Healing of excisional wound in alloxan induced diabetic sheep: A planimetric and histopathologic study

Healing of skin wound is a multi-factorial and complex process. Proper treatment of diabetic wounds is still a major clinical challenge. Although diabetes mellitus can occur in ruminants, healing of wounds in diabetic ruminants has not yet been investigated. The aim of this study was to evaluate healing of ovine excisional diabetic wound model. Eight 4-month-old Iranian Makoui wethers were equally divided to diabetic and nondiabetic groups. Alloxan monohydrate (60 mg kg-1, IV) was used for diabetes induction. In each wether, an excisional wound was created on the dorsum of the animal. Photographs were taken in distinct times for planimetric evaluation. Wound samples were taken on day 21 post-wounding for histopathologic evaluations of epidermal thickness, number of fibroblasts and number of new blood vessels. The planimetric study showed slightly delay in wound closure of diabetic animals, however, it was not significantly different from nondiabetic wounds (p ≥ 0.05). Furthermore, epidermal thickness, number of fibroblasts and number of blood vessels were significantly lower in diabetic group (p < 0.05). We concluded that healing of excisional diabetic wounds in sheep may be compromised, as seen in other species. However, contraction rate of these wounds may not be delayed due to metabolic features of ruminants and these animals might go under surgeries without any serious concern. However, healing quality of these wounds may be lower than normal wounds

Mechanism Underlying Defective Interferon Gamma-Induced IDO Expression in Non-obese Diabetic Mouse Fibroblasts

Indoleamine 2,3-dioxygenase (IDO) can locally suppress T cell-mediated immune responses. It has been shown that defective self-tolerance in early prediabetic female non-obese diabetic (NOD) mice can be attributed to the impaired interferon-gamma (IFN-γ)- induced IDO expression in dendritic cells of these animals. As IFN-γ can induce IDO in both dendritic cells and fibroblasts, we asked the question of whether there exists a similar defect in IFN-γ-induced IDO expression in NOD mice dermal fibroblasts. To this end, we examined the effect of IFN-γ on expression of IDO and its enzymatic activity in NOD dermal fibroblasts. The results showed that fibroblasts from either prediabetic (8 wks of age) female or male, and diabetic female or male (12 and 24 wks of age respectively) NOD mice failed to express IDO in response to IFN-γ treatment. To find underlying mechanisms, we scrutinized the IFN- γ signaling pathway and investigated expression of other IFN-γ-modulated factors including major histocompatibility complex class I (MHC-I) and type I collagen (COL-I). The findings revealed a defect of signal transducer and activator of transcription 1 (STAT1) phosphorylation in NOD cells relative to that of controls. Furthermore, we found an increase in MHC-I and suppression of COL-I expression in fibroblasts from both NOD and control mice following IFN-γ treatment; indicating that the impaired response to IFN-γ in NOD fibroblasts is specific to IDO gene. Finally, we showed that an IFN-γ-independent IDO expression pathway i.e. lipopolysaccharide (LPS)-mediated-c-Jun kinase is operative in NOD mice fibroblast. In conclusion, the findings of this study for the first time indicate that IFN-γ fails to induce IDO expression in NOD dermal fibroblasts; this may partially be due to defective STAT1 phosphorylation in IFN-γ-induced-IDO signaling pathway

Treatment of hyperfunctioning thyroid nodules by percutaneous ethanol injection

BACKGROUND: Autonomous thyroid nodules can be treated by a variety of methods. We assessed the efficacy of percutaneous ethanol injection in treating autonomous thyroid nodules. METHODS: 35 patients diagnosed by technetium-99 scanning with hyperfunctioning nodules and suppressed sensitive TSH (sTSH) were given sterile ethanol injections under ultrasound guidance. 29 patients had clinical and biochemical hyperthyroidism. The other 6 had sub-clinical hyperthyroidism with suppressed sTSH levels (<0.24 μIU/ml) and normal thyroid hormone levels. Ethanol injections were performed once every 1–4 weeks. Ethanol injections were stopped when serum T(3), T(4 )and sTSH levels had returned to normal, or else injections could no longer be performed because significant side effects. Patients were followed up at 3, 6 and, in 15 patients, 24 months after the last injection. RESULTS: Average pre-treatment nodule volume [18.2 ± 12.7 ml] decreased to 5.7 ± 4.6 ml at 6 months follow-up [P < 0.001]. All patients had normal thyroid hormone levels at 3 and 6 months follow-up [P < 0.001 relative to baseline]. sTSH levels increased from 0.09 ± 0.02 μIU/ml to 0.65 ± 0.8 μIU/ml at the end of therapy [P < 0.05]. Only 3 patients had persistent sTSH suppression at 6 months post-therapy. T(4 )and sTSH did not change significantly between 6 months and 2 years [P > 0.05]. Ethanol injections were well tolerated by the patients, with only 2 cases of transient dysphonia. CONCLUSION: Our findings indicate that ethanol injection is an alternative to surgery or radioactive iodine in the treatment of autonomous thyroid nodules

Development and application of a non-rejectable composite pancreatic islet allograft using indoleamine 2, 3 dioxygenase in a diabetic mouse model

Success of transplantation of pancreatic islets as a promising therapeutic method for restoring efficient regulated insulin secretion in type 1 diabetes depends on lifelong use of immunosuppressive drugs. With the goal of eliminating the necessity of systemic immunosuppressive agents after islet transplantation, in this doctoral research project we hypothesized that a novel non-rejectable islet graft through employing a local immunosuppressive factor, indoleamine 2, 3 dioxygenase (IDO) can be developed and applied. IDO is a tryptophan degrading enzyme and functions as a potent immunomodulatory factor. To address this hypothesis, we engineered a three-dimensional composite islet graft equipped with IDO expressing bystander cells as local immunosuppressive system. In this composite graft, expression of IDO in syngeneic fibroblasts provided a low tryptophan microenvironment within which T-cells could not proliferate and infiltrate islets. Three specific aims were accomplished in this study. We first showed that local expression of IDO in syngeneic bystander fibroblasts efficiently suppressed in vitro proliferation of lymphocytes stimulated with allogeneic pancreatic islets. In the next phase, the impact of IDO on viability and function of mouse islets embedded within IDO-expressing fibroblast-populated collagen matrix was investigated. The result showed no reduction in islet cells viability and comparable insulin content and secretion in IDO-expressing versus control preparations. In contrast to lymphocytes, a nutrient deficiency stress-responsive pathway was not activated in islets co-cultured with IDO-expressing fibroblasts confirming the selective suppressive effect of IDO on immune versus islet cells. Furthermore, when allogeneic immune response was eliminated by using a syngeneic transplant model, IDO-expressing composite islet grafts were functional in vivo for up to 100 days. Finally, to address the last specific aim, composite allogeneic islet grafts were transplanted into renal subcapsular space of streptozotocin-induced diabetic immunocompetent mice. IDO-expressing grafts survived significantly longer than controls without using any systemic immunosuppressive agent (41.2±1.64 vs. 12.9±0.73 days, p<0.001). Local IDO expression evidently prevented lymphocyte infiltration into allografts and delayed alloantibody production. The findings presented in this thesis collectively prove the potent local immunosuppressive activity of IDO in islet allografts and set the stage for development of a long-lasting non-rejectable islet allograft using stable IDO induction in bystander fibroblasts.Medicine, Faculty ofMedicine, Department ofExperimental Medicine, Division ofGraduat

Clinical Science- The Mutations Of Ret Proto-Oncogene Inmedullarythyroid Carcinomas In Iran

Medullary thyroid carcinoma (MTC) occurs both sporadically and in the autoso-mal dominantly inherited multiple endocrine neoplasia (MEN) type 2 syndromes. The distinction between true sporadic MTC and a new mutation familial case is important for future clinical management of both the patient and family.The susceptibility gene for hereditary MTC is the RET proto-oncogene. DNA analysis for germline mutations of the RET proto-oncogene was performed in a series of 24 patients with MTC [appar-ently sporadic MTC (20 cases), familial MTC (2 cases), MEN 2A (one case) and MEN 2B (one case)] to determine whether they were true sporadic cases or heredi-tary forms. Genomic DNA was amplified using polymerase chain reaction (PCR) and oligonucleotide primers for exons 10 & 11. The PCR products were examined by restriction enzymes analysis to detect the mutations. One of the 20 patients with appar-ent sporadic MTC had exon 10 mutation (Cys-620 Arg); and exon 11 mutation (Cys-634 Trp) was also found in the index case with MEN 2A. No mutation was detected in the other patients. Three of six evaluated members of the MEN 2A patient had the same mutation. We conclude that routine application of RET proto-oncogene testing should be included in all cases of apparent sporadic MTC

Treatment of hyperfunctioning thyroid nodules by percutaneous ethanol injection

Abstract Background Autonomous thyroid nodules can be treated by a variety of methods. We assessed the efficacy of percutaneous ethanol injection in treating autonomous thyroid nodules. Methods 35 patients diagnosed by technetium-99 scanning with hyperfunctioning nodules and suppressed sensitive TSH (sTSH) were given sterile ethanol injections under ultrasound guidance. 29 patients had clinical and biochemical hyperthyroidism. The other 6 had sub-clinical hyperthyroidism with suppressed sTSH levels (3, T4 and sTSH levels had returned to normal, or else injections could no longer be performed because significant side effects. Patients were followed up at 3, 6 and, in 15 patients, 24 months after the last injection. Results Average pre-treatment nodule volume [18.2 ± 12.7 ml] decreased to 5.7 ± 4.6 ml at 6 months follow-up [P 4 and sTSH did not change significantly between 6 months and 2 years [P > 0.05]. Ethanol injections were well tolerated by the patients, with only 2 cases of transient dysphonia. Conclusion Our findings indicate that ethanol injection is an alternative to surgery or radioactive iodine in the treatment of autonomous thyroid nodules.</p

IFN-γ-induced-STAT1 phosphorylation in C57BL/6 and NOD dermal fibroblasts.

<p>Following starvation for 18 hours, dermal fibroblasts from NOD (open bars) and C57BL/6 (solid bars) mice were remained untreated or treated with 1000 U IFN-γ per ml of DMEM plus 2% FBS for 15, 30 or 60 minutes. Cell lysates were collected for western blot analysis. <b>A</b>: STAT 1 phosphorylation shown by western blotting. <b>B</b>: the Mean±SEM ratio of phospho-STAT1 (P-STAT1), to the ratio of β-actin to total STAT1. Total STAT1 and β-actin expressions were used as loading controls. *denotes significant difference between related bars (p<0.05, n = 3). UT: untreated, ND: not detected.</p

COL-I expression in dermal fibroblasts from control and NOD mice.

<p>COL-I expression in dermal fibroblasts from C57BL/6 (solid bars) and NOD (open bars) mice was evaluated by western blot and RT-PCR analyses. Cells were exposed to 0 or 1000 U/ml of IFN-γ for 48 hours before analysis. <b>A</b>: COL-1 expression at the protein level. <b>C</b>: COL-1 expression at mRNA level. <b>B</b> and <b>D</b> represent the Mean±SEM ratio of COL-1 to β-actin at protein and mRNA levels respectively. β-actin was used as loading control in both western blotting and RT-PCR assays. *demonstrates significant difference between C57BL/6 and NOD fibroblasts treated with IFN-γ in terms of COL-1 expression. **corresponds to significant difference between cells from the same strain treated with 0 or 1000 U/ml of IFN-γ (n = 3, p<0.05).</p

Different effect of IFN-γ on IDO expression in dermal fibroblasts of C57BL/6 prediabetic NOD mice.

<p>Dermal fibroblasts from prediabetic (8 weeks of age) male and female NOD mice failed to respond to IFN-γ induced IDO. Dermal fibroblasts isolated from C57BL/6 male mice of 8 weeks of age as control (solid bars), and aged matched male (hatched bars) or female (open bars) prediabetic NOD mice were treated with 1000 U/ml of IFN-γ for 48 hours. <b>A</b>: Kyn levels in CM of treated cells, <b>B</b>: IDO expression at the protein level, <b>C</b>: the Mean±SEM ratio of densities of IDO to β-actin at protein control group treated with IFN-γ (n = 3, p<0.01). β-actin expression showed equal loading of proteins. ND: not detected.</p