In vitro studies of the dual properties of Allopurinol anti- and photo-oxidants Mechanisms / Estudio in vitro de dos propiedades del Alopurinol.

The objective of this study was to investigate the ability of allopurinol (1) to inhibit free radical or reactive oxygen species (.OH, ¹O2, H2O2) as well as the study of its photochemical activity. We investigated the ability of 1 to scavenge oxygen metabolites generated by cell-free systems using luminol enhanced-chemiluminescence and electronic absorption spectra as monitors. Both absorbance and fluorescence scans reveal that 1 is able to react with equimolar quantities of H2O2. In the presence of 1 a dose-dependent inhibition period was observed in this system as assayed by isoluminol-enhanced chemiluminescence (ILCL) in the presence of horseradish peroxidase (HRP), as well as by luminol-enhanced chemiluminescence (LCL) in the presence of H2O2 or ferrous ion. On the other hand, 1 did not show an efficient scavenging activity of galvanoxyl radical in ethanolic solutions. Furthermore, in a separate experiment, it was not observed trapping of singlet oxygen (¹O2) generated by Rose Bengal, in the presence of 1. The activity of 1 was compared with that of vitamins E and C. In vitro experiments of photohemolysis in presence of 1 and cinoxacin, a phototoxic antibacterial quinolone, the photohemolytic effect of cinoxacin was diminished. Allopurinol alone did not show any phototoxic effect under irradiation with UV-A or visible light but was photo-unstable and phototoxic in vitro with UV-B light.

Se estudió la habilidad del alopurinol (1) para inhibir radicales libres o especies reactivas de oxigeno (.OH, ¹O2, H2O2), igualmente se determinó su actividad fotoquímica. De otro lado se midió la habilidad de 1 para eliminar los metabolitos de oxígeno generados por un sistema libre de células basado en quimioluminicencia aumentada de luminol y se monitoreo el espectro de absorción electrónica. Las dos determinaciones, absorbancia y fluorescencia, revelan que 1 es capaz de reaccionar con cantidades equimoleculares de H2O2. En presencia de alopurinol se observan periodos de inhibición dosis dependiente al usar isoluminol como intensificador de luminiscencia (ILCL) en presencia de peroxidasa de rábano o ión ferroso. Por otro lado, 1 no mostró una eficiente actividad frente a radicales galvanoxil en solución etanólica. En otros experimentos en presencia de 1 no se observó bloqueo de especies de oxígeno singlete (¹O2) generado por rosa bengala. La habilidad de 1 fue comparada con la de vitaminas E y C. En experimentos de fotohemólisis in vitro en presencia de 1 y cinoxacin, quinolona fototóxico antibanterial, el efecto fotohemolítico del cinoxacin fue disminuido. El alopurinol no mostró efecto fototóxico por irradiación con luz UV-A o luz visible, sin embargo se mostró foto - inestable y fototóxico in vitro bajo irradiación con luz UV-B.

Photodegradation and in vitro phototoxicity of aceclofenac.

Aceclofenac (Airtal) (1) is a photoallergic and phototoxic anti-inflammatory and analgesic agent. This drug is photolabile under aerobic and anaerobic conditions. Irradiation of an ethanol-solution of aceclofenac under oxygen or argon at 290-320 nm affords via decarboxlation compound 2 as the main isolated and spectroscopically identified photoproduct, besides hydroxylamine derivates 3 and 4. A radical intermediate was evidenced spectrophotometrically with GSH and DTNB, as well as by the dimerization of cysteine. Red blood cell lysis photosensitized by 1-4 was investigated. Furthermore, in a lipid-photoperoxidation test with linoleic acid the in vitro phototoxicity of aceclofenac was also verified. The photoinduced generation of peroxide by compound 1 was determined during the irradiation in presence of NADPH by chemiluminescence. In relation to the photoallergic activity of this drug, the interaction of aceclofenac with human serum albumin (HSA) has been studied through fluorescence spectroscopy. No photoinduced binding was observed after irradiation of compounds 1 in the presence of human serum albumin.

[Effect of anti-retroviral therapy on body composition changes: a literature review]. / Variazioni della composizione corporea associate alla terapia antiretrovirale: una rassegna della letteratura.

Protein-energetic malnutrition, characterized by both lean mass and fat depletion, was common in the pre-HAART era, and was associated with shortened survival and diminished quality of life. The pathogenesis of protein-energy malnutrition was multifactorial, and nutritional treatments were largely ineffective in the absence of disease stabilization. The introduction of HAART brought markedly improved outcomes, including a decrease in the incidence of malnutrition. However, other nutritional and metabolic alterations were noticed, and included changes in body shape, both lipoatrophy and lipohypertrophy, as well as changes in metabolism, notably hyperlipidemia and insulin resistance. These conditions, though sometimes occurring together, may occur independently, suggesting a complex, multifactorial cause. Several mechanisms have been hypothesized, including impairment to adipocyte differentiation and adipokine regulation, production of proinflammatory cytokines and mitochondrial toxicity. The role of the single drug class is still unclear, because both PI and NRTI have been associated with the syndrome, and the therapeutic protocols include both groups. Most of the medical therapies proposed for lipodystrophy are ineffective, and even if surgery remains an alternative, it is not associated with long lasting outcomes.

Laparoscopic tubal anastomosis.

OBJECTIVES: To evaluate the pregnancy outcome after laparoscopic tubal anastomosis. METHODS: From December 1998 to December 2001, 26 patients with bilateral tubal ligation who underwent laparoscopic tubal anastomosis were prospectively evaluated. Patients' age varied from 28 to 37 years. RESULTS: Laparoscopic tubal reversal was performed in 23 patients. Bilateral reversal was possible in all but two patients. The operation time ranged from 95 to 155 min and all patients were discharged in the following morning after surgery. After 3 months, tubal patency was confirmed in 15 patients (15/23). Pregnancy rate was 56.5% (13/23), without ectopic pregnancies. The average time from tubal reversal and pregnancy was 6 months. CONCLUSIONS: In selected cases, laparoscopic tubal reversal can be offered to patients who had been submitted to tubal sterilization and desire new pregnancies. Patient selection as well as meticulous surgical technique are key factors in achieving satisfactory pregnancy rates.

[Genotoxic effects of pesticide-treated vegetable extracts using the Allium cepa chromosome aberration and micronucleus tests]. / Valutazione della genotossicità di frutta e verdura contaminata da pesticidi mediante il test dell'Allium cepa.

The presence of chemical residues in vegetables and fruit is a source of human exposure to toxic and genotoxic chemicals. The mutagenic and carcinogenic action of herbicides, insecticides and fungicides on experimental animals is already known. Several studies have shown that chronic exposure to low levels of pesticides can cause adverse health effects and that many pesticides are mutagenic/carcinogenic. In the present research we monitored concurrently the presence of pesticides and genotoxic compounds extracted from 21 treated vegetables and 8 types of grapes sampled from the markets of a region in Southern Italy. The extracts were analysed for pesticides by gas-chromatography and HPLC, and for genotoxicity with two plant tests in Allium cepa roots: the micronucleus test and the chromosomal aberration test. We found 33 pesticides, some of which are outlawed. Genotoxicity was found in some of the vegetables and grapes tested. Allium cepa tests were sensitive for monitoring genotoxicity in food extracts. The micronucleus test in interphase cells gave much higher mutagenicity than the chromosomal aberration test in anaphase-telophase cells.

Seroprevalence of chronic Chlamydia pneumoniae infection in patients affected by chronic stable asthma.

OBJECTIVE: To evaluate the seroprevalence of Chlamydia pneumoniae and age, gender and smoking habits in stable asthmatic patients. METHODS: Over a period of 3 months, 197 adult patients affected by intermittent-to-severe chronic asthma were enrolled from 16 respiratory disease units in the south of Italy. As a control group, we tested 185 healthy, non-asthmatic subjects matched for age and gender, recruited among hospital staff. All patients were submitted to clinical examination, spirometry and blood collection for C. pneumoniae serology. The presence of infection was investigated by microimmunofluorescence (Micro-IF Test) for C. pneumoniae-specific IgG, IgM and IgA antibodies. RESULTS: C. pneumoniae IgG titers > or =1 : 64 were detected in 30.4% of asthmatics and in 30.8% of controls. Correlation of age, gender and smoking habit with C. pneumoniae seropositivity was evaluated by linear regression analysis. Age was significantly associated with C. pneumoniae IgG titer > or =1 : 64 when seropositive asthmatics were tested. Moreover, C. pneumoniae seroprevalence was higher among smokers with a diagnosis of chronic asthma. CONCLUSIONS: The seroprevalence of C. pneumoniae in stable asthmatics was comparable with the controls; therefore, the study does not support the association between C. pneumoniae antibody titers and stable asthma. However, the analysis for likely confounders such as age, gender and smoking status suggests a possible association of enhanced susceptibility to C. pneumoniae infection with age and smoking habitus.

[Experience at a branch center of the National HIV Protease Inhibitor Registry]. / L'esperienza di un centro periferico sul registro nazionale degli inibitori delle proteasi di HIV.

In the present study we show our experience as one of the Centers (center 006) of the Italian Index of Protease Inhibitors (PI). In this multicentric, observational study we studied HIV-positive naive patients for antiretroviral therapy with protease inhibitors. From 8/1/97 to 31/1/98 in our department 64 patients were enrolled and followed up through an electronic medical sheet in order to evaluate efficacy, tolerability, toxicity and compliance of treatment with PI. Our results show a reduction in AIDS-related mortality and a decrease in AIDS defining diseases: in particular data show an increase in CNS pathologies in comparison with other opportunistic events. Moreover, in patients non complaints to therapy, the progression of the disease was more frequent. The side effects more frequently observed during PI treatment were hypertrigliceridaemia and lipodystrophy; gynaecomastia and hyperprolactinaemia were even present, perhaps as expression of direct metabolic action of HAART therapy.

Clinical features of women with resistance to luteinizing hormone.

OBJECTIVE: To review clinical and hormonal characteristics of new and published females with LH resistance. PATIENTS: Seven sisters of patients with male pseudohermaphroditism due to LH resistance. MEASUREMENTS: Clinical characteristics, hormonal levels, pelvic ultrasound and molecular studies. RESULTS: Patients had: (1) normal female external genitalia; (2) spontaneous breast and pubic hair development at ages 9-13 years; (3) menarche at 12-20 years, followed by irregular menstrual cycles (3 weeks to 15 months); (4) infertility; (5) withdrawal bleeding after progesterone administration; (6) elevated serum LH levels (10-38 IU/l, normal 0. 9-8.4) and elevated LH/FSH ratio with normal androgen levels; (7) low or normal oestradiol levels for the follicular phase; and (8) normal or small uterus and normal or increased ovary size with one or more cysts at ultrasound examination. Ovarian biopsy showed antral follicles and confirmed lack of ovulation. One homozygous mutation in exon 11 of the LH receptor gene was found in each of 4 patients: 1 nonsense (Arg554Stop) and 2 missense mutations (Glu354Lys and Ala593Pro) and 1 microdeletion (Leu-608, Val-609) were found. In 3 patients (1 family) with the same phenotypic characteristics, no mutations in the coding sequence of the LH receptor gene were found. CONCLUSIONS: We conclude that women with LH resistance have spontaneous breast development, primary or secondary amenorrhoea, infertility, elevated serum LH levels and LH/FSH ratio with normal androgen levels and normal or enlarged cystic ovaries. Therefore, in females, primary and secondary sexual characteristics develop independently of LH action. However, LH stimulation is necessary for normal ovarian steroidogenesis and ovulation.

[Tumors in newborn infants]. / Tumores en recién nacidos.

Fifty one cases of tumors detected along a 20 years period (1969 throughout 1989) in newborn infants are described. Most frequent kinds of neonatal tumors were teratomas (n: 30), followed by vascular tumors (n: 6), neuroblastomas IV-S (n: 5), hepatic hamartomas (n: 5), renal tumors (n: 3), soft tissue sarcomas (n: 2) and melanocytic melanoma (n: 1). Follow up was extended from 1 to 20 years. Death occurred in two patients of this series: one in a case of sacrococcygeal teratoma, who died of septicemia secondary to urinary tract obstruction and infection before any attempt of surgical treatment was possible, and by multiple pulmonary metastases one year after apparently satisfactory surgical treatment in another patient with neuroblastoma.

Effect of immunosuppressive therapy on HBsAg-positive chronic active hepatitis in relation to presence or absence of HBeAg and anti-HBe.

Two hundred and four patients with biopsy-proven HBsAg-positive chronic active hepatitis were treated for at least 2 years with azathioprine, prednisolone or a combination of both, or were untreated. Prevalences of HBeAg, anti-HBe, and anti-HBc were 26, 58, and 100%, respectively. Criteria for improvement or progression of disease were based on clinical, biochemical, and morphologic parameters. Prednisolone therapy and combination therapy did not modify the course of the disease in HBeAg-positive patients; azathioprine therapy may have been deleterious for these patients. Among HBeAg-negative patients, deterioration or death occurred more frequently in those who were untreated than in those who received the combination of azathioprine and prednisolone. Prednisolone therapy was of moderate effect, whereas azathioprine did not influence the outcome.

Treatment of HBsAg-positive chronic active hepatitis with corticosteroids and/or azathioprine. A prospective study.

204 patients with biopsy-proven CAH were observed for at least one year with periodic examinations including a second liver biopsy for each patient. Patients were assigned at random to a group left untreated or treated with either azathioprine 100 mg daily, or prednisolone (Dutimelan 8 15), or prednisolone plus azathioprine 50 mg daily. The outcome, assessed on clinical, biochemical and histological parameters, was correlated to the presence or absence of HBeAg and its antibody. No treatment was effective in HBeAg-positive patients whereas, among anti-HBe-positive patients, those who were treated with the association of steroids and azathioprine more frequently showed improvement and less frequently deteriorated (p less than 0.01) than untreated patients. Thus, HBsAg/anti-HBe-positive patients with CAH may be treated with the combination of these drugs, even though many side-effects may occur and there are frequent relapses after discontinuation of treatment.

Infantile embryonal carcinoma of testis.

Between 1968 and 1979 we treated 19 patients with infantile embryonal carcinoma of the testis. All 10 patients treated with orchiectomy plus chemotherapy (methotrexate, actinomycin D and cyclophosphamide) are well with no evidence of disease, whereas 2 of 4 who underwent orchiectomy alone and 2 of 5 treated with orchiectomy plus lymphadenectomy are dead. Infantile tumors with yolk sac elements have an apparent increase in hematogenous spread and, despite negative lymph nodes in the majority, a significant number have subsequent disseminated metastases. This suggests that infantile embryonal carcinoma is different from the adult type and implies that in clinical stage A disease retroperitoneal lymphadenectomy is unnecessary. The alpha-fetoprotein correlates with the presence of yolk sac tissue in biopsy and the radioimmunoassay level assists in monitoring the treatment.

Serum levels of hepatitis B surface and core antigens during immunosuppressive treatment of HBsAg-positive chronic active hepatitis.

One of the following treatments was randomly assigned to 101 consecutive patients with biopsy-proven chronic active hepatitis: prednisone 20 mg daily, azathioprine 100 mg daily, prednisone 20 mg and azathioprine 50 mg daily, or B vitamins 2 tablets daily (control group). Patients were observed at the beginning of the study, then at 2, 6, and 12 months after treatment. At each visit levels of HBsAg and Dane-particle-associated core-antigen (HBcAg) and their corresponding antibodies were determined. 42 patients were HBcAg positive at the beginning of the study. Of these 42 patients, the 34 who were under treatment remained HBcAg positive and showed a rise in HBcAg titre, while the 8 who were not treated had a fall in HBcAg titre (6 patients) or became HBcAg negative (2 patients) in 6 months. Among 59 patients who were HBcAg negative at the beginning of the study, this antigen became persistently detectable in 40% of the 42 patients who were treated, and was transiently present in 2 (12%) out of the 17 untreated patients (p < 0.05). Our data indicate that long-term prednisone and/or azathioprine treatments favour the replication of hepatitis-B virus in patients with HBsAg-positive chronic active hepatitis.

Metabolic impairment and membrane abnormality in red cells from Huntington's disease.

The following aspects have been investigated in 10 patients affected by Huntington's disease )HD): --extensive haematological investigations; --red cell enzyme activities and level of the most important glycolytic intermediate compounds; --protein, lipid and carbohydrate composition of the erythrocyte membrane and membrane polarity; --effects of in vitro aging on red cell membranes. Lack of 4.5 protein band in SDS-PAGE and 14-fold decrease in membrane-bound catalase were found in the in vitro aged red cells from the 10 HD patients examined. Na+ + K+ATPase was slightly higher than normal in all the patients. Red cells from 5 out of 8 patients showed a decrease in reduced glutathione and phosphoenolpyruvate levels and/or an increase in hexokinase, glucose-6-phosphate dehydrogenase, pyruvate kinase and glutathione reductase activities. The haematological investigations, the protein lipid and carbohydrate composition of the fresh red cells, the membrane polypeptide aggregates and the membrane polarity evaluated by microspectrofluorometric analysis were normal.

The first stable variant of erythrocyte glucose-phosphate isomerase associated with severe hemolytic anemia.

A new variant of glucosephosphate isomerase (GPI) associated with hemolytic anemia, mental retardation, and muscular hypotonia is described. The defective enzyme showed increased affinity for fructose-6-phosphate (F-6-P), decreased affinity for glucose-6-phosphate (G-6-P) altered electrophoretic and isoelectrofocusing patterns, and shift to the left of the precipitin curve. The enzyme was stable under all the conditions tested (heat, urea, guanidine-HCl, and storage). Optimum pH, Ki for 6-phosphogluconic acid (6-PGA) and for erythrose-4-phosphate (E-4-P), molecular weight, GPI-related antigen concentration, immunodiffusion pattern, and immunoinactivation were in the normal range. This is the first example of the association of a stable mutant GPI with severe hemolytic anemia. Enzyme instability has been present in all previously reported cases.

Membrane abnormalities of pyruvate kinase deficient red cells.

Two experimental systems have been applied to the study of PK-deficient red cells in an attempt to reveal possible membrane abnormalities of these erythrocytes: (1) the glycoprotein self-digestion of intact erythrocytes during in vitro incubation at 37 degrees C; (2) the red cell sensitivity to the lytic action of mouse macrophages. PK-deficient erythrocytes display a more precocious than normal membrane glycoprotein self-digestion and are much more susceptible than normal to the cytotoxic activity of mouse macrophages. This latter effect is more pronounced in young than in old PK-deficient red cells. These observations indicate the existence of membrane abnormality in PK-deficient red cells other than those so far suspected.