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Biomedical subjects

G Thibault

Publications and source records attributed to G Thibault.

At least 37 records · Page 2Linked to original sources
PubMed1999

Metabolic and antihypertensive effects of nebivolol and atenolol in normometabolic patients with mild-to-moderate hypertension.

This was a double-blind, randomized, two-center, active-controlled, prospective, parallel study designed to evaluate the effects of nebivolol at daily doses of 5 mg on lipid and carbohydrate metabolism and on blood pressure in comparison with atenolol at daily doses of 50 mg. Normometabolic subjects with mild-to-moderate essential hypertension were recruited for this study, which included a 4-week, single-blind placebo washout phase and a 12-week double-blind treatment phase. After 12 weeks of treatment, both drugs demonstrated a significant decrease from baseline in high-density lipoprotein (HDL) apolipoprotein A-I (HDL-apoA-I) (nebivolol, P <.02; atenolol, P <.05). A significant reduction in HDL cholesterol (HDL-C) from baseline was also observed with nebivolol (P <.05). There were no significant differences between the drugs for these parameters, and the ratio low-density lipoprotein cholesterol (LDL-C)-to-HDL-C did not change significantly after 12 weeks of active treatment with nebivolol or atenolol. There were no significant changes in total cholesterol, HDL (2) -C, HDL (3) -C, LDL-C, very-low-density lipoprotein cholesterol (VLDL-C), total triglycerides, HDL-triglycerides (TG), LDL-TG, VLDL-TG, total apoB, LDL-B, VLDL-B (including the ratio LDL-C-to-LDL-apoB), or Lp(a) during treatment with both drugs. No significant differences in plasma apoA-I and apoC-III as well as in apoA-I-, C-III-containing lipoprotein particles (including the apoC-III ratio) were observed between the drugs, neither before nor after each active treatment. There were no significant differences between the drugs or within each treatment group in plasma glucose, insulin, or C-peptide concentrations after a 2-hour oral glucose tolerance test. Mean clinic trough sitting systolic blood pressure (SBP)/diastolic blood pressure (DBP) significantly decreased from 150/98 mm Hg at baseline to 141/90 mm Hg at termination for nebivolol and from 160/99 mm Hg at baseline to 145/88 mm Hg at termination for atenolol. No significant between-treatment differences were observed for the mean clinic trough sitting SBP/DBP. Both drugs significantly increased the atrial natriuretic factor (ANF) N-terminal plasma levels, whereas no changes were observed in ANF C-terminal plasma concentrations. A significant decrease (P <. 05) in the plasma adrenocorticotropic hormone levels was observed after administration of both drugs. A significant decrease (P <.05) in plasma cortisol levels was observed only after atenolol treatment. The incidence of adverse events reported during nebivolol treatment was comparable to that observed during atenolol treatment. Heart rate was significantly reduced by both drugs. There were no significant changes in hematology, biochemistry, or urinalysis studies. Neither nebivolol nor atenolol adversely affected lipid or carbohydrate metabolism in normometabolic hypertensive patients. Both treatments demonstrated adequate and similar antihypertensive effects and were well tolerated.

Adrenocorticotropic Hormone↗
PubMed1999

Regulation of natriuretic peptide secretion by the heart.

Secreted by the heart, more specifically by atrial cardiomyocytes under normal conditions but also by ventricular myocytes during cardiac hypertrophy, natriuretic peptides are now considered important hormones in the control of blood pressure and salt and water excretion. Studies on natriuretic peptide secretagogues and their mechanisms of action have been complicated by hemodynamic changes and contractions to which the atria are constantly subjected. It now appears that atrial stretch through mechano-sensitive ion channels, adrenergic stimulation via alpha 1A-adrenergic receptors, and endothelin via its ETA receptor subtype are major triggering agents of natriuretic peptide release. With several other stimuli, such as angiotensin II and beta-adrenergic agents, modulation of natriuretic peptide release appears to be linked to local generation of prostaglandins. In all cases, intracellular calcium homeostasis, controlled by several ion channels, is considered a key element in the regulation of natriuretic peptide secretion.

Animals↗
PubMed1998

Human T lymphocyte proliferative response to resting porcine endothelial cells results from an HLA-restricted, IL-10-sensitive, indirect presentation pathway but also depends on endothelial-specific costimulatory factors.

To investigate the mechanisms of cellular rejection in pig-to-human xenotransplantation, the proliferation of different human purified lymphocyte subpopulations in response to swine leukocyte Ag class II-negative porcine aortic endothelial cells (PAEC) was measured in the presence or absence of human autologous adherent cells (huAPC). CD8+ lymphocytes proliferated moderately in the absence of huAPC, and the immune response was slightly increased when huAPC were added. CD56+ lymphocytes failed to proliferate in response to PAEC whether huAPC were present or not. CD4+ lymphocytes alone did not proliferate in response to PAEC, but a strong proliferative response was observed in the presence of metabolically active huAPC. This response was totally abolished by mAbs directed against HLA class II molecules or by pretreatment of huAPC by human IL-10. Even in the presence of huAPC, CD4+ lymphocytes failed to respond to fixed PAEC or to PAEC-lysates, suggesting that PAEC must be viable to support lymphocyte proliferation. Finally, none of the nonendothelial porcine adherent cells tested was able to induce human lymphocyte proliferation, despite the fact that they also provided a large set of xenogeneic peptides. Our results show that the indirect presentation pathway of xenoantigens by huAPC to CD4+ lymphocytes is crucial in the response to porcine endothelial cells, and that IL-10 could be of therapeutic interest to prevent human lymphocyte activation by this pathway. Furthermore, we demonstrated that stimulatory signals specifically provided by endothelial cells are also necessary for this huAPC-restricted proliferative response.

Animals↗
PubMed1998

Evidence for intracellular generation of angiotensin II in rat juxtaglomerular cells.

The formation of the vasoactive peptide angiotensin II (AII) is dependent on the sequential action of two enzymes, renin and angiotensin converting enzyme (ACE), on the substrate angiotensinogen. Although the renin-producing cells of the kidney do not express angiotensinogen, they contain large amounts of AII in the same storage granules that contain renin. When renin expression is suppressed in these cells, AII also disappears. In the current study, we have tested whether the renin-associated disappearance of AII in renal juxtaglomerular (JG) cells is due to a renin-dependent down-regulation of granule biosynthesis and whether receptor-mediated internalization of AII could account for its concentration in these cells. Our results support a model whereby AII peptides are generated within JG cells, presumably by a mechanism which involves the action of endogenous renin on internalized, exogenous angiotensinogen.

Angiotensin II↗
PubMed1998

Endothelin-1 signaling is altered in cardiac cells from deoxycorticosterone acetate-salt hypertensive rats.

The cardiac cellular effects of endothelin-1 (ET-1) on intracellular free Ca2+ concentration ([Ca2+]i) were investigated in deoxycorticosterone acetate (DOCA)-salt rats with severe cardiac hypertrophy. [Ca2+]i was measured by fura-2 methodology in ventricular cardiomyocytes and fibroblasts of DOCA-salt hypertensive and control unilaterally nephrectomized rats (Uni-Nx). Blood pressure and heart weight were increased (p < 0.01) in DOCA-salt rats compared to control rats. ET-1 (10(-12)-10(-6) M) increased [Ca2+]i in a dose-dependent manner in both cell types from control and hypertensive rats. However, ET-1-induced [Ca2+]i responses were significantly attenuated (p < 0.01) in cardiomyocytes and fibroblasts of DOCA-salt rats. Sarafotoxin S6c (S6c) increased [Ca2+]i in fibroblasts but not in cardiomyocytes. In conclusion, ET-1 dose-dependently increased [Ca2+]i in cardiomyocytes (primarily via ETA receptors) and fibroblasts (via ETA and ETB receptors). Cardiac cell ET-1 signaling pathways are blunted in DOCA-salt hypertensive rats. ET-1 may not play a critical role in the pathophysiology of the severe concentric cardiac hypertrophy present in DOCA-salt hypertensive rats.

Animals↗
PubMed1998

Immunolocalization of natriuretic peptide receptor B in the rat kidney.

The natriuretic peptide receptor (NPR) family consists of three receptor subtypes: two transmembrane forms that contain a guanylyl cyclase intracellular domain (NPR-A and NPR-B), and one truncated form (NPR-C). Because of the lack of specific agonists and antagonists for each receptor subtype and to the difficulty to detect the presence of small quantities of NPR-B by ligand binding studies, polyclonal antibodies against a peptide whose sequence was chosen from a region of the extracellular domain of rat NPR-B that is not homologous to sequences in NPR-A and NPR-C were developed. Western blotting with affinity-purified anti-NPR-B (413-426)-Tyr revealed a polypeptide of approximately 120 kD on COS-1 cell membranes transfected with rat NPR-B cDNA. The antibody recognized a second polypeptide, approximately 5 to 10 kD smaller, which probably represents the unglycosylated receptor. Anti-NPR-B (413-426)-Tyr did not show crossreactivity to any other NPR. Western blotting analysis with anti-NPR-B (413-426)-Tyr also identified a protein of appropriate size in renal vascular membranes. These results were supported by immunohistochemistry findings that demonstrated staining for NPR-B on papillary and medullary capillaries, glomeruli, and renal arteries. This study concludes that NPR-B is present in the rat kidney, although it was only detected in vascular structures.

Animals↗
PubMed1997

Glucose transporter (GLUT-4) is targeted to secretory granules in rat atrial cardiomyocytes.

The insulin-responsive glucose transporter GLUT-4 is found in muscle and fat cells in the trans-Golgi reticulum (TGR) and in an intracellular tubulovesicular compartment, from where it undergoes insulin-dependent movement to the cell surface. To examine the relationship between these GLUT-4-containing compartments and the regulated secretory pathway we have localized GLUT-4 in atrial cardiomyocytes. This cell type secretes an antihypertensive hormone, referred to as the atrial natriuretic factor (ANF), in response to elevated blood pressure. We show that GLUT-4 is targeted in the atrial cell to the TGR and a tubulo-vesicular compartment, which is morphologically and functionally indistinguishable from the intracellular GLUT-4 compartment found in other types of myocytes and in fat cells, and in addition to the ANF secretory granules. Forming ANF granules are present throughout all Golgi cisternae but only become GLUT4 positive in the TGR. The inability of cyclohexamide treatment to effect the TGR localization of GLUT-4 indicates that GLUT-4 enters the ANF secretory granules at the TGR via the recycling pathway and not via the biosynthetic pathway. These data suggest that a large proportion of GLUT-4 must recycle via the TGR in insulin-sensitive cells. It will be important to determine if this is the pathway by which the insulin-regulatable tubulo-vesicular compartment is formed.

Animals↗
PubMed1997

Tissue targeting of angiotensin peptides.

Angiotensin II (Ang II) is an octapeptide generated by the sequential proteolytic action of renin and angiotensin converting enzyme on the glycoprotein angiotensinogen. While numerous mammalian tissues have been shown to express some or all of the components of the renin-angiotensin system (RAS), the function of most of these tissue RAS remains a matter of conjecture. To test for tissue-specific functions of Ang II and as an alternative to co-expressing all the components of RAS, we have engineered a fusion protein that leads to direct Ang II release within specific tissues. The angiotensin peptide is cleaved from the fusion protein within the secretory pathway by the ubiquitous endoprotease furin and is released from the cell by constitutive secretion. Direct injection of an expression vector encoding such a fusion protein into rat cardiac ventricles results in a highly localized expression of atrial natriuretic peptide mRNA (an angiotensin responsive marker of cardiac hypertrophy), demonstrating the utility of this approach for local targeting of mature peptides to tissues in animal models.

Angiotensin II↗
PubMed1997

Adverse outcomes of underuse of beta-blockers in elderly survivors of acute myocardial infarction.

OBJECTIVES: To study determinants and adverse outcomes (mortality and rehospitalization) of beta-blocker underuse in elderly patients with myocardial infarction; and whether the relative risks (RRs) of survival associated with beta-blocker use were comparable to those reported in the large randomized controlled trials (RCTs). SETTING: New Jersey Medicare population. DESIGN: Retrospective cohort design using linked Medicare and drug claims data from 1987 to 1992. PATIENTS: Statewide cohort of 5332 elderly 30-day acute myocardial infarction (AMI) survivors with prescription drug coverage, of whom 3737 were eligible for beta-blockers. MAIN OUTCOME MEASURES: beta-Blocker and calcium channel blocker use in the first 90 days after discharge and mortality rates and cardiac hospital readmissions over the 2-year period after discharge, controlling for sociodemographic and baseline risk variables. RESULTS: Only 21% of eligible patients received beta-blocker therapy; this rate remained unchanged from 1987 to 1991. Patients were almost 3 times more likely to receive a new prescription for a calcium channel blocker than for a new beta-blocker after their AMIs. Advanced age and calcium channel blocker use predicted underuse of beta-blockers. Controlling for other predictors of survival, the mortality rate among beta-blocker recipients was 43% less than that for nonrecipients (RR=0.57; 95% confidence interval [CI], 0.47-0.69). Effects on mortality were substantial in all age strata (65-74 years, 75-84 years, and > or = 85 years) and consistent with the results for elderly subgroups of 2 large RCTs. beta-Blocker recipients were rehospitalized 22% less often than nonrecipients (RR=0.78; 95% CI, 0.67-0.90). Use of a calcium channel blocker instead of a beta-blocker was associated with a doubled risk of death (RR= 1.98; 95% CI, 1.44-2.72), not because calcium channel blockers had a demonstrable adverse effect, but because they were substitutes for beta-blockers. CONCLUSIONS: beta-Blockers are underused in elderly AMI survivors, leading to measurable adverse outcomes. These data suggest that the survival benefits of beta-blockade after an AMI may extend to eligible patients older than 75 years, a group that has been excluded from RCTs.

Adrenergic beta-Antagonists↗
PubMed1997

Discharge destination and repeat hospitalizations.

OBJECTIVES: Is discharge destination a determinant of readmission? Studies to date have been inconclusive. The primary purpose of this study was to identify the role of discharge destination in the occurrence of repeat hospitalizations for a national sample of patients discharged from Veterans Health Administration (VHA) hospitals. METHODS: The authors studied a 20% random sample of individual patients, 65 years of age or older, with either chronic obstructive pulmonary disease, stroke, or dementia who were discharged from a Veterans Health Administration hospital in 1988. All data for the study were obtained from secondary administrative sources. Multiple sources were used to determine discharge destination. The authors focused on personal home versus nursing home discharge destination. Both VHA and Medicare discharge data were used to track hospital readmissions. Proportional hazards regression models were used to examine the independent association of discharge destination with time to readmission within 30 days, 6 months, 1 year, and 2 years of discharge, adjusting for severity, other clinical and demographic characteristics, and censoring deaths. RESULTS: After adjustment and including out-of-system (Medicare) use, we found that patients with chronic obstructive pulmonary disease and patients with dementia who were discharged to nursing homes (community and Veterans Health Administration, respectively) were less likely to be readmitted within 30 days after discharge than patients discharged to personal homes. CONCLUSIONS: These findings have important implications for adjusting hospital performance profiles based on discharge destination and for focussing efforts to reduce the frequency and associated costs of hospital readmissions.

Aftercare↗
PubMed1997

Comparison of ANP binding and sensitivity in brains from hypertensive and normotensive rats.

We compared the abundance and sensitivity of atrial natriuretic peptides (ANP) receptors in the brains of spontaneously hypertensive (SHR) and Wistar-Kyoto (WKY) rats and examined the effect of blood pressure on the abundance of brain ANP receptors in several other experimental rat models. Brain slices from SHR generated more guanosine 3',5'-cyclic monophosphate in response to ANP than brain slices from WKY rats. No differences were found in brain particulate guanylate cyclase activity in both strains of rats. In rat brain homogenates, we observed that ANP bound in a specific and saturable fashion to samples from WKY rats, but not in samples from SHR. In vitro receptor autoradiography revealed that ANP binding was reduced in the subfornical organ, the choroid plexus, and the paraventricular nucleus of SHR compared with WKY rat brains. Correction of hypertension in SHR or induction of hypertension in other strains did not affect ANP binding in any of these brain regions. Altogether, our data suggest that the increased sensitivity of SHR brains to the action of ANP may be a consequence of factors other than the abundance of receptors and that it is not secondary to the elevation of blood pressure.

1-Methyl-3-isobutylxanthine↗
PubMed1997

Cardiac type-1 angiotensin II receptor status in deoxycorticosterone acetate-salt hypertension in rats.

The regulation of angiotensin II (Ang II) receptors and Ang II-induced modulation of intracellular Ca2+ concentration in cardiac cells from hearts of experimentally induced hypertensive deoxycorticosterone acetate (DOCA)-salt and control unilaterally nephrectomized (Uni-Nx) Sprague-Dawley rats was assessed. Ang II receptor density and intracellular Ca2+ concentration measurements were examined in adult ventricular myocytes and fibroblasts by radioligand binding assay and digital imaging using fura 2 methodology, respectively. Four-week DOCA-salt treatment induced hypertension associated with cardiac hypertrophy. Ang II binding studies demonstrated that adult ventricular myocytes and fibroblasts possess mainly the AT1 subtype receptor. Moreover, DOCA-salt hypertension was associated with a 1.8-fold increase in Ang II-specific binding compared with myocytes from Uni-Nx control rats. Intracellular Ca2+ responses induced by increasing Ang II concentrations (10[-12] to 10[-4] mol/L) were significantly enhanced in cardiomyocytes from DOCA-salt rats. The effects of Ang II on intracellular Ca2+ spike frequency were unaltered in cardiomyocytes from DOCA-salt-hypertensive rats. The density of AT1 subtype receptors was not modified in ventricular fibroblasts after DOCA-salt treatment. Ang II increased intracellular Ca2+ concentration similarly in ventricular fibroblasts from normal and hypertensive rats. In conclusion, DOCA-salt hypertension is characterized by an increased AT1 receptor density and intracellular calcium responses in ventricular myocytes, whereas in ventricular fibroblasts the AT1 receptor status is unaltered. These findings report for the first time the cardiac cell-specific implication of Ang II and the intracellular calcium signaling pathway stimulated by the AT1 receptor in cardiac hypertrophy in DOCA-salt-hypertensive rats.

Animals↗
PubMed1997

Clinical significance of endothelin in cardiovascular disease.

Endothelins are ubiquitously produced 21-amino-acid peptides that were discovered as an endothelial product and may play important roles in cardiovescular physiology and pathophysiology. The main endothelin produced by the endothelium is endothelin-1. The vasoconstrictor role of endothelins may participate in blood pressure elevation and vascular hypertrophy in salt-dependent models of hypertension (deoxycorticosterone acetate-salt hypertensive rats, spontaneously hypertensive rats treated with deoxycorticosterone, acetate and salt, and Dehl salt-sensitive rats), and in stroke-prone spontaneously hypertensive rats. In humans, endothelins may play important roles in moderate to severe essential hypertension, and in the hypertension of African-Americans. Endothelins may be involved in cardiac hypertrophy, and there is increasing evidence of their participation in heart failure, in which acute endothelin antagonism in humans exerts beneficial effects. Endothelin expression is enhanced in smooth muscle cells migrating into the intima of arteries in atherosclerosis, suggesting a role in atherogenesis. Endothelin may participate as a vasoconstrictor in coronary artery disease, and as a contributor to intimal proliferation in restenosis after coronary angioplasty. In patients with myocardial infarction, cardiac production of endothelin is increased, particularly in those with cardiogenic shock. There is a potential for participation of endothelins in vasospasm accompanying stroke or subarachnoid hemorrhage: in the latter, endothelin antagonism has shown beneficial effects in experimental models. In neonatal and in primary pulmonary hypertension, endothelin expression is enhanced, and in experimental models endothelin antagonism resulted in favorable responses. Systemic sclerosis is another, peripheral, form of vascular disease in which endothelin may play a role and in which endothelin antagonism may be an interesting therapeutic alternative. The pathophysiologic role of endothelins is becoming increasingly apparent in cardiovascular disease, generating interesting potential therapeutic targets for the use of endothelin antagonists or endothelin-converting enzyme inhibitors.

Animals↗
PubMed1996

Docosahexaenoic and eicosapentaenoic acids inhibit in vitro human lymphocyte-endothelial cell adhesion.

Dietary supplementation with fish oil, which contains high amounts of long chain omega 3 ((n-3)) polyunsaturated fatty acids (PUFAs), particularly docosahexaenoic acid (DHA) and eicosapentaenoic acid (EPA), has recently been shown to have protective and ameliorative effects on diseases characterized by chronic inflammatory reactions. Interactions between vascular endothelium, mononuclear cells, and cytokines are crucial steps in the course of inflammatory processes such as chronic graft rejection. We therefore studied the effects of DHA and EPA on both the adhesion of peripheral blood lymphocytes (PBL) to human endothelial cells (EC) in culture and the expression of EC-adhesion molecules and their counterreceptors on PBL. The addition of DHA or EPA to the adhesion assay significantly decreased the adhesion of PBL to untreated EC and tumor necrosis factor-alpha (TNF alpha)-, interleukin (IL) 4-, and lipopolysaccharide-stimulated EC. When EC were pretreated with (n-3) PUFAs for 18 hr, washed, and then stimulated by TNF alpha, IL-4, or lipopolysaccharide, PBL adhesion was also significantly reduced compared with controls. We also showed that PBL preincubated with DHA or EPA, and then washed and chromium radiolabeled, still exhibited an adhesion inhibition to TNF alpha- and IL-4-treated EC as well as untreated EC. Cytofluorometry and immunoenzymatic analyses indicated that pretreatment of EC with (n-3) PUFAs before their activation significantly reduced the EC-induced expression of vascular cell adhesion molecule 1, whereas the level of expression of intercellular adhesion molecule 1 and E-selectin was not modified. Furthermore, we showed that incubation of PBL with DHA or EPA moderately reduced the level of cell surface expression of L-selectin and leukocyte function-associated antigen 1, but not of very late antigen 4. In all cases, the inhibitory effect of (n-3) PUFAs was specific and dose dependent. In addition, DHA seems to be a more potent inhibitor than EPA, but the two compounds in association had an additive effect. Regardless of the mode of action, this inhibitory effect may explain the protective and ameliorative effects of (n-3) PUFAs on diseases involving chronic inflammatory reaction.

Cell Adhesion↗
PubMed1996

Secretory granule targeting of atrial natriuretic peptide correlates with its calcium-mediated aggregation.

Atrial natriuretic peptide (ANP) is a 28-aa peptide hormone secreted predominantly from atrial cardiocytes. ANP is first synthesized in the form of a 126-aa precursor (proANP) which is targeted to dense core granules of the regulated secretory pathway. ProANP is stored until the cell receives a signal that triggers the processing and release of the mature peptide (regulated secretion). Various models have been proposed to explain the targeting of selected proteins to the regulated secretory pathway, including specific "sorting receptors" and calcium-mediated aggregation. As potential calcium binding regions had previously been reported in the profragment of ANP, the current study was undertaken in an effort to determine the relationship between the ability of ANP to enter the regulated secretory pathway and its calcium-mediated aggregation. Deletion and site-directed mutagenesis of selected regions of the prosegment demonstrates that acidic amino acids at positions 23 and 24 are critical for both regulated secretion of proANP from transfected AtT-20 cells and calcium-mediated aggregation of purified recombinant proANP in vitro. These results demonstrate that the ability of certain proteins to enter secretory granules is directly linked to their calcium-mediated aggregation.

Amino Acid Sequence↗
PubMed1996

A protease processing site is essential for prorenin sorting to the regulated secretory pathway.

Transfected mouse pituitary AtT-20 cells were used to examine the sorting of human prorenin to dense core secretory granules and the regulated secretory pathway. These cells secrete prorenin constitutively and sort a portion of the prorenin to secretory granules, where it is converted to active renin by proteolytic processing. Pulse-chase labeling of transfected AtT-20 cells demonstrated that regulated secretion of prorenin was prevented by: 1) the mutagenic deletion of the prosegment, 2) the premature proteolytic removal of the prosegment by a Golgi-resident processing protease, or 3) the mutation of the native cleavage site so as to prevent removal of the prosegment. In addition, expression of fusion proteins containing portions of the prorenin prosegment demonstrated that exposure of potential proteolytic cleavage sites was sufficient to confer cleavage-dependent regulated secretion of the corresponding protein. These data implicate the protease cleavage event in the regulated secretion of prorenin and are consistent with the involvement of a subclass of processing proteases in the sorting of certain proteins to secretory granules in AtT-20 cells.

Amino Acid Sequence↗
PubMed1996

Removal of terminal alpha-galactosyl residues from xenogeneic porcine endothelial cells. Decrease in complement-mediated cytotoxicity but persistence of IgG1-mediated antibody-dependent cell-mediated cytotoxicity.

To determine the role of the terminal alpha-galactosyl residue in the endothelial damage mediated by human xenoreactive natural antibodies (IgM and IgG), we treated porcine endothelial cells in culture with green coffee bean alpha-galactosidase. A practically complete removal of terminal alpha-Gal residues (as evaluated by flow cytometry with Bandeiraea simplicifolia isolectin B4) and concomitant exposure of N-acetyllactosamine were obtained without altering cell viability. A dramatic decrease in IgM and IgG binding (from a pool of human sera) was observed, confirming the key role of the alpha-galactosyl residues. The enzyme treatment did not induce any nonspecific immunoglobulin binding sites, but led to the exposure of new epitopes for a minor fraction of IgM. The main residual IgM and IgG binding could be due to xenoantigens other than the alpha-galactosyl residues. When alpha-galactosidase-treated endothelial cells were used as targets in cytotoxicity experiments, they were less susceptible than untreated cells to complement-mediated cytotoxicity induced by fresh human serum. In contrast, they did not acquire resistance to human IgG-dependent cellular cytotoxicity, despite the decrease in IgG binding. Because it is known that antibody-dependent cytotoxicity mediated by CD16+ NK cells is dependent on IgG1 and IgG3, and not on IgG2 or IgG4, which was confirmed by blocking experiments, we studied the binding of all four subclasses to intact and alpha-galactosidase-treated endothelial cells. Two major subclasses, IgG1 and IgG2, bound to untreated endothelial cells, whereas IgG3 binding was low and IgG4 binding was negligible. A decrease in IgG1, IgG2, and IgG3 binding was observed upon alpha-galactosidase treatment, indicating that antibodies belonging to these three subclasses recognize alpha-galactosyl residues. The decrease in IgG2 binding was more pronounced than the decrease in IgG1 binding. Collectively, these data indicate that IgG1 xenoreactive natural antibodies, including those which are not directed at the alpha-galactosyl residues, could play a major role in the early delayed vascular rejection of pig xenografts.

Animals↗