Clusterin silencing restores myoblasts viability and down modulates the inflammatory process in osteoporotic disease.

BACKGROUND: Targeting new molecular pathways leading to Osteoporosis (OP) and Osteoarthritis (OA) is a hot topic for drug discovery. Clusterin (CLU) is a glycoprotein involved in inflammation, proliferation, cell death, neoplastic disease, Alzheimer disease and aging. The present study focuses on the expression and the role of CLU in influencing the decrease of muscle mass and fiber senescence in OP-OA condition. METHODS: Vastus lateralis muscle biopsies were collected from 20 women with OP undergoing surgery for fragility hip fracture and 20 women undergoing arthroplasty for hip osteoarthritis. RESULTS: We found an overexpression of CLU in degenerated fibers in OP closely correlated with interleukin 6 (IL6) and histone H4 acetylation level. Conversely, in OA muscle tissues we observed a weak expression of CLU but no nuclear histone H4 acetylation. Ex vivo studies on isolated human myoblasts confirmed CLU overexpression in OP as compared to OA (p < 0.001). CLU treatment of isolated OP and OA myoblasts showed: modulation of proliferation, morphological changes, increase of histone H4 acetylation and induction of myogenin (MYOG) activation in OP myoblast only. In OP condition, functional knockdown of CLU by siRNA restores proliferative myoblasts capability and tissue damage repair, carried out by an evident upregulation of Transglutaminase 2 (TGM2). We also observed downmodulation of CX3CR1 expression with consequent impairing of the inflammatory infiltrate recruitment. CONCLUSIONS: Results obtained suggest a potential role of CLU in OP by influencing myoblasts terminal differentiation, epigenetic regulation of muscle cell differentiation and senescence. Moreover, CLU silencing points out its role in the modulation of tissue damage repair and inflammation, proposing it as a new diagnostic marker for muscle degeneration and a potential target for specific therapeutic intervention in OP related sarcopenia.

Femoral cortical index: an indicator of poor bone quality in patient with hip fracture.

BACKGROUND: Osteoporosis is a common disease in elderly, characterized by poor bone quality as a result of alterations affecting trabecular bone. However, recent studies have described also an important role of alterations of cortical bone in the physiopathology of osteoporosis. Although dual-energy X-ray absorptiometry (DXA) is a valid method to assess bone mineral density, in the presence of comorbidities real bone fragility is unable to be evaluated. The number of hip fractures is rising, especially in people over 85 years old. AIMS: The aim is to evaluate an alternative method so that it can indicate fracture risk, independent of bone mineral density (BMD). Femoral cortical index (FCI) assesses cortical bone stock using femur X-ray. METHODS: A retrospective study has been conducted on 152 patients with hip fragility fractures. FCI has been calculated on fractured femur and on the opposite side. The presence of comorbidities, osteoporosis risk factors, vitamin D levels, and BMD have been analyzed for each patient. RESULTS: Average values of FCI have been 0.42 for fractured femurs and 0.48 at the opposite side with a statistically significant difference (p = 0.002). Patients with severe hypovitaminosis D had a minor FCI compared to those with moderate deficiency (0.41 vs. 0.46, p < 0.011). 42 patients (27.6%) with osteopenic or normal BMD have presented low values of FCI. DISCUSSION AND CONCLUSION: A significant correlation among low values of FCI, comorbidities, severe hypovitaminosis D. and BMD in patients with hip fractures has been found. FCI could be a useful tool to evaluate bone fragility and to predict fracture risk even in the normal and osteopenic BMD patients.

[Professional drivers and psychoactive substances consumption: results from medical surveillance at the workplace in Piedmont region]. / Guida professionale e consumo di sostanze stupefacenti, risultati relativi ala sorveglinza sanitaria nella regione Piemonnte.

BACKGROUND: The use of psychoactive substances has been shown to be a risk factor for accidents in professional drivers. According to an approved Italian law, in order to detect dependency at the workplace the occupational health physician is called to assess the use of illicit drugs among professional drivers. The main purpose of this study was to investigate the use of psychoactive substances among professional drivers. METHODS: From July to December 2008, rapid urine screening test was carried out on 198 professional drivers. All positive results from the screening stage were verified by specialized laboratories. RESULTS: We found 4 workers with a positive rapid urine screening test (7.1%), one of which was positive only for benzodiazepines and another positive test was not confirmed by specialized laboratory. By only considering illegal substances detected, 6.1% of the drivers tested positive. In this study, the high number of consumers among professional drivers ranged from 31 to 35 years old. Cannabis (THC) was the most frequently detected substance (seen in 10 over 12 cases,), after that was methadone (2/12 cases) and cocaine (1/12 case). We only had one case where more than one substance was found in the same subject (THC and cocaine). Five (41.7%) were former drug-addicts and public Pathological Addiction Services (Ser.T.) had previously followed them. CONCLUSIONS: Our results highlight the problem of drug consumption among professional drivers in Piedmont region. Health education and medical surveillance in workplace drug-testing may improve worker and third parties safety.

Comparison between immunoradiometric and fluorimetric brain natriuretic peptide determination in patients with congestive heart failure.

UNLABELLED: This study compared two different methods, namely the immunoradiometric (IRMA) and fluorimetric (FIA), in order to determine plasma brain natriuretic peptide (BNP) in congestive heart failure (CHF) patients. METHODS: CHF in-patients underwent echocardiography and plasma BNP determination using both two methods. The echocardiograms analysed left ventricular end-systolic (LVESV) and end-diastolic (LVEDV) volumes and systolic dysfunction [left ventricular ejection fraction (LVEF) <50%]. RESULTS: Seventy-three (71% males, age 67 ± 9.6 yr) patients were enrolled, 31.5% affected by valvular heart disease. The mean LVEF was 39.8 ± 14.1%; in 26 (35%) a hypertensive etiology emerged. The immunoradiometric assay (IRMA) BNP was found to be significantly lower than the FIA determination 116.5 ± 149 pg/ml vs 267.3 ± 285.6 pg/ml; p=0.0001) and the two methods were closely correlated (r=0.89; p=0.00001). Logistic regression demonstrated a significant correlation between BNP, LVEF, and LVESV/LVEDV (r=-0.45, p=0.0003; r=-0.48, p=0.00001; r=0.22 p=0.003; r=0.34 p=0.0001; r=0.13 p=0.02; r=0.28 p=0.001 IRMA and FIA, respectively). IRMA BNP and FIA BNP significantly increased according to the worsening functional class [from 34.3 ± 60.2 pg/ml in NYHA (New York Heart Association) I to 555.5 ± 273.1 pg/ml in NYHA IV; from 86.1 ± 162.1 pg/ml in NYHA I to 1070 ± 42.2 pg/ml in NYHA IV, respectively]. In severe systolic dysfunction (LVEF<30%), receiver operating characteristic analysis revealed a satisfactorily sensitivity and specificity using a cut-off point of 50.6 pg/ml with IRMA and 243 pg/ml with FIA. In mild systolic dysfunction (LVEF<50%), a good sensitivity and specificity using a cut-off point of 42 pg/ml with IRMA and 182 pg/ml with FIA emerged. CONCLUSIONS: In CHF patients both BNP methods correlated with NYHA class, LVEF, and ventricular volumes.

Performance of influenza rapid antigen testing in influenza in emergency department patients.

BACKGROUND: The use of rapid antigen tests to triage specimens for polymerase chain reaction (PCR) testing from emergency department patients with influenza-like illness during surveillance for novel influenza viruses has been suggested. OBJECTIVE: To measure the observed sensitivity and specificity for a widely used rapid antigen test (Binax) using a PCR-based assay (Medical Diagnostic Laboratories). METHODS: Nasopharyngeal samples were taken with flocked swabs (Copan Diagnostics) from patients presenting to the emergency department of a community hospital. Samples were analysed using a rapid antigen and a PCR-based test. PCR testing was used as the criterion reference. Sensitivity and specificity were calculated for influenza and influenza A. Positive predictive values were calculated over a range of possible prevalence. RESULTS: Samples from 566 unique patients were tested using both methods. Sensitivity was 69.1% (95% CI 58.9% to 78.1%) and specificity was 97.7% (95% CI 95.8% to 98.8%) for the detection of any influenza and 75.3% (95% CI 64.7% to 84.0%) and 97.8% (95% CI 95.9% to 98.9%), respectively, for influenza A only. The resultant positive predictive value ranges from 23% to 77% when the prevalence ranges from 1% to 10%. CONCLUSION: When planning early outbreak surveillance, provision of adequate PCR testing capacity rather than triaging specimens using rapid antigen testing for influenza is advisable.

B-type natriuretic peptide levels and insulin resistance in patients with severe ischemic myocardial dysfunction.

B-type natriuretic peptide (BNP) is an important clinical parameter of severity in congestive heart failure (CHF). Recent findings suggest a close relation between lipid and glucose metabolism and the natriuretic peptide axis, even if conflicting data exist on the relationship between natriuretic peptide levels and insulin resistance (IR). Thus, we sought to investigate potential relations between BNP level and IR in 134 patients with severe ischemic myocardial dysfunction [mean+/-SD: age =64.8+/-9.6 yr, male/female =104/30; body mass index (BMI) =25.5+/-4.05 kg/m2, 26.1% diabetics; ejection fraction (EF) = 30.2+/-7.7%]. In univariate analysis, an inverse relationship between BNP levels and EF% was observed (R=-0.43, p=0.0006). Moreover, we found an inverse association between BNP levels and BMI (R=-0.27, p=0.036), and also between BNP and homeostasis model assessment of insulin resistance (HOMA-IR) (R=-0.27, p=0.039). In multivariate analysis, EF% and HOMA-IR were significantly and independently associated with logarithmically transformed BNP levels (beta=-0.40, p=0.019 and beta=-0.26, p=0.042, respectively; R2=0.36). In conclusion, in patients with severe ischemic myocardial dysfunction EF and IR are independently associated with BNP levels explaining about 1/3 of the variability of this parameter. Multiple potential mechanisms may underlie this association, but it seems now clinically important to take into account also metabolic features when interpreting plasma natriuretic peptide concentrations obtained for diagnostic or prognostic purposes.

[Peculiarities and aims of first aid training in the road haulage sector]. / Peculiarità e obiettivi della formazione degli addetti al primo soccorso nel settore dell'autotrasporto.

BACKGROUND: Four years after the law concerning first aid in the workplace was passed in Italy (inter-ministerial decree 388/2003), which is a useful tool in improving safety at work, it is necessary to organize first aid courses specifically for those working in the road haulage sector which would also be effective in case of traffic accidents. In view of the characteristics of the working environment in this sector (the road) and of the working conditions (generally workers are isolated and far from the company's headquarters), it would be necessary to organize ad hoc first aid courses at the workplace for the category of professional drivers. OBJECTIVES AND METHODS: The aim of this article is to discuss some possible organizational aspects of first aid in the road haulage sector, such as: number of workers responsible for first aid, how to train workers for specific risks of traffic accidents, the requirements for teachers responsible for first aid and course targets that must be achieved. RESULTS AND CONCLUSIONS: A good level of training, achieved during the first aid course at the workplace, might therefore be useful to improve road safety and increase the quality of basic and advanced first aid in road traffic injuries.

[Neurally-mediated syncope and occupational accidents: prevention strategies and case report]. / Sincope neuromediata e infortuni sul lavoro: strategie preventive e considerazioni su un caso clinico.

A bus driver came to our observation after an occupational traffic accident due to a syncopal event. The positive result of the tilt testing demonstrated the neurally-mediated nature of the syncope. The accident involved approximately 40 people (all the bus passengers), fortunately without severe injuries or deaths. The described episode indicates the need for a procedural algorithm, commonly approved, applicable in the field of prevention, for those occupational categories with severe accident risk. Indeed, the possibility exists to identify at least a part of the subjects predisposed to neurally-mediated syncope. Fundamental steps for such screening are history taking (looking for previous events, familiarity), the physical examination (useful, for example, to exclude orthostatic hypotension or carotid sinus syncope), and, in particular, the tilt testing, a diagnostic investigation recommended for all the workers who have had a previous syncope and are at high occupational accident risk. Moreover, the reported case recalls the need to strengthen the collaboration between the cardiologist and the occupational health physician.

[Syncope and professional driving: the role of the occupational physician]. / Sincope e guida professionale, il ruolo del medico del lavoro.

BACKGROUND: Road traffic injuries constitute one of the main causes of death and disability in Italy and in the European Union. Although much scientific evidence exists on the important role of complete loss of consciousness in the dynamics of motor vehicle accidents, at present there is no specific legislation in Italy. Occupational medicine should take particular care in this field, because every year a large number of accidents with fatal outcomes occur among truck drivers. Via health surveillance, the occupational physician can play an important role in the prevention of such events. OBJECTIVES AND METHODS: The aim of this article is to discuss some possible strategies of health surveillance, comparing the current Italian regulations with the recent guidelines of the European Society of Cardiology (ESC) Task Force regarding the association of syncope and road transport. RESULTS AND CONCLUSION: Current Italian legislation does not include specific prescriptions in case of syncope in truck drivers. This deficiency leads to a lack of information for occupational physicians in order to define unanimous judgements of work fitness. The authors attempted to apply the ESC guidelines on syncope and truck driving to the Italian situation. Four cases of syncope were described, in which the judgement of work fitness for truck drivers proved to be especially complex and stressed the need for cooperation between occupational health physicians and cardiologists.

[Professional driving and safety, a target for occupational medicine]. / Guida professionale e sicurezza, un approccio obiettivo che coinvolge anche la medicina del lavoro.

BACKGROUND: Road traffic injuries constitute one of the main causes of death and disability in Italy and in the European Union. Occupational medicine should pay special attention to the field of road transport because every year a large number of road accidents occur with fatal outcomes. Via health surveillance the occupational physician can play an important role in the prevention of such events. OBJECTIVES: The aim of the article is to summarize the results of the most recent studies on the main risk factors for road transport safety and discuss possible strategies of health surveillance, according to the recent indications of the European Agency for Safety and Health at Work, Bilbao. METHODS: A review of the literature was made. RESULTS AND CONCLUSIONS: The scientific literature provides a large amount of interesting information on the most important risk factors for road accidents, such as drinking and drug abuse, sleepiness and other medical conditions, or excessive speed. The presence of numerous and varied hazards for road transport safety requires, as suggested by the Bilbao Agency, the adoption of occupational health measures, including risk assessment, health education, technical and environmental prevention, health surveillance and clinical interventions (diagnosis and rehabilitation of occupational accidents). Moreover, the paper stresses the need to strengthen collaboration between occupational health physicians and other medical specialists.

Comparison of thrombolytic therapy and primary coronary angioplasty with liberal stenting for inferior myocardial infarction with precordial ST-segment depression: immediate and long-term results of a randomized study.

OBJECTIVES: The aim of the study was to compare randomly assigned primary angioplasty and accelerated recombinant tissue plasminogen activator (rt-PA), in patients with "high-risk" inferior acute myocardial infarction (ST-segment elevation in the inferior leads and ST-segment depression in the precordial leads). BACKGROUND: The ST-segment depression in the precordial leads is a marker of severe prognosis in patients with inferior myocardial infarction. The comparative outcome of treatment with primary angioplasty or lysis with accelerated rt-PA has not been investigated. METHODS: One hundred and ten patients within 6 h of symptoms were randomized to either treatment. To assess the in-hospital and 1-year outcome of both treatments the following results were compared: death or nonfatal infarction, recurrence of angina, left ventricular ejection fraction (LVEF), and the need for repeat target vessel revascularization (TVR). RESULTS: In patients treated with angioplasty (55) and rt-PA (55) the rate of in-hospital mortality and reinfarction was 3.6% versus 9.1% (p=0.4). Recurrence of angina was 1.8% versus 20% (p=0.002), new TVR was used in 3.6% versus 29.1% (p=0.0003), and the LVEF (%) at discharge was 55.2+/-9.5 versus 48.2+/-9.9 (p=0.0001). There were no hemorrhagic strokes, no emergency coronary artery bypass graft (CABG) and identical (5.5%) need for blood transfusions. At 1 year, the incidence of death, reinfarction or repeat TVR was 11% in the percutaneous transluminal coronary angioplasty (PTCA) group versus 52.7% in the rt-PA group (log-rank 22.38, p < 0.0001). CONCLUSIONS: Primary angioplasty is superior to accelerated rt-PA in terms of both myocardial preservation and reduction of in-hospital complications in patients with inferior myocardial infarction and precordial ST-segment depression. Primary angioplasty also yields a better long-term event-free survival.

Improved survival after coronary bypass surgery in patients with poor left ventricular function: role of intraaortic balloon counterpulsation.

The operative mortality rate of aortocoronary bypass surgery in 23 patients with poor left ventricular function (ejection fraction 0.30 or less) operated on in 1973-74 was 34.7 percent. The incidence rate of operative myocardial infarction was 30.4 percent. In an attempt to improve survival, intraaortic balloon counterpulsation was used therafter in 25 similar patients. Counterpulsation was instituted preoperatively and continued intra- and postoperatively for 2 to 5 days. Preoperative studies revealed an "unloading" effect of the left ventricle, with significant reductions of systolic arterial blood pressure, end-diastolic pulmonary arterial pressure and end-diastolic left ventricular volume and pressure. Metabolic improvement was demonstrated by the lesser production of myocardial lactate after pacing-induced tachycardia when the ventricle was balloon-assisted. Intraoperatively, blood flow through the vein graft was found to increase with counterpulsation. The rate of operative myocardial infarction was reduced to 4 percent and the mortality rate to 8 percent. In patients who have sustained a significant loss of functioning myocardium, the beneficial hemodynamic and metabolic effects of intraaortic balloon counterpulsation appear to prevent furhter, possibly critical, myocardial damage in the perioperative period.

Monitoring tissue oxygenation of the heart after myocardial revascularization.

A polarographic technique capable of simultaneous monitoring of myocardial tissue oxygen tension (MPO2) and intramyocardial electrograms by way of the same electrodes has been developed. Initially, the method was evaluated in dogs to verify the appropriateness of the directional changes of MPO2 in function of selected determinants of myocardial oxygen supply (regional coronary blood flow, arterial blood oxygen tension) and demand (heart rate, force of ventricular contraction). A combined reduction of MPO2 and elevation of the S-T segment in the corresponding electrograms was observed only when a 50 percent or greater reduction of blood flow to the sampled area was effected. Subsequently, in nine patients undergoing aortocoronary bypass surgery, MPO2 was measured from 48 areas for 2 weeks postoperatively. In 11 normal and 31 revascularized areas, MPO2 increased during the postoperative period. In four areas subsequently found to be supplied by occluded grafts MPO2 decreased from 12.7 +/- 3.1 (mean +/- standard error) to 10.1 +/- 3.3 mm Hg (P less than 0.05). In two areas, MPO2 decreased during the 3rd postoperative day from 16 to 3 and from 14 to 4.2 mm Hg, respectively. This reduction was attended by a significant rise in the S-T segment of the corresponding electrograms. This finding preceded by 24 hours standard electrocardiographic evidence of myocardial infarction. This technique appears to be sensitive and reliable, and thereby capable of enhancing the management of patients during the high risk early postoperative period after coronary bypass surgery.

Improved oxygenation of ischemic myocardium by hemodilution with stroma-free hemoglobin solution.

Experiments were conducted in anesthetized open-chest dogs subjected to occlusion of the left anterior descending coronary artery for three hours. The oxygenation of myocardial tissue was monitored by a polarographic technique capable of recording simultaneously the oxygen tension (Po2) of myocardial tissue and electrograms. Ischemic injury was monitored by means of ST-segment elevations on myocardial and epicardial electrograms. The volume of the myocardial infarct was measured at the end of each experiment by incubation of transverse slices of left ventricle in a solution of nitroblue tetrazolium and by separation of the unstained (ischemic) from the stained (normal) portions. In one group of dogs, hemodilution was performed after 15 minutes of ischemia by exchanging blood with a stroma-free hemoglobin solution (from a hematocrit reading of 45 +/- 3 percent to 23 +/- 2 percent). Changes occurring in this group were compared with those occurring in dogs that did not undergo hemodilution, underwent hemodilution with dextran 75, or were transfused with whole blood. Hemodilution with hemoglobin reduced aortic and left ventricular filling pressures while increasing coronary blood flow, increased myocardial Po2 from 2 +/- 2 mm Hg to 8 +/- 2 mm Hg (P less than 0.005), lowered the ST-segment elevation of both myocardial and epicardial electrograms, and reduced the volume of the myocardial infarct. These effects were unmatched by hemodilution with dextran or infusion of whole blood.

Glucocorticoid protection of the myocardial cell membrane and the reduction of edema in experimental acute myocardial ischemia.

A possible protective effect of glucocorticoids on the ischemic myocardium was investigated in in situ dog hearts subjected to regional ischemia and in isolated rat hearts subjected to global ischemia. In the whole-animal preparation, the left anterior descending coronary artery (LAD) was occluded for 3 hours, or for 2 1/2 hours followed by 30 minutes of reperfusion. Dexamethasone phosphate was randomly administered (20 mg. per kilogram intravenously) after 15 minutes of ischemia. Its effects were studied on the following: (1) myocardial cell membrane integrity, using electron microscopic examination of tissue biopsies treated with colloidal lanthanum; (2) myocardial water content, measuring the wet/dry weight of myocardial tissue; (3) ischemic injury, by a count of fuchsinophilic cells at light microscopy. In isolated rat hearts, ischemia was produced by a 60 per cent reduction of coronary flow. Randomized hearts were perfused for 2 hours with dexamethasone, 15 mg. per milliliter in buffered salt solution. Study included determination of tissue water content and coronary vascular resistance. Lanthanum was confined to the extracellular spaces in normal dog myocardium, but it was found all intracellularly after 3 hours of ischemia or after reperfusion. This was associated with morphologic changes characteristic of irreversible cell injury. In the hearts treated with dexamethasone, lanthanum remained excluded from the cells, water content was less (p less than 0.005), and fuchsinophilia less severe (p less than 0.005). Likewise, water content was less (p less than 0.005) and the increase in coronary vascular resistance resulting from ischemia less severe (p less than 0.005) in the dexamethasone-treated isolated rat hearts. Thus dexamethasone administered in pharmacologic doses, early, appeared to stabilize the cell membrane, limit myocardial edema, and reduce the severity of injury, both during ischemia and upon reperfusion.

Nonthrombogenic small-caliber human umbilical vein vascular prosthesis.

The present investigation demonstrates covalent binding of heparin with carbodiimide to ammonium hydroxide-treated collagenous surfaces. Human umbilical vein grafts (HUVG) outperform carotid arteries of goat, porcine, and canine origin in both heparin loading and stability of the immobilized heparin preparation. The average heparin loading on the untreated carotid arteries and HUVGs and ammonium hydroxide-treated HUVGs were 18, 27, and 31.5 micrograms/cm2, respectively. There was negligible loss of heparin activity under in vitro and in vivo conditions. In vitro studies demonstrate that heparin-bound HUVGs discourage platelet adhesion and subsequent fibrin clot formation. In vivo studies with heparin-bound HUVGs show a significant increase in thrombus-free surface compared with control grafts. Heparin-bound HUVGs also show an enhanced patency rate in the two sets of protocols tested--one lasting for 3 in vivo days (seven grafts) and the other lasting for 7 (15 grafts) in vivo days. The studies conducted so far demonstrate the promise of developing a nonthrombogenic small-caliber HUVG prosthesis.

The terminal pathway of the lymphatic system of the human heart.

Aantomical dissections in 9 human cadavers revealed the terminal pathway of the lymphatic system of the left ventricle to be constituted mainly by channels emptying into the right angulus venosus (junction of the internal jugular and subclavian veins) at the base of the right side of the neck. This observation has clinical implications because it has been shown that a sampling of cardiac lymph provides the best method for analyzing myocardial metabolic abnormalities and that drainage of cardiac lymph alleviates the myocardial changes produced by ischemic injury.

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.

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.

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.