Neutralizing antibody to VEGF reduces intravitreous neovascularization and may not interfere with ongoing intraretinal vascularization in a rat model of retinopathy of prematurity.

PURPOSE: To study the effects of a neutralizing antibody to vascular endothelial growth factor (VEGF), given as an intravitreous injection, on intravitreous neovascularization (IVNV) and ongoing vascular development of avascular retina in a rat model relevant to human retinopathy of prematurity. METHODS: Newborn Sprague-Dawley rats were exposed to oxygen fluctuations alternating between 50% O(2) and 10% O(2) every 24 h. At postnatal day (p)12, rat pups received intravitreous injections of a neutralizing antibody to VEGF or control nonimmune rat IgG in one eye and were returned to oxygen cycling until p14, at which time they were placed into room air. At p18 (time of maximal IVNV) or p25 (time point in regression), animals were sacrificed. Their retinas were dissected, flat mounted, and stained with Alexa-isolectin for fluorescence microscopy. IVNV was measured as number of clock hours involved in injected VEGF antibody and control eyes. Mean clock hours of IVNV, avascular/total retinal areas and capillary densities within vascularized retinas were determined in injected eyes of control and treatment groups. Mean clock hours of IVNV in fellow noninjected eyes from control and treatment groups were analyzed by Student's t-tests to assess possible crossover effects from systemic absorption of antibody. Eyes from p13 rat pups were sectioned for immunohistochemistry or analyzed for VEGF receptor 2 (VEGFR2) phosphorylation by western blot. Free retinal VEGF at p13, one day following injections, was measured by ELISA. RESULTS: Neutralizing antibody to VEGF at 25 ng and 50 ng caused a modest but significant inhibition of IVNV compared to IgG injected controls at p18, but only the 50 ng dose decreased IVNV compared to control at p25 (one-way ANOVA p=0.003; posthoc Bonferroni t-test p=0.003). Neither dose caused a significant difference in avascular/total retinal area at p18 compared to control. However, at p25, the 50 ng dose caused a significant reduction in avascular/total retinal area compared to the 25 ng dose (ANOVA p=0.038; posthoc Student's t-test p=0.038). There was no difference in avascular/total retinal area between IgG and the 25 ng dose. At p13, qualitative analysis of immunohistochemical sections of retina showed the 50 ng dose of VEGF antibody reduced VEGFR2 phosphorylation within the retina and around blood vessels. Also at p13, there was a significant increase in free intraretinal VEGF protein in eyes that had been treated with 50 ng dose of VEGF antibody compared to IgG injected control (Student's t-test p=0.042). There were no differences in capillary densities in the vascularized retinas between eyes injected with the 50 ng dose of VEGF antibody and IgG control. There was also no difference in weight gain between treated and control groups. CONCLUSIONS: Neutralizing antibody to VEGF at a 50 ng dose caused a significant and sustained reduction in IVNV without interfering with ongoing retinal vascularization in a rat model of ROP, whereas a lower dose of antibody did not. These data also suggest that compensatory regulatory mechanisms may lead to increased VEGF concentration after intravitreous injection of a neutralizing antibody to VEGF. Further study is necessary for safety and for determination of drug dose of VEGF antibody, since dose of treatment appears important and may vary among infants with severe ROP. In this study, survival of already developed retinal capillaries did not appear affected. Neutralizing VEGF by an intravitreous injection of antibody may offer a treatment consideration for severe ROP, which fails current standard of care management.

Effect of prophylactic amoxicillin on endodontic flare-up in asymptomatic, necrotic teeth.

The purpose of this prospective, randomized, double-blind, placebo-controlled study was to determine the effect of prophylactic amoxicillin on the occurrence of endodontic flare-up in asymptomatic, necrotic teeth. Seventy patients participated and had a clinical diagnosis of an asymptomatic, necrotic tooth with associated periapical radiolucency. One hour before endodontic treatment, patients randomly received either 3 g of amoxicillin or 3 g of a placebo control in a double-blind manner. After endodontic treatment, each patient received: ibuprofen; acetaminophen with codeine (30 mg); and a 5 1/2-day diary to record pain, swelling, percussion pain, and number and type of pain medication taken. The results demonstrated 10% of the 70 patients had a flare-up characterized by moderate-to-severe postoperative pain or swelling that began approximately 30 h after endodontic treatment and persisted for an average of 74 h. Of the seven patients who had flare-ups, 4 were in the amoxicillin group and 3 were not. Prophylactic amoxicillin did not significantly (p = 0.80) influence the endodontic flare-up. We concluded that a prophylactic dose of amoxicillin before endodontic treatment of asymptomatic, necrotic teeth had no effect on the endodontic flare-up.

Differential engraftment of genetically modified CD34(+) and CD34(-) hematopoietic cell subsets in lethally irradiated baboons.

OBJECTIVE: To test gibbon ape leukemia virus (GALV) pseudotype vector transduction of marrow subpopulations that contribute to hematopoietic reconstitution in vivo. MATERIALS AND METHODS: Autologous CD34(+) Lin(-), CD34(+) Lin(+), and CD34(-) Lin(-) marrow cells, transduced by coculture with PG13/LN, PG13/LNX, and PG13/LNY vector-producing cells, respectively, were transplanted in three female baboons. Two female baboons also were transplanted with fresh allogeneic CD34(-)Lin(-) marrow cells from MHC-matched male siblings and, to ensure survival, with autologous CD34(+)Lin(-) and CD34(+)Lin(+) marrow cells transduced with PG13/LN and PG13/LNX, respectively. The LN, LNX, and LNY vectors are identical except for different length sequences at the 3' end of the bacterial neomycin phosphotransferase (neo) gene. RESULTS: LN(+) and LNX(+) cells from CD34(+)Lin(-) and CD34(+)Lin(+) cells, respectively, but no LNY(+) from CD34(-)Lin(-) cells were detectable in blood and marrow of all animals after transplant. LN(+), CD34(+)Lin(-) cells contributed to reconstitution of the T, B, and myeloid lineages. LNX(+), CD34(+)Lin(+) cells contributed only to B and myeloid lineages. Male cells, CD34(-)Lin(-), were detected by polymerase chain reaction in blood and marrow of the two allogeneic transplanted animals at estimated frequencies of

Practical methods for reducing radioactive contamination incidents in the nuclear cardiology laboratory.

OBJECTIVE: The purpose of this study was to determine the extent and cause of radioactive contamination in our nuclear cardiology laboratory, and to develop possible solutions to minimize future occurrence. METHODS: We conducted a retrospective review to determine the underlying causes of the 15 minor radioactive contamination events that have occurred in the exercise areas of our laboratory since 1986. Of the 15 documented events, 8 were caused by failure of intravenous apparatus and 7 were due to syringe mishandling. Based on a staff questionnaire, we determined the most prevalent causes of radioactive contamination. Other than problems associated with intravenous setup, the causes were lack of experience by the individual performing the injection, followed closely by radioactive syringe disposal problems, injection technique, and unclear designation of duties during the exercise procedure. RESULTS: Based on these findings, we formulated a 4-part plan: a training program; a closely inspected intravenous apparatus; a mobile radioactive waste container; and a clear designation of duties for personnel to be included in the exercise procedure protocol. CONCLUSION: We have implemented a sensible and practical plan for reducing radioactive contamination, which is currently being evaluated.

A prospective study of trephined bone grafts of the tibial shaft and iliac crest.

Autogenous cancellous bone grafting is a common procedure in maxillofacial surgery. Open harvesting usually results in a long scar and considerable morbidity, but harvesting using a trephine can be done through a smaller scar with minimal morbidity. The commonly used anatomical areas for trephine harvesting are the iliac crest and the tibial shaft. A prospective study was carried out in 30 patients to compare the technique and the morbidity as perceived by patients using a visual analogue scale (VAS) and by an independent observer. The surgical anatomy and techniques are described. The results show no significant difference between the two groups, but the tibial trephine procedure is easier, quicker, and causes less blood loss. The total scores for pain and difficulty in walking were much less for tibial than that for the iliac grafts.

Severe combined immunodeficient mice engrafted with human splenocytes have functional human T cells and reject human allografts.

Previous studies have shown that human hemopoietic cells can be adoptively transferred into immunodeficient C.B-17 scid/scid (SCID) mice that lack autologous T and B lymphocytes, to generate chimeric animals. The future development of novel immunomodulatory drugs in transplantation will depend increasingly on experimental animal models to investigate the properties of the agents on human cells before starting clinical trials. However, in previous models of SCID mice engrafted with human PBLs, human T cells have been found either to be in an unresponsive state, unable to respond to mitogenic stimulations in vitro, or to mediate skin graft rejection only when HLA-primed in vivo before their adoptive transfer into SCID mice. In addition, T cells and other leukocyte subsets engraft quite poorly in the lymphoid tissues of the animals. In an attempt to develop a useful model for transplantation research, we have inoculated SCID mice with fresh human splenocytes from cadaveric organ donors (hu-Spl-SCID mice). In this model, various leukocyte subsets engraft effectively in different lymphoid compartments. In addition, human T cells retain their proliferative responses to mitogens and to alloantigens when tested 3 wk after engraftment into SCID mice. Finally, mice engrafted with unprimed human spleen cells acutely reject human foreskin allografts. Treatment of hu-Spl-SCID mice with OKT3, an immunosuppressive mAb directed against the human CD3 complex associated with the TCR, prevents the rejection of most human skin allografts, indicating a major role for human T cells in this phenomenon. Thus, this hu-Spl-SCID model may be useful for the study of immunosuppressive therapies in a preclinical in vivo setting.