Phlegmasia Cerulea Dolens: a sudden unexpected death with hypothesis of medical malpractice.

ABSTRACT: Phlegmasia Cerulea Dolens (PCD) is a severe and rare form of venous thrombosis of the lower extremities, caused by a subtotal or complete occlusion of venous outflow by a thrombus. PCD should be considered a real medical emergency; complications include necrosis and gangrene of the affected limb, amputation, massive pulmonary embolism and, in extreme cases, the death of the patient. Case Report. A 63-years-old man was admitted to the Emergency room with localized pain on the right calf, hyperthermia, cold sweating and vomiting episodes. Five days prior he developed flu-like symptoms, joint pain and cold sensation unresponsive to treatment. Ultrasound examination showed a deep venous thrombosis of the lower right limb with partial occlusion of common iliac and femoral veins. The patient was treated with low molecular weight heparin given twice daily. He began to develop severe hypotension and metabolic acidosis, with tachycardia and atrial fibrillation. Despite the treatment, there was no improvement and he developed severe sinus node dysfunction. He failed to respond to all resuscitative efforts and died. Family members complained Authority, assuming it was a medical error. The clinical-forensic investigation is essential to determine the causes and manner of death and to assess medical responsibility and liability.

Methadone overdose in patients following methadone mainte- nance treatment:A three years overview in the district of Bari (South-Italy).

BACKGROUND: Methadone maintenance treatment (MMT) is reco-gnized as a reference treatment for opioid dependence. According to Italian Law, at the beginning of the treatment patients must receive medication under the supervision of a physician, to avoid overdose. After a period of stability, patients could be allowed to take methadone at home in pre-arranged and personalized concentrations, in order to empower their self-responsibility. The aim of the present investigation is to underline the presence of a "glitch in the system" of the MMT. METHODS: In the last three years, 7 forensic autopsies and toxi-cological analysis on corpses of regular opioid users were performed into the Institute of Legal Medicine of Bari. CONCLUSION: Therefor an improvement of the MMT's guideline is needed to reduce methadone overdose deaths in future. RESULTS: In all these cases very high methadone concentration in blood were found. All the 7 subjects were following a MMT in a Public Health Institute and the cause of death was respiratory depression by overdose of methadone in 6 cases.

Modulation of glucose-related metabolic pathways controls glucose level in airway surface liquid and fight oxidative stress in cystic fibrosis cells.

Direct and indirect evidences show that elevated glucose concentrations in airway surface liquid (ASL) promote lung infection by pathogens, playing a role in the progression of the Cystic Fibrosis (CF) disease. The joint action of transporter/s for glucose and of the cellular enzymes is essential in order to try to lower ASL glucose level. Inside the cell, the glycolysis and the pentose phosphate pathway (PPP) compete for the utilization of glucose-6-phosphate (G6P), the product in which glucose, after entry within the cell and phosphorylation, is trapped. The study aims to clarify whether, modulating the activity of enzymatic proteins and/or the level of metabolites/cofactors, involved in intracellular glucose utilization, a lowering of the extracellular glucose level in CF occurs. Biochemical approaches have enabled us to understand i) how G6P is shunted between glycolysis and PPP and ii) that mitochondria, more than enzymes/cofactors participating to the two cell glucose utilization pathways, are protagonists of the scene in counteracting the high ASL glucose level as well as oxidative stress in CF.

Aberrant GSH reductase and NOX activities concur with defective CFTR to pro-oxidative imbalance in cystic fibrosis airways.

Cystic fibrosis (CF) is associated to impaired Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) channel also causing decreased glutathione (GSH) secretion, defective airway bacterial clearance and inflammation. Here we checked the main ROS-producing and ROS-scavenging enzymes as potential additional factors involved in CF pathogenesis. We found that CFBE41o-cells, expressing F508del CFTR, have increased NADPH oxidase (NOX) activity and expression level, mainly responsible of the increased ROS production, and decreased glutathione reductase (GR) activity, not dependent on GR protein level decrease. Furthermore, defective CFTR proved to cause both extracellular and intracellular GSH level decrease, probably by reducing the amount of extracellular GSH-derived cysteine required for cytosolic GSH synthesis. Importantly, we provide evidence that defective CFTR and NOX/GR activity imbalance both contribute to NADPH and GSH level decrease and ROS overproduction in CF cells.

Longitudinal rotation: an unrecognised motion pattern in patients with dilated cardiomyopathy.

BACKGROUND: Heart failure patients who are candidates for CRT frequently display longitudinal rotation (LR) - a swinging motion of the heart when imaged in a horizontal long-axis plane. OBJECTIVES: To identify the magnitude and predictors of LR in patients with ischaemic (ICM) and idiopathic dilated (DCM) cardiomyopathy, and to assess predictive value of LR in patients undergoing cardiac resynchronisation therapy (CRT). DESIGN AND SETTING: A retrospective study in a tertiary heart care setting. METHODS: Echocardiography was performed in 45 ICM and 41 DCM patients who were CRT candidates and 16 control subjects. Global LR, segmental strains and segmental LR were assessed from echocardiograms using speckle tracking. Repeat echocardiography >40 days after the beginning of CRT was performed in 64 patients. RESULTS: While DCM patients with QRS duration of both <130 ms and > or =130 ms displayed significant clockwise LR (p<0.001 for both vs 0), ICM patients and control subjects had LR that did not differ from 0. The most significant LR predictor was end-diastolic volume (p<0.001) followed by the absence of ischaemia (p<0.001) and QRS duration (p = 0.05). DCM patients with prominent clockwise LR had lower septal but higher lateral strains than DCM patients with minimal LR, or ICM patients with counterclockwise LR. LR correlated with decrease of end-systolic volume in DCM (r = 0.49, p = 0.004), while no relationship was observed in ICM. CONCLUSION: Clockwise LR is linked to presence of DCM, with the small impact of QRS duration. LR is a moderately strong predictor of end-systolic volume decrease during CRT in DCM.

NHE3 inhibits PKA-dependent functional expression of CFTR by NHERF2 PDZ interactions.

It has been shown that when CFTR and NHE3 are co-expressed on the apical membrane of the A6-NHE3 cell monolayers, the two transporters interact via a shared regulatory complex composed of NHERF2, ezrin, and PKA. We observe here that co-expression of NHE3 reduced both PKA-dependent apical CFTR expression and its activation once in place by approximately 50%. To analyze the role of NHERF2 in this process, we transfected NHE3 expressing and non-expressing A6 monolayers with NHERF2 cDNA in which its binding domains had been deleted. When only CFTR is expressed on the apical membrane, deletion of any of the NHERF2 binding domains inhibited both PKA-dependent apical CFTR expression and its activation, while when NHE3 was co-expressed with CFTR PDZ2 deletion was without effect on CFTR sorting and activity. This suggests that when the PDZ2 domain is "sequestered" by interacting with NHE3 it can no longer participate in CFTR functional expression.

Stimulation of Xenopus P2Y1 receptor activates CFTR in A6 cells.

Nucleotide binding to purinergic P2Y receptors contributes to the regulation of a variety of physiological functions in renal epithelial cells. Here, we investigate the regulatory mechanism of the P2Y1 receptor agonist 2-methylthioadenosine diphosphate (2-MeSADP) on Cl- transport in A6 cells, a commonly used model of the distal section of the Xenopus laevis nephron. Protein and mRNA expression analysis together with functional measurements demonstrated the basolateral location of the Xenopus P2Y1 receptor. 2-MeSADP increased intracellular [Ca2+] and cAMP and Cl- efflux, responses that were all inhibited by the specific P2Y1 receptor antagonist MRS 2179. Cl- efflux was also inhibited by the cystic fibrosis transmembrane conductance regulator (CFTR) blocker glibenclamide. Inhibition of either protein kinase A (PKA) or the binding between A-kinase-anchoring proteins (AKAPs) and the regulatory PKA RII subunit blocked the 2-MeSADP-induced activation of CFTR, suggesting that PKA mediates P2Y1 receptor regulation of CFTR through one or more AKAPs. Further, the truncation of the PDZ1 domain of the scaffolding protein Na+/H+ exchanger regulatory factor-2 (NHERF-2) inhibited 2-MeSADP-dependent stimulation of Cl- efflux, suggesting the involvement of this scaffolding protein. Activation or inhibition of PKC had no effect per se on basal Cl- efflux but potentiated or reduced the 2-MeSADP-dependent stimulation of Cl- efflux, respectively. These data suggest that the X laevis P2Y1 receptor in A6 cells can increase both cAMP/PKA and Ca2+/PKC intracellular levels and that the PKC pathway is involved in CFTR activation via potentiation of the PKA pathway.

Extracellular adenine nucleotides regulate Na+/H+ exchanger NHE3 activity in A6-NHE3 transfectants by a cAMP/PKA-dependent mechanism.

As potential autocrine or paracrine factors, extracellular nucleotides are known to be important regulators of renal ion transporters by activating cell surface receptors and intracellular signaling pathways. We investigated the influence of extracellular adenine nucleotides on Na+/H+ exchanger isoform 3 (NHE3) activity in A6-NHE3 cells. This is a polarized cell line obtained by stable transfection of A6 cells with the cDNA encoding the rat isoform of NHE3, which is expressed on the apical membrane. Basolateral addition of the P2Y(1) agonist, 2-MeSADP, induced an inhibition of NHE3 activity, which was prevented by preincubation with selective P2Y(1) antagonists, MRS 2179 (N6-methyl-2'-deoxyadenosine-3',5'-bisphosphate) and MRS 2286 (2-[2-(2-chloro-6-methylamino-purin-9-yl)-ethyl]-propane-1,3-bisoxy(diammoniumphosphate)). NHE3 activity was also significantly inhibited by ATP and ATP-gamma-S but not by UTP. 2-MeSADP induced a P2Y(1) antagonist-sensitive increase in both [Ca2+]i and cAMP production. Pre-incubation with a PKC inhibitor, Calphostin C, or the calcium chelator BAPTA-AM, had no effect on the 2-MeSADP-dependent inhibition of NHE3 activity, whereas this inhibition was reversed by either incubation with the PKA inhibitor H89 or by mutation of two PKA target serines (S552 and S605) on NHE3. Pre-incubation of the A6-NHE3 cells with the synthetic peptide, Ht31, which prevents the binding between AKAPs and the regulatory PKA subunits RII, also prevented the 2-MeSADP-induced inhibition of NHE3. We conclude that only the cAMP/PKA pathway is involved in the inhibition of NHE3 activity.