In vitro studies will contribute significantly to an understanding of cell growth and differentiation in the adult pancreas. This thesis reports on the changes occurring when acinar cells, terminal ducts and islets are grown in culture. / A number of methods for adult pancreatic duct epithelial isolation and culture from different species have been reported. However, there are no reliable methods for the large scale isolation and culture of the terminal segments of the pancreatic duct system. Acinar fragments of the hamster pancreas are isolated by partial digestion with collagenase. Culture is achieved by embedding in a matrix of rat-tail collagen in DME:F12 medium and 10% NuSerum supplemented with epidermal growth factor (EGF) and cholera toxin (CT). The results show that ductal cysts arise within the areas of degenerated acinar tissue. The method developed in the preparation of this thesis gave a high yield of ductal cysts. Autoradiography indicates the duct cysts could have originated from either progenitor cells of ducts or those of acini. In addition, the question of whether these duct epithelial cysts originate from phenotypic transformation of acinar cells needs to be further evaluated. / Acinar fragments isolated from the hamster pancreas were embedded in type 1 collagen and grown in various media. After 34 days in culture, up to 80% of the acinar cells can be shown to be normal, while the remainder were severely degenerated. This occurred in media to which EGF and CT had not been added. When the latter were added, cystic structures developed. However, a few of the cells within the cystic wall still showed amylase positive immuno-reactivity. Cellular amylase activity decreased over time but a more rapid decline was shown in cells cultured with Media containing EGF and CT. It was also found that the duct-like cells had a limited capacity to redifferentiate into acinar cells. This study suggests that ductal-like epithelial structures may arise from transformation of acinar cells and/or proliferation of ductal cells. / Purified islets from human pancreas were placed into collagen gel matrix and cultured in DME:F12 medium and 10% NuSerum supplemented with EGF and CT. The results showed that the cultured islets underwent a cystic transformation that was associated with (i) a progressive loss of insulin gene expression, (ii) a loss of immunoreactivity for insulin protein, and (iii) the appearance of CK-19, a marker for ductal cells. After the transformation was complete, the cells had the ultrastructural appearance of primitive duct-like cells. Cysts showed a progressive enlargement with cell replication, as reflected in a 1500% increase in the incorporation of tritiated thymidine. These results are consistent with the transdifferentiation of islet cells to ductal cells.

Amyotrophic lateral sclerosis (ALS) is characterized by the progressive loss of upper and lower motor neurons. A subset of familial ALS cases (20%; FALS-1) are linked to mutations in the Cu/Zn-superoxide dismutase (SOD-1). In order to study the mechanisms of mutant SOD-1 (mSOD-1) toxicity and preferential vulnerability of motor neurons, human wild type and seven different mSOD-1 were expressed in motor neurons or dorsal root ganglion neurons of dissociated cultures of murine spinal cord-dorsal root ganglia, or in cultured hippocampal neurons. Expression was obtained by intranuclear microinjection of plasmid expression vectors. Many features of the motor neuron disease observed in humans with ALS and in mSOD-1 transgenic mice were reproduced in this new culture model, including preferential vulnerability of motor neurons and abnormal distribution of hyperphosphorylated neurofilament proteins. A novel observation was the formation of cytoplasmic SOD-1 aggregates in motor neurons expressing mSOD-1 followed by evidence of apoptotic cell death. Both formation of aggregates and motoneuronal death were reduced by coexpression of the stress-inducible heat shock protein, HSP70, indicating that insufficiency of molecular chaperones may be involved in FALS-1. Using specific antagonists of glutamate receptor subtypes, we have demonstrated that normally nontoxic glutamatergic input, particularly via calcium-permeable AMPA/kainate receptors, is a major factor in the vulnerability of motor neurons to the toxicity of mSOD-1. Partial protection was obtained by blockade of L-type voltage-gated calcium channels, implicating Ca2+ entry through these channels in the toxicity of mSOD-1. Dramatic neuroprotection was obtained by coexpressing the calcium-binding protein calbindin-D28k indicating that the toxicity of mSOD-1 is calcium-dependent. However, increasing intracellular glutathione levels or treatment with N-tert-butyl-alpha-phenylnitrone (PBN) had no significant effect on motone

The neuropathogenicity of two retroviruses was investigated. The human immunodeficiency virus, in addition to its profound effect on the immune system, also causes degenerative changes in the brain, the spinal cord and peripheral nerves. In order to elucidate how it affects the nervous system, transgenic mice were generated that express the entire HIV genome in neurons in the anterior thalamus and in the anterior horn of the spinal cord, and examined clinically, neuropsychologically, electrophysiologically and histologically. Animals developed a neurological syndrome characterized by hypoactivity and weakness, and by axonal degeneration in peripheral nerves. These results provide evidence for a role of HIV in affecting both the central and peripheral nervous systems. / In a second project, pathological effects associated with a disease determining region contained in the gp70 envelope protein of the Cas-Br-E murine leukemia virus, were investigated. In infected mice, this virus causes hind limb paralysis and a spongiform myeloencephalopathy with gliosis and neuronal loss. Stably transfected fibroblasts that express gp70 were injected into the brains of mice, and the animals were examined for histopathological changes attributable to the effects of gp70. While gp70 protein was detected at the implantation site, this was not accompanied by any specific histological changes. These data suggest that the intracerebral expression of the neuropathogenic gp70 protein alone is not sufficient to cause disease, and lend indirect support to a model according to which gp70 causes disease by altering the cytokine profile of infected mononuclear cells in the central nervous system.

Tuberculosis, caused by Mycobacterium tuberculosis, is globally a leading cause of morbidity and mortality. While a host genetic contribution to tuberculosis susceptibility is known to occur, the extent and nature of host genetic variability to tuberculosis pathogenesis are unknown. Thus, to better understand the role of host genetic factors in tuberculosis susceptibility, we have tested the contribution of candidate gene variants to tuberculosis susceptibility in three geographically, epidemically, and clinically distinct populations. We chose four candidate genes involved in the innate immune response, namely, NRAMP1, MBL, SFTPA1, and SFTPA2. / Since the global spread of tuberculosis is highly dependent on HIV/AIDS pandemic, we investigated the association of genetic MBL variants and HIV-1 infection in 278 Colombian HIV-infected and control individuals. MBL genotype frequencies were similar for both groups, and no association was detected between MBL alleles B, C, and D and susceptibility to HIV-1 infection (P = 1.0). These results do not support the hypothesis that MBL levels are a risk factor for HIV-1 infection in Colombia. Moreover, we were able to show that MBL variants do not contribute to tuberculosis susceptibility in this population. / In a pediatric population composed of 184 families we found allelic variants in the NRAMP1 gene to be associated with tuberculosis disease (P = 0.01; Odds ratio [OR] = 1.75 [95% confidence interval: 1.10--2.77]). Common NRAMP1 alleles were identified as risk factors for pediatric tuberculosis while these same alleles were reported to be protective in adult cases, suggesting that the common alleles promote rapid progression from infection to tuberculosis disease. Furthermore, the association of NRAMP1 with pediatric tuberculosis disease was significantly heterogeneous (P = 0.01) between simplex (P < 0.0008; OR = 3.13 [1.54--6.25]) and multiplex families (P = 1) suggesting an interplay between mechanisms of genetic control and exposure intensities. Finally, we tested the correlation between the NRAMP1 risk and NRAMP1 functional activity by measuring the recruitment efficiency of mannose-6-phosphate receptor (M6PR) to Salmonella containing vacuoles (SCV) in monocyte-derived-macrophages (MDM). We show that recruitment of M6PR to SCV is significantly lower ( P = 0.024) in MDM from patients homozygous for the risk allele as compared to MDM from heterozygous patients. Thus, altered function of NRAMP1 appears to modulate the rate of progression from infection to disease.

The apolipoprotein E (apoE) ɛ4 is a risk factor for AD. The mechanism by which apoE ɛ4 confers such a susceptibility to AD is unknown. We explored whether low levels of apoE may mediate the processes leading to neurodegeneration and cognitive decline in AD by using apoE knockout mice (apoEKO). The complementary hypothesis was also tested to determine whether apoE could be used as a therapeutic target in the treatment of AD. / We examined the effect of apoE deficiency on the reparative capacity and glial response following entorhinal cortex lesion (study 1), the effect of age and apoE deficiency on the integrity of the circuitry underlying hippocampal-dependent cognitive functioning in apoEKO mice (3 and 12 month-old) (study 2). Given that enhanced sensitivity to stress and/or anxiety can interfere with cognitive functioning, we assessed behavioral anxiety in apoEKO mice (12 month-old) using a battery of tests with varying degree of aversiveness and novelty (study 3). The forth study assessed the effect of pharmacological induction of apoE on alterations typically associated with age such as synaptic loss and glial reactivity in aged rats (26 month old) using Probucol. / The results revealed that apoE deficiency is associated with an altered pattern of glial reactivity that coincided with impaired sprouting responses, and with behavioral impairments (hippocampal-dependent procedural deficit and enhanced susceptibility to stress and anxiety) that mimic some aspects of the clinical phenotype of AD patients. Furthermore, chronic Probucol treatment in rats led to significant increases in apoE and its receptor the low density lipoprotein related protein receptor (LRP). In parallel, were increases in the synaptic marker SNAP-25 along with a drastic attenuation of the age-related astroglial reactivity. / The findings show that apoE plays an important role in the repair processes and its induction by Probucol could promote synaptic plasticity to counteract the synaptic deterioration and glial activation associated with age or damage. These findings are consistent with the hypothesis that part of the risk of developing AD may be related to low levels of apoE, and suggest a novel therapy for the treatment of AD based on apoE neurobiology.

Abstract not available.

Abstract not available.

Rundown of ionic gradients is a central feature of white matter anoxic injury. However little is known about the contribution of anions such as Cl -. Previous studies have shown that Na+-blockade during anoxia was in adequate in preserving K+-loss. We hypothesized that run down of the K+-gradient during anoxia/Na+-channel inhibition proceeds in conjunction with an anion and more specifically Cl -. We used the in vitro rat optic nerve to study the role of aberrant Cl- transport in anoxia/ischemia. After 30 min of anoxia (NaN3 2mM), axonal membrane potential (V m) decreased to 42 +/- 11% of control, and to 73 +/- 11% in the presence of TTX (1 muM). TTX + DIDS 500muM (a broad spectrum anion transport blocker) abolished anoxic depolarization (95 +/- 8%). Inhibition of the K-Cl cotransporter (KCC) (furosemide 100muM) together with TTX was also more effective than TTX alone (84 +/- 14%). Compound action potential (CAP) area recovered to 26 +/- 6% of control after 1 h anoxia. KCC blockade (furosemide 10muM) improved outcome (40 +/- 4%) and TTX (100nM) was even more effective (74 +/- 12%). In contrast, the Cl- channel blocker niflumic acid (50 muM) worsened injury (6 +/- 1%). Co-application of TTX (100 nM) + furosemide (10 muM) was more effective than either agent alone (91 +/- 9%). Furosemide was also very effective at normalizing the shape of the CAPs. The KCC3a isoform was localized to astrocytes. KCC3 and weaker KCC3a was detected in myelin of larger axons. KCC2 was seen in oligodendrocytes and within axon cylinders. Cl- gradients contribute to resting optic nerve membrane potential, and transporter and channel-mediated Cl- fluxes of during anoxia contribute to injury, possibly due cellular volume changes and disruption of axo-glial integrity, leading to propagation failure and distortion of fiber conduction velocities.

No description available.

No description available.