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ABSTRACT: Dendritic cell (DC) modification is a potential strategy to induce clinical transplantation tolerance. We compared two DC modification strategies to inhibit allogeneic T-cell proliferation. In the first strategy, murine DCs were transduced with a lentiviral vector expressing CTLA4-KDEL, a fusion protein which prevents surface CD80/86 expression by retaining the costimulatory molecules within the endoplasmic reticulum. In the second approach, DCs were transduced to express the tryptophan-catabolising enzyme indoleamine 2,3-dioxygenase (IDO). CTLA4-KDEL-expressing DCs induced anergy in alloreactive T cells and generated both CD4(+) CD25(+) and CD4(+) CD25(-) Treg cells (with direct and indirect donor allospecificity and capacity for linked suppression) both in vitro and in vivo. In contrast, T-cell unresponsiveness induced by IDO(+) DCs lacked donor specificity. In the absence of any immunosuppressive treatment, intravenous administration of CTLA4-KDEL-expressing DCs resulted in long-term survival of corneal allografts only when the DCs were capable of indirect presentation of alloantigen. This study demonstrates the therapeutic potential of CTLA4-KDEL-expressing DCs in tolerance induction.
European Journal of Immunology 12/2012; · 5.10 Impact Factor
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Albrecht von Graæes Archiv für Ophthalmologie 04/2012; 248(12):1849-1849. · 2.17 Impact Factor
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ABSTRACT: To measure the retinal blood flow velocity in patients with retinitis pigmentosa using the retinal function imaging technique.
The clinical observational investigation included a study group of five eyes of five patients (age: 55.7 ± 8.6 years) with retinitis pigmentosa (RP) and a control group of five eyes of five healthy subjects. We used a randomly chosen eye of the RP patients, and compared its results to the normal subjects using a mixed linear model, correcting for heart rate, age, and gender.
The mean blood velocity in the narrow retinal veins (1.7 ± 0.35 cm/s versus 3.0 ± 0.35 cm/s; P < 0.001) and wide retinal veins (1.5 ± 0.35 cm/s versus 3.1 ± 0.30 cm/s; P < 0.001) was significantly lower in the study group than in the control group not correcting for heart rate, age or gender. Correspondingly, the arterial blood flow velocity was significantly lower in the study group than in the control group for the narrow arterial vessels (2.3 ± 0.55 versus 4.2 ± 0.5; P = 0.006) and for the wide retinal arteries (2.5 ± 1.05 cm/s versus 4.8 ± 1.0 cm/s; P < 0.001).
Using the retinal function imaging technology revealed significantly lower retinal blood flow velocities in the small and large retinal vessels in patients with retinitis pigmentosa than in healthy subjects. This corresponds with the known decrease in the retinal vessel diameters as observed upon ophthalmoscopy in patients with retinitis pigmentosa. Retinal function imaging technology may hold promise for measurements of retinal blood flow parameters.
Albrecht von Graæes Archiv für Ophthalmologie 08/2011; 249(12):1855-8. · 2.17 Impact Factor
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ABSTRACT: Axonal loss is considered a key prognostic factor in diagnosing and monitoring the progress of multiple sclerosis (MS). The purpose of our research was to determine whether the measurement of retinal nerve fibre layer thickness (RNFLT) as measured with high-resolution spectral-domain optical coherence tomography (SD-OCT) differs between optic nerve injury following acute optic neuritis (ON) or following unregistered subclinical axonal damage in patients with MS.
High-resolution SD-OCT measurements of RNFLT were initially carried out in the acute phase of ON and again after 3 months, in 25 patients with clinical definite MS and 25 sex- and age-matched healthy controls, all at the University Eye Hospital, Vienna.
Conventional OCT-based RNFLT analysis correctly identified all three patients with initial RNFL swelling. However, only two of three acute ON eyes with a history of ON were registered with RNFLT decrease in seven peripapillary sectors (PPs). The remaining have only been revealed using RNFLT symmetry comparison. Two of 22 (9%) first-episode ON eyes were labelled as pathologic. The number and metric RNFL values of pathologically labelled PPs remained unchanged after 3 months. Our age- and sex-match-based measurement model, with patients with MS being plotted individually and towards the fellow eye, identified all acute ON eyes (with a history of prior ON) with RNFLT reduction in 11 PPs. A global RNFL loss was registered in 36.4% (eight of 22 eyes). However, in 72%, or 16 of 22 ON eyes presenting with first episode of acute ON, a segmental RNFL loss was initially registered in 39 PPs upon baseline examination. The number of PPs with identified axonal decrease increased to a total of 48 PPs within the observational period.
Spectral-domain optical coherence tomography imaging of identical scanning locations, combined with an optimized scan centring around the optic disc, offers the technological potential of detecting prior, subtle, clinically unregistered optic nerve injury within MS individuals. Significant discrepancy in RNFLT to the potential ON eye may be achieved by comparing OCT metrics with the fellow eye and a sufficient number of age and sex-matched controls.
Acta ophthalmologica 03/2011; 89(5):e451-60. · 2.44 Impact Factor
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ABSTRACT: To assess retinal blood flow in patients with central serous chorioretinopathy (CSR).
The hospital-based observational comparative study included a study group with 12 patients (age: 45 ± 13 years) with an acute onset of CSR and a control group of 12 subjects matched for age and gender with the study group. The diagnosis was substantiated by fluorescein angiography and optical coherence tomography. All study participants underwent measurement of retinal blood perfusion using the retinal function imager (RFI).
The retinal blood flow velocity in the retinal veins was significantly lower in the study group than in the control group (2.76 ± 0.53 mm/s versus 3.33 ± 0.76 mm/s; p = 0.03).The difference between the study group and control group was more marked for the larger retinal veins (2.87 ± 0.51 mm/s versus 3.47 ± 0.48 mm/s; p = 0.001) than for the smaller veins (2.69 ± 0.53 mm/s versus 3.42 ± 1.05 mm/s; p = 0.04). Both groups did not differ significantly in the data of the retinal arterial flow velocities.
The data suggest an abnormal retinal venous blood flow regulation in patients with active CSR.
Acta ophthalmologica 03/2011; 89(6):e479-82. · 2.44 Impact Factor
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ABSTRACT: "Non-invasive, faster and less expensive than MRI" and "the eye is a window to the brain" are recent slogans promoting optical coherence tomography (OCT) as a new surrogate marker in multiple sclerosis (MS). Indeed, OCT allows for the first time a non-invasive visualization of axons of the central nervous system (CNS). Reduction of retina nerve fibre layer (RNFL) thickness was suggested to correlate with disease activity and duration. However, several issues are unclear: Do a few million axons, which build up both optic nerves, really resemble billions of CNS neurons? Does global CNS damage really result in global RNFL reduction? And if so, does global RNFL reduction really exist in all MS patients, and follow a slowly but steadily ongoing pattern? How can these (hypothesized) subtle global RNFL changes be reliably measured and separated from the rather gross RNFL changes caused by optic neuritis? Before generally being accepted, this interpretation needs further critical and objective validation.
We prospectively studied 37 MS patients with relapsing remitting (n = 27) and secondary progressive (n = 10) course on two occasions with a median interval of 22.4±0.5 months [range 19-27]. We used the high resolution spectral domain (SD-)OCT with the Spectralis 3.5 mm circle scan protocol with locked reference images and eye tracking mode. Patients with an attack of optic neuritis within 12 months prior to the onset of the study were excluded.
Although the disease was highly active over the observation period in more than half of the included relapsing remitting MS patients (19 patients/32 relapses) and the initial RNFL pattern showed a broad range, from normal to markedly reduced thickness, no significant changes between baseline and follow-up examinations could be detected.
These results show that caution is required when using OCT for monitoring disease activity and global axonal injury in MS.
PLoS ONE 01/2011; 6(5):e19843. · 4.09 Impact Factor
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ABSTRACT: Purpose: Optical coherence tomography (OCT) has emerged as the technique of choice in measuring the retinal nerve fibre layer (RNFL) quantitatively. It is suggested that RNFL reduction may correlate with lesion burden and diffuse axonal degeneration in the whole CNS of patients with multiple sclerosis (MS). However, RNFL changes because of optic neuritis (ON) must be taken into account. Methods: Twenty-three patients with acute ON (46 eyes) associated with clinical definite MS (23 ON eyes, 23 fellow eyes) and 23 sex- and age-matched healthy controls were studied. Retinal nerve fibre layer thickness (RNFLT) was measured at baseline, using a high-resolution spectral domain OCT (SD-OCT) applying circular, peripapillary OCT scans with a novel eye-tracking mechanism. Results: The internal OCT software was able to identify RNFL atrophy in three out of five of the acute ON eyes and one out of four of the fellow eyes with previous ON episodes. Retinal nerve fibre layer thickness of two ON (8.7%) and five fellow eyes (21.7%) was overestimated, thus located within the 95% and 5% confidence interval of the company standard values (not marked pathologic). In contrast, our comparison with age- and sex-matched controls revealed RNFL atrophy suggestive of prior, clinically silent RNFL loss in ON and fellow eyes (30.4%). Conclusion: Retinal nerve fibre layer thickness measurements at a single time-point seem to have a limited role in detecting prior clinically silent optic nerve injury. Our data suggest that affected eyes should be compared with the fellow eyes and a sufficient number of age- and sex-matched controls to allow the detection of even subtle RNFL changes at baseline. The role of OCT for disease monitoring of MS must be evaluated in detail, as ON is often the initial symptom of MS.
Acta ophthalmologica 11/2010; 90(6):540-5. · 2.44 Impact Factor
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Nermin Serbecic,
Fahmy Aboul-Enein, Sven C Beutelspacher,
Martin Graf,
Karl Kircher,
Wolfgang Geitzenauer,
Werner Brannath,
Priska Lang,
Wolfgang Kristoferitsch,
Hans Lassmann,
Andreas Reitner,
Ursula Schmidt-Erfurth
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ABSTRACT: Recently the reduction of the retinal nerve fibre layer (RNFL) was suggested to be associated with diffuse axonal damage in the whole CNS of multiple sclerosis (MS) patients. However, several points are still under discussion. (1) Is high resolution optical coherence tomography (OCT) required to detect the partly very subtle RNFL changes seen in MS patients? (2) Can a reduction of RNFL be detected in all MS patients, even in early disease courses and in all MS subtypes? (3) Does an optic neuritis (ON) or focal lesions along the visual pathways, which are both very common in MS, limit the predication of diffuse axonal degeneration in the whole CNS? The purpose of our study was to determine the baseline characteristics of clinical definite relapsing-remitting (RRMS) and secondary progressive (SPMS) MS patients with high resolution OCT technique.
Forty-two RRMS and 17 SPMS patients with and without history of uni- or bilateral ON, and 59 age- and sex-matched healthy controls were analysed prospectively with the high resolution spectral-domain OCT device (SD-OCT) using the Spectralis 3.5mm circle scan protocol with locked reference images and eye tracking mode. Furthermore we performed tests for visual and contrast acuity and sensitivity (ETDRS, Sloan and Pelli-Robson-charts), for color vision (Lanthony D-15), the Humphrey visual field and visual evoked potential testing (VEP).
All 4 groups (RRMS and SPMS with or without ON) showed significantly reduced RNFL globally, or at least in one of the peripapillary sectors compared to age-/sex-matched healthy controls. In patients with previous ON additional RNFL reduction was found. However, in many RRMS patients the RNFL was found within normal range. We found no correlation between RNFL reduction and disease duration (range 9-540 months).
RNFL baseline characteristics of RRMS and SPMS are heterogeneous (range from normal to markedly reduced levels).
PLoS ONE 01/2010; 5(11):e13877. · 4.09 Impact Factor
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ABSTRACT: The expression of chemokines is central to the recruitment of inflammatory cells for graft rejection, and modulation of chemokine action is of potential in preventing graft rejection. We have examined chemokine expression in a murine model of corneal allograft rejection, and also determined the effect of expressing a broad acting chemokine antagonist, viral macrophage inflammatory protein II (vMIP II), on graft survival.
The expression of chemokines in a murine model of corneal transplantation was determined by real time RT-PCR and, in the case of regulated on activation normal T-cell expressed and secreted, by ELISA. The plasmid encoding the virally derived chemokine antagonist, vMIP II, was introduced into the corneal endothelial cells using a non-viral vector consisting of liposomes and transferrin. The expression and activity of vMIP II was determined by ELISA and functional assays, and the effect on graft survival noted.
After allotransplantation, there was up-regulation of all 11 chemokines examined. After gene delivery, there was expression of active vMIP II for more than 14 days and considerable prolongation of graft survival. This was associated with a decrease in leukocyte infiltration of the stroma of the cells.
As expected there was considerable up-regulation of chemokines during allograft rejection. The expression of vMIP II showed considerable prolongation of graft survival. This is the first time we have observed prolongation of graft survival after a non-viral (as opposed to viral) means of gene delivery and indicates the potential of interfering with chemokine action to prevent corneal graft failure.
Transplantation 07/2008; 85(11):1640-7. · 4.00 Impact Factor
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ABSTRACT: Indoleamine 2,3-dioxygenase (IDO) suppresses T cell responses by its action in catabolising tryptophan. It is important in maintenance of immune privilege in the placenta. We investigated the activity of IDO in the cornea, following corneal transplantation and the effect of IDO over-expression in donor corneal endothelium on the survival of corneal allografts. IDO expression was analysed and functional activity was quantified in normal murine cornea and in corneas following transplantation as allografts. Low levels of IDO, at both mRNA and protein levels, was detected in the normal cornea, up-regulated by IFN-gamma and TNF. Expression of IDO in cornea was significantly increased following corneal transplantation. However, inhibition of IDO activity in vivo had no effect on graft survival. Following IDO cDNA transfer, murine corneal endothelial cells expressed functional IDO, which was effective at inhibiting allogeneic T cell proliferation. Over-expression of IDO in donor corneal allografts resulted in prolonged graft survival. While, on one hand, our data indicate that IDO may augment corneal immune privilege, up-regulated IDO activity following cytokine stimulation may serve to inhibit inflammatory cellular responses. While increasing IDO mRNA expression was found in allogeneic corneas at rejection, over-expression in donor cornea was found to significantly extend survival of allografts.
European Journal of Immunology 04/2006; 36(3):690-700. · 5.10 Impact Factor
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ABSTRACT: Endothelium is an important target for gene therapy. We have investigated the effect of viral and nonviral vectors on the phenotype and function of endothelial cells (ECs) and developed methods to block any activation caused by these vectors.
Transduction of ECs with viral vectors, including adenovirus, lentiviruses, and Moloney murine leukemia virus, can induce a pro-inflammatory phenotype. This activation was reduced when nonviral vectors were used. We demonstrate that after transduction there is upregulation of dsRNA-triggered antiviral and PI3K/Akt signaling pathway. Blockade of the NFkappaB, PI3-K, or PKR signaling pathways all operated to inhibit partially virally induced activation, and inhibition of both PKR and PI3-K pathways totally blocked EC activation. Furthermore, inhibition of IFN-alpha/beta in addition to PI3-K was effective at preventing EC activation.
Viral vectors, although efficient at transducing ECs, result in their activation. Blockade of the signaling pathways involved in viral activation may be used to prevent such activation.
Arteriosclerosis Thrombosis and Vascular Biology 04/2006; 26(3):462-7. · 6.37 Impact Factor
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ABSTRACT: Dendritic cells (DCs) are central to T cell immunity, and many strategies have been used to manipulate DCs to modify immune responses. We investigated the effects of antioxidants ascorbate (vitamin C) and alpha-tocopherol (vitamin E) on DC phenotype and function. Vitamins C and E are both antioxidants, and concurrent use results in a nonadditive activity. We have demonstrated that DC treated with these antioxidants are resistant to phenotypic and functional changes following stimulation with proinflammatory cytokines. Following treatment, the levels of intracellular oxygen radical species were reduced, and the protein kinase RNA-regulated, eukaryotic translation initiation factor 2alpha, NF-kappaB, protein kinase C, and p38 MAPK pathways could not be activated following inflammatory agent stimulation. We went on to show that allogeneic T cells (including CD4(+)CD45RO, CD4(+)CD45RA, and CD4(+)CD25(-) subsets) were anergized following exposure to vitamin-treated DCs, and secreted higher levels of Th2 cytokines and IL-10 than cells incubated with control DCs. These anergic T cells act as regulatory T cells in a contact-dependent manner that is not dependent on IL-4, IL-5, IL-10, IL-13, and TGF-beta. These data indicate that vitamin C- and E-treated DC might be useful for the induction of tolerance to allo- or autoantigens.
The Journal of Immunology 07/2005; 174(12):7633-44. · 5.79 Impact Factor
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Peng H Tan, Sven C Beutelspacher,
Shao-An Xue,
Yao-He Wang,
Peter Mitchell,
James C McAlister,
D Frank P Larkin,
Myra O McClure,
Hans J Stauss,
Mary A Ritter,
Giovanna Lombardi,
Andrew J T George
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ABSTRACT: Genetic modification of dendritic-cell (DC) function is an attractive approach to treat disease, either using mature DCs (mDCs) to immunize patients, or immature DCs (iDCs) to induce tolerance. Viral vectors are efficient at transducing DCs, and we have investigated the effect of transduction with a variety of viral vectors on the phenotype and function of DCs. Adenovirus (Ad), human immunodeficiency virus (HIV), equine anemia virus (EIAV), and Moloney murine leukemia virus (MMLV) all up-regulate costimulatory molecules and major histocompatibility complex (MHC) class II expression on DCs, as well as, in the case of Ad and lentiviral vectors, inducing production of Th1 and proinflammatory cytokines. Following transduction there is activation of double-stranded (ds) RNA-triggered pathways resulting in interferon (IFN) alpha/beta production. In addition, the function of virally infected DCs is altered; iDCs have an increased, and mDCs a decreased, ability to stimulate a mixed lymphocyte reaction (MLR). Viral transduction of mDCs results in up-regulation of the indoleamine 2,3-dioxygenase (IDO) enzyme, which down-regulates T-cell responsiveness. Inhibition of IDO restores the ability of mDCs to stimulate an MLR, indicating that IDO is responsible for the modulation of mDC function. These data have important implications for the use of viral vectors in the transduction of DCs.
Blood 06/2005; 105(10):3824-32. · 9.90 Impact Factor
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ABSTRACT: Genetic manipulation of dendritic cells (DCs) is important in the context of using either mature DCs to immunize patients or immature DCs to induce tolerance. Here, we describe a novel method of transfecting monocyte-derived human DCs using immunolipoplexes containing anti-CD71 or anti-CD205 monoclonal Abs. This results in up to 20% transfection, which can be increased to 20-30% if the immunolipoplexes are used to transfect CD14+ monocytes prior to differentiation into DCs. Transfected DCs can be substantially enriched using a drug-selection protocol during differentiation. Unlike adenoviral transduction, this nonviral transfection does not alter the expression of costimulatory molecules or the production of proinflammatory cytokines by DCs. In addition, DC function is unaltered, as assessed by mixed lymphocyte reactions. To test the feasibility of the immunolipoplexes and selection protocol for therapeutic intervention, we transfected DCs with the immunomodulatory enzyme indoleamine 2,3-dioxygenase (IDO). Allogeneic T cells exposed to IDO-expressing DCs did not proliferate, secreted more IL-10 and less Th1 and Th2 cytokines, and had a higher amount of apoptosis than T cells incubated with control DCs. Furthermore the remaining T cells were rendered anergic to further stimulation by allogeneic DC. These immunolipoplexes, which can be easily and rapidly assembled, have potential for clinical immunization, in particular for tolerance induction protocols.
Molecular Therapy 06/2005; 11(5):790-800. · 6.87 Impact Factor
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ABSTRACT: Accurate measurement of central corneal thickness (CCT) is essential in refractive surgery and advanced glaucoma diagnostics. The gold standard for pachymetry is full-contact ultrasound-based pachymetry. As this method is associated with potential sources of error, noncontact methods have been introduced. The aim of this study was to compare CCT results measured using 4 different techniques.
In this analysis of 20 patients (40 eyes) at the University Eye Hospital Heidelberg, Germany, we compared a slit-lamp-mounted optical coherence tomography (OCT) system (SL-OCT, Heidelberg Engineering, Heidelberg, Germany), conventional ultrasound pachymetry (IOPac, Heidelberg Engineering), optical low coherence reflectometry (OLCR, Haag-Streit, Germany), and scanning-slit pachymetry (Orbscan).
Comparison among the 4 groups did not show significant differences, except the comparison of OLCR to Orbscan; the mean was significantly different (p=0.0247) and the Orbscan detected slightly thicker values than the other methods.
Orbscan, SL-OCT, and OLCR provide non-touch technology, without the need for local anesthesia, and limiting the risk of infection or artifacts. Extreme care must be used interpreting the results obtained from Orbscan, as this technique may overestimate the CCT significantly.
European journal of ophthalmology 21(2):132-7. · 0.96 Impact Factor
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ABSTRACT: Dendritic cells (DCs) are central to T cell immunity, and many strategies have been used to manipulate DCs to modify immune responses. We investigated the effects of antioxidants ascorbate (vitamin C) and -tocopherol (vitamin E) on DC phenotype and function. Vitamins C and E are both antioxidants, and concurrent use results in a nonadditive activity. We have demonstrated that DC treated with these antioxidants are resistant to phenotypic and functional changes following stimulation with proinflammatory cytokines. Following treatment, the levels of intracellular oxygen radical species were reduced, and the protein kinase RNA- regulated, eukaryotic translation initiation factor 2, NF-B, protein kinase C, and p38 MAPK pathways could not be activated following inflammatory agent stimulation. We went on to show that allogeneic T cells (including CD4CD45RO, CD4CD45RA, and CD4CD25 subsets) were anergized following exposure to vitamin-treated DCs, and secreted higher levels of Th2 cytokines and IL-10 than cells incubated with control DCs. These anergic T cells act as regulatory T cells in a contact-dependent manner that is not dependent on IL-4, IL-5, IL-10, IL-13, and TGF-. These data indicate that vitamin C- and E-treated DC might be useful for the induction of tolerance to allo- or autoantigens. The Journal of Immunology, 2005, 174: 7633-7644.
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