Characterization of Different Types of Epiretinal Proliferations by Synchrotron Radiation-Based Fourier Transform Infrared Micro-Spectroscopy.

Pathological tissue on the surface of the retina that can be of different etiology and pathogenesis can cause changes in the retina that have a direct consequence on vision. Tissues of different etiology and pathogenesis have different morphological structures and also different macromolecule compositions usually characteristic of specific diseases. In this study, we evaluated and compared biochemical differences among samples of three different types of epiretinal proliferations: idiopathic epiretinal membrane (ERMi), membranes in proliferative vitreoretinopathy (PVRm), and proliferative diabetic retinopathy (PDRm). The membranes were analyzed by using synchrotron radiation-based Fourier transform infrared micro-spectroscopy (SR-FTIR). We used the SR-FTIR micro-spectroscopy setup, where measurements were set to achieve a high resolution that was capable of showing clear biochemical spectra in biological tissue. We were able to identify differences between PVRm, PDRm, and ERMi in protein and lipid structure; collagen content and collagen maturity; differences in proteoglycan presence; protein phosphorylation; and DNA expression. Collagen showed the strongest expression in PDRm, lower expression in ERMi, and very low expression in PVRm. We also demonstrated the presence of silicone oil (SO) or polydimethylsiloxane in the structure of PVRm after SO endotamponade. This finding suggests that SO, in addition to its many benefits as an important tool in vitreoretinal surgery, could be involved in PVRm formation.

Biomacromolecular Profile in Human Primary Retinal Pigment Epithelial Cells-A Study of Oxidative Stress and Autophagy by Synchrotron-Based FTIR Microspectroscopy.

Synchrotron radiation-based Fourier Transform Infrared (SR-FTIR) microspectroscopy is a non-destructive and chemically sensitive technique for the rapid detection of changes in the different components of the cell's biomacromolecular profile. Reactive oxygen species and oxidative stress may cause damage to the DNA, RNA, and proteins in the retinal pigment epithelium (RPE), which can further lead to age-related macular degeneration (AMD) and visual loss in the elderly. In this study, human primary RPEs (hRPEs) were used to study AMD pathogenesis by using an established in vitro cellular model of the disease. Autophagy-a mechanism of intracellular degradation, which is altered during AMD, was studied in the hRPEs by using the autophagy inducer rapamycin and treated with the autophagy inhibitor bafilomycin A1. In addition, oxidative stress was induced by the hydrogen peroxide (H2O2) treatment of hRPEs. By using SR-FTIR microspectroscopy and multivariate analyses, the changes in the phosphate groups of nucleic acids, Amide I and II of the proteins, the carbonyl groups, and the lipid status in the hRPEs showed a significantly different pattern under oxidative stress/autophagy induction and inhibition. This biomolecular fingerprint can be evaluated in future drug discovery studies affecting autophagy and oxidative stress in AMD.

UV Effect on Human Anterior Lens Capsule Macro-Molecular Composition Studied by Synchrotron-Based FTIR Micro-Spectroscopy.

Ultraviolet (UV) irradiation is an important risk factor in cataractogenesis. Lens epithelial cells (LECs), which are a highly metabolically active part of the lens, play an important role in UV-induced cataractogenesis. The purpose of this study was to characterize cell compounds such as nucleic acids, proteins, and lipids in human UV C-irradiated anterior lens capsules (LCs) with LECs, as well as to compare them with the control, non-irradiated LCs of patients without cataract, by using synchrotron radiation-based Fourier transform infrared (SR-FTIR) micro-spectroscopy. In order to understand the effect of the UV C on the LC bio-macromolecules in a context of cataractogenesis, we used the SR-FTIR micro-spectroscopy setup installed on the beamline MIRAS at the Spanish synchrotron light source ALBA, where measurements were set to achieve a single-cell resolution with high spectral stability and high photon flux. UV C irradiation of LCs resulted in a significant effect on protein conformation with protein formation of intramolecular parallel ß-sheet structure, lower phosphate and carboxyl bands in fatty acids and amino acids, and oxidative stress markers with significant increase of lipid peroxidation and diminishment of the asymmetric CH3 band.

Cataract Progression Associated with Modifications in Calcium Signaling in Human Lens Epithelia as Studied by Mechanical Stimulation.

Ca2+ homeostasis and signaling disturbances are associated with lens pathophysiology and are involved in cataract formation. Here, we explored the spatiotemporal changes in Ca2+ signaling in lens epithelial cells (LECs) upon local mechanical stimulation, to better understand the LECs' intercellular communication and its association with cataractogenesis. We were interested in if the progression of the cataract affects the Ca2+ signaling and if modifications of the Ca2+ homeostasis in LECs are associated with different cataract types. Experiments were done on the human postoperative anterior lens capsule (LC) preparations consisting of the monolayer of LECs on the basement membrane. Our findings revealed that the Ca2+ signal spreads radially from the stimulation point and that the amplitude of Ca2+ transients decreases with increasing distance. It is noteworthy that a comparison of signaling characteristics with respect to the degree of cataract progression revealed that, in LCs from more developed cataracts, the Ca2+ wave propagates faster and the amplitudes of Ca2+ signals are lower, while their durations are longer. No differences were identified when comparing LCs with regard to the cataract type. Moreover, experiments with Apyrase have revealed that the Ca2+ signals are not affected by ATP-dependent paracrine communication. Our results indicated that cataract progression is associated with modifications in Ca2+ signaling in LECs, suggesting the functional importance of altered Ca2+ signaling of LECs in cataractogenesis.

Structural Characteristics of the Lens in Presenile Cataract.

The purpose of this work is to examine the structure of the anterior lens epithelial cells (aLECs) of presenile idiopathic cortical cataract to investigate the possible structural reasons for its development. The anterior lens capsules (aLCs: basement membrane and associated lens epithelial cells) were obtained from routine uneventful cataract surgery of 5 presenile cataract patients (16 and 41 years old women and 29, 39, and 45 years old men). None of the patients had family history of cataract, medication, or trauma and they were otherwise healthy. In addition, the patients did not have any other abnormal features in the ocular status except cataract. The aLCs were prepared for scanning electron microscopy (SEM) and transmission electron microscopy (TEM). The most prominent abnormal features observed by SEM for all 5 studied presenile cataract patients were the changes of the aLECs structure with the dents, the selective concavity of some LECs, at their apical side centrally toward the nucleus. In addition, TEM showed the thinning of the lens epithelium with the segmentally concave cells and the compressed and elongated nuclei. Abnormal and distinguishable structural features were observed in the anterior lens epithelium aLECs in all 5 patients with presenile cataract. Disturbed structure of aLECs, regularly present in presenile cataract type is shown that might be associated with water accumulation in the presenile idiopathic cortical cataract lens.

Synchrotron-based FTIR microspectroscopy of protein aggregation and lipids peroxidation changes in human cataractous lens epithelial cells.

Cataract is the leading cause of blindness worldwide but the mechanisms involved in the process of cataractogenesis are not yet fully understood. Two most prevalent types of age-related cataracts are nuclear (N) and cortical (C) cataracts. A common environmental factor in most age-related cataracts is believed to be oxidative stress. The lens epithelium, the first physical and biological barrier in the lens, is build from lens epithelial cells (LECs). LECs are important for the maintenance of lens transparency as they control energy production, antioxidative mechanisms and biochemical transport for the whole lens. The purpose of this study is to characterize compounds in LECs originated from N and C cataracts, by using the synchrotron radiation-based Fourier Transform Infrared (SR-FTIR) microspectroscopy, in order to understand the functional importance of their different bio-macromolecules in cataractogenesis. We used the SR-FTIR microspectroscopy setup installed on the beamline MIRAS at the Spanish synchrotron light source ALBA, where measurements were set to achieve single cell resolution, with high spectral stability and high photon flux. The results showed that protein aggregation in form of fibrils was notably pronounced in LECs of N cataracts, while oxidative stress and the lipids peroxidation were more pronounced in LECs of C cataracts.

Anterior lens epithelium in cataract patients with retinitis pigmentosa - scanning and transmission electron microscopy study.

PURPOSE: In retinitis pigmentosa (RP) patients, relatively minor lens opacity in central part of posterior pole of the lens may cause disproportionate functional symptoms requiring cataract operation. To investigate the possible structural reasons for this opacity development, we studied the structure of the lens epithelium of patients with RP. METHODS: The anterior lens capsule (aLC: basement membrane and associated lens epithelial cells, LECs) was obtained from cataract surgery and prepared for scanning and transmission electron microscopy (SEM and TEM). RESULTS: Both SEM and TEM show a number of abnormal features in the anterior lens epithelium of cataract patients with RP. The abnormalities appear mainly as holes, thinning and degradation of the epithelium, with the dimensions from <1 µm to more than 50 µm. Other types of holes in size up to 20 µm were seen that may be formed by gradual stretching of the lens epithelium. Another type of abnormalities was cracks that were seen between adjacent LECs, with dimensions 0.1-2 µm × up to 10 µm. CONCLUSIONS: Abnormal structural features were observed in the anterior lens epithelium that may cause water influx into the lens. This may lead to clouding along the water clefts leading towards the posterior pole in the RP cataractous lens. We suggest that the lens epithelium has a role in the development of the cataract in patients with RP.

Microplasma Induced Cell Morphological Changes and Apoptosis of Ex Vivo Cultured Human Anterior Lens Epithelial Cells - Relevance to Capsular Opacification.

Inducing selective or targeted cell apoptosis without affecting large number of neighbouring cells remains a challenge. A plausible method for treatment of posterior capsular opacification (PCO) due to remaining lens epithelial cells (LECs) by reactive chemistry induced by localized single electrode microplasma discharge at top of a needle-like glass electrode with spot size ~3 µm is hereby presented. The focused and highly-localized atmospheric pressure microplasma jet with electrode discharge could induce a dose-dependent apoptosis in selected and targeted individual LECs, which could be confirmed by real-time monitoring of the morphological and structural changes at cellular level. Direct cell treatment with microplasma inside the medium appeared more effective in inducing apoptosis (caspase 8 positivity and DNA fragmentation) at a highly targeted cell level compared to treatment on top of the medium (indirect treatment). Our results show that single cell specific micropipette plasma can be used to selectively induce demise in LECs which remain in the capsular bag after cataract surgery and thus prevent their migration (CXCR4 positivity) to the posterior lens capsule and PCO formation.

Anterior lens epithelium in intumescent white cataracts - scanning and transmission electron microscopy study.

PURPOSE: Our purpose was to study the structure of the lens epithelial cells (LECs) of intumescent white cataracts (IC) in comparison with nuclear cataracts (NC) in order to investigate possible structural reasons for development of IC. METHODS: The anterior lens capsule (aLC: basement membrane and associated LECs) were obtained from cataract surgery and prepared for scanning electron microscopy (SEM) and transmission electron microscopy (TEM). RESULTS: We observed by SEM that in IC, LEC swelling was pronounced with the clefts surrounding the groups of LECs. Another structural feature was spherical formations, that were observed on the apical side of LEC's, towards the fibre cell layer, both by SEM and TEM. Development of these structures, bulging out from the apical cell membrane of the LEC's and disrupting it, could be followed in steps towards the sphere formation. The degeneration of the lens epithelium and the structures of the aLC in IC similar to Morgagnian globules were also observed. None of these structural changes were observed in NC. CONCLUSIONS: We show by SEM and TEM that, in IC, LECs have pronounced structural features not observed in NC. This supports the hypothesis that the disturbed structure of LECs plays a role in water accumulation in the IC lens. We also suggest that, in IC, LECs produce bulging spheres that represent unique structures of degenerated material, extruded from the LEC.

Anterior lens epithelial cells attachment to the basal lamina.

PURPOSE: To study the structure of the anterior lens epithelial cells (aLECs) and the contacts of the aLECs with the basal lamina (BL) in order to understand their role in the lens epithelium's function. METHODS: The aLCs (BL and associated aLECs) were obtained from routine uneventful cataract surgery, prepared for and studied by scanning electron microscopy (SEM), transmission electron microscopy (TEM) and confocal microscopy. RESULTS: SEM shows that the basal surface of the aLECs (~10-15 µm) is with aLECs foldings (~1-3 µm) and extensions (~0.5-3 µm) attached to the BL. Confocal microscopy images of the basal sections of the aLECs after membrane staining also suggest that the basal part of aLECs has foldings (~1-3 µm). TEM shows in the aLECs basal parts, towards BL, the structures that look like entanglement (~1-4 µm). In cases where there is a swelling of the cytoplasm and offset of the aLECs from the BL, individual extensions (~0.5-2 µm) that extend to the BL are visible by TEM. CONCLUSIONS: We provide detail evidence about the structural organization of the aLECs, in particular about their basal side which is in contact with the BL. This is supported by the complementary use of three techniques, SEM, TEM and confocal microscopy, each of them showing the same morphological features, the extensions and the entanglements of the aLECs cytoplasmic membrane at the border with the BL. The basal surface of the aLECs is increased. It suggests the functional importance of the contact between aLECs and BL.

The Analysis of Intracellular and Intercellular Calcium Signaling in Human Anterior Lens Capsule Epithelial Cells with Regard to Different Types and Stages of the Cataract.

In this work we investigated how modifications of the Ca2+ homeostasis in anterior lens epithelial cells (LECs) are associated with different types of cataract (cortical or nuclear) and how the progression of the cataract (mild or moderate) affects the Ca2+ signaling. We systematically analyzed different aspects of intra- and inter-cellular Ca2+ signaling in the human LECs, which are attached to surgically isolated lens capsule (LC), obtained during cataract surgery. We monitored the temporal and spatial changes in intracellular Ca2+ concentration after stimulation with acetylcholine by means of Fura-2 fluorescence captured with an inverted microscope. In our analysis we compared the features of Ca2+ signals in individual cells, synchronized activations, spatio-temporal grouping and the nature of intercellular communication between LECs. The latter was assessed by using the methodologies of the complex network theory. Our results point out that at the level of individual cells there are no significant differences when comparing the features of the signals with regard either to the type or the stage of the cataract. On the other hand, noticeable differences are observed at the multicellular level, despite inter-capsule variability. LCs associated with more developed cataracts were found to exhibit a slower collective response to stimulation, a less pronounced spatio-temporal clustering of LECs with similar signaling characteristics. The reconstructed intercellular networks were found to be sparser and more segregated than in LCs associated with mild cataracts. Moreover, we show that spontaneously active LECs often operate in localized groups with quite well aligned Ca2+ activity. The presence of spontaneous activity was also found to affect the stimulated Ca2+ responses of individual cells. Our findings indicate that the cataract progression entails the impairment of intercellular signaling thereby suggesting the functional importance of altered Ca2+ signaling of LECs in cataractogenesis.

Long-Term Cultures of Human Cornea Limbal Explants Form 3D Structures Ex Vivo - Implications for Tissue Engineering and Clinical Applications.

Long-term cultures of cornea limbal epithelial stem cells (LESCs) were developed and characterized for future tissue engineering and clinical applications. The limbal tissue explants were cultivated and expanded for more than 3 months in medium containing serum as the only growth supplement and without use of scaffolds. Viable 3D cell outgrowth from the explants was observed within 4 weeks of cultivation. The outgrowing cells were examined by immunofluorescent staining for putative markers of stemness (ABCG2, CK15, CK19 and Vimentin), proliferation (p63α, Ki-67), limbal basal epithelial cells (CK8/18) and differentiated cornea epithelial cells (CK3 and CK12). Morphological and immunostaining analyses revealed that long-term culturing can form stratified 3D tissue layers with a clear extracellular matrix deposition and organization (collagen I, IV and V). The LESCs showed robust expression of p63α, ABCG2, and their surface marker fingerprint (CD117/c-kit, CXCR4, CD146/MCAM, CD166/ALCAM) changed over time compared to short-term LESC cultures. Overall, we provide a model for generating stem cell-rich, long-standing 3D cultures from LESCs which can be used for further research purposes and clinical transplantation.

Morphological and proliferative studies on ex vivo cultured human anterior lens epithelial cells - relevance to capsular opacification.

PURPOSE: To determine the structural characteristics of lens epithelial cells (LECs) found on the anterior portion of the lens capsule and their pluripotency, proliferating and migrating potential when grown ex vivo with relevance to posterior capsular opacification (PCO) after cataract surgery. METHODS: The explants of anterior portion of the lens capsule consisting of monolayer of LECs were obtained from uneventful cataract surgery and were cultivated under adherent conditions. The size and shape of the outgrowing cells were recorded by scanning electron microscopy (SEM), while their migration and proliferation potential were followed using light microscopy. Positivity for proliferation (Ki-67)- and pluripotency (Sox2)-specific markers were tested by immunofluorescent staining. RESULTS: The proliferation and migration of anterior portion of the lens capsule's LECs filling up the denuded and reverse side regions of the lens capsule as well as their growth on glass culture surfaces could be followed by light microscopy and SEM, while the distribution of LECs and their morphology could be analysed in detail by SEM. The expression of Ki-67 and Sox2 in LECs growing adherently on human anterior portion of the lens capsule could also be detected. CONCLUSIONS: Classic light microscopy and SEM can be used to show that human anterior portion of the lens capsule harbours LECs that can proliferate and migrate, suggesting their pluripotency or putative stem cell nature. Similarly, morphological techniques can be used to study PCO and the effect different drugs or physical treatments have against PCO development.

Orexin-dependent activation of layer VIb enhances cortical network activity and integration of non-specific thalamocortical inputs.

Neocortical layer VI is critically involved in thalamocortical activity changes during the sleep/wake cycle. It receives dense projections from thalamic nuclei sensitive to the wake-promoting neuropeptides orexins, and its deepest part, layer VIb, is the only cortical lamina reactive to orexins. This convergence of wake-promoting inputs prompted us to investigate how layer VIb can modulate cortical arousal, using patch-clamp recordings and optogenetics in rat brain slices. We found that the majority of layer VIb neurons were excited by nicotinic agonists and orexin through the activation of nicotinic receptors containing α4-α5-ß2 subunits and OX2 receptor, respectively. Specific effects of orexin on layer VIb neurons were potentiated by low nicotine concentrations and we used this paradigm to explore their intracortical projections. Co-application of nicotine and orexin increased the frequency of excitatory post-synaptic currents in the ipsilateral cortex, with maximal effect in infragranular layers and minimal effect in layer IV, as well as in the contralateral cortex. The ability of layer VIb to relay thalamocortical inputs was tested using photostimulation of channelrhodopsin-expressing fibers from the orexin-sensitive rhomboid nucleus in the parietal cortex. Photostimulation induced robust excitatory currents in layer VIa neurons that were not pre-synaptically modulated by orexin, but exhibited a delayed, orexin-dependent, component. Activation of layer VIb by orexin enhanced the reliability and spike-timing precision of layer VIa responses to rhomboid inputs. These results indicate that layer VIb acts as an orexin-gated excitatory feedforward loop that potentiates thalamocortical arousal.

Characterization of ex vivo cultured neuronal- and glial- like cells from human idiopathic epiretinal membranes.

BACKGROUND: Characterization of the neuro-glial profile of cells growing out of human idiopathic epiretinal membranes (iERMs) and testing their proliferative and pluripotent properties ex vivo is needed to better understand the pathogenesis of their formation. METHODS: iERMs obtained during uneventful vitrectomies were cultivated ex vivo under adherent conditions and assessed by standard morphological and immunocytochemical methods. The intracellular calcium dynamics of the outgrowing cells was assessed by fluorescent dye Fura-2 in response to acetylcholine (ACh)- or mechano- stimulation. RESULTS: The cells from the iERMs formed sphere-like structures when cultured ex vivo. The diameter of the spheres increased by 5% at day 6 and kept an increasing tendency over a month time. The outgrowing cells from the iERM spheres had mainly glial- and some neuronal- like morphology. ACh- or mechano- stimulation of these cells induced intracellular calcium propagation in both cell types; in the neuronal-like cells resembling action potential from the soma to the dendrites. Immunocytochemistry confirmed presence of glial- and neuronal cell phenotype (GFAP and Nestin-1 positivity, respectively) in the iERMs, as well as presence of pluripotency marker (Sox2). CONCLUSION: iERMs contain cells of neuronal- and glial- like origin which have proliferative and pluripotent potential, show functionality reflected through calcium dynamics upon ACh and mechano- stimulation, and a corresponding molecular phenotype.

A simple method for establishing adherent ex vivo explant cultures from human eye pathologies for use in subsequent calcium imaging and inflammatory studies.

A novel, simple, and reproducible method for cultivating pathological tissues obtained from human eyes during surgery was developed using viscoelastic material as a tissue adherent to facilitate cell attachment and expansion and calcium imaging of cultured cells challenged by mechanical and acetylcholine (ACh) stimulation as well as inflammatory studies. Anterior lens capsule-lens epithelial cells (aLC-LECs) from cataract surgery and proliferative diabetic retinopathy (PDR) fibrovascular epiretinal membranes (fvERMs) from human eyes were used in the study. We hereby show calcium signaling in aLC-LECs by mechanical and acetylcholine (ACh) stimulation and indicate presence of ACh receptors in these cells. Furthermore, an ex vivo study model was established for measuring the inflammatory response in fvERMs and aLC-LECs upon TNFα treatment.

The effect of gentian violet on human anterior lens epithelial cells.

PURPOSE: To evaluate whether the gentian violet staining of the anterior lens capsule during the cataract surgery is cytotoxic for the human lens epithelial cells, as an indirect indication of possible toxicity towards the corneal endothelium and the safety of gentian violet application. MATERIALS AND METHODS: Two groups of anterior lens capsules obtained during the cataract surgery, gentian violet stained and non-stained, were incubated with fluorescent dye Fura-2. Their fluorescence, upon excitation at 360 and 380 nm, was imaged to monitor changes in free intracellular calcium concentration ([Ca(2+)]i) in response to pharmacological stimulation by acetylcholine. The [Ca(2+)]i homeostasis is the indicator of cellular function. The changes in [Ca(2+)]i were compared between the two groups. RESULTS: Epithelial cells responded to acetylcholine in both groups of capsules - gentian violet stained (n = 17) and non-stained ones (n = 33). No significant differences of the elicited responses were found in rise time (p = 0.89), decay time (p = 0.61) or amplitude of [Ca(2+)]i (p = 0.96 for 63× and p = 0.26 for 40× objectives) between the two groups of capsules (Student t test). CONCLUSIONS: The staining of the anterior lens capsule with gentian violet during phacoemulsification in concentration of 0.01%, does not have detectable cytotoxic effects, which would affect the [Ca(2+)]i homeostasis in lens epithelial cells. The data, if extrapolated to corneal endothelium, exposed to the same concentration, suggest that gentian violet in concentration of 0.01% is safe as an adjunct for capsule visualization in cataract surgery.

Functional and molecular characterization of ex vivo cultured epiretinal membrane cells from human proliferative diabetic retinopathy.

Characterization of the cell surface marker phenotype of ex vivo cultured cells growing out of human fibrovascular epiretinal membranes (fvERMs) from proliferative diabetic retinopathy (PDR) can give insight into their function in immunity, angiogenesis, and retinal detachment. FvERMs from uneventful vitrectomies due to PDR were cultured adherently ex vivo. Surface marker analysis, release of immunity- and angiogenesis-pathway-related factors upon TNF α activation and measurement of the intracellular calcium dynamics upon mechano-stimulation using fluorescent dye Fura-2 were all performed. FvERMs formed proliferating cell monolayers when cultured ex vivo, which were negative for endothelial cell markers (CD31, VEGFR2), partially positive for hematopoietic- (CD34, CD47) and mesenchymal stem cell markers (CD73, CD90/Thy-1, and PDGFR ß ), and negative for CD105. CD146/MCAM and CD166/ALCAM, previously unreported in cells from fvERMs, were also expressed. Secretion of 11 angiogenesis-related factors (DPPIV/CD26, EG-VEGF/PK1, ET-1, IGFBP-2 and 3, IL-8/CXCL8, MCP-1/CCL2, MMP-9, PTX3/TSG-14, Serpin E1/PAI-1, Serpin F1/PEDF, TIMP-1, and TSP-1) were detected upon TNF α activation of fvERM cells. Mechano-stimulation of these cells induced intracellular calcium propagation representing functional viability and role of these cells in tractional retinal detachment, thus serving as a model for studying tractional forces present in fvERMs in PDR ex vivo.

Human anterior lens capsule epithelial cells contraction.

PURPOSE: Human anterior lens epithelial cells, attached to surgically isolated capsules, were found to contract upon stimulation. The purpose of this study was to characterize these contractions, which create gaps between cells, and to assess the underlying physiological mechanisms and their possible association with cataract formation. METHODS: Lens capsules obtained during cataract surgery were stained with fluorescent dye Fura-2. Its fluorescence, upon excitation at 360 and 380 nm, was imaged to monitor changes in cell morphology and cytosolic free Ca(2+) concentrations ([Ca(2+) ](i) ) in response to pharmacological stimulation by acetylcholine (ACh) and to mechanical stimulation by flow of saline or direct contact. RESULTS: Epithelial cells contracted in approximately a third of preparations when stimulated by either ACh application, fluid movement or direct mechanical contact. Contractions started either before or at best simultaneously with the rise in [Ca(2+) ](i). Contractions also occurred when there was hardly any change in [Ca(2+) ](i) upon application of physiological saline alone. The probability of contractions occurring did not differ significantly among cortical, nuclear and combined cortical + nuclear cataract. CONCLUSIONS: This study provides the evidence that contractions of the anterior lens epithelial cells take place in significant portion of human lens anterior capsule postoperative preparations after non-specific stimulation. Contractions are at least partially independent of changes in [Ca(2+) ](i). They can be mechanically induced, are localized and reversible and have a fast response and did not differ among different types of cataract. Physiological and clinical significance of this phenomenon remains to be elucidated.

Structural and immunogenicity studies of a cleaved, stabilized envelope trimer derived from subtype A HIV-1.

SOSIP gp140 trimers represent a soluble, stabilized, proteolytically cleaved form of the HIV-1 envelope (Env) glycoproteins. SOSIP gp140 derived from a subtype A HIV-1 isolate, KNH1144, forms exceptionally stable trimers that resemble virion-associated Env in antigenicity and topology. Here, we used electron microscopy to demonstrate that KNH1144 SOSIP gp140 trimers bound three soluble CD4 molecules in a symmetrical orientation similar to that seen for native Env. We compared the immunogenicities of KNH1144 SOSIP gp140 trimers and gp120 monomers in rabbits and found that the trimers were superior at eliciting neutralizing antibodies (NAbs) to homologous virus as well as neutralization-sensitive subtype B and C viruses. The NAb specificities for SOSIP antisera mapped in part to the CD4 binding site on gp120. We also observed adjuvant-dependent induction of antibodies to the residual levels of host cell proteins (HCPs) contained in the purified Env preparations. When present, HCP antibodies enhanced pseudovirus infection. Our findings are relevant for the further development of Env-based vaccines for HIV-1.