In situ RT-PCR can distinguish between productive and latent cytomegalovirus infection in the blood cells of bone marrow transplant recipients.

Thirty-four peripheral blood leukocyte samples from bone marrow transplant (BMT) recipients were examined for Human cytomegalovirus (HCMV) phosphoprotein 65 (pp65), DNA and late transcripts. Twenty seven samples were positive for pp65 in the cytoplasm by immunofluorescent assay (IFA). Viral DNA was confirmed in 26 samples by nested PCR (nPCR). Using in situ RT-PCR, viral late transcripts were found in 19 samples, positive also by IFA and nPCR; these samples were considered indicative of productive viral infection. Five samples, positive by nPCR but negative by IFA and in situ RT-PCR, were considered to represent latent viral infection. In 8 samples, positive by IFA and nPCR but negative by in situ RT-PCR, apparently phagocytosis of viral particles took place.

Comparative efficacy of expression of genes delivered to mouse sensory neurons with herpes virus vectors.

To achieve gene delivery to sensory neurons of the trigeminal ganglion, thymidine kinase-negative (TK-) herpes simplex viruses (HSV) containing the reporter gene lacZ (the gene for E. coli beta-galactosidase) downstream of viral (in vectors RH116 and tkLTRZ1) or mammalian (in vector NSE-lacZ-tk) promoters were inoculated onto mouse cornea and snout. Trigeminal ganglia were removed 4, 14, 30, and 60 days after inoculation with vectors and histochemically processed with 5-bromo-4-chloro-3 indolyl-beta-galactoside (X-Gal). With vector tkLTRZ1, large numbers of labeled neurons were observed in rostromedial and central trigeminal ganglion at 4 days after inoculation. A gradual decline in the number of labeled neurons was observed with this vector at subsequent time points. With vectors RH116 and NSE-lacZ-tk, smaller numbers of labeled neurons were seen at 4 days following inoculation than were observed with vector tkLTRZ1. No labeled neurons could be observed at 14 days after inoculation with vectors RH116 and NSE-lacZ-tk. Immunocytochemistry for E. coli beta-galactosidase and in situ hybridization to HSV latency-associated transcripts revealed labeled neurons in regions of the trigeminal ganglion similar to that observed with X-Gal staining. A comparable distribution of labeled neurons in trigeminal ganglion was also observed after application of the retrograde tracer Fluoro-Gold to mouse cornea and snout. These data provide evidence that retrogradely transported tk- herpes virus vectors can be used to deliver a functional gene to sensory neurons in vivo in an anatomically predictable fashion.

Alterations in morphology of REF cells induced by growth factors produced by ASV-transformed (GCA) cells.

Morphological changes (in shape and pattern of growth in culture) of REF cells, characteristic for transformed cells, appeared after incubation with AE-GCA (acid extract of GCA cells). The surface alterations of REF caused by growth factor produced by GCA cells, such a appearance of membrane ruffles, finger-like structures, increase in the number of coated pits and in the number of blebs varying in size were also described. These alterations in cell morphology and cell surface suggest that growth factors present in AE-GCA (obtained from ASV-transformed rat kidney line), have a biological activity characteristic for many polypeptide growth factors.

Anchorage-independent growth of RSV-transformed rat (GCA, W 12 and XC) cells.

Three RSV-transformed rat cell lines: GCA, W12 and XC were characterized as to their ability to anchorage-independent growth in comparison to normal rat kidney (NRK-49F) cells. Differences in the threshold density (TD) and colony forming efficiency (CFE) of the investigated cells are described. The ability of virally transformed cells to stimulation of soft agar colony formation of NRK cells in coculture assay was presented. The production of TGFs-like factors by GCA, W12 and XC cells was suggested.

The "comet" assay for detection of potential genotoxicity of polluted water.

The aim of this study was to determine the potential genotoxic activity of polluted water samples taken from wastewater from selected industrial plants in Kraków: 1. the Thermal-electric Power Station 2. the Institute of Metal Cutting. The recently developed single cell gel assay (SCG or comet assay), which is a quick and simple technique for the evaluation of DNA damage and repair in individual cells, was used. The assay was carried out on human hepatoma cells (Hep G2) as target cells. A greater number of cells with comets was observed in those treated in vitro with the polluted water samples (70%-88%) than in those in the control (22%, 33%). These preliminary results indicate that comet assay can have an application in biomonitoring studies for determining the potential genotoxicity of water pollutants.

Concomitant infections with human papillomavirus and various mycoplasma and ureaplasma species in women with abnormal cervical cytology.

PURPOSE: The objective of the present study is to verify possible association between infections with mycoplasmas and ureaplasmas and the presence of HPV infections in women diagnosed with abnormal cervical cytology. MATERIAL/METHODS: The investigation included 387 non-pregnant women among whom: 62 were diagnosed with ASCUS, 167 with LSIL, 27 with HSIL, 49 with cervical carcinomas, and 82 females with normal cytology.The presence of HPV infection and identification of both ureaplasma and mycoplasma were confirmed by PCR using specific primers. RESULTS: HPV infections were demonstrated in 156 females (40%), with mycoplasmas and/or ureaplasmas were confirmed in 93 cases (24%). In HPV-positive patients, infections with mycoplasmas/ureaplasmas were more frequent, particularly for ureaplasmas (U. urealyticum p=0.004, U. parvum p=0.027). The percentage of females infected with U. urealyticum significantly increased in women diagnosed with cervical carcinoma as compared to controls.The statistical analysis demonstrated that the risk of HPV infection while already infected with any of the four analyzed species of Mycoplasmataceae increased two-fold. With concomitant of U. urealyticum infection, the risk of HPV infection was 4.7-fold greater than in the absence U. urealyticum infection. CONCLUSION: Since the presence of U. urealyticum associates significantly with the HPV infection, genotyping of the ureaplasma species should be recomended.

Restricted expression of herpes simplex virus lytic genes during establishment of latent infection by thymidine kinase-negative mutant viruses.

Infection of cells by herpes simplex virus (HSV) can lead to either lytic, productive infection or nonlytic, latent infection. The factors influencing this infection pathway decision are largely unknown. Thymidine kinase-negative mutant viruses can establish latent infection in neurons of mouse trigeminal ganglia but do not replicate productively in these cells. We show that during the early stages of establishment of latency by these mutants, expression of viral lytic genes is drastically reduced or undetectable as assayed by in situ hybridization. Thus, establishment of latent infection by HSV can occur despite severely restricted levels of lytic gene expression. This suggests that the block to productive replication during establishment of latent infection by HSV occurs before or early during the expression of alpha genes.

Application of PCR in situ method for detection of human cytomegalovirus (HCMV) DNA.

Present HCMV diagnosis is relatively slow and inefficient. We applied PCR in situ to 15 samples of human salivary glands, 20 cytospins from blood, and 10 human fibroblast samples in order to detect the presence of HCMV DNA. The results indicate that PCR in situ is an effective and very sensitive method for detection of HCMV infections in variety of specimens.

Interleukin 1 receptor antagonist localization exclusively in non-parenchymal cells of the mouse liver.

The natural interleukin 1 receptor antagonist (IL-1ra) is induced in mouse liver after lipopolysaccharide (LPS) or recombinant interleukin 1 (IL-1) administration. Changes in hepatic IL-1ra mRNA concentration were measured following LPS injection to CBA mice. The results showed, that in vivo, IL-1ra transcripts were stimulated early at about 2h with maximal elevation at 4-6 h. Northern blot analysis of RNA extracted from primary cultures of mouse hepatocytes did not detect IL-1ra mRNA, although constitutive and stimulated contrapsin mRNA expression were easily demonstrated. Therefore, induction of IL-1ra mRNA in sinusoid lining cells of the mouse liver at 4 h after LPS injection was evidenced by in situ hybridization using the same specific cDNA probe as in the Northern blotting technique. Additionally, immunohistochemical studies revealed that IL-1 receptor antagonist protein was induced with LPS after 19 hours in sinusoid lining cells. The data deriving from Northern blotting, in situ hybridization and immunohistochemical studies, suggest that IL-1ra is formed exclusively in non-parenchymal liver cells.

Human glioblastoma cells persistently infected with simian virus 40 carry nondefective episomal viral DNA and acquire the transformed phenotype and numerous chromosomal abnormalities.

A stable, persistent infection of A172 human glioblastoma cells with simian virus 40 (SV40) was readily established after infection at an input of 450 PFU per cell. Only 11% of the cells were initially susceptible to SV40, as shown by indirect immunofluorescent staining for the SV40 T antigen at 48 h. However, all cells produced T antigen by week 11. In contrast, viral capsid proteins were made in only about 1% of the cells in the established carrier system. Weekly viral yields ranged between 10(4) and 10(6) PFU/ml. Most of the capsid protein-producing cells contained enormous aberrant (lobulated or multiple) nuclei. Persistent viral DNA appeared in an episomal or "free" state exclusively in Southern blots and was indistinguishable from standard SV40 DNA by restriction analysis. Viral autointerference activity was not detected, and yield reduction assays did not indicate defective interfering particle activity, further implying that variant viruses were not a factor in this carrier system. Interferon was also not a factor in the system, as shown by direct challenge with vesicular stomatitis virus. Persistent infection resulted in cellular growth changes (enhanced saturation density and plating efficiency) characteristic of SV40 transformation. Persistent infection also led to an increased frequency of cytogenetic effects. These included sister chromatid exchanges, a variety of chromosomal abnormalities (ring chromosomes, acentric fragments, breaks, and gaps), and an increase in the chromosome number. Nevertheless, the persistently infected cells continued to display a bipolar glial cell-like morphology with extensive process extension and intercellular contacts.

Evidence for a novel regulatory pathway for herpes simplex virus gene expression in trigeminal ganglion neurons.

Thymidine kinase (TK)-negative (TK-) mutant strains of herpes simplex virus type 1 (HSV-1) show reduced expression of alpha and beta viral genes during acute infection of trigeminal ganglion neurons following corneal infection (M. Kosz-Vnenchak, D. M. Coen, and D. M. Knipe, J. Virol. 64:5396-5402, 1990). It was surprising that a defect in a beta gene product would lead to decreased alpha and beta gene expression, given the regulatory pathways demonstrated for HSV infection of cultured cells. In this study, we have examined viral gene expression during reactivation from latent infection in explanted trigeminal ganglion tissue. In explant reactivation studies with wild-type virus, we observed viral productive gene expression over the first 48 h of explant incubation occurring in a temporal order (alpha, beta, gamma) similar to that in cultured cells. This occurred predominantly in latency-associated transcript-positive neurons but was limited to a fraction of these cells. In contrast, TK- mutant viruses showed greatly reduced alpha and beta gene expression upon explant of latently infected trigeminal ganglion tissue. An inhibitor of viral TK or an inhibitor of viral DNA polymerase greatly decreased viral lytic gene expression in trigeminal ganglion tissue latently infected with wild-type virus and explanted in culture. These results indicate that the regulatory mechanisms governing HSV gene expression are different in trigeminal ganglion neurons and cultured cells. We present a new model for viral gene expression in trigeminal ganglion neurons with implications for the nature of the decision process between latent infection and productive infection by HSV.

Herpes simplex virus thymidine kinase and specific stages of latency in murine trigeminal ganglia.

From marker rescue, sequencing, transcript, and latency analyses of the thymidine kinase-negative herpes simplex virus mutant dlsactk and studies using the thymidine kinase inhibitor Ro 31-5140, we infer that the virus-encoded thymidine kinase is required in murine trigeminal ganglia for acute replication and lytic gene expression, for increasing the numbers of cells expressing latency-associated transcripts, and for reactivation from latent infection.

Thymidine kinase-negative herpes simplex virus mutants establish latency in mouse trigeminal ganglia but do not reactivate.

Herpes simplex virus infection of mammalian hosts involves lytic replication at a primary site, such as the cornea, translocation by axonal transport to sensory ganglia and replication, and latent infection at a secondary site, ganglionic neurons. The virus-encoded thymidine kinase, which is a target for antiviral drugs such as acyclovir, is not essential for lytic replication yet evidently is required at the secondary site for replication and some phase of latent infection. To determine the specific stage in viral pathogenesis at which this enzyme is required, we constructed virus deletion mutants that were acyclovir resistant and exhibited no detectable thymidine kinase activity. After corneal inoculation of mice, the mutants replicated to high titers in the eye but were severely impaired for acute replication in trigeminal ganglia and failed to reactivate from ganglia upon cocultivation with permissive cells. Nevertheless, latency-associated transcripts were expressed in neuronal nuclei of ganglia from mutant-infected mice and superinfection of the ganglia with a second virus rescued the latent mutant virus. Thus, contrary to a widely accepted hypothesis, the thymidine kinase-negative mutants established latent infections, implying that neither thymidine kinase activity nor ganglionic replication is necessary for establishment of latency. Rather, thymidine kinase appears to be necessary for reactivation from latency. These results suggest that acyclovir-resistant viruses could establish latent infections in clinical settings and have implications for the use of genetically engineered herpesviruses to deliver foreign genes to neurons.

A deletion mutant of the latency-associated transcript of herpes simplex virus type 1 reactivates from the latent state with reduced frequency.

We have generated and characterized a deletion mutant of herpes simplex virus type-1, dlLAT1.8, which lacks the putative promoter region, transcriptional start site, and 1,015 base pairs of the DNA sequences specifying the latency-associated transcripts (LATs). When tested in a CD-1 mouse ocular model, dlLAT1.8 was replication competent in the eye and in ganglia during acute infection but reactivated from explant cultures of ganglia with reduced efficiency (49%) relative to those of wild-type and marker-rescued viruses (94 and 85%, respectively) despite the fact that levels of mutant viral DNA in ganglia during latent infection were comparable to wild-type levels. The neurovirulence of KOS was not significantly altered by the removal of sequences specifying the LATs, as judged by numbers of animals dying on or before 30 days postinfection. Examination of ganglia latently infected with dlLAT1.8 by in situ hybridization revealed no LAT expression. The genotype of reactivated virus was identical to that of input dlLAT1.8 virus as judged by Southern blot analysis. These studies suggest that although the LATs are not essential for the establishment and reactivation of latency in our model, they may play a role in determining the frequency of reactivation of virus from the latent state.