Patterns of keratin expression define distinct pathways of epithelial development and differentiation.
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Animals↗
Biomedical subjects
T T Sun
Publications and source records attributed to T T Sun.
Suprabasal expression of a 64-kilodalton keratin (no. 3) in developing human corneal epithelium.
We have previously shown that a basic 64-kilodalton (no. 3 in the catalog of Moll et al.) and an acidic 55-kilodalton (no. 12) keratin are characteristic of suprabasal cell layers in cultured rabbit corneal epithelial colonies, and therefore may be regarded as markers for an advanced stage of corneal epithelial differentiation. Moreover, using an AE5 mouse monoclonal antibody, we showed that the 64-kilodalton keratin marker is expressed suprabasally in limbal epithelium but uniformly (basal layer included) in central corneal epithelium, suggesting that corneal basal cells are in a more differentiated state than limbal basal cells. In conjunction with previous data implicating the centripetal migration of corneal epithelial cells, our data support a model of corneal epithelial maturation in which corneal epithelial stem cells are located in the limbus, the transitional zone between the cornea and conjunctiva. In the present study, we analyzed the expression of the 64-kilodalton keratin in developing human corneal epithelium by immunohistochemical staining. At 8 weeks of gestation, the presumptive corneal epithelium is composed of a single layer of cuboidal cells with an overlying periderm; neither of these cell layers is AE5 positive. At 12-13 weeks of gestation, some superficial cells of the three- to four-layered epithelium become AE5 positive, providing the earliest sign of overt corneal epithelial differentiation. At 36 weeks, although the epithelium is morphologically mature (four to six layers), AE5 produces a suprabasal staining pattern, this being in contrast to the adult epithelium which exhibits uniform staining.(ABSTRACT TRUNCATED AT 250 WORDS)
Aging↗
Monoclonal antibody analysis of bovine epithelial keratins. Specific pairs as defined by coexpression.
We have characterized the keratin proteins of various bovine epithelial tissues by one- and two-dimensional gel electrophoresis, coupled with the immunoblot technique using AE1, AE2, AE3, AE5, CA20, BE14, and 6.11 monoclonal antikeratin antibodies. The results indicate that all known bovine keratins can be divided into two subfamilies. The "acidic" (Type I) subfamily consists of 41-, 43-, 45-, 46-, 50-, 54-, 56-, and 56.5-kDa keratins, all of which have a pI of less than 5.6, and most of them are recognized by our AE1 antibody, whereas the "neutral-to-basic" (Type II) subfamily consists of 55-, 57-, 58-, 62-65-, 66-, and 67-kDa keratins, all of which have a pI of greater than 6.0 and are recognized by our AE3 antibody. Tissue distribution data and cell culture studies show that, within the two subfamilies, keratins with similar "size ranks" form a "pair" as defined by frequent co-expression. Furthermore, within most "keratin pairs," the basic keratin is larger than the acidic one by 8-10 kDa. These results provide further support for the concepts of "keratin subfamilies" and keratin pairs and are consistent with the possibility that the acidic and basic members of at least some keratin pairs may interact specifically during in vivo tonofilament assembly and/or function. Immunoblotting data derived from the use of several monospecific antibodies show that although the size, charge, and pattern of expression of most bovine keratins are similar to those of the human counterparts, there are important exceptions to this rule.
Animals↗
The role of keratin subfamilies and keratin pairs in the formation of human epidermal intermediate filaments.
The four major keratins of normal human epidermis (molecular mass 50, 56.5, 58, and 65-67 kD) can be subdivided on the basis of charge into two subfamilies (acidic 50-kD and 56.5-kD keratins vs. relatively basic 58-kD and 65-67-kD keratins) or subdivided on the basis of co-expression into two "pairs" (50-kD/58-kD keratin pair synthesized by basal cells vs. 56.5-kD/65-67-kD keratin pair expressed in suprabasal cells). Acidic and basic subfamilies were separated by ion exchange chromatography in 8.5 M urea and tested for their ability to reassemble into 10-nm filaments in vitro. The two keratins in either subfamily did not reassemble into 10-nm filaments unless combined with members of the other subfamily. While electron microscopy of acidic and basic keratins equilibrated in 4.5 M urea showed that keratins within each subfamily formed distinct oligomeric structures, possibly representing precursors in filament assembly, chemical cross-linking followed by gel analysis revealed dimers and larger oligomers only when subfamilies were combined. In addition, among the four major keratins, the acidic 50-kD and basic 58-kD keratins showed preferential association even in 8.5 M urea, enabling us to isolate a 50-kD/58-kD keratin complex by gel filtration. This isolated 50-kD/58-kD keratin pair readily formed 10-nm filaments in vitro. These results demonstrate that in tissues containing multiple keratins, two keratins are sufficient for filament assembly, but one keratin from each subfamily is required. More importantly, these data provide the first evidence for the structural significance of specific co-expressed acidic/basic keratin pairs in the formation of epithelial 10-nm filaments.
Cytoskeleton↗
Differentiation-dependent changes in the solubility of a 195-kD protein in human epidermal keratinocytes.
We have prepared a monoclonal antibody, AE11, that recognizes specifically a 195-kD protein (pI 5.4) of human keratinocytes. This antigen constitutes approximately 0.01-0.1% of total protein in keratinocytes of skin, esophagus, and cornea, and is readily detectable in these cells by immunofluorescent staining and immunoblotting. However, it is barely detectable in MCF mammary carcinoma cells and HeLa cells, and is undetectable in nonepithelial cell types. Results from serial extraction experiments have shown that this protein exists in two distinct pools: a Tris-soluble, and a Tris-insoluble but urea- or SDS-soluble one. The distribution of the 195-kD protein between these two pools appears to be differentiation-related, since relatively undifferentiated cells selected by a low-calcium medium contain primarily the soluble form, while highly differentiated cells contain mainly the insoluble form. Data from immunofluorescent staining and trypsin-sensitivity experiments suggest that the soluble form is cytoplasmic, whereas the insoluble form is submembranously located at the cell periphery of upper, differentiated cells. The insoluble, cell peripheral form of the 195-kD antigen increases progressively during epidermal differentiation; its insolubility appears to be related to the formation of disulfide-bond(s). These results indicate that the 195-kD protein, which has recently been suggested to be involved in cornified envelope formation (Simon, M., and H. Green, 1985, Cell, 36:827-834), undergoes significant changes in its solubility characteristics and intracellular location during keratinocyte maturation.
Antibodies, Monoclonal↗
Differentiation-related expression of a major 64K corneal keratin in vivo and in culture suggests limbal location of corneal epithelial stem cells.
In this paper we present keratin expression data that lend strong support to a model of corneal epithelial maturation in which the stem cells are located in the limbus, the transitional zone between cornea and conjunctiva. Using a new monoclonal antibody, AE5, which is highly specific for a 64,000-mol-wt corneal keratin, designated RK3, we demonstrate that this keratin is localized in all cell layers of rabbit corneal epithelium, but only in the suprabasal layers of the limbal epithelium. Analysis of cultured corneal keratinocytes showed that they express sequentially three major keratin pairs. Early cultures consisting of a monolayer of "basal" cells express mainly the 50/58K keratins, exponentially growing cells synthesize additional 48/56K keratins, and postconfluent, heavily stratified cultures begin to express the 55/64K corneal keratins. Cell separation experiments showed that basal cells isolated from postconfluent cultures contain predominantly the 50/58K pair, whereas suprabasal cells contain additional 55/64K and 48/56K pairs. Basal cells of the older, postconfluent cultures, however, can become AE5 positive, indicating that suprabasal location is not a prerequisite for the expression of the 64K keratin. Taken together, these results suggest that the acidic 55K and basic 64K keratins represent markers for an advanced stage of corneal epithelial differentiation. The fact that epithelial basal cells of central cornea but not those of the limbus possess the 64K keratin therefore indicates that corneal basal cells are in a more differentiated state than limbal basal cells. These findings, coupled with the known centripetal migration of corneal epithelial cells, strongly suggest that corneal epithelial stem cells are located in the limbus, and that corneal basal cells correspond to "transient amplifying cells" in the scheme of "stem cells----transient amplifying cells----terminally differentiated cells."
Animals↗
Acidic and basic hair/nail ("hard") keratins: their colocalization in upper cortical and cuticle cells of the human hair follicle and their relationship to "soft" keratins.
Although numerous hair proteins have been studied biochemically and many have been sequenced, relatively little is known about their in situ distribution and differential expression in the hair follicle. To study this problem, we have prepared several mouse monoclonal antibodies that recognize different classes of human hair proteins. Our AE14 antibody recognizes a group of 10-25K hair proteins which most likely corresponds to the high sulfur proteins, our AE12 and AE13 antibodies define a doublet of 44K/46K proteins which are relatively acidic and correspond to the type I low sulfur keratins, and our previously described AE3 antibody recognizes a triplet of 56K/59K/60K proteins which are relatively basic and correspond to the type II low sulfur keratins. Using these and other immunological probes, we demonstrate the following. The acidic 44K/46K and basic 56-60K hair keratins appear coordinately in upper corticle and cuticle cells. The 10-25K, AE14-reactive antigens are expressed only later in more matured corticle cells that are in the upper elongation zone, but these antigens are absent from cuticle cells. The 10-nm filaments of the inner root sheath cells fail to react with any of our monoclonal antibodies and are therefore immunologically distinguishable from the cortex and cuticle filaments. Nail plate contains 10-20% soft keratins in addition to large amounts of hair keratins; these soft keratins have been identified as the 50K/58K and 48K/56K keratin pairs. Taken together, these results suggest that the precursor cells of hair cortex and nail plate share a major pathway of epithelial differentiation, and that the acidic 44K/46K and basic 56-60K hard keratins represent a co-expressed keratin pair which can serve as a marker for hair/nail-type epithelial differentiation.
Antibodies, Monoclonal↗
Differentiation-dependent expression of keratins in human oral epithelia.
The polypeptide composition of epithelial keratins varies with the state of differentiation. The epithelia lining the human oral cavity show regional variations in their histology. In the present study, paired samples of nonkeratinized buccal epithelium and keratinized hard palate epithelium were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and by immunoblots with monoclonal antibodies AE1, AE2, and AE3, and results were correlated with immunofluorescence staining of tissue sections of the same samples. Keratins from hard palate (Mr 67K, 63-65K, 58K, 56.5K, 56K, 50K, 48K) and epidermis (Mr 67K, 63-65K, 58K, 56.5K, 50K) were similar to each other but distinctly different from those of buccal epithelium (major bands of Mr 52K and 59K, minor bands of 50K and 58K). The immunoblot analysis further indicated the similarity of hard palate and epidermal keratins, in contrast to those of buccal epithelium. Each oral tissue expressed keratins of the type I (AE1, acidic) subfamily and type II (AE3, basic) subfamily. In tissue sections, the predominant staining pattern for nonkeratinized buccal epithelium was: AE1, positive in the basal layer; AE2, negative; AE3, positive in all layers. In contrast, the staining pattern for keratinized palatal epithelium was: AE1 and AE2, positive in the suprabasal layers; AE3, positive in all layers. Strong suprabasal AE1 staining in palate may be related to the presence of the 48K keratin. Some buccal samples showed an alternate staining pattern of spotty suprabasal staining with AE1 and AE2 which was correlated with the expression of the 56.5K and 63-67K keratins, as well as filaggrin. These results suggest differentiation-specific expression of the keratins and show immunologically detectable variation in the apparently normal differentiation pattern of nonkeratinized buccal epithelium.
Antibodies, Monoclonal↗
Clinical, electron microscopic, and monoclonal antibody studies of intraocular epithelial downgrowth.
Epithelial downgrowth developed in three patients following cataract extraction and keratoplasty. Light and electron microscopy of the downgrowth tissue disclosed stratified squamous epithelium, but could not determine whether they were derived from conjunctival or corneal epithelium. The epithelial downgrowth contained cells that were connected laterally by desmosomal junctions and displayed well-defined basement membranes. Surface epithelial cells exhibited myriad microvillous processes scant to moderate mitochondria. In one case, prominent hemidesmosomal junctions were present. Immunohistochemical staining with monoclonal antikeratin antibodies revealed immunoreactivity with AE1, AE3, and AE11 in all cases, but with a cornea-specific antibody, AE5, in only one case. We concluded that in this last case, the epithelial downgrowth appears to have originated from the corneal epithelium.
Adult↗
A pilot study on universal immunization of newborn infants in an area of hepatitis B virus and primary hepatocellular carcinoma prevalence with a low dose of hepatitis B vaccine.
Hepatitis B virus (HBV) had been considered as the main causative factor of primary hepatocellular carcinoma and universal immunization of newborns was recommended as the major approach to control hepatitis and hepatoma in areas of prevalence. As the initial phase of the first vaccination program for such a purpose, a pilot study was done from September 1983 to May 1984 in a high incidence rural area of China. In an area of 214,343 inhabitants, 1,703 newborns (99% of all births) were vaccinated. Ninety-seven percent of all vaccinees were followed up at 1 year. The vaccine used was Hep-B Vax, given intramuscularly at 0, 1, and 6 months after birth. Four immunization regimes were used: 5-micrograms or 2.5-micrograms doses with or without hepatitis B immune globulin (HBIG) added in the case of carriers children. These groups were defined by drawing lots at community level. A matched control was selected on a voluntary basis. Each group consisted of 400 infants. Vaccination was proven to be very safe and well accepted by the public. The prevalence of HBV infection in the area was further demonstrated by the high HBsAg-positive rate measured: 14.2% of the 1,180 mothers (3.9% were also e-antigen positive), 7.6% and 10.1% of the unvaccinated children at 6.5 months and 1 year of age, respectively. It was shown that vaccination with a 5-micrograms or 2.5-micrograms dose significantly lowered the HBsAg positives to a level close to 1.5% versus 10% in the control group at 1 year. An 85% protection was thus achieved. A 5-micrograms dose plus HBIG did not show additional benefit. A 2.5-micrograms dose plus HBIG gave less protection, and anti-HB levels were also significantly lower than in other groups. Among the 12 failures found in the 5-micrograms and 2.5-micrograms groups, 11 were born to HBsAg-positive mothers, nine of whom also had e-antigen. Available data showed that 29% of children born by e-antigen-positive and 2.7% of children born by e-antigen-negative carriers had the risk of becoming carriers during the first year of life following vaccination. The present study demonstrated the feasibility and rationale of conducting universal immunization of newborns in endemic rural area for controlling hepatitis and hepatoma. The significance of the possible use of the vaccination at lower dose had also been stressed.
Adult↗
Interactive effects of chemical carcinogens and hepatitis B virus in the pathogenesis of hepatocellular carcinoma.
Liver cancer is one of the most prevalent forms of cancer in the world. Hepatitis B virus (HBV) is considered to be a major aetiological factor. Evidence from epidemiological studies has also indicated that environmental contaminants such as mycotoxins may, either in combination with HBV or independently, be important aetiological factors in the pathogenesis of primary hepatocellular carcinoma (PHC). Laboratory data also suggest an interplay between viral and chemical factors in the multifactorial aetiology of PHC. Aflatoxin B1, the chemical carcinogen most frequently implicated in the aetiology of hepatocellular carcinoma is a procarcinogen that must be activated by mixed-function oxidases to an electrophilic metabolite before it can exert its carcinogenic effects. Interindividual differences (greater than 10-fold) in the metabolic activation of aflatoxin B1 are observed. These differences may play a part in an individual's oncogenic susceptibility to aflatoxin B1. Chemical carcinogens and integrated HBV may activate cellular oncogenes, eg N-ras, and inactivate tumour suppressor genes. Recently developed methods that allow monitoring of aflatoxin B1 and HBV exposures and also genetic damage caused by these agents in individuals should help in biochemical and molecular epidemiological studies concerning the aetiology of hepatocellular carcinoma. We identify areas of uncertainties and of future experimentation and propose a hypothesis of liver carcinogenesis.
Aflatoxin B1↗
Comparison of 68Ga-EDTA, [1-11 C]alpha-aminoisobutyric acid, and [99mTc]sodium pertechnetate in an experimental blood-brain barrier lesion.
Unilateral opening of the blood-brain barrier (BBB) in rats, induced by infusion of a hyperosmotic solution of mannitol into the left internal carotid artery, was applied to compare 68Ga-EDTA, [1-11C]alpha-aminoisobutyric acid (using the long-lived 14C-labeled analog), and [9mTc]sodium pertechnetate as agents for diagnosis of BBB disruption. Of the three agents and two time intervals studied, 68Ga-EDTA at 30 min postinjection gave the highest target-to-nontarget ratio. In addition, 68Ga-EDTA, unlike commonly used [99mTc]sodium pertechnetate, can be used in conjunction with positron emission tomography, which could make possible earlier and better assessment of BBB defects using 68Ga-EDTA.
Aminoisobutyric Acids↗
Expression of epidermal keratins and filaggrin during human fetal skin development.
The major structural proteins of epithelia, the keratins, and the keratin filament-associated protein, filaggrin, were analyzed in more than 50 samples of human embryonic and fetal skin by one-dimensional SDS PAGE and immunoblotting with monoclonal and polyclonal antibodies. Companion samples were examined by immunohistochemistry and electron microscopy. Based on structural characteristics of the epidermis, four periods of human epidermal development were identified. The first is the embryonic period (before 9 wk estimated gestational age), and the others are within the fetal period: stratification (9-14 wk), follicular keratinization (14-24 wk), and interfollicular keratinization (beginning at approximately 24 wk). Keratin proteins of both the acidic (AE1-reactive, type I) and the basic (AE3-reactive, type II) subfamilies were present throughout development. Keratin intermediate filaments were recognized in the tissue by electron microscopy and immunohistochemical staining. Keratins of 50 and 58 kD were present in the epidermis at all ages studied (8 wk to birth), and those of 56.5 and 67 kD were expressed at the time of stratification and increased in abundance as development proceeded. 40- and 52-kD keratins were present early in development but disappeared with keratinization. Immunohistochemical staining suggested the presence of keratins of 50 and 58 kD in basal cells, 56.5 and 67 kD in intermediate cells, and 40 and 52 kD in the periderm as well as in the basal cells between the time of stratification and birth. Filaggrin was first detected biochemically at approximately 15 wk and was localized immunohistochemically in the keratinizing cells that surround hair follicles. It was identified 8-10 wk later in the granular and cornified cell layers of keratinized interfollicular epidermis. These results demonstrate the following. An intimate relationship exists between expression of structural proteins and morphologic changes during development of the epidermis. The order of expression of individual keratins is consistent with the known expression of keratins in simple vs. stratified vs. keratinized epithelia. Expression of keratins typical of stratified epithelia (50 and 58 kD) precedes stratification, and expression of keratins typical of keratinization (56.5 and 67 kD) precedes keratinization, which suggests that their expression marks the tissue commitment to those processes. Because only keratins that have been demonstrated in various adult tissues are expressed during fetal development, we conclude that there are no "fetal" keratins per se.(ABSTRACT TRUNCATED AT 400 WORDS)
Electrophoresis, Polyacrylamide Gel↗
Monoclonal antibody studies of mammalian epithelial keratins: a review.
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Animals↗
Change in epithelial keratin expression during healing of rabbit corneal wounds.
Corneal epithelial wound healing following full-thickness trephination and transcorneal freeze injury was studied by electron microscopy and immunofluorescent microscopy using monoclonal antibodies AE1, AE2, and AE3 to human epithelial keratin. Wounds were evaluated at various time intervals between 4 hr and 2 mo after injury. By scanning and transmission electron microscopy, epithelial migration was evident 4 hr after injury and was characterized by thinning of the epithelium and extension of filopodial processes. AE1 monoclonal antibody, which stains specifically the superficial cells of normal corneal epithelium, reacted to cells at the leading edge of the migrating epithelium. By 24 hr, all cells migrating over the wound displayed positive fluorescence with AE1 while the epithelium over the undamaged cornea exhibited normal fluorescence limited to the superficial epithelial cells. In full-thickness corneal wounds, reepithelialization was complete by 1-2 wk; however, all epithelial cells covering the wound remained positive for the AE1 antikeratin antibody. By 2 mo, the AE1 fluorescence returned to normal. In transcorneal freeze injuries, reepithelialization was complete by 4 to 7 days after injury, with all cells overlying the wound reacting with the AE1 antibody. By 2 wk after freeze injury, all epithelial cells appeared to express a normal AE1 staining pattern. No change was noted in the fluorescent distribution of either AE2 antibody, which did not react with the corneal epithelium, or AE3, which reacts with all corneal epithelial cells. These results suggest that healing of corneal epithelial wounds involves changes in keratin expression of the corneal epithelium.
Animals↗
Classification of human epithelia and their neoplasms using monoclonal antibodies to keratins: strategies, applications, and limitations.
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Antibodies, Monoclonal↗
Measurement of individual aflatoxin exposure among people having different risk to primary hepatocellular carcinoma.
The establishment of the carcinogenic role of aflatoxins has been impeded by the lack of suitable tests to measure individual exposure for long-term studies. It was shown that the use of immuno-concentration followed by high pressure liquid chromatography (HPLC) or immunoassay can regularly detect aflatoxins down to pg/ml in fluids including urine. Aflatoxin M1 (AFM1) in free form was identified as the major metabolite in urine suitable for use as an approximate dosimetric indicator of recent exposure to aflatoxin B1 (AFB1). A panel of monoclonal antibodies of IgG class were developed in the murine system against AFB1 and/or AFM1. Their affinity constants are at the level of 10(8)-10(9) L/mol, and they are suitable for use in either effective immuno-concentration or in high sensitivity immunoassays. It was shown in a high risk area of liver cancer (Qidong of China) that 10% or more of the local inhabitants had a urinary output of AFM1 one or two orders of magnitude higher than that of Beijing people, especially during the wet seasons. The ingestion of AFB1 among these local people was estimated to exceed 1 mg/year. The major source came from contaminated corn and rice, but local alcoholic beverages also contributed. The increased excretion of AFM1 was more pronounced in patients with chronic active hepatitis. This observation of very low AFM1 content in urine of a significant percentage of local people implies their food storage practices may offer a feasible method for such prevention. The value and possible problems in conducting long-term studies on primary prevention are discussed.
Aflatoxins↗
Antikeratin antibodies in tumor diagnosis. Distinction between seminoma and embryonal carcinoma.
The authors investigated the presence and distribution of keratin in germ cell tumors using a rabbit-anti-keratin antiserum and a monoclonal antikeratin antibody--which is specific for keratin classes of 40, 50, and 56.5 kdaltons--by various immunohistochemical methods on frozen sections, alcohol-fixed, and formalin-fixed paraffin-embedded tissues. Thirty-four germ cell tumors were studied. These were the following: 18 seminomas, 10 embryonal carcinomas, 2 teratocarcinomas, 3 yolk sac tumors and 1 choriocarcinoma. All seminomas, including four poorly differentiated (so-called anaplastic seminomas), gave negative results, regardless of the method employed. Embryonal carcinoma, the epithelial component of the teratocarcinoma, the yolk sac tumors, and choriocarcinoma were at least focally positive for keratin. The monoclonal antibody provided a cleaner background and stronger staining than the rabbit-anti-total-human-epidermal-keratin antibody. Best results were obtained from fresh-frozen sections or alcohol-fixed, paraffin-embedded materials. Formalin-fixed, nonseminomatous tumors, when predigested with trypsin and incubated overnight with primary antibody, gave no false-negative results but staining was often focal. The authors' results agree with the reported absence of detectable keratin in primordial germ cells of the normal testis, and with prevailing concepts of the histogenesis of germ cell tumors. These results indicate that the presence or absence of keratin by immunocytochemical methods can be helpful in distinguishing seminoma from embryonal carcinoma.
Antibodies, Monoclonal↗