Assignment of Opsonic Values to Pneumococcal Reference Serum 007sp for Use in Opsonophagocytic Assays for 13 Serotypes.

Opsonophagocytic assays (OPAs) are routinely used for assessing the immunogenicity of pneumococcal vaccines, with OPA data often being utilized for licensure of new vaccine formulations. However, no reference serum for pneumococcal OPAs is available, making evaluation of data among different laboratories difficult. This international collaboration was initiated to (i) assign consensus opsonic indexes (OIs) to FDA pneumococcal reference serum lot 007sp (here referred to as 007sp) and a panel of serum samples used for calibration of the OPA and (ii) determine if the normalization of the OPA results obtained with test samples to those obtained with 007sp decreases the variability in OPA results among laboratories. To meet these goals, six participating laboratories tested a panel of serum samples in five runs for 13 serotypes. For each serum sample, consensus OIs were obtained using a mixed-effects analysis of variance model. For the calibration serum samples, normalized consensus values were also determined on the basis of the results obtained with 007sp. For each serotype, the overall reduction in interlaboratory variability was calculated by comparing the coefficients of variation of the unadjusted and the normalized values. Normalization of the results substantially reduced the interlaboratory variability, ranging from a 15% reduction in variability for serotype 9V to a 64% reduction for serotype 7F. Normalization also increased the proportion of data within 2-fold of the consensus value from approximately 70% (average for all serotypes) to >90%. On the basis of the data obtained in this study, pneumococcal reference standard lot 007sp will likely be a useful reagent for the normalization of pneumococcal OPA results from different laboratories. The data also support the use of the 16 FDA serum samples used for calibration of the OPA as part of the initial evaluation of new assays or periodic assessment of established assays.

Trans-scleral dye injection during vitreous surgery to identify clinically undetectable retinal breaks causing retinal detachment.

BACKGROUND/AIMS: Finding all retinal breaks is a critical step in rhegmatogenous retinal detachment (RRD) surgery in order to prevent persistent/recurrent retinal detachment (RD). We describe a technique of trans-scleral dye injection into the subretinal fluid under the detached retina in the context of recurrent/persistent RD in vitrectomized eyes, in order to determine the location of clinically unidentified (occult) retinal breaks causing RD. METHODS: Retrospective consecutive single-surgeon case-series analysis of patients presenting with a repeat RRD after having been treated with pars plana vitrectomy (PPV) as the method of primary RRD repair. Trans-scleral injection of subretinal vision blue (TSVB) was used to help identify retinal breaks during repeat vitrectomy. OUTCOME MEASURES: successful detection of a break; location of breaks; persistent retinal attachment; final visual acuity (VA); complications. RESULTS: There were 395 cases of RRD during the 3-year period reviewed. TSVB was used for eight instances in seven eyes. All eight instances were repeat RRD. TSVB facilitated occult break detection in 7/8 instances of use. Breaks were at or adjacent to the previous cryo site in three instances. Persistent retinal attachment was achieved in 5/7 cases. Final VA increased in 5/7 cases. There was no evidence of complications as a result of TSVB injection. CONCLUSIONS: TSVB coupled with indentation to vent a plume of dye through an occult break during vitreous surgery is a relatively simple technique that may facilitate the identification of occult retinal breaks and help achieve anatomical success and functional success.

The immunogenicity and impact on nasopharyngeal carriage of fewer doses of conjugate pneumococcal vaccine immunization schedule.

The immunogenicity and impact on carriage of fewer doses of pneumococcal conjugate vaccine (PCV7) followed by booster with pneumococcal polysaccharide vaccine (PPV) were investigated. 684 infants were assigned randomly to one of the three groups that received one (A), two (B) or three (C) doses of PCV7 between 2 and 4 months of age, plus PPV at 10 months. Following primary vaccination protective antibody titers of >0.35 µg/ml against the PCV7 serotypes combined increased significantly with the number of PCV7 doses, 44% vs. 77% vs. 94% (p<0.001), and correlated positively with the opsonophagocytic indices, but negatively with nasopharyngeal carriage of pneumococcus. The differences in antibody responses and pneumococcal carriage between the groups diminished following booster with PPV, implying that administration of one or two doses of PCV7, with a booster dose of PPV might lower the cost of protection against IPD in young children in resource poor countries.

Opsonophagocytic activity following a reduced dose 7-valent pneumococcal conjugate vaccine infant primary series and 23-valent pneumococcal polysaccharide vaccine at 12 months of age.

Opsonophagocytic activity (OPA) was measured following reduced infant doses of 7-valent pneumococcal conjugate vaccine (PCV-7) with or without 23-valent pneumococcal polysaccharide vaccine (PPV-23) at 12 months, and subsequent re-exposure to a small dose of pneumococcal polysaccharide antigens (mPPS) at 17 months. Fijian infants were randomized to receive 0, 1, 2, or 3 PCV-7 doses. Half received PPV-23 at 12 months and all received mPPS at 17 months. OPA was performed on up to 14 serotypes. Three and 2 PCV-7 doses resulted in similar OPA for most PCV-7 serotypes up to 9 months and for half of the serotypes at 12 months. A single dose improved OPA compared with the unvaccinated group. PPV-23 significantly improved OPA for all serotypes tested but in general, was associated with diminished responses following re-challenge.

23-gauge sutureless vitrectomy and 20-gauge vitrectomy: a case series comparison.

PURPOSE: To directly compare the per-operative safety and efficacy of the 20- and 23-gauge vitrectomy systems as well as day 1 intraocular pressure (IOP). METHODS: Data were collected on 50 consecutive vitrectomy cases performed using the 20-gauge system and 23-gauge sutureless vitrectomy. All surgeries were carried out by one surgeon (RLB) at a single centre. Data collected prospectively included indication for surgery, iatrogenic retinal tears, and operating times. RESULTS: Most common indications for surgery were macular hole, rhegmatogenous retinal detachment, diabetic vitreous haemorrhage (no tractional retinal detachment), and macular pucker. Intraocular tamponade with air, sulphur hexafluoride (SF6), hexafluoroethane (C2F6) or octafluoropropane (C3F8), or silicone oil was used in 25 patients in the 20-gauge group and 46 patients in the 23-gauge group. One scleral port required suture in patients who underwent 23-gauge vitrectomy (0.67%). Every 20-gauge patient had all the three ports sutured. The mean first day IOP was 22.88 mm Hg in the 20-gauge vs 17.58 mm Hg in the 23-gauge (P<0.001). Four patients in the 20-gauge group had an IOP >40 mm Hg compared to none in the 23-gauge group. In contrast, four patients had postoperative hypotony in the 23-gauge group compared to none in the 20-gauge group. The mean operating time for all the 50 cases in each group was 39.4 (20 gauge) vs 29 min (23 gauge) P<0.001. CONCLUSION: Our study indicates less risk of considerably raised IOPs and reduced surgical operating time with the 23-gauge system. Additional advantages observed included faster wound healing, diminished conjunctival scarring, improved patient comfort, and decreased postoperative inflammation.

Surgery for bilateral macular holes.

OBJECTIVE: To report on bilateral sequential macular hole repair. DESIGN: Retrospective case series report. PARTICIPANTS: Sixteen eyes of eight patients with reduced visual acuity (VA) and metamorphopsia due to bilateral macular hole. INTERVENTION: Patients underwent vitrectomy surgery for bilateral macular hole. Seven patients had both eyes operated on consecutively at the same operating session. One patient had surgery to the fellow eye the next day. Strict facedown posturing was undertaken for 1 week by all patients. MAIN OUTCOME MEASURES: Closure of macular hole; final VA; complications. RESULTS: In 15 (94%) eyes the macular holes closed with resolution of symptoms; the mean VA at discharge was 6/15; two patients had reduced VA due to age-related macular degeneration (6/36, 6/18); five eyes had entry site tears; there was one case of lens touch with elevated intraocular pressure (IOP); in one (6%) patient the macular hole reopened after later cataract surgery (VA 6/24). CONCLUSIONS: Surgical closure of bilateral macular holes at the same operating session has distinct advantages but also considerable disadvantages. Case selection is paramount-patients require careful preoperative counselling and postoperative support, especially during the posturing week.

B lymphoblastoid cell lines as efficient APC to elicit CD8+ T cell responses against a cytomegalovirus antigen.

Potent and readily accessible APC are critical for development of immunotherapy protocols to treat viral disease and cancer. We have shown that B lymphoblastoid cell lines (BLCL) that stably express CMV phosphoprotein 65 (BLCLpp65), as a result of retroviral transduction, can be used to generate ex vivo CTL cultures that possess cytotoxicity against CMV and EBV. In this report, we demonstrate that the EBV-specific cytotoxicity in the BLCLpp65-primed culture had a spectrum of EBV-Ag recognition similar to that of the BLCL-primed counterpart, suggesting that retroviral transduction and expression of the CMV Ag would not compromise the Ag-presenting capacity of BLCL. In addition, BLCLpp65 appeared to present multiple natural pp65 epitopes, because pp65-specific CTL, which recognized different CMV clinical isolates, were generated in BLCLpp65-primed cultures from individuals with various HLA backgrounds. Consistent with a polyclonal expansion of virus-specific CTL, T cell lines established from the BLCLpp65-primed CTL cultures expressed different TCR-Vbeta Although most of the virus-specific T cell isolates were CD8+, EBV-specific CD4+ lines were also established from BLCLpp65-primed cultures. Western blot analysis revealed that the CD8+ lines, but not the CD4+ line, expressed granzyme B, consistent with features of classic CTL. Thus, our results suggested that BLCL stably expressing a foreign Ag might be used as a practical APC to elicit CD8+ T cell responses.

A phase I-II trial to examine the toxicity of CMV- and EBV-specific cytotoxic T lymphocytes when used for prophylaxis against EBV and CMV disease in recipients of CD34-selected/T cell-depleted stem cell transplants.

Epstein-Barr virus (EBV)-induced lymphoproliferative disease and cytomegalovirus (CMV) infection are major causes of morbidity and mortality in individuals with compromised cellular immunity. Although anti-viral pharmacological agents exist, severe side effects such as myelosuppression often limit the application of these medications. Infusion of ex vivo-expanded, virus-specific cytotoxic T-lymphocytes (CTL) has been proven to be safe and efficacious for the prophylaxis and treatment of EBV and CMV complications. While EBV-specific CTL can be readily and reliably produced with EBV-immortalized B-lymphoblastoid cell lines (BLCL) as stimulators, current protocols for CMV-specific CTL, which use CMV-infected fibroblasts as stimulators, may be associated with alloreactivity and the need for cloning, as well as the potential for exposure to human blood-born infectious agents. Our laboratory has developed a novel system to generate EBV/CMV-bi-specific CTL by co-culturing PBMC with autologous BLCL expressing a CMV protein pp65 (BLCLpp65) (Sun et al., 1999). pp65, an immunodominant CMV antigen, is transduced into BLCL by a recombinant retrovirus MSCVpp65. While low in alloreactivity, BLCLpp65-stimulated CTL are cytolytic to autologous cells infected with EBV or CMV, and this cytotoxicity is mediated by polyclonal, CD8+, MHC Class I-restricted T-cells. Further experiments revealed that retroviral transduction and expression of pp65 do not compromise the capacity of presenting EBV antigens, and T cells stimulated by BLCLpp65 recognize clinical strains of CMV (Sun et al., 2000). These data indicated that BLCLpp65 could substitute for BLCL as antigen presenting cells in adoptive immunotherapy against EBV-LPD, with the benefit of providing protection against CMV reactivation. This protocol is a Phase I/II study to examine the toxicity associated with and the immunologic effects of ex vivo simultaneously expanded EBV- and CMV-specific CTL for prophylaxis against EBV and CMV complications in recipients of CD34 selected/T-cell depleted stem cell transplants (SCT). EBV/CMV-specific CTL will be generated from peripheral blood mononuclear cells (PBMC) of EBV/CMV-seropositive donors in a course of from 21-28 days by weekly stimulation with autologous BLCLpp65. Qualified CTL will be administered to consenting patients at 40, 60, and 80 days post-transpOFF criteria of molecular virology and immunological reconstitution, which include blood levels of pp65 antigen and EBV viral DNA, and virus-specific CTL precursor frequency. Patients will also be tested for replication-competent retrovirus at 3, 6, and 12 month intervals post-transplant to ensure bio-safety.

Does preservative-free lignocaine 1% for hydrodissection reduce pain during phacoemulsification?

PURPOSE: To compare preservative-free 1% lignocaine with balanced salt solution (BSS) in alleviating pain during hydrodissection in phacoemulsification cataract surgery. SETTING: West Norwich Hospital, Norfolk, United Kingdom. METHODS: This prospective double-masked trial comprised 68 patients having day-case phacoemulsification cataract surgery. Patients were randomly divided into 2 groups, receiving either BSS or lignocaine 1% solution for hydrodissection during routine uneventful phacoemulsification using topical anesthesia. The level of intraoperative pain was scored on a scale of 0 (no pain) to 10 (severe pain), and the scores between the 2 groups were compared. RESULTS: Of the 68 patients, 33 (49%) received BSS and 35 (51%), lignocaine 1% solution. A pain score greater than 2 was considered clinically significant; 28 patients (85%) in the BSS group and 25 (71%) in the lignocaine 1% group scored 2 or less. The chi-square and Mann-Whitney tests found no significant difference between the BSS and lignocaine 1% groups (P = .30 and P = .432, respectively). CONCLUSION: There was no significant difference in the pain scores in patients who received BSS or lignocaine 1% solution. Thus, we conclude that hydrodissecting with lignocaine 1% solution does not provide added pain relief during phacoemulsification.

The phacoemulsification learning curve: per-operative complications in the first 3000 cases of an experienced surgeon.

PURPOSE: To assess the per-operative complications occurring during the first 3000 phacoemulsification cases performed by an experienced consultant surgeon. METHODS: A prospective analysis of 3000 consecutive cases performed without supervision between November 1992 and November 1998 was carried out. Data recorded for each case included details of per-operative complications, pre-operative best corrected visual acuity, nuclear density, history of previous pars plana vitrectomy, and whether phacoemulsification was performed as part of a phacotrabeculectomy procedure. RESULTS: The overall rate of vitreous loss was 1.3%. Nuclear fragments were lost to the vitreous in 6 cases (0.2%). The initial rate of vitreous loss was 4.0% in the first 300 cases falling to 0.7% in the last 300 cases. Capsulorhexis failure was the commonest per-operative complication observed, but the risk of subsequent posterior capsule rupture fell significantly from 9 of 45 (20.0%) in the first 100 cases to 1 of 49 (2.0%) in the next 2000 cases (p = 0.0061, Fisher's exact test). There was a significant increase in risk with denser cataracts, especially for capsulorhexis failure, rising to over 35% in the densest cases. The increases in posterior capsule rupture and vitreous loss were less dramatic but nonetheless very significant. There was no significant increase in the risk of per-operative complications with phacotrabeculectomy, and no increased risk in patients who had previously undergone pars plana vitrectomy. Posterior capsule rupture occurred in 22 of 612 (3.6%) local anaesthesia cases compared with 31 of 2269 (1.4%) topical anaesthesia cases. Per-operative best corrected visual acuity of 6/9 or better was recorded in 2.0% of the first 1000 cases compared with 13.9% of the last 1000 cases. CONCLUSIONS: (1) Per-operative surgical risks could be reduced to low levels during the learning curve, but complications continued to occur at a low frequency. (2) The risk of per-operative complications was not significantly elevated in previously vitrectomised eyes. (3) Nuclear density correlated significantly with per-operative complication risk. (4) The visual threshold for cataract surgery fell dramatically with increasing experience of phacoemulsification. (5) Topical anaesthesia was not associated with an increased risk of per-operative complications.

Phacoemulsification cataract surgery: is routine review necessary on the first post-operative day?

PURPOSE: To determine the value of routine review on the first post-operative day following phacoemulsification cataract surgery. METHODS: A prospective study was performed of 238 consecutive patients who underwent phacoemulsification cataract surgery. Local anaesthesia was used for 97% of patients and surgery was performed as a day-case procedure for 93% of patients. The findings at the first day post-operative review were analysed separately for patients who had undergone uncomplicated surgery and patients who had suffered an intraoperative complication. Four patients were excluded because of incomplete data collection. RESULTS: A total of 227 patients underwent uncomplicated phacoemulsification cataract surgery. Thirteen (5.7%, 95% confidence interval (CI) 3.1-9.6%) of these were found to have post-operative complications at their first day review which comprised corneal oedema (4.4%, 95% CI 2.1-8.0%), raised intraocular pressure > or = 30 mmHg (1.3%, 95% CI 0.3-3.8%), hyphaema (0.9%, 95% CI 0.1-3.1%), corneal abrasion (0.4%, 95% CI 0.0-2.4%) and anterior uveitis (0.4%, 95% CI 0.0-2.4%). These findings led to the standard post-operative management being altered for 5 (2.2%) patients. Intraoperative complications occurred in 7 (2.9%) patients during phacoemulsification cataract surgery. Five (71%) of these patients had post-operative complications at their first day review. CONCLUSIONS: Routine review on the first post-operative day following uncomplicated phacoemulsification cataract surgery could safely be withdrawn. A single post-operative review at 1-2 weeks after surgery would then be required, supplemented by patient-initiated post-operative review in the interim.

Simultaneous ex vivo expansion of cytomegalovirus and Epstein-Barr virus-specific cytotoxic T lymphocytes using B-lymphoblastoid cell lines expressing cytomegalovirus pp65.

Cytomegalovirus (CMV) infection and Epstein-Barr virus (EBV)-induced lymphoproliferative disease are serious complications associated with allogeneic stem cell transplantation. Immunotherapy using ex vivo expanded, virus-specific cytotoxic T lymphocytes (CTL) has been explored and proven to be effective in therapeutic or prophylactic regimens for CMV and EBV infections. To generate CTL specific for both CMV and EBV, we engineered EBV-transformed B-lymphoblastoid cell lines (BLCL) to express CMV pp65 for use as antigen-presenting cells (APC). BLCL were transduced with a recombinant retrovirus encoding pp65, the immunodominant CMV polypeptide. Western blot analysis and immunocytochemistry confirmed the expression of pp65 in the transduced cells. Peripheral blood mononuclear cells (PBMC) from healthy CMV seropositive donors were stimulated with autologous pp65-expressing BLCL weekly for 3 weeks. Chromium release assays showed that the resulting CTL cultures possessed specific cytotoxicity against EBV and CMV. Recombinant vaccinia viruses encoding individual CMV peptides were used to demonstrate that this CMV-specific cytotoxicity was specific for pp65. Assays on CD4- and CD8-depleted CTL fractions indicated that CD8(+) CTL mediated the pp65-specific cytotoxicity. These CMV/EBV-specific CTL recognized CMV- and EBV-infected targets sharing HLA class I antigens, but not HLA mismatched targets. Our results demonstrate that BLCL can be used as APC to stimulate expansion of EBV- and CMV-specific CTL simultaneously. These findings have potential implications for posttransplant CMV and EBV immunotherapy in recipients of allogeneic stem cell transplants.

CD4(+) Epstein-Barr virus-specific cytotoxic T-lymphocytes from human umbilical cord blood.

Umbilical cord blood (CB) is increasingly used for allogeneic hematopoietic stem cell transplantation. To determine whether viral antigen-specific cytotoxic T-lymphocytes (CTL) could be generated from the predominantly naive T-cell populations in CB, CB-derived mononuclear cells were stimulated with autologous Epstein-Barr virus (EBV) transformed B-lymphoblastoid cell lines over several weeks in the presence of recombinant human interleukin-2 (IL-2). By 28 days of culture, T-lymphocytes from all six CB that had been treated with IL-2 displayed EBV-specific cytotoxicity. These cells were largely CD4(+), with complete inhibition of cytotoxicity by anti-CD3 and variable inhibition by anti-HLA DR monoclonal antibodies. The EBV-specific effectors were cloned by limiting dilution, and most of the CTL clones were CD4(+). The cytotoxicity of the CB-derived CD4(+) CTL clones was inhibited by EGTA but not by anti-Fas ligand mAb, suggesting that this cytotoxicity was mediated by perforin/granzyme B. These data indicate that virus-specific CTL can be cultivated and cloned from CB, a human T-cell source that may not have prior in vivo antigenic exposure or reactivity. This finding may have applications in adoptive immunotherapy to recipients of CB transplants.

Primary vitrectomy for pseudophakic and aphakic retinal detachments.

PURPOSE: To evaluate primary vitrectomy for the treatment of pseudophakic and aphakic retinal detachments. Primary vitrectomy may enable better identification of retinal breaks than scleral buckling procedures. METHODS: A prospective study was performed of primary vitrectomy for the treatment of 25 consecutive cases of pseudophakic and aphakic retinal detachment. RESULTS: The primary retinal reattachment rate was 84% (21 eyes). Surgical failure resulted from new/missed retinal breaks (2 eyes) and proliferative vitreoretinopathy (2 eyes). The final retinal reattachment rate with further surgery was 96% (24 eyes). There were 7 macula-on detachments which all retained their presenting visual acuity. A visual acuity of 6/18 or better was achieved by 56% of the 18 macula-off detachments. Visualisation of the peripheral retina was impaired in 17 eyes and procedures to improve visualisation were performed in 7 eyes. Retinal breaks were detected in 16 eyes at surgery that had not been identified pre-operatively. Raised intraocular pressure was the most common complication, affecting 10 eyes in the early post-operative period. CONCLUSIONS: Primary vitrectomy offers certain advantages in the treatment of pseudophakic and aphakic retinal detachments. A controlled study is required to determine whether primary vitrectomy achieves a better outcome than scleral buckling procedures for these retinal detachments.

Contrast sensitivity after implantation of diffractive bifocal and monofocal intraocular lenses.

PURPOSE: To compare contrast sensitivity (CS) after implantation of a diffractive bifocal intraocular lens (IOL) and a monofocal IOL of similar design. SETTING: Seven European centers. METHODS: In this randomized, prospective study, CS was tested 5 months after cataract and IOL implantation surgery in 115 patients with a diffractive bifocal IOL and 106 patients with a monofocal IOL. It was also tested in a subgroup of 38 patients who had bilateral implantation of a diffractive bifocal IOL. Contrast sensitivity was tested using the Vision Contrast Test System (VCTS). RESULTS: In patients with a best corrected visual acuity (BCVA) of 1.0 or better, the CS at all spatial frequencies (1.5 to 18 cycles/degree), both at distance and near, was slightly lower in the bifocal IOL group than in the monofocal group. Mean values were within the normal range. In patients with a BCVA of less than 1.0, the CS was lower and the difference between the bifocal and monofocal groups was less. In patients with bilateral bifocal IOLs, CS was better when tested bilaterally than when testing the better eye alone. Pupil size affected the results to a small degree. Contrast sensitivity appeared to improve over time after implantation of a diffractive bifocal IOL. CONCLUSIONS: In patients with cataract and no other eye pathology, the diffractive bifocal IOL with slightly reduce the CS at all spatial frequencies. In those with reduced visual acuity after cataract surgery, CS will be reduced accordingly. In this situation, the reduction from the diffractive bifocal optic would be minor.