Properties of the blood group LW glycoprotein and preliminary comparison with Rh proteins.

The major component immunoprecipitated from human red cell membranes by murine monoclonal antibodies (BS46 and BS56) against the LW blood group antigens is a 42,000 mol. wt glycoprotein. Upon digestion by an N-glycanase the LW component migrated as a 25,000 mol. wt component on SDS gels, whereas treatment by an O-glycanase led only to a small size reduction (2000). These data suggest that the LW glycoprotein might carry approximately eight to nine N-linked sugar chains and only a few (one or two) O-linked oligosaccharide chains. A minor component of 31,000 mol. wt was also identified in the LW immunoprecipitate. Preliminary analyses by two-dimensional peptide mapping indicate that the 31,000 mol. wt polypeptide is identical to authentic Rh proteins, therefore raising the possibility that the Rh and LW antigens are associated in the membrane as a functional complex called Rh cluster. Since the N-deglycosylated form of the LW and RhD proteins have different sizes (25,000 vs 31,000-32,000 respectively) and since their externally 125I-labelled domains have different two-dimensional peptide maps, it is concluded that LW is probably not a simple glycosylated form of the Rh proteins.

Characterization of the D, c, E and G antigens of the Rh blood group system with human monoclonal antibodies.

The human MAbs, anti-D, -c, -E and -G of the Rh blood group system, produced by Epstein-Barr virus transformed B-cell lines, were purified by protein A-Sepharose chromatography and used to characterize the Rh antigens of human red cells. Scatchard plot analyses performed with the radiolabelled MABs indicated that each R2R2 red cell carries 0.43, 0.32 and 0.38 x 10(5) D, c and E binding sites, respectively. About half this number of antigen sites are present on erythrocytes from heterozygote individuals using the appropriate antibody. We found, however, that only 0.18 x 10(5)G antigenic sites were present on each R1R1 red cell. The affinity constants of the anti-D, -E and -G were similar varying from 0.6 to 1.5 x 10(8) M-1 whereas that of the anti-c was much lower (0.035 x 10(8) M-1). The blood group specificity and binding properties indicate that the MAbs behave like the polyclonal anti-Rh reagents. Immunoprecipitation experiments carried out with membranes from R2R2 red cells show that a 30-32 kDa component can be identified whatever the antibody used. The immune complexes involving anti-c, -E or -G antibodies could be formed with the detergent lysates from red cell membranes. In contrast, membrane integrity was a prerequisite for the binding of the anti-D antibodies. Finally, from extraction studies of immunocomplexes with non-ionic detergents it was concluded that all the Rh-active components are bound to the membrane skeleton, suggesting that these molecules may have important function for maintaining red cell shape and viability.

Evidence that the c, D and E epitopes of the human Rh blood group system are on separate polypeptide molecules.

The distribution of the epitopes, c, D, E and G of the human Rh system on red cells has been investigated using both 125I-labelled and fluorescently-labelled MAbs. There is a very wide range in the density of each epitope on individual red cells and the numbers of c, D and E epitopes on each cell are independent of each other. These observations taken together with the finding that there is no steric hindrance to binding between the antibodies, anti-c, anti-D and anti-E indicate that these epitopes are on separate Rh polypeptide molecules. The observations are taken to indicate that the total amount of Rh polypeptide on a single red cell is constant but that there is considerable heterogeneity between cells in the amount of each separate polypeptide carrying the different epitopes. In contrast, there was a mutual inhibition of binding of anti-G and anti-D monoclonals and direct positive correlation between the number of G and D sites on individual cells, indicating that the G and D epitopes are probably on the same Rh polypeptide.

ATR-FTIR spectroscopic investigation of E. coli transconjugants beta-lactams-resistance phenotype.

Hyphenation of attenuated total reflection Fourier transform infrared spectroscopy and cluster analysis has been used to characterise a susceptible Escherichia coli K12 strain and the transconjugants TEM-1, TEM-2, TEM-3, SHV-2, SHV-3, SHV-4. A good discrimination of the susceptible strain from the transconjugants was obtained. Although a limited success was achieved in the differentiation of SHV and TEM phenotypes in general, results obtained with TEM-2 and SHV-3 were convincing. Spectral differences observed are ascribed to the global effects of the conjugation process, particularly their repercussions in the nucleic acids and carbohydrate absorbing regions, rather than to beta-lactamase point-mutations.

Acute and chronic haemodynamic effects of naftazone in portal hypertensive rats.

It has been demonstrated that hyperproduction of nitric oxide (NO) plays a major role in the vasodilatation of cirrhosis; thus, the vasodilatation might be reversed by an inhibition of NO production. Experimental studies in isolated aortic rings showed that naftazone inhibits the effects of NO production. The aim of this study was to evaluate the haemodynamic effects of acute and chronic administration of naftazone in rats with portal hypertension. Haemodynamic values were measured either before and 10 min after intravenous administration of 432 microg/kg of naftazone or after 4 days of oral administration of 10 mg/kg per day. Acute administration of naftazone significantly reduced portal pressure in portal vein-stenosed and cirrhotic rats. This reduction was related to a decrease in the resistance of the liver and collateral circulation and it was associated with an increased cardiac output. Oral administration of naftazone significantly decreased portal pressure in rats with portal vein stenosis; this decrease depended on a significant reduction of portal blood flow. In both groups, arterial pressure did not change significantly. These haemodynamic effects differed from those observed following prazosin or propranolol administration. However, these effects were similar but less marked than those observed following N-nitro-L-arginine administration in systemic and splanchnic arterial territories. In conclusion, acute and oral administration of naftazone significantly reduces portal pressure by two different mechanisms in portal hypertensive rats. The exact mechanism has, however, to be elucidated.

Naftazone reduces glutamate cerebro spinal fluid levels in rats and glutamate release from mouse cerebellum synaptosomes.

It is well known that an excessive release of glutamate in the mammalian brain plays a major role in several neurological diseases. Naftazone (Etioven) is a currently used vasoprotectant drug that is metabolized in humans by reduction and glucuronidation. In the present study naftazone was found to decrease glutamate levels in the cerebro spinal fluid (CSF) of rats treated for 15 days, as determined by a chemiluminescent glutamate assay reaction. Naftazone and its glucuronide derivative also reduced respectively spontaneous and high K+-evoked glutamate release from mouse cerebellum synaptosomes. It is likely that naftazone and its glucuronide metabolite contribute in vivo to decrease glutamate levels in the CSF through their inhibitory actions on glutamate release.

In-vitro and ex-vivo inhibition of blood platelet aggregation by naftazone.

Because of the considerable interest in the role of platelets and antiplatelet therapy in cardiovascular disease, including the aggregation of platelets to each other during arterial thrombosis and atherogenesis, we have studied the effect of naftazone (Etioven), an original vasculotropic drug on platelet aggregation. Rat and human platelets were prepared and incubated in-vitro with different concentrations of naftazone. We found that naftazone inhibited both platelet secretion and aggregation in platelet-rich plasma (PRP) and washed platelets after stimulation with thrombin or ADP. Rats were also treated intraperitoneally for five days with various naftazone doses (0.125-10 mg kg-1) and ex-vivo platelet aggregation compared, at various times after the last injection, with that of control animals. Inhibition by naftazone was dose-dependent in both PRP and isolated platelets. The inhibition was transient, a maximum value (approximately 50%) being obtained about 3-6 h after the last injection, with a return to near-control values after 24 h. Naftazone also facilitated platelet deaggregation after in-vitro stimulation with thrombin or ADP. In another series of experiments, rats were treated intraperitoneally for five days with 10 mg kg-1 of aspirin, ticlopidine, dipyridamole or naftazone. Platelets were prepared and tested for aggregation 90 min after the last injection. Thrombin-induced aggregation in PRP and washed platelets was significantly reduced after in-vivo treatment with ticlopidine and naftazone. Except for dipyridamole, all the drugs inhibited ex-vivo ADP-induced aggregation in PRP. In isolated platelet preparation, only naftazone induced a significant inhibition of ADP- or thrombin-stimulated aggregation. We conclude that naftazone inhibits platelet aggregation in-vitro and ex-vivo.

CJD PrPsc removal by nanofiltration process: application to a therapeutic immunoglobulin solution (Lymphoglobuline).

The characteristic of transmissible spongiform encephalopathies (TSE) is an accumulation of partially protease resistant (PrP(res)) abnormal prion protein (PrP(sc)). This pathological prion protein is very resistant to conventional inactivation methods. The risk of transmission of TSE, such as Creutzfeldt-Jakob disease (CJD), by biopharmaceutical products prepared from human cells must be taken into account. The nanofiltration process has been proved to be effective in removing viruses and scrapie agent. The major advantages of this technique are flexibility and efficacy in removing infectious particles without altering biopharmaceutical characteristics and properties. This study focused on the removal of human PrP(sc) by means of a nanofiltration method after spiking a Lymphoglobuline solution with a CJD brain homogenate. Lymphoglobuline equine anti-human thymocyte immunoglobulin is a selective immunosuppressive agent acting mainly on human T lymphocytes. The therapeutic indications are: immunosuppression for transplantation: prevention and treatment of graft rejection; treatment of aplastic anemia. In our study, CJD homogenate was spiked at three different dilutions (low, moderate and high) in the Lymphoglobuline product. The nanofiltration process was performed on each sample. Using the western blot technique, the PrP(res) signal detected in nanofiltrates was compared to that obtained with a reference scale (dilution series of CJD brain homogenate in Lymphoglobuline detected by western blot and elaborated on 3.3 log). After nanofiltration, the PrP(res) western blot signal was detected with a significant reduction in the less dilute sample, whereas the signal was undetectable in the two other samples. These are the first data in CJD demonstrating a clearance between 1.6 and 3.3 log with a Lymphoglobuline recovery of over 93%. The nanofiltration process confirms its relative efficacy in removing human CJD PrP(sc).

Determination of the N-terminal sequence of human red cell Rh(D) polypeptide and demonstration that the Rh(D), (c), and (E) antigens are carried by distinct polypeptide chains.

Human monoclonal antibodies (MoAbs) directed against the blood group Rh(D), (c), and (E) antigens produced by Epstein-Barr virus (EBV)-transformed lymphoblastoid cell lines have been used to characterize the Rh components of human red cell membranes and to determine whether the Rh(D), (c), and (E) epitopes are carried by distinct polypeptides. After immunoprecipitation with the anti-Rh(D) antibody and preparative gel electrophoresis, a homogenous preparation of the Rh(D) protein was obtained from two different erythrocyte samples (Blo and D--/D--), which have an increased density of Rh(D) antigen. Both preparations exhibited the same N-terminal sequence (S)-(S)-K-Y-P-R-S-V-R-R-(L)-L-P-L-X-A, indicating that different Rh(D)-positive red cells are carrying a similar Rh(D) protein. Comparative immunoprecipitation studies with the human monoclonal anti-Rh(D), (c), and (E) antibodies have also shown that Rh components from intact and papain-treated erythrocytes have similar apparent mol wt of 30 to 32 Kd and are buried in the lipid bilayer and are not readily available to the proteolytic enzyme. Further investigations by indirect affinity chromatography and one-dimensional peptide mapping of the Rh(D), (c), and (E) molecules immunopurified from a single red cell sample demonstrate that a common Rh haplotype encodes three distinct polypeptide chains carrying the Rh(D), (c), and (E) epitopes, respectively.

The immunomodulating glycoprotein extract from Klebsiella pneumoniae RU 41740 exerts a suppressive effect on human monocyte death by apoptosis.

Programmed cell death or apoptosis is a physiological cell suicide process that can be suppressed by survival factors. Monocytes undergo rapid apoptosis in culture, unless signalled by cytokines or the bacterial lipopolysaccharide LPS. We have investigated the effect on monocyte apoptosis of the immunostimulating agent RU 41740 (Biostim), a glycoprotein extract from the Klebsiella pneumoniae K2O1 strain that is used for the prevention of recurrent infections. RU 41740, as LPS, strongly enhanced monocyte survival in vitro, an effect related to apoptosis suppression. RU 41740 at concentration ranging from 1 ng/ml to 10 microg/ml prevented apoptosis induced both by survival factor deprival and by gamma-irradiation. Our observation suggests that enhancement of monocyte survival may represent a component of the reported immunostimulating effect of this compound.

Comparative analysis by two-dimensional iodopeptide mapping of the RhD protein and LW glycoprotein.

The RhD polypeptide and LW glycoprotein were separately immunopurified with monoclonal antibodies and compared by two-dimensional (2-D) iodopeptide mapping after digestion with alpha-chymotrypsin. These proteins have distinct 2-D maps, as seen after 125I-labeling tyrosine residues (chloramine-T procedure), and even more strikingly after labeling primary amine residues (Bolton-Hunter procedure). Of the more than 20 iodopeptides visualized, only five migrated identically when preparations of RhD and LW were directly compared, suggesting that RhD and LW are different proteins that may share some common protein domains. N-glycanase treatment of the iodopeptides did not modify the 2-D map of the RhD protein but greatly affected the LW map, further indicating that LW, but not RhD, carries N-linked carbohydrate chains. After deglycosylation the LW map was different from the RhD map, confirming that the RhD and LW polypeptides are different proteins. These findings demonstrate that LW is neither a glycosylated form of Rh protein nor is Rh a precursor of LW.

S-s-U-phenotype in a Caucasian family.

The S-s-U-blood group phenotype, commonly detected in Black populations, was found in a Caucasian family in which 4 homozygous U-negative members exhibit this phenotype. The erythrocyte blood group antigens and membrane glycoproteins from these donors have been serologically and electrophoretically characterized and compared to the U-negative red cells from Black people. Caucasian and Black S-s-U-red blood cells behaved identically since both lack Ss and U antigens and the minor red-cell membrane glycophorin B.

Characterization of murine monoclonal antibodies directed against the Kell blood group glycoprotein.

Murine monoclonal antibodies (MoAbs) were produced against the blood group KEL1 glycoprotein (93 kD component) immunopurified from human erythrocytes. One monoclonal antibody, 5A11 (IgGa, kappa), detects by immunoblotting a 93 and 184 kD component from KEL: 1,-2 or KEL: -1,2 red cell membrane preparations, separated by SDS polyacrylamide gel electrophoresis (PAGE) under non-reducing conditions. The 184 kD component was not detected under reducing conditions, suggesting that it represented a dimer of the 93 kD KEL glycoprotein. Neither the 93 nor the 184 kD could be detected from K0 or McLeod erythrocyte membrane preparations, indicating that the monoclonal antibody reacts with the KEL glycoprotein previously identified as a 93 kD species. Since this antibody does not agglutinate native or protease-treated erythrocytes, it is likely that it reacts with the cytoplasmic domain of the KEL glycoprotein. This was also substantiated by showing that 5A11 could immunoprecipitate the 93 kD component from intact membranes and inside-out vesicles but not from right-side-out vesicles. Immunostaining of membrane proteins prepared from human blood cells (platelets, lymphocytes, monocytes and granulocytes) and non-human erythrocytes revealed that the 93 kD molecule was only present on human red cells. Several other murine monoclonal antibodies obtained from the same fusion experiment gave identical results, but competition analyses on immunoblots indicated that these antibodies reacted with distinct epitopes on the KEL glycoprotein.

Naftazone accelerates human saphenous vein endothelial cell proliferation in vitro.

Restoration of a haemocompatible surface after endothelial damage induced by treatments such as embolectomy, angioplasty, endarterectomy or irradiation or following vascular graft implantation is an important factor for the ultimate success of these interventions. The development of substances which enhance endothelial cell growth and accelerate their proliferation is therefore of great interest in such situations. In the present work naftazone was shown to accelerate human saphenous vein endothelial cell proliferation in vitro at concentrations which did not alter the hemostatic balance, resulting in a cell density at confluence 20% higher than in controls. This compound was able to partially substitute for serum requirements and further displayed additive effects in the presence of fibroblast growth factors. Thus naftazone, an original synthetic molecule distinct from growth factor peptides, is a promising candidate drug for the amelioration of vascular repair.

RU 41 740 (Biostim) and IL-4, or IL-13, have opposite effects on CD14, CD23, HLA-DR and HLA-DQ on monocytes.

RU 41 740 (Biostim) is a glycoprotein extract obtained from Klebsiella pneumoniae. Its immunostimulating properties on monocytes have been established in vivo and in vitro. To confirm its spectrum of action at molecular level we studied its role on the modulation of four molecules involved in antigen presentation (HLA-DR, HLA-DQ), uptake of endotoxin (CD14) and activation (CD23). These four molecules are known to be modulated by interleukins IL-4 and IL-13. We found that HLA-DR, HLA-DQ, CD14 and CD23 were differentially regulated by biostim and IL-4 or IL-13. Surprisingly, Biostim inhibited the IL-4 or IL-13-induced expression of CD23, HLA-DQ and HLA-DR, while it did not have any action on these molecules by itself. We therefore hypothesize that Biostim, through the action on its receptor, could interact with the IL-4 receptor and IL-13 receptor and/or inhibit the IL-4 and IL-13 receptor transducing signal.

Vascular endothelial growth factor is modulated in vascular muscle cells by estradiol, tamoxifen, and hypoxia.

Vascular endothelial growth factor (VEGF) promotes neovascularization, microvascular permeability, and endothelial proliferation. We described previously VEGF mRNA and protein induction by estradiol (E2) in human endometrial fibroblasts. We report here E2 induction of VEGF expression in human venous muscle cells [smooth muscle cells (SMC) from human saphenous veins; HSVSMC] expressing both ER-alpha and ER-beta estrogen receptors. E2 at 10(-9) to 10(-8) M increases VEGF mRNA in HSVSMC in a time-dependent manner (3-fold at 24 h), as analyzed by semiquantitative RT-PCR. This level of induction is comparable with E2 endometrial induction of VEGF mRNA. Tamoxifen and hypoxia also increase HSVSMC VEGF mRNA expression over control values. Immunocytochemistry of saphenous veins and isolated SMC confirms translation of VEGF mRNA into protein. Immunoblot analysis of HSVSMC-conditioned medium detects three bands of 18, 23, and 28 kDa, corresponding to VEGF isoforms of 121, 165, and 189 amino acids. Radioreceptor assay of the conditioned medium produced by E2-stimulated HSVSMC reveals an increased VEGF secretion. Our data indicate that VEGF is E2, tamoxifen, and hypoxia inducible in cultured HSVSMC and E2 inducible in aortic SMC, suggesting E2 modulation of VEGF effects in angiogenesis, vascular permeability, and integrity.

FT-IR spectroscopy as an emerging method for rapid characterization of microorganisms.

Statistical methods such as principal component analysis and cluster analysis were used to analyze ATR-FT-IR spectra obtained from bacterial whole cells. Both methods gave satisfactory results and are conclusive in showing that they can discriminate and classify bacterial strains of clinical origin exhibiting different resistance mechanisms. This approach places FT-IR spectroscopy at the forefront of those new potential techniques that could be used in the rapid screening of microorganisms.

Protective effects of RU 41740, a bacterial immunomodulator, against experimental infections: induction of cytokine and immunoglobulin release in mice after oral administration.

RU 41740 (Biostim) is an immunomodulator extracted from Klebsiella pneumoniae (strain O1:K2). In humans, it is able to reduce the number and duration of infectious exacerbations of chronic bronchitis. Using a mouse model of experimental infection, we found that oral RU 41740 administration strongly protected against gram-negative infections by preventing lethal septicemia, and, to a lesser extent, protected against the gram-positive intracellular pathogen L. monocytogenes. Oral administration of RU 41740 leads to the mobilization of newly dividing T and B cells in the thoracic duct lymph, reflecting the ability of the drug to induce an immune response in gut-associated lymphoid tissue. In cells isolated from mesenteric lymph nodes and spleen, RU 41740 leads to preferential release of the proinflammatory cytokines interleukin (IL)-12 and/or interferon (IFN)-gamma, as well as IL-10, a cytokine involved in inhibiting the synthesis of these latter cytokines. RU 41740 also increases the serum total immunoglobulin (Ig)M concentration and elicits IgM and IgG antibodies against the drug. Infection of mice with Klebsiella pneumoniae has similar functional consequences. Pretreatment of infected mice with RU 41740 leads to a fall in the high levels of proinflammatory cytokines (which could be detrimental), and to an increase in IgG antibodies (which are protective).

Effect of naftazone on in vivo platelet function in the rat.

The aim of this study was to investigate the in vivo effects of 50 mg/kg (i.p.) naftazone or ticlopidine on platelet functions in the rat. An automated isotope monitoring system (Aims plus) was used to determine the height of platelet aggregation and disaggregation (measured by the area under the curve, AUC) of 111indium-labelled platelets activated by ADP (10 microg/kg i.v.) or collagen (50 microg/kg i.v.). Fibrinogen-binding experiments were carried out with activated platelets in whole blood and measured by flow cytometry. Naftazone reduced the height of platelet aggregation induced by ADP compared with controls (P = 0.024). Ticlopidine-treated rats gave similar results (P = 0.008). Platelet disaggregation, following the aggregation induced by collagen, was significantly increased in naftazone-treated rats compared with controls (P = 0.003). Similar results were observed with ticlopidine-treated rats (P = 0.002). Fibrinogen binding to 2.5 or 5 microM ADP-stimulated platelets, from naftazone-treated rats, were significantly reduced compared with controls (P = 0.05 and 0.04 respectively). These results show that naftazone has similar inhibitory effects on rat platelet functions as ticloplidine. In conclusion, naftazone could be a useful agent to modulate platelet function in patients with cardiovascular disease.

Surface orientation and antigen properties of Rh and LW polypeptides of the human erythrocyte membrane.

Time course digestion of intact human erythrocytes and right side-out vesicles with carboxypeptidase Y altered the Rh polypeptides and removed the 125I label that is normally incorporated by cell-surface radioiodination, but did not affect the RhD, Rhc, or RhE antigens. Under the same conditions, however, the LW antigens were rapidly destroyed. Digestion of inside-out and right side-out vesicles with aminopeptidase M was without any detectable effect on the Rh and LW antigens or polypeptides, although glycophorin A was degraded from right side-out but not from inside-out vesicles. These findings demonstrate that the C-terminal domain of the Rh and LW polypeptides is exposed at the external surface of human erythrocytes and indicate, in addition, that the LW antigens and tyrosine residue(s) of the LW and Rh proteins, respectively, are located close to the C termini of these polypeptides. Further studies using monoclonal and polyclonal antibodies showed that LW antigen expression is inhibited by treatment of red cells with EDTA and is selectively restored by Mg2+, but not by Mn2+ or Ca2+, whereas the Rh antigens were not affected under these conditions. In addition, O- and N-glycanase digestion of the LW glycoprotein removed its sugar chains, but did not alter significantly the epitopes recognized by the monoclonal anti-LW antibody.