Gene therapy in peripheral artery disease

In the last decade, studies of the biological mechanisms underlying angiogenesis, i.e. the development of a new vasculature from pre-existing blood vessels, have suggested a new approach to peripheral obstructive artery disease based on the treatment of ischemic tissues with angiogenic growth factors. As demonstrated by experimental studies in animal models, a therapeutic effect can be reached as the newly formed vascular network, functioning as a biologic by-pass, restores a normal blood supply to the ischemic territories. New techniques of gene therapy proved effective in reaching sustained concentrations of angiogenic factors in the target tissues. This review concerns the pre-clinical background and the results of the early clinical trials of angiogenic gene therapy, which have shown the safety and feasibility of this new approach.     

<Emphasis Type="Italic">NRAS</Emphasis><Superscript><Emphasis Type="Italic">Q61K</Emphasis></Superscript> mutated diffuse leptomeningeal melanomatosis in an adult patient with a brief review of the so-called “forme fruste” of neurocutaneous melanosis

Primary melanocytic tumors of central nervous system represent rare tumors arising from melanocytes of the leptomeninges. These neoplasms include focal forms like melanocytoma and primary malignant melanoma and diffuse forms like leptomeningeal melanocytosis and primary leptomeningeal melanomatosis. The clinical diagnosis remains challenging, with clinical and radiologic features overlapping with other more common diseases. Here we present a case of a 38 years old male with primary diffuse leptomeningeal melanomatosis with presence of a NRAS^Q61K mutation without features of neurocutaneous melanosis.     

Adenovirus-mediated Wild-type p53 Expression Induces Apoptosis and Suppresses Tumorigenesis of Experimental Intracranial Human Malignant Glioma

Adenoviral-mediated gene transfer for the treatment of experimental intrinsic malignant brain neoplasms holds promise. The role, however, of intracellular, adenoviral-mediated p53 expression to inhibit growth of experimental human intracranial malignant gliomas remains largely unexplored. Using the AdCMV.p53 vector we measured the in vitro expression of p53 and the resultant effect upon U251 human malignant glioma cellular proliferation. We further measured the survival of nude mice after intracranial injection of the infected vs. control U251 cells. The growth of the infected U251 cells was inhibited when compared to both the uninfected cells and cells infected with the control vector (AdCMV.Null). Agarose gel electrophoresis confirmed the AdCMV.p53-dependent cellular apoptosis. Nude mice having intracranial injections of the U251 cells infected with the control (AdCMV.Null) vector showed diminished survival. In contrast, mice having intracranial injections of the cells infected with the AdCMV.p53 vector showed 100% survivorship measured 100 days after treatment. Gene therapy via the AdCMV.p53 viral vector holds promise for the clinical treatment of human malignant gliomas.     

PGE(1) short term therapy in critical lower limb ischemia.

AIM: Our study aims to evaluate the efficiency of short-term therapy with alprostadil (a PGE molecular derivative) on patients affected by critical ischemia of the lower limbs and unsuitable for surgical revascularization. The study was carried out on two groups of patients treated with the traditional long-term or a short-term protocol respectively. METHODS: The parameters evaluated and statistically compared to existing studies were, the side effects, subjective pain measured on an analogic scale, objective pain calculated according to analgesic intake, and change in trophic lesions. RESULTS: Our results revealed some differences between the two groups. The manifestation of side effects led to treatment suspension in 8% of long-term therapy cases only. Subjective pain was reduced or disappeared in 83.83% of cases (p<0.001) and there was no statistically significant difference between the two groups. The course of analgesic intake was again similar in both groups. Trophic lesions improved or completely healed in 64.7% (p<0.005) and although a greater tendency towards complete healing was evident in the short-term patients, it was not statistically significant. There was no significant difference between the two groups in the ankle/arm pressure index, but a significant improvement has been observed in 30.88% of cases. The results we obtained from both groups are similar and confirm the valid therapeutic use of alprostadil in patients with peripheral arterial occlusive disease (PAOD). CONCLUSION: Our study reveals the presence of intrinsic advantages to the physician with the short-term schedule, through its higher tolerability, improved and more frequent patient and therapy controls and shorter hospitalization.     

p53 and the hypoxia-induced apoptosis of cultured neonatal rat cardiac myocytes.

Myocyte cell loss is a prominent and important pathogenic feature of cardiac ischemia. We have used cultured neonatal rat cardiac myocytes exposed to prolonged hypoxia as an experimental system to identify critical factors involved in cardiomyocyte death. Exposure of myocytes to hypoxia for 48 h resulted in intranucleosomal cleavage of genomic DNA characteristic of apoptosis and was accompanied by increased p53 transactivating activity and protein accumulation. Expression of p21/WAF-1/CIP-1, a well-characterized target of p53 transactivation, also increased in response to hypoxia. Hypoxia did not cause DNA laddering or cell loss in cardiac fibroblasts. To determine whether the increase in p53 expression in myocytes was sufficient to induce apoptosis, normoxic cultures were infected with a replication-defective adenovirus expressing wild-type human p53 (AdCMV.p53). Infected cells expressed high intracellular levels of p53 protein and exhibited the morphological changes and genomic DNA fragmentation characteristic of apoptosis. In contrast, no genomic DNA fragmentation was observed in myocytes infected with the control virus lacking an insert (AdCMV.null) or in cardiac fibroblasts infected with AdCMV.p53. These results suggest that the intracellular signaling pathways activated by p53 might play a critical role in the regulation of hypoxia-induced apoptosis of cardiomyocytes.     

Incidence of myeloperoxidase deficiency in an area of northern Italy: histochemical, biochemical and functional studies

Forty-five subjects with a complete deficiency of myeloperoxidase were identified in an area of the region Friuli-Venezia Giulia in north-eastern Italy using the Hemalog D system as the screening technique. Histochemical and biochemical tests performed on the leucocytes of some of these subjects confirmed the defects shown by the Hemalog D system. The defect was of genetic origin in seven subjects. The genetic origin could be suspected in another eight subjects since more than two affected members were present in a given family. Eosinophil peroxidase, which is present in MPO deficient subjects, interfered with the guaiacol assay of MPO, and in several cases masked the genetic transmission. An assay was developed using o-dianisidine as the electron donor which considerably reduced the interference by EPO. With this assay an autosomal recessive pattern of inheritance was found. The MPO deficient leucocytes had a higher respiratory burst than control cells and an impaired bactericidal activity, at early post-phagocytic periods, which became comparable to that of control cells at later stages. Particle ingestion by the MPO-deficient cells was normal.     

Remifentanil conscious sedation during regional anaesthesia for carotid endarterectomy: rationale and safety.

OBJECTIVES: to prospectively evaluate the safety and efficacy of remifentanil during regional anaesthesia for carotid endarterectomy. METHODS: twenty-eight consecutive patients underwent carotid endarterectomy with combined superficial and deep cervical plexus block supplemented with continuous intravenous 0.04 microg.kg(-1).min(-1)remifentanil infusion. Depth of sedation was monitored using the Observer's Assessment of Alertness/Sedation Scale (OAA/S). The degree of pain, discomfort and anxiety was self-assessed by the patients using a horizontal visual analogue scale. RESULTS: all patients experienced adequate comfort and analgesia. No local anaesthetic supplementation was necessary. No patient had a OAA/S score lower than 4 (with 5=awake/alert to 1=asleep). Respiratory depression did not occur. Selective shunting was required in four cases. No patient was converted to general anaesthesia. There were no permanent neurological deficits, cardiopulmonary complications or deaths. CONCLUSION: remifentanil as a supplement to regional anaesthesia for carotid endarterectomy, provides comfort and analgesia without hampering mental status evaluation.     

Anti-tumor gene therapy

Gene therapy as an anti-tumor strategy is becoming a powerful tool for cytokine delivery to inhibit the growth of many tumors. Several delivery systems are being utilized and designed for the expression of specific genes to achieve a therapeutic result. Liposomes, retroviral vectors, and adenoviral vectors have all been used and eventual clinical application may depend on the type of tumor, the location, the specific gene carried, and the patients health status. Novel expression vectors may eventually achieve tissue-specific targeting and low immuno-reactivity. Inactivation of mutated oncogenes, such as ras, or re-expression of inactive suppressor genes, such as p53 have been used as strategies for anti-tumor therapy. Additionally, exogenous genes, su ch as viral thymidine kinase that metabolize chemotherapeutic agents to achieve local cytotoxicity have also been employed. Neuro-endocrine tumors are targets of these genetic strategies since they are often difficult to treat by conventional methods bec ause of their location (brain tumors) or because they have spread from the primary tumor (melanoma). Further advances in the design of these vectors may achieve safe targeting of a variety of malignant tumors.     

Adenovirus-mediated gene transfer of wild-type p53 results in melanoma cell apoptosis in vitro and in vivo

Gene transfer techniques may provide efficient treatment for a variety of malignant neoplasms. A replication-deficient adeno-virus (Ad) vector which carries the cDNA for wild-type p53 (AdCMV.p53) was tested for its in vitro and in vivo effects on the growth of murine melanoma cell line B16-G3.26 and human melanoma cell line SK-MEL-24. The growth of B16-G3.26 cells infected with AdCMV.p53 was inhibited when compared to the uninfected cells or cells infected with the control vector AdCMV.NLSβgal. Similarly, the growth of SK-MEL-24 cells infected with AdCMV.p53 was also below that of AdCMV.NLSβgal-infected and uninfected controls. DNA laddering using agarose gel electrophoresis and in situ labeling of DNA fragmentation (TUNEL) showed that AdCMV.p53-infected murine and human melanoma cells underwent apoptosis. Nude mice injected s.c. either with B16-G3.26 cells or with SK-MEL-24 cells developed localized tumors. These tumors were subsequently infiltrated with either AdCMV.p53, AdCMV.NLSβgal or saline alone. One week after infection, B16-G3.26 tumors exposed to AdCMV.p53 were 2.5 times smaller than control tumors and exhibited DNA fragmentation. A similar growth-inhibitory effect of AdCMV.p53 was observed with SK-MEL-24 tumors. Thus, Ad-mediated wild-type p53 overexpression resulted in melanoma cell apoptosis and inhibition of melanoma growth in vitro and in vivo. These gene therapy approaches may be useful in targeting rapidly growing, malignant melanomas in a clinical setting. © 1995 Wiley-Liss, Inc.