Quantification of Basal and Stimulated ROS Levels as Predictors of Islet Potency and Function

We have developed a luminol-based assay using intact islets, which allows for quantification of reactive oxygen species (ROS). In addition, an index capable of characterizing metabolic and mitochondrial integrity prior to transplantation was created based on the capacity of islets to respond to high glucose and rotenone (mitochondrial respiratory chain complex I inhibitor) by production of ROS. To validate this assay, lipid peroxidation and antioxidative defense capacity were evaluated by detection of malondialdehyde (MDA) levels and glutathione peroxidase activity (GPx), respectively. Also, flow cytometric analyses of ROS (dihydroethidine), apoptosis (Annexin V, active caspases), necrosis (Topro3), and mitochondrial membrane potential (JC-1) were done in parallel to correlate with changes in luminol-measured ROS. ATP/ADP ratios were quantified by HPLC and the predictive value of ROS measurement on islet functional potency was correlated with capacity to reverse diabetes in a streptozotocin-induced diabetic NOD.scid mouse model as well as in human transplant recipients. Our data demonstrate that levels of ROS in islets correlate with the percentage of apoptotic cells and their functional potency in vivo. The ROS indices following glucose and rotenone exposure are indicative of metabolic potency and mitochondrial integrity and can be used as surrogate markers to evaluate the quality of islets prior to transplantation.     

ES05ROUTINE PARAFIBROMIN IMMUNOHISTOCHEMISTRY OF LARGE PARATHYROID ADENOMAS IS NOT AN EFFECTIVE SCREENING STRATEGY FOR CARCINOMA

Purpose Parathyroid carcinoma (PC) is rare and accounts for less than 1% of all cases of primary hyperparathyroidism (PHPT). The definitive histopathologic diagnosis of PC requires unequivocal invasion or metastasis which may be absent at first presentation. As a result, many cases of PC can only be diagnosed retrospectively. Parafibromin is the protein encoded by HRPT2 which is mutated and not expressed in many parathyroid carcinomas. Given that PCs generally weigh more than parathyroid adenomas (PA)s, we hypothesized that amongst large PAs there may be a high incidence of occult PC which could be identified by negative staining for parafibromin. Methodology 57 parathyroid glands weighing greater than 2 grams excised from 1998–2006 were identified from the University of Sydney Endocrine Surgical Database. Two specimens with a histopathologic diagnosis of PC were excluded. Immunohistochemical staining for parafibromin was performed on the remaining 55 PAs. Results Of the 55 specimens stained for parafibromin only one definite negative stain was detected. This case was originally classified as an “atypical adenoma” because it showed nuclear and architectural atypia without unequivocal evidence of invasive growth. In view of the negative staining for parafibromin it therefore probably represents occult carcinoma. There has been no evidence of recurrence or metastasis after 6.5 years. Conclusions Complete loss of staining for parafibromin is very rare in giant parathyroid adenomas suggesting that occult carcinoma is equally rare. As a result routine immunohistochemical staining for parafibromin does not appear to be an effective screening test for carcinoma in large PA without histopathologic features of PC.     

Nontransplantation of Livers from Deceased Donors Who Are Able to Donate Another Solid Organ: How Often and Why It Happens

Deceased donor factors associated with poor graft outcome are well known, but how often these factors lead to livers left untransplanted is poorly defined. A nested, case-control study was conducted using the United Network for Organ Sharing (UNOS) database from 1987 to 2005. Only those donating >/=1 solid organ were included. Primary outcome was livers not transplanted (LNT, cases) versus transplanted (LT, controls). Primary variables for multivariate analysis were donor age and obesity. Covariates included donation after cardiac death (DCD), cerebral vascular accident death, viral serologies, cancer, ALT and bilirubin. There were 23 373 (26%) LNT's from 91 362 donors who donated at least one organ. Percent LNT fell over time (1987-1990: 48%; 1991-1995: 29%; 1996-2000: 21%; 2000-2005: 16%; p < 0.01). Increased age (odds ratio: 4.2, 95% confidence interval 3.6-4.9, p < 0.01) and obesity (2.1, 1.9-2.3, p < 0.01) were significantly associated with LNT across all time periods. Other significant factors included DCD and elevated ALT. For 2001-2005, population attributable risk indicate that age >40, abnormal ALT and obesity account for 32.6%, 25.3% and 9.2% of untransplanted livers, respectively. Use of expanded criteria livers has pushed LNT lower in spite of an aging and heavier donor population. Nevertheless, age and obesity still account for a significant portion of untransplanted livers.     

Association of ex-vivo expanded human mesenchymal stem cells and rhBMP-7 is highly effective in treating critical femoral defect in rats

Mesenchymal stem cells (MSC), easily culture-expanded from bone marrow, can significantly enhance bone defect healing. Several proteins, such as the bone morphogenetic proteins (BMPs) and in particular BMP-7, are involved in bone formation in vitro and in vivo. In this preclinical study, we evaluated if the association of human MSC (hMSC) with BMP-7 had synergic action on bone healing. Rat femoral defects (n=12) were treated with: autoclaved bone and mononucleated cells (MNC) as control group G1; bone and hMSC, group G2; bone with BMP-7, group G3; bone and hMSC plus BMP-7, group G4. Defect regeneration was evaluated with plain radiographs after 2, 4, 8 and 12 weeks and with histological analysis. We observed organized trabeculae bridging between the osteotomic ends of the host bone in rats treated with the association of hMSC and rhBMP-7. These trabeculae, formed by a core of devitalized tissue surrounded by osteoblasts, osteocytes and osteoclasts, were continuous with a cortical-like structure of bony tissue. Such new bone formation of the group treated with the association of hMSC and rhBMP-7 (G4) was clearly superior compared to rats treated with rhBMP-7 (G2) or hMSC (G3) alone, as shown by radiographic analysis and histological study. The present study suggests that the association of hMSC and BMP-7 is more effective than hMSC or BMP-7 alone in the healing of femoral defects in rats. Further studies with larger samples are required to confirm these results and to evaluate the best dosage.     

Combination of low-dose mirtazapine and ibuprofen for prophylaxis of chronic tension-type headache

Chronic headaches are difficult to treat and represent the biggest challenge in headache centres. Mirtazapine has a prophylactic and ibuprofen an acute effect in tension-type headache. Combination therapy may increase efficacy and lower side effects. We aimed to evaluate the prophylactic effect of a combination of low-dose mirtazapine and ibuprofen in chronic tension-type headache. Ninety-three patients were included in the double-blind, placebo-controlled, parallel trial. Following a 4-week run-in period they were randomized to four groups for treatment with a combination of mirtazapine 4.5 mg and ibuprofen 400 mg, placebo, mirtazapine 4.5 mg or ibuprofen 400 mg daily for 8 weeks. Eighty-four patients completed the study. The primary efficacy parameter, change in area under the headache curve from run-in to the last 4 weeks of treatment, did not differ between combination therapy (190) and placebo (219), P  = 0.85. Explanatory analyses revealed worsening of headache already in the third week of treatment with ibuprofen alone. In conclusion, the combination of low-dose mirtazapine and ibuprofen is not effective for the treatment of chronic tension-type headache. Moreover, the study suggests that daily intake of ibuprofen worsens headache already after few weeks in chronic tension-type headache.     

Delayed expression of NADPH-diaphorase in rat brain after administration of the cholinotoxin AF64A

Administration of cholinotoxin etylcholine aziridinium (AF64A) into the brain selectively induces nonrever-sible cholinergic deficit. Wistar rats were injected intracerebroventricularly bilaterally with AF64A at doses of 1–3 nmol/ventricle. 28 days later the number of neurons survived was counted in dorsolateral, intermediate and medial groups of cells of the medial septum. AF64A induced a decrease in neuronal density and expression of cholineacetyl transferase at all doses used as well as in all regions studied. Brain sections were also stained for NADPH-diaphorase representing neuronal NO-synthase. Effects of AF64A on NADPH-diaphorase expression depended on the region studied. The number of NADPH-diaphorase-positive cells increased in the medial cellular group where more cholineacetly transferase-positive cells survived. In contrast, decrease in NADPH-diaphorase expression in the dorsolateral group of cells coincided with low level of cholineacetyltransferase-po-sitive neurons. The data presented suggest that in the AF64A-dependent model of neurodegeneration NO may play a neuroprotective function.     

Diabetes mellitus, hypertension and medial temporal lobe atrophy: the LADIS study.

HYPOTHESIS: Based on recent findings on the association between vascular risk factors and hippocampal atrophy, we hypothesized that hypertension and diabetes mellitus (DM) are associated with medial temporal lobe atrophy (MTA) in subjects without disability, independent of the severity of white matter hyperintensities. METHODS: In the Leukoaraiosis And DISability in the elderly (LADIS) study, we investigated the relationships between DM, hypertension, blood pressure and MTA in 582 subjects, stratified by white matter hyperintensity severity, using multinomial logistic regression. MTA was visually scored for the left and right medial temporal lobe (score 0-4), and meaned. RESULTS: Mean age was 73.5 years (sd 5.1), 54% was female. Of the subjects, 15% had DM, and 70% had a history of hypertension. The likelihood of having MTA score 3 was significantly higher in subjects with DM (OR 2.9; 95% CI: 1.1-7.8) compared with an MTA score of 0 (no atrophy). The odds ratio for MTA score 2 was not significantly increased (OR 1.8; CI: 0.9-4). Systolic and diastolic blood pressure and a history of hypertension were not associated with MTA. There was no interaction between DM and hypertension. Stratification on white matter hyperintensities (WMH) did not alter the associations. CONCLUSION: Our study strengthens the observation that MTA is associated with DM, independently of the amount of small vessel disease as reflected by WMH.     

Histamine and tryptase modulate asthmatic airway smooth muscle GM-CSF and RANTES release.

Degranulating mast cells are increased in the airway smooth muscle (ASM) of asthmatics, where they may influence ASM function. The aim of the present study was to determine whether histamine and tryptase modulate ASM cell granulocyte-macrophage colony-stimulating factor (GM-CSF) and RANTES (regulated on activation, normal T-cell expressed and secreted) release and also to examine which receptors are involved in this release. Confluent, quiescent ASM cells from asthmatic and nonasthmatic donors were treated with histamine (1 microM-100 microM) with and without histamine receptor antagonist pre-treatment, or the protease-activated receptor (PAR)-2 agonists tryptase (0.5-5 nM) and SLIGKV (100 and 400 microM). The cells were then stimulated with interleukin (IL)-1beta and/or tumour necrosis factor (TNF)-alpha (10 ng.mL(-1)) or left unstimulated for 24 h. Release of GM-CSF and RANTES was determined by ELISA and prostaglandin (PG)E(2) measured by enzyme immunoassay. Neither histamine nor tryptase induced ASM GM-CSF or RANTES secretion. However, histamine increased IL-1beta-induced GM-CSF release and markedly reduced TNF-alpha-induced RANTES release by both asthmatic and nonasthmatic cells to a similar extent, but did not modulate PGE(2) release. All changes involved activation of the histamine H1 receptor as they were partially or fully blocked by chlorpheniramine, but not ranitidine. Tryptase, via its proteolytic activity, also potentiated GM-CSF, but not RANTES, release from asthmatic and nonasthmatic ASM cells induced by both cytokines. PAR-2 involvement in the tryptase potentiation was unlikely because SLIGKV had no effect. In conclusion, mast cells, through histamine and tryptase, may locally modulate airway smooth muscle-induced inflammation in asthma.