Development of fulminant Type 1 diabetes with thrombocytopenia after influenza vaccination: a case report.

BACKGROUND: Fulminant Type 1 diabetes was originally reported as idiopathic Type 1 diabetes. Involvement of viral infections in the pathogenesis of fulminant T1D has been suggested, but the development of fulminant Type 1 diabetes after influenza vaccination has not been reported. CASE REPORT: We report a case of fulminant Type 1 diabetes with thrombocytopenia following influenza vaccination. A 54-year-old man was admitted to hospital with hyperglycaemia and diabetic ketosis. Seven days before admission, he received a seasonal influenza vaccine for the prevention of influenza infection. On admission, blood glucose was 29 mmol/L and HbA1c 40 mmol/mol (5.9%). Fasting and 2-h C-peptide immunoreactivity were <0.0333 nmol/L and 0.0999 nmol/L, respectively. Anti-GAD and anti-IA-2 antibodies were negative, so no autoimmunity seemed to participate in the etiology. ELISPOT assay also showed no association with T cell-mediated autoimmunity. HLA genotypes were consistent with susceptibility to fulminant Type 1 diabetes. After the abrupt onset of diabetes, he showed mild thrombocytopenia, which has been observed for approximately 5 years after diabetes development. CONCLUSION: This is the first description of fulminant Type 1 diabetes after influenza vaccination. Our observation raises the possibility that influenza vaccination might trigger this condition via the TLR7 pathway.

A possible association of Pro12Ala polymorphism in peroxisome proliferator-activated receptor gamma2 gene with obesity in native Javanese in Indonesia.

BACKGROUND: Peroxisome proliferators-activated receptor gamma (PPAR gamma) is a nuclear hormone receptor that serves as a master regulator of adipocytes-specific genes contributing to adipocytes differentiation, susceptibility to obesity, and insulin sensitivity. The substitution of proline to alanine at codon 12 of the PPAR gamma2 gene (Pro12Ala polymorphism) is most frequently identified and the associations with diabetes, obesity, and other clinical parameters have been reported and discussed in several ethnic groups. Among native Javanese ethnicity, however, there is no report about this polymorphism. AIMS AND METHODS: Aims of this study were to estimate the allele frequency of the Pro12Ala polymorphism of PPAR gamma2 gene among native Javanese in Indonesia and to investigate the relationship between this polymorphism and obesity or diabetes. This study included 540 native Javanese subjects consisting of 337 diabetic patients and 203 normal glucose tolerance subjects. Both groups included totally 160 obese subjects (body mass index > or = 25 kg/m(2)). PCR-restriction fragment length polymorphism was used for the genotype determination. RESULTS: The allele frequency of Pro12Ala polymorphism in PPAR gamma2 gene among native Javanese is lower than that in other ethnic groups. No association is seen between the Pro12Ala and diabetes (0.01 vs 0.017%, p = 0.404), a trend of the higher BMI was observed in Pro12Ala carriers in nondiabetic subjects, although this association is limited by small numbers. CONCLUSION: In this study, no association is seen between the Pro12Ala polymorphism in PPAR gamma2 gene and diabetes; a weak association with obesity is seen.

T lymphocyte response against pancreatic beta cell antigens in fulminant Type 1 diabetes.

AIMS/HYPOTHESIS: Fulminant Type 1 diabetes is a novel subtype of Type 1 diabetes that involves the abrupt onset of insulin-deficient hyperglycaemia. This subtype appears to be non-autoimmune because of the absence of diabetes-related autoantibodies in the serum, and of insulitis in pancreatic biopsy specimens. The pathogenesis of the disease is still unknown. In this study, we investigated whether T cell autoimmune responses are involved in fulminant Type 1 diabetes. METHODS: Cellular immune responses to beta cell autoantigens were studied by enzyme-linked immunospot (ELISPOT) assay in 13 fulminant Type 1 diabetic patients and 49 autoantibody-positive autoimmune Type 1 diabetic patients. Results were compared with those of 18 Type 2 diabetic patients, six secondary diabetic patients (diabetes due to chronic pancreatitis) and 35 healthy controls. RESULTS: Nine of 13 (69.2%) GAD-reactive Th1 cells, and three of 12 (25%) insulin-B9-23-reactive Th1 cells were identified in fulminant Type 1 diabetic patients by ELISPOT, as in autoantibody-positive Type 1 diabetic patients. Four fulminant Type 1 diabetic patients possessed the highly diabetes-resistant allele DR2, three of whom had GAD-reactive Th1 cells in the periphery. CONCLUSIONS/INTERPRETATION: Peripheral immune reaction was observed in 69.2% of fulminant Type 1 diabetic patients, indicating that autoreactive T cells might contribute, at least in part, to the development of fulminant Type 1 diabetes.

Critical roles of CD30/CD30L interactions in murine autoimmune diabetes.

CD30/CD30L is a member of tumour necrosis factor (TNF) receptor/TNF superfamily and has been implicated in immune-regulation. A genetic study has also suggested a possible implication of CD30 in spontaneous autoimmune diabetes in NOD mice. In this study, we investigated the involvement of CD30/CD30L in the development of diabetes in NOD mice. Flow cytometric analysis showed that CD30 and CD30L were highly expressed on CD4+ or CD8+ T cells in the spleen and pancreatic lymph node of younger NOD mice. In addition, islet-specific CD4+ or CD8+ T cell lines expressed CD30 and CD30L. Administration of a neutralizing anti-CD30L monoclonal antibody (mAb) from 2 to 10 week of age completely suppressed the development of spontaneous diabetes in NOD mice. In addition, the treatment with anti-CD30L mAb also inhibited the development of diabetes induced by adoptive transfer of spleen cells from diabetic NOD mice or islet-specific CD4+ or CD8+ T cell lines into NOD-SCID mice. Furthermore, anti-CD30L mAb inhibited T cell proliferation in response to islet antigens. These results suggested that CD30/CD30L interaction plays important roles in both induction and effector phases of autoimmune diabetes in NOD mice.

[Recent trends of development of rapid-acting and long-acting human insulin analogues--its biological characteristics and clinical availability].

Modern diabetes therapy is positively introduced insulin administration including intensive insulin therapy to type 1 and type 2 diabetes to prevent or delay the development of long-term complications. However, conventional insulin derivatives could not mimic more closely physiological insulin secretion profiles, resulting in frequent hypoglycemia attacks and brittle diabetes. Recent advanced technologies have allowed to modify insulin molecules to obtain several insulin analogues. Lys.Pro insulin and insulin aspart, rapid-acting insulin analogues, have demonstrated improved post-prandial glucose control in comparison with regular insulin, even although they are usually administered immediately prior to the meal. Insulin glargine and SoLongln, long-acting insulin analogues, could preserve the stable basic insulin profiles during a day as compared with NPH insulin. These insulin analogues are very useful to supply the post-prandial and basic insulin secretions in type 1 and type 2 diabetics.

Effect of lactate on the synaptic potential, energy metabolism, calcium homeostasis and extracellular glutamate concentration in the dentate gyrus of the hippocampus from guinea-pig.

Towards understanding the role of glycolysis on synaptic function, we examined the effect of lactate on synaptic potential, energy metabolism, Ca(2+) homeostasis and extracellular glutamate in the dentate gyrus of guinea-pig hippocampus. Postsynaptic population spikes were recorded from the granule cell layer of the dentate gyrus in guinea-pig hippocampal slices after replacing glucose with lactate in the perfusion medium. Population spikes were not maintained and spontaneously recovered around 35min after the replacement of glucose with lactate. However, ATP levels of the dentate gyrus remained unchanged while those during the glucose-free condition decreased to 73% of the initial levels at 60min. Intracellular Ca(2+) was measured with the calcium indicator dye fura-2 AM, and the population spike was recorded simultaneously. Ca(2+) levels increased concomitantly with the early decay of synaptic potentials, and recovered partially with the spontaneous recovery of synaptic potentials. The time course of decay of population spikes and the increase of Ca(2+) levels during lactate replacement were similar to those during glucose deprivation. Increase in Ca(2+) levels during lactate replacement was completely blocked by the ryanodine receptor/calcium release channel antagonist dantrolene. Glutamate was released more significantly in the medium during lactate replacement than with normal Ringer solution, and less than that during glucose deprivation. Addition of the N-methyl-D-aspartate blocker, D-(-)-2-amino-5-phosphonovaleric acid, and the L-type calcium channel blocker, nimodipine, but not dantrolene blocked spontaneous recovery of population spikes. The results indicate that lactate can maintain energy levels in hippocampal slices, but cannot maintain ion homeostasis in granule cells of the dentate gyrus. Glycolysis plays an important role in maintaining ion homeostasis, and activation of N-methyl-D-aspartate and L-type calcium channels is necessary for support of synaptic function by lactate utilization.

Suppression and acceleration of autoimmune diabetes by neutralization of endogenous interleukin-12 in NOD mice.

A corpus of evidence suggests that T-helper type 1 (Th1)-dependent cellular immunity plays a pivotal role in the pathogenesis of autoimmune diabetes. This study was intended to find ways to prevent the development of NOD diabetes using a neutralizing anti-interleukin (IL)-12 antibody (C17.8) that inhibits Thl cell differentiation. When C17.8 was administered from 5 to 30 weeks of age, NOD mice exhibited suppression of both insulitis and diabetes. However, when C17.8 administration ceased at 15 weeks of age, 8 of 13 recipients showed diabetes at 30 weeks of age. These results suggest that IL-12 plays an important role not only in the development of effector cells but also in their activation. In contrast, when C17.8 was injected into 2-week-old female NOD mice for 6 consecutive days, all 16 recipients showed diabetes at 30 weeks of age, whereas 12 of 20 control mice became diabetic. This result suggests that depletion of endogenous IL-12 at a young age results in the enhancement of diabetes. Flow cytometric analysis indicated that activated memory T-cells were present in higher numbers after C17.8 treatment. Transfer of spleen cells from 15-week-old C17.8-treated NOD mice to NOD-scid mice resulted in an earlier onset and a higher incidence of diabetes. Furthermore, administration of C17.8 to 2-week-old NOD mice also resulted in a much earlier onset of diabetes. These results suggest that short-term treatment with anti-IL-12 antibody prohibits IL-2 production at a young age, which may influence the expansion and apoptosis of pathogenic T-cells, resulting in the acceleration of autoimmune diabetes.

Phosphofructokinase, a glycolytic regulatory enzyme has a crucial role for maintenance of synaptic activity in guinea pig hippocampal slices.

To clarify the importance of glucose metabolism in maintaining neural activity, we investigated the effects of lowering the concentration of glucose in perfusion medium on synaptic activity (population spikes, PS) and on the level of high energy phosphates in the region of dentate gyrus (DG), CA3 and CA1 area of hippocampal slices of guinea pig. Further we determined the activity of phosphofructokinase (PFK) and hexokinase (HK) of these areas. Lowering the concentration of glucose from 10 to 5, 4, 3, 1 and 0 (mM) caused a reduction in the PS amplitude. The PS in CA3 and CA1 region decayed and extinguished much faster than that in DG area. The ATP and CrP levels in these three areas were well preserved even after lowering the concentration of glucose to 3mM, and the patterns of reduction showed no difference in these three areas. On the other hand, there were significant differences in the activity of PFK in the DG, CA3 and CA1 areas, in contrast with HK which showed no significant differences. PFK activity was highest in the DG area and followed by CA3 and CA1 regions. The sensitivity in maintaining synaptic activity to lowered glucose concentrations showed good parallels with the activity of PFK in these regions, indicating that non-oxidative glycolytic metabolism regulated by PFK is crucial for the maintenance of synaptic activity

Anaerobic glycolysis is crucial for the maintenance of neural activity in guinea pig hippocampal slices.

To investigate the functional significance of anaerobic and aerobic glycolysis on neural activity and levels of high energy phosphates, we tested the effects of glucose, mannose, fructose lactate, and pyruvate on the maintenance of neural activity and on the levels of ATP and creatine-P (CrP) in hippocampal slices. For an index of neural activity, population spikes (PS) evoked in the granule cell layer were monitored. Immediately after deprivation of glucose, the PS amplitude was gradually reduced and extinguished within 30 mm. Replacement of glucose with either lactate or pyruvate resulted in a decay and loss of PS with a similar time-course as observed during glucose deprivation. However, after the complete loss of neural activity for 10-20 min the PS reappeared and recovered to normal levels. The replacement of glucose with either mannose or fructose resulted in a transient decrease of the PS to 80-70% of the original amplitude in 20 mm, followed by recovery. The time-course of the decrease of PS in the mannose-containing medium was slower than that in the medium containing fructose and the time-course of recovery was faster. ATP and CrP were reduced to 90 and 70% of original level in each slice after glucose deprivation for 30 and 100 mm, respectively. In media containing either lactate, pyruvate, mannose, or fructose, the level of ATP and CrP was maintained at the original level. The anaerobic metabolic rate of glucose, mannose and fructose, determined by the rate of lactate production during complete anoxia, was consistent with the order of the decay and recovery of the PS in mannose and fructose-containing medium. The mode of the transient decay or loss of PS with no apparent reduction in the levels of ATP and CrP in each slice, in the presence of either mannose, fructose or lactate, indicates that anaerobic glycolysis is crucial for the maintenance of PS. The results obtained in this experiment are not in accordance with the reports by Schurr and Fowler in which they showed that lactate can support neural activity, although they did not measure the levels of ATP and CrP in slices. The present experiment disclosed that this discrepancy was due to the difference of slice preparation; namely rapidly prepared slice with shorter period anoxia (1 mm after removal of hippocampal tissue block) gives the results mentioned in the present study whereas slowly prepared slices by vibratome or chopper method with longer period of ischemia (5-20 mm), did not show transient loss of PS after application of lactate. Thus present experiment indicates that glycolytic process is essential for maintaining neural activity for physiological state of slices, if it is admitted to say that rapidly prepared slices is more physiological because they are exposed on shorter period of ischemia, and that more careful attention should be paid for the interpretation of the results of slice experiment according to the method of slice preparation.

[Exogenous lactate sustains synaptic activity and neuronal viability, but fails to induce long-term potentiation (LTP)].

In Alzheimer's disease, brain glucose metabolic ratio decreases, whereas the brain lactate metabolic ratio increases. To investigate possible synaptic dysfunction in Alzheimer's disease, we examined the effects of exogenous glucose deprivation and replacement of glucose with lactate on the synaptic transmission, synaptic plasticity, and the morphological integrity of hippocampal neurons. Synaptic activity was estimated by the amplitude of the population spike (PS) recorded in the granular cell layer in the hippocampal slices from guinea pig and rat. Exogenous glucose deprivation caused the immediate depression of PS. Replacement of glucose with lactate induced transient decrease of PS, followed by spontaneous recovery of synaptic transmission. Neural activity recovered from transient glucose deprivation became resistant to the replacement of glucose with exogenous lactate. Glucose-supported synaptic transmission exhibited approximately 140% enhancement of PS (LTP). However, lactate-supported synaptic activity yielded approximately 110% potentiation of PS. Effects of exogenous glucose and lactate on the cell viability were examined by the propidum iodide uptake and LDH release in the organotypic hippocampal slice cultures. Hippocampal slice cultures incubated in medium containing 10 mM lactate suppressed the cell death during 48 h observation as well as those in the 10-30 mM glucose containing medium. These results indicate that lactate can sustain the neural transmission and support the morphological integrity of hippocampal neurons, but failed to induce LTP, which could at least in part, cause the memory impairment in Alzheimer's disease.

Serum lipids status in patients with diabetic uremia on 10 years of maintenance hemodialysis.

BACKGROUND: Dyslipidemia in patients with diabetic uremic patients remains unclear. We previously reported that lipid abnormalities in diabetic uremia on short-term (3 to 28 months) hemodialysis therapy were more severe than those in nondiabetic uremic patients. The object of this study is to investigate the serum lipid profiles in diabetic uremic patients on 10 years of maintenance hemodialysis treatment. METHODS: Thirty diabetic uremic subjects and 40 age-matched nondiabetic subjects on long-term hemodialysis therapy were selected, and their clinical characteristics and serum concentrations of lipids, apolipoproteins, lecithin cholesterol acyltransferase (LCAT) activity, and apolipoprotein (apo) E phenotype were evaluated. RESULTS: Patients with diabetic uremia had a higher prevalence of macrovascular complications, including ischemic heart diseases and cerebrovascular diseases. The mean levels of serum total cholesterol, triglyceride, and high-density lipoprotein cholesterol remained normal. Nondiabetic uremic patients exhibited a reduction in serum apo A-1 serum apo A-2, serum apo C-2, and LCAT activity and an increase in serum Apo C-3. Diabetic uremic patients showed a further reduction in serum apo A-1, serum apo A-2, serum apo E, and LCAT activity. Frequencies of apo E isoforms were not significantly different between two groups of uremic patients. CONCLUSIONS: These results clearly indicate that lipid abnormalities in diabetic uremic patients on long-term hemodialysis therapy are more enhanced than those in nondiabetic uremic patients, suggesting that diabetic hemodialyzed patients are more prone to increase the individual risk for accelerated atherosclerosis to cause a higher incidence of cardiovascular diseases.

Local expression of immunoregulatory IL-12p40 gene prolonged syngeneic islet graft survival in diabetic NOD mice.

Local production of immunosuppressive cytokines will be one of the most suitable therapeutic strategies against organ-specific autoimmune diabetes. To establish such a new therapy, we constructed recombinant adenoviral vectors with inserted mIL-12p40 (Ad.IL-12p40) and mIL-10 (Ad.IL-10). Sufficient amounts of IL-12p40 and IL-10 were secreted by relevant adenovirus-transfected nonobese diabetic (NOD) islets. Shortly after transfection, 400 NOD islets transfected with Ad.IL-12p40 or Ad.IL-10 were transplanted under the renal capsule of a newly diabetic NOD mouse. NOD mice with IL-12p40-producing islet grafts kept normoglycemia in all of 14 grafted mice for over 4 wk after transplantation. In contrast, NOD mice with IL-10-producing islet grafts became diabetic in all of six grafted mice within 2 wk af-ter transplantation. Reverse transcription-PCR analysis revealed that local production of IL-12p40 led to the decrease of interferon-gamma and the augmentation of transforming growth factor-beta at the graft site. These results suggest that IL-12 plays an important role in the destruction of islet cells at the inflamed site of autoimmunity. Such a local blockade of IL-12 would be a useful gene therapy for human autoimmune diabetes.

Administration of IL-4 prevents autoimmune diabetes but enhances pancreatic insulitis in NOD mice.

The present study demonstrated that the administration of recombinant interleukin-4 (rIL-4) prevented overt diabetes in nonobese diabetic (NOD) mice whose T cells produced relatively low amounts of IL-4. However, massive insulitis was observed in rIL-4-treated NOD mice. The flow cytometric analysis of islet-infiltrating T cells revealed that the number of CD45RBlowCD4+ T cells was significantly increased by in vivo administration of rIL-4. By measuring the cytokine production of splenic T cells after stimulation, it was shown that CD45RBlowCD4+ T cells predominantly produced IL-4 and IL-10 but produced less IL-2 and interferon-gamma (IFN-gamma). A semiquantitative reverse-transcriptase polymerase chain reaction assay revealed a higher expression of IL-4 and IL-10 mRNA and an apparent decrease in IFN-gamma mRNA in the islets of NOD mice which were administered rIL-4. These results suggested that autoreactive CD45RBlowCD4+ T helper 2 (Th2)-like cells which developed following rIL-4 administration were predominant in the infiltrate of the islets, and overt diabetes was prevented. On the other hand, when splenocytes from rIL-4-treated NOD mice were transferred to irradiated NOD recipients, along with splenocytes from diabetic NOD mice, all of the recipient mice became diabetic within 8 weeks after transfer. Considered together, a supplement of rIL-4 administered to NOD mice may protect against autoimmune diabetes by facilitating the development of Th2-like autoreactive T cells in the islets.

Development of an assay for bioactive insulin.

A method to detect the biological activity of serum insulin has been developed. This method, called a bioactive insulin assay, determines the ability of serum insulin to stimulate the autophosphorylation of insulin receptors in an intact cell system. For this, intact Chinese hamster ovary cells which overexpress the human insulin receptor are treated with serum and then lysed. Autophosphorylation of the insulin receptors is then measured by a two-site immunofluorometric assay using monoclonal anti-insulin receptor antibodies and europium-labeled anti-phosphotyrosine antibodies. The detection limit of this assay is 1 microU/ml of insulin. Dilution and recovery test inter- and intraassay coefficient variations are permissible. The amount of insulin determined by this assay correlated well with the amount of insulin detected by a traditional immunological assay for insulin (r = 0.94, P < 0.001). In the case of a mutant insulin, the insulin from a Wakayama subject, the biologically active insulin was found to constitute 9% of the immunologically reactive insulin. Since this assay specifically measures the amount of biologically active insulin present in serum, it should be particularly useful in monitoring active insulin in patients with various mutant insulins.