Successful Transplantation of Lungs From an Uncontrolled Donor After Circulatory Death Preserved In Situ by Alveolar Recruitment Maneuvers and Assessed by Ex Vivo Lung Perfusion.

We developed a protocol to procure lungs from uncontrolled donors after circulatory determination of death (NCT02061462). Subjects with cardiovascular collapse, treated on scene by a resuscitation team and transferred to the emergency room, are considered potential donors once declared dead. Exclusion criteria include unwitnessed collapse, no-flow period of >15 min and low flow >60 min. After death, lung preservation with recruitment maneuvers, continuous positive airway pressure, and protective mechanical ventilation is applied to the donor. After procurement, ex vivo lung perfusion (EVLP) is performed. From November 2014, 10 subjects were considered potential donors; one of these underwent the full process of procurement, EVLP, and transplantation. The donor was a 46-year-old male who died because of thoracic aortic dissection. Lungs were procured 4 h and 48 min after death, and deemed suitable for transplantation after EVLP. Lungs were then offered to a rapidly deteriorating recipient with cystic fibrosis (lung allocation score [LAS] 46) who consented to the transplant in this experimental setting. Six months after transplantation, the recipient is in good condition (forced expiratory volume in 1 s 85%) with no signs of rejection. This protocol allowed procurement of lungs from an uncontrolled donor after circulatory determination of death following an extended period of warm ischemia.

Leukotriene D4-induced activation of smooth-muscle cells from human bronchi is partly Ca2+-independent.

Cysteine-containing leukotrienes (cysteinyl-LTs) are potent bronchoconstrictors and play a key role in asthma. We found that histamine and LTD4 markedly constrict strips of human bronchi (HB) with similar efficacy. However, in human airway smooth-muscle (HASM) cells, LTD4, at variance with histamine, elicited only a small, transient change in intracellular calcium ion concentration. HASM cells express both Ca2+-dependent and -independent isoforms of protein kinase C (PKC) (i.e., PKC-alpha and PKC-alpha ). Western blot analysis showed that PKC-alpha is activated by histamine and, to a lesser extent, by LTD4, whereas only LTD4 translocates PKC-alpha. This translocation was specifically inhibited by the LTD4 antagonist pobilukast. Phorbol-dibutyrate ester (PDBu) (a PKC activator) contracted HB strips to the same extent in the presence as in the absence of extra- and intracellular Ca2+. In the absence of Ca2+, LTD4 contracted HB strips to the same extent as did PDBu, suggesting the involvement of a Ca2+-independent PKC in LTD4-mediated signal transduction. PDBu-induced desensitization and the PKC inhibitor H7 abolished the slow and sustained LTD4-triggered contraction of HB strips in the absence of Ca2+, although H7 did not greatly affect the response in the presence of the ion. Thus, in human airways, we identified a novel LTD4 transduction mechanism linked to bronchial smooth-muscle contraction, which is partly independent of Ca2+ and involves the activation of PKC-alpha.

Selectivity of recombinant human leukotriene D(4), leukotriene B(4), and lipoxin A(4) receptors with aspirin-triggered 15-epi-LXA(4) and regulation of vascular and inflammatory responses.

Aspirin-triggered lipoxin A(4) (ATL, 15-epi-LXA(4)) and leukotriene D(4) (LTD(4)) possess opposing vascular actions mediated via receptors distinct from the LXA(4) receptor (ALX) that is involved in leukocyte trafficking. Here, we identified these receptors by nucleotide sequencing and demonstrate that LTD(4) receptor (CysLT(1)) is induced in human vascular endothelia by interleukin-1beta. Recombinant CysLT(1) receptor gave stereospecific binding with both [(3)H]-LTD(4) and a novel labeled mimetic of ATL ([(3)H]-ATLa) that was displaced with LTD(4) and ATLa ( approximately IC(50) 0.2 to 0.9 nmol/L), but not with a bioinactive ATL isomer. The clinically used CysLT(1) receptor antagonist, Singulair, showed a lower rank order for competition with [(3)H]-ATLa (IC(50) approximately 8.3 nmol/L). In contrast, LTD(4) was an ineffective competitive ligand for recombinant ALX receptor with [(3)H]-ATLa, and ATLa did not compete for [(3)H]-LTB(4) binding with recombinant LTB(4) receptor. Endogenous murine CysLT(1) receptors also gave specific [(3)H]-ATLa binding that was displaced with essentially equal affinity by LTD(4) or ATLa. Systemic ATLa proved to be a potent inhibitor (>50%) of CysLT(1)-mediated vascular leakage in murine skin (200 microg/kg) in addition to its ability to block polymorphonuclear leukocyte recruitment to dorsal air pouch (4 microg/kg). These results indicate that ATL and LTD(4) bind and compete with equal affinity at CysLT(1), providing a molecular basis for aspirin-triggered LXs serving as a local damper of both vascular CysLT(1) signals as well as ALX receptor-regulated polymorphonuclear leukocyte traffic.

Effect of endogenous and exogenous prostaglandin E(2) on interleukin-1 beta-induced cyclooxygenase-2 expression in human airway smooth-muscle cells.

We studied the effect of endogenous and exogenous prostaglandin E(2) (PGE(2)), a metabolite of arachidonic acid through the cyclooxygenase (COX) pathway, on interleukin (IL)-1 beta-induced COX-2 expression, using primary cultures of human bronchial smooth-muscle cells (HBSMC). Treatment with exogenous PGE(2) resulted in enhanced expression of IL-1 beta-induced COX-2 protein and messenger RNA (mRNA) as compared with the effect of the cytokine per se. Inhibition of PGE(2) production with a nonselective COX inhibitor (flurbiprofen, 10 microM) resulted in a significant reduction in IL-1 beta- induced COX-2 expression, supporting a role of endogenous COX metabolites in the modulation of COX-2 expression. None of the experimental conditions used in the study affected the expression of constitutive cyclooxygenase (COX-1). Treatment with cycloheximide to inhibit translation, and with dexamethasone or actinomycin D to inhibit transcription, linked the effect of PGE(2) to the transcriptional level of COX-2 mRNA rather than to a potential effect on protein and/or mRNA stabilization. PGE(2) increased adenylate cyclase activity in a concentration dependent manner, and forskolin, a direct activator of adenylate cyclase, caused a marked increase in IL-1 beta-dependent COX-2, suggesting the existence of a causal relationship between the two events. The same results were observed with salbutamol, a bronchodilator that acts by increasing cyclic adenosine monophosphate. The effect of PGE(2) on COX-2 expression may contribute to the hypothesized antiinflammatory role of PGE(2) in human airways, providing a self-amplifying loop leading to increased biosynthesis of PGE(2) during an inflammatory event.

A kinetic binding study to evaluate the pharmacological profile of a specific leukotriene C(4) binding site not coupled to contraction in human lung parenchyma.

We report the identification of a novel pharmacological profile for the leukotriene (LT)C(4) binding site we previously identified in human lung parenchyma (HLP). We used a series of classic cysteinyl-LT (CysLT)(1) receptor antagonists belonging to different chemical classes and the dual CysLT(1)-CysLT(2) antagonist BAY u9773 for both binding and functional studies. Because the presence of (S)-decyl-glutathione interfered with cysteinyl-LT binding, with a kinetic protocol we avoided the use of this compound. By means of heterologous dissociation time courses, we demonstrated that zafirlukast, iralukast, and BAY u9773 selectively competed only for (3)H-LTD(4) binding sites, whereas pobilukast, pranlukast, and CGP 57698 dissociated both (3)H-LTC(4) and (3)H-LTD(4) from their binding sites. Thus, with binding studies, we have been able to identify a pharmacological profile for LTC(4) distinct from that of LTD(4) receptor (CysLT(1)) in HLP. On the contrary, in functional studies, all of the classic antagonists tested were able to revert both LTC(4)- and LTD(4)-induced contractions of isolated HLP strips. Thus, LTD(4) and LTC(4) contract isolated HLP strips through the same CysLT(1) receptor. The results of kinetic binding studies, coupled to a sophisticated data analysis, confirm our hypothesis that HLP membranes contain two cysteinyl-LT high-affinity binding sites with different pharmacological profiles. In functional studies, however, LTD(4)- and LTC(4)-induced contractions are mediated by the same CysLT(1) receptor. In conclusion, the specific LTC(4) high-affinity binding site cannot be classified as one of the officially recognized CysLT receptors, and it is not implicated in LTC(4)-induced HLP strip contractions.

Synthesis of 19-[(2-azido-5-iodo)-benzoyloxy]-LTA4 and enzymatic conversion to the LTC4 analogue.

New photoaffinity probes based on C-19 position of leukotriene A4 has been synthesized from 19-hydroxy-LTA4 methyl ester. Enzymatic conversion into the LTC4 analogue yielded a potential tool for the study of cys-LT2 receptors.

Detrusor instability as an energy-saving device in prostatic obstruction.

PURPOSE: We assessed whether voiding dynamics differ in patients with infravesical obstruction from benign prostatic hyperplasia between those with detrusor instability and those with stable bladders. MATERIALS AND METHODS: A total of 50 such patients with (25) and without (25) detrusor instability was investigated urodynamically by cystometry and pressure-flow study. RESULTS: In the unstable group there was greater obstruction (as assessed by the linear passive urethral resistance relation pressure-flow nomogram) but the detrusor expended less energy (p = 0.001) for voiding the unit volume (54.4 +/- 22.8 mJ./ml versus 74.4 +/- 31.8 mJ./ml. in the stable series). Spared energy was converted into more powerful micturition contractions and more efficient voiding, with a mean maximum contraction power and mean fraction of bladder volume voided of 17.9 +/- 4.9 microW./mm.2 and 93 +/- 11%, respectively, in the unstable population versus 12.7 +/- 2.1 microW./mm.2 (p < 0.001) and 79 +/- 13% (p < 0.001) in the stable subjects. CONCLUSIONS: Detrusor instability possibly works within physiological limits as an energy-saving device, preventing voiding efficiency in patients with prostate disease from decreasing too much with increasing obstruction.

Percutaneous ethanol injection of hepatic tumors: single-session therapy with general anesthesia.

OBJECTIVE: We studied the feasibility and the effectiveness of percutaneous ethanol injection, performed with general anesthesia in a single session, for treating malignant hepatic lesions. SUBJECTS AND METHODS: We treated 30 patients with sonographically guided percutaneous injection of ethanol. Twenty had hepatocellular carcinoma and cirrhosis, and 10 had hepatic metastases, principally from colon cancer. The mean volume of ethanol injected was 57 ml (range, 6-165 ml). RESULTS: CT showed complete necrosis (up to 8.2 cm) in seven of 10 patients with encapsulated hepatocellular carcinoma and about 90% necrosis in the remaining three patients. In four of these patients, the alpha-fetoprotein level fell from more than 200 ng/ml to less than 20 ng/ml during treatment. In 10 patients with infiltrating hepatocellular carcinoma, about 70-90% necrosis was achieved; in six of these patients, the alpha-fetoprotein level, which had been more than 200 ng/ml, decreased during treatment. In the 10 patients with metastases, more than 50% necrosis was always achieved. Levels of carcinoembryonic antigen decreased after treatment in all patients. In three patients who had cirrhosis with superficial hepatocellular carcinoma, peritoneal hemorrhage occurred but did not require transfusion. CONCLUSION: Our results show that percutaneous injection of ethanol in a single session with general anesthesia is feasible and effective and has several advantages over multisession therapy. These include shorter treatment time and the ability to treat larger and more numerous lesions.