Transfer factor: delayed hypersensitivity to Schistosoma mansoni and tuberculin in Macaca mulatta.

Delayed hypersensitivity in Macaca mulatta infected with either Schistosoma mansoni or mycobacteria was demonstrated by biopsies of skin test sites. Both dialyzable and nondialyzable leukocyte extracts from infected donors transferred delayed hypersensitivity to recipient monkeys. In two recipients, skin test conversion was associated with in vitro transformation of the recipients' lymphocytes.

Phase I trial of a melanoma vaccine with gp100(280-288) peptide and tetanus helper peptide in adjuvant: immunologic and clinical outcomes.

A melanoma vaccine composed of HLA-A2-restricted peptide YLEPGPVTA (gp100(280)), with or without a modified T-helper epitope from tetanus toxoid AQYIKANSKFIGITEL, has been evaluated in a Phase I trial to assess safety and immunological response. The vaccines were administered s.c. in either of two adjuvants, Montanide ISA-51 or QS-21, to 22 patients with high-risk resected melanoma (stage IIB-IV). Local and systemic toxicities were mild and transient. We detected CTL responses to the gp100(280) peptide in peripheral blood in 14% of patients. Helper T-cell responses to the tetanus helper peptide were detected in 79% of patients and had a Th1 cytokine profile. One patient with a CTL response to gp100 had a recurrence in a lymph node 2 years later; her nodes contained CD8+ cells reactive to gp100(280) (0.24%), which proliferated in response to peptide. The overall survival of patients is 75% (95% confidence interval, 57-94%) at 4.7 years follow-up, which compares favorably with expected survival. Four of 14 patients who completed at least six vaccines subsequently developed metastases, all of which were solitary and surgically resectable. They remain alive and clinically free of disease at last follow-up. Data from this trial demonstrate immunogenicity of the gp100(280) peptide and suggest that immune responses may persist long-term in some patients. The frequency and magnitude of the CTL response may be improved with more aggressive vaccination regimens. Although this Phase I study was not intended to evaluate clinical benefit, the excellent survival of patients on this protocol suggests the possibility of a benefit that should be assessed in future studies.

Rapid regression of pituitary prolactinomas during bromocriptine treatment.

Therapy for large prolactinomas remains controversial. Surgery is often unsuccessful in restoring endocrine function to normal. However, medical therapy with bromocriptine, a dopamine agonist, not only suppresses PRL levels, but may also lead to a reduction in tumor size. Previous reports have demonstrated radiographic evidence of tumor regression only after 3 or more months of bromocriptine therapy. We have now documented, for the first time, objective evidence of extremely rapid reduction in tumor size in two patients harboring large PRL-secreting pituitary tumors (mean pretreatment serum PRL levels, 2350 and 3900 ng/ml) who were prospectively treated with bromocriptine (7.5 mg/day) in preference to surgical intervention despite marked visual impairment in one of the patients. After 2 and 6 weeks of therapy, respectively, marked reduction in tumor size was demonstrated radiographically in both patients. Headache, visual acuity, and visual fields had improved after only 3 days. Although the mechanism of bromocriptine's antitumor activity is unclear, we believe that a large prospective trial to study the effects of bromocriptine therapy on the size of PRL-secreting macroadenomas is urgently needed to determine whether medical therapy should become the primary modality of treatment to reduce tumor size as well as restore endocrine function.

Ultrastructural and electron-immunocytochemical characterization of cells in epiretinal membranes.

Electron-immunocytochemical staining for three intermediate filament (IF) proteins, keratin (K), glial fibrillary acidic protein (GFAP), and vimentin (V), and for the macrophage marker, EBM/11 (E), was performed on epiretinal membranes obtained during vitrectomies performed for proliferative vitreoretinopathy (PVR), postdetachment macular puckers (PDMPs), idiopathic macular puckers (IMPs), or macular puckers associated with other disease processes. The ultrastructural and immunocytochemical characteristics of the cells were compared. Unstained cells outnumbered stained cells for each of the markers in almost all membranes. Six cell types, based on ultrastructure, were found in the majority of epiretinal membranes: 1) polarized cells with microvilli on the free border and foot processes anchoring them to extracellular matrix that consistently stained negative for all of the immunocytochemical markers; 2) spindle-shaped fibroblastlike cells that were generally negative for all markers, but rarely positive for V; 3) large undifferentiated cells with large, lightly stained nuclei and little cytoplasm that frequently expressed one of the intermediate filament (IF) proteins; 4) poorly differentiated cells that contained numerous mitochondria and frequently expressed one of the IF proteins; 5) undifferentiated, pigment-laden cells that rarely stained for any of the above IF proteins, but occasionally showed K or V positivity in a portion of the cell, suggesting that they may be losing or acquiring these proteins, and that rarely expressed GFAP; and 6) small, round, mononuclear cells with short processes that were sometimes, but not always, positive for E and that were consistently negative for K, V, and GFAP. In addition to these morphologic types, transitional cells demonstrating features of two or more of the above cell types were seen, suggesting that phenotypic changes between the various cell types can occur. The amount of extracellular matrix in epiretinal membranes showed a correlation with disease process (PVR greater than PDMP greater than IMP), and a negative correlation with the percentage of cells expressing a highly differentiated polarized morphology and with the percentage of cells staining for IF proteins. These data suggest that both cell morphology and IF protein expression may be dependent in part on microenvironment and that neither alone can be used to identify unequivocally the derivation of particular cells found in epiretinal membranes. The integration of ultrastructural and immunocytochemical data may provide a more accurate determination of the cell of origin and of phenotypic changes that have occurred. In some cases, however, both ultrastructural and IF protein composition taken together are insufficient for the precise identification of all cells.(ABSTRACT TRUNCATED AT 250 WORDS)

Retinoic acid promotes density-dependent growth arrest in human retinal pigment epithelial cells.

After retinal detachment the retinal pigment epithelium (RPE) undergoes a striking phenotypic change. It becomes dedifferentiated, proliferates to form multilayered colonies, and migrates into the subretinal space. These processes are important because they have been implicated in proliferative vitreoretinopathy and poor visual recovery after retinal reattachment; however the mechanisms by which they occur are unknown. In this study, the effect of retinoic acid on RPE cell morphology and growth in culture was examined. Cells grown in the presence of 1 microM retinoic acid do not exhibit cellular overgrowth and maintain characteristics associated with the morphologic appearance of mature RPE cells in vivo. Growth curves and 3H-thymidine incorporation suggest that retinoic acid inhibits RPE cell growth primarily after the cells have reached confluence. It may act by promoting density-dependent growth arrest. Dibutryl cyclic adenosine monophosphate also inhibits RPE cell growth and 3H-thymidine incorporation, but has little effect on cell morphology. However, in combination with retinoic acid it appears to have an additive effect on inhibition of cell growth and maintenance of a morphology like RPE in vivo. Retinoids have been demonstrated to modulate the growth and differentiation of several cell types. They are usually present in high levels in RPE cells. They become depleted in RPE in culture and such depletion may also occur in vivo after retinal detachment. This could play a role in the phenotypic alteration of RPE that occurs in association with retinal detachment.

Ultrastructural changes in rabbit ciliary body after extraocular mitomycin C.

PURPOSE: To study the ultrastructural changes in ciliary body epithelium of the rabbit eye after subconjunctival injections of mitomycin C. METHODS: One eye of six New Zealand white rabbits was given a subconjunctival injection at the 12-o'clock position with 0.005, 0.02, 0.08, 0.1, 0.12, or 0.16 mg mitomycin C. The fellow eye was given a subconjunctival injection of balanced salt solution. Two weeks after treatment, the eyes were enucleated, and the ciliary body was exposed and submerged in fresh 4% paraformaldehyde/2% glutaraldehyde in 0.1 M phosphate buffer, pH 7.4, at 4 degrees C. Electron microscopy of the ciliary body was performed at two sites: the injection site (12-o'clock position) and 180 degrees away (6-o'clock position). RESULTS: At dosages of 0.1 mg and higher, ciliary body epithelial cells beneath the injection site were thinned. There were vacuoles and expansion of intracellular and intercellular spaces. Plasma membrane infoldings were disrupted, and the apical membrane was thinned. Mitochondria and nuclei were normal. Ciliary body epithelium at 6-o'clock position showed only mild architectural distortion of the plasma membrane infoldings. Eyes that received lower doses of mitomycin C (0.005 mg, 0.02 mg, and 0.08 mg) and balanced salt solution showed normal ciliary body epithelium at the injection site and 180 degrees away. CONCLUSIONS: Subconjunctival injection of mitomycin C in the rabbit produces dose-dependent localized ultrastructural changes of the ciliary body epithelium.

11-cis retinol dehydrogenase mutations as a major cause of the congenital night-blindness disorder known as fundus albipunctatus.

PURPOSE: Patients with fundus albipunctatus uniformly experience difficulty with vision at night. Their retinas are spotted with characteristic light yellow flecks of unknown composition that typically spare the macula. A defect in the transport or utilization of visual cycle retinoids is thought to underlie this recessive disorder with variable clinical expression. To elucidate the molecular defect we considered the genes for interphotoreceptor retinoid-binding protein (RBP3) and 11-cis retinol dehydrogenase (RDH5) as candidates for this disease. METHODS: We examined two unrelated families with fundus albipunctatus. The diagnosis was determined clinically and RBP3 and RDH5 were analyzed by molecular screening methods and direct genomic sequencing. RESULTS: Each family had two affected members with typical fundus albipunctatus. The affected members were siblings born to unaffected parents who were seventh cousins in the first family and unrelated in the second family. The probands from both families were clinically similar except for the fundus dots that were more extensive in the second family to the point of involving the parafoveal region. In the initial phase of genetic screening RBP3 defects were ruled-out as the cause of the disease in both families. In contrast, RDH5 mutations were found in the affected siblings in both families. The proband in one had a homozygotic Gly238Trp missense mutation (GGG -> TGG) involving exon 4 and in the other carried compound heterozygotic changes Arg280His (CGC -> CAC) and Ala294Pro (GCC -> CCC) in exon 5. The disease phenotype was only manifested in family members with two abnormal RDH5 alleles consistent with autosomal recessive inheritance in both pedigrees. CONCLUSIONS: These findings strongly implicate defects of RDH5 as the cause of fundus albipunctatus and point to a heterogeneity of RDH5 mutations in this form of congenital stationary night blindness with variable expressivity.

Deletion (13)(q14.1q14.3) in two generations: variability of ocular manifestations and definition of the phenotype.

A 5-yr-old girl with unilateral retinoblastoma was found to have del(13)(q14.1q14.3). Her 16-month-old sister and 35-year-old mother, with retinal colobomata but without retinoblastoma, have the same deletion. Esterase D studies indicate reduced gene dose at this locus in the 3 females, consistent with a deletion of band 13q14. These patients are of apparently normal intelligence but have a mildly "coarse" facial appearance, a broad nasal bridge, upturned nares, and a long upper lip with thin upper lip vermillion similar to the phenotype suggested by Motegi et al [1983a] for patients with this deletion. Review of the literature documents 2 other patients with deletions of band 13q14 but without retinoblastoma, indicating that retinoblastoma is not a necessary consequence of this deletion. Of the 12 reported patients with deletions limited to band 13q14, seven had normal intelligence and five were macrocephalic. Insufficient clinical information is provided to draw conclusions about phenotype. The family which we describe and those reviewed by Motegi et al suggest that there may be a characteristic appearance in patients with this deletion.

Macular infarction after endophthalmitis treated with vitrectomy and intravitreal gentamicin.

We encountered two cases of macular infarction that occurred after vitrectomy performed for mild endophthalmitis, which, on culture, yielded Staphylococcus epidermidis. At the end of each vitrectomy, 0.4 mg of gentamicin, 1.0 mg of cefazolin, and 0.32 mg of dexamethasone were injected into the vitreous cavity. We are concerned that the macular lesion was a retinal toxic effect of gentamicin because of the recent description of similar lesions occurring after the inadvertent intraocular injection of massive doses of this drug.

Aminoglycoside toxicity--a survey of retinal specialists. Implications for ocular use.

Surveyed members of the Retina, Macula, and Vitreous Societies reported 93 cases of macular infarction they believed to be related to administration of gentamicin sulfate; five, to administration of amikacin sulfate; and three, to administration of tobramycin sulfate. Most cases of infarction after administration of gentamicin occurred in eyes that received an intravitreous injection of 0.4 mg after vitrectomy, but a surprisingly high number, 17, occurred after injection of 0.1 or 0.2 mg, doses that are considered safe by many ophthalmologists. Four additional cases of infarction occurred in eyes that did not undergo vitrectomy after injection of 0.1 or 0.2 mg. Four of the five cases related to administration of amikacin occurred after intravitreous injection of 0.4 mg, and one of these four occurred in an eye that did not undergo vitrectomy. Twenty-three cases of macular infarction occurred in eyes that were treated with prophylactic subconjunctival injections of aminoglycosides after routine ocular surgery. Responses from this survey suggest that aminoglycoside-induced retinal infarction is widely recognized and more common than indicated from the small number of cases reported in the literature. The role of aminoglycosides in the prophylaxis of ocular infections and the management of endophthalmitis should be reevaluated.

Hemangiomalike masses of the retina.

We have followed five patients, each of whom had a solitary unilateral vascular mass of the sensory retina. The lesions were white or pink and often had hemorrhage and exudate on their surface or surrounding area. They appeared to be acquired, as three of the five patients were specifically noted to have normal fundi at some time before presentation. Fluorescein angiography and angioscopy demonstrated the masses to be composed of numerous vessels supplied by the retinal circulation. Slow growth was documented in one patient. Visual loss was variable and was due to leakage-related complications, including exudative detachment, recurrent vitreous hemorrhage, and cystoid macular edema. Four patients were treated with cryopexy, and in all four, this successfully caused involution of the lesions. Two patients had improvement in vision after treatment: one had stabilization, and one had deterioration. This report provides support for the existence of a distinct entity that we have called hemangiomalike mass of the retina, since no histopathologic evidence is yet available (to our knowledge). It also supports the use of judicious cryopexy to lesions associated with visual loss.

Subretinal fluid stimulation of retinal pigment epithelial cell migration and proliferation is dependent on certain features of the detachment or its treatment.

Subretinal fluid was collected without contamination by blood by placing capillary tubes within drainage sclerotomies when retinal reattachment surgery was performed. All samples (N = 24) stimulated retinal pigment epithelial (RPE) cell migration and proliferation, but a wide range of activity existed (migration, 27.5% to 322.5% above baseline; proliferation, 7.5% to 318.3% above baseline). The correlation between migration- and proliferation-stimulating activity was not strong, suggesting the possibility that different agents are responsible for each. Several variables were examined for correlation with each activity, including age and sex of the patient; extent, duration, and height of the detachment; protein content of the fluid; and amount of cryopexy. It was found that proliferation-stimulating activity increased with the extent of detachment and duration of detachment, while migration-stimulating activity correlated best with amount of cryopexy given before drainage. These data suggest that retinal cryopexy enhances RPE cell migration activity in subretinal fluid, while RPE proliferation activity increases with the size and duration of detachment.

Cryotherapy causes extensive breakdown of the blood-retinal barrier. A comparison with argon laser photocoagulation.

Using computerized vitreous fluorophotometry (VFP) in pigmented rabbits, we examined two modalities frequently used in retinal reattachment surgery, cryotherapy and argon laser photocoagulation, for their effect on the blood-retinal barrier. The VFP readings were taken 2 mm posterior to the lens one hour after intravenous injection of 14 mg/kg of fluorescein sodium. After baseline fluorophotometry readings, rabbits were treated with confluent cryotherapy over the inferior 180 degrees in one eye and with confluent laser over an equivalent area of retina in the other eye. The VFP readings were taken 2, 4, 6, and 15 days after treatment. By day 6, the VFP reading had risen from a pretreatment value of 6.4 +/- 0.4 ng/mL to 41.8 +/- 7.9 ng/mL in the cryotherapy-treated eyes as opposed to 15.5 +/- 3.1 ng/mL in the laser-treated eyes. By day 15, the readings were almost back to baseline, and there was no significant difference between the two treatment groups. These data suggest that there is a significant breakdown of the blood-retinal barrier with both modalities, but that it is considerably more severe with cryotherapy.

Intravitreal chemotactic and mitogenic activity. Implication of blood-retinal barrier breakdown.

We produced breakdown of the blood-retinal barrier (BRB) in pigmented rabbits by several different mechanisms: retinal cryopexy, retinal laser, intravenous sodium iodate (30 mg/kg), or intravitreal injection of epinephrine (0.1 mL, 10(-3) M). The degree of BRB breakdown was monitored by computerized vitreous fluorophotometry. At 72 hours after treatment, rabbits were killed, and eyes were quickly removed, washed, and placed in liquid nitrogen. The vitreous was then carefully isolated free of contamination from other ocular tissues, homogenized, and aliquotted. For each sample we measured protein content, retinal pigment epithelial (RPE) cell chemotactic activity, and RPE mitogenic activity. Each of the above modalities produced significant breakdown of the BRB as measured by vitreous fluorophotometry and intravitreal protein concentration, while a pars plana intravitreal injection of saline (0.1 mL) did not. Vitreous from eyes treated with each of the above modalities caused significant stimulation of RPE migration (cryopexy, 684%; laser, 211%; sodium iodate, 480%; intravitreal epinephrine, 546%), while control vitreous and saline-injected vitreous caused only 89% and 77% stimulations, respectively. Similarly, vitreous from eyes treated with the above modalities caused significant stimulation of RPE proliferation (116% to 159%), while control vitreous caused only a 56% increase above baseline. Proliferative vitreoretinopathy, a disease process in which cellular migration and proliferation play important roles, occurs most commonly after surgical intervention. Breakdown of the BRB at the time of surgery may be a critical event due to intravitreal accumulation of serum-derived chemoattractants and mitogens.

Ciliary body adenoma in a 10-year-old girl who had a rhabdomyosarcoma.

A retrolenticular fibrovascular membrane occurred in the setting of a ciliary body mass in a 10-year-old black girl who had been successfully treated for a rhabdomyosarcoma of the lung. The results of a transscleral biopsy of the mass demonstrated it to be a pigmented adenoma of the ciliary body. A lensectomy and membranectomy were performed to aid in the follow-up and rehabilitation of the eye. At 9 months after surgery, the patient had 20/25 vision with no evidence of tumor growth. This case demonstrates an unusual cause of cyclitic membrane formation and suggests that transscleral biopsy may be a useful adjunct in the management of selected ciliary body masses.

Clear cell differentiation in choroidal melanoma. COMS report no. 8. Collaborative Ocular Melanoma Study Group.

OBJECTIVE: To describe 2 enucleated eyes of patients enrolled in the Collaborative Ocular Melanoma Study that contained primary choroidal melanoma with clear cell features. METHODS: During a 9-year period, 1493 eyes enucleated as part of the Collaborative Ocular Melanoma Study routinely processed for histologic examination were evaluated by the pathology review committee (H.E.G, D.M.A, and W.R.G). Two eyes with unusual variants of choroidal melanoma were identified and immunostained for S100 protein and HMB 45. Portions of the tumors were processed for electron microscopic examination. RESULTS: Results of electron microscopic examination of both tumors displayed malignant melanoma (mixed cell type with many malignant cells with clear cytoplasm). The cytoplasm of the clear cells stained with periodic acid-Schiff and failed to stain when pretreated with diastase. Results of immunohistochemical stains in both tumors were positive for S100 protein and HMB 45 in the tumor cells. Results of electron microscopic examination showed that the cytoplasm of the clear cells contained scattered glycogen granules, premelanosomes, and melanosomes. CONCLUSION: These cases represent a clear cell variant of malignant melanoma of the choroid. This tumor should not be confused with metastatic clear cell carcinoma to the choroid.

Clinical risk factors for proliferative vitreoretinopathy.

We reviewed the records of all patients with rhegmatogenous retinal detachments examined and treated by a single surgeon (B.P.C.) at the University of Virginia, Charlottesville, between 1978 and 1984. Of the 607 eyes that satisfied the selection criteria, a preliminary chart review of outcomes found that 65 (10.7%) had proliferative vitreoretinopathy and 34 had macular puckers. From the remaining 508 eyes, 325 controls were randomly selected to match each case from within a time window. Thirty-six (55.4%) of the 65 patients with proliferative vitreoretinopathy had had unequivocal reattachment after a single procedure before the onset of proliferative vitreoretinopathy, and the only clearly identified technical difficulty that was significantly more common in the patients with proliferative vitreoretinopathy was the inability to identify a retinal break. Several other features of the rhegmatogenous retinal detachments that correlated with the development of postoperative proliferative vitreoretinopathy were identified, and stepwise discriminant analysis was used to ascertain which of these were more important. The strongest predictor was use of vitrectomy in management of the detachment. Following this in order of importance were the presence of preoperative proliferative vitreoretinopathy, preoperative choroidal detachment, and the amount of cryopexy required. Vitrectomy remained a strong predictor even when considered after adjustment for all other characteristics. These data suggest that proliferative vitreoretinopathy is not simply an iatrogenic disease, but it is more likely to occur in association with certain detachment features that either by themselves or through their management require prolongation of the retinal wound healing process.

The role of breakdown of the blood-retinal barrier in cell-injection models of proliferative vitreoretinopathy.

Rabbits were given an intravitreous injection of 5.0 x 10(5) rabbit retinal pigment epithelial (RPE) cells, human RPE cells, or human dermal fibroblasts in one eye and an injection of vehicle alone in the other eye. Some rabbits were treated with retinal cryopexy or intravenous sodium iodate on the day before injection. Vitreous fluorophotometry (VFP) and fundus examinations were performed before and at various times after cell injections. Retinal detachments were graded by premortem ophthalmoscopic examinations and postmortem gross pathologic examinations. Eyes injected with cells had higher VFP readings than eyes injected with vehicle at all time points. Eyes injected with fibroblasts or rabbit RPE had significantly higher mean VFP values before the onset of retinal detachment than those injected with human RPE cells. Within each group, high levels of fluorescein leakage in the first week correlated well with severity of subsequent traction retinal detachment and the fibroblast and rabbit RPE groups had more severe detachments than the human RPE group. Treatment with cryopexy or sodium iodate resulted in higher VFP readings, a higher frequency of retinal detachments, and detachments that occurred earlier and that were more severe. These data demonstrate that intravitreous cells cause blood-retinal barrier breakdown in rabbits and that the amount and duration of this breakdown are important variables in retinal detachment formation.

Gentamicin toxicity in the primate retina.

To study the toxic effect of aminoglycoside antibiotics in the primate retina, gentamicin sulfate was injected into the center of the vitreous cavity of Cebus navrigatus monkeys. At a dose of 1000 to 3000 microgram, a picture consistent with apparent macular infarction appeared on fundus examination and fluorescein angiography by three days and gradually faded by 21 days. While light and electron microscopic examination of the retina showed no primary vascular lesions, striking damage to the inner retinal layers, mainly the nerve fiber layer, ganglion cell layer, and the inner plexiform and nuclear layer, was seen. Less severe effects in the outer retinal layers and the retinal pigment epithelium occurred. These observations suggest that the neurotoxic effect of intravitreal gentamicin was sufficient to cause a complete shutdown of the regional blood flow, perhaps by the mechanism of granulocytic plugging of the capillary bed. Although this toxic effect occurred at doses considerably in excess of what has been recommended for clinical use in humans, the "safe" dose of intravitreal gentamicin, nevertheless, remains to be established unequivocally.

X-chromosone-linked juvenile retinoschisis with hemorrhagic retinal cyst.

A 6-year-old boy with X-chromosome-linked juvenile retinoschisis had a large blood-filled retinal cyst surrounded by a rim of exudate which had infiltrated the macula. Spontaneous clearing of the blood and exudate was associated with improvement in vision.