The scanning ion-conductance microscope.

A scanning ion-conductance microscope (SICM) has been developed that can image the topography of nonconducting surfaces that are covered with electrolytes. The probe of the SICM is an electrolyte-filled micropipette. The flow of ions through the opening of the pipette is blocked at short distances between the probe and the surface, thus, limiting the ion conductance. A feedback mechanism can be used to maintain a given conductance and in turn determine the distance to the surface. The SICM can also sample and image the local ion currents above the surfaces. To illustrate its potential for imaging ion currents through channels in membranes, a topographic image of a membrane filter with 0.80-micrometer pores and an image of the ion currents flowing through such pores are presented.

Imaging and manipulating molecules on a zeolite surface with an atomic force microscope.

The adsorption of neutral molecules and ions on the surfaces of zeolites was observed in real time with an atomic force microscope (AFM). Direct imaging of the surface of the zeolite clinoptilolite was possible by using a diluted tert-butyl ammonium chloride solution as a medium. Images of the crystal in different liquids revealed that molecules could be bound to the surface in different ways; neutral molecules of tert-butanol formed an ordered array, whereas tert-butyl ammonium ions formed clusters. These absorbed molecules were not rearranged by the AFM tip when used in an imaging mode. However, when a sufficiently large force was applied, the tip of the AFM could rearrange the tert-butyl ammonium ions on the zeolite surface. This demonstration of molecular manipulation suggests new applications, including biosensors and lithography.

Imaging crystals, polymers, and processes in water with the atomic force microscope.

The atomic force microscope (AFM) can be used to image the surface of both conductors and nonconductors even if they are covered with water or aqueous solutions. An AFM was used that combines microfabricated cantilevers with a previously described optical lever system to monitor deflection. Images of mica demonstrate that atomic resolution is possible on rigid materials, thus opening the possibility of atomic-scale corrosion experiments on nonconductors. Images of polyalanine, an amino acid polymer, show the potential of the AFM for revealing the structure of molecules important in biology and medicine. Finally, a series of ten images of the polymerization of fibrin, the basic component of blood clots, illustrate the potential of the AFM for revealing subtle details of biological processes as they occur in real time.

Effects of recombinant human erythropoietin and interleukin-3 on erythropoietic recovery from acute anemia.

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.

Polymerized pyridoxylated hemoglobin: a red cell substitute with normal oxygen capacity.

Polymerization of pyridoxylated stroma free hemoglobin (poly SFH-P) yields a solution with a normal hemoglobin concentration, normal colloid osmotic pressure (COP), and a suitable P50. In the present study we compared its in vivo O2-carrying capacity and intravascular persistence with those of pyridoxylated hemoglobin (SFH-P), following a partial exchange transfusion (900 ml) in adult baboons (n = 4 in each group). Poly SFH-P in plasma had an O2-carrying capacity that was 77% greater than that achieved with SFH-P (8.0 versus 4.5 vol%). Furthermore, poly SFH-P provided 5.0 vol% at the end of 24 hours and 2.5 vol% at the end of 48 hours; in contrast SFH-P provided only 2.2 vol% at the end of 6 hours and none within 24 hours. A normal COP was maintained throughout the postexchange period with poly SFH-P despite a 68% increase in plasma protein concentration (from 7.1 to 11.9 gm/dl). The plasma P50 of poly SFH-P decreased from 16 torr immediately after exchange to 12 torr at the end of 48 hours. The in vivo rate of conversion to methemoglobin was similar with both solutions. Polymerized pyridoxylated hemoglobin is currently the only modification of hemoglobin solution that approximates the O2-carrying capacity of whole blood and can be infused without altering the plasma COP.

Hemoglobin solution--from tetramer to polymer.

Efforts to produce a clinically acceptable hemoglobin solution that could function as a temporary oxygen carrier have evolved through several stages. Beginning with the simple tetramer, we have lowered the affinity state by pyridoxylation and then polymerized the modified tetramer. This new substance, polyhemoglobin, has a normal oxygen capacity and a half-life of 38 hours. Issues of nephrotoxicity and immunocompetence after infusion remain to be defined.

Limits of cardiac compensation in anemic baboons.

The risk of homologous blood may cause physicians to withhold red cell treatment after acute blood loss. We believe that in the euvolemic patient with acute anemia, the heart is the principal organ at risk. The cardiac compensation to extreme anemia is unknown and is the purpose of this report. Fourteen adult baboons were anesthetized, paralyzed, and ventilated with room air. Left atrial and coronary sinus catheters were inserted surgically. Experimental animals (n = 7) were hemodiluted at constant left atrial pressure with 5% human serum albumin. Control animals (n = 7) underwent similar volume exchanges with fresh, cross-matched, homologous red blood cells resuspended in human serum albumin, also at constant left atrial pressure. Six of seven experimental animals survived until hematocrit levels were 4%. Adequate cardiac compensation was observed until hematocrit levels were less than 10%. Increased flow, without increases in the O2 extraction ratio, was the mechanism of compensation used by the healthy heart with patent coronary vessels.

Recombinant human erythropoietin and autologous blood donation.

Risks of transfusion are minimized with autologous blood. However, autologous donation programs require 2 to 5 weeks to yield only 2.2 units per patient. Recombinant human erythropoietin (r-HuEPO) has been shown to increase erythropoiesis. This study evaluated the effects of r-HuEPO on an aggressive autologous donation program. Twelve adult male baboons were randomized into two groups of six. All animals were studied three times per week for 5 weeks. A unit of blood was donated when on any study day the hematocrit was greater than 30%. Animals received intravenously either 750 units/kg of r-HuEPO (n = 6) or placebo (n = 6) on each study day. Iron dextran was given intravenously to replace 150% of shed iron. The r-HuEPO group had an earlier onset of reticulocytosis (2.7 vs 5.5 days, p less than 0.01) and donated 35% more blood (13.5 vs 10.0 units, p = 0.01) than the control group. No adverse reactions to r-HuEPO were observed. The data show that an aggressive autologous donation program can yield 10 units of blood over a 5-week period. Further, r-HuEPO increases that yield by an additional 35%. This aggressive autologous donation program with r-HuEPO may significantly reduce the need for homologous transfusion and its attendant risks.

Perioperative recombinant human erythropoietin.

The risks of transfusion-associated infectious disease have made increased efforts to avoid homologous transfusion imperative. Little attention has been focused on efforts to accelerate erythropoiesis as a method of reducing homologous blood use. Recombinant human erythropoietin (rHuEPO) has been shown to enhance erythropoiesis. The purpose of this study was to evaluate the effects of perioperative rHuEPO administration on postoperative erythropoiesis. Fifteen baboons were divided into three groups of five each. Group I received no rHuEPO. Group II received five daily preoperative doses of rHuEPO (1000 U/kg). Group III received five daily preoperative doses and 14 daily postoperative doses of rHuEPO (1000 U/kg). All animals underwent a laparotomy followed by an exchange transfusion to a final hematocrit of 15%. The time in days required to recover to hematocrits of 20% was significantly shorter in both groups that received preoperative doses of rHuEPO when compared with that of controls (3.3 vs 5.7 days, p less than 0.01). The recovery times to hematocrits of 25%, 30%, and baseline levels were all significantly shorter in the group that received both preoperative and postoperative doses of rHuEPO. The data show that perioperative dosage of rHuEPO significantly accelerates postoperative erythropoiesis. Perioperative administration of rHuEPO may reduce the requirements for homologous transfusion.

Effect of hemoglobin solution on compensation to anemia in the erythrocyte-free primate.

Hemoglobin solutions are undergoing clinical trials as erythrocyte substitutes. Some of these solutions have higher O2 affinities compared with normal erythrocyte hemoglobin. Also, they appear to interact with endothelial-derived smooth muscle relaxation. The purpose of this study was to evaluate the nature and limits of compensation to acute normovolemic anemia in the erythrocyte-free primate maintained with a hemoglobin solution. The experimental group consisted of six anesthetized paralyzed adult baboons (Papio anubis) that were exchange transfused (ET) with a pyridoxylated polymerized hemoglobin solution [hemoglobin concentration [( Hb]) = 14 g/dl, O2 half-saturation pressure of hemoglobin (P50) = 19.6 Torr] until a hematocrit less than 1% was achieved. They underwent a second ET with Dextran-70 until [Hb] = 1 g/dl. A control group (n = 6) underwent an ET with Dextran-70 until [Hb] = 1 g/dl. Both groups maintained O2 consumption (VO2) until [Hb] = 3 g/dl. Both groups were stable until [Hb] less than 1 g/dl, and both groups increased their cardiac output. The relation between VO2 and O2 delivery was similar for both groups. In vivo P50 and mixed venous O2 tension were significantly lower in the experimental group. The nature and limits of compensation to diminished O2 delivery due to anemia were similar in the two groups.

Erythropoietin deficiency after coronary artery bypass procedures.

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.

The first randomized trial of human polymerized hemoglobin as a blood substitute in acute trauma and emergent surgery.

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.

STM and AFM images of nucleosome DNA under water.

We have imaged DNA from the calf thymus nucleosome using a scanning tunneling microscope (STM) operated in water. The fragments are deposited onto the interface between a buffer solution and an epitaxially grown gold surface using an electrochemical tecnique. Most of the fragments are fairly straight, and when individual polymers can be identified, their length is consistent with the expected 146 basepairs (approximately 500 A). The resolution is often adequate to show signs of the 36 A helical pitch. Some images show a structure which appears to have abrupt kinks of the sort predicted by Crick and Klug (Nature 255, 530-533, 1975). In order to check that this shape is not a consequence of binding to underlying structure on the gold substrate, we have also made images of kinked structures using an atomic force microscope (AFM) with the DNA bound to glass.

Microporous Montmorillonites Expanded with Alumina Clusters and M[(&mgr;-OH)Cu(&mgr;-OCH(2)CH(2)NEt(2))](6)(ClO(4))(3), (M = Al, Ga, and Fe), or Cr[(&mgr;-OCH(3))(&mgr;-OCH(2)CH(2)NEt(2))CuCl](3) Complexes.

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.

Plasma expanders. An update.

This review of the literature has revealed that isotonic fluids, such as 0.9 percent sodium chloride and Ringer's lactate, are effective plasma volume expanders. Despite the continued use of a variety of colloid solutions in resuscitation, there is no good evidence to document a benefit of these solutions over the crystalloid solutions. The additional cost of colloid compared with crystalloid is another argument against colloid use. The most interesting solution currently being assessed is hypertonic saline solution. Its major benefit is that a small volume of fluid can achieve effective resuscitation. The smaller weight gain and lower incidence of peripheral edema may also prove to be significant benefits. Further evaluations are needed to verify the efficacy of this therapy. Finally, a recent National Institute of Health consensus panel identified the appropriate indications for fresh frozen plasma. They concluded that there is no indication for the use of fresh frozen plasma as a volume expander.

Hypovolemic shock.

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.

Artificial blood: current status of hemoglobin solutions.

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)

Practice parameter for the use of red blood cell transfusions: developed by the Red Blood Cell Administration Practice Guideline Development Task Force of the College of American Pathologists.

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.

Short and long range order of the morphology of silk from Latrodectus hesperus (Black Widow) as characterized by atomic force microscopy.

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.

The effects of embolectomy-thrombectomy catheters on vascular architecture.

The purpose of this report is to describe the evolution of an embolectomy-thrombectomy catheter (ETC) injury over a six week period. Carotid arteries and jugular veins in six adult dogs were subjected to ETC withdrawals at a standard velocity and balloon size. Vascular segments were excised as early as one hour and as late as six weeks. The specimens were prepared for light, scanning electron and transmission electron microscopic examination. In early specimens, arteries and veins showed endothelial denudation followed by regeneration. In later specimens, the arteries showed progressive disruption of the internal elastic lamina and marked subendothelial proliferation (arteriosclerosis). By the sixth week the artery's intima was equal in thickness to the media. The veins showed only regenerating endothelium without alterations of the subendothelium. Exposure of canine vasculature to ETC procedures caused pronounced transmural damage in the arteries and only endothelial alterations in the veins.