Endothelial Cells Potentiate Interferon-γ Production in a Novel Tripartite Culture Model of Human Cerebral Malaria

We have established a novel in vitro co-culture system of human brain endothelial cells (HBEC), Plasmodium falciparum parasitised red blood cells (iRBC) and peripheral blood mononuclear cells (PBMC), in order to simulate the chief pathophysiological lesion in cerebral malaria (CM). This approach has revealed a previously unsuspected pro-inflammatory role of the endothelial cell through potentiating the production of interferon (IFN)-γ by PBMC and concurrent reduction of interleukin (IL)-10. The IFN-γ increased the expression of CXCL10 and intercellular adhesion molecule (ICAM)-1, both of which have been shown to be crucial in the pathogenesis of CM. There was a shift in the ratio of IL-10:IFN-γ protein from >1 to <1 in the presence of HBEC, associated with the pro-inflammatory process in this model. For this to occur, a direct contact between PBMC and HBEC, but not PBMC and iRBC, was necessary. These results support HBEC playing an active role in the pathogenesis of CM. Thus, if these findings reflect the pathogenesis of CM, inhibition of HBEC and PBMC interactions might reduce the occurrence, or improve the prognosis, of the condition.     

Characterization and Modulation of Glucose Uptake in a Human Blood–Brain Barrier Model

The blood–brain barrier (BBB) plays a key role in limiting and regulating glucose access to glial and neuronal cells. In this work glucose uptake on a human BBB cell model (the hCMEC/D3 cell line) was characterized. The influence of some hormones and diet components on glucose uptake was also studied. ³H-2-deoxy-d-glucose ([³H]-DG) uptake for hCMEC/D3 cells was evaluated in the presence or absence of tested compounds. [³H]-DG uptake was sodium- and energy-independent. [³H]-DG uptake was regulated by Ca²⁺ and calmodulin but not by MAPK kinase pathways. PKC, PKA and protein tyrosine kinase also seem to be involved in glucose uptake modulation. Progesterone and estrone were found to decrease ³H-DG uptake. Catechin and epicatechin did not have any effect, but their methylated metabolites increased [³H]-DG uptake. Quercetin and myricetin decreased [³H]-DG uptake, and glucuronic acid-conjugated quercetin did not have any effect. These cells expressed GLUT1, GLUT3 and SGLT1 mRNA.     

Endothelial Vesicles in the Blood–Brain Barrier: Are They Related to Permeability?

1. Macromolecules cross capillary walls via large vascular pores that are thought to be formed by plasmalemmal vesicles. Early hypotheses suggested that vesicles transferred plasma constituents across the endothelial wall either by a shuttle mechanism or by fusing to form transient patent channels for diffusion. Recent evidence shows that the transcytotic pathway involves both movement of vesicles within the cell and a series of fusions and fissions of the vesicular and cellular membranes.2. The transfer of macromolecules across the capillary wall is highly specific and is mediated by receptors incorporated into specific membrane domains. Therefore, despite their morphological similarity, endothelial vesicles form heterogeneous populations in which the predominant receptor proteins incorporated in their membranes define the functions of individual vesicles.3. Blood–brain barrier capillaries have very low permeabilities to most hydrophilic molecules. Their low permeability to macromolecules has been presumed to be due to an inhibition of the transcytotic mechanism, resulting in a low density of endothelial vesicles.4. A comparison of vesicular densities and protein permeabilities in a number of vascular beds shows only a very weak correlation, therefore vesicle numbers alone cannot be used to predict permeability to macromolecules.5. Blood–brain barrier capillaries are fully capable of transcytosing specific proteins, for example, insulin and transferrin, although the details are still somewhat controversial.6. It has recently been shown that the albumin binding protein gp60 (also known as albondin), which facilitates the transcytosis of native albumin in other vascular beds, is virtually absent in brain capillaries.7. It seems likely that the low blood–brain barrier permeability to macromolecules may be due to a low level of expression of specific receptors, rather than to an inhibition of the transcytosis mechanism.     

Tissue Plasminogen Activator Enhances the Hypoxia/reoxygenation-induced Impairment of the Blood–brain Barrier in a Primary Culture of Rat Brain Endothelial Cells

Hemorrhagic transformation is a major complication associated with tissue plasminogen activator (tPA) therapy for ischemic stroke. We studied the effect of tPA on the blood–brain barrier (BBB) function with our in vitro monolayer model generated using rat brain microvascular endothelial cells subjected either to normoxia or to hypoxia/reoxygenation (H/R) with or without the administration of tPA. The barrier function was evaluated by the transendothelial electrical resistance (TEER), the permeability of sodium fluorescein and Evans’ blue-albumin (EBA), and the uptake of lucifer yellow (LY). The permeability of sodium fluorescein and EBA was used as an index of paracellular and transcellular transport, respectively. The administration of tPA increased the permeability of EBA and the uptake of LY under normoxia. It enhanced the increase in the permeability of both sodium fluorescein and EBA, the decrease in the TEER, and the disruption in the expression of ZO-1 under H/R conditions. Administration of tPA could cause an increase in the transcellular transport under normoxia, and both the transcellular and paracellular transport of the BBB under H/R conditions in vitro. Even in humans, tPA may lead to an opening of the BBB under non-ischemic conditions and have an additional effect on the ischemia-induced BBB disruption.     

Impact of Perinatal Systemic Hypoxic–Ischemic Injury on the Brain of Male Offspring Rats: An Improved Model of Neonatal Hypoxic–Ischemic Encephalopathy in Early Preterm Newborns

In this study, we attempted to design a model using Sprague-Dawley rats to better reproduce perinatal systemic hypoxic-ischemic encephalopathy (HIE) in early preterm newborns. On day 21 of gestation, the uterus of pregnant rats were exposed and the blood supply to the fetuses of neonatal HIE groups were thoroughly abscised by hemostatic clamp for 5, 10 or 15 min. Thereafter, fetuses were moved from the uterus and manually stimulated to initiate breathing in an incubator at 37 °C for 1 hr in air. We showed that survival rates of offspring rats were decreased with longer hypoxic time. TUNEL staining showed that apoptotic cells were significant increased in the brains of offspring rats from the 10 min and 15 min HIE groups as compared to the offspring rats in the control group at postnatal day (PND) 1, but there was no statistical difference between the offspring rats in the 5 min HIE and control groups. The perinatal hypoxic treatment resulted in decreased neurons and increased cleaved caspase-3 protein levels in the offspring rats from all HIE groups at PND 1. Platform crossing times and the percentage of the time spent in the target quadrant of Morris Water Maze test were significantly reduced in the offspring rats of all HIE groups at PND 30, which were associated with decreased brain-derived neurotrophic factor levels and neuronal cells in the hippocampus of offspring rats at PND 35. These data demonstrated that perinatal ischemic injury led to the death of neuronal cells and long-lasting impairment of memory. This model reproduced hypoxic ischemic encephalopathy in early preterm newborns and may be appropriate for investigating therapeutic interventions.     

Cell Therapy as a Way to Correct Impaired Neurogenesis in the Adult Brain in a Model of Alzheimer’s Disease

Journal of Evolutionary Biochemistry and Physiology - This work is based on the hypothesis of an impairment in neurogenesis during aging, which may be one of the causes of neurodegenerative...     

Protective Effects of Notoginsenoside R1 via Regulation of the PI3K-Akt-mTOR/JNK Pathway in Neonatal Cerebral Hypoxic–Ischemic Brain Injury

Notoginsenoside R1 (NGR1) is a predominant phytoestrogen extracted from Panax notoginseng that has recently been reported to play important roles in the treatment of cardiac dysfunction, diabetic kidney disease, and acute liver failure. Studies have suggested that NGR1 may be a viable treatment of hypoxic-ischemic brain damage (HIBD) in neonates by reducing endoplasmic reticulum stress via estrogen receptors (ERs). However, whether NGR1 has other neuroprotective mechanisms or long-term neuroprotective effects is unclear. In this study, oxygen-glucose deprivation/reoxygenation (OGD/R) in primary cortical neurons and unilateral ligation of the common carotid artery (CCL) in 7-day-old postnatal Sprague Dawley (SD) rats followed by exposure to a hypoxic environment were used to mimic an HIBD episode. We assessed the efficacy of NGR1 by measuring neuronal damage with MTT assay and assessed brain injury by TTC staining and brain water content detection 24–48 h after OGD/HIE. Simultaneously, we measured the long-term neurophysiological effects using the beam walking test (5 weeks after HI) and Morris water maze test 5–6 weeks after HI. Expression of PI3K-Akt-mTOR/JNK (24 h after HI or OGD/R) proteins was detected by Western blotting after stimulation with HI, NGR1, LY294002 (PI3K inhibitor), 740Y-P (PI3K agonist), or ICI 182780(estrogen receptors inhibitor). The results indicated that NGR1 exerted neuroprotective effects by inhibiting neuronal apoptosis and promoting cell survival via the PI3K-Akt-mTOR/JNK signaling pathways by targeting ER in neonatal hypoxic–ischemic injury.     

Apocynum venetum Leaf Extract Attenuates Disruption of the Blood–Brain Barrier and Upregulation of Matrix Metalloproteinase-9/-2 in a Rat Model of Cerebral Ischemia–Reperfusion Injury

We investigated the neuroprotective effects of Apocynum venetum leaf extract (AVLE) on a rat model of cerebral ischemia-reperfusion injury and explored the underlying mechanisms. Rats were randomly divided into five groups: sham, ischemia-reperfusion, AVLE125, AVLE250, and AVLE500. Cerebral ischemia was induced by 1.5 h of occlusion of the middle cerebral artery. Cerebral infarct area was measured by tetrazolium staining at 24 and 72 h after reperfusion, and neurological function was evaluated at 24, 48 and 72 h after reperfusion. Pathological changes on the ultrastructure of the blood-brain barrier (BBB) were observed by transmission electron microscopy. BBB permeability was assessed by detecting leakage of Evan's blue (EB) dye in brain tissue. The expression and activities of matrix metalloproteinase (MMP)-9/-2 were measured by western blot analyses and gelatin zymography at 24 h after reperfusion. AVLE (500 mg/kg/day) significantly reduced cerebral infarct area, improved recovery of neurological function, relieved morphological damage to the BBB, reduced water content and EB leakage in the brain, and downregulated the expression and activities of MMP-9/-2. These findings suggest that AVLE protects against cerebral ischemia-reperfusion-induced injury by alleviating BBB disruption. This action may be due to its inhibitory effects on the expression and activities of MMP-9/-2.     

Regulation of BCRP (ABCG2) and P-Glycoprotein (ABCB1) by Cytokines in a Model of the Human Blood–Brain Barrier

Brain capillary endothelial cells form the blood–brain barrier (BBB), a highly selective permeability membrane between the blood and the brain. Besides tight junctions that prevent small hydrophilic compounds from passive diffusion into the brain tissue, the endothelial cells express different families of drug efflux transport proteins that limit the amount of substances penetrating the brain. Two prominent efflux transporters are the breast cancer resistance protein and P-glycoprotein (P-gp). During inflammatory reactions, which can be associated with an altered BBB, pro-inflammatory cytokines are present in the systemic circulation. We, therefore, investigated the effect of the pro-inflammatory cytokines interleukin-1β (IL-1β), interleukin-6 (IL-6) and tumor necrosis factor-α (TNF-α) on the expression and activity of BCRP and P-gp in the human hCMEC/D3 cell line. BCRP mRNA levels were significantly reduced by IL-1β, IL-6 and TNF-α. The strongest BCRP suppression at the protein level was observed after IL-1β treatment. IL-1β, IL-6 and TNF-α also significantly reduced the BCRP activity as assessed by mitoxantrone uptake experiments. P-gp mRNA levels were slightly reduced by IL-6, but significantly increased after TNF-α treatment. TNF-α also increased protein expression of P-gp but the uptake of the P-gp substrate rhodamine 123 was not affected by any of the cytokines. This in vitro study indicates that expression levels and activity of BCRP, and P-gp at the BBB may be altered by acute inflammation, possibly affecting the penetration of their substrates into the brain.     

Lipopolysaccharide-Activated Microglia Induce Dysfunction of the Blood–Brain Barrier in Rat Microvascular Endothelial Cells Co-Cultured with Microglia

The blood–brain barrier (BBB) is formed by brain capillary endothelial cells, astrocytes, pericytes, microglia, and neurons. BBB disruption under pathological conditions such as neurodegenerative disease and inflammation is observed in parallel with microglial activation. To test whether activation of microglia is linked to BBB dysfunction, we evaluated the effect of lipopolysaccharide (LPS) on BBB functions in an in vitro co-culture system with rat brain microvascular endothelial cells (RBEC) and microglia. When LPS was added for 6 h to the abluminal side of RBEC/microglia co-culture at a concentration showing no effects on the RBEC monolayer, transendothelial electrical resistance was decreased and permeability to sodium-fluorescein was increased in RBEC. Immunofluorescence staining for tight junction proteins demonstrated that zonula occludens-1-, claudin-5-, and occludin-like immunoreactivities at the intercellular borders of RBEC were fragmented in the presence of LPS-activated microglia. These functional changes induced by LPS-activated microglia were blocked by the nicotinamide adenine dinucleotide phosphate (NADPH) oxidase inhibitor, diphenyleneiodonium chloride. The present findings suggest that LPS activates microglia to induce dysfunction of the BBB by producing reactive oxygen species through NADPH oxidase.     

In Search of the Astrocytic Factor(s) Modulating Blood–Brain Barrier Functions in Brain Capillary Endothelial Cells In Vitro

Summary 1. The blood–brain barrier (BBB) is formed by brain capillary endothelial cells (ECs). There are various cell types, in particular astrocytes, but also pericytes and neurons, located in close vicinity to the capillary ECs which may influence formation and function of the BBB. Based on this consideration, this paper discusses various aspects of the influence of the surrounding cells on brain capillary ECs with special focus on the role of astrocytes.2. Based on the morphology of the BBB, important aspects of brain EC functions are summarized, such as transport functions and maintenance of low paracellular permeability. Moreover, various facets are discussed with respect to the influence of astrocytes, pericytes, microglia, and neurons on the BBB. Data on the role of glial cells in the ontogenesis of the BBB are presented subsequently. The knowledge on this subject is far from being complete, however, these data imply that the neural/neuronal environment rather than glial cells may be of importance in the maturation of the barrier.3. The role of glial cells in the induction and maintenance of the BBB is discussed under physiological as well as pathological conditions. Although the literature presents manifold evidence for a great variety of effects induced by astroglia, there are also many controversies, which may result from different cellular models and experimental conditions used in the respective studies. Numerous factors secreted by astrocytes have been shown to induce a BBB phenotype. On the molecular level, increased expression of barrier-relevant proteins (e.g., tight junction proteins) is documented in the presence of astrocyte-derived factors, and many studies demonstrate the improvement of physiological parameters, such as increased transendothelial resistance and decreased paracellular permeability, in different in vitro models of the BBB. Moreover, one has to take into account that the interaction of brain ECs and astrocytes is bi-directional, and that the other cell types surrounding the brain microvasculature also contribute to BBB function or dysfunction, respectively.4. In conclusion, it is expected that the present and future research focused on molecular mechanisms and signaling pathways will produce new and exciting insights into the complex network of BBB regulation: the cornerstone is laid.This revised article was published online in May 2005 with a February 2005 cover date.     

Circulating Brain Microvascular Endothelial Cells (cBMECs) as Potential Biomarkers of the Blood–Brain Barrier Disorders Caused by Microbial and Non-Microbial Factors

Despite aggressive research, central nervous system (CNS) disorders, including blood-brain barrier (BBB) injury caused by microbial infection, stroke, abused drugs [e.g., methamphetamine (METH) and nicotine], and other pathogenic insults, remain the world''s leading cause of disabilities. In our previous work, we found that dysfunction of brain microvascular endothelial cells (BMECs), which are a major component of the BBB, could be caused by nicotine, meningitic pathogens and microbial factors, including HIV-1 virulence factors gp41 and gp120. One of the most challenging issues in this area is that there are no available cell-based biomarkers in peripheral blood for BBB disorders caused by microbial and non-microbial insults. To identify such cellular biomarkers for BBB injuries, our studies have shown that mice treated with nicotine, METH and gp120 resulted in increased blood levels of CD146+(endothelial marker)/S100B+ (brain marker) circulating BMECs (cBMECs) and CD133+[progenitor cell (PC) marker]/CD146+ endothelial PCs (EPCs), along with enhanced Evans blue and albumin extravasation into the brain. Nicotine and gp120 were able to significantly increase the serum levels of ubiquitin C-terminal hydrolase 1 (UCHL1) (a new BBB marker) as well as S100B in mice, which are correlated with the changes in cBMECs and EPCs. Nicotine- and meningitic E. coli K1-induced enhancement of cBMEC levels, leukocyte migration across the BBB and albumin extravasation into the brain were significantly reduced in alpha7 nAChR knockout mice, suggesting that this inflammatory regulator plays an important role in CNS inflammation and BBB disorders caused by microbial and non-microbial factors. These results demonstrated that cBMECs as well as EPCs may be used as potential cell-based biomarkers for indexing of BBB injury.     

Human–felid conflict as a further handicap to the conservation of the critically endangered Iberian lynx

Most studies on felid depredation of livestock have focused on big cats, and little attention has been paid to this type of conflict in smaller species. The medium-sized Iberian lynx (Lynx pardinus) is not thought to be affected by conflict with humans. However, parallel to an increase in the range of the Iberian lynx in Andújar-Cardeña, an increased incidence of Iberian lynx attacks on livestock has been recorded. A 6-year overview of Iberian lynx predation on livestock in this population shows a total of 40 attacks involving 716 kills (31 attacks on poultry and nine on sheep). Although the majority of these attacks (78 %) were carried out against poultry, sheep depredation resulted in higher economic losses, mainly in extensive flocks (4.6 times more than semi-intensive flocks). An effective compensation program has been implemented in order to mitigate the consequences of the human–lynx conflict in this area. Given that this sort of conflict could become an acute impediment to future conservation of the most endangered felid, managers should anticipate and prevent the potential conflicts that could arise as Iberian lynx colonizes more developed areas.     

Three-Dimensional Cell Cultures as a Model System to Evaluate the Biological Activity of Gemcitabine (2′, 2′-Difluoro-2′deoxycytidine)

Abstract

The cytotoxicity of Gemcitabine (dFdC) was 10 (ovarian cancer) to > 10,000 (colon cancer) fold higher in monolayer compared to three-dimensional multilayer cell cultures. This selectivity was related to marked differences in dFdC activation and effects on ribonucleotides.     

Gene Expression of the EGF System—a Prognostic Model in Non–Small Cell Lung Cancer Patients Without Activating EGFR Mutations

OBJECTIVES: Contradicting results have been demonstrated for the expression of the epidermal growth factor receptor (EGFR) as a prognostic marker in non–small cell lung cancer (NSCLC). The complexity of the EGF system with four interacting receptors and more than a dozen activating ligands is a likely explanation. The aim of this study is to demonstrate that the combined network of receptors and ligands from the EGF system is a prognostic marker. MATERIAL AND METHODS: Gene expression of the receptors EGFR, HER2, HER3, HER4, and the ligands AREG, HB-EGF, EPI, TGF-α, and EGF was measured by quantitative polymerase chain reaction in tumor samples from 100 NSCLC patients without EGFR activating mutations. Results were dichotomized into high or low levels of target expression. Coexpression of the ligands and receptors was observed, and a score was developed based on the summed effect of receptors and ligands. Akaike’s information criteria selected the optimal score. Results were correlated with age, sex, stage, histology, performance status, and overall survival. RESULTS: Patients were randomly split 1:1 to create test and validation cohorts. In multivariate analyses, the only individual prognostic marker was EPI (hazard ratio [HR] 0.38 [0.20-0.72], P = .003). The optimal score in the test cohort was validated as a marker of inferior survival in the validation cohort and by bootstrapping. Multivariate analysis confirmed the combined score as a prognostic marker of inferior survival (HR 3.75 [2.17-6.47], P < .00001). CONCLUSION: Our study has developed a model that takes the complexity of the EGF system into account and shows that this model is a strong prognostic marker in NSCLC patients.Despite advances in the treatment, non–small cell lung cancer (NSCLC) remains the leading cause of cancer-related death in the world [1]. In particular, the overall prognosis is poor for the metastatic stages, with a median overall survival (OS) of only 8 to 10 months. Even in the early nonmetastatic stages, the 5-year survival rate is as low as 50% [2], [3]. Prognostic markers are needed to stratify patients with different risk outcome. Several biomarkers have been evaluated in NSCLC, but only a few have proven to be clinically relevant. An activating mutation in the epidermal growth factor receptor (EGFR) is both a well-described predictive marker of benefit of EGFR-targeted tyrosine kinase inhibitors but also a debated prognostic marker of better OS [4], [5], [6], [7], [8], [9]. As EGFR expression has been associated with OS in head and neck, colorectal, and esophagus cancer [10], [11], [12], attention has been directed toward the use of EGFR expression as a prognostic marker in NSCLC, but contradicting results have been demonstrated [13], [14], [15], [16]. The EGF system is complex, and the effect of ligand-receptor interaction depends on a variety of different factors, which provides a plausible explanation for the divergence observed between studies that only evaluate EGFR expression in general. EGFR is one out of four related receptors from the EGF system and is capable of forming homodimers or heterodimers with one of the three other receptors when activated by a ligand. Several ligands from the EGF system such as amphiregulin (AREG), epidermal growth factor (EGF), and transforming growth factor–α (TGF-α) only activate EGFR, whereas some have the ability to activate several combinations of the four EGF receptors like heparin-binding epidermal growth factor (HB-EGF), epiregulin (EPI), and betacellulin (BCL). Most knowledge on the role of the ligands in NSCLC is from in vitro studies or from smaller clinical studies. In vitro studies have suggested that the biological effect of EGFR activation is dependent on the specific activating ligand as well as the dimerization partner [17]. Yet, no clinical studies have evaluated the effect of the network of receptors and ligands influencing EGFR in NSCLC. Furthermore, the majority of the clinical studies exploring EGFR expression are based on immunohistochemistry which is a semiquantitative method with a great risk of interobserver variability. Quantitative gene expression analyses provide a more accurate measure and are therefore more suitable for studies comparing expression levels. Prospectively, we have collected fresh tumor samples from patients suspected of lung cancer. Accordingly, the aim of this study is to evaluate the gene expression of the network of receptors and ligands of the EGF system affecting EGFR as a prognostic markers in NSCLC.     

The Equilibrium of Phosphatidylcholine–Amino Acid System in Monolayer at the Air/water Interface

Monolayers of phosphatidylcholine, tyrosine, and phenylalanine and binary mixtures phosphatidylcholine–tyrosine or phosphatidylcholine–phenylalanine were investigated at the air/water interface. Phosphatidylcholine (lecithin, PC), tyrosine (Tyr), and phenylalanine (Phe) were used in the experiment. The surface tension values of pure and mixed monolayers were used to calculate π–A isotherms. The surface tension measurements were carried out at 22°C using an improved Teflon trough and a Nima 9000 tensiometer. The Teflon trough was filled with a subphase of triple-distilled water. Known amounts of lipid dissolved in 1-chloropropane were placed at the surface using a syringe. The interactions between lecithin and amino acid result in significant deviations from the additivity rule. An equilibrium theory to describe the behavior of monolayer components at the air/water interface was developed in order to obtain the stability constants of PC–Tyr as well as PC–Phe complexes. We considered the equilibrium between the individual components and the complex and established that lecithin and amino acid formed highly stable 1:1 complex.     

All-Trans Retinoic Acid Ameliorates the Early Experimental Cerebral Ischemia–Reperfusion Injury in Rats by Inhibiting the Loss of the Blood–Brain Barrier via the JNK/P38MAPK Signaling Pathway

All-trans retinoic acid (ATRA) influences the outcomes of cerebral ischemic reperfusion (CIR) injury, but the mechanism remains unclear. The present study aimed to investigate the effects of ATRA on loss of the blood brain barrier (BBB) following CIR and to explore the possible mechanisms. Transient middle cerebral artery occlusion was performed on male SD rats to construct an in vivo CIR model. Neurological deficits, BBB permeability, brain edema, MRI and JNK/P38 MAPK proteins were detected at 24 h following CIR. We demonstrated that ATRA pretreatment could alleviate CIR-induced neurological deficits, increase of BBB permeability, infarct volume, degradation of tight junction proteins, inhibit MMP-9 protein expression and activity. ATRA treatment also reduced the p-P38 and p-JNK protein level. However the protective effect of ATRA on CIR could be reversed by administration of retinoic acid alpha receptor antagonist Ro41-5253. SP600125 and SB203580, which is the JNK/P38 pathway inhibitors has the same protective effect as ATRA. These results indicated that ATRA may inhibit the JNK/P38 MAPK pathway to alleviate BBB disruption and improve CIR outcomes.     

A Structure–Function Study of a Catalytic Antiidiotypic Antibody to the Human Erythrocyte Acetyl Cholinesterase

The catalytic monoclonal antibody 9A8 (MA 9A8), antiidiotypic to the antibody AE-2 (MA AE2) produced to the active site of acetyl cholinesterase from human erythrocytes, was subjected to a structure–function study. The specific binding of MA 9A8 to MA AE2 (K 2.26 × 10⁹ M^–1) was found by the method of surface plasmon resonance, and the functional activity of MA 9A8 was demonstrated. Unlike acetyl cholinesterase, this antibody specifically reacted with the irreversible phosphonate inhibitors of esterases. A peptide map of MA 9A8 was analyzed by MALDI mass spectrometry. The Ser99 residue of its heavy chain was shown to be within the active site of the catalytic antibody. A computer modeling of the MA 9A8 active site suggested the existence of a catalytic dyad formed by Ser99 and His35. A comparison of the tertiary structures of the MA 9A8 and the 17E8 monoclonal antibody, which also exhibited the esterase activity and was produced to the stable analogue of the reaction transition state, indicated a practically complete coincidence of the structures of their presumed active sites.