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Biomedical subjects

R Pacifici

Publications and source records attributed to R Pacifici.

At least 55 records · Page 3Linked to original sources
PubMed2000

[Role of laboratories in the clinical study of drug dependence].

Clinical toxicological laboratory is involved in two different topics related to drug addiction: tests devoted to diagnosis and treatment of addicted and analyses for administrative and forensic purposes. A laboratory which analyses drugs of abuse has to guarantee reliability and performance of its services working with a quality system. It can be possible to differentiate between laboratories which perform only screening tests with immunological methods and laboratories which execute confirmations with a forensic meaning using chromatographic techniques. The primary biological matrix for this kind of tests is urine, although in some specific cases hair analysis can be a support of traditional investigations. Internet sites on specific topics can be of assistance for questions of different nature to be faced by a clinical toxicological laboratory.

Humans↗
PubMed2000

[Analysis of drugs and abuse substances in the keratinic matrix].

Hair analysis of xenobiotics is an ideal addendum to blood and urine analysis because it can reveal information that is not available through analysis of the other two specimens. However, there are still some unresolved issues that may affect the legal acceptance of hair testing to quantify drug consumption or exposition. The present paper shows hypotheses on mechanisms of drug incorporation in the hair shaft, variability factors pending on incorporation, methodology for hair collection and extraction, analytes determination and finally data presentation and interpretation.

Hair↗
PubMed2000

[Immunomodulator properties of ecstasy (MDMA)].

In vitro exposure to ecstasy (3,4-methylenedioxymethamphetamine, MDMA) alters some immune parameters such as T-cell regulatory function, cytotoxic T-lymphocyte activity, natural killer cell activity and macrophage function. Administration of MDMA in rats produces a suppression of lympho-proliferation response and a decrease in circulating lymphocytes, accompanied by an increase in plasma corticosterone. It was postulated a direct action of MDMA on lymphocytes or rather an indirect action mediated by the hypothalamic pituitary adrenal axis (HPA-AXIS) and/or the sympathetic nervous system (SNS). Acute MDMA treatment effected on healthy-volunteers produces an immune dysfunction associated with pharmaceutical characteristics and so with MDMA plasma concentrations. There is a decrease in CD4+ T-cells and functional responsiveness of lymphocytes, while percentage of natural killer cells increases. A contemporary rise of cortisol plasma concentrations supports the hypothesis of MDMA-induced release of corticotrophin-releasing factor from the hypothalamus and subsequent HPA-axis and SNS activation.

Animals↗
PubMed1999

Determination of lorazepam in plasma and urine as trimethylsilyl derivative using gas chromatography-tandem mass spectrometry.

A procedure based on gas chromatography-tandem mass spectrometry for identification and quantitation of lorazepam in plasma and urine is presented. The analyte was extracted from biological fluids under alkaline conditions using solid-phase extraction with an Extrelut-1 column in the presence of oxazepam-d5 as the internal standard. Both compounds were then converted to their trimethylsilyl derivatives and the reaction products were identified and quantitated by gas chromatography-tandem mass spectrometry using the product ions of the two compounds (m/z 341, 306 and 267 for lorazepam derivative and m/z 346, 309 and 271 for oxazepam-d5 derivative) formed from the parent ions by collision-induced dissociation in the ion trap spectrometer. Limit of quantitation was 0.1 ng/ml. This method was validated for urine and plasma samples of individuals in treatment with the drug.

Anti-Anxiety Agents↗
PubMed1999

Immunomodulating properties of MDMA alone and in combination with alcohol: a pilot study.

Cell-mediated immune response after the administration of MDMA alone and in combination with alcohol was evaluated in a randomized, double-blind, double-dummy, cross-over pilot clinical trial conducted in four healthy MDMA consumers who received single oral doses of 75 mg MDMA (n = 2) or 100 mg MDMA (n = 2), alcohol (0.8 mg/kg), MDMA and alcohol, or placebo. Acute MDMA treatment produced a time-dependent immune dysfunction associated with MDMA plasma concentrations. Although total leukocyte count remained unchanged, there was a decrease in the CD4 T/CD8 T-cell ratio as well as in the percentage of mature T lymphocytes, probably because of a decrease in both the percentage and absolute number of T helper cells. The decrease in CD4 T-cell counts and in the functional responsiveness of lymphocytes to mitogenic stimulation was dose-dependent. The correlation between MDMA pharmacokinetics and the profile of MDMA-induced immune dysfunction suggests that alteration of the immune system may be mediated by the central nervous system. Alcohol consumption produced a decrease in T helper cells, B lymphocytes, and PHA-induced lymphocyte proliferation. Combined MDMA and alcohol produced the greatest suppressive effect on CD4 T-cell count and PHA-stimulated lymphoproliferation. Immune function was partially restored at 24 hours. These results provide the first evidence that recreational use of MDMA alone or in combination with alcohol alters the immunological status.

Adjuvants, Immunologic↗
PubMed1999

Immature germ cell separation using a modified discontinuous Percoll gradient technique in human semen.

The difficulty of identifying immature germ cells in unstained, fresh semen has led most laboratories to use the broad definition 'round cells' to indicate cells other than spermatozoa, thus grouping together both leukocytes and immature germ cells. This is also the case in research andrology, where very little attention has been given to immature germ cells in the semen apart from some rare exceptions, such as the attempts to study meiosis. Here we report on the use of a discontinuous Percoll gradient method modified to enable the best separation possible of immature germ cells from the other cells found in the ejaculate, in order to obtain a cellular suspension free of spermatozoa. Our technique (intra-assay variation in duplicates < 10%) demonstrated a high immature germ cell concentration in gradient fractions with 30% to 45% Percoll with a small contamination (1.5-6%) of leukocytes, confirmed by May-Grünwald-Giemsa staining, immunofluorescence and cytofluorimetry. The concentrations of immature germ cells ranged from zero in obstructive azoospermia to 2.0 x 10(6)/ml in oligozoospermia and genital tract infection. The purified immature germ cell suspensions obtained can be useful for diagnostic and research purposes.

Cell Separation↗
PubMed1999

Determination of opiates and cocaine in hair as trimethylsilyl derivatives using gas chromatography-tandem mass spectrometry.

An analytical method for the determination of heroin, 6-monoacetylmorphine, morphine, codeine, cocaine, benzoylecgonine, and cocaethylene in human hair using gas chromatography-tandem mass spectrometry is presented. The analytes were extracted from finely cut hair with methanol at 56 degrees C for 18 h in the presence of nalorphine as the internal standard. After the incubation, methanol was evaporated to dryness, and all the analytes, except heroin, cocaine, and cocaethylene, were converted to their trimethylsilyl derivatives. The reaction products were identified and quantitated using product ions formed from the parent ions by collision-induced dissociation in the ion-trap mass spectrometer. This method provided excellent sensitivity and specificity for analytes at the concentrations usually found in the keratin matrix.

Cocaine↗
PubMed1999

Estrogen decreases TNF gene expression by blocking JNK activity and the resulting production of c-Jun and JunD.

Central to the bone-sparing effect of estrogen (E(2)) is its ability to block the monocytic production of the osteoclastogenic cytokine TNF-alpha (TNF). However, the mechanism by which E(2) downregulates TNF production is presently unknown. Transient transfection studies in HeLa cells, an E(2) receptor-negative line, suggest that E(2) inhibits TNF gene expression through an effect mediated by estrogen receptor beta (ERbeta). We also report that in RAW 264.7 cells, an E(2) receptor-positive murine monocytic line, E(2) downregulates cytokine-induced TNF gene expression by decreasing the activity of the Jun NH(2)-terminal kinase (JNK). The resulting diminished phosphorylation of c-Jun and JunD at their NH(2)-termini decreases the ability of these nuclear proteins to autostimulate the expression of the c-Jun and JunD genes, thus leading to lower production of c-Jun and JunD. The consequent decrease in the nuclear levels of c-Jun and JunD leads to diminished binding of c-Jun/c-Fos and JunD/c-Fos heterodimers to the AP-1 consensus sequence in the TNF promoter and, thus, to decreased transactivation of the TNF gene.

Base Sequence↗
PubMed1998

Estrogen blocks M-CSF gene expression and osteoclast formation by regulating phosphorylation of Egr-1 and its interaction with Sp-1.

Central to the pathogenesis of osteoporosis is the ability of estrogen deficiency to increase osteoclast formation by enhancing stromal cell production of the osteoclastogenic cytokine macrophage colony-stimulating factor (M-CSF). We report that stromal cells from ovariectomized mice exhibit increased casein kinase II-dependent phosphorylation of the nuclear protein Egr-1. Phosphorylated Egr-1 binds less avidly to the transcriptional activator Sp-1 and the resulting higher levels of free Sp-1 stimulate transactivation of the M-CSF gene. Estrogen replacement fails to block M-CSF mRNA expression and osteoclast formation in ovariectomized mice lacking Egr-1, confirming the critical role played by this transcription factor in mediating the antiosteoclastogenic effects of estrogen. Thus, by downregulating formation of a novel Egr-1/Sp-1 complex in stromal cells, estrogen deficiency results in enhanced levels of free Sp-1 and increased M-CSF gene expression and osteoclast formation.

Animals↗
PubMed1998

Simultaneous determination of epirubicin, doxorubicin and their principal metabolites in human plasma by high-performance liquid chromatography and electrochemical detection.

A high-performance liquid chromatographic method with electrochemical detection has been developed for the simultaneous determination of epirubicin, 13-S-dihydroepirubicin, doxorubicin and 13-S-dihydrodoxorubicin in human plasma. An aliquot of 200 microl plasma, spiked with internal standard, was extracted by solid-phase extraction using polymeric adsorbent columns. Chromatography was performed using a C18 reversed-phase column with a mobile phase consisting of water-acetonitrile (71:29, v/v) containing 0.05 M Na2HPO4 and 0.05% v/v triethylamine adjusted to pH 4.6 with citric acid. Linearity of the method was obtained in the concentration range of 1-500 ng/ml for all the analytes. Analytical recoveries of the analytes ranged from 89 to 93%. The assay can be used for the simultaneous determination of the four analytes, or for epirubicin and its metabolite or doxorubicin and its metabolite, using the other parent drug as an internal standard. The method was applied to analyze human plasma samples from patients treated with epirubicin using doxorubicin as an internal standard.

Antibiotics, Antineoplastic↗
PubMed1998

Quantification of the variation due to lysing technique in immunophenotyping of healthy and HIV-infected individuals.

OBJECTIVE: We performed a side-by-side comparison between three stain-then-lyse commercially available methods (Ortho-mune Lysing solution, FACS lysing solution and ImmunoPrep reagent system) and a lyse-then-stain method using hypotonic NH4Cl. The major difference between these methods is that only in the latter the aliquots of sample to be distributed into diverse tubes for the various antibody combinations were obtained from a lysis step performed in the same tube. DESIGN AND METHODS: Lymphocytes from 20 healthy and 20 HIV+ subjects were phenotyped by dual color flow cytometry using a standard procedure that included the establishing of a lymphocyte gate on light scatter bit map and the use of the minimal acceptable antibody combinations, i.e., CD45/CD14, CD3/CD4 and CD3/CD8, according to CDC recommendations. All samples were processed in triplicate to assess tube-to-tube variability. RESULTS: In healthy subjects, erythrocytes pre-lysing provided the highest purity and recovery in the lymphocyte gate, and allowed the best identification of CD4+ lymphocytes. Most remarkably, erythrocytes pre-lysing significantly outdid all other methods in reducing tube-to-tube variability. This allowed the attainment of highest correlation between CD3+ cells identified by CD3/CD4 and CD3/CD8 antibody combinations and the minimum variability between the sum of the %CD3+CD4+ and %CD3+CD8+, and the total %CD3+. This higher reliability of the pre-lysis method was particularly evident with HIV+ patients in which the lymphocyte gate was often and unpredictably contaminated by debris and other cell types. CONCLUSIONS: The present study demonstrates that lysing erythrocytes in a single tube and distributing aliquots of lysed blood into different tubes for the various antibody combinations provides superior results for routine immunophenotyping.

CD4-Positive T-Lymphocytes↗
PubMed1998

Issues in methodology and applications for therapeutic drug monitoring of fluoxetine and norfluoxetine enantiomers.

A standardization of the analytical procedures for monitoring of fluoxetine and norfluoxetine enantiomers is described. Simultaneous determination of fluoxetine and norfluoxetine enantiomers in plasma and serum was performed by high-performance liquid chromatography with a chiral stationary phase, using ultraviolet absorbance detection. The analytes were extracted from the biologic matrix by alkalinization with NaOH and solid-phase extraction. Stability studies were conducted in EDTA, lithium-heparinized plasma and in serum spiked with the analytes stored at +4 degrees C for 1 week and at -20 degrees C for 1 month. Furthermore, stability studies in NaOH and in the extraction solvents were executed. Using this methodology, EDTA plasma is the most suitable matrix for drug monitoring, even if the storage should not exceed 3 weeks at -20 degrees C. Furthermore, the biologic sample should be left in NaOH for a short time before solid-phase extraction to prevent a degradation of matrix, which would interfere with the chromatographic analysis.

Chromatography, High Pressure Liquid↗
PubMed1998

Lamotrigine hypersensitivity in childhood epilepsy.

PURPOSE: To evaluate the effect of lamotrigine (LTG) on several humoral and cellular immune functions in children with epilepsy and the change in immunological status in patients with LTG-induced rash. METHODS: Sixteen children with epilepsy of unknown origin or secondary to various etiologies undergoing treatment with LTG participated in the humoral and cellular immunological study. Of these, 2 patients developed a rash during LTG treatment and are described in detail. RESULTS: No modifications of humoral or cellular immunity (measured at 1 and 3 months) were noted in 14 of the 16 patients during this treatment. In the 2 children who manifested rash, basal immune function was normal. In both, immediately after the skin rash appeared, there was a high increase in the percentage of activated T-helper lymphocytes (CD4-DR) and activated T-suppressor lymphocytes (CD8-DR), a slight increase in percentage of B lymphocytes (CD19), and a greater increase in serum concentration of IgE. In 1 of the 2 patients, reevaluation of immunity 20 days after the rash appeared and after LTG suspension showed normal percentages of CD4-DR, CD8-DR, and CD19, whereas the serum concentration of IgE had decreased. CONCLUSIONS: The observed immunological results indicate that LTG-induced rash may be considered an immune-mediated hypersensitivity reaction.

Antibody Formation↗
PubMed1998

The effect of zinc and vitamin A supplementation on immune response in an older population.

OBJECTIVE: To determine if either supplemental vitamin A, zinc, or both increases cell-mediated immune response in an older population. DESIGN: A double-blind, randomized, controlled trial of supplementation with vitamin A and zinc. SETTING: Casa Di Riposo Roma III, a public home for older people in Rome, Italy. SUBJECTS: The health and nutritional status of 178 residents were evaluated. One hundred thirty-six residents agreed to participate in the trial and were randomized into four treatment groups, and 118 of these residents completed the trial. INTERVENTION: The four treatments consisted of: (1) Vitamin A (800 micrograms retinol palmitate); (2) Zinc (25 mg as zinc sulfate); (3) Vitamin A and Zinc (800 micrograms retinol palmitate and 25 mg as zinc sulfate); (4) Placebo capsules containing starch. MAIN OUTCOME MEASUREMENTS: Immune tests-counts of leucocytes, lymphocytes, T-cell subsets, and lymphocyte proliferative response to mitogens-were measured before and after supplementation. RESULTS: Zinc increased the number of CD4 + DR + T-cells (P = .016) and cytotoxic T-lymphocytes (P = .005). Subjects treated with vitamin A experienced a reduction in the number of CD3 + T-cells (P = .012) and CD4 + T-cells (P = .012). CONCLUSIONS: These data indicate that zinc supplementation improved cell-mediated immune response, whereas vitamin A had a deleterious effect in this older population. Further research is needed to clarify the clinical significance of these findings.

Aged↗
PubMed1998

Management of postmenopausal osteoporosis for primary care.

OBJECTIVE: The shift in health care delivery from a subspecialty to primary care system has transferred the responsibility of preventing osteoporotic fractures from specialists in metabolic bone disease to the web of physicians--family practitioners, general internists, pediatricians, and gynecologists--who provide the bulk of primary care. The challenge for this group of physicians is to decrease the rising prevalence of osteoporotic hip and vertebral fractures while operating within the cost parameters. It is the goal of this brief summary to provide primary practitioners with focused guidelines for the management of postmenopausal osteoporosis based on new and exciting developments. Prevention and treatment will change rapidly over the next decade and these advances will require changes in these recommendations. DESIGN: We identified patients at risk for osteoporosis and provided indications for bone mass measurement, criteria for diagnosis of osteoporosis, therapeutic interventions, and biochemical markers of the disease. RESULTS: Prevention and treatment are discussed, including hormone replacement therapy and use of calcitonin, sodium fluoride, bisphosphonates, and serum estrogen receptor modulators. CONCLUSIONS: Postmenopausal osteoporosis should no longer be an accepted process of aging. It is both preventable and treatable. Primary care physicians must proactively prevent and treat osteoporosis in their daily practice, and combination therapies are suggested.

Absorptiometry, Photon↗
PubMed1997

Hematopoietic cell phosphatase negatively regulates erythropoietin-induced hemoglobinization in erythroleukemic SKT6 cells.

In an increasing number of hematopoietic cytokine receptor systems (T-cell receptor, B-cell receptor, and macrophage colony-stimulating factor, stem cell factor, interleukin-3, and erythropoietin [EPO] receptors), inhibitory roles for the protein tyrosine phosphatase hematopoietic cell phosphatase (HCP; SHPTP1, PTP1C, and SHP1) have been defined in proliferative signaling. However, evidence exists to suggest that HCP also may exert important effects on blood cell differentiation. To investigate possible roles for HCP during late erythroid differentiation, effects of manipulating HCP expression or recruitment on EPO-induced hemoglobinization in erythroleukemic SKT6 cells have been investigated. No effects of EPO on levels of HCP, Syp, Stat5, the EPO receptor, or GATA-1 expression were observed during induced differentiation. However, the tyrosine phosphorylation of JAK2, the EPO receptor, and Stat5 was efficiently activated, and HCP was observed to associate constitutively with the EPO receptor in this differentiation-specific system. In studies of HCP function, inhibition of HCP expression by antisense oligonucleotides enhanced hemoglobinization, whereas the enforced ectopic expression of wild-type (wt) HCP markedly inhibited EPO-induced globin expression and Stat5 activation. Based on these findings, epidermal growth factor (EGF) receptor/EPO receptor chimeras containing either the wt EPO receptor cytoplasmic domain (EECA) or a derived HCP binding site mutant (EECA-Y429,431F) were expressed in SKT6 cells, and their abilities to mediate differentiation were assayed. Each chimera supported EGF-induced hemoglobinization, but efficiencies for EECA-Y429,431F were enhanced 400% to 500%. Thus, these studies show a novel role for HCP as a negative regulator of EPO-induced erythroid differentiation. In normal erythroid progenitor cells, HCP may act to prevent premature commitment to terminal differentiation. In erythroleukemic SKT6 cells, this action also may enforce mitogenesis.

Animals↗