Service evaluation: orthoptic-led teaching of soft contact lens handling for parents in the management of pediatric aphakia.

To gauge parental satisfaction of an orthoptic-led specialist service for teaching soft contact lens (CL) handling in the management of children with pediatric aphakia. 20 families attending the contact lens clinic completed a satisfaction questionnaire to enquire about their experience of learning how to insert and remove their child's CL. Families were questioned on their experiences with preoperative counseling, practical teaching, additional support and the patient literature that was provided by the orthoptists in the CL clinic. Qualitative data and free comments were analyzed. 18/20 (90%) said they had received good practical insertion and removal teaching. 19/20 (95%) felt they received good emotional support. Only 6/20 (30%) families agreed with the statement that they found learning insertion and removal difficult. 15/20 (75%) families did not feel rushed, and 18/20 (90%) said they could learn at their own pace. All except one family (95%) achieved daily CL handling within a few months, with ongoing support from a multi-disciplinary team. One family surveyed was much earlier in their postoperative journey, but was on track to achieve this imminently. All families (100%) felt they were appropriately counselled preoperatively regarding the need for training and daily CL handling. 95% found the provided written information useful. 8/20 (40%) reported they found insertion harder than removal, 4/20(20%) reported they found removal harder. Teaching insertion and removal skills is an important aspect of managing paediatric aphakia and presents many challenges. Most parents eventually cope well and achieve daily CL handling within a few months, with support from a multi-disciplinary team. Families surveyed were all competent and were satisfied with their experience. The parental satisfaction survey gave our team confidence that our orthoptic-led service works well, orthoptists have the knowledge and skills to provide technical training to parents alongside vital emotional support, and contact lens handling is a rewarding extended role for orthoptists in a multi-disciplinary team.

Arthropathy in hereditary haemochromatosis segregates with elevated erythrocyte mean corpuscular volume.

Objective: To evaluate the relationship between erythrocyte parameters and the presence or absence of arthritis in HFE C282Y homozygous hereditary haemochromatosis (HH) subjects compared to control groups of non-HH subjects with arthritis.Method: Erythrocyte and arthritis parameters [mean corpuscular volume (MCV) and mean cell haemoglobin (MCH)] were obtained from consecutive HH subjects (n = 119) who were referred for initial evaluation and management. For comparison, MCV and MCH values were collected from randomly selected non-HH subjects with rheumatoid arthritis (n = 100) and osteoarthritis (n = 100), consisting of equal numbers of men and women. Two other comparison groups comprised 16 men and women who were heterozygous for C282Y with arthritis, and 38 non-HH subjects with type 2 polyarticular osteoarthritis (T2POA).Results: MCV values were significantly higher in HH subjects with arthritis (95 ± 0.56 fL) than in HH subjects without arthritis (92.75 ± 0.50 fL, p = 0.037). HH subjects with or without arthritis demonstrated a higher mean MCV than the control groups of non-HH osteoarthritis (90.12 ± 0.46 fL, p < 0.001) and non-HH rheumatoid arthritis (90.94 ± 0.57 fL, p < 0.001). HH subjects with arthritis also demonstrated a higher MCV than heterozygous C282Y subjects with arthritis (93.18 ± 1.55 fL, p = 0.025) and non-HH subjects with a similar pattern of arthritis, notably T2POA (91.13 ± 0.50 fL, p < 0.01). An MCV of ≥ 97.85 fL provided a likelihood ratio of 2.2 for development of arthritis in HH subjects.Conclusion: This study demonstrated a relationship between elevated MCV and arthritis in incident cases of HH.

The flicker response of venous oxygen saturation is significantly reduced in the early and late stages of age-related macular degeneration.

BACKGROUND: Retinal oxygen saturation (SO2) during flicker light stimulation was measured non-invasively in humans with age-related macular degeneration (AMD). Furthermore, the differences between early and late stages of AMD were evaluated. METHODS: In 60 eyes of 45 AMD patients (74 ± 8.3 years) and 23 eyes of 23 healthy controls (73.4 ± 7.4 years), the SO2 of arterioles and venules was measured with the oximetry module of the Retinal Vessel Analyzer. Arterial SO2, venous SO2 and arteriovenous SO2 difference at baseline and with the flicker were assessed and compared with controls. From the difference between the arteriovenous SO2 under flicker stimulation and at baseline, the parameter delta av. Diff was calculated. Subgroup analyses of non-exudative (dry) AMD, exudative (wet) AMD and their end stages, geographic atrophy (GA) and disciform scar (DS) were performed. RESULTS: In comparison with healthy subjects (mean - 4.90, CI [- 6.32, - 3.43]), the parameter delta av. Diff was significantly reduced in all AMD patients (mean - 2.20, CI - 3.15, -1.23, p = 0.003), dry AMD (mean - 1.97, CI - 3.31, -0.63, p = 0.013) and wet AMD (mean - 2.35, CI - 3.50, - 1.19, p = 0.025). The comparison between wet and dry AMD revealed no significant results (p = 1). The comparison between AMD subgroups and healthy controls (median (IQR) - 4.29 (- 8.32; - 2.42) %) showed significant differences in non-neovascular (early dry AMD) (median (IQR) - 2.43 (- 4.59; - 0.74) %, p = 0.038), GA (median (IQR) 0.10 (- 4.02; 3.15) %, p = 0.019) and DS (median (IQR) - 1.67 (- 3.52; - 0.12) %, p = 0.03). A nearly significant reduction was observed in exudative (early wet) AMD (median (IQR) - 2.71 (- 5.84; - 0.2) %, p = 0.055). Minimal, not statistically significant differences of delta av. Diff were found between AMD subgroups. None of the baseline parameters was significantly different between patients and healthy controls, even after flicker light stimulation. CONCLUSIONS: Non-invasive retinal oximetry with flicker light stimulation seems to be a suitable method to study the pathogenetic mechanisms of AMD. The mathematically derived parameter delta av. Diff appears to be more sensitive than arteriovenous SO2 difference. Results suggest that the regulation of retinal oxygen supply, oxygen consumption or both is impaired in AMD.

Investigation of blood flow regulation and oxygen saturation of the retinal vessels in primary open-angle glaucoma.

PURPOSE: To examine the supply of oxygen to the retina in primary open-angle glaucoma (POAG). METHODS: Forty-one patients with primary open-angle glaucoma (mean age 64.1 ± 12.9 years) and 40 healthy subjects (63.6 ± 14.1 years) were included. Fundus images, centered at the optic disc, were taken using the Retinal Vessel Analyzer (RVA). The vessel diameters were calculated as central retinal artery (CRAE) and vein equivalent (CRVE) from diameter measurements in the peripapillary vessels. The oxygen saturation of the arteries and veins was investigated employing a two-wavelengths technique. After the measurement at baseline, the vascular response to flicker light exposure was measured. RESULTS: In glaucoma patients the mean oxygen saturation of the retinal veins at baseline was higher than in the healthy controls (64.36 ± 7.11 vs. 59.78 ± 8.47, p = 0.01), whereas the mean arteriovenous oxygen saturation difference was lower (33.07 ± 5.24 vs. 37.53 ± 6.95, p = 0.002). The arterial oxygen saturation as well as the arterial and venous diameters showed no difference between the groups. The increase of the CRVE during flicker light stimulation (3.72 ± 3.29 % vs. 5.43 ± 4.04, p = 0.039), as well as the change of the venous oxygen saturation (2.08 ± 3.74 % vs. 4.18 ± 3.88 %, p = 0.016) and the arteriovenous saturation difference (-2.1 ± 3.31 % vs. -4.43 ± 3.6 %, p = 0.003) were smaller in POAG patients than in the healthy group. CONCLUSIONS: The reduction in the arteriovenous difference in oxygen saturation in POAG patients might show a decreased oxygen demand of the retina caused by the glaucomatous loss of neuroretinal tissue. The lower extent of the flicker light-induced change of the diameter of retinal veins and the venous oxygen saturation could indicate an impairment of blood flow regulation.

Single-cell analysis by RT-PCR reveals differential expression of multiple type 1 and 2 cytokine genes among cells within polarized CD4+ T cell populations.

RT-PCR was used to examine the expression of IFN-gamma, IL-2, IL-4, IL-5, IL-6 and IL-10 mRNAs by single murine CD4+ T cells activated either in a strongly type 1-polarized mixed lymphocyte reaction (MLR) or in the type 2-polarized response to immunization with keyhole limpet hemocyanin (KLH) in alum. The frequencies of expression of each cytokine differed markedly between the two responses, consistent with their polarization at the population level. However, most cells expressed only none to three of the six cytokines assayed, few displayed the canonical type 1 profile and none in either response expressed a full type 2 or type 0 profile. A significant fraction of cells co-expressed IFN-gamma with IL-4 and/or other type 2 cytokines at frequencies that suggested that most of these genes were independently regulated. Collectively, these single-cell expression patterns indicate that polarization at the population level can mask substantial intercellular heterogeneity, and show directly that multiple type 1 and 2 cytokines can be expressed simultaneously in an individual T cell.

The CD4+ T-cell response to protein immunization is independent of accompanying IFN-gamma-producing CD8+ T cells.

By virtue of their strong bias towards production of interferon-gamma (IFN-gamma), CD8+ T cells have the potential to promote the development of type 1 immune responses. We have previously shown that the CD4+ T-cell response to immunization with the protein antigen keyhole limpet haemocyanin (KLH) has a mixed interleukin-4 (IL-4)/IFN-gamma production profile. Here we show that this immunization regimen also stimulates accumulation in the draining lymph nodes of CD8+ T cells, which preferentially contain IFN-gamma mRNA ex vivo and secrete IFN-gamma protein in vitro. This provides a model to test whether CD8+ cell-derived IFN-gamma participates in the normal control of the immune response to a non-viable exogenous antigen. To investigate regulation of the anti-KLH response by the CD8+ population or IFN-gamma produced by this or other cell types, mice were administered depleting antibodies. Depletion of CD8+ cells had no effect on the frequency of clonogenic KLH-specific CD4+ T cells, the IL-4/IFN-gamma profiles of their progeny, or the isotype profiles of the serum antibody response to KLH. In contrast, IFN-gamma neutralization diminished cell accumulation in the lymph nodes and reduced both the frequency of KLH-specific CD4+ T cells that gave rise to IFN-gamma-producing clones and serum titres of KLH-specific IgG2a and IgG3. Therefore, despite the potential for cross-regulation, the CD4+ T-cell response to this immunogen is independent of the IFN-gamma-skewed CD8+ response.

Factors effecting the measurement of tumor-associated MUC1 mucins in serum.

MUC1 epithelial mucins are produced by both normal and malignant epithelial cells. Serum proteins reactive with monoclonal antibodies against MUC1 mucins were studied using several techniques. Separation of proteins by native PAGE showed that anti-MUC1 core protein antibodies reacted with a high M(r) mucin but also with a 70-kD protein (p70) in normal women and women with ovarian cancer. After purification by gel filtration, p70 was not reactive in a double-determinant ELISA (CASA) and only weakly reactive in an inhibition assay. Serum CASA levels increased during pregnancy but p70 disappeared. Neuraminidase treatment of serum resulted in a greater increase in CASA in normal women and in ovarian cancer patients with low initial CASA than in ovarian cancer patients with high CASA and pregnant women (p < 0.05). These findings suggest that pregnancy and ovarian cancer-derived mucins are less heavily sialylated than mucin derived from normal tissues. An inhibition assay was developed which was more sensitive, but provided no diagnostic advantage over CASA. MUC1 is present in the serum of all women, reactivity in assays utilizing core protein antibodies is probably dependent not only on molar concentration but on the degree of exposure of peptide epitopes.

The use of mucin-specific monoclonal antibodies and lectins in the detection of tumor-associated serum markers in gynecological cancer.

The use of the mucin-specific lectin from Sambucus sieboldiana (SSAM) in the detection of tumor-associated serum antigens produced by patients with ovarian, cervical, and uterine cancer was investigated. Two-site assays were developed which used either SSAM or the MUC1 core protein-specific monoclonal antibody (mab) BC2 as capture, and biotinylated SSAM to detect bound mucin (SSAM and BC2SSAM assays respectively). These new assays were compared to the CA125 assay, and another assay for MUC1 (CASA), which utilizes the core protein reactive mabs BC2 and BC3. some asymptomatic women and patients with benign disease showed very high levels in the SSAM assay, while this was not the case in the other assays. When cutoff levels were set to exclude healthy women and patients with benign disease, the levels of detection in patients with ovarian cancer were 51% with CASA (> 6.7 units/ml), 71% with CA125 (> 250 units/ml), and 38% with BC2SSAM (> 8.6 units/ml). The levels of detection in cervical and uterine cancer patients were 28% and 25% with CASA, 0% and 8% with CA125, and 28% and 25% with BC2SSAM respectively. Of particular interest was the very different spectrum of reactivity observed with the CASA and BC2SSAM assays which use the same capture mab, indicating that each assay detects different glycoforms of the MUC1 mucin. Indeed, when used in combination, the CASA and BC2SSAM assays gave 62% of ovarian cancer patients, and 50% of cervical or uterine cancer patients with elevated marker levels. The additional use of BC2SSAM gave no advantage over the combined use of the CASA and CA125 assays in ovarian cancer, with 80% of patients detected, but the CASA/BC2SSAM combination was particularly useful in the cervical and uterine cancers due to the low level of detection with CA125. In fact, the additional use of CA125 gave no advantage over the CASA/BC2SSAM combination in these patients. Furthermore, the BC2SSAM assay may also be useful in monitoring patients with high preoperative BC2SSAM levels (> 10 units/ml), since this assay predicted recurrence in 5/5 cases, and was negative in all cases with no evidence of disease. Furthermore, the performance of this assay in monitoring these patients was equal or superior to CA125 and CASA.

Serum mucin antigens CASA and MSA in tumors of the breast, ovary, lung, pancreas, bladder, colon, and prostate. A blind trial with 420 patients.

BACKGROUND: The tumor markers CASA (cancer-associated serum antigen) and MSA (mammary serum antigen) have previously been shown to be useful in the clinical management of ovarian and breast carcinoma, respectively, but have not been assessed in other types of cancer. These assays were compared with carcinoembryonic antigen (CEA) and prostate-specific antigen (PSA) in a blind trial using sera from the Mayo Clinic-National Cancer Institute (NCI) Diagnostic Serum Bank. METHODS: CASA and MSA were assessed retrospectively in a blind trial using 465 serum samples from the Mayo Clinic-NCI Diagnostic Serum Bank representing malignant and benign disease of the breast, ovary, lung, pancreas, bladder, colon, and prostate and age-matched and gender-matched healthy control donors. CASA, MSA, and PSA levels were determined using commercially available kits, and CEA values and clinical details were later provided by the Mayo Clinic. RESULTS: CASA and MSA showed good reproducibility in 45 duplicate samples. CASA values were significantly elevated in the serum of patients with malignant tumors of the breast (44%), ovary (58%), lung (56%), prostate (48%), and bladder (54%), but not in those with benign conditions of these organs or pancreatic or colon cancer. MSA levels were only elevated significantly in cancers of the breast (52%) and ovary (58%). CASA showed significantly better sensitivity than either CEA (20%) or MSA (25%) in the detection of lung cancer, whereas CEA showed significantly superior detection of colon cancers (78%). CASA was not as sensitive as PSA in prostate cancer (48% versus 96%), but gave superior specificity in nonmalignant conditions of the prostate (93% versus 70%), although this was not statistically significant. CONCLUSIONS: The commercial CASA and MSA assays are reliable and reproducible tests for these tumor markers. In addition to ovarian cancer, CASA is also elevated significantly in many patients with breast, lung, prostate, and bladder cancer and has potential clinical use in patients with these tumors. The use of the MSA assay appears restricted to breast cancer.

Circulating tumour-associated mucin concentrations, determined by the CASA assay, in healthy women.

This investigation was undertaken to establish a reference range for tumour-associated MUC1 mucins in the serum of healthy women of the ages at risk for adenocarcinoma of the ovary and breast. Blood samples and clinical information were obtained from 5,000 women attending a breast screening mammography clinic. Data from women diagnosed with breast carcinoma and those subsequently diagnosed with other cancers were omitted from the reference range. Mucin concentrations were measured using the CASA assay which detects the protein core of MUC1 encoded mucins. Multiple linear regression analysis showed no effect on CASA concentrations by non-malignant changes to the breast, menopausal status, presence/absence of the reproductive tract, parity or history of hormone use. However, CASA concentrations were significantly increased in smokers (P < 0.001) and progressively increased with age (P < 0.001). These data show that these factors must be given consideration when setting upper limits of normal using MUC1 protein core binding assays.

The management of ovarian carcinoma is improved by the use of cancer-associated serum antigen and CA 125 assays.

BACKGROUND: The new tumor-associated mucin assay, cancer-associated serum antigen (CASA), was assessed with the CA 125 assay for use in the management of patients with epithelial ovarian cancer. METHODS: CASA and CA 125 were assessed retrospectively for use in (1) monitoring 28 patients with Stage 3 or 4 ovarian carcinoma during therapy, (2) predicting the outcome of 41 second-look laparotomies (SLL), and (3) predicting the survival outcome by measuring these levels after surgery but before chemotherapy in 65 patients with Stage 3 disease. RESULTS: Of 20 patients with recurrence after an initial response, the presence of CASA levels detected recurrence in 65% before clinical detection; CA 125, 50%; and the combination of CASA and CA 125, 80%. Six patients whose disease was in long-term remission did not have elevations of either marker. When used to predict the results of SLL, the positive predictive values of CASA and CA 125 were 77% and 100%, respectively. The negative predictive values for CASA and CA 125 were 71% and 66%, respectively. CASA detected 50% of positive SLL where microscopic disease only was found; the CA 125 test did not. Multivariate analysis of survival rates using levels of CASA and CA 125, age, residual disease, tumor type and grade, or the presence or absence of cisplatin in the chemotherapeutic regimen found that postoperative CASA levels ranked above all prognostic factors except age. CASA levels may be more accurate than surgical reporting of residual disease or they may define a subset of patients with biologically more aggressive ovarian carcinoma. CONCLUSIONS: The CASA test is sensitive to ovarian carcinoma, and both CASA and CA 125 are more useful when used in conjunction.

Comparative study of mammography and mammary serum antigen estimation for breast cancer screening.

OBJECTIVE: To compare mammary serum antigen (MSA) levels with mammography as a screening test for breast cancer. To determine the value of MSA testing to decrease the need for women to undergo mammography. DESIGN: A blind prospective comparison of MSA levels and mammography to detect breast cancer. SETTING: Royal Women's Hospital Breast Cancer Screening Clinic. Women were mainly self-referred. RESULTS: MSA levels had a wide range in normal women and women with mammography-detected breast cancer. Mean MSA levels in women with breast cancer reflected tumour volume, but a wide range was again seen. At 60% specificity, the sensitivity of an elevated MSA level for breast cancer was 63% for invasive cancer and zero for in-situ disease. MSA levels were modestly but significantly elevated in smokers over non-smokers. CONCLUSION: The MSA level is an insufficiently sensitive or specific marker to have a role in screening for breast cancer.

Preoperative discrimination between ovarian carcinoma, non-ovarian gynecological malignancy and benign adnexal masses using serum levels of CA125 and the polymorphic epithelial mucin antigens CASA, OSA and MSA.

Serum levels of the tumor associated antigens CA125, CASA, OSA and MSA were determined preoperatively in a non-consecutive series of patients with: invasive epithelial ovarian cancer (OC, n = 87), ovarian tumors of low malignant potential (LMP, n = 9), benign adnexal masses (BAM, n = 48) and other peritoneal and pelvic malignancies (n = 48). In addition, serum levels of CASA, OSA, and MSA were determined in 3477 asymptomatic well women. Ninety-eight percent of the asymptomatic women had CASA levels < 6.0 U ml-1, OSA levels < 5.5 U ml-1 and MSA levels < 80.0 U ml-1. Serum CA125 levels were> 35 U ml-1 in 89% of OC, in 44% of LMP, and in 23% of BAM. Serum CASA levels were> 6.0 U ml-1 in 58% of OC, in 0% of LMP, and in 0% of BAM. Serum OSA levels were> 5.5 U ml-1 in 61% of OC in 0% of LMP and in 4% of BAM. Serum MSA levels were> 80.0 U ml-1 in 56% of OC, in 11% of LMP, and in 10% of BAM. When cut-off levels were set to exclude all patients with BAM, the best discrimination from OC using a single assay was achieved using CASA (58%). However, a combination of CASA and CA125 gave positive levels in 69% of OC at levels which precluded BAM. All markers were also elevated in some colon cancers, cervical cancers, uterine cancers and other peritoneal malignancies. A combination of CA125 and CASA levels, obtained preoperatively may assist the general gynecologist in avoiding potentially difficult oncologic surgery.

Expression of tumour markers CA125, CASA and OSA in minimal/mild endometriosis.

Ovarian cancer associated antigens CA125, CASA and OSA were measured in serum from 23 patients with mild endometriosis before, during and after medical therapy. Pretreatment CA125 levels were elevated above 35 mu/ml in 4(17%), and above 25 mu/ml in 7 (30%) patients. Mean CA125 levels decreased during treatment, but in only 10 patients did levels reflect disease response. There was no correlation between CA125 levels and disease severity as measured by modified American Fertility Society Scoring. Neither CASA nor OSA were detected in these women.

Quantitative analysis of adenine nucleotides during the prelytic phase of cell death mediated by C5b-9.

The nucleated cell death mediated by C5b-9 depends on the extent of C fixation and parameters that affect the ability of the cell to eliminate C5b-9. When C5b-9 formation exceeds elimination, cell death can be initiated. High Ca2+ in the medium accelerates Ehrlich ascites cell death induced by a large number of C5b-9, whereas osmotic prevention of cell swelling has little effect in protecting Ehrlich cells from killing by C5b-9. In the present study, we investigated the interrelationship between intracellular Ca2+, intra- and extracellular adenine nucleotides, and mitochondrial membrane potential, to understand the mechanism of acute cell death induced by C5b-9. When Ehrlich cells carrying C5b-8 were exposed to C9, rapid and profound ATP depletion in the cell was observed before cell death. Leakage of the adenine nucleotides ATP, ADP, and AMP also began during the prelytic phase. Studies using digital imaging fluorescence microscopy showed that loss of mitochondrial membrane potential was noted immediately after C9 addition but before nuclear staining with propidium iodide. These findings suggest that an increase in intracellular Ca2+ through C5b-9 channels and loss of mitochondrial membrane potential may initiate rapid cell death. The prelytic leakage of ATP precursors may also contribute to cell death by decreasing nucleotide pools, because recovery of ATP production was observed after a similar degree of ATP loss in cells exposed to sublethal doses of KCN, in which ADP and AMP leakage was not present.

Role of complement C9 and calcium in the generation of arachidonic acid and its metabolites from rat polymorphonuclear leukocytes.

We have previously shown that antibody-sensitized mouse peritoneal macrophages release arachidonic acid (C20:4) and its oxygenated derivatives when treated with complement, and that the major part of the release depended on the terminal complement complexes (TCC). To further delineate the process(es) responsible for this release we have extended our studies to rat peritoneal polymorphonuclear leukocytes (PMNs). Experiments were performed with antibody-sensitized rat PMNs labeled with [3H]C20:4 and carrying the TCC, C5b-7, C5b-8 or C5b-9. In contrast to the results of other studies, production of leukotriene B4 (LTB4), the major radiolabeled derivative, was strictly dependent on the presence of C9. However, low levels of C20:4 and prostaglandins (PGs) were produced prior to the C5b-9 stage. Kinetic studies demonstrated that release of LTB4 was rapid; the initial release occurred within 4-6 min and a second rise in release coincided with cell death. Virtually all the LTB4 produced was released as we found no evidence of retention of intracellular LTB4 at either the C5b-8 or C5b-9 stages. In the absence of extracellular calcium, the release of LTB4 was completely abolished and the release of C20:4 and PGs was drastically reduced. [3H]C20:4-labeled PMNs carrying C5b-9 did release substantial amounts of radiolabeled material in the presence of EGTA; however, the majority of this lipid was in the form of intact phospholipid and triglyceride. These results indicate that release of C20:4 and its oxygenated derivatives from rat PMNs is (1) dependent on the participation of C9 in the preexisting C5b-8 complex in the cell membrane, and (2) largely dependent on the presence of calcium.

Release of arachidonic acid and formation of oxygenated derivatives after complement attack on macrophages: role of channel formation.

Treatment of [3H]arachidonic acid ([3H]C20:4)-labeled, antibody-sensitized mouse resident peritoneal macrophages with rabbit serum complement, or C6-deficient rabbit serum + C6, caused hydrolytic release of incorporated [3H]C20:4 from phospholipids, followed by conversion to oxygenated derivatives. The C6 dose-response curve for release of C20:4 plus its metabolites was monotonic, which indicates dependence on channel formation, whereas the dose-response curve for lysis displayed multi-hit behavior. High-performance liquid chromatography demonstrated that the major radiolabeled products in the aqueous phase co-eluted with C20:4, 6-keto-prostaglandin F1 alpha (6-keto-PGF1 alpha), and prostaglandin E2. Kinetic studies of the release of 6-keto-PGF1 alpha, the major metabolite, displayed biphasic characteristics; a moderate amount of this prostaglandin was released before the onset of cell lysis. Experimental evidence obtained by freeze-thaw or by incubation of these cells with melittin or A23187 indicated that cell lysis does not necessarily result in the production of inflammatory mediators. Furthermore, when macrophages were treated with serum complement, it was apparent that the major part of the release was due to C5b-9 and not to the action of C5a. We conclude that release of C20:4 and its derivatives from complement-treated macrophages does not depend on cytolysis, but is a consequence of insertion and channel formation.

Complement lysis of U937, a nucleated mammalian cell line in the absence of C9: effect of C9 on C5b-8 mediated cell lysis.

Previous studies have demonstrated that in general, nucleated cells are more resistant to killing by serum complement than are erythrocytes. During studies aimed at defining the mechanisms of nucleated cell resistance, we found that the human histiocytic cell line U937 was easily lysed by homologous serum. U937 cells were also killed by serum depleted of C9, but not by serum depleted of C8, implying that the C5b-8 complex was sufficient to cause lysis of these cells. Enumeration of complexes on the cell surface demonstrated that approximately 40-fold more complexes were required to lyse U937 cells in the absence of C9 than in the presence of an excess of C9. Examination of the effects of small amounts of C9 on lysis of U937 cells by the C5b-8 complex demonstrated that at very low doses, C9 inhibited C5b-8 mediated lysis. The use of radiolabeled anti-C8 antibody showed that C5b-8 complexes were eliminated from the surface of U937 cells at 37 degrees C, and C9 at the dose causing inhibition of lysis accelerated the elimination of complexes. These results suggest that the increased lytic potential resulting from binding of small amounts of C9 to C5b-8 complexes is outweighed by enhanced elimination of complexes resulting in decreased cell death.