Infection of the mouse trigeminal ganglia (TG) is the most commonly used model for the study of herpes simplex virus type 1 (HSV-1) latency. Its popularity is caused, at least in part, by the perception that latent infection can be studied in this system in the absence of spontaneous viral reactivation. However, this perception has never been rigorously tested. To carefully study this issue, the eyes of Swiss-Webster mice were inoculated with HSV-1 (KOS), and 37-47 days later the TG were dissected, serial-sectioned, and probed for HSV-1 ICP4, thymidine kinase, glycoprotein C, and latency-associated transcript RNA by in situ hybridization. Serial sections of additional latently infected TG were probed with HSV-1-specific polyclonal antisera. Analysis of thousands of probed sections revealed abundant expression of viral transcripts, viral protein, and viral DNA replication in about 1 neuron per 10 TG tested. These same neurons were surrounded by a focal white cell infiltrate, indicating the presence of an antigenic stimulus. We conclude that productive cycle viral genes are abundantly expressed in rare neurons of latently infected murine TG and that these events are promptly recognized by an active local immune response. In the absence of detectable infectious virus in these ganglia, we propose the term ''spontaneous molecular reactivation'' to describe this ongoing process.H erpes simplex virus type 1 (HSV-1) infections of the skin or eye lead to invasion of the trigeminal ganglia (TG), where the virus follows one of two pathways (1-3). In some infected neurons, the virus replicates and destroys the cell. This lytic cycle path involves regulated viral gene expression in which immediate-early genes are required for the efficient expression of early and late genes. In other neurons, a latent infection is established in which lytic cycle genes are not expressed and infectious virus is not produced. Latent infection of neurons is characterized by the expression of the latency-associated transcripts (LAT), which are encoded in a single region of the HSV-1 genome and which accumulate to high levels in the nucleus of the host cell (4-10). In situ hybridization (ISH) studies of latently infected sensory ganglia have revealed numerous neurons expressing LAT but no other viral RNAs (4, 5, 7, 9-15). Thus, for many years a latent neuron has been defined in molecular terms as a neuron that is positive for LAT and negative for other viral RNAs.In humans, HSV-1 intermittently reactivates from the latent state, with peripheral shedding of infectious virus. Shedding of infectious virus is similarly observed in rabbit ocular models of HSV infection, both spontaneously and in response to local or systemic stimuli (16,17). In contrast, latent HSV-1 infection in the mouse seems to be more tightly regulated, and attempts to identify infectious virus or viral antigen in murine sensory ganglia after resolution of primary infection (indicative of a spontaneous reactivation localized to the sensory ganglion) have been unsuccessful (2,7,9,...

Productive infection with herpes simplex virus (HSV) type 1 is limited by both innate and adaptive immune mechanisms. The purpose of the current study was to determine whether these mechanisms also play a role in the establishment of latent HSV infection. First we examined the trigeminal ganglia (TG) of severe combined immunodeficiency (SCID), interferon-gamma knockout (GKO), and beige (a strain deficient in natural killer cell activity) mice following ocular inoculation with HSV. Although infection of SCID mice was invariably lethal, we consistently found latently infected neurons in the TG of these animals at 2-4 days postinoculation. HSV infection of GKO and beige mice, while not lethal, was characterized by a greater number of productively infected TG neurons and/or a delay in the time to peak productive infection compared to C57BL/6 controls. However, as assayed by both in situ hybridization for LAT expression and quantitative PCR (Q-PCR) for viral DNA, we found that HSV established a latent infection in GKO and beige mice as efficiently as in C57BL/6 controls. We subsequently examined the TG of "HSV-sensitive" strains of mice (Swiss-Webster, CBA, and BALB/c) following ocular infection with HSV. At the peak of acute ganglionic infection the number of productively infected TG neurons in each of these mouse strains was about sevenfold greater than in the "HSV-resistant" strain C57BL/6, consistent with previously reported differences in susceptibility to lethal challenge with HSV. However, as assayed by both in situ hybridization for LAT and Q-PCR for viral DNA, we found that HSV established a latent infection in Swiss-Webster, CBA, and BALB/c mice as efficiently as in C57BL/6 controls. We conclude that HSV efficiently establishes latent infection in the TG of mice in the absence of innate and adaptive immune mechanisms that are essential for limiting productive viral infection.

Developmental changes in the responsiveness of the fetal adrenals to corticotropin (ACTH) play an important role in the regulation of the fetal hypothalamic-pituitary-adrenal axis. Responsiveness of adrenal cortical cells to ACTH is dependent on the extent of ACTH receptor expression. Therefore, we examined the localization and regulation of ACTH receptor expression in the midgestation (16-24 weeks) human fetal adrenal cortex. In situ hybridization analysis was used to localize messenger RNA (mRNA) encoding the ACTH receptor in sections of human fetal adrenal glands. Messenger RNA encoding the ACTH receptor was localized in cells from all cortical zones; abundance was higher in definitive zone than in fetal zone cells and was least abundant in the more central portions of the cortex. Regulation of ACTH receptor expression was studied using Northern blot analysis of total RNA extracted from primary cultures of fetal and definitive zone cells. Two major (1.5 and 3.5 kilobases) and, upon stimulation with ACTH, 3 minor (4.0, 6.0 and 10.0 kb) ACTH receptor mRNA transcripts were detected in RNA from fetal and definitive zone cells. In both cell types, ACTH-(1-24) increased the abundance of mRNA encoding the ACTH receptor 10- to 20-fold compared with untreated cells. The effects of ACTH-(1-24) on ACTH receptor expression in fetal zone cells were time- and dose-dependent. The ED50 for the stimulation of ACTH receptor expression by ACTH-(1-24) was 1-10 pM, and maximal response to 0.1 nm ACTH-(1-24) was detected after 12-16 h. Eight-bromoadenosine cAMP and forskolin also stimulated ACTH receptor expression in fetal zone cells and closely mimicked the effects of ACTH-(1-24). In contrast, stimulation of protein kinase C with 12-O-tetradecanoyl phorbol 13-acetate had no effect on ACTH receptor expression. Changes in ACTH receptor expression in response to ACTH-(1-24), cAMP and forskolin were paralleled by changes in expression of the P450 cholesterol side chain cleavage (P450scc) enzyme. These data demonstrate that expression of the ACTH receptor by the human fetal adrenal cortex is up-regulated by its own ligand and that this effect is mediated by a cAMP-dependent mechanism. In addition, the coordinate stimulation of ACTH receptor and P450scc expression by ACTH indicates that the gene for the ACTH receptor is one of a specific cohort of genes regulated by ACTH that are required to facilitate fetal adrenal cortical response to ACTH. ACTH regulation of its own receptor may represent a mechanism by which fetal adrenal responsiveness to ACTH is maintained and possibly enhanced during fetal development.

Cortisol, produced by the primate fetal adrenal, regulates the maturation of organ systems necessary for extrauterine life. During most of primate pregnancy, however, the fetal adrenal lacks the enzyme 3 beta-hydroxysteroid dehydrogenase/isomerase (3 beta HSD), which is essential for cortisol synthesis. Therefore, we used immunohistochemistry and in situ hybridization techniques to investigate the developmental expression of 3 beta HSD in the fetal rhesus monkey adrenal from 109 days' gestation until term (165 +/- 5 days) and assessed the role of ACTH in the induction of its expression and localization. We also examined whether ACTH regulates the expression of two other steroidogenic enzymes, cytochrome P450 cholesterol side-chain cleavage (P450scc) and P450 17 alpha-hydroxylase, 17/20-lyase (P450c17), in the fetal rhesus monkey adrenal. To stimulate ACTH secretion from the fetal pituitary in vivo, we administered metyrapone to late gestation fetal rhesus monkeys for 3-7 days. Adrenals were collected from untreated fetuses at 109-125 days (n = 5), 130-148 days (n = 7), 155-172 days (n = 4), and after metyrapone treatment at 135-137 days (n = 4). The cortical width and total amount of 3 beta HSD staining were measured using an image analysis system. 3 beta HSD was localized primarily in the definitive zone cells of the adrenal from fetuses between 109-148 days, whereas at term (155-172 days), 3 beta HSD was localized in both definitive and transitional zone cells. The cortical width and total amount of 3 beta HSD staining in the adrenal increased significantly (P < 0.05) between 148 days (137 +/- 14 microns and 3,689 +/- 522 grains) and 155 days (315 +/- 61 microns and 7,321 +/- 2,008 grains). Interestingly, in metyrapone-treated fetuses at 135-137 days, 3 beta HSD messenger RNA (mRNA) and protein were localized extensively in both the definitive and transitional zones, a pattern seen only in term fetal adrenals in untreated animals. In addition, metyrapone treatment significantly (P < 0.05) increased cortical width (386 +/- 95 microns) and total 3 beta HSD immunostaining (29,063 +/- 13,692 grains) compared with age-matched controls. In contrast to 3 beta HSD, P450scc mRNA was detected in the definitive, transitional, and fetal zones, and its expression was not altered after metyrapone treatment. P450c17 mRNA was detected in the transitional and fetal zones, and the relative abundance was greater in the transitional zone. The relative abundance of P450c17 mRNA was increased in the fetal zone after metyrapone treatment. In summary, at term or after metyrapone treatment, expression of 3 beta HSD is induced in the transitional zone of the fetal rhesus monkey adrenal gland, an indication of functional maturation of the primate adrenal cortex. These data suggest that the ontogenetic increase in fetal pituitary ACTH secretion plays an important role in the induction of 3 beta HSD expression in the transitional zone.

The rapid growth of the primate fetal adrenal from midgestation until term is regulated by ACTH secreted by the fetal pituitary. Previous studies suggest that the trophic actions of ACTH are mediated by insulin-like growth factor II (IGF-II) synthesized by fetal adrenal cortical cells. To characterize further the role of IGF-II in the regulation of fetal adrenal growth, we investigated the expression of the messenger RNAs (mRNAs) encoding IGF-I, IGF-II, IGF-I receptor (IGF-IR) and IGF binding protein (IGFBP) 1-6 in the fetal rhesus monkey adrenal in vivo from 109 days of gestation until term (165 +/- 5 days) using in situ hybridization. To assess the role of ACTH in the regulation of expression of the IGF system in vivo, we administered metyrapone (3-7 days) to late gestation fetal rhesus monkeys (n = 4) in utero to increase fetal pituitary ACTH secretion. IGF-II mRNA was abundant in the definitive, transitional and fetal zones of the adrenal cortex from 109 days until term. IGF-IR mRNA was expressed in the definitive, transitional and fetal zones and decreased to nondetectable levels at term. IGFBP-2 and IGFBP-6 mRNAs were expressed in the definitive, transitional, and fetal zones, whereas IGFBP-1, -3, -4, and -5 were not detected in adrenal cells. The effects of increasing ACTH secretion on the growth of the specific zones of the adrenal were determined using morphometric techniques. Metyrapone treatment approximately doubled adrenal weight, which was due to an increase in the area of the definitive, transitional, and fetal zones with decreased cell density of the definitive, transitional, and fetal zones compared with controls and not due to a change in total cell number. Therefore, the increase in adrenal weight after metyrapone treatment was due to hypertrophy of the three cortical zones; there was no effect on adrenal medullary growth. The relative abundance of the mRNAs encoding IGF-II and the IGF-IR was increased after metyrapone treatment, whereas the localization and relative abundance of IGFBP 1-6 mRNAs were not altered by metyrapone treatment. We conclude that the ontogenetic increase in adrenal growth may be regulated, at least in part, by locally synthesized IGF-II, and the cessation of adrenal growth that occurs at term may be mediated by the decrease in the IGF-IR. The adrenal cortical expression of IGFBP-2 and IGFBP-6 suggests that these IGFBPs may modulate the IGF-IGF-IR interaction. Metyrapone treatment, which likely increased fetal pituitary ACTH secretion, causes a coordinated increase in expression of IGF-II and IGF-IR in fetal adrenal cortical cells, which may be an important mechanism of regulation of fetal adrenal cortical growth.