Extracorporeal photopheresis: a useful therapy for patients with steroid-refractory acute graft-versus-host disease but not for the prevention of the chronic form.

BACKGROUND: Extracorporeal photopheresis (ECP) has been used successfully to treat severe steroid-refractory acute and chronic graft-versus-host disease (aGVHD, cGVHD) since the late 1990s. OBJECTIVES: To evaluate retrospectively the efficacy and safety of ECP in patients with aGVHD. We also assessed whether ECP may play a role in the prevention of cGVHD. PATIENTS AND METHODS: Nine consecutive patients with allografts with aGVHD grade II-III, as defined by consensus criteria, and refractory to steroids, were treated with ECP. ECP was started at a median interval of 46·3 days (range 10-70) from aGVHD onset. Patients were treated initially on two consecutive days (one cycle) at 1-week intervals until improvement and then every 2 weeks. Treatment was then tapered off individually. To evaluate statistical relationships with outcome after 30, 60 and 90 days of ECP, all clinical and historical variables of the patients before treatment were analysed. RESULTS: All patients survived and responded within 90 days. The average aGVHD score was 1·72 at aGVHD onset, 2·44 when ECP was started and then gradually declined to 0·44 on day 90. At the same time, the average dose of methylprednisolone declined from 2·22 mg kg(-1) to 0·27 mg kg(-1) (day 90), while the average dose of ciclosporin declined from 2·46 mg kg(-1) to 0·77 mg kg(-1) (day 90). Six of nine patients showed a complete skin response after 90 days of treatment. All patients with liver and gastrointestinal tract involvement had complete responses after 90 days, apart from one patient. All our patients developed cGVHD, seven of nine while still on maintenance regimen (6-13 months after haematopoietic stem cell transplantation, HSCT) and the other two patients after suspension of ECP (6 and 9 months after HSCT). CONCLUSIONS: ECP is effective in patients with mild to moderate steroid-refractory aGVHD (grade II-III). On the other hand, ECP did not prevent the development of cGVHD in our patients.

Neutral loss and precursor ion scan tandem mass spectrometry for study of activated benzopyrene-DNA adducts.

Methodology for detection of activated benzo[a]pyrene (B[a]P)-nucleoside adducts by liquid chromatography-tandem mass spectrometry is reported. Adducts of B[a]P-dihydrodiol epoxide (B[a]PDE) with guanosine and adenosine have been detected for the first time by use of precursor ion scan and neutral loss scan. B[a]P was then activated by use of UV irradiation and some of the products obtained have been identified by taking advantage of the information obtained for B[a]PDE. Photoactivation has also been carried out in the presence of hydrogen peroxide; this resulted in a higher yield of products with increased production of BaP diones. The reactivity of these compounds toward nucleosides has been tested. The proposed method was successfully used for detection of one stable guanosine-B[a]P dione adduct.

Multiclass analysis of illicit drugs in plasma and oral fluids by LC-MS/MS.

An analytical procedure for the simultaneous determination in human plasma and oral fluids of several illicit drugs belonging to different chemical and toxicological classes is presented. Amphetamine, methamphetamine, morphine, 6-monoacetylmorphine, methylenedioxyamphetamine, methylenedioxyethylamphetamine, methylenedioxymethamphetamine, cocaine, benzoylecgonine, tetrahydrocannabinol, carboxytetrahydrocannabinol, ketamine, and phencyclidine have been quantified in real samples using a very rapid sample treatment, basically a protein precipitation. The quantitative analysis was performed by liquid chromatography-tandem mass spectrometry and has been fully validated. All the analytes were detected in positive ionization mode using a TurboIonSpray source, except carboxytetrahydrocannabinol, which was detected in negative ionization mode. The use of a diverter valve between the column and the mass spectrometer allows the preservation of the ion source performances for high-throughput analysis.

CD4+CD25+ lymphocyte subsets in chronic graft versus host disease patients undergoing extracorporeal photochemotherapy.

Extracorporeal photochemotherapy (ECP) has been used successfully for the treatment of chronic Graft versus Host Disease (cGvHD). However, the mechanism by which ECP exerts its protective effects remains elusive. Some recent observations have suggested a possible role of certain subsets of T lymphocytes with immunosuppressive properties (T-regulatory cells) that coexpress CD4 and high levels of the interleukin-2 receptor chain: CD4+CD25+ T lymphocytes. We studied whether ECP affects the percentage of these cells in the peripheral blood of patients with cGvHD. The study population consisted of 14 patients with cGvHD refractory to systemic steroids. On enrollment in each cycle of ECP, patients underwent clinical examination, blood chemistry analysis and other instrumental procedures to document and assess involvement of the various organs and systems. For cytofluorimetric identification and phenotyping of CD4+CD25+ T lymphocytes, peripheral blood samples were collected in EDTA anticoagulant before ECP, after 48 hours, and after 6 and 12 months from the start of treatment. The 14 patients in this study received a total of more than 300 cycles of ECP, with only minor side effects. The clinical outcome was negative in 2 patients and positive in 12 patients. Within subject analysis indicated that the percentage of CD4+CD25+ T lymphocytes before ECP and after 12 months of treatment was significantly increased. Our study confirms that changes in the percentage of CD4+CD25+ T cells induced by ECP could be a central aspect in the cascade of immune events leading to the immunological and clinical effects of this treatment in patients with cGvHD.

Ocular manifestations of chronic graft-versus-host disease in patients treated with extracorporeal photochemotherapy.

PURPOSE: Eye involvement has long been appreciated in patients with chronic graft versus host disease (cGVHD). In particular, ocular complications are frequent and can be potentially severe in patients with steroid-refractory cGVHD, and therefore necessitate close monitoring. This prospective study was designed to describe eye manifestations of cGVHD in a large series of patients monitoring them before and after 1 year of extracorporeal photochemotherapy (ECP). ECP is a relatively new therapeutic approach based on the biological effects of psoralen 8-methoxypsoralen (8-MOP) and ultraviolet A light (UVA) on mononuclear cells collected by apheresis, and reinfused into the patient. METHODS: Only patients with steroid-refractory cGVHD under treatment with ECP, who developed cGVHD-related eye symptoms, were selected for the study. Ophthalmologic examination was repeated every 3 months. Only patients with complete recovery of the ocular manifestations and symptoms were considered responsive. RESULTS: In our study we observed eye alterations in 24 out of 140 patients (17%) with cGVHD. After 12 months of ECP, 10 out of 21 patients (48%) completely responded to the therapy. In all these cases the contribution of ECP was also essential in all the other organs subject to cGVHD. CONCLUSIONS: Further studies are necessary to clarify the role of ECP in patients with cGVHD, especially in associated eye manifestations. Although our experience is limited, it suggests that ECP could be a safe and effective therapy for steroid-refractory eye manifestations of cGVHD.

Inhibition of maturation of human monocyte-derived dendritic cells in a patient with Mycobacterium avium infection.

Mycobacterium avium complex is a facultative intracellular pathogen that can cause pulmonary disease in immunocompromised individuals. Dendritic cells (DCs) play a central role in protective immunity against mycobacteria. Mycobacterium avium complex infects DCs but does not impair in vitro infected monocytes differentiation into DCs. A 54-year old woman affected by chronic graft-versus-host-disease (cGVHD) was referred to our Division of Dermatology. Immature DCs were generated from her monocytes. One week later she was hospitalized due to a lung infection with Mycobacterium avium complex. Monocyte-derived DCs during Mycobacterium avium infection expressed low levels of CD1a and CD80 as determined by flow cytometry. They also expressed high levels of CD83 and CD86, and when stimulated with LPS for 24 hrs they slightly up-regulated CD83 and did not produce IL12. When monocyte-derived DCs were obtained from the patient after having recovered from the Mycobacterium avium complex infection, they expressed normal levels of CD1a and CD80 and were negative both for CD83 and for CD86. IL12 production in response to LPS was restored. Inhibition of DC maturation by the in vivo infection with Mycobacterium avium may be an immune-evasion mechanism used by the pathogen because incompletely matured DCs may not activate effector T cells efficiently in vivo.

Development of a method based on liquid chromatography-electrospray mass spectrometry for analyzing imidazolinone herbicides in environmental water at part-per-trillion levels.

An evaluation was made of the feasibility of using reversed-phase liquid chromatography-mass spectrometry with an electrospray interface (LC-ESI-MS) to measure traces of imidazolinone herbicides in different natural water samples. The imidazolinones are a significant new class of low-use-rate, reduced-environmental-risk herbicides for the protection of a wide variety of agricultural commodities. The procedure used involved passing 0.5, 1, 2 1 of river, ground and drinking water samples, respectively, through a 0.5 g graphitized carbon black (GCB) extraction cartridge. Analytes were eluted from the GCB surface by 8 ml of a methylene chloride-methanol (80:20, v/v) solution acidified with formic acid, 25 mM. Recovery was higher than 89% irrespective of the aqueous matrix in which the analytes were dissolved. A conventional 4.6 mm I.D. reversed-phase LC C18 column operating with a mobile phase flow-rate of 1 ml/min was used to chromatograph the analytes. A flow of 50 microliters/min of the column effluent was diverted to the ESI source. The effects of acid concentration on ESI-MS detector response in the mobile phase were investigated. The effects on the production of diagnostic fragments produced by varying the orifice plate voltage and the response of the MS detector were also evaluated. For the analyte considered, the response of the mass detector was linearly related to the amount of analyte injected between 1 and 50 ng. The limit of detection (signal-to-noise ratio = 3) of the method for the pesticides considered in drinking water samples was estimated to be about 2-5 ng/l.

Determination of arylphenoxypropionic herbicides in water by liquid chromatography-electrospray mass spectrometry.

A very sensitive and specific analytical procedure for determining arylphenoxypropionic herbicides in aqueous environmental samples, using pneumatically assisted electrospray (ESI) liquid chromatography-mass spectrometry (LC-MS) is presented. Arylphenoxypropionic acids are a new class of herbicides used for the selective removal of most grass species from any nongrass crop. These herbicides are commercialized as herbicide esters. It has been shown that the ester derivatives undergo fast hydrolysis in the presence of vegetable tissues and soil bacteria, yielding the corresponding free acid. The analytical procedure involves passing 1l of surface or ground water and 2l of drinking-water samples, through a 0.5-g graphitized carbon black (GCB) extraction cartridge. A conventional 4.6-mm I.D. reversed-phase LC C18, operating with a 1 ml/min mobile phase flow-rate, was used for chromatographing the analytes. A flow of 200 microliters/min of the column effluent was diverted to the ESI source. The ESI source was operated in positive-ion mode for neutral pesticides and in negative-ion mode for acid pesticides. For ion-signal optimization, the effect of the concentration of the acid in the mobile phase on the response of the ESI-MS detector was investigated. By evaluating the specificity and sensitivity of the method, the effects of varying the orifice plate voltage on the production of the diagnostic fragment and the response of the MS detector were also investigated. For the analyte considered, the response of the mass detector was linearly related to the amount of the analyte injected between 1 and 200 ng. In all cases, recoveries of the analytes were better than 91%. The limit of detection (signal-to-noise ratio = 3) of the method for the pesticides considered in drinking water samples was estimated to be about 3-10 ng/l.

Head-space chromatography in determination of enzymatic activity: microdetermination of the urinary kallikrein as arginine esterases.

Many enzymatic reactions yield volatile products either directly or by cascade sequences, so it seems possible that head-space chromatography might be used to determine enzymatic activity. The activity of urinary kallikrein, as arginine esterase, has been determined in this way by using the N(alpha)-acetyl-L-phenylalanyl-L-arginine ethyl ester as substrate and measuring the ethanol yielded on incubation for 10 min at 30 degrees, followed by quenching of the reaction. The method has been applied to aqueous solutions and urine.

Potentiometric determination of bile phosphates using a lead selective electrode.

A potentiometric method based on phosphate precipitation with Pb2+ and on a lead ion selective electrode is applied to the determination of bile phosphates. The method is rapid and simple and does not require extensive sample pretreatment.

Total phosphorus determination in human bile. Comparison between two spectrometric methods.

The two most commonly used spectrometric methods for the determination of the phosphorus content of human bile are compared. The optimum experimental conditions are studied, and the analytical characteristics of the two methods, using both standard samples and human bile, are evaluated. The methods are compared on the basis of their sensitivity, precision and accuracy, and the correlation between the two techniques demonstrated using fifteen samples of human bile. Data obtained by both methods have been used to calculate lithogenic index values.

High-performance liquid chromatographic analysis of norfloxacin in human tissues and plasma with fluorescence detection.

A high-performance liquid chromatographic analysis with fluorimetric detection is described for the quantitative determination of norfloxacin in renal, prostatic tissues and in plasma. The analytical procedure in the tissue pretreatment, consists of purification of the obtained sample by a solid state extraction and quantitation by HPLC. The samples were chromatographed on a C(8) reversed-phase column. Analytical recoveries ranged from 95.2 to 97.6%. Within and between day precision were assessed by analysing serum containing 50 and 500 ng/ml norfloxacin. At each concentration, within day precision was < or = 3.6% (relative standard deviation, n = 10) and day-to-day precision was < or = 5.3% (n = 10). Limit of detection was ca 1 ng/ml.

Fate of natural estrogen conjugates in municipal sewage transport and treatment facilities.

The aim of this study was to investigate the fate of the conjugated forms of the three most common natural estrogens in the municipal aqueous environment. Levels of conjugated and free estrogens in (1) female urine; (2) a septic tank collecting domestic wastewater; (3) influents and effluents of six activated sludge sewage treatment plants (STPs) were measured. The analytical method was based on solid-phase extraction by using a Carbograph 4 cartridge and Liquid Chromatography-tandem Mass Spectrometry. On average, a group of 73 women selected to represent a typical cross section of the female inhabitants of a Roman condominium, excreted 106, 14 and 32 microg/day of conjugated estriol (E(3)), estradiol (E(2)) and estrone (E(1)), respectively. Apart from some E(3) in pregnancy urine, free estrogens were never detected in urine samples. Estrogen sulfates represented 21% of the total conjugated estrogens. This situation changed markedly in the condominium collecting tank. Here, significant amounts of free estrogens were observed and the estrogen sulfate to estrogen glucuronated ratio rose to 55/45. A laboratory biodegradation test confirmed that glucuronated estrogens are readily deconjugated in unmodified domestic wastewater, presumably due to the large amounts of the beta-glucuronidase enzyme produced by fecal bacteria (Escherichia coli). Deconjugation continued in sewer transit. At the STP entrance, free estrogens and sulfated estrogens were the dominant species. The sewage treatment completely removed residues of estrogen glucuronates and with good efficiency (84-97%) the other analytes, but not E(1) (61%) and estrone-3-sulfate (E(1)-3S) (64%). Considering that (1) E(1) has half the estrogenic potency of E(2), (2) the amount of the former species discharged from STPs into the receiving water was more than ten times larger than the latter one and (3) a certain fraction of E(1)-3S could be converted to E(1) in the aquatic environment, E(1) appears to be the most important natural endocrine disrupter.

Simultaneous determination of heroin 6-monoacetylmorphine, morphine, and its glucuronides by liquid chromatography--atmospheric pressure ionspray-mass spectrometry.

A new analytical technique has been developed for the simultaneous determination of heroin, 6-monoacetylmorphine, morphine, morphine-6- and 3-glucuronides, and codeine in serum using liquid chromatography coupled with ionspray mass spectrometry. The analytes and the internal standard, nalorphine, were subjected to solid-phase extraction (SPE) using ethyl SPE columns before chromatography. The chromatographic separation of the analytes was achieved using a normal phase column and a water-methanol-acetonitrile-formic acid mobile phase at a flow rate of 230 microL/min. The mass spectrometer was operated in selected-ion monitoring mode. Under these conditions, the limit of quantitation was 0.5 ng/ml for heroin, 4 ng/ml for 6-monoacetylmorphine, 4 ng/ml for morphine, 1 ng/ml for morphine-3-glucuronide, 4 ng/ml for morphine-6-glucuronide, and 4 ng/mL for codeine. Serum levels of heroin metabolites were determined in C57BL/6 inbred mice after a dose of 20 mg/kg heroin administered subcutaneously. 6-monoacetylmorphine showed a peak concentration of 0.93 micrograms/mL serum at 3 min, whereas morphine and morphine-3-glucuronide achieved their peak concentrations of 9.6 and 2.9 micrograms/mL serum at 10 and 20 min, respectively. Finally, the absence of morphine-6-glucuronide and codeine excluded the possibility of their formation from morphine in this animal model.

Effect of water of Anticolana Valley on urinary sediment of renal stone formers.

An investigation was carried out to ascertain the effect of drinking Fiuggi water on the microcrystalline structure of the calcium oxalate monohydrate present in urinary sediments provided from patients suffering from recurrent idiopathic oxalic calculosis. The experimental group was administered tap and Fiuggi water for ten days. The control group was administered tap and Fiuggi water according to the same procedure as for the experimental group. The comparative data show that drinking Fiuggi water leads to a strong reduction, and sometimes even to the elimination, of the calcium oxalate monohydrate present in the urinary sediment reducing the risk of oxalic calculosis. Fiuggi water contains organic molecules belonging to the fulvic acid family. These acids are capable of complexing the calcium ions and interact preferentially with the crystal lattice of the calcium oxalate monohydrate via the formation of a film and behave as pumping systems by linking the calcium ion, demolishing the crystal lattice and dissolving calcium and oxalate ions. Mineral water treatments must therefore be viewed as a function of the specific composition of the water administered. The ecosystem influences the composition of water, as a complex matrix containing a number of organic molecules which are potentially biologically active.