Lenore Rhodes

High glucose inhibits human epidermal keratinocyte proliferation for cellular studies on diabetes mellitus

In order to more clarify the delayed wound healing in diabetes mellitus, we cultured the human epidermal keratinocytes in both 6 mM (control group) and 12 mM glucose (high‐glucose group) of ‘complete’ MCDB 153 medium. Hyperglycaemia slowed the rate of their proliferation and inhibited their DNA synthesis and the production of total proteins. By 1 month after primary seeding in high‐glucose group, the cells ceased their proliferation, whereas the cells in control group grew for more than 40 days. Mean population doublings in high‐glucose group was 5·27 (vs. 7·25 in control, P = 0·001), and mean population doubling time during 1 month in high glucose group was 5·43 days (vs. 3·65 days in control, P = 0·02). They indicate that prolonged exposure to high glucose decreases the replicative life span of human epidermal keratinocytes in vitro. Furthermore, analysis of fatty acid contents in membrane phospholipids with thin‐layer and gas chromatography showed no difference between the cultured keratinocytes in both conditions. Immunocytochemical staining of glucose transporter 1 shows that 28·1% of cells in high‐glucose group were almost twice positive of those in control group (13·2%, P = 0·008). The mechanism of the ill effects of high glucose on epidermal keratinocytes is not so far clear, but it indicates the possibility of any direct effect of hyperglycaemia on glucose metabolism without changing lipid metabolism on cell membrane. The high‐glucose group presented in this report can be available as an in vitro valuable study model of skin epidermal condition on diabetes mellitus.

Human stratified squamous epithelia differ in cellular fatty acid composition

The phospholipid component of the cellular membrane is crucial to the structure and function of cells. Basal cells from three epithelial tissues, adult human skin epidermis, oral mucosa, and hair follicles, grow rapidly in serum- and lipid-free medium. Analysis of phospholipid extracts from the above three types of stratified squamous epithelium in both in vivo and in vitro was done to relate fatty acid cell composition to cell function. The fatty acid composition of hair follicles in vivo was analyzed in plucked scalp hairs, and those of skin epidermis and oral mucosa in vivo were analyzed after separating the tissue into suprabasal and basal layers. The fatty acid composition of the in vivo cells from hair follicles shows a partial essential fatty acid (EFA)-deficient state. There was no significant difference between the skin epidermis and the oral mucosa in the fatty acid composition of the in vivo cells from each basal layer. However, in the suprabasal layers, the percent of linoleic acid (18:2) from the skin epidermis was higher than that from the oral mucosa. This study shows that total fatty acid composition in cell membranes of stratified squamous epithelium varies with their keratinization pattern. When cultured, the three types of rapidly growing keratinocytes showed the same essential fatty acid deficient pattern in the membrane phospholipids.

EPR measurements showing that plasma membrane viscosity can vary from 30 to 100 cP in human epidermal cell strains

Abstract A rigorous technique for the measurement of human membrane viscosity by electron paramagnetic resonance (EPR) spectroscopy has been developed by designing a sample preparation procedure to optimize the spin labeling process and using a special (grown in essential fatty acid free medium) epidermal cell strain. The essential fatty acid deficient cell strains (keratinocytes) were also grown in fatty acid supplemented media formulated to alter the fatty acid composition of the phospholipids that form the cell membrane. Fatty acid free bovine serum albumin was used as a carrier for the spin label (16-doxyl stearate methyl ester) at an approximately equimolar ratio. Monolayers grown in T-75 flasks were labeled for 15 min at 4°C with 12 μM bovine serum albumin plus 20 μM spin label. The cells were then washed and transferred (at 4°C) to a flatcell for EPR studies at 37°C. The spectra were computer simulated and the results were interpreted by comparison with a “standard curve” obtained from the EPR spectra of the spin label in oil at multiple temperatures. Arguments are presented for preferring this measurement technique over the more conventional use of order parameters and over the use of some other spin labels. The EPR spectra were completely insensitive to the effects of molecular dioxygen in the growth medium and cytoplasm, but remarkabley sensitive to the fatty acid composition of the cellular phospholipids. Fatty acid modified epidermal cells showed a very strong correlation between membrane fluidity (a three-fold change in the membrane viscosity) and a fatty acid double bond index.

Detection of Coronavirus Infection of Man by Immunofluorescence

Summary An indirect fluorescent antibody technique has been developed for use with coronaviruses 229E and OC43. For the former virus, WI-38 cells were used as the host system, and for the latter LLC-MK2 cells were employed. Human serum pairs which were known to show a rise in antibody titer by other serologic techniques were tested by the FA method. Comparison of results obtained by different procedures showed that FA was specific and would be a useful addition to available methods for identifying infection serologically.

The effect of three Korean traditional medicines on the growth rate of cultured human keratinocytes

The effect of three different Korean Traditional Medicines (KTM) was studied on several functional parameters of adult human cells in culture. The cells were non-transformed strains of normal, skin epidermal cells (keratinocytes) from adult humans. Aqueous extracts of the herbal medicines were tested using two types of cell strains: one type was essential fatty acid deficient (EFAD) cells which grow rapidly in medium that was low in calcium and had no essential fatty acids; the second type was a cell strain grown in medium supplemented with essential fatty acid (EFA-supplemented). These cells had much slower, in vivo skin growth rates, and the fatty acid composition resembled that measured in epidermal biopsy tissue. The KTMs chosen for this study were tae-gang-hual-tang (for treating osteoarthritis), hual-ak-tang (for pain relief) and sip-zeon-tae-bo-tang (for fortifying immune systems). Because high proliferation rates usually correlate with skin inflammation and because many of the chemotactic agents mediating inflammatory response are modified fatty acids, this study focused on cell growth rate and membrane fatty acid composition as signals for the effects of the herbal medicines. By monitoring growth rate, these experiments measured both a stimulatory and a regulatory effect on the growth of keratinocytes. Some toxicity was seen at the highest doses of the KTMs. These effects were modeled mathematically, and the results showed varying effects on growth rate depending on dose and herbal recipe. The fitting parameters were discussed as they relate to biological function. The experimental design was also discussed and alternatives were suggested.