High MDR-1 expression by MAIT cells confers resistance to cytotoxic but not immunosuppressive MDR-1 substrates.

High expression of the ATP-binding cassette-multi-drug efflux protein 1 (MDR1) is a striking feature of mucosal-associated invariant T (MAIT) cells, a prominent human innate-like T cell subset. We demonstrate significantly higher MDR1 expression by CD8 + CD161 ++ Vα7.2 + MAIT cells than the phenotypically and functionally related CD8 + CD161 ++ Vα7.2-subset and show MDR1 expression to be similarly high throughout MAIT CD4 + , CD8 + , double-negative (DN) and double-positive (DP) cell subsets. We demonstrate the MAIT cell-predominant CD8+ CD161++ subset to uniquely and efficiently efflux the cytotoxic anthracycline daunorubicin, retain function on daunorubicin exposure and demonstrate MDR1-dependent protection from daunorubicin-induced apoptosis. By contrast, CD8+ CD161++ Vα7.2+ MAIT cells were not protected from the anti-proliferative and cytotoxic effects of the immunosuppressive MDR1 substrates tacrolimus and mycophenoic acid, although function following MAIT cell-specific T cell receptor (TCR)-dependent and -independent stimulation was preserved on in-vitro exposure to these agents. Overall, our data further define MDR1 expression by CD161++ T and MAIT cells and demonstrate the potential for high MDR1 expression by MAIT cells to confer resistance to cytotoxic MDR1 substrates in vivo . As our understanding of the importance of MAIT cells in human immunity and immunopathology grows, this is an important observation for clinical contexts such as the treatment of malignancy, autoimmunity and post-transplant immunosuppression.

CD161(int)CD8+ T cells: a novel population of highly functional, memory CD8+ T cells enriched within the gut.

The C-type lectin-like receptor CD161 is expressed by lymphocytes found in human gut and liver, as well as blood, especially natural killer (NK) cells, T helper 17 (Th17) cells, and a population of unconventional T cells known as mucosal-associated invariant T (MAIT) cells. The association of high CD161 expression with innate T-cell populations including MAIT cells is established. Here we show that CD161 is also expressed, at intermediate levels, on a prominent subset of polyclonal CD8+ T cells, including antiviral populations that display a memory phenotype. These memory CD161(int)CD8+ T cells are enriched within the colon and express both CD103 and CD69, markers associated with tissue residence. Furthermore, this population was characterized by enhanced polyfunctionality, increased levels of cytotoxic mediators, and high expression of the transcription factors T-bet and eomesodermin (EOMES). Such populations were induced by novel vaccine strategies based on adenoviral vectors, currently in trial against hepatitis C virus. Thus, intermediate CD161 expression marks potent polyclonal, polyfunctional tissue-homing CD8+ T-cell populations in humans. As induction of such responses represents a major aim of T-cell prophylactic and therapeutic vaccines in viral disease and cancer, analysis of these populations could be of value in the future.

Fluid administration, vasopressor use and patient outcomes in a group of high-risk cardiac surgical patients receiving postoperative goal-directed haemodynamic therapy: a pilot study.

The role of goal-directed therapy in high-risk cardiac surgical patients has not been determined. This study sought to observe the effect of a postoperative standardised haemodynamic protocol (SHP) on the administration of fluid and vasoactive drugs after high-risk cardiac surgery. This was an interventional pilot study. In 2010 to 2011, the SHP was introduced to the ICU at Wellington Regional Hospital, Wellington, New Zealand, for the perioperative management of patients undergoing high-risk cardiac surgery. A pulmonary artery catheter was inserted in the patients in the study group and fluids and supportive medications were provided in the ICU according to a protocol that targeted a cardiac index ≥ 2 l/min/m², mixed venous oxygen saturation ≥ 60% and a mean arterial pressure of 65 to 75 mmHg. Data from 40 consecutive high-risk cardiac surgical patients assigned to this protocol were compared with a matched cohort of 40 consecutive high-risk cardiac surgical patients receiving 'usual care' in 2009. Baseline characteristics were similar in the two groups. There was no significant difference in the duration of noradrenaline infusion in the SHP cohort compared to historical controls (median [IQR] 18.5 hours [31.63] versus 18 hours [18.3]; P=0.35), despite patients receiving more fluid in their first 12 hours in the ICU (mean 4687 ml [SD ± 2284 ml] versus 1889 ml [SD ± 1344 ml]; P <0.001). The SHP cohort had a higher rate of reintubation (4 in 37 [10.8%] versus 0 in 40 [0%]; P=0.049). The SHP delivered significantly more fluid, but did not reduce the duration of noradrenaline infusion, compared to usual care.

MAIT cells are licensed through granzyme exchange to kill bacterially sensitized targets.

Mucosal-associated invariant T (MAIT) cells are an innate-like T-cell population restricted by the non-polymorphic, major histocompatibility complex class I-related protein 1, MR1. MAIT cells are activated by a broad range of bacteria through detection of riboflavin metabolites bound by MR1, but their direct cytolytic capacity upon recognition of cognate target cells remains unclear. We show that resting human MAIT cells are uniquely characterized by a lack of granzyme (Gr) B and low perforin expression, key granule proteins required for efficient cytotoxic activity, but high levels of expression of GrA and GrK. Bacterial activation of MAIT cells rapidly induced GrB and perforin, licensing these cells to kill their cognate target cells. Using a novel flow cytometry-based killing assay, we show that licensed MAIT cells, but not ex vivo MAIT cells from the same donors, can efficiently kill Escherichia coli-exposed B-cell lines in an MR1- and degranulation-dependent manner. Finally, we show that MAIT cells are highly proliferative in response to antigenic and cytokine stimulation, maintaining high expression of GrB, perforin, and GrA, but reduced expression of GrK following antigenic proliferation. The tightly regulated cytolytic capacity of MAIT cells may have an important role in the control of intracellular bacterial infections, such as Mycobacterium tuberculosis.

CD8αα Expression Marks Terminally Differentiated Human CD8+ T Cells Expanded in Chronic Viral Infection.

The T cell co-receptor CD8αß enhances T cell sensitivity to antigen, however studies indicate CD8αα has the converse effect and acts as a co-repressor. Using a combination of Thymic Leukemia (TL) antigen tetramer, which directly binds CD8αα, anti-CD161, and anti-Vα7.2 antibodies we have been able for the first time to clearly define CD8αα expression on human CD8 T cells subsets. In healthy controls CD8αα is most highly expressed by CD161 "bright" (CD161++) mucosal associated invariant T (MAIT) cells, with CD8αα expression highly restricted to the TCR Vα7.2+ cells of this subset. We also identified CD8αα-expressing populations within the CD161 "mid" (CD161+) and "negative" (CD161-) non-MAIT CD8 T cell subsets and show TL-tetramer binding to correlate with expression of CD8ß at low levels in the context of maintained CD8α expression (CD8α+CD8ß(low)). In addition, we found CD161-CD8α+CD8ß(low) populations to be significantly expanded in the peripheral blood of HIV-1 and hepatitis B (mean of 47 and 40% of CD161- T cells respectively) infected individuals. Such CD8αα expressing T cells are an effector-memory population (CD45RA-, CCR7-, CD62L-) that express markers of activation and maturation (HLA-DR+, CD28-, CD27-, CD57+) and are functionally distinct, expressing greater levels of TNF-α and IFN-γ on stimulation and perforin at rest than their CD8α+CD8ß(high) counterparts. Antigen-specific T cells in HLA-B(∗)4201+HIV-1 infected patients are found within both the CD161-CD8α+CD8ß(high) and CD161-CD8α+CD8ß(low) populations. Overall we have clearly defined CD8αα expressing human T cell subsets using the TL-tetramer, and have demonstrated CD161-CD8α+CD8ß(low) populations, highly expanded in disease settings, to co-express CD8αß and CD8αα. Co-expression of CD8αα on CD8αß T cells may impact on their overall function in vivo and contribute to the distinctive phenotype of highly differentiated populations in HBV and HIV-1 infection.

Cowden syndrome.

Cowden syndrome (CS) is a rare inherited condition characterised by multiple hamartomas in a variety of tissues from all three embryonic layers. It is a cancer predisposition syndrome with an increased risk of developing malignancy in many tissues but especially breast, thyroid and endometrium. It is inherited in an autosomal dominant manner with ∼80% of patients having a germ-line mutation of the PTEN tumour suppressor gene. Presenting signs and symptoms are highly non-specific. Nevertheless clinicians should be able to recognise this syndrome so that patients may be screened for cancerous growths and afforded the opportunity to have genetic testing to assist them and their family members in making medical management decisions. We present a review of this unusual but important condition with particular emphasis on the diagnostic criteria, clinical features, genetics, management and surveillance.

T cell sensitivity and the outcome of viral infection.

The importance of CD8(+) T cells in the control of viral infections is well established. However, what differentiates CD8(+) T cell responses in individuals who control infection and those who do not is not well understood. 'Functional sensitivity' describes an important quality of the T cell response and is determined in part by the affinity of the T cell receptor for antigen. A more sensitive T cell response is generally believed to be more efficient and associated with better control of viral infection, yet may also drive viral mutation and immune escape. Various in vitro techniques have been used to measure T cell sensitivity; however, rapid ex vivo analysis of this has been made possible by the application of the 'magic' tetramer technology. Such tools have potentially important applications in the design and evaluation of vaccines.

Anti-Saccharomyces cerevisiae antibodies (ASCA) in Crohn's disease are associated with disease severity but not NOD2/CARD15 mutations.

Anti-Saccharomyces cerevisiae antibodies (ASCAs) have been proposed as serological markers, which may differentiate Crohn's disease (CD) from ulcerative colitis (UC) and predict disease phenotype. Their importance in pathogenesis is unproven. We investigated the relationship between ASCAs, disease phenotype and NOD2/CARD15 genotype in CD and whether ASCAs were related to antibodies to other fungal proteins. Serum from 228 patients [143 CD, 75 UC, 10 with indeterminate colitis (IC)] and 78 healthy controls (HC) were assayed for ASCA. Antibodies (IgA, IgG) to other fungal proteins (Fusarium species ATC20334, Mycoprotein) were measured in the same samples using an in-house enzyme-linked immunosorbent assay (ELISA) assay. ASCAs were present in 57% of CD, 19% of UC, 30% of IC and 8% of HCs. ASCA-positive status was a predictor for CD with sensitivity of 57%, specificity of 87%, positive predictive value of 78% and negative predictive value of 68%. ASCA was associated with proximal (gastroduodenal and small bowel involvement) rather than purely colonic disease (P < 0.001) and with a more severe disease phenotype and requirement for surgery over a median follow-up time of 9 years (P < 0.0001). No associations with NOD2/CARD15 mutations were seen. There was no association between ASCA and antibodies to MP (IgA or IgG). These data implicate ASCA as a specific marker of disease location and progression in CD, emphasizing the heterogeneity within IBD.

College students' attitudes about AIDS: 1986 to 2000.

College students' attitudes about AIDS and people with AIDS (PWAs) were measured over a 15-year period. The AIDS Attitude Scale, designed by Shrum, Turner, and Bruce (1989; AIDS Education and Prevention, 1, 222-230), was administered to introductory psychology students (n = 1,571) at one midsized southeastern university, thus allowing direct comparison of attitudes over time. Overall tolerance about AIDS and PWAs has increased from 1986 to 2000 and robust gender differences in attitudes have been apparent over time, with females expressing more tolerant attitudes. Concerns about contagion from casual contact are diminishing as well and perceived knowledge about AIDS has increased over time. Perceptions about personal susceptibility to HIV remain low and show little relationship to attitudes about AIDS and PWAs. These data may be used to help refine HIV prevention programs for college students and provide an example of a useful approach to monitor changes in attitudes over time.

Possible neuronal refractory or recovery artifacts associated with recording the mismatch negativity response.

The mismatch negativity (MMN) is an event-related potential that involves a negative voltage shift of baseline electroencephalographic (EEG) activity in the approximate latency window of the N1 and P2 cortical potentials in response to new or novel sounds. The MMN is present at birth and has been hypothesized to serve as an automatic preconscious detector of changes in the auditory environment. Research paradigms used to extract the MMN response from EEG activity have a potential problem related to neuronal refractoriness or recovery. Both N1 and P2 are known to increase in amplitude with longer interstimulus intervals (ISIs). The MMN extraction procedures involve mathematical subtraction of waveforms elicited by standard sounds (with short ISIs) from those recorded to rare deviant sounds (with longer ISIs). Any ISI-dependent amplitude changes in N1 and/or P2 could therefore alter the morphology of the resulting difference wave and lead to misinterpretation of the nature of the underlying MMN generators. We tested 12 young females and found that the MMN can be influenced by ISI-dependent refractory effects that may modify the waveform morphology. This has important clinical implications since the MMN is being investigated as an assessment tool.

The consolidation/transition model in moral reasoning development.

The consolidation/transition model conceptualizes development as entailing a cyclical pattern of alternating consolidation and transition phases and posits that stage advance is predicted by a specific distribution of reasoning across stages indicative of disequilibrium (more reasoning above than below the mode, with a high degree of mixture). The validity of this model was examined in the context of moral reasoning development with the use of standard statistical techniques as well as Bayesian techniques that can better account for classification error. In this longitudinal study. 64 children and adolescents participated in 5 annual administrations of the Moral Judgment Interview. The distribution of their reasoning across stages was used to predict subsequent development. The results support the hypotheses regarding cyclical patterns of change and predictors of stage transition and demonstrate the utility of Bayesian techniques for evaluating developmental change.

Parent and peer contexts for children's moral reasoning development.

This study addressed the polarization among theoretical perspectives in moral psychology regarding the relative significance of parents and peers in children's developing moral maturity. The sample was composed of 60 target children from late childhood and midadolescence, 60 parents, and 60 friends who participated in parent/child and friend/child dyadic discussions of a series of moral conflicts. The quality of parents' and friends' verbal interactions, ego functioning, and level of moral reasoning in these discussions was used to predict the rate of children's moral reasoning development over a 4-year longitudinal interval. Results revealed that interactions with both parents and peers were predictive of children's development but that these two types of relationships influence development in rather different ways. Implications of the findings for the understanding of these socialization agents' roles in moral development are discussed.

Naturalistic conceptions of moral maturity.

By examining naturalistic conceptions of moral maturity, this project sought a more comprehensive understanding of moral excellence than is evident in dominant theories of moral development. Studies 1 and 2 involved different samples of 120 adults (17-25, 35-55, and 65+ years). Study 3 involved a sample of 180 undergraduates. In Study 1, a free-listing procedure was used to generate the attributes of a highly moral person as well as those for two related person-concepts. In Study 2, a rating procedure for these attributes was used to generate a prototype of the moral person-concept. In Study 3, a similarity-sorting task was used to uncover people's implicit typology of moral maturity. The findings indicate that naturalistic notions of moral excellence not only contain themes of principled reasoning but also reference aspects of moral character and virtue that enlarge our understanding of the psychological functioning of the mature moral agent.

Identification of critical active-site residues in the multifunctional human DNA repair enzyme HAP1.

All organisms express dedicated repair enzymes for counteracting the cytotoxic and mutagenic potential of apurinic/apyrimidinic (AP) lesions, which would otherwise pose a serious threat to genome integrity. We present the predicted three-dimensional structure of the major human AP site-specific DNA repair endonuclease, HAP1, and show that an aspartate/histidine pair, in conjunction with a metal ion-coordinating glutamate residue, are critical for catalyzing the multiple repair activities of HAP1. We suggest that this catalytic mechanism is conserved in certain reverse transcriptases, but is distinct from the two metal ion-mediated mechanism defined for other hydrolytic nucleases.

Site-directed mutagenesis of the human DNA repair enzyme HAP1: identification of residues important for AP endonuclease and RNase H activity.

HAP1 protein, the major apurinic/apyrimidinic (AP) endonuclease in human cells, is a member of a homologous family of multifunctional DNA repair enzymes including the Escherichia coli exonuclease III and Drosophila Rrp1 proteins. The most extensively characterised member of this family, exonuclease III, exhibits both DNA- and RNA-specific nuclease activities. Here, we show that the RNase H activity characteristic of exonuclease III has been conserved in the human homologue, although the products resulting from RNA cleavage are dissimilar. To identify residues important for enzymatic activity, five mutant HAP1 proteins containing single amino acid substitutions were purified and analysed in vitro. The substitutions were made at sites of conserved amino acids and targeted either acidic or histidine residues because of their known participation in the active sites of hydrolytic nucleases. One of the mutant proteins (replacement of Asp-219 by alanine) showed a markedly reduced enzymatic activity, consistent with a greatly diminished capacity to bind DNA and RNA. In contrast, replacement of Asp-90, Asp-308 or Glu-96 by alanine led to a reduction in enzymatic activity without significantly compromising nucleic acid binding. Replacement of His-255 by alanine led to only a very small reduction in enzymatic activity. Our data are consistent with the presence of a single catalytic active site for the DNA- and RNA-specific nuclease activities of the HAP1 protein.