Pituitary tumours without distinct lineage differentiation express stem cell marker SOX2.

CONTEXT: The recent WHO 2022 Classification of pituitary tumours identified a novel group of 'plurihormonal tumours without distinct lineage differentiation (WDLD)'. By definition, these express multiple combinations of lineage commitment transcription factors, in a monomorphous population of cells. OBJECTIVES: To determine the expression of stem cell markers (SOX2, Nestin, CD133) within tumours WDLD, immature PIT-1 lineage and acidophil stem cell tumours, compared with committed cell lineage tumours. METHODS: Retrospective evaluation of surgically resected pituitary tumours from St Vincent's Hospital, Sydney. Patients were selected to cover a range of tumour types, based on transcription factor and hormone immunohistochemistry. Clinical data was collected from patient files. Radiology reports were reviewed for size and invasion. Samples were analysed by immunohistochemistry and RT-qPCR for SF-1, PIT-1, T-PIT, SOX2, Nestin and CD133. Stem cell markers were compared between tumours WDLD and those with classically "mature" types. RESULTS: On immunohistochemistry, SOX2 was positive in a higher proportion of tumours WDLD compared with those meeting WHO lineage criteria, 7/10 v 10/42 (70 v 23.4%, p = 0.005). CD133 was positive in 2/10 tumours WDLD but 0/41 meeting lineage criteria, P = 0.003. On RT-qPCR, there was no significant difference in relative expression of stem cell markers (SOX2, CD133, Nestin) between tumours with and WDLD. CONCLUSIONS: Our study is the first to biologically characterise pituitary tumours WDLD. We demonstrate that these tumours exhibit a higher expression of the stem cell marker SOX2 compared with other lineage-differentiated tumours, suggesting possible involvement of stem cells in their development.

Ophthalmic pterygium: a stem cell disorder with premalignant features.

Pterygia are common ocular surface lesions thought to originate from limbal stem cells altered by chronic UV exposure. Traditionally regarded as a degenerative condition, pterygia also display tumor-like features, such as a propensity to invade normal tissue and high recurrence rates following resection, and may coexist with secondary premalignant lesions. This study was initiated to determine the rate of concurrent ocular surface diseases in patients with pterygia recruited from the practice of a single surgeon operating in a Sydney metropolitan hospital. One hundred pterygium specimens were histopathologically reviewed and selected cases were immunohistochemically assessed to confirm diagnosis. Along with previously documented typical features including epithelial proliferation, goblet cell hyperplasia, angiogenesis, inflammation, elastosis, stromal plaques, and Bowman's membrane dissolution, we identified five cases of ocular surface squamous neoplasia, six cases of primary acquired melanosis, two compound nevi (one suspect invasive melanoma), and one dermoid-like lesion. In 18 specimens, clusters of basal epithelial cells that coexpressed cytokeratin-15/-19 and p63-α were identified at the head of the pterygium, coinciding with clinical observation of Fuchs' flecks. Our data show that significant preneoplastic lesions may be associated with pterygium and that all excised pterygia should undergo histological examination. The presence of p63-α-positive epithelial cell clusters supports the hypothesis that pterygia develop from limbal epithelial progenitors.

Stem cell activity in the developing human cornea.

The adult cornea harbors stem cells (SCs) in its periphery, in a niche known as the limbus. Over the past 2 decades there has been substantial research into these adult corneal SCs, their limbal niche, and their therapeutic applications. However, few studies have investigated how this niche and its SCs develop in humans. To better characterize this development, human fetal corneas from 8.5- to 22-weeks'-gestation (n = 173), neonatal (n = 2), and adult (n = 10) specimens were obtained. Histological and immunohistochemical assessments were conducted to determine embryological changes and expression of developmental and SC-related genes. Fresh fetal corneas were explanted to propagate corneal progenitors and cells characterized using reverse transcription-polymerase chain reaction, immunohistochemistry, flow cytometry, and colony-forming assays. A novel "ridge-like" structure was identified, circumscribing the fetal cornea, which we hypothesize represents the rudimentary SC niche. Immunohistochemistry disclosed "stem-like" cells across the cornea, becoming confined to this ridge with increasing gestational age. In addition, for the first time, pure long-term cultures of fetal corneal epithelium, which displayed phenotypical and functional properties similar to those of adult limbal SCs, were established. Optimization of culture techniques and purification of this SC population will allow for further investigation of their proliferative ability, with potential research and clinical applications. This study expands our understanding of limbal niche development and opens new avenues for investigation.

The pathogenesis of pterygium: current concepts and their therapeutic implications.

Pterygium is a disease of the ocular surface that is associated with chronic UV exposure and is characterized by proliferation, inflammatory infiltrates, fibrosis, angiogenesis and extracellular matrix breakdown. Although pterygium is not fully understood, significant progress has been made toward understanding the mechanisms involved in its pathogenesis. In this review, we provide an update on the signaling pathways activated by UV light that result in induction of mediators responsible for the growth of pterygium. We also review the recent genetic studies on hereditary factors and provide a brief overview of the role of epithelial mesenchymal transition, bone marrow progenitor cells, and neuronal signals that may also contribute to the pathogenesis of pterygium. Therapeutic options for pterygium are discussed based on the mechanisms that perpetuate its growth.

The role of substance P in the pathogenesis of pterygia.

PURPOSE: Pterygium is a prevalent ocular surface disorder thought to be triggered by chronic ultraviolet damage to the limbus. One of the enigmatic features of pterygium is its wing-like shape, and the mechanism(s) supporting its centripetal growth remain to be elucidated. Because the growth pattern of pterygia mirrors the radial arrangement of corneal nerves, the authors propose that neuropeptides may facilitate its directional growth. This hypothesis prompted an investigation of the role of the sensory neuropeptide substance P (SP) and its receptor (NK(1) receptor) in directing cell migration in pterygia that may explain the characteristic growth pattern. METHODS: Immunohistochemical analysis for SP and the NK(1) receptor was performed on five pterygium specimens with corresponding autologous conjunctiva and limbus. Migration of pterygium epithelium, fibroblasts, and vascular endothelial cells toward SP was assessed by using a modified Boyden chamber. RESULTS: SP and NK(1) receptors were localized to infiltrating fibroblasts, mononuclear cells and the epithelia of pterygium, conjunctiva, and limbus, with elevated NK(1) receptor staining observed in pterygia. SP at nanomolar concentrations induced cell migration in pterygium fibroblasts and vascular endothelium in a dose-dependent fashion, which was inhibited by an NK(1) receptor antagonist. Pterygium epithelial cells were not migratory in these experiments. CONCLUSIONS: For the first time, this study showed the presence of NK(1) receptor in pterygia and that SP is a potent chemoattractant for pterygium fibroblasts and vascular endothelial cells, implying that SP may contribute to the shape of pterygia through its profibrogenic and angiogenic action.

Cultured human ocular surface epithelium on therapeutic contact lenses.

BACKGROUND: This study was initiated after observation of some intriguing epithelial growth properties of contact lenses used as a bandage for patients after pterygium surgery. AIM: To determine the efficacy of culturing human ocular surface epithelial cells on therapeutic contact lenses in autologous serum with a view of using this system to transfer epithelial cells to patients with persistent corneal or limbal defects. METHODS: Excess graft tissue resected from patients undergoing pterygium surgery (n = 3) consisting of limbal epithelium was placed on siloxane-hydrogel contact lenses (lotrafilcon A and balafilcon A). Limbal explants were cultured in media with 10% autologous serum. Morphology, proliferative capacity and cytokeratin profile were determined by phase contrast, light and electron microscopy, and immunohistochemical analysis. RESULTS: Lotrafilcon A contact lenses sustained proliferation and migration from limbal tissue. Cells became confluent after 10-14 days and consisted of 2-3 layers with a corneal phenotype (CK3(+)/CK12(+)/CK19(-)) and a propensity to proliferate (p63(+)). Electron microscopy showed microvilli on the apical surface with adhesive projections, indicating that these cells were stable and likely to survive for a long term. Growth was not observed from limbal explants cultured on balafilcon A contact lenses. CONCLUSION: A method for culturing human ocular surface epithelium on contact lenses that may facilitate expansion and transfer of autologous limbal epithelial cells while avoiding the risks associated with transplanting allogeneic tissue has been developed. This technique may be potentially useful for the treatment of patients with limbal stem cell deficiency.

Pathogenesis of pterygia: role of cytokines, growth factors, and matrix metalloproteinases.

Pterygium is a common ocular surface disease apparently only observed in humans. Chronic UV exposure is a widely accepted aetiological factor in the pathogenesis of this disease and this concept is supported by epidemiological data, ray tracing models and histopathological changes that share common features with UV damaged skin. The mechanism(s) of pterygium formation is incompletely understood. Recent data have provided evidence implicating a genetic component, anti-apoptotic mechanisms, cytokines, growth factors, extracellular matrix remodelling (through the actions of matrix metalloproteinases), immunological mechanisms and viral infections in the pathogenesis of this disease. In this review, the current knowledge on pterygium pathogenesis is summarised, highlighting recent developments. In addition, we provide novel data further demonstrating the complexity of this intriguing disease.

Significance of Fuchs Flecks in Patients With Pterygium/Pinguecula: Earliest Indicator of Ultraviolet Light Damage.

PURPOSE: Fuchs flecks (FFs) have been previously identified at the leading edge of pterygia and may represent collections of epithelial stem-like cells that give rise to this condition. This study aims to evaluate the clinical significance of FFs in patients with ocular surface disorders, such as pterygium and pinguecula, by in vivo confocal microscopy (IVCM). METHODS: This study is a Single-center, retrospective, observational case series of 40 eyes from 20 patients with clinical diagnoses of pinguecula or pterygium, or both. IVCM (Rostock Cornea Module; Heidelberg Engineering, Heidelberg, Germany) was performed on patients with pinguecula or pterygium, or both. The presence of FFs on the ocular surface of patients with pterygium and pinguecula was assessed by IVCM and subsequently documented. RESULTS: FFs were present in 24 of 30 eyes (80.0%) in paired macroscopically normal nasal or limbal regions, 19 of 20 (95.0%) in pinguecula, 13 of 15 (86.7%) in primary pterygia, and 7 of 7 (100%) in recurrent pterygia. CONCLUSIONS: High rates of FFs were identified at the head of pinguecula, primary pterygium, recurrent pterygium, and macroscopically normal nasal and temporal limbus. We postulate that FFs may represent precursor lesions to UV-associated ocular surface pathology. Identification of Fuchs fleck by IVCM may permit clinicians to predict the patients who may progress to develop more advanced pathology.

Expression of classical components of the renin-angiotensin system in the human eye.

PURPOSE: The purpose of this study was to determine the relative expression of clinically-relevant components of the renin-angiotensin system (RAS) in the adult human eye. METHODS: We obtained 14 post-mortem enucleated human eyes from patients whom had no history of inflammatory ocular disease nor pre-mortem ocular infection. We determined the gene expression for prorenin, renin, prorenin receptor, angiotensin-converting enzyme, angiotensinogen and angiotensin II Type 1 receptor, on tissue sections and in cultured human primary retinal pigment epithelial and iris pigment epithelial (RPE/IPE) cell lines, using both qualitative and quantitative reverse transcription polymerase chain reaction (RT-PCR). Protein expression was studied using indirect immunofluorescence (IF). RESULTS: Almost all components of the classical RAS were found at high levels, at both the transcript and protein level, in the eyes' uvea and retina; and at lower levels in the cornea, conjunctiva and sclera. There was a much lower level of expression in the reference cultured RPE/IPE cells lines. CONCLUSION: This study describes the distribution of RAS in the normal adult human eye and demonstrates the existence of an independent ocular RAS, with uveal and retinal tissues showing the highest expression of RAS components. These preliminary findings provide scope for examination of additional components of this system in the human eye, as well as possible differential expression under pathological conditions.

Role of toll-like receptors in human iris pigment epithelial cells and their response to pathogen-associated molecular patterns.

BACKGROUND: Toll-like receptor (TLR) activation is hypothesized to contribute to inflammatory eye disease including uveitis, yet the distribution pattern of TLRs in human uveal tissues remains poorly described. The purpose of this study was to investigate the expression profile of TLRs in human iris pigment epithelial cells (IPE) at the gene and protein level and examine the effect of pathogen-associated molecular patterns (PAMPs), such as Pam3CSK4.3HCl, Poly(I:C), lipopolysaccharides (LPS from E. coli serotype O111:B4), Flagellin, MALP-2 (macrophage activating lipopeptide-2), Poly(U) and CpGODN2395 on the production of inflammatory mediators including interleukin-8 (IL-8) and monocyte chemotactic protein 1 (MCP-1) from human IPE and retinal pigment epithelial cells (RPE). METHODS: RT-PCR and Western blotting was employed to investigate the expression of TLRs 1-10 in primary IPE and RPE. Secretion of IL-8 or MCP-1 following treatment with PAMPs was measured by ELISA. The role of TLR2, TLR3 and TLR4 in mediating an inflammatory response was investigated using pharmacological TLR inhibitors. RESULTS: IPE and RPE expressed transcripts for TLR1-6 and 8-10; and proteins for TLR1-6 and 9. IPE secreted IL-8 or MCP-1 in response to Pam3CSK4.3HCl, Poly(I:C), LPS and MALP-2, whereas RPE produced IL-8 only after Poly(I:C), LPS or MALP-2 treatment. TLR inhibitors (OxPAPC, CI-095 and chloroquine) blocked IL-8 secretion in Poly(I:C), LPS or MALP-2-treated IPE and RPE. CONCLUSIONS: Ocular pigment epithelial cells respond to PAMPs through activation of TLRs, particularly TLR2, TLR3 and TLR4. Expression of TLRs in human IPE cells provides a basis for responses to many ocular pathogens and their activation may be involved in the pathogenesis of ocular inflammation.

Measurement of neopterin, TGF-ß1 and ACE in the exhaled breath condensate of patients with sarcoidosis.

Exhaled breath condensate (EBC) is a non-invasive method of sampling airway lining fluids in respiratory diseases. This may be useful in identifying exhaled biomarkers of granulomatous inflammation and pulmonary fibrosis in patients with sarcoidosis. The aim of this pilot study was to identify markers of granulomatous airway inflammation and disease activity including neopterin, transforming growth factor-ß1 (TGF-ß1) and angiotensin converting enzyme (ACE) in EBC. EBC was collected from 16 patients with sarcoidosis and 22 healthy control subjects. EBC neopterin, and active-TGF-ß1 were measured by ELISA. EBC-ACE activity was measured using a colorimetric assay. EBC neopterin was detectable in 3/20 controls and 7/16 patients with sarcoidosis. Patients with sarcoidosis had greater mean neopterin levels compared to control subjects (0.57 ± 0.45 nmol l(-1) versus 0.41 ± 0.22 nmol l(-1), p = 0.04). TGF-ß1 was detectable in the EBC of all subjects and concentrations were higher in patients with sarcoidosis compared with controls (115.5 ± 79.6 pg mol(-1) versus 82.3 ± 16.2 pg mol(-1), p = 0.048). There was no difference in EBC ACE activity, which was only detectable in 3/20 healthy controls and 2/16 patients (p = 0.91). EBC markers of granulomatous inflammation are detectable at greater levels in patients with sarcoidosis compared to healthy controls subjects. Larger studies and development of sensitive assays are warranted to examine the disease correlates and predictive utility of these markers.

Peripheral blood responses to specific antigens and CD28 in sarcoidosis.

BACKGROUND: Potential antigens inducing sarcoid inflammation include mycobacterial and auto-antigens. Paradoxically, peripheral anergy to common recall antigens also occurs, possibly due to impaired dendritic cell or regulatory T-cell responses, or impaired T-cell co-stimulation. The purpose of this study was to compare peripheral blood responses of patients with sarcoidosis to candidate antigens, and examine CD28 T-cell co-stimulation. METHODS: Peripheral blood mononuclear cell (PBMC) responses were examined from patients with sarcoidosis (n=16) and healthy control subjects (n=22) following PBMC stimulation with: anti-CD3/CD28 coated beads; Mycobacterium tuberculosis ESAT-6 and KatG peptides; vimentin and lysyl tRNA peptides; and common recall antigens, including cytomegalovirus (CMV) cell lysate as well as CMV, Epstein-Barr virus, influenza virus (CEF) peptides. RESULTS: ESAT-6/KatG peptide stimulation induced greater numbers of IFN-γ producing T-cells, and elevated IL-2, IL-6 and TNF-α production in sarcoidosis compared to purified protein derivative (PPD)-negative healthy control subjects. PBMCs from patients with sarcoidosis showed reduced IFN-γ producing T-cells following stimulation with CMV lysate, CEF peptides and CD3/CD28 beads; and reduced IL-4 and TNF-α production following CD3/CD28 activation. CONCLUSIONS: Patients with sarcoidosis exhibit greater PBMC responses to M. tuberculosis antigens compared to PPD-negative controls, but reduced T-cell responses to common recall antigens. One contributing mechanism may be impairment of T-cell CD28 co-stimulation.

Bluebottle envenomation-induced crystalline keratopathy.

PURPOSE: To report a patient who developed a crystalline keratopathy after bluebottle envenomation of the cornea. METHOD: Case report with histopathological correlation and literature review. RESULTS: A 61-year-old man presented to the Ophthalmology clinic after he was stung in the left eye by a bluebottle while swimming in the sea. He complained of ocular and facial pain, facial swelling, and transient blurred vision. First aid by the beach included a hot shower and methoxyflurane for the pain. Crystalline deposits and pseudodendritiform epithelial defects were noted on slit-lamp examination. Topical chloramphenicol was prescribed, and 2 days after the injury, the cornea was debrided of persisting crystalline material. The cornea healed quickly after debridement with visual acuity improving from 6/9 to 6/6 in the affected eye. Microscopic examination demonstrated the corneal crystals to be irregularly shaped and nonrefractile with squared off edges. Raman spectroscopy partially identified the crystals as calcium based. CONCLUSIONS: Although bluebottle stings of the cornea are infrequent, they may be challenging to manage. In addition to inactivation of the nematocysts and pain management, early debridement of the foreign matter may aid in the rapid resolution of the symptoms.

Iris pigment epithelial cells express a functional lipopolysaccharide receptor complex.

PURPOSE: Ocular pigment epithelial cells are hypothesized to play a role in the pathogenesis of acute anterior uveitis (AAU), where LPS activation of Toll-like receptors (TLRs) may serve as a trigger. In this study, the expression of LPS receptors in iris pigment epithelium (IPE) was determined. METHODS: RT-PCR, flow cytometry, Western blot, and immunohistochemistry were used to investigate the expression of the LPS receptor complex (TLR4, MD-2, and CD14) in primary human IPE. Cytokine secretion by LPS-treated IPE was measured by multiplex bead array and ELISA. The role of CD14 in modulating the LPS response was investigated by addition of soluble CD14 and by antibody neutralization studies. In vivo expression of CD14 was examined by immunohistochemistry and Western blot analysis. RESULTS: IPE expressed TLR4, MD-2, and CD14 in vitro and secreted a panel of proinflammatory cytokines (IL-6, CXCL8, CXCL10, CCL2, CCL4, and CCL5) when stimulated with LPS. CXCL8 secretion by LPS-treated IPE was dependent on CD14 and TLR4. CD14 was detected in CD68+ cells in the iris by immunohistochemistry and in normal aqueous by Western blot analysis. CONCLUSIONS: IPE cells express a functional LPS receptor complex and are capable of promoting ocular inflammation through secretion of an array of proinflammatory mediators. CD14 was identified as a key molecule that modulated the LPS response in IPE.

Localization of the low-affinity nerve growth factor receptor p75 in human limbal epithelial cells.

Biological effects of nerve growth factor (NGF) are mediated through receptors known as nerve growth factor receptors (NGFR), which include p75 and TrkA. This study was initiated after identifying NGFR as an up-regulated gene in the limbus by cDNA microarray analysis and we postulate that its expression may be indicative of a stem/progenitor cell phenotype. Immunohistochemistry was performed on normal human adult (n=5) and foetal (n=3) corneal tissue using antibodies directed against p75, TrkA, NGF, p63, ABCG2 and CK3/12. Limbal, conjunctival and pterygium tissue was obtained from patients (n=10) undergoing pterygium resection and used for immunohistochemical assessment. Paraffin-embedded archival human skin specimens (n=4) were also evaluated. In vitro expression of NGFR was determined in limbal, conjunctival and pterygium-derived epithelial cells. p75 was selectively expressed by basal epithelial cells in pterygia, conjunctiva and limbus, but was absent in the central cornea. These results were confirmed with two additional p75 specific antibodies. In contrast, TrkA was found in full-thickness pterygium, conjunctival, limbal and corneal epithelium in both adult and foetal eyes. p75 expression was identified in a small percentage, while TrkA was found on the entire population of cultured conjunctival, limbal and pterygium-derived epithelial cells. This receptor was also observed in selective regions of the human epidermis and hair follicle bulge. Our results illustrate the selective expression of p75 in basal pterygium, conjunctival and limbal epithelium, while staining was absent in adult and foetal central cornea. p75 may represent an additional ocular surface epithelial stem/progenitor cell signature gene.