Carl-David Agardh’s research while affiliated with Lund University and other places

Latent autoimmune diabetes of the adults (LADA) accounts for up to 12% of all patients with diabetes. Initially the disease resembles type 2 diabetes (T2D), however the typical presence of β-cell autoantibodies indicates an autoimmune basis of LADA. While dysfunctional regulatory T cells (Tregs) have been implicated in autoimmune diabetes, these cells have been scarcely studied in LADA. The aim of this study was to investigate the frequency and phenotype of circulating Tregs in LADA patients early during disease progression. Flow cytometric analysis was performed on whole blood and peripheral mononuclear cell (PBMC) from patients diagnosed with LADA prior to insulin deficiency (n. 39) and from healthy volunteers (n. 20). Overall, we found the frequency and activation status of peripheral putative Tregs, to be altered in LADA patients compared to healthy controls. While total T cells, and CD4+ T cells expressing high level of CD25 (CD4+CD25hi) were unchanged, the frequency and total numbers of CD4+ T cells expressing an intermediate level of CD25 (CD4+CD25int) were decreased in LADA patients. Interestingly, the expression of the Treg-specific marker FOXP3, as well as the activation and memory makers CD69, CTLA-4 CCR4 and CD45RO was increased in CD4+CD25+ T cells of the patients. Our data depict phenotypic changes in T cells of LADA patients that may reflect a derangement in peripheral immune-regulation contributing to the slow process leading to insulin-dependent diabetes in these patients. This article is protected by copyright. All rights reserved.

Epigenetic variation has been linked to several human diseases. Proliferative diabetic retinopathy (PDR) is a major cause of vision loss in subjects with diabetes. However, studies examining the association between PDR and the genome-wide DNA methylation pattern are lacking. Our aim was to identify epigenetic modifications that associate with and predict PDR in subjects with type 1 diabetes (T1D). DNA methylation was analyzed genome-wide in 485,577 sites in blood from cases with PDR (n = 28), controls (n = 30), and in a prospective cohort (n = 7). False discovery rate analysis was used to correct the data for multiple testing. Study participants with T1D diagnosed before 30 years of age and insulin treatment within 1 year from diagnosis were selected based on 1) subjects classified as having PDR (cases) and 2) subjects with T1D who had had diabetes for at least 10 years when blood DNA was sampled and classified as having no/mild diabetic retinopathy also after an 8.7-year follow-up (controls). DNA methylation was also analyzed in a prospective cohort including seven subjects with T1D who had no/mild diabetic retinopathy when blood samples were taken, but who developed PDR within 6.3 years (converters). The retinopathy level was classified by fundus photography. We identified differential DNA methylation of 349 CpG sites representing 233 unique genes including TNF, CHI3L1 (also known as YKL-40), CHN2, GIPR, GLRA1, GPX1, AHRR, and BCOR in cases with PDR compared with controls. The majority of these sites (79 %) showed decreased DNA methylation in cases with PDR. The Natural Killer cell-mediated cytotoxicity pathway was found to be significantly (P = 0.006) enriched among differentially methylated genes in cases with PDR. We also identified differential DNA methylation of 28 CpG sites representing 17 genes (e.g. AHRR, GIPR, GLRA1, and BCOR) with P <0.05 in the prospective cohort, which is more than expected by chance (P = 0.0096). Subjects with T1D and PDR exhibit altered DNA methylation patterns in blood. Some of these epigenetic changes may predict the development of PDR, suggesting that DNA methylation may be used as a prospective marker of PDR.

PurposeDespite the extensive use of retinal photocoagulation for ischaemia and vascular leakage in retinal vascular disease, the molecular mechanisms behind its clinical beneficial effects are still poorly understood. One important target of laser irradiation is the retinal pigment epithelium (RPE). In this study, we aimed at identifying the isolated effects of photocoagulation of RPE at both the mRNA and protein expression levels.Methods Human ARPE-19 cells were exposed to photocoagulation. Gene expression and protein expression were compared to untreated cells using microarray and liquid chromatography–mass spectrometry analysis. Genes and proteins queried by microarray and mass spectrometry were subjected to the Kyoto Encyclopedia of Genes and Genomes (KEGG) database pathway analyses.ResultsLaser irradiation resulted in an induction of the cytoprotective heat-shock protein subfamily Hsp70 as well as in a suppression of the vascular permeability factor carbonic anhydrase 9 (CA9). These expression patterns were evident at both the mRNA and protein levels. KEGG pathway analyses revealed genes and proteins involved in cellular turnover, repair and inflammation.Conclusions By characterizing the transcriptional and translational effects of laser coagulation on the RPE cells in culture, we have revealed responses, which might contribute to some of the beneficial effects obtained by photocoagulation for ischaemia and vascular leakage in retinal vascular disease.

OBJECTIVE Patients with latent autoimmune diabetes in adults (LADA) express autoantibodies against the 65-kDa isoform of GAD antibody (GADA). Intervention with recombinant human GAD65 formulated with aluminium hydroxide (GAD-alum) given twice subcutaneously to LADA patients at intervals of 4 weeks was safe and did not compromise β-cell function in a Phase II clinical trial. GADA affinity has been shown to predict progression to type 1 diabetes. Here, we asked whether GADA affinity was affected by the GAD65 antigen-specific vaccination and/or associated with β-cell function in participants of this trial.RESEARCH DESIGN AND METHODSGADA affinity was measured in sera of 46 LADA patients obtained prior to the 1st week and 20 weeks after the second injection with GAD-alum or placebo using competitive binding experiments with [(125)I]-labeled and unlabeled human GAD65.RESULTSAt baseline, GADA affinities ranged from 1.9 × 10(7) to 5.0 × 10(12) L/mol (median 2.8 × 10(10) L/mol) and were correlated with GADA titers (r = 0.47; P = 0.0009), fasting (r = -0.37; P = 0.01) and stimulated (r = -0.40; P = 0.006) C-peptide concentrations, and HbA1c (r = 0.39; P = 0.007). No significant changes in affinity were observed from baseline to week 24. Patients with GADA affinities in the lower first quartile (<4 × 10(9) L/mol) had better preserved fasting C-peptide concentrations at baseline than those with higher affinities (mean 1.02 vs. 0.66 nmol/L; P = 0.004) and retained higher concentrations over 30 months of follow-up (mean 1.26 vs. 0.62 nmol/L; P = 0.01).CONCLUSIONS Intervention with GAD-alum in LADA patients had no effect on GADA affinity. Our data suggest that patients with low GADA affinity have a prolonged preservation of residual β-cell function.

This study sought to compare type 2 diabetes (T2D) risk indicators in Iraqi immigrants with those in ethnic Swedes living in southern Sweden. Population-based, cross-sectional cohort study of men and women, aged 30-75 years, born in Iraq or Sweden conducted in 2010-2012 in Malmö, Sweden. A 75g oral glucose tolerance test was performed and sociodemographic and lifestyle data were collected. T2D risk was assessed by the Finnish Diabetes Risk Score (FINDRISC). In Iraqi versus Swedish participants, T2D was twice as prevalent (11.6 vs. 5.8%, p<0.001). A large proportion of the excess T2D risk was attributable to larger waist circumference and first-degree family history of diabetes. However, Iraqi ethnicity was a risk factor for T2D independently of other FINDRISC factors (odds ratio (OR) 2.5, 95% CI 1.6-3.9). The FINDRISC algorithm predicted that more Iraqis than Swedes (16.2 vs. 12.3%, p<0.001) will develop T2D within the next decade. The total annual costs for excess T2D risk in Iraqis are estimated to exceed 2.3 million euros in 2005, not accounting for worse quality of life. Our study suggests that Middle Eastern ethnicity should be considered an independent risk indicator for diabetes. Accordingly, the implementation of culturally tailored prevention programs may be warranted.

Type 2 diabetes is highly prevalent in immigrants to Sweden from Iraq, but the prevalence of cardiovascular disease (CVD) and its risk factors are not known. In this survey we aimed to compare the prevalence of CVD and CVD-associated risk factors between a population born in Iraq and individuals born in Sweden. This population-based, cross-sectional study comprised 1,365 Iraqi immigrants and 739 Swedes (age 30-75 years) residing in the same socioeconomic area in Malmo, Sweden. Blood tests were performed and socio-demography and lifestyles were characterized. To investigate potential differences in CVD, odds ratios (ORs) with 95% confidence intervals (CIs) were estimated by multivariate logistic regression analysis with adjustment for metabolic, lifestyle and psychosocial risk factors for CVD. Outcome measures were odds of CVD. There were no differences in self-reported prevalence of CVD between Iraqi- and Swedish-born individuals (4.0 vs. 5.5%, OR 0.9, 95% CI 0.4-1.8). However, the prevalence of type 2 diabetes was higher in Iraqi compared to Swedish participants (8.4 vs. 3.3%, OR = 4.2, 95% CI 2.6-6.7). Moreover, among individuals with type 2 diabetes, Iraqis had a higher prevalence of CVD (22.8 vs. 8.0%, OR = 4.2, 95% CI 0.9-20.0), after adjustment for age and sex. By contrast, among those without diabetes, immigrants from Iraq had a lower prevalence of CVD than Swedes (2.2 vs. 5.5%, OR = 0.6, 95% CI 0.3-0.9).Type 2 diabetes was an independent risk factor for CVD in Iraqis only (OR = 6.8, 95% CI 2.8-16.2). This was confirmed by an interaction between country of birth and diabetes (p = 0.010). In addition, in Iraqis, type 2 diabetes contributed to CVD risk to a higher extent than history of hypertension (standardized OR 1.5 vs. 1.4). This survey indicates that the odds of CVD in immigrants from Iraq are highly dependent on the presence or absence of type 2 diabetes and that type 2 diabetes contributes with higher odds of CVD in Iraqi immigrants compared to native Swedes. Our study suggests that CVD prevention in immigrants from the Middle East would benefit from prevention of type 2 diabetes.

Sight-threatening diabetic retinopathy has been treated with photocoagulation for decades but the mechanisms behind the beneficial clinical effects are poorly understood. One target of irradiation and a potential player in this process is the retinal pigment epithelium (RPE). Here we establish an in vitro model for photocoagulation of human RPE cells. ARPE-19 cells were exposed to photocoagulation and studied at various time points up to 168h. Lesion morphology, necrosis and apoptosis were investigated by light microscopy; LIVE/DEAD staining and measurements of lactate dehydrogenase activity; and TUNEL- and ELISA-based quantification of DNA fragments, respectively. Cell migration and proliferation were explored using docetaxel and mitomycin C; temporal and spatial changes in proliferation were assessed by confocal immunofluorescence of proliferating cell nuclear antigen. Gene expression was measured by qPCR. Photocoagulation of ARPE-19 resulted in denaturation of proteins and reproducible lesion formation. A transient peak in necrosis, followed by a peak in apoptosis was observed in cells within the lesions at 6h and 24h, respectively after photocoagulation. Cell proliferation was depressed during the first hours after photocoagulation, back to control levels at 24h and augmented in the following days. These effects were not limited to cells in the lesions, but also evident in neighbouring cells. Changes in cell proliferation during lesion repair were preceded by changes in cell migration. Altered mRNA expression of genes previously implicated in the regulation of cell proliferation (FOS, IL-1β, IL-8, HMGA2), migration and tissue repairing (TGFBR2, ADAMTS6, TIMP3, CTGF) was observed, as well as increased expression of the alarmin IL33 and the cytoprotective gene HSPA6. Using a laser system and experimental settings that comply with standards used in clinical practice, we have established a suitable model for in vitro photocoagulation of human RPE cells to isolate their contribution to the beneficial effects of laser treatment.

Photocoagulation results in changes in gene expression. FOS, CTGF, IL33 and HSPA6 mRNA expression in ARPE-19 cells 6 h after photocoagulation and in control non-irradiated cells. Measurements were performed by qPCR using cyclophilin B as an endogenous control. ***p<0.001 and **p<0.01 vs. control; n≥6. (TIF)

(A) Confocal immunofluorescence microscopy images showing merged VCAM-1 (red)- and nuclei (green) fluorescence in the left panel; single VCAM-1 fluorescence in the middle panel and single green fluorescence in the right panel. Images are from a retinal whole-mount from a non-diabetic ApoE−/− mouse. Note adjacent VCAM-1 positive and negative vessels (white arrows). Bars = 50 µm. (B) Summarized calculations from confocal immunofluorescence microscopy data showing percentage of VCAM-1 positive and VCAM-1 negative vessels in retinas from control non-diabetic and diabetic wt, ApoE−/−, TNFα−/− and ApoE−/−/TNFα−/− mice. The percentage of VCAM-1 positive vessels was reduced in diabetic animals from wt and ApoE−/− groups (*p<0.05 and ***p<0.001, respectively), but unaltered in the TNFα−/− genotypes. (7.14 MB TIF)

Cell death is not detected in control non-photocoagulated ARPE-19 cell preparations or in cells surrounding the laser lesions. Simultaneous staining with green-fluorescent calcein-AM (left panels) and red-fluorescent ethidium homodimer-1 (middle panels) to discriminate between live and dead cells demonstrated absence of dead cells (red) and only live cells (green) in A) areas in between laser lesions 0 and 24 h after photocoagulation; and in B) control cells on cover slips that have not been laser treated. Right panels show merged images for the two fluorophores. Scale bar represents 100 µm. (TIF)