Computer controlled expansion of equine cord blood mesenchymal stromal cells on microcarriers in 3 L vertical-wheel® bioreactors.

Mesenchymal stromal cells (MSCs) are an ideal cell source for allogenic cell therapy due to their immunomodulatory and differentiation properties. Equine MSCs (eMSCs) have been found to be a promising treatment for equine joint injuries including meniscal injuries, cartilage degradation, and osteoarthritis. Although the use of eMSCs has shown efficacy in preliminary studies, challenges associated with biomanufacturing remain. To achieve the required cell numbers for clinical application, bioreactor-based processes are required. Initial studies have shown that eMSCs can be cultivated in microcarrier-based, stirred suspension bioreactor culture at the laboratory 0.1 L scale using a Vertical-Wheel® (VW) bioreactor. However, investigations regarding scale up of these processes to the required biomanufacturing scales are required. This study investigated the scale-up of a equine cord blood MSC (eCB-MSC) bioprocess in VW bioreactors at three scales. This included scale-up from the 0.1-0.5 L bioreactor, scale-up from static culture to the 3 L computer-controlled bioreactor, and scale-up into the 3 L computer-controlled bioreactor using a mock clinical trial process. Results from the various scale-up experiments demonstrated similar cell expansion at the various tested scales. The 3 L computer-controlled system resulted in a final cell densities of 1.5 × 105 cells/cm2 on average, achieving 1.5 × 109 harvested cells. Biological testing of the cells showed that cell phenotype and functionality were maintained after scale-up. These findings demonstrate the scalability of an eCB-MSC bioprocess using microcarriers in VW bioreactors to achieve clinically relevant cell numbers, a critical step to translate MSC treatments from research to clinical applications. This study also represents the first known published study expanding any cell type in the 3 L VW bioreactor.

Proton Density MRI Increases Detection of Cervical Spinal Cord Multiple Sclerosis Lesions Compared with T2-Weighted Fast Spin-Echo.

BACKGROUND AND PURPOSE: There is a paucity of literature that supports the Consortium of Multiple Sclerosis Centers guideline that proton density MR imaging is a core spinal cord sequence. We hypothesized that proton density fast spin-echo imaging is superior to T2 fast spin-echo MR imaging for the detection of cervical cord MS lesions. This study compared the detection rate and conspicuity of cervical cord MS lesions on sagittal 1.5T proton density fast spin-echo and T2 fast spin-echo MR imaging. MATERIALS AND METHODS: One hundred consecutive patients with MS imaged with 1.5T sagittal proton density fast spin-echo and T2 fast spin-echo cervical cord MR imaging between September 2012 and October 2013 were retrospectively included. The number of MS lesions detected on each sequence was recorded; conspicuity was assessed quantitatively with the lesion-to-cord contrast ratio and lesion-contrast-to-noise ratio. Statistical analysis was performed by using the Wilcoxon signed rank test. RESULTS: Seventy-eight patients had MS cord lesions detected. Proton density fast spin-echo imaging detected a greater number of lesions (n = 181) compared with T2 fast spin-echo imaging (n = 137, P < .001). Fifteen patients (19%) with abnormal findings on proton density fast spin-echo imaging had normal findings on T2 fast spin-echo imaging; no patient with abnormal T2 fast spin-echo imaging findings had normal proton density fast spin-echo imaging findings. Although proton density fast spin-echo and T2 fast spin-echo imaging had similar lesion-to-cord contrast ratios (proton density fast spin-echo, 0.32 ± 0.01, versus T2 fast spin-echo, 0.33 ± 0.01; P = .43), proton density fast spin-echo had greater lesion-contrast-to-noise ratio (proton density fast spin-echo, 82 ± 3.0, versus T2 fast spin-echo, 64 ± 2.6; P < .001). CONCLUSIONS: Proton density fast spin-echo imaging is superior to T2 fast spin-echo MR imaging for the detection of cervical cord MS lesions. Proton density fast spin-echo detects cord lesions in patients in whom T2 fast spin-echo findings appear normal. This study forms the evidentiary base for the current Consortium of Multiple Sclerosis Centers guideline that proton density imaging is a core spinal cord sequence.

Plasma adenosine deaminase isoform 2 in cancer patients undergoing chemotherapy.

Adenosine deaminase (AD), a purine salvage enzyme, exists as AD isoform 1 (AD1) and AD isoform 2 (AD2). Plasma AD has been advocated for the screening and monitoring of cancer, as AD2 activity is increased in conditions associated with tumour growth. Plasma AD2 was measured before and seven to 10 days after the first dose of chemotherapy in patients with different tumours. A 'tumour regression score' was assessed independently based on radiological changes seen in the tumour following completion of chemotherapy. Changes in plasma AD2 were then compared with the tumour regression score. Following first-dose chemotherapy, plasma AD2 decreased on average from 22.7 +/- 10.5 U/L to 15.0 +/- 4.6 U/L. The percentage decrease in plasma AD2 correlated with the tumour regression score (r=0.5, P=0.028). These data suggest plasma AD2 may have a role in determining tumour response to treatment.

Guanosine deaminase in human serum and tissue extracts--a reappraisal of the products.

Of the human salvage enzymes that deaminate ribonucleosides, two--cytidine deaminase and adenosine deaminase--have been found particularly useful for diagnostic purposes. In humans, no enzymes are present that can directly deaminate the bases of these ribonucleosides. Indeed, the only enzyme present that can directly deaminate a base is guanine deaminase, and the diagnostic usefulness of this enzyme has been well documented. The aim of this study is to identify the origin of the ammonia formed when human sera and tissue extracts are incubated with buffered guanosine, and to clarify whether the ammonia comes from the deamination of guanosine by guanosine deaminase or is produced as a result of deamination of guanine formed as a breakdown product of guanosine by purine nucleoside phosphorylase (PNP). Apparent deamination of guanosine by guanosine deaminase in human sera and tissue extracts was found to be due to two enzymes acting in tandem when the products of the reaction were examined by HPLC. The ribose was first removed from guanosine by PNP to form guanine, which was then deaminated to xanthine by guanine deaminase.

Do active cerebral neurons really use lactate rather than glucose?

Glucose has long been considered the substrate for neuronal energy metabolism in the brain. Recently, an alternative explanation of energy metabolism in the active brain, the astrocyte-neuron lactate shuttle hypothesis, has received attention. It suggests that during neural activity energy needs in glia are met by anaerobic glycolysis, whereas neuronal metabolism is fueled by lactate released from glia. In this article, we critically examine the evidence supporting this hypothesis and explain, from the perspective of enzyme kinetics and substrate availability, why neurons probably use ambient glucose, and not glial-derived lactate, as the major substrate during activity.

Comparison of glucose and lactate as substrates during NMDA-induced activation of hippocampal slices.

It has been postulated that lactate released from astrocytes may be the preferred metabolic substrate for neurons, particularly during intense neuronal activity (the astrocyte-neuron lactate shuttle hypothesis). We examined this hypothesis by exposing rat hippocampal slices to artificial cerebrospinal fluid containing either glucose or lactate and either N-methyl-D-aspartate, which activates neurons without stimulating astrocytic glucose uptake, or alpha-cyano-4-hydroxycinnamate, which blocks monocarboxylate transport across plasma and mitochondrial membranes. When exposed to N-methyl-D-aspartate, slices lost synaptic transmission and K+ homeostasis more slowly in glucose-containing artificial cerebrospinal fluid than in lactate-containing artificial cerebrospinal fluid. After N-methyl-D-aspartate exposure, slices recovered synaptic transmission more completely in glucose. These results suggest that hippocampal neurons can use glucose more effectively than lactate when energy demand is high. In experiments with alpha-cyano-4-hydroxycinnamate, 500 microM alpha-cyano-4-hydroxycinnamate caused loss of K+ homeostasis and synaptic transmission in hippocampal slices during normoxia. When 200 microM alpha-cyano-4-hydroxycinnamate was used, synaptic activity and intracellular pH in slices decreased significantly during normoxia. These results suggest that alpha-cyano-4-hydroxycinnamate may have blocked mitochondrial oxidative metabolism along with lactate transport. Thus, studies using alpha-cyano-4-hydroxycinnamate to demonstrate the presence of a lactate shuttle in the brain tissue may need reevaluation. Our findings, together with observations in the literature that (1) glucose is available to neurons during activation, (2) heightened energy demand rapidly activates glycolysis in neurons, and (3) activation of glycolysis suppresses lactate utilization, suggests that glucose is the primary substrate for neurons during neuronal activation and do not support the astrocyte-neuron lactate shuttle hypothesis.

Rat hippocampal slices need bicarbonate for the recovery of synaptic transmission after anoxia.

The purpose of this study was to see how the nominal removal of bicarbonate (HCO(-)(3)) from the extracellular space of brain tissue influenced recovery of brain tissue from anoxia. Removal of HCO(-)(3) in HEPES-buffered artificial cerebrospinal fluid (aCSF) inhibited almost completely recovery of synaptic transmission in hippocampal slices after anoxia. Altered pH did not contribute to this finding because adjusting intracellular (pH(i)) and extracellular (pH(o)) pH to control levels did not reduce the effect of HCO(-)(3) removal. Our results suggest that HCO(-)(3) levels are important in determining the extent of anoxic or ischemic brain injury.

Non-ophthalmologist screening for retinopathy of prematurity.

AIM: To determine if a non-ophthalmologist can accurately screen for retinopathy of prematurity (ROP) by evaluating the posterior pole blood vessels of the retina. ROP is a common ocular disorder of premature infants and may require multiple screening examinations by an ophthalmologist to allow for timely intervention. Since there is a strong correlation between posterior pole vascular abnormalities and vision threatening ROP, screening examinations performed by non-ophthalmologist may yield useful clinical information in high risk infants. METHODS: Infants born at the Medical University of South Carolina who met screening criteria (n = 142) were examined by a single non-ophthalmologist using a direct ophthalmoscope to evaluate the posterior pole blood vessels for abnormalities of the venules and/or arterioles. To determine the accuracy of the non-ophthalmologist's clinical observations, infants were also examined by an ophthalmologist, using an indirect ophthalmoscope, who graded the posterior pole vessels as normal, dilated venules, or dilated and tortuous venules and arterioles (including "plus disease"). RESULTS: There was significant correlation (p <0.001) between the non-ophthalmologist's and ophthalmologist's diagnoses of posterior pole vascular abnormalities. 47 infants had normal posterior pole blood vessels by the non-ophthalmologist examination. Of these, 31 (66%) were considered to have normal vessels and 16 (34%) to have dilated venules by the ophthalmologist. The non-ophthalmologist correctly identified abnormal posterior pole vessels in all 21 infants diagnosed with abnormal arterioles and venules by the ophthalmologist. No infants with clinically important ROP ("prethreshold" or worse) would have failed detection by this screening method. CONCLUSION: Using a direct ophthalmoscope, a non-ophthalmologist can screen premature infants at risk for ROP by evaluating the posterior pole blood vessels of the retina. While not necessarily recommended for routine clinical practice, this technique may nevertheless be of value to those situations where ophthalmological consultation is unavailable or difficult to obtain.

Using hippocampal slices to study how aging alters ion regulation in brain tissue.

Aging alters ion regulation in brain tissue. This article describes methods useful for studying such age-related changes in the rat hippocampal slice preparation. Topics considered include (a) selection of appropriate age groups of rats for aging studies, (b) a description of methods for preparing and maintaining hippocampal slices, (c) measurement of intracellular pH with the H+-sensitive dye carboxy-SNARF-1, and (d) measurement of extracellular pH and K+ with cation-selective microelectrodes.

Solution structures of apo and holo biotinyl domains from acetyl coenzyme A carboxylase of Escherichia coli determined by triple-resonance nuclear magnetic resonance spectroscopy.

A subgene encoding the 87 C-terminal amino acids of the biotinyl carboxy carrier protein (BCCP) from the acetyl CoA carboxylase of Escherichia coli was overexpressed and the apoprotein biotinylated in vitro. The structures of both the apo and holo forms of the biotinyl domain were determined by means of multidimensional NMR spectroscopy. That of the holo domain was well-defined, except for the 10 N-terminal residues, which form part of the flexible linker between the biotinyl and subunit-binding domains of BCCP. In agreement with X-ray crystallographic studies [Athappilly, F. K., and Hendrickson, W. A. (1995) Structure 3, 1407-1419], the structure comprises a flattened beta-barrel composed of two four-stranded beta-sheets with a 2-fold axis of quasi-symmetry and the biotinyl-lysine residue displayed in an exposed beta-turn on the side of the protein opposite from the N- and C-terminal residues. The biotin group is immobilized on the protein surface, with the ureido ring held down by interactions with a protruding polypeptide "thumb" formed by residues 94-101. However, at the site of carboxylation, no evidence could be found in solution for the predicted hydrogen bond between the main chain O of Thr94 and the ureido HN1'. The structure of the apo domain is essentially identical, although the packing of side chains is more favorable in the holo domain, and this may be reflected in differences in the dynamics of the two forms. The thumb region appears to be lacking in almost all other biotinyl domain sequences, and it may be that the immobilization of the biotinyl-lysine residue in the biotinyl domain of BCCP is an unusual requirement, needed for the catalytic reaction of acetyl CoA carboxylase.

Aging and the effects of MK-801 on anoxic damage in rat hippocampal slices.

We examined whether age-related differences in N-methyl-D-aspartate (NMDA) receptor-mediated neurotoxicity contribute to the increased vulnerability of the aged brain to anoxic damage. In both adult and aged hippocampal slices, NMDA receptor blockade with MK-801 did not affect the onset of anoxic depolarization. MK-801 improved the postanoxic recovery of synaptic transmission by the same percentage in both age groups. Thus, the faster onset of anoxic depolarization and diminished postanoxic recovery of synaptic transmission seen in aged hippocampal slices cannot be attributed to age-related differences in NMDA receptor-mediated neurotoxicity.

The influence of glucose on intracellular and extracellular pH in rat hippocampal slices during and after anoxia.

In this study we investigated in rat hippocampal slices (1) how glucose availability affected tissue acidosis during and after anoxia, (2) whether the onset of anoxic depolarization was associated with a specific pH, (3) whether glycolysis was the major source of acidification before and during anoxic depolarization, and (4) whether improved recovery of synaptic function with elevated glucose levels was related to changes in tissue acidosis. Intracellular pH (pHi) and extracellular pH (pHo) were measured simultaneously before, during, and after anoxia in hippocampal slices bathed in 0, 5, 10, and 15 mM glucose. Slices exposed to 0 mM glucose were given 20 mM sodium lactate as a metabolic substrate. We found that the pHi and pHo at which anoxic depolarization occurred depended upon glucose concentration. We also found that elevated glucose availability increased acidification in both the intracellular and extracellular compartments during anoxia and delayed recovery of pH homeostasis after anoxia. Our results suggest that glycolysis is the primary source of acidosis before the onset of anoxic depolarization, but not during anoxic depolarization. Our results also suggest that moderate increases in acidosis resulting from increased glycolysis are potentially beneficial for anoxic survival.

The pH buffering capacity of hippocampal slices from young adult and aged rats.

We examined the hypothesis that aging alters the capacity of brain tissue to buffer intracellular pH changes. Intracellular buffering power was determined in hippocampal slices from young adult and aged rats by raising the partial pressure of CO2. Changes in intracellular and extracellular pH in response to increases and decreases in CO2 were measured simultaneously with spectrophotometry and pH-sensitive microelectrodes. The intrinsic buffering power did not differ between young adult (25.6 +/- 11.1 mM) and aged (28.2 +/- 8.6 mM) slices. However, the bicarbonate buffering power was higher in young adult slices (72.52 +/- 5.07 mM) compared with aged slices (58.67 +/- 5.78 mM) since the resting intracellular pH was about 0.1 unit lower in aged slices. This decreased bicarbonate buffering capacity may contribute to the increased vulnerability of the aged brain to metabolic stress.

The influence of age of pH regulation in hippocampal slices before, during, and after anoxia.

Changes in intracellular and extracellular pH may influence the vulnerability of brain tissue to anoxic or ischemic damage. In the present study, we investigated whether the increased vulnerability of aged brain tissue to anoxic damage is associated with age-related alterations in pH regulation. We obtained evidence for altered pH regulation by measuring concurrent changes in intracellular and extracellular pH before, during, and after anoxia in hippocampal slices from young adult (6-8 months old) and aged (24-27 months old) rats. We found indications of impaired pH regulation in aged hippocampal slices (a) before anoxia, as seen in a lower resting intracellular pH, (b) during anoxia, as seen in a slower decline in extracellular pH, and (c) during recovery after anoxia, as seen in a slower rate of recovery of intracellular pH. Age-related changes in pH regulation may contribute to the faster onset of anoxic depolarization in aged brain tissue during anoxia.

Three-dimensional structure of the lipoyl domain from the dihydrolipoyl succinyltransferase component of the 2-oxoglutarate dehydrogenase multienzyme complex of Escherichia coli.

A sub-gene encoding the lipoyl domain of the dihydrolipoyl succinyltransferase polypeptide chain of the 2-oxoglutarate dehydrogenase multienzyme complex of Escherichia coli was over-expressed and the protein was purified uniformly labelled with 15N. The three-dimensional structure of the domain was determined by means of nuclear magnetic resonance spectroscopy, based on 905 nuclear Overhauser effect inter-proton distance restraints, 42 phi torsion angle restraints and hydrogen bond restraints from 24 slowly exchanging amide protons. The structure of the 80-residue domain is that of a flattened beta-barrel surrounding a hydrophobic core in which Trp22 plays a central role in anchoring two four-stranded sheets together. The polypeptide backbone exhibits a 2-fold axis of quasi-symmetry, with the lipoylation site, Lys43, located at the tip of an exposed beta-turn in one beta-sheet and the N and C-terminal residues close together in space in the other beta-sheet. The atomic r.m.s. distribution about the mean coordinate is 0.46 A for the backbone atoms in the highly structured region and 0.88 A along the entire backbone (residues Ser1 to Asn80), including a less well-defined surface loop and the lipoyl-lysine beta-turn. The structure closely resembles that of the lipoyl domains from pyruvate dehydrogenase complexes, in accord with the existence of strongly conserved residues at critical positions in the domains. The structures of the lipoyl domains throw light on the requirements for the specificity of reductive acylation of their pendant lipoyl groups in the parent 2-oxo acid dehydrogenase complexes; an important aspect of the mechanisms underlying active site coupling and substrate channelling.

Aging impairs regulation of intracellular pH in rat hippocampal slices.

We investigated whether aging changed H+ homeostasis in hippocampal slices bathed in HEPES buffer. Intracellular pH in hippocampal slices from rats aged 26-27 months (7.06 +/- 0.02) was significantly lower compared with that in slices from rats aged 6-7 months (7.16 +/- 0.04). Age did not influence extracellular ph. Age-related reductions in intracellular pH may reflect altered pH regulation that potentially affects brain function and could contribute to the increased vulnerability of the aged brain to metabolic stress.

Surgery for vertical head position in null point nystagmus.

PURPOSE: We applied the general concepts of Anderson and Kestenbaum to the management of patients with chin up or chin down head posture caused by vertical or horizontal nystagmus. METHODS: We reviewed retrospective charts of seven patients with vertical plane torticollis who had surgical management of null point nystagmus. RESULTS: Three patients with chin down head posture underwent successful treatment using large recessions of the superior rectus muscles combined with inferior oblique muscle anterior transposition. The four patients with chin up head posture were more resistant to surgical treatment. Two of the four required multiple surgical procedures for adequate correction, but had residual torticollis. CONCLUSION: Bilateral superior rectus muscle recession combined with inferior oblique muscle anterior transposition is effective surgical management of vertical plane torticollis with chin down head posture caused by null point nystagmus. Although the treatment of patients with chin up head posture was less successful, surgically induced down-gaze deficits may be necessary to improve outcome.

Mechanized anterior capsulectomy as an alternative to manual capsulorhexis in children undergoing intraocular lens implantation.

BACKGROUND: Although manual capsulorhexis is an ideal anterior capsulectomy technique for adults, it is more difficult to perform in very young eyes because the capsular bag is more elastic. Our mechanized anterior capsulectomy technique has compared favorably with manual capsulorhexis in the laboratory using autopsy eyes. We now report the results of circular mechanized anterior capsulectomy in consecutive pediatric patients receiving a posterior chamber intraocular lens (IOL) after cataract extraction. METHODS: Twenty eyes of 17 consecutive patients between 6 months and 13 years of age underwent a mechanized anterior capsulectomy followed by IOL implantation into the capsular bag. The integrity of the anterior capsular edge was evaluated at the completion of capsulectomy, prior to IOL insertion, and at the end of surgery. RESULTS: A single radial tear developed in three eyes (15%) of two patients (12%). In the remaining 17 eyes (85%), an intact circular capsulectomy edge was verified at the completion of the mechanized capsulectomy, at the end of IOL insertion, and at the completion of surgery. Complete in-the-bag capsular fixation of the IOL was accomplished in all 20 eyes. Both patients who sustained radial tears were older children (age 11 years). All IOLs remain well centered behind a smooth circular anterior capsular edge after a minimum follow up of 6 months. CONCLUSION: A mechanized anterior capsulectomy technique can produce a circular capsular opening that resists tearing during lens aspiration and IOL insertion. The vitrector-cut capsulectomy performed well even in the youngest patients in whom manual capsulorhexis would have been difficult to control.