Macular translocation with chorioscleral outfolding: an experimental study.

PURPOSE: Macular translocation by chorioscleral infolding has been proposed as a surgical intervention for exudative age-related macular degeneration, but the surgery is unpredictable and can be associated with severe complications. We tested a new surgical technique, macular translocation with chorioscleral outfolding secured by neurosurgical clips. METHODS: This was a prospective interventional study in two parts; the first in human cadaver eyes and the second in pigs. Chorioscleral infolding was performed on six human donor eyes, and chorioscleral outfolding was performed on an additional six. The inner surface of the eye wall was measured, and then the fold was unfolded and the distance was measured again. In the second half of the study, macular translocation surgery was performed on 33 pig eyes with one of three sclera shortening methods: 1) a circumferential chorioscleral infolding using 5-0 nylon sutures, 2) a circumferential chorioscleral outfolding using scleral clips, or 3) a radial chorioscleral outfolding using scleral clips. Foveal translocation was measured. RESULTS: The inner wall of the human cadaver eye was shortened in the chorioscleral infolding group by a mean of 1.6 mm, and in the chorioscleral outfolding group by 3.0 mm. In the pig eyes, the fovea was translocated a mean 2377 microm by circumferential suturing, 2582 microm by circumferential clipping, and 3386 microm by radial clipping. Irregular deformation of the globe was more apparent in the circumferential suture group. Undesirable retinal folds often formed after circumferential infolding but not after radial clipping. CONCLUSION: Radial chorioscleral outfolding with clips is more predictable and effective than infolding. It produces more translocation and prevents folds across the fovea, one of the most undesirable complications in macular translocation surgery.

Echography of retinoblastoma: histopathologic correlation and serial evaluation after globe-conserving radiotherapy or chemotherapy.

PURPOSE: To assess the sensitivity of echography in detecting retinoblastoma, compare tumor features observed by echography with histopathology data, and assess the usefulness of echography in serially following retinoblastoma tumors after globe-conserving treatments. METHODS: The medical and echography records of all patients treated for retinoblastoma at the Bascom Palmer Eye Institute between 1991 and 1997 were reviewed. All eyes underwent pretreatment echographic evaluation, and eyes treated with external beam radiotherapy, brachytherapy, or chemotherapy underwent serial follow-up echography. RESULTS: Sixty-nine eyes of 48 patients were identified. Echography demonstrated evidence of retinoblastoma in 69 of 69 (100%) eyes and calcification in 63 (91.3%) eyes. Histopathology was superior to echography in detecting optic nerve invasion, extraocular extension, and presence of calcification. CONCLUSION: Echography is a useful adjunct to indirect ophthalmoscopy in establishing the diagnosis of retinoblastoma. While not as specific as histopathology, echographic evaluation before and after treatment of retinoblastoma permits monitoring of treatment response and may aid in detecting recurrent tumor growth or failure to respond to treatment.

Functional organization of single and paired V(D)J cleavage complexes.

RAG-1 and RAG-2 initiate V(D)J recombination by binding to specific recognition sequences (RSS) and then cleave the DNA in two steps: nicking and hairpin formation. Recent work has established that a dimer of RAG-1 and either one or two monomers of RAG-2 bind to a single RSS, but the enzymatic contributions of the RAG molecules within this nucleoprotein complex and its functional organization have not been elucidated. Using heterodimeric protein preparations containing both wild-type and catalytically deficient RAG-1 molecules, we found that one active monomer is sufficient for both nicking and hairpin formation at a single RSS, demonstrating that a single active site can carry out both cleavage steps. Furthermore, the mutant heterodimers efficiently cleaved both RSS in a synaptic complex. These results strongly suggest that two RAG-1 dimers are responsible for RSS cleavage in a synaptic complex, with one monomer of each dimer catalyzing both nicking and hairpin formation at each RSS.

RAG transposase can capture and commit to target DNA before or after donor cleavage.

The discovery that the V(D)J recombinase functions as a transposase in vitro suggests that transposition by this system might be a potent source of genomic instability. To gain insight into the mechanisms that regulate transposition, we investigated a phenomenon termed target commitment that reflects a functional association between the RAG transposase and the target DNA. We found that the V(D)J recombinase is quite promiscuous, forming productive complexes with target DNA both before and after donor cleavage, and our data indicate that the rate-limiting step for transposition occurs after target capture. Formation of stable target capture complexes depends upon the presence of active-site metal binding residues (the DDE motif), suggesting that active-site amino acids in RAG-1 are critical for target capture. The ability of the RAG transposase to commit to target prior to cleavage may result in a preference for transposition into nearby targets, such as immunoglobulin and T-cell receptor loci. This could bias transposition toward relatively "safe" regions of the genome. A preference for localized transposition may also have influenced the evolution of the antigen receptor loci.

Macular translocation for subfoveal choroidal neovascularization in angioid streaks.

PURPOSE: To report a case of visual improvement after macular translocation performed for a subfoveal choroidal neovascular membrane in a patient with pseudoxanthoma elasticum and angioid streaks. METHODS: The fovea was translocated inferiorly by scleral imbrication, intentional retinal detachment with a small posterior retinotomy, and partial fluid-air exchange. The choroidal neovascular membrane was photocoagulated 1 week later. RESULTS: The visual acuity of the patient improved from 20/125 to 20/40. The center of the foveal avascular zone was moved inferiorly 844 microm. The choroidal neovascular membrane was extrafoveal after translocation and was treated with laser photocoagulation. CONCLUSION: Macular translocation may be considered in the management of subfoveal choroidal neovascular membrane in patients with pseudoxanthoma elasticum and angioid streaks.

Ku80 is required for addition of N nucleotides to V(D)J recombination junctions by terminal deoxynucleotidyl transferase.

V(D)J recombination generates a remarkably diverse repertoire of antigen receptors through the rearrangement of germline DNA. Terminal deoxynucleotidyl transferase (TdT), a polymerase that adds random nucleotides (N regions) to recombination junctions, is a key enzyme contributing to this diversity. The current model is that TdT adds N regions during V(D)J recombination by random collision with the DNA ends, without a dependence on other cellular factors. We previously demonstrated, however, that V(D)J junctions from Ku80-deficient mice unexpectedly lack N regions, although the mechanism responsible for this effect remains undefined in the mouse system. One possibility is that junctions are formed in these mice during a stage in development when TdT is not expressed. Alternatively, Ku80 may be required for the expression, nuclear localization or enzymatic activity of TdT. Here we show that V(D)J junctions isolated from Ku80-deficient fibroblasts are devoid of N regions, as were junctions in Ku80-deficient mice. In these cells TdT protein is abundant at the time of recombination, localizes properly to the nucleus and is enzymatically active. Based on these data, we propose that TdT does not add to recombination junctions through random collision but is actively recruited to the V(D)J recombinase complex by Ku80.

Joining-deficient RAG1 mutants block V(D)J recombination in vivo and hairpin opening in vitro.

The RAG proteins cleave at V(D)J recombination signal sequences then form a postcleavage complex with the broken ends. The role of this complex in end processing and joining, if any, is undefined. We have identified two RAG1 mutants proficient for DNA cleavage but severely defective for coding and signal joint formation, providing direct evidence that RAG1 is critical for joining in vivo and strongly suggesting that the postcleavage complex is important in end joining. We have also identified a RAG1 mutant that is severely defective for both hairpin opening in vitro and coding joint formation in vivo. These data suggest that the hairpin opening activity of the RAG proteins plays an important physiological role in V(D)J recombination.

Separation-of-function mutants reveal critical roles for RAG2 in both the cleavage and joining steps of V(D)J recombination.

The only established physiological function of the V(D)J recombinase, comprising RAG1 and RAG2, is to perform DNA cleavage. The molecular roles of RAG2 in cleavage, the mechanisms used to join the broken DNA ends, and the identity of nuclease(s) that open the hairpin coding ends have been unknown. Site-directed mutagenesis targeting each conserved basic amino acid in RAG2 revealed several separation-of-function mutants that address these questions. Analysis of these mutants reveals that RAG2 helps recognize or cleave distorted DNA intermediates and plays an essential role in the joining step of V(D)J recombination. Moreover, the discovery that some mutants block RAG-mediated hairpin opening in vitro provides a critical link between this biochemical activity and coding joint formation in vivo.

Screening for retinopathy of prematurity employing the retcam 120: sensitivity and specificity.

OBJECTIVES: To compare the method of photographic screening using the RetCam 120 to the standard method of screening for retinopathy of prematurity (ROP) by ophthalmologic examination. METHODS: A total of 100 RetCam 120 photoscreening examinations of the retina were performed on 32 premature infants. These were stored in a separate file from which all identifying information was removed. At this same examination, a detailed ophthalmological evaluation, employing the indirect ophthalmoscope with scleral depression, was performed by an experienced examiner. Masked examiners performed an evaluation of the fundus photographs to identify presence or absence of ROP, the location and extent of the disease, and the presence or absence of plus disease. These data were then compared with the results of the ophthalmological examination to determine the specificity, sensitivity, and the positive and negative predictive value (PPV and NPV) of the method. RESULTS: Retinopathy of prematurity was detected in 68 of 100 subjects by ophthalmologic examinations and in 58 of 100 subjects' photoscreening examinations. No ROP was detected in 32 of 100 subjects. The sensitivity of the method was 56 (82.4%) of 68 and the specificity was 30 (93.8%) of 32. The PPV was 96.6%; NPV, 76.9%. CONCLUSIONS: The sensitivity of the method was low. The ROP that was missed was peripheral stage 1 or stage 2 disease in peripheral zone 2 or zone 3. This was largely due to the technical limitations of the speculum-camera interface preventing a better view of the periphery. The ROP cases that were missed by the photographic examination regressed spontaneously on follow-up. No disease more posterior to peripheral zone 2 was overlooked. These results detail the accuracy of the method employing the technique of photoscreening as a potential substitute for detailed ophthalmological examination. At present there are clear technical limitations to such a substitution. The study is part of an ongoing project to determine the feasibility of employing neonatal nurses trained to take digitized images of the premature infant's retina and telemeter the results to be read by an experienced ophthalmologist remote from the site.

Conditional RAG-1 mutants block the hairpin formation step of V(D)J recombination.

Hairpin formation serves an important regulatory role in V(D)J recombination because it requires synapsis of an appropriate pair of recombination sites. How hairpin formation is regulated and which regions of the RAG proteins perform this step remain unknown. We analyzed two conditional RAG-1 mutants that affect residues quite close in the primary sequence to an active site amino acid (D600), and we found that they exhibit severely impaired recombination in the presence of certain cleavage site sequences. These mutants are specifically defective for the formation of hairpins, providing the first identification of a region of the V(D)J recombinase necessary for this reaction. Substrates containing mismatched bases at the cleavage site rescued hairpin formation by both mutants, which suggests that the mutations affect the generation of a distorted or unwound DNA intermediate that has been implicated in hairpin formation. Our results also indicate that this region of RAG-1 may be important for coupling hairpin formation to synapsis.

Bilateral blindness as the initial presentation of lymphoma of the sphenoid sinus.

PURPOSE: To report a patient with large-cell lymphoma of the sphenoid sinus presenting with bilateral blindness and no other signs or symptoms. METHOD: Case report. A previously healthy 5-year-old boy complained of sudden vision loss without other systemic complaints. RESULTS: Ophthalmologic examination revealed no light perception bilaterally. The pupils of the patient were fixed at 8 mm without reaction to the brightest light stimulus. Systemic examination was unremarkable, and neuroimaging revealed a large sphenoid tumor extending intracranially. Biopsy of the tumor proved to be large-cell lymphoma. CONCLUSION: Large-cell lymphoma affecting children may present initially with blindness, without other systemic symptoms.

Differential requirements for cis and trans V(D)J cleavage: effects of substrate length.

The assembly of productive synaptic complexes is a critical, but poorly understood, regulatory step in V(D)J recombination. Several lines of evidence suggest that there may be important differences between recombination involving sites situated in cis (on the same DNA molecule) and in trans (on separate molecules). Because biochemical experiments using both purified RAG proteins and crude extracts have failed to detect trans cleavage of plasmid substrates it has been thought that there is a substantial bias against trans synapsis. In conflict with these results are more recent studies showing that purified RAG proteins can catalyze trans cleavage of short oligonucleotide substrates. Furthermore, recent experiments have detected efficient trans cleavage of plasmid substrates in vivo. We sought to investigate why these different systems yield such divergent results. We found that, unexpectedly, the ability of both purified RAG proteins and crude extracts to cleave DNA substrates in trans is a function of substrate length. Our data raise two critical issues: first, oligonucleotides, which are the most commonly used substrates to study V(D)J recombination in vitro, do not mimic the behavior of plasmid substrates; second, in the trans cleavage reaction current purified RAG systems do not accurately reflect the in vivo situation. We propose a unifying model to explain the effects of substrate length and coniguration (cis or trans) on the efficiency of synapsis.

From lymphocytes to sharks: V(D)J recombinase moves to the germline.

The antigen-receptor genes of vertebrates are rearranged by a specialized somatic recombination mechanism in developing lymphocytes - and, unexpectedly, also in the germline of cartilaginous fishes. The recombination system that carries out these DNA rearrangements may thus be a significant evolutionary force, perhaps not limited to rearrangements at antigen-receptor loci.

Mutational analysis of RAG1 and RAG2 identifies three catalytic amino acids in RAG1 critical for both cleavage steps of V(D)J recombination.

RAG1 and RAG2 initiate V(D)J recombination, the process of rearranging the antigen-binding domain of immunoglobulins and T-cell receptors, by introducing site-specific double-strand breaks (DSB) in chromosomal DNA during lymphocyte development. These breaks are generated in two steps, nicking of one strand (hydrolysis), followed by hairpin formation (transesterification). The nature and location of the RAG active site(s) have remained unknown. Because acidic amino acids have a critical role in catalyzing DNA cleavage by nucleases and recombinases that require divalent metal ions as cofactors, we hypothesized that acidic active site residues are likewise essential for RAG-mediated DNA cleavage. We altered each conserved acidic amino acid in RAG1 and RAG2 by site-directed mutagenesis, and examined >100 mutants using a combination of in vivo and in vitro analyses. No conserved acidic amino acids in RAG2 were critical for catalysis; three RAG1 mutants retained normal DNA binding, but were catalytically inactive for both nicking and hairpin formation. These data argue that one active site in RAG1 performs both steps of the cleavage reaction. Amino acid substitution experiments that changed the metal ion specificity suggest that at least one of these three residues contacts the metal ion(s) directly. These data suggest that RAG-mediated DNA cleavage involves coordination of divalent metal ion(s) by RAG1.

Irradiation-induced rescue of thymocyte differentiation and V(D)J recombination in mice lacking the catalytic subunit of DNA-dependent protein kinase.

Scid mice express a truncated form of the catalytic subunit of the DNA-dependent protein kinase (DNA-PKcs) and are unable to properly rearrange their Ig and TCR genes, resulting in a severe combined immunodeficiency that is characterized by arrested differentiation of B and T lymphocytes. Treatment of scid mice with low doses of gamma irradiation rescues rearrangements at several TCR loci and promotes limited thymocyte differentiation. The machinery responsible for sensing DNA damage and the mechanism by which irradiation compensates for the scid defect in TCR recombination remain unknown. Because DNA-PKcs is present in scid thymocytes, it may mediate some or all of the irradiation effects. To test this hypothesis, we examined the effects of irradiation on DNA-PKcs-deficient (slip) mice. Our data provide the first evidence that DNA-PKcs is not required for limited rescue of thymocyte differentiation or TCR rearrangements.

Irradiation-mediated rescue of T cell-specific V(D)J recombination and thymocyte differentiation in severe combined immunodeficient mice by bone marrow cells.

In SCID (severe combined immunodeficient) mice, proper assembly of immunoglobulin and T cell receptor (TCR) genes is blocked by defective V(D)J recombination so that B and T lymphocyte differentiation is arrested at an early precursor stage. Treating the mice with gamma irradiation rescues V(D)J rearrangement at multiple TCR loci, promotes limited thymocyte differentiation, and induces thymic lymphomas. These effects are not observed in the B cell lineage. Current models postulate that irradiation affects intrathymic T cell precursors. Surprisingly, we found that transfer of irradiated SCID bone marrow cells to unirradiated host animals rescues both TCR rearrangements and thymocyte differentiation. These data indicate that irradiation affects precursor cells at an earlier stage of differentiation than was previously thought and suggest new models for the mechanism of irradiation rescue.

Optic nerve avulsion from a golfing injury.

PURPOSE: To describe a patient with optic nerve avulsion after being struck in the eye with a golf club. METHODS: A 10-year-old male was hit in the left eye by a golf club. The patient underwent full ophthalmoscopic evaluation and neuroimaging. RESULTS: The patient had no light perception in the left eye when first seen. Avulsion of the optic nerve with vitreous hemorrhage was apparent on examination. Computed tomographic imaging of the brain and orbits revealed no abnormalities. CONCLUSIONS: Optic nerve avulsion from golf-related injury is more likely to occur when the impact site is between the globe and the orbital rim. Rupture of the globe is more likely to occur with direct impact to it.

Modulation of terminal deoxynucleotidyltransferase activity by the DNA-dependent protein kinase.

Rare Ig and TCR coding joints can be isolated from mice that have a targeted deletion in the gene encoding the 86-kDa subunit of the Ku heterodimer, the regulatory subunit of the DNA-dependent protein kinase (DNA-PK). However in the coding joints isolated from Ku86-/- animals, there is an extreme paucity of N regions (the random nucleotides added during V(D)J recombination by the enzyme TdT). This finding is consistent with a decreased frequency of coding joints containing N regions isolated from C.B-17 SCID mice that express a truncated form of the catalytic subunit of the DNA-PK (DNA-PKCS). This finding suggests an unexpected role for DNA-PK in addition of N nucleotides to coding ends during V(D)J recombination. In this report, we establish that TdT forms a stable complex with DNA-PK. Furthermore, we show that DNA-PK modulates TdT activity in vitro by limiting both the length and composition of nucleotide additions.