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Sample records for human peripheral benzodiazepine

  1. Characterization of peripheral benzodiazepine binding sites in human term placenta.

    PubMed

    Fares, F; Gavish, M

    1986-01-15

    Peripheral benzodiazepine binding sites were characterized in human term placental membranes using [3H]PK 11195, which is a ligand specific for peripheral benzodiazepine binding sites. Binding of [3H]PK 11195 to human term placental membranes was found to be saturable. Scatchard analysis revealed a single population of binding sites (r = 0.98). Equilibrium dissociation constant (KD) was 2.1 +/- 0.3 nM, and density of binding sites (Bmax) was 920 +/- 105 fmol/mg protein. The KD value calculated from kinetic experiments was 3.6 +/- 0.2 nM. The ability of various drugs to displace [3H]PK 11195 from human term placental binding sites was tested: the inhibition constants (KI) for PK 11195, Ro 5-4864, and diazepam were 2.9, 11.8, and 177 nM, respectively, whereas clonazepam, methyl-beta-carboline-3-carboxylate, Ro 15-1788, chlordiazepoxide, atropine, and estradiol were inefficient in displacing [3H]PK 11195 (KI greater than 10(-5) M).

  2. Peripheral benzodiazepine receptors are decreased during cocaine withdrawal in humans.

    PubMed

    Javaid, J I; Notorangelo, M P; Pandey, S C; Reddy, P L; Pandey, G N; Davis, J M

    1994-07-01

    In the present study, homovanillic acid in plasma (pHVA) and benzodiazepine receptors (3H-PK11195 binding) in neutrophil membranes were determined in blood obtained from cocaine-dependent (DSM-III-R) adult male inpatients at baseline-(within 72 hr of last cocaine use) and after 3 weeks of cocaine abstinence, and normal controls. The mean (+/- SEM) pHVA at baseline (10.3 ng/ml +/- 1.1) was similar to normals and did not change after 3 weeks of cocaine abstinence. Similarly, the binding indices of benzodiazepine receptors in cocaine-dependent subjects as a group were not significantly different than in normal controls. In 10 cocaine-dependent subjects, however, where both blood samples were available, the number of 3H-PK11195 binding sites was significantly (p < 0.05) decreased after 3 weeks of cocaine abstinence (mean +/- sem: Bmax = 6371 +/- 657 fmol/mg protein) compared with baseline (Bmax = 7553 +/- 925 fmol/mg protein), although there were no differences in the binding affinity (mean +/- sem: KD = 8.6 +/- 1.2 nmol/L after 3 weeks of abstinence compared with 8.1 +/- 1.0 nmol/L at baseline). These preliminary results suggest that peripheral benzodiazepine receptors may play an important role in the pathophysiology of cocaine withdrawal in cocaine-dependent human subjects.

  3. Imaging of a glioma using peripheral benzodiazepine receptor ligands

    SciTech Connect

    Starosta-Rubinstein, S.; Ciliax, B.J.; Penney, J.B.; McKeever, P.; Young, A.B.

    1987-02-01

    Two types of benzodiazepine receptors have been demonstrated in mammalian tissues, one which is localized on neuronal elements in brain and the other, on glial cells and in peripheral tissues such as kidney. In vivo administration of /sup 3/H-labeled PK 11195 (1-(2-chlorophenyl-N-methyl-N-(1-methylpropyl)-3-isoquinoline carboxamide) or (/sup 3/H)flunitrazepam with 5 mg of clonazepam per kg to rats with intracranial C6 gliomas resulted in high levels of tritiated-drug binding to the tumor as shown by quantitative autoradiography. Pharmacological studies indicated that the bound drugs labeled the peripheral benzodiazepine binding site. Binding to the peripheral benzodiazepine site was confirmed primarily to malignant cells with little binding to adjacent normal brain tissue or to necrotic tissue. Tumor cell binding was completely inhibited by preadministration of the peripheral benzodiazepine blocking agent PK 11195 at 5 mg/kg. The centrally selective benzodiazepine ligand clonazepam had no effect on PK 11195 binding to the tumor cells. When binding to other tumor cell lines grown in nude mice and nude athymic rats was evaluated, little or no peripheral benzodiazepine binding was detected on human pheochromocytoma (RN1) and neuroblastoma (SK-N-MC, SK-N-SH) tumor cells, respectively. However, high densities of peripheral benzodiazepine binding sites were observed on tumors derived from a human glioma cell line (ATCC HTB 14, U-87 MG). The presence of high concentrations of specific peripheral benzodiazepine receptors on glial tumors suggests that human primary central nervous system tumors could be imaged and diagnosed using peripheral benzodiazepine ligands labeled with positron- or gamma-emitting isotopes.

  4. Prenatal benzodiazepine immunosuppression: possible involvement of peripheral benzodiazepine site.

    PubMed

    Schlumpf, M; Parmar, R; Ramseier, H R; Lichtensteiger, W

    1990-01-01

    Treatment of pregnant Long Evans rats with a low dose of diazepam (1.25 mg/kg from gestational day 14-20) produced offspring suffering from suppression of cellular immune responses. Analogous effects were produced by clonazepam, a benzodiazepine (BDZ) with high affinity for the central-type, and Ro 5-4864, a BDZ with selective affinity for the peripheral-type BDZ receptor. Peripheral-type BDZ receptors are found to develop early in fetal life in peripheral organs including primary (thymus) and secondary (spleen) lymphoid organs, in the central nervous system and on immune cells (lymphocytes). In prenatally diazepam-exposed offspring the affinity constant is significantly changed. BDZ and PK 11195 also inhibit mitogen and alloantigen-induced T and B cell proliferation in vitro in adult murine lymphocytes. Diazepam, Ro 5-4864 and PK 11195 were found to be the most active compounds.

  5. Platelet peripheral benzodiazepine receptors in repeated stress.

    PubMed

    Dar, D E; Weizman, A; Karp, L; Grinshpoon, A; Bidder, M; Kotler, M; Tyano, S; Bleich, A; Gavish, M

    1991-01-01

    [3H]PK 11195 binding to platelet membranes and plasma stress hormones were studied in soldiers at the beginning of a parachute training course, following 6 days of preparatory exercises, and after the fourth actual parachute jump. A slight reduction (15%; NS) in the number of peripheral benzodiazepine receptors (PBR) was detected at the end of the exercise period, prior to the first jump. Reduced (26%; P less than 0.05) density of PBR was observed immediately after the repeated actual jumps. Equilibrium dissociation constants were not affected by the stressful situation. Plasma cortisol and prolactin levels remained unaltered during the entire study period.

  6. Platelet peripheral benzodiazepine receptors in repeated stress

    SciTech Connect

    Dar, D.E.; Bidder, M.; Gavish, M. ); Weizman, A.; Karp, L.; Tyano, S. ); Grinshpoon, A.; Bleich, A.

    1991-01-01

    ({sup 3}H)PK 11195 binding to platelet membranes and plasma stress hormones were studied in soldiers at the beginning of a parachute training course, following 6 days of preparatory exercises, and after the fourth actual parachute jump. A slight reduction (15%; NS) in the number of peripheral benzodiazepine receptors (PBR) was detected at the end of the exercise period, prior to the first jump. Reduced density of PBR was observed immediately after the repeated actual jumps. Equilibrium dissociation constants were not affected by the stressful situation. Plasma cortisol and prolactin levels remained unaltered during the entire study period.

  7. The human peripheral benzodiazepine receptor gene: Cloning and characterization of alternative splicing in normal tissues and in a patient with congenital lipoid adrenal hyperplasia

    SciTech Connect

    Lin, D.; Miller, W.L. ); Chang, Y.J.; Strauss, J.F. III )

    1993-12-01

    The mitochondrial benzodiazepine receptor (mBzR) appears to be a key factor in the flow of cholesterol into mitochondia to permit the initiation of steroid hormone synthesis. The mBzR consists of three components; the 18-kDa component on the outer mitochondrial membrane appears to contain the benzodiazepine binding site, and is hence often termed the peripheral benzodiazepine receptor (PBR). Using a cloned human PBR cDNA as probe, the authors have cloned the human PBR gene. The 13-kb gene is divided into four exons, with exon 1 encoding only a short 5[prime] untranslated segment. The 5[prime] flanking DNA lacks TATA and CAAT boxes but contains a cluster of SP-1 binding sites, typical of [open quotes]housekeeping[close quotes] genes. The encoded PBR mRNA is alternately spliced into two forms: [open quotes]authentic[close quotes] PBR mRNA retains all four exons, while a short form termed PBR-S lacks exon 2. While PBR-S contains a 102-codon open reading frame with a typical initiator sequence, the reading frame differs from that of PBR, so that the encoded protein is unrelated to PBR. RT-PCR and RNase protection experiments confirm that both PBR and PBR-S are expressed in all tissues examined and that expression of PBR-S is about 10 times the level of PBR. Expression of PBR cDNA in pCMV5 vectors transfected into COS-1 cells resulted in increased binding of [[sup 3]H]PK11195, but expression of PBR-S did not. It has been speculated that patients with congenital lipoid adrenal hyperplasia, who cannot make any steroids, might have a genetic lesion in mBzR. RT-PCR analysis of testicular RNA from such a patient, sequencing of the cDNA, and blotting analysis of genomic DNA all indicate that the gene and mRNA for the PBR component of mBzR are normal in this disease. 36 refs., 6 figs.

  8. The bovine peripheral-type benzodiazepine receptor: A receptor with low affinity for benzodiazepines

    SciTech Connect

    Parola, A.L.; Laird, H.E. II )

    1991-01-01

    The density of bovine peripheral-type benzodiazepine receptors (PBR) in four tissues was highest in adrenal cortex. The adrenal cortex PBR cofractionated with a mitochondrial membrane marker enzyme and could be solubilized with intact ligand binding properties using digitonin. The membrane bound and soluble mitochondrial receptors were pharmacologically characterized and showed the rank order of potency to inhibit ({sup 3}H)PK 11195 binding was PK 11195 > protoporphyrin IX > benzodiazepines. ({sup 3}H)PK 11195 binding to bovine adrenal mitochondria was unaffected by diethylpyrocarbonate, a histidine residue modifying reagent that decreased binding to rat liver mitochondria by 70%. ({sup 3}H)PK 14105 photolabeled the bovine PBR and the Mr was estimated under nondenaturing and denaturing conditions. These results demonstrate the bovine peripheral-type benzodiazepine receptor is pharmacologically and biochemically distinct from the rat receptor, but the receptor component photolabeled by an isoquinoline ligand has a similar molecular weight.

  9. Late evolutionary appearance of 'peripheral-type' binding sites for benzodiazepines.

    PubMed

    Bolger, G T; Weissman, B A; Lueddens, H; Basile, A S; Mantione, C R; Barrett, J E; Witkin, J M; Paul, S M; Skolnick, P

    1985-07-15

    Four classes of non-mammalian vertebrates were examined for the presence of both 'brain-specific' and 'peripheral-type' binding sites for benzodiazepines in the central nervous system. 'Brain-specific' binding sites for benzodiazepines were found in the central nervous systems of all non-mammalian vertebrates studied. However, in contrast to mammals, either very low or undetectable levels of 'peripheral-type' binding sites for benzodiazepines were observed in the central nervous systems of these non-mammalian vertebrates. Furthermore, the density of 'peripheral-type' binding sites for benzodiazepines in non-mammalian vertebrate heart was less than or equal to 2% of that found in mammalian cardiac tissue. These findings suggest a very late evolutionary appearance of 'peripheral-type' binding sites for benzodiazepines, implying that these sites may have (a) highly specialized function(s) in both peripheral tissues and the central nervous system.

  10. PET study of carbon-11-PK 11195 binding to peripheral type benzodiazepine sites in glioblastoma: A case report

    SciTech Connect

    Pappata, S.; Cornu, P.; Samson, Y.; Prenant, C.; Benavides, J.; Scatton, B.; Crouzel, C.; Hauw, J.J.; Syrota, A. )

    1991-08-01

    The utility of the peripheral type benzodiazepine site ligand 11C-PK 11195, for imaging human glioma in conjunction with Positron Emission Tomography, relies on a high specific binding of the tracer to tumoral peripheral type benzodiazepines sites. In a patient with glioblastoma, the authors found that 11C-PK 11195 binding was two-fold higher in the tumor than in normal gray matter and that 30% of tumoral binding could be displaced by a large excess of unlabeled drug. These findings suggest that tumoral retention of the ligand is due, in part, to specific binding.

  11. Platelet peripheral benzodiazepine receptors are decreased in Parkinson's disease

    SciTech Connect

    Bonuccelli, U.; Nuti, A.; Del Dotto, P.; Piccini, P.; Martini, C.; Giannacccini, G.; Lucacchini, A.; Muratorio, A. )

    1991-01-01

    Peripheral benzodiazepine (BDZ) receptors are located in a variety of tissues, including platelets, in the nuclear and/or mitochondrial membranes. The authors studied the density of peripheral BDZ receptors in platelets of 10 de novo Parkinson's disease (PD) patients, 18 PD patients treated with a levodopa/carbidopa combination, and in 15 healthy subjects matched for sex and age. The binding assay was conducted using ({sup 3}H)PK 11195, a specific ligand for peripheral BDZ receptors. A significant decrease in the density of ({sup 3}H)PK 11195 binding sites has been observed in PD patients with respect to controls but not between de novo and treated PD patients. No correlation has been found between the decrease in density of ({sup 3}H)PK 11195 binding sites in platelets and either the duration or severity of PD. Peripheral BDZ receptors are implicated in the regulation of mitochondrial respiratory function. Thus, their decrease in PD might parallel the abnormalities in mitochondrial function recently found in this neurologic disease.

  12. Benzodiazepines

    MedlinePlus

    ... The most common benzodiazepines are the prescription drugs Valium ® , Xanax ® , Halcion ® , Ativan ® , and Klonopin ® . Tolerance can develop, ... benzodiazepines include alprazolam (Xanax ® ), chlordiazepoxide(Librium ® ), clorazepate (Tranxene ® ), diazepam (Valium ® ), halazepam (Paxipam ® ), lorzepam (Ativan ® ), oxazepam (Serax ® ), prazepam ( ...

  13. Regulation of renal peripheral benzodiazepine receptors by anion transport inhibitors

    SciTech Connect

    Basile, A.S.; Lueddens, W.M.; Skolnick, P.

    1988-01-01

    The in vitro and in vivo regulation of (/sup 3/H)Ro 5-4864 binding to peripheral benzodiazepine receptors (PBR) by ion transport/exchange inhibitors was studied in the kidney. The potencies of 9-anthroic acid, furosemide, bumetanide, hydrochlorothiazide and SITS as inhibitors of (/sup 3/H)Ro 5-4864 binding to renal membranes were consistent with their actions as anion transport inhibitors (Ki approx. = 30 - 130 ..mu..M). In contrast, spironolactone, amiloride, acetazolamide, and ouabain were less potent (Ki=100-1000 ..mu..M). Administration of furosemide to rats for five days resulted in a profound diuresis accompanied by a significant increase in PBR density (43%) that was apparent by the fifth day of treatment. Administration of hydrochlorothiazide or Ro 5-4864 for five days also caused diuresis and increased renal PBR density. Both the diuresis and increased density of PBR produced by Ro 5-4864 were blocked by coadministration of PK 11195, which alone had no effect on either PBR density or urine volume. The equilibrium binding constants of (/sup 3/H)Ro 5-4864 to cardiac membranes were unaffected by administration of any of these drugs. These findings suggest that renal PBR may be selectively modulated in vivo and in vitro by administration of ion transport/exchange inhibitors. 36 references, 4 tables.

  14. Benzodiazepines

    PubMed Central

    Greenblatt, D. J.; Shader, R. I.; Divoll, M.; Harmatz, J. S.

    1981-01-01

    1 The onset and duration of action of benzodiazepines after single oral doses depend largely on absorption rate and the rate and extent of distribution, respectively. 2 The rate and extent of accumulation during multiple dosage depend on elimination half-life and clearance. A framework is proposed for classification of benzodiazepines according to elimination half-life. 3 Long-acting benzodiazepines have half-life values usually exceeding 24 hours. Drugs in this category have long-acting pharmacologically active metabolites (often desmethyldiazepam), accumulate extensively during multiple dosage, and may have impaired clearance in the elderly and those with liver disease. 4 Intermediate and short-acting benzodiazepines have half-life values from 5-24 hours. Active metabolites are uncommon. Accumulation during multiple dosage is less extensive than with the long-acting group, and diminishes as the half-life becomes shorter. Age and liver disease have a small influence on metabolic clearance. 5 The half-life of ultrashort acting benzodiazepines is less than 5 hours. These drugs are essentially non-accumulating. 6 Pharmacokinetic classification may assist in understanding of differences among benzodiazepines, but does not explain all of their clinical actions. PMID:6133528

  15. Peripheral benzodiazepine receptor ligands reverse apoptosis resistance of cancer cells in vitro and in vivo.

    PubMed

    Decaudin, Didier; Castedo, Maria; Nemati, Fariba; Beurdeley-Thomas, Arnaud; De Pinieux, Gonzague; Caron, Antoine; Pouillart, Pierre; Wijdenes, John; Rouillard, Dany; Kroemer, Guido; Poupon, Marie-France

    2002-03-01

    The mitochondrial peripheral benzodiazepine receptor (mPBR) is involved in a functional structure designated as the permeability transition pore, which controls apoptosis. Binding of Fas/APO-1/CD95 triggers a prototypic apoptosis-inducing pathway. Using four different human tumor cell lines (T-cell Jurkat, neuroblastoma SHEP, osteosarcoma 143N2, and glioblastoma SNB79 cell lines), all of which express CD95 and mPBR, we investigated the potential role of mPBR ligands in CD95-induced apoptosis. We show that, in vitro, the three mPBR ligands tested (RO5-4864, PK11195, and diazepam) enhanced apoptosis induced by anti-CD95 antibody in Jurkat cells, as demonstrated by mitochondrial transmembrane potential drop and DNA fragmentation. In contrast, RO5-4864, but not PK11195 or diazepam, enhanced anti-CD95 apoptosis in all other cell lines. These effects were obtained in Bcl-2-overexpressing SHEP cell lines, but not in Bcl-X(L) SHEP cell lines. Enhancement of anti-CD95 antibody-induced apoptosis by RO5-4864 was characterized by an increased mitochondrial release of cytochrome c and Smac/DIABLO proteins and an enhanced activation of caspases 9 and 3, suggesting a mitochondrion-dependent mechanism. Preincubation of cells with the different mPBR ligands or anti-CD95 did not affect the levels of expression of either mPBR or CD95. In vivo, we found that the RO5-4864 mPBR ligand significantly increased the growth inhibition induced by two chemotherapeutic agents, etoposide and ifosfamide, using two human small cell lung cancers xenografted into nude mice. Peripheral benzodiazepine receptor ligands may therefore act as chemosensitizing agents for the treatment of human neoplasms.

  16. Peripheral-type benzodiazepine receptor: a protein of mitochondrial outer membranes utilizing porphyrins as endogenous ligands

    SciTech Connect

    Snyder, S.H.; Verma, A.; Trifiletti, R.R.

    1987-10-01

    The peripheral-type benzodiazepine receptor is a site identified by its nanomolar affinity for (/sup 3/H)diazepam, similar to the affinity of diazepam for the central-type benzodiazepine receptor in the brain. The peripheral type benzodiazepine receptor occurs in many peripheral tissues but has discrete localizations as indicated by autoradiographic studies showing uniquely high densities of the receptors in the adrenal cortex and in Leydig cells of the testes. Subcellular localization studies reveal a selective association of the receptors with the outer membrane of mitochondria. Photoaffinity labeling of the mitochondrial receptor with (/sup 3/H)flunitrazepam reveals two discrete labeled protein bands of 30 and 35 kDa, respectively. The 35-kDa band appears to be identical with the voltage-dependent anion channel protein porin. Fractionation of numerous peripheral tissues reveals a single principal endogenous ligand for the receptor, consisting of porphyrins, which display nanomolar affinity. Interactions of porphyrins with the mitochondrial receptor may clarify its physiological role and account for many pharmacological actions of benzodiazepines.

  17. Thiamine deficiency in cultured neuroblastoma cells: effect on mitochondrial function and peripheral benzodiazepine receptors.

    PubMed

    Bettendorff, L; Goessens, G; Sluse, F; Wins, P; Bureau, M; Laschet, J; Grisar, T

    1995-05-01

    When neuroblastoma cells were transferred to a medium of low (6 nM) thiamine concentration, a 16-fold decrease in total intracellular thiamine content occurred within 8 days. Respiration and ATP levels were only slightly affected, but addition of a thiamine transport inhibitor (amprolium) decreased ATP content and increased lactate production. Oxygen consumption became low and insensitive to oligomycin and uncouplers. At least 25% of mitochondria were swollen and electron translucent. Cell mortality increased to 75% within 5 days. [3H]PK 11195, a specific ligand of peripheral benzodiazepine receptors (located in the outer mitochondrial membrane) binds to the cells with high affinity (KD = 1.4 +/- 0.2 nM). Thiamine deficiency leads to an increase in both Bmax and KD. Changes in binding parameters for peripheral benzodiazepine receptors may be related to structural or permeability changes in mitochondrial outer membranes. In addition to the high-affinity (nanomolar range) binding site for peripheral benzodiazepine ligands, there is a low-affinity (micromolar range) saturable binding for PK 11195. At micromolar concentrations, peripheral benzodiazepines inhibit thiamine uptake by the cells. Altogether, our results suggest that impairment of oxidative metabolism, followed by mitochondrial swelling and disorganization of cristae, is the main cause of cell mortality in severely thiamine-deficient neuroblastoma cells.

  18. Increase of peripheral type benzodiazepine binding sites in kidney and olfactory bulb in acutely stressed rats.

    PubMed

    Novas, M L; Medina, J H; Calvo, D; De Robertis, E

    1987-03-17

    Fifteen minutes after the initiation of swimming stress in the rat we observed a 50% increase in the number of [3H]RO 5-4864 binding sites in kidney and a 37% increase in the olfactory bulb, without change in affinity. The binding in heart and cerebral cortex remained unchanged after the stress. These results are discussed in relation to previous work on both the action of an acute stress in central benzodiazepine receptors and the possible modulation of peripheral benzodiazepine receptors of the kidney by adrenocortical hormones.

  19. Aldosterone-reversible decrease in the density of renal peripheral benzodiazepine receptors in the rat after adrenalectomy

    SciTech Connect

    Basile, A.S.; Ostrowski, N.L.; Skolnick, P.

    1987-03-01

    A statistically significant decrease in the density of peripheral benzodiazepine receptors was observed in renal membranes of rats beginning 2 weeks after adrenalectomy when compared with sham-operated controls. This decrease in peripheral benzodiazepine receptor density was manifest as a decrease in the maximum binding of two ligands, (/sup 3/H)Ro 5-4864 and (/sup 3/H)PK 11195, without accompanying changes in their Kd for this site. Similar changes were not seen in another aldosterone-sensitive organ, the submandibular salivary gland. The decrease in peripheral benzodiazepine receptor density observed in adrenalectomized rat renal membranes was restored to control levels after 1 week of aldosterone administration using a dose (12.5 micrograms/kg/day) that had no effect on peripheral benzodiazepine receptor density in sham-operated animals. In contrast, dexamethasone administration (50 micrograms/kg/day, 1 week) had no effect on renal peripheral benzodiazepine receptor density when administered to either adrenalectomized or sham-operated rats. Further, adrenal demedullation had no effect on renal peripheral benzodiazepine receptor density or affinity. The decrease in peripheral benzodiazepine receptor density was localized to the renal cortex and the outer stripe of the medulla by gross dissection of renal slices and renal tissue section autoradiography. The specific effect of adrenalectomy on renal peripheral benzodiazepine receptor density, the lack of direct effect of aldosterone on (/sup 3/H) Ro 5-4864 binding and the localization of the change in peripheral benzodiazepine receptor density to the renal cortex and outer stripe suggest that these changes may reflect an adaptation of the renal nephron (possibly the distal convoluted tubule, intermediate tubule and/or the collecting duct) to the loss of mineralocorticoid hormones.

  20. Comparison of (/sup 14/C)-deoxyglucose metabolism and peripheral benzodiazepine receptor binding in rat C6 glioma

    SciTech Connect

    Richfield, E.K.; Ciliax, B.J.; Starosta-Rubinstein, S.R.; McKeever, P.E.; Penney, J.B.; Young, A.B.

    1988-08-01

    A rat C6 glioma tumor model has demonstrated that C6 tumor cells express a high density of peripheral benzodiazepine (BDZ) receptors. We report a poor correlation between the density of peripheral BDZ receptors and tumor metabolism using (/sup 14/C)-deoxyglucose in this C6 glioma model. We also compared the abilities of in vivo and in vitro BDZ receptor binding and tumor metabolism to measure the tumor area. All imaging methods were capable of estimating both the gross and total tumor area and had similar specificities and sensitivities. Because of a high signal-to-noise ratio, in vivo peripheral BDZ receptor binding may be a useful method for determining tumor size in human tumors expressing the receptor using PET.

  1. Treatment with dehydroepiandrosterone increases peripheral benzodiazepine receptors of mitochondria from cerebral cortex in D-galactose-induced aged rats.

    PubMed

    Chen, Chunfu; Lang, Senyang; Zuo, Pingping; Yang, Nan; Wang, Xiangqing

    2008-12-01

    The aim of this study was to determine whether dehydroepiandrosterone (DHEA) could regulate the expression of peripheral benzodiazepine receptors of mitochondria in cerebral cortex. The rats were divided into five groups. Those, in the vehicle-physiological or senescent group, received physiological or d-galactose (subcutaneously) once a day. Rats, in the vehicle-dimethyl sulfoxide- or DHEA-treated senescent group, received 2% of dimethyl sulfoxide or DHEA (intraperitoneally) every other day besides D-galactose (subcutaneously) once a day. Rats in the DHEA-treated normal group received physiological once a day and DHEA every other day. After 8-week, spatial learning was assessed for 5 days by water maze methods. Following behavioural testing, the cerebral cortex mitochondria were purified for PK11195 binding analysis. When compared to the respective vehicle, D-galactose alone induced a significant impairment in water maze performance accompanied by a reduction (30.7%) in peripheral benzodiazepine receptor density of mitochondria, and DHEA displayed a significant enhancement in learning memory accompanied by the elevation (18.3%) of peripheral benzodiazepine receptor density but not affinity in senescent rats. DHEA showed insignificant effects on both learning/memory ability and peripheral benzodiazepine receptors in normal rats when compared to physiological saline. These results suggest that chronic treatment with DHEA enhance cognitive function and increase peripheral benzodiazepine receptor density in cerebral cortex mitochondria in middle-aged senescent rats.

  2. Differential expression of the peripheral benzodiazepine receptor and gremlin during adipogenesis.

    PubMed

    Wade, F Marlene; Wakade, Chandramohan; Mahesh, Virendra B; Brann, Darrell W

    2005-05-01

    This study used the mRNA differential display technique to identify differentially expressed genes during the process of adipogenesis in the preadipocyte cell line, 3T3-L1. 3T3-L1 cells were treated with dexamethasone, isobutyl-1-methylxanthine, and insulin to induce differentiation into mature adipocytes. Cells were collected at three time-points during differentiation: Day 0 (d0), or nondifferentiated; Day 3 (d3), during differentiation; and Day 10 (d10), >90% of the cells had differentiated into mature adipocytes. Initial studies yielded 18 potentially differentially regulated cDNA candidates (8 down-regulated and 10 up-regulated). Reverse Northern and Northern blots confirmed differential expression of six of the candidates. Four of the candidates up-regulated on d3 and d10 were identified by sequence analysis to be lipoprotein lipase, a well-known marker of adipocyte differentiation. A fifth candidate that was expressed in d0, but not d3 or d10, was identified as DRM/gremlin, a bone morphogenetic protein antagonist. Finally, a sixth candidate that was increased at d3 and d10 was identified as the peripheral benzodiazepine receptor, which has been implicated in proliferation, differentiation, and cholesterol transport in cells. This study is the first to show that peripheral benzodiazepine receptor and DRM/gremlin are expressed in preadipocyte cell lines and that they are differentially regulated during adipogenesis.

  3. Visualization of specific binding sites of benzodiazepine in human brain

    SciTech Connect

    Shinotoh, H.; Yamasaki, T.; Inoue, O.; Itoh, T.; Suzuki, K.; Hashimoto, K.; Tateno, Y.; Ikehira, H.

    1986-10-01

    Using 11C-labeled Ro15-1788 and positron emission tomography, studies of benzodiazepine binding sites in the human brain were performed on four normal volunteers. Rapid and high accumulation of 11C activity was observed in the brain after i.v. injection of (11C)Ro15-1788, the maximum of which was within 12 min. Initial distribution of 11C activity in the brain was similar to the distribution of the normal cerebral blood flow. Ten minutes after injection, however, a high uptake of 11C activity was observed in the cerebral cortex and moderate uptake was seen in the cerebellar cortex, the basal ganglia, and the thalamus. The accumulation of 11C activity was low in the brain stem. This distribution of 11C activity was approximately parallel to the known distribution of benzodiazepine receptors. Saturation experiments were performed on four volunteers with oral administration of 0.3-1.8 mg/kg of cold Ro15-1788 prior to injection. Initial distribution of 11C activity following injection peaked within 2 min and then the accumulation of 11C activity decreased rapidly and remarkably throughout the brain. The results indicated that (11C) Ro15-1788 associates and dissociates to specific and nonspecific binding sites rapidly and has a high ratio of specific receptor binding to nonspecific binding in vivo. Carbon-11 Ro15-1788 is a suitable radioligand for the study of benzodiazepine receptors in vivo in humans.

  4. Effect of Diazepam on Severity of Acute Pancreatitis: Possible Involvement of Peripheral Benzodiazepine Receptors

    PubMed Central

    Abed, Alireza; Minaiyan, Mohsen; Safaei, Azadeh; Taheri, Diana

    2013-01-01

    Acute pancreatitis is a lethal inflammatory condition of pancreas with high mortality rate. There is a pressing need for research to explore active agents and novel mechanisms involving in the treatment of pancreatitis. Clinical studies have shown after the initial acinar cell injury plasma levels of pro-inflammatory cytokines are elevated in patients with acute pancreatitis and the degree of cytokine elevation correlates with disease severity. Diazepam may decrease interleukin release from macrophages, suppress neutrophil activities, and exhibit anti-inflammatory effects. So it is expected that in vivo pretreatment of acute pancreatitis with different doses of diazepam can attenuate its severity. Thus, we evaluated the effects of diazepam, intraperitoneally (5, 10, and 20 mg/kg i.p.), intracerebroventricularly (ICV 10 μg), and concurrently with flumazenil (1 mg/kg) on cerulein-induced acute pancreatitis in mice. Interestingly, the pretreatment with diazepam (5 mg/kg i.p.) reduced significantly the inflammatory response of acute pancreatitis by ameliorating pancreatic edema, amylase and lipase serum levels, myeloperoxidase activity, pancreatic TNF-alpha, and pathological alteration compared to control group. Diazepam i.c.v. was ineffective, suggesting that central benzodiazepine receptors have no significant role in this property. These results demonstrate that pretreatment with diazepam exhibits anti-inflammatory property in cerulein-induced acute pancreatitis possibly through peripheral benzodiazepine receptors. PMID:23956866

  5. The role of the peripheral benzodiazepine receptor in the apoptotic response to photodynamic therapy.

    PubMed

    Kessel, D; Antolovich, M; Smith, K M

    2001-08-01

    Several previous studies have suggested that the peripheral benzodiazepine receptor (PBR) on the mitochondrial surface was an important target for photodynamic therapy (PDT). In this study we compared PBR affinity vs photodynamic efficacy of protoporphyrin-IX (PP-IX) and two structural analogs, PP-III and PP-XIII, using murine leukemia L1210 cells in culture. The results indicate that the three agents have approximately equal hydrophobicity, affinity for L1210 cells and ability to initiate photodamage leading to an apoptotic response. But only PP-IX had significant affinity for the PBR. These data indicate that the relationship between PDT efficacy and PBR affinity may hold only for sensitizers with the PP-IX configuration.

  6. Preservation of peripheral benzodiazepine receptors: differential effects of freezing on (/sup 3/H)Ro 5-4864 and (3H)PK 11195 binding

    SciTech Connect

    Basile, A.S.; Ostrowski, N.L.; Skolnick, P.

    1987-04-01

    A statistically significant decrease in the density of peripheral benzodiazepine receptors was observed in renal membranes of rats beginning 2 weeks after adrenalectomy when compared with sham-operated controls. This decrease in peripheral benzodiazepine receptor density was manifest as a decrease in the Bmax of two ligands (/sup 3/H)Ro 5-4864 and (/sup 3/H)PK 11195, without accompanying changes in their apparent affinity (Kd) for this site. Similar changes were not seen in another aldosterone-sensitive organ, the submandibular salivary gland. The decrease in peripheral benzodiazepine receptor density in observed in adrenalectomized rat renal membranes was restored to control levels after 1 week of aldosterone administration using a dose (12.5 micrograms/kg/day) that had no effect on peripheral benzodiazepine receptor density in sham-operated animals. In contrast, dexamethasone administration (50 micrograms/kg/day, 1 week) had no effect on renal peripheral benzodiazepine receptor density when administered to either adrenalectomized or sham-operated rats. Further, adrenal demedullation had no effect on renal peripheral benzodiazepine receptor density or affinity. The decrease in peripheral benzodiazepine receptor density was localized to the renal cortex and the outer stripe of the medulla by gross dissection of renal slices and renal tissue section autoradiography. The specific effect of adrenalectomy on renal peripheral benzodiazepine receptor density, the lack of direct effect of aldosterone on (/sup 3/H)Ro 5-4864 binding, and the localization of the change in peripheral benzodiazepine receptor density to the renal cortex and outer stripe suggests that these changes may reflect an adaptation of the renal nephron (possibly the distal convoluted tubule, intermediate tubule and/or the collecting duct) to the loss of mineralocorticoid hormones.

  7. Central- and peripheral-type benzodiazepine receptors: similar regulation by stress and GABA receptor agonists.

    PubMed

    Rägo, L; Kiivet, R A; Harro, J; Pŏld, M

    1989-04-01

    Central- and peripheral-type benzodiazepine (BD) receptors were labelled either by 3H-flunitrazepam or 3H-Ro 5-4864 in vitro after stress and in vivo administration of GABAA and GABAB agonists. A significant increase in the density of cerebral cortex and kidney BD binding sites was observed in rats after forced swimming stress. Similar changes in both type of BD receptors were also followed when naive (stressed) and handling-habituated (unstressed) rats were used. Stress in both models was unable to change the affinity of BD receptors in cerebral cortex, but significantly lowered it in kidneys. Acute treatment of rats with muscimol (1.5 mg/kg) or (-)baclofen (5 mg/kg) resulted in marked increase in the affinity of BD binding not only in cerebral cortex but also in kidneys. After (-)baclofen treatment the number of BD binding sites was lowered in the structures studied. In a separate study mice selected according to their behavioral response to (-)baclofen (1 mg/kg) were studied. Two weeks after the selection it appeared that baclofen responders were behaviorally more "anxious" than baclofen nonresponders. The number of BD binding sites was reduced in cerebral cortex, cerebellum, heart and kidneys in baclofen responders as compared to baclofen nonresponders. In several cases the changes in peripheral BD binding sites were even more pronounced than those in central ones. The data presented here evidence that peripheral- and central-type BD receptors are regulated similarly by GABA and some models of stress. The physiological mechanisms involved in similar regulation of central- and peripheral-type BD receptors are yet unknown.

  8. Tissue specific regulation of peripheral-type benzodiazepine receptor density after chemical sympathectomy

    SciTech Connect

    Basile, A.S.; Skolnick, P.

    1988-01-01

    The characteristics of (/sup 3/H)Ro 5-4864 binding to peripheral benzodiazepine receptors (PBR) in the central nervous system and peripheral tissues were examined after chemical sympathectomy with 6-hydroxydopamine (6-OHDA). One week after the intracisternal administration of 6-OHDA, the number of (/sup 3/H)Ro 5-4864 binding sites (Bmax) in the hypothalamus and striatum increased 41 and 50% respectively, concurrent with significant reductions in catecholamine content. An increase (34%) in the Bmax of (/sup 3/H)Ro 5-4864 to cardiac ventricle was observed one week after parenteral 6-OHDA administration. In contrast, the B/sub max/ of (/sup 3/H)Ro 5-4684 to pineal gland decreased 48% after 6-OHDA induced reduction in norepinephrine content. The Bmax values for (/sup 3/H)Ro 5-4864 binding to other tissues (including lung, kidney, spleen, cerebral cortex, cerebellum, hippocampus and olfactory bulbs) were unaffected by 6-OHDA administration. The density of pineal, but not cardiac PBR was also reduced after reserpine treatment, an effect reversed by isoproterenol administration. These findings demonstrate that alterations in sympathetic input may regulate the density of PBR in both the central nervous system and periphery in a tissue specific fashion. 33 references, 4 tables.

  9. Development of a unique 3D interaction model of endogenous and synthetic peripheral benzodiazepine receptor ligands

    NASA Astrophysics Data System (ADS)

    Cinone, Nunzia; Höltje, Hans-Dieter; Carotti, Angelo

    2000-11-01

    Different classes of Peripheral-type Benzodiazepine Receptor (PBR) ligands were examined and common structural elements were detected and used to develop a rational binding model based on energetically allowed ligand conformations. Two lipophilic regions and one electrostatic interaction site are essential features for high affinity ligand binding, while a further lipophilic region plays an important modulator role. A comparative molecular field analysis, performed over 130 PBR ligands by means of the GRID/GOLPE methodology, led to a PLS model with both high fitting and predictive values (r2 = 0.898, Q2 = 0.761). The outcome from the 3D QSAR model and the GRID interaction fields computed on the putative endogenous PBR ligands DBI (Diazepam Binding Inhibitor) and TTN (Tetracontatetraneuropeptide) was used to identify the amino acids most probably involved in PBR binding. Three amino acids, bearing lipophilic side chains, were detected in DBI (Phe49, Leu47 and Met46) and in TTN (Phe33, Leu31 and Met30) as likely residues underlying receptor binding. Moreover, a qualitative comparison of the molecular electrostatic potentials of DBI, TTN and selected synthetic ligands indicated also similar electronic properties. Convergent results from the modeling studies of synthetic and endogenous ligands suggest a common binding mode to PBRs. This may help the rational design of new high affinity PBR ligands.

  10. Nootropic nefiracetam inhibits proconvulsant action of peripheral-type benzodiazepines in epileptic mutant EL mice.

    PubMed

    Nakamoto, Yurie; Shiotani, Tadashi; Watabe, Shigeo; Nabeshima, Toshitaka; Yoshii, Mitsunobu

    2004-10-01

    Piracetam and structurally related nootropics are known to potentiate the anticonvulsant effects of antiepileptic drugs. It remains to be seen, however, whether these nootropics inhibit proconvulsant actions of many toxic agents including Ro 5-4864, a specific agonist for peripheral-type benzodiazepine receptors (PBR). The present study was designed to address this issue using EL mice, an animal model of epilepsy. In behavioral pharmacological experiments, EL mice were highly susceptible to convulsions induced by Ro 5-4864 (i.p.) in comparison with nonepileptic DDY mice. Nefiracetam administered orally to EL mice inhibited spontaneous seizures. In DDY mice, convulsions induced by Ro 5-4864 were prevented by nefiracetam when administered by i.v. injection. Aniracetam (i.v.) was partially effective, but piracetam and oxiracetam were ineffective as anticonvulsants. Binding assay for brain tissues revealed a higher density of mitochondrial PBR in EL mice compared with DDY mice. Binding of the PBR ligands Ro 5-4864 to either EL or DDY mouse brain was inhibited by micromolar concentrations of these nootropic agents in the sequence of nefiracetam > aniracetam > oxiracetam, piracetam. This rank order is identical to potency as anticonvulsants. These data suggest that nefiracetam may prevent toxic effects of PBR agonists through interacting with PBR.

  11. Interactions of pyrethroid insecticides with GABA sub A and peripheral-type benzodiazepine receptors

    SciTech Connect

    Devaud, L.L.

    1988-01-01

    Pyrethroid insecticides are potent proconvulsants in the rat. All pyrethroids evincing proconvulsant activity elicited a similar 25-30% maximal reduction of seizure threshold. The Type II pyrethroids were the most potent proconvulsants with 1R{alpha}S, cis cypermethrin having an ED{sub 50} value of 6.3 nmol/kg. The proconvulsant activity of both Type I and Type II pyrenthroids was blocked by pretreatment with PK 11195, the peripheral-type benzodiazepine receptor (PTBR) antagonist. In contrast, phenytoin did not antagonize the proconvulsant activity of either deltamethrin or permethrin. Pyrethroids displaced the specific binding of ({sup 3}H)Ro5-4864 to rat brain membranes with a significant correlation between the log EC{sub 50} values for their activities as proconvulsants and the log IC{sub 50} values for their inhibition of ({sup 3}H)Ro5-4864 binding. Both Ro5-4864 and pyrethroid insecticides were found to influence specific ({sup 35}S)TBPS binding in a GABA-dependent manner. PK 11195 and the Type II pyrethroid, deltamethrin antagonized the Ro5-4864-induced modulation of ({sup 35}S)TBPS binding. Pyrethroid insecticides, Ro5-4864 and veratridine influenced GABA-gated {sup 36}Chloride influx. Moreover, the Type II pyrethroids elicited an increase in {sup 36}chloride influx in the absence of GABA-stimulation. Both of these actions were antagonized by PK 11195 and tetrodotoxin.

  12. Development of a monoclonal anitbody to immuno-cytochemical analysis of the cellular localization of the peripheral benzodiazepine receptor

    SciTech Connect

    Dussossoy, D.; Carayon, P.; Feraut, D.

    1996-05-01

    Based on the amino acid sequence deduced from the cloned human peripheral benzodiazepine receptor (PBR) gene, monoclonal antibody (Mab 8D7) was produced against the C-terminal fragment of the receptor. Immunoblot experiments, performed against purified PBR, indicated that the antipeptide antibody recognized, under denaturing conditions, the corresponding amino acid sequence of the PBR. When mitochondrial membranes form PBR transfected yeast or from THP1 and U937 cells were used on immunoblot analysis, a high level of immunoreactivity was observed at 18 kDa, the PBR molecular mass deduced from cDNA, establishing the specificity of the antibody for the receptor. Moreover, binding experiments realized with intact mitochondria demonstrated that the immunogenic sequence was accessible to the antibody indicating that the C-terminal fragment of the PBR faces the cytosol. Using this Mab we developed a technique which allowed precise quantification of PBR density per cell. Furthermore, cellular localization studies by flow cytometric analysis and confocal microscopy on cell lines displaying different levels of PBR showed that Mab 8D7 was entirely colocalized with an antimitochondria Mab. 34 refs., 7 figs.

  13. Environmentally induced changes in peripheral benzodiazepine receptors are stressor and tissue specific.

    PubMed

    Drugan, R C; Holmes, P V; Scher, D M; Luczak, S; Oh, H; Ferland, R J

    1995-04-01

    The stress-induced changes in peripheral benzodiazepine receptors (PBR) can be observed in a number of different tissues, depending upon the nature and chronicity of the aversive experience. In addition, virtually all stress procedures that cause rapid changes in PBR simultaneously increase the physical activity or metabolic rate of the subjects. The present study analyzed the contributions of rapid alterations in activity or metabolic rate with and without aversive stimulation and their subsequent impact on PBR. Mechanically induced increases in activity by forced running stress results in a significant reduction in [3H]Ro 5-4864 binding to PBR in olfactory bulb, opposite to the PBR changes in this tissue following forced cold-water swim stress. Pharmacological induction of increased locomotor activity as well as metabolic rate by d-amphetamine causes a significant increase in cardiac PBR binding, again, opposite to the response typically observed following inescapable shock stress. Finally, administration of the anxiogenic beta-carboline, FG-7142, causes increases in both hippocampus and adrenal gland PBR binding reminiscent of acute noise stress exposure. These experiments demonstrate that increased locomotor activity or metabolic rate alone is not a necessary and sufficient condition for previous stress-induced changes in PBR. Conversely, increased metabolic rate coupled with an aversive stimulus appears to be an important factor for inducing stress-like changes in PBR. This data, coupled with previous reports, suggests that rapid alterations in these sites are stressor and tissue dependent. Finally, we propose that the PBR may be involved in many aspects of the stress response including: a) a blowarning system in adrenal gland, b) participation in stress-induced hypertension via renal PBR, and c) a modulator of stress-induced immunosuppression and subsequent recovery of function or recuperation by actions on immune cells.

  14. Selective pharmacological modulation of renal peripheral-type benzodiazepine binding by treatment with diuretic drugs

    SciTech Connect

    Lukeman, D.S.; Vaughn, D.A.; Fanestil, D.D.

    1988-01-01

    The authors have assessed the effects of in vivo administration of different classes of diuretic drugs on the expression of the peripheral-type benzodiazepine binding site (PBBS) in crude membranes derived from the cortex and outer medulla of rat kidney by saturation analysis with the PBBS-selective ligands (/sup 3/H)RO5-4864 and (/sup 3/H)PH 11195 in cortex and (/sup 3/H)RO5-4864 in outer medulla. Administration for 14-15 days of furosemide, a drug that blocks NaCl-KCl coupled transport in the thick ascending limb of the loop of Henle, produced a significant doubling in the PBBS density (B/sub max/) in outer medulla, a region of the kidney rich in thick ascending limbs, and produced a lesser but significant increase in PBBS density in the cortex. Conversely, administration for 14-15 days of the carbonic anhydrase inhibitor acetazolamide, which acts predominantly in the proximal tubule, and hydrochlorothiazide, which acts predominantly in the early distal tubule, elicited statistically significant increases in PBBS density in renal cortex but not in renal outer medulla. Furthermore, all drug treatments were without effect on the equilibrium dissociation constants (K/sub d/s) of (/sup 3/H)RO5-4864 and (/sup 3/H)PK 11195 binding to cortical and outer medullary membrane preparations. These findings demonstrate that the PBBS can be selectively up-regulated in different regions of the kidney by diuretic drugs with different modes/sites of action. 50 references, 1 table.

  15. Radiosynthesis and initial evaluation of [18F]-FEPPA for PET imaging of peripheral benzodiazepine receptors.

    PubMed

    Wilson, Alan A; Garcia, Armando; Parkes, Jun; McCormick, Patrick; Stephenson, Karin A; Houle, Sylvain; Vasdev, Neil

    2008-04-01

    A novel [18F]-radiolabelled phenoxyanilide, [18F]-FEPPA, has been synthesized and evaluated, in vitro and ex vivo, as a potential positron emission tomography imaging agent for the peripheral benzodiazepine receptor (PBR). [18F]-FEPPA and two other radiotracers for imaging PBR, namely [11C]-PBR28 and [11C]-PBR28-d3, were synthesised and evaluated in vitro and ex vivo as potential PBR imaging agents. [18F]-FEPPA is efficiently prepared in one step from its tosylate precursor and [18F]-fluoride in high radiochemical yields and at high specific activity. FEPPA displayed a Ki of 0.07 nM for PBR in rat mitochondrial membrane preparations and a suitable lipophilicity for brain penetration (log P of 2.99 at pH 7.4). Upon intravenous injection into rats, [18F]-FEPPA showed moderate brain uptake [standard uptake value (SUV) of 0.6 at 5 min] and a slow washout (SUV of 0.35 after 60 min). Highest uptake of radioactivity was seen in the hypothalamus and olfactory bulb, regions previously reported to be enriched in PBR in rat brain. Analysis of plasma and brain extracts demonstrated that [18F]-FEPPA was rapidly metabolized, but no lipophilic metabolites were observed in either preparation and only 5% radioactive metabolites were present in brain tissue extracts. Blocking studies to determine the extent of specific binding of [18F]-FEPPA in rat brain were problematic due to large perturbations in circulating radiotracer and the lack of a reference region. Further evaluation of the potential of [18F]-FEPPA will require the employment of rigorous kinetic models and/or appropriate animal models.

  16. PET imaging of brain macrophages using the peripheral benzodiazepine receptor in a macaque model of neuroAIDS

    PubMed Central

    Venneti, Sriram; Lopresti, Brian J.; Wang, Guoji; Bissel, Stephanie J.; Mathis, Chester A.; Meltzer, Carolyn C.; Boada, Fernando; Capuano, Saverio; Kress, Geraldine J.; Davis, Denise K.; Ruszkiewicz, James; Reynolds, Ian J.; Murphey-Corb, Michael; Trichel, Anita M.; Wisniewski, Stephen R.; Wiley, Clayton A.

    2004-01-01

    HIV infection in humans and simian immunodeficiency virus (SIV) infection in macaques result in encephalitis in approximately one-quarter of infected individuals and is characterized by infiltration of the brain with infected and activated macrophages. 1-(2-chlorphenyl)-N-methyl-N-(1-methylpropyl)-3-isoquinoline-carboxamide (PK11195) is a ligand specific for the peripheral benzodiazepine receptor abundant on macrophages and is expressed in low levels in the noninfected brain. We hypothesized that positron-emission tomography (PET) with the carbon-11–labeled, R-enantiomer form of PK11195 ([11C](R)-PK11195) could image brain macrophages and hence the development of encephalitis in vivo. [11C](R)-PK11195 binding was assessed in the brain using PET in 11 SIV infected macaques, six of which showed increased binding in vivo. Postmortem examination of the brain in these six macaques demonstrated encephalitis, while macaques that did not show an increase in [11C](R)-PK11195 binding did not develop SIV encephalitis. Brain tissue from SIV encephalitic macaques also showed increased [3H](R)-PK11195 binding compared with binding in nonencephalitic macaques. Increased PK11195 binding in vivo and in postmortem brain tissue correlated with abundance of macrophages but not astrocytes. Our results suggest that PET [11C](R)-PK11195 imaging can detect the presence of macrophages in SIV encephalitis in vivo and may be useful to predict the development of HIV encephalitis and in studies of the pathogenesis and treatment of HIV dementia. PMID:15057304

  17. Regulation of the mitochondrial permeability transition pore by the outer membrane does not involve the peripheral benzodiazepine receptor (Translocator Protein of 18 kDa (TSPO)).

    PubMed

    Šileikytė, Justina; Blachly-Dyson, Elizabeth; Sewell, Randall; Carpi, Andrea; Menabò, Roberta; Di Lisa, Fabio; Ricchelli, Fernanda; Bernardi, Paolo; Forte, Michael

    2014-05-16

    Translocator protein of 18 kDa (TSPO) is a highly conserved, ubiquitous protein localized in the outer mitochondrial membrane, where it is thought to play a key role in the mitochondrial transport of cholesterol, a key step in the generation of steroid hormones. However, it was first characterized as the peripheral benzodiazepine receptor because it appears to be responsible for high affinity binding of a number of benzodiazepines to non-neuronal tissues. Ensuing studies have employed natural and synthetic ligands to assess the role of TSPO function in a number of natural and pathological circumstances. Largely through the use of these compounds and biochemical associations, TSPO has been proposed to play a role in the mitochondrial permeability transition pore (PTP), which has been associated with cell death in many human pathological conditions. Here, we critically assess the role of TSPO in the function of the PTP through the generation of mice in which the Tspo gene has been conditionally eliminated. Our results show that 1) TSPO plays no role in the regulation or structure of the PTP, 2) endogenous and synthetic ligands of TSPO do not regulate PTP activity through TSPO, 3) outer mitochondrial membrane regulation of PTP activity occurs though a mechanism that does not require TSPO, and 4) hearts lacking TSPO are as sensitive to ischemia-reperfusion injury as hearts from control mice. These results call into question a wide variety of studies implicating TSPO in a number of pathological processes through its actions on the PTP.

  18. Downregulation of (3H)Ro5-4864 binding sites after exposure to peripheral-type benzodiazepines in vitro

    SciTech Connect

    Johnson, M.D.; Wang, J.K.; Morgan, J.I.; Spector, S.

    1986-09-01

    Peripheral-type benzodiazepine (BZD) binding sites undergo a rapid and pronounced downregulation after exposure to these compounds in vitro. Friend erythroleukemia cells were incubated with micromolar concentrations of BZD after which they were washed thoroughly and the binding of the specific peripheral-type BZD radioligand (/sup 3/H)Ro5-4864 was determined. Exposure to the peripheral-type BZD Ro7-3351 decreased the number of (/sup 3/H)Ro5-4864 binding sites from 324 to 41 fmol/10(6) cells with no change in affinity. Downregulation appears to require active cellular processes because it is blocked when exposure to BZD is at 4/sup 0/C rather than at 37/sup 0/C. Furthermore, whereas (/sup 3/H)Ro5-4864 binding is decreased substantially in membrane preparations made from downregulated cells, it is not altered when membrane preparations from control cells are exposed to BZD. The time course of downregulation is quite rapid, as it occurs within minutes. In contrast, the return of sites requires days and there is a close relationship between return of sites and growth of new cells. The ability of BZDs to downregulate correlates more closely with affinity for the peripheral-type site than with biological activity. The ability to undergo downregulation is characteristic of receptors and its occurrence suggests that peripheral-type BZD binding sites are functional receptors.

  19. DMT1 (IRE) expression in intestinal and erythroid cells is regulated by peripheral benzodiazepine receptor-associated protein 7

    PubMed Central

    Okazaki, Yasumasa; Ma, Yuxiang; Yeh, Mary; Yin, Hong; Li, Zhen; Yeh, Kwo-yih

    2012-01-01

    The divalent metal transporter 1 (DMT1) is essential for cellular uptake of iron, mediating iron absorption across the duodenal brush border membrane. We have previously shown that with iron feeding DMT1 in the brush border membrane undergoes endocytosis into the subapical compartment of enterocytes. To understand the mechanisms of iron-induced endocytosis of DMT1, we used the yeast two-hybrid system to find proteins that interact with DMT1 and isolated from a rat duodenal cDNA library a protein that interacts specifically with the IRE containing isoform of DMT1 {DMT1 [iron-responsive element (IRE)]}. The protein (Genbank AY336075) is 97.5% identical with peripheral benzodiazepine receptor-associated protein 7 (PAP7), a protein that interacts with the peripheral benzodiazepine receptor. PAP7 is ubiquitously expressed in the rat and in multiple cell lines with consensus sequences including a nuclear localization signal and a Golgi dynamic domain. PAP7, expressed on the brush border of rat duodenum, copurified with DMT1 in brush border membrane vesicles, and following iron feeding, was internalized in parallel with the internalization of DMT1. To determine if PAP7 plays a role in cellular iron metabolism, we downregulated PAP7 expression in K562 cells with small interfering RNA. Following the decrease in PAP7 protein, DMT1 (IRE) protein but not mRNA was significantly downregulated but without effect on DMT1 (non-IRE), transferin (Tf)R1, or ferritin expression. Lowered levels of PAP7 resulted also in decreased cell proliferation and G1 cell cycle arrest. These data are consistent with PAP7 interacting with DMT1 (IRE) and regulating DMT1 (IRE) expression in K562 cells by modulating expression of DMT1 (IRE) protein. PMID:22383495

  20. Early ontogeny of the central benzodiazepine receptor in human embryos and fetuses

    SciTech Connect

    Hebebrand, J.; Hofmann, D.; Reichelt, R.; Schnarr, S.; Knapp, M.; Propping, P.; Foedisch, H.J.

    1988-01-01

    The early ontogeny of the central benzodiazepine receptor (BZR) was investigated in human embryos and fetuses between 7 and 26 weeks of gestation. Brain tissue was gained from terminated pregnancies or spontaneous abortions. Binding studies, which were performed with /sup 3/H-flunitrazepam (FNZ), revealed that specific benzodiazepine binding is already detectable at an embryonal age of 7 weeks post conception. Binding at this early stage can be displaced potently by clonazepam and the inverse agonist ..beta..-CCE. Additionally, /sup 3/H-FNZ binding is enhanced by GABA. Thus, benzodiazepine binding is of the central type. Receptor density increases steeply in whole brain between weeks 8 and 11 of gestation. In frontal cortex receptor density increases gradually between weeks 12 and 26 of gestation. No specific fetal disease entity (including trisomy 21) was consistently associated with exceptionally high or low B/sub max/-values.

  1. Modulation of cholinephosphotransferase activity in breast cancer cell lines by Ro5-4864, a peripheral benzodiazepine receptor agonist

    SciTech Connect

    Akech, Jacqueline; Roy, Somdutta Sinha; Das, Salil K. . E-mail: sdas@mmc.edu

    2005-07-22

    Changes in phospholipid and fatty acid profile are hallmarks of cancer progression. Increase in peripheral benzodiazepine receptor expression has been implicated in breast cancer. The benzodiazepine, Ro5-4864, increases cell proliferation in some breast cancer cell lines. Biosynthesis of phosphatidylcholine (PC) has been identified as a marker for cells proliferating at high rates. Cholinephosphotransferase (CPT) is the terminal enzyme for the de novo biosynthesis of PC. We have addressed here whether Ro5-4864 facilitates some cancer causing mechanisms in breast cancer. We report that cell proliferation increases exponentially in aggressive breast cancer cell lines 11-9-1-4 and BT-549 when treated with nanomolar concentrations of Ro5-4864. This increase is seen within 24 h of treatment, consistent with the cell doubling time in these cells. Ro5-4864 also upregulates c-fos expression in breast cancer cell lines 11-9-1-4 and BT-549, while expression in non-tumorigenic cell line MCF-12A was either basal or slightly downregulated. We further examined the expression of the CPT gene in breast cancer (11-9-1-4, BT-549) and non-tumorigenic cell lines (MCF-12A, MCF-12F). We found that the CPT gene is overexpressed in breast cancer cell lines compared to the non-tumorigenic cell lines. Furthermore, the activity of CPT in forming PC is increased in the breast cancer cell lines cultured for 24 h. Additionally, we examined the CPT activity in the presence of nanomolar concentrations of Ro5-4864. Biosynthesis of PC was increased in breast cancer cell lines upon treatment. We therefore propose that Ro5-4864 facilitates PC formation, a process important in membrane biogenesis for proliferating cells.

  2. Modulatory action of benzodiazepines on human term placental steroidogenesis in vitro.

    PubMed

    Barnea, E R; Fares, F; Gavish, M

    1989-07-01

    Peripheral benzodiazepine (BZ) binding sites (PBzS) were characterized on placental explant membranes. [3H]PK 11195, an isoquinoline carboxamide derivative, which is a ligand specific for PBzS, labeled these sites with an equilibrium dissociation constant of 2.1 nM; the maximal number of binding sites was 396 fmol/mg protein. The effect of various BZ ligands and PK 11195 on the secretion of progesterone (P4) and estradiol-17 beta (E2) from human term placental explants was studied. Exposure of placental explants to low doses (10(-8) M) of Ro 5-4864, a BZ ligand which binds with high affinity to PBzS, caused a significant (P less than 0.05) increase in the secretion of P4 and E2 into the media (2.4- and 1.4-fold, respectively). On the other hand, high doses (10(-5) M) of Ro 5-4864 caused a significant (P less than 0.05) decrease in the secretion of P4 and E2 into the media. Also, exposure of explants to diazepam (10(-7) M) and PK 11195 (10(-6) M) caused a significant increase in P4 and E2 secretion into the media. In contrast, clonazepam, a BZ ligand specific for the central-type receptors, had no effect on the secretion of either steroid. The combination of diazepam (10(-7) M) or Ro 5-4864 (10(-8) M) with PK 11195 (10(-6) M) did not enhance the stimulatory effects obtained with each agent alone. The effects exerted by Ro 5-4864, PK 11195, and diazepam may be mediated via PBzS.

  3. Peripheral benzodiazepine receptor ligand PK11195 reduces microglial activation and neuronal death in quinolinic acid-injected rat striatum.

    PubMed

    Ryu, Jae K; Choi, Hyun B; McLarnon, James G

    2005-11-01

    The effects of the peripheral benzodiazepine receptor (PBR) ligand, PK11195, were investigated in the rat striatum following the administration of quinolinic acid (QUIN). Intrastriatal QUIN injection caused an increase of PBR expression in the lesioned striatum as demonstrated by immunohistochemical analysis. Double immunofluorescent staining indicated PBR was primarily expressed in ED1-immunoreactive microglia but not in GFAP-immunoreactive astrocytes or NeuN-immunoreactive neurons. PK11195 treatment significantly reduced the level of microglial activation and the expression of pro-inflammatory cytokines and iNOS in QUIN-injected striatum. Oxidative-mediated striatal QUIN damage, characterized by increased expression of markers for lipid peroxidation (4-HNE) and oxidative DNA damage (8-OHdG), was significantly diminished by PK11195 administration. Furthermore, intrastriatal injection of PK11195 with QUIN significantly reduced striatal lesions induced by the excitatory amino acid and diminished QUIN-mediated caspase-3 activation in striatal neurons. These results suggest that inflammatory responses from activated microglia are damaging to striatal neurons and pharmacological targeting of PBR in microglia may be an effective strategy in protecting neurons in neurological disorders such as Huntington's disease.

  4. Regulation of the inner membrane mitochondrial permeability transition by the outer membrane translocator protein (peripheral benzodiazepine receptor).

    PubMed

    Sileikyte, Justina; Petronilli, Valeria; Zulian, Alessandra; Dabbeni-Sala, Federica; Tognon, Giuseppe; Nikolov, Peter; Bernardi, Paolo; Ricchelli, Fernanda

    2011-01-14

    We studied the properties of the permeability transition pore (PTP) in rat liver mitochondria and in mitoplasts retaining inner membrane ultrastructure and energy-linked functions. Like mitochondria, mitoplasts readily underwent a permeability transition following Ca(2+) uptake in a process that maintained sensitivity to cyclosporin A. On the other hand, major differences between mitochondria and mitoplasts emerged in PTP regulation by ligands of the outer membrane translocator protein of 18 kDa, TSPO, formerly known as the peripheral benzodiazepine receptor. Indeed, (i) in mitoplasts, the PTP could not be activated by photo-oxidation after treatment with dicarboxylic porphyrins endowed with protoporphyrin IX configuration, which bind TSPO in intact mitochondria; and (ii) mitoplasts became resistant to the PTP-inducing effects of N,N-dihexyl-2-(4-fluorophenyl)indole-3-acetamide and of other selective ligands of TSPO. Thus, the permeability transition is an inner membrane event that is regulated by the outer membrane through specific interactions with TSPO.

  5. Synthesis and evaluation in monkey of two sensitive 11C-labeled aryloxyanilide ligands for imaging brain peripheral benzodiazepine receptors in vivo.

    PubMed

    Briard, Emmanuelle; Zoghbi, Sami S; Imaizumi, Masao; Gourley, Jonathan P; Shetty, H Umesha; Hong, Jinsoo; Cropley, Vanessa; Fujita, Masahiro; Innis, Robert B; Pike, Victor W

    2008-01-10

    We sought to develop (11)C-labeled ligands for sensitive imaging of brain peripheral benzodiazepine receptors (PBR) in vivo. Two aryloxyanilides with high affinity for PBR were identified and synthesized, namely, N-acetyl- N-(2-methoxycarbonylbenzyl)-2-phenoxyaniline ( 3, PBR01) and N-(2-methoxybenzyl)- N-(4-phenoxypyridin-3-yl)acetamide ( 10, PBR28). 3 was hydrolyzed to 4, which was esterified with [ (11)C]iodomethane to provide [ (11)C] 3. The O-desmethyl analogue of 10 was converted into [ (11)C] 10 with [ (11)C]iodomethane. [ (11)C] 3 and [ (11)C] 10 were each injected into monkey to assess their brain kinetics with positron emission tomography (PET). After administration of either radioligand there was moderately high brain uptake of radioactivity. Receptor blocking and displacement experiments showed that a high proportion of this radioactivity was bound specifically to PBR. In monkey and rat, 3 and 10 were rapidly metabolized by ester hydrolysis and N-debenzylation, respectively, each to a single polar radiometabolite. [ (11)C] 3 and [ (11)C] 10 are effective for imaging PBR in monkey brain. [ (11)C] 10 especially warrants further evaluation in human subjects.

  6. Single-step High-yield Radiosynthesis and Evaluation of a Sensitive 18F-Labeled Ligand for Imaging Brain Peripheral Benzodiazepine Receptors with PET

    PubMed Central

    Briard, Emmanuelle; Zoghbi, Sami S.; Siméon, Fabrice G.; Imaizumi, Masao; Gourley, Jonathan P.; Shetty, H. Umesha; Lu, Shuiyu; Fujita, Masahiro; Innis, Robert B.; Pike, Victor W.

    2009-01-01

    Elevated levels of peripheral benzodiazepine receptors (PBR) are associated with activated microglia in their response to inflammation. Hence, PBR imaging in vivo is valuable for investigating brain inflammatory conditions. Sensitive, easily prepared and readily available radioligands for imaging with positron emission tomography (PET) are desirable for this purpose. We describe a new 18F-labeled PBR radioligand, namely [18F]N-fluoroacetyl-N-(2,5-dimethoxybenzyl)-2-phenoxyaniline ([18F]9). [18F]9 was produced easily through a single and highly efficient step, the reaction of [18F]fluoride ion with the corresponding bromo precursor, 8. Ligand 9 exhibited high affinity for PBR in vitro. PET showed that [18F]9 was avidly taken into monkey brain and gave a high ratio of PBR-specific to nonspecific binding. [18F]9 was devoid of defluorination in rat and monkey and gave predominantly polar radiometabolite(s). In rat, a low level radiometabolite of intermediate lipophilicity was identified as [18F]2-fluoro-N-(2-phenoxyphenyl)acetamide ([18F]11). [18F]9 is a promising radioligand for future imaging of PBR in living human brain. PMID:19119848

  7. Benzodiazepine self-administration in humans and laboratory animals--implications for problems of long-term use and abuse.

    PubMed

    Griffiths, R R; Weerts, E M

    1997-11-01

    Drug reinforcement may represent the primary behavioral-pharmacological mechanism underlying two types of problematic use of benzodiazepines--recreational abuse by polydrug abusers and inappropriate chronic use by patients. High dose polydrug abuse for the purpose of getting high is readily recognized as a significant social problem. Inappropriate chronic benzodiazepine use is more subtle but relatively common: for anxiolytics, 36% of past-year users (3% of the adult population in the US) report using these drugs for 4 consecutive months or longer. The risks of such long-term use are much better documented than the benefits. This paper provides a current review of various problems that have been identified with the long-term use and the recreational abuse of benzodiazepines, including memory impairment, risk of accidents, falls and hip fractures in the elderly, a withdrawal syndrome, brain damage, overuse in the elderly, overuse by chronic pain patients, overuse by alcoholics and recreational abuse among alcoholics and polydrug abusers. A comprehensive review of the literature on benzodiazepine reinforcing effects in humans and laboratory animals is also provided. Drug self-administration studies in humans and laboratory animals provide models of both types of problematic benzodiazepine use. Recreational abuse of benzodiazepines has been modeled in human research with polydrug abusers and in laboratory animal studies, which show that the reinforcing effect of benzodiazepines is intermediate relative to other sedative compounds and is increased in subjects with histories of previous sedative drug self-administration. The problem of inappropriate long-term use of benzodiazepines by people without histories of drug abuse has been partially modeled in human studies showing that benzodiazepines function as reinforcers in subjects with anxiety, insomnia, and histories of moderate alcohol consumption, and in preclinical studies showing stable, low-rate benzodiazepine self

  8. Human biodistribution and dosimetry of the SPECT benzodiazepine receptor radioligand iodine-123-iomazenil

    SciTech Connect

    Dey, H.M.; Seibyl, J.P.; Stubbs, J.B.

    1994-03-01

    SPECT imaging of the brain with [{sup 123}I]iomazenil has shown avid uptake of the radioligand in a distribution consistent with benzodiazepine receptor binding. The purposes of this study were to measure the whole-body distribution of activity following i.v. adminstration of [{sup 123}I]iomazenil and to evaluate the resulting organ radiation burdens. Serial total body scans were obtained in healthy volunteers after thyroid blockade and demonstrated avid brain uptake of radioligand. Abdominal imaging showed significant activity retention within the urinary and gastrointestinal tracts consistent with excretion via these routes. Absorbed dose to the urinary bladder was calculated to be 0.19 mGy/MBq, to the lower large intestine 0.079 mGy/MBq, to the upper large intestine 0.066 mGy/MBq, and to the thyroid 0.063 mGy/MBq. Thyroid uptake may in part have represented binding to benzodiazepine receptors, since radioligand binding to tissue homogenates prepared from human thyroid showed the presence of benzodiazepine binding sites. 20 refs., 4 figs., 2 tabs.

  9. Peripheral Benzodiazepine Receptor/Translocator Protein Global Knock-out Mice Are Viable with No Effects on Steroid Hormone Biosynthesis*♦

    PubMed Central

    Tu, Lan N.; Morohaku, Kanako; Manna, Pulak R.; Pelton, Susanne H.; Butler, W. Ronald; Stocco, Douglas M.; Selvaraj, Vimal

    2014-01-01

    Translocator protein (TSPO), previously known as the peripheral benzodiazepine receptor, is a mitochondrial outer membrane protein implicated as essential for cholesterol import to the inner mitochondrial membrane, the rate-limiting step in steroid hormone biosynthesis. Previous research on TSPO was based entirely on in vitro experiments, and its critical role was reinforced by an early report that claimed TSPO knock-out mice were embryonic lethal. In a previous publication, we examined Leydig cell-specific TSPO conditional knock-out mice that suggested TSPO was not required for testosterone production in vivo. This raised controversy and several questions regarding TSPO function. To examine the definitive role of TSPO in steroidogenesis and embryo development, we generated global TSPO null (Tspo−/−) mice. Contrary to the early report, Tspo−/− mice survived with no apparent phenotypic abnormalities and were fertile. Examination of adrenal and gonadal steroidogenesis showed no defects in Tspo−/− mice. Adrenal transcriptome comparison of gene expression profiles showed that genes involved in steroid hormone biosynthesis (Star, Cyp11a1, and Hsd3b1) were unchanged in Tspo−/− mice. Adrenocortical ultrastructure illustrated no morphological alterations in Tspo−/− mice. In an attempt to correlate our in vivo findings to previously used in vitro models, we also determined that siRNA knockdown or the absence of TSPO in different mouse and human steroidogenic cell lines had no effect on steroidogenesis. These findings directly refute the dogma that TSPO is indispensable for steroid hormone biosynthesis and viability. By amending the current model, this study advances our understanding of steroidogenesis with broad implications in biology and medicine. PMID:24936060

  10. Benzodiazepines have no effect on fear-potentiated startle in humans.

    PubMed

    Baas, Johanna M P; Grillon, Christian; Böcker, Koen B E; Brack, Anouk A; Morgan, Charles A; Kenemans, J Leon; Verbaten, Marinus N

    2002-05-01

    Pre-clinical and clinical investigations have provided a great deal of evidence that the fear-potentiated startle paradigm represents a valid model for the objective assessment of emotional states of anxiety and fear. The four studies presented in this report sought to further validate the "threat of shock" paradigm as a human analogue to fear-potentiated startle in rats, by examining the effect of benzodiazepine administration on both baseline and fear-potentiated startle. Three studies, conducted at Utrecht University, evaluated the effects of oxazepam and of diazepam on baseline and fear-potentiated startle, whereas a fourth study, conducted at Yale University, evaluated the effect of diazepam on baseline, contextual and cue-specific fear-potentiated startle. The threat of shock paradigm consisted of verbal instruction about two visual cues (the threat cue predicted the possible administration of electric shock, the other predicted a safe period), followed by a series of presentations of these cues. During these conditions, acoustic startle stimuli were presented in order to elicit startle responses. The magnitude of the startle response was used to index the degree of fear or alarm experienced during the periods of threat and safety. The fourth study examined the effect of IV administration of diazepam in a similar threat of shock paradigm except that there were two additional context manipulations: electrode placement and darkness. None of the drug manipulations affected specific threat-cue potentiation of startle. However, reductions in baseline startle were observed. Further, startle potentiation by darkness was inhibited by diazepam. At least one type of fear-potentiated startle, i.e. potentiation by a cue-specific fear manipulation, is not susceptible to benzodiazepine treatment. In contrast, effects of manipulations more akin to anxiety (darkness, context) appear sensitive to benzodiazepines. Human experimental models differentiating between these cue

  11. Benzodiazepines counteract rostral anterior cingulate cortex activation induced by cholecystokinin-tetrapeptide in humans.

    PubMed

    Leicht, Gregor; Mulert, Christoph; Eser, Daniela; Sämann, Philipp G; Ertl, Matthias; Laenger, Anna; Karch, Susanne; Pogarell, Oliver; Meindl, Thomas; Czisch, Michael; Rupprecht, Rainer

    2013-02-15

    Benzodiazepines modulate γ-aminobutyric acid type A (GABA(A)) receptors throughout the brain. However, it is not fully understood which brain regions within anxiety-related brain circuits are really responsible for their anxiolytic effects and how these regions interact. We investigated whether the benzodiazepine alprazolam affects activity in distinct brain regions within anxiety-related circuits during an experimental anxiety paradigm by means of functional magnetic resonance imaging (fMRI). Panic symptoms were elicited by a bolus injection of the neuropeptide cholecystokinin-tetrapeptide (CCK-4) in 16 healthy male subjects in a double-blind, placebo-controlled design. Functional brain activation patterns were determined before and during the CCK-4-challenge without pretreatment and after treatment with either placebo or 1 mg alprazolam. The CCK-4 induced anxiety and elicited widely distributed activation patterns in anxiety-related brain circuits, especially in the rostral anterior cingulate cortex (rACC), which was attenuated after alprazolam treatment. In contrast to placebo, alprazolam abolished the activation of the rACC after challenge with CCK-4 (p<.005, corrected for multiple comparisons) and increased functional connectivity between the rACC and other anxiety-related brain regions such as amygdala and prefrontal cortex. Moreover, the reduction in the CCK-4 induced activation of the rACC correlated with the anxiolytic effect of alprazolam (r(p) = .52; p = .04). These findings put forward the rACC as a target for benzodiazepines and suggest that the CCK-4/fMRI paradigm might represent a human translational model for the investigation of anxiolytic drugs. Copyright © 2013 Society of Biological Psychiatry. Published by Elsevier Inc. All rights reserved.

  12. Design and synthesis of benzodiazepine analogs as isoform-selective human lysine deacetylase inhibitors.

    PubMed

    Reddy, D Rajasekhar; Ballante, Flavio; Zhou, Nancy J; Marshall, Garland R

    2017-02-15

    A comprehensive investigation was performed to identify new benzodiazepine (BZD) derivatives as potent and selective human lysine deacetylase inhibitors (hKDACis). A total of 108 BZD compounds were designed, synthesized and from that 104 compounds were biologically evaluated against human lysine deacetylases (hKDACs) 1, 3 and 8 (class I) and 6 (class IIb). The most active compounds showed mid-nanomolar potencies against hKDACs 1, 3 and 6 and micromolar activity against hKDAC8, while a promising compound (6q) showed selectivity towards hKDAC3 among the different enzyme isoforms. An hKDAC6 homology model, refined by molecular dynamics simulation was generated, and molecular docking studies performed to rationalize the dominant ligand-residue interactions as well as to define structure-activity-relationships. Experimental results confirmed the usefulness of the benzodiazepine moiety as capping group when pursuing hKDAC isoform-selectivity inhibition, suggesting its continued use when designing new hKDACis.

  13. The influence of clozapine treatment and other antipsychotics on the 18 kDa translocator protein, formerly named the peripheral-type benzodiazepine receptor, and steroid production.

    PubMed

    Danovich, Lena; Veenman, Leo; Leschiner, Svetlana; Lahav, Michal; Shuster, Vered; Weizman, Abraham; Gavish, Moshe

    2008-01-01

    It has been shown that the atypical antipsychotic drug clozapine increases the levels of the neurosteroid allopregnanolone in the rat brain. The 18 kDa translocator protein (TSPO), formerly known as the peripheral-type benzodiazepine receptor, has been demonstrated to be involved in the process of steroid biosynthesis, in peripheral steroidogenic tissues as well as in glia cells in the brain. In the current study, we investigated the influence of chronic treatment with clozapine and other antipsychotics (thioridazine,sulpiride and risperidone) on TSPO binding in cell cultures and rat tissues. Clozapine significantly increased TSPO binding density in C6 rat glioma cells and in MA-10 mouse Leydig tumor cells, while the antipsychotic sulpiride had no effect on TSPO binding density in both cell lines. In addition, clozapine, but not sulpiride, significantly increased progesterone synthesis by MA-10 Leydig tumor cells. In an animal experiment, male Sprague-Dawley rats were treated with clozapine (20 mg/kg), risperidone (0.5 mg/kg), thioridazine (20 mg/kg), or sulpiride (20 mg/kg) for 21 days, followed by 7 days of withdrawal. Clozapine induced significant increases in TSPO binding in brain and peripheral steroidogenic tissues, whereas the other antipsychotics did not show such pronounced effects on TSPO binding. Our results suggest that TSPO may be involved in the modulation of steroidogenesis by clozapine.

  14. Benzodiazepines and anterior pituitary function.

    PubMed

    Arvat, E; Giordano, R; Grottoli, S; Ghigo, E

    2002-09-01

    Benzodiazepines (BDZ) are one of the most prescribed classes of drugs because of their marked anxiolytic, anticonvulsant, muscle relaxant and hypnotic effects. The pharmacological actions of BDZ depend on the activation of 2 specific receptors. The central BDZ receptor, present in several areas of the central nervous system (CNS), is a component of the GABA-A receptor, the activation of which increases GABAergic neurotransmission and is followed by remarkable neuroendocrine effects. The peripheral benzodiazepine receptors (PBR), structurally and functionally different from the GABA-A receptor, have been shown in peripheral tissues but also in the CNS, in both neurones and glial cells, and in the pituitary gland. BDZ receptors bind to a family of natural peptides called endozepines, firstly isolated from neurons and glial cells in the brain and then in several peripheral tissues as well. Endozepines modulate several central and peripheral biological activities, including some neuroendocrine functions and synthetic BDZ are likely to mimic them, at least partially. BZD, especially alprazolam (AL), possess a clear inhibitory influence on the activity of the HPA axis in both animals and humans. This effect seems to be mediated at the hypothalamic and/or suprahypothalamic level via suppression of CRH. The strong negative influence of AL on hypothalamicpituitary-adrenal (HPA) axis agrees with its peculiar efficacy in the treatment of panic disorders and depression. BZD have also been shown to increase GH secretion via mechanisms mediated at the hypothalamic or supra-hypothalamic level, though a pituitary action cannot be ruled out. Besides the impact on HPA and somatotrope function, BDZ also significantly affect the secretion of other pituitary hormones, such as gonadotropins and PRL, probably acting through GABAergic mediation in the hypothalamus and/or in the pituitary gland. In all, BDZ are likely to represent a useful tool to investigate GABAergic activity and clarify

  15. Quantitation of drugs via molecularly imprinted polymer solid phase extraction and electrospray ionization mass spectrometry: benzodiazepines in human plasma.

    PubMed

    Figueiredo, Eduardo Costa; Sparrapan, Regina; Sanvido, Gustavo Braga; Santos, Mariane Gonçalves; Arruda, Marco Aurélio Zezzi; Eberlin, Marcos Nogueira

    2011-09-21

    The association of solid phase extraction with molecularly imprinted polymers (MIP) and electrospray ionization mass spectrometry (ESI-MS) is applied to the direct extraction and quantitation of benzodiazepines in human plasma. The target analytes are sequestered by MIP and directly analyzed by ESI-MS. Due to the MIP highly selective extraction, ionic suppression during ESI is minimized; hence no separation is necessary prior to ESI-MS, which greatly increases analytical speed. Benzodiazepines (medazepam, nitrazepam, diazepam, chlordiazepoxide, clonazepam and midazolam) in human plasma were chosen as a proof-of-principle case of drug analyses by MIP-ESI-MS in a complex matrix. MIP-ESI-MS displayed good figures of merits for medazepam, nitrazepam, diazepam, chlordiazepoxide and midazolam, with analytical calibration curves ranging from 10 to 250 μg L(-1) (r > 0.98) with limit of quantification <10 μg L(-1) and acceptable within-day and between-day precision and accuracy.

  16. Benzodiazepines and Pregnancy

    MedlinePlus

    ... Psychiatr Serv. 2002;53(1):39-49. Wikner BN, et al. 2007. Use of benzodiazepines and benzodiazepine ... Pharmcoepidemiol Drug Saf 16(9):988-994. Wikner BN, et al. 2007. Use of benzodiazepines and benzodiazepine ...

  17. Inhibitory effects of benzodiazepines on the adenosine A(2B) receptor mediated secretion of interleukin-8 in human mast cells.

    PubMed

    Hoffmann, Kristina; Xifró, Rosa Altarcheh; Hartweg, Julia Lisa; Spitzlei, Petra; Meis, Kirsten; Molderings, Gerhard J; von Kügelgen, Ivar

    2013-01-30

    The activation of adenosine A(2B) receptors in human mast cells causes pro-inflammatory responses such as the secretion of interleukin-8. There is evidence for an inhibitory effect of benzodiazepines on mast cell mediated symptoms in patients with systemic mast cell activation disease. Therefore, we investigated the effects of benzodiazepines on adenosine A(2B) receptor mediated interleukin-8 production in human mast cell leukaemia (HMC1) cells by an enzyme linked immunosorbent assay. The adenosine analogue N-ethylcarboxamidoadenosine (NECA, 0.3-3 μM) increased interleukin-8 production about 5-fold above baseline. This effect was attenuated by the adenosine A(2B) receptor antagonist MRS1754 (N-(4-cyanophenyl)-2-{4-(2,3,6,7-tetrahydro-2,6-dioxo-1,3-dipropyl-1H-purin-8-yl)phenoxy}-acetamide) 1 μM. In addition, diazepam, 4'-chlorodiazepam and flunitrazepam (1-30 μM) markedly reduced NECA-induced interleukin-8 production in that order of potency, whereas clonazepam showed only a modest inhibition. The inhibitory effect of diazepam was not altered by flumazenil 10 μM or PK11195 (1-(2-chlorophenyl)-N-methyl-N-(1-methylpropyl)-3-isoquinolinecarboxamide) 10 μM. Diazepam attenuated the NECA-induced expression of mRNA encoding for interleukin-8. Moreover, diazepam and flunitrazepam reduced the increasing effects of NECA on cAMP-response element- and nuclear factor of activated t-cells-driven luciferase reporter gene activities in HMC1 cells. Neither diazepam nor flunitrazepam affected NECA-induced increases in cellular cAMP levels in CHO Flp-In cells stably expressing recombinant human adenosine A(2B) receptors, excluding a direct action of benzodiazepines on human adenosine A(2B) receptors. In conclusion, this is the first study showing an inhibitory action of benzodiazepines on adenosine A(2B) receptor mediated interleukin-8 production in human mast (HMC1) cells. The rank order of potency indicates the involvement of an atypical benzodiazepine binding site.

  18. Peripheral-type benzodiazepine receptor binding 4{prime}-IODO-PK11195: A new radioiodinated ligand for detecting lesioned brain areas

    SciTech Connect

    Saji, H.; Iida, Y.; Nakatsuka, I.

    1995-05-01

    An increase in the peripheral-type benzodiazepine binding sites (PBBS) has recently been reported in excitotoxic and ischaemic lesions in the brain. Thus, PBBS visualization has been of greater interest due to the possibility of imaging the lesioned area as positive image. In this study, our interest is focussed in the development of a radioiodinated compound for the SPECT study of PBBS function. Taking account of the environment of binding sites and the stability in vivo, we selected the 4{prime}position of C-1 phenyl moiety of the isoqunoline derivative PK11195 as the best exploitable site for the iodination. The no-carrier-added I-125 labeled 4{prime}-iodo-PK11195 (IPK) was synthesized by the bromine-iodine exchange reaction in 60% radiochemical yield and > 98% radiochemical purity. In vitro competitive binding studies with H-3-PI11195 using rat kidney membranes shows that IPK has high affinity for PBBS as much as PK11195. The in vivo biodistribution in mice showed high uptake of I-125-IPK in the kidney, lung, heart and adrenal, organs reported as containing high PBBS, which were reduced by the treatment with cold PK11195. Furthermore, autoradiographic studies in transient middle cerebral arteries occlusion in rats showed high accumulation of I-125-IPK in lesioned sites, in contrast to the decease of radioactivity of Tc-99m-HM-PAO. Gathered data indicated that the newly designed IPK holds to great potential for detecting the lesioned brain areas as positive image.

  19. Direct interactions of androgenic/anabolic steroids with the peripheral benzodiazepine receptor in rat brain: implications for the psychological and physiological manifestations of androgenic/anabolic steroid abuse.

    PubMed

    Masonis, A E; McCarthy, M P

    1996-08-01

    The peripheral benzodiazepine receptor (PBR) is a mitochondrial protein involved in regulating steroid synthesis and transport. We report here the effects of androgenic/anabolic steroids (AAS) on the binding of the PBR-specific ligand [3H] PK11195 to male rat brain cortical synaptoneurosomes. Two synthetic AAS, stanozolol and 17beta-testosterone cypionate (17beta-cyp), significantly inhibited 1 nM [3H] PK11195 binding at concentrations greater than 5 and 25 microM, respectively. Stanozolol was the most effective inhibitor, reducing [3H] PK11195 binding by up to 75%, compared to only 40% inhibition by 17beta-cyp, at 50 microM AAS concentration. Two other AAS, 17alpha-methyltestosterone and nortestosterone decanoate, were incapable of inhibiting [3H] PK11195 binding at concentrations up to 50 microM. On the basis of Scatchard/Rosenthal analysis, [3H] PK11195 binds to two classes of binding sites, and the inhibition of [3H] PK11195 binding by stanozolol appears to be allosteric, primarily reducing binding to the higher affinity [3H] PK11195 binding site. These results, in combination with earlier studies indicating the direct effects of AAS on the function of additional central nervous system receptor complexes, suggest that the behavioral and psychological effects of AAS result from the interactions of AAS with multiple regulatory systems in the brain.

  20. The high affinity peripheral benzodiazepine receptor ligand DAA1106 binds to activated and infected brain macrophages in areas of synaptic degeneration

    PubMed Central

    Venneti, Sriram; Wang, Guoji; Wiley, Clayton A.

    2008-01-01

    HIV encephalitis (HIVE) is characterized by neurodegeneration mediated by toxins derived from infected and activated brain macrophages. Since the peripheral benzodiazepine receptor (PBR) is abundant on brain macrophages, we hypothesized that [3H]DAA1106, a new PBR ligand, can label infected and activated brain macrophages in HIVE. Using cell culture and postmortem brain tissues from HIVE and a macaque model of HIVE, we show that [3H]DAA1106 binds with high affinity to activated and infected macrophages in regions of synaptic damage. Further, binding affinity reflected by lower KD (dissociation constant) values and the Bmax (total number of binding sites) to KD ratios reflective of ligand-binding potential, were significantly higher with [3H]DAA1106 compared to the extensively characterized PBR ligand [3H](R)-PK11195. These data suggest that DAA1106 binds with high affinity to activated and infected brain macrophages and possesses binding characteristics beneficial for in vivo use in the detection and clinical monitoring of HIVE using positron emission tomography. PMID:17920902

  1. Imaging of peripheral-type benzodiazepine receptor in tumor: carbon ion irradiation reduced the uptake of a positron emission tomography ligand [11C]DAC in tumor.

    PubMed

    Yamasaki, Tomoteru; Koike, Sachiko; Hatori, Akiko; Yanamoto, Kazuhiko; Kawamura, Kazunori; Yui, Joji; Kumata, Katsushi; Ando, Koichi; Zhang, Ming-Rong

    2010-01-01

    We aimed to determine the effect of carbon ion irradiation on the uptake of N-benzyl-N-11C-methyl-2-(7-methyl-8-oxo-2-phenyl-7,8-dihydro-9H-purin-9-yl)acetamide ([(11)C]DAC), a positron emission tomography (PET) ligand for the peripheral-type benzodiazepine receptor (PBR), in tumor cells and tumor-bearing mice. Spontaneous murine fibrosarcoma (NFSa) cells were implanted into the right hind legs of syngeneic C3H male mice. Conditioning irradiation with 290 MeV/u carbon ions was delivered to the 7- to 8-mm tumors In vitro uptake of [(11)C]DAC was measured in single NFSa cells isolated from NFSa-bearing mice after irradiation. In vivo biodistribution of [(11)C]DAC in NFSa-bearing mice was determined by small animal PET scanning and dissection. In vitro autoradiography was performed using tumor sections prepared from mice after PET scanning. In vitro and in vivo uptake of [(11)C]DAC in single NFSa cells and NFSa-bearing mice was significantly reduced by carbon ion irradiation. The decrease in [(11)C]DAC uptake in the tumor sections was mainly due to the change in PBR expression. In conclusion, [(11)C]DAC PET responded to the change in PBR expression in tumors caused by carbon ion irradiation in this study. Thus, [(11)C]DAC is a promising predictor for evaluating the effect of carbon ion radiotherapy.

  2. The peripheral clock regulates human pigmentation.

    PubMed

    Hardman, Jonathan A; Tobin, Desmond J; Haslam, Iain S; Farjo, Nilofer; Farjo, Bessam; Al-Nuaimi, Yusur; Grimaldi, Benedetto; Paus, Ralf

    2015-04-01

    Although the regulation of pigmentation is well characterized, it remains unclear whether cell-autonomous controls regulate the cyclic on-off switching of pigmentation in the hair follicle (HF). As human HFs and epidermal melanocytes express clock genes and proteins, and given that core clock genes (PER1, BMAL1) modulate human HF cycling, we investigated whether peripheral clock activity influences human HF pigmentation. We found that silencing BMAL1 or PER1 in human HFs increased HF melanin content. Furthermore, tyrosinase expression and activity, as well as TYRP1 and TYRP2 mRNA levels, gp100 protein expression, melanocyte dendricity, and the number gp100+ HF melanocytes, were all significantly increased in BMAL1 and/or PER1-silenced HFs. BMAL1 or PER1 silencing also increased epidermal melanin content, gp100 protein expression, and tyrosinase activity in human skin. These effects reflect direct modulation of melanocytes, as BMAL1 and/or PER1 silencing in isolated melanocytes increased tyrosinase activity and TYRP1/2 expression. Mechanistically, BMAL1 knockdown reduces PER1 transcription, and PER1 silencing induces phosphorylation of the master regulator of melanogenesis, microphthalmia-associated transcription factor, thus stimulating human melanogenesis and melanocyte activity in situ and in vitro. Therefore, the molecular clock operates as a cell-autonomous modulator of human pigmentation and may be targeted for future therapeutic strategies.

  3. The 18 kDa translocator protein (peripheral benzodiazepine receptor) expression in the bone of normal, osteoprotegerin or low calcium diet treated mice.

    PubMed

    Kam, Winnie Wai-Ying; Meikle, Steven R; Zhou, Hong; Zheng, Yu; Blair, Julie M; Seibel, Marcus; Dunstan, Colin R; Banati, Richard B

    2012-01-01

    The presence of the translocator protein (TSPO), previously named as the mitochondrial or peripheral benzodiazepine receptor, in bone cells was studied in vitro and in situ using RT-qPCR, and receptor autoradiography using the selective TSPO ligand PK11195.In vitro, the TSPO is highly expressed in osteoblastic and osteoclastic cells.In situ, constitutive expression of TSPO is found in bone marrow and trabecular bone, e.g., spongiosa. Mice with a reduction of bone turnover induced by a 4-day treatment of osteoprotegerin reduces [(3)H]PK11195 binding in the spongiosa (320±128 Bq x mg(-1), 499±106 Bq x mg(-1) in saline-treated controls). In contrast, mice with an increase in bone turnover caused by a 4-day low calcium diet increases [(3)H]PK11195 binding in the spongiosa (615±90 Bq x mg(-1)). Further, our study includes technical feasibility data on [(18)F]fluoride microPET imaging of rodent bone with altered turnover. Despite [(18)F]fluoride having high uptake, the in vivo signal differences were small. Using a phantom model, we describe the spillover effect and partial volume loss that affect the quantitative microPET imaging of the small bone structures in experimental mouse models. In summary, we demonstrate the expression of TSPO in small rodent bone tissues, including osteoblasts and osteoclasts. A trend increase in TSPO expression was observed in the spongiosa from low to high bone turnover conditions. However, despite the potential utility of TSPO expression as an in vivo biomarker of bone turnover in experimental rodent models, our small animal PET imaging data using [(18)F]fluoride show that even under the condition of a good biological signal-to-noise ratio and high tracer uptake, the currently achievable instrument sensitivity and spatial resolution is unlikely to be sufficient to detect subtle differences in small structures, such as mouse bone.

  4. Omega 3 (peripheral type benzodiazepine binding) site distribution in the rat immune system: an autoradiographic study with the photoaffinity ligand (/sup 3/H)PK 14105

    SciTech Connect

    Benavides, J.; Dubois, A.; Dennis, T.; Hamel, E.; Scatton, B.

    1989-04-01

    The anatomical distribution of omega 3 (peripheral type benzodiazepine binding) sites in the immune system organs of the rat has been studied autoradiographically at both macroscopic and microscopic levels of resolution using either reversible or irreversible (UV irradiation) labeling with (/sup 3/H)PK 14105. In thymus sections, (/sup 3/H)PK 14105 labeled with high affinity (Kd, derived from saturation experiments = 10.8 nM) a single population of sites which possessed the pharmacological characteristics of omega 3 sites. In the thymus gland, higher omega 3 site densities were detected in the cortex than in the medulla; in these subregions, silver grains were associated to small (10-18 microns diameter) cells. In the spleen, omega 3 sites were more abundant in the white than in the red pulp. In the white pulp, silver grains were denser in the marginal zone than in the vicinity of the central artery and labeling was, as in the thymus, associated to small cytoplasm-poor cells. In the red pulp, omega 3 site associated silver grains were observed mainly in the Bilroth cords. In the lymph nodes, the medullary region showed a higher labeling than the surrounding follicles and paracortex. A significant accumulation of silver grains was observed in the lymph node medullary cords. In the intestine, Peyer patches were particularly enriched in omega 3 sites (especially in the periphery of the follicles). The distribution of omega 3 sites in the immune system organs suggests a preferential labeling of cells of T and monocytic lineages. This is consistent with the proposed immunoregulatory properties of some omega 3 site ligands.

  5. The high affinity peripheral benzodiazepine receptor ligand DAA1106 binds specifically to microglia in a rat model of traumatic brain injury: implications for PET imaging

    PubMed Central

    Venneti, Sriram; Wagner, Amy K.; Wang, Guoji; Slagel, Susan L.; Chen, Xiangbai; Lopresti, Brian J.; Mathis, Chester A.; Wiley., Clayton A.

    2007-01-01

    Traumatic brain injury (TBI) is a significant cause of mortality, morbidity, and disability. Microglial activation is commonly observed in response to neuronal injury which, when prolonged, is thought to be detrimental to neuronal survival. Activated microglia can be labeled using PK11195, a ligand that bind the peripheral benzodiazepine receptor (PBR), receptors which are highly expressed in activated microglia and sparse in the resting brain. We compared the binding properties of two PBR ligands PK11195 and DAA1106 in rats using the controlled cortical impact (CCI) model of experimental TBI. While both ligands showed relative increases with specific binding in the cortex ipsilateral to injury compared to the contralateral side, [3H]DAA1106 showed higher binding affinity compared with [3H](R)-PK11195. Combined immunohistochemistry and autoradiography in brain tissues near the injury site showed that [3H]DAA1106 binding co-registered with activated microglia more than astrocytes. Further, increased [3H]DAA1106 specific binding positively correlated with degree of microglial activation, and to a lesser degree with reactive astrocytosis. Finally, in vivo administration of each ligand in rats with TBI showed greater retention of [11C]DAA1106 compared to [11C](R)-PK11195 at the site of the contusion as assessed by ex vivo autoradiography. These results in a rat model of TBI indicate that [11C]DAA1106 binds with higher affinity to microglia when compared with PK11195, suggesting that [11C]DAA1106 may represent a better ligand than [11C](R)-PK11195 for in vivo PET imaging of activated microglia in TBI. PMID:17658516

  6. The 18 kDa Translocator Protein (Peripheral Benzodiazepine Receptor) Expression in the Bone of Normal, Osteoprotegerin or Low Calcium Diet Treated Mice

    PubMed Central

    Kam, Winnie Wai-Ying; Meikle, Steven R.; Dunstan, Colin R.; Banati, Richard B.

    2012-01-01

    The presence of the translocator protein (TSPO), previously named as the mitochondrial or peripheral benzodiazepine receptor, in bone cells was studied in vitro and in situ using RT-qPCR, and receptor autoradiography using the selective TSPO ligand PK11195. In vitro, the TSPO is highly expressed in osteoblastic and osteoclastic cells. In situ, constitutive expression of TSPO is found in bone marrow and trabecular bone, e.g., spongiosa. Mice with a reduction of bone turnover induced by a 4-day treatment of osteoprotegerin reduces [3H]PK11195 binding in the spongiosa (320±128 Bq.mg−1, 499±106 Bq.mg−1 in saline-treated controls). In contrast, mice with an increase in bone turnover caused by a 4-day low calcium diet increases [3H]PK11195 binding in the spongiosa (615±90 Bq.mg−1). Further, our study includes technical feasibility data on [18F]fluoride microPET imaging of rodent bone with altered turnover. Despite [18F]fluoride having high uptake, the in vivo signal differences were small. Using a phantom model, we describe the spillover effect and partial volume loss that affect the quantitative microPET imaging of the small bone structures in experimental mouse models. In summary, we demonstrate the expression of TSPO in small rodent bone tissues, including osteoblasts and osteoclasts. A trend increase in TSPO expression was observed in the spongiosa from low to high bone turnover conditions. However, despite the potential utility of TSPO expression as an in vivo biomarker of bone turnover in experimental rodent models, our small animal PET imaging data using [18F]fluoride show that even under the condition of a good biological signal-to-noise ratio and high tracer uptake, the currently achievable instrument sensitivity and spatial resolution is unlikely to be sufficient to detect subtle differences in small structures, such as mouse bone. PMID:22295097

  7. Genetic markers of a Munc13 protein family member, BAIAP3, are gender specifically associated with anxiety and benzodiazepine abuse in mice and humans.

    PubMed

    Wojcik, Sonja M; Tantra, Martesa; Stepniak, Beata; Man, Kwun-Nok M; Müller-Ribbe, Katja; Begemann, Martin; Ju, Anes; Papiol, Sergi; Ronnenberg, Anja; Gurvich, Artem; Shin, Yong; Augustin, Iris; Brose, Nils; Ehrenreich, Hannelore

    2013-07-24

    Anxiety disorders and substance abuse, including benzodiazepine use disorder, frequently occur together. Unfortunately, treatment of anxiety disorders still includes benzodiazepines, and patients with an existing comorbid benzodiazepine use disorder or a genetic susceptibility for benzodiazepine use disorder may be at risk of adverse treatment outcomes. The identification of genetic predictors for anxiety disorders, and especially for benzodiazepine use disorder, could aid the selection of the best treatment option and improve clinical outcomes. The brain-specific angiogenesis inhibitor I-associated protein 3 (Baiap3) is a member of the mammalian uncoordinated 13 (Munc13) protein family of synaptic regulators of neurotransmitter exocytosis, with a striking expression pattern in amygdalae, hypothalamus and periaqueductal gray. Deletion of Baiap3 in mice leads to enhanced seizure propensity and increased anxiety, with the latter being more pronounced in female than in male animals. We hypothesized that genetic variation in human BAIAP3 may also be associated with anxiety. By using a phenotype-based genetic association study, we identified two human BAIAP3 single-nucleotide polymorphism risk genotypes (AA for rs2235632, TT for rs1132358) that show a significant association with anxiety in women and, surprisingly, with benzodiazepine abuse in men. Returning to mice, we found that male, but not female, Baiap3 knockout (KO) mice develop tolerance to diazepam more quickly than control animals. Analysis of cultured Baiap3 KO hypothalamus slices revealed an increase in basal network activity and an altered response to diazepam withdrawal. Thus, Baiap3/BAIAP3 is gender specifically associated with anxiety and benzodiazepine use disorder, and the analysis of Baiap3/BAIAP3-related functions may help elucidate mechanisms underlying the development of both disorders.

  8. Genetic Markers of a Munc13 Protein Family Member, BAIAP3, Are Gender Specifically Associated with Anxiety and Benzodiazepine Abuse in Mice and Humans

    PubMed Central

    Wojcik, Sonja M; Tantra, Martesa; Stepniak, Beata; Man, Kwun-nok M; Müller-Ribbe, Katja; Begemann, Martin; Ju, Anes; Papiol, Sergi; Ronnenberg, Anja; Gurvich, Artem; Shin, Yong; Augustin, Iris; Brose, Nils; Ehrenreich, Hannelore

    2013-01-01

    Anxiety disorders and substance abuse, including benzodiazepine use disorder, frequently occur together. Unfortunately, treatment of anxiety disorders still includes benzodiazepines, and patients with an existing comorbid benzodiazepine use disorder or a genetic susceptibility for benzodiazepine use disorder may be at risk of adverse treatment outcomes. The identification of genetic predictors for anxiety disorders, and especially for benzodiazepine use disorder, could aid the selection of the best treatment option and improve clinical outcomes. The brain-specific angiogenesis inhibitor I–associated protein 3 (Baiap3) is a member of the mammalian uncoordinated 13 (Munc13) protein family of synaptic regulators of neurotransmitter exocytosis, with a striking expression pattern in amygdalae, hypothalamus and periaqueductal gray. Deletion of Baiap3 in mice leads to enhanced seizure propensity and increased anxiety, with the latter being more pronounced in female than in male animals. We hypothesized that genetic variation in human BAIAP3 may also be associated with anxiety. By using a phenotype-based genetic association study, we identified two human BAIAP3 single-nucleotide polymorphism risk genotypes (AA for rs2235632, TT for rs1132358) that show a significant association with anxiety in women and, surprisingly, with benzodiazepine abuse in men. Returning to mice, we found that male, but not female, Baiap3 knockout (KO) mice develop tolerance to diazepam more quickly than control animals. Analysis of cultured Baiap3 KO hypothalamus slices revealed an increase in basal network activity and an altered response to diazepam withdrawal. Thus, Baiap3/BAIAP3 is gender specifically associated with anxiety and benzodiazepine use disorder, and the analysis of Baiap3/BAIAP3-related functions may help elucidate mechanisms underlying the development of both disorders. PMID:23698091

  9. Human skin: an independent peripheral endocrine organ.

    PubMed

    Zouboulis, C C

    2000-01-01

    factor-binding proteins. Therefore, the human skin fulfils all requirements for being the largest, independent peripheral endocrine organ.

  10. Purification of basophils from peripheral human blood.

    PubMed

    Falcone, Franco H; Gibbs, Bernhard F

    2014-01-01

    The purification of basophils from peripheral blood has represented a formidable challenge for researchers since they were discovered by Paul Ehrlich in 1879. From the first published attempts in the late 1960s, it took half a century to develop robust protocols able to provide sufficient numbers of pure, functionally unimpaired basophils. The existing protocols for basophil purification exploit those properties of basophils which distinguish them from other cell types such as their localization in blood, density, and the presence or absence of surface markers. Purification techniques have been used in various combinations and variations to achieve a common goal in mind: to obtain a pure population of human basophils in sufficient numbers for downstream studies. The arduous way leading up to the modern protocols is summarized in this historical retrospective. A fast protocol for purification of basophils to near homogeneity is also described.

  11. [Enterobacterial antigen in human peripheral blood lymphocytes].

    PubMed

    Faure-Fontenla, M A; García-Tamayo, F

    1989-11-01

    The following study has as prior history the research reports which have shown the existence of an antigenic tissue deposit in gram-negative enterobacteria. The antigens of the enterobacteria have also been found in the lymphocytic membranes and cytoplasm. Since intestinal lymphoid tissue cells can recirculate by means of the thoracic duct to the peripheral venous system, it was proposed that the circulating lymphocytes in healthy people could also contain small amounts of a common enterobacterial antigen. The study was carried out in 15 human venous blood samples, of which the lymphocytic population was separated to later be used in the preparation of 15 alcohol soluble extracts. This material was used for inhibiting the immuno-hemolysis assay in three occasions in order to show the presence of antigens shared by different enterobacterias, using as reference a fraction separated from the LPS of Escherichia coli 08. The results showed that the human lymphocytes also had antigenic determinants common to gram-negative bacteria.

  12. Mitochondrial benzodiazepine receptors regulate steroid biosynthesis

    SciTech Connect

    Mukhin, A.G.; Papadopoulos, V.; Costa, E.; Krueger, K.E. )

    1989-12-01

    Recent observations on the steroid synthetic capability within the brain open the possibility that benzodiazepines may influence steroid synthesis in nervous tissue through interactions with peripheral-type benzodiazepine recognition sites, which are highly expressed in steroidogenic cells and associated with the outer mitochondrial membrane. To examine this possibility nine molecules that exhibit a greater than 10,000-fold difference in their affinities for peripheral-type benzodiazepine binding sites were tested for their effects on a well-established steroidogenic model system, the Y-1 mouse adrenal tumor cell line. 4{prime}-Chlorodiazepam, PK 11195, and PK 14067 stimulated steroid production by 2-fold in Y-1 cells, whereas diazepam, flunitrazepam, zolpidem, and PK 14068 displayed a lower (1.2- to 1.5-fold) maximal stimulation. In contrast, clonazepam and flumazenil did not stimulate steroid synthesis. The potencies of these compounds to inhibit {sup 3}H-labeled PK 11195 binding to peripheral-type benzodiazepine recognition sites correlated with their potencies to stimulate steroid production. Similar findings were observed in bovine and rat adrenocortical cell preparations. These results suggest that ligands of the peripheral-type benzodiazepine recognition site acting on this mitochondrial receptor can enhance steroid production. This action may contribute specificity to the pharmacological profile of drugs preferentially acting on the benzodiazepine recognition site associated with the outer membrane of certain mitochondrial populations.

  13. Human peripheral blood eosinophils induce angiogenesis.

    PubMed

    Puxeddu, Ilaria; Alian, Akram; Piliponsky, Adrian Martin; Ribatti, Domenico; Panet, Amos; Levi-Schaffer, Francesca

    2005-03-01

    Eosinophils play a crucial role in allergic reactions and asthma. They are also involved in responses against parasites, in autoimmune and neoplastic diseases, and in fibroses. There is increasing evidence that angiogenesis plays an important role in these processes. Since eosinophils are known to produce angiogenic mediators, we have hypothesized a direct contribution of these cells to angiogenesis. The effect of human peripheral blood eosinophil sonicates on rat aortic endothelial cell proliferation (in vitro), rat aorta sprouting (ex vivo) and angiogenesis in the chick embryo chorioallantoic membrane (in vivo) have been investigated. To determine whether eosinophil-derived vascular endothelial growth factor influences the eosinophil pro-angiogenic activity, eosinophil sonicates were incubated with anti-vascular endothelial growth factor antibodies and then added to the chorioallantoic membrane. Vascular endothelial growth factor mRNA expression and vascular endothelial growth factor receptor density on the endothelial cells were also evaluated. Eosinophils were found to enhance endothelial cell proliferation and to induce a strong angiogenic response both in the aorta rings and in the chorioallantoic membrane assays. Pre-incubation of eosinophil sonicates with anti-vascular endothelial growth factor antibodies partially reduced the angiogenic response of these cells in the chorioallantoic membrane. Eosinophils also increased vascular endothelial growth factor mRNA production on endothelial cells. Eosinophils are able to induce angiogenesis and this effect is partially mediated by their pre-formed vascular endothelial growth factor. This strongly suggests an important role of eosinophils in angiogenesis-associated diseases such as asthma.

  14. Modulation of the immunologic response to acute stress in humans by beta-blockade or benzodiazepines.

    PubMed

    Benschop, R J; Jacobs, R; Sommer, B; Schürmeyer, T H; Raab, J R; Schmidt, R E; Schedlowski, M

    1996-03-01

    Acute stress evokes immediate responses in the cardiovascular endocrine, and immune systems. In particular, the number and activity of natural killer (NK) lymphocytes increase after stress. Here, we investigate the possibility to pharmacologically interfere with these stress-induced immunologic changes. Twenty-five healthy males were subjected to an acute stressor, a first-time tandem parachute jump. Subjects were randomly assigned to a beta-adrenoceptor antagonist (propranolol), a benzodiazepine (alprazolam), or placebo group. To analyze the role of the spleen in lymphocyte redistribution, splenectomized subjects performed a parachute jump. Propranolol, but no alprazolam, inhibited the heart rate increase during jumping. Increases in epinephrine and cortisol in the propranolol group were comparable to placebo, but were attenuated by alprazolam. The number and activity of NK cells significantly increased in the placebo group but not in the propranolol group immediately after stress. Alprazolam treatment did not alter the increase in NK cell numbers but did inhibit the increase in NK activity. In splenectomized subjects, NK cell numbers, but not NK activity, increased as in placebo subjects. We conclude that stress-induced changes in the immune system are controlled by beta-adrenergic mechanisms and only partly depend on the spleen; central interference with alprazolam differentially affects stress-induced changes in the NK cell compartment.

  15. Functional characterization of the 1,5-benzodiazepine clobazam and its major active metabolite N-desmethylclobazam at human GABA(A) receptors expressed in Xenopus laevis oocytes.

    PubMed

    Hammer, Harriet; Ebert, Bjarke; Jensen, Henrik Sindal; Jensen, Anders A

    2015-01-01

    The 1,5-benzodiazepine clobazam is indicated for the adjunctive treatment of seizures associated with Lennox-Gastaut syndrome in patients 2 years of age or older in the United States, and for treatment of anxiety and various forms of epilepsy elsewhere. Clobazam has been reported to exhibit different in vivo adverse effects and addiction liability profile than the classic 1,4-benzodiazepines. In this study, it was investigated whether the in vitro pharmacological properties of clobazam and its major active metabolite N-desmethylclobazam could explain some of these clinical differences. The functional properties of the two 1,5-benzodiazepines were characterized at the human γ-aminobutyric acid type A receptor (GABA(A)R) subtypes α1β2γ(2S), α2β2γ(2S), α3β2γ(2S), α5β2γ(2S) and α6β2δ expressed in Xenopus laevis oocytes by use of two-electrode voltage-clamp electrophysiology and compared to those exhibited by the 1,4-benzodiazepine clonazepam. All three compounds potentiated GABA EC20-evoked responses through the α(1,2,3,5)β2γ(2S) GABA(A)Rs in a reversible and concentration-dependent manner, with each displaying similar EC50 values at the four subtypes. Furthermore, the degrees of potentiation of the GABA EC20 currents through the four receptors mediated by saturating modulator concentrations did not differ substantially for any of the three benzodiazepines. The three compounds were substantially less potent (200-3900 fold) as positive allosteric modulators at the α6β2δ GABA(A)R than at the α(1,2,3,5)β2γ(2S) receptors. Interestingly, however, clobazam and especially N-desmethylclobazam were highly efficacious potentiators of α6β2δ receptor signaling. Although this activity component is unlikely to contribute to the in vivo effects of clobazam/N-desmethylclobazam, the 1,5-benzodiazepine could constitute an interesting lead for novel modulators targeting this low-affinity binding site in GABAARs. In conclusion, the non-selective modulation

  16. Preferential affinity of /sup 3/H-2-oxo-quazepam for type I benzodiazepine recognition sites in the human brain

    SciTech Connect

    Corda, M.G.; Giorgi, O.; Longoni, B.; Ongini, E.; Montaldo, S.; Biggio, G.

    1988-01-01

    The hypnotic drug quazepam and its active metabolite 2-oxo-quazepam (2-oxo-quaz) are two benzodiazepines (BZ) containing a trifluoroethyl moiety on the ring nitrogen at position 1, characterized by their preferential affinity for Type I BZ recognition sites. In the present study we characterized the binding of /sup 3/H-2-oxo-quaz in discrete areas of the human brain. Saturation analysis demonstrated specific and saturable binding of /sup 3/H-2-oxo-quaz to membrane preparations from human cerebellum. Hill plot analysis of displacement curves of /sup 3/H-flunitrazepam binding by 2-oxo-quaz yielded Hill coefficients of approximately 1 in the cerebellum and significantly less than 1 in the cerebral cortex, hippocampus, caudate nucleus, thalamus and pons. Self and cross displacement curves for /sup 3/H-FNT and /sup 3/H-2-oxo-quaz binding in these brain areas indicated that 2-oxo-quaz binds with different affinities to two populations of binding sites. High affinity binding sites were more abundant in the cerebellum, cerebral cortex, hippocampus and thalamus, whereas low affinity sites were predominant in the caudate nucleus and pons. Competition studies of /sup 3/H-2-oxo-quaz and /sup 3/H-FNT using unlabelled ligands indicated that compounds which preferentially bind to Type I sites are more potent at displacing /sup 3/H-2-oxo-quaz than /sup 3/H-FNT from cerebral cortex membrane preparations. 26 references, 2 figures, 3 tables.

  17. Detection and validated quantification of 21 benzodiazepines and 3 "z-drugs" in human hair by LC-MS/MS.

    PubMed

    Rust, Kristina Y; Baumgartner, Markus R; Meggiolaro, Natascha; Kraemer, Thomas

    2012-02-10

    A method for detection and quantification of 21 benzodiazepines and the pharmacologically related "z-drugs" in human hair samples was developed and fully validated using liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS). After methanolic and methanolic/aqueous extraction, the analytes were separated using two different LC-MS systems (AB Sciex 3200 QTRAP and AB Sciex 5500 QTRAP). Separation columns, mobile phases and MS modes for both systems were: Phenomenex Kinetex, 2.6 μm, 50/2.1; 5mM ammonium formate buffer pH 3.5/methanol, total flow 0.75 mL/min; electrospray ionization (ESI), multiple reaction monitoring (MRM), information dependent acquisition (IDA), enhanced product ion scan (EPI). The assays were found to be selective for the tested compounds (alprazolam, 7-aminoclonazepam, 7-aminoflunitrazepam, bromazepam, chlordiazepoxide, clonazepam, N-desalkylflurazepam, diazepam, flunitrazepam, flurazepam, alpha-hydroxymidazolam, lorazepam, lormetazepam, midazolam, nitrazepam, nordazepam, oxazepam, phenazepam, prazepam, temazepam, triazolam, zaleplon, zolpidem and zopiclone), all validation criteria were in the required ranges according to international guidelines, except for bromazepam. Matrix effects, and process efficiencies were in the acceptable ranges evaluated using the post-extraction addition approach. Lower limits of quantification were between 0.6 and 16 pg/mg of hair. The LC-MS/MS assay has proven to be applicable for determination of the studied analytes in human hair in numerous authentic cases (n=175). Copyright © 2011 Elsevier Ireland Ltd. All rights reserved.

  18. Amygdala Lesions Reduce Anxiety-like Behavior in a Human Benzodiazepine-Sensitive Approach-Avoidance Conflict Test.

    PubMed

    Korn, Christoph W; Vunder, Johanna; Miró, Júlia; Fuentemilla, Lluís; Hurlemann, Rene; Bach, Dominik R

    2017-10-01

    Rodent approach-avoidance conflict tests are common preclinical models of human anxiety disorder. Their translational validity mainly rests on the observation that anxiolytic drugs reduce rodent anxiety-like behavior. Here, we capitalized on a recently developed approach-avoidance conflict computer game to investigate the impact of benzodiazepines and of amygdala lesions on putative human anxiety-like behavior. In successive epochs of this game, participants collect monetary tokens on a spatial grid while under threat of virtual predation. In a preregistered, randomized, double-blind, placebo-controlled trial, we tested the effect of a single dose (1 mg) of lorazepam (n = 59). We then compared 2 patients with bilateral amygdala lesions due to Urbach-Wiethe syndrome with age- and gender-matched control participants (n = 17). Based on a previous report, the primary outcome measure was the effect of intra-epoch time (i.e., an adaptation to increasing potential loss) on presence in the safe quadrant of the spatial grid. We hypothesized reduced loss adaptation in this measure under lorazepam and in patients with amygdala lesions. Lorazepam and amygdala lesions reduced loss adaptation in the primary outcome measure. We found similar results in several secondary outcome measures. The relative reduction of anxiety-like behavior in patients with amygdala lesions was qualitatively and quantitatively indistinguishable from an impact of anterior hippocampus lesions found in a previous report. Our results establish the translational validity of human approach-avoidance conflict tests in terms of anxiolytic drug action. We identified the amygdala, in addition to the hippocampus, as a critical structure in human anxiety-like behavior. Copyright © 2017 Society of Biological Psychiatry. Published by Elsevier Inc. All rights reserved.

  19. Differential effect of detergents on (/sup 3/H)Ro 5-4864 and (/sup 3/H)PK 11195 binding to peripheral-type benzodiazepine-binding sites

    SciTech Connect

    Awad, M.; Gavish, M.

    1988-01-01

    The present study demonstrates a differential effect of various detergent treatments on (/sup 3/H)Ro 5-4864 and (/sup 3/H)PK 11195 binding to peripheral benzodiazepine binding sites (PBS). Triton X-100 caused a decrease of about 70% in (/sup 3/H)Ro 5-4864 binding to membranes from various peripheral tissues of rat, but had only a negligible effect on (/sup 3/H)PK 11195 binding. A similar effect of Triton X-100 was observed on guinea pig and rabbit kidney membranes. The decrease in (/sup 3/H)Ro 5-4864 binding after treatment with Triton X-100 was apparently due to a decrease in the density of PBS, since the affinity remained unaltered. The detergents 3-((3-cholamidopropyl)-dimethylammonio)-1-propane sulfonate (CHAPS), Tween 20, deoxycholic acid, or digitonin (0.0125%) caused only a minor change in (/sup 3/H)Ro 5-4864 and (/sup 3/H)PK 11195 binding to rat kidney membranes; but when concentrations were substantially increased (0.1%), all detergents caused a decrease of at least 50% in (/sup 3/H)Ro 5-4864 binding, while (/sup 3/H)PK 11195 binding to rat kidney membranes remained unaffected by the first three detergents, with only a minor decrease (15%) after treatment with digitonin.

  20. The history of benzodiazepines.

    PubMed

    Wick, Jeannette Y

    2013-09-01

    After more than 50 years of experience with benzodiazepines, the American health care system has a love-hate relationship with them. In 1955, Hoffmann-La Roche chemist Leo Sternbach serendipitously identified the first benzodiazepine, chlordiazepoxide (Librium). By 1960, Hoffmann-La Roche marketed it as Librium, and it pursued molecular modifications for enhanced activity. Valium (diazepam) followed in 1963. Hoffmann-La Roche's competitors also began looking for analogues. Initially, benzodiazepines appeared to be less toxic and less likely to cause dependence than older drugs. A specific improvement was their lack of respiratory depression, a safety concern with barbiturates. Medical professionals greeted benzodiazepines enthusiastically at first, skyrocketing their popularity and patient demand. In the mid-to-late 1970s, benzodiazepines topped all "most frequently prescribed" lists. It took 15 years for researchers to associate benzodiazepines and their effect on gamma-aminobutyric acid as a mechanism of action. By the 1980s, clinicians' earlier enthusiasm and propensity to prescribe created a new concern: the specter of abuse and dependence. As information about benzodiazepines, both raising and damning, accumulated, medical leaders and legislators began to take action. The result: individual benzodiazepines and the entire class began to appear on guidelines and in legislation giving guidance on their use. Concurrently, clinicians began to raise concerns about benzodiazepine use by elderly patients, indicating that elders'lesser therapeutic response and heightened sensitivity to side effects demanded prescriber caution. The benzodiazepine story continues to evolve and includes modern-day issues and concerns beyond those ever anticipated.

  1. Anti-conflict effects of benzodiazepines in rhesus monkeys: relationship with therapeutic doses in humans and role of GABAA receptors.

    PubMed

    Rowlett, James K; Lelas, Snjezana; Tornatzky, Walter; Licata, Stephanie C

    2006-01-01

    Conflict procedures are used to study mechanisms underlying the anxiolytic effects of benzodiazepines (BZs). We established a conflict procedure with rhesus monkeys in order to examine the role of GABAA receptors in the anxiolytic-like effects of BZs. Four rhesus monkeys responded under a two-component multiple schedule in which responding was maintained under a fixed-ratio schedule of food delivery in the absence (non-suppressed responding) and presence (suppressed responding) of response-contingent electric shock. Conventional BZs (alprazolam, flunitrazepam, clonazepam, nitrazepam, lorazepam, bromazepam, diazepam, flurazepam, clorazepate, chlordiazepoxide) engendered increases in the average rates of suppressed responding at low to intermediate doses and decreased the average rates of non-suppressed responding at higher doses. Positive correlations were observed when the therapeutic potencies of BZs in humans were compared with potencies to increase the rates of suppressed responding (R2=0.83) or decrease the rates of non-suppressed responding (R2=0.60). The 5-HT1A agonist buspirone increased the rates of suppressed responding, although the effects were modest, whereas the opioid morphine lacked anti-conflict effects. The BZ antagonist flumazenil also modestly increased the rates of suppressed responding. A relatively low dose of flumazenil enhanced, while a high dose blocked, alprazolam's anti-conflict effects. Compounds selective for alpha1 subunit-containing GABAA receptors (zolpidem, zaleplon, CL218,872) engendered relatively weak increases in the rates of suppressed responding. A rhesus monkey conflict procedure was established with predictive validity for therapeutic doses in people and provided evidence that anxiolytic-like effects of BZs can occur with relatively low intrinsic efficacy at GABAA receptors and are reduced by alpha1GABAA receptor selectivity.

  2. Human urinary metabolic patterns of the designer benzodiazepines flubromazolam and pyrazolam studied by liquid chromatography-high resolution mass spectrometry.

    PubMed

    Pettersson Bergstrand, Madeleine; Meyer, Markus R; Beck, Olof; Helander, Anders

    2017-07-05

    Over the past ~8 years, hundreds of unregulated new psychoactive substances (NPS) of various chemical categories have been introduced as recreational drugs through mainly open online trade. This study was performed to further investigate the human metabolic pattern of the NPS, or designer benzodiazepines flubromazolam and pyrazolam, and to propose analytical targets for urine drug testing of these substances. The urine samples originated from patient samples confirmed by liquid chromatography-high-resolution tandem mass spectrometry (LC-HRMS/MS) analysis to contain flubromazolam or pyrazolam. The LC-HRMS/MS system consisted of a YMC-UltraHT Hydrosphere C18 column (YMC, Dinslaken, Germany) coupled to a Thermo Scientific (Waltham, MA, USA) Q Exactive Orbitrap MS operating in positive electrospray mode. The samples were analyzed both with and without enzymatic hydrolysis using β-glucuronidase. Besides the parent compounds, the main urinary excretion products were parent glucuronides, mono-hydroxy metabolites, and mono-hydroxy glucuronides. In samples prepared without hydrolysis, the most common flubromazolam metabolites were 1 of the mono-hydroxy glucuronides and 1 of the parent glucuronides. For pyrazolam, a parent glucuronide was the most common metabolite. These 3 metabolites were detected in all samples and were considered the primary targets for urine drug testing and confirmation of intake. After enzymatic hydrolysis of the urine samples, a 2-19-fold increase in the concentration of flubromazolam was found, highlighting the value of hydrolysis for this analyte. With hydrolysis, the flubromazolam hydroxy metabolites should be used as target metabolites. Copyright © 2017 John Wiley & Sons, Ltd.

  3. MRI-constrained spectral imaging of benzodiazepine modulation of spontaneous neuromagnetic activity in human cortex.

    PubMed

    Ahveninen, Jyrki; Lin, Fa-Hsuan; Kivisaari, Reetta; Autti, Taina; Hämäläinen, Matti; Stufflebeam, Steven; Belliveau, John W; Kähkönen, Seppo

    2007-04-01

    Spontaneous electromagnetic brain rhythms have been widely used in human neuropharmacology, but their applicability is complicated by the difficulties to localize their origins in the human cortex. Here, we used a novel multi-modal non-invasive imaging approach to localize lorazepam (30 microg/kg i.v.) modulation of cortical generators of spontaneous brain rhythms. Eight healthy subjects were measured with 306-channel magnetoencephalography (MEG) in a double-blind, randomized, placebo-controlled (saline), crossover design. For anatomically realistic source modeling, wavelet-transformed MEG data were combined with high-resolution MRI to constrain the current locations to the cortical mantle, after which individual data were co-registered to surface-based coordinate system for the calculation of group statistical parametric maps of drug effects. The distributed MRI-constrained MEG source estimates demonstrated decreased alpha (10 Hz) activity in and around the parieto-occipital sulcus and in the calcarine sulcus of the occipital lobe, following from increased GABA(A)-inhibition by lorazepam. Anatomically constrained spectral imaging displays the cortical loci of drug effects on oscillatory brain activity, providing a novel tool for human pharmacological neuroimaging.

  4. Benzodiazepines: Are We Overprescribing?

    PubMed Central

    Schiralli, Vanna; McIntosh, Marion

    1987-01-01

    The authors made a survey of benzodiazepine use in the Family Practice Units at Toronto General Hospital and report the findings. They have examined, among other factors, drugs used, reasons for use, and perceived withdrawal symptoms. The results indicated that 24.3% of respondents had taken benzodiazepines in the previous year, and 12.2% in the previous two weeks. There was no difference in the percentage of use of benzodiazepines by males and females. This study confirms that diazepam was the most common drug used in all age ranges. Finally, 6.1% of benzodiazepine users stated that they had attempted an overdose. PMID:21263905

  5. Benzodiazepines: are we overprescribing?

    PubMed

    Schiralli, V; McIntosh, M

    1987-04-01

    The authors made a survey of benzodiazepine use in the Family Practice Units at Toronto General Hospital and report the findings. They have examined, among other factors, drugs used, reasons for use, and perceived withdrawal symptoms. The results indicated that 24.3% of respondents had taken benzodiazepines in the previous year, and 12.2% in the previous two weeks. There was no difference in the percentage of use of benzodiazepines by males and females. This study confirms that diazepam was the most common drug used in all age ranges. Finally, 6.1% of benzodiazepine users stated that they had attempted an overdose.

  6. Gnathic and peripheral ameloblastomas lack human papillomavirus DNA.

    PubMed

    Verduin, Lindsey; Bishop, Justin; Mills, Stacey E

    2015-10-01

    Human papillomavirus (HPV) has been associated with a variety of head and neck neoplasms, including squamous cell carcinomas and Schneiderian papillomas. Ameloblastomas can arise from either the gnathic bones or peripheral soft tissues. Peripheral sinonasal ameloblastomas share clinical features with Schneiderian papillomas. A small number of reports have described detection of HPV DNA within ameloblastomas. However, Most of these cases was reported in the 1990s, used the polymerase chain reaction technique, and only examined gnathic tumors. The current study was designed to determine whether low- or high-risk HPV DNA could be detected in gnathic or peripheral ameloblastomas using in situ hybridization. Twenty-nine examples of gnathic osseous and peripheral head and neck ameloblastomas were obtained from the authors' archives (University of Virginia and the Johns Hopkins Hospital). High-risk HPV DNA was not detected in any of the 29 tumors analyzed. Low-risk HPV DNA was identified in only 1 tumor, which was peripheral in origin, and from an immunocompromised patient. We believe that the HPV in this case represents a background "passenger" infection. This study demonstrates that HPV of either high- or low-risk subtypes is unlikely to play a role in the pathogenesis of sinonasal ameloblastomas. Copyright © 2015 Elsevier Inc. All rights reserved.

  7. Mouse forward genetics in the study of the peripheral nervous system and human peripheral neuropathy

    PubMed Central

    Douglas, Darlene S.; Popko, Brian

    2009-01-01

    Forward genetics, the phenotype-driven approach to investigating gene identity and function, has a long history in mouse genetics. Random mutations in the mouse transcend bias about gene function and provide avenues towards unique discoveries. The study of the peripheral nervous system is no exception; from historical strains such as the trembler mouse, which led to the identification of PMP22 as a human disease gene causing multiple forms of peripheral neuropathy, to the more recent identification of the claw paw and sprawling mutations, forward genetics has long been a tool for probing the physiology, pathogenesis, and genetics of the PNS. Even as spontaneous and mutagenized mice continue to enable the identification of novel genes, provide allelic series for detailed functional studies, and generate models useful for clinical research, new methods, such as the piggyBac transposon, are being developed to further harness the power of forward genetics. PMID:18481175

  8. Photoacoustic tomography of small-animal and human peripheral joints

    NASA Astrophysics Data System (ADS)

    Wang, Xueding; Chamberland, David L.; Fowlkes, J. Brian; Carson, Paul L.; Jamadar, David A.

    2008-02-01

    As an emerging imaging technology that combines the merits of both light and ultrasound, photoacoustic tomography (PAT) holds promise for screening and diagnosis of inflammatory joint diseases such as rheumatoid arthritis. In this study, the feasibility of PAT in imaging small-animal joints and human peripheral joints in a noninvasive manner was explored. Ex vivo rat tail and fresh cadaveric human finger joints were imaged. Based on the intrinsic optical contrast, intra- and extra-articular tissue structures in the joints were visualized successfully. Using light in the near-infrared region, the imaging depth of PAT is sufficient for cross-sectional imaging of a human peripheral joint as a whole organ. PAT, as a novel imaging modality with unique advantages, may contribute significantly to the early diagnosis of inflammatory joint disorders and accurate monitoring of disease progression and response to therapy.

  9. Peripheral fat loss and decline in adipogenesis in older humans.

    PubMed

    Caso, Giuseppe; McNurlan, Margaret A; Mileva, Izolda; Zemlyak, Alla; Mynarcik, Dennis C; Gelato, Marie C

    2013-03-01

    Aging is associated with a redistribution of body fat including a relative loss of subcutaneous peripheral fat. These changes in body fat can have important clinical consequences since they are linked to increased risk of metabolic complications. The causes and mechanisms of loss of peripheral fat associated with aging are not clear. The aim of this study was to assess whether defects in adipogenesis contribute to fat loss in aging humans, as suggested from animal studies, and to evaluate the role of inflammation on pathogenesis of fat loss. Preadipocytes isolated from subcutaneous peripheral fat of healthy young and elderly subjects were compared in their ability to replicate and differentiate. The results show that both the rate of replication and differentiation of preadipocytes are reduced in older subjects. The reduction in adipogenesis is accompanied by a higher plasma level of the inflammatory marker, soluble tumor necrosis factor receptor 2, and greater release of tumor necrosis factor α from fat tissue. Thus, the gradual relative loss of peripheral fat in aging humans may in part result from a defect in adipogenesis, which may be linked to inflammation and increased release of proinflammatory cytokines from fat tissue. Copyright © 2013 Elsevier Inc. All rights reserved.

  10. Activation of the peripheral endocannabinoid system in human obesity.

    PubMed

    Engeli, Stefan; Böhnke, Jana; Feldpausch, Mareike; Gorzelniak, Kerstin; Janke, Jürgen; Bátkai, Sándor; Pacher, Pál; Harvey-White, Judy; Luft, Friedrich C; Sharma, Arya M; Jordan, Jens

    2005-10-01

    Obesity is the main risk factor for the development of type 2 diabetes. Activation of the central endocannabinoid system increases food intake and promotes weight gain. Blockade of the cannabinoid type 1 (CB-1) receptor reduces body weight in animals by central and peripheral actions; the role of the peripheral endocannabinoid system in human obesity is now being extensively investigated. We measured circulating endocannabinoid concentrations and studied the expression of CB-1 and the main degrading enzyme, fatty acid amide hydrolase (FAAH), in adipose tissue of lean (n = 20) and obese (n = 20) women and after a 5% weight loss in a second group of women (n = 17). Circulating levels of anandamide and 1/2-arachidonoylglycerol were increased by 35 and 52% in obese compared with lean women (P < 0.05). Adipose tissue mRNA levels were reduced by -34% for CB-1 and -59% for FAAH in obese subjects (P < 0.05). A strong negative correlation was found between FAAH expression in adipose tissue and circulating endocannabinoids. Circulating endocannabinoids and CB-1 or FAAH expression were not affected by 5% weight loss. The expression of CB-1 and FAAH was increased in mature human adipocytes compared with in preadipocytes and was found in several human tissues. Our findings support the presence of a peripheral endocannabinoid system that is upregulated in human obesity.

  11. Benzodiazepine poisoning in elderly.

    PubMed

    Vukcević, Natasa Perković; Ercegović, Gordana Vuković; Segrt, Zoran; Djordjević, Snezana; Stosić, Jasmina Jović

    2016-03-01

    Benzodiazepines are among the most frequently ingested drugs in self-poisonings. Elderly may be at greater risk compared with younger individuals due to impaired metabolism and increased sensitivity to benzodiazepines. The aim of this study was to assess toxicity of benzodiazepines in elderly attempted suicide. A retrospective study of consecutive presentations to hospital after self-poisoning with benzodiazepines was done. Collected data consisted of patient's characteristics (age, gender), benzodiazepine ingested with its blood concentrations at admission, clinical findings including vital signs and Glasgow coma score, routine blood chemistry, complications of poisoning, details of management, length of hospital stay and outcome. According the age, patients are classified as young (15-40-year old), middle aged (41-65-year old) and elderly (older than 65). During a 2-year observational period 387 patients were admitted because of pure benzodiazepine poisoning. The most frequently ingested drug was bromazepam, the second was diazepam. The incidence of coma was significantly higher, and the length of hospital stay significantly longer in elderly. Respiratory failure and aspiration pneumonia occurred more frequently in old age. Also, flumazenil was more frequently required in the group of elderly patients. Massive benzodiazepines overdose in elderly may be associated with a significant morbidity, including deep coma with aspiration pneumonia, respiratory failure, and even death. Flumazenil is indicated more often to reduce CNS depression and prevent complications of prolonged unconsciousness, but supportive treatment and proper airway management of comatose patients is the mainstay of the treatment of acute benzodiazepine poisoning.

  12. Peripheral-type benzodiazepine receptor (PBR) aggregation and absence of steroidogenic acute regulatory protein (StAR)/PBR association in the mitochondrial membrane as determined by bioluminescence resonance energy transfer (BRET).

    PubMed

    Bogan, Randy L; Davis, Tracy L; Niswender, Gordon D

    2007-04-01

    The steroidogenic acute regulatory protein (StAR) is responsible for acute control of cholesterol transport across the mitochondrial membrane, however the mechanism of StAR-associated cholesterol transport is unknown and may involve the peripheral-type benzodiazepine receptor (PBR)/endozepine system. Several molecules of PBR may associate to form a channel through which cholesterol passes to the inner mitochondrial membrane, and endozepine is the natural ligand for PBR. Bioluminescence resonance energy transfer (BRET) was used to test StAR/PBR/endozepine interactions, PBR aggregation, and the effect of second messengers on interactions. There was no evidence of StAR/PBR, StAR/endozepine, or PBR/endozepine interactions. The StAR and PBR fusion proteins were trafficking to the mitochondria as expected, but the endozepine fusion protein was not localized to the mitochondria indicating that it was not biologically active. Data were obtained indicating that PBR forms aggregates in the mitochondrial membrane. Energy transfer between PBR fusion proteins was dose and time dependent, but there was no effect induced by PK11195 ligand binding or pharmacologic activation of PKA or PKC second messenger pathways. It appears that PBR aggregates in the mitochondrial membrane, however there was no evidence that PBR aggregation is regulated in the acute control of steroidogenesis, or that PBR and StAR interact.

  13. 2-Phenyl-imidazo[1,2-a]pyridine compounds containing hydrophilic groups as potent and selective ligands for peripheral benzodiazepine receptors: synthesis, binding affinity and electrophysiological studies.

    PubMed

    Denora, Nunzio; Laquintana, Valentino; Pisu, Maria Giuseppina; Dore, Riccardo; Murru, Luca; Latrofa, Andrea; Trapani, Giuseppe; Sanna, Enrico

    2008-11-13

    A series of imidazopyridine acetamides were synthesized to evaluate the effects of structural changes at both central (CBRs) and peripheral benzodiazepine receptors (PBRs). These changes include the introduction of polar substituents or ionizable functional groups at the 2- and 8-position of the imidazopyridine skeleton. The results suggest that substituents endowed with hydrogen bonding acceptor and/or donor properties in the para position of the phenyl ring lead to high affinity for PBR. In electrophysiological studies, it was found that compounds 9, 12, 13, and 28 markedly enhanced GABA-evoked Cl (-) currents in Xenopus oocytes expressing alpha 1beta 2gamma 2 GABA A receptors. The capability of flumazenil to reduce the stimulatory effect exerted by compound 9 supports the conclusion that the modulatory effects of the examined compounds occur involving the CBR. The ability of compound 16 to increase GABA A receptor-mediated miniature inhibitory postsynaptic currents in CA1 pyramidal neurons is indicative of its ability to stimulate the local synthesis and secretion of neurosteroids.

  14. Human beta-mannosidase deficiency associated with peripheral neuropathy.

    PubMed

    Levade, T; Graber, D; Flurin, V; Delisle, M B; Pieraggi, M T; Testut, M F; Carrière, J P; Salvayre, R

    1994-01-01

    Human beta-mannosidosis is an inherited lysosomal storage disorder described in only seven families. We present a further case in a black African 14-year-old boy with severely deficient beta-mannosidase activity, bilateral thenar and hypothenar amyotrophy, electrophysiologically demonstrable demyelinating peripheral neuropathy, and cytoplasmic vacuolation of skin fibroblasts and lymphoid cells. The clinical and biochemical features of our patient are compared to those of previously reported patients.

  15. Are Human Peripheral Nerves Sensitive to X-Ray Imaging?

    PubMed Central

    Scopel, Jonas Francisco; de Souza Queiroz, Luciano; O’Dowd, Francis Pierce; Júnior, Marcondes Cavalcante França; Nucci, Anamarli; Hönnicke, Marcelo Gonçalves

    2015-01-01

    Diagnostic imaging techniques play an important role in assessing the exact location, cause, and extent of a nerve lesion, thus allowing clinicians to diagnose and manage more effectively a variety of pathological conditions, such as entrapment syndromes, traumatic injuries, and space-occupying lesions. Ultrasound and nuclear magnetic resonance imaging are becoming useful methods for this purpose, but they still lack spatial resolution. In this regard, recent phase contrast x-ray imaging experiments of peripheral nerve allowed the visualization of each nerve fiber surrounded by its myelin sheath as clearly as optical microscopy. In the present study, we attempted to produce high-resolution x-ray phase contrast images of a human sciatic nerve by using synchrotron radiation propagation-based imaging. The images showed high contrast and high spatial resolution, allowing clear identification of each fascicle structure and surrounding connective tissue. The outstanding result is the detection of such structures by phase contrast x-ray tomography of a thick human sciatic nerve section. This may further enable the identification of diverse pathological patterns, such as Wallerian degeneration, hypertrophic neuropathy, inflammatory infiltration, leprosy neuropathy and amyloid deposits. To the best of our knowledge, this is the first successful phase contrast x-ray imaging experiment of a human peripheral nerve sample. Our long-term goal is to develop peripheral nerve imaging methods that could supersede biopsy procedures. PMID:25757086

  16. A rapid ultrasound-assisted dispersive liquid-liquid microextraction followed by ultra-performance liquid chromatography for the simultaneous determination of seven benzodiazepines in human plasma samples.

    PubMed

    Fernández, Purificación; González, Cristina; Pena, M Teresa; Carro, Antonia M; Lorenzo, Rosa A

    2013-03-12

    A simple and efficient ultrasound-assisted dispersive liquid-liquid microextraction (UA-DLLME) method has been developed for the determination of seven benzodiazepines (alprazolam, bromazepam, clonazepam, diazepam, lorazepam, lormetazepam and tetrazepam) in human plasma samples. Chloroform and methanol were used as extractant and disperser solvents, respectively. The influence of several variables (e.g., type and volume of dispersant and extraction solvents, pH, ultrasonic time and ionic strength) was carefully evaluated and optimized, using an asymmetric screening design 3(2)4(2)//16. Analysis of extracts was performed by ultra-performance liquid chromatography coupled with photodiode array detection (UPLC-PDA). Under the optimum conditions, two reversed-phases, Shield RP18 and C18 columns were successfully tested, obtaining good linearity in a range of 0.01-5μgmL(-1), with correlation coefficients r>0.996. Quantification limits ranged between 4.3-13.2ngmL(-1) and 4.0-14.8ngmL(-1), were obtained for C18 and Shield RP18 columns, respectively. The optimized method exhibited a good precision level, with relative standard deviation values lower than 8%. The recoveries studied at two spiked levels, ranged from 71 to 102% for all considered compounds. The proposed method was successfully applied to the analysis of seven benzodiazepines in real human plasma samples.

  17. Characterization of muscarinic cholinergic receptor subtypes in human peripheral lung

    SciTech Connect

    Bloom, J.W.; Halonen, M.; Yamamura, H.I.

    1988-02-01

    The authors have characterized the muscarinic cholinergic receptor subtypes in human peripheral lung membranes using the selective muscarinic antagonist (/sup 3/H)pirenzepine ((/sup 3/H)PZ) and the classical muscarinic antagonist (/sup 3/H)(-)-quinuclidinyl benzilate. High-affinity binding with pharmacologic specificity was demonstrated for both radioligands. The high affinity Kd for (/sup 3/H)PZ binding determined from saturation isotherms was 5.6 nM, and the Kd for (/sup 3/H)(-)-quinuclidinyl benzilate binding was 14.3 pM. Approximately 62% of the total muscarinic binding sites in human peripheral lung bind (/sup 3/H)PZ with high affinity. There was no significant effect of the guanine nucleotide, guanyl-5'-yl imidodiphosphate, on the inhibition of (/sup 3/H)(-)-quinyclidinyl benzilate binding by the muscarinic agonist carbachol in peripheral lung membranes. If the muscarinic receptor with high affinity for PZ has an important role in bronchoconstriction, its characterization could result in the development of more selective bronchodilators.

  18. [Benzodiazepines in geriatrics].

    PubMed

    Hofmann, W

    2013-12-01

    About 10 % of community dwelling elderly people are chronically consuming benzodiazepines. This proportion rises to 30 % in nursing homes or hospitals. Particularly in older patients, this usage leads to a higher risk of adverse drug reactions. Exposure contributes to delirium and falls with subsequent femoral neck fractures. The WHO has classified the risk potential of the new z-drugs to be the same as that associated with benzodiazepines. It is recommended that benzodiazepines should be discontinued step by step under supervision of a doctor or the dosage should be reduced.

  19. Benzodiazepines: Sedation and Agitation.

    PubMed

    Gallagher, Catherine

    2016-01-01

    Dental anxiety is common and frequently poses a barrier to necessary dental treatment. The increasing availability of conscious sedation in dental practice has made treatment much more accessible for anxious patients. At present, benzodiazepines are the most commonly used drugs in sedation practice and provide a pleasant experience for most, but not all, patients. An understanding of the mechanism of action of benzodiazepines should inform our practice and deepen our understanding of why and how sedation may fail. CPD/CLINICAL RELEVANCE: As an increasing number of dentists provide sedation for their patients an update on benzodiazepines is timely.

  20. Regional specific binding of (/sup 11/C)RO 15 1788 to central type benzodiazepine receptors in human brain: quantitative evaluation by PET

    SciTech Connect

    Pappata, S.; Samson, Y.; Chavoix, C.; Prenant, C.; Maziere, M.; Baron, J.C.

    1988-06-01

    The central type benzodiazepine receptors were studied in 17 healthy human subjects with /sup 11/C-RO 15 1788 and positron emission tomography (PET). The brain regional distribution of the tracer in eight control studies performed after injection of trace doses of /sup 11/C-RO 15 1788 was consistent with that of benzodiazepine receptors. Saturation studies with co-injected cold RO 15 1788 in the remaining subjects showed a dose-dependent decrease of brain radiotracer until full inhibition of specific binding was achieved with doses above 0.1 mg/kg (four studies). Based on the results, a simple method to estimate the specifically bound /sup 11/C-RO 15 1788 regionally in a single PET study is proposed, using the data from the full-saturation studies as a stable estimate of the nondisplaceable radioligand concentration. Using this method, it was found that quasiequilibrium between the estimated specifically bound and nondisplaceable components was achieved at times equal to or longer than 20 min after tracer administration. The validity of this method was partly supported by further results, showing a good agreement between the regional specific binding so calculated and postmortem data of receptor density.

  1. The use of microcomputer-based psychomotor tests for the evaluation of benzodiazepine effects on human performance: a review with emphasis on temazepam.

    PubMed Central

    Kunsman, G W; Manno, J E; Manno, B R; Kunsman, C M; Przekop, M A

    1992-01-01

    1. The literature relating to the effects of benzodiazepines in general, and temazepam in particular, on human psychomotor performance as assessed using microcomputer-based testing batteries is surveyed. 2. The adverse effects of central nervous system depressants on performance is an important mediocolegal issue and frequently comes into question in on-the-road and on-the-job accidents. The use of microcomputer-based testing batteries allows for performance evaluation both in the laboratory and at-the-scene, as well as providing the opportunity to model a large number of different behaviours required in routine yet complex psychomotor tasks. 3. The conclusions in general are: (1) The benzodiazepines as a class of drugs impair both cognitive and motor performance. These effects are often subtle when low doses are involved or when testing occurs the morning following evening administration of the medication. (2) No single psychomotor task adequately simulates complex daily tasks such as automobile driving. A battery of tests that evaluates a number of the components of such tasks is necessary to determine adequately the full range of effects of these medications. PMID:1457261

  2. Gamma-aminobutyric acid-modulated benzodiazepine binding sites in bacteria

    SciTech Connect

    Lummis, S.C.R.; Johnston, G.A.R. ); Nicoletti, G. ); Holan, G. )

    1991-01-01

    Benzodiazepine binding sites, which were once considered to exist only in higher vertebrates, are here demonstrated in the bacteria E. coli. The bacterial ({sup 3}H)diazepam binding sites are modulated by GABA; the modulation is dose dependent and is reduced at high concentrations. The most potent competitors of E.Coli ({sup 3}H)diazepam binding are those that are active in displacing ({sup 3}H)benzodiazepines from vertebrate peripheral benzodiazepine binding sites. These vertebrate sites are not modulated by GABA, in contrast to vertebrate neuronal benzodiazepine binding sites. The E.coli benzodiazepine binding sites therefore differ from both classes of vertebrate benzodiazepine binding sites; however the ligand spectrum and GABA-modulatory properties of the E.coli sites are similar to those found in insects. This intermediate type of receptor in lower species suggests a precursor for at least one class of vertebrate benzodiazepine binding sites may have existed.

  3. Benzodiazepine metabolism: an analytical perspective.

    PubMed

    Mandrioli, Roberto; Mercolini, Laura; Raggi, Maria Augusta

    2008-10-01

    Benzodiazepines are currently among the most frequently prescribed drugs all over the world. They act as anxiolytics, sedatives, hypnotics, amnesics, antiepileptics and muscle relaxants. Despite their common chemical scaffold, these drugs differ in their pharmacokinetic and metabolic properties. In particular, they are biotransformed by different cytochrome P450 isoforms and also by different UDP-glucuronosyltransferase subtypes. The most important studies on the metabolic characteristics of several 1,4-benzodiazepines, carried out from 1998 onwards, are reported and briefly discussed in this review. Moreover, the analytical methods related to these studies are also described and commented upon and their most important characteristics are highlighted. Most methods are based on liquid chromatography, which provides wide applicability and good analytical performance granting high precision, accuracy and feasibility. Mass spectrometry is gaining widespread acceptance, particularly if the matrix is very complex and variable, such as human or animal blood. However, spectrophotometric detection is still used for this purpose and can grant sufficient selectivity and sensitivity when coupled to suitable sample pre-treatment procedures. A monograph is included for each of the following benzodiazepines: alprazolam, bromazepam, brotizolam, clotiazepam, diazepam, etizolam, flunitrazepam, lorazepam, midazolam, oxazepam and triazolam.

  4. Evaluation of Anti-Hyperalgesic and Analgesic Effects of Two Benzodiazepines in Human Experimental Pain: A Randomized Placebo-Controlled Study

    PubMed Central

    Vuilleumier, Pascal H.; Besson, Marie; Desmeules, Jules; Arendt-Nielsen, Lars; Curatolo, Michele

    2013-01-01

    Background and Aims Compounds that act on GABA-receptors produce anti-hyperalgesia in animal models, but little is known on their effects in humans. The aim of this study was to explore the potential usefulness of GABA-agonism for the control of pain in humans. Two agonists at the benzodiazepine-binding site of GABAA-receptors (clobazam and clonazepam) were studied using multiple experimental pain tests. Positive results would support further investigation of GABA agonism for the control of clinical pain. Methods In a randomized double-blind crossover design, 16 healthy male volunteers received clobazam 20 mg, clonazepam 1 mg and tolterodine 1 mg (active placebo). The area of static hyperalgesia after intradermal capsaicin injection was the primary endpoint. Secondary endpoints were: area of dynamic hyperalgesia, response to von Frey hair stimulation, pressure pain thresholds, conditioned pain modulation, cutaneous and intramuscular electrical pain thresholds (1, 5 and 20 repeated stimulation), and pain during cuff algometry. Results For the primary endpoint, an increase in the area of static hyperalgesia was observed after administration of placebo (p<0.001), but not after clobazam and clonazepam. Results suggestive for an anti-hyperalgesic effect of the benzodiazepines were obtained with all three intramuscular pain models and with cuff algometry. No effect could be detected with the other pain models employed. Conclusions Collectively, the results are suggestive for a possible anti-hyperalgesic effect of drugs acting at the GABAA-receptors in humans, particularly in models of secondary hyperalgesia and deep pain. The findings are not conclusive, but support further clinical research on pain modulation by GABAergic drugs. Because of the partial results, future research should focus on compounds acting selectively on subunits of the GABA complex, which may allow the achievement of higher receptor occupancy than unselective drugs. Our data also provide information

  5. Human peripheral nerve macrophages in normal and pathological conditions.

    PubMed

    Bonetti, B; Monaco, S; Giannini, C; Ferrari, S; Zanusso, G; Rizzuto, N

    1993-09-01

    We investigated, by immunocytochemistry and immune electron microscopy, the immunophenotype, morphology and functional properties of human peripheral nervous system (PNS) macrophages (M phi) under normal and pathological conditions. Endoneurial M phi disclosed an elongated, ramified morphology, with the main processes oriented along the major axis of nerve fibers; they shared several lineage-related and functional markers with monocyte/macrophages and central nervous system (CNS) microglia, including CD4, CR3, CR4 and FcRIII. In addition, basal expression of HLA-DR antigens was exclusively confined to M phi in normal PNS. In the course of unrelated pathological conditions, resident M phi underwent activation with transformation to hypertrophic cells or foamy phagocytes and up-regulation of the markers expressed in normal conditions; new expression of a macrophagic antigen was detected on activated M phi. In different neuropathies, HLA-DR expression was also detected on non-myelin forming Schwann cells with ultrastructural features indicative of denervation. The present results demonstrate that the human PNS is provided with an intrinsic population of immunocompetent and potentially phagocytic M phi, which represent the peripheral counterpart of CNS microglia.

  6. Chronic use of benzodiazepines among older adults

    PubMed Central

    Alvarenga, Jussara Mendonça; Giacomin, Karla Cristina; de Loyola, Antônio Ignácio; Uchoa, Elizabeth; Firmo, Josélia Oliveira Araújo

    2014-01-01

    OBJECTIVE To analyze the perception of and motivation for the chronic use of benzodiazepine among older adults. METHODS A qualitative study was conducted on 22 older adults living in Bambuí, MG, Southeastern Brazil, who were taking benzodiazepines and had the clinical and cognitive ability to respond to interview questions. The collected data were analyzed on the basis of the “signs, meanings, and actions” model. RESULTS The main reasons pointed out for the use of benzodiazepines were “nervousness”, “sleep problems”, and “worry” due to family and financial problems, everyday problems, and existential difficulties. None of the interviewees said that they used benzodiazepines in a dose higher than that recommended or had been warned by health professionals about any risks of their continuous use. Different strategies were used to obtain the prescription for the medication, and any physician would prescribe it, indicating that a bond was established with the drug and not with the health professional or healthcare service. Obtaining and consuming the medication turned into a crucial issue because benzodiazepine assumes the status of an essential food, which leads users to not think but sleep. It causes a feeling of relief from their problems such as awareness of human finitude and fragility, existential difficulties, and family problems. CONCLUSIONS Benzodiazepine assumes the characteristics of polyvalence among older adults, which extrapolate specific clinical indications, and of essentiality to deal with life’s problems in old age. Although it relieves the “nerves”, the chronic use of benzodiazepines buffers suffering and prevents older adults from going through the suffering. This shows important difficulties in the organization and planning of strategies that are necessary for minimizing the chronic use in this population. PMID:26039388

  7. Chronic use of benzodiazepines among older adults.

    PubMed

    Alvarenga, Jussara Mendonça; Giacomin, Karla Cristina; Loyola Filho, Antônio Ignácio de; Uchoa, Elizabeth; Firmo, Josélia Oliveira Araújo

    2014-12-01

    OBJECTIVE To analyze the perception of and motivation for the chronic use of benzodiazepine among older adults. METHODS A qualitative study was conducted on 22 older adults living in Bambuí, MG, Southeastern Brazil, who were taking benzodiazepines and had the clinical and cognitive ability to respond to interview questions. The collected data were analyzed on the basis of the "signs, meanings, and actions" model. RESULTS The main reasons pointed out for the use of benzodiazepines were "nervousness", "sleep problems", and "worry" due to family and financial problems, everyday problems, and existential difficulties. None of the interviewees said that they used benzodiazepines in a dose higher than that recommended or had been warned by health professionals about any risks of their continuous use. Different strategies were used to obtain the prescription for the medication, and any physician would prescribe it, indicating that a bond was established with the drug and not with the health professional or healthcare service. Obtaining and consuming the medication turned into a crucial issue because benzodiazepine assumes the status of an essential food, which leads users to not think but sleep. It causes a feeling of relief from their problems such as awareness of human finitude and fragility, existential difficulties, and family problems. CONCLUSIONS Benzodiazepine assumes the characteristics of polyvalence among older adults, which extrapolate specific clinical indications, and of essentiality to deal with life's problems in old age. Although it relieves the "nerves", the chronic use of benzodiazepines buffers suffering and prevents older adults from going through the suffering. This shows important difficulties in the organization and planning of strategies that are necessary for minimizing the chronic use in this population.

  8. A comparison of the effects of a subtype selective and non-selective benzodiazepine receptor agonist in two CO(2) models of experimental human anxiety.

    PubMed

    Bailey, J E; Papadopoulos, A; Seddon, K; Nutt, D J

    2009-03-01

    Studies in human volunteers that can demonstrate proof of concept are attractive in that possible mechanisms and potential new drug treatments can be examined. We have been developing models of anxiety disorders using the inhalation of 7.5% CO(2) for 20 min to model generalised anxiety disorder, as well as using the previously reported 35% CO(2) as a model for panic anxiety. In a double-blind, placebo-controlled, three-way crossover study in 12 healthy volunteer subjects, we compared a full agonist at the benzodiazepine receptor that binds to four alpha-subtypes of the receptor (alpha-1,-2,-3,-5) (alprazolam 1 mg), with zolpidem (5 mg), an agonist selective for the alpha-1 subtype of the gamma amino butyric acid-receptor subtype A (GABA-A) receptor, which is a widely used hypnotic drug. Compared with placebo, both drugs significantly attenuated peak CO(2)-induced changes in subjective feelings after the inhalation of 7.5% CO(2) for 20 min. However, there were fewer significant differences after a single vital capacity inhalation of 35% CO(2), where zolpidem was less efficacious than alprazolam at reducing CO(2)-induced symptoms. In conclusion, our results show that zolpidem shows some anxiolytic efficacy in the 7.5% CO(2) model, similar to alprazolam, and this is the first report of such an effect of zolpidem in a model of anxiety. These and other studies of benzodiazepines in clinical and volunteer studies suggest a definite role of the GABA-A receptor in CO(2)-induced anxiety, and it would be of interest to examine other GABA-A receptor subtype selective drugs, which are now in early phase clinical studies and are showing selective efficacy in pharmacodynamic studies.

  9. [Benzodiazepine and nonbenzodiazepine hypnotics].

    PubMed

    Nakamura, Masaki; Inoue, Yuichi

    2015-06-01

    The prevalence of insomnia shows an age-associated increase. Especially, persons with age over 60 years frequently suffer from arousal during sleep and early-morning awakening. The reason of this phenomenon can be explained by age-related change in sleepwake regulation, comorbid diseases and psycho-social status. Benzodiazepine derivatives and benzodiazepine agonists have been widely used for treatment of insomnia. These GABA-A receptor agonist hypnotics have sedative effect, possibly causing various adverse events, i.e. falls and hip fracture, anterograde amnesia, next morning hangover especially in the elderly. When making a choice of treatment drugs for the elderly, low dose benzodiazepine hypnotics with relatively high Ω1-selectivity, and newer hypnotics including melatonic receptor agonist or orexin receptor antagonist can become important candidates considering their comorbid diseases or drug interaction with other medications.

  10. Expression of human peripheral cannabinoid receptor for structural studies

    PubMed Central

    Yeliseev, Alexei A.; Wong, Karen K.; Soubias, Olivier; Gawrisch, Klaus

    2005-01-01

    Human peripheral-type cannabinoid receptor (CB2) was expressed in Escherichia coli as a fusion with the maltose-binding protein, thioredoxin, and a deca-histidine tag. Functional activity and structural integrity of the receptor in bacterial protoplast membranes was confirmed by extensive binding studies with a variety of natural and synthetic cannabinoid ligands. E. coli membranes expressing CB2 also activated cognate G-proteins in an in vitro coupled assay. Detergent-solubilized receptor was purified to 80%–90% homogeneity by affinity chromatography followed by ion-exchange chromatography. By high-resolution NMR on the receptor in DPC micelles, it was determined that purified CB2 forms 1:1 complexes with the ligands CP-55,940 and anandamide. The receptor was successfully reconstituted into phosphatidylcholine bilayers and the membranes were deposited into a porous substrate as tubular lipid bilayers for structural studies by NMR and scattering techniques. PMID:16195551

  11. [Benzodiazepines and forensic aspects].

    PubMed

    Michel, L; Lang, J-P

    2003-01-01

    Adverse effects of benzodiazepines are well known since the first one was used in 1958 (chlordiazepoxide). The literature collects study-cases or rarely controlled studies concerning side effects or paradoxical reactions to benzodiazepines. They mostly described drowsiness and behavioral disinhibition, including increased well-being feeling but also hostility, rage access with feeling of invulnerability, serious crimes and sometimes homicides. Delusional, manic, confusional or depressive states are also pointed out. Rate for aggressive behaviour is 0.3 to 0.7% but distinction should be done between accidental or "idiosyncratic" reaction and voluntary sought disinhibition, clearly more frequent. No benzodiazepine has any specificity for these adverse effects but pharmacology, doses, associated drugs (or alcohol) and psychopathology interact to produce hazardous psychic states. Pharmacology: GABA induces a decrease in serotonin compound and vigilance. Pharmacokinetic: first dose effect or over-dose effect, short half-life, lipophily, affinity, digestive absorption, active metabolites interact. Psychopathology: age, alcohol association, psychological status (high initial level of hostility, impulsivity, frustration, personality disorder and depressive status). External conditions: chronic illness, affective and professional frustrations, physical or psychic exhaustion contribute also. Some benzodiazepines (flunitrazepam, diazepam, clorazepate, triazolam, alprazolam, lorazepam, for example) are more often concerned for pharmacokinetics characteristics but also prescription habits. Forensic aspects should be considered in case of homicide. Especially, reality of benzodiazepines consumption and awareness of the potential paradoxical reaction should be precisely evaluated. Special focus on voluntary induced disinhibition has to be done for forensic considerations. Relationship but also crime facilitations are sometimes consciously sought. Some benzodiazepines have already

  12. Benzodiazepines and memory

    PubMed Central

    Roth, T.; Roehrs, T.; Wittig, R.; Zorick, F.

    1984-01-01

    1 Benzodiazepines possess anterograde amnesic properties, disrupting both short-term and long-term memory function. 2 The amount of amnesia is systematically related to dose effects and half-life differences among the benzodiazepines. 3 Memory deficits are found for episodic, semantic, and iconic memory function. 4 The deficits in long-term memory are probably the result of a disruption of consolidation of information in memory and not retrieval from memory. The disruption is produced by rapid sleep onset. 5 Thus the long-term amnesia is really a retrograde effect of sleep and not the anterograde effect of the drug. PMID:6151849

  13. Prokineticins in central and peripheral control of human reproduction.

    PubMed

    Traboulsi, Wael; Brouillet, Sophie; Sergent, Frederic; Boufettal, Houssine; Samouh, Naima; Aboussaouira, Touria; Hoffmann, Pascale; Feige, Jean Jacques; Benharouga, Mohamed; Alfaidy, Nadia

    2015-11-01

    Prokineticin 1 (PROK1) and (PROK2), are two closely related proteins that were identified as the mammalian homologs of their two amphibian homologs, mamba intestinal toxin (MIT-1) and Bv8. PROKs activate two G-protein linked receptors (prokineticin receptor 1 and 2, PROKR1 and PROKR2). Both PROK1 and PROK2 have been found to regulate a stunning array of biological functions. In particular, PROKs stimulate gastrointestinal motility, thus accounting for their family name "prokineticins". PROK1 acts as a potent angiogenic mitogen, thus earning its other name, endocrine gland-derived vascular endothelial factor. In contrast, PROK2 signaling pathway has been shown to be a critical regulator of olfactory bulb morphogenesis and sexual maturation. During the last decade, strong evidences established the key roles of prokineticins in the control of human central and peripheral reproductive processes. PROKs act as main regulators of the physiological functions of the ovary, uterus, placenta, and testis, with marked dysfunctions in various pathological conditions such as recurrent pregnancy loss, and preeclampsia. PROKs have also been associated to the tumor development of some of these organs. In the central system, prokineticins control the migration of GnRH neurons, a key process that controls reproductive functions. Importantly, mutations in PROK2 and PROKR2 are associated to the development of Kallmann syndrome, with direct consequences on the reproductive system. This review describes the finely tuned actions of prokineticins in the control of the central and peripheral reproductive processes. Also, it discusses future research directions for the use of these cytokines as diagnostic markers for several reproductive diseases.

  14. Benzodiazepines and Pregnancy

    MedlinePlus

    ... to suggest that they have long-term harmful effects on the child’s brain or development. Someone told me not to take ... case-control teratologic study of nitrazepam, medazepam, tofisopam, ... L, et al.1992. The effect of benzodiazepines on the fetus and the newborn. ...

  15. Sex differences in the human peripheral blood transcriptome

    PubMed Central

    2014-01-01

    Background Genomes of men and women differ in only a limited number of genes located on the sex chromosomes, whereas the transcriptome is far more sex-specific. Identification of sex-biased gene expression will contribute to understanding the molecular basis of sex-differences in complex traits and common diseases. Results Sex differences in the human peripheral blood transcriptome were characterized using microarrays in 5,241 subjects, accounting for menopause status and hormonal contraceptive use. Sex-specific expression was observed for 582 autosomal genes, of which 57.7% was upregulated in women (female-biased genes). Female-biased genes were enriched for several immune system GO categories, genes linked to rheumatoid arthritis (16%) and genes regulated by estrogen (18%). Male-biased genes were enriched for genes linked to renal cancer (9%). Sex-differences in gene expression were smaller in postmenopausal women, larger in women using hormonal contraceptives and not caused by sex-specific eQTLs, confirming the role of estrogen in regulating sex-biased genes. Conclusions This study indicates that sex-bias in gene expression is extensive and may underlie sex-differences in the prevalence of common diseases. PMID:24438232

  16. Simultaneous Separation of Eight Benzodiazepines in Human Urine Using Field-Amplified Sample Stacking Micellar Electrokinetic Chromatography.

    PubMed

    Oledzka, Ilona; Kulińska, Zofia; Prahl, Adam; Baczek, Tomasz

    2015-01-01

    A novel approach for the simultaneous quantification of eight benzodiazepines (BZDs) using dispersive liquid-liquid microextraction (DLLME) and field-amplified sample stacking (FASS) combined with micellar electrokinetic chromatography (MEKC) was investigated and evaluated in the context of precision, accuracy, sensitivity, linearity, detection and limits of quantification (LOQ). The absolute recovery rates of BZDs were above 90.65%. The limits of detection (LOD) were 20 ng/mL for chlordiazepoxide, estazolam, temazepam and midazolam, and 30 ng/mL for clonazepam, lorazepam, lormetazepam and medazepam, while the LOQ was set at 50 ng/mL for chlordiazepoxide, estazolam, temazepam and midazolam, and 100 ng/mL for clonazepam, lorazepam, lormetazepam and medazepam. Linearity was confirmed in the range of 50-2,000 ng/mL for chlordiazepoxide, estazolam, temazepam and midazolam, and 100-2,000 ng/mL for clonazepam, lorazepam, lormetazepam and medazepam, with a correlation coefficient greater than 0.9987 for all analytes. The elaborated procedure meets all the requirements of analytical methods. During the extraction procedure, a mixture of 1 mL of ethanol and 500 µL of dichloromethane, used as the disperser and extraction solvent, respectively, was rapidly injected into 3 mL of a urine sample. A significant improvement in sensitivity was achieved when DLLME was used to extract BZDs from the urine sample and FASS as an on-line preconcentration technique was developed. For the best separation of analytes, the running buffer was composed of 30 mM SDS, 10 mM sodium tetraborate and 15% methanol (pH 8.8), whereas a sample buffer was composed of 10 mM SDS and 2 mM sodium tetraborate. Moreover, a fused-silica capillary [inner diameter (i.d.) of 75 µm and length of 50 cm], photodiode array detection, pneumatic injection for 15 s and a voltage of 23 kV were applied. The applicability of the method has been confirmed for the analysis of BZD in urine samples collected from patients who

  17. Development of the MAE/UHPLC-MS-TOF method for determination of benzodiazepines in human bio-fluids for toxicological analysis.

    PubMed

    Woźniakiewicz, Aneta; Wietecha-Posłuszny, Renata; Woźniakiewicz, Michał; Nowak, Julia; Kościelniak, Paweł

    2015-04-10

    A rapid method of microwave-assisted extraction (MAE) followed by ultrahigh performance liquid chromatography with mass spectrometry with time of flight detection (UHPLC-MS-TOF) was optimized and validated for the purpose of determination of five benzodiazepines in human serum and blood samples. Extraction parameters and conditions of the UHPLC-MS-TOF method were defined. Validation of the developed method was performed at three concentration levels: 10, 100 and 250 ng/mL of each drug for both serum and blood samples. For serum and blood the limit of detection was found in the ranges 0.46-2.58 ng/mL and 0.43-1.87 ng/mL, precision (RSD): 0.3-6.7% and 0.9-8.4%, accuracy of the assay (RE): -5.3 to +2.4% and -5.7 to +7.6%, recovery: 80.5-104.3% and 79.9-106.9%, matrix effects: 95.9-110.5% and 97.5-114.2%, respectively. Moreover, the optimized and validated MAE/UHPLC-MS-TOF method was applied to analysis of blood samples.

  18. Benzodiazepines for schizophrenia.

    PubMed

    Dold, Markus; Li, Chunbo; Tardy, Magdolna; Khorsand, Vesal; Gillies, Donna; Leucht, Stefan

    2012-11-14

    Because of the high number of people with schizophrenia not responding adequately to monotherapy with antipsychotic agents, the evidence regarding the efficacy and safety of additional medication was examined in a number of clinical trials. One approach to this research question was the use of benzodiazepines, as monotherapy as well as in combination with antipsychotics. To determine the efficacy, acceptability, and tolerability of benzodiazepines in people with schizophrenia and schizophrenia-like psychoses. In February 2011, we updated the literature search of the previous version of this systematic review (last search March 2005). We searched the trial register of the Cochrane Schizophrenia Group (containing methodical searches of BIOSIS, CINAHL, Dissertation abstracts, EMBASE, LILACS, MEDLINE, PSYNDEX, PsycINFO, RUSSMED, Sociofile, supplemented with hand searching of relevant journals and numerous conference proceedings). Additionally, we inspected references of all identified studies for further relevant studies and contacted authors of relevant publications in order to obtain missing data from existing trials. We applied no language restrictions. We included all randomised controlled trials comparing benzodiazepines (as monotherapy or as adjunctive agent) with antipsychotic drugs or placebo for the pharmacological management of schizophrenia and/or schizophrenia-like psychoses. Review authors (MD and CL) analysed independently the new references of the update-search referring to the inclusion criteria. MD and CL extracted all data from the included trials. For dichotomous outcomes we calculated risk ratios (RR) and their 95% confidence intervals (CI). We analysed continuous data by using mean differences (MD) and their 95% CI. We assessed each pre-selected outcome from the included trials with the risk of bias tool. The 2011 update search yielded three further randomised controlled trials. The review currently includes 34 studies with 2657 participants. Most

  19. Benzodiazepine use and aggressive behaviour: a systematic review.

    PubMed

    Albrecht, Bonnie; Staiger, Petra K; Hall, Kate; Miller, Peter; Best, David; Lubman, Dan I

    2014-12-01

    The relationship between benzodiazepine consumption and subsequent increases in aggressive behaviour in humans is not well understood. The current study aimed to identify, via a systematic review, whether there is an association between benzodiazepine consumption and aggressive responding in adults. A systematic review was conducted and reported in line with the PRISMA statement. English articles within MEDLINE, PsycARTICLES, PsycINFO, Academic Search Complete, and Psychology and Behavioural Sciences Collection databases were searched. Additional studies were identified by searching reference lists of reviewed articles. Only articles that explicitly investigated the relationship between benzodiazepine consumption and subsequent aggressive behaviour, or a lack thereof, in human adults were included. Forty-six studies met the inclusion criteria. It was not possible to conduct a meta-analysis due to the heterogeneity of study design and benzodiazepine type and dose. An association between benzodiazepine use and subsequent aggressive behaviour was found in the majority of the more rigorous studies, although there is a paucity of high-quality research with clinical or forensic populations. Diazepam and alprazolam have received the most attention. Dose-related findings are inconsistent: therapeutic doses may be more likely to be associated with aggressive responding when administered as a once-off, whereas higher doses may be more risky following repeated administration. Trait levels of anxiety and hostility may indicate a vulnerability to the experience of benzodiazepine-related aggression. There appears to be a moderate association between some benzodiazepines and subsequent aggressive behaviour in humans. The circumstances under which aggressive responding may be more likely to follow benzodiazepine use remain unclear, although some evidence suggests dose and/or personality factors may influence this effect. © The Royal Australian and New Zealand College of

  20. Identification of interleukin-2 in human peripheral blood eosinophils.

    PubMed Central

    Levi-Schaffer, F; Barkans, J; Newman, T M; Ying, S; Wakelin, M; Hohenstein, R; Barak, V; Lacy, P; Kay, A B; Moqbel, R

    1996-01-01

    Interleukin-2 (IL-2) is an essential growth factor for T cells. Previous studies have shown that human peripheral eosinophils respond to IL-2 in chemotaxis and express the IL-2 receptor (CD25). In addition, eosinophils have been shown to transcribe messenger RNA for IL-2. The aim of the present study was to determine whether eosinophils translate mRNA for IL-2 and to determine the site of intracellular localization. By immunocytochemistry, an average of 9% of cells showed cytoplasmic staining for IL-2 in freshly isolated unstimulated blood eosinophils obtained from asthmatic subjects who were not receiving oral corticosteroid treatment (n = 5). Freshly isolated, disrupted, highly purified eosinophils (> 99%, by CD16- immunomagnetic selection) contained an average of 6 pg/10(6) cells of IL-2 measured by a specific enzyme linked immunosorbent assay (ELISA) (n = 7). Purified eosinophil incubated with serum-coated Sephadex beads showed an increase in the amount of intracellularly-retained IL-2 (26.2 +/- 7.2 pg/10(6) cells) with some evidence for release of this cytokine but only in three out of six eosinophil preparations (range 1.3-5.8 pg/10(6) cells). The intracellular localization of IL-2 was determined by fractionation of the cells on a linear (0-45%) Nycodenz gradient in sucrose buffer followed by detection of IL-2 in the fractions using an IL-2-specific ELISA and dot blotting. The majority of the IL-2 detected co-eluted with known eosinophil granule markers (i.e. major basic protein (MBP), eosinophil cationic protein (ECP), eosinophil peroxidase (EPO) and beta-hexosaminidase) but small quantities were also detected in the cytosolic (lactate dehydrogenase-(LDH) associated) and membrane (CD9+) fractions. Immunogold labelling of intact eosinophils using an anti-IL-2 monoclonal antibody confirmed IL-2 immunoreactivity in association with the eosinophil crystalline granule cores. These data are consistent with the hypothesis that eosinophils synthesize, release and

  1. Drugs of abuse and benzodiazepines in the Madrid Region (Central Spain): seasonal variation in river waters, occurrence in tap water and potential environmental and human risk.

    PubMed

    Mendoza, A; Rodríguez-Gil, J L; González-Alonso, S; Mastroianni, N; López de Alda, M; Barceló, D; Valcárcel, Y

    2014-09-01

    This work analyzes the seasonal variation (winter and summer) of ten drugs of abuse, six metabolites and three benzodiazepines in surface waters from the Jarama and Manzanares Rivers in the Madrid Region, the most densely populated area in Spain. The occurrence of these compounds in tap water in this region is also investigated and a preliminary human health risk characterization performed for those substances found in tap water. Finally, a screening level risk assessment that combines the measured environmental concentrations (MECs) with dose-response data to estimate Hazard Quotients (HQs) for the compounds studied is also presented. The results of this study show the presence of fourteen out of the nineteen compounds analyzed in winter and twelve of them in summer. The most ubiquitous compounds, with a frequency of detection of 100% in both seasons, were the cocaine metabolite benzoylecgonine (BE), the amphetamine-type stimulant (ATS) ephedrine (EPH), the opioid methadone (METH), the METH metabolite 2-ethylene-1,5-dimethyl-3,3-diphenylpyrrolidine (EDDP), and the three benzodiazepines investigated, namely alprazolam (ALP), diazepam (DIA) and lorazepam (LOR). The highest concentrations observed corresponded to EPH (1020ngL(-1) in winter and 250ngL(-1) in summer). The only compounds not detected in both seasons were heroin (HER) and its metabolite 6-acetylmorphine (6ACM), lysergic acid diethylamide (LSD) and its metabolite 2-oxo-3-hydroxy-LSD (O-H-LSD), and Δ(9)-tetrahydrocannabinol (THC). In terms of overall concentration, all sampling points presented higher concentrations in winter than in summer. Statistical analyses performed to gather evidence concerning occasional seasonal differences in the concentrations of individual substances between summer and winter showed statistically significantly higher concentrations (p<0.05) of BE, EPH and the opioid morphine (MOR) in winter than in summer. Two out of the nineteen compounds studied, namely cocaine (CO) and EPH

  2. Tolerance to benzodiazepines among long-term users in primary care.

    PubMed

    Willems, Inge A T; Gorgels, Wim J M J; Oude Voshaar, Richard C; Mulder, Jan; Lucassen, Peter L B J

    2013-08-01

    Tolerance towards the effects of benzodiazepines is observed in various animal and human studies. Therefore, it is assumed that patients who use benzodiazepines for a longer period of time need to increase their dose over time to experience the same effect. To observe whether long-term benzodiazepine users increase their dose over time. From the Dutch National Information Network of Family Practices, a group of long-term benzodiazepine users was identified. This group was divided into an incident long-term benzodiazepine users group (N = 113) and a prevalent long-term benzodiazepine users group (N = 992). Long-term use of benzodiazepines was defined as usage for at least 6 months. The main outcome was a change in prescribed dose from baseline until 24 months after baseline. Linear regression analysis was performed to evaluate dose change. Neither incident long-term benzodiazepine users nor prevalent long-term benzodiazepine users were prescribed increasing dosages during follow-up. There is no increase in prescribed dose among long-term users, as might be expected due to the development of tolerance to the effects of benzodiazepines.

  3. The stimulatory effect of canrenoate, a mineralocorticoid antagonist, on the activity of the hypothalamus-pituitary-adrenal axis is abolished by alprazolam, a benzodiazepine, in humans.

    PubMed

    Grottoli, S; Giordano, R; Maccagno, B; Pellegrino, M; Ghigo, E; Arvat, E

    2002-10-01

    Mineralocorticoid receptors (MR) in the hippocampus play a major role in the control of the hypothalamus-pituitary-adrenal (HPA) axis, mediating the proactive feedback of glucocorticoids in the maintenance of basal activity. Intracerebroventricular and intrahippocampal MR blockade stimulates HPA axis in animals; the systemic administration of mineralocorticoid antagonists enhances spontaneous and CRH-stimulated ACTH and cortisol secretion in humans. Benzodiazepines, namely alprazolam, activate central gamma-aminobutyric acid (GABA)ergic receptors, which are mainly distributed in the hippocampus. Alprazolam has a inhibitory effect on HPA axis either in basal conditions or after central nervous system-mediated stimuli. In humans, alprazolam strongly reduces the corticotroph responsiveness to removal of glucocorticoid feedback by metyrapone. We studied the effect of alprazolam (0.02 mg/kg, orally) on the effect of canrenoate (CAN), an MR antagonist (200 mg as an iv bolus, followed by 200 mg infused in 250 ml saline) or placebo on ACTH, cortisol, and dehydroepiandrosterone (DHEA) secretion in six normal young women (aged 25-32 yr; body mass index, 19-23 kg/m(2)). During placebo, ACTH, cortisol, and DHEA secretion showed a progressive decrease (baseline vs. nadir, mean +/- SEM, from 1830-2400 h, 2.6 +/- 0.3 vs. 1.4 +/- 0.3 pmol/liter, 133.2 +/- 16.4 vs. 46.9 +/- 5.2 nmol/liter, and 22.6 +/- 2.3 vs. 18.6 +/- 2.3 nmol/liter, respectively), although statistical significance was obtained for ACTH and cortisol only (P < 0.05). During CAN treatment, ACTH, cortisol, and DHEA secretion showed a progressive rise, which began at approximately 2100 h and peaked between 2300 and 2400 h (2.9 +/- 0.3 pmol/liter, 172.6 +/- 27.9 nmol/liter, and 45.3 +/- 10.7 nmol/liter, respectively; P < 0.05). Alprazolam abolished the CAN-induced increases in ACTH, cortisol, and DHEA levels (1.8 +/- 0.1 pmol/liter, 59.7 +/- 8.6 nmol/liter, and 19.8 +/- 6.7 nmol/liter; P < 0.05), inducing hormonal

  4. [239Pu and chromosomal aberrations in human peripheral blood lymphocytes].

    PubMed

    Okladnikova, N D; Osovets, S V; Kudriavtseva, T I

    2009-01-01

    The genome status in somatic cells was assessed using the chromosomal aberration (CA) test in peripheral blood lymphocytes from 194 plutonium workers exposed to occupational radiation mainly from low-transportable compounds of airborne 230Pu. Pu body burden at the time of cytogenetic study varied from values close to the method sensitivity to values multiply exceeding the permissible level. Standard (routine) methods of peripheral blood lymphocytes cultivation were applied. Chromatid- and chromosomal-type structural changes were estimated. Aberrations were estimated per 100 examined metaphase cells. The quantitative relationship between the CA frequency and Pu body burden and the absorbed dose to the lung was found. Mathematical processing of results was carried out based on the phenomenological model. The results were shown as theoretical and experimental curves. The threshold of the CA yield was 0.43 +/- 0.03 kBq (Pu body burden) and 6.12 +/- 1.20 cGy (absorbed dose to the lung).

  5. Pilot trial of gabapentin for the treatment of benzodiazepine abuse or dependence in methadone maintenance patients.

    PubMed

    Mariani, John J; Malcolm, Robert J; Mamczur, Agnieszka K; Choi, Jean C; Brady, Ronald; Nunes, Edward; Levin, Frances R

    2016-05-01

    Benzodiazepine use disorders are a common clinical problem among methadone maintenance treatment patients and have adverse effects on clinical outcomes. To evaluate gabapentin for the outpatient treatment of benzodiazepine abuse or dependence in methadone maintenance patients. Participants (n = 19) using benzodiazepines at least 4 days per week were enrolled into an 8-week randomized double-blind placebo-controlled outpatient pilot trial. All participants received a manual-guided supportive psychotherapy aimed to promote abstinence. Study medication was titrated over a 2-week period to a maximum dose of gabapentin 1200 mg or placebo three times a day. Benzodiazepine use was assessed using urine toxicology confirmed self-report. Benzodiazepines were not provided as part of study participation; participants were provided guidance to gradually reduce benzodiazepine intake. Sixteen participants had post-randomization data for analysis. Retention at week eight was 50%. The mean dose of gabapentin achieved by titration was 2666 mg/day (SD = ± 1446). There were no significant between group differences on benzodiazepine use outcomes (amount benzodiazepine per day [Mann-Whitney U = 27, p = 0.745], abstinent days per week [U = 28, p = 0.811]) and Clinical Instrument Withdrawal Assessment (CIWA)-benzodiazepines scale (U = 29.0, p = 0.913). One participant in the gabapentin group discontinued study medication because of peripheral edema. Two participants in the placebo group requested admission for inpatient detoxification treatment. In outpatient methadone-maintained patients with benzodiazepine use disorder, gabapentin did significantly decrease benzodiazepine use relative to placebo. The small sample recruited for this trial may have limited the ability to detect a group difference.

  6. Benzodiazepines for restless legs syndrome.

    PubMed

    Carlos, Karla; Prado, Gilmar F; Teixeira, Camila Dm; Conti, Cristiane; de Oliveira, Marcio M; Prado, Lucila Bf; Carvalho, Luciane Bc

    2017-03-20

    Restless legs syndrome (RLS) is a common disease affecting about 5% to 15% of the population. Symptoms of RLS can be severe in a minority of and can have a major impact on sleep, mostly sleep initiation, and quality of life. Benzodiazepines are drugs that can induce and maintain sleep and, hence, intuitively are thought to be beneficial to people with RLS. Altough benzodiazepines, particularly clonazepam, are used to treat RLS symptoms, a systematic review done by the American Academy of Sleep Medicine stated that benzodiazepines should not be used as a first-line treatment, although could be used as a coadjuvant therapy. To evaluate the efficacy and safety of benzodiazepine compared to placebo or other treatment for idiopathic RLS, including unconfounded trials comparing benzodiazepines versus open control. In March 2016 we searched CENTRAL, MEDLINE, Embase and LILACS We checked the references of each study and contacted study authors to identify any additional studies. We considered studies published in any language. Randomised clinical trials of benzodiazepine treatment in idiopathic RLS. We did not perform data collection and analysis, since we did not include any studies, MAIN RESULTS: We did not identify any studies that met the inclusion criteria of the review. Two cross-over studies are awaiting classification because the cross-over trials did not give data at the end of the first cross-over period. The effectiveness of benzodiazepines for RLS treatment is currently unknown.

  7. Relaxant effect of benzodiazepines on uterine rings isolated from estrogen-treated rats.

    PubMed

    Kazanietz, M G; Elgoyhen, A B

    1990-08-28

    K(+)-contracted rat uterine rings were relaxed in a concentration-dependent manner by the benzodiazepines Ro 5-4864, diazepam and clonazepam, as well as by the putative peripheral benzodiazepine antagonist PK 11195. The relaxation induced by diazepam was not counteracted by the central antagonist Ro 15-1788 (10 microM), and the relaxant effects of Ro 5-4864 and of diazepam were not prevented by either the GABAA antagonist bicuculline (10 microM) or the beta-adrenoceptor antagonist propranolol (1 microM). The mechanism underlying the relaxant effects of benzodiazepines on K(+)-contracted uterine rings is still under study.

  8. [Long-term prescription of benzodiazepines and non-benzodiazepines].

    PubMed

    Verthein, U; Martens, M S; Raschke, P; Holzbach, R

    2013-07-01

    The number of persons with a dependence on prescription drugs such as sedatives or tranquilizers in Germany is estimated at between 1.4 and 1.9 million. According to national addiction treatment documentations only very few of them seek help in specialised treatment services. The majority of prescription drug-dependent people use benzodiazepines. This medication is usually prescribed by physicians and according to German guidelines it should be prescribed only for limited, short periods and in low doses. This study aims to determine the extent of the problematic prescription of benzodiazepines and non-benzodiazepines. We used prescription data from the Northern Germany Computing Centre for Pharmacies registered between 2005 and 2007. For the German regions of Hamburg, Bremen and Schleswig-Holstein, benzodiazepine prescriptions during an individual prospective period of 12 months were analysed. From July 2005 to June 2006, 294 143 prescriptions of benzodiazepines and non-benzodiazepines were recorded for 78 456 citizens of Hamburg and billed at the expenses of the governmental health insurance funds. In the course of one observed patient year, 51.1% of benzodiazepine prescriptions were in accordance with the German guidelines. 15.6% of the patients were supplied on a long-term basis (0.5-1 DDD during at least 2 months). Prescriptions for women and persons older than 70 years were disproportionately high. Compared with the Federal states of Bremen and Schleswig-Holstein, Hamburg does not show an exceptional position. The prescription of benzodiazepines which is not in accordance with the relevant national guidelines is widespread and calls for discussion and education among physicians and pharmacists. Furthermore, professional addiction services should reconsider ways to help and attract prescription drug-dependent people to cover their needs, as their numbers will grow in an aging society. © Georg Thieme Verlag KG Stuttgart · New York.

  9. The induction of human peripheral blood lymphoid colonies by conditioned media from human tumour cell lines.

    PubMed Central

    Vesole, D H; Moore, G E

    1980-01-01

    Conditioned medium (CM) from 29 human tumour cell lines and 3 malignant pleural fluids were tested for their ability to stimulate lymphoid colony formation in semi-solid agar; 9 of 14 malignant melanomas, 3 of 6 colonic carcinomas, 2 of 5 ovarian carcinomas, 3 of 4 breast carcinomas and 1 of 3 pleural fluids from breast cancer patients contained colony-stimulating activity (CSA) for human peripheral blood lymphoid cells (PBL) in semi-solid agar. Conditioned media also stimulated PBL proliferation in liquid medium; these effects were dose dependent. With the exception of one pleural fluid, extensive dialysis of CM did not significantly increase colony formation; CM from two tumour cell lines demonstrated a significant decrease in the induction of colony formation after dialysis. PMID:6970165

  10. The inhibitory effect of alprazolam, a benzodiazepine, overrides the stimulatory effect of metyrapone-induced lack of negative cortisol feedback on corticotroph secretion in humans.

    PubMed

    Arvat, E; Maccagno, B; Ramunni, J; Di Vito, L; Giordano, R; Gianotti, L; Broglio, F; Camanni, F; Ghigo, E

    1999-08-01

    Alprazolam (ALP), a benzodiazepine that activates gamma-aminobutyric acid-ergic receptors, inhibits the activity of hypothalamo-pituitary-adrenal (HPA) axis, probably via inhibition of hypothalamic CRH and/or arginine vasopressin release. To further clarify the effects of ALP on the HPA axis in humans, in six normal young women (26-34 yr old) we studied the effects of 0.02 mg/kg ALP (administered orally at 0700 h) or placebo on ACTH, cortisol (F), and 11-deoxycortisol (S) levels assayed after placebo or metyrapone (MET; 0.04 g/kg administered orally at 2300 h the night before). After placebo administration, ACTH, F, and S levels showed a progressive decrease from 0700-1200 h (P < 0.03). At 0700 h, ACTH, F, and S levels before ALP overlapped with those after placebo. At 1200 h, ACTH, F, and S levels after ALP were lower than those after placebo (P < 0.03). MET pretreatment strongly increased ACTH (P < 0.03) and S (P < 0.02) while clearly inhibiting F (P < 0.03) levels at 0700 h. After MET, ACTH levels did not show any decrease up to 1200 h; similarly, S levels persisted similar up to 1200 h, whereas F levels at 1200 h were significantly increased (P < 0.03). At 0700 h, MET-induced ACTH and F levels before ALP overlapped with those after MET alone. The MET-induced ACTH levels at 1200 h were markedly inhibited by ALP (P < 0.05). At 1200 h after MET and ALP, a clear reduction of S levels (P < 0.02) and an insignificant F reduction were also found. In conclusion, our present data show that ALP inhibits basal and, much more, metyrapone-induced corticotroph secretion. These findings indicate that the inhibitory effect of central gamma-aminobutyric acid-ergic activation by ALP overrides the stimulatory effect of the MET-induced lack of negative F feedback on corticotroph secretion. These results also point toward potential contraindication of ALP administration in patients with suspected hypoadrenalism.

  11. Peripheral opioid analgesia in experimental human pain models.

    PubMed

    Tegeder, Irmgard; Meier, Silke; Burian, Maria; Schmidt, Helmut; Geisslinger, Gerd; Lötsch, Jörn

    2003-05-01

    This placebo-controlled, double-blind crossover study assessed whether exclusive activation of peripheral opioid receptors results in significant pain reduction. To achieve opioid activity restricted to the periphery, we used a short-term (2 h) low dose infusion of morphine-6-beta-glucuronide (M6G) because M6G does not pass the blood-brain barrier during this time in amounts sufficient to induce CNS effects. The lack of central opioid effects of M6G was confirmed by a lack of change of the pupil size and absence of other opioid-related CNS effects. As a positive control, morphine was infused at a dosage that definitely produced CNS effects. This was evident by a rapid decrease of the pupil size and by other typical opioid-related side effects including nausea, vomiting, itchiness, hiccup and sedation. Three different pain models were employed to evaluate the analgesic effects: (i) cutaneous inflammatory hyperalgesia induced by briefly freezing a small skin area to -30 degrees C ('freeze lesion'); (ii) muscle hyperalgesia induced by a series of concentric and eccentric muscle contractions (DOMS model; delayed onset of muscle soreness); and (iii) pain induced by electrical current (5 Hz sinus stimuli of 0-10 mA). M6G significantly reduced cutaneous hyperalgesia in the 'freeze lesion' model as assessed with von Frey hairs. It also reduced muscle hyperalgesia in the DOMS model. Electrical pain, however, was not affected by M6G. Morphine was significantly more active in the 'freeze lesion' and DOMS model, and also significantly increased the electrical pain threshold and tolerance. Subcutaneous tissue concentrations of M6G and morphine as assessed with microdialysis were about half those of the respective plasma concentrations. The results of the study indicate that M6G has antihyperalgesic effects in inflammatory pain through activation of peripheral opioid receptors. Since this occurs at concentrations that do not cause central opioid effects, M6G might be useful as a

  12. Stem Cell-Derived Immature Human Dorsal Root Ganglia Neurons to Identify Peripheral Neurotoxicants

    PubMed Central

    Klima, Stefanie; Karreman, Christiaan; Grinberg, Marianna; Meisig, Johannes; Henry, Margit; Rotshteyn, Tamara; Rahnenführer, Jörg; Blüthgen, Nils; Sachinidis, Agapios; Waldmann, Tanja; Leist, Marcel

    2016-01-01

    Safety sciences and the identification of chemical hazards have been seen as one of the most immediate practical applications of human pluripotent stem cell technology. Protocols for the generation of many desirable human cell types have been developed, but optimization of neuronal models for toxicological use has been astonishingly slow, and the wide, clinically important field of peripheral neurotoxicity is still largely unexplored. A two-step protocol to generate large lots of identical peripheral human neuronal precursors was characterized and adapted to the measurement of peripheral neurotoxicity. High content imaging allowed an unbiased assessment of cell morphology and viability. The computational quantification of neurite growth as a functional parameter highly sensitive to disturbances by toxicants was used as an endpoint reflecting specific neurotoxicity. The differentiation of cells toward dorsal root ganglia neurons was tracked in relation to a large background data set based on gene expression microarrays. On this basis, a peripheral neurotoxicity (PeriTox) test was developed as a first toxicological assay that harnesses the potential of human pluripotent stem cells to generate cell types/tissues that are not otherwise available for the prediction of human systemic organ toxicity. Testing of more than 30 chemicals showed that human neurotoxicants and neurite growth enhancers were correctly identified. Various classes of chemotherapeutic agents causing human peripheral neuropathies were identified, and they were missed when tested on human central neurons. The PeriTox test we established shows the potential of human stem cells for clinically relevant safety testing of drugs in use and of new emerging candidates. Significance The generation of human cells from pluripotent stem cells has aroused great hopes in biomedical research and safety sciences. Neurotoxicity testing is a particularly important application for stem cell-derived somatic cells, as

  13. Genotoxicity in primary human peripheral lymphocytes after exposure to lithium titanate nanoparticles in vitro.

    PubMed

    Akbaba, Giray B; Turkez, Hasan; Sönmez, Erdal; Tatar, Abdulgani; Yilmaz, Mehmet

    2016-08-01

    Lithium titanate (Li2TiO3) nanoparticles (LTT NPs; <100 nm) are widely used in battery technology, porcelain enamels, and ceramic insulating bodies. With the increased applications of LTT NPs, the concerns about their potential human toxicity effects and their environmental impact were also increased. However, toxicity data for LTT NPs relating to human health are very limited. Therefore, the purpose of this study was to evaluate whether LTT NPs are able to induce genetic damage in human peripheral lymphocytes in vitro when taking into consideration that DNA damage plays an important role in carcinogenesis. With this aim, the chromosome aberrations (CA), sister chromatid exchanges (SCE), and micronucleus (MN) assays were used as genotoxicity end points. Human peripheral lymphocytes obtained from five healthy male volunteers were exposed to LTT NPs at final dispersed concentrations ranging from 0 to 1000 μg/mL for 72 h at 37°C. The obtained results indicated that LTT NPs compound did not induce DNA damage in human peripheral lymphocytes as depicted by CA/cell, SCE/cell, and MN/1000 cell values in all concentrations tested. In summary, our results revealed that exposure to LTT NPs is not capable of inducing DNA lesions in human peripheral lymphocytes for the first time. © The Author(s) 2014.

  14. Pharmacologic strategies for discontinuing benzodiazepine treatment.

    PubMed

    Rickels, K; DeMartinis, N; Rynn, M; Mandos, L

    1999-12-01

    Benzodiazepines have been shown to have broad-spectrum activity, rapid onset of action, and a wide therapeutic window compared with other anxiolytic medications. Yet the use of benzodiazepines has been limited by concern regarding dependence, withdrawal, and abuse. Agents such as antidepressants, serotonergic anxiolytics, anticonvulsants, and beta-blockers have been used with varying degrees of success to help facilitate the tapering of benzodiazepines. Carbamazepine, imipramine, valproate, and trazodone have been beneficial in the management of benzodiazepine discontinuation, but not in decreasing the severity of benzodiazepine withdrawal. A stepwise approach to discontinuing benzodiazepines is offered.

  15. [Benzodiazepines in the treatment of catatonia].

    PubMed

    Van Dalfsen, A N; Van Den Eede, F; Van Den Bossche, B; Sabbe, B G C

    2006-01-01

    A patient who developed acute catatonia during benzodiazepine withdrawal is discussed. The case prompted us to review the literature on the role of benzodiazepines in the treatment of acute catatonia. Only retrospective and open studies were found which indicate that benzodiazepines do have a beneficial effect. Lorazepam is the most widely studied benzodiazepine and at present is the best treatment option. In the specific case of acute catatonia brought on by benzodiazepine withdrawal the recommended dosage is the same as for acute catatonia caused by something other than benzodiazepine withdrawal.

  16. Radioiodinated benzodiazepines: agents for mapping glial tumors

    SciTech Connect

    Van Dort, M.E.; Ciliax, B.J.; Gildersleeve, D.L.; Sherman, P.S.; Rosenspire, K.C.; Young, A.B.; Junck, L.; Wieland, D.M.

    1988-11-01

    Two isomeric iodinated analogues of the peripheral benzodiazepine binding site (PBS) ligand Ro5-4864 have been synthesized and labeled in high specific activity with iodine-125. Competitive binding assays conducted with the unlabeled analogues indicate high affinity for PBS. Tissue biodistribution studies in rats with these /sup 125/I-labeled ligands indicate high uptake of radioactivity in the adrenals, heart, and kidney--tissues known to have high concentrations of PBS. Preadministration of the potent PBS antagonist PK 11195 blocked in vivo uptake in adrenal tissue by over 75%, but to a lesser degree in other normal tissues. In vivo binding autoradiography in brain conducted in C6 glioma bearing rats showed dense, PBS-mediated accumulation of radioactivity in the tumor. Ligand 6 labeled with /sup 123/I may have potential for scintigraphic localization of intracranial glioma.

  17. Characterization of two classes of benzodiazepine binding sites in Schistosoma mansoni.

    PubMed

    Noël, F; Mendonça-Silva, D L; Thibaut, J-P B; Lopes, D V S

    2007-07-01

    As we have recently shown that GABA should be considered a putative neurotransmitter in Schistosoma mansoni, the present work aimed to search for GABAA receptors in adult worms using [3H]-flunitrazepam to label the allosteric benzodiazepine binding site which is classically present on GABAA receptor complexes. We detected a large population (Bmax=8.25+/-1.1 pmol x mg protein(-1)) of high affinity (Kd=33.6+/-1.5 nM) binding sites for flunitrazepam. These sites harboured a singular pharmacological modulation that does not fit well with a mammalian central benzodiazepine receptor, mainly due to a very high affinity for Ro5-4864 and a very low affinity for clonazepam. We also detected a second population of benzodiazepine binding sites labelled with high affinity (IC50=85 nM) by [3H]-PK11195, a selective ligand of the mammalian peripheral benzodiazepine receptor. In conclusion, this work describes the pharmacological properties of a large population of central-like benzodiazepine receptors supporting their study as putative new targets for the development of anti-parasitic agents. We also describe, for the first time, the presence of peripheral benzodiazepine receptors in this parasite.

  18. The use of benzodiazepines in clinical practice

    PubMed Central

    Shader, R. I.; Greenblatt, D. J.

    1981-01-01

    1 The pharmacokinetic characteristics of the six benzodiazepine anxiolytic drugs available in the United States are reviewed. 2 Concern over the abuse potential of the benzodiazepine class of drugs is discussed. PMID:6133535

  19. Induction of tumor necrosis factor in human peripheral-blood mononuclear cells by proteolytic enzymes.

    PubMed

    Desser, L; Rehberger, A

    1990-01-01

    We could demonstrate that polyenzyme preparations as well as bromelain and papain stimulate the production of tumor necrosis factor-alpha in human peripheral-blood mononuclear cell cultures in a time-dependent manner. We give evidence that immunomodulation and especially the release of cytokines may contribute to the therapeutic effect of these preparations.

  20. Differential Infectivity and Division of Toxoplasma gondii in Human Peripheral Blood Leukocytes

    PubMed Central

    Channon, Jacqueline Y.; Seguin, Rosanne M.; Kasper, Lloyd H.

    2000-01-01

    When tachyzoites were incubated with human peripheral blood leukocytes in vitro, more monocytes and dendritic cells than neutrophils or lymphocytes were infected. Although tachyzoites were able to divide in each of these cell types, monocytes and dendritic cells were more permissive to rapid tachyzoite division than neutrophils or lymphocytes. PMID:10899898

  1. Genotoxic effect of ozone in human peripheral blood leukocytes.

    PubMed

    Díaz-Llera, Silvia; González-Hernández, Yanela; Prieto-González, E A; Azoy, Angel

    2002-05-27

    The genotoxic effect of ozone was studied in human leukocytes in vitro, using the single cell gel electrophoresis (SCGE) assay. Cell treatment for 1 h at 37 degrees C with 0.9-5.3 mM O(3) resulted in a dose-dependent increase of DNA damage, comparable to that induced by 4-40 mM of H(2)O(2), used as a positive control. This effect of ozone was reversed by post-treatment incubation of the cells for 45-90 min at 37 degrees C, and prevented by pre-incubation of the cells with catalase (20 microg/ml). These results demonstrate that O(3) induces DNA-damage in primary human leukocytes. The damage is rapidly repaired, and probably mediated by the formation of H(2)O(2).

  2. Use and abuse of benzodiazepines*

    PubMed Central

    1983-01-01

    Benzodiazepines are widely used for the treatment of anxiety, insomnia, and certain neuromuscular and convulsive disorders. However, their widespread availability has given rise to fears that they are over-prescribed. The problem is compounded by the fact that there is no universal agreement among medical practitioners as to the clinical indications warranting the use of these drugs. Although most industrialized countries exercise control over the sale and manufacture of benzodiazepines, many developing countries do not have sufficient control of these drugs. As a result, information on drug utilization and associated problems is difficult to obtain and there is a lack of comparative data on drug consumption in different countries. The present article describes the current knowledge on the pharmacological, clinical, and epidemiological characteristics of benzodiazepines, and the problems associated with their use, and indicates areas where more research is needed. Recommendations are made for future work. PMID:6605211

  3. Interaction of Choriocarcinoma Cells and Human Peripheral Blood Lymphocytes

    PubMed Central

    August, Charles S.; Cox, Sheila T.; Naughton, Michael A.

    1979-01-01

    Cultured choriocarcinoma (Be Wo) cells exist that share many of the morphologic and bio-synthetic properties of normal human trophoblasts. In an attempt to develop a model for the immunologic relationship between a sensitized mother and fetus, we mixed Be Wo cells with mitogen-activated cytotoxic lymphocytes in vitro. Be Wo cells were resistant to the cytolytic effects of the activated lymphocytes despite 24-h exposure and intimate cell-to-cell contact as determined by microscopy. Control target cells, a line of human hepatoma cells, were readily destroyed. Cytotoxicity was measured by determining residual radioactivity of [3H]thymidine-labeled target cells after exposure to activated lymphocytes. Employing the quantitative assay, we confirmed the morphologic results and showed that Be Wo and a number of other choriocarcinoma cell lines were resistant to the cytotoxic effects of lymphocytes activated by phytohemagglutinin, pokeweed mitogen, and allogeneic cells in mixed lymphocyte cultures. Moreover, Be Wo cells were resistant to injury over a wide range of killer to target cell ratios. Significant killing of the Be Wo cells occurred only after prolonged exposure (48 and 72 h) to the activated lymphocytes. We suggest that one mechanism that may assist the fetus (or a choriocarcinoma) in its immunologic survival is the intrinsic resistance of trophoblast cells to lymphocyte-mediated cytotoxicity. Images PMID:570981

  4. Recent development in [1,4]benzodiazepines as potent anticancer agents: a review.

    PubMed

    Gill, Rupinder Kaur; Kaushik, Shiv Om; Chugh, Jasreen; Bansal, Sumit; Shah, Anamik; Bariwal, Jitender

    2014-01-01

    The [1,4]benzodiazepine is an important class of heterocyclic compounds and clinically used for many ailments in humans. The [1,4]benzodiazepine has unique structure that mimics the peptide linkage. This interesting observation completely shifted the interest of medicinal chemist for [1,4]benzodiazepine from CNS acting drugs to anticancer agents. During last few decades, a large number of reports have appeared in the literature highlighting the anticancer activity of [1,4]benzodiazepines. Here, in this article, we have discussed the brief synthesis, origin of [1,4]benzodiazepines as anticancer agent, their mechanism of action and latest developments in this field. We have compiled the most important literature reports from last few decades till date.

  5. Evaluation of genetic damage in human peripheral lymphocytes exposed to mineral trioxide aggregate and Portland cements.

    PubMed

    Braz, M G; Camargo, E A; Salvadori, D M F; Marques, M E A; Ribeiro, D A

    2006-03-01

    summary Mineral trioxide aggregate (MTA) and Portland cement are being used in dentistry as root-end-filling material for periapical surgery and for the sealing of communications between the root canal system and the surrounding tissues. However, genotoxicity tests for complete risk assessment of these compounds have not been conducted up to now. In the present study, the genotoxic effects of MTA and Portland cements were evaluated in peripheral lymphocytes from 10 volunteers by the alkaline single cell gel (comet) assay. The results pointed out that the single cell gel (comet) assay failed to detect the presence of DNA damage after a treatment of peripheral lymphocytes by MTA and Portland cements for concentrations up to 1000 mug mL(-1). In summary, our results indicate that exposure to MTA or Portland cements may not be a factor that increases the level of DNA lesions in human peripheral lymphocytes as detected by single cell gel (comet) assay.

  6. Genotoxicity test of self-renovated ceramics in primary human peripheral lymphocytes.

    PubMed

    Hua, Nan; Zhu, Huifang; Zhuang, Jing; Chen, Liping

    2014-12-01

    Zirconia-based ceramics is widely used in dentistry. Different compositions of ceramics have different features. Our self-renovated ceramics become more machinable without scarifying its dental restoration properties after adjusting ratio of lanthanum phosphate (LaPO4)/yttrium oxide (Y2O3). In order to evaluate its safety, here, we tested its genotoxicity in primary human peripheral lymphocytes. The human lymphocytes cultured on three groups of different ratios of LaPO4/Y2O3 diphase ceramics for 6 days showed little effect of growth inhibition and similar effect of growth trend to the negative control. Furthermore, single-cell gel electrophoresis (comet assay) indicated that there was no significant difference of the value of tail moment between the tested ceramics and negative control, the IPS Empress II (P > 0.05). Our findings implicate that our self-renovated ceramics do not induce DNA damages in human peripheral lymphocytes and support their future clinic application.

  7. Myokymia and neuromyotonia in veterinary medicine: a comparison with peripheral nerve hyperexcitability syndrome in humans.

    PubMed

    Vanhaesebrouck, An E; Bhatti, Sofie F M; Franklin, Robin J M; Van Ham, Luc

    2013-08-01

    Involuntary muscle hyperactivity can result from muscle or peripheral nerve hyperexcitability or central nervous system dysfunction. In humans, diseases causing hyperexcitability of peripheral nerves are grouped together under the term 'peripheral nerve hyperexcitability' (PNH). Hyperexcitability of the peripheral motor nerve can result into five different phenotypic main variants, i.e. fasciculations, myokymia, neuromyotonia, cramps and tetany, each with their own clinical and electromyographic characteristics. This review focuses on the most commonly described expressions of PNH in veterinary medicine, i.e. myokymia and neuromyotonia, in particular in young Jack Russell terriers. Data from 58 veterinary cases with generalized myokymia and neuromyotonia were analyzed, including unpublished treatment and follow-up data on eight Jack Russell terriers from a previous study and seven additional Jack Russell terriers. A dysfunction of the potassium channel or its associated proteins has been found in many human syndromes characterized by PNH, in particular in generalized myokymia and neuromyotonia, and is suspected to occur in veterinary medicine. Potential pathomechanisms of potassium channel dysfunction leading to signs of PNH are broad and include genetic mutations, antibody-mediated attack or ion channel maldistribution due to axonal degeneration or demyelination. A more accurate classification of the different PNH syndromes will facilitate a more rapid diagnosis and guide further research into natural occurring PNH in animals.

  8. Ectopic lymphokine gene expression in human peripheral blood lymphocytes in vivo

    SciTech Connect

    Chambers, C.A.; Kang, Joonsoo; Hozumi, Nobumichi Mount Sinai Hospital, Toronto, Ontario )

    1992-02-01

    An animal model to study the effects of ectopic expression of cytokines involved in cell growth and differentiation has been established. Retrovirus vectors containing the human interleukin 6 cDNA were used to produce high titer virus-producing lines. Human peripheral blood lymphocytes (hPBLs) were successfully infected with the retrovirus and engrafted into severe combined immunodeficient mice. The majority of the animals were engrafted with hPBLs, as determined by the presence of human glucose phosphate isomerase. Furthermore, six of seven mice engrafted with hPBLs infected with high titer virus and detectable hPBLs present in the spleen expressed the retroviral human interleukin 6 gene. Importantly, human interleukin 6 protein was expressed at physiologically significant levels in these mice. These results demonstrate that models for human disease and immunotherapy involving retrovirus-mediated gene transfer into human cells can be developed in mice.

  9. A new surface marker on T lymphocytes of human peripheral blood.

    PubMed

    Hammarström, S; Hellström, U; Perlmann, P; Dillner, M L

    1973-11-01

    Neuraminidase treatment of human peripheral blood lymphocytes uncovers cell surface receptors that bind purified A hemagglutinin from the snail Helix pomatia. No hemagglutinin was bound to untreated lymphocytes. Binding studies with (125)I-labeled hemagglutinin suggested that the number of receptors on neuraminidase-treated lymphocytes was approximately 1.10(6)/cell. The apparent association constant for hemagglutinin binding to lymphocytes, as calculated from Scatchard's plots, was 5-7.10(8) liters/mol. Immunofluorescent staining with FITC-conjugated hemagglutinin gave positive reactions with approximately 60% of the lymphocytes from normal donors. Positive staining was inversely related to the number of lymphocytes with Fc or complement receptors or with surface immunoglobulin, thus suggesting that See PDF for Structure the lymphocytes with receptors for Helix pomatia A hemagglutinin are T cells. Cell fractionation on columns charged with hemagglutinin indicate that these receptors may be used for separating subpopulations of human peripheral lymphocytes.

  10. The cytogenetic effects of food sweetener maltitol in human peripheral lymphocytes.

    PubMed

    Canimoğlu, Semir; Rencüzoğullari, Eyyüp

    2006-01-01

    The effects of the low-calorie artifical sweetener maltitol (E965), a sugar alcohol (Polyol), on sister chromatid exchange (SCE), chromosome aberration (CA), and micronucleus formation (MN) were investigated in human peripheral lymphocytes. Maltitol did not induce SCE at all concentrations (1.25, 2.5, and 5 mg/mL) and treatment periods (24 and 48 h). Maltitol induced CA, although not statistically significantly. Maltitol induced the frequency of MN at 24 and 48 h in a non-dose-dependent manner. In addition, maltitol did not decrease the replication index (RI) and the mitotic index (MI) at all concentrations and treatment periods. Maltitol did not alter the pH and osmolality of the medium. In conclusion, it can be concluded that maltitol has a weak genotoxic potential and it appears non-cytotoxic to human peripheral lymphocytes in vitro.

  11. Peripheral Nerve Regeneration by Secretomes of Stem Cells from Human Exfoliated Deciduous Teeth

    PubMed Central

    Sugimura-Wakayama, Yukiko; Osugi, Masashi; Kawai, Takamasa; Ogata, Kenichi; Sakaguchi, Kohei; Hibi, Hideharu

    2015-01-01

    Peripheral nerve regeneration across nerve gaps is often suboptimal, with poor functional recovery. Stem cell transplantation-based regenerative therapy is a promising approach for axon regeneration and functional recovery of peripheral nerve injury; however, the mechanisms remain controversial and unclear. Recent studies suggest that transplanted stem cells promote tissue regeneration through a paracrine mechanism. We investigated the effects of conditioned media derived from stem cells from human exfoliated deciduous teeth (SHED-CM) on peripheral nerve regeneration. In vitro, SHED-CM-treated Schwann cells exhibited significantly increased proliferation, migration, and the expression of neuron-, extracellular matrix (ECM)-, and angiogenesis-related genes. SHED-CM stimulated neuritogenesis of dorsal root ganglia and increased cell viability. Similarly, SHED-CM enhanced tube formation in an angiogenesis assay. In vivo, a 10-mm rat sciatic nerve gap model was bridged by silicon conduits containing SHED-CM or serum-free Dulbecco's modified Eagle's medium. Light and electron microscopy confirmed that the number of myelinated axons and axon-to-fiber ratio (G-ratio) were significantly higher in the SHED-CM group at 12 weeks after nerve transection surgery. The sciatic functional index (SFI) and gastrocnemius (target muscle) wet weight ratio demonstrated functional recovery. Increased compound muscle action potentials and increased SFI in the SHED-CM group suggested sciatic nerve reinnervation of the target muscle and improved functional recovery. We also observed reduced muscle atrophy in the SHED-CM group. Thus, SHEDs may secrete various trophic factors that enhance peripheral nerve regeneration through multiple mechanisms. SHED-CM may therefore provide a novel therapy that creates a more desirable extracellular microenvironment for peripheral nerve regeneration. PMID:26154068

  12. The human BDNF gene: peripheral gene expression and protein levels as biomarkers for psychiatric disorders

    PubMed Central

    Cattaneo, A; Cattane, N; Begni, V; Pariante, C M; Riva, M A

    2016-01-01

    Brain-derived neurotrophic factor (BDNF) regulates the survival and growth of neurons, and influences synaptic efficiency and plasticity. The human BDNF gene consists of 11 exons, and distinct BDNF transcripts are produced through the use of alternative promoters and splicing events. The majority of the BDNF transcripts can be detected not only in the brain but also in the blood cells, although no study has yet investigated the differential expression of BDNF transcripts at the peripheral level. This review provides a description of the human BDNF gene structure as well as a summary of clinical and preclinical evidence supporting the role of BDNF in the pathogenesis of psychiatric disorders. We will discuss several mechanisms as possibly underlying BDNF modulation, including epigenetic mechanisms. We will also discuss the potential use of peripheral BDNF as a biomarker for psychiatric disorders, focusing on the factors that can influence BDNF gene expression and protein levels. Within this context, we have also characterized, for we believe the first time, the expression of BDNF transcripts in the blood, with the aim to provide novel insights into the molecular mechanisms and signaling that may regulate peripheral BDNF gene expression levels. PMID:27874848

  13. Receptor expression and responsiveness of human peripheral blood mononuclear cells to a human cytomegalovirus encoded CC chemokine.

    PubMed

    Zheng, Qi; Xu, Jun; Gao, Huihui; Tao, Ran; Li, Wei; Shang, Shiqiang; Gu, Weizhong

    2015-01-01

    Human cytomegalovirus is a ubiquitous pathogen that infects the majority of the world's population. After long period of time co-evolving with human being, this pathogen has developed several strategies to evade host immune surveillance. One of the major trick is encoding homologous to those of the host organism or stealing host cellular genes that have significant functions in immune system. To date, we have found several viral immune analogous which include G protein coupled receptor, class I major histocompatibility complex and chemokine. Chemokine is a small group of molecules which is defined by the presence of four cysteines in highly conserved region. The four kinds of chemokines (C, CC, CXC, and CX3C) are classified based on the arrangement of 1 or 2 N-terminal cysteine residues. UL128 protein is one of the analogous that encoded by human cytomegalovirus that has similar amino acid sequences to the human CC chemokine. It has been proved to be one of the essential particles that involved in human cytomegalovirus entry into epithelial/endothelial cells as well as macrophages. It is also the target of potent neutralizing antibodies in human cytomegalovirus-seropositive individuals. We had demonstrated the chemotactic trait of UL128 protein in our previous study. Recombinant UL128 in vitro has the ability to attract monocytes to the infection region and enhances peripheral blood mononuclear cell proliferation by activating the MAPK/ERK signaling pathway. However, the way that this viral encoded chemokine interacting with peripheral blood mononuclear cells and the detailed mechanism that involving the virus entry into host cells keeps unknown. Here we performed in vitro investigation into the effects of UL128 protein on peripheral blood mononuclear cell's activation and receptor binding, which may help us further understand the immunomodulatory function of UL128 protein as well as human cytomegalovirus diffusion mechanism.

  14. Self-injection of barbiturates and benzodiazepines in baboons.

    PubMed

    Griffiths, R R; Lukas, S E; Bradford, L D; Brady, J V; Snell, J D

    1981-01-01

    Self-injection of three barbiturates, six benzodiazepines, and chlorpromazine was examined in baboons. Intravenous injections of drug were dependent upon completion of 160 lever presses (a 160-response fixed-ratio schedule). A 3-h time-out period followed each injection, permitting a maximum of eight injections per day. Prior to testing each dose of drug, self-injection performance was established with cocaine. Subsequently, a test dose was substituted for cocaine. Amobarbital, pentobarbital, and secobarbital maintained the highest levels of self-injection, which were similar to those maintained by cocaine. Clonazepam, clorazepate, diazepam, flurazepam, medazepam, and midazolam maintained relatively modest levels of self-injection, while chlorpromazine maintained only low levels, which were in the range of vehicle control. Of the six benzodiazepines, midazolam produced the highest levels of self-injection. At the highest self-injected doses, the barbiturates produced anesthesia in contrast to the benzodiazepines, which produced only sedation. None of the drugs affected food intake except for chlorpromazine, which produced dose-related decreases. The differences among the drug classes (i.e., barbiturate, benzodiazepine, phenothiazine) with respect to the maintenance of self-injection correspond well with the results of previous animal and human drug self-administration studies.

  15. Fasting levels of monoketonic bile acids in human peripheral and portal circulation.

    PubMed

    Björkhem, I; Angelin, B; Einarsson, K; Ewerth, S

    1982-09-01

    It has been suggested that large amounts of ketonic bile acids may be present in portal venous blood. We have therefore determined the approximate concentration of 3-oxo-, 7-oxo-, and 12-oxo-bile acids (monoketonic bile acids) in human peripheral and portal circulation. These compounds were converted into the corresponding 3alpha-, 7alpha-, and 12alpha-hydroxy bile acids by treatment with sodium borodeuteride, thus increasing the molecular weight of each bile acid formed by one mass unit. The ratio between deuterated and nondeuterated bile acid was determined by combined gas-liquid chromatography-mass spectrometry with use of selected ion monitoring. From the ratio obtained and from the concentration of unlabeled bile acid, determined by isotope dilution-mass spectrometry, the approximate concentration of the different ketonic bile acids could be calculated. This method underestimates 3-oxygenated bile acids by 4-8%, 7-oxygenated bile acids by 2-3%, and 12-oxygenated bile acids by about 25%. The approximate concentration of monoketonic 3,7-oxygenated bile acids was found to be 0.08 +/- 0.02 and 0.37 +/- 0.25 micro mol/l in the peripheral venous serum and the portal venous serum, respectively. The approximate concentration of monoketonic 3,12-oxygenated bile acids was found to be 0.07 +/- 0.02 and 0.32 +/- 0.12 micro mol/l in the peripheral venous serum and the portal venous serum, respectively. The approximate concentration of monoketonic 3,7,12-oxygenated bile acids was found to be 0.03 +/- 0.01 and 0.14 +/- 0.05 micro mol/l in the peripheral venous serum and in the portal venous serum, respectively. The total concentration of the ketonic bile acids constituted only 9 +/- 1% and 8 +/- 3% of the nonoxidized bile acids in the peripheral venous serum and in the portal venous serum, respectively. Thus it seems less likely that the portal inflow of ketonic bile acids is of significant physiological importance under normal conditions.-Björkhem, I., B. Angelin, K

  16. Do benzodiazepines contribute to respiratory problems?

    PubMed

    Vozoris, Nicholas T

    2014-12-01

    Non-selective benzodiazepines are a class of sedative and anxiolytic medication that are commonly prescribed. Physiology studies and animal studies suggest that non-selective benzodiazepines may adversely impact respiration through a variety of mechanisms. Several recent, well-designed, population-based observational studies confirm that benzodiazepine-related negative respiratory outcomes are a concern. In this article, the mechanisms and clinical evidence for non-selective benzodiazepine-related adverse respiratory outcomes, as well as the methodological issues relating to the evaluation of adverse drug effects are reviewed.

  17. [Pharmacology and clinical aspects of benzodiazepines].

    PubMed

    Mendlewicz, J; Sevy, S

    1985-01-01

    The widespread use of benzodiazepines has led the authors to review the pharmacological and clinical aspects of these substances. On a molecular level, the benzodiazepines have an effect on receptors in relation with the GABA system. Presently, endogenous ligand(s) to these receptors have not yet been fully demonstrated. The main benzodiazepines are also compared for their kinetics which is function of absorption, metabolisation and various factors such as binding to the receptor, age, hepatic and renal disorders. These pharmacological studies have clinical implications. The authors finally make a brief review of the clinical indications of the benzodiazepines.

  18. Neural Basis of Benzodiazepine Reward: Requirement for α2 Containing GABAA Receptors in the Nucleus Accumbens

    PubMed Central

    Engin, Elif; Bakhurin, Konstantin I; Smith, Kiersten S; Hines, Rochelle M; Reynolds, Lauren M; Tang, Wannan; Sprengel, Rolf; Moss, Stephen J; Rudolph, Uwe

    2014-01-01

    Despite long-standing concerns regarding the abuse liability of benzodiazepines, the mechanisms underlying properties of benzodiazepines that may be relevant to abuse are still poorly understood. Earlier studies showed that compounds selective for α1-containing GABAA receptors (α1GABAARs) are abused by humans and self-administered by animals, and that these receptors may underlie a preference for benzodiazepines as well as neuroplastic changes observed in the ventral tegmental area following benzodiazepine administration. There is some evidence, however, that even L-838, 417, a compound with antagonistic properties at α1GABAARs and agonistic properties at the other three benzodiazepine-sensitive GABAA receptor subtypes, is self-administered, and that the α2GABAARs may have a role in benzodiazepine-induced reward enhancement. Using a two-bottle choice drinking paradigm to evaluate midazolam preference and an intracranial self-stimulation (ICSS) paradigm to evaluate the impact of midazolam on reward enhancement, we demonstrated that mice carrying a histidine-to-arginine point mutation in the α2 subunit which renders it insensitive to benzodiazepines (α2(H101R) mice) did not prefer midazolam and did not show midazolam-induced reward enhancement in ICSS, in contrast to wild-type controls, suggesting that α2GABAARs are necessary for the reward enhancing effects and preference for oral benzodiazepines. Through a viral-mediated knockdown of α2GABAARs in the nucleus accumbens (NAc), we demonstrated that α2 in the NAc is necessary for the preference for midazolam. Findings imply that α2GABAARs in the NAc are involved in at least some reward-related properties of benzodiazepines, which might partially underlie repeated drug-taking behavior. PMID:24553732

  19. Sleep Deprivation Impairs the Human Central and Peripheral Nervous System Discrimination of Social Threat

    PubMed Central

    Goldstein-Piekarski, Andrea N.; Greer, Stephanie M.; Saletin, Jared M.

    2015-01-01

    Facial expressions represent one of the most salient cues in our environment. They communicate the affective state and intent of an individual and, if interpreted correctly, adaptively influence the behavior of others in return. Processing of such affective stimuli is known to require reciprocal signaling between central viscerosensory brain regions and peripheral-autonomic body systems, culminating in accurate emotion discrimination. Despite emerging links between sleep and affective regulation, the impact of sleep loss on the discrimination of complex social emotions within and between the CNS and PNS remains unknown. Here, we demonstrate in humans that sleep deprivation impairs both viscerosensory brain (anterior insula, anterior cingulate cortex, amygdala) and autonomic-cardiac discrimination of threatening from affiliative facial cues. Moreover, sleep deprivation significantly degrades the normally reciprocal associations between these central and peripheral emotion-signaling systems, most prominent at the level of cardiac-amygdala coupling. In addition, REM sleep physiology across the sleep-rested night significantly predicts the next-day success of emotional discrimination within this viscerosensory network across individuals, suggesting a role for REM sleep in affective brain recalibration. Together, these findings establish that sleep deprivation compromises the faithful signaling of, and the “embodied” reciprocity between, viscerosensory brain and peripheral autonomic body processing of complex social signals. Such impairments hold ecological relevance in professional contexts in which the need for accurate interpretation of social cues is paramount yet insufficient sleep is pervasive. PMID:26180190

  20. Characterization of Endoneurial Fibroblast-like Cells from Human and Rat Peripheral Nerves.

    PubMed

    Richard, Laurence; Védrenne, Nicolas; Vallat, Jean-Michel; Funalot, Benoît

    2014-06-01

    Endoneurial fibroblast-like cells (EFLCs) are one of the cell populations present in the peripheral nervous system. The role and immunophenotypic characteristics of EFLCs are not well known and led us to perform a histological and cytological study of EFLCs in normal human and rat peripheral nerves. We found that all EFLCs express CD34, neural/glial antigen 2 (NG2), and prolyl-4-hydrolase-beta. In addition, half of the EFLCs in normal peripheral nerves express platelet-derived growth factor receptor-β (PDGFR-β) and some also express the intermediate filament nestin in vivo (at a lower level than Schwann cells, which express high levels of nestin). Using cell cultures of purified EFLCs, we characterized subpopulations of EFLCs expressing PDGFR-β alone or PDGFR-β and nestin. Experimental nerve lesions in rat resulted in an increase in nestin-positive EFLCs, which returned to normal levels after 8 days. This suggests that some EFLCs could have a different proliferative and/or regenerative potential than others, and these EFLCs may play a role in the initial phase of nerve repair. These "activated" EFLCs share some immunophenotypic similarities with pericytes and Interstitial cells of Cajal, which have progenitor cell potentials. This raises the questions as to whether a proportion of EFLCs have a possible role as endoneurial progenitor cells. © The Author(s) 2014.

  1. Sleep Deprivation Impairs the Human Central and Peripheral Nervous System Discrimination of Social Threat.

    PubMed

    Goldstein-Piekarski, Andrea N; Greer, Stephanie M; Saletin, Jared M; Walker, Matthew P

    2015-07-15

    Facial expressions represent one of the most salient cues in our environment. They communicate the affective state and intent of an individual and, if interpreted correctly, adaptively influence the behavior of others in return. Processing of such affective stimuli is known to require reciprocal signaling between central viscerosensory brain regions and peripheral-autonomic body systems, culminating in accurate emotion discrimination. Despite emerging links between sleep and affective regulation, the impact of sleep loss on the discrimination of complex social emotions within and between the CNS and PNS remains unknown. Here, we demonstrate in humans that sleep deprivation impairs both viscerosensory brain (anterior insula, anterior cingulate cortex, amygdala) and autonomic-cardiac discrimination of threatening from affiliative facial cues. Moreover, sleep deprivation significantly degrades the normally reciprocal associations between these central and peripheral emotion-signaling systems, most prominent at the level of cardiac-amygdala coupling. In addition, REM sleep physiology across the sleep-rested night significantly predicts the next-day success of emotional discrimination within this viscerosensory network across individuals, suggesting a role for REM sleep in affective brain recalibration. Together, these findings establish that sleep deprivation compromises the faithful signaling of, and the "embodied" reciprocity between, viscerosensory brain and peripheral autonomic body processing of complex social signals. Such impairments hold ecological relevance in professional contexts in which the need for accurate interpretation of social cues is paramount yet insufficient sleep is pervasive.

  2. Biomarkers for the effects of benzodiazepines in healthy volunteers

    PubMed Central

    de Visser, S J; van der Post, J P; de Waal, P P; Cornet, F; Cohen, A F; van Gerven, J M A

    2003-01-01

    Studies of novel centrally acting drugs in healthy volunteers are traditionally concerned with kinetics and tolerability, but useful information may also be obtained from biomarkers of clinical endpoints. A useful biomarker should meet the following requirements: a consistent response across studies and drugs; a clear response of the biomarker to a therapeutic dose; a dose–response relationship; a plausible relationship between biomarker, pharmacology and pathogenesis. In the current review, all individual tests found in studies of benzodiazepine agonists registered for anxiety in healthy volunteers since 1966 were progressively evaluated for compliance with these requirements. A MedLine search yielded 56 different studies, investigating the effects of 16 different benzodiazepines on 73 different (variants of) neuropsychological tests, which could be clustered into seven neuropsychological domains. Subjective and objective measures of alertness were most sensitive to benzodiazepines. The most consistent effects were observed on saccadic peak velocity (SPV) and visual analogue scores ( VAS) of alertness, where 100% and 79% of all studies respectively showed statistically significant effects. A dose–response relationship could be constructed for temazepam and SPV, which was used to determine dose equivalencies relative to temazepam, for seven different benzodiazepines. These dose equivalencies correlated with the lowest recommended daily maintenance dose (r2 = 0.737, P < 0.05). This relationship between SPV reduction and clinical efficacy could reflect the clinical practice of aiming for maximum tolerated levels, or it could represent a common basis behind SPV reduction and anxiolytic activity for benzodiazepines (probably sedation). The number of tests used in human psychopharmacology appears to be excessive and their sensitivity and reproducibility low. PMID:12534639

  3. Activation of axonal Kv7 channels in human peripheral nerve by flupirtine but not placebo - therapeutic potential for peripheral neuropathies: results of a randomised controlled trial

    PubMed Central

    2013-01-01

    Background Flupirtine is an analgesic with muscle-relaxing properties that activates Kv7 potassium channels. Kv7 channels are expressed along myelinated and unmyelinated peripheral axons where their activation is expected to reduce axonal excitability and potentially contribute to flupirtine’s clinical profile. Trial design To investigate the electrical excitability of peripheral myelinated axons following orally administered flupirtine, in-vitro experiments on isolated peripheral nerve segments were combined with a randomised, double-blind, placebo-controlled, phase I clinical trial (RCT). Methods Threshold tracking was used to assess the electrical excitability of myelinated axons in isolated segments of human sural nerve in vitro and motoneurones to abductor pollicis brevis (APB) in situ in healthy subjects. In addition, the effect of flupirtine on ectopic action potential generation in myelinated axons was examined using ischemia of the lower arm. Results Flupirtine (3-30 μM) shortened the relative refractory period and increased post-conditioned superexcitability in human myelinated axons in vitro. Similarly, in healthy subjects the relative refractory period of motoneurones to APB was reduced 2 hours after oral flupirtine but not following placebo. Whether this effect was due to a direct action of flupirtine on peripheral axons or temperature could not be resolved. Flupirtine (200 mg p.o.) also reduced ectopic axonal activity induced by 10 minutes of lower arm ischemia. In particular, high frequency (ca. 200 Hz) components of EMG were reduced in the post-ischemic period. Finally, visual analogue scale ratings of sensations perceived during the post-ischemic period were reduced following flupirtine (200 mg p.o.). Conclusions Clinical doses of flupirtine reduce the excitability of peripheral myelinated axons. Trial registration ClinicalTrials registration is NCT01450865. PMID:23394517

  4. Aluminum exposure for 60days at an equivalent human dietary level promotes peripheral dysfunction in rats.

    PubMed

    Martinez, Caroline Silveira; Vera, Gema; Ocio, José Antonio Uranga; Peçanha, Franck Maciel; Vassallo, Dalton Valentim; Miguel, Marta; Wiggers, Giulia Alessandra

    2017-08-25

    Aluminum (Al) is a neurotoxic associated with a number of chronic human diseases. We investigated the effects of Al exposure at doses similar to human dietary levels and at a high level exposure to Al on the peripheral nervous system. Wistar male rats were divided into two major groups and received orally: 1) First group - Low level - rats were subdivided and treated for 60days: a) Control - received ultrapure water; b) AlCl3 - received Al at 8.3mg/kg body weight (bw) for 60days; and 2) Second group - High level - rats were subdivided and treated for 42days: C) Control - received ultrapure water through oral gavage; d) AlCl3 - received Al at 100mg/kg bw for 42days. Von Frey hair test, plantar test, the presence of catalepsy and the spontaneous motor activity were investigated. Reactive oxygen species, lipid peroxidation and total antioxidant capacity, immunohistochemistry to investigate the nerve inflammation and, the specific presence of Al in the sciatic nerve fibers were investigated. Al exposure at a representative human dietary level promotes the development of mechanical allodynia, catalepsy, increased inflammation in the sciatic nerve, systemic oxidative stress and, is able to be retained in the sciatic nerve. The effects of low-dose Al were similar to those found in rats exposed to Al at a dose much higher (100mg/kg). Our findings suggest that Al may be considered toxic for the peripheral nervous system, thus inducing peripheral dysfunction. Copyright © 2017 Elsevier Inc. All rights reserved.

  5. Benzodiazepine inhibition of adenosine uptake is not prevented by benzodiazepine antagonists.

    PubMed

    Morgan, P F; Lloyd, H G; Stone, T W

    1983-01-28

    Uptake of [3H]adenosine into rat cerebral cortex synaptosomes was studied. Hexobendine (10(-5) M) and the benzodiazepine agonists diazepam (10(-5) M) and flurazepam (10(-4) M) significantly inhibited this uptake, but only if the compounds were pre-incubated for 10 min in the case of the benzodiazepines. The benzodiazepine antagonists Ro15-1788 (10(-5) M) and CGS 8216 (10(-5) M) failed to reverse the action of benzodiazepine agonists or hexobendine on [3H]adenosine uptake. The results add weight to the view that inhibition of adenosine uptake processes by benzodiazepines do not contribute to their behavioural effects.

  6. Chlorobenzenes, lindane and dieldrin induce apoptotic alterations in human peripheral blood lymphocytes (in vitro study).

    PubMed

    Michałowicz, Jaromir; Mokra, Katarzyna; Rosiak, Karolina; Sicińska, Paulina; Bukowska, Bożena

    2013-11-01

    In this study, we have assessed apoptotic effect of 1,2,4-trichlorobenzene, hexachlorobenzene, lindane and dieldrin on human peripheral blood lymphocytes. We observed an increase in ROS formation and a decrease in mitochondrial transmembrane potential in the cells incubated with low concentrations of all compounds studied, in particular lindane and dieldrin. ROS formation and changes in mitochondrial transmembrane potential may have influenced caspase-3 activation, a crucial enzyme in the apoptotic process. Moreover, chlorobenzenes, and in particular lindane and dieldrin changed cells' membrane permeability and induced phosphatidylserine translocation, which confirmed that they are capable of inducing apoptosis in human lymphocytes. Apoptotic changes in human lymphocytes provoked by biologically relevant concentrations of these substances suggest that they may disturb function of immunological system especially among people occupationally exposed to their action. Copyright © 2013 Elsevier B.V. All rights reserved.

  7. Decreased chemotaxis of human peripheral phagocytes exposed to a strong static magnetic field.

    PubMed

    Sipka, S; Szöllosi, I; Batta, Gy; Szegedi, Gy; Illés, A; Bakó, Gy; Novák, D

    2004-01-01

    The chemotaxis of human peripheral phagocytes, neutrophils and monocytes was examined in a strong static magnetic field (0.317+/-0.012 Tesla). The chemotaxis of the suspension of purified neutrophils and monocytes was tested in the Boyden chamber using C5a as a chemotactic signal. The chambers were placed into a temperature regulated (36.6 degrees C) equipment producing a strong static magnetic field (0.317 Tesla) for 60 minutes. The movement of cells proceeded into a nitrocellulose membrane toward the north-pole of the magnet, i.e. in the direction of the Earth's gravitational pull. The C5a induced chemotaxis of human neutrophils decreased significantly in the strong static magnetic field. Monocytes were not significantly effected. The strong static magnetic field decreased the chemotactic movement of neutrophils and this phenomenon may have implications when humans are exposed to magnetic resonance imaging for extended periods of time.

  8. Genotoxicity evaluation of drinking water sources in human peripheral blood lymphocytes using the comet assay.

    PubMed

    Wu, Yulin; Chen, Haigang; Li, Zhaoli; Sun, Liwei; Qu, Mengmeng; Li, Mei; Kong, Zhiming

    2008-01-01

    The potential harm of organic pollutants in drinking water to human health is widely focused on in the world; more and more pollutants with genotoxic substances are released into the aquatic environment. Water source samples were collected from 7 different localities of Nanjing City. The potential genotoxicity of organic extracts from drinking water sources were investigated by means of the comet assay in human peripheral lymphocytes. The results showed that all the organic extracts from all the water source samples could induce DNA damages of human peripheral blood lymphocytes at different levels. A significant difference (P < 0.01) was observed when compared with the solvent control. The DNA damage increased with the increase of the dosage of the original water source. Significant differences of DNA damage were observed in different drinking water sources, as shown by the multiple comparisons analysis at the dosage of 100x; the degree of DNA damage treated by Hushu waterworks (at town level) was the most serious, the arbitrary units (AU) was 141.62 +/- 6.96, however, that of Shangyuanmen waterworks (at city level) was only 109.64 +/- 2.97. The analysis also revealed that the genotoxicity of town's water sources was higher than that of the city. The results demonstrated that the comet assay can be successfully applied to the genotoxicity monitoring programs of drinking water sources.

  9. The effect of stress on core and peripheral body temperature in humans.

    PubMed

    Vinkers, Christiaan H; Penning, Renske; Hellhammer, Juliane; Verster, Joris C; Klaessens, John H G M; Olivier, Berend; Kalkman, Cor J

    2013-09-01

    Even though there are indications that stress influences body temperature in humans, no study has systematically investigated the effects of stress on core and peripheral body temperature. The present study therefore aimed to investigate the effects of acute psychosocial stress on body temperature using different readout measurements. In two independent studies, male and female participants were exposed to a standardized laboratory stress task (the Trier Social Stress Test, TSST) or a non-stressful control task. Core temperature (intestinal and temporal artery) and peripheral temperature (facial and body skin temperature) were measured. Compared to the control condition, stress exposure decreased intestinal temperature but did not affect temporal artery temperature. Stress exposure resulted in changes in skin temperature that followed a gradient-like pattern, with decreases at distal skin locations such as the fingertip and finger base and unchanged skin temperature at proximal regions such as the infra-clavicular area. Stress-induced effects on facial temperature displayed a sex-specific pattern, with decreased nasal skin temperature in females and increased cheek temperature in males. In conclusion, the amplitude and direction of stress-induced temperature changes depend on the site of temperature measurement in humans. This precludes a direct translation of the preclinical stress-induced hyperthermia paradigm, in which core temperature uniformly rises in response to stress to the human situation. Nevertheless, the effects of stress result in consistent temperature changes. Therefore, the present study supports the inclusion of body temperature as a physiological readout parameter of stress in future studies.

  10. Effects of cadmium on metallothionein levels in human peripheral blood leukocytes: a comparison with zinc.

    PubMed

    Yurkow, E J; DeCoste, C J

    1999-11-12

    Metallothioneins (MT) are low-molecular-weight, cysteine-rich proteins that are induced in response to a variety of chemical stresses and therefore can be used to assess human exposure to environmental agents. In the current study, flow cytometry was used to characterize the basal and cadmium-induced expression of MT in the three major leukocyte populations of human peripheral blood. In the analysis, monocytes were the most sensitive leukocytes to this toxic metal, with significant increases in cellular MT levels being detected at concentrations of cadmium as low as 0.1 microM (24 h). The lymphocyte population also exhibited pronounced treatment-associated elevations in cellular MT, while the granulocyte population was found to be nonresponsive. Although both CdCl2 (3 microM) and ZnCl2 (50 microM) induced MT expression in monocytes to a similar degree and did not affect the expression of this protein in granulocytes, cadmium but not zinc treatment induced dramatic increases in MT levels of lymphocytes. Our results indicate that cellular MT protein levels, as determined by this flow cytometric method, may be used to characterize the differential responsiveness of the major human leukocyte subpopulations to transitional metals. It is evident from the current work that the responsiveness of all peripheral blood leukocyte populations should be analyzed in exposure assessment studies.

  11. Changes of Dietary Fat and Carbohydrate Content Alter Central and Peripheral Clock in Humans.

    PubMed

    Pivovarova, Olga; Jürchott, Karsten; Rudovich, Natalia; Hornemann, Silke; Ye, Lu; Möckel, Simona; Murahovschi, Veronica; Kessler, Katharina; Seltmann, Anne-Cathrin; Maser-Gluth, Christiane; Mazuch, Jeannine; Kruse, Michael; Busjahn, Andreas; Kramer, Achim; Pfeiffer, Andreas F H

    2015-06-01

    The circadian clock coordinates numerous metabolic processes with light-dark and feeding regimens. However, in humans it is unknown whether dietary patterns influence circadian rhythms. We examined the effects of switching from a high-carbohydrate, low-fat diet to a low-carbohydrate, high fat (LC/HFD) isocaloric diet on the central and peripheral circadian clocks in humans. Diurnal patterns of salivary cortisol and gene expression were analyzed in blood monocytes of 29 nonobese healthy subjects before and 1 and 6 weeks after the dietary switch. For this, we established a method of rhythm prediction by 3-time point data. The centrally driven cortisol rhythm showed a phase delay 1 and 6 weeks after the dietary switch to a LC/HFD as well as an amplitude increase. The dietary switch altered diurnal oscillations of core clock genes (PER1, PER2, PER3, and TEF) and inflammatory genes (CD14, CD180, NFKBIA, and IL1B). The LC/HFD also affected the expression of nonoscillating genes contributing to energy metabolism (SIRT1) and fat metabolism (ACOX3 and IDH3A). Expression of clock genes but not of salivary cortisol in monocytes tightly correlated with levels of blood lipids and with expression of metabolic and inflammatory genes. Our results suggest that the modulation of the dietary fat and carbohydrate content alters the function of the central and peripheral circadian clocks in humans.

  12. Photoacoustic and ultrasound dual-modality imaging of human peripheral joints

    NASA Astrophysics Data System (ADS)

    Xu, Guan; Rajian, Justin R.; Girish, Gandikota; Kaplan, Mariana J.; Fowlkes, J. Brian; Carson, Paul L.; Wang, Xueding

    2013-01-01

    A photoacoustic (PA) and ultrasound (US) dual modality system, for imaging human peripheral joints, is introduced. The system utilizes a commercial US unit for both US control imaging and PA signal acquisition. Preliminary in vivo evaluation of the system, on normal volunteers, revealed that this system can recover both the structural and functional information of intra- and extra-articular tissues. Confirmed by the control US images, the system, on the PA mode, can differentiate tendon from surrounding soft tissue based on the endogenous optical contrast. Presenting both morphological and pathological information in joint, this system holds promise for diagnosis and characterization of inflammatory joint diseases such as rheumatoid arthritis.

  13. Expression of extracellular calcium (Ca2+o)-sensing receptor in human peripheral blood monocytes

    NASA Technical Reports Server (NTRS)

    Yamaguchi, T.; Olozak, I.; Chattopadhyay, N.; Butters, R. R.; Kifor, O.; Scadden, D. T.; Brown, E. M.; O'Malley, B. W. (Principal Investigator)

    1998-01-01

    The calcium-sensing receptor (CaR) is a G protein-coupled receptor playing key roles in extracellular calcium ion (Ca2+o) homeostasis in parathyroid gland and kidney. Macrophage-like mononuclear cells appear at sites of osteoclastic bone resorption during bone turnover and may play a role in the "reversal" phase of skeletal remodeling that follows osteoclastic resorption and precedes osteoblastic bone formation. Bone resorption produces substantial local increases in Ca2+o that could provide a signal for such mononuclear cells present locally within the bone marrow microenvironment. Indeed, previous studies by other investigators have shown that raising Ca2+o either in vivo or in vitro stimulated the release of interleukin-6 (IL-6) from human peripheral blood monocytes, suggesting that these cells express a Ca2+o-sensing mechanism. In these earlier studies, however, the use of reverse transcription-polymerase chain reaction (RT-PCR) failed to detect transcripts for the CaR previously cloned from parathyroid and kidney in peripheral blood monocytes. Since we recently found that non-specific esterase-positive, putative monocytes isolated from murine bone marrow express the CaR, we reevaluated the expression of this receptor in human peripheral blood monocytes. Immunocytochemistry, flow cytometry, and Western blot analysis, performed using a polyclonal antiserum specific for the CaR, detected CaR protein in human monocytes. In addition, the use of RT-PCR with CaR-specific primers, followed by nucleotide sequencing of the amplified products, identified CaR transcripts in the cells. Therefore, taken together, our data show that human peripheral blood monocytes possess both CaR protein and mRNA very similar if not identical to those expressed in parathyroid and kidney that could mediate the previously described, direct effects of Ca2+o on these cells. Furthermore, since mononuclear cells isolated from bone marrow also express the CaR, the latter might play some role in

  14. The effects of lipid A on gamma-irradiated human peripheral blood lymphocytes in vitro

    NASA Astrophysics Data System (ADS)

    Dubničková, M.; Kuzmina, E. A.; Chausov, V. N.; Ravnachka, I.; Boreyko, A. V.; Krasavin, E. A.

    2016-03-01

    The modulatory effects of lipid A (diphosphoryl lipid A (DLA) and monophosphoryl lipid A (MLA)) on apoptosis induction and DNA structure damage (single and double-strand breaks (SSBs and DSBs, respectively)) in peripheral human blood lymphocytes are studied for 60Co gamma-irradiation. It is shown that in the presence of these agents the amount of apoptotic cells increases compared with the irradiated control samples. The effect is most strongly pronounced for DLA. In its presence, a significant increase is observed in the number of radiation-induced DNA SSBs and DSBs. Possible mechanisms are discussed of the modifying influence of the used agents on radiation-induced cell reactions

  15. Expression of extracellular calcium (Ca2+o)-sensing receptor in human peripheral blood monocytes

    NASA Technical Reports Server (NTRS)

    Yamaguchi, T.; Olozak, I.; Chattopadhyay, N.; Butters, R. R.; Kifor, O.; Scadden, D. T.; Brown, E. M.; O'Malley, B. W. (Principal Investigator)

    1998-01-01

    The calcium-sensing receptor (CaR) is a G protein-coupled receptor playing key roles in extracellular calcium ion (Ca2+o) homeostasis in parathyroid gland and kidney. Macrophage-like mononuclear cells appear at sites of osteoclastic bone resorption during bone turnover and may play a role in the "reversal" phase of skeletal remodeling that follows osteoclastic resorption and precedes osteoblastic bone formation. Bone resorption produces substantial local increases in Ca2+o that could provide a signal for such mononuclear cells present locally within the bone marrow microenvironment. Indeed, previous studies by other investigators have shown that raising Ca2+o either in vivo or in vitro stimulated the release of interleukin-6 (IL-6) from human peripheral blood monocytes, suggesting that these cells express a Ca2+o-sensing mechanism. In these earlier studies, however, the use of reverse transcription-polymerase chain reaction (RT-PCR) failed to detect transcripts for the CaR previously cloned from parathyroid and kidney in peripheral blood monocytes. Since we recently found that non-specific esterase-positive, putative monocytes isolated from murine bone marrow express the CaR, we reevaluated the expression of this receptor in human peripheral blood monocytes. Immunocytochemistry, flow cytometry, and Western blot analysis, performed using a polyclonal antiserum specific for the CaR, detected CaR protein in human monocytes. In addition, the use of RT-PCR with CaR-specific primers, followed by nucleotide sequencing of the amplified products, identified CaR transcripts in the cells. Therefore, taken together, our data show that human peripheral blood monocytes possess both CaR protein and mRNA very similar if not identical to those expressed in parathyroid and kidney that could mediate the previously described, direct effects of Ca2+o on these cells. Furthermore, since mononuclear cells isolated from bone marrow also express the CaR, the latter might play some role in

  16. Bacillus anthracis’ lethal toxin induces broad transcriptional responses in human peripheral monocytes

    PubMed Central

    2012-01-01

    Background Anthrax lethal toxin (LT), produced by the Gram-positive bacterium Bacillus anthracis, is a highly effective zinc dependent metalloprotease that cleaves the N-terminus of mitogen-activated protein kinase kinases (MAPKK or MEKs) and is known to play a role in impairing the host immune system during an inhalation anthrax infection. Here, we present the transcriptional responses of LT treated human monocytes in order to further elucidate the mechanisms of LT inhibition on the host immune system. Results Western Blot analysis demonstrated cleavage of endogenous MEK1 and MEK3 when human monocytes were treated with 500 ng/mL LT for four hours, proving their susceptibility to anthrax lethal toxin. Furthermore, staining with annexin V and propidium iodide revealed that LT treatment did not induce human peripheral monocyte apoptosis or necrosis. Using Affymetrix Human Genome U133 Plus 2.0 Arrays, we identified over 820 probe sets differentially regulated after LT treatment at the p <0.001 significance level, interrupting the normal transduction of over 60 known pathways. As expected, the MAPKK signaling pathway was most drastically affected by LT, but numerous genes outside the well-recognized pathways were also influenced by LT including the IL-18 signaling pathway, Toll-like receptor pathway and the IFN alpha signaling pathway. Multiple genes involved in actin regulation, signal transduction, transcriptional regulation and cytokine signaling were identified after treatment with anthrax LT. Conclusion We conclude LT directly targets human peripheral monocytes and causes multiple aberrant gene responses that would be expected to be associated with defects in human monocyte’s normal signaling transduction pathways and function. This study provides further insights into the mechanisms associated with the host immune system collapse during an anthrax infection, and suggests that anthrax LT may have additional downstream targets outside the well-known MAPK

  17. Complications during the management of pediatric refractory status epilepticus with benzodiazepine and pentobarbital infusions.

    PubMed

    Patten, William; Naqvi, Sayed Z; Raszynski, Andre; Totapally, Balagangadhar R

    2015-05-01

    The objective of this retrospective study was to evaluate complications in the management of refractory status epilepticus (RSE) treated with benzodiazepine and pentobarbital infusions. Of 28 children with RSE, eleven (39%) were treated with a pentobarbital infusion after failure to control RSE with a benzodiazepine infusion; while17 children (61%) required only a benzodiazepine infusion. The mean maximum pentobarbital infusion dosage was 5.2 ± 1.8 mg/kg/h. Twenty-five patients received a continuous midazolam infusion with an average dosage of 0.41 ± 0.43 mg/kg/h. The median length of stay was longer for the pentobarbital group. Children requiring pentobarbital therapy were more likely to develop hypotension, require inotropic support, need intubation, mechanical ventilation, peripheral nutrition, and blood products; furthermore, they were more likely to develop hypertension and movement disorder after or during weaning. In conclusion, children with RSE who required pentobarbital therapy had a longer hospital stay with more complications.

  18. Benzodiazepines: risks and benefits. A reconsideration.

    PubMed

    Baldwin, David S; Aitchison, Katherine; Bateson, Alan; Curran, H Valerie; Davies, Simon; Leonard, Brian; Nutt, David J; Stephens, David N; Wilson, Sue

    2013-11-01

    Over the last decade there have been further developments in our knowledge of the risks and benefits of benzodiazepines, and of the risks and benefits of alternatives to benzodiazepines. Representatives drawn from the Psychopharmacology Special Interest Group of the Royal College of Psychiatrists and the British Association for Psychopharmacology together examined these developments, and have provided this joint statement with recommendations for clinical practice. The working group was mindful of widespread concerns about benzodiazepines and related anxiolytic and hypnotic drugs. The group believes that whenever benzodiazepines are prescribed, the potential for dependence or other harmful effects must be considered. However, the group also believes that the risks of dependence associated with long-term use should be balanced against the benefits that in many cases follow from the short or intermittent use of benzodiazepines and the risk of the underlying conditions for which treatment is being provided.

  19. Outpatient benzodiazepine detoxification procedure for methadone patients.

    PubMed

    McDuff, D R; Schwartz, R P; Tommasello, A; Tiegel, S; Donovan, T; Johnson, J L

    1993-01-01

    Benzodiazepines are used by a substantial minority of opioid addicts on methadone maintenance. Alprazolam, now the most widely prescribed benzodiazepine in the United States, appears to have supplanted diazepam as the benzodiazepine drug of choice in this population. Its greater addiction liability, shorter half-life, and more intense withdrawal symptoms make addiction to alprazolam more likely and its management in methadone patients more complicated. This article describes a slow outpatient tapered reduction procedure that was utilized to detoxify benzodiazepine dependent methadone patients seen over a two-year period. The reduction procedure was offered to 22 opioid addicts on methadone maintenance who were regularly ingesting low to moderate amounts of benzodiazepines, primarily alprazolam. Of the 22 patients, 4 patients refused outpatient detoxification, and 18 were started on a reduction procedure. Twelve patients completed the detoxification procedure which averaged 7.8 weeks. Comparisons are made between completers and non-completers and essential design features of the procedure are discussed.

  20. Effect of drinking water disinfection by-products in human peripheral blood lymphocytes and sperm.

    PubMed

    Ali, Aftab; Kurzawa-Zegota, Malgorzata; Najafzadeh, Mojgan; Gopalan, Rajendran C; Plewa, Michael J; Anderson, Diana

    2014-12-01

    Drinking water disinfection by-products (DBPs) are generated by the chemical disinfection of water and may pose hazards to public health. Two major classes of DBPs are found in finished drinking water: haloacetic acids (HAAs) and trihalomethanes (THMs). HAAs are formed following disinfection with chlorine, which reacts with iodide and bromide in the water. Previously the HAAs were shown to be cytotoxic, genotoxic, mutagenic, teratogenic and carcinogenic. To determine the effect of HAAs in human somatic and germ cells and whether oxidative stress is involved in genotoxic action. In the present study both somatic and germ cells have been examined as peripheral blood lymphocytes and sperm. The effects of three HAA compounds: iodoacetic acid (IAA), bromoacetic acid (BAA) and chloroacetic acid (CAA) were investigated. After determining appropriate concentration responses, oxygen radical involvement with the antioxidants, butylated hydroxanisole (BHA) and the enzyme catalase, were investigated in the single cell gel electrophoresis (Comet) assay under alkaline conditions, >pH 13 and the micronucleus assay. In the Comet assay, BHA and catalase were able to reduce DNA damage in each cell type compared to HAA alone. In the micronucleus assay, micronuclei (MNi) were found in peripheral lymphocytes exposed to all three HAAs and catalase and BHA were in general, able to reduce MNi induction, suggesting oxygen radicals play a role in both assays. These observations are of concern to public health since both human somatic and germ cells show similar genotoxic responses. Copyright © 2014. Published by Elsevier B.V.

  1. Novel Immunohistochemical Techniques Using Discrete Signal Amplification Systems for Human Cutaneous Peripheral Nerve Fiber Imaging

    PubMed Central

    Wang, Ningshan; Gibbons, Christopher H.; Freeman, Roy

    2011-01-01

    Confocal imaging uses immunohistochemical binding of specific antibodies to visualize tissues, but technical obstacles limit more widespread use of this technique in the imaging of peripheral nerve tissue. These obstacles include same-species antibody cross-reactivity and weak fluorescent signals of individual and co-localized antigens. The aims of this study were to develop new immunohistochemical techniques for imaging of peripheral nerve fibers. Three-millimeter punch skin biopsies of healthy individuals were fixed, frozen, and cut into 50-µm sections. Tissues were stained with a variety of antibody combinations with two signal amplification systems, streptavidin-biotin-fluorochrome (sABC) and tyramide-horseradish peroxidase-fluorochrome (TSA), used simultaneously to augment immunohistochemical signals. The combination of the TSA and sABC amplification systems provided the first successful co-localization of sympathetic adrenergic and sympathetic cholinergic nerve fibers in cutaneous human sweat glands and vasomotor and pilomotor systems. Primary antibodies from the same species were amplified individually without cross-reactivity or elevated background interference. The confocal fluorescent signal-to-noise ratio increased, and image clarity improved. These modifications to signal amplification systems have the potential for widespread use in the study of human neural tissues. PMID:21411809

  2. ERP topography and human perceptual learning in the peripheral visual field.

    PubMed

    Shoji, Hiroaki; Skrandies, Wolfgang

    2006-08-01

    We studied human perceptual learning in the peripheral visual field in 16 healthy adults. Horizontal or vertical vernier stimuli were presented simultaneously at 8 locations at an eccentricity of 4 degrees . One of the stimuli displayed an offset, and subjects were asked to detect the target offset. Training was performed with either vertical or horizontal stimuli by the repeated presentation of stimuli. Discrimination performance was also measured with the untrained stimuli. Before and after the psychophysical experiment, EEG was recorded from 30 electrodes over the occipital areas (between the inion and Cz) while targets were presented at all locations as vernier onset/offset stimuli. The EEG was averaged for each orientation separately. Improvement in discrimination performance was observed in about 70% of the subjects with the trained orientation only. The evoked potential maps displayed three components occurring between 80 and 160, 180 and 260, and 280 and 340 ms. The potential field topography of the first and third component showed significant differences before and after learning. In addition, field strength (global field power) of the second and third component increased with learning. No effects were seen with the untrained stimuli in the psychophysical and electrophysiological experiments. Our findings suggest that perceptual learning in the peripheral visual field is specifically related to neurophysiological changes induced by training, and it is not caused by unspecific changes of spatial attention. The changes of electrical brain activity reflect short-term plasticity related to human perceptual learning.

  3. Accumulation of peripheral autoreactive B cells in the absence of functional human regulatory T cells

    PubMed Central

    Kinnunen, Tuure; Chamberlain, Nicolas; Morbach, Henner; Choi, Jinyoung; Kim, Sangtaek; Craft, Joseph; Mayer, Lloyd; Cancrini, Caterina; Passerini, Laura; Bacchetta, Rosa; Ochs, Hans D.; Torgerson, Troy R.

    2013-01-01

    Regulatory T cells (Tregs) play an essential role in preventing autoimmunity. Mutations in the forkhead box protein 3 (FOXP3) gene, which encodes a transcription factor critical for Treg function, result in a severe autoimmune disorder and the production of various autoantibodies in mice and in IPEX (immune dysregulation, polyendocrinopathy, enteropathy, X-linked) patients. However, it is unknown whether Tregs normally suppress autoreactive B cells. To investigate a role for Tregs in maintaining human B-cell tolerance, we tested the reactivity of recombinant antibodies isolated from single B cells isolated from IPEX patients. Characteristics and reactivity of antibodies expressed by new emigrant/transitional B cells from IPEX patients were similar to those from healthy donors, demonstrating that defective Treg function does not impact central B-cell tolerance. In contrast, mature naive B cells from IPEX patients often expressed autoreactive antibodies, suggesting an important role for Tregs in maintaining peripheral B-cell tolerance. T cells displayed an activated phenotype in IPEX patients, including their Treg-like cells, and showed up-regulation of CD40L, PD-1, and inducibl T-cell costimulator (ICOS), which may favor the accumulation of autoreactive mature naive B cells in these patients. Hence, our data demonstrate an essential role for Tregs in the establishment and the maintenance of peripheral B-cell tolerance in humans. PMID:23223361

  4. Genotoxic evaluation of Halfenprox using the human peripheral lymphocyte micronucleus assay and the Ames test.

    PubMed

    Akyıl, Dilek; Eren, Yasin; Konuk, Muhsin; Dere, Hatice; Serteser, Ahmet

    2017-04-01

    The genotoxicity and mutagenicity of Halfenprox, a synthetic pyrethroid insecticide and acaricide, was assessed using two standard genotoxicity assays of the Salmonella typhimurium mutagenicity assay (Ames test) and in vitro micronucleus (MN) assay in human peripheral lymphocytes. In the Ames test, Salmonella strains TA98 and TA100 were treated with or without S9 fraction. The doses of Halfenprox were 6.25, 12.5, 25, 50, and 100 μg/plate and test materials were dissolved in DMSO. The concentrations of Halfenprox did not show mutagenic activity on both strains with and without S9 fraction. The MN assay was used to investigate the genotoxic effects of Halfenprox in human peripheral lymphocytes treated with 250, 500, 750, and 1000 μg/ml concentrations of Halfenprox for 24 and 48 h, and at 1000 μg/ml the concentration was significantly increased and the MN formation was compared with the negative control for both treatment periods. In addition, a significant decrease of the nuclear devision index (NDI) values at the higher concentrations of Halfenprox and at both treatment periods was observed.

  5. Immunohistochemical methods for semiquantitative analysis of collagen content in human peripheral nerve

    PubMed Central

    LOWRY, A.; WILCOX, D.; MASSON, E. A.; WILLIAMS, P. E.

    1997-01-01

    Methods are described for the semiquantitative analysis of the connective tissue components of human peripheral nerve using light microscopy. General histological preservation was assessed using haematoxylin and eosin staining and the distribution of collagen type IV was investigated using immunohistochemistry. Several techniques were investigated to establish the one giving optimum structural preservation, immunobinding and greatest contrast for image analysis. Frozen sections were unsuitable for this tissue and paraffin wax sections were therefore used. Alcohol fixation was rejected due to poor preservation of the endoneurium, although immunobinding was excellent. Ice-cold formalin fixation for 24 h was found to be adequate for structural preservation and antibody binding, provided that a protease step was introduced. Trypsin was found to be superior to pepsin for exposing collagen type IV epitopes. Of the detection systems investigated indirect immunofluorescence was not suitable due to considerable autofluorescence of the nerve. The avidin-biotin method provided the greatest contrast, and was therefore the detection method of choice for image analysis. The optimum techniques for image analysis were then used on control human sural nerve to ascertain the best comparative method for collagen type IV in the perineurium. A method of semiquantitative analysis is described which takes into account the fact that there is a close linear relationship between collagen content per unit of perineurium and perineurial perimeter as fascicle size increases in peripheral nerve. This means that data from 2 different sample groups can easily be compared, provided that a range of fascicle sizes is analysed in each case. PMID:9418993

  6. A novel splice variant of human gene NPL, mainly expressed in human liver, kidney and peripheral blood leukocyte.

    PubMed

    Wu, Maoqing; Gu, Shaohua; Xu, Jian; Zou, Xianqiong; Zheng, Huarui; Jin, Zhe; Xie, Yi; Ji, Chaoneng; Mao, Yumin

    2005-04-01

    From the human fetal brain cDNA library constructed by our lab, a novel variant cDNA of a human gene was successfully cloned and identified. Because the gene has been named N-acetylneuraminate pyruvate lyase (NPL), accordingly we term our splice variant NPL_v2. The cDNA of NPL_v2 has a full-length open reading frame (ORF) from the nucleotide position 320 to 1225 that encodes a protein comprising 301 amino acids. SMART analysis showed that our hypothetical protein has one dihydrodipicolinate synthase (DHDPS) domain. Phosphorylation analysis of the deduced protein show that there are five phosphorylation sites including three "serine" and two "threonine" at the region that are not found in other splice variant. RT-PCR experiment revealed that our splice variant of the gene is mainly expressed in human placenta, liver, kidney, pancreas, spleen, thymus, ovary, small intestine and peripheral blood leukocyte.

  7. Phases of daylight and the stability of color perception in the near peripheral human retina.

    PubMed

    Panorgias, Athanasios; Kulikowski, Janus J; Parry, Neil R A; McKeefry, Declan J; Murray, Ian J

    2012-03-01

    Typical daylight extends from blue (morning sky) to orangey red (evening sky) and is represented mathematically as the Daylight Locus in color space. In this study, we investigate the impact of this daylight variation on human color vision. Thirty-eight color normal human observers performed an asymmetric color match in the near peripheral visual field. Unique hues were identified using a naming paradigm. The observers' performance for matching was almost perfectly coincident with the Daylight Locus but declined markedly in other regions. Interobserver variability reached a conspicuous minimum adjacent to the Daylight Locus and was maximal in the red and yellowish-green regions. In the naming task, unique blue and yellow were virtually coincident with the Daylight Locus. The results suggest that the mechanisms of color perception mediated by the phylogenetically older (blue-yellow) color pathway have been strongly influenced by the different phases of daylight.

  8. Problems with measuring peripheral oxytocin: can the data on oxytocin and human behavior be trusted?

    PubMed

    McCullough, Michael E; Churchland, Patricia Smith; Mendez, Armando J

    2013-09-01

    Research on the neurobiological and behavioral effects of oxytocin (OT), as well as on its possible therapeutic applications, has intensified in the past decade. Accurate determination of peripheral OT levels is essential to reach meaningful conclusions and to motivate, support and inform clinical interventions. Different, but concordant, methods for measuring plasma OT have been developed over the past four decades, but since 2004 several commercially available methods have been favored in research with humans. Evaluation of these methods reveals that they lack reliability when used on unextracted samples of human fluids, and that they tag molecules in addition to OT, yielding estimates that are wildly discrepant with an extensive body of earlier findings that were obtained using methods that are well validated, but more laborious. An accurate, specific, and readily available method for measuring OT that can be adopted as the standard in the field is urgently needed for advances in our understanding of OT's roles in cognition and behavior.

  9. Modulation of Mitogen-Induced Proliferation of Autologous Peripheral Blood Lymphocytes by Human Alveolar Macrophages

    PubMed Central

    Yeager, Henry; Sweeney, Jan A.; Herscowitz, Herbert B.; Barsoum, Ibrahim S.; Kagan, Elliott

    1982-01-01

    Experiments were carried out to determine the effect of cocultivation of T-cell-enriched human peripheral blood lymphocytes with autologous alveolar macrophages on mitogen-induced proliferation as determined by [3H]thymidine uptake. Cells obtained by fiberoptic bronchoscopy and saline bronchial lavage from 14 normal volunteers were enriched for macrophages by adherence in plastic dishes for 1 h in RPMI 1640 medium supplemented with 10% fetal calf serum. Nonadherent mononuclear cells were prepared from heparinized venous blood after Ficoll-Hypaque sedimentation by passage over nylon wool columns. T-cell-enriched populations were incubated with and without alveolar macrophages, either in the presence or absence of phytohemagglutinin. In these experiments, the number of lymphocytes was held constant (105 per well), while the number of alveolar macrophages was varied (0.1 × 105 to 4.0 × 105 per well). Alveolar macrophages generally tended to stimulate phytohemagglutinin-induced lymphoproliferation at lymphocyte/macrophage ratios of 10:1 but consistently and significantly suppressed proliferation at ratios which approach those usually observed in recovered human bronchial lavage fluid, namely, 1:4. The suppressive effect of alveolar macrophages was observed as early as 48 h after culture initiation, while the magnitude of suppression increased with time. Suppression did not appear to be due to alteration in lymphocyte viability, nor was it sensitive to indomethacin. These results indicate that human alveolar macrophages can modulate the in vitro proliferative response of autologous peripheral blood lymphocytes. This observation may have relevance to interactions between alveolar macrophages and bronchial lymphocytes in the human lung in vivo. PMID:6982862

  10. Differential expression of the granzymes A, K and M and perforin in human peripheral blood lymphocytes.

    PubMed

    Bade, Britta; Boettcher, Heidrun Elise; Lohrmann, Jens; Hink-Schauer, Clara; Bratke, Kai; Jenne, Dieter E; Virchow, J Christian; Luttmann, Werner

    2005-11-01

    Granzymes (Gzm) are a group of serine proteases which are stored in the granules of cytotoxic lymphocytes. In humans, five granzymes have been characterized to date at the molecular level. While GzmA and GzmB have been extensively studied, little is known about GzmH, GzmK and GzmM. In this study, we describe the generation of mAbs against human GzmK and GzmM by genetic immunization. The obtained anti-GzmK and anti-GzmM mAbs are not cross-reactive with GzmA, GzmB, GzmM and GzmA, GzmB, GzmK, respectively, and show a granular staining pattern in human lymphocytes. Flow cytometric analysis of peripheral blood lymphocytes revealed that GzmA, GzmM and perforin show a similar distribution. They are expressed in almost all CD16+CD56+ NK cells, CD3+CD56+ NKT cells and gammadelta T cells as well as in 20-30% of all CD3+CD8+ TC cells. Surprisingly, GzmK was not detected in the highly cytotoxic CD16+CD56+ NK cells but was preferentially expressed in lymphocytes of the T cell lineage, staining 20% of CD3+CD8+ TC cells, 50% of CD3+CD56+ NKT cells and 40% of gammadelta T cells, as well as 60% of the small sub-population of CD56bright+ NK cells. Our data suggest that human granzymes are differentially expressed in distinct sub-populations of peripheral blood lymphocytes.

  11. LC-(TOF) MS analysis of benzodiazepines in urine from alleged victims of drug-facilitated sexual assault.

    PubMed

    ElSohly, Mahmoud A; Gul, Waseem; Murphy, Timothy P; Avula, Bharathi; Khan, Ikhlas A

    2007-10-01

    The present study employs a recently reported liquid chromatography-(time of flight) mass spectrometry procedure for the simultaneous analysis of 22 benzodiazepines in human urine specimens. The analysis focused on the most commonly prescribed benzodiazepines and/or their metabolites. Using this method, the limit of quantitation for the benzodiazepines tested ranged from 2 to 10 ng/mL, while the limit of detection range was 0.5 to 3.0 ng/mL. Urine specimens collected from alleged victims of drug-facilitated sexual assault (156 specimens) were tested. Only 19 out of the 22 benzodiazepines analyzed were detected in these specimens. These same specimens were previously screened for benzodiazepines by various immunoassay techniques using a 50 ng/mL cut-off level and confirmed by a gas chromatography-mass spectrometry method after acid hydrolysis to their benzophenone skeletons, thus making the identification of the specific benzodiazepine(s) involved impossible for most specimens. This study aims to offer an alternative methodology that would allow such identification for similar specimens. Additionally, the distribution of the individual benzodiazepines of interest among the 156 specimens as well as their prevalence in specimens originating in different U.S. states is presented.

  12. Peripheral inflammation acutely impairs human spatial memory via actions on medial temporal lobe glucose metabolism.

    PubMed

    Harrison, Neil A; Doeller, Christian F; Voon, Valerie; Burgess, Neil; Critchley, Hugo D

    2014-10-01

    Inflammation impairs cognitive performance and is implicated in the progression of neurodegenerative disorders. Rodent studies demonstrated key roles for inflammatory mediators in many processes critical to memory, including long-term potentiation, synaptic plasticity, and neurogenesis. They also demonstrated functional impairment of medial temporal lobe (MTL) structures by systemic inflammation. However, human data to support this position are limited. Sequential fluorodeoxyglucose positron emission tomography together with experimentally induced inflammation was used to investigate effects of a systemic inflammatory challenge on human MTL function. Fluorodeoxyglucose positron emission tomography scanning was performed in 20 healthy participants before and after typhoid vaccination and saline control injection. After each scanning session, participants performed a virtual reality spatial memory task analogous to the Morris water maze and a mirror-tracing procedural memory control task. Fluorodeoxyglucose positron emission tomography data demonstrated an acute reduction in human MTL glucose metabolism after inflammation. The inflammatory challenge also selectively compromised human spatial, but not procedural, memory; this effect that was independent of actions on motivation or psychomotor response. Effects of inflammation on parahippocampal and rhinal glucose metabolism directly mediated actions of inflammation on spatial memory. These data demonstrate acute sensitivity of human MTL to mild peripheral inflammation, giving rise to associated functional impairment in the form of reduced spatial memory performance. Our findings suggest a mechanism for the observed epidemiologic link between inflammation and risk of age-related cognitive decline and progression of neurodegenerative disorders including Alzheimer's disease. Copyright © 2014 Society of Biological Psychiatry. Published by Elsevier Inc. All rights reserved.

  13. 21 CFR 862.3170 - Benzodiazepine test system.

    Code of Federal Regulations, 2013 CFR

    2013-04-01

    ... 21 Food and Drugs 8 2013-04-01 2013-04-01 false Benzodiazepine test system. 862.3170 Section 862....3170 Benzodiazepine test system. (a) Identification. A benzodiazepine test system is a device intended to measure any of the benzodiazepine compounds, sedative and hypnotic drugs, in blood, plasma, and...

  14. 21 CFR 862.3170 - Benzodiazepine test system.

    Code of Federal Regulations, 2011 CFR

    2011-04-01

    ... 21 Food and Drugs 8 2011-04-01 2011-04-01 false Benzodiazepine test system. 862.3170 Section 862....3170 Benzodiazepine test system. (a) Identification. A benzodiazepine test system is a device intended to measure any of the benzodiazepine compounds, sedative and hypnotic drugs, in blood, plasma, and...

  15. 21 CFR 862.3170 - Benzodiazepine test system.

    Code of Federal Regulations, 2012 CFR

    2012-04-01

    ... 21 Food and Drugs 8 2012-04-01 2012-04-01 false Benzodiazepine test system. 862.3170 Section 862....3170 Benzodiazepine test system. (a) Identification. A benzodiazepine test system is a device intended to measure any of the benzodiazepine compounds, sedative and hypnotic drugs, in blood, plasma, and...

  16. 21 CFR 862.3170 - Benzodiazepine test system.

    Code of Federal Regulations, 2014 CFR

    2014-04-01

    ... 21 Food and Drugs 8 2014-04-01 2014-04-01 false Benzodiazepine test system. 862.3170 Section 862....3170 Benzodiazepine test system. (a) Identification. A benzodiazepine test system is a device intended to measure any of the benzodiazepine compounds, sedative and hypnotic drugs, in blood, plasma, and...

  17. 21 CFR 862.3170 - Benzodiazepine test system.

    Code of Federal Regulations, 2010 CFR

    2010-04-01

    ... 21 Food and Drugs 8 2010-04-01 2010-04-01 false Benzodiazepine test system. 862.3170 Section 862....3170 Benzodiazepine test system. (a) Identification. A benzodiazepine test system is a device intended to measure any of the benzodiazepine compounds, sedative and hypnotic drugs, in blood, plasma, and...

  18. Post-prandial rise of microvesicles in peripheral blood of healthy human donors.

    PubMed

    Suštar, Vid; Bedina-Zavec, Apolonija; Stukelj, Roman; Frank, Mojca; Ogorevc, Eva; Janša, Rado; Mam, Keriya; Veranič, Peter; Kralj-Iglič, Veronika

    2011-03-21

    Microvesicles isolated from body fluids are membrane - enclosed fragments of cell interior which carry information on the status of the organism. It is yet unclear how metabolism affects the number and composition of microvesicles in isolates from the peripheral blood. To study the post - prandial effect on microvesicles in isolates from the peripheral blood of 21 healthy donors, in relation to blood cholesterol and blood glucose concentrations. The average number of microvesicles in the isolates increased 5 hours post - prandially by 52%; the increase was statistically significant (p = 0.01) with the power P = 0.68, while the average total blood cholesterol concentration, average low density lipoprotein cholesterol concentration (LDL-C) and average high density lipoprotein cholesterol concentration (HDL-C) all remained within 2% of their fasting values. We found an 11% increase in triglycerides (p = 0.12) and a 6% decrease in blood glucose (p < 0.01, P = 0.74). The post - prandial number of microvesicles negatively correlated with the post - fasting total cholesterol concentration (r = - 0.46, p = 0.035) while the difference in the number of microvesicles in the isolates between post - prandial and post - fasting states negatively correlated with the respective difference in blood glucose concentration (r = - 0.39, p = 0.05). In a population of healthy human subjects the number of microvesicles in isolates from peripheral blood increased in the post - prandial state. The increase in the number of microvesicles was affected by the fasting concentration of cholesterol and correlated with the decrease in blood glucose.

  19. Peripheral inflammatory disease associated with centrally activated IL-1 system in humans and mice.

    PubMed

    Lampa, Jon; Westman, Marie; Kadetoff, Diana; Agréus, Anna Nordenstedt; Le Maître, Erwan; Gillis-Haegerstrand, Caroline; Andersson, Magnus; Khademi, Mohsen; Corr, Maripat; Christianson, Christina A; Delaney, Ada; Yaksh, Tony L; Kosek, Eva; Svensson, Camilla I

    2012-07-31

    During peripheral immune activation caused by an infection or an inflammatory condition, the innate immune response signals to the brain and causes an up-regulation of central nervous system (CNS) cytokine production. Central actions of proinflammatory cytokines, in particular IL-1β, are pivotal for the induction of fever and fatigue. In the present study, the influence of peripheral chronic joint inflammatory disease in rheumatoid arthritis (RA) on CNS inflammation was investigated. Intrathecal interleukin (IL)-1β concentrations were markedly elevated in RA patients compared with controls or with patients with multiple sclerosis. Conversely, the anti-inflammatory IL-1 receptor antagonist and IL-4 were decreased in RA cerebrospinal fluid (CSF). Tumor necrosis factor and IL-6 levels in the CSF did not differ between patients and controls. Concerning IL-1β, CSF concentrations in RA patients were higher than in serum, indicating local production in the CNS, and there was a positive correlation between CSF IL-1β and fatigue assessments. Next, spinal inflammation in experimental arthritis was investigated. A marked increase of IL-1β, IL-18, and tumor necrosis factor, but not IL-6 mRNA production, in the spinal cord was observed, coinciding with increased arthritis scores in the KBxN serum transfer model. These data provide evidence that peripheral inflammation such as arthritis is associated with an immunological activation in the CNS in both humans and mice, suggesting a possible therapeutic target for centrally affecting conditions as fatigue in chronic inflammatory diseases, for which to date there are no specific treatments.

  20. Identification, characterization, and purification of a 65,000 dalton protein in rat brain is photolabeled by nitro-containing benzodiazepines

    SciTech Connect

    Bowling, A.C.

    1988-01-01

    Benzodiazepines bind to two well-characterized classes of nanomolar-affinity binding sites, the central and the peripheral types. Although these sites appear to mediate many of the effects of these compounds, they cannot account for all of the biochemical and physiologic effects of the benzodiazepines. In this investigation, a protein that is photolabeled by NO{sub 2}-containing benzodiazepines was identified and characterized in rat brain by performing photaffinity labeling experiments with ({sup 3}H)-clonazepam and ({sup 3}H)-flunitrazepam. These experiments demonstrate that this photolabeled protein has a molecular weight of 65,000 daltons. Photolabeling of the protein was saturable, inhibited in a stereoselective manner by benzodiazepine enantiomers, inhibited by therapeutically-relevant concentrations of many different NO{sub 2}-containing benzodiazepines, and was not inhibited by more than 70 non-benzodiazepine compounds. The photolabeled protein is distinct from the central and peripheral sites on the basis of molecular weight, benzodiazepine inhibitory potencies, subcellular localization, and tissue distribution.

  1. Cytogenetic Low-Dose Hyperradiosensitivity Is Observed in Human Peripheral Blood Lymphocytes

    SciTech Connect

    Seth, Isheeta; Joiner, Michael C.; Tucker, James D.

    2015-01-01

    Purpose: The shape of the ionizing radiation response curve at very low doses has been the subject of considerable debate. Linear-no-threshold (LNT) models are widely used to estimate risks associated with low-dose exposures. However, the low-dose hyperradiosensitivity (HRS) phenomenon, in which cells are especially sensitive at low doses but then show increased radioresistance at higher doses, provides evidence of nonlinearity in the low-dose region. HRS is more prominent in the G2 phase of the cell cycle than in the G0/G1 or S phases. Here we provide the first cytogenetic mechanistic evidence of low-dose HRS in human peripheral blood lymphocytes using structural chromosomal aberrations. Methods and Materials: Human peripheral blood lymphocytes from 2 normal healthy female donors were acutely exposed to cobalt 60 γ rays in either G0 or G2 using closely spaced doses ranging from 0 to 1.5 Gy. Structural chromosomal aberrations were enumerated, and the slopes of the regression lines at low doses (0-0.4 Gy) were compared with doses of 0.5 Gy and above. Results: HRS was clearly evident in both donors for cells irradiated in G2. No HRS was observed in cells irradiated in G0. The radiation effect per unit dose was 2.5- to 3.5-fold higher for doses ≤0.4 Gy than for doses >0.5 Gy. Conclusions: These data provide the first cytogenetic evidence for the existence of HRS in human cells irradiated in G2 and suggest that LNT models may not always be optimal for making radiation risk assessments at low doses.

  2. THE SIGNIFICANCE OF LESIONS IN PERIPHERAL GANGLIA IN CHIMPANZEE AND IN HUMAN POLIOMYELITIS

    PubMed Central

    Bodian, David; Howe, Howard A.

    1947-01-01

    1. The peripheral ganglia of eighteen inoculated chimpanzees and thirteen uninoculated controls, and of eighteen fatal human poliomyelitis cases, were studied for histopathological evidence of the route of transmission of virus from the alimentary tract to the CNS. 2. Lesions thought to be characteristic of poliomyelitis in inoculated chimpanzees could not be sharply differentiated from lesions of unknown origin in uninoculated control animals. Moreover, although the inoculated animals as a group, in comparison with the control animals, had a greater number of infiltrative lesions in sympathetic as well as in sensory ganglia, it was not possible to make satisfactory correlations between the distribution of these lesions and the routes of inoculation. 3. In sharp contrast with chimpanzees, the celiac and stellate ganglia of the human poliomyelitis cases were free of any but insignificant infiltrative lesions. Lesions in human trigeminal and spinal sensory ganglia included neuronal damage as well as focal and perivascular inflitrative lesions, as is well known. In most ganglia, as in monkey and chimpanzee sensory ganglia, these were correlated in intensify with the degree of severity of lesions in the region of the CNS receiving their axons. This suggested that lesions in sensory ganglia probably resulted from spread of virus centrifugally from the CNS, in accord with considerable experimental evidence. 4. Two principal difficulties in the interpretation of histopathological findings in peripheral ganglia were revealed by this study. The first is that the specificity of lesions in sympathetic ganglia has not been established beyond doubt as being due to poliomyelitis. The second is that the presence of characteristic lesions in sensory ganglia does not, and cannot, reveal whether the virus reached the ganglia from the periphery or from the central nervous system, except in very early preparalytic stages or in exceptional cases of early arrest of virus spread and of

  3. Magnetic targeting of human peripheral blood CD133+ cells for skeletal muscle regeneration.

    PubMed

    Ohkawa, Shingo; Kamei, Naosuke; Kamei, Goki; Shi, Ming; Adachi, Nobuo; Deie, Masataka; Ochi, Mitsuo

    2013-08-01

    Skeletal muscle injuries often leave lasting functional damage or pain. Muscle injuries are routinely treated conservatively, but the most effective treatment to promote the repair of injured muscles has not yet been established. Our previous report demonstrated that human peripheral blood-derived CD133(+) cell transplantation to rat skeletal muscle injury models inhibited fibrosis and enhanced myogenesis after injury. However, the acquisition of a sufficient number of cells remains the limitation for clinical application, as the CD133(+) population is rare in human blood. In this study, we applied a magnetic cell targeting system to accumulate transplanted cells in the muscle injury site and to enhance the regenerative effects of CD133(+) cell transplantation, focusing on the fact that CD133(+) cells are labeled with a magnetic bead for isolation. For the magnetic cell targeting, the magnet field generator was set up to adjust the peak of the magnetic gradient to the injury site of the tibialis anterior muscle, and 1×10(4) human peripheral blood CD133(+) cells were locally injected into the injury site. This cell number is 10% of that used in the previous study. In another group, the same number of CD133(+) cells was injected without magnetic force. The CD133(+) cells transplanted with the magnetic force were more accumulated in the muscle injury site compared with the CD133(+) cells transplanted without the magnetic force. In addition, the transplantation of CD133(+) cells under the magnetic control inhibited fibrous scar formation and promoted angiogenesis and myogenesis, and also upregulated the mRNA expression of myogenic transcription factors, including Pax7, MyoD1 and Myogenin. However, the transplantation of CD133(+) cells without the magnetic force failed to demonstrate these effects. Thus, our magnetic cell targeting system enables transplantation of a limited number of CD133(+) cells to promote the repair of skeletal muscle injury.

  4. Characteristics of nucleoplasmic bridges induced by 60Co γ-rays in human peripheral blood lymphocytes.

    PubMed

    Zhao, Hua; Lu, Xue; Li, Shuang; Chen, De-Qing; Liu, Qing-Jie

    2013-12-16

    Few studies have shown that the yields of ionising-radiation-induced nucleoplasmic bridges (NPBs) in human cells are dose dependent. However, a dose-response curve between the NPB frequency and the absorbed dose of ionising radiation has not yet been established. This study aimed to investigate NPB frequencies in human peripheral blood lymphocytes induced by cobalt-60 ((60)Co) γ-rays and to establish a dose-response curve. Human peripheral blood samples were collected from three healthy males, and some of these samples were irradiated with 0-6 Gy (60)Co γ-rays. A cytokinesis-block micronucleus cytome assay was then carried out to analyse NPBs and micronuclei (MN) in binucleated cells. The remaining blood samples were irradiated with 0, 2 and 5 Gy of γ-rays, and unstable chromosome aberrations (dicentric chromosome, ring chromosome and acentric chromosome fragment) were analysed. The correlation between NPBs and dicentric plus ring chromosome (dic+r) induced by the same γ-ray dose was also analysed. Results showed that the NPB yields among the three subjects at each dose level were not significantly different. NPBs in binucleated cells at all γ-ray doses conformed to Poisson distribution. The dose-response curve of the γ-ray-induced NPB frequencies followed the linear-quadratic model y = (1.39×10(-3))x (2) + (4.94×10(-3))x. A positive correlation was observed between the frequencies of NPB and dic+r, as well as between the frequencies of MN and acentric fragments. Therefore, NPB is an important biomarker of early chromosome damage event induced by ionising radiation.

  5. [Consumption of benzodiazepines: good and bad uses].

    PubMed

    Ginestet, D

    1983-01-01

    In both general practice and psychiatry, the indication for benzodiazepines recognized by the French Technical Commissions--"all forms of anxiety"--remains very broad, and absolute contraindications are limited to severe respiratory insufficiency and, for certain drugs, myasthenia. Responsibility for deciding at what moment it becomes necessary to prescribe benzodiazepines therapy lies with the individual doctor--he is sole judge in his relationship with the patient: reactional anxiety, anxiety or distress accompanying somatic disease, insomnia. Benzodiazepines should not be prescribed to children, unless there are exceptional reasons for doing so. In psychiatry, higher doses are authorized for neurotic or psychotic anxiety, which are considered to be more serious forms. It is more or less accepted practice to prescribe benzodiazepines in combination therapy of various kinds with other psychotropic drugs. There are certain indications for which benzodiazepines in monotherapy are wrongly used, such as depressive states, and others where their use is contested, schizophrenic states, for example. Pharmacokinetic data are now beginning to provide a guide to dosage scheduling and to the choice of drug for the different indications. Maximum duration of benzodiazepine therapy should not exceed 4 months in certain cases. Risk of withdrawal syndrome appears to be slight for usual therapeutic doses. Finally, benzodiazepines are not the first choice of the drug addicts.

  6. Regulation of Exacerbated Immune Responses in Human Peripheral Blood Cells by Hydrolysed Egg White Proteins

    PubMed Central

    Lozano-Ojalvo, Daniel; Molina, Elena; López-Fandiño, Rosina

    2016-01-01

    The anti-allergic potential of egg white protein hydrolysates (from ovalbumin, lysozyme and ovomucoid) was evaluated as their ability to hinder cytokine and IgE production by Th2-skewed human peripheral blood mononuclear cells (PBMCs), as well as the release of pro-inflammatory factors and generation of reactive oxygen species from Th1-stimulated peripheral blood leukocytes (PBLs). The binding to IgE of egg allergic patients was determined and the peptides present in the hydrolysates were identified. The hydrolysates with alcalase down-regulated the production of Th2-biased cytokines and the secretion of IgE to the culture media of Th2-skewed PBMCs, and they significantly neutralized oxidative stress in PBLs. The hydrolysates of ovalbumin and ovomucoid with pepsin helped to re-establish the Th1/Th2 balance in Th2-biased PBMCs, while they also inhibited the release of pro-inflammatory mediators and reduced oxidative stress in PBLs treated with inflammatory stimuli. The hydrolysates with alcalase, in addition to equilibrating Th2 differentiation, exhibited a low IgE-binding. Therefore, they would elicit mild allergic reactions while retaining T cell-stimulating abilities, which might correlate with an anti-allergic benefit. PMID:27007699

  7. Characterization of peripheral blood human immunodeficiency virus isolates from Hispanic women with cognitive impairment.

    PubMed

    Nieves, Dianedis M Toro; Plaud, Marinés; Wojna, Valerie; Skolasky, Richard; Meléndez, Loyda M

    2007-08-01

    Human immunodeficiency virus type 1 (HIV-1) tropism plays an important role in HIV-associated dementia. In this study, aimed at determining if the tropism and coreceptor usage of circulating viruses correlates with cognitive function, the authors isolated and characterized HIV from the peripheral blood of 21 Hispanic women using antiretroviral therapy. Macrophage tropism was determined by inoculation of HIV isolates onto monocyte-derived macrophages and lymphocyte cultures. To define coreceptor usage, the HIV isolates were inoculated onto the U87.CD4 glioma cell lines with specific CCR5 and CXCR4 coreceptors. HIV isolates from cognitively impaired patients showed higher levels of replication in mitogen-stimulated peripheral blood mononuclear cells than did isolates from patients with normal cognition (P < .05). The viral growth of HIV primary isolates in macrophages and lymphocytes did not differ between patients with and those without cognitive impairment. However, isolates from the cognitively impaired women preferentially used the X4 coreceptor (P < .05). These phenotypic studies suggest that cognitively impaired HIV-infected women receiving treatment may have a more highly replicating and more pathogenic X4 virus in the circulation that could contribute to their neuropathogenesis.

  8. The "Intermediate" CD14(++)CD16(+) monocyte subset increases in severe peripheral artery disease in humans.

    PubMed

    Wildgruber, Moritz; Aschenbrenner, Teresa; Wendorff, Heiko; Czubba, Maria; Glinzer, Almut; Haller, Bernhard; Schiemann, Matthias; Zimmermann, Alexander; Berger, Hermann; Eckstein, Hans-Henning; Meier, Reinhard; Wohlgemuth, Walter A; Libby, Peter; Zernecke, Alma

    2016-12-19

    Monocytes are key players in atherosclerotic. Human monocytes display a considerable heterogeneity and at least three subsets can be distinguished. While the role of monocyte subset heterogeneity has already been well investigated in coronary artery disease (CAD), the knowledge about monocytes and their heterogeneity in peripheral artery occlusive disease (PAOD) still is limited. Therefore, we aimed to investigate monocyte subset heterogeneity in patients with PAOD. Peripheral blood was obtained from 143 patients suffering from PAOD (Rutherford stage I to VI) and three monocyte subsets were identified by flow cytometry: CD14(++)CD16(-) classical monocytes, CD14(+)CD16(++) non-classical monocytes and CD14(++)CD16(+) intermediate monocytes. Additionally the expression of distinct surface markers (CD106, CD162 and myeloperoxidase MPO) was analyzed. Proportions of CD14(++)CD16(+) intermediate monocyte levels were significantly increased in advanced stages of PAOD, while classical and non-classical monocytes displayed no such trend. Moreover, CD162 and MPO expression increased significantly in intermediate monocyte subsets in advanced disease stages. Likewise, increased CD162 and MPO expression was noted in CD14(++)CD16(-) classical monocytes. These data suggest substantial dynamics in monocyte subset distributions and phenotypes in different stages of PAOD, which can either serve as biomarkers or as potential therapeutic targets to decrease the inflammatory burden in advanced stages of atherosclerosis.

  9. Consequences of peripheral chemoreflex inhibition with low-dose dopamine in humans

    PubMed Central

    Niewinski, Piotr; Tubek, Stanislaw; Banasiak, Waldemar; Paton, Julian F R; Ponikowski, Piotr

    2014-01-01

    Low-dose dopamine inhibits peripheral chemoreceptors and attenuates the hypoxic ventilatory response (HVR) in humans. However, it is unknown: (1) whether it also modulates the haemodynamic reactions to acute hypoxia, (2) whether it also modulates cardiac baroreflex sensitivity (BRS) and (3) if there is any effect of dopamine withdrawal. We performed a double-blind, placebo-controlled study on 11 healthy male volunteers. At sea level over 2 days every subject was administered low-dose dopamine (2 μg kg–1 min–1) or saline infusion, during which we assessed both ventilatory and haemodynamic responses to acute hypoxia. Separately, we evaluated effects of initiation and withdrawal of each infusion and BRS. The initiation of dopamine infusion did not affect minute ventilation (MV) or mean blood pressure (MAP), but increased both heart rate (HR) and cardiac output. Concomitantly, it decreased systemic vascular resistance. Dopamine blunted the ventilatory, MAP and HR reactions (hypertension, tachycardia) to acute hypoxia. Dopamine attenuated cardiac BRS to falling blood pressure. Dopamine withdrawal evoked an increase in MV. The magnitude of the increment in MV due to dopamine withdrawal correlated with the size of the HVR and depended on the duration of dopamine administration. The ventilatory reaction to dopamine withdrawal constitutes a novel index of peripheral chemoreceptor function. PMID:24396060

  10. The Immunomodulatory Effects of Nidus Vespae on Human Peripheral Blood Immune Cells In Vitro

    PubMed Central

    Zhu, Ming; Ling, Yang; Qi, Qiufeng; Zhang, Yaping; Bao, Yanqing; Liu, Yongping

    2015-01-01

    Nidus Vespae has been used in traditional Chinese medicine (TCM) to treat various cancers, but the underlying mechanisms were not yet clarified. This study was to investigate the effect of Nidus Vespae decoction (NVD) on tumor cell viability and immunoregulating functions of human peripheral blood immune cells. The effects on tumor cell viability, peripheral blood mononuclear cell (PBMC) proliferation activity, and the tumor cell phagocytosis of monocytes were evaluated by cell counting kit-8. Tumor-killing activity of cytotoxic T lymphocyte (CTL) was analyzed by 51Cr releasing assay. IgG production of B cells and cytokine (TNF-α and IL-6) secretion of monocytes were determined by ELISA method. Data showed that NVD has no significant inhibiting effects on gastric cancer cells growth. Nevertheless, it could obviously promote PBMC proliferation in a time- and concentration-dependent manner. After treatment with NVD, the CTL cytotoxicity against SGC-7901 was significantly greater than control. The TNF-α and IL-6 secretion of monocytes and the IgG production of B cells also increased remarkably. Furthermore, NVD could significantly promote the phagocytosis of monocytes on tumor cells. These results suggest that NVD appears to have an immunoenhancing effect on immune cells, indicating that Nidus Vespae is worth exploring for immunomodulatory effects in tumor treatment. PMID:26339270

  11. Putative Epimutagens in Maternal Peripheral and Cord Blood Samples Identified Using Human Induced Pluripotent Stem Cells

    PubMed Central

    Arai, Yoshikazu; Hayakawa, Koji; Arai, Daisuke; Ito, Rie; Iwasaki, Yusuke; Saito, Koichi; Akutsu, Kazuhiko; Takatori, Satoshi; Ishii, Rie; Hayashi, Rumiko; Izumi, Shun-Ichiro; Sugino, Norihiro; Kondo, Fumio; Horie, Masakazu; Nakazawa, Hiroyuki; Makino, Tsunehisa; Hirosawa, Mitsuko; Shiota, Kunio; Ohgane, Jun

    2015-01-01

    The regulation of transcription and genome stability by epigenetic systems are crucial for the proper development of mammalian embryos. Chemicals that disturb epigenetic systems are termed epimutagens. We previously performed chemical screening that focused on heterochromatin formation and DNA methylation status in mouse embryonic stem cells and identified five epimutagens: diethyl phosphate (DEP), mercury (Hg), cotinine, selenium (Se), and octachlorodipropyl ether (S-421). Here, we used human induced pluripotent stem cells (hiPSCs) to confirm the effects of 20 chemicals, including the five epimutagens, detected at low concentrations in maternal peripheral and cord blood samples. Of note, these individual chemicals did not exhibit epimutagenic activity in hiPSCs. However, because the fetal environment contains various chemicals, we evaluated the effects of combined exposure to chemicals (DEP, Hg, cotinine, Se, and S-421) on hiPSCs. The combined exposure caused a decrease in the number of heterochromatin signals and aberrant DNA methylation status at multiple gene loci in hiPSCs. The combined exposure also affected embryoid body formation and neural differentiation from hiPSCs. Therefore, DEP, Hg, cotinine, Se, and S-421 were defined as an “epimutagen combination” that is effective at low concentrations as detected in maternal peripheral and cord blood. PMID:26339649

  12. Benzodiazepines consumption: does dependence vary with age?

    PubMed

    Gérardin, Marie; Victorri-Vigneau, Caroline; Guerlais, Marylène; Guillou-Landreat, Morgane; Grall-Bronnec, Marie; Jolliet, Pascale

    2014-09-01

    We have compared two groups of chronic benzodiazepines (or zolpidem/zopiclone) users: "Seniors," aged 65 years or more, and "Adults," aged less than 65 years. The study took place in the Pays de Loire region. The questionnaire assesses dependence based on items from the DSM-IV. The analysis was based on 176 Senior questionnaires and 212 Adult questionnaires. Whereas Senior patients take benzodiazepines routinely with little negative consequences, Adults suffer from underlying psychological trouble, mention a higher consumption than planned, which causes negative consequences. 35.2% of Seniors are dependent on benzodiazepines versus 49.8% of Adults.

  13. Grass immunotherapy induces inhibition of allergen-specific human peripheral blood mononuclear cell proliferation.

    PubMed

    Baskar, S; Hamilton, R G; Norman, P S; Ansari, A A

    1997-02-01

    The peripheral blood mononuclear cells (PBMC) from humans allergic to grass pollens (GR+ subjects) show strong in vitro proliferative responses to purified allergens from Lolium perenne pollen Lol p 1, and to a lesser extent to Lol p 2 and Lol p 3. By contrast, PBMC from grass allergic patients undergoing immunotherapy (GR + IT subjects) exhibit a very poor Lol p-specific proliferative response, similar to that observed in nongrass allergic subjects (GR-subjects). Unlike GR-subjects, both GR+ and GR + IT subjects have high levels of antigen-specific serum IgG and IgE antibodies to Lol p 1, Lol p 2 and Lol p 3. While GR+ subjects exhibit a significant correlation between antigen-specific serum antibody and PBMC responses, GR + IT subjects do not show a correlation between the two responses. The possible mechanisms by which immunotherapy may modulate allergen-specific T cell proliferative response are discussed.

  14. Plasma and mitochondrial membrane perturbation induced by aluminum in human peripheral blood lymphocytes.

    PubMed

    Skarabahatava, Aliaksandra Sergeevna; Lukyanenko, Ludmila Michaylovna; Slobozhanina, Ekaterina Ivanovna; Falcioni, Maria Letizia; Orlando, Patrick; Silvestri, Sonia; Tiano, Luca; Falcioni, Giancarlo

    2015-01-01

    Aluminum is a redox-inert element that could induce cell damage via activation of oxidative stress. In this work, the effect of aluminum on different cellular compartments of human peripheral blood lymphocytes was studied. The presence of aluminum induced a lipid peroxidation and physico-chemical modifications at the membrane level. A decrease in fluorescence anisotropy of TMA-DPH and in the polarity of the lipid bilayer with a concomitant shift toward a gel phase was observed, while the pyrene excimerization coefficient (Kex) increased. Flow cytometry measurements, using JC-1, Rhodamine 123 and H2-DCFDA as fluorescent probes, indicated that aluminum induces a slight mitochondrial membrane depolarization that was associated with a moderate increase in reactive oxygen species production. A significative influence on these parameters was measured only at high aluminum concentration. Copyright © 2015 Elsevier GmbH. All rights reserved.

  15. In vitro study of interactions between silicon-containing nanoparticles and human peripheral blood leukocytes.

    PubMed

    Andreeva, E R; Rudimov, E G; Gornostaeva, A N; Beklemyshev, V I; Makhonin, I I; Maugeri, U O G; Buravkova, L B

    2013-07-01

    The effects of silicon dioxide-based nanoparticles on the viability and proliferative activity of human peripheral blood cultured lymphocytes were studied. All nanoparticles in a concentration of 100 μg/ml produced a significant cytotoxic effect, its intensity depending on particles' structure: SiO2 nanoparticles were least toxic, while Ce3(+)-intercaled montmorillonite nanoparticles were most toxic. The cells died mainly by apoptosis and postapoptotic necrosis. Incubation with nanoparticles in a concentration of 100 μg/ml for 72 h caused death of all phytohemagglutinin-activated lymphocytes, while in concentrations of 1 and 10 μg/ml the nanoparticles had no effect of proliferative activity of cells. The results suggest that the effects of nanoparticles on cells are determined by the nanoparticle concentration and size, as well as by their structure.

  16. Specific high-affinity binding sites for a synthetic gliadin heptapeptide of human peripheral blood lymphocytes

    SciTech Connect

    Payan, D.G.; Horvath, K.; Graf, L.

    1987-03-23

    The synthetic peptide containing residues 43-49 of ..cap alpha..-gliadin, the major protein component of gluten, has previously been shown to inhibit the production of lymphokine activities by mononuclear leukocytes. The authors demonstrate using radiolabeled ..cap alpha..-gliadin(43-49) that human peripheral blood lymphocytes express approximately 20,000-25,000 surface receptors for this peptide, with a dissociation constant (K/sub D/) of 20 nM. In addition, binding is inhibited by naloxone and an enkephalin analog, thus confirming the functional correlate which demonstrates inhibition by these agents of ..cap alpha..-gliadin(43-49) functional effects. Furthermore, B-lymphocytes bind specifically a greater amount of (/sup 125/I)..cap alpha..-gliadin(43-49) than T-lymphocytes. The lymphocyte ..cap alpha..-gliadin(43-49) receptor may play an important role in mediating the immunological response to ..cap alpha..-gliadin. 16 references, 4 figures.

  17. Neutron scattering studies on protein dynamics using the human myelin peripheral membrane protein P2

    NASA Astrophysics Data System (ADS)

    Laulumaa, Saara; Kursula, Petri; Natali, Francesca

    2015-01-01

    Myelin is a multilayered proteolipid membrane structure surrounding selected axons in the vertebrate nervous system, which allows the rapid saltatory conduction of nerve impulses. Deficits in myelin formation and maintenance may lead to chronic neurological disease. P2 is an abundant myelin protein from peripheral nerves, binding between two apposing lipid bilayers. We studied the dynamics of the human myelin protein P2 and its mutated P38G variant in hydrated powders using elastic incoherent neutron scattering. The local harmonic vibrations at low temperatures were very similar for both samples, but the mutant protein had increased flexibility and softness close to physiological temperatures. The results indicate that a drastic mutation of proline to glycine at a functional site can affect protein dynamics, and in the case of P2, they may explain functional differences between the two proteins.

  18. Altered Distribution of Peripheral Blood Memory B Cells in Humans Chronically Infected with Trypanosoma cruzi

    PubMed Central

    Fernández, Esteban R.; Olivera, Gabriela C.; Quebrada Palacio, Luz P.; González, Mariela N.; Hernandez-Vasquez, Yolanda; Sirena, Natalia María; Morán, María L.; Ledesma Patiño, Oscar S.; Postan, Miriam

    2014-01-01

    Numerous abnormalities of the peripheral blood T cell compartment have been reported in human chronic Trypanosoma cruzi infection and related to prolonged antigenic stimulation by persisting parasites. Herein, we measured circulating lymphocytes of various phenotypes based on the differential expression of CD19, CD4, CD27, CD10, IgD, IgM, IgG and CD138 in a total of 48 T. cruzi-infected individuals and 24 healthy controls. Infected individuals had decreased frequencies of CD19+CD27+ cells, which positively correlated with the frequencies of CD4+CD27+ cells. The contraction of CD19+CD27+ cells was comprised of IgG+IgD-, IgM+IgD- and isotype switched IgM-IgD- memory B cells, CD19+CD10+CD27+ B cell precursors and terminally differentiated CD19+CD27+CD138+ plasma cells. Conversely, infected individuals had increased proportions of CD19+IgG+CD27-IgD- memory and CD19+IgM+CD27-IgD+ transitional/naïve B cells. These observations prompted us to assess soluble CD27, a molecule generated by the cleavage of membrane-bound CD27 and used to monitor systemic immune activation. Elevated levels of serum soluble CD27 were observed in infected individuals with Chagas cardiomyopathy, indicating its potentiality as an immunological marker for disease progression in endemic areas. In conclusion, our results demonstrate that chronic T. cruzi infection alters the distribution of various peripheral blood B cell subsets, probably related to the CD4+ T cell deregulation process provoked by the parasite in humans. PMID:25111833

  19. Altered distribution of peripheral blood memory B cells in humans chronically infected with Trypanosoma cruzi.

    PubMed

    Fernández, Esteban R; Olivera, Gabriela C; Quebrada Palacio, Luz P; González, Mariela N; Hernandez-Vasquez, Yolanda; Sirena, Natalia María; Morán, María L; Ledesma Patiño, Oscar S; Postan, Miriam

    2014-01-01

    Numerous abnormalities of the peripheral blood T cell compartment have been reported in human chronic Trypanosoma cruzi infection and related to prolonged antigenic stimulation by persisting parasites. Herein, we measured circulating lymphocytes of various phenotypes based on the differential expression of CD19, CD4, CD27, CD10, IgD, IgM, IgG and CD138 in a total of 48 T. cruzi-infected individuals and 24 healthy controls. Infected individuals had decreased frequencies of CD19+CD27+ cells, which positively correlated with the frequencies of CD4+CD27+ cells. The contraction of CD19+CD27+ cells was comprised of IgG+IgD-, IgM+IgD- and isotype switched IgM-IgD- memory B cells, CD19+CD10+CD27+ B cell precursors and terminally differentiated CD19+CD27+CD138+ plasma cells. Conversely, infected individuals had increased proportions of CD19+IgG+CD27-IgD- memory and CD19+IgM+CD27-IgD+ transitional/naïve B cells. These observations prompted us to assess soluble CD27, a molecule generated by the cleavage of membrane-bound CD27 and used to monitor systemic immune activation. Elevated levels of serum soluble CD27 were observed in infected individuals with Chagas cardiomyopathy, indicating its potentiality as an immunological marker for disease progression in endemic areas. In conclusion, our results demonstrate that chronic T. cruzi infection alters the distribution of various peripheral blood B cell subsets, probably related to the CD4+ T cell deregulation process provoked by the parasite in humans.

  20. Flow cytometric assay for analysis of cytotoxic effects of potential drugs on human peripheral blood leukocytes

    NASA Astrophysics Data System (ADS)

    Nieschke, Kathleen; Mittag, Anja; Golab, Karolina; Bocsi, Jozsef; Pierzchalski, Arkadiusz; Kamysz, Wojciech; Tarnok, Attila

    2014-03-01

    Toxicity test of new chemicals belongs to the first steps in the drug screening, using different cultured cell lines. However, primary human cells represent the human organism better than cultured tumor derived cell lines. We developed a very gentle toxicity assay for isolation and incubation of human peripheral blood leukocytes (PBL) and tested it using different bioactive oligopeptides (OP). Effects of different PBL isolation methods (red blood cell lysis; Histopaque isolation among others), different incubation tubes (e.g. FACS tubes), anticoagulants and blood sources on PBL viability were tested using propidium iodide-exclusion as viability measure (incubation time: 60 min, 36°C) and flow cytometry. Toxicity concentration and time-depended effects (10-60 min, 36 °C, 0-100 μg /ml of OP) on human PBL were analyzed. Erythrocyte lysis by hypotonic shock (dH2O) was the fastest PBL isolation method with highest viability (>85%) compared to NH4Cl-Lysis (49%). Density gradient centrifugation led to neutrophil granulocyte cell loss. Heparin anticoagulation resulted in higher viability than EDTA. Conical 1.5 mL and 2 mL micro-reaction tubes (both polypropylene (PP)) had the highest viability (99% and 97%) compared to other tubes, i.e. three types of 5.0 mL round-bottom tubes PP (opaque-60%), PP (blue-62%), Polystyrene (PS-64%). Viability of PBL did not differ between venous and capillary blood. A gentle reproducible preparation and analytical toxicity-assay for human PBL was developed and evaluated. Using our assay toxicity, time-course, dose-dependence and aggregate formation by OP could be clearly differentiated and quantified. This novel assay enables for rapid and cost effective multiparametric toxicological screening and pharmacological testing on primary human PBL and can be adapted to high-throughput-screening.°z

  1. Differential expression of intracellular and extracellular CB(2) cannabinoid receptor protein by human peripheral blood leukocytes.

    PubMed

    Castaneda, Julie T; Harui, Airi; Kiertscher, Sylvia M; Roth, Jeffrey D; Roth, Michael D

    2013-03-01

    mRNA encoding for the CB(2) cannabinoid receptor is expressed by many subsets of human peripheral blood leukocytes (PBL), but little is known about the resulting protein expression and function. Employing clones from the A549 and 293T cell lines that were constructed to express both full-length human CB(2) and GFP, we developed a flow cytometry assay for characterizing CB(2) protein expression. A monoclonal antibody directed against human CB(2) selectively stained the surface of transduced but not parental cell lines. When cells were fixed and permeabilized, imaging flow cytometry identified large stores of intracellular protein. Total cellular staining for CB(2) corresponded closely with the level of GFP expression. When exposed to Δ(9)-tetrahydrocannabinol, CB(2)-expressing cells internalized cell surface CB(2) receptors in a time- and dose-dependent manner. Applying these approaches to human PBL, CB(2) protein was identified on the surface of human B cells but not on T cells or monocytes. In contrast, when PBL were fixed and permeabilized, intracellular CB(2) expression was readily detected in all three subsets by both conventional and imaging flow cytometry. Similar to the protein expression pattern observed in fixed and permeabilized PBL, purified B cells, T cells, and monocytes expressed relatively equal levels of CB(2) mRNA by quantitative real-time RT-PCR. Our findings confirm that human PBL express CB(2) protein but that its distribution is predominantly intracellular with only B cells expressing CB(2) protein at the extracellular membrane. The differential role of intracellular and extracellular CB(2) receptors in mediating ligand signaling and immune function remains to be determined.

  2. A permethrin/allethrin mixture induces genotoxicity and cytotoxicity in human peripheral blood lymphocytes.

    PubMed

    Ramos-Chavez, Lucio A; Sordo, Monserrat; Calderon-Aranda, Emma; Castañeda-Saucedo, Eduardo; Ostrosky-Wegman, Patricia; Moreno-Godinez, Ma Elena

    2015-01-01

    Two pyrethroids, permethrin and allethrin, are often combined for large-scale use in public health programs to control vector-borne diseases. In this study, the genotoxic potential of a commercial formulation of permethrin and allethrin was examined using cultured human peripheral blood lymphocytes (PBL). Genotoxicity was evaluated using the cytokinesis-block micronucleus cytome (CBMN cyt) assay by measuring the frequency of micronuclei (MN), nuclear division index (NDI), formation of nucleoplasmic bridges (NPB) and nuclear buds (NBUD), as well as apoptotic and necrotic cells. Human PBL were treated with different concentrations of a permethrin/allethrin mixture (1/0.01, 5/0.07, and 10/0.14 μg/ml) for 24 or 36 h. The highest concentration (10/0.14 μg/ml) of permethrin/allethrin mixture significantly increased MN frequency and percent apoptotic cells after incubations for 24 or 36 h. The NDI was markedly decreased in response to treatment with 5/0.07 or 10/0.14 μg/ml permethrin/allethrin for both 24 and 36 h. Exposure to the permethrin/allethrin mixture did not significantly alter formation of NBUD, NPB, or percent necrotic cells. The MN frequency was significantly correlated with the number of apoptotic and necrotic cells but inversely correlated with NDI. Data demonstrated that a mixture of permethrin and allethrin induced concentration- and time-dependent cytotoxic and genotoxic damage to human PBL in vitro.

  3. Cytotoxic and genotoxic effects of dioxacarb by human peripheral blood lymphocytes CAs and Allium test.

    PubMed

    Eren, Yasin; Erdoğmuş, Sevim Feyza; Akyıl, Dilek; Özkara, Arzu; Konuk, Muhsin; Sağlam, Esra

    2015-12-01

    Dioxacarb (Elecron, Famid) is a phenyl methylcarbamate insecticide and in vitro cytotoxic and genotoxic effects of this pesticide on human peripheral blood lymphocytes and Allium root meristematic cells were investigated by chromosomal aberrations (CAs) and Allium test. Human lymphocytes were treated with 62.5, 125, 250 and 500 ppm doses of dioxacarb for CAs. CA/cell, abnormal cell % and mitotic index % (MI %) data were obtained from these concentrations in 24 and 48 h treatment periods. Dioxacarb did not increase the CA/cell frequency significantly, so this insecticide was not identified as genotoxic. But it was found cytotoxic especially at 250 and 500 ppm concentrations because of the reduced the MI % and increased the abnormal cell %. In Allium test, 25 ppm (EC50/2), 50 ppm (EC50) and 100 ppm (EC50 × 2) concentrations were used for root growth inhibition (EC50 determination) and Allium mitotic index (MI) determination tests. The used concentrations of dioxacarb induced dose-dependent inhibition of MI and root growth on root meristems. Mitotic inhibition of dioxacarb was found significantly higher than for the positive control. These Allium results indicated the high cytotoxicity of dioxacarb. The present study is the first research on cytotoxicity and genotoxicity of dioxacarb by human lymphocyte CAs and Allium test.

  4. Deoxynivalenol induced oxidative stress and genotoxicity in human peripheral blood lymphocytes.

    PubMed

    Yang, Wei; Yu, Miao; Fu, Juan; Bao, Wei; Wang, Di; Hao, Liping; Yao, Ping; Nüssler, Andreas K; Yan, Hong; Liu, Liegang

    2014-02-01

    Deoxynivalenol (DON) is one of the most common mycotoxins. The aim of this study consists in using diverse cellular and molecular assays to evaluate cytotoxicity, genotoxicity as well as oxidative damage and to investigate their mechanisms in human peripheral blood lymphocytes. The human lymphocytes were cultured in eight different doses of DON (0, 6.25, 12.5, 25, 50, 100, 250 and 500 ng/mL) during 6, 12 and 24 h. DON was able to decrease cell viability and cause damage to the membrane, the chromosomes or the DNA at all times of culture. It was also able to induce lipid peroxidation and raise the levels of 8-OHdG and ROS in 6, 12 and 24 h. The results of the RT-PCR and the Western Blot indicated that DON is able to enhance mRNA or protein expressions of DNA repair genes and HO-1 in 6 h and to inhibit these expressions in 24 h. DON potentially triggers genotoxicity in human lymphocytes. This mechanism is probably related to depletion of antioxidase and oxidative damage to the DNA that reduced expression of HO-1, thereby inhibiting the ability of DNA repair.

  5. Inter-Individual Differences in RNA Levels in Human Peripheral Blood

    PubMed Central

    Chomczynski, Piotr; Wilfinger, William W.; Eghbalnia, Hamid R.; Kennedy, Amy; Rymaszewski, Michal; Mackey, Karol

    2016-01-01

    Relatively little is known about the range of RNA levels in human blood. This report provides assessment of peripheral blood RNA level and its inter-individual differences in a group of 35 healthy humans consisting of 25 females and 10 males ranging in age from 50 to 89 years. In this group, the average total RNA level was 14.59 μg/ml of blood, with no statistically significant difference between females and males. The individual RNA level ranged from 6.7 to 22.7 μg/ml of blood. In healthy subjects, the repeated sampling of an individual’s blood showed that RNA level, whether high or low, was stable. The inter-individual differences in RNA level in blood can be attributed to both, differences in cell number and the amount of RNA per cell. The 3.4-fold range of inter-individual differences in total RNA levels, documented herein, should be taken into account when evaluating the results of quantitative RT-PCR and/or RNA sequencing studies of human blood. Based on the presented results, a comprehensive assessment of gene expression in blood should involve determination of both the amount of mRNA per unit of total RNA (U / ng RNA) and the amount of mRNA per unit of blood (U / ml blood) to assure a thorough interpretation of physiological or pathological relevance of study results. PMID:26863434

  6. Characterization of T-bet and eomes in peripheral human immune cells.

    PubMed

    Knox, James J; Cosma, Gabriela L; Betts, Michael R; McLane, Laura M

    2014-01-01

    The T-box transcription factors T-bet and Eomesodermin (Eomes) have been well defined as key drivers of immune cell development and cytolytic function. While the majority of studies have defined the roles of these factors in the context of murine T-cells, recent results have revealed that T-bet, and possibly Eomes, are expressed in other immune cell subsets. To date, the expression patterns of these factors in subsets of human peripheral blood mononuclear cells beyond T-cells remain relatively uncharacterized. In this study, we used multiparametric flow cytometry to characterize T-bet and Eomes expression in major human blood cell subsets, including total CD4(+) and CD8(+) T-cells, γδ T-cells, invariant NKT cells, natural killer cells, B-cells, and dendritic cells. Our studies identified novel cell subsets that express T-bet and Eomes and raise implications for their possible functions in the context of other human immune cell subsets besides their well-known roles in T-cells.

  7. Characterization of T-Bet and Eomes in Peripheral Human Immune Cells

    PubMed Central

    Knox, James J.; Cosma, Gabriela L.; Betts, Michael R.; McLane, Laura M.

    2014-01-01

    The T-box transcription factors T-bet and Eomesodermin (Eomes) have been well defined as key drivers of immune cell development and cytolytic function. While the majority of studies have defined the roles of these factors in the context of murine T-cells, recent results have revealed that T-bet, and possibly Eomes, are expressed in other immune cell subsets. To date, the expression patterns of these factors in subsets of human peripheral blood mononuclear cells beyond T-cells remain relatively uncharacterized. In this study, we used multiparametric flow cytometry to characterize T-bet and Eomes expression in major human blood cell subsets, including total CD4+ and CD8+ T-cells, γδ T-cells, invariant NKT cells, natural killer cells, B-cells, and dendritic cells. Our studies identified novel cell subsets that express T-bet and Eomes and raise implications for their possible functions in the context of other human immune cell subsets besides their well-known roles in T-cells. PMID:24860576

  8. Influence of calorie reduction on DNA repair capacity of human peripheral blood mononuclear cells.

    PubMed

    Matt, Katja; Burger, Katharina; Gebhard, Daniel; Bergemann, Jörg

    2016-03-01

    Caloric restrictive feeding prolongs the lifespan of a variety of model organisms like rodents and invertebrates. It has been shown that caloric restriction reduces age-related as well as overall-mortality, reduces oxidative stress and influences DNA repair ability positively. There are numerous studies underlining this, but fewer studies involving humans exist. To contribute to a better understanding of the correlation of calorie reduction and DNA repair in humans, we adapted the host cell reactivation assay to an application with human peripheral blood mononuclear cells. Furthermore, we used this reliable and reproducible assay to research the influence of a special kind of calorie reduction, namely F. X. Mayr therapy, on DNA repair capacity. We found a positive effect in all persons with low pre-existing DNA repair capacity. In individuals with normal pre-existing DNA repair capacity, no effect on DNA repair capacity was detectable. Decline of DNA repair, accumulation of oxidative DNA damages, mitochondrial dysfunction, telomere shortening as well as caloric intake are widely thought to contribute to aging. With regard to that, our results can be considered as a strong indication that calorie reduction may support DNA repair processes and thus contribute to a healthier aging.

  9. Geno- and cytotoxicity of salinomycin in human nasal mucosa and peripheral blood lymphocytes.

    PubMed

    Scherzad, Agmal; Hackenberg, Stephan; Schramm, Carolin; Froelich, Katrin; Ginzkey, Christian; Hagen, Rudolf; Kleinsasser, Norbert

    2015-06-01

    Salinomycin is usually applied in stock breading but has also been described as a promising agent against cancer stem cells (CSC). However, knowledge about the toxicity of this ionophor substance is incomplete. The aim of this study was to investigate cyto- and genotoxic effects of salinomycin in human non-malignant cells. Primary human nasal mucosa cells (monolayer and mini organ cultures) and peripheral blood lymphocytes from 10 individuals were used to study the cytotoxic effects of salinomycin (0.1-175 μM) by annexin-propidiumiodide- and MTT-test. The comet assay was performed to evaluate DNA damage. Additionally, the secretion of interleukin-8 was analyzed by ELISA. Flow cytometry and MTT assay revealed significant cytotoxic effects in nasal mucosa cells and lymphocytes at low salinomycin concentrations of 10-20 μM. No genotoxic effects could be observed. IL-8 secretion was elevated at 5 μM. Salinomycin-induced cytotoxic and pro-inflammatory effects were seen at concentrations relevant for anti-cancer treatment. Concurrent to the evaluation of salinomycin application in experimental oncology, adverse effects in non-malignant cells need to be monitored and reduced as much as possible. Further studies are also warranted to evaluate the toxic effects in a variety of human cell systems, e.g., liver, kidney and muscle cells.

  10. Benzodiazepines in panic disorder and agoraphobia.

    PubMed

    Sheehan, D V

    1987-01-01

    Benzodiazepines, particularly alprazolam, are quickly becoming the drugs of first choice in the treatment of many cases of panic and agoraphobia. The reason for this choice is that these drugs are safer to use, quicker in onset of action, easier for the physician to prescribe and more pleasant for the patient to take than the alternatives. Although the treatment of panic disorder and agoraphobia has been best studied with the benzodiazepine alprazolam, it now appears likely that other benzodiazepines, for example diazepam, lorazepam and chlorazepam, may also be effective when correctly used. There is no reason at this point to believe that any of the benzodiazepines are unique in this regard. Future research will undoubtedly clarify this observation. In the meantime, it is hoped that some of the guidelines in this paper will help the practicing clinician in the management of his patient with this disabling and neglected disease.

  11. Quantitative peripheral blood perturbations of γδ T cells in human disease and their clinical implications.

    PubMed

    Bank, Ilan; Marcu-Malina, Victoria

    2014-12-01

    Human γδ T cells, which play innate and adaptive, protective as well as destructive, roles in the immune response, were discovered in 1986, but the clinical significance of alterations of the levels of these cells in the peripheral blood in human diseases has not been comprehensively reviewed. Here, we review patterns of easily measurable changes of this subset of T cells in peripheral blood from relevant publications in PubMed and their correlations with specific disease categories, specific diagnoses within disease categories, and prognostic outcomes. These collective data suggest that enumeration of γδ T cells and their subsets in the peripheral blood of patients could be a useful tool to evaluate diagnosis and prognosis in the clinical setting.

  12. The Use of Electrical Impedance to Identify Intraneural Needle Placement in Human Peripheral Nerves: A Study on Amputated Human Limbs.

    PubMed

    Vydyanathan, Amaresh; Kosharskyy, Boleslav; Nair, Singh; Gritsenko, Karina; Kim, Ryung S; Wang, Dan; Shaparin, Naum

    2016-07-01

    Even as the use of peripheral nerve blockade in the perioperative setting is increasing, neural injury secondary to accidental intraneural injection remains a significant patient safety concern. Current modalities, including electrical stimulation and ultrasound imaging, still lack consistency and absolute reliability in both the detection and prevention of this complication. The measurement of electrical impedance (EI) could be an easy and valuable additional tool to detect intraneural needle placement. Our objectives in this study were to measure the change in EI with intraneural needle advancement in recently amputated human limbs. The study was conducted within 45 minutes of amputation. The nerves that were studied were the sciatic nerve in the popliteal fossa in above-knee amputations or the tibial nerve below the calf in below-knee amputations. The amputated limb was placed on a tray and under ultrasound imaging guidance, an insulated peripheral block needle connected to a nerve stimulator was placed extraneurally and subsequently advanced intraneurally. The experiment was repeated on the same nerve after exposure by surgical dissection. The differences in impedance measurements between intraneural and extraneural needle placement were compared. In the below-knee amputated extremity (tibial nerve, n = 6) specimens based on the ultrasound methods, mean ± SD for ultrasound-guided intraneural impedance was 10 ± 2 kΩ compared with an extraneural impedance of 6 ± 1.6 kΩ (P = 0.005). The difference between intraneural and extraneural impedance after open dissection was also significant when we repeated the analysis based on the same specimens (P = 0.005). Similarly, in the above-the-knee amputated extremity (sciatic nerve, n = 5) specimens, mean intraneural impedance was 35.2 ± 7.9 kΩ compared with an extraneural impedance of 25.2 ± 5.3 kΩ (P = 0.037). The difference between intraneural and extraneural impedance obtained after open dissection was also

  13. Dynamic changes in the proteome of human peripheral blood mononuclear cells with low dose ionizing radiation.

    PubMed

    Nishad, S; Ghosh, Anu

    2016-02-01

    Humans are continually exposed to ionizing radiation from natural as well as anthropogenic sources. Though biological effects of high dose radiation exposures have been well accepted, studies on low-to-moderate dose exposures (in the range of 50-500 mGy) have been strongly debated even as researchers continue to search for elusive 'radiation signatures' in humans. Proteins are considered as dynamic functional players that drive cellular responses. However, there is little proteomic information available in context of human exposure to ionizing radiation. In this study, we determined differential expressed proteins in G0 peripheral blood mononuclear cells (PBMCs) from healthy individuals 1h and 4h after 'ex vivo' exposure with two radiation doses (300 mGy and 1 Gy). Twenty-three proteins were found to be significantly altered in irradiated cells when compared to sham irradiated cells with fold change ± 1.5-fold (p ≤ 0.05), with only three proteins showing ≥ 2.5-fold change, either with dose or with time. Mass spectrometry analyses identified redox sensor protein, chloride intracellular channel protein 1 (CLIC-1), the antioxidant protein, peroxiredoxin-6 and the pro-survival molecular chaperone 78 KDa glucose regulated protein (GRP78) among the 23 modulated proteins. The mean coefficient of variation (CV) for the twenty-three radiation responsive protein spots was found to be 33.7% for 300 mGy and 48.3% for 1 Gy. We thus, conclude that the radiation proteomic response of G0 human PBMCs, which are in the resting stage of the cell cycle, involves moderate upregulation of protective mechanisms, with low inter-individual variability. This study will help further our understanding of cellular effects of low dose acute radiation in humans and contribute toward differential biomarker discovery. Copyright © 2016 Elsevier B.V. All rights reserved.

  14. Design, synthesis and biological evaluation of sulfamide and triazole benzodiazepines as novel p53-MDM2 inhibitors.

    PubMed

    Yu, Zhiliang; Zhuang, Chunlin; Wu, Yuelin; Guo, Zizhao; Li, Jin; Dong, Guoqiang; Yao, Jianzhong; Sheng, Chunquan; Miao, Zhenyuan; Zhang, Wannian

    2014-09-05

    A series of sulfamide and triazole benzodiazepines were obtained with the principle of bioisosterism. The p53-murine double minute 2 (MDM2) inhibitory activity and in vitro antitumor activity were evaluated. Most of the novel benzodiazepines exhibited moderate protein binding inhibitory activity. Particularly, triazole benzodiazepines showed good inhibitory activity and antitumor potency. Compound 16 had promising antitumor activity against the U-2 OS human osteosarcoma cell line with an IC50 value of 4.17 μM, which was much better than that of nutlin-3. The molecular docking model also successfully predicted that this class of compounds mimicked the three critical residues of p53 binding to MDM2.

  15. The effects of benzodiazepine use during pregnancy and lactation.

    PubMed

    McElhatton, P R

    1994-01-01

    Although there are a number of studies and individual case reports concerning the use of benzodiazepines in human pregnancy, the data concerning teratogenicity and effects on postnatal development and behaviour are inconsistent. There is evidence from studies in the 1970s that first trimester exposure to benzodiazepines in utero has resulted in the birth of some infants with facial clefts, cardiac malformations, and other multiple malformations, but no syndrome of defects. Diazepam and chlordiazepoxide are amongst the drugs most frequently implicated in the earlier studies. However, data from later studies provide no clear evidence of significant increase in either the overall incidence of malformations or of any particular type of defect. Many of the women included in these studies has psychiatric illnesses, epilepsy, or diabetes all of which have an intrinsic risk in pregnancy, and some were on multidrug therapy. Medical-obstetric histories and family history of malformations were not always presented in the publications, which makes assessment of risk associated with benzodiazepine use per se difficult. Nevertheless, in most of the studies involving first trimester use of benzodiazepines, the majority of infants were normal at birth and had normal postnatal development. Late third trimester use and exposure during labour seems to be associated with much greater risks to the fetus/neonate. Some, but by no means all infants exposed at this time, exhibit either the floppy infant syndrome, or marked neonatal withdrawal symptoms. Symptoms vary from mild sedation, hypotonia, and reluctance to suck, to apnoeic spells, cyanosis, and impaired metabolic responses to cold stress. These symptoms have been reported to persist for periods from hours to months after birth. This correlates well with the pharmacokinetic and placental transfer of the benzodiazepines and their disposition in the neonate. However, there has been no significant increase in the incidence of neonatal

  16. Growth and differentiation of eosinophils from human peripheral blood CD 34+ cells.

    PubMed

    Shalit, M

    1997-01-01

    Small numbers of CD34+ primitive hematopoietic progenitors are found in normal human peripheral blood. These cells differentiate to myeloid or lymphoid lineage under the influence of growth factors. We investigated the effects of IL5 and other growth factors on the production of eosinophils from peripheral blood CD34+ cells. CD34+ cells were plated in agarose with different combinations of cytokines. At 14 days of growth a triple stain technique was used to identify eosinophil, monocyte and neutrophil colonies. IL5 alone did not support colony growth. In contrast GM-CSF and IL3 alone or together supported the generation of more than 50% eosinophil colonies. Addition of IL5 increased the fraction of eosinophil colonies to over 70%. Under the best conditions (IL3 + GM-CSF + IL5), the addition of interferon-a or LPS inhibited colony growth by 51% and 58%, respectively. Since IL5 alone did not support colony growth from CD34+ cells, we determined when IL5 responsive cells appeared in culture. Cells were grown initially with IL3 + GM-CSF, washed, and plated with IL5 alone. Only when progenitors were grown at least 3 days, could IL5 serve as the single growth factor supporting pure eosinophil colony growth (47 colonies/104 cells plated at day 3 and 134 colonies/104 cells at day 7). Growth of CD34+ in liquid culture for 28 days in the presence of IL3, GM-CSF and IL5 resulted in almost 250 fold increase in cell number, yielding a population of 83% maturing eosinophils. We used our culture system and the sensitive technique of RT-PCR to analyze the kinetics of production of mRNA transcripts encoding several eosinophil proteins. Freshly isolated CD34+ cells contained no eosinophil granule protein transcripts and barely detectable amounts of some oxidase protein transcripts. At day 3 of culture no cells recognizable by histochemical staining as eosinophils could be detected, but transcripts for all five eosinophil granule proteins were present. These transcripts increased

  17. Peripheral vasodilatation determines cardiac output in exercising humans: insight from atrial pacing.

    PubMed

    Bada, A A; Svendsen, J H; Secher, N H; Saltin, B; Mortensen, S P

    2012-04-15

    In dogs, manipulation of heart rate has no effect on the exercise-induced increase in cardiac output. Whether these findings apply to humans remain uncertain, because of the large differences in cardiovascular anatomy and regulation. To investigate the role of heart rate and peripheral vasodilatation in the regulation of cardiac output during steady-state exercise, we measured central and peripheral haemodynamics in 10 healthy male subjects, with and without atrial pacing (100–150 beats min(−1)) during: (i) resting conditions, (ii) one-legged knee extensor exercise (24 W) and (iii) femoral arterial ATP infusion at rest. Exercise and ATP infusion increased cardiac output, leg blood flow and vascular conductance (P < 0.05), whereas cerebral perfusion remained unchanged. During atrial pacing increasing heart rate by up to 54 beats min(−1), cardiac output did not change in any of the three conditions, because of a parallel decrease in stroke volume (P < 0.01). Atrial pacing increased mean arterial pressure (MAP) at rest and during ATP infusion (P < 0.05), whereas MAP remained unchanged during exercise. Atrial pacing lowered central venous pressure (P < 0.05) and pulmonary capillary wedge pressure (P < 0.05) in all conditions, whereas it did not affect pulmonary mean arterial pressure. Atrial pacing lowered the left ventricular contractility index (dP/dt) (P < 0.05) in all conditions and plasma noradrenaline levels at rest (P < 0.05), but not during exercise and ATP infusion. These results demonstrate that the elevated cardiac output during steady-state exercise is regulated by the increase in skeletal muscle blood flow and venous return to the heart, whereas the increase in heart rate appears to be secondary to the regulation of cardiac output.

  18. Molybdate modulates mitogen and cyclosporin responses of human peripheral blood lymphocytes.

    PubMed

    Michelis, Fotios V; Delitheos, Andreas; Tiligada, Ekaterini

    2011-07-01

    The trace element molybdenum (Mo) is an essential component of key physiological systems in animals, plants and microorganisms. The molybdate oxoanion MoO(4)(2-) has been demonstrated to cause diverse yet poorly understood biochemical and pharmacological effects, such as non-specific inhibition of phosphatases and stabilization of steroid receptors. This study aimed to investigate the effects of molybdate on the activation of human peripheral blood lymphocytes (hPBLs) ex vivo and its potential interaction with the widely used immunosuppressant drug cyclosporin A (CsA). Lymphocyte activation was evaluated by performing multiple experiments determining blastogenesis in cultured peripheral blood lymphocytes obtained from 5 healthy volunteers, following stimulation induced by phytohemagglutinin (PHA), in the absence or presence of 0.05-10 mM sodium molybdate or/and 2.5-30 μg/mL CsA. Blastogenesis was assessed by a morphometric assay based on the relative proportions of unactivated lymphocytes, activated lymphoblasts and cells with aberrant morphology after PHA-induced activation. Molybdate concentrations up to 1 mM showed no effect on lymphocyte blastogenesis, while higher concentrations exerted immunosuppressive actions on cultured hPBLs. Co-administration of 0.1 mM sodium molybdate with CsA, at doses up to 20 μg/mL, induced no alteration in the response of cultured hPBLs to CsA. However, molybdate potentiated the immunosuppressive action of higher CsA concentrations, implying a likely dose-related synergistic interaction of the two agents in PHA-stimulated blood lymphocytes. These observations are indicative of the possible biological importance of molybdate oxoanions in the modulation of hPBL activation that may have pharmacological consequences during the therapeutic application of immunomodulatory drugs.

  19. Stability of Radiation Induced Chromosome Damage in Human Peripheral Blood Lymphocytes

    NASA Technical Reports Server (NTRS)

    Cucinotta, F. A.; George, K.; Willingham, V.

    2006-01-01

    Chromosome damage in an individual's peripheral blood lymphocytes can be an indicator of radiation exposure and this data can be used to evaluate dose after accidental or occupational exposure. Evidence suggests that the yield of chromosome damage in lymphocytes is also a relevant biomarker of cancer risk in humans that reflects individual cancer susceptibility. It follows that biomonitoring studies can be used to uncover subjects who are particularly susceptible to radiation damage and therefore at higher risk of cancer. Translocations and other stable aberrations are commonly believed to persist in peripheral blood cells for many years after exposure, and it has been suggested that translocations can be used for assessing retrospective radiation doses or chronic exposures. However, recent investigations suggest that translocations might not always persist indefinitely. We measured chromosome aberrations in the blood lymphocytes of six astronauts before their respective missions of approximately 3 to 6 months onboard the international space station, and again at various intervals up to 5 years after flight. In samples collected a few days after return to earth, the yield of chromosome translocations had significantly increased compared with preflight values, and results indicate that biodosimetry estimates lie within the range expected from physical dosimetry. However, for five of the astronauts, follow up analysis revealed a temporal decline in translocations with half-lives ranging from 10 to 58 months. The yield of exchanges remained unchanged for the sixth astronaut during an observation period of 5 months post-flight. These results may indicate complications with the use of stable aberrations for retrospective dose reconstruction and could affect cancer risk predictions that are estimated from yields of chromosome damage obtained shortly after exposure.

  20. Diurnal Preference Predicts Phase Differences in Expression of Human Peripheral Circadian Clock Genes.

    PubMed

    Ferrante, Andrew; Gellerman, David; Ay, Ahmet; Woods, Kerri Pruitt; Filipowicz, Allan Michael; Jain, Kriti; Bearden, Neil; Ingram, Krista Kenyon

    2015-06-05

    Circadian rhythms play an integral role in human behavior, physiology and health. Individual differences in daily rhythms (chronotypes) can affect individual sleep-wake cycles, activity patterns and behavioral choices. Diurnal preference, the tendency towards morningness or eveningness among individuals, has been associated with interpersonal variation in circadian clock-related output measures, including body temperature, melatonin levels and clock gene mRNA in blood, oral mucosa, and dermal fibroblast cell cultures. Here we report gene expression data from two principal clock genes sampled from hair follicle cells, a peripheral circadian clock. Hair follicle cells from fourteen individuals of extreme morning or evening chronotype were sampled at three time points. RNA was extracted and quantitative PCR assays were used to measure mRNA expression patterns of two clock genes, Per3 and Nr1d2. We found significant differences in clock gene expression over time between chronotype groups, independent of gender or age of participants. Extreme evening chronotypes have a delay in phase of circadian clock gene oscillation relative to extreme morning types. Variation in the molecular clockwork of chronotype groups represents nearly three-hour phase differences (Per3: 2.61 hours; Nr1d2: 3.08 hours, both: 2.86) in circadian oscillations of these clock genes. The measurement of gene expression from hair follicles at three time points allows for a direct, efficient method of estimating phase shifts of a peripheral circadian clock in real-life conditions. The robust phase differences in temporal expression of clock genes associated with diurnal preferences provide the framework for further studies of the molecular mechanisms and gene-by-environment interactions underlying chronotype-specific behavioral phenomena, including social jetlag.

  1. Reversible Folding of Human Peripheral Myelin Protein 22, a Tetraspan Membrane Protein†

    PubMed Central

    Schlebach, Jonathan P.; Peng, Dungeng; Kroncke, Brett M.; Mittendorf, Kathleen F.; Narayan, Malathi; Carter, Bruce D.; Sanders, Charles R.

    2013-01-01

    Misfolding of the α-helical membrane protein peripheral myelin protein 22 (PMP22) has been implicated in the pathogenesis of the common neurodegenerative disease known as Charcot-Marie-Tooth disease (CMTD) and also several other related peripheral neuropathies. Emerging evidence suggests that the propensity of PMP22 to misfold in the cell may be due to an intrinsic lack of conformational stability. Therefore, quantitative studies of the conformational equilibrium of PMP22 are needed to gain insight into the molecular basis of CMTD. In this work, we have investigated the folding and unfolding of wild type (WT) human PMP22 in mixed micelles. Both kinetic and thermodynamic measurements demonstrate that the denaturation of PMP22 by n-lauroyl sarcosine (LS) in dodecylphosphocholine (DPC) micelles is reversible. Assessment of the conformational equilibrium indicates that a significant fraction of unfolded PMP22 persists even in the absence of the denaturing detergent. However, we find the stability of PMP22 is increased by glycerol, which facilitates quantitation of thermodynamic parameters. To our knowledge, this work represents the first report of reversible unfolding of a eukaryotic multispan membrane protein. The results indicate that WT PMP22 possesses minimal conformational stability in micelles, which parallels its poor folding efficiency in the endoplasmic reticulum. Folding equilibrium measurements for PMP22 in mixed micelles may provide an approach to assess the effects of cellular metabolites or potential therapeutic agents on its stability. Furthermore, these results pave the way for future investigation of the effects of pathogenic mutations on the conformational equilibrium of PMP22. PMID:23639031

  2. Utilization of iPSC-derived human neurons for high-throughput drug-induced peripheral neuropathy screening.

    PubMed

    Rana, Payal; Luerman, Gregory; Hess, Dietmar; Rubitski, Elizabeth; Adkins, Karissa; Somps, Christopher

    2017-08-24

    As the number of cancer survivors continues to grow, awareness of long-term toxicities and impact on quality of life after chemotherapy treatment in cancer survivors has intensified. Chemotherapy-induced peripheral neuropathy (CIPN) is one of the most common side effects of modern chemotherapy. Animal models are used to study peripheral neuropathy and predict human risk; however, such models are labor-intensive and limited translatability between species has become a major challenge. Moreover, the mechanisms underlying CIPN have not been precisely determined and few human neuronal models to study CIPN exist. Here, we have developed a high-throughput drug-induced neurotoxicity screening model using human iPSC-derived peripheral-like neurons to study the effect of chemotherapy agents on neuronal health and morphology using high content imaging measurements (neurite length and neuronal cell viability). We utilized this model to test various classes of chemotherapeutic agents with known clinical liability to cause peripheral neuropathy such as platinum agents, taxanes, vinca alkaloids, proteasome inhibitors, and anti-angiogenic compounds. The model was sensitive to compounds that cause interference in microtubule dynamics, especially the taxane, epothilone, and vinca alkaloids. Conversely, the model was not sensitive to platinum and anti-angiogenic chemotherapeutics; compounds that are not reported to act directly on neuronal processes. In summary, we believe this model has utility for high-throughput screening and prediction of human risk for CIPN for novel chemotherapeutics. Copyright © 2017 Elsevier Ltd. All rights reserved.

  3. Cerium Dioxide Nanoparticles Induce Apoptosis and Autophagy in Human Peripheral Blood Monocytes

    PubMed Central

    Hussain, Salik; Al-Nsour, Faris; Rice, Annette B.; Marshburn, Jamie; Yingling, Brenda; Ji, Zhaoxia; Zink, Jeffrey I.; Walker, Nigel J.; Garantziotis, Stavros

    2015-01-01

    Cerium dioxide nanoparticles (CeO2 NPs) have diversified industrial uses and novel therapeutic applications are actively being pursued. There is lack of mechanistic data concerning the effects of CeO2 NPs on primary human cells. We aimed at characterizing the cytotoxic effects of CeO2 NPs in human peripheral blood monocytes. CeO2 NPs and their suspensions were thoroughly characterized, including using transmission electron microscopy (TEM), dynamic light scattering (DLS), and zeta potential analysis. Blood from healthy human volunteers was drawn through phlebotomy and CD14+ cells were isolated. Cells were exposed to CeO2 NPs (0.5–10 μg/mL) for 20 or 40 hours and mechanisms of cell injury were studied. TEM revealed that CeO2 NPs are internalized by monocytes and are found either in vesicles or free in the cytoplasm. CeO2 NP exposure leads to decrease in cell viability and treated cells exhibit characteristic hallmarks of apoptosis (activation of Bax, loss of mitochondrial membrane potential, DNA fragmentation). CeO2 NP toxicity is caused by mitochondrial damage leading to apoptosis inducing factor (AIF) release, but not due to caspase activation or reactive oxygen species production. Moreover, CeO2 NP exposure leads to autophagy, which is further increased after pharmacological inhibition of tumour suppressor protein p53. Inhibition of autophagy partially reverses cell death by CeO2 NPs. It is concluded that CeO2 NPs are toxic to primary human monocytes at relatively low doses. PMID:22717232

  4. Visualization of peripheral vasodilative indices in human skin by use of red, green, blue images

    NASA Astrophysics Data System (ADS)

    Nishidate, Izumi; Tanaka, Noriyuki; Kawase, Tatsuya; Maeda, Takaaki; Yuasa, Tomonori; Aizu, Yoshihisa; Yuasa, Tetsuya; Niizeki, Kyuichi

    2013-06-01

    We propose a method to visualize the arterial inflow, the vascular resistance, and the venous capacitance in the skin tissue from red, green, blue (RGB) digital color images. The arterial inflow and the venous capacitance in the skin tissue are visualized based on an increase in the rate of change in the total blood concentration and the change of the total blood concentration during upper limb occlusion at a pressure of 50 mmHg. The resultant arterial inflow with the measured mean arterial pressure also provides an image of the vascular resistance in human skin. The arterial inflow, the vascular resistance, and the venous capacitance acquired by the method are well correlated with those obtained from the conventional strain-gauge plethysmograph. The correlation coefficients R between the estimated values by the method and the measurements by the SPG are calculated to be 0.83 (P<0.001) for the arterial inflow, 0.77 (P<0.01) for the vascular resistance, and 0.77 (P<0.01) for the venous capacitance. The arterial inflow and the venous capacitance in the skin tissue are significantly higher in active subjects compared with the sedentary subjects, whereas the vascular resistance was significantly lower in the active subjects compared with the sedentary subjects. The results of the present study indicate the possibility of using the proposed method for evaluating the peripheral vascular functions in human skin.

  5. Protective effect of quercetin against oxidative stress caused by dimethoate in human peripheral blood lymphocytes

    PubMed Central

    2011-01-01

    Background The aim of this study is to investigate the effect of quercetin in alleviating the cytotoxic effects of Dimethoate in human peripheral blood lymphocytes. Methods Lymphocytes were divided into too groups. The first group, lymphocytes were incubated for 4 h at 37°C with different concentrations (0, 40, 60 and 100 mM) of Dimethoate. The second group was preincubated with quercetin for 30 min and followed by Dim incubation for 4 h at 37°C. Results Following in vitro incubation, Dimethoate caused a significant increase in malondialdehyde levels, a significant decrease in thiol levels, as well as a significant increase in superoxide dismutase, and catalase activities in lymphocytes at different concentrations. Quercetin pretreated lymphocytes showed a significant protection against the cytotoxic effects inducted by Dimethoate on the studied parameters. Conclusion In conclusion, antioxidant quercetin could protect against Dimethoate-induced oxidative stress by decreasing lipid peroxidation, protein oxidation and increasing superoxide dismutase and catalase activities in human lymphocytes. PMID:21861917

  6. Sulforaphane mitigates cadmium-induced toxicity pattern in human peripheral blood lymphocytes and monocytes.

    PubMed

    Alkharashi, Nouf Abdulkareem Omer; Periasamy, Vaiyapuri Subbarayan; Athinarayanan, Jegan; Alshatwi, Ali A

    2017-10-01

    Cadmium (Cd) is a highly toxic and widely distributed heavy metal that induces various diseases in humans through environmental exposure. Therefore, alleviation of Cd-induced toxicity in living organisms is necessary. In this study, we investigated the protective role of sulforaphane on Cd-induced toxicity in human peripheral blood lymphocytes and monocytes. Sulforaphane did not show any major reduction in the viability of lymphocytes and monocytes. However, Cd treatment at a concentration of 50μM induced around 69% cell death. Treatment of IC10-Cd and 100μM sulforaphane combination for 24 and 48h increased viability by 2 and 9% in cells subjected to Cd toxicity, respectively. In addition, IC25 of Cd and 100μM sulforaphane combination recovered 17-20% of cell viability. Cd induced apoptotic and necrotic cell death. Sulforaphane treatment reduced Cd-induced cell death in lymphocytes and monocytes. Our results clearly indicate that when the cells were treated with Cd+sulforaphane combination, sulforaphane decreased the Cd-induced cytotoxic effect in lymphocytes and monocytes. In addition, sulforaphane concentration plays a major role in the alleviation of Cd-induced toxicity. Copyright © 2017 Elsevier B.V. All rights reserved.

  7. Hericium erinaceum induces maturation of dendritic cells derived from human peripheral blood monocytes.

    PubMed

    Kim, Sun Kyung; Son, Chang Gue; Yun, Cheol-Heui; Han, Seung Hyun

    2010-01-01

    Hericium erinaceum, a medicinal mushroom, has long been used as a therapeutic due to its immuno-regulating potentials eliciting anticarcinogenic and antimicrobial efficacies. Since maturation of dendritic cells (DC) is an important process in the initiation and regulation of immune responses, the ability of water-soluble components from H. erinaceum (WEHE) to regulate DC maturation was investigated. Immature DC were prepared by differentiating human peripheral blood CD14-positive cells with GM-CSF and IL-4. DC were stimulated with WEHE at 2-20 microg/mL for 48 h and subjected to flow cytometric analysis to determine the expression of indicative maturation markers. The endocytic capacity of WEHE-stimulated DC was examined by a Dextran-FITC uptake assay. An enzyme-linked immunosorbent assay was performed to examine the secretion of TNF-alpha and IL-12p40. DC stimulated with WEHE showed representative features upon DC maturation: enhanced expression of CD80, CD83 and CD86, and both MHC class I and II molecules, decreased endocytic capacity of DC, increased expression of CD205, and decreased expression of CD206. However, interestingly, WEHE could not induce the production of TNF-alpha and IL-12p40, whereas lipopolysaccharide substantially increased the production of both cytokines. Collectively, these results suggest that H. erinaceum induces the maturation of human DC, which might reinforce the host innate immune system.

  8. Long-term immunologically competent human peripheral lymphoid tissue cultures in a 3D bioreactor

    PubMed Central

    Kuzin, Igor; Sun, Hongliang; Moshkani, Safiekhatoon; Feng, Changyong; Mantalaris, Athanasios; Wu, JH David; Bottaro, Andrea

    2011-01-01

    Peripheral lymphoid organs (PLOs), the primary sites of development of adaptive immune responses, display a complex structural organization reflecting separation of cellular subsets (e.g. T and B lymphocytes) and functional compartments which is critical for immune function. The generation of in vitro culture systems capable of recapitulating salient features of PLOs for experimental, biotechnological and clinical applications would be highly desirable, but has been hampered so far by the complexity of these systems. We have previously developed a three-dimensional bioreactor system for long-term, functional culture of human bone marrow cells on macroporous microspheres in a packed-bed bioreactor with frequent medium change. Here we adapt the same system for culture of human primary cells from PLOs (tonsil) in the absence of specific exogenous growth factors or activators. Cells in this system displayed higher viability over several weeks, and maintain population diversity and cell surface markers largely comparable to primary cells. Light microscopy showed cells organizing in large diverse clusters within the scaffold pores and presence of B cell-enriched areas. Strikingly, these cultures generated a significant number of antibody-producing B cells when challenged with a panel of diverse antigens, as expected from a lymphoid tissue. Thus the three-dimensional tonsil bioreactor culture system may serve as a useful model of PLOs by recapitulating their structural organization and function ex vivo. PMID:21309085

  9. Widespread Decreased Expression of Immune Function Genes in Human Peripheral Blood Following Radiation Exposure

    PubMed Central

    Paul, Sunirmal; Smilenov, Lubomir B.; Amundson, Sally A.

    2014-01-01

    We report a large-scale reduced expression of genes in pathways related to cell-type specific immunity functions that emerges from microarray analysis 48 h after ex vivo γ-ray irradiation (0, 0.5, 2, 5, 8 Gy) of human peripheral blood from five donors. This response is similar to that seen in patients at 24 h after the start of total-body irradiation and strengthens the rationale for the ex vivo model as an adjunct to human in vivo studies. The most marked response was in genes associated with natural killer (NK) cell immune functions, reflecting a relative loss of NK cells from the population. T- and B-cell mediated immunity genes were also significantly represented in the radiation response. Combined with our previous studies, a single gene expression signature was able to predict radiation dose range with 97% accuracy at times from 6–48 h after exposure. Gene expression signatures that may report on the loss or functional deactivation of blood cell subpopulations after radiation exposure may be particularly useful both for triage biodosimetry and for monitoring the effect of radiation mitigating treatments. PMID:24168352

  10. In vitro transdifferentiation of human peripheral blood mononuclear cells to photoreceptor-like cells.

    PubMed

    Komuta, Yukari; Ishii, Toshiyuki; Kaneda, Makoto; Ueda, Yasuji; Miyamoto, Kiyoko; Toyoda, Masashi; Umezawa, Akihiro; Seko, Yuko

    2016-06-15

    Direct reprogramming is a promising, simple and low-cost approach to generate target cells from somatic cells without using induced pluripotent stem cells. Recently, peripheral blood mononuclear cells (PBMCs) have attracted considerable attention as a somatic cell source for reprogramming. As a cell source, PBMCs have an advantage over dermal fibroblasts with respect to the ease of collecting tissues. Based on our studies involving generation of photosensitive photoreceptor cells from human iris cells and human dermal fibroblasts by transduction of photoreceptor-related transcription factors via retrovirus vectors, we transduced these transcription factors into PBMCs via Sendai virus vectors. We found that retinal disease-related genes were efficiently detected in CRX-transduced cells, most of which are crucial to photoreceptor functions. In functional studies, a light-induced inward current was detected in some CRX-transduced cells. Moreover, by modification of the culture conditions including additional transduction of RAX1 and NEUROD1, we found a greater variety of retinal disease-related genes than that observed in CRX-transduced PBMCs. These data suggest that CRX acts as a master control gene for reprogramming PBMCs into photoreceptor-like cells and that our induced photoreceptor-like cells might contribute to individualized drug screening and disease modeling of inherited retinal degeneration.

  11. Adaptive response to low dose of EMS or MMS in human peripheral blood lymphocytes.

    PubMed

    Harish, S K; Guruprasad, K P; Mahmood, R; Vasudev, V; Manjunath, K R; Chethan, G K

    1998-11-01

    Human peripheral blood lymphocytes stimulated in vitro for 6 hr were exposed to a low (conditioning) dose of ethyl methanesulfonate (EMS; 1.5 x 10(-4) M) or methyl methanesulfonate (MMS; 1.5 x 10(-5) M). After 6 hr, the cells were treated with a high (challenging) concentration of the same agent (1.5 x 10(-3) M EMS or 1.5 x 10(-4) M MMS). The cells that received both conditioning and challenging doses became less sensitive to the induction of sister chromatid exchanges (SCEs) than those which did not receive the pretreatment with EMS or MMS. They responded with lower frequencies of SCEs. This suggests that conditioning dose of EMS or MMS has offered the lymphocytes to have decreased SCEs. This led to the realization that pre-exposure of lymphocytes to low dose can cause the induction of repair activity. This is a clear indication of the existence of adaptive response induced by alkylating agents whether it is ethylating or methylating in human lymphocytes in vitro.

  12. Uric acid increases cellular and humoral alloimmunity in primary human peripheral blood mononuclear cells.

    PubMed

    Eleftheriadis, Theodoros; Pissas, Georgios; Sounidaki, Maria; Antoniadi, Georgia; Antoniadis, Nikolaos; Liakopoulos, Vassilios; Stefanidis, Ioannis

    2017-05-05

    Hyperuricemia is common among kidney transplant recipients and has been associated with worse graft outcome. Since episodes of acute cellular rejection and chronic humoral rejection contribute to decreased graft survival, in this study the effect of uric acid on cellular and humoral alloimmunity was evaluated. Cellular alloimmunity was assessed by cell proliferation in two-way mixed lymphocyte reaction (MLR) with human peripheral blood mononuclear cells (PBMC). For assessing humoral alloimmunity we developed a method in which humoral alloimmunity was induced in one-way MLR. Then the de novo production of alloantibodies was measured with an antibody-mediated complement-dependent cytotoxicity assay, in which supernatants from the above MRLs were used against resting PBMC similar to the stimulator cells of the above MLRs. Uric acid at a concentration above its crystallization threshold increased cellular proliferation in two-way MLRs. Supernatants from one-way MLRs performed in the presence of uric acid were more cytotoxic against PBMC from individuals that had conferred the stimulator cells for the above MLRs. Uric acid increases both cellular and humoral alloimmunity in human PBMC. These results offer a possible pathogenetic mechanism for the observed relation between hyperuricemia and worse kidney allograft survival. This article is protected by copyright. All rights reserved.

  13. CD1d-restricted peripheral T cell lymphoma in mice and humans

    PubMed Central

    Bachy, Emmanuel; Urb, Mirjam; Chandra, Shilpi; Robinot, Rémy; Bricard, Gabriel; de Bernard, Simon; Traverse-Glehen, Alexandra; Gazzo, Sophie; Blond, Olivier; Khurana, Archana; Baseggio, Lucile; Heavican, Tayla; Ffrench, Martine; Crispatzu, Giuliano; Mondière, Paul; Schrader, Alexandra; Taillardet, Morgan; Thaunat, Olivier; Martin, Nadine; Dalle, Stéphane; Le Garff-Tavernier, Magali; Salles, Gilles; Lachuer, Joel; Hermine, Olivier; Asnafi, Vahid; Roussel, Mikael; Lamy, Thierry; Herling, Marco; Iqbal, Javeed; Buffat, Laurent; Marche, Patrice N.; Gaulard, Philippe; Kronenberg, Mitchell; Defrance, Thierry

    2016-01-01

    Peripheral T cell lymphomas (PTCLs) are a heterogeneous entity of neoplasms with poor prognosis, lack of effective therapies, and a largely unknown pathophysiology. Identifying the mechanism of lymphomagenesis and cell-of-origin from which PTCLs arise is crucial for the development of efficient treatment strategies. In addition to the well-described thymic lymphomas, we found that p53-deficient mice also developed mature PTCLs that did not originate from conventional T cells but from CD1d-restricted NKT cells. PTCLs showed phenotypic features of activated NKT cells, such as PD-1 up-regulation and loss of NK1.1 expression. Injections of heat-killed Streptococcus pneumonia, known to express glycolipid antigens activating NKT cells, increased the incidence of these PTCLs, whereas Escherichia coli injection did not. Gene expression profile analyses indicated a significant down-regulation of genes in the TCR signaling pathway in PTCL, a common feature of chronically activated T cells. Targeting TCR signaling pathway in lymphoma cells, either with cyclosporine A or anti-CD1d blocking antibody, prolonged mice survival. Importantly, we identified human CD1d-restricted lymphoma cells within Vδ1 TCR-expressing PTCL. These results define a new subtype of PTCL and pave the way for the development of blocking anti-CD1d antibody for therapeutic purposes in humans. PMID:27069116

  14. Perceptions of Benzodiazepine Dependence Among Women Age 65 and Older

    PubMed Central

    Canham, Sarah L.; Gallo, Joseph; Simoni-Wastila, Linda

    2014-01-01

    A phenomenological study explored whether older women who are chronic benzodiazepine users identified themselves as dependent, how dependence was perceived, and how meanings and understandings shaped experiences of benzodiazepine use. Self-reported benzodiazepine dependence was associated with being unable to reduce use or a desire to discontinue use and reliance on benzodiazepines to remain comfortable and able to handle daily life. Themes included: 1) benzodiazepine dependence is similar to dependence to diabetes or blood pressure medications; 2) dependence is distinctive from addiction/abuse; 3) addiction/abuse is perceived as worse than dependence; and 4) concerns of addiction/abuse result in low-dose benzodiazepine use. PMID:24918963

  15. Alcohol Withdrawal Syndrome: Benzodiazepines and Beyond

    PubMed Central

    Sachdeva, Ankur; Chandra, Mina

    2015-01-01

    Alcohol dependence is an increasing and pervasive problem. Alcohol withdrawal symptoms are a part of alcohol dependence syndrome and are commonly encountered in general hospital settings, in most of the departments. Alcohol withdrawal syndrome ranges from mild to severe. The severe complicated alcohol withdrawal may present with hallucinations, seizures or delirium tremens. Benzodiazepines have the largest and the best evidence base in the treatment of alcohol withdrawal, and are considered the gold standard. Others, such as anticonvulsants, barbiturates, adrenergic drugs, and GABA agonists have been tried and have evidence. Supportive care and use of vitamins is essential in the management. Symptom triggered regime is favoured over fixed tapering dose regime, although monitoring through scales is cumbersome. This article aims to review the evidence base for appropriate clinical management of the alcohol withdrawal syndrome. We searched Pubmed for articles published in English on ‘Alcohol withdrawal syndrome’ in humans during the last 10 years. A total of 1182 articles came up. Articles not relevant to clinical utility and management were excluded based on the titles and abstract available. Full text articles, meta-analyses, systematic reviews and randomized controlled trials were obtained from this list and were considered for review. PMID:26500991

  16. Docking and molecular dynamics studies of peripheral site ligand-oximes as reactivators of sarin-inhibited human acetylcholinesterase.

    PubMed

    de Almeida, Joyce S F D; Cuya Guizado, Teobaldo R; Guimarães, Ana P; Ramalho, Teodorico C; Gonçalves, Arlan S; de Koning, Martijn C; França, Tanos C C

    2016-12-01

    In the present work, we performed docking and molecular dynamics simulations studies on two groups of long-tailored oximes designed as peripheral site binders of acetylcholinesterase (AChE) and potential penetrators on the blood brain barrier. Our studies permitted to determine how the tails anchor in the peripheral site of sarin-inhibited human AChE, and which aminoacids are important to their stabilization. Also the energy values obtained in the docking studies corroborated quite well with the experimental results obtained before for these oximes.

  17. In vitro expansion of Lin+ and Lin- mononuclear cells from human peripheral blood

    NASA Astrophysics Data System (ADS)

    Norhaiza, H. Siti; Rohaya, M. A. W.; Zarina, Z. A. Intan; Hisham, Z. A. Shahrul

    2013-11-01

    Haematopoietic stem cells (HSCs) are used in the therapy of blood disorders due to the ability of these cells to reconstitute haematopoietic lineage cells when transplanted into myeloablative recipients. However, substantial number of cells is required in order for the reconstitution to take place. Since HSCs present in low frequency, larger number of donor is required to accommodate the demand of transplantable HSCs. Therefore, in vitro expansion of HSCs will have profound impact on clinical purposes. The aim of this study was to expand lineage negative (Lin-) stem cells from human peripheral blood. Total peripheral blood mononuclear cells (PBMNCs) were fractionated from human blood by density gradient centrifugation. Subsequently, PBMNCs were subjected to magnetic assisted cell sorter (MACS) which depletes lineage positive (Lin+) mononuclear cells expressing lineage positive markers such as CD2, CD3, CD11b, CD14, CD15, CD16, CD19, CD56, CD123, and CD235a to obtained Lin- cell population. The ability of Lin+ and Lin- to survive in vitro was explored by culturing both cell populations in complete medium consisting of Alpha-Minimal Essential Medium (AMEM) +10% (v/v) Newborn Calf Serum (NBCS)+ 2% (v/v) pen/strep. In another experiment, Lin+ and Lin- were cultured with complete medium supplemented with 10ng/mL of the following growth factors: stem cell factor (SCF), interleukin (IL)-3, granulocyte-macrophage colony stimulating factor (GM-CSF), 2IU/mL of Erythropoietin (Epo) and 20ng/mL of IL-6. Three samples were monitored in static culture for 22 days. The expansion potential was assessed by the number of total viable cells, counted by trypan blue exclusion assay. It was found that Lin+ mononuclear cells were not able to survive either in normal proliferation medium or proliferation medium supplemented with cytokines. Similarly, Lin- stem cells were not able to survive in proliferation medium however, addition of cytokines into the proliferation medium support Lin

  18. HCG-Activated Human Peripheral Blood Mononuclear Cells (PBMC) Promote Trophoblast Cell Invasion.

    PubMed

    Yu, Nan; Yan, Wenjie; Yin, Tailang; Wang, Yaqin; Guo, Yue; Zhou, Danni; Xu, Mei; Ding, Jinli; Yang, Jing

    2015-01-01

    Successful embryo implantation and placentation depend on appropriate trophoblast invasion into the maternal endometrial stroma. Human chorionic gonadotropin (hCG) is one of the earliest embryo-derived secreted signals in the peripheral blood mononuclear cells (PBMC) that abundantly expresses hCG receptors. The aims of this study were to estimate the effect of human embryo-secreted hCG on PBMC function and investigate the role and underlying mechanisms of activated PBMC in trophoblast invasion. Blood samples were collected from women undergoing benign gynecological surgery during the mid-secretory phase. PBMC were isolated and stimulated with or without hCG for 0 or 24 h. Interleukin-1β (IL-1β) and leukemia inhibitory factor (LIF) expressions in PBMC were detected by enzyme-linked immunosorbent assay and real-time polymerase chain reaction (PCR). The JAR cell line served as a model for trophoblast cells and was divided into four groups: control, hCG only, PBMC only, and PBMC with hCG. JAR cell invasive and proliferative abilities were detected by trans-well and CCK8 assays and matrix metalloproteinase (MMP)-2 (MMP-2), MMP-9, vascular endothelial growth factor (VEGF), tissue inhibitor of metalloproteinase (TIMP)-1, and TIMP-2 expressions in JAR cells were detected by western blotting and real-time PCR analysis. We found that hCG can remarkably promote IL-1β and LIF promotion in PBMC after 24-h culture. PBMC activated by hCG significantly increased the number of invasive JAR cells in an invasion assay without affecting proliferation, and hCG-activated PBMC significantly increased MMP-2, MMP-9, and VEGF and decreased TIMP-1 and TIMP-2 expressions in JAR cells in a dose-dependent manner. This study demonstrated that hCG stimulates cytokine secretion in human PBMC and could stimulate trophoblast invasion.

  19. HCG-Activated Human Peripheral Blood Mononuclear Cells (PBMC) Promote Trophoblast Cell Invasion

    PubMed Central

    Wang, Yaqin; Guo, Yue; Zhou, Danni; Xu, Mei; Ding, Jinli; Yang, Jing

    2015-01-01

    Successful embryo implantation and placentation depend on appropriate trophoblast invasion into the maternal endometrial stroma. Human chorionic gonadotropin (hCG) is one of the earliest embryo-derived secreted signals in the peripheral blood mononuclear cells (PBMC) that abundantly expresses hCG receptors. The aims of this study were to estimate the effect of human embryo–secreted hCG on PBMC function and investigate the role and underlying mechanisms of activated PBMC in trophoblast invasion. Blood samples were collected from women undergoing benign gynecological surgery during the mid-secretory phase. PBMC were isolated and stimulated with or without hCG for 0 or 24 h. Interleukin-1β (IL-1β) and leukemia inhibitory factor (LIF) expressions in PBMC were detected by enzyme-linked immunosorbent assay and real-time polymerase chain reaction (PCR). The JAR cell line served as a model for trophoblast cells and was divided into four groups: control, hCG only, PBMC only, and PBMC with hCG. JAR cell invasive and proliferative abilities were detected by trans-well and CCK8 assays and matrix metalloproteinase (MMP)-2 (MMP-2), MMP-9, vascular endothelial growth factor (VEGF), tissue inhibitor of metalloproteinase (TIMP)-1, and TIMP-2 expressions in JAR cells were detected by western blotting and real-time PCR analysis. We found that hCG can remarkably promote IL-1β and LIF promotion in PBMC after 24-h culture. PBMC activated by hCG significantly increased the number of invasive JAR cells in an invasion assay without affecting proliferation, and hCG-activated PBMC significantly increased MMP-2, MMP-9, and VEGF and decreased TIMP-1 and TIMP-2 expressions in JAR cells in a dose-dependent manner. This study demonstrated that hCG stimulates cytokine secretion in human PBMC and could stimulate trophoblast invasion. PMID:26087261

  20. IL-4 induces neutrophilic maturation of HL-60 cells and activation of human peripheral blood neutrophils.

    PubMed Central

    Bober, L A; Waters, T A; Pugliese-Sivo, C C; Sullivan, L M; Narula, S K; Grace, M J

    1995-01-01

    IL-4 is a T-helper cell derived cytokine that has effects on myelomonocytic cell maturation and activation. We have studied the effect of IL-4 on neutrophilic maturation using the cell line HL-60 and found that it has a profound effect on the maturation and activation of the cell line. The treatment of HL-60 cells with recombinant hu IL-4 (0.15 to 15.0 ng/ml) induced a shift in the percentage of HL-60 cells staining positive for chloroacetate esterase enzyme activity (indicating commitment to the neutrophilic lineage). IL-4 increased surface expression of the neutrophil-lineage antigen WEM G11, the complement receptors CR3 (CD11b) and CR1 (CD35), but not for the monocyte differentiation antigen CD14. IL-4 treated HL-60 cells demonstrated enhanced Fc- and complement-mediated phagocytic capacity and increased hexose-monophosphate shunt activity. In addition, IL-4 was capable of sustaining the neutrophil maturation of HL-60 cells that had been pre-treated for 24 h with DMSO. To investigate the effect of IL-4 on the mature neutrophil, we studied freshly isolated and rested human peripheral blood neutrophils. In the absence of other stimuli, neutrophils were induced by IL-4 to have significantly elevated phagocytic responses. The response was specific since treatment with anti-human IL-4 abolished phagocytic stimulation. Finally, IL-4 treatment also stimulated resting neutrophils to migrate toward zymosan-activated serum (ZAS) and human IL-5. The results demonstrate that IL-4 is a potent maturation factor for myelocytes to become neutrophils and that IL-4 can stimulate resting mature neutrophils. PMID:7529148

  1. Persistent alterations of gene expression profiling of human peripheral blood mononuclear cells from smokers.

    PubMed

    Weng, Daniel Y; Chen, Jinguo; Taslim, Cenny; Hsu, Ping-Ching; Marian, Catalin; David, Sean P; Loffredo, Christopher A; Shields, Peter G

    2016-10-01

    The number of validated biomarkers of tobacco smoke exposure is limited, and none exist for tobacco-related cancer. Additional biomarkers for smoke, effects on cellular systems in vivo are needed to improve early detection of lung cancer, and to assist the Food and Drug Administration in regulating exposures to tobacco products. We assessed the effects of smoking on the gene expression using human cell cultures and blood from a cross-sectional study. We profiled global transcriptional changes in cultured smokers' peripheral blood mononuclear cells (PBMCs) treated with cigarette smoke condensate (CSC) in vitro (n = 7) and from well-characterized smokers' blood (n = 36). ANOVA with adjustment for covariates and Pearson correlation were used for statistical analysis in this study. CSC in vitro altered the expression of 1 178 genes (177 genes with > 1.5-fold-change) at P < 0.05. In vivo, PBMCs of heavy and light smokers differed for 614 genes (29 with > 1.5-fold-change) at P < 0.05 (309 remaining significant after adjustment for age, race, and gender). Forty-one genes were persistently altered both in vitro and in vivo, 22 having the same expression pattern reported for non-small cell lung cancer. Our data provides evidence that persistent alterations of gene expression in vitro and in vivo may relate to carcinogenic effects of cigarette smoke, and the identified genes may serve as potential biomarkers for cancer. The use of an in vitro model to corroborate results from human studies provides a novel way to understand human exposure and effect. © 2015 Wiley Periodicals, Inc.

  2. Persistent Alterations of Gene Expression Profiling of Human Peripheral Blood Mononuclear Cells From Smokers

    PubMed Central

    Weng, Daniel Y.; Chen, Jinguo; Taslim, Cenny; Hsu, Ping-Ching; Marian, Catalin; David, Sean P.; Loffredo, Christopher A.; Shields, Peter G.

    2016-01-01

    The number of validated biomarkers of tobacco smoke exposure is limited, and none exist for tobacco-related cancer. Additional biomarkers for smoke, effects on cellular systems in vivo are needed to improve early detection of lung cancer, and to assist the Food and Drug Administration in regulating exposures to tobacco products. We assessed the effects of smoking on the gene expression using human cell cultures and blood from a cross-sectional study. We profiled global transcriptional changes in cultured smokers’ peripheral blood mononuclear cells (PBMCs) treated with cigarette smoke condensate (CSC) in vitro (n = 7) and from well-characterized smokers’ blood (n = 36). ANOVA with adjustment for covariates and Pearson correlation were used for statistical analysis in this study. CSC in vitro altered the expression of 1 178 genes (177 genes with > 1.5-fold-change) at P < 0.05. In vivo, PBMCs of heavy and light smokers differed for 614 genes (29 with > 1.5-fold-change) at P < 0.05 (309 remaining significant after adjustment for age, race, and gender). Forty-one genes were persistently altered both in vitro and in vivo, 22 having the same expression pattern reported for non-small cell lung cancer. Our data provides evidence that persistent alterations of gene expression in vitro and in vivo may relate to carcinogenic effects of cigarette smoke, and the identified genes may serve as potential biomarkers for cancer. The use of an in vitro model to corroborate results from human studies provides a novel way to understand human exposure and effect. PMID:26294040

  3. Alteration of peripheral blood monocyte gene expression in humans following diesel exhaust inhalation.

    PubMed

    Pettit, Ashley P; Brooks, Andrew; Laumbach, Robert; Fiedler, Nancy; Wang, Qi; Strickland, Pamela Ohman; Madura, Kiran; Zhang, Junfeng; Kipen, Howard M

    2012-02-01

    Epidemiologic associations between acutely increased cardiorespiratory morbidity and mortality and particulate air pollution are well established, but the effects of acute pollution exposure on human gene expression changes are not well understood. In order to identify potential mechanisms underlying epidemiologic associations between air pollution and morbidity, we explored changes in gene expression in humans following inhalation of fresh diesel exhaust (DE), a model for particulate air pollution. Fourteen ethnically homogeneous (white males), young, healthy subjects underwent 60-min inhalation exposures on 2 separate days with clean filtered air (CA) or freshly generated and diluted DE at a concentration of 300 μg/m(3) PM(2.5). Prior to and 24 h following each session, whole blood was sampled and fractionated for peripheral blood mononuclear cell (PBMC) isolation, RNA extraction, and generation of cDNA, followed by hybridization with Agilent Whole Human Genome (4X44K) arrays. Oxidative stress and the ubiquitin proteasome pathway, as well as the coagulation system, were among hypothesized pathways identified by analysis of differentially expressed genes. Nine genes from these pathways were validated using real-time polymerase chain reaction (PCR) to compare fold change in expression between DE exposed and CA days. Quantitative gene fold changes generated by real-time PCR were directionally consistent with the fold changes from the microarray analysis. Changes in gene expression connected with key oxidative stress, protein degradation, and coagulation pathways are likely to underlie observed physiologic and clinical outcomes and suggest specific avenues and sensitive time points for further physiologic exploration.

  4. Alteration of peripheral blood monocyte gene expression in humans following diesel exhaust inhalation

    PubMed Central

    Pettit, Ashley P.; Brooks, Andrew; Laumbach, Robert; Fiedler, Nancy; Wang, Qi; Strickland, Pamela Ohman; Madura, Kiran; Zhang, Junfeng; Kipen, Howard M.

    2013-01-01

    Context Epidemiologic associations between acutely increased cardiorespiratory morbidity and mortality and particulate air pollution are well established, but the effects of acute pollution exposure on human gene expression changes are not well understood. Objective In order to identify potential mechanisms underlying epidemiologic associations between air pollution and morbidity, we explored changes in gene expression in humans following inhalation of fresh diesel exhaust (DE), a model for particulate air pollution. Materials and methods Fourteen ethnically homogeneous (white males), young, healthy subjects underwent 60-min inhalation exposures on 2 separate days with clean filtered air (CA) or freshly generated and diluted DE at a concentration of 300 μg/m3 PM2.5. Prior to and 24 h following each session, whole blood was sampled and fractionated for peripheral blood mononuclear cell (PBMC) isolation, RNA extraction, and generation of cDNA, followed by hybridization with Agilent Whole Human Genome (4X44K) arrays. Results Oxidative stress and the ubiquitin proteasome pathway, as well as the coagulation system, were among hypothesized pathways identified by analysis of differentially expressed genes. Nine genes from these pathways were validated using real-time polymerase chain reaction (PCR) to compare fold change in expression between DE exposed and CA days. Quantitative gene fold changes generated by real-time PCR were directionally consistent with the fold changes from the microarray analysis. Discussion and conclusion Changes in gene expression connected with key oxidative stress, protein degradation, and coagulation pathways are likely to underlie observed physiologic and clinical outcomes and suggest specific avenues and sensitive time points for further physiologic exploration. PMID:22369193

  5. Modulation of the Host Environment by Human Cytomegalovirus with Viral Interleukin 10 in Peripheral Blood.

    PubMed

    Young, Vivian P; Mariano, Margarette C; Tu, Carolyn C; Allaire, Kathryn M; Avdic, Selmir; Slobedman, Barry; Spencer, Juliet V

    2017-03-15

    Human cytomegalovirus (HCMV) is a herpesvirus with both lytic and latent life cycles. Human cytomegalovirus encodes 2 viral cytokines that are orthologs of human cellular interleukin 10 (cIL-10). Both cytomegalovirus interleukin 10 (cmvIL-10) and Latency-associated cytomegalovirus interleukin 10 (LAcmvIL-10) (collectively vIL-10) are expressed during lytic infection and cause immunosuppressive effects that impede virus clearance. LAcmvIL-10 is also expressed during latent infection of myeloid progenitor cells and monocytes and facilitates persistence. Here, we investigated whether vIL-10 could be detected during natural infection. Plasma from healthy blood donors was tested by enzyme-linked immunosorbent assay for anti-HCMV immunoglobulin G and immunoglobulin M and for cIL-10 and vIL-10 levels using a novel vIL-10 assay that detects cmvIL-10 and LAcmvIL-10, with no cross-reactivity to cIL-10. vIL-10 was evident in HCMV+ donors (n = 19 of 26), at levels ranging 31-547 pg/mL. By comparison, cIL-10 was detected at lower levels ranging 3-69 pg/mL. There was a strong correlation between vIL-10 and cIL-10 levels (P = .01). Antibodies against vIL-10 were also detected and neutralized vIL-10 activity. vIL-10 was detected in peripheral blood of healthy blood donors. These findings suggest that vIL-10 may play a key role in sensing or modifying the host environment during latency and, therefore, may be a potential target for intervention strategies.

  6. Disability pension as predictor of later use of benzodiazepines among benzodiazepine users.

    PubMed

    Hartz, Ingeborg; Tverdal, Aage; Skille, Eivind; Skurtveit, Svetlana

    2010-03-01

    The proportion of Norwegians on disability pensions has doubled since the 1980s. The Norwegian Government wants action to stimulate the working capacity in those disability pensioners who have the potential to work. Information on factors that may impair rehabilitation efforts, including the unfavourable use of benzodiazepines, may be useful in this context. A longitudinal design, including data on 40-42 year old participants in Norwegian health surveys (year 1985-1989) linked to a prescription database (year 2004-2006), was used to describe risk of long-term use of benzodiazepines among disability pension recipients. The study population constituted benzodiazepine users at baseline. More than half of those on disability pensions, 57% of all men and 65% of all women, retrieved benzodiazepine prescriptions 20 years later, a span covering a large part of the potential active workforce period. Further, the observed amount of benzodiazepines dispensed over a three-year period indicated more than sporadic use e.g. half of the female disability pensioners were dispensed an amount of benzodiazepines corresponding to the use of a daily dose every second day over a three year period (median 450 daily doses). The majority of those who were dispensed benzodiazepines, were dispensed opioids as well: half of all men and 3 out of four women. And last, being on a disability pension was a predictor of benzodiazepine use 20 years later. Our study suggests that benzodiazepines are extensively and unfavourably used among disability pensioners, and that disability pension may have an independent effect on long-term use. Improved management of benzodiazepine use may be one alternative to get disability pensioners with the potential to work back into employment.

  7. Use of benzodiazepines and detoxification with methadone.

    PubMed

    Popescu, G; Negrei, C; Bălălău, D; Ciobanu, A M; Baconi, D; Bălălău, C

    2014-01-01

    Benzodiazepines are used as anti anxiety drugs, as well as in adjunct treatment for a range of neurological and psychiatric disorders. Abusive patterns of use were increasingly reported and building evidence points to prevalence of benzodiazepines abuse, on one hand as well as to their common abuse in combination with other drugs such as opioids, most frequently. The main objective of this research is to conduct a systematic study on the behavior profile of a patient admitted to a prison hospital, who is a benzodiazepines user consecutive to admission into a methadone administration program. Statistic values have been taken into account describing the distribution and the distribution form of the various variables studied to find the normality degree of distributions, regarding three measurements at the three moments: before the administration of methadone, immediately after its completion and two months after completion. The statistic results obtained speak of a strong positive correlation, allowing the support of the fact that persons diagnosed with prescribed/ unprescribed benzodiazepine, use the display association with the admission into a methadone administration program, based on the assumption which concerns a significant positive association between the use of reported benzodiazepine and the administration of methadone in the questioned patients on admission. As far as the second premise regarding the administration of methadone is concerned it brings about an improvement in the level of benzodiazepines used in research patients, which one may assert that, according to the results obtained, the initiation of methadone therapy in the detoxification program is conducive to the reduction of benzodiazepines use.

  8. Use of benzodiazepines and detoxification with methadone

    PubMed Central

    Popescu, G; Negrei, C; Bălălău, D; Ciobanu, AM; Baconi, D; Bălălău, C

    2014-01-01

    Rationale: Benzodiazepines are used as anti anxiety drugs, as well as in adjunct treatment for a range of neurological and psychiatric disorders. Abusive patterns of use were increasingly reported and building evidence points to prevalence of benzodiazepines abuse, on one hand as well as to their common abuse in combination with other drugs such as opioids, most frequently. Objective: The main objective of this research is to conduct a systematic study on the behavior profile of a patient admitted to a prison hospital, who is a benzodiazepines user consecutive to admission into a methadone administration program. Methods and results: Statistic values have been taken into account describing the distribution and the distribution form of the various variables studied to find the normality degree of distributions, regarding three measurements at the three moments: before the administration of methadone, immediately after its completion and two months after completion. Conclusions and discussions: The statistic results obtained speak of a strong positive correlation, allowing the support of the fact that persons diagnosed with prescribed/ unprescribed benzodiazepine, use the display association with the admission into a methadone administration program, based on the assumption which concerns a significant positive association between the use of reported benzodiazepine and the administration of methadone in the questioned patients on admission. As far as the second premise regarding the administration of methadone is concerned it brings about an improvement in the level of benzodiazepines used in research patients, which one may assert that, according to the results obtained, the initiation of methadone therapy in the detoxification program is conducive to the reduction of benzodiazepines use. PMID:25870711

  9. In Vivo Confocal Microscopy of the Human Cornea in the Assessment of Peripheral Neuropathy and Systemic Diseases

    PubMed Central

    Wang, Ellen F.; Misra, Stuti L.; Patel, Dipika V.

    2015-01-01

    In vivo confocal microscopy (IVCM) of the living human cornea offers the ability to perform repeated imaging without tissue damage. Studies using corneal IVCM have led to significant contributions to scientific and clinical knowledge of the living cornea in health and pathological states. Recently the application of corneal IVCM beyond ophthalmology to wider clinical and research fields has been demonstrated. Abnormalities of the corneal subbasal nerve plexus have been associated with many forms of peripheral neuropathy and Langerhans cells correlate with systemic inflammatory states. There is a rapidly growing evidence base investigating the use of corneal IVCM in many systemic conditions and a well-established evidence base for IVCM imaging of the corneal subbasal plexus in diabetic peripheral neuropathy. This paper reviews the potential use of corneal IVCM in general clinical practice as a noninvasive method of assessing peripheral neuropathies, monitoring inflammatory states and clinical therapeutic response. PMID:26770980

  10. Human peripheral blood granulocytes and myeloid leukemic cell lines express both transcripts encoding for stem cell factor.

    PubMed

    Ramenghi, U; Ruggieri, L; Dianzani, I; Rosso, C; Brizzi, M F; Camaschella, C; Pietsch, T; Saglio, G

    1994-09-01

    Stem cell factor (SCF), the ligand for the c-kit proto-oncogene, has been shown to play a critical role in the migration of melanocytes and germ cells during embryogenesis as well as in the proliferative control of the hematopoietic compartment. In this study we investigated the expression of both the soluble and transmembrane SCF forms in purified peripheral blood populations and in several hematopoietic cell lines. Expression of both transcripts, though in different ratios, was identified in whole bone marrow, in bone marrow stromal cells and in human peripheral blood. In peripheral blood, SCF expression could be ascribable to polymorphonuclear leukocytes (PMN), whereas no SCF expression was detected in isolated lymphocytes, monocytes and in some T lymphoid cell lines. Conversely, some hematopoietic myeloid cell lines, such as HL-60, KG1 and K562, express SCF with similar patterns.

  11. Modest stimulatory effect of recombinant human GM-CSF on colony growth from peripheral blood human megakaryocyte progenitor cells.

    PubMed

    Mazur, E M; Cohen, J L; Wong, G G; Clark, S C

    1987-12-01

    Recombinant human granulocyte-macrophage colony-stimulating factor (rGM-CSF) has been previously demonstrated to stimulate colony formation from human myeloid, erythroid, and multipotential stem cells. In this investigation, we evaluated the effects of rGM-CSF on colony growth by human megakaryocyte progenitors (CFU-Meg). rGM-CSF was tested at concentrations of 0.1-100 U/ml in plasma clot cultures of adherent-depleted normal peripheral blood mononuclear cells. Control cultures were concurrently prepared containing either no stimulator or megakaryocyte colony-stimulating factor (Meg-CSF) partially purified from aplastic canine serum. rGM-CSF increased megakaryocyte colony numbers from a baseline of 4.3 +/- 1.4 (+/- SEM) in the unstimulated cultures to a maximum of 21.0 +/- 5.3 colonies at an rGM-CSF concentration of 1.0 U/ml. Corresponding megakaryocytic colony size increased from 4.4 to 8.3 cells/colony. Further increasing the rGM-CSF concentration resulted in decreasing megakaryocyte colony growth, reaching 6.8 +/- 2.9 colonies at 100 U/ml. The maximum number of megakaryocyte colonies stimulated by rGM-CSF was only 23.3% of that achieved in the control cultures containing optimal concentrations of serum-derived Meg-CSF protein (2.0 mg/ml). Megakaryocyte colonies stimulated by rGM-CSF consisted of predominantly low ploidy cells approximately equally distributed in 2N, 4N, and 8N ploidy classes. There was no increase in ploidy with any rGM-CSF concentration. These data indicate that rGM-CSF has modest activity in stimulating human megakaryocyte colony growth that is substantially less than that present in serum-derived Meg-CSF. rGM-CSF appears to primarily affect the early mitotic phase of megakaryocyte colony development with little influence on megakaryocyte endoreduplication.

  12. Phospholipase A2 Inhibitor from Crotalus durissus terrificus rattlesnake: Effects on human peripheral blood mononuclear cells and human neutrophils cells.

    PubMed

    Xavier, Caroline V; da S Setúbal, Sulamita; Lacouth-Silva, Fabianne; Pontes, Adriana S; Nery, Neriane M; de Castro, Onassis Boeri; Fernandes, Carla F C; Soares, Andreimar M; Fortes-Dias, Consuelo L; Zuliani, Juliana P

    2017-07-23

    Crotalus Neutralizing Factor (CNF) is an inhibitor of phospholipase A2 (PLA2), present in the blood plasma of Crotalus durissus terrificus snake. This inhibitor neutralizes the lethal and enzymatic activity of crotoxin, the main neurotoxin from this venom. In this study, we investigated the effects of CNF on the functionality of human peripheral blood mononuclear cells (PBMCs) and human neutrophils. The following parameters were evaluated: viability and proliferation, chemotaxis, cytokines and LTB4 production, cytosolic PLA2s activity, myeloperoxidase (MPO) and superoxide anion (O2(-)) production. CNF showed no toxicity on PBMCs or neutrophils, and acts by stimulating the release of TNF-α and LTB4, but neither stimulates IL-10 and IL-2 nor affects PBMCs proliferation and O2(-) release. In neutrophils, CNF induces chemotaxis but does not induce the release of both MPO and O2(-). However, it induces LTB4 and IL-8 production. These data show the influence of CNF on PBMCs' function by inducing TNF-α and LTB4 production, and on neutrophils, by stimulating chemotaxis and LTB4 production, via cytosolic PLA2 activity, and IL-8 release. The inflammatory profile produced by CNF is shown for the first time. Our present results suggest that CNF has a role in activation of leukocytes and exert proinflammatory effects on these cell. Copyright © 2017 Elsevier B.V. All rights reserved.

  13. Differences in vanadocene dichloride and cisplatin effect on MOLT-4 leukemia and human peripheral blood mononuclear cells.

    PubMed

    Havelek, Radim; Siman, Pavel; Cmielova, Jana; Stoklasova, Alena; Vavrova, Jirina; Vinklarek, Jaromir; Knizek, Jiri; Rezacova, Martina

    2012-07-01

    Modern chemotherapy is interested in developing new agents with high efficiency of treatment in low-dose medication strategies, lower side toxicity and stronger specificity to the tumor cells. Vanadocene dichloride (VDC) belongs to the group of the most promising metallocene antitumor agents; however, its mechanism of action and cytotoxicity profile are not fully understood. In this paper we assess cytotoxic effects of VDC in comparison to cisplatin using opposite prototype of cells; human peripheral blood mononuclear (PBMCs) cells and human acute lymphoblastic leukemia cell line (MOLT-4). Our findings showed cytotoxic effect of VDC on leukemia cells, but unfortunately on human peripheral blood mononuclear cells as well. VDC induces apoptosis in leukemia cells; the induction is, however, lower than that of cisplatin, and in contrary to cisplatin, VDC does not induce p53 up-regulation. Cytotoxic effect of VDC on leukemia cells is less pronounced than that of cisplatin and more pronounced in PBMCs than in MOLT-4 cells.

  14. Catabolism of exogenously supplied thymidine to thymine and dihydrothymine by platelets in human peripheral blood

    SciTech Connect

    Pero, R.W.; Johnson, D.; Olsson, A.

    1984-11-01

    The interference of platelets with the estimation of unscheduled DNA synthesis in human peripheral mononuclear leukocytes following genotoxic exposure was studied. A 96% reduction in the unscheduled DNA synthesis value was achieved by incubating (/sup 3/H)thymidine with platelet-rich plasma for 5 hr at 37 degrees. Using radioactive thymine-containing compounds, together with quantitative analyses based on thin-layer and ion-exchange chromatographies, we have shown that thymidine was converted to thymine which, in turn, was converted to dihydrothymine in platelet-rich plasma. The enzymes responsible were separated from platelet lysates by gel filtration and were identified as thymidine phosphorylase and dihydrothymine dehydrogenase. The phosphorylase reversibly catalyzed the formation of thymine from thymidine and converted bromodeoxyuridine to bromouracil. The dehydrogenase reversibly catalyzed the interconversion of thymine and dihydrothymine in a reaction dependent on NADP(H), and it was inhibited by diazouracil and by thymine. Nearly all the thymidine-catabolizing activity found in whole blood samples supplied exogenously with thymidine was accounted for by the platelets. Since most genetic toxicological tests that use blood samples do not involve removing platelets from the blood cell cultures, then it is concluded that precautions should be taken in the future to determine the influence of platelets on these test systems. This is particularly true for methods dependent on thymidine pulses such as unscheduled DNA synthesis, or those dependent on bromodeoxyuridine, such as sister chromatid exchanges, since this nucleoside is also a substrate for thymidine phosphorylase.

  15. Antigenotoxic Effect of Trametes spp. Extracts against DNA Damage on Human Peripheral White Blood Cells

    PubMed Central

    Knežević, Aleksandar; Živković, Lada; Stajić, Mirjana; Vukojević, Jelena; Milovanović, Ivan; Spremo-Potparević, Biljana

    2015-01-01

    Trametes species have been used for thousands of years in traditional and conventional medicine for the treatment of various types of diseases. The goal was to evaluate possible antigenotoxic effects of mycelium and basidiocarp extracts of selected Trametes species and to assess dependence on their antioxidant potential. Trametes versicolor, T. hirsuta, and T. gibbosa were the species studied. Antigenotoxic potentials of extracts were assessed on human peripheral white blood cells with basidiocarp and mycelium extracts of the species. The alkaline comet test was used for detection of DNA strand breaks and alkali-labile sites, as well as the extent of DNA migration. DPPH assay was used to estimate antioxidative properties of extracts. Fruiting body extracts of T. versicolor and T. gibbosa as well as T. hirsuta extracts, except that at 20.0 mg/mL, were not genotoxic agents. T. versicolor extract had at 5.0 mg/mL the greatest antigenotoxic effect in both pre- and posttreatment of leukocytes. The mycelium extracts of the three species had no genotoxic activity and significant antigenotoxic effect against H2O2-induced DNA damage, both in pre- and posttreatment. The results suggest that extracts of these three species could be considered as strong antigenotoxic agents able to stimulate genoprotective response of cells. PMID:26258163

  16. Separation of hematopoietic stem cells from human peripheral blood through modified polyurethane foaming membranes.

    PubMed

    Higuchi, Akon; Sekiya, Mayu; Gomei, Yumiko; Sakurai, Masaru; Chen, Wen-Yih; Egashira, Satsuki; Matsuoka, Yuki

    2008-06-15

    Cell separation from peripheral blood was investigated using polyurethane (PU) foam membranes having 5.2 mum pore size and coated with Pluronic F127 or hyaluronic acid. The permeation ratio of hematopoietic stem cells (CD34(+) cells) and lymphocytes through the membranes was lower than for red blood cells and platelets. Adhered cells were detached from membrane surfaces using human serum albumin (HSA) solution after permeation of blood through the membranes, allowing isolation of CD34(+) cells in the permeate (recovery) solution. High-yield isolation of CD34(+) cells was achieved using Pluronic-coated membranes. This was because the Pluronic coating dissolved into the recovery solution at 4 degrees C, releasing adhered cells from the surfaces of the membranes during permeation of HSA solution through these membranes. Dextran and/or bovine serum albumin solutions were also evaluated for use as recovery solutions after blood permeation. A high recovery ratio of CD34(+) cells was achieved at 4 degrees C in a process using 20% dextran solution through PU membranes having carboxylic acid groups.

  17. Antigenotoxic Properties of Agaricus blazei against Hydrogen Peroxide in Human Peripheral Blood Cells.

    PubMed

    Živković, Lada; Borozan, Sunčica; Čabarkapa, Andrea; Topalović, Dijana; Ciptasari, Ummi; Bajić, Vladan; Spremo-Potparević, Biljana

    2017-01-01

    The ability of Agaricus blazei mushroom in its dried and powdered mycelial form was evaluated for its antigenotoxic properties for the first time. Antigenotoxic effects in human peripheral blood cells against H2O2-induced DNA damage were examined in pretreatment and posttreatment protocol by comet assay. The results showed better antigenotoxic properties of Agaricus blazei on the interventional level, respectively, after treatment. Agaricus blazei in concentration of 250 μg/mL after treatment was most efficient in regard to its action against DNA damage. The evaluation of repair kinetics showed decrease in H2O2 induced DNA damage 15 min after the application of A. blazei, reaching the maximum potency after 30 min. Analysis of antioxidant properties of Agaricus blazei revealed strong (•)OH scavenging properties and moderate reducing power, while its DPPH scavenging ability was weak. In regard to our findings, we can conclude that our preliminary results demonstrated antigenotoxic properties of Agaricus blazei and its strong (•)OH scavenging ability. Mechanisms underlying its properties should be further evaluated in in vivo studies.

  18. Human peripheral blood monocytes display surface antigens recognized by monoclonal antinuclear antibodies

    SciTech Connect

    Holers, V.M.; Kotzin, B.L.

    1985-09-01

    The authors used monoclonal anti-nuclear autoantibodies and indirect immunofluorescence to examine normal human peripheral blood mononuclear leukocytes for the presence of cell surface nuclear antigens. Only one monoclonal anti-histone antibody (MH-2) was found to bind to freshly isolated PBL, staining approximately 10% of large cells. However, after cells were placed into culture for 16-24 h, a high percentage (up to 60%) of large-sized cells were recognized by an anti-DNA (BWD-1) and several different antihistone monoclonal antibodies (BWH-1, MH-1, and MH-2). These antibodies recognize separate antigenic determinants on chromatin and histones extracted from chromatin. The histone antigen-positive cells were viable, and the monoclonal antibodies could be shown to be binding to the cell surface and not to the nucleus. Using monoclonal antibodies specific for monocytes and T cells, and complement-mediated cytotoxicity, the cells bearing histone antigens were shown to be primarily monocytes. The appearance of histone and DNA antigen-positive cells was nearly completely inhibited by the addition of low concentrations of cycloheximide at initiation of the cultures. In contrast, little effect on the percentage of positive cells was detected if cells were exposed to high doses of gamma irradiation before culture. These data further support the existence of cell surface nuclear antigens on selected cell subsets, which may provide insight into the immunopathogenesis of systemic lupus erythematosus and related autoimmune diseases.

  19. Differential Behaviour of Selenium Analogs against Anticancer Drug Induced Apoptosis of Lymphocytes in Human Peripheral Blood.

    PubMed

    Elango, Sonaa; Subbiah, Usha; Jain, Jeong

    2016-01-01

    Sensitising cancer cells and at the same time desensitizing normal cells is a double task in cancer management. Agents which can combat the debilitating side effects of cancer therapeutics and simultaneously synergize with anticancer agents in specifically targeting cancer cells are needed. Selenium, a proven anticarcinogen, gains due importance in terms of its efficacy to combat the side effects of cancer therapy. This study is a comparative analysis of the chemoprotective effects of selenium compounds, methyl selenol (generated from organic selenomethionine (5mmol/L ; METase 40U/L)) and sodium selenite (inorganic form)(30μM) in peripheral blood human lymphocytes exposed to cisplatin and mitomycin. Biochemical alterations occurring in many cells during apoptosis include loss of plasma membrane phospholipid asymmetry, DNA fragmentation, and activation of caspase-3. The present study demonstrated that the selenium metabolite and selenite are efficient in protecting lymphocytes undergoing DNA damage and exerted their activity by reducing caspase 3 expression. Interestingly organic methylselenol (MeSe) was found to offer more protective effects compared to inorganic selenite (SeL), by reducing the induction of apoptosis by the cytotoxic agents. This suggests that MeSe and to a lesser extent selenite might have potential for assessment in clinical trials and could be considered as strong candidates in pharmacogenomics or in the nutriprotective arena.

  20. The human peripheral subunit-binding domain folds rapidly while overcoming repulsive Coulomb forces

    PubMed Central

    Arbely, Eyal; Neuweiler, Hannes; Sharpe, Timothy D; Johnson, Christopher M; Fersht, Alan R

    2010-01-01

    Peripheral subunit binding domains (PSBDs) are integral parts of large multienzyme complexes involved in carbohydrate metabolism. PSBDs facilitate shuttling of prosthetic groups between different catalytic subunits. Their protein surface is characterized by a high density of positive charges required for binding to subunits within the complex. Here, we investigated folding thermodynamics and kinetics of the human PSBD (HSBD) using circular dichroism and tryptophan fluorescence experiments. HSBD was only marginally stable under physiological solvent conditions but folded within microseconds via a barrier-limited apparent two-state transition, analogous to its bacterial homologues. The high positive surface-charge density of HSBD leads to repulsive Coulomb forces that modulate protein stability and folding kinetics, and appear to even induce native-state movement. The electrostatic strain was alleviated at high solution-ionic-strength by Debye-Hückel screening. Differences in ionic-strength dependent characteristics among PSBD homologues could be explained by differences in their surface charge distributions. The findings highlight the trade-off between protein function and stability during protein evolution. PMID:20662005

  1. Methamidophos induces cytotoxicity and oxidative stress in human peripheral blood mononuclear cells.

    PubMed

    Ramirez-Vargas, Marco Antonio; Huerta-Beristain, Gerardo; Guzman-Guzman, Iris Paola; Alarcon-Romero, Luz Del Carmen; Flores-Alfaro, Eugenia; Rojas-Garcia, Aurora Elizabeth; Moreno-Godinez, Ma Elena

    2017-01-01

    Previous studies have shown that organophosphate pesticide (OP) exposure is associated with oxidative stress. Methamidophos (MET) is an OP widely used in agriculture, which is regarded as a highly toxic pesticide and it is a potent inhibitor of acetylcholinesterase. The aim of this study was to evaluate whether MET can induce oxidative stress at low concentrations in primary cultures of human peripheral blood mononuclear cells (PBMCs). PBMCs from healthy individuals were exposed to MET (0-80 mg/L) for 0-72 h. We performed the MTT and neutral-red assays to assess the cytotoxicity. As indicators of oxidative stress, the levels of reactive oxygen species (ROS) were assessed using flow cytometry, and the malondialdehyde (MDA) and reduced glutathione (GSH) levels were determined. MET decreased the viability of PBMCs in a dose-dependent manner. At concentrations of 3, 10, or 20 mg/L for 24 h, MET increased the ROS production significantly compared with the vehicle control. Similarly, MET increased the levels of MDA at the same concentrations that increased ROS (10 and 20 mg/L); however, no changes in GSH levels were observed. These results suggest that MET increased the generation of oxidative stress in PBMCs. © 2015 Wiley Periodicals, Inc. Environ Toxicol 32: 147-155, 2017.

  2. Defining the restriction point in normal asynchronous human peripheral blood lymphocytes.

    PubMed

    Jiang, Jianwu; Liu, Liang; Li, Xiaolan; Tao, Deding; Hu, Junbo; Qin, Jichao

    2013-10-01

    Although the restriction point (R-point) was proposed in animal cells several decades ago, its existence in normal cells is still controversial, because, in most studies, long-term cultured cell lines rather than primary normal cells were used. Furthermore, cell synchronization was generally applied, resulting in growth imbalance between DNA synthesis and protein expression in cells. Finally, R-point was originally proposed as a unique arrest point that may be in G0 phase; however, generally believed R-point locates within G1 phase. Thus, up to now, there is no solid experimental evidence that supports the existence of R-point in asynchronous primary normal cells. In this study, we used freshly purified peripheral human blood lymphocytes, as asynchronous primary normal cells, to confirm the existence of restriction point in G1 not G0 phase. Our findings may help uncover the mystery of the deregulation of cell cycle progression in malignant tumors. © 2013 International Society for Advancement of Cytometry.

  3. Antigenotoxic Properties of Agaricus blazei against Hydrogen Peroxide in Human Peripheral Blood Cells

    PubMed Central

    Borozan, Sunčica; Topalović, Dijana; Ciptasari, Ummi; Bajić, Vladan

    2017-01-01

    The ability of Agaricus blazei mushroom in its dried and powdered mycelial form was evaluated for its antigenotoxic properties for the first time. Antigenotoxic effects in human peripheral blood cells against H2O2-induced DNA damage were examined in pretreatment and posttreatment protocol by comet assay. The results showed better antigenotoxic properties of Agaricus blazei on the interventional level, respectively, after treatment. Agaricus blazei in concentration of 250 μg/mL after treatment was most efficient in regard to its action against DNA damage. The evaluation of repair kinetics showed decrease in H2O2 induced DNA damage 15 min after the application of A. blazei, reaching the maximum potency after 30 min. Analysis of antioxidant properties of Agaricus blazei revealed strong •OH scavenging properties and moderate reducing power, while its DPPH scavenging ability was weak. In regard to our findings, we can conclude that our preliminary results demonstrated antigenotoxic properties of Agaricus blazei and its strong •OH scavenging ability. Mechanisms underlying its properties should be further evaluated in in vivo studies. PMID:28316757

  4. Dose rate effect of pulsed electron beam on micronucleus frequency in human peripheral blood lymphocytes.

    PubMed

    Acharya, Santhosh; Sanjeev, Ganesh; Bhat, Nagesh N; Narayana, Yerol

    2010-03-01

    The micronucleus assay in human peripheral blood lymphocytes is a sensitive indicator of radiation damage and could serve as a biological dosimeter in evaluating suspected overexposure to ionising radiation. Micronucleus (MN) frequency as a measure of chromosomal damage has also extensively been employed to quantify the effects of radiation dose rate on biological systems. Here we studied the effects of 8 MeV pulsed electron beam emitted by Microtron electron accelerator on MN induction at dose rates between 35 Gy min-1 and 352.5 Gy min-1. These dose rates were achieved by varying the pulse repetition rate (PRR). Fricke dosimeter was employed to measure the absorbed dose at different PRR and to ensure uniform dose distribution of the electron beam. To study the dose rate effect, blood samples were irradiated to an absorbed dose of (4.7+/-0.2) Gy at different rates and cytogenetic damage was quantified using the micronucleus assay. The obtained MN frequency showed no dose rate dependence within the studied dose rate range. Our earlier dose effect study using 8 MeV electrons revealed that the response of MN was linear-quadratic. Therefore, in the event of an accident, dose estimation can be made using linear-quadratic dose response parameters, without adding dose rate as a correction factor.

  5. Bone resorptive activity of human peripheral blood mononuclear cells after fusion with polyethylene glycol.

    PubMed

    Manrique, Edwin; Castillo, Luz M; Lazala, Oswaldo; Guerrero, Carlos A; Acosta, Orlando

    2017-03-01

    The bone remodeling process occurs through bone formation by osteoblasts and bone resorption by osteoclasts, a process involving the contribution of endocrine and nervous systems. The mechanisms associated to differentiation and proliferation of osteoclasts and osteoblasts are considered a potential therapeutic target for treating some erosive bone diseases. The aim of the present study is to explore the feasibility of generating active osteoclast-like cells from peripheral blood mononuclear cells (PBMCs) following polyethylene glycol (PEG)-induced fusion. PEG-fused PBMCs showed TRAP(+)-multinucleated cells and bone resorption activity, and were also positive for osteoclast markers such as carbonic anhydrase II, calcitonin receptor, vacuolar ATPase, and cathepsin K, when examined by reverse transcription-polymerase chain reaction, immunochemistry and Western blotting. TRAP expression and bone resorptive activity were higher in whole PEG-fused PBMCs than in separated T lymphocytes, B lymphocytes or monocytes. Both TRAP expression and bone resorptive activity were also higher in osteogenesis imperfecta patients compared to PEG-fused PBMCs from healthy individuals. PEG-induced fusion was more efficient in inducing TRAP and bone resorptive activities than macrophage colony-stimulating factor or dexamethasone treatment. Bone resorptive activity of PEG-fused PMBCs was inhibited by bisphosphonates. Evidence is provided that the use of PEG-based cell fusion is a straightforward and amenable method for studying human osteoclast differentiation and testing new therapeutic strategies.

  6. Chromosomal Aberrations in Human Peripheral Blood Lymphocytes after Exposure to Ionizing Radiation

    PubMed Central

    Ryu, Tae Ho; Kim, Jin-Hong; Kim, Jin Kyu

    2016-01-01

    Biological dosimetry using chromosome aberration analyses in human peripheral blood lymphocytes is suitable and useful tool for estimating the dose when a nuclear or radiological emergency is investigated. Blood samples from five healthy donors were obtained to establish dose-response calibration curves for chromosomal aberrations after exposure to ionizing radiation. In this work, dicentric assay and CBMN assay were compared considering the sensitivity and accuracy of dose estimation. In a total of 21,688 analyzed metaphase spreads, 10,969 dicentric chromosomes, 563 centric rings and 11,364 acentric chromosomes were found. The number of metaphase cells decreased with increasing radiation dose. The centric rings were not found in the non-irradiated control. There was no relationship between radiation dose and acentric ring induction. The frequency of total MN increased in a dose-dependent manner. In comparison with the control value, MN increased about 9, 32, 75, 87, and 52 fold higher after treatment with 1, 2, 3, 4, and 5 Gy, respectively. The results revealed that the mean frequency of chromosomal aberrations, both in dicentric and in micronuclei analyses increased with increasing radiation dose. PMID:28217281

  7. Induction of sister chromatid exchanges by coal dust and tobacco snuff extracts in human peripheral lymphocytes

    SciTech Connect

    Tucker, J.D.; Ong, T.

    1985-01-01

    The organic solvent extracts of sub-bituminous coal dust and tobacco snuff, both together and separately, were tested for the induction of sister chromatid exchanges (SCEs) in human peripheral lymphocytes. The results indicate that these extracts induced SCEs, and that when tested together synergistically induced SCEs in two of three donors. Studies with the organic solvent extracts of all five ranks of coal indicate that the extracts of bituminous, lignite, and peat, but not anthracite, induced SCEs. Similar experiments conducted with water extracts, induced SCEs, and that anthracite was equivocal. To determine whether individuals differed in their SCE responses to coal dust extracts, lymphocytes from five donors were tested with organic solvent extracts of bituminous and sub-bituminous coal. An analysis of variance indicates that the SCE response was significantly influenced by the donor and each of the two coal extracts. The findings presented here suggest that coal dust, with or without tobacco snuff, may play a role in the elevated incidence of gastric cancer in coal miners. Because water extracts of some ranks of coal induced SCEs, there exists the possibility of adverse environmental effects due to coal leachates.

  8. Analysis of cytotoxic effects of silver nanoclusters on human peripheral blood mononuclear cells 'in vitro'.

    PubMed

    Orta-García, Sandra Teresa; Plascencia-Villa, Germán; Ochoa-Martínez, Angeles Catalina; Ruiz-Vera, Tania; Pérez-Vázquez, Francisco Javier; Velázquez-Salazar, J Jesús; Yacamán, Miguel José; Navarro-Contreras, Hugo Ricardo; Pérez-Maldonado, Iván N

    2015-10-01

    The antimicrobial properties of silver nanoparticles (AgNPs) have made these particles one of the most used nanomaterials in consumer products. Therefore, an understanding of the interactions (unwanted toxicity) between nanoparticles and human cells is of significant interest. The aim of this study was to assess the in vitro cytotoxicity effects of silver nanoclusters (AgNC, < 2 nm diameter) on peripheral blood mononuclear cells (PBMC). Using flow cytometry and comet assay methods, we demonstrate that exposure of PBMC to AgNC induced intracellular reactive oxygen species (ROS) generation, DNA damage and apoptosis at 3, 6 and 12 h, with a dose-dependent response (0.1, 1, 3, 5 and 30 µg ml(-1)). Advanced electron microscopy imaging of complete and ultrathin-sections of PBMC confirmed the cytotoxic effects and cell damage caused by AgNC. The present study showed that AgNC produced without coating agents induced significant cytotoxic effects on PBMC owing to their high aspect ratio and active surface area, even at much lower concentrations (<1 µg ml(-1)) than those applied in previous studies, resembling what would occur under real exposure conditions to nanosilver-functionalized consumer products.

  9. Fu-Ling, a Chinese herbal drug, modulates cytokine secretion by human peripheral blood monocytes.

    PubMed

    Yu, S J; Tseng, J

    1996-01-01

    Fu-Ling, the sclederma of Poria cocos (Schw.) Wolf, has long been used as a sedative and diuretic in traditional Chinese herbal medicine. Our study demonstrated that the substances extracted from Fu-Ling by 50% hot ethanol significantly augmented the secretion of interleukins IL-1 beta and IL-6 6 h after in vitro cultivation of human peripheral blood monocytes. The augmented effect was dose dependent. Tumour necrosis factor-alpha (TNF-alpha) secretion was also increased as the cells were treated with 0.4 mg/ml or higher doses of Fu-Ling extract. By contrast, Fu-Ling extract significantly suppressed the secretion of transforming growth factor-beta (TGF-beta) 3 h after the in vitro drug treatment. The suppressive effect was shown at doses as low as 0.2 mg/ml of Fu-Ling extract. Since Fu-Ling extract enhanced the secretion of immune stimulators (IL-1 beta, IL-6 and TNF-alpha) but suppressed the secretion of an immune suppressor (TGF-beta), the substance in 50% hot ethanol extract of Fu-Ling might have potentiated the immune response. Fu-Ling extract was further fractionated by reverse-phase column chromatography and high-performance liquid chromatography (HPLC). The components showing activity in modulating the cytokine secretion were relatively high in hydrophobicity.

  10. Antioxidant enzyme activities of human peripheral blood mononuclear cells exposed to trace elements.

    PubMed

    Kuppusamy, U R; Dharmani, M; Kanthimathi, M S; Indran, M

    2005-07-01

    The trace elements copper, zinc, and selenium are important immune modulators and essential cofactors of the antioxidant enzymes. In the present study, the proliferative effect of human peripheral mononuclear cells (PBMCs) that have been exposed to copper, zinc, and selenium and the corresponding activities of antioxidant enzymes, namely superoxide dismutase (SOD), glutathione peroxidase (GPx), and catalase, were determined. Zinc and copper stimulated the PBMC proliferation in a dose-dependent manner within the dose range 25-200 micromol/L. SOD and GPx activities in PBMCs exposed to zinc were inhibited, whereas catalase activity was unaffected. All the three antioxidant enzymes in the cells exposed to copper were inhibited. Selenium exerted more potent inhibition of the cell proliferation while causing stimulation of the antioxidant enzymes at the lowest dose (25 micromol/L) than at the highest dose (200 micromol/L) tested. A significant negative correlation was observed between proliferation and antioxidant enzyme (SOD and GPx) activities in trace-element-exposed PBMC. The present findings substantiate the importance of trace elements as immune modulators and the involvement of enzymatic antioxidant system in the immune cell regulation.

  11. Anterior corneal and internal contributions to peripheral aberrations of human eyes

    NASA Astrophysics Data System (ADS)

    Atchison, David A.

    2004-03-01

    Anterior corneal and internal component contributions to overall peripheral aberrations of five human eyes were determined, based on corneal topography and overall aberration measurements. Anterior corneal position and orientation (tilt) were referenced to the line of sight. Ray tracing was performed through the anterior cornea for 6-mm-diameter pupils at angles out to 40° in both the temporal and the nasal visual fields. In general, both component and overall Zernike aberrations were greater for the nasal than for the temporal visual field. In general, the anterior corneal aberration components were considerably higher than the overall aberrations across the visual field and were balanced to a considerable degree by the internal ocular aberration components. The component and overall levels of Zernike third-order aberrations showed linear trends away from the fixation axis, and the component levels of Zernike fourth-order aberrations showed quadratic trends away from the fixation axis. The second-order, but not higher-order, aberration components were susceptible to the choice of image radius of curvature, while disregarding corneal position and orientation affected second- and higher-order aberration components.

  12. Recombinant human endostatin in combination with CHOP regimen for peripheral T cell lymphoma

    PubMed Central

    Zhang, Qunling; Cao, Junning; Xue, Kai; Liu, Xiaojian; Ji, Dongmei; Guo, Ye; Hong, Xiaonan

    2017-01-01

    Peripheral T cell lymphoma (PTCL) has a poor prognosis. Overexpression of vascular endothelial growth factor (VEGF) might contribute to the poor prognosis of PTCL and could be the target of novel therapy. The efficacy and safety of recombinant human endostatin (Endostar) in combination with cyclophosphamide, doxorubicin, vincristine and prednisone (ECHOP) have been explored in 15 PTCL patients. The objective response rate was 80%, with 53.3% patients having achieved complete response (CR) rate. The CR rate was 100% (3/3) in angioimmunoblastic T cell lymphoma (AITL) patients compared to only 36.4% (4/11) in PTCL not otherwise specified (PTCL-NOS) patients. With a median follow-up of 69 months, the 5-year progression-free survival and overall survival (OS) were 53% and 60%, respectively. The 5-year OS was 100% in AITL but was only 45% in PTCL-NOS. Seven out of 11 patients showed overexpression of VEGFR2 in their tumor vessels and had a better efficacy than those with low expression of VEGFR2. Grade 3 or 4 neutropenia is the most common toxicity observed. ECHOP was safe and might display potential benefit in AITL patients. PMID:28053548

  13. [Association of peripheral facial paralysis in patients with human immunodeficiency virus infection].

    PubMed

    Casanova-Sotolongo, P; Casanova-Carrillo, P

    Neurological disorders are common in patients infected with the human immunodeficiency virus (HIV). The objective of this study is to show the possible association of peripheral facial paralysis (PFP) in persons who test positive for HIV, which because of the characteristics of outpatient management is important in basic medical attention. The scope of our study included all patients observed in 1998 and the first six months of 1999 who had PFP, and in whom detailed clinical history, physical examination by a neurologist, laboratory tests and HIV serological tests were done. We found that 89.1% of the patients with PFP were seropositive. The facial paralysis showed similar characteristics to Bell s palsy, with complete recovery after four weeks in 66.6% of this group of patients. The seronegative patients also recovered completely but took longer to do so. In two cases the PFP was associated with obvious features of AIDS. In the literature reviewed we have not found such a large group of patients with PFP associated with HIV+. However, it is obvious that there is an increase in facial paralysis which is in proportion to the increase in HIV+ persons. The results of our study suggest to us that HIV should be tested for in patients with PFP.

  14. Eosinophils Regulate Peripheral B Cell Numbers in Both Mice and Humans

    PubMed Central

    Wong, Tina W.; Doyle, Alfred D.; Lee, James J.; Jelinek, Diane F.

    2014-01-01

    The view of eosinophils (Eos) as solely effector cells involved in host parasite defense and in the pathophysiology of allergic diseases has been challenged in recent years. In fact, there is a growing realization that these cells interact with other components of innate and adaptive immunity. For example, mouse Eos were recently demonstrated to promote plasma cell retention in the bone marrow. However, it remains unknown whether Eos influence the biology of normal B lymphocytes. In this study, we specifically assessed the effect of Eos on B cell survival, proliferation, and immunoglobulin secretion. Our data first revealed that the genetic deletion of Eos from NJ1638 IL-5 transgenic hypereosinophilic mice (previously shown to display profound B cell expansion) resulted in the near abolishment of the B cell lymphocytosis. In vitro studies using human tissues demonstrated Eos’ proximity to B cell follicles and their ability to promote B cell survival, proliferation, and immunoglobulin secretion via a contact-independent mechanism(s). Additionally, this ability of Eos to enhance B cell responsiveness was observed in both T-independent and T-dependent B cell activation and appears to be independent of the Eos’ activation state. Finally, a retrospective clinical study of hypereosinophilic patients revealed for the first time a direct correlation between peripheral blood eosinophil levels and B cell numbers. Taken together, our study identifies a novel role for Eos in the regulation of humoral immunity via their impact on B cell homeostasis and proliferation upon activation. PMID:24616476

  15. Allograft inflammatory factor-1 stimulates chemokine production and induces chemotaxis in human peripheral blood mononuclear cells.

    PubMed

    Kadoya, Masatoshi; Yamamoto, Aihiro; Hamaguchi, Masahide; Obayashi, Hiroshi; Mizushima, Katsura; Ohta, Mitsuhiro; Seno, Takahiro; Oda, Ryo; Fujiwara, Hiroyoshi; Kohno, Masataka; Kawahito, Yutaka

    2014-06-06

    Allograft inflammatory factor-1 (AIF-1) is expressed by macrophages, fibroblasts, endothelial cells and smooth muscle cells in immune-inflammatory disorders such as systemic sclerosis, rheumatoid arthritis and several vasculopathies. However, its molecular function is not fully understood. In this study, we examined gene expression profiles and induction of chemokines in monocytes treated with recombinant human AIF (rhAIF-1). Using the high-density oligonucleotide microarray technique, we compared mRNA expression profiles of rhAIF-1-stimulated CD14(+) peripheral blood mononuclear cells (CD14(+) PBMCs) derived from healthy volunteers. We demonstrated upregulation of genes for several CC chemokines such as CCL1, CCL2, CCL3, CCL7, and CCL20. Next, using ELISAs, we confirmed that rhAIF-1 promoted the secretion of CCL3/MIP-1α and IL-6 by CD14(+) PBMCs, whereas only small amounts of CCL1, CCL2/MCP-1, CCL7/MCP-3 and CCL20/MIP-3α were secreted. Conditioned media from rhAIF-1stimulated CD14(+) PBMCs resulted in migration of PBMCs. These findings suggest that AIF-1, which induced chemokines and enhanced chemotaxis of monocytes, may represent a molecular target for the therapy of immune-inflammatory disorders. Copyright © 2014 Elsevier Inc. All rights reserved.

  16. Current use of benzodiazepines in anxiety disorders.

    PubMed

    Cloos, Jean-Marc; Ferreira, Valérie

    2009-01-01

    The aim of this study is to provide a review of articles published between July 2007 and August 2008 on the current use and rationale of benzodiazepines in anxiety disorders. Recent review articles confirm selective serotonin reuptake inhibitors as first-choice drugs for treating anxiety disorders, alongside newer agents such as pregabalin or serotonin-norepinephrine reuptake inhibitors, and combined with cognitive-behavioural therapy. Benzodiazepines are still widely used by clinicians for these disorders, as shown by recent surveys, even though their anxiolytic effectiveness is questioned. Newer agents are in development and may in the future resolve the therapeutic dilemma. Despite current guidelines, benzodiazepines are still considered by many clinicians to remain good treatment options, in both the acute and the chronic phase of the treatment of anxiety disorders, partially because of their rapid onset of action and their efficacy with a favourable side effect profile, and also because of the sometimes only incomplete therapeutic response and the emergence of side effects of alternative medications. Having experienced good initial symptom relief with benzodiazepine treatment, patients may also be reluctant to taper it down. Clinicians should, however, bear in mind the frequent physiological dependence associated with these substances, and suggest both pharmacological and psychological treatment alternatives before opting for a long-term benzodiazepine treatment, which may remain necessary in certain clinical conditions.

  17. Neural bases for addictive properties of benzodiazepines.

    PubMed

    Tan, Kelly R; Brown, Matthew; Labouèbe, Gwenaël; Yvon, Cédric; Creton, Cyril; Fritschy, Jean-Marc; Rudolph, Uwe; Lüscher, Christian

    2010-02-11

    Benzodiazepines are widely used in clinics and for recreational purposes, but will lead to addiction in vulnerable individuals. Addictive drugs increase the levels of dopamine and also trigger long-lasting synaptic adaptations in the mesolimbic reward system that ultimately may induce the pathological behaviour. The neural basis for the addictive nature of benzodiazepines, however, remains elusive. Here we show that benzodiazepines increase firing of dopamine neurons of the ventral tegmental area through the positive modulation of GABA(A) (gamma-aminobutyric acid type A) receptors in nearby interneurons. Such disinhibition, which relies on alpha1-containing GABA(A) receptors expressed in these cells, triggers drug-evoked synaptic plasticity in excitatory afferents onto dopamine neurons and underlies drug reinforcement. Taken together, our data provide evidence that benzodiazepines share defining pharmacological features of addictive drugs through cell-type-specific expression of alpha1-containing GABA(A) receptors in the ventral tegmental area. The data also indicate that subunit-selective benzodiazepines sparing alpha1 may be devoid of addiction liability.

  18. The effect of catechol on human peripheral blood mononuclear cells (in vitro study).

    PubMed

    Bukowska, Bożena; Michałowicz, Jaromir; Marczak, Agnieszka

    2015-01-01

    Catechol also known as pyrocatechol or 1,2-dihydroxybenzene is formed endogenously in the organism from neurotransmitters including adrenaline, noradrenaline, and dopamine. It is also a metabolite of many drugs like DOPA, isoproterenol or aspirin and it is also formed in the environment during transformation of various xenobiotics. We evaluated in vitro the effect of catechol on the structure and function of human peripheral blood mononuclear cells (PBMCs). The cells were incubated with xenobiotic at concentration range from 2 to 500μg/mL for 1h. Human blood mononuclear cells were obtained from leucocyte-platelet buffy coat taken from healthy donors in the Blood Bank of Łódź, Poland. Using flow cytometry we have evaluated necrotic, apoptotic and morphological changes in PBMCs incubated with catechol. Moreover, we have estimated changes in reactive oxygen species (ROS) formation, protein carbonylation and lipid peroxidation in the cells studied. The compound studied provoked necrotic (from 250μg/mL), apoptotic (from 100μg/mL), and morphological changes (from 250μg/mL) in the incubated cells. We have also noted that catechol decreased H2DCF oxidation at 2 and 10μg/mL but at higher concentrations of 250 and 500μg/mL it caused statistically significant increase in the oxidation of this probe. We also observed an increase in lipid peroxidation (from 250μg/mL) and protein carbonylation (from 50μg/mL) of PBMCs. It was observed that catechol only at high concentrations was capable of inducing changes in PBMCs. The obtained results clearly showed that catechol may induce change in PBMCs only in the caste of poisoning with this compound.

  19. Microcystin-LR induced DNA damage in human peripheral blood lymphocytes.

    PubMed

    Zegura, B; Gajski, G; Straser, A; Garaj-Vrhovac, V; Filipič, M

    2011-12-24

    Human exposure to microcystins, which are produced by freshwater cyanobacterial species, is of growing concern due to increasing appearance of cyanobacterial blooms as a consequence of global warming and increasing water eutrophication. Although microcystins are considered to be liver-specific, there is evidence that they may also affect other tissues. These substances have been shown to induce DNA damage in vitro and in vivo, but the mechanisms of their genotoxic activity remain unclear. In human peripheral blood lymphocytes (HPBLs) exposure to non-cytotoxic concentrations (0, 0.1, 1 and 10μg/ml) of microcystin-LR (MCLR) induced a dose- and time-dependent increase in DNA damage, as measured with the comet assay. Digestion of DNA from MCLR-treated HPBLs with purified formamidopyrimidine-DNA glycosylase (Fpg) displayed a greater number of DNA strand-breaks than non-digested DNA, confirming the evidence that MCLR induces oxidative DNA damage. With the cytokinesis-block micronucleus assay no statistically significant induction of micronuclei, nucleoplasmic bridges and nuclear buds was observed after a 24-h exposure to MCLR. At the molecular level, no changes in the expression of selected genes involved in the cellular response to DNA damage and oxidative stress were observed after a 4-h exposure to MCLR (1μg/ml). After 24h, DNA damage-responsive genes (p53, mdm2, gadd45a, cdkn1a), a gene involved in apoptosis (bax) and oxidative stress-responsive genes (cat, gpx1, sod1, gsr, gclc) were up-regulated. These results provide strong support that MCLR is an indirectly genotoxic agent, acting via induction of oxidative stress, and that lymphocytes are also the target of microcystin-induced toxicity.

  20. Erythropoietin priming improves the vasculogenic potential of G-CSF mobilized human peripheral blood mononuclear cells.

    PubMed

    Kang, Jeehoon; Yun, Ji-Yeon; Hur, Jin; Kang, Jin-A; Choi, Jae-Il; Ko, Seung Bum; Lee, Jaewon; Kim, Ju-Young; Hwang, In-Chang; Park, Young-Bae; Kim, Hyo-Soo

    2014-10-01

    From our previous clinical trials, intracoronary infusion of granulocyte-colony stimulating factor (G-CSF)-mobilized peripheral blood mononuclear cells ((mob)PBMCs) proved to be effective in improving myocardial contractility and reducing infarct volume in acute myocardial infarction. We tested the effect of priming (mob)PBMCs with erythropoietin (EPO) to augment its therapeutic efficacy. (mob)PBMCs were obtained from healthy volunteers after a 3-day subcutaneous injection of G-CSF (10 μg/kg). About 40% of (mob)PBMCs were EPO receptor (EPOR) (+) and responded to 6 h EPO-priming (10 IU/mL) by increasing the expression of vasculogenic factors (i.e. IL8, IL10, bFGF, PDGF, MMP9) and adhesion molecules (i.e. integrin αV, β1, β2, β8) through the JAK2 and Akt pathway. These responses were also observed in PBMCs from elderly patients with coronary disease. The conditioned media from EPO-primed (mob)PBMCs contained various cytokines such as IL8, IL10, TNFα, and PDGF, which enhanced the migration and tube formation capability of endothelial cells. EPO-primed (mob)PBMCs also showed increased adhesion on endothelial cells or fibronectin. Augmented vasculogenic potential of EPO-primed (mob)PBMCs was confirmed in a Matrigel plug assay, ischaemic hindlimb, and myocardial infarction models of athymic nude mice. There were two action mechanisms: (i) cellular effects confirmed by direct incorporation of human (mob)PBSCs into mouse vasculature and (ii) indirect humoral effects confirmed by the therapeutic effect of the supernatant of EPO-primed (mob)PBMCs. Brief ex vivo EPO-priming is a novel method to augment the vasculogenic potential of human (mob)PBMCs, which would help to achieve better results after intracoronary infusion in myocardial infarction patients. Published on behalf of the European Society of Cardiology. All rights reserved. © The Author 2014. For permissions please email: journals.permissions@oup.com.

  1. Dissecting Interferon-Induced Transcriptional Programs in Human Peripheral Blood Cells

    PubMed Central

    Waddell, Simon J.; Popper, Stephen J.; Rubins, Kathleen H.; Griffiths, Michael J.; Brown, Patrick O.; Levin, Michael; Relman, David A.

    2010-01-01

    Interferons are key modulators of the immune system, and are central to the control of many diseases. The response of immune cells to stimuli in complex populations is the product of direct and indirect effects, and of homotypic and heterotypic cell interactions. Dissecting the global transcriptional profiles of immune cell populations may provide insights into this regulatory interplay. The host transcriptional response may also be useful in discriminating between disease states, and in understanding pathophysiology. The transcriptional programs of cell populations in health therefore provide a paradigm for deconvoluting disease-associated gene expression profiles. We used human cDNA microarrays to (1) compare the gene expression programs in human peripheral blood mononuclear cells (PBMCs) elicited by 6 major mediators of the immune response: interferons α, β, ω and γ, IL12 and TNFα; and (2) characterize the transcriptional responses of purified immune cell populations (CD4+ and CD8+ T cells, B cells, NK cells and monocytes) to IFNγ stimulation. We defined a highly stereotyped response to type I interferons, while responses to IFNγ and IL12 were largely restricted to a subset of type I interferon-inducible genes. TNFα stimulation resulted in a distinct pattern of gene expression. Cell type-specific transcriptional programs were identified, highlighting the pronounced response of monocytes to IFNγ, and emergent properties associated with IFN-mediated activation of mixed cell populations. This information provides a detailed view of cellular activation by immune mediators, and contributes an interpretive framework for the definition of host immune responses in a variety of disease settings. PMID:20339534

  2. Proteoglycans regulate the chemotaxis of dendritic cells derived from human peripheral blood monocytes.

    PubMed

    Yoshino, Hironori; Takahashi, Kenji; Monzen, Satoru; Kashiwakura, Ikuo

    2010-01-01

    Dendritic cells (DCs) are a type of antigen-presenting cell which play an essential role in the immune system. The transition from immature DC (iDCs) to mature DCs (mDCs) requires appropriate maturation stimuli, such as pro-inflammatory cytokines or pathogen-derived components. Proteoglycans (PGs), which are composed of core proteins and the glycosaminoglycans that bind to them, are one of the main components of the extracellular matrix around pathogens such as bacteria. This study investigated the effects of PG extracted from the nasal septum cartilage of whale (W-PG) on the maturation of DCs derived from human peripheral blood monocytes. iDCs were prepared from human monocytes using granulocyte-macrophage colony-stimulating factor (GM-CSF) and interleukin-4 (IL-4). The iDCs were stimulated by W-PG alone. In another type of experiment, the iDCs were stimulated by MIX (tumor necrosis factor-alpha (TNF-alpha), IL-1beta, IL-6 and prostaglandin E(2) (PGE(2))) or a combination of MIX plus W-PG. The stimulation of W-PG alone did not induce the phenotypic maturation from iDCs. However, W-PG promoted the up-regulation of chemokine receptor CCR7-surface expression and the chemotactic responsiveness to CCR7 ligand macrophage inflammatory protein-3beta on MIX-stimulated mDCs although W-PG did not influence matrix metalloproteinase-9 activity which is an important factor in DC migration through the extracellular matrix. The findings that W-PG can selectively regulate the chemotactic activity of DCs in vitro under inflammatory conditions therefore indicate that the interaction of PGs with immune cells including DCs plays an important role in the immune response under the milieu of innate immunity.

  3. Human Enterovirus in the Gastrocnemius of Patients With Peripheral Arterial Disease

    PubMed Central

    Kim, Julian K. S.; Zhu, Zhen; Casale, George; Koutakis, Panagiotis; McComb, Rodney D.; Swanson, Stanley; Thompson, Jonathan; Miserlis, Dimitrios; Johanning, Jason M.; Haynatzki, Gleb; Pipinos, Iraklis I.

    2013-01-01

    Background Peripheral arterial disease (PAD) is characterized by myofiber degeneration and loss of function in muscles of the lower limbs. Human enterovirus (HEV) infection has been implicated in the pathogenesis of a number of muscle diseases. However, its association with PAD has not been studied. In this study, we tested the hypothesis that infectious HEV is present in skeletal muscle of patients with PAD and is associated with severity of disease. Methods and Results Gastrocnemius biopsies from 37 patients with PAD and 14 controls were examined for the presence of HEV RNA, viral capsid protein, viral RNA copy number, and viral infectivity. HEV RNA was detected in 54% of the biopsies from patients with PAD but was not detected in muscle biopsies from control patients. This difference in prevalence among PAD and control patients was significant at P<0.001. Viral RNA copy numbers were increased significantly at the later stages of disease; Fontaine Stage IV (105.50 copies/mg muscle wet weight, at P<0.005) and Stage III (104.87 copies/mg, at P<0.010) compared to Stage II (102.50 copies/mg). Viral replication was confirmed by the presence of the negative‐strand of viral RNA in all specimens positive for HEV RNA. Cultures of HeLa and human skeletal muscle cells treated with muscle homogenates showed HEV replication and the presence of HEV capsid protein. Conclusion Our data identified infectious HEV in the gastrocnemius of PAD patients but not in controls. Viral copy number and prevalence of infection were higher in the later stages of disease. Our data point to the need for further studies to determine the contribution of HEV infection to the pathophysiology of PAD. PMID:23920231

  4. Combustible and non-combustible tobacco product preparations differentially regulate human peripheral blood mononuclear cell functions.

    PubMed

    Arimilli, Subhashini; Damratoski, Brad E; Prasad, G L

    2013-09-01

    Natural killer (NK) cells and T cells play essential roles in innate and adaptive immune responses in protecting against microbial infections and in tumor surveillance. Although evidence suggests that smoking causes immunosuppression, there is limited information whether the use of smokeless tobacco (ST) products affects immune responses. In this study, we assessed the effects of two preparations of cigarette smoke, ST extract and nicotine on T cell and NK cell responses using Toll-like receptor-ligand stimulated human peripheral blood mononuclear cells (PBMCs). The tobacco product preparations (TPPs) tested included whole smoke conditioned media (WS-CM), total particulate matter (TPM) and a ST product preparation in complete artificial saliva (ST/CAS). The PBMCs were stimulated with polyinosinic:polycytidylic acid (poly I:C) and lipopolysaccharide (LPS). A marked reduction of the expression of intracellular IFN-γ and TNF-α was evident in NK cells and T cells treated with WS-CM and TPM. Consistently, attenuation of ligand-induced secretion of cytokines (IL-1β, IL-10, IL-12 and TNF-α) from PBMCs treated with WS-CM and TPM were observed. While the treatment with TPPs did not alter the expression of the maturation marker CD69, WS-CM and TPM inhibited the cytolytic activity of human PBMCs. Suppression of perforin by WS-CM was also detected. Although interference from the vehicle confounded the interpretation of effects of ST/CAS, some effects were evident only at high concentrations. Nicotine treatment minimally impacted expression of cytokines and cytolytic activity. Data presented herein suggests that the function of NK cells and T cells is influenced by exposure to TPPs (based on equi-nicotine units) in the following order: WS-CM>TPM>ST/CAS. These findings are consistent with the hypothesis put forward by others that chronic smoking leads to immunosuppression, an effect that may contribute to increased microbial infections and cancer incidence among smokers.

  5. Identification of Lactobacillus plantarum genes modulating the cytokine response of human peripheral blood mononuclear cells

    PubMed Central

    2010-01-01

    Background Modulation of the immune system is one of the most plausible mechanisms underlying the beneficial effects of probiotic bacteria on human health. Presently, the specific probiotic cell products responsible for immunomodulation are largely unknown. In this study, the genetic and phenotypic diversity of strains of the Lactobacillus plantarum species were investigated to identify genes of L. plantarum with the potential to influence the amounts of cytokines interleukin 10 (IL-10) and IL-12 and the ratio of IL-10/IL-12 produced by peripheral blood mononuclear cells (PBMCs). Results A total of 42 Lactobacillus plantarum strains isolated from diverse environmental and human sources were evaluated for their capacity to stimulate cytokine production in PBMCs. The L. plantarum strains induced the secretion of the anti-inflammatory cytokine IL-10 over an average 14-fold range and secretion of the pro-inflammatory cytokine IL-12 over an average 16-fold range. Comparisons of the strain-specific cytokine responses of PBMCs to comparative genome hybridization profiles obtained with L. plantarum WCFS1 DNA microarrays (also termed gene-trait matching) resulted in the identification of 6 candidate genetic loci with immunomodulatory capacities. These loci included genes encoding an N-acetyl-glucosamine/galactosamine phosphotransferase system, the LamBDCA quorum sensing system, and components of the plantaricin (bacteriocin) biosynthesis and transport pathway. Deletion of these genes in L. plantarum WCFS1 resulted in growth phase-dependent changes in the PBMC IL-10 and IL-12 cytokine profiles compared with wild-type cells. Conclusions The altered PBMC cytokine profiles obtained with the L. plantarum WCFS1 mutants were in good agreement with the predictions made by gene-trait matching for the 42 L. plantarum strains. This study therefore resulted in the identification of genes present in certain strains of L. plantarum which might be responsible for the stimulation of anti

  6. Corticotropin releasing hormone- and adreno-corticotropin-like immunoreactivity in human placenta, peripheral and uterine vein plasma.

    PubMed

    Schulte, H M; Healy, D L

    1987-01-01

    The presence of corticotropin releasing hormone (CRH)-like immunoreactivity (IR) in human placenta and maternal peripheral blood has been reported by many investigators. However, its physiological role has not yet been defined. We investigated plasma and placental tissue from women at different times of pregnancy and performed peripheral and uterine vein sampling during caesarean section before and after removal of the placenta. Beside IR-CRH, IR-GRF and -GnRH as well as -ACTH and cortisol were measured. The highest content of CRH was found in placental extracts from end term (40 weeks) pregnancies and lower levels at an earlier stage (10 weeks). Plasma CRH from peripheral blood could be detected in some samples and was higher as pregnancy advanced. Thirty minutes after removal of the placenta CRH levels dropped in peripheral plasma and could not be detected in uterine vein samples. IR-ACTH plasma levels were within the range of normals, cortisol was elevated. Gel- and HPLC-chromatographie revealed that placental extracts coeluted with synthetic human CRH. The material from endterm placenta showed full bioactivity in the rat pituitary bio-assay. IR-GRF could only be detected in 10 weeks placental tissue and no IR-GnRH was measured. We conclude that CRH from the placenta is biologically active, however, cannot stimulate the maternal pituitary-adrenal-axis.

  7. Metabolic profile of oxazepam and related benzodiazepines: clinical and forensic aspects.

    PubMed

    Dinis-Oliveira, Ricardo Jorge

    2017-09-14

    Anxiolytic drugs, namely benzodiazepines, are the most commonly used psychoactive substances since anxiety disorders are prevalent mental disorders particularly in the Western world. Oxazepam is a short-acting benzodiazepine and one of the most frequently prescribed anxiolytic drugs. It is also the active metabolite of a wide range of other benzodiazepines, such as diazepam, ketazolam, temazepam, chlordiazepoxide, demoxazepam, halazepam, medazepam, prazepam, pinazepam, and chlorazepate. Therefore, relevant clinical and forensic outocomes may arise, namely those related to interference in driving performance. It is clinically available as a racemic formulation, with S-enantiomer being more active than R-enantiomer. In humans, it is mainly polimorphically metabolized by glucuronide conjugation at the 3-carbon hydroxyl group, yielding stable diastereomeric glucuronides (R- and S-oxazepam glucuronide). Relevant metabolic and stereoselective interspecies differences have been reported. In this work, the pharmacokinetics of oxazepam with particular focus on metabolic pathways is fully reviewed. Moreover, the metabolic profile of other prescribed benzodiazepines that produce oxazepam as a metabolite is also discussed. It is aimed that knowing the metabolism of oxazepam and related benzodiazepines may lead to the development of new analytical strategies for its early detection and help in further toxicological and clinical interpretations.

  8. IL-7 enhances thymic human T cell development in "human immune system" Rag2-/-IL-2Rgammac-/- mice without affecting peripheral T cell homeostasis.

    PubMed

    van Lent, Anja U; Dontje, Wendy; Nagasawa, Maho; Siamari, Rachida; Bakker, Arjen Q; Pouw, Stephan M; Maijoor, Kelly A; Weijer, Kees; Cornelissen, Jan J; Blom, Bianca; Di Santo, James P; Spits, Hergen; Legrand, Nicolas

    2009-12-15

    IL-7 is a central cytokine in the development of hematopoietic cells, although interspecies discrepancies have been reported. By coculturing human postnatal thymus hematopoietic progenitors and OP9-huDL1 stromal cells, we found that murine IL-7 is approximately 100-fold less potent than human IL-7 for supporting human T cell development in vitro. We investigated the role of human IL-7 in newborn BALB/c Rag2(-/-)gamma(c)(-/-) mice transplanted with human hematopoietic stem cells (HSC) as an in vivo model of human hematopoiesis using three approaches to improve IL-7 signaling: administration of human IL-7, ectopic expression of human IL-7 by the transplanted human HSC, or enforced expression of a murine/human chimeric IL-7 receptor binding murine IL-7. We show that premature IL-7 signaling at the HSC stage, before entrance in the thymus, impeded T cell development, whereas increased intrathymic IL-7 signaling significantly enhanced the maintenance of immature thymocytes. Increased thymopoiesis was also observed when we transplanted BCL-2- or BCL-x(L)-transduced human HSC. Homeostasis of peripheral mature T cells in this humanized mouse model was not improved by any of these strategies. Overall, our results provide evidence for an important role of IL-7 in human T cell development in vivo and highlight the notion that IL-7 availability is but one of many signals that condition peripheral T cell homeostasis.

  9. TRPA1 receptor localisation in the human peripheral nervous system and functional studies in cultured human and rat sensory neurons.

    PubMed

    Anand, U; Otto, W R; Facer, P; Zebda, N; Selmer, I; Gunthorpe, M J; Chessell, I P; Sinisi, M; Birch, R; Anand, P

    2008-06-20

    TRPA1 is a receptor expressed by sensory neurons, that is activated by low temperature (<17 degrees C) and plant derivatives such as cinnamaldehyde and isoeugenol, to elicit sensations including pain. Using immunohistochemistry, we have, for the first time, localised TRPA1 in human DRG neurons, spinal cord motoneurones and nerve roots, peripheral nerves, intestinal myenteric plexus neurones, and skin basal keratinocytes. TRPA1 co-localised with a subset of hDRG neurons positive for TRPV1, the heat and capsaicin receptor. The number of small/medium TRPA1 positive neurons (< or =50 microm) was increased after hDRG avulsion injury [percentage of cells, median (range): controls 16.5 (7-23); injured 46 (34-55); P<0.005], but the number of large TRPA1 neurons was unchanged [control 19.5 (13-31); injured 21 (11-35)]. Similar TRPA1 changes were observed in cultured hDRG neurons, after exposure to a combination of key neurotrophic factors NGF, GDNF and NT-3 (NTFs) in vitro. We used calcium imaging to examine responses of HEK cells transfected with hTRPA1 cDNA, and of human and rat DRG neurons cultured with or without added NTFs, to cinnamaldehyde (CA) and isoeugenol (IE). Exposure to NTFs in vitro sensitized cultured human sensory neuronal responses to CA; repeated CA exposure produced desensitisation. In rDRG neurons, low (225 microM) CA preincubation enhanced capsaicin responses, while high (450 microM and 2mM) CA caused inhibition which was partially reversed in the presence of 8 bromo cAMP, indicating receptor dephosphorylation. While TRPA1 localisation is more widespread than TRPV1, it represents a promising novel drug target for the treatment of chronic pain and hypersensitivity.

  10. Adherence of human peripheral blood lymphocytes to measles-virus infected cells: modulation by solubilized rhesus erythrocyte membranes and carbohydrates.

    PubMed Central

    Bankhurst, A D; Maki, D; Sanchez, M; McLaren, L

    1979-01-01

    The adherence of human peripheral blood lymphocytes to HeLa cells persistently infected with measles virus (HeLa-K11) was studied. The following data were observed. (i) The proportion of HeLa-K11 cells with adherent human peripheral blood lymphocytes of rhesus monkey erythrocytes was similar over a wide range of ratios of HeLa-K11 cells to lymphocytes or erythrocytes. (ii) The great majority of human peripheral blood lymphocytes and erythrocytes reacted with the same HeLa-K11 cell (iii). The adherence of lymphocytes or erythrocytes to HeLa-K11 cells was blocked by rabbit anti-measles virus antibody or solubilized monkey erythrocyte membranes. The pretreatment of erythrocytes or lymphocytes with receptor-destroying enzyme did not alter their adherence properties. (iv) The pattern of inhibition observed with several carbohydrates was similar in both the erythrocyte and the lymphocyte adherence assays. These data are consistent with the possibility that the receptor present on both rhesus monkey erythrocytes and human lymphocytes has similar specificities and biochemical composition. PMID:572346

  11. Still no evidence that benzodiazepines cause depression.

    PubMed

    Patten, Scott B

    2008-01-01

    A large number of drugs have been implicated in causing depression by case reports and case series. For a few specific drugs, the association has subsequently been confirmed by appropriately designed studies. In other instances, a lack of substantiating evidence has lead to a gradual disappearance of concern about a potential association. The benzodiazepines represent a deviation from this pattern: they are widely believed to cause depression, but there is a lack of evidence to substantiate this claim. In DSM-IV, there is a category of mood disorder for drug-induced depression (substance-induced mood disorder), and the text of the manual specifically refers to benzodiazepines as a potential cause. Despite the apparently entrenched nature of this belief, there continues to be a lack of credible evidence that benzodiazepines can cause depression as a side effect.

  12. Measurement of the Phagocytic Activity of Human Peripheral Blood Using a Highly Sensitive Fluorometric Detection Device Without Hemolysis.

    PubMed

    Zhang, Ran; Inagawa, Hiroyuki; Takahashi, Masaru; Kawanishi, Hisami; Kazumura, Kimiko; Tsuchiya, Hiroshi; Morishita, Naokazu; Kobayashi, Yutaro; Masaki, Tsutomu; Kobara, Hideki; Soma, Gen-Ichiro

    2017-07-01

    Phagocytes recognize pathogens that enter the body as well as other abnormal and foreign materials that may exist within an organism (such as dead cells, oxidized lipids, and denatured proteins), and phagocytose and eliminate them to maintain a healthy state. In a previous study a simple prototype device was used, under development by Hamamatsu Photonics (Prototype), that detects fluorescence to determine the phagocytic activity of the murine macrophage cell line J774.1. The present study aimed to determine whether it was possible to detect phagocytic activity in a slight amount of human peripheral blood without using hemolysis. Three microliters of human peripheral blood was drawn from the fingertip and mixed with 30 μg of pH-sensitive fluorescent particles. The fluorescence intensity of the human peripheral blood sample was then measured using the Prototype in development, cultured for 2 h at 37°C, and then re-measured. The phagocytes were observed under fluorescence microscopy and the phagocytosis rate of CD11b-positive cells was verified with a flow cytometer. The phagocytic activity of non-hemolyzed human peripheral blood was measured using the Prototype under development; fluorescence after phagocytosis was detected. Furthermore, this was confirmed by both fluorescence microscopy and flow cytometry. The precision of the measurements of human peripheral blood phagocytic activity was verified with the Prototype using samples from three healthy individuals. The relationship between blood sugar levels and phagocytic activity before and after meal times was determined. Concerning exercise, phagocytic activity tended to decrease, although salivary amylase level increased in the healthy individual examined after exercise. The simple Prototype can measure phagocytic activity in a small amount of peripheral blood without hemolysis. The device allows for rapid and minimally-invasive detection of changes in phagocytic activity, which has conventionally been difficult

  13. Translocator protein (18 kD) as target for anxiolytics without benzodiazepine-like side effects.

    PubMed

    Rupprecht, Rainer; Rammes, Gerhard; Eser, Daniela; Baghai, Thomas C; Schüle, Cornelius; Nothdurfter, Caroline; Troxler, Thomas; Gentsch, Conrad; Kalkman, Hans O; Chaperon, Frederique; Uzunov, Veska; McAllister, Kevin H; Bertaina-Anglade, Valerie; La Rochelle, Christophe Drieu; Tuerck, Dietrich; Floesser, Annette; Kiese, Beate; Schumacher, Michael; Landgraf, Rainer; Holsboer, Florian; Kucher, Klaus

    2009-07-24

    Most antianxiety drugs (anxiolytics) work by modulating neurotransmitters in the brain. Benzodiazepines are fast and effective anxiolytic drugs; however, their long-term use is limited by the development of tolerance and withdrawal symptoms. Ligands of the translocator protein [18 kilodaltons (kD)] may promote the synthesis of endogenous neurosteroids, which also exert anxiolytic effects in animal models. Here, we found that the translocator protein (18 kD) ligand XBD173 enhanced gamma-aminobutyric acid-mediated neurotransmission and counteracted induced panic attacks in rodents in the absence of sedation and tolerance development. XBD173 also exerted antipanic activity in humans and, in contrast to benzodiazepines, did not cause sedation or withdrawal symptoms. Thus, translocator protein (18 kD) ligands are promising candidates for fast-acting anxiolytic drugs with less severe side effects than benzodiazepines.

  14. The Role of Human Adult Peripheral and Umbilical Cord Blood Platelet-Rich Plasma on Proliferation and Migration of Human Skin Fibroblasts.

    PubMed

    Hashemi, Seyedeh-Sara; Mahmoodi, Mahdokht; Rafati, Ali Reza; Manafi, Farzad; Mehrabani, Davood

    2017-05-01

    Wound healing is a complex and dynamic process following damage in tissue structures. Due to extensive skin damage caused by burn injuries, this study determined the role of human adult peripheral and umbilical cord blood platelet-rich plasma on proliferation and migration in human skin fibroblasts. Platelet-rich plasma (5, 10, 15, 20 and 50% PRP) from human umbilical cord blood and adult peripheral blood were provided and added to fibroblasts cultured from a human skin sample. Migration and proliferation of fibroblasts were assessed in comparison to 10% FBS and by the fibroblast responses to a concentration gradient. All components of the umbilical cord blood PRP significantly stimulated the growth of fibroblasts when compared to the negative control. Fibroblast growth was enhanced in a dose dependent manner. All fibroblast cultures retained normal morphology. No significant difference was noted between umbilical cord blood and adult peripheral blood PRP preparations regarding cell proliferation and migration, but the difference to 10% FBS was significant. 1% and 50% PRP reduced cellular proliferation. The 20% umbilical cord blood PRP and 10% adult peripheral blood PRP had a significant stimulatory effect on the migration of the skin fibroblast cells in comparison with 10% FBS. As PRP could promote the migration and proliferation of dermal fibroblasts, it can be safely added in cultures when treatment of chronic wounds without triggering the immune response is needed.

  15. Peripheral viewing during exposure to a 2D/3D video clip: effects on the human body.

    PubMed

    Takada, Masumi; Fukui, Yuta; Matsuura, Yasuyuki; Sato, Motohiko; Takada, Hiroki

    2015-03-01

    Symptoms of three-dimensional (3D) sickness, such as intoxication and eye fatigue, have been observed in subjects viewing 3D films and vary according to the image quality and visual environment. In addition, the influence of stereoscopic vision on the incidence of 3D sickness has not been explored sufficiently. Therefore, it is important to examine the safety of viewing virtual 3D content. The present study examines the effects of peripheral vision on reported motion sickness during exposure to 2D/3D video clips for 1 min and for 1 min afterwards in human subjects. Stabilograms were recorded during exposure to video clips with or without visual pursuit of a 3D object and compared, and subjects were administered the simulator sickness questionnaire after stabilometry. There were no significant main effects of solidity of the visual stimulous (2D/3D) and the vision method (visual pursuit/peripheral viewing) in accordance with the two-way analysis of variance of the sway values, although the sway values during the 2D/3D video clips were higher than in control subjects. A consistent trend in the main effect of stability was observed. Further, the sway values changed remarkably after the 3D video clip was viewed peripherally and produced a persistent instability in equilibrium function. The questionnaire findings also significantly changed after the subjects viewed the video clips peripherally. Subjective exacerbation and deterioration of equilibrium function were observed after peripheral viewing of 3D video clips. This persistent influence may result when subjects view a poorly depicted background element peripherally, which generates depth perception that contradicts daily experience.

  16. Screening of benzodiazepines in thirty European rivers.

    PubMed

    Fick, Jerker; Brodin, Tomas; Heynen, Martina; Klaminder, Jonatan; Jonsson, Micael; Grabicova, Katerina; Randak, Tomas; Grabic, Roman; Kodes, Vit; Slobodnik, Jaroslav; Sweetman, Andrew; Earnshaw, Mark; Barra Caracciolo, Anna; Lettieri, Teresa; Loos, Robert

    2017-06-01

    Pharmaceuticals as environmental contaminants have received a lot of interest over the past decade but, for several pharmaceuticals, relatively little is known about their occurrence in European surface waters. Benzodiazepines, a class of pharmaceuticals with anxiolytic properties, have received interest due to their behavioral modifying effect on exposed biota. In this study, our results show the presence of one or more benzodiazepine(s) in 86% of the analyzed surface water samples (n = 138) from 30 rivers, representing seven larger European catchments. Of the 13 benzodiazepines included in the study, we detected 9, which together showed median and mean concentrations (of the results above limit of quantification) of 5.4 and 9.6 ng L(-1), respectively. Four benzodiazepines (oxazepam, temazepam, clobazam, and bromazepam) were the most commonly detected. In particular, oxazepam had the highest frequency of detection (85%) and a maximum concentration of 61 ng L(-1). Temazepam and clobazam were found in 26% (maximum concentration of 39 ng L(-1)) and 14% (maximum concentration of 11 ng L(-1)) of the samples analyzed, respectively. Finally, bromazepam was found only in Germany and in 16 out of total 138 samples (12%), with a maximum concentration of 320 ng L(-1). This study clearly shows that benzodiazepines are common micro-contaminants of the largest European river systems at ng L(-1) levels. Although these concentrations are more than a magnitude lower than those reported to have effective effects on exposed biota, environmental effects cannot be excluded considering the possibility of additive and sub-lethal effects. Copyright © 2017 Elsevier Ltd. All rights reserved.

  17. Microneurography as a tool in clinical neurophysiology to investigate peripheral neural traffic in humans.

    PubMed

    Mano, Tadaaki; Iwase, Satoshi; Toma, Shinobu

    2006-11-01

    Microneurography is a method using metal microelectrodes to investigate directly identified neural traffic in myelinated as well as unmyelinated efferent and afferent nerves leading to and coming from muscle and skin in human peripheral nerves in situ. The present paper reviews how this technique has been used in clinical neurophysiology to elucidate the neural mechanisms of autonomic regulation, motor control and sensory functions in humans under physiological and pathological conditions. Microneurography is particularly important to investigate efferent and afferent neural traffic in unmyelinated C fibers. The recording of efferent discharges in postganglionic sympathetic C efferent fibers innervating muscle and skin (muscle sympathetic nerve activity; MSNA and skin sympathetic nerve activity; SSNA) provides direct information about neural control of autonomic effector organs including blood vessels and sweat glands. Sympathetic microneurography has become a potent tool to reveal neural functions and dysfunctions concerning blood pressure control and thermoregulation. This recording has been used not only in wake conditions but also in sleep to investigate changes in sympathetic neural traffic during sleep and sleep-related events such as sleep apnea. The same recording was also successfully carried out by astronauts during spaceflight. Recordings of afferent discharges from muscle mechanoreceptors have been used to understand the mechanisms of motor control. Muscle spindle afferent information is particularly important for the control of fine precise movements. It may also play important roles to predict behavior outcomes during learning of a motor task. Recordings of discharges in myelinated afferent fibers from skin mechanoreceptors have provided not only objective information about mechanoreceptive cutaneous sensation but also the roles of these signals in fine motor control. Unmyelinated mechanoreceptive afferent discharges from hairy skin seem to be

  18. Selected scorpion toxin exposures induce cytokine release in human peripheral blood mononuclear cells.

    PubMed

    Corzo, Gerardo; Espino-Solis, Gerardo Pavel

    2017-03-01

    A cytokine screening on human peripheral blood mononuclear cells (PBMCs) stimulated with selected scorpion toxins (ScTx's) was performed in order to evaluate their effect on human immune cells. The ScTx's chosen for this report were three typical buthid scorpion venom peptides, one with lethal effects on mammals Centruroides suffussus suffusus toxin II (CssII), another, with lethal effects on insects and crustaceans Centruroides noxius toxin 5 (Cn5), and one more without lethal effects Tityus discrepans toxin (Discrepin). A Luminex multiplex analysis was performed in order to determine the amounts chemokines and cytokines IL-1β, IL-2, IL-4, IL-5, IL-6, IL-10, IL-12-p40, IL-13, interferon alpha (IFN-α), interferon gamma (IFN-γ), tumor necrosis factor alpha TNF-α, and interferon-inducible protein-10 (IP-10) secreted from human PBMCs exposed to these toxins. Although, the ScTx Cn5 is not lethal for mammals, it was able to induce the secretion of cytokines IL-1β, IL-6, and TNF-α, IL-10 and IP-10 in comparison to the lethal CssII, which was able to induce only IP-10 secretion. Discrepin also was able to induce only IP-10. Interestingly, only low amounts of interferons α and β were induced in the presence of the ScTx's assayed. In a synergic experiment, the combination of Discrepin and Cn5 displayed considerable reverse effects on induction of IL-1β, IL-6, IL-10 and TNF-α, but they had a slight synergic effect on IP-10 cytokine production in comparison with the single effect obtained with the Cn5 alone. Thus, the results obtained suggest that the profile of secreted cytokines promoted by ScTx Cn5 is highly related with a cytokine storm event, and also it suggests that the mammalian lethal neurotoxins are not solely responsible of the scorpion envenomation symptomatology. Copyright © 2017 Elsevier Ltd. All rights reserved.

  19. Effect of sulfur mustard exposure on protease activity in human peripheral blood lymphocytes. (Reannouncement with new availability information)

    SciTech Connect

    Cowan, F.M.; Broomfield, C.A.; Smith, W.J.

    1991-12-31

    Sulfur mustard is a chemical warfare blistering agent for which neither the mechanism of action nor an antidote is known. Papirmeister et al. (1985) have postulated a biochemical hypothesis for mustard-induced cutaneous injury involving a sequelae of DNA alkylation, metabolic disruption and activation of protease. Human peripheral blood lymphocytes in cell cultures were employed as an in vitro model for alkylating agent toxicity. A chromogenic peptide substrate assay was used for detection of protease in lymphocytes treated with sulfur mustard or chloroethyl sulfide. Exposure of human peripheral blood lymphocytes from normal donors to these alkylating agents resulted in an increase in cell associated protease activity. This increase in protease activity may contribute to the pathology or act as an indicator to predict methods of therapeutic intervention for sulfur mustard toxicity.

  20. Sensitivity of PCR and real-time PCR for the diagnosis of human visceral leishmaniasis using peripheral blood

    PubMed Central

    da Costa Lima, Manoel Sebastião; Zorzenon, Denielly Christina Rodrigues; Dorval, Maria Elizabeth Cavalheiros; Pontes, Elenir Rose Jardim Cury; Oshiro, Elisa Teruya; Cunha, Rodrigo; Andreotti, Renato; Matos, Maria de Fatima Cepa

    2013-01-01

    Objective To evaluate the effectiveness of PCR and real-time PCR for the diagnosis of human visceral leishmaniasis using peripheral blood samples. Methods DNA extraction was performed using Promega Wizard® Genomic kits. PCR employing RV1/RV2 primers yielded 145-bp amplicons. Real-time PCR was performed with the same primers and SYBR Green ROX Plus mix. These techniques were used to analyze 100 peripheral blood samples from patients with clinical signs of the disease. Results The sensitivity and specificity levels were 91,3%% and 29,6%, respectively, for real-time PCR and 97.78% and 61.82%, respectively, for PCR. Conclusions Real-time PCR proved to be a satisfactory method for the diagnosis of human visceral leishmaniasis.

  1. Cysteamine suppresses human peripheral blood mononuclear cells – human corneal endothelial cell reaction via reactive oxygen species reduction

    PubMed Central

    Hyon, Joon Young; Kim, Seonhowa; Koh, Jae Woong; Kwon, Soon Il; Wee, Won Ryang

    2011-01-01

    Purpose To investigate the effect of cysteamine (CYS) on mixed peripheral blood mononuclear cells (PBMCs) - human corneal endothelial cell (HCEC) reaction (MLER). Methods PBMC stimulation assay was performed using cultured HCEC. MLERs were treated with various concentrations of CYS (0–20 mM). The proliferation rate and secretion profiles of transforming growth factor-β1 (TGF-β1) and interleukin-6 (IL-6) of PBMCs stimulated by cultured HCEC were determined using bromodeoxyuridine proliferation assay and enzyme-linked immunosorbent assay, respectively. Results CYS suppressed PBMC proliferation in a dose-dependent manner (p<0.001). The intracellular reactive oxygen species (ROS) levels decreased with an increase in CYS concentration (p<0.001). The levels of TGF-β1 and IL-6 decreased in a dose-dependent manner as well (p=0.011 and 0.003, respectively). Conclusions This study showed that CYS decreased PBMC proliferation, IL-6 and TGF-β1 levels via ROS formation. Our results suggest that CYS could suppress inflammation associated with PBMCs to corneal endothelial cells. PMID:22219632

  2. Change in peripheral refraction and curvature of field of the human eye with accommodation

    NASA Astrophysics Data System (ADS)

    Ho, Arthur; Zimmermann, Frederik; Whatham, Andrew; Martinez, Aldo; Delgado, Stephanie; Lazon de la Jara, Percy; Sankaridurg, Padmaja

    2009-02-01

    Recent research showed that the peripheral refractive state is a sufficient stimulus for myopia progression. This finding led to the suggestion that devices that control peripheral refraction may be efficacious in controlling myopia progression. This study aims to understand whether the optical effect of such devices may be affected by near focus. In particular, we seek to understand the influence of accommodation on peripheral refraction and curvature of field of the eye. Refraction was measured in twenty young subjects using an autorefractor at 0° (i.e. along visual axis), and 20°, 30° and 40° field angles both nasal and temporal to the visual axis. All measurements were conducted at 2.5 m, 40 cm and 30 cm viewing distances. Refractive errors were corrected using a soft contact lens during all measurements. As field angle increased, refraction became less hyperopic. Peripheral refraction also became less hyperopic at nearer viewing distances (i.e. with increasing accommodation). Astigmatism (J180) increased with field angle as well as with accommodation. Adopting a third-order aberration theory approach, the position of the Petzval surface relative to the retinal surface was estimated by considering the relative peripheral refractive error (RPRE) and J180 terms of peripheral refraction. Results for the estimated dioptric position of the Petzval surface relative to the retina showed substantial asymmetry. While temporal field tended to agree with theoretical predictions, nasal response departed dramatically from the model eye predictions. With increasing accommodation, peripheral refraction becomes less hyperopic while the Petzval surface showed asymmetry in its change in position. The change in the optical components (i.e. cornea and/or lens as opposed to retinal shape or position) is implicated as at least one of the contributors of this shift in peripheral refraction during accommodation.

  3. Enriched population of PNS neurons derived from human embryonic stem cells as a platform for studying peripheral neuropathies.

    PubMed

    Valensi-Kurtz, Moran; Lefler, Sharon; Cohen, Malkiel A; Aharonowiz, Michal; Cohen-Kupiec, Rachel; Sheinin, Anton; Ashery, Uri; Reubinoff, Benjamin; Weil, Miguel

    2010-02-18

    The absence of a suitable cellular model is a major obstacle for the study of peripheral neuropathies. Human embryonic stem cells hold the potential to be differentiated into peripheral neurons which makes them a suitable candidate for this purpose. However, so far the potential of hESC to differentiate into derivatives of the peripheral nervous system (PNS) was not investigated enough and in particular, the few trials conducted resulted in low yields of PNS neurons. Here we describe a novel hESC differentiation method to produce enriched populations of PNS mature neurons. By plating 8 weeks hESC derived neural progenitors (hESC-NPs) on laminin for two weeks in a defined medium, we demonstrate that over 70% of the resulting neurons express PNS markers and 30% of these cells are sensory neurons. Our method shows that the hNPs express neuronal crest lineage markers in a temporal manner, and by plating 8 weeks hESC-NPs into laminin coated dishes these hNPs were promoted to differentiate and give rise to homogeneous PNS neuronal populations, expressing several PNS lineage-specific markers. Importantly, these cultures produced functional neurons with electrophysiological activities typical of mature neurons. Moreover, supporting this physiological capacity implantation of 8 weeks old hESC-NPs into the neural tube of chick embryos also produced human neurons expressing specific PNS markers in vivo in just a few days. Having the enriched PNS differentiation system in hand, we show for the first time in human PNS neurons the expression of IKAP/hELP1 protein, where a splicing mutation on the gene encoding this protein causes the peripheral neuropathy Familial Dysautonomia. We conclude that this differentiation system to produce high numbers of human PNS neurons will be useful for studying PNS related neuropathies and for developing future drug screening applications for these diseases.

  4. Enriched Population of PNS Neurons Derived from Human Embryonic Stem Cells as a Platform for Studying Peripheral Neuropathies

    PubMed Central

    Cohen, Malkiel A.; Aharonowiz, Michal; Cohen-Kupiec, Rachel; Sheinin, Anton; Ashery, Uri; Reubinoff, Benjamin; Weil, Miguel

    2010-01-01

    Background The absence of a suitable cellular model is a major obstacle for the study of peripheral neuropathies. Human embryonic stem cells hold the potential to be differentiated into peripheral neurons which makes them a suitable candidate for this purpose. However, so far the potential of hESC to differentiate into derivatives of the peripheral nervous system (PNS) was not investigated enough and in particular, the few trials conducted resulted in low yields of PNS neurons. Here we describe a novel hESC differentiation method to produce enriched populations of PNS mature neurons. By plating 8 weeks hESC derived neural progenitors (hESC-NPs) on laminin for two weeks in a defined medium, we demonstrate that over 70% of the resulting neurons express PNS markers and 30% of these cells are sensory neurons. Methods/Findings Our method shows that the hNPs express neuronal crest lineage markers in a temporal manner, and by plating 8 weeks hESC-NPs into laminin coated dishes these hNPs were promoted to differentiate and give rise to homogeneous PNS neuronal populations, expressing several PNS lineage-specific markers. Importantly, these cultures produced functional neurons with electrophysiological activities typical of mature neurons. Moreover, supporting this physiological capacity implantation of 8 weeks old hESC-NPs into the neural tube of chick embryos also produced human neurons expressing specific PNS markers in vivo in just a few days. Having the enriched PNS differentiation system in hand, we show for the first time in human PNS neurons the expression of IKAP/hELP1 protein, where a splicing mutation on the gene encoding this protein causes the peripheral neuropathy Familial Dysautonomia. Conclusions/Significance We conclude that this differentiation system to produce high numbers of human PNS neurons will be useful for studying PNS related neuropathies and for developing future drug screening applications for these diseases. PMID:20174633

  5. Human severe combined immunodeficiency disease: phenotypic and functional characteristics of peripheral B lymphocytes.

    PubMed

    Gougeon, M L; Drean, G; Le Deist, F; Dousseau, M; Fevrier, M; Diu, A; Theze, J; Griscelli, C; Fischer, A

    1990-11-01

    Human severe combined immunodeficiency disease (SCID) includes an X-chromosome-linked type characterized by a complete absence of mature T cells, hypogammaglobulinemia but normal or elevated number of B cells, suggesting that the disease results from a block in early T cell differentiation. It has been shown that B cells from obligate carrier women of this disorder exhibit the preferential use of the nonmutant X chromosome as the active X (as shown for T cells), suggesting that the SCID gene product has a direct effect on B cells as well as on T cells. To examine this question, we analyzed the phenotypic and functional characteristics of peripheral B cells from nine infants with SCID. We found a constant absence of spontaneously expressed activation Ag on B cell membrane from all SCID patients tested which contrasts with the phenotypic pattern exhibited by age-matched infants whom all cells bearing surface Ig express the 4F2 Ag and to a lesser extent the transferrin receptor. Concurrently, B cells from SCID patients have a profound impairment in their responses to stimuli that induce in vitro B cell proliferation and differentiation. Although rIL-2 and low-Mr B cell growth factor are potent inducers of proliferation on age-matched infants' B cells, they are poorly efficient in inducing proliferation of anti-mu-activated SCID B cells. This impairment is not related to the resting B cell phenotype of SCID B cells as shown by comparison with normal resting B cells. Furthermore, we observed an apparent block in B cell differentiation inasmuch as neither rIL-2 nor rIL-6 could support SAC-activated SCID B cell differentiation, both lymphokines being very efficient in inducing SAC-activated age-matched infants' B cell or purified resting B cell differentiation. These results suggest that the SCID gene defect has a direct effect on B cells and is required during B cell maturation.

  6. Electrical Stimulation to Enhance Axon Regeneration After Peripheral Nerve Injuries in Animal Models and Humans.

    PubMed

    Gordon, Tessa

    2016-04-01

    Injured peripheral nerves regenerate their lost axons but functional recovery in humans is frequently disappointing. This is so particularly when injuries require regeneration over long distances and/or over long time periods. Fat replacement of chronically denervated muscles, a commonly accepted explanation, does not account for poor functional recovery. Rather, the basis for the poor nerve regeneration is the transient expression of growth-associated genes that accounts for declining regenerative capacity of neurons and the regenerative support of Schwann cells over time. Brief low-frequency electrical stimulation accelerates motor and sensory axon outgrowth across injury sites that, even after delayed surgical repair of injured nerves in animal models and patients, enhances nerve regeneration and target reinnervation. The stimulation elevates neuronal cyclic adenosine monophosphate and, in turn, the expression of neurotrophic factors and other growth-associated genes, including cytoskeletal proteins. Electrical stimulation of denervated muscles immediately after nerve transection and surgical repair also accelerates muscle reinnervation but, at this time, how the daily requirement of long-duration electrical pulses can be delivered to muscles remains a practical issue prior to translation to patients. Finally, the technique of inserting autologous nerve grafts that bridge between a donor nerve and an adjacent recipient denervated nerve stump significantly improves nerve regeneration after delayed nerve repair, the donor nerves sustaining the capacity of the denervated Schwann cells to support nerve regeneration. These reviewed methods to promote nerve regeneration and, in turn, to enhance functional recovery after nerve injury and surgical repair are sufficiently promising for early translation to the clinic.

  7. Impact of Neutron Exposure on Global Gene Expression in a Human Peripheral Blood Model.

    PubMed

    Broustas, Constantinos G; Xu, Yanping; Harken, Andrew D; Chowdhury, Mashkura; Garty, Guy; Amundson, Sally A

    2017-04-01

    The detonation of an improvised nuclear device would produce prompt radiation consisting of both photons (gamma rays) and neutrons. While much effort in recent years has gone into the development of radiation biodosimetry methods suitable for mass triage, the possible effect of neutrons on the endpoints studied has remained largely uninvestigated. We have used a novel neutron irradiator with an energy spectrum based on that 1-1.5 km from the epicenter of the Hiroshima blast to begin examining the effect of neutrons on global gene expression, and the impact this may have on the development of gene expression signatures for radiation biodosimetry. We have exposed peripheral blood from healthy human donors to 0.1, 0.3, 0.5 or 1 Gy of neutrons ex vivo using our neutron irradiator, and compared the transcriptomic response 24 h later to that resulting from sham exposure or exposure to 0.1, 0.3, 0.5, 1, 2 or 4 Gy of photons (X rays). We identified 125 genes that responded significantly to both radiation qualities as a function of dose, with the magnitude of response to neutrons generally being greater than that seen after X-ray exposure. Gene ontology analysis suggested broad involvement of the p53 signaling pathway and general DNA damage response functions across all doses of both radiation qualities. Regulation of immune response and chromatin-related functions were implicated only following the highest doses of neutrons, suggesting a physiological impact of greater DNA damage. We also identified several genes that seem to respond primarily as a function of dose, with less effect of radiation quality. We confirmed this pattern of response by quantitative real-time RT-PCR for BAX, TNFRSF10B, ITLN2 and AEN and suggest that gene expression may provide a means to differentiate between total dose and a neutron component.

  8. Proteolytic enzymes and amylase induce cytokine production in human peripheral blood mononuclear cells in vitro.

    PubMed

    Desser, L; Rehberger, A; Paukovits, W

    1994-01-01

    In vitro treatment of human peripheral blood mononuclear cells (PBMNC) with proteolytic enzymes (bromelain, papain) and amylase leads to the production of large amounts of tumor necrosis factor-alpha (TNF-alpha), interleukin-1-beta (IL-1 beta), and interleukin-6 (IL-6) in a time and dose dependent manner. Increased TNF-alpha and IL-6 production was already found after 4-6 hours of incubation, and plateau levels were reached after 12-16 hours. Plateau levels up to 1500 pg TNF-alpha/ml/10(6) PBMNC, 13000 pg IL-1 beta/ml/10(6) PBMNC, and 23000 pg IL-6/ml/10(6) PBMNC were observed. Control cultures contained below 35 pg/ml/10(6) PBMNC of TNF-alpha, IL-1 beta or IL-6. In contrast to TNF-alpha which was undetectable after more than 24 hours, peak levels of IL-1 beta and IL-6 were still present at 24 hours. After incubation of the enzyme solution for some hours at 56 degrees C the cytokine inducing capacity disappeared. Neutralization experiments with inactivating antibodies, radioimmunoassay, and western blotting after electrophoretic separation showed that the TNF-like activity found in the lytic assay was due to TNF-alpha. Interferon-alpha (IFN-alpha) and Interferon-gamma (IFN-gamma), which had no effect alone, synergistically increased TNF-alpha production when applied together with the enzymes. A commercial mixture of these enzymes (Wobenzym), which was also investigated, showed a similar concentration and time dependence, as well as synergism with the interferons. A synergistic effect on TNF-alpha production was also found with the enzymes and phorbol ester (PMA).

  9. Lipopolysaccharide Stimulates Butyric Acid-Induced Apoptosis in Human Peripheral Blood Mononuclear Cells

    PubMed Central

    Kurita-Ochiai, Tomoko; Fukushima, Kazuo; Ochiai, Kuniyasu

    1999-01-01

    We previously reported that butyric acid, an extracellular metabolite from periodontopathic bacteria, induced apoptosis in murine thymocytes, splenic T cells, and human Jurkat T cells. In this study, we examined the ability of butyric acid to induce apoptosis in peripheral blood mononuclear cells (PBMC) and the effect of bacterial lipopolysaccharide (LPS) on this apoptosis. Butyric acid significantly inhibited the anti-CD3 monoclonal antibody- and concanavalin A-induced proliferative responses in a dose-dependent fashion. This inhibition of PBMC growth by butyric acid depended on apoptosis in vitro. It was characterized by internucleosomal DNA digestion and revealed by gel electrophoresis followed by a colorimetric DNA fragmentation assay to occur in a concentration-dependent fashion. Butyric acid-induced PBMC apoptosis was accompanied by caspase-3 protease activity but not by caspase-1 protease activity. LPS potentiated butyric acid-induced PBMC apoptosis in a dose-dependent manner. Flow-cytometric analysis revealed that LPS increased the proportion of sub-G1 cells and the number of late-stage apoptotic cells induced by butyric acid. Annexin V binding experiments with fractionated subpopulations of PBMC in flow cytometory revealed that LPS accelerated the butyric acid-induced CD3+-T-cell apoptosis followed by similar levels of both CD4+- and CD8+-T-cell apoptosis. The addition of LPS to PBMC cultures did not cause DNA fragmentation, suggesting that LPS was unable to induce PBMC apoptosis directly. These data suggest that LPS, in combination with butyric acid, potentiates CD3+ PBMC T-cell apoptosis and plays a role in the apoptotic depletion of CD4+ and CD8+ cells. PMID:9864191

  10. Impact of Neutron Exposure on Global Gene Expression in a Human Peripheral Blood Model

    PubMed Central

    Broustas, Constantinos G.; Xu, Yanping; Harken, Andrew D.; Chowdhury, Mashkura; Garty, Guy; Amundson, Sally A.

    2017-01-01

    The detonation of an improvised nuclear device would produce prompt radiation consisting of both photons (gamma rays) and neutrons. While much effort in recent years has gone into the development of radiation biodosimetry methods suitable for mass triage, the possible effect of neutrons on the endpoints studied has remained largely uninvestigated. We have used a novel neutron irradiator with an energy spectrum based on that 1–1.5 km from the epicenter of the Hiroshima blast to begin examining the effect of neutrons on global gene expression, and the impact this may have on the development of gene expression signatures for radiation biodosimetry. We have exposed peripheral blood from healthy human donors to 0.1, 0.3, 0.5 or 1 Gy of neutrons ex vivo using our neutron irradiator, and compared the transcriptomic response 24 h later to that resulting from sham exposure or exposure to 0.1, 0.3, 0.5, 1, 2 or 4 Gy of photons (X rays). We identified 125 genes that responded significantly to both radiation qualities as a function of dose, with the magnitude of response to neutrons generally being greater than that seen after X-ray exposure. Gene ontology analysis suggested broad involvement of the p53 signaling pathway and general DNA damage response functions across all doses of both radiation qualities. Regulation of immune response and chromatin-related functions were implicated only following the highest doses of neutrons, suggesting a physiological impact of greater DNA damage. We also identified several genes that seem to respond primarily as a function of dose, with less effect of radiation quality. We confirmed this pattern of response by quantitative real-time RT-PCR for BAX, TNFRSF10B, ITLN2 and AEN and suggest that gene expression may provide a means to differentiate between total dose and a neutron component. PMID:28140791

  11. Sodium tungstate (Na2WO4) exposure increases apoptosis in human peripheral blood lymphocytes.

    PubMed

    Osterburg, Andrew R; Robinson, Chad T; Schwemberger, Sandy; Mokashi, Vishwesh; Stockelman, Michael; Babcock, George F

    2010-01-01

    The potential for adverse health effects of using tungsten and its alloys in military munitions are an important concern to both civilians and the US military. The toxicological implications of exposure to tungsten, its alloys, and the soluble tungstate (Na(2)WO(4)) are currently under investigation. To examine tungstate toxicity, a series of experiments to determine its in vitro effects on cells of the immune system were performed. We identified alterations in isolated human peripheral blood lymphocytes (PBL) treated in vitro with sodium tungstate (0.01, 0.1, 1.0, and 10 mM). Analyses of apoptosis with annexin V and propidium iodide revealed a dose- and time-dependent increase in the quantity of cells in early apoptosis after tungstate exposure. Reductions in the number of cells entering into the cell cycle were also noted. Exposure of PBL to tungstate (1 mM) and Concanavalin A (ConA) for 72 h reduced the number of cells in S and G(2)/M phases of the cell cycle. There were alterations in the numbers of cells in G(0)/G(1), S, and G(2)/M phases of the cell cycle in long-term THP-1 (acute leukemic monocytes) cultures treated with tungstate (0.01, 0.1, 1.0, and 10 mM). Gel electrophoresis, silver staining, and LC-MS/MS showed the cytoplasmic presence of histone H1b and H1d after 72 h of tungstate exposure. The addition of tungstate to cultures resulted in significant reductions in the quantity of interleukin-10 (IL-10), tumor necrosis factor-alpha (TNF-alpha), and IL-6 produced by stimulated [CD3/CD28, ConA, or lipopolysaccharide (LPS)] and tungstate-treated lymphocytes. Taken together, these data indicate that tungstate increases apoptosis of PBL, alters cell cycle progression, reduces cytokine production, and therefore warrants further investigation.

  12. Comparative genotoxicity of cobalt nanoparticles and ions on human peripheral leukocytes in vitro.

    PubMed

    Colognato, R; Bonelli, A; Ponti, J; Farina, M; Bergamaschi, E; Sabbioni, E; Migliore, L

    2008-09-01

    Owing to the increasing development of nanotechnology, there is a need to assess how engineered nanomaterials can interact with living cells. The main purpose of the present study was to assess whether metal cobalt nanoparticles (CoNP 100-500 nm) are genotoxic compared to cobalt ions (Co(2+)). Uptake experiments were carried out by incubating peripheral blood leukocytes (PBLs) with (57)Co(2+) (added to stable Co(2+) 10(-2) M to obtain concentrations in the range of 10(-5) to 10(-4) M) or with (60)CoNP for 24 and 48 h. Whereas intracellular Co(2+) showed slight or no variations over the baseline levels, CoNP were taken up efficiently leading to intracellular CoNP concentrations of 485 +/- 106.1 and 970 +/- 99 fg per cell after 24 and 48 h, respectively. The genotoxicity end points considered in this study were the frequency of binucleated micronucleated (BNMN) cells and the percentage of tail DNA (% Tail DNA) fragmentation by means of the comet assay. Genotoxic effects were evaluated by incubating PBLs of three healthy donors with subtoxic concentrations (10(-5) to 8 x 10(-5)M) of Co(2+) in the form of cobalt chloride, CoNP and 'washed' CoNP, the latter to exclude any interference by Co(2+). On a group basis, Co(2+) induced a clear trend in the increase of the BNMN frequency, whereas CoNP showed only minor changes. Moreover, we observed a high variability among donors in the induction of micronuclei. The comet assay showed a statistically significant dose-related increase in % Tail DNA for CoNP (P < 0,001), whereas Co(2+) did not induce significant changes over control values. These findings suggest that nanosized Co can be internalized by human leukocytes and can interact with DNA leading to the observed genotoxic effects, which are, however, modulated both by donor's characteristics and/or by Co(2+) release.

  13. Dysregulation of the peripheral and adipose tissue endocannabinoid system in human abdominal obesity.

    PubMed

    Blüher, Matthias; Engeli, Stefan; Klöting, Nora; Berndt, Janin; Fasshauer, Mathias; Bátkai, Sándor; Pacher, Pál; Schön, Michael R; Jordan, Jens; Stumvoll, Michael

    2006-11-01

    The endocannabinoid system has been suspected to contribute to the association of visceral fat accumulation with metabolic diseases. We determined whether circulating endocannabinoids are related to visceral adipose tissue mass in lean, subcutaneous obese, and visceral obese subjects (10 men and 10 women in each group). We further measured expression of the cannabinoid type 1 (CB(1)) receptor and fatty acid amide hydrolase (FAAH) genes in paired samples of subcutaneous and visceral adipose tissue in all 60 subjects. Circulating 2-arachidonoyl glycerol (2-AG) was significantly correlated with body fat (r = 0.45, P = 0.03), visceral fat mass (r = 0.44, P = 0.003), and fasting plasma insulin concentrations (r = 0.41, P = 0.001) but negatively correlated to glucose infusion rate during clamp (r = 0.39, P = 0.009). In visceral adipose tissue, CB(1) mRNA expression was negatively correlated with visceral fat mass (r = 0.32, P = 0.01), fasting insulin (r = 0.48, P < 0.001), and circulating 2-AG (r = 0.5, P < 0.001), whereas FAAH gene expression was negatively correlated with visceral fat mass (r = 0.39, P = 0.01) and circulating 2-AG (r = 0.77, P < 0.001). Our findings suggest that abdominal fat accumulation is a critical correlate of the dysregulation of the peripheral endocannabinoid system in human obesity. Thus, the endocannabinoid system may represent a primary target for the treatment of abdominal obesity and associated metabolic changes.

  14. [Effect of nicotine on the secretion of TNF of human peripheral blood mononuclear cells in vitro].

    PubMed

    Lei, Guang-hua; Li, Kang-hua; Zhou, Jiang-nan

    2002-06-28

    To observe the effect of nicotine on the secretion of TNF of human peripheral blood mononuclear cells (PBMC) in vitro, and explore the possible mechanism of the high level of TNF caused by smoking. Twenty-one non-smokers and 19 smokers were studied. The PBMCs isolated from each volunteer's blood sample were distributed to three groups, and cultured for 72 hours in supplemented RMPI 1640 alone or in supplemented RPMI 1640 containing 50 ng.ml-1 nicotine or 500 ng.ml-1 nicotine respectively. The level of TNF in supernate was quantified with the immuno-radioassay, and PBMC proliferation was observed. The level of TNF spontaneously secreted by PBMCs in smokers was significantly higher than that in non-smokers (P < 0.05). When the concentrations of nicotine were 50 ng.ml-1 and 500 ng.ml-1, the level of TNF increased significantly in non-smokers (P < 0.05, P < 0.01). In smokers, the level of TNF significantly increased when the concentration of nicotine was 50 ng.ml-1; however, the level of TNF significantly decreased (P < 0.05), and the proliferation of PBMCs was inhibited when the concentration of nicotine was 500 ng.ml-1 (P < 0.05, P < 0.05). The level of TNF was positively correlated to the multiple of PBMC proliferation (r = 0.93, P < 0.05). The linear regression equation was Y = 2.913X - 2.955. Nicotine can induce PBMCs to secrete more TNF, and the magnitude of the effect is strongly related to the dosage of nicotine, but a great dose of nicotine will inhibit the production of TNF.

  15. Ex Vivo Expansion of Human Mobilized Peripheral Blood Stem Cells Using Epigenetic Modifiers

    PubMed Central

    Saraf, Santosh; Araki, Hiroto; Petro, Benjamin; Park, Youngmin; Taioli, Simona; Yoshinaga, Kazumi G; Koca, Emre; Rondelli, Damiano; Mahmud, Nadim

    2014-01-01

    Background Epigenetic modifications likely control fate of hematopoietic stem cells (HSC). The chromatin modifying agents (CMA), 5-aza-2’-deoxyctidine (5azaD) and trichostatin A (TSA) have previously been shown to expand HSC from cord blood and bone marrow. Here we assessed whether CMA can also expand HSCs present in growth factor mobilized human peripheral blood (MPB). Study Design & Methods 5azaD and TSA were sequentially added to CD34+ MPB cells in the presence of cytokines and the cells were cultured for nine days. Results Following culture, a 3.6 ± 0.5 fold expansion of CD34+CD90+ cells, a 10.1 ± 0.5 fold expansion of primitive colony forming unit (CFU)-mix, and a 2.2 ± 0.5 fold expansion of long-term cobble stone-area forming cells (CAFC) was observed in 5azaD/TSA expanded cells. By contrast, cells cultured in cytokines without 5azaD/TSA displayed no expansion; rather a reduction in CD34+CD90+ cells (0.7 ± 0.1 fold) and CAFCs (0.3 ± 0.1) from their initial numbers was observed. Global hypomethylation corresponding with increased transcript levels of several genes implicated in HSC self-renewal, including HOXB4, GATA2, and EZH2, was observed in 5azaD/TSA expanded MPB cells in contrast to controls. 5azaD/TSA expanded MPB cells retained in vivo hematopoietic engraftment capacity. Conclusion MPB CD34+ cells from donors can be expanded using 5azaD/TSA and these expanded cells retain in vivo hematopoietic reconstitution capacity. This strategy may prove to be potentially useful to augment HSCs numbers for patients who fail to mobilize. PMID:25363624

  16. Tetrazepam: a benzodiazepine which dissociates sedation from other benzodiazepine activities. II. In vitro and in vivo interactions with benzodiazepine binding sites

    SciTech Connect

    Keane, P.E.; Bachy, A.; Morre, M.; Biziere, K.

    1988-05-01

    Tetrazepam is a 1,4-benzodiazepine (BZD) derivative which, in rodents, appears to have very little sedative and ataxic effects. In an attempt to identify the molecular mechanisms underlying this particular pharmacological profile we examined the interaction of tetrazepam with BZD binding sites. Tetrazepam interacted competitively with central and peripheral BZD binding sites and exhibited comparable affinities for both sites. Tetrazepam was approximately one-seventh as potent as diazepam at the central receptor and as potent as diazepam at the peripheral binding site. Tetrazepam did not distinguish type I from type II central BZD receptors, as evidenced by comparable affinities for the cerebellar and hippocampal receptors. In vitro autoradiographic studies showed that tetrazepam displaced (3H)flunitrazepam from rat brain membranes without any clear regional specificity. Like all BZD receptor agonists, tetrazepam exhibited a gamma-aminobutyric acid shift, a photoaffinity shift and potentiated the binding of 35S-t-butyl-bicyclophosphorothionate to rat brain membranes. However, the latter effect was observed at relatively high concentrations of tetrazepam. In vivo, tetrazepam displaced specifically bound (3H)flunitrazepam from mouse brain (ID50, 37 mg/kg p.o. vs 3.5 mg/kg p.o. for diazepam) and from mouse kidney (ID50, 38 mg/kg p.o. vs. 21 mg/kg p.o. for diazepam). It is concluded that tetrazepam is a BZD receptor agonist; the molecular mechanisms which underly the low sedative potential of the drug cannot at present be explained by a particular interaction with either central or peripheral BZD binding sites, but may be related to the drug's relatively weak effect on 35S-t-butyl-bicyclophosphorothionate binding.

  17. Sporothrix schenckii sensu stricto and Sporothrix brasiliensis Are Differentially Recognized by Human Peripheral Blood Mononuclear Cells.

    PubMed

    Martínez-Álvarez, José A; Pérez-García, Luis A; Mellado-Mojica, Erika; López, Mercedes G; Martínez-Duncker, Iván; Lópes-Bezerra, Leila M; Mora-Montes, Héctor M

    2017-01-01

    Sporothrix schenckii sensu stricto and S. brasiliensis are usually associated to sporotrichosis, a subcutaneous mycosis worldwide distributed. Comparative analyses between these two species indicate they contain genetic and physiological differences that are likely to impact the interaction with host cells. Here, we study the composition of the cell wall from conidia, yeast-like cells and germlings of both species and found they contained the same sugar composition. The carbohydrate proportion in the S. schenckii sensu stricto wall was similar across the three cell morphologies, with exception in the chitin content, which was significantly different in the three morphologies. The cell wall from germlings showed lower rhamnose content and higher glucose levels than other cell morphologies. In S. brasiliensis, the wall sugars were constant in the three morphologies, but glucose was lower in yeast-like cells. In S. schenckii sensu stricto cells most of chitin and β1,3-glucan were underneath wall components, but in S. brasiliensis germlings, chitin was exposed at the cell surface, and β1,3-glucan was found in the outer part of the conidia wall. We also compared the ability of these cells to stimulate cytokine production by human peripheral blood mononuclear cells. The three S. schenckii sensu stricto morphologies stimulated increased levels of pro-inflammatory cytokines, when compared to S. brasiliensis cells; while the latter, with exception of conidia, stimulated higher IL-10 levels. Dectin-1 was a key receptor for cytokine production during stimulation with the three morphologies of S. schenckii sensu stricto, but dispensable for cytokine production stimulated by S. brasiliensis germlings. TLR2 and TLR4 were also involved in the sensing of Sporothrix cells, with a major role for the former during cytokine stimulation. Mannose receptor had a minor contribution during cytokine stimulation by S. schenckii sensu stricto yeast-like cells and germlings, but S. schenckii

  18. Sporothrix schenckii sensu stricto and Sporothrix brasiliensis Are Differentially Recognized by Human Peripheral Blood Mononuclear Cells

    PubMed Central

    Martínez-Álvarez, José A.; Pérez-García, Luis A.; Mellado-Mojica, Erika; López, Mercedes G.; Martínez-Duncker, Iván; Lópes-Bezerra, Leila M.; Mora-Montes, Héctor M.

    2017-01-01

    Sporothrix schenckii sensu stricto and S. brasiliensis are usually associated to sporotrichosis, a subcutaneous mycosis worldwide distributed. Comparative analyses between these two species indicate they contain genetic and physiological differences that are likely to impact the interaction with host cells. Here, we study the composition of the cell wall from conidia, yeast-like cells and germlings of both species and found they contained the same sugar composition. The carbohydrate proportion in the S. schenckii sensu stricto wall was similar across the three cell morphologies, with exception in the chitin content, which was significantly different in the three morphologies. The cell wall from germlings showed lower rhamnose content and higher glucose levels than other cell morphologies. In S. brasiliensis, the wall sugars were constant in the three morphologies, but glucose was lower in yeast-like cells. In S. schenckii sensu stricto cells most of chitin and β1,3-glucan were underneath wall components, but in S. brasiliensis germlings, chitin was exposed at the cell surface, and β1,3-glucan was found in the outer part of the conidia wall. We also compared the ability of these cells to stimulate cytokine production by human peripheral blood mononuclear cells. The three S. schenckii sensu stricto morphologies stimulated increased levels of pro-inflammatory cytokines, when compared to S. brasiliensis cells; while the latter, with exception of conidia, stimulated higher IL-10 levels. Dectin-1 was a key receptor for cytokine production during stimulation with the three morphologies of S. schenckii sensu stricto, but dispensable for cytokine production stimulated by S. brasiliensis germlings. TLR2 and TLR4 were also involved in the sensing of Sporothrix cells, with a major role for the former during cytokine stimulation. Mannose receptor had a minor contribution during cytokine stimulation by S. schenckii sensu stricto yeast-like cells and germlings, but S. schenckii

  19. Immunocapture and microplate-based activity and quantity measurement of pyruvate dehydrogenase in human peripheral blood mononuclear cells

    PubMed Central

    Liu, Xiaowen; Pervez, Hira; Andersen, Lars W; Uber, Amy; Montissol, Sophia; Patel, Parth; Donnino, Michael W

    2015-01-01

    Background Pyruvate dehydrogenase (PDH) activity is altered in many human disorders. Current methods require tissue samples and yield inconsistent results. We describe a modified method for measuring PDH activity from isolated human peripheral blood mononuclear cells (PBMCs). Results/Methodology We found that PDH activity and quantity can be successfully measured in human PBMCs. Freeze-thaw cycles cannot efficiently disrupt the mitochondrial membrane. Processing time of up to 20 h does not affect PDH activity with proteinase inhibitor addition and a detergent concentration of 3.3% showed maximum yield. Sample protein concentration is correlated to PDH activity and quantity in human PBMCs from healthy subjects. Conclusion Measuring PDH activity from PBMCs is a novel, easy and less invasive way to further understand the role of PDH in human disease. PMID:25826140

  20. Immunocapture and microplate-based activity and quantity measurement of pyruvate dehydrogenase in human peripheral blood mononuclear cells.

    PubMed

    Liu, Xiaowen; Pervez, Hira; Andersen, Lars W; Uber, Amy; Montissol, Sophia; Patel, Parth; Donnino, Michael W

    2015-01-01

    Pyruvate dehydrogenase (PDH) activity is altered in many human disorders. Current methods require tissue samples and yield inconsistent results. We describe a modified method for measuring PDH activity from isolated human peripheral blood mononuclear cells (PBMCs). RESULTS/METHODOLOGY: We found that PDH activity and quantity can be successfully measured in human PBMCs. Freeze-thaw cycles cannot efficiently disrupt the mitochondrial membrane. Processing time of up to 20 h does not affect PDH activity with proteinase inhibitor addition and a detergent concentration of 3.3% showed maximum yield. Sample protein concentration is correlated to PDH activity and quantity in human PBMCs from healthy subjects. Measuring PDH activity from PBMCs is a novel, easy and less invasive way to further understand the role of PDH in human disease.

  1. Genotoxicity and carcinogenicity studies of benzodiazepines.

    PubMed

    Brambilla, Giovanni; Carrozzino, Roberto; Martelli, Antonietta

    2007-12-01

    This survey is a compendium of genotoxicity and carcinogenicity information of benzodiazepines and benzodiazepine analogues. Data from 51 drugs were collected; 41 of them are still in the market. Of the 51 drugs, 12 (23.5%) do not have retrievable genotoxicity or carcinogenicity data. The remaining 39 (76.5%) have at least one genotoxicity or carcinogenicity test result. Of these 39, 12 (30.8%) have at least one positive finding: 9 tested positive in at least one genotoxicity assay, 8 in at least one carcinogenicity assay, and 5 gave a positive result in both at least one genotoxicity assay and at least one carcinogenicity assay. In terms of correlation between results of the various genotoxicity assays and absence of carcinogenic activity in mice and/or rats or in other species, none of the 11 non-carcinogenic drugs tested positive in one of the various genotoxicity assay systems. Concerning the predictivity of genetic toxicology findings for the result(s) of long-term carcinogenesis assays, 18 drugs had both genotoxicity and carcinogenicity data; of these 11 (61.1%) were neither genotoxic nor carcinogenic, 2 (11.1%) were carcinogenic in at least one sex of mice or rats but tested negative in genotoxicity assays, and 5 (27.8%) gave a positive response in at least one genotoxicity assay and in at least one carcinogenicity assay. Only 8 (19.5%) of the 41 marketed benzodiazepines and benzodiazepine analogues had all data required by current guidelines for testing of pharmaceuticals.

  2. Autoradiographic localization of benzodiazepine receptor downregulation

    SciTech Connect

    Tietz, E.I.; Rosenberg, H.C.; Chiu, T.H.

    1986-01-01

    Regional differences in downregulation of brain benzodiazepine receptors were studied using a quantitative autoradiographic method. Rats were given a 4-week flurazepam treatment known to cause tolerance and receptor downregulation. A second group of rats was given a similar treatment, but for only 1 week. A third group was given a single acute dose of diazepam to produce a brain benzodiazepine-like activity equivalent to that found after the chronic treatment. Areas studied included hippocampal formation, cerebral cortex, superior colliculus, substantia nigra, dorsal geniculate nucleus, lateral amygdala and lateral hypothalamus. There was a regional variation in the degree of downregulation after 1 week of flurazepam treatment, ranging from 12% to 25%. Extending the flurazepam treatment to 4 weeks caused little further downregulation in those areas studied, except for the pars reticulata of the substantia nigra, which showed a 13% reduction in (/sup 3/H)flunitrazepam binding after 1 week and a 40% reduction after 4 weeks of treatment. In a few areas, such as the lateral hypothalamus, no significant change in binding was found after 4 weeks. Acute diazepam treatment caused no change in binding. This latter finding as well as results obtained during the development of the methodology show that downregulation was not an artifact due to residual drug content of brain slices. The regional variations in degree and rate of downregulation suggest areas that may be most important for benzodiazepine tolerance and dependence and may be related to the varying time courses for tolerance to different benzodiazepine actions.

  3. Benzodiazepine Synthesis and Rapid Toxicity Assay

    ERIC Educational Resources Information Center

    Fletcher, James T.; Boriraj, Grit

    2010-01-01

    A second-year organic chemistry laboratory experiment to introduce students to general concepts of medicinal chemistry is described. Within a single three-hour time window, students experience the synthesis of a biologically active small molecule and the assaying of its biological toxicity. Benzodiazepine rings are commonly found in antidepressant…

  4. Benzodiazepine Synthesis and Rapid Toxicity Assay

    ERIC Educational Resources Information Center

    Fletcher, James T.; Boriraj, Grit

    2010-01-01

    A second-year organic chemistry laboratory experiment to introduce students to general concepts of medicinal chemistry is described. Within a single three-hour time window, students experience the synthesis of a biologically active small molecule and the assaying of its biological toxicity. Benzodiazepine rings are commonly found in antidepressant…

  5. Benzodiazepines, benzodiazepine-like drugs, and typical antipsychotics impair manual dexterity in patients with schizophrenia.

    PubMed

    Sasayama, Daimei; Hori, Hiroaki; Teraishi, Toshiya; Hattori, Kotaro; Ota, Miho; Matsuo, Junko; Kinoshita, Yukiko; Okazaki, Mitsutoshi; Arima, Kunimasa; Amano, Naoji; Higuchi, Teruhiko; Kunugi, Hiroshi

    2014-02-01

    Impaired dexterity is a major psychomotor deficit reported in patients with schizophrenia. In the present study, the Purdue pegboard test was used to compare the manual dexterity in patients with schizophrenia and healthy controls. We also examined the influence of antipsychotics, benzodiazepines, and benzodiazepine-like drugs on manual dexterity. Subjects were 93 patients with schizophrenia and 93 healthy controls, matched for sex and age distributions. Control subjects scored significantly higher on all scores of Purdue pegboard than patients with schizophrenia. Age, PANSS negative symptom scale, typical antipsychotic dose, and use of benzodiazepines and/or benzodiazepine-like drugs were negatively correlated with the pegboard scores in patients with schizophrenia. The present results indicate that patients with schizophrenia have impaired gross and fine fingertip dexterity compared to healthy controls. The use of typical antipsychotics and benzodiazepines and/or benzodiazepine-like drugs, but not atypical antipsychotics, had significant negative impact on dexterity in patients with schizophrenia. Psychiatrists should be aware that some psychotropic medications may enhance the disability caused by the impairment of dexterity in patients with schizophrenia. Copyright © 2013 Elsevier Ltd. All rights reserved.

  6. Treatment of human immunodeficiency virus-related peripheral neuropathy with Scrambler Therapy: a case report.

    PubMed

    Smith, Thomas J; Auwaerter, Paul; Knowlton, Amy; Saylor, Deanna; McArthur, Justin

    2017-02-01

    Peripheral neuropathy is one of the most common neurological complications of HIV infection with a 30-60% lifetime prevalence. Newer HIV drugs cause less peripheral neuropathy, but patients are now living long enough to develop concomitant diabetes-related, vascular-related, and chemotherapy-related neuropathy so it continues as a major debilitating issue. Recent national CDC guidelines have stressed the importance of non-opioid therapies, especially in this population that may have had drug abuse problems. We treated a 52-year-old man who had severe disabling classic peripheral neuropathy since 1998 with Scrambler Therapy (Calmare), an FDA-cleared peripheral non-invasive neuromodulation device. His pain rapidly improved, as did his motor and sensory function, with just four 45-min treatments, and he was able to come off opioids for the first time in years. When his pain returned six months later, only two treatments were needed to resolve it. This represents the first published use of this novel, inexpensive, and non-invasive pain modality in HIV peripheral neuropathy, and should engender further trials.

  7. Assessment of Potential In vitro Genotoxic and Cytotoxic Effects of Bupropion Hydrochloride (Wellbutrin) in Human Peripheral Lymphocytes and Human Cortical Neuron.

    PubMed

    Bhattacharya, Saurabh Kumar; Nathawat, Lokendra Singh; Damani, Priyankka; Choksi, Arpan Kumar; Banik, Arpita; Sinha, Kriti; Bhattacharya, Aditi Sarkar

    2013-01-01

    Wellbutrin (bupropion hydrochloride; WB), an anti-depressant of the aminoketone class is new highly selective norepinephrine and dopamine reuptake inhibitor; it is effective in the treatment of patients with major depression. To investigate the in vitro effects of WB in human cultured peripheral blood lymphocytes and human cortical neural (HCN2) cell lines, micronucleus, sister chromatid exchange analysis, cellular viability, and comet assays were employed. The present study is to our knowledge, the first report on WB genotoxicity in cultured human peripheral blood lymphocytes and its cytotoxicity in the HCN2 cell line. We have also investigated the genotoxic potential of WB to induce chromosomal aberrations. WB-induced cytotoxicity (measured as reduction of the nuclear division index) possibly prevented the division of damaged cells. We conclude that although, WB exerts potential genotoxic effects in cultured lymphocytes, its cytogenetic effects are very unlikely to occur in blood cultures of WB-administered subjects.

  8. Autoradiographic localization of benzodiazepine receptors in the rat kidney

    SciTech Connect

    Beaumont, K.; Healy, D.P.; Fanestil, D.D.

    1984-11-01

    The localization of benzodiazepine (BZD) receptors in the rat kidney was studied by autoradiography after in vitro labeling of kidney slices with flunitrazepam. The affinity, density, and rank order of displacement of (/sup 3/H)-flunitrazepam by several BZDs (RO 5-4864 > diazepam > clonazepam) demonstrated that binding was to BZD receptors of the peripheral type. In autoradiograms obtained with tritium-sensitive film, a high density of silver grains was obtained in the outer medulla, with lower densities in the cortex. Binding was absent from the inner medulla (papilla). In higher resolution autoradiograms obtained with an emulsion-coated cover slip procedure, silver grains were seen to be concentrated over a tubular element in both outer medulla and cortex, identifiable by morphology and distribution as the thick ascending limb of the loop of Henle and the distal convoluted tubule. The identity of the labeled tubules was confirmed by immunofluorescent localization in adjacent slices of Tamm-Horsfall protein, a specific marker for these segments of tubules. Investigation of the effects of peripherally specific BZDs such as RO 5-4864 on distal tubule function is indicated.

  9. The modulating effect of royal jelly consumption against radiation-induced apoptosis in human peripheral blood leukocytes.

    PubMed

    Rafat, Navid; Monfared, Ali Shabestani; Shahidi, Maryam; Pourfallah, Tayyeb Allahverdi

    2016-01-01

    The present work was designed to assess the radioprotective effect of royal jelly (RJ) against radiation-induced apoptosis in human peripheral blood leukocytes. In this study, peripheral blood samples were obtained on days 0, 4, 7, and 14 of the study from six healthy male volunteers taking a 1000 mg RJ capsule orally per day for 14 consecutive days. On each sampling day, all collected whole blood samples were divided into control and irradiated groups which were then exposed to the selected dose of 4 Gy X-ray. Percentage of apoptotic cells (Ap %) was evaluated for all samples immediately after irradiation (Ap0) and also after a 24 h postirradiation incubation at 37°C in 5% CO2 (Ap24) by the use of neutral comet assay. Concerning Ap0, collected data demonstrated that the percentage of apoptotic cells in both control and irradiated groups did not significantly change during the study period. However, with respect to Ap24, the percentage of apoptotic cells in irradiated groups gradually reduced during the experiment, according to which a significant decrease was found after 14 days RJ consumption (P = 0.002). In conclusion, the present study revealed the protective role of 14 days RJ consumption against radiation-induced apoptosis in human peripheral blood leukocytes.

  10. [Cytogenetic analysis of the effects of selected 2d generation cytostatics (iproplatin and oxoplatin) on human peripheral lymphocytes in vitro].

    PubMed

    Srb, V; Vancurová, R; Kubzová, E

    1989-01-01

    Cytostatic effect of Iproplatinum (CHIP, cis-dichlorotrans-dihydroxy-bis-isopropylaminoplatinic complex) and Oxoplatinum (oxo-Pt, cis-diamin-dichloro-trans-dihydroxyplatinic complex) is studied as influencing genetic structures of in vitro human peripheral lymphocytes. Both mentioned substances are classed as prospective cytostatics with satisfactory effect on various tumors, and both undergo now preclinical tests in our country. They are supposed to cause less undesired side effects in comparison with previous preparation of this range--cisplatinum (cis-DDP; Platidiam). The genotoxicity of both substances is examined using the short-term test (72 hrs.), which means a cultivation of raw human peripheral blood modified according to Macek (1965). To set the testing scheme, five concentrations of substances (0, 5, 12, 60 and 120 mumol.l-1) were selected as well as three time intervals of action of a substance (3, 6 and 24 hrs.) prior the expiration of cultivation time, i.e. before the mitotic cycle stop in c-metaphase. Concentrations were determined estimating cisplatinum's dosage to patients. The concentration value 120 mumol.l-1 responds in theory to a single therapeutic dose administration of Platidiam. However, in praxis this concentration is never achieved in organism (resp. protein-binding effect). In accordance with mice LD50 values, both the Iproplatinum and Oxoplatinum showed experimentally 10 times less toxicity than cis-DDP. Cytogenetic changes were evaluated by microscopy in peripheral lymphocytes (predominantly the occurrence of chromosome abnormalities in metaphase), and mitotic activity was as well identified.

  11. Chronic inorganic arsenic exposure in vitro induces a cancer cell phenotype in human peripheral lung epithelial cells

    SciTech Connect

    Person, Rachel J.; Olive Ngalame, Ntube N.; Makia, Ngome L.; Bell, Matthew W.; Waalkes, Michael P.; Tokar, Erik J.

    2015-07-01

    Inorganic arsenic is a human lung carcinogen. We studied the ability of chronic inorganic arsenic (2 μM; as sodium arsenite) exposure to induce a cancer phenotype in the immortalized, non-tumorigenic human lung peripheral epithelial cell line, HPL-1D. After 38 weeks of continuous arsenic exposure, secreted matrix metalloproteinase-2 (MMP2) activity increased to over 200% of control, levels linked to arsenic-induced cancer phenotypes in other cell lines. The invasive capacity of these chronic arsenic-treated lung epithelial (CATLE) cells increased to 320% of control and colony formation increased to 280% of control. CATLE cells showed enhanced proliferation in serum-free media indicative of autonomous growth. Compared to control cells, CATLE cells showed reduced protein expression of the tumor suppressor gene PTEN (decreased to 26% of control) and the putative tumor suppressor gene SLC38A3 (14% of control). Morphological evidence of epithelial-to-mesenchymal transition (EMT) occurred in CATLE cells together with appropriate changes in expression of the EMT markers vimentin (VIM; increased to 300% of control) and e-cadherin (CDH1; decreased to 16% of control). EMT is common in carcinogenic transformation of epithelial cells. CATLE cells showed increased KRAS (291%), ERK1/2 (274%), phosphorylated ERK (p-ERK; 152%), and phosphorylated AKT1 (p-AKT1; 170%) protein expression. Increased transcript expression of metallothioneins, MT1A and MT2A and the stress response genes HMOX1 (690%) and HIF1A (247%) occurred in CATLE cells possibly in adaptation to chronic arsenic exposure. Thus, arsenic induced multiple cancer cell characteristics in human peripheral lung epithelial cells. This model may be useful to assess mechanisms of arsenic-induced lung cancer. - Highlights: • Chronic arsenic exposure transforms a human peripheral lung epithelia cell line. • Cells acquire characteristics in common with human lung adenocarcinoma cells. • These transformed cells provide a

  12. Diazepam binding inhibitor gene expression: Location in brain and peripheral tissues of rate

    SciTech Connect

    Alho, H.; Fremeau, R.T. Jr.; Tiedge, H.; Wilcox, J.; Bovolin, P.; Brosius, J.; Roberts, J.L.; Costa, E.

    1988-09-01

    Diazepam binding inhibitor (DBI), an endogenous 10-kDa polypeptide was isolated from rat and human brain by monitoring displacement of radioactive diazepam bound to specific recognition sites in brain synaptic and mitochondrial membranes. The cellular location of DBI mRNA was studied in rat brain and selected peripheral tissues by in situ hybridization histochemistry with a /sup 35/S-labeled single-stranded complementary RNA probe. DBI mRNA was heterogeneously distributed in rat brain, with particularly high levels in the area postrema, the cerebellar cortex, and ependyma of the third ventricle. Intermediate levels were found in the olfactory bulb, pontine nuclei, inferior colliculi, arcuate nucleus, and pineal gland. Relatively low but significant levels of silver grains were observed overlying many mesencephalic and telencephalic areas that have previously been shown to contain numerous DBI-immunoreactive neurons and a high density of central benzodiazepine receptors. In situ hybridizations also revealed high levels of DBI mRNA in the posterior lobe of the pituitary gland, liver, and germinal center of the white pulp of spleen, all tissues that are rich in peripheral benzodiazepine binding sites. The tissue-specific pattern of DBI gene expression described here could be exploited to further understand the physiological function of DBI in the brain and periphery.

  13. Determination of intracellular fludarabine triphosphate in human peripheral blood mononuclear cells by LC-MS/MS

    PubMed Central

    Huang, Liusheng; Lizak, Patricia; Aweeka, Francesca; Long-Boyle, Janel

    2013-01-01

    Fludarabine is a nucleoside analog routinely used in conditioning regimens of pediatric allogeneic stem cell transplantation to promote stem cell engraftment. In children, it remains a challenge to accurately and precisely quantify the active intracellular triphosphate species of fludarabine in vivo, primarily due to limitations on blood volume and inadequate assay sensitivity. Here we report a liquid chromatography tandem mass spectrometry (LC-MS/MS) method for determination of fludarabine triphosphate in human peripheral blood mononuclear cells (PBMC). PBMC (~5 million cells) were collected and lysed in 1 mL 70% methanol containing 1.2 mM tris buffer (pH7.4). The lysate (80 uL) was mixed with internal standard (2-chloro-adenosine triphosphate, 150ng/mL, 20 µL) and injected onto an API5000 LC-MS/MS system. Separation was achieved on a hypercarb column (100 × 2.1 mm, 3 µm) eluted with 100mM ammonium acetate (pH9.8) and acetonitrile in a gradient mode at a flow rate of 0.4 mL/min. Multiple reactions monitoring (MRM) and electrospray ionization in negative mode (ESI−) were used for detection. The ion pairs 524.0/158.6 for the drug and 540.0/158.8 for the IS were selected for quantification and 524.0/425.7 used for confirmation. Retention time was 3.0 and 3.4 min for fludarabine triphosphate and the IS, respectively. The concentration range for the calibration curve was 1.52 to 76 nM. Our method is simple, fast, and has been successfully applied in a clinical dose-concentration study in children to quantify intracellular fludarabine in low volume clinical samples. The median concentration was 1.03 and 3.19 pmole/million PBMC at trough and peak time points, respectively. Fludarabine triphosphate is degraded in water within hours but relatively stable in 70% methanol-tris (1.2mM, pH7.4). One limitation is that the hypercarb column takes a longer time to equilibrate than conventional reverse phase columns, and peaks become broad and distorted if the column is not

  14. Determination of intracellular fludarabine triphosphate in human peripheral blood mononuclear cells by LC-MS/MS.

    PubMed

    Huang, Liusheng; Lizak, Patricia; Aweeka, Francesca; Long-Boyle, Janel

    2013-12-01

    Fludarabine is a nucleoside analog routinely used in conditioning regimens of pediatric allogeneic stem cell transplantation to promote stem cell engraftment. In children, it remains a challenge to accurately and precisely quantify the active intracellular triphosphate species of fludarabine in vivo, primarily due to limitations on blood volume and inadequate assay sensitivity. Here we report a liquid chromatography tandem mass spectrometry (LC-MS/MS) method for determination of fludarabine triphosphate in human peripheral blood mononuclear cells (PBMC). PBMC (∼5 million cells) were collected and lysed in 1mL 70% methanol containing 1.2mM tris buffer (pH 7.4). The lysate (80μL) was mixed with internal standard (2-chloro-adenosine triphosphate, 150ng/mL, 20μL) and injected onto an API5000 LC-MS/MS system. Separation was achieved on a hypercarb column (100mm×2.1mm, 3μm) eluted with 100mM ammonium acetate (pH 9.8) and acetonitrile in a gradient mode at a flow rate of 0.4mL/min. Multiple reactions monitoring (MRM) and electrospray ionization in negative mode (ESI(-)) were used for detection. The ion pairs 524.0/158.6 for the drug and 540.0/158.8 for the IS were selected for quantification and 524.0/425.7 used for confirmation. Retention time was 3.0 and 3.4min for fludarabine triphosphate and the IS, respectively. The concentration range for the calibration curve was 1.52-76nM. Our method is simple, fast, and has been successfully applied in a clinical dose-concentration study in children to quantify intracellular fludarabine in low volume clinical samples. The median concentration was 1.03 and 3.19pmole/million PBMC at trough and peak time points, respectively. Fludarabine triphosphate is degraded in water within hours but relatively stable in 70% methanol-tris (1.2mM, pH 7.4). One limitation is that the hypercarb column takes a longer time to equilibrate than conventional reverse phase columns, and peaks become broad and distorted if the column is not washed

  15. Influence of GSM signals on human peripheral lymphocytes: study of genotoxicity.

    PubMed

    Waldmann, Petra; Bohnenberger, Susanne; Greinert, Rüdiger; Hermann-Then, Beate; Heselich, Anja; Klug, Stefanie J; Koenig, Jochem; Kuhr, Kathrin; Kuster, Niels; Merker, Mandy; Murbach, Manuel; Pollet, Dieter; Schadenboeck, Walter; Scheidemann-Wesp, Ulrike; Schwab, Britt; Volkmer, Beate; Weyer, Veronika; Blettner, Maria

    2013-02-01

    Exposure to radiofrequency (RF) electromagnetic fields (EMF) is continuously increasing worldwide. Yet, conflicting results of a possible genotoxic effect of RF EMF continue to be discussed. In the present study, a possible genotoxic effect of RF EMF (GSM, 1,800 MHz) in human lymphocytes was investigated by a collaboration of six independent institutes (institutes a, b, c, d, e, h). Peripheral blood of 20 healthy, nonsmoking volunteers of two age groups (10 volunteers 16-20 years old and 10 volunteers 50-65 years old) was taken, stimulated and intermittently exposed to three specific absorption rates (SARs) of RF EMF (0.2 W/kg, 2 W/kg, 10 W/kg) and sham for 28 h (institute a). The exposures were performed in a setup with strictly controlled conditions of temperature and dose, and randomly and automatically determined waveguide SARs, which were designed and periodically maintained by ITIS (institute h). Four genotoxicity tests with different end points were conducted (institute a): chromosome aberration test (five types of structural aberrations), micronucleus test, sister chromatid exchange test and the alkaline comet assay (Olive tail moment and % DNA). To demonstrate the validity of the study, positive controls were implemented. The genotoxicity end points were evaluated independently by three laboratories blind to SAR information (institute c = laboratory 1; institute d = laboratory 2; institute e = laboratory 3). Statistical analysis was carried out by institute b. Methods of primary statistical analysis and rules to adjust for multiple testing were specified in a statistical analysis plan based on a data review before unblinding. A linear trend test based on a linear mixed model was used for outcomes of comet assay and exact permutation test for linear trend for all other outcomes. It was ascertained that only outcomes with a significant SAR trend found by at least two of three analyzing laboratories indicated a substantiated suspicion of an exposure effect

  16. Allelic Exclusion and Peripheral Reconstitution by TCR Transgenic T Cells Arising From Transduced Human Hematopoietic Stem/Progenitor Cells

    PubMed Central

    Giannoni, Francesca; Hardee, Cinnamon L; Wherley, Jennifer; Gschweng, Eric; Senadheera, Shantha; Kaufman, Michael L; Chan, Rebecca; Bahner, Ingrid; Gersuk, Vivian; Wang, Xiaoyan; Gjertson, David; Baltimore, David; Witte, Owen N; Economou, James S; Ribas, Antoni; Kohn, Donald B

    2013-01-01

    Transduction and transplantation of human hematopoietic stem/progenitor cells (HSPC) with the genes for a T-cell receptor (TCR) that recognizes a tumor-associated antigen may lead to sustained long-term production of T cells expressing the TCR and confer specific antitumor activity. We evaluated this using a lentiviral vector (CCLc-MND-F5) carrying cDNA for a human TCR specific for an HLA-A*0201-restricted peptide of Melanoma Antigen Recognized by T cells (MART-1). CD34+ HSPC were transduced with the F5 TCR lentiviral vector or mock transduced and transplanted into neonatal NSG mice or NSG mice transgenic for human HLA-A*0201 (NSG-A2). Human CD8+ and CD4+ T cells expressing the human F5 TCR were present in the thymus, spleen, and peripheral blood after 4–5 months. Expression of human HLA-A*0201 in NSG-A2 recipient mice led to significantly increased numbers of human CD8+ and CD4+ T cells expressing the F5 TCR, compared with control NSG recipients. Transduction of the human CD34+ HSPC by the F5 TCR transgene caused a high degree of allelic exclusion, potently suppressing rearrangement of endogenous human TCR-β genes during thymopoiesis. In summary, we demonstrated the feasibility of engineering human HSPC to express a tumor-specific TCR to serve as a long-term source of tumor-targeted mature T cells for immunotherapy of melanoma. PMID:23380815

  17. Modeling leukocyte trafficking at the human blood-nerve barrier in vitro and in vivo geared towards targeted molecular therapies for peripheral neuroinflammation.

    PubMed

    Greathouse, Kelsey M; Palladino, Steven P; Dong, Chaoling; Helton, Eric S; Ubogu, Eroboghene E

    2016-01-06

    Peripheral neuroinflammation is characterized by hematogenous mononuclear leukocyte infiltration into peripheral nerves. Despite significant clinical knowledge, advancements in molecular biology and progress in developing specific drugs for inflammatory disorders such as rheumatoid arthritis, inflammatory bowel disease, and multiple sclerosis, there are currently no specific therapies that modulate pathogenic peripheral nerve inflammation. Modeling leukocyte trafficking at the blood-nerve barrier using a reliable human in vitro model and potential intravital microscopy techniques in representative animal models guided by human observational data should facilitate the targeted modulation of the complex inflammatory cascade needed to develop safe and efficacious therapeutics for immune-mediated neuropathies and chronic neuropathic pain.

  18. Effects of oral eicosapentaenoic acid versus docosahexaenoic acid on human peripheral blood mononuclear cell gene expression

    USDA-ARS?s Scientific Manuscript database

    Objective: Eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) have beneficial effects on inflammation and cardiovascular disease (CVD). Our aim was to assess the effect of a six-week supplementation with either olive oil, EPA, or DHA on gene expression in peripheral blood mononuclear cells (...

  19. Induction of chromosome aberrations by Fusarium T-2 toxin in cultured human peripheral blood lymphocytes and Chinese hamster fibroblasts

    SciTech Connect

    Hsia, C.C.; Gao, Y.; Wu, J.L.; Tzian, B.

    1986-01-01

    T-2 toxin is an important representative of trichothecenes produced by various species of imperfect fungi, mainly Fusarium genus. No definite data demonstrating the carcinogenic potential of T-2 toxin had been reported up to now. The authors demonstrated that T-2 toxin reproducibly induced chromosomal structural aberrations both in cultured human peripheral blood lymphocytes as well as in V/sub 79/ Chinese hamster fibroblasts. The mean percentage of cells with aberration of human lymphocytes from normal individuals induced by T-2 toxin is 49-fold (9.8%) of the mean percentage of corresponding control cultures without T-2 toxin (0.2%). T-2 toxin induced chromosome type (76%) as well as chromatid type (24%) of aberrations; among them, acentric fragment (46%) was the most common type, and chromatid gap, deletion, and chromosome gap were the next most common. T-2 toxin can induce aberrations in cells at different phases of the cell cycle. There are definite dose-effect relationships within a certain range of dosage of T-2 toxin in experiments with both human peripheral blood lymphocytes and V/sub 79/ cells. T-2 toxin exhibited three types of effects on cells, namely, mitogenic at lowest concentration, clastogenic (chromosome aberration) at median concentration, and cytotoxic at higher concentration. The dose-effect curves of these three effects are partly overlapping. Sex or age effect was not observed. The results suggest that T-2 toxin has carcinogenic potentials. The dosage of aflatoxin that can induce chromosomal aberration of human peripheral blood lymphocytes is thousands-fold of the dosage of T-2 toxin as shown in this report.

  20. Peripheral CD4+ T cell cytokine responses following human challenge and re-challenge with Campylobacter jejuni.

    PubMed

    Fimlaid, Kelly A; Lindow, Janet C; Tribble, David R; Bunn, Janice Y; Maue, Alexander C; Kirkpatrick, Beth D

    2014-01-01

    Campylobacter jejuni is a leading cause of human gastroenteritis worldwide; however, our understanding of the human immune response to C. jejuni infection is limited. A previous human challenge model has shown that C. jejuni elicits IFNγ production by peripheral blood mononuclear cells, a response associated with protection from clinical disease following re-infection. In this study, we investigate T lymphocyte profiles associated with campylobacteriosis using specimens from a new human challenge model in which C. jejuni-naïve subjects were challenged and re-challenged with C. jejuni CG8421. Multiparameter flow cytometry was used to investigate T lymphocytes as a source of cytokines, including IFNγ, and to identify cytokine patterns associated with either campylobacteriosis or protection from disease. Unexpectedly, all but one subject evaluated re-experienced campylobacteriosis after re-challenge. We show that CD4+ T cells make IFNγ and other pro-inflammatory cytokines in response to infection; however, multifunctional cytokine response patterns were not found. Cytokine production from peripheral CD4+ T cells was not enhanced following re-challenge, which may suggest deletion or tolerance. Evaluation of alternative paradigms or models is needed to better understand the immune components of protection from campylobacteriosis.

  1. Induction of endometriosis alters the peripheral and endometrial regulatory T cell population in the non-human primate

    PubMed Central

    Braundmeier, A.; Jackson, K.; Hastings, J.; Koehler, J.; Nowak, R.; Fazleabas, A.

    2012-01-01

    BACKGROUND Endometriosis is a gynecological condition that is characterized by extreme abdominal pain and also decreased fertility. Regulatory T cells (Tregs) have immunosuppressive activity critical for embryonic implantation and likewise the acceptance of tissue engraftment. Utilizing the induced non-human primate (Papio anubis) model of endometriosis, we hypothesize that endometriosis decreases the peripheral and endomet rial Treg profile, whereas ectopic lesions have increased Treg localization. METHODS Peripheral blood and endometrium were obtained throughout the menstrual cycle prior to and after induction of disease. Animals were randomly assigned to control (n = 7) or diseased (n = 16) treatment groups. Endometriosis was induced by i.p. injection of autologous menstrual tissue for 2 consecutive months during menses. Peripheral blood and endometrial tissue were collected at d9-11PO at 1, 3, 6, 9, 12 and 15 months post-induction of disease for fluorescence-activated cell sorting, quantitative RT–PCR and immunohistochemistry. Ectopic lesions were excised at 1 and 6 months post-inoculation and also harvested at necropsy (15 months) and processed for RNA of IHC. Identification of Tregs through analysis of FOXP3 expression was conducted utlilizing several methodologies. Differences were determined by non-parametric statistical analysis between all treatment groups and time points. RESULTS In control animals, the proportion of peripheral natural Tregs (nTregs) was reduced (P < 0.05) during the mid- and late secretory stages of the menstrual cycle compared with menses. The induction of disease decreased peripheral Treg expression at early time points (P < 0.05) and this remained low throughout the time course, compared with the pre-inoculatory level of an individual. FOXP3 gene expression and Treg populations were also decreased in the eutopic endometrium (P < 0.05) compared with control animals, whereas these parameters were increased in ectopic lesions (P < 0

  2. Bioavailability of insulin detemir and human insulin at the level of peripheral interstitial fluid in humans, assessed by open-flow microperfusion.

    PubMed

    Bodenlenz, M; Ellmerer, M; Schaupp, L; Jacobsen, L V; Plank, J; Brunner, G A; Wutte, A; Aigner, B; Mautner, S I; Pieber, T R

    2015-12-01

    To find an explanation for the lower potency of insulin detemir observed in humans compared with unmodified human insulin by investigating insulin detemir and human insulin concentrations directly at the level of peripheral insulin-sensitive tissues in humans in vivo. Euglycaemic-hyperinsulinaemic clamp experiments were performed in healthy volunteers. Human insulin was administered i.v. at 6 pmol/kg/min and insulin detemir at 60 pmol/kg/min, achieving a comparable steady-state pharmacodynamic action. In addition, insulin detemir was doubled to 120 pmol/kg/min. Minimally invasive open-flow microperfusion (OFM) sampling methodology was combined with inulin calibration to quantify human insulin and insulin detemir in the interstitial fluid (ISF) of subcutaneous adipose and skeletal muscle tissue. The human insulin concentration in the ISF was ∼115 pmol/l or ∼30% of the serum concentration, whereas the insulin detemir concentration in the ISF was ∼680 pmol/l or ∼2% of the serum concentration. The molar insulin detemir interstitial concentration was five to six times higher than the human insulin interstitial concentration and metabolic clearance of insulin detemir from serum was substantially reduced compared with human insulin. OFM proved useful for target tissue measurements of human insulin and the analogue insulin detemir. Our tissue data confirm a highly effective retention of insulin detemir in the vascular compartment. The higher insulin detemir relative to human insulin tissue concentrations at comparable pharmacodynamics, however, indicate that the lower potency of insulin detemir in humans is attributable to a reduced effect in peripheral insulin-sensitive tissues and is consistent with the reduced in vitro receptor affinity. © 2015 John Wiley & Sons Ltd.

  3. Ex vivo measurement of calpain activation in human peripheral blood lymphocytes by detection of immunoreactive products of calpastatin degradation.

    PubMed

    Mikosik, Anna; Zaremba, Anna; Puchalska, Zofia; Daca, Agnieszka; Smolenska, Zaneta; Lopatniuk, Paulina; Mital, Andrzej; Hellman, Andrzej; Bryl, Ewa; Witkowski, Jacek M

    2007-01-01

    Limited proteolysis of multiple intracellular proteins by endogenous Ca-dependent cysteine proteases--calpains--is an important regulatory mechanism for cell proliferation, apoptosis etc. Its importance for cellular functions is stressed by existence of endogenous calpain inhibitors--calpastatins. The calpain-calpastatin system within living cells is in a fragile balance, which depends on both partners. The interdependence of calpain--a protease--and calpastatin--an endogenous inhibitor and at the same time a substrate for this enzyme makes any assessment of actual activity of this enzyme in the cells very difficult. In this work we made an attempt to estimate and compare the activity of calpain in human peripheral blood lymphocytes by assessing the levels of limited proteolysis of calpastatin in these cells by western blot, while at the same time the levels of calpain protein inside these cells was measured by flow cytometry. Our results indicate that it is possible to compare (semi-quantitatively) the activities of calpain in peripheral blood CD4+ and CD19+ lymphocytes from various donors that way. Preliminary results showed that calpain activity is increased in the CD4+ T cells isolated from peripheral blood of rheumatoid arthritis patients as compared to control lymphocytes. Extremely high intrinsic activity of calpain was detected in chronic lymphocytic leukemia (CD19+) cells. All this confirms the detection of immunoreactive products of calpastatin as a good maker of endogenous calpain activity.

  4. Method validation and reference range values for a peripheral blood immunophenotyping assay in non-human primates.

    PubMed

    Caldwell, Robert G; Marshall, Peggy; Fishel, Jared

    2016-01-01

    A peripheral blood immunophenotyping assay was developed and validated for determination of total T-lymphocytes, helper T-lymphocytes, cytotoxic T-lymphocytes, B-lymphocytes, and natural killer cells in cynomolgus monkeys. Validation parameters included assessment of precision, linearity, antibody optimization, stability of peripheral blood samples, and stability of fixed immunophenotyping samples. Total lymphocyte populations were determined using a heterogeneous lymphocyte gating strategy consisting of CD45 fluorescent staining and side-scatter demarcation. Relative lymphocyte subset values were determined using antigen-specific gating strategies. Absolute subset concentrations for each lymphocyte subset were subsequently determined using a dual-platform methodology wherein relative lymphocyte subset values (via flow cytometry analyses) were multiplied by the absolute total lymphocyte (via hematology analyses) values. Reference ranges are presented for cynomolgus monkey, rhesus monkey, and baboon. Additional 1-year longitudinal immunophenotyping values are presented for the cynomolgus monkey. The method validation and reference ranges presented in this research provide a robust analytical methodology for determination of peripheral blood lymphocyte subsets in various non-human primate species.

  5. Extracellular matrix from human umbilical cord-derived mesenchymal stem cells as a scaffold for peripheral nerve regeneration

    PubMed Central

    Xiao, Bo; Rao, Feng; Guo, Zhi-yuan; Sun, Xun; Wang, Yi-guo; Liu, Shu-yun; Wang, Ai-yuan; Guo, Quan-yi; Meng, Hao-ye; Zhao, Qing; Peng, Jiang; Wang, Yu; Lu, Shi-bi

    2016-01-01

    The extracellular matrix, which includes collagens, laminin, or fibronectin, plays an important role in peripheral nerve regeneration. Recently, a Schwann cell-derived extracellular matrix with classical biomaterial was used to mimic the neural niche. However, extensive clinical use of Schwann cells remains limited because of the limited origin, loss of an autologous nerve, and extended in vitro culture times. In the present study, human umbilical cord-derived mesenchymal stem cells (hUCMSCs), which are easily accessible and more proliferative than Schwann cells, were used to prepare an extracellular matrix. We identified the morphology and function of hUCMSCs and investigated their effect on peripheral nerve regeneration. Compared with a non-coated dish tissue culture, the hUCMSC-derived extracellular matrix enhanced Schwann cell proliferation, upregulated gene and protein expression levels of brain-derived neurotrophic factor, glial cell-derived neurotrophic factor, and vascular endothelial growth factor in Schwann cells, and enhanced neurite outgrowth from dorsal root ganglion neurons. These findings suggest that the hUCMSC-derived extracellular matrix promotes peripheral nerve repair and can be used as a basis for the rational design of engineered neural niches. PMID:27630705

  6. Effects of Dietary n-3 Fatty Acids on Hepatic and Peripheral Insulin Sensitivity in Insulin-Resistant Humans

    PubMed Central

    Lalia, Antigoni Z.; Johnson, Matthew L.; Jensen, Michael D.; Hames, Kazanna C.; Port, John D.

    2015-01-01

    OBJECTIVE Dietary n-3 polyunsaturated fatty acids, including eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA), prevent insulin resistance and stimulate mitochondrial biogenesis in rodents, but the findings of translational studies in humans are thus far ambiguous. The aim of this study was to evaluate the influence of EPA and DHA on insulin sensitivity, insulin secretion, and muscle mitochondrial function in insulin-resistant, nondiabetic humans using a robust study design and gold-standard measurements. RESEARCH DESIGN AND METHODS Thirty-one insulin-resistant adults received 3.9 g/day EPA+DHA or placebo for 6 months in a randomized double-blind study. Hyperinsulinemic-euglycemic clamp with somatostatin was used to assess hepatic and peripheral insulin sensitivity. Postprandial glucose disposal and insulin secretion were measured after a meal. Measurements were performed at baseline and after 6 months of treatment. Abdominal fat distribution was evaluated by MRI. Muscle oxidative capacity was measured in isolated mitochondria using high-resolution respirometry and noninvasively by magnetic resonance spectroscopy. RESULTS Compared with placebo, EPA+DHA did not alter peripheral insulin sensitivity, postprandial glucose disposal, or insulin secretion. Hepatic insulin sensitivity, determined from the suppression of endogenous glucose production by insulin, exhibited a small but significant improvement with EPA+DHA compared with placebo. Muscle mitochondrial function was unchanged by EPA+DHA or placebo. CONCLUSIONS This study demonstrates that dietary EPA+DHA does not improve peripheral glucose disposal, insulin secretion, or skeletal muscle mitochondrial function in insulin-resistant nondiabetic humans. There was a modest improvement in hepatic insulin sensitivity with EPA+DHA, but this was not associated with any improvements in clinically meaningful outcomes. PMID:25852206

  7. Reactive oxygen species formation and apoptosis in human peripheral blood mononuclear cell induced by 900 MHz mobile phone radiation.

    PubMed

    Lu, Yao-Sheng; Huang, Bao-Tian; Huang, Yao-Xiong

    2012-01-01

    We demonstrate that reactive oxygen species (ROS) plays an important role in the process of apoptosis in human peripheral blood mononuclear cell (PBMC) which is induced by the radiation of 900 MHz radiofrequency electromagnetic field (RFEMF) at a specific absorption rate (SAR) of ~0.4 W/kg when the exposure lasts longer than two hours. The apoptosis is induced through the mitochondrial pathway and mediated by activating ROS and caspase-3, and decreasing the mitochondrial potential. The activation of ROS is triggered by the conformation disturbance of lipids, protein, and DNA induced by the exposure of GSM RFEMF. Although human PBMC was found to have a self-protection mechanism of releasing carotenoid in response to oxidative stress to lessen the further increase of ROS, the imbalance between the antioxidant defenses and ROS formation still results in an increase of cell death with the exposure time and can cause about 37% human PBMC death in eight hours.

  8. Derivation of Neural Stem Cells from Human Adult Peripheral CD34+ Cells for an Autologous Model of Neuroinflammation

    PubMed Central

    Wang, Tongguang; Choi, Elliot; Monaco, Maria Chiara G.; Campanac, Emilie; Medynets, Marie; Do, Thao; Rao, Prashant; Johnson, Kory R.; Elkahloun, Abdel G.; Von Geldern, Gloria; Johnson, Tory; Subramaniam, Sriram; Hoffman, Dax; Major, Eugene; Nath, Avindra

    2013-01-01

    Proinflammatory factors from activated T cells inhibit neurogenesis in adult animal brain and cultured human fetal neural stem cells (NSC). However, the role of inhibition of neurogenesis in human neuroinflammatory diseases is still uncertain because of the difficulty in obtaining adult NSC from patients. Recent developments in cell reprogramming suggest that NSC may be derived directly from adult fibroblasts. We generated NSC from adult human peripheral CD34+ cells by transfecting the cells with Sendai virus constructs containing Sox2, Oct3/4, c-Myc and Klf4. The derived NSC could be differentiated to glial cells and action potential firing neurons. Co-culturing NSC with activated autologous T cells or treatment with recombinant granzyme B caused inhibition of neurogenesis as indicated by decreased NSC proliferation and neuronal differentiation. Thus, we have established a unique autologous in vitro model to study the pathophysiology of neuroinflammatory diseases that has potential for usage in personalized medicine. PMID:24303066

  9. Antigen exposure shapes the ratio between antigen-specific Tregs and conventional T cells in human peripheral blood

    PubMed Central

    Su, Laura F.; del Alcazar, Daniel; Stelekati, Erietta; Wherry, E. John; Davis, Mark M.

    2016-01-01

    The T-cell receptor (TCR) is required for maturation and function of regulatory T cells (Tregs), but the ligand specificities of Tregs outside the context of transgenic TCRs are largely unknown. Using peptide–MHC tetramers, we isolated rare specific Foxp3+ cells directly ex vivo from adult peripheral blood and defined their frequency and phenotype. We find that a proportion of circulating Tregs recognize foreign antigens and the frequency of these cells are similar to that of self-reactive Tregs in the absence of cognate infection. In contrast, the frequencies of Tregs that recognize some common microbial antigens are significantly reduced in the blood of most adults. Exposure to peripheral antigens likely has a major influence on the balance between Tregs and conventional T-cell subsets because a larger proportion of flu-specific T cells has a regulatory cell phenotype in the cord blood. Consistent with this finding, we show that lymphocytic choriomeningitis virus infection can directly modulate the ratio of virus-specific effectors and Tregs in mice. The resulting change in the balance within an antigen-specific T-cell population further correlates with the magnitude of effector response and the chronicity of infection. Taken together, our data highlight the importance of antigen specificity in the functional dynamics of the T-cell repertoire. Each specific population of CD4+ T cells in human peripheral blood contains a subset of Tregs at birth, but the balance between regulatory and effector subsets changes in response to peripheral antigen exposure and this could impact the robustness of antipathogen immunity. PMID:27681619

  10. Frequency and genetic characterization of V(DD)J recombinants in the human peripheral blood antibody repertoire.

    PubMed

    Briney, Bryan S; Willis, Jordan R; Hicar, Mark D; Thomas, James W; Crowe, James E

    2012-09-01

    Antibody heavy-chain recombination that results in the incorporation of multiple diversity (D) genes, although uncommon, contributes substantially to the diversity of the human antibody repertoire. Such recombination allows the generation of heavy chain complementarity determining region 3 (HCDR3) regions of extreme length and enables junctional regions that, because of the nucleotide bias of N-addition regions, are difficult to produce through normal V(D)J recombination. Although this non-classical recombination process has been observed infrequently, comprehensive analysis of the frequency and genetic characteristics of such events in the human peripheral blood antibody repertoire has not been possible because of the rarity of such recombinants and the limitations of traditional sequencing technologies. Here, through the use of high-throughput sequencing of the normal human peripheral blood antibody repertoire, we analysed the frequency and genetic characteristics of V(DD)J recombinants. We found that these recombinations were present in approximately 1 in 800 circulating B cells, and that the frequency was severely reduced in memory cell subsets. We also found that V(DD)J recombination can occur across the spectrum of diversity genes, indicating that virtually all recombination signal sequences that flank diversity genes are amenable to V(DD)J recombination. Finally, we observed a repertoire bias in the diversity gene repertoire at the upstream (5') position, and discovered that this bias was primarily attributable to the order of diversity genes in the genomic locus.

  11. Ultra high performance liquid chromatographic-tandem mass spectrometric multi-analyte procedure for target screening and quantification in human blood plasma: validation and application for 31 neuroleptics, 28 benzodiazepines, and Z-drugs.

    PubMed

    Remane, Daniela; Meyer, Markus R; Wissenbach, Dirk K; Maurer, Hans H

    2011-09-01

    For fast and reliable screening, identification, and quantification of as many analytes as possible, multi-analyte approaches are very useful in clinical and forensic toxicology. Using ultra high performance liquid chromatography-tandem mass spectrometry, such an approach has been developed for blood plasma analysis after simple liquid-liquid extraction. In the present paper, validation and application is described for 31 neuroleptics, 28 benzodiazepines, and Z-drugs (zaleplone, zolpidem, and zopiclone). The validation parameters included recovery, matrix effects, process efficiency, ion suppression/enhancement of co-eluting analytes, selectivity, crosstalk, accuracy and precision, stabilities, and limits of quantification and detection. The results showed that the approach was selective, sensitive, accurate, and precise for 24 neuroleptics and 21 benzodiazepines and Z-drugs. The remaining analytes were unstable and/or too low dosed. Cost- and time-saving one-point calibration was applicable only for half of the analytes. The applicability was successfully shown for most of the drugs by analyzing authentic plasma samples and external quality control samples.

  12. Magnetic stimulation of the human brain and peripheral nervous system: an introduction and the results of an initial clinical evaluation.

    PubMed

    Barker, A T; Freeston, I L; Jalinous, R; Jarratt, J A

    1987-01-01

    This report describes a novel method of stimulating the motor cortex and deep peripheral nerves in humans. The technique, developed in the Department of Medical Physics of Sheffield University, uses a large pulse of magnetic field to induce currents within the body and is painless. The basic principles of magnetic stimulation are described, and the technique is compared with conventional electrical stimulation. Safety aspects are discussed with reference to established clinical electrical and magnetic procedures. The results of the first clinical study using magnetic stimulation are described and show clear central motor pathway slowing in multiple sclerosis patients.

  13. Fetal toxicity of benzodiazepines in rats.

    PubMed

    Saito, H; Kobayashi, H; Takeno, S; Sakai, T

    1984-12-01

    This study was undertaken to evaluate fetal toxicity of benzodiazepine derivatives, nitrazepam, oxazepam, nimetazepam, clonazepam, diazepam and chlordiazepoxide. In fetotoxicities induced by the oral administration of these six drugs (100 mg/kg), nitrazepam (NTZ) and nimetazepam caused a great damage to the fetus compared with other analogues. The fetotoxicity of NTZ was dose-dependent at a dose of 50 to 100 mg/kg p.o. and its peak toxicity was observed in the group treated from the 8th to 10th day of gestation. Maternal serum and whole fetus concentration of NTZ were higher than that of diazepam which showed weak toxicity. It was suggested that the intensity of fetotoxicity induced by benzodiazepines was related to the amount of drug in blood of dam.

  14. Withdrawal from long-term benzodiazepine treatment.

    PubMed Central

    Petursson, H; Lader, M H

    1981-01-01

    Long-term, normal-dose benzodiazepine treatment was discontinued in 16 patients who were suspected of being dependent on their medication. The withdrawal was gradual, placebo-controlled, and double-blind. All the patients experienced some form of withdrawal reaction, which ranged from anxiety and dysphoria to moderate affective and perceptual changes. Symptom ratings rose as the drugs were discontinued, but usually subsided to prewithdrawal levels over the next two to four weeks. Other features of the withdrawal included disturbance of sleep and appetite and noticeable weight loss. Electroencephalography showed appreciable reduction in fast-wave activity as the drugs were withdrawn, and an improvement in psychological performance was recorded by the Digit Symbol Substitution Test. Because of the risk of dependence on benzodiazepines these agents should probably not be given as regular daily treatment for chronic anxiety. PMID:6114776

  15. [Role of benzodiazepine receptors in regulating aggressive behavior].

    PubMed

    Vasar, E E; Riago, L K; Allikmets, L Kh

    1983-01-01

    In experiments of male Wistar rats, it was established that a long-term apomorphine treatment caused changes in density of benzodiazepine receptors in forebrain structures opposite to those elicited by acute administration. The changes in benzodiazepine binding correlated with decrease of antiaggressive effect of diazepam and Ro 15-1788 after long-term apomorphine administration. The action of apomorphine on the benzodiazepine receptors was not direct, as apomorphine did not change the benzodiazepine agonist-antagonist interaction, and naloxone, opiate blocator, was a more powerful antagonist of antiaggressive action of diazepam than Ro 15-1788. The involvement of the two types of benzodiazepine receptors in the regulation of aggressive behavior is suggested, the first being apparently linked with GABA and opiate receptors and the other one--with the serotoninergic system. Ro 15-1788 was able to antagonize the effects of diazepam on the first but not on the second type of benzodiazepine receptors.

  16. Effect of oxidatively modified and non-modified human serum albumin on luminol-dependent chemiluminescence of human peripheral blood leukocytes stimulated with opsonized zymosan.

    PubMed

    Piryazev, A P; Azizova, A P; Aseichev, A V; Sergienko, V I

    2014-07-01

    We studied the effects of native and oxidized human serum albumin on luminol-dependent chemiluminescence of human peripheral blood leukocytes stimulated with opsonized zymosan. Human serum albumin was added simultaneously with opsonized zymosan at the beginning of the chemiluminescent reaction. Otherwise, leukocytes were incubated with human serum albumin at 37°C for various periods before addition of opsonized zymosan. Oxidized human serum albumin was obtained by the method of metal-catalyzed oxidation. In control to non-modified albumin, oxidized albumin produced an inhibitory effect on luminol-dependent chemiluminescence of leukocytes. These changes were observed in experiments with addition of oxidized albumin at the beginning of a chemiluminescent reaction and after incubation of study agent with cells.

  17. Imaging benzodiazepine receptors in man with C-11-suriclone and positron emission tomography

    SciTech Connect

    Frost, J.J.; Dannals, R.F.; Ravert, H.T.; Wilson, A.A.; Links, J.M.; Trifiletti, R.; Snyder, S.H.; Wagner, H.N. Jr.

    1985-05-01

    Suriclone is a potent cyclopyrrolone, anti-anxiety drug which binds to the benzodiazepine receptor complex (BZR) with high affinity. Suriclone binds to a site on the BZR distinct from the site where benzodiazepines bind. The K/sub D/ of suriclone at 37oC is 0.03 nM. C-11-suriclone (SUR) was synthesized by reacting C-CH3I with the appropriate amine precursor. SUR (1 ..mu..g/kg) was injected IV into a baboon alone or with 1 mg/kg of Ro-151788, a benzodiazepine antagonist, and serial PET scans of the brain were obtained. High radioactivity concentrations were observed in the cerebral cortex and cerebellum which contain high densities of BZR, intermediate concentrations in thalamus and low concentrations in the striatum. When Ro-151788 was given a uniform distribution of radioactivity was observed; the radioactivity was reduced to ca. 25% of control values in the brain which was contained within the PET slice. SUR (0.2 ..mu..g/kg) was next administered to a human subject. From 30-60 minutes after injection high radioactivity concentrations were observed in the cerebral cortex and cerebellum, intermediate concentrations in the thalamus and a low concentration in the caudate. Radioactivity in the cerebral cortex and cerebellum decreased slowly with time, implying that binding of SUR to a high affinity site had occurred. These results demonstrate utility of SUR for measuring binding to the benzodiazepine receptor complex non-invasively in man.

  18. Disinhibitory reactions to benzodiazepines: a review.

    PubMed

    van der Bijl, P; Roelofse, J A

    1991-05-01

    This article reviews some of the important aspects of benzodiazepine-induced disinhibitory reactions. Although reactions of this type are relatively rare, they may sometimes manifest themselves in aggressive behavior accompanied by suicidal or homicidal tendencies. It appears that these reactions occur more commonly in younger patients, although the elderly (above 65 years) may also be at risk. Many mechanisms have been postulated, but none truly explain how these reactions arise. The concept that central cholinergic mechanisms may play a role, however, remains attractive and stems primarily from physostigmine's ability to successfully reverse this type of reaction. The potential role of the benzodiazepine antagonists, eg, flumazenil, in reversing disinhibitory reactions is also discussed. Apart from patients who previously exhibited poor impulse control, there are no reliable indicators for recognizing potential candidates for this type of reaction. To minimize the occurrence of disinhibitory reactions, some guidelines, which include the avoidance of certain drug combinations, the use of low doses of benzodiazepines, slow incremental intravenous administration, and good rapport with patients, are presented.

  19. The Use of Benzodiazepines to Facilitate Sexual Assault.

    PubMed

    Montgomery, M A

    2010-01-01

    Benzodiazepines are one of the classes of drugs most commonly associated with drug-facilitated sexual assault. As a widely prescribed class of medications and abused drugs, benzodiazepines are extensively available. Their sedating and amnesic effects make them effective candidates for use in drug-facilitated assaults. Detection methods for benzodiazepines and their metabolites in biological fluids are plentiful, but methods must be tailored to the low concentrations of drugs and metabolites expected to be encountered in these cases.

  20. Alprazolam is relatively more toxic than other benzodiazepines in overdose

    PubMed Central

    Isbister, Geoffrey K; O'Regan, Luke; Sibbritt, David; Whyte, Ian M

    2004-01-01

    Aims To describe alprazolam poisoning and the relative toxicity of alprazolam compared with other benzodiazepines. Methods A database of consecutive poisoning admissions to a regional toxicology service was searched to identify consecutive benzodiazepine deliberate self poisonings, which were coded as alprazolam, diazepam or other benzodiazepine. Major outcomes used were length of stay (LOS), intensive care (ICU) admission, coma (GCS < 9), flumazenil administration and requirement for mechanical ventilation. Prescription data were obtained for benzodiazepines for the study period. Results There were 2063 single benzodiazepine overdose admissions: 131 alprazolam overdoses, 823 diazepam overdoses and 1109 other benzodiazepine overdoses. The median LOS for alprazolam overdoses was 19 h which was 1.27 (95% CI 1.04, 1.54) times longer compared with other benzodiazepines by multiple linear regression. For patients with alprazolam overdoses, 22% were admitted to ICU which was 2.06 (95% CI 1.27, 3.33) times more likely compared with other benzodiazepines after multivariate analysis adjusting for age, dose, gender, time to ingestion and co-ingested drugs. Flumazenil was administered to 14% of alprazolam patients and 16% were ventilated, which was significantly more than for other benzodiazepine overdoses (8% and 11%, respectively). Twelve percent of alprazolam overdoses had a GCS < 9 compared with 10% for other benzodiazepines. From benzodiazepine prescription data, total alprazolam prescriptions in Australia increased from 0.13 million in 1992 to 0.41 million in 2001. Eighty five percent of prescriptions were for panic disorder, anxiety, depression or mixed anxiety/depression. Conclusions Alprazolam was significantly more toxic than other benzodiazepines. The increased prescription of alprazolam to groups with an increased risk of deliberate self poisoning is concerning and needs review. PMID:15206998

  1. Benzodiazepines do not potentiate GABA responses in neonatal hippocampal neurons.

    PubMed

    Rovira, C; Ben-Ari, Y

    1991-09-16

    Benzodiazepines (midazolam; flunitrazepam) and pentobarbital increase the response to exogenous gamma-aminobutyric acid (GABA) in adult hippocampal cells. We report in this paper that in contrast pentobarbital but not benzodiazepine potentiate the effects of exogenous (GABA) in neurons recorded from slices of less than two weeks old. This finding suggests that the functional association of benzodiazepine and GABAA receptors is changed during early postnatal life.

  2. A novel method to generate and culture human mast cells: Peripheral CD34+ stem cell-derived mast cells (PSCMCs).

    PubMed

    Schmetzer, Oliver; Valentin, Patricia; Smorodchenko, Anna; Domenis, Rossana; Gri, Giorgia; Siebenhaar, Frank; Metz, Martin; Maurer, Marcus

    2014-11-01

    The identification and characterization of human mast cell (MC) functions are hindered by the shortage of MC populations suitable for investigation. Here, we present a novel technique for generating large numbers of well differentiated and functional human MCs from peripheral stem cells (=peripheral stem cell-derived MCs, PSCMCs). Innovative and key features of this technique include 1) the use of stem cell concentrates, which are routinely discarded by blood banks, as the source of CD34+ stem cells, 2) cell culture in serum-free medium and 3) the addition of LDL as well as selected cytokines. In contrast to established and published protocols that use CD34+ or CD133+ progenitor cells from full blood, we used a pre-enriched cell population obtained from stem cell concentrates, which yielded up to 10(8) differentiated human MCs per batch after only three weeks of culture starting with 10(6) total CD34+ cells. The total purity on MCs (CD117+, FcεR1+) generated by this method varied between 55 and 90%, of which 4-20% were mature MCs that contain tryptase and chymase and show expression of FcεRI and CD117 in immunohistochemistry. PSCMCs showed robust histamine release in response to stimulation with anti-FcεR1 or IgE/anti-IgE, and increased proliferation and differentiation in response to IL-1β or IFN-γ. Taken together, this new protocol of the generation of large numbers of human MCs provides for an innovative and suitable option to investigate the biology of human MCs. Copyright © 2014 Elsevier B.V. All rights reserved.

  3. Atomic resolution view into the structure–function relationships of the human myelin peripheral membrane protein P2

    SciTech Connect

    Ruskamo, Salla; Yadav, Ravi P.; Sharma, Satyan; Lehtimäki, Mari; Laulumaa, Saara; Aggarwal, Shweta; Simons, Mikael; Bürck, Jochen; Ulrich, Anne S.; Juffer, André H.; Kursula, Inari; Kursula, Petri

    2014-01-01

    The structure of the human myelin peripheral membrane protein P2 has been refined at 0.93 Å resolution. In combination with functional experiments in vitro, in vivo and in silico, the fine details of the structure–function relationships in P2 are emerging. P2 is a fatty acid-binding protein expressed in vertebrate peripheral nerve myelin, where it may function in bilayer stacking and lipid transport. P2 binds to phospholipid membranes through its positively charged surface and a hydrophobic tip, and accommodates fatty acids inside its barrel structure. The structure of human P2 refined at the ultrahigh resolution of 0.93 Å allows detailed structural analyses, including the full organization of an internal hydrogen-bonding network. The orientation of the bound fatty-acid carboxyl group is linked to the protonation states of two coordinating arginine residues. An anion-binding site in the portal region is suggested to be relevant for membrane interactions and conformational changes. When bound to membrane multilayers, P2 has a preferred orientation and is stabilized, and the repeat distance indicates a single layer of P2 between membranes. Simulations show the formation of a double bilayer in the presence of P2, and in cultured cells wild-type P2 induces membrane-domain formation. Here, the most accurate structural and functional view to date on P2, a major component of peripheral nerve myelin, is presented, showing how it can interact with two membranes simultaneously while going through conformational changes at its portal region enabling ligand transfer.

  4. Efforts of the human immune system to maintain the peripheral CD8+ T cell compartment after childhood thymectomy.

    PubMed

    Zlamy, Manuela; Almanzar, Giovanni; Parson, Walther; Schmidt, Christian; Leierer, Johannes; Weinberger, Birgit; Jeller, Verena; Unsinn, Karin; Eyrich, Matthias; Würzner, Reinhard; Prelog, Martina

    2016-01-01

    Homeostatic mechanisms to maintain the T cell compartment diversity indicate an ongoing process of thymic activity and peripheral T cell renewal during human life. These processes are expected to be accelerated after childhood thymectomy and by the influence of cytomegalovirus (CMV) inducing a prematurely aged immune system. The study aimed to investigate proportional changes and replicative history of CD8+ T cells, of recent thymic emigrants (RTEs) and CD103+ T cells (mostly gut-experienced) and the role of Interleukin-(IL)-7 and IL-7 receptor (CD127)-expressing T cells in thymectomized patients compared to young and old healthy controls. Decreased proportions of naive and CD31 + CD8+ T cells were demonstrated after thymectomy, with higher proliferative activity of CD127-expressing T cells and significantly shorter relative telomere lengths (RTLs) and lower T cell receptor excision circles (TRECs). Increased circulating CD103+ T cells and a skewed T cell receptor (TCR) repertoire were found after thymectomy similar to elderly persons. Naive T cells were influenced by age at thymectomy and further decreased by CMV. After childhood thymectomy, the immune system demonstrated constant efforts of the peripheral CD8+ T cell compartment to maintain homeostasis. Supposedly it tries to fill the void of RTEs by peripheral T cell proliferation, by at least partly IL-7-mediated mechanisms and by proportional increase of circulating CD103+ T cells, reminiscent of immune aging in elderly. Although other findings were less significant compared to healthy elderly, early thymectomy demonstrated immunological alterations of CD8+ T cells which mimic features of premature immunosenescence in humans.

  5. LC-MS-(TOF) analysis method for benzodiazepines in urine samples from alleged drug-facilitated sexual assault victims.

    PubMed

    ElSohly, Mahmoud A; Gul, Waseem; ElSohly, Kareem M; Avula, Bharathi; Khan, Ikhlas A

    2006-10-01

    In recent years there has been an increase in the number of reports in the U.S. of the use of drugs to commit sexual assault. In 1994, a nationwide urine testing program was developed to assess the incidence of the use of drugs to facilitate sexual assault and provide information for use in the investigation of these crimes. Urine samples were collected from victims of suspected drug-induced sexual assault by law enforcement agencies, emergency rooms, and rape crisis centers. The most implicated drug class was benzodiazepines, either alone or in combination with alcohol. In this report, a procedure was developed for the screening of 22 benzodiazepines in human urine by liquid chromatography-time of flight-mass spectrometry [LC-MS-(TOF)]. The limit of quantitation for all benzodiazepines ranged from 2 to 10 ng/mL, and the limit of detection was 0.5 to 3.0 ng/mL. These results suggest that the method sensitivity is suitable to screen for all 22 benzodiazepines in human urine at low levels. The method was used to analyze samples previously reported to have screened positive for benzodiazepines by immunoassay at 50 ng/mL cut off but failed to confirm by a gas chromatography-MS method. The results of reanalysis of these samples using this LC-MS method are reported.

  6. Benzodiazepines and amphetamine on avoidance behaviour in mice.

    PubMed

    Sansone, M

    1975-11-01

    Six benzodiazepine derivatives, given alone or in combination with amphetamine, were tested in mice subjected to five 100-trial avoidance sessions in the shuttle-box. All derivatives, execpt bromazepam, showed some facilitating effects on avoidance responding when given alone. Facilitation was particularly evident following the administration of chlordiazepoxide (2.5 mg/kg), medazepam (10 mg/kg) and nitrazepam (0.25, 0.5 and 1 mg/kg). Favourable effects were obtained by combining each benzodiazepine compound with amphetamine. The levels of avoidance respinses were usually higher under benzodiazepine-amphetamine combinations than under benzodiazepines alone.

  7. Benzodiazepine dependence and its treatment with low dose flumazenil.

    PubMed

    Hood, Sean David; Norman, Amanda; Hince, Dana Adelle; Melichar, Jan Krzysztof; Hulse, Gary Kenneth

    2014-02-01

    Globally benzodiazepines remain one of the most prescribed medication groups, especially in the primary care setting. With such high levels of prescribing it is not surprising that benzodiazepine dependence is common, cutting across all socioeconomic levels. Despite recognition of the potential for the development of iatrogenic dependence and the lack of any effective treatment, benzodiazepines continue to be widely prescribed in general practice. Conventional dependence management, benzodiazepine tapering, is commonly a protracted process over several weeks or months. It is often associated with significant withdrawal symptoms and craving leading to patient drop out and return to use. Accordingly, there is a worldwide need to find effective pharmacotherapeutic interventions for benzodiazepine dependence. One drug of increasing interest is the GABAA benzodiazepine receptor antagonist/partial agonist, flumazenil. Multiple bolus intravenous infusions of low dose flumazenil used either with or without benzodiazepine tapering can reduce withdrawal sequelae, and/or longer term symptoms in the months following withdrawal. Preliminary data suggest that continuous intravenous or subcutaneous flumazenil infusion for 4 days significantly reduces acute benzodiazepine withdrawal sequelae. The subcutaneous infusion was shown to be tissue compatible so the development of a longer acting (i.e. several weeks) depot flumazenil formulation has been explored. This could be capable of managing both acute and longer term benzodiazepine withdrawal sequelae. Preliminary in vitro water bath and in vivo biocompatibility data in sheep show that such an implant is feasible and so is likely to be used in clinical trials in the near future.

  8. Triazolam, an anomalous benzodiazepine receptor ligand: in vitro characterization of alprazolam and triazolam binding.

    PubMed

    Clow, A; Glover, V; Sandler, M

    1985-08-01

    Both alprazolam and triazolam displaced clonazepam (but not Ro 5-4864) from rat brain membranes with high affinity, showing them to act at central but not peripheral benzodiazepine receptors. At 0 degrees C, 10 microM gamma-aminobutyric acid (GABA) increased the ability of alprazolam, but not of triazolam, to displace ethyl-beta-carboline-3-carboxylate (beta-CCE) and Ro 15-1788 from these receptors. At 37 degrees C, GABA increased the affinity of the receptors for both drugs, with a +GABA/-GABA ratio of 1.5 for each in promoting Ro 15-1788 binding displacement. As both triazolam and alprazolam act as anxiolytics in vivo, the results at 37 degrees C would be compatible with the hypothesis that GABA causes an increase in affinity of drugs that act in this way, but the results at 0 degrees C would not be compatible. At 37 degrees C, alprazolam had a higher IC50 for the benzodiazepine receptor than at 0 degrees C, whereas triazolam showed the reverse effect. The relative IC50 values in vitro at 37 degrees C correlated better with the potency in vivo than those obtained at 0 degrees C. At 0 degrees C, both drugs showed Hill plots with slopes of 0.9-1 with beta-CCE and Ro 15-1788. At 37 degrees C, the slopes with triazolam were much reduced, indicating that the drug may have a selective action on a subclass of central benzodiazepine receptors. In the studies reported here, alprazolam behaved like other benzodiazepines, whereas triazolam showed several anomalous properties. It would be of interest if these properties could be related either to the drug's use as a hypnotic or to the side effects it sometimes induces.

  9. Human Monoclonal Antibodies against Clostridium difficile Toxins A and B Inhibit Inflammatory and Histologic Responses to the Toxins in Human Colon and Peripheral Blood Monocytes

    PubMed Central

    Koon, Hon Wai; Shih, David Q.; Hing, Tressia C.; Yoo, Jun Hwan; Ho, Samantha; Chen, Xinhua; Kelly, Ciarán P.; Targan, Stephan R.

    2013-01-01

    Clostridium difficile infection (CDI) is a common and debilitating nosocomial infection with high morbidity and mortality. C. difficile mediates diarrhea and colitis by releasing two toxins, toxin A and toxin B. Since both toxins stimulate proinflammatory signaling pathways in human colonocytes and both are involved in the pathophysiology of CDI, neutralization of toxin A and B activities may represent an important therapeutic approach against CDI. Recent studies indicated that human monoclonal antibodies (MAbs) against toxins A and B reduce their cytotoxic and secretory activities and prevent CDI in hamsters. Moreover, anti-toxin A and anti-toxin B MAbs together with antibiotics also effectively reduced recurrent CDI in humans. However, whether these MAbs neutralize toxin A- and toxin B-associated immune responses in human colonic mucosa or human peripheral blood monocyte cells (PBMCs) has never been examined. We used fresh human colonic biopsy specimens and peripheral blood monocytes to evaluate the effects of these antibodies against toxin A- and B-associated cytokine release, proinflammatory signaling, and histologic damage. Incubation of anti-toxin A (MK3415) or anti-toxin B (MK6072) MAbs with human PBMCs significantly inhibited toxin A- and toxin B-mediated tumor necrosis factor alpha (TNF-α) and interleukin-1β (IL-1β) expression. MK3415 and MK6072 also diminished toxin A- and toxin B-mediated NF-κB p65 phosphorylation in human monocytes, respectively, and significantly reduced toxin A- and B-induced TNF-α and IL-1β expression as well as histologic damage in human colonic explants. Our results underline the effectiveness of MK3415 and MK6072 in blocking C. difficile toxin A- and toxin B-mediated inflammatory responses and histologic damage. PMID:23629713

  10. LDL and HDL transfer rates across peripheral microvascular endothelium agree with those predicted for passive ultrafiltration in humans.

    PubMed

    Michel, C Charles; Nanjee, M Nazeem; Olszewski, Waldemar L; Miller, Norman E

    2015-01-01

    The mechanisms by which LDLs and HDLs cross the vascular endothelium from plasma into interstitial fluid are not understood, and have never been studied in humans in vivo. We determined whether the plasma-to-lymph clearance rates of LDL and HDL conform with those predicted by passive ultrafiltration through intercellular pores, or if it is necessary to invoke an active process such as receptor-mediated transcytosis. Plasma and afferent peripheral lymph were collected under steady-state conditions from 30 healthy men, and assayed for seven globular proteins of molecular radii 2.89-8.95 nm, complement C3, and apo AI, apo AII, and apo B. Plasma-to-lymph clearance rates of the seven proteins fitted the relation expected for molecules of their size when transported through two populations of pores of radius 4.95 and 20.1 nm. The same model parameters were then found to accurately predict the clearance rates of both HDL and LDL. The apparent clearance of complement C3, previously shown to be secreted by cultured endothelium, exceeded that predicted by the model. We conclude that the transport of HDL and LDL from plasma into interstitial fluid across the peripheral vascular endothelium in healthy humans can be explained by ultrafiltration without invoking an additional active process such as transcytosis.

  11. X-ray-induced changes in the expression of inflammation-related genes in human peripheral blood.

    PubMed

    Wang, Ping; Guo, Fei; Han, Lin; Wang, Xi'ai; Li, Jie; Guo, Yan; Lü, Yumin

    2014-10-27

    Using quantitative real-time polymerase chain reaction (PCR) array, we explored and compared the expression changes of inflammation-related genes in human peripheral blood irradiated with 0.5, 3, and 10 Gy doses of X-rays 24 h after exposure. Results indicated that the expression of 62 out of 84 genes was significantly altered after X-ray radiation. Among these 62 genes, 35 (such as TNFSF4) are known to be associated with radiation response, but others are novel. At a low radiation dose (0.5 Gy), 9 genes were up-regulated and 19 were down-regulated. With further increased dose to 3 Gy, 8 unique genes were up-regulated and 19 genes were down-regulated. We also identified 48 different genes that were differentially expressed significantly after 10 Gy of irradiation, and among these transcripts, up-regulated genes accounted for only one-third (16 genes) of the total. Of the 62 genes, 31 were significantly altered only at a specific dose, and a total of 10 genes were significantly expressed at all 3 doses. The dose- and time-dependent expression of CCL2 was confirmed by quantitative real-time reverse-transcription PCR. A number of candidate genes reported herein may be useful molecular biomarkers of radiation exposure in human peripheral blood.

  12. LDL and HDL transfer rates across peripheral microvascular endothelium agree with those predicted for passive ultrafiltration in humans

    PubMed Central

    Michel, C. Charles; Nanjee, M. Nazeem; Olszewski, Waldemar L.; Miller, Norman E.

    2015-01-01

    The mechanisms by which LDLs and HDLs cross the vascular endothelium from plasma into interstitial fluid are not understood, and have never been studied in humans in vivo. We determined whether the plasma-to-lymph clearance rates of LDL and HDL conform with those predicted by passive ultrafiltration through intercellular pores, or if it is necessary to invoke an active process such as receptor-mediated transcytosis. Plasma and afferent peripheral lymph were collected under steady-state conditions from 30 healthy men, and assayed for seven globular proteins of molecular radii 2.89–8.95 nm, complement C3, and apo AI, apo AII, and apo B. Plasma-to-lymph clearance rates of the seven proteins fitted the relation expected for molecules of their size when transported through two populations of pores of radius 4.95 and 20.1 nm. The same model parameters were then found to accurately predict the clearance rates of both HDL and LDL. The apparent clearance of complement C3, previously shown to be secreted by cultured endothelium, exceeded that predicted by the model. We conclude that the transport of HDL and LDL from plasma into interstitial fluid across the peripheral vascular endothelium in healthy humans can be explained by ultrafiltration without invoking an additional active process such as transcytosis. PMID:25398615

  13. Validation of a simultaneous analytical method for the detection of 27 benzodiazepines and metabolites and zolpidem in hair using LC-MS/MS and its application to human and rat hair.

    PubMed

    Kim, Jihyun; Lee, Sooyeun; In, Sanghwan; Choi, Hwakyung; Chung, Heesun

    2011-04-15

    Benzodiazepines and zolpidem are controlled in many countries due to their inherent adverse effects of a high degree of tolerance and dependence. Recently, as some of these drugs have become distributed illegally and available through media such as the Internet, their abuse is becoming a serious social problem. Hair is a useful specimen to prove chronic drug use. In the present study, a simultaneous analytical method for the detection of 27 benzodiazepines and metabolites and zolpidem in hair was established and validated using liquid chromatography-tandem mass spectrometry (LC-MS/MS). The drugs and their metabolites in hair were extracted using methanol, filtered and injected on the LC-MS/MS. The following validation parameters of the method were satisfactory: selectivity, linearity, matrix effect, recovery, process efficiency, intra- and inter-assay precision and accuracy and processed sample stability. The limit of detection (LOD) and the limit of quantification (LOQ) were the total drug detected from the sample. The LODs ranged from 0.005 ng (zolpidem) to 0.5 ng (bromazepam and chlordiazepoxide) and the LOQs were 0.25 ng in every analyte except for bromazepam and chlordiazepoxide, for which they were 0.5 ng. The developed method was successfully applied to five legal cases involving use of benzodiazepines and zolpidem and to an animal study on drug incorporation into hair. Diazepam and its three metabolites, as well as lorazepam, were detected in hair from both the multiple- and single-dose administration groups of lean Zucker rats. The concentration of diazepam was higher than those of its metabolites in both dark grey and white hair from the multiple-dose administration groups, with the mean concentration ranges from 0.16 to 0.51 ng/mg and from 0.10 to 0.24 ng/mg, respectively. The mean concentration ranges of lorazepam were from 0.05 to 0.37 ng/mg in dark grey hair and from 0.11 to 0.45 ng/mg in white hair from the multiple-dose administration groups. Hair

  14. A Semi-automated Approach to Preparing Antibody Cocktails for Immunophenotypic Analysis of Human Peripheral Blood

    PubMed Central

    Koguchi, Yoshinobu; Gonzalez, Iliana L.; Meeuwsen, Tanisha L.; Miller, William L.; Haley, Daniel P.; Tanibata-Branham, Alice N.; Bahjat, Keith S.

    2016-01-01

    Immunophenotyping of peripheral blood by flow cytometry determines changes in the frequency and activation status of peripheral leukocytes during disease and treatment. It has the potential to predict therapeutic efficacy and identify novel therapeutic targets. Whole blood staining utilizes unmanipulated blood, which minimizes artifacts that can occur during sample preparation. However, whole blood staining must also be done on freshly collected blood to ensure the integrity of the sample. Additionally, it is best to prepare antibody cocktails on the same day to avoid potential instability of tandem-dyes and prevent reagent interaction between brilliant violet dyes. Therefore, whole blood staining requires careful standardization to control for intra and inter-experimental variability. Here, we report deployment of an automated liquid handler equipped with a two-dimensional (2D) barcode reader into a standard process of making antibody cocktails for flow cytometry. Antibodies were transferred into 2D barcoded tubes arranged in a 96 well format and their contents compiled in a database. The liquid handler could then locate the source antibody vials by referencing antibody names within the database. Our method eliminated tedious coordination for positioning of source antibody tubes. It provided versatility allowing the user to easily change any number of details in the antibody dispensing process such as specific antibody to use, volume, and destination by modifying the database without rewriting the scripting in the software method for each assay. A proof of concept experiment achieved outstanding inter and intra- assay precision, demonstrated by replicate preparation of an 11-color, 17-antibody flow cytometry assay. These methodologies increased overall throughput for flow cytometry assays and facilitated daily preparation of the complex antibody cocktails required for the detailed phenotypic characterization of freshly collected anticoagulated peripheral blood

  15. Stimulation through CD50 preferentially induces apoptosis of TCR1+ human peripheral blood lymphocytes.

    PubMed

    López-Briones, S; Portales-Pérez, D P; Baranda, L; de la Fuente, H; Rosenstein, Y; González-Amaro, R

    1998-01-01

    Apoptosis has an important role in several key immunological phenomena such as regulation of the immune response, and deletion of auto-reactive cells. This phenomenon is induced following the interaction of several cell membrane receptors with their respective ligands or after cell activation. We have studied the possible effect of signaling through CD50/ICAM-3 and CD69/AIM on apoptosis of peripheral blood lymphocytes. Apoptosis was assessed by both flow cytometry analysis (content of cell DNA and binding to annexin V), and detection of DNA fragmentation by agarose gel electrophoresis. We found that a stimulatory anti-CD50 mAb was able to induce a small but significant degree of apoptosis in resting peripheral blood mononuclear cells from most donors; this effect was dose-dependent and was evident as early as at 12 h, with a maximal induction at 48 h. Studies with T and non-T cells showed that only the former cell population was sensitive to the induction of apoptosis through CD50. Further experiments revealed that the anti-ICAM-3 mAb preferentially induced apoptosis of TCR gamma delta-bearing cells. In addition, we found a significant increase in Cai2+ in PBMC stimulated with an anti-CD50 mAb, suggesting the involvement of this signaling pathway in the induction of apoptosis through this adhesion receptor. In contrast, under our experimental conditions, stimulation through CD69 did not have any effect on the induction of apoptosis on either cultured T lymphoblasts or PMA-stimulated PBMC. Our findings suggest that the interaction of CD50 with its natural ligand LFA-1 results in the induction of apoptosis in a significant fraction of resting PBMC. This phenomenon may be involved in immune regulation, lymphocyte turnover and peripheral deletion of auto-reactive cells.

  16. Peripheral venous congestion causes time- and dose-dependent release of endothelin-1 in humans.

    PubMed

    Lin, Jeffrey; Chudasama, Neelesh; Hayashi, Yacki; Hawk, Christopher; Ramnauth, Sahadeo D; Wong, Ka Yuk; Harxhi, Ante; Onat, Duygu; Wakabayashi, Michiyori; Uriel, Nir; Jorde, Ulrich P; LeJemtel, Thierry H; Sabbah, Hani N; Demmer, Ryan T; Colombo, Paolo C

    2017-03-01

    Endothelin-1 (ET-1) is a pivotal mediator of vasoconstriction and inflammation in congestive states such as heart failure (HF) and chronic kidney disease (CKD). Whether peripheral venous congestion (VC) increases plasma ET-1 at pressures commonly seen in HF and CKD patients is unknown. We seek to characterize whether peripheral VC promotes time- and dose-dependent increases in plasma ET-1 and whether these changes are sustained after decongestion. We used a randomized, cross-over design in 20 healthy subjects (age 30 ± 7 years). To experimentally model VC, venous pressure was increased to either 15 or 30 mmHg (randomized at first visit) above baseline by inflating a cuff around the subject's dominant arm; the nondominant arm served as a noncongested control. We measured plasma ET-1 at baseline, after 20, 60 and 120 min of VC, and finally at 180 min (60 min after cuff release and decongestion). Plasma ET-1 progressively and significantly increased over 120 min in the congested arm relative to the control arm and to baseline values. This effect was dose-dependent: ET-1 increased by 45% and 100% at VC doses of 15 and 30 mmHg, respectively (P < 0.05), and declined after 60 min of decongestion though remaining significantly elevated compared to baseline. In summary, peripheral VC causes time- and dose-dependent increases in plasma ET-1. Of note, the lower dose of 15 mmHg (more clinically relevant to HF and CKD patients) was sufficient to raise ET-1. These findings support the potentially contributory, not merely consequential, role of VC in the pathophysiology of HF and CKD.

  17. Barriers of the peripheral nerve

    PubMed Central

    Peltonen, Sirkku; Alanne, Maria; Peltonen, Juha

    2013-01-01

    This review introduces the traditionally defined anatomic compartments of the peripheral nerves based on light and electron microscopic topography and then explores the cellular and the most recent molecular basis of the different barrier functions operative in peripheral nerves. We also elucidate where, and how, the homeostasis of the normal human peripheral nerve is controlled in situ and how claudin-containing tight junctions contribute to the barriers of peripheral nerve. Also, the human timeline of the development of the barriers of the peripheral nerve is depicted. Finally, potential future therapeutic modalities interfering with the barriers of the peripheral nerve are discussed. PMID:24665400

  18. Selective effects of tea extract and its phenolic compounds on human peripheral blood mononuclear cell cytokine secretions.

    PubMed

    Neyestani, Tirang R; Gharavi, A'azam; Kalayi, Ali

    2009-01-01

    The effects of black tea extract (BTE) and some of its pure phenolics on human peripheral blood mononuclear cell (PBMC) cytokine secretion were examined in vitro. The main steps of the study included chromatographic analysis of BTE and commercial green tea extract, Polyphenon 60, determination of the total antioxidant capacity of both the extracts and their phenolics, and finally evaluation of their effects on PBMCs. Four major peaks in the chromatogram of BTE belonged to caffeine, gallic acid, epigallocatechin and epigallocatechin gallate, and the latter showed the highest antioxidant capacity. While pure phenolics at the concentration of 20 mM did not significantly affect PBMC cytokine secretion, BTE and Polyphenon 60 suppressed interleukin-1 beta, tumor necrosis factor-alpha and interleukin-6. Interestingly, the secretion of interferon-gamma after 24 h was slightly, but significantly, boosted by the extracts. BTE has selective pro-inflammatory cytokine-suppressing effects on human PBMCs.

  19. Integrative genomic analysis implicates limited peripheral adipose storage capacity in the pathogenesis of human insulin resistance.

    PubMed

    Lotta, Luca A; Gulati, Pawan; Day, Felix R; Payne, Felicity; Ongen, Halit; van de Bunt, Martijn; Gaulton, Kyle J; Eicher, John D; Sharp, Stephen J; Luan, Jian'an; De Lucia Rolfe, Emanuella; Stewart, Isobel D; Wheeler, Eleanor; Willems, Sara M; Adams, Claire; Yaghootkar, Hanieh; Forouhi, Nita G; Khaw, Kay-Tee; Johnson, Andrew D; Semple, Robert K; Frayling, Timothy; Perry, John R B; Dermitzakis, Emmanouil; McCarthy, Mark I; Barroso, Inês; Wareham, Nicholas J; Savage, David B; Langenberg, Claudia; O'Rahilly, Stephen; Scott, Robert A

    2017-01-01

    Insulin resistance is a key mediator of obesity-related cardiometabolic disease, yet the mechanisms underlying this link remain obscure. Using an integrative genomic approach, we identify 53 genomic regions associated with insulin resistance phenotypes (higher fasting insulin levels adjusted for BMI, lower HDL cholesterol levels and higher triglyceride levels) and provide evidence that their link with higher cardiometabolic risk is underpinned by an association with lower adipose mass in peripheral compartments. Using these 53 loci, we show a polygenic contribution to familial partial lipodystrophy type 1, a severe form of insulin resistance, and highlight shared molecular mechanisms in common/mild and rare/severe insulin resistance. Population-level genetic analyses combined with experiments in cellular models implicate CCDC92, DNAH10 and L3MBTL3 as previously unrecognized molecules influencing adipocyte differentiation. Our findings support the notion that limited storage capacity of peripheral adipose tissue is an important etiological component in insulin-resistant cardiometabolic disease and highlight genes and mechanisms underpinning this link.

  20. Human plasma enhances the infectivity of primary human immunodeficiency virus type 1 isolates in peripheral blood mononuclear cells and monocyte-derived macrophages.

    PubMed Central

    Wu, S C; Spouge, J L; Conley, S R; Tsai, W P; Merges, M J; Nara, P L

    1995-01-01

    Physiological microenvironments such as blood, seminal plasma, mucosal secretions, or lymphatic fluids may influence the biology of the virus-host cell and immune interactions for human immunodeficiency virus type 1 (HIV-1). Relative to media, physiological levels of human plasma were found to enhance the infectivity of HIV-1 primary isolates in both phytohemagglutinin-stimulated peripheral blood mononuclear cells and monocyte-derived macrophages. Enhancement was observed only when plasma was present during the virus-cell incubation and resulted in a 3- to 30-fold increase in virus titers in all of the four primary isolates tested. Both infectivity and virion binding experiments demonstrated a slow, time-dependent process generally requiring between 1 and 10 h. Human plasma collected in anticoagulants CPDA-1 and heparin, but not EDTA, exhibited this effect at concentrations from 90 to 40%. Furthermore, heat-inactivated plasma resulted in a loss of enhancement in peripheral blood mononuclear cells but not in monocyte-derived macrophages. Physiological concentrations of human plasma appear to recruit additional infectivity, thus increasing the infectious potential of the virus inoculum. PMID:7666510

  1. A new machine classification method applied to human peripheral blood leukocytes

    NASA Technical Reports Server (NTRS)

    Rorvig, Mark E.; Fitzpatrick, Steven J.; Vitthal, Sanjay; Ladoulis, Charles T.

    1994-01-01

    Human beings judge images by complex mental processes, whereas computing machines extract features. By reducing scaled human judgments and machine extracted features to a common metric space and fitting them by regression, the judgments of human experts rendered on a sample of images may be imposed on an image population to provide automatic classification.

  2. Increased intracellular levels of lysosomal beta-glucuronidase in peripheral blood PMNs from humans with rapidly progressive periodontitis.

    PubMed

    Pippin, D J; Cobb, C M; Feil, P

    1995-01-01

    Release of potent lysosomal enzymes by degranulation of polymorphonuclear leukocytes (PMNs) in host gingiva may contribute significantly to tissue destruction and the pathogenesis of periodontal disease. A pilot study established that peripheral blood PMNs from humans with rapidly progressive periodontitis (RPP) contained significantly increased amounts of intracellular lysosomal beta-glucuronidase as compared to healthy controls. This investigation gained insight into the question: are the increased levels of beta-glucuronidase in persons with RPP an a priori genetically determined PMN characteristic, or a reactive phenomenon induced by the periodontal disease process during granulopoiesis? Twelve healthy controls and twelve otherwise healthy individuals with RPP participated in a repeated measures design to T0 (initial, baseline), T1 (four weeks after disease control therapy), and T2 (two months later). At each visit clinical indices (GI, pocket depths, GCF flow, plaque index) were performed and peripheral blood obtained. PMNs were isolated and suspended as 5 x 10(6) cells in 2.0 ml of HBSS. PMN suspensions were tested for total intracellular beta-glucuronidase, degranulation induced by 1 x 10(-6)M and 5 x 10(-7) M FMLP challenges, and unchallenged for non-specific enzyme release. PMNs from individuals with RPP contained significantly higher absolute amounts of beta-glucuronidase and released greater absolute amounts at FMLP challenge at T0, T1, and T2 compared to controls. No relationship was found between any of the clinical indices and beta-glucuronidase levels and no pattern was discovered relating to the repeated measures over time. We conclude that RPP peripheral blood PMNs contain elevated levels of beta-glucuronidase that are not induced by the periodontal disease process.

  3. Peripheral autonomic nerves of human pineal organ terminate on vessels, their supposed role in the periodic secretion of pineal melatonin.

    PubMed

    Manzano E Silva, Maria Joao; Singh, Royana; Haldar, Chandana; Vigh, Béla; Szél, Ágoston

    2012-08-01

    Nonvisual pineal and retinal photoreceptors are synchronizing circadian and circannual periodicity to the environmental light periods in the function of various organs. Melatonin of the pineal organ is secreted at night and represents an important factor of this periodic regulation. Night illumination suppressing melatonin secretion may result in pathological events like breast and colorectal cancer. Experimental works demonstrated the role of autonomic nerves in the pineal melatonin secretion. It was supposed that mammalian pineals have lost their photoreceptor capacity that is present in submammalians, and sympathetic fibers would mediate light information from the retina to regulate melatonin secretion. Retinal afferentation may reach the organ by central nerve fibers via the pineal habenulae as well. In our earlier works we have found that the pineal organ developing from lobular evaginations of the epithalamus differs from peripheral endocrine glands and is composed of a retina-like central nervous tissue that is comprised of cone-like pinealocytes, secondary pineal neurons and glial cells. Their autonomic nerves in submammalians as well as in mammalian animals do not terminate on pineal cells, rather, they run in the meningeal septa among pineal lobules and form vasomotor nerve endings. Concerning the adult human pineal there are no detailed fine structural data about the termination of autonomic fibers, therefore, in the present work we investigated the ultrastructure of the human pineal peripheral autonomic nerve fibers. It was found, that similarly to other parts of the brain, autonomic nerves do not enter the human pineal nervous tissue itself but separated by glial limiting membranes take their course in the meningeal septa of the organ and terminate on vessels by vasomotor endings. We suppose that these autonomic vasomotor nerves serve the regulation of the pineal blood supply according to the circadian and circannual changes of the metabolic activity

  4. Thymosin increases production of T-cell growth factor by normal human peripheral blood lymphocytes.

    PubMed Central

    Zatz, M M; Oliver, J; Samuels, C; Skotnicki, A B; Sztein, M B; Goldstein, A L

    1984-01-01

    The in vitro incubation of phytohemagglutinin-stimulated peripheral blood lymphocytes with thymosin results in a marked and reproducible increase in production of T-cell growth factor, which is dose dependent and most pronounced in the first 24 hr of culture. Incubation of lymphocytes with thymosin alone failed to induce any production of T-cell growth factor. The biological activity of thymosin fraction 5 cannot be attributed to the activity of thymosin alpha 1, one of the well-characterized peptide components of fraction 5. These data provide the basis for (i) a potential mechanism for the in vivo immunorestorative effects of thymosin in primary and secondary immunodeficiencies and (ii) identification of an additional, but as yet undefined, immunoregulatory component of thymosin fraction 5. PMID:6609371

  5. Human central nervous system response to peripheral action of low-intensity millimeter waves

    SciTech Connect

    Lebedeva, N.N.

    1994-07-01

    We employ a modification of a psychophysical method developed by Kholodov to study electromagnetic field induced skin sensations. The main part of the setup is a program controller, which provides for the timed delivery of EMF signals and also false presentations. The EMF signals are fed in random order with a uniform distribution. The response-strength index and the false-alarm level were used to evaluate MF sensitivity. In addition, the subjects determined the presence or absence of a field according to four criteria. Forty healthy subjects ages 17 to 35 were tested on their right and left hands. Sensory data was analyzed via computer. EEG responses to peripheral action were studied in depth.

  6. PSP activates monocytes in resting human peripheral blood mononuclear cells: immunomodulatory implications for cancer treatment.

    PubMed

    Sekhon, Bhagwant Kaur; Sze, Daniel Man-Yuen; Chan, Wing Keung; Fan, Kei; Li, George Qian; Moore, Douglas Edwin; Roubin, Rebecca Heidi

    2013-06-15

    Polysaccharopeptide (PSP), from Coriolus versicolor, has been used as an adjuvant to chemotherapy, and has demonstrated anti-tumor and immunomodulating effects. However its mechanism remains unknown. To elucidate how PSP affects immune populations, we compared PSP treatments both with and without prior incubation in phytohaemagglutinin (PHA) - a process commonly used in immune population experimentation. We first standardised a capillary electrophoresis fingerprinting technique for PSP identification and characterisation. We then established the proliferative capability of PSP on various immune populations in peripheral blood mononuclear cells, using flow cytometry, without prior PHA treatment. It was found that PSP significantly increased the number of monocytes (CD14(+)/CD16(-)) compared to controls without PHA. This increase in monocytes was confirmed using another antibody panel of CD14 and MHCII. In contrast, proliferations of T-cells, NK, and B-cells were not significantly changed by PSP. Thus, stimulating monocyte/macrophage function with PSP could be an effective therapeutic intervention in targeting tumors.

  7. Allosteric modulation by benzodiazepines of GABA-gated chloride channels of an identified insect motor neurone.

    PubMed

    Buckingham, Steven D; Higashino, Yoshiaki; Sattelle, David B

    2009-11-01

    The actions of benzodiazepines were studied on the responses to GABA of the fast coxal depressor (D(f)) motor neurone of the cockroach, Periplaneta americana. Ro5-4864, diazepam and clonazepam were investigated. Responses to GABA receptors were enhanced by both Ro5-4864 and diazepam, whereas clonazepam, a potent-positive allosteric modulator of human GABA(A) receptors, was ineffective on the native insect GABA receptors of the D(f) motor neurone. Thus, clear pharmacological differences exist between insect and mammalian native GABA-gated chloride channels with respect to the actions of benzodiazepines. The results enhance our understanding of invertebrate GABA-gated chloride channels which have recently proved important in (a) comparative studies aimed at identifying human allosteric drug-binding sites and (b) understanding the actions of compounds used to control ectoparasites and insect crop pests.

  8. Impairment of peripheral Vdelta2 T cells in human cystic echinococcosis.

    PubMed

    Wang, Hao; Li, Ming; Zhang, Xiaoxia; He, Fang; Zhang, Shengbin; Zhao, Jiaqing

    2017-03-01

    Cystic echinococcosis (CE) induced by metacestodes (larval stages) of Echinococcus granulosus (E.granulosus) represents a severe endemic disease worldwide. Gamma delta (γδ) T cells, one of innate immune cells, play pivotal role in pathogenic infections. However, whether γδ T cells are involved in CE remains unclear. This study firstly investigated the role of peripheral γδ T cells in CE. The results showed that the percentage of peripheral γδ T cells from CE patients was decreased, compared with healthy controls (HC) (p < 0.01). This decrease was primarily due to a reduction in Vδ2 subset. Furthermore, Vδ2 T cells in CE expressed lower Natural Killer Group 2D (NKG2D) (p < 0.01). The abundance of Vδ2 T cells correlated negatively with cyst burden. To further identify the function of decreased Vδ2 T cells in CE, proliferation rate, cytokine secretion and cytotoxin were detected subsequently in vitro. As a result, the proliferation rate of Vδ2 cells in CE patients was lower than that in HC (p < 0.01). Meanwhile, Vδ2 T cells from CE patients released significantly decreased interferon (IFN)-γ, compared with HC (p < 0.05). Moreover, the levels of perforin and granzyme B of Vδ2 T cells from the patients were decreased significantly (p < 0.05), suggesting impaired cytotoxin generation of Vδ2 cells in CE. Collectively, our findings indicated that circulating Vδ2 T cells in CE was impaired, and these aberrations may contribute to disease pathogenesis.

  9. Changes in transcriptional output of human peripheral blood mononuclear cells following resistance exercise.

    PubMed

    Carlson, Lara A; Tighe, S W; Kenefick, R W; Dragon, J; Westcott, N W; Leclair, R J

    2011-12-01

    Various types of exercise alter the population of circulating peripheral blood mononuclear cells (PBMCs) and change their transcriptional output. This work examines changes in PBMC populations and transcription in response to resistance exercise training (RET), and identify key transcriptional changes in PBMCs that may play a role in altering peripheral tissues in response to RET. Ten resistance-trained men (20-24 years), performed an acute bout of RET for ~30 min following a 12 h fast. Venous blood was sampled at rest, immediately following exercise, and at 2 h post-exercise and analyzed for total and differential leukocytes and global gene expression using Affymetrix Genechips. Results showed elevated leukocytes, monocytes, lymphocytes, and lactate values immediately post-exercise (P < 0.05) over baseline. At 2 h post-exercise, leukocytes, and granulocytes remained elevated (P < 0.05), whereas lymphocytes were lower than (P < 0.05) baseline values. Initial microarray results showed the greatest transcriptional changes in pathways related to immune response, inflammation, and cellular communication. The change in PBMC population (2 h time point) correlated with a dramatic decrease in the expression of CD160, and XCL1, markers of lymphocyte populations. At the 2 h recovery time point upregulation of matrix metalloproteinase 9, orosomucoid 1, dishevelled-associated activator of morphogenesis 2, and arginase 1 suggest an induction in muscle damage and repair during this time frame. These results demonstrate that an acute bout of RET disrupts cellular homeostasis, induces a transient redistribution of certain leukocytes, and results in transcriptional changes in PBMCs translating into systemic changes in response to RET.

  10. Extended Interferon-Alpha Therapy Accelerates Telomere Length Loss in Human Peripheral Blood T Lymphocytes

    PubMed Central

    O'Bryan, Joel M.; Potts, James A.; Bonkovsky, Herbert L.; Mathew, Anuja; Rothman, Alan L.

    2011-01-01

    Background Type I interferons have pleiotropic effects on host cells, including inhibiting telomerase in lymphocytes and antiviral activity. We tested the hypothesis that long-term interferon treatment would result in significant reduction in average telomere length in peripheral blood T lymphocytes. Methods/Principal Findings Using a flow cytometry-based telomere length assay on peripheral blood mononuclear cell samples from the Hepatitis-C Antiviral Long-term Treatment against Cirrhosis (HALT-C) study, we measured T cell telomere lengths at screening and at months 21 and 45 in 29 Hepatitis-C virus infected subjects. These subjects had failed to achieve a sustained virologic response following 24 weeks of pegylated-interferon-alpha plus ribavirin treatment and were subsequently randomized to either a no additional therapy group or a maintenance dose pegylated-IFNα group for an additional 3.5 years. Significant telomere loss in naïve T cells occurred in the first 21 months in the interferon-alpha group. Telomere losses were similar in both groups during the final two years. Expansion of CD8+CD45RA+CD57+ memory T cells and an inverse correlation of alanine aminotransferase levels with naïve CD8+ T cell telomere loss were observed in the control group but not in the interferon-alpha group. Telomere length at screening inversely correlated with Hepatitis-C viral load and body mass index. Conclusions/Significance Sustained interferon-alpha treatment increased telomere loss in naïve T cells, and inhibited the accumulation of T cell memory expansions. The durability of this effect and consequences for immune senescence need to be defined. PMID:21829595

  11. Effect of 900 MHz Electromagnetic Radiation on the Induction of ROS in Human Peripheral Blood Mononuclear Cells

    PubMed Central

    Kazemi, E.; Mortazavi, S. M. J.; Ali-Ghanbari, A.; Sharifzadeh, S.; Ranjbaran, R.; Mostafavi-pour, Z.; Zal, F.; Haghani, M.

    2015-01-01

    Background Despite numerous studies over a decade, it still remains controversial about the biological effects of RF EMF emitted by mobile phone telephony. Objective Here we investigated the effect of 900 MHz GSM on the induction of oxidative stress and the level of intracellular reactive oxygen species (ROS) in human mononuclear cells, monocytes and lymphocytes as defence system cells. Method 6 ml Peripheral Blood samples were obtained from 13 healthy volunteers (21-30 year-old). Each sample was devided into 2 groups: one was exposed RF radiation emitted from a mobile phone simulator for 2 hour and the other used as control group which was not exposed to any fields. After that, mononuclear cells were isolated from peripheral blood by density gradient centrifugation in Ficoll-Paque. The intracellular ROS content in monocytes and lymphocytes was measured by the CM-H2DCFDA fluorescence probe using flowcytometry technique. Results Our results showed significant increase in  ROS production after exposure in population rich in monocytes. This effect was not significant in population rich in lymphocytes in comparison with non exposed cells. Conclusion The results obtained in this study clearly showed the oxidative stress induction capability of RF electromagnetic field in the portion of PBMCs mostly in monocytes, like the case of exposure to micro organisms, although the advantages or disadvantages of this effect should be evaluated. PMID:26396966

  12. The “Intermediate” CD14++CD16+ monocyte subset increases in severe peripheral artery disease in humans

    PubMed Central

    Wildgruber, Moritz; Aschenbrenner, Teresa; Wendorff, Heiko; Czubba, Maria; Glinzer, Almut; Haller, Bernhard; Schiemann, Matthias; Zimmermann, Alexander; Berger, Hermann; Eckstein, Hans-Henning; Meier, Reinhard; Wohlgemuth, Walter A.; Libby, Peter; Zernecke, Alma

    2016-01-01

    Monocytes are key players in atherosclerotic. Human monocytes display a considerable heterogeneity and at least three subsets can be distinguished. While the role of monocyte subset heterogeneity has already been well investigated in coronary artery disease (CAD), the knowledge about monocytes and their heterogeneity in peripheral artery occlusive disease (PAOD) still is limited. Therefore, we aimed to investigate monocyte subset heterogeneity in patients with PAOD. Peripheral blood was obtained from 143 patients suffering from PAOD (Rutherford stage I to VI) and three monocyte subsets were identified by flow cytometry: CD14++CD16− classical monocytes, CD14+CD16++ non-classical monocytes and CD14++CD16+ intermediate monocytes. Additionally the expression of distinct surface markers (CD106, CD162 and myeloperoxidase MPO) was analyzed. Proportions of CD14++CD16+ intermediate monocyte levels were significantly increased in advanced stages of PAOD, while classical and non-classical monocytes displayed no such trend. Moreover, CD162 and MPO expression increased significantly in intermediate monocyte subsets in advanced disease stages. Likewise, increased CD162 and MPO expression was noted in CD14++CD16− classical monocytes. These data suggest substantial dynamics in monocyte subset distributions and phenotypes in different stages of PAOD, which can either serve as biomarkers or as potential therapeutic targets to decrease the inflammatory burden in advanced stages of atherosclerosis. PMID:27991581

  13. Expression of the Kynurenine Pathway in Human Peripheral Blood Mononuclear Cells: Implications for Inflammatory and Neurodegenerative Disease.

    PubMed

    Jones, Simon P; Franco, Nunzio F; Varney, Bianca; Sundaram, Gayathri; Brown, David A; de Bie, Josien; Lim, Chai K; Guillemin, Gilles J; Brew, Bruce J

    2015-01-01

    The kynurenine pathway is a fundamental mechanism of immunosuppression and peripheral tolerance. It is increasingly recognized as playing a major role in the pathogenesis of a wide variety of inflammatory, neurodegenerative and malignant disorders. However, the temporal dynamics of kynurenine pathway activation and metabolite production in human immune cells is currently unknown. Here we report the novel use of flow cytometry, combined with ultra high-performance liquid chromatography and gas chromatography-mass spectrometry, to sensitively quantify the intracellular expression of three key kynurenine pathway enzymes and the main kynurenine pathway metabolites in a time-course study. This is the first study to show that up-regulation of indoleamine 2,3-dioxygenase (IDO-1), kynurenine 3-monoxygenase (KMO) and quinolinate phosphoribosyltransferase (QPRT) is lacking in lymphocytes treated with interferon gamma. In contrast, peripheral monocytes showed a significant elevation of kynurenine pathway enzymes and metabolites when treated with interferon gamma. Expression of IDO-1, KMO and QPRT correlated significantly with activation of the kynurenine pathway (kynurenine:tryptophan ratio), quinolinic acid concentration and production of the monocyte derived, pro-inflammatory immune response marker: neopterin. Our results also describe an original and sensitive methodological approach to quantify kynurenine pathway enzyme expression in cells. This has revealed further insights into the potential role of these enzymes in disease processes.

  14. Atomic resolution view into the structure–function relationships of the human myelin peripheral membrane protein P2

    PubMed Central

    Ruskamo, Salla; Yadav, Ravi P.; Sharma, Satyan; Lehtimäki, Mari; Laulumaa, Saara; Aggarwal, Shweta; Simons, Mikael; Bürck, Jochen; Ulrich, Anne S.; Juffer, André H.; Kursula, Inari; Kursula, Petri

    2014-01-01

    P2 is a fatty acid-binding protein expressed in vertebrate peripheral nerve myelin, where it may function in bilayer stacking and lipid transport. P2 binds to phospholipid membranes through its positively charged surface and a hydrophobic tip, and accommodates fatty acids inside its barrel structure. The structure of human P2 refined at the ultrahigh resolution of 0.93 Å allows detailed structural analyses, including the full organization of an internal hydrogen-bonding network. The orientation of the bound fatty-acid carboxyl group is linked to the protonation states of two coordinating arginine residues. An anion-binding site in the portal region is suggested to be relevant for membrane interactions and conformational changes. When bound to membrane multilayers, P2 has a preferred orientation and is stabilized, and the repeat distance indicates a single layer of P2 between membranes. Simulations show the formation of a double bilayer in the presence of P2, and in cultured cells wild-type P2 induces membrane-domain formation. Here, the most accurate structural and functional view to date on P2, a major component of peripheral nerve myelin, is presented, showing how it can interact with two membranes simultaneously while going through conformational changes at its portal region enabling ligand transfer. PMID:24419389

  15. Human neurocysticercosis: in vivo expansion of peripheral regulatory T cells and their recruitment in the central nervous system.

    PubMed

    Adalid-Peralta, Laura; Fleury, Agnes; García-Ibarra, Teresa M; Hernández, Marisela; Parkhouse, Michael; Crispín, José Carlos; Voltaire-Proaño, Jefferson; Cárdenas, Graciela; Fragoso, Gladis; Sciutto, Edda

    2012-02-01

    Human neurocysticercosis (NC) is caused by Taenia solium larvae lodged in the central nervous system. Most cases occur with no, or mild, neurological symptoms. However, in some patients, neuroinflammation is exacerbated, leading to severe forms of the disease. Considering the critical role of regulatory T cells (Tregs) in balancing inflammation in chronic diseases, their participation in restraining the inflammatory response in NC was explored in the present study. The frequency of Tregs and their relationship with the level of the proliferative response, the level of activated lymphocytes, and the cytokines expressed were determined in severe NC patients compared with those from healthy donors. Significantly increased peripheral Tregs (CD4(+)CD25(high) and CD4(+)CD25(high)FoxP3(+), CD4(+)CD25(high)CTLA4(+), and CD4(+)CD25(high) IL10(+)) and a significant decrease in activated (CD38(+) and CD69(+)) T cells were observed in 19 NC patients versus 10 healthy subjects. Significantly increased Tregs in NC are accompanied by a depressed specific, and non-specific, lymphocyte proliferative response, and they negatively correlate with activated CD4(+)CD69(+) lymphocytes. Treg frequencies were also determined in cerebral spinal fluid for 8 of the 19 NC patients. A positive significant correlation between peripheral and local Tregs was observed. Here, we report for the first time data that support the possible contribution of local and systemic Tregs in limiting neuroinflammation in NC.

  16. Comparison of complement-dependent cytotoxicity and indirect immunofluorescence for enumeration of T-cell subpopulations in human peripheral blood.

    PubMed

    Gratama, J W; Schuurman, R K; Van Leeuwen, A; Jansen, J; Oljans, P; Tanke, H J; Van Rood, J J

    1983-11-11

    The monitoring of T-lymphocyte subsets of recipients of organ grafts enables studies on immune reconstitution (after bone-marrow transplantation) and may predict graft rejection (after kidney transplantation). Quantitation of human peripheral T-lymphocyte subsets from healthy volunteers and from recipients of a bone-marrow graft by a complement dependent cytotoxicity (CDC) assay, based on the use of propidium iodide, and by an indirect immunofluorescence (IIF) technique has been compared using the monoclonal antibodies OKT3, OKT4 and OKT8. Except for OKT3 in healthy individuals--for which no significant difference was found between CDC and IIF--CDC detected significantly more cells of each subset than IIF. Furthermore, the CDC results indicated the presence of low numbers of OKT4+8+ cells in the peripheral blood of healthy individuals and--with higher numbers--following marrow transplantation. Results of depletion experiments, obtained by fluorescence activated cell sorting (FACS) for either OKT4 or OKT8, supported this conclusion. OKT4/OKT8 ratios were calculated from enumerations by the CDC assay and by the IIF assay and found to be linearly related, both in healthy persons and in marrow-graft recipients. Thus, the CDC assay is a reliable method for monitoring T-cell subsets, allowing detection of lymphocytes carrying low densities of membrane determinants.

  17. Accumulation of defective viral genomes in peripheral blood mononuclear cells of human immunodeficiency virus type 1-infected individuals.

    PubMed Central

    Sanchez, G; Xu, X; Chermann, J C; Hirsch, I

    1997-01-01

    Human immunodeficiency virus type 1 (HIV-1) genomes present in peripheral blood mononuclear cells (PBMCs) of infected persons or in lymphocytes infected in vitro were studied by long-distance PCR (LD-PCR) using primers localized in the HIV-1 long terminal repeats. The full-length 9-kb DNA was the only LD-PCR product obtained in peripheral and cord blood lymphocytes from seronegative donors infected in vitro. However, a high proportion (27% to 66%) of distinct populations of extensively deleted HIV-1 genomes of variable size was detected in PBMCs of 15 of 16 HIV-1-infected persons. Physical mapping of defective genomes showed that the frequency of deletions is proportional to their proximity to the central part of HIV-1 genome, which is consistent with a deletion mechanism involving a single polymerase jump during reverse transcription. Sequencing of deletion junctions revealed the presence of short direct repeats of three or four nucleotides. The number of defective HIV-1 genomes decreased after in vitro activation of PBMCs. Persistence of full-length and deleted genomes in in vitro activated PBMCs correlated with isolation of an infectious virus. Our results represent the first quantitative assessment of intragenomic rearrangements in HIV-1 genomes in PBMCs of infected persons and demonstrate that, in contrast to in vitro infection, defective genomes accumulate in PBMCs of infected persons. PMID:9032358

  18. Effect of 900 MHz Electromagnetic Radiation on the Induction of ROS in Human Peripheral Blood Mononuclear Cells.

    PubMed

    Kazemi, E; Mortazavi, S M J; Ali-Ghanbari, A; Sharifzadeh, S; Ranjbaran, R; Mostafavi-Pour, Z; Zal, F; Haghani, M

    2015-09-01

    Despite numerous studies over a decade, it still remains controversial about the biological effects of RF EMF emitted by mobile phone telephony. Here we investigated the effect of 900 MHz GSM on the induction of oxidative stress and the level of intracellular reactive oxygen species (ROS) in human mononuclear cells, monocytes and lymphocytes as defence system cells. 6 ml Peripheral Blood samples were obtained from 13 healthy volunteers (21-30 year-old). Each sample was devided into 2 groups: one was exposed RF radiation emitted from a mobile phone simulator for 2 hour and the other used as control group which was not exposed to any fields. After that, mononuclear cells were isolated from peripheral blood by density gradient centrifugation in Ficoll-Paque. The intracellular ROS content in monocytes and lymphocytes was measured by the CM-H2DCFDA fluorescence probe using flowcytometry technique. Our results showed significant increase in  ROS production after exposure in population rich in monocytes. This effect was not significant in population rich in lymphocytes in comparison with non exposed cells. The results obtained in this study clearly showed the oxidative stress induction capability of RF electromagnetic field in the portion of PBMCs mostly in monocytes, like the case of exposure to micro organisms, although the advantages or disadvantages of this effect should be evaluated.

  19. Expression of the Kynurenine Pathway in Human Peripheral Blood Mononuclear Cells: Implications for Inflammatory and Neurodegenerative Disease

    PubMed Central

    Jones, Simon P.; Franco, Nunzio F.; Varney, Bianca; Sundaram, Gayathri; Brown, David A.; de Bie, Josien; Lim, Chai K.; Guillemin, Gilles J.; Brew, Bruce J.

    2015-01-01

    The kynurenine pathway is a fundamental mechanism of immunosuppression and peripheral tolerance. It is increasingly recognized as playing a major role in the pathogenesis of a wide variety of inflammatory, neurodegenerative and malignant disorders. However, the temporal dynamics of kynurenine pathway activation and metabolite production in human immune cells is currently unknown. Here we report the novel use of flow cytometry, combined with ultra high-performance liquid chromatography and gas chromatography-mass spectrometry, to sensitively quantify the intracellular expression of three key kynurenine pathway enzymes and the main kynurenine pathway metabolites in a time-course study. This is the first study to show that up-regulation of indoleamine 2,3-dioxygenase (IDO-1), kynurenine 3-monoxygenase (KMO) and quinolinate phosphoribosyltransferase (QPRT) is lacking in lymphocytes treated with interferon gamma. In contrast, peripheral monocytes showed a significant elevation of kynurenine pathway enzymes and metabolites when treated with interferon gamma. Expression of IDO-1, KMO and QPRT correlated significantly with activation of the kynurenine pathway (kynurenine:tryptophan ratio), quinolinic acid concentration and production of the monocyte derived, pro-inflammatory immune response marker: neopterin. Our results also describe an original and sensitive methodological approach to quantify kynurenine pathway enzyme expression in cells. This has revealed further insights into the potential role of these enzymes in disease processes. PMID:26114426

  20. Anticonvulsive Activity in Audiogenic DBA/2 Mice of 1,4-Benzodiazepines and 1,5-Benzodiazepines with Different Activities at Cerebellar Granule Cell GABAA Receptors.

    PubMed

    Gatta, Elena; Cupello, Aroldo; Di Braccio, Mario; Grossi, Giancarlo; Robello, Mauro; Scicchitano, Francesca; Russo, Emilio; De Sarro, Giovambattista

    2016-12-01

    Herein, we tested in a model of generalized reflex epilepsy in mice different 1,4-benzodiazepines and 1,5-benzodiazepines with agonistic activity at the GABAA receptor population contributing to the peak component of the chloride current elicited by GABA in cerebellar granule cells (CGCs) in culture. The substances have all higher lipophilia than clobazam, an antiepileptic drug well known and used in human therapy. This ensures that they all can pass relatively easily the blood-brain barrier (BBB). The benzodiazepines were administered intraperitoneally (i.p.) and tested for their activity against sound-induced tonic and clonic seizures in a genetic model of experimental epilepsy, the DBA/2 mouse. Our data demonstrates an interesting inverse correlation between the ED50s and the efficacy (E %) of the drugs in increasing the peak chloride current elicited by GABA in cerebellar granule cells in culture. There is indication of the existence of a threshold of E % above which the increase of ED50 with increasing E % becomes linear. This is statistically significant for the clonic phase, whereas it is at the limit of significance for the tonic one. A possible interpretation of these results is that in this epilepsy model, projections from the cerebellum exert a convulsion prevention activity.

  1. Equilibrium constants and assay of benzodiazepines in acetic acid.

    PubMed

    Barbosa, J; Barrón, D

    1989-04-01

    The over-all dissociation constants, in anhydrous acetic acid, of a series of benzodiazepines have been determined. A method is described for evaluating the formation constants of the perchlorate salts. From these values simple potentiometric and visual titration methods for the assay of benzodiazepines in acetic acid are described.

  2. Benzodiazepines and vigilance performance: a review.

    PubMed

    Koelega, H S

    1989-01-01

    The literature on the effects of anxiolytic and hypnotic drugs on performance in tasks requiring sustained attention is confusing. This review is an attempt to evaluate the usefulness of vigilance tasks in the assessment of adverse effects of benzodiazepines. The long, monotonous, character of these tasks may be more relevant to many tasks performed in everyday life than the short, and often stimulating, tasks commonly employed in test batteries. From 37 available studies, 26 were examined in detail. In young, normal volunteers, vigilance tasks were found to be sensitive, often dose dependently, to the impairing effects of drugs, even in low doses (2.5 mg diazepam). With these subjects the tasks may be successfully used to compare different benzodiazepines with respect to residual activity. Both accuracy and speed of performance appear to be affected. However, in people actually using the drugs ("patients"), adverse effects on performance are usually not found. There is no evidence that benzodiazepines aggravate the vigilance decrement occurring under normal conditions. They do affect overall level of perceptual sensitivity, but show less effects on response criterion. The drugs do not seem to interact with anxiety or sleep quality in their effect on performance, but there are few studies with patients, and the assessment of anxiety is not without problems. It is unlikely that impairments in vigilance are simply a byproduct of global, sedative effects, but there is uncertainty regarding measures of general sedation. Developing tolerance with repeated doses has been noted only occasionally, but the opposite of tolerance, aggravated impairment, has also been reported.

  3. A novel thyroid stimulating hormone beta-subunit isoform in human pituitary, peripheral blood leukocytes, and thyroid.

    PubMed

    Schaefer, Jeremy S; Klein, John R

    2009-07-01

    Thyroid stimulating hormone (TSH) is produced by the anterior pituitary and is used to regulate thyroid hormone output, which in turn controls metabolic activity. Currently, the pituitary is believed to be the only source of TSH used by the thyroid. Recent studies in mice from our laboratory have identified a TSHbeta isoform that is expressed in the pituitary, in peripheral blood leukocytes (PBL), and in the thyroid. To determine whether a human TSHbeta splice variant exists that is analogous to the mouse TSHbeta splice variant, and whether the pattern of expression of the splice variant is similar to that observed in mice, PCR amplification of RNAs from pituitary, thyroid, PBL, and bone marrow was done by reverse-transcriptase PCR and quantitative realtime PCR. Human pituitary expressed a TSHbeta isoform that is analogous to the mouse TSHbeta splice variant, consisting of a 27 nucleotide portion of intron 2 and all of exon 3, coding for 71.2% of the native human TSHbeta polypeptide. Of particular interest, the TSHbeta splice variant was expressed at significantly higher levels than the native form or TSHbeta in PBL and the thyroid. The TSHalpha gene also was expressed in the pituitary, thyroid, and PBL, but not the BM, suggesting that the TSHbeta polypeptide in the thyroid and PBL may exist as a dimer with TSHalpha. These findings identify an unknown splice variant of human TSHbeta. They also have implications for immune-endocrine interactions in the thyroid and for understanding autoimmune thyroid disease from a new perspective.

  4. Cytotoxic effect of wine polyphenolic extracts and resveratrol against human carcinoma cells and normal peripheral blood mononuclear cells.

    PubMed

    Matić, Ivana; Zizak, Zeljko; Simonović, Mladen; Simonović, Branislav; Godevac, Dejan; Savikin, Katarina; Juranić, Zorica

    2010-08-01

    Red and white wine polyphenols have been reported to provide substantial health benefits. In this study, the cytotoxic activity of red and white wine polyphenolic extracts and of resveratrol was evaluated against different cancer cell lines--human cervix adenocarcinoma HeLa, human breast adenocarcinoma MDA-MB-361, and human breast carcinoma MDA-MB-453--and normal human peripheral blood mononuclear cells (PBMCs). Qualitative and quantitative compositions of wine polyphenolic extracts obtained by fractional vacuum distillation of corresponding wines were determined using spectrophotometric methods and high-performance liquid chromatography with diode array detection and liquid chromatography with electrospray ionization-time of flight mass spectrometry analysis. It was demonstrated that wine polyphenolic extracts and resveratrol exerted higher cytotoxic activity against HeLa and MDA-MB-453 cells in comparison to MDA-MB-361 cells and unstimulated and stimulated PBMCs. Furthermore, white wine polyphenolic extract exhibited a significantly higher antiproliferative action on cancer cell lines than red wine extract. The presence of condensed or fragmented nuclei in HeLa cells, pretreated with extract of white wine and stained with a mixture of acridine orange and ethidium bromide, pointed to the morphological signs of apoptosis. In addition, HeLa cells in late stages of apoptosis or secondary necrosis were also observed. Results from our study suggest that polyphenolic extracts from red and white wine may have anticarcinogenic potential.

  5. Comparison of two radiolabeled quinuclidinyl benzilate ligands for the characterization of the human peripheral lung muscarinic receptor

    SciTech Connect

    Joad, J.P.; Casale, T.B.

    1987-09-28

    Quinuclidinyl benzilate, a muscarinic antagonist, has previously been used in its tritiated form ((/sup 3/H)-QNB) to study the lung muscarinic receptor. The authors investigated whether a newer iodinated form of QNB ((/sup 125/I)-QNB) of higher specific activity would be an appropriate ligand to study the human peripheral lung muscarinic receptor. Both the tritiated and iodinated ligands bound specifically to human lung at 23/sup 0/C. At 37/sup 0/C the specific binding of (/sup 3/H)-QNB increased slightly, but no specific binding of (/sup 125/I)-QNB was found. The data from multiple equilibrium binding experiments covering a wide range of radiolabeled QNB concentrations were combined and analyzed using the computer modeling program, LIGAND. The tritiated QNB identified a single affinity human lung binding site with a Kd of 46 +/- 9 pM. The iodinated QNB identified a single higher affinity human lung binding site of much smaller quantity. Competition studies comparing the binding of unlabeled QNB relative to labeled QNB indicated that unlabeled QNB had the same Kd as that measured for (/sup 3/H)-QNB, but a 5 log greater Kd than that measured for (/sup 125/I)-QNB. Other muscarinic receptor agonists and antagonists competed with (/sup 3/H)QNB, but not (/sup 125/I)-QNB for binding to muscarinic receptors with the expected magnitude and rank order of potency. 17 references, 2 figures, 2 tables.

  6. KANK1 inhibits cell growth by inducing apoptosis though regulating CXXC5 in human malignant peripheral nerve sheath tumors

    PubMed Central

    Cui, Zhibin; Shen, Yingjia; Chen, Kenny H.; Mittal, Suresh K.; Yang, Jer-Yen; Zhang, GuangJun

    2017-01-01

    Malignant peripheral nerve sheath tumors (MPNSTs) are a type of rare sarcomas with a poor prognosis due to its highly invasive nature and limited treatment options. Currently there is no targeted-cancer therapy for this type of malignancy. Thus, it is important to identify more cancer driver genes that may serve as targets of cancer therapy. Through comparative oncogenomics, we have found that KANK1 was a candidate tumor suppressor gene (TSG) for human MPNSTs. Although KANK1 is known as a cytoskeleton regulator, its tumorigenic function in MPNSTs remains largely unknown. In this study, we report that restoration of KANK1 in human MPNST cells inhibits cell growth both in human cell culture and xenograft mice by increasing apoptosis. Consistently, knockdown of KANK1 in neurofibroma cells promoted cell growth. Using RNA-seq analysis, we identified CXXC5 and other apoptosis-related genes, and demonstrated that CXXC5 is regulated by KANK1. Knockdown of CXXC5 was found to diminish KANK1-induced apoptosis in MPNST cells. Thus, KANK1 inhibits MPNST cell growth though CXXC5 mediated apoptosis. Our results suggest that KANK1 may function as a tumor suppressor in human MPNSTs, and thus it may be useful for targeted therapy. PMID:28067315

  7. Cytogenetic comparison of the responses of mouse and human peripheral blood lymphocytes to /sup 60/Co gamma radiation

    SciTech Connect

    Kligerman, A.D.; Halperin, E.C.; Erexson, G.L.; Honore, G.; Westbrook-Collins, B.; Allen, J.W.

    1988-08-01

    Experiments were conducted to compare the chromosome damaging effects of /sup 60/Co gamma radiation on mouse and human peripheral blood lymphocytes (PBLs). Either whole blood or isolated and pelleted mononuclear leucocytes (MNLs) were irradiated with a /sup 60/Co unit to yield exposures of 1, 2, 3, or 4 Gy. In addition, mice were whole-body irradiated in vivo with the same doses so that an in vitro-in vivo comparison could be made. The results indicate that mouse PBLs irradiated in whole blood, whether in vivo or in vitro, respond similarly to /sup 60/Co gamma rays as measured by dicentric chromosome formation. In addition, mouse and human PBLs showed a similar radiosensitivity, but because the mouse PBL data were best fitted to an exponential function and the human PBL data to a quadratic function, direct comparisons were difficult to make. Pelleted MNLs from mice were much less sensitive to the clastogenic effects of gamma radiation than whole blood. This is believed to be due to hypoxic conditions that developed during irradiation and transport. Human PBLs did not show a marked difference whether irradiated in whole blood or as pelleted MNLs in tissue culture medium.

  8. Cytogenetic observations in human peripheral blood leukocytes following in vitro exposure to THz radiation: a pilot study.

    PubMed

    Zeni, O; Gallerano, G P; Perrotta, A; Romanò, M; Sannino, A; Sarti, M; D'Arienzo, M; Doria, A; Giovenale, E; Lai, A; Messina, G; Scarfì, M R

    2007-04-01

    Emerging technologies are considering the possible use of Terahertz radiation in different fields ranging from telecommunications to biology and biomedicine. The study of the potential effects of Terahertz radiation on biological systems is therefore an important issue in order to safely develop a variety of applications. This paper describes a pilot study devoted to determine if Terahertz radiation could induce genotoxic effects in human peripheral blood leukocytes. For this purpose, human whole blood samples from healthy donors were exposed for 20 min to Terahertz radiation. Since, to our knowledge, this is the first study devoted to the evaluation of possible genotoxic effects of such radiation, different electromagnetic conditions were considered. In particular, the frequencies of 120 and 130 GHz were chosen: the first one was tested at a specific absorption rate (SAR) of 0.4 mW g-1, while the second one was tested at SAR levels of 0.24, 1.4, and 2 mW g-1. Chromosomal damage was evaluated by means of the cytokinesis block micronucleus technique, which also gives information on cell cycle kinetics. Moreover, human whole blood samples exposed to 130 GHz at SAR levels of 1.4 and 2 mW g-1 were also tested for primary DNA damage by applying the alkaline comet assay immediately after exposure. The results obtained indicate that THz exposure, in the explored electromagnetic conditions, is not able to induce either genotoxicity or alteration of cell cycle kinetics in human blood cells from healthy subjects.

  9. Pharmacodynamic and antiretroviral activities of combination nanoformulated antiretrovirals in HIV-1-infected human peripheral blood lymphocyte-reconstituted mice.

    PubMed

    Roy, Upal; McMillan, JoEllyn; Alnouti, Yazen; Gautum, Nagsen; Smith, Nathan; Balkundi, Shantanu; Dash, Prasanta; Gorantla, Santhi; Martinez-Skinner, Andrea; Meza, Jane; Kanmogne, Georgette; Swindells, Susan; Cohen, Samuel M; Mosley, R Lee; Poluektova, Larisa; Gendelman, Howard E

    2012-11-15

    Lack of adherence, inaccessibility to viral reservoirs, long-term drug toxicities, and treatment failures are limitations of current antiretroviral therapy (ART). These limitations lead to increased viral loads, medicine resistance, immunocompromise, and comorbid conditions. To this end, we developed long-acting nanoformulated ART (nanoART) through modifications of existing atazanavir, ritonavir, and efavirenz suspensions in order to establish cell and tissue drug depots to achieve sustained antiretroviral responses. NanoART's abilities to affect immune and antiviral responses, before or following human immunodeficiency virus type 1 infection were tested in nonobese severe combined immune-deficient mice reconstituted with human peripheral blood lymphocytes. Weekly subcutaneous injections of drug nanoformulations at doses from 80 mg/kg to 250 mg/kg, 1 day before and/or 1 and 7 days after viral exposure, elicited drug levels that paralleled the human median effective concentration, and with limited toxicities. NanoART treatment attenuated viral replication and preserved CD4(+) Tcell numbers beyond that seen with orally administered native drugs. These investigations bring us one step closer toward using long-acting antiretrovirals in humans.

  10. Design, Synthesis and Biological Evaluation of Sulfamide and Triazole Benzodiazepines as Novel p53-MDM2 Inhibitors

    PubMed Central

    Yu, Zhiliang; Zhuang, Chunlin; Wu, Yuelin; Guo, Zizhao; Li, Jin; Dong, Guoqiang; Yao, Jianzhong; Sheng, Chunquan; Miao, Zhenyuan; Zhang, Wannian

    2014-01-01

    A series of sulfamide and triazole benzodiazepines were obtained with the principle of bioisosterism. The p53-murine double minute 2 (MDM2) inhibitory activity and in vitro antitumor activity were evaluated. Most of the novel benzodiazepines exhibited moderate protein binding inhibitory activity. Particularly, triazole benzodiazepines showed good inhibitory activity and antitumor potency. Compound 16 had promising antitumor activity against the U-2 OS human osteosarcoma cell line with an IC50 value of 4.17 μM, which was much better than that of nutlin-3. The molecular docking model also successfully predicted that this class of compounds mimicked the three critical residues of p53 binding to MDM2. PMID:25198897

  11. Hyperammoneic encephalopathy, valproic acid, and benzodiazepine withdrawal: a case series.

    PubMed

    Starer, Jacquelyn; Chang, Grace

    2010-03-01

    Benzodiazepine withdrawal is accompanied by a risk of seizures, delirium, and death. While a gradual outpatient taper off of benzodiazepines is the most commonly recommended method for discontinuation, acute inpatient detoxification and seizure prophylaxis may be necessary for some. Complications related to the use of valproic acid for seizure prophylaxis are presented. The study's objectives are to highlight an uncommon and possibly unrecognized complication of valproic acid when used for seizure prophylaxis during acute inpatient detoxification from benzodiazepines in the context of current practice. Case series. Three patients with hyperammoneic encephalopathy are described. Hyperammoneic encephalopathy can occur as a distinct entity separate from hepatotoxicity with the use of valproic acid and may be an unrecognized complication among patients receiving this drug during benzodiazepine detoxification. A previously unreported complication among the addiction patient population is reported. This underscores the need for a better evidence base regarding the prevention of seizures during acute benzodiazepine detoxification, particularly in terms of indications, safety, and efficacy.

  12. Using Tutte polynomials to analyze the structure of the benzodiazepines

    NASA Astrophysics Data System (ADS)

    Cadavid Muñoz, Juan José

    2014-05-01

    Graph theory in general and Tutte polynomials in particular, are implemented for analyzing the chemical structure of the benzodiazepines. Similarity analysis are used with the Tutte polynomials for finding other molecules that are similar to the benzodiazepines and therefore that might show similar psycho-active actions for medical purpose, in order to evade the drawbacks associated to the benzodiazepines based medicine. For each type of benzodiazepines, Tutte polynomials are computed and some numeric characteristics are obtained, such as the number of spanning trees and the number of spanning forests. Computations are done using the computer algebra Maple's GraphTheory package. The obtained analytical results are of great importance in pharmaceutical engineering. As a future research line, the usage of the chemistry computational program named Spartan, will be used to extent and compare it with the obtained results from the Tutte polynomials of benzodiazepines.

  13. Hooked on benzodiazepines: GABAA receptor subtypes and addiction

    PubMed Central

    Tan, Kelly R.; Rudolph, Uwe; Lüscher, Christian

    2011-01-01

    Benzodiazepines are widely used clinically to treat anxiety and insomnia. They also induce muscle relaxation, control epileptic seizures, and can provoke amnesia. Moreover, benzodiazepines are often abused after chronic clinical treatment but also for recreational purposes. Within weeks, tolerance to the pharmacological effects can develop, in addition to dependence and even addiction in vulnerable individuals. Here, we review recent observations from animal models regarding the cellular and molecular basis that may underlie the addictive properties of benzodiazepines. These data reveal how benzodiazepines, acting through specific GABAA receptor subtypes, activate midbrain dopamine neurons and how this may hijack the mesolimbic reward system. Such findings have important implications for the future design of benzodiazepines with reduced or even absent addiction liability. PMID:21353710

  14. Relation between clinical mature and immature lymphocyte cells in human peripheral blood and their spatial label free scattering patterns

    NASA Astrophysics Data System (ADS)

    Zhang, Lu; Zhao, Xin; Zhang, Zhenxi; Zhao, Hong; Chen, Wei; Yuan, Li

    2016-07-01

    A single living cell's light scattering pattern (LSP) in the horizontal plane, which has been denoted as the cell's "2D fingerprint," may provide a powerful label-free detection tool in clinical applications. We have recently studied the LSP in spatial scattering planes, denoted as the cell's "3D fingerprint," for mature and immature lymphocyte cells in human peripheral blood. The effects of membrane size, morphology, and the existence of the nucleus on the spatial LSP are discussed. In order to distinguish clinical label-free mature and immature lymphocytes, the special features of the spatial LSP are studied by statistical method in both the spatial and frequency domains. Spatial LSP provides rich information on the cell's morphology and contents, which can distinguish mature from immature lymphocyte cells and hence ultimately it may be a useful label-free technique for clinical leukemia diagnosis.

  15. Interferon-α curbs production of interleukin-22 by human peripheral blood mononuclear cells exposed to live Borrelia burgdorferi.

    PubMed

    Berner, Anika; Bachmann, Malte; Pfeilschifter, Josef; Kraiczy, Peter; Mühl, Heiko

    2015-10-01

    Cytokine networks initiated by means of innate immunity are regarded as a major determinant of host defence in response to acute infection by bacteria including Borrelia burgdorferi. Herein, we demonstrate that interferon (IFN)-α, either endogenously produced after exposure of cells to toll-like receptor-9-activating CpG oligonucleotides or provided as recombinant cytokine, weakens activation of the anti-bacterial interleukin (IL)-1/IL-22 axis in human peripheral blood mononuclear cells exposed to viable B. burgdorferi. As IFN-α has been related to pathological dissemination of the spirochaete, data suggest an immunoregulatory role of type I IFN in this context that is able to significantly modify cytokine profiles thereby possibly determining early course of B. burgdorferi infection.

  16. Estrogenic xenobiotics affect the intracellular activation signal in mitogen-induced human peripheral blood lymphocytes: immunotoxicological impact.

    PubMed

    Sakabe, K; Okuma, M; Kazuno, M; Yamaguchi, T; Yoshida, T; Furuya, H; Kayama, F; Suwa, Y; Fujii, W; Fresa, K L

    1998-01-01

    The present study was an attempt to elucidate the effect of estrogenic xenobiotics on the proliferation of mitogen-stimulated human peripheral blood lymphocyte (PBL). Our findings follow: (a) the proliferation of PBL in response to phytohemagglutinin (PHA) was mediated by protein kinase C activity, but estrogenic xenobiotics had a strong inhibitory effect on protein kinase C activity of PHA-stimulated PBL; (b) cytoplasmic extracts from PHA-stimulated PBL greatly activated DNA replication, but estrogenic xenobiotics had a strong inhibitory effect on these activities. The results suggest that the cytoplasmic signal-generating system in mitogen-treated PBL is inhibited by estrogenic xenobiotics, and that the defect occurs at all stages in the sequence of events leading to DNA synthesis and cell proliferation.

  17. The effect of age, gender, diet and lifestyle on DNA damage measured using micronucleus frequency in human peripheral blood lymphocytes.

    PubMed

    Fenech, Michael; Bonassi, Stefano

    2011-01-01

    Micronucleus (MN) frequency in cytokinesis-blocked peripheral blood lymphocytes (PBL) has become one of the best-established biomarkers for studying DNA damage occurring in vivo in humans. The application of this method in population biomonitoring studies requires a deep understanding of how lifestyle and common host variables may influence MN frequency in PBL. In this mini-review, an update is provided on results from studies reporting on the impact of age, gender, diet and lifestyle factors (e.g. exercise, alcohol, smoking and recreational drugs) on this biomarker. Evidence from these studies shows that each of these factors, either in isolation or in combination, can significantly influence MN frequency. Proper control for these factors is required to enable better measurement of the impact of other conditions, such as environmental exposure to genotoxins or a susceptible genetic background, on MN frequency in PBL.

  18. Clinical trials for human T-cell lymphotropic virus type I-associated peripheral T-cell lymphoma in Japan.

    PubMed

    Tobinai, Kensei

    2010-04-01

    The most common subtype of T-/natural killer (NK) cell lymphoma in Japan is adult T-cell leukemia-lymphoma (ATL), which is associated with the human T-cell lymphotropic virus type I (HTLV-1). The investigators in Japan have conducted several clinical trials on multi-agent chemotherapy and stem cell transplantation for patients with ATL. They have also initiated several new clinical trials with a number of agents: an anti-CCR4 antibody, KW-0761; forodesine, a purine nucleoside phosphorylase inhibitor; and lenalidomide, an immunomodulatory agent. Clinical trials with pralatrexate, a folate analog, and denileukin diftitox, an immunoconjugate, are under discussion for patients with ATL and peripheral T-cell lymphoma (PTCL).

  19. Growth inhibition, cell-cycle alteration and apoptosis in stimulated human peripheral blood lymphocytes by multiwalled carbon nanotube buckypaper.

    PubMed

    Zeni, Olga; Sannino, Anna; Romeo, Stefania; Micciulla, Federico; Bellucci, Stefano; Scarfi, Maria Rosaria

    2015-02-01

    This study was designed to investigate the cytotoxicity of multiwalled carbon nanotube buckypaper (BP) in stimulated human peripheral blood lymphocytes. Materials & methods & results: BP treatment led to a delay in the cell growth, as proven by a minor increase in the cell number over time relative to that seen in untreated cells, assessed by trypan blue, resazurin and neutral red assays. The analysis of cell-cycle profile, by propidium iodide staining, indicated that BP treatment blocked cell-cycle progression by arresting cells at the G0/G1 phase. Moreover, increased apoptosis was also recorded by Annexin V-fluorescein isothiocyanate/propidium iodide staining. The results presented here demonstrate an inhibitor effect of BP on cell growth that was likely through cytostatic and cytotoxic events.

  20. Characterisation of the endogenous human peripheral serotonin transporter SLC6A4 reveals surface expression without N-glycosylation.

    PubMed

    Chamba, Anita; Holder, Michelle J; Barnes, Nicholas M; Gordon, John

    2008-11-15

    RT-PCR confirmed that human cell lines of diverse peripheral origins express transcripts for the serotonin transporter (sert/slc6a4). Molecular weights reported for the translated protein appear to be quite variable however. Here we compared directly immunoreactive protein generated from cloned sert transfected into HEK293 with that carried endogenously among the cell lines. The dominant glycosylated 85-95 kDa immunoreactive species contained in HEK-sert transfectants was poorly represented in any native cell: instead, discrete 70 and 60 kDa bands were universally detected. Biotinylation of lymphoid cells revealed that the endogenous non-glycosylated 60 kDa but not 70 kDa protein was available at the surface to access exogenous ligands.

  1. Altered cytokine production by specific human peripheral blood cell subsets immediately following space flight

    NASA Technical Reports Server (NTRS)

    Crucian, B. E.; Cubbage, M. L.; Sams, C. F.

    2000-01-01

    In this study, flow cytometry was used to positively identify the specific lymphocyte subsets exhibiting space flight-induced alterations in cytokine production. Whole blood samples were collected from 27 astronauts at three points (one preflight, two postflight) surrounding four space shuttle missions. Assays performed included serum/urine stress hormones, white blood cell (WBC) phenotyping, and intracellular cytokine production following mitogenic stimulation. Absolute levels of peripheral granulocytes were significantly elevated following space flight, but the levels of circulating lymphocytes and monocytes were unchanged. Lymphocyte subset analysis demonstrated a decreased percentage of T cells, whereas percentages of B cells and natural killer (NK) cells remained unchanged after flight. Nearly all the astronauts exhibited an increased CD4/CD8 T cell ratio. Assessment of naive (CD45RA+) vs. memory (CD45RO+) CD4+ T cell subsets was ambiguous, and subjects tended to group within specific missions. Although no significant trend was seen in absolute monocyte levels, a significant decrease in the percentage of the CD14+ CD16+ monocytes was seen following space flight in all subjects tested. T cell (CD3+) production of interleukin-2 (IL-2) was significantly decreased after space flight, as was IL-2 production by both CD4+ and CD8+ T cell subsets. Production of interferon-gamma (IFN-gamma) was not altered by space flight for the CD8+ cell subset, but there was a significant decrease in IFN-gamma production for the CD4+ T cell subset. Serum and urine stress hormone analysis indicated significant physiologic stresses in astronauts following space flight. Altered peripheral leukocyte subsets, altered serum and urine stress hormone levels, and altered T cell cytokine secretion profiles were all observed postflight. In addition, there appeared to be differential susceptibility to space flight regarding cytokine secretion by T cell subsets. These alterations may be the

  2. Altered cytokine production by specific human peripheral blood cell subsets immediately following space flight

    NASA Technical Reports Server (NTRS)

    Crucian, B. E.; Cubbage, M. L.; Sams, C. F.

    2000-01-01

    In this study, flow cytometry was used to positively identify the specific lymphocyte subsets exhibiting space flight-induced alterations in cytokine production. Whole blood samples were collected from 27 astronauts at three points (one preflight, two postflight) surrounding four space shuttle missions. Assays performed included serum/urine stress hormones, white blood cell (WBC) phenotyping, and intracellular cytokine production following mitogenic stimulation. Absolute levels of peripheral granulocytes were significantly elevated following space flight, but the levels of circulating lymphocytes and monocytes were unchanged. Lymphocyte subset analysis demonstrated a decreased percentage of T cells, whereas percentages of B cells and natural killer (NK) cells remained unchanged after flight. Nearly all the astronauts exhibited an increased CD4/CD8 T cell ratio. Assessment of naive (CD45RA+) vs. memory (CD45RO+) CD4+ T cell subsets was ambiguous, and subjects tended to group within specific missions. Although no significant trend was seen in absolute monocyte levels, a significant decrease in the percentage of the CD14+ CD16+ monocytes was seen following space flight in all subjects tested. T cell (CD3+) production of interleukin-2 (IL-2) was significantly decreased after space flight, as was IL-2 production by both CD4+ and CD8+ T cell subsets. Production of interferon-gamma (IFN-gamma) was not altered by space flight for the CD8+ cell subset, but there was a significant decrease in IFN-gamma production for the CD4+ T cell subset. Serum and urine stress hormone analysis indicated significant physiologic stresses in astronauts following space flight. Altered peripheral leukocyte subsets, altered serum and urine stress hormone levels, and altered T cell cytokine secretion profiles were all observed postflight. In addition, there appeared to be differential susceptibility to space flight regarding cytokine secretion by T cell subsets. These alterations may be the

  3. Initiation of Benzodiazepines in the Elderly After Hospitalization

    PubMed Central

    Fischer, Hadas D.; Gill, Sudeep S.; Zagorski, Brandon; Sykora, Kathy; Wodchis, Walter P.; Herrmann, Nathan; Bronskill, Susan E.; Lee, Phil E.; Anderson, Geoff M.; Rochon, Paula A.

    2007-01-01

    Objective To estimate the rate of new chronic benzodiazepine use after hospitalization in older adults not previously prescribed with benzodiazepines. Design Retrospective cohort study using linked, population-based administrative data. Setting Ontario, Canada between April 1, 1992 and March 31, 2005. Participants Community-dwelling seniors who had not been prescribed benzodiazepine drugs in the year before hospitalization were selected from all 1.4 million Ontario residents aged 66 years and older. Main Outcome Measures New chronic benzodiazepine users, defined as initiation of benzodiazepines within 7 days after hospital discharge and an additional claim within 8 days to 6 months. We used multivariate logistic regression to examine for the effect of hospitalization on the primary outcome after adjusting for confounders. Results There were 405,128 patient hospitalizations included in the cohort. Benzodiazepines were prescribed to 12,484 (3.1%) patients within 7 days of being discharged from hospital. A total of 6,136 (1.5%) patients were identified as new chronic benzodiazepine users. The rate of new chronic benzodiazepine users decreased over the study period from 1.8% in the first year to 1.2% in the final year (P < .001). Multivariate logistic regression found that women, patients admitted to the intensive care unit or nonsurgical wards, those with longer hospital stays, higher overall comorbidity, a prior diagnosis of alcoholism, and those prescribed more medications had significantly elevated adjusted odds ratios for new chronic benzodiazepine users. Older individuals had a lower risk for the primary outcome. Conclusion New benzodiazepine prescription after hospitalization occurs frequently in older adults and may result in chronic use. A systemic effort to address this risky practice should be considered. PMID:17453266

  4. Protection of resveratrol and its analogues against ethanol-induced oxidative DNA damage in human peripheral lymphocytes.

    PubMed

    Yan, Yu; Yang, Jingyu; Chen, Guoliang; Mou, Yanhua; Zhao, Yanan; Pan, Leyang; Ma, Chaohui; Liu, Xinwei; Wu, Chunfu

    2011-04-03

    Diseases related to ethanol abuse, especially binge drinking, are becoming one of the most costly health problems in the world. Ethanol-induced DNA damage plays a key role in the etiology of these diseases. New compounds are expected to offer new options against ethanol-induced genotoxicity. It was found, for the first time, that resveratrol and three analogues with 3,5-dimethoxyl groups in the A-ring, such as (E)-4-(3,5-dimethoxystyryl)phenol (RV32), or with a quinolyl in the B-ring, such as (E)-5-[2-(quinolin-4-yl)vinyl]benzene-1,3-diol (RV01) and (E)-4-(3,5-dimethoxystyryl)quinoline (RV02), strongly inhibited ethanol-induced oxidative DNA damage in human peripheral lymphocytes in vitro. Resveratrol and RV32 with more hydroxyl groups in structures showed stronger direct scavenging activity of hydroxyl radicals than RV01 and RV02. Moreover, all compounds reduced hydroxyl radical generation by regulating the mRNA expression of alcohol dehydrogenase 1B and acetaldehyde dehydrogenase 2. Further studies proved resveratrol and three analogues activated the base excision repair system in transcriptional and protein levels in DNA repair process. Both 3,5-dimethoxyl groups and quinolyl modification may enhance such activity. In summary, resveratrol and its three analogues revealed significant protective activity against ethanol-induced oxidative DNA damage in human peripheral lymphocytes, which demonstrates their potential for use in prevention and treatment of the diseases related to ethanol abuse. Copyright © 2011 Elsevier B.V. All rights reserved.

  5. Critical role of peripheral vasoconstriction in fatal brain hyperthermia induced by MDMA (Ecstasy) under conditions that mimic human drug use.

    PubMed

    Kiyatkin, Eugene A; Kim, Albert H; Wakabayashi, Ken T; Baumann, Michael H; Shaham, Yavin

    2014-06-04

    MDMA (Ecstasy) is an illicit drug used by young adults at hot, crowed "rave" parties, yet the data on potential health hazards of its abuse remain controversial. Here, we examined the effect of MDMA on temperature homeostasis in male rats under standard laboratory conditions and under conditions that simulate drug use in humans. We chronically implanted thermocouple microsensors in the nucleus accumbens (a brain reward area), temporal muscle, and facial skin to measure temperature continuously from freely moving rats. While focusing on brain hyperthermia, temperature monitoring from the two peripheral locations allowed us to evaluate the physiological mechanisms (i.e., intracerebral heat production and heat loss via skin surfaces) that underlie MDMA-induced brain temperature responses. Our data confirm previous reports on high individual variability and relatively weak brain hyperthermic effects of MDMA under standard control conditions (quiet rest, 22-23°C), but demonstrate dramatic enhancements of drug-induced brain hyperthermia during social interaction (exposure to male conspecific) and in warm environments (29°C). Importantly, we identified peripheral vasoconstriction as a critical mechanism underlying the activity- and state-dependent potentiation of MDMA-induced brain hyperthermia. Through this mechanism, which prevents proper heat dissipation to the external environment, MDMA at a moderate nontoxic dose (9 mg/kg or ~1/5 of LD50 in rats) can cause fatal hyperthermia under environmental conditions commonly encountered by humans. Our results demonstrate that doses of MDMA that are nontoxic under cool, quiet conditions can become highly dangerous under conditions that mimic recreational use of MDMA at rave parties or other hot, crowded venues.

  6. Expression of doublecortin in tumours of the central and peripheral nervous system and in human non-neuronal tissues.

    PubMed

    Bernreuther, Christian; Salein, Nora; Matschke, Jakob; Hagel, Christian

    2006-03-01

    Doublecortin is a microtubule-associated phosphoprotein involved in neuronal migration and differentiation expressed in migrating neuroblasts in the central nervous system. We systematically analysed doublecortin expression in 179 tumours of the central and 65 tumours of peripheral nervous system as well as in 74 different non-neuronal tissues to evaluate the specificity of doublecortin as a marker for neuronal differentiation in glioneuronal tumours. Glioneuronal tumours and oligodendrogliomas grade II and III uniformly showed a high intensity and frequency of doublecortin staining, whereas intermediate doublecortin expression was observed in astrocytic tumours of grade II-IV. In pilocytic astrocytomas and ependymomas only scattered doublecortin positive cells were detected. In the peripheral nervous system, doublecortin expression was found in neurofibroma but was absent in schwannoma. Double staining of tumour tissue revealed co-expression of doublecortin and neurofilament in cells of gangliocytomas and gangliogliomas and co-expression of doublecortin with S100 protein or GFAP in glial tumours, respectively. In a tissue array comprised of 74 different normal non-neuronal human tissues, doublecortin expression was demonstrated in epithelia of the kidney, liver, salivary glands and duodenum among others. Interestingly, doublecortin expression could not be shown in brain metastases of tumours originating from these tissues. Immunohistochemical data was further corroborated by Western blot analysis and reverse transcription polymerase chain reaction. In conclusion, doublecortin can be regarded as specific neuronal marker only in normal developing brain, but lacks specificity in glioneuronal and glial tumours and other non-neuronal human tissues where it is expressed in a wide variety of tumours and tissues.

  7. Short-term in vitro responses of human peripheral blood monocytes to ferritic stainless steel fiber networks.

    PubMed

    Spear, Rose L; Brooks, Roger A; Markaki, Athina E

    2013-05-01

    Beneficial effects on bone-implant bonding may accrue from ferromagnetic fiber networks on implants which can deform in vivo inducing controlled levels of mechanical strain directly in growing bone. This approach requires ferromagnetic fibers that can be implanted in vivo without stimulating undue inflammatory cell responses or cytotoxicity. This study examines the short-term in vitro responses, including attachment, viability, and inflammatory stimulation, of human peripheral blood monocytes to 444 ferritic stainless steel fiber networks. Two types of 444 networks, differing in fiber cross section and thus surface area, were considered alongside austenitic stainless steel fiber networks, made of 316L, a widely established implant material. Similar high percent seeding efficiencies were measured by CyQuant® on all fiber networks after 48 h of cell culture. Extensive cell attachment was confirmed by fluorescence and scanning electron microscopy, which showed round monocytes attached at various depths into the fiber networks. Medium concentrations of lactate dehydrogenase (LDH) and tumor necrosis factor alpha (TNF-α) were determined as indicators of viability and inflammatory responses, respectively. Percent LDH concentrations were similar for both 444 fiber networks at all time points, whereas significantly lower than those of 316L control networks at 24 h. All networks elicited low-level secretions of TNF-α, which were significantly lower than that of the positive control wells containing zymosan. Collectively, the results indicate that 444 networks produce comparable responses to medical implant grade 316L networks and are able to support human peripheral blood monocytes in short-term in vitro cultures without inducing significant inflammatory or cytotoxic effects. Copyright © 2012 Wiley Periodicals, Inc.

  8. Evaluation of γ-Induced Apoptosis in Human Peripheral Blood Lymphocytes

    NASA Astrophysics Data System (ADS)

    Baranova, Elena; Boreyko, Alla; Ravnachka, Ivanka; Saveleva, Maria

    2010-01-01

    Several experiments have been performed to study regularities in the induction of apoptotic cells in human lymphocytes by 60Co γ-rays at different times after irradiation. Apoptosis induction by 60Co γ-rays in human lymphocytes in different cell cycle phases (G0, S, G1, and G2) has been studied. The maximal apoptosis output in lymphocyte cells was observed in the S phase. Modifying effect of replicative and reparative DNA synthesis inhibitors—1- β -D-arabinofuranosylcytosine (Ara-C) and hydroxyurea (Hu)—on the kinetics of 60Co γ-rays induced apoptosis in human lymphocytes has been studied.

  9. Evaluation of gamma-Induced Apoptosis in Human Peripheral Blood Lymphocytes

    SciTech Connect

    Baranova, Elena; Boreyko, Alla; Ravnachka, Ivanka; Saveleva, Maria

    2010-01-05

    Several experiments have been performed to study regularities in the induction of apoptotic cells in human lymphocytes by {sup 60}Cogamma-rays at different times after irradiation. Apoptosis induction by {sup 60}Cogamma-rays in human lymphocytes in different cell cycle phases (G{sub 0}, S, G{sub 1}, and G{sub 2}) has been studied. The maximal apoptosis output in lymphocyte cells was observed in the S phase. Modifying effect of replicative and reparative DNA synthesis inhibitors - 1-beta-D-arabinofuranosylcytosine (Ara-C) and hydroxyurea (Hu) - on the kinetics of {sup 60}Cogamma-rays induced apoptosis in human lymphocytes has been studied.

  10. An implantable, designed-for-human-use peripheral nerve stimulation and recording system for advanced prosthetics.

    PubMed

    Lachapelle, John R; Bjune, Caroline K; Kindle, Alexander L; Czarnecki, Andrew; Burns, John R; Grainger, Julianne E; Segura, Carlos A; Nugent, Brian D; Sriram, Tirunelveli S; Parks, Philip D; Keefer, Edward; Cheng, Jonathan

    2016-08-01

    Complex suture prostheses that deliver sensory and position feedback require a more sophisticated integration with the human user. Here a micro-size active implantable system that provides many-degree-of-freedom neural feedback in both sensory stimulation and motor control is shown, as one potential human-use solution in DARPA's HAPTIX program. Various electrical and mechanical challenge and solutions in meeting both sensory /motor performance as well as ISO 14708 FDA-acceptable human use in an aspirin-size active implementation are discussed.

  11. Modulation of Human Peripheral Blood Mononuclear Cell Signaling by Medicinal Cannabinoids

    PubMed Central

    Utomo, Wesley K.; de Vries, Marjan; Braat, Henri; Bruno, Marco J.; Parikh, Kaushal; Comalada, Mònica; Peppelenbosch, Maikel P.; van Goor, Harry; Fuhler, Gwenny M.

    2017-01-01

    Medical marijuana is increasingly prescribed as an analgesic for a growing number of indications, amongst which terminal cancer and multiple sclerosis. However, the mechanistic aspects and properties of cannabis remain remarkably poorly characterized. In this study we aimed to investigate the immune-cell modulatory properties of medical cannabis. Healthy volunteers were asked to ingest medical cannabis, and kinome profiling was used to generate comprehensive descriptions of the cannabis challenge on inflammatory signal transduction in the peripheral blood of these volunteers. Results were related to both short term and long term effects in patients experimentally treated with a medical marijuana preparation for suffering from abdominal pain as a result of chronic pancreatitis or other causes. The results reveal an immunosuppressive effect of cannabinoid preparations via deactivation of signaling through the pro-inflammatory p38 MAP kinase and mTOR pathways and a concomitant deactivation of the pro-mitogenic ERK pathway. However, long term cannabis exposure in two patients resulted in reversal of this effect. While these data provide a powerful mechanistic rationale for the clinical use of medical marijuana in inflammatory and oncological disease, caution may be advised with sustained use of such preparations. PMID:28174520

  12. RANK Expression and Osteoclastogenesis in Human Monocytes in Peripheral Blood from Rheumatoid Arthritis Patients

    PubMed Central

    Kobashigawa, Tsuyoshi

    2016-01-01

    Rheumatoid arthritis (RA) appears as inflammation of synovial tissue and joint destruction. Receptor activator of NF-κB (RANK) is a member of the TNF receptor superfamily and a receptor for the RANK ligand (RANKL). In this study, we examined the expression of RANKhigh and CCR6 on CD14+ monocytes from patients with RA and healthy volunteers. Peripheral blood samples were obtained from both the RA patients and the healthy volunteers. Osteoclastogenesis from monocytes was induced by RANKL and M-CSF in vitro. To study the expression of RANKhigh and CCR6 on CD14+ monocytes, two-color flow cytometry was performed. Levels of expression of RANK on monocytes were significantly correlated with the level of osteoclastogenesis in the healthy volunteers. The expression of RANKhigh on CD14+ monocyte in RA patients without treatment was elevated and that in those receiving treatment was decreased. In addition, the high-level expression of RANK on CD14+ monocytes was correlated with the high-level expression of CCR6 in healthy volunteers. Monocytes expressing both RANK and CCR6 differentiate into osteoclasts. The expression of CD14+RANKhigh in untreated RA patients was elevated. RANK and CCR6 expressed on monocytes may be novel targets for the regulation of bone resorption in RA and osteoporosis. PMID:27822475

  13. Cytokine synthesis in human peripheral blood mononuclear cells after oral administration of polyenzyme preparations.

    PubMed

    Desser, L; Rehberger, A; Kokron, E; Paukovits, W

    1993-01-01

    Pharmaceutical preparations containing mixtures of various proteolytic and nonproteolytic enzymes have been suggested for use in the treatment of malignant diseases. However, the mode of action of such preparations was not clear. We have shown before that intact bromelain, papain or amylase, which are components of a commercial polyenzyme preparation, induce cytokine production in peripheral blood mononuclear cells in vitro. IFN-alpha and IFN-gamma which had no effect alone, synergistically increased TNF production when applied together with the enzymes. Here we show that trypsin alone had only a small inducing effect. The tryptic but not the autolytic fragments of papain and bromelain have a higher (10- to 40-fold) inducing capacity for TNF production than the untreated enzyme. Additionally we demonstrate that after ingestion of milligram doses of the polyenzyme preparation (as recommended for clinical use), PBMNC of healthy donors acquire the ability to produce TNF-alpha, IL-1 beta and IL-6 when incubated ex vivo with IFN-gamma. Our results indicate that the biological effects observed after oral administration of polyenzyme preparations are related to their ability to induce cytokine production. This may explain the antitumor effects of such enzymes. Our results also suggest that polyenzyme preparations may have a stronger immunomodulary effect when used in combination with IFN-gamma.

  14. Detection of circulating tumor cells from cryopreserved human sarcoma peripheral blood mononuclear cells.

    PubMed

    Li, Heming; Meng, Qing H; Noh, Hyangsoon; Batth, Izhar Singh; Somaiah, Neeta; Torres, Keila E; Xia, Xueqing; Wang, Ruoyu; Li, Shulin

    2017-09-10

    Circulating tumor cells (CTCs) enter the vasculature or lymphatic system after shedding from the primary tumor. CTCs may serve as "seed" cells for tumor metastasis. The utility of CTCs in clinical applications for sarcoma is not fully investigated, partly owing to the necessity for fresh blood samples and the lack of a CTC-specific antibody. To overcome these drawbacks, we developed a technique for sarcoma CTCs capture and detection using cryopreserved peripheral blood mononuclear cells (PBMCs) and our proprietary cell-surface vimentin (CSV) antibody 84-1, which is specific to tumor cells. This technique was validated by sarcoma cell spiking assay, matched CTCs comparison between fresh and cryopreserved PBMCs, and independent tumor markers in multiple types of sarcoma patient blood samples. The reproducibility was maximized when cryopreserved PBMCs were prepared from fresh blood samples within 2 h of the blood draw. In summary, as far as we are aware, ours is the first report to capture and detect CTCs from cryopreserved PBMCs. Further validation in other types of tumor may help boost the feasibility and utility of CTC-based diagnosis in a centralized laboratory. Copyright © 2017 Elsevier B.V. All rights reserved.

  15. Cardiotrophin-1 induces tumor necrosis factor alpha synthesis in human peripheral blood mononuclear cells.

    PubMed

    Fritzenwanger, Michael; Meusel, Katharina; Jung, Christian; Franz, Marcus; Wang, Zhenhua; Foerster, Martin; Figulla, Hans-R

    2009-01-01

    Chronic heart failure (CHF) is associated with elevated concentrations of tumor necrosis factor (TNF) alpha and cardiotrophin-1 (CT-1) and altered peripheral blood mononuclear cell (PBMC) function. Therefore, we tested whether CT-1 induces TNFalpha in PBMC of healthy volunteers. CT-1 induced in PBMC TNFalpha protein in the supernatant and TNFalpha mRNA in a concentration- and time-dependent manner determined by ELISA and real-time PCR, respectively. Maximal TNFalpha protein was achieved with 100 ng/mL CT-1 after 3-6 hours and maximal TNFalpha mRNA induction after 1 hour. ELISA data were confirmed using immunofluorescent flow cytometry. Inhibitor studies with actinomycin D and brefeldin A showed that both protein synthesis and intracellular transport are essential for CT-1 induced TNFalpha expression. CT-1 caused a dose dependent nuclear factor (NF) kappaB translocation. Parthenolide inhibited both NFkappaB translocation and TNFalpha protein expression indicating that NFkappaB seems to be necessary. We revealed a new mechanism for elevated serum TNFalpha concentrations and PBMC activation in CHF besides the hypothesis of PBMC activation by bacterial translocation from the gut.

  16. Evaluating the role of low-speed centrifugation towards transfecting human peripheral blood mononuclear cell culture.

    PubMed

    Majumdar, M; Ratho, R; Chawla, Y; Singh, M P

    2014-01-01

    The conventional method of transfection of suspension cells by chemical has proven to be very difficult. We present a new transfection protocol, wherein, low-speed centrifugation of cell culture plates immediately after adding the lipid: DNA complex significantly enhances the transfection efficiency. Peripheral blood mononuclear cells (PBMCs) were transfected with BLOCK-iT™ Fluorescent Oligo (scrambled siRNA) and lipofectamine complex using conventional and low-speed centrifugation modified transfection protocols. The efficiency of transfection was determined using flowcytometer and cell viability was checked using MTT assay. Incorporation of low-speed centrifugation significantly enhances the transfection efficiency of BLOCK-iT™ in the suspension culture of PBMCs as compared to conventional transfection method (99.8% vs 28.3%; P < 0.0001), even at a low concentration of 40 picomoles without affecting the cell viability. Centrifugation enhanced transfection (CET) technique is simple, time-saving and novel application without compromising the cell viability in the context of recently popular RNA interference in suspension cultures of PBMCs. This undemanding modification might be applicable to a wide variety of cell lines and solve crucial problem of researchers working with RNA interference in suspension cultures.

  17. Associating peripheral and foveal visual input across saccades: a default mode of the human visual system?

    PubMed

    Weiß, Katharina; Schneider, Werner X; Herwig, Arvid

    2014-09-09

    Spatial processing resolution of a particular object in the visual field can differ considerably due to eye movements. The same object will be represented with high