Localizing brain interactions from rhythmic EEG/MEG data.

The interpretation of MEG/EEG data in terms of brain connectivity is largely obscured by artefacts of volume conduction, i.e. by the fact that a single source is observable in many channels. Here, we analyze a measure which is insensitive to spurious connectivity arising from volume conducted "self-interaction". For rhythmic data such a measure can be given by the imaginary part of the cross-spectrum between EEG/MEG channels. For the derivation we essentially exploit that a signal is not time-lagged to itself. To localize the sources of this observed interaction we fit a model cross-spectrum consisting of N interacting dipoles to the sample cross-spectrum. The relation to the maximum likelihood estimator will be discussed in detail. The method is illustrated for MEG data of human alpha rhythm in eyes closed condition. The eigenvalues of the imaginary cross-spectrum clearly indicate the presence of at least 4 necessarily interacting sources. Fits of 2 to 6 dipoles in a realistic volume conductor all resulted in locations scattered in the mesial part of the occipital lobe.

Insulin resistance in adult rat offspring associated with maternal dietary fat and alcohol consumption.

Maternal diet during pregnancy has been reported to alter the offspring's ability to respond to a glucose challenge. The current studies report changes in basal and insulin-stimulated, in vitro glucose uptake in red (soleus) and white (extensor digitorum longus) muscle fiber types, as well as whole body insulin responsiveness of adult rat offspring associated with their mother's dietary fat and alcohol content during pregnancy. The offspring of Harlan-derived Sprague-Dawley female rats, dosed during pregnancy with ethanol (ETOH) via a liquid diet (35% of calories as ETOH) with either 12% or 35% of calories as fat, were compared with offspring from litters whose mothers were pair-fed an isocaloric amount of the liquid diet without ETOH. Maternal access to the liquid diets was terminated on day 20 of the pregnancies (sperm plug=day 0). The offspring were surrogate fostered within 48 h of birth to mothers which had consumed commercial chow throughout their pregnancy. Following weaning at 21 days of age, the offspring consumed only commercial rat chow and they were examined over the next 14 months for changes in glucose homeostasis as a consequence of in utero exposure to maternal dietary fat and/or alcohol. The 35% maternal fat diet resulted in both in vivo and in vitro decreases in insulin sensitivity. Thus, compared with adults whose mother's diet contained 12% fat, significant, in vitro muscle and in vivo whole body insulin resistance (measured by hyperinsulinemic-euglycemic clamping) was observed in adult rats whose mothers consumed 35% of dietary calories as fat. The addition of ethanol to the maternal 35% fat diet further reduced the offspring's red muscle tissues in vitro response to insulin, but did not affect whole body insulin sensitivity. Muscle basal and insulin-stimulated receptor tyrosine kinase activity were significantly decreased (approximately -50%) by the 35% fat maternal diet but there was no compensatory increase in serum insulin or glucose levels. Based upon both in vivo and in vitro data, these studies suggested that in utero exposure to 35% fat has a sustained effect on the adult offspring's glucose uptake/insulin sensitivity and that the effect is paralleled, at least in part, by decreased insulin receptor tyrosine kinase activity. In utero ETOH exposure resulted in the loss of basal and insulin-stimulated, in vitro glucose uptake in red muscle fibers but maternal dietary ETOH had no detectable effect on either in vivo insulin sensitivity or muscle tyrosine kinase activity.

Alcohol-induced modulation of the insulin-like growth factor system in early chick embryo cranial tissue.

BACKGROUND: Fetal alcohol exposure has been shown to reduce fetal/embryonic growth. The insulin-like growth factor (IGF) system plays a major role in normal growth and development of the embryo. The purpose of this study was to gain a better understanding of the effects of alcohol (ethanol, EtOH) exposure on the insulin-like growth factors, their binding proteins, and receptors during embryonic development. METHODS: After the administration of either alcohol or chick Ringer's solution to individual eggs at the start of incubation, type-1 IGF receptors, IGF-binding proteins (IGFBPs) as well as IGF-1 and IGF-2 levels were measured in chick embryo craniums on days 5, 6, 7, and 8 of incubation. RESULTS: Levels of the IGF-1 receptor protein were not significantly different between treatment groups on any day studied. In EtOH-treated embryos, the 30 kDa IGFBP levels were significantly higher than vehicle levels on days 5 and 6. On day 6, IGF-1 levels were significantly lower in the alcohol-treated embryos compared with levels in vehicle-treated embryos of the same age. By day 8 of incubation, IGF-1 levels were significantly higher and the 30 kDa IGFBP levels were significantly lower in the alcohol-treated group compared with vehicles. These results indicate an initial EtOH-associated reduction in the amount of IGF-1 available to bind to its receptor (bioavailability), followed by increased IGF-1 bioavailability by day 8. CONCLUSIONS: The elevated IGFBP levels and reduced IGF-1 levels on days 5 and 6 of incubation are congruent with an overall reduction in the bioavailability of IGF-1 during this period and correlate with the decreased embryo weight observed in the alcohol-treated embryos. An increased bioavailability of IGF-1 observed by day 8 may represent a rebound effect and is associated with increases in ornithine decarboxylase activity, a marker of increased growth.

Differential expression of glucose transporters during chick embryogenesis.

The patterns of Glut1 and Glut3 glucose transporter protein and mRNA expression were assessed during embryogenesis of chicken brain and skeletal muscle, Glut4 protein levels were also evaluated in skeletal muscle and heart, and Glut1 was examined in the developing heart and liver. Glut1 protein expression was detectable throughout brain ontogeny but was highest during early development. Glut1 mRNA levels in the brain remained very high throughout development. Glut3 protein was highest very early and very late and mRNA was highest during the last half of development. In embryonic skeletal muscle, the levels of Glut1 and Glut3 proteins and mRNA were highest very early, and declined severely by mid-development. Glut1 protein and mRNA in the heart also peaked early and then decreased steadily. Although Glut1 mRNA levels were consistently high in the embryonic liver, Glut1 protein expression was not detected. These results suggest that (1) Glut1 is developmentally regulated in chick brain, skeletal muscle, and heart, (2) Glut1 mRNA is present in liver but does not appear to be translated, (3) Glut3 in brain increases developmentally but is virtually absent in muscle, and (4) Glut4 protein and mRNA appear to be absent from chick heart and skeletal muscle.

Quantitative measurements of edge-to-face aromatic interactions by using chemical double-mutant cycles.

Synthetic H-bonded zipper complexes have been used to quantify the magnitude of an edge-to-face aromatic interaction between a benzoyl group and an aniline ring. Four chemical double-mutant cycles were constructed by using a matrix of nine closely related complexes in which the aromatic rings were sequentially substituted for alkyl substituents. The stability constants and three-dimensional structures of the complexes were determined by using 1H NMR titrations in deuterochloroform at room temperature. The value of the interaction energy is similar in all cases, the average is -1.4 +/- 0.5 kJ mol(-1). The scope and limitations of the double-mutant approach are explored, and the consequences of conformational equilibria are discussed.

Increased intracellular localization of brain GLUT-1 transporter in response to ethanol during chick embryogenesis.

Fetal exposure to ethanol is associated with growth retardation of the developing central nervous system. We have previously described a chick model to study the molecular mechanism of ethanol effects on glucose metabolism in ovo. Total membrane fractions were prepared from day 4, day 5, and day 7 chick embryos exposed in ovo to ethanol or to vehicle. By Western blotting analysis, ethanol exposure caused a mean 7- to 10-fold increase in total GLUT-1 and a 2-fold increase in total GLUT-3. However, glucose uptake by ethanol-treated cells increased by only 10%. Analysis of isolated plasma (PM) and intracellular (IM) membranes from day 5 cranial tissue revealed a mean 25% decrease in GLUT-1 in the PM and a 66% increase in the IM in the ethanol group vs. control. The amount of PM GLUT-3 was unchanged but that of IM GLUT-3 was significantly decreased. The data suggest that GLUT-3 cell surface expression may be resistant to the suppressive effects of ethanol in the developing brain of ethanol-treated embryos. The overall increase in GLUT-1 may reflect a deregulation of the transporter induced by ethanol exposure. The increased IM localization and decreased amount of PM GLUT-1 may be a mechanism used by the ethanol-treated cell to maintain normal glucose uptake despite the overall increased level of the transporter.

Spermatophore production and spermatheca content in Tenebrio molitor infected with Hymenolepis diminuta.

Male and female Tenebrio molitor act as intermediate hosts for metacestodes of the rat tapeworm, Hymenolepis diminuta. It is known that the bean-shaped accessory glands of infected males exhibit an extended growth period and are significantly larger than those from noninfected males by day 10 after emergence. We wished to determine whether more material is transferred from these glands into the spermatophores. Here we report that the protein content and trehalase activity of spermatophores produced by bean-shaped accessory glands from infected males is elevated. However, protein transferred to the female spermatheca during mating was not affected by the infection status of the male. No evidence of transfer of trehalase to the spermatheca was detected but spermatheca from virgin, infected females contained significantly greater trehalase activity than those from noninfected females.

The effect of metacestodes of Hymenolepis diminuta on the bean-shaped accessory glands in male Tenebrio molitor.

Metacestodes of Hymenolepis diminuta affect several aspects of female reproductive physiology in Tenebrio molitor and such effects are mediated via the endocrine system. The effects on male reproduction are less well known and were studied with respect to the Bean-Shaped Accessory Glands (BAGs). The size and wet and dry weight of BAGs from infected and uninfected beetles were compared and rose to a plateau from 0-6 days post-emergence in uninfected beetles but in infected individuals continued to increase in both size and weight. These effects were density independent. Glands from both infected and uninfected beetles were assayed for trehalase activity measured by its ability to convert the sugar trehalose to glucose. The activity of this enzyme, per mg wet weight, was not affected by the parasite. However, total activity per gland increased in infected males. Total protein content and electrophoretic profiles of BAGs from infected and uninfected individuals showed no change in profile but showed an increase in all protein subunits per gland over a broad molecular weight range.

Changes in brain glucose levels and glucose transporter protein isoforms in alcohol- or nicotine-treated chick embryos.

Suppression of fetal brain growth during pregnancy as the result of maternal smoking or alcohol consumption leads to significant problems for the offspring as well as for the society who must care for these individuals. Chronic maternal intake of cigarette smoke is frequently observed in humans and studies using animal models suggest that in utero nicotine exposure is an important component of the growth suppression that results. Similarly, maternal consumption of alcohol (ethanol) has a profound, negative effect on fetal growth. The developing fetal central nervous system (CNS) is sensitive to the growth inhibitory effect of nicotine or alcohol and morphological as well as functional CNS deficits may result from fetal exposure. Using an embryonic chick model which minimizes drug-induced changes in maternal nutrition and behavior, the studies presented here indicate that nicotine or alcohol exposure during early embryonic development inhibits brain growth to a degree comparable to that seen in the rest of the organism, i.e., there was no 'brain sparing' in this model. Glucose content per milligram tissue was markedly decreased in brains of the nicotine-treated embryos but was not significantly different in the alcohol-exposed embryos. Western blots of fetal brain glucose transporter protein isoforms showed no change in the Glut 3 transporter content in the growth suppressed brains compared to vehicle-treated brains. The Glut 1 55 kilodalton (kd) isoform protein content was significantly decreased in the nicotine-treated brains but unchanged in the ethanol-treated brains, while the reverse was true for the Glut 1 45 kd isoform. Thus, the changes in the 55 kd isoform protein content were correlated with tissue glucose levels in the ethanol- and nicotine-treated embryos.

Ethanol differentially affects metabolic and mitotic processes in chick embryonic cells.

Our laboratory has been investigating the mechanisms by which ethanol-induced growth inhibition occurs in a developing embryo, and our studies have focused on disruption of cellular signaling pathways. Previous work on ethanol-induced changes in signaling systems that regulate ornithine decarboxylase activity indicated that the pathways containing protein kinase A, protein kinase C (PKC), and insulin-dependent tyrosine kinase were important for the control of ornithine decarboxylase in chick embryonic cells. Herein, we report ethanol's effect on the regulation of glucose uptake and thymidine uptake by these same kinase pathways. A pronounced increase in glucose uptake was associated with PKC downregulation in both vehicle- and ethanol-exposed cells, with the larger increase occurring in ethanol-exposed cells. An increase in thymidine uptake was associated with an activation of all three kinases, as well as with downregulation of PKC. Because previous work on signaling pathways has looked for changes in the insulin signaling pathway, the work herein focuses on the signaling pathways involving protein kinase A and PKC. cAMP levels were increased by ethanol treatment, but the increase was relatively small. Analysis of changes in PKC activity induced by ethanol exposure showed a significant suppression of PKC activity in the ethanol-treated cells and suggested that, overall, ethanol treatment affects the regulation of glucose uptake in embryonic cells predominantly by PKC downregulation.

The facilitating effect of one-DR antigen sharing in renal allograft tolerance induced by donor bone marrow in rhesus monkeys.

Infusion of donor bone marrow cells (DBMC), a long-standing, successful strategy for inducing tolerance in experimental rodent transplantation models, can promote long-term acceptance of life-sustaining renal allografts in rhesus monkeys with no maintenance immunosuppression. To investigate the immunological basis for heterogeneity in duration of long-term graft acceptance following infusion of the DR-/dim fraction of DBMC into RATG-treated rhesus monkeys, we examined the relationship of recipient-donor major histo-compatibility class I and II DR matching to the development of antidonor antibody-dependent cellular cytotoxicity (ADCC) and renal allograft survival. The findings indicate a requirement for sharing one DR allele to achieve long-term graft acceptance. The observed immunological consequence of DR sharing that correlated with functional graft tolerance in this model was the suppression of early antidonor ADCC+ IgG antibody responses. Significant associations were observed between graft survival and suppression of ADCC antibody (P < 0.0005), graft survival and DR sharing (P < 0.005), and DR sharing and suppression of ADCC (P < 0.02). Early antidonor ADCC antibody responses associated with failure to maintain graft tolerance and were most consistently directed to donor class I. The required one DR antigen sharing in DBMC-induced suppression of antidonor class I antibody suggests a restriction for recipient DR, implying critical regulation of a response to donor antigen presented on recipient cells. We hypothesize a DBMC tolerogenic mechanism in which presentation of donor class I peptide by a shared DR allele configuration allows a veto effect by DBMC. Thus DR sharing would allow DBMC veto cells to reduce clonal expansion elicited by both the direct and indirect antigen presentation pathways.

Veto cells in transplantation tolerance.

Advances in immunosuppressive management for transplantation have improved graft survival. However, lasting success will probably depend on the induction of donor-specific unresponsiveness, avoiding chronic immunosuppressive drug therapy and its debilitating side effects. Tolerance strategies have been developed in rodents, but applicability to human organ transplantation has not been achieved. We have established a preclinical allogeneic kidney transplant model in unrelated outbred rhesus monkeys and have investigated a tolerance-inducing strategy in which posttransplant administration of rabbit antithymocyte globulin and infusion of a subpopulation of donor bone marrow cells yields long-term graft acceptance in the absence of chronic immunosuppressive drugs. Recent studies of the immunological mechanisms by which induction and maintenance of transplant tolerance is achieved in this model are presented within the framework of a veto hypothesis.

A role for transforming growth factor-beta in the veto mechanism in transplant tolerance.

We have studied the veto cell-mediated induction of transplant tolerance by allogeneic donor bone marrow cells and have achieved kidney allograft tolerance in a preclinical rhesus monkey model. Here we extend these studies to investigate the veto mechanism of CTLp suppression and the role of CD8 and TGF-beta in these events. Infusion of DR-/dim donor BMC into RATG-treated rhesus monkeys induced functional deletion of donor-specific CTLp and prolongation of kidney allograft survival, whereas depletion of the CD8+ subset from BMC ablated these effects. A role of CD8 in the veto effect was further implicated by rhesus MLR-induced CML experiments in which pretreatment of normal responder cells with MAb to MHC class I, the natural ligand of CD8, blocked the suppressive activity of allogeneic BMC. In addition, pretreatment of the BMC with anti-CD8 MAbs blocked strong veto activity significantly, suggesting that CD8 functions as an accessory or adhesion ligand. In contrast, anti-CD8 treatment significantly enhanced weak BMC-mediated veto activity, suggesting that CD8 might additionally serve as a signal transducer to increase veto activity, perhaps by the induction of cytokine release. The cytokine TGF-beta was studied because it has immunosuppressive properties that are shared by veto cells. Human TGF-beta, like BMC veto cells, inhibited MLR-induced CML in a dose-dependent manner, and anti-TFB-beta Ig relieved the BMC-mediated veto suppressive effect. Active TGF-beta was detected only in the supernatants of CML cultures containing BMC. Pretreatment of BMC with L-leucyl-leucine methyl ester (Leu-leu-OMe), which eliminates cytotoxic precursor and effector lymphocytes and monocytes, did not affect levels of active TGF-beta. In previous studies, the veto effect of BMC was also shown to be Leu-leu-OMe-resistant. Finally, treatment of isolated DR-/dim BMC cultures with anti-CD8 elicited TGF-beta secretion, whereas anti-CD2 or anti-CD3 had no effect. When isolated after stimulation with anti-CD8, only the CD8+ subset of DR-/dim BMC produced detectable levels of active TGF-beta. In summary, these studies demonstrate that CD8 functions as an immunoregulatory molecule in veto effects by freshly isolated rhesus BMC and suggest that CD8-ligand interactions may induce low-level secretion of TGF-beta to mediate or facilitate the veto mechanism of CTLp inactivation in a paracrine manner.

Further studies of veto activity in rhesus monkey bone marrow in relation to allograft tolerance and chimerism.

Infusing the DR-/dim fraction of bone marrow cells (BMC) from an allogeneic kidney donor into rabbit antithymocyte globulin-treated transplant recipients delivers a tolerogenic signal, leading to functional allograft tolerance in rhesus monkeys without additional drug therapy. Our updated results in an expanded series show a median 131-day graft survival of recipients given DR-/dim donor BMC with a 23% 1-year survival (P < 0.00001 vs. rabbit antithymocyte globulin controls). Removing DRbright cells from donor BMC appeared to have a significant effect (P < 0.05). We have further investigated the tolerogenic mechanism within the experimental framework of the veto hypothesis in this preclinical model. In limiting dilution assays, we demonstrated the donor specificity of clonal inactivation of CTL precursors (CTLp) after in vitro or in vivo exposure to DR-/dim donor BMC, confirming specific tolerance. Additionally, in vitro studies confirmed the allogeneic specificity of CTLp inactivation in 3-cell MLR assays; minimal bystander effects were seen on normal CTLp responses to third party stimulator cells, while CTLp responses to the BMC donor's cells were abrogated in the same cultures. BMC mediating the veto effect were found to be resistant to L-leucyl-L-leucine methyl ester (Leu-leu-OMe), which excluded BMC-mediated cytotoxicity by NK or lymphokine-activated killer cells, CTL, or activated macrophages. In contrast, veto activity was abolished if the BMC were pretreated with either high dose UV-B light irradiation, mitomycin, or gamma-irradiation, indicating that BMC contained a UV-B-sensitive precursor of the veto effector, and that a proliferative step separated the two. Irradiation of DR-/dim donor BMC or administration of cyclophosphamide after infusion of nonirradiated BMC prevented the tolerogenic effect. Only recipients given nonirradiated DR-/dim donor BMC demonstrated PBL chimerism, which associated with functional deletion of antidonor CTLp and duration of graft survival. The Leu-leu-OMe resistance and the other properties of the allogeneic monkey CD3- CD2+ CD8+ BMC subpopulation that exhibits tolerance-promoting activity in vitro and in vivo lead us to postulate that a donor BMC-derived precursor population, possibly a dendritic cell population, may induce allogeneic unresponsiveness in this model.

Kidney allograft tolerance in primates without chronic immunosuppression--the role of veto cells.

Posttransplant infusion of specific donor bone marrow cells into ATG-treated recipients promotes long survival of allografts in the absence of immunosuppressive drug therapy. DBMC infusion also inhibits antidonor CTL activation in allograft recipients, a trend analogous to the veto phenomenon. The present studies investigated a hypothesis that veto activity in DBMC is involved in the induction of donor-specific unresponsiveness in rhesus monkey kidney transplant recipients given ATG and DBM. Normal monkey BMC were fractionated into subpopulations by depletion with mAbs and immunomagnetic beads. The unfractionated BMC and BMC subsets were tested for veto activity in in vitro MLR-induced CTL and for in vivo tolerance-promoting activity in ATG-treated monkey kidney transplant recipients. In MLR-induced CTL assays, BMC specifically suppressed CTL activity to PBL from the BMC donor. The suppression was mediated by a small population of BMC that expressed a CD2+, CD8+, CD16+, DR-, CD3-, CD38- phenotype. Results of in vivo studies showed a striking correlation with the in vitro results. ATG-treated recipients given either DR- DBMC or DR- CD3- DBMC infusions had significantly prolonged graft survival and a 40-50% incidence of long survivors over 150 days (P less than 0.001 vs. ATG controls). In contrast, in recipients given CD2- DBMC or DR- CD16- DBMC infusions, the tolerance-promoting effect of DBMC was absent, and there were no long-term survivors. The results also showed an association between long survival and suppressed in vivo antidonor CTL activity. Thus acute rejection and in vivo and in vitro antidonor CTL responses were suppressed by a minor subpopulation of DBMC with similar phenotypic markers. We suggest that a veto mechanism may control the induction phase of allograft tolerance in this model, providing a critical period of CTL silence to allow development of host immunoregulatory mechanisms necessary for maintaining graft tolerance.