Pathophysiology of Mild Hypercortisolism: From the Bench to the Bedside

Mild hypercortisolism is defined as biochemical evidence of abnormal cortisol secretion without the classical detectable manifestations of overt Cushing’s syndrome and, above all, lacking catabolic characteristics such as central muscle weakness, adipose tissue redistribution, skin fragility and unusual infections. Mild hypercortisolism is frequently discovered in patients with adrenal incidentalomas, with a prevalence ranging between 5 and 50%. This high variability is mainly due to the different criteria used for defining this condition. This subtle cortisol excess has also been described in patients with incidentally discovered pituitary tumors with an estimated prevalence of 5%. To date, the mechanisms responsible for the pathogenesis of mild hypercortisolism of pituitary origin are still not well clarified. At variance, recent advances have been made in understanding the genetic background of bilateral and unilateral adrenal adenomas causing mild hypercortisolism. Some recent data suggest that the clinical effects of glucocorticoid (GC) exposure on peripheral tissues are determined not only by the amount of the adrenal GC production but also by the peripheral GC metabolism and by the GC sensitivity. Indeed, in subjects with normal cortisol secretion, the combined estimate of cortisol secretion, cortisone-to-cortisol peripheral activation by the 11 beta-hydroxysteroid dehydrogenase enzyme and GC receptor sensitizing variants have been suggested to be associated with the presence of hypertension, diabetes and bone fragility, which are three well-known consequences of hypercortisolism. This review focuses on the pathophysiologic mechanism underlying both the different sources of mild hypercortisolism and their clinical consequences (bone fragility, arterial hypertension, subclinical atherosclerosis, cardiovascular remodeling, dyslipidemia, glucose metabolism impairment, visceral adiposity, infections, muscle damage, mood disorders and coagulation).

Brain Kynurenine Pathway and Functional Outcome of Rats Resuscitated From Cardiac Arrest

Background Brain injury and neurological deficit are consequences of cardiac arrest (CA), leading to high morbidity and mortality. Peripheral activation of the kynurenine pathway (KP), the main catabolic route of tryptophan metabolized at first into kynurenine, predicts poor neurological outcome in patients resuscitated after out‐of‐hospital CA. Here, we investigated KP activation in hippocampus and plasma of rats resuscitated from CA, evaluating the effect of KP modulation in preventing CA‐induced neurological deficit. Methods and Results Early KP activation was first demonstrated in 28 rats subjected to electrically induced CA followed by cardiopulmonary resuscitation. Hippocampal levels of the neuroactive metabolites kynurenine, 3‐hydroxy‐anthranilic acid, and kynurenic acid were higher 2 hours after CA, as in plasma. Further, 36 rats were randomized to receive the inhibitor of the first step of KP, 1‐methyl‐DL‐tryptophan, or vehicle, before CA. No differences were observed in hemodynamics and myocardial function. The CA‐induced KP activation, sustained up to 96 hours in hippocampus (and plasma) of vehicle‐treated rats, was counteracted by the inhibitor as indicated by lower hippocampal (and plasmatic) kynurenine/tryptophan ratio and kynurenine levels. 1‐Methyl‐DL‐tryptophan reduced the CA‐induced neurological deficits, with a significant correlation between the neurological score and the individual kynurenine levels, as well as the kynurenine/tryptophan ratio, in plasma and hippocampus. Conclusions These data demonstrate the CA‐induced lasting activation of the first step of the KP in hippocampus, showing that this activation was involved in the evolving neurological deficit. The degree of peripheral activation of KP may predict neurological function after CA.

Characterizing the NLRP3 Inflammasome in Mood Disorders: Overview, Technical Development, and Measures of Peripheral Activation in Adolescent Patients

The NOD-, LRR-, and pyrin-domain-containing protein 3 (NLRP3) inflammasome is a node of intracellular stress pathways and a druggable target which integrates mitochondrial stress and inflammatory cascades. While a body of evidence suggests the involvement of the NLRP3 inflammasome in numerous diseases, a lack of reliable measurement techniques highlights the need for a robust assay using small quantities of biological samples. We present a literature overview on peripheral activation of the NLRP3 inflammasome in mood disorders, then outline a process to develop and validate a robust assay to measure baseline and activated intracellular levels of “apoptosis-associated speck-like protein containing a CARD” (ASC) as a key component of an inflammatory profile in peripheral blood mononuclear cells (PBMC). A consistent association between high NLRP3 mRNA levels and relevant cytokines was seen in the literature. Using our method to measure ASC, stimulation of PBMC with lipopolysaccharide and nigericin or adenosine triphosphate resulted in microscopic identification of intracellular ASC specks, as well as interleukin 1 (IL-1) beta and caspase-1 p10 in the periphery. This was abolished by dose-dependent pre-treatment with 100 nM MCC950. We also report the use of this technique in a small pilot sample from patients with bipolar disorder and depressive disorders. The results show that levels of intracellular ASC and IL-1 beta are sensitive to change upon activation and maintained over time, which may be used to improve the detection of NLRP3 activation and guide personalized therapeutic strategy in the treatment of patients.

Distinct neocortical mechanisms underlie human SI responses to median nerve and laser evoked peripheral activation

Magneto- and/or electro-encephalography (M/EEG) are non-invasive clinically-relevant tools that have long been used to measure electromagnetic fields in somatosensory cortex evoked by non-painful and painful somatosensory stimuli. Two commonly applied stimulation paradigms that produce distinct responses in primary somatosensory cortex (SI) linked to non-painful and painful sensations are electrical median nerve (MN) stimulation and cutaneous laser-evoked (LE) stimulation to the dorsum of the hand, respectively. Despite their prevalence, the physiological mechanisms that produce stereotypic macroscale MN and LE responses have yet to be fully articulated, limiting their utility in understanding brain dynamics associated with non-painful or painful somatosensation. We examined the neocortical circuit mechanisms contributing to MN and LE responses in SI using the Human Neocortical Neurosolver (HNN) neural modeling software tool. HNN was specifically designed for biophysically principled interpretation of the cell and circuit origin of M/EEG signals (Neymotin et al., 2020). Detailed analysis of the timing and orientation of peaks in source localized SI current dipole responses from MN and laser-evoked (LE) stimulation showed that these features were robust and conserved across prior studies. The first peak in the MN response at ∼20 ms corresponds to outward-directed deep-to-superficial electrical current flow through the cortical laminae, while the initial LE response occurs later at ∼170 ms and is oriented in the opposite direction. Historically, these peaks have both been labeled N20 and N1, despite their opposite current orientations. Simulating the cellular and circuit-level mechanisms accounting for these and later peaks with HNN’s detailed laminar neocortical column model revealed that the MN response can be simulated with a sequence of layer-specific exogenous excitatory feedforward and feedback synaptic drive. This sequence was similar to that previously reported for tactile evoked responses (Jones et al., 2007; Neymotin et al., 2020), with the novel discovery of an early excitatory feedback input to superficial layers at ∼30 ms post-stimulus that facilitated generation of the MN response’s first prominent inward-oriented deflection, known historically as the P30. Simulations of the LE response revealed that the initial ∼170 ms inward-deflection required a burst of repetitive gamma-frequency (∼40 Hz) excitatory supragranular feedback drives, consistent with prior reports of LE gamma-frequency activity. These results make novel and detailed multiscale predictions about the dynamic laminar circuit mechanisms underlying temporal and spectral features of MN and LE responses in SI, and can guide further investigations in follow-up studies. Ultimately, these findings may help with the development of targeted therapeutics for pathological somatosensation, such as chronic and neuropathic pain.

Nociceptors and Chronic Pain

Chronic pain lasting months or longer is very common, poorly treated, and sometimes devastating. Nociceptors are sensory neurons that usually are silent unless activated by tissue damage or inflammation. In humans their peripheral activation evokes conscious pain, and their spontaneous activity is highly correlated with spontaneous pain. Persistently hyperactive nociceptors mediate increased responses to normally painful stimuli (hyperalgesia) in chronic conditions and promote the sensitization of central pain pathways that allows low-threshold mechanoreceptors to elicit painful responses to innocuous stimuli (allodynia). Investigations of rodent models of neuropathic pain and hyperalgesic priming have revealed many alterations in nociceptors and associated cells that are implicated in the development and maintenance of chronic pain. These include chronic nociceptor hyperexcitability and spontaneous activity, sprouting, synaptic plasticity, changes in intracellular signaling, and modified responses to opioids, along with alterations in the expression and translation of thousands of genes in nociceptors and closely linked cells.

Cannabinoid Receptor Agonist Inhibits Atrial Electrical Remodeling in a Tachypaced Ex Vivo Rat Model

Introduction: Atrial fibrillation (AF) leads to rate-dependent atrial changes collectively defined as atrial remodelling (AR). Shortening of the atrial effective refractory period (AERP) and decreased conduction velocity are among the hallmarks of AR. Pharmacological strategies to inhibit AR, thereby reducing the self-perpetual nature of AF, are of great clinical value. Cannabinoid receptor (CBR) ligands may exert cardioprotective effects; CB13, a dual CBR agonist with limited brain penetration, protects cardiomyocytes from mitochondrial dysfunction induced by endothelin-1. Here, we examined the effects of CB13 on normal physiology of the rat heart and development of tachypacing-induced AR.Methods: Rat hearts were perfused in a Langendorff set-up with CB13 (1 µM) or vehicle. Hemodynamic properties of non-paced hearts were examined conventionally. In a different set of hearts, programmed stimulation protocol was performed before and after atrial tachypacing for 90 min using a mini-hook platinum quadrupole electrode inserted on the right atrium. Atrial samples were further assessed by western blot analysis.Results: CB13 had no effects on basal hemodynamic properties. However, the compound inhibited tachypacing-induced shortening of the AERP. Protein expression of PGC1α was significantly increased by CB13 compared to vehicle in paced and non-paced hearts. Phosphorylation of AMPKα at residue threonine 172 was increased suggesting upregulation of mitochondrial biogenesis. Connexin43 was downregulated by tachypacing. This effect was diminished in the presence of CB13.Conclusion: Our findings support the notion that peripheral activation of CBR may be a new treatment strategy to prevent AR in patients suffering from AF, and therefore warrants further study.

CXCL10+ peripheral activation niches couple preferred sites of Th1 entry with optimal APC encounter

SUMMARYCorrect positioning of T cells within infected tissues is critical for T cell activation and pathogen control. Upon tissue entry, effector T cells must efficiently locate antigen presenting cells (APC) for peripheral activation. We reveal that tissue entry and initial peripheral activation of Th1 effector T cells are tightly linked to perivascular positioning of chemokine-expressing APCs. Dermal inflammation induced tissue-wide de novo generation of discrete perivascular CXCL10+ cell clusters, enriched for CD11c+MHC-II+ monocyte-derived dendritic cells. These chemokine clusters were ‘hot spots’ for both Th1 extravasation and activation in the inflamed skin. CXCR3-dependent Th1 localization to the cluster micro-environment prolonged T-APC interactions and boosted function. Both the frequency and range of these clusters were enhanced via a Th1-intrinsic, IFNγ-dependent positive feedback loop. Thus, the perivascular CXCL10+ clusters act as initial peripheral activation niches, optimizing controlled activation broadly throughout the tissue by coupling Th1 tissue entry with enhanced opportunities for Th1-APC encounter.

CD5 dynamically calibrates basal NF-κB signaling in T cells during thymic development and peripheral activation

Immature T cells undergo a process of positive selection in the thymus when their new T cell receptor (TCR) engages and signals in response to self-peptides. As the T cell matures, a slew of negative regulatory molecules, including the inhibitory surface glycoprotein CD5, are up-regulated in proportion to the strength of the self-peptide signal. Together these regulators dampen TCR-proximal signaling and help avoid any subsequent peripheral activation of T cells by self-peptides. Paradoxically, antigen-specific T cells initially expressing more CD5 (CD5hi) have been found to better persist as effector/memory cells after a peripheral challenge. The molecular mechanisms underlying such a duality in CD5 function is not clear. We found that CD5 alters the basal activity of the NF-κB signaling in resting peripheral T cells. When CD5 was conditionally ablated, T cells were unable to maintain higher expression of the cytoplasmic NF-κB inhibitor IκBα. Consistent with this, resting CD5hiT cells expressed more of the NF-κB p65 protein than CD5locells, without significant increases in transcript levels, in the absence of TCR signals. This posttranslationally stabilized cellular NF-κB depot potentially confers a survival advantage to CD5hiT cells over CD5loones. Taken together, these data suggest a two-step model whereby the strength of self-peptide–induced TCR signal lead to the up-regulation of CD5, which subsequently maintains a proportional reserve of NF-κB in peripheral T cells poised for responding to agonistic antigen-driven T cell activation.

SAT-LB100 Central and Peripheral Endocannabinoid Levels in Obese Versus Lean Women

The endocannabinoid system (ECS) is thought to be involved in obesity because its activation increases appetite and weight gain (Pagotto et al 2006). The ECS is hyperactivated in the hypothalamus of obese mice, and peripheral overactivation has been observed in humans; circulating 2-arachidonoyl glycerol (2-AG) levels positively correlated with body fat, visceral fat and fasting glucose (Osei-Hyiaman et al 2005) (Bluher et al 2006) (Motaghedi and McGraw 2008) (Cavuoto et al 2007) (Artmann et al 2008) (Bermudez-Silva 2009). The aim of this study was to evaluate whether differential activation of the peripheral versus the central ECS occurred in humans and to test the hypothesis that the ECS is hyperactivated in the human central nervous system (CNS).Cerebral spinal fluid (CSF) and blood samples were collected from 13 obese and 11 lean control women to measure 2-AG and anandamide (AEA) levels.AEA levels were higher in the plasma of obese women (obese: 4.03 ± 0.91 pmol/mL, N=13; lean: 1.84 ± 0.21 pmol/mL, N=10; p<0.05) but were lower in the CSF of obese women. The plasma/CSF ratio was 41.58 ± 5.78 (N=10) in lean women and 103.0 ± 37.36 (N=6) in obese women (p=0.054). There were no correlations between plasma and CSF AEA levels or with any biochemical parameter. The 2-AG analysis was not possible because of technical problems.Our data suggested that in human obesity, the peripheral ECS may be more active than the central ECS. Indeed, the system appeared to be suppressed in the CNS of obese women. Therefore, the peripheral activation of the ECS may be more relevant for obesity. Keywords: Anandamide, 2-arachidonoyl glycerol, obesity, endocannabinoid, endocannabinoid system, cb1 receptor.