Association of a variable number tandem repeat in the NLRP3 gene in women with susceptibility to RVVC.

Vaginal infections with Candida spp. frequently occur in women of childbearing age. A small proportion of these women experience recurrent vulvovaginal candidosis (RVVC), which is characterized by at least three episodes of infection in one year. In addition to known risk factors such as antibiotics, diabetes, or pregnancy, host genetic variation and inflammatory pathways such as the IL-1/Th17 axis have been reported to play a substantial role in the pathogenesis of RVVC. In this study, we assessed a variable number tandem repeat (VNTR) polymorphism in the NLRP3 gene that encodes a component of the inflammasome, processing the proinflammatory cytokines IL-1ß and IL-18. A total of 270 RVVC patients and 583 healthy controls were analyzed, and increased diseases susceptibility was associated with the presence of the 12/9 genotype. Furthermore, functional studies demonstrate that IL-1ß production at the vaginal surface is higher in RVVC patients bearing the 12/9 genotype compared to controls, whereas IL-1Ra levels were decreased and IL-18 levels remained unchanged. These findings suggest that IL-1ß-mediated hyperinflammation conveyed by the NLRP3 gene plays a causal role in the pathogenesis of RVVC and may identify this pathway as a potential therapeutic target in the disease.

A missense mutation underlies defective SOCS4 function in a family with autoimmunity.

OBJECTIVE: The aim of this study was to determine the genetic and immunological defects underlying familial manifestations of an autoimmune disorder. METHODS: Whole-exome sequencing was performed on the index patient with various manifestations of autoimmunity, including hypothyroidism, vitiligo and alopecia. Peripheral blood mononuclear cells and DNA of family members were used for functional and genetic testing of the candidate variants obtained by Sanger sequencing. RESULTS: Exome sequencing identified 233 rare, coding and nonsynonymous variants in the index patient; five were highly conserved and affect genes that have a possible role in autoimmunity. Only a heterozygous missense mutation in the suppressor of cytokine signalling 4 gene (SOCS4) cosegregated with the autoimmune disorder in the family. SOCS4 is a known inhibitor of epidermal growth factor (EGF) receptor signalling, and functional studies demonstrated specific upregulation of EGF-dependent immune stimulation in affected family members. CONCLUSION: We present a family with an autoimmune disorder, probably resulting from dysregulated immune responses due to mutations in SOCS4.

Defective trained immunity in patients with STAT-1-dependent chronic mucocutaneaous candidiasis.

Patients with signal transducer and activator of transcription-1 (STAT1)-dependent chronic mucocutaneous candidiasis (CMC) and patients with STAT3-dependent hyper-immunoglobulin (Ig)E syndrome (HIES) display defects in T helper type 17 (Th17) cytokine production capacity. Despite this similar immune defect in Th17 function, they show important differences in the type of infections to which they are susceptible. Recently, our group reported differential regulation of STAT-1 and STAT-3 transcription factors during epigenetic reprogramming of trained immunity, an important host defence mechanism based on innate immune memory. We therefore hypothesized that STAT1 and STAT3 defects have different effects on trained immunity, and this may partly explain the differences between CMC and HIES regarding the susceptibility to infections. Indeed, while trained immunity was normally induced in cells isolated from patients with HIES, the induction of innate training was defective in CMC patients. This defect was specific for training with Candida albicans, the main pathogen encountered in CMC, and it involved a type II interferon-dependent mechanism. These findings describe the role of STAT-1 for the induction of trained immunity, and may contribute to the understanding of the differences in susceptibility to infection between CMC and HIES patients. This study could also provide directions for personalized immunotherapy in patients suffering from these immunodeficiencies.

Association of autophagy-related 16-like 1 (ATG16L1) gene polymorphism with sepsis severity in patients with sepsis and ventilator-associated pneumonia.

Autophagy is a highly conserved mechanism of eukaryotic cells implicated in cell homeostasis and elimination of intracellular pathogens. Functional polymorphisms in genes encoding for autophagy have been associated with susceptibility to inflammatory and infectious diseases, but data on severe infections are missing. The aim of the present study was to assess whether polymorphisms in genes encoding proteins involved in autophagy influence susceptibility to ventilator-associated pneumonia (VAP). Mechanically ventilated patients with VAP were studied. Genotyping for autophagy-related 16-like 1 (ATG16L1, rs2241880) functional polymorphism was performed using the TaqMan single-nucleotide assay. Monocytes were isolated from patients and stimulated with lipopolysaccharide (LPS). Tumor necrosis factor-α (TNF-α) was measured in the supernatants of monocytes using an enzyme-linked immunosorbent assay. Procalcitonin (PCT) was also measured in the serum of patients by an immuno-time-resolved amplified cryptate technology assay. A total of 155 patients with VAP were enrolled in the study. Carriage of the minor A allele of ATG16L1 was associated with septic shock with at least one organ failure (odds ratio (OR): 2.40, p: 0.036). TNF-α production was significantly greater among the carriers of the polymorphism presenting with at least one organ failure (p: 0.040). PCT was increased upon worsening to septic shock and organ failure only among carriers of the minor frequency A alleles. In a homogeneous cohort of septic patients with VAP, the carriage of autophagy polymorphisms predisposes to VAP severity and septic shock development. This may be related with predisposition to immunoparalysis.

Polymorphisms in cytokine genes IL6, TNF, IL10, IL17A and IFNG influence susceptibility to complicated skin and skin structure infections.

Complicated skin and skin structure infections (cSSSIs) are caused by Gram-positive and Gram-negative, aerobic and anaerobic pathogens, with a polymicrobial aetiology being frequent. Recognition of invading pathogens by the immune system results in the production of pro- and anti-inflammatory cytokines, which are extremely important for intercellular communication and control of infection. This study assessed whether genetic variation in genes encoding cytokines influences the susceptibility to cSSSIs. For the association study, 318 patients with cSSSI and 328 healthy controls were genotyped for single nucleotide polymorphisms (SNPs) in cytokine genes IL1A, IL1B, IL1RN, TNF, IL10, IL17A, IL17F and IFNG. For immunological validation, peripheral blood mononuclear cells (PBMCs) from 74 healthy individuals, genotyped for SNPs of interest, were stimulated with Staphylococcus aureus or Escherichia coli and corresponding cytokine levels were determined by enzyme-linked immunosorbent assay (ELISA). Polymorphisms IL6 rs1800797, TNF rs1800629, IL10 rs1800871, IL17A rs8193036 and IFNG rs2069705 influenced susceptibility to cSSSIs. No differences in cytokine responses, stratified for genotype, were detected after PBMC stimulation. No association with cSSSIs was observed for polymorphisms IL1A rs17561 and rs1800587, IL1B rs16944 and rs1143627, IL1RN rs4251961, TNF rs361525, IL10 rs1800896, IL17A rs2275913 and IL17F rs763780. In conclusion, polymorphisms in IL6, TNF, IL10, IL17A and IFNG are associated with susceptibility to cSSSIs.

Autophagy is redundant for the host defense against systemic Candida albicans infections.

Autophagy has been demonstrated to play an important role in the immunity against intracellular pathogens, but very little is known about its role in the host defense against fungal pathogens such as Candida albicans. Therefore, the role of autophagy for the host defense against C. albicans was assessed by complementary approaches using mice defective in autophagy, as well as immunological and genetic studies in humans. Although C. albicans induced LC3-II formation in macrophages, myeloid cell-specific ATG7(-/-) mice with defects in autophagy did not display an increased susceptibility to disseminated candidiasis. In in vitro experiments in human blood mononuclear cells, blocking autophagy modulated cytokine production induced by lipopolysaccharide, but not by C. albicans. Furthermore, autophagy modulation in human monocytes did not influence the phagocytosis and killing of C. albicans. Finally, 18 single-nucleotide polymorphisms in 13 autophagy genes were not associated with susceptibility to candidemia or clinical outcome of disease in a large cohort of patients, and there was no correlation between these genetic variants and cytokine production in either candidemia patients or healthy controls. Based on these complementary in vitro and in vivo studies, it can be concluded that autophagy is redundant for the host response against systemic infections with C. albicans.

Different patterns of Toll-like receptor 2 polymorphisms in populations of various ethnic and geographic origins.

Upon the invasion of the host by microorganisms, innate immunity is triggered through pathogen recognition by pattern recognition receptors (PRRs). Toll-like receptors (TLRs) are the best-studied class of PRRs, and they recognize specific pathogen-associated molecular patterns (PAMPs) from various microorganisms. A large number of studies have shown that genetic variation in TLRs may influence susceptibility to infections. We assessed the genetic variation of TLR2, which encodes one of the most important TLRs, in various populations around the globe and correlated it with changes in the function of the molecule. The three best-known nonsynonymous TLR2 polymorphisms (1892C>A, 2029C>T, and 2258G>A) were assessed in different populations from the main continental masses: Romanians, Vlax-Roma, Dutch (European populations), Han Chinese (East Asia), Dogon, Fulani (Africa), and Trio Indians (America). The 2029C>T polymorphism was absent in both European and non-European populations, with the exception of the Vlax-Roma, suggesting that this polymorphism most likely arose in Indo-Aryan people after migration into South Asia. The 1892C>A polymorphism that was found exclusively in European populations, but not in Asian, African, or American volunteers, probably occurred in proto-Indo-Europeans. Interestingly, 2258G>A was present only in Europeans, including Vlax-Roma, but at a very low frequency. The differential pattern of the TLR2 polymorphisms in various populations may explain some of the differences in susceptibility to infections between these populations.

The impact of caspase-12 on susceptibility to candidemia.

Candida is one of the leading causes of sepsis, and an effective host immune response to Candida critically depends on the cytokines IL-1ß and IL-18, which need caspase-1 cleavage to become bioactive. Caspase-12 has been suggested to inhibit caspase-1 activation and has been implicated as a susceptibility factor for bacterial sepsis. In populations of African descent, CASPASE-12 is either functional or non-functional. Here, we have assessed the frequencies of both CASPASE-12 alleles in an African-American Candida sepsis patients cohort compared to uninfected patients with similar predisposing factors. African-American Candida sepsis patients (n = 93) and non-infected African-American patients (n = 88) were genotyped for the CASPASE-12 genotype. Serum cytokine concentrations of IL-6, IL-8, and IFNγ were measured in the serum of infected patients. Statistical comparisons were performed in order to assess the effect of the CASPASE-12 genotype on susceptibility to candidemia and on serum cytokine concentrations. Our findings demonstrate that CASPASE-12 does not influence the susceptibility to Candida sepsis, nor has any effect on the serum cytokine concentrations in Candida sepsis patients during the course of infection. Although the functional CASPASE-12 allele has been suggested to increase susceptibility to bacterial sepsis, this could not be confirmed in our larger cohort of fungal sepsis patients.

Targetable gene fusions identified in radioactive iodine refractory advanced thyroid carcinoma.

Objective Gene alterations leading to activation of the MAPK pathway are of interest for targeted therapy in patients with advanced radioactive iodine refractory (RAI-R) thyroid carcinoma. Due to technical reasons gene fusion analysis in RNA isolated from formalin-fixed tumor tissues has till now been limited. The objective of the present study was to identify targetable gene rearrangements in RNA isolated from formalin-fixed RAI-R thyroid carcinomas. Design Retrospective study in 132 patients with RAI-R thyroid carcinoma (59 papillary-, 24 follicular-, 35 Hürthle cell- and 14 anaplastic thyroid carcinoma). Methods Total nucleic acid (undivided DNA and RNA) was isolated from formalin-fixed tissue. Extensive gene fusion analysis was performed in all samples that tested negative for pathogenic BRAF, NRAS, HRAS and KRAS variants. Results Seven targetable gene fusions were identified in the remaining 60 samples without known DNA variants. This includes frequently reported gene fusions such as CCDC6/RET (PTC1), PRKAR1A/RET (PTC2) and ETV6/NTRK3 , and gene fusions that are less common in thyroid cancer (TPM3/NTRK1, EML4/ALK and EML4/NTRK3). Of note, most gene fusions were detected in papillary thyroid carcinoma and MAPK-associated alterations in Hürthle cell carcinomas are rare (2/35). Conclusion Targetable gene fusions were found in 12% of RAI-R thyroid carcinoma without DNA variants and can be effectively identified in formalin-fixed tissue. These gene fusions might provide a preclinical rationale to include specific kinase inhibitors in the treatment regimen for these patients. The latter intends to restore iodine transport and/or take advantage of the direct effect on tumor cell vitality once progressive disease is seen.

Rare NOX3 Variants Confer Susceptibility to Agranulocytosis During Thyrostatic Treatment of Graves' Disease.

Agranulocytosis is a rare and serious adverse effect of antithyroid drugs, with unknown etiology. The present study aimed to uncover genetic susceptibility and underlying mechanisms of antithyroid drug-induced agranulocytosis (ATDAC). We studied two independent families with familial Graves' disease, of which several members developed ATDAC. In addition, six sporadic ATDAC patients with Graves' disease were investigated. Whole exome sequencing analysis of affected and unaffected family members was performed to identify genetic susceptibility variants for ATDAC, followed by functional characterization of primary granulocytes from patients and unrelated healthy controls. Whole exome sequencing, cosegregation analysis, and stringent selection criteria of candidate gene variants identified NOX3 as a genetic factor related to ATDAC. Functional studies revealed increased apoptosis of methimazole-treated granulocytes from patients carrying NOX3 variants. In conclusion, genetic variants in NOX3 may confer susceptibility to antithyroid drug-induced apoptosis of granulocytes. These findings contribute to the understanding of the mechanisms underlying ATDAC.

Glucocorticoid receptor polymorphisms modulate cardiometabolic risk factors in patients in long-term remission of Cushing's syndrome.

CONTEXT: Glucocorticoid receptor (GR) polymorphisms modulate glucocorticoid (GC) sensitivity and are associated with altered metabolic profiles. OBJECTIVE: To evaluate the presence of GR polymorphisms (BclI (rs41423247), N363S (rs56149945), ER22/23EK (rs6189/rs6190), and 9ß (rs6198) and investigate their associations with metabolic alterations in patients in long-term remission of Cushing's syndrome (CS). DESIGN AND SETTING: Cross-sectional case-control study. PATIENTS AND METHODS: Sixty patients in long-term remission of CS were genotyped. Associations between GR polymorphisms and multiple vascular, body composition and metabolic parameters were investigated. MAIN OUTCOME MEASURES: Allelic frequencies of the polymorphisms and their associations with several cardiometabolic risk factors. RESULTS: This study shows that carriers of the 9ß polymorphism have a higher systolic blood pressure and lower resistin levels. The GC sensitizing BclI polymorphism is associated with an adverse cardiometabolic risk factor profile: higher fat percentages of extremities and legs, higher serum leptin and E-selectin levels, and higher intima media thickness in carriers versus non-carriers. CONCLUSIONS: The 9ß and BclI polymorphisms of the GR adversely affect the cardiometabolic profile in patients who are in remission after the treatment of CS. This suggests that genetically altered GC sensitivity modulates the long-term adverse cardiometabolic effects resulting from (endogenous) hypercortisolism.

Vascular Health in Patients in Remission of Cushing's Syndrome Is Comparable With That in BMI-Matched Controls.

CONTEXT: In active Cushing's syndrome (CS), patients suffer from endothelial dysfunction and premature atherosclerosis. However, it is uncertain to what extent vascular health recovers after long-term remission. This is highly relevant because this topic relates to future development of cardiovascular disease. OBJECTIVE: The objective of the study was to investigate whether micro- and macrovascular health is impaired after long-term remission of CS in patients with no or adequately treated comorbidities. DESIGN AND SETTING: This was a cross-sectional case-control study in two tertiary referral centers. PATIENTS AND MAIN OUTCOME MEASURES: Sixty-three patients (remission of CS for ≥ 4 y) and 63 healthy, well-matched controls were compared. In group A (58 patients and 58 controls), serum biomarkers associated with endothelial dysfunction, intima media thickness, pulse wave velocity, and pulse wave analysis were studied. In group B (14 patients and 14 controls), endothelium-dependent and -independent vasodilatation was studied in conduit arteries (flow mediated dilation of the brachial artery) and forearm skeletal muscle resistance arteries (vasodilator response to intraarterial acetylcholine, sodium-nitroprusside, and NG-monomethyl-L-arginine using venous occlusion plethysmography). RESULTS: There were no significant differences between the outcome measures of vascular health of patients and controls in groups A and B. CONCLUSION: The vascular health of patients in long-term remission of CS seems to be comparable with that of healthy gender-, age-, and body mass index-matched controls, provided that the patients have no, or adequately controlled, comorbidities. Therefore, the effects of hypercortisolism per se on the vasculature may be reversible. This accentuates the need for the stringent treatment of metabolic comorbidities in these patients.