1,25-Dihydroxyvitamin D3 Protects against Immune-Mediated Killing of Neurons in Culture and in Experimental Autoimmune Encephalomyelitis

Several studies have reported that low vitamin D levels are associated with an increased risk of developing multiple sclerosis (MS). As MS is an inflammatory disorder with degeneration of axons and neurons, we examined whether the biologically active form of vitamin D, 1,25-dihydroxyvitamin D₃ (1,25D₃), could protect against the T cell-mediated killing of human neurons in culture, and the axonal loss seen in mice with experimental autoimmune encephalomyelitis (EAE). Human neurons were exposed to activated human T lymphocytes and the loss of neurons was documented 24 hours later by counting the number of microtubule-associated protein-2 positive cells. Mice with EAE were harvested for counts of axonal profiles in the spinal cord. 1,25D₃ was exposed to T cells in culture or administered to mice from peak EAE clinical severity when axonal loss was already evolving. Activated T lymphocytes killed human neurons prominently within 24 hours but toxicity was significantly attenuated when T cells were exposed to 1,25D₃ prior to the co-culture. In EAE, 1,25D₃ treatment initiated from peak clinical severity reduced the extent of clinical disability and mitigated the progressive loss of axons. The reduction of axonal and neuronal loss by 1,25D₃ in the context of an inflammatory assault to the central nervous system is a potential contributor to the putative benefits of vitamin D in MS.     

Cigarette Smoking and Fat Distribution in 21, 828 British Men and Women: A Population‐based Study

Objective: To examine the relationship between cigarette smoking habits and fat distribution in a population‐based cohort of men and women. Research Methods and Procedures: We analyzed cross‐sectional data from 21, 828 men and women who were 45 to 79 years of age, residents in Norfolk, United Kingdom, and were recruited between 1993 and 1997. Cigarette smoking habits and other lifestyle factors were assessed using self‐reported questionnaires. Anthropometric measures were obtained during a health examination. Results: Waist‐hip ratio was highest among current smokers and least among never smokers after adjusting for age, BMI, alcohol intake, total energy intake, physical activity, and education. Higher waist‐hip ratio was directly associated with higher smoking pack‐years in current and former smokers and inversely with duration since quitting smoking in former smokers. Adjusting for age, BMI, and other covariates, current smokers had higher waist circumference but lower hip circumference compared with former or never smokers. Discussion: Cigarette smoking habits seem to influence fat distribution patterns. Although smokers have lower mean BMI compared with nonsmokers, they have a more metabolically adverse fat distribution profile, with higher central adiposity. The explanation for this association may help elucidate the mechanisms underlying the adverse health consequences of cigarette smoking and abdominal obesity.     

Purification and Characterization of Microbial Protease Produced Extracellularly from <Emphasis Type="Italic">Bacillus subtilis</Emphasis> FBL-1

An ammonium sulfate precipitation of fermentation broth produced by Bacillus subtilis FBL-1 resulted in 2.9-fold increase of specific protease activity. An eluted protein fraction from the column chromatographies using DEAE-Cellulose and Sephadex G-75 had 94.2- and 94.9-fold higher specific protease activity, respectively. An SDS-PAGE revealed a band of purified protease at approximately 37.6 kDa. Although purified protease showed the highest activity at 45°C and pH 9.0, the activity remained stable in temperature range from 30 to 50°C and pH range from 7.0 to 9.0. Protease activity was activated by metal ions such as Ca²⁺, Mg²⁺, Mn²⁺, Fe²⁺, Ca²⁺ and K⁺, but 10 mM Fe³⁺ significantly inhibited enzyme activity (53%). Protease activity was inhibited by 2 mM EDTA as a metalloprotease inhibitor, but it showed good stability against surfactants and organic solvents. The preferred substrates for protease activity were found to be casein (100%) and soybean flour (71.6%).     

Establishment and physiological analyses of photoautotrophic callus cultures of the fern Platycerium coronarium (Koenig) Desv. under CO2 enrichmen

Gametophyte-derived callus cultures of Platycerium coronariumcould be maintained under photoautotrophic conditions on Murashigeand Skoog medium supplemented with 2µM 2,4-dichlorophenoxyaceticacid (2,4-D) and with CO₂ enrichment. Progressive reductionof sucrose from the medium resulted in a reduction in growth,but an increase in total chlorophyll content. When subculturingwas delayed beyond 2 weeks, callus cells differentiated intogametophytes on the medium with 0.2 sucrose and no CO₂ enrichment.Enriching the photoautotrophic cultures on 2µM 2, 4-Dwith 1% CO₂ resulted in about 1.7-fold increase in fresh weightwithin 42 d. Total chlorophyll content was generally higherwith 1% CO₂ enrichment than with 10%. F_v/F_m ratio was higherfor callus on low levels of sucrose (>0.5%) than that onsucrose 1.0%. An increase in autofluorescence of chloroplasts,but not the size, was observed with decreasing sucrose levelsin the medium. Autofluorescence decreased with increase in CO₂from 0.03%. Our data are in agreement with the view that long-termexposure to high levels of decrease in photosynthetic capacity. Key words: Platycerium coronarium, stag's horn fern, autofluorescence of chloroplasts, confocal laser scanning microscope, F_v/F_m ratio, photoautotrophic callus     

Ammonium and nitrate uptake and nitrate reductase activity of photoautotrophic callus cultures of the fernPlatycerium coronarium (Koenig) DESV

Summary The uptake of nitrate and ammonium by callus ofPlatycerium coronarium from the culture medium was examined. Nitrate reductase activity of photoautotrophic callus cultures under CO₂ enrichment was significantly lower compared to the cultures without CO₂ enrichment, but higher than that of heterotrophic callus cultured on medium with 2% (wt/vol) sucrose. When sucrose concentration of the heterotrophic culture was lowered to 0.2%, nitrate reductase activity increased. The level of nitrate reductase activity increased by about 25% in the heterotrophic callus with an increase in 2,4-D from 2 μM to 10 μM, despite a decline in fresh weight gain. However, photoautotrophic cultures with 1% CO₂ enrichment showed 20% decline in nitrate reductase activity and 45% decline in fresh weight gain with a similar increase in 2,4-D level. The rate of uptake of nitrate from the culture medium was unrelated to the level of nitrate reductase activity in the callus. For photoautotrophic callus under CO₂ enrichment, the presence of 1% (vol/vol) CO₂ generally resulted in the highest rate of nitrate uptake. The rate of uptake of ammonium was higher for callus cultured on 2 μM 2,4-D compared to that on 10 μM 2,4-D.     

Immune complexes and human neoplasia

Summary We have investigated the membrane-binding properties of fetal liver cells (FLC) and developed an assay to quantitate circulating immune complexes (CIC) based on complement (C) receptor binding on FLC. Both binding and blocking studies identified FLC membrane receptors for IgG-Fc, C, and antifetal antibodies (FL-Ab), but not IgG F(ab)2. Fc binding of IgG or aggregated human IgG (AHG) was relatively weak, with an association constant of 1.5×10⁷ l/mol. In contrast, there was a six- to seven-fold increase in binding of AHG by C receptors, with an association constant of 10⁸ l/mol. A simple and sensitive procedure for detecting CIC in the sera of patients with various disease states has been developed by the use of C receptors on FLC. Reference to an AHG standard curve permits quantitation of CIC in micrograms of AHG equivalent per milliliter of serum. Clinical evaluation in patients with active collagen vascular disease and in cancer patients confirmed the reliability, specificity of binding, and sensitivity of the FLC method. Although there was overall agreement with the Raji cell method for CIC detection, FLC-RIA quantiation of CIC was found to be more sensitive than the Raji cell assay. Other discrepancies could be explained by differing sensitivity to CIC size.Preliminary results were presented at the Seventieth Annual Meeting of the American Association for Cancer Research, May 1979 [40]     

Morphogenetic plasticity of callus reinitiated from cell suspension cultures of the fern Platycerium coronarium

Cell suspension cultures were initiated from gametophyte-derived callus of the fern Platycerium coronarium. Two distinct types of callus masses, distinguished by their colouration, were obtained when the cells from suspension culture were plated on semisolid Murashige and Skoog (MS) medium containing 10 M kinetin. The two types of callus masses had distinct morphogenetic capacities despite their common origin. Morphogenesis into either gametophytes or sporophytes occurred when these callus masses were cultured on phytohormone-free MS medium depending on the type of callus used. The dark-green gametophytic callus showed a faster rate of growth and morphogenesis as compared to the pale-green sporophytic callus. Total chlorophyll content and autofluorescence and size of chloroplasts of the sporophytic callus and cell suspension cultures were lower than that of the gametophytic callus. Observations from confocal laser scanning microscopy were in agreement with the physiological parameters measured. The availability of cell cultures of the same ploidy level, but with two distinct pathways of development will be useful for comparative studies of developmental plasticity.     

Ribulose-1,5-bisphosphate carboxylase and phosphoenolpyruvate carboxylase activities of photoautotrophic callus of Platycerium coronarium (Koenig ex O.F. Muell.) Desv. under CO2 enrichment

The in vitro activities of ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) and phosphoenolpyruvate carboxylase (PEPC) were measured in cell-free extracts of Platycerium coronarium callus cultured for up to 42 days under photoautotrophic conditions with CO₂ enrichment. With an increase in CO₂ in the culture environment to 10% (v/v) at low light, the apparent photoautotrophic fixation of CO₂ by Rubisco declined, whereas the non-photoautotrophic CO₂ fixation by PEPC activity was enhanced. Hence, photosynthesis appears to play a lesser role in providing carbon skeletons and energy with prolonged culture in a CO₂-enriched environment. Instead, the anaplerotic supply of C-skeletons by PEPC may be important under such a situation. Short-term H¹⁴CO₃-fixation experiments indicated that photoautotrophic callus cultured for 3 weeks with 10% CO₂ enrichment assimilated less ¹⁴CO₂ than the control (0.03% CO₂). Analyses of ¹⁴C-metabolites indicated that about 50% of the total soluble ¹⁴CO₂ fixed was in the organic acid fraction and 35% in the amino acid fraction. Despite the changes in the in vitro Rubisco/PEPC activity-ratio, no significant change in the ¹⁴C distribution pattern was apparent in response to increasing sucrose or CO₂ concentrations. The suppression of Rubisco activity and total chlorophyll content in high sucrose or elevated CO₂ concentrations suggests an inhibition of the capacity for photoautotrophic callus growth under these conditions. This revised version was published online in June 2006 with corrections to the Cover Date.     

Comparative Analysis of Substrate-Free Cultured Oral Mucosal Epithelial Cell Sheets from Cells of Subjects with and without Stevens—Johnson Syndrome for Use in Ocular Surface Reconstruction

Purpose

To compare the regenerative potential of cultured oral mucosal epithelial cells sheets (COMECs) from Stevens-Johnson syndrome (SJS) subjects with those from non-SJS subjects.

Methods

Human oral mucosal epithelial cells from SJS and non-SJS subjects were cultured, and colony-forming efficiency (CFE), proliferative and migration potential, expression of cytokines/growth factors and stem cells were compared. COMECs from SJS and non-SJS subjects were transplanted into 12 limbal stem cell-deficient rabbits, and their regenerative potential was analyzed at 1 week after transplantation.

Results

CFE (p>0.05, student’s t test), cell proliferation potential (p>0.05, two-way ANOVA) and expression of the cytokeratins (K3, K4, K13, K19) in the oral mucosal epithelial cells from SJS subjects were similar to those of the cells from non-SJS subjects. The initial migratory potential of SJS cells was delayed compared to that of non-SJS cells (p <0.05, RM two-way ANOVA). The SJS cells expressed lower levels of EGF and higher levels of VEGF compared to that of non-SJS cells (p<0.05, one-way ANOVA). In vivo transplanted SJS-COMECs showed similar expression of K3, K4, and K13, proliferation markers (Ki-67; p>0.05, Mann-Whitney U test), and stem cell markers (p63; p>0.05, Mann-Whitney U test) compared to non-SJS COMECs. The initial epithelial defects in vivo were larger in the eyes treated with SJS-COMECs on day 3 (p<0.01, RM two-way ANOVA), but no differences were observed by day 7 between SJS- and non-SJS-COMECs.

Conclusions

These results suggest that, aside from differences in migratory potential, oral mucosal epithelial cells from SJS and non-SJS subjects are comparable in their regeneration potential in treating limbal stem cell deficiency.