PubMed Health⌕ Search

Biomedical subjects

K A Harrison

Publications and source records attributed to K A Harrison.

At least 19 recordsLinked to original sources

Oxidized alkyl phospholipids are specific, high affinity peroxisome proliferator-activated receptor gamma ligands and agonists.

Synthetic high affinity peroxisome proliferator-activated receptor (PPAR) agonists are known, but biologic ligands are of low affinity. Oxidized low density lipoprotein (oxLDL) is inflammatory and signals through PPARs. We showed, by phospholipase A(1) digestion, that PPARgamma agonists in oxLDL arise from the small pool of alkyl phosphatidylcholines in LDL. We identified an abundant oxidatively fragmented alkyl phospholipid in oxLDL, hexadecyl azelaoyl phosphatidylcholine (azPC), as a high affinity ligand and agonist for PPARgamma. [(3)H]azPC bound recombinant PPARgamma with an affinity (K(d)((app)) approximately 40 nm) that was equivalent to rosiglitazone (BRL49653), and competition with rosiglitazone showed that binding occurred in the ligand-binding pocket. azPC induced PPRE reporter gene expression, as did rosiglitazone, with a half-maximal effect at 100 nm. Overexpression of PPARalpha or PPARgamma revealed that azPC was a specific PPARgamma agonist. The scavenger receptor CD36 is encoded by a PPRE-responsive gene, and azPC enhanced expression of CD36 in primary human monocytes. We found that anti-CD36 inhibited azPC uptake, and it inhibited PPRE reporter induction. Results with a small molecule phospholipid flippase mimetic suggest azPC acts intracellularly and that cellular azPC accumulation was efficient. Thus, certain alkyl phospholipid oxidation products in oxLDL are specific, high affinity extracellular ligands and agonists for PPARgamma that induce PPAR-responsive genes.

Animals↗

Identification of platelet-activating factor as the inflammatory lipid mediator in CCl4-metabolizing rat liver.

Unmitigated oxidative stress is deleterious, as epitomized by CCl4 intoxication. In this well-characterized model of free radical-initiated damage, liver metabolism of CCl4 to CCl3. causes lipid peroxidation, F-ring isoprostane formation, and pathologic leukocyte activation. The nature of the mediator that couples oxidation to the hepatotoxic inflammatory response is uncharacterized. We found that oxidatively modified phosphatidylcholines were present in the livers of CCl4-exposed rats and not in livers from control animals, that CCl4 metabolism generated lipids that activated 293 cells stably transfected with the human platelet-activating factor (PAF) receptor, and that this PAF-like activity was formed as rapidly as isoprostane-containing phosphatidylcholine (iPC) during oxidation. iPC and the PAF-like activity also had similar chromatographic properties. The potential for iPC activation of the PAF receptor has been unexplored, but we conclude that iPC themselves did not activate the PAF receptor, as phospholipase A1 hydrolysis completely destroyed iPC, but none of the PAF-like bioactivity. Oxidatively fragmented phospholipids are potent agonists of the PAF receptor, but mass spectrometry characterized PAF as the major inflammatory component coeluting with iPC. Oxidatively fragmented phospholipids and iPC are markers of free radical generation in CCl4-intoxicated liver, but PAF generation by activated hepatic cells generated the inflammatory agent.

Animals↗

Bioactive phospholipid oxidation products.

Oxidation of phospholipids results in chain-shortened fragments and oxygenated derivatives of polyunsaturated sn-2 fatty acyl residues, generating a myriad of phospholipid products. Certain oxidation products of phosphatidylcholine bind to and activate the human receptor for PAF, and these PAF-like lipids are potent, selective inflammatory mediators. Formation of PAF-like lipids is nonenzymatic and so their accumulation is unregulated. PAF-like lipids are produced in vivo in response to oxidative stresses and are responsible for attendant acute inflammatory responses. PAF-like lipids almost exclusively contain an ether-linked alkyl residue at the sn-1 position of the phosphatidylcholine backbone and molecular identification of these is facilitated by phospholipase A(1) treatment to remove the bulk of the inactive phospholipids. The identity of biologically active species generated by oxidative fragmentation and oxidation can be elucidated by understanding relevant reactions leading to the formation of PAF-like lipids, and then their structure can be established by tandem mass spectrometry and chemical synthesis.

Animals↗

Analysis of oxidized glycerophosphocholine lipids using electrospray ionization mass spectrometry and microderivatization techniques.

Oxidized low-density lipoprotein (LDL) is thought to play an important role in atherogenesis and cardiovascular disease in humans. Oxidized LDL is a complex mixture of many oxidized species, including numerous oxidized glycerophospholipids. Electrospray ionization and tandem mass spectrometry as well as microchemical derivatization of high-performance liquid chromatographically purified fractions derived from oxidized LDL were investigated as means to determine the structure of individual components present in oxidized LDL. One major oxidized phosphocholine lipid had an [M + H](+) ion at m/z 650. Derivatization to the trimethylsilyl ether and methoxime caused shifts in mass which, along with negative ion collision-induced dissociation mass spectra, were consistent with the presence of three species, 1-palmitoyl-2-(9-oxononanoyl)glycerophosphocholine and two isomeric 1-octadecanoyl-2-(hydroxyheptenoyl)glycerophosphocholines. These species were chemically synthesized. Trimethylsilylation of free hydroxyl groups increased the mass of the phospholipid acyl chains containing hydroxyl groups by 72 u. Conversion of carbonyl groups to the methoxylamine derivative increased the mass by 29 u. Ozonolysis of those products which contained double bonds proved to be a facile technique to determine the position and number of double bonds present. The use of these techniques was illustrated in the structural characterization of one major component (m/z 650, positive ions) in oxidized LDL as 1-octadecanoyl-2-(7-hydroxyhepta-5-enoyl)glycerophosphocholi ne. A possible mechanism for the formation of this unique chain-shortened glycerophospholipid is proposed.

Chromatography, High Pressure Liquid↗

Role of STAT3 and C/EBP in cytokine-dependent expression of the mouse serum amyloid P-component (SAP) and C-reactive protein (CRP) genes.

Inflammation is accompanied by a rapid increase in blood levels of acute phase proteins synthesized by hepatocytes in response to cytokines. Although C-reactive protein (CRP) levels increase dramatically in most mammals, the major acute phase protein in the mouse is the homologous pentraxin, serum amyloid P-component (SAP), whereas CRP is a minor acute phase reactant. The molecular basis for the pronounced difference in SAP and CRP gene expression in the mouse is unknown. Transfection of ++/Li mouse hepatoma cells with CAT-reporter constructs containing the 5'-flanking region of the mouse CRP gene indicated that transcription was stimulated by either IL-6, or IL-6 plus IL-1, when > or =360 bp of the 5'-proximal DNA was present. Examination of the 5'-flanking region of the mouse SAP gene revealed that the region between -433 and -397 from the transcription start site responded to IL-1 and IL-6 by binding both STAT3 and C/EBPbeta. This responsive region consisted of two adjacent C/EBPbeta consensus sites that overlap with two STAT3 consensus sites and was found to bind C/EBPbeta at an upstream site of -427 to -409 and STAT3 at a downstream site of -415 to -397. By contrast, the 360 bp promoter of the CRP gene was bound only by STAT3 at consensus sites at -93, -142, -173, and -287 from the start site; however, a single consensus site for C/EBP at -75 was not recognized. STAT3 appears to be necessary for both mouse SAP and CRP gene transcription since overexpression of an inactive, deletion mutant of STAT3 inhibited transcription of both genes. The results indicate that both STAT3 and C/EBPbeta participate in mouse SAP gene expression, whereas only STAT3 is involved in mouse CRP gene expression. The findings for mouse SAP gene expression are consistent with the reported interaction between these two transcription factors for human CRP gene transcription.

Animals↗

Inflammatory platelet-activating factor-like phospholipids in oxidized low density lipoproteins are fragmented alkyl phosphatidylcholines.

Oxidation of human low density lipoprotein (LDL) generates proinflammatory mediators and underlies early events in atherogenesis. We identified mediators in oxidized LDL that induced an inflammatory reaction in vivo, and activated polymorphonuclear leukocytes and cells ectopically expressing human platelet-activating factor (PAF) receptors. Oxidation of a synthetic phosphatidylcholine showed that an sn-1 ether bond confers an 800-fold increase in potency. This suggests that rare ether-linked phospholipids in LDL are the likely source of PAF-like activity in oxidized LDL. Accordingly, treatment of oxidized LDL with phospholipase A(1) greatly reduced phospholipid mass, but did not decrease its PAF-like activity. Tandem mass spectrometry identified traces of PAF, and more abundant levels of 1-O-hexadecyl-2-(butanoyl or butenoyl)-sn-glycero-3-phosphocholines (C(4)-PAF analogs) in oxidized LDL that comigrated with PAF-like activity. Synthesis showed that either C(4)-PAF was just 10-fold less potent than PAF as a PAF receptor ligand and agonist. Quantitation by gas chromatography-mass spectrometry of pentafluorobenzoyl derivatives shows the C(4)-PAF analogs were 100-fold more abundant in oxidized LDL than PAF. Oxidation of synthetic alkyl arachidonoyl phosphatidylcholine generated these C(4)-PAFs in abundance. These results show that quite minor constituents of the LDL phosphatidylcholine pool are the exclusive precursors for PAF-like bioactivity in oxidized LDL.

Chromatography, High Pressure Liquid↗

Negative ion electrospray and tandem mass spectrometric analysis of platelet activating factor (PAF) (1-hexadecyl-2-acetyl-glycerophosphocholine).

The analysis of 1-hexadecyl-2-acetyl-glycerophosphocholine (platelet activating factor, PAF) by negative ion and normal-phase liquid chromatography/tandem mass spectrometry (LC/MS/MS) was investigated as an alternative technique to the currently used gas chromatography/MS and positive ion LC/MS/MS procedures. The positive ion [M + H]+ derived from PAF and generated by electrospray ionization is abundant, but the potential presence of isobaric 1-octadecanoyl-2-lyso-glycerophosphocholine (stearoyl-lyso-GPC) and 1-hexadecanoyl-2-formyl-glycerophosphocholine (PFPC) in biological samples limits the use of the most abundant collision-induced decomposition (CID) transition (formation of the phosphocholine ion, m/z 524-->184) if chromatographic separation is not achieved. Less abundant CID product ions, such as loss of the neutral ketene molecule derived from the respective fatty acyl groups, provide the requisite specificity, but the intensity of these transitions yields a signal-to-noise ratio that greatly diminishes the analytical sensitivity. With negative ion LC/MS/MS, however, the molecular anions [M - 15]- derived from PAF, stearoyl-lyso-GPC and PFPC decompose to the carboxylate anions at m/z 59, 283 and 255, respectively, permitting discrimination of these isobaric molecules even without chromatographic separation. In addition, the CID of [M - 15]- was favorable, yielding ion currents of sufficient intensity to permit the measurement of PAF when isolated from small quantities of biological material. With the use of a stable isotopically labeled variant of PAF and isotope dilution, negative ion LC/MS/MS was found to measure PAF reliably even in the presence of the isobaric stearoyl-lyso-GPC and permitted the use of non-chlorinated mobile phases for normal-phase high-performance LC.

Humans↗

Pancreas dorsal lobe agenesis and abnormal islets of Langerhans in Hlxb9-deficient mice.

In most mammals the pancreas develops from the foregut endoderm as ventral and dorsal buds. These buds fuse and develop into a complex organ composed of endocrine, exocrine and ductal components. This developmental process depends upon an integrated network of transcription factors. Gene targeting experiments have revealed critical roles for Pdx1, Isl1, Pax4, Pax6 and Nkx2-2 (refs 3,4,5,6,7, 8,9,10). The homeobox gene HLXB9 (encoding HB9) is prominently expressed in adult human pancreas, although its role in pancreas development and function is unknown. To facilitate its study, we isolated the mouse HLXB9 orthologue, Hlxb9. During mouse development, the dorsal and ventral pancreatic buds and mature beta-cells in the islets of Langerhans express Hlxb9. In mice homologous for a null mutation of Hlxb9, the dorsal lobe of the pancreas fails to develop. The remnant Hlxb9-/- pancreas has small islets of Langerhans with reduced numbers of insulin-producing beta-cells. Hlxb9-/- beta-cells express low levels of the glucose transporter Glut2 and homeodomain factor Nkx 6-1. Thus, Hlxb9 is key to normal pancreas development and function.

Animals↗

Tumor localization and systemic absorption of intravesical instillation of radio-iodinated iododeoxyuridine in patients with bladder cancer.

PURPOSE: We evaluated tumor uptake and systemic distribution of intravesically instilled iododeoxyuridine (IUdR) in patients with superficial bladder cancer. MATERIALS AND METHODS: We performed 24 intravesical instillation studies in 11 patients with a mean age of 71 years. Radio-iodinated IUdR was administered through a Foley catheter. Gamma camera imaging was done after instillation and after 5 to 7 bladder irrigations. Tumor uptake was estimated by region of interest analysis. Bladder biopsy samples and surgical tumor specimens were tested for acid insoluble (deoxyribonucleic acid incorporated) radioactivity. Blood samples were obtained and analyzed for systemic absorption. RESULTS: Imaging was positive in all patients with bladder cancer. Average tumor uptake plus or minus standard deviation was 0.185+/-0.120% of the instilled dose. Preferential uptake of IUdR in the tumor was observed in all 6 patients undergoing tissue analysis. The tumor-to-normal bladder ratio ranged from 3.2 to 74,000 (median 202). Systemic absorption of IUdR was minimal. Blood sample analysis performed after intravesical instillation in all 11 cases revealed an average uptake of 3.2x10(-5)% instilled dose per ml. (range 0.69x10(-5) to 6.7x10(-5)) in the systemic circulation. Instillation within 24 hours after transurethral bladder tumor resection in 5 cases resulted in a higher but not dangerous average systemic uptake of 7.3x10(-4)% instilled dose per ml. (range 1.3x10(-5) to 2.6x10(-3)). Instillation 1 to 4 weeks after transurethral surgery in 8 cases resulted in no increased systemic absorption with an average blood level of 3.4+/-1.8x10(-5)% instilled dose per ml. There was no detectable distribution of radioactivity into other organs, including the thyroid. We noted no evidence of systemic toxicity in the study. CONCLUSIONS: Intravesical instillation of radio-iodinated IUdR achieves selective localization in the bladder tumor with minimal uptake by the normal bladder and minimal systemic absorption. The use of intravesical IUdR therapy for bladder cancer appears to be promising and requires further study.

Absorption↗

Palliation of pain associated with metastatic bone cancer using samarium-153 lexidronam: a double-blind placebo-controlled clinical trial.

PURPOSE: To evaluate the effectiveness and safety of samarium-153 (153Sm) lexidronam (EDTMP) in a double-blind, placebo-controlled study. PATIENTS AND METHODS: Patients with painful bone metastases secondary to a variety of primary malignancies were randomized to receive 153Sm-EDTMP 0.5 or 1.0 mCi/kg, or placebo. Treatment was unblinded for patients who did not respond by week 4, with those who had received placebo eligible to receive 1.0 mCi/kg of active drug in an open-label manner. Patient and physician evaluations were used to assess pain relief, as was concurrent change in opioid analgesia. RESULTS: One hundred eighteen patients were enrolled onto the study. Patients who received 1.0 mCi/kg of active drug had significant reductions in pain during each of the first 4 weeks in both patient-rated and physician-rated evaluations. Pain relief was observed in 62% to 72% of those who received the 1.O-mCi/kg dose during the first 4 weeks, with marked or complete relief noted in 31% by week 4. Persistence of pain relief was seen through week 16 in 43% of patients who received 1.0 mCi/kg, of active drug. A significant correlation (P = .01) was observed between reductions in opioid analgesic use and pain scores only for those patients who received 1.0 mCi/kg 153Sm-EDTMP. Bone marrow suppression was mild, reversible, and not associated with grade 4 toxicity. CONCLUSION: A single dose of 1.0 mCi/kg of 153Sm-EDTMP provided relief from pain associated with bone metastases. Pain relief was observed within 1 week of administration and persisted until at least week 16 in the majority of patients who responded.

Adult↗

Patient-specific dosimetry of indium-111- and yttrium-90-labeled monoclonal antibody CC49.

UNLABELLED: The objective of this work was to develop patient-specific dosimetry for patients with metastatic gastrointestinal tract cancers who received 111In-CC49 IgG for imaging before therapy with 90Y-CC49 IgG. METHODS: Whole-body imaging of 12 patients, who received 111-185 MBq (3-5 mCi) of 111In-CC49, commenced in < 2 hr postinfusion and was continued daily for 4-5 days. SPECT data were acquired at 24 and 72 hr to determine the range of 111In-CC49 activity concentrations in tumors and normal organs. Time-activity curves were generated from the image data and scaled from 111In-CC49 to 90Y-CC49 for dosimetric purposes. Absorbed-dose calculations for 90Y-CC49 included the mean and range in tumor and normal organs. Computed 90Y-CC49 activity concentrations were compared with measurements on 10 needle biopsies of normal liver and four tumor biopsies. RESULTS: In 9 of 10 normal liver samples, the range of computed 90Y-CC49 activity concentrations bracketed measured values. This was also the case for 3 of 4 tumor biopsies. Absorbed-dose calculations for 90Y-CC49 were based on patients' images and activities in tissue samples and, hence, were patient-specific. CONCLUSION: For the radiolabeled antibody preparations used in this study, quantitative imaging of 111In-CC49 provided the data required for 90Y-CC49 dosimetry. The range of activities in patients' SPECT images was determined for a meaningful comparison of measured and computed values. Knowledge of activity distributions in tumors and normal organs was essential for computing mean values and ranges of absorbed dose and provided a more complete description of the absorbed dose from 90Y-CC49 than was possible with planar methods.

Absorption↗

Indium-111-leukocyte imaging: a case of peritonitis mimicking inflammatory bowel disease.

Leukocytes labeled with 99mTc-HMPAO and 111In have been used extensively in imaging inflammatory disorders, including inflammatory bowel disease (IBD), which has the appearance of tubular bowel activity. Peritonitis is inflammation of the serosal surfaces lining the peritoneal cavity which envelopes the bowel, giving a pattern of diffuse abdominal uptake on imaging. We present a case of an elderly man with surgically and pathologically confirmed peritonitis whose preoperative leukocyte scan mimicked the findings of IBD. Our findings suggest that diffuse peritonitis can mimic IBD on an 111In-leukocyte scan.

Abdomen↗

Direct mass spectrometric analysis of ozonides: application to unsaturated glycerophosphocholine lipids.

The reaction of ozone with double bonds present in glycerophosphocholine lipids results in formation of ozonides that can be directly analyzed by mass spectrometry as either positive or negative molecular ion species generated by electrospray ionization. Polyunsaturated fatty acyl groups esterified to the phospholipid yielded a mixture of ozonide species with the maximum number of ozone molecules added equal to the total number of double bonds. Ozonide decomposition resulted in omega-aldehyde and omega-carboxylic acid products as revealed by ESI-MS. Collisional activation of the ozone adducts for mono- and polyunsaturated phospholipids gave rise to fragment ions indicative of the position of the double bonds in these molecules. The major decomposition pathway for either positive or negative ozonide ion species involved charge remote fragmentation of the ozonide initiated by homolytic cleavage of the peroxide bridge followed by rearrangement to form the omega-aldehyde and omega-carboxylate acyl species. The reaction of ozone with phospholipids containing polyunsaturated fatty acyl groups is a useful method to probe the position of double bonds by electrospray ionization mass spectrometry.

Lipids↗

Platelet-activating factor biosynthesis induced by various stimuli in human HaCaT keratinocytes.

Platelet-activating factor (PAF) is a potent inflammatory mediator that is thought to play a role in cutaneous inflammation. These studies used mass spectrometry to examine the molecular species of PAF precursor glycerophosphocholine lipids (GPC) as well as the biosynthesis of PAF and other sn-2 acetyl-GPC in a human keratinocyte-derived cell line (HaCaT keratinocytes). Approximately 28% of HaCaT keratinocyte GPC consisted of 1-alkyl species, and the relative amounts of the sn-1 alkyl constituents of the PAF precursor 1-alkyl-2-acyl-GPC were as follows: hexadecyl > octadecenyl > octadecyl. Ionophore (A23187)-stimulated HaCaT keratinocytes synthesized both PAF (1-hexadecyl, 1-octadecenyl, and 1-octadecyl species) and less potent 1-acyl analogs (1-palmitoyl, 1-oleoyl, and 1-stearoyl species). PAF production was rapid and maximal by 10 min. The major species of sn-2acetyl-GPC at 2.5 min were 1-hexadecyl-2-acetyl-GPC (2.2 ng/10(6) cells) and 1-palmitoyl-2-acetyl-GPC (2.4 ng/10(6) cells). HaCaT keratinocytes also synthesized PAF and 1-acyl PAF analogs when stimulated with the peptide growth factor endothelin-1 and the nonhydrolyzable PAF receptor agonist carbamyl-PAF. Both 1-hexadecyl-2- acetyl-GPC and 1-palmitoyl-2-acetyl-GPC stimulated intracellular calcium mobilization in HaCaT cells, indicating that these sn-2 acetyl-GPC act in autocrine fashion. These studies revealed that the human keratinocyte-derived cell line HaCaT can synthesize significant amounts of PAF and 1-acyl analogs in vitro from both nonspecific (A23187) and specific (endothelin-1, carbamyl-PAF) stimulation, suggesting a role for this inflammatory lipid mediator in keratinocyte pathophysiology.

Calcimycin↗

Single-photon emission computed tomography gallium imaging versus computed tomography: predictive value in patients undergoing high-dose chemotherapy and autologous stem-cell transplantation for non-Hodgkin's lymphoma.

PURPOSE: To evaluate the predictive value of computed tomography (CT) scanning and single-photon emission computed tomography (SPECT) gallium (Ga) scanning in the disease-free survival of patients receiving high-dose chemotherapy and autologous stem-cell transplantation for non-Hodgkin's lymphoma (NHL). PATIENTS AND METHODS: One hundred forty-three patients undergoing transplant for NHL underwent CT scanning of chest, abdomen, and pelvis, and a SPECT Ga scan before transplantation and at day + 100 after transplant. The failure-free survival (FFS) by scan result was analyzed. RESULTS: In the diffuse aggressive lymphoma patients, the 1-year FFS for patients having a positive SPECT Ga scan at day + 100 was 15% compared with a 3-year FFS of 47% for those with a negative scan (P < .001). Patients with a positive CT scan at day + 100 had a 36% 3-year FFS, and those with a negative CT scan had a 39% 3-year FFS (P = not significant [NS]). An analysis of the combination of CT scan and SPECT Ga scan results at day + 100 posttransplant demonstrated a 3-year FFS of 14% if they were both abnormal; if the CT was positive and Ga was negative, the 3-year FFS was 68%; positive Ga with a negative CT was 25%; and both negative was 34% (P = .0015). For the patients with follicular NHL, those with a positive SPECT Ga at day + 100 had a 14% 1-year FFS compared with those with a negative scan, who had a 45% 3-year FFS (P < .001). In the follicular NHL patients, the 3-year FFS of those with a positive CT was 17% compared with a 64% 3-year FFS for patients with a negative CT scan (P < .001). CONCLUSION: The use of SPECT Ga scan at day + 100 posttransplant for evaluation of disease activity in patients with diffuse aggressive NHL was highly predictive of eventual outcome and was more predictive than the CT scan results. However, for patients with follicular NHL, the addition of SPECT Ga scanning to CT scanning did not add substantially to the evaluation of transplant outcome.

Adolescent↗