Of the 12 million people who inject drugs worldwide, 13% are chronically infected with Human Immunodeficiency Virus (HIV), i.e., they live with HIV. Chronic opioid use affects the host immune system and increases an individual’s susceptibility to HIV infection. However, it is unclear how opioid use changes the course of HIV pathogenesis. Particularly, there is a gap in understanding how opioids impact HIV latency. Latency results in a reservoir of infected quiescent cells that evade antiviral immune responses, are not targeted by antiretroviral therapy (ART), and allow HIV viremia to rebound upon treatment interruption. While in vitro studies show that opioids modulate the activity of transcription factors involved in T-cell activation and HIV transcription, few studies have investigated whether opioid use impacts HIV latency in vivo in HIV-infected people. In this research, peripheral blood mononuclear cells (PBMCs) were utilized from People with HIV (PWH) with or without recent opioid use or opioid use disorder (OUD) who were enrolled in the Linking Infection and Narcology Care-Part II (LINC-II) and Studying Partial Agonists for Ethanol and Tobacco Elimination in Russians with HIV (St PETER HIV ARCH) studies conducted in St. Petersburg, Russia. Intact proviral DNA digital droplet PCR (ddPCR) assays were performed on PBMCs from antiretroviral treated PWH, with (n=8) or without (n=11) current OUD, to quantify intact and defective proviral genomes. Samples from ART-treated PWH with OUD compared to those without OUD had similar levels of intact and defective proviruses. To evaluate latency reversal, PBMCs from ART-treated PWH with or without OUD, were activated with anti-CD3/28 beads and RT-ddPCR assays were performed to measure HIV LTR-gag RNA. A variable response in PWH without OUD was seen where half of the samples showed an increase in HIV RNA upon activation. Interestingly, only 1 of 8 samples from PWH with OUD showed an increase in HIV transcription. However, no suppression of HIV reactivation was found in vitro from latent cells generated using a primary CD4+ T-cell latency model in the presence or absence of morphine. Similarly, no differences in HIV integration and transcription in vitro were observed between morphine and control conditions. Additionally, expression of opioid receptors was not detected in primary PBMCs, CD4 T cells, or macrophages. These results show that PWH with OUD have a pool of persistent HIV proviruses that are refractive to reactivation, although opioids did not affect HIV replication and latency reactivation in vitro. The discrepancy in these in vitro and in vivo results and the lack of expression of opioid receptors in immune cells suggests that while opioids do not directly impact HIV replication, latency, and reactivation in target CD4+ cells, opioids could indirectly shape the HIV reservoir in vivo by modulating general immune functions, neuroderived factors or other cells that are responsive to opioids. Eradication of the latent HIV reservoir is necessary to achieve a cure for HIV/AIDS. One approach for latency eradication is the “shock and kill” approach that entails stimulating viral production with latency-reversing agents followed by the killing of cells actively producing the virus by immune clearance. However, this approach does not induce all intact proviruses, leaving a residual reservoir. An alternative approach is to permanently repress HIV expression precluding viral rebound after ART discontinuation. Here, a nuclease-deficient disabled Cas9 (dCas9) coupled with a transcriptional repressor domain derived from Kruppel-associated box (KRAB) was used to epigenetically silence the proviral DNA. I show that specific guide RNAs (gRNAs) and dCas9-KRAB repress HIV-1 transcription and reactivation of latent HIV-1 provirus. This repression is correlated with chromatin changes, including decreased H3 histone acetylation and increased histone H3 lysine 9 trimethylation, which are histone marks that are associated with transcriptional repression. dCas9-KRAB-mediated inhibition of HIV-1 transcription suggests that CRISPR can be engineered as a tool for block-and-lock strategies. The research presented here provides evidence of opioid-mediated modulation of HIV-1 latency reactivation in PWH with opioid dependency. Additionally, we show that HIV-1 reactivation can be suppressed by epigenetic remodeling of the HIV-1 promoter using a repurposed CRISPR/Cas9 system.

Bakgrund: Att lindra smärta är en av sjuksköterskans vanligaste arbetsuppgifter. Opioider är en vanlig ångest och smärtlindring men är problematisk då det finns ett omfattande substansmissbruk världen över. Palliativ vård förekommer när sjukdomen ej går att bota och att sjukdomen kommer leda till patientens död. Det finns ett utbrett användande av opioider inom den palliativa vården. Syfte: Studien avser att ge en översikt kring sjuksköterskans erfarenheter av smärtbehandling med opioider inom palliativ vård. Metod: Allmän litteraturstudie med kvalitativ ansats med systematisk dataanalys. Studien följde Polit & Becks tiostegsmodell (2020) och analyserades med innehållsanalys enligt Graneheim & Lundman (2003). Resultat: Efter den tematiska analysen framkom teman innehållande ”Komplexiteten med palliativ vård”, ”Opioiders potential och risker”, ”Förutsättningar och kompetenser”. Subteman framkom innehållande “Svårnavigerade etiska dilemman”, “Utmaningar i livets slutskede”, “Potentiellt riskfylld administrering”, “I syfte att minska lidande”, “Vikten av lyhördhet från läkarna”, “Komplexa smärtbedömningar”, “Trygghet och kompetens genom arbetserfarenhet”. Slutsats: Mer kunskap och kompentensutveckling för att sjuksköterskan ska kunna behandla patientens behov av smärtlindring utan att känna rädsla och okunskap inför risken att administrera en felaktig dos. / Background: Relieving pain is one of the nurse's most common tasks. Opioids are a common anxiety and pain reliever but are problematic as there is extensive substance abuse worldwide. Palliative care occurs when the disease cannot be cured and the disease will lead to the patient's death. There is widespread use of opioids in palliative care. Purpose: This study aims to provide an overview of what the research says about the nurse's experiences of pain treatment with opioids in palliative care. Method: General literature study with a qualitative approach with systematic data analysis. The study followed Polit & Beck's ten-step model (2020) and qualitative thematic analysis according to Graneheim & Lundman's (2003) analysis method. Results: After the thematic analysis, themes emerged containing "The complexity of palliative care", "Opioids' potential and risks", "Prerequisites and competences". Subthemes emerged containing "Difficult to navigate ethical dilemmas", "Challenges at the end of life", "Potentially risky administration", "In order to reduce suffering", "The importance of responsiveness from doctors", "Complex pain assessments", "Safety and competence through work experience". Conclusion: More knowledge and competence development so that the nurse can treat the patient's need for pain relief without feeling fear and ignorance about the risk of administering an incorrect dose.

The purpose of this integrative literature review was to understand the effects of medical cannabis for chronic pain management in adults. Anecdotal reports suggest the use of medical marijuana as a pain management therapy could be an alternative to opioids and other medications which have long term consequences. Potential uses span the health care continuum, from prescribed outpatient symptom management, to acute care, extended care, home care, and hospice treatment settings. The methodology included a review and synthesis of relevant research articles from 2012 to 2018, written in the English language. The findings suggest medical cannabis has the potential of effectively managing chronic pain in older adults. Adverse effects, if present, are mild and resolve without intervention. Lower doses of medical cannabis were reported to be more effective in treating chronic pain compared to higher doses. Inconsistencies in the efficacy of THC were noted compared to CBD for managing neuropathic pain. Implication for nursing practice, policy, education, and recommendation for future research were discussed along with study limitations.

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<p>Catecholamine (CAT) secretion from adrenomedullary chromaffin cells (AMCs) is essential for survival of the fetus and for adaptation of the newborn to extrauterine life. CAT secretion protects the fetus from intrauterine hypoxia (low O₂) and is required for maintaining cardiac conduction and preparing the lungs for air breathing. Asphyxial stressors (e.g. hypoxia, hypercapnia (high PCO₂), and acidosis (low pH)) arising from labor contractions and postnatal apneas, are the main stimuli for the ‘non-neurogenic’ CAT release from perinatal AMCs. In the rat, the mechanisms of hypoxia chemosensitivity in AMCs involve inhibition of a variety of K⁺ channels, leading to membrane depolarization, voltage-gated Ca²⁺ entry, and CAT secretion. The magnitude of this depolarization is regulated by the simultaneous activation of ATP-sensitive K⁺ (K_ATP) channels, which tends to hyperpolarize the membrane potential during hypoxia. Interestingly, chemosensitivity of rat AMCs and CAT secretion in response to asphyxial stressors are markedly reduced postnatally following the development of functional innervation of these cells by the splanchnic nerve.</p> <p>The primary purpose of this thesis was to delineate molecular mechanisms involved in the suppression of hypoxia and hypercapnia chemosensitivity following splanchnic innervation in neonatal rat AMCs. Experiments were designed to test the general hypothesis that the ontogeny of O₂ and CO₂ sensitivity in AMCs is regulated by the activation of postsynaptic nicotinic ACh and opioid receptor signalling pathways following innervation. Previous studies in this laboratory showed that exposure of perinatal rat AMCs to nicotine <em>in utero </em>and <em>in vitro</em> resulted in the selective blunting of hypoxia (but <em>not</em> hypercapnia) chemosensitivity. The underlying mechanism was attributable to the increased membrane hyperpolarization caused by the functional upregulation of K_ATP channels. In Chapter 2, I report the results of investigations of molecular mechanisms involved in the nicotine-induced upregulation of K_ATP channels, using a rat fetal-derived, O₂- and CO₂-sensitive immortalized chromaffin cell line (MAH cells), as a model. Exposure of MAH cells to chronic nicotine (50 μM) for 7 days in culture caused an increase in the expression of the K_ATP channel subunit, Kir6.2. This effect was blocked by α-bungarotoxin, a blocker of homomeric α7 nicotinic acetylcholine receptors (α7 nAChRs). The upregulation of Kir6.2 in MAH cells was also dependent on the transcription factor, hypoxia inducible factor (HIF)-2α. First, whereas the upregulation of Kir6.2 was present in wild type and scrambled control MAH cells, it was absent in HIF-2α-deficient (shHIF-2α) MAH cells. Second, chronic nicotine caused a progressive, time-dependent increase in HIF-2α accumulation that occurred in parallel with the increase in Kir6.2 expression. Third, chromatin immunoprecipitation (ChIP) assays revealed the binding of HIF-2α to a hypoxia response element (HRE) in the promoter region of the Kir6.2 gene. These data suggest that chronic nicotine causes the accumulation of HIF-2α which results in the transcriptional upregulation of the Kir6.2 gene. These observations were validated in an <em>in vivo</em> model where rat pups were exposed to nicotine <em>in utero</em>. Western blot analysis of adrenal gland tissues from nicotine-exposed (relative to saline-exposed) pups revealed a significant increase in Kir6.2 subunit expression and HIF-2α accumulation, and both were restricted to the medullary (but not cortical) tissue.</p> <p>Chapter 3 tested the hypothesis that postnatal innervation causes the suppression of O₂- and CO₂-chemosensitivity in neonatal AMCs via opioid receptor signalling. It was found that chronic μ- and δ-opioid agonists (2 μM) <em>in vitro </em>led to the suppression of both O₂- and CO₂-chemosensitivity; this was correlated with the upregulation of K_ATP channel expression and the downregulation of carbonic anhydrase (CA) I and II respectively. The underlying molecular and signalling mechanisms were further investigated in Chapter 4. Using the MAH cell model, it was found that exposure to a combination of μ- and δ-opioid agonists for 7 days resulted in the naloxone-sensitive upregulation of Kir6.2 subunit and the downregulation of CAII. Similar to chronic nicotine exposure, the effects of chronic opioids on the upregulation of Kir6.2 and downregulation of CAII were HIF-2α-dependent. Western blot analysis revealed that HIF-2α accumulation in opioid-treated MAH cells occurred along a time-course that paralleled the upregulation of Kir6.2 subunit. ChIP assays demonstrated the binding of HIF-2α to the promoter region of the Kir6.2 subunit gene in opioid-treated MAH cells. Moreover, PKA activity (but not PKC or CaMK) was found to be required for the effects of opioids on Kir6.2 and CAII expression, but not HIF-2α accumulation. In complementary <em>in vivo</em> studies, adrenomedullary tissues from morphine-exposed rat pups showed an increased expression of both HIF-2α and Kir6.2, and decreased expression of CA1 and II protein. These findings have uncovered novel mechanisms by which postnatal innervation contributes to the ontogeny of O₂- and CO₂-chemosensitivity in rat adrenal chromaffin cells. They also suggest mechanisms by which exposure of the fetus to nicotine in cigarette smoke or opioids from drug abuse might contribute to abnormal arousal reflexes, and pathophysiological conditions such as Sudden Infant Death Syndrome (SIDS).<strong></strong></p> / Doctor of Philosophy (PhD)

Opioids have been shown to modulate immune function in a variety of assays and animal models. In a more limited number of studies, opioids have been shown to sensitize to infection. Heroin, the prototypical opioid drug of abuse, is rapidly metabolized to morphine in the body. Morphine has been used as an analgesic for hundreds of years, and continues to be a drug of choice for treating pain in ICU and trauma patients. The continued use of these opioid compounds in humans warrants further investigation of their effect on immune responses against, and progression of, common bacterial infections. Two infections were investigated in this thesis using murine models, Acinetobacter baumannii and Salmonella typhimurium. A recent increase in the prevalence of A. baumannii infections among healthy, but wounded, military personnel, lead to the hypothesis that analgesic morphine might sensitize to infection with this multiply-drug resistant bacterium. A systemic, intraperitoneal A. baumannii infection model was established in mice that resulted in rapid, disseminated disease where animals became septic as organisms replicated in the blood, lungs, and other organs. This model was used to investigate the role of various parameters of innate immune defenses to Acinetobacter. Neutralization of neutrophils by antibody depletion greatly sensitized to this infection. Infection resulted in a rapid, biphasic induction of both IL-17 and the chemokine, KC/CXCL1, a major chemotactic factor for neutrophils, that continued to rise through 18h after bacterial inoculation. However, depletion of either IL-17 or KC/CXCL1 using monoclonal antibodies failed to sensitize to Acinetobacter infection. Further, IL-17 receptor KO mice were not sensitized to this infection. Collectively, these results suggest that there must be other chemotactic factors for neutrophils that can compensate for the absence of IL-17 and KC. Morphine, delivered by extended release pellet, sensitized two strains of mice to two strains of Acinetobacter, as measured by mortality to a sublethal challenge dose, and this effect was blocked by administration of the opioid-receptor antagonist, naltrexone. . Morphine increased Acinetobacter burdens in the organs and blood of infected mice, and increased the levels of pro-inflammatory cytokines. Evidence for an effect of morphine on neutrophil infiltration was obtained. Morphine decreased the total numbers of cells, as well as the total numbers of neutrophils and macrophages infiltrating into the peritoneal cavity. This inhibition of neutrophil accumulation correlated with suppression of levels of both IL-17 and KC/CXCL1. The evidence supports the conclusion that morphine sensitizes to Acinetobacter infection by suppressing the response of neutrophils, potentially via depression of neutrophil chemotactic factors IL-17 and KC. However, taken together with the data above there are probably additional factors in addition to IL-17 and KC that are sensitizing the animals to infection in the presence of morphine. In addition to these studies, the opioid-receptor dependency of morphine-mediated sensitization to Salmonella enteric serovar Typhimurium was examined. Previous experiments had determined that extended release morphine pellets sensitized mice to a sublethal dose of Salmonella, as determined by survival and bacterial burdens in the organs of infected mice, but naltrexone resulted in only incomplete reversal of the morphine-mediated effects. To further characterize the receptor dependency of the observed phenomenon, mu-opioid receptor knockout (MORKO) mice were used. MORKO mice were found to be completely resistant to the lethal effects of morphine plus infection observed in wild-type (WT) mice. In addition, MORKO mice showed greatly reduced bacterial burdens and pro-inflammatory cytokine levels when treated with morphine and challenged with a sublethal challenge dose of Salmonella, in comparison to WT mice. In summary, the studies presented in this thesis explored basic mechanisms of innate immunity to A. baumannii using a systemic model of infection. The work provides additional evidence that morphine sensitizes to infection, using models of Acinetobacter and Salmonella in mice. An implication of this work is use of caution in the administration of opioids in patients that are susceptible to opportunistic infections. / Microbiology and Immunology

Study Background and Objectives:Excessive perioperative opioid prescribing has been detrimental to public health, contributing to the elevated prevalence of opioid use disorder. Since 2016, rigorous regulation of opioid prescribing has reduced over-prescription, but has also led to opioid-phobia. The 2022 CDC guideline promotes person-centered decisions on pain management by relaxing restrictions on opioid prescription. The determination of opioid requirements for surgical pain management is influenced by various factors and stakeholders. Despite extensive research, the mechanisms underlying perioperative pain management and the persistence of opioid use after surgery remain unclear. Clinicians currently lack tools to guide opioid prescription in clinical settings, and patients often face a dearth of information regarding expected pain levels, proper opioid use, and options for surgical pain management. The main objective of my doctoral project is to disentangle the intricate relationships among patients, healthcare providers, and policy changes in perioperative opioid prescription for pain management and to identify key intervention points to balance the beneficial effects of proper opioids use against the risks of addition. Another objective is to develop a risk score algorithm for perioperative opioid requirements to help with decision-making in clinical practice. Materials and Methods:In chapter 1, I undertook a systematic review and meta-analysis, and investigated the percentage of adult patients scheduled for general surgeries who received opioid analgesia for perioperative pain management, the quantities of opioids prescribed to patients, the actual quantities consumed, the percentage of patients without prior opioid exposure experiencing prolonged opioid use, and the evolution of perioperative opioid prescription patterns since the policy changes. A causal loop diagram was used to visualize the complex conceptual framework of perioperative pain management and post-surgical prolonged use of opioids based on insights derived from the systematic review and meta-analysis. In chapter 2 and 3, data from patients aged 18-64 years undergoing one of 12 commonly performed procedures (e.g., laparoscopic cholecystectomy) from 2015 to 2018 at a single institution were analyzed. Perioperative opioid requirements (none/low, medium, high) were determined based on patients’ self-reported pain scores and opioid prescription/administration from 30 days before to 2 weeks after surgery. Patients’ clinical and procedure-related factors were collected as potential predictors. Random forest, the Least Absolute Shrinkage and Selection Operator (LASSO), and multinomial logistic regression were used to develop prediction models. Models’ performance, including discrimination, calibration, classification measures were evaluated. A nomogram based on multinomial logistic regression was generated as a score tool, and decision curve analysis was used to examine the clinical utility of the final prediction model dichotomizing the opioid prescription as none/sparing versus medium/high requirements. Results: My systematic review and meta-analysis revealed that around 85% of surgical patients received opioids perioperatively. The pooled mean total amount of opioids dispensed was 210 MME per patient per surgical procedure. Notably, only approximately 44% of the prescribed opioids were consumed. Among opioid-naïve patients who initiated opioid use perioperatively, 7.1% persisted in opioid use beyond the conventional three-month postoperative recovery timeframe. Intervention programs (such as setting up maximum limits of opioids prescription, providing trainings to health providers, monitoring opioids prescription behaviors, providing health education to patients, etcetera) reduced perioperative opioid prescription by 38% and opioid consumption by 63.2%. The causal loop diagram illustrates a balancing feedback loop between policy and over-prescription, highlighting the pivotal role of a decision tool in reducing the over-prescription of perioperative opioids while ensuring the fulfillment of opioid needs for effective perioperative pain management. To develop a decision-aid tool based on prediction models, I included 2733 patients in the training dataset and 1081 in the testing dataset, all of whom underwent general surgeries. All prediction models demonstrated moderate discrimination in the testing dataset. The null hypothesis of perfect calibration intercepts and calibration slopes was rejected. In analyses restricted to patients undergoing laparoscopic cholecystectomy, model discrimination remained similar while model calibration improved. The revised LASSO model had an accuracy of around 65% in the testing dataset, classifying future cases correctly into opioid requirements groups in laparoscopic cholecystectomy cohort. Features in the final laparoscopic cholecystectomy model included the use of opioid/NSAID/anti-depressant before surgery, emergency surgery, anesthesia type, and surgical indication for cholelithiasis/cholecystitis. A nomogram was created to guide perioperative opioids use among laparoscopic cholecystectomy patients, and the decision curve analysis demonstrated the clinical utility of the prediction model; it generated higher net benefits than the strategy of prescribing no opioids or opioid sparing to surgical patients and the strategy of prescribing medium or high opioids doses to all patients, with a broad threshold probability from 18% to 92%. Conclusions:In summary, this dissertation described the historically high levels of perioperative opioid prescriptions and highlighted their adverse impacts: persistent opioid use and community diversion. Although the implementation of guidance and policies has significantly reduced nationwide over-prescriptions of opioids, it is essential to recognize the potential benefits of appropriate opioid use in perioperative pain management. The incorporation of a machine-learning approach with subject-matter knowledge may achieve more accurate predictions of opioid requirements than employing machine-learning techniques alone and increase the interpretability of the prediction model. Notably, the surgery-specific model demonstrated superior performance than the model for general surgeries. Future studies should further validate the conceptual model of perioperative opioid prescription and misuse in real-world scenarios, enhance model discrimination, extend external validation efforts, and develop electronic applications tailored to contemporary medical practices.