T-cell defects and postpartum depression.

BACKGROUND: Most studies of immune dysregulation in perinatal mood and anxiety disorders have focused on peripheral cytokines, but literature from non-perinatal mood disorders also implicates T-cell defects. We sought to characterize proportions of T-cell subtypes in women with postpartum depression. MATERIALS AND METHODS: We enrolled 21 women with postpartum depression (PPD), 39 healthy postpartum controls, and 114 healthy non-postpartum women. Blood was collected in sodium-heparin EDTA tubes and was analyzed using flow cytometry. We conducted statistical tests including linear regression analysis that were aimed at determining differences in proportions of T cell populations among groups. RESULTS: Mean counts of T-cells (all CD3+ T cells), T-helper cells, (CD3+CD4+ T cells), and T-cytotoxic cells (CD3+CD8+ T cells) were significantly increased in healthy postpartum women compared to healthy non-postpartum controls (p < 0.001, p = 0.007, and p = 0.002, respectively), but not in women with PPD. The increases in healthy postpartum women were driven by increases in TH1 cells and T regulatory cells, increases that were nonexistent or attenuated in women with postpartum depression. Mean counts of CD4+ T-helper memory cells were also increased in healthy postpartum women (p = 0.009), but slightly decreased in women with PPD (p = 0.066), when compared to healthy non-postpartum controls. CONCLUSIONS: Our study confirms that the postpartum period in healthy women is a time of enhanced T cell activity. Women with postpartum depression failed to show physiological enhanced T-cell activity postpartum, and future research is needed to elucidate etiological mechanisms and consequences.

Ambulatory Blood Pressure Trajectory and Perceived Stress in Relation to Birth Outcomes in Healthy Pregnant Adolescents.

OBJECTIVE: An early decline in resting blood pressure (BP), followed by an upward climb, is well documented and indicative of a healthy pregnancy course. Although BP is considered both an effector of stress and a clinically meaningful measurement in pregnancy, little is known about its trajectory in association with birth outcomes compared with other stress effectors. The current prospective longitudinal study examined BP trajectory and perceived stress in association with birth outcomes (gestational age (GA) at birth and birth weight (BW) percentile corrected for GA) in pregnant adolescents, a group at risk for stress-associated poor birth outcomes. METHODS: Healthy pregnant nulliparous adolescents (n = 139) were followed from early pregnancy through birth. At three time points (13-16, 24-27, and 34-37 gestational weeks ±1 week), the Perceived Stress Scale was collected along with 24-hour ambulatory BP (systolic and diastolic) and electronic diary reporting of posture. GA at birth and BW were abstracted from medical records. RESULTS: After adjustment for posture and pre-pregnancy body mass index, hierarchical mixed-model linear regression showed the expected early decline (B = -0.18, p = .023) and then increase (B = 0.01, p < .001) of diastolic BP approximating a U-shape; however, systolic BP displayed only an increase (B = 0.01, p = .010). In addition, the models indicated a stronger systolic and diastolic BP U-shape for early GA at birth and lower BW percentile and an inverted U-shape for late GA at birth and higher BW percentile. No effects of perceived stress were observed. CONCLUSIONS: These results replicate the pregnancy BP trajectory from previous studies of adults and indicate that the degree to which the trajectory emerges in adolescence may be associated with variation in birth outcomes, with a moderate U-shape indicating the healthiest outcomes.

Benzenesulphonamide inhibitors of the cytolytic protein perforin.

The pore-forming protein perforin is a key component of mammalian cell-mediated immunity and essential to the pathway that allows elimination of virus-infected and transformed cells. Perforin activity has also been implicated in certain auto-immune conditions and therapy-induced conditions such as allograft rejection and graft versus host disease. An inhibitor of perforin activity could be used as a highly specific immunosuppressive treatment for these conditions, with reduced side-effects compared to currently accepted therapies. Previously identified first-in-class inhibitors based on a 2-thioxoimidazolidin-4-one core show suboptimal physicochemical properties and toxicity toward the natural killer (NK) cells that secrete perforin in vivo. The current benzenesulphonamide-based series delivers a non-toxic bioisosteric replacement possessing improved solubility.

Systemic and Brain Pharmacokinetics of Perforin Inhibitor Prodrugs.

We have recently reported that by converting a perforin inhibitor into an l-type amino acid transporter 1 (LAT1)-utilizing prodrug its cellular uptake can be greatly increased. The aim of the present study was to determine the in vivo and brain pharmacokinetics of two perforin inhibitors and their LAT1-utilizing prodrugs 1 and 2. In addition, the brain uptake mechanism and entry into primary mouse cortical neurons and astrocytes were evaluated. After 23 µmol/kg i.p. bolus injection, the prodrugs' unbound area under the concentration curve in brain was 0.3 nmol/g × min, whereas the parent drugs could not reach the brain. The unbound brain concentrations of the prodrugs after 100 µM in situ mouse brain perfusion were 521.4 ± 46.9 and 126.9 ± 19.9 pmol/g for prodrugs 1 and 2, respectively. The combination of competing transporter substrates for LAT1, l-tryptophan, and for organic anion transporting polypeptides, probenecid, decreased the brain concentrations to 352.4 ± 44.5 and 70.9 ± 7.0 pmol/g, respectively. In addition, in vitro uptake studies showed that at 100 µM prodrug 1 had 3.4-fold and 4.5-fold higher uptake rate into neurons and astrocytes, respectively, compared to its parent drug. Thus, the prodrugs enhance significantly the therapeutic potential of the parent drugs for the treatment of disorders of central nervous system in which neuroinflammation is involved.

Maternal prenatal iron status and tissue organization in the neonatal brain.

BACKGROUND: Children prenatally exposed to inadequate iron have poorer motor and neurocognitive development. No prior study to our knowledge has assessed the influence of maternal prenatal iron intake on newborn brain tissue organization in full-term infants. METHODS: Third trimester daily iron intake was obtained using the Automated Self-Administered 24-h Dietary Recall with n = 40 healthy pregnant adolescents (aged 14-19 y). Cord blood ferritin was collected in a subsample (n = 16). Newborn (mean = 39 gestational weeks at birth; range 37-41) magnetic resonance imaging scans were acquired on a 3.0 Tesla MR Scanner. Diffusion Tensor Imaging (DTI) slices were acquired to measure the directional diffusion of water indexed by fractional anisotropy (FA). RESULTS: Reported iron intake was inversely associated with newborn FA values (P ≤ 0.0001) predominantly in cortical gray matter. FA findings were similar using cord blood ferritin values. CONCLUSION: Higher maternal prenatal iron intake accentuates, and lower intake attenuates, the normal age-related decline in FA values in gray matter, perhaps representing increasing dendritic arborization and synapse formation with higher iron intake. These DTI results suggest that typical variation in maternal iron outside the scope of standard clinical surveillance exerts subtle effects on infant brain development.

Diarylthiophenes as inhibitors of the pore-forming protein perforin.

Evolution from a furan-containing high-throughput screen (HTS) hit (1) resulted in isobenzofuran-1(3H)-one (2) as a potent inhibitor of the function of both isolated perforin protein and perforin delivered in situ by intact KHYG-1 NK cells. In the current study, structure-activity relationship (SAR) development towards a novel series of diarylthiophene analogues has continued through the use of substituted-benzene and -pyridyl moieties as bioisosteres for 2-thioxoimidazolidin-4-one (A) on a thiophene (B) -isobenzofuranone (C) scaffold. The resulting compounds were tested for their ability to inhibit perforin lytic activity in vitro. Carboxamide (23) shows a 4-fold increase over (2) in lytic activity against isolated perforin and provides good rationale for continued development within this class.

The neural bases of distracter-resistant working memory.

A major difference between humans and other animals is our capacity to maintain information in working memory (WM) while performing secondary tasks, which enables sustained, complex cognition. A common assumption is that the lateral prefrontal cortex (PFC) is critical for WM performance in the presence of distracters, but direct evidence is scarce. We assessed the relationship between fMRI activity and WM performance within subjects, with performance matched across distracter and no-distracter conditions. Activity in the ventrolateral PFC during WM encoding and maintenance positively predicted performance in both conditions, whereas activity in the presupplementary motor area (pre-SMA) predicted performance only under distraction. Other parts of the dorsolateral and ventrolateral PFCs predicted performance only in the no-distracter condition. These findings challenge a lateral-PFC-centered view of distracter resistance, and suggest that the lateral PFC supports a type of WM representation that is efficient for dealing with task-irrelevant input but is, nonetheless, easily disrupted by dual-task demands.

Capacity estimates in working memory: Reliability and interrelationships among tasks.

The concept of capacity has become increasingly important in discussions of working memory (WM), in so far as most models of WM conceptualize it as a limited-capacity mechanism for maintaining information in an active state, and as capacity estimates from at least one type of WM task-complex span-are valid predictors of real-world cognitive performance. However, the term capacity is also often used in the context of a distinct set of WM tasks, change detection, and may or may not refer to the same cognitive capability. We here develop maximum-likelihood models of capacity from each of these tasks-as well as from a third WM task that places heavy demands on cognitive control, the self-ordered WM task (SOT)-and show that the capacity estimates from change detection and complex span tasks are not correlated with each other, although capacity estimates from change detection tasks do correlate with those from the SOT. Furthermore, exploratory factor analysis confirmed that performance on the SOT and change detection load on the same factor, with performance on our complex span task loading on its own factor. These findings suggest that at least two distinct cognitive capabilities underlie the concept of WM capacity as it applies to each of these three tasks.

Effects of a Small-Molecule Perforin Inhibitor in a Mouse Model of CD8 T Cell-Mediated Neuroinflammation.

BACKGROUND AND OBJECTIVES: Alteration of the blood-brain barrier (BBB) at the interface between blood and CNS parenchyma is prominent in most neuroinflammatory diseases. In several neurologic diseases, including cerebral malaria and Susac syndrome, a CD8 T cell-mediated targeting of endothelial cells of the BBB (BBB-ECs) has been implicated in pathogenesis. METHODS: In this study, we used an experimental mouse model to evaluate the ability of a small-molecule perforin inhibitor to prevent neuroinflammation resulting from cytotoxic CD8 T cell-mediated damage of BBB-ECs. RESULTS: Using an in vitro coculture system, we first identified perforin as an essential molecule for killing of BBB-ECs by CD8 T cells. We then found that short-term pharmacologic inhibition of perforin commencing after disease onset restored motor function and inhibited the neuropathology. Perforin inhibition resulted in preserved BBB-EC viability, maintenance of the BBB, and reduced CD8 T-cell accumulation in the brain and retina. DISCUSSION: Therefore, perforin-dependent cytotoxicity plays a key role in the death of BBB-ECs inflicted by autoreactive CD8 T cells in a preclinical model and potentially represents a therapeutic target for CD8 T cell-mediated neuroinflammatory diseases, such as cerebral malaria and Susac syndrome.

Fragment-based and structure-guided discovery of perforin inhibitors.

Perforin is a pore-forming protein whose normal function enables cytotoxic T and natural killer (NK) cells to kill virus-infected and transformed cells. Conversely, unwanted perforin activity can also result in auto-immune attack, graft rejection and aberrant responses to pathogens. Perforin is critical for the function of the granule exocytosis cell death pathway and is therefore a target for drug development. In this study, by screening a fragment library using NMR and surface plasmon resonance, we identified 4,4-diaminodiphenyl sulfone (dapsone) as a perforin ligand. We also found that dapsone has modest (mM) inhibitory activity of perforin lytic activity in a red blood cell lysis assay in vitro. Sequential modification of this lead fragment, guided by structural knowledge of the ligand binding site and binding pose, and supported by SPR and ligand-detected 19F NMR, enabled the design of nanomolar inhibitors of the cytolytic activity of intact NK cells against various tumour cell targets. Interestingly, the ligands we developed were largely inert with respect to direct perforin-mediated red blood cell lysis but were very potent in the context of perforin's action on delivering granzymes in the immune synapse, the context in which it functions physiologically. Our work indicates that a fragment-based, structure-guided drug discovery strategy can be used to identify novel ligands that bind perforin. Moreover, these molecules have superior physicochemical properties and solubility compared to previous generations of perforin ligands.

Estimating and testing variance components in a multi-level GLM.

Most analysis of multi-subject fMRI data is concerned with determining whether there exists a significant population-wide 'activation' in a comparison between two or more conditions. This is typically assessed by testing the average value of a contrast of parameter estimates (COPE) against zero in a general linear model (GLM) analysis. However, important information can also be obtained by testing whether there exist significant individual differences in effect magnitude between subjects, i.e. whether the variance of a COPE is significantly different from zero. Intuitively, such a test amounts to testing whether inter-individual differences are larger than would be expected given the within-subject error variance. We compare several methods for estimating variance components, including a) a naïve estimate using ordinary least squares (OLS); b) linear mixed effects in R (LMER); c) a novel Matlab implementation of iterative generalized least squares (IGLS) and its restricted maximum likelihood variant (RIGLS). All methods produced reasonable estimates of within- and between-subject variance components, with IGLS providing an attractive balance between sensitivity and appropriate control of false positives. Finally, we use the IGLS method to estimate inter-subject variance in a perfusion fMRI study (N=18) of social evaluative threat, and show evidence for significant inter-individual differences in ventromedial prefrontal cortex (VMPFC), amygdala, hippocampus and medial temporal lobes, insula, and brainstem, with predicted inverse coupling between VMPFC and the midbrain periaqueductal gray only when high inter-individual variance was used to define the seed for functional connectivity analyses. In sum, tests of variance provides a way of selecting regions that show significant inter-individual variability for subsequent analyses that attempt to explain those individual differences.

Activation of a PAK-MEK signalling pathway in malaria parasite-infected erythrocytes.

Merozoites of malaria parasites invade red blood cells (RBCs), where they multiply by schizogony, undergoing development through ring, trophozoite and schizont stages that are responsible for malaria pathogenesis. Here, we report that a protein kinase-mediated signalling pathway involving host RBC PAK1 and MEK1, which do not have orthologues in the Plasmodium kinome, is selectively stimulated in Plasmodium falciparum-infected (versus uninfected) RBCs, as determined by the use of phospho-specific antibodies directed against the activated forms of these enzymes. Pharmacological interference with host MEK and PAK function using highly specific allosteric inhibitors in their known cellular IC50 ranges results in parasite death. Furthermore, MEK inhibitors have parasiticidal effects in vitro on hepatocyte and erythrocyte stages of the rodent malaria parasite Plasmodium berghei, indicating conservation of this subversive strategy in malaria parasites. These findings have profound implications for the development of novel strategies for antimalarial chemotherapy.

Inhibition of the pore-forming protein perforin by a series of aryl-substituted isobenzofuran-1(3H)-ones.

An aryl-substituted isobenzofuran-1(3H)-one lead compound was identified from a high throughput screen designed to find inhibitors of the lymphocyte pore-forming protein perforin. A series of analogs were then designed and prepared, exploring structure-activity relationships through variation of 2-thioxoimidazolidin-4-one and furan subunits on an isobenzofuranone core. The ability of the resulting compounds to inhibit the lytic activity of both isolated perforin protein and perforin delivered in situ by intact KHYG-1 natural killer effector cells was determined. Several compounds showed excellent activity at concentrations that were non-toxic to the killer cells. This series represents a significant improvement on previous classes of compounds, being substantially more potent and largely retaining activity in the presence of serum.

Linking prenatal maternal adversity to developmental outcomes in infants: the role of epigenetic pathways.

Prenatal exposure to maternal stress, anxiety, and depression can have lasting effects on infant development with risk of psychopathology. Although the impact of prenatal maternal distress has been well documented, the potential mechanisms through which maternal psychosocial variables shape development have yet to be fully elucidated. Advances in molecular biology have highlighted the role of epigenetic mechanisms in regulating gene activity, neurobiology, and behavior and the potential role of environmentally induced epigenetic variation in linking early life exposures to long-term biobehavioral outcomes. In this article, we discuss evidence illustrating the association between maternal prenatal distress and both fetal and infant developmental trajectories and the potential role of epigenetic mechanisms in mediating these effects. Postnatal experiences may have a critical moderating influence on prenatal effects, and we review findings illustrating prenatal-postnatal interplay and the developmental and epigenetic consequences of postnatal mother-infant interactions. The in utero environment is regulated by placental function and there is emerging evidence that the placenta is highly susceptible to maternal distress and a target of epigenetic dysregulation. Integrating studies of prenatal exposures, placental function, and postnatal maternal care with the exploration of epigenetic mechanisms may provide novel insights into the pathophysiology induced by maternal distress.

Prenatal ambient temperature and risk for schizophrenia.

OBJECTIVE: We conducted a systematic review of the published literature to test the hypothesis that maternal exposure to extremes of ambient temperatures during pregnancy is associated with the risk for psychiatric disorders or congenital malformations in offspring, both of which are indicative of perturbations of fetal neurodevelopment. METHOD: This study was conducted in accordance with the recommendations outlined in the Meta-analysis of Observational Studies in Epidemiology (MOOSE) reporting proposal. Electronic databases (Ovid MEDLINE, Ovid Embase, Ovid PsycINFO, Ovid Global Health, Web of Science, and Cochrane Library) were searched. Four independent reviewers selected studies with the following criteria: (1) prenatal maternal ambient temperature exposure; (2) outcome of offspring psychiatric disorder or congenital defects; (3) empirical study; (4) full-length article, no conference presentations or abstracts. RESULTS: Twenty-two studies met criteria and one was added from a reference list (n = 23). Of these, schizophrenia (n = 5), anorexia nervosa (n = 3) and congenital cardiovascular malformations (n = 6) studies were the most common. Each of these categories showed some evidence of association with an early pregnancy maternal ambient heat exposure effect, with other evidence for a late pregnancy cold effect. CONCLUSION: Some evidence supports a role for early pregnancy maternal exposure to extreme ambient heat in the development of psychiatric disorders, but large-scale, prospective cohort data on individual births is essential. Optimal studies will be conducted in seasonally variable climates, accounting for actual maternal residence over the pregnancy and at parturition, local environmental temperature records, and appropriate covariates, similar to studies identified by this systematic review for congenital malformations.

Small Molecule Inhibitors of Lymphocyte Perforin as Focused Immunosuppressants for Infection and Autoimmunity.

New drugs that precisely target the immune mechanisms critical for cytotoxic T lymphocyte (CTL) and natural killer (NK) cell driven pathologies are desperately needed. In this perspective, we explore the cytolytic protein perforin as a target for therapeutic intervention. Perforin plays an indispensable role in CTL/NK killing and controls a range of immune pathologies, while being encoded by a single copy gene with no redundancy of function. An immunosuppressant targeting this protein would provide the first-ever therapy focused specifically on one of the principal cell death pathways contributing to allotransplant rejection and underpinning multiple autoimmune and postinfectious diseases. No drugs that selectively block perforin-dependent cell death are currently in clinical use, so this perspective will review published novel small molecule inhibitors, concluding with in vivo proof-of-concept experiments performed in mouse models of perforin-mediated immune pathologies that provide a potential pathway toward a clinically useful therapeutic agent.

Third Trimester Fetuses Demonstrate Priming, a Form of Implicit Memory, In Utero.

Research examinations of changes in fetal heart rate (HR) to operationalize fetal memory suggests that human memory capacities emerge in utero. However, there is little evidence for a form of implicit memory or priming. The present aim was to determine if priming is evident in utero. Fetal HR, maternal HR and maternal respiratory rate (RR) were examined in 105 women during the third trimester of pregnancy. Women experienced two counterbalanced laboratory tasks, the Stroop task and the paced breathing task, and their cardiorespiratory activity functioned as a stimulus for fetuses. Repeated measures ANOVAs revealed maternal HR increased during the Stroop task but only when the Stroop task was presented first (89.64 bpm to 92.39 bpm) (p = 0.04). Maternal RR increased during the Stroop task, regardless of task order (17.72 bpm to 21.11 bpm; 18.50 bpm to 22.60 bpm) (p < 0.01). Fetal HR increased during the paced breathing task, but only when it followed maternal exposure to the Stroop task (141.13 bpm to 143.97 bpm) (p < 0.01). Fetuses registered maternal HR and RR reactivity to the Stroop task, which influenced their response during maternal engagement with a related task, suggesting priming. Further study of fetal memory may suggest another pathway by which prenatal exposures impact future development.

IFN-γ+ cytotoxic CD4+ T lymphocytes are involved in the pathogenesis of colitis induced by IL-23 and the food colorant Red 40.

The food colorant Red 40 is an environmental risk factor for colitis development in mice with increased expression of interleukin (IL)-23. This immune response is mediated by CD4+ T cells, but mechanistic insights into how these CD4+ T cells trigger and perpetuate colitis have remained elusive. Here, using single-cell transcriptomic analysis, we found that several CD4+ T-cell subsets are present in the intestines of colitic mice, including an interferon (IFN)-γ-producing subset. In vivo challenge of primed mice with Red 40 promoted rapid activation of CD4+ T cells and caused marked intestinal epithelial cell (IEC) apoptosis that was attenuated by depletion of CD4+ cells and blockade of IFN-γ. Ex vivo experiments showed that intestinal CD4+ T cells from colitic mice directly promoted apoptosis of IECs and intestinal enteroids. CD4+ T cell-mediated cytotoxicity was contact-dependent and required FasL, which promoted caspase-dependent cell death in target IECs. Genetic ablation of IFN-γ constrained IL-23- and Red 40-induced colitis development, and blockade of IFN-γ inhibited epithelial cell death in vivo. These results advance the understanding of the mechanisms regulating colitis development caused by IL-23 and food colorants and identify IFN-γ+ cytotoxic CD4+ T cells as a new potential therapeutic target for colitis.

Preclinical Activity and Pharmacokinetic/Pharmacodynamic Relationship for a Series of Novel Benzenesulfonamide Perforin Inhibitors.

Perforin is a key effector of lymphocyte-mediated cell death pathways and contributes to transplant rejection of immunologically mismatched grafts. We have developed a novel series of benzenesulfonamide (BZS) inhibitors of perforin that can mitigate graft rejection during allogeneic bone marrow/stem cell transplantation. Eight such perforin inhibitors were tested for their murine pharmacokinetics, plasma protein binding, and their ability to block perforin-mediated lysis in vitro and to block the rejection of major histocompatibility complex (MHC)-mismatched mouse bone marrow cells. All compounds showed >99% binding to plasma proteins and demonstrated perforin inhibitory activity in vitro and in vivo. A lead compound, compound 1, that showed significant increases in allogeneic bone marrow preservation was evaluated for its plasma pharmacokinetics and in vivo efficacy at multiple dosing regimens to establish a pharmacokinetic/pharmacodynamic (PK/PD) relationship. The strongest PK/PD correlation was observed between perforin inhibition in vivo and time that total plasma concentrations remained above 900 µM, which correlates to unbound concentrations similar to 3× the unbound in vitro IC90 of compound 1. This PK/PD relationship will inform future dosing strategies of BZS perforin inhibitors to maintain concentrations above 3× the unbound IC90 for as long as possible to maximize efficacy and enhance progression toward clinical evaluation.