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HIV-1 infection requires interaction of viral envelope protein gp160 with CD4 and a chemokine receptor, CCR5 or CXCR4 as entry coreceptor. We designed HIV-inhibitory peptides targeted to CCR5 using a novel computer program (ANTIS), which searched all possible sense-antisense amino acid pairs between proteins. Seven AHBs were found in CCR5 receptor. All AHB peptides were synthesized and tested for their ability to prevent HIV-1 infection to human T cells. A peptide fragment (LC5) which is a part of the CCR5 receptor corresponding to the loop between the fifth and sixth transmembrane regions (amino acids 222-240) proved to inhibit HIV-1IIIB infection of MT-4 cells. Interaction of these antisense peptides could be involved in sustaining HIV-1 infectivity. LC5 effectively indicated dose-dependent manner, and the suppression was enhanced additively by T20 peptide, which inhibits infection in vitro by disrupting the gp41 conformational changes necessary for membrane fusion. Thus, these results indicate that CCR5-derived AHB peptides could provide a useful tool to define the mechanism(s) of HIV infection, and may provide insight which will contribute to the development of an anti-HIV-1 reagent.

Cardiac anaphylaxis is a severe, life-threatening manifestation of acute hypersensitivity reactions to allergens and drugs. Earlier studies highlighted an amplifying effect of locally applied C5a on the process; however, the role of systemic complement (C) activation with C5a liberation in blood has not been explored to date. In the present study, we used the porcine liposome-induced cardiopulmonary distress model for 1) characterizing and quantifying peripheral C activation-related cardiac dysfunction; 2) exploring the role of C5a in cardiac abnormalities and therapeutic potential of C blockage by soluble C receptor type 1 (sCR1) and an anti-C5a antibody (GS1); and 3) elucidating the role of adenosine and adenosine receptors in paradoxical bradycardia, one of the symptoms observed in this model. Pigs were injected intravenously with different liposomes [Doxil and multilamellar vesicles (MLV)], zymosan, recombinant human (rhu) C5a, and adenosine, and the ensuing hemodynamic and cardiac changes (hypotension, tachy- or bradycardia, arrhythmias, ST-T changes, ventricular fibrillation, and arrest) were quantified by ranking on an arbitrary scale [cardiac abnormality score (CAS)]. There was significant correlation between CAS and C5a production by liposomes in vitro, and the liposome-induced cardiac abnormalities were partially or fully reproduced with zymosan, rhuC5a, adenosine, and the selective adenosine A1receptor agonist cyclopentyl-adenosine. The use of C nonactivator liposomes or pretreatment of pigs with sCR1 or GS1 attenuated the abnormalities. The selective A1blocker cyclopentyl-xanthine inhibited bradycardia without influencing hypotension, whereas the A2blocker 4-(2-{7-amino-2-(2-furyl)[1,2,4]triazolo[2,3-a][1,3,5]triazin-5-ylamino}ethyl)phenol (ZM-24135) had no such effect. These data suggest that 1) systemic C activation can underlie cardiac anaphylaxis, 2) C5a plays a causal role in the reaction, 3) adenosine action via A1receptors may explain paradoxical bradycardia, and 4) inhibition of C5a formation or action or of A1-receptor function may alleviate the acute cardiotoxicity of liposomal drugs and other intravenous agents that activate C.

We designed a new computer program (MIMETIC), which generates a series of peptides for interaction with a target peptide sequence. The genetic algorithm employed ranks the sequences obtained from one generation to the next by "goodness of fit" to the target. MIMETIC designed recognition peptides to various regions of HIV-1 reverse transcriptase. Among ten peptide candidates synthesized, three inhibited reverse transcription in vitro. TLMA2993 and PSTW1594 both targeted the connection domain of reverse transcriptase and ESLA2340 targeted the thumb domain.

Activation of the complement system on the cell surface results in the insertion of pore forming membrane attack complexes (MAC, C5b-9). In order to protect themselves from the complement attack, the cells express several regulatory molecules, including the terminal complex regulator CD59 that inhibits assembly of the large MACs by inhibiting the insertion of additional C9 molecules into the C5b-9 complex. Using the whole cell patch clamp method, we were able to measure accumulation of homologous MACs in the membrane of CD59(-) human B-cells, which formed non-selective ion channels with a total conductance of 360 +/- 24 pS as measured at the beginning of the steady-state phase of the inward currents. C5b-8 and small-size MAC (MAC containing only a single C9) can also form ion channels. Nevertheless, in CD59(+) human B-cells in spite of small-size MAC formation, an ion current could not be detected. In addition, restoring CD59 to the membrane of the CD59(-) cells inhibited the serum-evoked inward current. The ion channels formed by the small-size MAC were therefore sealed, indicating that CD59 directly interfered with the pore formation of C5b-8 as well as that of small-size C5b-9. These results offer an explanation as to why CD59-expressing cells are not leaky in spite of a buildup of homologous C5b-8 and small-size MAC. Our experiments also confirmed that ion channel inhibition by CD59 is subject to homologous restriction and that CD59 cannot block the conductivity of MAC when generated by xenogenic (rabbit) serum.

Large anionic multilamellar liposomes containing 71% membrane cholesterol (MLV) caused complement (C) activation in human serum in vitro, as reflected in significant rises in S protein-bound terminal complex (SC5b-9) and C3a-desarg levels. Increasing the albumin content in serum by 1–4 g/100 ml led to 50–100% further increase in MLV-induced C activation, while higher amounts of exogenous human serum albumin (HSA) gradually lost the capability to potentiate liposomal C activation. HSA alone had no influence on SC5b-9 formation at any level below 12%. Complement activation by liposomes and the potentiating effect of supplemental HSA were greatly reduced or eliminated in the absence of C1q or in the presence of 10 mM EGTA/2.5 mM Mg2+, pointing to the involvement of the classical pathway. Potentiation of C activation by supplemental HSA was not unique to MLV-induced activation, as deposition of HSA on the membrane of ‘Centricon’ ultrafiltration units also potentiated the C-activating effect of the polycarbonate membrane. Fatty acid (FA) or non-monomeric protein contamination in HSA were unlikely to be playing a role in the described effects, as 96% pure, FA-rich (Buminate) and 99% pure, FA-free HSA had identical effects on liposomal C activation. While highlighting a new modulatory mechanism on liposomal C activation, the above data raise the possibility that deposition of extravasated HSA at sites of tissue injury may serve a hitherto unrecognized proinflammatory function.

Item does not contain fulltext 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.

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.

Liposomal formulations of some drugs, most importantly pegylated liposomal doxorubicin (Doxil®), have been reported to cause immediate hypersensitivity reactions that cannot be explained with the conventional paradigm of IgE-mediated (type I) allergy. Here we present a rationale and experimental evidence for the concept that these reactions represent a novel type of drug-induced hypersensitivity that can be called complement (C) activation-related pseudoallergy (CARPA). The theoretical foundation includes the facts that 1) some liposomes have been known to activate C, 2) most of the clinical symptoms of liposome-induced reactions coincide with those caused by C activation by other activators, and 3) the C mechanism explains those manifestations which are atypical for type 1 reactions. The experimental evidence includes the observations that 1) Doxil caused massive C activation in a high ratio (4/10) of normal human sera, 2) high dose IgG attenuated Doxil-induced C activation in serum and prevented...

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 Ca 2+ 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.

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.

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.

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 ET A receptor–selective antagonist BQ 610, the novel ET A -receptor antagonist ETR-Pl/fl peptide, and the ET B -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-Pl/fl peptide inhibited leukocyte rolling by 88%, BQ 610 by 73%, and IRL 1038 by 30%. Both ET A -receptor antagonists prevented ET-1–induced firm adhesion. The ET A -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 ET A receptor–mediated mechanism in the submucosal venules of the intestinal microcirculation. GASTROENTEROLOGY 1998;114:103-114

Recent advances in the genetic modification of primary cells for ex vivo gene therapy have brought the field close to the promise envisioned almost a quarter of a century ago. The integration of vector systems that can stably transduce cells without genotoxicity, combined with superior cell processing methods, has been largely responsible for these advances. The result has been a series of ongoing clinical trials that are showing promising outcome for patients. Among the successes are adoptive T cell immunotherapy and stem cell therapies for cancer and other diseases.

The field of possible application of mesenchymal stem cells in medicine and research expanded tremendously with the advent of improved Lentiviral-vectors capable of inserting stable copies of genes of interest and expressing proteins or biologically active RNA species ad libitum, performing delicate gene editing or active gene silencing or serving as advanced drug delivery systems utilized in ex vivo cell therapy. The combination of these two fields has created a number of new areas of research in the landscape of modern medicine which are now extensively studied and discussed here. These areas include tissue engineering, tissue repair, wound healing and tissue implants, anticancer therapies, angiogenesis, myocardial infarction and repair as well as understanding and treating acute lung damage and injury. In addition, genetically modified, tagged MSCs are being intensively deployed in research and therapeutic attempts of the various ailments of the central nervous system including Parkinson’s disease, Alzheimer’s disease, various phases of acute ischemia and trauma. The emergence of new and important data for type II diabetes research is being followed with treatment suggestions and studies of senescence to find novel applications for genetically engineered MSCs. We find in ­general that genetically modified MSCs are at the cusp of breaking through from basic research into the next phase of clinical trials.

Transduction with Lentiviral vectors has been shown to be the most efficient method for the stable delivery of nucleic acid sequences into mammalian cells. Lentiviral vectors have been widely used in research and have recently shown success in clinical trials for human gene therapy. In this paper, we describe the use of lentiviral vectors to generate genetically modified cell substrates for the manufacture of proteins and other complex biologics. The use of lentiviral vectors for the generation of genetically modified cell substrates for the production of biologic material has several advantages over other systems: (1) highly productive mammalian cell lines can be rapidly generated without selection or gene amplification; (2) the high number of vector copies are distributed throughout the open chromatin of the genome, resulting in cell lines that are extremely stable for high levels of gene expression and, consequently, protein production; and (3) high levels of protein glycosylation are maintained despite very high levels of protein production. These advantages offer the potential to significantly improve the quality, time-to-market, and manufacturing cost of biologics for human use.

We have reported that, in A/J (A) (H-2a) mice, a partial tolerance to C57BL/10ScSn (B10) (H-2b) skin allografts and a high incidence of lethal lymphoproliferative disorders (LPD) can be induced by the neonatal i.v. injection of 2 x 10(7) semiallogeneic (B10 x A)F1 spleen cells (SC) (Végh, P., Baranyi, L. and Jánossy, T., Cell. Immunol. 1990, 129: 56). In this study, we show that the incidence and mortality of LPD were continuously growing from 1 month of age until the end of the experiment at 1 year (64% and 36%, respectively). Based on histology, 27% of the diseased mice suffered from lymphoid malignancies. In the remaining cases (73%), reactive histopathological changes were seen in the spleen, lymph nodes (LN), liver and kidneys. The proportion of CD4+ T cells in the spleen and LN as well as that of splenic B cells decreased, while the percentages of mature and immature myeloid cells doubled. The total cell number of each (sub)population, however, was elevated in both lymphoid organs. The cells taking part in the lymphoproliferation were of host (A) and not of donor (F1) origin. Preceding the development of apparent LPD, the SC, LN cell and thymus cell suspensions of 1-month-old tolerized mice showed reduced in vitro proliferative responses to T cell and T cell-dependent B cell mitogens (Con A or PWM), while their reactivity to a T cell-independent B cell mitogen (lipopolysaccharide) was essentially unimpaired. This hyporeactivity seems to be functional, because neither histology nor immunophenotyping by flow cytometry revealed significant alterations in the spleen and thymus of such animals, apart from a slight reduction in the ratio of CD4+/CD8+ T cell subpopulations in the spleen. The in vivo T cell-mediated immune response of the tolerized mice was practically normal to third party CBA/Ca (H-2k) allografts. Antithymocyte autoantibodies (ATA) were detected in the sera of 76% of the tolerized mice at 1 month of age (i.e., even before the mass appearance of LPD). ATA as well as antinuclear Ab were present in 65% of the adult tolerized mice, independently of the presence of LPD. Taken together, in A mice neonatally injected with (B10 x A)F1 SC, a partial, specific allograft tolerance and a chronic host-vs.-graft disease-like syndrome developed. The latter is manifested in hyporeactivity to T cell mitogens, development of autoantibodies and, subsequently, in progressive LPD and lymphoid malignancies.

Two lymphomas were found in, and isolated from A (H-2a) mice in which permanent transplantation tolerance was induced to CBA (H-2k) histocompatibility antigens by the neonatal injection of (CBAxA)F1 spleen cells. They proved to be of recipient origin and were transferable to syngeneic A mice, growing as disseminated lymphomas (L33 and L46) and killing the recipients rapidly. Analysis of the cell surface antigens disclosed that both lymphomas had an immature T cell phenotype [Thy-1+, CD5+, CD3low, TCR alpha beta low, CD4low, CD8high, heat-stable antigen (HSA) positive, and CD44-, MHC class II-, CD45R-, sIg-, Gr-1-, CD11b-]. Intraperitoneal (i.p.) injection of syngeneic A mice with viable L33 lymphoma cells resulted in a dose-dependent, significant prolongation of the mean survival times of "specific" CBA and MHC-identical B10.BR skin allografts as compared to the survival of appropriate grafts in non-lymphoma-bearing controls. The survival times of third party MHC-incompatible B10 (H-2b) and B10.D2 (H-2d) allografts were only slightly prolonged in A mice inoculated with L33 cells. The graft-protective effect was not abrogated if the proliferative capacity of the L33 cells was blocked by in vitro mitomycin C (MMC) pretreatment. Furthermore, the inoculation of L33 lymphoma into A mice significantly inhibited their DTH response to the sensitizing CBA histocompatibility antigens. In contrast, the L46 lymphoma had no effect on the survival of CBA allografts and the DTH reactivity. These data suggest that the CD4+CD8+TCR alpha beta + L33 T cell lymphoma originating from a neonatally tolerant mouse has a specific immunosuppressive effect on the in vivo reactivity of syngeneic mice to the tolerance-inducing (MHC class I) alloantigens.

Patients with hemophilia A present with spontaneous and sometimes life-threatening bleeding episodes that are treated using blood coagulation factor VIII (fVIII) replacement products. Although effective, these products have limited availability worldwide due to supply limitations and product costs, which stem largely from manufacturing complexity. Current mammalian cell culture manufacturing systems yield around 100 µg/l of recombinant fVIII, with a per cell production rate of 0.05 pg/cell/day, representing 10,000-fold lesser production than is achieved for other similar-sized recombinant proteins (e.g. monoclonal antibodies). Expression of human fVIII is rate limited by inefficient transport through the cellular secretory pathway. Recently, we discovered that the orthologous porcine fVIII possesses two distinct sequence elements that enhance secretory transport efficiency. Herein, we describe the development of a bioengineered fVIII product using a novel lentiviral-driven recombinant protein manufacturing platform. The combined implementation of these technologies yielded production cell lines that biosynthesize in excess of 2.5 mg/l of recombinant fVIII at the rate of 9 pg/cell/day, which is the highest level of recombinant fVIII production reported to date, thereby validating the utility of both technologies.

Intravenous injection of some liposomal drugs, diagnostic agents, micelles and other lipid-based nanoparticles can cause acute hypersensitivity reactions (HSRs) in a high percentage (up to 45%) of patients, with hemodynamic, respiratory and cutaneous manifestations. The phenomenon can be explained with activation of the complement (C) system on the surface of lipid particles, leading to anaphylatoxin (C5a and C3a) liberation and subsequent release reactions of mast cells, basophils and possibly other inflammatory cells in blood. These reactions can be reproduced and studied in pigs, dogs and rats, animal models which differ from each other in sensitivity and spectrum of symptoms. In the most sensitive pig model, a few miligrams of liposome (phospholipid) can cause anaphylactoid shock, characterized by pulmonary hypertension, systemic hypotension, decreased cardiac output and major cardiac arrhythmias. Pigs also display cutaneous symptoms, such as flushing and rash. The sensitivity of dogs to hemodynamic changes is close to that of pigs, but unlike pigs, dogs also react to micellar lipids (such as Cremophor EL) and their response includes pronounced blood cell and vegetative neural changes (e.g., leukopenia followed by leukocytosis, thrombocytopenia, fluid excretions). Rats are relatively insensitive inasmuch as hypotension, their most prominent response to liposomes, is induced only by one or two orders of magnitude higher phospholipid doses (based on body weight) compared to the reactogenic dose in pigs and dogs. It is suggested that the porcine and dog models are applicable for measuring and predicting the (pseudo)allergic activity of particulate "nanodrugs".

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.

Activation of the complement (C) cascade is known to play a key role in the adverse immune consequences of hemorrhagic trauma with subsequent shock and resuscitation. However, it is not clear whether hypovolemia per se, without trauma and resuscitation, can also lead to C activation. To address this question, we studied the presence, kinetics, and cause of C activation in a porcine model of hemorrhagic shock and resuscitation in the absence of trauma. Pigs were bled to and kept at 35 mmHg for 90 min, followed by hypotensive resuscitation with different fluids and, finally, with shed blood. The animals developed severe lactic acidosis between 30 and 90 min, which was accompanied by a trend for initial rise and subsequent 40% drop of CH50/mL, indicating massive C activation even before resuscitation, i.e., before reperfusion damage could have occurred. Resuscitation with plasma expanders caused 20% additional C consumption, whereas whole blood raised CH50/mL. Plasma C5a decreased initially and then significantly increased at 60 and 180 min, whereas thromboxane B2 showed a 3-fold increase at 30 and 60 min. Plasma LPS was also increased above baseline at 90 and 180 min. In in vitro studies with pig blood, spontaneous C5a formation, as well as zymosan-induced C consumption, was significantly enhanced under the conditions of lactic acidosis. Our data suggest that lactic acidosis, endotoxemia, and possibly other ischemia-related tissue alterations act in a vicious cycle in inducing C activation and, hence, aggravation of shock. The biphasic course of CH50/mL and C5a changes may reflect yet unrecognized physiological responses to hemorrhage-related C activation.

Publisher Summary This chapter describes some basic principles and details of the most widely used experimental methods in studying the interaction of liposomes with the complement (C) system. Liposomes can interact with all four “rms” of the immune system: the cellular and humoral arms of acquired immunity and the corresponding parts of native immunity. The first two interactions underlie the attempts to use ‘‘immune’’ or ‘‘adjuvant’’ liposomes as antigen carriers to augment T-cell and antibody responses to liposomal vaccines. The third interaction is studied widely as the major clearance mechanism of most ‘‘non-stealth’’ drug carrier liposomes. The fourth type of interaction of liposomes with the immune system, referred to as “nonspecific humoral,” is manifested in liposome-induced C activation with consequent opsonization of vesicles by C split products, mainly C3b and its by-products.

High-dose intravenous immunoglobulin (IVIG) prevents immune damage by scavenging complement fragments C3b and C4b. We tested the hypothesis that exogenous immunoglobulin molecules also bind anaphylatoxins C3a and C5a, thereby neutralizing their pro-inflammatory effects. Single-cell calcium measurements in HMC-1 human mast cells showed that a rise in intracellular calcium caused by C3a and C5a was inhibited in a concentration-dependent manner by IVIG, F(ab)2-IVIG and irrelevant human monoclonal antibody. C3a- and C5a-induced thromboxane (TXB2) generation and histamine release from HMC-1 cells and whole-blood basophils were also suppressed by exogenous immunoglobulins. In a mouse model of asthma, immunoglobulin treatment reduced cellular migration to the lung. Lethal C5a-mediated circulatory collapse in pigs was prevented by pretreatment with F(ab)2-IVIG. Molecular modeling, surface plasmon resonance (SPR) and western blot analyses suggested a physical association between anaphylatoxins and the constant region of F(ab)2. This binding could interfere with the role of C3a and C5a in inflammation.

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.

Baranyi, L., W. Campbell, K. Ohshima, S. Fujimoto, M. Boros, J. Kaszaki and H. Okada. Antisense homology box-derived peptides represent a new class of endothelin receptor inhibitors. Peptides 19(2) 211–223, 1998.—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 ETA receptor fragment peptides ETR-P1 (“CALSVDRYRAVASW”), ETR-P3 (“QGIGPLITAIEI”), ETR-P4 (“IADNAERYSANLSSHV”) and ETR-P6 (“LNRRNGSLRIALSEHLKNRREVA”) reported here can inhibit ET-1 activity.

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.

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.

The monoclonal antibody 5I2 recognizes and functionally inhibits a Crry-like complement regulator molecule on rat cells (corresponding to human decay accelerating factor and membrane cofactor protein activity). The inhibition of complement regulatory activity by 5I2 antibody results in complement deposition on rat cell membranes exposed to homologous complement. Two subclones of KDH-8 rat hepatoma were selected for the experiments; one expressing high (CrryH) and the second low (CrryL) amounts of Crry-like antigen. Both sublines grow in vivo in syngeneic rats, but at lower cell doses (< 10(5) cells/rat) the survival time of rats injected with CrryL cells is prolonged. Injection of tumour-bearing rats with 5I2 monoclonal antibody intraperitoneally did not influence the tumour growth. However, it resulted in 50% mortality within a few hours, accompanied by severe haemorrhage in the peritoneal cavity. Pretreatment of the tumour cells with 5I2 monoclonal antibody or its F(ab')2 fragment substantially increased the survival time of recipient rats. Even permanent survivors were found, indicating that cell membrane-associated complement regulatory activity, which provides protection against attack of homologous complement, is necessary for in vivo tumour growth.

The possibility, conditions, and quantitative aspects of eliciting GVHD in CBA (H-2k) mice made neonatally tolerant to the alloantigens of the donor A (H-2a) strain were studied. The intravenous injection of different doses (10(7)-2 x 10(8)) of A spleen cells caused a severe, often fatal, systemic GVHD in 12-month-old tolerant mice. The GVHD was found to be specific: spleen cells of a third party strain (B10) did not induce any disease. The intensity and the mortality of the GVHD depended on the cell dose and on the age of the recipients. In contrast, unirradiated (CBA x A)F1 recipients proved to be resistant to the lethal disease. In spite of their different susceptibility to the systemic GVHD, the tolerant and F1 hybrid recipients showed equally strong local GVH reactivity in the popliteal lymph node enlargement assay. Neonatally tolerant mice offer a new, sensitive model for the induction of lethal GVHD without the need of immunosuppression or irradiation.

We examined, on the basis of the fact that sense and antisense peptides have affinity for each other, whether any relationship exists between homologous restriction factor (HRF20), a membrane inhibitor of the terminal stage of homologous complement attack, and the antisense sequence of the terminal complement components C8 and C9. In this article, we demonstrate that there are two regions of C9 that contain antisense sequences to one continuous region of HRF20 and that this relationship exists between human HRF20 and human C9, but not mouse C9. We also found one region of the C8 beta chain that contains an antisense sequence to HRF20.

Acute management of patients with traumatic brain/blast injury is a challenge. To minimize secondary injury and improve outcome, it is critical to detect neurological deterioration early, when it is potentially reversible. One potential monitoring method is cerebral electrical impedance (rheoencephalography-REG) because of its non-invasiveness and good time resolution. Reported here are the results of cerebral blood flow (CBF) manipulations comparing electroencephalogram (EEG) with REG (both intra-cerebral) and measuring with surface and skull REG electrodes. Our hypothesis was that REG would reflect spreading depression and CBF autoregulation. Animal experiments were performed using one rat (four trials with intracerebral electrodes), monkeys (n=8, with surface electrodes) and pigs (n = 24 pigs with skull electrodes; 57 trials, 19 types of liposomes). Challenges included intracranial pressure (ICP) elevation, liposome infusion, and hemorrhage. Data were stored on a PC and evaluated off line. CBF autoregulation was evaluated both by visual inspection and by a Matlab script. These studies confirmed that REG reflects CBF autoregulation and that REG is useful for detecting spreading depression (SD), vasospasm and the lower limit of CBF autoregulation. These findings have clinical relevance for use in noninvasive neuro-monitoring in the neurosurgery intensive care and during transportation of patients with brain injury.

The increased cost of goods associated with low-level expression of factor VIII (fVIII) is considered a significant factor in the pricing of recombinant fVIII products. Worldwide treatment of hemophilia A is limited due to the cost of fVIII products, which easily can exceed $100,000 per year. Using state-of-the-art commercial production facilities and technology, recombinant human (h)-fVIII expresses at 100 – 1000-fold lower levels than other plasma proteins. We have developed a novel recombinant fVIII product that overcomes the primary limitation to the manufacture of currently available hemophilia A pharmaceuticals. There are two components to this product: a humanized highexpression hybrid-human/porcine (HP) fVIII transgene, designated ET-801i, and the LENTIMAX™ lentiviral production system. The ET-801i transgene contains elements of porcine fVIII (p-fVIII) sequence in the A1 and A3 domains that facilitate more efficient secretion from production cell lines and is 90% identical in amino acid sequence to h-fVIII. Additionally, lentiviral introduction of the transgene allows achievement of an optimal number of transgene copies and the lentiviral vectors typically integrate into sites of active chromatin, thus facilitating high-levels of transgene transcription. In the current study, we demonstrated that high-expression porcine sequence elements enable 20 – 100- fold superior expression over any previously described fVIII transgene variant. Using a stable baby hamster kidney cell-derived expression system, ET-801i was expressed at a level indistinguishable from p-fVIII, which both were significantly greater than h-fVIII (

The optimal life sign monitor in the practice of emergency medicine would be a technique to detect the earliest pathological change. Since the brain is the most sensitive organ in response to hypoxia/ischemia, "cardio-pulmonary resuscitation should be brain-oriented" (Peter Safar, 1998). Nevertheless, monitoring of cerebral blood flow (CBF) is not performed in the practice of emergency medicine in either civilian or military environments. 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. Unlike CBF monitoring this technique of CBF reactivity is being proposed as a non-invasive, mobile, and non-operator dependant means of evaluating an unconscious patient. The ideal life sign monitor would be one that reflects CBF non-invasively, continuously and globally. Because the most common cause of death following serious military and civilian injuries is hemorrhage, the goal of this study was to describe CBF changes during hemorrhage and to compare various CBF modalities in order to identify the optimal CBF vital sign monitor. A computer controlled rat hemorrhage model was introduced for resuscitation purposes. In anesthetized rats blood was removed at a rate required to achieve a mean arterial blood pressure (MABP) of 40 mmHg over 15 min. Shed blood was measured and used as an indicator of compensation/decompensation phase. Continuous recording was taken during 30 minutes of compensatory phase with intracranial rheoencephalogram – (iREG; n = 14), laser Doppler flowmetry (LDF; n = 3), and carotid flow by ultrasound (n = 11). Data were stored and processed off-line. During the initial phase of hemorrhage, when MABP was close to 40 mmHg, intracranial REG amplitude transiently increased (81 %), while LDF (78 %) and carotid flow (52 %) decreased and changed with systemic arterial pressure. Intracranial REG amplitude change suggests classical CBF autoregulation, indicating its close relationship to arteriolar changes 1. The studies indicated that 1) iREG may reflect cerebrovascular responses more accurately than changes in local CBF measured by LDF and carotid flow; CBF autoregulation was present at the beginning of SAP 40 mmHg. The physiological background or resolution of this virtual conflict is: "Flow through large arteries may be constant despite changes of regional flow in tissue" 2 REG may indicate promise as a continuous, non-invasive life sign monitoring tool with potential advantages over EKG, ultrasound, and other measurement techniques normally applied in clinical practice. Additional quantitative studies are in progress; the following studies are needed: 1) to test CBF autoregulation during the entire period of hemorrhage (with CO2 inhalation); 2) to estimate REG during graded increases in intracranial pressure; 3) to estimate REG during graded decreases in SAP; and 4) to test various data processing methods of the REG signal.

Recent findings indicate that two novel theories, the Molecular Recognition Theory (MRT) based on the structure of the genetic code and on a novel motif termed antisense homology box, found in proteins and the Resonant Recognition Model, (RRM theory) based on resonant energy distribution of delocalised electrons between classes of proteins having similar functions may have common roots. The authors found that in contrast to a statistically expected 20% of overlap in the regions involved in the antisense homology boxes and hot "spots" predicted by RRM there is more than 60% prevalence of co-localization in proteins analyzed so far.

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.

In our earlier results, we demonstrated that cells expressing the complement C5aR are vulnerable since abnormal activation of C5aR caused apoptosis of these cells. In this study, we demonstrate that activation of C5aR by antisense homology box (AHB) peptides synthesized in multiple antigenic peptide form and representing putative interaction sites of the C5a/C5aR evoked calcium influx in TGW neuroblastoma cells. Dose-dependent inhibition of the response was found when the cells were pretreated with C5a, suggesting that C5aR was involved in this process. In addition, pretreatment with monomeric forms of the AHB peptides resulted in attenuation of the calcium signals, supporting the idea of the role of C5aR in this process. Cells of a neuron-rich primary culture and pyramidal cells of rat brain slices also responded to the AHB peptide activation with an increase in the intracellular calcium level, showing that calcium metabolism might be affected in these cells. TUNEL staining demonstrated that C5aR-mediated apoptosis could be induced both in cells of the primary culture as well as in cortical pyramidal neurons of the rat brain. In addition, we investigated expression of C5aR in the hippocampal and cortical neurons of human brains of healthy and demented patients using two anti-human C5aR Abs. Pyramidal cells of the hippocampus and cortex and granular cells of the hippocampus were immunopositive on staining. Although staining was also positive in the vascular dementia brain, it disappeared in the brain with Alzheimer’s disease. These results provide further support that C5aR may be involved in neurodegeneration.