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Glioblastoma multiforme (GBM) is the most common highly malignant brain tumor and is also one among the most therapy-resistant human neoplasias. At the University Hospital in Lund, a group of patients with GBM were treated with a new therapy form attempting immunization by glioma cells transfected to produce interferon-γ. The purpose of this report was to evaluate tumor material from the first nine patients treated with this therapy, assessing the levels of inflammatory/reactive cells (lymphocytes and macrophages). Tumor biopsies from surgery performed at different time points during treatment were analyzed with conventional histotechnical methods and immunohistochemistry. A post-mortem neuropathological investigation with a whole brain assessment was achieved in 5 immunized patients. The results show that cytotoxic T lymphocytes exhibited a mild increase during immunotreatment. This increase indicates an invoked stimulation of a cytotoxic T cell reaction, which may prove beneficial when immunization is adequately manipulated in dosage and timing. [ABSTRACT FROM AUTHOR]

Glioblastoma multiforme (GBM) is an aggressive primary brain malignancy with a very poor prognosis. Researchers employ animal models to develop potential therapies. It is important that these models have clinical relevance. This means that old models, propagated for decades in cultures, should be questioned. Parameters to be evaluated include whether animals are immune competent or not, the infiltrative growth pattern of the tumor, tumor volume resulting in symptoms and growth rate. We here describe the growth pattern of an experimental glioblastoma model in detail with GFP positive glioblastoma cells in fully immune competent animals and study tumor growth rate and tumor mass as a function of time from inoculation. We were able to correlate findings made with classical immunohistochemistry and MR findings. The tumor growth rate was fitted by a Gompertz function. The model predicted the time until onset of symptoms for 5000 inoculated cells to 18.7±0.4 days, and the tumor mass at days 10 and 14, which are commonly used as the start of treatment in therapeutic studies, were 5.97±0.62 mg and 29.1±3.0 mg, respectively. We want to raise the question regarding the clinical relevance of the outline of glioblastoma experiments, where treatment is often initiated at a very early stage. The approach presented here could potentially be modified to gain information also from other tumor models.

Background: Coping with the immune rejection of allotransplants or autologous cells in patients with an active sensitization towards their autoantigens and autoimmunity presently necessitates life-long immune suppressive therapy acting on the immune system as a whole, which makes the patients vulnerable to infections and increases their risk of developing cancer. New technologies to induce antigen selective long-lasting immunosuppression or immune tolerance are therefore much needed., Methodology/principal Findings: The DNA demethylating agent Zebularine, previously demonstrated to induce expression of the genes for the immunosuppressive enzymes indolamine-2,3-deoxygenase-1 (IDO1) and kynureninase of the kynurenine pathway, is tested for capacity to suppress rejection of allotransplants. Allogeneic pancreatic islets from Lewis rats were transplanted under the kidney capsule of Fischer rats previously made diabetic by a streptozotocin injection (40 mg/kg). One group was treated with Zebularine (225 mg/kg) daily for 14 days from day 6 or 8 after transplantation, and a control group received no further treatment. Survival of the transplants was monitored by blood sugar measurements. Rats, normoglycemic for 90 days after allografting, were subjected to transplant removal by nephrectomy to confirm whether normoglycemia was indeed due to a surviving insulin producing transplant, or alternatively was a result of recovery of pancreatic insulin production in some toxin-treated rats. Of 9 Zebularine treated rats, 4 were still normoglycemic after 90 days and became hyperglycemic after nephrectomy. The mean length of normoglycemia in the Zebularine group was 67±8 days as compared to 14±3 days in 9 controls. Seven rats (2 controls and 5 Zebularine treated) were normoglycemic at 90 days due to pancreatic recovery as demonstrated by failure of nephrectomy to induce hyperglycemia., Conclusions/significance: Zebularine treatment in vivo induces a long-lasting suppression of the immune destruction of allogeneic pancreatic islets resulting in protection of allograft function for more than 10 weeks after end of treatment.

Elevated telomerase activity is an important molecular signature of cancer cells and primitive cells in regenerative tissues. However, isolation of single living cells with endogenous telomerase activity has not yet been possible. Here, we developed adenovirus serotype 35 tropism-based vectors encoding destabilized enhanced green fluorescence protein with a half-life of 2 h (d2EGFP) driven by the human telomerase reverse transcriptase (hTERT) promoter. As assessed in telomerase-positive or -negative cell lines, the d2EGFP expression positively correlated with hTERT transcript content and telomerase activity. In retinoic acid-induced differentiating HL-60 cells, the d2EGFP expression is diminished in the same manner as the hTERT expression. Individual cells from HeLa and HL-60 cell lines exhibited heterogeneous d2EGFP expression, which was cell cycle dependent, as the sorted d2EGFP+ HL-60 cells contained twice as many cells in S/G2/M phase of the cell cycle compared with the d2EGFP- HL-60 cells. However, both cell populations exhibited the same proliferation and regeneration capacities. Heterogeneous d2EGFP expression was also detected in xenograft glioblastoma multiforme cells with tumor formation capacity. Thus, d2EGFP expression reported cell cycle- and differentiation stage-dependent hTERT expression. Our study facilitates isolation and characterization of single living cells with telomerase activity.

Aiming to map the distribution of spinally projecting, hypothalamic neurons containing neuronal nitric oxide synthase (nNOS), True Blue (TB) is injected into the rat spinal cord. After survival times of 7-14 days the animals are anaesthetized and perfused transcardially with a solution containing paraformaldehyde and sucrose. After dissection, the injection site is further fixed for 4-8 h, cut in a cryostat, and documented by computer-assisted digital photography. The brain region of interest is fixed for 4 h, rinsed in phosphate buffer for 48 h, sectioned, and photographically documented utilizing filter settings for visualization of TB. The brain sections are then immunohistochemically processed using a primary antibody against nNOS and a Texas Red (TR)-labelled secondary antibody and once again photographically documented, now using filter settings for visualization of TB and TR, respectively. Utilizing the Photoshop program, the TB containing cells can then be exactly aligned and the presence of TB and/or TR fluorescence in the same cell bodies are evaluated. This method for neurotransmitter-specific retrograde tracing derives its high sensitivity from the optimization of fixation/rinsing parameters, the use of appropriate fluorophores, and sequential digital microphotography.

The anatomical distribution and quantitative relations of cell bodies containing neuronal nitric oxide synthase (nNOS), 8-arginine vasopressin (AVP) and oxytocin (OT) were examined throughout the supraoptic nucleus (SON) of the female rat by means of immunocytochemical and NADPH-diaphorase (NADPH-d) histochemical techniques using a triple labelling methodology. Seven chemically defined populations of neurons containing all combinations of either nNOS, AVP or OT were identified. nNOS-containing (NADPH-d positive) neurons, amounting to about 40% of all neurons counted, were most frequent in central and dorsal regions, and were evenly distributed along the rostro-caudal axis. Two small nNOS-positive neuronal populations were preferentially located dorso-centrally in the nucleus: nNOS-positive neurons containing both AVP- and OT-immunoreactivity, and neurons only containing nNOS. Slightly less than half of all nNOS-positive neurons contained AVP, and a similar share of nNOS-positive neurons contained OT. The occurrence of nNOS-positive/AVP-containing neurons was highest in the caudal half, whereas that of nNOS-positive/OT-neurons was highest in the rostral half of SON. The data demonstrate new findings concerning the anatomical organization and co-localization patterns of nNOS-, AVP- and OT-containing neuronal populations in SON. We conclude that the absolute and relative occurrence of the identified neuronal populations vary markedly in different parts of SON. This is important to take into consideration when performing, and evaluating experimental investigations concerned with neurochemical changes in SON.

Background and Purpose: MR imaging has made it easier to distinguish among the different types of intracranial mass lesions. Nevertheless, it is sometimes impossible to base a diagnosis solely on clinical and neuroradiologic findings, and, in these cases, biopsy must be performed. The purpose of this study was to evaluate the hypothesis that proton MR spectroscopy is able to improve preoperative diagnostic accuracy in cases of intracranial tumors and may therefore obviate stereotactic biopsy., Methods: Twenty-six patients with intracranial tumors underwent MR imaging, proton MR spectroscopy, and stereotactic biopsy. MR spectroscopic findings were evaluated for the distribution pattern of pathologic spectra (NAA/Cho ratio < 1) across the lesion and neighboring tissue, for signal ratios in different tumor types, and for their potential to improve preoperative diagnostic accuracy., Results: Gliomas and lymphomas showed pathologic spectra outside the area of contrast enhancement while four nonastrocytic circumscribed tumors (meningioma, pineocytoma, metastasis, and germinoma) showed no pathologic spectra outside the region of enhancement. No significant correlation was found between different tumor types and signal ratios. MR spectroscopy improved diagnostic accuracy by differentiating infiltrative from circumscribed tumors; however, diagnostic accuracy was not improved in terms of differentiating the types of infiltrative or circumscribed lesions., Conclusion: MR spectroscopy can improve diagnostic accuracy by differentiating circumscribed brain lesions from histologically infiltrating processes, which may be difficult or impossible solely on the basis of clinical or neuroradiologic findings.

The distribution of immunoreactivity to neuronal nitric oxide synthase (nNOS) and vasopressin (AVP) was studied in the circumventricular organs of the female rat. The occurrence of NOS immunoreactivity showed correspondence to nicotinamide dinucleotide phosphate diaphorase reactivity, a previously used but less specific marker for neuronal NOS. nNOS immunolabeling was detected in the two most rostrally located circumventricular organs - the organum vasculosum of the lamina terminalis and the subfornical organ. In the latter, AVP immunoreactivity was observed in some cell bodies, which also were nNOS-immunoreactive. In the median eminence and the neurohypophysis there were large amounts of nNOS- and AVP-immunoreactive nerve fibers, which often displayed similarities in distribution and morphology. Within the pineal gland, only very few nNOS-immunoreactive varicose terminals were observed, which ran along blood vessels. nNOS immunoreactivity was also seen in the epithelium of the choroid plexus, whereas no nNOS immunoreactivity could be found in the subcommissural organ or in the area postrema. The present demonstration of nNOS and AVP immunoreactivity in the subfornical organ, median eminence, and neurohypophysis, and the occurrence of nNOS immunoreactivity also in the choroid plexus and organum vasculosum of the lamina terminalis, provides a morphological background for a functional role for nitric oxide in water homeostatic mechanisms, both as executed through the hypothalamohypophyseal system and via the production of cerebrospinal fluid.

The distribution of delta sleep-inducing peptide immunoreactivity (DSIP-IR) was studied in the rat diencephalon. Varicose nerve fibers exhibiting DSIP-IR were found throughout the mediobasal hypothalamus, most frequently in the hypothalamic arcuate nucleus and in the adjoining median eminence and pituitary stalk. This innervation provides a basis for the involvement of DSIP in neuroendocrine regulation at the hypothalamic level. In the hypothalamus, DSIP-IR innervation was also observed close to the third ventricle and within the mamillary complex. Despite pretreatment with colchicine, no evidence of immunoreactive cell bodies containing DSIP-IR could be found.

The brainstem origin of preganglionic fibers to the sphenopalatine ganglion in rat was revealed by the aid of the retrograde axonal tracer True Blue (which does not traverse to a second order neuron) applied deep in the sphenopalatine ganglion or the Vidian nerve on one side. The majority of fibers originate in the ipsilateral lacrimo-muconasal nucleus in the ventrolateral rostral medulla oblongata and caudal pons. A smaller number of fibers originate more dorsomedially and caudally in the medullary reticular formation. After application to the ganglion a third small group of labelled neurons was found more rostrally in the brainstem, in the reticular formation ventrolateral to the caudal part of the dorsal raphe nucleus. Simultaneous visualization of catecholaminergic nerves revealed that the labelled neurons in the lacrimo-muconasal nucleus were heavily innervated by catecholaminergic fibers. It appears from previous studies that the preganglionic neurons may not be cholinergic. None of the labelled neurons in the brainstem stained positively for catecholamines. Thus, further studies are required to elucidate the transmitter(s) used in these neurons.

The presence and distribution of adrenergic nerves in the developing calvarium of the newborn rat documented by means of the formaldehyde-induced fluorescence technique in rats aged 2 or 7 days. Nerve fibres exhibiting catecholamine-specific fluorescence were seen within the developing calvarium of all animals. In coronal sections, these fibres could be seen in the developing bone, especially in the lamina interna, while in sagittal sections, they were seen to traverse the tissue to reach the central of the diploë. These fibres originate from a denser plexus within the dura mater. Especially in the younger age group, the fluorescent fibres often exhibited an immature appearance, being coarse and devoid of varicosities. In the older animals the fibres were often varicose. The sutural tissue proper was always found to be devoid of adrenergic innervation. The possible origin and functional significance of the adrenergic innervation in the developing bone in relation to skull growth and sutural closure are discussed.

Immunocytochemical studies have documented the presence of neuropeptide Y (NPY) in the hypothalamic paraventricular nucleus (PVN) which harbours a large number of neurones that contain corticotrophin-releasing factor (CRF). In this study the close morphological association between NPY fibres and CRF cell bodies in the PVN was confirmed. The localization of NPY terminals in the vicinity of CRF neurones forms a morphological basis for an action of NPY in the hypothalamic control of the pituitary-adrenocortical axis. We therefore microinjected NPY into the area of the PVN of both conscious, freely moving and anaesthetized rats and noted a powerful stimulatory effect on adrenocorticotropic hormone (ACTH) and corticosterone release as measured by radioimmunoassay. In experiments with conscious, freely moving rats, higher ACTH and corticosterone levels were detected following injection of NPY into the area of the PVN than following control injection (desamidated NPY). Intracerebroventricular injection of NPY produced a small, albeit significant, increase in circulating corticosterone levels as compared to control (saline-injected) rats. Anaesthetized rats responded to NPY (but not to saline) injected into the area of the PVN with elevated ACTH and corticosterone levels, while injection of NPY into the neocortex failed to affect the blood concentration of either ACTH or corticosterone. In conclusion, we have demonstrated an activating effect of NPY on the pituitary-adrenocortical axis both in conscious and anaesthetized rats which may reflect the anatomical relationship between NPY fibres and CRF neurones in the PVN.

By means of the aluminum-formaldehyde (ALFA) fluorescence technique for monoamine visualization the distribution of catecholamines was studied in the caudal spinal cord, particularly in relation to motoneurons innervating pelvic structures. In the lumbosacral cord all parts of the spinal gray matter were found to contain catecholamines. In the dorsal horn the most intense fluorescence was seen in the superficial layers. The motoneuron neuropil exhibited the most prominent catecholamine-fluorescence of the ventral horn layers. In the sixth lumbar segment, which contains the motor nuclei that innervate the pelvic striated muscles as well as one innervating muscles in the lower limb, a differential distribution of the density of catecholamine fluorescence was presented by the individual nuclei. The catecholamine fibers in the motoneuron neuropil were seen closely surrounding the motoneuron somata, suggesting the existence of axosomatic contacts, and by utilizing the fluorescent retrograde tracer True Blue in combination with the ALFA method tentative axosomatic noradrenergic synapses on identified neurons innervating small striated pelvic muscles could be visualized in the light microscope. In the intermediate gray the intermediolateral nucleus in thoracic and upper lumbar segments was the most heavily innervated area, followed by the medial lumbar sympathetic group, which contains the majority of the sympathetic preganglionic neurons innervating the pelvic organs. The parasympathetic intermediolateral nucleus in the upper sacral segments received a catecholamine innervation of moderate density. The catecholamine innervation pattern is discussed in relation to the patterns of other putative transmitters. The distribution of catecholamine fluorescence in relation to nuclei that control the pelvic organs differs from the arrangement of other transmitters in this region. The complexity of the innervation of the pelvic organs and their related striated muscles is thus further stressed.

The anatomy of the recently discovered diencephalospinal dopaminergic system is summarized and its possible role in physiological and pathological processes suggested. The cell bodies of origin of this system are localized periventricularly in the dorsal hypothalamus and caudal thalamus, and the terminal innervations are found in the dorsal horn at all spinal levels and around the preganglionic sympathetic neurons in the thoracolumbar spinal cord. Available data favor the participation of the spinal dopaminergic system in pain modulation and autonomic and motor responses. Dysfunction of spinal dopaminergic neurons could be involved in the pathophysiology of certain conditions, such as Parkinson's disease. It appears possible that the beneficial effects of dopamine agonists in this condition as well as some of the side effects of neuroleptics are mediated through their actions on spinal dopaminergic mechanisms.

Using a combination of neonatal 6-hydroxydopamine and adult 5,7-dihydroxytryptamine treatment we have been able to achieve a 94-99% depletion of noradrenaline in the spinal cord. In such animals the dopamine levels are only marginally affected in the dorsal horn (at all levels) and in the intermediate zone at thoraco-lumbar levels. This combined treatment thus offers new possibilities for selective studies of the spinal dopamine projection. In agreement with the biochemical data the fluorescence histochemistry shows that the spinal dopamine innervation is mainly confined to the dorsal horn, the intermediolateral cell column and associated parts of the intermediate and central gray. Injections of fluorescent retrograde tracer combined with monoamine fluorescence histochemistry reveal that the diencephalic A11 cell group is the principal, and perhaps exclusive, source of this innervation. The area of termination, as well as the organizational similarities with certain diencephalic peptide-containing projections to the spinal cord, suggest that the diencephalo-spinal dopamine system may be importantly involved in autonomic regulatory processes.

This study describes the organization of the mesohabenular dopamine (DA) system in the rat as revealed by fluorescence histochemistry in combination with lesions, DA uptake experiments and injections of a retrograde tracer. The DA axons were found to be aggregated in a dense terminal field located in the caudal two thirds of the medial part of the lateral habenular nucleus. Microknife lesions of the stria medullaris left this DA innervation unaffected while cuts through the fasciculus retroflexus resulted in the virtual disappearance of the DA innervation. Injections of the fluorescent retrograde tracer True Blue (TB) into the lateral habenula produced labeling of both DA and non-DA-containing cells in the ventral mesencephalon, mainly in the interfascicular nucleus ipsilateral to the injection. This study thus documents the existence of a mesohabenular DA pathway whose cell bodies are located in the ventral mesencephalon and whose axons ascend with the fasciculus retroflexus to terminate in the caudomedial part of the lateral habenular nucleus. This information, taken together with insights gained from other studies, suggests a role for the mesohabenular DA system in modulating telencephalic feedback onto the mesencephalic DA-neurons and also in regulating the output from the dorsal raphe nucleus.

Some basic methodological aspects on the use of the retrograde fluorescent tracer True Blue (TB) was studied in freeze-dried material in the rat CNS. On the basis of fluorescence morphology and intensity at different filter settings it was possible to objectively delineate 3 distinct zones of the TB injection site, and define degrees of retrograde labelling from the different zones. Correlative studies of injection sites in subportions of the spinal cord and retrograde labelling in cell bodies of defined nuclei in the brainstem and cortex showed that effective uptake occurred only from the central area of the injection (zone I), while the uptake from the less intensely fluorescent zone II was variable and could only be documented for systems terminating within this zone. Time-course studies revealed that the size of the injection site and the resulting retrograde labelling is stable up to at least two months after injection and that relatively long survival times are often needed for optimal labelling. For practical purposes a bulk transport velocity of 20 mm/day can be used for estimating the survival time required for reasonable retrograde labelling. The accumulation of TB did not interfere with the visualization of monoamines in the same neurones, and the tracer was never seen to be anterogradely or transcellularly transported.

The distribution of dopamine (DA)-containing fibers in the virtual absence of noradrenaline (NA)-containing ones has been mapped by aldehyde fluorescence histochemistry in rats subjected to a combined neurotoxin treatment (intracerebral 6-hydroxydopamine injections plus systemic injections of the selective NA neurotoxin DSP-4). This pretreatment left di- and telencephalic DA levels largely unaffected, but reduced the NA levels by at least 86-96%. The resulting DA:NA ratios suggested that the catecholamine-containing structures, demonstrable by fluorescence histochemistry in the di- and telencephalic regions, were predominantly the DA-containing ones. While the distribution of DA terminal systems in the neo- and allocortical regions conformed well to previous results, the combined neurotoxin treatment revealed new features of the distribution of DA fibers in the diencephalon. In addition to the previously described innervations of the tubero-hypophyseal system, the incerto-hypothalamic system, and the mesohabenular pathway, previously unknown innervations were revealed in the supraoptic, paraventricular and dorsomedial nuclei of the hypothalamus, and in the paraventricular nucleus of the thalamus. Apart from some scattered fibers in the periventricular and lateral hypothalamic areas and medical zona incerta, other diencephalic nuclei seemed to be devoid of any significant DA terminal networks. The dopaminergic nature of these innervations is supported by DA uptake experiments (evaluated by fluorescence histochemistry) as well as by independent biochemical and immunohistochemical evidence. It is suggested that the DA innervations of the hypothalamic neurosecretory nuclei originate in cell bodies of the diencephalic A11-A14 cell groups and that such intradiencephalic DA projections participate in the regulation of oxytocin and vasopressin release from the pituitary.

Using the aluminium-formaldehyde method for visualization of catecholamines in combination with injections of the fluorescent retrograde tracer True Blue we have studied those diencephalic dopamine (DA)-containing cell groups which have been proposed to give rise to the DA innervation of the spinal cord and investigated the organization of the diencephalospinal DA system in detail. The A13 cell group was found to contain 370, and the A11 cell group 140, DA-producing cells on each side, whereas only very few such cells were found in the paraventricular hypothalamic nucleus. Tracer injections into the spinal cord labelled only DA cells within the A11 group. The overall majority of labelled cells were found ipsilaterally but some cells were also found contralaterally indicating the existence of a minor crossed dopaminergic projection to the spinal cord. Large tracer injections which covered the hemicord at different levels generally resulted in very similar distributions and numbers of retrogradely-labelled DA cells. The labelled DA-containing cells constituted 30-50% of the total number of labelled neurones in the ipsilateral A11 area and about 20-40% of the total number of DA containing cells in this area were labelled. Small injections that did not extend into the nucleus reticularis or the adjacent part of the lateral funiculus failed to label any diencephalic DA cells but usually labelled some non-DA cells in the A11 area. It is concluded that the diencephalospinal DA neurones have long axons that extend over several segments and possibly traverse the entire length of the spinal cord, giving off collateral branches at various levels. From the anatomical data of the present study and previous pharmacological and electrophysiological findings it seems possible that diencephalospinal DA neurones could modulate both sympathetic activity and nociception.

Dopamine (DA) was early demonstrated in the arcuate nucleus by means of the formaldehyde-induced histofluorescence method. In the present study we have investigated the distribution of cell bodies in the arcuate nucleus with antisera against tyrosine hydroxylase (TH), aromatic L-amino acid decarboxylase (AADC) and DA. The results indicate that TH-immunoreactive cells in the dorsomedial part of the arcuate nucleus also contain immunoreactivity for both AADC and DA. However, TH-positive cells in the ventrolateral arcuate nucleus lacked AADC- and DA-immunoreactivity with the sensitivity of the present methods. The findings raise the question whether the ventrolateral cells synthesize L-DOPA or DA as endproducts.

A procedure is described for the use of fluorescent retrograde tracers in conjunction with monamine fluorescence histochemistry for single or double labeling studies of the projections of identified catecholamine or indoleamine neurons in the CNS. A number of fluorescent tracers, including Evans Blue, bisbenzimide, DAPI, 'True Blue', 'Granular Blue' and propidium iodide, have been tested and characterized microspectrofluorometrically. Of these, 'True Blue', propidium iodide and Evans Blue were found to have the most suitable properties to be studied concomitant with the monoamine fluorphores. A combination of two of these tracers--'True Blue' fluorescing blue and propidium iodide or Evans Blue fluorescing orange to red--and the catecholamines and indoleamines fluorescing yellow-green and yellow to brownish yellow, respectively, makes possible simultaneous use of four different fluorescent markers in one and the same section. The present procedure has great practical advantages over available HRP techniques and should in modified form also be applicable to other types of transmitter-specific neuronal tracing based on immunocytochemistry or enzyme histochemistry.