Inhibition of herpes simplex virus reactivation by dipyridamole.

Herpes simplex virus (HSV) reactivation from latency was investigated. Reactivation of thymidine kinase-negative HSV, which is defective for reactivation, was greatly enhanced by thymidine (TdR). The reactivation-enhancing effect of TdR was blocked by dipyridamole (DPM), a known nucleoside transport inhibitor. DPM also inhibited wild-type HSV reactivation, suggesting potential antiviral use.

Spatial orientation adjustments in children with autism in Hong Kong.

Abnormal spatial orientation and body postures in children with autism often interfere with visual abilities to attend tasks and social interactions. Twenty-four children diagnosed with autism from Kowloon, Hong Kong were assessed for spatial orientation and spatial management abilities. Positive changes in spatial orientation were evident when the children wore ambient prism lenses and included changes in posture from slanted to erect. Adjustments in spatial management were evident in improved ball catching ability, a task requiring visual tracking and eye-hand coordination. The findings suggest that alterations to the sensory systems may lead to behavioral change in some children.

Herpes simplex virus latency after direct ganglion virus inoculation.

Herpes simplex virus (HSV) latent infection of ganglion neurons follows axoplasmic transport of HSV, probably in the form of nucleocapsid from peripheral sites of infection (e.g. footpad). This raises the possibility that latency is dependent on this particular means of presenting HSV to ganglion neurons. To investigate this, we directly infected ganglia of mice with HSV and evaluated latency. Initially, ganglia were surgically exposed in intact mice, infected with HSV and after 4 weeks evaluated for HSV latency-associated transcript (LAT) expression. LAT expression suggested latency. To more fully evaluate latency after direct ganglion inoculation, a transplant model was developed. In this model, ganglia were removed from mice, inoculated with HSV, transplanted into syngeneic recipients and evaluated for latency after several weeks. Latency was evident in transplanted ganglia by (1) the presence of LAT in neurons; (2) the lack of HSV ICP4 RNA or viral antigen, and (3) the isolation of HSV from explants of transplants but not from direct homogenates. The transplant model was then used to evaluate the effect of inhibition of HSV replication on latency. Antivirals which inhibited HSV replication markedly decreased the number of LAT-positive neurons in transplants, suggesting a role for HSV replication mechanisms and latency. It is thought that direct ganglion inoculation and ganglion transplant methods will permit unique investigations of mechanisms of latency.

Inhibition of herpes simplex virus reactivation by dipyridamole in a mouse model.

Herpes simplex virus (HSV) thymidine kinase (TK) has been demonstrated to be important for reactivation from latency. Specifically, HSV latency-associated transcripts (LAT) are expressed during latent infection established by TK-negative (TK-) HSV mutants, but reactivation is minimal. TK- HSV, however, readily reactivated in the presence of exogenous thymidine (TdR) in explant medium [Tenser et al. (1996): Journal of Virology 70:1271-1276]. In the present report this was further studied by evaluating the effect of dipyridamole (DPM) on HSV reactivation. DPM is known to interfere with nucleoside transport. Inhibition of TdR-enhanced reactivation of TK- HSV and inhibition of reactivation of wild-type TK+ HSV were evaluated in an experimental mouse model of latency. Without DPM, TK- HSV reactivation was increased from 0% to 88% with TdR in explant medium, demonstrating TdR-enhanced reactivation of TK- HSV (as seen previously), TdR-enhanced reactivation of TK- HSV was decreased when DPM (25 or 50 microM) was also present, to 30%-60% and to 0%, respectively. Secondly, DPM also decreased reactivation of wild-type TK+ HSV. The reactivation frequency of latently infected dorsal root ganglia was 90% in standard medium (no added TdR), and this was decreased by DPM to 9% and 0%, respectively. Reactivation of trigeminal ganglia in standard medium was 100%, and this decreased to 59% and 23%, respectively. The possibility of a direct toxic effect of DPM on ganglion neurons to explain the results was unlikely. DPM had a modest antiviral effect on HSV replication in cell culture, and its efficacy in blocking reactivation may be related to this activity, probably by inhibition of nucleoside transport.

Reactivation of thymidine kinase-defective herpes simplex virus is enhanced by nucleoside.

Herpes simplex virus (HSV) mutants defective for thymidine kinase expression (TK-) have been reported to establish latent infection of sensory ganglia of mice, in that HSV latency-associated transcript is expressed, but to be defective for reactivation. In the present study, the mechanism of defective reactivation by TK- HSV was investigated. Latent infection established by each of three reactivation-defective HSV type 1 mutants was studied. Reactivation in explant culture was markedly enhanced by the addition of thymidine (dTdR) to the explant culture medium. Without added dTdR, reactivation occurred in 0 of 32 ganglia, while when dTdR (200 microM) was present, reactivation occurred in 32 of 37 ganglia (86%). Reactivation was minimal or did not occur after treatment with other nucleosides; specificity for dTdR would suggest the importance of dTdR nucleotide levels rather than more general nucleotide pool imbalance. Enhanced reactivation by dTdR was dose dependent and was blocked by acyclovir. While some degree of inhibition of TK- HSV by acyclovir may be expected, the complete block of dTdR-enhanced reactivation was unexpected. This result may suggest that HSV is particularly vulnerable during initial reactivation events. The mechanism of dTdR-enhanced reactivation of TK- HSV was further evaluated during in vivo infection by TK- HSV. For mice infected with TK- HSV, virus was undetectable in ganglia 3 days later. However, for mice infected with TK- HSV and treated with dTdR, virus was readily detected (2.8 x 10(3) PFU per ganglion). This result suggested that in vivo treatment with dTdR enhanced replication of TK- HSV in ganglion neurons. In turn, this suggests that in latently infected ganglia, dTdR-enhanced reactivation of TK- HSV occurred as a result of viral replication in neurons following initial reactivation events.

Postural orientation modifications in autism in response to ambient lenses.

Autistic children often display abnormal postures, head tilts, and other spatial management dysfunctions. Methods were introduced to measure spatial orientation in tasks in a group of fourteen autistic children in Montreal, Canada. Ambient lenses were found to improve posture, correct head tilts, and improve ball catching abilities. A model of spatial orientation is described and recommendations are made to incorporate ambient lenses in treatment programs.

The role of herpes simplex thymidine kinase expression in neurovirulence and latency in newborn vs. adult mice.

Infection by standard thymidine kinase-positive (TK+) and TK- mutant herpes simplex virus (HSV) was performed in order to evaluate the role of HSV TK expression in neurovirulence and in HSV latency. In newborn mice, mortality and trigeminal ganglion (TG) HSV titer correlated (both were high) for TK+ and TK- HSV. In adult mice after TK- HSV infection they also correlated (both were low). After TK+ infection of adult mice, correlation was not present; mortality was low while HSV titer was moderately high. During the period of HSV latent infection (> 28 days after HSV infection), the number of neurons expressing HSV latency-associated transcript (LAT) was much greater for TK- HSV newborn-inoculated mice (average of 943/ganglion) than adult-inoculated mice (average of 138/ganglion). In addition, total amount of TG LAT was greater in the former than the latter. Reactivation from latency was restricted, however, for both groups. This result supported the important role of HSV TK expression in HSV reactivation, even when the number of LAT-positive neurons was greatly increased. The following conclusions were drawn from the study of TK- HSV in newborn mice: (i) HSV TK expression was of limited importance for neurovirulence and in vivo HSV TG infection (but was of importance in adult mice); (ii) increased in vivo HSV TG infection correlated with increased number of LAT-positive neurons, so that HSV replication and establishment of latency were not completely separable; and (iii) even with greatly increased numbers of latently infected neurons, HSV TK expression was important for reactivation from latency. Results in newborn mice suggested that the role of HSV TK expression in reactivation from latency and in neurovirulence were separable. To further investigate HSV replication in newborn and adult mice, ganglia were infected with HSV in vitro and either maintained in vitro or transplanted beneath the renal capsule of adult recipients. In both of these studies, HSV titers in ganglia were much higher in newborn than adult ganglia. This suggested that in addition to the well-know role of the immune system in HSV neurovirulence in newborn mice, it is likely that HSV replication per se in neural tissue is greater in newborn than adult mice. This may be related to the high level of HSV neurovirulence in newborn mice.

Secondary herpes simplex virus latent infection in transplanted ganglia.

Sensory ganglia latently infected with herpes simplex virus (HSV) were transplanted beneath the renal capsule of syngeneic recipients, and the latent infection remaining was investigated. HSV latency-associated transcript (LAT) expression and reactivation of HSV after explant of transplanted dorsal root ganglia were monitored as markers of latency. Two to four weeks after transplantation, both indicated evidence of HSV latency in transplants. At those times, infectious virus was not detected in direct ganglion homogenates. In addition, viral antigen and infected cell polypeptide 4 RNA were not detected. Taken together, the results suggested that HSV latent infection rather than persistent infection was present in transplants. From these results, two explanations seemed possible: latency was maintained in transplanted neurons, or alternatively, latency developed after transplantation, in neurons not previously latently infected. The latter was considered putative secondary latency and was investigated in three ways. First, evidence of reactivation which might serve as a source for secondary latency was evaluated. Reactivation of HSV in transplants was evident from HSV antigen expression (52% of transplants) and the presence of cell-free virus (38% of transplants) 3 to 5 days after transplantation. Second, putative secondary latency was investigated in recipients immunized with HSV prior to receiving latently infected ganglia. Reactivation was not detected 3 to 5 days after transplantation in immunized recipients, and LAT expression was rare in these recipients after 3 to 4 weeks. Lastly, the possibility of secondary latency was investigated by comparing results obtained with standard HSV and with reactivation-defective thymidine kinase-negative (TK-) HSV. Defective reactivation of TK- HSV was demonstrated by immunohistochemistry and by the inability to isolate infectious virus. Donor dorsal root ganglia latently infected with TK+ HSV showed many LAT-positive neurons 2 or more weeks after transplantation (average, 26 per transplant). However, LAT expression was undetectable or minimal > 2 weeks after transplantation in donor ganglia latently infected with TK- HSV (average, 0.2 per transplant). Impaired reactivation of TK- HSV-infected donor ganglia after transplantation, therefore, was correlated with subsequent limited LAT expression. From these results, the occurrence of secondary latency was concluded for ganglia latently infected with TK+ HSV and transplanted beneath the kidney capsule. In vivo reactivation in this transplant model may provide a more useful means to investigate HSV reactivation than in usual in vitro explant models and may complement other in vivo reactivation models. The occurrence of secondary latency was unique. The inhibition of secondary latency by the immune system may provide an avenue to evaluate immunological control of HSV latency.

Effect of dimethylsulfoxide on human gliomas: correlations between the nuclear magnetic resonance spectra and the transformed phenotypes of the tumor cells.

Prolonged in vitro induction of six established human glioma cell lines with dimethylsulfoxide (DMSO) generated an adherent human fibroblastoid phenotype. The development of contact-inhibited cell growth coincided with the decreased colony-forming potential of these cells in semisolid medium and with the reduction or elimination of tumorigenicity when transplanted in athymic nude mice. These DMSO-induced changes persisted for at least 19 passages after removal of the inducer from the medium. High-resolution natural-abundance 13C nuclear magnetic resonance spectroscopy showed specific spectral differences between the cell lines with more or less malignant transformed phenotypes: the glioma cells with a higher degree of tumorigenicity and colony-forming potential exhibited more intense myoinositol signals than those with the more benign phenotype.