Changes in the intracranial rheoencephalogram at lower limit of cerebral blood flow autoregulation.

Cerebral blood flow (CBF) reactivity monitoring is an appropriate primary parameter to evaluate cerebral resuscitation due to a systemic or regional cerebral injury leading to possible irreversible brain injury. Use of the electrical impedance method to estimate CBF is rare, as the method's anatomical background is not well understood. Use of intracranial rheoencephalography (iREG) during hemorrhage and comparison of iREG to other CBF measurements have not been previously reported. Our hypothesis was that iREG would reflect early cerebrovascular alteration (CBF autoregulation). Studies comparing iREG, laser Doppler flowmetry and ultrasound were undertaken on anesthetized rats to define CBF changes during hemorrhage. Blood was removed at a rate required to achieve a mean arterial blood pressure (MABP) of 40 mm Hg over 15 min. Estimation of CBF was taken with intracranial, bipolar REG (REG I; n=14), laser Doppler flowmetry (LDF; n=3) and carotid flow by ultrasound (n=11). Data were processed off-line. During the initial phase of hemorrhage, when MABP was close to 40 mm Hg, intracranial REG amplitude transiently increased (80.94%); LDF (77.92%) and carotid flow (52.04%) decreased and changed with systemic arterial pressure. Intracranial REG amplitude change suggests classical CBF autoregulation, demonstrating its close relationship to arteriolar changes. The studies indicate that iREG might reflect cerebrovascular responses more accurately than changes in local CBF measured by LDF and carotid flow. REG may indicate promise as a continuous, non-invasive life-sign monitoring tool with potential advantages over ultrasound, the CBF measurement technique normally applied in clinical practice. REG has particular advantages in non-hospital settings such as military and emergency medicine.

Role of complement activation in hypersensitivity reactions to doxil and hynic PEG liposomes: experimental and clinical studies.

Pegylated liposomal doxorubicin (Doxil) and 99mTc-HYNIC PEG liposomes (HPL) were reported earlier to cause hypersensitivity reactions (HSRs) in a substantial percentage of patients treated i.v. with these formulations. Here we report that (1) Doxil, HPL, pegylated phosphatidylethanolamine (PEG-PE)-containing empty liposomes matched with Doxil and HPL in size and lipid composition, and phosphatidylglycerol (PG)-containing negatively charged vesicles were potent C activators in human serum in vitro, whereas small neutral liposomes caused no C activation. (2) Doxil and other size-matched PEG-PE and/or PG-containing liposomes also caused massive cardiopulmonary distress with anaphylactoid shock in pigs via C activation, whereas equivalent neutral liposomes caused no hemodynamic changes. (3) A clinical study showed more frequent and greater C activation in patients displaying HSR than in non-reactive patients. These data suggest that liposome-induced HSRs in susceptible individuals may be due to C activation, which, in turn, is due to the presence of negatively charged PEG-PE in these vesicles.

Liposome-induced pulmonary hypertension: properties and mechanism of a complement-mediated pseudoallergic reaction.

Intravenous injection of liposomes can cause significant pulmonary hypertension in pigs, a vasoconstrictive response that provides a sensitive model for the cardiopulmonary distress in humans caused by some liposomal drugs. The reaction was recently shown to be a manifestation of "complement activation-related pseudoallergy" (CARPA; Szebeni J, Fontana JL, Wassef NM, Mongan PD, Morse DS, Dobbins DE, Stahl GL, Bünger R, and Alving CR. Circulation 99: 2302-2309, 1999). In the present study we demonstrate that the composition, size, and administration method of liposomes have significant influence on pulmonary vasoactivity, which varied between instantaneously lethal (following bolus injection of 5 mg lipid) to nondetectable (despite infusion of a 2,000-fold higher dose). Experimental conditions augmenting the pulmonary hypertensive response included the presence of dimyristoyl phosphatidylglycerol, 71 mol% cholesterol, distearoyl phosphatidylcholine, and hemoglobin in liposomes, increased vesicle size and polydispersity, and bolus injection vs. slow infusion. The vasoactivity of large multilamellar liposomes was reproduced with human C3a, C5a, and xenoreactive immunoglobulins, and it correlated with the complement activating and natural antibody binding potential of vesicles. Unilamellar, monodisperse liposomes with 0.19 +/- 0.10 microm mean diameter had no significant vasoactivity. These data indicate that liposome-induced pulmonary hypertension in pigs is multifactorial, it is due to natural antibody-triggered classic pathway complement activation and it can be prevented by appropriate tailoring of the structure and administration method of vesicles.

Heterogeneous nuclear ribonucleoprotein D0 contains transactivator and DNA-binding domains.

Heterogeneous nuclear ribonucleoprotein D0 (hnRNP D0) is an abundant, ubiquitous protein that binds RNA and DNA sequences specifically, and has been implicated in the transcriptional regulation of the human complement receptor 2 gene. We found that in vivo expression of hnRNP D0-GAL4 fusion proteins increased the transcriptional activity of a GAL4-driven reporter gene, providing direct proof that hnRNP D0 possesses a transactivator domain. We found, using truncated hnRNP D0 proteins fused to GAL4, that 29 amino acids in the N-terminal region are critical for transactivation. We established, using a series of recombinant truncated hnRNP D0 proteins, that the tandem RNA-binding domains alone were not able to bind double-stranded DNA. Nevertheless, 24 additional amino acids of the C-terminus imparted sequence-specific DNA binding. Experiments using peptide-specific antisera supported the importance of the 24-amino-acid region in DNA binding, and suggested the involvement of the 19-amino-acid alternative insert which is present in isoforms B and D. The N-terminus had an inhibitory effect on binding of hnRNP D0 to single-stranded, but not to double-stranded, DNA. Although both recombinant hnRNP D0B and D0D bound DNA, only the B isoform recognized DNA in vivo. We propose that the B isoform of hnRNP D0 functions in the nucleus as a DNA-binding transactivator and has distinct transactivator and DNA-binding domains.

C5a receptor expression by TGW neuroblastoma cells.

We recently reported that not only lymphoid cells, but cells of neuronal origin may harbor C5a receptors (C5aR) as suggested by results of RT-PCR testing and that an apoptotic pathway is associated with the C5aR. To determine whether C5aR is expressed as an integral membrane protein, we generated mono- and polyclonal anti-C5aR antibodies. Flow cytometry showed a low-level expression of C5aR in TGW neuroblastoma cells. Epitope mapping suggested that a conformation change in C5aR occurs when exposed to C5a. Although an aphysiologically high concentration of C5a is necessary for inducing a transient increase in the intracellular Ca2+ level, TGW cells do employ the signal transduction pathway associated with C5aR, suggesting that these cells may serve as putative model for C5aR-expressing neurons.

Estrogen receptor immunoreactivity is present in the majority of central histaminergic neurons: evidence for a new neuroendocrine pathway associated with luteinizing hormone-releasing hormone-synthesizing neurons in rats and humans.

The central regulation of the preovulatory LH surge requires a complex sequence of interactions between neuronal systems that impinge on LH-releasing hormone (LHRH)-synthesizing neurons. The reported absence of estrogen receptors (ERs) in LHRH neurons indicates that estrogen-receptive neurons that are afferent to LHRH neurons are involved in mediating the effects of this steroid. We now present evidence indicating that central histaminergic neurons, exclusively located in the tuberomammillary complex of the caudal diencephalon, serve as an important relay in this system. Evaluation of this system revealed that 76% of histamine-synthesising neurons display ERalpha-immunoreactivity in their nucleus; furthermore histaminergic axons exhibit axo-dendritic and axo-somatic appositions onto LHRH neurons in both the rodent and the human brain. Our in vivo studies show that the intracerebroventricular administration of the histamine-1 (H1) receptor antagonist, mepyramine, but not the H2 receptor antagonist, ranitidine, can block the LH surge in ovariectomized estrogen-treated rats. These data are consistent with the hypothesis that the positive feedback effect of estrogen in the induction of the LH surge involves estrogen-receptive histamine-containing neurons in the tuberomammillary nucleus that relay the steroid signal to LHRH neurons via H1 receptors.

Complement C5a anaphylatoxin fragment causes apoptosis in TGW neuroblastoma cells.

Human neuroblastoma TGW cells express a C5a anaphylatoxin receptor-like molecule termed neuronal C5a receptor. A C5a-receptor fragment peptide (termed PR226-multiple antigenic peptide) can induce rapid apoptosis in TGW cells via neuronal C5a receptor-associated signal transduction pathways. In order to analyse role of activated complement system in neurodegeneration, TGW cells were exposed to an oligomer form of a C5a fragment (amino acids: 37-53) peptide termed PL37-multiple antigenic peptide. Upon treatment with PL37-multiple antigenic peptide, an increased nuclear c-fos expression was shown within 30 min. DNA fragmentation, a hallmark of apoptosis, was noted within 4 h. Extracellular administration of 100 nM PL37-multiple antigenic peptide evoked inward calcium current pulses. At higher doses (0.5 microM-1 microM), PL37-multiple antigenic peptide evoked higher current pulses, followed by an irreversible, high inward current. To exert its apoptotic effect, PL37-multiple antigenic peptide utilizes a pertussis toxin-sensitive signal transduction pathway associated with the neuronal C5a receptor. Activation of the complement system and therefore release of C5a has already been reported in Alzheimer's disease. In addition, the presence of the Kunitz-type proteinase inhibitors indicates an impaired protease function and a possible abnormal fragmentation of C5a anaphylatoxin. Our data suggest that neurons expressing neuronal C5a receptor are more vulnerable to the apoptosis associated with the neuronal C5a receptor and the possibility that abnormal activation of C5a receptor and C5a anaphylatoxin fragments might be involved in the pathogenesis of Alzheimer's disease.

A neuronal C5a receptor and an associated apoptotic signal transduction pathway.

1. We report the first experimental evidence of a neuronal C5a receptor (nC5aR) in human cells of neuronal origin. Expression of nC5aR mRNA was demonstrated by the reverse transcriptase-polymerase chain reaction (RT-PCR) in TGW human neuroblastoma cells. 2. Expression of a functional C5aR was supported by the finding that C5a evoked a transient increase in the intracellular calcium level as measured by flow cytometry (FACS). 3. To analyse the function of the nC5aR, an antisense peptide fragment of the C5aR was used. Previous data showed that a C5aR fragment (a peptide termed PR226) has C5aR agonist and antagonist effects in U-937 cells depending on the concentration of the peptide. We found that a multiple antigenic peptide (MAP) form of the same peptide (termed PR226-MAP) induced rapid elevation of nuclear c-fos immunoreactivity and resulted in DNA fragmentation, a characteristic sign of apoptosis, in TGW cells. 4. Early electrophysiological events characteristic of apoptosis were also detected: intermittent calcium current pulses were recorded within 1-2 min of peptide administration. C5a pretreatment delayed the onset of this calcium influx. 5. We also demonstrated that the apoptotic pathway is linked to nC5aR via pertussis toxin-sensitive G-proteins. 6. Although the function of C5a and its receptor on neurons is unknown, these results suggest that an abnormal activation of this signal transduction pathway can result in apoptosis and, subsequently, in neurodegeneration.

Endothelin (ET)-1 induced mucosal damage in the rat small intestine: role of ET(A) receptors.

The role of endothelin (ET)-1 as a mediator of small intestinal mucosal perfusion failure and tissue damage was investigated in the rat using intravital fluorescence videomicroscopy. The effects of intravenous infusion of ET-1 (3 nmol/kg) on functional capillary density, mucosal thickness, and the degree of mucosal damage were evaluated. Administration of ET-1 caused pronounced mucosal injury with a significant reduction of mucosal thickness compared with vehicle-treated control animals. Concomitantly, villous functional capillary density was markedly reduced 30 and 90 min after the infusion of ET-1. ET(A) receptor blockade by pretreatment with BQ 610 or with the novel ET(A) receptor antagonist ETR-P1/FL peptide prevented ET-1 induced capillary perfusion failure and mucosal damage. In contrast, the ET(B) receptor antagonist IRL 1038 was not effective. These results indicate that, acting via the ET(A) receptor, elevated levels of circulating ET-1 under various pathophysiological conditions, such as septic or hemorrhagic shock, might impair nutritive perfusion of the intestinal mucosa and contribute to tissue injury.

Antisense homology box-derived peptides represent a new class of endothelin receptor inhibitors.

Several peptides encoded by the sense and corresponding antisense DNA have been found to recognize and bind to each other. We developed software to search for sense-antisense regions within proteins taking into account the degeneracy of the genetic code, i.e., one amino acid can have several "antisense" counterparts. Using this approach, we searched endothelin receptor type A for intramolecular regions related in sense-antisense fashion. After locating these regions (termed "antisense homology boxes"), several corresponding peptides were synthesized. The four new ET(A) receptor fragment peptides ETR-P1 ("CALSVDRYRAVASW"), ETR-P3 ("QGIGPLITAIEI"), ETR-P4 ("IADNAERYSANLSSHV") and ETR-P6 ("LNRRNGSLRIALSEHLKNRREVA") reported here can inhibit ET-1 activity.

Endothelin 1 induces leukocyte adhesion in submucosal venules of the rat small intestine.

BACKGROUND & AIMS: The release of endothelin 1 (ET-1) and the activation of leukocytes are involved in the pathophysiology of gastrointestinal ischemia/reperfusion injuries. The aim of this study was to define the in vivo relation between ET-1 and endothelial cell-leukocyte interactions. METHODS: Anesthetized rats were studied to characterize the microvascular effects of increasing doses of local and systemic infusions of ET-1 in all layers of an ileal segment. Leukocyte-endothelial interactions were monitored with intravital fluorescence videomicroscopy. The ETA receptor-selective antagonist BQ 610, the novel ETA-receptor antagonist ETR-PI/fl peptide, and the ETB-receptor antagonist IRL 1038 were used to investigate the roles of receptor subtypes. RESULTS: The functional capillary density of the mucosa was significantly decreased by 3 nmol/kg intravenous ET-1. After 30 minutes the rolling fraction of leukocytes reached 90% in the postcapillary venules, and the number of adherent leukocytes was significantly increased after 90 minutes. ETR-PI/fl peptide inhibited leukocyte rolling by 88%, BQ 610 by 73%, and IRL 1038 by 30%. Both ETA-receptor antagonists prevented ET-1-induced firm adhesion. The ETA-receptor antagonists but not IRL 1038 inhibited the ET-1-induced lymphatic muscle and mucosal capillary perfusion failure. CONCLUSIONS: ET-1 induces leukocyte rolling and adherence through a predominantly ETA receptor-mediated mechanism in the submucosal venules of the intestinal microcirculation.

Effects of antiendothelin treatment on the early hemodynamic changes in hyperdynamic endotoxemia.

We have performed a series of experiments to study the effects of a newly developed antisense homology box-derived endothelin (ET) antagonist peptide (ETR-P1/fl) on the early hemodynamic changes in a hyperdynamic endotoxemic dog model. Mean arterial pressure (MAP), cardiac output (CO) and myocardial contractility (MC) were measured in closed-chest animals. Plasma levels of ET-1,2 were determined by radioimmunassay. A hyperdynamic circulatory response was elicited with a 2-hour infusion of 5.3 micrograms/kg of E. coli endotoxin (ETX). Control and ETX-treated animals received an infusion of ETR-P1/fl (0.1 mg/kg) i.v. ETX treatment decreased MAP and MC, increased initially CO, and a long lasting elevation in the plasma ET level was observed. In ETX-treated animals the administration of ETR-P1/fl significantly prolonged the increase in CO and inhibited the depression of MC. Our results suggest that treatment with the ET antagonist ETR-P1/fl may be advantageous in the early phase of endotoxemia.

Endothelin-1-induced circulatory response in the rat: the role of ETA and ETB receptors.

Production of the powerful vasoconstrictor endothelin-1 (ET1) is increased in a number of pathological conditions. This study was performed 1. to assess the effects of a twofold elevation of circulating ET1 on global hemodynamics and cardiac function, and 2. to determine the ET receptor subtypes that are responsible for this action. We have used the ETA receptor-selective antagonist BQ 610, the novel ETA receptor antagonist ETR-Pl/fl peptide and the specific ETB receptor antagonist IRL 1038 to investigate the role of these receptor subtypes in mediating circulatory changes induced by ET1 in anesthetized Wistar rats. ET1 infusion produced a significant rise in mean arterial pressure (MAP), elevated total peripheral resistance (TPR), and decreased cardiac output (CO). BQ 610 and ETR-Pl/fl pretreatment significantly attenuated the ET1-induced hemodynamic changes. Pretreatment with IRL 1038 had no effect on CO, but significantly reduced MAP and TPR elevation 20 min after ET1 infusion. These results suggest that ET1 may contribute to circulatory failure in conditions with increased ET1 production via a mechanism involving ETA receptors. ETB receptors, albeit to a lesser extent than ETA receptors, are also involved in mediating ET1-induced peripheral vasoconstriction in the rat.

Antisense homology boxes in C5a receptor and C5a anaphylatoxin: a new method for identification of potentially active peptides.

A computer program was designed to locate regions termed antisense homology boxes (AHB), i.e., 8-15 amino acid regions corresponding to complementary DNA strands. Two AHBs were found in C5a and eight within the C5a receptor. The majority of intermolecular AHBs were found to overlap those found in the intramolecular AHBs. Several AHB peptides were synthesized and tested for their ability to interfere with C5a receptor functions. A peptide fragment of the C5a receptor corresponding to the loop between the fifth and sixth hypothetical transmembrane regions (amino acids 226-245) antisense to C5a and an intramolecular AHB in C5a receptor proved to be an antagonist of C5a when preincubated with C5a at high concentrations (>0.5 microM). However, when U937 cells bearing the C5a receptor were preincubated with this peptide at a much lower concentration (even as little as 40 pmol), the AHB peptide behaved as an agonist. Another AHB peptide corresponding to region 10-27 in the C5a receptor bound to two of its corresponding antisense peptides derived from C5a anaphylatoxin, corresponding to amino acids 37-43 and 61-74. This observation raises the possibility that the C5a receptor may bind C5a with two distinct orientations. Two other AHB peptides derived from C5a, PL12 (amino acids 12-27), and PL61 (amino acids 61-74), were also shown to inhibit activity. Incubation of dibutyryl cyclic AMP-stimulated U937 cells with PL37 (amino acids 37-51) resulted in increased intracellular Ca2+ levels and an anergy to subsequent challenge with C5a. Locating regions with sense-antisense relationships in proteins might help in identification of peptides that can interfere with the function of target proteins.

Antagonistic peptides against human anaphylatoxin C5a.

Multivalent synthetic peptides derived from C5a were prepared in order to examine their effects on the C5a receptor (C5aR). Multiple antigen peptide (MAP) of the C5a C-terminal region (MAP61-74) bound to cells expressing C5aR with high affinity. On the other hand, N-terminal peptides (MAP3-16 and MAP12-26) and one with a sequence from the mid-portion of C5a (MAP37-53) did not bind to the cells. In addition, MAP61-74 inhibited Ca2+ mobilization and release of beta-hexosaminidase by C5a from dibutyryl cAMP-activated U937 cells. This Ca2+ mobilization was also inhibited by MAP12-26 and Mono61-74, the monomeric C-terminal peptide. Taken together, these data indicate that C5a binds to the C5aR via its C-terminal region. Furthermore, MAP61-74, a 14mer peptide that has additional amino acids at the N-terminal compared with the C-terminal octapaptide, can bind to C5aR and can be considered an antagonist of C5a which may prove useful as an agent for controlling the allergic response caused by complement activation.

The antisense homology box: a new motif within proteins that encodes biologically active peptides.

Amphiphilic peptides approximately fifteen amino acids in length and their corresponding antisense peptides exist within protein molecules. These regions (termed antisense homology boxes) are separated by approximately fifty amino acids. Because many sense-antisense peptide pairs have been reported to recognize and bind to each other, antisense homology boxes may be involved in folding, chaperoning and oligomer formation of proteins. The antisense homology box-derived peptide CALSVDRYRAVASW, a fragment of human endothelin A receptor, proved to be a specific inhibitor of endothelin peptide (ET-1) in a smooth muscle relaxation assay. The peptide was able to block endotoxin-induced shock in rats as well. Our finding of endothelin receptor inhibitor among antisense homology box-derived peptides indicates that searching proteins for this new motif may be useful in finding biologically active peptides.

The role of a complement regulatory protein in rat mesangial glomerulonephritis.

The host cells are protected from the indiscriminate attack of homologous complement by the membrane-associated complement regulatory proteins. A mouse monoclonal antibody (mAb) 512 (immunoglobulin G1 subclass) has recently been described that recognizes and inhibits the function of a rat complement regulatory protein, a rat homologue of mouse Crry/p65. The aim of this work is to assess the role of a complement regulatory protein (512Ag) recognized by mAb 512 in the complement-dependent glomerular injury induced by mAb OX7 against rat Thy-1.1. For the induction of mesangial injury, the left kidney of a rat was perfused with a combination of OX7 and 512 and the perfusate was discarded from the renal vein (Group I). After the renal artery and vein were restored, the left kidney was connected to the systemic circulation. Rats were euthanized 3 h, 2 days, and 14 days later. Rats perfused either with OX7 (Group II) or with 512 (Group III) or with vehicle only (Group IV) were used as controls. At 3 h, rats of Group I showed more prominent cellular infiltration and mesangial lysis and more C3 deposition in the glomeruli than rats of Group II. Rats of Groups III and IV showed no significant changes. At Day 2, there was still significant mesangial lysis and leukocyte infiltration in Group I rats, whereas rats in other groups showed an almost normal appearance. Glomerular injury in Group I rats returned to normal by Day 14.

Partial characterization of a low molecular weight phagocytosis inhibitory factor obtained from human erythrocyte membranes.

Phagocytosis Inhibitory Factor (PIF), a small (< 3000 D) molecule, was partially purified from human red blood cell membranes. This factor inhibits latex phagocytosis by monocytic cells. PIF is not toxic under the experimental conditions employed and the phagocytosis inhibitory activity is reversible since removal of this factor restores the phagocytic capability of cells. The phagocytic activity of murine macrophages was not affected by PIF.

In vivo effects of monoclonal antibodies that functionally inhibit complement regulatory proteins in rats.

The present work was designed to evaluate the effects of functional suppression of complement regulatory proteins in vivo. Male Wistar rats were anesthetized with Nembutal and were intravenously injected with 1 mg/kg of F(ab')2 or Fab fraction of either monoclonal antibody 5I2, which inhibits the function of rat counterpart of mouse Crry/p65, or monoclonal antibody 6D1, which inhibits the rat counterpart of CD59. Mean arterial pressure was continuously measured for 30 min. When 5I2 was injected, there was a biphasic change of mean arterial pressure, namely, the rapid increase immediately after the injection (approximately 2 min, phase 1) and the subsequent fall and slow recovery (approximately 4-30 min, phase 2). These effects were completely abrogated by pretreatment of rats with cobra venom factor. Pretreatment with carboxypeptidase inhibitor, which inhibits inactivation of anaphylatoxins C3a and C5a, induced enhanced reduction of blood pressure. Circulating leukocytes and platelets were rapidly decreased 5 min after antibody injection and became normal by 2 h. Hematocrit and erythrocyte count were continuously increased up to 2 h after injection, suggesting that there was hemoconcentration due to increased vascular permeability. Immunofluorescence study revealed binding of antibody fragments and rat C3 along the capillaries of lung, heart, and liver 5 min after injection. In contrast to 5I2, F(ab')2 fraction of 6D1, though localized to the same areas and in similar amounts, had no significant effect on the parameters measured. These data suggest that the rat counterpart of mouse Crry/p65 plays a vital role in vivo by preventing the activation of autologous complement on vascular endothelium.