Interferon alpha-2a for subfoveal neovascularization in age-related macular degeneration.

BACKGROUND: The current study is a prospective randomized clinical trial to determine the effect of interferon alpha-2a on eyes with subfoveal subretinal neovascularization secondary to age-related macular degeneration (AMD). METHODS: Twenty eyes of 19 patients with subfoveal neovascularization secondary to AMD were prospectively evaluated. Ten eyes were randomized to subcutaneous interferon alpha-2a (3 million units/m2) every other day for 8 weeks, whereas 10 eyes were randomized to observation alone as controls. Fluorescein angiography, best-corrected visual acuity tests, and macular visual field assessments were performed, and all eyes were followed for a minimum of 6 months. RESULTS: At the 2-month follow-up visit, the interferon group manifested somewhat slower neovascular growth than controls, but the results were not statistically significant. At the 6-month follow-up visit, there was no difference in visual acuity, average macular sensitivities, or extent of neovascularization. The rate of neovascular progression was significantly related to the extent of previous macular photocoagulation in both groups. CONCLUSION: Though the rate of neovascular progression was slowed during the second month of interferon treatment, the effect did not persist once interferon was discontinued. No long-term benefit appeared to be present. Unfortunately, lengthening the time of administration, increasing the dosage, or increasing the frequency of administration would likely give rise to unacceptable side effects.

Genetic analysis of human B cell hybridomas expressing a cross-reactive idiotype.

We have examined the genetic basis for the expression of a human cross-reactive idiotype (CRI) commonly found on monoclonal IgM rheumatoid factors. The CRI was identified with a monoclonal antibody (17.109) and has been localized previously to the kappa-variable region. By using the human lymphoblastoid cell line WI-L2-729-HF2, and mononuclear cells from several sources, a panel of hybridomas was generated that produced 17.109 CRI-positive Ig. A recently cloned human germ-line V kappa III gene, Humkv305, served as a probe to identify genes which were rearranged and expressed in 17.109 CRI-positive and -negative hybridomas. This probe, when hybridized to human genomic DNA under stringent conditions, identified only two to five germ-line bands. In 10 separate 17.109 CRI-positive hybridoma clones, an additional rearranged V kappa band was identified. The probe did not anneal to rearranged V kappa bands in hybridoma clones that produced kappa-chains lacking the CRI. RNA dot-blot studies provided evidence for expression of genes hybridizing to the Humkv305 probe. The results indicate that the 17.109 CRI is a serologic marker for a single V kappa gene, or a small family of closely related V kappa genes, which is identified by the Humkv305 probe.

The WI-L2-729-HF2 human hybridoma system. Arrangement of lambda genes in monoclonal hybrids.

Fusion of WI-L2-729-HF2 human lymphoblastoid cells and human B-cell blasts provides a very efficient and rapid means of isolating stable B-cell hybridomas that secrete high levels of new human immunoglobulins. By titrating the plating density of fused cells into microwells immediately following fusion, it has been possible to obtain monoclonal hybrids. In this communication, proof of monoclonality is provided based on subcloning, karyotyping, and Southern blot analyses of lambda light chain immunoglobulin genes. The results reveal rearranged lambda genes in hybridoma subclones that produce both kappa (the WI-L2-729-HF2 isotype) and new lambda light chains. In contrast, the WI-L2-729-HF2 parental cell line and kappa-producing hybrids exhibit a germline configuration of lambda genes. The results provide evidence that stable, monoclonally derived hybridomas may be obtained upon initial plating of fused cells, without subsequent subcloning. The data further demonstrate the WI-L2-729-HF2 system to be ideal for rapidly generating, at very high frequency, clonal human B-cell hybridomas that stably secrete human monoclonal antibodies.

Cloning and sequence determination of a human rheumatoid factor light-chain gene.

The contribution of germ-line variable regions to autoantibody formation in humans is poorly understood. To study the gene structure of a human autoantibody, chronic lymphatic leukemia (CLL) cells from a patient with an IgM anti-IgG (rheumatoid factor, RF) paraprotein were utilized. The rearranged immunoglobulin gene encoding the kappa light chain for the RF was cloned, and the nucleic acid sequence of its variable region was determined. As demonstrated by Southern blot analysis using a kappa joining-region probe, the CLL cells, stable CLL-WIL2-729-HF2 RF-secreting hybridomas, and the cloned light-chain gene all had an identical restriction fragment containing the rearranged light-chain gene. The CLL RF light chains reacted weakly with an antipeptide antibody against a primary structure-dependent idiotype present on the light chains of the majority of IgM RF paraproteins. The nucleotide and predicted amino acid sequences of the CLL light-chain gene place it in the kappa III variable-region subgroup, and a comparison to known RF paraproteins reveals marked homology to the light-chain amino acid sequence of the IgM RF paraprotein Pom. Both Pom and the CLL light chain appear to identify a second kappa III gene or gene group that is able to encode RF paraprotein light chains.

Presence of a novel epithelial antigen on rat cerebellar cell lines as detected by a monoclonal antibody.

We have derived a monoclonal antibody, MCAb 51, following immunization of BALB/c mice with a Rous sarcoma virus-transformed rat cerebellar cell line. When assayed by immunofluorescence on primary rat cerebellar cultures MCAb 51 recognizes only islands of cells with an epitheloid morphology. Double-label immunofluorescence experiments with MCAb 51 and antisera to tetanus toxin, glial fibrillary acidic protein, galactocerebroside and fibronectin reveal that these cells do not appear to be neurons, astrocytes, oligodendrocytes, or fibroblasts, respectively. In contrast, cells from kidney, liver, tongue and choroid plexus epithelium are positive for the antigen. Of 12 Rous sarcoma virus-transformed cell lines, in contrast to 2 out of 9 chemically transformed lines, 11 exhibit the MCAb 51 antigen. These findings demonstrate that MCAb 51 recognizes an epithelial cell surface marker. Possible explanations for the difference in the expression of the antigen on Rous sarcoma virus and chemically transformed neural lines are discussed.

The WI-L2-729-HF2 human hybridoma system. Stable hybrids at high frequency.

A new system for the rapid production at high frequency (greater than 10(-5] of stable human hybridomas is described. This system is based on a high efficiency fusion variant human lymphoblastoid cell line designated WI-L2-729-HF2 and is comparable in many ways to commonly used murine hybridoma systems. WI-L2-729-HF2 cells fuse at high frequency with mitogen-stimulated human b cells resulting in the rapid appearance (within 2 weeks) of stable hybridomas in hypoxanthine/aminopterin/thymidine (HAT) medium. Whereas the WI-L2-729-HF2 cell line secretes only trace levels (50 to 100 ng/ml) of an IgG,kappa and has surface IgM,kappa, HAT-resistant hybridomas secrete high levels (10 to 20 micrograms/ml) of new human immunoglobulins, including immunoglobulins containing alpha or lambda chains not found in the tumor parental cell line. These hybridomas have remained stable for over four months in continuous culture, secreting high levels of new immunoglobulins in both conventional and serum-free medium, a feature which makes possible large-scale production and purification of human antibodies. This system has the potential to provide tools and reagents for investigations involving, for example, the diagnosis and treatment of human autoimmune disease and cancer.