A rapid and sensitive method for detecting human cells in xenogeneic hosts.

Xenografts, specifically transplantation of human cells into other species, are a valuable tool in preclinical transplantation experiments. A central issue is accurate identification of the grafted cells, particularly in cases in which cellular migration has occurred. We report that detection of grafted human cells can be achieved by in situ hybridization techniques using human centromeric probes which result in unambiguous nuclear labeling. The resulting reaction can be combined with immunocytochemical or histochemical techniques for cell-type characterization.

Transplant therapy: recovery of function after spinal cord injury.

Spinal cord injuries (SCI) result in devastating loss of function and altered sensation. Presently, victims of SCI have few remedies for the loss of motor function and the altered sensation often experienced subsequent to the injury. A goal in SCI research is to improve function in both acute and chronic injuries. Among the most successful interventions is the utilization of transplanted tissues toward improved recovery. The theory is that the transplanted tissue could (1) bridge the spinal lesion and provide chemical and/or mechanical guidance for host neurons to grow across the lesion, (2) bridge the spinal lesion and provide additional cellular elements to repair the damaged circuitry, (3) provide factors that would rescue neurons that would otherwise die and/or modulate neural circuits to improve function. A variety of tissues and cells have been added to the adult mammalian spinal cord to encourage restoration of function. These include Schwann cells, motor neurons, dorsal root ganglia, adrenal tissue, hybridomas, peripheral nerves, and fetal spinal cord (FSC) tissue en bloc or as disassociated cells. It is postulated that these tissues would rescue or replace injured adult neurons, which would then integrate or promote the regeneration of the spinal cord circuitry and restore function. In some instances, host-appropriate circuitry is supplied by the transplant and functional improvement is demonstrated. In this presentation, specific examples of recent work with transplanted tissue and cells that demonstrate improved behavioral outcome are presented. New recent work describing the in vitro propagation and characterization of human fetal spinal cord multipotential progenitor cells are also described in the context of a potential resource for transplantable cells. Additionally, data from transplantation experiments of human FSC cells into nonimmunosuppressed rat spinal cord are described, and the resultant improvements in behavioral outcome reported. Lastly, directions for future SCI research are proposed.

Basic fibroblast growth factor (bFGF) increases the survival of embryonic and postnatal basal forebrain cholinergic neurons in primary culture.

Basic fibroblast growth factor (bFGF) is found in high concentrations in the mammalian central nervous system. It is a mitogen for glia and it influences the development and survival of specific populations of neurons. In this study, we investigated the effect of various concentrations of bFGF on the survival of embryonic and postnatal cholinergic basal forebrain neurons plated at low and high density in the presence and absence of glia. We observed that 50 and 100 ng/ml of bFGF increased the survival of embryonic cholinergic neurons plated at high density. This effect was observed only in the presence of glia. Lower concentrations of 10 and 20 ng/ml had no effect on cholinergic neuronal survival. The number of GFAP (glial fibrillary acidic protein)-positive cells in high-density embryonic cultures was increased by all concentrations of bFGF. In low-density embryonic cultures, an increase in cholinergic neuron survival was observed at concentrations ranging from 20 to 100 ng/ml. The number of GFAP-positive cells in low-density cultures was also increased by all concentrations of bFGF. Similar to low-density embryonic cultures, the survival of cholinergic neurons from postnatal day 2 cultures was significantly increased in the presence of glia at concentrations of 20, 50 and 100 ng/ml of bFGF. Postnatal glia was affected by all concentrations of bFGF, as was observed in embryonic cultures. This study indicates that high concentrations of bFGF can influence cholinergic neuronal survival by stimulating and increasing glia, which may produce factor(s) that are necessary for cholinergic neuron survival.

D-xylose absorption as a measurement of malabsorption in poult enteritis and mortality syndrome.

Severe wasting of body tissues, diarrhea, high morbidity and mortality, and stunting are all characteristics of poult enteritis and mortality syndrome (PEMS). The wasting of musculature and loss of nearly all adipose tissue suggested that even though the PEMS-infected poults were eating some feed, nutrient intake was not sufficient to meet body requirements for maintenance and growth. Because epithelial cells in the gastrointestinal tract appeared to be a target of the undefined etiological agent (or agents) that causes PEMS, a study was conducted in which PEMS-infected poults were evaluated for malabsorption through 3 wk of age. D-Xylose, a poorly metabolized pentose, was given per os as a bolus, and blood samples were obtained from the ulnar vein in the wing of control and PEMS-infected poults over a 3-h period to estimate intestinal absorption. D-Xylose absorption in control poults peaked 30 to 60 min after the oral treatment, similar to results reported earlier. The PEMS-infected poults did not show a peak in absorption. The PEMS-infected poults showed significant delays in D-xylose absorption at 4, 7, and 11 d after PEMS challenge. The severe malabsorption and metabolic deficiency problem associated with PEMS was postulated to be a direct effect of the undefined infectious agent or agents that cause the disease.

The peripheral cytoplasm of adrenocortical cells: zone-specific responses to ACTH.

BACKGROUND: Differences in the cytoskeletal protein actin in cells from the zona glomerulosa and zona fasciculata would be of considerable interest because there is persuasive evidence that rat corticosteroids are secreted by mechanisms that are somewhat zone-specific. We have previously shown evidence that actin may be involved in steroid secretion, possibly in connection with changes in adrenocortical microvilli. However, the cells upon which the data were based were not separated according to zone of origin. METHODS: Immunogold electron microscopy and morphometric procedures were used to determine whether ACTH-induced changes in the peripheral cytoplasm of isolated adrenocortical cells occur in both zona fasciculata and zona glomerulosa cells. RESULTS: Actin immunoreactivity was more concentrated in the cytoplasm adjacent to the plasma membrane (including the cytoplasm within the microvilli) than it was in the internal cytoplasm in cells from both zones (4-6 times more concentrated in zona glomerulosa cells and 3-6 times more concentrated in zona fasciculata cells). However, the mean aggregate microvillar surface length (microvillar index) of untreated zona fasciculata cells (previously reported (Loesser and Malamed, 1987)) was 23% greater than that of untreated zona glomerulosa cells. Although ACTH (at a maximal steroidogenic concentration) had no effect on the peripheral cytoplasmic actin concentration of zona glomerulosa cells, there was a 24% increase in the aggregate microvillar length. In contrast, in zona fasciculata cells, ACTH treatment was accompanied by an increase in peripheral cytoplasmic actin concentration of 58-64% and an increase in aggregate microvillar surface length of 40% (previously reported (Loesser and Malamed, 1987)), almost twice that for zona glomerulosa cells. CONCLUSION: The results suggest that ACTH-induced hormone release from zona fasciculata cells is mediated by increases in peripheral cytoplasmic actin and aggregate microvillar length; in zona glomerulosa cells such changes are small or absent.

Immunocytochemical studies of chicken somatotrophs and somatotroph granules before and after hatching.

Immunocytochemical methods were used to gain information about the embryonic development of chicken somatotrophs before and after hatching. To localize growth hormone, anterior pituitary sections were incubated with growth-hormone antibody, and then an indirect peroxidase method was used for light microscopy and an immunogold method for electron microscopy. The earliest evidence of embryonic somatotrophs was seen at 12 days. At this stage somatotrophs were sparse (0.2% of parenchymal cells) and their granules were pleomorphic with elongated ovoid and lozenge shapes predominating. Few of the immunogold-labeled somatotroph granules of the embryo were spherical until 15 days after fertilization. At 18 days, most of the granules were spherical (their shape in the adult chicken). During the six days between the 15-day-old embryo and the 1-day-old chick, the number of gold particles per granule section approximately doubled suggesting an increase in growth hormone content of the granules. This rise was the result of increases in the size of the granule sections and in the concentration of gold particles in the sections. During the embryonic period of 12-20 days, somatotrophs were not more than 3.6% of the anterior pituitary cell population. During the following two days, between the 20-day-old embryo and the 1-day-old chick, the percentage of somatotrophs in the pituitary parenchymal cell population rose rapidly from 3.6% to 20.7% and then increased slowly to 24.6% during the period of 1-5 days after hatching.(ABSTRACT TRUNCATED AT 250 WORDS)

Differential actions of neurotrophins in the locus coeruleus and basal forebrain.

The neurotrophin gene family, including nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF), neurotrophin-3 (NT-3), and NT-4/NT-5, supports the survival of distinct peripheral neurons, however, actions upon central neurons are relatively undefined. In this study we have compared different neurotrophins in the regulation of neuronal survival and function using dissociated embryonic cell cultures from two brain regions, the basal forebrain (BF) and locus coeruleus (LC). In the BF, NGF increased choline acetyl transferase (ChAT) activity, but did not influence cholinergic cell survival. In contrast to NGF, BDNF, NT-3, and the novel neurotrophin, NT-4, all increased ChAT activity and cholinergic cell survival. We also examined embryonic LC neurons in culture. LC neurons are unresponsive to NGF. In contrast, NT-3 and NT-4 elicited significant increases in survival of noradrenergic LC neurons, the first demonstration of trophic effects in this critical brain region. Identification of factors supporting coeruleal and basal forebrain neuronal survival may provide insight into mechanisms mediating degeneration of these disparate structures in clinical disorders.