A perspective on NETosis in diabetes and cardiometabolic disorders.

AIMS: To review the significance of a new type of neutrophil cell death (NETosis) in diabetes and cardiometabolic diseases. DATA SYNTHESIS: Diabetes and the metabolic syndrome are characterized by activation of the innate immune system. In this framework, neutrophils are front line defences against infections, but can also turn deleterious if abnormally stimulated. NETosis refers to a type of cell death whereby neutrophils release nuclear material and granule enzymes that together form the NETs (neutrophil extracellular traps). As NETs entrap bacteria, NETosis is instrumental to the clearance of microorganisms, but an exaggerated NETosis response can also lead to tissue damage in several pathological conditions. In diabetes, the finely tuned balance of NETosis required to protect the human body from microorganisms yet avoiding self-damage seems to be lost. In fact, in vitro induction of NETosis and circulating concentrations of NET-associated proteins appear to be enhanced in diabetic patients. Furthermore, NETs contribute to endothelial damage, thrombosis, and ischemia/reperfusion injury, making it a novel player in the pathobiology of cardiovascular disease. Though the cellular events taking place during NETosis have been described and directly visualized, its molecular machinery is still incompletely understood. Protein kinase C (PKC) and NADPH oxidase (NOX) are two important targets to counter NETosis in the setting of diabetes. CONCLUSIONS: NETosis appears to be part of an abnormal response to damage in diabetes that, in turn, can promote or aggravate end-organ complications. We suggest that this will be a hot topic of investigation in diabetology in the near future.

The relevance of echocardiography heart measures for breeding against the risk of subaortic and pulmonic stenosis in Boxer dogs.

The aims of this study were to investigate the role and relative importance of auscultation and echocardiography traits as risk factors for the diagnosis of subaortic (SubAS) and pulmonic (PS) stenosis and to estimate the heritability (h(2)) of cardiac measurements taken through echocardiography for a random sample of Italian Boxer dogs. The data were cardiovascular examination results of 1,283 Italian Boxer dogs (686 females and 597 males) enrolled in the national screening program for heart defects arranged by the Italian Boxer Club. Examinations were performed during a 6-yr period by a group of 7 veterinary cardiologists following a standard protocol. Occurrence and severity of SubAS and PS were diagnosed, taking into account clinical and echocardiography findings such as the grade of cardiac murmur, direct ultrasound imaging of the anatomic obstructive lesions, and values of aortic or pulmonary blood flow velocities. A Bayesian logistic regression analysis was performed to identify clinical and echocardiography variables related to SubAS and PS diagnosis. Estimation of variance components for clinical and echocardiography traits was performed using a mixed linear animal model, Bayesian procedures, and the Gibbs sampler. Prevalence of SubAS (PS) was 8.4% (2.2) and 10.7% (6.4) for female and male dogs, respectively. Cardiac murmur, peak velocities, and annulus areas behaved as risk factors for SubAS and PS. The risk of a positive diagnosis for SubAS was 3 times greater for dogs with aortic annulus area <2.1 cm(2) relative to dogs with areas >2.37 cm(2), 84 times greater for dogs showing aortic peak velocities >2.19 m/s relative to dogs with peak velocities <1.97 m/s, and 41 times greater for dogs with moderate to severe murmur grades relative to dogs with absent murmur. Similar results were obtained for PS. The estimated h(2) for the occurrence of cardiac defects was 23.3% for SubAS and 8.6% for PS. Echocardiography and cardiac murmur grades exhibited moderate h(2) estimates and exploitable additive genetic variation. The estimated h(2) was 36, 24, and 20% for aortic annulus area, aortic peak velocity, and cardiac murmur score, respectively. For the area of the pulmonary annulus and peak pulmonary velocity, the estimated h(2) were smaller, ranging from 9.5 to 12.8%. These measures are candidate indicator traits that might be effectively used in dog breeding to reduce the prevalence and severity of cardiac defects.

The increased dipeptidyl peptidase-4 activity is not counteracted by optimized glucose control in type 2 diabetes, but is lower in metformin-treated patients.

AIM: Dipeptidyl peptidase (DPP)-4 in responsible for incretin degradation and some observations suggest that DPP-4 activity is increased in type 2 diabetes (T2D). We aimed to assess the effect of T2D and glucose control on DPP-4 activity. METHODS: In the first set (SET1) of patients, we compared plasma DPP-4 activity between 30 T2D and 20 age- and sex-matched non-diabetic subjects. In the second set (SET2), we measured serum DPP-4 activity in 42 T2D patients before and after a trial of glucose control achieved by add-on basal insulin therapy (NCT00699686). Serum/plasma DPP-4 activity was determined using chromogenic and fluorigenic substrates, as well as several positive and negative controls. RESULTS: In SET1, plasma DPP-4 activity was significantly higher in T2D vs. controls (32.2 ± 1.2 U/l vs. 21.2 ± 1.1 U/l, p < 10(-6)). From a meta-analysis of the literature, we found that T2D is associated with a 33% increase in DPP-4 activity compared to controls. In SET2, serum DPP-4 activity was not lowered by intensified glucose control, despite an average haemoglobin A1c (HbA1c) reduction of 1.5%. In both sets of diabetic patients, the use of metformin was associated with a significantly lower DPP-4 activity, independently of age, sex, body mass index and HbA1c. CONCLUSION: DPP-4 activity is increased in T2D, but is not lowered by glucose control, suggesting that hyperglycaemia is not a direct determinant of DPP-4 activity. However, metformin may indirectly reduce DPP-4 activity.

Defective recruitment, survival and proliferation of bone marrow-derived progenitor cells at sites of delayed diabetic wound healing in mice.

AIMS/HYPOTHESIS: Bone marrow (BM)-derived endothelial progenitor cells (EPC) promote tissue healing and angiogenesis, whereas altered EPC biology may favour diabetic complications. We tested the hypothesis that diabetes impairs the contribution of BM-derived cells at sites of wound healing. METHODS: Four weeks after induction of diabetes in C57BL/6 mice, hindlimb skin wounds were created and monitored by digital imaging and histology. Circulating EPCs were quantified by flow cytometry before and after wounding. In separate experiments, bone marrow from C57BL/6 mice constitutively producing green fluorescent protein (GFP) was transplanted into myeloablated wild-type mice before induction of diabetes. We quantified proliferation, apoptosis and endothelial differentiation of tissue GFP(+) cells. Net recruitment of GFP(+) cells was estimated by correcting the number of tissue GFP(+) cells at each time point for basal levels, apoptosis and proliferation rates. RESULTS: Diabetes delayed wound healing, with reduced granulation tissue thickness and vascularity, and increased apoptosis. Circulating EPC levels were not modified by 4 week diabetes and/or skin wounding. BM-derived EPCs (GFP(+)vWf(+) [von Willebrand factor] cells) within the granulation tissue were significantly reduced in diabetic compared with control mice. BM-derived GFP(+) cells showed increased apoptosis and decreased proliferation in diabetic versus non-diabetic wound tissues. Estimated net recruitment of BM-derived GFP(+) cells was reduced on day 1 after wounding in diabetic mice. CONCLUSIONS/INTERPRETATION: Diabetic-delayed wound healing was associated with defective recruitment, survival and proliferation of BM-derived progenitor cells. Local treatments aimed at restoring EPC homing and survival might improve tissue healing in diabetes.

Behaviour during attacks and assessment of intensity in primary headaches of children and adolescents.

Assessment of attack intensity in primary headaches of paediatric age has not received great attention in the literature to date and in the international classification the criteria to define pain intensity are also not specified. The purpose of this research was to evaluate whether behaviour during attacks, reported by the child or a parent, can be used as a measure of attack disability, and so as an indirect measure of attack intensity in primary headaches of children and adolescents. The subjects were 320 patients aged between 3 and 14 years (mean age 9.9 years, SD 2.6 years) affected by primary recurrent headaches and first seen at a headache clinic. Twelve variables taken from their history were considered and sequentially analysed with multiple correspondence analysis and cluster analysis. Five types of behaviour during attacks were identified: (i) the child (or the parent) is unable to answer the questions or the child has no limitation in activities; (ii) the child may have some activity limitation, but only in lively games; (iii) the child has limitations in daily life with regard also to quiet activities; (iv) at least during some attacks the child lies down with closed eyes or in the dark; (v) during each attack the child lies down with closed eyes or in the dark. The least important variables for the identification of the five behaviour types were studying at school and absence from school. There are some limitations in considering child's behaviour as a measure of attack intensity/disability; one of these is the fact that it was found to be related to the educational level of the mother. However, behaviour during attacks, reported by the child or the parent, provides useful information independently of child's age and, together with the score of pain, when this is given, it can be used as measure of attack intensity.

Vertebral mineral density measured by dual-energy X-ray absorptiometry (DEXA) in a group of healthy Italian boxer dogs.

We studied the feasibility of using dual-energy X-ray absorptiometry (DEXA) to obtain reference bone density values in relation to age, gender and body weight in growing and young adult Italian boxer dogs. The study was performed on eight animals (three males and five females) at 7, 12 and 18 months of age. Animals were carefully examined and blood samples were collected from each dog to detect any sign of metabolic and/or endocrine disease. Each subject underwent radiographs to evaluate growth of the spine and hip. One female was not considered in the statistical model because of the development of grade 4 spondylosis deformans during the study period. All animals were serially scanned using DEXA; the region of interest was the whole spine T12-L2, while the subregions of interest were the four vertebrae (T12-T13-L1-L2) within the scanned spine. Statistical analysis was performed separately for each region of interest. Age had the strongest relationship with bone density (P < 0.001). Gender effect on spinal mineral density was not significant while vertebral site effect was highly significant. Average bone mineral density (BMD) +/- SD for the whole spine trait was 0.862 +/- 0.108 g/cm2 while average BMD +/- SD for subregions of interest ranged from 0.836 +/- 0.141 g/cm2 for T12 to 0.928 +/- 0.119 g/cm2 for L2. Estimated reference BMD values at 7, 12 and 18 months of age for each vertebral site in males and females are provided.

Enteric conversion after bladder drained pancreas transplantation experience of 14 cases.

The method of exocrine diversion in pancreas allograft continues to be controversial due to the advantages versus disadvantages of bladder versus enteric techniques. Bladder drainage (BD) exposes the patient to urological and metabolic problems that may require conversion to enteric drainage (ED). The purpose of this study was to review our initial experience of conversion from BD to ED for patients who underwent pancreas transplantation originally with bladder diversion. Among 114 pancreas transplantation performed with BD, from January 1996 to April 2003, 60 were simultaneous pancreas-kidney transplantation (SPKT), 35 were pancreas transplantation alone (PA), and 19 were pancreas after kidney transplantations (PAK). Twenty-three (20.2%) cases were excluded due to early death of the patient or the graft, yielding an analyses of 91 patients. Enteric conversion (EC) was performed in 14 (15.4%) patients with a mean follow-up of 15.7 months (range, 3-51 months) after transplantation including 8 (8.8%) SPKT, 4 (4.4%) PAK, and 2 (2.2%) PA. No surgical morbidity or mortality was observed related to EC. All patients had complete resolution of the initial problem with preservation of pancreatic function. EC represents an easy, safe procedure with low morbidity and mortality rates, representing the option of choice for patients with persistent urological or metabolic disturbances.

Laser enhancement in hepatic regeneration for partially hepatectomized rats.

BACKGROUND AND OBJECTIVE: The bio-stimulation effect of laser has been observed in many areas of Medicine. However, there are a few works which investigate its use for liver regeneration. Most of their results were inconclusive due to the use of high power lasers. This work was carried out to investigate the bio-stimulation effect of laser in liver regeneration using low power lasers. STUDY DESIGN/MATERIALS AND METHODS: We used Wistar male rats, which were irradiated with laser light (wavelength 590 nm and intensity of 50 mW/cm(2)) for 5 minutes after 70% hepatectomy. The respiratory mitochondrial activity, the serum level of aminotransferase and the PCNA were measured. RESULTS: Our results show a dramatic increase in the mitochondrial activity for the laser treated group at 24 hours after the hepatectomy. CONCLUSIONS: We conclude that the laser promotes a bio-stimulation effect on the early stages of liver regeneration without any detectable damage of the cells.

Classification criteria and distinction between migraine and tension-type headache in children.

The International Headache Society (IHS) classification system (1988) was developed primarily for headache disorders in adults and its validity for paediatric age is currently under discussion; in 1995 Seshia et al. proposed a revision of the criteria for migraine without aura to make diagnostic criteria more applicable to children. The purposes of the current study were to: (1) compare the IHS classification with the Seshia proposal, (2) compare the children affected by migraine without aura (MO) with the children affected by tension headache (TH) as defined by Seshia, for characteristics which are not included in the classification. The patients are a series of 320 children (mean age 9.9, SD 2.6 years; 144 males, 176 females) with recurrent or chronic headaches referred to a headache clinic in Milan, Italy. Using the Seshia criteria instead of the IHS criteria a higher number of children were included in the MO category: bilateral pain and family history of migraine were the most important factors which allowed a shift of children into this category. However, with the Seshia classification there was no reduction in the number of unclassifiable children. The reason why some children could not be classified was a short duration of attacks; the majority of unclassifiable children were 6 years old or less. No relevant difference was found between children with MO and children with TH for the following variables: occurrence of attacks in the afternoon or evening after school, reduction of attacks during school holidays, full-time schooling, after-school activities on school days, disordered daily life. On the contrary children with MO when compared with those with TH showed a higher number of precipitating factors and for the following factors a significant difference was found: exposure to TV or a computer, sleep deficiency, and strong emotions. Furthermore, children with MO showed a greater severity of attacks.

Mechanism of the diabetogenic action of cyclosporin A.

To investigate the mechanism of diabetogenic action of cyclosporin A (CsA), 7 male Wistar albino rats received 10 mg/kg/day of the drug for 4 weeks (CsA). The results were compared with controls (C); blood CsA levels measured weekly remained stable throughout the experiment (mean +/- SEM) (X = 2657.9+/-155.1 ng/ml). Intravenous glucose load (0.75 g/kg) performed after 2 weeks of CsA therapy showed glucose intolerance in treated animals as evaluated by the glucose area under the curve (CsA = 409.2+/-17.8 vs. C = 313.3+/-12.6 umol x ml(-1) x min(-1)) (p < 0.05) with insulin levels being similar in the two groups (CsA = 8603.9+/-1645.5 vs. C = 9571.9+/-828.5 pmol x ml(-1) x min(-1)). After 4 weeks of CsA administration, glucose intolerance was maintained (CsA = 398.6+/-35.6 vs. C = 301.7+/-23.0 umol x ml(-1) x min(-1)) (p < 0.05) associated with a significant decrease in insulin secretion (CsA = 4404.9+/-2392.0 vs. C = 10075.9+/-2861.0 pmol x ml(-1) x min(-1) (p < 0.05). These results suggest that CsA induced a state of insulin resistance preceding the failure of insulin secretion. After 4 weeks, the pancreatic insulin content was also decreased (CsA = 0.7+/-0.1 vs. C = 1.4+/-0.5 mU/mg) (p < 0.05). Maximal insulin binding to isolated adipocytes was not affected by CsA (CsA = 7.4+/-2.6 vs. C = 6.4+/-2.0%), although glucose transport and oxidation decreased after CsA treatment (p < 0.05). In conclusion, glucose intolerance induced by CsA in Wistar albino rats is due to decreased insulin production and impaired insulin action by a post-binding mechanism.