An axis of genetic heterogeneity in autism is indexed by age at diagnosis and is associated with varying developmental and mental health profiles.

There is growing recognition that earliest signs of autism need not clearly manifest in the first three years of life. To what extent is this variation in developmental trajectories associated with age at autism diagnosis? Does the genetic profile of autism vary with age at autism diagnosis? Using longitudinal data from four birth cohorts, we demonstrate that two different trajectories of socio-emotional behaviours are associated with age at diagnosis. We further demonstrate that the age at autism diagnosis is partly heritable (h2 SNP = 0.12, s.e.m = 0.01), and is associated with two moderately correlated (rg = 0.38, s.e.m = 0.07) autism polygenic factors. One of these factors is associated with earlier diagnosis of autism, lower social and communication abilities in early childhood. The second factor is associated with later autism diagnosis, increased socio-emotional difficulties in adolescence, and has moderate to high positive genetic correlations with Attention-Deficit/Hyperactivity Disorder, mental health conditions, and trauma. Overall, our research identifies an axis of heterogeneity in autism, indexed by age at diagnosis, which partly explains heterogeneity in autism and the profiles of co-occurring neurodevelopmental and mental health profiles. Our findings have important implications for how we conceptualise autism and provide one model to explain some of the diversity within autism.

Polygenic scores for autism are associated with neurite density in adults and children from the general population.

Genetic variants linked to autism are thought to change cognition and behaviour by altering the structure and function of the brain. Although a substantial body of literature has identified structural brain differences in autism, it is unknown whether autism-associated common genetic variants are linked to changes in cortical macro- and micro-structure. We investigated this using neuroimaging and genetic data from adults (UK Biobank, N = 31,748) and children (ABCD, N = 4,928). Using polygenic scores and genetic correlations we observe a robust negative association between common variants for autism and a magnetic resonance imaging derived phenotype for neurite density (intracellular volume fraction) in the general population. This result is consistent across both children and adults, in both the cortex and in white matter tracts, and confirmed using polygenic scores and genetic correlations. There were no sex differences in this association. Mendelian randomisation analyses provide no evidence for a causal relationship between autism and intracellular volume fraction, although this should be revisited using better powered instruments. Overall, this study provides evidence for shared common variant genetics between autism and cortical neurite density.

Ethanol extracts of chickpeas alter the total lipid content and expression levels of genes related to fatty acid metabolism in mouse 3T3-L1 adipocytes.

Desi-type chickpeas, which have long been used as a natural treatment for diabetes, have been reported to lower visceral adiposity, dyslipidemia and insulin resistance induced by a chronic high-fat diet in rats. In this study, in order to examine the effects of chickpeas of this type in an in vitro system, we used the 3T3-L1 mouse cell line, a subclone of Swiss 3T3 cells, which can differentiate into cells with an adipocyte-like phenotype, and we used ethanol extracts of chickpeas (ECP) instead of chickpeas. Treatment of the 3T3-L1 cells with ECP led to a decrease in the lipid content in the cells. The desaturation index, defined as monounsaturated fatty acids (MUFAs)/saturated fatty acids (SFAs), was also decreased by ECP due to an increase in the cellular content of SFAs and a decrease in the content of MUFAs. The decrease in this index may reflect a decreased reaction from SFA to MUFA, which is essential for fat storage. To confirm this hypothesis, we conducted a western blot analysis, which revealed a reduction in the amount of stearoyl-CoA desaturase 1 (SCD1), a key enzyme catalyzing the reaction from SFA to MUFA. We observed simultaneous inactivations of enzymes participating in lipogenesis, i.e., liver kinase B1 (LKB1), acetyl-CoA carboxylase (ACC) and AMPK, by phosphorylation, which may lead to the suppression of reactions from acetyl-CoA to SFA via malonyl-CoA in lipogenesis. We also investigated whether lipolysis is affected by ECP. The amount of carnitine palmitoyltransferase 1 (CPT1), an enzyme important for the oxidation of fatty acids, was increased by ECP treatment. ECP also led to an increase in uncoupling protein 2 (UCP2), reported as a key protein for the oxidation of fatty acids. All of these results obtained regarding lipogenesis and fatty acid metabolism in our in vitro system are consistent with the results previously shown in rats. We also examined the effects on SCD1 and lipid contents of ethanol extracts of Kabuli-type chickpeas, which are used worldwide. The effects were similar, but of much lesser magnitude compared to those of ECP described above. Thus, Desi-type chickpeas may prove to be effective for the treatment of diabetes, as they can alter the lipid content, thus reducing fat storage.

The effect of fibrin on the survival of ischemic skin flaps in rats.

BACKGROUND: Skin flap necrosis is one of the hazards encountered in plastic and reconstructive surgery. Angiogenic agents may be useful for treating it by increasing blood flow. The angiogenic effect of fibrin in vitro has been demonstrated, but little is known about its in vivo effect. Te authors tested the hypothesis that local application of fibrin can improve the survival of ischemic skin flaps. METHODS: A cranially based dorsal skin flap (3 x 7 cm) was made in each rat. Fibrin (8 mg suspended in 400 microl of phosphate-buffered saline) was applied to the subcutaneous side of elevated skin flaps in the experimental group (n = 15), and phosphate-buffered saline alone was delivered in the control group (n = 15). Tissue blood flow of the skin flaps was measured four times (before the operation and on days 1, 3, and 7) at 1, 3, and 5 cm distal to the baseline of the skin flap. The survival rate of the skin flaps was measured on day 7 and histologic assessments were performed. RESULTS: The blood flow change rate at 5 cm in the experimental group was significantly higher than that in the control group on day 7 (60.9 +/- 5.7 percent versus 13.7 +/- 4.8 percent, p < 0.001). The survival rate of skin flaps was also significantly improved in the experimental group (77.0 +/- 2.0 percent) in comparison with the control group (54.7 +/- 2.2 percent, p < 0.01). Histologic analysis showed many more blood vessels in the experimental group in comparison with the control group. CONCLUSION: The local application of fibrin could improve the blood flow and survival of ischemic skin flaps.

Dietary chickpeas reverse visceral adiposity, dyslipidaemia and insulin resistance in rats induced by a chronic high-fat diet.

The improved effects of dietary chickpeas on visceral adiposity, dyslipidaemia and insulin resistance were examined. Rats were fed a normal-fat diet (NFD), a high-fat diet (HFD) or a high-fat plus chickpea diet (HFD+CP) for 8 months. The epididymal fat pad weight v. total body weight of rats was higher in the HFD group (0.032 (sd 0.0042) g/g) than in the NFD group (0.015 (sd 0.0064) g/g) and smaller in the HFD+CP group (0.023 (sd 0.0072) g/g) compared with the HFD group (P < 0.05). Chickpea treatment also induced a favourable plasma lipid profile reflecting decreased TAG, LDL-cholesterol (LDL-C) and LDL-C:HDL-cholesterol levels (P < 0.05). HFD-fed rats had higher TAG concentration in muscle and liver, whereas the addition of chickpeas to the HFD drastically lowered TAG concentration (muscle, 39 %; liver, 23 %). The activities of lipoprotein lipase (LPL) in epididymal adipose tissue and hepatic TAG lipase in liver recorded a 40 and 23 % increase respectively in HFD rats compared with those in NFD rats; dietary chickpeas completely normalised the levels. Furthermore, chickpea-treated obese rats also showed a markedly lower leptin and LPL mRNA content in epididymal adipose tissue. An insulin tolerance test, oral glucose tolerance test and insulin-releasing test showed that chickpeas significantly improved insulin resistance, and prevented postprandial hyperglycaemia and hyperinsulinaemia induced by the chronic HFD. The present findings provide a rational basis for the consumption of chickpeas as a functional food ingredient, which may be beneficial for correcting dyslipidaemia and preventing diabetes.

Hyperglycemia and diabetic renal change in a model of polyvinyl alcohol bioartificial pancreas transplantation.

OBJECTIVES: We have developed a bioartificial pancreas transplantation method using polyvinyl alcohol. Using this model, the relationship between hyperglycemia and parameters that represent renal function was investigated. METHODS: Plasma glucose, 1,5-anhydro-d-glucitol (1,5-AG), and renal factors including plasma urea nitrogen and creatinine levels, urine volume, glucose, and albumin were examined once a week for 9 weeks in diabetic mice with or without transplantation of encapsulated rat islets, and in normal C57BL/6 mice. The mesangial matrix fraction of the glomerulus was measured histologically. The mice were classified into 3 groups according to their mean plasma glucose levels as either severe (n = 17) or mild (n = 23) hyperglycemia or normoglycemia (n = 11). The plasma glucose, renal factors, and mesangial matrix fraction were tested by single and multiple regression analyses. RESULTS: Almost all the renal factors correlated significantly with mean plasma glucose and 1,5-AG levels. The level and duration of hyperglycemia estimated by the area under the curve above basal correlated most significantly with mesangial matrix fraction. CONCLUSIONS: Bioartificial pancreas transplantation significantly reduced the deterioration of renal factors. The 1,5-AG was useful to predict urine albumin loss. The level and duration of hyperglycemia determined the degree of renal damage, which were reduced by bioartificial pancreas transplantation.

Therapeutic angiogenesis by intramuscular injection of fibrin particles into ischaemic hindlimbs.

1. Fibrin gel has been used as a carrier of angiogenic molecules to promote neovascularization in animal models of limb ischaemia. However, little is known about the effects of fibrin itself under such pathological conditions. Accordingly, the present study tested the efficacy of fibrin in a rabbit model of acute hindlimb ischaemia. 2. Unilateral ischaemia was induced by resection of the left femoral artery. Seven days after surgery, fibrin particles (FP), which were free of fibrinogen, thrombin and vascular endothelial growth factor, were injected directly into the ischaemic thigh muscles. Twenty-four rabbits were divided into four groups, namely a control group receiving phosphate-buffered saline and three FP-treated groups receiving 5, 10 or 20 mg FP. 3. Collateral vessel development and limb perfusion were assessed by angiography, measuring the calf blood pressure ratio (BPR), thermographic scanning and the histological determination of capillary density. 4. At day 35 post-surgery, the treatment with 5 mg FP produced an augmentation of collateral vessel development (P < 0.01), increased numbers of capillaries (P < 0.05) and improved perfusion manifested by a higher blood flow (P < 0.01) and calf BPR (P < 0.05) compared with controls. Treatment with 10 and 20 mg FP had similar effects to those observed with 5 mg FP. 5. The present study reveals that FP promotes angiogenesis in a rabbit model of hindlimb ischaemia, thus providing a feasible approach to therapeutic angiogenesis in ischaemic diseases.

Effect of rat-to-mouse bioartificial pancreas xenotransplantation on diabetic renal damage and survival.

OBJECTIVE: Diabetic nephropathy is a life-threatening complication of diabetes mellitus. Bioartificial pancreas transplantation is becoming a therapeutic option for diabetes mellitus as it protects both allogeneic and xenogeneic islets from the host immune system. This study was undertaken to determine the effectiveness of bioartificial pancreas transplantation to improve or prevent diabetic renal damage. METHODS: Approximately 800 rat islets were macroencapsulated in polyvinyl alcohol gel and then transplanted into the peritoneal cavity of diabetic mice (transplantation group [Tx group]). Diabetic mice transplanted with a capsule without islets served as a sham operation group. After transplantation, the following data were collected: survival, body weight, blood glucose, blood urea nitrogen, serum creatinine levels, urinalysis, water intake, and histological changes in the kidney. RESULTS: There was a significant improvement in survival, blood glucose, blood urea nitrogen, and creatinine in the Tx group compared with the sham operation group. No remarkable changes were seen in urinary parameters between the 2 groups, and there was also no significant difference in water intake. Histological examination revealed that mesangial matrix expansion was decreased in the Tx group. CONCLUSIONS: This study demonstrated that polyvinyl alcohol gel bioartificial pancreas transplantation can protect the kidney from diabetic damage.

Cold preservation of islets in UW solution--with special reference to apoptosis.

BACKGROUND: Apoptosis progresses in cultured islets. Little is known with regard to apoptosis under cold preservation. We examined viability and function of islets in University of Wisconsin (UW) solution. MATERIALS AND METHODS: Isolated rat islets were cultured overnight (overnight group) and further treated with 7-day culture in RPMI 1640 medium at 37 degrees C (culture group) or 7-day preservation in UW solution at 4 degrees C (preservation group). They were evaluated by glucose-stimulated insulin secretion test. Apoptosis was examined by TdT-mediated dUTP-biotin nick end-labeling (TUNEL) assay. Expression of caspase mRNA and the ratio of Bax to Bcl-2 were evaluated by reverse-transcriptase polymerase chain reaction (RT-PCR). RESULTS: Islet recovery after 7 days was significantly lower in culture group than in preservation group (44.0 +/- 3.7% versus 75.0 +/- 4.9%, P < 0.05). The stimulation index in the culture group was significantly lower than in the overnight group (2.1 +/- 0.2 versus 4.1 +/- 0.4, P < 0.05). The apoptotic index in the culture group was significantly higher than both in the overnight group and in the preservation group (38.0 +/- 3.0% versus 10.8 +/- 2.0 and 27.0 +/- 4.0%, P < 0.05). Caspase 3, 8, and 9 mRNA in the culture group expressed more than in the other groups. Bax/Bcl-2 in the culture group was significantly lower than in the overnight group (3.2 +/- 0.66 versus 8.1 +/- 0.95, P < 0.05), suggesting that apoptosis had been already destined early after isolation. CONCLUSIONS: The preservation group showed better recovery and function than the culture group. Apoptosis contributed to islet loss under culture and it was significantly suppressed under cold preservation.

Globular adiponectin augments insulin secretion from pancreatic islet beta cells at high glucose concentrations.

Adiponectin plays an important role in improving insulin resistance and preventing atherosclerosis. However it has been rarely reported that adiponectin influences insulin secretion because its receptor was identified in human islet beta cells. In order to investigate the direct effect of adiponectin on pancreatic islet beta cells, we performed an insulin secretion test in purified rat islets, which were incubated with adiponectin (100 ng/mL) at low (3.3 mM) and high (16.7 mM) glucose concentrations. Furthermore, cell lysates were extracted from the adiponectin-treated islets for p-AMPKalpha assay. RTPCR and immunohistochemical examination showed both adiponectin receptor 1 (AdipoR1) and receptor 2 (AdipoR2) were expressed in islet cells and AdipoR1 was predominantly expressed. Insulin secretion was significantly increased in the presence of adiponectin for 6 h at high glucose concentration. Meanwhile, the levels of phosphorylated AMPK increased with adiponectin treatment at high glucose concentrations. It is concluded that adiponectin augments insulin secretion from pancreatic islet beta cells at high glucose concentration through AMPK activation.

Stem cells and regenerative medicine for diabetes mellitus.

A profound knowledge of the development and differentiation of pancreatic tissues, especially islets of Langerhans, is necessary for developing regenerative therapy for severe diabetes mellitus. A recent developmental study showed that PTF-1a is expressed in almost all parts of pancreatic tissues, in addition to PDX-1, a well-known transcription factor that is essential for pancreas development. Another study suggested that alpha cells and beta cells individually, but not sequentially, differentiated from neurogenin-3--expressing precursor cells. Under strong induction of pancreas regeneration, it is likely that pancreatic duct cells dedifferentiate to grow, express PDX-1, and re-differentiate toward other cell types including islet cells. Duct epithelium-like cells can be cultivated from crude pancreatic exocrine cells and can be induced to differentiate toward islet-like cell clusters under some culture conditions. These cell clusters made from murine pancreas have been shown to control hyperglycemia when transplanted into diabetic mice. Liver-derived oval cells and their putative precursor H-CFU-C have been shown to differentiate toward pancreatic cells. Furthermore, extrapancreatic cells contained in bone marrow and amniotic membrane are reported to become insulin-producing cells. However, their exact characterization and relationship between these cell types remain to be elucidated. Our recent study has shown that islet-like cell clusters can be differentiated from mouse embryonic stem cells. Transplantation of these clusters could ameliorate hyperglycemia of STZ-induced diabetic mice without forming teratomas. Interestingly, these cells expressed several genes specific to exocrine pancreatic tissue in addition to islet-related genes, suggesting that stable and efficient differentiation toward certain tissues can only be achieved through a process mimicking normal development of the tissue. Perhaps recent developments in these fields may rapidly lead to an established regenerative therapy for diabetes mellitus.

PVA hydrogel sheet macroencapsulation for the bioartificial pancreas.

We newly developed a sheet-type macroencapsulation device entrapping rat islets from 3% polyvinyl alcohol (PVA) dissolved in Euro-Collins solution containing 10% fetal bovine serum and 5% dimethyl sulfoxide (PVA + EC) using a freezing/thawing technique. The same encapsulation technique but with 3% PVA dissolved only in double-distilled water (PVA) and a culture of free islets were served as controls. After 14-day culture in the CMRL-1066 medium, the islet recovery rate, morphological changes, insulin content, and insulin secretion were evaluated in vitro to prove the feasibility of this method of encapsulation. We also xenotransplanted the device into the peritoneal cavity of diabetic C57BL/6 mice to check its function in vivo. After 1-day culture, the islet recovery rate and insulin content in the PVA group were significantly lower than that in the PVA + EC and free islet groups. After 14-day culture, only the islets in the PVA+EC group maintained a normal morphology and effective insulin secretory response to high glucose while the response was not observed in the PVA group after 1-day culture and no longer observed in the free islets after 7-day culture. After transplantation of rat islets encapsulated in the PVA + EC device to diabetic C57BL/6 mice, nonfasting blood glucose levels showed a rapid decrease from high glucose levels of pre-transplantation, maintaining significantly lower glucose levels during the whole course of study in comparison with the sham-operated group. Our results indicated that this freezing/thawing macroencapsulation technique using 3% PVA + EC was effective for xenotransplantation of islet cells.

The development of new immunoisolatory devices possessing the ability to induce neovascularization.

The transplantation of a bioartificial pancreas has been regarded as a potential method for successful islet transplantation without any immunosuppressive agents. The subcutaneous site is a very attractive site for transplantation of a bioartificial pancreas because of its advantage of an easy operation site. Our group has been reporting that transplantation of a bioartificial pancreas to the subcutaneous site can reverse hyperglycemia in diabetic recipients. Regarding shapes of a bioartificial pancreas, it is believed that a bag form has an advantage because it is easy to prepare a large quantity. Our group previously reported successful transplantation of a bioartificial pancreas in bag form, a mesh-reinforced polyvinyl alcohol bag (MRPB), implanted in the peritoneal cavity. We also reported that the effect of subcutaneous islet transplantation can be greatly improved with prevascularization treatment. In the present study, we attempted to combine MRPB to our protocol of subcutaneous prevascularization. The main problem of this trial is that the procedure of MRPB implantation injures the prevascularized blood vessel networks. To solve this problem, we made a slight alternation in our protocol, and designed new devices on the basis of MRPB. The new devices, possessing the ability to induce neovascularization, were prepared by collagen coating on the surface of MRPB and were implanted with/without different doses of FGF-2 impregnated in gelatin microspheres. When using 5 microg of FGF-2, more blood vessels were observed on the surface of type I/IV collagen-coated MRPB compared with the original MRPB and type I collagen-coated MRPB. Quite a few blood vessels were observed either around the injection site of 50 microg of FGF-2 impregnated in gelatin microspheres alone or around the implantation site of FGF-2-free gelatin microspheres and type I collagen-coated MRPB or type I/IV collagen-coated MRPB. Here we demonstrated that the combination of both FGF-2 impregnated in gelatin microspheres and collagen-coated MRPB could give an effective system of neovascularization suitable for subcutaneous implantation of a bioartificial pancreas.

Subcutaneous transplantation of macroencapsulated porcine pancreatic endocrine cells normalizes hyperglycemia in diabetic mice.

BACKGROUND: The ultimate goal of islet transplantation is the unlimited availability of insulin-secreting cells to be transplanted in a simple procedure that requires no use of immunosuppressive drugs. Immunoisolation of xenogeneic pig islets for transplantation has great potential therapeutic benefits for treatment of diabetes. METHODS: Approximately 4 x 10(6) porcine pancreatic endocrine cells (PEC) isolated from 6-month-old pigs were macroencapsulated in agarose-poly(styrene sulfonic acid) mixed gel and implanted into a prevascularized subcutaneous site in streptozotocin-induced C57BL/6 diabetic mice. Animals receiving an equal number of free porcine PEC were used as controls. After transplantation, nonfasting blood glucose, body weight, intraperitoneal glucose tolerance test, and immunohistologic evaluations were processed. RESULTS: All 10 animals receiving the subcutaneous xenografts of the macroencapsulated porcine PEC normalized hyperglycemia within 5 days after transplantation, maintained the duration of normoglycemia for 24 to 76 days, and gradually gained weight. The subcutaneous xenografts of free porcine PEC could not reverse hyperglycemia. The recipient became hyperglycemic again when the implanted graft was retrieved at day 45 after transplantation. The glucose clearances were significantly ameliorated at day 21 and day 45 after transplantation when compared with those in diabetic mice. The immunohistochemical results revealed an inherent intact structure of the macroencapsulated porcine PEC and positive double-immunofluorescence staining for insulin and glucagon. CONCLUSIONS: Subcutaneous transplantation of macroencapsulated porcine PEC normalized hyperglycemia in diabetic mice. Our results identified a potential for a favorable development of subcutaneous transplantation of porcine PEC as a cure for diabetes.

In vivo functioning and transplantable mature pancreatic islet-like cell clusters differentiated from embryonic stem cell.

INTRODUCTION: Although the differentiation of embryonic stem (ES) cells to islet like clusters using differentiation method without employing gene transfer technique has been recently reported, neither endocrine granules in the cytoplasm nor in vivo function of differentiated islet like clusters has been demonstrated. AIMS: To investigate whether ES cells could be differentiated to mature islet like clusters which show in vivo function after transplantation as well as retain endocrine granules in the cytoplasm by electron microscopic observation. METHODOLOGY: In this experiment, using mouse embryonic stem (mES) cells as a model system for lineage specific differentiation, we tried to differentiate mES cells to pancreatic islet-like cell clusters (PICCs) through a series of treatments (4-step procedure). Differentiated PICCs were analyzed and characterized by various techniques, such as RT-PCR, immunohistochemistry, electron microscopic observation, in vitro static incubation test, and in vivo transplantation to diabetic animals. RESULTS: Differentiated islet-like cell clusters from ES cells using our newly developed method (four-step procedure) showed strong expression of essential specific genes to the endocrine pancreas and also specific genes to the exocrine pancreas demonstrating that these islet-like clusters were mature from the developmental biologic point of view. These differentiated cells clearly revealed many mature insulin secretory granules of pleomorphic shape in the cytoplasm as well as well-developed rough endoplasmic reticulum. In vitro study indicated that differentiated cells retain a potent insulin secretory responsiveness to glucose stimulation. Furthermore, the islet-like cell clusters significantly decreased high blood glucose levels almost to normal levels when grafted to streptozotocin-induced diabetic mice without induction of any teratoma formation after transplantation. CONCLUSION: Our results provide evidence that ES cells could differentiate to functioning and transplantable mature pancreatic islet-like cell clusters using our newly developed differentiation method without employing gene transfer technique. This study may lead to a basis for production of indefinite sources of islets that could be applicable for future clinical trial.

Bioartificial pancreas transplantation at prevascularized intermuscular space: effect of angiogenesis induction on islet survival.

INTRODUCTION: Bioartificial pancreas (BAP) transplantation offers a potential treatment of diabetes mellitus. The optimal site for BAP transplantation has not yet been established. AIM: To monitor the effect of induction of neovascularization at the intermuscular space on islet survival after allogenic transplantation of BAP. METHODOLOGY: Angiogenesis was induced at the intermuscular space of diabetic Lewis rats by implanting a polyethylene terephthalate (PET) mesh bag, which enclosed a collagen sponge and biodegradable gelatin microspheres containing basic fibroblast growth factor. After confirmation of angiogenesis, BAP was prepared by mixing of 5% agarose with approximately 2,800 isolated rat (Sprague-Dawley) islets and transplanted into the prevascularized PET mesh bag. RESULTS: Neovascularization was observed in and around the PET mesh bag within 10 days after implantation as confirmed by macroscopic and microscopic examinations. In the presence of a collagen sponge, new blood vessels penetrated into the PET mesh bag and formed a vascular bed. After transplantation, normoglycemia was achieved in the rats within 3 days and maintained for >35 days. The rats gradually gained body weight, and the results of intravenous glucose tolerance test showed normal patterns of blood glucose clearance 1 month after transplantation. CONCLUSION: It can be concluded that the prevascularized PET mesh bag enabled transplanted BAP to survive and maintain function, thus indicating a potential site for BAP transplantation.

Reversal of diabetes in mice by xenotransplantation of a bioartificial pancreas in a prevascularized subcutaneous site.

BACKGROUND: The subcutaneous site has been regarded as a potential site for a bioartificial pancreas. Transplantation of islets, encapsulated by the development of diverse biocompatible materials and structural designs, can reverse hyperglycemia in diabetic recipients. METHODS: Approximately 750 Sprague-Dawley rat islets macroencapsulated in an agarose/poly (styrene sulfonic acid) mixed gel were implanted into a prevascularized subcutaneous site. The site was constructed by subcutaneous injection of basic fibroblast growth factor (bFGF)-impregnated gelatin microspheres in streptozotocin-induced C57BL/6 diabetic mice. Diabetic mice treated with bFGF-free gelatin microspheres and diabetic mice without any treatment undergoing the same subcutaneous transplantation were used as controls. After transplantation, non-fasting blood glucose, body weight, intraperitoneal glucose tolerance test, and histologic evaluations were processed. RESULTS: All the recipients undergoing the subcutaneous xenograft returned to normoglycemia within 1 week after transplantation. Eight of 10 recipients in the bFGF+ group maintained normoglycemia for a period of 38-101 days and gradually gained increase of body weight. Two of 10 recipients became hyperglycemic again when the grafts were respectively retrieved at days 31 and 63. Intraperitoneal glucose tolerance tests at month 1 and 2 revealed significant ameliorated glucose tolerance but a tendency to reduced glucose tolerance when compared respectively with those of the streptozotocin-induced diabetic mice and normal mice. Histologic examination revealed that islets within the retrieved grafts at days 31 and 63 were viable and intact; no fibrotic overgrowth was present around the surface of grafts. CONCLUSIONS: A successfully prevascularized subcutaneous site could be constructed by a tissue bioengineering approach. Xenotransplantation of the agarose/poly (styrene sulfonic acid) mixed gel-based bioartificial pancreas in the prevascularized subcutaneous site could reverse diabetes in mice.