<Emphasis Type="Italic">Gcg</Emphasis><Superscript><Emphasis Type="Italic">CreERT2</Emphasis></Superscript> knockin mice as a tool for genetic manipulation in pancreatic alpha cells

Aims/hypothesis

The Cre/loxP system, which enables tissue-specific manipulation of genes, is widely used in mice for diabetes research. Our aim was to develop a new Cre-driver mouse line for the specific and efficient manipulation of genes in pancreatic alpha cells.

Methods

A Gcg ^CreERT2 knockin mouse, which expresses a tamoxifen-inducible form of Cre from the endogenous preproglucagon (Gcg) gene locus, was generated by homologous recombination. The new Gcg ^CreERT2 mouse line was crossed to the Rosa26 ^tdTomato (R26 ^tdTomato ) Cre reporter mouse line in order to evaluate the tissue specificity, efficiency and tamoxifen dependency of Gcg ^CreERT2 -mediated recombination. Cell types of pancreatic islets were identified using immunohistochemistry. Biochemical and physiological data, including blood glucose levels, plasma glucagon and glucagon-like peptide (GLP)-1 levels, and pancreatic glucagon content, were collected and used to assess the overall effect of Gcg gene targeting on Gcg ^CreERT2/w heterozygous mice.

Results

Tamoxifen-treated Gcg ^CreERT2/w ;R26 ^tdTomato/w mice displayed Cre reporter activity, i.e. expression of tdTomato red fluorescent protein (RFP) in all known cells that produce proglucagon-derived peptides. In the adult pancreas, RFP was detected in 94–97% of alpha cells, whereas it was detected in a negligible (~ 0.2%) proportion of beta cells. While more than 98% of cells labelled with tamoxifen-induced RFP were glucagon-positive cells, 14–25% of pancreatic polypeptide (PP)-positive cells were also positive for RFP, indicating the presence of glucagon/PP bihormonal cell population. Tamoxifen-independent expression of RFP occurred in approximately 6% of alpha cells. In contrast to alpha cells and GLP-1-producing neurons, in which RFP expression persisted for at least 5 months after tamoxifen administration (presumably due to rare neogenesis in these cell types in adulthood), nearly half of RFP-positive intestinal L cells were replaced with RFP-negative L cells over the first 2 weeks after tamoxifen administration. Heterozygous Gcg ^CreERT2/w mice showed reduced Gcg mRNA levels in islets, but maintained normal levels of pancreatic and plasma glucagon. The mice did not exhibit any detectable baseline physiological abnormalities, at least in young adulthood.

Conclusions/interpretation

The newly developed Gcg ^CreERT2 knockin mouse shows faithful expression of CreER^T2 in pancreatic alpha cells, intestinal L cells and GLP-1-producing neurons. This mouse line will be particularly useful for manipulating genes in alpha cells, due to highly specific and efficient CreER^T2-mediated recombination in this cell type in the pancreas.

     

Analysis of PD-1 and Tim-3 expression on CD4<Superscript>+</Superscript> T cells of patients with rheumatoid arthritis; negative association with DAS28

Expression of T cell immunoglobulin and mucin-domain containing-3 (Tim-3) and programmed cell death-1 (PD-1) was studied on CD4⁺ T cells of patients with rheumatoid arthritis (RA). Association of Tim-3 and PD-1 expression with disease activity of RA patients was also addressed. A total of 37 RA patients and 31 sex- and age-matched healthy controls were included in this study. Disease activity of RA patients was determined by Disease Activity Score of 28 joints scoring system (DAS28). A three-color flow cytometry method was applied to determine the frequency of Tim-3⁺/PD-1⁺/CD4⁺ T cells. To measure the cytokine production, peripheral blood mononuclear cells (PBMCs) were stimulated with PMA/ionomycin. Concentrations of IL-17, IL-10, IFN-γ, and TNF-α were measured in culture supernatants by ELISA. The frequency of PD-1⁺/CD4⁺ and Tim-3⁺/PD-1⁺/CD4⁺ T cells was significantly higher in patients with RA compared to that in controls (p?=?0.0013 and p?=?0.050, respectively). The percentage of Tim-3⁺/CD4⁺ T cells was similar in patients and controls (p?=?0.4498). The RA patients have produced significant higher levels of TNF-α, IL-17, and IFN-γ than those of healthy controls (p?=?0.0121, p?=?0.0417, and p?=?0.0478, respectively). Interestingly, an inverse correlation was found between the frequency of Tim-3⁺/CD4⁺ cells and DAS28 of RA patients (r?=???0.4696, p?=?0.0493). Similarly, the percentage of Tim-3⁺/PD-1⁺/CD4⁺ T cells was also revealed an inverse correlation with DAS28 (r?=???0.5268, p?=?0.0493). Moreover, significant positive correlations were detected between the concentrations of TNF-α (r?=?0.6418, p?=?0.0023) and IL-17 (r?=?0.4683, p?=?0.0373) with disease activity of RA patients. Our results indicate that Tim-3 and PD-1 are involved in immune dysregulation mechanisms of rheumatoid arthritis and could be considered as useful biomarkers for determination of disease activity and progression.     

Association Between CD4<Superscript>+</Superscript>, Viral Load,and Pulmonary Function in HIV

Purpose

The antiretroviral therapy era has shifted the epidemiology of HIV-associated diseases, increasing the recognition of non-infectious pulmonary complications secondary to HIV. We aimed to determine the association between CD4⁺, viral load, and pulmonary function in individuals with uncontrolled HIV, and determine how changes in these parameters are associated with pulmonary function longitudinally.

Methods

This is a retrospective observational study of individuals with HIV who underwent pulmonary function testing in an urban medical center between August 1997 and November 2015.

Results

Of the 146 participants (mean age 52 ± 10 years), 49% were Hispanic, 56% were men, and 44% were current smokers. CD4⁺ <200 cells/μl was associated with significant diffusion impairment compared to CD4⁺ ≥200 cells/μl (DL_CO 56 vs. 70%, p = <0.01). VL (viral load) ≥75 copies/ml was associated with significant diffusion impairment compared to VL <75 copies/ml (DL_CO 60 vs. 71%, p = <0.01). No difference in FEV1, FEV1/FVC, or TLC was noted between groups. In univariate analysis, CD4⁺ and VL correlated with DL_CO (r = +0.33; p = <0.01; r = ?0.26; p = <0.01) and no correlation was noted with FEV₁, FEV₁/FVC, or TLC. Current smoking and history of AIDS correlated with DL_CO (r = ?0.20; p = 0.03; r = ?0.20; p = 0.04). After adjusting for smoking and other confounders, VL ≥75 copies/ml correlated with a 11.2 (CI 95% [3.03–19.4], p = <0.01) decrease in DL_CO. In Spearman’s Rank correlation, there was a negative correlation between change in VL and change in DL_CO over time (ρ = ?0.47; p = <0.01).

Conclusion

The presence of viremia in individuals with HIV is independently associated with impaired DL_CO. Suppression of VL may allow for recovery in diffusing capacity over time, though the degree to which this occurs requires further investigation.

     

Cytotoxic isolates of <Emphasis Type="Italic">Helicobacter pylori</Emphasis> from Peptic Ulcer Diseases decrease K<Superscript>+</Superscript>-dependent ATPase Activity in HeLa cells

Background

Helicobacter pylori is a Gram negative bacterium that plays a central role in the etiology of chronic gastritis and peptic ulcer diseases. However, not all H. pylori positive cases develop advanced disease. This discriminatory behavior has been attributed to the difference in virulence of the bacteria. Among all virulence factors, cytotoxin released by H. pylori is the most important factor. In this work, we studied variation in H. pylori isolates from Indian dyspeptic patients on the basis of cytotoxin production and associated changes in K⁺-dependent ATPase (one of its targets) enzyme activity in HeLa cells.

Methods

The patients were retrospectively grouped on the basis of endoscopic and histopathological observation as having gastritis or peptic ulcer. The HeLa cells were incubated with the broth culture filtrates (BCFs) of H. pylori isolates from patients of both groups and observed for the cytopathic effects: morphological changes and viability. In addition, the K⁺-dependent ATPase activity was measured in HeLa cells extracts.

Results

The cytotoxin production was observed in 3/7 (gastritis) and 4/4 (peptic ulcer) H. pylori isolates. The BCFs of cytotoxin producing H. pylori strains reduced the ATPase activity of HeLa cells to 40% of that measured with non-cytotoxin producing H. pylori strains (1.33 μmole Pi/mg protein and 3.36 μmole Pi/mg protein, respectively, p < 0.05). The decreased activity of ATPase enzyme or the release of cytotoxin also correlated with the increased pathogenicity indices of the patients.

Conclusions

Our results suggest that the isolation of cytotoxic H. pylori is more common in severe form of acid peptic diseases (peptic ulcer) than in gastritis patients from India. Also the cytotoxin released by H. pylori impairs the ion-transporting ATPase and is a measure of cytotoxicity.

     

A <Emphasis Type="Italic">Tbc1d1</Emphasis><Superscript>Ser231Ala</Superscript>-knockin mutation partially impairs AICAR- but not exercise-induced muscle glucose uptake in mice

Aims/hypothesis

TBC1D1 (tre-2/USP6, BUB2, cdc16 domain family member 1) is a Rab GTPase-activating protein (RabGAP) that has been implicated in regulating GLUT4 trafficking. TBC1D1 can be phosphorylated by the AMP-activated protein kinase (AMPK) on Ser²³¹, which consequently interacts with 14-3-3 proteins. Given the key role for AMPK in regulating insulin-independent muscle glucose uptake, we hypothesised that TBC1D1-Ser²³¹ phosphorylation and/or 14-3-3 binding may mediate AMPK-governed glucose homeostasis.

Methods

Whole-body glucose homeostasis and muscle glucose uptake were assayed in mice bearing a Tbc1d1 ^Ser231Ala-knockin mutation or harbouring skeletal muscle-specific Ampkα1/α2 (also known as Prkaa1/2) double-knockout mutations in response to an AMPK-activating agent, 5-aminoimidazole-4-carboxamide-1-β-d-ribofuranoside (AICAR). Exercise-induced muscle glucose uptake and exercise capacity were also determined in the Tbc1d1 ^Ser231Ala-knockin mice.

Results

Skeletal muscle-specific deletion of Ampkα1/a2 in mice prevented AICAR-induced hypoglycaemia and muscle glucose uptake. The Tbc1d1 ^Ser231Ala-knockin mutation also attenuated the glucose-lowering effect of AICAR in mice. Glucose uptake and cell surface GLUT4 content were significantly lower in muscle isolated from the Tbc1d1 ^Ser231Ala-knockin mice upon stimulation with a submaximal dose of AICAR. However, this Tbc1d1 ^Ser231Ala-knockin mutation neither impaired exercise-induced muscle glucose uptake nor affected exercise capacity in mice.

Conclusions/interpretation

TBC1D1-Ser²³¹ phosphorylation and/or 14-3-3 binding partially mediates AMPK-governed glucose homeostasis and muscle glucose uptake in a context-dependent manner.

     

Reduced glutamate in white matter of male neonates exposed to alcohol <Emphasis Type="Italic">in utero</Emphasis>: a <Superscript>1</Superscript>H-magnetic resonance spectroscopy study

In utero exposure to alcohol leads to a spectrum of fetal alcohol related disorders (FASD). However, few studies used have used proton magnetic resonance spectroscopy (¹H-MRS) to understand how neurochemical disturbances relate to the pathophysiology of FASD. Further, no studies to date have assessed brain metabolites in infants exposed to alcohol in utero. We hypothesize that neonates exposed to alcohol in utero will show decreased glutamatergic activity, pre-emptive of their clinical diagnosis or behavioural phenotype. Single voxel ¹H-MRS data, sampled in parietal white and gray matter, were acquired from 36 neonates exposed to alcohol in utero, and 31 control unexposed healthy neonates, in their 2nd-4th week of life. Metabolites relative to creatine with phosophocreatine and metabolites absolute concentrations using a water reference are reported. Male infants exposed to alcohol in utero were found to have reduced concentration of glutamate with glutamine (Glx) in their parietal white matter (PWM), compared to healthy male infants (p = 0.02). Further, male infants exposed to alcohol in utero had reduced concentration and ratio for glutamate (Glu) in their PWM (p = 0.02), compared to healthy male infants and female infants exposed to alcohol in utero. Female infants showed higher relative Glx and Glu ratios for parietal gray matter (PGM, p < 0.01), compared to male infants. We speculate that the decreased Glx and Glu concentrations in PWM are a result of delayed oligodendrocyte maturation, which may be a result of dysfunctional thyroid hormone activity in males exposed to alcohol in utero. Further study is required to elucidate the relationship between Glx and Glu, thyroid hormone activity, and oligodendrocyte maturation in infants exposure to alcohol in utero.     

Characterization and CRISPR-based genotyping of clinical <Emphasis Type="Italic">trh</Emphasis>-positive <Emphasis Type="Italic">Vibrio parahaemolyticus</Emphasis>

Background

Vibrio parahaemolyticus is a causative agent of gastroenteritis. Most of the clinical isolates carry either tdh and/or trh genes which are considered as the major virulence genes of this pathogen. In this study, the clinical isolates of V. parahaemolyticus carrying trh gene (n?=?73) obtained from 1886 to 2012 from various countries were investigated for the urease production, haemolytic activity, and biofilm formation. In addition, the potential of clustered regularly interspaced short palindromic repeats (CRISPR)-based genotyping among these isolates was investigated.

Results

In this study, no significant differences were observed in the urease production between tdh⁺ trh1⁺ and tdh⁺ trh2⁺ isolates (p?=?0.063) and between the tdh^? trh1⁺ and tdh^? trh2⁺ isolates (p?=?0.788). The isolates carrying only the trh gene showed variation in their haemolytic activity. The ratio of urease production and haemolytic activity between the trh1⁺ and trh2⁺ isolates and biofilm formation of trh⁺ V. parahaemolyticus isolates were not significantly different. Sixteen of thirty-four tested isolates (47.0%) of trh⁺ V. parahaemolyticus were positive for CRISPR detection. The discriminatory power index (DI) of CRISPR-virulence typing was higher than the DI obtained by CRISPR typing alone.

Conclusion

The tdh and trh genes were not involved in urease production in the trh⁺ V. parahaemolyticus, and variation of haemolytic activity detected in V. parahaemolyticus carrying only the trh gene might be correlated to the sequence variation within trh1 and trh2 genes. Additionally, biofilm production of V. parahaemolyticus was not associated with harboring of virulence genes. For genotyping, CRISPR sequences combined with virulence genes can be used as genetic markers to differentiate trh⁺ V. parahaemolyticus strains.

     

<Emphasis Type="Italic">Fusobacterium nucleatum</Emphasis> Potentiates Intestinal Tumorigenesis in Mice via a Toll-Like Receptor 4/p21-Activated Kinase 1 Cascade

Background

The underlying pathogenic mechanism of Fusobacterium nucleatum in the carcinogenesis of colorectal cancer has been poorly understood.

Methods

Using C57BL/6-Apc^Min/+ mice, we investigated gut microbial structures with F. nucleatum, antibiotics, and Toll-like receptor 4 (TLR4) antagonist TAK-242 treatment. In addition, we measured intestinal tumor formation and the expression of TLR4, p21-activated kinase 1 (PAK1), phosphorylated-PAK1 (p-PAK1), phosphorylated-β-catenin S675 (p-β-catenin S675), and cyclin D1 in mice with different treatments.

Results

Fusobacterium nucleatum and antibiotics treatment altered gut microbial structures in mice. In addition, F. nucleatum invaded into the intestinal mucosa in large amounts but were less abundant in the feces of F. nucleatum-fed mice. The average number and size of intestinal tumors in F. nucleatum groups was significantly increased compared to control groups in Apc^Min/+ mice (P?<?0.05). The expression of TLR4, PAK1, p-PAK1, p-β-catenin S675, and cyclin D1 was significantly increased in F. nucleatum groups compared to the control groups (P?<?0.05). Moreover, TAK-242 significantly decreased the average number and size of intestinal tumors compared to F. nucleatum groups (P?<?0.05). The expression of p-PAK1, p-β-catenin S675, and cyclin D1 was also significantly decreased in the TAK-242-treated group compared to F. nucleatum groups (P?<?0.05).

Conclusions

Fusobacterium nucleatum potentiates intestinal tumorigenesis in Apc^Min/+ mice via a TLR4/p-PAK1/p-β-catenin S675 cascade. Fusobacterium nucleatum-induced intestinal tumorigenesis can be inhibited by TAK-242, implicating TLR4 as a potential target for the prevention and therapy of F. nucleatum-related colorectal cancer.

     

A novel duplex real-time PCR permits simultaneous detection and differentiation of <Emphasis Type="Italic">Borrelia</Emphasis><Emphasis Type="Italic">miyamotoi</Emphasis> and <Emphasis Type="Italic">Borrelia burgdorferi</Emphasis> sensu lato

Purpose

For simultaneous detection of Borrelia miyamotoi (relapsing fever spirochete) and Borrelia burgdorferi sensu lato, we have developed a duplex real-time PCR targeting the flagellin gene (flaB; p41), a locus frequently used in routine diagnostic PCR for B. burgdorferi s.l. detection.

Methods

Primers and probes were designed using multiple alignments of flaB sequences of B. miyamotoi and B. burgdorferi s.l. species. The sensitivity and specificity of primers and probes were determined using serial dilutions (ranging from 10⁴ to 10^?1) of B. miyamotoi and B. burgdorferi s.l. DNA and of several species of relapsing fever spirochetes. Conventional PCR on recG and glpQ and sequencing of p41 PCR products were used to confirm the species assignment.

Results

The detection limit of both singleplex and duplex PCR was 10 genome equivalents except for B. spielmanii and two B. garinii genotypes which showed a detection limit of 10² genome equivalents. There was no cross reactivity of the B. miyamotoi primers/probes with B. burgdorferi s.l. DNA, while the B. burgdorferi s.l. primer/probe generated a signal with B. hermsii DNA. Out of 2341 Ixodes ricinus ticks from Germany and Slovakia that were screened simultaneously for the presence of B. miyamotoi and B. burgdorferi s.l., 52 were positive for B. miyamotoi and 276 for B. burgdorferi s.l., denoting an average prevalence of 2.2 % for B. miyamotoi and 11.8 % for B. burgdorferi s.l., and B. miyamotoi DNA was also detectable by PCR using artificial clinical samples.

Conclusion

The duplex real-time PCR developed here represents a method that permits simultaneous detection and differentiation of B. burgdorferi s.l. and B. miyamotoi in environmental and potentially clinical samples.

     

Septic pulmonary emboli detected by <Superscript>18</Superscript>F-FDG PET/CT in children with <Emphasis Type="Italic">S. aureus</Emphasis> catheter-related bacteremia

Purpose

The role of ¹⁸F-fluorodeoxyglucose positron emission tomography-computed tomography (¹⁸F-FDG PET/CT) in the diagnosis of metastatic infectious foci in children with catheter-related blood stream infection has been hardly studied, although some authors have reported it benefit in the screening of metastatic foci in adult population. Septic pulmonary emboli are among the most difficult to identify, because many cases do not present pulmonary complaints or abnormal chest radiography. However, diagnosis of these foci has important therapeutic consequences. The purpose of this article is to describe the role of ¹⁸F-FDG PET/CT in the diagnosis of septic pulmonary embolism in children with S. aureus catheter-related bacteremia.

Methods

We report 3 children with S. aureus catheter-related bacteremia and normal chest X-ray at admission, in whom ¹⁸F-FDG PET/CT led to the diagnosis of unsuspected septic pulmonary emboli, with an impact on clinical management.

Results

All patients had hemophilia and implantable venous access ports and presented with fever and normal lung auscultation. Only 1 reported non-specific symptoms (undifferentiated left chest pain). All patients had normal chest X-ray on admission. Catheters were removed within 48 h after admission in 2 cases, and 5 days after admission in the last case, subsiding fever. In 2 children, paired blood cultures were not able to identify bacteremia. However, in all cases catheter tip and subcutaneous port cultures yielded S. aureus and PET/CT detected unsuspected pulmonary metastatic emboli.

Conclusions

¹⁸F-FDG PET/CT should be considered as a useful tool to diagnose septic pulmonary embolism in S. aureus catheter-related bacteremia, especially if conventional diagnostic imaging techniques have failed to reveal possible metastatic foci. Further studies are needed to clarify the usefulness of PET/CT performance in children with CRBSI.

     

Validation of [<Superscript>18</Superscript>F]fluorodeoxyglucose and positron emission tomography (PET) for the measurement of intestinal metabolism in pigs,and evidence of intestinal insulin resistance in patients with morbid obesity

Aims/hypothesis

The role of the intestine in the pathogenesis of metabolic diseases is gaining much attention. We therefore sought to validate, using an animal model, the use of positron emission tomography (PET) in the estimation of intestinal glucose uptake (GU), and thereafter to test whether intestinal insulin-stimulated GU is altered in morbidly obese compared with healthy human participants.

Methods

In the validation study, pigs were imaged using [¹⁸F]fluorodeoxyglucose ([¹⁸F]FDG) and the image-derived data were compared with corresponding ex vivo measurements in tissue samples and with arterial–venous differences in glucose and [¹⁸F]FDG levels. In the clinical study, GU was measured in different regions of the intestine in lean (n?=?8) and morbidly obese (n?=?8) humans at baseline and during euglycaemic hyperinsulinaemia.

Results

PET- and ex vivo-derived intestinal values were strongly correlated and most of the fluorine-18-derived radioactivity was accumulated in the mucosal layer of the gut wall. In the gut wall of pigs, insulin promoted GU as determined by PET, the arterial–venous balance or autoradiography. In lean human participants, insulin increased GU from the circulation in the duodenum (from 1.3?±?0.6 to 3.1?±?1.1 μmol [100 g]^?1?min^?1, p?<?0.05) and in the jejunum (from 1.1?±?0.7 to 3.0?±?1.5 μmol [100 g]^?1?min^?1, p?<?0.05). Obese participants failed to show any increase in insulin-stimulated GU compared with fasting values (NS).

Conclusions/interpretation

Intestinal GU can be quantified in vivo by [¹⁸F]FDG PET. Intestinal insulin resistance occurs in obesity before the deterioration of systemic glucose tolerance.

     

Associations between <Emphasis Type="Italic">ERAP1</Emphasis> polymorphisms and susceptibility to ankylosing spondylitis: a meta-analysis

The aim of this study was to determine whether 11 polymorphisms of endoplasmic reticulum aminopeptidase 1 (ERAP1) confer susceptibility to ankylosing spondylitis (AS). The authors conducted meta-analyses on associations between ERAP1 polymorphisms and AS susceptibility by using fixed and random effects models. A total of 19 articles were included in this meta-analysis, which comprised a total of 19,902 AS patients and 39,750 controls. The meta-analysis revealed a significant association between AS and the minor alleles of the rs30187 polymorphism in all study subjects (OR?=?1.255, 95 % CI?=?1.147–1.373, P?=?8.0?×?10^?8). Stratification by ethnicity led to the identification of a significant association between this polymorphism and AS in European patients (OR?=?1.283, 95 % CI?=?1.237–1.331, P?<?1.0?×?10^?9). Meta-analyses of the results for the rs27044, rs10050860, rs2287987, rs17482078, and rs26653 polymorphisms showed the same pattern that was found for rs30187. Interestingly, the rs27037 polymorphism was significantly associated with AS susceptibility in both European and Asian patients. Meta-analysis showed a significant association between AS and the minor alleles of the rs27980 and rs27582 polymorphisms in the East Asian patients (OR?=?0.904, 95 % CI?=?0.818–0.999, P?=?0.047; OR?=?0.871, 95 % CI?=?0.772–0.982, P?=?0.024, respectively) (Table 2). However, these polymorphisms have not been studied in Europeans. This meta-analysis shows that the ERAP1 polymorphisms are associated with the development of AS in Europeans and East Asians.     

A proinflammatory CD4<Superscript>+</Superscript> T cell phenotype in gestational diabetes mellitus

Aims/hypothesis

Numerous adaptations of the maternal immune system are necessary during pregnancy to maintain immunological tolerance to the semi-allogeneic fetus. Several complications of pregnancy have been associated with dysregulation of these adaptive mechanisms. While gestational diabetes mellitus (GDM) has been associated with upregulation of circulating inflammatory factors linked to innate immunity, polarisation of the adaptive immune system has not been extensively characterised in this condition. We aimed to characterise pro- and anti-inflammatory CD4⁺ (T helper [Th]) T cell subsets in women with GDM vs women without GDM (of similar BMI), during and after pregnancy, and examine the relationship between CD4⁺ subsets and severity of GDM.

Methods

This is a prospective longitudinal case–control study of 55 women with GDM (cases) and 65 women without GDM (controls) at a tertiary maternity hospital. Quantification of proinflammatory (Th17, Th17.1, Th1) and anti-inflammatory (regulatory T cell [Treg]) CD4⁺ T cell subsets was performed on peripheral blood at 37 weeks gestation and 7 weeks postpartum, and correlated with clinical characteristics and measures of blood glucose.

Results

Women with GDM had a significantly greater percentage of Th17 (median 2.49% [interquartile range 1.62–4.60] vs 1.85% [1.13–2.98], p?=?0.012) and Th17.1 (3.06% [1.30–4.33] vs 1.55% [0.65–3.13], p?=?0.006) cells compared with the control group of women without GDM. Women with GDM also had higher proinflammatory cell ratios (Th17:Treg, Th17.1:Treg and Th1:Treg) in pregnancy compared with the control group of women without GDM. In the control group, there was a statistically significant independent association between 1 h glucose levels in the GTT and Th17 cell percentages, and also between 2 h glucose levels and percentage of Th17 cells. The percentage of Th17 cells and the Th17:Treg ratio declined significantly after delivery in women with GDM, whereas this was not the case with the control group of women. Nevertheless, a milder inflammatory phenotype persisted after delivery (higher Th17:Treg ratio) in women with GDM vs women without.

Conclusions/interpretation

Dysregulation of adaptive immunity supports a novel paradigm of GDM that extends beyond hyperglycaemia and altered innate immunity.

     

A somatic <Emphasis Type="Italic">GNA11</Emphasis> mutation is associated with extremity capillary malformation and overgrowth

Background

Capillary malformation is a cutaneous vascular anomaly that is present at birth, darkens over time, and can cause overgrowth of tissues beneath the stain. The lesion is caused by a somatic activating mutation in GNAQ. In a previous study, we were unable to identify a GNAQ mutation in patients with a capillary malformation involving an overgrown lower extremity. We hypothesized that mutations in GNA11 or GNA14, genes closely related to GNAQ, also may cause capillary malformations.

Methods

Human capillary malformation tissue obtained from 8 patients that had tested negative for GNAQ mutations were studied. Lesions involved an extremity (n = 7) or trunk (n = 1). Droplet digital PCR (ddPCR) was used to detect GNA11 or GNA14 mutant cells (p.Arg183) in the specimens. Single molecule molecular inversion probe sequencing (smMIP-seq) was performed to search for other mutations in GNA11. Mutations were validated by subcloning and sequencing amplimers.

Results

We found a somatic GNA11 missense mutation (c.547C > T; p.Arg183Cys) in 3 patients with a diffuse capillary malformation of an extremity. Mutant allelic frequencies ranged from 0.3 to 5.0%. GNA11 or GNA14 mutations were not found in 5 affected tissues or in unaffected tissues (white blood cell DNA).

Conculsions

GNA11 mutations are associated with extremity capillary malformations causing overgrowth. Pharmacotherapy that affects GNA11 signaling may prevent the progression of capillary malformations.