Redistribution of accumulated cell iron: a modality of chelation with therapeutic implications

Various pathologies are characterized by the accumulation of toxic iron in cell compartments. In anemia of chronic disease, iron is withheld by macrophages, leaving extracellular fluids iron-depleted. In Friedreich ataxia, iron levels rise in the mitochondria of excitable cells but decrease in the cytosol. We explored the possibility of using deferiprone, a membrane-permeant iron chelator in clinical use, to capture labile iron accumulated in specific organelles of cardiomyocytes and macrophages and convey it to other locations for physiologic reuse. Deferiprone's capacity for shuttling iron between cellular organelles was assessed with organelle-targeted fluorescent iron sensors in conjunction with time-lapse fluorescence microscopy imaging. Deferiprone facilitated transfer of iron from extracellular media into nuclei and mitochondria, from nuclei to mitochondria, from endosomes to nuclei, and from intracellular compartments to extracellular apotransferrin. Furthermore, it mobilized iron from iron-loaded cells and donated it to preerythroid cells for hemoglobin synthesis, both in the presence and in the absence of transferrin. These unique properties of deferiprone underlie mechanistically its capacity to alleviate iron accumulation in dentate nuclei of Friedreich ataxia patients and to donate tissue-chelated iron to plasma transferrin in thalassemia intermedia patients. Deferiprone's shuttling properties could be exploited clinically for treating diseases involving regional iron accumulation.     

Toad venom poisoning: resemblance to digoxin toxicity and therapeutic implications

A healthy man developed gastrointestinal symptoms after ingesting purported aphrodisiac pills. He had severe unrelenting bradycardia, hyperkalaemia, and acidosis. He rapidly developed severe life threatening cardiac arrhythmias and died after a few hours. He was found to have positive serum digoxin concentrations, although he was not taking digoxin. Toad venom poisoning is similar to digitalis toxicity and carries a high mortality. Cardiac glycoside poisoning can occur from ingestion of various plants and animal toxins, and the venom gland of cane toad (Bufo marinus) contains large quantities of cardiac glycosides. Toad venom, a constituent of an aphrodisiac, was considered responsible for the development of clinical manifestations and death in this patient. Digoxin specific Fab fragment has been reported to be beneficial in the treatment of toad venom poisoning. This report alerts physicians to the need to be aware of a new community toxic exposure, as prompt treatment with digoxin specific Fab fragment may be life saving. The treatment approach to patients with suspected toad venom poisoning is described.     

Genetic analysis of iron citrate toxicity in yeast: implications for mammalian iron homeostasis

Deletion of the yeast homologue of frataxin, YFH1, results in mitochondrial iron accumulation and respiratory deficiency (petite formation). We used a genetic screen to identify mutants that modify iron-associated defects in respiratory activity in Deltayfh1 cells. A deletion in the peroxisomal citrate synthase CIT2 in Deltayfh1 cells decreased the rate of petite formation. Conversely, overexpression of CIT2 in Deltayfh1 cells increased the rate of respiratory loss. Citrate toxicity in Deltayfh1 cells was dependent on iron but was independent of mitochondrial respiration. Citrate toxicity was not restricted to iron-laden mitochondria but also occurred when iron accumulated in cytosol because of impaired vacuolar iron storage. These results suggest that high levels of citrate may promote iron-mediated tissue damage.     

Genetic disorders affecting proteins of iron and copper metabolism: clinical implications

Iron and copper are essential transition metals that permit the facile transfer of electrons in a series of critical biochemical pathways. Recent work has identified the specific proteins involved in the absorption, transport, utilization, and storage of iron and copper. Remarkable progress is being made in understanding the molecular basis of disorders of human iron and copper metabolism. This review describes these proteins and examines the clinical consequences of new insights into the pathophysiology of genetic abnormalities affecting iron and copper metabolisms. Hereditary hemochromatosis is the most common genetic disorder of iron metabolism caused by mutations in the HFE gene. Aceruloplasminemia is a rare iron metabolic disorder that results from deficiency of ceruloplasmin ferroxidase activity as a consequence of mutations in the ceruloplasmin gene. Menkes disease and Wilson's disease are inherited disorders of copper metabolism resulting from the absence or dysfunction of homologous copper-transporting ATPases.     

Interactions between microvascular and macrovascular disease in diabetes: pathophysiology and therapeutic implications

Convention partitions the complications of diabetes into two main subtypes. First are the diabetes-specific microvascular complications of retinopathy, nephropathy and neuropathy; second are the atherothrombotic macrovascular complications that account for the majority of premature deaths. Pathological interactions between microvascular and macrovascular complications, for example, nephropathy and macrovascular disease, are common. Similar mechanisms and shared risk factors drive the development and progression of both small and large vessel disease. This concept has therapeutic implications. Mounting evidence points to the need for multifactorial strategies to prevent vascular complications in subjects with diabetes and/or the metabolic syndrome. We advocate a combined therapeutic approach that addresses small and large vessel disease. Preferential use should be made of drug regimens that (i) maximize vascular protection, (ii) reduce the risk of iatrogenic vascular damage and (iii) minimize the increasing problem of polypharmacy.     

Acute leukaemia after hydroxyurea therapy in polycythaemia vera and allied disorders: Prospective study of efficacy and leukaemogenicity with therapeutic implications

Abstract: Fifty consecutive patients, 30 of whom had polycythaemia vera (PV), 10 essential thrombocythaemia (ET), and 10 myelofibrosis (MF), entered a long-term prospective study of hydroxyurea (HU) therapy. The indication for treatment was mainly thrombocytosis or symptomatic splenomegaly. Control of erythrocytosis and thrombocytosis was achieved in 70% of the patients. Continuous maintenance treatment was required. In 15% of responding patients with thrombocytosis, unexpected rises of the platelet count occurred during maintenance therapy. Severe thrombo-embolic events occurred in 6 patients. The size of the spleen decreased in all patients who did not develop thrombocytopenia and could absorb adequate HU doses. Acute leukaemia (AL) was diagnosed in 9 patients and a myelodysplastic syndrome in one. Seven of them had been treated with HU alone. Among the patients with PV and ET, 6 developed AL and 4 of them were treated with HU alone (3 PV and 1 ET), giving an incidence of 10.5%. In previously untreated patients with initially normal karyotypes (n = 19), chromosome abnormalities developed during HU therapy in 7 (37%). Our results indicate that HU should be regarded as leukaemogenic, at least when used for treatment of PV and allied diseases. Since myelosuppressive treatment of PV does not prolong survival, the use of HU should be restricted to patients in whom the treatment indication outweighs the risk of leukaemia induction.     

Immunopathogenesis of acute graft-versus-host disease: implications for novel preventive and therapeutic strategies

Acute graft-versus-host disease (GVHD) is a primary T-cell-mediated complication of allogeneic hematopoietic stem cell transplantation (HSCT), occurring when donor-derived T cells are stimulated by host antigen-presenting cells (APCs), enhanced by proinflammatory cytokines such as interleukin (IL)-1, IL-6, IL-8, and tumor necrosis factor (TNF)-alpha. Recent data indicate that besides differences in major histocompatibility and minor histocompatibility antigens, cytokine gene polymorphisms have a predictive value for the complication of GVHD. Patients with a high anti-inflammatory IL-10 production have been demonstrated to be protected from GVHD while patients with high TNF-alpha serum levels were more at risk for GVHD. Pharmacological immunosuppression for GVHD prophylaxis and therapy, including unspecific approaches with corticosteroids or methotrexate (MTX), as well as more specific therapy with cyclosporin A (CsA), tacrolimus (FK506), sirolimus, mycophenolate mofetil (MMF), antithymocyte globulin (ATG), and monoclonal antibodies (MAbs) directed against CD3, CD25, CD52, cytotoxic T-lymphocyte antigen (CTLA)-4, CD40 ligand, or TNF-alpha, have been proven to be effective. Recent data on novel techniques to selectively deplete alloreactive T cells by removal, destruction, or anergy induction while preserving leukemia-specific T-cell clones suggest a clinical benefit from these approaches. Gene-modified T cells that can selectively be depleted and CD4⁺CD25⁺ regulatory T cells are under investigation for their ability to modulate alloreactivity after HSCT. With a better understanding of the immunopathogenesis of acute GVHD and the technical improvement of recently described therapeutic approaches, such as removal of naive T cells, selection of Th2 cells, suicide gene transduced T cells, and adoptive transfer of regulatory T cells, the use of alloreactivity as a treatment modality may be expanded to nonhematological disease entities such as solid tumors or autoimmune disorders.     

Molecular pathogenesis of chronic myeloid leukemia: implications for new therapeutic strategies

 With an annual incidence of about ten in 1,000,000 people, chronic myeloid leukemia (CML) accounts for most cases of myeloproliferative disease and for 20% of all leukemias. While novel therapies such as treatment with interferon-α or bone marrow transplantation have successively improved the outcome of CML treatment, hope for future progress in the therapy of CML lies in an almost unique feature of this hematological malignancy. In contrast to many other forms or subforms of leukemias which display a great diversity in chromosomal alterations, most cases (>95%) of CML seem to be caused by an almost invariably found cytogenetic aberration, the so-called Philadelphia chromosome (Ph), resulting in the bcr-abl fusion gene. Its gene product, p210^bcr-abl (Bcr-Abl), is believed to be essential for hematopoietic cell transformation and seems to exert its effects by interfering with cellular signal transduction pathways, normally involved in the control of cell death and proliferation. Several partially interacting pathways have been shown to be induced by Bcr-Abl. The role of most of them is still unclear and, as understanding their biological functions should lead to novel therapeutic strategies on a molecular basis, much effort is spent on identifying their precise roles in CML. This review focuses on our current understanding of Bcr-Abl-induced signal transduction and outlines its importance for the biological effects of Bcr-Abl. Received: July 7, 1998 / Accepted: November 26, 1998     

New therapeutic strategies in secondary hyperparathyroidism on dialysis (II): vitamin D analogues and calcium-mimetics

Secondary hyperparathyroidism (SHP) is a frequent complication of dialysis patients. In this second article we will analyze the new vitamin D analogs, capable of decreasing parathyroid hormone (PTH) levels with a lower effect on intestinal calcium and phosphorus absorption. Among other advantages described in the experimental setting, paricalcitol shows a survival benefit in dialysis patients as compared to calcitriol, at least in retrospective studies, and thus it became our first-line vitamin D derivative. Calcimimetics are unique since they decrease PTH levels without increasing serum calcium and phosphorus. Actually, calcium and phosphorus decrease in a significant number of patients. These drugs will soon be authorized in Spain, and we describe the better achievement of K/DOQI guidelines as well as other beneficial effects observed in the experimental animal with them. Finally, we mention the potential benefit of mild metabolic acidosis, the use of bisphosphonates, the role of bone morphogenetic protein BMP-7, and the use of teriparatide. The future treatment of SHP will probably require the independent management of calcium, phosphorus, vitamin D and PTH. Thus, low-dose combined treatments with selective drugs may prove more effective than sequential therapies.     

Cholelithiasis and acromegaly: therapeutic strategies

OBJECTIVE The aims of this study were (i) to evaluate gall-bladder form and contents, (ii) to assess the prevalence of gallstones in acromegalic patients before octreotide treatment and the incidence of gallstone formation in patients with acromegaly during long-term (6–90 months, mean 44 months) octreotide treatment, and (iii) to test the efficacy of ursodeoxycholic acid in preventing and treating octreotide-induced cholelithiasis. DESIGN Forty-nine patients (23 men and 26 women, aged 19–81 years) were studied by repeated gall-bladder ultrasonography performed at baseline and then every 6 months during octreotide therapy. All ultrasound scans were evaluated by the same radiologist. Statistical analysis was performed using the Chi-squared and regression analysis tests. RESULTS Asymptomatic stones were recorded in 13/49 patients (26·5%) prior to octreotide treatment (the prevalence of cholelithiasis in the Italian population is 9·5% in men and 18·9% in women). During octreotide therapy gallstones developed in 10/36 patients (27·7%). No significant correlations with sex, age, body mass index, duration of the disease, daily dose and duration of octreotide therapy, altered gall-bladder form, family history of gallbladder stones, basal plasma values of cholesterol and triglycerides were found between the patients (10/36) who developed stones during octreotide treatment and the ones who did not (26/36). Fourteen patients (10 with newly developed stones and four with cholelithiasis diagnosed prior to octreotide) were put on ursodeoxycholic acid at the daily dose of 10 mg/kg. Gallstones completely disappeared in 6/14 patients (42·8%; five patients with newly developed stones and one with stones prior to octreotide therapy) after a mean of 30·8 months of ursodeoxycholic acid treatment. In addition, seven patients were treated with ursodeoxycholic acid at the preventive dose of 450 mg, administered as a once-a-day oral preparation in the evening. However, stones developed in one of these seven patients who was thereafter cured (gallstones completely disappeared) by the therapeutic dose of ursodeoxycholic acid of 10 mg/kg/day after 23 months of treatment. CONCLUSIONS This study indicates that (i) acromegaly by itself is correlated with a high prevalence of gallbladder stones, (ii) the long-term treatment with octreotide increases the incidence of cholelithiasis, and (iii) ursodeoxycholic acid is useful in the treatment of gallstones in acromegalic patients but its prophylactic effect in patients on octreotide treatment requires further assessment.     

Pathogenesis of metastatic disease: implications for current therapy and for the development of new therapeutic strategies

Different tumor cell subpopulations coexisting within the same tumor exhibit varied susceptibilities to antineoplastic agents. Tumor cell heterogeneity is now recognized as the principal cause of treatment failure in cancer, and is a formidable obstacle to effective therapy and to the development of drug delivery systems for selective targeting of antineoplastic agents to tumor cells. Recent insights into the genesis of tumor cell heterogeneity during progressive tumor growth reveal new complexities that raise challenging questions about the adequacy of certain approaches to the current therapy of metastatic disease and impose challenging criteria for the development of improved therapeutic strategies. Many of the experimental approaches used in the search for new antineoplastic agents and targeted drug delivery systems ignore the pathogenesis of metastasis and the problem of tumor cell heterogeneity. The adoption of more relevant assay systems is an urgent priority. These include the greater use of metastatic tumor models and the increased use of human tumor cells to replace rodent cell systems which have been of limited predictive value in identifying effective anticancer agents. In contrast to current strategies for the development of new antineoplastic drugs which seek to identify agents with activity against a broad range of histologically diverse tumors, greater success may be achieved by seeking agents active only against specific cell lineages. Many established human tumor cell lines may not be suitable for this purpose because of extensive phenotypic change produced by prolonged passage ex vivo. Development of histiotype-specific human tumor cell screens will require an extensive research effort to identify target cells that display demonstrable phenotypic relatedness to tumor cells in neoplastic lesions. Major advances in the therapy of metastatic disease are considered unlikely in the next few years, and progress will stem from improved use of existing agents in refined combination therapy protocols in which greater attention is given to the duration, frequency, and sequence of therapy with different agents to limit emergence of tumor cell variants resistant to one or more antineoplastic agents. Advances in molecular biology offer exciting prospects for the identification of new therapeutic targets in human tumor cells, for the induction of alterations in tumor cells that could serve as therapeutic targets, and for the elucidation of the mechanisms responsible for the rapid phenotypic diversification of tumor cells.(ABSTRACT TRUNCATED AT 400 WORDS)     

Interactions among iron(II) bleomycin, Lewis bases, and DNA.

The interaction of sulfur-containing ligands with complexes of Fe(II) bleomycin [Fe(II)Blm] was investigated by comparing DNA strand-scission activity with the structural characteristics of the complex. The bithiazole ring of excess Blm can form intermolecular complexes with Fe(II)Blm and with NO-Fe(II)Blm, and the bithiazole groups of CO-Fe(II)Blm interact with the metal center. This binding can be reversed by glutathione, CO-Fe(II)Blm binds to poly(dA-dT) . poly(dA-dT) via its bithiazole group to change the environment of the Fe2+ ion. Glutathione binds to the CO-Fe(II)Blm-poly(dA-dT) . poly(dA-dT) complex under conditions in which it stimulates DNA strand scission by Fe(II)Blm.     

Low incidence of bleeding from HIV-related thrombocytopenia in drug addicts and hemophiliacs: implications for therapeutic strategies

The records of 608 intravenous drug addicts and 124 hemophiliacs diagnosed in 1985 to be seropositive for HIV were reviewed to identify patients with thrombocytopenia. The records of 54 drug addicts (8.9%) and 14 hemophiliacs (11.2%) who had chronic thrombocytopenia were then followed through June 1989 to estimate the incidence of bleeding episodes. The records of 55 HIV-seronegative patients with idiopathic thrombocytopenic purpura (ITP), comparable for age, platelet count at diagnosis and duration of follow-up, were used for comparison. Patients with ITP were significantly different from those with HIV-associated thrombocytopenia in having a higher proportion of females (M/F ratio 13/42 v 53/15, p less than 0.001) and more intensive treatment (treated: 62% v 21%, p less than 0.001; splenectomized 37% v 1.5%, p less than 0.001). The incidence of major bleeding was not significantly different in drug addicts, hemophiliacs and ITP patients (0.98, 1.82 and 1.17/100 patients/year). Among drug addicts, bleeding was significantly more frequent in women than in men (3.4 v 0/100 patients/year, p less than 0.05). Our results indicate that the incidence of major bleeding in patients with HIV-associated thrombocytopenia is low and similar to that observed in more intensively treated ITP patients.     

Artificial miRNAs mitigate shRNA-mediated toxicity in the brain: implications for the therapeutic development of RNAi

Huntington's disease (HD) is a fatal, dominant neurodegenerative disease caused by a polyglutamine repeat expansion in exon 1 of the HD gene, which encodes the huntingtin protein. We and others have shown that RNAi is a candidate therapy for HD because expression of inhibitory RNAs targeting mutant human HD transgenes improved neuropathology and behavioral deficits in HD mouse models. Here, we developed shRNAs targeting conserved sequences in human HD and mouse HD homolog (HDh) mRNAs to initiate preclinical testing in a knockin mouse model of HD. We screened 35 shRNAs in vitro and subsequently narrowed our focus to three candidates for in vivo testing. Unexpectedly, two active shRNAs induced significant neurotoxicity in mouse striatum, although HDh mRNA expression was reduced to similar levels by all three. Additionally, a control shRNA containing mismatches also induced toxicity, although it did not reduce HDh mRNA expression. Interestingly, the toxic shRNAs generated higher antisense RNA levels, compared with the nontoxic shRNA. These results demonstrate that the robust levels of antisense RNAs emerging from shRNA expression systems can be problematic in the mouse brain. Importantly, when sequences that were toxic in the context of shRNAs were placed into artificial microRNA (miRNA) expression systems, molecular and neuropathological readouts of neurotoxicity were significantly attenuated without compromising mouse HDh silencing efficacy. Thus, miRNA-based approaches may provide more appropriate biological tools for expressing inhibitory RNAs in the brain, the implications of which are crucial to the development of RNAi for both basic biological and therapeutic applications.     

Relapsing polychondritis: new therapeutic strategies with biological agents

Relapsing polychondritis (RP) is a rare disease of unknown etiology characterized by recurrent episodes of inflammation resulting in the destruction of cartilaginous tissues. We describe a young girl with RP unresponsive to conventional therapy.