Ординатура / Офтальмология / Английские материалы / Ocular Disease Mechanisms and Management_Levin, Albert_2010

retinal neurons.92 The intensity of light needed to drive the retinal neurons was in fact similar to the brightness of the summer sun at noon.93 Most subjects reported improvement in acuity, sensitivity, visual field size, or motion perception. Chow’s group then reported that the visual benefits experienced by their patients resulted from a “trophic” effect that was unrelated to point-to-point electrical stimulation.94 The trophic effect might be similar to that originally discovered in experiments on retinal transplantation, that is believed to be related to a soluble factor produced by the RPE.95–98 Perhaps any type of subretinal implant might benefit from some tropism that might occur secondary to the surgery to introduce the device.

Second Sight Medical Products

This company is now in a second phase I trial of their second-generation device, which utilizes modified electronics from a cochlear prosthesis.

Second Sight has performed two types of psychophysical experiment on their chronically implanted patients. The first type of experiment used stimuli unrelated to the environment but allowed the researchers to study the relationship between stimulus parameters and visual perception. The second type of experiment assessed the degree to which patients could appreciate the presence of objects imaged by a camera within their environment. With the first set of studies, electrical stimulation of the retina allowed patients to identify: (1) which of the two electrodes had been activated in a two-point discrimination task; (2) the direction of “movement” when electrodes were activated sequentially; and (3) whether electrodes were activated in rows or columns.31,89,99,100 With the second type of experiment, the subjects failed to perform better than chance when they had to keep their head stationary while a bright object was moved across the visual field. However, when allowed to scan the visual field, the subjects could variably: (1) identify which quadrant the object was in; (2) count the number of objects presented on a screen; (3) identify the orientation of the long limb of the letter “L”; and (4) choose between a knife, cup, and plate that were randomly presented on a flat

surface.31,89,99,100

Patients are now being allowed to use the secondgeneration device outside the laboratory. Scientific data have not yet been presented on this device, although nonscientific statements by some of the patients are clearly very positive.

Intelligent Medical Implants

Intelligent Medical Implants (IMI, based in Zug, Switzerland) utilizes a microfilm epiretinal implant introduced through the pars plana.101 Their first-generation electrode array included 19 electrodes; the current array includes 49 electrodes. The initial publication of their human studies reported that the patients were able to ascertain different impressions of their brightness, shape, color, and duration of stimuli, and with practice, the patients could differentiate the relative localization of stimuli, recognize basic patterns such as lines and spots, and detect motion. In their acute trial consisting of 20 RP patients, visual percepts appeared

What has been achieved to date?

like little stars, circles, triangles, rectangles, half-moons, and solar eclipses in white, yellow, and blue colors, and had the brightness of candle light or lamp.88 IMI is conducting a multicenter trial across Europe.

Retina Implant AG

This company emerged from research led by E. Zrenner, based in Tübingen, Germany. Their implant contains 1500 light-sensitive elements that are designed to act as a photodetecting array and a 4 × 4 array of hard-wired electrodes. Like the Second Sight device, hard-wired electrodes are connected to the electronic elements positioned behind the ear. The surgical group, led by Helmut Sachs, has been successful in (at least) 7 of 7 patients using a transchoroidal approach to the subretinal space. This surgical approach is more challenging (but potentially more advantageous) compared to the pars plana approach in which foreign elements are left in position toward the front of the eye, as this latter technique can predispose to extrusion of the devices through the conjunctiva and possibly an infection. One patient has elected to keep his device long-term and has provided very useful psychophysical information.

Psychophysical results have mostly been obtained from the 16 hard-wired electrodes. Four of the 7 patients reported some degree of pattern recognition and 2 of these patients reported the perception of pea-sized objects with singleelectrode stimulation.102 Patients were able to report variation of brightness and sizes of percepts with varying strength of stimulation, orientation of horizontal and vertical lines, the relative position of electrodes, and the direction of movement following sequential activation of the electrodes.87,103,104

Experiments conducted by photic activation of the photodiode array allowed 3 patients to perceive light in certain shapes and patterns. Some patients detected single spots of light of only 100–400 µm diameter. One patient was able to locate white dinner plates on a dark tablecloth.103

Optic nerve prostheses

Veraart and coworkers have performed implants of a cuff with four electrodes around the optic nerves of two RP patients. Using a camera-based system; their patients reported some characteristics of the electrically induced phosphenes such as their shape, size, basic structure, location, and brightness. These researchers reported an 87% success rate with pattern recognition of basic forms, but with a relatively long time delay of 53 seconds for patients to discern the perception of objects. With training, the subjects learned to scan more effectively, discriminate objects on a table (e.g., cup, eating utensil) more quickly, and were able to reach out and grasp them. The appearance of the phosphenes could be modified by varying the strength of the electrical pulses and one patient reported being able to generate a letter “C” by stimulating in a particular way through three electrodes.30,105,106 More recently, a Chinese program was formed under the direction of Drs. Lee (surgeon) and Q. Ren (engineer) to develop an optic nerve prosthesis with penetrating electrodes.

Summary of human implantation with retinal prosthetic devices

The collective outcome of human testing for visual prosthetic devices has demonstrated that: (1) patients who have been legally blind for decades can reliably see phosphenes in response to electrical stimulation; and (2) modulation of electrical stimulation can create multiple images that are geometrically similar to the pattern of electrical stimulation. Although the induced percepts have generally been crude, the more recent chronic experiments have shown that blind patients can detect large moving objects, identify large letters by scanning, and report the orientation of lines generated by electrical stimulation. These outcomes, which have been found to a similar degree by multiple groups, are one of the most encouraging aspects of the psychophysical results obtained to date. It is also notable, however, that no retinal group has yet provided a sufficiently detailed description of their tests (including number of raw trials, standard errors, false-positive and false-negative responses across a reasonably large number of trials) to allow an independent researcher the opportunity to assess the validity of the conclusions of their published works.

The variability in testing methods also makes it difficult to draw comparisons of the qualitative differences in results across groups. One less encouraging result from the collective body of work is the general lack of very obvious improvements in the spatial quality of perception over the last couple of years. Perhaps this is just the result of the normal delay in reporting, or perhaps the field has hit a psychophysical plateau.

The challenge of creating “useful” and spatially more detailed visual perceptions at least partly relates to the need to develop substantially better knowledge about improved methods for stimulating the afferent visual pathway. In this regard, the status of the visual prosthetic projects is not unlike the history of cochlear implants, which required many years of work to discover methods that ultimately produced great success in helping many deaf patients. Overall, the results of human testing to date support the hope that artificial stimulation will one day provide “useful” vision for the blind. It should be remembered that, for severely blind patients, even crude visual assistance that would help patients navigate more safely in an unfamiliar environment would be a substantial improvement in their quality of life.

1.Rizzo JF 3rd, Wyatt J, Humayun M,

et al. Retinal prosthesis: an encouraging first decade with major challenges ahead. Ophthalmology 2001;108:13.

3.Congdon N, O’Colmain B, Klaver CC, et al. Causes and prevalence of visual impairment among adults in the United States. Arch Ophthalmol 2004;122:477– 485.

7.Adler R, Curcio C, Hicks D, et al. Cell death in age-related macular degeneration. Mol Vision 1999;5:31.

17.Jones BW, Marc RE. Retinal remodeling during retinal degeneration. Exp Eye Res 2005;81:123–137.

29.Rizzo JF 3rd, Wyatt J, Loewenstein J, et al. Methods and perceptual thresholds for short-term electrical stimulation of human retina with microelectrode arrays. Invest Ophthalmol Vis Sci 2003;44:5355– 5361.

30.Veraart C, Wanet-Defalque MC, Gerard B, et al. Pattern recognition with the optic nerve visual prosthesis. Artif Organs 2003;27:996–1004.

31.Mahadevappa M, Weiland JD, Yanai D, et al. Perceptual thresholds and electrode impedance in three retinal prosthesis subjects. IEEE Trans Neural Syst Rehabil Eng 2005;13:201–206.

41.Rizzo JF, Wyatt JL. Retinal prosthesis. In: Berger J, Fine S, Maguire M (eds) Age-Related Macular Degeneration. St. Louis, MO: Mosby, 1999:429–430.

51.Bainbridge JW, Smith AJ, Barker SS, et al. Effect of gene therapy on visual function in Leber’s congenital amaurosis. N Engl J Med 2008;358: 2231–2239.

65.LaVail MM, Unoki K, Yasumura D, et al. Multiple growth factors, cytokines, and neurotrophins rescue photoreceptors from the damaging effects of constant light. Proc Natl Acad Sci USA 1992;89: 11249–11253.

74.Dobelle WH, Mladejovsky MG. Phosphenes produced by electrical stimulation of human occipital cortex, and their application to the development of a prosthesis for the

blind. J Physiol 1974;243:553– 576.

78.Rizzo J. Embryology, anatomy and physiology of the afferent visual pathway. In: Miller NR, Newman NJ, Kerrison JB, et al (eds) Walsh and Hoyt’s Clinical Neuro-Ophthalmology. Philadelphia, PA: Lippincott Williams and Wilkins, 2005:3–82.

88.Hornig R, Zehnder T, Velikay-Parel M, et al. The IMI retinal implant system. In: Humayun MS, Weiland JD, Chader G, et al (eds) Artificial Sight Basic Research, Biomedical Engineering, and Clinical Advances. New York: Springer, 2007:111–128.

89.Yanai D, Weiland J, Mahadevappa M, et al. Visual performance using a retinal

prosthesis in three subjects with retinitis pigmentosa. Am J Ophthalmol 2007;5:820–827.

103.Zrenner E. Restoring neuroretinal function: new potentials. Doc Ophthalmol 2007;115:56–59.

Overview

Paraneoplastic retinopathies (PRs) represent visual dysfunctions and retinal degeneration associated with known or suspect malignancies, without direct involvement of the eye. PRs are believed to originate from an autoimmune process involving circulating autoantibodies elicited against cancer antigens that also recognize retinal antigens. PRs include cancer-associated retinopathy, melanoma-associated retinopathy, and bilateral diffuse uveal melanocytic proliferation syndrome (Table 76.1).

Clinical background

Key symptoms and signs

Cancer-associated retinopathy (CAR) syndrome

CAR was first recognized as a paraneoplastic disorder, in association with small cell lung cancer (SCCL) and retinal dysfunction and degeneration. Besides SCCL, CAR occurs with other associated tumors, mostly carcinomas of the breast, endometrium, ovary, colon, bladder, and prostate,1 as well as lymphomas and thymoma.

In CAR, the progression of retinal degeneration is examined by electroretinogram (ERG), visual field, and fundus examination.2 Patients frequently describe the sudden onset of a sensation of flickering or shimmering lights and night blindness, with additional visual loss over weeks to several months. Rod dysfunction is characterized by night blindness and prolonged dark adaptation. Cone degeneration features include decreased visual acuity, sensitivity to light and glare, reduced color vision, and central and ring scotomas. The fundus can initially appear normal, but narrowed retinal arteries, retinal pigment epithelial mottling, and optic nerve pallor can be observed on ophthalmic examination. Vision loss is associated with decreased responses of cone and rod responses. Ocular symptoms may precede the diagnosis of cancer by months to years; thus, recognition of CAR facilitates early diagnosis of malignancy.

Antiretinal CAR autoantibodies are detected in blood by Western blotting (Figure 76.1).1 The most recognized autoantibodies are against recoverin and α-enolase, but

Paraneoplastic retinal degeneration

Grazyna Adamus

autoantibodies with other specificities can also be detected (Table 76.2). Patients with different antiretinal autoantibodies may have different clinical manifestations (Box 76.1). Antirecoverin CAR presents with symptoms of night blindness, photopsias, loss of peripheral or pericentral visual field, reduced central acuity, and widespread rod and cone dysfunction.2 Anti-α-enolase CAR presents with varying degrees of central or pericentral visual field loss, shimmering photopsias, loss of color vision, reduced vision in bright light, and night blindness.3 Thus, autoantibodies can serve as a biomarker for the prognosis and management of CAR.

Melanoma-associated retinopathy (MAR) syndrome

MAR is associated with visual loss, antiretinal autoantibodies, and diagnosed cutaneous malignant melanoma, often at the end-stage of metastasis.4 MAR was first described in 1988 by Berson and Lessell.5 Patients with MAR have nearly normal vision but develop night blindness, shimmering light, photopsia, and loss of peripheral vision that appear months to years after diagnosis of a tumor. The average time from the diagnosis of cancer to diagnosis of MAR averages 3.6 years.4 The typical ERG pattern in MAR shows a slight reduction in scotopic a-wave, and a marked reduction or absence of dark-adapted b-wave (Box 76.2).6 Autoantibodies against bipolar cells were first reported in patients with MAR,7 and recently, autoantibodies against other retinal proteins have also been found in these patients (Table 76.2).

Autoimmune retinopathy (AR)

Patients can have symptoms resembling CAR or MAR, and antiretinal autoantibodies, but have no tumor at the time of initial evaluation. AR is the preferred term for this acquired autoimmune-mediated condition (Table 76.1). The presence of autoantibodies without known malignancy leads to several speculations on the origin of autoimmune responses. Most likely, they originate from a very small tumor, too small to be detected by conventional methods, but the immune system detects it. The tumor might be detected clinically in the subsequent years. The length of the diagnostic follow-up period necessary to detect the tumor in clinical PR is under investigation. Thus far, it ranges from months up to 5 years, but it can be longer.1 Antirecoverin antibodies are typically associated with CAR and have rarely been reported in AR patients. In such cases, antiretinal autoanti-

Figure 76.1  Autoantibody testing by Western blotting and immunocytochemistry. (A) Western blotting showing immunostaining of human retinal proteins by patient’s serum: 1, molecular standards; 2, retinal proteins; 3, human serum recognizing α-enolase; 4, human serum recognizing recoverin on the blot. (B) Immunohistochemistry: antirecoverin serum antibodies bind to photoreceptor cell layer (outer retina), and anti-α-enolase serum binds to the inner and ganglion cell layers, and also stains neuronal fibers. OS, outer segments; ONL, outer nuclear layer; INL, inner nuclear layer; GCL, ganglion cell layer.

Box 76.1  Anti-retinal autoantibodies

Different autoantibodies, such as antirecoverin and anti-α- enolase, can be associated with different clinical presentations, phenotypic findings, and electroretinogram patterns

Box 76.2  Paraneoplastic retinopathies

Electroretinograms, visual field analysis, and autoantibody detection, in combination with an evaluation for a systemic malignancy, are essential for diagnosing cancerand melanomaassociated retinopathy

bodies may be involved in tumor regression. This “new class” of retinopathies with autoantibodies cannot be ignored and needs to be followed up in ophthalmic examination. The condition can be classified as CAR or AR (= probable PR), based on clinical symptoms, the presence or absence of autoantibodies, and the presence or absence of cancer (Box 76.3).

Bilateral diffuse uveal melanocytic proliferation (BDUMP)

BDUMP is a rare syndrome that involves painless, bilateral vision loss, serous retinal detachment, and a systemic malignant neoplasm. Visual loss is due to the proliferation of

uveal melanocytes occurring throughout the uvea without metastasis outside the eye.8 The most common tumors are ovary and uterus in women and the lung in men. Most patients die from metastasis of their primary tumor within a year of BDUMP diagnosis. Autoantibodies against retinal or uveal antigens are not usually tested.

Historical development

In 1976, Sawyer et al9 first described PR as the progressive loss of vision with ring scotomas and nearly absent ERG in 3 women with SCCL. Interestingly, visual symptoms preceded the cancer diagnosis, leading them to postulate that

Box 76.3  Autoimmune retinopathy

Patients can have symptoms resembling canceror melanomaassociated retinopathy and antiretinal autoantibodies, but without diagnosed tumor

Box 76.4  Diagnostic autoantibodies

Various antiretinal autoantibodies can be found in sera from patients with paraneoplastic retinopathies; thus, the absence of autoantibodies against recoverin (23 kDa) does not exclude a paraneoplastic retinopathy diagnosis

photoreceptor degeneration was caused by remote effects from the cancer. In later years, circulating antibodies against retinal proteins were reported in sera from cancer patients with visual problems, suggesting that antiretinal autoantibodies may play a role in PR pathogenicity.10,11 Immuno­ staining of the outer retina with CAR antibodies further supported the autoimmune nature of the syndrome, as did beneficial effects of steroids on vision. The hypothesis was that vision loss was due to serum autoantibodies, originating from the immune response against tumor antigens and cross-reacting with retinal proteins, which led to rod and cone dysfunction. The syndrome was named “cancerassociated retinopathy,” and the associated 23-kDa reactive protein was called “the CAR antigen.”11 The CAR antigen has since been sequenced and identified as “recoverin.”12–14 However, 50% of symptomatic patients have high titers of autoantibodies against proteins other than recoverin (Table 76.2).1 Therefore, the absence of antibodies to recoverin does not exclude a diagnosis of PR (Box 76.4).

Epidemiology

PRs are rare. It is estimated that paraneoplastic diseases occur in fewer than 1% of cancer patients.15 Although more PR cases have been reported in recent years and the number is growing, the incidence of CAR and MAR remains unknown. Moreover, most patients with cancers, including melanoma, may not consider their vision symptoms due to their malignancy, and consequently, they do not report vision problems. The average age of a CAR patient is 63 years, with women affected more than men at a ratio of 2 : 1.1 At an average age of 57.5 years, males with melanoma appear to have a higher risk of developing MAR than women, even though cutaneous malignant melanoma affects men and women equally.4,16 AR affects twice as many women as men, at an average age of 55. BDUMP is very rare and also affects more women than men, at a ratio of 2 at an average of 63 years.8 Thus far, there is no known genetic involvement in CAR and MAR development.

Diagnostic workup

Diagnosis of PRs is difficult (Table 76.3), and patients are frequently misdiagnosed. Ocular symptoms may develop

Table 76.3  Clinical criteria for paraneoplastic retinopathy

1.Unexplained, painless, and progressive retinal dysfunction

2.Abnormal electroretinogram

3.Patients may have the following clinical symptoms (these symptoms may be restricted or multifocal):

a.Photopsia

b.Blurred vision

c.Progressive worsening of visual acuity and visual fields

d.Ring scotomas

4.Suspected or diagnosed cancer

5.Exclusion criteria

a.No known genetic (familial) causes

b.No ocular infection

c.No ocular trauma

d.No intraocular surgery (other than cataract surgery)

e.No drug toxicity

f.No retinal detachment

g.No typical age-related macular degeneration

h.No active ocular inflammation

Table 76.4  Enolase in disease

1.Autoantibodies against α-enolase reported in inflammatory, degenerative, and psychiatric disorders

2.Anti-α-enolase antibodies associated with systemic and invasive autoimmune disorders

3.High incidence of autoantibodies in autoimmune diseases:

a.Cancer-associated retinopathy (68.8%, 11 of 16)

b.Polyglandular syndrome type 1 (80%, 35 of 44)

c.Primary (69%, 60 of 87) and secondary (58%, 14 of 24) membranous nephropathy

d.Autoimmune hepatitis type 1 (60%, 12 of 20)

e.Mixed cryoglobulinemia with renal involvement (63.6%, 7 of 11)

f.Cystoid macular edema (60%, 6 of 10)

g.Endometriosis (50%, 21 of 41)

h.Rheumatoid arthritis (25%, 36 of 145)

4.Healthy subjects have autoantibodies ranging from 0% (0 of 91) to 11.7% (7 of 60)

Box 76.5  Diagnostic criteria

Based on clinical presentation and findings, and on the presence or absence of antibodies and cancer, patients can be classified as having a paraneoplastic retinopathy or autoimmune retinopathy (= possible paraneoplastic retinopathy)

before diagnosis of cancer, and CAR and MAR can occur in patients without tumors (Table 76.1). About 50% of patients have retinopathy symptoms as the first manifestation.17 Nearly 50% of symptomatic patients initially have negative antibody tests, and few of the seropositive patients have antirecoverin antibodies.1 Importantly, the presence of antirecoverin autoantibodies indicates a high likelihood of associated neoplasm, particularly SCCL and gynecological cancers in women (Box 76.5). Anti-α-enolase CAR occurs predominantly with cancers with endocrine features, and visual symptoms typically develop months to years after discovery of the malignancy. Antienolase autoantibodies have also been reported in a diverse range of inflammatory, degenerative, and psychiatric disorders (Table 76.4), and in

~10% of normal subjects (0–11%).18,19 The production of MAR autoantibodies has rarely been linked to the remission or stabilization of melanoma, indicating that an antibody response is usually insufficient to protect against spreading. If ophthalmologic findings, including ERG and antibodies, are indicative of CAR and MAR, a cancer workup is recommended. A worsening of symptoms may precede a recurrence or metastasis.

Treatment

There is no established protocol for PR treatment. Without treatment, antirecoverin CAR almost always progresses rapidly to severe vision loss, often to no light perception. Management of CAR is generally ineffective, although some patients benefit from systemic corticosteroids, plasmapheresis, or intravenous immunoglobulins (IVIg).20–22 Recently, a beneficial effect from alemtuzumab (anti-CD52 monoclonal antibody) has been reported.23 In MAR patients, prednisone seems not to improve ocular symptoms, and plasmapheresis reduces antibody titer without improving vision.4 Clinical trials evaluating the benefits of IVIg therapy, alone or with corticosteroids, are needed to manage PRs and AR better.

Prognosis and complications

Prognosis depends on the type of serum autoantibodies present, the underlying tumor, and its stage of development. Patients with antirecoverin antibodies have the worst prognosis because their disease is typically progressive and advances to complete loss of vision. However, there are patients with antirecoverin autoantibodies whose retinal degeneration follows a slow course. Unlike antirecoverin CAR, which often precedes a cancer diagnosis by months to over a year, PR associated with anti-α-enolase autoantibodies usually develops months to years after the discovery of the malignancy and accompanies a slower vision loss with bouts of stable vision.3 MAR patients have the worst survival prognosis24 because MAR presents after the melanoma is diagnosed, often at the metastatic stage.

Pathology

In histopathological studies of CAR, postmortem eyes revealed extensive rod and cone degeneration.10,25,26 The striking disappearance of photoreceptor cells and of the outer nuclear layer (ONL) in the retina was evident, but the inner retina was preserved (Figure 76.2). No or very low inflammation in the uvea, retina, and vitreous was found, suggesting a noninflammatory aspect to retinal degeneration. The molecular mechanism by which cell loss occurs in the retina will be described later in this chapter. Histopathological examination of postmortem MAR eyes showed a marked reduction in the density of bipolar neurons in the inner nuclear layer (INL), but photoreceptor cell neurons in the ONL were normal. Retinal ganglion cells were present, although many showed evidence of transsynaptic atrophy. These histopathological changes were consistent with clinical, immunologic, and electrophysiologic findings that implicate photoreceptor or bipolar cells as the site of the paraneoplastic process in PRs.27

Etiology

PR etiology and the source of antigenic stimulation are largely unknown. The first cases of CAR were associated with SCCL. Since recoverin is a photoreceptor-specific, calciumbinding protein, there was a question as to whether autoantibodies that bind to the photoreceptor recoverin bind to a “cross-reactive antigen” in tumor tissue. Indeed, unaltered recoverin was expressed in malignant cells of SCCL in a CAR patient who had circulating antirecoverin autoantibodies.28 This study strongly implicated an immune mechanism initiated by antitumor recoverin responses in a subset of patients.29 Recoverin is expressed in ~70% of lung cancer tissue, not only in tumors of CAR patients but also in tumors of patients without ocular symptoms.30–32 Serum autoantibodies were present in 20% of lung cancer patients, and CAR occurred in only 1% of these patients, suggesting that recoverin expression is not strongly coupled with cancer cell differentiation.29 Recoverin can be found in tumors during neoplastic development, and this might be related to the

location of the recoverin gene at chromosome 17p13.1, in proximity to the tumor suppressor gene p53.33 A single mutation may activate the recoverin gene and deactivate the p53 gene, in effect causing the aberrant expression of recoverin in tumor cells. During tumor turnover, the immune system could elicit antirecoverin immune responses. Thus, autoantibodies originating against tumor recoverin may cross-react with the photoreceptor recoverin, causing immune-mediated photoreceptor degeneration. Identification of immunogenic, tumor-associated molecules is not only a crucial step in understanding molecular mechanisms of CAR, but also in understanding the role of these molecules in clinical diagnosis or therapy.

α-Enolase is a common autoantigen found in the retina and in malignant cells in lung cancer.34 Autoantibodies against α-enolase have been found in 13.8–65% of patients with different subtypes of lung cancer. Such a high enolase overexpression and high autoantibody levels usually correlate with a poor prognosis.34,35 Moreover, the triggering of distinct immune responses by tumor antigens emerging during the course of disease has been observed in a CAR patient.36 Initially, the serum showed antiretinal 35-kDa autoantibodies before SCCL surgery, antiretinal 35-kDa and 46-kDa (α-enolase) autoantibodies 1 week after surgery, and only antienolase 1 month after surgery. These findings suggest “epitope spreading,” which refers to the development of an immune response to epitopes, distinct from and noncross-reactive with the initial disease-causing epitope.37 Epitope spreading enhances the heterogeneity and pathogenicity of CAR antibodies and can only be discovered in follow-up examination.

Antibipolar cell autoantibodies were originally demonstrated in MAR,7 but an autoantigen in the bipolar cell was not identified. In addition, MAR autoantibodies against photoreceptor proteins were detected in a subset of melanoma patients (Table 76.2). Photoreceptor proteins – such as rhodopsin, transducin, cGMP-phosphodiesterase 6, cGMPdependent channels, guanylyl cyclase, rhodopsin kinase, recoverin, and arrestin – can be expressed in melanoma cells, which can potentially induce autoantibody responses in melanoma patients, leading to an immune attack on the retina.38 Taken together, these findings provide a sound foundation for immunological mechanisms of PRs (Box 76.6).

Pathophysiology

In the last several years, studies of PR pathological mechanisms have been undertaken by several groups. Based on clinical presentation, presence of autoantibodies and extraocular tumors, a possible mechanism causing retinal degeneration has been proposed, in which serum autoantibodies against recoverin and other retinal antigens play a

Box 76.6  Cancer and retinopathy

Paraneoplastic retinopathies are autoimmune-mediated disorders triggered by aberrant expression of retinal antigens in distant tumors

central role in pathogenicity. The molecular pathology of CAR caused by autoantibody occurs in two steps (Figure 76.3): (1) antitumor response, involving an immune response that is elicited against aberrantly expressed antigens in tumor, during which the autoantibodies, when they reach high titers, gain access to the retina through the blood–retina barrier; and (2) antiretinal response, involving autoantibodies penetrating retinal layers and being taken up into target cells, where they block the target antigen metabolic function, which in effect induces apoptosis and cell death. Because recoverin and α-enolase have been initially linked to CAR,

Bax

Bax

Bcl-2 Bcl-x

Cyt c

Apaf-1 Cyt c

Cyt c Pro-caspase 9

Cell death

Figure 76.3  Illustration depicting molecular aspects of apoptosis in retinal cells induced by autoantibodies. Antibody entry is necessary to start the apoptotic process in retinal cells. After antirecoverin and anti-α-enolase antibodies translocate to the cytoplasm by endocytosis, they bind to the appropriate autoantigen in the cytoplasm and block the target antigen metabolic function, in effect leading to an increase in intracellular Ca2+. Blocking of the α-enolase function decreases glycolytic adenosine triphosphate (ATP) production and increases intracellular Ca2+. Antirecoverin antibodies inhibit recoverin function, which leads to higher levels of rhodopsin phosphorylation and the continuous opening of cGMP-gated channels, resulting in the accumulation of intracellular Ca2+ within cells. Blocking of other antigen (Ag) function may also lead to intracellular Ca2+. The rise in Ca2+ induces an increase in proapoptotic Bcl-xs and Bax proteins and a decrease in antiapoptotic Bcl-xL and Bcl-2, which is followed by the release of cytochrome c from the mitochondria and the downregulation of apaf-1. Cytochrome c initiates apoptosis by inducing formation of the caspase 9–apaf-1 complex. All of these events correlate with the sequential activation of caspase 9 and caspase 3, as well as the degradation of the caspase substrate poly (ADP-ribose) polymerase (PARP) and the fragmentation of DNA, a hallmark of apoptosis.

Table 76.5  Recoverin as an autoantigen

1.A 23-kDa calcium-binding protein

2.Present in the cytoplasm of photoreceptor and bipolar cells of the retina

3.Regulates (inhibits) rhodopsin phosphorylation in a calcium-dependent manner

4.Also found in various tumors (cancer-associated retinopathy (CAR) and50% non-CAR tumors)

5.Regulates cell proliferations of the tumors (?)

6.A potent antigen and pathogen

7.Induces experimental uveitis in rats

8.The sequence 64–70 in proximity of the first calcium-binding motif EF-hand 2 is highly antigenic and pathogenic

most mechanistic studies have focused on them.39–41 Below we discuss in vitro and in vivo evidence underlying the molecular mechanism of retinal degeneration associated with autoantibodies against recoverin and α-enolase.

Recoverin

Recoverin is a 23-kDa, calcium-binding protein present mostly in photoreceptor cells: it regulates rhodopsin phosphorylation in visual transduction (Table 76.5). Recoverin is highly immunogenic and pathogenic. Two linear stretches of amino acids within the human recoverin sequence form the major epitopes for autoantibodies: residues 48–52 (QFQSI) and residues 64–70 (KAYAQHV) in proximity to the calcium-binding domain EF-hand 2.42 The binding of autoantibodies has been found to be dependent on recoverin calcium-binding properties since conformational changes induced by the bound calcium enhance antibody binding. Moreover, the sequence 64–70 has been found to be a major pathogenic site, causing intraocular inflammation and the degeneration of photoreceptor cells in Lewis rats immunized with recoverin or its peptides.28,43 In addition, this immunogenic sequence induces peptide-specific Th and cytotoxic T-lymphocyte responses.28,44

α-Enolase

Enolase plays a role in glycolysis (Table 76.6). About 40% of retinopathy patients have anti-α-enolase autoantibodies, and half of seropositive patients are cancer survivors.1,18 Anti-α-enolase autoantibodies can also be found in a number of inflammatory, degenerative, and neurologic diseases, and in healthy subjects (Table 76.4).45 Because of such a wide prevalence of antienolase autoantibodies, fine epitope maps were studied in human α-enolase to determine their role in pathogenicity. Epitope mapping revealed three epitopes within the residues 31–38 (FRAAVPSG), 176–183 (ANFREAMR), and 421–428 (AKFAGRNF) for all CAR autoantibodies tested.46 However, 70% of CAR patients, particularly those with breast or bladder cancer, recognized a unique epitope sequence 56–63 (RYMGKGVS).47 The epitope sequences are located in proximity to external loops of the enolase molecule: loop 1: 37–43, the catalytic site; loop 2: 153–166; and loop 3: 251–276, the plasminogen-binding site.45 Depending on specificity, anti-α-enolase autoantibod-

Pathophysiology

Table 76.6  Enolase – multifunctional protein

1.A 46-kDa protein

2.Has glycolytic (metabolic sensor) and nonglycolytic functions (transcriptional regulation)

3.Belongs to a novel class of surface proteins without classical machinery for surface transport

4.Forms three heterodimeric forms with tissue-specific distributions

a.α-enolase, the embryonic form, all tissues (nonneuronal enolase)

b.β-enolase is expressed in skeletal and cardiac muscle

c.γ-enolase in nervous tissue (neuronal-specific enolase)

5.Has multiple locations in the cell (cytosolic, membrane, nuclear)

6.Has 100% homology with myc promoter-binding protein (MBP-1)

7.Serves as a plasminogen receptor on the surface of a variety of hematopoetic, epithelial, and endothelial cells

8.Stimulates immunoglobulin production

9.Functions as a heat shock protein

10.Binds cytoskeletal and chromatin structures

ies may label several layers and cell types within the retina, whereas antirecoverin autoantibodies bind exclusively to rods and cones and to some bipolar cells (Figure 76.2).

Apoptosis of retinal cells

Antiretinal autoantibodies, including recoverin, are cytotoxic to E1A.NR3 retinal cells.48 When living cells are grown with these antibodies, destruction of these cells in a doseand time-dependent manner occurs only in cells expressing the autoantigen.48 Antirecoverin IgG, as well as its Fab fragments, penetrates retinal cells by an active process of endocytosis.39 This antibody-mediated destruction of retinal cells is independent of complement and IgG from normal subjects is not cytotoxic. Importantly, the antibodies induce morphological changes in cells that are characteristic of apoptosis. Moreover, antibodies injected intravenously into Lewis rats penetrated the blood–ocular barrier, reached the retina, and caused the death of some photoreceptors.40 Direct intravitreal injection of antirecoverin autoantibodies into the vitreous of rat eyes caused apoptotic cell death within the outer and bipolar cell layers, as evidenced by DNA fragmentation, nuclear chromatin condensation, and increased vacuolization of photoreceptor outer segments.49 Rats receiving antirecoverin antibodies lost 25–30% of nuclei in the ONL and INL, whereas all controls had unchanged numbers of nuclei. Antibodies against α-enolase that specifically labeled the ganglion cell layer and INL, when injected into the vitreous, penetrated these target layers and, consequently, cell death was induced through an apoptotic process.50 The apoptotic nuclei detected by a DNA fragmentation assay and caspase 3-positive cells were colocalized in these layers. Additional studies showed that when antirecoverin and anti-α-enolase antibodies translocated to the cytoplasm, they induced an increase in proapoptotic Bcl-xS and Bax proteins, and decreased antiapoptotic Bcl-xL which was followed by the release of cytochrome c from mitochondria and the downregulation of apaf-1.39,51 These events correlated with the sequential activation of caspase 9 and caspase 3, as well as the degradation of the

Box 76.7  Apoptosis

Antiretinal autoantibodies are inducers of apoptosis in retinal cells through the mitochondrial pathway involving caspases 9 and 3

caspase substrate poly (ADP-ribose) polymerase (PARP) and the fragmentation of DNA, a hallmark of apoptosis (Box 76.7).39 Thus, autoantibodies are inducers of apoptosis through the mitochondrial pathway involving caspases 9 and 3 (Figure 76.3).

Once antirecoverin antibodies access rod photoreceptor cells, they block recoverin function, which leads to an increase in intracellular free Ca2+.52 This increase and the inhibition of rhodopsin phosphorylation activate caspasedependent apoptotic pathways.41,53 In vivo studies showed that Ca2+-dependent suppression of rhodopsin phosphory­ lation was abolished, and the level of rhodopsin phosphory­ lation was significantly enhanced in the presence of antirecoverin IgG, but not in the presence of normal IgG.41 These effects likely occur from the inhibition of recoverin function, leading to higher levels of rhodopsin phosphorylation and continuous opening of cGMP-gated channels, in turn resulting in an accumulation of intracellular Ca2+ within photoreceptor cells. Therefore, uncontrolled states of the phototransduction pathway and the rise in free Ca2+ by blocking the recoverin function with autoantibodies may be an important, mechanistic step in the degeneration of photoreceptor cells. Also, antienolase antibodies penetrate retinal cells and cause apoptosis through the inhibition of the enolase catalytic function, resulting in the depletion of glycolytic adenosine triphosphate and leading to an increase in intracellular Ca2+.50,51 L-type voltage-gated Ca2+ channel blockers (nifedipine, d-cis-diltiazem, and verapamil) are effective in blocking the antibody-induced intracellular Ca2+ rise and in suppressing proapoptotic Bax. Thus, chronic access of autoantibodies to the retina may result in inhibition of the antigen function and elevation of intracellular

Box 76.8  Calcium and retinopathy

An increase in intracellular free Ca2+ may constitute a common mechanism in photoreceptor cell death induced by different antiretinal autoantibodies

Ca2+, leading to the activation of apoptosis pathways and subsequent cell death.51 Since the increase in intracellular Ca2+ appears to be a key element in the induction of apoptosis, this increase likely constitutes a common mechanism in photoreceptor cell death that is induced by antiretinal autoantibodies (Box 76.8).

In MAR, evidence of autoantibody involvement in bipolar cell inactivation is supported by results obtained from injecting MAR autoantibodies into the vitreous of monkeys, resulting in a transient alteration of ERG patterns, similar to those of MAR.54 The proposed mechanism involves an autoantibody response against yet-to-be-identified melanoma antigens that cross-react with specific antigens in the retina. This immunological reaction causes the depolarization of bipolar cells of rod and cone systems of the ON pathway.7 Immunohistochemistry showed positive staining of the outer plexiform and nerve fiber layers, but the significance of such reactivity in pathogenesis was not explained.4 Also, the aberrant expression of MAR antigens in melanoma may underlie a similar mechanism, as in CAR. The heterogeneity in autoantibody specificity may explain the variation and complexity of clinical symptoms in retinopathy patients.

Acknowledgments

This study was supported by a grant EY13053 from the National Institutes of Health, by an unrestricted grant to the OHSU Casey Eye Institute from Research to Prevent Blindness, New York, NY, and by the Foundation Fighting Blindness.

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