Precision Measurement of Vibrational Quanta in Tritium Hydride.

Saturated absorption measurements of transitions in the (2-0) band of radioactive tritium hydride are performed with the ultrasensitive noise-immune cavity-enhanced optical-heterodyne molecular spectroscopy intracavity absorption technique in the range 1460-1510 nm. The hyperfine structure of rovibrational transitions of tritium hydride, in contrast to that of hydrogen deuteride, exhibits a single isolated hyperfine component, allowing for the accurate determination of hyperfineless rovibrational transition frequencies, resulting in R(0)=203 396 426 692(22) kHz and R(1)=205 380 033 644(21) kHz. This corresponds to an accuracy 3 orders of magnitude better than previous measurements in tritiated hydrogen molecules. Observation of an isolated component in P(1) with reversed signal amplitude contradicts models for line shapes in hydrogen deuteride based on crossover resonances.

Autoantibodies against zinc transporter 8 further stratify the autoantibody-defined risk for type 1 diabetes in a general population of schoolchildren and have distinctive isoform binding patterns in different forms of autoimmune diabetes: results from the Karlsburg Type 1 Diabetes Risk Study.

AIMS: To evaluate the diagnostic relevance of autoantibodies against zinc transporter 8 (ZnT8) in schoolchildren from the general population as well as in people with autoimmune diabetes. METHODS: A total of 137 schoolchildren positive for at least one of the three major diabetes-associated autoantibodies, without diabetes heredity or preselection on HLA typing, from the Karlsburg Type 1 Diabetes Risk Study, as well as 102 people at type 1 diabetes onset, 88 people with latent autoimmune diabetes in adults and 119 people with type 2 diabetes, were analysed for different ZnT8 autoantibody variants. RESULTS: Zinc transporter 8 autoantibody positivity was found in 18% of autoantibody-positive schoolchildren, with a noticeable association with other autoantibodies associated with type 1 diabetes and disease progression. Furthermore, ZnT8 autoantibody positivity was associated with diabetes progression in schoolchildren positive for autoantibodies against insulinoma-associated antigen-2 (IA-2) and, importantly, in seven IA-2 autoantibody-negative schoolchildren. Additionally, ZnT8 autoantibodies were found in 56% of people with type 1 diabetes, predominantly directed against all three ZnT8 variants and comparable to schoolchildren with multiple autoantibodies. In contrast, ZnT8 autoantibodies were detected in 10% of people with latent autoimmune diabetes in adults, none of them with reactivity to all three isoforms. CONCLUSION: Zinc transporter 8 autoantibodies are useful markers for prediction of type 1 diabetes in a general population, further stratifying the risk of progression in autoantibody-positive children. ZnT8 autoantibodies are also important markers in adult-onset diabetes, with a completely different reaction pattern in type 1 diabetes in comparison to latent autoimmune diabetes in adults, and may therefore help to differentiate between the two forms.

Digital laser frequency and intensity stabilization based on the STEMlab platform (originally Red Pitaya).

We report on the development, implementation, and characterization of digital controllers for laser frequency stabilization as well as intensity stabilization and control. Our design is based on the STEMlab (originally Red Pitaya) platform. The presented analog hardware interfaces provide all necessary functionalities for the designated applications and can be integrated in standard 19-in. rack mount units. Printed circuit board layouts are made available as an open-source project (T. Preuschoff et al., https://github.com/TU-Darmstadt-APQ/RedPitaya-Lockbox, 2020 and T. Preuschoff et al., https://github.com/TU-Darmstadt-APQ/RedPitaya-IntStab, 2020). A detailed characterization shows that the bandwidth (1.25 MHz) and the noise performance of the controllers are limited by the STEMlab system and not affected by the supplementary hardware. Frequency stabilization of a diode laser system resulting in a linewidth of 52(1) kHz (FWHM) is demonstrated. Intensity control to the 1 × 10-3 level with sub-microsecond rise and fall times based on an acousto-optic modulator as actuator is achieved.

Precision measurement of the fundamental vibrational frequencies of tritium-bearing hydrogen molecules: T2, DT, HT.

High-resolution coherent Raman spectroscopic measurements of all three tritium-containing molecular hydrogen isotopologues T2, DT and HT were performed to determine the ground electronic state fundamental Q-branch (v = 0 → 1, ΔJ = 0) transition frequencies at accuracies of 0.0005 cm-1. An over hundred-fold improvement in accuracy over previous experiments allows the comparison with the latest ab initio calculations in the framework of non-adiabatic perturbation theory including nonrelativisitic, relativisitic and QED contributions. Excellent agreement is found between experiment and theory, thus providing a verification of the validity of the NAPT-framework for these tritiated species. While the transition frequencies were corrected for ac-Stark shifts, the contributions of non-resonant background as well as quantum interference effects between resonant features in the nonlinear spectroscopy were quantitatively investigated, also leading to corrections to the transition frequencies. Methods of saturated CARS with the observation of Lamb dips, as well as the use of continuous-wave radiation for the Stokes frequency were explored, that might pave the way for future higher-accuracy CARS measurements.

Relativistic and QED Effects in the Fundamental Vibration of T_{2}.

The hydrogen molecule has become a test ground for quantum electrodynamical calculations in molecules. Expanding beyond studies on stable hydrogenic species to the heavier radioactive tritium-bearing molecules, we report on a measurement of the fundamental T_{2} vibrational splitting (v=0→1) for J=0-5 rotational levels. Precision frequency metrology is performed with high-resolution coherent anti-Stokes Raman spectroscopy at an experimental uncertainty of 10-12 MHz, where sub-Doppler saturation features are exploited for the strongest transition. The achieved accuracy corresponds to a 50-fold improvement over a previous measurement, and it allows for the extraction of relativistic and QED contributions to T_{2} transition energies.

Cyclooxygenase-2 contributes to the selective induction of cell death by the endocannabinoid 2-arachidonoyl glycerol in hepatic stellate cells.

The endogenous cannabinoid 2-arachidonoyl glycerol (2-AG) is an anti-fibrotic lipid mediator that induces apoptosis in hepatic stellate cells (HSCs), but not in hepatocytes. However, the exact molecular mechanisms of this selective induction of HSC death are still unresolved. Interestingly, the inducible isoform of cyclooxygenase, COX-2, can metabolize 2-AG to pro-apoptotic prostaglandin glycerol esters (PG-GEs). We analyzed the roles of COX-2 and endocannabinoid-derived PG-GEs in the differential susceptibility of primary activated HSCs and hepatocytes toward 2-AG-induced cell death. HSCs displayed significant COX-2 expression in contrast to hepatocytes. Similar to 2-AG, treatment of HSCs with PGD2-GE dose-dependently induced cell death independently from cannabinoid receptors that was accompanied by PARP- and caspase 3-cleavage. In contrast to 2-AG, PGD2-GE failed to induce significant ROS formation in HSCs, and depletion of membrane cholesterol did not rescue HSCs from PGD2-GE-induced apoptosis. These findings indicate differential engagement of initial intracellular signaling pathways by 2-AG and its COX-2-derived metabolite PGD2-GE, but similar final cell death pathways. Other PG-GEs, such as PGE2-or PGF2α-GE did not induce apoptosis in HSCs. Primary rat hepatocytes were mainly resistant against 2-AG- and PGD2-GE-induced apoptosis. HSCs, but not hepatocytes were able to metabolize 2-AG to PGD2-GE. As a proof of principle, HSCs from COX-2(-/-) mice lacked PDG2-GE production after 2-AG treatment. Accordingly, COX-2(-/-) HSCs were resistant against 2-AG-induced apoptosis. In conclusion, the divergent expression of COX-2 in HSCs and hepatocytes contributes to the different susceptibility of these cell types towards 2-AG-induced cell death due to the generation of pro-apoptotic PGD2-GE by COX-2 in HSCs. Modulation of COX-2-driven metabolization of 2-AG may provide a novel physiological concept allowing the specific targeting of HSCs in liver fibrosis.

Autoantibody-defined risk for Type 1 diabetes mellitus in a general population of schoolchildren: results of the Karlsburg Type 1 Diabetes Risk Study after 18 years.

AIMS: To investigate the occurrence of diabetes-associated autoantibodies and cumulative Type 1 diabetes risk over 18 years in a general population of schoolchildren. METHODS: In the Karlsburg Type 1 Diabetes Risk Study, 11 986 schoolchildren from north-eastern Germany without a family history of diabetes were screened for glutamic acid decarboxylase antibodies, insulinoma-associated antigen-2 antibodies and insulin autoantibodies by radioligand binding assay. Those children found to be autoantibody-positive were invited to follow-up examinations and HLA-DQB1 genotyping, and were followed for progression to Type 1 diabetes. RESULTS: At first follow-up, 119 children had single and 36 children had multiple autoantibodies. Of the multiple autoantibody-positive children, 33 had at least one diabetes-associated HLA-DQB1 allele (*02 and/or *0302). A total of 26 children progressed to Type 1 diabetes, of whom 22 had multiple autoantibodies. The male-to-female ratio of those who progressed to Type 1 diabetes was 1.6. The positive predictive value of multiple autoantibodies was 61.1% compared with only 23.7% for diabetes-associated HLA-DQB1 genotypes among all those who were autoantibody-positive. The cumulative risk was 59.7% after 10 years and 75.1% after 18 years for children with multiple autoantibodies compared with 1.2 and 22.6%, respectively, for children with single autoantibodies (P<0.001). Among the three examined autoantibodies, insulinoma-associated antigen-2 antibodies conferred the highest risk. CONCLUSIONS: The diabetes risk in schoolchildren with multiple autoantibodies was similar to the risk reported in other studies for genetically preselected probands; thus, a combined autoantibody-based screening could effectively identify at-risk individuals from the general population for future intervention trials.

ETS-dependent p16INK4a and p21waf1/cip1 gene expression upon endothelin-1 stimulation in malignant versus and non-malignant proximal tubule cells.

AIM: Cellular senescence, leading to cell death through prevention of regular cell renewal, is associated with the upregulation of the tumor suppressor gene p16(INK4a). While this mechanism has been described as leading to progressive nephron loss, p16(INK4a) upregulation in renal cell carcinoma has been linked to a disease-specific improved patient survival rate. While in both conditions endothelin-1 is also upregulated, the signaling pathway connecting ET-1 to p16(INK4a) has not been characterized until this study. MAIN METHODS: Cell culture, qRT-PCR, Western Blot, immunoprecipitation (IP), proximity ligation assay (PLA), and non-radioactive electrophoretic mobility shift assay (EMSA). KEY FINDINGS: In malignant renal proximal tumor cells (Caki-1), an activation of p16(INK4a) and p21(waf1/cip1) was observed. An increased expression of E-26 transformation-specific (ETS) transcription factors was detectable. Using specific antibodies, a complex formation between ETS1 and extracellular signal-regulated kinase-2 (ERK2) was shown. A further complex partner was Mxi2. EMSA with supershift analysis for ETS1 and Mxi2 indicated the involvement of both factors in the protein-DNA interaction. After specifically blocking the endothelin receptors, ETS1 expression was significantly downregulated. However, the endothelin B receptor dependent downregulation was stronger than that of the A receptor. In contrast, primary proximal tubule cells showed a nuclear decrease after ET-1 stimulation. This indicates that other ETS members may be involved in the observed p16(INK4a) upregulation (as described in the literature). SIGNIFICANCE: ETS1, ERK2 and Mxi2 are important complex partners initiating increased p16(INK4a) and p21w(af1/cip1) activation in renal tumor cells.

Quantitative immunohistochemical examination of the local cellular reactions following implantation of biomaterials.

Examining the biocompatibility of implant materials includes the in vivo investigation of the local tissue response following implantation in experimental animals. By contrast to qualitative and semi-quantitative approaches often used in this field, a quantitative technique would facilitate a more accurate determination and better comparability of different studies. Therefore, this study aimed at evaluating the applicability of the free image analysis software ImageJ for fast, easy and reproducible quantification of the tissue response following implantation of titanium samples in rats with subsequent immunohistochemical examination of peri-implant tissue samples for monocytes and macrophages (ED1) and MHC class II positive antigen presenting cells (OX6). The quantification of positively stained cells in the vicinity of the implant pockets was based on a grid-supported manual count carried out using two ImageJ plugins (CellCounter, Grid) and resulted in a mean coefficient of variation of 13.8% (ED1) and 19.6% (OX6) between different investigators and 10.0% (ED1) and 13.8% (OX6) for repeated counting by the same investigator. In conclusion, ImageJ was found to be suitable for morphometric evaluation of the tissue response following implantation, particularly the analysis of discrete cellular events at the tissue-biomaterial interface. The procedure which was used is described in detail, and its advantages and disadvantages are discussed.

Diabetes Antibody Standardization Program: evaluation of assays for insulin autoantibodies.

AIMS/HYPOTHESIS: Insulin autoantibodies (IAA) are important in type 1 diabetes risk assessment. However, their determination varies more between laboratories than other diabetes autoantibodies. The Diabetes Antibody Standardization Program (DASP) aims to improve and standardise measurement of autoantibodies associated with type 1 diabetes. We report the results of measurement of IAA from DASP workshops in 2002, 2003 and 2005. METHODS: Up to 32 laboratories in 14 countries participated in each workshop. Aliquots of coded sera from 50 patients with newly diagnosed type 1 diabetes and 100 blood donor controls were circulated to participating laboratories. Reported results were analysed using receiver operator characteristic (ROC) curves. We compared concordance of antibody levels by ranking, IAA and insulin antibody (IA) indices and units derived from an IA standard curve. RESULTS: In all three workshops IAA assay performance had improved compared with DASP 2000. The median area under the ROC curve was 0.73 in DASP 2002, 0.78 in 2003 and 0.80 in 2005 (p = 0.0012), and median laboratory-assigned sensitivity was 26% in 2002, 36% in 2003 and 45% in 2005 (p < 0.0001). There was, however, marked variation between assays. The range of AUC was 0.36-0.91 and that of laboratory-assigned sensitivity was 22-57%. Concordance of ranking of patient serum samples was related to AUC (p < 0.001). Using an index related to common IAA and IA-positive or -negative control sera improved the concordance between assays (p < 0.0001). CONCLUSIONS/INTERPRETATION: The overall performance of IAA assays has improved but there is still wide variation between laboratories. Concordance between assays would be improved by the use of a common reference reagent.

Coherent transport of atomic quantum states in a scalable shift register.

We demonstrate the coherent transport of 2D arrays of small ensembles of neutral atoms in a shift register architecture based on 2D arrays of microlenses. We show the scalability of the transport process by presenting the repeated hand over of atoms from site to site. We prove the conservation of coherence during transport, reloading, and a full shift register cycle. This shows that the fundamental shift sequence can be cascaded and thus scaled to complex and versatile 2D architectures for atom-based quantum information processing, quantum simulation, and the investigation of quantum degenerate gases.

In vivo investigation of the inflammatory response against allylamine plasma polymer coated titanium implants in a rat model.

Titanium (Ti) is an established biomaterial for bone replacement. However, facilitation of osteoblast attachment by surface modification with chemical groups could improve the implant performance. Therefore, this study aimed to evaluate the effect of a plasma polymerized allylamine (PPAAm) layer on the local inflammation in a rat model. Three series (RM76AB, RM78AB, RM77AB) of PPAAm-treated Ti plates were prepared using different plasma conditions. Twelve male LEW.1A rats received one plate of each series and one uncoated control plate implanted into the back musculature. After 7, 14 and 56 days, four rats were euthanized to remove the implants with surrounding tissue. Total monocytes/macrophages, tissue macrophages, T-cells and MHC-class-II-positive cells were morphometrically counted. On day 14, the macrophage/monocyte number was significantly higher for the controls than for the PPAAm samples. On day 56, the RM76AB and RM78AB samples had significantly lower numbers than RM77AB and the controls. The same was found for the tissue macrophages. No change over time and no differences between the implants were found for the T-cells. For the number of MHC-class-II-positive cells, a significant decrease was found only for the RM78AB implants between day 14 and day 56. Physico-chemical analysis of the PPAAm implants revealed that the RM77AB implants had the lowest water absorption, the highest nitrogen loss and the lowest oxygen uptake after sonication. These results demonstrate that the PPAAm samples and the controls were comparable regarding local inflammation, and that different plasma conditions lead to variations in the material properties which influence the tissue reaction.

[Indication for CRT]. / Indikationen zur CRT.

Since the first studies on cardiac resynchronization therapy (CRT), the evidence for the benefit of this electrical therapy in heart failure has continuously grown. Thus, CRT has been firmly implemented in current therapy guidelines for heart failure. However, there are distinct differences between the different guidelines published. In addition, indications for CRT are still evolving in certain patient groups. This article aims to give an overview of the current guidelines for CRT and also discusses some of the differences between the different guidelines. It also provides an outlook for potential candidates for CRT in the future where current guidelines do not yet define a clear indication for implantation of such a device.

Diabetes Antibody Standardization Program: evaluation of assays for autoantibodies to glutamic acid decarboxylase and islet antigen-2.

AIMS/HYPOTHESIS: Islet autoantibodies are important in diabetes classification and risk assessment, and as endpoints in observational studies. The Diabetes Autoantibody Standardization Program (DASP) aims to improve and standardise measurement of autoantibodies associated with type 1 diabetes. We report results for glutamic acid decarboxylase autoantibodies (GADA) and islet antigen-2 autoantibodies (IA-2A) from three DASP workshops (2002--2005). METHODS: Up to 60 laboratories in 18 countries participated in each workshop. Participants received coded serum aliquots from 50 patients with newly diagnosed type 1 diabetes (median age 18 years, range 9-35 years) and 100 blood donor controls. Results were analysed using receiver operator characteristic (ROC) curves with sensitivity adjusted to 95% specificity in workshop controls. RESULTS: GADA assays performed well in all three workshops (median area under the ROC curve [AUC] 0.94; interquartile range 0.91-0.95) and performance was similar to DASP 2000. Performance of IA-2A assays improved over the workshop programme. Median AUC was 0.81 (interquartile range 0.79-0.83) in DASP 2002, 0.82 (interquartile range 0.78-0.84) in 2003, and 0.85 (interquartile range 0.82-0.87) in 2005 (p < 0.0001). Performance of GADA ELISA improved between 2002 and 2005, and, in DASP 2005, achieved higher median AUC and adjusted sensitivity than RIA. IA-2A ELISA improved and, in DASP 2005, achieved AUCs equivalent to in-house RIA. Assays using IA-2ic or full length IA-2 clones were more sensitive than those using IA-2bdc, with higher AUC (p = 0.004). CONCLUSIONS/INTERPRETATION: GADA and IA-2A assays perform well in discriminating health and disease. The workshop format highlights systematic differences related to assay method and allows full evaluation of novel methods. The programme of autoantibody workshops in type 1 diabetes provides a model for other autoimmune diseases.

Insulin VNTR I/III genotype is associated with autoantibodies against glutamic acid decarboxylase in newly diagnosed type 1 diabetes.

BACKGROUND: In type 1 diabetes (T1D), the influence of age at diagnosis and of the IDDM1 and IDDM2 genetic susceptibility loci on the profile of beta-cell autoantibodies has been demonstrated. We studied these associations in a group of 92 patients (children, adolescents and adults, aged 2-62 years) with newly diagnosed T1D. METHODS: The prevalence of the HLA-DQB1*02 and *0302 alleles and of the classes of variable number of tandem repeats (VNTR) of the insulin gene (INS), and of beta-cell autoantibodies (GADA, IA-2A, ICA and IAA) was determined. Statistical analysis was performed using linear and logistic regression models. RESULTS: The presence of IAA, IA-2A and ICA, but not of GADA, was negatively associated with age at diagnosis. Younger patients were more likely to have multiple autoantibodies. There was a tendency of a higher prevalence of IAA in patients with the HLA-DQB1*02/0302 genotype or with the DQB1*0302 allele compared to patients lacking these markers. As a novel observation, the INS VNTR I/III genotype was significantly associated with the presence of GADA (OR = 4.79; p = 0.018). CONCLUSION: The association between the INS VNTR I/III genotype and GADA may suggest that in patients with T1D lacking the INS VNTR I/I genotype, the effect of other susceptibility factors prevails, which promotes the development of autoimmunity to beta-cell antigens other than insulin.

Enterovirus RNA sequences in sera of schoolchildren in the general population and their association with type 1-diabetes-associated autoantibodies.

Type 1 diabetes (T1D) is an autoimmune disease linked with genetic factors as well as with environmental triggers, such as virus infections, but the aetiology is still unclear. The authors analysed serum from autoantibody-positive (n=50) and autoantibody-negative (n=50) schoolchildren as well as children newly diagnosed with T1D (n=47; time from diagnosis, median 5 days, interquartile range 1-12 days) for the presence and frequency of enterovirus (EV) and adenovirus sequences. The autoantibody-positive and -negative groups were part of the Karlsburg Type 1 Diabetes Risk Study of a Normal Schoolchild Population, which represents a general population without T1D first-degree relatives. There was no significant seasonality of sampling in any of the three groups investigated. EV RNA sequences were detected in 10 of 50 (20%) autoantibody-positive children and in 17 of 47 (36%) children newly diagnosed with T1D, but only in two of 50 (4%) of the age- and sex-matched controls (P<0.05, P<0.001). Characterization of the EV amplicons by direct sequencing revealed high homology with coxsackievirus B group. For adenovirus we found no data to support an association with T1D. The data support the hypothesis that different enteroviruses may be aetiologically important as a trigger and/or accelerating factor in the process of T1D development.

In insulin-autoantibody-positive children from the general population, antibody affinity identifies those at high and low risk.

AIMS/HYPOTHESIS: Insulin autoantibodies (IAA) precede and predict the onset of type 1 diabetes, but not all children with IAA develop the disease. In affected families, IAA affinity can identify IAA-positive children who are more likely to progress to diabetes. The purpose of this study was to determine whether affinity is a useful marker to stratify type 1 diabetes risk in IAA-positive children from the general population. METHODS: IAA affinity was determined by competitive binding to 125I-insulin with increasing concentrations of cold insulin and with cold proinsulin in sera from 46 IAA-positive children identified in the Karlsburg Type 1 Diabetes Risk Study of a Normal Schoolchild Population in north-eastern Germany. RESULTS: IAA affinity ranged between 5 x 10(6) and 1.2 x 10(11) l/mol. IAA affinity was higher in 24 children who developed multiple islet autoantibodies or diabetes (median 3.5 x 10(9) l/mol; interquartile range [IQR] 2.1x10(9) to 2.1 x 10(10) l/mol) than in 22 children who did not develop multiple islet autoantibodies or diabetes (median 1.3 x 10(8) l/mol; IQR 3.8 x 10(7) to 7.2 x 10(8) l/mol; p<0.0001). Using a threshold of > or = 10(9) l/mol, 22 of the 24 children who developed multiple islet autoantibodies or diabetes were correctly identified by high-affinity IAA and 18 of 22 who did not develop multiple islet autoantibodies or diabetes were correctly identified by low-affinity IAA. IAA affinity was significantly higher in samples with proinsulin reactive IAA (p<0.0001). CONCLUSIONS/INTERPRETATION: IAA affinity measurement provides robust identification of IAA associated with high diabetes risk.

Quantitative evaluation of a monoclonal antibody and its fragment as potential markers for pancreatic beta cell mass.

Antibodies, due to their high specificities and retention, represent potential beta cell imaging agents, however their slow clearance from the blood may preclude their use. Antibody fragments (Fabs) have much higher clearance and if they can be made with similar binding characteristics, would be more efficacious agents. An existing beta cell specific antibody (K14D10) and its Fab were evaluated with a previously developed screening assay. The Fab and the intact immunoglobulin (IgG) had similar affinities (6 - 20 nM), binding sites (300 000 - 700 000 sites/cell), and binding kinetics (t (1/2) = 8 - 18 minutes) for beta cells. However, the cellular specificity was far below the estimated requisite values needed to overcome the very low beta cell mass in the pancreas. The Fab cleared the blood twice as fast as the IgG, but did not preferentially accumulate into pancreas. Thus, generation of Fabs from IgGs with high beta cell binding and blood clearance appears feasible, but in order for molecules to be useful for tracking beta cell mass, antibodies of greater cellular specificity will have to be used.

Dynamic changes of GAD65 autoantibody epitope specificities in individuals at risk of developing type 1 diabetes.

AIMS/HYPOTHESIS: Progression to type 1 diabetes is associated with intramolecular epitope spreading to disease-specific antibody epitopes located in the middle region of glutamic acid decarboxylase 65 (GAD65). METHODS: The relationship between intramolecular epitope spreading of autoantibodies specific to GAD65 in relation to the risk of developing type 1 diabetes was tested in 22 high-risk individuals and 38 low-risk individuals. We determined the conformational epitopes in this longitudinal study by means of competition experiments using recombinant Fab of four GAD65-specific monoclonal antibodies. RESULTS: Sera from high-risk children in the preclinical stage recognise a specific combination of GAD65 antibody epitopes located in the middle and the C-terminus of GAD65. High risk of progressing to disease is associated with the emergence of antibodies specific for conformational epitopes at the N-terminus and the middle region. Binding to already established antibody epitopes located in the middle and at the N-terminus increases and shows a significant relation (p=0.005) with HLA, which confers risk of developing diabetes. CONCLUSIONS/INTERPRETATION: In type 1 diabetes, GAD65 antibodies are initially generated against the middle and C-terminal regions of GAD65. In genetically predisposed subjects the autoimmune response may then undergo intramolecular epitope spreading towards epitopes on the N-terminus and further epitopes located in the middle. These findings clearly demonstrate that the GAD65 autoantibody response in the preclinical stage of type 1 diabetes is dynamic and related to the HLA genotypes that confer risk of diabetes. GAD65-specific Fab should prove useful in predicting progression from islet autoimmunity to clinical onset of type 1 diabetes.

Antibody response to collagen after functional implantation of different polyester vascular prostheses in pigs.

Besides inflammation, specific immune responses are seen also after implantation of biomaterials. The aim was to investigate the humoral response to bovine collagen type I following implantation of various polyester (Dacron) prostheses into pigs. In 24 randomized pigs, the infrarenal aorta was replaced with a segment of collagen-impregnated, woven polyester prosthesis of low, medium, or high porosity. IgG antibodies were detected by immunoassay using native and denatured collagen type I as a target for blood samples taken on day 1 (implantation), 10, 17, 24, 62, and 116. As generally observed, antibodies to native and denatured collagen are of low titer and were significantly correlated with enhanced binding to the denatured form (p < 0.001). The highest overall antibody prevalence to native and denatured collagen was obtained on day 116 with 68% and on day 62 with 59%, respectively. Prostheses with high porosity induced an early immune response on day 10; those with low and medium porosity induced the highest antibody levels later after 2 months. Collagen antibodies neither correlated with serum IgG contents nor with antibodies to the prosthesis polyester matrix. Thus, humoral immune response against implant components may provide a further parameter in describing biocompatibility but also a potential marker that may facilitate monitoring of individual perigraft reaction.