Identification of HPV16 E1 and E2-specific T cells in the oropharyngeal cancer tumor microenvironment.

BACKGROUND: High-risk human papillomavirus (HPV) is a primary cause of an increasing number of oropharyngeal squamous cell carcinomas (OPSCCs). The viral etiology of these cancers provides the opportunity for antigen-directed therapies that are restricted in scope compared with cancers without viral components. However, specific virally-encoded epitopes and their corresponding immune responses are not fully defined. METHODS: To understand the OPSCC immune landscape, we conducted a comprehensive single-cell analysis of HPV16+ and HPV33+ primary tumors and metastatic lymph nodes. We used single-cell analysis with encoded peptide-human leukocyte antigen (HLA) tetramers to analyze HPV16+ and HPV33+ OPSCC tumors, characterizing the ex vivo cellular responses to HPV-derived antigens presented in major Class I and Class II HLA alleles. RESULTS: We identified robust cytotoxic T-cell responses to HPV16 proteins E1 and E2 that were shared across multiple patients, particularly in HLA-A*01:01 and HLA-B*08:01. Responses to E2 were associated with loss of E2 expression in at least one tumor, indicating the functional capacity of these E2-recognizing T cells and many of these interactions validated in a functional assay. Conversely, cellular responses to E6 and E7 were limited in quantity and cytotoxic capacity, and tumor E6 and E7 expression persisted. CONCLUSIONS: These data highlight antigenicity beyond HPV16 E6 and E7 and nominate candidates for antigen-directed therapies.

Childhood Physical Neglect Is Associated With Exaggerated Systemic and Intracellular Inflammatory Responses to Repeated Psychosocial Stress in Adulthood.

Experiences of child maltreatment are associated with a host of adverse mental and physical health outcomes in adulthood. Altered reactivity to psychosocial stress exposure may partially explain known associations between early experiences of maltreatment and later life health. The present study focuses on examining whether experiences of child maltreatment are associated with physiological reactions to initial and repeated psychosocial stress in adulthood. To this end, 44 healthy adults (52% male, aged 18-65) completed the Childhood Trauma Questionnaire to provide information about exposure to child maltreatment and completed the Trier Social Stress Test (TSST) on 2 consecutive days. Peripheral blood was collected prior to as well as 30 and 120 min following the TSST on each day. Plasma Interleukin-6 (IL-6) and gene expression of IL-6, IL-1ß, nuclear factor-kB (NF-kB), and inhibitor of kB (IkB) were measured from each blood sample. Total CTQ scores were unrelated to plasma IL-6 and gene expression (ps > .10) but a history of childhood physical neglect was associated with increased interleukin-1ß (ß =.35; p =.02; R2 =.19) and nuclear factor-kB (ß =.30; p =.046; R2 =.13) expression following initial stress. Following repeated exposure to the TSST, childhood physical neglect was associated with increased plasma IL-6 reactivity (ß =.34; p =.02; R2 =.16) and increased expression of nuclear factor-kB (ß =.31; p =.04; R2 =.08). Finally, childhood physical neglect was associated with decreased habituation following repeated exposure to the TSST. Other CTQ subscales were not related to plasma IL-6 and gene expression when considered individually. Results from this study are suggestive of a unique effect of childhood physical neglect on the physiological stress response following initial and repeated exposure to a common psychosocial stressor. This provides important directions for future research because the effect of childhood physical neglect on long-term neglect are not well understood and in need of further investigation.

Inhibition of Inflammatory Gene Transcription by IL-10 Is Associated with Rapid Suppression of Lipopolysaccharide-Induced Enhancer Activation.

IL-10 limits the magnitude of inflammatory gene expression following microbial stimuli and is essential to prevent inflammatory disease; however, the molecular basis for IL-10-mediated inhibition remains elusive. Using a genome-wide approach, we demonstrate that inhibition of transcription is the primary mechanism for IL-10-mediated suppression in LPS-stimulated macrophages and that inhibited genes can be divided into two clusters. Genes in the first cluster are inhibited only if IL-10 is included early in the course of LPS stimulation and is strongly enriched for IFN-inducible genes. Genes in the second cluster can be rapidly suppressed by IL-10 even after transcription is initiated, and this is associated with suppression of LPS-induced enhancer activation. Interestingly, the ability of IL-10 to rapidly suppress active transcription exhibits a delay following LPS stimulation. Thus, a key pathway for IL-10-mediated suppression involves rapid inhibition of enhancer function during the secondary phase of the response to LPS.

Increased alpha-amylase response to an acute psychosocial stress challenge in healthy adults with childhood adversity.

Childhood adversity is highly prevalent and linked to lasting psychological and physiological consequences. A potential mechanism for negative health outcomes is altered stress reactivity. While previous research has addressed associations of childhood adversity with stress system reactivity, sympathetic nervous system (SNS) stress reactivity is understudied. We therefore set out here to examining salivary alpha-amylase (sAA) reactivity in relation with childhood adversity. Forty-one healthy adult subjects (n = 24 male; n = 17 female) aged 18-34 years underwent the Trier Social Stress Test (TSST) and completed the Childhood Trauma Questionnaire (CTQ). Saliva for measurement of sAA was collected at three time points; before the TSST, immediately after, and 10 min post-TSST. We found that those with childhood trauma had a higher overall sAA response to the TSST, as seen in a repeated measures ANOVA (CTQ by time interaction: F(1.8,71.5) = 6.46, p = .01) and an independent samples t-test indicating higher sAA baseline to peak response (t = 3.22, p = .003). There was also a positive correlation between sAA reactivity and the CTQ subscales of childhood physical abuse (r = .46, p = .005) and emotional abuse (r = .37, p = .024). Healthy adults with low-to-moderate childhood adversity had a heightened sAA response immediately following the stressor. Higher SNS reactivity could be a link to negative health outcomes in adults with early adversity. Future research should address whether altered sAA reactivity is predictive of negative health outcomes in those with childhood adversity.

Steroid-independent male sexual behavior in B6D2F2 male mice.

It is well established that male sexual behavior (MSB) is regulated by gonadal steroids; however, individual differences in MSB, independent of gonadal steroids, are prevalent across a wide range of species, and further investigation is necessary to advance our understanding of steroid-independent MSB. Studies utilizing B6D2F1 hybrid male mice in which a significant proportion retain MSB after long-term orchidectomy, identified as steroid-independent-maters (SI-maters), have begun to unravel the genetic underpinnings of steroid-independent MSB. A recent study demonstrated that steroid-independent MSB is a heritable behavioral phenotype that is mainly passed down from B6D2F1 hybrid SI-maters when crossed with C57BL6J female mice. To begin to uncover whether the strain of the dam plays a role in the inheritance of steroid-independent MSB, B6D2F1 hybrid females were crossed with B6D2F1 hybrid males. While the present study confirms the finding that steroid-independent MSB is a heritable behavioral phenotype and that SI-mater sires are more likely to pass down some components of MSB than SI-non-maters to their offspring, it also reveals that the B6D2F2 male offspring that were identified as SI-maters that displayed the full repertoire of steroid-independent MSB had the same probability of being sired from either a B6D2F1 SI-mater or SI-non-mater. These results, in conjunction with previous findings, indicate that the specific chromosomal loci pattern that codes for steroid-independent MSB in the B6D2F2 male offspring may result regardless of whether the father was a SI-mater or SI-non-mater, and that the maternal strain may be an important factor in the inheritance of steroid-independent MSB.

Inheritance of steroid-independent male sexual behavior in male offspring of B6D2F1 mice.

The importance of gonadal steroids in modulating male sexual behavior is well established. Individual differences in male sexual behavior, independent of gonadal steroids, are prevalent across a wide range of species, including man. However, the genetic mechanisms underlying steroid-independent male sexual behavior are poorly understood. A high proportion of B6D2F1 hybrid male mice demonstrates steroid-independent male sexual behavior (identified as "maters"), providing a mouse model that opens up avenues of investigation into the mechanisms regulating male sexual behavior in the absence of gonadal hormones. Recent studies have revealed several proteins that play a significant factor in regulating steroid-independent male sexual behavior in B6D2F1 male mice, including amyloid precursor protein (APP), tau, and synaptophysin. The specific goals of our study were to determine whether steroid-independent male sexual behavior was a heritable trait by determining if it was dependent upon the behavioral phenotype of the B6D2F1 sire, and whether the differential expression of APP, tau, and synaptophysin in the medial preoptic area found in the B6D2F1 sires that did and did not mate after gonadectomy was similar to those found in their male offspring. After adult B6D2F1 male mice were bred with C57BL/6J female mice, they and their male offspring (BXB1) were orchidectomized and identified as either maters or "non-maters". A significant proportion of the BXB1 maters was sired only from B6D2F1 maters, indicating that the steroid-independent male sexual behavior behavioral phenotype of the B6D2F1 hybrid males, when crossed with C57BL/6J female mice, is inherited by their male offspring. Additionally, APP, tau, and synaptophysin were elevated in in the medial preoptic area in both the B6D2F1 and BXB1 maters relative to the B6D2F1 and BXB1 non-maters, respectively, suggesting a potential genetic mechanism for the inheritance of steroid-independent male sexual behavior.

Self-compassionate young adults show lower salivary alpha-amylase responses to repeated psychosocial stress.

In this study we tested the hypothesis that participants higher in dispositional self-compassion would show lower stress-induced reactivity of salivary alpha-amylase (sAA), a marker of sympathetic nervous system activation. Thirty-three healthy participants (18-34 years old) were exposed to a standardized laboratory stressor on two consecutive days. Self-compassion, self-esteem, and demographic factors were assessed by questionnaire and sAA was assessed at baseline and at 1, 10, 30, and 60 minutes following each stressor. Self-compassion was a significant negative predictor of sAA responses on both days. This relationship remained significant when controlling for self-esteem, subjective distress, age, gender, ethnicity, and Body Mass Index (BMI). These results suggest that self-compassion may serve as a protective factor against stress-induced physiological changes that have implications for health.

Response and habituation of pro- and anti-inflammatory gene expression to repeated acute stress.

INTRODUCTION: Acute stress induces increases in plasma inflammatory mediators, which do not habituate to repeated stress. Inflammation is a risk factor for age-related illnesses, highlighting the need to understand factors controlling inflammation. No studies have examined changes in pro- and anti-inflammatory gene expression in response to repeated acute stress in humans. METHODS: RNA was isolated from peripheral blood before, 30 and 120min after exposure of n=32 healthy human participants to the Trier Social Stress Test (TSST) on two days. Gene expression of interleukin (IL)-6, IL-1ß, nuclear factor (NF)-κB and IκB was measured repeatedly on both days. We further assessed leukocyte numbers, plasma IL-6, and salivary cortisol. RESULTS: Stress induced IL-6 (F=44.7; p<0.001) and cortisol responses (F=18.6; p<0.001). Cortisol responses habituated (F=5.1, p=0.003), but IL-6 responses did not (n.s.). All genes increased in response to initial stress (IL-6: F=3.8; p=0.029; IL-1ß: F=7.1; p=0.008; NF-κB: F=5.1; p=0.009; IκB: F=4.7; p=0.013) and showed habituation to repeated stress (IL-6: t=2.3; p=0.03; IL-1ß: t=3.9; p=0.001; NF-κB: t=2.1; p=0.041; IκB: t=3.1; p=0.005). Day 1 responses of IL-1ß and IκB were not explained by changes in leukocyte populations, but IL-6 and NF-κB, as well as most day 2 changes were not independent of leukocyte populations. CONCLUSIONS: Stress response and habituation of pro- and anti-inflammatory gene expression as found here might indicate that even on an intracellular level, inflammatory responses to acute stress are adaptive in that they respond to initial, but habituate to repeated, similar stress. Future studies will need to test whether non-habituation is predictive of disease.

Unraveling the contributions of hydrogen-bonding interactions to the activity of native and non-native ligands in the quorum-sensing receptor LasR.

Quorum sensing (QS) via the synthesis and detection of N-acyl L-homoserine lactone (AHL) signals regulates important pathogenic and mutualistic phenotypes in many bacteria. Over the past two decades, the development of non-native molecules that modulate this cell-cell signaling process has become an active area of research. The majority of these compounds were designed to block binding of the native AHL signal to its cognate LuxR-type receptor, and much effort has focused on LasR in the opportunistic pathogen Pseudomonas aeruginosa. Despite a small set of reported LasR structural data, it remains unclear which polar interactions are most important for either (i) activation of the LasR receptor by its native AHL signal, N-(3-oxo)-dodecanoyl L-homoserine lactone (OdDHL), or (ii) activation or inhibition of LasR by related AHL analogs. Herein, we report our investigations into the activity of OdDHL and five synthetic analogs in wild-type LasR and in nine LasR mutants with modifications to key polar residues in their ligand binding sites. Our results allowed us to rank, for the first time, the relative importance of each LasR:OdDHL hydrogen bond for LasR activation and provide strong evidence for the five synthetic ligands binding LasR in a very similar orientation as OdDHL. By delineating the specific molecular interactions that are important for LasR modulation by AHLs, these findings should aid in the design of new synthetic modulators of LasR (and homologous LuxR-type receptors) with improved potencies and selectivities.

Mutational analysis of the quorum-sensing receptor LasR reveals interactions that govern activation and inhibition by nonlactone ligands.

Gram-negative bacteria use N-acyl L-homoserine lactone (AHL) quorum-sensing (QS) signals to regulate the expression of myriad phenotypes. Non-native AHL analogs can strongly attenuate QS receptor activity and thereby QS signaling; however, we currently lack a molecular understanding of the mechanisms by which most of these compounds elicit their agonistic or antagonistic profiles. In this study, we investigated the origins of striking activity profile switches (i.e., receptor activator to inhibitor, and vice versa) observed upon alteration of the lactone head group in certain AHL analogs. Reporter gene assays of mutant versions of the Pseudomonas aeruginosa QS receptor LasR revealed that interactions between the ligands and Trp60, Tyr56, and Ser129 govern whether these ligands behave as LasR activators or inhibitors. Using this knowledge, we propose a model for the modulation of LasR by AHL analogs-encompassing a subtly different interaction with the binding pocket to a global change in LasR conformation.

Measures of adiposity predict interleukin-6 responses to repeated psychosocial stress.

OBJECTIVE: Overweight and obese individuals, who comprise approximately two-thirds of the U.S. population, are at increased risk for developing a range of diseases. This increased risk may be due in part to maladaptive stress responses within this group, including heightened low-grade inflammation and HPA axis non-habituation. In this study we tested the relationship between adiposity, plasma interleukin-6 (IL-6) and HPA axis responses to repeated stress. METHODS: Sixty-seven healthy participants were exposed to the Trier Social Stress Test (TSST) on two consecutive days. We collected saliva for cortisol measurements at baseline and at 1, 10, 30, 60 and 120min post-TSST, and blood for plasma IL-6 measurements at baseline and 30 and 120min post-TSST. RESULTS: Stress exposure induced significant increases of cortisol and IL-6 on both days (cortisol: F=38, p<0.001; IL-6: F=90.8; p<0.001), and repeated exposure was related with cortisol habituation (F=8.2; p<0.001) and IL-6 sensitization (F=5.2; p=0.022). BMI and body fat were related with higher cortisol responses to repeated stress (BMI: beta=0.34; p=0.014; body fat: beta=0.29; p=0.045), and with higher IL-6 responses to repeated stress (BMI: beta=0.27, p=0.044; body fat: beta=0.37; p=0.006). CONCLUSIONS: Taken together, individuals with higher measures of adiposity showed less efficient HPA axis habituation as well as sensitization of IL-6 responses to repeated acute stress. These findings point to maladaptive stress response patterns in overweight humans, which, through exposure to higher levels of inflammatory mediators, might partially explain diseases related with overweight and/or obesity.

Non-native N-aroyl L-homoserine lactones are potent modulators of the quorum sensing receptor RpaR in Rhodopseudomonas palustris.

Quorum sensing (QS) is a process by which bacteria use low-molecular-weight signaling molecules (or autoinducers) to assess their local population densities and alter gene expression levels at high cell numbers. Many Gram-negative bacteria use N-acyl L-homoserine lactones (AHLs) with aliphatic acyl groups as signaling molecules for QS. However, bacteria that utilize AHLs with aroyl acyl groups have been recently discovered; they include the metabolically versatile soil bacterium Rhodopseudomonas palustris, which uses p-coumaroyl HL (p-cAHL) as its QS signal. This autoinducer is especially unusual because its acyl group is believed to originate from a monolignol (i.e., p-coumarate) produced exogenously by plants in the R. palustris environment, rather than through the endogenous fatty acid biosynthesis pathway like other native AHLs. As such, p-cAHL could signal not only bacterial density, but also the availability of an exogenous plant-derived substrate and might even constitute an interkingdom signal. Like other Gram-negative bacteria, QS in R. palustris is controlled by the p-cAHL signal binding its cognate LuxR-type receptor, RpaR. We sought to determine if non-native aroyl HLs (ArHLs) could potentially activate or inhibit RpaR in R. palustris, and thereby modulate QS in this bacterium. Herein, we report the testing of a set of synthetic ArHLs for RpaR agonism and antagonism by using a R. palustris reporter strain. Several potent non-native RpaR agonists and antagonists were identified. Additionally, the screening data revealed that lower concentrations of ArHL are required to strongly agonize RpaR than to antagonize it. Structure-activity relationship analyses of the active ArHLs indicated that potent RpaR agonists tend to have sterically small substituents on their aryl groups, most notably in the ortho position. In turn, the most potent RpaR antagonists were based on either the phenylpropionyl HL (PPHL) or the phenoxyacetyl HL (POHL) scaffold, and many contained an electron-withdrawing group at either the meta or para positions of the aryl ring. To our knowledge, the compounds reported herein represent the first abiotic chemical modulators of RpaR, and more generally, the first abiotic ligands capable of intercepting QS in bacteria that utilize native ArHL signals. In view of the origins of the p-cAHL signal in R. palustris, the largely unknown role of QS in this bacterium, and R. palustris' unique environmental lifestyles, we anticipate that these compounds could be valuable as chemical probes to study QS in R. palustris in a range of fundamental and applied contexts.

Increased dendritic spine density and tau expression are associated with individual differences in steroidal regulation of male sexual behavior.

Male sexual behavior (MSB) is modulated by gonadal steroids, yet this relationship is highly variable across species and between individuals. A significant percentage (~30%) of B6D2F1 hybrid male mice demonstrate MSB after long-term orchidectomy (herein after referred to as "maters"), providing an opportunity to examine the mechanisms that underlie individual differences in steroidal regulation of MSB. Use of gene expression arrays comparing maters and non-maters has provided a first pass look at the genetic underpinnings of steroid-independent MSB. Surprisingly, of the ~500 genes in the medial preoptic area (MPOA) that differed between maters and non-maters, no steroid hormone or receptor genes were differentially expressed between the two groups. Interestingly, best known for their association with Alzheimer's disease, amyloid precursor protein (APP) and the microtubule-associated protein tau (MAPT) were elevated in maters. Increased levels of their protein products (APP and tau) in their non-pathological states have been implicated in cell survival, neuroprotection, and supporting synaptic integrity. Here we tested transgenic mice that overexpress tau and found facilitated mounting and intromission behavior after long-term orchidectomy relative to littermate controls. In addition, levels of synaptophysin and spinophilin, proteins generally enriched in synapses and dendritic spines respectively, were elevated in the MPOA of maters. Dendritic morphology was also assessed in Golgi-impregnated brains of orchidectomized B6D2F1 males, and hybrid maters exhibited greater dendritic spine density in MPOA neurons. In sum, we show for the first time that retention of MSB in the absence of steroids is correlated with morphological differences in neurons.

Environmental enrichment effects on the neurobehavioral profile of selective outbred trait anxiety rats.

Environmental enrichment attenuates the response to psychostimulants and has been shown to reduce both anxiety and stress-related behaviors. Since stress is a major vulnerability factor for addiction, we investigated whether enrichment could reverse stress profiles in high anxious rats as well as reduce their amphetamine sensitivity. Using selectively-bred high and low anxiety males (filial 3) from enriched, social or isolated environments, we tested elevated plus maze exploration, novelty place preference and amphetamine (AMPH; 0.5mg/kg, IP)-induced hyperactivity. We measured plasma corticosterone (CORT) response after forced novel object exposure, phosphorylation of the tropomyosin-related kinase B receptor (pTrkB) in the hippocampus and striatum, and dopamine (D2) receptor mRNA levels in the striatum and nucleus accumbens. Results indicate that high anxiety animals reared in social or enriched environments spent more time on open arms of the EPM while low anxiety animals raised in enriched environments spent more time on open arms when compared to either isolated or social groups. There were no group differences or interactions found for novelty place preference. Enriched environments decreased the response to AMPH and stress-induced CORT regardless of trait but selectively decreased pTrkB and increased D2 mRNA levels in high anxiety animals. The results suggest that selectively-bred trait anxiety rats show state anxiety that is influenced by rearing environments, and D2 protein levels and BDNF/TrkB signaling may differentially contribute to integrating these effects.

Thiolactone modulators of quorum sensing revealed through library design and screening.

Quorum sensing (QS) is a process by which bacteria use small molecules or peptidic signals to assess their local population densities. At sufficiently high density, bacteria can alter gene expression levels to regulate group behaviors involved in a range of important and diverse phenotypes, including virulence factor production, biofilm formation, root nodulation, and bioluminescence. Gram-negative bacteria most commonly use N-acylated l-homoserine lactones (AHLs) as their QS signals. The AHL lactone ring is hydrolyzed relatively rapidly at biological pH, and the ring-opened product is QS inactive. We seek to identify AHL analogues with heightened hydrolytic stability, and thereby potentially heightened activity, for use as non-native modulators of bacterial QS. As part of this effort, we probed the utility of thiolactone analogues in the current study as QS agonists and antagonists in Gram-negative bacteria. A focused library of thiolactone analogs was designed and rapidly synthesized in solution. We examined the activity of the library as agonists and antagonists of LuxR-type QS receptors in Pseudomonas aeruginosa (LasR), Vibrio fischeri (LuxR), and Agrobacterium tumefaciens (TraR) using bacterial reporter strains. The thiolactone library contained several highly active compounds, including some of the most active LuxR inhibitors and the most active synthetic TraR agonist reported to date. Analysis of a representative thiolactone analog revealed that its hydrolysis half-life was almost double that of its parent AHL in bacterial growth medium.

Design, synthesis, and biological evaluation of abiotic, non-lactone modulators of LuxR-type quorum sensing.

Quorum sensing (QS) is a cell-cell signaling mechanism that allows bacteria to monitor their population size and alter their behavior at high cell densities. Gram-negative bacteria use N-acylated L-homoserine lactones (AHLs) as their primary signals for QS. These signals are susceptible to lactone hydrolysis in biologically relevant media, and the ring-opened products are inactive QS signals. We have previously identified a range of non-native AHLs capable of strongly agonizing and antagonizing QS in Gram-negative bacteria. However, these abiotic AHLs are also prone to hydrolysis and inactivation and thereby have a relatively short time window for use (∼12-48 h). Non-native QS modulators with reduced or no hydrolytic instability could have enhanced potencies and would be valuable as tools to study the mechanisms of QS in a range of environments (for example, on eukaryotic hosts). This study reports the design and synthesis of two libraries of new, non-hydrolyzable AHL mimics. The libraries were screened for QS modulatory activity using LasR, LuxR, and TraR bacterial reporter strains, and several new, abiotic agonists and antagonists of these receptors were identified.

Copper-catalyzed rearrangement of vinyl oxiranes.

A novel copper-catalyzed vinyl oxirane ring expansion protocol has been developed. A wide range of vinyl oxiranes can be rearranged to 2,5-dihydrofurans in excellent yields in the presence of electrophilic copper(II) acetylacetonate catalysts. Regioisomeric vinyl oxiranes can be converted to a single dihydrofuran product using these conditions. This method uses low catalyst loadings (0.5-5 mol %), has good tolerance of substitution patterns, and can be done in the absence of solvent.

Molecular structure of lathyritol, a galactosylbornesitol from Lathyrus odoratus seeds, by NMR.

The molecular structure of galactosyl-D-(-)-bornesitol, a novel compound isolated from sweet pea seeds, was determined to be alpha-D-galactopyranosyl-(1-->3)-1-O-methyl-1D-myo-inositol by 1D and 2D NMR spectroscopy and is assigned the trivial name lathyritol.