Recombinant Treponema pallidum protein Tp47 promoted the phagocytosis of macrophages by activating NLRP3 inflammasome induced by PKM2-dependent glycolysis.

BACKGROUND: Glycolysis is a critical pathway in cellular glucose metabolism that provides energy and participates in immune responses. However, whether glycolysis is involved in NOD-like receptor family protein 3 (NLRP3) inflammasome activation and phagocytosis of macrophages in response to Treponema pallidum infection remains unclear. OBJECTIVES: To investigate the role of glycolysis in activating the NLRP3 inflammasome for regulating phagocytosis in macrophages in response to T. pallidum protein Tp47 and its associated mechanisms. METHODS: Interactions between activation of the NLRP3 inflammasome and phagocytosis and the role of glycolysis in Tp47-treated macrophages were investigated through experiments on peritoneal macrophages and human monocytic cell line-derived macrophages. RESULTS: Activation of phagocytosis and NLRP3 inflammasome were observed in Tp47-treated macrophages. Treatment with NLRP3 inhibitor MCC950 or si-NLRP3 attenuated Tp47-induced phagocytosis. Glycolysis and glycolytic capacity were enhanced by Tp47 stimulation in macrophages, and a change in the levels of glycolytic metabolites (phosphoenolpyruvate, citrate and lactate) was induced by Tp47 in macrophages. Inhibition of glycolysis with 2-deoxy-D-glucose, a glycolysis inhibitor, decreased the activation of NLRP3. Expression of M2 isoform of pyruvate kinase (PKM2), an enzyme catalysing a rate-limiting reaction in the glycolytic pathway, was upregulated in Tp47-stimulated macrophages. Inhibition of PKM2 with shikonin or si-PKM2 decreased glycolysis and NLRP3 activation. CONCLUSION: Tp47 promotes phagocytosis in macrophages by activating the NLRP3 inflammasome, which is induced by the enhancement of PKM2-dependent glycolysis.

[A case report of autosomal recessive bestrophinopathy].

The patient is a 40-year-old male who presented to the ophthalmology clinic due to easy visual fatigue for the past 3 months. Two months ago, the patient was misdiagnosed with "bilateral posterior uveitis", but the diagnosis was ruled out after ineffective treatment with corticosteroids. During the current visit, fundus examination revealed yellow-white material exudation below the macular center in both eyes. Considering the results of the ophthalmic examination and the genetic testing of the patient and his son, the patient was diagnosed with autosomal recessive bestrophinopathy.

Autophagy promotes phagocytosis and clearance of Treponema pallidum via the NLRP3 inflammasome in macrophages.

BACKGROUND: Elucidating the mechanism of the macrophage phagocytic response will improve our knowledge of host defence against Treponema pallidum. OBJECTIVE: To explore whether autophagy promotes T. pallidum phagocytosis and clearance via the NLRP3 inflammasome in macrophages. METHODS: The interactions between autophagy and phagocytosis and the role of NLRP3 in these processes in T. pallidum-treated macrophages were investigated through experiments using human monocytic cell line (THP-1)-derived macrophages. Treponema pallidum clearance after phagocytosis was evaluated by inoculating rabbits with macrophage-treponeme mixtures. RESULTS: Activation of autophagy and phagocytosis in T. pallidum-treated macrophages occurred in a dose- and time-dependent manner. The percentage of spirochete-positive macrophages (22.34% vs. 70.93%, P < 0.001) and spirochete internalization (MFI: 9.62 vs. 20.33, P < 0.001) were notably reduced by silencing Beclin1. Inoculation of macrophage-treponeme mixtures into rabbits showed a 3.00-day delay in lesion development (17.55 ± 3.73 vs. 14.55 ± 1.99 days) and decreased lesion numbers [11 (36.7%) vs. 20 (66.7%) of 30; χ2  = 5.406, P = 0.020] in the control compared with the si-Beclin1 group. Furthermore, silencing NLRP3 decreased the mRNA and protein levels of Beclin-1 and LC3B [mRNA: 49.86% and 43.02%; protein: 22.31% and 24.24%, respectively, differing significantly from the control group (P < 0.001)] and reduced the percentage of spirochete-positive macrophages (30.29% vs. 70.53%, P < 0.001) and spirochete internalization (MFI: 9.82 vs. 19.33, P < 0.001). CONCLUSION: Treponema pallidum induces autophagy in macrophages to promote phagocytosis and clearance. The NLRP3 inflammasome modulates autophagy and phagocytosis in vitro. These data may be useful for understanding the host-pathogen relationship and establish the groundwork for strategies to combat syphilis.

Recombinant Treponema pallidum protein Tp0136 promotes fibroblast migration by modulating MCP-1/CCR2 through TLR4.

BACKGROUND: Chancre self-healing is an important clinical feature in the early stages of syphilis infection. Wound healing may involve an important mechanism by the migration of fibroblasts filling the injured lesion. However, the specific mechanism underlying this process is still unknown. OBJECTIVES: We aimed to analyse the role of Tp0136 in the migration of fibroblasts and the related mechanism. METHODS: The migration ability of fibroblasts was detected by a wound-healing assay. RT-PCR and ELISA detected the expression of MCP-1, IL-6 and MMP-9. TLR4 expression was detected by RT-PCR. The protein levels of CCR2 and relevant signalling pathway molecules were measured by Western blotting. RESULTS: Tp0136 significantly promoted fibroblast migration. Subsequently, the levels of MCP-1 and its receptor CCR2 were increased in this process. The migration of fibroblasts was significantly inhibited by an anti-MCP-1 neutralizing antibody or CCR2 inhibitors. Furthermore, studies demonstrated that Tp0136 could activate the ERK/JNK/PI3K/NF-κB signalling pathways through TLR4 activity and that signalling pathways inhibitors could weaken MCP-1 secretion and fibroblast migration. CONCLUSIONS: These findings demonstrate that Tp0136 promotes the migration of fibroblasts by inducing MCP-1/CCR2 expression through signalling involving the TLR4, ERK, JNK, PI3K and NF-κB signalling pathways, which could contribute to the mechanism of chancre self-healing in syphilis.

Recombinant Treponema pallidum protein Tp47 induces angiogenesis by modulating the matrix metalloproteinase/tissue inhibitor of metalloproteinase balance in endothelial cells.

BACKGROUND: Although angiogenesis is an obvious pathological manifestation in the pathogenesis of syphilis, little is known about the underlying mechanisms of angiogenesis induced by reactions to Treponema pallidum antigens. OBJECTIVE: In this study, we sought to determine the role of recombinant T. pallidum Tp47 in promoting angiogenesis in endothelial cells and the related mechanism. METHODS: Evaluation of the pro-angiogenic activity of recombinant T. pallidum Tp47 in human umbilical vein endothelial cells (HUVECs) was assessed, and the balance of matrix metalloproteinase (MMP)/tissue inhibitor of metalloproteinase (TIMP) and the mechanisms underlying the involvement of Akt/mTOR/S6 pathways in this process were explored. RESULTS: Under stimulation by Tp47, HUVECs exhibited obvious proliferation, migration and tube formation. In addition, the apparent promotion of angiogenesis by Tp47 was observed using a zebrafish embryo model. During angiogenesis, the levels of MMP-1 and MMP-10 were significantly elevated, whereas those of TIMP-1 and TIMP-2 did not change. In addition, after transfection with siRNAMMP-1 and siRNAMMP-10, migration and tube formation were significantly inhibited. Akt/mTOR/S6 signalling was found to be involved in upregulating MMP-1 and MMP-10 expression, and the sequential blockade of steps in the pathways effectively prevented Tp47-induced angiogenesis. CONCLUSION: The results reveal the underlying mechanism of angiogenesis promoted by Tp47, namely, upregulating MMP-1 and MMP-10 expression to disrupt the MMP/TIMP balance through the Akt/mTOR/S6 pathway. These findings contribute to our understanding of the pathophysiology of syphilis.

Factors associated with syphilis infection: a comprehensive analysis based on a case-control study.

This study aimed to comprehensively evaluate factors that influence the likelihood of syphilis infection from risk-taking behaviours and medical conditions. A retrospective case-control study was conducted by enrolling 664 syphilis inpatients (excluding 11 congenital syphilis patients) and 800 sex- and age-matched controls. Medical histories, clinical data and patient interview data were collected and subjected to logistic regression analyses. The prevalence of syphilis in the study population was 3·9% (675/17,304). By univariate analysis, syphilis infection was associated with migration between cities, marital status, smoking, reproductive history, hypertension, elevated blood urea nitrogen (BUN) and infection with hepatitis B virus (HBV) (P < 0·05). A high rate of syphilis-HBV co-infection was observed in HIV-negative patients and further research revealed an association between syphilis and specific HBV serological reactivity. Syphilis was also associated with the frequency, duration and status of tobacco use. Multivariate analysis indicated that syphilis infection was independently associated with migration between cities [adjusted odds ratio (aOR) 1·368, 95% confidence interval (CI) 1·048-1·785], current smoking (aOR 1·607, 95% CI 1·177-2·195), elevated BUN (aOR 1·782, 95% CI 1·188-2·673) and some serological patterns of HBV infection. To prevent the spread of infectious diseases, inpatients and blood donors should be tested for HIV, syphilis, HBV and HCV simultaneously.

Laboratorial characteristics of patients with diarrhoea suffering from egg white allergy.

BACKGROUND: Egg allergy is associated with diarrhoeal symptoms. However, the mechanism underlying allergic diarrhoea remains unclear. OBJECTIVE: To determine whether egg white-specific IgE antibodies coexist with egg white-specific IgG antibodies in patients with egg allergy featuring diarrhoeal symptoms, and whether there is any relationship between these two antibody types. METHODS: A total of 89 patients with egg allergy featuring diarrhoeal symptoms (average age, 23.2 years; range, 1-78 years), all of whom tested positive for egg white-specific IgG, were enrolled in this study. The concentration of total IgE, egg white-specific IgE and number of eosinophils in the serum were determined. RESULTS: Among the 89 egg white allergic patients tested, 49 (55.1%) patients showed high reactivity to egg white-specific IgG, 48 (53.9%) patients had elevated serum total IgE levels, and 25 (28.1%) patients had elevated absolute eosinophil numbers. Out of the 89 egg white allergic patients, 25 showed elevated egg white-specific IgE antibody levels. Of the 25 patients who were positive for egg white-specific IgE antibody, 21 presented high sensitive reaction to egg white-specific IgG, three presented moderate sensitive reaction to egg white-specific IgG, and one presented mild sensitive reaction to egg white-specific IgG. A moderate correlation between egg white-specific IgG and egg white-specific IgE, egg white-specific IgG and absolute eosinophil number was found in the egg white allergic patients (r=0.438, P=0.000; r=0.322, P=0.002). Egg white-specific IgE levels varied in different age groups; the egg white-specific IgE concentration of younger patients (age≤18 years, mean rank 54.29) was significantly higher than that of the adult patients (age>18 years, mean rank 34.61) (Z=-3.629, P=0.000). CONCLUSION: Egg white-specific IgE antibody could coexist with egg white-specific IgG antibody in patients suffering from egg white allergy. Aberrant changes in the concentration of egg white-specific IgE antibody were associated with the presence of egg white-specific IgG antibody.

Tp47-Induced Monocyte-Derived Microvesicles Promote the Adherence of THP-1 Cells to Human Umbilical Vein Endothelial Cells via an ERK1/2-NF-κB Signaling Cascade.

The Treponema pallidum membrane protein Tp47 induces immunocyte adherence to vascular cells and contributes to vascular inflammation. However, it is unclear whether microvesicles are functional inflammatory mediators between vascular cells and immunocytes. Microvesicles that were isolated from Tp47-treated THP-1 cells using differential centrifugation were subjected to adherence assays to determine the adhesion-promoting effect on human umbilical vein endothelial cells (HUVECs). Intercellular adhesion molecule 1 (ICAM-1) and vascular cell adhesion molecule 1 (VCAM-1) levels in Tp47-induced microvesicle (Tp47-microvesicle)-treated HUVECs were measured, and the related intracellular signaling pathways of Tp47-microvesicle-induced monocyte adhesion were investigated. Tp47-microvesicles promoted THP-1 cell adhesion to HUVECs (P < 0.01) and upregulated ICAM-1 and VCAM-1 expression in HUVECs (P < 0.001). The adhesion of THP-1 cells to HUVECs was inhibited by anti-ICAM-1 and anti-VCAM-1 neutralizing antibodies. Tp47-microvesicle treatment of HUVECs activated the extracellular signal-regulated kinase 1/2 (ERK1/2) and NF-κB signaling pathways, whereas ERK1/2 and NF-κB inhibition suppressed the expression of ICAM-1 and VCAM-1 and significantly decreased the adhesion of THP-1 cells to HUVECs. IMPORTANCE Tp47-microvesicles promote the adhesion of THP-1 cells to HUVECs through the upregulation of ICAM-1 and VCAM-1 expression, which is mediated by the activation of the ERK1/2 and NF-κB pathways. These findings provide insight into the pathophysiology of syphilitic vascular inflammation.

Are nontreponemal tests suitable for monitoring syphilis treatment efficacy? Evidence from rabbit infection models.

OBJECTIVES: We aimed to characterize kinetics of non-treponamal antibody titres during the natural course of syphilis and explore their roles in monitoring syphilis treatment efficacy. METHODS: Sixty New Zealand white male rabbits were challenged with Nichols or Amoy Treponema pallidum strains, and the rapid plasma reagin (RPR) test was performed to quantify non-treponemal antibody titres during the infection course. Viable T. pallidum in the challenged rabbits was assessed with rabbit infectivity tests. RESULTS: The RPR titres of the Nichols or Amoy strain between no benzathine penicillin G (BPG) and BPG treatment subgroups displayed a similar trend: first ascending and then descending. Compared with baseline, the proportions of fourfold decline in RPR titres in the Nichols or Amoy group presented a similar result on days 30, 60 and 180 between the no BPG and BPG treatment subgroups (0%, 0/5; 80%, 4/5; 100%, 5/5; vs. 0%, 0/5; 80%, 4/5; 100%, 5/5; p 0.999; 0%, 0/5; 80%, 4/5; 80%, 4/5; vs. 40%, 2/5; 100%, 5/5; 100%, 5/5; p 0.098, respectively). Compared with the maximum baseline titre, the proportion of fourfold decline in PRR titre also showed a similar result in the two groups on days 30, 60 and 180 between the no BPG and the BPG treatment subgroups (0%, 0/5; 100%, 5/5; 100%, 5/5, vs. 40%, 2/5; 100%, 5/5; 100%, 5/5; p 0.129; 0%, 0/5; 100%, 5/5; 100%, 5/5, vs. 80%, 4/5; 100%, 5/5; 100%, 5/5; p 0.091, respectively. Moreover, regardless of whether the RPR titres presented a fourfold decline, viable T. pallidum could be detected in untreated rabbits' lymph nodes at 30, 60 and 180 days post infection, while viable T. pallidum was not detected in any of the treated rabbits' lymph nodes. CONCLUSIONS: The RPR titre increased and then decreased (even became negative) during the natural course of syphilis, similar to that seen after BPG treatment. The RPR tetre is thus a questionable indicator of syphilis treatment efficacy.

Effects of growth factors on proliferation and differentiation in human lens epithelial cells in early subculture.

PURPOSE: A successful method to subculture human lens epithelial (HLE) cells that retain their intrinsic characteristics is of great importance. This study examines the effects of four different growth factors on proliferation and differentiation in HLE cells in early subcultures. METHODS: Specimens of HLE cells were obtained from infants. First- or second-passage cells were cultured in the presence of 10(-2) to 10(2) ng/ml acidic and basic fibroblast growth factor (aFGF, bFGF), epidermal growth factor (EGF), or insulin-like growth factor-I (IGF-I). Cell proliferation was determined from cell number, and fiber differentiation was assessed from the time of appearance, the number of lentoids formed, and the expression of gamma-crystallin. RESULTS: Cell proliferation was increased by EGF, bFGF, and IGF-I at concentrations greater than 10(-1) ng/ml; the most effective concentration was 10 ng/ml. The effect of aFGF on proliferation appeared only at a concentration of 10(2) ng/ml. EGF, bFGF, or IGF-I at 10 ng/ml affected the time of appearance and the number of lentoids formed within 5 to 7 days. In contrast, lentoids were observed after 42 days without the addition of growth factors. Lentoid formation was accompanied by the expression of gamma-crystallin. CONCLUSIONS: EGF, aFGF, bFGF, and IGF-I stimulated cell proliferation and fiber differentiation in early subcultures.

The effect of hypertonicity on aldose reductase, alpha B-crystallin, and organic osmolytes in the retinal pigment epithelium.

PURPOSE: Aldose reductase (AR), an enzyme implicated in diabetic complications of ocular tissues, has been suggested to play a physiologic role in kidney and, possibly, other tissues by elevating the organic osmolytes in conditions of heightened extracellular tonicity. Hypertonicity has been shown to induce AR and alpha B-crystallin in some cells. To examine if similar mechanisms are operating in the retinal pigment epithelium (RPE), another target tissue of diabetic complications, we studied the effect of hypertonic media on the induction of AR, alpha B-crystallin, myoinositol, taurine, and other free amino acids. METHODS: Human RPE cells were cultured in normal and hypertonic media containing 150 mmol/l NaCl or 200 mmol/l cellobiose in combination with 30 mmol/l galactose from 0-8 days. Western blot analysis with antibodies were used to measure the expression of AR and alpha B-crystallin. Hybridization of northern blots using AR and alpha B-crystallin complementary DNA probes were employed for the measurement of the respective messenger RNA for these proteins. Changes in the levels of myoinositol, galactitol, taurine, and other free amino acids were determined biochemically. RESULTS: AR and alpha B-crystallin messenger RNA levels rose 16-fold and 4-fold, respectively, when human RPE cells were cultured for 3 days in media supplemented with either 150 mmol/l NaCl or 200 mmol/l cellobiose. AR and alpha B-crystallin protein levels also increased significantly, as seen by western blots. Consistent with the increased AR, galactitol accumulated to a greater extent when human RPE cells were grown in media containing 30 mmol/l galactose plus 150 mmol/l NaCl compared with cells grown in 30 mmol/l galactose alone. An 11-fold increase in cellular myoinositol and a 1.4-fold increase in taurine was observed in cells exposed to hypertonic media. CONCLUSIONS: These findings suggest that human RPE cells are responsive to hypertonic stress by elevating AR activity and use intracellular organic solutes in an interactive manner to help regulate intracellular tonicity.

Hypertonic stress increases NaK ATPase, taurine, and myoinositol in human lens and retinal pigment epithelial cultures.

PURPOSE: Recent evidence suggests that taurine and myoinositol may serve as organic osmolytes in a number of cells, including lens and retinal pigment epithelia, but the mechanism for their increased accumulation in response to hypertonic stress is not known. To assess whether NaK ATPase contributed to the elevated levels of taurine and myoinositol in cells exposed to hypertonic media, we measured the activity of NaK ATPase, which is known to be implicated in the transport of these substances, in human lens and retinal pigment epithelia cultured in isotonic and hypertonic media. METHODS: Primary cultures of human lens epithelial (HLE) and human retinal pigment epithelial (HRPE) cells were maintained in isotonic and hypertonic media for varying periods of time, and the activity of NaK ATPase and the levels of taurine and myoinositol were measured in cells cultured under two different conditions. The possible involvement of the transport enzyme in the accumulation of the two osmolytes was also investigated by inhibiting the enzyme with ouabain. RESULTS: When primary cultures of HLE and HRPE were exposed to hypertonic medium containing NaCl (600 mOsm) or cellobiose (500m Osm) for 72 hours, the concentration of taurine and myoinositol in HLE cells increased by 218% and 558% of control, respectively, in NaCl medium, whereas the corresponding increases in cellobiose medium were 147% and 439%. In HRPE cells, the increase in myoinositol levels in the two hypertonic media was more dramatic than that in taurine. Concomitant with the increase in the concentration of the osmolytes, there was an increase in NaK ATPase activity in both cell types. Although the accumulation of taurine in HLE cells in hypertonic media in a 6-hour culture was essentially prevented by 10(-8) mmol/l ouabain, myoinositol levels were affected to a lesser, but still significant, extent. In HRPE cells, which were cultured for 24 hrs in the presence of 10(-6) mmol/l ouabain, there was a more direct correlation between the inhibition of NaK ATPase and the decreased accumulation of taurine and myoinositol in the hypertonic media. CONCLUSION: Although the exact mechanism by which NaK ATPase activity increases in response to hypertonic stress remains to be established, the increased activity of the enzyme is related to the enhanced accumulation of the organic osmolytes, taurine, and myoinositol, in HLE and HRPE cells cultured in hypertonic medium.

The effect of aqueous humor ascorbate on ultraviolet-B-induced DNA damage in lens epithelium.

PURPOSE: High levels of ascorbic acid are known to be present in the aqueous humor of many diurnal species, whereas nocturnal animals have low concentrations of the compound. The purpose of this study was to test the hypothesis that the high concentration of aqueous ascorbate in diurnal animals protects the lens against ultraviolet (UV)-induced damage to the eye. This study compares the effect of UV-B-induced DNA strand breaks on the lens epithelia of guinea pigs and rats after depletion or elevation of aqueous humor ascorbate, respectively. METHODS: Eyes of guinea pigs and rats were exposed to UV-B radiation (0.25-0.75 J/cm2 on the cornea) for 10 minutes, and DNA strand breaks in lens epithelium were measured by single-cell gel electrophoresis. Ascorbic acid concentration in the aqueous humor, lens, and lens-capsule epithelium were assayed by spectrophotometric and electrochemical methods. For depletion of aqueous humor and lens ascorbate in guinea pigs, the animals were maintained on an ascorbate-deficient diet. Aqueous ascorbic acid was elevated in the rat by intraperitoneal injections of sodium ascorbate (1 g/kg). RESULTS: The ascorbate concentration in the aqueous humor of the normal rat was approximately 3% that of the guinea pig, whereas the concentration of the compound in the lens of the normal rat was 10% that of the guinea pig. Guinea pigs fed an ascorbate-deficient diet showed a dramatic drop of more than 80% in aqueous humor ascorbate in the first week, whereas lens ascorbate decreased by approximately 25% during this time period. After a single intraperitoneal injection of sodium ascorbate in the rat, aqueous humor ascorbic acid increased nearly 30 times that in the control, whereas lens ascorbate increased by approximately 30%. The extent of DNA damage in the lens epithelium of a normal rat exposed to UV-B was significantly greater than that occurring in lenses of normal guinea pigs after exposure to the same dose of radiation. Lenses from ascorbate-deficient guinea pigs showed 50% more DNA damage than those from normal guinea pigs after UV exposure, whereas the lenses in ascorbate-injected rats exhibited significant protection against UV-induced DNA strand breaks. CONCLUSIONS: High levels of ascorbic acid in the aqueous humor had a protective effect against UV-induced DNA damage to lens epithelium. The results were consistent with the hypothesis that high ascorbic acid in diurnal animals protects the lens against the cataractogenic effect of UV radiation in sunlight.

Enhancement of differentiation of human lens epithelium in tissue culture by changes in cell-substrate adhesion.

Differentiation of human lens epithelial (HLE) cells cultured in vitro was drastically accelerated when the cells were cultured on cell-substrate adhesion-free surfaces. Spontaneous differentiation of the lens epithelial cells in monolayer cultures could be recognized with the appearance of lentoid bodies after 40-50 days if maintained without further passage. Although dissociated HLE cells reconstituted into monolayers consistently on the haptotactic substrates (either gold-coated biopore membrane or regular plastic dishes), the cells from the same batches exclusively formed cell aggregates when cultured on either biopore membrane or agarose-coated plastic dishes (nonhaptotactic). The cells on nonadhesion substrate first aggregate, then synchronously develop into lentoids by the 10th day of culture. The differentiation of HLE cells into lentoid structures with lens-fiber characteristics was documented by both ultrastructural and biochemical markers, such as loss of cytoplasmic organelles, formation of gap junctions, and the expression of gamma-crystallin and MP26. The system, in which differentiation of epithelial cells can be induced predictably in a short period of time, provides an excellent model for the study of differentiation and gene expression in human lens cells cultured in vitro.

Synthesis of lens capsule in long-term culture of human lens epithelial cells.

PURPOSE: This study examined the extent to which human lens epithelial (HLE) cells in tissue culture retain the potential for differentiation, expression of lens-specific marker proteins, and the synthesis of lens capsule, the major characteristics of lens epithelium in vivo. METHODS: Primary cultures of HLE cells were maintained for up to 450 days. Transmission and immunoelectron microscopy were used to study the thickness of the synthesized capsule and the formation of type IV collagen and laminin, two major protein components of the basement membrane of lens capsule in vivo. RESULTS: In a long-term HLE culture system, without subcloning, lens fiber differentiation and capsular synthesis were maintained over a period of 450 days. In these cultures, the cell sheet showed three distinct zones: (1) a central zone with tight monolayer; (2) a mild peripheral zone with irregularly aggregated multilayer; and (3) a peripheral zone with loose monolayer. The basement membrane-like material was synthesized in the central zone and lentoids, which serve as a model for fiber differentiation, developed primarily in the mid peripheral zone. No capsular material or lentoids were observed in the peripheral zone. The capsule-like material was 2 to 2.5 microns thick and showed the presence of type IV collagen and laminin, as detected by antibody reaction. CONCLUSION: This study demonstrates for the first time that HLE cells in long-term cultures synthesize a continuous sheet of capsule-like material. The findings also suggest that reformation of a tight cell-to-cell relationship or generation of high cell density similar to that found in vivo may be an important factor for the synthesis of lens capsule.

Study of the polyol pathway and cell permeability changes in human lens and retinal pigment epithelium in tissue culture.

The polyol pathway was investigated in primary cultures of human retinal pigment epithelial (HRPE) cells and the results were compared with those in human lens epithelial (HLE) cells cultured under similar conditions. Significant levels of galactitol were formed in HRPE cells cultured for 72 hr in a medium containing 30 mmol/l D-galactose. Polyol accumulation was accompanied by the appearance of vacuoles as seen by transmission electron microscopy (TEM). Biochemical analysis revealed a significant depletion of cellular myoinositol, taurine, and a number of other free amino acids similar to those in HLE cells. These morphologic and biochemical changes observed in HRPE cells cultured in high galactose medium were inhibited or prevented by the inclusion of an aldose reductase inhibitor in the medium, further supporting the view that vacuole formation is due to the osmotic effect of polyol formation mediated by aldose reductase. The similarity of intracellular vacuole formation resulting from polyol accumulation and the biochemical changes in HRPE and HLE cells strongly suggests that a common mechanism is involved.

TEMPOL protects against lens DNA strand breaks and cataract in the x-rayed rabbit.

PURPOSE: To investigate the ability of the nitroxide free radical and superoxide dismutase mimic 4-hydroxy-2,2,6,6-tetramethylpiperidine-n-oxyl (TEMPOL) to protect against x-ray-induced lens DNA damage and cataract formation in the rabbit. METHODS: Eleven gray (Gy) x-rays was delivered twice, with a 48-hour interval, to the same eye of 5-week-old rabbits. Fifteen minutes before each x-ray, 150 microliters aqueous humor was removed from the anterior chamber and replaced with 150 microliters citrate buffer containing 0 mM or 100 mM TEMPOL. The development of cataract was classified into seven stages by slit-lamp examination. DNA strand breaks were measured in the lens epithelium of x-rayed rabbits using a single-cell gel electrophoresis method. RESULTS: The level of total TEMPOL in the aqueous humor of rabbits at 15 minutes after intracameral injection of the compound was 21 mM with 84% present in the oxidized form (determined by electron paramagnetic resonance spectroscopy). At 19 weeks after x-ray, rabbits irradiated without TEMPOL showed either stage 5 (complete posterior subcapsular opacity) or stage 6 (mature) cataracts, whereas the animals that had first been injected with TEMPOL developed only stage 2 to stage 4 cataracts (the difference between the two groups was significant at P < 0.01). Intracameral injection of TEMPOL resulted in a decrease of nearly 70% in the level of DNA strand breaks produced by a single 11-Gy dose of x-ray. In vitro studies showed that TEMPOL was reduced rapidly by ascorbic acid but not by reduced glutathione. Oxidized but not reduced TEMPOL (TEMPOL-H) was an effective radioprotector in cultured rabbit lenses, and TEMPOL was nearly completely bioreduced in the plasma and aqueous humor of animals that were fed the compound in drinking water. CONCLUSIONS: TEMPOL was effective in protecting against lens epithelial DNA damage and cataract formation in x-rayed rabbits. Although a number of mechanisms are possible, the protective effect may be associated with the ability of TEMPOL to protect against radiation-produced peroxides by acting as a superoxide dismutase mimic and to oxidize Fe2+ bound to DNA, thus preventing formation of the hydroxyl radical and subsequent DNA damage through the Haber-Weiss mechanism.

Lens epithelium-derived growth factor: increased survival and decreased DNA breakage of human RPE cells induced by oxidative stress.

PURPOSE: Lens epithelium-derived growth factor (LEDGF) has been shown to be a growth and survival factor and to be present in a wide variety of cell types. The purpose of this study was to determine whether LEDGF enhances survival of human retinal pigment epithelial (RPE) cells when challenged by oxidative stress or by ultraviolet (UVB) irradiation in a culture system. METHODS: Primary RPE cells were cultured in standard Dulbecco's modified Eagle's medium (DMEM) containing 15% fetal bovine serum. Protein blot analysis with antibodies to LEDGF was used to detect LEDGF in RPE cells. Initially, RPE cells were cultured in the standard medium for 1 day to allow attachment to the culture plates and then cultured in serum-free DMEM, with and without LEDGF. The trypan blue exclusion method was used to test RPE cell viability. Single-cell electrophoresis was used to evaluate single strand breaks of genomic DNA after exposure to H(2)O(2) or irradiation by UVB. RESULTS: LEDGF was present in RPE cells, predominantly in the nucleus. RPE cells grew for 1 week and survived for 3 weeks in the presence of LEDGF. In the absence of LEDGF, they increased in number for the first week and gradually died in the following 2 weeks. LEDGF protected RPE cells against H(2)O(2) exposure and UVB irradiation. DNA damage induced by H(2)O(2) exposure or UVB irradiation was lower in the presence than in the absence of LEDGF. The expression of heat shock protein (Hsp)27 was elevated by LEDGF. CONCLUSIONS: LEDGF enhanced survival of RPE cells in culture when challenged by oxidative stress and UVB irradiation. LEDGF protected DNA from single-strand breakage and upregulated the expression of Hsp27. These results suggest that LEDGF may be a potential agent for protecting RPE cells under various stress conditions.

Impact of aging and hyperbaric oxygen in vivo on guinea pig lens lipids and nuclear light scatter.

PURPOSE: To measure lipid compositional and structural changes in lenses as a result of hyperbaric oxygen (HBO) treatment in vivo. HBO treatment in vivo has been shown to produce increased lens nuclear light scattering. METHODS: Guinea pigs, approximately 650 days old at death, were given 30 and 50 HBO treatments over 10- and 17-week periods, respectively, and the lenses were sectioned into equatorial, cortical, and nuclear regions. Lipid oxidation, composition, and structure were measured using infrared spectroscopy. Phospholipid composition was measured using (31)P-NMR spectroscopy. Data were compared with those obtained from lenses of 29- and 644-day-old untreated guinea pigs. RESULTS: The percentage of sphingolipid approximately doubled with increasing age (29-544 days old). Concomitant with an increase in sphingolipid was an increase in hydrocarbon chain saturation. The extent of normal lens lipid hydrocarbon chain order increased with age from the equatorial and cortical regions to the nucleus. These order data support the hypothesis that the degree of lipid hydrocarbon order is determined by the amount of lipid saturation, as regulated by the content of saturated sphingolipid. Products of lipid oxidation (including lipid hydroxyl, hydroperoxyl, and aldehydes) and lipid disorder increased only in the nuclear region of lenses after 30 HBO treatments, compared with control lenses. Enhanced oxidation correlated with the observed loss of transparency in the central region. HBO treatment in vivo appeared to accelerate age-related changes in lens lipid oxidation, particularly in the nucleus, which possesses less antioxidant capability. CONCLUSIONS: Oxidation could account for the lipid compositional changes that are observed to occur in the lens with age and cataract. Increased lipid oxidation and hydrocarbon chain disorder correlate with increased lens nuclear opacity in the in vivo HBO model.

Role of small GTP-binding proteins in lovastatin-induced cataracts.

PURPOSE: To investigate the biochemical mechanisms involved in the cataract induced by lovastatin, a commonly used cholesterol-lowering agent. METHODS: The effects of lovastatin on lens transparency and on lens epithelial cell proliferation and structure have been investigated using organ-cultured rat lenses and cultured epithelial cells from human and rabbit lenses, respectively. Lens histologic and morphologic changes were recorded microscopically. Small GTP-binding protein profiles were determined by [alpha-32P] GTP overlay assays. RESULTS: Rat lenses organ cultured for 7 days with lovastatin, a 3-hydroxy-3-methylglutaryl CoA reductase inhibitor, developed frank subcapsular opacity. Lens epithelial cells (both human and rabbit) demonstrated extensive morphologic changes and inhibition of proliferation when treated with lovastatin. Histologic sections of lovastatin-treated lenses showed partial to complete degeneration of the central epithelium, distortion of elongating epithelial cells, and extensive vacuole formation in the equatorial regions of the cortex. Supplementation of the medium with DL-mevalonic acid (a precursor of isoprenoids whose synthesis is inhibited by lovastatin) prevented the lovastatin-induced changes in whole lenses or in lens epithelial cell cultures, whereas supplementation with cholesterol had no such effect. GTP-binding proteins accumulated in the soluble fractions of lovastatin-treated lens epithelial cells. This was consistent with a blockade in isoprenylation preventing normal association with membranes. CONCLUSIONS: The findings suggest that impairment of the function of small GTP-binding proteins, due to a lovastatin-induced blockade in their isoprenylation, affects lens cell structure and proliferation in tissue culture and induces lens opacity in organ culture. These findings are consistent with the proposed roles of small GTP-binding proteins as molecular switches that regulate fundamental cellular processes, including growth, differentiation, and maintenance of cell structure.