Emerging synthetic cannabinoids detected by a drug checking service in Toronto, Canada.

BACKGROUND: Toronto's Drug Checking Service (DCS) provides people who use drugs with information on the chemical composition of their substances and conducts real-time monitoring of the unregulated drug supply. Presented are first known data of three newly detected synthetic cannabinoids (SCs) in Toronto, Ontario. METHODS: The present data are from samples analyzed between April and November 2020. Samples were collected at partnering harm reduction agencies in Toronto and analyzed using gas or liquid chromatography-mass spectrometry. An intake survey queried about the sample characteristics on submission, including expected drug(s). RESULTS: Samples were analyzed between 1 April and 20 November 2020 (N = 19), which marks the period immediately following imposed COVID-19 border and movement restrictions in Canada. The newly detected, unexpected SCs were ACHMINACA (n = 15), AB-FUBINACA (n = 3), and 4-fluoro-MDMB-BUTINACA (n = 1). Fentanyl was expected in 74% (n = 14). Most SCs were detected in samples containing fentanyl or related analogues (n = 18; 95%), or benzodiazepine-related drugs (i.e., etizolam and flualprazolam) (n = 15; 79%). CONCLUSIONS: This information can inform overdose prevention efforts and drug market monitoring of SCs in Toronto and regions served by the same drug trafficking routes. The detection of SCs during a period marked by COVID-19-related restrictions can contribute to efforts to identify global drug market trends during this time.

Evaluating networked drug checking services in Toronto, Ontario: study protocol and rationale.

BACKGROUND: The increasing incidence of fatal opioid overdose is a public health crisis in Canada. Given growing consensus that this crisis is related to the presence of highly potent opioid adulterants (e.g., fentanyl) in the unregulated drug supply, drug checking services (DCS) have emerged as part of a comprehensive approach to overdose prevention. In Canada's largest city, Toronto, a network of DCS launched in 2019 to prevent overdose and overdose-related risk behaviors. This network employs mass spectrometry technologies, with intake sites co-located with supervised consumption services (SCS) at three frontline harm reduction agencies. The protocol and rationale for assessing the impact of this multi-site DCS network in Toronto is described herein. The aims of this study are to (1) evaluate the impact of DCS access on changes in and factors influencing overdose and related risk behaviors, (2) investigate the perceived capacity of DCS to prevent overdose, and (3) identify composition (qualitative and quantitative) trends in Toronto's unregulated drug supply. METHODS: We will use a parallel-mixed-methods design with complementary data sources (including data from chemical analysis of drug samples, quantitative intake and post-test surveys, SCS, coroners, paramedic services, and qualitative interviews), followed by a meta-inference process wherein results from analyses are synthesized. RESULTS: Whereas most DCS globally target "recreational drug users," in Toronto, this networked DCS will primarily target marginalized people who use drugs accessing frontline services, many of whom use drugs regularly and by injection. This evolution in the application of DCS poses important questions that have not yet been explored, including optimal service delivery models and technologies, as well as unique barriers for this population. Increasing information on the unregulated drug supply may modify the risk environment for this population of people who use drugs. CONCLUSIONS: This study addresses evidence gaps on the emerging continuum of overdose prevention responses and will generate critical evidence on a novel approach to reducing the ongoing high incidence of drug-related morbidity and mortality in Canada and elsewhere.

Programmed cell death and apoptosis--where it came from and where it is going: from Elie Metchnikoff to the control of caspases.

The story of cell death began with the origins of cell biology, including important observations by Elie (Ilya) Metchnikoff, who realized that phagocytes engulfed dying cells. Most of the early studies were observational. By the middle of the 20th C, researchers were beginning to explore how cells died, had recognized that cell death was a physiologically controlled process, that the most common mode of death ("shrinkage necrosis", later apoptosis) was tightly controlled, and were speculating whether lysosomes were "suicide bags". Just prior to 1990 several discoveries led to rapid expansion of interest in the field and elucidation of the mechanisms of apoptosis. Closer to the beginning of the 21st C comprehensive analysis of the molecules that controlled and effected apoptosis led to the conclusion that autophagic processes were linked to apoptosis and could serve to limit or increase cell death. Today, realizing that knowledge of the components of cell death has not yet produced pharmaceuticals of therapeutic value, research is turning to questions of what metabolic or other mechanisms indirectly control the activation or suppression of the cell death positive feedback loop. This article is part of a Special Issue entitled "Apoptosis: Four Decades Later"

Increased serum IL-6 level time-dependently regulates hyperalgesia and spinal mu opioid receptor expression during CFA-induced arthritis.

Interleukin (IL)-6 is known to cause pro- and anti-inflammatory effects during different stages of inflammation. Recent therapeutic investigations have focused on treatment of various inflammatory disorders with anti-cytokine substances. As a result, the aim of this study was to further elucidate the influence of IL-6 in hyperalgesia and edema during different stages of Complete Freund's Adjuvant (CFA)-induced arthritis (AA) in male Wistar rats. AA was induced by a single subcutaneous injection of CFA into the rats' hindpaw. Anti-IL-6 was administered either daily or weekly during the 21 days of study. Spinal mu opioid receptor (mOR) expression was detected by Western blotting. Daily and weekly treatment with an anti-IL-6 antibody significantly decreased paw edema in the AA group compared to the AA control group. Additionally, daily and weekly anti-IL-6 administration significantly reduced hyperalgesia on day 7 in the AA group compared to the AA control group; however, there were significant increases in hyperalgesia in the antibody-treated group on days 14 and 21 compared to the AA control group. IL-6 antibody-induced increases in hyperalgesia on the 14th and 21st days after CFA injection correlated with a time-dependent, significant reduction in spinal mOR expression during anti-IL-6 treatment. Our study confirmed the important time-dependent relationship between serum IL-6 levels and hyperalgesia during AA. These results suggest that the stages of inflammation in AA must be considered for anti-hyperalgesic and anti-inflammatory interventions via anti-IL-6 antibody treatment.

Comparison of changes in mRNA expression of spinal glutamate transporters following induction of two neuropathic pain models.

OBJECTIVES: It is now suspected that different kinds of neuropathic pain syndromes may have significantly different mechanisms. To date, much effort has been made to investigate the function of glutamate transporters (GTs) after nerve injury. The aim of this study is to compare the changes in GTs' mRNA expression levels between two distinct models of peripheral neuropathic pain: chronic constriction nerve injury (CCI) and spared nerve injury (SNI). METHODS: Experiments were performed on animal models of mononeuropathy. Several groups of rats were subjected to behavioral experiments before and 4, 7, and 14 days after the induction of mononeuropathy following the CCI and SNI. Allodynia was assessed by Von Frey filaments, and thermal hyperalgesia was assessed by the paw withdrawal tests. To study molecular experiments, the mRNA expression of (GTs) in CCI and SNI rats, reverse transcription polymerase chain reaction (RT-PCR) were used on days 4 and 14. RESULTS AND CONCLUSION: The maximum responses of mechanical allodynia and heat hyperalgesia in two distinct neuropathic pain models were detected on day 14. CCI and SNI induced upregulation of three GTs on day 4, which were followed by GTs downregulation in CCI and downregulation of glutamate aspartate transporter (GLAST) and glutamate transporter (GLT)1 in SNI when examined on day 14. These results indicate that there is an inverse correlation between pain responses and expression of GTs, and also changes in expression of spinal GTs may have a critical function in both the induction and maintenance of neuropathic pain in independent peripheral neuropathic pain models.

Insulin protects against stress-induced impairments in water maze performance.

The presence of insulin receptor in the hippocampus suggests that this organ is a target for insulin. However, unlike the classic peripheral insulin target tissues such as adipocyte, muscle and liver, where the primary function of insulin is to regulate glucose homeostasis, insulin in the central nervous system (CNS) exhibits more diverse actions, most of which have not been clearly understood. A direct role of hippocampal insulin receptor signaling in improving cognitive functions, including learning and memory, and the association of insulin receptor deterioration with brain degenerative dementia (e.g., Alzheimer's disease) have attracted increasing interest. Additionally it has been shown that insulin can be a neuroprotective agent against memory loss induced by ischemia, lesions and some pharmacological agents. In the present study we evaluate the hypothesis that the bilateral intra CA1 insulin injection can protects against stress-induced memory deficit. Chronic restraint stress (2h per day x 7 days) significantly impaired spatial performance in Morris water maze and elevated serum corticosterone level. Intrahippocampal insulin microinjection was done 15-20 min before every stress episode. Insulin in low dose (0.5 MU) had no significant effect on memory deficit induced by stress. But in higher doses (6 and 12 MU) insulin protects animals against the deleterious effect of stress. Insulin alone daily injection had no effect on water maze performance. These results suggest that spatial learning and memory is compromised during chronic stress and insulin may protect against this effect.

The effect of intrahippocampal insulin microinjection on spatial learning and memory.

Insulin is best known for its action on peripheral target tissues such as the adipocyte, muscle and liver to regulate glucose homeostasis. Insulin and its receptor are found in specific area of CNS with a variety of region-specific functions different from its direct glucose regulation in the periphery. The hippocampus and cerebral cortex distributed insulin/insulin receptor has been shown to be involved in brain cognitive functions. Previous studies about the effect of insulin on memory are controversial. In the present study, the effect of insulin microinjection into CA1 region of rat hippocampus on water maze performance has been investigated. Insulin had a discrepant effect dose dependently. The spatial learning and memory were impaired with lower dose of insulin, had not changed with intermediate doses, while they improved with higher doses. These results suggest that insulin may have a dose-dependent effect on spatial learning and memory.

Identification of peptides specific for antibodies in vitiligo using a phage library.

Patients with vitiligo produce specific autoantibodies that can be detected in their sera. These antibodies are believed to play a role in the pathogenesis of this disease. A random peptide library displayed on phage is a technique that can be used to identify the epitopes that react with monoclonal and polyclonal antibodies. We used this technique to identify the epitopes that react specifically with the vitiligo autoantibodies. By screening the random peptide phage library and using ELISA, two clones that showed a higher frequency of reactivity with the antibodies in the sera of patients with vitiligo were identified. The peptides do not show any similarity with the autoantigens so far implicated in vitiligo, indicating that these epitopes may mimic conformational epitopes in proteins.

Process development for production of recombinant human interferon-gamma expressed in Escherichia coli.

A simple fed-batch process was carried out using constant and variable specific growth rates for high-cell-density cultivation of Escherichia coli BL21 (DE3) expressing human interferon-gamma(hIFN-gamma). The feeding rate was adjusted to achieve an appropriate specific growth rate. The dissolved oxygen level was maintained at 20-30% of air saturation by control of airflow and stirrer speed and, where necessary, by enrichment of inlet air with pure oxygen. Glucose was the sole source of carbon and energy and was provided by following a simple exponential feeding rate. The final cell density in the fed-batch fermentation with constant and variable specific growth rate feeding strategies was ~100 g dry cell wt l(-1) after 36 and 20 h, respectively. The final specific yield and overall productivity of recombinant hIFN-gamma in the variable specific growth rate strategy were 0.35 g rHu-IFN-gamma g(-1) dry cell wt and 0.9 g rHu-IFN-gamma l(-1) h(-1), respectively. A new chromatographic purification procedure involving anion exchange and cation exchange chromatographies was developed for purification of rHu-IFN-gamma from inclusion bodies. The established purification process is reproducible and the total recovery of rHu-IFN-gamma was ~30% (100 mg rHu-IFN-gamma g(-1) dry cell wt). The purity of the rHu-IFN-gamma was determined using HPLC. Sterility, pyrogenicity, and DNA content tests were conducted to assure the absence of toxic materials and other components of E. coli in the final product. The final purified rHu-IFN-gamma has a specific antiviral activity of ~2 x 10(7) IU/mg protein, as determined by viral cytopathic effect assay. These results certify the product for clinical purposes.

Over-expression of recombinant human interferon-gamma in high cell density fermentation of Escherichia coli.

Human interferon-gamma (hIFN-gamma) was expressed in Escherichia coli BL21(DE3) under the control of the T7 promoter. Glucose was used as the sole source of carbon and energy with simple exponential feeding rate in fed-batch process. Cell density of recombinant E. coli was reached to 100 g dry wt l(-1) under both constant (0.12 h(-1)) and variable (0.12-0.52 h(-1)) specific growth rates. In the variable specific growth rate fed-batch process, plasmid stability and specific yield of rhIFN-gamma were greater than constant specific growth rate fed-batch process. The final specific yield and overall productivity of rhIFN-gamma were 0.35 +/- 0.02 g rhIFN-gamma g(-1) dry cell wt and 0.9 +/- 0.05 g rhIFN-gamma l(-1) h(-1) in the variable specific growth rate fed-batch process, respectively.