Profile repeatability: A new method for evaluating repeatability of individual hormone response profiles.

There is broad interest in determining repeatability of individual responses. Current methods calculate repeatability of individual points (initial and/or peak), time to peak value, or a single measure of the integrated total response (area under the curve), rather than the shape of the response profile. Repeatability estimates of response profiles using linear mixed models (LMM) generate an average repeatability for an aggregate of individuals, rather than an estimate of individual repeatability. Here we use a novel ad hoc method to calculate repeatability of individual response profiles and demonstrate the need for a more rigorous assessment protocol. Response profile repeatability has not been defined at the individual level. We do this using a new metric, Profile Repeatability (PR), which incorporates components of variance and the degree to which response profiles cross each other in a time series. Values range from 0 (no repeatability) to 1 (complete repeatability). We created synthetic data to represent a range of apparent time series repeatability, and 20 independent observers visually ranked those data sets by degree of repeatability. We also applied the method to real data on stress responses of European starlings Sturnus vulgaris. We then computed PR scores for the synthetic data and for real data from European starling corticosterone responses over time, and contrast the results to those from LMM. Finally, we assessed the sensitivity of PR to reductions in the number of time points in the corticosterone response, as well as reductions in the number of replicates per individual. We found the average PR scores for a group of individuals to be somewhat robust to reductions in points in the time series; however, the ranks of individuals (PR values relative to one another) could change substantially with reduction in the number of values in a time series. PR showed threshold sensitivity to losing replicate time series between 6 and 4 replicates. Surprisingly, human observers fell into two disparate groups when ranking repeatability of the synthetic data, and the PR score indicated that human observers may underestimate repeatability of data where replicates cross each other. In contrast to the average profile repeatability estimated using LMMs, our approach calculates individual repeatability. From our perspective, LMM does not provide a definitive idea of repeatability at the individual level; in essence, it concludes that suites of time series with low within-individual variance has high repeatability, regardless of replicate trajectories. LMM and PR have non-linear relationships between 0 and 1, but PR has greater discrimination for mid-values of repeatability. Consistent average group repeatability can be associated with substantial differences in individual ranks suggests that estimating individual repeatability is critical. The PR score should be useful in comparing repeatability of any type of nonlinear, including non-monotonic, response profiles over time, which are common in both physiology and behavior, and it demonstrates the specific needs for future improvements of a profile repeatability metric.

Leishmaniasis: Recent epidemiological studies in the Middle East.

Leishmaniasis, one of the most neglected tropical diseases (NTDs), is the third most important vector-borne disease worldwide. This disease has a global impact and severity of the infection and is greatest in the Middle East. The agent of infection is a protozoan parasite of the genus, Leishmania, and is generally transmitted by blood-sucking female sandflies. In humans, there are three clinical forms of infection: (1) cutaneous (CL), (2) mucocutaneous (ML), and (3) visceral leishmaniasis (VL). This review aims to discuss the current epidemiological status of leishmaniasis in Saudi Arabia, Iraq, Syria, and Yemen with a consideration of treatment options. The elevated risk of leishmaniasis is influenced by the transmission of the disease across endemic countries into neighboring non-infected regions.

Structure of Greyhound hemoglobin: origin of high oxygen affinity.

This study presents the crystal structure of Greyhound hemoglobin (GrHb) determined to 1.9 Šresolution. GrHb was found to crystallize with an α1ß1 dimer in the asymmetric unit and belongs to the R2 state. Oxygen-affinity measurements combined with the fact that GrHb crystallizes in the R2 state despite the high-salt conditions used for crystallization strongly indicate that GrHb can serve as a model high-oxygen-affinity hemoglobin (Hb) for higher mammals, especially humans. Structural analysis of GrHb and its comparison with the R2-state of human Hb revealed several regions that can potentially contribute to the high oxygen affinity of GrHb and serve to rationalize the additional stability of the R2-state of GrHb. A previously well studied hydrophobic cluster of bar-headed goose Hb near α119 was also incorporated in the comparison between GrHb and human Hb. Finally, a structural comparison with generic dog Hb and maned wolf Hb was conducted, revealing that in contrast to GrHb these structures belong to the R state of Hb and raising the intriguing possibility of an additional allosteric factor co-purifying with GrHb that can modulate its quaternary structure.

The Alabama Drug Discovery Alliance: a collaborative partnership to facilitate academic drug discovery.

The Alabama Drug Discovery Alliance is a collaboration between the University of Alabama at Birmingham and Southern Research Institute that aims to support the discovery and development of therapeutic molecules that address an unmet medical need. The alliance builds on the expertise present at both institutions and has the dedicated commitment of their respective technology transfer and intellectual property offices to guide any commercial opportunities that may arise from the supported efforts. Although most projects involve high throughput screening, projects at any stage in the drug discovery and development pathway are eligible for support. Irrespective of the target and stage of any project, well-functioning interdisciplinary teams are crucial to a project's progress. These teams consist of investigators with a wide variety of expertise from both institutions to contribute to the program's success.

Small-volume resuscitation from hemorrhagic shock using high-molecular-weight tense-state polymerized hemoglobins.

BACKGROUND: The objective of this study was to determine the role of plasma oxygen carrying capacity during resuscitation from hemorrhagic shock (HS). METHODS: Hemodynamic responses to small-volume resuscitation from HS with hypertonic saline followed by infusion of ultrahigh-molecular-weight tense-state polymerized hemoglobins (PolyHbs) were studied in the hamster window chamber model. HS was induced by withdrawing 50% of the blood volume (BV), and hypovolemic state was maintained for 1 hour. Resuscitation was implemented by infusion of hypertonic saline (3.5% of BV) followed by 10% of BV infusion of polymerized human Hb (PolyHbhum, P50=49 mm Hg), polymerized bovine Hb (PolyHbbov, P50=40 mm Hg), or human serum albumin (HSA), all at 10 g/dL. Resuscitation was monitored over 90 minutes. RESULTS: PolyHbhum elicited higher arterial pressure, produced vasoconstriction, and decreased perfusion. In contrast, PolyHbbov and HSA exhibited lower blood pressure and partially restored perfusion and functional capillary density compared with PolyHbhum. Blood gas parameters showed a pronounced recovery after resuscitation with PolyHbbov compared with both PolyHbhum and HSA. Tissue PO2 was significantly improved in the PolyHbbov group, showing that the moderate increase in P50 of PolyHbbov compared with hamster blood (P50=32 mm Hg) was beneficial during resuscitation. However, an excessive increase in oxygen release between the central and peripheral circulation, as induced by PolyHbhum produced vasoconstriction and hypoperfusion, limiting the benefits of additional oxygen carrying capacity. CONCLUSIONS: Appropriately engineered PolyHb will enhance/reinstate oxygenation, without hypertension or vasoconstriction, to be used in situations where blood transfusion is not logistically feasible.

Tissue oxygenation after exchange transfusion with ultrahigh-molecular-weight tense- and relaxed-state polymerized bovine hemoglobins.

Hemoglobin (Hb)-based O(2) carriers (HBOCs) constitute a class of therapeutic agents designed to correct the O(2) deficit under conditions of anemia and traumatic blood loss. The O(2) transport capacity of ultrahigh-molecular-weight bovine Hb polymers (PolybHb), polymerized in the tense (T) state and relaxed (R) state, were investigated in the hamster chamber window model using microvascular measurements to determine O(2) delivery during extreme anemia. The anemic state was induced by hemodilution with a plasma expander (70-kDa dextran). After an initial moderate hemodilution to 18% hematocrit, animals were randomly assigned to exchange transfusion groups based on the type of PolybHb solution used (namely, T-state PolybHb and R-state PolybHb groups). Measurements of systemic parameters, microvascular hemodynamics, capillary perfusion, and intravascular and tissue O(2) levels were performed at 11% hematocrit. Both PolybHbs were infused at 10 g/dl, and their viscosities were higher than nondiluted blood. Restitution of the O(2) carrying capacity with T-state PolybHb exhibited lower arterial pressure and higher functional capillary density compared with R-state PolybHb. Central arterial O(2) tensions increased significantly for R-state PolybHb compared with T-state PolybHb; conversely, microvascular O(2) tensions were higher for T-state PolybHb compared with R-state PolybHb. The increased tissue Po(2) attained with T-state PolybHb results from the larger amount of O(2) released from the PolybHb and maintenance of macrovascular and microvascular hemodynamics compared with R-state PolybHb. These results suggest that the extreme high O(2) affinity of R-state PolybHb prevented O(2) bound to PolybHb from been used by the tissues. The results presented here show that T-state PolybHb, a high-viscosity O(2) carrier, is a quintessential example of an appropriately engineered O(2) carrying solution, which preserves vascular mechanical stimuli (shear stress) lost during anemic conditions and reinstates oxygenation, without the hypertensive or vasoconstriction responses observed in previous generations of HBOCs.

Functional comparison of hemoglobin purified by different methods and their biophysical implications.

Hemoglobin (Hb) that is purified from red blood cells (RBCs) is commonly subjected to harsh processing conditions, such as high temperatures and extensive column separation, which may damage the Hb by altering the heme prosthetic group and/or the Hb protein structure. In this study, bovine and human Hb purified by tangential flow filtration (TFF) was compared to commercial preparations of human Hb (Hemosol, Inc., Toronto, Canada) and bovine Hb (Biopure, Inc., Cambridge, MA). Purified Hbs were characterized by measuring their overall purity (SDS-PAGE, SEC, and ESI-MS), susceptibility to oxidation (k(ox)), responses to physiological conditions (pH, [Cl(-)], [IHP], and T), and ligand binding kinetics (O(2), NO, and CO). All Hbs evaluated possessed comparable biophysical properties, however, a small amount of catalase was detected in the TFF-purified Hbs that reduced the rate of autoxidation. Mass changes observed by mass spectrometry suggest that structural alterations may be introduced into Hbs that are purified by extensive chromatographic separations. These results demonstrate that TFF is a suitable process for the purification of Hb from RBCs with a quality equivalent to that of commercial Hb preparations that employ more extensive purification strategies. This work also shows that TFF can yield highly pure Hb which can be used for Hb-based O(2) carrier synthesis.

Convection and hemoglobin-based oxygen carrier enhanced oxygen transport in a hepatic hollow fiber bioreactor.

Hepatic hollow fiber bioreactors are a promising class of bioartificial liver assist device (BLAD). The development of this type of device is currently hindered by limited oxygen transport to cultured hepatocytes, due to low solubility of oxygen in aqueous media. In order to increase the oxygen spectrum to cultured hepatocytes housed within a hollow fiber bioreactor, several different engineering strategies were explored in this study. These included: supplementing the circulating media stream of the hollow fiber bioreactor with a hemoglobin-based oxygen carrier (bovine red blood cells) with defined oxygen binding and release kinetics and operating the bioreactor with media flow through the hollow fiber membrane into the extracapillary space (ECS). We hypothesize that these two strategies can be used to improve hepatocyte oxygenation and possibly attain an in vivo-like pO(2) spectrum, similar to that observed in vivo in the liver sinusoid. This work is significant, since provision of an in vivo-like pO(2) spectrum should create a fully functional BLAD that could potentially bridge thousands of liver failure patients towards native liver regeneration of damaged tissue or, if necessary, orthotopic liver transplantation.

Hemoglobin regulates the migration of glioma cells along poly(ε-caprolactone)-aligned nanofibers.

Aligned fibers have been shown to facilitate cell migration in the direction of fiber alignment while oxygen (O2 )-carrying solutions improve the metabolism of cells in hypoxic culture. Therefore, U251 aggregate migration on poly(ε-caprolactone) (PCL)-aligned fibers was studied in cell culture media supplemented with the O2 storage and transport protein hemoglobin (Hb) obtained from bovine, earthworm and human sources at concentrations ranging from 0 to 5 g/L within a cell culture incubator exposed to O2 tensions ranging from 1 to 19% O2 . Individual cell migration was quantified using a wound healing assay. In addition, U251 cell aggregates were developed and aggregate dispersion/cell migration quantified on PCL-aligned fibers. The results of this work show that the presence of bovine or earthworm Hb improved individual cell viability at 1% O2 , while human Hb adversely affected cell viability at increasing Hb concentrations and decreasing O2 levels. The control data suggests that decreasing the O2 tension in the incubator from 5 to 1% O2 decreased aggregate dispersion on the PCL-aligned fibers. However, the addition of bovine Hb at 5% O2 significantly improved aggregate dispersion. At 19% O2 , Hb did not impact aggregate dispersion. Also at 1% O2 , aggregate dispersion appeared to increase in the presence of earthworm Hb, but only at the latter time points. Taken together, these results show that Hb-based O2 carriers can be utilized to improve O2 availability and the migration of glioma spheroids on nanofibers.

Down selection of polymerized bovine hemoglobins for use as oxygen releasing therapeutics in a guinea pig model.

Hemoglobin (Hb)-based oxygen carriers (HBOCs) are being developed as resuscitative fluids for use in multiple medical applications and in lieu of blood transfusion. However, cardiovascular, central nervous system, and renal adverse events have largely impeded progress. This has prompted a need to evaluate novel down selection approaches for HBOCs prior to in-depth preclinical and clinical safety testing. In the present study, polymerized bovine Hbs (PolybHbs) were prepared with increasing ratios of glutaraldehyde to bovine Hb (10:1, 20:1, 30:1, and 40:1). The optimal PolybHb candidate selection was based on a priori determined in vivo response to include a long circulating PolybHb with no measurable renal exposure, minimal cardiovascular response, limited oxidation to metHb in vitro, or in circulation and absence of acute end organ toxicity. Guinea pigs were dosed via a 50% blood for PolybHb exchange transfusion. Data suggested that the 30:1 preparation exhibited maximum circulatory exposure (AUC(0)(-∞)) with the lowest level of oxidation (plasma metHb formation) and minimal (< 10%) blood pressure elevation. Additionally, the 30:1 preparation was absent renal iron deposition as well as abnormal glomerular/tubular histopathology or serum creatinine elevation. Clearance pathways predominantly followed those consistent with endogenous Hb clearance based pathways. Therefore, data confirmed the ability to select a single PolybHb from a small library of HBOCs based on a priori determined characteristics. Moreover, the approach to down selection described could be applied to enhance the early predictability of human safety for this class of biological therapeutics to optimize for specific indications.

Synthesis, biophysical properties and pharmacokinetics of ultrahigh molecular weight tense and relaxed state polymerized bovine hemoglobins.

Hemoglobin-based oxygen carriers (HBOC) are currently being developed as red blood cell (RBC) substitutes for use in transfusion medicine. Despite significant commercial development, late stage clinical results of polymerized hemoglobin (PolyHb) solutions hamper development. We synthesized two types of PolyHbs with ultrahigh molecular weights: tense (T) state PolyHb (M(W)=16.59 MDa and P(50)=41 mmHg) and relaxed (R) state PolyHb (M(W)=26.33 MDa and P(50)=0.66 mmHg). By maintaining Hb in either the T- or R-state during the polymerization reaction, we were able to synthesize ultrahigh molecular weight PolyHbs in distinct quaternary states with no tetrameric Hb, high viscosity, low colloid osmotic pressure and the ability to maintain O(2) dissociation, CO association and NO dioxygenation reactions. The PolyHbs elicited some in vitro RBC aggregation that was less than 6% dextran (500 kDa) but more than 5% human serum albumin. In vitro, T-state PolybHb autoxidized faster than R-state PolybHb as expected from previously reported studies, conversely, when administered to guinea pigs as a 20% exchange transfusion, R-state PolybHb oxidized faster and to a greater extent than T-state PolybHb, suggesting a more complex oxidative processes in vivo. Our findings also demonstrate that T-state PolybHb exhibited a longer circulating half-life, slower clearance and longer systemic exposure time compared to R-state PolybHb.

Tangential flow filtration of hemoglobin.

Bovine and human hemoglobin (bHb and hHb, respectively) was purified from bovine and human red blood cells via tangential flow filtration (TFF) in four successive stages. TFF is a fast and simple method to purify Hb from RBCs using filtration through hollow fiber (HF) membranes. Most of the Hb was retained in stage III (100 kDa HF membrane) and displayed methemoglobin levels less than 1%, yielding final concentrations of 318 and 300 mg/mL for bHb and hHb, respectively. Purified Hb exhibited much lower endotoxin levels than their respective RBCs. The purity of Hb was initially assessed via SDS-PAGE, and showed tiny impurity bands for the stage III retentate. The oxygen affinity (P(50)) and cooperativity coefficient (n) were regressed from the measured oxygen-RBC/Hb equilibrium curves of RBCs and purified Hb. These results suggest that TFF yielded oxygen affinities of bHb and hHb that are comparable to values in the literature. LC-MS was used to measure the molecular weight of the alpha (alpha) and beta (beta) globin chains of purified Hb. No impurity peaks were present in the HPLC chromatograms of purified Hb. The mass of the molecular ions corresponding to the alpha and beta globin chains agreed well with the calculated theoretical mass of the alpha- and beta- globin chains. Taken together, our results demonstrate that HPLC-grade Hb can be generated via TFF. In general, this method can be more broadly applied to purify Hb from any source of RBCs. This work is significant, since it outlines a simple method for generating Hb for synthesis and/or formulation of Hb-based oxygen carriers.

The quaternary structure of tetrameric hemoglobin regulates the oxygen affinity of polymerized hemoglobin.

This study focuses on the effect of the initial quaternary structure of bovine hemoglobin (Hb) on the physical properties of glutaraldehyde polymerized Hb (PolyHb) solutions. Tense (T) state PolyHb was synthesized by maintaining the pO(2) of Hb before and after polymerization at 0 mm Hg. In contrast, relaxed (R) state PolyHb was generated by maintaining the pO(2) of Hb before and after polymerization to >749 mm Hg. PolyHb solutions were characterized by measuring the pO(2), methemoglobin (metHb) level, molecular weight distribution, O(2) affinity and cooperativity coefficient. The metHb level of all PolyHb solutions was low (<2%). Analysis of the molecular weight distribution of PolyHb solutions indicates that in general, the molecular weight of PolyHb solutions increased with increasing cross-link density. T-state PolyHb solutions exhibited lower O(2) affinity compared to unmodified Hb, whereas R-state PolyHb solutions exhibited higher O(2) affinity compared to unmodified Hb. In addition, the polymerization reaction resulted in a significant decrease in cooperativity that was more pronounced at higher cross-link densities. All of these results were explained in terms of the quaternary structure of Hb. Taken together, our results yield more insight into the importance Hb's quaternary structure plays in defining the physical properties of glutaraldehyde PolyHb solutions. This information will be useful in designing optimized glutaraldehyde PolyHb oxygen carriers for various applications in transfusion medicine.

Purification of hemoglobin by tangential flow filtration with diafiltration.

A recent study by Palmer, Sun, and Harris (Biotechnol. Prog., 25:189-199, 2009) demonstrated that tangential flow filtration (TFF) can be used to produce HPLC-grade bovine and human hemoglobin (Hb). In this current study, we assessed the quality of bovine Hb (bHb) purified by introducing a 10 L batch-mode diafiltration step to the previously mentioned TFF Hb purification process. The bHb was purified from bovine red blood cells (RBCs) by filtering clarified RBC lysate through 50 nm (stage I) and 500 kDa (stage II) hollow fiber (HF) membranes. The filtrate was then passed through a 100 kDa (stage III) HF membrane with or without an additional 10 L diafiltration step to potentially remove additional small molecular weight impurities. Protein assays, SDS-PAGE, and LC-MS of the purified bHb (stage III retentate) reveal that addition of a diafiltration step has no effect on bHb purity or yield; however, it does increase the methemoglobin level and oxygen affinity of purified bHb. Therefore, we conclude that no additional benefit is gained from diafiltration at stage III and a three stage TFF process is sufficient to produce HPLC-grade bHb.

Effects of the molecular mass of tense-state polymerized bovine hemoglobin on blood pressure and vasoconstriction.

Despite recent advances in the design of hemoglobin (Hb)-based oxygen carriers (HBOCs), vasoconstriction, presumably caused by nitric oxide (NO) scavenging, vessel wall hyperoxygenation, and/or extravasation, has been identified as the principal road block hampering commercial development of HBOCs. This study was designed to analyze systemic and microvascular responses to the molecular mass and plasma concentration of tense (T)-state polymerized bovine Hb (PolybHb) solutions. Experiments were performed using the hamster window chamber model subjected to successive hypervolemic infusions of T-state PolybHb solutions. PolybHb plasma concentrations were evaluated, namely, 0.5, 1.0 and 1.5 g/dl, respectively. Infusion of PolybHb solutions with molecular mass >500 kDa elicited hypertension and vasoconstriction proportional to the plasma concentration and inversely proportional to the PolybHb cross-link density. However, two high-molecular mass PolybHb solutions, PolybHb(40:1)(high) PolybHb(50:1)(high), did not elicit vasoconstriction at all concentrations studied, whereas PolybHb(50:1)(high) only elicited moderate hypertension at the highest concentration studied. In contrast, infusion of PolybHb solutions with molecular mass <500 kDa elicited significant hypertension and vasoconstriction compared with PolybHb solutions with molecular mass >500 kDa that was proportional to the plasma concentration and inversely proportional to the PolybHb cross-link density. We present promising results for highly cross-linked T-state PolybHb solutions with molecular mass >500 kDa [PolybHb(40:1)(high) PolybHb(50:1)(high)], which supports the concept that HBOC size/molecular mass influences its proximity to the vascular endothelium and molecular diffusivity. The hemodynamics of HBOC within the plasma layer surrounding the abluminal side endothelium regulates NO production and consumption, vessel oxygen flux, and extravasation. Although mechanistically attractive, neither of these hypotheses can be directly tested in vivo and will require further investigation.

The laser-assisted neck lift: modifications in technique and postoperative care to improve results.

BACKGROUND: Conventional submental tumescent liposuction has proved disappointing for some patients with anterior neck laxity, ptotic platysma muscles, and increased subplatysmal fat. Many of these patients are facelift candidates but are unwilling to undergo this extensive procedure. We describe our hybrid approach, which offers consistently improved results and enhanced patient satisfaction. OBJECTIVE: To establish a sharper cervicomental angle by more completely removing subplatysmal fat. We also wished to achieve more consistent, smoother results, minimizing ripples, folds, and hematomas with a novel postoperative dressing system. METHODS: Extensive tumescent liposuction of the lower face, jowls, and anterior neck was performed. Following this, subplatysmal fat was removed by dissection, the platysma muscle was imbricated, and the CO2 laser utilized in a defocused, low-power mode to partially treat the dermal undersurface and underlying muscle. Our postoperative dressing included a 10 cm mineral oil polymer gel disc in the submental location, covered by tape, silicone foam, and a lower face and neck garment to provide both support and even compression over the entire neck for at least the first 24 hours. RESULTS: Results proved uniformly satisfying for most patients, even those in their senior years. Benefits included an improved cervicomental angle, a decrease in jowling, and a marked reduction in the laxity and wrinkling of the neck skin and horizontal neck creases. Problems related to postoperative rippling or folding of the redraped skin and hematoma formation were minimized. conclusion. Laser neck tightening combined with tumescent liposuction and an advanced postoperative dressing for superior support and uniform compression has resulted in consistently excellent outcomes with improved patient satisfaction.