Insulin secretion is regulated by the glucose-dependent production of islet beta cell macrophage migration inhibitory factor.

Macrophage migration inhibitory factor (MIF), originally identified as a cytokine secreted by T lymphocytes, was found recently to be both a pituitary hormone and a mediator released by immune cells in response to glucocorticoid stimulation. We report here that the insulin-secreting beta cell of the islets of Langerhans expresses MIF and that its production is regulated by glucose in a time- and concentration-dependent manner. MIF and insulin colocalize by immunocytochemistry within the secretory granules of the pancreatic islet beta cells, and once released, MIF appears to regulate insulin release in an autocrine fashion. In perifusion studies performed with isolated rat islets, immunoneutralization of MIF reduced the first and second phase of the glucose-induced insulin secretion response by 39% and 31%, respectively. Conversely, exogenously added recombinant MIF was found to potentiate insulin release. Constitutive expression of MIF antisense RNA in the insulin-secreting INS-1 cell line inhibited MIF protein synthesis and decreased significantly glucose-induced insulin release. MIF is therefore a glucose-dependent, islet cell product that regulates insulin secretion in a positive manner and may play an important role in carbohydrate metabolism.

Islet Brain 1 Protects Insulin Producing Cells against Lipotoxicity.

Chronic intake of saturated free fatty acids is associated with diabetes and may contribute to the impairment of functional beta cell mass. Mitogen activated protein kinase 8 interacting protein 1 also called islet brain 1 (IB1) is a candidate gene for diabetes that is required for beta cell survival and glucose-induced insulin secretion (GSIS). In this study we investigated whether IB1 expression is required for preserving beta cell survival and function in response to palmitate. Chronic exposure of MIN6 and isolated rat islets cells to palmitate led to reduction of the IB1 mRNA and protein content. Diminution of IB1 mRNA and protein level relied on the inducible cAMP early repressor activity and proteasome-mediated degradation, respectively. Suppression of IB1 level mimicked the harmful effects of palmitate on the beta cell survival and GSIS. Conversely, ectopic expression of IB1 counteracted the deleterious effects of palmitate on the beta cell survival and insulin secretion. These findings highlight the importance in preserving the IB1 content for protecting beta cell against lipotoxicity in diabetes.

Primary isolated aortic valve surgery in octogenarians.

We reviewed our surgery registry, to identify predictive risk factors for operative results, and to analyse the long-term survival outcome in octogenarians operated for primary isolated aortic valve replacement (AVR). A total of 124 consecutive octogenarians underwent open AVR from January 1990 to December 2005. Combined procedures and redo surgery were excluded. Selected variables were studied as risk factors for hospital mortality and early neurological events. A follow-up (FU; mean FU time: 77 months) was obtained (90% complete), and Kaplan-Meier plots were used to determine survival rates. The mean age was 82+/-2.2 (range: 80-90 years; 63% females). Of the group, four patients (3%) required urgent procedures, 10 (8%) had a previous myocardial infarction, six (5%) had a previous coronary angioplasty and stenting, 13 patients (10%) suffered from angina and 59 (48%) were in the New York Heart Association (NYHA) class III-IV. We identified 114 (92%) degenerative stenosis, six (5%) post-rheumatic stenosis and four (3%) active endocarditis. The predicted mortality calculated by logistic European System for Cardiac Operative Risk Evaluation (EuroSCORE) was 12.6+/-5.7%, and the observed hospital mortality was 5.6%. Causes of death included severe cardiac failure (four patients), multi-organ failure (two) and sepsis (one). Complications were transitory neurological events in three patients (2%), short-term haemodialysis in three (2%), atrial fibrillation in 60 (48%) and six patients were re-operated for bleeding. Atrio-ventricular block, myocardial infarction or permanent stroke was not detected. The age at surgery and the postoperative renal failure were predictors for hospital mortality (p value <0.05), whereas we did not find predictors for neurological events. The mean FU time was 77 months (6.5 years) and the mean age of surviving patients was 87+/-4 years (81-95 years). The actuarial survival estimates at 5 and 10 years were 88% and 50%, respectively. Our experience shows good short-term results after primary isolated standard AVR in patients more than 80 years of age. The FU suggests that aortic valve surgery in octogenarians guarantees satisfactory long-term survival rates and a good quality of life, free from cardiac re-operations. In the era of catheter-based aortic valve implantation, open-heart surgery for AVR remains the standard of care for healthy octogenarians.

Transport of methotrexate by the in vitro isolated rabbit proximal tubule.

The tubular transport of [3H]methotrexate was studied in isolated nonperfused and perfused superficial proximal tubular segments of rabbit kidneys. Reabsorption represented only 5% of perfused methotrexate, and appeared to be mostly of passive nature inasmuch as it was not modified by reducing the temperature or by ouabain. Cellular accumulation in nonperfused segments and secretion in perfused tubules were highest in the S2 segment and lower in the S3 and S1 segments. Secretion against a bath-to-lumen concentration gradient was observed only in S2 segments (with a maximum methotrexate secretory rate of 478 +/- 48 fmol/mm.min and an apparent Km of transport of 363 +/- 32 microM), and was inhibited by probenecid and folate. The low capacity for methotrexate secretion may be explained by a low capacity of transport across the basolateral membrane of the proximal cell as methotrexate was accumulated only to a low extent in nonperfused tubules (tissue water to medium concentration ratio of 8.2 +/- 1 in S2 segments). During secretion a small amount of methotrexate was metabolized; the nature of the metabolite(s) remains to be defined.

Performance of matrix-assisted laser desorption ionization-time of flight mass spectrometry for identification of bacterial strains routinely isolated in a clinical microbiology laboratory.

Matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) has recently been introduced in diagnostic microbiology laboratories for the identification of bacterial and yeast strains isolated from clinical samples. In the present study, we prospectively compared MALDI-TOF MS to the conventional phenotypic method for the identification of routine isolates. Colonies were analyzed by MALDI-TOF MS either by direct deposition on the target plate or after a formic acid-acetonitrile extraction step if no valid result was initially obtained. Among 1,371 isolates identified by conventional methods, 1,278 (93.2%) were putatively identified to the species level by MALDI-TOF MS and 73 (5.3%) were identified to the genus level, but no reliable identification was obtained for 20 (1.5%). Among the 1,278 isolates identified to the species level by MALDI-TOF MS, 63 (4.9%) discordant results were initially identified. Most discordant results (42/63) were due to systematic database-related taxonomical differences, 14 were explained by poor discrimination of the MALDI-TOF MS spectra obtained, and 7 were due to errors in the initial conventional identification. An extraction step was required to obtain a valid MALDI-TOF MS identification for 25.6% of the 1,278 valid isolates. In conclusion, our results show that MALDI-TOF MS is a fast and reliable technique which has the potential to replace conventional phenotypic identification for most bacterial strains routinely isolated in clinical microbiology laboratories.

Development and preclinical assessment of a bioartificial pancreas.

Transplantation of insulin secreting cells is regarded as a possible treatment for type 1 diabetes. One major difficulty in this approach is, however, that the transplanted cells are exposed to the patient's inflammatory and autoimmune environment, which originally destroyed their own beta-cells. Therefore, even if a good source of insulin-secreting cells can be identified for transplantation therapy, these cells need to be protected against these destructive influences. The aim of this project was to evaluate, using a clonal mouse beta-cell line, whether genetic engineering of protective genes could be a viable option to allow these cells to survive when transplanted into autoimmune diabetic mice. We demonstrated that transfer of the Bcl-2 anti-apoptotic gene and of several genes specifically interfering with cytokines intracellular signalling pathways, greatly improved resistance of the cells to inflammatory stresses in vitro. We further showed that these modifications did not interfere with the capacity of these cells to correct hyperglycaemia for several months in syngeneic or allogeneic streptozocin-diabetic mice. However, these cells were not protected against autoimmune destruction when transplanted into type 1 diabetic NOD mice. This suggests that in addition to inflammatory attacks by cytokines, autoimmunity very efficiently kills the transplanted cells, indicating that multiple protective mechanisms are required for efficient transplantation of insulin-secreting cells to treat type 1 diabetes.

The plasminogen system in microdissected colonic mucosa distant from an isolated adenoma.

In the colon, the urokinase-type plasminogen activator (uPA), its receptor (uPAR), and plasminogen activator inhibitors, PAI-1 and PAI-2, are implicated in the transition from mucosa to adenoma and tumour progression. However, expression in the mucosa adjacent, or distant, to an adenoma has not yet been investigated. Three biopsies from mucosae adjacent (20 cm, ipsilateral) and distant (contralateral) to an isolated tubular adenoma were analysed in 14 patients and 8 controls. Laser microdissection isolated stromal and epithelial crypt components, and quantitative RT-PCR analyses of uPA, uPAR, PAI-1 and PAI-2 mRNA levels were performed. Among controls, no significant differences in the markers were noted. With left colon isolated tubular adenoma, uPA, uPAR, and PAI-2 mRNA levels were significantly increased in the adjacent mucosal stroma compared to epithelial crypt levels (p < 0.05). In right colon adenoma, the mRNA levels of these 3 molecular markers were significantly increased only in the adjacent mucosal stromal samples (p < 0.05). Isolated tubular adenoma in the colon increases significantly the mRNA levels of 3 proteolysis-associated molecular markers in the stromal, but not in the epithelial, components of adjacent mucosa. These results suggest the presence of regional and dynamic interactions in apparently non-involved mucosae.

In vitro functionality of isolated embryonic hypothalamic vasopressinergic and oxytocinergic neurons: modulatory effects of brain-derived neurotrophic factor and angiotensin II.

There are only a few studies on the ontogeny and differentiation process of the hypothalamic supraoptic-paraventriculo-neurohypophysial neurosecretory system. In vitro neuron survival improves if cells are of embryonic origin; however, surviving hypothalamic neurons in culture were found to express small and minimal amounts of arginine-vasopressin (AVP) and oxytocin (OT), respectively. The aim of this study was to develop a primary neuronal culture design applicable to the study of magnocellular hypothalamic system functionality. For this purpose, a primary neuronal culture was set up after mechanical dissociation of sterile hypothalamic blocks from 17-day-old Sprague-Dawley rat embryos (E17) of both sexes. Isolated hypothalamic cells were cultured with supplemented (B27)-NeuroBasal medium containing an agent inhibiting non-neuron cell proliferation. The neurosecretory process was characterized by detecting AVP and OT secreted into the medium on different days of culture. Data indicate that spontaneous AVP and OT release occurred in a culture day-dependent fashion, being maximal on day 13 for AVP, and on day 10 for OT. Interestingly, brain-derived neurotrophic factor (BDNF) and Angiotensin II (A II) were able to positively modulate neuropeptide output. Furthermore, on day 17 of culture, non-specific (high-KCl) and specific (Angiotensin II) stimuli were able to significantly (P < 0.05) enhance the secretion of both neuropeptides over respective baselines. This study suggests that our experimental design is useful for the study of AVP- and OT-ergic neuron functionality and that BDNF and A II are positive modulators of embryonic hypothalamic cell development.

Red blood cell-derived microparticles isolated from blood units initiate and propagate thrombin generation.

BACKGROUND: Red blood cell-derived microparticles (RMPs) are small phospholipid vesicles shed from RBCs in blood units, where they accumulate during storage. Because microparticles are bioactive, it could be suggested that RMPs are mediators of posttransfusion complications or, on the contrary, constitute a potential hemostatic agent. STUDY DESIGN AND METHODS: This study was performed to establish the impact on coagulation of RMPs isolated from blood units. Using calibrated automated thrombography, we investigated whether RMPs affect thrombin generation (TG) in plasma. RESULTS: We found that RMPs were not only able to increase TG in plasma in the presence of a low exogenous tissue factor (TF) concentration, but also to initiate TG in plasma in absence of exogenous TF. TG induced by RMPs in the absence of exogenous TF was neither affected by the presence of blocking anti-TF nor by the absence of Factor (F)VII. It was significantly reduced in plasma deficient in FVIII or F IX and abolished in FII-, FV-, FX-, or FXI-deficient plasma. TG was also totally abolished when anti-XI 01A6 was added in the sample. Finally, neither Western blotting, flow cytometry, nor immunogold labeling allowed the detection of traces of TF antigen. In addition, RMPs did not comprise polyphosphate, an important modulator of coagulation. CONCLUSIONS: Taken together, our data show that RMPs have FXI-dependent procoagulant properties and are able to initiate and propagate TG. The anionic surface of RMPs might be the site of FXI-mediated TG amplification and intrinsic tenase and prothrombinase complex assembly.

Simultaneous acute pulmonary embolism and isolated septal myocardial infarction in a young patient

While the overall incidence of myocardial infarction (MI) has been decreasing since 2000 [1], there is an increasing number of younger patients presenting with MI [2]. Few studies have focused on MI in very young patients, aged 35 years or less, as they only account for a minority of all patients with myocardial infarction [3]. According to the age category, MI differs in presentation, treatment and outcome, as illustrated in table 1. Echocardiography is considered mandatory according to scientific guidelines in the management and diagnosis of MI [4,5,6]. However, new imaging techniques such as cardiac magnetic resonance (CMR) and computed tomography (CT) are increasingly performed and enable further refinement of the diagnosis of MI. These techniques allow, in particular, precise location and quantification of MI. In this case, MI was located to the septum, which is an unusual presentation of MI. The incidence of pulmonary embolism (PE) has also increased in young patients over the past years [7]. Since symptoms and signs of PE may be non-specific, establishing its diagnosis remains a challenge [8]. Therefore, PE is one of the most frequently missed diagnosis in clinical medicine. Because of the widespread use of CT and its improved visualization of pulmonary arteries, PE may be discovered incidentally [9]. In the absence of a congenital disorder, multiple and/or simultaneous disease presentation is uncommon in the young. We report the rare case of a 35 year old male with isolated septal MI and simultaneous PE. The diagnosis of this rare clinical entity was only possible by means of newer imaging techniques.