Necrobiotic xanthogranuloma occurring in an eyelid scar.

We present a case report of necrobiotic xanthogranuloma (NXG) in a 76-year-old Caucasian lady occurring as a nodule in a blepharoplasty scar. NXG is a rare histiocytic disease with progressive orbital and systemic features. Management options of excision biopsy or chemotherapy are discussed.

TNF receptor secretion after ex vivo adenoviral gene transfer to cornea and effect on in vivo graft survival.

PURPOSE: To explore the potential for adenovirus-mediated ex vivo gene transfer of a soluble tumor necrosis factor (TNF) receptor and evaluate the effect of transplanting the adenovirally transplanted corneas in vivo. METHODS: Rabbit corneal segments were transfected with replication-deficient adenovirus (AdTNFR) encoding a soluble TNF receptor fusion protein (TNFR-Ig). Production of TNFR-Ig was measured by using ELISA and bioassay. Corneas were transfected ex vivo with AdTNFR and then transplanted in vivo. Survival of AdTNFR-transfected corneas was compared with that of those treated either with a null vector control adenovirus (Ad0) or nontransfected control corneas. RESULTS: Ex vivo production of a molecule with TNF blocking bioactivity from AdTNFR-transfected corneas was demonstrated over a period of 4 weeks. Transplanted AdTNFR-transfected corneas showed a marginally increased survival time in vivo over nontransfected control corneas, but a significantly increased survival time over Ad0-treated control corneas. Ad0 treatment of corneal allografts before transplantation had a proinflammatory effect and accelerated the onset of corneal endothelial rejection. CONCLUSIONS: Adenoviral gene transfer is an effective means of transferring a gene encoding soluble TNFR-Ig to corneal endothelium, and ex vivo production of a biologically active secreted molecule was demonstrated for 4 weeks. However, in vivo, only a marginally increased survival was seen compared with control corneas. The introduction of this transgene using a less immunogenic vector may demonstrate prolongation of corneal allograft survival.

Gene delivery to the corneal endothelium.

Gene transfer to the corneal endothelium has potential for modulating rejection of corneal grafts. It can also serve as a convenient and useful model for gene therapy of other organs. In this article we review the work carried out in our laboratory using both viral and nonviral vectors to obtain gene expression in the cornea.

Local bioactive tumour necrosis factor (TNF) in corneal allotransplantation.

The aim of this study was to examine the kinetic profile of bioactive TNF levels in aqueous humour of rabbit eyes undergoing corneal allograft rejection and to investigate the effect of locally blocking TNF activity after corneal transplantation. In a rabbit corneal transplantation, endothelial allograft rejection was identified and correlated with increase in central graft thickness. Samples of aqueous humour obtained on alternate days following transplantation were tested for TNF mRNA and bioactive TNF protein. To investigate the effect of locally blocking TNF activity in allograft recipients, the fusion protein TNFR-Ig was administered by injections into the anterior chamber after transplantation. Pulsatile increases in levels of this cytokine were found in 14 of 15 allograft recipients. Peaks of TNF bioactivity preceded by varying intervals the observed onset of rejection in allograft recipients. TNF levels were not elevated in aqueous humour from corneal autograft recipient controls or in serum of allografted animals. mRNA levels were elevated before onset of and during clinically observed allograft rejection. In three of seven animals receiving TNFR-Ig injections on alternate days from day 8 to day 16 post-transplant, clear prolongation of corneal allograft survival was demonstrated. Bioactive TNF is present in aqueous humour following rabbit corneal allotransplantation. Rather than correlating directly with endothelial rejection onset, pulsatile peak levels of TNF precede and follow the observed onset of endothelial rejection. Blockade of TNF activity prolongs corneal allograft survival in some animals, indicating that this cytokine may be a suitable target in local therapy of corneal allograft rejection.

Adeno-associated and herpes simplex viruses as vectors for gene transfer to the corneal endothelium.

PURPOSE: We examined the efficacy and cytopathogenicity of adeno-associated (AAV) and herpes simplex viruses (HSV) as vectors for gene transfer to corneal endothelial cells (CECs). METHODS: Recombinant AAV and HSV were examined for their ability to deliver a lacZ histochemical marker gene to whole-thickness rabbit and human corneas ex vivo. Transgene expression was detected with histochemistry and quantified by a colorimetric assay. RESULTS: Rabbit and human corneas transduced with AAV showed increasing numbers of cells expressing marker gene over a 3- to 4-week period. Using 2.5 x 10(6) or 1.5 x 10(7) infective units for rabbit and human corneal specimens, respectively, approximately 2% of CECs expressed the reporter gene. HSV (10(6) plaque-forming units/specimen) transduced approximately 5% of rabbit and human CECs but showed cytotoxicity. In contrast to the duration of recombinant AAV-mediated lacZ expression, recombinant HSV expression was maximal at day 1 and declined to low levels at day 7. CONCLUSION: AAV is a promising vector, but its usefulness for corneal transduction is currently limited by the technical difficulties preparing high titres. The HSV vector examined is efficient but needs further genetic modification to prolong transgene expression and reduce its toxicity.

Adhesion molecule expression in a rat model of Staphylococcus aureus endophthalmitis.

PURPOSE: To determine whether Staphylococcus aureus and its components induce expression of E-selectin and intercellular adhesion molecule (ICAM)-1 in rat ocular tissues and on human endothelial cells in culture. METHODS: Experimental and control rat eyes were injected with 80 colony-forming units of viable S. aureus and lipopolysaccharide-free sterile saline (NS), respectively. Eyes were enucleated and immediately frozen. E-selectin and ICAM-1 expression were evaluated on frozen sections by using standard immunohistochemical techniques. Using an enzyme-linked immunoassay, in vitro expression of E-selectin and ICAM-1 was evaluated on macrovascular endothelial cells after stimulation with S. aureus and selected purified components. RESULTS: In S. aureus-injected eyes, E-selectin and ICAM-1 expression peaked at six to 24 hours, decreased slightly at 24 and 48 hours, and further declined by 72 hours. However, in NS-injected eyes, peak levels of E-selectin and ICAM-1 were seen at 6 hours, after which expression declined in the areas in which an increase was previously observed. In in vitro assays, peptidoglycan (0.01 microg/ml) induced a fourfold increase in E-selectin (P < 0.0001) and a twofold increase in ICAM-1 (P < 0.002) expression. Ribitol teichoic acid (RTA) (1 microg/ml) induced a twofold increase in E-selectin (P < 0.0001) and a threefold increase in ICAM-1 (P < 0.0001) expression. CONCLUSIONS: Eyes injected with S. aureus demonstrated a more intense and prolonged expression of both E-selectin and ICAM-1 than did eyes injected with NS. In addition, S. aureus components induced the in vitro expression of these adhesion molecules on macrovascular endothelial cells. The relevance of these findings to microvascular endothelial cells is yet to be determined.

Activated polyamidoamine dendrimers, a non-viral vector for gene transfer to the corneal endothelium.

We investigated the efficiency of activated polyamidoamine dendrimers, a new class of nonviral vectors, to transfect rabbit and human corneas in ex vivo culture. In addition to assessing the expression of a marker gene we have demonstrated that this approach can be used to induce the production of TNF receptor fusion protein (TNFR-Ig), a protein with therapeutic potential. Whole thickness rabbit or human corneas were transfected ex vivo with complexes consisting of dendrimers and plasmids containing lacZ or TNFR-Ig genes. Following optimisation 6-10% of the corneal endothelial cells expressed the marker gene. Expression was restricted to the endothelium and was maximal after transfection with 18:1 (w/w) activated dendrimer:plasmid DNA ratio and culture for 3 days. The supernatant of corneas transfected with TNFR-Ig plasmid contained TNFR-Ig protein which was able to inhibit TNF-mediated cytotoxicity in a bioassay. We have therefore shown that activated dendrimers are an efficient nonviral vector capable of transducing corneal endothelial cells ex vivo. They may have applications in gene-based approaches aimed at prevention of corneal allograft rejection or in treatment of other disorders of corneal endothelium.

Quantitative estimation of RNA transcripts suggests persistence of Pax-6 expression in the postembryonic chick retina.

Pax-6 is expressed during early embryonic development of the eye. Very little is known about its expression in the functionally mature retina. We have detected Pax-6 transcripts in the ganglion cell- and amacrine cell layers at days 3, 10, 17 and 31 posthatching and in 2- to 3-month-old chick retina by in situ hybridization. These observations were confirmed by a quantitative analysis. Competitive RT-PCR with a homologous internal control revealed a significant reduction (p < 0.001) in the number of Pax-6 transcripts in day 17 retina [(0.39 +/- 0.13) x 10(10)/mg tissue] compared to day 3 retina [(1.65 +/- 0.48) x 10(10)/mg tissue]. Although significantly lower than at day 3, the day 31 retina [(0.7 +/- 0.16) x 10(10)/mg tissue] and retina from 2- to 3-month-old chicks [(0.9 +/- 0.28) x 10(10)/mg tissue] contained an increased number of Pax-6 transcripts in comparison to day 17. On the basis of the amount of RNA, the number of Pax-6 transcripts in the day 3 retina [(0.45 +/- 0.14) x 10(10)/microg RNA] relative to day 17 retina [(0.4 +/- 0.08) x 10(10)/microg RNA] did not change significantly (p = 0.29). However, at day 31 and at 2-3 months of age an increased number of Pax-6 transcripts [(0.65 +/- 0.14) x 10(10)/microg RNA and (0.65 +/- 0.2) x 10(10)/microg RNA, respectively)] were found. In view of the known association of Pax-6 expression with proliferation and emergence of different cell types, these data suggest that cell types in ganglion and inner nuclear cell layers may retain proliferative potential for an extended period in the young adult retina.

Botulinum toxin in childhood strabismus.

The use of botulinum toxin A (BTXA) in childhood strabismus is still a matter of debate. This study investigates the indications for and outcome of BTXA therapy in children at our institution. From 1985 to 1995, 237 children up to and including 16 years of age were treated with BTXA for strabismus. We undertook a retrospective study of 163 (69%) children from this group. Factors considered were age; anaesthesia; number of, indication for and outcome of injections; complications and follow-up. There were three major indications for the use of BTXA in children: firstly to improve binocular function, secondly as a post-operative diplopia test or for cosmetic reasons, and thirdly in the investigation or treatment of paralytic and restrictive strabismus. In the first group (54 children), BTXA produced improved binocular function in 54% of all patients treated and in 49% of those with a minimum follow-up of 12 months. In the second group (82 children), 88% showed informative post-operative diplopia tests and 44% had more than one injection to maintain improved cosmetic alignment. The third group comprised 27 children with a range of diagnoses, including 1 third nerve paresis, 12 unilateral or bilateral sixth nerve pareses, 7 unilateral or bilateral Duane's syndromes, 5 lost or fibrosed muscles and 2 others. This group had a range of outcomes which are discussed in the text. BTXA is useful in the treatment of a select group of children with strabismus. If there is evidence of threatened or recently lost binocularity, or risk of creating or worsening diplopia after surgery, it is a useful therapeutic tool. In children with strabismus of unusual cause it has diagnostic value.

Distribution of integrins alpha v beta 5, alpha v beta 3 and alpha v in normal human cornea: possible implications in clinical and therapeutic adenoviral infection.

PURPOSE: Integrins are heterodimeric cell surface molecules involved in cell-cell and cell-matrix interactions. Adenoviral entry into human cells has been shown to be dependent on integrins alpha v beta 5 and alpha v beta 3 that promote viral internalisation. We studied the distribution of integrins alpha v beta 5, alpha v beta 3 and the alpha v chain in normal human cornea to investigate possible mechanisms of adenoviral entry to specific corneal cell types. METHODS: We used immunohistochemistry with monoclonal antibodies to study the distribution of alpha v beta 5, alpha v beta 3 and alpha v in normal human corneas maintained for up to 4 days in corneal storage medium (Optisol) at 4 degrees C (n = 9). RESULTS: Both alpha v beta 5 and alpha v were present to a variable extent on the corneal epithelium and corneal endothelium of most specimens. In some specimens staining of both alpha v beta 5 and alpha v in the epithelium was graded, with more basal than superficial staining, alpha v beta 3 was not detectable in either the corneal epithelium or the corneal endothelium in those specimens tested. CONCLUSIONS: The integrin alpha v beta 5 is present on both epithelium and endothelium in the normal human cornea. The role of alpha v integrins in clinical infection and in adenoviral entry for gene transfer is discussed.

Gene transfer to ex vivo stored corneas.

PURPOSE: We examined the efficiency and kinetics of recombinant adenovirus vector-mediated gene transfer to rat and rabbit cornea in culture ex vivo. METHODS: A recombinant replication-defective adenovirus was used to transfer a lacZ marker gene to whole rat and rabbit corneas in culture. Histochemistry was used to localise transgene expression and a colorimetric assay to quantify recombinant protein expression. RESULTS: After infection with recombinant virus and culture for 3 days, high-efficiency gene transfer was found, with expression in most endothelial cells of both species. Minimal expression was found in other corneal cell types. On histochemistry, longer duration of expression was found in rat than in rabbit endothelium. In both rat and rabbit cornea, highest levels of recombinant protein were found at days 3-7 after incubation with virus, decreasing to low or undetectable levels at 21 days. CONCLUSION: Adenovirus vectors allow high-efficiency transgene expression in cornea, largely restricted to the endothelial cells of ex vivo cultured cornea. Kinetics of expression differ according to the species of cornea studied, a factor that must be considered if this vector is used in further studies.

Ocular chloramphenicol and aplastic anaemia. Is there a link?

The question of a link between the use of topical ocular chloramphenicol and the incidence of aplastic anaemia continues to be a controversial issue in ophthalmological spheres. At present topical ocular chloramphenicol is widely used in the UK for the treatment of conjunctivitis, whereas it is very rarely prescribed for this indication in the U.S. Individual policies vary around the rest of the world. The evidence for and against any association between topical and ocular chloramphenicol and an increased risk of aplastic anaemia is reviewed, and the reasons behind the current prescribing policies are clarified. The discussion generated in the literature over the past 2 years over this issue is considered, along with the published debate from the past 3 decades. The debate is not conclusive, but by presenting or referencing the specific case reports and the published opinions of various experts, we hope to enable the reader to make his or her own informed decision as to whether use of the topical preparation of chloramphenicol should be considered by the ophthalmological community.

Human neuroblastoma cell lines that express N-myc without gene amplification.

BACKGROUND: About one half of aggressive neuroblastomas lack N-myc amplification. Cell lines from such tumors are needed to determine the biological basis of aggressive tumor behavior. METHODS: Neuroblastoma cell lines were established from a primary tumor (SMS-LHN) and a bone marrow metastasis (LA-N-6) of two children with Stage IV neuroblastoma. Although both cell lines and their original tumors lacked N-myc genomic amplification, these patients died of progressive disease. RESULTS: SMS-LHN and LA-N-6 can be distinguished from primitive neuroectodermal tumor (PNET) lines by cell surface antigen expression and catecholamine production. Cytogenetic analysis of each cell line revealed unique genetic rearrangements, whereas both lines showed abnormalities involving chromosome 2. Neither cell line contained double-minute chromosomes, homogeneously staining regions, a 1p chromosomal deletion, or t(11;22). The growth rates of these two new lines in vitro and in vivo (as xenografts in nude mice) are slower than N-myc amplified neuroblastoma lines. Both lines express greater amounts of N-myc RNA and protein relative to nonneuroblastoma cell lines (including PNET), although not to the extent of cell lines with N-myc genomic amplification. CONCLUSIONS: The relatively large amount of N-myc expression in these two new cell lines suggests that N-myc expression without amplification could play a role in the pathogenesis of some neuroblastomas. These cell lines should be useful for investigating mechanisms and consequences of N-myc gene activation other than genomic amplification.

Morphology, growth, chromosomal pattern and fibrinolytic activity of two new human neuroblastoma cell lines.

Neuroblastoma cell lines LA-N-1 and LA-N-2 were extablished from neuroblastoma cells in the bone marrow and in the primary tumor, respectively, of two children with metastatic neuroblastoma. Morphology, growth in vitro and in athymic nude mice, chromosomal patter, and fibrinolytic activity of these cell lines and of previously extablished human neuroblastoma cell lines IMR-32, SK-N-MC, and SK-N-SH were compared. Most LA-N-1 cells were tear-drop shaped, small cells with processes; they tended to grow in clusters. LA-N-2 was comprised of elongated cells and small round cells, the latter growing in dense clumps on the former. Electron microscopy revealed numerous cytoplasmic dense cores in many LA-N-1 cells but none in LA-N-2 CELLS. During logarithmic growth in vitro, doubling times for LA-N-1, LA-N-2, SK-N-MC, SK-N-SH, and IMR-32 cells were 32,56, 23, 36, and 26 hr, respectively. Cells of all lines formed colonies in soft agar, and, after variable latency periods, LA-N-1, LA-N-2, SK-N-MC, and IMR-32 cells formed tumors in athymic nude mice. The marker chromosome(s) characteristic of each cell line was present in more than 90% of cells of given line. Significant plasminogen-dependent fibrinolytic activity was present in cells of all lines. These studies indicate that LA-N-1 and LA-N-2 cells arose from single but different aberrant progenitor cells and that they have properties of neuroblastoma cells. They also demonstrate that cell lines derived from human neuroblastomas are heterogenous as are the tumors in children.