Romuald Bellmann

Neuropeptide Y biosynthesis is markedly induced in mossy fibers during temporal lobe epilepsy of the rat

Neuropeptide Y (NPY) immunoreactivity and gene expression was investigated in the hippocampus after kainic acid-induced seizures and pentylenetetrazol kindling in the rat. Pronounced increases of NPY immunoreactivity were found in the terminal field of mossy fibers in both animal models. In kainic acid-treated rats the peptide progressively accumulated in the hilus and the stratum lucidum of CA3, 5-60 days after injection of the toxin and, at the later intervals, extended to the supragranular molecular layer of the dentate gyrus indicating sprouting of these neurons. Unilateral injection of colchicine into the hilus abolished NPY staining of the mossy fibers. Using in situ hybridization, in both animal models markedly enhanced expression of prepro-NPY mRNA was observed in the granular layer, containing the perikarya of the mossy fibers. It is suggested that sustained expression of the neuromodulatory neuropeptide NPY, in addition to the observed plastic changes, may contribute to altered excitability of hippocampal mossy fibers in epilepsy. Neither somatostatin immunoreactivity nor gene expression were enhanced in granule cells/mossy fibers.

ENHANCED RATE OF EXPRESSION AND BIOSYNTHESIS OF NEUROPEPTIDE Y AFTER KAINIC ACID-INDUCED SEIZURES

Abstract: Recent studies have shown marked increases in brain content of neuropeptide Y (NPY) after seizures induced by intraperitoneal injection of kainic acid and after pentylenetetrazole kindling in the rat. We have now investigated possible changes in the rate of biosynthesis of NPY after kainic acid treatment, by using pulse‐labeling of the peptide and by determining prepro‐NPY mRNA concentrations. For pulse labeling experiments, [3H]tyrosine was injected into the frontal cortex, and the incorporation of the amino acid into NPY was determined after purifying the peptide by gel filtration chromatography, antibody affinity chromatography, and reversed‐phase HPLC. At 2 and 30 days after kainic acid treatment, the rate of tyrosine incorporation was enhanced by ∼380% in the cortex. In addition, concentrations of prepro‐NPY mRNA were determined in four different brain areas by hybridization of Northern blots with a complementary 32P‐labeled RNA probe 2, 10, 30, and 60 days after kainic acid treatment. Marked increases were observed in the frontal cortex (by up to 350% of controls), in the dorsal hippocampus (by 750%), and in the amygdala/pyriform cortex (by 280%) at all intervals investigated. In the striatum only a small, transient increase was observed. The data demonstrate increased expression of prepro‐NPY mRNA and an enhanced rate of in vivo synthesis of NPY as a result of seizures induced by the neurotoxin kainic acid.

Treatment of refractory cholestatic pruritus after liver transplantation with albumin dialysis

Albumin dialysis has been shown to improve the outcome in patients with cholestatic liver failure caused by chronic liver disease. This study reports 7 liver transplant recipients who were treated with albumin dialysis for intractable pruritus of different origin (ductopenic graft rejection, non‐anastomotic strictures, and recurrence of hepatitis C). Treatment with histamine (H1) blockers, opioid antagonists, and cholestyramine had not been effective. The Molecular Adsorbent Recirculating System (MARS; Teraklin, Rostock, Germany) was used for albumin dialysis. All patients presented with numerous scratch marks, 6 of whom had a pronounced icterus. Six patients (86%) responded to 3 consecutive treatments with significant reduction of pruritus. The mean pruritus score, which was quantified by a visual analog scale (VAS), decreased from 9.7 ± 0.5 to 3.7 ± 0.8 (SD). The mean duration of 1 treatment was 15.6 hours. The procedure was well tolerated by all patients. The mean total serum bilirubin in patients who responded to therapy declined from 19.11 ± 16.96 mg/dL (SD) before MARS therapy to 9.24 ± 3.52 mg/dL after treatment. The mean serum concentration of 3α‐hydroxy bile acids decreased from 192.67 ± 58.12 μmol/L (SD) to 42.33 ± 31.58 μmol/L (SD). Follow‐up in 3 cases showed sustained improvement of pruritus lasting for more than 3 months. In 3 patients, however, pruritus relapsed. One patient, who showed severe pruritus, without relevant elevation of serum bile acids before treatment, did not respond to albumin dialysis. Our data indicate that MARS is an effective therapeutic option for patients with intractable cholestatic pruritus. (Liver Transpl 2004;10:107–114.)

CHROMOGRANINS IN RAT BRAIN : CHARACTERIZATION, TOPOGRAPHICAL DISTRIBUTION AND REGULATION OF SYNTHESIS

The properties and distribution of chromogranins A, B and secretogranin II in rat brain were analyzed by quantitative immunoblotting. In contrast to endocrine tissues brain contains a significant amount of the proteoglycan form of chromogranin A. For secretogranin II a significant degree of endogenous proteolytic processing is apparent. Chromogranin A and secretogranin II had a similar topographical distribution with the highest concentrations found in the hypothalamus, amygdala/piriform cortex and hippocampus, whereas for chromogranin B by far the highest concentration was found in the cerebellum. Compared with adrenal medulla the concentration of all three proteins is low, however, secretogranin II appears relatively enriched. The synthesis of chromogranin A in brain does not depend on glucocorticoids since neither adrenalectomy nor dexamethasone treatment changed its levels. This is in contrast to adrenal medulla and to the anterior pituitary. Three days after kainic acid-induced seizures the levels of chromogranin A in frontal cortex and hippocampus were significantly elevated. For frontal cortex there was also an increase of the respective mRNA. This result establishes that the synthesis of chromogranin A can be regulated like that of neuropeptides.

Effect of Molecular Weight and Substitution on Tissue Uptake of Hydroxyethyl Starch A Meta-Analysis of Clinical Studies

BackgroundIntravenously infused hydroxyethyl starch (HES) can be found in urine, plasma and tissues. HES remaining in plasma and tissues is thought to increase the risk of clinical complications. HES solutions of lower molecular weight and substitution have been developed to increase urinary excretion and reduce plasma persistence. However, their effect on tissue uptake of HES has not been investigated in human subjects.ObjectiveOur objective was to test the hypothesis that lower molecular weight and substitution decrease tissue uptake of HES.Data sourcesComputer searches were performed of MEDLINE; EMBASE; the Cochrane Library; meeting abstract databases in surgery, anaesthesiology and intensive care; ClinicalTrials.gov; and Google. Supplementary sources were reference lists and electronic tables of journal contents. No time period or language restrictions were imposed.Study SelectionClinical studies were eligible for inclusion in the meta-analysis, if data were reported both for cumulative urinary excretion of HES over 24 hours after infusion and for plasma HES concentration at 24 hours.Data ExtractionData were extracted on 24-hour urinary excretion of HES, 24-hour HES plasma concentration, plasma volume, HES molecular weight and substitution, study design, type and demographics of subjects, indication for fluid infusion, and HES infusion regimen. Tissue uptake of HES was computed as the difference between the infused dose and the sum of urinary excretion and residual plasma HES at 24 hours.Data SynthesisTwenty-five clinical studies totalling 287 subjects were included. Tissue uptake of low-molecular-weight HES (≤200 kD) was 42.3% (95% confidence interval [CI] 39.6, 45.0) compared with 24.6% (CI 17.8, 31.4) for high-molecular-weight HES (p<0.001). Similarly, tissue uptake of lower-substitution HES (≤0.5) was 42.4% (CI 39.5, 45.3) versus 26.6% (CI 19.6, 33.6) for higher-substitution HES (p<0.001). Among the three most often investigated single HES solutions, tissue uptake of 130/0.4 (42.6%; CI 35.0, 50.2) and HES 200/0.5 (43.3%; CI 39.4, 47.2) closely coincided, whereas uptake of HES 450/0.7 (22.2%; CI 14.8, 29.6) was lower (p = 0.001 and p<0.001, respectively).ConclusionsThis meta-analysis did not support the hypothesis that lower molecular weight and substitution decrease tissue uptake of HES. Further clinical studies of HES tissue uptake are needed.

Limbic seizures cause pronounced changes in the expression of neurokinin B in the hippocampus of the rat

Immunohistological and in situ hybridization techniques were used to study the influence of kainic acid-induced seizures and of pentylenetetrazol kindling on neurokinin B immunoreactivity and neurokinin B mRNA in the rat hippocampus. Pronounced increases in neurokinin B immunoreactivity were observed in the terminal field of mossy fibres 10-60 days after intraperitoneal injection of kainic acid. These slow but persistent increases in immunoreactivity were accompanied by markedly enhanced expression of neurokinin B mRNA in the granule cells and in hilar interneurons adjacent to the granule cell layer. These changes were preceded by transient increases in neurokinin B mRNA and immunoreactivity in CA1 pyramidal cell layer two and 10 days after kainic acid, which, however, subsided later on. Pentylenetetrazol kindling caused similar increases in neurokinin B mRNA expression in granule cells and in CA1 pyramidal cells, but not in hilar interneurons. In CA1, increased neurokinin B message was present two days after termination of the kindling procedure but not after 10 days. Sixty days after kainic acid injection, neurokinin B immunoreactivity extended to the inner-third of the molecular layer of the dentate gyrus. After pentylenetetrazol kindling, a neurokinin B-immunoreactive band was observed in the infrapyramidal region of CA3. Lesions of the dentate granule cells by local injection of colchicine in kainic acid-treated rats abolished the supragranular neurokinin B-positive staining, whereas it was almost unchanged after transection of the ventral hippocampal commissure. These observations suggest that neurokinin B immunoreactivity may be located in ipsilateral mossy fibres undergoing collateral sprouting to the inner molecular layer or to the infrapyramidal region in CA3, respectively. Preprotachykinin A mRNA, which encodes for neurokinin A and substance P, and substance P immunoreactivity were not changed in the hippocampus of epileptic rats compared with untreated animals. The observed changes in neurokinin B immunoreactivity and mRNA indicate that specific functional and morphological changes may be induced in hippocampal neurons by recurrent limbic seizures.

Determination of amphotericin B, liposomal amphotericin B, and amphotericin B colloidal dispersion in plasma by high-performance liquid chromatography

Amphotericin B is a potent polyene antifungal drug for intravenous treatment of severe infections. It is used as amphotericin B-deoxycholate and in order to reduce amphotericin B toxicity as lipid-formulated complex (liposomal or colloidal dispersion). A sensitive and specific analytical method is presented for the separation of lipid-complexed and plasma protein-bound amphotericin B in human heparinized plasma. This separation, which is required for pharmacokinetic studies, is achieved by solid-phase extraction (SPE) via Bond Elut C18. The protein-bound amphotericin B has a higher affinity to the SPE material and is therefore retained, whereas the lipid-complexed amphotericin B is eluted in the first step. The recovery of the SPE was >75% for high concentrations and >95% for low concentrations. Quantification was performed by reversed-phase HPLC using a LiChrosorb-RP-8 column, UV detection (lambda=405 nm) and a mixture of acetonitrile-methanol-0.010 M NaH2PO4 buffer (41:10:49, v/v) as mobile phase. The retention time for amphotericin B under the given conditions was 6.7 min. The calibration curves were found to be linear (r > or = 0.999) in two different ranges (5.0-0.50 microg/ml and 0.50-0.005 microg/ml). Intra- and inter-day precision and accuracy fulfilled the international requirements. No interference from other drugs (typical broad medication for intensive-care patients) or common plasma components was detected in >400 samples analyzed.

Pharmacokinetics of Caspofungin in Critically Ill Patients on Continuous Renal Replacement Therapy

ABSTRACT Caspofungin pharmacokinetics was assessed in 27 critically ill patients, including 7 on continuous venovenous hemofiltration (CVVH), 8 on continuous venovenous hemodialysis (CVVHD), and 13 not requiring continuous renal replacement therapy (CRRT). Caspofungin exposure during CRRT was very similar to that of the control group and comparable to that in healthy volunteers. Caspofungin clearance by CRRT was very low. Therefore, the standard dosage of caspofungin is probably adequate for critically ill patients undergoing CVVH or CVVHD.

Levosimendan inhibits release of reactive oxygen species in polymorphonuclear leukocytes in vitro and in patients with acute heart failure and septic shock: a prospective observational study

IntroductionLevosimendan is an extensively investigated inodilator showing also cardioprotective and antiinflammatory effects. The aim of our study was to explore the influence of levosimendan on polymorphonuclear leucocytes (PMN), a main source of reactive oxygen species, in vitro and in patients with acute heart failure or septic myocardial depression.MethodsPMN isolated from healthy volunteers were incubated with levosimendan in vitro. After stimulation with N-formyl-Met-Leu-Phe (fMLP) or phorbol 12-myristate 13-acetate (PMA) respiratory burst was quantified using a fluorescent dye. Apoptosis and expression of cell adhesion molecules of PMN were measured by flow cytometry. For determination of in vivo effects patients with acute heart failure (n = 16) or septic cardiac failure (n = 9) receiving levosimendan treatment were enrolled consecutively. PMN were isolated to measure respiratory burst activity before treatment as well as one and two hours after initiation of levosimendan administration. Furthermore inflammatory, hemodynamic and renal function parameters were obtained.ResultsIn vitro, levosimendan suppressed respiratory burst activity in fMLP or PMA stimulated PMN in a dose dependent manner by 30 ± 11% (P < 0.001) at 100 ng/mL and by 27 ± 17% (P < 0.001) at 1000 ng/mL respectively. Markers of apoptosis and PMN cell adhesion molecule expression remained unaffected by levosimendan treatment.In vivo, levosimendan treatment for two hours resulted in a significant reduction of PMA stimulated oxidative burst by 45% (P < 0.01) and fMLP stimulated oxidative burst by 49% (P < 0.05) in patients with acute heart failure. In patients suffering from septic shock levosimendan treatment decreased oxidative burst activity in unstimulated, fMLP and PMA stimulated PMN by 48% (P < 0.05), 46% (P < 0.01) and 43% (P < 0.01) respectively.ConclusionsLevosimendan appears to exert distinct immunomodulatory effects by decreasing oxidative burst activity of PMN. This property might contribute to the previously described cardioprotective effects of the drug.

Stimulation of human skin fibroblast migration by the neuropeptide secretoneurin

Fibroblasts, besides other cells, are called upon when tissue sustains an immunological, mechanical or chemical injury. Fibroblasts migrate into the site of inflammation, proliferate and synthesize and remodel a new matrix. These cellular responses are mediated locally by the release of neuropeptides from sensory nerve endings. Secretoneurin is a newly discovered 33-amino acid neuropeptide derived from secretogranin II (chromogranin C), which is found in sensory afferent C-fibers. We show here that secretoneurin triggers the selective migration of human skin fibroblasts in vitro, but does not stimulate their proliferation. The attraction of human skin fibroblasts toward secretoneurin could be blocked by specific anti-secretoneurin antibodies and is mediated by the C-terminal fragment of the peptide. The observed activity of this sensory neuropeptide is the first description of a specific effect on human skin fibroblasts and suggests a role for secretoneurin in inflammation and wound healing.