Gonadotropin-releasing hormone and its receptor in normal and malignant cells.

Gonadotropin-releasing hormone (GnRH) is the hypothalamic factor that mediates reproductive competence. Intermittent GnRH secretion from the hypothalamus acts upon its receptor in the anterior pituitary to regulate the production and release of the gonadotropins, LH and FSH. LH and FSH then stimulate sex steroid hormone synthesis and gametogenesis in the gonads to ensure reproductive competence. The pituitary requires pulsatile stimulation by GnRH to synthesize and release the gonadotropins LH and FSH. Clinically, native GnRH is used in a pump delivery system to create an episodic delivery pattern to restore hormonal defects in patients with hypogonadotropic hypogonadism. Agonists of GnRH are delivered in a continuous mode to turn off reproductive function by inhibiting gonadotropin production, thus lowering sex steroid production, resulting in medical castration. They have been used in endocrine disorders such as precocious puberty, endometriosis and leiomyomata, but are also studied extensively in hormone-dependent malignancies. The detection of GnRH and its receptor in other tissues, including the breast, ovary, endometrium, placenta and prostate suggested that GnRH agonists and antagonists may also have direct actions at peripheral targets. This paper reviews the current data concerning differential control of GnRH and GnRH receptor expression and signaling in the hypothalamic-pituitary axis and extrapituitary tissues. Using these data as a backdrop, we then review the literature about the action of GnRH in cancer cells, the utility of GnRH analogs in various malignancies and then update the research in novel therapies targeted to the GnRH receptor in cancer cells to promote anti-proliferative effects and control of tumor burden.

Management of acute epididymitis: are European guidelines being followed?

OBJECTIVE: Acute epididymitis is increasing in men aged 35 years or under due to sexually-transmitted Chlamydia trachomatis. This study examines whether Urological surgeons are following European guidelines for the management of acute epididymitis in these patients. METHODS: A postal questionnaire survey was conducted of specialists in Urology in two regions of the UK. RESULTS: Of 79 completed replies, 41 (52%) take a detailed sexual history but only 34 (43%) refer patients to a Genitourinary medicine clinic. Quinolones are the most commonly prescribed first-line antibiotic by 56 (71%) respondents, principally ciprofloxacin. CONCLUSIONS: The current management of acute epididymitis in young men must be improved. Ciprofloxacin is not the optimal antimicrobial for the treatment of urogenital chlamydial infection. We recommend that all such patients be referred to local Genitourinary medicine services for contact-tracing and treatment of their sexual partners.

Prospective randomized studies for carotid endarterectomy.

The clinical trial has become the standard method used to evaluate surgical procedures. Regarding carotid endarterectomy, clinical trials have reformed the indications for surgery as a means of decreasing the risk of stroke. The methodology and results from significant trials for the symptomatic and asymptomatic patient with carotid stenosis are described. Critical evaluation of these trials is necessary for the discerning surgeon to form a rational approach to clinical practice in carotid disease.

Hemifacial spasm due to a large distant ipsilateral posterior fossa meningioma.

A rare case of hemifacial spasm due to an ipsilateral foramen magnum/clival meningioma is described. Magnetic resonance imaging demonstrated that the tumor was located distant to the cranial nerve VII/VIII complex. Resolution of the ipsilateral hemifacial spasm was noted after complete resection of the tumor. The mechanism of hemifacial spasm was likely due to displacement and distortion of the brain stem from the lesion distant to the cranial nerve VII/VIII complex. In our review of the literature this is the first reported case of an ipsilateral posterior fossa meningioma causing hemifacial spasm from indirect mass effect.

Depletion of CD34+ CD4+ cells in bone marrow from HIV-1-infected individuals.

Pancytopenia as a consequence of bone marrow abnormalities is commonly seen in HIV-infected individuals. To examine the effect that HIV-1 has on hematopoietic cells, we compared hematopoietic properties of bone marrow samples from HTV+ patients at various stages of disease with bone marrow samples from uninfected donors. While the absolute number of recovered CD34+ cells and the cloning efficiency of these cells did not differ significantly in HIV+ donors, the percentage of CD34+ CD4+ cells was significantly depleted in late-stage HIV+ patients. We observed a direct correlation between the numbers of CD34+ CD4+ cells in the bone marrow and the peripheral CD4 count. Further characterization of the CD34+ CD4+ subpopulation demonstrated that these cells expressed lower levels of HLA-DR on their surface compared with CD34+ CD4- cells, suggesting an immature phenotype. We also found evidence for expression of HIV-1 coreceptors CXCR-4 and CKR-5 message and protein in CD34+ bone marrow cells. While this finding suggested that hematopoietic cells might be susceptible to HIV infection at an early stage of maturation, thus affecting different cell lineages as they matured, we did not find any evidence for infection of HIV in these cells. These data suggest that HIV affects early hematopoietic progenitor cells either directly or indirectly, and in particular CD34+ CD4+ cells. This finding has important implications for disease pathogenesis and for application of gene therapy approaches that use CD34+ hematopoietic cells.

Diphtheria toxin A gene-mediated HIV-1 protection of cord blood-derived T cells in the SCID-hu mouse model.

The reconstitutive potential of CD34+-derived cord blood (CB) cells, transduced with a regulated diphtheria toxin A (DT-A) chain gene, was examined in SCID-hu mice harboring a conjoint organ composed of human thymus and liver (thy/liv). The DT-A-transduced cells, injected directly into the thy/liv organ, showed the same engraftment potential as control CB cells transduced with the non-DT-A parental vector. CB cells, distinguishable from the thy/liv cells by the HLA marker B7, were preferentially maintained in ex vivo culture. In the thy/liv organ, the engrafted CB cells represented >80% of the total cells. A majority of cells (>70%) in the thy/liv organ were also CD4+CD8+, as would be expected of maturing thymocytes. The incidence of double-positive cells was highest at 44 days (compared with 30 days and 80 days) after injection of CB cells. This suggested that a minimum time was required to achieve optimal proliferation of cells in the thy/liv organ but that, at later times, all of the early cells had matured. Thus, the population used for engraftment contained early cells but not self-renewing cells. The double-positive cells matured rapidly into single-positive cells (either CD4+ or CD8+) when placed in ex vivo culture. Marked cells (neo+) could readily be detected in the thy/liv-derived cells. The cells transduced with DT-A showed long-term protection in ex vivo culture against HIV T lymphotropic isolate NL4-3. This study shows that DT-A-transduced cells had no apparent disadvantage in engraftment of the thy/liv organ and did not have any toxic effects in vivo. Such cells were protected against HIV infection even when challenged more than 2 months after transduction and after a 44-day engraftment period in the thy/liv mice. These data support the feasibility of toxin gene therapy as a strategy for HIV infection.

Lipid- and adenoviral-mediated gene transfer into AIDS-Kaposi's sarcoma cell lines.

Kaposi's sarcoma (KS) is the most frequent malignancy occurring in HIV-positive individuals. AIDS-KS is a more aggressive disease than the classical form, frequently having a rapid clinical course with numerous serious complications. Current systemic treatments for KS, such as chemotherapy and the administration of biological modifiers, are complicated by both the drug resistance of the tumor and the dose-limiting toxicity of the reagents. The relative accessibility of many KS lesions makes the disease a particularly attractive candidate for in vivo gene therapy protocols. In this regard, we are interested in delivering conditionally toxic suicide and/or antiangiogenic vectors to accomplish targeted cell death selectively in AIDS-KS cells. To this end, we examined both cationic lipid- and adenoviral-mediated DNA transfection methods. Using the firefly luciferase reporter gene, we optimized numerous variables known to be important in lipid-mediated DNA transfection, including lipid formulation, the amount of lipid and DNA, lipid/DNA ratio, and cell concentration. Under optimal transfection conditions, approximately 5-25% of KS cells expressed the introduced DNA sequences. Adenoviral-mediated DNA delivery was more efficient than lipid delivery in 4 of 5 primary KS cell lines. Two of the lines (RW248 and RW376) were transduced by adenovirus at frequencies approaching 100%; two cell lines (CVU-1 and RW80) gave efficiencies of 20-35%. Two immortalized KS cell lines (KS Y-1 and KS SLK) were poorly infected, giving a transduction efficiency of <5%. These findings demonstrate that gene transfer into AIDS-KS cells is feasible, and suggest that vector strategies may be permissive for translating gene therapy approaches for the disease.

Current challenges of gene therapy for prostate cancer.

Gene therapy for prostate cancer faces hurdles similar to those being encountered for other cancers and nonmalignant processes. The greatest obstacle is the identification of efficient delivery systems, since numerous animal models and cell culture systems have shown potential efficacy when most cells express the introduced genetic material. Early prostate cancers are easily accessible to gene vector introduction, and the predictable metastatic patterns of this cancer may offer additional advantages for gene therapy. This article reviews gene vectors and gene products, as well as ongoing trials of gene therapy that have recently begun in prostate cancer.

Cationic liposome-mediated expression of HIV-regulated luciferase and diphtheria toxin a genes in HeLa cells infected with or expressing HIV.

HIV-regulated expression of the diphtheria toxin A fragment gene (HIV-DT-A) is a potential gene therapy approach to AIDS. Since cationic liposomes are safe and non-immunogenic for in vivo gene delivery, we examined whether LipofectAMINE or DMRIE reagent could mediate the transfection of HIV-DT-A (pTHA43) or the HIV-regulated luciferase gene (pLUCA43) into HIV-infected or uninfected HeLa cells. pLUCA43 was expressed at a 10(3)-fold higher level in HeLa/LAV cells than in uninfected HeLa cells, while the extent of expression of RSV-regulated luciferase was the same in both cell lines. Co-transfection of HeLa cells with pTHA43 and the proviral HIV clone, HXB deltaBgl, resulted in complete inhibition of virus production. In contrast, the delivery of HIV-DT-A to chronically infected HeLa/LAV or HeLa/IIIB cells, or to HeLa CD4+ cells before infection, did not have a specific effect on virus production, since treatment of cells with control plasmids also reduced virus production. This reduction could be ascribed to cytotoxicity of the reagents. The efficiency of transfection, as measured by the percentage of cells expressing beta-gal, was approximately 5%. Thus, cationic liposome-mediated transfection was too inefficient to inhibit virus production when the DT-A was delivered by cationic liposomes to chronically- or de novo- infected cells. However, when both the virus and DT-A genes were delivered into the same cells by cationic liposomes, DT-A was very effective at inhibiting virus production. Our results indicate that the successful use of cationic liposomes for gene therapy will require the improvement of their transfection efficiency.

Improved production of recombinant AAV by transient transfection of NB324K cells using electroporation.

Adeno-associated virus (AAV) is useful as an integrating vector for gene transfer. AAV recombinants are generally produced by transient co-transfection methods since it has proven difficult to generate stable packaging cell lines. Acceptable titers of transducing recombinants should be obtainable by optimizing conditions for transient co-transfection. Here, using a luciferase reporter derivative of the AAV infectious plasmid psub201, we show that substantially higher yields of transducing virus can be obtained using electroporation, rather than calcium phosphate transfection. Furthermore, we observed that electroporation of NB324K cells (an SV40-transformed human cell line) with the helper plasmid, pAAV/Ad, with concomitant adenovirus dl309 infection, gave yields of luciferase transducing recombinant AAV equal or superior to those obtained from the more commonly employed 293 cells. NB324K cells are easier to manipulate and show increased cell-association of the recombinant virus (facilitating its concentration and purification). We also adapted an in situ infected cell hybridization procedure, using a digoxigenin labeled probe, as a general method for determining infectious titer. Titers thus estimated were similar for luciferase-transducing and for alkaline phosphatase-transducing AAV vectors: the estimated titer of the latter agreed with that determined by in situ expression of alkaline phosphatase. We also describe a multiple cloning site derivative of psub201 which should facilitate generation of further AAV recombinants.

Comparison of retroviral-mediated gene transfer into cultured human CD34+ hematopoietic progenitor cells derived from peripheral blood, bone marrow, and fetal umbilical cord blood.

Genetic alteration of stem cells ex vivo followed by bone marrow transplantation could potentially be used in the treatment of numerous diseases and malignancies. However, there are many unanswered questions as to the best source of hematopoietic cells for long-term reengraftment and the most effective way to introduce foreign genes into this target cell. We have compared retroviral-mediated gene transfer into CD34+-enriched cells derived from peripheral blood (PB), bone marrow (BM), or fetal umbilical cord blood (CB). Cells from all three sources that had been expanded ex vivo in the presence of stem cell factor (SCF), interleukin-3 (IL-3), IL-6, and granulocyte colony-stimulating factor (G-CSF) showed transduction efficiencies ranging from 5-45%, as measured by acquisition of G418 resistance. The average efficiencies of gene transfer from multiple experiments for PB, BM, and CB were not statistically different. To determine the effect of ex vivo expansion on gene transfer into CB CD34+ cells, we compared the transduction efficiencies of cells exposed to virus immediately after harvest and CD34 selection or after 6 days of culture CD34+ CB cells were more effectively transduced after expansion in culture, showing gene transfer efficiencies 3- to 5-fold higher on day 6 compared with day 0. Last, we examined retroviral transduction via spinoculation of CB CD34+ cells and found it to be approximately as effective as our standard transduction with no significant loss of cell viability as measured by colony formation in semi-solid medium.

HIV-regulated diphtheria toxin A chain gene confers long-term protection against HIV type 1 infection in the human promonocytic cell line U937.

Gene therapy approaches have recently been investigated for the treatment of acquired immunodeficiency syndrome (AIDS), both in preclinical and clinical studies, because more traditional antiviral agents have proven to be of limited effectiveness. We have previously shown that long-term protection against both laboratory and clinical isolates of human immunodeficiency virus type 1 (HIV-1) was conferred by HIV-regulated diphtheria toxin A (DT-A) chain in a human T cell line. Because the monocyte/macrophage cell is an important reservoir for HIV-1 in infected individuals, we sought here to determine whether HIV-regulated DT-A would also be effective in the promonocytic cell line U937. We report here that long-term protection, conferred by HIV-regulated DT-A, was observed in U937 cells, but that protection was dependent on the stock of HIV IIIB used for challenge. HIV production was measured by p24 assays, polymerase chain reaction (PCR) for HIV vif, gag, and reverse transcriptase (RT) sequences, and cocultivation with peripheral blood mononuclear cells (PBMCs). Complete protection was seen in DT-A-transduced cells with a stock of IIIB propagated on H9 cells and titered on peripheral blood mononuclear cells (PBMCs), while protection in these same cells with a second stock of IIIB, propagated and titered on H9 cells, was only partial and dose dependent.

Optimization of gene transfer using cationic lipids in cell lines and primary human CD4+ and CD34+ hematopoietic cells.

Cationic lipids offer several advantages for gene delivery, both in vitro and in vivo. However, high-efficiency gene transfer has been demonstrated only for limited cell types. Here, we examine the level of expression of a luciferase reporter gene, delivered using cationic lipids, in both cell lines and primary human cells including peripheral blood mononuclear cells and CD34(+)-enriched hematopoietic cells. Variables shown to affect the efficiency of gene expression included the type of lipid, the amounts of DNA and lipid, the day of assay following transfection, the media used for lipid:DNA complex formation, the cell number, the promoter driving expression of the reporter gene and the physiological state of the cells (e.g., whether or not cells were differentiated). The maximal luciferase expression observed with the primary cells was one to two orders of magnitude lower than that seen in cell lines. Further studies, possibly involving altering the growth conditions for the cells, or using episomal vectors that will allow extrachromosomal maintenance of the DNA, are required to improve the level of transgene expression in the primary human cell types used here.

Efficient foreign gene expression in Epstein-Barr virus-transformed human B-cells.

Epstein-Barr virus (EBV) is a herpesvirus that transforms B-cells (B-LCL) and has undergone intense scrutiny owing to its association with Burkitt's lymphoma, nasopharyngeal carcinoma, and immunoblastic lymphomas. B-LCL have also proven useful in the study of human immunology. We describe a novel system for inducing efficient foreign gene expression in B-LCL using biotinylated adenovirus as an endosome-disrupting agent. Plasmid DNA is coupled to the exterior of viral particles by streptavidin-polylysine chimeric proteins. Up to 67% of B-LCL may be induced to express foreign genes in vitro in transient expression systems, and gene expression lasts for at least 17 days. We have expressed firefly luciferase, beta-galactosidase (beta-gal), chloramphenicol acetyltransferase, HIV gag, and env genes, as well as infectious HIV, and the EBV-specific BZLF gene in B-LCL with this system. In vivo delivery of a beta-gal reporter gene to B-LCL was documented in a SCID mouse model. Potential applications include study of genetic regulation of EBV infection and transformation events, study of potential gene therapies for EBV-related B-cell tumors, and production of antigen-presenting cells for use in immunologic assays. Because of the high percentage of cells transformed and the length of foreign gene expression, the possibility of examining foreign gene expression in transient assays, without selection for clonal populations, exists.

Long-term inhibition of clinical and laboratory human immunodeficiency virus strains in human T-cell lines containing an HIV-regulated diphtheria toxin A chain gene.

The human immunodeficiency virus (HIV) causes persistent infection of T cells. Chemotherapy for infection in humans may slow HIV-related disease progression, but it does not eradicate virus. Thus, other treatment modalities are warranted. We have previously demonstrated that the human T cell line H9, ordinarily permissive for HIV infection, may be protected against infection with the LAI strain of HIV by intracellular immunization with the gene encoding diphtheria toxin A chain (DT-A) under the control of HIV Tat and Rev. Cloned cells were protected for up to 6 days in vitro. We now report protection against the LAI laboratory isolate for up to 59 days, and against clinical HIV strains of differing phenotypic properties and cell tropisms for up to 59 days. In some cases, protection was complete in that no residual HIV was detected by HIV p24 antigen production, co-culture with parental H9 cells, or the polymerase chain reaction (PCR). CD4+ surface expression of DT-A transduced cloned H9 cells was similar to parental H9 in most cases. These results suggest that toxin gene therapy for HIV infection may ultimately be feasible.

Effects of enhancer mutations on the expression of human immunodeficiency virus 1-regulated luciferase and diphtheria toxin A chain genes in transfected cells.

This study explores human immunodeficiency virus 1 (HIV-1)-regulated diphtheria toxin A (DT-A) gene expression as a means of eradicating HIV-infected cells. Previously, we constructed luciferase and DT-A plasmids, containing cis-acting Tat and Rev responsive elements, which showed low basal expression and required both Tat and Rev for maximal expression. Cell lines which had stably integrated the DT-A constructs were resistant to HIV production. To reduce toxicity due to basal expression, this study investigates the effect of mutations in the HIV enhancer on expression of luciferase and DT-A plasmids. Some mutations were found to substantially reduce basal expression while still allowing for trans-activation. Such mutations, in combination with attenuated versions of DT-A, may make regulated toxin gene expression feasible as a therapy for AIDS.

Inhibition of human immunodeficiency virus-1 production resulting from transduction with a retrovirus containing an HIV-regulated diphtheria toxin A chain gene.

Expression of a gene encoding the diphtheria toxin A (DT-A) chain, under the control of human immunodeficiency virus-1 (HIV-1) proteins Tat and Rev, has previously been shown to confer on cells an impaired ability to produce HIV. That work was done in HeLa cell lines that had stably integrated the regulated DT-A gene in a plasmid context. To increase the efficiency with which the HIV-regulated DT-A gene could be introduced into cells, we studied a recombinant, amphotropic murine leukemia virus containing the HIV-regulated DT-A transcription unit. Here we demonstrate that such recombinant retroviruses can be packaged, for both wild-type DT-A and an attenuated version, tox 176. In transient transfection assays, the proviral constructs exhibited similar basal and trans-activated levels of DT-A expression to the parental plasmids. Transduced H9 cells expressed the integrated DT-A gene upon transfection with plasmids encoding Tat and Rev, as assayed by decreased expression of a cotransfected luciferase reporter gene. Furthermore, the transduced H9 cells were substantially impaired in their ability to produce HIV, as demonstrated by p24 assays of culture supernatants following either transfection with an HIV proviral clone or infection with HIV-IIIB. These data demonstrate that basal expression of the regulated DT-A gene has been reduced to a tolerable level, both in packaging cells and transduced H9 cells. The use of HIV-regulated retroviruses encoding the highly lethal DT-A product may eventually be applicable as a gene therapy approach for the acquired immunodeficiency syndrome (AIDS).