A computer-controlled spraying-freezing apparatus for millisecond time-resolution electron cryomicroscopy

The application of gene therapy techniques to the clinical problem of coronary restenosis has generated tremendous attention and enthusiasm. Use of gene transfer technology to prevent a common intractable illness would represent a watershed event for human gene therapy. However, the time is not yet right to initiate gene therapy trials for restenosis. The biology of restenosis is incompletely understood, catheter-based gene delivery is poorly adapted to the coronary circulation, and current gene transfer vectors are ill-suited for safe and effective gene delivery to the coronary artery wall. Basic research designed to overcome these obstacles is currently more appropriate than the initiation of clinical trials.     

The road toward human gene therapy--a 25-year perspective

The concept of human gene therapy arose in the early 1970s and has now been the subject of many clinical trials in human patients. To date, there have been no truly convincing therapeutic successes, but any disappointment with that fact is due more to the exaggerated levels of our expectations than to any failure of concept. The concept of human gene therapy has been a startling success and it is now one of the most important driving forces in medicine. Current tools have simply not been adequate for the difficult job of efficient and stable gene delivery, faithful and regulated gene expression and clinical correction of a human disease. Nevertheless, progress toward human gene therapy is rapid and inexorable and the second phase of human gene therapy, the implementation phase, will soon also succeed.     

Gene therapy of single-gene disorders: preface to the special section

Gene therapy was introduced into clinical practice with great excitement, much publicity and considerable optimism in the early 1990s. Scientific evaluation of the early clinical trials has, however, greatly reduced the initial optimism. Follow-up studies have revealed that many early gene therapy trials mainly represented gene transfer into patients, possibly with short-term effects, but not true gene therapy where the course of the disease is permanently affected. This has lead to critical re-evaluation of the approaches taken. Clearly, more basic understanding is needed of the molecular mechanisms of the diseases treated. For this purpose, better animal models for human diseases are necessary. One of the biggest obstacles for gene therapy has been the lack of adequate vector systems. Development of new vectors for efficient and targeted delivery and uptake of therapeutic genes is a crucial area where progress needs to be made. The rationale for gene therapy depends largely on the type of disease to be treated. Recessively inherited single-gene disorders represent diseases where the concept of gene therapy--addition of a therapeutic gene to restore the lost function of two mutant alleles--is easily understood and rarely questioned. However, most gene therapy protocols are focused on multifactorial diseases such as malignancies where the therapeutic approach is quite different. While gene transfer technologies are being developed into truly effective gene therapy, the fight against inherited single-gene disorders also continues at population level by carrier screening and prenatal diagnostics where rapid methodological developments are taking place.     

Muscle-based gene therapy: realistic possibilities for the future

The past five years have witnessed tremendous growth in the field of gene therapy, with pre-clinical and clinical gene therapy trials for diseases as diverse as cancer, AIDS and atherosclerosis. These studies have utilized many different vectors and target organs in order to achieve therapeutic effects. In this review, we examine the rationale for using skeletal muscle as a target tissue for gene therapy, discuss the wide array of vectors that have been used for muscle-based gene therapy, summarize the disease-targets that have been approached using these techniques, and discuss some of the obstacles that remain to be overcome en route to successful muscle-based human gene therapy.     

The influence of progress in the treatment of severe burns on the quality of life

The problems related to burns treatment can be considered among the oldest and most passionating in history of medicine. Since the early forties amazing progresses have been done in the comprehension of the physiopathology of burns. The fast development of resuscitating techniques determined a remarkable reduction of mortality in the first phase; in a similar way through new concepts in the project and construction of intensive care facilities dedicated to burns, where patients can be isolated and a high standard of environmental control can be guaranteed, together with new topical and systemic antibacterial treatment protocols, a significant reduction of infectious complications has been achieved. Concerning surgical treatment early tangential excision and cultured epidermal grafts can be considered the cornerstones of burn therapy. Quality of life of burnt patients have been greatly ameliorated by these technical advances. Burn sequelae however remain the main concern of survivors because of the many controversial aspects of burn scar physiopathology and treatment. Along my career many endeavours I dedicated in this important research field. I will then report the results of most interesting clinical and experimental studies carried out in the last 30 years by our group in collaboration with basic researchers. All the work done in this domain enhance our hope that good results can really improve quality of life in burns: this is the goal for those who dedicated the whole life to relieve the suffering of these badly injured patients.     

Inhibition of apoptosis in chlamydia-infected cells: blockade of mitochondrial cytochrome c release and caspase activation

Current clinical gene therapy protocols for the treatment of human immunodeficiency virus type 1 (HIV-1) infection often involve the ex vivo transduction and expansion of CD4+ T cells derived from HIV-positive patients at a late stage in their disease (CD4 count <400). These protocols involve the transduction of T cells by murine leukemia virus (MLV)-based vectors encoding antiviral constructs such as the rev m10 dominant negative mutant or a ribozyme directed against the CAP site of HIV-1 RNA. We examined the efficiency and stability of transduction of CD4+ T cells derived from HIV-infected patients at different stages in the progression of their disease, from seroconversion to AIDS. CD4+ T cells from HIV-positive patients and uninfected donors were transduced with MLV-based vectors encoding beta-galactosidase and an intracellular antibody directed against gp120 (sFv 105) or Tat. (sFvtat1-Ckappa). The expression of marker genes and the effects of the antiviral constructs were monitored in vitro in unselected transduced CD4+ T cells. Efficiency and stability of transduction varied during the course of HIV infection; CD4+ T cells derived from asymptomatic patients were transducible at higher efficiencies and stabilities than CD4+ T cells from patients with acquired immunodeficiency syndrome (AIDS). Expression of the anti-tat intracellular antibody was more effective at stably inhibiting HIV-1 replication in transduced cells from HIV-infected individuals than was sFv 105. The results of this study have important implications for the development of a clinically relevant gene therapy for the treatment of HIV-1 infection.     

Is gene therapy in cystic fibrosis a realistic expectation?

To date there are 11 human protocols either ongoing or approved for gene therapy for cystic fibrosis (CF) in the United States. There are also two protocols in the United Kingdom and one in France. Of these, results have been published in four. The protocols vary in the cells targeted, the vectors used, and the frequency of administration, but despite these differences all have contributed toward answering the key questions that will determine the future of gene therapy for CF: the questions of efficacy and safety. These studies have demonstrated that it is feasible to transfer the normal human CF transmembrane conductance regulator complementary DNA to the respiratory epithelium and that this will lead to production of normal CF transmembrane conductance regulator protein and in some cases to a physiologic response. The most frequently used vector is the adenovirus. Obstacles to be overcome with this system include the need for improved and prolonged expression, efficacy on repeat administration, and decreased inflammation. Recent work on the immune response to adenoviral vectors may help achieve these goals. The cationic lipid method of gene delivery is less likely than adenovirus to cause inflammation, at least in the nose, but improved efficacy of gene transfer is necessary as well as improved complex stability. Furthermore, this system has yet to be tested in the lungs of individuals with CF. Finally, the adeno-associated virus, the other vector approved for human gene therapy studies in CF, has shown some promise in preclinical studies but remains to be tested in humans. The results of these studies give some cause for guarded optimism, but point out a number of problems that must be overcome before gene therapy for CF delivers on the promise of a safe effective treatment for this condition.     

Long-term in vitro correction of alpha-L-iduronidase deficiency (Hurler syndrome) in human bone marrow

Allogeneic bone marrow transplantation is the most effective treatment for Hurler syndrome but, since this therapy is not available to all patients, we have considered an alternative approach based on transfer and expression of the normal gene in autologous bone marrow. A retroviral vector carrying the full-length cDNA for alpha-L-iduronidase has been constructed and used to transduce bone marrow from patients with this disorder. Various gene-transfer protocols have been assessed including the effect of intensive schedules of exposure of bone marrow to viral supernatant and the influence of growth factors. With these protocols, we have demonstrated successful gene transfer into primitive CD34+ cells and subsequent enzyme expression in their maturing progeny. Also, by using long-term bone marrow cultures, we have demonstrated high levels of enzyme expression sustained for several months. The efficiency of gene transfer has been assessed by PCR analysis of hemopoietic colonies as 25-56%. No advantage has been demonstrated for the addition of growth factors or intensive viral exposure schedules. The enzyme is secreted into the medium and functional localization has been demonstrated by reversal of the phenotypic effects of lysosomal storage in macrophages. This work suggests that retroviral gene transfer into human bone marrow may offer the prospect for gene therapy of Hurler syndrome in young patients without a matched sibling donor.     

Effect of a traditional Chinese medicine, Bu-zhong-yi-qi-tang on the protection against an oral infection with Listeria monocytogenes

Gene therapy for vascular disease is in the beginning stages. Each year investigators are increasing our understanding of the molecular and cellular biology of vascular disease and its complications. Our genes exert exquisite control over the expressed molecular pattern that results in biological function and pathology. Gene transfer techniques can be used to affect the pattern of gene expression. Gene therapy is a powerful tool that will allow specific manipulation of the genetic cascade that determines biological function. Gene transfer techniques should help to define the molecular mechanisms involved in vascular pathology, such as atherosclerosis and its complications. Currently, gene therapy has only reached clinical trials, but this new technology will likely play a major role in our treatment of vascular problems in the future. An understanding of the significance of this new technology is important for both health care providers and patients.     

Prospects for gene therapy in human prostate cancer

Prostate cancer is the most common neoplasm in men and a significant cause of mortality in affected patients. Despite significant advances, current methods of treatment are effective only in the absence of metastatic disease. Gene therapy offers a renewed hope of using the differential characteristics of normal and malignant tissue in constructing treatment strategies. Several clinical trials in prostate cancer gene therapy are currently under way, using immunomodulatory genes, anti-oncogenes, tumor suppressor genes and suicide genes. A continued understanding of the etiological mechanisms involved in the establishment and progression of prostate cancer, along with advances in gene therapy technology, should make gene therapy for prostate cancer therapeutically valuable in the future.     

An aggressive approach to HIV antiretroviral therapy

The introduction of highly active antiretroviral therapy offers the first real hope of durable control of HIV infection and prevention of clinical sequelae. But success hinges on early and complete suppression of viral replication. That demands near-perfect adherence to a complex regimen involving three or more drugs, each with its own dosage and dietary requirements.     

Inhaled glucocorticosteroids in treatment of bronchial asthma

Studies of bronchial asthma pathogenesis, conducted over many years, have shown that inflammatory process plays a major role in the development of this disease. Glucocorticosteroids are agents with strongest antiinflammatory activity and together with beta 2-agonists are thought of as firstline therapy in the treatment of bronchial asthma. New inhaled glucocorticosteroids are preparations with small systemic activity, despite the fact, that some of them are more lipophyllic than the others, despite the differences in solubility, affinity to lung tissue, different serum concentrations and affinity to receptors. Even with the tremendous progress in increasing efficacy and lowering side effects of inhaled glucocorticosteroids, no ideal preparation was found so far, however the antiinflammatory activity was increased and the dose lowered, which means increasing clinical efficacy and lowering the systemic effects. Only in the future we can hope for developing an ideal preparation of inhaled glucocorticosteroid with common specificity towards lung tissue and direct influence on the target cell, and maybe even on a specific gene.     

Gene therapy of central nervous system tumors

Recently, remarkable advances have been achieved in molecular and genetic researches of different kinds of general diseases, as well as in basic and clinical studies using gene therapy for central nervous system diseases. For brain tumors, clinical trials have been already started in more than 10 clinical protocols and more than 100 patients with malignant brain tumors. Nevertheless, there are still major issues that remain to be resolved for achieving better clinical results, such as delivery system of genetic material, regulatory methods of the intracellular expression of the transgene, antitumor efficacy, and tumor selectivity. In this paper, molecular genetic studies and the current state of gene therapy for neurological diseases, especially brain tumors, are described, and the future direction of this fascinating approach is discussed.     

Gene therapy of cancer

Retroviral-mediated gene transfer has permitted the development of clinical protocols for the study and treatment of cancer. These protocols can be divided into gene-labeling and gene therapy proposals. Labeling studies include the tracking of tumor infiltrating lymphocytes (TIL) following the administration of those cells, and the detection, at the time of relapse, of tumor cells from transplanted autologous bone marrow. Most gene therapy protocols are designed to induce an immune attack against the tumor by inserting genes into tumor cells themselves. Although uncertainty about the safety of the procedure still exists, gene therapy of cancer holds much promise as an effective treatment modality.     

Toxicity of CNS prophylaxis for childhood leukemia

Long-term neurotoxicity associated with central nervous system (CNS) prophylaxis for childhood acute lymphoblastic leukemia (ALL), primarily involving physical growth and cognitive development, is an ongoing concern. Although contemporary treatment protocols are associated with less severe toxicity than was commonly observed with earlier protocols, there continue to be late effects. When treatment includes cranial radiation therapy (CRT), linear growth is likely to be impaired. Female patients are more vulnerable to late cognitive effects than male patients, particularly when treated with CRT and high doses of methotrexate. Young age at treatment is a risk factor as well, particularly for girls. In order to treat medical and psychological problems early, a commitment to long-term follow-up is essential.     

Gene testings in relation with gene diagnosis and therapy

Recent advances in the molecular biology research have devoted greatly to the clinical applications of genetic informations. Gene diagnosis and therapy is a new medical field to deal with these applications. Nowadays diseases related with gene abnormalities have been shown to reach 8,450, and gene testings to cover all these should be very wide and variable. So that any one of the clinical laboratories in a hospital or even research institutes and laboratory centers should not be able to handle all the testings. A model system to promote gene testings effectively in Japan, which includes a special department in a hospital and limited specifications to the individual laboratories or centers, were presented. In the case of monogenic genetic disorders, gene testing results are useful not only to confirm clinical diagnosis but to predict future development of disorders preclinically and even during the prenatal or pregestational period. Accordingly, these may evoke critical ethical and social problems. Many of common diseases are polygenic in nature and gene testings in these disorders are not directed to the diagnosis but are quite useful to see the individual characteristics of the patients related to the special phenotypes or even the fate of disease process. Acquired malignancies are known to be resulted from a somatic gene mutation and progress by the associations of further abnormalities. Only limited gene testings are already now in use. Wide future development in this field is expected after getting detailed meanings of gene testings in the individual cancers. Gene testings for infectious diseases are known to be quite effective on the early and accurate diagnosis, and many of these are already covered by the health insurance. In conclusion, gene testings are quite important but variable and laboratory personnel should not be able to deal all of these properly. Considering future development of gene therapy, rapid establishment in a hospital of a special department, Department of Gene Diagnosis and Therapy, which is consisted from 3 divisions such as genetic counseling, gene testing and management, and monitoring and adviser of gene therapy, is highly recommended.     

Molecular surgery for human colorectal cancer with tumor suppressor p53 gene transfer

Recent advances in molecular biology have demonstrated that multistep genetic alterations are involved in the carcinogenesis of human colorectal cancer and that alteration of the p53 gene by mutation, deletion, or rearrangement is a major factor in this process. Human gene therapy has become a reality with the development of effective techniques for delivering the gene to the target cells. The efficacy of gene therapy for various types of genetic disease now being evaluated in clinical trials. These findings led us to develop a novel gene therapeutic strategy for human colorectal cancer that could replace the abnormal p53 gene using a recombinant, replication-defective adenoviral vector (termed Adp53). Infection with Adp53 induced rapid apoptotic cell death in DLD-1 and LoVo human colorectal cancer cell lines differing in their p53 status. Treatment with cisplatin following infection with Adp53 significantly suppressed the growth of WiDr colorectal cancer cells compared to single treatments alone. Thus restoration of wild-type p53 function exhibited an antitumor effect by inducing apoptosis as well as by markedly enhancing the effect of common chemotherapeutic agents in human colorectal cancer cells. In addition, Adp53 infection was antiangiogenic in SW620 human colorectal cancer cells. The application of this technology to human cancer therapy is now in progress. The article reviews recent highlights in this rapidly evolving field.     

Radiotherapy of follicle center lymphoma. Results of a German multicenter and prospective study. Members of the Study Group "NHL-early stages"

PURPOSE: Follicle centre lymphoma grade I, II (REAL) or centroblastic-centrocytic lymphoma (Kiel classification) present a well defined clinical entity from a clinical point of view. These lymphomas are not curable by chemotherapy in early or advanced stages. They are treated by radiation therapy in early stages, but up to now the curative potency of radiotherapy has not been confirmed by prospective clinical trials. PATIENTS AND METHODS: Between January 1986 and August 1993 117 adults with follicle centre lymphoma were recruited from 24 institutions to enter the multicentric prospective, not randomised clinical trial. Patients with histologically proven nodal follicle centre lymphoma of stages I, II and limited III were included. They were treated by a standardised radiotherapy regimen, in stage I by extended field and in stages II and III by total nodal irradiation. Dose per fraction was 1.8 to 2.0 Gy, in the abdominal bath 1.5 Gy up to a total dose of 26 Gy in adjuvant situation and 36 Gy to enlarged lymphoma. RESULTS: All patients developed a complete remission at the end of radiotherapy. Median follow-up is 68 months. Overall survival of all patients in 86 +/- 3% at 5 and 8 years. Stage adjusted survival at 5 and 8 years was 89% for stage I, 86% for stage II and 81% for III. Patients in stages I and II < 60 years had survival rates of 94% at 5 and 8 years, patients > 60 years 63% (p < 0.0001). Recurrence free survival of all patients is 70% at 5 and 60 +/- 5% at 8 years. The number of recurrences is high with 29% at 5 and 41% at 8 years. All recurrences were seen within 7 years. The probability of localised nodal in-field recurrences is 11% and 22% at 5 and 8 years, respectively. Adverse prognostic factors were identified by multivariate analysis: age > 60 years, treatment breaks > or = 7 days and dose deviations > 20% from prescribed doses. Acute side effects of extended field irradiation were moderate. CONCLUSIONS: On the basis of these results radiotherapy is a potentially curative therapeutic approach in stages I, II and limited III of follicle centre lymphoma. The optimal technique is total lymphoid irradiation with doses of 30 Gy in the adjuvant situation and 40 to 44 Gy in enlarged lymphomas. The number of local recurrences leads to the assumption, that the extension of radiotherapy to the total lymphoid system might reduce their frequency.     

Human somatic cell gene therapy

The prelude to successful human somatic gene therapy, i.e. the efficient transfer and expression of a variety of human genes into target cells, has already been accomplished in several systems. Safe methods have been devised to do this using non-viral and viral vectors. Potentially therapeutic genes have been transferred into many accessible cell types, including hematopoietic cells, hepatocytes and cancer cells, in several different approaches to ex vivo gene therapy. Successful in vivo gene therapy requires improvements in tissue-targeting and new vector design, which are already being sought. Gene-transfer protocols have been approved for human use in inherited diseases, cancer and acquired disorders. Although the results of these trials to date have been somewhat disappointing, human somatic cell gene therapy promises to be an effective addition to the arsenal of approaches to the therapy of many human diseases in the 21st century if not sooner.