Retrovirus receptor mRNA expression correlates with gene transfer efficiency in pluripotent hematopoietic stem cells.

Hematopoietic stem cells (HSC) from bone marrow, peripheral blood and cord blood are important in clinical transplantation. However, their use in gene therapy protocols is still limited by a low level of transduction efficiency. In addition to the cell cycling block to retrovirus transduction, we recently demonstrated that the low level of retrovirus receptor mRNA in mouse HSC correlated with the low level of amphotropic retrovirus transduction in these cells. Similarly, we found low levels of mRNA encoding the amphotropic retrovirus receptor in human bone marrow Lin CD34+ CD38- HSC. In an effort to identify an alternative population of human HSC that might be more efficiently transduced, we assayed HSC populations from cord blood for mRNA encoding the amphotropic retrovirus receptor. High levels of receptor mRNA were present in HSC from previously cryopreserved cord blood compared with HSC from fresh bone marrow and fresh cord blood. The HSC from cryopreserved cord blood are excellent candidates for gene therapy protocols.

Interleukin-7R alpha mRNA expression increases as stem cells differentiate into T and B lymphocyte progenitors.

The gamma common (gamma c) chain is a partner in several interleukin receptor complexes, including the interleukin-2 receptor (IL-2R), IL-4R, and IL-7R. Mutations in the gamma c gene are associated with X-linked severe combined immunodeficiency (SCID). Using reverse transcriptase-PCR, we examined the level of mRNA-encoding gamma c and its partners in mouse pluripotent hematopoietic stem cells (PHSCs), which repopulate both bone marrow and thymus. We also assayed developing lymphocytes to define which, if any, IL-R complexes are expressed at the earliest stage of T and B lymphocyte maturation. RNA extracted from bone marrow-derived PHSCs did not contain detectable levels of mRNA-encoding IL-7R alpha. However, the most primitive (CD4- CD8-) T cells from the thymus and the most primitive (c-kit+ B220+) B cells from bone marrow contained high levels of IL-7R alpha mRNA. There were no detectable differences between PHSCs and primitive or more mature T and B cells for expression of gamma c mRNA. We conclude that the onset of IL-7R formation occurs at the earliest stage of differentiation of T and B lymphocytes. Our findings are consistent with the hypothesis that the absence of an intact IL-7R (IL-7R alpha and gamma c) may be a critical loss that interrupts lymphopoiesis.

Granulocyte colony-stimulating factor mobilized peripheral blood stem cells enter into G1 of the cell cycle and express higher levels of amphotropic retrovirus receptor mRNA.

We compared the cell cycle status and expression of mRNA for the amphotropic retroviral receptor in hematopoietic stem cells isolated from bone marrow and cytokine mobilized peripheral blood. CD34+ cells from six normal volunteers were enriched by immune selection from steady-state bone marrow and granulocyte colony-stimulating factor (G-CSF) mobilized peripheral blood (10 microg/kg/day for 5 days). Cell cycle status of the phenotypically primitive CD34+CD38- hematopoietic stem cell population was analyzed using a four-color flow cytometry technique that distinguished the G0, G1, and S/IG2/M phases of the cell cycle. Semiquantitative reverse transcriptase-polymerase chain reaction was performed to measure mRNA expression of the amphotropic retroviral receptor. Peripheral blood hematopoietic stem cells had 2.6-fold more cells in the G1 phase of the cell cycle compared to steady-state bone marrow. Furthermore, lineage CD34+CD38- cells from G-CSF mobilized peripheral blood had a fourfold higher level of amphotropic retrovirus receptor mRNA. In conclusion, we found that CD34+ CD38- hematopoietic stem cells isolated from G-CSF mobilized peripheral blood differ from those isolated from steady-state bone marrow in that a significant proportion have entered the G1 phase of the cell cycle and express higher levels of amphotropic receptor mRNA. These biologic properties are consistent with the reported rapid recovery of hematopoietic function following transplantation with peripheral blood hematopoietic stem cells and make these cells a preferred target for retroviral-based gene transfer.

Illuminated orbital retractor.

An illuminated orbital retractor that has been designed especially for microsurgery is made of methylmethacrylate and is shaped like an Arruga retractor. It is illuminated by fiber optics. Indications for the use of the illuminated retractor include exposure for repairing scleral lacerations, decompressions of the optic nerve, and orbital fractures.

Amphotropic retrovirus transduction of hematopoietic stem cells.

Mice treated with cytokines for 5 days have large numbers of hematopoietic stem cells (HSCs) in their peripheral blood and bone marrow at 1 and 14 days after the last injection. We fractionated the HSCs from the bone marrow of these mice using elutriation at flow rates of 25, 30 and 35 ml/min. The subpopulations of HSCs from cytokine-treated mice show a 3- to 8-fold higher level of mRNA encoding the amphotropic retrovirus receptor (amphoR) compared with the corresponding HSC subpopulation from untreated mouse bone marrow. In an earlier study with mouse HSCs we showed a direct correlation between high levels of amphoR mRNA and efficient retrovirus transduction. We have now utilized our gene transfer protocol to assay amphotropic retrovirus transduction efficiency using HSCs from the bone marrow of mice treated with granulocyte-colony stimulating factor/stem cell factor (G-CSF/SCF). To extend these findings to a more clinically relevant protocol we analyzed the amphoR mRNA levels in HSCs from human cord blood and adult bone marrow. The amphoR mRNA level in HSCs from human bone marrow and fresh cord blood was detectable at an extremely low level compared with the HSC population in cryopreserved cord blood samples. The 12- to 22-fold increase in amphoR mRNA in HSCs from cryopreserved cord blood renders these HSCs likely candidates for high efficiency, gene transfer.

Improved amphotropic retrovirus-mediated gene transfer into hematopoietic stem cells.

The efficiency of amphotropic retrovirus-mediated gene transfer into human Hematopoietic Stem Cells (HSC) is less than 1%. This has impeded gene therapy for hematopoietic diseases. In this study we demonstrate that populations of mouse and human HSC contain low to undetectable levels of the amphotropic virus receptor mRNA (ampho R mRNA), and are resistant to transduction with amphotropic retroviral vectors. In a subpopulation of mouse HSC expressing 7-fold higher levels of ampho R mRNA, transduction with amphotropic retrovirus vectors was 30-fold higher. We conclude that retrovirus transduction of HSC correlates with ampho R mRNA levels. Our results predict that alternative sources of HSC or retroviruses will be required for human gene therapy of hematopoietic diseases. One alternative source of stem cells is from individuals treated with cytokines. We have previously shown that mice treated with G-CSF and SCF have an immediate increase in peripheral blood HSC immediately after treatment, followed by a 10-fold increase in bone marrow HSC 14 days after treatment. In this report we show that when rhesus monkey bone marrow cells collected 14 days after G-CSF and SCF treatment were transduced with amphotropic retroviruses, gene transfer levels were approximately 10%, which was easily detected by Southern blot analysis. We conclude that the increased gene transfer may be the result of increased expression of the amphotropic retrovirus receptor, increased numbers of cycling HSC or both.

Pars plana lensectomy by ultrasonic fragmentation. I. Versatile and simple technique for cataract removal.

A comparison has been made between pars plana lansectomy and intracapsular cataract extraction Pars plana lensectomy has been shown to have many advantages. It is applicable to any age, while intracapsular cataract extraction is restricted to patients over 30 years old. It can be used in any type of cataract, whereas intracapsular cataract extraction is restricted to senile cataracts. It requires less patient preparation, less instrumentation, and less surgical dexterity than does intracapsular cataract extraction. It allows intraocular examination at the time of surgery. It can be combined with other surgical procedures (intraocular lens implantation, vitrectomy, cryoretinopexy, scleral buckle, etc.). It leaves the eyes in better condition postoperatively with fewer postoperative complications, and it gives good or better visual results than intracapsular cataract extraction. It is now unnecessary to learn (and teach) multiple techniques to handle different ages and types of cataracts since pars plana lensectomy manages them all with fewer complications.

Reducing surgically induced astigmatism by using a scleral tunnel.

We conducted a prospective study of 36 eyes undergoing pars plana lensectomy, vitrectomy, and implantation of an intraocular lens. We used a scleral tunnel begun 3 mm posterior to the corneoscleral limbus and entered the anterior chamber through clear cornea. We found that this technique produced minimal postoperative astigmatism. After follow-up periods ranging from three to 15 months, the average change in the flatter meridian was -0.07 diopter and that in the steeper meridian was +0.26 diopter. The mean induced astigmatism was -0.33 diopter. A group of nine eyes undergoing pars plana lensectomy and vitrectomy alone showed no significant changes in the postoperative astigmatism. Statistical comparisons established that there was no significant difference between eyes undergoing intraocular lens implantation in addition to pars plana lensectomy and vitrectomy and those not undergoing intraocular lens implantation.

Effect of penetrating keratoplasty using grafts of various sizes on keratoconic myopia and astigmatism.

The records of 72 consecutive keratoconic eyes undergoing penetrating keratoplasty were reviewed for changes in myopia and astigmatism. Ages of the patients averaged 32.7 years. All sutures were removed after three months. Follow-up average was 40.2 months. Results showed an average decrease in myopia of 6.63 diopters (D) in 60 eyes (82.86%) and an average increase in myopia of 1.88 D in 12 of 70 eyes (17.14%). The decrease/increase in myopia and postoperative astigmatism was compared for grafts equal to the opening, grafts smaller than the opening, and grafts larger than the opening. The largest average decrease in myopia was 13.86 D (range 6.63 to 20.00), which occurred when a graft smaller than the opening was used (P less than .01). This group also showed the least postoperative astigmatism (2.82 D) (P less than or equal to .01). From this study, it appears that the use of a graft 0.25 mm smaller than the trephine opening in the host (i.e., 7.50 mm graft/7.75 mm opening) for penetrating keratoplasty in keratoconus is justified. A prospective study is now in progress.

Scleral fixation of a subluxated posterior chamber intraocular lens.

A surgical technique for the treatment of a subluxated intraocular lens is described. It consists of a scleral incision 2 mm from the limbus, hooking the loop haptic of the implant into the scleral wound, and imbricating the loop into the sclera for permanent fixation. Four eyes in which this technique was applied successfully are reported.

Use of grafts smaller than the opening for keratoconic myopia and astigmatism. A prospective study.

A prospective study was conducted on 15 consecutive keratoconic eyes to evaluate the use of grafts smaller than the opening in keratoconic myopia and astigmatism. All surgeries were performed by the senior author. Average age of the patients was 41.1 years. Average follow-up was 1.6 years. After all sutures were removed, results showed an average decrease in myopia of 13.24 diopters (D) (range 1.75 to 23.25) principally from corneal flattening and a small reduction in axial length. Average postoperative spherical equivalent was -2.17 D (range +1.50 to -7.25). The average postoperative astigmatism was 3.78 D (range 1.75 to 6.00). This study and a previous retrospective study suggest that the use of grafts 0.25 mm smaller than the opening, i.e., 7.50/7.75 mm, for penetrating keratoplasty in keratoconus is justified.

Ligneous conjunctivitis after pingueculae removal in an adult.

A 45-year-old man who had uneventful excision of bilateral pingueculae developed bilateral membranous lesions involving the bulbar conjunctivae and corneas. Histologically, the membranes were composed mainly of large fibrinous deposits intermixed with acute and chronic inflammatory cells with areas of fibroblastic and capillary proliferation resembling granulation tissue. By electronmicroscopy the amorphous acidophilic masses were composed of electron-dense, fibrillar material with a periodicity of 10-12 mm, which was consistent with fibrin. Despite mechanical removal of the membranes, they continued to recur rapidly over a period of several months. The lesions apparently responded slowly to topical enzymatic therapy that consisted of hyaluronidase (175 U/ml) and alpha-chymotrypsin (1:5000) drops. Follow-up examination, approximately 1 year after surgery, revealed that the patient was asymptomatic. Ocular examination disclosed slight persistence of gelatinous membranes on the bulbar conjunctivae, most prominent in the left eye.

Nystagmus reduction by horizontal rectus retroplacements.

Seven patients with nystagmus underwent large retroplacements of the four horizontal rectus muscles. Nystagmus was horizontal in all seven patients, but three had a vertical component. The horizontal nystagmus improved in all seven patients. All improved at least one line for distance and from three to five lines for near. Retroplacement of the four horizontal rectus muscles is a safe and effective procedure for the reduction of nystagmus and the improvement of vision, particularly for near vision, producing little or no induced strabismus or limitation of motility.