Rapid Mass Spectrometry Imaging to Assess the Biochemical Profile of Pituitary Tissue for Potential Intraoperative Usage.

Pituitary adenomas are relatively common intracranial neoplasms that are frequently treated with surgical resection. Rapid visualization of pituitary tissue remains a challenge as current techniques either produce little to no information on hormone-secreting function or are too slow to practically aid in intraoperative or even perioperative decision-making. Matrix-assisted laser desorption/ionization mass spectrometry imaging (MALDI MSI) represents a powerful method by which molecular maps of tissue samples can be created, yielding a two-dimensional representation of the expression patterns of small molecules and proteins from biologic samples. In this chapter, we review the use of MALDI MSI, its application to the characterization of the pituitary gland, and its potential applications for guiding the management of pituitary adenomas.

Delayed re-opening of an STA-MCA bypass graft.

We describe the case of a 47-year-old female with symptomatic right MCA stenosis who had undergone cerebral revascularization through a superficial temporal artery-to-middle cerebral artery (STA-MCA) bypass. Despite clear patency in the operating room, post-operative angiography showed no flow in the bypass. Her ipsilateral internal carotid artery (ICA) was widely patent. She remained asymptomatic and follow-up angiography four years later showed a widely patent bypass graft in the setting of critical stenosis of the ipsilateral ICA. That the graft was found opened up and supplying the hemisphere was presumably stimulated by an increased "demand" and flow gradient promoting its patency.

TRAF1 is a negative regulator of TNF signaling. enhanced TNF signaling in TRAF1-deficient mice.

TNF receptor-associated factor 1 (TRAF1) is a unique TRAF protein because it lacks a RING finger domain and is predominantly expressed in activated lymphocytes. To elucidate the function of TRAF1, we generated TRAF1-deficient mice. TRAF1(-/-) mice are viable and have normal lymphocyte development. TRAF1(-/-) T cells exhibit stronger than wild-type (WT) T cell proliferation to anti-CD3 mAb, which persisted in the presence of IL-2 or anti-CD28 antibodies. Activated TRAF1(-/-) T cells, but not TRAF1(+/+) T cells, responded to TNF by proliferation and activation of the NF-kappa B and AP-1 signaling pathways. This TNF effect was mediated by TNFR2 (p75) but not by TNFR1 (p55). Furthermore, skin from TRAF1(-/-) mice was hypersensitive to TNF-induced necrosis. These findings suggest that TRAF1 is a negative regulator of TNF signaling.

Continuous release of endostatin from microencapsulated engineered cells for tumor therapy.

Research studies suggest that tumor-related angiogenesis contributes to the phenotype of malignant gliomas. We assessed the effect of local delivery of the angiogenesis inhibitor endostatin on human glioma cell line (U-87MG) xenografts. Baby hamster kidney (BHK) cells were stably transfected with a human endostatin (hES) expression vector and were encapsulated in alginate-poly L-lysine (PLL) microcapsules for long-term delivery of hES. The release of biologically active endostatin was confirmed using assays of bovine capillary endothelial (BCE) proliferation and of tube formation. Human endostatin released from the microcapsules brought about a 67. 2% inhibition of BCE proliferation. Furthermore, secreted hES was able to inhibit tube formation in KDR/PAE cells (porcine aortic endothelial cells stably transfected with KDR, a tyrosine kinase) treated with conditioned U-87MG medium. A single local injection of encapsulated endostatin-secreting cells in a nude mouse model resulted in a 72.3% reduction in subcutaneous U87 xenografts' weight 21 days post treatment. This inhibition was achieved by only 150.8 ng/ml human endostatin secreted from 2 x 10(5) encapsulated cells. Encapsulated endostatin-secreting cells are effective for the treatment of human glioblastoma xenografts. Continuous local delivery of endostatin may offer an effective therapeutic approach to the treatment of a variety of tumor types.

Assessment of alterations in gene expression in recurrent malignant glioma after radiotherapy using complementary deoxyribonucleic acid microarrays.

OBJECTIVE: We used complementary deoxyribonucleic acid expression microarrays to assess the effects of radiotherapy on gene expression in glioblastoma multiforme. We hypothesized that postradiation recurrent tumors may demonstrate alterations in gene expression from the primary tumor specimen. METHODS: Patients were diagnosed with glioblastoma multiforme at resection of the initial tumor, and they received 60 Gy of fractionated radiotherapy before recurrence. Ribonucleic acid samples from both the primary and the postradiation recurrent tumor in each patient were screened and compared using complementary deoxyribonucleic acid expression arrays and Northern blot analysis. RESULTS: Messenger ribonucleic acid levels of growth factors participating in paracrine loops, such as vascular endothelial growth factor and platelet-derived growth factor receptor beta, were decreased in postradiation recurrent tumors as compared with primary tumors in three of four patients. However, messenger ribonucleic acid levels of growth factors involved in autocrine loops, such as epidermal growth factor receptor, platelet-derived growth factor alpha, platelet-derived growth factor A, and basic fibroblast growth factor, were decreased in two of four, two of four, three of four, and three of four patients' recurrent tumors, respectively. Microvessel counts demonstrated that blood vessel growth was decreased significantly in postradiation recurrent tumor specimens. CONCLUSION: After radiotherapy of glioblastoma multiforme, levels of paracrine-acting growth factors are diminished in correspondence with the reduction in vascular density. In contrast, growth factors that participate in autocrine loops demonstrate elevated levels of gene expression. These results suggest that maintenance of autocrine loops may be important in tumor regrowth after radiotherapy.

A lipopolysaccharide-specific enhancer complex involving Ets, Elk-1, Sp1, and CREB binding protein and p300 is recruited to the tumor necrosis factor alpha promoter in vivo.

The tumor necrosis factor alpha (TNF-alpha) gene is rapidly activated by lipopolysaccharide (LPS). Here, we show that extracellular signal-regulated kinase (ERK) kinase activity but not calcineurin phosphatase activity is required for LPS-stimulated TNF-alpha gene expression. In LPS-stimulated macrophages, the ERK substrates Ets and Elk-1 bind to the TNF-alpha promoter in vivo. Strikingly, Ets and Elk-1 bind to two TNF-alpha nuclear factor of activated T cells (NFAT)-binding sites, which are required for calcineurin and NFAT-dependent TNF-alpha gene expression in lymphocytes. The transcription factors ATF-2, c-jun, Egr-1, and Sp1 are also inducibly recruited to the TNF-alpha promoter in vivo, and the binding sites for each of these activators are required for LPS-stimulated TNF-alpha gene expression. Furthermore, assembly of the LPS-stimulated TNF-alpha enhancer complex is dependent upon the coactivator proteins CREB binding protein and p300. The finding that a distinct set of transcription factors associates with a fixed set of binding sites on the TNF-alpha promoter in response to LPS stimulation lends new insights into the mechanisms by which complex patterns of gene regulation are achieved.

Growth factors in glioma angiogenesis: FGFs, PDGF, EGF, and TGFs.

It has become well accepted that solid tumors must create a vascular system for nutrient delivery and waste removal in order to grow appreciably. This process, angiogenesis, is critical to the progression of gliomas, with vascular changes accompanying the advancement of these tumors. The cascade of events in this process of blood vessel formation involves a complex interplay between tumor cells, endothelial cells, and their surrounding basement membranes in which enzymatic degradation of surrounding ground substance and subsequent endothelial cell migration, proliferation, and tube formation occurs. It is likely that a host of growth factors is responsible for mediating these key events. To date, a role for Vascular Endothelial Growth Factor (VEGF) in glioma angiogenesis has been convincingly demonstrated. This review explores the contribution of other growth factors--Fibroblast Growth Factors (FGFs), Platelet-Derived Growth Factor (PDGF), Epidermal Growth Factor (EGF), and Transforming Growth Factors (TGFs)--to glioma angiogenesis. These growth factors may influence glioma angiogenesis by directly stimulating endothelial cell proliferation, by mediating the expression of key proteases on endothelial cells necessary for angiogenesis, or by regulating the expression of VEGF and of each other.

Identification and characterization of two CD40-inducible enhancers in the mouse TRAF1 gene locus.

We have shown that CD40 engagement induces TRAF1 gene expression in B lymphocytes. Here we report that CD40-dependent TRAF1 gene transcription in murine B cells is controlled by two enhancer regions. One region is located approximately 2 kb upstream of the transcription start site and the other lies in the intron between exons 5 and 6. The upstream enhancer contains a single NF-kappaB site in addition to sites that bind constitutive transcription factors. Mutation of this NF-kappaB site completely abrogates CD40-driven TRAFl transcription. The intronic enhancer contains two sites that strongly bind the CD40-inducible factors NF-kappaB and AP-1. Simultaneous mutation of the AP-1 site and of the NF-kappaB site abolishes transcription driven by this enhancer. When cloned together into reporter constructs, the two TRAF1 enhancers do not synergize, suggesting that each enhancer may separately participate in the induction of TRAF1 transcription in B cells following CD40 activation.

Structure of the murine TRAF1 gene.

We have cloned, characterized and sequenced the murine TNF Receptor Associated Factor 1 (TRAF1) gene. Restriction mapping and Southern blotting analysis revealed that the TRAF1 gene comprises 10 exons and 9 intervening introns and spreads over 18 kb of genomic DNA. 5'-RACE analysis of the TRAF1 transcript using mRNA from activated spleen B cells revealed several transcription start sites between positions -42 to +4 relative to the 5'end of the murine TRAF1 cDNA sequence. We also isolated and sequenced the 5'-upstream promoter region, which lacks TATA-like and CAAT-like sites but contains GC-rich sequences. Taken together, these results suggest that the TRAF1 gene promoter is a member of the class of Sp-1-dependent promoters. Near the transcription initiation start site we identified three identical decanucleotide repeats (CCAGCCCAGC) which may play a role in the transcriptional regulation of TRAF1 expression. In addition we show that TRAF1 mRNA is not expressed in non-stimulated lymphocytes but can be induced upon activation with different stimuli, including anti-CD3, anti-IgM, anti-CD40 antibodies, LPS, or a combination of phorbol-12-myristate-13-acetate and ionomycin.

Heterotrimeric G proteins physically associated with the lipopolysaccharide receptor CD14 modulate both in vivo and in vitro responses to lipopolysaccharide.

Septic shock induced by lipopolysaccharide (LPS) triggering of cytokine production from monocytes/macrophages is a major cause of morbidity and mortality. The major monocyte/macrophage LPS receptor is the glycosylphosphatidylinositol (GPI)-anchored glycoprotein CD14. Here we demonstrate that CD14 coimmunoprecipitates with Gi/Go heterotrimeric G proteins. Furthermore, we demonstrate that heterotrimeric G proteins specifically regulate CD14-mediated, LPS-induced mitogen-activated protein kinase (MAPK) activation and cytokine production in normal human monocytes and cultured cells. We report here that a G protein binding peptide protects rats from LPS-induced mortality, suggesting a functional linkage between a GPI-anchored receptor and the intracellular signaling molecules with which it is physically associated.