G. A. Limb

MIO‐M1 Cells and Similar Müller Glial Cell Lines Derived from Adult Human Retina Exhibit Neural Stem Cell Characteristics

Growing evidence suggests that glial cells may have a role as neural precursors in the adult central nervous system. Although it has been shown that Müller cells exhibit progenitor characteristics in the postnatal chick and rat retinae, their progenitor‐like role in developed human retina is unknown. We first reported the Müller glial characteristics of the spontaneously immortalized human cell line MIO‐M1, but recently we have derived similar cell lines from the neural retina of several adult eye donors. Since immortalization is one of the main properties of stem cells, we investigated whether these cells expressed stem cell markers. Cells were grown as adherent monolayers, responded to epidermal growth factor, and could be expanded indefinitely without growth factors under normal culture conditions. They could be frozen and thawed without losing their characteristics. In the presence of extracellular matrix and fibroblast growth factor‐2 or retinoic acid, they acquired neural morphology, formed neurospheres, and expressed neural stem cell markers including βIII tubulin, Sox2, Pax6, Chx10, and Notch 1. They also expressed markers of postmitotic retinal neurons, including peripherin, recoverin, calretinin, S‐opsin, and Brn3. When grafted into the subretinal space of dystrophic Royal College of Surgeons rats or neonatal Lister hooded rats, immortalized cells migrated into the retina, where they expressed various markers of retinal neurons. These observations indicate that adult human neural retina harbors a population of cells that express both Müller glial and stem cell markers and suggest that these cells may have potential use for cell‐based therapies to restore retinal function.

Cytokines in proliferative vitreoretinopathy

This study determined the presence of interleukin 1 (IL-1), interleukin 6 (IL-6), tumour necrosis factor α (TNFa), tumour necrosis factor β (TNFβ), interferon γ (IFNγ), transforming growth factor β2 (TGFβ2) and fibroblast proliferation activity (FPA) in vitreous aspirates from eyes undergoing vitrectomy for the treatment of retinal detachment complicated by proliferative vitreoretinopathy (PVR) or uncomplicated retinal detachment (RD). Cadaveric vitreous from normal subjects were used as controls. The results showed that IL-1 and IL-6 predominated in vitreous from eyes with PVR or RD, and that concentrations of IL-6 >20 pg/ml were more frequently found in PVR than in RD (p = 0.031) or control specimens (p = 0.006). Low levels of TNFa were observed in 4/18 eyes with PVR, 1/15 eyes with RD and 1/15 control vitreous, and small concentrations of TNFα were seen in 3/18 eyes with PVR, 1/15 eyes with RD and 2/15 control vitreous. IFNγ was detected in 12/18 eyes with PVR, but only in 5/15 eyes with RD (p = 0.048) and 6/15 control specimens. TGFβ2 was present in all vitreous samples at concentrations ranging from 100 to 4,500 pg/ml with no significant differences among the three groups. Control vitreous possessed the greatest FPA when compared with vitreous from eyes with PVR (p = 0.031) or RD (p = 0.048). These observations provide further evidence that cytokine-mediated pathways of inflammation are involved in the pathogenesis of PVR and point to the possible involvement of IL-1, IL-6 and IFNγ in cellular interactions leading to chronicity.

Distribution of TNF alpha and its reactive vascular adhesion molecules in fibrovascular membranes of proliferative diabetic retinopathy.

AIMS: This study investigated the presence of the cytokine tumour necrosis factor alpha (TNF alpha) and the vascular adhesion glycoproteins ICAM-1, VCAM-1, E-selectin, P-selectin, and PECAM within fibrovascular membranes of eyes with proliferative diabetic retinopathy (PDR). METHODS: The presence of these molecules was determined by immunohistochemical staining using monoclonal antibodies and the APAAP technique. RESULTS: Staining for TNF alpha was observed on the retinal vascular endothelium of five of 12 specimens, on infiltrating cells within all membranes, and on the extracellular matrix of nine specimens. This staining wa abolished by absorption of the monoclonal antibody with human recombinant TNF alpha. Likewise, ICAM-1 staining was given by infiltrating cells and extracellular matrix of nine membranes and by the endothelium of three of the specimens. VCAM-1, E-selectin, and P-selectin staining was observed on the vascular endothelium of 5/12, 4/12, and 3/12 epiretinal membranes respectively. PECAM was expressed by the endothelium of 4/12 specimens, by infiltrating cells of 8/12 membranes, and also by the extracellular matrix of two of the specimens. CONCLUSION: The widespread distribution of TNF alpha and the nature of the adhesion molecules expressed by vascular endothelial cells in PDR membranes suggest that local activation of TNF alpha and enhanced expression of vascular cell adhesion molecules may play an important role in the development of the proliferative phase of diabetic retinopathy.

CYTOKINES IN HUMAN INTRAOCULAR INFLAMMATION

The presence of interleukin 6 (IL-6), interleukin 1 (IL-1), interleukin 2 (IL-2) and tumour necrosis factor (TNF) was investigated in vitreous and aqueous aspirates from eyes undergoing vitrectomy for the treatment of different inflammatory conditions. Cadaveric vitreous from 10 normal subjects were used as controls. IL-6 was observed in 5 specimens from eyes with idiopathic uveitis (range = 26-264 pg/ml), in 2 specimens from eyes with uveitis complicated with retinal detachment (28 and 279 pg/ml, respectively), in 6 samples from eyes with diabetic retinopathy (range = 5-480 pg/ml), in one sample from an eye with phacolytic glaucoma (1190 pg/ml) and in one specimen from an eye with Behçets disease (366 pg/ml). Although IL-1 was detected in 80% of all the samples investigated, concentrations of this cytokine greater than 3 pg/ml were only observed in 2 specimens from eyes with uveitis (5 and 20 pg/ml, respectively) and 2 samples from eyes with diabetic retinopathy (3 and 31 pg/ml, respectively). TNF was present in 3 specimens from eyes with uveitis (range = 2-24 pg/ml) and 1 sample from eyes with diabetic retinopathy (4 pg/ml), but was not detected in the eyes with phacolytic glaucoma or Behçets disease. IL-2 (less than 0.1 U/ml) was detected in one sample from an eye with uveitis, one specimen from an eye with uveitis complicated with retinal detachment and 2 samples from eyes with diabetic retinopathy. None of the cytokines measured were detected in any of the control vitreous. The present observations suggest that cytokines, particularly IL-6 and IL-1, may act as local amplification signals in pathological processes associated with chronic eye inflammation.

Cytokine mRNA expression in the mucosa of treated coeliac patients after wheat peptide challenge.

This study investigated the presence of mRNA coding for interferon gamma (IFN gamma), tumour necrosis factor alpha (TNF alpha), and interleukins 2 (IL2) and 6 (IL6), in the mucosa of four coeliac patients in remission who had been challenged with either gliadin or synthetic gliadin oligopeptides. Jejunal biopsy specimens from these patients, taken before and at two, four, and six hours after challenge, were hybridised with specific 35S-labelled DNA oligonucleotide probes. The lamina propria of all the patients contained significantly increased numbers of cytokine mRNA expressing cells four hours after challenge with gliadin or an oligopeptide corresponding to amino acids 31-49 of A-gliadin (peptide A). No significant changes were seen with the peptides corresponding to aminoacids 202-220 (peptide B) or 3-21 (peptide C) of A-gliadin, with the exception of one patient who showed a significant increase in the number of TNF alpha mRNA expressing cells four hours after challenge with peptide B. In vivo studies in coeliac disease have shown that significant histological changes occur in the mucosa of treated coeliac patients four hours after challenge with either gliadin or peptide A. These findings suggest that the histological changes seen previously in the mucosa of coeliac patients after wheat peptide challenge may be caused by increased expression of cytokines within the mucosa.

Distribution of cytokine proteins within epiretinal membranes in proliferative vitreoretinopathy.

This study reports on the immunohistochemical staining for cytokine proteins of 26 epiretinal membranes obtained from eyes undergoing surgery for the treatment of proliferative vitreoretinopathy. All specimens were investigated for the distribution of staining for interleukin-1 alpha (IL-1 alpha), interleukin-1 beta (IL-1 beta), interleukin-6 (IL-6), tumor necrosis factor-alpha (TNF alpha), interferon-gamma (IFN gamma) and interleukin-2 (IL-2). The results showed that 22 of the membranes (85%) stained for TNF alpha not only intracellularly but also in the extracellular matrix. This contrasts with the findings that only 2 membranes stained for IL-1 alpha and that another 3 were positive for IL-1 beta. Staining for the cytokines IL-6 and IFN gamma was also observed in 9 and 7 membranes respectively. None of the specimens investigated stained with antibodies to IL-2 or control antibodies, and none of three normal retinas stained with any of the antibodies used. Pre-absorption of anti-cytokine antibodies with the corresponding human recombinant cytokines abolished staining of cells and extracellular matrix. The present findings support growing evidence that cytokine-mediated pathways of inflammation are involved in the pathogenesis of proliferative vitreoretinopathy, and draw attention to the possibility that interaction between extracellular matrix-bound cytokine and inflammatory leucocytes or resident cells of the retina may promote the development and perpetuation of this condition.

Silicone oil concentrates fibrogenic growth factors in the retro-oil fluid

Aim: To determine whether silicone oil concentrates protein and growth factors in the retro-oil fluid. Methods: A laboratory analysis of intraocular fluid and vitreous specimens obtained from patients undergoing removal of silicone oil, revision vitrectomy, or primary vitrectomy for macular hole, proliferative vitreoretinopathy (PVR), or retinal detachment. Patients were prospectively recruited from routine vitreoretinal operating lists. Vitreous cavity fluid and vitreous samples were analysed for the presence of transforming growth factor beta (TGF-β2), basic fibroblast growth factor (bFGF), interleukin 6 (IL-6), and total protein using either commercially available enzyme linked immunosorbent assays (ELISA) or protein assay kits. Results: The median levels of bFGF, IL-6, and protein in the retro-oil fluid were raised (p<0.05) compared to all the other vitreous and vitreous cavity fluid samples. bFGF, IL-6, and protein levels were raised in PVR vitreous compared to non-PVR vitreous. TGF-β2 levels were not significantly raised in retro-oil fluid or in PVR vitreous. Conclusions: The concentration of fibrogenic (bFGF) and inflammatory (IL-6) growth factors and protein is raised in retro-silicone oil fluid. This may contribute to the process of retro-oil perisilicone proliferation and subsequent fibrocellular membrane formation.

Detection of interferon gamma mRNA in the mucosa of patients with coeliac disease by in situ hybridisation.

In situ hybridisation has been used to study interferon gamma (IFN gamma) mRNA expression in the small intestine of patients with coeliac disease. Sections of jejunal biopsies were obtained from five patients with treated and five with untreated coeliac disease and five disease controls. These sections were hybridised with radiolabelled specific DNA oligonucleotide probes. The lamina propria of untreated coeliac disease patients contained a significantly increased number of IFN gamma producing cells compared with controls but there was no significant difference between the coeliac patients treated with a gluten free diet and controls. The results suggest that IFN gamma may play a part in the immunopathogenesis of coeliac disease.

Activation of nuclear factor kappa B in Crohn's disease

Abstract.Objectives and Design: The location and degree of activation of nuclear factor kappa (NFκB), a primary transcription factor that plays a regulating role in immune and inflammatory responses, was determined in Crohns disease using full thickness specimens of bowel collected at surgery.¶Materials and Methods: Resected specimens of inflamed and non-inflamed bowel were collected from thirteen patients with Crohns disease and non-inflamed bowel from eleven control subjects. Prepared frozen sections were immunostained using a monoclonal antibody to the activated form of the p65 subunit of NFκB and the number of positive staining cells counted using a Lennox graticule.¶Results: The number of cells positive for activated NFκB was significantly increased (pu2009=u20090.001) in all layers of inflamed Crohns disease bowel, compared to non-inflamed bowel from controls. There was also a significant increase (pu2009=u20090.009) in the number of positive cells, when compared to non-inflamed bowel from control subjects, in the submucosa of non-inflamed areas of Crohns disease bowel. Cells positive for activated NFκB were provisionally identified by morphological criteria as mostly macrophages with some lymphocytes. There was no activation in endothelia.¶Conclusion: NFκB is activated within large mononuclear cells in all layers of inflamed areas of the bowel in Crohns disease and may represent key events in the inflammatory process. Increased activation in the submucosa of non-inflamed Crohns disease bowel provides further evidence of early immunological activation in macroscopically and microscopically uninvolved areas and an underlying abnormal immune system in Crohns disease.

Platelet expression of tumour necrosis factor-alpha (TNF-α), TNF receptors and intercellular adhesion molecule-1 (ICAM-1) in patients with proliferative diabetic retinopathy

Microvascular complications of insulin‐dependent diabetes mellitus (IDDM) have been strongly associated with platelet abnormalities, whilst TNF‐α has been implicated in the pathogenesis of this condition. However, at present it is not clear whether human circulating platelets express TNF‐α or TNF receptors (TNF‐R) or whether impaired expression of these molecules and of the TNF‐reactive adhesion molecule ICAM‐1 may be associated with platelet abnormalities in patients with IDDM. On this basis we investigated the platelet expression of these molecules in patients with IDDM complicated or uncomplicated by proliferative diabetic retinopathy (PDR) and in healthy subjects. We observed that the proportion of platelets staining for TNF‐α was significantly higher in IDDM patients with active PDR than in patients without microvascular complications (Pu2003=u20030.0078), quiescent PDR (Pu2003=u20030.003) or healthy subjects (Pu2003=u20030.0013). Patients with active PDR also showed a higher proportion of platelets expressing TNF‐RI (Pu2003=u20030.0052) and TNF‐RII (Pu2003=u20030.015) than healthy controls or patients with quiescent PDR (Pu2003=u20030.009 and 0.0006, respectively). In addition, the percentage of ICAM‐1+ platelets was significantly higher in patients with active PDR than in patients with quiescent PDR (Pu2003=u20030.0065) or normal subjects (Pu2003=u20030.013). There was a direct correlation between platelet expression of TNF‐α and that of TNF‐R in PDR patients, indicating that platelet staining for TNF‐α may be due to binding of this cytokine to its receptors. The results suggest that increased platelet expression of TNF‐α, TNF‐R and ICAM‐1 in IDDM patients may constitute important markers of thrombocyte abnormalities during the development of microvascular complications of diabetes mellitus.