92 research outputs found
Functional rescue of inactivating mutations of the human neurokinin 3 receptor using pharmacological chaperones
G protein-coupled receptors (GPCRs) facilitate the majority of signal transductions across
cell membranes in humans, with numerous diseases attributed to inactivating GPCR mutations.
Many of these mutations result in misfolding during nascent receptor synthesis in the endoplasmic
reticulum (ER), resulting in intracellular retention and degradation. Pharmacological chaperones
(PCs) are cell-permeant small molecules that can interact with misfolded receptors in the ER and
stabilise/rescue their folding to promote ER exit and trafficking to the cell membrane. The neurokinin
3 receptor (NK3R) plays a pivotal role in the hypothalamicβpituitaryβgonadal reproductive axis.
We sought to determine whether NK3R missense mutations result in a loss of cell surface receptor
expression and, if so, whether a cell-permeant small molecule NK3R antagonist could be repurposed
as a PC to restore function to these mutants. Quantitation of cell surface expression levels of seven
mutant NK3Rs identified in hypogonadal patients indicated that five had severely impaired cell
surface expression. A small molecule NK3R antagonist, M8, increased cell surface expression in four
of these five and resulted in post-translational receptor processing in a manner analogous to the
wild type. Importantly, there was a significant improvement in receptor activation in response to
neurokinin B (NKB) for all four receptors following their rescue with M8. This demonstrates that M8
may have potential for therapeutic development in the treatment of hypogonadal patients harbouring
NK3R mutations. The repurposing of existing small molecule GPCR modulators as PCs represents
a novel and therapeutically viable option for the treatment of disorders attributed to mutations in
GPCRs that cause intracellular retention.The National Research Foundation South Africa; Competitive Support for Unrated Researchers; the SwissβSouth African Joint Research Programme; Competitive Support for Rated Researchers; the NRF National Equipment Program and the National Institutes of Health.https://www.mdpi.com/journal/ijmsAnatomy and PhysiologyImmunologyPhysiolog
Inhibition of apoptosis prevents West Nile virus induced cell death
We found that WNV infection induces cell death in the brain-derived tumour cell line T98G by apoptosis under involvement of constituents of the extrinsic as well as the intrinsic apoptotic pathways. Our results illuminate the molecular mechanism of WNV-induced neural cell death
Differential Role for CD80 and CD86 in the Regulation of the Innate Immune Response in Murine Polymicrobial Sepsis
Inflammation in the early stages of sepsis is governed by the innate immune response. Costimulatory molecules are a receptor/ligand class of molecules capable of regulation of inflammation in innate immunity via macrophage/neutrophil contact. We recently described that CD80/86 ligation is required for maximal macrophage activation and CD80/86(-/-) mice display reduced mortality and inflammatory cytokine production after cecal ligation and puncture (CLP). However, these data also demonstrate differential regulation of CD80 and CD86 expression in sepsis, suggesting a divergent role for these receptors. Therefore, the goal of this study was to determine the individual contribution of CD80/86 family members in regulating inflammation in sepsis.CD80(-/-) mice had improved survival after CLP when compared to WT or CD86(-/-) mice. This was associated with preferential attenuation of inflammatory cytokine production in CD80(-/-) mice. Results were confirmed with pharmacologic blockade, with anti-CD80 mAb rescuing mice when administered before or after CLP. In vitro, activation of macrophages with neutrophil lipid rafts caused selective disassociation of IRAK-M, a negative regulator of NF-kappaB signaling from CD80; providing a mechanism for preferential regulation of cytokine production by CD80. Finally, in humans, upregulation of CD80 and loss of constitutive CD86 expression on monocytes was associated with higher severity of illness and inflammation confirming the findings in our mouse model.In conclusion, our data describe a differential role for CD80 and CD86 in regulation of inflammation in the innate immune response to sepsis. Future therapeutic strategies for blockade of the CD80/86 system in sepsis should focus on direct inhibition of CD80
Baicalin Improves Survival in a Murine Model of Polymicrobial Sepsis via Suppressing Inflammatory Response and Lymphocyte Apoptosis
BACKGROUND: An imbalance between overwhelming inflammation and lymphocyte apoptosis is the main cause of high mortality in patients with sepsis. Baicalin, the main active ingredient of the Scutellaria root, exerts anti-inflammatory, anti-apoptotic, and even antibacterial properties in inflammatory and infectious diseases. However, the therapeutic effect of baicalin on polymicrobial sepsis remains unknown. METHODOLOGY/PRINCIPAL FINDINGS: Polymicrobial sepsis was induced by cecal ligation and puncture (CLP) in C57BL/6 mice. Mice were infused with baicalin intraperitoneally at 1 h, 6 h and 12 h after CLP. Survival rates were assessed over the subsequent 8 days. Bacterial burdens in blood and peritoneal cavity were calculated to assess the bacterial clearance. Neutrophil count in peritoneal lavage fluid was also calculated. Injuries to the lung and liver were detected by hematoxylin and eosin staining. Levels of cytokines, including tumor necrosis factor (TNF)-alpha, interleukin (IL)-6, IL-10 and IL-17, in blood and peritoneum were measured by enzyme-linked immunosorbent assay. Adaptive immune function was assessed by apoptosis of lymphocytes in the thymus and counts of different cell types in the spleen. Baicalin significantly enhanced bacterial clearance and improved survival of septic mice. The number of neutrophils in peritoneal lavage fluid was reduced by baicalin. Less neutrophil infiltration of the lung and liver in baicalin-treated mice was associated with attenuated injuries to these organs. Baicalin significantly reduced the levels of proinflammatory cytokines but increased the level of anti-inflammatory cytokine in blood and peritoneum. Apoptosis of CD3(+) T cell was inhibited in the thymus. The numbers of CD4(+), CD8(+) T lymphocytes and dendritic cells (DCs) were higher, while the number of CD4(+)CD25(+) regulatory T cells was lower in the baicalin group compared with the CLP group. CONCLUSIONS/SIGNIFICANCE: Baicalin improves survival of mice with polymicrobial sepsis, and this may be attributed to its antibacterial property as well as its anti-inflammatory and anti-apoptotic effects
An Observational Cohort Study of the Kynurenine to Tryptophan Ratio in Sepsis: Association with Impaired Immune and Microvascular Function
Both endothelial and immune dysfunction contribute to the high mortality rate in human sepsis, but the underlying mechanisms are unclear. In response to infection, interferon-Ξ³ activates indoleamine 2,3-dioxygenase (IDO) which metabolizes the essential amino acid tryptophan to the toxic metabolite kynurenine. IDO can be expressed in endothelial cells, hepatocytes and mononuclear leukocytes, all of which contribute to sepsis pathophysiology. Increased IDO activity (measured by the kynurenine to tryptophan [KT] ratio in plasma) causes T-cell apoptosis, vasodilation and nitric oxide synthase inhibition. We hypothesized that IDO activity in sepsis would be related to plasma interferon-Ξ³, interleukin-10, T cell lymphopenia and impairment of microvascular reactivity, a measure of endothelial nitric oxide bioavailability. In an observational cohort study of 80 sepsis patients (50 severe and 30 non-severe) and 40 hospital controls, we determined the relationship between IDO activity (plasma KT ratio) and selected plasma cytokines, sepsis severity, nitric oxide-dependent microvascular reactivity and lymphocyte subsets in sepsis. Plasma amino acids were measured by high performance liquid chromatography and microvascular reactivity by peripheral arterial tonometry. The plasma KT ratio was increased in sepsis (median 141 [IQR 64β235]) compared to controls (36 [28β52]); p<0.0001), and correlated with plasma interferon-Ξ³ and interleukin-10, and inversely with total lymphocyte count, CD8+ and CD4+ T-lymphocytes, systolic blood pressure and microvascular reactivity. In response to treatment of severe sepsis, the median KT ratio decreased from 162 [IQR 100β286] on day 0 to 89 [65β139] by day 7; pβ=β0.0006) and this decrease in KT ratio correlated with a decrease in the Sequential Organ Failure Assessment score (p<0.0001). IDO-mediated tryptophan catabolism is associated with dysregulated immune responses and impaired microvascular reactivity in sepsis and may link these two fundamental processes in sepsis pathophysiology
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