Uveitis-mediated immune cell invasion through the extracellular matrix of the lens capsule.

While the eye is considered an immune privileged site, its privilege is abrogated when immune cells are recruited from the surrounding vasculature in response to trauma, infection, aging, and autoimmune diseases like uveitis. Here, we investigate whether in uveitis immune cells become associated with the lens capsule and compromise its privilege in studies of C57BL/6J mice with experimental autoimmune uveitis. These studies show that at D14, the peak of uveitis in these mice, T cells, macrophages, and Ly6G/Ly6C+ immune cells associate with the lens basement membrane capsule, burrow into the capsule matrix, and remain integrated with the capsule as immune resolution is occurring at D26. 3D surface rendering image analytics of confocal z-stacks and scanning electron microscopy imaging of the lens surface show the degradation of the lens capsule as these lens-associated immune cells integrate with and invade the lens capsule, with a subset infiltrating both epithelial and fiber cell regions of lens tissue, abrogating its immune privilege. Those immune cells that remain on the surface often become entwined with a fibrillar net-like structure. Immune cell invasion of the lens capsule in uveitis has not been described previously and may play a role in induction of lens and other eye pathologies associated with autoimmunity.

Immune cells in lens injury repair and fibrosis.

Immune cells, both tissue resident immune cells and those immune cells recruited in response to wounding or degenerative conditions, are essential to both the maintenance and restoration of homeostasis in most tissues. These cells are typically provided to tissues by their closely associated vasculatures. However, the lens, like many of the tissues in the eye, are considered immune privileged sites because they have no associated vasculature. Such absence of immune cells was thought to protect the lens from inflammatory responses that would bring with them the danger of causing vision impairing opacities. However, it has now been shown, as occurs in other immune privileged sites in the eye, that novel pathways exist by which immune cells come to associate with the lens to protect it, maintain its homeostasis, and function in its regenerative repair. Here we review the discoveries that have revealed there are both innate and adaptive immune system responses to lens, and that, like most other tissues, the lens harbors a population of resident immune cells, which are the sentinels of danger or injury to a tissue. While resident and recruited immune cells are essential elements of lens homeostasis and repair, they also become the agents of disease, particularly as progenitors of pro-fibrogenic myofibroblasts. There still remains much to learn about the function of lens-associated immune cells in protection, repair and disease, the knowledge of which will provide new tools for maintaining the core functions of the lens in the visual system.

An immune response to the avascular lens following wounding of the cornea involves ciliary zonule fibrils.

The lens and central cornea are avascular. It was assumed that the adult lens had no source of immune cells and that the basement membrane capsule surrounding the lens was a barrier to immune cell migration. Yet, microfibril-associated protein-1 (MAGP1)-rich ciliary zonules that originate from the vasculature-rich ciliary body and extend along the surface of the lens capsule, form a potential conduit for immune cells to the lens. In response to cornea debridement wounding, we find increased expression of MAGP1 throughout the central corneal stroma. The immune cells that populate this typically avascular region after wounding closely associate with this MAGP1-rich matrix. These results suggest that MAGP1-rich microfibrils support immune cell migration post-injury. Using this cornea wound model, we investigated whether there is an immune response to the lens following cornea injury involving the lens-associated MAGP1-rich ciliary zonules. Our results provide the first evidence that following corneal wounding immune cells are activated to travel along zonule fibers that extend anteriorly along the equatorial surface of the lens, from where they migrate across the anterior lens capsule. These results demonstrate that lens-associated ciliary zonules are directly involved in the lens immune response and suggest the ciliary body as a source of immune cells to the avascular lens.

Mechanism of atopic cataract caused by eosinophil granule major basic protein.

Atopic cataracts develop under the ages of 40 years, after which visual acuity rapidly declines. However, the mechanism underlying the development of atopic cataracts is not yet clear. We focused on the eosinophil granule major basic protein (MBP), which was detected in the aqueous humor of atopic cataracts previously, and which was cytotoxic. Specifically, we investigated its origin in this fluid and its effects on lens epithelial cells (LECs). MBP immunostaining was positive in atopic cataract-derived LECs, but negative in age-related cataract-derived LECs. MBP mRNA was not detected in either type of cataract, but protein was detected in the aqueous humor. Furthermore, the flare values associated with atopic cataracts were higher than those with age-related cataracts. When MBP was purified from eosinophils or recombinant MBP was added to LEC culture medium, cell viability decreased in a concentration-dependent manner, but an MBP antibody neutralized the cytotoxic effect of this protein towards these cells. These results were consistent with the flow of MBP into the aqueous humor from the blood due to a compromised blood-aqueous barrier. Thus, MBP could further penetrate the lens capsule and adhere to LECs, resulting in decreased cell viability and the development of atopic cataracts.

Role of innate immunity and oxidative stress in steroid-induced cataracts in developing chick embryos.

PURPOSE: The exact etiopathogenesis of steroid-induced cataracts (SIC) is not known. Although oxidative stress is one of the most acceptable hypotheses, the exact molecular events in steroid-induced oxidative events in the lens need to be clarified. In addition, to the best of our knowledge, the innate immune system components in SIC formation have not been studied previously. The aim of the present study was to study the oxidative system and the innate immune system components in the cataractous lenses of a developing chick embryo SIC model. MATERIALS AND METHODS: The study included 20 specific pathogen-free (SPF) fertilized eggs divided into two groups, with one hydrocortisone (HC)-treated group (G1) and one non-HC-treated control group (G2). On the 15th day of incubation, the SPF eggs in the two groups were removed from the incubator; HC was injected into G1, and phosphate-buffered saline (PBS) was injected into G2 using insulin injectors into the chorioallantoic membrane. On day 17, all of the chick embryos were removed from the eggs, and all lens and liver tissues were dissected from the embryos. Cataract formation was evaluated in all lenses, and total antioxidant status (TAS), total oxidant status (TOS), reduced glutathione (GSH), oxidized glutathione (GSSG), interleukin-6 (IL-6), and interleukin-1ß (IL-1ß) levels were measured in all lens and liver tissues. RESULTS: The lenses in the SIC group had lower levels of GSH, GSSG, TAS, and IL-6 (p = 0.016, 0.022, 0.000, 0.000, respectively), and higher levels of TOS (p = 0.000) than the control group. Furthermore, the liver tissues in the SIC group had decreased levels of TAS and IL-6, and increased levels of TOS compared to the control tissues (p = 0.000). Although the IL-1ß levels in the lens and liver tissues in the HC-induced cataract group were lower than in the control group, these differences were not statistically significant. In addition, the GSH levels in the liver tissues did not statistically differ between the two groups despite the significant GSH difference in the lens tissues. CONCLUSIONS: Steroid therapy causes a decrease in GSH, GSSG, and TAS levels and an increase in TOS levels in lens tissues, which means there is increased oxidative stress and decreased antioxidant defence. Furthermore, lenses with SIC have lower IL-6 levels compared to non-cataractous lenses. The interaction between lenticular IL-6 and antioxidant defence needs to be further studied.

Human cytokines activate JAK-STAT signaling pathway in porcine ocular tissue.

BACKGROUND: The JAK/STAT (Janus Tyrosine Kinase, Signal Transducers and Activators of Transcription) pathway is associated with cytokine or growth factor receptors and it is critical for growth control, developmental regulation and homeostasis. The use of porcine ocular cells as putative xenotransplants appears theoretically possible. The aim of this study was to investigate the response of various porcine ocular cells in vitro to human cytokines in regard to the activation of JAK-STAT signaling pathways. METHODS: Porcine lens epithelial cells, pigmented iris epithelial cells and pigmented ciliary body cells were used in this study. These cells were isolated from freshly enucleated porcine eyes by enzymatic digestion. Cultured cells between passages 3-8 were used in all experiments. Electromobility shift assay (EMSA), proliferation assay, immunofluorescence staining and flow cytometry were used to evaluate the JAK-STAT signaling pathway in these cells. RESULTS: JAK/STAT signaling pathways could be activated in porcine pigmented epithelial ciliary body cells, in pigmented iris epithelial cells and in lens epithelial cells in response to porcine and human interferons and cytokines. All cells showed very strong STAT1 activation upon stimulation with porcine interferon-gamma. Porcine ocular cells also respond to human cytokines; IFN-alpha induced strong activation of STAT1 in EMSA, flow cytometry and immunofluorescence experiments whereas activation of STAT3 was less strong in EMSA, but strong in flow cytometry and immunofluorescence. Human recombinant IL-6 activated STAT3 and human IL-4 activated STAT6. With the help of immunofluorescence assay and flow cytometry we observed nuclear localization of STAT proteins after activation of porcine ocular cells with cytokines and interferons. Human IFN-α had an inhibitory effect on porcine ocular cells in proliferation assays. CONCLUSION: Our study demonstrated that some types of human cytokines and interferon activate intracellular JAK-STAT signaling pathways in porcine ocular cells. We hypothesize that direct stimulation of the JAK-STAT pathway in porcine cells in response to human cytokines will lead to complications or failure, if pig-to-human ocular tissue xenotransplantation were to be carried out. For successful xenotransplantation among other obstacles there must be new approaches developed to regulate signaling pathways.

Characterization of an antibody that recognizes peptides containing D-ß-aspartyl residues.

PURPOSE: Biologically uncommon D-ß-aspartyl (D-ß-Asp) residues have been detected in proteins from various human tissues from elderly donors and are connected with cataract, age-related macular degeneration, Alzheimer disease and UV-irradiated skin. In a previous study, we prepared a highly specific antibody against the peptide Gly-Leu-D-ß-Asp-Ala-Thr-Gly-Leu-D-ß-Asp-Ala-Thr-Gly-Leu-D-ß-Asp-Ala-Thr (designated peptide 3R) that corresponds to three repeats of positions 149-153 of human lens αA-crystallin. This antibody clearly distinguishes between the different configurations of the Asp residue in that it reacted strongly with the D-ß-Asp-containing peptides but did not react with L-α-Asp-, L-ß-Asp-, or D-α-Asp-containing peptides. However, it remains unclear whether the antibody recognizes the amino acid sequences surrounding the D-ß-Asp residue. The purpose of the present study is to elucidate the sequence dependency of the epitope of the antigen. METHODS: To clarify the properties of the anti-peptide 3R antibody, we used F-moc (9-fluorenylmethoxycarbonyl) solid phase chemistry to synthesize various peptides and analogs based on the peptides T18 (I(146)QTGLDATHAER(157)) and T6 (T(55)VLDAGISEVR(65)) which correspond to amino acid sequences 146-157 and 55-65, respectively of human αA-crystallin. The specificity of antibody was confirmed by ELISA (enzyme-linked immunosorbent assay) using these peptides. RESULTS: The anti peptide 3R antibody specifically recognized D-ß-Asp residues and does not react with other configurations of Asp such as the L-α, L-ß, D-α isomers in peptides. When the Ala in the peptide was replaced by other amino acid residues, the antibody did not react with the antigen. The antibody requires the sequence Leu-D-ß-Asp-Ala to detect D-ß-Asp containing proteins in living tissue. CONCLUSIONS: The anti peptide 3R antibody is a powerful and easy tool for detection of D-ß-Asp containing proteins in living tissues from patients with age-related diseases. However, to detect the D-ß-Asp containing proteins in the living tissues using the anti-peptide 3R antibody, the protein must contain the sequence Leu-D-ß-Asp-Ala.

Klotho ameliorates the onset and progression of cataract via suppressing oxidative stress and inflammation in the lens in streptozotocin-induced diabetic rats.

Increased oxidative stress and inflammation play an important role in the pathogenesis of diabetic cataract. Klotho, known as an anti-ageing protein, has antioxidative and anti-inflammatory properties. Klotho is expressed in limited tissues including the lens. Here we examined whether klotho expression is decreased in diabetic lens and, if so, whether klotho treatment can prevent diabetic cataract formation. Streptozotocin (STZ)-induced diabetic rats and age-matched control rats were treated with vehicle or klotho protein, starting at 1 week after STZ injection. Twelve weeks after treatment, cataract formation was observed in diabetic rats but not control rats. Cataract formation and scores were significantly less in klotho-treated diabetic rats than vehicle-treated diabetic rats. Levels of klotho in plasma, aqueous humor and lens were significantly decreased in vehicle-treated diabetic rats, compared with control rats, but were restored in klotho-treated diabetic rats. Additionally, vehicle-treated diabetic rats had increased oxidative stress and inflammation in the lens, which were associated with decreased antioxidant transcriptional master regulator Nrf2 activity and increased transcription factor NF-κB activity. All of these findings were ameliorated in klotho-treated diabetic rats. Notably, klotho treatment did not alter blood glucose in diabetic rats. These results indicate that klotho reduction may increase susceptibility of the lens to oxidative and inflammatory insults, promoting cataract formation under diabetic conditions. Klotho treatment can ameliorate the onset and progression of diabetic cataract via enhancing Nrf2-mediated antioxidant defense and suppressing NF-κB-mediated inflammatory responses. Klotho in the lens may be a novel therapeutic target for prevention of cataract formation in diabetes.

Immunocytochemical localization of the lens main intrinsic polypeptide (MIP26) in communicating junctions.

Plasma membranes of vertebrate lens fiber cells contain a major intrinsic polypeptide with an apparent molecular weight of 26,000 (MIP26). These plasma membranes are extremely rich in communicating junctions, and it has been suggested that MIP26 is a component of them. MIP26 was purified from cow lenses using preparative SDS gel electrophoresis followed by hydroxylapatite column chromatography. From gel electrophoresis patterns and aggregational properties it was concluded that the MIP26 preparation was homogeneous. The purified MIP26 was used to produce monospecific antibodies in rabbits as assessed by double immunodiffusion and crossed immunoelectrophoresis of purified MIP26 and solubilized lens plasma membranes against the antiserum. Indirect immunocytochemical studies were performed on open and closed lens plasma membrane vesicles by incubation in anti-MIP antiserum followed by ferritin-conjugated goat antirabbit IgG. The conjugate bound unequivocally to lens communicating junctions, indicating that MIP26 is a component of these structures.

Expression of complement components coincides with early patterning and organogenesis in Xenopus laevis.

The complement system is the central component of innate immunity and an important player in the adaptive immunity of vertebrates. We analyzed the expression patterns of several key members of the complement cascade during Xenopus development. We found extensive expression of these molecules already during gastrula/early neurula stage. Remarkably, several genes also showed an organ-specific expression pattern during early organogenesis. Early expression is notable for two different expression patterns in the neuroectoderm. In one group, there is early strong neural plate and neural precursor expression. This is the case of properdin, C1qA, C3 and C9. The second pattern, seen with C1qR and C6, is noteworthy for its expression at the periphery of the neural plate, in the presumptive neural crest. Two genes stand out for their predominantly mesodermal expression. C3aR, the message for the cognate receptor for C3 in the complement cascade, is expressed at the same time as C3, but in a complementary, reciprocal pattern in the mesoderm. C1qA expression also predominates in somites, pronephros, visceral mesoderm and ventral blood islands. Finally, several genes are characterized by later expression in developing organs. C1qR displays a reticular pattern consistent with expression in the developing vasculature. The late expression of C1qA and C3bC4b is strongest in the pronephros. Finally, the expression of properdin in the hindbrain and in the developing lens are novel findings. The expression patterns of these molecules suggest that these components of the complement system may have in Xenopus a so far undefined developmental role.

Phaco-antigenic glaucoma following uncomplicated cataract surgery.

INTRODUCTION: Phaco-antigenic glaucoma occurs in less than 1% of cataract surgeries. Managing this rare complication is challenging, especially when there are few documented cases reported. We describe the challenges of managing a case of bilateral phaco-antigenic glaucoma following uncomplicated cataract surgery requiring viscocanalostomy. CASE DESCRIPTION: An 82-year-old atopic lady presented with a 2-day history of painful injected right eye. She was 4 days post left and 8 days post right uncomplicated cataract surgery. On examination, the anterior chambers were deep with no hypopyon. Intraocular pressure was raised at 38 mmHg in the right eye and 24 mmHg in the left eye initially. However, intraocular pressure remained uncontrolled despite maximum medical treatment; she attended A + E six times within 11 days with intraocular pressures of up to 48 mmHg in the right eye and 46 mmHg in the left eye. A vitreous biopsy was reported negative for infective organisms. Eventually, bilateral viscocanalostomies were performed and vision improved to 0.24 logMAR in both eyes with intraocular pressures of 8 mmHg in the right eye and 10 mmHg in the left eye. CONCLUSION: We present a rare presentation of phaco-antigenic glaucoma following an uncomplicated cataract surgical procedure with good results following timely intervention.

Different factors promote axonal regeneration of adult rat retinal ganglion cells after lens injury and intravitreal peripheral nerve grafting.

We have investigated the differential mediators of the neurotrophic effects of intravitreal peripheral nerve grafting and lens injury on adult rat retinal ganglion cells (RGC). Lens injury and intravitreal peripheral nerve grafting both stimulated RGC neurite growth in vitro and axon regeneration past the optic nerve lesion site in vivo concomitant with activation of retinal glia and invasion of macrophages into the eye. These observations, together with the results of coculture studies using a macrophage-free intact peripheral nerve segment, a macrophage-free intact lens, a macrophage-rich peripheral nerve segment, or a macrophage-rich injured lens in retinal cultures suggest that the stimulation of RGC axon regeneration by lens injury and intravitreal peripheral nerve grafting share a common macrophage-derived component overlain by distinct lens-derived and peripheral nerve-derived neurotrophic factors, respectively. RGC axon regeneration following lens injury and intravitreal peripheral nerve grafting was similar in vivo, correlating with similar retinal glia activation whereas, in vitro, the level of RGC neurite outgrowth was significantly higher following intravitreal peripheral nerve grafting compared with lens injury, concomitant with the presence of increased numbers of activated retinal glia. This suggests that in vivo RGC axon regeneration induced by lens injury and peripheral nerve grafting may be limited, in part, by factors derived from activated retinal glia.

Immunochemical detection of glycated lens crystallins and their circulating autoantibodies in human serum during aging.

PURPOSE: The aim of this investigation was to exploit lens-specific glycated crystallins as an immunogen to detect human glycated crystallins and their circulating autoantibodies in human serum during aging in relation to the development of cataract. METHODS: Polyclonal antibodies were produced against human total lens proteins (40-80 years) in rabbits. The specificity of the antibodies produced were determined by antibody capture assay using purified human lens crystallins (high molecular weight fraction [HMW]+alpha, HMW+alpha-glycated, beta, beta-glycated, gamma, and gamma-glycated) as antigens. The cross-reactivity of these lens specific antibodies against rat beta-, beta-glycated, gamma-, and gamma-glycated lens crystallins was also analyzed. A non-competitive enzyme linked immunosorbent assay (ELISA) methodology was developed for the detection of circulating lens crystallins in human sera using HMW+alpha, HMW+alpha-glycated, beta-, and beta-glycated crystallins from humans and gamma- and gamma-glycated crystallins from rats as immobilized antigens. Circulating autoantibodies were also detected in human sera by antibody capture assay. The methodology was validated by evaluating 60 human serum samples collected from cataract patients and 30 human serum samples from apparently normal subjects belonging to the same age group. RESULTS: The polyclonal antibodies raised against human total lens proteins showed 90% and 65% cross-reactivity with rat gamma- and beta-crystallins, respectively, by ELISA. Further, these polyclonal antibodies were capable of detecting both native and in vitro synthesized glycated crystallins. Their IC50 values were observed to be (i) human total lens proteins (55 ng), (ii) human HMW+alpha (16.45 ng), (iii) human HMW+alpha-glycated (273 ng), (iv) human beta- (37.82 ng), (v) human beta-glycated (260 ng), (vi) rat gamma- (105.34 ng), and (vii) rat gamma-glycated (313 ng). The immunochemical analysis of human serum indicated a significant change (p<0.001) in the levels of circulating beta-glycated and gamma-glycated crystallins in the age group of 40-80 years with respect to their control groups. However, there was no statistically significant change in the levels of HMW+alpha-glycated crystallins in the age group of 40-80 years as compared to their age-matched controls. Notably, the levels of serum gamma-glycated crystallins were found to be threefold higher than that of HMW+alpha-glycated and beta-glycated crystallins in the age group of 70-80 years. Circulating autoantibodies to HMW+alpha-glycated, beta-glycated, and gamma-glycated crystallins were detected in the serum of both apparently normal and cataract patients in the age group of 40-80 years by antibody capture assay. The levels of these autoantibodies were significantly higher at every time point compared to their respective controls. Autoantibodies to gamma-glycated crystallins were found to be twofold and 3.2 fold higher as compared to the levels of autoantibodies to beta-glycated and HMW+alpha-glycated crystallins, respectively. Western blot and immunohistochemical analysis substantiated the observations made in non-competitive ELISA. CONCLUSIONS: During the course of aging, leakage of lens crystallins (HMW+alpha, HMW+alpha-glycated, beta, beta-glycated, gamma, and gamma-glycated) elicit an immune response resulting in the formation of autoantibodies in cataract patients (40-80 years) as compared to age matched controls. This is the first experimental report where polyclonal antibodies raised against lens-specific glycated crystallins were capable of detecting the early leakage of glycated crystallins in human subjects. This immunochemical approach has implications in the early detection of senile cataract.

Altered Expression Patterns of the Sumoylation Enzymes E1, E2 and E3 Are Associated with Glucose Oxidase- and UVA-Induced Cataractogenesis.

PURPOSE: Protein sumoylation is a well established regulatory mechanism that regulates chromatin structure and dynamics, cell proliferation and differentiation, stress response and cell apoptosis. In the vertebrate eye, we and others have shown that sumoylation plays an indispensable role in regulating eye development. During stress induction and aging process, the ocular tissues gradually loss their normality and develop major ocular diseases such as cataract and aging-related macular degeneration. We have recently demonstrated that sumoylation actively regulates differentiation of lens cells, whether this process is implicated in lens pathogenesis remains to be investigated. In this study, we have demonstrated that transparent mouse lenses treated with glucose oxidase and UVA irradiation undergo in vitro cataract formation, and associated with this process, the expression patterns of the 3 sumoylation enzymes have been found significantly altered. METHODS: Four-week-old C57BL/6J mice were used in our experiment. Lenses were carefully excised from eyes and cultured in M199 medium (Sigma 3769) for at least 12 hours. Transparent lenses (without surgical damage) were selected for experimentation. The lenses were exposed to UVA for 60 min or treated with 30 mU/mL glucose oxidase (GO, MP Biomedicals, 1673) to induce cataract formation. The mRNA levels were analysed with qRT-PCR. The protein levels were determined with western blot analysis and quantitated with Image J. RESULTS: we have obtained the following results: 1) Both GO treatment and UVA irradiation can induce cataract formation in the in vitro cultured mouse lenses; 2) With GO treatment, the mRNAs and proteins for the 5 sumoylation enzymes were all significantly downregulated; 3) With UVA irradiation, the changes in the expression patterns of the mRNAs and proteins for the SAE1, UBA2 , UBC9 and PIAS1 were opposite, while the mRNAs were upregulated either significantly (for SAE1, UBA2 and UBC9) or slightly (PIAS1), the proteins for all 4 sumoylation enzymes were downregulated; For RanBP2, the UVA induced changes in both mRNA and protein are consist with the GO treatment. CONCLUSION: Under GO and UVA irradiation conditions, the expression levels of both mRNA and protein for the three major sumoylation enzymes were significantly changed. Our results suggest that altered expression patterns of the sumoylation enzymes are associated with oxidative stressinduced cataractogenesis.

Detection of kynurenine modifications in proteins using a monoclonal antibody.

N-formylkynurenine and kynurenine are oxidation products of tryptophan formed from the reaction catalyzed by indoleamine 2,3-dioxygenase. These kynurenines react with proteins to produce chemical modifications in the lens. We developed a novel monoclonal antibody that detects a kynurenine modification in proteins. The antibody recognized proteins (human lens proteins, RNase A and BSA) that were modified by either kynurenine or N-formylkynurenine. The antibody also reacted strongly with N-formylkynurenine-modified N(alpha)-acetyl histidine and weakly with N-formylkynurenine-modified N(alpha)-acetyl lysine, N(alpha)-acetyl cysteine and N(alpha)-acetyl arginine. The antibody recognized kynurenine and N-formylkynurenine but not other tryptophan oxidation products. We isolated and purified a major antigen from the reaction mixture of N(alpha)-acetyl histidine and N-formylkynurenine and identified the product as N-acetyl-1-[3-(2-aminophenyl)-1-carboxy-3-oxopropyl]-histidine. We then used our purified antibody to detect kynurenine modifications in kynurenine-treated human lens epithelial cells and human lens. We found epithelial immunoreactivity in a lens from an aged donor but not in one from a very young donor. This would suggest that the antibody detects age-related changes in lens proteins altered by kynurenines. We believe that our antibody could be used to establish the importance of kynurenine modifications in diseases where tryptophan oxidation is enhanced.

Antibodies to the lens and cornea in anti-DFS70-positive subjects.

Autoantibodies against DFS70 (dense fine speckles 70) antigen have recently been identified among antinuclear antibodies (ANA) in patients with various inflammatory diseases and in patients with different types of cancer. These antibodies are recognized using indirect immunofluorescence (IIF) on HEp-2 cells, by a fine speckled nuclear staining in interphase HEp-2 cells and a positive reaction in the chromosome region of mitotic cells. Given that the DFS70 protein is also known as the lens epithelium-derived growth factor, this study was performed with two objectives: (a) to assess the prevalence of these antibodies in patients sent for ANA testing and in 334 patients with different types of neoplasia and (b) to determine whether the lens tissue was a suitable substrate for the detection of antibodies specific to lens proteins. During routine workup for ANA detection by the IIF method, we found 172 DFS70-positive sera among 21,516 consecutive samples (prevalence, 0.8%). In the group of patients with neoplastic disease, 6 of 334 (1.8%) were anti-DFS70-positive. DFS70-positive sera were then assayed by the IIF method on cryostatic sections of mouse eye at a dilution of 1:40 with an anti-human IgG conjugate. Among the 172 DFS70-positive samples detected by the ANA screening, 32 (19%) were strongly reactive against the reticular fibers of the lens; 8 (5%) were positive only to the corneal epithelium (nuclear negative); 5 (3%) were positive both for the cornea and the lens fibers; 13 (7%) stained only the nuclei of lens and cornea cells, and 4 (2%) were positive against the ciliary muscle. Among the patients with neoplastic diseases, only one with lung cancer reacted weakly with the reticular fibers of the lens. Sera from 20 healthy blood donors were negative. In this preliminary study, we have shown that the prevalence of anti-DFS70 antibodies is much lower than previously reported, both in patients screened for ANA and in patients with cancer. We have also seen that some DFS70-positive sera have antibodies that recognize antigens of the lens. Further studies are needed to investigate the fine specificity and the possible significance of these new autoantibodies.

[Experimental study on 5-fluourouracil-loaded immunonanoparticles targeting to lens epithelial cells].

OBJECTIVE: To evaluate the immunological characteristics of an immunonanoparticles targeting to human lens epithelial cells and to study if it could internalize into and inhibit the proliferation of target cells in vitro. METHODS: Crosslinker carbodiimide was used to couple McAb HILE6 (anti-lens epithelial cells antibody) with 5-fluourouracil-loaded polyactic acid nanoparticles PLA (5-FU)-NP to prepare the immunonanoparticles HILE6-PLA (5-FU)-NP. The molar ratio of HILE6 and 5-FU in the immunonanoparticles were observed. The immunological activity of the antibodies in the immunonanoparticles was assessed by ELISA. The third passage lens epithelial cells were divided into four groups; immunonanoparticles, 5-FU nanoparticles, mixed HILE6 and 5-FU nanoparticles and the controls. The proliferation of lens epithelial cells were evaluated by MTT analysis. Internalization of immunonanoparticles, 5-FU nanoparticles and McAb HILE6 were observed by indirect immunofluorescence study of lens epithelial cells. RESULTS: The molar ratio of 5-FU to HILE6 in the immunonanoparticles was 1809:1 and 84% of the original immunological activity of antibodies could be retained. The immunonanoparticles inhibited the proliferation of lens epithelial cells, which was significantly greater than original 5-FU nanoparticles or the blend group. The IC(50) inhibition of immunonanoparticles was 5.0 microg/ml 2 hours after treatment. The immunonanoparticles were bound to the cells surface in 30 minutes; internalized in 2 hours and accumulated around the nucleus after 4 hours. CONCLUSION: The immunonanoparticles retain immunological activity and could specifically internalize into the lens epithelial cells to inhibit their proliferation.

Immunochemical comparison of the major intrinsic protein of eye-lens fibre cell membranes in mice with hereditary cataracts.

Expression of the major intrinsic protein (MIP) of eye-lens fibre cell membranes was compared in normal (DBA), cataractous (CAT, LOP, NCT) and chimaeric (CBA-LOP) mice at different stages of development using immunofluorescence microscopy and immunoblotting techniques. MIP of apparent molecular mass 26 kDa was detected in extracts of adult DBA, LOP and CBA-LOP lenses, but only low molecular mass (less than 26 kDa) immunoreactive proteins were detected in similar extracts from adult CAT and NCT lenses. The corresponding MIP distribution patterns confirmed the highly organised fibre-cell histology in embryonic DBA and adult CBA-LOP lenses and also highlighted the severe fibre-cell degeneration in the LOP lens. In contrast, however, no immunoreactive MIP was detected in situ in embryonic CAT and NCT lenses. These results suggest that a structural alteration of MIP occurs during embryonic lens development in the cataractous CAT (dominant) and NCT (recessive) mutant mice.

A micro-procedure for quantitative precipitin tests.

A new method for the quantitative analysis of antigens and antibodies has been based on (1) ultrafiltration of the antigen-antibody precipitates through silver membranes of 0.2 micrometer pore size in a specially designed multisample apparatus, and (2) spectrophotometric determination at 210 nm of the amount of proteins in the antigen-antibody precipitates dissolved in 0.01 N HCl. At this wavelength, the = C = O group of the polypeptide chains constitutes the main chromophoric group. In comparisons with the ninhydrin color reaction, protein determination by low UV spectrophotometry, e.g., at 210 nm, was shown to be about 6 times more sensitive, permitting analysis of samples containing from 1.0 to 35.0 microgram of antigen. The concentration range of protein solutions in 0.01 N HCl which is measured at low UV can be regulated by a factor of 8--10 by changing absorption between 200 and 230 nm. Comparison of the ultrafiltration microtechnique with the standard quantitative precipitin microtechnique involving centrifugation of precipitates was made in 4 different antigen-antibody systems. The new technique was found to be as accurate as the standard technique. It allows completion of analysis within only 5--6 h. The standard precipitin technique, by contrast, requires a 5--7-day reaction period for completion.

Synthesis of gamma crystallin by a cloned cell line from Nakano mouse lens.

A cloned cell line was derived from a culture of Nakano mouse lens epithelial cells. The cloned cells grew vigorously and produced large numbers of lentoid bodies. Sodium dodecyl sulfate (SDS) and non-SDS slab-gel electrophoresis of the soluble proteins from the cultured cell revealed protein bands identical in pattern to those of purified gamma crystallin. Antibody to mouse gamma crystallin reacted to the soluble protein fraction of the cultured cells, indicating the synthesis in culture of gamma crystallin by this cloned cell line.