Urodilatin: a new peptide with beneficial effects in the postoperative therapy of cardiac transplant recipients

Summary Renal failure after heart transplantation (HTx) still remains a serious problem, especially when cyclosporin A is used for immunosuppression in the early postoperative therapy. To preserve good renal function without reducing immunosuppressive cyclosporin A treatment, we administered urodilatin (CDD/ANP-95-126) in a long-term, low-dose infusion in addition to the usual medication after heart transplantation. From November 1990 to June 1991, 51 patients (46 male and 5 female; mean age 48 years) were treated with a 620 ng/kg bw·min infusion for 96 h after HTx. The renal function and hemodynamic parameters of these urodilatin-treated patients were compared in this sequential study with 40 patients (33 male and 7 female; mean age 49 years) who had undergone HTx previously from May to November, 1990, as controls. In this phase IIa study, both groups did not differ significantly with respect to age, sex, indication for HTx, and preoperative renal function. In comparison with controls patients treated with urodilatin had a significantly better renal function: a reduction in the peak plasma creatinine (PC values day 4 : 1.5 ± 0.11 vs. 2.19 ± 0.19 mg/dl; P = 0.002), a lower peak serum urea (SU values day 4 : 109 ± 8 vs. 154.7 ± 8.94 mg/dl ; P = 0.0036), and a lower incidence of hemodialysis (6% vs. 10%) were observed. Adequate diuresis was maintained in spite of the reduction of furosemide by more than 60% (P = 0.005) on each day of urodilatin infusion in comparison with controls. The mean central venous pressure was significantly lower by about 50% (P = 0.02) during the administration of urodilatin in spite of reduced vasodilator medication with nitroglycerin. From this phase IIa study, we may conclude that urodilatin could be an important drug in intensive care treatment. For patients undergoing HTx, this peptide seems to be indicated for the improvement of renal function and cardiovascular status, especially in postoperative therapy using high-dose cyclosporin A treatment.Abbreviations ACE angiotensin converting enzyme - ANP atrial natriuretic polypeptide - ATG antithymocyte globulin - bpm beats per minute - bw body weight - CDD cardiodilatin - CDD/ANP-99-126 circulating form of vasorelaxant cardiac peptide - CHD coronary heart disease - CyA cyclosporin A - DCM dilated cardiomyopathy - GLM general linear model - hANP human atrial natriuretic polypeptide - HTx heart transplantation - NTG nitroglycerine - PC plasma creatinine - SU serum urea - SAS statistical analysing system     

Endogene Natriuretische und Ouabain-ähnliche Faktoren

Summary The existence of an endogenous natriuretic hormone and ouabain-like factors (OLF) has been postulated for many years. This postulate was based on our original observation that a small M.W. fraction in the serum after acute expansion of the extracellular fluid volume (ECFV) not only exhibited natriuretic activity but also inhibited the Na-K-ATPase enzyme in vitro similar to ouabain. Since then, numerous studies confirmed the presence of OLFs in serum, urine, cerebrospinal fluid, and various organs including the heart and hypothalamus. Some of these OLFs are well-known endogenous compounds, such as free unsaturated fatty acids, which inhibit in vitro transmembranous sodium transport, Na-K-ATPase and³H-ouabain binding to its membrane receptor or crossreact with digoxin antibodies. Chemically yet undefined OLFs of potentially hypothalamic origin were detected in various models of experimental and clinical hypertension and are suggested to play a pathophysiological role especially in salt- and volume-dependent forms of hypertension. Our results show that OLFs isolated from the urine of salt-loaded healthy subjects strongly enhance basal and vasopressin-stimulated release of calcium in vascular smooth muscle cells and platelets similar to the effects we had observed with endothelin. This urine fraction also exhibits natriuretic activity which increases in parallel with sodium intake. Further chromatographic separation and amino acid analysis confirmed the peptidic nature (M.W.<1000) of the natriuretic factor(s). However, the two biological activities, namely natriuretic and ouabain-like activities, reside in distinct and chemically different compounds. In face of the previous discovery of the atrial natriuretic peptides (ANP) it is of special interest that very recent observations strongly suggest a natriuretic factor of non-cardiac origin to play an important role in the natriuresis that follows ECFV expansion. In addition, numerous experimental data point to an interaction between the ANP and OLF systems. They should stimulate once again the final identification of these yet unknown endogenous natriuretic and ouabain-like factors.

Die in dieser übersicht zitierten eigenen Untersuchungen wurden von der Deutschen Forschungsgemeinschaft, Bonn, dem Ministerium für Wissenschaft und Forschung des Landes Nordrhein-Westfalen (FA-2914, FA-8871, IVA6-402-046-87), Düsseldorf, und der Konrad-Adenauer-Stiftung, Bonn, unterstützt     

一种合成多肽——PLNG自由穿膜现象初探

目的 研究人工设计合成的多肽PLNG在不同作用环境条件下的跨膜运动现象。方法 体外培养不同组织来源的鼠细胞及CHO细胞、BEL细胞,用免疫荧光观察不同浓度、不同温度、不同反应时间条件下,PLNG的穿膜能力及PLNG对不同类型的细胞（CHO细胞、BEL细胞、成年大鼠肝细胞、幼大鼠肝细胞、成年大鼠心肌细胞、幼大鼠心肌细胞、成年大鼠神经细胞、幼大鼠神经细胞）的穿膜特性。结果 PLNG在不同作用环境条件下对细胞膜都有穿透作用,且进入细胞的量近乎相同。结论 实验观察到PLNG具有广谱的穿膜能力,这种穿膜能力在一定范围内对温度、时间及PLNG浓度不敏感;而且这种穿膜能力不受组织特异性的限制。     

脑梗死患者血浆神经肽Y、降钙素基因相关肽、神经降压素、胃动素动态变化及其临床意义

观察脑梗死(CI)患者神经肽Y(NPY)、神经降压素(NT)、胃动素(MTL)、降钙素基因 相关肽(CGRP)浓度变化及其临床意义。方法选择C162例,用放免法检测血浆NPY、NT、MTL与CGRP浓度。结果NPY、NT与MTL浓度显著高于对照组(P<0.0001);于发病后24h内显著升高,7d内达高峰,8～15d开始下降,15d后仍在较高水平。NPY浓度重型与大灶组显著高于轻型(P<0.05)与小灶组(P<0.01);发病积分≥6分组显著高于<6分组(P<0.05);高血压组显著高于正常血压组(P<0.05)。NT与MTL浓度重型组显著高于中(P<0.05)、轻型组(P<0.01);高血糖组显著高于正常血糖组(P<O.01)。CGRP浓度显著低于对照组(P<0.0001),发病24h内即显著降低,2～7d进一步降低,8～15d开始升高,15d后逐渐升至正常水平。重型与大灶组显著高于轻型(P<0.0001)、中型(P<0.01)与小灶组(P<0.01);伴发病积分≥6分组显著低于<6分组(P<0.05)。结论CI患者NPY、NT、MTL、CGRP浓度变化以7d内最显著,15d后逐渐恢复正常水平;四种浓度监测可作为判断CI患者病情严重程度、病灶大小及伴发病的实验室指标。     

Pilot study of an HLA-A2 peptide vaccine using flt3 ligand as a systemic vaccine adjuvant

A pilot vaccine study was conducted to test the safety and immunological efficacy of four monthly immunizations of an MHC class I peptide vaccine, the E75 HLA-A2 epitope from HER-2/neu, using flt3 ligand as a systemic vaccine adjuvant. Twenty HLA-A2-expressing subjects with advanced stage prostate cancer were randomly assigned to one of four immunization or treatment schedules: (a) Flt3 ligand (20 g/kg per day) administered subcutaneously daily for 14 days on a 28-day cycle, monthly for four months; (b) flt3 ligand course as above with the E75 peptide vaccine administered on day 7 of each flt3 ligand cycle; (c) flt3 ligand course as above with the E75 peptide vaccine administered on day 14 of each flt3 ligand cycle; or (d) E75 peptide admixed with granulocyte–macrophage colony-stimulating factor and administered intradermally once every 28 days, as has previously been reported. The primary endpoints of the study were the determination of safety and immunological efficacy in generating E75-specific T cells as determined by peptide-specific interferon-gamma ELIspot. Adverse events included one grade 3 skin reaction and the development of grade 2 autoimmune hypothyroidism in two subjects with preexisting subclinical autoimmune hypothyroidism. Dendritic cells were markedly increased in the peripheral blood of subjects receiving flt3 ligand with each repetitive cycle, but augmentation of antigen-presenting cells within the dermis was not observed. Apart from a single subject, no significant peptide-specific T-cell responses were detected by ELIspot, whereas delayed-type hypersensitivity responses were detectable in control subjects and in subjects receiving peptide vaccine early in the course of flt3 ligand administration. The absence of robust peripheral immune responses in the current study may be attributable to the small numbers of subjects or differences in the subject population. In addition, the inability of flt3 ligand to augment the number of peripheral skin antigen-presenting cells may have contributed to the absence of robust peptide-specific immunity detectable in the peripheral blood of immunized subjects treated with flt3 ligand.     

The renal nerves in the newborn rat

Immunocytochemical methods were used to investigate the distribution of afferent [calcitonin gene-related peptide-(CGRP) immunoreactive and substance P-immunoreactive] nerves and efferent (neuropeptide Y-immunoreactive and dopamine -hydroxylase-immunoreactive) nerves in the kidneys of rats within the 1st day of life. The newborn rat kidney possesses an afferent and efferent innervation. Both afferent and efferent nerves reach the kidney in the same bundles. The afferent sensory fibers predominate overwhelmingly in the renal pelvis and ureter while the efferent fibers clearly predominate in the vasculature. The corticomedullary connective tissue contains both types of innervation with a more prominent afferent innervation (CGRP immunoreactive). Only afferent arterioles of perihilar nephrons were innervated by efferent sympathetic fibers. The distribution and extent of afferent and efferent innervation is consistent with the renal nerves playing a significant role in the transition from fetal to newborn life. The close proximity between afferent and efferent fibers suggests a possible interaction between the two systems.     

Evaluation of Genetic Stability of Recombinant Human Factor VIII by Peptide Mapping and On-line Mass Spectrometric Analysis

Purpose. The genetic stability of a recombinant human factor VIII (rhFVIII) product expressed in Chinese hamster ovary cells (Recombinate) has been evaluated through comparisons of the protein produced at the beginning, middle and end of a typical production campaign. Methods. Recombinant human factor VIII was incubated with thrombin, the resulting four polypeptides were isolated by RP-HPLC, subjected to proteolysis with trypsin, and the peptide mixtures were resolved by RP-HPLC. Tryptic peptide mixtures were subjected to online mass spectrometric analysis using an electrospray ionization source interfaced to a quadrupole mass analyzer scanning from 1950–200 amu, and the peptide ion data were compared for three lots produced from the beginning, middle and end of a production campaign. Results. The UV elution profiles for each of the rhFVIIIa polypeptides were highly similar for factor VIII isolated from the beginning, middle and end of production. Total ion data from the peptide maps derived from three lots of rhFVIII were compared by MH¹⁺ values as a function of scan range. A total of 918 ions were analyzed for the four polypeptides of rhFVIII produced at the beginning, middle and end of a production campaign. The ions were detected at the same relative retention times, as indicated by the similar scan numbers for the three lots. Conclusions. These observations support that rhFVIII preparations produced from the beginning, middle and end of a production campaign were highly similar, and demonstrate genetic stability in the manufacturing process of Recombinate.     

Rat Jejunal Permeability and Metabolism of μ-Selective Tetrapeptides in Gastrointestinal Fluids from Humans and Rats

Purpose. To study intestinal transport and metabolism of three new -selective tetrapeptide enkephalin analogues, LEF537, LEF553 and TAPP These peptides are stabilized against enzymatic hydrolysis by having a D-aminoacid in position 2 and a blocked COOH-terminal. Methods. We used a single-pass perfusion technique to study the transport of the peptides in rat jejunum. To reduce luminal and/or brush-border metabolism during the perfusion we used protease inhibitors (Pefabloc^® SC, bestatin and thiorphan). The rate of metabolism was studied by incubations in rat jejunal homogenate, rat jejunal fluid and human gastric and jejunal fluid with and without these inhibitors. Results. The jejunal permeabilities (P_eff) of the peptides were 0.43–0.78 10^–4 cm/s without inhibitors and 0.09–0.45 10^–4 cm/s in presence of the inhibitors. All three peptides were rather rapidly degraded by enzymes in rat jejunal homogenate with half-lives of between 11.9 ± 0.5 and 31.7 ± 1.5 min. The addition of inhibitors to the homogenate prolonged the half-lives substantially for LEF553 (167 ± 35 min) and TAPP (147 ± 2 min), but only slightly for LEF537 (16.4 ± 0.5 min). LEF553 and TAPP were both hydrolyzed in rat and human jejunal fluid, while LEF537 was metabolized less in these fluids. When LEF553 and TAPP were incubated with intestinal fluid in the presence of inhibitors, metabolism was almost completely inhibited. There was no metabolism for any of the peptides in human gastric juice. Conclusions. The replacement of the terminal free carboxylic group with an amide group did not increase the stability of the peptides in jejunal tissue enough to allow successful oral drug delivery.     

Ultrastructural neuropathologic effects of Taxol on neurons of the freshwater snailLymnaea stagnalis

Summary Cerebral ganglia of the freshwater snailLymnaea stagnalis were incubatedin vitro in 10 M Taxol for 8 and 24 h. Cremophor EL (0.1%) was used as a diluant. The tissue was processed for electron microscopy. Various ultrastructural parameters were assessed quantitatively. Cremophor EL appeared to seriously affect the cell somata of the multipeptidergic caudodorsal cells. In the Cremophor-controls the mean area of Golgi zones, the percentage dense material (neuropeptides) in these zones, the number of large electron dense granules (these are involved in neuropeptide processing) and the mean nuclear heterochromatin clump size, were significantly smaller than in the Ringer-controls, whereas the number of lipid droplets was higher. All these parameters, except for the lipid droplets, were not different in the Cremophor-controls and the Taxol-treated specimens. After 24 h treatment, but not after 8 h, Cremophor EL furthermore induced an increase in the number of axonal microtubules. It is argued that the results might signify activation of the neurons by Cremophor EL. Taxol induced a significant increase in the number of microtubules in axons and cell somata. Furthermore an increase in the number of Golgi zones was observed, suggesting activated neuropeptide synthesis. In all groups immunostaining with antibodies to neuropeptides produced by the caudodorsal cells was normal. Release of neuropeptide (exocytosis) from axon endings was elevated after Taxol treatment, and exceptionally high in specimens cotreated with Taxol and Org 2766 (incubation time 22 h). The effect of Org 2766 and Taxol on the number of microtubules was cumulative. It is argued that transport of neuropeptide granules from the cell somata to the axon terminals was not affected by Taxol. It is concluded that Taxol neurotoxicity is probably not due to impeded microtubular axonal transport.