Heritable gene silencing in Drosophila using double-stranded RNA.

RNA-mediated interference (RNAi) is a recently discovered method to determine gene function in a number of organisms, including plants, nematodes, Drosophila, zebrafish, and mice. Injection of double-stranded RNA (dsRNA) corresponding to a single gene into organisms silences expression of the specific gene. Rapid degradation of mRNA in affected cells blocks gene expression. Despite the promise of RNAi as a tool for functional genomics, injection of dsRNA interferes with gene expression transiently and is not stably inherited. Consequently, use of RNAi to study gene function in the late stages of development has been limited. It is particularly problematic for development of disease models that reply on post-natal individuals. To circumvent this problem in Drosophila, we have developed a method to express dsRNA as an extended hairpin-loop RNA. This method has recently been successful in generating RNAi in the nematode Caenorhabditis elegans. The hairpin RNA is expressed from a transgene exhibiting dyad symmetry in a controlled temporal and spatial pattern. We report that the stably inherited transgene confers specific interference of gene expression in embryos, and tissues that give rise to adult structures such as the wings, legs, eyes, and brain. Thus, RNAi can be adapted to study late-acting gene function in Drosophila. The success of this approach in Drosophila and C. elegans suggests that a similar approach may prove useful to study gene function in higher organisms for which transgenic technology is available.

The 64-kilodalton eye muscle protein is the flavoprotein subunit of mitochondrial succinate dehydrogenase: the corresponding serum antibodies are good markers of an immune-mediated damage to the eye muscle in patients with Graves' hyperthyroidism.

Thyroid-associated ophthalmopathy (TAO) is a progressive eye disorder associated with thyroid autoimmunity, particularly Graves' hyperthyroidism, which is generally considered to have an autoimmune etiology. Eye muscle membrane proteins reportedly of 55 and 64 kDa are the best markers of the ophthalmopathy. The main focus of our recent studies has been to purify the pertinent proteins from porcine eye muscle membranes and characterize them. The 64-kDa protein is now shown from a partial sequence and by Western blotting using specific antibody probes to be the flavoprotein (Fp) subunit of succinate dehydrogenase and to have a correct molecular mass of 67 kDa. The protein was purified and cleaved with cyanogen bromide, and the N-terminal region of an immunoreactive partial peptide was determined. The 20-amino acid porcine sequence so obtained matched one within the Fp subunits of human and bovine succinate dehydrogenases in 20 and 18 of these positions, respectively. Succinate dehydrogenase is both a citric acid cycle enzyme and a component (complex II) of the mitochondrial respiratory chain. It is thus essential for aerobic energy production and is highly conserved. The mature human and bovine Fp subunits are 92% homologous and have a molecular mass of approximately 67 kDa, the same as our redetermined value for the 64-kDa marker protein. Sera from patients with TAO and from those with Graves' hyperthyroidism without evident ophthalmopathy highlighted the 64-kDa marker protein in crude porcine eye muscle membranes and the Fp subunit of highly purified bovine succinate dehydrogenase at the identical position on Western blots. Anti-beef Fp antibodies were detected in sera from 67% of patients with active TAO of more than 1-yr duration, in 30% with stable TAO of more than 3-yr duration, and in 30% of patients with Graves' hyperthyroidism without ophthalmopathy, but in only 7% of age- and sex-matched normal subjects. As succinate dehydrogenase is bound to the matrix (inside) surface of the mitochondrial inner membrane, it is unlikely to be accessible to circulating autoantibodies. We would postulate that eye muscle damage in ophthalmopathy is probably caused by cytotoxic antibodies or CD+ T lymphocytes targeting a cell membrane antigen, such as the thyroid and eye muscle shared protein G2s, and that presentation of succinate dehydrogenase is secondary. On the other hand, an autoantibody response to succinate dehydrogenase may be a good marker of immune-mediated damage to the eye muscle fiber and may support the idea that the extraocular muscles are targets of the autoimmune reactions of TAO.

Progressive visual loss due to glioblastoma: normal neuroroentgenorgraphic studies.

A case of progressive bilateral visual loss of abrupt onset an rapid course is described. Extensive neurorentgenographic evaluation was normal, and the results of exploratory craniotomy were unremarkable, except for a slight ovality of the left optic nerve. A biopsy of the left optic nerve showed a glioblastoma, apparently originating in the anterior optic pathways.

Dysthyroid orbitopathy.

Dysthyroid orbitopathy often presents with the classical signs and symptoms of hyperthyroidism with or without pretibial myxedema. However, it may start insidiously without a history of thyroid disease and without clinical signs of hyperthyroidism. The signs or symptoms may be confusing and mimic neurologic disease. The therapy of dysthyroid orbitopathy is becoming more accurately individualized, but is not yet directed at the immunologic cause.

Diagnostic approaches to pituitary adenomas.

The utility of diagnostic tests for pituitary adenoma was compared in 12 patients. Endocrine and radiologic studies were always abnormal, even when neuro-ophthalmologic tests were normal. In most cases, both tomography of the sella turcica and CT scan revealed a mass in the region of the sella turcica. For more precise definition of suprasellar extension of the mass, metrizamide CT cisternography or tomographic pneumoencephalography with metrizamide provided similar details, although CT cisternography was much more easily done. Angiography provides a means to exclude an aneurysm or vascular malformation, a vascular tumor, or an aberrant course of the internal carotid artery.

Dysthyroid and myasthenic myopathy of the medial rectus: a clinical pathologic report.

A patient with simultaneous involvement of the right medial rectus by dysthyroid and myasthenic myopathy is presented. These abnormalities were confirmed by a positive Tensilon test, computerized tomography demonstrating an enlarged right medial rectus, and a right medial rectus muscle biopsy demonstrating histopathology typical of both diseases.

Re-examination of peripheral blood T cell subsets in dysthyroid orbitopathy.

Patients with dysthyroid orbitopathy (DO) were grouped according to a multifactorial assessment of disease severity and the rate of disease progression. Using this system and flow cytometric measurements of T cell subsets in the peripheral blood, a significant increase in the percentage of CD4+ lymphocytes correlated with disease severity in DO patients with progressive disease. These observations are consistent with the hypothesis that the CD4+ peripheral blood T helper cells play a significant role in the progression of DO.

A 63 kDa skeletal muscle protein associated with eye muscle inflammation in Graves' disease is identified as the calcium binding protein calsequestrin.

It is generally accepted that thyroid-associated ophthalmopathy (TAO) is an autoimmune disease of the eye muscle (EM) and the surrounding orbital connective tissue in which circulating antibodies play an important role. Antibodies against EM membrane proteins of 63-67kDa mol. wt. seem to be the best markers of ophthalmopathy in patients with autoimmune thyroid disease. We purified a 63 kDa EM protein using SDS-polyacrylamide gel electrophoresis technology and TAO patients' sera as probes, digested the protein with cyanogen bromide and sequenced immunoreactive peptides. We also screened a human EM library with a rabbit antiserum against 63-65 kDa proteins and affinity purified antibodies from a TAO patient's serum that reacted with a 55 kDa EM membrane protein. From partial sequence information and from DNA sequencing of positive cDNA clones, the protein was identified as calsequestrin, a 63 kDa calcium binding protein localized in the sarcoplasmic reticulum of the muscle fiber. As determined by Northern blotting, calsequestrin was expressed in EM and other skeletal muscle but not thyroid or fibroblasts. Calsequestrin is different from the "64 kDa protein", which has been identified as succinate dehydrogenase flavoprotein subunit, which has a corrected mol. wt. of 67 kDa. Serum antibodies against calsequestrin were found in 40% of patients with clinically active TAO, but in only 4% of those with stable eye disease, and in 5% of normal subjects, by immunoblotting. Although it is possible that autoimmunity against calsequestrin plays a role in the progressive EM damage that characterizes ophthalmopathy it is more likely that the antibodies are secondary to a reaction against some other cell membrane protein, such as the novel thyroid and eye muscle shared protein G2s or the TSH receptor.

T cell interactions with extracellular matrix proteins in patients with thyroid-associated ophthalmopathy.

Although thyroid-associated ophthalmopathy (TAO) is now generally accepted as an autoimmune inflammatory disorder of the extraocular muscles and the orbital connective tissue, its aetiopathogenesis remains poorly understood. Recent data indicate that impaired interactions between T cells and extracellular matrix (ECM) proteins may play an important role in development and maintaining of an inflammatory process. We report here results of the study focusing on interactions between T lymphocytes and collagen-I (Coll-I), collagen-IV (Coll-IV), fibronectin (FN), laminin (LM) in patients with TAO. Using a standard peripheral blood mononuclear cells (PBMC) proliferation assay, we observed a markedly enhanced T cell response to Coll-I in patients with active TAO (mean SI=4.5). The proliferatory response to Coll-I was significantly greater (Wilcoxon test; p < 0.001) than in normal subjects (mean SI=1.88), patients with stable TAO (mean SI=2.05) and patients with thyroid autoimmune diseases (AITD) without ophthalmopathy (mean SI=2.49). PBMC stimulation by Coll-I is likely to be antigen-dependent requiring engagement of the T cell receptor with collagen peptides, rather than mediated via integrins. The percentage of circulating CD29+ (beta1 integrin chain) T cells was not increased in patients with active TAO. Additionally in the assay of costimulation of CD3-mediated proliferation, we found that peripheral blood T cells from patients with TAO and AITD were costimulated only by FN. On the other hand a markedly enhanced costimulation of CD3-mediated proliferative responses by Coll-I, Coll-IV, FN and LM were observed in a retrobulbar T cell line. We conclude that abnormalities in T cell interactions with ECM proteins, especially Coll-I may play a role in the pathogenesis of TAO.

Microsurgical approach to intraorbital tumors. Technique and instrumentation.

We believe that, with the modified Krönlein lateral orbitotomy microsurgical approach, most orbital tumors can be successfully removed with reduced morbidity and complications, compared with the frontotemporal craniotomy or the standard Krönlein lateral orbitotomy. The cosmetic result is quite satisfactory, and the hospital stay should be brief. The use of the operating microscope and the specifically designed and modified instruments for retraction and dissection in the orbit, combined with the efficiency of the self-irrigating bipolar coagulating forceps, add a new dimension to the safety and efficiency of orbital surgery.

Medial rectus injury from intranasal surgery.

Severe medial rectus injury occurred in two cases following intranasal sinus surgery. In the first case a scarred medial rectus muscle was found at the time of corrective surgery. Reexamination of the surgical specimen in the second case confirmed our finding of medial rectus transection. Both patients had good cosmetic and functional improvement by means of vertical recti transposition to correct their large angle, fixed exotropias. A review of the literature indicates that this is, to our knowledge, a previously unreported complication of intranasal sinus surgery.

Transantral-ethmoidal decompression of optic canal fracture.

A case of indirect trauma to the optic nerve was successfully treated with transethmoidal decompression. The literature was reviewed, and reports of poor results from neurosurgical procedures are cited. Improved results from transethmoidal decompression of the optic nerve have been reported by Japanese authors but, in our opinion, without proper selection of cases. Early diagnosis of traumatic compression of the intracanalicular optic nerve as evidenced by delayed and/or progressive vision loss following injury, coupled with prompt transantral-ethmoidal surgical decompression, should yield gratifying results in the treatment of this uncommon condition.

Graves' ophthalmopathy in childhood and adolescence.

Thirty-four patients with childhood Graves' disease were examined to determine the incidence and severity of Graves' ophthalmopathy in children and adolescents. Twenty-three of the 34 patients formed a population of childhood Graves' disease unselected for ocular disease; 11 were unaffected and 12 (52.2%) had findings considered to be of mild functional importance. The remaining 11 patients were referred for evaluation because of a history of Graves' ophthalmopathy; eight (72.7%) exhibited findings of mild severity and three the more meaningful complications of ophthalmoplegia. There were no cases of dysthyroid optic neuropathy or sight-threatening corneal involvement. Both the presence of asymmetric ocular manifestations and increasing severity of disease seem to correlate with increasing age. Although ophthalmoplegia does occur infrequently, Graves' ophthalmopathy in children and adolescents seems to be a substantially more benign process than that seen in adults.

Fine-needle aspiration biopsy. Its use in orbital tumors.

Fifteen orbital tumors have been evaluated with the fine-needle aspiration biopsy (FNAB) technique. The best indication for FNAB is supposed malignant orbital disease. The technique has not been helpful in tumors or inflammatory disease with a high fibrous content. Lesions that are suspected of being pseudotumors are not recommended for FNAB since, even in histologic sections, they are notoriously difficult to distinguish from well-differentiated lymphocytic malignant lymphoma. Benign encapsulated tumors should not be subjected to FNAB.

Choristoma of the optic nerve and chiasm.

A 21-year-old patient with a biopsy-proved choristomatous malformation of the optic nerve and chiasm is presented. The patient experienced progressive unilateral vision loss and optic atrophy. A gadolinium-enhanced magnetic resonance image demonstrated chiasmal involvement that was not evident on computed tomographic scan or visual field testing. We believe this is the third reported case of a choristomatous malformation of the optic nerve and chiasm and the first report of magnetic resonance imaging findings.

Apical optic nerve compression of dysthyroid optic neuropathy on computed tomography.

Computed assisted tomography (CT) of seven cases of dysthyroid optic neuropathy showed moderate to severe thickening of the extraocular muscles at the orbital apex, white CT of two control cases of dysthyroid orbitopathy without neuropathy showed minimal or fusiform thickening. The consequences include probable apical compression with optic nerve neuropathy either by direct pressure on the nerve or its blood supply. The management of these cases is either by oral administration of corticosteroids in high dosage with gradual tapering or radiation therapy to the orbital apex (approximately 3,000 rad over a ten-day period). If surgical decompression is to be attempted, it should be designed to decompress the optic nerve at the orbital apex.

Intrapapillary refractile bodies in optic nerve sheath meningioma.

Intrapapillary refractile bodies were observed in seven of 13 patients with chronic optic disc edema secondary to optic nerve sheath meningioma. The observation of refractile bodies preceded or coincided with decline in vision and disappeared as optic atrophy supervened. Intrapapillary refractile bodies appear to be a nonspecific sign of chronic optic disc edema.

The nonspecific orbital inflammatory syndromes.

The nonspecific orbital inflammatory syndromes are a peculiar group of inflammations that may occur in acute or subacute forms and can become chronic. They may be diffuse or primarily localized to a specific tissue of the orbit. The nonspecific inflammations that are targeted toward specific tissues are myositis, dacryoadenitis, perineuritis, and periscleritis. Each of these syndromes has definite signs, symptoms, ultrasonic and radiologic findings. Therefore, this group of nonspecific inflammations should be more clearly classified for purposes of better understanding and better management. All of these inflammatory syndromes in the acute form respond well to high doses of oral corticosteroids tapered gradually over a period of months, but may be reoccurrent or become chronic. The subacute form responds less well. Occasionally, patients require radiation to stop the inflammation in the subacute or chronic state, but these patients are often left with a functional deficit. The cause, although presumed to be an immune disorder involving the orbital tissues, remains unknown.

The clinical spectrum of schwannomas presenting with visual dysfunction: a clinicopathologic study of three cases.

Schwannomas (neurilemomas) are benign tumors that arise from Schwann cells in the peripheral nervous system. The most commonly involved nerves that cause neuro-ophthalmic manifestations are cranial nerves V and VIII. In this series of three women, schwannomas presented as intraconal masses that mimicked a cavernous hemangioma, a superior orbital mass transgressing the superior orbital fissure, and an expansive frontal lobe mass with clinical symptoms and signs of increased intracranial pressure. Although all three complained of visual blurring, none of our patients presented with Vth or VIIIth cranial nerve dysfunction. Histopathologic studies demonstrated well-circumscribed, encapsulated spindle-cell lesions with classic Antoni A and B patterns. Histopathologic examination is essential to confirm the diagnosis of a schwannoma that may be otherwise clinically confusing. Direct optic nerve compression, globe indentation with induced hyperopia, or increased intracranial pressure with optic nerve compromise may be responsible for visual symptoms. A multidisciplinary approach is often required because of the size and location of schwannomas.

Orbital apex syndrome secondary to herpes zoster ophthalmicus.

Two women, one with Hodgkin's disease and the other with no malignancy, developed herpes zoster with optic neuropathy and total ophthalmoplegia. Both patients developed an associated mild meningoencephalitis with a predominantly lymphocytic spinal fluid reaction that cleared spontaneously. The patient with Hodgkin's disease suffered a protracted course of the disease and developed a secondary bacterial endophthalmitis that necessitated an envisceration of the left eye. The patient without evidence of immunologic deficit recovered quickly with administration of corticosteroids.