Slug expression inhibits calcitriol-mediated sensitivity to radiation in colorectal cancer.

Recently, a reciprocal relationship between calcitriol and epithelial-to-mesenchymal transition has been described. Therefore, we hypothesized that calcitriol (1α,25-dihydroxyvitamin D3) would enhance radiation sensitivity in colorectal cancer regulated by epithelial mesenchymal transition. Vitamin-D receptor, E-cadherin and vimentin protein as well as E-cadherin, Snail and Slug mRNA levels were assessed in a panel of human colorectal cancer cell lines at baseline and in response calcitriol. We defined cell lines as calcitriol sensitive based on demonstrating an enhanced epithelial phenotype with increased E-cadherin, reduced vimentin and decreased expression of Snail and Slug as well as decreased cellular migration in response to calcitriol. In calcitriol sensitive cells, including DLD-1 and HCT116, 24 h calcitriol pre-treatment enhanced the radiation sensitivity by 2.3- and 2.6-fold, respectively, at 4 Gy (P < 0.05). In contrast, SW620 cells with high baseline mesenchymal features including high Slug and vimentin expression with low E-cadherin expression demonstrated no significant radiation sensitizing response to calcitriol treatment. Similarly, transfection of Slug in the calcitriol sensitive colon cancer cell lines, DLD-1 and HCT 116, completely inhibited the radiation sensitizing effect of calcitriol. Collectively, we demonstrate that calcitriol can enhance the therapeutic effects of radiation in colon cancer cells and Slug expression mitigates this observed effect potentially representing an effective biomarker for calcitriol therapy.

Transponder-induced sarcoma in the heterozygous p53+/- mouse.

Heterozygous p53+/- transgenic mice are being studied for utility as a short-term alternative model to the 2-yr rodent carcinogenicity bioassay. During a 26-wk study to assess the potential carcinogenicity of oxymetholone using p-cresidine as a positive control, glass/polypropylene microchips (radio transponder identification devices) were subcutaneously implanted into male and female p53+/- mice. During week 15, the first palpable mass was clinically observed at an implant site. This rapidly growing mass virtually quadrupled in size by week 25. Microscopic examination of all implant sites revealed that 18 of 177 animals had a subcutaneous histologically malignant sarcoma. The neoplasms were characterized as undifferentiated sarcomas unrelated to drug treatment, as indicated by the relatively even distribution among dose groups, including controls. An unusual preneoplastic mesenchymal change characterized by the term "mesenchymal dysplasia" was present in most groups and was considered to be a prodromal change to sarcoma development. The tumors were observed to arise from dysplastic mesenchymal tissue that developed within the tissue capsule surrounding the transponder. The preneoplastic changes, including mesenchymal dysplasia, appeared to arise at the transponder's plastic anchoring barb and then progressed as a neoplasm to eventually surround the entire microchip. Capsule membrane endothelialization, inflammation, mesenchymal basophilia and dysplasia, and sarcoma were considered unequivocal preneoplastic/neoplastic responses to the transponder and were not related to treatment with either oxymetholone or p-cresidine.

Dynamic expression of L-selectin in cell-to-cell interactions between neutrophils and endothelial cells in vitro.

Neutrophil-endothelial cell interactions are regulated by cell adhesion molecules and their cognate ligands. It has been proposed that L-selectin and Mac-1 (CD11b/CD18), two neutrophil adhesion receptors, have sequential roles in neutrophil extravasation during inflammation. In this model, L-selectin mediates rolling and initial adherence of neutrophils to endothelial cells, while Mac-1 strengthens this initial adherence and also facilitates migration of neutrophils through endothelial cells. L-selectin and Mac-1 expression are known to be inversely regulated. Here an in vitro culture system has been developed to investigate in situ expression of L-selectin during cell-to-cell interactions between neutrophils and endothelial cell monolayers by confocal immunofluorescence analysis. Neutrophils underwent profound cell shape change from round to polarized cell morphology with pseudopod formation after 5 to 15 min coculture with IL-1-stimulated human endothelial cells. L-selectin was redistributed to the pseudopod of the polarized neutrophils in correlation with such cellular changes. During initial cell attachment, neutrophils bound to IL-1-stimulated endothelial cells expressed a high level of L-selectin in a polarized pattern. L-selectin expression decreased over time during neutrophil-endothelial cell interactions.

Mechanisms of inflammatory cell attachment in chronic relapsing experimental allergic encephalomyelitis: a scanning and high-voltage electron microscopic study of the injured mouse blood-brain barrier.

Brain and spinal cord blood vessels from mice subjected to chronic relapsing experimental allergic encephalomyelitis were examined by scanning (SEM) and high-voltage electron microscopy (HVEM). SEM analysis of veins and venules from affected tissue regions demonstrated inflammatory cells (ICs), primarily lymphocytes or monocytes, attached to the luminal endothelial cell (EC) surface adjacent to the junctional complexes. In transverse section these cells were shown by HVEM to extend and to insert filopodia (lymphocytes) or flap-like lamellapodia (monocytes) into the luminal EC surfaces. Affected ECs often expressed increased microvillar projection as well as parajunctional crater-like structures on their luminal surfaces. Based on scanning and high-voltage electron microscopy, we present morphological evidence that some populations of sensitized ICs do not penetrate the EC junctions initially during EC attachment but instead insert pseudopodial projections into specialized openings in the ECs that are formed in response to chronic inflammation.

Laminar analysis of the number of neurons, astrocytes, oligodendrocytes and microglia in the visual cortex (area 17) of 3-month-old rhesus monkeys fed a human infant soy-protein formula with or without taurine supplementation from birth.

The effect of taurine addition to a commercial (taurine-free) soy-protein formula on the development of the visual cortex has been examined in 3-month-old rhesus monkeys. The thickness, number of neurons, astrocytes, oligodendrocytes and microglia have been measured in the different laminae. There were no significant differences in cortical thickness, total number of neurons, astrocytes, oligodendrocytes, or microglia between the groups, but there were some significant differences in some laminae as well as some qualitative differences. The numerical density of neurons in layers IV-C alpha and V-2 was significantly greater in the taurine-supplemented monkeys than in those fed the taurine-free formula; the numerical density of astrocytes in layer IV-A was significantly greater and in layer IV-C alpha was significantly smaller in the taurine-supplemented monkeys than in those fed the taurine-free formula. A number of other measurements in the two groups approached significance. These differences indicate that there are developmental disadvantages in the visual cortex of rhesus monkeys raised on a taurine-free human infant formula in addition to those previously reported.

Combined conventional transmission, scanning, and high-voltage electron microscopy of the same blood vessel for the study of targeted inflammatory cells in blood-brain barrier inflammation.

The microvasculature of brains and spinal cords from mice subjected to chronic relapsing experimental autoimmune encephalomyelitis (CREAE) was studied using three different electron microscopic techniques. Blood vessels were initially examined by scanning electron microscopy. This allowed for the investigation of topographical changes of the luminal aspects of endothelial cells (ECs) and identification of targeted inflammatory cells (ICs) attached to the ECs. The same blood vessel areas with attached ICs examined by scanning electron microscopy were subsequently trimmed, processed for routine conventional transmission electron microscopy, and plastic embedded. Thin (80 nm) sections were cut and evaluated. Semithick (0.5-0.75 microns) serial sections of this material were examined by high-voltage electron microscopy. Data presented here described a useful technique for combining several ultrastructural techniques that permits simultaneous topographic and cross-sectional examination of selected regions of individual blood vessels or specifically targeted ICs.

Microanalysis of Alzheimer disease NFT and plaques.

Electron probe energy dispersive microanalysis of isolated andin situ neurofibrillary tangles (NFT) and neuritic (senile) plaque cores have been done to investigate the levels of Al, Si, Ca and Fe in the leading lesions of Alzheimer disease neuropathology. Varying levels of Si and Al, and to a lesser extent Ca, have been co-localized in about one half of the NFT and plaques examined using X-ray mapping. The variability of detection and the low levels of Al present indicates that aluminum is not required for the formation of the NFT and that aluminosilicates are not involved in the formation of the plaque core.

Aluminum neurotoxicity in mammals.

Although aluminum comprises a large percentage of the Earth's crust, it is excluded from body tissues, and especially from the central nervous system. When aluminum is experimentally introduced to the central nervous system, several neurotoxic effects are observed:i.e. neurofibrillary changes, behavioral and cognitive deficits and enzymatic and neurotransmitter changes, as well as certain types of epileptic seizures.The localization of relatively high levels of aluminum in Alzheimer disease, Guamanian amyotrophic lateral sclerosis and Parkinsonism-dementia has led to the implication of aluminum as a pathogenic factor in these diseases. Recent studies have shown that microtubule-associated proteins are part of the paired helical filaments which make up the intraneuronal neurofibrillary tangle. Other studies have identified the protein making the vascular and neuritic (senile) plaque amyloid and located the gene responsible for this protein to chromosome 21.Our electron microprobe analysis studies have not found the levels of aluminum or silicon in either the neurofibrillary tangles or amyloid cores reported elsewhere, nor have the levels of aluminum been elevated in approximately one half of the tangles and plaque cores examined to date.

Sites of egress of inflammatory cells and horseradish peroxidase transport across the blood-brain barrier in a murine model of chronic relapsing experimental allergic encephalomyelitis.

Results are reported of experiments designed to focus at attachment sites of inflammatory cells (ICs) on the luminal surface of brain endothelial cells (ECs) and on the mechanisms of horseradish peroxidase (HRP) transport across the altered blood-brain barrier (BBB) in a murine model of chronic relapsing experimental allergic encephalomyelitis. Cationized ferritin (CF) served as a marker for evaluating the electrostatic nature of brain microblood vessels (MBVs) on the plasma membranes of ICs or normal mouse peripheral white blood cells and erythrocytes. SJL/J mice demonstrating clinical illness were given HRP or CF, in vivo or in situ, respectively. Light microscopy and conventional transmission electron microscopy of cerebellum or thoracic and lumbar spinal cord regions demonstrated HRP leakage most pronounced in MBVs with perivascular infiltrates. HRP traversed across the ECs via numerous vesicles and tubular profiles located mostly in the parajunctional regions, while EC junctions appeared closed. Scanning electron microscopy demonstrated that IC attachment was primarily at parajunctional sites on the EC surface. We also observed increased microvillar projections extending from the EC surface into the lumen. CF demonstrated a patchy decoration on both the luminal EC surface and IC membranes but did not label uncoated invaginating membrane pits or tubular structures. Our data indicate that the points of attachment of the ICs on the EC surface may reflect specific receptor sites where the ICs eventually gain entrance into CNS across the BBB during brain inflammation.

Genetic control of scrapie: incubation period and plaque formation in I mice.

The host component of control of scrapie incubation period in the mouse is manifested largely through the action of the Sinc gene. Only one mouse strain (VM) has been found that is p7p7 (prolonged incubation for ME7 agent) and two other strains have been derived from VM. All other strains, designated s7s7, have a short incubation for ME7. In the present study, the I strain was shown to fulfil the criteria that are characteristic of mouse strains with the p7 allele of Sinc: a comparatively long incubation period for ME7 and a short incubation period for 22A, the incubation period for F1 hybrid mice (s7s7 X p7p7) either fell between the incubation periods for the parental strains (with ME7) or were longer than either parent (with 139A and 22A), amyloid plaques occurred following injection of ME7 and 87V but not after 22A or 139A, lesion profiles for four scrapie strains were similar in I mice and p7p7 mouse strains, and injection of 87V led to disease in less than 300 days. Finally, allelism tests using F1 hybrid mice (I X a p7p7 mouse strain) and progeny of backcrosses between these F1 mice and I mice failed to reveal the segregation of additional major genes affecting scrapie incubation period.

Ultrastructural observations of spinal cord lesions and blood-brain barrier changes in scrapie-infected mice.

Spinal cord samples from IM or VM mice injected intracerebrally with the 87V scrapie agent were examined ultrastructurally at the clinical stage of disease for changes in blood vessel permeability and for pathological alterations. In several animals, (3 of 16), massive changes were noted in the cervical spinal cords in the subependymal area of the cortical gray matter immediately surrounding the central canal including ependymal cell changes, the presence of amyloid plaque in close association with microglial cells, extensive neuropil vacuolation, the appearance of reactive astrocytes, degenerating neurites and vacuolated neurons. In those regions showing structural damage, localized increased permeability to horseradish peroxidase across the blood-brain barrier was noticed along with the appearance of numerous vesiculo-canalicular profiles in micro-blood vessel endothelial cells with extravasation of the tracer to the neuropil. Some damaged neurons appeared flooded with this tracer. These changes were not observed in either the thoracic or lumbar spinal cord regions. The occurrence of pathological changes in the spinal cords of a small percentage of intracerebrally injected mice was probably due to a high concentration of the scrapie agent which localized in the cervical spinal cord, presumably after entering the spinal fluid via the lateral ventricle at the time of injection.

Retinal degeneration in 3-month-old rhesus monkey infants fed a taurine-free human infant formula.

Rhesus monkey infants were raised from birth on a taurine-free soy protein-based human infant formula or on the same formula supplemented with taurine. The monkeys were killed 3 months after birth and the retinas examined by light and electron microscopy. All of the monkeys raised on formula alone showed degenerative ultrastructural changes in photoreceptor outer segments that ranged from swelling and disorientation to fragmentation and disorganization. Cones were more severely affected than rods, and changes were most pronounced in the foveal region. Changes were also noted in the fine structure of the retinal pigment epithelium. These changes were prevented in all but one monkey fed the same formula supplemented with taurine. These results provide further support for the addition of taurine to commercial human infant formulas.

Feline maternal taurine deficiency: effects on retina and tapetum of the offspring.

The retina and tapetum of kittens born to taurine-deficient and taurine-supplemented mothers were compared. Retinal taurine concentrations typically reach adult levels 6 weeks postnatally. When measured at weaning at 8 postnatal weeks, the taurine concentrations in retina and tapetum of taurine-deficient kittens were 40% of normal levels. An ultrastructural correlate found in the retinas of taurine-deficient kittens was the presence of photoreceptor outer segments that were reduced in length and altered from the typical columnar configuration. Tapetal cells of taurine-deficient kittens were distinguished by accumulations of electron-dense droplets, the presence of tapetal rods with dilated limiting membranes and the presence of amorphous vesicles.

Abnormal visual cortex development in the kitten associated with maternal dietary taurine deprivation.

We have examined the visual cortex of newborn and 8-week-old kittens born to mothers consuming a taurine-supplemented or taurine-free diet using the rapid Golgi technique. Kittens from taurine-supplemented mothers exhibited normal development of the visual cortex. Kittens from taurine-depleted mothers showed striking differences. In newborn kittens, neuroblasts are aggregated both at the ventricular and pial zones, having failed to migrate and differentiate normally. Eight weeks after birth, only few pyramidal and nonpyramidal neurons are found. Those present have heavily spined dendritic processes indicative of poor arborization. Protoplasmic astrocytes are represented by undifferentiated cellular masses. The taurine concentration in the cortex of such kittens is four- to fivefold smaller than in kittens from taurine-supplemented mothers. These results suggest that normal concentrations of taurine in visual cortex, as well as cerebellum, are required for normal ontogeny of neurons. Once such deficits are established in the prenatal and immediate postnatal period, they result in permanent abnormalities. These findings have clear implications for vegetarian women who intend to have children, since virtually no taurine is present in plants and vegetables.

Postnatal taurine deficiency in the kitten results in a persistence of the cerebellar external granule cell layer: correction by taurine feeding.

Dietary deprivation of taurine in pregnant cats from approximately 1 week prior to giving birth is sufficient to reduce substantially the taurine concentration in feline milk but does not result in any abnormalities in kittens at birth. Kittens nursing on this low taurine milk have a lower growth rate than normal, have lower tissue taurine concentrations, and 8 weeks after birth have a persistence of cells in the cerebellar external granule cell layer. Mitotic figures are present also, indicating that cell division is occurring still, an event which normally is completed 3-4 weeks after birth. Daily oral supplementation with 40 mumoles taurine increases the growth rate almost to the level of normally nurtured kittens and results in normal tissue taurine concentrations and apparently normal migration of cells in the cerebellum. These findings indicate that nutritional taurine supplied in the milk is involved in the normal ontogeny of the cerebellum and that a taurine deficiency at this stage of development results in a maturational delay.

Taurine deficiency in the developing cat: persistence of the cerebellar external granule cell layer.

Dietary taurine deprivation adversely affects feline pregnancy and is associated with the frequent occurrence of fetal resorption, abortion, stillbirth, and low birthweight of live kittens at term. Taurine-deprived, live-born kittens have a poor postnatal survival rate and grow less well than kittens from taurine-supplemented queens. The postnatal dietary taurine intake of such kittens is reduced if they are nursed by their biologic mothers; the concentration of taurine in milk of taurine-deprived mothers is less than 10% of that in milk from taurine-supplemented queens. Surviving kittens from taurine-deprived mothers exhibit a constellation of neurological abnormalities (abnormal hind leg development, a peculiar gait characterized by excessive abduction and paresis, and thoracic kyphosis readily visible by X-ray). These findings suggest the presence of a developmental cerebellar deficit. Histological examination of the pre- and postnatally taurine-deprived kitten's cerebellum reveals a persistence of the external granule cell layer, which was confirmed by electron-microscopic examination. Numerous mitotic figures are present in the cells in the external granule cell layer of the cerebellum of kittens born from the nursed by taurine-deprived queens, but not in those from taurine-supplemented mothers. These findings suggest a maturational delay.