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In JoVE (1)

Other Publications (19)

Articles by Andrew C. Harris in JoVE

 JoVE Medicine

High Throughput Sequential ELISA for Validation of Biomarkers of Acute Graft-Versus-Host Disease

1Pediatric Blood and Marrow Transplant Program, University of Michigan

JoVE 4247

High throughput validation of multiple candidate biomarkers can be performed by sequential ELISA in order to minimize freeze/thaw cycles and use of precious plasma samples. Here, we demonstrate how to sequentially perform ELISAs for six different validated plasma biomarkers1-3 of graft-versus-host disease (GVHD)4 on the same plasma sample.

Other articles by Andrew C. Harris on PubMed

Elevated Startle During Withdrawal from Acute Morphine: a Model of Opiate Withdrawal and Anxiety

An elevated startle response has been observed in humans and animals during withdrawal from multiple substances of abuse, a phenomenon thought to reflect the anxiogenic effects of withdrawal. Although anxiety is a common symptom of opiate withdrawal, few studies have examined the effects of morphine withdrawal on acoustic startle.

Potentiated Startle and Hyperalgesia During Withdrawal from Acute Morphine: Effects of Multiple Opiate Exposures

Administration of an opiate antagonist following acute morphine exposure elevates the startle response in rodents, a phenomenon that may reflect the anxiogenic effects of withdrawal. Previous acute dependence studies have demonstrated escalated withdrawal severity following multiple withdrawal episodes.

HPA Axis Dysregulation Following Prenatal Opiate Exposure and Postnatal Withdrawal

We examined the effects of prenatal exposure to the long acting opiate l-alpha-acetylmethadol (LAAM) followed by postnatal withdrawal on hypothalamic-pituitary-adrenal (HPA) axis reactivity in neonatal and adult rats and anxiety-like behavior in adult rats. Female rats were treated with LAAM (0, 0.2, or 1.0 mg/kg/day) via oral gavage for 28 days prior to and continuing throughout pregnancy. Pups were fostered at birth to nontreated, lactating dams and underwent opiate withdrawal. On postnatal day (PND) 18, prenatal opiate-exposed male and female rat pups displayed a decreased corticosterone response 2 h after the application of an immunological stressor and 15 min following a social stressor compared to controls. In contrast, in adulthood, prenatal opiate-treated rats showed a heightened corticosterone response compared to prenatal water-treated controls at 3 h, but not 8 h, following an immunological stressor. Males prenatally treated with 1.0 mg/kg LAAM displayed elevated startle responding compared to the other prenatally treated male groups, but there was no effect of prenatal treatment in females. There were no effects of prenatal treatment in the open field test in either sex. These results suggest that prenatal opiate exposure followed by postnatal withdrawal dysregulated the HPA axis response to stressors in the neonate and adult and differentially affected adult anxiety-like behavior in males and females.

Acute Opioid Dependence: Characterizing the Early Adaptations Underlying Drug Withdrawal

While opioid withdrawal is typically studied under conditions of chronic (i.e., continuous) drug administration, withdrawal signs can also be demonstrated in both humans and animals after a single opioid exposure. This phenomenon, termed acute dependence, may be useful in understanding the early stages of opioid dependence and addiction.

Prevalence, Magnitude, and Correlates of an Extinction Burst in Drug-seeking Behavior in Rats Trained to Self-administer Nicotine During Unlimited Access (23 H/day) Sessions

Animals trained to self-administer a variety of addictive drugs exhibit a temporary increase in response rate when saline is substituted for the drug (i.e., an "extinction burst"). However, the presence of an extinction burst in animal models of nicotine self-administration (NSA) has not been studied extensively.

Effects of the NMDA Receptor Antagonist Memantine on the Expression and Development of Acute Opiate Dependence As Assessed by Withdrawal-potentiated Startle and Hyperalgesia

While the N-methyl-D: -aspartate (NMDA) glutamate receptor has been strongly implicated in chronic opiate dependence, relatively few studies have examined the effects of NMDA receptor antagonists on withdrawal from acute opiate exposure.

Compensatory Nicotine Self-administration in Rats During Reduced Access to Nicotine: an Animal Model of Smoking Reduction

The ability of smoking reduction (e.g., decreasing cigarettes per day) to produce significant reductions in toxin exposure is limited by compensatory increases in smoking behavior. Characterizing factors contributing to the marked individual variability in compensation may be useful for understanding this phenomenon. The goal of the current study was to develop an animal model of smoking reduction and to begin to examine potential behavioral and pharmacokinetic contributors to compensation. Rats trained for nicotine self-administration (NSA) in unlimited access sessions were exposed to a progressive decrease in duration of access to nicotine from 23-hr/day to 10-, 6-, and 2-hr/day. Following a return to 23 hr/day access and extinction, single-dose nicotine pharmacokinetic parameters were determined. Rats exhibited a reduction in total daily nicotine intake during reduced access to NSA, but decreases in nicotine intake were not proportional to decreases in access duration. Compensatory increases in hourly infusion rate were also observed when access was decreased. The magnitude of compensation differed considerably among animals. Early session infusion rate during baseline was significantly correlated, while nicotine clearance was moderately correlated, with 1 measure of compensation. Infusion rates were transiently increased compared to prereduction levels when unlimited access was restored, and this effect was greatest in animals that had exhibited the greatest levels of compensation. These findings indicate that rats exhibit compensatory increases in NSA during reduced access to nicotine, with substantial individual variability. This model may be useful for characterizing underlying factors and potential consequences of compensatory smoking.

Combined Active and Passive Immunization Enhances the Efficacy of Immunotherapy Against Nicotine in Rats

Vaccination against nicotine reduces the behavioral effects of nicotine in rats, and it is under clinical evaluation as a treatment for tobacco addiction. Efficacy is limited by the need for high serum nicotine-specific antibody (NicAb) levels, and currently available nicotine vaccines do not uniformly generate the required NicAb levels. Passive immunization with a nicotine-specific monoclonal antibody (Nic311) has also shown efficacy in rats. The principal aim of this study was to determine whether the combined use of vaccination and passive immunization would produce greater effects than vaccination alone on nicotine pharmacokinetics and locomotor sensitization (LMS) to nicotine. Rats were treated with vaccination alone, Nic311 alone, both, or neither, and then they were administered 10 daily injections of 0.3 mg/kg nicotine s.c. Treatment with Nic311 or vaccination alone increased the binding of nicotine in serum, reduced the unbound serum nicotine concentration and nicotine distribution to brain, and attenuated the development of LMS. Combined use of vaccination and passive immunization produced higher total serum NicAb levels, greater changes in nicotine pharmacokinetics, and a greater attenuation of LMS than either treatment alone. The total serum NicAb concentration was significantly correlated with brain nicotine levels and locomotor activity. These data indicate that providing higher serum NicAb concentrations improves the efficacy of immunotherapy against nicotine and that supplementing vaccination with passive immunization is a potential strategy to accomplish this.

Passive Immunization with a Nicotine-specific Monoclonal Antibody Decreases Brain Nicotine Levels but Does Not Precipitate Withdrawal in Nicotine-dependent Rats

Vaccination against nicotine is under investigation as a treatment for tobacco dependence. Passive immunization with nicotine-specific antibodies represents a complementary strategy to vaccination. A potential adverse effect of passive immunization in nicotine-dependent individuals is that it may lead to a rapid reduction in brain nicotine levels and trigger withdrawal. The goal of this study was to determine if passive immunization with the nicotine-specific monoclonal antibody Nic311 precipitated withdrawal in nicotine-dependent rats as measured by increases in brain reward thresholds and somatic signs. Another cohort of rats was used to measure brain nicotine levels after Nic311 administration. Nic311 30, 80 or 240 mg/kg reduced brain nicotine concentrations by 45, 83 or 92% compared to controls. None of these Nic311 doses precipitated withdrawal measured at intervals up to 72 h following antibody administration. Administration of the nicotinic antagonist mecamylamine precipitated a robust nicotine withdrawal syndrome. Therefore, a substantial, but not complete, acute reduction in brain nicotine levels following passive immunization was not sufficient to precipitate nicotine withdrawal in nicotine-dependent rats. The Nic311 doses used have been shown to attenuate the behavioral effects of nicotine, suggesting that the use of passive immunization to treat nicotine addiction is not likely to precipitate withdrawal.

Correlates of Individual Differences in Compensatory Nicotine Self-administration in Rats Following a Decrease in Nicotine Unit Dose

The ability of tobacco harm reduction strategies to produce significant reductions in toxin exposure is limited by compensatory increases in smoking behavior. Characterizing factors contributing to the marked individual variability in compensation may be useful for understanding this phenomenon and assessing the feasibility of harm reduction interventions.

Comparison of the Behavioral Effects of Cigarette Smoke and Pure Nicotine in Rats

Animal models of tobacco dependence typically rely on parenteral administration of pure nicotine. Models using cigarette smoke inhalation might more accurately simulate nicotine exposure in smokers. The primary goal of this study was to validate methods for administering cigarette smoke to rats using exposure conditions that were clinically relevant and also produced brain nicotine levels similar to those produced by behaviorally active doses of pure nicotine. A secondary goal was to begin examining the behavioral effects of smoke. Nose-only exposure (NOE) to smoke for 10-45min or whole-body exposure (WBE) to smoke for 1-4h produced serum nicotine concentrations similar to those in smokers (14-55ng/ml), without excessive carbon monoxide exposure. Daily nicotine (0.1mg/kg, s.c.) induced locomotor sensitization whereas 45-min NOE producing brain nicotine levels within the same range did not. Nicotine 0.125mg/kg s.c. reversed withdrawal from a chronic nicotine infusion as measured by elevations in intracranial self-stimulation thresholds whereas 4-h WBE producing similar brain nicotine levels did not. These data demonstrate the feasibility of delivering cigarette smoke to rats at clinically relevant doses, and provide preliminary evidence that the behavioral effects of nicotine delivered in smoke may differ from those of pure nicotine.

A Lack of Association Between Severity of Nicotine Withdrawal and Individual Differences in Compensatory Nicotine Self-administration in Rats

Compensatory smoking may represent an adverse consequence of smoking reduction or the use of reduced-nicotine tobacco products. Factors contributing to individual variability in compensation are poorly understood.

Combined Active and Passive Immunization Against Nicotine: Minimizing Monoclonal Antibody Requirements Using a Target Antibody Concentration Strategy

Nicotine vaccines have shown preliminary evidence of efficacy for enhancing smoking cessation rates, but the serum nicotine-specific antibody (NicAb) concentrations produced are highly variable and many subjects do not develop effective levels. As an alternative to vaccination, passive immunization with nicotine-specific monoclonal antibodies could produce more uniform serum NicAb concentrations, but its use is limited by their high cost and shorter elimination half-life. This study investigated supplementing vaccination with monoclonal antibodies in a targeted fashion to increase vaccine efficacy while minimizing the required monoclonal antibody dose. Rats were vaccinated and then given individualized supplemental doses of the nicotine-specific monoclonal antibody Nic311 to achieve a target total serum NicAb concentration known to be effective for blocking locomotor sensitization (LMS) to nicotine. Rats received vaccine, Nic311, both, or neither, followed by 0.3 mg/kg nicotine s.c. for 10 days to produce LMS. Combination immunotherapy completely blocked the development of LMS, while monotherapy with vaccine or Nic311 alone was only minimally effective. Lower brain nicotine levels were associated with reduced locomotor activity averaged over days 7-10. Despite its greater efficacy, combination immunotherapy did not reduce the variability in the resulting total serum NicAb concentrations. Variability in total serum NicAb concentrations was contributed to by both vaccine-generated antibody and by Nic311. These data show that combination immunotherapy, using a Nic311 dose that is by itself only minimally effective, can substantially enhance nicotine vaccine efficacy. However, variability in serum NicAb levels with combination immunotherapy may make translation of this approach challenging.

Regenerating Islet-derived 3-alpha is a Biomarker of Gastrointestinal Graft-versus-host Disease

There are no plasma biomarkers specific for GVHD of the gastrointestinal (GI) tract, the GVHD target organ most associated with nonrelapse mortality (NRM) following hematopoietic cell transplantation (HCT). Using an unbiased, large-scale, quantitative proteomic discovery approach to identify candidate biomarkers that were increased in plasma from HCT patients with GI GVHD, 74 proteins were increased at least 2-fold; 5 were of GI origin. We validated the lead candidate, REG3α, by ELISA in samples from 1014 HCT patients from 3 transplantation centers. Plasma REG3α concentrations were 3-fold higher in patients at GI GVHD onset than in all other patients and correlated most closely with lower GI GVHD. REG3α concentrations at GVHD onset predicted response to therapy at 4 weeks, 1-year NRM, and 1-year survival (P ≤ .001). In a multivariate analysis, advanced clinical stage, severe histologic damage, and high REG3α concentrations at GVHD diagnosis independently predicted 1-year NRM, which progressively increased with higher numbers of onset risk factors present: 25% for patients with 0 risk factors to 86% with 3 risk factors present (P < .001). REG3α is a plasma biomarker of GI GVHD that can be combined with clinical stage and histologic grade to improve risk stratification of patients.

Delivery of Nicotine in an Extract of a Smokeless Tobacco Product Reduces Its Reinforcement-attenuating and Discriminative Stimulus Effects in Rats

Animal models of tobacco addiction rely on administration of nicotine alone or nicotine combined with isolated constituents. Models using tobacco extracts derived from tobacco products and containing a range of tobacco constituents might more accurately simulate tobacco exposure in humans.

Soluble Tumor Necrosis Factor Receptor: Enbrel (etanercept) for Subacute Pulmonary Dysfunction Following Allogeneic Stem Cell Transplantation

Subacute lung disease, manifested as either obstructive (OLD) or restrictive (RLD) lung dysfunction, is a common complication following allogeneic stem cell transplantation. In each case, therapeutic options are limited, morbidity remains high, and long-term survival is poor. Between 2001 and 2008, 34 patients with noninfectious, obstructive (25) or RLD restrictive lung dysfunction (nine) received etanercept (Enbrel®, Amgen Inc.) 0.4 mg/kg/dose, subcutaneously, twice weekly, for 4 (group A) or 12 weeks (group B). Corticosteroids (if present at study entry) were kept constant for the initial 4 weeks of therapy and then tapered as tolerated. Thirty-one of 34 (91%) subjects were evaluable for response, and 10 (32%) met primary response criteria. There was no difference in response based on the duration of treatment (29% group A versus 35% group B; P = .99), the presence of RLD or OLD (33% versus 32%; P = .73), or the severity of pulmonary disease at study onset. Estimated 5-year overall survival rates following therapy were 61% (95% confidence interval, 46%-80%) for all subjects and 90% (95% confidence level, 73%-100%) for the 10 who met the primary response criteria. Five-year survival estimates for subjects treated with RLD was 44%, compared with 67% for those treated for OLD (P = .19). Etanercept was well tolerated, with no bacteremia or viremia observed. Pathogens were noted on posttherapy bronchoalveolar lavage in two cases. These data support the development of expanded clinical trials to study etanercept as a therapeutic agent for subacute lung injury after allogeneic stem cell transplantation.

Plasma Biomarkers of Lower Gastrointestinal and Liver Acute GVHD

The lower gastrointestinal tract (LGI) and liver are the GVHD target organs most associated with treatment failure and nonrelapse mortality. We recently identified regenerating islet-derived 3-α (REG3α) as a plasma biomarker of LGI GVHD. We compared REG3α with 2 previously reported GI and liver GVHD diagnostic biomarkers, hepatocyte growth factor (HGF) and cytokeratin fragment 18, in 954 hematopoietic cell transplantation patients. All 3 biomarkers were significantly elevated in LGI GVHD compared with non-GVHD diarrhea; REG3α discerned LGI GVHD from non-GVHD diarrhea better than HGF and cytokeratin fragment 18. Although all 3 biomarkers predicted nonresponse to therapy at day 28 in LGI GVHD patients, only REG3α and HGF concentrations predicted 1-year nonrelapse mortality (P = .01 and P = .02, respectively). Liver GVHD without GI involvement at GVHD onset and non-GVHD liver complications were uncommon; all 3 biomarkers were elevated in liver GVHD, but did not distinguish GVHD from other causes of hyperbilirubinemia.

Mecamylamine Elicits Withdrawal-like Signs in Rats Following a Single Dose of Nicotine

RATIONALE: The ability of nicotine to induce dependence (result in a withdrawal syndrome) is typically thought to require long-term, daily smoking. Emerging evidence suggests that symptoms of nicotine withdrawal may occur following only a few cigarettes. Whether acute exposure to nicotine can induce dependence in animals has not been well established. OBJECTIVE: The objective of this paper is to examine whether the nicotinic acetylcholine receptor antagonist mecamylamine elicits withdrawal-like signs in rats following acute nicotine exposure. METHODS AND RESULTS: Mecamylamine (3.0 mg/kg, s.c.) administered ≈2 h after a single dose of nicotine (0.5 mg/kg, s.c.) elicited increases in intracranial self-stimulation (ICSS) thresholds and somatic signs, two well-established effects of withdrawal from long-term (chronic) nicotine exposure. The magnitude of these effects remained constant across five daily test sessions. A lower dose of mecamylamine (1.5 mg/kg, s.c.) had little or no effect on ICSS thresholds or somatic signs following acute nicotine exposure, but precipitated robust increases in these measures during a chronic nicotine infusion. Finally, rats exhibited a small increase in ICSS thresholds over time following a single nicotine injection (0.5 mg/kg, s.c.), possibly reflecting a modest spontaneous withdrawal-like effect. CONCLUSIONS: Mecamylamine elicited withdrawal-like signs in rats following a single dose of nicotine. The different effects of mecamylamine 1.5 mg/kg following acute versus chronic nicotine exposure supports the notion that these models simulate the early and more advanced stages of nicotine dependence, respectively. While further optimization and validation of these models is necessary, they may provide a novel approach for studying the earliest stages of nicotine dependence.

Extramedullary Relapse of Acute Myeloid Leukemia Following Allogeneic Hematopoietic Stem Cell Transplantation: Incidence, Risk Factors and Outcomes

Background: Extramedullary relapse after allogeneic hematopoietic stem cell transplantation for acute myeloid leukemia is a contributor to post-transplant mortality but risk factors for, and outcomes, are not well characterized. Design and methods: We analyzed 257 consecutive patients receiving allogeneic stem cell transplantation for acute myeloid leukemia at our institution to characterize extramedullary relapse, identify predictive variables and assess outcomes. Results: The 5-year cumulative incidence of isolated extramedullary or marrow relapse was 9% and 29%, respectively. Extramedullary relapse occurred later than marrow relapse and most frequently involved skin and soft tissue. Factors predictive of extramedullary relapse post-transplant included previous extramedullary disease, French-American-British classification M4/M5 leukemia, high risk cytogenetics, and advanced disease status at time of transplant. Children were more likely to develop extramedullary relapse than adults, a finding probably explained by an overrepresentation of extramedullary disease prior to transplant and M4/M5 leukemia in children. Acute graft-versus-host disease was not protective against relapse. Unlike medullary relapse, chronic graft-versus-host disease was not protective against extramedullary relapse. Survival after extramedullary relapse was 30% at 1-yr and 12% at 2-yrs. Conclusions: Extramedullary relapse is a significant contributor to mortality after allogeneic transplantation for acute myeloid leukemia that appears to be resistant to the immunotherapeutic effect of allo-transplantation. Effective strategies for these patients are needed to improve outcomes after transplant.

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