Efficacy of a new delivery system based on solid lipid microparticles for the oral administration of the non-conventional antioxidant IAC on a diabetes mouse model.

PURPOSE: We previously showed the positive effects of the new antioxidant molecule bis(1-hydroxy-2,2,6,6-tetramethyl-4-piperidinyl)-decandioate (IAC) in reducing basal hyperglycaemia and relieving glucose intolerance in a diabetes model. However, the chemical properties of IAC did not allow an efficient oral administration, thus representing the main failing of that study. Here, we tested the effect of a new oral delivery system based on solid lipid microparticles (SLMs) in a diabetes mouse model. METHODS: The diabetes model was induced in C57B1/6J mice using streptozotocin and nicotinamide. Only the animals that overcame the glycaemic threshold of 180 mg/dL were enrolled in the study. Diabetic animals were then randomly assigned to 4 groups (n = 9) and treated once a day for 5 consecutive weeks with IAC (50, 100, and 150 mg/kg b.w.). The control group was composed of (n = 7) healthy mice that received only the vehicle. Glucose level was weekly monitored during the treatment period and up to 3 weeks after the suspension of the treatment. Glucose tolerance and insulin-resistance test were carried out. RESULTS: Our results showed that SLMs maintained the IAC effect in reducing basal hyperglycaemia as well as improving the insulin sensitivity and glucose tolerance. CONCLUSION: The present study confirms that SLMs are promising drug carriers, which allow the oral administration of IAC ensuring its therapeutic efficacy. The concrete possibility to administer IAC per os represents a significant breakthrough in the putative consideration of this multi-radical scavenger in the diabetes therapeutic approach.

Persistent correction of hyperglycemia in streptozotocin-nicotinamide-induced diabetic mice by a non-conventional radical scavenger.

We previously reported that in a diabetes mouse model, characterised by moderate hyperglycaemia and reduced beta-cell mass, the radical scavenger bis(1-hydroxy-2,2,6,6-tetramethyl-4-piperidinyl)decandioate di-hydrochloride (IAC), a non-conventional cyclic hydroxylamine derivative, improves metabolic alterations by counteracting beta-cell dysfunction associated with oxidative stress. The aims of this study were to ascertain whether the beneficial effects of IAC treatment could be maintained after its discontinuation and further elucidate the underlying mechanisms. Diabetes was induced in C57Bl/6J mice by streptozotocin (STZ) and nicotinamide (NA) administration. Diabetic mice were treated for 7 weeks with various doses of IAC (7.5, 15, or 30 mg/kg b.w./die i.p.) and monitored for additional 8 weeks after suspension of IAC. Then, pancreatic tissue was used for determination of beta-cell mass by immunohistochemistry and beta-cell ultrastructural analysis. STZ-NA mice showed moderate hyperglycaemia, glucose intolerance and reduced beta-cell mass (25% of controls). IAC-treated STZ-NA mice (at both doses of 15 and 30 mg/kg b.w.) showed long-term reduction of hyperglycaemia even after discontinuation of treatment, attenuation of glucose intolerance and partial preservation of beta-cell mass. The lowest IAC dose was much less effective. Plasma nitrotyrosine levels (an oxidative stress index) significantly increased in untreated diabetic mice and were lowered upon IAC treatment. At ultrastructural level, beta cells of IAC-treated diabetic mice were protected against degranulation and mitochondrial alterations. In the STZ-NA diabetic mouse model, the radical scavenger IAC induces a prolonged reduction of hyperglycaemia associated with partial restoration of beta-cell mass and function, likely dependent on blockade of oxidative stress-induced damaging mechanisms.

Insulin secretion defects of human type 2 diabetic islets are corrected in vitro by a new reactive oxygen species scavenger.

Oxidative stress is a putative mechanism leading to beta-cell damage in type 2 diabetes. We studied isolated human pancreatic islets from type 2 diabetic and non-diabetic subjects, matched for age and body mass index. Evidence of increased oxidative stress in diabetic islets was demonstrated by measuring nitrotyrosine concentration and by electron paramagnetic resonance. This was accompanied by reduced glucose-stimulated insulin secretion, as compared to non-diabetic islets (Stimulation Index, SI: 0.9 +/- 0.2 vs. 2.0 +/- 0.4, P<0.01), and by altered expression of insulin (approximately -60%), catalase (approximately +90%) and glutathione peroxidase (approximately +140%). When type 2 diabetic islets were pre-exposed for 24 h to the new antioxidant bis(1-hydroxy-2,2,6,6-tetramethyl-4-piperidinyl)decandioate di-hydrochloride, nitrotyrosine levels, glucose-stimulated insulin secretion (SI: 1.6+/-0.5) and gene expressions improved/normalized. These results support the concept that oxidative stress may play a role in type 2 diabetes beta-cell dysfunction; furthermore, it is proposed that therapy with antioxidants could be an interesting adjunctive pharmacological approach to the treatment of type 2 diabetes.

Quantitative evaluation of oxidative stress status on peripheral blood in beta-thalassaemic patients by means of electron paramagnetic resonance spectroscopy.

High oxidative stress status (OSS) is known to be one of the most important factors determining cell injury and consequent organ damage in thalassaemic patients with secondary iron overload. Using an innovative hydroxylamine 'radical probe' capable of efficiently trapping majority of oxygen-radicals including superoxide we measured, by electron paramagnetic resonance (EPR) spectroscopy, OSS in peripheral blood of 38 thalassaemic patients compared with sex-/age-matched healthy controls. Thalassaemic patients showed sixfold higher EPR values of OSS than controls. Significantly higher EPR values of OSS were observed in those with a severe phenotype (thalassaemia major, transfusion-dependent) with respect to mild phenotype (sickle-cell/beta-thalassaemia, not transfusion-dependent) or thalassaemia intermedia. In patients with thalassaemia major, EPR values of OSS were positively correlated with serum ferritin and with alanine aminotransferase levels. In patients with sickle cell/beta-thalassaemia, there was no correlation between EPR value of OSS and all parameters considered. The type of chelating therapy (desferrioxamine or deferiprone) did not have an effect on EPR value of OSS. In conclusion, EPR 'radical probe' seems to be a valid innovative method to determine total OSS in patients affected by thalassaemia and might be used for evaluating new strategies of chelation, new chelators, or the efficacy of antioxidant formula.

Reactivity to p52 and CM2 recombinant proteins in primary human cytomegalovirus infection with a microparticle agglutination assay.

We evaluated the reactivities of sera against p52 and CM2 recombinant antigens of human cytomegalovirus (HCMV), coated on microparticles, for the differentiation of primary HCMV infection from an established infection. Two different test formats of the CMV Multiplex Copalis assay were evaluated. The 214 serum samples tested were immunoglobulin M (IgM) positive or equivocal by our reference assay. Reactivities against p52 and CM2 antigens were tested for sera from 37 patients with a well-documented seroconversion within the preceding 3 months (119 serum specimens), 31 patients known to have had a seroconversion at least 8 months earlier (31 serum specimens), and 57 patients without a documented seroconversion (64 serum specimens). The assay had a sensitivity for the detection of a primary infection of 70 or 86% by the first test format and a sensitivity of 88 or 94% by the second test format, according to the criteria used to indicate a primary infection by this test. A good correlation of the results of the assay with our in-house avidity index was found. The specificity of the assay warrants further evaluation. With IgM-positive sera, the assay was not sufficiently specific to make a distinction between a primary infection and an established infection.

Selective transformation of host lymphocytes in vivo by retrovirus-producing macrophages.

Macrophage-mediated retroviral transformation of host cells was studied in vivo utilizing the cloned murine macrophage-line GG2EE, generated by in vitro infection of bone marrow cells from C3H/HeJ mice (H-2k) with the acute transforming retrovirus J2 bearing the v-myc and v-raf oncogenes. Because GG2EE macrophages produce the J2 retrovirus, the development of secondary, J2 virus-induced tumors after the injection of the cell line into several strains of mice was evaluated. GG2EE cells proliferated and gave rise to histiocytic tumors in syngeneic mice and in allogeneic athymic Swiss mice. The inoculum of GG2EE cells in allogeneic DBA/2 mice (H-2d) and, to a lesser extent, in BALB/c (H-2d) and BALB/k (H-2k) mice gave rise to a small, solid mass at the injection site. Although the initial tumor was slowly rejected, secondary lymphomas belonging to the B or T cell lineage developed, leading to mouse death. Extensive phenotypic, functional, and chromosomal analyses proved that lymphomas were derived from host T and B cell transformation. Southern and Northern blot studies showed that J2 virus was integrated and expressed in lymphoma cells, demonstrating that the virus was transmitted to the host lymphocytes and suggesting that it was causal in lymphoma development. The existence of close and protracted interactions between GG2EE macrophages and allogeneic host lymphocytes and the presence of viral particles in the area of macrophage-lymphocyte contact were demonstrated by histologic and ultrastructural analysis. Rejection of J2 virus-infected lymphocytes in allogeneic mice suggested that host lymphocyte transformation was dependent upon the macrophage cell type. These results demonstrate that macrophage-derived J2 retrovirus transforms host lymphocytes in vivo in allogeneic mice and that a condition of host alloreactivity is critical for such event.

Growth and spread of human malignant T lymphoblasts in immunosuppressed nude mice: a model for meningeal leukemia.

Previous work has shown that nude (nu/nu) mice additionally immunosuppressed by splenectomy, sublethal irradiation, and treatment with antiasialo GM1 antiserum (SIA-nu/nu mice) have no detectable natural killer activity and allow the growth of human malignant lymphoblasts. We show here that all SIA-nu/nu mice engrafted intravenously with 5 x 10(6) malignant lymphoblasts originally derived from a child with a T-cell acute lymphoblastic leukemia (PF382) and from a boy with a T-cell lymphoma (ST-4) develop lethal meningeal leukemia and die within 35 days. Histologic examination of moribund SIA-nu/nu mice showed that vertebral and skull bone marrow was always replaced by proliferating human T lymphoblasts. From the spinal canal, lymphoblasts spread to the meninges, causing hind leg paralysis. Leaving the skull, they permeated the meninges and then invaded the nervous parenchyma. This efficient and reproducible experimental model may be suitable for experimental studies on the pathogenesis of meningeal leukemia.

Growth and dissemination of human malignant lymphoblasts in immunosuppressed nu/nu mice.

Athymic nu/nu mice are commonly employed for the heterotransplantation of solid human tumors. Leukemias, however, have consistently proved difficult to transplant and, to enhance their take, recipient nu/nu mice have been variously immunosuppressed. In this study, the natural reactivity against human malignant T lymphoblast (PF382) of splenectomized nu/nu mice (S-nu/nu), nu/nu mice splenectomized and treated with polyinosinic-polycytidylic acid (SIC-nu/nu), and nu/nu mice splenectomized, irradiated and repeatedly injected with antiasialo GM1 antiserum (SIA-nu/nu) has been correlated with the in vivo growth of subcutaneous and intravenous PF382 cell challenges. SIC-nu/nu mice display a marked natural killer (NK) activity, quickly clear 125I-Urd-labelled PF382 cells injected intravenously and do not allow the growth of subcutaneous nor intravenous PF382 cell challenges. S-nu/nu mice display a slightly lower NK activity and slower clearance of 125I-Urd-labelled PF382 cells. Moreover, an intravenous PF382 cell challenge kills 56% of S-nu/nu mice. SIA-nu/nu mice have no NK activity, slowly clear 125I-Urd-labelled PF382 cells and always allow the growth of PF382 cells injected either subcutaneously or intravenously with a consistent pattern. Following the intravenous challenge, PF382 cells first metastasize to liver and kideny, then focal or diffuse infiltrations of the bone marrow and menings become evident. SIA-nu/nu mice thus offer an interesting experimental model for study of the pathogenesis of leukemic infiltration of the meninges, and the exploration of possible therapeutic approaches.