Isolating a pure population of epidermal stem cells for use in tissue engineering.

Continuously renewing tissues, such as the epidermis, are maintained by stem cells that slowly proliferate and remain in the tissue for life. Although it has been known for decades that epithelial stem cells can be identified as label-retaining cells (LRCs) by long term retention of a nuclear label, isolating a pure population of stem cells has been problematic. Using a Hoechst and propidium iodide dye combination and specifically defined gating, we sorted mouse epidermal basal cells into three fractions, which we have now identified as stem, transient amplifying (TA), and non-proliferative basal cells. More than 90% of freshly isolated stem cells showed a G0/G1 cell cycle profile, while greater than 20% of the TA cells were actively dividing. Both stem and TA cells retained proliferative capacity, but the stem cells formed larger, more expandable colonies in culture. Both populations could be transduced with a retroviral vector and used to bioengineer an epidermis. However, only the epidermis from the stem cell population continued to grow and express the reporter gene for 6 months in organotypic culture. The epidermis from the transient amplifying cell fraction completely differentiated by 2 months. This novel sorting method yields pure viable epithelial stem cells that can be used to bioengineer a tissue and to test permanent recombinant gene expression.

Look who's driving now--visual standards for driver licensing in the United States.

The current visual standards for driver licensing of the general population in all 50 states and the District of Columbia are described. Minimum visual acuity standards range from 20/40 (73%) to vision less than 20/200 (Washington). The majority of states (92%) offer some type of restricted license which provide an opportunity for low vision drivers to keep their independence while at the same time affording society a high level of safety in the driving system. Tips for the ophthalmic nurse are provided.

Subconjunctival hemorrhage--something more you should know.

Subconjunctival hemorrhage is a common disorder that can occur in all subspecialty areas. An area of sudden and painless bright red blood appears, often during sleep (Berson, 1993), under the clear conjunctiva when a subconjunctival blood vessel breaks.

Negotiating the nurse research coordinator role.

Ophthalmic nurses may be asked to be part of a research team. This article suggests ways the nurse can prepare for the research coordinator role and negotiate added compensation. Without preparation, the nurse's workload may not be adjusted to accommodate additional tasks and there may be a lack of remuneration. At worst, the nurse may fail to meet the expectations of the investigator and sponsor. Nurse research coordinators can learn new marketable skills, increase income, create opportunities for publications and speaking engagements, and widen their professional network. Sources of additional information about the research coordinator role are provided.

Linkage of posterior polymorphous corneal dystrophy to 20q11.

Posterior polymorphous dystrophy (PPMD) is an autosomal dominant disorder of the cornea that is clinically recognized by the presence of vesicles on the endothelial surface of the cornea. The corneal endothelium is normally a single layer of cells that lose their mitotic potential after development is complete. In PPMD, the endothelium is often multi-layered and has several other characteristics of an epithelium including the presence of desmosomes, tonofilaments, and microvilli. These abnormal cells retain their ability to divide and extend onto the trabecular meshwork to cause glaucoma in up to 40% of cases. A large family with 21 members affected with PPMD was genotyped with short tandem repeat polymorphisms distributed across the autosomal genome. Linkage was established with markers on the long arm of chromosome 20. The highest observed LOD score was 5.54 (theta = 0) with marker D20S45. Analysis of recombination events in four affected individuals revealed that the disease gene lies within a 30cM interval between markers D20S98 and D20S108.

Overview and new technology in cyclodestructive procedures.

When all medical and surgical measures have failed to lower intraocular pressure (IOP) in patients with severe and uncontrolled glaucoma, ophthalmologists must turn to cyclodestructive procedures to decrease aqueous production. Several months ago, the FDA (Food and Drug Administration) came through with an approval for a new technology in cyclophotocoagulation therapy. The IRIS Medical G-Probe used in conjunction with a semiconductor diode laser may be the superior alternative to previously available cyclodestructive techniques for which ophthalmologists have been searching.

Cornea: confocal microscopy.

The confocal microscope opens a whole new window in early diagnosis of ocular conditions. Previously, details at the cellular level could only be viewed with conventional microscopes in a laboratory setting. By using confocal microscopy, results can now be obtained instantaneously in the living human eye. This non-invasive high magnification technique provides real-time images of cornea morphology.

Decreased membrane phospholipid packing and decreased cell size precede DNA cleavage in mature mouse B cell apoptosis.

Mature resting mouse spleen B cells progress stochastically into apoptosis at a uniform rate over the first 16 h in vitro in 3 stages. In stage 1, early apoptotic B cells decreased the normal phospholipid packing of their plasma membranes, detected as increased binding of the lipophilic dye merocyanine 540, and also decreased in volume, detected as decreased forward scatter. In stage 2 there was abrupt internucleosomal cleavage of DNA, quantitated as hypodiploid nuclei by flow cytometry. Some stage 2 cells entered stage 3, where the plasma membrane became permeable to propidium iodide. B cells in later stages of this sequence retained the characteristics of earlier stages, whereas nonapoptotic B cells remained in their original state. Cycloheximide increased the progression of B cells through these three stages, whereas dextran sulfate inhibited stage 1 more effectively than stages 2 or 3. Increased orthogonal scatter also occurred late in some of the cells that had passed through stage 1, but did not correlate well with propidium iodide permeability. Fresh small dense spleen B cells contained 5% to 7% stage 1 cells but only about 1% stage 2 cells. Macrophages have been reported to destroy preferentially apoptotic thymocytes by recognizing plasma membrane alterations deriving from loose packing of phospholipid head groups. The recognition of stage 1 rather than stage 2 B cells by macrophages may help to keep the proportion of apoptotic cells low in vivo.

Lessons on dilation.

Mydriatics cause pupil dilation by blocking the responses to the sphincter muscle of the iris from cholinergic stimulation. Cycloplegics paralyze the action of the ciliary muscles thereby prohibiting accommodation of the lens.

Nine point corneal thickness measurements and keratometry readings in normal corneas using ultrasound pachymetry.

Central, paracentral, and peripheral corneal thickness measurements using ultrasound pachymetry and keratometry readings were performed in 303 normal corneas. The mean central corneal thickness was 515 +/- 34 microns (standard deviation) ranging from 410 to 625 microns. Mean paracentral thickness measurements ranged from 522 +/- 40 microns inferiorly to 574 +/- 41 microns superiorly; mean peripheral thickness measurements ranged from 633 +/- 50 microns inferiorly to 673 +/- 49 microns superiorly. Central corneal thickness measurements were not found to be correlated with age, however, paracentral and peripheral thickness measurements tended to become thinner with age. There was also a trend that central corneal thickness decreased as the average keratometry reading increased. These trends, however, were not statistically significant. The mean difference between right and left central corneal thicknesses was 25 microns, ranging from zero to 136 microns. No significant differences in corneal thickness or keratometry readings were found between males and females or right and left eyes. No differences in these measurements were found with regard to time of day, month of the year, or systemic medication use. We conclude that there is a wide range of corneal thickness centrally, paracentrally, and peripherally in normal corneas.

Reproducibility and variation of corneal thickness in different locations in the cornea as measured by an ultrasonic pachymeter.

Corneal thickness was measured in nine positions of nineteen normal eyes with an ultrasonic pachymeter. A standardized protocol was used to examine for intraobserver and interobserver variation, day to day variation, and time of day variation in the measurement of corneal thickness. No statistically significant difference was noted for most points in intraobserver, interobserver, day to day, or different time of day variation measurements. However, it was noted that central measurements of corneal thickness tended to be less variable (more reproducible) than paracentral (p = .0366) and peripheral (.0032) measurements. Paracentral measurements were less variable than peripheral measurements (p = .0323). While this was a small study, 95% confidence intervals were also calculated for each of the nine corneal positions where measurements were performed.

Visual acuity, residual astigmatism, and graft clarity following penetrating keratoplasty for keratoconus.

In summary, these finding show us several things. First, we know that with surgery keratoconus patients can be rehabilitated to achieve excellent vision. A postoperative average visual acuity of 20/20 after an average preoperative visual acuity of 20/500 is a most positive outcome. Secondly, the average amount of postoperative astigmatism of 3.5 diopters shows that even after totally removing a corneal button and replacing it with 360 degrees of sutures we can still achieve a relatively small amount of residual astigmatism and a relatively spherical cornea. Thirdly, with the exception of one primary donor failure, a total of one hundred percent clear grafts, even after the occurrence of two graft rejection episodes, indicated an extremely high success rate of keratoplasty in keratoconus.

Ethanol induced growth inhibition and growth adaptation in vitro. Cell cycle delay in late G1.

We have characterized the growth responses of HTC rat hepatoma sublines after exposure to clinically relevant concentrations of ethanol. These experiments demonstrate growth inhibition by ethanol, and both adaptive and non-adaptive growth responses after chronic exposure. Examination of the cell cycle compartmentation of HTC lines shows that a rapid accumulation of G0/G1 cells is induced by ethanol. Estimates of cellular G1 RNA content by flow cytometry reveal increases in mean G1 RNA and in late G1 cells in the line which growth adapts, and decreases in these parameters in a line which does not adapt to ethanol. Both the growth responses and the timing of cell cycle restriction by ethanol in the adapting line suggest parallels with the reported data for regenerating rat liver. Ethanol induced late G1 restriction appears to be of significant interest in the study of cellular mechanisms which are disturbed by ethanol in proliferating tissues.

Induction of HL-60 monocytic cell differentiation promoted by a perturbation of DNA synthesis: hydroxyurea promotes action of TPA.

Control of terminal cell differentiation was studied using the human promyelocytic leukemia cell line, HL-60. HL-60 cells are known to undergo terminal monocytic differentiation when continuously exposed to 1.6 nM tetradecanoylphorbol acetate (TPA). The dose-response relationship between TPA concentration and induced differentiation is relatively steep. TPA (1.1 nM) induces little G1/0 specific growth inhibition or phenotypic differentiation. In contrast, pretreating the cells with a pulse exposure to hydroxyurea promotes their capability to terminally differentiate in response to TPA. Initially exponentially proliferating cells exposed for 20 h, approximately one doubling time, to 0.3 mM hydroxyurea, a subcytotoxic dose, underwent rapid G1/0 specific growth arrest and cell differentiation in response to subsequent exposure to 1.1 nM TPA. The extent of terminal differentiation was comparable to that induced by 1.6 nM TPA. The results support the hypothesis that early events in induction of terminal HL-60 cell differentiation depend on an S phase-specific process which may involve gene amplification.

Precommitment states induced during HL-60 myeloid differentiation: possible similarities of retinoic acid- and DMSO-induced early events.

The possible relationship of the pathways by which two inducers, retinoic acid and DMSO, cause myeloid differentiation of HL-60 promyelocytic leukemia cells was studied. HL-60 cells were first exposed to retinoic acid and then washed free of it. As reported previously, this brief exposure results in no subsequent G0 growth arrest or phenotypic differentiation. When these cells were subsequently exposed to DMSO, onset of G1/0 growth arrest but not phenotypic differentiation occurred within 24 h. Since in these cells retinoic acid or DMSO normally requires 48 h of continuous exposure for onset of significant G0 growth arrest and phenotypic differentiation, it appears that retinoic acid and DMSO induce similar early cellular events needed for subsequent G0 growth arrest but not for phenotypic differentiation. While onset of growth arrest and differentiation occur together when the cells are exposed for 48 h to retinoic acid, the present results indicate that their occurrence can be uncoupled by this split dosage to inducers. The results are discussed in terms of a previously hypothesized model of cellular response to the inducers.