Auto- and alloimmune reactivity to human islet allografts transplanted into type 1 diabetic patients

Allogeneic islet transplantation can restore an insulin-independent state in C-peptide-negative type 1 diabetic patients. We recently reported three cases of surviving islet allografts that were implanted in type 1 diabetic patients under maintenance immune suppression for a previous kidney graft. The present study compares islet graft-specific cellular auto- and alloreactivity in peripheral blood from those three recipients and from four patients with failing islet allografts measured over a period of 6 months after portal islet implantation. The three cases that remained C-peptide-positive for >1 year exhibited no signs of alloreactivity, and their autoreactivity to islet autoantigens was only marginally increased. In contrast, rapid failure (<3 weeks) in three other cases was accompanied by increases in precursor frequencies of graft-specific alloreactive T-cells; in one of them, the alloreactivity was preceded by a sharply increased autoreactivity to several islet autoantigens. One recipient had a delayed loss of islet graft function (33 weeks); he did not exhibit signs of graft-specific alloimmunity, but developed a delayed increase in autoreactivity. The parallel between metabolic outcome of human beta-cell allografts and cellular auto- and alloreactivity in peripheral blood suggests a causal relationship. The present study therefore demonstrates that T-cell reactivities in peripheral blood can be used to monitor immune mechanisms, which influence survival of beta-cell allografts in diabetic patients.     

Peripheral blood mononuclear cell responses from type 1 diabetic patients and subjects at-risk for type 1 diabetes to human fetal pancreatic tissue proteins

BACKGROUND: Fetal pancreatic tissue has been suggested to be less immunogenic than adult islets. Thus, transplantation of human fetal pancreatic tissue as treatment for type 1 diabetes has been gaining interest. To investigate this question, we tested the peripheral blood mononuclear cell (PBMC) responses from different subject populations to human adult islet proteins (AIP) versus human fetal pancreatic proteins (FPP). METHODS: PBMC responses to FPP and AIP from normal controls (n=14), newly diagnosed type 1 diabetes patients (n=5), long-term type 1 diabetes patients (n=9), and subjects at-risk for development of type 1 diabetes (n=3) were studied. RESULTS: We observed that normal controls demonstrated PBMC reactivity to 0-3 molecular weight regions (mwr) for both the AIP (mean+/-SD, 0.8+/-1.1) and the FPP (0.6+/-0.7). In contrast, newly diagnosed type 1 diabetic patients (<1 year) demonstrated PBMC responses to 9-16 mwr for the AIP (12.8+/-2.5) and 0-14 mwr for the FPP (6.8+/-5.0). The PBMCs from long-term type 1 diabetes patients (> 3 years) were responsive to 2-11 mwr for AIP (6.0+/-2.8) and 0-11 mwr for FPP (4.9+/-4.0). Three nondiabetic ICA positive subjects at-risk for development of type 1 diabetes demonstrated positive PBMC reactivity to 9-18 mwr for the AIP (12.7+/-3.9) and 4-18 mwr for the FPP (10.0+/-5.9). CONCLUSIONS: We conclude that human fetal pancreatic proteins are not significantly less stimulatory than human adult islet proteins to PBMCs of subjects with or at risk for type 1 diabetes.     

Compromised arterial function in human type 2 diabetic patients

Diabetes is associated with a perturbation of signaling pathways in vascular tissue, which causes vasomotor dysfunction such as hypertension and accelerated atherosclerosis. In the present study, the mechanisms of vasomotor dysfunction, Akt (Thr308 and Ser473) phosphorylation and expression of endothelial NO (nitric oxide) synthase, and inducible NO synthase were investigated in human diabetic internal mammary arteries. The phospho-Akt (Thr308) level in arteries from diabetic patients was reduced to about one-half of the level in nondiabetic patients, suggesting impaired insulin signaling in human diabetic vascular tissue. Augmented vasoconstriction was observed in diabetic arteries, due in part to deficiency of basal and stimulated NO production. This correlated with decreased endothelial NO synthase expression and activity in diabetic vessels. The sensitivity of diabetic vessels to the NO donor, sodium nitroprusside, was reduced as well, suggesting that NO breakdown and/or decreased sensitivity of smooth muscle to NO are also responsible for abnormal vasoconstriction. In addition, the abnormal vasoconstriction in diabetic vessels was not completely abolished in the presence of Nomega-nitro-L-arginine methyl ester, revealing that NO-independent mechanisms also contribute to vasomotor dysfunction in diabetes. In conclusion, diabetes downregulates the Akt-signaling pathway and compromises human arterial function through a decrease in NO availability as well as through NO-independent mechanisms.     

Cellular immune response following thermal injury in human patients

Cellular immune response was studied longitudinally for a period of 1 month in a group of 51 patients sustaining burns between 20 and 55 per cent of their body surface area. The results indicated lymphocytopenia and significant depression in the total T cells in all the patients. T cells showed a significant rise in their levels in those patients who showed clinical improvement. Further, the loss of expression of 'E' receptor correlated with the lowering of the protein-synthesizing capacity of the mononuclear cells. The functional integrity of the lymphocytes also showed an inability to recognize and stimulate normal alloantigens in an MLR reaction. Investigation of the mechanisms responsible for immunosuppression revealed the generation of T suppressor activity.     

Islet cell hormonal responses to hypoglycemia after human islet transplantation for type 1 diabetes

Islet transplantation can eliminate severe hypoglycemic episodes in patients with type 1 diabetes; however, whether intrahepatic islets respond appropriately to hypoglycemia after transplantation has not been fully studied. We evaluated six islet transplant recipients, six type 1 diabetic subjects, and seven nondiabetic control subjects using a stepped hyperinsulinemic-hypoglycemic clamp. Also, three islet transplant recipients and the seven control subjects underwent a paired hyperinsulinemic-euglycemic clamp. In response to hypoglycemia, C-peptide was similarly suppressed in islet transplant recipients and control subjects and was not detectable in type 1 diabetic subjects. Glucagon was significantly more suppressed in type 1 diabetic subjects than in islet transplant recipients (P < 0.01), although the glucagon in islet transplant recipients failed to activate as in the control subjects (P < 0.01). Pancreatic polypeptide failed to activate in both type 1 diabetic subjects and islet transplant recipients compared with control subjects (P < 0.01). In islet transplant recipients, glucagon was suppressed normally by hyperinsulinemia during the euglycemic clamp and was significantly greater during the paired hypoglycemic clamp (P < 0.01). These results suggest that after islet transplantation and in response to insulin-induced hypoglycemia, endogenous insulin secretion is appropriately suppressed and glucagon secretion may be partially restored.     

Relationship between islet α - cell function and glomerular filtration rate in type 2 diabetic patients

Objective To analyze the islet α-cell function in type 2 diabetic patients with different levels of glomerular filtration rate(eGFR). Methods Three hundred and eighty-eight cases of type 2 diabetic patients were classified into four groups according to eGFR: glomerular hyperfiltration group, normal renal function group, mild renal dysfunction group and moderate-severe renal dysfunction group. Oral glucose tolerance test, insulin releasing test and glucagon releasing test were conducted to compare the changes of glucagon(GLA), insulin/glucagon ratio（INS/GLA）, glucose/glucagon ratio（GLU/GLA）, the area under the curve of glucagon(AUCGLA) among the groups and correlation analysis were performed among glucagon and glomerular filtration rate and other indicators. Results With the decline of eGFR, the change curve of glucagon level was“J”shaped; the level of fasting glucagon in each group were（82.21±15.06）ng/L,（74.25±15.34）ng/L,（81.16±20.18）ng/L, （100.21 ± 24.73）ng/L, respectively. Compared with normal renal function group, GLA, AUCGLA in glomerular hyperfiltration group and renal dysfunction group increased significantly(P＜0.05), GLU/GLA, INS/GLA decreased significantly. Pearson correlation coefficient analysis showed that fasting glucagon had a negative correlation with eGFR（r＝-0.360,P＜0.01）, plasma albumin (ALB)（r＝ -0.170,P＜0.01）and high - density lipoprotein cholesterol (HDL - C)（r＝-0.128,P＜0.05）, had a positive correlation with fasting venous blood glucose (FPG)（r＝0.339,P＜0.01）, postprandial 2 hours venous blood glucose(2hPG)（r＝0.443,P＜0.01）, the area under the curve of blood glucose (AUCG)（r＝0.475,P＜0.01）, duration（r＝0.257,P＜0.01）and glycosylated hemoglobin(HbA1c) （r＝0.202,P＜0.01）. Multiple stepwise regression analysis showed that fasting glucagon was negatively correlated with eGFR（β ＝-0.290,t＝-5.393,P＜0.01） and HDL - C（β ＝-0.157,t＝ -3.026,P＜0.01）. Conclusions Glucagon level is influenced by eGFR in type 2 diabetic patients. Glucagon in patients with glomerular hyperfiltration or renal dysfunction is significantly higher than those with normal renal function. The inhibition effect of blood glucose and insulin to glucagon are both weakened.     

Nutritional status in type 2 diabetic patients requiring haemodialysis.

BACKGROUND: Type 2 diabetic patients with end-stage renal disease are often overweight (BMI > 24) at the start of dialysis therapy. However, there are very few reports in the literature concerning the nutritional status of these patients after prolonged haemodialysis treatment. Therefore, we compared nutritional parameters in type 2 diabetic patients and age-matched non-diabetic patients after at least 18 months of renal replacement therapy with haemodialysis. METHODS: In a cross-sectional study, we measured BMI, serum albumin, total protein, serum cholesterol and interdialytic weight gain (IWG), and performed a subjective global assessment (SGA) in 14 patients with type 2 diabetes and 16 non-diabetic patients (aged > or = 50 years, haemodialysis therapy > or = 18 months). Protein intake was estimated using the protein catabolic rate (PCR) and Kt/V was calculated to compare the dose of dialysis. RESULTS: BMI was significantly higher in patients with type 2 diabetes (30+/-7 vs 24+/-3, P<0.01). In contrast, the concentration of serum albumin was significantly lower (3180+/-499 mg/dl vs 3576+/-431 mg/dl, P<0.05), but six of the diabetic patients had signs of chronic inflammation. All other nutritional parameters did not differ between the two groups. In addition, there were no significant differences in the intake of protein (PCR 0.93+/-0.19 vs 0.92+/-0.22) and the dose of dialysis (Kt/V 1.13+/-0.19 vs 1.2+/-0.2). CONCLUSION: After > or = 18 months of haemodialysis therapy, the majority of type 2 diabetic patients (9/14) were still overweight (BMI > 24). The nutritional status of diabetic patients was similar to that of age-matched non-diabetic patients on prolonged haemodialysis, but serum albumin levels were significantly lower in diabetics. The lower albumin levels in the diabetic patients may be explained by a state of subclinical chronic inflammation.     

Cellular immune response to phogrin in the NOD mouse: cloned T-cells cause destruction of islet transplants.

The ability of nonobese diabetic (NOD) mice to mount a cellular immune response to the secretory granule protein tyrosine phosphatase (PTP), phogrin was evaluated by immunization of 8- to 12-week-old animals with recombinant phogrin in complete Freund's adjuvant. Draining lymph nodes displayed a robust proliferative response to the protein, as did derived T-cell lines and clones. Ten clones obtained by limiting dilution were all CD4+ and of a T-helper-1-like phenotype, but showed variation in their Vbeta usage. Of the 10 clones, 3 responded to endogenous antigens in rat islets. Two of these caused the destruction of rat islets that had been transplanted under the kidney capsule of streptozotocin-treated NOD scid mice without affecting adjacent thyroid implants. The results demonstrate the feasibility of generating antigen-specific diabetes-inducing CD4+ cells by direct immunization of NOD mice and their potential use for further studies of the antigenic epitopes in the PTP family members. The conclusion, based on serological studies, that PTP members do not play a role in the pathogenesis of type 1 diabetes in rodent models needs reevaluation in light of these findings.     

Bladder dysfunction in type 2 diabetic patients

AIMS: To reevaluate urodynamic findings of bladder dysfunction (BD) in type 2 diabetic patients with patient characteristics and concommittant chronic complications. METHODS: Patients (M/F:27/27) with lower urinary tract symptoms (LUTS) underwent a detailed urodynamic investigation. Urodynamic findings were classified as diabetic cystopathy [DC, characterized by impaired bladder sensation, increased post-void residual urine (PVR) and increased bladder capacity and decreased bladder contractility], detrusor overactivity, bladder outlet obstruction (BOO), urge and stress urinary incontinence or BD in which one of the alterations was included. Glycated hemoglobin (HbA1C), diabetic retinopathy, nephropathy, sensorimotor, and autonomic neuropathies were evaluated. RESULTS: BD was present in 74.07% of men (DC, 50%; BOO, 25%; detrusor overactivity, 25%) and in 59.26% of diabetic women (DC, 43.75%; detrusor overactivity, 31%; urge incontinence, 12.5%; stress urinary incontinence 12.5%). In men, age, duration of diabetes and HbA1C threshold values predicting BD were >64 years, >9 year, >7.9%, while in women, they were >56 years, >8 years, >7%, respectively. Prolongation of QTc, abnormal esophageal transit and gastric emptying times, diabetic retinopathy, and microalbuminuria were associated with an increased risk of PVR >or= 100 ml. CONCLUSIONS: DC was the most frequent finding in patients. Ageing, duration of diabetes, worse metabolic control, PVR 100 ml, cardiac, esophageal and gastric parasympathetic autonomic neuropathies, retinopathy, and microalbuminuria provided a means to predict BD in patients in order to investigate by urodynamics. The establishment of DC in at least 8-9 years after the diagnosis of type 2 DM was an important parameter to inform our diabetic patients.     

Structural alterations to the podocyte are related to proteinuria in type 2 diabetic patients.

BACKGROUND: The podocyte is believed to play a key role in maintaining the integrity of the glomerular filtration barrier, and damage or loss has been linked to the development of albuminuria. METHODS: Renal biopsies from 16 type 2 diabetic patients with nephropathy and 28 non-diabetic controls were analysed using light and electron microscopy. RESULTS: Podocyte number per glomerulus was significantly lower in the type 2 patients compared with controls [mean (95% confidence interval) 464 (382-546) vs 589 (543-635), P = 0.004]. Mean glomerular volume was significantly increased in diabetic patients compared with controls [5.5 (4.9-6.1) vs 3.1 (2.7-3.5) x 10(6) microm(3), P<0.001], thus the diabetic patients demonstrated an even greater proportional reduction in podocyte density per glomerulus [88 (68-108) vs 201 (182-220)/10(6) microm(3), P<0.001]. Podocyte foot process width on both the filtration surface (FPWgbm) and mesangial surface (FPWmes) was significantly increased compared with controls [796 (708-884) vs 556 (460-908) nm, P = 0.001; 1108 (821-1394) vs 760 (555-1078) nm, P = 0.029, respectively]. There was a significant negative correlation between proteinuria and both podocyte number and podocyte density per glomerulus (r = -0.63, P = 0.009; r = -0.58, P = 0.018, respectively). There was a significant positive correlation between proteinuria and both FPWgbm and FPWmes (r = 0.64, P = 0.008, for both). CONCLUSION: Podocyte loss occurs in type 2 diabetic nephropathy and is related to increasing proteinuria. Whether the accompanying glomerular enlargement and widening of foot processes are a cause of podocyte loss is uncertain. Longitudinal studies are required to determine the sequence of events leading to podocyte loss in diabetic nephropathy.     

Pancreatic islet cells antibodies in diabetic patients submitted to pancreas transplantation

Several pieces of evidence suggest an autoimmune etiology of diabetes mellitus type 1. To trace patients who are susceptible to the disease, we utilized islet cells antibodies (ICAs). The aim of this study was to evaluate the presence of ICAs among diabetic patients undergoing simultaneous transplantation of the pancreas and kidney (SPK). Twenty-six diabetic patients received an SPK, 12 of whom were included in this analysis. The indirect immunofluorescence method was utilized for quantitation of ICAs. The types of ICAs were no different following transplantation of the pancreas. The serum levels of pre-existent ICAS in diabetic patients undergoing SPK with immunosuppression were not reduced, and they did not interfere with the function of the implanted pancreas over a period of 60 days.     

Higher urinary IgM excretion in type 2 diabetic nephropathy compared to type 1 diabetic nephropathy.

BACKGROUND: Proteinuria, due to impairment of the charge- and/or size selectivity of the glomerular capillary wall (GCW) is the earliest clinical evidence of diabetic nephropathy (DN). To study the pathophysiological differences between patients with DN in type 1 diabetes mellitus (type 1 DN) and type 2 diabetes mellitus (type 2 DN), we compared the patterns of urinary proteins of different size and charge in the two entities of diabetic kidney disease. METHODS: Urine concentrations of albumin, IgG2, IgG4 and IgM were assessed in 22 (15 males and 7 females) patients with type 1 DN, and in 20 (18 males and 2 females) patients with type 2 DN. Comparisons with one control group of 13 (12 males and one female) patients with nephrosclerosis due to systemic hypertension and a second control group of 16 (14 males and 2 females) healthy controls were made. RESULTS: The urine excretion of IgG2 and IgM and the ratio of IgG2 to IgG4 (IgG2/IgG4), were significantly higher in type 2 DN compared to type 1 DN (P < 0.01). Patients with type 2 DN and patients with nephrosclerosis had significantly higher urine excretion of IgG and IgM compared to the age-matched healthy subjects (P < 0.001). The IgG2/IgG4 ratio was higher in type 2 DN compared to nephrosclerosis and healthy controls (P < 0.01). CONCLUSION: The increased urine excretion of IgG and IgM that accompanies albuminuria in type 2 DN suggests that the dominant pathophysiological mechanism of proteinuria in type 2 DN might be an alteration of the size selective properties of the glomerular capillary wall, including the occurrence of non-discriminatory "shunt pathways." The charge selective properties of the glomerular capillary wall seem to be intact in type 2 DN, as indicated by the high IgG2/IgG4 ratio. The mechanisms of proteinuria in type 1 DN seem to be merely a consequence of an impaired charge selectivity of the glomerular capillary wall.     

Rosiglitazone decreases albuminuria in type 2 diabetic patients

Thiazolidinediones are insulin-sensitizing compounds that reduce plasma glucose and improve the lipid profile of type 2 diabetic patients. We determined the effect of rosiglitazone in 15 type 2 diabetic patients and compared these results to 14 randomly assigned placebo patients. After 3 months, the urinary albumin to creatinine ratio was significantly decreased, while the glucose metabolic clearance rate, during insulin clamp, was significantly increased by rosiglitazone compared to the placebo group. Fasting free fatty acid and tumor necrosis factor-alpha (TNF-alpha) levels were significantly decreased, while the adiponectin concentration was significantly increased by rosiglitazone treatment. The percentage decrease in albuminuria correlated with the decrease in fasting plasma glucose, free fatty acids TNF-alpha and the increase in fat mass, plasma adiponectin, and glucose metabolic clearance rate. Stepwise linear regression analysis showed the decrease in TNF-alpha and the increase in adiponectin were independently associated with decreased albuminuria. Our study indicates that thiazolidinediones may be useful to prevent nephropathy in type 2 diabetic patients.