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Infectious bovine keratoconjunctivitis (IBK) is a common ocular disease in cattle, associated with a 6.8 to 13.6 kg decrease in weaning weight. Antibiotic therapy is available but it is unclear if pain mitigation as an adjunct therapy would reduce the weight loss associated with IBK. Before assessing the impact of pain mitigation therapies, it is first necessary to validate approaches to qualifying ocular pain. The objective of this study was to evaluate approaches to qualifying ocular pain in bovine calves (Bos taurus) with IBK. Our a priori assumption was that scarification or corneal ulcerations consistent with IBK are painful compared to normal eyes. To quantify this difference in pain, we assessed 4 tools: pressure algometry-mechanical nociceptive threshold (PA-MNT), corneal touch thresholds (CTT) obtained with the use of a Cochet-Bonnet aesthesiometer, and assessment for the presence of blepharospasm and photophobia as metrics for pain. Using a 1-eye randomized controlled challenge trial, 31 calves with healthy eyes were randomly allocated to treatment groups, and then a left or right eye was randomly assigned for corneal scarification and inoculation with Moraxella bovoculi or Moraxella bovis. A repeated measures analysis of variance was used for PA-MNT, with significance set at P < 0.05. A log (base 10) transformation was used to stabilize the variance, and Tukey's t tests were used to test differences between assessment days for each landmark. Calves had statistically significantly lower PA-MNT scores (which indicates more pain) the day after scarification relative to baseline measurements (4 d before scarification). For example, at 1 landmark the median PA-MNT (kg/force) prescarification was 4.82 (95% confidence interval [CI]: 3.92-5.93) and 3.43 (95% CI: 2.79-4.22) postscarification. These data suggest PA-MNT may be a tool for quantifying ocular pain in calves. No differences (P < 0.1) in PA-MNT scores between scarified and not-scarified eyes were detected for any landmark on any day. This result suggests that the pain response occurs over the entire face, not just the affected eye. Corneal ulcerations consistent with IBK were not associated with statistically significant differences in PA-MNT or CTT at eye or calf levels. Not surprisingly, scarified eyes were more likely to exhibit blepharospasm and photophobia compared to healthy eyes. Due to blepharospasm, the use of the Cochet-Bonnet to evaluate corneal sensitivity by CTT was of limited value.

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The surfaces of both stretched and unstretched silk threads from the cobweb weaver, Latrodectus hesperus (Black Widow) have been examined by atomic force microscopy (AFM). AFM images of cobweb scaffolding threads show both unordered and highly ordered regions. Two types of fibers within the threads were observed: thicker (approximately 300 nm in diameter) fibers oriented parallel to the thread axis and thinner (10-100 nm) fibrils oriented across the thread axis. While regions which lacked parallel fibers or fibrils were observed on threads at all strain values, the probability of observing fibers and/or fibrils increased with strain. High-resolution AFM images show that with increasing strain, both mean fiber and fibril diameters decrease and that fibrils align themselves more closely with the thread axis. The observation of fibers and fibrils within the cobweb threads has implications for current models of the secondary and tertiary structure and organization of spider silk.

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Expanded clays bipillared with [Al(13)O(4)(OH)(24)(H(2)O)(12)](7+) ions and with hexameric Cu complexes such as M[(&mgr;-OH)Cu(&mgr;-OCH(2)CH(2)NEt(2))](6)(ClO(4))(3), or with M[(&mgr;-OH)Cu(&mgr;-OCH(2)CH(2)NEt(2))](6)(PF(6))(3) where M = Fe, Al, Ga, form microporous materials whose stability and microporosity depend mainly on the identity of the hexamer central metal atom. In fact, a general decrease in thermal stability, interlamellar heights, surface areas, and pore volumes was noted when, in the (Cu,M) hexamer, M changed from gallium to aluminum to iron. Mossbauer results have indicated that only Fe(3+) in octahedral coordination is present in the iron-containing bi-PILC samples (bi-PILC = bipillared interlayered clays). It is believed that metals such as Fe(3+) and Cu(2+) can interact with the interlamellar Keggin ions thereby decreasing the stability of the alumina pillars. In contrast, the intermediate Al(13)-PILC structure is least affected when the more stable Cr complex is used. Bi-PILC materials containing 2.7-3.4% Cr stable to 500 degrees C have been obtained. The low polarity of the chosen solvent (acetonitrile) appears to inhibit the back-exchange of the intermediate PILC's Keggin ions with the hexameric Cu complexes. Elemental analysis together with XRD results suggests that the primary intercalation pathway was diffusion or ion exchange when Cr[(&mgr;-OCH(3))(&mgr;-OCH(2)CH(2)NEt(2))CuCl](3) or M[(&mgr;-OH)Cu(&mgr;-OCH(2)CH(2)NEt(2))](6)(ClO(4))(3), respectively, was used. In all preparations, bi-PILC were produced containing complexes that suffered ligand losses during the synthesis reaction. Molecular scale AFM images have shown that these complexes can be found also outside the clay interlamellar space.

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BACKGROUND: Human polymerized hemoglobin (PolyHeme) is a universally compatible, disease-free, oxygen-carrying resuscitative fluid. This is the first prospective, randomized trial to compare directly the therapeutic benefit of PolyHeme with that of allogeneic red blood cells (RBCs) in the treatment of acute blood loss. STUDY DESIGN: Forty-four trauma patients (33 male, 11 female) aged 19-75 years with an average Injury Severity Score (ISS) score of 21+/-10 were randomized to receive red cells (n = 23) or up to 6 U (300 g) of PolyHeme (n = 21) as their initial blood replacement after trauma and during emergent operations. RESULTS: There were no serious or unexpected adverse events related to PolyHeme. The PolyHeme infusion of 4.4+/-2.0 units (mean +/- SD) resulted in a plasma [Hb] of 3.9+/-1.3 g/dL, which accounted for 40% of the total circulating [Hb]. There was no difference in total [Hb] between the groups before infusion (10.4+/-2.3 g/dL control vs. 9.4+/-1.9 g/dL experimental). At end-infusion the experimental RBC [Hb] fell to 5.8+/-2.8 g/dL vs. 10.6+/-1.8 g/dL (p < 0.05) in the control, although the total [Hb] was not different between the groups or from pre-infusion. The total number of allogeneic red cell transfusions for the control and experimental groups was 10.4+/-4.2 units vs. 6.8+/-3.9 units (p < 0.05) through day 1, and 11.3+/-4.1 units vs. 7.8 +/-4.2 units (p = 0.06) through day 3. CONCLUSIONS: PolyHeme is safe in acute blood loss, maintains total [Hb] in lieu of red cells despite the marked fall in RBC [Hb], and reduces the use of allogeneic blood. PolyHeme appears to be a clinically useful blood substitute.

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A practice parameter has been developed to assist physicians in the therapeutic use of red blood cell transfusions. The developers of this parameter used the best available information from the medical literature, as well as clinical experience and the extensive reality testing required by the College of American Pathologists for approval. In acute anemia, a fall in hemoglobin values below 6 g/dL or a rapid blood volume loss of more than 30% to 40% requires red blood cell transfusions in most patients. However, tissue oxygenation provides a better indication of physiologic need in situations where invasive monitoring provides this information. When these data are not available, heart rate and blood pressure measurements and the nature of bleeding (active, controlled, uncontrolled) supplement the hemoglobin value in guiding the transfusion decision. In sickle cell disease and thalassemias, red blood cells are transfused to prevent acute or chronic complications. Red blood cell transfusions are used in chronic anemias unresponsive to pharmacologic agents based on the patient's symptoms. Guidelines must be altered for neonates who require an increase in hematocrit to above 0.30 to 0.35 when respiratory distress is present. Indications for red blood cell transfusion for the pregnant or postpartum patient are similar to those for the nonpregnant patient. Risks of transfusion, particularly transmissible disease and incompatibility, remain but have been reduced. Thus, red blood cell transfusion continues to be a powerful therapeutic tool when used judiciously and carries less risk than in the recent past.

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We have previously documented the safety of 1 unit (50 gram) of human polymerized hemoglobin (Poly SFH-P) in healthy volunteers. This report describes the first patient trial to assess the therapeutic benefit of Poly SFH-P in acute blood loss. Thirty-nine patients received 1 (n = 14), 2 (n = 2), 3 (n = 15), or 6 (n = 8) units of Poly SFH-P instead of red cells as part of their blood replacement after trauma and urgent surgery. There were no safety issues related to the infusion of Poly SFH-P. The plasma hemoglobin concentration ([Hb]) after the infusion of 6 units (300 gram) of Poly SFH-P was 4.8 +/- 0.8 g/dL (mean +/- SD). Although the red cell [Hb] fell to 2.9 +/- 1.2 g/dL, the total [Hb] was maintained at 7.5 +/- 1.2 g/dL. Poly SFH-P maintained total [Hb], despite the marked fall in red cell [Hb] due to blood loss. The utilization of O2 (extraction ratio) was 27 +/- 16% from the red cells and 37 +/- 13% from the Poly SFH-P. Twenty-three patients (59%) avoided allogeneic transfusions during the first 24 hours after blood loss. Poly SFH-P effectively loads and unloads O2 and maintains total hemoglobin in lieu of red cells after acute blood loss, thereby reducing allogeneic transfusions. Poly SFH-P seems to be a clinically useful blood substitute.

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Although the efficacy of hemoglobin-based oxygen carriers was established more than 60 years ago, all prior clinical trials have demonstrated significant toxicity characterized by renal dysfunction, gastrointestinal distress, and systemic vasoconstriction. The mechanisms of these toxicities now appear to be understood. Tetrameric forms of the hemoglobin molecule extravasate from the circulation and interact with endothelium-derived relaxing factor, leading to unopposed vasoconstriction. Although numerous efforts are under way to chemically modify the native tetramer, it is likely that all tetrameric forms of the hemoglobin molecule will continue to extravasate. We have focused on developing a polymerized form of hemoglobin that is virtually free of unreacted tetramer. The development and characterization of this polymerized pyridoxylated hemoglobin solution (Poly SFH-P) is described. Clinical trials have been completed successfully in volunteers and are now under way to assess the safety and efficacy of Poly SFH-P as a clinically useful red blood cell substitute for treatment of acute blood loss in the setting of trauma and surgery.

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OBJECTIVE: Limited cardiac reserve, secondary to coronary disease, may be associated with end organ morbidity. In this study, we investigate the significance of anemia in the pathogenesis of this phenomenon. DESIGN: Nonrandomized controlled animal trial. SETTINGS: Animal laboratory in a university hospital. SUBJECT: Anesthetized dogs. INTERVENTIONS/MEASUREMENTS: Fourteen anesthetized dogs underwent isovolemic hemodilution with 6% hetastarch from a baseline hematocrit of 40 to 20%. Radioactive microspheres were used to evaluate regional blood flow and cardiac index. Systemic oxygen delivery, consumption, serum lactate, and systemic vascular resistance were recorded during each experiment. Arterial venous oxygen difference was determined from arterial and mixed venous blood. Seven dogs had an iatrogenic critical stenosis of their left anterior descending coronary artery (experimental group); seven dogs did not (control). MAIN RESULTS: Only in the control animals, the cardiac index increased by 35% with hemodilution to 20%. Systemic oxygen delivery decreased in both the control and the experimental animals. Systemic oxygen consumption and lactate levels were unchanged in both groups. In the renal cortex, spleen, distal colon, ileum, gallbladder, and stomach body, regional O2 delivery was significantly decreased with hemodilution to 20% in both groups. This finding was also observed in the left ventricle and cervical spinal cord in the experimental group. In addition, regional O2 delivery was reduced in the spleen, distal colon, and gallbladder with hemodilution to only 30%. Regional blood flow in the stomach body, gallbladder, ileum, renal cortex, and distal colon, in both groups, and the spleen in the control group was unchanged from baseline with hemodilution to 20%. However, regional blood flow under all other circumstances (control or experimental) was significantly increased with hemodilution to 20% with the exception of the spleen, which showed significant regional blood flow decrease in the experimental group only. CONCLUSIONS: These data suggest that with limited cardiac reserve, anemia may compromise aerobic splanchnic circulation. These observations may further our understanding of the pathogenesis of cholecystitis, gastric stress ulcers, ileal endotoxin translocation, and ischemic colitis in critically ill patients with coronary artery disease.

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Although the efficacy of hemoglobin-based oxygen carriers was established more than 60 years ago, all prior clinical trials have demonstrated significant toxicity characterized by renal dysfunction, gastrointestinal distress, and systemic vasoconstriction. The mechanisms of these toxicities now appear to be understood. Tetrameric forms of the hemoglobin molecule extravasate from the circulation and interact with endothelial derived relaxing factor, leading to unopposed vasoconstriction. Although numerous efforts are underway to chemically modify the native tetramer, it is likely that all tetrameric forms of the hemoglobin molecule will continue to extravasate. We have focused on developing a polymerized form of hemoglobin that is virtually free of unreacted tetramer. The development and characterization of this polymerized pyridoxylated hemoglobin solution (Poly SFH-P) is described. Clinical trials have been completed successfully in volunteers, and are now underway to assess the safety and efficacy of Poly SFH-P as a clinically useful red cell substitute in the treatment of acute blood loss in the setting of trauma and surgery.

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Hospitals are required by accrediting agencies to perform blood utilization review. Specific areas that must be addressed are the ordering, distribution, handling, dispensing, and administration of blood components. Monitoring the effects of transfusion on patients is also required. The format of the review process and the criteria for appropriate blood utilization must be developed by each institution. This article provides examples of areas that can be reviewed and procedures that may be used. However, the suggested laboratory values must not be interpreted as defining indications or criteria for transfusion. Each transfusion committee, or its equivalent, is responsible for developing its own institutional blood utilization procedures and audit criteria. Review and approval by the medical staff prior to implementation are essential. The procedures must also be reviewed and revised on a regular basis.

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The risks inherent in the use of homologous blood products have increased efforts toward identifying alternatives to transfusion. We have previously shown that the administration of recombinant human erythropoietin (rhEpo) enhances the erythropoietic response to acute blood loss. Recombinant human interleukin-3 (rh-IL-3) is a hematopoietic growth factor that has been shown to act synergistically with rhEpo in accelerating erythropoiesis in vitro. The purpose of this study in a primate model was to determine if the administration of rhIL-3 in combination with rhEpo could augment the erythropoietic response to acute blood loss more than rhEpo therapy alone. Twenty-four adult male baboons were randomized into four groups. The induction of acute normovolemic anemia to a hematocrit of 20% was accomplished via exchange-transfusion with 6% hetastarch. The groups were then treated for 7 consecutive days with the following growth factors: group I (n = 7), no growth factors; group II (n = 5), rhIL-3 alone (100 micrograms/kg/d); group III (n = 6), rhEpo alone (1000 U/kg/d); group IV (n = 6), rhEpo (1000 U/kg/d) plus rhIL-3 (100 micrograms/kg/d). All animals received folate, vitamin B12, and intravenous iron-dextran immediately following the exchange-transfusion. Response to therapy was monitored for 35 days. There were no adverse reactions following growth factor administration. The analysis of erythropoietic rates between study days 1 through 11, as determined via linear regression analysis, revealed that hematocrits increased significantly faster in the groups receiving rhEpo compared to controls. The administration of rhIL-3, however, did not increase the rate of erythropoiesis when compared to controls, nor did it augment response when added to the rhEpo regimen. The results of this study demonstrate that the administration of rhIL-3 alone had no significant effect on erythropoiesis in this setting of acute blood loss. Further, despite promising in vitro data, rhIL-3 provided no additional stimulation of erythropoiesis in animals receiving rhEpo. Nevertheless, the study confirms that the pharmacologic acceleration of erythropoiesis by rhEpo alone remains an attractive alternative to homologous transfusion.

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We assessed limit to cardiac compensation during isovolemic hemodilution (HD) in 14 anesthetized dogs. Radioactive microspheres were used to evaluate myocardial blood flow (MBF) and its transmural distribution (endo/epi). Myocardial O2 consumption (MVO2) and percent lactate extraction were determined. Coronary vasodilator reserve was assessed from reactive hyperemic responses. Dogs were divided into group 1, with intact left anterior descending coronary artery (LAD), and group 2, with critical stenosis of LAD. Measurements were obtained at baseline and during graded HD (Hespan) until cardiac failure (CF). CF occurred at lower hematocrit in group 1 compared with group 2 (9 +/- 1 vs. 17 +/- 1%). In group 1, MBF increased during HD to maintain MVO2 constant; increases in MBF were transmurally uniform until CF, when decreased endo/epi and lactate production suggested subendocardial ischemia. Coronary vasodilator reserve decreased progressively during HD and was absent at CF. In group 2, stenotic LAD demonstrated constant MBF (resulting in decreased MVO2) during HD. At CF, these responses along with reduced endo/epi and lactate production indicated local myocardial ischemia. We conclude that 1) with normal coronary circulation, cardiac function was well maintained over a wide range of hematocrits because increases in MBF were transmurally uniform and sufficient to maintain myocardial oxygenation: CF occurred during extreme HD when MBF became maldistributed, resulting in subendocardial ischemia; 2) critical coronary stenosis impaired coronary vascular adjustment to HD and reduced significantly tolerance of left ventricle to HD; and 3) present findings underscore the importance of recruitment of coronary vasodilator reserve in preserving total and regional myocardial oxygenation during HD.

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This article defines a rational approach to the treatment of hemorrhagic shock. All patients that are hypovolemic following hemorrhage require fluid resuscitation. Some patients require red cell restoration and very few require correction of any clotting deficiencies. A physiologic approach to these problems will lead to optimal patient care in these circumstances.

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Stimulation of erythropoiesis is an attractive alternative to the risks of homologous transfusion. The availability of rHuEPO has made erythropoietic acceleration possible. The use of rHuEPO perioperatively and in preoperative autologous donation will likely find firm indications. Combinations of present methods of autologous blood use with the various rHuEPO regimens will be the best methods of minimizing perioperative homologous blood exposure.

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We have described whole body oxygen (O2) extraction ratio (ER) as a reliable indicator of transfusion need in acute normovolemic anemia. In normal hearts, myocardial lactate production (-LACT), indicating anaerobic metabolism, does not occur until the ER greater than 50% and Hct less than 10%. It is not known if the ER is valid in the setting of limited coronary vascular reserve. This study assesses the effect of a critical left anterior descending (LAD) coronary stenosis on the compensation to acute blood loss anemia. Adult dogs were anesthetized, paralyzed, and mechanically ventilated. A critical LAD stenosis was created in seven animals (STEN). There were seven controls (CON). Animals underwent isovolemic exchange transfusion with 6% HES until cardiac failure (CF). Catheters were placed in the aorta, pulmonary artery, and anterior interventricular coronary vein. Cardiac failure occurred at Hct = 8.6% +/- 0.4% in the CON and 17.0% +/- 0.5% in the STEN animals. Cardiac output increased in the CON, but not in the STEN animals. Blood flow in the LAD increased in the CON but not the STEN animals. -LACT began in the CON and STEN animals at Hct less than 20% and coincided with an ER greater than 50% in both groups. We conclude that CF occurs at a higher hematocrit with a critical LAD stenosis. The whole body ER greater than 50% remains a valid indicator of myocardial metabolism in anemia in the presence of limited coronary vascular reserve. The ER may be a useful guide to transfusion therapy.

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Attempts to develop a hemoglobin-based red cell substitute have spanned many decades, but no clinically useful product has been produced to date. The issues preventing clinical application primarily are ones of safety--not efficacy. Numerous animal studies have documented the efficacy of SFH. Although effective, the solution has limitations that have caused concern. Oncotic considerations limit the concentration of the infusate SFH to 6 to 8 g/dL, or half-normal. Owing to the loss of organic phosphate modulators of P50, such as 2,3-DPG, the P50 of SFH is typically between 12 and 14 mm Hg, which is also half the normal value. And finally, the intravascular half-life of SFH is too short, ranging only from 2 to 6 hr. Polymerization provides a means of correcting these limitations. The high oxygen affinity can be greatly diminished by covalent binding of pyridoxal-5'-phosphate to the N-terminal of the chains. Colloid osmotic pressure exerted by a protein solution is proportional to the number of discrete colloid particles. Through polymerization, the number of colloid particles is reduced, leading to a decrease in COP. Data show that this can be achieved in a reproducible fashion. The rate at which COP diminishes determines the yield of polymeric species, as well as their molecular weight distribution. Polymerization can be controlled to result in a yield of 75% to 85% polymers with a molecular weight distribution of 128 to 400 kd. The number average and the weight average molecular weights indicate that the large proportion of polymers represent the cross linking of two tetramers. The data that reflect the interaction of oxygen with poly-SFH-P indicate that the oxygen carrying function of hemoglobin has not been significantly altered by the chemical modifications. The binding coefficient of oxygen is unchanged. As anticipated, there is a loss of cooperativity (diminished Hill coefficient) between the hemoglobin chains, suggesting structural restrictions in the polymeric species because of cross linking. A reduced alkaline Bohr effect is the expected result, and data confirm this. Finally, some increase in oxygen affinity is to be expected with polymerization. This is indeed the case, although the P50 of poly-SFH-P is comparable to banked blood (18 to 22 mm Hg). To be clinically useful, a modified hemoglobin solution requires a reasonable shelf-life.(ABSTRACT TRUNCATED AT 400 WORDS)

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Erythropoietin is the primary regulator of erythropoiesis. Erythropoietin has been shown to increase exponentially in response to linear decreases in hematocrit in normal, unstressed animals. However, the effect of operation, with its attendant stress, on erythropoietin levels is unknown. The purpose of this study is to evaluate the effect of surgical stress on erythropoietin. Twenty otherwise healthy patients scheduled for elective surgical procedures were studied. The cholecystectomy group included 10 patients who underwent cholecystectomy for documented stone disease. Ten patients who underwent coronary artery bypass procedures constituted the coronary artery bypass grafting group. Patients were studied preoperatively as well as on the first and second postoperative days. The hematocrit and erythropoietin levels were similar in both groups preoperatively. The hematocrit in the coronary artery bypass grafting group was lower than that of the cholecystectomy group on postoperative day 1 (0.31 versus 0.36; p less than 0.003) and postoperative day 2 (0.30 versus 0.36; p less than 0.001). During the first two postoperative days the erythropoietin levels were similar between groups. The data show that postoperative erythropoietin levels are similar after coronary artery bypass grafting, despite more severe anemia, when compared with cholecystectomy. This suggests that after coronary artery bypass grafting there is a relative deficiency of erythropoietin. Administration of recombinant human erythropoietin to patients undergoing surgical procedures could correct the erythropoietin deficiency and accelerate postoperative erythropoiesis.

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We present images of the polar or headgroup regions of bilayers of dimyristoyl-phosphatidylethanolamine (DMPE), deposited by Langmuir-Blodgett deposition onto mica substrates at high surface pressures and imaged under water at room temperature with the optical lever atomic force microscope. The lattice structure of DMPE is visualized with sufficient resolution that the location of individual headgroups can be determined. The forces are sufficiently small that the same area can be repeatedly imaged with a minimum of damage. The DMPE molecules in the bilayer appear to have relatively good long-range orientational order, but rather short-range and poor positional order. These results are in good agreement with x-ray measurements of unsupported lipid monolayers on the water surface, and with electron diffraction of adsorbed monolayers.

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