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.

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.

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.

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.

Role of extracorporeal photochemotherapy in patients with refractory chronic graft-versus-host disease.

Recent studies suggest that extracorporeal photochemotherapy (ECP) may be beneficial in patients with steroid-refractory chronic graft-versus-host disease (cGvHD). However, it is not yet clear whether certain conditions, such as age, mode of onset of cGvHD etc., influence clinical response and whether certain affected organs are more sensitive to ECP than others. We analysed the main clinical and laboratory parameters related to evolution of the disease in 32 steroid-refractory cGvHD patients, to identify any useful response predictors to ECP. ECP affected the course of the disease positively in 78% (25/32) of our cases.

Extracorporeal photochemotherapy in the treatment of eosinophilic fasciitis.

BACKGROUND: Eosinophilic fasciitis (EF) is a rare connective tissue disorder characterized clinically by symmetrical swelling, induration and thickening of the skin and histologically by thickening of the fascia with chronic inflammatory infiltrate containing eosinophils. The disease is classified in the spectrum morphea/systemic sclerosis and treated with systemic steroids and other immunosuppressant drugs. OBJECTIVE: The purpose of this study was to use extracorporeal photochemotherapy (ECP) in patients with EF to evaluate the effectiveness of this therapy. SUBJECTS AND METHODS: Three patients affected by EF were treated with ECP because they failed to respond or with contraindications to immunosuppressant treatment. The patients underwent ECP with a UVAR XTS apparatus. Subjects were treated on two consecutive days at 2-week intervals for the first 3 months and thereafter every 4 weeks on the basis of clinical response. The patients were assessed before therapy and then monthly by means of a clinical score. Changes in affected areas were evaluated at predetermined points by computerized skin elastometry (Cutometer SEM 474). RESULT: After 1 year of therapy we found considerable improvement of clinical parameters in two cases. There was less striking improvement in the other case. These clinical results were confirmed by the elastometry measurements. All patients reported improved quality of life, which enabled a reduction in the dose of immunosuppressants. CONCLUSION: ECP emerged as a safe and effective therapy in association with low doses of immunosuppressants in our three patients. A randomized comparative multicentre study between ECP as single therapy and ECP plus immunosuppressants and conventional therapies is required to firmly establish photopheresis as a possible basic treatment to combine with conventional therapies for EF.

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.

Food analyses: a new calorimetric method for ascorbic acid (vitamin C) determination.

The analysis of the ascorbic acid (vitamin C; A.A.) contained in some foodstuff and pharmaceutical samples was performed by a new microcalorimetric method. It uses the oxidation of the vitamin C catalysed by the enzyme ascorbate oxidase (A.O.), which gets the specificity of the reaction. The calibration curve was built under the following operative conditions: 25.00 degrees C, pH 5.6, [A.O.]=11 IU ml(-1), the linearity range is: 3

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.

Chemical analysis of water of the Anticolana Valley: isolation of humic compounds.

The purpose of this study was to identify and characterize humic compounds in Anticolana Valley (Fiuggi) water. The capacity of this class of compounds to mobilize metals from solid phases could have an important role in calculi solubilization. Humic compounds were isolated, purified and characterised by FTIR, 1H-NMR and 13C-NMR spectroscopy, thermogravimetry and elemental analysis. Only fulvic acids were found. They are mainly composed of aliphatic chains, made of six -CH2O- groups and contain a number of carboxylic groups, responsible for their metal complexing capacity.

Solvent effect in vitro of Anticolana Valley water on renal stones: analytical-instrumental study.

The presence of humic and fulvic acids in the Anticolana Valley (Fiuggi) water has been established. On the basis of this evidence we investigated the capacity of Anticolana Valley (Fiuggi) water to dissolve renal calculi in vitro. Crystals of calcium oxalate monohydrate to simulate a kidney stone were prepared. Human renal stones of calcium oxalate monohydrate were obtained by courtesy of the Division of Urology of 'La Sapienza' University (Rome), the Division of Urology of the University of Havana (Cuba) and the ASTIF of Fiuggi. The study was performed using the Anticolana Valley (Fiuggi) water, distilled water and tap water (ACEA, Rome), in a specially designed Perspex apparatus. Each calculus was subjected to a water flow of 2 liters/24 h. The capacity of the Anticolana Valley (Fiuggi) water to dissolve human and synthetic calculi was found to be much higher than that of distilled water which in turn was significantly more effective than tap (ACEA) water.

Multiresidue herbicide analysis in soil: subcritical water extraction with an on-line sorbent trap.

We evaluated the feasibility of extracting selectively and rapidly herbicide residues in soils by hot water and collecting analytes with a Carbograph 4 solid-phase extraction (SPE) cartridge set on-line with the extraction cell. Phenoxy acid herbicides and those nonacidic and acidic herbicides which are often used in combination with phenoxy acids were selected for this study. Five different soil samples were fortified with target compounds at levels of 100 and 10 ng/g (30 ng/g of clopyralid and picloram) by following a procedure able to mimic weathered soils. Herbicides were extracted with water at 90 °C and collected on-line by the SPE cartridge. After the cartridge was disconnected from the extraction apparatus, analytes were recovered by stepwise elution to separate nonacidic herbicides from acidic ones. The two final extracts were analyzed by liquid chromatography/mass spectrometry with an electrospray ion source. At the lowest spike level considered, analyte recoveries ranged between 81 and 93%, except those for 2,4-DB and MCPB, which were 63%. For 16 herbicides out of 18, the ANOVA test showed recoveries were not dependent on the type of soil. The method detection limit was in the 1.7-10 ng/g range. For the analytes considered, method comparison showed this extraction method was overall more efficient than Soxhlet and sonication extraction techniques.

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.