B cells facilitate platelet production mediated by cytokines in patients with essential thrombocythaemia.

We investigated the role of activated B cells in thrombopoiesis through the production of interleukin (IL)-1beta and IL-6 in patients with essential thrombocythaemia. The number of B cells did not differ between essential thrombocythaemia patients, irrespective of the presence of Janus activated kinase-2 V617F mutation or wild type, and age-matched healthy adults. However, the number of IL-1beta/IL-6-producing B cells was significantly higher in essential thrombocythaemia patients than that in healthy controls. The relatively high level of IL-1beta/IL-6 production by B cells was associated with serum B cell-activating factor and expression of Toll-like receptor 4 on B cells. A high level of B cell-activating factor was present in essential thrombocythaemia patients with both Janus activated kinase-2 genotypes. Incubation with B cell-activating factor enhanced the expression of Toll-like receptor 4 on B cells. IL-1beta and IL-6 production was not stimulated by B cell-activating factor alone; Toll-like receptor 4 was activated by lipopolysaccharide or patients' sera to produce IL-1beta and IL-6 in B cells. Moreover, essential thrombocythaemia patient B cells facilitated megakaryocyte differentiation when co-cultured with CD34+ haematopoietic stem cells. Antibody neutralisation of IL-1beta and IL-6 attenuated megakaryocyte differentiation. These data suggest that B cells play a crucial role in thrombopoiesis in essential thrombocythaemia patients.

Mutation of a Nopp140 gene dao-5 alters rDNA transcription and increases germ cell apoptosis in C. elegans.

Human diseases of impaired ribosome biogenesis resulting from disruption of rRNA biosynthesis or loss of ribosomal components are collectively described as 'ribosomopathies'. Treacher Collins syndrome (TCS), a representative human ribosomopathy with craniofacial abnormalities, is attributed to mutations in the tcof1 gene that has a homologous gene called nopp140. Previous studies demonstrated that the dao-5 (dauer and aged animal overexpression gene 5) of Caenorhabditis elegans is a member of nopp140 gene family and plays a role in nucleogenesis in the early embryo. Here, we established a C. elegans model for studying Nopp140-associated ribosomopathy. A null dao-5 mutant ok542 with a semi-infertile phenotype showed a delay in gonadogenesis, as well as a higher incidence of germline apoptosis. These phenotypes in dao-5(ok542) are likely resulted from inefficient rDNA transcription that was observed by run-on analyses and chromatin immunoprecipitation (ChIP) assays measuring the RNA Pol I occupancy on the rDNA promoter. ChIP assays further showed that the modifications of acetylated histone 4 (H4Ac) and dimethylation at the lysine 9 of histone 3 (H3K9me2) around the rDNA promoter were altered in dao-5 mutants compared with the N2 wild type. In addition, activated CEP-1 (a C. elegans p53 homolog) activity was also linked to the loss of DAO-5 in terms of the transcriptional upregulation of two CEP-1 downstream effectors, EGL-1 and CED-13. We propose that the dao-5 mutant of C. elegans can be a valuable model for studying human Nopp140-associated ribosomopathy at the cellular and molecular levels.

Pain management of living liver donors with morphine with or without ketorolac.

BACKGROUND: To compare the efficacy and dose requirements for intravenous (IV) patient-controlled analgesia (PCA) with morphine only versus morphine with ketorolac for living liver donors after partial hepatectomy. PATIENTS AND METHODS: Eighty living liver donors who had undergone partial hepatectomy received 3 days of IV PCA for postoperative pain control. Some were prescribed a PCA with morphine alone (group I) or morphine with ketorolac (group II), while both had a rescue dose of IV fentanyl (25 µg). The daily consumption of morphine, pain score, and frequency of rescue fentanyl doses were compared retrospectively using the Mann-Whitney U test and the incidence of side effects with chi-square tests; a P value of .05 was regarded as significant. All the data are shown as mean values±standard deviations. RESULTS: The 80 subjects were distributed as 57 group I and in 23 group II patients. The daily consumption of morphine, Visual Analogue Scale (VAS) and side effects were not different between the groups, but group II required significantly fewer rescue doses to achieve pain relief. CONCLUSION: Both regimens provided acceptable pain control with daily VAS less than 3. The use of ketorolac in the PCA did not reduce the daily total morphine requirements with a similar incidence of side effects but a significantly reduced requirement for rescue doses, which subsequently reduced the work load of personnel in the pain control service.

Effect of university of wisconsin and histidine-tryptophan-ketoglutarate preservation solutions on blood potassium levels of patients undergoing living-donor liver transplantation.

BACKGROUND: The potassium content of University of Wisconsin solution (UW) is 125 mEq/L and that of histidine-tryptophan-ketoglutarate solution (HTK) only 9 mEq/L. The aim of the present study was to analyze their effects to change potassium levels on reperfusion among patients undergoing living-donor liver transplantation. METHODS: Anesthesia records of adult living-donor liver transplant patients were reviewed retrospectively. Patients received liver graft preserved in UW were grouped in group I (GI) and HTK in group II (GII). The potassium levels in the anheptic phase were compared with those 5 minutes after reperfusion using paired Student t tests. P values of <.05 were regarded to be significant. RESULTS: Eighty-five GI patients showed the potassium significantly decreased from 3.76±0.70 to 3.60±0.74, whereas the change among 355 GII patients was almost unremarkable: 4.00±0.57 to 3.96±0.06 mEq/L. CONCLUSIONS: Although UW contains a higher potassium content, it seems to have no negative impact on changes in serum potassium levels; in contrast it decreased the potassium level significantly at 5 minutes after reperfusion. Both preservation solutions maintain the patients' potassium levels within the normal range.

Quality of life and psychological status of patients with implantable cardioverter defibrillators.

INTRODUCTION: Implantable cardioverter defibrillators (ICDs) are effective at reducing mortality in patients at high risk for sudden cardiac death (SCD) but can cause psychological distress and reduce quality of life (QOL). The full benefits of ICDs can only be achieved when the patient's QOL and psychological status are maintained. We examined psychological status and QOL post ICD implantation; the relationship of psychological status to QOL; the relationship of time since implantation to psychological status and QOL; and the relationship of time since ICD implantation and age of patient to these variables. METHODS AND RESULTS: A cross-sectional self-administered assessment of QOL, depression, anxiety, demographic characteristics and cardiovascular health history of patients (n = 48) who had received ICDs within the past 10 years at an urban hospital. Patients who had ICDs for longer experienced worse depression and QOL. Patients who were younger had worse depression, anxiety, and QOL. The combination of anxiety, depression, age, and time since ICD implant significantly predicted overall QOL and the psychosocial and physical dimensions of QOL explaining 55.5, 54, and 34.9% of the variance, respectively. CONCLUSION: Younger ICD patients are at highest risk for psychological distress and poor QOL. Longitudinal research would facilitate determination of the trajectory of changes in psychological status and QOL over the duration of the ICD experience.

Tubedown-1 in remodeling of the developing vitreal vasculature in vivo and regulation of capillary outgrowth in vitro.

Tubedown-1 (tbdn-1) is a mammalian homologue of the N-terminal acetyltransferase subunit NAT1 of Saccharomyces cerevisiae and copurifies with an acetyltransferase activity. Tbdn-1 expression in endothelial cells becomes downregulated during the formation of capillary-like structures in vitro and is regulated in vivo in a manner which suggests a functional role in dampening blood vessel development. Here we show that tbdn-1 is expressed highly in the vitreal vascular network (tunica vasculosa lentis and vasa hyaloidea propria) during the pruning and remodeling phases of this transient structure. The vitreal blood vessels of mice harboring a targeted inactivation of TGF-beta2 fail to remodel and abnormally accumulate, a phenomenon reminiscent of the ocular pathology resembling persistent fetal vasculature (PFV) in humans. Since suppression of normal tbdn-1 expression has been previously observed in retinal vessel proliferation, we analyzed vitreal vascular changes and tbdn-1 expression in TGF-beta2(-/-) eyes. The nuclei of vitreal vessel endothelial cells in TGF-beta2(-/-) eyes express proliferating cell nuclear antigen (PCNA) and exhibit increased levels of active (P42/44)mitogen-activated protein kinase (phospho-(P42/44)MAPK), characteristics consistent with proliferative endothelial cells. In contrast to normal vitreal vessels, collagen IV expression exhibited a disorganized pattern in the TGF-beta2(-/-) vitreal vessels, suggesting vessel disorganization and possibly a breakdown of vessel basal laminae. Moreover, vitreal vessels of TGF-beta2(-/-) mice lack expression of pericyte markers (CD13, alpha smooth muscle actin) and show ultrastructural changes consistent with pericyte degeneration. The accumulating vitreal blood vessels of TGF-beta2(-/-) mice, while maintaining expression of the endothelial marker von Willebrand Factor, show a significant decrease in the expression of tbdn-1. We addressed the functional role of tbdn-1 in the regulation of vitreal blood vessels using an in vitro model of choroid-retina capillary outgrowth. Clones of the RF/6A fetal choroid-retina endothelial cell line showing suppression of tbdn-1 levels after overexpression of an antisense TBDN-1 cDNA display a significant increase in the formation of capillary-like structures in vitro compared with controls. These findings suggest that tbdn-1 inhibits capillary-like formation in vitro and may serve to dampen vitreal blood vessel formation preceding the regression of the vitreal vasculature during development. Our results also suggest that tbdn-1 may participate with TGF-beta2 in regulating normal development of the vitreal vasculature.

Characterization of Corn1 mice: Alteration of epithelial and stromal cell gene expression.

PURPOSE: Corn1 is an autosomal recessive mutation characterized by corneal epithelial hyperplasia and stromal neovascularization. The aim of the present study is to examine the expression patterns of specific epithelial and stromal proteins in corn/corn1 mutant mice. METHODS: Immunohistochemistry with antibodies directed against keratins 1, 4, 5, 12, and 14 as well as loricrin, filaggrin, and involucrin were performed in corn1/corn1 and wild type, A.By/SnJ strain, mice at 4 weeks of age. Western blot hybridization was performed to confirm the presence of involucrin in corneas. In situ and northern blot hybridization were used to evaluate the expression of keratin 12, lumican, and keratocan in these mice. RESULTS: In corn1/corn1 mice, focal areas of corneal epithelial hyperplasia alternate with epithelium with normal appearance. Both regions of normal and hyperplastic corneal epithelium were labeled by anti-keratin 12 antibodies through all corneal epithelial layers. The anti-keratin 14 antibody only labeled the basal cell layer in normal epithelial areas, whereas it labeled both basal and suprabasal cell layers in hyperplastic areas. In wild type mice, anti-keratin 12 antibodies labeled all corneal epithelial layers, whereas anti-keratin 14 labeled the basal corneal epithelial cells only. Positive staining by anti-involucrin antibody was demonstrated in the basal corneal epithelial layer of wild type mice and normal areas of corn1/corn1 mice. Similarly, as observed with anti-keratin 14 antibody, the anti-involucrin antibody labeled both basal and suprabasal cell layers of hyperplastic corneal epithelium of corn1/corn1 mice. Antibodies against keratin 1, keratin 4, loricrin, and fillagrin did not label the corneas of wild type mice or corn1/corn1 mice. Northern hybridization indicated that the expressions of keratocan and lumican mRNA levels were up regulated in corn1/corn1 mice, but keratin 12 mRNA remained similar to that of the wild type mice. In situ hybridization revealed that the lumican mRNA was detected in epithelial and stromal cells of corn1/corn1 mice, whereas keratocan mRNA was only detected in stromal cells. CONCLUSIONS: Hyperproliferative epithelial cells of corn1/corn1 mice have increased levels of expression of keratin 14 and involucrin, but do not exhibit the phenotypical characteristics of cornification. These observations indicate that factors associated with the phenotypes of corn1/corn1 mice do not alter the cornea-type epithelial differentiation of keratin 12 expression, but cause aberrant expression of lumican by corneal epithelial cells.

TGFbeta2 in corneal morphogenesis during mouse embryonic development.

To examine the roles of TGFbeta isoforms on corneal morphogenesis, the eyes of mice that lack TGFbetas were analyzed at different developmental stages for cell proliferation, migration and apoptosis, and for expression patterns of keratin 12, lumican, keratocan and collagen I. Among the three Tgfb(-/-) mice, only Tgfb2(-/-) mice have abnormal ocular morphogenesis characterized by thin corneal stroma, absence of corneal endothelium, fusion of cornea to lens (a Peters'-like anomaly phenotype), and accumulation of hyaline cells in vitreous. In Tgfb2(-/-) mice, fewer keratocytes were found in stroma that has a decreased accumulation of ECM; for example, lumican, keratocan and collagen I were greatly diminished. The absence of TGFbeta2 did not compromise cell proliferation, nor enhance apoptosis. The thinner stroma resulting from decreased ECM synthesis may account for the decreased cell number in the stroma of Tgfb2 null mice. Keratin 12 expression was not altered in Tgfb2(-/-) mice, implicating normal corneal type epithelial differentiation. Delayed appearance of macrophages in ocular tissues was observed in Tgfb2(-/-) mice. Malfunctioning macrophages may account for accumulation of cell mass in vitreous of Tgfb2 null mice.

Role of lumican in the corneal epithelium during wound healing.

Lumican regulates collagenous matrix assembly as a keratan sulfate proteoglycan in the cornea and is also present in the connective tissues of other organs and embryonic corneal stroma as a glycoprotein. In normal unwounded cornea, lumican is expressed by stromal keratocytes. Our data show that injured mouse corneal epithelium ectopically and transiently expresses lumican during the early phase of wound healing, suggesting a potential lumican functionality unrelated to regulation of collagen fibrillogenesis, e. g. modulation of epithelial cell adhesion or migration. An anti-lumican antibody was found to retard corneal epithelial wound healing in cultured mouse eyes. Healing of a corneal epithelial injury in Lum(-/-) mice was significantly delayed compared with Lum(+/-) mice. These observations indicate that lumican expressed in injured epithelium may modulate cell behavior such as adhesion or migration, thus contributing to corneal epithelial wound healing.

Identification of the cornea-specific keratin 12 promoter by in vivo particle-mediated gene transfer.

PURPOSE: Keratin 12 (K12) is a cornea epithelial cell-specific intermediate filament component. To provide a better understanding of its expression, it is necessary to identify and characterize the promoter of Krt1.12 gene. METHODS: The 2.5-kb DNA 5' to Krt1.12 gene was sequenced. Krt1.12 promoter-beta-gal DNA constructs were prepared and used in vivo to transfect rabbit corneas, conjunctivas, and skin by particle-mediated gene transfer (Gene Gun). In vitro, the DNA constructs were transfected into cultured T-antigen-transformed rabbit corneal epithelial (RCE-T) cells and human fibrosarcoma HT-1080 fibroblasts with lipofectamine. The promoter activity was assessed by measuring beta-gal (beta-galactosidase) activity using histochemical staining with 5-Bromo-4-chloro-3-indolyl-beta-D-galactoside and enzyme assay with o-nitrophenyl beta-D-galactopyranoside. RESULTS: There are four Pax-6 pair box binding elements found between -910 and -2000 bp 5'-flanking the transcription initiation site of the Krt1.12 gene. None of promoter constricts can be expressed by HT-1080 cells. Cotransfection of Pax-6 cDNA with K12 promoter-beta-gal constructs containing Pax-6 elements results in a fourfold increase of beta-gal activities in RCE-T cells but not HT-1080 fibroblasts. The data of in vivo transfection in the rabbit by Gene Gun indicate that reporter gene constructs containing 0.6-kb and longer DNA fragments 5'-flanking Krt1.12 gene are effectively expressed in corneal, but not conjunctival or epidermal epithelial cells. CONCLUSIONS: The particle-mediated gene transfer is a suitable technique for in vivo delivery of transgenes to corneal epithelial cells. The 2.5-kb DNA fragment 5'-flanking Krt1.12 contains corneal epithelial cell-specific regulatory cis-DNA elements. Pax-6 is a positive transcription factor essential for keratin 12 expression.

A CAT reporter construct containing 277bp GNAT2 promoter and 214bp IRBP enhancer is specifically expressed by cone photoreceptor cells in transgenic mice.

PURPOSE: The alpha-subunit of human cone transducin plays an important role in interacting with visual pigment and activating the cGMP-dependent phosphodiesterase (cGMP-PDE). The human GNAT2 gene (cone transducin alpha-subunit) has been cloned and characterized by Fong et al. In this report, we describe the use of transgenic mice to characterize the tissue specificity of the GNAT2 promoter. METHODS: A chimeric reporter gene construct which consists of a 277 bp 5'-flanking fragment of the GNAT2 gene at 5' end of the chloramphenicol acetyltransferase (CAT) gene and a 214 bp enhancer region from the human interphotoreceptor retinoid-binding protein (IRBP) gene at the 3' end of the CAT gene was used to generate transgenic mice. Transgenic mice were identified by Southern blot hybridization and polymerase chain reaction (PCR) analysis using tail DNA from experimental animals. Immunostaining was used to study the developmental expression of CAT and the endogenous GNAT2 gene. RESULTS: Analysis of four transgenic mouse lines revealed that three lines had low CAT activity in the retina. The CAT gene, along with the endogenous GNAT2 gene, was expressed at high levels in cone photoreceptor cells in the fourth transgenic mouse line as determined by CAT enzyme assays and immunostaining. CONCLUSION: The results show that the 277 bp 5'-flanking sequence from the human GNAT2 gene coupled with the 214 bp IRBP enhancer can direct a tissue-specific expression pattern of CAT reporter gene in mouse retina, which parallels the expression pattern of endogenous GNAT2.

The cloning of mouse keratocan cDNA and genomic DNA and the characterization of its expression during eye development.

Keratan sulfate proteoglycans (KSPGs) play a pivotal role in the development and maintenance of corneal transparency. Keratocan, lumican, and mimecan (osteoglycin) are the major KSPGs in vertebrate corneas. To provide a better understanding of the structure/function relationship of keratocan, we have cloned both the mouse keratocan gene and its cDNA. We have also examined its expression during embryonic development. The mouse keratocan gene spans approximately 6.5 kilobases of the mouse genome and contains three exons and two introns. Northern blotting and in situ hybridization were employed to examine keratocan gene expression during mouse development. Unlike lumican gene, which is expressed by many tissues other than cornea, keratocan mRNA is more selectively expressed in the corneal tissue of the adult mouse. During embryonic development, keratocan mRNA was first detected in periocular mesenchymal cells migrating toward developing corneas on embryonic day 13.5 (E13.5). Its expression was gradually restricted to corneal stromal cells on E14. 5 approximately E18.5. Interestingly, keratocan mRNA can be detected in scleral cells of E15.5 embryos, but not in E18.5 embryos. In adult eyes, keratocan mRNA can be detected in corneal keratocytes, but not in scleral cells.

Characterization of Bsk mice: I. The Bsk mutation does not involve a recombination of cornea-specific keratin 12 and skin-specific hair keratin genes.

PURPOSE: Bsk (bare skin) is an autosomal dominant mutation linked to the Krt 1 (type 1 keratin) locus of mouse chromosome 11. The adult Bsk mouse manifests hair loss and corneal opacity. To identify and characterize the keratin genes involved in this mutation, we examined the hypothesis proposing that the Bsk mutation might involve a recombination event between cornea-specific (K12) and hair-specific (mHa 1, 2, 3 and 4) type I keratin genes. METHODS: The Bsk phenotype was examined by histochemical analysis, using light and electron microscopy. RFLP was used for their genotyping, and possible keratin gene expression was examined by immunohistochemical staining, Western analysis, RT-PCR and Northern hybridization. RESULTS: Northern hybridization, RT-PCR and Western blot analysis revealed that mHa 1, 2, 3 and 4 keratins are expressed in the skin, but not in cornea, whereas the expression of K12 is limited to the corneas of the Bsk mice. These data ruled out the hypothesis that Bsk phenotype results from a recombination event between K12 and mHa 1, 2, 3 and 4. Ultrastructural and biochemical analyses also indicated that Bsk does not involve negative dominant mutations of keratin 12, mHa 1, 2, 3 and 4, epidermal-specific keratin 10, or basal cell-specific keratin 14. Expression of an acidic 50 kD keratin, recognized by monoclonal antibody AK 2, was up-regulated in the injured corneas of normal mice as well as Bsk corneas. CONCLUSION: The gene linked to the Bsk mutation remains unknown. The pathological changes in the skin and corneas may be secondary to the loss of protecting hairs and lashes by an unknown mechanism.

Healing of corneal epithelial defects in plasminogen- and fibrinogen-deficient mice.

PURPOSE: The local deposition of fibrinogen and other plasma products from tears within corneal wounds and the expression of plasminogen activator by corneal epithelial cells suggest that the coagulation and fibrinolytic systems play an important role in corneal wound healing. The authors used mouse lines deficient in plasminogen (Plg), fibrinogen (Fib), or both to elucidate the roles of these key fibrinolytic and coagulation factors in the healing of corneal epithelial defects. METHODS: Mice were anesthetized, and corneal epithelial defects (3 mm) were created with a blade. The authors conducted histologic examination and immunohistochemical analysis on the healing of injured corneas. RESULTS: The corneal epithelial defects of wild-type mice with transparent corneas healed quickly in 7 days, whereas the healing of plasminogen-deficient mice was impaired and complicated by severe and persistent inflammatory responses, the formation of retrocorneal fibrin deposits, corneal cloudiness caused by scar-tissue formation, and often stromal neovascularization. To determine whether these defects in corneal wound repair were specifically related to an impediment in fibrinolysis, corneal wound healing was compared in mice with a combined deficiency in plasminogen and fibrinogen. The loss of fibrinogen in mice lacking plasminogen resulted in the restoration of normal healing with transparent corneas in 7 days, similar to that of wild-type mice. CONCLUSIONS: These results provide direct evidence that hemostatic factors play a crucial role in corneal wound repair despite the lack of local hemorrhage. Furthermore, they demonstrate that the essential role of plasmin in corneal would healing is fibrinolysis. It prevents the adverse inflammatory responses caused by prolonged fibrin and fibrinogen deposition in injured corneas.

Characterization and expression of the mouse lumican gene.

Lumican is one of the major keratan sulfate proteoglycans (KSPG) in vertebrate corneas. We previously cloned the murine lumican cDNA. This study determines the structure of murine lumican gene (Lum) and its expression during mouse embryonic developments. The mouse lumican gene was isolated from a bacterial artificial chromosome mouse genomic DNA library and characterized by polymerase chain reaction and Southern hybridization. The lumican gene spans 6.9 kilobase pairs of mouse genome. The gene consists of three exons and two introns. Exon 1 constitutes 88 bases (b) of untranslated sequence. Exon 2 is 883 b and contains most of the coding sequence of lumican mRNA, and exon 3 has 152 b of coding sequence and 659 b of 3' noncoding sequence. The mouse lumican gene has a TATCA element, a presumptive TATA box, which locates 27 b 5'-upstream from the transcription initiation site. Northern hybridization and in situ hybridization indicate that in early stages of embryonic development, day 7 post coitus the embryo expresses little or no lumican. Thereafter, different levels of lumican mRNA can be detected in various organ systems, such as cornea stroma, dermis, cartilage, heart, lung, and kidney. The cornea and heart are the two tissues that have the highest expression in adult. Immunoblotting studies found that KSPG core proteins became abundant in the cornea and sclera by postnatal day 10 but that sulfated KSPG could not be detected until after the eyes open. These results indicate that lumican is widely distributed in most interstitial connective tissues. The modification of lumican with keratan sulfates in cornea is concurrent with eye opening and may contribute to corneal transparency.

Stromal fibroblasts are associated with collagen IV in scar tissues of alkali-burned and lacerated corneas.

PURPOSE: Corneal wound healing frequently leads to the formation of opaque scar tissue. We examined whether stromal fibroblastic cells of injured corneas express collagen IV and contributes to the formation of a basal lamina-like structure. METHODS: Rabbits were anesthetized, and central corneal alkali burn (8 mm in diameter; 1 M NaOH, 1 min) or laceration (8 mm long) were produced. The injured corneas, which had healed for 1, 7, 21 and 45 days, were subjected to histological and immunohistochemical studies with goat anti-collagen IV antibodies, using light and electron microscopy, and in situ hybridization with an antisense digoxigenin-labeled riboprobe of collagen alpha 1(IV) mRNA. For comparison, twenty-day-old fetal corneas were subjected to immunohistochemical study and transmission electron microscopy (TEM). RESULTS: TEM examinations revealed that the stromal collagenous matrix was organized in orthogonal lamellae during corneal development, whereas that of alkali-burned cornea, which had healed for 3 weeks, was disorganized. The stroma of twenty-day-old fetal cornea was not labeled by the anti-collagen IV antibodies. In contrast, one week after injury, specific collagen IV immunostaining was detected in the injured stroma. As the healing proceeded (21-45 days), the antibodies reacted with fibroblastic cells and the extracellular matrix of scar tissues located in the anterior portion of alkali-burned corneas, as well as the posterior portion of lacerated corneas. The middle portion of the stromal tissues was weakly labeled by the anti-collagen IV antibodies with the exception of the blood vessel wall. Immuno-electron microscopic study showed that collagen IV and fibronectin were closely associated with the fibroblastic cells. In situ hybridization demonstrated that epithelial and endothelial cells and fibroblastic cells in the wounded corneal stroma and retro-corneal membrane expressed alpha 1(IV) mRNA, whereas in normal corneas the expression of alpha 1(IV) mRNA was limited to epithelial and endothelial cells. CONCLUSIONS: The enhanced expression of collagen IV by the fibroblastic cells in the stroma of injured corneas is consistent with the notion that they may contribute to the formation of basal lamina-like structures in injured corneas.

Keratin 12-deficient mice have fragile corneal epithelia.

PURPOSE: Expression of the K3-K12 keratin pair characterizes the corneal epithelial differentiation. To elucidate the role of keratin 12 in the maintenance of corneal epithelium integrity, the authors bred mice deficient in keratin 12 by gene-targeting techniques. METHODS: One allele of murine Krt1.12 gene was ablated in the embryonic stem cell line, E14.1, by homologous recombination with a DNA construct in which the DNA element between intron 2 and exon 8 of the keratin 12 gene was replaced by a neo-gene. The homologous recombinant embryonic stem cells were injected to mouse blastocysts, and germ lines of chimeras were obtained. The corneas of heterozygous and homozygous mice were characterized by clinical observations using stereomicroscopy, histology with light and electron microscopy, Western immunoblot analysis, immunohistochemistry, in situ hybridization, and Northern hybridization. RESULTS: The heterozygous mice (+/-) one allele of the Krt1.12 gene appear normal and do not develop any clinical manifestations (e.g., corneal epithelial defects). Homozygous mice (-/-) develop normally and suffer mild corneal epithelial erosion. Their corneal epithelia are fragile and can be removed by gentle rubbing of the eyes or brushing with a Microsponge. The corneal epithelium of the homozygote (-/-) does not express keratin 12 as judged by immunohistochemistry, Western immunoblot analysis with epitope-specific anti-keratin 12 antibodies, Northern hybridization with 32P-labeled keratin 12 cDNA, and in situ hybridization with an anti-sense keratin 12 riboprobe. Light and electron microscopy revealed subtle abnormalities in the corneal epithelia of -/- mice (i.e., a decrease in number of cell layers) and cytolysis of superficial cells, but the number of hemidesmosomes and desmosomes are normal in basal and suprabasal cells. The number of keratin intermediate filaments in basal and suprabasal corneal epithelial cells in -/- mice decreases, and they appear as dense bundles. This morphology is similar to that of keratin intermediate filaments in epidermal epithelial, cells but differs from that of normal corneal epithelial cells in which the keratins form fine filamentous networks. The superficial epithelial cells are devoid of keratin intermediate filaments and often detach from the corneal surface of -/- mice. CONCLUSIONS: The presence of cornea-specific K3-K12 keratin pairs is essential for the maintenance of corneal epithelium integrity.

Appearance of immune cells and expression of MHC II DQ molecule by fibroblasts in alkali-burned corneas.

Corneal alkali burns are characterized by persistent inflammatory response and recurrent epithelial erosions. We examine whether immune cell types, i.e., T-cells and B-cells, play a role in this devastating process. Rabbit alkali-burned corneas that healed for 1-49 days were subjected to immunostaining with monoclonal antibodies (mAb) L11/135 (anti-T-cells), and 2C4 (anti-MHC II DQ). Serum was collected weekly and subjected to Western blot immunostaining to detect antibodies against denatured corneal proteins. Our observations demonstrated that all injured corneas reepithelialized within 3 days but then developed recurrent erosions. Immunohistochemical studies revealed that PMN, monocytes, and B-cells labeled by 2C4 mAb and T-cells labeled by L11/135 mAb appeared in the periphery to the cornea at 1 day after alkali burn. Many of these myeloid and lymphoid cells invaded the central stroma after 2 weeks of injuries when the alkali-burned corneas were heavily vascularized. In addition, some fibroblastic cells also expressed the MHC II DQ molecules in the alkali-burned corneas that had healed for > 2 weeks. Plasma cells appeared in granulation tissue of injured corneas that had healed for > 3 weeks. Western blot analysis demonstrated a production of heterogeneous antibodies in a majority of the rabbits (11 of 14) to various denatured corneal proteins (between 80 kDa and 25 kDa) at 5 weeks of alkali burn. Inflammatory cell types, i.e., PMN, macrophages could be found underneath the detached epithelium. These observations are consistent with the notion that the myeloid and lymphoid cells may participate in and complicate the healing of corneal alkali burns.

Conjunctival epithelial cells can resurface denuded cornea, but do not transdifferentiate to express cornea-specific keratin 12 following removal of limbal epithelium in mouse.

Limbal stem cell deficiency contributes to recurrent corneal epithelial defects. We examined whether the conjunctival epithelium can transdifferentiate to corneal epithelium following surgically induced limbal stem cell deficiency. Mice were anesthetized by intraperitoneal injection of sodium pentobarbital. Partial or total epithelial removal was produced with a no. 69 Beaver blade under a dissecting microscope. The wounds were allowed to heal for 0-28 days, and the mice were examined every other day to evaluate re-epithelialization. Corneas were then subjected to histological, immunohistochemical studies and Western blot analysis with epitope-specific anti-keratin 12 antibodies. Partial epithelial defects re-epithelialized within 2 days and were normal in appearance and expressed cornea-specific keratin 12. In eyes with limbal deficiency, re-epithelialization progressed more slowly and was characterized by opacification; epithelial closure usually occurred by the 7th day. This epithelium differed from normal corneal epithelium in basic morphology, cell shape, and the presence of goblet cells at 2 weeks after injury. The epithelium at the center of injured corneas with total defect at 4 weeks had cornealike morphology and was devoid of goblet cells. These epithelial cells derived from conjunctiva did not express the cornea-specific keratin 12 as determined by immunohistochemistry, Western blot analysis and in situ hybridization. As evidenced by differences in morphology and the expression of cornea-specific keratin 12, conjunctival transdifferentiation does not occur in conjunctical overgrowth after the removal of limbal epithelium.

Characterization and chromosomal localization of the cornea-specific murine keratin gene Krt1.12.

Keratins are a group of water-insoluble proteins constituting paired acidic and basic keratin molecules that form 10-nm intermediate filaments in epithelial cells. Expression of the K3/K12 keratin pair characterizes the cornea-type differentiation. However, the mechanism that regulates this cornea-specific K12 expression remains unknown. To provide a better understanding of the cornea-specific expression, we have cloned the K12 cDNA (Liu, C.-Y., Zhu, G., Westerhausen-Larson, A., Converse, R., Kao, C. W.-C., Sun, T.-T., and Kao, W. W.-Y. (1993) Curr. Eye Res. 12, 963-974). In present studies, the murine K12 keratin gene (Krt1.12) was isolated and characterized. The murine Krt1.12 gene spans 6,567 base pairs of genomic DNA, and the mRNA encoding K12 keratin is distributed into eight exons. Chromosome mapping reveals that murine Krt1.12 is located within the Krt1 complex of mouse chromosome 11. In addition to the production of authentic K12 mRNA, the Krt1.12 gene gives rise to several alternate poly(A)+ RNAs by the use of alternative splicing in intron 2, an alternative promoter in intron 1, and/or both. Sequence analysis indicates that the transcripts derived from alternative splicing and/or the alternative promoter do not have a long open reading frame for keratin or keratin-like molecules. It is not known whether these alternate K12 poly(A)+ RNAs have any biological functions, e.g. regulation of K12 gene expression.