Impact of Surfactant Protein-A on Immunomodulatory Properties of Murine and Human Breast Milk.

OBJECTIVES: Human milk reduces the incidence of necrotizing enterocolitis (NEC). Prior studies have demonstrated that exogenous surfactant protein-A (SP-A) modulates intestinal inflammation, reduces NEC-like pathology in SP-A-deficient (SPAKO) pups, and may contribute to breast milk's immunomodulatory potential. We hypothesize that SP-A is present in milk and impacts inflammatory responses in the terminal ileum of neonatal mice. METHODS: Human milk was collected at postpartum days 1-3 and 28. Mouse milk was collected at postpartum days 1-10. SP-A was detected in milk through immunoprecipitation and western blot analysis. The impact of murine wild-type (WT) milk on SPAKO pup ileum was evaluated in a model of intestinal inflammation via cross-rearing experiments. Terminal ileum was evaluated for inflammatory cytokine and toll-like receptor 4 (TLR4) mRNA expression via quantitative real-time RT-PCR. RESULTS: SP-A was detected in human milk and wild type (WT) mouse milk, but not in SPAKO mouse milk. Expression of TLR4, interleukin (IL)-1ß, IL-6, and tumor necrosis factor (TNF)-α was decreased in SPAKO pups reared with WT dams compared to SPAKO pups reared with SPAKO dams, with a peak effect at day of life 14. When inflammation was induced using a lipopolysaccharide-induced model of inflammation, expression of TLR4, IL-1ß, IL-6, CXCL-1, and TNF-α was significantly lower in SPAKO pups reared with WT dams compared to SPAKO pups reared with SPAKO dams. CONCLUSIONS: SP-A is present in human and murine milk and plays a role in lowering inflammation in murine pup terminal ileum. Both baseline inflammation and induced inflammatory responses are reduced via exposure to SP-A in milk with the effect amplified in inflammatory conditions.

A primate-specific RNA-binding protein (RBMXL3) is involved in glucocorticoid regulation of human pulmonary surfactant protein B (SP-B) mRNA stability.

The ability of pulmonary surfactant to reduce alveolar surface tension requires adequate levels of surfactant protein B (SP-B). Dexamethasone (DEX) increases human SP-B expression, in part, through increased SP-B mRNA stability. A 30-nt-long hairpin element (RBE) in the 3'-untranslated region of human SP-B mRNA mediates both DEX-induced and intrinsic mRNA stabilities, but the mechanism is unknown. Proteomic analysis of RBE-interacting proteins identified a primate-specific protein, RNA-binding motif X-linked-like-3 (RBMXL3). siRNA directed against RBMXL3 reduces DEX-induced SP-B mRNA expression in human bronchoalveolar cells. Human SP-B mRNA stability, measured by our dual cistronic plasmid assay, is unaffected by DEX in mouse lung epithelial cells lacking RBMXL3, but DEX increases human SP-B mRNA stability when RBMXL3 is expressed and requires the RBE. In the absence of DEX, RBE interacts with cellular proteins, reducing intrinsic SP-B mRNA stability in human and mouse lung epithelial cells. RBMXL3 specifically binds the RBE in vitro, whereas RNA immunoprecipitation and affinity chromatography analyses indicate that binding is enhanced in the presence of DEX. These results describe a model where intrinsic stability of human SP-B mRNA is reduced through binding of cellular mRNA decay factors to RBE, which is then relieved through DEX-enhanced binding of primate-specific RBMXL3.

An internal amino-terminal FLAG-tag octapeptide alters oligomerization of expressed surfactant protein-A.

Pulmonary surfactant protein-A (SP-A) is expressed by lung alveolar and bronchiolar epithelial cells and plays a critical role in innate immunity of the lung. Exposure of the lung to various environmental insults alters SP-A homeostasis. To investigate the cellular mechanisms involved in these alterations, we added the FLAG octapeptide (DYKDDDDK) to the carboxy-terminus (SP-A/C-FLAG) or near the amino-terminus (SP-A/N-FLAG) of mouse SP-A (WT-SP-A) to tag specific pools of protein. We hypothesized that addition of FLAG would have negligible effects on SP-A expression, oligomerization and secretion. Analysis of Chinese hamster ovary cells expressing these proteins indicated that tagged SP-A mRNA could be distinguished from WT-SP-A by northern analysis and RT-PCR using sequence-specific oligonucleotides. Tagged SP-A protein could be differentiated from WT-SP-A by western analysis using antibodies specific for the FLAG epitope. Subcellular fractionation and immunocytochemistry indicated the majority of each protein was present in punctuate (presumably endocytic) vesicles, and all forms of SP-A protein were secreted. These results suggest that a FLAG epitope added to the carboxy-terminus or inserted into the amino-terminus of the mature SP-A protein has little effect on its expression and cellular processing. However, disruptions of the amino-terminal end of SP-A prevents proper oligomerization, suggesting that this region of mature SP-A is critical in proper oligomeric assembly and is not useful for studies intended to define mechanisms underlying SP-A homeostasis.

Antisense Reduction of Mutant COMP Reduces Growth Plate Chondrocyte Pathology.

Mutations in cartilage oligomeric matrix protein cause pseudoachondroplasia, a severe disproportionate short stature disorder. Mutant cartilage oligomeric matrix protein produces massive intracellular retention of cartilage oligomeric matrix protein, stimulating ER and oxidative stresses and inflammation, culminating in post-natal loss of growth plate chondrocytes, which compromises linear bone growth. Treatments for pseudoachondroplasia are limited because cartilage is relatively avascular and considered inaccessible. Here we report successful delivery and treatment using antisense oligonucleotide technology in our transgenic pseudoachondroplasia mouse model. We demonstrate delivery of human cartilage oligomeric matrix protein-specific antisense oligonucleotides to cartilage and reduction of cartilage oligomeric matrix protein expression, which largely alleviates pseudoachondroplasia growth plate chondrocyte pathology. One antisense oligonucleotide reduced steady-state levels of cartilage oligomeric matrix protein mRNA and dampened intracellular retention of mutant cartilage oligomeric matrix protein, leading to a reduction of inflammatory markers and cell death and partial restoration of proliferation. This novel and exciting work demonstrates that antisense-based therapy is a viable approach for treating pseudoachondroplasia and other human cartilage disorders.

Antioxidant and anti-inflammatory agents mitigate pathology in a mouse model of pseudoachondroplasia.

Pseudoachondroplasia (PSACH), a severe short-limb dwarfing condition, results from mutations that cause misfolding of the cartilage oligomeric matrix protein (COMP). Accumulated COMP in growth plate chondrocytes activates endoplasmic reticulum stress, leading to inflammation and chondrocyte death. Using a MT-COMP mouse model of PSACH that recapitulates the molecular and clinical PSACH phenotype, we previously reported that oxidative stress and inflammation play important and unappreciated roles in PSACH pathology. In this study, we assessed the ability of antioxidant and anti-inflammatory agents to affect skeletal and cellular pathology in our mouse model of PSACH. Treatment of MT-COMP mice with aspirin or resveratrol from birth to P28 decreased mutant COMP intracellular retention and chondrocyte cell death, and restored chondrocyte proliferation. Inflammatory markers associated with cartilage degradation and eosinophils were present in the joints of untreated juvenile MT-COMP mice, but were undetectable in treated mice. Most importantly, these treatments resulted in significantly increased femur length. This is the first and only therapeutic approach shown to mitigate both the chondrocyte and long-bone pathology of PSACH in a mouse model and suggests that reducing inflammation and oxidative stress early in the disease process may be a novel approach to treat this disorder.

Cigarette Cue Attentional Bias in Cocaine-Smoking and Non-Cocaine-Using Cigarette Smokers.

INTRODUCTION: Cigarette smoking in cocaine users is nearly four times higher than the national prevalence and cocaine use increases cigarette smoking. The mechanisms underlying cigarette smoking in cocaine-using individuals need to be identified to promote cigarette and cocaine abstinence. Previous studies have examined the salience of cigarette and cocaine cues separately. The present aim was to determine whether cigarette attentional bias (AB) is higher in cigarettes smokers who smoke cocaine relative to individuals who only smoke cigarettes. METHODS: Twenty cigarette smokers who smoke cocaine and 20 non-cocaine-using cigarette smokers completed a visual probe task with eye-tracking technology. During this task, the magnitude of cigarette and cocaine AB was assessed through orienting bias, fixation time, and response time. RESULTS: Cocaine users displayed an orienting bias towards cigarette cues. Cocaine users also endorsed a more urgent desire to smoke to relieve negative affect associated with cigarette craving than non-cocaine users (g = 0.6). Neither group displayed a cigarette AB, as measured by fixation time. Cocaine users, but not non-cocaine users, displayed a cocaine AB as measured by orienting bias (g = 2.0) and fixation time (g = 1.2). There were no significant effects for response time data. CONCLUSIONS: Cocaine-smoking cigarettes smokers display an initial orienting bias toward cigarette cues, but not sustained cigarette AB. The incentive motivation underlying cigarette smoking also differs. Cocaine smokers report more urgent desire to smoke to relieve negative affect. Identifying differences in motivation to smoke cigarettes may provide new treatment targets for cigarette and cocaine use disorders. IMPLICATIONS: These results suggest that cocaine-smoking cigarette smokers display an initial orienting bias towards cigarette cues, but not sustained attention towards cigarette cues, relative to non-cocaine-using smokers. Smoked cocaine users also report a more urgent desire to smoke to relieve negative affect than non-cocaine users. Identifying differences in motivation to smoke cigarettes may provide new treatment targets for both cigarette and cocaine use disorders.

Resistin deficiency in mice has no effect on pulmonary responses induced by acute ozone exposure.

Acute exposure to ozone (O3), an air pollutant, causes pulmonary inflammation, airway epithelial desquamation, and airway hyperresponsiveness (AHR). Pro-inflammatory cytokines-including IL-6 and ligands of chemokine (C-X-C motif) receptor 2 [keratinocyte chemoattractant (KC) and macrophage inflammatory protein (MIP)-2], TNF receptor 1 and 2 (TNF), and type I IL-1 receptor (IL-1α and IL-1ß)-promote these sequelae. Human resistin, a pleiotropic hormone and cytokine, induces expression of IL-1α, IL-1ß, IL-6, IL-8 (the human ortholog of murine KC and MIP-2), and TNF. Functional differences exist between human and murine resistin; yet given the aforementioned observations, we hypothesized that murine resistin promotes O3-induced lung pathology by inducing expression of the same inflammatory cytokines as human resistin. Consequently, we examined indexes of O3-induced lung pathology in wild-type and resistin-deficient mice following acute exposure to either filtered room air or O3. In wild-type mice, O3 increased bronchoalveolar lavage fluid (BALF) resistin. Furthermore, O3 increased lung tissue or BALF IL-1α, IL-6, KC, TNF, macrophages, neutrophils, and epithelial cells in wild-type and resistin-deficient mice. With the exception of KC, which was significantly greater in resistin-deficient compared with wild-type mice, no genotype-related differences in the other indexes existed following O3 exposure. O3 caused AHR to acetyl-ß-methylcholine chloride (methacholine) in wild-type and resistin-deficient mice. However, genotype-related differences in airway responsiveness to methacholine were nonexistent subsequent to O3 exposure. Taken together, these data demonstrate that murine resistin is increased in the lungs of wild-type mice following acute O3 exposure but does not promote O3-induced lung pathology.

A site-specific genetic modification for induction of pluripotency and subsequent isolation of derived lung alveolar epithelial type II cells.

Human induced pluripotent stem cells (hiPSCs) have great therapeutic potential in repairing defective lung alveoli. However, genetic abnormalities caused by vector integrations and low efficiency in generating hiPSCs, as well as difficulty in obtaining transplantable hiPSC-derived cell types are still major obstacles. Here we report a novel strategy using a single nonviral site-specific targeting vector with a combination of Tet-On inducible gene expression system, Cre/lox P switching gene expression system, and alveolar epithelial type II cell (ATIIC)-specific Neomycin(R) transgene expression system. With this strategy, a single copy of all of the required transgenes can be specifically knocked into a site immediately downstream of ß-2-microglobulin (B2M) gene locus at a high frequency, without causing B2M dysfunction. Thus, the expression of reprogramming factors, Oct4, Sox2, cMyc, and Klf4, can be precisely regulated for efficient reprogramming of somatic cells into random integration-free or genetic mutation-free hiPSCs. The exogenous reprogramming factor transgenes can be subsequently removed after reprogramming by transient expression of Cre recombinase, and the resulting random integration-free and exogenous reprogramming factor-free hiPSCs can be selectively differentiated into a homogenous population of ATIICs. In addition, we show that these hiPSC-derived ATIICs exhibit ultrastructural characteristics and biological functions of normal ATIICs. When transplanted into bleomycin-challenged mice lungs, hiPSC-derived ATIICs efficiently remain and re-epithelialize injured alveoli to restore pulmonary function, preventing lung fibrosis and increasing survival without tumorigenic side effect. This strategy allows for the first time efficient generation of patient-specific ATIICs for possible future clinical applications.

Effects of oxytocin on aggressive responding in healthy adult men.

This study investigated the acute effects of oxytocin (OT) on human aggression using a well-established laboratory measure of state (reactive) aggression to test the hypothesis that OT would decrease the frequency of aggressive responding. In a within-subject design, 17 healthy male volunteers received placebo or 24 IU of intranasal OT. Aggression was measured using the Point Subtraction Aggression Paradigm at 30 min before and 30, 60, and 90 min after dose. Acute OT did not produce a significant main effect on aggressive behavior. OT attenuated the expected rise in diastolic blood pressure from morning to early afternoon observed under placebo, providing a possible indication of biological activity. Examination of individual differences showed that aggressive responding following OT dosing (but not placebo) was positively correlated with psychometric measures of interpersonal manipulation and anger (Pearson's r=0.57), indicating that higher scores on these antisocial personality traits were related to increased aggressive behavior following OT administration. These preliminary results stand in contrast to previous work on the prosocial effects of OT and highlight the need for further understanding of individual differences in aggression following OT administration. Such individual differences may have implications for the therapeutic use of OT in individuals with psychiatric disorders and dysfunctional social behavior.

Oral administration of surfactant protein-a reduces pathology in an experimental model of necrotizing enterocolitis.

OBJECTIVES: Necrotizing enterocolitis (NEC) frequently results in significant morbidity and mortality in premature infants. Others reported that mice deficient in pulmonary surfactant protein-A (SP-A) born and raised in a nonhygienic environment succumb to significant gastrointestinal tract pathology, and enteral administration of purified SP-A significantly reduced mortality. We hypothesized that oral administration of purified SP-A can ameliorate pathology in an experimental model of neonatal NEC. METHODS: Experimental NEC was induced in newborn Sprague-Dawley rat pups by daily formula gavage and intermittent exposure to hypoxia. Purified human SP-A (5 µg/day) was administered by oral gavage. After 4 days, surviving pups were sacrificed, and intestinal pathology was assessed by histological examination of distal terminal ileal sections. Intestinal levels of inflammatory cytokines (IL-1ß, IFN-γ, and TNF-α) were assessed by enzyme-linked immunosorbent assay and levels of Toll-like receptor 4 (TLR4) by Western analysis. RESULTS: Sixty-one percent of the gavaged rat pups that survived to day 4 met the criteria for experimental NEC after hypoxia, whereas treatment with SP-A significantly reduced mortality and assessment of NEC. Intestinal levels of proinflammatory cytokines were significantly increased in pups exposed to hypoxia. Administration of SP-A to pups exposed to hypoxia significantly reduced IL-1ß and TNF-α levels, but had little effect on elevated levels of IFN-γ. SP-A treatment of hypoxia-exposed pups significantly reduced expression of intestinal TLR4, key in NEC pathogenesis. CONCLUSIONS: In a rat model of experimental neonatal NEC, oral administration of SP-A reduces intestinal levels of proinflammatory cytokines and TLR4 protein and ameliorates adverse outcomes associated with gastrointestinal pathologies.

Effects of Intranasal Oxytocin on Aggressive Responding in Antisocial Personality Disorder.

The oxytocin receptor is important in several domains of social behavior, and administration of oxytocin modulates social responding in several mammalian species, including humans. Oxytocin has both therapeutic and scientific potential for elucidating the neural and behavioral mechanisms governing social behavior. In the present study, operationally-defined aggressive behavior of six males with Antisocial Personality Disorder (ASPD) was measured following acute intranasal oxytocin dosing (12, 24, and 48 international units) and placebo, using a well-validated laboratory task of human aggression (Point-Subtraction Aggression Paradigm, or PSAP). The PSAP provides participants with concurrently available monetary-earning and operationally-defined aggressive response options, maintained by fixed ratio schedules of consequences. Shifts in response rates and inter-response time (IRT) distributions were observed on the aggressive response option following oxytocin doses, relative to placebo. Few changes were observed in monetary-reinforced responding. However, across participants the direction and magnitude of changes in aggressive responding were not systematically related to dose. No trends were observed between psychometric or physiological data and oxytocin dosing or aggressive behavior. While this report is to our knowledge the first to examine the acute effects of oxytocin in this population at high risk for violence and other forms of antisocial behavior, several limitations in the experimental design and the results cast the study as a preliminary report. Strategies for more extensive future projects are discussed.

Utilization of an ex vivo human placental perfusion model to predict potential fetal exposure to carboplatin during pregnancy.

OBJECTIVE: The objective of this study was to determine the fetal drug compartment concentrations when various concentrations of carboplatin cross the placental-trophoblastic barrier and the effect on the fetal kidneys. STUDY DESIGN: An ex vivo human placenta perfusion model was utilized. Term human placentae (n = 9) were collected immediately after delivery and then reperfused with plasma concentrations achieved with carboplatin an area under the curve of 5 (1000 ng/mL), 7.5 (5000 ng/mL), or 11 (11,000 ng/mL). Antipyrine was used as a reference compound. Samples were collected over 2 hours. Placental transfer was evaluated by computation of transport fraction and clearance index. Primary cells isolated by explant culture of 16-18 week old fetal organ tissues were incubated with carboplatin for up to 48 hours with untreated cell as controls. Immunohistochemical, flow cytometry analysis, and immunoblotting were applied for the expression of apoptosis-related proteins. RESULTS: Mean transport fractions for carboplatin at low, middle, and high concentrations were 0.05 ± 0.02, 0.04 ± 0.01, and 0.10 ± 0.01, respectively, with clearance indexes of 0.22 ± 0.01, 0.14 ± 0.08, and 0.50 ± 0.07, respectively. The fetal peak concentrations of carboplatin achieved were 61 ± 39 ng/mL (low), 375 ± 248 ng/mL (middle), and 2081 ± 529 ng/mL (high). Fetal kidney cells exposed to carboplatin showed a concentration-dependent increased expression of apoptosis-inducing factor and p53 apoptosis proteins and a time-dependent increase in expression Bax apoptosis protein expression. Apoptosis was confirmed at the high concentration by flow cytometry. CONCLUSION: Doses of carboplatin up to an area under the curve of 7.5 were not associated with significant placental transfer, fetal exposure, or fetal toxic effects. This suggests it might not be necessary to empirically reduce carboplatin doses in pregnant women.

Intermittent theta burst stimulation and functional connectivity in people living with HIV/AIDS who smoke tobacco cigarettes: a preliminary pilot study.

Background: People living with HIV (PLWHA) smoke at three times the rate of the general population and respond poorly to cessation strategies. Previous studies examined repetitive transcranial magnetic stimulation (rTMS) over left dorsolateral prefrontal cortex (L. dlPFC) to reduce craving, but no studies have explored rTMS among PLWHA who smoke. The current pilot study compared the effects of active and sham intermittent theta-burst stimulation (iTBS) on resting state functional connectivity (rsFC), cigarette cue attentional bias, and cigarette craving in PLWHA who smoke. Methods: Eight PLWHA were recruited (single-blind, within-subject design) to receive one session of iTBS (n=8) over the L. dlPFC using neuronavigation and, four weeks later, sham iTBS (n=5). Cigarette craving and attentional bias assessments were completed before and after both iTBS and sham iTBS. rsFC was assessed before iTBS (baseline) and after iTBS and sham iTBS. Results: Compared to sham iTBS, iTBS enhanced rsFC between the L. dlPFC and bilateral medial prefrontal cortex and pons. iTBS also enhanced rsFC between the right insula and right occipital cortex compared to sham iTBS. iTBS also decreased cigarette craving and cigarette cue attentional bias. Conclusion: iTBS could potentially offer a therapeutic option for smoking cessation in PLWHA.

D469del-COMP retention in chondrocytes stimulates caspase-independent necroptosis.

Mutations in the cartilage oligomeric matrix protein gene (COMP) cause pseudoachondroplasia (PSACH). This dysplasia results from the intracellular retention of mutant COMP protein and premature death of growth-plate chondrocytes. Toward better understanding of these underlying mechanisms, we examined D469del-COMP activation of the unfolded protein response and cell death pathways in rat chondrosarcoma cells. Using an inducible expression system, we examined the effects of D469del-COMP retention after 4 days of mRNA expression and then 5 days without inducing agent. Retention of D469del-COMP stimulated Chop (Ddit3) and Gadd34 (Ppp1r15a) and triggered reactivation of protein translation that exacerbated intracellular retention. High levels of Nox4 and endoplasmic reticulum receptor stress-inducible Ero1ß generated reactive oxygen species, causing oxidative stress. Increased expression of Gadd genes and presence of γH2AX indicated that DNA damage was occurring. The presence of cleaved apoptosis inducing factor (tAIF) and the absence of activated caspases indicated that retention of D469del-COMP triggers cell death in chondrocytes by necroptosis, a caspase-independent programmed necrosis. Loss of growth-plate chondrocytes by necroptosis was also found in our pseudoachondroplasia mouse model. These results suggest a model in which D469del-COMP expression induces persistent endoplasmic reticulum stress, oxidative stress, and DNA damage, thus priming chondrocytes for necroptosis. We define for the first time the precise mechanisms underlying D469del-COMP pathology in pseudoachondroplasia and suggest that oxidative stress and AIF may be promising therapeutic targets.

Chop (Ddit3) is essential for D469del-COMP retention and cell death in chondrocytes in an inducible transgenic mouse model of pseudoachondroplasia.

Cartilage oligomeric matrix protein (COMP), a secreted glycoprotein synthesized by chondrocytes, regulates proliferation and type II collagen assembly. Mutations in the COMP gene cause pseudoachondroplasia and multiple epiphyseal dysplasia. Previously, we have shown that expression of D469del-COMP in transgenic mice causes intracellular retention of D469del-COMP, thereby recapitulating pseudoachondroplasia chondrocyte pathology. This inducible transgenic D469del-COMP mouse is the only in vivo model to replicate the critical cellular and clinical features of pseudoachondroplasia. Here, we report developmental studies of D469del-COMP-induced chondrocyte pathology from the prenatal period to adolescence. D469del-COMP retention was limited prenatally and did not negatively affect the growth plate until 3 weeks after birth. Results of immunostaining, transcriptome analysis, and qRT-PCR suggest a molecular model in which D469del-COMP triggers apoptosis during the first postnatal week. By 3 weeks (when most chondrocytes are retaining D469del-COMP), inflammation, oxidative stress, and DNA damage contribute to chondrocyte cell death by necroptosis. Importantly, by crossing the D469del-COMP mouse onto a Chop null background (Ddit3 null), thereby eliminating Chop, the unfolded protein response was disrupted, thus alleviating both D469del-COMP intracellular retention and premature chondrocyte cell death. Chop therefore plays a significant role in processes that mediate D469del-COMP retention. Taken together, these results suggest that there may be an optimal window before the induction of significant D469del-COMP retention during which endoplasmic reticulum stress could be targeted.

Aggression, impulsivity, and psychopathic traits in combined antisocial personality disorder and substance use disorder.

Aggression, impulsivity, and psychopathic traits are prominent in both antisocial personality disorder (ASPD) and substance use disorders (SUD), but have rarely been examined collectively. The authors' results show that all three variables were elevated in adults with comorbid ASPD/SUD, relative to SUD-only and control subjects.

Protocol for expression of murine milk using modified human breast pump parts.

Understanding the nutritional and immunomodulatory components of breast milk is crucial to developing novel mechanisms to optimize neonatal health. Here, we present a protocol to express and isolate murine milk in sufficient quantities for further analysis of components and bioactivity. We describe steps for separating dams from pups, administering intraperitoneal anesthetic and oxytocin, and expressing milk using a minimally modified and readily available commercial breast pump parts. For complete details on the use and execution of this protocol, please refer to Meyer et al. (2022).1.

Surfactant protein a attenuates generalized and localized neuroinflammation in neonatal mice.

Surfactant protein A (SP-A) has important roles in innate immunity and modulation of pulmonary and extrapulmonary inflammation. Given SP-A has been detected in rat and human brain, we sought to determine if SP-A has a role in modulating inflammation in the neonatal mouse brain. Neonatal wildtype (WT) and SP-A-deficient (SP-A-/-) mice were subjected to three models of brain inflammation: systemic sepsis, intraventricular hemorrhage (IVH) and hypoxic-ischemic encephalopathy (HIE). Following each intervention, RNA was isolated from brain tissue and expression of cytokine and SP-A mRNA was determined by real-time quantitative RT-PCR analysis. In the sepsis model, expression of most cytokine mRNAs was significantly increased in brains of WT and SP-A-/- mice with significantly greater expression of all cytokine mRNA levels in SP-A-/- mice compared to WT. In the IVH model, expression of all cytokine mRNAs was significantly increased in WT and SP-A-/- mice and levels of most cytokine mRNAs were significantly increased in SP-A-/- mice compared to WT. In the HIE model, only TNF-α mRNA levels were significantly increased in WT brain tissue while all pro-inflammtory cytokine mRNAs were significantly increased in SP-A-/- mice, and all pro-inflammatory cytokine mRNA levels were significantly higher in SP-A-/- mice compared to WT. SP-A mRNA was not detectable in brain tissue of adult WT mice nor in WT neonates subjected to these models. These results suggest that SP-A-/- neonatal mice subjected to models of neuroinflammation are more susceptible to both generalized and localized neuroinflammation compared to WT mice, thus supporting the hypothesis that SP-A attenuates inflammation in neonatal mouse brain.

Comparing cigarette-cue attentional bias between people with HIV/AIDS and people with opioid use disorder who smoke.

Background: Special populations like people living with HIV/AIDS (PLWHA) and people with opioid use disorder (OUD) smoke tobacco cigarettes at rates three to four times greater than the general population. Patients with tobacco use disorder exhibit attentional bias (AB) for cigarette cues. Eye tracking can quantify this bias by measuring fixation time (FT) on cigarette and matched neutral cues, to calculate an AB score. Although previous studies have measured this bias in people who smoke without any other comorbid conditions, no study, to our knowledge, has measured or compared this bias in special populations. Methods: We performed exploratory analyses on eye tracking data collected in two separate randomized clinical trials (RCTs) (NCT05049460, NCT05295953). We compared FT and cigarette-cue AB score (measured by subtracting FT on neutral cues from FT on cigarette cues) between PLWHA and people with OUD who smoke, using a visual probe task and Tobii Pro Fusion eye tracker. We used two cigarette cue types, one encompassing people smoking cigarettes and the other consisting of cigarette paraphernalia. We used two cue presentation times, 1000 and 2000 milliseconds (ms). Results: Cues of people smoking cigarettes elicited greater AB than cues of cigarette paraphernalia across both subject groups when cues were presented for 2000 ms, but not 1000 ms. PLWHA who smoke exhibited greater AB for cues of people smoking cigarettes than cigarette paraphernalia when presented for 2000 ms compared to people with OUD who smoke. Conclusion: We use cigarette-cue AB to quantify craving and cigarette consumption in two populations smoking at elevated rates. The addition of social cues potentiates cigarette cue AB, based on cue type and stimulus presentation time. Understanding the neurobiology of this relationship can help design novel smoking cessation treatments that target AB and prevent relapse in these populations with suboptimal response to smoking cessation treatments. Trial registration: Clinical trials that provided the data for post hoc analyses are NCT05049460 and NCT05295953.

Naltrexone-bupropion combinations do not affect cocaine self-administration in humans.

The FDA has not yet approved a pharmacotherapy for cocaine use disorder despite nearly four decades of research. This study determined the initial efficacy, safety, and tolerability of naltrexone-bupropion combinations as a putative pharmacotherapy for cocaine use disorder. Thirty-one (31) non-treatment seeking participants with cocaine use disorder completed a mixed-design human laboratory study. Participants were randomly assigned to the naltrexone conditions (i.e., 0, 50 mg/day; between-subject factor) and maintained on escalating doses of bupropion (i.e., 0, 100, 200, 400 mg/day; within-subject factor) for at least four days prior to the conduct of experimental sessions. Cocaine self-administration (IN, 0, 40, 80 mg) was then determined using a modified progressive ratio and relapse procedure. Subjective and cardiovascular effects were also measured. Cocaine produced prototypical dose-related increases in self-administration, subjective outcomes (e.g., "Like Drug"), and cardiovascular indices (e.g., heart rate, blood pressure) during placebo maintenance. Naltrexone and bupropion alone, or in combination, did not significantly decrease self-administration on either procedure. Low doses of bupropion (i.e., 100 mg) blunted the effects of the cocaine on subjective measures of "Like Drug" and "Stimulated". No unexpected adverse effects were observed with naltrexone and bupropion, alone and combined, in conjunction with cocaine. Together, these results do not support the use of these bupropion-naltrexone combinations for the treatment of cocaine use disorder. Future research should determine if novel drug combinations may decrease cocaine self-administration.