects of contingent exposure, the present study examined the acute impact of METH self-administration on these transporters. Results revealed that five days of METH self-administration (4 h/session; 0.06 mg/infusion) decreased DAT and VMAT-2 activity, as assessed in synaptosomes and vesicles, respectively, prepared from striatal tissue 1 h after the final self-administration session. METH self-administration increased core body temperatures as well. Brain METH and amphetamine (AMPH) levels, assessed 1 h after the final self-administration session, were approximately twice greater in high-pressing rats compared to low-pressing rats despite similar changes in DAT function. In conclusion, the present manuscript is the first to describe transporter function and METH/AMPH levels after self-administration in rodents. These data provide a foundation to investigate complex questions including how the response of dopaminergic systems to METH self-administration contributes to contingent-related processes such as dependence.

Methamphetamine self-administration causes persistent striatal dopaminergic alterations and mitigates the deficits caused by a subsequent methamphetamine exposure.

Preclinical studies have demonstrated that repeated methamphetamine (METH) injections (referred to herein as a "binge" treatment) cause persistent dopaminergic deficits. A few studies have also examined the persistent neurochemical impact of METH self-administration in rats, but with variable results. These latter studies are important because: 1) they have relevance to the study of METH abuse; and 2) the effects of noncontingent METH treatment do not necessarily predict effects of contingent exposure. Accordingly, the present study investigated the impact of METH self-administration on dopaminergic neuronal function. Results revealed that self-administration of METH, given according to a regimen that produces brain METH levels comparable with those reported postmortem in human METH abusers (0.06 mg/infusion; 8-h sessions for 7 days), decreased striatal dopamine transporter (DAT) uptake and/or immunoreactivity as assessed 8 or 30 days after the last self-administration session. Increasing the METH dose per infusion did not exacerbate these deficits. These deficits were similar in magnitude to decreases in DAT densities reported in imaging studies of abstinent METH abusers. It is noteworthy that METH self-administration mitigated the persistent deficits in dopaminergic neuronal function, as well as the increases in glial fibrillary acidic protein immunoreactivity, caused by a subsequent binge METH exposure. This protection was independent of alterations in METH pharmacokinetics, but may have been attributable (at least in part) to a pretreatment-induced attenuation of binge-induced hyperthermia. Taken together, these results may provide insight into the neurochemical deficits reported in human METH abusers.

Simultaneous quantification of nicotine and metabolites in rat brain by liquid chromatography-tandem mass spectrometry.

A liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for simultaneous quantification of nicotine (NIC), cotinine (COT), nornicotine (NNIC), norcotinine (NCOT), nicotine-N-ß-D-glucuronide (NIC GLUC), cotinine-N-ß-D-glucuronide (COT GLUC), nicotine-1'-oxide (NNO), cotinine-N-oxide (CNO), trans-3'-hydroxycotinine (3-HC), anabasine (AB) and anatabine (AT) was modified and validated for quantification of these selected analytes in rat brain tissue. This analytical method provides support for preclinical NIC pharmacokinetic and toxicological studies after controlled dosing protocols. After brain homogenization and solid-phase extraction, target analytes and corresponding deuterated internal standards were chromatographically separated on a Discovery(®) HS F5 HPLC column with gradient elution and analyzed by LC-MS/MS in positive electrospray ionization (ESI) mode with multiple reaction monitoring (MRM) data acquisition. Method linearity was assessed and calibration curves were determined over the following ranges: 0.1-7.5 ng/mg for NIC, COT GLUC and AB; and 0.025-7.5 ng/mg for COT, NNIC, NCOT, NIC GLUC, NNO, CNO, 3-HC and AT (R(2)≥0.99 for all analytes). Extraction recoveries ranged from 64% to 115%, LC-MS/MS matrix effects were ≤21%, and overall process efficiency ranged from 57% to 93% at low and high quality control concentrations. Intra- and inter-assay imprecisions and accuracy for all analytes were ≤12.9% and ≥86%, respectively. The method was successfully applied to quantification of NIC and metabolites in the brain of post-natal day 90 rats that were sacrificed 2-h after a single 0.8 mg/kg s.c. administration of (-)NIC. In these tissues, striatal concentrations were 204.8±49.4, 138.2±14.2 and 36.1±6.1 pg/mg of NIC, COT and NNIC, respectively. Concentrations of NIC, COT and NNIC in the remaining whole brain (RWhB) were 183.3±68.0, 130.0±14.1 and 46.7±10.3 pg/mg, respectively. Quantification of these same analytes in plasma was also performed by a previously validated method. NIC, COT, NNIC, NCOT, NNO and CNO were detected in plasma with concentrations comparable to those reported in previous studies. However, and in contrast to brain tissues, COT concentrations in plasma were significantly higher than were those of NIC (194.6±18.6 ng/mL versus 52.7±12.9 ng/mL). Taken together, these results demonstrate that a sensitive and selective method has been developed for the determination of NIC biomarkers in rat brain.

4-Methylmethcathinone (mephedrone): neuropharmacological effects of a designer stimulant of abuse.

The designer stimulant 4-methylmethcathinone (mephedrone) is among the most popular of the derivatives of the naturally occurring psychostimulant cathinone. Mephedrone has been readily available for legal purchase both online and in some stores and has been promoted by aggressive Web-based marketing. Its abuse in many countries, including the United States, is a serious public health concern. Owing largely to its recent emergence, there are no formal pharmacodynamic or pharmacokinetic studies of mephedrone. Accordingly, the purpose of this study was to evaluate effects of this agent in a rat model. Results revealed that, similar to methylenedioxymethamphetamine, methamphetamine, and methcathinone, repeated mephedrone injections (4× 10 or 25 mg/kg s.c. per injection, 2-h intervals, administered in a pattern used frequently to mimic psychostimulant "binge" treatment) cause a rapid decrease in striatal dopamine (DA) and hippocampal serotonin (5-hydroxytryptamine; 5HT) transporter function. Mephedrone also inhibited both synaptosomal DA and 5HT uptake. Like methylenedioxymethamphetamine, but unlike methamphetamine or methcathinone, repeated mephedrone administrations also caused persistent serotonergic, but not dopaminergic, deficits. However, mephedrone caused DA release from a striatal suspension approaching that of methamphetamine and was self-administered by rodents. A method was developed to assess mephedrone concentrations in rat brain and plasma, and mephedrone levels were determined 1 h after a binge treatment. These data demonstrate that mephedrone has a unique pharmacological profile with both abuse liability and neurotoxic potential.

Methamphetamine treatment during development attenuates the dopaminergic deficits caused by subsequent high-dose methamphetamine administration.

Administration of high doses of methamphetamine (METH) causes persistent dopaminergic deficits in both nonhuman preclinical models and METH-dependent persons. Noteworthy, adolescent [i.e., postnatal day (PND) 40] rats are less susceptible to this damage than young adult (PND90) rats. In addition, biweekly treatment with METH, beginning at PND40 and continuing throughout development, prevents the persistent dopaminergic deficits caused by a "challenge" high-dose METH regimen when administered at PND90. Mechanisms underlying this "resistance" were thus investigated. Results revealed that biweekly METH treatment throughout development attenuated both the acute and persistent deficits in VMAT2 function, as well as the acute hyperthermia, caused by a challenge METH treatment. Pharmacokinetic alterations did not appear to contribute to the protection afforded by the biweekly treatment. Maintenance of METH-induced hyperthermia abolished the protection against both the acute and persistent VMAT2-associated deficits suggesting that alterations in thermoregulation were caused by exposure of rats to METH during development. These findings suggest METH during development prevents METH-induced hyperthermia and the consequent METH-related neurotoxicity.