Melanin content and downregulation of glutathione S-transferase contribute to the action of L-buthionine-S-sulfoximine on human melanoma.

L-buthionine-S,R-sulfoximine (L-S,R-BSO) was enriched for the active L-buthionine-S-sulfoximine (L-S-BSO) diastereomer. Comparative analysis was performed to determine if this enriched form possessed an increased capacity to deplete glutathione (GSH), and to inhibit the proliferation of tumor cell lines and fresh human tumor samples. Increased activity was observed for the enriched preparation of L-S-BSO in direct proportion to its increased L-S-diastereomeric percentage. Significant antitumor activity towards melanoma, breast and ovarian carcinoma specimens was noted, with the greatest activity directed against malignant melanoma. The activity of BSO on melanoma specimens was found to be correlated with their melanin content, suggesting that free radicals generated during melanin synthesis may become cytotoxic after GSH-dependent scavenging has been eliminated by BSO treatment. The antimelanoma activity of melphalan and BCNU were found to be significantly enhanced in combination with L-S-BSO. With respect to the mechanism of L-S-BSO synergy with alkylators, L-S-BSO treatment of M14 and ZAZ human melanoma cell lines resulted in decreased GSH levels and glutathione S-transferase (GST) activity. Western and Northern blot analyses indicated that GST-mu was the predominant isozyme downregulated after L-S-BSO treatment. Both M14 and ZAZ cell lines selected for resistance to L-S-BSO also showed decreased levels of GST-mu expression. However, in drug free media GST enzyme activity returned to pre-treatment levels without altering the BSO-resistance status of the cell lines. We conclude that L-S-BSO may be an active agent in the treatment of melanoma, and that it may enhance alkylator activity on melanoma through depletion of GSH and down-regulation of GST expression. Purified L-S-BSO should be explored clinically as an active agent for the treatment of melanoma.

Levels of multidrug resistance (MDR1) P-glycoprotein expression by human breast cancer correlate with in vitro resistance to taxol and doxorubicin.

To determine whether multidrug resistance (MDR1) P-glycoprotein (Pgp) expression correlated with clinical MDR1-related drug resistance, we established a protocol for quantitative measurement of Pgp expression and in vitro drug resistance in doxorubicin resistant MCF7 breast cancer cell lines and 359 freshly resected specimens of breast carcinoma. Pgp expression was detected with 4E3, UIC2, and JSB-1 monoclonal antibodies using flow cytometry and immunohistochemistry (IHC). Pgp function was determined using PSC833 in a drug resistance-reversal assay and with a three-dimensional agarose-based extreme drug resistance assay. MCF7 calibrator cell lines expressed Pgp, which was functional and in proportion to the degree of drug resistance. Flow cytometry, UIC2 shift assays, IHC scores, and determination of absorbance products by image analysis were all highly correlated (r > 0.9). Overall Pgp expression increased from 11% in untreated patients to 30% in patients who had previously received chemotherapy. Compared with Pgp-negative tumors, a significant increase in doxorubicin and Taxol resistance was seen for breast cancers that expressed Pgp, regardless of prior treatment. A strong correlation between the degree of Pgp expression and in vitro resistance to Taxol and doxorubicin (but not to 5-fluorouracil) was found when either IHC scores or image analysis-based methods were used to quantify Pgp expression (n = 185, P < 0.0001). The degree of Pgp expression strongly correlated with the degree of drug resistance in the clinical specimens studied. These data suggest that (a) Pgp contributes to clinical MDR1-related drug resistance, and (b) both intrinsic and acquired expression of Pgp in breast cancer may contribute in part to therapeutic failure and relapse.

Selective and synergistic activity of L-S,R-buthionine sulfoximine on malignant melanoma is accompanied by decreased expression of glutathione-S-transferase.

L-buthionine-S,R-sulfoximine (BSO) selectivley inhibits glutathione (GSH) synthesis. Malignant melanoma may be uniquely dependent on GSH and its linked enzymes, glutathione S-transferase (GST) and GSH-peroxidase, for metabolism of reactive orthoquinones and peroxides produced during melanin synthesis. We compared the in vitro effects of BSO on melanoma cell lines and fresh melanoma specimens (n = 118) with breast and ovarian cell lines and solid tumors (n = 244). IC50 values (microM) for BSO on melanoma, breast and ovarian tumor specimens were 1.9, 8.6, and 29, respectively. The IC90 for melanoma was 25.5 microM, a level 20-fold lower than steady state levels achieved clinically. The sensitivity of individual specimens of melanoma correlated with their melanin content (r = 0.63). BSO synergistically enhanced BCNU activity against melanoma cell lines and human tumors. We followed GSH levels, GST enzyme activity, GST isoenzyme profiles and mRNA levels after BSO. BSO (50 microM) treatment for 48 hr resulted in a 95% decrease in ZAZ and M14 melanoma cell line GSH levels, and a 60% decrease in GST enzyme activity. GST-mu protein and mRNA levels were significantly reduced in both cell lines. GST-pi expression was unaffected. These data suggest that BSO action on melanoma may be related to GSH depletion, diminishing the capacity to scavenge toxic metabolites produced during melanin synthesis. We report here for the first time that BSO enhancement of alkylator action may be related in part to down regulation of GST. BSO may be a clinically useful adjunct in the treatment of malignant melanoma.

Hereditary microphthalmia with colobomatous cyst.

We examined five members of a highly inbred kinship who had isolated microphthalmia associated with colobomatous cysts and various other ocular lesions. They were all offspring of consanguineous (first cousins) and unaffected parents. Microphthalmia in this kindred was transmitted as an autosomal recessive trait. Ultrasonography was effective for prenatal diagnosis in two pregnancies at risk.

[Traumatic myopia due to ciliary body detachment].

A 20-year-old man developed myopia immediately after blunt trauma to the eye. The main finding was a shallow anterior chamber. Ultrasonography revealed anterior displacement of the iris lens diaphragm without change in lens thickness or axial length of the eye. After 1 week intraocular pressure decreased to a very low level, and edema of the optic disc and macula developed. Since hypotony and shallowness of the anterior chamber persisted for many months, we assume the cause to be detachment of the ciliary body. This is an uncommon mechanism for traumatic myopia, spasm of the ciliary body being more common.

Collagen fibril abnormalities in the extraocular muscles in Ehlers-Danlos syndrome.

The dermal collagen fibrils and the fibrils of the extraocular muscles and the conjunctivas of both eyes of a child afflicted with Ehlers-Danlos syndrome (EDS) were studied ultrastructurally. The results were evaluated statistically. Biopsy material was used in all instances. The collagen fibrils from all involved tissues showed a high percentage of size variation. Abnormalities of shape were noted. In the reticular dermis, 48% of fibrils were of normal diameter, 23% were enlarged, and 29% were thinner than normal. In the extraocular muscles, 77% were of normal size, 14.5% were larger, and 8.5% were smaller. In the conjunctivas, 73% of fibrils were normal-sized, 22% were enlarged, and 5% were of small diameter. No small-sized fibrils were encountered in control extraocular muscles.

Recovery of the electroretinogram in rabbits after argon laser photocoagulation.

Argon laser is widely used to coagulate the diabetic retina in order to inhibit the proliferative stage of diabetic retinopathy. Ten pigmented rabbits underwent retinal photocoagulation with argon laser. Retinal function was assessed electroretinographically before treatment and at different time intervals after treatment. The ERG responses measured 4-7 days after treatment were reduced in amplitude by a degree which was proportional to the number of laser applications. In five eyes that were treated with at least 1000 applications the ERG responses were very small when measured 4 days post-treatment. However, a gradual recovery was observed and within 2 months the ERG responses approached the normal pretreatment amplitudes. Histological findings from light and electron-microscopy suggested that the ERG recovery could not be solely explained by healing of the coagulated areas. Structural differences were seen in the pigment epithelial layer between a retina obtained immediately after treatment and one studied after ERG recovery. It is suggested that changes in the electrical resistance of the pigment epithelium may contribute to the ERG reduction seen immediately after laser treatment in the rabbit and to the increase in the ERG amplitude observed during the apparent functional recovery of the retina.

Efficacy of antiprostaglandin therapy in vernal conjunctivitis.

Twenty-seven patients with vernal conjunctivitis who remained symptomatic following treatment with corticosteroid drops and/or Opticrom drops (sodium cromoglycate 2%, benzalkonium chloride 0.01%) were treated with aspirin. Aspirin produced marked improvement in all patients apart from three. The possible mechanism of the efficacy of aspirin in vernal conjunctivitis is discussed.

Optic neuropathy and central retinal artery occlusion in a case of herpes zoster ophthalmicus.

In this case report the authors present a patient in whom loss of vision developed in the left eye in the early stage of herpes zoster ophthalmicus. He suffered from optic neuropathy followed by central retinal artery occlusion. Ophthalmologists should be aware of this rare and early complication of herpes zoster.

The role of carcinoembryonic antigen in surveillance of patients with choroidal malignant melanoma: a prospective study.

Serum carcinoembryonic antigen levels were measured in nine patients with choroidal malignant melanoma prior to enucleation and at three-month intervals thereafter for three years. Raised levels were found preoperatively in four of the nine patients. One of these patients had persistent and progressive elevation of serum carcinoembryonic antigen levels postoperatively, and clinical evidence of metastases subsequently became apparent. In the remaining eight patients, carcinoembryonic antigen levels remained at or returned to normal values and no clinical evidence of progression of the disease was detected. These results suggest that although serum carcinoembryonic antigen estimation is not a reliable diagnostic screening test for choroidal malignant melanoma, it may be useful for follow-up and monitoring of recurrences of the disease.

Electron microscopic study of Bitot's spots.

The histological and structural features of Bitot's spots are described. Light microscopy revealed squamous metaplasia with keratinization. Electron microscopy showed severe loss and attenuation in height of microvilli as well as widening of the intercellular spaces. The amorphous material overlying the conjunctiva was composed of cellular debris containing edemetous endoplasmic reticular membranes and lysozomes.

Sudden loss of vision in a monocular patient, caused by visual cortex infarction.

Infarction of the occipital cortex usually causes loss of the contralateral half of the visual field in both eyes. Although cases with complete transient loss of vision have been described, this is due to involvement of the second half of the cortex in the pathologic process. We report a case of a one-eyed patient with almost complete loss of vision in her only eye because of an infarction of the ipsilateral occipital cortex. During the patient's hospitalization, the temporal half of the visual field recovered. The case is of interest because of the difficulties in diagnosing cortical blindness in a one-eyed patient.

Effects of immunosuppressive drugs on the morphology of the microvilli in Sjögren's syndrome.

Electron microscopic study of conjunctiva in two cases of Sjögren's syndrome showed decrease in height and number of microvilli and lack of branching. Following immunosuppressive treatment, an increase in height, number and branching of conjunctival microvilli was observed. Notwithstanding the lack of influence on tear secretion, immunosuppressive drugs are of benefit in the treatment of ocular manifestations of Sjögren's syndrome. The increase in number, height and branching of microvilli enlarge the surface of the conjunctiva and thus improve the efficacy of artificial tears.

Retinal function of the diabetic retina after argon laser photocoagulation assessed electroretinographically.

Electroretinographic (ERG) responses were measured in diabetic patients before, during, and after panretinal photocoagulation treatment with argon laser. The laser applications reduced considerably the amplitudes of the a and b waves of the ERG. Moreover, the relationship between the amplitude of the b wave and that of the a wave was severely affected, resulting in ERG responses of abnormal pattern. The b waves were smaller than expected from the a waves. These findings indicated that the photocoagulation treatment not only destroyed the retinal areas directly illuminated by the laser beam, but also affected the functional integrity of adjacent areas. These additional effects resulted in subnormal signal transmission from the photoreceptors to the proximal retina.

Light and electron microscopical study of the conjunctiva in sicca syndrome.

Conjunctival biopsies of 11 patients with sicca syndrome were studied under light and transmission electron microscopy. The notable findings were vasculitis of the conjunctival vessels. There were severe changes in the microvilli, namely a loss of microvilli and decrease in their height, measuring approximately 1,000-2,000 A, in comparison with microvilli of the control conjunctiva, measuring 4,000 A in height. There was no branching yet or fusion of microvilli.

Capillaries of human overacting extraocular muscles.

The capillaries of overacting inferior oblique muscles from children with strabismus and of dog inferior oblique muscles were ultrastructurally described. Biopsy material was used in all instances. The mean diameter of the capillary lumen was 2.03 microns for the human inferior oblique and 2.76 microns for the dog inferior oblique. Quantitative estimates of capillary basement membrane width were obtained by two different methods of measurement. The capillaries of human and dog inferior oblique muscles were compared with capillaries of human gastrocnemius muscle and of dog quadriceps muscle. The data were statistically evaluated. It was found that the mean basement membrane width of capillaries of overacting inferior oblique muscles is quite similar to that of the dog normal inferior oblique muscles, showing 2,049 A in overacting muscles and 1,976 A in normal muscles. With both methods of measurement, the mean basement membrane width of inferior oblique muscles appeared thicker than the mean basement membrane of capillaries of the leg muscles in man and in dog as well. It is suggested that the thickening of the capillary basement membrane of inferior oblique muscles represents a regional characteristic. Marked variation in the thickness of the basement membrane was encountered in all muscles.

Diabetic neovascularization: permeability and ultrastructure.

The permeability of newly formed blood vessels in proliferative diabetic retinopathy at different stages of development was correlated with their ultrastructural features. The young proliferating vessels were much more permeable to fluorescein than were the older vessels. Transmission electron microscopy revealed that the blood vessels within the young intravitreal proliferating fronds had intermediate interendothelial junctions, whereas the blood vessels within the old neovascular preretinal membranes had tight interendothelial junctions of the macula occludens type. Fenestrations were observed only rarely in the endothelial wall of either the young or the older proliferating vessels. We conclude that the principal route of fluorescein leakage from the newly formed blood vessels in proliferative diabetic retinopathy is via the interendothelial clefts.

Marginal melting of cornea in rheumatoid arthritis.

There was marginal melting of the cornea in a patient with inactive rheumatoid arthritis. Conjunctival biopsy revealed vasculitis which disappeared after treatment with topical steroids and systemic steroids and azathioprine. Conjunctival biopsy can serve as a diagnostic tool and for evaluation of treatment in marginal ulceration of cornea associated with rheumatoid disease.