Varicella-zoster immunization in pediatric liver transplant recipients: safe and immunogenic.

Varicella can have a severe course in immunosuppressed patients. Although prevention is fundamental, live-attenuated varicella-zoster (VZV) vaccine is not currently recommended in transplant recipients. Our aims were to (1) evaluate VZV immunity in pediatric liver transplant (LT) recipients; (2) immunize (two doses) seronegative patients post-LT; (3) monitor vaccine safety, (4) assess B and T cell vaccine responses. All patients followed at the Swiss National Pediatric LT Center were approached and 77/79 (97.5%) were enrolled (median age 7.8 years). Vaccine safety was monitored by standardized diary cards and phone calls. VZV-specific serology and CD4(+) T cells were assessed before and after immunization. Thirty-nine patients (51.1%) were seronegative including 14 children immunized pre-LT. Thirty-six of 39 seronegative patients were immunized post-LT (median 3.0 years post LT). Local (54.8%) and systemic (64.5%) reactions were mild and transient. The frequency of VZV-specific CD4(+) T cells and antibody titers increased significantly (respectively from 0.085% to 0.16%, p = 0.04 and 21.0 to 1134.5 IU/L, p < 0.001). All children reached seroprotective titers and 31/32 (97%) patients assessed remained seroprotected at follow-up (median 1.7 years). No breakthrough disease was reported during follow-up (median 4.1 years). Thereby, VZV vaccine appears to be safe, immunogenic and provide protection against disease in pediatric LT patients.

Impaired antibody memory to varicella zoster virus in HIV-infected children: low antibody levels and avidity*.

OBJECTIVE: HIV-infected children have impaired antibody responses after exposure to certain antigens. Our aim was to determine whether HIV-infected children had lower varicella zoster virus (VZV) antibody levels compared with HIV-infected adults or healthy children and, if so, whether this was attributable to an impaired primary response, accelerated antibody loss, or failure to reactivate the memory VZV response. METHODS: In a prospective, cross-sectional and retrospective longitudinal study, we compared antibody responses, measured by enzyme-linked immunosorbent assay (ELISA), elicited by VZV infection in 97 HIV-infected children and 78 HIV-infected adults treated with antiretroviral therapy, followed over 10 years, and 97 age-matched healthy children. We also tested antibody avidity in HIV-infected and healthy children. RESULTS: Median anti-VZV immunoglobulin G (IgG) levels were lower in HIV-infected children than in adults (264 vs. 1535 IU/L; P<0.001) and levels became more frequently unprotective over time in the children [odds ratio (OR) 17.74; 95% confidence interval (CI) 4.36-72.25; P<0.001]. High HIV viral load was predictive of VZV antibody waning in HIV-infected children. Anti-VZV antibodies did not decline more rapidly in HIV-infected children than in adults. Antibody levels increased with age in healthy (P=0.004) but not in HIV-infected children. Thus, antibody levels were lower in HIV-infected than in healthy children (median 1151 IU/L; P<0.001). Antibody avidity was lower in HIV-infected than healthy children (P<0.001). A direct correlation between anti-VZV IgG level and avidity was present in HIV-infected children (P=0.001), but not in healthy children. CONCLUSION: Failure to maintain anti-VZV IgG levels in HIV-infected children results from failure to reactivate memory responses. Further studies are required to investigate long-term protection and the potential benefits of immunization.

Normoxic expression of hypoxia-inducible factor 1 in rat Leydig cells in vivo and in vitro.

Hypoxia-inducible factors (HIF) are transcription factors that serve essential regulatory roles in cellular and molecular responses to oxygen debt. HIFs are composed of hypoxia-dependent α subunits (1α, 2α, 3α) and an oxygen-independent ß subunit. Previously we demonstrated that HIF-1, the master regulator of hypoxic responses, is expressed in the adult rat testis. We hypothesized that HIF-1 is involved in regulating responses to oxygen tension in the testis. Goals of this study were to determine if HIF-2α and HIF-3α are expressed in rat testis, identify testis cell types that express HIF-1α, and examine patterns of testicular HIF-1α protein expression under conditions of ischemia and hypoxia in vivo and in vitro. Reverse transcriptase polymerase chain reaction revealed that mRNA for Hif-1α, Hif-2α, and Hif-3α is expressed in the testis. The HIF-1α protein is the predominant subunit in testis. HIF-1α protein was abundant in normoxic testis, and its levels remained unchanged following ischemia created by surgically induced testicular torsion and reperfusion. Immunoblot and immunocytochemical experiments demonstrated that Leydig cells are the major source of HIF-1α in normoxic and hypoxic testes. To examine potential mechanisms of testicular HIF-1 stabilization, nuclear proteins from Leydig cells cultured in 5% or 21% oxygen, or cells cultured with H2O2, were analyzed by immunoblotting. Levels of HIF-1α were significantly diminished in 5% or 21% oxygen cultures compared with freshly isolated cells. Treating Leydig cells with H2O2 as a source of reactive oxygen species did not affect HIF-1α levels. High levels of constitutively expressed HIF-1α in normoxic Leydig cells suggest potentially unique roles for HIF-1 in Leydig cell responsiveness to oxygen.

Prenatal exposure to dexamethasone alters Leydig cell steroidogenic capacity in immature and adult rats.

This study examines the effects of prenatal exposure to dexamethasone (DEX) on postnatal testosterone production in male rats. Pregnant female rats were treated on gestation days 14-19 with DEX (100 microg/kg body weight per day; n = 9) or vehicle (n = 9). Results show that 35-day-old male offspring from DEX-treated pregnant females (n = 42) had decreased levels of serum testosterone (45.6% lower, P < .05) compared with control offspring (n = 43), although serum luteinizing hormone (LH) levels were not significantly altered. These findings suggest that a direct programming of developing gonadal cells occurs in response to high levels of maternal glucocorticoid. Indeed, testosterone production was significantly reduced in Leydig cells isolated from immature offspring of DEX-treated pregnant females compared with controls (48.3%, P < .001), and LH stimulation of these cells did not compensate for the lowered steroidogenic capacity. The hypothalamic-pituitary-adrenal axis was also affected, because significant reductions in both serum adrenocorticotropic hormone (ACTH; 26.2%, P < .001) and corticosterone (CORT; 32.3%, P < .001) were measured in DEX-exposed immature male offspring. In contrast, adult male offspring from DEX-treated dams had significantly higher levels of serum ACTH (39.2%, P <. 001) and CORT (37.8%, P < .001). These same animals had higher serum testosterone (31.6%, P < or = .05) and a significant reduction in serum LH (30.8%, P < .001). Moreover, Leydig cells isolated from these adult offspring exhibited an increased capacity for testosterone biosynthesis under basal (38.6%, P < .001) and LH-stimulated conditions (33.5%, P < .001). In summary, sustained changes in steroidogenic capacity were observed in male rats exposed to high levels of glucocorticoid during prenatal development. More specifically, DEX exposure in utero perturbed Leydig cell testosterone production in both pubertal and adult rats.

Modulation of rat Leydig cell steroidogenic function by di(2-ethylhexyl)phthalate.

Exposure of rodents to phthalates is associated with developmental and reproductive anomalies, and there is concern that these compounds may be causing adverse effects on human reproductive health. Testosterone (T), secreted almost exclusively by Leydig cells in the testis, is the primary steroid hormone that maintains male fertility. Leydig cell T biosynthesis is regulated by the pituitary gonadotropin LH. Herein, experiments were conducted to investigate the ability of di(2-ethylhexyl)phthalate (DEHP) to affect Leydig cell androgen biosynthesis. Pregnant dams were gavaged with 100 mg(-1) kg(-1) day(-1) DEHP from Gestation Days 12 to 21. Serum T and LH levels were significantly reduced in male offspring, compared to control, at 21 and 35 days of age. However, these inhibitory effects were no longer apparent at 90 days. In a second set of experiments, prepubertal rats, from 21 or 35 days of age, were gavaged with 0, 1, 10, 100, or 200 mg(-1) kg(-1) day(-1) DEHP for 14 days. This exposure paradigm affected Leydig cell steroidogenesis. For example, exposure of rats to 200 mg(-1) kg(-1) day(-1) DEHP caused a 77% decrease in the activity of the steroidogenic enzyme 17beta-hydroxysteroid dehydrogenase, and reduced Leydig cell T production to 50% of control. Paradoxically, extending the period of DEHP exposure to 28 days (Postnatal Days 21-48) resulted in significant increases in Leydig cell T production capacity and in serum LH levels. The no-observed-effect-level and lowest-observed-effect-level were determined to be 1 mg(-1) kg(-1) day(-1) and 10 mg(-1) kg(-1) day(-1), respectively. In contrast to observations in prepubertal rats, exposure of young adult rats by gavage to 0, 1, 10, 100, or 200 mg(-1) kg(-1) day(-1) DEHP for 28 days (Postnatal Days 62-89) induced no detectable changes in androgen biosynthesis. In conclusion, data from this study show that DEHP effects on Leydig cell steroidogenesis are influenced by the stage of development at exposure and may occur through modulation of T-biosynthetic enzyme activity and serum LH levels.

Müllerian-inhibiting substance type II receptor expression and function in purified rat Leydig cells.

Müllerian-inhibiting substance (MIS), a gonadal hormone in the transforming growth factor-beta superfamily, induces Müllerian duct involution during male sexual differentiation. Mice with null mutations of the MIS ligand or receptor develop Leydig cell hyperplasia and neoplasia in addition to retained Müllerian ducts, whereas MIS-overexpressing transgenic mice have decreased testosterone concentrations and Leydig cell numbers. We hypothesized that MIS directly modulates Leydig cell proliferation and differentiated function in the maturing testis. Therefore, highly purified rat Leydig and Sertoli cells were isolated to examine cell-specific expression, binding, and function of the MIS type II receptor. These studies revealed that this receptor is expressed abundantly in progenitor (21-day) and immature (35-day) Leydig cells as well as in Sertoli cells. Prepubertal progenitor Leydig cells exhibit high affinity (Kd = 15 nM), saturable binding of MIS. No binding, however, is detected with either peripubertal immature Leydig cells or Sertoli cells at either age. Moreover, progenitor, but not immature Leydig cells, respond to MIS by decreasing DNA synthesis. These data demonstrate that functional MIS type II receptors are expressed in progenitor Leydig cells and support the hypothesis that MIS has a direct role in the regulation of postnatal testicular development.

Increased proliferation of Leydig cells induced by neonatal hypothyroidism in the rat.

Treatment of male rat pups with the reversible goitrogen 6-n-propyl-2-thiouracil (PTU), administered by adding 0.1% PTU to the mother's drinking water from birth through weaning, increases adult testis size, number of Sertoli cells, and daily sperm production. Adult Leydig cell numbers are also increased by 70%, despite permanent suppression of serum luteinizing hormone (LH). The present study was designed to test whether this effect results from an increase in proliferation of Leydig cells or the mesenchymal precursors. The labeling indices (LI) of six interstitial cell types were measured by tritiated thymidine autoradiography. With the exception of Leydig cells, LI declined after birth for all interstitial cell types in both control and PTU-treated rats, but mesenchymal cell proliferation was not significantly different in control and treated rats. In contrast, the LI of Leydig cells from PTU-treated rats rose by day 10, remained elevated through day 45 (highest on day 35 at 3.6-fold higher than control, P < 0.05), and declined to control values by day 50. The LI of Leydig cells in control rats was unchanged during the experiment. Proliferating Leydig cells were immature, as shown by their cytoplasmic lipid droplets. Adult Leydig cells, which lack lipid droplets, did not proliferate. Mesenchymal and immature Leydig cells contained thyroid hormone receptor mRNA; levels in adult Leydig cells were reduced but detectable. In conclusion, proliferation of Leydig cells, rather than increased proliferation of their mesenchymal precursors, is the principal mechanism responsible for the increase in Leydig cell number after neonatal hypothyroidism. Increased Leydig cell proliferation could result from direct effects of hypothyroidism on these cells. Alternatively, the increased Sertoli cell population that results from PTU treatment could also stimulate increased Leydig cell proliferation, or both of these mechanisms could contribute to the Leydig cell increase.

Characterization of AGT1 encoding a general alpha-glucoside transporter from Saccharomyces.

Molecular genetic analysis is used to characterize the AGT1 gene encoding an alpha-glucoside transporter. AGT1 is found in many Saccharomyces cerevisiae laboratory strains and maps to a naturally occurring, partially functional allele of the MAL1 locus. Agt1p is a highly hydrophobic, postulated integral membrane protein. It is 57% identical to Mal61p, the maltose permease encoded at MAL6, and is also a member of the 12 transmembrane domain superfamily of sugar transporters. Like Mal61p, Agt1p is a high-affinity, maltose/proton symporter, but Mal61p is capable of transporting only maltose and turanose, while Agt1p transports these two alpha-glucosides as well as several others including isomaltose, alpha-methylglucoside, maltotriose, palatinose, trehalose and melezitose. AGT1 expression is maltose inducible and induction is mediated by the Mal-activator. The sequence of the upstream region of AGT1 is identical to that of the maltose-inducible MAL61 gene over a 469 bp region containing the UASMAL but the 315 bp sequence immediately upstream of AGT1 shows no significant homology to the sequence immediately upstream of MAL61. The evolutionary origin of the MAL1 allele to which AGT1 maps and the relationship of AGT1 to other alpha-glucoside fermentation genes is discussed.