Characterization of bacterial DNA binding to human neutrophil surface

Bacterial DNA activates neutrophils through a CpG- and TLR9-independent mechanism. Neutrophil activation does not require DNA internalization, suggesting that it results from the interaction of bacterial DNA with a neutrophil surface receptor. The aim of this study was to characterize the interaction of bacterial DNA with the neutrophil surface. Bacterial DNA binding showed saturation and was inhibited by unlabeled DNA but not by other polyanions like yeast tRNA and poly-A. Resembling the conditions under which bacterial DNA triggers neutrophil activation, binding was not modified in the presence or absence of calcium, magnesium or serum. Treatment of neutrophils with proteases not only dramatically reduced bacterial DNA binding but also inhibited neutrophil activation induced by bacterial DNA. Experiments performed with DNA samples of different lengths obtained after digestion of bacterial DNA with DNase showed that only DNA fragments greater than ≈170-180 nucleotides competed bacterial DNA binding and retained the ability to trigger cell activation. Treatment of neutrophils with chemoattractants or conventional agonists significantly increased bacterial DNA binding. Moreover, neutrophils that underwent transmigration through human endothelial cell monolayers even in the absence of chemoattractants, exhibited higher binding levels of bacterial DNA. Together, our findings provide evidence that binding of bacterial DNA to neutrophils is a receptor-mediated process that conditions the ability of DNA to trigger cell activation. We speculate that neutrophil recognition of bacterial DNA might be modulated by the balance of agonists present at inflammatory foci. This effect might be relevant in bacterial infections with a biofilm etiology, in which extracellular DNA could function as a potent neutrophil agonist. © 2008 USCAP, Inc All rights reserved.Fil:Fuxman Bass, J.I. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales; Argentina.Fil:Gabelloni, M.L. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales; Argentina.Fil:Vermeulen, M.E. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales; Argentina.Fil:Russo, D.M. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales; Argentina.Fil:Zorreguieta, Á. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales; Argentina.Fil:Trevani, A.S. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales; Argentina

Compound heterozygosity of a frameshift mutation in the coding region and a single base substitution in the promoter of the ACTH receptor gene in a family with isolated glucocorticoid deficiency

Isolated glucocorticoid deficiency (IGD) is an autosomal recessive syndrome characterized by glucocorticoid insufficiency without mineralocorticoid deficiency. Mutations in the coding region of the ACTH receptor (MC2R) have been reported in several families with IGD. We amplified and sequenced the entire MC2R coding region in a new family with IGD. The proband was found to be heterozygous (paternal allele) for the mutation Gly217fs, which changes the open reading frame of the MC2R protein resulting in a truncated receptor. No other abnormality was found in the MC2R coding region. However, sequencing of the promoter region of the MC2R gene (-1017/44 bp) of the proband revealed a heterozygous T→C substitution in the maternal allele at -2 bp position from initiation of the transcription start site. This substitution was found in only 6.5% in a healthy unrelated population. Constructs containing this polymorphism consistently showed a significant 15% decrease in promoter activity compared to wild type. In conclusion, we provide evidence that the IGD in this previously unreported family with ACTH resistance appears to be secondary to compound heterozygosity of a coding region and a promoter mutation in the MC2R gene. © Freund Publishing House Ltd., London

Flagellin delays spontaneous human neutrophil apoptosis

Neutrophils are short-lived cells that rapidly undergo apoptosis. However, their survival can be regulated by signals from the environment. Flagellin, the primary component of the bacterial flagella, is known to induce neutrophil activation. In this study we examined the ability of flagellin to modulate neutrophil apoptosis. Neutrophils cultured for 12 and 24 h in the presence of flagellin from Salmonella thyphimurim at concentrations found in pathological situations underwent a marked prevention of apoptosis. In contrast, Helicobacter pylori flagellin did not affect neutrophil survival, suggesting that Salmonella flagellin exerts the antiapoptotic effect by interacting with TLR5. The delaying in apoptosis mediated by Salmonella flagellin was coupled to higher expression levels of the antiapoptotic protein Mcl-1 and lower levels of activated caspase-3. Analysis of the signaling pathways indicated that Salmonella flagellin induced the activation of the p38 and ERK1/2 MAPK pathways as well as the PI3K/Akt pathway. Furthermore, it also stimulated IBα degradation and the phosphorylation of the p65 subunit, suggesting that Salmonella flagellin also triggers NF-B activation. Moreover, the pharmacological inhibition of ERK1/2 pathway and NF-B activation partially prevented the antiapoptotic effects exerted by flagellin. Finally, the apoptotic delaying effect exerted by flagellin was also evidenced when neutrophils were cultured with whole heat-killed S. thyphimurim. Both a wild-type and an aflagellate mutant S. thyphimurim strain promoted neutrophil survival; however, when cultured in low bacteria/neutrophil ratios, the flagellate bacteria showed a higher capacity to inhibit neutrophil apoptosis, although both strains showed a similar ability to induce neutrophil activation. Taken together, our results indicate that flagellin delays neutrophil apoptosis by a mechanism partially dependent on the activation of ERK1/2 MAPK and NF-B. The ability of flagellin to delay neutrophil apoptosis could contribute to perpetuate the inflammation during infections with flagellated bacteria. © 2010 USCAP, Inc All rights reserved.Fil:Salamone, G.V. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales; Argentina.Fil:Gabelloni, M.L. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales; Argentina.Fil:Vermeulen, M.E. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales; Argentina.Fil:Trevani, A.S. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales; Argentina

Supplementary Material for: Long-Term Retention of Young Adult Study Participants with Youth-Onset Type 2 Diabetes: Results from the TODAY2 Study

Introduction: The Treatment Options for type 2 Diabetes in Adolescents and Youth (TODAY) trial examined the effects of three treatment arms in a group of racially and ethnically diverse adolescents and youth with type 2 diabetes mellitus. TODAY2 was an observational follow-up study reporting outcomes and complications in these participants after having diabetes for approximately 13 years. Participant retention was essential to fulfill this objective. This report describes motivations and problems participants self-reported related to continuing in this study. Methods: The TODAY2 retention survey was administered to participants, mean age 27 years, 36% non-Hispanic Black, 18% non-Hispanic white, 39% Hispanic, 52% public and 35% private healthcare coverage, who completed the last study visit (63.8% of original TODAY cohort). The survey listed potential benefits and barriers to staying in the trial. Participants indicated agreement or disagreement with each statement using a four point Likert-type scale. Results: More than 93% of survey responders agreed with benefits listed for staying in TODAY2. The most cited reason for staying in the study was related to the strong relationship that participants had with study staff. The common barriers to attending trial visits were: tending to other medical problems, fear of disappointing study staff, and school/work scheduling conflicts. Participants with public healthcare coverage were more likely to endorse benefits related to diabetes care (e.g. getting latest test results, staying motivated to care for my diabetes) and monetary compensation, whereas participants with poor glycemic control cited that a barrier to attending study visits was “fear of disappointing” study staff. Discussion/Conclusion: In a racially and ethnically diverse population of youth-onset type 2 diabetes, benefits and barriers associated with long-term retention are described. These findings can be used to help inform future retention strategies for young adults in clinical trials

Supplementary Material for: Infants with Congenital Adrenal Hyperplasia Exhibit Thalamic Discrepancies in Early Brain Structure

Introduction: Patients with classical congenital adrenal hyperplasia (CAH) have prenatal and postnatal hormonal imbalances. To characterize the ontogeny of reported brain and behavior changes in older children with CAH, we aimed to study brain structure in infants with CAH compared to healthy controls. Methods: We performed neuroimaging in 16 infants with classical CAH due to 21-hydroxylase deficiency [8 males, gestational age 38.2 ± 1.7 weeks, post-conceptional age (PCA) 42.2 ± 3.0 weeks] and 14 control infants (9 males, gestational age 38.5 ± 1.8 weeks, PCA 42.5 ± 2.4 weeks) utilizing 3-Tesla magnetic resonance imaging. Regional brain volumes were adjusted for PCA and sex, along with an additional adjustment for total brain volume (TBV), for group comparisons by regression analyses [mean, 95% confidence interval (CI)]. The degree to which each brain region was differentiated between CAH and control infants was examined by relaimpo analyses, adjusting for all other brain regions, PCA, and sex. Results: Infants with CAH had significantly smaller thalamic volumes [8606 mm3, 95% CI (8209, 9002)] compared to age-matched control infants [9215 mm3, 95% CI (8783, 9647); β = -609; p = 0.02], which remained smaller after further adjustment for TBV. Upon further adjustment for TBV, the temporal lobe was larger in infants with CAH [66817 mm3, CI (65957, 67677)] compared to controls [65616 mm3, CI (64680, 66551); β = 1202, p = 0.03]. The brain regions most differentiated between CAH vs controls were the thalamus (22%) and parietal lobe (10%). Conclusions: Infants with CAH exhibit smaller thalamic regions from early life, suggesting a prenatal influence on brain development in CAH. Thalamic emergence at 8-14 weeks makes the region particularly vulnerable to changes in the intrauterine environment, with potential implications for later maturing brain regions. These changes may take time to manifest, meriting longitudinal study through adolescence in CAH