Thymoglobulin-associated Cd4+ T-cell depletion and infection risk in HIV-infected renal transplant recipients.

HIV-infected patients are increasingly referred for kidney transplantation, and may be at an increased risk for rejection. Treatment for rejection frequently includes thymoglobulin. We studied thymoglobulin's effect on CD4+ T-cell count, risk of infection and rejection reversal in 20 consecutive HIV-infected kidney recipients. All patients used antiretroviral therapy and opportunistic infection prophylaxis. Maintenance immunosuppression consisted of prednisone, mycophenolate mofetil and cyclosporine. Eleven patients received thymoglobulin (7 for rejection and 4 for delayed/slow graft function) while 9 did not. These two groups were similar in age, gender, race, donor characteristics and immunosuppression. Mean CD4+ T-cell counts remained stable in patients who did not receive thymoglobulin, but became profoundly suppressed in those who did, decreasing from 475 +/- 192 to 9 +/- 10 cells/microL (p < 0.001). Recovery time ranged from 3 weeks to 2 years despite effective HIV suppression. Although opportunistic infections were successfully suppressed, low CD4+ T-cell count was associated with increased risk of serious infections requiring hospitalization. Rejection reversed in 6 of 7 patients receiving thymoglobulin. We conclude that thymoglobulin reverses acute rejection in HIV-infected kidney recipients, but produces profound and long-lasting suppression of the CD4+ T-cell count associated with increased risk of infections requiring hospitalization.

Health-related fitness and quality of life in organ transplant recipients.

BACKGROUND: The purpose of this study was to describe the levels of health-related fitness and quality of life in a group of organ transplant recipients who participated in the 1996 U.S. Transplant Games. METHODS: A total of 128 transplant recipients were selected on a first reply basis for testing. Subjects with the following organ types were tested: kidney (n=76), liver (n=16), heart (n=19), lung (n=6), pancreas/kidney (n=7), and bone marrow (n=4). Cardiorespiratory fitness (peak oxygen uptake) was measured using symptom-limited treadmill exercise tests with expired gas analysis. The percentage of body fat was measured using skinfold measurements, and the Medical Outcomes Short Form questionnaire (SF-36) was used to evaluate health-related quality of life. RESULTS: Participants achieved near age-predicted cardiorespiratory fitness (94.7+/-32.5% of age-predicted levels). Scores on the SF-36 were near normal. The active subjects (76% of total sample) had significantly higher levels of peak VO2 and quality of life and a lower percentage of body fat compared with inactive subjects (P<0.01). CONCLUSIONS: Although this is a highly select group which is not representative of the general transplant population, the data suggest that near-normal levels of physical functioning and quality of life are possible after transplantation and that those who participate in regular physical activity may achieve even higher levels.

Properties and localization of DNA methyltransferase in preimplantation mouse embryos: implications for genomic imprinting.

Preimplantation mouse embryos contain very high levels of DNA methyltransferase activity. We show here that the form of DNA methyltransferase (DNA MTase) in early embryos differs from the form found in other cells and tissues by a slightly higher mobility on gel electrophoresis. Levels of DNA MTase were found to be very high throughout preimplantation development even though levels of 5-methylcytosine (m5C) in nuclear DNA are known to undergo a substantial decline in the same period. Confocal laser scanning microscopy of mouse embryos stained with DNA MTase-specific antibodies showed striking developmentally regulated changes in the distribution of DNA MTase. From the oocyte stage to the four-cell-stage, most DNA MTase was concentrated in peripheral cytoplasm, and nuclei did not contain detectable DNA MTase. In four- and eight-cell embryos, DNA MTase was seen in cytoplasmic granules; and in eight-cell embryos, DNA MTase was also present in large amounts in nuclei. Nuclei of blastocysts stained only faintly, whereas the cytoplasmic granules remained prominent. Paradoxically, DNA MTase was found to be at its highest levels in nuclei at a developmental stage where levels of m5C in DNA are decreasing most rapidly. Changes in methylation patterns in preimplantation embryos are therefore proposed to be under the control of unidentified regulatory factors rather than DNA MTase itself; these regulatory factors could be members of the group that contains the products of the Ssm-1 and Imp-1 genes, which are involved in the regulation of genomic imprinting.

Melatonin receptors and signal transduction during development in Siberian hamsters (Phodopus sungorus).

Maternal melatonin communicates daylength information to the fetus in Siberian hamsters. Fetal sensitivity to melatonin declines near birth. In this report, we describe melatonin receptor distribution and a second messenger response to melatonin in Siberian hamsters during the perinatal period. The sites of high-affinity 2-[125I]iodomelatonin ([125I]MEL) binding were generally similar throughout the perinatal period. The non-hydrolyzable GTP analog, guanosine-5'-O-(3-thiotriphosphate) (100 microM) inhibited [125I]MEL binding at each age, suggesting the melatonin receptors are associated with guanine nucleotide binding proteins (G proteins). Furthermore, melatonin (10 nM) inhibited forskolin-stimulated cAMP accumulation in median eminence/pars tuberalis (ME/PT) explants as early as 4 days before birth, when sensitivity to melatonin in vivo is high. The cAMP regulatory system appeared disrupted on the day of birth, in that forskolin (10 microM) stimulation of cAMP accumulation was reduced, and melatonin did not inhibit cAMP accumulation stimulated by forskolin. A higher forskolin dose (100 microM) elevated cAMP levels more clearly on the day of birth, and melatonin inhibited forskolin-stimulated cAMP accumulation. These results suggest that the decreased physiological responsiveness to melatonin at the end of gestation may be due to alterations in the cAMP regulatory system.

Melatonin receptors and signal transduction in photorefractory Siberian hamsters (Phodopus sungorus).

In seasonally breeding mammals, seasonal alterations in day length are perceived via the pineal hormone melatonin. When exposed to short day lengths, hamsters and other long-day breeders undergo gonadal regression. With chronic exposure (greater than 20 weeks) to short days, however, the animals become photorefractory, or insensitive to the inhibitory influence of short-day melatonin patterns, and gonadal recrudescence occurs. In this report, we examined photorefractory Siberian hamsters and long-day housed control hamsters to examine whether this apparent insensitivity to melatonin is due to alterations in melatonin receptors or signal transduction. In vitro autoradiographic assessment of melatonin receptors using 125I-labeled 2-iodomelatonin (I-MEL) revealed that melatonin receptor distribution, affinity, density, and G protein coupling are unaltered in photorefractory animals. In each animal, high-affinity (dissociation constant approximately 40 pM) 2-iodomelatonin binding sites were observed in the hypophysial pars tuberalis, in the suprachiasmatic nucleus of the hypothalamus, and in the thalamus (paraventricular nucleus, reuniens nucleus, and nucleus of the stria medullaris). The nonhydrolyzable GTP analog, GTP gamma S (100 microM) caused a 10-fold reduction in melatonin receptor affinity in the pars tuberalis in both photorefractory and control hamsters, demonstrating receptor-G protein coupling in both groups. Furthermore, melatonin (10 nM) inhibited forskolin-stimulated cAMP accumulation in median eminence/pars tuberalis explants in photorefractory animals, just as previously observed in explants from long-day hamsters. These results suggest that melatonin receptors, receptor-G protein coupling, and inhibition of adenylyl cyclase by melatonin are not altered in photorefractory hamsters.

Melatonin receptors and signal transduction in melatonin-sensitive and melatonin-insensitive populations of white-footed mice (Peromyscus leucopus).

The pineal hormone melatonin times seasonal alterations in reproductive function in photoperiodic mammals. In white-footed mice, there is variation in responsiveness to the reproductive effects of melatonin between populations originating in different locations; mice from Connecticut (CT) respond normally to melatonin, while mice from Georgia (GA) appear insensitive to melatonin. In the present paper, we compare melatonin receptor distribution and a second messenger response to melatonin in white-footed mice from CT and GA. Specific binding of 125I-labeled melatonin (I-MEL) was observed in a variety of brain regions in each population, but there were no consistent differences in the distribution or intensity of I-MEL binding between the populations. Furthermore, melatonin inhibited forskolin-stimulated cAMP accumulation in median eminence/pars tuberalis explants from both populations. These results suggest that insensitivity to melatonin in GA mice is not due to a gross defect in melatonin receptors or receptor-effector coupling.

Geographic differences for delay of sexual maturation in Peromyscus leucopus: effects of photoperiod, pinealectomy, and melatonin.

Effects of short-day photoperiod, pinealectomy, and melatonin on sexual maturation were tested in Peromyscus leucopus from either Connecticut (CT) or Georgia (GA). Laboratory reared-stocks from CT and GA were exposed to short daylength (photoperiod) from birth or 25 days of age. At 12 wk of age, delay in sexual maturation was indicated in most CT mice by decreased testis length, combined testes weight, and seminal vesicle weight. Conversely, GA animals did not delay sexual maturation when exposed to short-day photoperiod from either birth or 25 days of age. These results indicate that responses to short daylengths differ for juvenile CT and GA populations. In a second experiment, pinealectomized or sham-operated CT males were exposed to short-day (9L:15D) or long-day (16L:8D) photoperiod from birth. Pinealectomy blocked the effect of short daylength on reproduction. Therefore, the pineal must be involved in the delay of sexual maturation observed for short-day CT mice. The effects of melatonin, a pineal gland hormone, were tested with chronic s.c. implants or daily injections. In CT mice given either melatonin implants or afternoon injections, sexual maturation was delayed. GA mice were insensitive to all melatonin treatments. Further, no differences in circadian organization (phase angle, duration of activity, period under constant dark) between GA and CT animals were apparent. Collectively, these studies indicate that melatonin is involved in the mechanism responsible for delay of sexual maturation in CT mice. Short-day insensitivity of GA Peromyscus leucopus probably results from a deficiency in the melatonin effector pathway and is not due to a disruption of circadian organization.

Melatonin signal transduction in hamster brain: inhibition of adenylyl cyclase by a pertussis toxin-sensitive G protein.

Melatonin signal transduction was examined in median eminence/pars tuberalis (ME/PT) explants from Djungarian hamsters. High affinity melatonin receptors in hamster ME/PT were first quantified by in vitro autoradiography using the potent melatonin agonist 125I-labeled melatonin ([125I]MEL). Scatchard analysis of [125I]MEL binding in ME/PT revealed high affinity receptors [dissociation constant (Kd) = 2.75 X 10(-11) M]. [125I]MEL binding was markedly reduced by guanine nucleotides; treatment with the nonhydrolyzable GTP analog guanosine 5'-O-(3-thiotriphosphate) caused a 10-fold decrease in receptor affinity. Melatonin (10 nM) significantly inhibited forskolin-stimulated cAMP accumulation in ME/PT, but not in pituitary or pineal glands. In ME/PT explants, melatonin and 6-chloromelatonin inhibited forskolin-stimulated cAMP accumulation in a dose-dependent manner with similar potency (significant inhibition for each at concentrations greater than or equal to 100 pM). Serotonin significantly inhibited forskolin-stimulated cAMP levels only at doses greater than or equal to 100 microM. Inhibition of [125I]MEL binding in ME/PT by these three indolamines paralleled that determined for inhibition of forskolin-stimulated cAMP accumulation. Pertussis toxin treatment (1 microgram/ml) blocked the ability of melatonin (10 nM) to inhibit forskolin-stimulated cAMP accumulation and significantly reduced [125I]MEL binding. Pertussis toxin ADP-ribosylated the alpha-subunits of at least two guanine nucleotide-binding proteins in ME/PT explants with molecular weights of approximately 40 K. Melatonin did not increase phosphodiesterase activity in ME/PT explants. The results strongly suggest that a signal transduction pathway for melatonin in mammals involves inhibition of adenylyl cyclase by a pertussis toxin-sensitive guanine nucleotide-binding protein.

Guanine nucleotide-binding protein regulation of melatonin receptors in lizard brain.

Melatonin receptors were identified and characterized in crude membrane preparations from lizard brain by using 125I-labeled melatonin (125I-Mel), a potent melatonin agonist. 125I-Mel binding sites were saturable; Scatchard analysis revealed high-affinity and lower affinity binding sites, with apparent Kd of 2.3 +/- 1.0 x 10(-11) M and 2.06 +/- 0.43 x 10(-10) M, respectively. Binding was reversible and inhibited by melatonin and closely related analogs but not by serotonin or norepinephrine. Treatment of crude membranes with the nonhydrolyzable GTP analog guanosine 5'-[gamma-thio]triphosphate (GTP[gamma S]), significantly reduced the number of high-affinity receptors and increased the dissociation rate of 125I-Mel from its receptor. Furthermore, GTP[gamma S] treatment of ligand-receptor complexes solubilized by Triton X-100 also led to a rapid dissociation of 125I-Mel from solubilized ligand-receptor complexes. Gel filtration chromatography of solubilized ligand-receptor complexes revealed two major peaks of radio-activity corresponding to Mr greater than 400,000 and Mr ca. 110,000. This elution profile was markedly altered by pretreatment with GTP[gamma S] before solubilization; only the Mr 110,000 peak was present in GTP[gamma S]-pretreated membranes. The results strongly suggest that 125I-Mel binding sites in lizard brain are melatonin receptors, with agonist-promoted guanine nucleotide-binding protein (G protein) coupling and that the apparent molecular size of receptors uncoupled from G proteins is about 110,000.