Analyses of segment-specific expression of alkaline phosphatase activity in the mesoderm of the oligochaete annelid Tubifex: implications for specification of segmental identity.
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In the embryos of the oligochaete annelid Tubifex, segments VII and VIII specifically express mesodermal alkaline phophatase (ALP) activity in the ventrolateral region. In this study, we examined whether this segment-specific expression of ALP activity depends on external cues. Cell lineage analyses show that the ALP-expressing cells originate from M teloblasts. Furthermore, a set of teloblast-ablation experiments demonstrated that the seventh and eighth primary m blast cells (m7 and m8) produced from M teloblasts give rise to ALP-expressing cells in segments VII and VIII, respectively, and that primary m blast cells other than m7 and m8 lack the ability to generate ALP-expressing progeny cells. The results of another set of blastomere-ablation experiments suggest that ALP-expressing cells emerge independently of interactions with surrounding tissues. Teloblast-transplantation experiments demonstrated that m8 can generate ALP-expressing cells in an ectopical position, suggesting that it is unlikely that ALP activity emerges in response to the positional cues residing in the embryo. These results suggest that m7 and m8 are exclusively specified as precursors of ALP-expressing cells at the time of their birth from M teloblasts. We propose that segmental identities in primary m blast cells of the Tubifex embryo are determined according to the genealogical position in the M lineage and that the M teloblast possesses a developmental program through which the sequence of blast cell identities is determined. (+info)
Transmission of human T-cell lymphotropic virus type 1 tax to rabbits by tax-only-positive human cells.
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The human T-cell lymphrotropic virus type 1 (HTLV-1) is causally related to adult T-cell leukemia and lymphoma and the neurodegenerative diseases tropical spastic paraparesis and HTLV-1-associated myelopathy. In the United States the prevalence of infection has been estimated to range from 0.016 to 0.1% on the basis of serologic tests for antibodies to the viral structural proteins. Blood from donors positive for antibodies to HTLV-1 or HTLV-2 is not used for transfusion. However, patients with the cutaneous T-cell lymphoma mycosis fungoides (MF) are HTLV-1 and -2 seronegative yet harbor proviral sequences identical to those that encode the HTLV-1 transactivating and transforming gene product p40tax in their peripheral blood mononuclear cells (PBMCs), and they usually have antibodies to p40(tax). Moreover, a study of 250 randomly selected blood donors revealed that approximately 8% of these seronegative individuals also had HTLV-1 tax sequences and antibodies to p40(tax), while they lacked sequences and antibodies related to gag, pol, or env. Thus, it seemed important to determine whether the "tax-only" state can be transmitted by transfusion. To this end, PBMCs from HTLV-1 and -2 seronegative tax-only-positive MF patients or from healthy tax-only-positive blood donors were injected into adult rabbits, an established animal model for HTLV-1 infection. The PBMCs of all injected rabbits became tax sequence positive. These observations suggest that HTLV-1 tax can be transmitted by tax-only-positive mononuclear cells. (+info)
Cultured corneal epithelia for ocular surface disease.
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PURPOSE: To evaluate the potential efficacy for autologous and allogeneic expanded corneal epithelial cell transplants derived from harvested limbal corneal epithelial stem cells cultured in vitro for the management of ocular surface disease. METHODS: Human Subjects. Of the 19 human subjects included, 18 (20 procedures) underwent in vitro cultured corneal epithelial cell transplants using various carriers for the epithelial cells to determine the most efficacious approach. Sixteen patients (18 procedures on 17 eyes) received autologous transplants, and 2 patients (1 procedure each) received allogeneic sibling grafts. The presumed corneal epithelial stem cells from 1 patient did not grow in vitro. The carriers for the expanded corneal epithelial cells included corneal stroma, type 1 collagen (Vitrogen), soft contact lenses, collagen shields, and amniotic membrane for the autologous grafts and only amniotic membrane for the allogeneic sibling grafts. Histologic confirmation was reviewed on selected donor grafts. Amniotic membrane as carrier. Further studies were made to determine whether amniotic membrane might be the best carrier for the expanding corneal epithelial cells. Seventeen different combinations of tryspinization, sonication, scraping, and washing were studied to find the simplest, most effective method for removing the amniotic epithelium while still preserving the histologic appearance of the basement membrane of the amnion. Presumed corneal epithelial stem cells were harvested and expanded in vitro and applied to the amniotic membrane to create a composite graft. Thus, the composite graft consisted of the amniotic membrane from which the original epithelium had been removed without significant histologic damage to the basement membrane, and the expanded corneal epithelial stem cells, which had been applied to and had successfully adhered to the denuded amniotic membrane. Animal model. Twelve rabbits had the ocular surface of 1 eye damaged in a standard manner with direct removal of the presumed limbal stem cells, corneal epithelium, and related epithelium, followed by the application of n-heptanol for 60 seconds. After 6 weeks, all damaged eyes were epithelialized and vascularized. Two such treated eyes were harvested without further treatment, to be used for histologic study as damaged controls. The remaining 10 rabbits received composite grafts (consisting of amniotic membrane with expanded allogeneic rabbit corneal epithelial cell transplants) applied to the ocular surface in a standard manner followed by the application of a contact lens. At 16 days following transplantation, 5 of the rabbits were sacrificed and the corneal rims were removed for histologic study. At 28 days, the remaining rabbits were sacrificed and the previously damaged eyes were harvested for histologic and immunohistochemical study. RESULTS: Human subjects. Of the 19 total patients admitted to the study, the presumed corneal epithelial stem cells of 1 patient did not grow in vitro. Of the remaining 18 patients (20 procedures, 19 eyes), 3 patients had unsuccessful results (3 autologous procedures), 1 patient had a partially successful procedure (allogeneic procedure), and 1 patient had a procedure with an undetermined result at present (allogeneic procedure). One unsuccessful patient had entropion/trichiasis and mechanically removed the graft and eventually went into phthisis. The other 2 unsuccessful patients suffered presumed loss of autologous donor epithelium and recurrence of the ocular surface disease (pterygium). The partially successful patient receiving an allogeneic transplant had infectious keratitis delay of his re-epithelialization; he has only minimal visual improvement but has re-epithelialized. The patient receiving the second allogeneic graft lost his donor epithelium at day 4. Additional donor epithelium was reapplied, but the result is undetermined at present. Amniotic membrane as carrier. The in vitro preparation of the amniotic membrane with corneal epithelial stem cell graft overlay was successful. Histology documented removal of the amniotic epithelium and reapplication of corneal epithelial cells. Animal model. The 2 rabbits that had no reparative surgery following standard ocular surface injury had histology and immunopathology consistent with incomplete corneal epithelial stem cell failure with vascularization and scarring of the ocular surface. Light microscopy and immunohistologic staining with AE5 confirmed the conjunctival phenotype of the ocular surface repair but also documented the incomplete model. The allogeneic stern cell transplants had varying results. One rabbit had a suppurative infection and lost the graft. Reparative surgery failed in 2 of the rabbits, failed partially in 3 of the rabbits, was partially successful in 3 others, and was successful in 1 rabbit at 28 days. Histologic and immunopathologic study documented successful growth of corneal epithelium onto the recipient surface. CONCLUSIONS: 1. Presumed corneal epithelial stem cells can be harvested safely from the limbus and expanded successfully in vitro. 2. Expanded corneal epithelial cell cultures can be grown onto various carriers, but currently denuded amniotic membrane seems to be the best carrier for ocular surface repair. 3. Expanded corneal epithelial cell transplants appear to resurface damaged ocular surfaces successfully, but cellular tracking and further confirmation are required. 4. Expanded allogeneic corneal epithelial cell transplants are technically possible and may represent alternative treatment modalities for selected ocular surface problems. 5. These techniques potentially offer a new method of restoring a normal ocular surface while minimizing the threat of damage or depletion to the contralateral or sibling limbal corneal epithelial stem cells. 6. The rabbit model was probably incomplete and should be interpreted with caution. The complete eradication of all corneal epithelial stem cells from any eye is difficult, making confirmation of such work challenging. 7. The results of the rabbit model suggest that allogeneic grafts may restore a nearly normal ocular epithelial surface to certain ocular surface injuries. (+info)
Transplantation of ex vivo expanded endothelial progenitor cells for therapeutic neovascularization.
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Animal studies and preliminary results in humans suggest that lower extremity and myocardial ischemia can be attenuated by treatment with angiogenic cytokines. The resident population of endothelial cells that is competent to respond to an available level of angiogenic growth factors, however, may potentially limit the extent to which cytokine supplementation enhances tissue neovascularization. Accordingly, we transplanted human endothelial progenitor cells (hEPCs) to athymic nude mice with hindlimb ischemia. Blood flow recovery and capillary density in the ischemic hindlimb were markedly improved, and the rate of limb loss was significantly reduced. Ex vivo expanded hEPCs may thus have utility as a "supply-side" strategy for therapeutic neovascularization. (+info)
Irradiated mammary gland stroma promotes the expression of tumorigenic potential by unirradiated epithelial cells.
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We have shown that ionizing radiation, a known carcinogen of human breast, elicits rapid, persistent, and global changes in the mammary microenvironment as evidenced by altered extracellular matrix composition and growth factor activities. To address whether these events contribute to radiogenic carcinogenesis, we evaluated the effect of irradiated mammary stroma on the neoplastic potential of COMMA-D mammary epithelial cells. Although COMMA-D cells harbor mutations in both alleles of p53, they are nontumorigenic when injected s.c. into syngeneic hosts. Unirradiated COMMA-D cells transplanted to mammary fat pads cleared previously of epithelia preferentially formed tumors in irradiated hosts. Tumor incidence at 6 weeks was 81% +/- 12 SE when animals were irradiated with 4 Gy, 3 days prior to transplantation, compared with 19% +/- 2 SE (P < 0.005) in sham-irradiated hosts. This effect was evident when cells were transplanted 1 to 14 days after irradiation. Furthermore, tumors were significantly larger (243.1 +/- 61.3 mm3 versus 30.8 +/- 8.7 mm3) and arose more quickly (100% by 6 weeks versus 39% over 10 weeks in sham hosts) in fat pads in irradiated hosts. The contribution of local versus systemic effects was evaluated using hemibody (left versus right) irradiation; tumors formed only in fat pads on the irradiated side. These data indicate that radiation-induced changes in the stromal microenvironment can contribute to neoplastic progression in vivo. Disruption of solid tissue interactions is a heretofore unrecognized activity of ionizing radiation as a carcinogen. (+info)
A clinical trial combining donor bone marrow infusion and heart transplantation: intermediate-term results.
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BACKGROUND: Donor chimerism (the presence of donor cells of bone marrow origin) is present for years after transplantation in recipients of solid organs. In lung recipients, chimerism is associated with a lower incidence of chronic rejection. To augment donor chimerism with the aim to enhance graft acceptance and to reduce immunosuppression, we initiated a trial combining infusion of donor bone marrow with heart transplantation. Reported herein are the intermediate-term results of this ongoing trial. METHODS: Between September 1993 and August 1998, 28 patients received concurrent heart transplantation and infusion of donor bone marrow at 3.0 x 10(8) cells/kg (study group). Twenty-four contemporaneous heart recipients who did not receive bone marrow served as controls. All patients received an immunosuppressive regimen consisting of tacrolimus and steroids. RESULTS: Patient survival was similar between the study and control groups (86% and 87% at 3 years, respectively). However, the proportion of patients free from grade 3A rejection was higher in the study group (64% at 6 months) than in the control group (40%; P =.03). The prevalence of coronary artery disease was similar between the two groups (freedom from disease at 3 years was 78% in study patients and 69% in controls). Similar proportions of study (18%) and control (15%) patients exhibited in vitro evidence of donor-specific hyporesponsiveness. CONCLUSIONS: The infusion of donor bone marrow reduces the rate of acute rejection in heart recipients. Donor bone marrow may play an important role in strategies aiming to enhance the graft acceptance. (+info)
Functional analysis after auto iris pigment epithelial cell transplantation in patients with age-related macular degeneration.
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Recent transplantation studies indicate that subretinal space is not always an immunologically privileged site and non-autologous cells may be rejected in patients with exudative age-related macular degeneration (AMD). We performed autologous iris pigment epithelial (IPE) cell transplantation by cell suspension after autologous IPE cell culture in 8 patients with AMD. These patients were followed without immunosuppression between 1.5 and 8 months and the retinal function was analyzed. No cystoid macular edema or fluorescein leakage was observed. Six of the 8 patients improved visual acuity of more than two lines and the other two patients retained preoperative visual acuity. Five patients had increased visual field sensitivity, one patient retained pretransplantation sensitivity, and one patient showed a gradual decrease in sensitivity (one patient was not examined). Although 2 of the 8 patients showed decreased amplitude of flicker electroretinography (ERG) (about 60 to 70% as that of preoperative level), the average improvement of each amplitude of a single white flash (a wave), photopic, or flicker ERG was 123, 102, and 107%, respectively. No proliferative change in the submacular lesion or vitreous cavity was observed after transplantation. From this functional analysis, transplanted autologous IPE may have, in part, an alternative function in regard to the retinal pigment epithelium in the subretinal space. (+info)
Alteration of the retinotectal projection map by the graft of mesencephalic floor plate or sonic hedgehog.
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The floor plate plays crucial roles in the specification and differentiation of neurons along the dorsal-ventral (DV) axis of the neural tube. The transplantation of the mesecephalic floor plate (mfp) into the dorsal mesencephalon in chick embryos alters the fate of the mesencephalon adjacent to the transplant from the tectum to the tegmentum, a ventral tissue of the mesencephalon. In this study, to test whether the mfp is involved in the specification of the DV polarity of the tectum and affects the projection patterns of retinal fibers to the tectum along the DV axis, we transplanted quail mfp into the dorsal mesencephalon of chick embryos, and analyzed projection patterns of dorsal and ventral retinal fibers to the tectum. In the embryos with the mfp graft, dorsal retinal fibers grew into the dorsal part of the tectum which is the original target for ventral but not dorsal retinal fibers and formed tight focuses there. In contrast, ventral retinal fibers did not terminate at any part of the tectum. Transplantation of Sonic hedgehog (Shh)-secreting quail fibroblasts into the dorsal mesencephalon also induced the ectopic tegmentum and altered the retinotectal projection along the DV axis, as the mfp graft did. These results suggest that some factors from the mesencephalic floor plate or the tegmentum, or Shh itself, play a crucial role in the establishment of the DV polarity of the tectum and the retinotectal projection map along the DV axis. (+info)