The chaotic nature of tumor vessels forms physiological barriers to treatments. Tumor vessels have tortuous shapes, irregular surfaces and diameters, and heterogeneous spatial distribution. Instead of forming functional networks, tumor vessels form an immature mesh-like network similar to the primary vascular plexus in early stage embryos. Tumor blood flow is often sluggish and static, and even changes the direction over time. Furthermore, some tumor vessels are lacking oxygen despite of perfusion. As a result, tumors are often hypoxic and acidic. Tumor vessels have high vascular permeability and low leukocyte endothelial interaction in general. However, blood flow and vascular permeability in tumors are spatially and temporally heterogeneous. Altogether, these characteristics of tumor vessels form a unique microenvironment of tumors and hinder the efficacy of various therapies for tumors. Tumors consist of not only neoplastic cells but also host stromal cells, such as endothelial cells, peri-vascular cells, fibroblasts, macrophages, and mast cells. These are all embedded within a protein-rich extracellular matrix forming specific local microenvironments. We have found that expression of pro- and anti- angiogenic factors, angiogenesis and the microcirculatory functions were significantly different when we grew the same tumor cells in different host organs. These differences should be due to differences in stromal cells and local microenvironment of different organs. Indeed, we found that host stromal cells significantly contributed to the expression of vascular endothelial growth factor, one of the most potent angiogenic factors, in tumors depending on their context by means of intravital microscopy, novel green fluorescent protein reporter gene system and targeted deletion of specific genes. The tumor-host interaction influences both tumor cells and host stromal cells in their biology including the expression of positive and negative regulators of angiogenesis. This interaction clearly depends on tumor-type and organs, may vary during treatment, and influences the efficiency of various treatment modalities. A better understanding of tumor-host interaction especially during tumor growth and response to treatments should improve future tumor treatment strategies. (Presented at the 1194th Meeting, January 30, 2001.)

The formation of hematopoietic organs is dependent on the angiogenesis, indicating the existence of common progenitors, hemangioblasts, which are able to differentiate into both hematopoietic stem cells (HSCs) and endothelial cells (ECs). We have shown that the first site where HSCs proliferate is the omphalomesenteric artery (OA) besides para-aortic splanchnopleural region (P-Sp). HSCs form aggregates, adhere to ECs and express TIE2/TEK, c-Kit and Flk-1. Using the culture system of P-Sp explants on stromal cells, we demonstrate that proliferation of HSCs is associated with the growth of angioblasts. From P-Sp explants, vascular bed formation (vasculogenesis) and network formation (angiogenesis) are observed in addition to definitive hematopoiesis. P-Sp culture from TIE2 deficient embryos revealed that the TIE2 signalling is critical for definitive hematopoiesis as well as angiogenesis. Angiopoietin-1 (Ang-1), a ligand for TIE2 promoted the adhesion of TIE2+ HSCs to fibronectin through integrins. These HSCs remarkably proliferated in the presence of Ang-1 and SCF. Interestingly, Ang-1 is selectively expressed on HSCs such as c-Kit+TIE2+ cells. TIE2+ cells remained to be undifferentiated in the stromal layer, and when TIE2 signaling was blocked by the addition of TIE2-Fc protein, differentiation was induced on stromal cells. It is suggested that the autocrine loop of Ang-1, is involved in self-renewing of HSCs. On the other hand, we have also shown that HSCs are essential for angiogenesis during embryogenesis using AML1 deficient embryos. Taken together, we want to propose a model of vascular niche for hematopoietic stem cells. (Presented at the 1195th Meeting, January 31, 2001.)

The mechanisms allowing the gastrointestinal immune system to avoid an inappropriate inflammatory response to non-pathogenic luminal antigens are poorly understood. We have previously described a role for cyclooxygenase-2 (Cox-2) dependent arachidonic acid metabolites produced by the murine small intestine lamina propria in controlling the immune response to dietary antigen (Nature Medicine 5 : 900, 1999). To better understand the role of Cox-2 dependent arachidonic acid metabolites produced by the lamina propria, we examined the pattern of expression and the cellular source of Cox-2 and Cox-2 dependent prostaglandin E₂ (PGE₂). We now demonstrate that non-bone marrow derived lamina propria stromal cells have basal Cox-2 expression and that Cox-2 dependent PGE₂ production by these cells is spontaneous and continuous. The other mucosal and non-mucosal lymphoid compartments examined do not share this phenotype. In contrast to the majority of descriptions of Cox-2 expression, Cox-2 expression by lamina propria stromal cells is not dependent upon exogenous stimuli including adhesion, LPS signaling, or the pro-inflammatory cytokines TNF-α. IFN-γ and interleukin-1β. These findings, in conjunction with the known immunomodulatory capacities of prostaglandins, suggest that Cox-2 expression by the small intestine lamina propria is a basal state contributing to the hyporesponsiveness of the intestinal immune response. (Presented at the 1196th Meeting, January 26, 2001.)

We encountered a 43-year-old female patient who had suffered from mild nail candidiasis, insulin-dependent diabetes with a high level of anti-GAD autoantibody, and autoimmune hepatitis, and she was diagnosed as having type 1 autoimmune polyendocrine syndrome (APS-1; or autoimmune polyendocrinopathy candidiasis-ectodermal dystrophy: APECED). It was found that she possessed the compound heterozygous mutations of the AIRE gene. These newly found mutations included an 86T>C(L29P) mutation at exon 1 and an IVS9-1G>C mutation at the acceptor site of intron 9. Her brother, who also developed severe mucocutaneous candidiasis in his early infancy, hypoparathyroidism, Addison's disease, and pure red cell aplasia, was found to have the same mutations. The prevalence of these newly found mutations and most frequent mutations (R257X and dell3) were screened in 396 subjects including 178 healthy controls, 155 patients with non-insulin dependent diabetes and 63 patients with insulin-dependent diabetes using the PCR-RFLP method. As a result, only one heterozygous mutation of L29P was found in a patient with non-insulin dependent diabetes, suggesting that these mutations are rare in Japanese. The expression of the AIRE gene was predominantly observed in peripheral monocytes and dendritic cells, which may suggest that the AIRE gene may play a role in antigen-presentation. Further examinations to find the patients with APS-1 and to clarify the exact mechanisms responsible for the development of APS-1 associated with mutations of AIRE gene are essentially important. (Presented at the 1198th Meeting, February 9, 2001.)

There are three criteria for factors that are crucially related to ocular dominance plasticity: 1) that antagonists or inhibitors of the factor should block ocular dominance plasticity; 2).that the factor should be more concentrated or active at the peak of the critical period; and 3) that dark rearing, which makes the cortex less plastic early in the critical period and more plastic late in the critical period, should have a similar effect on the factor. The last criterion can be used to distinguish activity-related factors that may simply increase or decrease with development from factors that are more specifically related to plasticity. Two factors currently fulfill these criteria, namely NMDA receptors and protein kinase A (PKA). This suggests that the pattern of signals from the retina activates NMDA receptors, Ietting calcium into the cell, and producing cyclic AMP which activates PKA, eventually leading to growth of some synapses and decay of others. PKG and PKC antagonists do not inhibit ocular dominance plasticity, as PKA antagonists do. This result agrees with our previous observation that nitric oxide synthase inhibitors, which activate PKG, also do not inhibit ocular dominance plasticity. However, NMDA agonists and PKA activators by themselves do not bring back plasticity. This leads to the hypothesis that there may be two or more pathways for ocular dominance plasticity acting in parallel with each other. For example, metabotropic glutamate receptors may act in parallel with NMDA receptors to change calcium within the cell. (Presented at the 1199th Meeting, February 28, 2001.)

As we enter the new millennium, a look to the future is speculative but exciting. The field of gene therapy has not yet achieved its potential in the areas of muscular dystrophy, cystic fibrosis, and cancer treatment. However, there is great opportunity for progress in treatment of musculoskeletal problems, and particularly disorders related to the spine. There are three primary areas ripe for advance with regard to gene therapy and spinal disorders. First, nerve and/or spinal cord regeneration has great potential since nerves seem to take up DNA readily and be susceptible to transduction with a variety of genes. The future challenges lie in identifying the grighth gene or genes and scaffolds necessary to regenerate nerve tissue. The second opportunity resides in the management/prevention of intervertebral disc disorders. There are three areas within the intervertebral disc worth noting: 1) annular repair; 2) slowing of nuclear degeneration; and, 3) nuclear regeneration. These tasks are listed in order of increasing difficulty. A potential limitation for all three of these applications is the fact that adult animals, including humans, do not show evidence of meaningful cartilage repair or regeneration. Thus, the signals to coordinate these events may be more of a challenge to decode. Nevertheless, experiments already show that the nucleus pulposus cells are an excellent target for gene therapy. In addition, the intradiscal environment is relatively immune-privileged making use of conventional gene transfer vectors such as the adenovirus very feasible. The third opportunity, and most immediately attainable, is the use of local gene therapy to generate bone for more reliable spine fusions and avoidance of the pain, limited supply, and complications associated with autogenous bone graft harvest. Several groups are investigating the use of gene therapy to deliver growth factors or proteins to initiate bone formation. Our laboratory has identified, sequenced, and cloned a novel intracellular transcription-like factor called LIM Mineralization Protein-1 (LMP-1). Small quantities of LMP-1 cDNA inserted into cells results in the initiation of the bone formation cascade. This presentation will summarize currently available bone growth factors and the potential opportunity for using local gene therapy to generate spine fusion. Additional@opportunities exist for the enhancement of vertebral bone density and treatment/prevention of osteoporotic vertebral fractures. (Presented at the 1200th Meeting, March 15, 2001.)

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