Autophagy supports survival and phototransduction protein levels in rod photoreceptors.

Damage and loss of the postmitotic photoreceptors is a leading cause of blindness in many diseases of the eye. Although the mechanisms of photoreceptor death have been extensively studied, few studies have addressed mechanisms that help sustain these non-replicating neurons for the life of an organism. Autophagy is an intracellular pathway where cytoplasmic constituents are delivered to the lysosomal pathway for degradation. It is not only a major pathway activated in response to cellular stress, but is also important for cytoplasmic turnover and to supply the structural and energy needs of cells. We examined the importance of autophagy in photoreceptors by deleting the essential autophagy gene Atg5 specifically in rods. Loss of autophagy led to progressive degeneration of rod photoreceptors beginning at 8 weeks of age such that by 44 weeks few rods remained. Cone photoreceptor numbers were only slightly diminished following rod degeneration but their function was significantly decreased. Rod cell death was apoptotic but was not dependent on daily light exposure or accelerated by intense light. Although the light-regulated translocation of the phototransduction proteins arrestin and transducin were unaffected in rods lacking autophagy, Atg5-deficient rods accumulated transducin-α as they degenerated suggesting autophagy might regulate the level of this protein. This was confirmed when the light-induced decrease in transducin was abolished in Atg5-deficient rods and the inhibition of autophagy in retinal explants cultures prevented its degradation. These results demonstrate that basal autophagy is essential to the long-term health of rod photoreceptors and a critical process for maintaining optimal levels of the phototransduction protein transducin-α. As the lack of autophagy is associated with retinal degeneration and altered phototransduction protein degradation in the absence of harmful gene products, this process may be a viable therapeutic target where rod cell loss is the primary pathologic event.

PKCη promotes senescence induced by oxidative stress and chemotherapy.

Senescence is characterized by permanent cell-cycle arrest despite continued viability and metabolic activity, in conjunction with the secretion of a complex mixture of extracellular proteins and soluble factors known as the senescence-associated secretory phenotype (SASP). Cellular senescence has been shown to prevent the proliferation of potentially tumorigenic cells, and is thus generally considered a tumor suppressive process. However, some SASP components may act as pro-tumorigenic mediators on premalignant cells in the microenvironment. A limited number of studies indicated that protein kinase C (PKC) has a role in senescence, with different isoforms having opposing effects. It is therefore important to elucidate the functional role of specific PKCs in senescence. Here we show that PKCη, an epithelial specific and anti-apoptotic kinase, promotes senescence induced by oxidative stress and DNA damage. We further demonstrate that PKCη promotes senescence through its ability to upregulate the expression of the cell cycle inhibitors p21(Cip1) and p27(Kip1) and enhance transcription and secretion of interleukin-6 (IL-6). Moreover, we demonstrate that PKCη creates a positive loop for reinforcing senescence by increasing the transcription of both IL-6 and IL-6 receptor, whereas the expression of IL-8 is specifically suppressed by PKCη. Thus, the presence/absence of PKCη modulates major components of SASP. Furthermore, we show that the human polymorphic variant of PKCη, 374I, that exhibits higher kinase activity in comparison to WT-374V, is also more effective in IL-6 secretion, p21(Cip1) expression and the promotion of senescence, further supporting a role for PKCη in senescence. As there is now considerable interest in senescence activation/elimination to control tumor progression, it is first crucial to reveal the molecular regulators of senescence. This will improve our ability to develop new strategies to harness senescence as a potential cancer therapy in the future.

Association of complement factor H and LOC387715 genotypes with response of exudative age-related macular degeneration to photodynamic therapy.

AIM: To determine whether there is an association between complement factor H (CFH) or LOC387715 genotypes and response to treatment with photodynamic therapy (PDT) for exudative age-related macular degeneration (AMD). METHODS: Sixty-nine patients being treated for neovascular AMD with PDT were genotyped for the CFH Y402H and LOC387715 A69S polymorphisms by allele-specific digestion of PCR products. AMD phenotypes were characterized by clinical examination, fundus photography, and fluorescein angiography. RESULTS: Adjusting for age, pre-PDT visual acuity (VA), and lesion type, mean VA after PDT was significantly worse for the CFH TT genotype than for the TC or CC genotypes (P=0.05). Post-PDT VA was significantly worse for the CFH TT genotype in the subgroup of patients with predominantly classic choroidal neovascular lesions (P=0.04), but not for the patients with occult lesions (P=0.22). For the LOC387715 A69S variant, there was no significant difference among the genotypes in response to PDT therapy. CONCLUSIONS: The CFH Y402H variant was associated with a response to PDT treatment in this study. Patients with the CFH TT genotype fared significantly worse with PDT than did those with the CFH TC and CC genotypes, suggesting a potential relationship between CFH genotype and response to PDT.

Angiostatin produced by certain primary uveal melanoma cell lines impedes the development of liver metastases.

OBJECTIVES: To evaluate the ability of human uveal melanomas to produce angiostatin in vitro and the effect of angiostatin on the development of liver metastases in vivo. METHODS: Human uveal melanoma cell lines (OCM1, OCM3, MEL202, MEL285, 92-1, OM431, and OMM1) were assayed for their ability to produce angiostatin in vitro by an angiostatin bioassay and by Western blot analysis. The OCM3 and OMM1 tumor cells were inoculated either in the posterior or the anterior segment of nude mice. One group of mice in each experiment underwent enucleation and hepatic metastases were assayed by histopathologic and liver function analysis. RESULTS: OCM1, OCM3, and 92-1 cell lines significantly inhibited bovine endothelial cell proliferation in vitro and generated 38-Kd angiostatin molecules. Enucleation of eyes containing OCM3 in the posterior segment resulted in a higher number of metastatic foci (26.5) in that group compared with the nonenucleated group of mice (11.17). After enucleation, elevated levels of serum aspartate transaminase and alanine aminotransferase were observed in mice bearing OCM3 in either anterior or posterior segments. The enucleation of eyes containing OMM1 (nonangiostatin-producing cells) had no significant effect on liver metastasis. CONCLUSION: By removing a source of angiostatin, enucleation of melanoma-containing eyes may unwittingly exacerbate the metastatic potential of uveal melanomas. CLINICAL RELEVANCE: In certain circumstances, enucleating a melanoma-containing eye may unwittingly exacerbate metastatic disease. The results also suggest that exogenous angiostatin may have potential therapeutic implications in the management of patients with primary intraocular melanomas.

Etiology of blindness in an urban community hospital setting.

OBJECTIVE: To determine the cause of monocular and binocular blindness in a predominantly nonwhite urban community hospital setting. DESIGN: Retrospective hospital-based cross-sectional study. PARTICIPANTS: All 3562 unique subjects examined in the New and General Ophthalmology clinic at Parkland Memorial Hospital, Dallas, Texas, from July 1 to September 30, 1998. METHODS: The EYEstation program by Datamedic was queried to conduct a detailed review of electronic medical records of the participants listed previously. MAIN OUTCOME MEASURES: Blindness was defined as visual acuity

Enhancing the immunogenicity of liposomal hepatitis B surface antigen (HBsAg) by controlling its delivery from polymeric microspheres.

Microencapsulated liposome systems (MELs) were investigated as a potential immunization carrier for a recombinant 22-nm hepatitis B surface antigen (HBsAg) particle. MELs were prepared by first entrapping the HBsAg particles within liposomes composed of phosphatidylcholine:cholesterol (1:1 molar ratio), which were then encapsulated within alginate-poly(L-lysine) (PLL) hydrogel microspheres. The entrapped HBsAg particles retained immunoreactivity, as judged by an enzyme-linked immunosorbent assay (ELISA). Direct imaging of HBsAg particles and HBsAg incorporated into liposomes by cryo-transmission electron microscopy (cryo-TEM) indicated that HBsAg is embedded in the liposomal membrane. The antigenic particles were released from MELs mainly within the context of liposomes. The release rates in vitro and in vivo depended on the molecular weight of PLL used for MEL coating; MELs-214, coated with 214 kDa PLL, released the liposomal HBsAg at much higher rates than MELs-25, which was coated with 25 kDa PLL. Concomitantly, the specific anti-HBsAg titers in mice receiving HBsAg in MELs-214 were higher than those induced by MELs-25. MELs-214 were more efficient than conventional liposomes or alum in eliciting higher and prolonged antibody levels in mice. The ability of MELs to provide an HBsAg depot as well as a sustained release of liposomal HBsAg suggests that these carriers may be an ideal immunoadjuvant.

Macrophage activation for the production of immunostimulatory cytokines by delivering interleukin 1 via biodegradable microspheres.

Interleukin 1alpha (IL-1alpha), a pleiotropic cytokine with multiple anti-tumour activities, has been investigated in our laboratory for its potential to serve as an immunotherapeutic agent. In the present study, an attempt was made to direct IL-1alpha to macrophages, in order to induce their immunoregulatory activities. For that purpose, IL-1alpha was encapsulated within biodegradable poly(lactic/glycolic acid) microspheres, 1-5 microm diameter in size. The microspheres were efficiently taken-up by macrophages in culture and after intraperitoneal injection into mice. In culture, phagocytosis of the microspheres reached saturation within 3 h and there was no apparent effect of polymer type on the extent of uptake. In vivo uptake of human IL-1alpha-microspheres by the macrophages lead to cell activation, as evidenced by the enhanced production of murine IL-1alpha, IL-6 and IL-12. Control microspheres, containing bovine serum albumin, induced only background to low levels of cytokine production. These cytokines, when expressed by or secreted from macrophages, may stimulate in situ diverse immune and inflammatory responses, including T cell-mediated immune responses, such as the development of Th(1)cells and cytotoxic lymphocytes. Thus, directing IL-1alpha into macrophages, via the appropriate microspheres, may serve as a unique mean to activate these cells to participate in anti-tumour immune responses in situ.

Interleukin 1alpha activity of peritoneal and bone marrow macrophages infected with Leishmania major and Leishmania donovani in vitro.

In this study, the pattern of interleukin-1alpha (IL-1alpha) production by both peritoneal (PM) and bone marrow macrophages (BMM) from resistant (C3H/HeJ) and susceptible (BALB/c) mice was investigated, using a bioassay and an IL-1alpha-specific ELISA kit. PM from normal uninfected mice showed either an initial high (C3H/HeJ) or a neglected (BALB/c) level of IL-1alpha activity, respectively, probably due to thioglycollate stimulation. Infection with Leishmania major induced only a marginal effect on IL-1 production by both cells. Normal, uninfected and unstimulated BMM from both mice did not produce IL-1alpha over a 7-day period of cultivation in vitro. Upon stimulation with either lipopolysaccharide (LPS) (BALB/c) or concanavalin A (Con A) (C3H/HeJ), both cell types produced IL-1alpha that peaked within the first 12-24 h following stimulation. BMM from C3H/HeJ and BALB/c mice failed to produce IL-1alpha when infected in vitro with L. major or L. donovani promastigotes. However, infection with these two parasites did not interfere with the capability of the host cell to produce IL-1alpha when stimulated with LPS or Con A. The level of IL-1alpha production was independent of the degree of parasitization of the macrophages. Similar results were observed with IL-1beta and IL-6 production by BMM, even though their levels were generally slightly higher than those obtained with IL-1alpha.

Expression of PKCeta in NIH-3T3 cells promotes production of the pro-inflammatory cytokine interleukin-6.

Protein kinase C encodes a family of enzymes implicated in cellular differentiation, growth control and tumor promotion. The generation and characterization of NIH-3T3 cells which stably overexpress the PKCeta isoform has been previously described by us. In these cells, overexpression of PKCeta altered the expression of specific cell cycle regulators and promoted differentiation [20]. Since PKC has been implicated in the regulation of gene expression, including that of various cytokines, we examined the production of several cytokines in these cells. We report here that out of the major pro-inflammatory cytokines examined, IL-1alpha, IL-1beta, TNF-alpha and IL-6, only IL-6 was generated and secreted in PKCeta -expressing cells without any additional inducer in serum-supplemented cultures (10% FCS). IL-6 was not detected in the control cell line, transfected with the same vector, but lacking the cDNA coding for PKCeta. Moreover, the production of IL-6 on serum stimulation correlated with the levels of PKCeta expressed in these cells. This implies that factors in the serum activate PKCeta and induce IL-6 production. We have examined several growth factors and cytokines for their ability to induce IL-6 production in our PKCeta-expressing cells. Among the growth factors tested (EGF, PDGF, FGF, insulin, IGF-1 and IL-1), PDGF and FGF were the most potent IL-6 inducers. The effects of FGF and PDGF on IL-6 production were blocked in the presence of PKC inhibitors. We also examined the signaling pathways that mediate production of IL-6 in PKCeta-expressing cells. Using specific inhibitors of the MAPK pathway, we have shown a role for ERK and p38 MAPK in FGF- and serum-stimulated IL-6 production, but only for p38 MAPK in PDGF-stimulated IL-6 production. Our studies provide evidence that PDGF and FGF can serve as upstream regulators of PKCeta and that PKCeta is involved in the expression of IL-6. This suggests that inhibition of PKC may provide a basis for the development of drugs for the treatment of disorders in which IL-6 is pathologically involved.

Inhibition of intraocular tumor growth by topical application of the angiostatic steroid anecortave acetate.

PURPOSE: This study examined the effect of an angiostatic agent on the growth of a highly vascularized intraocular tumor. METHODS: A murine uveal melanoma cell line (99E1) was transplanted intracamerally into athymic nude BALB/c mice. Mice were treated topically three times per day beginning on the day of tumor transplantation and continuing through day 28. Groups included (a) 1% anecortave acetate, (b) vehicle control, or (c) no treatment. Tumor growth was scored clinically according to the volume of anterior chamber occupied by tumor. Intraocular tumor weights were determined on days 10, 14, 21, and 28. The effect of the test agents on tumor cell proliferation was examined in vitro by [3H]thymidine incorporation. RESULTS: Tumors grew progressively in untreated mice and mice treated with the vehicle; tumors filled the entire eye by day 20 and frequently perforated the globe by day 21. By contrast, tumors treated with anecortave acetate grew significantly slower (P < 0.025) and did not perforate the eye. On days 21 and 28 the net tumor weight of the AL-3789-treated animals was 40% to 30% of controls (P < 0.05). Tumor inhibition was presumably due to the angiostatic properties of anecortave acetate because the compound did not affect tumor cell proliferation in vitro. CONCLUSIONS: The topical ocular administration of anecortave acetate restricted the growth of a highly vascularized angiogenic intraocular tumor.

Spontaneous intracranial hypotension.

PURPOSE: To describe a patient with classic presentation of spontaneous intracranial hypotension and subsequent improvement with targeted epidural blood patch. METHODS: Report of one case and review of the literature. RESULTS: Examination of cerebrospinal fluid after lumbar puncture disclosed a reduced opening pressure, an increased level of protein, and lymphocytic pleocytosis. Magnetic resonance imaging of the brain with gadolinium showed diffuse enhancement of the pachymeninges, no evidence of leptomeningeal enhancement, and chronic subdural fluid collection. Radionuclide cisternography demonstrated reduced activity over the cerebral convexities, early accumulation of radiotracer in the urinary bladder, and direct evidence of leakage at the cervicothoracic junction (C7-T1). Clinical, laboratory, and radiologic features were consistent with the diagnosis of spontaneous intracranial hypotension. Therapy with a targeted epidural blood patch resulted in the rapid resolution of symptoms. CONCLUSIONS: In this report, we describe a classic case of spontaneous intracranial hypotension in a 63-year-old man with an initial presentation of postural headaches, blurred vision, pain in the left eye, diplopia on left gaze, and neck soreness.

Antitumor and immunotherapeutic effects of activated invasive T lymphoma cells that display short-term interleukin 1alpha expression.

Expression of cytokines in malignant cells represents a novel approach for therapeutic treatment of tumors. Previously, we demonstrated the immunostimulatory effectiveness of interleukin 1alpha (IL-1alpha) gene transfer in experimental fibrosarcoma tumors. Here, we report the antitumor and immunotherapeutic effects of short-term expression of IL-1alpha by malignant T lymphoma cells. Activation in culture of T lymphoma cells with lipopolysaccharide-stimulated macrophages induces the expression of IL-1alpha. The short-term expression of IL-1alpha persists in the malignant T cells for a few days (approximately 3-6 days) after termination of the in vitro activation procedure and, thus, has the potential to stimulate antitumor immune responses in vivo. As an experimental tumor model, we used the RO1 invasive T lymphoma cell line. Upon i.v. inoculation, these cells invade the vertebral column and compress the spinal cord, resulting in hind leg paralysis and death of the mice. Activated RO1 cells, induced to express IL-1alpha in a short-term manner, manifested reduced tumorigenicity: approximately 75% of the mice injected with activated RO1 cells remained tumor free. IL-1 was shown to be essential for the eradication of activated T lymphoma cells because injection of activated RO1 cells together with IL-1-specific inhibitors, i.e., the IL-1 receptor antagonist or the M 20 IL-1 inhibitor, reversed reduced tumorigenicity patterns and led to progressive tumor growth and death of the mice. Furthermore, activated RO1 cells could serve as a treatment by intervening in the growth of violent RO1 cells after tumor take. Thus, when activated RO1 cells were injected 6 or 9 days after the inoculation of violent cells, mortality was significantly reduced. IL-1alpha, in its unique membrane-associated form, in addition to its cytosolic and secreted forms, may represent a focused adjuvant for potentiating antitumor immune responses at low levels of expression, below those that are toxic to the host. Further assessment of the immunotherapeutic potential of short-term expression of IL-1alpha in activated tumor cells may allow its improved application in the treatment of malignancies.

Cutting edge: role of macrophage migration inhibitory factor in inhibiting NK cell activity and preserving immune privilege.

The absence of MHC class I Ags on the corneal endothelium, which lines the anterior chamber of the eye, makes this cell layer potentially vulnerable to lysis by NK cells. However, aqueous humor (AH), which bathes the corneal endothelium, contains a 12-kDa protein which inhibits the NK-mediated lysis of corneal endothelial cells. An amino acid sequence analysis of AH revealed that this factor shared >90% homology with macrophage migration inhibitory factor (MIF). The NK inhibitory effect of AH was neutralized with anti-human MIF Ab. Moreover, mouse rMIF produced a similar inhibition of NK cell activity. However, neither rMIF nor AH inhibited the CTL-mediated Lysis of allogeneic cells. rMIF prevented the release of perforin granules by NK cells but not CTLs. Although MIF displays proinflammatory properties, these results indicate that it can also inhibit at least one immune effector element, NK cells, and thereby contribute to immune privilege in the eye.