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J Simoni

Publications and source records attributed to J Simoni.

35 records · Page 2Linked to original sources
PubMed1988

Complement activation and the toxicity of stroma-free hemoglobin solutions in primates.

The toxicity of hemoglobin solutions was studied in the context of their ability to activate serum complement (C). Three bovine polymerized hemoglobin solutions (BPHSs) with different degrees of purity were used for experiments in vitro and in vivo. BPHS-1 contained bacterial endotoxins (E) (5 EU/ml) and stromal phospholipids (PLs) (1.2 mg/dl), BPHS-2 contained only PLs (2.0 mg/dl), while BPHS-3 was completely free of both contaminants. C-activation was studied by the direct measurement of C3a, C4a, and C5a des Arg fragments, using commercially available RIA kits. During 1 hour of incubation with fresh monkey plasma, BPHS-1 and -2 activated both pathways of C, while BPHS-3 caused no activation of any factor. In vivo, Hb solutions were used to replace one-third of blood volume in three groups of six Coebus monkeys each, while fresh homologous plasma was used in a control group of four animals. Impure solutions activated the alternative pathway of C and caused significant reactions of the circulating blood (thrombocytopenia, leukopenia, and disseminated intravascular coagulation) associated with multiorgan dysfunction (cardiac arrhythmias, hypoxemia, reduction of renal clearance of endogenous creatinine, and elevation of liver enzyme SGPT). The pure solution neither activated C nor caused any reaction in the circulating blood. However, it caused a moderate degree of direct tissue injury, evidenced by transient reduction of creatinine clearance and elevation of SGPT. These observations suggest that impure and pure Hb solutions carry separate mechanisms of toxicity. Complement, activated by toxic impurities, plays an active role in the toxicity of impure solutions. C-activation in vitro could be used as a screening test of biocompatibility.

Animals↗
PubMed1988

Toxicity of polymerized hemoglobin solutions.

Four solutions of bovine polymerized hemoglobin (BPHS) and rabbit plasma were used to replace one-third of the blood volume in five groups of rabbits. The first three solutions were "impure" because of the presence of stromal phosphatidyl-ethanolamine and phosphatidyl-serine in BPHS-1, environmental endotoxins in BPHS-2, and a large amount of higher molecular weight hemoglobin-glutaraldehyde polymers in BPHS-3. These solutions caused a 33 per cent mortality rate and significant morbidity which was characterized by hemodynamic instability, respiratory and renal insufficiency, elevation of hepatic enzyme levels, thrombocytopenia, leukopenia, disseminated intravascular coagulation (DIC) and activation of the alternate pathway of complement. Histopathologic changes found in the heart, lungs, liver, spleen and kidney were characterized by a combination of ischemic and inflammatory lesions. Fibrin thrombi were visible by immunofluorescence in the microcirculation. In contrast, the fourth solution (BPHS-4) was free of the aforementioned impurities; caused no deaths and minimal morbidity, which was limited to elevated levels of serum glutamic pyruvic transaminase and reduction of creatinine clearance; no DIC or complement activation, and mild histopathologic changes which were exclusively ischemic in nature. The results of this study indicated that the toxicity of polymerized hemoglobin solutions is due principally to the presence of impurities. Pure hemoglobin does exhibit mild toxicity when compared with a control solution which is most likely due to a vasoconstrictor effect of oxyhemoglobin.

Animals↗
PubMed1987

Mouse embryo growth in different culture media: selection of a medium for quality control cross-testing of human in vitro fertilization conditions.

A total of 2070 two-cell mouse embryos were recovered from 89 superovulated female hybrid mice. Six different culture media were tested. The various media supported mouse embryo development as follows (percentage mean +/- SD, n = 10): Hopp and Pitts medium (H&P) 87 +/- 5 Dulbecco's modified; Eagle's medium supplemented with 10% (volume/volume, v/v) fetal bovine serum (DMEM) 80 +/- 4; Ham's F-10 +/- 15.0% (v/v) human fetal cord serum (hFCS) 79 +/- 3; Whittingham's T-6 medium (WT-6) 60 +/- 4; Ham's F-10 +/- 7.5% (v/v) hFCS 55 +/- 5; Krebs-Ringer low bicarbonate buffer (KRLBB) 42 +/- 6. In H&P, DMEM, WT-6, and Ham's F-10 medium supplemented with hFCS, the pH was maintained within a narrow range of 7.30-7.45 and adequate level of oxygenation was achieved during 72 h in culture. KRLBB had poor buffering capacity and attained ineffective levels of oxygenation during culture. Superior mouse embryo development from two-cells to morulae and hollow blastocysts occurred in H&P, Ham's F-10 + 15% hFCS, and DMEM. Ham's F-10 medium supplemented with hFCS is routinely checked for its ability to support mouse two-cell embryo development to morulae and blastocysts. This is done in conjunction with H&P medium as the control.

Animals↗
PubMedDate not supplied

Evidence for the direct inhibition of endothelin-1 secretion by hemoglobin in human endothelial cells.

The actual hemoglobin (Hb) contribution to endothelin-1 (ET-1) production in human umbilical vein endothelial cells (EC) was investigated. Cells were incubated with 0.1 mmol or 0.3 mmol of bovine: 1) unmodified (U) ferrous-Hb; 2) U-ferric-Hb; 3) U-ferryl-Hb; 4) polymerized low molecular weight (m.w.) Hb with chemically modified surface (< 400 kDa); and 5) glutaraldehyde polymerized, high m.w. Hb (< 1020 kDa). The incubation medium was tested at 6 and 24 hr for lactate dehydrogenase (index of cellular injury), and for ET-1 release by the cells. Before radioimmunoassay, the ET-1 was extracted from cell culture medium by a two-step purification procedure: 1) ultrafiltration, and 2) column extraction with C18 cartridges. The data suggested that the oxidation status of Hb and its concentration play an important role in causing EC injury. The highest toxicity was observed when EC were incubated with 0.1 mmol of ferryl-Hb, and there was no toxicity with 0.3 mmol of ferric-Hb. These results indicate that the ferric-Hb and low m.w. polymerized Hb at a concentration of 0.1 mmol did not alter ET-1 synthesis and produced a level similar to that of the control. However, it was found that ferryl-Hb and ferrous-Hb in a concentration of 0.1 mmol significantly reduced ET-1 release. All Hbs at a concentration of 0.3 mmol markedly inhibited the production of ET-1. The greatest decrease in ET-1 levels was produced by ferryl-Hb, and the lowest by ferric-Hb and low m.w. polymerized Hb. The Hb's inhibitory effect was more pronounced at 24 hr of incubation. It was also found that although Hb molecules showed a high degree of cross-reactivity with polyclonal anti ET-1 antibodies, the presence of different Hb solutions in the EC culture medium did not change the immunologic properties of ET-1 peptide. In conclusion, Hb inhibitory activity toward ET-1 production might be related to Hb mediated endothelial oxidative injury.

Animals↗
PubMedDate not supplied

A novel hemoglobin-adenosine-glutathione based blood substitute: evaluation of its effects on human blood ex vivo.

Chemically modified hemoglobin (Hb) solutions are under current investigation as potential red cell substitutes. Researchers at Texas Tech University have developed a novel free Hb based blood substitute product. This blood substitute is composed of purified bovine Hb cross-linked intramolecularly with o-adenosine-5'-triphosphate and intermolecularly with o-adenosine, and conjugated with reduced glutathione (GSH). In this study, we compared the effects of our novel blood substitute and unmodified (U) Hb, by using allogenic plasma as the control, on human blood components: red blood cells (RBCs), platelets, monocytes (Mo), and low-density lipoproteins (LDLs). The pro-oxidant potential of both Hb solutions on RBCs was examined by the measurement of osmotic and mechanical fragility, conjugated dienes (CD), lipid hydroperoxides (LOOH), thiobarbituric acid reactants (TBAR-S), isoprostanes (8-iso PGF2alpha) and intracellular GSH. The oxidative modification of LDLs was assessed by CD, LOOH, and TBAR-S, and the degree of apolipoprotein (apo) B cross-linking. The effects of Hb on platelets have been studied by monitoring their responses to the aggregation agonists: collagen, ADP, epinephrine, and arachidonic acid. Monocytes were cultured with Hb solutions or plasma and tested for TNF-alpha and IL-1beta release, then examined by electron microscopy. Results indicate that native UHb initiates oxidative stress of many blood components and aggravates inflammatory responses of Mo. It also caused an increase in RBC osmotic and mechanical fragility (p < 0.001). While the level of GSH was slightly changed, the lipid peroxidation of RBC increased (p < 0.001). UHb was found to be a stimulator of 8-iso PGF2alpha synthesis, a potent modulator of LDLs, and an effective potentiator of agonist induced platelet aggregation. Contrarily, our novel blood substitute did not seem to induce oxidative stress nor to increase Mo inflammatory reactions. The osmotic and mechanical fragility of RBCs was similar to that of the control. Such modified Hb failed to alter LDLs, increase the production of 8-iso PGF2alpha, but markedly inhibited platelet aggregation. The effect of this novel blood substitute can be linked with the cytoprotective and anti-inflammatory properties of adenosine, which is used as a cross-linker and surface modifier, and a modification procedure that lowers the hemoglobin pro-oxidant potential.

Adenosine↗
PubMedDate not supplied

Endotoxemia after burn injury: effect of early excision on circulating endotoxin levels.

Serial circulating endotoxin measurements (quantitative chromogenic limulus assay) were performed in sera from 19 burned patients to determine the profile of circulating serial circulating endotoxin after burn and the effect of early wound excision on serial circulating endotoxin level. Results indicate an early endotoxemia with the peak serial circulating endotoxin levels 7 to 12 hours and 4 days after burn. More importantly, the level of circulating serial circulating endotoxin can be diminished by early excision; late wound excision was associated with a transient increase of serial circulating endotoxin level. Early excision, therefore, may play a critical role in limiting endotoxemia after burn injury.

Adult↗
PubMedDate not supplied

Modified hemoglobin solution, with desired pharmacological properties, does not activate nuclear transcription factor NF-kappa B in human vascular endothelial cells.

The aim of the present study was to evaluate the role of hemoglobin (Hb) and the contribution of chemically modified Hb solutions on the activation of nuclear transcription factor. NF-kappa B, and propagation of oxidative stress within human vascular endothelial cells. The activation of an oxidative stress-sensitive NF-kappa B can be linked with the propagation of an inflammatory state via rapid induction of genes for several pro-inflammatory mediators. Human coronary artery endothelial cells (HCAEC) were cultured on glass coverslips or cell culture plates to confluence. Then, the cells were incubated for up to 18 hours with endothelial basal medium (EBM) supplemented with 5% FBS and test agents in a concentration of 0.1 and 0.2 mmol: 1) unmodified bovine Hb (UHb): 2) modified Hb solution polymerized with glutaraldehyde (GLUT-Hb), and 3) a novel modified Hb solution (Hb-PP-GSH) prepared according to our patented procedure (U.S. Patent No. 5,439,882). The positive control for the NF-kappa B activation study included a treatment of the cells with: I) endotoxin: IL-1; TNF; and H2O2. Results indicate that Hb's pro-oxidant potential was influenced by the type of chemical modification procedure. The GLUT-Hb autoxidation rate, peroxidase-like activity and reactivity with H2O2/ferryl species formation were higher as compared to UHb, by 15%, 35% and 30%, respectively. However, pro-oxidant potential of Hb-PP-GSH was significantly lower than that of UHb (by 22%, 12% and 28%, respectively). The extent of oxidative stress of the HCAECs was found to be the Hb modification-type and concentration dependent. Although the highest endothelial lipid peroxidation and the largest depletion of intracellular GSH was associated with 0.2 mmol of GLUT-Hb, the Hb-PP-GSH did not produce significant changes when compared to the control cells. The UHb generated a moderate oxidative stress to the endothelium. The immunofluorescent and EMSA results indicate a correlation between the type of Hb chemical modification and the induction of NF-kappa B nuclear translocation. We found that GLUT-Hb rapidly activated NF-kappa B and induced nuclear translocation. Treatment of the cells with an increasing amount of UHb leads to the partial nuclear induction of NF-kappa B. However, Hb-PP-GSH did not activate NF-kappa B directly. In this study, the positive control cells treated with endotoxin, IL-1 or TNF demonstrated full nuclear translocations, whereas H2O2 caused only partial induction. In conclusion, nuclear translocation of NF-kappa B by Hb solutions might be dependent on Hb's pro-oxidant potential and extent of Hb-mediated endothelial oxidative stress. Besides the low oxidative potential of Hb-PP-GSH, the observed lack of NF-kappa B activation by this Hb solution can be also related to the anti-inflammatory properties of adenosine which is used in our novel modification procedure. In this study, only the Hb-PP-GSH, cross-linked intramolecularly with o-adenosine triphosphate and intermolecularly with o-adenosine, and combined with reduced glutathiore, was shown to be non-toxic to the endothelium and promises to be an effective free-Hb based blood substitute.

Animals↗
PubMedDate not supplied

Expression of adhesion molecules and von Willebrand factor in human coronary artery endothelial cells incubated with differently modified hemoglobin solutions.

Previous studies have established a linkage between free Hb molecules and the production of inflammatory mediators by the reticuloendothelial cells. An important aspect of the endothelial response to the inflammatory stimuli is the expression of adhesion molecules on the luminal surface. Therefore, the present study was designed to investigate the effects of various free-Hb based oxygen carrying solutions on the intracellular adhesion molecule-1 (ICAM-1), the vascular cell adhesion molecule-1 (VCAM-1) and also von Willebrand factor (vWF) expression by human endothelium. Human coronary artery endothelial cells (HCAEC) were cultured on glass coverslips until they reached confluence, then incubated for 18 hours with endothelial basal medium (EBM) supplemented with 5% FBS and a 0.1 mmol or 0.2 mmol of the bovine Hb solutions: 1) pure unmodified bovine Hb (UHb); 2) modified bovine Hb solution (Hb-PP-GSH) prepared according to our newly developed procedure (U.S. Patent No. 5,439,882); and 3) modified bovine Hb solution polymerized with glutaraldehyde (GLUT-Hb). The HCAECs were also incubated with EBM (negative control) and EBM containing bacterial endotoxins in a concentration of 50 EU/ml (positive control). After treatment, cells were exposed to primary antibodies; anti-human ICAM-1, anti-human VCAM-1 or anti-human vWF, and consequently to the secondary antibody (fluorescein isothiocyanate-conjugated F(ab)2). Immunofluorescence analysis revealed different expressions of ICAM-1 and VCAM-1 on the surface membranes of variously treated cells. Although negative control cells had an undetectable level of adhesion molecules, the positive control cells, activated by endotoxin, exhibited high immunoreactivity for ICAM-1 and VCAM-1. The Hb's treated cells demonstrated differing degrees of activation. An insignificant expression of ICAM-1 was observed in HCAEC, following treatment with a 0.1 or 0.2 mmol of Hb-PP-GSH and 0.1 mmol of UHb. Cell treated with 0.2 mmol of UHb and both concentrations of GLUT-Hb demonstrated a massive expression of this adhesion molecule. A similar effects was observed during induction of VCAM-1. While a lack of expression was noted with both concentrations of Hb-PP-GSH and 0.1 mmol of UHb, the GLUT-Hb stimulated significant VCAM-1 induction at all tested concentrations. Immunofluorescence analysis confirmed the expression of vWF uniformly in HCAEC from the different experimental groups. The data suggest, vWF expression was unaffected by all but the GLUT-Hb treatment. In conclusion, the Hb stimulatory activity toward ICAM-1 and VCAM-1 inductions were related with the type of Hb chemical modification method. Although modification of Hb with glutaraldehyde potentiates adhesion molecules expression, our novel Hb modification procedure, which comprises intramolecular cross-linking with o-adenosine triphosphate and intermolecular with o-adenosine, and combined with reduced glutathione, apparently prevents these inflammatory events.

Animals↗
PubMedDate not supplied

Toxic epidermal necrolysis complicated by severe wound sepsis: a case study.

The case of a 74-year-old female who developed toxic epidermal necrolysis (30% TBSA) is presented. Despite early, aggressive treatment, the patient developed severe wound sepsis and multiple-system organ failure. The fulminant wound sepsis was preceded by early endotoxemia. Patient serum suppressed T lymphocyte blastogenesis in the mixed lymphocyte reaction test and neutrophil chemotaxis in vitro. Endotoxin removal (affinity chromatography) resulted in a reverse of serum suppressive activity against T lymphocytes but did not reverse inhibition of PMN chemotaxis. Circulating endotoxin levels remained detectable during the entire course while blood cultures remained negative. It is our belief that early endotoxemia (detected before clinical signs of wound sepsis becomes apparent) leads to immunosuppression and other systemic complications.

Aged↗
PubMedDate not supplied

An improved blood substitute. In vivo evaluation of its hemodynamic effects.

The purpose of the present study was to assess the ability of an improved free hemoglobin based blood substitute to serve as a resuscitative fluid in the treatment of hemorrhagic shock. Comparison studies were performed by using blood autotransfusion as a positive control. The hemodynamic parameters studied included cardiac index, mean arterial pressure, pulse pressure, heart rate, stroke volume index, and total peripheral resistance. Tissue oxygenation was measured in the biceps femori muscle by polarography. Hemorrhagic shock (at 40% of the total blood volume) in anesthetized rats caused severe disturbances in hemodynamic parameters and tissue oxygenation. Shock was characterized by a 66% drop in cardiac index, a 67% drop in mean arterial pressure with a significant increase in total peripheral resistance, and a 78% reduction in tissue oxygenation, all lasting 30 min. Resuscitation from shock with the blood substitute was effective in restoring hemodynamic parameters, producing vasodilation, and improving tissue oxygenation. Autotransfusion with blood also restored hemodynamics. However, lower tissue oxygenation and lack of vasodilation were noted. Therefore, the modified hemoglobin solution yielded better results than blood in the resuscitation of rats after hemorrhagic shock. The vasodilatory activity and the reduction of vasoconstriction that followed hemorrhage can be primarily linked with adenosine, which possesses vasodilatory and anti-inflammatory properties, and is used in our technology as an intermolecular cross linking reagent and hemoglobin surface modifier.

Animals↗
PubMedDate not supplied

An improved blood substitute. In vivo evaluation of its renal effects.

Nephrotoxicity of free hemoglobin (Hb) based blood substitutes still awaits full elucidation. Previous reports attributed Hb passage through the renal glomeruli to a tendency of the Hb tetramer to dissociate into dimers. Now it has become more evident that the Hb tetramer is able to extravasate. It appears that the electrical charge of proteins plays an important role, with electronegativity and a low isoelectric point favoring intravascular persistence. This effect was utilized in the development of an improved blood substitute, comprising Hb reacted with o-ATP and o-adenosine, to form an intra- and intermolecularly cross linked product, which is reduced with glutathione. The modification reagents possess the desired pharmacologic activities and produce an increase in the electronegative charges on the Hb surface. All Hb polymers and chemically modified tetramers present in this solution have a uniform electronegative charge, with a pl of 6.1-6.2. In this present study, unmodified bovine Hb and an improved blood substitute were used for the replacement of 40% of the total blood volume in rats. The nephrotoxic effect was investigated by the determination of urinary output, glomerular filtration rate (GFR), fractional excretion of sodium (FENa), potassium (FEK), and chloride (FECl), urine/plasma osmolality ratio, and urine N-acetyl-beta-D-glucosaminidase (NAG) level. The free Hb and non heme protein contents in the urine were analyzed by using isoelectric focusing and size exclusion liquid chromatography methods. The results indicate that unmodified Hb is nephrotoxic. An initially elevated urinary output was followed by a significant oliguria associated with decreased GFR, FEK, and FECl and elevated FENa and NAG. Severe hemoglobinuria was associated with proteinuria. Analysis of urine from unmodified Hb treated rats revealed the presence of Hb tetramers. Histopathological examination of the kidneys showed cytoplasmic vacuolization of proximal tubular epithelium. On the contrary, an improved blood substitute did not produce any nephrotoxic reactions. It was found that this Hb solution did not pass through the renal glomerular barrier and was not present in urine samples. In conclusion, such a chemical and pharmacological alteration of Hb molecules reduced their interaction with renal glomeruli and suspended nephrotoxicity.

Animals↗
PubMedDate not supplied

Improved blood substitute: evaluation of its effects on human endothelial cells.

The authors have previously documented that appropriate chemical and pharmacologic modification of the hemoglobin molecule are required to attenuate certain pathophysiologic reactions of the reticuloendothelium. The current study further investigates the molecular responses of human coronary artery endothelial cells to a high concentration (0.4 mmol) of 1) unmodified bovine hemoglobin; and 2) an improved blood substitute that comprises hemoglobin cross-linked intramolecularly with o-adenosine triphosphate and intermolecularly with o-adenosine, and conjugated with reduced glutathione. In this study, the scavenging effect of hemoglobins toward nitric oxide (NO) was evaluated by the measurement of nitrite (NO2-) and nitrate (NO3-) formation. The pro-oxidant effect of hemoglobin on endothelial cells was examined by the measurement of intracellular reduced glutathione, and by monitoring the formation of lipid hydroperoxides and 8-iso prostaglandin F2alpha, a novel potent vasoconstrictor, which is produced by a noncyclooxygenase mechanism involving free radical catalyzed peroxidation of arachidonic acid. The inflammatory reactions of endothelial cells were evaluated by the expression of the adhesion molecule, intracellular adhesion molecule-1, and the activation of nuclear transcription factor, nuclear factor kappaB. In additional, endothelial cell responses were investigated by analysis of intracellular ionized calcium concentrations. Results indicate that unmodified hemoglobin in a concentration of 0.4 mmol/L can aggravate endothelial cell oxidative and inflammatory responses. This hemoglobin produced a significant (p < 0.01) depletion of reduced glutathione, acceleration of lipid peroxidation, and a greater influx of Ca2+. The formation of 8-iso prostaglandin F2alpha increased compared with the control cells (p < 0.01). Unmodified hemoglobin was found to be a potent scavenger of NO, great activator of nuclear factor kappaB, and a stimulator of intracellular adhesion molecule-1 expression. Contrarily, the improved blood substitute did not appear to induce oxidative stress nor to increase the intracellular Ca2+. The concentration of 8-iso prostaglandin F2alpha was similar to that in the control cells, whereas the formation of NO2-/NO3- was much lower (p < 0.05) than in the unmodified hemoglobin group. The effect of an improved blood substitute can be linked with the anti-inflammatory and cytoprotective properties of adenosine, which is used as a cross-linker and surface modifier, and the type of the chemical modification procedure that lowers hemoglobin pro-oxidant potential.

Arachidonic Acid↗