Therapeutic efficacy of the thymidine kinase/ganciclovir system on large experimental gliomas: a nuclear magnetic resonance imaging study.
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Contradictory experimental results and human trials have questioned the clinical relevance of the HSVtk/ganciclovir system. To bypass the problem of transfection efficiency, we used a glioma cell line stably expressing the HSVtk gene, which was also fully characterized from gene to protein. We also designed a more clinically relevant experimental protocol, consisting of late GCV delivery on large tumor formations. In short-term studies, histological examination revealed a significant decrease in tumor volume in GCV-treated animals from day 1 or from day 10 after cell inoculation. We observed that late GCV delivery is as efficient as early delivery, probably because GCV can reach tumor cells more easily when neoangiogenesis occurs. In long-term experiments, the survival of treated rats bearing 15-day tumors was improved by 60% compared with C6 control animals. Surprisingly, a 30% survival rate was observed in C6TK control animals. Nuclear magnetic resonance imaging demonstrated, in all surviving animals, a complete regression of tumors without mass effect. These results clearly demonstrate that the HSVtk/GCV system remains a potent therapeutic strategy, even when tested in large tumors, in contrast with the microscopic tumor formations previously reported. (+info)
Ganciclovir-mediated in vivo elimination of myeloid leukemic cells expressing the HSVtk gene induces HSVtk loss variants.
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The in vivo elimination of suicide gene-expressing tumor cells with prodrug treatment can induce protective immunity against wild-type tumors. In this study, we determined the efficacy and safety of the in vivo elimination of HSVtk expressing cells with ganciclovir treatment of a bystander cell killing-insensitive leukemic cell line. The retroviral construct pLTk+NeoDeltaMo, containing the HSVtk gene and the NeoR gene in a bicistronic unit, was introduced into rat leukemic LT12 cells. LT12/Tk+N cells showed a 1000- to 10 000-fold increased sensitivity to ganciclovir in vitro. In vitro mixing experiments demonstrated that LT12 cells were not susceptible to bystander cell lysis by LT12/Tk+N-2 cells exposed to ganciclovir. Rats injected s.c. with cloned LT12/Tk+N-2 cells developed tumors reaching a diameter of 3-4 cm after 40 days. Rats treated with gan- ciclovir twice daily for 5 consecutive days starting at day 7 did not develop s.c. tumors. Large established s.c. LT12/Tk+N-2 tumors completely regressed after ganciclovir treatment. However, recurrences of s.c. tumors were observed that were no longer sensitive to ganciclovir treatment. In vitro analysis of aspirates from the recurrent tumors demonstrated loss of HSVtk expression. In vitro culture of LT12/Tk+N-2 cells in soft agar in the presence of ganciclovir indicated that the frequency with which HSVtk-loss variants occurred is approximately one per 104 cells. The in vivo occurrence of HSVtk-loss variants escaping ganciclovir-induced elimination may have important implications for vaccination protocols using HSVtk gene expressing tumor cells that are not susceptible to bystander cell killing. (+info)
Targeted ablation of secretin-producing cells in transgenic mice reveals a common differentiation pathway with multiple enteroendocrine cell lineages in the small intestine.
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The four cell types of gut epithelium, enteroendocrine cells, enterocytes, Paneth cells and goblet cells, arise from a common totipotent stem cell located in the mid portion of the intestinal gland. The secretin-producing (S) cell is one of at least ten cell types belonging to the diffuse neuroendocrine system of the gut. We have examined the developmental relationship between secretin cells and other enteroendocrine cell types by conditional ablation of secretin cells in transgenic mice expressing herpes simplex virus 1 thymidine kinase (HSVTK). Ganciclovir-treated mice showed markedly increased numbers of apoptotic cells at the crypt-villus junction. Unexpectedly, ganciclovir treatment induced nearly complete ablation of enteroendocrine cells expressing cholecystokinin and peptide YY/glucagon (L cells) as well as secretin cells, suggesting a close developmental relationship between these three cell types. In addition, ganciclovir reduced the number of enteroendocrine cells producing gastric inhibitory polypeptide, substance-P, somatostatin and serotonin. During recovery from ganciclovir treatment, the enteroendocrine cells repopulated the intestine in normal numbers, suggesting that a common early endocrine progenitor was spared. Expression of BETA2, a basic helix-loop-helix protein essential for differentiation of secretin and cholecystokinin cells was examined in the proximal small intestine. BETA2 expression was seen in all enteroendocrine cells and not seen in nonendocrine cells. These results suggest that most small intestinal endocrine cells are developmentally related and that a close developmental relationship exists between secretin-producing S cells and cholecystokinin-producing and L type enteroendocrine cells. In addition, our work shows the existence of a multipotent endocrine-committed cell type and locates this hybrid multipotent cell type to a region of the intestine populated by relatively immature cells. (+info)
Identification and sequence analysis of the Marek's disease virus serotype 2 homologous genes of the herpes simplex virus type 1 UL25, UL26 and UL26.5 genes.
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We identified and determined the nucleotide sequence of Marek's disease virus serotype 2 (MDV2) UL25, UL26 and UL26.5 homologous genes of herpes simplex virus type 1 (HSV-1). The UL25, UL26 and UL26.5 genes of HSV-1 encode virion proteins (UL25 and UL26.5) and serine protease (UL26). The deduced amino acid sequences of the three proteins show a high degree of homology to counterparts of HSV-1. By northern blot analyses we found that four transcripts whose sizes are 4.9, 3.9, 2.0 and 1.3 kb are transcribed from the domains of MDV2 genome containing the three genes. This is the first report dealing with UL25, UL26 and UL26.5 homologues of HSV-1 in MDV serotypes. (+info)
Distribution of retroviral vectors and vector producer cells using two routes of administration in rats.
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The clinical use of retroviral vector producer cells (VPCs) to deliver retroviral vectors efficiently to target cells has been investigated as a method to increase efficiency of gene delivery, presumably as a result of continued vector production in vivo. Studies were conducted in rats to evaluate the distribution of vector to distal organs and tissues as measured by transduction. Rats were treated with two doses of VPCs using two routes of administration: (1) subcutaneous injection, chosen to maximize both the dose and exposure of animals, thereby enabling identification of potential target organs under worst-case conditions; and (2) direct injection into brain parenchyma, chosen to mimic the intended clinical route of administration and provide an estimate of risk to patients receiving this therapy. Twelve organs or tissues were collected 7 days after administration of VPCs and analyzed by PCR for the presence of vector and vector producer cell sequences. Vector was detected most frequently at the site of injection by either route of administration. Less frequently, vector was detected in draining lymph nodes at the higher dose only using either route of injection. Single specimens of lung and contralateral skin were positive for vector following subcutaneous administration only. Vector was detected in gonadal tissue from a single low-dose male following subcutaneous administration, but this finding was not reproduced in any high-dose male or any males injected intracerebrally. In contrast, VPCs were detected only at the site of administration. The frequency of detection of VPCs 7 days after administration was higher when rats were injected by the intracerebral route. Based on these studies, gene transfer to distal organs or gonadal tissue following intracerebral administration of VPCs is not considered to be a risk to patients undergoing retroviral vector gene therapy for the treatment of brain cancer (glioblastoma multiforme; GBM). (+info)
Hepatoma-specific antitumor activity of an albumin enhancer/promoter regulated herpes simplex virus in vivo.
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Targeting viral vectors to appropriate cell types so that normal cells are not adversely affected is an important goal for gene therapy. Previously, we described a novel approach to viral gene therapy using a conditional, replication-competent herpes simplex virus (HSV), where replication and associated cytotoxicity are limited to a specific cell-type by the regulated expression of an essential immediate-early viral gene product. In this report we analyze the hepatoma-specific replication, cytotoxicity and anti-tumor effect of recombinant HSV G92A, regulated by the albumin enhancer/promoter. G92A efficiently replicated in vitro in two human hepatoma cell lines expressing albumin, but not in four human non-hepatoma, albumin-non-expressing tumor cell lines, while all cell lines were equally susceptible to a tissue nonspecific HSV recombinant, hrR3. In vivo, G92A replicated well in subcutaneous xenografts of human hepatoma cells (Hep3B) in athymic mice, but not in non-hepatoma subcutaneous tumors (PC3 and HeLa), whereas, hrR3 replicated well in both tumor types. Intratumoral inoculation of G92A inhibited the growth of established subcutaneous hepatoma tumors in nude mice, but not prostate tumors. Replication-competent viral vectors controlled by cell-specific transcriptional regulatory sequences provide a new therapeutic strategy for tumor therapy. (+info)
Chancroid, primary syphilis, genital herpes, and lymphogranuloma venereum in Antananarivo, Madagascar.
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Ulcer material from consecutive patients attending clinics in Antananarivo, Madagascar, was tested using multiplex polymerase chain reaction (M-PCR) to detect Treponema pallidum, Haemophilus ducreyi, and herpes simplex virus. Sera were tested for syphilis and for IgG and IgM antibodies to Chlamydia trachomatis by microimmunofluorescence testing (MIF). By M-PCR, 33% of 196 patients had chancroid, 29% had syphilitic ulcers, and 10% had genital herpes; 32% of the ulcer specimens were M-PCR negative. Compared with M-PCR, syphilis serology was 72% sensitive and 83% specific. The sensitivity of clinical diagnosis of syphilis, chancroid, and genital herpes was 93%, 53%, and 0% and specificity was 20%, 52%, and 99%, respectively. Less schooling was associated with increased prevalence of syphilitic ulcers (P=.001). Sixteen patients (8%) were clinically diagnosed with lymphogranuloma venereum (LGV); 1 plausible case of LGV was found by MIF. In Madagascar, primary care of genital ulcers should include syndromic treatment for syphilis and chancroid. (+info)
Effect of distance between homologous sequences and 3' homology on the frequency of retroviral reverse transcriptase template switching.
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Deletion of direct repeats in retroviral genomes provides an in vivo system for analysis of reverse transcriptase (RT) template switching. The effect of distance between direct repeats on the rate of deletion was determined for 16 murine leukemia virus (MLV)-based vectors containing a 701-bp direct repeat of overlapping fragments of the herpes simplex virus thymidine kinase gene (HTK). The direct repeats were separated by spacer fragments of various lengths (0.1 to 3.5 kb). Southern analysis of infected cells after one replication cycle indicated that all vectors in which the distance between homologous sequences was >1,500 bp deleted at very high rates (>90%). In contrast, vectors containing <1,500 bp between homologous sequences exhibited lower frequencies of deletion (37 to 82%). To analyze the pattern of locations at which RT switched templates, restriction site markers were introduced to divide the downstream direct repeat into five regions. RT switched templates within all five regions of the 701-bp direct repeat and the frequency of template switching was greater within the 5' regions in comparison to the 3' regions. The probability of RT switching templates within the 5' regions doubled when the MLV packaging sequence (Psi) was placed between the 701-bp direct repeats. However, Psi did not increase the rate of template switching for shorter direct repeats. These results indicate that linear distance between homologous sequences increases the rate of template switching and suggest that duplex formation between nascent DNA and homologous template sequences 3' of RT promote template switching. (+info)