Injury and response of the shoulder in lateral sled tests.

The biomechanical response and injury tolerance of the shoulder in lateral impacts is not well understood. These data are needed to better understand human injury tolerance, validate finite element models and develop biofidelic shoulders in side impact dummies. Seventeen side impact sled tests were performed with unembalmed human cadavers. Data analyzed for this study include T1-Y acceleration, shoulder and thoracic load plate forces, upper sternum x and y accelerations, and struck side acromion x, y and z accelerations. One dimensional deflection at the shoulder level was determined from high-speed film by measuring the distance between a target on T1 and the impacted wall. Force-time response corridors were obtained for tests with 9 m/s pelvic offset, 10.5 m/s pelvic offset, 9 m/s unpadded flat wall, 6.7 m/s unpadded flat wall, 9 m/s soft padding and 9 m/s stiff padding. Maximum shoulder plate forces in unpadded 9 m/s tests (5.5 kN) were larger than in 6.7 m/s tests (3.3 kN). The peak force at the shoulder was larger than at the thorax plate in unpadded and soft padded tests. T1-Y accelerations were larger in unpadded 9 m/s flat wall tests and unpadded pelvic offset 10.5 m/s tests (peak values of 130 and 145 g's) than in other test conditions. Deflections between T1 and the struck wall ranged from 88 to 154 mm in unpadded tests and 95 to 128 mm in stiff and soft padded tests. Eighteen AIS 2 level shoulder injuries occurred in 11 test subjects. These injuries included left acromion fracture in five subjects, left acromioclavicular separation in ten subjects and left clavicle fracture in three subjects. Average MAIS to the shoulder was 0.86 in seven subjects which impacted 4 to 6 inches (101.6 to 152.4 mm) of soft or stiff padding and 1.6 in ten subjects which impacted no padding or 3 inches (76.2 mm) of stiff padding. Previous findings from this test series were reported by Irwin et al. (1993) for seven tests and focused on detailed analysis of shoulder deflection (T5 to shoulder edge). The current study is expanded to 17 tests and includes force, acceleration response and analysis of shoulder deflection (T1 to shoulder edge). Padding of 4 to 6 inches (101.6 mm to 152.4 mm) reduced shoulder injury approximately one AIS level. A combination of ASA10 and deflection was the best shoulder injury predictor. Shoulder deflection of 106 mm predicts 50% probability of MAIS 2.

Corneal endothelial cell survival in organ cultures under acute oxidative stress: effect of VIP.

PURPOSE: Human corneal endothelium, a neural crest-derived tissue, has a very limited regenerative capacity and may depend on trophic factors for its survival throughout life, as well as after injury and during storage before transplantation. The purpose of this study was to determine whether vasoactive intestinal peptide (VIP), a 28-amino acid neurotrophic factor present in human aqueous humor, promotes the survival of corneal endothelium in corneal organ cultures, and whether VIP is produced by the corneal endothelium. METHODS: Thirteen viable human donor corneas that had been received from the Central Florida Lions Eye Bank and stored in preservation medium (Optisol-GS; Chiron Vision, Irvine, CA) at 4 degrees C for 8 to 17 days were bisected. Each half was treated with either 0 or 10 nM VIP (15 minutes) and subjected to H(2)O(2) (1.4 mM, 30 minutes) treatment at 37 degrees C. The numbers of live and dead corneal endothelial (CE) cells isolated from the corneas were then determined under fluorescence microscopy using a live-dead viability-cytotoxicity assay conducted by an observer uninformed of the treatment. The effect of VIP (10(-16) to 10(-6) M) on CE cell survival was also studied in fresh bovine corneas in situ, by using the same assay. The presence of VIP in the corneal endothelium in fresh human donor and bovine eyes was examined by immunocytochemistry, in situ hybridization, and Western blot analysis, whereas VIP in the bovine aqueous humor was assessed by radioimmunoassay. RESULTS: VIP (10 nM) significantly increased CE survival in 10 of 13 human corneas. The mean survival of CE cells (+/-SEM) was 42% +/- 3% in control corneas versus 59% +/- 3% in VIP-treated corneas (P < 0.001). In bovine corneas, VIP at concentrations as low as 10(-10) M demonstrated a significant protective effect. The mean number of dead CE cells on bovine corneas was maximally decreased by 10(-6) M VIP from 46 +/- 5 to 18 +/- 3 per field. In CE cells from fresh human and bovine corneas, VIP immunoreactivity and mRNA were detected. VIP was also present in bovine aqueous humor at 40 +/- 8 pM. CONCLUSION: VIP may be an autocrine trophic factor that protects CE cells from H(2)O(2) in normal aqueous humor and possibly from other oxidative insults.

Frequency of spinocerebellar ataxia types 1,2,3,6,7 and dentatorubral pallidoluysian atrophy mutations in Korean patients with spinocerebellar ataxia.

Autosomal dominant cerebellar ataxias (ADCAs) are a heterogeneous group of disorders characterized by degenerative symptoms in the cerebellum, spinal cord, and brain stem. Six different genes have been reported to be associated with ADCA, and the length of trinucleotide repeats of these genes is correlated with the age at onset and severity of symptoms. Although there are strong hereditary effects in these disorders, most of the studies carried out in heterogeneous populations and in small groups obscure the true incidence of these diseases. We examined the frequency of six types of ADCAs in 87 unrelated Korean patients with progressive ataxia and compared the results to the frequencies in other ethnic groups. Spinocerebellar ataxia (SCA) type 2 was the most frequent hereditary ataxia (12.6%) and types 3 and 6 accounted for 4.6% and 6.9% of ataxia patients, respectively. Dentatorubral pallidoluysian atrophy was also found in three patients (3.4%). No instances of SCA types 1 or 7 were detected. These findings show the striking contrast to the white population and a difference from Japanese findings. Our results demonstrate that dentatorubral pallidoluysian atrophy should be included in the differential diagnosis of Korean patients with spinocerebellar ataxia, and that there are strong hereditary effects in patients with ADCAs.

Bombesin activates MAP kinase in non-small cell lung cancer cells.

The effects of bombesin (BB) on mitogen activated protein (MAP) kinase were investigated using non-small cell lung cancer (NSCLC) cells. By Western blot, both 42 and 44 kDalton forms of MAP kinase were present in NCI-H1299 and NCI-H838 cells. Addition of BB to NCI-H1299 cells resulted in phosphorylation of the MAP kinase substrate myelin basic protein (MBP). Phosphorylation of MBP was maximal 6 min after the addition of 10 nM BB to NCI-H1299 cells. Addition of gastrin releasing peptide (GRP) or GRP14-27 but not GRP1-16 to NCI-H 1299 cells caused MBP phosphorylation. The effects of BB were inhibited by BW2258U89, a BB receptor antagonist, and PD98059, a MAP kinase kinase inhibitor. Also, PD98059 inhibited the clonal growth of NCI-H1299 cells. These data suggest that MAP kinase may be an important regulatory enzyme in NSCLC.

Vasoactive intestinal peptide stimulation of human trabecular meshwork cell growth.

PURPOSE: To demonstrate that vasoactive intestinal peptide (VIP), a 28-amino acid neuropeptide, is a growth factor of human trabecular meshwork (TM) cells in culture and in a corneoscleral explant organ culture treated with laser trabeculoplasty (LTP). METHODS: Proliferating human TM cells in cell cultures were incubated with VIP for 20 hours, followed by total cell number determination, using a Coulter counter. The percentage of proliferating TM cells was assessed, using an antibody against the proliferating cell nuclear antigen (PCNA). To test the growth effect of VIP on TM cells in situ, corneoscleral explants in organ cultures were first treated with argon LTP to initiate TM-cell proliferation and then were exposed to VIP for 48 hours. The mitotic TM cells were demonstrated immunocytochemically, using anti-PCNA in paraffin sections of the explants; and the total number of TM cells was determined after paraffin sections were counterstained by hematoxylin. RESULTS: Vasoactive intestinal peptide dose-dependently stimulated the proliferation of TM cells in cell culture. Treatment with 5 x 10(-10) M VIP resulted in a maximal increase of 40% in cell number. The effect of VIP was blocked by a VIP antagonist. The number of PCNA-stained TM cells and the total cell number in the TM in LTP-treated corneoscleral explants were increased by VIP. CONCLUSIONS: Exogenously applied VIP stimulated the proliferation of human TM cells in subconfluent cultures and in LTP-treated corneoscleral explants. In that LTP has been shown to increase the number of TM cells in situ, the growth stimulatory effect of VIP may help enhance this therapy.

Evidence of a functional VIP receptor in cultured human retinal pigment epithelium.

The effect of VIP on the intracellular cyclic AMP of human retinal pigment epithelium cultures has been studied. Functional VIP receptor has been demonstrated in cultures from eyes given by five normal donors (age 16-64) (N-HRPE). But it has been found to be absent from high passage number cultures obtained from a retinitis pigmentosa eye of an 84-year-old patient (RP-HRPE). After 3 min of reaction with 1 x 10(-6) M VIP, the intracellular cyclic AMP level has increased to 5-15-fold over the basal level. The maximal effect of VIP (20-fold over the basal level) has been observed at 1 x 10(-7) M VIP. The half maximal activity of VIP is 3-5 x 10(-8) M. The present study also demonstrates the inducibility of the VIP responsiveness in RP-HRPE cultures after they have been treated with butyrate. Curr. Eye Res. 14: 1009-1014, 1995.

The pp60c-src in retinal pigment epithelium and its modulation by vasoactive intestinal peptide.

The pp60c-src is present at high level in differentiated retinal pigment epithelium (RPE) in culture. Immunofluorescence microscopy using GD11 (anti-pp60c-src) shows intense staining in the plasma membrane, especially at the cell-cell junctions, and diffuse staining in the cytoplasma. Western blot analysis shows that the majority of the GD11-reactive molecules is localized in the membrane. The pp60c-src does not translocate between membrane and cytoplasma when the RPE was reacted with vasoactive intestinal peptide (VIP), which is previously shown to stimulate phosphorylation of the pp60c-src in the membranes (Biochem. Biophys. Res. Commun. 174, 452-8, 1991). Here, VIP modulation is shown to alter the reactivity of pp60c-src with a monoclonal anti-phosphotyrosine antibody.

VIP stimulates proliferation and differentiation of the cultured retinal pigment epithelium with disparate potencies.

Previous studies showed that VIP modulates mediators of two signal transduction pathways, namely the adenylate cyclase and the nonreceptor tyrosine protein kinase pp60c-src in cultured chick retinal pigment epithelium (RPE). Here we show that VIP modulates simultaneously two disparate cellular events, namely the cell proliferation and differentiation of the RPE, however, with different potencies. The maximal effects on proliferation and differentiation are observed at 5 x 10(-9)M and 5 x 10(-7)M, respectively. Treatment with the maximally effective concentrations of VIP for 10 days increases the cell numbers and the melanin contents to 150% and 200% of the controls, respectively. The lowest concentrations of VIP showing significant stimulatory effect on cell proliferation and melanin synthesis are 5 x 10(-11) M and 5 x 10(-9)M, respectively.

VIP stimulation of polarized macromolecule secretion in cultured chick embryonic retinal pigment epithelium.

Vasoactive intestinal peptide (VIP) stimulated macromolecule secretion at the apical membranes of the chick embryonic retinal pigment epithelium cultured on permeable supports in a time- and concentration-dependent manner. VIP stimulated secretion of molecules with MW of 80, 74, 70, 60, 42, 35, 24, 20, and 14 kDa. A 1.9- to 2.6-fold stimulation in secretion of molecules with MW greater than 10 kDa precipitable by 10% trichloroacetic acid was observed after treatment with 1 microM VIP for 15 min. The effect of 1 microM VIP was mimicked by 10 microM dibutyryl cyclic AMP and attenuated by dopamine (1 x 10(-4) M), while colchicine, beta-lumicolchicine, and monensin, all at 1 microM, had no effect.

Signal transduction through the vasoactive intestinal peptide receptor stimulates phosphorylation of the tyrosine kinase pp60c-src.

The present study demonstrates that signal transduction through a receptor lacking intrinsic tyrosine protein kinase activity involves a rapid and potent phosphorylation of a non-receptor tyrosine protein kinase in the membranes. Vasoactive intestinal peptide (VIP) stimulates phosphorylation of a membrane protein with a M.W. of 56 KD (pp60) in the cultured chick embryonic retinal pigment epithelium. VIP stimulates phosphorylation of the pp60 with such efficiency and potency that the maximal phosphorylation has been observed at the earliest time (3 minutes at 1 x 10(-6)M VIP) and the lowest concentration (1 x 10(-11)M for 20 minutes) examined. Western blot analysis with a monoclonal antibody anti-pp60src (GD11, Parsons et al., J. Virol. 51, 272-282, 1984) indicates that the pp60 is the pp60c-src, a normal cell oncogene product with intrinsic tyrosine protein kinase activity.

VIP modulation of cultured glial cells of the rat retina.

Cultured retinal glial cells from the rat are responsive to modulation by vasoactive intestinal peptide (VIP). VIP (1 X 10(-6) M) elevated the intracellular cyclic AMP concentration from the basal level of (4.4-11.1) p mole/mg protein to (354-440) p mole/mg protein in three minutes at 25 degrees C. The half-maximal concentration is 4.8 X 10(-8) M, which is similar to that observed in the cultured retinal glial cells from the chick embryo.

Modulation of a 190-kD microtubule-associated protein in pigment epithelium by VIP.

Vasoactive intestinal peptide stimulates phosphorylation of six high molecular weight cytosolic proteins in the cultured retinal pigment epithelium (RPE). Of these, the 190-kD phosphoprotein is associated with the microtubules assembled by taxol/GTP and is immunologically related to the brain microtubule-associated protein 2 (mol.wt. = 280 kD). VIP is also shown here to stimulate secretion in the cultured RPE. VIP-stimulated phosphorylation of a 190-kD microtubule-associated protein is also demonstrated here in the retinal glia.

The chick retinal pigment epithelium grown on permeable support demonstrates functional polarity.

The retinal pigment epithelium (RPE) from the chick embryo was cultured on permeable support. Using confluent cultures and analysis of the incubation medium, the present study demonstrates that RPE cells cultured on permeable membrane retain functional polarity, a characteristic of the RPE in vivo. The degree of intercellular permeability in the confluent RPE cultures was estimated by following [3H]inulin movement from the apical side to the basal side of the cultures. Twenty-four hours after exposure of the apical side of the culture to [3H]inulin, the 3H concentration in the apical medium remained at 3.4 to 4.4 times of that in the basal medium. The barrier function of RPE disappears in the presence of EDTA. Net unidirectional fluid movement from the apical side of the cultures to the basal side of the cultures is regularly observed in confluent RPE cultures. The rate varies among different preparations of cultures and the highest is 1.60-1.84 microliters/cm2/h. When cultures are given 26 h of [35S]methionine, more than 20 bands with molecular weights ranging from 20,000 to greater than 250,000 Da can be detected in the medium as assessed by autoradiography of SDS-polyacrylamide gels. While six macromolecules appear to be equally concentrated in the basal medium and the apical medium, the majority are in higher concentration in the basal medium. Analysis of the 10% TCA-precipitable fraction of the medium showed that the specific activities in the apical medium and basal medium were 24.0 +/- 0.4 X 10(6) and 46.4 +/- 0.2 X 10(6) (mean +/- SEM, N = 8) cpm/ml/mg RPE protein, respectively. When cultures react with VIP (vasoactive intestinal peptide), the elevated intracellular cyclic AMP is extruded into the medium bathing the cells. However, the rate of extrusion into the basal medium is twice as fast as that into the apical medium. Electron microscopy of the confluent RPE cultures shows morphological polarization of the cells. The intercellular spaces appear to be closed at the apical side of the cells by junctional complexes consisting of tight junctions, zonular adherens junctions, and gap junctions.

Immunosuppression after injection of Friend virus into C57BL/6 mice of different ages.

Injection with Friend virus (FV) causes immunosuppression in young and old C57BL/6 mice, i.e. it occurs whether or not the virus replicates very briefly or for a long period. There are only minor age-related differences in the extent of immunosuppression, except that suppression appears to persist somewhat longer in old than in young animals.

Effects of agonists on the intracellular cyclic AMP concentration in monkey trabecular meshwork cells.

We examined the effects of several neuroendocrine agents on the intracellular cyclic AMP level in cultured monkey trabecular meshwork cells. Among the peptide agonists studied, only the vasoactive intestinal peptide elevated the cyclic AMP level. Alpha-melanocyte stimulating hormone, glucagon, and others showed no stimulation. Prostaglandin E1 and L-isoproterenol also enhanced the intracellular cyclic AMP level. Such effects may implicate physiologic roles of these agonists in the trabecular meshwork.

Lymphokine-activated killer (LAK) cells: I. Age-dependent decline of LAK cell-mediated cytotoxicity.

Experiments were designed to assess age-related changes in generation of lymphokine-activated killer (LAK) cells and to test whether these changes can be modified by diets differing in the proportion of polyunsaturated to saturated fatty acids (P/S). Ficoll-Hypaque-isolated spleen lymphocytes of rodent chow-fed, 6-85-week-old C57BL/6 (H-2b), 8-81-week-old C57BL/10 (H-2b) and 6-62-week-old SJL (H-2s) mice were cultured in IL-2-containing medium and examined in 51Cr cytotoxicity assay. Similarly, Ficoll-Hypaque-isolated spleen lymphocytes of 6-36-week-old SJL mice fed diets which differed in the ratio of polyunsaturated/saturated fatty acids were cultured in IL-2-containing medium and assayed for cytotoxicity. Age-related decline of LAK cell-mediated cytolysis was observed in mice of both H-2b and H-2s haplotype. The age-related decline of LAK cell-mediated cytolysis was the consequence of age-related decrease in the rate of LAK cell precursor maturation. SJL mice fed from birth with diets differing in P/S did not differ in LAK cell-mediated cytolysis.

Epinephrine regulates cholinergic transmission mediated by rat retinal neurons in culture.

The purpose of this study was to investigate the effects of epinephrine on neurotransmission mediated by cholinergic neurons derived from the rat retina. We used a culture system in which striated muscle cells served as postsynaptic targets for cholinergic neurons of the embryonic retina. This culture system permitted the physiological monitoring of acetylcholine released by retinal neurons. Here, we report that epinephrine facilitates evoked transmission across retina-muscle synapses. This facilitation of cholinergic transmission by epinephrine is reversible, can be mimicked by isoproterenol (a beta adrenoceptor agonist) and blocked by propranolol (a beta adrenoceptor antagonist). Neither the alpha-2 adrenoceptor blocker, yohimbine, nor the dopamine receptor antagonist, haloperidol, blocked this effect of epinephrine. Since epinephrine was found not to influence the membrane potential of muscle cells nor the responses of myotubes to acetylcholine, epinephrine appeared to have mediated its facilitatory effect on cholinergic transmission by affecting retinal cells. Because previous findings indicated that adenosine 3',5'-cyclic monophosphate may be involved in the modulation of transmission at retina-muscle synapses, the effect of epinephrine on adenosine 3',5'-cyclic monophosphate levels was investigated. Our biochemical studies demonstrated that epinephrine could increase adenosine 3',5'-cyclic monophosphate levels markedly in cultured retinal cells. The accumulation of adenosine 3',5'-cyclic monophosphate induced by epinephrine could be blocked by propranolol, but not by yohimbine nor haloperidol. Taken together, the results indicate that the facilitatory effect of epinephrine is mediated via a beta adrenoceptor and may involve an increase in adenosine 3',5'-cyclic monophosphate levels. Our findings are in agreement with the hypothesis that epinephrine may be a modulatory neurotransmitter in the rat retina.

Dietary fat alters progression of some age-related changes of the immune system.

SJL mice develop resistance against tolerance between the 9th and 25th wk of life. This resistance is linked with a loss of suppressor capacity in the thymus. We have shown here that contact photosensitivity (CPS) decreases as a function of age and that this is due to an age-dependent increase in suppressor capacity. Diet fats have a differential effect on age-dependent changes in suppressor activity; a low P/S diet prevents or delays loss of suppressor activity for antibody formation and a high P/S diet prevents or delays the development of suppressor activity in CPS reactions.

Elevation of intracellular cyclic AMP and stimulation of adenylate cyclase activity by vasoactive intestinal peptide and glucagon in the retinal pigment epithelium.

Both vasoactive intestinal peptide (VIP) and glucagon rapidly elevated cyclic AMP levels in embryonic chick retinal pigment epithelium (RPE), in culture as well as in freshly dissected tissue. In cultured cells, the half-maximal activities of VIP and glucagon were 5 X 10(-8) M and 3 X 10(-8) M, respectively. After 3 min of reaction, VIP elevated intracellular cyclic AMP by 100-fold; elevation with glucagon was up to 10-fold. Both neuropeptides stimulated adenylate cyclase activity in RPE membranes. Glucagon showed a half-maximal activity of 1 X 10(-8) M. VIP remained more effective than glucagon in stimulating adenylate cyclase activity, but the dose-response curve was shifted to a higher concentration range when compared to that of the VIP-elevated intracellular cyclic AMP.