Mouse papillary lung tumors transplacentally induced by N-nitrosoethylurea: evidence for alveolar type II cell origin by comparative light microscopic, ultrastructural, and immunohistochemical studies.

A histogenetic study was designed to evaluate controversial findings on the cell of origin of tubular/papillary lung tumors in mice, i.e., bronchiolar Clara cell versus alveolar type II cell. N-Nitrosoethylurea (0.5 mmol or 0.74 mmol/kg) was given to pregnant C3H (C3H/HeNCr MTV-) and Swiss Webster [Tac:(SW)fBR] mice as a single i.p. injection on Day 14, 15, 16, or 18 of gestation. The offspring were studied at various ages ranging from 7 days to 52 wk. Serial sections of the whole lung (100 to 200 sections per mouse) showed that solid/alveolar and papillary tumors arose from the pulmonary acinus, invading the bronchioles only as the tumors grew. Furthermore, a mixture of solid and papillary patterns within a single module did not represent a merging of two tumors but a progression from the solid to the papillary form. By use of two rabbit antisera against mouse lung surfactant apoproteins found in normal alveolar type II cells, it was shown by the avidin-biotin peroxidase complex procedure, by the peroxidase-antiperoxidase technique, and by indirect immunofluorescence that both solid and papillary tumors contained these proteins that are specific markers for alveolar type II cells. With a rabbit anti-rat Clara cell antiserum, none of the tumors studied was immunoreactive while normal Clara cells were reactive. The nitroblue tetrazolium formazan stain for dehydrogenase enzymes, found particularly in Clara cells, did not reveal these enzymes in any lung tumors from either strain. Ultrastructurally, no typical features of the mature Clara cell were detected in papillary or other pulmonary neoplasms. However, all tumors showed characteristic alveolar type II cell structures such as various stages of lamellar body formation, although these features were less well differentiated in the papillary tumors. Argentaffin dense bodies, representing lysosomes and immature forms of lamellar bodies, were commonly observed in papillary tumors. Some features of the papillary tumors such as cell shape, high glycogen content, and primary cilia were equivalent to those seen in pulmonary epithelial precursor cells during fetal development. With age, the papillary tumors became invasive, accumulated neutral lipids, and developed bizarre cleaved nuclei and lamellated nuclear pseudoinclusions. In conclusion, the papillary lung tumors of the mouse, at least those induced transplacentally by N-nitrosoethylurea, constitute less well-differentiated or poorly differentiated alveolar type II cell adenomas or carcinomas with fetal morphological and biochemical properties.

Isolation of alveolar type II cells from fetal rat lung by differential adherence in monolayer culture.

Type II alveolar epithelial cells were isolated from fetal rat lung by differential adherence in monolayer culture. The preparation had a high degree of purity, as assessed by phase contrast microscopy and immunocytochemistry. Purity, based on reactivity with specific anti-adult lung serum (SAALS), which recognizes only type II cells, was 91% for cells isolated from 19-day fetal lungs and 79% for cells isolated from 21-day fetal lungs. The lower purity of type II cells in cultures derived from 1-day postnatal rat lungs (51% cells reactive with SAALS) is probably due to a lower tendency of the type II cells from neonatal rats to adhere to culture dishes than of type II cells from fetal rats. Type II cells isolated from 21-day fetal lungs contained a higher percentage phosphatidylglycerol and incorporated [Me-3H]choline faster into phosphatidylcholine (PC) than type II cells isolated from 19-day fetal lungs. Moreover, in cell preparations derived from lungs at fetal day 21, a higher percentage of epithelial cells contained lamellar bodies than in preparations derived from lungs at fetal day 19. The observation of these differences in the stage of maturation indicates that these differences, which are typical features of the original material, are not obliterated by differentiation during the culture. Type II cells isolated according to the present procedure were capable of synthesizing PC with a high percentage of the disaturated species. This method for the isolation of fetal type II cells may be a useful tool in studies concerning surfactant synthesis and its regulation in the fetal lung.

Inhibition of T3-receptor binding by Nitrofen.

Lung development is controlled by various hormones, including thyroid hormone. The herbicide 2,4-dichlorophenyl-p-nitrophenyl ether (Nitrofen) induces lung hypoplasia in fetal rats, when administered to the mother during gestation. Nitrofen might be teratogenic by an anti-thyroid activity. The present study shows that Nitrofen decreases the binding of T3 to the alpha 1 and beta 1 form of the thyroid hormone receptor in a non-competitive way. Consequently, rat lung hypoplasia might result from the decreased binding of T3 to its receptor, via exposure to Nitrofen during fetal development.

Expression of Tcf/Lef and sFrp and localization of beta-catenin in the developing mouse lung.

Recent evidence that Wnts and other genes in the Wnt signaling pathway are expressed in embryonic and adult mouse lung suggests that this pathway is important for cell fate decisions and differentiation of lung cell types. We therefore examined the expression and protein distribution of several Wnt pathway components during prenatal mouse lung development using whole-mount in situ hybridization and immunohistochemistry. Between embryonic days 10.5 and 17.5 (E10.5-E17.5), beta-catenin was localized in the cytoplasm, and often also the nucleus, of the undifferentiated primordial epithelium (PE), differentiating alveolar epithelium (AE; present from E14.5 onward), and adjacent mesenchyme. Tcf1, Lef1, Tcf3, Tcf4, sFrp1, sFrp2 and sFrp4 were also expressed in the PE, AE, and adjacent mesenchyme in specific spatio-temporal patterns.

Expression of the major surfactant-associated protein, SP-A, in type II cells of human lung before 20 weeks of gestation.

In a previous paper (Otto-Verberne et al., Anat. Embryol. 178, 29-39 (1988) we reported that the type II alveolar epithelial cell can be identified in fetal human lung on the basis of morphological and immunological characteristics from 10 to 12 weeks after conception (a.c.) onward. For immunological recognition we used a lung-specific antibody, called SALS-Hu (specific anti-lavage serum, rabbit antihuman). The present immunoblotting experiments, after one-and two-dimensional electrophoresis, showed that SALS-Hu-reactive proteins in lavage fractions obtained from alveolar proteinosis patients exhibited molecular masses of mainly 29, 31 to 36, and 62 to 66 kDa. All SALS-Hu-reactive proteins migrated in the same acidic isoelectric point range (pI 4.4-5.1) and were almost undetectable when we used SALS-Hu preabsorbed with recombinant surfactant-associated protein A. We concluded that SALS-Hu recognizes exclusively isoforms of the major surfactant-associated protein, SP-A. In vitro translation assays in which we used mRNA isolated from adult human lung confirmed that SALS-Hu recognized the 29 to 31 kDa SP-A precursor proteins. These SALS-Hu-immunoreactive precursors for SP-A were already detectable (though in much lower amounts) in human fetuses aged 17 to 18 weeks, indicating that mRNA coding for SP-A is present at that time. We concluded that the cytoplasmic staining of fetal (from 10-12 weeks a.c. onward) and adult human type II cells by SALS-Hu is due to the presence of SP-A.

Bronchiolo-alveolar regions in adenocarcinoma arising from canine segmental bronchus.

Adenocarcinomas induced in canine bronchial segments placed subcutaneously have bronchiolo-alveolar regions. Immunocytochemistry and routine staining of adjacent sections strongly suggests that the lining of these regions consists of type II cells. These regions may thus represent true prospective alveolar regions, as also seen in embryonic lungs. This first observation of bronchoalveolar cancer arising from a major bronchus indicates that the carcinogen-induced neoplastic progression in bronchial epithelium may lead to type II cell differentiation and type II cell tumor development. The preservation of cell properties in serial nude mouse transplants suggests that it is a stable type II cell population.

Alveolar stem cells in canine bronchial carcinogenesis.

Alveolar type II cells are not present in normal epithelium of canine segmental bronchi but after carcinogen exposure they do occur in intra-epithelial lesions with all degrees of atypia and in invasive lesions with different glandular growth patterns. Immunohistochemistry for proliferation markers (PCNA; Ki-67) strongly suggest that such novel type II cells are pluripotential stem cells in canine bronchial carcinogenesis. Very likely, bronchial carcinogenesis is subject to an oncofetal mechanism of differentiation: bronchial epithelial retrodifferentiation followed by novel differentiation of alveolar tumor stem cells.

The alveolar type II cell is a pluripotential stem cell in the genesis of human adenocarcinomas and squamous cell carcinomas.

Studies in a canine bronchogenic carcinoma model indicate that alveolar type II cells may differentiate from carcinogen-exposed epithelium of larger bronchi and generate adenocarcinomas with bronchioloalveolar and other growth patterns. In this study, we investigated whether type II cells are one of the major proliferating cells (= stem cells) in the genesis of two major subsets of bronchogenic carcinoma in humans. Adenocarcinomas (17 bronchioloalveolar; 3 papillary; and 10 other) and squamous cell carcinomas (n = 27) as well as (pre)neoplastic lesions in adjacent bronchi and bronchioles were examined for the presence of type II cell markers and cellular proliferation markers (PCNA; Ki-67) using light and electron microscopy and immunohistochemistry. Distinctive features of type II cells, which do not depend upon the degree of cell maturity, are the approximately cuboid shape, large and roundish nucleus, cytoplasmic staining for surfactant protein A (SP-A), and presence of multilamellar bodies or their precursory forms. Cells with this phenotype were found in early progressive (i.e., dysplastic, in situ, microinvasive) lesions in conducting airways and in all the carcinomas investigated, although with a much greater abundance among glandular lesions compared to squamous lesions. The most consistent sites of type II cells were the basal and adjacent epithelial layers. Nuclear PCNA (Ki-67) expression usually predominated in the same region. None of the lesions displayed specific Clara cell features. Our findings strongly suggest that the type II cell is a pluripotential stem cell in human lung carcinogenesis. Based on our findings in humans and dogs, we postulate that type II tumor stem cells may originate from one of two sources: (1) normal bronchial epithelium (by an oncofetal mechanism of differentiation); and (2) normal alveolar type II cells.

In favour of an oncofoetal concept of bronchogenic carcinoma development.

Our recent studies in a heterotopic model of non-small cell lung cancer in dogs (subcutaneous bronchial autografts treated with 3-methylcholanthrene) have provided evidence that alveolar type II cells may newly arise during initial phases of bronchial carcino-genesis. In the light of these novel findings, which are in agreement with our observations in human non-small cell lung cancer, and in view of present insights into embryonic lung differentiation, we discuss evidence that favours a new, oncofoetal concept of bronchogenic carcinoma development. According to this concept, the primary cells of origin for these tumors are undifferentiated primordial-like cells that derive from bronchial epithelial cells present in major bronchi or their divisions by retrodifferentiation. Such primordial-like cells of origin undergo novel differentiation into the potential (alveolar, bronchial or primordial) tumor stem cells, which occupy the dividing cellular layers of the (pre)neoplastic lesions and constitute the actively dividing and invading part of the neoplasm. Examples of tumors that may originate from alveolar tumor stem cells are carcinomas of the bronchiolo-alveolar, papillary, acinar, and adenoid-cystic types. Squamous cell carcinomas could possibly belong to this group as well, but much more evidence is required to reach conclusions regarding this type of cancer. We suggest that epithelial retrodifferentiation followed by novel differentiation (oncofoetal mechanism) is fundamental in bronchial carcinogenesis.

Clara cell differentiation in the mouse: ultrastructural morphology and cytochemistry for surfactant protein A and Clara cell 10 kD protein.

The morphologic and functional differentiation of the nonciliated columnar (Clara) cell, one of two secretory cell types in distalmost bronchioles in mammals, was studied in the mouse. Lungs from embryos (16-19 days of developmental age, dDA; birth on day 19), postnatal animals (5-20 days postnatally dPN), and adult animals were investigated by transmission electron microscopy, using standard staining procedures and immunogold (GAR-Au10) labeling for SP-A and Clara cell 10 kD antigen (CCA). At 16 dDa, all the columnar epithelial cells lining prospective distalmost bronchioles lacked distinctive features. By 17 dDa, some cells displayed a few cilia or apical dense granules. At 18 dDa, many nonciliated columnar cells had apical protrusions, as are seen in adult Clara cells. Apical concentrations of glycogen observed in nonciliated columnar cells perinatally were absent by 5 dPN, whereas apical dense granules became more abundant. Profiles of smooth and rough endoplasmic reticulum (ER), which had been randomly distributed, exhibited a selective, adult distribution at 20 dPN (apical vs. basal cytoplasmic domains). Labeling for SP-A and CCA, which was almost absent between 17 and 19 dDa, reached adult levels at the same time. The two proteins differed in distribution. SP-A predominated in adluminal cytoplasmic areas, where it was found over dense granules, vesicles, and multivesicular bodies; it was also present in bronchiolar lumens and intercellular spaces but not in rough ER or Golgi apparatus. In contrast, CCA showed a more uniform distribution; it was present over the same structures as SP-A and in the synthetic organelles. Ciliated columnar cells were virtually devoid of SP-A and CCA. We conclude that mouse Clara cells acquire a mature phenotype by 20 dPN. They are likely to be involved in recycling and/or degradation of SP-A that is internalized from airway lumens through their apical or lateral cell borders; furthermore, they synthesize the Clara cell 10 kD protein. These two Clara cell functions (first detectable late prenatally) reach mature levels by 20 dPN.

Stereological methods: a new approach in the assessment of pulmonary emphysema.

In order to develop a reliable and sensitive method for studying the development and progression of pulmonary emphysema, we compared stereological indices with the usual index for grade of emphysema, i.e., the mean linear intercept (Lm), in elastase-induced emphysema in mice. The Lm and stereological indices, including volumes of total lung tissue (V(lt)), airspaces (V(air)), and surface area of alveolar walls (S(alv)), were determined in 5-microns, H&E-stained, paraffin-embedded lung sections from elastase- (n = 7) or saline-treated (n = 8) mice. The indices were measured by point counting, using Cavalieri's principle (V(lt)) and V(air)) or by counting intersections of alveolar walls with test lines of a known length (S(alv) and Lm). Elastase treatment resulted in a significant increase of Lm and of V(air), both indicating airspace enlargement, and in a significant decrease of V(lt) and S(alv), indicating destruction of alveolar walls. Between each of the stereological indices and the Lm, significant correlations were found when all lungs were included, but not when the emphysematous lungs were considered separately. We conclude that stereological methods can be powerful morphometric tools for studying pulmonary emphysema development and progression, since they give information not only about the grade of airspace enlargement but also about the grade of destruction of alveolar walls. Based on this unique property, stereological methods also allow a distinction between pulmonary emphysema and unrelated conditions with dilatation of airspaces only.

Ultrastructural features of alveolar epithelial cells in the late fetal pulmonary acinus: a comparison between normal and hypoplastic lungs using a rat model of pulmonary hypoplasia and congenital diaphragmatic hernia.

The aim of this study was to describe and compare the ultrastructural features and functional maturity of alveolar epithelial cells in hypoplastic and normal fetal rat lungs. Pulmonary hypoplasia in association with congenital diaphragmatic hernia was induced in fetuses by administration of 2,4-dichlorophenyl-p-nitrophenylether (Nitrofen) to pregnant Sprague Dawley rats (100 mg on day 10 of gestation). Lung tissue of Nitrofen-exposed and control fetal rats aged 19-22 days (vaginal plug day 1, birth day 23) was embedded in Epon. Semithin (1 micron) toluidine blue-stained sections were examined by light microscopy; ultrathin sections (approximately 80 nm) were studied via transmission electron microscopy. In bronchoalveolar lavage fluid from control and Nitrofen-exposed fetuses (day 22), phospholipid fractions and surfactant protein A content were measured semiquantitatively. On day 19 both control and Nitrofen-exposed lungs contained only cuboid alveolar epithelial cells; from day 20 there were cuboid, low cuboid, and thinner epithelial cells. The (low) cuboid cells contained large glycogen fields, some precursory stages of multilamellar bodies (MLBs), and just a few mature MLBs on day 19 and 20; smaller glycogen fields, more precursory stages, and more mature MLBs on day 21; and little or no glycogen but many precursory stages and mature MLBs on day 22. The thinner cells contained little or no glycogen and a few precursory stages of MLBs on days 20-22; very thin cells on day 22 contained neither glycogen nor any precursory stages of MLBs. MLBs and tubular myelin were seen in the lumens of future air spaces from day 20 onward. Nitrofen-exposed lungs differed from control lungs in that inclusion bodies (IBs) were less numerous in (low) cuboid alveolar cells on days 19 and 20, and more glycogen was seen on day 22. In addition intra- and extracellular "MLBs" in exposed lungs more often had an unusual appearance, i.e., a confluent structure and higher electron density. However, despite morphologic differences, there was no clear difference in phospholipid composition and SP-A content per mol phospholipid in bronchoalveolar lavage fluid. We conclude that morphologically hypoplastic lungs are less mature near term, without an apparent effect on surfactant composition.

Altered gelatinolytic activities in an apparent unilateral keratoconus patient. A case report.

Recent studies have suggested that one possible cause of keratoconus involves increased degradation of the corneal extracellular matrix. Most studies have examined corneas from patients with bilateral, sporadic keratoconus. In this report, both corneas from a 70-year-old man with a history of familial keratoconus and clinical signs of unilateral keratoconus were examined for gelatinase activity. Our results demonstrated a qualitative (three additional activity bands of 88, 92, and 100 kDa molecular weights) and quantitative differences (an apparent increase in overall amounts) in the gelatinolytic enzyme profiles of the affected keratoconus cornea as compared with his unaffected (nonkeratoconus) cornea, normal corneas, bilateral sporadic keratoconus corneas, and cultured keratocytes. In addition, gelatinolytic enzymes were more readily extracted from the affected cornea compared with controls. These data lend support to the hypothesis of a heterogeneous etiology for keratoconus and to previous suggestions that this disorder may be related to an alteration in extracellular matrix degradation.

Increased gelatinolytic activity in keratoconus keratocyte cultures. A correlation to an altered matrix metalloproteinase-2/tissue inhibitor of metalloproteinase ratio.

Keratoconus is a noninflammatory corneal disorder characterized by gradual stromal thinning and astigmatism. Altered degradation of corneal extracellular matrix is a suggested etiology for this disorder. In the present study we established keratocyte cultures from normal and keratoconus corneas and investigated the roles that matrix metalloproteinases (MMPs) and tissue inhibitors of metalloproteinases (TIMP, TIMP-2) may play. After chemical modification (reduction and alkylation) to remove the inhibitor and activation of enzyme with p-aminophenylmercuric acetate (APMA), keratoconus-conditioned media displayed a significant increase (p < 0.05) in the total potential gelatinolytic activity when compared with normal culture media treated in a similar manner. Basal levels of gelatinolytic activity in keratoconus culture media (no reduction, alkylation, or APMA treatment), determined by two different assay methods, tended to be about twice that of normal cell cultures. By zymography, both keratoconus and normal cultures showed identical enzyme patterns, which represented MMP-2 (72 kDa) in its proform and, depending on the treatment of the media, varying amounts of activated MMP-2 (65 kDa). This suggests that the increased gelatinolytic activity in keratoconus was not correlated with an increased appearance of either the 65-kDa-activated form of MMP-2 or a new MMP species. In addition, no differences in the amount of MMP-2 were detected that could account for the increased activities in keratoconus cultures. However, a relative decline in the detectable TIMP levels in keratoconus cultures resulted in an apparent three-fold increase in the ratio of MMP-2/TIMP. Northern blots showed no significant changes in mRNA levels for MMP-1, MMP-2, MMP-3, TIMP, or TIMP-2. These data suggest that a possible alteration in the interaction between MMP-2 and TIMP may play a role in the increased gelatinolytic activity seen in keratoconus tissues.

The structural composition of the pulmonary acinus in the mouse. A scanning electron microscopical and developmental-biological analysis.

For analysis of the structural composition of the pulmonary acinus in the mouse, fixed lungs of adult mice were examined by scanning and transmission electron microscopy. The murine acinus proved to consist of one or two generations of rather short respiratory bronchioles and about three generations of alveolar ducts opening into alveolar sacs. The epithelial cells lining the respiratory bronchioles (except for a sharply demarcated zone in the initial portion of the first-order branch) have a cuboidal or squamous shape and are very probably of the same type as those lining the alveolar ducts and sacs, i.e., small and great alveolar cells and/or their precursor cells. Therefore, this respiratory bronchiole portion may be considered part of the respiratory unit or pulmonary acinus. The initial zone of the first-order branch is lined by columnar epithelium, i.e., bronchial epithelium (mainly Clara cells), and should be assigned to the bronchial system of the lung.

Development of the pulmonary acinus in fetal rat lung: a study based on an antiserum recognizing surfactant-associated proteins.

In this study on the development of the pulmonary acinus in fetal rat lung use was made of an antiserum, rabbit anti-mouse, that recognizes the type II alveolar epithelial cell or its precursor (a cuboidal cell lacking multilamellar bodies) by the presence of a cell-specific antigen. This serum had already been used in studies on mouse-lung development in our laboratory. Immunoblotting experiments showed that this serum reacts with surfactant-associated proteins in the pellet fraction of rat-lung lavage fluid having molecular weights of about 26,000, 32,000, and 38,000 daltons. In adult and fetal rat-lung homogenates the antiserum reacts with proteins with apparent molecular weights of about 40,000 and 42,000 daltons, probably also surfactant-associated proteins. No reaction with serum proteins was seen. Use of this antiserum in immuno-incubations of frozen sections of lungs of 15- to 21-day-old rat embryos showed that the type II epithelial cell or its precursor first appears on day 16 in embryos weighing 349-398 mg. Our results indicate that in the rat - as in the mouse - the bronchial and respiratory portions develop from morphologically and immunologically different parts of the tubular system in the fetal lung. The basic structure in the genesis of the pulmonary acinus is a tubule, called the acinar tubule, which is lined by the type II epithelial cell or its precursor.

Detection of the type II cell or its precursor before week 20 of human gestation, using antibodies against surfactant-associated proteins.

The present study was performed to find out whether the type II alveolar epithelial cell or its precursor (an approximately cuboidal cell lacking multilamellar bodies) is present before the twentieth week of human gestation. For this purpose we used an antibody, SALS-Hu(E), which recognizes the human type II cell on the basis of surfactant-associated proteins. Application of SALS-HuE (by indirect immunofluorescence) to acetone-fixed frozen sections of fetal lung tissue gave a distinct staining of the cuboidal or low columnar epithelial cells lining the end-pieces of the tubular system of fetal lung (initially only a few): this staining started around weeks 10 to 12 after conception. Around week 16 some of the labeled epithelial cells appeared to be rather flat and by week 19 a combined cellular and linear fluorescence pattern was seen. Columnar epithelial cells of the prospective bronchial portion did not show this specific staining. Our results indicate that the type II cell or its precursor cell is indeed present in the pseudoglandular period of human lung development, i.e., starting around the tenth to twelfth week. This cell type lines the acinar tubule, the basic structure of the pulmonary acinus. Transformation of this cell type into the type I alveolar epithelial cell seems to start in week 16.

Ultrastructural characteristics of inclusion bodies of type II cells in late embryonic mouse lung.

As we reported earlier, type II alveolar epithelial cells make their appearance in the early embryonic mouse lung around day 14.2, and show distinctive ultrastructural features. The present study focuses on the ultrastructural characteristics of the inclusion bodies by investigating embryos aged 17-19 days (birth on day 19), using transmission electron microscopy. Late embryonic type II cells appear also as low-columnar or cuboid cells having large, approximately round nuclei and cytoplasm displaying typical features of a differentiated cell. The inclusion bodies show a widespread distribution and are extremely variable in appearance. Schematically we discern five main types, namely cytoplasmic, granular/flocculent, multivesicular, dense, and (multi)lamellar, which occur with intermediate and composite forms. All these inclusion bodies frequently contain glycogen particles, and show a structural relation to profiles of endoplasmic reticulum which are wrapped around them. Other distinctive properties are the osmiophily of multivesicular inclusion bodies, and the presence of vesicles in many dense inclusion bodies. The possible interrelationship, and the differences in various aspects of electron density, suggest that the five main types of inclusion bodies may represent different stages in the formation of mature multilamellar bodies.

Characterization of a human corneal metalloproteinase inhibitor (TIMP-1).

The gradual corneal thinning seen in keratoconus may be due to altered degradation of the corneal extracellular matrix. Studies have shown that human keratocytes produce matrix metalloproteinase-2 (MMP-2) and two proteins (28 kDa and 21 kDa) that are capable of inhibiting the activity of MMP-2. In the present study, the 28 kDa inhibitor from keratoconus keratocyte cultures has been characterized as it may be important to the elevated MMP-2 activity seen in these cultures. Biochemical analyses indicated that this keratoconus corneal inhibitor was similar to TIMP-1 from other sources. Oligonucleotides to the reported sequence of human tumor cell TIMP-1 were used for reverse-transcriptase PCR to generate a 700 bp clone of the 28 kDa inhibitor from keratoconus keratocyte cytoplasmic RNA. Sequence analysis verified that the clone was nearly identical to the reported human TIMP-1 with a single base substitution that did not affect the predicted amino acid sequence. In addition, protein translated from the clone corresponded to the expected size. This data suggests that the elevated levels of gelatinolytic activity in these keratoconus keratocyte cultures is not due to a primary alteration of the TIMP-1 molecule. Protein expression studies of the TIMP-1 clone are currently underway.

Keratoconus corneas: increased gelatinolytic activity appears after modification of inhibitors.

We examined the metalloproteinase activity from normal and keratoconus corneal extracts. No differences were detected in the total amount of the metalloproteinase or its physical form of activation. However, there was a significant elevation in enzymatic activity in the keratoconus extracts after chemical modification of inhibitory elements. This suggests either a difference in the enzymatic capabilities of keratoconus corneas or, as suggested previously, a decrease in the amount of TIMP (tissue inhibitor of metalloproteinase) present in the tissue.