Beta oscillations in cortical-basal ganglia (BG) circuits have been implicated in normal movement suppression and motor impairment in Parkinson’s disease. To dissect the functional correlates of these rhythms we compared neural activity during four distinct variants of a cued choice task in rats. Brief beta (~20 Hz) oscillations occurred simultaneously throughout the cortical-BG network, both spontaneously and at precise moments of task performance. Beta phase was rapidly reset in response to salient cues, yet increases in beta power were not rigidly linked to cues, movements, or movement suppression. Rather, beta power was enhanced after cues were used to determine motor output. We suggest that beta oscillations reflect a postdecision stabilized state of cortical-BG networks, which normally reduces interference from alternative potential actions. The abnormally strong beta seen in Parkinson’s Disease may reflect overstabilization of these networks, producing pathological persistence of the current motor state.
Measurement of red blood cell (RBC) survival (RCS) is important for investigating pathophysiology and treatment of anemia. Our objective was to validate the multidensity biotin method for RCS determination in sheep, a commonly used model of RBC physiology. [14C]Cyanate served as the reference method for long-term RCS because the 51Cr method (the reference method for humans) is not reliable in sheep.
STUDY DESIGN AND METHODS
Aliquots of autologous RBCs from eight adult sheep were labeled with [14C]cyanate and four separate densities of biotin (BioRBCs) and reinfused. Short-term RCS was assessed by posttransfusion recovery at 24 hours (PTR24); long-term RCS was assessed by the time to 50% survival (T50) and mean potential life span (MPL).
Values for PTR24 of the four BioRBC densities were not different. Values for RCS as reflected by T50 and MPL were nearly identical for [14C]cyanate and the two intermediate-density BioRBC populations. In contrast, the lowest-density BioRBC population survived slightly longer (p < 0.01), but with a difference of no clinical significance. The highest-density BioRBC population importantly shortened RCS (p < 0.01 compared to the two intermediate densities).
This study provides evidence that BioRBCs labeled at four biotin densities can be used to independently and simultaneously measure short-term RCS and that BioRBCs labeled at the three lowest biotin densities can be used to accurately and simultaneously measure long-term RCS. Because the sheep RBC model is comparable to humans, this nonradioactive method has promise for use in RBC kinetic studies in neonates and pregnant women.
The Akita mouse is a robust model of diabetic autonomic neuropathy which develops severe diabetes following beta cell death, which occurs reproducibly at 3-4 weeks of age, and maintains the diabetic state without therapy for as long as 11 additional months. Neuritic dystrophy and neuronopathy involving prevertebral sympathetic superior mesenteric and celiac ganglia begin to develop within the first two months of onset of diabetes and are progressive with time. We have examined the effect of insulin implants resulting in normoglycemia and injections of ARA290, a small erythropoietin peptide which has no effect on glycemic parameters, on the reversal of established neuritic dystrophy and neuronopathy. We have found that 4 weeks of insulin therapy beginning at 2 months of diabetes resulted in normalization of blood glucose, body weight and HbA1c. Insulin therapy successfully reversed established neuritic dystrophy and neuronopathy to control levels. Numbers of sympathetic neurons were not significantly changed in either 3 month diabetic or insulin treated Akita mice. Treatment with ARA290 for 7 weeks beginning at 4 months of diabetes did not result in altered metabolic severity of diabetes as measured by blood glucose, body weight or HbA1c levels. ARA290 treatment was able to decrease neuritic dystrophy by 55-74% compared to untreated diabetics or in comparison to a separate group of diabetic animals representing the 4 month treatment onset point. Surprisingly, there was no effect of ARA290 on ganglionic neuron number or ongoing neuronopathy (pale/degenerating neurons) in diabetic Akita mice during this time period. The development of neuroprotective EPO-like peptides may provide a possible future therapy for this debilitating complication of diabetes; however, it appears that discrete elements may be differentially targeted by the diabetic state and may require selective therapy.
diabetes; Akita mouse; neuritic dystrophy; neuronopathy; erythropoietin; insulin; sympathetic ganglia
We describe the electronic structure and the origin of ferromagnetic exchange coupling in two new metal complexes, NN-SQ-CoIII(py)2Cat-NN (1) and NN-Ph-SQ-CoIII(py)2Cat-Ph-NN (2) (NN = nitronylnitroxide radical, Ph = 1,4-phenylene, SQ = S = 1/2 semiquinone radical, Cat = S = 0 catecholate, and py = pyridine). Near-IR electronic absorption spectroscopy for 1 and 2 reveals a low energy optical band that has been assigned as a Ψu → Ψg transition involving bonding and antibonding linear combinations of delocalized dioxolene (SQ/Cat) valence frontier molecular orbitals. The ferromagnetic exchange interaction in 1 is so strong that only the high-spin quartet state (ST = 3/2) is thermally populated at temperatures up to 300 K. The temperature-dependent magnetic susceptibility data for 2 reveals that an excited state spin doublet (ST = 1/2) is populated at higher temperatures, indicating that the phenylene spacer modulates the magnitude of the magnetic exchange. The valence delocalization within the dioxolene dyad of 2 results in ferromagnetic alignment of two localized NN radicals separated by over 22 Å. The ferromagnetic exchange in 1 and 2 results from a spin-dependent delocalization (double exchange type) process and the origin of this strong electron correlation has been understood in terms of a valence bond configuration interaction (VBCI) model. We show that ferromagnetic coupling promoted by organic mixed-valency provides keen insight into the ability of single molecules to communicate spin information over nanoscale distances. Furthermore, the strong interaction between the itinerant dioxolene electron and localized NN electron spins impacts our ability to understand the exchange interaction between delocalized electrons and pinned magnetic impurities in technologically important dilute magnetic semiconductor materials. The long correlation length (22 Å) of the itinerant electron that mediates this coupling indicates that high-spin π-delocalized organic molecules could find applications as nanoscale spin-polarized electron injectors and molecular wires.
Regulated protein degradation by the proteasome plays an essential role in the enhancement and suppression of signaling pathways in the nervous system. Proteasome-associated factors are pivotal in ensuring appropriate protein degradation, and we have previously demonstrated that alterations in one of these factors, the proteasomal deubiquitinating enzyme ubiquitin-specific protease 14 (Usp14), can lead to proteasome dysfunction and neurological disease. Recent studies in cell culture have shown that Usp14 can also stabilize the expression of over-expressed, disease-associated proteins such as tau and ataxin-3. Using Usp14-deficient axJ mice, we investigated if loss of Usp14 results in decreased levels of endogenous tau and ataxin-3 in the nervous system of mice. Although loss of Usp14 did not alter the overall neuronal levels of tau and ataxin-3, we found increased levels of phosphorylated tau that correlated with the onset of axonal varicosities in the Usp14-deficient mice. These changes in tau phosphorylation were accompanied by increased levels of activated phospho-Akt, phosphorylated MAPKs, and inactivated phospho-GSK3β. However, genetic ablation of tau did not alter any of the neurological deficits in the Usp14-deficient mice, demonstrating that increased levels of phosphorylated tau do not necessarily lead to neurological disease. Due to the widespread activation of intracellular signaling pathways induced by the loss of Usp14, a better understanding of the cellular pathways regulated by the proteasome is required before effective proteasomal-based therapies can be used to treat chronic neurological diseases.
To describe the development of progressive multifocal leukoencephalopathy (PML) in patients with rheumatoid arthritis (RA) treated with rituximab.
Clinical care for patients with rheumatologic diseases. Most were referred to academic centers for care after diagnosis (Washington University, St Louis, Missouri; Karolinska Insitute, Stockholm, Sweden; and Royal Melbourne Hospital, Melbourne, Australia) while one was cared for in a neurology practice in Dallas, Texas, with consultation by an academic neurovirologist from the University of Colorado in Denver.
Four patients developing PML in the setting of rituximab therapy for RA.
Main Outcome Measures
Clinical and pathological observations.
Four patients from an estimated population of 129 000 exposed to rituximab therapy for RA are reported in whom PML developed after administration of this drug. All were women older than 50 years, commonly with Sjögren syndrome and a history of treatment for joint disease ranging from 3 to 14 years. One case had no prior biologic and minimal immunosuppressive therapy. Progressive multifocal leukoencephalopathy presented as a progressive neurological disorder, with diagnosis confirmed by detection of JC virus DNA in the cerebrospinal fluid or brain biopsy specimen. Two patients died in less than 1 year from PML diagnosis, while 2 remain alive after treatment withdrawal. Magnetic resonance scans and tissue evaluation confirmed the frequent development of inflammatory PML during the course of the disease.
These cases suggest an increased risk, about 1 case per 25 000 individuals, of PML in patients with RA being treated with rituximab. Inflammatory PML may occur in this setting even while CD20 counts remain low.
Neuronal oscillations allow for temporal segmentation of neuronal spikes. Interdependent oscillators can integrate multiple layers of information. We examined phase-phase coupling of theta and gamma oscillators in the CA1 region of rat hippocampus during maze exploration and REM sleep. Hippocampal theta waves were asymmetric, and estimation of the spatial position of the animal was improved by identifying the waveform-based phase of spiking, compared to traditional methods used for phase estimation. Using the waveform-based theta phase, 3 distinct gamma bands were identified: slow gammaS (30-50 Hz), mid-frequency gammaM (50-90 Hz) and fast gammaF (90-150 Hz or epsilon band). The amplitude of each sub-band was modulated by the theta phase. In addition, we found reliable phase-phase coupling between theta and both gammaS and gammaM but not gammaF oscillators. We suggest that cross-frequency phase coupling can support multiple time-scale control of neuronal spikes within and across structures.
Safe, accurate methods permitting simultaneous and/or repeated measurement of red blood cell (RBC) survival (RCS) are important to investigate pathophysiology and therapy of anemia. Methods using chromium 51 (51Cr) -labeled RBCs are unacceptable for infants, children, and pregnant women. We report RCS measured in vivo using RBCs labeled with several densities of biotin (BioRBCs).
STUDY DESIGN AND METHODS
Aliquots of autologous RBCs from eight healthy adult subjects were labeled separately at four discrete biotin densities, mixed, and infused. The proportion of each population of BioRBCs circulating was determined serially by flow cytometry over 20 weeks. For each population, RCS was assessed by the following: 1) post-transfusion BioRBC recovery at 24 hour (PTR24); 2) time to decrease to 50% of the enrichment at 24 hours (T50); and 3) mean potential lifespan (MPL).
Among the four BioRBC densities, no significant differences in PTR24 were observed. T50 and MPL were similar for the two lowest BioRBC densities. In contrast, the two highest BioRBC densities demonstrated progressively decreased T50 and MPL.
RBCs labeled at four biotin densities can be used to independently and accurately measure PTR24 and two lowest biotin densities can accurately quantitate long-term RCS. This method provides a tool for investigating anemia in infants, fetuses, and pregnant women with the following advantages over the standard 51Cr method: 1) study subjects are not exposed to radiation; 2) small blood volumes (e.g., 20 μL) are required; and 3) multiple independent RCS measurements can be made simultaneously in the same individual.
Correlation of diffusion tensor imaging (DTI) with histochemical staining for demyelination and axonal damage in multiple sclerosis (MS) ex vivo human cervical spinal cords.
In MS, demyelination, axonal degeneration, and inflammation contribute to disease pathogenesis to variable degrees. Based upon in vivo animal studies with acute injury and histopathologic correlation, we hypothesized that DTI can differentiate between axonal and myelin pathologies within humans.
DTI was performed at 4.7 Tesla on 9 MS and 5 normal control fixed cervical spinal cord blocks following autopsy. Sections were then stained for Luxol fast blue (LFB), Bielschowsky silver, and hematoxylin and eosin (H&E). Regions of interest (ROIs) were graded semi-quantitatively as normal myelination, mild (<50%) demyelination, or moderate-severe (>50%) demyelination. Corresponding axonal counts were manually determined on Bielschowsky silver. ROIs were mapped to co-registered DTI parameter slices. DTI parameters evaluated included standard quantitative assessments of apparent diffusion coefficient (ADC), relative anisotropy (RA), axial diffusivity and radial diffusivity. Statistical correlations were made between histochemical gradings and DTI parameters using linear mixed models. Results: Within ROIs in MS subjects, increased radial diffusivity distinguished worsening severities of demyelination. Relative anisotropy was decreased in the setting of moderate-severe demyelination compared to normal areas and areas of mild demyelination. Radial diffusivity, ADC, and RA became increasingly altered within quartiles of worsening axonal counts. Axial diffusivity did not correlate with axonal density (p=0.091).
Increased radial diffusivity can serve as a surrogate for demyelination. However, radial diffusivity was also altered with axon injury, suggesting that this measure is not pathologically specific within chronic human MS tissue. We propose that radial diffusivity can serve as a marker of overall tissue integrity within chronic MS lesions. This study provides pathologic foundation for on-going in vivo DTI studies in MS.
Multiple Sclerosis; Diffusion Tensor Imaging; Post mortum; Spinal cord
Mitochondrial dysfunction is a common cause of peripheral neuropathies. While the role of neuron and axonal mitochondria in peripheral nerve disease is well appreciated, whether Schwann cell (SC) mitochondrial deficits contribute to peripheral neuropathies is unclear. Here we examine how SC mitochondrial dysfunction affects axonal survival and contributes to the decline of peripheral nerve function by generating mice with SC-specific mitochondrial deficits. These mice (Tfam-SCKOs) were produced through the tissue-specific deletion of the mitochondrial transcription factor A gene (Tfam), which is essential for mitochondrial DNA (mtDNA) transcription and maintenance. Tfam-SCKOs were viable but, as they aged, they developed a progressive peripheral neuropathy characterized by nerve conduction abnormalities as well as extensive muscle denervation. Morphological examination of Tfam-SCKO nerves revealed early preferential loss of small unmyelinated fibers followed by prominent demyelination and degeneration of larger-caliber axons. Tfam-SCKOs displayed sensory and motor deficits consistent with this pathology. Remarkably, the severe mtDNA depletion and respiratory chain abnormalities in Tfam-SCKO mice did not affect SC proliferation or survival. Mitochondrial function in SCs is therefore essential for maintenance of axonal survival and normal peripheral nerve function, suggesting that SC mitochondrial dysfunction contributes to human peripheral neuropathies.
TFAM; metabolism; mitochondrial dysfunction; axonal degeneration
Anemia is a serious problem in critically ill neonates. To investigate the pathophysiology of anemia and responses to red blood cell (RBC) transfusions and erythropoietin therapy, repeated measurement of red cell volume (RCV) and blood volume are useful. To extend our previous sheep study in which RBCs were labeled at four different biotin densities, we assessed the validity of this multidensity method for in vivo measurement of circulating RCV in humans.
STUDY DESIGN AND METHODS
In eight healthy adults, autologous RBCs were biotinylated at each of four biotin densities (6, 18, 54, and 162 µg biotinylation reagent per mL RBC), mixed, and infused intravenously; blood was sampled at 10, 20, and 60 minutes. At each time, RCV was calculated from dilution of individual RBC populations enumerated by flow cytometry. RCV measurements from the population of RBCs biotinylated at 6 µg/mL were chosen as the reference values because this density had been previously validated against the 51Cr method in vitro and in vivo in humans.
Values for RCVs were not significantly different among the four densities of biotinylated RBCs at any of the three time points and did not change over 60 minutes.
These studies provide evidence that four densities of biotinylated RBCs can be used in vivo for simultaneous, independent, accurate measurements of RCV in humans. We speculate that this method will also be useful for repeated measurement of RCV and blood volume in infants and other patient populations in whom radioactive labels should be avoided.
To investigate the pathophysiology of anemia and responses to RBC transfusions and erythropoietin, repeated measurement of the circulating red cell volume (RCV) would be useful. Ovine erythropoiesis is similar to human erythropoiesis. Accordingly, a method for measuring RCV using either human or sheep RBCs labeled at different biotin densities has been previously validated in vitro. Here preclinical studies validating this method for in vivo measurement of circulating RCV in sheep are reported.
STUDY DESIGN AND METHODS
For each sheep, autologous RBCs were biotinylated were at four discrete densities (12, 24, 48, and 96 µg biotinylation reagent per mL RBC). The densities were mixed and infused intravenously. Blood was sampled five times over one hour beginning at 4 minutes. RCV values were determined based on dilution of each population of biotinylated RBCs and by the 14C-cyanate method.
There was no difference among RCVs measured at all densities through 16 minutes; however, by 60 minutes, RBCs biotinylated at the highest density overestimated RCV by 7.6%. RCV values increased 41% over the hour, consistent with equilibration with a pool of RBCs sequestered in the spleen. RCV by the 14C-cyanate method paralleled results by the biotin method but averaged 8% greater.
These studies provide evidence that all four densities of biotinylated RBCs can be used in sheep to simultaneously and independently determine RCV. We speculate that the multidensity biotinylation method will be useful both for multiple simultaneous measurements and for repeated measurement of circulating RCV and blood volume in humans.
circulating red cell volume; blood volume; biotin; multiple densities; sheep; spleen
The effect of the presentation of similar images for distinction between benign and malignant masses on mammograms was evaluated in the observer performance study. Images of masses were obtained from the Digital Database for Screening Mammography. We selected 50 benign and 50 malignant masses by a stratified randomization method. For each case, similar images were selected based on the size of masses and the similarity measures. Radiologists were shown images with unknown masses and asked to provide their confidence level that the lesions were malignant before and after the presentation of the similar images. Eleven observers, including three attending breast radiologists, three breast imaging fellows, and five residents, participated. The average areas under the receiver operating characteristic curves without and with the presentation of the similar images were almost equivalent. However, there were many cases in which the similar images caused beneficial effects to the observers, whereas there were a small number of cases in which the similar images had detrimental effects. From a detailed analysis of the reasons for these detrimental effects, we found that the similar images would not be useful for diagnosis of rare and very difficult cases, i.e., benign-looking malignant and malignant-looking benign cases. In addition, these cases should not be included in the reference database, because radiologists would be confused by these unusual cases. The results of this study could be very important and useful for the future development and improvement of a computer-aided diagnosis system.
Similar images; computer-aided diagnosis; breast masses; mammograms
The purpose of this study was to apply an empirical mathematical model (EMM) to kinetic data acquired under a clinical protocol to determine if the sensitivity and specificity can be improved compared with qualitative BI-RADS descriptors of kinetics. 3D DCE-MRI data from 100 patients with 34 benign and 79 malignant lesions were selected for review under an Institutional Review Board (IRB)-approved protocol. The sensitivity and specificity of the delayed phase classification were 91% and 18%, respectively. The EMM was able to accurately fit these curves. There was a statistically significant difference between benign and malignant lesions for several model parameters: the uptake rate, initial slope, signal enhancement ratio, and curvature at the peak enhancement (at most P = 0.04). These results demonstrated that EMM analysis provided at least the diagnostic accuracy of the kinetic classifiers described in the BI-RADS lexicon, and offered a few key advantages. It can be used to standardize data from institutions with different dynamic protocols and can provide a more objective classification with continuous variables so that thresholds can be set to achieve desired sensitivity and specificity. This suggests that the EMM may be useful for analysis of routine clinical data.
malignant; breast; DCE-MRI; sensitivity
Par-1 is an evolutionarily conserved protein kinase required for polarity in worms, flies, frogs, and mammals. The mammalian Par-1 family consists of four members. Knockout studies of mice implicate Par-1b/MARK2/EMK in regulating fertility, immune homeostasis, learning, and memory as well as adiposity, insulin hypersensitivity, and glucose metabolism. Here, we report phenotypes of mice null for a second family member (Par-1a/MARK3/C-TAK1) that exhibit increased energy expenditure, reduced adiposity with unaltered glucose handling, and normal insulin sensitivity. Knockout mice were protected against high-fat diet-induced obesity and displayed attenuated weight gain, complete resistance to hepatic steatosis, and improved glucose handling with decreased insulin secretion. Overnight starvation led to complete hepatic glycogen depletion, associated hypoketotic hypoglycemia, increased hepatocellular autophagy, and increased glycogen synthase levels in Par-1a−/− but not in control or Par-1b−/− mice. The intercrossing of Par-1a−/− with Par-1b−/− mice revealed that at least one of the four alleles is necessary for embryonic survival. The severity of phenotypes followed a rank order, whereby the loss of one Par-1b allele in Par-1a−/− mice conveyed milder phenotypes than the loss of one Par-1a allele in Par-1b−/− mice. Thus, although Par-1a and Par-1b can compensate for one another during embryogenesis, their individual disruption gives rise to distinct metabolic phenotypes in adult mice.
We evaluated the potential utility of a newly developed liquid-crystal display (LCD), which used an independent sub-pixel drive (ISD) technique for increasing the spatial resolution of a standard LCD three times in one direction, by use of receiver operating characteristic (ROC) analysis and a two-alternative-forced-choice (2AFC) method to determine improvement in radiologists’ accuracy in the detection of clustered microcalcifications (MCLs) on digital mammograms. We used a standard LCD without and with the ISD technique, which can increase the spatial resolution of the LCD three times in one direction from three mega- to nine megapixels without changes in the size of the display. We used 60 single views of digital mammograms (30 with and 30 without clustered MCLs) for ROC studies and 60 regions of interest (ROIs) with clustered MCLs for 2AFC studies. In the ROC study, seven radiologists attempted to detect clustered MCLs without and with the ISD on the same LCD. In the 2AFC study, the same observer group compared the visibility of MCLs by use of the LCD without and with the ISD. Our institutional review board approved the use of this database and the participation of radiologists in this study. The accuracy in detecting clustered MCLs in the ROC study was improved by use of the LCD with the ISD, but the improvement was not statistically significant (p = 0.08). However, the superiority of the LCD with the ISD was demonstrated as significant (p < 0.001) in the 2AFC study. An LCD with ISD can improve the visibility of clustered MCLs when high-resolution digital mammograms are available.
Digital mammography; observer performance; display device; receiver operating characteristic curve; digital display
Accurate diagnosis of spinal cord injury (SCI) severity must be achieved before highly aggressive experimental therapies can be tested responsibly in the early phases after trauma. These studies demonstrate for the first time that axial diffusivity (λ||), derived from diffusion tensor imaging (DTI) within 3 h after SCI, accurately predicts long-term locomotor behavioral recovery in mice. Female C57BL/6 mice underwent sham laminectomy or graded contusive spinal cord injuries at the T9 vertebral level (5 groups, n = 8 for each group). In-vivo DTI examinations were performed immediately after SCI. Longitudinal measurements of hindlimb locomotor recovery were obtained using the Basso mouse scale (BMS). Injured and spared regions of ventrolateral white matter (VLWM) were reliably separated in the hyperacute phase by threshold segmentation. Measurements of λ|| were compared with histology in the hyperacute phase and 14 days after injury. The spared normal VLWM determined by hyperacute λ|| and 14-day histology correlated well (r = 0.95). A strong correlation between hindlimb locomotor function recovery and λ||-determined spared normal VLWM was also observed. The odds of significant locomotor recovery increased by 18% with each 1% increase in normal VLWM measured in the hyperacute phase (odds ratio = 1.18, p = 0.037). The capability of measuring subclinical changes in spinal cord physiology and murine genetic advantages offer an early window into the basic mechanisms of SCI that was not previously possible. Although significant obstacles must still be overcome to derive similar data in human patients, the path to clinical translation is foreseeable and achievable.
axial diffusion; diffusion tensor imaging; hyperacute; magnetic resonance imaging; recovery; spinal cord injury
The objective of the study is to report 2 new genotypic forms of protease-sensitive prionopathy (PSPr), a novel prion disease described in 2008, in 11 subjects all homozygous for valine at codon 129 of the prion protein (PrP) gene (129VV). The 2 new PSPr forms affect individuals who are either homozygous for methionine (129MM) or heterozygous for methionine/valine (129MV).
Fifteen affected subjects with 129MM, 129MV, and 129VV underwent comparative evaluation at the National Prion Disease Pathology Surveillance Center for clinical, histopathologic, immunohistochemical, genotypical, and PrP characteristics.
Disease duration (between 22 and 45 months) was significantly different in the 129VV and 129MV subjects. Most other phenotypic features along with the PrP electrophoretic profile were similar but distinguishable in the 3 129 genotypes. A major difference laid in the sensitivity to protease digestion of the disease-associated PrP, which was high in 129VV but much lower, or altogether lacking, in 129MV and 129MM. This difference prompted the substitution of the original designation with “variably protease-sensitive prionopathy” (VPSPr). None of the subjects had mutations in the PrP gene coding region.
Because all 3 129 genotypes are involved, and are associated with distinguishable phenotypes, VPSPr becomes the second sporadic prion protein disease with this feature after Creutzfeldt-Jakob disease, originally reported in 1920. However, the characteristics of the abnormal prion protein suggest that VPSPr is different from typical prion diseases, and perhaps more akin to subtypes of Gerstmann-Sträussler-Scheinker disease.
Sepsis continues to cause significant morbidity and mortality in critically ill patients. Studies of patients and animal models have revealed that changes in the immune response during sepsis play a decisive role in the outcome. Using a clinically relevant two-hit model of sepsis, i.e., cecal ligation and puncture (CLP) followed by the induction of Pseudomonas aeruginosa pneumonia, we characterized the host immune response. Second, AS101 [ammonium trichloro(dioxoethylene-o,o′)tellurate], a compound that blocks interleukin 10 (IL-10), a key mediator of immunosuppression in sepsis, was tested for its ability to reverse immunoparalysis and improve survival. Mice subjected to pneumonia following CLP had different survival rates depending upon the timing of the secondary injury. Animals challenged with P. aeruginosa at 4 days post-CLP had ∼40% survival, whereas animals challenged at 7 days had 85% survival. This improvement in survival was associated with decreased lymphocyte apoptosis, restoration of innate cell populations, increased proinflammatory cytokines, and restoration of gamma interferon (IFN-γ) production by stimulated splenocytes. These animals also showed significantly less P. aeruginosa growth from blood and bronchoalveolar lavage fluid. Importantly, AS101 improved survival after secondary injury 4 days following CLP. This increased survival was associated with many of the same findings observed in the 7-day group, i.e., restoration of IFN-γ production, increased proinflammatory cytokines, and decreased bacterial growth. Collectively, these studies demonstrate that immunosuppression following initial septic insult increases susceptibility to secondary infection. However, by 7 days post-CLP, the host's immune system has recovered sufficiently to mount an effective immune response. Modulation of the immunosuppressive phase of sepsis may aid in the development of new therapeutic strategies.
Diabetic neuropathy is a major complication of diabetes that affects the sensory and autonomic nervous systems and leads to significant morbidity and impact on quality of life of patients. Mitochondrial stress has been proposed as a major mediator of neurodegeneration in diabetes. This review briefly summarizes the nature of sensory and autonomic nerve dysfunction and presents these findings in the context of diabetes-induced nerve degeneration mediated by alterations in mitochondrial ultrastructure, physiology and trafficking. Diabetes-induced dysfunction in calcium homeostasis is discussed at length and causative associations with sub-optimal mitochondrial physiology are developed. It is clear that across a range of complications of diabetes that mitochondrial physiology is impaired, in general a reduction in electron transport chain capability is apparent. This abnormal activity may predispose mitochondria to generate elevated reactive oxygen species (ROS), although experimental proof remains lacking, but more importantly will deleteriously alter the bioenergetic status of neurons. It is proposed that the next five years of research should focus on identifying changes in mitochondrial phenotype and associated cellular impact, identifying sources of ROS in neurons and analyzing mitochondrial trafficking under diabetic conditions.
PMID: 20729997 CAMSID: cams804
calcium; dorsal root ganglia; electron transport chain; mitochondrial trafficking; reactive oxygen species; respiration; sensory polyneuropathy; sympathetic neuropathy
In this study, axial (λ∥) and radial (λ⊥) diffusivities derived from diffusion tensor imaging (DTI) were used to evaluate white matter injury in brains of mice affected by experimental autoimmune encephalomyelitis (EAE). Sixteen female C57BL/6 mice were immunized with amino acids 35-55 of myelin oligodendrocyte glycoprotein (MOG35-55). Three months after immunization, optic nerve and tract were severely affected with 19% and 18% decrease in λ∥ respectively, suggesting the presence of axonal injury. In addition, a 156% and 86% increase in λ⊥ was observed in optic nerve and tract respectively, suggestive of myelin injury. After in vivo DTI, mice were perfusion fixed and immunohistochemistry for the identification of myelin basic protein (MBP) and phosphorylated neurofilament (pNF) was performed to verify the presence of axonal and myelin injury. The present study demonstrated that the visual pathway is selectively affected in MOG35-55 induced murine EAE and these injuries are non-invasively detectable using λ∥ and λ⊥.
EAE; MOG; DTI; visual pathway; optic nerve; optic tract; mouse brain; axonal damage; myelin damage; directional diffusivity
Most pharmacodynamic (PD) models of cellular response assume a time-invariant (i.e., constant) cellular disposition despite known changes in the disposition with time, such as the reticulocyte residence time in the systemic circulation during stress erythropoiesis. To account for changes in cellular disposition, a comprehensive PD model that involves endogenous erythropoietin (Epo), reticulocytes, and hemoglobin responses was developed in phlebotomized sheep that considers a time-variant reticulocyte residence time and allows for the simultaneous determination of changes in the cellular disposition and cellular production. Five sheep were phlebotomized to hemoglobin concentrations of approximately 4 g/dl. Epo concentrations, reticulocytes, and hemoglobin concentrations were frequently sampled for 5–7 days prior to and 25–30 days following the phlebotomy. Initial steady-state conditions were assumed and the time-variant reticulocyte residence time in the systemic circulation was semiparametrically represented using a constrained spline function. Hemoglobin production was modeled using a Hill function via an effect site compartment. The initial steady state reticulocyte residence time in the systemic circulation was estimated as 0.477 (0.100) (mean (SD)) days, which maximally increased 2.01- to 2.64-fold higher than the initial steady-state residence time 5.95 (0.899) days post-phlebotomy (P < 0.01). On average, the residence time returned to steady-state values 15.4 (2.36) days post-phlebotomy, which was not significantly different from the initial steady-state value (P > 0.05). The baseline hemoglobin production rate was estimated at 0.0929 (0.0472) g/kg/day and the maximum production rate under stress phlebotomy was estimated at 0.504 (0.0422) g/kg/day. These data indicate that endogenously released Epo under acute anemic conditions can increase hemoglobin production approximately 5-fold. The determined increase in reticulocyte residence time produced under stress erythropoiesis is similar to the commonly reported 2- to 3-fold increase observed in human patients.
Reticulocyte maturation; Erythropoiesis; Hemapoietic cells; Hemoglobin production; Anemia; Time varient kinetics; Erythropoietin; Cellular transformations; Endogenous pharmacodynamics
The feasibility, efficacy, and safety of transfusing stored allogeneic RBCs has been demonstrated for small-volume transfusions given to infants. We measured the posttransfusion recovery and intravascular survival of allogeneic RBCs stored up to 42 days to further elucidate their efficacy.
Study Design and Methods
Preterm infants were transfused with 1.0 mL of biotinylated RBCs plus 15 mL per kg of unlabeled allogeneic RBCs. Posttransfusion infant blood samples obtained at 10 minutes, and at 1, 2, 4, 7, 10, 14, and 21 days were used to determine the 24-hour posttransfusion recovery (PTR24), mean potential life span (MPL), and time to disappearance of 50 percent of biotinylated RBCs (T50).
No significant differences were found between allogeneic RBCs stored 1 to 21 days versus 22 to 42 days for PTR24, MPL, or T50, indicating comparable posttransfusion circulation, regardless of storage age. Although MPL and T50 values in infants using biotinylated RBCs were short, compared to those expected using chromium-labeled RBCs in adults, they agreed with results reported by others using biotinylated RBCs.
Satisfactory posttransfusion RBC recovery and survival, measured with biotinylated RBCs, support earlier clinical trials that established the efficacy and safety of stored allogeneic RBCs for small-volume transfusions given to infants. The relatively short MPL and T50 values in some infants may underestimate true survival due to ongoing erythropoiesis and infant growth with commensurate increase in blood volume during the time of RBC survival studies. Because values in infants differ from those expected using chromium-labeled RBCs in adults, and the number of posttransfusion determinations was few, additional studies are needed to define the mechanisms involved.
The establishment of effective treatment of neonatal anemia using recombinant human erythropoietin (r-HuEPO) requires a thorough understanding of the physiology and mechanism of EPO’s pharmacologic effect. The purpose of the present preclinical study in sheep was to elucidate the stimulatory effect of EPO on erythroid progenitors and their differentiation into reticulocytes useful in predicting optimal r-HuEPO dosing.
Five young adult sheep each underwent two phlebotomies spaced 4–6 weeks apart in which their hemoglobin levels were reduced from 12 g/dL to 3–4 g/dL. Endogenous EPO levels and reticulocyte counts produced in response to anemia were sampled throughout the study and analyzed using a pharmacokinetic/pharmacodynamic (PK/PD) model.
The phlebotomy-induced drop in hemoglobin resulted in a increase in EPO levels, which reached a maximum of 764 ± 55 mU/mL (mean ± %CV) in 0.5–2.6 days. The reticulocyte counts increased from baseline values of 76.9 × 103 ± 67/μL to 619 × 103 ± 30/μL in 8 days. The PK/PD analysis indicated an increased maturation time for the reticulocytes (4.88 ± 35 days) and demonstrated that the Emax model for EPO’s activation of the progenitors did not show significant effect saturation at the endogenous EPO levels reached.
In extrapolating from the animal pilot experiment, the present study provides a case for the use of higher r-HuEPO doses in human studies to determine if higher doses are more effective in treatment of neonatal anemia to reduce, and in some less severe cases, eliminate, the need for blood transfusions.
anemia of prematurity; erythropoietin; pharmacokinetic/pharmacodynamic analysis; progenitor activation; reticulocyte
To determine by pharmacokinetic (PK) means the role of erythropoietin-receptor (EPO-R) upregulation in fetuses on the elimination of erythropoietin (EPO).
Materials and Methods
Six fetal sheep were catheterized at a gestational age of 125–127 days and phlebotomized daily for 6 days. Paired tracer PK studies using recombinant human EPO (rHuEPO) were conducted in the sheep fetuses at baseline and post-phlebotomy, 7 days later. A PK model with Michaelis-Menten elimination was simultaneously fit to the PK data at baseline and post-phlebotomy for each fetus.
Daily phlebotomies reduced the hemoglobin levels from baseline values of 10.8 (5%) (mean (C.V.)) g/dl to a nadir of 4.5 (17%) g/dl post-phlebotomy. The endogenous EPO concentration rapidly increased after the first phlebotomy and remained elevated, although variable, thereafter. The Michaelis-Menten maximal rHuEPO elimination rate parameter, Vmax, was significantly greater post-phlebotomy than at baseline (p < 0.05), increasing 1.31 fold. The fetal baseline “linear” clearance at very low concentrations of rHuEPO was determined to be 117 ml/kg/h, similar to that determined in newborn sheep but 2–3 fold higher than that determined in adult sheep.
The observed increase in Vmax is consistent with an up-regulation of EPO-R due to a positive feedback resulting from the phlebotomy-induced anemia.
developmental comparison; erythropoietin receptors; fetus; pharmacokinetics; receptor regulation