Cytotoxic effects of FGF2-saporin on bovine epithelial lens cells in vitro.

PURPOSE: To test the ability of two preparations of FGF2-saporin, either FGF2 chemically conjugated to saporin (FGF2-SAP) or genetically engineered FGF2-saporin (rFGF2-SAP) to inhibit the growth of bovine epithelial lens (BEL) cells in vitro when in solution and when immobilized on heparin surface-modified (HSM) polymethylmethacrylate (PMMA) intraocular lenses (IOLs). METHOD: Bovine epithelial lens cells were incubated with various concentrations FGF2-saporin for as long as 4 days. The number of surviving cells was determined by counting the number of nuclei. Because FGF2 binds to heparin, FGF2-saporin was incubated with HSM PMMA IOLs; excess toxin was washed off, and the BEL cells were grown on the FGF2-saporin-treated IOLs (HSM and non-HSM) for 4 days. Cell density was determined by image analysis. RESULTS: Both FGF2-SAP and rFGF2-SAP were highly cytotoxic (nM range), with rFGF2-SAP 10 times less active than FGF2-SAP. FGF2-saporin bound to the surface of HSM IOLs and eluted by 2M NaCl retained its activity. Toxin bound to HSM IOLs killed more than 90% of the BEL cells placed on the IOL surface within 4 days. The ability of FGF2-saporin to prevent the growth of cells on the IOL surface was strictly dependent on the presence of heparin on the IOL. CONCLUSIONS: FGF2-saporin is bound to HSM PMMA IOLs and prevents the growth of epithelial cells on the surface of the lens.

In vivo inhibition of lens regrowth by fibroblast growth factor 2-saporin.

PURPOSE: To investigate the ability of fibroblast growth factor (FGF) 2-saporin to prevent lens regrowth in the rabbit. METHODS: Chemically conjugated and genetically fused FGF2-saporin (made in Escherichia coli) were used. Extracapsular extraction of the lens was performed on the rabbit, and the cytotoxin either was injected directly into the capsule bag or was administered by FGF2-saporin-coated, heparin surface-modified (HSM) polymethylmethacrylate intraocular lenses. The potential of the conjugate was checked by slit lamp evaluation of capsular opacification and by measuring crystallin synthesis. Toxin diffusion and sites of toxin binding were assessed by immunohistochemistry. Possible toxicity was determined by histologic analysis of ocular tissues. RESULTS: FGF2-saporin effectively inhibited lens regrowth when it was injected directly into the capsular bag. However, high concentration of the toxin induced transient corneal edema and loss of pigment in the iris. Intraocular lenses coated with FGF2-saporin reduced lens regrowth and crystallin synthesis without any detectable clinical side effect. After implantation, FGF2-saporin was shown to have bound to the capsules and, to a lesser extent, to the iris; no histologic damage was found on ocular tissues as a result of implantation of drug-loaded HSM intraocular lenses. CONCLUSIONS: Chemically conjugated (FGF2-SAP) and genetically fused FGF2-saporin (rFGF2-SAP) bound to HSM intraocular lenses can prevent lens regrowth in the rabbit.

Encoded chemical synthesis coupled to screening: "Pot Assay".

A variety of screening methodologies is available to identify lead compounds. Screening methods that would permit the direct use of libraries made via the Radiofrequency Encoded Combinatorial chemistry paradigm (each individual small molecule in the library is presented separately on an individual encoded support) have the potential to diminish burdensome steps in this process. Here we report on our studies leading to such a direct method, which we have termed a Pot Assay. Pot Assay is a multiplex assay, which simultaneously measures specific binding of a number of ligands to at least one target. Pot Assay uses specific radiofrequency signals to decode compounds that are high affinity binders. We validated this approach by evaluating the interaction of biotin and its analogs with labeled streptavidin. This report introduces Pot Assay as a rapid, simple, sensitive and accurate format for identifying active members of libraries synthesized on solid supports. The success of this study demonstrates the power of coupling Radiofrequency Encoded Combinatorial chemistry and screening. This assay format may be applied to a wide range of screens that are based on binding events: ligand/receptor, inhibitor/enzyme, antigen/antibody, protein/protein, DNA/protein, and RNA/DNA.

A fluorometric assay for the measurement of endothelial cell density in vitro.

A fluorometric assay for determining endothelial cell numbers based on the endogenous enzyme acid phosphatase is described. In preliminary studies, three substrates--p-nitrophenyl phosphate, 4-methylumbelliferyl phosphate, and 2'-[2-benzthiazoyl]-6'-hydroxy-benthiazole phosphate (AttoPhos)--were compared with respect to their kinetic, optimum assay conditions, sensitivity, and detection limits. Only AttoPhos was found to have a high degree of sensitivity, reliability, and reproducibility for measuring both high and low cell numbers in the same plate. In subsequent experiments, assay conditions were validated for measuring endothelial cell density in response to basic fibroblast growth factor and fumagillin. Furthermore, the AttoPhos assay revealed a linear correlation between acid phosphatase activity and cell number in many cell types, including BALB/3T3, CHO-K1, A431, MCF7, 2008, SK-OV-3, T47-D, and OVCAR-3. This assay is potentially valuable for use in many in vitro systems in which the quantitation of cell density and proliferation is necessary. The practical advantages of AttoPhos assay for measuring endothelial cell numbers include (1) nonradioactivity, (2) simplicity, (3) economy, (4) speed of assessment of proliferation of large number of samples, and (5) amenability to high-throughput drug screening.

Scleroderma-like skin indurations in a child with phenylketonuria: a clinicopathologic correlation and review of the literature.

Scleroderma-like skin lesions are one sequela of phenylketonuria. We describe a child with phenylketonuria and associated dermal connective tissue changes consistent with early inflammatory scleroderma. The severity of the skin lesions apparently waned with dietary restriction of phenylalanine.

Neurofollicular hamartoma. A clinicopathological study.

We review two patients with unusual pilosebaceous and spindle cell neoplasms. Both lesions were on the nose and were clinically similar to angiofibroma. Immunoperoxidase (S-100 protein, vimentin, immunostain for actin) and special stains (Masson's trichrome, periodic acid-Schiff, and Bielschowsky silver stain) were used to evaluate the lesions. The histologic differential diagnosis included neural nevi, trichogenic myxoma, trichodiscoma, benign neural and vascular tumors, and dermal scar formation. Our patients, we believe, had single asymptomatic smooth, skin-colored papules; this condition was recently termed "neurofollicular hamartoma."

Multinucleate cell angiohistiocytoma: a distinct entity diagnosable by clinical and histologic features.

BACKGROUND: Multinucleate cell angiohistiocytoma is a newly described entity; examples from the United States have not yet been reported. OBJECTIVE: Our purpose was to analyze the clinical and histologic features of this entity and confirm or refute its existence. METHODS: Seven cases were analyzed clinically and by light microscopy. RESULTS: Multinucleate cell angiohistiocytoma typically occurs in middle-aged women and consists of multiple, grouped, red-brown to violaceous papules that are dome-shaped or flat-topped, roundish and smooth in outline, sharply circumscribed, and occasionally coalescent. Typical sites are the legs, thighs, and backs of hands and fingers. Microscopic features are an increased number of blood vessels (usually capillaries and venules) that are small, rounded, and not well grouped, together with multinucleated histiocyte-like cells with scalloped borders. CONCLUSION: Multinucleate cell angiohistiocytoma is a distinct entity that is diagnosable clinically and histopathologically.

Porokeratosis arising in a burn scar.

A histologically verified porokeratosis arose in a burn scar on the back of a 47-year-old man. This phenomenon has not been previously reported. We discuss the literature on porokeratosis, the relation between epidermal and dermal injury, and the genesis of this lesion.