HSC extrinsic sex-related and intrinsic autoimmune disease-related human B-cell variation is recapitulated in humanized mice.

B cells play a major role in antigen presentation and antibody production in the development of autoimmune diseases, and some of these diseases disproportionally occur in females. Moreover, immune responses tend to be stronger in female vs male humans and mice. Because it is challenging to distinguish intrinsic from extrinsic influences on human immune responses, we used a personalized immune (PI) humanized mouse model, in which immune systems were generated de novo from adult human hematopoietic stem cells (HSCs) in immunodeficient mice. We assessed the effect of recipient sex and of donor autoimmune diseases (type 1 diabetes [T1D] and rheumatoid arthritis [RA]) on human B-cell development in PI mice. We observed that human B-cell levels were increased in female recipients regardless of the source of human HSCs or the strain of immunodeficient recipient mice. Moreover, mice injected with T1D- or RA-derived HSCs displayed B-cell abnormalities compared with healthy control HSC-derived mice, including altered B-cell levels, increased proportions of mature B cells and reduced CD19 expression. Our study revealed an HSC-extrinsic effect of recipient sex on human B-cell reconstitution. Moreover, the PI humanized mouse model revealed HSC-intrinsic defects in central B-cell tolerance that recapitulated those in patients with autoimmune diseases. These results demonstrate the utility of humanized mouse models as a tool to better understand human immune cell development and regulation.

Prevalence of detectable C-Peptide according to age at diagnosis and duration of type 1 diabetes.

OBJECTIVE: It is generally accepted that complete ß-cell destruction eventually occurs in individuals with type 1 diabetes, which has implications for treatment approaches and insurance coverage. The frequency of residual insulin secretion in a large cohort of individuals at varying ages of diagnosis and type 1 diabetes duration is unknown. RESEARCH DESIGN AND METHODS: The frequency of residual insulin secretion was determined by measurement of nonfasting serum C-peptide concentration in 919 individuals with type 1 diabetes according to prespecified groups based on age at diagnosis and duration of disease (from 3 to 81 years' duration). Stimulated C-peptide was measured in those with detectable nonfasting values and a group of those with undetectable values as control. RESULTS: The overall frequency of detectable nonfasting C-peptide was 29%, decreasing with time from diagnosis regardless of age at diagnosis. In all duration groups, the frequency of C-peptide was higher with diagnosis age >18 years compared with ≤18 years. Nineteen percent of those with undetectable nonfasting C-peptide were C-peptide positive upon stimulation testing. CONCLUSIONS: The American Diabetes Association's definition of type 1 diabetes as "usually leading to absolute insulin deficiency" results in clinicians often considering the presence of residual insulin secretion as unexpected in this population. However, our data suggest that residual secretion is present in almost one out of three individuals 3 or more years from type 1 diabetes diagnosis. The frequency of residual C-peptide decreases with time from diagnosis regardless of age at diagnosis, yet at all durations of disease, diagnosis during adulthood is associated with greater frequency and higher values of C-peptide.

Human oocytes reprogram adult somatic nuclei of a type 1 diabetic to diploid pluripotent stem cells.

The transfer of somatic cell nuclei into oocytes can give rise to pluripotent stem cells that are consistently equivalent to embryonic stem cells, holding promise for autologous cell replacement therapy. Although methods to induce pluripotent stem cells from somatic cells by transcription factors are widely used in basic research, numerous differences between induced pluripotent stem cells and embryonic stem cells have been reported, potentially affecting their clinical use. Because of the therapeutic potential of diploid embryonic stem-cell lines derived from adult cells of diseased human subjects, we have systematically investigated the parameters affecting efficiency of blastocyst development and stem-cell derivation. Here we show that improvements to the oocyte activation protocol, including the use of both kinase and translation inhibitors, and cell culture in the presence of histone deacetylase inhibitors, promote development to the blastocyst stage. Developmental efficiency varied between oocyte donors, and was inversely related to the number of days of hormonal stimulation required for oocyte maturation, whereas the daily dose of gonadotropin or the total number of metaphase II oocytes retrieved did not affect developmental outcome. Because the use of concentrated Sendai virus for cell fusion induced an increase in intracellular calcium concentration, causing premature oocyte activation, we used diluted Sendai virus in calcium-free medium. Using this modified nuclear transfer protocol, we derived diploid pluripotent stem-cell lines from somatic cells of a newborn and, for the first time, an adult, a female with type 1 diabetes.

Recruitment and retention of participants for an international type 1 diabetes prevention trial: a coordinators' perspective.

BACKGROUND: The Trial to Reduce Insulin Dependent Diabetes Mellitus in the Genetically at Risk (TRIGR) is the first multicenter international type 1 diabetes (T1D) prevention trial to be undertaken. A unique feature of TRIGR has been recruitment of eligible pregnant women and enrollment of newborns for long-term follow-up assessments. PURPOSE: Our purpose is to summarize the recruitment and retention strategies used to conduct TRIGR from the perspective of the study coordinators. METHODS: TRIGR was designed to test whether weaning to formula containing hydrolyzed versus intact cow's milk protein would be efficacious in decreasing risk for development of T1D-associated autoantibodies and T1D among infants identified to be at increased risk for T1D based on their human leukocyte antigen (HLA) profile and family history. Multiple strategies tailored to local issues were required to enroll and follow the target number of infants. RESULTS: This study was conducted in the United States, Canada, Australia, and 12 countries in Europe. Of the 5606 mothers registered worldwide, 5000 of their infants were randomized. Of these, 2159 were HLA eligible and enrolled in the 8-month intervention and 10-year follow-up phases of this study. The TRIGR study met the accrual goal after 4.7 years of recruitment, 2.7 years longer than projected initially. Challenges included difficulty in finding fathers with T1D, a higher than expected rate of premature delivery among T1D mothers, and implementation of new privacy regulations mid-trial. The majority of participants were recruited from primary care antenatal clinics located near the study centers and from a general hospital or pediatric center that was affiliated with a TRIGR Study center. Internet and magazine advertisements were found to be useful for recruitment of families. Alternative follow-up strategies are offered to families who wish to reduce or discontinue participation. LIMITATIONS: Our experience is limited to a single international multicenter trial. CONCLUSIONS: TRIGR coordinators played key roles in the recruitment and intervention periods and continue to be instrumental in retaining families and children during the 10-year follow-up period for each child.

iPSC-derived ß cells model diabetes due to glucokinase deficiency.

Diabetes is a disorder characterized by loss of ß cell mass and/or ß cell function, leading to deficiency of insulin relative to metabolic need. To determine whether stem cell-derived ß cells recapitulate molecular-physiological phenotypes of a diabetic subject, we generated induced pluripotent stem cells (iPSCs) from subjects with maturity-onset diabetes of the young type 2 (MODY2), which is characterized by heterozygous loss of function of the gene encoding glucokinase (GCK). These stem cells differentiated into ß cells with efficiency comparable to that of controls and expressed markers of mature ß cells, including urocortin-3 and zinc transporter 8, upon transplantation into mice. While insulin secretion in response to arginine or other secretagogues was identical to that in cells from healthy controls, GCK mutant ß cells required higher glucose levels to stimulate insulin secretion. Importantly, this glucose-specific phenotype was fully reverted upon gene sequence correction by homologous recombination. Our results demonstrate that iPSC-derived ß cells reflect ß cell-autonomous phenotypes of MODY2 subjects, providing a platform for mechanistic analysis of specific genotypes on ß cell function.

A model for personalized in vivo analysis of human immune responsiveness.

Studies of human immune diseases are generally limited to the analysis of peripheral blood lymphocytes of heterogeneous patient populations. Improved models are needed to allow analysis of fundamental immunologic abnormalities predisposing to disease and in which to assess immunotherapies. Immunodeficient mice receiving human fetal thymus grafts and fetal CD34(+) cells intravenously produce robust human immune systems, allowing analysis of human T cell development and function. However, to use humanized mice to study human immune-mediated disorders, immune systems must be generated from adult hematopoietic cells. Here, we demonstrated robust immune reconstitution in mice with hematopoietic stem cells (HSCs) aspirated from bone marrow of adults with type 1 diabetes (T1D) and healthy control volunteers. In these humanized mice, cryopreservation of human leukocyte antigen allele-matched fetal thymic tissue prevented allogeneic adult HSC rejection. Newly generated T cells, which included regulatory T cells (T(regs)), were functional and self-tolerant and had a diverse repertoire. The immune recognition of these mice mimicked that of the adult CD34(+) cell donor, but the T cell phenotypes were more predominantly "naïve" than those of the adult donors. HSCs from T1D and control donors generated similar numbers of natural T(regs) intrathymically; however, peripheral T cells from T1D subjects showed increased proportions of activated or memory cells compared to controls, suggesting possible HSC-intrinsic differences in T cell homeostasis that might underlie immune pathology in T1D. This "personalized immune" mouse provides a new model for individualized analysis of human immune responses that may provide new insights into not only T1D but also other forms of immune function and dysfunction as well.

Human oocytes reprogram somatic cells to a pluripotent state.

The exchange of the oocyte's genome with the genome of a somatic cell, followed by the derivation of pluripotent stem cells, could enable the generation of specific cells affected in degenerative human diseases. Such cells, carrying the patient's genome, might be useful for cell replacement. Here we report that the development of human oocytes after genome exchange arrests at late cleavage stages in association with transcriptional abnormalities. In contrast, if the oocyte genome is not removed and the somatic cell genome is merely added, the resultant triploid cells develop to the blastocyst stage. Stem cell lines derived from these blastocysts differentiate into cell types of all three germ layers, and a pluripotent gene expression program is established on the genome derived from the somatic cell. This result demonstrates the feasibility of reprogramming human cells using oocytes and identifies removal of the oocyte genome as the primary cause of developmental failure after genome exchange.

Factors for radical creativity, incremental creativity, and routine, noncreative performance.

This study extends theory and research by differentiating between routine, noncreative performance and 2 distinct types of creativity: radical and incremental. We also use a sensemaking perspective to examine the interplay of social and personal factors that may influence a person's engagement in a certain level of creative action versus routine, noncreative work. Results demonstrate that willingness to take risks, resources for creativity, and career commitment are associated primarily with radical creativity; that the presence of creative coworkers and organizational identification are associated with incremental creativity; and that conformity and organizational identification are linked with routine performance. Theoretical and managerial implications are discussed.

Accelerated tooth eruption in children with diabetes mellitus.

OBJECTIVE: The objective of this study was to evaluate tooth eruption in 6- to 14-year-old children with diabetes mellitus. METHODS: Tooth eruption status was assessed for 270 children with diabetes and 320 control children without diabetes. Data on important diabetes-related variables were collected. Analyses were performed using logistic regression models. RESULTS: Children with diabetes exhibited accelerated tooth eruption in the late mixed dentition period (10-14 years of age) compared to healthy children. For both case patients and control subjects the odds of a tooth being in an advanced eruptive stage were significantly higher among girls than boys. There was also a trend associating gingival inflammation with expedited tooth eruption in both groups. No association was found between the odds of a tooth being in an advanced stage of eruption and hemoglobin A(1c) or duration of diabetes. Patients with higher body mass index percentile demonstrated statistically higher odds for accelerated tooth eruption, but the association was not clinically significant. CONCLUSIONS: Children with diabetes exhibit accelerated tooth eruption. Future studies need to ascertain the role of such aberrations in dental development and complications such as malocclusion, impaired oral hygiene, and periodontal disease. The standards of care for children with diabetes should include screening and referral programs aimed at oral health promotion and disease prevention.

Gingival bleeding in 6- to 13-year-old children with diabetes mellitus.

PURPOSE: This study assessed gingival bleeding in diabetic children during the mixed dentition period. METHODS: Three hundred fifty-five 6- to 13-year-old diabetic (99% type 1) and nondiabetic control children in the mixed dentition stage were evaluated from a total cohort of 700 6- to 18-year-old children. Gingival status was assessed, and data on important diabetes-related variables were collected. Analyses were performed using Poisson's regression. RESULTS: Diabetic children had significantly more gingival bleeding than controls for both primary and permanent teeth. The risk of gingival bleeding around the primary teeth in cases was 35% more than in the control group (P=.001); and the risk of gingival bleeding around the permanent teeth in cases was 57% more than in the controls (P<.001). The number of teeth with bleeding had a very modest, but statistically significant, association with: (1) mean HbA1c; (2) body mass index (BMI)-for-age percentile; and (3) duration of diabetes. CONCLUSIONS: These findings demonstrate that diabetic children are at a significantly higher risk for gingival bleeding. Diabetes-related oral complications affect the primary periodontium as early as age 6 and possibly earlier. The emphasis on oral hygiene may be valuable in preventing future periodontal complications in diabetic patients.

Diabetes mellitus promotes periodontal destruction in children.

AIM: The association between diabetes mellitus and periodontal attachment and bone loss is well established. Most of the prior literature has focused on adults, and studies in children have mostly reported gingival changes. Our aim was to assess the periodontal status of a large cohort of children and adolescents with diabetes. MATERIAL AND METHODS: We examined 350 children with diabetes (cases) and 350 non-diabetic controls (6-18 years of age). Using three different case definitions for periodontal disease, which incorporated gingival bleeding and/or attachment loss findings, multiple logistic regression analyses adjusting for age, gender, ethnicity, frequency of prior dental visits, dental plaque, and examiner were performed. RESULTS: Subjects with diabetes had increased gingival inflammation and attachment loss compared with controls. Regression analyses revealed statistically significant differences in periodontal destruction between cases and controls across all disease definitions tested (odds ratios ranging from 1.84 to 3.72). The effect of diabetes on periodontal destruction remained significant when we separately analysed 6-11 and 12-18 year old subgroups. CONCLUSIONS: These findings demonstrate an association between diabetes and an increased risk for periodontal destruction even very early in life, and suggest that programmes to address periodontal needs should be the standard of care for diabetic youth.

Periodontal infection profiles in type 1 diabetes.

OBJECTIVES: We investigated the levels of subgingival plaque bacteria and serum IgG responses in patients with type 1 diabetes and non-diabetic controls of comparable periodontal status. MATERIAL AND METHODS: Fifty type 1 diabetes patients (mean duration 20.3 years, range 6-41) were age-and gender-matched with 50 non-diabetic individuals with similar levels of periodontal disease. Full-mouth clinical periodontal status was recorded, and eight plaque samples/person were collected and analysed by checkerboard hybridization with respect to 12 species. Homologous serum IgG titres were assessed by checkerboard immunoblotting. In a sub-sample of pairs, serum cytokines and selected markers of cardiovascular risk were assessed using multiplex technology. RESULTS: Among the investigated species, only levels of Eubacterium nodatum were found to be higher in diabetic patients, while none of the IgG titres differed between the groups, both before and after adjustments for microbial load. Patients with diabetes had significantly higher serum levels of soluble E-selectin (p=0.04), vascular cell adhesion molecule-1 (VCAM-1; p=0.0008), adiponectin (p=0.01) and lower levels of plasminogen activator inhibitor-1 (PAI-1; p=0.02). CONCLUSIONS: After controlling for the severity of periodontal disease, patients with type 1 diabetes and non-diabetic controls showed comparable subgingival infection patterns and serum antibody responses.

Periodontal changes in children and adolescents with diabetes: a case-control study.

OBJECTIVE: To evaluate the level of oral disease in children and adolescents with diabetes. RESEARCH DESIGN AND METHODS: Dental caries and periodontal disease were clinically assessed in 182 children and adolescents (6-18 years of age) with diabetes and 160 nondiabetic control subjects. RESULTS: There were no differences between case and control subjects with respect to dental caries. Children with diabetes had significantly higher plaque and gingival inflammation levels compared with control subjects. The number of teeth with evidence of attachment loss (the hallmark of periodontal disease) was significantly greater in children with diabetes (5.79 +/- 5.34 vs. 1.53 +/- 3.05 in control subjects, unadjusted P < 0.001). When controlling for age, sex, ethnicity, gingival bleeding, and frequency of dental visits, diabetes remained a highly significant correlate of periodontitis, especially in the 12- to 18-year-old subgroup. In the case group, BMI was significantly correlated with destruction of connective tissue attachment and bone, but duration of diabetes and mean HbA(1c) were not. CONCLUSIONS: Our findings suggest that periodontal destruction can start very early in life in diabetes and becomes more prominent as children become adolescents. Programs designed to promote periodontal disease prevention and treatment should be provided to young patients with diabetes.

Patterns of colonization with Ureaplasma urealyticum during neonatal intensive care unit hospitalizations of very low birth weight infants and the development of chronic lung disease.

BACKGROUND: Ureaplasma urealyticum and its association with chronic lung disease (CLD) of prematurity has remained a controversial topic. To readdress this question, we performed a longitudinal study using culture and polymerase chain reaction to detect U urealyticum in the respiratory tract of very low birthweight infants throughout their neonatal intensive care unit hospitalizations. METHODS: We screened 125 infants weighing <1500 g and/or <32 weeks' gestational age over a 12-month period, collecting endotracheal, nasopharyngeal, and throat specimens on days of age 1, 3, 7, and weekly thereafter. CLD was defined as dependency on supplemental oxygen at 28 days and at 36 weeks' postconceptional age. RESULTS: Forty infants (32%) had 1 or more positive specimens by culture or polymerase chain reaction. We identified 3 patterns of U urealyticum colonization: persistently positive (n = 18), early transient (n = 14), and late acquisition (n = 8). We compared the rates of CLD in each of the 3 colonized groups with the rate of CLD in the noncolonized group. We found a significantly higher rate of CLD at 28 days of age (odds ratio: 8.7; 95% confidence interval: 3.3, 23) and at 36 weeks' postconception (odds ratio: 38.5, 95% confidence interval: 4.0, 374) only for infants with persistently positive colonization. CONCLUSIONS: This study demonstrates that the risk of developing CLD varies with the pattern of U urealyticum colonization. Only the persistently positive colonization pattern, which accounted for 45% of the U urealyticum-positive infants, was associated with a significantly increased risk of development of CLD.