Stress as a determinant of saliva-mediated adherence and coadherence of oral and nonoral microorganisms

Objective:: The mucosal secretory proteins, such as the salivary proteins, play a key role in the acquisition and regulation of the mucosal microflora. Most notably, some microorganisms utilize the host's secretory proteins to adhere to the mucosa; a first step in colonization and infection. The secretory proteins also influence colonization by affecting the binding among microorganisms, a process denoted as coadherence. Previously we reported that acute stressors cause specific changes in saliva composition. The present study investigated to what extent these changes influence saliva-mediated microbial adherence and coadherence (ex vivo). Methods:: Thirty-two male undergraduates provided unstimulated saliva before and during a control condition and two stressors: A memory test and a surgery video presentation. We used saliva-coated microplates to test the adherence of bacteria for which the oral cavity is either a natural reservoir (eg, viridans streptococci) or a portal of entry (eg, Helicobacter pylori). We also tested the saliva-mediated co-adherence between Streptococcus gordonii and the yeast Candida albicans. Correlation analyses were performed to determine the relationships between changes in microbial adherence and the concentrations of potential salivary ligands, viz. cystatin S, the mucins MUC5B and MUC7, S-IgA, lactoferrin, [alpha]-amylase, and total salivary protein. Results:: During the memory test, saliva-mediated adhesion of Streptococcus sanguis, Streptococcus gordonii, and H. pylori increased, whereas the coadherence of C. albicans with S. gordonii decreased. During the surgical video presentation the saliva-mediated adherence of H. pylori, S. sanguis, and Streptococcus mitis increased. These changes were independent of salivary flow rate, but correlated with specific changes in salivary protein composition. Conclusion:: The results show that even moderate stressors, by altering the activity of the mucosal secretory glands, may affect microbial colonization processes such as adherence and coadherence. This study hereby presents a mechanism by which stress may affect the mucosal microflora and susceptibility to infectious disease

PPAR alpha-activation results in enhanced carnitine biosynthesis and OCTN2-mediated hepatic carnitine accumulation

In fasted rodents hepatic carnitine concentration increases considerably which is not observed in PPAR alpha-/- mice, indicating that PPAR alpha is involved in carnitine homeostasis. To investigate the mechanisms underlying the PPAR alpha-dependent hepatic carnitine accumulation we measured carnitine biosynthesis enzyme activities, levels of carnitine biosynthesis intermediates, acyl-carnitines and OCTN2 mRNA levels in tissues of untreated, fasted or Wy-14643-treated wild type and PPAR alpha-/- mice. Here we show that both enhancement of carnitine biosynthesis (due to increased gamma-butyrobetaine dioxygenase activity), extra-hepatic gamma-butyrobetaine synthesis and increased hepatic carnitine import (OCTN2 expression) contributes to the increased hepatic carnitine levels after fasting and that these processes are PPAR alpha-dependen

DataSheet_1_Mevalonate kinase-deficient THP-1 cells show a disease-characteristic pro-inflammatory phenotype.zip

ObjectiveBi-allelic pathogenic variants in the MVK gene, which encodes mevalonate kinase (MK), an essential enzyme in isoprenoid biosynthesis, cause the autoinflammatory metabolic disorder mevalonate kinase deficiency (MKD). We generated and characterized MK-deficient monocytic THP-1 cells to identify molecular and cellular mechanisms that contribute to the pro-inflammatory phenotype of MKD.MethodsUsing CRISPR/Cas9 genome editing, we generated THP-1 cells with different MK deficiencies mimicking the severe (MKD-MA) and mild end (MKD-HIDS) of the MKD disease spectrum. Following confirmation of previously established disease-specific biochemical hallmarks, we studied the consequences of the different MK deficiencies on LPS-stimulated cytokine release, glycolysis versus oxidative phosphorylation rates, cellular chemotaxis and protein kinase activity.ResultsSimilar to MKD patients’ cells, MK deficiency in the THP-1 cells caused a pro-inflammatory phenotype with a severity correlating with the residual MK protein levels. In the MKD-MA THP-1 cells, MK protein levels were barely detectable, which affected protein prenylation and was accompanied by a profound pro-inflammatory phenotype. This included a markedly increased LPS-stimulated release of pro-inflammatory cytokines and a metabolic switch from oxidative phosphorylation towards glycolysis. We also observed increased activity of protein kinases that are involved in cell migration and proliferation, and in innate and adaptive immune responses. The MKD-HIDS THP-1 cells had approximately 20% residual MK activity and showed a milder phenotype, which manifested mainly upon LPS stimulation or exposure to elevated temperatures.ConclusionMK-deficient THP-1 cells show the biochemical and pro-inflammatory phenotype of MKD and are a good model to study underlying disease mechanisms and therapeutic options of this autoinflammatory disorder.</p

Manipulation of isoprenoid biosynthesis as a possible therapeutic option in mevalonate kinase deficiency

OBJECTIVE: In cells from patients with the autoinflammatory disorder mevalonate kinase (MK) deficiency, which includes the hyperimmunoglobulin D with periodic fever syndrome, MK becomes the rate-limiting enzyme in the isoprenoid biosynthesis pathway. This suggests that up-regulation of residual MK activity in these patients could be a way in which to prevent or alleviate the associated symptoms. We studied the effect of 2 specific inhibitors of isoprenoid biosynthetic enzymes on the residual activity of MK in cells from patients with MK deficiency. METHODS: Skin fibroblasts from MK-deficient patients and from controls were cultured for 7 days with either simvastatin, an inhibitor of 3-hydroxy-3-methylglutaryl coenzyme A reductase, or zaragozic acid A, an inhibitor of squalene synthase. Following culture, MK activity, MK protein levels, MVK messenger RNA levels, and the effect on the pathway flux toward non-sterol isoprenoid biosynthesis were determined. RESULTS: Treatment of the fibroblasts with either of the inhibitors led to a marked increase in residual MK enzyme activity, which was largely attributable to increased MVK gene transcription. This effect was even more pronounced when the cells were cultured in lipoprotein-depleted medium. The flux toward nonsterol isoprenoid end-product synthesis was reduced when cells were treated with simvastatin but was partly restored by concomitant treatment with zaragozic acid A. CONCLUSION: Our results indicate that manipulations of the isoprenoid biosynthesis pathway that promote the synthesis of nonsterol isoprenoids may provide an interesting therapeutic option for the treatment of MK deficienc

Fasting adaptation in idiopathic ketotic hypoglycemia: a mismatch between glucose production and demand

In order to study the pathophysiology of hypoglycemia in idiopathic ketotic hypoglycemia (KH), glucose kinetics during fasting in patients with KH were determined. A fasting test was performed in 12 children with previously documented KH. Besides determination of glucoregulatory hormones, plasma ketones, FFA and alanine, the rates of endogenous glucose production (EGP), glucose uptake, gluconeogenesis (GNG) and glycogenolysis (GGL) were quantified using the [6,6-H-2(2)] glucose isotope dilution method and the deuterated water method. The five youngest subjects (age 2.5-3.9 years) became hypoglycemic (glucose <3.0 mmol/l) during the test. Mean differences in glucose kinetics between overnight fasting and the end of the test in the hypoglycemic vs. the normoglycemic subjects were: EGP: -31.9% vs. -17.9% (p=0.007), GGL: -66.2% vs. -50.8% (p=0.465) and GNG 6.8% vs. 19.5% (p=0.465). Plasma alanine levels were significantly lower (p=0.028) at the end of the test in the hypoglycemic subjects. Plasma ketones and FFA levels were in the normal range for fasting duration in all subjects. We conclude that hypoglycemia in KH is caused by the inability to sustain an adequate EGP during fasting in view of the higher glucose requirement in young children. The decrease in GGL is not accompanied by a significant increase in GNG, possibly because of a limitation in the supply of alanine. Our results support the hypothesis that KH represents the lower tail of the Gaussian distribution of fasting tolerance in childre

Mevalonate kinase-deficient THP-1 cells show a disease-characteristic pro-inflammatory phenotype

ObjectiveBi-allelic pathogenic variants in the MVK gene, which encodes mevalonate kinase (MK), an essential enzyme in isoprenoid biosynthesis, cause the autoinflammatory metabolic disorder mevalonate kinase deficiency (MKD). We generated and characterized MK-deficient monocytic THP-1 cells to identify molecular and cellular mechanisms that contribute to the pro-inflammatory phenotype of MKD.MethodsUsing CRISPR/Cas9 genome editing, we generated THP-1 cells with different MK deficiencies mimicking the severe (MKD-MA) and mild end (MKD-HIDS) of the MKD disease spectrum. Following confirmation of previously established disease-specific biochemical hallmarks, we studied the consequences of the different MK deficiencies on LPS-stimulated cytokine release, glycolysis versus oxidative phosphorylation rates, cellular chemotaxis and protein kinase activity.ResultsSimilar to MKD patients’ cells, MK deficiency in the THP-1 cells caused a pro-inflammatory phenotype with a severity correlating with the residual MK protein levels. In the MKD-MA THP-1 cells, MK protein levels were barely detectable, which affected protein prenylation and was accompanied by a profound pro-inflammatory phenotype. This included a markedly increased LPS-stimulated release of pro-inflammatory cytokines and a metabolic switch from oxidative phosphorylation towards glycolysis. We also observed increased activity of protein kinases that are involved in cell migration and proliferation, and in innate and adaptive immune responses. The MKD-HIDS THP-1 cells had approximately 20% residual MK activity and showed a milder phenotype, which manifested mainly upon LPS stimulation or exposure to elevated temperatures.ConclusionMK-deficient THP-1 cells show the biochemical and pro-inflammatory phenotype of MKD and are a good model to study underlying disease mechanisms and therapeutic options of this autoinflammatory disorder

The enigmatic role of tafazzin in cardiolipin metabolism

The mitochondrial phospholipid cardiolipin plays an important role in cellular metabolism as exemplified by its involvement in mitochondrial energy production and apoptosis. Following its biosynthesis, cardiolipin is actively remodeled to achieve its final acyl composition. An important cardiolipin remodeling enzyme is tafazzin, of which several mRNA splice variants exist. Mutations in the tafazzin gene cause the X-linked recessive disorder Barth syndrome. In addition to providing an overview of the current knowledge in literature about tafazzin, we present novel experimental data and use this to discuss the functional role of the different tafazzin variants in cardiolipin metabolism in relation to Barth syndrome. We developed and performed specific quantitative PCR analyses of different tafazzin mRNA splice variants in 16 human tissues and correlated this with the tissue cardiolipin profile. In BTHS fibroblasts we showed that mutations in the tafazzin gene affected both the level and distribution of tafazzin mRNA variants. Transient expression of selected human tafazzin variants in BTHS fibroblasts showed for the first time in a human cell system that tafazzin lacking exon5 indeed functions in cardiolipin remodelin

The role of ELOVL1 in very long-chain fatty acid homeostasis and X-linked adrenoleukodystrophy

X-linked adrenoleukodystrophy (X-ALD) is caused by mutations in the ABCD1 gene encoding the peroxisomal ABC transporter adrenoleukodystrophy protein (ALDP). X-ALD is characterized by the accumulation of very long-chain fatty acids (VLCFA; >= C24) in plasma and tissues. In this manuscript we provide insight into the pathway underlying the elevated levels of C26:0 in X-ALD. ALDP transports VLCFacyl-CoA across the peroxisomal membrane. A deficiency in ALDP impairs peroxisomal beta-oxidation of VLCFA but also raises cytosolic levels of VLCFacyl-CoA which are substrate for further elongation. We identify ELOVL1 (elongation of very-long-chain-fatty acids) as the single elongase catalysing the synthesis of both saturated VLCFA (C26:0) and mono-unsaturated VLCFA (C26:1). ELOVL1 expression is not increased in X-ALD fibroblasts suggesting that increased levels of C26:0 result from increased substrate availability due to the primary deficiency in ALDP. Importantly, ELOVL1 knockdown reduces elongation of C22:0 to C26: 0 and lowers C26:0 levels in X-ALD fibroblasts. Given the likely pathogenic effects of high C26:0 levels, our findings highlight the potential of modulating ELOVL1 activity in the treatment of X-AL