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Background/aims: Adrenomedullin (AM) is a recently purified hypotensive peptide and its encoding gene has been sequenced from a human pheochromocytoma. High levels of AM have been shown in Addison's disease (AD). X-linked adrenoleukodystrophy/adrenomyeloneuropathy (ALD/AMN) is a peculiar adrenal insufficiency due to an accumulation of very-long chain fatty acid in adrenal cells and it is very often associated with a devastating demyelination of the central nervous system., Methods: We studied the AM plasma levels of 22 patients with ALD/AMN (18 with hypoadrenalism, ALDa, and 4 with normal adrenal function, ALDb) and compared them with 18 males with classical AD and 16 normal male subjects. All patients with hyposurrenalism were studied before treatment with hydrocortisone., Results: Both patients with ALD/AMN and AD showed increased levels of AM and all of them showed a significant difference from the control group (p < 0.0001). The plasma renin activity was higher in all patient groups than in the control group (p <0.001 ALDa, ALDb and AD vs. control group). The aldosterone levels were higher in ALDa and ALDb groups than AD (ALDa vs. AD p < 0.01; ALDb vs. control group p < 0.05; AD vs. controls p < 0.01). ACTH plasma levels were higher in ALDa and AD than ALDb and the control group (ALDa vs. AD not significant while ALDa and AD vs. control p <0.0001)., Conclusions: Our data indicate that plasma AM levels in ALDa, ALDb and AD are higher than controls. These results were previously described in untreated AD. While classical AD patients show complete adrenal insufficiency (both mineralocorticoid and glucocorticoid defects), ALD/AMN patients show a less compromised glomerular function, indicating that AM is not completely correlated with mineralocorticoid insufficiency, and that the exact mechanism responsible for the increased AM levels in ALD/AMN is still unknown., (Copyright (c) 2005 S. Karger AG, Basel.)

Purpose of Review: This review outlines recent advances in the clinical, genetic and molecular aspects of laminopathies, an expanding group of disorders caused by mutations of the lamin A/C gene., Recent Findings: Mutations in lamin A/C were originally described in skeletal and cardiac muscle disorders. It has subsequently been shown that partial lipodystrophy syndromes with or without developmental abnormalities and premature ageing are also associated with lamin A/C alterations. Concomitantly, peripheral nerve involvement with autosomal recessive and dominant inheritance is adding to the picture. The clinical heterogeneity of laminopathies ranges from intrafamilial variability to the description of overlapping phenotypes. A large variability in clinical presentation and the course of cardiomyopathy occurs, including sudden death despite pacemaker implant and embolic stroke in young patients. Similarly, premature ageing syndromes encompass classic and atypical forms of varying severity with the involvement of diverse tissues. In addition, an association of myopathic and neuropathic phenotypes is now emerging., Summary: Advances in molecular genetics of apparently unrelated disorders, involving muscle, heart, nerve, fat, bone, liver, skin tissues and premature ageing, have enriched our knowledge of the diverse phenotypes associated with lamin A/C mutations. Nevertheless, the understanding of pathogenetic mechanisms still remains speculative. More basic and clinical research is needed in order to identify genes concurring in determining the lamin A/C phenotypes and to envisage proper treatment strategies.

Aphanizomenon flos-aquae (AFA) is a fresh water unicellular blue-green alga (cyanophyta) rich in phycocyanin (PC), a photosynthetic pigment with antioxidant and anti-inflammatory properties. The purpose of this study was to evaluate the ability of a novel natural extract from AFA enriched with PC to protect normal human erythrocytes and plasma samples against oxidative damage in vitro. In red blood cells, oxidative hemolysis and lipid peroxidation induced by the aqueous peroxyl radical generator [2,2'-Azobis (2-amidinopropane) dihydrochloride, AAPH] were significantly lowered by the AFA extract in a time- and dose-dependent manner; at the same time, the depletion of cytosolic glutathione was delayed. In plasma samples, the natural extract inhibited the extent of lipid oxidation induced by the pro-oxidant agent cupric chloride (CuCl2); a concomitant increase of plasma resistance to oxidation was observed as evaluated by conjugated diene formation. The involvement of PC in the antioxidant protection of the AFA extract against the oxidative damage was demonstrated by investigating the spectral changes of PC induced by AAPH or CuCl2. The incubation of the extract with the oxidizing agents led to a significant decrease in the absorption of PC at 620 nm accompanied with disappearance of its blue color, thus indicating a rapid oxidation of the protein. In the light of these in vitro results, the potential clinical applications of this natural compound are under investigation.

Objectives: To evaluate the condition of oxidative stress in patients undergoing prolonged exposure to hyperbaric oxygen (HBO) and the possible modifications of the antioxidant defense systems in the absence of antioxidant supplementation., Design and Methods: Twelve patients exposed to 15 HBO treatments for pathological conditions related to hypoxia were included in the study. Oxidative stress indices as well as plasma and erythrocyte antioxidant levels were measured in blood samples collected both at the 1st and 15th HBO session., Results: The repeated exposures to HBO led to a significant accumulation of plasmatic reactive oxygen metabolites (ROM) and malondialdehyde (MDA). After 15 HBO sessions, no relevant differences were detected for reduced glutathione (GSH), alpha-tocopherol, and retinol plasma levels; however, a significant decrease in erythrocyte superoxide dismutase (SOD) and catalase (CAT) activity was observed when compared to the 1st HBO exposure; glutathione peroxidase (GPx) activity remained almost unchanged., Conclusions: In the absence of antioxidant supplementation, the prolonged HBO treatment leads to a condition of oxidative stress that seems to affect in particular the response of the enzymatic antioxidant defense system; the possible relationship between the chemical modifications of the enzymes caused by oxygen reactive species and the consequent inactivation of the proteins is under investigation.

We have previously reported the presence of dying cells in the granule cell layer (GCL) of adult rat dentate gyrus (DG), where neurogenesis occurs. In particular, we found that cell death in the GCL increased in vitamin E deficiency and decreased in vitamin E supplementation. These findings were regarded as related to changes in neurogenesis rate, which in turn was influenced by vitamin E availability; a neuroprotective effect of vitamin E on cell death was also proposed. In order to verify this latter hypothesis, we have studied cell death in all layers of DG in vitamin E-deficient and vitamin E-supplemented rats and in control rats at different ages, using TUNEL and nick translation techniques. The phenotype of TUNEL-positive cells was characterized and the existence of dying BrdU-positive cells was investigated. Dying cells with neuronal phenotype were observed throughout the DG in all experimental groups. The number of TUNEL-positive cells decreased from juvenile to adult age. A higher number of TUNEL-positive cells in vitamin E-deficient rats and a lower number in vitamin E-supplemented rats, with respect to age-matched controls, were found; moreover, in these groups, TUNEL-positive cells had a different percentage distribution in the different layers of the DG. Our results confirm the occurrence of cell death in DG, demonstrate that cell death affects neuronal cells and support the hypothesis that the effect of vitamin E on cell death is not related to neurogenesis.

Background: Hyperhomocysteinemia is a well-recognized independent risk factor for cardiovascular disease in end-stage renal disease (ESRD) patients. Since homocysteine (Hcy) largely binds to serum proteins (80 to 90%), in this study we investigated the possibility that polymethylmethacrylate (PMMA)-based protein-leaking dialyzers could reduce total plasma Hcy (tHcy) levels in ESRD patients., Methods: Two matched groups of patients (N = 13) showing mild to intermediate hyperhomocysteinemia on standard hemodialysis (HD) with conventional non-protein-leaking dialyzers were included. In the control group membranes were maintained the same, while the study group was switched to protein-leaking dialyzers (BK-F series; Toray, Japan) and studied for 6 months. tHcy was measured by high performance liquid chromatography (HPLC) at baseline and after 1, 3, and 6 months. Proteins and Hcy were also measured in the spent dialysate., Results: The pre-HD levels of tHcy in the control group remained close to baseline values (26.6 +/- 5.0 micromol/L), while in the study group at 1, 3, and 6 months they decreased from a baseline value (in micrormol/L) of 25.3 +/- 5.9 to 21.5 +/- 4.5, 16.9 +/- 4.0, and 17.2 +/- 4.2, respectively (P < 0.01 for values at 3 and 6 months vs. baseline). The intra-HD drop of tHcy (Delta HDHcy) slightly but progressively decreased during the 3 steps on protein-leaking dialyzers and a positive correlation was found between Delta HDHcy and pre-HD levels of tHcy. In spent dialysate samples from protein-leaking dialyzer-treated patients, the amount of protein-bound Hcy (bHcy) was approximately 10 times higher than in non-protein-leaking dialyzers, but the Delta HDHcy observed in non-protein-leaking dialyzers and protein-leaking dialyzers was comparable. Serum proteins and albumin were only slightly affected by protein-leaking dialyzers., Conclusion: This study demonstrates that protein-leaking dialyzers used with a pure diffusive technique significantly lower pre-HD tHcy (approximately 33% of starting levels after 3 months of treatment) in ESRD patients. A possible underlying mechanism for this effect could be the removal of large molecular weight solutes responsible for a defective metabolism of the Hcy, as the removal of bHcy with protein-leaking dialyzers seems not sufficient, per se, to explain this steady reduction of tHcy levels in pre-HD.

Background: An increased apoptotic rate of peripheral blood mononuclear leukocytes (PBMLs) in haemodialysis (HD) patients has been reported in several studies, but its underlying mechanisms remain poorly understood. Oxidant stress is a well known cause of cell damage, and several lines of evidence suggest that it might influence the induction and signalling steps of mononuclear cell apoptosis through different mechanisms so as to provoke disturbances of the intracellular pool of thiols (SHi). In this study, we investigated the in vitro apoptotic rate and SHi of PBMLs in end-stage renal disease (ESRD) patients on HD or peritoneal dialysis (PD)., Methods: Apoptosis and SHi were evaluated in vitro in PBMLs obtained from 40 ESRD patients (HD, n = 30 and PD, n = 10) and 10 healthy controls. A subgroup of HD patients was also studied before and after 1 month of treatment with a vitamin E-coated dialyser (CL-E). Cell thiols and viability were also assessed in the monocyte-like cell line U937 and PBMLs after incubation in the presence of uraemic plasma with or without supplementation of the antioxidants vitamin E (70 micro M) or N-acetyl-cysteine (NAC) (0.5 mM)., Results: After 24 h in culture, the PBMLs of HD patients, but not those of CAPD patients, showed an apoptotic rate twice that of healthy controls and a 40% decrease of SHi levels (P < 0.01 in both). A negative correlation between the apoptotic rate and SHi was observed in both patients and controls (r = 0.648, P < 0.001). Plasma and ultrafiltrate samples from HD patients contained solutes (mainly in the low-middle molecular weight range) able to trigger apoptosis and oxidative stress in U937 cells. The treatment of HD patients with CL-E, as well as the in vitro supplementation of U937 cells with vitamin E or NAC during the exposure to uraemic plasma, decreased the rate of apoptosis and partially restored SHi., Conclusions: This study showed an association between an increased apoptotic rate and decreased SHi in PBML of HD patients, but not of CAPD patients. These changes are partially due to different pro-apoptogens that accumulate in the plasma and are at least partially prevented by exogenous antioxidants able to restore SHi, such as vitamin E or thiol suppliers.

Two experiments were performed to examine sow fertility following the pre-weaning injection of gonadotrophin. In experiment 1, sows received PG600 2-days pre-weaning (PW2), on the day of weaning (W) or received no injection and served as controls (CT1). Compared to controls, the injection of PG600 resulted in shorter (P<0.02) wean-estrus intervals and more (P<0.05) sows exhibiting estrus by 7 days after weaning (4.5+/-0.2, 5.1+/-0.3, 6.1+/-0.3 days, and 81.3, 82.9, 68.8%, for PW2, W, and CT1, respectively). No effect of treatment was apparent for farrowing rate or subsequent litter size. In experiment 2, PG600 was injected at 4 days pre-weaning (PW4) and then either GnRH or hCG was injected at the time of weaning. Control (CT2) sows received no injections. Injection of PG600 was associated with shorter (P<0.0001) wean-estrus intervals and a higher (P<0.003) farrowing rate (1.1+/-0.2, 5.1+/-0.2 days, and 94.8, 78.5% for PW4 and CT2, respectively). There was no effect of treatment on subsequent litter size and results were the same for GnRH and hCG treated sows. We conclude that injection of gonadotrophins prior to weaning will result in very short wean-estrus intervals and that, when combined with a hormonally controlled ovulation, sow fertility may be enhanced.

Numerous industrial and experimental facilities have been set up, particularly in the last ten years, as a result of studies and researches on treating asbestos-containing waste (ACW) to stabilise it and to enable its reuse. Some of the stabilisation processes reduce the hazards of ACW by imprisoning in a cement or resonoid matrix. Other processes modify the fibrous structure of asbestos and transform it into an inert substance. One such inactivation process is mechanochemical transformation. This new technology is extremely interesting both economically and industrially, especially in view of the European Directive 1999/3/CE of 24/4/99, which provides for the obligatory treatment of all types of waste material before its disposal.

Retroviral-mediated gene transfer of the IL-4 gene into experimental gliomas can cause tumor rejection, supporting the clinical use of this form of gene therapy for glioblastomas (GBM). In a clinical setting, the administration of dexamethasone (dex) is a standard procedure for GBM patients. This led us to examine the effects of dex on IL-4 gene therapy. We injected intracranially Fischer 344 rats with phosphate-buffered saline, 9L gliosarcoma cells mixed with E86.L4SN(200) cells (retroviral producer cells, RPC, transducing IL-4 cDNA) and 9L cells mixed with PA317.STK.SBA cells (control RPC expressing the HSV-tk gene). The rats from each group were treated with 0, 50, 100 or 250 microg dex/kg/day released by osmotic pumps implanted subcutaneously. While 80-100% of rats receiving 9L cells mixed with IL-4 RPC and not treated by dex survived for at least 2 months following tumor injection, only 50% and 17% of rats receiving 50 or 100 microg/kg/day of dex, respectively, reached this time point. These results indicate that dex significantly diminished the anti-tumor effect of IL-4. Thus, in a clinical setting, IL-4 gene transfer should be performed when low levels of dex are administered or in the absence of dex.

The adult hippocampal neurogenesis is affected by vitamin E deficiency. In the present investigation we examined if neural precursor proliferation, newborn cell survival or both are altered by vitamin E deficiency. 5-Bromo-2'-deoxyuridine (BrdU) was employed as a marker of proliferating cells. BrdU-labelled cells were revealed 1 and 30 days after BrdU administration in order to evaluate proliferation and newborn cell survival, respectively. Cell proliferation decreased in controls from juvenile to adult age, and the decrease was lesser in vitamin E deficiency. Thus we found a higher number of proliferating cells in vitamin E-deficient rats than in age-matched controls at 5 months of age. Comparing the number of BrdU-positive cells between 1 and 30 days after the last BrdU injection revealed a remarkable decrease in all groups; this is the greatest in vitamin E-deficient rats and the lowest in control rats. Consistently cell death in the dentate gyrus, assessed by TUNEL technique, was found to decrease from 1 to 5 months of age, but at 5 months it was significantly higher in vitamin E-deficient rats than in age-matched controls. These data show that vitamin E deficiency enhances neural precursor proliferation and cell death during adult neurogenesis.

In this paper a rapid method based on a FIA (flow injection analysis) system with amperometric detection for the evaluation of ascorbyl 6-palmitate in foods is described. The selectivity of the proposed method is related to the low anodic potential applied to the working glassy carbon electrode (+0.1 V vs. Ag/AgCl) that leaves out interferences from ascorbic acid and phenolic compounds. By flow injection analysis, under optimised conditions, the calibration curve was linear in the range 0-20 mg l(-1) and the detection limit was 0.2 mg l(-1).

Background: Preliminary evidence on the accumulation of polyamine-protein conjugates (PPCs) was obtained in uremic patients. The presence of these substances in the plasma of hemodialysis (HD) patients was evaluated, and their possible contribution to uremic anemia was investigated by testing the effect of PPC synthesized in vitro on erythroid cell proliferation., Methods: Plasma PPC was measured by high-performance liquid chromatography. The in vitro synthesis of PPC from human plasma was carried out by means of the enzyme transglutaminase in the presence of either [3H]-labeled or unlabeled spermidine (SPD). After gel filtration chromatography and detection of the fractions containing [3H]SPD, the latter were tested for their effect on mononuclear bone marrow cell proliferation., Results: In three out of four patients examined, mainly SPD-protein conjugates (SPD-PC) were observed to accumulate during HD. The levels ranged from 0.17 to 4.93 pmol/mg proteins before dialysis, and these values increased at 30 minutes and at the end of the dialysis up to levels 11.90 pmol/mg. SPD-PC levels in healthy controls were 1.46 +/- 0.82. SPD-PCs synthesized in vitro were recovered in two main fractions showing a molecular weight of> 100 kD (peak 1) and of approximately 30 to 50 kD (peak 3), respectively. The SPD-PC contained in peak 1 showed the greatest inhibitory effect on colony-forming units-erythroid (CFU-E) proliferation without any appreciable effect on burst-forming units-erythroid (BFU-E)., Conclusion: We demonstrate that SPD-PC can accumulate in HD patients. These substances, which affect CFU-E proliferation, can be considered as an at yet unrevealed class of uremic toxins contributing to the onset of the uremic anemia.

Glioblastomas, the most frequent and malignant of primary brain tumors, have a very poor prognosis. Gene therapy of glioblastomas is limited by the short survival of viral vectors and by their difficulty in reaching glioblastoma cells infiltrating the brain parenchyma. Neural stem/progenitor cells can be engineered to produce therapeutic molecules and have the potential to overcome these limitations because they may travel along the white matter, like neoplastic cells, and engraft stably into the brain. Retrovirus-mediated transfer of the gene for interleukin-4 is an effective treatment for rat brain glioblastomas. Here, we transferred the gene for interleukin-4 into C57BL6J mouse primary neural progenitor cells and injected those cells into established syngeneic brain glioblastomas. This led to the survival of most tumor-bearing mice. We obtained similar results by implanting immortalized neural progenitor cells derived from Sprague-Dawley rats into C6 glioblastomas. We also documented by magnetic resonance imaging the progressive disappearance of large tumors, and detected 5-bromodeoxyuridine-labeled progenitor cells several weeks after the injection. These findings support a new approach for gene therapy of brain tumors, based on the grafting of neural stem cells producing therapeutic molecules.

Gene therapy approaches to the treatment of experimental cancer are usually based on established neoplastic cell lines which are manipulated in vitro and subsequently transplanted in host animals. However, the relevance of these artificial models to the biology and therapy of human tumors is uncertain. We have previously validated an experimental model based on MMTV-neu transgenic mice in which breast tumors arise spontaneously in 100% of animals and have many features in common with their human counterpart, including the involvement of the neu oncogene and the ability to metastatize. In this article we report the effect of intratumoral, retrovirus-mediated, IL-4 expression on the growth of breast tumors arising in these mice. The size of IL-4 inoculated tumors on the right side was significantly smaller than that of controlateral untreated tumors, suggesting a local effect of IL-4. In addition, the non-injected tumors on the left side of treated animals were significantly smaller than those arising in control transgenic mice, suggesting that IL-4 can also inhibit tumor growth systemically. These findings suggest that IL-4 gene transfer can significantly reduce the growth rate of spontaneously arising breast tumors and that immune-based gene therapy could efficiently complement other approaches based on different mechanisms, such as suicide gene transfer or antisense technology.

Background and Aims: Recent studies have underlined a significant incidence of peripheral arterial occlusive disease (PAOD) of the lower limbs in the general population which is often wrongly diagnosed. The "classic" risk factors--like dyslipidemia--are not always present in significant percentages in patients suffering from PAOD of the lower limbs. The aim of this study was to evaluate the incidence of the most common vascular risk factors (smoking, hypertension, hyperglycemia, dyslipidemia) in patients suffering from stenosing lesions of the extracranial carotid axes, comparing the data with similar findings in lower limbs. Moreover, the authors evaluated the association between these risk factors, carotid atheromatous lesions and ischemic cardiomyopathy (CI)., Methods: A retrospective study was performed to evaluate the data from 1643 patients examined consecutively. A total of 636 (age > 40, carotid stenosis > 40%, presence of risk factors) were considered eligible., Results: The results showed that, contrary to the findings in patients suffering from PAOD, diabetes was not among the most frequently associated risk factors, whereas a relatively large number of patients had a history of smoking, including both smokers and ex-smokers., Conclusions: The difference in the most frequent risk factors identified for PAOD and carotid lesions suggests different etiopathogenetic mechanisms for the two districts.

Background: Overexpression of glutathione S-transferase (GST; EC 2.5. 1.18) has been documented in the erythrocytes of patients with chronic renal failure, and this event may well be of relevance from a clinical standpoint. In fact, it could serve as a marker of uremic toxicity overall, which can contribute to impair the function and survival of the erythrocytes. However, the biochemical details of this phenomenon are poorly understood., Methods: In this study, we characterized the expression of GST in erythrocytes of 118 uremic patients under different clinical conditions. The mechanisms responsible for the regulation of protein expression and enzyme activity were investigated in light of different dialysis approaches, oxidative stress, uremic toxins, erythrocyte age, and erythropoietin (EPO) supplementation., Results: Mean GST activity in uremic patients was highly overexpressed with respect to controls, and this phenomenon was exclusively attributable to an increased expression of GST. Overexpression of GST did not appear to be dependent on oxidative stress and was not influenced by vitamin E supplementation. In the same manner, both erythrocyte age and EPO supplementation apparently did not interfere with the GST concentrations, which were the same in controls and patients. Preliminary experiments suggested that high-molecular weight or protein-bound toxins could play some role in the overexpression of GST., Conclusions: GST expression may be a useful marker for the individual accumulation of uremic toxins as well as of the efficiency of new dialysis strategies in removing them.

Overexpression of interleukin 4 (IL-4) can impair the tumorigenicity of glioma cells, but direct evidence of its antitumor efficacy after in vivo gene transfer into malignant gliomas has not been provided. To test this, we first injected into the brain of Sprague Dawley rats a 1:1 mixture of C6 rat glioblastoma cells and psi2.L4SN20 or E86.L4SN50 retroviral producer cells (RPCs), secreting 20 and 50 ng of IL-4/5 x 10(5) cells/48 h, respectively. Twenty-seven and 56% of rats receiving injections with these low- or medium-level IL-4 RPCs, respectively, survived tumor injection, whereas control rats died in about 1 month. E86.L4SN50 RPCs coinjected with 9L gliosarcoma cells into syngeneic Fischer 344 rats yielded similar results. A novel IL-4 RPC clone expressing higher levels of IL-4, E86.L4SN200, coinjected with 9L cells increased to 75% the fraction of long-term survivors and induced tumor regression in 50% of rats when injected into established 9L gliosarcomas. Cured rats developed an immunological memory because they rejected a challenge of wild-type 9L cells into the contralateral hemisphere. Magnetic resonance imaging was used to monitor 9L and C6 gliomas and gave direct evidence for tumor rejection in treated rats. Immunohistology showed inflammatory infiltrates in IL-4-treated tumors in which CD8+ T lymphocytes were more abundant, although CD4+ T lymphocytes, B lymphocytes, and macrophages were also present. Overall, these findings suggest that IL-4 gene transfer is a new, promising approach for treating malignant gliomas.

In tumors, gap junctional intercellular communication (GJIC) is usually down-regulated and the expression of connexins, membrane proteins constituting gap junction channels, is often low or altered. GJIC, allowing the intercellular diffusion of ganciclovir (GCV) triphosphate, is also one mediator of the 'bystander effect', the phenomenon by which herpes simplex virus thymidine kinase (HSVtk)-transduced, neoplastic cells kill surrounding HSVtk-negative cells when treated with GCV. We set up experiments to evaluate the effects of retrovirus-mediated in vivo gene transfer of connexin 43 in malignancies with low GJIC capacity. We found that U-87 human glioblastoma cells transfected in vitro by the human Cx43 cDNA grow significantly more slowly than control U-87 cells and lose their tumorigenicity when injected subcutaneously in nude mice. When the Cx43 gene was transduced in vitro in U-87 cells by a retroviral producer cell line (N3.2.ii, titer 1.5 x 10(6) c.f.u./ml) in vivo results were similar. However, only when U-87 cells were co-injected with N3.2.ii cells in nude mice in a 1:5 ratio, a 50% reduction in tumor size was obtained during the first 3 weeks. Moreover the coinjection of U-87 cells with N3.2.ii and SBA cells (a retroviral producer cell line expressing the HSVtk gene), was not able to potentiate the effects of GCV administration, suggesting that Cx43 gene transfer requires more efficient vectors to increase the bystander effect in vivo.

A promising strategy in the treatment of malignant gliomas involves the creation of Herpes Simplex Virus-thymidine kinase (HSV-tk) modified tumor cells. Some authors have observed complete tumor regression after ganciclovir (GCV) treatment of established gliomas transduced in vivo by the HSV-tk gene. Yet, further investigations did not confirm completely these results, even if confirmed the therapeutic potential of such a therapy. Using the rat C6 glioblastoma as a model of malignant brain tumor, we investigated the efficacy of in vivo and in vitro transduction of growing brain tumors with the HSV-tk gene, followed by GCV administration. The stereotactic injection into the left striatum of C6 cells mixed with retroviral producer cells and GCV treatment did not improve significantly the animal survival compared to controls. On the contrary, there was a significant prolongation of the survival of rats inoculated with C6 cells engineered in vitro to express the HSV-tk gene. Nevertheless, complete eradication of the tumors was not achieved. We also injected a group of six rats with a mixture of cells expressing the murine Interleukin-4 (IL-4) gene and C6 cells. IL-4 has been shown to produce the regression of experimental brain tumors. Our preliminary experience seems to confirm that hypothesis. Our results indicate that the outcome of HSV-tk gene therapy can be limited not only by low gene transfer but also by insufficient delivery of GCV to tumor cells. Combined strategies, based on contemporary transduction of HSV-tk and IL-4, may enhance the therapeutic perspectives of such a therapy.

The growth of U-87 or C6 gliomas co-implanted in nude mice with retroviral producer cells (VPC) expressing the herpes simplex virus-thymidine kinase (HSV-tk) gene is only partially impaired by treatment with ganciclovir (GCV). The effect of GCV is even less evident when C6 and VPC are co-implanted into the rat brain. Furthermore, tumors from C6 cells carrying the HSV-tk gene are not eradicated by GCV, although they remain sensitive to GCV when replated in vitro. These limits of the HSV-tk/GCV system in glioma gene therapy may be due to insufficient gene transfer and/or insufficient delivery of GCV to glioma cells. Combination of HSV-tk and one or more cytokines may improve the antitumor efficacy. Among cytokines, interleukin-4 (IL-4) has already been shown to be active against gliomas. In nude mice, GCV treatment inhibited tumor growth more effectively after co-injection of C6 cells with a mixture of VPC transducing IL-4 and HSV-tk genes than after co-injection with either IL-4 or HSV-tk VPC only. In immunocompetent Sprague-Dawley rats, co-injection of IL-4 VPC and C6 cells was also effective in inhibiting the growth of C6 brain tumors, 38% of the animals surviving for at least 2 months. Furthermore, increased and prolonged antitumor efficacy was obtained by transducing both IL-4 and HSV-tk genes.

The potential of gene therapy strategies for malignant gliomas that are based on retroviral-mediated transfer of a "suicide gene" such as Herpes Simplex Virus-thymidine kinase HSV-tk and subsequent treatment by a prodrug (ganciclovir, for example), has been emphasized by the promising results obtained by several groups. However, further experimental data as well as preliminary clinical results indicate that the low efficiency of retroviral-mediated gene transfer in vivo as well as difficulties for the diffusion of the prodrug inside the tumour mass can limit the efficacy of this form of gene therapy. To achieve a more effective limitation of tumour growth other approaches may be combined with the "suicide gene" strategy and the enhancement of the immunological response to the tumour by cytokine gene transfer is prominent among these approaches. The authors' experiments in nude mice confirm the antineoplastic role of IL-4 and encourage testing the effects of the simultaneous transfer of IL-4 and HSV-tk genes in immunocompetent animals.

Mice transgenic for the activated rat neu oncogene under the control of the mouse mammary tumor virus long terminal repeat (MMTV-LTR) (neu+ mice), develop breast tumors in 100% of cases. We have previously reported that double transgenic mice obtained from crossing neu+ mice with mice transgenic for the herpes simplex virus thymidine kinase (HSVtk) gene can be used as a suitable model to test the 'suicide gene' strategy for mammary tumor gene therapy in vivo. In the present study, we evaluated the efficacy of the HSVtk/ganciclovir (GCV) system in the neu+ mice by inoculating cells producing a retroviral vector bearing the HSVtk gene in the mammary tumors on one side of the animals, and comparing their weight with that of the contralateral tumors, after systemic GCV administration. A statistically significant effect of this therapy was clearly seen (P < 0.001) but complete eradication of the tumors could not be achieved. This was not due to the inefficient delivery of GCV, as no HSVtk expression was detected in the residual tumors, but could be related to the low transduction efficiency (< 10%) and to inability of the 'bystander effect' (probably due to the absence of functional gap-junctions among mammary tumor cells) to kill nontransduced neoplastic cells. These data suggest that results obtained by in vivo models using transplanted tumor cell lines as targets for gene therapy might not be immediately transferable to spontaneously arising tumors in animals or humans.

Purpose: To determine the feasibility of using posterior chamber intraocular contact lenses to treat myopia., Setting: Ambulatory surgery centers in Torino, Ancona, and Ivrea, Italy., Methods: The phakic intraocular contact lens is made of a collagen copolymer. We implanted 15 of these lenses in 14 patients with preoperative spherical equivalents ranging from -10.8 to -24.0 diopters (D); average myopia was -15.3 D +/- 3.1 (SD). Average follow-up was 7.0 +/- 1.95 months., Results: Mean postoperative spherical equivalent was -2.0 +/- 1.6 D. Uncorrected visual acuity improved in 14 eyes. Best spectacle-corrected acuity was maintained or improved in all but one eye. No iritis or cataracts were observed. Six months postoperatively, the mean endothelial cell loss was 4%. A pupillary block requiring a superior peripheral iridectomy occurred in one eye., Conclusions: Because of the incomplete follow-up, we cannot draw conclusions about the long-term safety of the implantable contact lens. The intimate contact between the contact lens and the natural lens raises the possibility of cataract formation. However, examination by Scheimpflug photography and ultrasound biomicroscopy showed no progressive lens opacities.

We have investigated the status of the MTS2 gene, encoding the cyclin-dependent kinase (CDK) inhibitor p15, in 32 glioblastomas. Semi-quantitative PCR identified 7 tumors in which the amplified material was 18.6% of controls and 7 in which was 48.0%, suggesting the occurrence of homozygous and hemizygous deletions, respectively. Single strand conformation polymorphism analysis identified one polymorphism but no mutations. We also expressed MTS2 and MTS1, encoding the contiguous and highly homologous CDK inhibitor p16, in U-87 human glioblastoma cells. Both genes, either separately or in combination, inhibit significantly the proliferation rate of U-87 cells but such inhibition is progressively lost. As a whole, the data assign a tumor suppressor role to p15 and confirm homozygous deletions as the favorite mechanism for the inactivation of MTS1 and MTS2 in glioblastomas.

Cells expressing the herpes simplex-thymidine kinase (HS-TK) gene as a consequence of retroviral transduction, as well as TK-negative (TK-) bystander cells, can be killed by treatment with ganciclovir (GCV). In vitro, this "bystander effect," has been attributed to metabolic cooperation through gap junctions or to the uptake of apoptotic vesicles. We show that GCV treatment kills TK-negative U-87 glioma cells cocultured with cells that express TK (TK+) but that have lost the capacity for releasing retroviral particles. A photometric enzyme immunoassay identifies histone-associated DNA fragments, typical of apoptosis, in the cytosol of GCV-treated TK+ cells, and apoptotic features are also demonstrated by ultrastructural studies. Northern blot analysis and the reverse transcription polymerase chain reaction (PCR) show that connexin 43, a major constituent of gap junctions, is expressed in TK+ and U-87 cells. The size of U-87 tumors in nude mice subsequently injected with TK+ cells and GCV is not significantly different than in untreated animals; whereas, after injecting 1:1 mixtures of U-87 and TK+ cells, GCV treatment only causes a temporary regression of tumor growth. On the contrary, when the injected mixtures contain PA317.STK.SBA (a retroviral producer cell line that can transduce efficiently the HS-TK gene) and U-87 cells, tumors are destroyed effectively by GCV treatment. Thus, an experimental setting in which U-87 gliomas are matched with cells that are able to express, but not to transduce, the HS-TK gene indicates that the bystander effect kills U-87 cells in vitro by mechanisms associated with apoptotic death. In vivo, this effect is not sufficient to restrain the tumor growth taking place in immunodeficient animals.

Background: Retrobulbar anesthesia is considered an effective method of obtaining anesthesia and akinesia in eye surgery. It can give rise to serious complications, however, and these complications could be considerably reduced with peribulbar anesthesia., Methods: After using peribulbar anesthesia for 2 years in surgical treatment of the anterior segment, the authors began a study to test its efficacy in posterior segment surgery. Retinal and vitreoretinal surgery with the peribulbar anesthesia technique were performed in 32 consecutive patients., Results: In all cases anesthesia was complete with one preoperative block. In 2 patients, however, completion of akinesia required a supplemental 2- to 3-cc injection of the anesthetic solution. Surgery was performed with preoperative block only in 31 cases. In the remaining case, a supplemental peribulbar injection of the anesthetic solution was given intraoperatively for pain. In 5 cases (15%) pain at the end of surgery from conjunctiva was controlled by anesthetic drops., Conclusion: It is recommended that this method be used only when the duration of the operation is expected to be brief.

We have evaluated the effect of pyridostigmine (PD), a cholinergic agonist, on the growth hormone (GH) response to physical exercise (EXC) in nine healthy volunteers. PD administration and EXC caused a similar increase of GH secretion to mean (+/- SE) peak values of 5.3 +/- 0.9 and 6.5 +/- 1.2 micrograms/l, respectively. Pretreatment with PD caused a significant augmentation of the EXC-induced GH release evaluated both as maximum peak (13.5 +/- 2.1 micrograms/l, p < 0.01 vs EXC) and as area under the secretory curve (EXC = 292.6 +/- 41.9 micrograms.min.l; PD + EXC = 587.3 +/- 68.9 micrograms.min.l, p < 0.005). The action of PD on GH secretion was additive to that of EXC since the sum of the GH responses to PD and EXC was not significantly different from the response obtained during PD + EXC. Whether PD and EXC act through a common final pathway, i.e. inhibition of endogenous somatostatin release, or the EXC-induced GH secretion involves stimulation of endogenous GHRH remains matter of investigation.

We evaluated the GH response to combined administration of pyridostigmine (PD), a cholinergic agonist, and GH-releasing hormone (GHRH) (60 mg PD given orally 60 min before the GHRH bolus) as well as baseline IGF-I concentrations in 10 patients (5 males and 5 females, age 6.0-24 years) with Prader-Labhard-Willi (PLW) syndrome, 8 prepubertal obese children (4 males and 4 females, age 5.6-12.0 years) and 9 prepubertal short normal children (7 males and 2 females, age 8.0-12.8 years). Mean GH responses to PD+GHRH were significantly lower (p < 0.0001) in the PLW patients (13.8 +/- 3.3 micrograms/l) than in the short normal children (52.2 +/- 9.0 micrograms/l) and similar to those of the obese children (14.3 +/- 3.2 micrograms/l). Mean serum IGF-I levels were significantly lower (p < 0.05) in the PLW patients (117.5 +/- 26.4 micrograms/l) than in the obese (329.3 +/- 88.0 micrograms/l) and the short normal children (214.3 +/- 38.3 micrograms/l). Two of the PLW patients had absent GH responses to PD+GHRH associated with subnormal IGF-I concentrations, indicating pituitary GH deficiency. When these 2 cases were excluded from the statistical calculation, mean peak GH responses to PD+GHRH remained significantly lower (p < 0.0001) in the PLW patients (17.1 +/- 3.0 micrograms/l), while their mean serum IGF-I concentrations (143.4 +/- 71.5 micrograms/l) were not significantly different from those of the other two groups. These results indicate that patients with the PLW syndrome have a reduced or absent GH secretory reserve associated in some cases with low levels of IGF-I.(ABSTRACT TRUNCATED AT 250 WORDS)

1. A study was carried out to find out if digoxin and acipimox interact. 2. Six elderly patients on digoxin were each given acipimox 150 mg three daily for a week, after informed consent. Digoxin and acipimox plasma concentrations and urinary excretion were measured after the first dose of acipimox and after a week of treatment. 3. Data were fitted to a one-compartment oral absorption model. Areas under the plasma concentration-time curve, plasma and renal clearances, and elimination half-life were computed. 4. There was no significant difference in digoxin plasma concentrations and kinetic parameters before and after acipimox administration. Acipimox kinetics were not affected by the concomitant ingestion of digoxin. 5. The patients' clinical condition remained stable during the study. 6. Thus there was no evidence for an adverse interaction between digoxin and acipimox in human subjects under the conditions of this study.

The future will bring an increase in the economic forces which drive the succession of organizational forms. Migrating to integrated models of medical practice will become a survival technique for more and more physicians. This is a frightening prospect for individuals trained to be independent and self reliant. Fortunately, there are a variety of organizational models through which physicians can progress to ease the transition from solo to larger integrated practice forms. Regardless of the evolutionary course, successful physician organizations of the future will be primary care-based, geographically distributed, meaningfully partnered with hospital entities, equipped to accept risk, and lead by strong, business-savvy physician leaders and administrators. This organization will have the ability to flourish within a radically changing climate and will result in the delivery of measurably superior health care to its community.

The role of somatostatinergic tone (SST) in growth hormone (GH) neuroregulation in children with chronic renal failure (CRF) and short stature (mean height standard deviation score -3.47) was investigated. Ten children (9 males, 1 female), mean age 13.4 years (range 8-17 years), five with renal transplants (TP) and five on chronic haemodialysis (HD), underwent three separate investigations: (1) measurement of spontaneous GH secretion: (2) measurement of GH after infusion of GH releasing hormone (GHRH); (3) measurement of GH following treatment with pyridostigmine bromide (PD) and subsequent infusion of GHRH. All patients showed normal or exaggerated spontaneous nocturnal GH secretion (mean concentration values ranging between 3.8 and 19.07 ng/ml). In four of ten patients GHRH was not able to cause an increase in GH levels (mean peak GH 7.35 +/- 2.05 ng/ml) while PD pretreatment reinstated the GH response to GHRH (mean peak GH 55.25 +/- 17.23 ng/ml) in these children. In the other patients in whom GHRH-induced GH release was normal or exaggerated (mean peak GH 42.0 +/- 13.8 ng/ml). PD did not potentiate the GH response to GHRH (mean peak GH 54.83 +/- 7.88 ng/ml). These different types of responses were observed both in TP and HD patients. Our data indicate that: (1) PD potentiates the response to GHRH only when GHRH alone is not able to cause GH release, suggesting that SST is already reduced in patients with a normal or exaggerated GH response to GHRH; (2) in CRF patients the SST can be either reduced or increased, at least during the daytime.

Due to the need to epistemologically delimitate the theretical instrument, in order to understand the phenomena from a psycho-analitical reading, 65 complete psychological studies and 12 psychotherapeutic developments in a group of patients with psoriasis symptoms are analysed. The psychodiagnostic constants at the intellectual area, in the perceptivo-motor integration and the structure and the dynamics of the personality are proved, where there appears the constitution of a symbiotic link and the formation of a psychic apparatus with a feeble Ego, and very old defense mechanisms which form an ambiguous personality with identity problems. The authors try to understand theoretically the material accounting for the skin function and its relationship with the body Ego and the frustrated processus of the propioceptive sensibilization which fall upon the frustrating phase of the development that later, by a compulsory repetition, would be reproduced in the psoriasis symptoms. The behaviour which give character to the psoriasic management, both from the patient point of view and the physician's are analysed, in order to consider the psychotherapy in Psoriasic patients.