Samppa J. Ryhänen

Natural history of the infant gut microbiome and impact of antibiotic treatment on bacterial strain diversity and stability

A longitudinal strain-level analysis of the infant gut microbiome after repeated antibiotic treatments reveals decreased diversity and stability, as well as transient increases in antibiotic resistance genes. Elucidating the effects of drugs on bugs Despite widespread use of antibiotics in children, the effects of antibiotic exposure on the developing infant gut microbiome have remained underexplored. Here, Yassour et al. present a longitudinal study capturing how the gut microbiome responds to and recovers from antibiotic perturbations. Antibiotic-treated children had less stable and less diverse bacterial communities. Antibiotic resistance genes within the guts of these children peaked after antibiotic treatment but generally returned rapidly to baseline. Delivery mode (vaginal versus cesarean) also had strong long-term effects on microbial diversity. These data give insights into the consequences of early life factors such as birth mode and antibiotic treatment on the infant gut microbiome. The gut microbial community is dynamic during the first 3 years of life, before stabilizing to an adult-like state. However, little is known about the impact of environmental factors on the developing human gut microbiome. We report a longitudinal study of the gut microbiome based on DNA sequence analysis of monthly stool samples and clinical information from 39 children, about half of whom received multiple courses of antibiotics during the first 3 years of life. Whereas the gut microbiome of most children born by vaginal delivery was dominated by Bacteroides species, the four children born by cesarean section and about 20% of vaginally born children lacked Bacteroides in the first 6 to 18 months of life. Longitudinal sampling, coupled with whole-genome shotgun sequencing, allowed detection of strain-level variation as well as the abundance of antibiotic resistance genes. The microbiota of antibiotic-treated children was less diverse in terms of both bacterial species and strains, with some species often dominated by single strains. In addition, we observed short-term composition changes between consecutive samples from children treated with antibiotics. Antibiotic resistance genes carried on microbial chromosomes showed a peak in abundance after antibiotic treatment followed by a sharp decline, whereas some genes carried on mobile elements persisted longer after antibiotic therapy ended. Our results highlight the value of high-density longitudinal sampling studies with high-resolution strain profiling for studying the establishment and response to perturbation of the infant gut microbiome.

Surface Charge Density Determines the Efficiency of Cationic Gemini Surfactant Based Lipofection

The efficiencies of the binary liposomes composed of 1,2-dimyristoyl-sn-glycero-3-phosphocholine and cationic gemini surfactant, (2S,3R)-2,3-dimethoxy-1,4-bis(N-hexadecyl-N,N-dimethylammonium)butane dibromide as transfection vectors, were measured using the enhanced green fluorescent protein coding plasmid and COS-1 cells. Strong correlation between the transfection efficiency and lipid stoichiometry was observed. Accordingly, liposomes with X(SR-1) > or = 0.50 conveyed the enhanced green fluorescent protein coding plasmid effectively into cells. The condensation of DNA by liposomes with X(SR-1) > 0.50 was indicated by static light scattering and ethidium bromide intercalation assay, whereas differential scanning calorimetry and fluorescence anisotropy of diphenylhexatriene revealed stoichiometry dependent reorganization in the headgroup region of the liposome bilayer, in alignment with our previous Langmuir-balance study. Surface charge density and the organization of positive charges appear to determine the mode of interaction of DNA with (2S,3R)-2,3-dimethoxy-1,4-bis(N-hexadecyl-N,N-dimethylammonium)butane dibromide/1,2-dimyristoyl-sn-glycero-3-phosphocholine liposomes, only resulting in DNA condensation when X(SR-1) > 0.50. Condensation of DNA in turn seems to be required for efficient transfection.

Characterization of Mixed Monolayers of Phosphatidylcholine and a Dicationic Gemini Surfactant SS-1 with a Langmuir Balance: Effects Of DNA

Monolayers of a cationic gemini surfactant, 2,3-dimethoxy-1,4-bis(N-hexadecyl-N;N-dimethyl-ammonium)butane dibromide (abbreviated as SS-1) and its mixtures with 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) were studied using a Langmuir balance. More specifically, we measured the force-area (pi-A) curves and determined the elastic area compressibility modulus (C) as a function of lateral packing pressure and the mole fraction of the cationic lipid (X(SS-1)), with and without DNA in the subphase. Both SS-1 and POPC exhibited smooth compression isotherms, indicating their monolayers to be in the liquid expanded state. Even low contents (X(SS-1) < 0.05) of SS-1 in a POPC monolayer condensed the film dramatically, up to 20% at 30 mN/m. This effect is suggested to reflect reorientation of the P(-)-N(+) dipole of the POPC headgroup. Accordingly, the magnitude of the condensing effect diminishes with X(SS-1) and is not observed for mixed films of dioleoylglycerol and SS-1. Reorientation of the P(-)-N(+) dipole is further supported by the pronounced increase in monolayer dipole potential psi due to SS-1. The presence of DNA in the subphase affected the mixed POPC/SS-1 monolayers differently depending on the constituent lipid stoichiometry as well as on the DNA/SS-1 charge ratio. At a DNA concentration of 0.63 microM (in base pairs) condensation of neat POPC monolayers was evident, and this effect remained up to X(SS-1) < 0.5, corresponding to DNA/SS-1 charge ratio of 1.25. An expansion due to DNA, evident as an increase in DeltaA/molecule, was observed at X(SS-1) > 0.5. At a higher concentration of DNA (1.88 microM base pairs) in the subphase corresponding to DNA/SS-1 charge ratio of 3.75 at X(SS-1) = 0.5, condensation was observed at all values of X(SS-1).

Th1/Th17 Plasticity Is a Marker of Advanced β Cell Autoimmunity and Impaired Glucose Tolerance in Humans

Upregulation of IL-17 immunity and detrimental effects of IL-17 on human islets have been implicated in human type 1 diabetes. In animal models, the plasticity of Th1/Th17 cells contributes to the development of autoimmune diabetes. In this study, we demonstrate that the upregulation of the IL-17 pathway and Th1/Th17 plasticity in peripheral blood are markers of advanced β cell autoimmunity and impaired β cell function in human type 1 diabetes. Activated Th17 immunity was observed in the late stage of preclinical diabetes in children with β cell autoimmunity and impaired glucose tolerance, but not in children with early β cell autoimmunity. We found an increased ratio of IFN-γ/IL-17 expression in Th17 cells in children with advanced β cell autoimmunity, which correlated with HbA1c and plasma glucose concentrations in an oral glucose tolerance test, and thus impaired β cell function. Low expression of Helios was seen in Th17 cells, suggesting that Th1/Th17 cells are not converted thymus-derived regulatory T cells. Our results suggest that the development of Th1/Th17 plasticity may serve as a biomarker of disease progression from β cell autoantibody positivity to type 1 diabetes. These data in human type 1 diabetes emphasize the role of Th1/Th17 plasticity as a potential contributor to tissue destruction in autoimmune conditions.

Autoantibodies against zinc transporter 8 are related to age, metabolic state and HLA DR genotype in children with newly diagnosed type 1 diabetes

We set out to define the characteristics of humoral autoimmunity against ZnT8 in children and adolescents with newly diagnosed T1D in relation to age and metabolic status at diagnosis, human leucocyte antigen (HLA) genotype and family history of T1D.

Extended Family History of Type 1 Diabetes and Phenotype and Genotype of Newly Diagnosed Children

OBJECTIVE To determine the frequency of newly diagnosed diabetic children with first- and second-degree relatives affected by type 1 diabetes and to characterize the effects of this positive family history on clinical markers, signs of β-cell autoimmunity, and HLA genotype in the index case. RESEARCH DESIGN AND METHODS Children (n = 1,488) with type 1 diabetes diagnosed under 15 years of age were included in a cross-sectional study from the Finnish Pediatric Diabetes Register. Data on family history of diabetes and metabolic decompensation at diagnosis were collected using a questionnaire. Antibodies to β-cell autoantigens (islet cell antibodies, insulin autoantibodies, GAD antibodies, and antibodies to the islet antigen 2 molecule) and HLA genotypes were analyzed. RESULTS A total of 12.2% of the subjects had a first-degree relative with type 1 diabetes (father 6.2%, mother 3.2%, and sibling 4.8%) and 11.9% had an affected second-degree relative. Children without affected relatives had lower pH (P < 0.001), higher plasma glucose (P < 0.001) and β-hydroxybutyrate concentrations (P < 0.001), a higher rate of impaired consciousness (P = 0.02), and greater weight loss (P < 0.001). There were no differences in signs of β-cell autoimmunity. The familial cases carried the HLA DR4-DQ8 haplotype more frequently than sporadic cases (74.0 vs. 67.0%, P = 0.02). CONCLUSIONS When the extended family history of type 1 diabetes is considered, the proportion of sporadic diabetes cases may be reduced to <80%. A positive family history for type 1 diabetes associates with a less severe metabolic decompensation at diagnosis, even when only second-degree relatives are affected. Autoantibody profiles are similar in familial and sporadic type 1 diabetes, suggesting similar pathogenetic mechanisms.

Impact of Intranasal Insulin on Insulin Antibody Affinity and Isotypes in Young Children With HLA-Conferred Susceptibility to Type 1 Diabetes

OBJECTIVE Despite promising results from studies on mouse models, intranasal insulin failed to prevent or delay the development of type 1 diabetes in autoantibody-positive children with HLA-conferred disease susceptibility. To analyze whether the insulin dose was inadequate to elicit an immunomodulatory response, we compared the changes observed in insulin antibody (IA) affinity and isotypes after treatment with nasal insulin or placebo. RESEARCH DESIGN AND METHODS Ninety-five children (47 in the placebo group and 48 in the insulin group of the total of 224 children randomized for the trial) with HLA-conferred susceptibility to type 1 diabetes derived from the intervention arm of the Finnish Type 1 Diabetes Prediction and Prevention study were included in these analyses. Blood samples drawn before or at the beginning of the treatment and after treatment for 3 and 6 months were analyzed for IA affinity and isotype-specific IAs (IgG1–4, IgA, IgM, and IgE). RESULTS IgG3- and IgA-IA levels (P = 0.031 and 0.015, respectively) and the number of IgG3-IA–positive subjects (P = 0.022) were significantly higher at 6 months after the initiation of the treatment in the insulin group. No significant differences were observed between the two groups in IA affinity or other IA isotypes. CONCLUSIONS The insulin dose administered induced a modest change in the IA isotype profile. The lack of impact of nasal insulin on IA affinity implies that the immune response of study subjects was already mature at the beginning of the intervention.

Cationic lipid membranes-specific interactions with counter-ions.

Lipids bearing net electric charges in their hydrophilic headgroups are ubiquitous in biological membranes. Recently, the interest in cationic lipids has surged because of their potential as non-viral transfection vectors. In order to utilize cationic lipids in transfer of nucleic acids and to elucidate the role of charged lipids in cellular membranes in general, their complex interactions within the membrane and with the molecules in the surrounding media need to be thoroughly characterized. Yet, even interactions between monovalent counter-ions and charged lipids are inadequately understood. We studied the interactions of the cationic gemini surfactant (2R,3R)-2,3-dimethoxy-1,4- bis(N-hexadecyl-N,N-dimethylammonium)butane dibromide (RR-1) with chloride, bromide, fluoride, and iodide as counter-ions by differential scanning calorimetry and Langmuir balance. Chloride interacts avidly with RR-1, efficiently condensing the monolayer, decreasing the collapse pressure, and elevating the main transition temperature. With bromide and iodide clearly different behaviour was observed, indicating specific interactions between RR-1 and these counter-ions. Moreover, with fluoride as a counter-ion and in pure water identical results were obtained, demonstrating inefficient electrostatic screening of the headgroups of RR-1 and suggesting fluoride being depleted on the surface of RR-1 membranes.

Circulating Concentrations of Soluble Receptor for AGE Are Associated With Age and AGER Gene Polymorphisms in Children With Newly Diagnosed Type 1 Diabetes

OBJECTIVE We analyzed the relationship among soluble receptor for advanced glycation end products (sRAGEs), the clinical phenotype, HLA genotype, and risk-associated single nucleotide polymorphisms (SNPs) in the AGER gene in a large population of Finnish children with newly diagnosed type 1 diabetes. RESEARCH DESIGN AND METHODS Samples from 2,115 clinically phenotyped children <15 years of age in whom type 1 diabetes was diagnosed and 316 control subjects were analyzed for sRAGEs. Three SNPs of AGER, previously associated with HLA-DR/DQ haplotype independent diabetes risk (rs2070600, rs9469089, and rs17493811), were analyzed in 1,390 affected subjects. RESULTS Children with type 1 diabetes and control subjects had similar sRAGE concentrations (1,171 vs. 1,153 pg/mL, P = 0.48). There was a correlation between age at diagnosis and serum sRAGE concentrations (r = 0.10, P < 0.001) among the patients but not among the control subjects. Children <2 years of age had the lowest concentrations in the diabetic population (1,027 vs. 1,181 pg/mL, P < 0.001) and the highest among the control subjects (1,329 vs. 1,140 pg/mL, P = 0.04). Ketoacidosis at diagnosis was associated with reduced concentrations (1,086 vs. 1,190 pg/mL, P < 0.001). HLA DR3/DR4 heterozygosity and the DR3 allele were associated with reduced sRAGE concentrations. The predisposing AA genotype of rs2070600 was associated with decreased sRAGE concentrations, while the protective CC genotype of rs9469089 was linked to increased concentrations. CONCLUSIONS Age and AGER polymorphisms are associated with the circulating sRAGE concentration among children with type 1 diabetes. The observations of reduced sRAGE concentrations in young children, in those with ketoacidosis, and in carriers of the high-risk HLA DR3/DR4 genotype suggest that decreased sRAGE concentration reflects a more aggressive disease phenotype.

R705H mutation of MYH9 is associated with MYH9-related disease and not only with non-syndromic deafness DFNA17

MYH9‐related disease (MYH9‐RD) is a rare autosomal dominant disease caused by mutation of MYH9, the gene encoding for the heavy chain of non‐muscle myosin IIA (NMMHC‐IIA). MYH9‐RD patients have macrothrombocytopenia and granulocyte inclusions (pathognomonic sign of the disease) containing wild‐type and mutant NMMHC‐IIA. During life they might develop sensorineural hearing loss, cataract, glomerulonephritis, and elevation of liver enzymes. One of the MYH9 mutations, p.R705H, was previously reported to be associated with DFNA17, an autosomal dominant non‐syndromic sensorineural hearing loss without any other features associated. We identified the same mutation in two unrelated families, whose four affected individuals had not only hearing impairment but also thrombocytopenia, giant platelets, leukocyte inclusions, as well as mild to moderate elevation of some liver enzymes. Our data suggest that DFNA17 should not be a separate genetic entity but part of the wide phenotypic spectrum of MYH9‐RD characterized by congenital hematological manifestations and variable penetrance and expressivity of the extra‐hematological features.