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Biomedical subjects

Terry M Phillips

Publications and source records attributed to Terry M Phillips.

16 recordsLinked to original sources
PubMed2005

Mass of the postsynaptic density and enumeration of three key molecules.

The total molecular mass of individual postsynaptic densities (PSDs) isolated from rat forebrain was measured by scanning transmission EM. PSDs had a mean diameter of 360 nm and molecular mass of 1.10 +/- 0.36 GDa. Because the mass represents the sum of the molecular masses of all of the molecules comprising a PSD, it becomes possible to derive the number of copies of each protein, once its relative mass contribution is known. Mass contributions of PSD-95, synapse-associated protein (SAP)97, and alpha-Ca2+/calmodulin-dependent protein kinase II (CaMKII) were determined by quantitative gel electrophoresis of PSD fractions. The number of PSD-95 molecules per average PSD, contributing 2.3% of the mass of the PSD, was calculated to be 300, whereas the number of SAP97 molecules, contributing 0.9% of the mass of the PSD, was 90. The alpha-CaMKII holoenzymes, which contribute 6% of the mass when brains are homogenized within 2 min of interrupting blood flow, have 80 holoenzymes associated with a typical PSD. When blood flow is interrupted 15 min before homogenization, the average mass of PSDs increases by approximately 40%. The additional alpha-CaMKII associated with PSDs accounts for up to 20% of this mass increase, representing the addition of 100-200 alpha-CaMKII holoenzymes.

Adaptor Proteins, Signal Transducing↗
PubMed2005

An in vivo microanalytical technique for measuring the local biochemical milieu of human skeletal muscle.

Myofascial pain associated with myofascial trigger points (MTrPs) is a common cause of nonarticular musculoskeletal pain. Although the presence of MTrPs can be determined by soft tissue palpation, little is known about the mechanisms and biochemical milieu associated with persistent muscle pain. A microanalytical system was developed to measure the in vivo biochemical milieu of muscle in near real time at the subnanogram level of concentration. The system includes a microdialysis needle capable of continuously collecting extremely small samples (approximately 0.5 microl) of physiological saline after exposure to the internal tissue milieu across a 105-microm-thick semi-permeable membrane. This membrane is positioned 200 microm from the tip of the needle and permits solutes of <75 kDa to diffuse across it. Three subjects were selected from each of three groups (total 9 subjects): normal (no neck pain, no MTrP); latent (no neck pain, MTrP present); active (neck pain, MTrP present). The microdialysis needle was inserted in a standardized location in the upper trapezius muscle. Due to the extremely small sample size collected by the microdialysis system, an established microanalytical laboratory, employing immunoaffinity capillary electrophoresis and capillary electrochromatography, performed analysis of selected analytes. Concentrations of protons, bradykinin, calcitonin gene-related peptide, substance P, tumor necrosis factor-alpha, interleukin-1beta, serotonin, and norepinephrine were found to be significantly higher in the active group than either of the other two groups (P < 0.01). pH was significantly lower in the active group than the other two groups (P < 0.03). In conclusion, the described microanalytical technique enables continuous sampling of extremely small quantities of substances directly from soft tissue, with minimal system perturbation and without harmful effects on subjects. The measured levels of analytes can be used to distinguish clinically distinct groups.

Bradykinin↗
PubMed2005

Importance of the surface area ratio on cytokines production by human monocytes in vitro induced by various hydroxyapatite particles.

A possible complication associated with the implantation of hydroxyapatite (HA)-based prosthesis is the release of particles. Those particles can be phagocyted by monocytes that are among the first cells to colonize the inflammatory site. The activated monocytes produce inflammatory mediators, such as cytokines, which cause osteoclasts activation. It has previously been demonstrated using a surface area ratio (ratio of the total surface of the given particles to the surface area of cells) of 1 to 1 that there was a correlation between the expression and production of cytokines induced by HA. The present work studies the effect of physical characteristics of HA particles on the production of various inflammatory cytokines (tumour necrosis factor-alpha, interleukin (IL)-6, and IL-8) and anti-inflammatory cytokine (IL-10). However, the experiments were performed using a surface area ratio of 10 to 1. Our data demonstrate that all the particles, whatever their characteristics, induced a high expression of cytokines but the production was different, meaning that there was a post-transcriptional regulation. The size and sintering temperature seemed to be a characteristics that were less important compared to the shape; the needle particles appeared to induce the most important production of all the cytokines studied.

Biocompatible Materials↗
PubMed2004

Rapid analysis of inflammatory cytokines in cerebrospinal fluid using chip-based immunoaffinity electrophoresis.

A chip-based capillary electrophoresis system has been designed for rapidly measuring the concentrations of inflammatory cytokines in the cerebrospinal fluid of patients with head trauma. Isolation of the reactive cytokines was achieved by immunoaffinity capture using a panel of six immobilized antibodies, directly attached to the injection port of the chip. The captured cytokines were labeled in situ with a red light-emitting laser dye and electroeluted into the separation channel. Separation of the isolated cytokines was achieved by electrophoresis in under 2 min with quantification of the resolved peaks being achieved by on-line laser-induced fluorescence and integration of each peak area. Comparison of the results to commercially available high-sensitivity immunoassays demonstrates that the chip-based assay provides a fast, accurate procedure for studying the concentrations of these analytes in complex biological materials. The degree of accuracy and precision achieved by the chip-based CE is comparable to conventional immunoassays, the system being able to analyze between 10-12 samples per hour. With the ever-expanding array of antibodies that are commercially available, this chip-based system can be applied to a wide variety of different analyses.

Antibodies↗
PubMed2004

The effect of the physical characteristics of hydroxyapatite particles on human monocytes IL-18 production in vitro.

Hydroxyapatite (HA) is widely used to coat the metal parts of prosthetic implants in order to improve their biocompatibility and as a bone defect filling material. HA has been demonstrated to produce particles at the prosthetic interface that lead to an activation of phagocytic cells that induce a cascade reaction leading to bone resorption and aseptic loosening. Monocytes/macrophages are commonly observed in the interface tissue, and are among the first cells to colonize the inflammatory site where they play a key role in the immune response. IL-18 is a pro-inflammatory cytokine. Monocytes/macrophages were described as IL-18 producing cells. IL-18 works antagonistically to IL-6, which activates osteoclastogenesis. In the present study, we investigated the ability of HA particles to induce the production of active IL-18 by human monocytes according to particle characteristics (size, sintering temperature and shape). Our study demonstrates, for the first time, that HA particles are capable of stimulating the production of the proinflammatory cytokine IL-18 in human monocytes according to their particle characteristics. The expression and the production of IL-18 was modified by the parameter studied. The difference observed between the expression and the production could be explain by the production of ICE. The needle shaped particles induced the larger production of IL-18.

Base Sequence↗
PubMed2003

Neonatal cytokines and cerebral palsy in very preterm infants.

To examine the relationship of cytokines in blood of very preterm neonates with later diagnosis of spastic cerebral palsy (CP) compared with infants of similar gestational age without CP, we measured concentrations of inflammatory cytokines and other substances in archived neonatal blood by recycling immunoaffinity chromatography. Subjects were surviving children born before 32 wk gestational age (GA) to women without preeclampsia, 64 with later diagnoses of CP and 107 control children. The initial analyses were augmented by measurement of 11 cytokines by a bead-based flow analytic system (Luminex) in an additional 37 children with CP and 34 control children from the same cohort. Concentrations of examined substances did not differ by presence of indicators of infection in mother, infant, or placenta. On ANOVA, concentrations of a number of cytokines were significantly related to neonatal ultrasound abnormalities (periventricular leukomalacia, ventricular enlargement, or moderate or severe germinal matrix hemorrhage). None of the substances measured either by immunoaffinity chromatography or flow analytic methods, including IL-1, -6, and -8 and tumor necrosis factor-alpha, was related to later diagnosis of CP or its subtypes. Inflammatory cytokines in neonatal blood of very premature infants did not distinguish those with later diagnoses of CP from control children.

Adult↗
PubMed2003

Complex array of cytokines released by vasoactive intestinal peptide.

A complex mixture of five cytokines has been shown to be released by vasoactive intestinal peptide (VIP). Cytokines were measured in paired samples of culture medium and astroglial cytosol by capillary electrophoresis. This is the first description of VIP-mediated release for TNF-alpha, IL-3, G-CSF and M-CSF from astrocyte cultures. Kinetic studies after VIP treatment demonstrated a gradual but incomplete depletion of cytosolic cytokine levels, with differences observed among the cytokines. Significant increases in release were apparent within 15-30 min for all cytokines. As the recognized VIP receptors (VPAC1 and VPAC2) are linked to adenylate cyclase and also interact with pituitary adenylate cyclase activating polypeptide-38 (PACAP-38), both this homologous peptide and 8-bromo cAMP were investigated and compared to VIP-mediated release. Treatment with 1 mM 8-bromo cAMP produced cytokine release similar in amount to 0.1 nM PACAP-38, but significantly less (<50%) in comparison to 0.1 nM VIP. PACAP-38 and VIP exhibited similar EC(50)'s for the release of G-CSF and TNF-alpha; however, the maximal release was 4-6 times greater for VIP than for PACAP-38. This similarity in potency suggested a VPAC-like receptor; however, the greater efficacy for VIP in comparison to PACAP-38, combined with a lack of cAMP production at subnanomolar concentrations of VIP, suggested a mechanism not currently associated with VPAC receptors. For M-CSF, IL-3 and IL-6, the EC(50)'s of VIP were 3-30 times more potent than those of PACAP-38 in producing release. These studies suggested that multiple mechanisms mediate cytokine release in astrocytes: (1) a low efficacy release produced by PACAP-38 that is cAMP-mediated and (2) a high efficacy, VIP-preferring mechanism that was not linked to cAMP. In summary, subnanomolar concentrations of VIP released a complex array of cytokines from astrocytes that may contribute to the mitogenic and neurotrophic properties of this neuropeptide in the central nervous system.

Adenylyl Cyclases↗
PubMed2003

Upregulation of MHC class II, interferon-alpha and interferon-gamma receptor protein expression in HIV-associated nephropathy.

BACKGROUND: Renal cellular HIV infection has been linked to the pathogenesis of HIV-associated nephropathy (HIVAN), but mediators of its development are unknown. HIV infection is associated with disordered cytokine metabolism, and chemokine receptors are coreceptors for HIV immune cellular infection. Chemokines such as interleukin (IL)-8, monocyte chemoattractant protein-1 (MCP-1) and RANTES, and interferons (IFNs) have been implicated in the progression of nephropathy. Renal major histocompatibility complex (MHC) protein expression is involved in antigen presentation and modulating tissue cellular immune responses. Their relative importance in HIVAN pathogenesis is unknown. METHODS: We measured levels of chemokines, IFN-alpha, IFN-gamma receptor and non-polymorphic MHC Class II protein by high performance capillary electrophoresis, and incubation with antibodies for quantification by chemiluminesce in renal tissue of patients with HIVAN, compared with tissue without HIV infection, in the presence and absence of nephropathy. Renal biopsy tissue protein levels were correlated with the number and type of infiltrating tissue immune cells. RESULTS: Mean renal interstitial and glomerular MCP-1, RANTES and IL-8 tissue levels were higher in patients with HIV infection compared with tissue without HIV infection, regardless of the presence of renal disease. In contrast, mean renal interstitial and glomerular non-polymorphic MHC Class II, IFN-alpha and IFN-gamma receptor protein were higher in patients with HIVAN compared with all other groups. Tissue MHC Class II and IFN-gamma receptor protein levels did not correlate with immune cellular infiltration in patients with HIV infection and renal disease. CONCLUSIONS: The data suggest an upregulated renal immune microenvironment, capable of antigen presentation, exists in HIVAN. MHC Class II proteins and IFNs, and the capacity to present antigen may be crucial in HIVAN pathogenesis.

AIDS-Associated Nephropathy↗
PubMed2003

Dietary magnesium intake influences circulating pro-inflammatory neuropeptide levels and loss of myocardial tolerance to postischemic stress.

Severe dietary Mg restriction (Mg(9), 9% of recommended daily allowance [RDA], plasma Mg = 0.25 mM) induces a pro-inflammatory neurogenic response in rats (substance P [SP]), and the associated increases in oxidative stress in vivo and cardiac susceptibility to ischemia/reperfusion (I/R) injury were previously shown to be attenuated by SP receptor blockade and antioxidant treatment. The present study assessed if less severe dietary Mg restriction modulates the extent of both the neurogenic/oxidative responses in vivo and I/R injury in vitro. Male Sprague-Dawley rats maintained on Mg(40) (40% RDA, plasma Mg = 0.6 mM) or Mg(100) (100% RDA, plasma Mg = 0.8 mM) diets were assessed for plasma SP levels (CHEM-ELISA) during the first 3 weeks and were compared with the Mg(9) group; red blood cell (RBC) glutathione and plasma malondialdehyde levels were compared at 3 weeks in Mg(9), Mg(20) (plasma Mg = 0.4 mM), Mg(40), and Mg(100) rats; and 40-min global ischemia/30-min reperfusion hearts from 7-week-old Mg(20), Mg(40), and Mg(100) rats were compared with respect to functional recovery (cardiac work, and diastolic, systolic, and developed pressures), tissue LDH release, and free radical production (ESR spectroscopy and alpha-phenyl-N-tert butylnitrone [PBN; 3 mM] spin trapping). The Mg(40) diet induced smaller elevations in plasma SP (50% lower) compared with Mg(9), but with a nearly identical time course. RBC glutathione and plasma malondialdehyde levels revealed a direct relationship between the severity of oxidative stress and hypomagnesemia. The dominant lipid free radical species detected in all I/R groups was the alkoxyl radical (PBN/alkoxyl: alpha(H) = 1.93 G, alpha(N) = 13.63 G); however, Mg(40) and Mg(20) hearts exhibited 2.7- and 3.9-fold higher alkoxyl levels, 40% and 65% greater LDH release, and lower functional recovery (Mg(20) < Mg(40)) compared with Mg(100). Our data suggest that varying dietary Mg intake directly influences the magnitude of the neurogenic/oxidative responses in vivo and the resultant myocardial tolerance to I/R stress.

Animals↗
PubMed2003

Immune profiling of plasma and cervical secretions using recycling immunoaffinity chromatography.

Small volumes of cervical secretions have limited measurements of immunity at the cervix, which may be important to studies of human papillomavirus (HPV). We report the use of recycling immunoaffinity chromatography to efficiently study immune profiles in cervical secretions. Frozen pairs of plasma and cervical secretions (collected on ophthalmic sponges) were selected randomly from women with normal cervical cytology (n = 50) participating in a natural history study of HPV in Guanacaste, Costa Rica. Single 25- micro l aliquots of plasma and (diluted) cervical secretions were assayed for interleukin (IL) -1 beta, -2, -4, -6, -8, -10, -12, -13, -15, IFN-alpha, -beta, -gamma, tumor necrosis factor-alpha, -beta, RANTES (regulated on activation normal T-cell express and secreted), MCP-1 (monocyte chemoattractant protein), -2, -3, macrophage inflammatory protein-1 alpha, -1 beta (regulated on activation normal T-cell express and secreted), macrophage colony-stimulating factor, IgG, IgA, and cyclooxygenase 2. All of the specimens were tested as blind replicates, and refrozen plasma was retested 4 months later. To evaluate the reproducibility of the repeat measurements and to examine the correlation between plasma and cervical secretions, we calculated kappa values with 95% confidence intervals among categorized analyte values and Spearman correlation coefficients (rho) among detectable, continuous analyte values. Measurements of all of the analytes in either plasma or cervical secretions were highly reproducible, with all of the kappa > or = 0.78 (70% above 0.90), and all of the rho > or = 0.88 (96% above 0.90). Only IL-1 beta (kappa = 0.60 and rho = 0.82) and IL-6 (kappa = 0.50 and rho = 0.78) levels were strongly correlated between plasma and cervical secretions. IFN-gamma, tumor necrosis factor-beta, RANTES, MCP-1, MCP -2, macrophage inflammatory protein-1 alpha, and macrophage colony-stimulating factor levels were especially poorly correlated between plasma and cervical secretions (kappa < or = 0.25 and rho < or = 0.25). We conclude that recycling immunoaffinity chromatography is a reproducible method of measuring immune profiles from biological specimens, and immune profiles are not well correlated between plasma and cervical secretions, perhaps necessitating cervical collections to study cervix-specific immunity in HPV natural history studies.

Adult↗
PubMed2002

Changes in astroglial GLT-1 expression after neural transplantation or stab wounds.

Uncontrolled release of glutamate from damaged brain initiates events that result in excitotoxic neuronal death. Glutamate uptake by specialized astroglial transporters is essential for control of extracellular glutamate levels. Many studies have demonstrated a reduction in astrocytic GLT-1 expression after different forms of injury. Because extensive neuronal death does not occur after direct cortical stab wounds and viable developing neurons populate fetal CNS grafts, we hypothesized that reactive astroglia associated with these procedures might maintain or up-regulate GLT-1. We examined the temporal and spatial distribution of GLT-1, GFAP and nestin proteins by confocal double-label immunohistochemistry combined with a new methodology in which precise brain areas are microdissected and analyzed for protein content by immunoaffinity chromatography. In stab wounds, GLT-1 protein content did not change compared to normal cortex, as determined by direct protein measurements; GLT-1 colocalized with nestin- and GFAP(+) astroglia adjacent to the lesion. In contrast, host reactive astroglia adjacent to grafts significantly upregulated GLT-1 by 3 days postoperative. The GFAP protein analysis suggests that increased GLT-1 is not the result of greater numbers of activated astroglia around grafts, but that developing graft tissue influences adjacent host astroglia to upregulate GLT-1. GLT-1 protein within grafts was rapidly accelerated to mature levels by just three days, and was expressed by the nestin(+) cell population. These data, which demonstrate immunoexpression of GLT-1 protein combined with a new method for protein measurement in situ indicate that, in contrast to other injury models, astroglial GLT-1 is upregulated or maintained following invasive CNS procedures. (c)2002 Elsevier Science (USA).

Animals↗
PubMed2002

Chemokine release is associated with the protective action of PACAP-38 against HIV envelope protein neurotoxicity.

The envelope protein (gp120) of the human immunodeficiency virus produces neuronal cell death in cultures that can be prevented by co-treatment with pituitary adenylate activating peptide-38 (PACAP-38) or chemokines. To investigate the hypothesis that a functional relationship exists between these two protectants, the release of chemokines was measured in rat astrocyte cultures after PACAP-38 treatment. Chemokine analyses were performed by immunoaffinity capillary electrophoresis. Bell-shaped dose-responses for PACAP-mediated release of chemokines into the culture medium were observed with EC(50)'s of 3 x 10(15) M (RANTES: regulated upon activation normal T cell expressed and secreted), 3 x 10(-11) M (MIP-1 beta) and 10(-7)M (MIP-1 alpha). In addition, PACAP-mediated depletion of chemokines from cultured astrocytes exhibited inverted bell-shaped curves, with similar EC(50)'s to those observed for chemokine measurements of the medium. Comparative studies with structurally related peptides (vasoactive intestinal peptide [VIP] and secretin) revealed that PACAP was the most potent secretagogue for RANTES on astrocyte cultures. Gp120-mediated neuronal cell death was prevented by co-treatment with PACAP-38, although the efficacy of protection varied significantly among the gp120 isolates. A bi-model dose-response was observed with EC(50)'s of 3 x 10(-15) and 3 x 10(-11) M. Co-treatment with neutralizing antiserum to RANTES attenuated PACAP-mediated protection from toxicity associated with gp120. In contrast to previous studies with VIP and gp120 toxicity, co-treatment with anti-MIP-1 alpha did not affect PACAP-induced protection. These studies support the hypothesis that PACAP produces neuroprotection from gp120 toxicity, in part, through the release of RANTES and this mechanism is distinct from that observed with VIP.

Animals↗
PubMed2002

Specific up-regulation of CRH or AVP secretion by acetylcholine or lipopolysaccharide in inflammatory susceptible Lewis rat fetal hypothalamic cells.

Lewis (LEW/N) rats, compared to Fischer (F344/N) rats, are susceptible to inflammatory/autoimmune diseases, in part, as a result of their blunted hypothalamic-pituitary-adrenal (HPA) axis responses. We examined regulation of LEW/N and F344/N fetal hypothalamic cell secretion of corticotropin-releasing hormone (CRH) and arginine vasopressin (AVP), two major HPA axis mediators, by inflammatory and neurotransmitter stimuli. Interleukin-1beta (IL-1beta), interleukin-6 (IL-6), tumor necrosis factor-alpha (TNF-alpha), and protein kinase A (PKA) and protein kinase C (PKC) activators did not affect LEW/N basal secretion. Compared to F344/N, LEW/N cells were hyporesponsive to lipopolysaccharide (LPS), serotonin (5-HT), and acetylcholine chloride (ACh). However, LPS-induced AVP release and ACh-evoked CRH secretion in LEW/N were comparable with those of F344/N. Our findings suggest that the blunted LEW/N neuropeptide response was more likely related to components of second messenger systems, rather than to any one specific stimulus.

Acetylcholine↗
PubMed2000

Dyadic relationship conflict, gender, and mortality in urban hemodialysis patients.

The effects of dyadic satisfaction and conflict have not been well defined in the hemodialysis (HD) population. The aim of this study was to determine whether the perception of decreased dyadic satisfaction was associated with mortality in patients treated with HD, and if so, whether there were different relationships between risk factors, and differential outcomes in men and women. A total of 174 HD patients, primarily African-Americans, involved in dyadic relationships for more than 6 mo had indices of dyadic satisfaction, depression, perception of illness effects, social support, behavioral compliance with the dialysis prescription, and plasma interleukin-1 (IL-1) and beta-endorphin levels measured. Cox proportional hazards models assessed relative mortality risks. Patients' dyadic satisfaction scores correlated with beta-endorphin levels. There was no correlation of IL-1 or beta-endorphin with any psychosocial or behavioral compliance measure in the group as a whole. Correlations between psychosocial, medical, and neuroimmunologic variables were different in men and women. For women, dyadic satisfaction correlated with beta-endorphin levels, depression, and perception of illness. Women with higher dyadic satisfaction and decreased dyadic conflict were at decreased mortality risk, but dyadic adjustment indices were unassociated with differential survival in the larger group of men. Correlations between neuroendocrine and immune markers are different in African-American male and female HD patients. Greater dyadic satisfaction and lower dyadic conflict are independently associated with decreased mortality in female African-American HD patients, of the same order of magnitude as medical risk factors. Such effects may be attributable to a relationship between dyadic satisfaction and conflict and health-related behaviors, or through an effect on neuroendocrine or immunologic status.

Adult↗
PubMedDate not supplied

Analysis of intracellular regulatory proteins by immunoaffinity capillary electrophoresis coupled with laser-induced fluorescence detection.

Measurement of intracellular regulatory proteins is of great importance in many areas of biomedical research. In this communication we describe an antibody-based capillary electrophoresis system equipped with an on-line laser-induced fluorescence detector capable of measuring intracellular proteins in cultures as low as 100 cells. The system demonstrated a high degree of precision and accuracy, being capable of detecting the fluorochrome-labeled analytes of interest at concentration of approximately 0.5 pg. We have used this instrument to study concentrations of the intracellular regulatory proteins STAT-1 and STAT-3, following stimulation of lymphocyte cultures with the inflammatory cytokine, IL-6. Using a combination of four antibodies specific for either STAT-1 or STAT-3 in both their nonphosphorylated and phosphorylated forms, we were able to demonstrate the differential expression of these proteins over time.

Cells, Cultured↗