Interleukin (IL)-12, interferon (IFN)-gamma, and other inflammatory cytokines play an important role in the pathogenesis of autoimmune insulitis and diabetes in NOD mice, and inhibition of these cytokines is likely to be beneficial. In this study, we found that Pentoxifylline (PTX) and Rolipram (phosphodiesterase [PDE] inhibitors that induce increased intracellular cAMP) can block inflammatory cytokine production. Inhibition of IL-12 and IFN-gamma secretion was demonstrated in macrophages activated with lipopolysaccharide or T-cells stimulated through the CD3/T-cell receptor complex, respectively. Moreover, strong inhibition of IL-12 was demonstrated in vivo in superantigen-immunized mice. Rolipram was inhibitory at concentrations as low as 10-8 to 10-7 mol/l, and on molar basis, it was 100-fold more effective than PTX. Tumor necrosis factor (TNF)-alpha was also inhibited, but IL-4 was less sensitive to suppression. In NOD mice, both PTX and Rolipram reduced the severity of insulitis and prevented cyclophosphamide-accelerated diabetes. It appears that blocking the activity of type IV PDE is sufficient to mediate the effects reported in this study, since Rolipram inhibits only this isoform, unlike PTX (a general inhibitor). / Significance. PTX and Rolipram may be effective in the treatment of autoimmune diabetes or other conditions characterized by excessive production of inflammatory cytokines.

Heterozygous 1Neu mice transmit a single base pair insertion mutation within the paired domain (exon 2) of the PAX2 gene; this mutation is identical to that in humans with Renal-Coloboma Syndrome. In both mice and humans, PAX2 heterozygotes are born with optic colobomas and renal hypoplasia. Previously, we have shown that kidneys of fetal 1Neu mice have increased apoptosis of ureteric bud (UB) cells and reduced UB branching. However, it is unclear whether increased susceptibility to apoptosis is the critical determinant of renal hypoplasia in PAX2 haploinsufficiency. / In this study, E13.5 fetal kidney pairs were microdissected from wildtype and heterozygous PAX2 mutant embryos and cultured +/- the caspase inhibitor, Z-VAD-fmk, for 50 hours. Explants were immunostained with Dolichos biflorus lectin, visualized by fluorescent microscopy and scored for the number of terminal UB branches. Mutant kidneys had fewer UB terminal branches compared to wildtype littermates (-20%, p = 0.04). (Abstract shortened by UMI.)

Using longitudinal MRI volumetry we examined the relationship between the degree of cerebral atrophy and cognitive function in Mild Cognitively Impaired (MCI) subjects during the progression to Dementia of the Alzheimer's type (DAT). We used these patterns of atrophy to examine the Dichotomous Groups and Accelerated Aging theories of progression to DAT. We acquired MRI scans on 20 subjects with Mild Cognitive Impairment (MCI), 3 subjects with DAT, and 19 normal elderly controls. Scanning was carried out at two times with an average of 73 months between scans. At follow-up, 10 of the MCI subjects had progressed to dementia (Progressors) while 10 had remained stable (Non-Progressors). We found that the Progressors had a significant increase in medial temporal atrophy between Time 1 and Time 2, while the Non-Progressors remained stable. This data supports the Dichotomous Groups theory. Cognitively we found that delayed verbal recall followed the same pattern of change as medial temporal atrophy, but that language ability did not follow the same pattern of change as lateral temporal atrophy.

Under some circumstances CsA induces autoimmune phenomena. A lethal form of graft-versus-host disease occurs in irradiated recipients of syngeneic or autologous BM treated with CsA, following withdrawal of the drug. We could induce this disease in all rat strains tested (Lewis, ACI, BN), but in only one mouse strain, i.e. DBA/2. The AKR, C57BR, CBA/J, C57B1/6 and SJL mouse strains were resistant to disease induction. We studied the effects of CsA on T cell maturation and clonal deletion in rats and mice. We found (as have others) that in the thymus CsA partially blocks the generation of mature-type T cells. In the periphery, we observed the transient appearance of an unusual population of CD4+CD8+ T cells. The T cells of CsA-treated mice of several strains, including diseased DBA/2 mice, showed a normal pattern of clonal deletion when analyzed for expression of TcR Vbeta6 and Vbeta17. Disease could be prevented in rats and mice by injecting normal spleen cells. We postulate that CsA causes autoimmune disease by altering a population of suppressor cells, rather than by blocking clonal deletion.

A three-dimensional structural analysis model of the human thoracolumbar spine and rib cage has been developed in order to investigate its stability in relation to adolescent idiopathic scoliosis. Idiopathic scoliosis is one of the most puzzling deformities of the spine, due to the fact that there is no known initiating cause. From the viewpoint that it can be explained in a purely biomechanical manner, one particular hypothesis as to its etiology is investigated in this thesis. The hypothesis is that a lordosis-inducing growth of the thoracic spine [34,100,115] in conjunction with spinal asymmetries in the lateral or horizontal plane [34] is the primary cause of the deformity. / Analyses are performed on the constructed model using the MSC/NASTRAN finite element program. The model consists primarily of interconnected beam elements to represent a realistic geometry of the spine and rib cage. The various stiffness properties needed in the model were obtained from the published literature. Simulations of analyses and experiments performed by other researchers produced comparable results, thereby validating the present model, which is then used to investigate the above hypothesis. / Lordosis-inducing growth, in which the anteriors of the thoracic vertebrae grow faster than the posteriors, is simulated in a geometric nonlinear analysis by differential thermal loading of these parts. Results show that under such loading, the model of the normal spine with its natural asymmetries of the thoracic region, gradually deforms into a shape with displacements and rotations typical of thoracic idiopathic scoliosis. These results therefore constitute a validation of the stated hypothesis, and indicate that a lordosis-inducing growth of the thoracic vertebrae is a likely cause of thoracic idiopathic scoliosis.

We review the salient clinical and laboratory features of cyclical neutropenia (CN) and other periodic hematological disorders, and the insight into these diseases afforded by mathematical modeling. Using Lomb periodogram analysis, we show that occurrence of significant cycling in the serial blood counts of neutropenic patients is very prevalent and the dynamics of all the cell lineages in CN can be modified by the administration of recombinant granulocyte colony stimulating factor (G-CSF). The analysis of the serial blood counts in the animal model of CN---the grey collie (GC), reveals a complex pattern of oscillations that we could reproduce with a model of hematopoiesis combining a peripheral control of granulopoiesis through G-CSF, together with an oscillatory input from the pluripotential stem cell. Both the human and GC data analysis suggests that CN results from a complex interaction between the stem cells, the mature cells and G-CSF, and that the regulation of the different blood cell lineages are strongly linked together.

Breast cancer is the second most frequent cancer in middle-aged women. Control of this disease requires identification of the various factors involved in the invasive-metastatic cascade, facilitating the subsequent development of relevant blocking compounds. / Tumor cell invasion is a complex sequence of events where cell adhesion molecules exert determinant roles. CD44 is a family of cell adhesion glycoproteins generated by alternative splicing of up to ten variant exons. Discrete CD44 isoforms are overexpressed in different human cancers, including breast cancer. CD44 is expressed on the plasma membrane of cells and binds mainly to hyaluronan. Hyaluronan is a negatively charged high-molecular-weight glycosaminoglycan conspicuously present in the extracellular matrix ant its concentration is increased at sites of tissue remodeling. / This study tested the hypothesis that CD44 and its main ligand, hyaluronan participate in the invasive properties of breast cancer cells. The hypothesis is based on the following rationale: the previously documented upregulation of CD44 expression in human breast cancer tumors, the role of CD44 in the invasive properties of nonepithelial cells in vitro, and the high concentrations of hyaluronan present in human breast cancer tumors. / The experiments were performed with different human breast cancer cell lines. In one cell line, CD44 and its v6 variant isoform participate directly in tumor cell invasion in vitro. Moreover, hyaluronan alters cell behavior in vitro, mainly by changing CD44 expression, adhesion and motility of breast cancer cells with high CD44 expression. In addition, it is shown that intratumoral injection of hyaluronan reduces the volume of tumors produced by orthotopically xenotransplanted breast cancer cells. / The contribution of these studies to the advance of knowledge in breast cancer is based on a role of CD44 and hyaluronan in the interactions between tumor cells and the extracellular matrix, offering an opportunity to develop a new therapeutic strategy.

The Wilms' tumor suppressor gene 1 (wt1) encodes a transcription factor belonging to the Early Growth Response (EGR) family of proteins. The gene produces an mRNA that is alternatively spliced to yield four different species. In this thesis, it is shown that the wt1 gene has two sites of translational initiation, an AUG codon and a CUG codon that is upstream of and in frame with the AUG codon. The combination of alternative initiation sites and alternatively spliced exons allows the gene to encode eight different isoforms. It was previously shown that heterozygous mutations of wt1, predicted to result in a reduction in dosage of WT1, are associated with a predisposition to Wilms' tumor and mild abnormalities of the genitourinary system. However, other mutations of wt1, primarily missense mutations predicted to disrupt the ability of WT1 to bind to DNA, are associated with the severe disease of Denys-Drash syndrome (DDS). To explain the different disease states associated with mutations of wt1, it has been suggested that the mutations in DDS individuals must be acting other than as null alleles, i.e. in a dominant or dominant-negative fashion. In this thesis, an individual with DDS is reported to have a splicing mutation of wt1, predicted to drastically alter the ratio of WT1 isoforms. Because no altered forms of WT1 are produced in this individual, we were able to eliminate the possibility that DDS is caused by a gain of function of mutated WT1. In this thesis it is also shown that WT1 can form dimers through a domain encoded by its first two exons, and that mutated WT1 proteins are able to antagonize the function of wild-type WT1 by binding to and inactivating the wild-type proteins. This dominant-negative activity is hypothesized to reduce the dosage of functional WT1 to very low levels and to thus cause the severe disease of DDS. The work reported in this thesis also shows that WT1 is imported into the nucleus by a signal contained within its first zinc finger

No description available.

Three known mammalian endothelins, ET-1, ET-2, and ET-3, are each encoded by separate genes and expressed in a variety of vascular and nonvascular tissues. Two subtypes of endothelin receptors have been identified and termed endothelin-A and endothelin-B receptors (ET-A and ET-B). In the first part of this study, the ET-A gene was disrupted in mouse embryonic stem cells to generate mice deficient in ET-A. / In the second part of this study, we investigated a targeted disruption of the mouse ET-B gene that results in aganglionic megacolon associated with coat color spotting, resembling a hereditary syndrome of mice, humans and other mammalian species (Waardenburg syndrome). / In the third part of this study, we demonstrated that a targeted disruption of the mouse endothelin-3 ligand (ET-3) gene produces a similar recessive phenotype of megacolon and coat color spotting. / Two types of endothelin converting enzymes (ECE-1 and ECE-2) have been recently identified. In the last part of this study, we investigated the expression of ECE-1 in human tissues using immunohistochemistry and in situ hybridization, and compared it to those of ET-1 and big ET-1. (Abstract shortened by UMI.)