Cross-Reactivity of 24 Cannabinoids and Metabolites in blood using the immunalysis cannabinoids direct enzyme-linked immunosorbent assay kit.

With a wider availability of synthetic and semi-synthetic cannabinoids in the consumer space, there is a growing impact on public health and safety. Forensic toxicology laboratories should keep these compounds in mind as they attempt to remain effective in screening for potential sources of human performance impairment. Enzyme-linked immunosorbent assay (ELISA) is a commonly utilized tool in forensic toxicology, as its efficiency and sensitivity make it useful for rapid and easy screening for a large number of drugs. This screening technique has lower specificity, which allows for broad cross-reactivity among structurally-similar compounds. In this study, the Cannabinoids Direct ELISA kit from Immunalysis was utilized to assess the cross-reactivities of 24 cannabinoids and metabolites in whole blood. The assay was calibrated with 5 ng/mL of 11-nor-9-carboxy-Δ9-tetrahydrocannabinol and the analytes of interest were evaluated at concentrations ranging from 5 to 500 ng/mL. Most parent compounds demonstrated cross-reactivity ≥ 20 ng/mL, with increasing alkyl side chain length relative to Δ9-tetrahydrocannabinol resulting in decreased cross-reactivity. Of the 24 analytes, only the carboxylic acid metabolites, 11-nor-9-carboxy-Δ8-tetrahydrocannabinol, 11-nor-9(R)-carboxy-hexahydrocannabinol, and 11-nor-9(S)-carboxy-hexahydrocannabinol, were cross-reactive at levels ≤ 10 ng/mL. Interestingly, 11-nor-9(R)-carboxy-hexahydrocannabinol demonstrated cross-reactivity at 5 ng/mL, where its stereoisomer 11-nor-9(S)-carboxy-hexahydrocannabinol, did not. As more information emerges about the prevalence of these analytes in blood specimens, it is important to understand and characterize their impact on current testing paradigms.

Screening and confirmation of psilocin, mitragynine, phencyclidine, ketamine and ketamine metabolites by liquid chromatography-tandem mass spectrometry.

A safe and productive workplace requires a sober workforce, free from substances that impair judgment and concentration. Although drug monitoring programs already exist, the scope and loopholes of standard workplace testing panels are well known, allowing other substances to remain a source of risk. Therefore, a high-throughput urine screening method for psilocin, mitragynine, phencyclidine, ketamine, norketamine and dehydronorketamine was developed and validated in conjunction with a urine and blood confirmation method. There are analytical challenges to overcome with psilocin and mitragynine, particularly when it comes to drug stability and unambiguous identification in authentic specimens. Screening and confirmation methods were validated according to the American National Standards Institute/Academy Standards Board (ANSI/ASB) Standard 036, Standard Practices for Method Validation in Forensic Toxicology. An automated liquid handling system equipped with dispersive pipette extraction tips was utilized for preparing screening samples, whereas an offline solid-phase extraction method was used for confirmation sample preparation. Both methods utilized liquid chromatography-tandem mass spectrometry to achieve limits of detection between 1-5 ng/mL for the screening method and 1 ng/mL for the confirmation method. Automation allows for faster throughput and enhanced quality assurance, which improves turnaround time. Compared to previous in-house methods, specimen volumes were substantially decreased for both blood and urine, which is an advantage when volume is limited. This screening technique is well suited for evaluating large numbers of specimens from those employed in safety-sensitive workforce positions. This method can be utilized by workplace drug testing, human performance and postmortem laboratories seeking robust qualitative screening and confirmation methods for analytes that have traditionally been challenging to routinely analyze.

Quantitative Analysis of Lysergic Acid Diethylamide and Metabolite in Urine by Automated Extraction and Liquid Chromatography-Tandem Mass Spectrometry.

Recently, lysergic acid diethylamide (LSD) has become a resurgent drug of abuse. The detection of LSD is problematic because of the low dosage taken by users, light and heat sensitivity of the analyte and the lack of efficient analytical methods. Presented here is the validation of an automated sample preparation method for the analysis of LSD and its primary urinary metabolite, 2-oxo-3-hydroxy-LSD (OHLSD), in urine samples by liquid chromatography-tandem mass spectrometry. Analytes were extracted from urine using an automated Dispersive Pipette XTRaction method on Hamilton STAR and STARlet liquid handling systems. The limit of detection for both analytes was administratively defined at the lowest calibrator used in the experiments, and the limit of quantitation was 0.05 ng/mL for both analytes. All validation criteria were acceptable per Department of Defense Instruction 1010.16 requirements. This method offers an efficient, sensitive analytical solution to routinely evaluate large numbers of urine specimens for LSD in workplace drug deterrence programs.

An Enhanced LC-MS-MS Technique for Distinguishing Δ8- and Δ9-Tetrahydrocannabinol Isomers in Blood and Urine Specimens.

Among the abundance of cannabinoids identified in cannabis, the active parent drug, Δ9-tetrahydrocannabinol (Δ9-THC), and its oxidized metabolite, 11-nor-9-carboxy-Δ9-THC (Δ9-THCCOOH), are attractive analytical targets to detect cannabis use. More recently, confirmation of these analytes may be hindered by a related interfering compound. Forensic toxicology laboratories attribute this phenomenon to an increase in cases containing Δ8-tetrahydrocannabinol (Δ8-THC) and 11-nor-9-carboxy-Δ8-THC (Δ8-THCCOOH). It is technically challenging to chromatographically resolve and accurately quantify Δ8- and Δ9-THC and THCCOOH in toxicology specimens due to their structural resemblance. This study describes a validated method to resolve and quantify active Δ8-THC and Δ9-THC in blood while qualitatively confirming the inactive metabolites Δ8-THCCOOH and Δ9-THCCOOH in blood and urine. Analytes are extracted and concentrated by solid-phase extraction and analyzed by liquid chromatography--electrospray ionization tandem mass spectrometry, which is amenable to modern toxicology laboratory routine workflows. This procedure offers a clear solution to untangling mixtures of these isomers, particularly in cases where Δ8-THC and its metabolite are the sole or dominant form.

Screening and confirmation methods for the qualitative identification of nine phytocannabinoids in urine by LC-MS/MS.

Qualitative liquid chromatography tandem mass spectrometry (LC-MS/MS) methods were developed and validated to screen and confirm the presence of nine phytocannabinoids in urine. The nine phytocannabinoids targeted in the methods included Δ9-tetrahydrocannabinol (THC), 11-hydroxy-THC, 11-nor-9-carboxy-THC, cannabidiol, 7-carboxy cannabidiol, cannabinol, cannabigerol, Δ9-tetrahydrocannabivarin (THCV), and 11-nor-9-carboxy-THCV. The methods presented use a rapid, single-step enzymatic hydrolysis followed by solid-phase extraction and LC-MS/MS analysis. Limits of detection were established at 1 µg/L for non-carboxylated analytes and 5 µg/L for carboxylated analytes. The screening and confirmation methods were validated and implemented in the analysis of authentic case samples. These methods can assist forensic, medicolegal, or medical compliance investigations as the presence of phytocannabinoids, or lack there-of, may be used to help differentiate cannabis (hemp, marijuana) use from synthetic THC (dronabinol) exposure.

A Case Report of Fatal Desmethyl Carbodenafil Toxicity.

We present the case report of a 34-year-old Hispanic male who was found unresponsive in the carport of his residence. Surveillance video footage from a security camera showed that he collapsed as he was walking to his vehicle. The decedent had no medical history and no history of illicit drug use. Initial toxicology testing revealed no alcohol or illicit drugs. Autopsy findings indicated a need for additional toxicological analysis due to a lack of trauma and the paucity of pathophysiologically significant natural disease. Liquid chromatography time-of-flight mass spectrometry of postmortem blood revealed the presence of two large peaks corresponding to desmethyl carbodenafil, an unapproved sildenafil analogue and its hydroxy metabolite. Species that are probable desmethyl and hydroxydesmethyl metabolites of desmethyl carbodenafil were also found. The mass and retention time of the parent compound in the decedent's sample were matched to those of a commercial standard. Based on this preliminary match, a method was developed and validated to quantify desmethyl carbodenafil in human blood. This is the first known case of fatal intoxication by desmethyl carbodenafil, a phosphodiesterase-5 inhibitor that is not approved for use in the United States. Over the past several years, retailers have issued voluntary recalls for dietary supplements marketed as sexual performance enhancers on the basis that these supplements may contain undeclared desmethyl carbodenafil.

Detection and Characterization of the Effect of AB-FUBINACA and Its Metabolites in a Rat Model.

Synthetic cannabinoids were originally developed by academic and pharmaceutical laboratories with the hope of providing therapeutic relief from the pain of inflammatory and degenerative diseases. However, recreational drug enthusiasts have flushed the market with new strains of these potent drugs that evade detection yet endanger public health and safety. Although many of these drug derivatives were published in the medical literature, others were merely patented without further characterization. AB-FUBINACA is an example of one of the new indazole-carboxamide synthetic cannabinoids introduced in the past year. Even though AB-FUBINACA has become increasingly prominent in forensic drug and toxicology specimens analyses, little is known about the pharmacology of this substance. To study its metabolic fate, we utilized Wistar rats to study the oxidative products of AB-FUBINACA in urine and its effect on gene expressions in liver and heart. Rats were injected with 5 mg/kg of AB-FUBINACA each day for 5 days. Urine samples were collected every day at the same time. On day 5 after treatment, we collected the organs such as liver and heart. The urine samples were analyzed by mass spectrometry, which revealed several putative metabolites and positioning of the hydroxyl addition on the molecule. We used quantitative PCR gene expression array to analyze the hepatotoxicity and cardiotoxicity on these rats and confirmed by real-time quantitative RT-PCR. We identified three genes significantly associated with dysfunction of oxidation and inflammation. Our study reports in vivo metabolites of AB-FUBINACA in urine and its effect on the gene expressions in liver and heart.

Poor Man's Methadone: A Case Report of Loperamide Toxicity.

Loperamide, a common over-the-counter antidiarrheal drug and opioid derivative, is formulated to act upon intestinal opioid receptors. However, at high doses, loperamide crosses the blood-brain barrier and reaches central opioid receptors in the brain, leading to central opiate effects including euphoria and respiratory depression. We report the case of a young man found dead in his residence with a known history of drug abuse. At autopsy, the only significant findings were a distended bladder and bloody oral purge. Drug screening found nontoxic levels of alprazolam, fluoxetine, and marijuana metabolites. Liquid chromatography time-of-flight mass spectrometry found an unusual set of split isotope peaks consistent with chlorine. On the basis of autopsy and toxicological findings, loperamide toxicity was suspected because of its opioid properties and molecular formula containing chlorine. A sample of loperamide was analyzed by liquid chromatography time-of-flight mass spectrometry, resulting in a matching mass and retention time to the decedent's sample. Subsequently, quantitative testing detected 63 ng/mL of loperamide or more than 6 times of therapeutic peak concentration. Cause of death was determined as "toxic effects of loperamide with fluoxetine and alprazolam." Because of its opioid effects and easy accessibility, loperamide is known as "poor man's methadone" and may go undetected at medical and forensic drug screening.

Pathological findings in 2 cases of fatal 25I-NBOMe toxicity.

The research compound 25I-NBOMe, also known as CIMBI-5 or INBMeO, was created in academic laboratories as a potent serotonin 2A receptor agonist. Because of its high affinity and ambiguous legal status, recreational drug enthusiasts have used this compound as a powerful alternative to other hallucinogenic drugs such as lysergic acid diethylamide. We report 2 deaths after 25I-NBOMe ingestion by decedents who attended separate "rave" parties. The first case involved a 21-year-old male who admitted taking "acid" to his friend. A sudden violent rage caused him to flail about, and he subsequently became unresponsive. The postmortem examination revealed numerous external injuries that were consistent with physical aggression. The second case involved a 15-year-old female who was socializing outside a rave party, became ill, and rapidly deteriorated as her friend transported her to the hospital. The postmortem assessment was similar to the first case in that external contusions featured prominently. Comprehensive toxicology screens in both cases revealed only evidence of marijuana use. A deeper analysis using time-of-flight mass spectrometry revealed the presence of 25I-NBOMe, which was further confirmed by tandem-mass spectrometry. The behavior and injuries in these cases reveal a consistent pattern preceding fatal 25I-NBOMe toxicity.

Validation of LC-TOF-MS screening for drugs, metabolites, and collateral compounds in forensic toxicology specimens.

Liquid chromatography time-of-flight mass spectrometry (LC-TOF-MS) analysis provides an expansive technique for identifying many known and unknown analytes. This study developed a screening method that utilizes automated solid-phase extraction to purify a wide array of analytes involving stimulants, benzodiazepines, opiates, muscle relaxants, hypnotics, antihistamines, antidepressants and newer synthetic "Spice/K2" cannabinoids and cathinone "bath salt" designer drugs. The extract was applied to LC-TOF-MS analysis, implementing a 13 min chromatography gradient with mobile phases of ammonium formate and methanol using positive mode electrospray. Several common drugs and metabolites can share the same mass and chemical formula among unrelated compounds, but they are structurally different. In this method, the LC-TOF-MS was able to resolve many isobaric compounds by accurate mass correlation within 15 ppm mass units and a narrow retention time interval of less than 10 s of separation. Drug recovery yields varied among spiked compounds, but resulted in overall robust area counts to deliver an average match score of 86 when compared to the retention time and mass of authentic standards. In summary, this method represents a rapid, enhanced screen for blood and urine specimens in postmortem, driving under the influence, and drug facilitated sexual assault forensic toxicology casework.

Mediation of electronegative low-density lipoprotein signaling by LOX-1: a possible mechanism of endothelial apoptosis.

The lectin-like oxidized LDL receptor LOX-1 mediates endothelial cell (EC) uptake of experimentally prepared copper-oxidized LDL (oxLDL). To confirm the atherogenic role of this receptor cloned against copper-oxLDL, we examined whether it mediates EC uptake of L5, an electronegative LDL abundant in dyslipidemic but not normolipidemic human plasma. Hypercholesterolemic (LDL-cholesterol, >160 mg/dL) human LDL was fractionated into L1-L5, increasingly electronegative, by ion-exchange chromatography. In cultured bovine aortic ECs (BAECs), L5 upregulated LOX-1 and induced apoptosis. Transfection of BAECs with LOX-1-specific small interfering RNAs (siLOX-1) minimized baseline LOX-1 production and restrained L5-induced LOX-1 upregulation. Internalization of labeled L1-L5 was monitored in BAECs and human umbilical venous ECs by fluorescence microscopy. LOX-1 knockdown with siLOX-1 impeded the endocytosis of L5 but not L1-L4. In contrast, blocking LDL receptor with RAP (LDL receptor-associated protein) stopped the internalization of L1-L4 but not L5. Although chemically different, L5 and oxLDL competed for EC entry through LOX-1. Via LOX-1, L5 signaling hampered Akt phosphorylation and suppressed EC expression of fibroblast growth factor-2 and Bcl-2. L5 also selectively inhibited Bcl-xL expression and endothelial nitric oxide synthase phosphorylation but increased synthesis of Bax, Bad, and tumor necrosis factor-alpha. Blocking Akt phosphorylation with wortmannin increased LOX-1 expression, suggesting a modulatory role of Akt in LOX-1 synthesis; L5 upregulated LOX-1 by dephosphorylating Akt. Because endothelial nitric oxide synthase and Bcl-2 activities are Akt-dependent, L5 impairs Akt-mediated growth and survival signals in vascular ECs by way of LOX-1. Thus, the L5/LOX-1 complex may play a critical role in atherogenesis and illuminate important targets for disease intervention.

Electronegative LDL impairs vascular endothelial cell integrity in diabetes by disrupting fibroblast growth factor 2 (FGF2) autoregulation.

OBJECTIVE: L5, a circulating electronegative LDL identified in patients with hypercholesterolemia or type 2 diabetes, induces endothelial cell (EC) apoptosis by suppressing fibroblast growth factor (FGF)2 expression. FGF2 plays a pivotal role in endothelial regeneration and compensatory arteriogenesis. It is likely that vasculopathy and poor collateralization in diabetes is a result of FGF2 dysregulation. RESEARCH DESIGN AND METHODS: To investigate this mechanism, we isolated L5 from type 2 diabetic patients. In cultured bovine aortic ECs (BAECs), L5 inhibited FGF2 transcription and induced apoptosis. Because FGF2 stimulates the phosphatidylinositol 3-kinase (PI3K)-Akt pathway, we examined whether FGF2 transcription is regulated by Akt through a feedback mechanism. RESULTS: Diabetic L5 reduced FGF2 release to the medium but enhanced caspase-3 activity, with resultant apoptosis. Inhibition of PI3K with wortmannin or suppression of Akt activation with dominant-negative Akt inhibited FGF2 expression. Transfection of BAECs with FGF2 antisense cDNA depleted endogenous FGF2 protein. In these cells, not only was Akt phosphorylation inhibited, but FGF2 transcription was also critically impaired. In contrast, transfecting BAECs with FGF2 sense cDNA augmented Akt phosphorylation. Treatment with constitutively active Akt enhanced FGF2 expression. Augmentation of either FGF2 transcription or Akt phosphorylation rendered BAECs resistant to L5. CONCLUSIONS: These findings suggest that FGF2 is the primary initiator of its own expression, which is autoregulated through a novel FGF2-PI3K-Akt loop. Thus, by disrupting FGF2 autoregulation in vascular ECs, L5 may impair reendothelialization and collateralization in diabetes.

Electronegative LDL circulating in smokers impairs endothelial progenitor cell differentiation by inhibiting Akt phosphorylation via LOX-1.

Endothelial progenitor cells (EPCs), important for endothelial regeneration and vasculogenesis, are reduced by cigarette smoking. To elucidate the mechanisms, we examined the effects of electronegative LDL, circulating in chronic smokers, on EPC differentiation. Using ion-exchange chromatography, we purified smoker LDL into five subfractions, L1-L5. In matched, nonsmoking healthy subjects, L5, the most electronegative subfraction, was either absent or scanty. Sustained L5 treatment inhibited CD31 and KDR expression and EPC differentiation, whereas L1-L4 had no effect. L5 also inhibited telomerase activity to accelerate EPC senescence in correlation with reduced Akt phosphorylation. Transfection of day 3 EPCs with dominant negative Akt constructs inhibited CD31 and KDR expression, stalled EPC differentiation, and promoted early senescence. In contrast, transfection with constitutively active Akt rendered the EPCs resistant to L5, allowing normal maturation. L5 upregulated the lectin-like oxidized low density lipoprotein receptor 1 (LOX-1), and pretreatment of EPCs with TS20, a LOX-1-neutralizing antibody, blocked internalization of L5 by EPCs and prevented L5-mediated inhibition of EPC differentiation. Mixing L5 with L1 to physiological L5/L1 ratios did not attenuate L5's effects. These findings suggest that cigarette smoking is associated with the formation of L5, which inhibits EPC differentiation by impairing Akt phosphorylation via the LOX-1 receptor.

Cis-urocanic acid, a sunlight-induced immunosuppressive factor, activates immune suppression via the 5-HT2A receptor.

Exposure to UV radiation induces skin cancer and suppresses the immune response. To induce immune suppression, the electromagnetic energy of UV radiation must be absorbed by an epidermal photoreceptor and converted into a biologically recognizable signal. Two photoreceptors have been recognized: DNA and trans-urocanic acid (UCA). Trans-UCA is normally found in the outermost layer of skin and isomerizes to the cis isomer upon exposure to UV radiation. Although UCA was identified as a UV photoreceptor years ago, and many have documented its ability to induce immune suppression, its exact mode of action remains elusive. Particularly vexing has been the identity of the molecular pathway by which cis-UCA mediates immune suppression. Here we provide evidence that cis-UCA binds to the serotonin [5-hydroxytryptamine (5-HT)] receptor with relatively high affinity (Kd = 4.6 nM). Anti-cis-UCA antibody precipitates radiolabeled 5-HT, and the binding is inhibited by excess 5-HT and/or excess cis-UCA. Similarly, anti-5-HT antibody precipitates radiolabeled cis-UCA, and the binding is inhibited by excess 5-HT or excess cis-UCA. Calcium mobilization was activated when a mouse fibroblast line, stably transfected with the human 5-HT2A receptor, was treated with cis-UCA. Cis-UCA-induced calcium mobilization was blocked with a selective 5-HT2A receptor antagonist. UV- and cis-UCA-induced immune suppression was blocked by antiserotonin antibodies or by treating the mice with 5-HT2A receptor antagonists. Our findings identify cis-UCA as a serotonin receptor ligand and indicate that the immunosuppressive effects of cis-UCA and UV radiation are mediated by activation of the 5-HT2A receptor.

Platelet-activating factor is crucial in psoralen and ultraviolet A-induced immune suppression, inflammation, and apoptosis.

Psoralen plus UVA (PUVA) is used as a very effective treatment modality for various diseases, including psoriasis and cutaneous T-cell lymphoma. PUVA-induced immune suppression and/or apoptosis are thought to be responsible for the therapeutic action. However, the molecular mechanisms by which PUVA acts are not well understood. We have previously identified platelet-activating factor (PAF), a potent phospholipid mediator, as a crucial substance triggering ultraviolet B radiation-induced immune suppression. In this study, we used PAF receptor knockout mice, a selective PAF receptor antagonist, a COX-2 inhibitor (presumably blocking downstream effects of PAF), and PAF-like molecules to test the role of PAF receptor binding in PUVA treatment. We found that activation of the PAF pathway is crucial for PUVA-induced immune suppression (as measured by suppression of delayed type hypersensitivity to Candida albicans) and that it plays a role in skin inflammation and apoptosis. Downstream of PAF, interleukin-10 was involved in PUVA-induced immune suppression but not inflammation. Better understanding of PUVA's mechanisms may offer the opportunity to dissect the therapeutic from the detrimental (ie, carcinogenic) effects and/or to develop new drugs (eg, using the PAF pathway) that act like PUVA but have fewer side effects.

Platelet activating factor receptor binding plays a critical role in jet fuel-induced immune suppression.

Applying military jet fuel (JP-8) or commercial jet fuel (Jet-A) to the skin of mice suppresses the immune response in a dose-dependent manner. The release of biological response modifiers, particularly prostaglandin E2 (PGE2), is a critical step in activating immune suppression. Previous studies have shown that injecting selective cyclooxygenase-2 inhibitors into jet fuel-treated mice blocks immune suppression. Because the inflammatory phospholipid mediator, platelet-activating factor (PAF), up-regulates cyclooxygenase-2 production and PGE2 synthesis by keratinocytes, we tested the hypothesis that PAF-receptor binding plays a role in jet fuel-induced immune suppression. Treating keratinocyte cultures with PAF and/or jet fuel (JP-8 and Jet-A) stimulates PGE2 secretion. Jet fuel-induced PGE2 production was suppressed by treating the keratinocytes with specific PAF-receptor antagonists. Injecting mice with PAF, or treating the skin of the mice with JP-8, or Jet-A, induced immune suppression. Jet fuel-induced immune suppression was blocked when the jet fuel-treated mice were injected with PAF-receptor antagonists before treatment. Jet fuel treatment has been reported to activate oxidative stress and treating the mice with anti-oxidants (Vitamins C, or E or beta-hydroxy toluene), before jet fuel application, interfered with immune suppression. These findings confirm previous studies showing that PAF-receptor binding can modulate immune function. Furthermore, they suggest that PAF-receptor binding may be an early event in the induction of immune suppression by immunotoxic environmental agents that target the skin.

Ultraviolet radiation-induced immunosuppression of delayed-type hypersensitivity in mice.

Ultraviolet (UV) radiation present in sunlight plays a critical role in the initiation and promotion of nonmelanoma skin carcinogenesis and immune suppression. The immune suppressive effects of UV have been identified as a risk factor for skin cancer induction. For these reasons, scientists have focused on elucidating the mechanisms of UV-induced immune suppression to better understand the pathogenesis of skin cancer induction. A hallmark of UV-induced immune suppression is the generation of antigen-specific suppressor T cells. These suppressor cells have been shown to suppress antitumor immunity as well as other cell-mediated responses such as delayed-type hypersensitivity (DTH) reactions. Due to the excessive cost and time involved in traditional UV carcinogenic experiments, scientists have opted to use UV-induced suppression of DTH reactions as a surrogate model. DTH has been, and continues to be, a widely used assay system to measure in vivo immune function. Although somewhat unsophisticated by today's standards, this assay has great advantages because it presents a fast, inexpensive, and reliable model system to help dissect the mechanisms involved in UV-induced immune suppression. Furthermore, the murine model of DTH enables scientists to perform additional procedures, such as adoptive transfer studies with suppressor T cells, which are currently unavailable with human subjects.

Determining the role of cytokines in UV-induced immunomodulation.

Ultraviolet radiation exposure damages DNA and promotes the development of skin cancer. In addition, UV exposure suppresses the immune response. Although the mechanism by which epidermal exposure to UV induces systemic immune suppression is not fully understood, it is clear that cytokines are involved. Therefore, quantitative measurement of cytokines is a critical aspect of modern research techniques. Determining the level of synthesis and secretion of cytokines in vivo or in vitro can be achieved through several possible techniques, depending on the sampling size, its physical state, and the type of answers required to test the hypothesis. When studying transcriptional activation, the level of cytokine mRNA is often determined using reverse transcription polymerase chain reaction (RT-PCR), ribonuclease protection assay (RPA), or Northern blot. Quantitative determinations of specific protein levels require a capture ELISA. As with any analytical technique, there are compromises among expense of sensitivity, labor, and time. These methods are discussed as they pertain to surveying cytokine induction and their relative usefulness to the laboratory scientist.

Dermal application of jet fuel suppresses secondary immune reactions.

Applying military jet fuel (JP-8) to the skin of mice activates systemic immune suppression. In all of our previous experiments, JP-8 was applied to immunologically naïve mice. The effect of jet fuels on established immune reactions, such as immunological memory, is unknown. The focus of the experiments presented here was to test the hypothesis that jet fuel exposure [both JP-8 and commercial jet fuel (Jet-A)] suppresses established immune reactions. Mice were immunized with the opportunistic fungal pathogen Candida albicans and, at different times after immunization (10 to 30 days), various doses of undiluted JP-8 or Jet-A were applied to their skin. Both the elicitation of delayed-type hypersensitivity (DTH) (mice challenged 10 days after immunization) and immunological memory (mice challenged 30 days after immunization) were significantly suppressed in a dose-dependent manner. Dermal exposure to either multiple small doses (50 microl over 4 days) or a single large dose (approximately 200-300 microl) of JP-8 and/or Jet-A suppressed DTH to C. albicans. The mechanism by which dermal application of JP-8 and Jet-A suppresses immunological memory involves the release of immune biologic response modifiers. Blocking the production of prostaglandin E(2) by a selective cyclooxygenase-2 inhibitor (SC 236) significantly reversed jet fuel-induced suppression of immunologic memory. These findings indicate, for the first time, that dermal exposure to commercial jet fuel (Jet-A) suppresses the immune response. In addition, the data reported here expand on previous findings by suggesting that jet fuel exposure may depress the protective effect of prior vaccination.