Sahay, Khushboo; Department of Physiology (7/26/2018)
      Liver disease is an important health concern and a significant source of morbidity and mortality in the United States and worldwide. NEDD8 (neural-precursor-cell-expressed developmentally down-regulated 8) is a novel ubiquitin-like protein modifier. The conjugation of NEDD8 to target proteins, termed neddylation, requires NEDD8 specific E1, E2 and E3 ligases. Neddylation participates in various cellular processes. However, whether neddylation regulates liver development and function is completely unknown. We created mice with hepatocyte specific deletion of NAE1, a subunit of the only NEDD8 E1 enzyme, and identified that they display severe hepatomegaly, hypertriglyceridemia, and hypercholesterolemia from 10 days after birth. By postnatal 14 days, their liver cytoarchitecture is completely disrupted, along with formation of numerous biliary cysts, fibrosis and hypoglycemia, which ultimately result in liver failure and premature death by 6 weeks. Mechanistically, NAE1 deficiency in hepatocytes caused reduced hepatocytespecific gene expression but increased biliary/oval cell gene expression in liver. In vitro, NAE1 inhibition by MLN4924 and CRISPR/Cas9-mediated NAE1 deletion in HepG2 cells recapitulated in vivo findings with repressed expression of hepatocyte specific genes but elevated biliary/oval cell gene expression. Together, these data highlight an essential role for neddylation in regulating hepatocyte lineage commitment and function as well as polycyst formation through trans/de-differentiation of hepatocytes.
    • Needle Bore Size and The Degree of Hemolysis During Blood Specimen Acquisition

      Carr, Rebecca Lamb; Department of Physiological and Technological Nursing (1983-05)
      This study examined the degree of detectable hemolysis in- blood specimens _as-pirated with needles'p~ :small and 1.arge bore_ size. A quasiexperimental design was used to test the resea;ch hypothesis that blood . specimens. aspira~ed_~tth a 22~gauge_needle will have a higher degree of -detectable-hemolysis than blood specimens asp:irated .with a 20-gauge needle. Data collectionconsisted of aspirating from each subject - (n=31) one blood-·speci~en--wi-th a 22-gauge-needle and-one-:.blood specimen·: _ _, . ,.,. : . . . . ' . . with a 20~gauge ·needle~- .. Th~- -plasma -hemoglobin df each specimen was . . . . . . '• . mea~ured and data were 'analyzed using the independent t~test~ The - results- indicated- that the mean difference in the plasma hemogl_obin· . ' . . . . . - ·between the two blood specimens w·as not stattstically · si'gnif1cant', -t-hus ·· .. the research hypothesis was not supported .. Thefindi~gs sug\Jested that . . '·· heme lysis was not influenced by the bore si z~ of the needle us.ed during blood specimen acq-uisition.
    • Neonatal Nurses' Knowledge of Their Standards of Practice: A Reflection of Accountability

      Chadwich, Jean; Department of Nursing (1984-02)
      The purpose of this study was to examine the neonatal nurses'- knowledge of standards of nursing practice. It was post~lated the degree to which a nurse understands and accepts her professional . accou~ta.bility depend·s :upon her own understanding of her status~ The level of knowledge was obtained from the investigator's selfdesigned tool. Additionally, the study .examined··whether or not a ' . ' ' . difference existed between the knowledge of nu'rses who care for . . . infants who require varying degree~ of 'nursing care, as evidenced by the t:y-pe of hospital ·they are employed in· und~r :regionalization guide~ lines. It further examined the relationship be~ween ·the personal -· variable of type of basic nursing education with the t·otal and subscale scores on the tool. A descriptive--correlational design was ·used to examine the study data generated from· 61 registered nurses, distri~uted _among 15 hospitals in one Southeastern stat_e. Analysis of the study .data using descriptive stati.stics indicated that the neonatal nurses wereknowledgeable of their standards of practice. Pe~rson correlation coefficients revealed no significant difference exis~ed amongst the nurses in Level I,. II _and III neonatal care facilities, nor was there any correlation between the nurses' educational preparation and their level of knowledge of the standards of practice. Additionally~ Pearson correlation coefficients· and Cronhach' s alpha ·were. used to determine the statistic:al validity and r~liabi.li ty es-timates of the tool •..
    • Neuregulin1 promotes excitatory synapse development specifically in GABAergic interneurons

      Tin, Kin Lai; Department of Neuroscience and Regenerative Medicine (2010-03)
      Neuregulin 1 (NRG1) and its receptor ErbB4 are both susceptibility genes of schizophrenia. However, little is known about the underlying mechanisms of their malfunction. Although ErbB4 is enriched in GABAergic interneurons, the role of NRG1 in excitatory synapse formation in these neurons remains poorly understood. We showed that NRG1 increased both the number and size of PSD- 95 puncta in GABAergic interneurons, indicating that NRG1 stimulates the formation of new synapses and strengthens existing synapses. In contrast, NRG1 treatment had no consistent effect on either the number or size of excitatory synapses in glutamatergic neurons, suggesting its synaptogenic effect is specific to GABAergic interneurons. Ecto-ErbB4 treatment diminished both the number and size of excitatory synapses, suggesting that endogenous NRG1 may be critical for basal synapse formation. NRG1 could stimulate the stability of PSD-95 in the manner that requires tyrosine kinase activity of ErbB4. Finally, deletion of ErbB4 in parvalbumin-positive interneurons led to reduced amplitude of mEPSCs, providing in vivo evidence that ErbB4 is important in postsynaptic differentiation in interneurons. Taken together, our findings suggested a novel synaptogenic role of NRG1 in excitatory synapse development, possibly via stabilizing PSD-95, and this effect is specific to GABAergic interneurons. In light of the association of the genes of both NRG1 and ErbB4 with schizophrenia and dysfunction of GABAergic system in this disorder, these results provide insight into its potential pathological mechanism.
    • Neuregulin3 Regulation of Glutamatergic Transmission

      Figueiredo, Dwight; Department of Neuroscience and Regenerative Medicine (2015-12)
      Synapses are fundamental communication units in the brain, essential for meaningful response to stimuli received from the environment. Abnormal synaptic communication leads to mental disorders. My studies focus on Neuregulin3, a member of the Neuregulin family. Single Nucleotide Polymorphisms (SNPs) within the NRG3 gene are associated with schizophrenia in different populations. Analysis of postmortem human brain samples of schizophrenia patients revealed abnormal levels of NRG3. However, unlike its well-studied family member NRG1, NRG3’s role in synaptic transmission is not understood. I studied how depletion of Nrg3 protein in the brain could affect synaptic transmission. I measured the amplitude and the frequency of spontaneous as well as miniature Excitatory Post Synaptic Currents (sEPSC and mEPSC, respectively) at hippocampal CA1 neurons of GFAP
    • Neuro-vascular Communication in the Hypothalamic Supraoptic Nucleus in Rats. Do nitric oxide and vasopressin play a role?

      Du, Wenting; Department of Physiology (2015-03)
      The classical model of neurovascular coupling (NVC) proposes that activity-dependent synaptically released glutamate dilates arterioles. However, whether this model is also applicable to brain areas that use less conventional neurotransmitters, such as neuropeptides, is currently unknown. To this end, we studied NVC in the hypothalamic magnocellular neurosecretory system (MNS) of the supraoptic nucleus (SON), in which dendritically released vasopressin (VP) can be found. Bath-applied VP significantly constricted SON arterioles via activation of the V ia receptor subtype. Vasoconstriction was also observed in response to single VP neuronal stimulation, an effect prevented by V ia receptor blockade (V2255). Conversely, osmotically-driven magnocellular neurosecretory neuronal population activity leads to a predominant nitric oxide (NO)- mediated vasodilation. Activity-dependent vasodilation was followed by a VP-mediated vasoconstriction, which acted to reset vascular tone. Taken together, our results unveiled a unique and complex form of NVC in the MNS, supporting a competitive balance between activity-dependent dendritic released VP and NO, in the generation of proper NVC responses.
    • Neuron-derived estrogen and neural function

      Lu, Yujiao; Department of Neuroscience and Regenerative Medicine (Augusta University, 2020-05)
      17β-estradiol (E2) is produced from androgens via the action of the enzyme aromatase. E2 is known to be made in neurons in the brain, but its precise functions in the brain are unclear. We created a forebrain neuron-specific aromatase knockout (FBN-ARO-KO) mouse model to deplete neuron-derived E2 in the forebrain of mice. Under normal conditions, FBN-ARO-KO mice showed a 70-80% decrease in aromatase and forebrain E2 levels. Male and female FBN-ARO-KO mice exhibited significant deficits in forebrain spine and synaptic density, as well as hippocampal-dependent cognitive functions. Reinstating forebrain E2 levels via exogenous in vivo E2 administration was able to rescue both the molecular and behavioral defects in FBN-ARO-KO mice. Furthermore, electrophysiological study suggested normal long-term potentiation (LTP) induction, but significantly decreased amplitude in FBN-ARO-KO mice which could be fully rescued by acute E2 treatment in vitro. Mechanistic studies revealed that FBN-ARO-KO mice had compromised rapid kinase (AKT, ERK) and CREB-BDNF signaling in the hippocampus and cerebral cortex. After global cerebral ischemia (GCI), ovariectomized female FBN-ARO-KO mice had significantly attenuated aromatase and hippocampal E2 levels. Intriguingly, FBN-ARO-KO mice exhibited a robust reduction in astrocyte activation, as well as exacerbated neuronal damage and worse cognitive dysfunction after GCI. Similar results were observed in intact male mice. RNA-seq analysis revealed alterations in pathways and genes associated with astrocyte activation, neuroinflammation and oxidative stress in FBN-ARO-KO mice. The compromised astrocyte activation in FBN-ARO-KO mice was associated with robust downregulation of the astrocyte-derived neurotrophic factors, BDNF and IGF-1, as well as the astrocytic glutamate transporter, GLT-1. In vivo E2 replacement rescued the compromised reactive astrogliosis and cognitive deficits. Moreover, neuronal FGF2, which acts in a paracrine manner to suppress astrocyte activation, was dramatically increased in FBN-ARO-KO neurons. Interestingly, blocking FGF2 signaling in astrocytes by central injection of an FGFR3 antibody was able to reverse the diminishment in neuroprotective astrocyte reactivity, and attenuate neuronal damage in FBN-ARO-KO mice. Collectively, our data provides novel genetic evidence for the roles of neuron-derived E2 in regulating synaptic plasticity, cognitive function in the non-injured brain, and astrocyte activation and neuroprotection in the injured brain.
    • Neuron-Glia interaction and role of Nrf2 in hyperhomocysteinemic retina

      Navneet, Soumya; Biomedical Sciences (Augusta University, 2019-05)
      Elevated level of the excitatory amino acid homocysteine (Hcy) or hyperhomocysteinemia (Hhcy) has been reported in patients with glaucoma, a disease characterized by increased oxidative stress and retinal ganglion cell (RGC) degeneration. Whether Hhcy is causative or merely a biomarker for glaucoma is not known. Primary RGCs exhibit acute sensitivity to Hcy exposure, while in vivo murine models of Hhcy demonstrate a more modest RGC loss (∼20%) over a period of several months. This differential response to Hhcy in isolated cells and the intact retina suggests the presence a buffering mechanism invoked by the retinal milieu. Oxidative stress has been implicated as a mechanism of Hcy-induced neuronal loss. Owing to the key role of Müller glial cells (MCs) in retinal antioxidant defense we hypothesized that MCs protect RGCs under conditions of Hhcy via the NRF2 antioxidant pathway. Compared to RGCs, MCs were less sensitive to Hcy. Hcy exposure increased oxidative stress and induced apoptosis in RGCs, whereas in MCs Hcy evoked several cytoprotective responses including reduced oxidative stress, increased antioxidant levels and improved mitochondrial function. Hcy upregulated the expression of Nrf2 and several downstream antioxidant targets including glutathione in MCs. To investigate the role of NRF2 in Hcy-induced RGC degeneration, we crossed Nrf2-/- mice with two mouse models of chronic Hhcy (Cbs+/- and Mthfr+/- mice) and generated Cbs+/-Nrf2-/- and Mthfr+/-Nrf2-/- mouse models and analyzed their retinas. Absence of NRF2 reduced inner retinal thickness and visual acuity, accelerated RGC loss and increased gliosis in Hhcy mice. To understand the role of Müller glial specific NRF2 in RGC survival, we established an ex-vivo indirect co-culture system using primary RGCs and MCs. Apoptosis induced by Hcy exposure in primary RGCs were abrogated when the RGCs were co-cultured with wild type (WT) MCs but not with Nrf2-/- MCs. Hhcy induced robust mitochondrial and glycolytic response in WT MCs, but not in Nrf2-/- MCs. Altogether, the in vivo and in vitro data here suggest that the deleterious effects of Hhcy on RGCs are likely dependent upon the health of retinal glial cells and the availability of retinal antioxidant response mechanism.
    • Neuronal and astrocytic disruption during traumatic brain injury described using longitudinal imagin

      Sword, Jeremy Jon; Medical College of Georgia (Augusta University, 2012)
      In Traumatic Brain Injury (TBI) mechanical forces applied to the cranium and brain cause irreversible primary neuronal and astroglial damage associated with terminal dendritic beading and spine loss. Edema, which quickly develops after TBI, causes an increase in intracranial pressure, which can cause secondary injury in the peri-contusional area. Spreading depolarizations have also been shown to occur after TBI in humans as well as in animal models, contributing to secondary injury. However, the impact of spreading depolarizations on real-time injury to dendrites and astrocytes in the pericontusional area during edema development is unknown. We used in vivo twophoton laser scanning microscopy (2PLSM) in mouse somatosensory cortex via an optical window to monitor yellow fluorescent protein expressing neurons and enhanced green fluorescent protein expressing astrocytes in the peri-contusional area after mild TBI. Loss of capillary blood flow preceded dendritic beading, which developed slowly over the ·next several hours. This indicated that acute injury to dendrites was gated by the degree of ischemia as a result of developing edema. Astrocytes were swollen (cytotoxic edema) due to ion and 'rater uptake and remained swollen up to 24 hours. When spreading depolarizations were repeatedly induced by pressure-injecting 1 M KCI with a Picospritzer outside the imaging area, dendrites underwent rapid beading and recovery coinciding with passage of spreading depolarizations, as was confirmed with electrophysiological recordings in the vicinity of imaged dendrites. However, each s:ubsequent I spreading depolarization resulted in a smaller percentage of dendrites that were I able to fully recover until ultimately all were terminally injured. Thus our data suggests that accumulated stress resulting from spreading depolarizations expedites acute dendritic injury in the peri-contusional area, worsening secondary damage following TBI. We also propose that persistent astroglial swelling in the peri-contusional area may be harmful and decreases astroglial maintenance of normal homeostatic function and possibly contributes greatly to the negative outcome of TBI as astrocytes fail to provide neuronal support.
    • Neuronal and Astroglial Injury and Recovery from Stroke-Induced Depolarizations

      Risher, William Christopher; Department of Neurosurgery (2010-12)
    • Neuronal and astroglial injury and recovery from stroke-induced depolarizations

      Risher, William Christopher; Medical College of Georgia (Augusta University, 2010-12)
    • Neuronal death in lewy body disease

      Tompkins, Margaret Marie; Medical College of Georgia (Augusta University, 1997)
      In Parkinson's disease and other Lewy body-associated disorders, neurons in the substantia nigra pars compacta (SNpc) undergo degeneration, but the mechanism of cell death has not been previously described. The substantia nigra of normal and Alzheimer's disease cases were compared with substantia nigra from patients with Lewy bodyassociated disorders (Parkinson's disease, concomitant Alzheimer's/Parkinson's disease, and diffuse Lewy body disease) using in situ end-labeling to detect fragmented DNA. In situ end-labeled neurons demonstrated changes resembling apoptosis: nuclear condensation, chromatin fragmentation and formation of apoptotic-like bodies. Ultrastructural analysis confirmed nuclear condensation and formation of apoptotic-like bodies. Apoptotic-like changes were seen in the substantia nigra of both normal and disea·sed cases; concomitant Alzheimer's/Parkinson's disease and diffuse Lewy body disease cases had significantly higher amounts of apoptotic-like changes than normal controls or Alzheimer patients. Parkinson's disease cases were not significantly different from controls, probably due to high variation among cases of Parkinson's disease. Control and Alzheimer's disease patients had the same percentage of apoptotic-Iike changes. Lewy bodies (LBs) are abnormal inclusions found in the SNpc neurons of patients with Lewy body-associated disorders. It is not known what role LBs play in the disease process. We sought to discover whether apoptotic-like changes were more common in SNpc neurons with or without somal· LBs. In SNpc tissue from cases with Lewy body-associated disorders, .cells were double-labeled to colocalize apoptotic-like changes and LBs with in 'Situ .endlabeling and anti-ubiquitin antibody. Nigral neurons with LBs showed the same amount of apoptotic-like changes as nigral neurons without LBs. The majority of SNpc neurons undergoing apoptotic-like cell death did not appear to contain somal LBs. These results support the theory that the presence of a somal LB does not predispose a neuron to undergo apoptoticlike cell death.
    • Neuropathology in non-human primates

      Summers, James Bradley; Medical College of Georgia (Augusta University, 1997-08)
      Non-human primates develop senile plaques that are similar to those observed in humans, in addition to experiencing age-related declines in cognitive ability which may be similar to the cognitive deficits observed in elderly humans with dementia In the last decade, information regarding the biochemical properties ofbeta-amyloid, the primary constituent of senile plaques, has become available at a brisk pace. In addition, numerous proteins have been recently identified in plaques, although the role of such proteins in the pathogenesis of plaques or Alzheimer's dfsease remains unknown. Investigation of these plaque-associated proteins may offer clues to the events responsible for the accumulation ofbeta-amyloid in the brain. One protein implicated in the pathogenesis of Alzheimer's disease and other neurodegenerative diseases is ubiquitin. This protein is involved in the cytosolic, ATP-dependent degradation of abnormal and short-lived proteins in all eukaryotic cells examined thus far. Studies of humans with neurological disorders, such as Alzheimer's disease and Creutzfeldt-Jakob disease, have identified ubiquitinated neurites surrounding plaques. Although the mechanism responsible for the accumulation of ubiquitinated products within dystrophic neurites is currently unknown, it has been suggested that protein turnover in these neurons may be altered by neurotoxic beta-amyloid in the surrounding neuropil. Although numerous studies utilizing human brain tissue have demonstrated ubiquitin around plaques, very few studies in non-human primates have investigated the association ofubiquitin with plaques. To investigate ubiquitin in the brains of non-human primates, dual-label immunohistochemistry was performed on sections from 15 monkeys (age range: 4 to 32 years) using primary antibodies against beta-amyloid and ubiquitin along with fluorescent secondary antibodies. Ubiquitin was observed to be localized in the majority (range: 55.7 to 100%) of plaques in the five aged monkeys possessing these lesions. Ubiquitin immunoreactivity generally appeared as granular structures with either the same orientation as plaques or localized in the periphery. These structures appear similar to the ubiquitinated neurites reported around the plaques in patients with Alzheimer's disease and Creutzfeldt-Jakob disease. The five plaque-containing monkeys also possessed varying degrees of cerebrovascular amyloid, but only two monkeys possessed granular ubiquitin immunoreactivity in the surroun.ding neuropil adjacent to these vascular lesions. In addition, an apparent age-related accumulation of dot-like ubiquitinated material was observed in these 15 monkeys that was not associated with plaques. These results suggest that perturbations in ubiquitin-mediated protein turnover exist within some neurons of the aging monkey brain. Furthermore, similar mechanisms may be responsible for the neuronal degeneration and accumulation ofubiquitinated products within dystrophic neurites of both humans and monkeys. Another protein recently implicated in the pathogenesis of Alzheimer's disease is apolipoprotein E, a plasma protein which plays a central role in cholesterol and triglyceride metabolism throughout the body. In the nervous system, apolipoprotein Eis believed to participate in the mobilization and redistribution of lipids during development and in neurons undergoing repair or remodeling processes. A polymorphic gene on chromosome 19 codes for apolipoprotein E, and three alleles are associated with the production of three major isoforms of apolipoprotein E. Epidemiologic studies demonstrating that the frequency of the apolipoprotein E e4 allele is significantly increased in patients with Alzheimer's disease suggest that apolipoprotein E may be an important etiologic agent in the pathogenesis of Alzheimer's disease, and the localization of apolipoprotein E in the major neuropathological lesions of Alzheimer's disease further supports this hypothesis. Despite numerous studies undertaken in humans, few studies have investigated the localization of apolipoprotein E in the plaques in non-human primates. Therefore, to study apolipoproteinE in the brains of these 15 non-human primates, sections were processed for dual-label immunohistochemistry against apolipoprotein E and beta-amyloid. Also, some sections were immunostained for glial fibrillary acidic protein and beta-amyloid to investigate the orientation of astrocytes to the plaques in these monkeys. Other sections were immunostained for apolipoprotein E and glial fibrillary acidic protein to determine if astrocytes in these monkeys demonstrate apoE immunoreactivity, since astrocytes have been suggested to be the primary source of the apolipoprotein E found in the brain. Immunoreactivity for apolipoprotein E was associated with all plaques and cerebrovascular amyloid in the five aged monkeys possessing these lesions. Astrocytes immunoreactive for glial fibrillary acidic protein were generally observed in the neuropil surrounding plaques, but none of the astrocytes examined in these monkeys were immunoreactive for apolipoprotein E. These results support a role for apolipoprotein E in the development of plaques and cerebrovascular amyloid in the brains of nonhuman primates. Furthermore, the failure to demonstrate apolipoprotein E immunoreactivity in the astrocytes in these monkeys suggests that the apolipoprotein E may be principally derived from cells in the brain other than astrocytes, such as microglial cells which also known to express apolipoprotein E and have been localized within the core of plaques. In summary, this study has shown that both ubiquitin and apolipoprotein E are-important constituents of plaques, in agreement with previous studies in non-human primates. The roles ubiquitin and apolipoprotein E play in the development of plaques await elucidation, and nonhuman primates provide the opportunity to expand upon the current body of information regarding the mechanisms of these proteins in disease processes. The non-human primate is a valuable model for which to study neuropathological and cognitive changes because it is so closely related to humans, as opposed to rats and mice, and monkeys possess a broad behavioral repertoire which easily lends itself to investigation.
    • Neurotransmitter gases as modulators of GnRH and the preovulatory LH surge

      Lamar, Charisee; School of Graduate Studies (1998-04)
    • Neurotransmitter receptor binding and protein phosphorylation in the rabbit iris smooth muscle

      Taft, William C; Department of Cell and Molecular Biology (1982-06)
    • Neurotransmitters and the phospholipid effect in rabbit iris muscle

      Owen, Mary Pruitt; Department of Cell and Molecular Biology (1976-06)

      Shosha, Esraa; Department of Cellular Biology and Anatomy (2017)
      Ischemic retinopathies such as retinopathy of prematurity, central retinal artery occlusion and diabetic retinopathy are leading causes of visual impairment and blindness. These pathologies share common features of oxidative stress, activation of inflammatory pathways and neurovascular damage. There is no clinically effective treatment for these conditions because the underlying mechanisms are still not fully understood. In the current study, we used a mouse model of retinal ischemia reperfusion (I/R) insult to explore the underlying mechanisms of neurovascular degeneration in ischemic retinopathies. The arginase enzyme utilizes the L-arginine amino acid for the production of L-ornithine and urea. Here, we investigated the role of the mitochondrial arginase isoform, arginase 2 (A2) in retinal I/R induced neurovascular injury. We found that retinal I/R induced neurovascular degeneration, superoxide and nitrotyrosine formation, glial activation, cell death by necroptosis and impairment of inner retinal function in wild type (WT) mice. A2 homozygous deletion (A2-/-) significantly protected against the neurovascular degeneration after retinal I/R. That was attributed to decreased oxidative stress and glial activation. A2 deletion protected against I/R induced retinal function impairment. Using Optical coherence tomography (OCT), we evaluated the retinal structure in live animals and found that A2-/- retinas showed a more preserved structure and less retinal detachment. To investigate the underlying mechanisms of A2 induced vascular damage after I/R, we used an in vitro model of oxygen glucose deprivation/ reperfusion (OGD/R) in bovine retinal endothelial cells (BRECs). Analysis of oxidative metabolism showed impaired mitochondrial function. We also found an increase in dynamin elated protein 1 (Drp1), a mitochondrial fission marker. Mitochondria labeling studies showed fragmented mitochondria after OGD/R. Arginase inhibition reduced mitochondrial fragmentation in OGD/R insult. This dissertation presents A2 as a new therapeutic target in reducing neurovascular damage in ischemic retinopathies.
    • Neutrophil function studies with respect to antibiotic tolerant staphylococcus aureus and patients with recurring infection

      Raynor, Robert H; Depatment of Cell and Molecular Biology (1980-08)
      Polymorphonuclear neutrophils (PMNs) from s~ven patients who experien<;: ed recurring staphylococcal infections, were tested in order to establish if measurements of phagocytic and bactericidal capacity would reveal a functional cause for the repeated episodes of disease. Using a microprocedure developed for this 'purpose, the results for six of these patients were found to be comparable to normal values. PMNs from one patient, however, showed a deficiency in their ability to both phagocytize and kill s. aureus. This defect could not be attributed to serum deficiencies or ascribed to any previously defined class of nel.ltrophil function. In other studies, six clinical isolates of Staphylococcus aureus were compared for their relative susceptibilities to the killing effects of oxacillin. Three of the strains had minimum bactericidal concentrations which were >10 times the minimum bacteriostatic concentration for this antibiotic and were designated tolerant (Tol+). The other st;r-ains had minimum bactericidal concentrations which were comparable to the minimum bacteriostatic concentration (Tol-). Lysis curves of these strains revealed that the Tal+ strains exhibited a diminished rate of lysis when inhibited by oxacillin. This reduced rate of lysis was reflected also in a reduced rate of viability loss when the cells were exposed to oxacillin. Cells of each phenotype, previously labeled with [ 14C]Glycerol, secreted radioactivity when inhibited by oxacillin. However,. the Tal+ strains rele.ased over twice as much labe 1 as the Talstrains. The behavior of 60 to 65%_ of the labeled material released by. inhibited cells during both sodium dodecyl sulfate gel electrophoresis and Sepharose 6B chromatography corresponded to that of lipoteichoic acid. When the major component of secreted material was added to oxacillin-inhibited Tol- strains, an inhibition of the lytic response was observed. These results suggest that oxacillin tolerance in S. aureus could be related to the enhanced secretion of an autolysin inhibitor, such as lipoteichoic acid. Several investigators have recently reported an increase in the severity of infections due to Tol+ strains. Since the enhanced'excretion of LTA by Tol+ staphylococci is the only knownphenotypJ.c difference between these two strains, the effect of this molecule on neutrophil function was measured using the microprocedure. The addition of LTA to the incubation mix·ture res1..1lted in a dose dependent inhibition of phagocytosis •. The decrease~ uptake of. S. aureus in the presence of LTA was accompanied by a corresponding increase in the number of organisms surviving in the presence of the phagocytes. Additional studies suggested that LTA interferes with the process' of opsonization, prior to engulfment.
    • Neutrophil-mediated endothelial dysfunction

      Chen, Xilin; Department of Pharmacology and Toxicology (1992-08)