Biomedical subjects

M Levi

Publications and source records attributed to M Levi.

At least 235 records · Page 13Linked to original sources

PubMed1995

Inhibition of the release of soluble tumor necrosis factor receptors in experimental endotoxemia by an anti-tumor necrosis factor-alpha antibody.

The role of tumor necrosis factor-alpha in the shedding of soluble tumor necrosis factor receptors in endotoxemia was investigated. The appearance of the soluble tumor necrosis factor receptors was assessed in four healthy volunteers following an intravenous injection of tumor necrosis factor-alpha and in eight chimpanzees after intravenous administration of endotoxin in the absence or presence of concurrent treatment with a neutralizing anti-tumor necrosis factor-alpha monoclonal antibody. Injection of tumor necrosis factor-alpha in humans elicited a significant, instantaneous (after 15 min) increase in the plasma concentrations of both types of soluble tumor necrosis factor receptors. In chimpanzees, treatment with the anti-tumor necrosis factor-alpha antibody completely neutralized endotoxin-induced tumor necrosis factor-alpha activity. The release of soluble tumor necrosis factor receptors was strongly (80-90%) inhibited in the presence of the neutralizing antibody. Our results indicate that tumor necrosis factor-alpha is a prime mediator of endotoxin-induced release of its own soluble receptors.

Adult↗

PubMed1995

Molecular characterization of a human anti-HIV 1 monoclonal antibody revealed a CD26-related motif in CDR2.

Genes encoding the immunoglobulin variable regions of a human anti-HIV-1 IgG1 kappa monoclonal antibody were rescued from a hybridoma, derived from a sero-negative donor, using PCR cloning and expression in Escherichia coli. The ELISA binding results obtained from the expressed Fab fragment confirmed the anti-V3 loop specificity for HIV-1 (LAI) of the original antibody. In addition, an amino acid sequence derived from the second complementarity determining region (CDRH2) of the heavy chain was found to be very similar to the catalytic motif of CD26, a T-cell activation antigen. Furthermore, synthetic peptides containing both the catalytic domain of CD26 and CDRH2 of the antibody showed specific binding to an HIV peptide representing the V3 region in a dose-dependent manner. This suggests an involvement of CD26 as a possible coreceptor for HIV-1.

Amino Acid Sequence↗

PubMed1995

Plasminogen activator and plasminogen activator inhibitor I release during experimental endotoxaemia in chimpanzees: effect of interventions in the cytokine and coagulation cascades.

1. Disseminated intravascular coagulation frequently accompanies Gram-negative sepsis and may contribute to widespread deposition of microthrombi. Besides the endotoxin-induced activation of coagulation, an important role for the fibrinolytic system has been postulated. The precise mechanisms underlying these fibrinolytic changes during endotoxaemia are not known but have been suggested to be mediated directly by cytokines or secondary to thrombin generation. 2. In the present study we have delineated in detail the fibrinolytic response to a bolus injection of endotoxin in non-human primates and analysed the contribution of cytokines and thrombin generation to the endotoxin-induced release of tissue-type plasminogen activator and plasminogen activator inhibitor 1. Chimpanzees received a bolus injection of endotoxin alone or in combination with blocking monoclonal antibodies directed against tumour necrosis factor or interleukin 6 or in combination with pentoxifylline. Furthermore, to assess the effect of coagulation activation on the activation of fibrinolysis, another group of chimpanzees received endotoxin in combination with either anti-tissue factor antibodies or recombinant hirudin. 3. Infusion of endotoxin induced a rapid increase in plasminogen activator activity and tissue-type plasminogen activator antigen levels and subsequent plasmin generation, reaching peak levels 2h after endotoxin administration. Plasminogen activator inhibitor 1 levels remained constant for the first 2 h, after which time a steep increase was observed. Plasminogen activator activity and plasmin generation decreased simultaneously with the rise in plasminogen activator inhibitor 1 levels. Fibrinolytic activity remained suppressed during the remainder of the study owing to sustained increased levels of plasminogen activator inhibitor 1. The administration of pentoxifylline strongly attenuated the release of tissue-type plasminogen activator and plasminogen activator inhibitor 1, whereas the antitumour necrosis factor antibodies blocked the fibrinolytic response entirely. In contrast, interleukin 6-neutralizing antibodies did not affect the fibrinolytic response. Although endotoxin-induced generation of thrombin was completely prevented by the administration of tissue factor-neutralizing antibodies or by hirudin, no effect on the fibrinolytic response was observed.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMed1995

Effect of postponed treatment with an anti-tumour necrosis factor (TNF) F(ab')2 fragment on endotoxin-induced cytokine and neutrophil responses in chimpanzees.

TNF is considered to be an intermediate factor in endotoxin-induced release of other cytokines and endotoxin-induced neutrophil degranulation. Little is known about the effect of postponed treatment with anti-TNF in primate endotoxin models. To assess the effect of delayed treatment with anti-TNF in endotoxaemia, six healthy adult chimpanzees were intravenously injected with Escherichia coli endotoxin (4 ng/kg). In three of these animals the administration of endotoxin was followed after 30 min by a bolus i.v. injection of the anti-TNF F(ab')2 fragment MAK 195F (0.1 mg/kg). Post-treatment with MAK 195F completely prevented the appearance of TNF activity in serum elicited by endotoxin, and markedly reduced the rises in the serum concentrations of IL-6 and IL-8. In addition, the endotoxin-induced increases in the type I and type II soluble TNF receptors were also profoundly inhibited by MAK 195F, suggesting that TNF is involved in the release of its own soluble receptors in endotoxaemia. Neutrophilic leucocytosis was not affected by MAK 195F. In contrast, MAK 195F did significantly abrogate neutrophil degranulation, as measured by the plasma concentrations of lactoferrin. These results indicate that treatment with anti-TNF 30 min after the administration of endotoxin is still effective in attenuating the induction of the cytokine network and of neutrophil degranulation.

Animals↗

PubMed1995

Epidermal growth factor inhibits Na-Pi cotransport and mRNA in OK cells.

The present study examined the effect of epidermal growth factor (EGF) on Na-Pi cotransport in a tubular epithelial cell line derived from the opossum kidney (OKP cells). EGF caused a time- and dose-dependent decrease in Na-Pi cotransport. The inhibition of Na-Pi cotransport by 10(-8) M EGF was first demonstrable after 18 h with maximal effect seen at 24 h. EGF inhibited Na-Pi cotransport by decreasing the maximal velocity (10.8 +/- 0.9 in control vs. 4.9 +/- 0.8 nmol 32Pi.4 min-1.mg protein-1 in EGF, P < 0.001). Northern blot analysis indicated that EGF caused a significant decrease in NaPi-4 mRNA abundance. The abundance of NaPi-4 mRNA relative to beta-actin and/or glyceraldehyde-3-phosphate dehydrogenase mRNA was decreased by twofold in OK cells treated with EGF for 4 h and threefold in OKP cells treated with EGF for 24 h. Thus the decrease in NaPi-4 mRNA abundance preceded the decrease in Na-Pi cotransport activity. Inhibition of transcription with actinomycin D and protein synthesis with cycloheximide prevented the inhibition of Na-Pi cotransport. Furthermore, inhibition of phospholipase C activity with U-73,122 also significantly blocked the inhibitory effect of EGF on Na-Pi cotransport. The results indicate that EGF-induced decrease in OKP Na-Pi cotransport is mediated through a decrease in NaPi-4 mRNA and activation of the phospholipase C signaling pathway.

Animals↗

PubMed1995

Parathyroid hormone action on phosphate transporter mRNA and protein in rat renal proximal tubules.

The inhibitory action of parathyroid hormone (PTH) on Pi reabsorption in the renal proximal tubule is accompanied by a specific decrease in Na-Pi cotransport at the apical brush-border membrane (BBM). It is not known whether this decrease represents decreased activity of Na-Pi cotransporters already present in the BBM or whether the number of cotransporters is decreased. The present study of the molecular mechanism of PTH action made use of a specific cDNA probe and antiserum to a rat renal Na-Pi cotransporter (NaPi-2). Three groups of rats were used: intact controls, chronically parathyroidectomized (PTX), and PTX rats treated acutely (2 h) with bovine PTH-(1--34). Na-Pi cotransport by isolated renal BBM vesicles was increased to 1,315 +/- 44 in PTX rats, compared with 721 +/- 94 pmol.mg-1.10 s-1 in controls (P < 0.002), and was returned to control levels by PTH. Western blots of these BBM showed that PTX caused a 2.8-fold increase in NaPi-2 protein content, which was reduced to control levels by PTH. Immunohistochemistry of perfusion-fixed kidneys showed NaPi-2-specific immunofluorescence exclusively in apical BBM of proximal tubules. Expression of NaPi-2 protein at these sites was increased in PTX rats and decreased after PTH treatment. Northern analysis of total RNA showed that the abundance of NaPi-2-specific mRNA was not changed by PTX but there was a small decrease in response to PTH. The data indicate that PTH regulation of renal Na-Pi cotransport is determined by changes in expression of NaPi-2 protein in the renal BBM.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMed1995

Characterization of Na(+)-phosphate cotransporters in renal cortical endosomes.

To determine the role of membrane recycling in proximal tubular P(i) transport, we studied the transport functions of simultaneously prepared rat renal cortical endosomal vesicles (EV) and brush-border membrane vesicles (BBMV). Initial P(i) uptake was Na+ gradient-dependent in both vesicles. Kinetic studies showed a lower apparent maximal rate (Vmax) for P(i) uptake in EV compared with BBMV (446 +/- 69 vs. 1493 +/- 117 pmol.mg protein-1 x 15 s-1, respectively; n = 4, P < 0.05), with no difference in the apparent Michaelis constant (Km). Endosomal Na(+)-P(i) cotransport was inhibited by phosphate analogues, phosphonoformic acid and arsenate, but not by the anion-exchange inhibitor, 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid (DIDS). Na(+)-P(i) cotransport was electroneutral in both vesicles, with a stoichiometry of 2 for BBMV and 1 for EV. The nonpermeant sulfhydryl reagent, 5,5'-dithiobis(2-nitrobenzoic acid) (DTNB), produced a marked inhibition of Na(+)-P(i) cotransport in EV but not in BBMV, suggesting accessible sulfhydryl groups, an "inside-out" orientation in EV, and "right-side-out" orientation in BBMV. The EV and the BBMV differed significantly in their phospholipid composition and lipid fluidity. The Na(+)-P(i) cotransporter protein (NaPi-2) abundance, determined by Western blots, was five times lower in EV than in BBMV (0.25 +/- 0.05 vs. 1.36 +/- 0.20 arbitrary units). Renal cortical endosomes contain Na(+)-P(i) cotransporters, albeit at a lower density, suggesting involvement of membrane recycling in the regulation of proximal tubular P(i) transport.

Animals↗

PubMed1995

Dexamethasone modulates rat renal brush border membrane phosphate transporter mRNA and protein abundance and glycosphingolipid composition.

Glucocorticoids are important regulators of renal phosphate transport. This study investigates the role of alterations in renal brush border membrane (BBM) sodium gradient-dependent phosphate transport (Na-Pi cotransporter) mRNA and protein abundance in the dexamethasone induced inhibition of Na-Pi cotransport in the rat. Dexamethasone administration for 4 d caused a 1.5-fold increase in the Vmax of Na-Pi cotransport (1785 +/- 119 vs. 2759 +/- 375 pmol/5 s per mg BBM protein in control, P < 0.01), which was paralleled by a 2.5-fold decrease in the abundance of Na-Pi mRNA and Na-Pi protein. There was also a 1.7-fold increase in BBM glucosylceramide content (528 +/- 63 vs. 312 +/- 41 ng/mg BBM protein in control, P < 0.02). To determine whether the alteration in glucosylceramide content per se played a functional role in the decrease in Na-Pi cotransport, control rats were treated with the glucosylceramide synthase inhibitor, D-threo-1-phenyl-2-decanoyl-amino-3-morpholino-1-propanol (PDMP). The resultant 1.5-fold decrease in BBM glucosylceramide content (199 +/- 19 vs. 312 +/- 41 ng/mg BBM protein in control, P < 0.02) was associated with a 1.4-fold increase in Na-Pi cotransport activity (1422 +/- 73 vs. 1048 +/- 85 pmol/5 s per mg BBM protein in control, P < 0.01), and a 1.5-fold increase in BBM Na-Pi protein abundance. Thus, dexamethasone-induced inhibition of Na-Pi cotransport is associated with a decrease in BBM Na-Pi cotransporter abundance, and an increase in glucosylceramide. Since primary alteration in BBM glucosylceramide content per se directly and selectively modulates BBM Na-Pi cotransport activity and Na-Pi protein abundance, we propose that the increase in BBM glucosylceramide content plays an important role in mediating the inhibitory effect of dexamethasone on Na-Pi cotransport activity.

Animals↗

PubMed1995

Complete inhibition of endotoxin-induced coagulation activation in chimpanzees with a monoclonal Fab fragment against factor VII/VIIa.

Gram-negative sepsis is oftentimes complicated by activation of coagulation with disseminated intravascular coagulation and microthrombosis. This may contribute to the associated morbidity, multiple organ failure and death. Recent studies have established that the tissue factor-dependent pathway of blood coagulation has a significant participatory role in the initial endotoxin-induced activation of coagulation. Tissue factor (TF), expressed on the surface of activated monocytes and endothelial cells forms cell surface complexes with free circulating factors VII and VIIa. The latter complex proteolytically activates factors X and IX. Recent in vivo experiments have shown that a rapidly neutralizing TF monoclonal antibody prevents and arrests the endotoxin-induced activation of coagulation and similar studies have shown to reduce mortality in baboons. In this study we describe the preparation of a factor VII/VIIa neutralizing monoclonal Fab fragment and characterize its effect on in vivo activation of coagulation during experimental endotoxemia in chimpanzees. Four chimpanzees received a bolus intravenous injection of 4 ng/kg endotoxin in combination with Fab fragments of a factor VII/VIIa neutralizing murine monoclonal antibody (12D10) at a dose of either 50 micrograms/kg (n = 2) or 100 micrograms/kg (n = 2). Four control animals received a bolus injection of endotoxin alone. Administration of the 12D10 Fab fragments, immediately preceding the endotoxin bolus injection, effectively blocked the endotoxin-induced activation of coagulation. Plasma levels of products of in vivo activation, namely F1 + 2, TAT complexes and FpA remained at baseline values. The administration of 12D10 resulted in a rapid decline in factor VII/VIIa antigen levels which remained below 5 ng/ml for 180-240 min, followed by a rapid return to baseline levels.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMed1995

Balanced activation of coagulation and fibrinolysis after a 2-h triathlon.

To examine whether exercise-induced thrombin formation is accompanied by increased in vivo plasmin formation, we measured molecular markers and neoantigens of the hemostatic system in 10 male subjects (mean 29 yr. range 19-38) before, immediately after, and 2, 8, and 21 h after a triathlon lasting 128-163 min. Thrombin-antithrombin (TAT) complexes, fibrinopeptide A (FPA), and tissue plasminogen activator (t-PA) antigen were maximally increased immediately after exercise and decreased thereafter rapidly. Prothrombin fragment 1 + 2 (PTF1 + 2), fibrin degradation products (FbDP) and plasmin-antiplasmin (PAP) complexes rose to a similar extent 0 and 2 h after exercise decreased thereafter. The maximal levels of PTF1 + 2, TAT, FPA, and FbDP were 1.5-, 2.1-, 1.8-, and 1.9-fold above baseline, respectively. This investigation shows that strenuous prolonged exercise leads to a moderate activation of blood coagulation resulting in thrombin and fibrin formation which is accompanied by a greatly enhanced plasmin generation. It is concluded that the hemostatic of healthy individuals is well kept in balance when stimulated by prolonged strenuous exercise.

Adult↗

PubMed1994

Regulation of interleukin 10 release by tumor necrosis factor in humans and chimpanzees.

Interleukin 10 (IL-10) has been shown to inhibit endotoxin-induced tumor necrosis factor (TNF) production. To assess the role of TNF in the induction of IL-10 in endotoxemia, four healthy men were studied after a bolus intravenous injection of recombinant human TNF (50 micrograms/m2). In addition, 13 healthy chimpanzees were investigated after a bolus intravenous injection of Escherichia coli endotoxin (4 ng/kg), 6 animals received endotoxin only, 4 animals received a simultaneous intravenous injection of a monoclonal anti-TNF antibody, whereas 3 chimpanzees were treated with an anti-TNF F(ab')2 fragment 30 min after the administration of endotoxin. TNF induced a modest rise in IL-10 concentrations peaking after 45 min (47 +/- 32 pg/ml; p < 0.05). IL-10 peaked 2 h after injection of endotoxin (202 +/- 61 pg/ml; p < 0.005). In both anti-TNF-treated groups, the early endotoxin-induced TNF activity was completely neutralized. Simultaneous anti-TNF treatment attenuated endotoxin-induced IL-10 release (73 +/- 13 pg/ml; p < 0.01 versus endotoxin alone), whereas postponed anti-TNF treatment did not significantly affect this response (p = 0.21). These results indicate that TNF, in part, mediates the induction of IL-10 in endotoxemia, resulting in an autoregulatory feedback loop.

Adult↗

PubMed1994

Enhanced bioavailability of phosphonoformic acid by dietary phosphorus restriction.

Phosphonoformic acid (PFA, foscarnet) is a potent inhibitor of Na(+)-P(i) cotransport in intestinal and renal brush border membranes (BBM). We have studied the effect of dietary phosphorus restriction on intestinal PFA absorption and bioavailability. Rats were placed on low (0.04% P(i), LPD) or normal (0.95% P(i), NPD) phosphorus diets for 5 days, followed by administration of an oral bolus of [14C]PFA (100 mg/kg). Of the oral PFA dose, 60 +/- 4% was absorbed in LPD rats, compared with 43 +/- 3% in NPD rats (P < 0.05, N = 5). This was associated with higher plasma PFA concentrations in LPD compared with NPD rats (44.2 +/- 2.0 and 17.9 +/- 4.3 micrograms/mL, respectively). [14C]PFA uptake, determined in intestinal BBM vesicles (BBMV), was Na+ gradient (Na+out > Na+in) dependent. Dietary phosphorus restriction resulted in a 39.8% increase in the initial (1 min) Na(+)-dependent [14C]PFA uptake by intestinal BBMV. We conclude that PFA absorption is enhanced by dietary phosphorus restriction.

Animals↗

PubMed1994

Elimination of interleukin 6 attenuates coagulation activation in experimental endotoxemia in chimpanzees.

The role of interleukin 6 (IL-6) in the toxic sequelae of sepsis is controversial. To assess the part of IL-6 in inflammatory responses to endotoxin, we investigated eight chimpanzees after either a bolus intravenous injection of Escherichia coli endotoxin (n = 4; 4 ng/kg) or after the same dose of endotoxin with a simultaneous bolus intravenous injection of an anti-IL-6 mAb (30 mg; n = 4). Anti-IL-6 did not affect the induction of the cytokine network (tumor necrosis factor [TNF], soluble TNF receptors types I and II, and IL-8) by endotoxin, nor did it influence the occurrence of a neutrophilic leukocytosis and neutrophil degranulation, as monitored by the measurement of elastase-alpha 1-antitrypsin complexes. In contrast, anti-IL-6 markedly attenuated endotoxin-induced activation of coagulation, monitored with the plasma levels of the prothrombin fragment F1+2 and thrombin-antithrombin III complexes, whereas activation of fibrinolysis, determined with the plasma concentrations of plasmin-alpha 2-antiplasmin complexes, remained unaltered. We conclude that IL-6 does not have a feedback effect on the release of other cytokines after injection of endotoxin, and that it is not involved in endotoxin-induced neutrophilia or neutrophil degranulation. IL-6 is, however, an important intermediate factor in activation of coagulation in low grade endotoxemia in chimpanzees.

Animals↗

PubMed1994

Platelet-activating factor antagonist TCV-309 attenuates the induction of the cytokine network in experimental endotoxemia in chimpanzees.

Platelet-activating factor (PAF) has been postulated to play a role in the pathogenesis of sepsis. Additionally, in vitro studies have revealed tight interactions between PAF and the cytokine network, and PAF is considered to be an important stimulator of neutrophil functions. To assess the intermediate role of PAF in the induction of cytokines and neutrophil degranulation in endotoxemia in vivo, 12 healthy adult chimpanzees were i.v. injected with a bolus dose of Escherichia coli endotoxin (4 ng/kg); four animals received endotoxin alone, whereas the other chimpanzees were infused with the specific and potent PAF antagonist TCV-309 (bolus of 100 micrograms/kg, followed by either 100 micrograms/kg/h (n = 4) or 500 micrograms/kg/h (n = 4) for 5 h). At both doses TCV-309 significantly inhibited the endotoxin-induced rise in cytokine levels. Peak TNF concentrations after injection of endotoxin alone were 366 +/- 96 pg/ml, vs 105 +/- 47 and 115 +/- 56 pg/ml after administration of endotoxin together with the lower or higher dose of TCV-309, respectively (p < 0.05). TCV-309 also reduced the appearance of soluble TNFRs. Maximal levels of the type I soluble TNFR were diminished from 2.53 +/- 0.27 ng/ml (endotoxin alone) to 1.69 +/- 0.36 ng/ml (high dose TCV-309; p < 0.05); peak values of the type II soluble TNFR were diminished from 8.62 +/- 1.19 ng/ml to 5.76 +/- 0.92 ng/ml (p < 0.05). Furthermore, TCV-309 attenuated the endotoxin-induced release of IL-6 (160 +/- 82 pg/ml after endotoxin alone, vs 63 +/- 30 pg/ml in the low dose TCV-309 group (p < 0.05) and 65 +/- 29 pg/ml in the high dose group (p = 0.07) as well as that of IL-8 (279 +/- 168, vs 71 +/- 15 and 46 +/- 17 pg/ml, respectively; both p < 0.05). TCV-309 tended to reduce the endotoxin-provoked rise in serum IL-1R antagonist levels. In contrast, TCV-309 did not affect the neutrophilic leukocytosis elicited by endotoxin, nor did it inhibit endotoxin-induced neutrophil degranulation, as monitored by the plasma levels of elastase-alpha 1-antitrypsin complexes. We conclude that PAF plays a role, either directly or indirectly, in the stimulation of the cytokine network and in the shedding of soluble TNFR in endotoxemia. PAF does not seem to be an important intermediate factor in endotoxin-induced neutrophilia or neutrophil degranulation.

Animals↗

PubMed1994

Differential effects of anti-tumor necrosis factor monoclonal antibodies on systemic inflammatory responses in experimental endotoxemia in chimpanzees.

Tumor necrosis factor (TNF) is considered to be a pivotal mediator of endotoxin-induced lethality. To assess the intermediate role of TNF in specific systemic inflammatory responses known to contribute to tissue injury in endotoxemia, eight healthy adult chimpanzees were intravenously injected with Escherichia coli endotoxin (4 ng/kg). In four of these animals the administration of endotoxin was followed immediately by a bolus intravenous injection of an anti-TNF monoclonal antibody (15 mg/kg). Treatment with anti-TNF completely prevented the endotoxin-induced increase in serum TNF activity, and profoundly reduced the appearance of interleukin-6 and -8 (both P < .05). Neutrophilia and lymphopenia were not affected by anti-TNF, whereas neutrophil degranulation, as measured by the plasma concentrations of elastase-alpha 1-antitrypsin complexes, was only slightly reduced (peak levels after endotoxin alone 31.0 +/- 3.4 ng/mL, versus 25.5 +/- 3.4 ng/mL after endotoxin with anti-TNF; P < .05). Anti-TNF did not influence endotoxin-induced activation of the coagulation system, as reflected by unchanged increases in the plasma concentrations of the prothrombin fragment F1 + 2 and thrombin-antithrombin III complexes. In contrast, anti-TNF strongly attenuated the activation of the fibrinolytic system, ie, peak plasma levels of plasmin-alpha 2-antiplasmin were 33.8 +/- 11.1 nmol/L after endotoxin alone and 17.0 +/- 2.9 nmol/L after endotoxin with anti-TNF (P < .05). These results suggest that TNF is not the common mediator of systemic inflammatory changes in low-grade endotoxemia. Moreover, the finding that in this mild model anti-TNF specifically inhibited fibrinolysis suggests that treatment with anti-TNF potentially may enhance the tendency towards microvascular thrombosis in sepsis.

Animals↗

PubMed1994

Low-Pi diet increases the abundance of an apical protein in rat proximal-tubular S3 segments.

Dietary phosphate (Pi) restriction is associated with an adaptive increase in proximal-tubular apical brush-border membrane (BBM) sodium-dependent Pi transport (Na-Pi cotransport). Adaptation to Pi restriction is dependent on de novo protein synthesis; however, it is not known whether the proteins involved represent newly synthesized Na-Pi cotransporters or some other (regulatory) proteins. Recently the cDNA for a Na-Pi cotransport system of rabbit kidney cortex (system NaPi-1) has been identified by expression cloning. The purpose of this study was to determine if the adaptive increase in Na-Pi cotransport in response to dietary Pi restriction in the rat is associated with an increase in the abundance of a NaPi-1-related protein. To answer this question we took advantage of the cross-reactivity of polyclonal antibodies raised against a C-terminal peptide of the NaPi-1 protein with a protein of BBM isolated from rat kidney cortex. On Western blots, a positive reaction with a protein with an apparent molecular mass of 100 kDa was observed in BBM isolated from juxtamedullary cortex and, to a lesser extent, in BBM isolated from superficial cortex. In immunohistochemical studies anti-(NaPi-1)-antiserum-mediated immunofluorescence was observed predominantly in S3 segments where the immunoreaction was restricted to the brush borders. Compared to control BBM, in BBM isolated from the juxtamedullary cortex of rats fed a low-Pi diet, there was a twofold increase in the abundance of the 100-kDa protein. In the same membrane vesicles Na-Pi cotransport was increased threefold. The results of this study demonstrate specific expression of a 100-kDa apical protein in S3 cells of rat proximal tubules. Increased abundance of the 100-kDa protein due to chronic Pi restriction suggests an involvement of this protein in the (chronic) adaptive response of S3 cells to a low-Pi diet.

Adaptation, Physiological↗

PubMed1994

Impaired fibrinolysis in the hemolytic-uremic syndrome of childhood.

Thrombotic obstruction of glomerular capillaries causes acute renal failure in patients with hemolytic-uremic syndrome (HUS). Recanalization of occluded vessels normally occurs by activation of the endogenous fibrinolytic system, mediated by plasminogen activators, which are stored and synthesized in the endothelial cells. However, endothelial injury is considered the primary event in the pathogenesis of HUS, and this may result in impaired fibrinolysis. In five children with HUS we performed a prospective study of plasminogen activator activity and two plasminogen activator antigens: tissue-type plasminogen activator and urokinase-type plasminogen activator before and after intravenous desmopressin. Plasminogen activator inhibitor type-1 antigen was also studied. In the acute stage of HUS plasminogen activating activity was low, in spite of elevated levels of total plasminogen activator antigens. This decrease of plasminogen activating activity was due to high levels of the plasminogen activator inhibitor. Improvement of fibrinolysis paralleled recovery from HUS. We conclude that decreased fibrinolysis is an important pathophysiologic feature of HUS.

Adolescent↗

PubMed1994

Linear epitopes of HIV-1, presented as hybrids with Escherichia coli beta-galactosidase or synthetic peptides.

HIV-1 B cell epitopes from gp41, the T cell epitope of p34pol, and a cluster of B and T epitopes from p17gag were selected. The epitopes were presented as synthetic peptides and as either N- or C-terminal insertions into beta-galactosidase. Hybrids were efficiently expressed in E. coli and easily purified when epitopes were inserted at the beta-galactosidase C terminus. Sera from HIV-1-infected individuals reacted in peptide- and hybrid protein-based enzyme-linked immunosorbent assays (ELISAs) mostly with the immunodominant site of gp41. The second site of gp41 and also sites from p17 and p34 appeared to be immunorecessive. A few of the HIV-1-positive sera exhibited several immunorecessive reactivities. HIV-1-positive sera from the former Soviet Union and Cuba had reactivities similar to those of American, African, and west European sera. Some sera could not be evaluated as specifically HIV-1 seropositive because of their broad reactivities with a multitude of peptides and proteins, unrelated to HIV-1. Extensive tests were performed to define unspecific reactivities by absorption, blocking, and sandwich ELISAs. The application of the hybrid protein assay substantially improved the specificity of the ELISA tests. Thus, hybrid protein-based ELISAs appeared to be more suitable than peptide-based ELISAs, especially for the evaluation of immunorecessive reactivities.

Amino Acid Sequence↗