Resolvin E1 protects the rat heart against reperfusion injury.

The purpose of the present study was to assess whether resolvin E1 (RvE1), an anti-inflammatory mediator derived from eicosapentaenoic acid, would limit myocardial infarct size in the rat. The H9c2 cell line was used to assess whether RvE1 has direct protective effects on cardiomyocytes. In in vivo experiments, Male Sprague-Dawley rats underwent 30 min of ischemia/4 h of reperfusion. Before reperfusion, rats received intravenous RvE1 (0, 0.03, 0.1, or 0.3mg/kg). In in vitro experiments, H9c2 cells were incubated with RvE1 (0, 1, 10, 100, or 1000 nM). Cells were subjected to 18 h of incubation under normoxic conditions, 16 h of hypoxia, or 16 h of hypoxia and 2 h of reoxygenation. In vivo, RvE1 dose dependently reduced infarct size (30.7 +/- 1.7% of the area at risk in the control group and 29.1 +/- 1.6%, 14.7 +/- 1.3%, and 9.0 +/- 0.6% in the 0.03, 0.1, and 0.3 mg/kg groups, respectively, P < 0.001). In vitro, RvE1 increased viability and decreased apoptosis in a dose-dependent fashion in cells exposed to hypoxia or hypoxia/reoxygenation. A maximal effect was achieved at a concentration of 100 nM. RvE1 augmented phosphoinositide 3-kinase activity, attenuated caspase-3 activity, and augmented calcium-dependent nitric oxide synthase activity in cells exposed to hypoxia or hypoxia/reoxygenation. RvE1 increased Akt, ERK1/2, and endothelial nitric oxide synthase phosphorylation and attenuated the levels of activated caspase-3 and phosphorylated p38 levels. AG-1478, an EGF receptor tyrosine kinase inhibitor, blocked the protective effect of RvE1 both in vivo and in vitro and attenuated the RvE1-induced increase in Akt and ERK1/2 phosphorylation. In conclusion, RvE1, an anti-inflammatory mediator derived from eicosapentaenoic acid, has a direct protective effect on cardiomyocytes against ischemia-reperfusion injury and limits infarct size when administered intravenously before reperfusion.

Resolvin E1 reduces proinflammatory markers in human pancreatic islets in vitro.

BACKGROUND: In clinical islet transplantation, inflammatory responses initiated by the transplanted islets and by the host immune system cause acute and chronic graft loss. The resolution of acute inflammation is an active process mediated by specific signals and mediators such as resolvin E1 (RvE1). We investigated the effect of RvE1 on i) the inflammatory status of human pancreatic islets, ii) islet viability and apoptosis, and iii) the instant blood-mediated inflammatory reaction (IBMIR) IN VITRO. METHODS: Pro-inflammatory cytokines and tissue factor (TF) in isolated human islets were determined by real-time RT-qPCR (mRNA levels), CBA and Gyrolab bioaffy (protein levels) after lipopolysaccaride (LPS) stimulation. Islet viability was measured using insulin secretion in a dynamic model, ADP/ATP ratio and total ATP content. Apoptosis was measured using commercial kits after stimulation with proinflammatory cytokines. To assess effect on IBMIR, human islets were mixed with non-anticoagulated, RvE1 or vehicle pretreated ABO-compatible blood in heparin-coated tubing loops. RESULTS: Treatment of human islets with RvE1 (500 nM) for 24 h reduced LPS-induced increase in mRNA and protein levels of selected pro-inflammatory markers (IL-8, MCP-1, and TF). RvE1 lowered the ADP/ATP ratio, but had no effect on insulin secretion. RvE1 reduced the apoptotic effect of proinflammatory cytokines. Additionally, RvE1 reduced platelet consumption and TAT complex formation during the first 5 min after islet-blood contact. CONCLUSIONS: RvE1 suppresses proinflammatory markers and lowers the ADP/ATP ratio in human islets IN VITRO. RvE1 demonstrates anti-apoptotic effects in a proinflammatory milieu. Additionally, RvE1 has modest dampening effects on IBMIR. We conclude that RvE1 may have potential in clinical islet transplantation.

Relationship of urinary myelin basic protein-like material with cranial magnetic resonance imaging in advanced multiple sclerosis.

BACKGROUND: A significant correlation exists between disability and the volume of black holes (BHL VOL), defined as hypointense lesions on T1-weighted cranial magnetic resonance imaging. A consistent correlation has also been reported between urinary myelin basic protein-like material (MBPLM) and the transition toward secondary progression (SP) from relapsing-remitting (RR) multiple sclerosis (MS). OBJECTIVE: To improve the management of MS through a noninvasive and cost-effective test for monitoring disease activity or disease status. DESIGN AND METHODS: From 662 patients with MS (86 with RR MS, 259 with SP MS without continued attacks, and 317 with SP MS with continued attacks), 24-hour urine samples were obtained at enrollment in the phase 3 Linomide (roquinimex) drug study. The urine specimens were analyzed for MBPLM and correlated with clinical features and findings on cranial magnetic resonance imaging. RESULTS: Significant but weak correlations existed between urinary MBPLM and BHL VOL in all patients with MS (r = 0.114, P =.003; n = 662), patients with SP MS without attacks (r = 0.185, P =.003; n = 259), and all patients with SP MS (r = 0.122, P =.003; n = 576). No significant correlations were detected in the RR MS group or any of the disease groups or subgroups whose Expanded Disability Status Scale score was 5.0 or lower. In subgroup analysis, the most significant correlation was detected between urinary MBPLM after adjustment for creatinine and BHL VOL in patients with SP MS with an Expanded Disability Status Scale score of 5.5 or higher but without continued relapses (r = 0.417, P<.001; n = 138). CONCLUSIONS: In patients with advanced SP MS, urinary MBPLM may possibly serve as an indicator of failed remission and axonal damage. Urinary MBPLM correlates with disease status in MS, especially the transition of RR MS to SP MS with advancing disability.

Linomide in relapsing and secondary progressive MS: part I: trial design and clinical results. North American Linomide Investigators.

OBJECTIVE: To determine whether linomide (roquinimex) is better than placebo in slowing the time to confirmed clinical worsening in patients with relapsing-remitting (RR) and secondary progressive (SP) MS. METHODS: In this 27-center, randomized, double-blind, placebo-controlled, multiple-dose, phase III trial, 715 patients with active RRMS (n = 90) or SPMS (n = 625) were randomized to receive either linomide (1.0, 2.5, or 7.5 mg orally daily) or placebo. Patients were evaluated at 3-month intervals clinically and with MRI. The planned primary outcome was the time to the development of "confirmed" clinical worsening (increase of >/= 1.0 Expanded Disability Status Scale [EDSS] score for an enrollment EDSS score /= 0.5 point for an enrollment EDSS score of >/= 5.5) not associated with an acute relapse. RESULTS: The trial was terminated 1 month after it became fully enrolled due to unanticipated serious cardiopulmonary toxicities (pericarditis, pleural effusion, myocardial infarction, and possible pulmonary embolism), pancreatitis, and death. Notable arthralgia, myalgia, bursitis, and facial and peripheral edema were common adverse events. The high dose of linomide (7.5 mg) was not well tolerated. The trial was too brief to determine unequivocal clinical benefits. Trends suggested an unconfirmed early effect on change in EDSS score at 6 months for the medium dose (2.5 mg daily). CONCLUSION: MS patients may be more prone to develop important linomide treatment-related adverse events than other previously studied patients. However, linomide may be a potentially more toxic drug than was suspected from observations made in smaller studies for other indications. Phase III trials may identify infrequent and important toxicities that may not be anticipated by phase I and II trials.

Linomide in relapsing and secondary progressive MS: part II: MRI results. MRI Analysis Center of the University of Texas-Houston, Health Science Center, and the North American Linomide Investigators.

OBJECTIVE: To determine the safety and efficacy of roquinimex (linomide) in the management of relapsing-remitting and secondary progressive MS as monitored by MRI. BACKGROUND: Preclinical studies and several short term randomized trials of linomide suggested clinical and MRI-measured benefits with acceptable risk for closely followed MS patients. METHODS: The North American Linomide Trial formally screened 853 individuals for relapsing or secondary progressive, clinically definite MS; recent disease activity or progression; and an Expanded Disability Status Scale score at entry of 3.0 to 6.5 inclusive. MRI was obtained on 811 subjects at pre-enrollment, 718 cases at enrollment, and then at three monthly intervals until the trial was prematurely terminated for unacceptable toxicity. RESULTS: Enhancement was found on 40.2% of 718 entry scans. Statistically robust correlations were found between clinical demographic data and several entry MRI measures including CSF volume, a reflection of brain atrophy. Assessment of the effect of treatment on MRI-measured disease was limited by early trial termination. However, active treatment for 3 months reduced the proportion of patients with one or more enhancements. An exploratory analysis suggested that 2.5 mg was the most active of three doses tested in limiting the total volume of enhanced tissue, the proportion of MRI-defined lesions designated as "black holes," and by a novel MRI composite disease measure. CONCLUSIONS: The short term signature of the effect of linomide on MRI-measured aspects of the disease suggests that safer drugs of this class might be useful in the management of MS. The use of a composite index of the heterogeneous nature of the pathology of MS as captured by MRI may have merit as an outcome measure in clinical trials.

Linomide reduces the rate of active lesions in relapsing-remitting multiple sclerosis.

The synthetic immunomodulator Linomide, a quinoline-3-carboxamide, has a profound inhibitory influence in several experimental autoimmune diseases, including acute and chronic experimental allergic encephalomyelitis. In a double-blind trial, 31 patients with relapsing-remitting multiple sclerosis were randomized to oral doses of 2.5 mg Linomide or placebo once a day for six months. Fourteen patients receiving Linomide and 14 receiving placebo completed the trial, and the results were based on this population. The mean number of active (new and enlarged T2 weighted) lesions per monthly MRI scan was 1.37 in the patients receiving Linomide and 4.22 in the patients receiving placebo (p = 0.043). The percentage of scans with active MRI lesions was lower in the Linomide-treated group (p = 0.0064). When neurologic deficit was assessed by the Regional Functional Scoring System (RFSS), the Linomide group showed an improvement of 1% of the maximal RFSS range and the placebo group a deterioration of 0.2% (p = 0.14). There were three patients with relapses in the Linomide-treated group and six in the placebo group (p = 0.22). A slightly decreased proportion of natural killer cells in cerebrospinal fluid and peripheral blood was noted in the Linomide group. A severe adverse event of pleuropericarditis occurred in one of the Linomide-treated patients. The most frequent adverse event was musculoskeletal pain, of mild to severe degree, which tended to diminish after three months on Linomide therapy.

Treatment of rapidly progressive glomerulonephritis by extracorporeal immunoadsorption, prednisolone and cyclophosphamide.

Ten patients with rapidly progressive glomerulonephritis and acute renal failure were treated with extracorporeal immunoadsorption, prednisolone, and cyclophosphamide. Three patients had systemic lupus erythematosus, five had microscopic polyarteritis and two had Wegener's granulomatosis. All ten patients were dialysis-dependent prior to immunoadsorption. Nine of ten patients rapidly regained renal function and seven continue to have independent renal function between 9 and 30 months after immunoadsorption. Three patients at presentation were not dialysis dependent. Despite treatment with methylprednisolone, cyclophosphamide, and oral prednisolone, renal function continued to deteriorate and they required dialysis. Immunoadsorption was then started without alteration in baseline immunosuppression. Within a mean of 4.6 days, range 3-7 days, renal function improved and the patients no longer required dialysis. Antineutrophil cytoplasmic antibodies and double-stranded DNA antibodies were rapidly removed by immunoadsorption. Only one patient with systemic lupus erythematosus and two with microscopic polyarteritis had significant resynthesis of antibody at 1 month post-immunoadsorption. Renal biopsy before and after immunoadsorption and immunosuppressive therapy showed resolution of glomerular crescents and no evidence of active disease. Immunoadsorption coupled with prednisolone and cyclophosphamide may be of value in the treatment of rapidly progressive glomerulonephritis.

Kinetic aspects of the removal of IgG and inhibitors in hemophiliacs using protein A immunoadsorption.

Seven patients with hemophilia A and B and 3 patients with acquired hemophilia were treated on 13 and 4 occasions, respectively, with protein A immunoadsorption to reduce anti-factor VIII or IX antibodies. Usually the patients were treated on 2 consecutive days. On each treatment day an average of 3 (range: 1.02-5.83) plasma volumes were processed in the congenital patients and 1.5 (range: 1.02-2.90) in those with acquired hemophilia. Plasma levels of IgG decreased from 18.9 +/- 1.9 to 3.1 +/- 1.2 g/l in the congenital group, and from 11.5 +/- 2.3 to 2.3 +/- 0.6 g/l in the acquired group. In the congenital hemophiliacs a corresponding reduction in inhibitor level of 70-95% was regularly seen; in 1 exceptional patient the inhibitor was reduced from 4,350 to 12 Bethesda Units/ml (BU/ml) during 5 days of treatment. In the congenital hemophiliacs immunoadsorption was followed by factor infusion to peak levels between 8 and 215 IU/dl. In the patients with acquired hemophilia a satisfactory reduction in inhibitor levels was obtained in 2 of the 4 treatments, which were followed by DDAVP or factor infusion. Some recommendations for the use of protein A immunoadsorption in the treatment of hemophilic patients will be given.

Removal of anti-HLA antibodies by extracorporeal immunoadsorption to enable renal transplantation.

10 highly and persistently sensitised patients awaiting renal transplantation underwent extracorporeal immunoadsorption to remove anti-HLA antibodies. 7 patients have since received transplants. Only 1 allograft has been lost because of rejection, and there have been no serious side-effects attributable to treatment. Extracorporeal immunoadsorption may therefore be of considerable value in the management of highly sensitised patients.

Structural and functional studies of the effects of sympathetic nerve stimulation on rabbit submandibular salivary glands.

Continuous sympathetic stimulation at 8-10 Hz caused intense vasoconstriction in the gland, so stimulation was generally given in an interrupted pattern to minimize this detrimental effect on secretion. Only a small increase in fluid secretion occurred; it became thick and tended to block the cannula; therefore in later experiments the main duct was not cannulated. After sympathetic stimulation there was substantial degranulation of acinar cells. However, as this was accompanied by little movement of water, the secreted mucosubstance distended the ductal lumina. The granular tubule cells were unchanged by sympathetic stimulation. Use of selective blocking agents revealed that the sympathetically-evoked secretion of acinar mucin was mediated mainly via beta-adrenoreceptor activation. As stimulation of the sympathetic nerves alone caused little additional formation of fluid, the effects of superimposing continuous low frequency sympathetic stimulation onto a background of low parasympathetic secretion were compared with similar parasympathetic stimulation alone of the contralateral gland. These double nerve stimulations did not augment the volume of fluid secreted, or cause morphological changes additional to those from parasympathetic stimulation alone. Nevertheless, it is likely that, under natural reflex conditions, sympathetic impulses can increase the amount of acinar mucosubstance secreted.

Synthesis and secretion of amylase in the rat parotid gland following autonomic nerve stimulation in vivo.

Parasympathetic and sympathetic nerve stimulation in vivo, individually and in combination, was used to study secretion and synthesis of amylase in the rat parotid gland. After 30 min with sympathetic nerve stimulation (3 Hz) a decrease in glandular amylase was seen, which corresponded approximately to the salivary output. On the other hand, after parasympathetic stimulation (10 Hz), chosen to obtain comparable amylase output, there was no decrease in glandular amylase, which points to synthesis during such activation. Experiments with incorporation of [3H]leucine, reflecting amylase synthesis, showed that both types of nerve stimulations increased such uptake in parotid protein. These results indicate that beside sympathetic activity, which is the main stimulus for granular amylase secretion, parasympathetic nerve impulses can evoke considerable amylase secretion because amylase synthesis is stimulated and amylase is rapidly available from a special, possibly non-granular pool. As expected from previous experiments an augmentation of amylase secretion was found, and the present experiments also indicated an augmentation at the level of synthesis when the two nerves were stimulated at the same time.

The effects of the cardiotonic dihydropyridine derivatives Bay k 8644 and H160/51 on post-rest adaptation of guinea-pig papillary muscles.

In isolated guinea-pig papillary muscle, the effect of the two dihydropyridine derivatives Bay k 8644 (0.03-10 mumol/l) and H160/51 (0.1-3 mumol/l) on transmembrane action potentials and force of contraction were investigated at regular stimulation (1 Hz) and after a period of rest (10 min). The following results were obtained: At regular stimulation of the preparations, Bay k 8644 and H160/51 enhanced force of contraction without affecting time-to-peak tension. The time required for relaxation and the action potential duration were prolonged. These effects were transient with exposure to high concentrations of Bay k 8644 (greater than 3 mumol/l). The amplitude of the post-rest contraction thought to depend entirely on transmembrane calcium influx was small under control conditions and increased because of prolongation in time-to-peak tension in the presence of either dihydropyridine derivative. Isoprenaline (30 nmol/l) - as opposed to Bay k 8644 and H160/51 - increased the rate of force development of post-rest contractions. Bay k 8644 and H160/51 prolonged the duration of the first action potential after 10 min of rest. In the course of adaptation to steady state stimulation this prolongation transiently increased further resulting in a biphasic pattern which was attenuated by addition of nifedipine. With isoprenaline the biphasic pattern changed into a monotonous adaptation to pre-rest control. Our results show that the small enhancement of the post-rest contraction in the presence of Bay k 8644 or H160/51 is due to prolonged action potential duration after rest, whereas isoprenaline enhances the intensity of post-rest activation.(ABSTRACT TRUNCATED AT 250 WORDS)

Muscarinic receptors in mammalian myocardium: effects of atrial and ventricular receptors on phosphatidylinositol metabolism and adenylate cyclase.

Cat myocardium was used to investigate muscarinic receptor function in atria and ventricles. Carbachol and oxotremorine were used to determine agonist binding to the muscarinic receptors. It was found that carbachol was bound with almost the same characteristics in atria (KdH 1 microM; KdL 150 microM) as in ventricles (KdH 3 microM; KdL 150 microM). However, in the presence of guanylylimidodiphosphate Gpp(NH)p a difference was apparent so that the ventricular curve was shifted to a majority of low affinity sites whereas in atria two affinity sites remained even if the guanine nucleotide concentration was increased to 10 mM. Oxotremorine was bound with almost equal affinity in both atria and ventricles. The addition of Gpp(NH)p left all the receptors in the low affinity state irrespective of receptor localisation. The two agonists were also used to determine inhibition of adenylate cyclase activity. It was shown that the magnitude of adenylate cyclase inhibition was more pronounced in ventricles than in atria whether it was induced by oxotremorine or carbachol. When the effects of muscarinic agonists were determined on phosphatidylinositol metabolism it was shown that carbachol mediated a greater effect in atria than in ventricles. Almost no effect was seen with oxotremorine on phosphatidylinositol breakdown. Pirenzipine binding showed the presence of M1 receptors both in atria and ventricles. On the basis of diversity of muscarinic agonists on function and receptor occupancy it is suggested that heterogeneity exists for muscarinic receptors in both atria and ventricles.

The enantiomers of the dihydropyridine derivative H 160/51 show opposite effects of stimulation and inhibition.

H 160/51 is a calcium agonistic 1,4-dihydropyridine. Its optical isomers were now found to have opposing actions in the cat papillary muscle and rat portal vein. The inhibitory (+)-enantiomer had potencies of the same order as those of the stimulatory (-)-enantiomer. However the racemate and the (-)-enantiomer had approximately the same potencies and intrinsic activities. A model is presented to account for the fact that the effects of the inhibitory (+)-enantiomer can be concealed in the effects of the racemate of H 160/51.

Studies on cardiac distribution and function of neuropeptide Y.

High concentrations of a novel peptide, neuropeptide Y, have been demonstrated in the guinea-pig and canine heart and in the latter, a particularly high concentration was found in the region of the coronary vasculature (126 +/- 31 pmol g-1). Intra-arterial infusion of neuropeptide Y for 30 s into the coronary artery of the intact, innervated dog heart resulted in a rapid and short-lasting reduction of blood flow from 38 +/- 4 to 31 +/- 3 ml min-1 (P less than 0.05) to resume control level, 39 +/- 5 ml min-1, within 5 min. These injections were unaccompanied by changes in heart rate and aortic pressure, while there was an associated small reduction in dP/dt, used as a measure for changes in contractility. In vitro studies using the isolated, paced papillary muscle from cat, guinea-pig and rat, and spontaneously beating right atria from the guinea-pig, demonstrated no effect of NPY on active tension or beating frequency. The results indicate that NPY has vasoconstrictor properties, but under the test circumstances to lack both positive and negative inotropic and chronotropic effects.

Adrenergic nerves to the rat parotid gland originating in the contralateral sympathetic chain.

The parotid gland of the rat seems to receive some adrenergic nerves from the sympathetic chain of the opposite side. This is suggested by the following evidence: after unilateral removal of the superior cervical ganglion, parotid tissue from the contralateral gland shows degeneration secretion of amylase in vitro similar to, but much smaller than that known to occur ipsilaterally. When parotid secretion is evoked parasympathetically in the anesthetized rat, superimposed stimulation of the contralateral cervical sympathetic trunk can be shown to increase the secretion of amylase into this parasympathetic saliva; as it does, much more, ipsilaterally. It may also cause an evanescent decrease of the salivary flow, suggesting that not only secretory, but also vasoconstrictor nerves had been activated. After removal of one sympathetic ganglion, some undergenerated adrenergic nerves remain ipsilaterally, as earlier demonstrated; but no such fibers can be detected when the ganglion has been removed on both sides.