Ibrutinib monotherapy for relapse or refractory primary CNS lymphoma and primary vitreoretinal lymphoma: Final analysis of the phase II 'proof-of-concept' iLOC study by the Lymphoma study association (LYSA) and the French oculo-cerebral lymphoma (LOC) network.

BACKGROUND: Primary central nervous system lymphomas (PCNSLs) are mainly diffuse large B-cell lymphomas (DLBCLs) of the non-germinal centre B-cell subtype, with unmet medical needs. This study aimed to evaluate the efficacy and toxicity of ibrutinib in DLBCL-PCNSL PATIENTS AND METHODS: This prospective, multicentre, phase II study involved patients with relapse or refractory(R/R) DLBCL-PCNSL or primary vitreoretinal lymphoma. The treatment consisted of ibrutinib (560 mg/day) until disease progression or unacceptable toxicity occurred. The primary outcome was the disease control (DC) rate after two months of treatment (P0 < 10%; P1 > 30%). RESULTS: Fifty-two patients were recruited. Forty-four patients were evaluable for response. After 2 months of treatment, the DC was 70% in evaluable patients and 62% in the intent-to-treat analysis, including 10 complete responses (19%), 17 partial responses (33%) and 5 stable diseases (10%). With a median follow-up of 25.7 months (range, 0.7-30.5), the median progression-free and overall survivals were 4.8 months (95% confidence interval [CI]; 2.8-12.7) and 19.2 months (95% CI; 7.2-NR), respectively. Thirteen patients received ibrutinib for more than 12 months. Two patients experienced pulmonary aspergillosis with a favourable (n = 1) or fatal outcome (n = 1). Ibrutinib was detectable in the cerebrospinal fluid (CSF). The clinical response to ibrutinib seemed independent of the gene mutations in the BCR pathway. CONCLUSION: Ibrutinib showed clinical activity in the brain, the CSF and the intraocular compartment and was tolerated in R/R PCNSL. The addition of ibrutinib to standard methotrexate-base induction chemotherapy will be further evaluated in the first-line treatment. CLINICAL TRIAL NUMBER: NCT02542514.

Phase I study of lapatinib plus vinorelbine in patients with locally advanced or metastatic breast cancer overexpressing HER2.

BACKGROUND: To determine the recommended doses of lapatinib (LPT) combined with vinorelbine (VNR) in women with human epidermal growth factor receptor 2-overexpressing advanced breast cancer pretreated with trastuzumab. METHODS: In this phase I study, women were treated with oral daily LPT and i.v. VNR infused on days 1 and 8 every 3 weeks. Dose levels (DL) of LPT (mg)/VNR (mg m(-2)) ranged from 750/20 to 1250/30. The primary end point was feasibility based on maximal tolerated dose (MTD) and maximum administered dose (MAD). Pharmacokinetic interactions were investigated. RESULTS: Of 33 patients included, 29 were evaluable. Two DLT occurred at DL4 (1000/25) meeting the MAD criteria. Despite an additional intermediate DL3' (1250/22.5), MTD was reached at DL3 (1000/22.5). Grade 3-4 neutropenia was the most common toxicity (34% and 38% of patients, respectively). Other significant toxicities included grade 3-4 diarrhoea (3% each), and grade 3 asthenia (10%). Although not statistically significant, LPT (at 1000 or 1250 mg) decreased the VNR clearance by 30-40% compared with DL1. CONCLUSION: The MTD LPT 1000 mg/VNR 22.5 mg m(-2) (DL3) is recommended for additional development. Pharmacokinetic interactions might increase the exposure to VNR and consequently alter the hematological tolerance.

Effect of ABCB1 C3435T polymorphism on docetaxel pharmacokinetics according to menopausal status in breast cancer patients.

BACKGROUND: It can be hypothesised that inherited polymorphisms in the drug-transporter ABCB1 gene may interfere with interindividual variations in drug response in breast cancer patients. Docetaxel is a substrate for ABCB1 whose function has been shown to be modulated by oestrogen and progesterone. METHODS: Whether ABCB1 polymorphisms including T-129C, A61G, C1236T, G2677T/A and C3435T polymorphisms could account for variations in the disposition of docetaxel and whether menopausal status at the time of diagnosis might interact with this effect were analysed in women receiving neoadjuvant chemotherapy for breast cancer (n=86). RESULTS: A highly significant association was observed, but restricted to premenopausal women (n=53), between the pharmacokinetics of docetaxel and C3435T polymorphism, as patients with CC genotype had lower mean values of the area under the plasma concentration-time curve (AUC) of docetaxel than patients with CT and TT genotypes (P<0.0001). Comparison between pre- and postmenopausal women with the same C3435T genotype yielded a significant difference in docetaxel AUC only for CC genotype (P<0.0001). CONCLUSION: These results suggest that C3435T polymorphism genotyping and menopausal status at the time of diagnosis might be useful when considering chemotherapy regimens including docetaxel in breast cancer patients.

Variations in schedules of ifosfamide administration: a better understanding of its implications on pharmacokinetics through a randomized cross-over study.

PURPOSE: The metabolism of ifosfamide is a delicate balance between a minor activation pathway (4-hydroxylation) and a mainly toxification pathway (N-dechloroethylation), and there remains uncertainty as to the optimal intravenous schedule. METHODS: This study assesses ifosfamide pharmacokinetics (PK) according to two standard schedules. Using a 1:1 randomized trial design, we prospectively evaluated ifosfamide PK on two consecutive cycles of 3 g/m2/day for 3 days (9 g/m2/cycle) given in one of two schedules either by continuous infusion (CI) or short (3 h) infusion. Highly sensitive analytical methods allowed determination of concentrations of ifosfamide and the key metabolites 4-hydroxy-ifosfamide, 2- and 3-dechloroethyl-ifosfamide. RESULTS: Extensive PK analysis was available in 12 patients and showed equivalence between both schedules (3 h versus CI) based on area under the curves (micromol/l x h) for ifosfamide, 4-hydroxy-ifosfamide, 2- and 3-dechloroethyl-ifosfamide (9,379 +/- 2,638 versus 8,307 +/- 1,995, 152 +/- 59 versus 161 +/- 77, 1,441 +/- 405 versus 1,388 +/- 393, and 2,808 +/- 508 versus 2,634 +/- 508, respectively, all P > 0.2). The classical auto-induction of metabolism over the 3 days of infusion was confirmed for both schedules. CONCLUSION: This study confirms similar PK for both active and toxic metabolites of ifosfamide in adult cancer patients when 9 g/m2 of ifosfamide is administered over 3 days by CI or daily 3-h infusions.

Development of Daptomycin resistance in vivo in methicillin-resistant Staphylococcus aureus.

Daptomycin is a new lipopeptide antibiotic that is rapidly bactericidal against Staphylococcus aureus. We report daptomycin resistance and treatment failure in 2 patients with osteomyelitis due to methicillin-resistant S. aureus. Disk diffusion susceptibility testing failed to detect resistance. Daptomycin at high concentration retained bactericidal activity against resistant isolates.

Factors influencing visualization of vertebral metastases on MR imaging versus bone scintigraphy.

OBJECTIVE: The purpose of this study was to investigate whether the location and size of vertebral body metastases influence the difference in detection rates between MR imaging and bone scintigraphy. MATERIALS AND METHODS: We retrospectively evaluated the vertebral body lesions detected on MR imaging in 74 patients with known widely disseminated metastatic disease. Three radiologists independently reviewed the MR images and bone scintigraphs. MR imaging findings included lesion size and its spatial relationship to the bony cortex (intramedullary, subcortical, and transcortical) and results were correlated with those of planar technetium 99m bone scintigraphy. RESULTS: Findings on bone scans were negative for all intramedullary lesions without cortical involvement shown on MR imaging, regardless of their size. Findings on bone scans (71.3% for transcortical and 33.8% for subcortical) were frequently positive for lesions with cortical involvement (trans- or subcortical), and the probability of positive findings on bone scans was also influenced by the lesion size. Statistical analysis showed a positive correlation among cortical involvement, lesion size, and positive findings on bone scintigraphy (p < 0.0001). CONCLUSION: Location (the presence of cortical bone involvement on MR imaging) and size of the vertebral body metastases appear to be important contributing factors to the difference in detection rates between MR imaging and bone scintigraphy. Cortical involvement is likely the cause of positive findings on bone scans. Early vertebral metastases tend to be small and located in the medullary cavity without cortical involvement, and therefore, findings may be positive on MR images but negative on bone scans.

Cytokine modulation of costimulatory molecules on human fetal retinal pigment epithelial cells.

PURPOSE: Experiments were performed to evaluate the effect of various pro- and anti-inflammatory cytokines on the human fetal retinal pigment epithelium's (HFRPE) expression of major histocompatibility complex (MHC) and costimulatory molecules. METHODS: Pure cultures of HFRPE cells were isolated. HFRPE cells were incubated in the presence of Interferon-gamma (IFN-gamma), IFN-beta, Tumor Necrosis Factor-alpha (TNF-alpha), Interleukin-1beta (IL-1beta), Tumor Growth Factor-beta (TGF-beta), and a combination of IFN-gamma and TGF-beta (pre-incubation and simultaneously incubated). The expression of MHC class I and class II, Intercellular cell adhesion molecule (ICAM-1), B7-1 (CD80), and B7-2 (CD86) molecules was quantitatively analyzed by flow cytometry. RESULTS: The cultured HFRPE cells expressed high levels of MHC class I and low levels of MHC class II and ICAM-1 molecules. After culture with the above mentioned cytokines, IFN-gamma up-regulated the HFRPE's expression of MHC class II and ICAM-1. IFN-beta and IL-beta1 only up-regulated the expression of ICAM-1. TGF-beta was unable to suppress the up-regulatory effect of IFN-gamma in HFRPE cells (pre-incubated and simultaneously incubated). The other cytokines did not have any significant effect on HFRPE's expression of MHC I and II or the selected costimulatory molecules. CONCLUSIONS: Our findings indicate that TGF-beta cannot suppress up-regulating effects of IFNgamma- on HFRPE's expression of MHC and costimulatory molecules. Overall, the weak or lack of expression of costimulatory molecules after stimulation with various cytokines further confirms that HFRPE cells are weak antigen presenting cells.

Human fetal retinal pigment epithelium-induced cell cycle arrest, loss of mitochondrial membrane potential and apoptosis.

PURPOSE: To investigate the mechanism of action of the soluble immune suppressive product secreted by human fetal retinal pigment epithelial (HFRPE) cells in a model system using the human T-cell line Jurkat (Jkt). METHODS: Pure HFRPE cells were isolated and cultured. The supernatants of both nonactivated and IFN-gamma-activated HFRPE cells were isolated. Cells from the human T-cell line Jkt were incubated either in standard culture medium or in the supernatant isolated from HFRPE cells. In the first assay Jkt cell proliferation was measured by [(3)H]thymidine incorporation. In the second assay Jkt cell apoptosis was examined for annexin V staining by flow cytometry. In the third assay Jkt cell division was evaluated with carboxyfluorescein succinimidyl ester (CFSE) fluorescent dye. In the last assay the mitochondrial transmembrane potential of Jkt cells was measured with the cationic lipophilic fluorochrome 3,3'-dihexyloxacarbocyanine iodide [DiOC(6)]. In all the assays the effect of supernatants isolated from both nonactivated and IFN-gamma-activated HFRPE cells were compared with standard culture medium. The involvement of antiapoptotic human gene bcl-x(L:)was determined by using a Jkt cell line that was stably transfected with bcl-x(L:). RESULTS: The supernatant isolated from HFRPE cells significantly suppressed the cell division in Jkt cells and induced apoptosis. These effects were stronger when the supernatant was isolated from IFN-gamma-activated HFRPE cells. The apoptosis pathway induced by the secreted product of HFRPE cells involved the early disruption of mitochondrial transmembrane potential. Although the overexpression of bcl-x(L) gene rescued the Jkt cells from supernatant-induced apoptosis, it could not restore the proliferation of Jkt cells. CONCLUSIONS: These data suggest that HFRPE cells secrete a product that initiates an early cell cycle arrest in the human T-cell line Jkt, which is followed by the activation of an apoptotic pathway that involves the loss of mitochondrial membrane potential. The latter could be prevented by bcl-x(L) overexpression. Also these data suggest that the HFRPE-induced T-cell apoptosis may play a significant role in maintaining the immune privilege in the subretinal space.

Iris pigment epithelial cell translocation in exudative age-related macular degeneration. A pilot study in patients.

BACKGROUND: This prospective, non-controlled pilot study investigates the practicability of IPE translocation and functional outcome in ARMD patients. Removal of submacular choroidal neovascularization (CNV) in age-related macular degeneration (ARMD) is usually associated with RPE damage and poor visual prognosis. Homologous RPE transplants fail to preserve macular function, possibly due to immune rejection. Instead of homologous RPE, we suggest translocating autologous iris pigment epithelium (IPE), building on earlier evidence from animal and in vitro investigations that IPE can substitute RPE functions in the experimental animal. Immunological cell rejection is avoided. METHODS: Four eyes with well-defined and eight eyes with ill-defined subfoveal CNV were submitted to operation and followed up for a minimum of 6 months. IPE cells were harvested from a peripheral iridectomy. A vitrectomy was performed. Submacular membranes were removed, and isolated IPE cells were injected into the subretinal space. Examinations included ETDRS visual acuity, fluorescein angiography, and SLO microperimetry. RESULTS: All patients underwent successful surgical removal of CNV and subretinal IPE injection. Compared to preoperative visual acuity (20/400-20/100) no significant change was observed after 6 months (20/320-16/80). A change of more than two ETDRS chart lines was defined as significant. One eye with preoperative ill-defined CNV developed a recurrence, leading to reduced visual acuity. In all patients, postoperative fluorescence angiography revealed early hyperfluorescence (window defect) in the surgically denuded area. Central fixation was demonstrated in 50% of eyes. CONCLUSIONS: Preliminary data suggests that IPE translocation in submacular surgery for ARMD can preserve but not improve preoperative visual acuity over 6 months. Functional results are promising compared to submacular membrane extraction alone and RPE transplantation. Continued research on improvement of IPE translocation seems justified.

A model for xenogenic immune response.

PURPOSE: To develop a model for analyzing the immune response after xenogenic human fetal retinal pigment epithelium (HFRPE) transplantation. MATERIALS AND METHODS: Pure sheets of HFRPE cells were isolated and attached to poly (DL-lactide-co-glycolide) polymer films and HFRPE spheroids were formed. The spheroids were transplanted into the subretinal space of New Zealand albino rabbits and were observed for 5 months. Bare polymer films were transplanted into the subretinal space of Dutch Belted pigmented rabbits, serving as control. RESULTS: The polymer film was biodegraded within 3 weeks in the subretinal space. No signs of inflammation in the retina or choroid were observed. The HFRPE spheroids were easily transplanted into the subretinal space. The immune response was followed with ophthalmoscopy. Light microscopy indicated a localized immune response in the transplanted area in which the retina and the choroid were infiltrated with immune cells. This infiltration was denser in the choroid. CONCLUSIONS: HFRPE spheroid transplantation may be utilized as a model for studying the xenogenic immune response after HFRPE transplantation. This model may also have applications in evaluating the role of immune suppressive agents in preventing rejection after HFRPE transplantation.

Human fetal retinal pigment epithelium suppresses the activation of CD4(+) and CD8(+) T-cells.

BACKGROUND: The suppressive effect of human fetal retinal pigment epithelium (HFRPE) on the activation of human CD4(+) and CD8(+) T-cells was evaluated in vitro. METHODS: Pure populations of CD4(+) and CD8(+) T-cells were isolated from human peripheral blood-derived buffy coats by negative immunomagnetic selection. The purity of the cells was examined by flow cytometry using anti-CD3-FITC, anti-CD4-FITC, anti-CD8-PE, and anti-CD20-PE mAbs. HFRPE cells were isolated from fetal eyes and pure cultures were obtained. The effect of normal or IFN-gamma-activated HFRPE cells at early (P3) or late (P6) passages on the activation of CD4(+) and CD8(+) T-cells was assessed in two different T-cell activation assays. In both activation models anti-CD3 mAb (OKT3) provided the antigen-specific signal. The secondary signal for the activation of CD4(+) and CD8(+) T-cells was provided with anti-CD18 mAb (TS1/18) and anti-CD28 mAb (9.3) in the first and the second assay respectively. Cross-linking of these soluble mAbs was performed with sheep-anti-mouse IgG-coated latex beads. The T-cell activation was determined by cell proliferation measured by [(3)H]thymidine incorporation. In each activation assay T-cells were incubated with HFRPE cells in a ratio of T-cells to HFRPE of 1:1 or 1:4. RESULTS: CD4(+) and CD8(+) T-cells were activated by cross-linking CD3 and CD18 in the first assay (CD3/CD18) and CD3 and CD28 in the second assay (CD3/CD28). In both assays HFRPE inhibited the activation of CD4(+) and CD8(+) T-cells. IFN-gamma-activated HFRPE cells totally suppressed the T-cell activation at a 1:1 ratio. This suppressive effect was weaker at lower cell ratios. Some donor variation was observed in the inhibition at the lower cell ratios, especially for the inhibition of CD8(+) T-cell activation with anti-CD3/CD18. The passaging of HFRPE cells did not alter their suppressive effect on CD4(+) and CD8(+) T-cells. CONCLUSIONS: HFRPE cells suppressed the activation of both CD4(+) and CD8(+) T-cells in vitro. These findings suggest that RPE-induced immune suppression may play a significant role in maintaining immune privilege in the subretinal space.

Human fetal retinal pigment epithelial cells induce apoptosis in allogenic T-cells in a Fas ligand and PGE2 independent pathway.

PURPOSE: To evaluate the inhibitory effect of human fetal retinal pigment epithelium (HFRPE) on the activation of human T-cells. METHODS: Pure cultures of HFRPE cells were incubated with purified human T-cells in three different activation assays: 1) allogenic peripheral blood mononuclear cells; 2) OKT3 coated beads in the presence of accessory cells; and 3) stimulation with phorbol ester and phytohemagglutinin. RESULTS: HFRPE cells suppressed the activation of T-cells in all three assays. The mechanism of HFRPE mediated T-cell suppression was apoptosis. The role of Fas ligand(FasL)/Fas-mediated T-cell suppression was excluded, since FasL protein or mRNA could not be detected on HFRPE cells with flow cytometry and by reverse transcriptase polymerase chain reaction, respectively. Additionally, the inhibitory effect of HFRPE cells could not be blocked by anti-Fas ligand or antagonistic anti-Fas antibodies. Moreover, HFRPE cells suppressed the proliferation of anti-CD3 mAb mediated T-cell proliferation of murine splenocytes isolated from lpr mice. The inhibitory effect of HFRPE cells was not PGE2 mediated, since indomethacin could not restore the T-cell activation. Although the HFRPE mediated T-cell apoptosis was cell-cell contact independent, it was not induced by secretion of TNF-alpha, TGF-beta, or IL-10. The ratio between HFRPE and T-cells had a major impact on the HFRPE's inhibitory effect. CONCLUSIONS: HFRPE cells suppressed the activation of human T-cells by induction of T-cell apoptosis through a process that involves the secretion of soluble factors. The HFRPE mediated T-cell suppression was dependent on the ratio between HFRPE and T-cells. This undefined pathway of T-cell apoptosis may play a role in the maintenance of immune privilege in the subretinal space and may reduce the severity of the immune response after HFRPE transplantation.

Cryoprecipitate: An autologous substrate for human fetal retinal pigment epithelium.

PURPOSE: To harvest thin membranes from cryoprecipitates isolated from human blood donors and utilize them as substrates for the adhesion of human fetal retinal pigment epithelial (HFRPE) cells. METHODS: Frozen human cryoprecipitates from anonymous blood donors were obtained from the blood bank. Thin cryo-membranes were harvested by their mixture with riboflavin-5-phosphate (R5P) and overnight exposure to ultra-violet light. Sheets of retinal pigment epithelium (RPE) were isolated from fetal eyes at 17-22 weeks gestational age. The sheets were subsequently attached to cryo-membranes. The morphology of the cells was examined with phase contrast and electron microscope. Cell proliferation was evaluated by their incorporation of 5-bromo-2'-deoxyuridine (BrdU). Functional viability was assessed by rod outer segment (ROS) phagocytosis. RESULTS: Thin membrane films were made from isolated human cryoprecipitates. Isolated sheets of pure HFRPE cells were attached to the membranes. The cells maintained their cuboidal morphology and did not dedifferentiate. The cells subsequently proliferated and migrated onto the culture plate, forming cellular monolayers. The cultured cells phagocytized isolated ROS. CONCLUSIONS: Cryoprecipitate membranes may provide an ideal source for the adhesion, cultivation, and transfer of HFRPE cells. Their autologous isolation from the recipient's blood grants an additional advantage for their application as a carrier for HFRPE transplantation into the subretinal space.

Human fetal retinal pigment epithelial cells induce apoptosis in the T-cell line Jurkat.

PURPOSE: To investigate the mechanism(s) involved in human fetal retinal pigment epithelium (HFRPE)-mediated T-cell death. METHODS: Pure HFRPE cells were isolated and cultured. Normal and interferon (IFN)-gamma-activated HFRPE from early and late in vitro passages were incubated with cells from the human T-cell leukemia line Jurkat (Jkt). Cultures were pulsed with [3H]-thymidine to measure Jkt cell proliferation. Jkt cells were evaluated for apoptosis either by staining with an ethidium bromide/acridine orange mixture (AO/EB) or with Annexin V-phycoerythrin. The role of Fas ligand (FasL) molecule in HFRPE-mediated apoptosis was assessed by using a mutant Jkt cell line (DD3), which is deficient in Fas-mediated signaling. The involvement of the antiapoptotic human gene bcl-xL was determined by using Jkt cells that were stably transfected with bcl-x(L). The role of cell- cell contact in the induction of apoptosis was evaluated in a transwell system in the presence or absence of neutralizing antibodies against IFN-gamma and tumor necrosis factor (TNF)-alpha. RESULTS: HFRPE cells inhibited the proliferation of Jkt cells by inducing apoptosis through a FasL-independent pathway. Passaging and IFN-gamma activation strengthened the inhibitory effect of HFRPE cells on the proliferation of Jkt cells. At lower HFRPE passages (P2), bcl-alphaL, overexpression rescued the HFRPE cell-mediated apoptosis. The separation of the cells by the transwell system did not affect the HFRPE cell-mediated suppression. This suppressive effect was not mediated by the secretion of IFN-gamma or TNF-alpha molecules. CONCLUSIONS: HFRPE cells suppressed the proliferation of Jkt cells by inducing apoptosis. HFRPE cells induced a stronger inhibitory effect on Jkt cells at higher in vitro passages. The HFRPE-induced apoptosis was not mediated through the FasL/Fas pathway or through the secretion of the apoptosis-inducing cytokines IFN-gamma and TNF-alpha. The bcl-xL gene may play a role in preventing HFRPE cell-induced apoptosis in Jkt cells. These combined results suggest that the HFRPE-mediated suppression of primary T cells may also be mediated by the induction of apoptosis. Therefore, the retinal pigment epithelium may play a role in the induction of immune privilege in the subretinal space.

Biodegradable polymer film as a source for formation of human fetal retinal pigment epithelium spheroids.

PURPOSE: To evaluate the attachment of human fetal rctinal pigment epithelial (HFRPE) cells to a biodegradable polymer film with subsequent formation of spheroids in vitro. METHODS: Ten biodegradable polymer films with different compositions were examined for their physical properties and ease of manipulation under a dissecting microscope. The film with the most suitable handling characteristics was chosen, and a purely isolated sheet of HFRPE cells was attached to it. The purity of the cells was assessed by their pigmentation and expression of cytokeratin. Proliferation was assessed by incorporation of 5-bromo-2'-deoxyuridine (BrdtJ). Cellular structure was analyzed under light and electron microscopes, and the functional capability of the cells was evaluated by rod outer segment (ROS) phagocytosis. RESULTS: The polymer film with composition 50:50 poly (DL-lactide) (PLA)/poly (DL-lactide-co-glycolide) (PLG) with an inherent viscosity of 1.03 dl/g was found to be the most suitable for handling under the microscope. Sheets of HFRPE cells attached to the polymer films within 48 hours and began to form spheroids. All the isolated cells were pigmented and expressed cytokeratin. They possessed a cuboidal morphology, numerous apical microvilli, and no sign of dedifferentiation. HFRPE cells produced extracellular matrix (collagen filaments) on their basal side, filling the cavities of the polymer film. The cells subsequently proliferated, incorporated BrdU, migrated onto the culture plate to form monolayers, and phagocytized ROS. CONCLUSIONS: Biodegradable polymer films can be used as a scaffold for the adhesion of the HFRPE sheet and formation of spheroids. Spheroids represent a source of high density and well-differentiated HFRPE cells that are easy to transfer. Furthermore, the stricture of the membrane makes it suitable for additional applications.

Growth of human fetal retinal pigment epithelium as microspheres.

BACKGROUND: The aim was to develop a three-dimensional cell culture system for human fetal retinal pigment epithelial (HFRPE) cells for in vitro cellular studies and for possible application in subretinal transplantation. METHODS: Pieces of freshly isolated HFRPE monolayer tissue were grown on crosslinked fibrinogen (CLF) films. The growth pattern and morphologic characteristics of the implanted tissue were studied using phase-contrast microscopy, photography, and light and electron microscopy. The cells were screened immunohistochemically for HLA-ABC, HLA-DR, ICAM-1, B7, and Cytokeratin. Cell proliferation was studied using 5-bromo-2-deoxyuridine incorporation. RESULTS: After attachment to CLF, HFRPE monolayer tissue formed small tumor-like formations, i.e. microspheres. HFRPE microspheres survived and proliferated in a floating state for at least 4 months. After attachment of the microspheres to the culture dish floor, formation of a confluent HFRPE cell monolayer with high proliferative activity was noted around the microspheres. HFRPE cells stained positive for HLA-ABC, ICAM-1, and cytokeratin and negative for B7 and HLA-DR. The microspheres could be easily detached from the dish and they were able to initiate similar growth after reattachment. CONCLUSION: HFRPE grown on CLF resemble a three-dimensional culture system with high yield of pure cells that can be useful for a wide variety of in vitro studies. Because of their adjustable size, spherical shape, and ability to initiate growth of cells with a high proliferative potential, HFRPE microspheres may be successfully utilized as a source of donor cells for subretinal transplantation.

The immunogenic potential of human fetal retinal pigment epithelium and its relation to transplantation.

PURPOSE: To perform a quantitative analysis of the expression of major histocompatibility molecules (MHC classes I and II) and costimulatory molecules by human fetal retinal pigment epithelial (HFRPE) cells and to evaluate their potential role in providing costimulatory signals for the activation of human T cells in vitro. METHODS: Pure HFRPE cells were isolated and cultured. The ability of HFRPE cells to express MHC class I and II and costimulatory molecules before and after incubation with interferon (IFN)-gamma was quantitatively analyzed by flow cytometry. The potential of HFRPE cells to activate human T cells was assessed in three different lymphocyte activation models. In the first model, anti-CD3 (OKT3)-coated beads were used to provide the T-cell receptor (TcR) signal. In the second model, the allogenic potential of HFRPE cells was assessed, and in the third assay a potent superantigen (SEA) was used to provide the TcR signal. T-cell activation was determined by cell proliferation, measured by [3H]-thymidine incorporation. RESULTS: The cultured HFRPE cells expressed low levels of MHC class I and ICAM-1 molecules. After incubation with IFN-gamma, the expression of MHC class I and ICAM-1 molecules was further upregulated, and the expression of MHC class II and VCAM-1 molecules was induced. The expression of the costimulatory molecules B7-1 and B7-2 was not observed in normal or activated HFRPE cells. In the first T-cell activation model, neither normal nor IFN-gamma-activated HFRPE cells could provide T-cell costimulation for anti-CD3 (OKT3)-coated beads. However, the autologous peripheral blood mononuclear cells (PBMCs; used here as the source of antigen-presenting cells) could provide costimulation for the T cells, inducing their proliferation. In the second T-cell activation assay, normal or IFN-gamma-activated HFRPE cells could not stimulate an alloresponse from the T cells, but they could induce a significant alloimmune T-cell response in the presence of PBMCs. In the third T-cell activation assay, the IFN-gamma-activated HFRPE cells were able to provide T-cell costimulation for the SEA-mediated activation. CONCLUSIONS: In these in vitro experiments, the IFN-gamma-activated HFRPE cells stimulated only the T cells with the potent superantigen SEA. In the absence of antigen-presenting cells, the HFRPE cells did not provide T-cell costimulation in an anti-CD3 mAb-coated bead system or induce significant alloimmune response. These results suggest that in transplantation between donors and recipients with different MHC molecules, the direct MHC peptide presentation by HFRPE cells may not induce a significant allospecific immune response. Nevertheless, an allospecific immune response could occur as a consequence of the indirect presentation, to the host T cells by the host antigen-presenting cells, of the HFRPE cells' derived MHC alloantigens.

Effect of antipsychotics on regional cerebral blood flow measured with positron emission tomography.

Positron Emission Tomography (PET) imaging of regional cerebral blood flow (rCBF) provides an in vivo method for studying brain function. We used [15O]H20 PET to assess the effect of antipsychotic medications on rCBF in 17 subjects with schizophrenia. Each subject was scanned while receiving antipsychotic medication, and after having been withdrawn from antipsychotic medication for a 3-week period. The two scans were subtracted from one another, using a within subjects design, and the areas of difference were identified using the Montreal method. Subjects treated with antipsychotic medication had significantly higher rCBF in the left basal ganglia and left fusiform gyrus compared with the "off-medication" condition. Significantly higher relative rCBF in the anterior cingulate, left dorsolateral and inferior frontal cortex, and left and right cerebellum was observed when off antipsychotic medication. Upregulation of dopamine D2 receptors may lead to a regional increase of blood flow and metabolism in the basal ganglia, which may explain recently reported anatomical enlargement in these regions.

Iris pigment epithelium transplantation.

BACKGROUND: Iris pigment epithelium (IPE) cells and retinal pigment epithelium (RPE) cells possess the same embryonic origin. It is also known that the pigmented epithelial cells in the eye have a high transdifferentiation potential. In this study we transplanted IPE cells into the subretinal space of albino Royal College of Surgeons (RCS) rats and evaluated their influence on the degeneration of the photoreceptors. METHODS: IPE cells of Long Evans rats were isolated and pure cultures were obtained. The isolated cells were transplanted into the subretinal space of RCS rats. Light microscopic and morphometric analysis were carried out. RESULTS: The IPE transplants survived in the subretinal space and attached themselves to the Bruch's membrane. The transplanted cells were able to delay the degeneration of the photoreceptors for up to 3 months. CONCLUSION: These results suggest that IPE cells could be successfully transplanted and survive in the subretinal space. In the transplanted eyes the photoreceptors were preserved for a period of 3 months. Further studies are needed to explore the capability of IPE cells to assume the main functions of RPE cells in the subretinal space and their potential in the therapy of selective degenerative diseases of the retina.