Pathways for Memory, Cognition and Emotional Context: Hippocampal, Subgenual Area 25, and Amygdalar Axons Show Unique Interactions in the Primate Thalamic Reuniens Nucleus.

The reuniens nucleus (RE) is situated at the most ventral position of the midline thalamus. In rats and mice RE is distinguished by bidirectional connections with the hippocampus and medial prefrontal cortex (mPFC) and a role in memory and cognition. In primates, many foundational questions pertaining to RE remain unresolved. We addressed these issues by investigating the composition of the rhesus monkey RE in both sexes by labeling for GABA, a marker of inhibitory neurons, and for the calcium-binding proteins parvalbumin (PV), calbindin (CB), and calretinin (CR), which label thalamic excitatory neurons that project to cortex. As in rats and mice, the macaque RE was mostly populated by CB and CR neurons, characteristic of matrix-dominant nuclei, and had bidirectional connections with hippocampus and mPFC area 25 (A25). Unlike rodents, we found GABAergic neurons in the monkey RE and a sparser but consistent population of core-associated thalamocortical PV neurons. RE had stronger connections with the basal amygdalar complex than in rats or mice. Amygdalar terminations were enriched with mitochondria and frequently formed successive synapses with the same postsynaptic structures, suggesting an active and robust pathway to RE. Significantly, hippocampal pathways formed multisynaptic complexes that uniquely involved excitatory projection neurons and dendrites of local inhibitory neurons in RE, extending this synaptic principle beyond sensory to high-order thalamic nuclei. Convergent pathways from hippocampus, A25, and amygdala in RE position it to flexibly coordinate activity for memory, cognition, and emotional context, which are disrupted in several psychiatric and neurologic diseases in humans.SIGNIFICANCE STATEMENT The primate RE is a central node for memory and cognition through connections with the hippocampus and mPFC. As in rats or mice, the primate RE is a matrix-dominant thalamic nucleus, suggesting signal traffic to the upper cortical layers. Unlike rats or mice, the primate RE contains inhibitory neurons, synaptic specializations with the hippocampal pathway, and robust connections with the amygdala, suggesting unique adaptations. Convergence of hippocampal, mPFC, and amygdalar pathways in RE may help unravel a circuit basis for binding diverse signals for conscious flexible behaviors and the synthesis of memory with affective significance in primates, whereas disruption of distinct circuit nodes may occur in psychiatric disorders in humans.

Trust and Communication: Cornerstones of Care.

In this issue of the North Carolina Medical Journal, authors explore and debate the ways communication can influ-ence how North Carolinians access health care in our state. Articles illustrate the importance of increasing trust in health care providers and systems at a time when care is changing dramatically, and patients have more access to information than they ever have.

Broad-spectrum in vitro activity of macrophage infectivity potentiator inhibitors against Gram-negative bacteria and Leishmania major.

BACKGROUND: The macrophage infectivity potentiator (Mip) protein, which belongs to the immunophilin superfamily, is a peptidyl-prolyl cis/trans isomerase (PPIase) enzyme. Mip has been shown to be important for virulence in a wide range of pathogenic microorganisms. It has previously been demonstrated that small-molecule compounds designed to target Mip from the Gram-negative bacterium Burkholderia pseudomallei bind at the site of enzymatic activity of the protein, inhibiting the in vitro activity of Mip. OBJECTIVES: In this study, co-crystallography experiments with recombinant B. pseudomallei Mip (BpMip) protein and Mip inhibitors, biochemical analysis and computational modelling were used to predict the efficacy of lead compounds for broad-spectrum activity against other pathogens. METHODS: Binding activity of three lead compounds targeting BpMip was verified using surface plasmon resonance spectroscopy. The determination of crystal structures of BpMip in complex with these compounds, together with molecular modelling and in vitro assays, was used to determine whether the compounds have broad-spectrum antimicrobial activity against pathogens. RESULTS: Of the three lead small-molecule compounds, two were effective in inhibiting the PPIase activity of Mip proteins from Neisseria meningitidis, Klebsiella pneumoniae and Leishmania major. The compounds also reduced the intracellular burden of these pathogens using in vitro cell infection assays. CONCLUSIONS: These results indicate that Mip is a novel antivirulence target that can be inhibited using small-molecule compounds that prove to be promising broad-spectrum drug candidates in vitro. Further optimization of compounds is required for in vivo evaluation and future clinical applications.

Duration of American Football Play and Chronic Traumatic Encephalopathy.

OBJECTIVE: Chronic traumatic encephalopathy (CTE) is a neurodegenerative disease associated with exposure to contact and collision sports, including American football. We hypothesized a dose-response relationship between duration of football played and CTE risk and severity. METHODS: In a convenience sample of 266 deceased American football players from the Veterans Affairs-Boston University-Concussion Legacy Foundation and Framingham Heart Study Brain Banks, we estimated the association of years of football played with CTE pathological status and severity. We evaluated the ability of years played to classify CTE status using receiver operating characteristic curve analysis. Simulation analyses quantified conditions that might lead to selection bias. RESULTS: In total, 223 of 266 participants met neuropathological diagnostic criteria for CTE. More years of football played were associated with having CTE (odds ratio [OR] = 1.30 per year played, 95% confidence interval [CI] = 1.19-1.41; p = 3.8 × 10-9 ) and with CTE severity (severe vs mild; OR = 1.14 per year played, 95% CI = 1.07-1.22; p = 3.1 × 10-4 ). Participants with CTE were 1/10th as likely to have played <4.5 years (negative likelihood ratio [LR] = 0.102, 95% CI = 0.100-0.105) and were 10 times as likely to have played >14.5 years (positive LR = 10.2, 95% CI = 9.8-10.7) compared with participants without CTE. Sensitivity and specificity were maximized at 11 years played. Simulation demonstrated that years played remained adversely associated with CTE status when years played and CTE status were both related to brain bank selection across widely ranging scenarios. INTERPRETATION: The odds of CTE double every 2.6 years of football played. After accounting for brain bank selection, the magnitude of the relationship between years played and CTE status remained consistent. ANN NEUROL 2020;87:116-131.

Early specialist palliative care on quality of life for malignant pleural mesothelioma: a randomised controlled trial.

PURPOSE: Malignant pleural mesothelioma (MPM) has a high symptom burden and poor survival. Evidence from other cancer types suggests some benefit in health-related quality of life (HRQoL) with early specialist palliative care (SPC) integrated with oncological services, but the certainty of evidence is low. METHODS: We performed a multicentre, randomised, parallel group controlled trial comparing early referral to SPC versus standard care across 19 hospital sites in the UK and one large site in Western Australia. Participants had newly diagnosed MPM; main carers were additionally recruited. INTERVENTION: review by SPC within 3 weeks of allocation and every 4 weeks throughout the study. HRQoL was assessed at baseline and every 4 weeks with the European Organization for Research and Treatment of Cancer (EORTC) Quality of Life Questionnaire Core 30. PRIMARY OUTCOME: change in EORTC C30 Global Health Status 12 weeks after randomisation. RESULTS: Between April 2014 and October 2016, 174 participants were randomised. There was no significant between group difference in HRQoL score at 12 weeks (mean difference 1.8 (95% CI -4.9 to 8.5; p=0.59)). HRQoL did not differ at 24 weeks (mean difference -2.0 (95% CI -8.6 to 4.6; p=0.54)). There was no difference in depression/anxiety scores at 12 weeks or 24 weeks. In carers, there was no difference in HRQoL or mood at 12 weeks or 24 weeks, although there was a consistent preference for care, favouring the intervention arm. CONCLUSION: There is no role for routine referral to SPC soon after diagnosis of MPM for patients who are cared for in centres with good access to SPC when required. TRIAL REGISTRATION NUMBER: ISRCTN18955704.

Development, synthesis and structure-activity-relationships of inhibitors of the macrophage infectivity potentiator (Mip) proteins of Legionella pneumophila and Burkholderia pseudomallei.

The bacteria Burkholderia pseudomallei and Legionella pneumophila cause severe diseases like melioidosis and Legionnaire's disease with high mortality rates despite antibiotic treatment. Due to increasing antibiotic resistances against these and other Gram-negative bacteria, alternative therapeutical strategies are in urgent demand. As a virulence factor, the macrophage infectivity potentiator (Mip) protein constitutes an attractive target. The Mip proteins of B. pseudomallei and L. pneumophila exhibit peptidyl-prolyl cis/trans isomerase (PPIase) activity and belong to the PPIase superfamily. In previous studies, the pipecolic acid moiety proved to be a valuable scaffold for inhibiting this PPIase activity. Thus, a library of pipecolic acid derivatives was established guided by structural information and computational analyses of the binding site and possible binding modes. Stability and toxicity considerations were taken into account in iterative extensions of the library. Synthesis and evaluation of the compounds in PPIase assays resulted in highly active inhibitors. The activities can be interpreted in terms of a common binding mode obtained by docking calculations.

Perils and pitfalls for clinicians embarking on qualitative research in physiotherapy.

AIM: To consider the pros and cons of focus groups versus interviews for studies interested in examining patient experiences of clinical interventions. The paper looks at the hazards of being a clinician when collecting qualitative data and shares these experiences to provide useful learning for other clinicians embarking on qualitative approaches. BACKGROUND: Sub-acromial decompression surgery (SAD) is the accepted, surgical intervention for shoulder impingement syndrome. Evidence suggests that outcomes from SAD are no more superior to conservative management. This raises questions as to whether alternative explanations such as patient experience are at play when considering patient outcomes. DATA SOURCES: A study looking at patients' experiences of subacromial decompression surgery six months after the operation. REVIEW METHODS: One small focus group and one individual interview took place to explore patient experience following sub-acromial decompression shoulder surgery. DISCUSSION: Focus groups risk producing competitive and comparative discussions, and clinical researchers require sufficient training and mentoring to recognise and assist group dynamics. The study exposed ways in which clinicians involved in collecting data may be injured to aspects of patients' experiences, and accordingly may not explore in depth aspects important to patients. CONCLUSION: This paper highlights the importance of novice researchers thinking carefully about the capacity for 'practitioner eyes' to influence analytical decisions about study design and the direction of data collection. Focus group interactions are complex and risk being underestimated by inexperienced clinical researchers. IMPLICATIONS FOR RESEARCH/PRACTICE: Novice researchers are advised to be open to the possibility that unpredictable situations are likely to occur and expose assumptions about the study design. Preparation and guidance is required to avoid some of the challenges and manage group dynamics effectively in the first instance.

CASPASE-12 and rheumatoid arthritis in African-Americans.

CASPASE-12 (CASP12) has a downregulatory function during infection and thus may protect against inflammatory disease. We investigated the distribution of CASP12 alleles (#rs497116) in African-Americans (AA) with rheumatoid arthritis (RA). CASP12 alleles were genotyped in 953 RA patients and 342 controls. Statistical analyses comparing genotype groups were performed using Kruskal-Wallis non-parametric ANOVA with Mann-Whitney U tests and chi-square tests. There was no significant difference in the overall distribution of CASP12 genotypes within AA with RA, but CASP12 homozygous patients had lower baseline joint-narrowing scores. CASP12 homozygosity appears to be a subtle protective factor for some aspects of RA in AA patients.

The serine protease inhibitor Ecotin is required for full virulence of Burkholderia pseudomallei.

Burkholderia pseudomallei is a Gram negative soil saprophyte that causes the disease melioidosis where clinical symptoms can vary from localised infection to pneumonia and septic shock. Ecotin is a potent periplasmic serine protease inhibitor first identified in Escherichia coli. Ecotin, although present in only a small subset of genera, can inhibit a broad range of serine proteases including those typically associated with the innate immune system such as neutrophil elastase and cathepsin G. An Ecotin orthologue identified in B. pseudomallei was recombinantly expressed and found to inhibit elastase. To study the role of Ecotin in B. pseudomallei virulence an in-frame unmarked deletion mutant was created. Infection of a murine macrophages-like cell line revealed Ecotin was necessary for the early stages of colonisation allowing replication following cell entry. Attenuation of the Δeco mutant strain in the murine model of melioidosis further supported Ecotin as a virulence factor of B. pseudomallei.

Documentation of guideline adherence in antenatal records across maternal weight categories: a chart review.

BACKGROUND: Documentation in medical records fulfills key functions, including management of care, communication, quality assurance and record keeping. We sought to describe: 1) rates of standard prenatal care as documented in medical charts, and given the higher risks with excess weight, whether this documentation varied among normal weight, overweight and obese women; and 2) adherence to obesity guidelines for obese women as documented in the chart. METHODS: We conducted a chart review of 300 consecutive charts of women who delivered a live singleton at an academic tertiary centre from January to March 2012, computing Analysis of Variance and Chi Square tests. RESULTS: The proportion of completed fields on the mandatory antenatal forms varied from 100% (maternal age) to 52.7% (pre-pregnancy body mass index). Generally, documentation of care was similar across all weight categories for maternal and prenatal genetic screening tests, ranging from 54.0% (documentation of gonorrhea/chlamydia tests) to 85.0% (documentation of anatomy scan). Documentation of education topics varied widely, from fetal movement in almost all charts across all weight categories but discussion of preterm labour in only 20.6%, 12.7% and 13.4% of normal weight, overweight and obese women's charts (p = 0.224). Across all weight categories, documentation of discussion of exercise, breastfeeding and pain management occurred in less than a fifth of charts. CONCLUSION: Despite a predominance of excess weight in our region, as well as increasing perinatal risks with increasing maternal weight, weight-related issues and other elements of prenatal care were suboptimally documented across all maternal weight categories, despite an obesity guideline.

Pooling robustness in distance sampling: Avoiding bias when there is unmodelled heterogeneity.

The pooling robustness property of distance sampling results in unbiased abundance estimation even when sources of variation in detection probability are not modeled. However, this property cannot be relied upon to produce unbiased subpopulation abundance estimates when using a single pooled detection function that ignores subpopulations. We investigate by simulation the effect of differences in subpopulation detectability upon bias in subpopulation abundance estimates. We contrast subpopulation abundance estimates using a pooled detection function with estimates derived using a detection function model employing a subpopulation covariate. Using point transect survey data from a multispecies songbird study, species-specific abundance estimates are compared using pooled detection functions with and without a small number of adjustment terms, and a detection function with species as a covariate. With simulation, we demonstrate the bias of subpopulation abundance estimates when a pooled detection function is employed. The magnitude of the bias is positively related to the magnitude of disparity between the subpopulation detection functions. However, the abundance estimate for the entire population remains unbiased except when there is extreme heterogeneity in detection functions. Inclusion of a detection function model with a subpopulation covariate essentially removes the bias of the subpopulation abundance estimates. The analysis of the songbird point count surveys shows some bias in species-specific abundance estimates when a pooled detection function is used. Pooling robustness is a unique property of distance sampling, producing unbiased abundance estimates at the level of the study area even in the presence of large differences in detectability between subpopulations. In situations where subpopulation abundance estimates are required for data-poor subpopulations and where the subpopulations can be identified, we recommend the use of subpopulation as a covariate to reduce bias induced in subpopulation abundance estimates.

The effect of infliximab dose escalation in inflammatory bowel disease patients with antibodies to infliximab.

BACKGROUND: Infliximab dose escalation (DE) can be used in inflammatory bowel disease patient; however, the long-term benefit remains unclear, especially in those with antibodies to infliximab (ATI). The aim was to assess the effect of DE in patients with ATI on drug level, clinical response and ATI status. METHODS: All patients undergoing infliximab DE (a reduction in dose interval between infusions <8 weeks ± an increase in dose up to 10 mg/kg) at a referral centre between April 2016 and August 2019 were included. RESULTS: Ninety-two patients were DE: 51 were men, 50 had CD and 63 were receiving immunosuppression. A total of 87 people received DE for a median of 44 weeks (range 4-176). Five stopped infliximab after 1 dose of DE: 2 for loss of response and 3 for infusion reaction. In patients with ATI ≤10 vs. >10 AU/mL, DE significantly increased drug levels: median infliximab levels of 1.4 and 0.9 at baseline, respectively, to 3.2 and 3.5 at week 24. After DE, 21/35 ATI-positive patients had a fall in ATI ≤10 AU/mL. At week 24 following DE 62/92 patients were in clinical remission. Duration of clinical remission was shorter in those with ATI >10 AU/mL (median 24 weeks, range 0-88) than in those with transient/ATI ≤10 AU/mL (median 36 weeks, range 0-126, P = 0.06). CONCLUSIONS: A strategy of DE for selected patients receiving infliximab is associated with an increase in drug levels and reduced ATI positivity. This is associated with clinical remission in approximately 70% of patients at 6 months.

Processing of tumor antigen differentially impacts the development of helper and effector CD4+ T-cell responses.

CD4(+) T cells contribute to the antitumor T-cell response as both effectors that promote tumor rejection and helpers that facilitate the activation of other antitumor effector cells, such as CD8(+) T cells. Maximal engagement of both effector and helper CD4(+) T-cell responses is a desirable attribute of cancer vaccines. We have employed the B16F10 murine melanoma model and a series of recombinant adenovirus (Ad) vaccines expressing mutant forms of the tumor antigen, dopachrome tautomerase, to investigate the relationship between antigen processing and the antitumor CD4(+) T-cell response. Our results have revealed an unexpected dichotomy in the generation of helper and effector CD4(+) T-cell responses where CD4(+) T effector responses are dependent upon protein processing and trafficking, whereas CD4(+) T helper responses are not. The results have important implications for strategies aimed at augmenting antigen immunogenicity by altering intracellular processing and localization.

Retrospective evaluation of hyperproteinorrachia without pleocytosis (albuminocytologic dissociation) and survival in dogs.

BACKGROUND: Hyperproteinorrachia (raised cerebrospinal fluid total protein [CSF-TP]) without pleocytosis (HP) (also known as albuminocytologic dissociation) is identified in dogs with different neurologic diseases. However, the association between survival and increased CSF-TP is unknown. OBJECTIVES: (a) Identify conditions commonly associated with HP in dogs and (b) investigate whether higher CSF-TP concentrations or other relevant factors are associated with 1-year survival. METHODS: This is a retrospective study that identified dogs with HP (Cisternal CSF-TP >0.30 g/L, Lumbar CSF-TP >0.45 g/L with total nucleated cell concentrations [TNCCs] and RBC counts within RIs) from 2008 to 2019: recording signalment, weight, vital parameters, inflammation, neuroanatomic localization, CSF-TP, sampling site, final diagnosis, etiologic classification, and 1-year survival. Corrected CSF-TP was calculated as CSF-TP minus 0.3 (cisternal) or 0.45 (lumbar or unknown). Descriptive statistics were produced, CSF-TP differences between groups (eg, neuroanatomic localizations) were evaluated using the Mann-Whitney U test or Kruskal-Wallis test (post-hoc testing). The Cox proportional hazards model was used for survival data. Statistical significance was set at a P < 0.05. RESULTS: In all, 39 dogs had HP, associated with 17 conditions, including neoplasia (n = 6), meningoencephalitis of unknown origin (n = 4) (MUO), and intervertebral disc disease (n = 4) (IVDD) as the most common conditions. There was no significant difference between the CSF-TP/corrected CSF-TP between 1-year survivors and non-survivors, nor was there a difference between different neuroanatomic localizations or etiologic classifications (P > 0.05). Neoplasia, after adjustment for age, was the only variable associated with a worse survival (P = 0.01 HR: 2.08 (95% CI: 1.65-39.2). CSF-TP was not associated with age (P > 0.05). CONCLUSIONS: HP in dogs is associated with a wide range of conditions; the most common conditions are neoplasia, MUO, and IVDD. Higher CSF-TP levels do not correlate with a worse 1-year survival; however, they do correlate with neoplastic lesions.

Effects of Ketoprofen and Morphine on Pain-Related Depression of Nestlet Shredding in Male and Female Mice.

A primary goal in pain treatment is restoration of behaviors that are disrupted by pain. Measures of pain interference indicate the degree to which pain interferes with activities in pain patients, and these measures are used to evaluate the effects of analgesic drugs. As a result of the emphasis on the expression and treatment of functional impairment in clinical settings, preclinical pain researchers have attempted to develop procedures for evaluation of pain-related functional impairment in laboratory animals. The goal of the present study was to develop and validate a low cost procedure for the objective evaluation of pain-related depression of home cage behavior in mice. On test days, a 5 × 5 cm Nestlet was weighed prior to being suspended from the wire lid of the home cage of individually housed male and female ICR mice. Over the course of experimental sessions, mice removed pieces of the suspended Nestlet, and began to build a nest with the material they removed. Thus, the weight of the pieces of Nestlet that remained suspended at various time points in the session provided an indicator of the rate of this behavior. The results indicate that Nestlet shredding was stable with repeated testing, and shredding was depressed by intra-peritoneal injection of 0.32% lactic acid. The non-steroidal anti-inflammatory drug ketoprofen blocked 0.32% lactic acid-induced depression of shredding, but did not block depression of shredding by a pharmacological stimulus, the kappa opioid receptor agonist U69,593. The µ-opioid receptor agonist morphine did not block 0.32% lactic acid-induced depression of shredding when tested up to doses that depressed shredding in the absence of lactic acid. When noxious stimulus intensity was reduced by decreasing the lactic acid concentration to 0.18%, morphine was effective at blocking pain-related depression of behavior. In summary, the data from the present study support consideration of the Nestlet shredding procedure for use in studies examining mechanisms, expression, and treatment of pain-related functional impairment.

Values of cancer survivors and the supportive role of recreational video games.

Value-affirming activities have been linked to positive health outcomes and improved ability to cope. For cancer survivors who regularly play video games, might the games have potential to affirm values? We surveyed gameplaying survivors and included an open-ended question asking about values and the extent to which they perceived gameplaying as supporting values. A content analysis of responses (N = 533) using Schwartz's value typology revealed that a majority perceived gameplaying as supporting values or offering other benefits. Self-transcendence followed by openness to change were the most frequently coded higher-order categories. The results contribute to a richer understanding of survivors who gameplay.

Predicting survival in malignant pleural mesothelioma using routine clinical and laboratory characteristics.

INTRODUCTION: The prognosis of malignant pleural mesothelioma (MPM) is poor, with a median survival of 8-12 months. The ability to predict prognosis in MPM would help clinicians to make informed decisions regarding treatment and identify appropriate research opportunities for patients. The aims of this study were to examine associations between clinical and pathological information gathered during routine care, and prognosis of patients with MPM, and to develop a 6-month mortality risk prediction model. METHODS: A retrospective cohort study of patients diagnosed with MPM at Queen Alexandra Hospital, Portsmouth, UK between December 2009 and September 2013. Multivariate analysis was performed on routinely available histological, clinical and laboratory data to assess the association between different factors and 6-month survival, with significant associations used to create a model to predict the risk of death within 6 months of diagnosis with MPM. RESULTS: 100 patients were included in the analysis. Variables significantly associated with patient survival in multivariate analysis were age (HR 1.31, 95% CI 1.09 to 1.56), smoking status (current smoker HR 3.42, 95% CI 1.11 to 4.20), chest pain (HR 2.14, 95% CI 1.23 to 3.72), weight loss (HR 2.13, 95% CI 1.18 to 3.72), platelet count (HR 1.05, 95% CI 1.00 to 1.10), urea (HR 2.73, 95% CI 1.31 to 5.69) and adjusted calcium (HR 1.47, 95% CI 1.10 to 1.94). The resulting risk model had a c-statistic value of 0.76. A Hosmer-Lemeshow test confirmed good calibration of the model against the original dataset. CONCLUSION: Risk of death at 6 months in patients with a confirmed diagnosis of MPM can be predicted using variables readily available in clinical practice. The risk prediction model we have developed may be used to influence treatment decisions in patients with MPM. Further validation of the model requires evaluation of its performance on a separate dataset.

The memory T cell response to West Nile virus in symptomatic humans following natural infection is not influenced by age and is dominated by a restricted set of CD8+ T cell epitopes.

We examined the West Nile virus (WNV)-specific T cell response in a cohort of 52 patients with symptomatic WNV infections, including neuroinvasive and non-invasive disease. Although all virus proteins were shown to contain T cell epitopes, certain proteins, such as E, were more commonly targeted by the T cell response. Most patients exhibited reactivity toward 3-4 individual WNV peptides; however, several patients exhibited reactivity toward >10 individual peptides. The relative hierarchy of T cell reactivities in all patients showed a fixed pattern that was sustained throughout the 12-mo period of the current study. Surprisingly, we did not observe any relationship between age and either the breadth or magnitude of the T cell response following infection. We also did not observe a relationship between disease severity and either the breadth or magnitude of the T cell response. The T cell epitopes were distributed in a non-random fashion across the viral polyprotein and a limited number of epitopes appeared to dominate the CD8(+) T cell response within our cohort. These data provide important new insight into the T cell response against WNV in humans.

First synthesis of [1,3,5-(13)C3]gallic acid.

An efficient and high-yielding synthesis for [1,3,5-(13)C(3)]gallic acid from non-aromatic precursors is presented. [3,5-(13)C(2)]4H-Pyran-4-one was first prepared from the reaction between triethyl orthoformate and [1,3-(13)C(2)]acetone. The third (13)C-atom was introduced into the ring by reaction of the pyranone with diethyl [2-(13)C]malonate. The resulting ethyl 4-hydroxy-[1,3,5-(13)C(3)]benzoate was brominated in the 3- and 5-positions to give ethyl 3,5-dibromo-4-hydroxy-[1,3,5-(13)C(3)]benzoate. Subsequent hydrolysis of the ester and substitution of the bromine atoms with hydroxyl groups was achieved under basic conditions in a single step to yield the desired [1,3,5-(13)C(3)]gallic acid. The synthesis of [2,6-(13)C(2)]4H-pyran-4-one is also presented to demonstrate the potential of the methodology for the regioselective placement of (13)C-atoms into benzene rings.

Elevated frequencies of self-reactive CD8+ T cells following immunization with a xenoantigen are due to the presence of a heteroclitic CD4+ T-cell helper epitope.

Immunization of mice with human dopachrome tautomerase (hDCT) provides greater protection against melanoma than immunization with the murine homologue (mDCT). We mapped the CD8(+) and CD4(+) T-cell epitopes in both proteins to better understand the mechanisms of the enhanced protection. The dominant CD8(+) T-cell epitopes were fully conserved between both proteins, yet immunization with hDCT produced frequencies of CD8(+) T cells that were 5- to 10-fold higher than immunization with mDCT. This difference was not intrinsic to the two proteins because comparable frequencies of CD8(+) T cells were elicited by both antigens in DCT-deficient mice. Strikingly, only hDCT elicited a significant level of specific CD4(+) T cells in wild-type (WT) mice. The murine protein was not devoid of CD4(+) T-cell epitopes because immunization of DCT-deficient mice with mDCT resulted in robust CD4(+) T-cell immunity directed against two epitopes that were not identified in WT mice. These results suggested that the reduced immunogenicity of mDCT in WT mice may be a function of insufficient CD4(+) T-cell help. To address this possibility, the dominant CD4(+) T-cell epitope from hDCT was introduced into mDCT. Immunization with the mutated mDCT evoked CD8(+) T-cell frequencies and protective immunity comparable with hDCT. These results reveal a novel mechanism by which xenoantigens overcome tolerance. Our data also suggest that immunologic tolerance is more stringent for CD4(+) T cells than CD8(+) T cells, providing a mechanism of peripheral tolerance where autoreactive CD8(+) T cells fail to be activated due to a lack of autoreactive CD4(+) T cells specific for the same antigen.