Ocular Cicatricial Pemphigoid

Figure 1 presents the case and control selection process. Study subjects were initially selected from a population of singleton births between 1982 and 1999 with AF samples available as part of the Historic Birth Cohort (HBC) stored at the Statens Serum Institute (SSI) in Copenhagen, Denmark [47 (link)]. The HBC is based on a collection of antenatal biological samples obtained during screening/diagnostic procedures performed mainly in three Danish regions. The collection of samples goes from the late 1970s until 2004 and includes more than 100,000 samples of AF, bloodspots, and maternal serum samples (Fig. 1) [14 (link), 48 (link)]. The AF samples from the HBC were centrifuged after routine screening or diagnostic amniocentesis, and samples were kept frozen at − 20 °C until further analyzed [49 (link)]. The Danish nation-wide health registers were employed to follow-up individuals in the HBC until 2009. All psychiatric diagnoses were identified utilizing the Danish Psychiatric Central Register (DPCR) which has high diagnosis validity of infantile autism diagnoses [50 (link)]. All singleton ASD cases born during 1982–1999 were identified according to the International Classification of Diseases (ICD)-8 codes 299.xx up to 1993 and ICD-10 codes DF84.xx since 1994. Furthermore, the Danish National Hospital Register (DNHR) primary diagnoses [51 (link)] were applied to complement diagnoses of congenital malformations and other psychiatric comorbidities. The birth record data of the study subjects were retrieved from the Danish medical birth registry [52 (link)]. The controls were non-ASD individuals randomly retrieved from the HBC and frequency-matched with cases on gender and year of birth [47 (link)].Fig. 1

Flow chart of autism disorders (ASD) and controls selection process

The present study aims to examine whether EDCs in AF influence individuals diagnostics with ASD later in life using the case-control design. We first performed a pilot study on pooled AF to establish methods for the EDC-receptor function analyses and measurement of POPs such as PCBs, OCPs, and PFAS as well as elements including heavy metals. The pilot study showed that the levels of lipophilic POPs (PCBs, OCPs, dioxins, PBDEs) in AF samples were below the detection limits while PFAS, elements/metals, and AF-induced combined receptor transactivities could be detected in AF samples. Therefore, 1032 individual AF samples including 332 ASD cases and 700 controls were obtained from the SSI and stored at − 20 °C in Centre for Arctic Health & Molecular Epidemiology, Department of Public Health, Aarhus University, Denmark, for the determination of PFAS, elements, and the combined ex vivo receptor transactivities (ER, AR, AhR, and TH). Since many parameters had to be determined in each AF sample, the first selection of samples was based on available samples having adequate volume. AF samples of ASD cases and controls with adequate volume were thus selected and frequency-matched by gender and year of birth. Due to the possible influence of maternal age on ASD, the ASD cases and controls were further matched by maternal age with approximately 1:2 case-control match (Fig. 1).
Changes in concentrations of analytes over prolonged times are a known issue [53 ], and the way the samples were stored pre- and post-1993 was different. Baron-Cohen reported the evidence of evaporation and the concentrations of various analytes of pre-1993 samples were higher than those of post-1993 samples [14 (link)]. In addition, after 1993, the timing of amniocentesis in Denmark was standardized using ultrasound to mark gestational age and diagnostic information after 1993 became much more reliable by switching to ICD-10 [50 (link), 54 (link)]. Therefore, the present study was restricted to individuals born between 1993 and 1999. However, several samples were stored in tubes with blue rubber caps containing cell toxic compounds affecting cell culture growth. Those samples were excluded from the present study. Finally, 75 ASD cases (62 boys, 13 girls) and 135 frequency-matched controls (109 boys, 26 girls) born during 1995–1999 with adequate AF volume were included in the study (Fig. 1).

All statistical analyses were performed using SAS V.9.4 (SAS Institute, Cary, NC, USA). The variables were assessed for normality and log transformed where relevant. Mean differences between sexes for continuous variables were measured by independent samples t-test and analysis of variance; and Pearson χ² test for categorical variables. We performed univariable linear regression analysis to explore the association between explanatory variables and serum 25(OH)D₂, 25(OH)D₃ and total 25(OH)D concentrations. We log transformed 25(OH)D₂, 25(OH)D₃ and 25(OH)D, and expressed these on standardised scales (z-scores). To examine whether sex was an effect modifier of associations, an interaction term (sex × explanatory variable) was additionally included in univariable analyses. We conducted multivariable analyses aiming to examine mutually adjusted associations of different exposures with 25(OH)D₂, 25(OH)D₃ and 25(OH)D measures, namely season of blood sampling (low and high sunlight period), latitude, BMI, waist circumference, SEP, smoking status, alcohol consumption, leisure time computer use, physical activity, diet score and contraception status. In addition, we examined serum 25(OH)D₂, 25(OH)D₃ and 25(OH)D concentrations by excluding women using OCPs.
Following examination of the determinants associated with 25(OH)D₂, 25(OH)D₃ and 25(OH)D concentrations, we performed multinomial ordinal logistic regression analysis to assess the risk factors associated with being in the lower tertile (reference: tertile III) of vitamin D. Owing to equivocal definitions of cut-off values for vitamin D status in the general population, we categorised the analysis sample into tertiles of 25(OH)D. Statistical significance was set at global p<0.05 using two-tailed test.

Cells were lysed on ice for 20 min in a buffer containing 1% Triton X-100, 10 mM Tris (pH 7.6), 50 mM NaCl, 30 mM sodium pyrophosphate, 50 mM NaF, 5 mM EDTA, protease inhibitor cocktail (Roche), and phosphatase inhibitor cocktail (Roche). The resulting lysates were centrifuged at 16,000 × g at 4 °C, and the supernatant fraction was incubated with antibodies against FLAG (Cell Signaling Technology), and HDAC6 (Cell Signaling Technology) for 2 h at 4 °C, followed by incubation with Protein G Plus Agarose (Merck Millipore). Immunoprecipitants were washed two times with the lysis buffer, boiled in 5× SDS loading buffer for 10 min, followed by separation on SDS-PAGE and analysis by western blot. Western blot analysis was performed as described above. For inflammatory IL-1β-induced protein interaction, WT or Parkin^−/− OCPs were cultured for 3 days with 10 ng/ml of IL-1β in the presence of RANKL and M-CSF. Then, the anti-HDAC6 immunoprecipitation was performed.

WT- or Parkin^−/−-derived macrophages (1 × 10⁵ cells) were cultured on dentine slices for 24 h in α-MEM medium containing 30 ng/ml M-CSF and 10 ng/ml RANKL and further incubated for 7 days. The medium was then removed, and 1 M NH₄OH was added to the wells for 30 min. Adherent cells were removed from the dentine slices by ultrasonication; the resorption areas were visualized by staining with 1% toluidine blue. The resorbed pit area on the dentine was then photographed using an image analysis system (NIS-Elements Imaging Software) linked to a light microscope (Nikon). For inflammatory IL-1β-induced OC bone resorption, WT or Parkin^−/− OCPs primed with RANKL on a bone slice for 24 h were further incubated for 7 days in the presence of 10 ng/ml IL-1β, and the resorbed pit area sizes were evaluated, as described above. Resorption pit areas were quantified by ImageJ software.