beta-Arrestin 1 and 2 stabilize the angiotensin II type I receptor in distinct high-affinity conformations.

BACKGROUND AND PURPOSE: The angiotensin II type 1 (AT(1)) receptor belongs to family A of 7 transmembrane (7TM) receptors. The receptor has important roles in the cardiovascular system and is commonly used as a drug target in cardiovascular diseases. Interaction of 7TM receptors with G proteins or beta-arrestins often induces higher binding affinity for agonists. Here, we examined interactions between AT(1A) receptors and beta-arrestins to look for differences between the AT(1A) receptor interaction with beta-arrestin1 and beta-arrestin2. EXPERIMENTAL APPROACH: Ligand-induced interaction between AT(1A) receptors and beta-arrestins was measured by Bioluminescence Resonance Energy Transfer 2. AT(1A)-beta-arrestin1 and AT(1A)-beta-arrestin2 fusion proteins were cloned and tested for differences using immunocytochemistry, inositol phosphate hydrolysis and competition radioligand binding. KEY RESULTS: Bioluminescence Resonance Energy Transfer 2 analysis showed that beta-arrestin1 and 2 were recruited to AT(1A) receptors with similar ligand potencies and efficacies. The AT(1A)-beta-arrestin fusion proteins showed attenuated G protein signalling and increased agonist binding affinity, while antagonist affinity was unchanged. Importantly, larger agonist affinity shifts were observed for AT(1A)-beta-arrestin2 than for AT(1A)-beta-arrestin1. CONCLUSION AND IMPLICATIONS: beta-Arrestin1 and 2 are recruited to AT(1A) receptors with similar ligand pharmacology and stabilize AT(1A) receptors in distinct high-affinity conformations. However, beta-arrestin2 induces a receptor conformation with a higher agonist-binding affinity than beta-arrestin1. Thus, this study demonstrates that beta-arrestins interact with AT(1A) receptors in different ways and suggest that AT(1) receptor biased agonists with the ability to recruit either of the beta-arrestins selectively, would be possible to design.

Human responses in assessing object relations subscales of the Psychoanalytic Rorschach Profile.

The Rorschach test is often used to assess object relations. This research examined whether human responses are necessary for a valid assessment.

TNF alpha and IL-1beta mediate intercellular adhesion molecule-1 induction via microglia-astrocyte interaction in CNS radiation injury.

Radiation injury to the central nervous system (CNS) results in glial activation accompanied by expression of pro-inflammatory cytokines and adhesion molecules. In this study we demonstrate intercellular adhesion molecule-1 (ICAM-1) induction in the irradiated mouse brain at the mRNA and protein levels. Immunocytochemical analysis revealed that ICAM-1 protein was primarily expressed in endothelial cells and microglia. In vitro, ionizing radiation significantly induces TNF alpha, IL-1beta and ICAM-1 mRNA in primary microglia cultures. Interestingly, although ionizing radiation activated primary astrocyte cultures, it did not induce ICAM-1 expression. However, exposure of astrocytes to conditioned medium collected from irradiated microglia resulted in ICAM-1 induction, which was abrogated when the conditioned medium was pre-incubated with neutralizing antibodies raised against murine TNF alpha and IL-1beta. These results indicate that pro-inflammatory cytokines may be necessary for ICAM-1 expression in astrocytes in CNS radiation injury.

Clinical anatomy in the oncology patient: a preclinical elective that reinforces cross-sectional anatomy using examples of cancer spread patterns.

A preclinical elective entitled Oncologic Anatomy is described which emphasizes the study of anatomy from a three-dimensional approach using examples of cancer spread patterns. Features of this elective course include patient presentations, a review of the anatomy, a diagnostic presentation and overview of radiologic imaging techniques, and "hands on" experience where students are introduced to aspects of the physical examination. This course format encourages active learning, facilitates basic and clinical science integration, offers early exposure to patients, and validates the importance of the psychosocial aspects of the medical profession.

ICAM-1 induction in the mouse CNS following irradiation.

Injury to the central nervous system (CNS) results in inflammation, increased trafficking of leukocytes into the CNS, induction of cytokines, and exacerbation of the primary injury. The increased trafficking of neutrophils into the CNS has been described following a number of injury models including stab, stroke, and excitotoxin-induced injury. This enhanced trafficking has largely been ascribed to the adhesion molecule intercellular adhesion molecule-1 (ICAM-1, CD54). In the current study, we wished to determine if the inflammation caused by irradiation of the CNS resulted in a similar induction of ICAM-1. C3H/HeJ mice were irradiated using gamma irradiation aimed over the right cerebral hemisphere. The relative induction of ICAM-1 mRNA levels was determined using quantitative RT-PCR 6 hours following irradiation with either 0, 5, 15, 25 or 35 Gy. ICAM-1 message was seen to exhibit a normal dose response curve with increasing mRNA levels seen at 15 Gy and higher. To determine the cellular distribution of the ICAM-1 protein following irradiation, mice were sacrificed at 4 hrs, 24 hrs, 48 hrs and 7 days following 25 Gy irradiation and the tissue was processed for ICAM-1 immunocytochemistry. ICAM-1 staining was seen to increase in both endothelial cells and astrocytes beginning as early as 4 hrs. The staining intensity continued to increase throughout the 7 day period observed. Together, these results suggest that irradiation of the CNS causes a rapid induction of both ICAM-1 mRNA and protein. This suggests that increased leukocyte trafficking into the CNS may exacerbate the inflammation induced by radiation injury.

The implementation of therapeutic community principles in acute care psychiatric hospital settings: an empirical analysis and recommendations to clinicians.

We applied therapeutic community principles to a contemporary acute care psychiatric treatment program and investigated the effects upon milieu treatment variables using the Ward Atmosphere Scale (WAS) as a measure. Significant differences were found between experimental and control units on several scales of the WAS following the introduction of program changes. These differences indicated that the introduction of therapeutic community principles in treatment programming enhanced milieu factors that are conducive to patient recovery.

Sprouting of dopaminergic fibers from spared mesencephalic dopamine neurons in the unilateral partial lesioned rat.

A unilateral partially lesioned rat model of Parkinson's disease was developed following selective lesioning of the dopamine neurons of the substantia nigra pars compacta by stereotactic injection of the neurotoxin 6-hydroxydopamine. In this animal model the dopamine neurons of the ventral tegmental area and medial substantia nigra are spared. The neuronal loss in such partial lesioned models mimics more closely that seen in human mid-stage parkinsonism. Cografts of adrenal medullary cells and sciatic nerve to the partially lesioned striatum induced a sprouting response in grafted animals that was confirmed by immunocytochemical staining with antibodies to tyrosine hydroxylase (TH) and by quantification of the high affinity dopamine uptake complex using [3H]GBR 12935 binding. Enhanced TH fiber immunostaining was evident even in the presence of poor cograft survival. The origin of the TH-like immunostained fibers in the striatum was determined using Lucifer yellow retrograde axonal transport. Following discrete tracer injections into the striatum adjacent to a cograft, neurons in the medial substantia nigra and ventral tegmental area (areas A9 and A10, respectively) were labeled with Lucifer yellow. These labelled neurons displayed a morphology characteristic of dopamine neurons and, in double-labelling experiments, also immunostained for TH. These results support the utility of unilateral partially lesioned rat models of Parkinson's disease for studies investigating a host sprouting or upregulation response and confirm that the immunostained striatal fibers originate from spared dopamine neurons in the ventromedial midbrain.

Adrenal medulla in neural grafting and neural plasticity.

The recent history of neural transplantation using the adrenal medulla parallels an evolution in our thinking about neural grafting as a therapeutic approach to treat neurodegenerative diseases such as Parkinson's disease. Initially, neural grafting was an approach to study development and regeneration. With the discovery that adrenal chromaffin cell grafts would ameliorate some of the motor deficits associated with the loss of striatal dopamine, adrenal grafts were used to provide dopamine to the dopamine-depleted striatum. However, subsequent studies showed poor chromaffin cell survival unless trophic factors were present at the site of transplantation. These experiments lead to the appreciation of the complex interactions between neurotrophic factors, inflammatory cytokines, the grafted tissue, and the host brain's response. Thus, we find ourselves again using neural transplantation as an approach to help us better understand central nervous system plasticity and the features this plasticity shares in common with development and regeneration.

Disruption of the blood-brain barrier as the primary effect of CNS irradiation.

The blood-brain barrier (BBB) is believed to be unique in organ microcirculation due to the 'tight junctions' which exist between endothelial cells and, some argue, the additional functional components represented by the perivascular boundary of neuroglial cells; these selectively exclude proteins and drugs from the brain parenchyma. This study was designed to examine the effects of irradiation on the BBB and determine the impact of the altered pathophysiology on the production of central nervous system (CNS) late effects such as demyelination, gliosis and necrosis. Rats, irradiated at 60 Gy, were serially sacrificed at 2, 6, 12 and 24 weeks. Magnetic resonance image analysis (MRI) was obtained prior to sacrifice with selected animals from each group. The remaining animals underwent horse-radish peroxidase (HRP) perfusion at the time of sacrifice. The serial studies showed a detectable disruption of the BBB at 2 weeks post-irradiation and this was manifested as discrete leakage; late injury seen at 24 weeks indicated diffuse vasculature leakage, severe loss of the capillary network, cortical atrophy and white matter necrosis. Reversal or repair of radiation injury was seen between 6 and 12 weeks, indicating a bimodal peak in events. Blood-brain barrier disruption is an early, readily recognizable pathophysiological event occurring after radiation injury, is detectable in vivo/in vitro by MRI and HRP studies, and appears to precede white matter necrosis. Dose response studies over a wide range of doses, utilizing both external and interstitial irradiation, are in progress along with correlative histopathologic and ultrastructural studies.

The use of genetically altered astrocytes to provide nerve growth factor to adrenal chromaffin cells grafted into the striatum.

Transplantation of adrenal chromaffin cells into the striatum of Parkinson's disease patients is unlikely to become a reliable therapy unless techniques are devised to improve cell survival. To address this issue, we investigated the use of genetically altered astrocytes that constitutively secrete beta-nerve growth factor (NGF) to provide trophic support for adrenal chromaffin cells grafted into the dopamine-denervated striatum of the rat. Primary rat astrocytes were altered genetically in vitro by infection with a retroviral vector harboring a mouse beta-NGF transgene under constitutive long terminal repeat transcriptional control. Confluent cultures of these genetically altered astrocytes secrete NGF into their culture medium at a rate of approximately 9 pg/10(5) cells/h. This rate of NGF secretion is at least 10-fold higher than that of confluent sister cultures of uninfected astrocytes. The effects of the NGF-secreting astrocytes on the survival and neuronal transformation of dissociated adrenal chromaffin cells were assessed in vitro and following transplantation into the dopamine-denervated striatum of the adult rat. In vitro experiments demonstrated that neuritic outgrowth is stimulated when postnatal day 12 chromaffin cells are grown on a monolayer of the genetically altered astrocytes. When co-grafted with genetically altered astrocytes, young postnatal chromaffin cells displayed extensive neuritic outgrowth within the host brain 2 weeks postimplantation, whereas chromaffin cells grafted alone or with normal astrocytes retain an endocrine-like morphology. Survival of the chromaffin cells is also enhanced 3-6-fold when co-grafted with the genetically altered astrocytes. In addition, the neuronally transformed chromaffin cells appear to lose adrenergic properties as assessed by diminished immunoreactivity to the adrenergic marker, phenylethanolamine-N-methyltransferase. Although their survival is also enhanced approximately 4-fold relative to controls, adult chromaffin cells do not convert to a neuronal morphology when co-grafted with the genetically altered astrocytes. These studies demonstrate that rat astrocytes carrying a mouse NGF transgene provide trophic support for intrastriatal chromaffin cell grafts.

Functional aspects of mammalian neural transplantation.

Although initially perceived as a method to study neural development and regeneration, neural transplantation has recently become a very promising approach in its own right as a therapeutic tool to treat neurodegenerative disorders. The development of several animal models which mimic aspects of clinical disorders such as Parkinson's disease, Alzheimer's disease, and Huntington's chorea, provides systems in which to study the potential benefits of grafts derived from different sources. Both fetal and adult donor tissues presently are under investigation. Additionally, cell lines and genetically engineered cells also are being developed as suitable graft material. Important aspects of graft-host interaction, including cell survival, host regeneration, immune interactions, improved behavior, and blood-brain barrier phenomena, may be studied in these transplant models. Advances in this field of biomedical research have led to clinical trials in patients afflicted with Parkinson's disease who now are undergoing transplantation therapy. Thus far, the results have been equivocal, raising important ethical questions about continued clinical studies until more is understood about how neural transplants function and interact with the host. Nevertheless, neural transplantation holds tremendous promise as a future therapeutic tool to treat progressive and irreversible neural disorders.

Effect of MPTP on primate chromaffin cells in vitro: relevance for adrenal medullary cell transplantation.

Primate adrenal medullary cells were exposed to l-methyl-4-phenyl-l,2,3,6-tetrahydropyridine (MPTP) in vitro to examine the effect of this neurotoxic agent on chromaffin cells. Chromaffin cells from monkey and humans were cultured in the presence of 100 ng/ml nerve growth factor for 1 week and then exposed to 150 µM MPTP or its active metabolite methylpyridinium ion (MPP+) for an additional week. Cells which had extended neurites in the presence of NGF showed no morphological effect in response to MPTP or MPP+ at the light microscopic level. However, there was a significant loss in catecholamines as seen by histofluorescence and high performance liquid chromotography (HPLC). Electron microscopy revealed a depletion in dense-core vesicles in chromaffin cells after chronic exposure to MPTP while the mitochondria appeared similar to those observed in control cells. Replacement of MPTP medium with standard medium stimulated restoration of catecholamine histofluorescence after 7 days. An acute 15 min pretreatment of chromaffin cells with MPTP or MPP+ induced secretion of catecholamines over a 1 h pulse, with MPP+ producing the maximum and more rapid secretion as determined by HPLC. These data indicate that MPTP induces a dramatic loss in catecholamines in primate chromaffin cells in vitro after both acute and chronic exposures; however, removal of the toxic agent permits restoration of catecholamines without permanent effect on the integrity of these cells.

NGF-like trophic support from peripheral nerve for grafted rhesus adrenal chromaffin cells.

Autopsy results on patients and corresponding studies in nonhuman primates have revealed that autografts of adrenal medulla into the striatum, used as a treatment for Parkinson's disease, do not survive well. Because adrenal chromaffin cell viability may be limited by the low levels of available nerve growth factor (NGF) in the striatum, the present study was conducted to determine if transected peripheral nerve segments could provide sufficient levels of NGF to enhance chromaffin cell survival in vitro and in vivo. Aged female rhesus monkeys, rendered hemiparkinsonian by the drug MPTP (n-methyl-4-phenyl-1,2,3,6 tetrahydropyridine), received autografts into the striatum using a stereotactic approach, of either sural nerve or adrenal medulla, or cografts of adrenal medulla and sural nerve (three animals in each group). Cell cultures were established from tissue not used in the grafts. Adrenal chromaffin cells either cocultured with sural nerve segments or exposed to exogenous NGF differentiated into a neuronal phenotype. Chromaffin cell survival, when cografted with sural nerve into the striatum, was enhanced four- to eightfold from between 8000 and 18,000 surviving cells in grafts of adrenal tissue only up to 67,000 surviving chromaffin cells in cografts. In grafts of adrenal tissue only, the implant site consisted of an inflammatory focus. Surviving chromaffin cells, which could be identified by both chromogranin A and tyrosine hydroxylase staining, retained their endocrine phenotype. Cografted chromaffin cells exhibited multipolar neuritic processes and numerous chromaffin granules, and were also immunoreactive for tyrosine hydroxylase and chromogranin A. Blood vessels within the graft were fenestrated, indicating that the blood-brain barrier was not intact. Additionally, cografted chromaffin cells were observed in a postsynaptic relationship with axon terminals from an undetermined but presumably a host origin.

Effects of NGF and dibutyryl cAMP on neuronal differentiation of embryonal carcinoma cells.

The P19S18O1A1 embryonal carcinoma cell line is capable of neuronal differentiation and is therefore useful in studying neuronal development and the influence of growth modulators on neuronal differentiation. We report here on the effects of nerve growth factor (NGF) and dibutyryl cyclic adenosine monophosphate (db cAMP), individually and combined, on differentiation of P19S18O1A1 cells. NGF alone did not induce any significant neuron-like changes in cultures exposed to NGF for as long as 12 days. Treatment with db cAMP resulted in changes in a significant population of the cells, including development of a neuron-like morphology, seen at both the light and electron microscopic level, loss of stage-specific embryonic antigen expression and the appearance of two neuronal markers, neurofilament protein and neuron-specific enolase. These changes were similar to changes seen when embryonal carcinoma (EC) cells are treated with retinoic acid. NGF in combination with dibutyryl cyclic adenosine monophosphate brought about similar changes as dibutyryl cyclic adenosine monophosphate alone, and was therefore not synergistic for induction of neuronal properties. In retinoic acid-treated cultures, the neuron-like cells had ultrastructural features very similar to neurons in non-tumorous, normal tissue, with typical organelles, such as one nucleolus, neurotubules and neurofilaments, while db cAMP-treated EC cells showed similar findings at the electronmicroscopic level. The results suggest that db cAMP can induce the neuronal phenotype in EC cells alone without pre-treatment with retinoic acid.

Cografts of adrenal medulla with C6 glioma cells in rats with 6-hydroxydopamine-induced lesions.

Amitotic [3H]thymidine-labeled C6 glioma cells, which are known to produce neurotrophic factor(s), were grafted alone and with adrenal chromaffin cells in an attempt to improve chromaffin cell survival and phenotypic differentiation. Long-Evans rats with unilateral 6-hydroxydopamine-induced lesions of the nigrostriatal pathway were divided into four groups: (1) those receiving adrenal medullary cells co-transplanted with C6 glioma cells; (2) those receiving adrenal medullary graft alone; (3) those receiving C6 glioma grafts alone; and (4) those serving as a vehicle control group. All rats were killed one month after transplantation. Immunohistochemical, neurochemical, and autoradiographic methods were used to identify and characterize the grafted cells. Tyrosine hydroxylase-immunoreactive cells were found in all animals that received grafts of the adrenal medulla alone or of adrenal medulla co-transplanted with C6 glioma cells. The cograft recipients had more tyrosine hydroxylase-immunoreactive cells than the hosts receiving just adrenal chromaffin cells (P less than 0.05). Additionally, more grafted chromaffin cells formed processes in the former group. All three tissue recipient groups (adrenal medullary, C6 glioma cell, and cografted animals) had a significant reduction (P less than 0.05) in ipsilateral rotations after amphetamine (0.5 mg/kg i.p.) injections as compared to the control vehicle recipient group. Moreover, the reduction in rotation was more marked in the cografted hosts than in the other two implanted groups (P less than 0.05). Significantly higher dopamine levels were found in the transplant sites of both cograft and adrenal medullary graft recipients than in sham grafted control animals.

Fetal research.

This article reviews some of the significant contributions of fetal research and fetal tissue research over the past 20 years. The benefits of fetal research include the development of vaccines, advances in prenatal diagnosis, detection of malformations, assessment of safe and effective medications, and the development of in utero surgical therapies. Fetal tissue research benefits vaccine development, assessment of risk factors and toxicity levels in drug production, development of cell lines, and provides a source of fetal cells for ongoing transplantation trials. Together, fetal research and fetal tissue research offer tremendous potential for the treatment of the fetus, neonate, and adult.