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PubMed2002

Left ventricular diastolic dysfunction as a predictor of the first diagnosed nonvalvular atrial fibrillation in 840 elderly men and women.

OBJECTIVES: The objective of this study was to determine whether diastolic dysfunction is associated with increased risk of nonvalvular atrial fibrillation (NVAF) in older adults with no history of atrial arrhythmia. BACKGROUND: Few data exist regarding the relationship between diastolic function and NVAF. METHODS: The clinical and echocardiographic characteristics of patients age > or =65 years who had an echocardiogram performed between 1990 and 1998 were reviewed. Exclusion criteria were history of atrial arrhythmia, stroke, valvular or congenital heart disease, or pacemaker implantation. Patients were followed up in their medical records to the last clinical visit or death for documentation of first AF. RESULTS: Of 840 patients (39% men; mean [+/- SD] age, 75 +/- 7 years), 80 (9.5%) developed NVAF over a mean (+/- SD) follow-up of 4.1 +/- 2.7 years. Abnormal relaxation, pseudonormal, and restrictive left ventricular diastolic filling were associated with hazard ratios of 3.33 (95% confidence interval [CI], 1.5 to 7.4; p = 0.003), 4.84 (95% CI, 2.05 to 11.4; p < 0.001), and 5.26 (95% CI, 2.3 to 12.03; p < 0.001), respectively, when compared with normal diastolic function. After a number of adjustments, diastolic function profile remained incremental to history of congestive heart failure and previous myocardial infarction for prediction of NVAF. Age-adjusted Kaplan-Meier five-year risks of NVAF were 1%, 12%, 14%, and 21% for normal, abnormal relaxation, pseudonormal, and restrictive diastolic filling, respectively. CONCLUSIONS; The presence and severity of diastolic dysfunction are independently predictive of first documented NVAF in the elderly.

Age Factors↗

PubMed1996

Bladder instability and incontinence after radical prostatectomy.

Thirty-nine patients were evaluated after retropubic prostatectomy, by urodynamic studies. They were divided into three groups depending upon the degree of urinary continence. A statistically significant difference was found between different groups in mean functional profile length and maximal urethral closure pressure. Differences in age, previous prostatic surgery, tumour extension, or preservation of the neurobundles did not have any significant influence on recovery of continence.

Aged↗

PubMed1998

P50 abnormalities in schizophrenia: relationship to clinical and neuropsychological indices of attention.

While the P50 component (50-60-ms latency) of the auditory evoked potential has been reported as abnormal in schizophrenia, few studies have examined the relationship between this abnormality and clinical or neuropsychological measures. To examine these possible relationships, mid-latency auditory evoked potentials were recorded at the CZ recording site of 47 patients with schizophrenia in response to binaural clicks presented at three stimulus rates: 1, 5 and 10/sec. A sub-sample of patients were then divided into high- (n = 15) and low-P50 abnormality (n = 16) groups based on a median split of the P50 amplitude at a rate of 10/sec (a greater amplitude at this rate suggests a greater abnormality in recovery) of the entire sample. Only those patients with complete neuropsychological and clinical data and who were reasonably matched on demographic dimensions were included. A multivariate analysis of variance of 11 neuropsychological function profile scores showed a significant group x global score interaction (Hotelling t = 3.97, p < 0.005). The high-abnormality group had relatively greater deficits for attention profile scores than for the remaining neuropsychological measures. An analysis of global subscores for SAPS and SANS clinical measures revealed a significant difference only for the SANS attention subscale (p < 0.05). The high-abnormality group was rated as more severe on the attention measure. These convergent findings across both phenomenological and neuropsychological measures suggest that abnormalities in P50 recovery may be linked to deficits in attention processes in schizophrenia.

Adult↗

PubMed1998

A secreted streptococcal cysteine protease can cleave a surface-expressed M1 protein and alter the immunoglobulin binding properties.

Previous studies of recent clinical isolates of serotype M1 group A streptococci indicated that they display two patterns of non-immune human IgG subclass binding reactivity associated with their M1 protein. One group reacted with all four IgG subclasses (type IIo), while the second group expressed an M1 protein reacting preferentially with human IgG3 (type IIb). In this study, we have demonstrated that a cysteine protease, SpeB, present in culture supernatants of M1 serotype group A streptococcal isolates expressing type IIb IgG binding protein, can convert a recombinant Emm1 protein from a type IIo functional profile to a type IIb profile by removal of 24 amino acids from the N-terminus of the mature M1 protein. Furthermore, SpeB can convert bacteria expressing IgG binding proteins of the type IIo phenotype into those expressing type IIb proteins. The role of the cysteine protease as the central bacterial enzyme in this posttranslational modification event was confirmed by generation of an isogenic SpeB-negative mutant.

Amino Acid Sequence↗

PubMed2002

New insights into the mechanism of purple acid phosphatase through (1)H NMR spectroscopy of the recombinant human enzyme.

Proton NMR spectra of FeIII-FeII recombinant single polypeptide human PAP (recHPAP) have been measured at, above, and below its pH optimum, as have the spectra of inhibited forms containing fluoride and phosphate, analogues of the substrates hydroxide and phosphate esters, respectively. The results demonstrate that binding of inhibitory anions to the dinuclear mixed-valent site of recHPAP is controlled by protonation of a ligand to the dinuclear center. Thus, the group that is responsible for pKa,1 in the enzymatic activity versus pH profile functions as a "gatekeeper", whose protonation state controls anion binding to the mixed-valent dinuclear site. The correlation between the pKa values observed in kinetics studies and for the spectroscopic changes strongly suggests that this group is the nucleophilic hydroxide that attacks the phosphate ester substrate.

Acid Phosphatase↗

PubMed1999

Design and synthesis of novel alpha(1)(a) adrenoceptor-selective antagonists. 3. Approaches to eliminate opioid agonist metabolites by using substituted phenylpiperazine side chains.

Dihydropyrimidinones, such as 1, represent a novel class of alpha(1a) adrenoceptor antagonists with potential for the treatment of benign prostatic hyperplasia (BPH) (see part 1 of this series). Analysis of the metabolites of 1 revealed that 4-methoxycarbonyl-4-phenylpiperidine is formed as the major metabolite and is an agonist at the mu-opioid receptor. To circumvent any potential liability resulting from the metabolite, we decided to identify alternate templates devoid of agonist activity at the mu-opioid receptor to replace the 4-methoxycarbonyl-4-phenylpiperidine moiety. The present study describes the synthesis and SAR of dihydropyrimidinones linked to substituted 4-phenylpiperazine containing side chains. Compound (+)-38 was identified as a lead compound with a binding and functional profile comparable to that of 1. The putative metabolite 2-carboxamidophenylpiperazine has negligible affinity for the mu-opioid receptor.

Adrenergic alpha-Antagonists↗

PubMedDate not supplied

Proteome analysis of tobacco bright yellow-2 (BY-2) cell culture plastids as a model for undifferentiated heterotrophic plastids.

We analyzed the proteome of undifferentiated plastids from a tobacco BY-2 cell culture by shotgun proteomics following multidimensional protein fractionation. The fractionation strategy initiated with the serial extraction of proteins from membranes which allowed us to distinguish soluble, peripheral, and integral membrane proteins. The majority of the identified proteins have a function in the cellular metabolism and most of them are active in amino acid synthesis pathways. A significant number of the identified proteins was not identified in chloroplast proteome analyses before. This suggests BY-2 plastid specific functions that differ from the major activities of chloroplasts. We have used the BY-2 plastid proteins reported here to assess the metabolic activities of undifferentiated heterotrophic plastids and compared the functional profile with that of differentiated heterotrophic amyloplasts. Comparative shotgun proteome analyses as reported here provide information about prevalent metabolic activities of different plastid types.

Cell Differentiation↗

PubMedDate not supplied

Phase II trial of uracil/tegafur (UFT) plus leucovorin in patients with advanced hepatocellular carcinoma.

UNLABELLED: Although UFT 300 mg/m2/day and leucovorin 90 mg/day administered orally in divided doses administered every 8 hours for 28 days repeated every 35 days could be administered safely to patients with advanced hepatomas and good performance status, this combination and schedule has limited activity in treating advanced hepatoma. BACKGROUND/PURPOSE: Biochemical modulation of 5-fluorouracil has yielded higher response rates in hepatoma when compared to treatment with 5-fluorouracil as a single agent, although the impact on survival has been negligible. This study was conducted to determine the activity and evaluate the toxicity of uracil and tegafur in a 4:1 molar concentration ratio (UFT; Bristol-Myers Squibb, Wallingford, CT) plus oral calcium leucovorin in the treatment of patients with advanced hepatocellular carcinoma (hepatoma). PATIENTS AND METHODS: Sixteen patients with advanced measurable hepatocellular carcinoma were enrolled onto the trial. All patients had a Karnofski performance status > or = 60%, platelet count > or = 75,000/micro L, total bilirubin < or = 2.0 x institutional upper limit of normal but otherwise normal liver and kidney function profile and bidimensionally measurable disease by CT or ultrasound examination. None of these patients received prior cytotoxic chemotherapy or radiation therapy for advanced disease. Fourteen patients received 300 mg/m2/d UFT plus 90 mg/d leucovorin administered orally in divided daily doses every 8 hours for 28 days repeated every 35 days. Two patients registered for the trial but did not receive study medication. Objective tumor response, the primary purpose of this trial, was evaluated after two courses of therapy. Other end-points included toxicity, time to progression, and overall survival. RESULTS: Fourteen patients were evaluable for response and toxicity, respectively. No complete or partial responders were observed in this trial. Three patients had stable disease lasting 17 to 22 weeks. Toxicity was mild with severe (grade 3 or 4) liver pain, diarrhea, anorexia/nausea, fatigue, dyspnea, hyperbilirubinemia, anemia, and edema seen in 3 (21%), 2 (14%), 3 (21%), 2 (14%), 1 (7%), 1 (7%), 1 (7%) and 1 (7%) patients, respectively. The most frequent grade I and 2 toxic effects included fever of unknown origin, dyspnea, nausea, vomiting and diarrhea. CONCLUSION: UFT 300 mg/m2/d plus oral leucovorin 90 mg/d administered for 28 days did not demonstrate antitumor activity against advanced hepatomas. Further treatment using this regimen is not recommended for this disease.

Adult↗

PubMed1999

Phase II trial of uracil/tegafur (UFT) plus leucovorin in patients with advanced biliary carcinoma.

PRECIS: UFT 300 mg/m2/day and leucovorin 90 mg/day could be administered safely to patients with advanced biliary cancer with good performance status; however, this combination and schedule of 28-day administration has no activity in this disease. PURPOSE: To determine the activity and evaluate the toxicity of uracil and tegafur in a 4:1 molar concentration ratio (UFT; Bristol-Myers Squibb, Wallingford, CT) plus oral calcium leucovorin in the treatment of patients with advanced biliary (gallbladder and bile duct) carcinoma. PATIENTS AND METHODS: Thirteen patients with advanced measurable biliary carcinoma were enrolled onto the trial. All patients had a Karnofsky performance status > or = 60%, platelet count > or = 75,000/microL, total bilirubin < or = 2.0x institutional upper limit of normal but otherwise normal liver and kidney function profile and bidimensionally measurable disease by CT scan or ultrasound examination. None of these patients previously received cytotoxic chemotherapy or radiation therapy for advanced disease. Patients received 300 mg/m2/d UFT plus 90 mg/d leucovorin administered orally in divided daily doses every 8 hours for 28 days repeated every 35 days. Objective tumor response, the primary endpoint of this trial, was evaluated after two courses of therapy. Other endpoints included toxicity, time to progression, and overall survival. RESULTS: All patients were evaluable for response and toxicity. No complete or partial responses were observed in this trial. Four patients had stable disease lasting 17, 30, 33, and 35 weeks, respectively. The median (range) time to progression and survival were 9 (1-35) and 28 (1-61) weeks, respectively. Treatment-related toxicity was mild with severe (grade 3 or 4) diarrhea seen in 2 (15%). Grade 3-4 hyperbilirubinemia (31%) and nausea/vomiting (31%) were observed and likely related to the underlying disease. Grade 1 and 2 toxic effects included mainly anorexia and fatigue. CONCLUSION: UFT 300 mg/m2/d plus oral leucovorin 90 mg/d administered for 28 days repeated every 35 days is ineffective in the treatment of advanced biliary carcinoma.

Aged↗

PubMed1998

The genome sequence of Rickettsia prowazekii and the origin of mitochondria.

We describe here the complete genome sequence (1,111,523 base pairs) of the obligate intracellular parasite Rickettsia prowazekii, the causative agent of epidemic typhus. This genome contains 834 protein-coding genes. The functional profiles of these genes show similarities to those of mitochondrial genes: no genes required for anaerobic glycolysis are found in either R. prowazekii or mitochondrial genomes, but a complete set of genes encoding components of the tricarboxylic acid cycle and the respiratory-chain complex is found in R. prowazekii. In effect, ATP production in Rickettsia is the same as that in mitochondria. Many genes involved in the biosynthesis and regulation of biosynthesis of amino acids and nucleosides in free-living bacteria are absent from R. prowazekii and mitochondria. Such genes seem to have been replaced by homologues in the nuclear (host) genome. The R. prowazekii genome contains the highest proportion of non-coding DNA (24%) detected so far in a microbial genome. Such non-coding sequences may be degraded remnants of 'neutralized' genes that await elimination from the genome. Phylogenetic analyses indicate that R. prowazekii is more closely related to mitochondria than is any other microbe studied so far.

DNA Replication↗

PubMed2002

A proteomic view of the Plasmodium falciparum life cycle.

The completion of the Plasmodium falciparum clone 3D7 genome provides a basis on which to conduct comparative proteomics studies of this human pathogen. Here, we applied a high-throughput proteomics approach to identify new potential drug and vaccine targets and to better understand the biology of this complex protozoan parasite. We characterized four stages of the parasite life cycle (sporozoites, merozoites, trophozoites and gametocytes) by multidimensional protein identification technology. Functional profiling of over 2,400 proteins agreed with the physiology of each stage. Unexpectedly, the antigenically variant proteins of var and rif genes, defined as molecules on the surface of infected erythrocytes, were also largely expressed in sporozoites. The detection of chromosomal clusters encoding co-expressed proteins suggested a potential mechanism for controlling gene expression.

Animals↗

PubMed2026

Whole-genome sequencing reveals hidden antimicrobial resistance genes in phenotypically susceptible probiotic candidate lactic acid bacteria.

Phenotypic assays commonly used to evaluate probiotic safety may fail to detect clinically relevant antimicrobial resistance (AMR), potentially allowing genetically concerning strains to appear acceptable based on MIC testing alone. To explore this issue, we applied whole-genome sequencing (WGS) to three lactic acid bacteria (LAB) isolates previously identified as probiotic candidates based on acid and bile tolerance, antagonism against enteric pathogens, and biofilm formation in vitro: Lactiplantibacillus plantarum L25F and L22F (from pigs) and Ligilactobacillus salivarius AF2319 (from a chicken). Genome annotation identified extensive repertoires of probiotic-associated genes (46-47 per strain) linked to stress tolerance, adhesion, immunomodulation, and quorum sensing, supporting functional potential. The two L. plantarum strains exhibited broader predicted metabolic capacities than L. salivarius AF2319. However, genomic analysis revealed acquired AMR genes with complex genotype-phenotype relationships not fully apparent from phenotypic testing. The L. plantarum strains harbored lnu(A) (99.79% identity) on extrachromosomal DNA, conferring the L-phenotype (lincomycin resistance, clindamycin susceptibility); clindamycin MICs (1 mg/L) were concordant with this genotype, though lincomycin MICs were not determined. L. salivarius AF2319 carried tet(M), tet(L), and erm(C) (99.48%, 99.49%, and 99.45% identity by ResFinder, respectively) on extrachromosomal DNA; notably, the erythromycin MIC (1 mg/L) was precisely at the EFSA breakpoint (≤ 1 mg/L), representing borderline genotype-phenotype discordance potentially due to silent gene expression. Under current EFSA QPS criteria, these acquired ARGs would preclude all three strains from approval as probiotic feed additives despite favorable functional profiles, underscoring the indispensable role of WGS-based AMR gene detection in modern probiotic safety evaluation.

Probiotics↗

PubMed1998

Metabolism of the vitamin D3 analogue EB1089 alters receptor complex formation and reduces promoter selectivity.

1. 1alpha,25-dihydroxyvitamin3 (VD) is a nuclear hormone that has important cell regulatory functions but also a strong calcemic effect. EB1089 is a potent antiproliferative VD analogue, which has a modified side chain resulting in increased metabolic stability and a selective functional profile. Since EB1089 is considered for potential systemic application, it will be investigated to what extent its recently identified metabolites (hydroxylated at positions C26 and C26a) contribute to biological profile of the VD analogue. 2. Limited protease digestion analysis demonstrated that EB1089 is able to stabilize the high affinity ligand binding conformation of the VDR, starting at concentrations of 0.1 nM and affecting up to 80% of all receptor molecules. The metabolites EB1445 and EB1470 showed to be 100 fold less potent than EB1089, whereas the remaining three metabolites (EB1435, EB1436 and EB1446) showed a clearly reduced ability to stabilize the high affinity ligand binding conformation. Interestingly, at pharmacological concentrations all EB1089 metabolites stabilized a second, apparently lower affinity conformation to a much higher extent than EB1089. 3. In reporter gene assays all metabolites showed lower potency than EB1089. Moreover, the preference of EB1089 for activation of VDR binding to sites formed by inverted palindromic arrangements spaced by nine nucleotide (IP9-type VD response elements) appeared to be reduced (with EB1445 and EB1470) or completely lost (with EB1435, EB1436 and EB1446). The ranking of EB1089 and its metabolites that was obtained by limited protease digestion and reporter gene assays was confirmed by an analysis of their antiproliferative effect in breast cancer cells. . The potency and selectivity of the EB1089 metabolites in mediating gene regulatory effects was found to be drastically reduced in comparison to the parent compound suggesting that the contribution of the metabolites to the biological effect of EB1089 is minor. However, the compounds showed to be interesting tools for understanding the selective biological profile of EB1089.

Antineoplastic Agents↗

PubMed2002

Suppression of basic fibroblast growth factor-induced angiogenesis by a specific chymase inhibitor, BCEAB, through the chymase-angiotensin-dependent pathway in hamster sponge granulomas.

1. We investigated the profound involvement of mast cell chymase, an alternative angiotensin II-generating enzyme, in angiogenesis using a specific chymase inhibitor. We also studied the functional profiles of this novel inhibitor in basic fibroblast growth factor (bFGF)-induced angiogenesis. 2. In this study, angiogenesis was induced by daily injections of bFGF (0.3 micro g site(-1) day(-1)), angiotensin I (2 nmol site(-1) day(-1)) or angiotensin II (2 nmol site(-1) day(-1)) into sponges implanted to male hamsters subcutaneously for 7 days. Angiogenesis in the granulation tissue surrounding sponges was evaluated by measuring the haemoglobin (Hb) content and local blood flow as the parameters for angiogenesis. 3. A chymase inhibitor, BCEAB (4-[1-[[bis-(4-methyl-phenyl)-methyl]-carbamoyl]-3-(2-ethoxy-benzyl)-4-oxo-azetidine-2-yloxy]-benzoic acid), was simultaneously administered into the implanted sponges (2 or 5 nmol site(-1) day(-1), for 7 days) treated with bFGF and strongly suppressed the haemoglobin contents in sponge granulomas. In the studies using a laser doppler perfusion imager, BCEAB (5 nmol site(-1) day(-1)) also attenuated the bFGF-induced increase of local blood flow around the implanted sponge granuloma. 4. In bFGF-induced angiogenesis, chymase activity in sponge granulomas was substantially increased. It was also confirmed that the chymase activity increased by bFGF was significantly and dose-dependently inhibited by BCEAB (2, 5 nmol site(-1) day(-1)). 5. BCEAB inhibited the Hb contents and the expression of vascular endothelial growth factor (VEGF) mRNA induced by angiotensin I but not by angiotensin II. 6. These results suggest that the significance of chymase in bFGF-induced angiogenesis was confirmed, and a novel inhibitor, BCEAB, strongly suppresses the bFGF-induced angiogenesis through the chymase-angiotensin II-VEGF dependent pathway.

Angiotensins↗

PubMed2003

Nrf2 is an inhibitor of the Fas pathway as identified by Achilles' Heel Method, a new function-based approach to gene identification in human cells.

Here we describe the Achilles' Heel Method (AHM), a new function-based approach for identification of inhibitors of signaling pathways, optimized for human cells. The principle of AHM is the identification of 'sensitizing' cDNAs based on their decreased abundance following selection. As a proof of principle, we have employed AHM for the identification of Fas/CD95/APO-1 pathway inhibitors. HeLa cells were transfected with an antisense cDNA expression library in an episomal vector followed by selection with a suboptimal dose of the apoptotic inducer. Antisense inactivation of Fas inhibitors rendered the cells more sensitive to apoptosis resulting in their preferential death and consequent loss of their sensitizing episomes that were identified by subtraction. We show that the resulting products were enriched for sensitizing cDNAs as seven out of eight candidates tested were confirmed as inhibitors of Fas-induced killing either by transfection or by pharmacological inhibition. Furthermore, we demonstrate by multiple approaches that one candidate, NF-E2 related factor 2 (Nrf2), is an inhibitor of Fas-induced apoptosis. Inactivation of Nrf2 by antisense or by a membrane permeable dominant-negative polypeptide sensitized cells while overexpression of Nrf2 protected cells from Fas-induced apoptosis. In addition, dicumarol, an inhibitor of the phase II detoxifying enzyme NQO1, a downstream target of Nrf2, sensitized cells. Nrf2 induces the production of Glutathione (GSH) and we demonstrated that N-acetyl L-cysteine (NAC), a precursor to GSH, protected cells from Fas-mediated killing. Taken together, AHM is a powerful approach for the identification of inhibitors of a signaling pathway with a low rate of false positives that opens new avenues for function profiling of human genes and discovery of new drug targets.

Apoptosis↗

PubMed1989

Functional properties of membrane cofactor protein of complement.

Membrane cofactor protein (MCP or gp45-70) of the complement system is a cofactor for factor I-mediated cleavage of fluid-phase C3b and C3b-like C3, which opens the thioester bond. In the present study the activity of MCP was further characterized. Unexpectedly, in the absence of factor I, MCP stabilized the alternative- and, to a lesser extent, the classical-pathway cell-bound C3 convertases and thereby enhanced C3b deposition. Soluble MCP, if added exogenously, hardly functioned as cofactor for the cleavage of erythrocyte-bound C3b to iC3b; i.e. its activity, compared with the cofactor activity of factor H, was inefficient, since less than 10% of the bound C3b was MCP-sensitive. Further, exogenously added soluble MCP was also a weak cofactor for the cleavage of C3b bound to zymosan. Likewise, factor I, in the presence of cells bearing MCP, cleaved fluid-phase C3b inefficiently. These results imply that MCP has very little extrinsic cofactor activity for factor I. In contrast, exogenously added MCP and factor I mediated efficient cleavage of erythrocyte-bound C3b if the concentration of Nonidet P40 was sufficient to solubilize the cells. Interestingly, soluble MCP and factor I degraded C3b attached to certain solubilized acceptor membrane molecules more readily than others. The cleavage reaction of fluid-phase and cell-bound C3b by soluble MCP and factor I produced iC3b, but no C3c and C3dg. These and prior data indicate that soluble MCP has potent cofactor activity for fluid-phase C3b or C3b bound to solubilized molecules, but acts inefficiently towards C3b on other cells. This functional profile is unique for a C3b/C4b binding protein and, taken together with its wide tissue distribution, suggests an important role for MCP in the regulation of the complement system.

Animals↗

PubMed1998

Mammalian expression of transmembrane receptors for pharmaceutical applications.

Three mammalian expression systems suitable for expressing recombinant receptors have been described. Each is suited to a different aspect of the study of receptors and their behaviour. IRES-based vectors are ideal for creating stable mammalian cell lines suitable for screening receptors using a signalling readout. Unlike traditional vectors they result in almost 100% of cell lines generated expressing a particular receptor, thus increasing the efficiency of cell line generation and increasing the chance of higher expression-level cell lines being generated. They may also be utilized to express more than one protein of interest, for example it is possible to co-express a particular receptor with a particular signalling protein or trafficking protein from a single RNA, thus ensuring that both are expressed simultaneously in the same cell. The ecdysone-inducible expression system is ideal for studying receptor signalling and behaviour. It is possible to alter receptor expression levels in an identical cellular background thus making it possible to study phenomena such as constitutive receptor activity in the absence of agonist. The SFV expression system is ideal for expressing receptors at high levels of a mammalian cell. It is thus a good system for purifying receptors for structural analysis and for providing material for binding assays. All of the expression systems described above have been demonstrated to express seven-transmembrane receptors with the expected pharmacological and functional profile.

Animals↗

PubMed2001

Functional heterogeneity of anti-endothelial cell antibodies.

While it has been claimed that some anti-endothelial cell antibodies (AECA) activate EC, there is also evidence that others trigger apoptosis. To address the issue of whether activation is a prerequisite for AECA-mediated apoptosis of EC, 23 AECA-positive sera were evaluated for their ability to induce activation and/or apoptosis. Activation was defined as an over-expression of E-selectin and intercellular adhesion molecule 1. Optical microscopy, annexin V binding, hypoploid cell enumeration, and determination of poly (ADP-ribose) polymerase cleavage-related products were used to assess apoptosis. Four functional profiles were defined: 10 sera promoted activation and apoptosis (act+/apo+), one was act+/apo-, six act-/apo+, and the remaining six act-/apo-. The reduced membrane expression of thrombomodulin was associated with apoptosis, rather than activation. Caspase-3 was implicated in the two models of apoptosis, the ratios of several survival proteins to Bax decreased, regardless of the ability of apo+ AECA to activate the cells, while radical oxygen species did not appear to be involved. Furthermore, it occurred that macrophages engulfed EC treated with apoptosis-promoting AECA, but not those incubated with AECA that did not induce apoptosis. Hence, AECA represent an extremely heterogeneous family of autoantibodies, not only because of the variety of their target antigens, but also the subsequent diversity of their effects.

Antibody Diversity↗