Expression of HB-EGF by retinal pigment epithelial cells in vitreoretinal proliferative disease

The heparin-binding epidermal growth factor-like growth factor (HB-EGF) has been implicated in wound-healing processes of various tissues. However, it is not known whether HB-EGF may represent a factor implicated in overstimulated wound-healing processes of the retina during proliferative retinopathies. Therefore, we investigated whether human retinal pigment epithelial (RPE) cells, which are crucially involved in proliferative retinopathies, express and respond to HB-EGF. RPE cells express mRNAs for various members of the EGF-related growth factor family, among them for HB-EGF, as well as for the EGF receptors ErbB1, -2, -3, and -4. The gene expression of HB-EGF is stimulated in the presence of transforming and basic fibroblast growth factors and by oxidative stress and is suppressed during chemical hypoxia. Exogenous HB-EGF stimulates proliferation and migration of RPE cells and the gene and protein expression of the vascular endothelial growth factor (VEGF). HB-EGF activates at least three signal transduction pathways in RPE cells including the extracellular signal-regulated kinases (involved in the proliferation-stimulating action of HB-EGF), p38 (mediates the effects on chemotaxis and secretion of VEGF), and the phosphatidylinositol-3 kinase (necessary for the stimulation of chemotaxis). In epiretinal membranes of patients with proliferative retinopathies, HB-EGF immunoreactivity was partially colocalized with the RPE cell marker, cytokeratins; this observation suggests that RPE cell-derived HB-EGF may represent one factor that drives the uncontrolled wound-healing process of the retina. The stimulating effect on the secretion of VEGF may suggest that HB-EGF is also implicated in the pathological angiogenesis of the retina.

Enhancement of titanium implants via bioengineered periodontal tissues

Osseointegration of titanium dental implants into the jaw bone, which is required for maintenance of the implant in the jaw, results in ankylosis. Dental implants are therefore very unlike natural teeth, which exhibit significant movement in response to mechanical forces. The ability to generate periodontal ligament (PDL) tissues onto dental implants would better mimic the functional characteristics of natural teeth, and would likely improve implant duration and function. OBJECTIVES: The objective of this study was to investigate the feasibility of bioengineering PDL tissues onto titanium implant surfaces. METHODS: Bilateral maxillary first and second molars of 8-week old rats were extracted and used to generate single cell suspensions of PDL tissues, which were expanded in culture. Immunohistochemistry and RT-PCR were used to identify putative PDL progenitor/stem cell populations and characterize stem cell properties, including self-renewal, multipotency and stem cell maker expression. Cultured rPDL cells were harvested at third passage, seeded onto Matrigel-coated titanium implants (1.75 mm x 1 mm), and placed into healed M1/M2 extraction sites. Non-cell seeded Matrigel-coated titanium implants served as negative controls. Implants were harvested after 8, 12, or 18 weeks. RESULTS: Cultured rPDL cells expressed the mesenchymal stem-cell marker STRO-1. Under defined culture conditions, PDL cells differentiated into adipogenic, neurogenic and osteogenic lineages. While control implants were largely surrounded by alveolar bone, experimental samples exhibited fibrous PDL-like tissues, and perhaps cementum, on the surface of experimental implants. CONCLUSIONS: PDL contains stem cells that can generate cementum/PDL-like tissue in vivo. Transplantation of these cells might hold promise as a therapeutic approach for the bioengineering of PDL tissues onto titanium implant. Further refinement of this method will likely result in improved dental implant strategies for use of autologous PDL tissue regeneration in humans. This research was supported by CIMIT, and NIH/NIDCR grant DE016132 (PCY), and TEACRS (YL).

Culture-induced changes in blood-brain barrier transcriptome: implications for amino-acid transporters in vivo

Tight homeostatic control of brain amino acids (AA) depends on transport by solute carrier family proteins expressed by the blood-brain barrier (BBB) microvascular endothelial cells (BMEC). To characterize the mouse BMEC transcriptome and probe culture-induced changes, microarray analyses of platelet endothelial cell adhesion molecule-1-positive (PECAM1(+)) endothelial cells (ppMBMECs) were compared with primary MBMECs (pMBMEC) cultured in the presence or absence of glial cells and with b.End5 endothelioma cell line. Selected cell marker and AA transporter mRNA levels were further verified by reverse transcription real-time PCR. Regardless of glial coculture, expression of a large subset of genes was strongly altered by a brief culture step. This is consistent with the known dependence of BMECs on in vivo interactions to maintain physiologic functions, for example, tight barrier formation, and their consequent dedifferentiation in culture. Seven (4F2hc, Lat1, Taut, Snat3, Snat5, Xpct, and Cat1) of nine AA transporter mRNAs highly expressed in freshly isolated ppMBMECs were strongly downregulated for all cultures and two (Snat2 and Eaat3) were variably regulated. In contrast, five AA transporter mRNAs with low expression in ppMBMECs, including y(+)Lat2, xCT, and Snat1, were upregulated by culture. We hypothesized that the AA transporters highly expressed in ppMBMECs and downregulated in culture have a major in vivo function for BBB transendothelial transport.

Ataxia due to vertebral hemangiosarcoma with evidence of disseminated disease in a 20-year old Swiss Warmblood mare

A 20-year old Swiss Warmblood mare was referred to the Swiss Institute of Equine Medicine with a history of poor performance, coughing and ataxia and hindlimb weakness which progressed to recumbency. Lung auscultation revealed pronounced wheezing, blood work showed signs of chronic inflammation and increased bone turnover and thoracic ultrasound indicated patchy pulmonary consolidation. Cerebrospinal fluid revealed only mild, unspecific changes allowing exclusion of meningoencephalomyelitis and clinically relevant bleeding. Despite medical treatment and support in a sling the mare did not improve and was euthanized. Necropsy revealed a poorly demarcated, non-encapsulated and invasively growing mass dorsally in the musculature at the level of the forth cervical vertebra (C4) infiltrating the vertebral body and the spinal canal at the level of C1–C2. Multiple nodular, firm masses were present in all lobes of the lung and appeared to be mainly located in vessels. Histologically the masses were composed of spindle cells with marked anisocytosis, anisocaryosis, a high mitotic activity and showed invasive growth. These neoplastic cells stained positive for CD31, an endothelial cell marker, which confirmed diagnosis of a hemangiosarcoma. Definite ante mortem diagnosis of hemangiosarcoma, which is rare in horses, is challenging. Besides the vertebral localization, disseminated, locally invasive and cutaneous forms of hemangiosarcoma exist and can be either acquired or congenital. Prognosis for equine hemangiosarcoma and response to treatment are usually poor and progression of clinical signs is rapid. Vertebral hemangiosarcoma is an uncommon cause of spinal ataxia in horses.

Immunopathology of postprimary tuberculosis: increased T-regulatory cells and DEC-205-positive foamy macrophages in cavitary lesions.

Postprimary tuberculosis occurs in immunocompetent people infected with Mycobacterium tuberculosis. It is restricted to the lung and accounts for 80% of cases and nearly 100% of transmission. Little is known about the immunopathology of postprimary tuberculosis due to limited availability of specimens. Tissues from 30 autopsy cases of pulmonary tuberculosis were located. Sections of characteristic lesions of caseating granulomas, lipid pneumonia, and cavitary stages of postprimary disease were selected for immunohistochemical studies of macrophages, lymphocytes, endothelial cells, and mycobacterial antigens. A higher percentage of cells in lipid pneumonia (36.1%) and cavitary lesions (27.8%) were positive for the dendritic cell marker DEC-205, compared to granulomas (9.0%, P < .05). Cavities contained significantly more T-regulatory cells (14.8%) than found in lipid pneumonia (5.2%) or granulomas (4.8%). Distribution of the immune cell types may contribute to the inability of the immune system to eradicate tuberculosis.

High expression of FOXP3 in primary melanoma is associated with tumour progression.

BACKGROUND The antitumour immune response plays an important role in the prognosis of melanoma. High numbers of circulating regulatory T cells have been associated with rapid disease progression. OBJECTIVES To assess the influence of forkhead box protein (FOXP)3, CD1a and langerin expression on the prognosis of primary melanoma. METHODS We analysed 185 primary melanomas by immunohistochemical staining for expression of the regulatory T-cell marker FOXP3 and the dendritic cell markers langerin and CD1a, and correlated marker expression with clinical outcome. RESULTS Disease-free survival and overall survival were significantly longer in patients expressing low levels of FOXP3 in the primary melanoma, whereas they were associated with high expression of CD1a. The negative prognostic value of FOXP3 expression was independent of the Breslow tumour thickness. Langerin expression did not correlate with the clinical outcome. CONCLUSIONS High expression of FOXP3 in the primary melanoma may be used as an additional independent prognostic marker for early tumour progression in patients with melanoma.

Materno-fetal nutrient transfer across primary human trophoblast layer.

MATERNO-FETAL NUTRIENT TRANSFER ACROSS PRIMARY HUMAN TROPHOBLAST MONOLAYER Objectives: Polarized trophoblasts represent the transport and metabolic barrier between the maternal and fetal circulation. Currently human placental nutrient transfer in vitro is mainly investigated unidirectionallyon cultured primary trophoblasts, or bidirectionally on the Transwell® system using BeWo cells treated with forskolin. As forskolin can induce various gene alterations (e.g. cAMP response element genes), we aimed to establish a physiological primary trophoblast model for materno-fetal nutrient exchange studies without forskolin application. Methods: Human term cytotrophoblasts were isolated by enzymatic digestion and Percoll® gradient separation. The purity of the primary cells was assessed by flow cytometry using the trophoblast-specific marker cytokeratin-7. After screening different coating matrices, we optimized the growth conditions for the primary cytotrophoblasts on Transwell/ inserts. The morphology of 5 days cultured trophoblasts was determined by scanning electron microscopy (SEM) and transmission electron microscopy (TEM). Membrane makers were visualized using confocal microscopy. Additionally transport studies were performed on the polarized trophoblasts in the Transwell® system. Results: During 5 days culture, the trophoblasts (>90% purity) developed a modest trans-epithelial electrical resistance (TEER) and a sizedependent apparent permeability coefficient (Papp) to fluorescently labeled compounds (MW ~400-70’000D). SEM analyses confirmed a confluent trophoblast layer with numerous microvilli at day six, and TEM revealed a monolayer with tight junctions. Immunocytochemistry on the confluent trophoblasts showed positivity for the cell-cell adhesion molecule E-cadherin, the tight junction protein ZO-1, and the membrane proteins ABCA1 and Na+/K+-ATPase. Vectorial glucose and cholesterol transport studies confirmed functionality of the cultured trophoblast barrier. Conclusion: Evidence from cell morphology, biophysical parameters and cell marker expressions indicate the successful and reproducible establishment of a primary trophoblast monolayer model suitable for transport studies. Application of this model to pathological trophoblasts will help to better understand the mechanism underlying gestational diseases, and to define the consequences of placental pathology on materno-fetal nutrient transport.

PHENOTYPIC CHARACTERIZATION OF SPONTANEOUS AND IN VITRO-MAINTAINED AKR LYMPHOMAS

T-cell lymphomas from AKR mice were studied to determine their potential as a model of T-cell differentiation. Homogeneous tumor cell lines have been used as model to study normal lymphocyte subpopulations, including differentiation lineages, functional properties, and the inducibility to maturation. The underlying concept is that each lymphoid tumor represents a monoclonal neoplastic proliferation of a discrete lymphoid subpopulation arrested at a particular differentiation stage.^ Individual tumors were analyzed to determine the extent of intertumor heterogeneity, and to determine whether lymphomas represented different thymocyte subsets, by determining the cell-surface antigenic phenotype, PNA-binding capacity, and terminal deoxynucleotidyl transferase (TdT) activity. Splenic and thymic tumor cells were compared to determine if the particular lymphoid microenvironment influenced T-cell marker expression. Several of the lymphomas were passaged in syngeneic hosts to verify the original tumor phenotype and to assess the stability of the cell surface and TdT phenotype after transplantation.^ Lymphomas were adapted to in vitro culture to determine whether the T-cell phenotype was maintained in the absence of the host microenvironment. Clonal progeny were analyzed and compared with each other and with parent cell lines to determine the extent of intratumor heterogeneity in this lymphoma system. Parent and cloned cell lines were passaged in vivo to determine whether alterations in surface phenotype occurred after transplantation.^ Our investigation has verified that most spontaneous AKR lymphomas phenotypically resemble known T-cell subsets, including both immature and mature thymic subpopulations. The in vitro lines, however, expressed a highly unstable phenotype in culture that included loss of Ly-1 and Ly-2 antigen expression. After transplantation in vivo, the in vitro lines exhibited alterations in phenotype, including re-expression of Ly antigen on some lymphomas. The inducibility of T-cell antigen markers on tumor cell lines passaged in vivo suggests that the in vitro lines may serve as a possible model system to study the molecular events involved in gene expression in the T-cell system. ^

Unique molecular surface features of in vivo tolerized T cells

Differential expression of surface markers can frequently be used to distinguish functional subsets of T cells, yet a surface phenotype unique to T cells induced into an anergic state has not been described. Here, we report that CD4 T cells rendered anergic in vivo by superantigen can be identified by loss of the 6C10 T cell marker. Inoculation of Vβ8.1 T cell antigen receptor (TCR) transgenic mice with a Vβ8.1-reactive minor lymphocyte-stimulating superantigen (Mls-1a) induces tolerance to Mls-1a by clonal anergy. CD4 lymph node T cells from Mls-1a inoculated transgenic mice enriched for the 6C10− phenotype neither proliferate nor produce interleukin-2 upon TCR engagement, whereas 6C10+ CD4 T cells retain responsiveness. Analysis of T cell memory markers demonstrate that 6C10− T cells remain 3G11hi but express heterogeneous levels of CD45RB, CD62L, CD44, and the CD69 early activation marker, suggesting that T cells at various degrees of activation can be functionally anergic. These studies demonstrate that anergic T cells can be purified based on 6C10 expression permitting examination of issues concerning biochemical and biological features specific to T cell anergy.

Detection of targeted GFP-Hox gene fusions during mouse embryogenesis

The ability to use a vital cell marker to study mouse embryogenesis will open new avenues of experimental research. Recently, the use of transgenic mice, containing multiple copies of the jellyfish gene encoding the green fluorescent protein (GFP), has begun to realize this potential. Here, we show that the fluorescent signals produced by single-copy, targeted GFP in-frame fusions with two different murine Hox genes, Hoxa1 and Hoxc13, are readily detectable by using confocal microscopy. Since Hoxa1 is expressed early and Hoxc13 is expressed late in mouse embryogenesis, this study shows that single-copy GFP gene fusions can be used through most of mouse embryogenesis. Previously, targeted lacZ gene fusions have been very useful for analyzing mouse mutants. Use of GFP gene fusions extends the benefits of targeted lacZ gene fusions by providing the additional utility of a vital marker. Our analysis of the Hoxc13GFPneo embryos reveals GFP expression in each of the sites expected from analysis of Hoxc13lacZneo embryos. Similarly, Hoxa1GFPneo expression was detected in all of the sites predicted from RNA in situ analysis. GFP expression in the foregut pocket of Hoxa1GFPneo embryos suggests a role for Hoxa1 in foregut-mediated differentiation of the cardiogenic mesoderm.

Removal of LIF (leukemia inhibitory factor) results in increased vitamin A (retinol) metabolism to 4-oxoretinol in embryonic stem cells

Retinoids, vitamin A (retinol) and its metabolic derivatives, are required for normal vertebrate development. In murine embryonic stem (ES) cells, which remain undifferentiated when cultured in the presence of LIF (leukemia inhibitory factor), little metabolism of exogenously added retinol takes place. After LIF removal, ES cells metabolize exogenously added retinol to 4-hydroxyretinol and 4-oxoretinol and concomitantly differentiate. The conversion of retinol to 4-oxoretinol is a high-capacity reaction because most of the exogenous retinol is metabolized rapidly, even when cells are exposed to physiological (≈1 μM) concentrations of retinol in the medium. No retinoic acid or 4-oxoRA synthesis from retinol was detected in ES cells cultured with or without LIF. The cytochrome P450 enzyme CYP26 (retinoic acid hydroxylase) is responsible for the metabolism of retinol to 4-oxoretinol, and CYP26 mRNA is greatly induced (>15-fold) after LIF removal. Concomitant with the expression of CYP26, differentiating ES cells grown in the absence of LIF activate the expression of the differentiation marker gene FGF-5 whereas the expression of the stem cell marker gene FGF-4 decreases. The strong correlation between the production of polar metabolites of retinol and the differentiation of ES cells upon removal of LIF suggests that one important action of LIF in these cells is to prevent retinol metabolism to biologically active, polar metabolites such as 4-oxoretinol.

p63 identifies keratinocyte stem cells

The proliferative compartment of stratified squamous epithelia consists of stem and transient amplifying (TA) keratinocytes. Some polypeptides are more abundant in putative epidermal stem cells than in TA cells, but no polypeptide confined to the stem cells has yet been identified. Here we show that the p63 transcription factor, a p53 homologue essential for regenerative proliferation in epithelial development, distinguishes human keratinocyte stem cells from their TA progeny. Within the cornea, nuclear p63 is expressed by the basal cells of the limbal epithelium, but not by TA cells covering the corneal surface. Human keratinocyte stem and TA cells when isolated in culture give rise to holoclones and paraclones, respectively. We show by clonal analysis that p63 is abundantly expressed by epidermal and limbal holoclones, but is undetectable in paraclones. TA keratinocytes, immediately after their withdrawal from the stem cell compartment (meroclones), have greatly reduced p63, even though they possess very appreciable proliferative capacity. Clonal evolution (i.e., generation of TA cells from precursor stem cells) is promoted by the sigma isoform of the 14-3-3 family of proteins. Keratinocytes whose 14-3-3σ has been down-regulated remain in the stem cell compartment and maintain p63 during serial cultivation. The identification of p63 as a keratinocyte stem cell marker will be of practical importance for the clinical application of epithelial cultures in cell therapy as well as for studies on epithelial tumorigenesis.

Transferring an inborn auditory perceptual predisposition with interspecies brain transplants

Inborn species' perceptual preferences are thought to serve as important guides for neonatal learning in most species of higher vertebrates. Although much work has been carried out on experiential contributions to the expression of such preferences, their neural and developmental correlates remain largely unexplored. Here we use embryonic neural transplants between two bird species, the Japanese quail and the domestic chicken, to demonstrate that an inborn auditory perceptual predisposition is transferable between species. The transfer of the perceptual preference was dissociated from changes to the vocalizations of the resulting animals (called chimeras), suggesting that experiential differences in auditory self-stimulation cannot explain the perceptual change. A preliminary localization of the effective brain region for the behavioral transfer by using a naturally occurring species-cell marker revealed that it is not contained within the major avian auditory pathways. To our knowledge, this is the first demonstration that abstract aspects of auditory perception can be transferred between species with transplants of the central nervous system.

Pulmonary malformation in transgenic mice expressing human keratinocyte growth factor in the lung.

Expression of human keratinocyte growth factor (KGF/FGF-7) was directed to epithelial cells of the developing embryonic lung of transgenic mice disrupting normal pulmonary morphogenesis during the pseudoglandular stage of development. By embryonic day 15.5(E15.5), lungs of transgenic surfactant protein C (SP-C)-KGF mice resembled those of humans with pulmonary cystadenoma. Lungs were cystic, filling the thoracic cavity, and were composed of numerous dilated saccules lined with glycogen-containing columnar epithelial cells. The normal distribution of SP-C proprotein in the distal regions of respiratory tubules was disrupted. Columnar epithelial cells lining the papillary structures stained variably and weakly for this distal respiratory cell marker. Mesenchymal components were preserved in the transgenic mouse lungs, yet the architectural relationship of the epithelium to the mesenchyme was altered. SP-C-KGF transgenic mice failed to survive gestation to term, dying before E17.5. Culturing mouse fetal lung explants in the presence of recombinant human KGF also disrupted branching morphogenesis and resulted in similar cystic malformation of the lung. Thus, it appears that precise temporal and spatial expression of KGF is likely to play a crucial role in the control of branching morphogenesis during fetal lung development.

HMG box transcription factor gene Hbp1 is expressed in germ cells of the developing mouse testis

HMG box containing protein 1 (HBP1) is a high mobility group domain transcriptional repressor that regulates proliferation in differentiated tissues. We have found mouse Hbp1 to be expressed strongly in the embryonic mouse testis from approximately 12.5 days post coitum, compared with low levels of expression in the embryonic ovary. Expression of Hbp1 is maintained in the developing testis beyond the onset of spermatogenesis after birth. Whole-mount in situ hybridisation analysis showed that expression of Hbp1 in the XY gonad is localized within the developing testis cords, the precursors of the seminiferous tubules. Expression of Hbp1 is not apparent in testis cords of gonads from homozygous We mutant embryos, which lack germ cells. In situ hybridisation analysis on cryosectioned embryonic testis indicated that Hbp1 expression resembles that of the germ cell marker Oct4. We conclude that Hbp1 is up-regulated specifically in germ cells of the developing XY gonad. The expression of Hbp1 in XY germ cells appears to correlate with the onset of mitotic arrest in these cells. (C) 2004 Wiley-Liss, Inc.