Use of primary health care services and mortality in older patients with type 2 diabetes with or without comorbidities.

OBJECTIVE: This study aimed to examine primary health care (PHC) service utilization and mortality in older patients with type 2 diabetes (T2D) with or without comorbidities. DESIGN AND SETTING: A cohort study in PHC in the city of Vantaa, Finland. Follow-up period was set between the years 2011 and 2018. SUBJECTS: PHC patients aged 60 years or more with a T2D were included. MAIN OUTCOME MEASURES: Service utilization was defined as the number of face-to-face appointments and telephone contacts between a patient and general practitioner (GP) or nurse. The presence of comorbidities was defined using the Charlson Comorbidity Index (CCI). Mortality was assessed using hazard ratio (HR) and standardized mortality ratio (SMR). RESULTS: In total, 11,020 patients were included and followed for 71,596 person years. Mean age of the women and men in the beginning of follow-up were 71 and 69 years, respectively. The patients in the study cohort had a mean of eight appointments per person year to the GPs or nurses. Patients with T2D with comorbidities had more appointments than patients with T2D without comorbidities (incidence rate ratio (IRR) 1.44 [95% CI 1.39-1.49]). Increase in the number of all appointments reduced mortality in patients with T2D with and without comorbidities. Between patients with T2D with comorbidities and patients with T2D without comorbidities, the age and sex adjusted HR for death was 1.50 (95% CI 1.39-1.62). The SMR was higher in patients with T2D with comorbidities (1.83 [95% CI 1.74-1.92]) than in patients with T2D without comorbidities (0.91 [95% CI 0.86-0.96]). CONCLUSIONS: In older patients with T2D, the presence of comorbidities was associated with increased use of PHC services and increased mortality. Increase in the number of appointments was associated with reduced mortality in patients with T2D with or without comorbidities.Key PointsIn older patients with T2D, it has not been studied whether and to what extend multimorbidity affects use of PHC services and mortality.The presence of comorbidities according to the Charlson Comorbidity Index (CCI) was associated with increased use of PHC services.The number of appointments to GPs or nurses was associated with reduced mortality in patients with T2D with or without comorbidities according to the CCI.

The association between bacterial infections and the risk of coronary heart disease in type 1 diabetes.

BACKGROUND: Diabetes increases the risk of infections and coronary heart disease (CHD). Whether infections increase the risk of CHD and how this applies to individuals with diabetes is unclear. OBJECTIVES: To investigate the association between bacterial infections and the risk of CHD in type 1 diabetes. METHODS: Individuals with type 1 diabetes (n = 3781) were recruited from the Finnish Diabetic Nephropathy Study (FinnDiane), a prospective follow-up study. CHD was defined as incident events: fatal or nonfatal myocardial infarction, coronary artery bypass surgery or percutaneous coronary intervention, identified through national hospital discharge register data. Infections were identified through national register data on all antibiotic purchases from outpatient care. Register data were available from 1 January 1995 to 31 December 2015. Bacterial lipopolysaccharide (LPS) activity was measured from serum samples at baseline. Data on traditional risk factors for CHD were collected during baseline and consecutive visits. RESULTS: Individuals with an incident CHD event (n = 370) had a higher mean number of antibiotic purchases per follow-up year compared to those without incident CHD (1.34 [95% CI: 1.16-1.52], versus 0.79 [0.76-0.82], P < 0.001), as well as higher levels of LPS activity (0.64 [0.60-0.67], versus 0.58 EU mL-1 [0.57-0.59], P < 0.001). In multivariable-adjusted Cox proportional hazards models, the mean number of antibiotic purchases per follow-up year was an independent risk factor for incident CHD (HR 1.21, 95% CI: 1.14-1.29, P < 0.0001). High LPS activity was a risk factor for incident CHD (HR 1.93 [1.34-2.78], P < 0.001) after adjusting for static confounders. CONCLUSION: Bacterial infections are associated with an increased risk of incident CHD in individuals with type 1 diabetes.

A novel glycocluster molecule prevents timothy-induced allergic airway inflammation in mice.

BACKGROUND: Allergen-specific immunotherapy (SIT) effectively alleviates type I allergic diseases characterized by T helper (Th)2-type immunity. Our recent studies have shown that a synthetic trivalent glycocluster, triacedimannose (TADM), suppresses the Th2-type allergic inflammation. The aim of this study was to compare TADM with two well-known adjuvants, unmethylated cytosine-phosphate-guanine oligodeoxynucleotide (CpG) and monophosphoryl lipid A (MPLA) in a grass allergen-induced chronic allergic inflammation model in mice. METHODS: Female BALB/c mice were intranasally sensitized with 50 µL of timothy grass pollen extract (TE) twice a week for a period of 15 weeks. Therapeutic intranasal treatments were then performed once a week after the tenth intranasal TE instillation using TADM (10 or 25 µg/50 µL), CpG-ODN (20 µg/50 µL) or MPLA (2 µg/50 µL). Groups of 9-10 animals per treatment were killed 24 hours after the last timothy dosage. Blood, bronchoalveolar lavage (BAL) fluids and lung biopsies were taken for subsequent analysis. RESULTS: When mice were repeatedly exposed to TE for 15 weeks, the number of eosinophils and lymphocytes increased in the BAL fluids. The eosinophil and lymphocyte counts decreased dose-dependently and were practically abolished in the mice treated with TADM. Treatments with MPLA or CpG significantly increased the numbers of neutrophils, while CpG nonsignificantly decreased eosinophilia compared to timothy exposure. CONCLUSIONS: A novel synthetic glycocluster molecule inhibited the development of grass-induced eosinophilic pulmonary inflammation in mice when administrated in the airways. This compound could be a candidate to be used either as an adjuvant in SIT or as a topical anti-inflammatory treatment.

Embolic strokes of undetermined source in young adults: baseline characteristics and long-term outcome.

BACKGROUND AND PURPOSE: Embolic strokes of undetermined source (ESUS) are a recent entity, not yet thoroughly investigated in young stroke patients. The clinical characteristics and long-term risks of vascular events and all-cause mortality between young-onset ESUS and other aetiological subgroups were compared. METHODS: Patients with ESUS were identified amongst the 1008 patients aged 15-49 years with first-ever ischaemic stroke in Helsinki Young Stroke Registry, and primary end-points were defined as recurrent stroke, composite vascular events and all-cause mortality. Cumulative 15-year risks for each end-point were analysed with life tables and adjusted risks were based on Cox proportional hazard analyses. RESULTS: Of the 971 eligible patients, 203 (20.9%) were classified as ESUS. They were younger (median age 40 years, interquartile range 32-46 vs. 45 years, 39-47), more often female (43.3% vs. 35.7%) and had fewer cardiovascular risk factors than other modified TOAST groups. With a median follow-up time of 10.1 years, ESUS patients had the second lowest cumulative risk of recurrent stroke and composite vascular events and lowest mortality compared to other TOAST groups. Large-artery atherosclerosis and small vessel disease carried significantly higher risk for recurrent stroke than did ESUS, whilst no difference appeared between cardioembolism from high-risk sources and ESUS. CONCLUSIONS: In our cohort, ESUS patients were younger and had milder cardiovascular risk factor burden and generally better long-term outcome compared to other causes of young-onset stroke. The comparable risk of recurrent stroke between ESUS and high-risk sources of cardioembolism might suggest similarities in their pathophysiology.

Nasal mucosa and blood cell transcriptome profiles do not reflect respiratory symptoms associated with moisture damage.

Upper and lower respiratory symptoms and asthma are adverse health effects associated with moisture-damaged buildings. Quantitative measures to detect adverse health effects related to exposure to dampness and mold are needed. Here, we investigate differences in gene expression between occupants of moisture-damaged and reference buildings. Moisture-damaged (N = 11) and control (N = 5) buildings were evaluated for dampness and mold by trained inspectors. The transcriptomics cohort consisted of nasal brushings and peripheral blood mononuclear cells (PBMCs) from 86 teachers, with/without self-perceived respiratory symptoms. Subject categories comprised reference (R) and damaged (D) buildings with (S) or without (NS) symptoms, that is, R-S, R-NS, DS, and D-NS. Component analyses and k-means clustering of transcriptome profiles did not distinguish building status (R/D) or presence of respiratory symptoms (S/NS). Only one nasal mucosa gene (YBX3P1) exhibited a significant change in expression between D-S and D-NS. Nine other nasal mucosa genes were differentially expressed between R-S and D-S teachers. No differentially expressed genes were identified in PBMCs. We conclude that the observed mRNA differences provide very weak biological evidence for adverse health effects associated with subject occupancy of the specified moisture-damaged buildings. This emphasizes the need to evaluate all potential factors (including those not related to toxicity) influencing perceived/self-reported ill health in moisture-damaged buildings.

Intestinal alkaline phosphatase at the crossroad of intestinal health and disease - a putative role in type 1 diabetes.

BACKGROUND: Patients with type 1 diabetes have shown an increase in circulating cytokines, altered lipoprotein metabolism and signs of vascular dysfunction in response to high-fat meals. Intestinal alkaline phosphatase (IAP) regulates lipid transport and inflammatory responses in the gastrointestinal tract. We therefore hypothesized that changes in IAP activity could have profound effects on gut metabolic homeostasis in patients with type 1 diabetes. METHODS: Faecal samples of 41 nondiabetic controls and 46 patients with type 1 diabetes were analysed for IAP activity, calprotectin, immunoglobulins and short-chain fatty acids (SCFAs). The impact of oral IAP supplementation on intestinal immunoglobulin levels was evaluated in C57BL/6 mice exposed to high-fat diet for 11 weeks. RESULTS: Patients with type 1 diabetes exhibited signs of intestinal inflammation. Compared to controls, patients with diabetes had higher faecal calprotectin levels, lower faecal IAP activities accompanied by lower propionate and butyrate concentrations. Moreover, the amount of faecal IgA and the level of antibodies binding to oxidized LDL were decreased in patients with type 1 diabetes. In mice, oral IAP supplementation increased intestinal IgA levels markedly. CONCLUSION: Deprivation of protective intestinal factors may increase the risk of inflammation in the gut - a phenomenon that seems to be present already in patients with uncomplicated type 1 diabetes. Low levels of intestinal IgA and antibodies to oxidized lipid epitopes may predispose such patients to inflammation-driven complications such as cardiovascular disease and diabetic nephropathy. Importantly, oral IAP supplementation could have beneficial therapeutic effects on gut metabolic homeostasis, possibly through stimulation of intestinal IgA secretion.

A novel mannoside-glycocluster adjuvant: Compared in vitro to CpG ODN and MPL and tested in vivo in mouse asthma model.

BACKGROUND: Allergen-specific immunotherapy balances the Th2-biased immunity towards Th1 and Treg responses. Adjuvants are used in allergen preparations to intensify the immune responses. The increased prevalence of allergies in developed societies has been associated with decreased microbial load during childhood. This has initiated a search for microbial structures to be used as adjuvants. Our study has shown that a synthetic triacedimannose (TADM) may suppress the Th2-type allergic inflammatory response. The aim of this study was to compare the properties of TADM with capacities of other adjuvants, CpG ODN and MPL, to modulate cytokine production in PBMC and regulate sensitisation in an OVA-sensitised mouse asthma model. METHODS: The effects of TADM were studied in vitro on birch stimulated PBMC cultures of birch allergic rhinitis patients with other known adjuvants. Cytokines in supernatants were measured by Luminex. Effects of TADM were analysed in vivo in a mouse model of OVA-induced allergic asthma by analysing BAL, cytokine mRNA and serum antibodies. RESULTS: TADM was the only adjuvant that significantly suppressed the production of all birch induced Th2-type cytokines. In a murine model, TADM significantly suppressed the specific IgE production and enhanced IFN-γ production. CONCLUSIONS: TADM suppresses the birch allergen induced Th2-type cytokine responses in allergic subjects more efficiently than the two other adjuvants, MPL and CpG ODN. TADM is immunomodulatory also in vivo and decreases the IgE levels and increases the IFN-γ responses in a murine model. These results suggest that TADM may be a promising candidate for novel adjuvants in immunotherapy.

Mapping of a gene for type 2 diabetes associated with an insulin secretion defect by a genome scan in Finnish families.

Non-insulin dependent diabetes mellitus (NIDDM) affects more than 100 million people worldwide and is associated with severe metabolic defects, including peripheral insulin resistance, elevated hepatic glucose production, and inappropriate insulin secretion. Family studies point to a major genetic component, but specific susceptibility genes have not yet been identified-except for rare early-onset forms with monogenic or mitochondrial inheritance. We have screened over 4,000 individuals from a population isolate in western Finland, identified 26 families (comprising 217 individuals) enriched for NIDDM and performed a genome-wide scan using non-parametric linkage analysis. We found no significant evidence for linkage when the families were analysed together, but strong evidence for linkage when families were classified according to mean insulin levels in affecteds (in oral glucose tolerance tests). Specifically, families with the lowest insulin levels showed linkage (P = 2 x 10(-6)) to chromosome 12 near D12S1349. Interestingly, this region contains the gene causing the rare, dominant, early-onset form of diabetes MODY3. Unlike MODY3 families, the Finnish families with low insulin have an age-of-onset typical for NIDDM (mean = 58 years). We infer the existence of a gene NIDDM2 causing NIDDM associated with low insulin secretion, and suggest that NIDDM2 and MODY3 may represent different alleles of the same gene.

Soluble receptor for AGE (RAGE) is a novel independent predictor of all-cause and cardiovascular mortality in type 1 diabetes.

AIMS/HYPOTHESIS: Activation of the receptor for AGE (RAGE) is implicated in the development and progression of vascular complications of diabetes. In this study, we explore factors and mortality outcomes associated with soluble RAGE (sRAGE) in a multicentre nationwide cohort of Finnish adults with type 1 diabetes. METHODS: Baseline sRAGE concentrations were estimated in 3,100 adults with type 1 diabetes. Clinical and biological variables independently associated with sRAGE were identified using multivariate regression analysis. Independent predictors of mortality were determined using Cox and Fine-Gray proportional-hazards models. RESULTS: The main independent determinants of sRAGE concentrations were estimated glomerular filtration rate, albuminuria, body mass index, age, duration of diabetes, HbA(1c) and insulin dose (all p < 0.05). During a median of 9.1 years of follow-up there were 202 deaths (7.4 per 1,000 patient years). sRAGE was independently associated with all-cause (Cox model: HR 1.03) and cardiovascular mortality (Fine-Gray competing risks model: HR 1.06) such that patients with the highest sRAGE concentrations had the greatest risk of mortality, after adjusting for age, sex, macrovascular disease, HDL-cholesterol, HbA(1c), triacylglycerol, high-sensitivity C-reactive protein (hsCRP) and the presence and severity of chronic kidney disease. Although polymorphisms in the gene coding for RAGE were significantly associated with sRAGE concentrations, none were associated with mortality outcomes. CONCLUSIONS/INTERPRETATION: Increased concentrations of sRAGE are associated with increased all-cause and cardiovascular mortality in type 1 diabetes, potentially reflecting the activation and production of RAGE in the context of accelerated vascular disease. These novel findings highlight the importance of the RAGE activation in the prevention and management of diabetic complications.

Defective central tolerance in Aire-deficient mice is not sufficient to induce symptomatic autoimmunity during lymphopenia-induced T cell proliferation.

Transcriptional regulator autoimmune regulator (AIRE) controls thymic negative selection but it is also expressed in secondary lymphoid organs. The relative contribution of AIRE's central and peripheral function to the maintenance of tolerance is unclear. We transferred mature lymphocytes from Aire(-/-) or wild-type donors to Aire(+/+) lymphopenic recipients, which allowed us to gauge the autoreactivity inherent in the cells originating in an Aire(-/-) thymus. In the ensuing lymphopenia-induced proliferation (LIP), the recipients of cells from Aire(-/-) showed definite T cell hyperproliferation and developed autoantibodies at a higher frequency than the recipients of wild-type cells. However, neither of the recipient groups developed clinical symptoms, and pathological tissue infiltrates were also absent. The recipients of Aire(-/-) cells showed hyperproliferation and increased accumulation of regulatory T cells (Tregs), especially in tissues susceptible to inflammation triggered by LIP. These data are consistent with the view that T cells developing in the absence of Aire are autoreactive. However, overt autoimmunity was prevented, most likely by the suppressive function of Treg cells in the Aire-sufficient recipients. Our results support the importance of the peripheral AIRE expression in the maintenance of immunological tolerance.

A combined Fuzzy and Naive Bayesian strategy can be used to assign event codes to injury narratives.

BACKGROUND: Bayesian methods show promise for classifying injury narratives from large administrative datasets into cause groups. This study examined a combined approach where two Bayesian models (Fuzzy and Naïve) were used to either classify a narrative or select it for manual review. METHODS: Injury narratives were extracted from claims filed with a worker's compensation insurance provider between January 2002 and December 2004. Narratives were separated into a training set (n=11,000) and prediction set (n=3,000). Expert coders assigned two-digit Bureau of Labor Statistics Occupational Injury and Illness Classification event codes to each narrative. Fuzzy and Naïve Bayesian models were developed using manually classified cases in the training set. Two semi-automatic machine coding strategies were evaluated. The first strategy assigned cases for manual review if the Fuzzy and Naïve models disagreed on the classification. The second strategy selected additional cases for manual review from the Agree dataset using prediction strength to reach a level of 50% computer coding and 50% manual coding. RESULTS: When agreement alone was used as the filtering strategy, the majority were coded by the computer (n=1,928, 64%) leaving 36% for manual review. The overall combined (human plus computer) sensitivity was 0.90 and positive predictive value (PPV) was >0.90 for 11 of 18 2-digit event categories. Implementing the 2nd strategy improved results with an overall sensitivity of 0.95 and PPV >0.90 for 17 of 18 categories. CONCLUSIONS: A combined Naïve-Fuzzy Bayesian approach can classify some narratives with high accuracy and identify others most beneficial for manual review, reducing the burden on human coders.

The effect of waiting time on health and quality of life outcomes and costs of medication in hip replacement patients: a randomized clinical trial.

OBJECTIVE: To evaluate the effect of waiting time on health and quality of life outcomes and costs of medication in total hip replacement (THR) patients in a randomized clinical trial. METHODS: 395 THR patients were recruited into the study. When placed on the waiting list, patients were randomized into a short (< or =3 months) or a non-fixed waiting time (NFWT) (>3 months) group. In the final analyses 309 patients (179 women) with a mean age of 65 years were included. Health-related quality of life (HRQoL) (generic 15D), and pain and function (modified Harris Hip Score (HHS)) were calculated when placed on the waiting list, at hospital admission, and at 3 and 12 months postoperatively. The costs of disease-specific medication were calculated at the same measurement points. All analyses were performed using the intention-to-treat (ITT) principal. RESULTS: Of the recruited patients, 309 (78%) completed the follow-up (short group 140 and non-fixed group 169 patients). The mean waiting time was 74 days in the short and 194 days in the NFWT groups. In the ITT analyses there were no statistically significant differences between the groups in the weekly use and costs of medication, HRQoL or HHS at baseline, at admission, or 3 or 12 months after surgery. The only difference was in total medication costs during the waiting time period, at EUR 83 and 171, respectively. CONCLUSIONS: The length of the waiting time did not generate different effects on the studied health and quality of life outcomes of the randomized groups. However, those in short waiting time group reached earlier better HRQoL.

Bayesian methods: a useful tool for classifying injury narratives into cause groups.

To compare two Bayesian methods (Fuzzy and Naïve) for classifying injury narratives in large administrative databases into event cause groups, a dataset of 14 000 narratives was randomly extracted from claims filed with a worker's compensation insurance provider. Two expert coders assigned one-digit and two-digit Bureau of Labor Statistics (BLS) Occupational Injury and Illness Classification event codes to each narrative. The narratives were separated into a training set of 11 000 cases and a prediction set of 3000 cases. The training set was used to develop two Bayesian classifiers that assigned BLS codes to narratives. Each model was then evaluated for the prediction set. Both models performed well and tended to predict one-digit BLS codes more accurately than two-digit codes. The overall sensitivity of the Fuzzy method was, respectively, 78% and 64% for one-digit and two-digit codes, specificity was 93% and 95%, and positive predictive value (PPV) was 78% and 65%. The Naïve method showed similar accuracy: a sensitivity of 80% and 70%, specificity of 96% and 97%, and PPV of 80% and 70%. For large administrative databases, Bayesian methods show significant promise as a means of classifying injury narratives into cause groups. Overall, Naïve Bayes provided slightly more accurate predictions than Fuzzy Bayes.

Characterization of the MODY3 phenotype. Early-onset diabetes caused by an insulin secretion defect.

Maturity-onset diabetes of the young (MODY) type 3 is a dominantly inherited form of diabetes, which is often misdiagnosed as non-insulin-dependent diabetes mellitus (NIDDM) or insulin-dependent diabetes mellitus (IDDM). Phenotypic analysis of members from four large Finnish MODY3 kindreds (linked to chromosome 12q with a maximum lod score of 15) revealed a severe impairment in insulin secretion, which was present also in those normoglycemic family members who had inherited the MODY3 gene. In contrast to patients with NIDDM, MODY3 patients did not show any features of the insulin resistance syndrome. They could be discriminated from patients with IDDM by lack of glutamic acid decarboxylase antibodies (GAD-Ab). Taken together with our recent findings of linkage between this region on chromosome 12 and an insulin-deficient form of NIDDM (NIDDM2), the data suggest that mutations at the MODY3/NIDDM2 gene(s) result in a reduced insulin secretory response, that subsequently progresses to diabetes and underlines the importance of subphenotypic classification in studies of diabetes.

A comparison of survival of moulded monoblock and modular tibial components of 751 AGC total knee replacements in the treatment of rheumatoid arthritis.

We evaluated the survival of moulded monoblock and modular tibial components of the AGC total knee replacement in patients with rheumatoid arthritis. Between 1985 and 1995, 751 knees with this diagnosis were replaced at our institution. A total of 256 tibial components were of the moulded design and 495 of the modular design. The mean follow-up of the moulded subgroup was 9.6 years (0.5 to 14.7), and that of the modular group 7.0 years (0.1 to 14.7). The groups differed significantly from each other in Larsen grade, cementing of components and patellar resurfacing, but no statistically significant difference between the survival of the components was found (Log rank test, p = 0.91). The cumulative success rate of the moulded group was 96.8% (95% confidence interval 93.6% to 98.4%) at five years and 94.4% (95% confidence interval 90.4% to 96.7%) at ten years, and of the modular group 96.2% (95% confidence interval 94% to 97.6%) and 93.6% (95% confidence interval 89.7% to 96%), respectively. Revision was required in 37 total knee replacements, the main causes were infection, pain, loosening of the tibial component and patellar problems. Survival rates for both components were satisfactory.

Biochemical pathways of breath ammonia (NH3) generation in patients with end-stage renal disease undergoing hemodialysis.

Breath ammonia (NH3) has been proposed as a potential biomarker in monitoring hemodialysis (HD) adequacy, since a strong correlation between blood urea and mouth-exhaled breath NH3 has been observed in patients with end-stage renal disease (ESRD) undergoing HD. However, the biochemical pathways for breath NH3 generation from blood urea have not been demonstrated. In this study, we show a strong correlation (r s = 0.77, p < 0.001) between blood and salivary urea, indicating that salivary urea levels reflect blood urea levels. Salivary urea is in turn strongly correlated to salivary ammonia ([Formula: see text] + NH3) in most of the patients. This confirms that the hydrolysis of urea by urease generates ammonia in the oral cavity. A further strong correlation between salivary ammonia and breath NH3 indicates that salivary ammonia evaporates into gas phase and turns to breath NH3. Therefore, blood urea is a major biochemical source of breath NH3. Since breath NH3 is generated predominantly in the oral cavity, the levels of breath NH3 are influenced significantly by the patient's oral condition including urease activity and salivary pH. Our results agree with previous studies that have shown a connection between salivary urea and breath NH3.

An EBV-based mammalian cell expression vector for efficient expression of cloned coding sequences.

The construction of a mammalian cell expression vector using human cytomegalovirus immediate early gene enhancer to initiate transcription of inserted coding sequences is described. The vector also carries Epstein-Barr virus EBNA-1 nuclear antigen gene, ori-P sequences and hygromycin B resistance gene hph from E. coli. The expression capacity of this construct was tested by inserting the chloramphenicol acetyltransferase (CAT) gene into the vector. The EBV-CAT construct was transfected into various cell lines and high levels of CAT activity were obtained in human and monkey cells. In these cells, the vector DNA also replicates as an extrachromosomal element having 1 to 20 copies per cell. In most cases, the vector copy number and the expression level of inserted gene was in positive correlation in different cell clones.

Search for a third susceptibility gene for maturity-onset diabetes of the young. Studies with eleven candidate genes.

Maturity-onset diabetes of the young (MODY) is a model for genetic studies of non-insulin-dependent diabetes mellitus. We have identified 15 MODY families in which diabetes is not the result of mutations in the glucokinase gene. This cohort of families will be useful for identifying other diabetes-susceptibility genes. Nine other candidate genes potentially implicated in insulin secretion or insulin action have been tested for linkage with MODY in these families, including glucokinase regulatory protein, hexokinase II, insulin receptor substrate 1, fatty acid-binding protein 2, glucagon-like peptide-1 receptor, apolipoprotein C-II, glycogen synthase, adenosine deaminase (a marker for the MODY gene on chromosome 20), and phosphoenolpyruvate carboxykinase. None of these loci showed evidence for linkage with MODY, implying that mutations in these genes do not make a major genetic contribution to the development of MODY. In addition to these linkage analyses, one or two affected subjects from each family were screened for the presence of the A to G mutation at nucleotide 3,243 of the mitochondrial tRNA(Leu(UUR)) gene. This mutation was not found in any of these subjects. Finally, we report the localization of the gene encoding the regulatory protein of glucokinase to chromosome 2, band p22.3 and the identification of a restriction fragment length polymorphism at this locus.

Novel mutations and a mutational hotspot in the MODY3 gene.

Maturity-onset diabetes of the young 3 (MODY3) is a type of NIDDM caused by mutations in the transcription factor hepatocyte nuclear factor-1alpha (HNF-1alpha) located on chromosome 12q. We have identified four novel HNF-1alpha missense mutations in MODY3 families. In four additional and unrelated families, we observed an identical insertion mutation that had occurred in a polycytidine tract in exon 4. Among those families, one exhibited a de novo mutation at this location. We propose that instability of this sequence represents a general mutational mechanism in MODY3. We observed no HNF-1alpha mutations among 86 unrelated late-onset diabetic patients with relative insulin deficiency. Hence mutations in this gene appear to be most strongly associated with early-onset diabetes.

Enrichment of dendritic cells from human peripheral blood.

A simplified method is described for purification of dendritic cells from human peripheral blood. The method is based on depletion of phagocytes with carbonyl iron and magnet, followed by centrifugation of nonphagocytic cells on Percoll and elimination of contaminating T lymphocytes, B lymphocytes, natural killer cells, and monocytes from the low-density cell fraction by treatment with monoclonal antibodies and complement. The purity of enriched dendritic cells was about 80% and these cells represented 0.2% of the starting mononuclear cell population. Dendritic cells were potent autologous and allogeneic stimulators in mixed leukocyte cultures.