Biomedical subjects
Publications and source records attributed to J C Kvedar.
Nail involvement in cutaneous disease provides the clinician with diagnostic clues, and may be of great cosmetic significance to patients. Nail manifestations of the most common and/or important cutaneous diseases are discussed. Photographic illustrations of these changes are also presented.
A monoclonal and two polyclonal antibodies to human involucrin were used to look for involucrin epitopes in other species. All antibodies react strongly with the same proteins of monkey, and both polyclonal antibodies react with specific proteins of cow and dog. One of the polyclonal antibodies also reacts with proteins of sheep, guinea pig, rat, and finback whale. The immunoreactive proteins from cow and dog could be purified using a procedure developed for human involucrin. The reaction with the purified dog protein could be blocked by purified human involucrin. The results suggest that involucrin-like proteins have a wider species distribution than originally appreciated.
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The effect of minoxidil on arachidonic acid metabolism by cells in culture was studied. In bovine aorta endothelial cells, treatment with minoxidil in the presence of various stimulators of arachidonic acid metabolism was accompanied by a dose-dependent inhibition of prostacyclin production (measured as 6-keto-prostaglandin F1 alpha). Synthesis of the other cyclooxygenase products (prostaglandins E2, F2 alpha and thromboxane) was not inhibited. When the bovine aorta endothelial cells were stimulated by the Ca2+ ionophore A-23187, the inhibition was seen as early as 2 min. Minoxidil also inhibited prostacyclin production by a second cell line of bovine aorta endothelial cells (the established CPAE cell line), bovine aorta smooth muscle cells, porcine aorta endothelial cells, and rat liver cells (the C-9 cell line)--the latter, less effectively. Again, formation of all the other cyclooxygenase products studied was not inhibited. Minoxidil did not affect significantly prostaglandin E2 and F2 alpha production by newborn rat keratinocytes (the NBR cell line)--a cell that does not produce PGI2. The clinical, biochemical, and pharmacologic implications are discussed.
The cornified envelope has been shown to be formed beneath the plasma membrane as a result of the cross-linking of soluble and membrane-associated precursor proteins by transglutaminase. We have obtained a monoclonal antibody which reacts with the periphery of cells in the upper layers of human epidermis by indirect immunofluorescence (IIF) following immunization of mice with cornified envelopes of cultured human keratinocytes. The antibody also stained the cell peripheries of bovine, rat and mouse epidermis as well as stratified epithelium. Neutral buffer extracts of human cultured keratinocytes and epidermis examined under denaturing conditions contained polypeptides of molecular weight 14,900 and 16,800 which reacted with the antibody, and an additional component of molecular weight 24,800 was found in cultured cells. The polypeptides were shown to have a pI of about 9.0. Under non-denaturing conditions the two lower-molecular-weight polypeptides had an apparent molecular weight of 30,000, while the 24,800 protein had one of 60,000. Incubation of the polypeptides under conditions that activate transglutaminase resulted in a disappearance of the polypeptides or the formation of cross-linked products. Basic polypeptides with somewhat different pI values and molecular weights were identified in neutral buffer extracts of bovine and rat epidermis. The HCE-2 antibody appears to identify a new class of basic protein precursors of mammalian cornified envelope.
Male pattern hair loss (androgenetic alopecia) is a common problem. In fact, it affects nearly all males to some degree. Expression of the disorder is variable, and while it is never life-threatening, it often becomes a major source of consternation. The biology of the process is poorly understood, and no current therapy can halt or reverse the process. Only cosmetic surgery, which is painful, time consuming, and expensive, has been effective. In the past 7 years, since it was noted that a patient taking minoxidil for hypertension had reversal of male pattern hair loss, awareness of a possible therapeutic role for topical minoxidil in the management of this disorder has grown among physicians, scientists, and the general public. It can be concluded from available data that topical application of minoxidil is effective in providing cosmetically satisfying thickening of hair in a select group of individuals with male pattern hair loss. The drug's mechanism of action remains obscure. No serious side effects have been demonstrated with its use, however, and it is therefore advised in selected patients.
The long-lived keratinocyte line, NM 1, was isolated from the epidermis of a pool of foreskins obtained from apparently normal neonates at the time of circumcision. Cultures were initiated in Dulbecco's minimal essential medium containing 20% fetal bovine serum, 0.4 micrograms/ml hydrocortisone, 10(-9) M cholera toxin, and 10 ng/ml epidermal growth factor using mitomycin C-treated 3T3 cells as a feeder layer. Unlike normal keratinocytes which survive for only 150 generations these cells have been in culture for more than a year and have been carried for more than 400 doublings. The cells seem to follow a pathway of growth and differentiation that is very similar to normal keratinocytes. Cytokeratin fibrils, intercellular attachments, and cornified envelopes were observed. The keratin polypeptides isolated from the NM 1 cells were similar to those previously described in normal cultured cells; the presence of profilaggrin and involucrin was demonstrated by sodium dodecyl sulfate electrophoresis and immunoblotting with monoclonal antibodies specific to these proteins. The NM 1 cells showed a reduced dependency on 3T3 feeder cells but did not form tumors when placed into athymic nude mice. Screening of the cells for SV40, BK, HPV 16, and HPV 18 viruses was negative. The NM 1 cells showed trisomy of chromosome 8. The long-lived nature of these cells makes them a valuable model for studying growth and differentiation of keratinocytes.
Studies in our laboratory have shown that minoxidil prolongs the life of keratinocytes in culture and extends the time after confluence that cells can be subcultured. These data suggest that the drug reduces the rate at which cells are lost from the germinative pool and hence slows senescence. In a dose-response study with minoxidil, the maximal effect of the drug was seen at doses from 6 to 12 micrograms/ml; however, activity could be detected at doses below 1 microgram/ml. Cells subcultured during log growth failed to demonstrate that minoxidil increased the total number of generations attainable under these conditions, although as expected, epidermal growth factor extended the life span of cells. When the experiments were repeated in a keratinocyte cell line that does not require a fibroblast feeder layer, the same results were obtained, indicating that the difference observed between log phase and postconfluence growth cannot be explained by the presence of fibroblasts. Minoxidil's effect on postconfluent cells was blunted by the addition of cholera toxin to the medium, suggesting that elevation of cyclic adenosine monophosphate cannot be a mechanism, although reduction of cyclic adenosine monophosphate is a possibility. Finally, maintaining keratinocytes at a 20 to 40 mM calcium concentration greatly reduced the ability of postconfluent cells to be subcultured, in comparison with the normal calcium concentration of 2 mM. That minoxidil almost completely reversed this inhibitory effect suggests it may work by preventing cross-linking by transglutaminase, which is activated by elevated calcium concentrations.
We have previously described a human keratinocyte line, NM1, which had been carried for more than 400 doublings, was trisomic for chromosome 8, and appeared to make a number of structural proteins characteristic of keratinocytes. This line has now been carried for more than 800 doublings and grows with the same vigor. It reaches confluence in 7 to 10 days and can be grown without a feeder layer for more than 15 passages. Its karyotype has remained 47,XY, +8. The current NM1 cells make readily detectable amounts of 67 kd and 48 kd keratins, and it has been established that the previously poorly resolved 58 kd band actually consists of 58 kd and 59 kd bands. We have also found that the apparent 56 kd band consists of the 56 kd and 56.5 kd bands. A unique basic polypeptide precursor of the cornified envelope has been discovered in the NM1 line. Although similar in charge to one in normal cells it is lower in molecular weight.
The bovine hoof has been examined as a model for the study of keratinized skin appendages. We characterized the keratin polypeptides of hoof bed and matrix and compared them to epidermis using two-dimensional electrophoresis and immunoblot techniques. Both hoof tissues express keratins 6 and 16 (as described by Franke et al. (1981) J. Mol. Biol. 153, 933-959) and b2 and a1-4 which are previously undescribed proteins unique to the bovine hoof. Keratins of hoof matrix and bed share one or more common antigenic components as defined by immunoblot analysis. Hoof matrix expresses keratins 7 and 14, which are absent in hoof bed, and also expresses a greater number of isoelectric variants of keratin 6. Biopsies of hoof bed and matrix transplanted onto athymic mice both made hard hoof and underwent active keratin synthesis as evidenced by incorporation of [3H]leucine. Indirect immunofluorescence studies of the grafts showed that they had the histology and immunoreactivity previously noted for hoof bed and matrix. The two-dimensional gel electrophoretic patterns of both grafts were similar and expressed keratins b2 and a1-4. We conclude that a unique group of keratins exists in hoof. Furthermore, while hoof matrix is the major contributor to hard hoof, hoof bed epidermis maintains the capacity to make hard hoof and may contribute to the synthesis of the hoof plate in vivo. The ability to graft hoofs onto athymic mice provides an opportunity for the study of a number of aspects of hoof formation.
We examined seven invasive squamous cell carcinomas, five squamous cell carcinomas in situ, four keratoacanthomas, two actinic keratoses, and two seborrheic keratoses by indirect immunofluorescence. We used a panel of three antibodies: one directed against filaggrin, one against involucrin, and one against peptidylarginine deiminase. Anti-involucrin stained all the lesions studied, but the pattern within a given category of lesions was variable and consistent differences between the categories were not observed. Similarly, the antibodies against peptidylarginine deiminase and filaggrin were not able to distinguish differences between the various types of tumors. We conclude that in tumors of epidermis, benign or malignant, products of differentiation are expressed independently of histologic atypia or clinical aggressiveness. Therefore, markers of differentiation do not appear to be reliable indexes for distinguishing benign from malignant lesions.
Hirsutism, a troublesome cosmetic problem, may also be a sign of serious systemic disease that can be screened for by means of the patient's history, physical examination, and certain basic blood tests. Most patients with functional hirsutism have elevated production rates of testosterone, elevated metabolic clearance rates of testosterone, depressed levels of sex hormone-binding globulin, and elevated levels of serum-free testosterone and hair follicle sensitivity. Once a neoplastic source is ruled out and hirsutism established as functional, essentially empiric treatment, including low-dose dexamethasone, oral contraceptives, and spironolactone, has proved to be effective.
The efficacy of electronic monitoring in the home care of heart failure (HF) patients has not been widely reported. We developed a Vital Sign System (VSS) monitoring device capable of measuring the weight, blood pressure, and heart rate of congestive heart failure (CHF) patients in the home and transmitting these measurements via modem to a World Wide Web server. In this study of 22 CHF patients, we tested the reliability of the VSS electronic measurements compared to manual measurements taken by visiting home care nurses and ease of use of the VSS units as rated by both patients and home care nurses. The correlation of electronic to manual measurements was high (weight r=0.99; systolic blood pressure [SBP] r=0.84; diastolic blood pressure [DBP] r=0.54; heart rate [HR] r=0.88). The mean difference between electronic and manual measurements was within an acceptable range for clinical surveillance and care of CHF patients (weight 1.6 lbs; SBP 8.8 mm Hg; DBP 9.2 mm Hg; HR 0.7 bpm) The devices were rated favorably by both nurses and patients. The VSS monitoring device is a reliable, feasible, and favorably rated technology for home surveillance of CHF patients. (c)1999 by CHF, Inc.