Antibodies to Helminth Defense Molecule-1 are associated with inflammation, organomegaly, and decreased nutritional status in schistosomiasis japonica.

Immunomodulation enhances parasite fitness by reducing inflammation-induced morbidity in the mammalian host, as well as by attenuating parasite-targeting immune responses. Using a whole proteome differential screening method, we identified Schistosoma japonicum Helminth Defense Molecule (SjHDM-1) as a target of antibodies expressed by S. japonicum resistant, but not susceptible, individuals. In a longitudinal cohort study (N=644) conducted in a S. japonicum endemic region of the Philippines, antibody levels to SjHDM-1 did not predict resistance to reinfection but were associated with increased measures of inflammation. Individuals with high levels of anti-SjHDM-1 IgG had higher levels of C-reactive protein compared to individuals with low anti-SjHDM-1. High anti-SjHDM-1 IgG responses were also associated with reduced biomarkers of nutritional status (albumin), as well as decreased anthropometric measures of nutritional status (WAZ and HAZ) and increased measures of hepatomegaly. Our results suggest that anti-SjHDM-1 responses inhibit the immunomodulatory function of SjHDM-1, resulting in increased morbidity.

Immunization with PfGBP130 generates antibodies that inhibit RBC invasion by P. falciparum parasites.

Background: Despite decades of effort, Plasmodium falciparum malaria remains a leading killer of children. The absence of a highly effective vaccine and the emergence of parasites resistant to both diagnosis as well as treatment hamper effective public health interventions. Methods and results: To discover new vaccine candidates, we used our whole proteome differential screening method and identified PfGBP130 as a parasite protein uniquely recognized by antibodies from children who had developed resistance to P. falciparum infection but not from those who remained susceptible. We formulated PfGBP130 as lipid encapsulated mRNA, DNA plasmid, and recombinant protein-based immunogens and evaluated the efficacy of murine polyclonal anti-PfGBP130 antisera to inhibit parasite growth in vitro. Immunization of mice with PfGBP130-A (aa 111-374), the region identified in our differential screen, formulated as a DNA plasmid or lipid encapsulated mRNA, but not as a recombinant protein, induced antibodies that inhibited RBC invasion in vitro. mRNA encoding the full ectodomain of PfGBP130 (aa 89-824) also generated parasite growth-inhibitory antibodies. Conclusion: We are currently advancing PfGBP130-A formulated as a lipid-encapsulated mRNA for efficacy evaluation in non-human primates.

Evaluation of sensitivity and specificity of Kato-Katz and circulating cathodic antigen in terms of Schistosoma japonicum using latent class analysis.

Schistosoma japonicum is endemic in the Philippines. The Kato-Katz (KK) method was used to diagnose S. japonicum. This is impractical, particularly when the sample size is limited. Knowledge on point-of-care circulating cathodic antigen (CCA) test performance for S. japonicum is limited. Determining the sensitivity and specificity of new diagnostics is difficult when the gold standard test is less effective or absent. Latent class analysis (LCA) can address some limitations. A total of 484 children and 572 adults from the Philippines were screened for S. japonicum. We performed Bayesian LCA to estimate the infection prevalence, sensitivity and specificity of each test by stratifying them into two age groups. Observed prevalence assessed by KK was 50.2% and 31.8%, and by CCA was 89.9% and 66.8%, respectively. Using Bayesian LCA, among children, the sensitivity and specificity of CCA were 94.8% (88.7-99.4) and 21.5% (10.5-36.1) while those of KK were 66.0% (54.2-83.3) and 78.1% (61.1-91.3). Among adults, the sensitivity and specificity of CCA were 86.4% (76.6-96.9) and 62.8% (49.1-81.1) while those of KK were 43.6% (35.1-53.9) and 85.5% (75.8-94.6). Overall, CCA was more sensitive than KK, regardless of the age group at diagnosis, as KK was more specific. KK and CCA have different diagnostic performance, which should inform their use in the planning and implementation of S. japonicum control programs.

T follicular helper cell profiles differ by malaria antigen and for children compared to adults.

Background: Circulating T-follicular helper (cT FH ) cells have the potential to provide an additional correlate of protection against Plasmodium falciparum ( Pf) as they are essential to promote B cell production of long-lasting antibodies. Assessing the specificity of cT FH subsets to individual malaria antigens is vital to understanding the variation observed in antibody responses and identifying promising malaria vaccine candidates. Methods: Using spectral flow cytometry and unbiased clustering analysis we assessed antigen-specific cT FH cell recall responses in vitro to malaria vaccine candidates Pf SEA-1A and Pf GARP within a cross-section of children and adults living in a malaria holoendemic region of western Kenya. Findings: In children, a broad array of cT FH subsets (defined by cytokine and transcription factor expression) were reactive to both malaria antigens, Pf SEA-1A and Pf GARP, while adults had a narrow profile centering on cT FH 17- and cT FH 1/17-like subsets following stimulation with Pf GARP only. Interpretation: Because T FH 17 cells are involved in the maintenance of memory antibody responses within the context of parasitic infections, our results suggest that Pf GARP might generate longer lived antibody responses compared to Pf SEA-1A. These findings have intriguing implications for evaluating malaria vaccine candidates as they highlight the importance of including cT FH profiles when assessing interdependent correlates of protective immunity.

Higher platelet counts and platelet factors are associated with a reduction in Plasmodium falciparum parasite density in young Malian children.

OBJECTIVES: The association between thrombocytopenia and parasite density or disease severity is described in numerous studies. In recent years, several studies described the protective role of platelets in directly killing Plasmodium parasites, mediated by platelet factor 4 (PF4) binding to Duffy antigen. This study aimed to evaluate the protective role of platelets in young children who are Duffy antigen-negative, such as those in sub-Saharan Africa. METHODS: A zero-inflated negative binomial model was used to relate platelet count and parasite density data collected in a longitudinal birth cohort. Platelet factors were measured by enzyme-linked immunosorbent assay in samples collected from malaria-infected children who participated in a cross-sectional study. RESULTS: We described that an increase of 10,000 platelets/µl was associated with a 2.76% reduction in parasite count. Increasing levels of PF4 and CXCL7 levels were also significantly associated with a reduction in parasite count. CONCLUSIONS: Platelets play a protective role in reducing parasite burden in Duffy-negative children, possibly mediated through activation of the innate immune system.

Single cell transcriptional changes across the blood stages of artemisinin resistant K13C580Y mutant Plasmodium falciparum upon dihydroartemisinin exposure.

Artemisinins have been a cornerstone of malaria control, but resistance in Plasmodium falciparum, due to mutations in the Kelch 13 gene, threaten these advances. Artemisinin exposure results in a dynamic transcriptional response across multiple pathways, but most work has focused on ring stages and ex vivo transcriptional analysis, limiting evaluation of all life cycle stages. We applied single cell RNAseq to two unsynchronized isogenic parasite lines (K13C580 and K13580Y) over 6 hrs after a pulse exposure to dihydroartemisinin (DHA). Transcription was altered across all stages, with the greatest occurring at the early trophozoite and mid ring stage in both lines. This response involved the arrest of metabolic processes and the enhancement of protein trafficking and the unfolded protein response. While similar, the response was enhanced in the K13580Y mutant, which may lead to the dormancy phenomenon upon treatment. Increased surface protein expression was seen in mutant parasites at baseline and upon drug exposure, highlighted by the increased expression of PfEMP1 and GARP, a potential therapeutic target. Antibody targeting GARP maintained anti-parasitic efficacy in mutant parasites. This work provides single cell insight of gene transcription across all life cycle stages revealing transcriptional changes that could initiate dormancy state and mediate survival.

Natural History of Malaria Infections During Early Childhood in Twins.

BACKGROUND: The frequency and clinical presentation of malaria infections show marked heterogeneity in epidemiological studies. However, deeper understanding of this variability is hampered by the difficulty in quantifying all relevant factors. Here, we report the history of malaria infections in twins, who are exposed to the same in utero milieu, share genetic factors, and are similarly exposed to vectors. METHODS: Data were obtained from a Malian longitudinal birth cohort. Samples from 25 twin pairs were examined for malaria infection and antibody responses. Bayesian models were developed for the number of infections during follow-up. RESULTS: In 16 of 25 pairs, both children were infected and often developed symptoms. In 8 of 25 pairs, only 1 twin was infected, but usually only once or twice. Statistical models suggest that this pattern is not inconsistent with twin siblings having the same underlying infection rate. In a pair with discordant hemoglobin genotype, parasite densities were consistently lower in the child with hemoglobin AS, but antibody levels were similar. CONCLUSIONS: By using a novel design, we describe residual variation in malaria phenotypes in naturally matched children and confirm the important role of environmental factors, as suggested by the between-twin pair heterogeneity in malaria history.

Protocol for Differential Biopanning of P. falciparum Phage Display cDNA Library to Identify Parasite Targets of Protective Antibodies.

Malaria remains a significant global health burden, killing hundreds of thousands of children annually (WHO, The world malaria report. WHO, Geneva, 2019). Despite decades of effort, no broadly effective vaccine exists. Differential screening of parasite phage display libraries is a promising approach to identify the targets of human antibodies expressed by resistant but not by susceptible individuals (Raj et al., Nature, 582, 104-108, 2020; Science, 344, 871-877, 2014). Our whole proteome differential screening (WPDS) approach consists of positive selection to capture phage that bind antibodies expressed by malaria-resistant individuals, followed by negative selection to remove phage that bind antibodies expressed by malaria-susceptible individuals, and amplification of differentially recognized clones.

Assessing PfGARP-Mediated Apoptosis of Blood-Stage Plasmodium falciparum Parasites.

Apoptosis is conventionally regarded as an evolutionarily conserved and genetically controlled process of programmed cell death confined to metazoan organisms. However, recently, conserved features of apoptosis have also been demonstrated in unicellular eukaryotes (Holzmuller et al. Parasitology 132:S19-S32, 2006; Le Chat et al. Mol Biochem Parasitol 153:41-47, 2007; Madeo et al. Curr Opin Microbiol 7:655-660, 2004; Welburn et al. Parasitology 132:S7-S18, 2006; Jensen et al. Science 216:1230-1233, 1982) including malaria parasites (Al-Olayan et al. Int J Parasitol 32:1133-1143, 2002; Ch'ng et al. Cell Death Dis 1:e26, 2010; Meslin et al. J Infect Dis 195:1852-1859, 2007; Picot et al. Trans R Soc Trop Med Hyg 91:590-591, 1997; Raj et al. Nature 582:104-108, 2020). P. falciparum glutamic-acid-rich protein (PfGARP) is an antigen of 80 kDa that is uniquely expressed on the exofacial surface of red blood cells (RBCs) infected by early-to-late-trophozoite-stage P. falciparum parasites (Raj et al. Nature 582:104-108, 2020). We have recently demonstrated that antibodies against PfGARP bind to the PfGARP displayed on the surface of P. falciparum trophozoite-infected RBCs and trigger apoptosis in the intracellular parasites (Raj et al. Nature 582:104-108, 2020). This is the first demonstration of antibody-induced apoptosis in blood-stage malaria parasites and is characterized by several conserved features such as crisis form morphology, loss of mitochondrial membrane potential, loss of integrity of food vacuole, activation of caspase-like cysteine proteases, and fragmentation of chromosomal DNA. Here we describe the assays used to detect these features of apoptosis in the mature blood stage of malaria parasites.

Pediatric Participant Retention Rates in a Longitudinal Malaria Immunology Study.

The resurgence of drug-resistant Plasmodium falciparum parasites continues to motivate the development of a safe and efficacious malaria vaccine. Immuno-epidemiologic studies of naturally acquired immunity (NAI) have been a useful strategy to identify new malaria vaccine targets. However, retention of pediatric participants throughout longitudinal studies is essential for gathering comprehensive exposure and outcome data. Within the context of a 3-year cohort (N = 400) study involving monthly finger prick and bi-annual venous blood sample collections, we conducted qualitative surveys to assess factors impacting participant retention. Phase 1 was conducted 3 months after enrollment in July 2018 and phase 2, 12 months later. In phase 1, 236 parents/guardians participated in focus groups and three withdrawn participants and 10 community health volunteers (CHVs) in key informant interviews. Qualitative analysis indicated overall satisfaction with the study, with 61.8% (136/220 respondents) reporting no concerns. Focus group discussants associated attendance with benefits such as improved access to comprehensive healthcare services. Community health volunteers reported concerns over village rumors of inappropriate use of blood samples and dangers associated with venous blood draws. Phase 2 involved 205 parents/guardians and revealed continued satisfaction, with 46.3% (95/205) identifying no concerns, but expressed increasing worries regarding the amount of venous blood sample. This concern was reflected in an uptick of missed visits when venous blood samples were scheduled. Future studies will address parental concerns to determine whether community engagement and education measures increase study retention until completion.

Comparison of self-reported and observed water contact measures in Schistosoma japonicum-endemic villages in Leyte, The Philippines.

BACKGROUND: We compared individuals' self-reported water contact from a questionnaire to direct observation of water contact. Questionnaires that accurately capture water contact are necessary to risk-stratify individuals and communities at high risk for schistosomiasis. METHODS: Individuals (N=677) ages 7-30 y were included from three Schistosoma japonicum-endemic villages in Leyte, The Philippines. Each individual was observed for 12 d over the course of the 18-month study and the questionnaire was administered six times. A questionnaire index was derived that captured the number of self-reported contacts with water bodies for any purpose. An exposure index was created based on the sum of contacts that was weighted by the percentage of body surface area (BSA) exposed and exposure duration. RESULTS: Of 16 water contact activities, only bathing and washing clothes exhibited a significant, positive correlation between self-reported contacts and the observed exposure index related to those contacts. CONCLUSIONS: We found that only the reported frequencies of bathing and washing clothes were significantly related to an individual's overall observed exposure index, while use of all reported contacts was not related to the observed exposure. This study further supports the need for questionnaires to be augmented by some measure of how much BSA is exposed and/or time is spent in the water on average for a specific activity.

Interplay between IL-10, IFN-γ, IL-17A and PD-1 Expressing EBNA1-Specific CD4+ and CD8+ T Cell Responses in the Etiologic Pathway to Endemic Burkitt Lymphoma.

Children diagnosed with endemic Burkitt lymphoma (eBL) are deficient in interferon-γ (IFN-γ) responses to Epstein-Barr Nuclear Antigen1 (EBNA1), the viral protein that defines the latency I pattern in this B cell tumor. However, the contributions of immune-regulatory cytokines and phenotypes of the EBNA1-specific T cells have not been characterized for eBL. Using a bespoke flow cytometry assay we measured intracellular IFN-γ, IL-10, IL-17A expression and phenotyped CD4+ and CD8+ T cell effector memory subsets specific to EBNA1 for eBL patients compared to two groups of healthy children with divergent malaria exposures. In response to EBNA1 and a malaria antigen (PfSEA-1A), the three study groups exhibited strikingly different cytokine expression and T cell memory profiles. EBNA1-specific IFN-γ-producing CD4+ T cell response rates were lowest in eBL (40%) compared to children with high malaria (84%) and low malaria (66%) exposures (p < 0.0001 and p = 0.0004, respectively). However, eBL patients did not differ in CD8+ T cell response rates or the magnitude of IFN-γ expression. In contrast, eBL children were more likely to have EBNA1-specific CD4+ T cells expressing IL-10, and less likely to have polyfunctional IFN-γ+IL-10+ CD4+ T cells (p = 0.02). They were also more likely to have IFN-γ+IL-17A+, IFN-γ+ and IL-17A+ CD8+ T cell subsets compared to healthy children. Cytokine-producing T cell subsets were predominantly CD45RA+CCR7+ TNAIVE-LIKE cells, yet PD-1, a marker of persistent activation/exhaustion, was more highly expressed by the central memory (TCM) and effector memory (TEM) T cell subsets. In summary, our study suggests that IL-10 mediated immune regulation and depletion of IFN-γ+ EBNA1-specific CD4+ T cells are complementary mechanisms that contribute to impaired T cell cytotoxicity in eBL pathogenesis.

High prevalence of Schistosoma japonicum by perfusion in naturally exposed water buffalo in a region of the Philippines endemic for human schistosomiasis.

In the past decade, ecological surveys emphasized rats and dogs as the most significant animal reservoirs for Schistosoma japonicum (S.j) in the Philippines. However, recent studies demonstrated 51-91% prevalence of schistosomiasis among water buffalo using qPCR in the Sj endemic regions in the Philippines. In order to resolve the inconsistency of reported surveys regarding Sj endemicity among carabao, a domestic water buffalo that is the most important draught animal, we introduced 42 schistosome negative water buffalo to Macanip, Jaro municipality, Leyte, the Philippines, a subsistence rice-farming village that has been the focus of schistosomiasis japonica studies of our group for the past 20 years. We conducted perfusion to the remaining 34 buffalo that survived 10 months of nature exposure and Typhoon Haiyan. Thirty-three water buffalo were found to be positive with at least 1 pair of worms from the mesenteric vein. The infection rate is 97%, with the worm burden of 94 (95% confidence interval, 49-138 worms) worms. To our knowledge, this is the first report about S. japonicum worm burden in naturally infected water buffalo in the Philippines. The fact that with less than one-year of exposure, in this human schistosomiasis endemic area, only 1 out of 34 water buffalo was uninfected is striking. Urgent attention is needed for a cost-effective technique for monitoring Sj infection in animals and humans. Meanwhile, intervention implementation, including water buffalo treatment and vaccination, should be taken into consideration.

A newly characterized malaria antigen on erythrocyte and merozoite surfaces induces parasite inhibitory antibodies.

We previously identified a Plasmodium falciparum (Pf) protein of unknown function encoded by a single-copy gene, PF3D7_1134300, as a target of antibodies in plasma of Tanzanian children in a whole-proteome differential screen. Here we characterize this protein as a blood-stage antigen that localizes to the surface membranes of both parasitized erythrocytes and merozoites, hence its designation as Pf erythrocyte membrane and merozoite antigen 1 (PfEMMA1). Mouse anti-PfEMMA1 antisera and affinity-purified human anti-PfEMMA1 antibodies inhibited growth of P. falciparum strains by up to 68% in growth inhibition assays. Following challenge with uniformly fatal Plasmodium berghei (Pb) ANKA, up to 40% of mice immunized with recombinant PbEMMA1 self-cured, and median survival of lethally infected mice was up to 2.6-fold longer than controls (21 vs. 8 d, P = 0.005). Furthermore, high levels of naturally acquired human anti-PfEMMA1 antibodies were associated with a 46% decrease in parasitemia over 2.5 yr of follow-up of Tanzanian children. Together, these findings suggest that antibodies to PfEMMA1 mediate protection against malaria.

Fetal Cytokine Balance, Erythropoietin and Thalassemia but Not Placental Malaria Contribute to Fetal Anemia Risk in Tanzania.

Fetal anemia is common in malaria-endemic areas and a risk factor for anemia as well as mortality during infancy. Placental malaria (PM) and red cell abnormalities have been proposed as possible etiologies, but the relationship between PM and fetal anemia has varied in earlier studies, and the role of red cell abnormalities has not been studied in malaria-endemic areas. In a Tanzanian birth cohort study designed to elucidate the pathogenesis of severe malaria in young infants, we performed a cross-sectional analysis of risk factors for fetal anemia. We determined PM status, newborn red cell abnormalities, and maternal and cord blood levels of iron regulatory proteins, erythropoietin (EPO), cytokines and cytokine receptors. We examined the relationship between these factors and fetal anemia. Fetal anemia was present in 46.2% of the neonates but was not related to PM. Maternal iron deficiency was common (81.6%), most frequent in multigravidae, and interacted with parity to modify risk of fetal anemia, but it was not directly related to risk. Among offspring of iron-deficient women, the odds of fetal anemia increased with fetal α+-thalassemia, as well as these patterns of cord blood cytokines: increased cord IL-6, decreased TNF-RI, and decreased sTfR. The EPO response to fetal anemia was low or absent and EPO levels were significantly decreased in newborns with the most severe anemia. This study from an area of high malaria transmission provides evidence that 1) fetal α+-thalassemia and cytokine balance, but not PM at delivery, are related to fetal anemia; 2) maternal iron deficiency increases the risk that other factors may cause fetal anemia; and 3) fetal anemia has a multifactorial etiology that may require a variety of interventions, although measures that reduce maternal iron deficiency may be generally beneficial.

Effect of maternal praziquantel treatment for Schistosoma japonicum infection on the offspring susceptibility and immunologic response to infection at age six, a cohort study.

In areas endemic to schistosomiasis, fetal exposure to schistosome antigens prime the offspring before potential natural infection. Praziquantel (PZQ) treatment for Schistosoma japonicum infection in pregnant women has been demonstrated to be safe and effective. Our objectives were to evaluate whether maternal PZQ treatment modifies the process of in utero sensitization to schistosome antigens potentially impacting later risk of infection, as well as immune response to S. japonicum. We enrolled 295 children at age six, born to mothers with S. japonicum infection who participated in a randomized control trial of PZQ versus placebo given at 12-16 weeks gestation in Leyte, The Philippines. At enrollment, we assessed and treated current S. japonicum infection and measured serum cytokines. During a follow-up visit four weeks later, we assessed peripheral blood mononuclear cell (PBMC) cytokine production in response to soluble worm antigen preparation (SWAP) or soluble egg antigen (SEA). Associations between maternal treatment group and the child's S. japonicum infection status and immunologic responses were determined using multivariate linear regression analysis. PZQ treatment during pregnancy did not impact the prevalence (P = 0.12) or intensity (P = 0.59) of natural S. japonicum infection among children at age six. Among children with infection at enrollment (12.5%) there were no significant serum cytokine concentration differences between maternal treatment groups. Among children with infection at enrollment, IL-1 production by PBMCs stimulated with SEA was higher (P = 0.03) in the maternal PZQ group compared to placebo. Among children without infection, PBMCs stimulated with SEA produced greater IL-12 (P = 0.03) and with SWAP produced less IL-4 (P = 0.01) in the maternal PZQ group compared to placebo. Several cytokines produced by PBMCs in response to SWAP and SEA were significantly higher in children with S. japonicum infection irrespective of maternal treatment: IL-4, IL-5, IL-10, and IL-13. We report that maternal PZQ treatment for S. japonicum shifted the PBMC immune response to a more inflammatory signature but had no impact on their offspring's likelihood of infection or serum cytokines at age six, further supporting the safe use of PZQ in pregnant women. Trial Registration: ClinicalTrials.gov NCT00486863.

Immune-Based Interventions Against Infectious Disease - Impact of a Phase I Center for Biomedical Research Excellence in Translational Infectious Diseases Immunology.

In 2011, faculty from the University of Rhode Island (URI)'s Institute for Immunology and Informatics and Lifespan's Center for International Health Research collaborated to develop a successful application for a Phase I Center of Biomedical Research Excellence around the scientific theme of translational infectious diseases immunology. From 2013 to 2020, this COBRE supported significant discoveries in research on dengue, HIV, and malaria, among other diseases, and facilitated the career development of several independent Rhode Island (RI)-based early-stage investigators. Our experience illustrates both the potential and challenges for investigators with shared scientific interests to leverage the NIH COBRE program to enhance cross-institutional interactions.

Whole-Proteome Differential Screening Identifies Novel Vaccine Candidates for Schistosomiasis japonica.

Schistosomiasis remains a leading cause of chronic morbidity in endemic regions despite decades of widespread mass chemotherapy with praziquantel. Using our whole proteome differential screening approach, and plasma and epidemiologic data from a longitudinal cohort of individuals living in a Schistosoma japonicum-endemic region of the Philippines, we interrogated the parasite proteome to identify novel vaccine candidates for Schistosoma japonicum. We identified 16 parasite genes which encoded proteins that were recognized by immunoglobulin G or immunoglobulin E antibodies in the plasma of individuals who had developed resistance to reinfection, but were not recognized by antibodies in the plasma of individuals who remained susceptible to reinfection. Antibody levels to Sj6-8 and Sj4-1 measured in the entire cohort (N = 505) 1 month after praziquantel treatment were associated with significantly decreased risk of reinfection and lower intensity of reinfection over 18 months of follow-up.

Brief Report: Relationship Between Cotinine Levels and Peripheral Endogenous Concentrations of Oxytocin, ß-Endorphin, and Orexin in Individuals With Both Alcohol and Nicotine Use Disorders.

BACKGROUND AND OBJECTIVES: In this secondary analysis of a pilot clinical trial with individuals with alcohol and nicotine use disorders, we investigate the relationship between serum concentrations of oxytocin, ß-endorphin, melatonin, α-melanocyte-stimulating hormone, substance P, and orexin, with objective biomarkers (salivary cotinine and serum γ-glutamyl transferase [GGT]) as well as with self-reported smoking and alcohol drinking. METHODS: Biomarkers for a total of N = 19 participants were analyzed using multiplexed, competitive format immune-assay (peptides) and enzyme competitive immunoassay (saliva). A regression analysis using Pearson's correlation coefficient was utilized to determine correlations. We controlled for multiple comparisons, checked for collinearities, and ran two-sided statistical tests. RESULTS: We found significant positive correlations for cotinine and oxytocin (P = .002), ß-endorphin (P = .008), and orexin (P < .001), but not for either GGT or self-reported smoking or alcohol drinking. CONCLUSION AND SCIENTIFIC SIGNIFICANCE: These preliminary results suggest a relationship between cotinine and oxytocin, ß-endorphin, and orexin, which opens up new potential hypotheses on the potential role of these endocrine pathways in tobacco smokers. (Am J Addict 2021;30:88-91).