Long-acting PEGylated recombinant human growth hormone (Jintrolong) for children with growth hormone deficiency: phase II and phase III multicenter, randomized studies.

OBJECTIVE: We assessed the efficacy and safety of a weekly pegylated human growth hormone (PEG-rhGH) (Jintrolong) vs daily rhGH for children with growth hormone deficiency (GHD). DESIGN: Phase II and III, multicenter, open-label, randomized controlled trials. METHODS: 108 and 343 children with treatment-naive GHD from 6 hospitals in China were enrolled in the phase II and III studies respectively. Patients in the phase II study were randomized 1:1:1 to weekly Jintrolong (0.1 mg/kg/week PEG-rhGH complex), weekly Jintrolong (0.2 mg/kg/week PEG-rhGH complex) or daily rhGH (0.25 mg/kg/week) for 25 weeks. Patients in the phase III study were randomized in a 2:1 ratio to weekly Jintrolong (0.2 mg/kg/week) or daily rhGH (0.25 mg/kg/week) for 25 weeks. The primary endpoint for both studies was height velocity (HV) increase at the end of treatment. Other growth-related parameters, safety and compliance were also monitored. RESULTS: The phase II study established the preliminary efficacy, safety and recommended dose of Jintrolong PEG-rhGH. In the phase III study, we demonstrated significantly greater HV increases in patients receiving Jintrolong treatment (from 2.26 ± 0.87 cm/year to 13.41 ± 3.72 cm/year) vs daily rhGH (from 2.25 ± 0.82 cm/year to 12.55 ± 2.99 cm/year) at the end of treatment (P < 0.05). Additionally, significantly greater improvement in the height standard deviation scores was associated with Jintrolong throughout the treatment (P < 0.05). Adverse event rates and treatment compliance were comparable between the two groups. CONCLUSION: Jintrolong PEG-rhGH at a dose of 0.2 mg/kg/week for 25 weeks is effective and safe for GHD treatment and is non-inferior to daily rhGH.

Sulfonylurea in the treatment of neonatal diabetes mellitus children with heterogeneous genetic backgrounds.

OBJECTIVE: The pathogenic base of neonatal diabetes mellitus (NDM) is highly heterogeneous. Sulfonylurea (SU) has been successfully applied in majority of NDM patients with KATP channel mutations; however, its rationality and effectiveness among patients with NDM stemmed from other genetic mutations have not been established. The objective of the present study was to investigate the effectiveness of SU therapy in NDM patients with heterogeneous genetic backgrounds. METHODS: We identified 16 patients with NDM. These patients underwent SU titration and were followed after successful SU monotherapy. All patients were sequenced for all exons and adjacent intron-exon junctions of ABCC8, KCNJ11, and INS, and analyzed for 6q24 methylation defects. SU regimens were applied and glycemic status was evaluated in each patient. RESULTS: Of the 16 patients, 15 (94%) reached glycemic goal (7-10 mmol/L) after SU monotherapy except one patient with the INS mutation. No significant side effects or organ damage were found in any of the 16 patients. Among these patients, five were found to harbor ABCC8 mutations, another five had mutations in KCNJ11, two had INS gene mutations, one with 6q24 hypomethylation, and three were absent for defects in genes tested. CONCLUSION: Our study showed that SU monotherapy resulted in satisfactory glycemic control in most of the patients with NDM whose genetic defects are heterogeneous. The usage of SU may be considered as first-line therapy for patients with NDM in developing countries where effective genetic screening is not established.

Clinical and biochemical consequences of an intragenic growth hormone receptor (GHR) deletion in a large Chinese pedigree.

OBJECTIVE: Growth hormone insensitivity (GHI) may be caused by failure of GH receptor function. Some patients bearing specific GHR mutations differ from classical GHI individuals by extremely elevated GH-binding protein (GHBP) serum concentrations. We investigated clinical, genetic and biochemical characteristics of a severely growth-retarded Chinese boy with classical Laron syndrome manifestations. PATIENTS AND MEASUREMENTS: DNA and mRNA from blood cells of the patient and 11 family members were investigated for GHR mutations. Basal GH, GHBP, IGF-1 and IGFBP-3 concentrations were determined in serum samples. The impact of the aberrant mRNA on GHR protein expression and secretion was analysed in vitro by transfection studies in HEK293 cells. RESULTS: The proband and seven relatives had excessively elevated GHBP serum concentration. Basal GH in these individuals was significantly greater compared with family members with normal GHBP. The GHBP increase originated from a novel GHR intragenic deletion comprising parts of exon and intron 8 that caused exon 8 skipping from the GHR mRNA transcript. Transfection studies revealed that the predicted loss of plasma membrane anchorage results in direct secretion of the mutant GHR. CONCLUSIONS: The partial GHR deletion causes excessively elevated GHBP serum concentrations regardless of the state of zygosity of the mutation. The increase in GHBP is associated with significantly elevated basal GH levels. Clinically, only homozygous carriers exhibit classical GHI manifestations. The truncated GHR protein resulting from exon 8 skipping is directly secreted out of the cell.

SNPs in the exons of Toll-like receptors are associated with susceptibility to type 1 diabetes in Chinese population.

OBJECTIVE: Toll-like receptors (TLRs) recognize a wide range of pathogen-associated molecular patterns (PAMP) and mount the initiation of immune response. Single nucleotide polymorphisms (SNPs) in exons of genes encoding TLRs might be responsible for the generation of an abnormal immune response which could lead to autoimmune diseases. In this study, we investigated the SNPs in TLRs in a Chinese population, and we hypothesized that SNPs in TLRs are associated with type 1 diabetes (T1D), an autoimmune disease caused by destruction of insulin producing pancreatic ß-cells, in the studied population. RESEARCH DESIGN AND METHODS: We selected 28 SNPs in exons of TLRs with an aim to identify those that might have a direct correlation with T1D etiology and many have not been included in previous GWAS studies. Genotyping of those SNPs in TLRs was performed in 429 T1D patients and 300 age and gender-matched healthy controls in Chinese Han population which was not included in the earlier GWAS studies. RESULTS: Among the SNPs genotyped, the T allele of TLR1-626 was positively associated with T1D (OR=1.98, Pc=0.01). We identified another T1D association locus in TLR6: the homozygous AA genotype of TLR6-1329 was negatively and heterozygous GA was positively associated with T1D (OR=0.54, Pc=0.02 and OR=1.70, Pc=0.03, respectively). We also identified the haplotype T-G-A in TLR1 gene to be positively associated with T1D (OR=2.22, Pc=0.03). Additional haplotypes in TLR-6 also showed significant positive and negative association. In addition, our haplotype analysis and conditional analysis showed that these two SNPs are the primary T1D associated loci among the SNPs tested in our cohort in each TLR gene. CONCLUSION: SNPs and haplotypes in TLR1 and TLR6 gene were associated with T1D in Chinese Han population. Our study, for the first time, indicates that TLR1 and TLR6 gene might play important roles in the etiology of T1D.

Rapidly rising incidence of childhood type 1 diabetes in Chinese population: epidemiology in Shanghai during 1997-2011.

The aim of this study was to investigate incidence trend of childhood type 1 diabetes in Shanghai, a megalopolis in east China. We established a population-based retrospective registry for the disease in the city's registered population during 1997-2011 and collected 622 incident type 1 diabetes in children aged 0-14 years. Standardized incidence rates and 95 % CI were estimated by applying the capture-recapture method and assuming Poisson distribution. Incidence trend was analyzed using the Poisson regression model. The mean annual incidence of childhood type 1 diabetes was 3.1 per 100,000 person-years. We did not observe significant difference in incidence between boys and girls. The incidence is unstable and had a mean annual increase 14.2 % per year during the studied period. A faster annual increase was observed in boys, warmer seasons, and in the outer regions of the city. If present trends continue, the number of new type 1 diabetes cases will double from 2016 to 2020, and prevalent cases will sextuple by 2025. Our results showed the incidence of childhood type 1 diabetes was rising rapidly in Shanghai. More studies are needed to analyze incidence changes in other regions of China for appropriate allocation of healthcare resources.

[Protective effect of cotransfection of A20 and HO-1 gene against the apoptosis induced by TNF-α in rat islets in vitro].

OBJECTIVE: To establish the method for cotransferring human A20 gene and human heme oxygenase-1 (HO-1) gene into the isolated rat islets using lentiviral transfection system, and to study the protective effect of A20 and HO-1 protein against the apoptosis induced by cycloheximide (CHX) and TNF-α, and finally to explore the underlying mechanism. METHOD: The A20 gene and HO-1 gene were cloned and inserted into the lentiviral transfection system. The efficacy of gene transfer was measured by the intensity of the enhanced green fluorescent protein (EGFP) fluorescence-positive islets. Western blot was applied to verify the expression of the A20 and HO-1 genes. To induce apoptosis in vitro, the isolated islets were treated with CHX+TNF-α, terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) and the fluorescence-activated cell sorting (FACS) methods were used to evaluate the apoptosis of the islet cells and Western blot was used to detect caspase-3 activation. RESULT: (1) A20 and HO-1 genes were introduced into the isolated islets by lentiviral transfection, both of the genes were highly expressed in the islets after 96 hours culture detected by Western blot method. (2) The insulin levels in the cell culture medium from A20 and/or HO-1 transgenic islets were significantly higher than that in non-transgenic controls (P < 0.01). (3)After CHX + TNF-alpha treatment, the cell culture medium insulin concentration in the A20 gene transfected group [(93.58 ± 4.12)µg/ml], HO-1 gene transfected group [(88.98 ± 4.77) µg/ml ] and A20/HO-1 co-transfected group [(103.33 ± 3.16) µg/ml] were significantly higher than that in the EGFP group [(9.03 ± 0.65) µg/ml ] and the control group [(8.86 ± 0.38) µg/ml] (P < 0.001). Minimum expression level of the activated caspase-3 was found in the A20/HO-1 co-transfected group. CONCLUSION: The lentiviral gene transfer system was an efficient and stable gene transfer vector, the over-expressed A20 and HO-1 protein delivered via lentivirus could preserve rats' islets function and act against the apoptosis induced by CHX and TNF-α.

[The establishment of "two-step sequential filtration method" on the yield rate of purified islets in rats].

OBJECTIVE: To develop a simple, rapid and reliable method of purifying Sprague-Dawley (SD) rat islets by sequential filtration through two cell strainers of different sizes and to evaluate the efficacy of the method. METHODS: Islets were isolated from 8 to 12-week-old clean grade Sprague-Dawley rat pancreases using the standard collagenase digestion procedure and purified with either the generally used Ficoll density gradient method or the innovative two-step sequential filtration method. The purity and vitality of the isolated islets were visualized and assessed with DTZ and AO/PI staining. Glucose stimulating tests were performed to assay cell activity, and immunohistochemical staining was used to evaluate the synthesis function of islet cells. RESULTS: The yield of islets in the two-step filtration method group was 782±115 IEQ per rat, which was significantly higher than in the conventional Ficoll density gradient method group (598 ± 135 IEQ per rat, P < 0.01). Purity of the isolated islets in the two-step filtration method group was 90%-100% and vitality was over 95%. In the conventional Ficoll density gradient method group, islet purity was 65%-85% and vitality was 85%-95%. With regard to the high-sugar stimulation test in the two-step filtration method group, insulin concentrations in islets cultured for 24 hours were significantly higher than in those that were freshly purified (76.9 ± 6.1 µg/L vs 49.4 ± 3.9 µg/L; P < 0.01). CONCLUSIONS: A two-step sequential filtration method for rat islet purification was developed and the method was simple and reliable, with high islet vitality, purity and yield.

Comparative evaluation of short-term biomarker response to treatment for growth hormone deficiency in Chinese children with growth hormone deficiency born small for or appropriate for gestational age: a randomized phase IV open-label study.

OBJECTIVES: To compare the response between Chinese children with growth hormone deficiency (GHD) born either small for gestational age (SGA) or appropriate for gestational age (AGA) after 4 weeks of recombinant human growth hormone (r-hGH) therapy. METHODS: This was a phase IV, open-label, multicenter, interventional study (NCT01187550). Prepubertal children with GHD received open-label treatment with daily r-hGH (0.033 mg/kg) for 4 weeks. Serum levels of insulin-like growth factor I (IGF-I) and insulin-like growth factor-binding protein 3 (IGFBP3), and metabolic markers (including fasting glucose, insulin, total cholesterol, and homeostasis model assessment of insulin resistance) were assessed at baseline and after 4 weeks of treatment, and were analyzed according to patient subgroup (SGA or AGA). RESULTS: A total of 205 children with GHD (mean age 10.4 years; 175 AGA, 30 SGA) were included in the analysis. Mean baseline serum IGF-I and IGFBP3 standard deviation scores (SDS) across the whole patient population were lower than the population norms (mean values: -2.1 SDS for IGF-I and -1.2 SDS for IGFBP3), with no significant differences between the two patient subgroups. After 4 weeks, IGF-I and IGFBP3 levels increased by 1.0 SDS (p < 0.001) and 0.34 SDS (p < 0.001), respectively, but no significant differences were found between the two patient subgroups for growth-related or metabolic markers. CONCLUSIONS: For children with GHD born SGA, IGF-I and IGFBP3 are short-term biomarkers of responsiveness to treatment with growth hormone, as for children with GHD born AGA.

Killer cell immunoglobulin-like receptor along with HLA-C ligand genes are associated with type 1 diabetes in Chinese Han population.

OBJECTIVE: Killer cell immunoglobulin-like receptor (KIR) genes and their putative ligands human leukocyte antigen (HLA)-C genes have been associated with type 1 diabetes (T1D). We hypothesize that KIR genes and their ligands HLA-C genes are important in T1D aetiology. RESEARCH DESIGN AND METHODS: KIR and HLA-C ligand genotyping was performed in 259 T1D patients and 262 healthy children. RESULTS: No significant difference was observed in the distribution of KIR genes between T1D patients and healthy controls. However, frequency of HLA-C1 gene and HLA-C2 gene (marginal association) was higher in patient group. The combinations 2DL2-/HLA-C1+; 2DL3+/HLA-C1+; 2DS2-/HLAC1+ were positively associated with T1D. The combinations 2DL1+/HLA-C2-; 2DL2-/HLA-C1-; 2DL3+/HLA-C1-; 2DS2-/HLAC1- were found to be negatively associated with T1D. Among the genes we tested, a combination of HLA-C1 and -C2 conferred the strongest association with T1D and the strength of this association was higher than that of HLA-C1 alone. The frequencies of KIR 2DL1, 2DL2 and 2DL3 and HLA-C1 were higher in T1D patients positive for GAD65 autoantibody; frequency of KIR 2DS4 is higher in T1D patients positive for IA-2 autoantibody. The association between KIR/HLA-C gene and autoantibody status was not statistically significant after applying Bonferroni correction. CONCLUSION: In our study of a Han population (East China), we found no direct association of KIR genes with T1D. However, a combination of HLA-C1 and -C2 showed a positive association with T1D. Different combinations of HLA-C and KIR showed positive and negative association with T1D.

The growth hormone receptor (GHR) exon 3 polymorphism and its correlation with metabolic profiles in obese Chinese children.

OBJECTIVE: We investigated the correlation between the growth hormone receptor (GHR) exon 3 polymorphism and the metabolic profiles of Chinese children with obesity. SUBJECTS AND METHODS: A total of 409 obese/overweight children and 206 normal weight children were recruited. Anthropological and biochemical indexes including insulin and lipid profiles were measured. Genomic DNA was extracted from the peripheral blood leukocytes, and the GHR exon 3 polymorphism was genotyped by polymerase chain reaction. Homeostasis model of assessment for insulin resistance index (HOMA-IR) and insulin sensitivity index (ISI) were calculated using the homeostasis model. RESULTS: The frequency of the exon 3-deleted GHR (d3-GHR) polymorphism within the obese group was significantly higher than that of the control group (p < 0.05). Body mass index (BMI), fasting insulin (FIns), HOMA-IR, total cholesterol, and triglycerides were significantly lower in the d3-GHR (d3/d3 and d3/fl) group than in the full-length GHR (fl/fl, fl-GHR) group (p < 0.05). After adjustment for BMI, cholesterol level was still significantly lower and HOMA-IR was marginally lower (p = 0.079) in the d3-GHR obese group. There was no statistically significant difference in BMI, FIns, HOMA-IR, ISI, total cholesterol, or triglyceride levels between the two genotypes in the control group. CONCLUSION: We report that the d3-GHR polymorphism has a significant effect on BMI and the metabolic parameters of Chinese children with obesity. The d3 allele may have a protective effect on the development of metabolic syndrome by increasing insulin sensitivity.

[Lipoprotein lipase gene mutations and the risk of cardiovascular diseases in children with obesity].

OBJECTIVE: To inquire into the relationship between lipoprotein lipase (LPL) gene D9N, N291S and S447X polymorphisms and the development of cardiovascular diseases in children with obesity. METHODS: The polymerase chain reaction (PCR) and restriction fragment length polymorphism (RLFP) techniques were used to detect three common mutations of LPL gene exon D9N, N291S and S447X in 157 obese children and 175 normal controls. Plasma lipid and lipoprotein levels between children with different genotypes were compared. RESULTS: The D9N and N291S gene mutations were not detected in either the obese or the control groups. There were no significant differences in the frequency of S447X gene mutation between the two groups. There were no significant differences in the levels of plasma lipid and lipoprotein between children with S447 and X447 genotypes. CONCLUSIONS: D9N and N291S gene mutations may not be risk factors associated with cardiovascular diseases in children with obesity. S447X gene mutation might not play an important role in the development of cardiovascular diseases in childhood.

[Efficacy and safety of recombinant human growth hormone solution in children with growth hormone deficiency in China: a multicenter trial].

OBJECTIVE: Human growth hormone (hGH) is an essential therapeutic drug for the treatment of growth hormone (GH) deficiency (GHD). However, the process of dissolving hGH of the powder form is complicated and potentially hazardous. In the present study, we evaluated the efficacy and safety of preparation in the replacement therapy for children with GH deficiency. METHODS: A 12-month randomized, open-label, multicenter trial was conducted in 31 previously untreated children with growth failure secondary to GH deficiency [20 boys and 11 girls, mean age (10.5 +/- 4.1) years]. An recombined human growth hormone (rhGH) solution (Iintropin AQ) was given via subcutaneous injection daily in every evening at a weekly dose of 0.25 mg/kg. The patients were followed up at 3, 6, 9, and 12 months of the treatment, and the course of treatment was 12 months. Body height was measured 3-monthly and height velocity (HV) and mean height standard deviation score (HT SDS) were calculated. Serum Insulin-like growth factor I (IGF-1), Insulin-like growth factor binding protein 3 (IGFBP-3), GH antibodies and safety parameters were assessed at the baseline and at 3-month intervals. Bone age (BA) was assessed at the baseline and the rate of skeletal maturation (DeltaBA/DeltaCA) was calculated after 6 and 12 months of rhGH treatment by a central bone age reader. Moreover, the safety of rhGH solution treatment was assessed. RESULTS: After 12 months of liquid rhGH therapy, growth parameters were significantly increased over baseline. (1) The mean (+/- SD) height increment DeltaHT (cm) was 4.0 +/- 1.3, 7.0 +/- 2.0, 10.3 +/- 2.6 and 12.9 +/- 3.3 after 3, 6, 9, and 12 months of treatment, respectively (P < 0.01), which indicated linear growth after treatment. The GV (cm/years) was 2.7 +/- 0.9 before treatment and increased to 16.0 +/- 5.1, 14.1 +/- 4.0, 13.7 +/- 3.5, and 12.9 +/- 3.3 after treatment, suggesting that catch-up growth was significant after treatment as compared to the pre-treatment status (P < 0.01). Accordingly, post-treatment catch-up growth was obvious, significant differences were observed in HT SDS, which was -4.62 +/- 1.46 at the onset of therapy and increased significantly after the treatment to -3.80 +/- 1.53, -3.28 +/- 1.60, -2.86 +/- 1.75 and -2.47 +/- 1.86, respectively (P < 0.01). The height difference between GH deficient children and unimpaired children of the same age and gender gradually decreased after treatment, which was significantly different from that seen before treatment (P < 0.01). (2) The levels of serum IGF-1 and IGFBP-3 were increased comparably for the treatment. IGF-1 level (microg/L) was 41 +/- 64 at baseline and increased to 179 +/- 155, 202 +/- 141, 156 +/- 155 and 159 +/- 167 after 3, 6, 9, 12 months of treatment. IGFBP-3 level (mg/L) was 1540 +/- 1325 at baseline, and increased to 3891 +/- 1815, 4051 +/- 1308, 3408 +/- 1435 and 3533 +/- 1413, respectively, suggesting that with the increases in height, IGF-1, and IGFBP-3 were significantly activated to relatively high levels by the medication and reached peak values between 3 and 6 months of treatment. The levels of IGF-1 and IGFBP-3 were significantly different before and after treatment (P < 0.01). The IGF-1/IGFBP-3 molar ratio significantly increased during GH therapy (0.143 +/- 0.013 pre-therapy up to 0.240 +/- 0.055 post-therapy, P < 0.01). The IGF-1/IGFBP-3 molar ratio tended to stabilize after 3-month GH therapy. (3) The bone age assessment carried out 6 and 12 months after treatment showed that the bone maturity (DeltaBA/DeltaCA) was 1.01 +/- 0.57 and 1.07 +/- 0.75, respectively, suggesting that there was no speed-up development in the bone age. No severe adverse events were observed during the trial and the most frequent accompanying event was mild hypothyroidism. CONCLUSIONS: rhGH solution (Iintropin AQ) is a safe and effective preparation in the replacement therapy for children with GH deficiency.

Novel BSCL2 gene mutation E189X in Chinese congenital generalized lipodystrophy child with early onset diabetes mellitus.

CONTEXT: Congenital generalized lipodystrophy (CGL) is a rare and heterogeneous disease of autosomal recessive inheritance. Until now, no genetic findings had been reported in Chinese patients with CGL. OBJECTIVE: To analyze Berardinelli-Seip congenital lipodystrophy type 2 (BSCL2) and 1-acylglycerol-3-phosphate O-acyltransferase 2 (AGPAT2) gene variation in a Chinese boy with CGL and his family. DESIGN, SETTING, AND PARTICIPANTS: All exons of BSCL2 and AGPAT2 with adjacent intron-exon junctions were analyzed using direct sequencing. MAIN OUTCOME MEASURES: Sequences of each exon and nearby intron of the BSCL2 and AGPAT2 genes of the family members were compared with the gene bank genomic sequences. RESULTS: DNA sequence analysis of the entire coding regions and surrounding uncoding regions disclosed a novel homozygous G-->T mutation at nucleotide 909 in exon 5 of the BSCL2 gene in the affected child. A heterozygous state of the G-->T mutation of the BSCL2 gene was also found in other family members. This mutation predicts the substitution of glutamic acid at codon 189 by the stop codon (Glu189X or E189X). No variation was found in the AGPAT2 gene. Conclusion E189X is a novel BSCL2 gene mutation that contributes to CGL formation in a family of Chinese origin.

Infant diet and type 1 diabetes in China.

Infant milk and food introduction may be linked to type 1 diabetes risk in high incidence populations. Dietary data through age 12 months was collected for 247 type 1 diabetic cases and 443 controls in China, a low incidence population, to determine if milk and solid food intake differed. Age range at introduction to milk and formulas was similar in cases and controls but solid food introduction more often occurred before age 3 months in cases. Logistic regression analyses showed soy milk formula consumption at 4-6 (OR = 2.0; 95% CI: 1.1-3.4) and 7-12 months of age (OR = 1.5; 95% CI: 1.0-2.1) was associated with a twofold higher risk of type 1 diabetes, while steamed bread consumption (4-6 months, OR = 0.44; 95% CI: 0.28-0.68; 7-12 months, OR = 0.48; 95% CI: 0.34-0.69) and higher SES (4-6 months, OR = 0.55; 95% CI: 0.39-0.78; 7-12 months, OR = 0.57; 95% CI: 0.40-0.83) were negatively associated. Drinking cow's milk at 7-12 months (OR = 0.60; 95% CI: 0.43-0.85) was negatively associated with type 1 diabetes while consuming vegetables at 4-6 months (OR = 1.5; 95% CI: 1.0-2.2) was positively associated. Results suggest that infant milk and solid food intake are associated with type 1 diabetes in China. Prospective studies may determine how these dietary factors impact disease etiology, particularly for at-risk-populations.

Gene therapy of experimental autoimmune thyroiditis mice by in vivo administration of plasmid DNA coding for human interleukin-10.

AIM: To investigate the effect of interleukin-10 (IL-10) gene on experimental autoimmune thyroiditis mice. METHODS: Mice were immunized to induce autoimmune thyroiditis with porcine thyroglobulin (pTg), and thyroids of mice were injected with IL-10 DNA. On d 28 after immunization with pTg, mRNA expression of IL-10 in thyroid glands was detected and thyroid specimens were histopathological studied. RESULTS: The mRNA expression of IL-10 was detected in thyroid glands on d 7 and 14 after injection of IL-10 plasmid DNA or on COS-7 cells 48 h after IL-10 plasmid DNA transfection. In addition, hIL-10 levels in culture media significantly increased 48 h and 72 h after IL-10 plasmid DNA transfection. Infiltration index of lymphocytes (1.1+/-0.4) in thyroids of IL-10-treated mice was significantly lower than that of pcDNA3-null-treated mice (2.2+/-0.5) (P<0.01). Compared with pcDNA3-null control mice, IL-10-treated mice had lower levels of serum IFN-gamma (P<0.01). CONCLUSION: The direct injection of DNA expression vectors encoding IL-10 into thyroid significantly inhibited development of lymphocytic infiltration of thyroid of autoimmune thyroiditis mice, and alleviated the progression of this disease.

Intramuscular injection of interleukin-10 plasmid DNA prevented autoimmune diabetes in mice.

AIM: To investigate the effect of plasmid coding interleukin-10 (IL-10) DNA on the development of autoimmune diabetes induced by multiple low doses of streptozotocin (STZ) in mice. METHODS: Injection of STZ (40 mg/kg, i.p.) was given daily for five consecutive days. pcDNA3-IL-10 plasmid (IL-10-treated group) or pcDNA3-null plasmid (pcDNA3-null-treated group) (100 microg DNA once a day) were injected into skeletal muscles of mice on d 1 and d 14. Blood glucose concentration was measured. After mice were killed on d 28, serum IFN-gamma level was measured by ELISA, and pancreatic IL-1beta and TNF-alpha mRNA expression was detected by semi-quantitative reverse-transcription PCR (RT-PCR). The number of CD4+ and CD8+ lymphocytes from spleen was detected using FACS. In addition, pancreatic histology was measured for determination of insulitis grades. RESULTS: Treatment with pcDNA3-IL-10 resulted in the retention and expression of the vector in skeletal muscle, associated with a considerable elevation in the plasma level of IL-10, which was not observed in pcDNA3-null-treated mice. In IL-10-treated diabetic mice induced by STZ, delay-type hypersensitivity responses were suppressed and the glucose level was greatly lower on d 14, 21, and 28 than pcDNA3-null-treated group (P<0.05 or P<0.01). On d 21 and 28 the incidence of diabetes was 33.3% and 40.0%, respectively, which was markedly lower than that of pcDNA3-null-treated group (P<0.05). In IL-10-treated mice pancreatic IL-1beta and TNF-alpha mRNA expression was depressed, and serum IFN-gamma concentration and the number of spleen CD4+ or CD8+ lymphocytes were decreased on d 28. The insulitis grades of IL-10-treated mice were lower than that of pcDNA3-null-treated group (P<0.01). CONCLUSION: Systemic administration of IL-10 plasmid DNA can alleviate insulitis of experimental autoimmune diabetes in mice and reduce incidence of diabetes.