Pancreatic islet transplantation using vascularised chambers containing nerve growth factor ameliorates hyperglycaemia in diabetic mice.

Intraportal islet transplantation has shown initial promise for the treatment of type 1 diabetes. However, the portal vein site is associated with complications such as thrombosis and hepatic steatosis, leading to transplant failure. The aims of this study were to (1) test the feasibility of an alternative islet transplantation method that utilises a FDA-approved gelatin sponge as a novel islet carrier and (2) assess if exogenous addition of nerve growth factor (NGF) has any additional beneficial effects on graft performance in diabetic mice. Mice were rendered diabetic by a single intraperitoneal injection of streptozotocin. Five hundred syngeneic islets were seeded onto a Gelitaspon((R)) disc in the presence or absence of NGF, and placed into a silicone chamber surrounding the femoral neurovascular pedicle. Islet function was assessed by weekly monitoring of blood glucose levels and an intraperitoneal glucose tolerance test performed at the end of the study. Chambers were harvested for further histological analysis. Four of five mice transplanted with islets seeded onto Gelitaspon with NGF showed a significant reduction in blood glucose levels by 4 weeks after transplantation, and demonstrated a response similar to non-diabetic mice when tested with an intraperitoneal glucose tolerance test. Chamber tissue from this group contained islets with insulin-producing beta cells adjacent to the vascular pedicle. Islets seeded onto Gelitaspon with NGF and sited on femoral vessels using a tissue-engineering chamber offer an alternative method for islet transplantation in diabetic mice. This may have potential as a method for clinical islet transplantation.

Evaluation of ICA512As in combination with other islet cell autoantibodies at the onset of IDDM.

OBJECTIVE: The ICA512 pancreatic islet autoantigen is a putative tyrosine phosphatase that is co-identified with the earlier described 40-kDa autoantigen. We report the frequency of autoantibodies to islet cell antigen 512 (ICA512As) in recent-onset IDDM and compare this with other islet cell autoantibodies, including those to GAD (GADAs), insulin (IAAs), and islet cell cytoplasm (ICAs) identified by immunofluorescence. RESEARCH DESIGN AND METHODS: Sera from 232 children aged between 9 months and 14.9 years collected within 14 days of diagnosis were tested for ICA512As by a radioimmunoprecipitation assay. The results were compared with previously reported data for GADAs (n = 232), IAAs (n = 167), and ICAs (n = 230). RESULTS: The frequency of a positive result for ICA512As in children with newly diagnosed IDDM was 60%. The frequency was greater for children with an age of onset between 5 and 10 years (69%) than for children aged < 5 years (49%) and aged between 10 and 15 years (56%). The frequencies for other autoantibody reactivities were 69% for GADAs, 65% for IAAs, and 70% for ICAs. A combination of positive results for ICA512As, GADAs, and IAAs gave a sensitivity for the diagnosis of childhood IDDM of 95%, which was not significantly increased by a positive result for ICAs (96%). CONCLUSIONS: Our results further establish that positivity in a combination of tests is more valuable for the prediction of IDDM than a result for any single autoantibody and that the age of the patient should be considered when selecting the combination of tests to use.

LIF (AM424), a promising growth factor for the treatment of ALS.

Growth factors are theoretically promising agents for ALS therapy, but have been disappointing in subcutaneous delivery due to either toxicity or lack of major efficacy. Leukaemia inhibitory factor (LIF), was named after its effect on haemopoietic cells, and belongs to a group of cytokines which includes CNTF, IL-6, CT-1, OM and IL-11. All group members use the gp130 signal transducing subunit for intracellular signalling, but show differences in biological effect. In vitro and in vivo studies on axotomy and nerve crush models demonstrate a powerful effect of LIF in the survival of both motor and sensory neurones, while reducing denervation induced muscle atrophy. Its effects in muscle also include stimulating myoblast proliferation in vitro, and up-regulation after muscle injury. LIF will also stimulate muscle regeneration in vivo when applied exogenously after injury. In published studies of both axotomy induced neuronal death and in the Wobbler mouse models LIF is active at doses of 10 microg/kg delivered systemically, well below the expected maximum tolerated dose suggested by primate safety studies. LIF is expressed in low levels by spinal cord neurones with significant up-regulation when the neurones are damaged by BOAA toxin, an excitatory amino acid associated with a form of ALS. This augments other evidence suggesting LIF is a trauma factor playing a role in the injury response of adult neuronal tissue, and may be more effective than related growth factors. Taken together, the data suggests LIF is a physiologically relevant trophic factor with implications in clinical medicine as a therapy for ALS, and a human recombinant form (AM424), entered human clinical trials during 1998.

Immunoreactivity of antimitochondrial autoantibodies in Japanese patients with primary biliary cirrhosis.

The incidence and prevalence of primary biliary cirrhosis show wide geographic differences. The frequency of this disease in Japan is lower than in Northern Europe. To elucidate the immunoreactivity of serum with enzymes of the 2-oxo-acid dehydrogenase complex (2-OADC) and the M2 mitochondrial antigenic complex in Japanese patients, we examined sera from 107 patients with primary biliary cirrhosis from three geographically different regions of Japan. The sera were assayed by immunofluorescence on frozen tissue sections, immunoblotting on bovine heart mitochondria and recombinant E2 subunit of branched chain oxo-acid dehydrogenase complex (BCOADC-E2), ELISA using recombinant E2 subunit of human pyruvate dehydrogenase complex (PDC-E2) and purified porcine 2-oxoglutarate dehydrogenase complex (OGDC), and enzyme inhibition assay using procine PDC and OGDC. Of the 107 sera, 95 (88%) reacted by immunofluorescence, 102 (95%) by immunoblotting with at least one of the M2 autoantigens, although only 78 (73%) reacted with PDC-E2; 72 (67%) by ELISA with PDC-E2; and 81 (76%) with PDC by the enzyme inhibition assay. Thus, the frequency of reactivity with PDC-E2 by all assays was lower for Japanese than the reported frequency for Caucasian patients with primary biliary cirrhosis, whereas the frequency of reactivity by immunoblotting and ELISA against 2-OADC enzymes other than PDC was relatively higher. The relative frequency of reactivity of autoantibodies to the M2 autoantigens was similar for the three different regions of Japan. The different autoantibody profiles for Japanese and Caucasian patients with primary biliary cirrhosis point to immunogenetic and environmental determinants of this disease, which should provide new insights into its autoimmune origins.

Autoimmune cholangitis syndrome with a bias towards primary biliary cirrhosis.

The apparent coexistence of primary biliary cirrhosis (PBC) and autoimmune hepatitis in the same patient raises unresolved problems for nosology and therapy. These are exemplified by a 45-year-old Japanese woman with overlapping clinical, serological and histological features of autoimmune cholangitis and autoimmune hepatitis. The classical serological test for PBC, antimitochondrial antibody (AMA) by immunofluorescence, was atypical. By immunoblotting there was reactivity with one of the enzymes of the 2-oxo-acid dehydrogenase complex (2-OADC) family, now recognized as autoantigens responsible for AMA reactivity. Also there was reactivity by immunofluorescence for antinuclear antibodies (ANA), one showing the typical speckled pattern of anti-Sp-100 and the other the peripheral pattern of antinuclear membrane antibody, both with titres > 10(6). There was also a positive result to the lupus erythematosus (LE) cell test. Treatment with ursodeoxycholic acid was beneficial. Thus while the clinical presentation suggested the overlapping syndrome of autoimmune hepatitis and PBC, PBC eventually proved to be the likely diagnosis. We suggest that apparent cases of overlapping PBC-autoimmune cholangitis-hepatitis syndromes, after detailed testing, will mostly align with PBC.

Antibodies to ICA512/IA-2 in rodent models of IDDM.

Antibodies to ICA512/IA-2 are a well established marker of human IDDM and can be detected prior to and soon after the onset of insulin dependency. The non-obese diabetic (NOD) mouse and the diabetes-prone BB rat develop spontaneous diabetes as a consequence of T-cell mediated autoimmune destruction of islet beta-cells, but the occurrence of autoantibodies is controversial. We tested sera from NOD mice and BB-rats for anti-ICA512 by a radioimmunoprecipitation assay (RIP). In sequential serum samples from 20 NOD mice, of which 15 developed diabetes, low levels of anti-ICA512 were demonstrable. Anti-ICA512 appeared close to the onset of hyperglycaemia and was usually transient. Non-diabetic NOD mice also produced anti-ICA512, but at a later age and at lower levels than the diabetic NOD mice. In a cross-sectional analysis of sera from BB rats, low levels of anti-ICA512 were present in 11/20 (55%) of non-diabetic-diabetes prone (DP) BB rats, 0/4 (0%) of diabetic DP BB rats, and 1/6 (17%) of diabetes-resistant BB rats. Anti-ICA512 was not detected in rats of other strains, including three Sprague-Dawley rats with streptozotocin-induced diabetes. In both NOD mice and BB rats the anti-ICA512 reactivity was directed to the cytoplasmic domain of the protein. The transient appearance of anti-ICA512 close to the onset of diabetes in NOD mice and the loss of these antibodies after diabetes onset is consistent with the occurrence of anti-ICA512 in human IDDM. Thus in both human IDDM and rodent models, anti-ICA512 is a marker of the impending onset of diabetes and disappears after diabetes onset.

Presymptomatic motor neuron loss and reactive astrocytosis in the SOD1 mouse model of amyotrophic lateral sclerosis.

In familial amyotrophic lateral sclerosis (fALS), there is a need to establish more precisely the progression of the disease, particularly whether there is gradual presymptomatic neuronal loss or an abrupt loss coinciding with the symptomatic stage. To elucidate this, we investigated the progression of motor neuron loss through morphological techniques, reactive astrocytosis, and expression of ubiquitin and neurofilament proteins, by immunohistochemistry, in SOD1 G93A mice with a protracted disease course and control mice. Loss of motor neurons in SOD1 G93A mice followed a biphasic progression, with an initial loss at 126 days of age, followed by a gradual loss from onset of symptoms through to end-stage disease. Reactive astrocytosis was first observed at 70 days of age and showed a gradual increase through to end-stage disease. This suggests that there is a need for early detection of fALS cases, and potential therapeutic treatments may be more beneficial if administered at an early stage.

Autoantibodies associated with presymptomatic insulin-dependent diabetes mellitus in women.

Presymptomatic autoantibody markers of insulin-dependent (Type 1) diabetes mellitus (IDDM) are less well characterized in adults than in children. We quantitated anti-GAD, anti-ICA512 and ICA by titration to endpoint and compared frequencies and levels in 139 Finnish women from whom 390 serum samples had been archived during antecedent pregnancies for 10 years before and up to 1 year after diagnosis of diabetes. Also, we compared the autoantibody status in adults with IDDM with that of children with newly diagnosed IDDM. Of the 35 women seropositive for 1 or more autoantibodies, 77% developed IDDM, 11% non-insulin-dependent (Type 2) diabetes mellitus (NIDDM), 9% gestational diabetes mellitus requiring insulin (GDM-ins) and 3% GDM controlled by diet. The frequency of antibodies during the 10-year presymptomatic period was 83% for anti-glutamic acid decarboxylase (GAD), 52% for anti-ICA512 and 41% for islet cell antibodies (ICA) for those who developed IDDM, 25%, 17%, and 0% for NIDDM, 12%, 4%, and 8% for GDM-ins and 1%, 0%, and 1% for GDM-diet. Anti-GAD was found most consistently in early samples; 13 of 15 with a single autoantibody at their first test had anti-GAD. Among those who developed IDDM, the frequency of anti-GAD was constant, anti-ICA512 increased threefold, and ICA increased slightly before diagnosis. Levels of the autoantibodies varied between subjects, but were relatively stable in individual subjects. Comparison of tests on the women, and children after diagnosis of IDDM, showed the frequencies and levels to be the same for anti-GAD but lower for anti-ICA512 and ICA in adults. Our observations show in women the long latency of seropositivity before overt IDDM, the predominance of anti-GAD among these three serological markers, and the presence of these markers in NIDDM presumably representing a NIDDM phase of autoimmune insulitis.