Baculovirus-Mediated Expression of Human 65 kDa and 67 kDa Glutamic Acid Decarboxylases in SF9 Insect Cells and Their Relevance in Diagnosis of Insulin-Dependent Diabetes Mellitus

cDNAs coding for the full-length human 65 and 67 kDa glutamic acid decarboxylases (GAD65 and GAD67) were amplified from pancreas and hippocampus cDNA libraries by polymerase chain reaction, respectively. Both cDNAs were inserted into a baculovirus vector which mediated highly efficient expression of the human GAD65 and GAD67 with histidine-hexapeptides as affinity ligands at their C-termini in Spodoptera frugiperda (Sf9) cells. The recombinant GAD proteins were purified to homogeneity by affinity chromatography using a metal-chelating matrix. The infected Sf9 insect cells expressed the recombinant human GAD65 and GAD67 with natural-like conformations, as confirmed by measurement of their enzyme activities as well as their fully restored autoantigenicities. Immunoprecipitation of metabolically labeled infected Sf9 cells demonstrated the autoantigenic potential of the recombinant GAD proteins. The practicability of using recombinant GAD65 and GAD67 derived from the baculovirus expression system for the development of an immunoassay for the diagnosis of insulin-dependent diabetes mellitus is discussed

Recombinant human preproinsulin expression, purification and reaction with insulin autoantibodies in sera from patients with insulin-dependent diabetes mellitus

A novel prokaryotic expression vector pGEX-6T was designed for high-level expression of recombinant fusion protein with a histidine-hexapeptide and glutathione-S-transferase at its N-terminus and the recombinant human preproinsulin at its C-terminus. Efficiency of expression was investigated in the Escherichia coli strain CAG456. The synthesized protein was sequestered in an insoluble form in inclusion bodies and was purified to homogeneity by one-step affinity chromatography based on the specific complex formation of the histidine-hexapeptide and a chelating matrix which was charged with Ni2+ ions. The antigenic nature of the purified recombinant preproinsulin fusion protein was evaluated by ELISA screening for insulin autoantibodies in selected sera from patients with recent-onset type 1 (insulin-dependent) diabetes mellitus classified by the existence of additional autoantibodies reactive against glutamic acid decarboxylase. 14% of the tested sera (n=43) conttained insulin autoantibodies which strongly recognized the recombinant human preproinsulin. Comparable measurements with both recombinant human preproinsulin and mature insulin suggested that the observed autoantigenicity of preproinsulin was mediated by the C-peptide or/and signal peptide

Association between Antibodies to the MR 67,000 Isoform of Glutamate Decarboxylase (GAD) and Type 1 (Insulin-Dependent) Diabetes Mellitus with Coexisting Autoimmune Polyendocrine Syndrome Type II

By using an immunoprecipitation assay, we analysed reactivity of autoantibodies to human recombinant GAD65 and GAD67 in sera from patients with autoimmune polyendocrine syndrome Type II (APS II) with and without Type 1 (insulin-dependent) diabetes mellitus (IDDM) compared to patients with organ-specific autoimmunity. Overall antibodies to GAD65 were correlated with IDDM in all study groups, whereas GAD67 antibodies were associated with IDDM when APS II coexists. Antibodies to GAD65 and GAD67 were detected in 13 (44.8%) and 7 (24.1%) out of 29 APS II patients with IDDM, but in only 4 (13.8%) and 2 (6.9%) out of 29 APS II patients without IDDM, respectively (p < 0.05). In short-standing IDDM (< 1 year), antibodies to GAD67 were significantly more frequent in patients with APS II (5 of 9 [55.6%] subjects) compared to matched diabetic patients without coexisting polyendocrinopathy (1 of 18 [5.6%] subjects) (p < 0.02). The levels of GAD65 (142 ± 90 AU) and GAD67 antibodies (178 ± 95 AU) were significantly higher in patients with polyglandular disease than in patients with isolated IDDM (91 ± 85 AU and 93 ± 57 AU) (p < 0.02). Interestingly, all 11 GAD67 antibody positive subjects also had GAD65 antibodies (p < 0.0001), and in 10 of 11 anti-GAD67 positive sera the GAD67 antibodies could be blocked by either GAD67 or GAD65, suggesting the presence of cross-reactive autoantibodies. No correlation was observed between GAD antibodies and age, sex or any particular associated autoimmune disease, besides IDDM. GAD antibodies were present in only 1 of 6 (16.7%) patients with APS Type I, in 1 of 26 (3.9%) patients with autoimmune thyroid disease but in none of the patients with Addison's disease (n = 16), pernicious anaemia (n = 7) or normal controls (n = 50). Our data suggest distinct antibody specificities reactive to GAD isoforms in APS II and IDDM, which might reflect different mechanisms of autoimmune response in IDDM with coexisting autoimmune polyendocrine autoimmunity

Interdisciplinary Surveillance of Ocular Melanomas: Experiences in a German Tertiary Centre

Background In contrast to cutaneous melanoma, there are no uniform guidelines regarding surveillance of ocular (uveal, conjunctival) melanomas. A consented standard operating procedure (SOP) by the Netzwerk onkologische Spitzenzentren Germany only exists for conjunctival melanoma but not for uvealmelanoma. Surveillance is partially based on German S3-guidelines for cutaneous melanoma and is carried out by oncologic centres in a multidisciplinary approach. This study aims to evaluate patients' adherence to surveillance programs and whether surveillance recommendations (examinations, intervals) can be realised. Methods Retrospective analysis of all ocular melanoma patients overseen at the University Hospital of Cologne between 2008 and 2019. The study evaluates rates of successful patient integration into a standardized surveillance program and patients ' surveillance adherence, subject to age, gender, primary therapy and tumour entity, respectively. Results 99 patients were included (56 female, 43male), 83 of which had uveal melanomas and 16 conjunctival melanomas. Mean follow-up was 59 months. 81% of patients were integrated into a surveillance program. Surveillance was performed according to published recommendations in 78%. 13% of patients dropped out of the surveillance program after a mean period of 38 months. Conclusions Our data show a growing number of patients in standardized surveillance. However, there are still gaps. There is a need for guidelines specifically designed for ocular melanomas, allowing centres to offer patients an individualized approach

Ocular diseases in metastatic cutaneous melanoma: review of 108 consecutive patients in two German tertiary centers

Purpose To analyze the incidence and spectrum of ocular disease in patients with metastatic cutaneous melanoma. Methods One hundred and eight consecutive patients with metastatic cutaneous melanoma were screened for ocular diseases using standardized eye examination, including measurement of visual acuity and intraocular pressure, slit-lamp examination, funduscopy in mydriasis, and spectral-domain optical coherence tomography (SDOCT) of the retina. Selected cases with atypical findings underwent electrophysiological studies. One patient was examined for hypercortisolism by a dexamethasone suppression test. Results Ocular diseases were found in 65 out of 108 patients (60 %) with metastatic cutaneous melanoma, significantly more often in older patients (p=0.004). Cataract was present in 27 patients (25 %), pseudophakia in 22 patients (20 %), macular disease in 29 patients (28 %), diabetic retinopathy in ten patients (9 %), hypertensive retinal disease in 14 patients (13 %), retinal venous and arterial occlusive disease in three patients (3 %), optic neuropathy in four patients (4 %), and uveitis in one patient (1%). Eight patients (8%) had choroidal or iridal nevi, one patient (1 %) choroidal hemangioma, and one patient (1 %) choroidal metastasis. No patient had periocular neoplastic lesions. Paraneoplastic retinopathy manifesting as acute exudative polymorphous vitelliform maculopathy (AEPVM)-like disease was diagnosed in two patients (2 %) with multifocal central serous chorioretinopathy and development of vitelliform or fibrin-like subretinal deposits in one patient. Conclusions Patients with metastatic cutaneous melanoma reveal ocular diseases with a spectrum similar to the normal population of this age range. Very rarely, uveal metastasis as well as paraneoplastic retinopathy can occur

Prevalence of autoantibodies to the 65- and 67-kD isoforms of glutamate decarboxylase in insulin-dependent diabetes mellitus.

We investigated the presence of autoantibodies to baculovirus-expressed human recombinant 65- and 67-kD isoforms of glutamate decarboxylase (GAD65 and GAD67) in insulin-dependent diabetes mellitus (IDDM). In the immunoprecipitation test using [35S]methionine-labeled GADs antibodies to GAD65 were detected in 13/15 (87%) islet cell antibody (ICA)-positive and in 1/35 (2.9%) ICA-negative first-degree relatives of patients with IDDM, in 6/11 (54.5%) ICA-positive nondiabetic schoolchildren, and in 35/50 (70%) patients with newly diagnosed IDDM. GAD67 antibodies were positive only in five (33%) of the ICA-positive relatives (P < 0.05) and in nine (18%) IDDM patients at onset (P < 0.00001). After onset of IDDM antibodies to GAD65 and GAD67 declined but were still positive in 25 and 9.4% of subjects with long-standing IDDM (> 10 yr). In all study groups antibodies to GAD67 were only detected in GAD65 antibody-positive sera. An immunotrapping enzyme activity assay for GAD65 antibodies was positive in 64/75 (85.3%) of sera that were GAD antibody positive in the immunoprecipitation test (r = 0.870, P < 0.0001). In two (2.7%) sera GAD65 antibodies that block GAD enzyme activity were found. Our data suggest that antibodies to GAD65 but not to GAD67 represent sensitive markers for preclinical and overt IDDM. The immunotrapping assay here described represents a valuable technique for specific and sensitive screening for GAD antibodies