BACKGROUND:Many adults living with sickle cell disease (SCD) suffer from chronic pain, turning to cannabis for relief. However, there are few studies that examine the efficacy of cannabis in treating pain associated with SCD. Prior to an efficacy study, a feasibility study is necessary to evaluate if such a study would be acceptable to patients, including those who use cannabis; if participants would be able to abstain from other cannabinoid-containing substances during the study; if masking would be feasible; and if dronabinol would prove safe. METHODS:We approached outpatients with SCD of any genotype at healthy baseline and asked about interest in such a study. If eligible enrolled participants received dronabinol or placebo for two, 2-week periods. Feasibility outcomes were acceptability (patient interest and enrollment rates), protocol adherence (completion of stud procedures), cannabinoid avoidance, and masking effectiveness. Patient-reported outcomes (PRO), laboratory markers of inflammation, and urine tests for the presence of cannabinoids were collected after each exposure period. RESULTS:A total of 27 patients were approached; 23 (85%) were interested, 13 (48%) signed consents, and 6 (22%) were enrolled so the study was determined to be acceptable. Patients who used unregulated cannabis and medical cannabis also found the study acceptable. All enrolled participants successfully completed all study procedures. Urine testing revealed no cannabinoid use except for study drug, so the study was determined to be feasible. While 4 out of 6 (67%) participants correctly identified their exposure assignment after the first study period, all 6 (100%) identified the exposure assignment after the second treatment period, so masking after the second period was not feasible. No serious adverse events were attributed to dronabinol. CONCLUSION:In conclusion, a controlled study of dronabinol is acceptable, feasible, and safe to participants. However, a crossover design compromises participant masking. A larger, longer, controlled efficacy study without the crossover component is now being performed (NCT05519111). Trial registration NCT03978156. Date of registration: 07/26/2019.

Purpose: O6-methylguanine DNA methyltransferase (MGMT)-silenced tumors reveal sensitivity to temozolomide (TMZ), which may be enhanced by PARP inhibitors. Approximately 40% of colorectal cancer has MGMT silencing and we aimed to measure antitumoral and immunomodulatory effects from TMZ and olaparib in colorectal cancer. Experimental Design: Patients with advanced colorectal cancer were screened for MGMT promoter hypermethylation using methylation-specific PCR of archival tumor. Eligible patients received TMZ 75 mg/m2 days 1–7 with olaparib 150 mg twice daily every 21 days. Pretreatment tumor biopsies were collected for whole-exome sequencing (WES), and multiplex quantitative immunofluorescence (QIF) of MGMT protein expression and immune markers. Results: MGMT promoter hypermethylation was detected in 18/51 (35%) patients, 9 received study treatment with no objective responses, 5/9 had stable disease (SD) and 4/9 had progressive disease as best response. Three patients had clinical benefit: carcinoembryonic antigen reduction, radiographic tumor regression, and prolonged SD. MGMT expression by multiplex QIF revealed prominent tumor MGMT protein from 6/9 patients without benefit, while MGMT protein was lower in 3/9 with benefit. Moreover, benefitting patients had higher baseline CD8+ tumor-infiltrating lymphocytes. WES revealed 8/9 patients with MAP kinase variants (7 KRAS and 1 ERBB2). Flow cytometry identified peripheral expansion of effector T cells. Conclusions: Our results indicate discordance between MGMT promoter hypermethylation and MGMT protein expression. Antitumor activity seen in patients with low MGMT protein expression, supports MGMT protein as a predictor of alkylator sensitivity. Increased CD8+ TILs and peripheral activated T cells, suggest a role for immunostimulatory combinations. Significance: TMZ and PARP inhibitors synergize in vitro and in vivo in tumors with MGMT silencing. Up to 40% of colorectal cancer is MGMT promoter hypermethylated, and we investigated whether TMZ and olaparib are effective in this population. We also measured MGMT by QIF and observed efficacy only in patients with low MGMT, suggesting quantitative MGMT biomarkers more accurately predict benefit to alkylator combinations.

Supplementary Figure 2: Molecular Analysis

Supplementary Table 2: A classification of the immune cell subtypes.

Supplementary Figure 4: The change in PBMCs for patients with clinical benefit (blue) and no benefit (red).

Supplementary Table 1: Study representativeness table.

Supplementary Figure 3: Changes in MGMT expression, yH2AX and CD8+ T cells in pre-treatment and post-treatment biopsies of patients who derived clinical benefit from the trial.

Supplementary Methods 1: Clinical trial protocol.

Seasonal influenza causes mild to severe respiratory infections and significant morbidity, especially in older adults. Transcriptomic analysis in populations across multiple flu seasons has provided insights into the molecular determinants of vaccine response. Still, the metabolic changes that underlie the immune response to influenza vaccination remain poorly characterized. We performed untargeted metabolomics to analyze plasma metabolites in a cohort of younger and older subjects before and after influenza vaccination to identify vaccine-induced molecular signatures. Metabolomic and transcriptomic data were combined to define networks of gene and metabolic signatures indicative of high and low antibody response in these individuals. We observed age-related differences in metabolic baselines and signatures of antibody response to influenza vaccination and the abundance of α-linolenic and linoleic acids, sterol esters, fatty-acylcarnitines, and triacylglycerol metabolism. We identified a metabolomic signature associated with age-dependent vaccine response, finding increased tryptophan and decreased polyunsaturated fatty acids (PUFAs) in young high responders (HRs), while fatty acid synthesis and cholesteryl esters accumulated in older HRs. Integrated metabolomic and transcriptomic analysis shows that depletion of PUFAs, which are building blocks for prostaglandins and other lipid immunomodulators, in young HR subjects at Day 28 is related to a robust immune response to influenza vaccination. Increased glycerophospholipid levels were associated with an inflammatory response in older HRs to flu vaccination. This multi-omics approach uncovered age-related molecular markers associated with influenza vaccine response and provides insight into vaccine-induced metabolic responses that may help guide development of more effective influenza vaccines.

Background: Chronic red blood cell transfusions reduce acute care utilization for sickle cell disease (SCD) pain. However, little is known about whether chronic transfusions treat or prevent the development of non-crisis pain. We investigated patient-report of pain in adults with SCD receiving chronic exchange transfusions (CET) compared to adults not on CET with similar disease characteristics. Study Method and Design: Eleven participants receiving chronic exchange transfusion (CET) for at least one year were compared to 33 participants not receiving CET. Participants completed validated patient-reported outcomes regarding pain impact and quality of life at regularly scheduled visits or before CET. One year of health care utilization and opioid prescriptions were examined. Results: After 1:1 propensity matching was performed for age, genotype, WBC and neutrophil counts, patients on CET had lower Pain Impact scores (-5.1, p = 0.03) and higher Neuropathic (7.4, p < 0.001) and Nociceptive Pain Quality (3.7, p < 0.001) scores, all indicating worse pain. However, CET was associated with a reduction in annual all cause admissions (-3.1, p < 0.001), length of stay (-2.1 days, p < 0.001) and ED visits (-2.7, p < 0.001). CET was not associated with differences in opioids dispensed. Conclusions: After adjusting for disease characteristics, CET was associated with worse pain impact and neuropathic and nociceptive pain quality, lower health care utilization and with similar levels of opioids dispensed. This data suggest that CET may reduce hospitalizations for acute pain but may not adequately treat nociceptive or neuropathic pain in SCD.

Introduction:Previous studies have shown that cannabis use is common in adults with sickle cell disease (SCD), and that many patients report using cannabis to treat pain. Methods:We performed a cross-sectional study of adults with SCD and compared daily users of cannabis with others using validated patient-reported measures of pain and quality of life as well as opioid and health care utilization. Results:Daily cannabis users with SCD had worse pain episode severity scores than others (56.7 vs. 48.8,p=0.02) yet had 1.8 fewer annual admissions (p=0.01) and 1.2 fewer annual emergency room (ER) visits (p=0.01), and similar amounts of opioids dispensed to others after matching for age, gender, SCD genotype, hydroxyurea use, and pain impact scores. Conclusions:We show that people with SCD with more severe pain crisis are more likely to use daily cannabis, yet have lower rates of hospital admission and ER use as compared with others with similar disease severity and pain impact. Randomized controlled trials should be performed.

BACKGROUND:People living with sickle cell disease (SCD) are prone to red blood cell (RBC) alloimmunization. We hypothesized that subjects with alloantibodies (responders) would have differences in circulating T-follicular helper (Tfh)-like cells compared to subjects without alloantibodies (non-responders).MATERIALS AND METHODS:Peripheral blood mononuclear cells were collected from 28 subjects, including those with SCD and controls. Circulating CD4 T-cell subsets were first evaluated at baseline. CD4 T-cell subsets were also evaluated after naïve CD4 T-cells were differentiated into Tfh-like cells following in vitro culture with CD3/CD28 beads, IL-7, IL-12, and Activin A. Transfusion and alloantibody histories were extracted from the electronic medical record.RESULTS:Non-responders had a lower percentage of CD45RA negative Tmemory cells than responders or controls (p<0.05). Notably, there were no differences in circulating Tfh-like cells between any group. However, naïve CD4 T-cells from subjects with SCD were more likely to express CXCR5 after in vitro culture than cells from controls. After culture, CXCR5 expressing cells from responders were more likely to express PD1 and ICOS (16.43 %, sd. 20.23) compared to non-responders (3.69 %, s.d. 3.09) or controls (2.78 %, s.d. 2.04).DISCUSSION:The tendency for naïve CD4 T-cells from responders to differentiate into Tfh-like cells after in vitro culture may suggest these cells are prepared to assist B-cells with antibody production regardless of antigen specificity. Further studies are needed, but it is possible that these results may explain why some responders form RBC alloantibodies with multiple specificities, in addition to RBC autoantibodies and HLA alloantibodies.

Type 1 CD8α+ conventional dendritic cells (cDC1s) are required for CD8+ T cell priming but, paradoxically, promote splenic Listeria monocytogenes infection. Using mice with impaired cDC2 function, we ruled out a role for cDC2s in this process and instead discovered an interleukin-10 (IL-10)-dependent cellular crosstalk in the marginal zone (MZ) that promoted bacterial infection. Mice lacking the guanine nucleotide exchange factor DOCK8 or CD19 lost IL-10-producing MZ B cells and were resistant to Listeria. IL-10 increased intracellular Listeria in cDC1s indirectly by reducing inducible nitric oxide synthase expression early after infection and increasing intracellular Listeria in MZ metallophilic macrophages (MMMs). These MMMs trans-infected cDC1s, which, in turn, transported Listeria into the white pulp to prime CD8+ T cells. However, this also facilitated bacterial expansion. Therefore, IL-10-mediated crosstalk between B cells, macrophages, and cDC1s in the MZ promotes both Listeria infection and CD8+ T cell activation.

Background: Acute alcohol consumption triggers release of cytokines, which are immune signaling molecules. Dysregulated cytokine levels are associated with impaired immune function, and peripheral cytokine levels may communicate with the brain to propagate drinking-related behaviors. This exploratory study aims to characterize the peripheral cytokine response to an alcohol challenge in a well-controlled laboratory setting. Methods: Moderate alcohol drinkers (n = 17), abstinent for >5 days, consumed alcohol calibrated to achieve blood concentrations of 120 mg/dL. Serum cytokine levels (IL-6, IL-8, IL-12, IFN-gamma, TNF-alpha) were measured prior to drinking, 6 h after drinking, and 24 h after drinking. Linear mixed models evaluated within-subject differences in cytokine levels over time. Results: The pro-inflammatory chemokine IL-8 significantly increased 6 h after alcohol [F(1,34) = 4.13, p = 0.0002, d' = 0.5]. In contrast, the pro-inflammatory cytokine TNF-alpha significantly decreased 6 h after alcohol [F(1,34) = -3.07, p = 0.004, d' = 0.3]. No cytokines were significantly different from baseline 24 h after alcohol. Conclusions: In our exploratory data, acute alcohol challenge (120 mg/dL) elicits dynamic changes in the pro-inflammatory molecules IL-8 and TNF-alpha. The findings help inform the temporal profile of cytokine response to alcohol, and identify IL-8 as a cytokine of interest for future studies of periphery-brain immune communication. (C) 2019 Elsevier Inc. All rights reserved.

BACKGROUNDDespite the clinical significance of red blood cell (RBC) alloantibodies, there are currently no laboratory tests available to predict which patients may be at risk of antibody formation after transfusion exposure. Given their phagocytic and inflammatory functions, we hypothesized that differences in circulating monocytes may play a role in alloimmunization.STUDY DESIGN AND METHODSForty‐two adults with sickle cell disease (SCD) were recruited, with data extracted from the electronic medical record and peripheral blood analyzed by flow cytometry for total monocytes, monocyte subsets (CD14 high/CD16 low+ classical monocytes, CD14 high/CD16 high+ intermediate monocytes, and CD14 intermediate/CD16 high+ non‐classical/inflammatory monocytes), and FcγR1 (CD64) expression. Thirteen “non‐responder” patients (non‐alloimmunized patients with documented RBC transfusion at the study institution) were compared to 20 alloimmunized “responder” patients, who had a total of 44 RBC alloantibodies identified.RESULTSThere were no significant differences in the percentages of total monocytes, monocyte subsets, or measured cytokines between non‐responders and responders. However, non‐responders had higher CD64 expression on classical monocytes (MFI mean 3424 ± standard deviation 1141) compared to responders (MFI mean 2285 ± 1501), p = 0.029, and on intermediate monocytes (MFI mean 3720 ± 1191) compared to responders (MFI mean 2497 ± 1640), p = 0.033.CONCLUSIONSMonocytes and the inflammatory milieu increasingly are being appreciated to play a role in some complications of SCD. The differences in FcγR1 expression on monocyte subsets noted between responders and non‐responders, which cannot be directly explained by the serum cytokines evaluated, warrant further investigation.

Rationale Alcohol has both acute and chronic effects on neuroimmune signaling, including triggering pro-inflammatory cytokine release by microglia. Minocycline, a second-generation tetracycline antibiotic, inhibits microglial activation and reduces neuroinflammation in preclinical studies. In mice, minocycline also reduces ethanol intake, attenuates ethanol-induced conditioned place preference, and inhibits ethanol-induced microglial activation and pro-inflammatory cytokine release. Objective Here, for the first time, we tested the effects of minocycline on subjective response to ethanol and acute ethanol-induced inflammation in humans. Methods Forty-eight heavy drinkers participated in a double-blind, placebo-controlled trial in which they were randomized to receive placebo, 100 mg, or 200 mg of minocycline for 10 days. Each subject then underwent two experimental sessions in which they were given a fixed dose of intravenous ethanol using a "clamp" procedure (100 mg%) or placebo (normal saline) on days 8 and 10 of treatment. Results Minocycline was well tolerated, but there was no effect of either dose of minocycline on subjective response to ethanol or ethanol-induced craving; minocycline effects on cognitive function seem to interact with age. Minocycline treatment did not alter serum cytokine levels at baseline or during ethanol-exposure, although certain baseline cytokine levels predict sedative response to ethanol. Conclusion These findings indicate that a short-term treatment with minocycline may not alter ethanol-related inflammation or subjective response to ethanol in humans. Further research is needed to identify pharmacological agents with robust effects on ethanol-induced inflammation to determine whether neuroimmune modulation represents a viable treatment strategy for alcohol use disorder.

Background: Ischemic stroke is a cause of morbidity and mortality in children and adults with sickle cell disease (SCD). Recent studies showed that non-classical monocytes may be protective against vaso-occlusive crisis. Monocyte subsets have been examined in non-SCD stroke, but little is known about the role of monocyte or their subsets in ischemic stroke in SCD. CD64 (FCγRI), a high affinity receptor for IgG, is a marker of endothelial adherence on immature neutrophils in SCD, however it is more classically exhibited on activated pro-inflammatory monocytes. We hypothesized that higher levels of CD64 mean fluorescence index (MFI) on monocyte subsets may be associated with ischemic stroke due to increased adherence.

Introduction: Chronic red blood cell exchange transfusion (CET) therapy is currently used to prevent neurologic and other complications of sickle cell disease (SCD) in children and adults. Despite past pediatric studies showing that CET decreases stroke risk and hospital admissions, few studies have evaluated whether CET decreases hospital utilization or pain in adults with SCD. Data support health care utilization is a poor marker of pain frequency so other ways of assessing the impact of therapy on pain outcomes is necessary and important. We noted that despite receiving CET, our adult patients experienced daily pain requiring home opioid use and intermittent hospitalization for pain crisis. Thus, we conducted a study to examine the effects of CET on the pain experience. Our primary hypothesis was that adults on CET will have similar levels of pain as defined by Adult Sickle Cell Quality of Life Measurement Information System (ASCQ-Me) pain domains as compared to adults not on CET. Our secondary hypothesis was that adults on CET will have similar levels of hospital utilization, opioid use, QOL measures of domains noted to be affected by SCD or inflammatory markers shown to be correlated with pain and SCD pain crisis.