Effects of Glucocorticoids in the Immune System.

Glucocorticoids (GCs) are steroid hormones with widespread effects. They control intermediate metabolism by stimulating gluconeogenesis in the liver, mobilize amino acids from extra hepatic tissues, inhibit glucose uptake in muscle and adipose tissue, and stimulate fat breakdown in adipose tissue. They also mediate stress response. They exert potent immune-suppressive and anti-inflammatory effects particularly when administered pharmacologically. Understanding these diverse effects of glucocorticoids requires a detailed knowledge of their mode of action. Research over the years has uncovered several details on the molecular action of this hormone, especially in immune cells. In this chapter, we have summarized the latest findings on the action of glucocorticoids in immune cells with a view of identifying important control points that may be relevant in glucocorticoid therapy.

Localization and dynamics of glucocorticoid receptor at the plasma membrane of activated mast cells.

In addition to their actions in the cell nucleus, glucocorticoids exhibit rapid non-nuclear responses that are mechanistically not well understood. To explain these effects, the localization of a glucocorticoid receptor (GR) expressed in mast cells as a GFP fusion was analyzed after activation of the cells on allergenic lipid arrays. These arrays were produced on glass slides by dip-pen nanolithography (DPN) and total internal reflection (TIRF) microscopy was used to visualize the GR. A rapid glucocorticoid-independent and -dependent recruitment of the GR-GFP to the plasma cell membrane was observed following contact of the cells with the allergenic array. In addition, the mobility of the GR at the membrane was monitored by fluorescence recovery after photobleaching (FRAP) and shown to follow binding kinetics demonstrating interactions of the receptor with membrane-bound factors. Furthermore the recruitment of the GR to the cell membrane was shown to result in a glucocorticoid-mediated increase in Erk phosphorylation. This is evidenced by findings that destruction of the membrane composition of the mast cells by cholesterol depletion impairs the membrane localization of the GR and subsequent glucocorticoid-mediated enhancement of Erk phosphorylation. These results demonstrate a membrane localization and function of the GR in mast cell signaling.

Sexual dimorphism, weight gain and glucose intolerance in a B- and T-cell deficient mouse model.

BACKGROUND: Estrogen is thought to aid maintenance of insulin sensitivity potentially through modulation of a counter-regulatory mechanism that interferes with the contribution of adaptive and innate immune systems to visceral fat deposition. We evaluated the impact of estrogen on long-term high fat diet (HFD) intake in B- and T-cell deficient and immunocompetent animals comparatively. METHODS: A total of 16 BALB and 16 SCID mice, 8 of each sex and strain, were randomized to receive low fat diet, 4.1% fat or HFD, 35% fat, such that there was a group of both each sex and each strain receiving each diet. Biweekly levels of adiponectin, leptin and insulin levels were assessed and a glucose tolerance test (GTT) was performed after 13 weeks. RESULTS: Unlike their male counterparts, HFD-fed SCID females neither gained weight, nor became insulin resistant. Meanwhile, in the HFD-fed BALB groups both males and females gained weight similarly, but remarkable sexual dimorphism was nonetheless observed. The females had notable higher adiponectin levels as compared to males (10-60 µg/mL vs. 6-10 µg/mL respectively) causing the adiponectin-to-leptin (A/L) ratio to reach 80 one week after HFD initiation. The A/L dropped to 10, still higher than males, by week 13, but dropped to 2 by the end of the study in agreement with inverse insulin trends. None of the HFD-fed female groups developed insulin resistance (IR) by week 13, while all male counterparts had. Similar results were observed in the HFD-fed SCID groups whereby the females did not develop IR and had a higher A/L; however, adiponectin levels were comparable between groups (5-11 µg/mL). CONCLUSIONS: The present study provides lacking evidence indicating that estrogen may be sufficient to prevent weight gain and development of glucose intolerance in high-fat fed B- and T-cell deficient mice.

Spinal muscular atrophy in Ghanaian children confirmed by molecular genetic testing: a case series.

Spinal muscular atrophy (SMA) is an autosomal recessive inherited motor neuron disease characterized by progressive muscle weakness due to degeneration and loss of the anterior horn cells in the spinal cord and the brain stem nuclei from foetal life through infancy and childhood. SMA is prevalent in Ghanaian children, though not widely reported. Cases are likely missed or misdiagnosed due to lack of expertise and investigations. Newborn screening is not currently available in Ghana. The management remains supportive as newly approved genetic modifications therapies are currently not available. We present a retrospective folder review of children attending a tertiary pediatric neurology clinic who were diagnosed with SMA and confirmed by molecular genetic testing. Between January 2018 and August 2021, five (5) children from three families had molecular genetic tests confirming their diagnosis of SMA. Three (3) children had SMA I phenotype while 2 had SMA III phenotype. Two (2) of the 3 children with SMA I died from respiratory complications. The last surviving child with SMA I was diagnosed through newborn screening program overseas and received gene modification therapy. Careful history and physical examination remain the best approach to diagnosis as confirmatory genetic testing and supplemental investigations are not readily available. The current management of the children with SMA in Ghana include respiratory care, physiotherapy, and genetic counselling. Genetic modification therapies are currently not available.

Allergen arrays for antibody screening and immune cell activation profiling generated by parallel lipid dip-pen nanolithography.

Multiple-allergen testing for high throughput and high sensitivity requires the development of miniaturized immunoassays that allow for a large test area and require only a small volume of the test analyte, which is often available only in limited amounts. Developing such miniaturized biochips containing arrays of test allergens needs application of a technique able to deposit molecules at high resolution and speed while preserving its functionality. Lipid dip-pen nanolithography (L-DPN) is an ideal technique to create such biologically active surfaces, and it has already been successfully applied for the direct, nanoscale deposition of functional proteins, as well as for the fabrication of biochemical templates for selective adsorption. The work presented here shows the application of L-DPN for the generation of arrays of the ligand 2,4-dinitrophenyl[1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine-N-[6-[(2,4-dinitrophenyl)amino]hexanoyl] (DNP)] onto glass surfaces as a model system for detection of allergen-specific Immunoglobin E (IgE) antibodies and for mast cell activation profiling.

Atypical Kawasaki syndrome in COVID-19 infection: a case report of a multisystem inflammatory syndrome in a child (MIS-C).

The emergence of COVID-19 by a novel severe acute respiratory syndrome coronavirus (SARS-CoV-2) in 2019 has seen evolving data reporting infrequent infection in children and mostly mild disease for children who contract the infection. A severe form of COVID-19 in children recently reported in Europe and North America describes a multisystem inflammation syndrome in children (MIS-C), presenting as toxic-shock-like and Kawasaki-like syndromes. Data on MIS-C in Africa is being documented with recent reports from South Africa and Nigeria in black children, but information on MIS-C in Ghana is yet to be characterized. We report the first case of multisystem inflammatory syndrome in a child who tested PCR positive to SARS-CoV2 in a tertiary hospital in Ghana. The case describes a 10-year-old boy who reported Kawasaki-like syndrome without shock but with moderate respiratory distress requiring supportive acute care without the need for intensive care. FUNDING: None declared.

An Amyloidogenic Sequence at the N-Terminus of the Androgen Receptor Impacts Polyglutamine Aggregation.

The human androgen receptor (AR) is a ligand inducible transcription factor that harbors an amino terminal domain (AR-NTD) with a ligand-independent activation function. AR-NTD is intrinsically disordered and displays aggregation properties conferred by the presence of a poly-glutamine (polyQ) sequence. The length of the polyQ sequence as well as its adjacent sequence motifs modulate this aggregation property. AR-NTD also contains a conserved KELCKAVSVSM sequence motif that displays an intrinsic property to form amyloid fibrils under mild oxidative conditions. As peptide sequences with intrinsic oligomerization properties are reported to have an impact on the aggregation of polyQ tracts, we determined the effect of the KELCKAVSVSM on the polyQ stretch in the context of the AR-NTD using atomic force microscopy (AFM). Here, we present evidence for a crosstalk between the amyloidogenic properties of the KELCKAVSVSM motif and the polyQ stretch at the AR-NTD.

Molecular mechanisms of glucocorticoid action in mast cells.

Glucocorticoids are compounds that have successfully been used over the years in the treatment of inflammatory disorders. They are known to exhibit their effects through the glucocorticoid receptor (GR) that acts to downregulate the action of proinflammatory transcription factors such as AP-1 and NF-κB. The GR also exerts anti-inflammatory effects through activation of distinct genes. In addition to their anti-inflammatory actions, glucocorticoids are also potent antiallergic compounds that are widely used in conditions such as asthma and anaphylaxis. Nevertheless the mechanism of action of this hormone in these disorders is not known. In this article, we have reviewed reports on the effects of glucocorticoids in mast cells, one of the important immune cells in allergy. Building on the knowledge of the molecular action of glucocorticoids and the GR in the treatment of inflammation in other cell types, we have made suggestions as to the likely mechanisms of action of glucocorticoids in mast cells. We have further identified some important questions and research directions that need to be addressed in future studies to improve the treatment of allergic disorders.

Stimulated emission depletion-based raster image correlation spectroscopy reveals biomolecular dynamics in live cells.

Raster image correlation spectroscopy is a powerful tool to study fast molecular dynamics such as protein diffusion or receptor-ligand interactions inside living cells and tissues. By analysing spatio-temporal correlations of fluorescence intensity fluctuations from raster-scanned microscopy images, molecular motions can be revealed in a spatially resolved manner. Because of the diffraction-limited optical resolution, however, conventional raster image correlation spectroscopy can only distinguish larger regions of interest and requires low fluorophore concentrations in the nanomolar range. Here, to overcome these limitations, we combine raster image correlation spectroscopy with stimulated emission depletion microscopy. With imaging experiments on model membranes and live cells, we show that stimulated emission depletion-raster image correlation spectroscopy offers an enhanced multiplexing capability because of the enhanced spatial resolution as well as access to 10-100 times higher fluorophore concentrations.

A potent and selective small-molecule inhibitor for the lymphoid-specific tyrosine phosphatase (LYP), a target associated with autoimmune diseases.

Lymphoid-specific tyrosine phosphatase (LYP), a member of the protein tyrosine phosphatase (PTP) family of signaling enzymes, is associated with a broad spectrum of autoimmune diseases. Herein we describe our structure-based lead optimization efforts within a 6-hydroxy-benzofuran-5-carboxylic acid series culminating in the identification of compound 8b, a potent and selective inhibitor of LYP with a K(i) value of 110 nM and more than 9-fold selectivity over a large panel of PTPs. The structure of LYP in complex with 8b was obtained by X-ray crystallography, providing detailed information about the molecular recognition of small-molecule ligands binding LYP. Importantly, compound 8b possesses highly efficacious cellular activity in both T- and mast cells and is capable of blocking anaphylaxis in mice. Discovery of 8b establishes a starting point for the development of clinically useful LYP inhibitors for treating a wide range of autoimmune disorders.