Direct method for detecting small quantities of hepatitis B virus DNA in serum and plasma using the polymerase chain reaction.

Serum components inhibit DNA polymerase, thereby obviating direct detection of serum viral DNA sequences by the polymerase chain reaction (PCR). This has necessitated extraction of nucleic acid from sera before performing PCR and has resulted in loss of sensitivity. By adsorbing virus to a solid surface (microcentrifuge tubes or antibody coated microparticles) followed by proteinase K digestion, as little as three viruses per 200 microliters serum may be directly detected by PCR without nucleic acid extraction. The sensitivity is dependent on the surface area of the adsorptive surface and is increased by having antibodies on the adsorptive surface. The nucleic acid sequence of the amplified DNA fragments may be directly determined by the dideoxy method. Of 24 plasma samples from HBsAg+ volunteer blood donors, HBV DNA was detected in 7 by dot blot assay, 7 by liquid hybridization, and 9 by PCR. PCR detected DNA in every sample that was positive by another assay. Analysis of serial samples of two patients with acute self-limited hepatitis B found detectable HBsAg and pre-S2 antigenemia before HBV DNA by the PCR method. These results suggest that surface antigenemia may precede viremia during acute hepatitis.

An anti-CD30 chimeric receptor that mediates CD3-zeta-independent T-cell activation against Hodgkin's lymphoma cells in the presence of soluble CD30.

Hodgkin's lymphoma patients fail to establish an efficient cellular response against CD30+ Hodgkin/Reed-Sternberg cells. An impaired T-cell receptor/CD3-zeta-mediated activation of T cells is thought to be involved in this situation. We here present a chimeric anti-CD30 receptor that mediates MHC and T-cell receptor/CD3-zeta-independent T-cell activation against CD30+ lymphoma cells even in the presence of soluble CD30. The receptor consists of the binding domain of the monoclonal antibody HRS3 and the signaling unit of the Fc epsilonRI-receptor gamma-chain. After expression in MD45 T cells, receptor cross-linking with immobilized anti-idiotypic monoclonal antibody and CD30+ cells, respectively, results in increased interleukin 2 secretion and specific cytolysis of CD30+ Hodgkin's lymphoma cells. Soluble CD30 in concentrations up to 6000 units/ml did not interfere with cellular activation induced by membrane-bound antigen. This demonstrates the feasibility of the chimeric anti-CD30-scFv-gamma receptor in CD30+ lymphoma cell targeting, even in the presence of as high concentrations of soluble CD30 as are found in patients during progression of the disease.

Isolation of single chain antibody fragments with specificity for cell surface antigens by phage display utilizing internal image anti-idiotypic antibodies.

Recombinant single chain antibody fragments (scFv) with specificity for membrane-bound antigens can be isolated by phage display techniques. The strategy involves selection of recombinant phage antibodies by binding to cells expressing the respective antigen. This results frequently in high nonspecific adherence of phages to cellular membranes. To resolve the problem we have made use of an internal image anti-idiotypic antibody mimicking the membrane-bound CD30 antigen and successfully isolated scFv fragments with specificity for CD30. The cDNA coding for the immunoglobulin heavy and light chain variable regions of the anti-CD30 monoclonal antibody (mAb) HRS3 was expressed by phage display techniques. Recombinant HRS3-scFv phages were efficiently enriched by one cycle of panning on the internal image anti-idiotypic mAb 9G10. The isolated HRS3-scFv clone retained the binding specificity of the parental mAb HRS3 to the internal image anti-idiotypic mAb 9G10 as well as to an anti-idiotypic mAb without the internal image. Furthermore HRS3-scFv reacted with recombinant and cell-bound CD30 antigen, respectively. Binding of scFv fragments to anti-idiotypic mAbs will provide a versatile strategy for the efficient isolation of recombinant antibody fragments.

Hepatitis B disease in dialysis and transplant patients. Further epidemiologic and serologic studies.

As hepatitis B virus (HBV) infection in renal transplant recipients is associated with a high incidence of progressive liver disease it may be inadvisable to transplant hemodialysis patients with hepatitis B antigenemia. To determine the natural history of HBV disease in hemodialysis patients, all 49 patients on hemodialysis treatment for at least 1 year, at 3 centers, who developed circulating hepatitis B surface antigen (HBsAG), were studied. A subgroup of these patients (n = 31) aged less than or equal to 50 years, followed for 55 +/- 6 months after detection of HBsAg was compared with 22 previously studied HBsAg-positive transplant patients followed for 81 +/- 9 months. Significantly more transplant patients developed chronic hepatitis defined biochemically (P less than .001) and none of the transplant patients became HBsAg-negative compared with 19% of the hemodialysis group. Taking difference in follow-up into account, mortality was significantly higher in the transplant recipients (P less than .005) following development of HBsAg antigenemia, and the mortality difference was attributable to deaths from liver disease. A total of 36 serum samples from 14 of the 22 HBsAg-positive renal transplant recipients was analyzed for hepatitis B e antigen (HBeAg), antibody to hepatitis D virus (anti-HD), and hepatitis B virus deoxyribonucleic acid (HBVDNA) concentration. No serum sample was anti-HD-positive. Twelve of the 14 patients were HBeAg-positive. Five patients became HBeAg-negative, 3 of whom developed aggressive liver disease. One HBeAg-negative anti-HBe-positive patient had progression of liver disease from asymptomatic carrier status to chronic active hepatitis (CAH). Of 14 patients, 9 developed progressive CAH. HBVDNA concentration was not diagnostic of disease activity on liver biopsy. However only 1 sample of 10 measured in 5 patients with nonprogressive disease had a level greater than 100 pg/L, compared with 9 of 17 in the group who progressed to CAH. During the interval when the liver histology progressed from asymptomatic carriage or chronic persistent hepatitis (CPH) to CAH, the HBVDNA concentration increased by greater than 10 times baseline in 4 of 5 patients who had serial samples, whereas this did not occur in 4 patients with nonprogressive disease. We conclude that the long-term outcome of hepatitis B infection in transplant recipients is significantly worse than in hemodialysis patients. Therefore it may be inadvisable to transplant HBsAg-positive hemodialysis patients.(ABSTRACT TRUNCATED AT 400 WORDS)

Postnatal phencyclidine treatment differentially regulates N-methyl-D-aspartate receptor subunit mRNA expression in developing rat cerebral cortex.

The present study was designed to determine the effects of chronic neonatal exposure to the NMDA receptor antagonist phencyclidine (PCP) on [3H]MK-801 binding and on gene expression of NMDA receptor subunits in juvenile male rats. Rat pups were injected daily with PCP from day 5 to 15 and killed on day 21. [3H]MK-801 binding was measured by quantitative autoradiography. A sensitive RNase protection assay was employed to determine simultaneously the mRNA levels of NR1 subunit (comprising all different splice variants) and three NR2 subunits (NR2A-NR2C). The relative distribution profile of NMDA receptor subunits in the cerebral cortex was NR2B > NR1 > NR2A > NR2C and in the cerebellum NR2C = NR1 > NR2A = NR2B. Chronic PCP administration in postnatal rats produced significant reduction in both [3H]MK-801 binding and mRNA level of the NR2B subunit in the cerebral cortex. Expression of the other NMDA receptor subunits in the cerebral cortex did not change following the drug treatment. In the cerebellum, neither [3H]MK-801 binding nor any of the NMDA receptor subunit expression levels showed any alteration. Together, these data provide a molecular correlate for chronic postnatal PCP-induced down-regulation of [3H]MK-801 binding in rat cerebral cortex and suggest that the NR2B subunit plays an important role in developmental plasticity.

Repeated neonatal phencyclidine treatment impairs performance of a spatial task in juvenile rats.

The present study was designed to determine whether repeated postnatal blockade of N-methyl-D-aspartate (NMDA) channel produces cognitive deficit in juvenile rats. Rats receive phencyclidine (PCP) intraperitoneally (i.p.) from postnatal Day 5 and continued daily till Day 15. On Day 28, saline- and PCP-treated rats were trained in the Morris water maze task. PCP-treated rats performed as well as the saline-treated rats on the first day of testing, but on the second day of testing they did significantly poorly compared to saline-treated controls. These data suggest that chronic postnatal NMDA channel blockade by PCP impairs processes that enable rats to retain spatial information.

Repeated postnatal phencyclidine administration in female juvenile rat delays onset of puberty but has no effect on pentylenetetrazol-induced seizure-susceptibility.

The effects of chronic phencyclidine (PCP) treatment on sexual maturation and seizure-susceptibility in the developing female rat were studied. Postnatal female rats were injected once daily with 5 mg/kg PCP from Day 5 till Day 15 and were observed for sexual maturation--vaginal opening and estrus cyclicity were used as indices of sexual maturation. PCP-treated rats showed a delay in both vaginal opening and in estrus cyclicity compared to saline-treated pair-fed controls. On postnatal Day 21, PCP-treated rats and saline-treated controls were tested for seizure-susceptibility using the pentylenetetrazol (PTZ)-induced seizure paradigm. PCP-treated female rats did not differ from saline-treated controls in their susceptibility to PTZ-induced seizures. In conclusion, chronic PCP administration during development in female rats disrupts sexual maturation but has no short-term effect on seizure susceptibility.

Effect of melatonin on cocaine-induced behavioral sensitization.

Melatonin, a pineal hormone and a potent free radical scavenger with neuroprotective actions, has been reported to act as an inhibitor of nitric oxide synthase (NOS). We have earlier shown that inhibitors of NOS (N(omega)-nitro-L-arginine methyl ester [L-NAME], 7-nitroindazole [7-NI]) block cocaine-induced behavioral sensitization. In the present study, the effects of melatonin on cocaine behavior were studied. A single injection of melatonin markedly augmented cocaine-induced locomotor activity. Rats injected daily with melatonin prior to cocaine injections failed to elicit cocaine sensitization. These behavioral effects of melatonin do not completely mimic those of other NOS inhibitors, suggesting that the effects of melatonin on cocaine behavior are mediated by both NOS-dependent as well as NOS-independent mechanisms.

Kinetic mechanisms of glycine requirement for N-methyl-D-aspartate channel activation.

Glycine potentiates N-methyl-D-aspartate (NMDA) receptor-mediated responses via its interaction with a strychnine-insensitive glycine recognition site. We have previously shown that the potent glycine receptor antagonist 7-chlorokynurenic acid (7Cl-KYN) dose-dependently inhibits [3H]MK-801 binding to the PCP receptor and that this effect is reversed by glycine. [3H]MK-801 binding to the PCP receptor within the NMDA receptor-gated ion channel is a measure of channel activation. Association of PCP receptor ligands is biexponential with the fast component of binding serving as a marker of activated NMDA channels. In the present study we utilize 7Cl-KYN as a probe of the kinetic mechanism of the glycine effect upon NMDA receptor functioning. In the presence of L-glutamate, incubation with 7Cl-KYN completely abolished the fast component of [3H]MK-801 association in 4 out of 5 experiments. In the fifth experiment where the fast component was detected, it accounted for less than half of that seen in the presence of L-glutamate alone. 7Cl-KYN-induced inhibition of the fast component of [3H]MK-801 association was reversed by the addition of glycine. Since the fast component represents ligand binding to the PCP receptor via the open NMDA channel, selective reduction of this component by 7Cl-KYN indicates that glycine receptor antagonists reduce the probability of channel opening, and also that the selective reduction in the component of [3H]MK-801 binding that manifests fast kinetics can serve as a marker for glycine antagonists.

Quantitative localization of [3H]TCP binding in rat brain by light microscopy autoradiography.

The anatomical localization of phencyclidine (PCP)/sigma-opiate receptors in rat brain was determined by quantitative light microscopy autoradiography using the new ligand N-(1-[2-thienyl]cyclohexyl) [3H]piperidine ([3H]TCP). TCP is a potent analog of PCP which possesses a higher affinity for PCP/sigma-opiate receptor than does PCP itself. The highest level of [3H]TCP binding was detected in the hippocampus. Intermediate levels were found in frontal cortex, striatum, amygdala and cerebellum. Specific [3H]TCP binding was undetectable in anterior commissure and corpus callosum. The distribution pattern of [3H]TCP binding sites is similar to the pattern obtained with [3H]PCP but more sharply defined. On the basis of its greater potency and specificity, [3H]TCP may prove superior to [3H]PCP as a molecular probe for the study of brain sigma opiate/phencyclidine receptors.

Effects of nitric oxide synthase inhibitors on cocaine sensitization.

Behavioral sensitization to cocaine was tested for in rats pretreated with a nitric oxide (NO) synthase inhibitor, N omega-nitro-L-arginine methyl ester (L-NAME) or 7-nitro indazole (7-NI). A 5-day pre-exposure to once daily cocaine (15 mg/kg, i.p.) injections yielded sensitization to cocaine (15 mg/kg)-induced behavioral activation. Pretreatment injections of L-NAME (100 mg/kg) or 7-NI (30 mg/kg), administered 30 min before each cocaine pre-exposure injection, acutely inhibited cocaine-induced behavioral activation. No sensitization was found after L-NAME pretreatment in a protocol with a 3-day withdrawal between pre-exposure and test cocaine injections. With a 10-day withdrawal period, cocaine sensitization was prevented by L-NAME or 7-NI pretreatment. These results after a 10-day withdrawal are unlikely to arise from deficient brain NO synthase activity on the test day. Instead, these findings suggest a role for NO in mechanisms underlying the development of cocaine sensitization. We conclude that NO participates in both the development of sensitization as well as the expression of cocaine-induced behavior in previously drug-naive animals.

The novel anticonvulsant MK-801: a potent and specific ligand of the brain phencyclidine/sigma-receptor.

MK-801 (5-methyl-10,11-dihydro-5H-dibenzo[a,d]cyclohepten-5,10-imine maleate) is a novel anticonvulsant agent reported to antagonize certain N-methyl-D-aspartate (NMDA)-mediated effects non-competitively. The question arises of the mechanism underlying the anti-NMDA and anticonvulsant effects of MK-801. In the present study MK-801 is shown to be an extremely potent inhibitor of the binding of N-[3H] (1-[2-thienyl]cyclohexyl)piperidine ([3H]TCP) to brain phencyclidine (PCP)/sigma-receptors. Its IC50 value of 3.8 +/- 0.8 nM in this assay ranks it as the most potent known ligand of brain PCP/sigma-receptors. Addition of MK-801 altered the apparent Kd but not the apparent Bmax values for [3H]TCP binding, indicating a competitive interaction. The specificity of action of MK-801 is supported by the finding that MK-801 strongly inhibited the binding of (+)-N-[3H]allylnormetazocine ((+)-[3H]SKF 10,047) to the PCP/sigma-receptor but its effect on (+)-[3H]SKF 10,047 binding to the non-PCP, haloperidol-sensitive sigma-binding site was weaker by several orders of magnitude. Furthermore, MK-801 exerts PCP-like antagonistic effects upon NMDA-induced [3H]norepinephrine release. These findings support the concept that the anticonvulsant and anti-NMDA effects of MK-801 result from its being the most potent known ligand of PCP/sigma-receptors.

Glycine reverses 7-chlorokynurenic acid-induced inhibition of [3H]MK-801 binding.

7-Chlorokynurenic acid (7-Cl KYNA) has been reported to attenuate N-methyl-D-aspartate (NMDA) receptor functioning by a potent and selective inhibitory action mediated at the strychnine-insensitive glycine recognition site of the NMDA complex. Here we report that 7-Cl KYNA dose-dependently inhibits [3H]MK-801 binding to the PCP receptor, and that this effect is reversed by addition of glycine. Since [3H]MK-801 binding is a measure of channel activation, our results are consistent with the hypotheses that 7-Cl KYNA exerts its NMDA receptor antagonism by acting at the glycine site, and that activation of the glycine site is required for NMDA channel activity to occur.

Postnatal stress selectively upregulates striatal N-methyl-D-aspartate receptors in male rats.

Early life events have been thought to contribute towards vulnerability to drug addiction later in life. In the present investigation, the effect of daily neonatal maternal isolation stress on NMDA channel activity was studied. [3H]MK-801 binding was measured in several brain regions from neonatally isolated (ISO) and nonhandled (NH) adult male and female rats. Maximal [3H]MK-801 binding in the caudate-putamen of male ISO rats was 58% higher compared to same sex NH rats. Unlike male rats, maximal [3H]MK-801 binding in the caudate-putamen of female ISO rats was lower than female NH rats. No other brain region showed any significant difference in maximal [3H]MK-801 binding between ISO and NH male and female rats, respectively. There was no effect of pup isolation on the binding affinity (K(d) value) in either sex. Repeated maternal isolation is associated with alterations in the NMDA channel activity in the caudate-putamen of adult rats, and may be responsible for the augmentation in the addictive behavior reported.

Polyamine effects upon N-methyl-D-aspartate receptor functioning: differential alteration by glutamate and glycine site antagonists.

Polyamines such as spermidine potentiate activation of the N-methyl-D-aspartate (NMDA)-type excitatory amino acid receptor. The goal of the present study was to investigate interactions between the putative polyamine binding site and previously described sites for glutamate and glycine. Binding of the high-potency PCP receptor ligand [3H]MK-801 to well-washed rat brain membranes was used as an in vitro probe of NMDA receptor activation. Spermidine concentration-response studies were performed in the absence and presence of both glutamate and glycine, with and without D-(-)-2-amino-5-phosphonovaleric acid (D(-)-AP-5) or 7-chlorokynurenic acid (7Cl-KYN). Incubation in the presence of spermidine alone induced a 20.4-fold increase in [3H]MK-801 binding with an EC50 value of 13.3 microM. The mean concentration of spermidine which induced maximal stimulation of binding was 130 microM (n = 10, S.E.M. = 24.66, range = 25-250 microM). Glutamate (10 microM) decreased the EC50 value for spermidine-induced stimulation of [3H]MK-801 binding to 3.4 microM. Glycine (10 microM) did not significantly alter either maximum spermidine-induced [3H]MK-801 binding or the EC50 value for spermidine-induced stimulation of [3H]MK-801 binding. Incubation in the presence of the specific glutamate antagonist D(-)AP-5 attenuated [3H]MK-801 binding in a glutamate-reversible fashion. The competitive glycine antagonist 7Cl-KYN decreased maximum spermidine-induced [3H]MK-801 binding in a glycine-reversible fashion. In addition, 7Cl-KYN increased the EC50 value for spermidine-induced stimulation of [3H]MK-801 binding while D(-)AP-5 was without effect.(ABSTRACT TRUNCATED AT 250 WORDS)

Developmental maturation of the N-methyl-D-aspartic acid receptor channel complex in postnatal rat brain.

The N-methyl-D-aspartate (NMDA) receptor plays an important role in developmental plasticity. Previous studies have reported differences between the NMDA receptor-channel complex in the rat pup brain and the adult brain. In the present study, modulation of the NMDA channel complex as a function of age was measured to determine when the temporal switching of the NMDA receptor from the immature form to the adult mature form takes place. [(3)H]MK-801 binding was measured in the rat forebrain from postnatal day 1 to day 21. Our data suggest the presence of two types of NMDA receptors - an immature type and a mature type. The immature NMDA receptor, seen during the early postnatal period (day 1-day 14) is highly sensitive to spermidine, L-glutamate alone potentiates [(3)H]MK-801 binding, and glycine failed to potentiate an L-glutamate-induced increase in [(3)H]MK-801 binding. During the late postnatal period (after day 14) spermidine alone did not increase [(3)H]MK-801 binding as potently as it did during the early postnatal period, high-affinity [(3)H]MK-801 binding was not seen in the presence of L-glutamate alone, and L-glutamate and glycine or L-glutamate and spermidine or L-glutamate, glycine and spermidine together, significantly increased [(3)H]MK-801 binding in a manner similar to that reported in the adult brain. Together, the pharmacology of the NMDA receptor during the early postnatal period differs from the adult-like receptor seen during the late postnatal period, and that in rats the apparent switching of the NMDA receptor from the immature type to the mature type takes place after the second postnatal week.

Chronic neonatal phencyclidine treatment produces age-related changes in pentylenetetrazol-induced seizures.

Although excitatory amino acids are known to play a critical role in the plasticity of developing brain, the behavioral effects of blocking the N-methyl-D-aspartate (NMDA) receptor-gated ion channel during development are not clear. Here we report the effects of chronic postnatal administration of 1-phenylcyclohexylpiperidine (phencyclidine or PCP), a NMDA channel blocker, on seizure susceptibility. To study the short-term effects of chronic PCP administration on pentylenetetrazol (PTZ)-induced seizures, rats were treated with PCP (5 mg/kg, i.p.) for 11 days from postnatal days 5-15, 24-34 or 44-54 and tested in the PTZ-induced seizure paradigm on postnatal days 21, 40 and 60, respectively. Administration of PCP in 5-15-day-old rats resulted in increased seizure susceptibility at day 21, while administration of PCP in postweanling rats (days 24-34) markedly attenuated their susceptibility to seizures at day 40. PCP injection had little effect on the seizure susceptibility of older rats. To study the long-term effects of postnatal PCP treatment, rats were injected with PCP (5 mg/kg from postnatal day 5-15, i.p.) and were tested for PTZ-induced seizures on postnatal days 40 and 60; each rat was tested only once. When tested for PTZ-induced seizure on day 40, PCP-treated rats did not differ from saline-treated controls. When tested on day 60, PCP-treated rats had a lower incidence of seizures and in the rats that did have seizures their latencies were significantly prolonged compared to controls. Together, our data suggest that chronic PCP administration alters PTZ-induced seizure susceptibility in an age-dependent manner and chronic PCP administration in postnatal rats produces long-term changes that persist into adulthood.

Chronic postnatal phencyclidine treatment on [3H](+) pentazocine binding in juvenile rat brain.

Chronic phencyclidine (PCP) administrations in postnatal rats produce long-term behavioral changes. Here we report the effects of repeated postnatal PCP treatment on [3H](+)pentazocine binding in juvenile rats. Saturation analyses of the binding data showed no significant difference in any of the binding characteristics between chronic PCP-related rats and saline-treated controls suggesting that mechanisms other than alterations in sigma1 binding underlie the behavioral effects of repeated postnatal PCP administration in immature rats.

Non-competitive regulation of phencyclidine/sigma-receptors by the N-methyl-D-aspartate receptor antagonist D-(-)-2-amino-5-phosphonovaleric acid.

D-(-)-2-Amino-5-phosphonovaleric acid (D(-)AP5), a selective, potent competitive antagonist of N-methyl-D-aspartate (NMDA)-type excitatory amino acid receptors was used to investigate the relationship between NMDA receptors and phencyclidine (PCP) binding. Incubation of rat brain membranes with D(-)AP5 decreased the apparent number of PCP/sigma-receptors in dose-dependent fashion without affecting their affinity for [2-thienyl-3H]cyclohexylpiperidine (TCP). These data, taken together with electrophysiological evidence that PCP non-competitively antagonizes NMDA receptor-mediated transmission, are consistent with the hypothesis that the PCP/sigma-receptor may be situated in or near a channel regulated by an NMDA receptor complex.

Ontogeny of sigma opiate/phencyclidine-binding sites in rat brain.

The development of stereospecific sigma opiate/phencyclidine (PCP)-binding sites in fetal and neonatal rat brain was examined. Displaceable PCP binding was detected from day 13 of gestation. Bmax values increased to adult levels just prior to birth. Apparent KD values did not change. Stereospecific binding became detectable after day 19, concomitant with a rapid increase in Bmax. Despite the early appearance of binding, pharmacologically relevant stereospecific sigma opiate/PCP-binding sites do not become evident until shortly prior to parturition.