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Strict control of major histocompatibility complex class II gene expression is essential for proper functioning of the immune system. Recent transgenic mouse studies have revealed an intricate fine-tuning of class II gene transcription in microenvironments such as the germinal centers and thymic cortex and medulla.
OBJECTIVES: To present our recent observations obtained from the continuing characterization of the TSP50 gene pertaining to its evolutionary importance and behavior in human testicular germ cell tumors. Previous studies have reported that expression of the human TSP50 gene is testes specific. Its product is similar to many serine proteases but possesses its own unique features. In addition, TSP50 is abnormally activated in most tested patients with breast cancer. METHODS: Testicular tissue from rats, mice, and humans was obtained through biopsy or orchiectomy. The expression of the TSP50 protein was determined using immunohistochemical staining and Western blotting techniques. RESULTS: The Western blot results showed that the polyclonal anti-human TSP50antibody reaction pattern in both rodent testes was the same as that observed in the human testes. In addition, the immunohistochemical staining patterns in the human, mouse, and rat testes were similar. We also discovered that expression of TSP50 was largely downregulated in all testicular germ cell tumors examined by immunohistochemical analysis. CONCLUSIONS: The results of our studies suggest that the TSP50 gene could be of evolutionary importance in mammalian reproduction. Unlike the results generated from patients with breast cancer, in whom upregulation of the TSP50 gene correlates with disease development, the TSP50 gene was downregulated in patients with seminoma. This information indicates that altered expression levels of the TSP50 gene in different microenvironments are associated with different or distinct types of human cancer.
Here we report that glioblastoma multiforme (GBM) mediates immunosuppression by promoting T-cell death via tumor-associated CD70 and gangliosides that act through receptor-dependent and receptor-independent pathways, respectively. GBM lines cocultured with T cells induced lymphocyte death. The GBM lines were characterized for their expression of CD70, Fas ligand (FasL), and tumor necrosis factor-alpha (TNF-alpha), and the possible participation of those molecules in T-cell killing was assessed by doing GBM/T cell cocultures in the presence of anti-CD70 antibodies, Fas fusion proteins, or anti-TNF-alpha antibodies. CD70 but not TNF-alpha or FasL is responsible for initiating T-cell death via the receptor-dependent pathway. Of the four GBM cell lines that induced T-cell death, three highly expressed CD70. Two nonapoptogenic GBM lines (CCF3 and U138), on the other hand, had only minimally detectable CD70 expression. Blocking experiments with the anti-CD70 antibody confirmed that elevated CD70 levels were involved in the apoptogenicity of the three GBM lines expressing that molecule. Gangliosides were found to participate in the induction of T-cell apoptosis, because the glucosylceramide synthase inhibitor (PPPP) significantly reduced the abilities of all four apoptogenic lines to kill the lymphocytes. High-performance liquid chromatography (HPLC) and mass spectroscopy revealed that GM2, GM2-like gangliosides, and GD1a were synthesized in abundance by all four apoptogenic GBM lines but not by the two GBMs lacking activity. Furthermore, gangliosides isolated from GBM lines as well as HPLC fractions containing GM2 and GD1a were directly apoptogenic for T cells. Our results indicate that CD70 and gangliosides are both products synthesized by GBMs that may be key mediators of T-cell apoptosis and likely contribute to the T-cell dysfunction observed within the tumor microenvironment.
Seizures are known to induce dramatic alterations in neuronal gene expression. These changes may play a role in the genesis of an epileptic state. The present report describes another consequence of seizures-a dramatic induction of glial fibrillary acidic protein (GFAP) expression in astrocytes. Using a hippocampal kindling model, we demonstrate that kindled seizures lead to many fold increases in mRNA for GFAP in structures which experience electrographic seizures. The increases can be detected 1 day following a single seizure. If seizures are induced repetitively (every other day for many days), levels of GFAP mRNA remain elevated. However, when kindled seizures are not induced, levels of GFAP mRNA return to near control levels within a few days. The increases in GFAP mRNA levels are not in response to decreases in neuronal activity (as a result of postictal depression), because GFAP mRNA levels are unaffected when neuronal activity is decreased by blocking afferent drive (with tetrodotoxin). The induction of GFAP expression by seizures may reflect the first step in a process in which seizures induce astrocytic hypertrophy. The changes in astrocytes could in turn modify the way in which astrocytes maintain homeostasis in the extracellular microenvironment in ways that could contribute to the development of an epileptic state.
Allergic reactions seem to be under a variety of different control systems. Concerning the onset of allergic disease, it has become clear that cytokines play an important regulatory role. Specific T-Helper cells subsets produce a pattern of cytokines that preferentially induces IgE synthesis. As IgE synthesis always occurs in the normal individual, we propose that an additional control system is needed. Naturally occurring anti-IgE autoantibodies may represent such a control system that are capable to neutralise IgE or to eliminate IgE producing B cells. This hypothesis is also supported by our recent cloning of autoantibodies against IgE from the human genome. Interestingly, the brain seems to represent a superseding control system that is also capable of modulating allergic reactions and this even at the level of IgE synthesis. This control is mediated by neuroendocrine factors that influence the cytokine microenvironment as if a shift in the TH1/TH2 phenotype had occurred.
Large concentrations of ethanol in vitro decreased ligand binding to mu and delta opiate receptors in the frontal cortex of the C57BL mouse, but did not alter binding to kappa opiate receptors. Mu and delta receptors were equally sensitive to the inhibitory effect of ethanol. Since the effects of ethanol were significant only in large concentrations, ethanol may alter opiate binding through its membrane lipid-perturbing actions, and the selectivity of the effects of ethanol may reflect differences in the microenvironments of the opiate receptor subtypes in membranes. After chronic ingestion of ethanol by mice, in vivo, there was a selective decrease in the number of mu receptors in the frontal cortex. This change may result from indirect effects of ethanol on the opiate receptor and may contribute to specific central effects of ethanol.
The nervous system of adult mammals, unlike the rest of the organs in the body, has been considered unique in its apparent inability to replace neurons following injury. However, in certain regions of the brain, neurogenesis occurs postnatally and continues through adulthood. The nature, fate, and longevity of cells undergoing proliferation within the CNS are unknown. These cells are increasingly becoming the focus of intense scrutiny; this is a recent development that has led to considerable controversy over the appropriate terminology to describe neural cells as they pass through different stages of proliferation, migration, and differentiation. Continuing studies detailing the properties of mitotic populations in the adult CNS will provide a better understanding of the nature of these cells during their development and should lead to a more consistent nomenclature. Studies of neural precursors isolated from the embryonic brain have indicated that many subgroups of cells undergo mitosis and subsequent differentiation into neurons and glia in vitro. A number of substances, such as growth factors and substrate molecules, are essential for these processes and also for lineage restriction and fate determination of these cells. Recent studies have shown that cells with proliferative capabilities can also be isolated from the adult brain. The nature of these cells is unknown, but there is evidence that both multipotent cells (stem cells) and lineage-restricted cells (neuroblasts or glioblasts) are resident within the mature CNS and that they can be maintained and induced to divide and differentiate in response to many of the same factors that influence their embryonic counterparts. Presently, it is unclear how many potentially quiescent precursor cells exist in the adult brain or what combination of growth factors and substrate molecules is involved in the proliferation and differentiation of these cells. Some of these questions are currently being addressed by using immortalized neural precursors or growth factor-expanded populations of primary precursors to model precursor responsiveness to environmental manipulations. Because in vitro culture conditions are unlikely to provide all of the factors necessary for inducing the proliferation and differentiation of neural precursors, recent studies have explored the properties of well-characterized precursor populations after implantation back into specific regions of the developing or adult CNS. These studies have highlighted the importance of the microenvironment in precursor differentiation and further suggested that precursor plasticity is a characteristic that is probably common to neural precursors throughout the CNS.(ABSTRACT TRUNCATED AT 400 WORDS)
To promote both efficiency and selectivity, many protein kinases and phosphatases are maintained in specific subcellular microenvironments through their association with anchoring proteins. In this study, we describe a new class of proteins, called GKAPS, that specifically bind the Type II cGMP-dependent protein kinase (PKG). GKAPs were detected in rat aorta, brain, and intestine using a protein overlay technique. The PKG binding proteins were distinct from AKAPs, proteins known to bind the cAMP-dependent protein kinase (PKA). Furthermore, a synthetic peptide that blocks association of PKA with AKAPs did not affect the PKG-GKAP interaction. Deletion mutagenesis was used to map the GKAP binding determinants within PKG to the N-terminal regulatory region. While most GKAPs were tissue-specific, a ubiquitous PKG-binding protein was detected and identified as myosin. Analysis of myosin fragments revealed that PKG binds within Subfragment 2. The results define a novel class of anchoring proteins that may target PKG for specific functional roles.
The mechanism through which VIP prevents neurotoxicity associated with HIV envelope protein has been shown to involve the release of a beta-chemokine, MIP-1 alpha. Astrocytes stimulated with subnanomolar concentrations of VIP caused the release of MIP-1 alpha and RANTES, both of which have been shown to prevent neuronal cell death associated with gp120. It is further proposed that gp120 causes neuronal cell death, in part, by competing with endogenous chemokines at various chemokines receptors in the brain that are necessary for neuronal survival. Although the chemokines are known to be mediators of inflammation, our studies suggest that these compounds have additional roles as neuroprotective agents that depend on the concentration of chemokine, cellular microenvironment, and stage of development of target neurons. Our studies further imply that in a developing system, stimulation with a MIP-1 alpha like substance is necessary for neuronal survival and interference with this action results in neuronal cell death.
Transplantation of neural stem/progenitor cells has been proposed as a novel approach for the replacement and repair of damaged CNS tissues. We have evaluated the influence of the host cellular microenvironment upon the survival, differentiation, and integration of neural progenitor cells transplanted into the CNS. Using this approach, we have investigated the fate of neural progenitor cells in vivo following transplantation into the developing mammalian eye. Murine brain progenitor cells (mBPCs) isolated from neonatal mice expressing the green fluorescent protein (GFP) transgene were transplanted into the eyes of Brazilian opossums (Monodelphis domestica). Monodelphis pups are born in an extremely immature, fetal-like state. The eyes of neonatal pups provide a fetal-like environment in which to study cellular interactions between host tissues and transplanted neural progenitor cells. mBPCs were transplanted by intraocular injection in hosts ranging in age from 5 days postnatal to adult. The transplanted cells were easily identified because of their GFP fluorescence. Extensive survival, differentiation, and morphological integration of mBPCs within the host tissue was observed. We found that the younger retinas provided a more supportive environment for the morphological integration of the transplanted mBPCs. Cells with morphologies characteristic of specific retinal cell types were observed. Moreover, some transplanted mBPCs were labeled with antibodies characteristic of specific neural/retinal phenotypes. These results suggest that the host environment strongly influences progenitor cell differentiation and that transplantation of neural progenitor cells may be a useful approach aimed at treating degeneration and pathology of the CNS.
Astrocytes, once relegated to a mere supportive role in the central nervous system, are now recognized as a heterogeneous class of cells with many important and diverse functions. Major astrocyte functions can be grouped into three categories: guidance and support of neuronal migration during development, maintenance of the neural microenvironment, and modulation of immune reactions by serving as antigen-presenting cells. The concept of astrocytic heterogeneity is critical to understanding the functions and reactions of these cells in disease. Astrocytes from different regions of the brain have diverse biochemical characteristics and may respond in different ways to a variety of injuries. Astrocytic swelling and hypertrophy-hyperplasia are two common reactions to injury. This review covers the morphologic and pathophysiologic findings, time course, and determinants of these two responses. In addition to these common reactions, astrocytes may play a primary role in certain diseases, including epilepsy, neurological dysfunction in liver disease, neurodegenerative disorders such as Parkinson's and Huntington's diseases, and demyelination. Evidence supporting primary involvement of astrocytes in these diseases will be considered.
Tissues in the body are maintained by somatic stem cells. This has been demonstrated both in organs with high cell turnover rate, such as the bone marrow, colon and skin, and in organs with low cell turnover rate, such as the brain. To maintain homeostasis in the body it is important to keep tight control over stem cell fate. Stem cells are under strict control from both intrinsic and extrinsic factors and loss of this control has been postulated to be a key step in the carcinogenic process. There is increasing evidence that cancer initiation results from accumulative oncogenic mutations (intrinsic loss of control) in long-lived stem cells or their immediate progenitor, followed by modification of the surrounding microenvironment (loss of extrinsic control). Decades ago, studies on teratocarcinoma led to the hypothesis that a small subset of self-renewing cancer stem cells with differentiation potential exists within tumors. These studies showed that teratocarcinomas contain undifferentiated embryonic carcinoma cells that are able to give rise to differentiated cells which belong to all three germ layers. More recent studies have confirmed cancer stem cells in such diverse cancers as leukemia, brain and breast cancer. It is, however, unclear whether cancer stem cells originate from resident stem cells or whether they arise as a result of an acquired gain of self-renewal capacity in tissue progenitor cells or even more differentiated cells. The characterization of a cancer stem cell profile within diverse cancer types may open up new avenues for cancer treatment. In this review we discuss the concept of cancer stem cells and focus on examples where these cells have been identified.
The origin of spontaneous human brain tumors has been recently debated as originating from a stem rather than a progenitor cell. Based on the statistical distribution of a selected group of primary brain tumors, and on the clonality and field theory for cancer growth, we propose as plausible the contribution of progenitor cells in the origin of the most common brain tumors such as, pediatric, mixed (glial and neuronal), and the aggressive glioblastoma multiforme in adults. Operationally, stem cell is defined as having a renewal probability of p=1, a progenitor 0<p<1, and a terminally differentiated cell as p=0. The space-time field location (microenvironment, cell-cell interactions...) and mitotic potential of a cell limits the distinguishability between stem and progenitors, due to the interconvertibility of these states via the process of retrodifferentiation (dedifferentiation). If excessively iterated retrodifferentiation of a mitotically competent cell prompts such a cell to organize programs that are atypical vis-à-vis the standard ones. Regarding specifically malignant cells, aperiodic cycles of dedifferentiation favour emergence of subclonal populations whose anachronistic evolution might become idiosyncratically resistant to therapies previously efficient against the initial tumoral mass. Unfortunately the "ab initio" identification of such subclonal "drifters" is impaired by systematic uncertainties as quali-quantified in appendix.
Cholecystokinin (CCK) acts as an anti-opioid peptide. The mechanisms of CCK-opioid interaction under normal and pathological conditions were examined with various techniques. Nerve injury induces upregulation of CCK mRNA and CCK2 receptors in sensory neurons. The involvement of CCK in spinal nociception in normal and axotomized rats was examined. The CCK2 receptor antagonist CI-988 did not reduce spinal hyperexcitability following repetitive C-fiber stimulation in normal or axotomized rats, suggesting that CCK is probably not released from injured primary afferents. With in vivo microdialysis intravenous (i.v.) or intrathecal (i.t.) morphine increased the extracellular level of CCK in the dorsal horn in a naloxone reversible manner. Morphine also released CCK after axotomy, but not during carrageenan-induced inflammation. In contrast, K(+)-stimulation failed to increase extracellular levels of CCK in axotomized rats, but did so in inflamed rats. Double-coloured immunofluorescence technique revealed partial co-localization between CCK-like immunoreactivity (LI) and mu-opioid receptor (MOR)-LI in superficial dorsal horn neurons. The presence of MOR in CCK containing neurons suggests a possible direct influence of opioids on CCK release in the spinal cord. Axotomy, but not inflammation, induced a moderate decrease in CCK- and MOR-LI in the dorsal horn. I.v. morphine further temporarily reduced CCK- and MOR-LIs in axotomized, but not in normal or inflamed, rats. While the effect of morphine on CCK-LI can be interpreted as the result of increased CCK release, the effect on MOR-LI may be related to changes in the microenvironment of the dorsal horn induced by nerve injury.
By combining the partition method for enrichment of sulfatides without any chromatographic procedures and the preparation method of lysosulfatides, we succeeded in analyzing these sulfated glycosphingolipids from biological materials by matrix-assisted laser desorption and ionization time-of-flight mass spectrometry (MALDI-TOF MS) to reduce the complexity of mass fragmentation patterns within a day. We found that sulfated GalCer (HSO3-3Gal beta 1Cer) (SM4s [galactosylsulfatide]) was composed of different species. While composition of SM4s specifically depended on source materials, it always contained hydroxy fatty acids of various degrees. In addition to the common sphingoid 4-sphingenine (d18:1), uncommon/unusual sphingoids phytosphingosine (4-hydroxysphinganine) (t18:0), eicosasphinganine (d20:0), 4-eicosasphingenine (d20:1), and sphingadienine (d18:2) were easily detected. Finally, in addition to SM4s, sulfatide sulfated LacCer (HSO3-3Gal beta 4Glc beta 1Cer) (SM3 [sulfated lactosylceramide]) and sulfated Gg3Cer (GalNAc beta 4(HSO3-3)Gal beta 4Glc beta 1Cer) (SM2 [sulfated gangliotriaosylceramide]) were clearly detected in renal tubule cells. The major SM4s was composed of ceramides possessing d18:1 with C22 hydroxy fatty acids (C22:0 h), C23:0 h, and C24:0 h, whereas the major SM3/SM2 were composed of ceramides possessing t18:0 with C22 normal fatty acids (C22:0), C23:0, C24:0. Namely, in these two series of sulfatides, either fatty acids or sphingoids were hydroxylated, and chain lengths of these components were exactly the same, consequently resulting in a similar polarity of ceramide moieties in these sulfatide species. These results demonstrated diversities of sulfatide molecular species, not only with respect to sugar moieties but also to ceramide moieties, which are probably important for specific effective functions in particular microenvironments such as lipid membrane microdomains.
Tumor necrosis factor-alpha (TNF-alpha), a cytokine produced by astrocytes in vivo and in vitro, was tested for its effects on two malignant astrocytoma cell lines (A-172, U-87). Both lines were immunoreactive for glial fibrillary acidic protein, vimentin, Class I antigens, and interleukin-6. The lines differed in their expression of Class II and intercellular adhesion molecule-1 (ICAM-1) antigenic determinants: A-172 cells were negative for both Class II and ICAM-1 antigens, while U-87 cells were intensely positive for Class II and weakly positive for ICAM-1. When these astrocytoma cell lines were exposed to TNF-alpha, A-172 growth was stimulated while U-87 growth was inhibited. Furthermore, in U-87 cells, TNF-alpha enhanced both ICAM-1 and interleukin-1 beta (IL-1 beta) expression, and decreased immunoreactivity for transforming growth factor-beta (TGF-beta) protein. In contrast, in the presence of TNF-alpha, A-172 cells remained negative for IL-1 beta and TGF-beta, but showed an increased expression of ICAM-1. These results demonstrate that TNF-alpha can induce changes in growth rate, cytokine production, and surface antigen expression in malignant astrocytomas; however, the nature of these changes is dependent on the specific characteristics of these malignant astrocytomas. The resultant variability in the immunological microenvironment of these tumors may reflect differences in their growth potential.
The results obtained with neuronal grafting in an animal model of heredo-degenerative ataxia (the pcd mutant mouse) have been extremely useful to unmask new aspects of neural plasticity. The grafted embryonic Purkinje cells invade the deficient molecular layer of the host by migrating radially through adult Bergmann fibers. There, they start building their dendritic trees and, by promoting the axonal sprouting of specific adult neuronal population in a timed sequence, they receive appropriate synaptic contacts, starting ten days after grafting. Twenty-one days after grafting, the grafted Purkinje cells have acquired their adult dendritic pattern and synaptic investment. Both the detailed timetable and the nature of the cellular interactions between embryonic and adult neural cells are remarkably similar to those occurring during normal development. These results raise the possibility that embryonic Purkinje cells can induce in adult neural cells a new type of plasticity, that of recreating a permissive microenvironment for the synaptic integration of the grafted neurons, leading to the anatomical restoration of the cortical circuit of the mutant cerebellum.
The effectiveness of radiation therapy for human brain tumors is limited by the presence of radiation-resistant hypoxic cells. In order to improve patient outcomes, therapeutic methods that increase hypoxic cell killing must be developed. To investigate the possibility of using the hypoxic tumor microenvironment itself as a target for gene therapy, we stably transfected U-251 MG human glioblastoma cells with constructs containing the suicide gene Bax under the regulation of a nine-copy concatemer of hypoxia responsive elements (HREs). Previously, we demonstrated that the expression of BAX protein under anoxic conditions in transfected U-251 MG clones leads to increased cell killing in vitro. Our recent studies revealed that HIF-1alpha induction under anoxic conditions occurs prior to the increase in BAX expression, thereby implicating HIF-1 induction as the basis of BAX upregulation. To test the effect of BAX-mediated cell killing in vivo, we implanted five stably transfected clones subcutaneously into the flanks of athymic mice. Compared to nontransfected controls, tumor growth in four of five clones was significantly retarded. Histopathological analysis demonstrated decreased hypoxic fractions and increased amounts of apoptosis in clone-derived tumors. These results suggest that the tumor microenvironment is sufficiently hypoxic to trigger HRE-mediated cell killing via the BAX apoptotic pathway.